High Proliferation Predicts Pathological Complete Response to Neoadjuvant Chemotherapy in Early Breast Cancer

PubMed Central

Lluch, Ana; Ribelles, Nuria; Anton-Torres, Antonio; Sanchez-Rovira, Pedro; Albanell, Joan; Calvo, Lourdes; García-Asenjo, Jose Antonio Lopez; Palacios, Jose; Chacon, Jose Ignacio; Ruiz, Amparo; De la Haba-Rodriguez, Juan; Segui-Palmer, Miguel A.; Cirauqui, Beatriz; Margeli, Mireia; Plazaola, Arrate; Barnadas, Agusti; Casas, Maribel; Caballero, Rosalia; Carrasco, Eva; Rojo, Federico

2016-01-01

Background. In the neoadjuvant setting, changes in the proliferation marker Ki67 are associated with primary endocrine treatment efficacy, but its value as a predictor of response to chemotherapy is still controversial. Patients and Methods. We analyzed 262 patients with centralized basal Ki67 immunohistochemical evaluation derived from 4 GEICAM (Spanish Breast Cancer Group) clinical trials of neoadjuvant chemotherapy for breast cancer. The objective was to identify the optimal threshold for Ki67 using the receiver-operating characteristic curve method to maximize its predictive value for chemotherapy benefit. We also evaluated the predictive role of the defined Ki67 cutoffs for molecular subtypes defined by estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). Results. A basal Ki67 cutpoint of 50% predicted pathological complete response (pCR). Patients with Ki67 >50% achieved a pCR rate of 40% (36 of 91) versus a pCR rate of 19% in patients with Ki67 ≤50% (33 of 171) (p = .0004). Ki67 predictive value was especially relevant in ER-HER2− and ER-HER2+ patients (pCR rates of 42% and 64%, respectively, in patients with Ki67 >50% versus 15% and 45%, respectively, in patients with Ki67 ≤50%; p = .0337 and .3238, respectively). Both multivariate analyses confirmed the independent predictive value of the Ki67 cutpoint of 50%. Conclusion. Basal Ki67 proliferation index >50% should be considered an independent predictive factor for pCR reached after neoadjuvant chemotherapy, suggesting that cell proliferation is a phenomenon closely related to chemosensitivity. These findings could help to identify a group of patients with a potentially favorable long-term prognosis. Implications for Practice: The use of basal Ki67 status as a predictive factor of chemotherapy benefit could facilitate the identification of a patient subpopulation with high probability of achieving pathological complete response when treated with primary chemotherapy, and thus with a potentially favorable long-term prognosis. PMID:26786263

Quantitative contrast-enhanced ultrasound evaluation of pathological complete response in patients with locally advanced breast cancer receiving neoadjuvant chemotherapy.

PubMed

Wan, Cai-Feng; Liu, Xue-Song; Wang, Lin; Zhang, Jie; Lu, Jin-Song; Li, Feng-Hua

2018-06-01

To clarify whether the quantitative parameters of contrast-enhanced ultrasound (CEUS) can be used to predict pathological complete response (pCR) in patients with locally advanced breast cancer receiving neoadjuvant chemotherapy (NAC). Fifty-one patients with histologically proved locally advanced breast cancer scheduled for NAC were enrolled. The quantitative data for CEUS and the tumor diameter were collected at baseline and before surgery, and compared with the pathological response. Multiple logistic regression analysis was performed to examine quantitative parameters at CEUS and the tumor diameter to predict the pCR, and receiver operating characteristic (ROC) curve analysis was used as a summary statistic. Multiple logistic regression analysis revealed that PEAK (the maximum intensity of the time-intensity curve during bolus transit), PEAK%, TTP% (time to peak), and diameter% were significant independent predictors of pCR, and the area under the ROC curve was 0.932(Az 1 ), and the sensitivity and specificity to predict pCR were 93.7% and 80.0%. The area under the ROC curve for the quantitative parameters was 0.927(Az 2 ), and the sensitivity and specificity to predict pCR were 81.2% and 94.3%. For diameter%, the area under the ROC curve was 0.786 (Az 3 ), and the sensitivity and specificity to predict pCR were 93.8% and 54.3%. The values of Az 1 and Az 2 were significantly higher than that of Az 3 (P = 0.027 and P = 0.034, respectively). However, there was no significant difference between the values of Az 1 and Az 2 (P = 0.825). Quantitative analysis of tumor blood perfusion with CEUS is superior to diameter% to predict pCR, and can be used as a functional technique to evaluate tumor response to NAC. Copyright © 2018. Published by Elsevier B.V.

A central review of histopathology reports after breast cancer neoadjuvant chemotherapy in the neo-tango trial

PubMed Central

Provenzano, E; Vallier, A-L; Champ, R; Walland, K; Bowden, S; Grier, A; Fenwick, N; Abraham, J; Iddawela, M; Caldas, C; Hiller, L; Dunn, J; Earl, H M

2013-01-01

Background: Neo-tAnGo, a National Cancer Research Network (NCRN) multicentre randomised neoadjuvant chemotherapy trial in early breast cancer, enroled 831 patients in the United Kingdom. We report a central review of post-chemotherapy histopathology reports on the surgical specimens, to assess the presence and degree of response. Methods: A central independent two-reader review (EP and HME) of histopathology reports from post-treatment surgical specimens was performed. The quality and completeness of pathology reporting across all centres was assessed. The reviews included pathological response to chemotherapy (pathological complete response (pCR); minimal residual disease (MRD); and lesser degrees of response), laterality, the number of axillary metastases and axillary nodes, and the type of surgery. A consensus was reached after discussion. Results: In all, 825 surgical reports from 816 patients were available for review. Out of 4125 data items there were 347 discrepant results (8.4% of classifications), which involved 281 patients. These involved grading of breast response (169 but only 9 involving pCR vs MRD); laterality (6); presence of axillary metastasis (35); lymph node counts (108); and type of axillary surgery (29). Excluding cases with pCR, only 45% of reports included any comment regarding response in the breast and 30% in the axillary lymph nodes. Conclusion: We found considerable variability in the completeness of reporting of surgical specimens within this national neoadjuvant breast cancer trial. This highlights the need for consensus guidelines among trial groups on histopathology reporting, and the participation of histopathologists throughout the development and analysis of neoadjuvant trials. PMID:23299526

Comprehensive Review on the Surrogate Endpoints of Efficacy Proposed or Hypothesized in the Scientific Community Today.

PubMed

von Minckwitz, Gunter; Fontanella, Caterina

2015-05-01

An intermediate endpoint is a surrogate marker of treatment efficacy assessed earlier than the true outcome of interest. A suitable intermediate endpoint in neoadjuvant trials of specific breast cancer subtypes is pathological complete response (pCR) rate, defined as no invasive (+/-noninvasive) residual cancer in breast and nodes at surgery. On the basis of available evidence, Food and Drug Administration the US allowed to use of pCR as a surrogate endpoint for accelerated approval process. However, surrogacy to long-term outcome remains an unresolved issue. Literature data provide indications that triple-negative, HER2-positive, and high-grade hormone receptor-positive breast cancer subtypes achieved the highest pCR rate; the prognostic impact of pCR on survival is established only for these aggressive subtypes. In the German experience, early response after two to four cycles of neoadjuvant treatment strongly correlated with both pCR rate and long-term outcome. Therefore, early response may be considered a predictive marker for pCR and used for driving clinical trial design. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Definition and impact of pathologic complete response on prognosis after neoadjuvant chemotherapy in various intrinsic breast cancer subtypes.

PubMed

von Minckwitz, Gunter; Untch, Michael; Blohmer, Jens-Uwe; Costa, Serban D; Eidtmann, Holger; Fasching, Peter A; Gerber, Bernd; Eiermann, Wolfgang; Hilfrich, Jörn; Huober, Jens; Jackisch, Christian; Kaufmann, Manfred; Konecny, Gottfried E; Denkert, Carsten; Nekljudova, Valentina; Mehta, Keyur; Loibl, Sibylle

2012-05-20

The exact definition of pathologic complete response (pCR) and its prognostic impact on survival in intrinsic breast cancer subtypes is uncertain. Tumor response at surgery and its association with long-term outcome of 6,377 patients with primary breast cancer receiving neoadjuvant anthracycline-taxane-based chemotherapy in seven randomized trials were analyzed. Disease-free survival (DFS) was significantly superior in patients with no invasive and no in situ residuals in breast or nodes (n = 955) compared with patients with residual ductal carcinoma in situ only (n = 309), no invasive residuals in breast but involved nodes (n = 186), only focal-invasive disease in the breast (n = 478), and gross invasive residual disease (n = 4,449; P < .001). Hazard ratios for DFS comparing patients with or without pCR were lowest when defined as no invasive and no in situ residuals (0.446) and increased monotonously when in situ residuals (0.523), no invasive breast residuals but involved nodes (0.623), and focal-invasive disease (0.727) were included in the definition. pCR was associated with improved DFS in luminal B/human epidermal growth factor receptor 2 (HER2) -negative (P = .005), HER2-positive/nonluminal (P < .001), and triple-negative (P < .001) tumors but not in luminal A (P = .39) or luminal B/HER2-positive (P = .45) breast cancer. pCR in HER2-positive (nonluminal) and triple-negative tumors was associated with excellent prognosis. pCR defined as no invasive and no in situ residuals in breast and nodes can best discriminate between patients with favorable and unfavorable outcomes. Patients with noninvasive or focal-invasive residues or involved lymph nodes should not be considered as having achieved pCR. pCR is a suitable surrogate end point for patients with luminal B/HER2-negative, HER2-positive (nonluminal), and triple-negative disease but not for those with luminal B/HER2-positive or luminal A tumors.

Effect of increasing radiation dose on pathologic complete response in rectal cancer patients treated with neoadjuvant chemoradiation therapy.

PubMed

Hall, Matthew D; Schultheiss, Timothy E; Smith, David D; Fakih, Marwan G; Wong, Jeffrey Y C; Chen, Yi-Jen

2016-12-01

Neoadjuvant chemoradiation therapy (CRT) increases pathological complete response (pCR) rates compared to radiotherapy alone in patients with stage II-III rectal cancer. Limited evidence addresses whether radiotherapy dose escalation further improves pCR rates. Our purpose is to measure the effects of radiotherapy dose and other factors on post-therapy pathologic tumor (ypT) and nodal stage in rectal cancer patients treated with neoadjuvant CRT followed by mesorectal excision. A non-randomized comparative effectiveness analysis was performed of rectal cancer patients treated in 2000-2013 from the National Oncology Data Alliance™ (NODA), a pooled database of cancer registries from >150 US hospitals. The NODA contains the same data submitted to state cancer registries and SEER combined with validated radiotherapy and chemotherapy records. Eligible patients were treated with neoadjuvant CRT followed by proctectomy and had complete data on treatment start dates, radiotherapy dose, clinical tumor (cT) and ypT stage, and number of positive nodes at surgery (n = 3298 patients). Multivariable logistic regression was used to assess the predictive value of independent variables on achieving a pCR. On multivariable regression, radiotherapy dose, cT stage, and time interval between CRT and surgery were significant predictors of achieving a pCR. After adjusting for the effect of other variates, patients treated with higher radiotherapy doses were also more likely to have negative nodes at surgery and be downstaged from cT3-T4 and/or node positive disease to ypT0-T2N0 after neoadjuvant CRT. Our study suggests that increasing dose significantly improved pCR rates and downstaging in rectal cancer patients treated with neoadjuvant CRT followed by surgery.

RNA-based determination of ESR1 and HER2 expression and response to neoadjuvant chemotherapy.

PubMed

Denkert, C; Loibl, S; Kronenwett, R; Budczies, J; von Törne, C; Nekljudova, V; Darb-Esfahani, S; Solbach, C; Sinn, B V; Petry, C; Müller, B M; Hilfrich, J; Altmann, G; Staebler, A; Roth, C; Ataseven, B; Kirchner, T; Dietel, M; Untch, M; von Minckwitz, G

2013-03-01

Hormone and human epidermal growth factor receptor 2 (HER2) receptors are the most important breast cancer biomarkers, and additional objective and quantitative test methods such as messenger RNA (mRNA)-based quantitative analysis are urgently needed. In this study, we investigated the clinical validity of RT-PCR-based evaluation of estrogen receptor (ESR1) and HER2 mRNA expression. A total of 1050 core biopsies from two retrospective (GeparTrio, GeparQuattro) and one prospective (PREDICT) neoadjuvant studies were evaluated by quantitative RT-PCR for ESR1 and HER2. ESR1 mRNA was significantly predictive for reduced response to neoadjuvant chemotherapy in univariate and multivariate analysis in all three cohorts. The complete pathologically documented response (pathological complete response, pCR) rate for ESR1+/HER2- tumors was 7.3%, 8.0% and 8.6%; for ESR1-/HER2- tumors it was 34.4%, 33.7% and 37.3% in GeparTrio, GeparQuattro and PREDICT, respectively (P < 0.001 in each cohort). In the Kaplan-Meier analysis in GeparTrio patients with ESR1+/HER2- tumors had the best prognosis, compared with ESR1-/HER2- and ESR1-/HER2+ tumors [disease-free survival (DFS): P < 0.0005, overall survival (OS): P < 0.0005]. Our results suggest that mRNA levels of ESR1 and HER2 predict response to neoadjuvant chemotherapy and are significantly associated with long-term outcome. As an additional option to standard immunohistochemistry and gene-array-based analysis, quantitative RT-PCR analysis might be useful for determination of the receptor status in breast cancer.

Outcomes of Hispanic women with lymph-node positive, HER2 positive breast cancer treated with neoadjuvant chemotherapy and trastuzumab in Mexico.

PubMed

Villarreal-Garza, Cynthia; Soto-Perez-de-Celis, Enrique; Sifuentes, Erika; Ruano, Santiago; Baez-Revueltas, Berenice; Lara-Medina, Fernando; Arce-Salinas, Claudia; Alvarado-Miranda, Alberto; Chavarri-Guerra, Yanin; Caro-Sanchez, Claudia; Castañeda-Soto, Noel; Bargallo-Rocha, Enrique; Mohar, Alejandro

2015-06-01

Evidence regarding the outcomes of Hispanic women with breast cancer is lacking. We analyzed women with HER2+ disease treated with trastuzumab-based neoadjuvant chemotherapy in Mexico. 244 patients were included. Outcomes were compared between patients who achieved pathologic complete response (pCR) (n = 119), or less than pCR (n = 125). Patients with noninvasive (ypT0/is, ypN0) residual disease were also analyzed. 119 (48.8%) patients achieved pCR. pCR was the only factor associated with improved 3 year survival (98.1% vs 92.3%: P = 0.02). Survival was better in patients with ypT0/is, ypN0 response than in those with residual invasive disease (p < 0.01). 3 year survival was 98.1% for patients with pCR and 92.6% for patients with ypTis, ypN0 response (p = 0.64). Response rates to trastuzumab based neoadjuvant chemotherapy in Hispanics mimic that of other ethnic groups. This underlines the fact that access to treatment, rather than ethnicity, is the main prognostic factor in this population. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phase II Study of Neoadjuvant Anthracycline-Based Regimens Combined With Nanoparticle Albumin-Bound Paclitaxel and Trastuzumab for Human Epidermal Growth Factor Receptor 2-Positive Operable Breast Cancer.

PubMed

Tanaka, Satoru; Iwamoto, Mitsuhiko; Kimura, Kosei; Matsunami, Nobuki; Morishima, Hirotaka; Yoshidome, Katsuhide; Nomura, Takashi; Morimoto, Takashi; Yamamoto, Daigo; Tsubota, Yu; Kobayashi, Toshihiro; Uchiyama, Kazuhisa

2015-06-01

We treated patients with operable human epidermal growth factor receptor 2-positive breast cancer with neoadjuvant anthracycline regimens followed by nanoparticle albumin-bound paclitaxel plus trastuzumab. Of the 44 patients, 49% achieved a pathologic complete response (pCR). The pCR rate was 36% and 71% in the patients with estrogen receptor-positive and -negative cancer, respectively. Neoadjuvant therapy using this combination appears to be effective and safe. Introduction: Neoadjuvant chemotherapy plus trastuzumab. Neoadjuvant chemotherapy plus trastuzumab results in a 30% to 50% pathologic complete response (pCR) rate in human epidermal growth factor receptor 2 (HER2)-positive breast cancer and has been associated with improved therapeutic outcomes. Thus, the pCR rate can be useful in evaluating novel agents in this patient population. Nanoparticle albumin-bound (nab)-paclitaxel (PTX) can reduce the toxicity of PTX while maintaining its efficacy. The present study evaluated the activity and safety of nab-PTX as a neoadjuvant treatment of HER2(+) breast cancer. We treated patients with stage I to IIIA breast cancer using neoadjuvant epirubicin/cyclophosphamide (EC) or 5-fluorouracil/epirubicin/cyclophosphamide every 3 weeks (q3w) for 4 cycles, followed by nab-PTX (260 mg/m(2)) plus trastuzumab q3w for 4 cycles. The primary endpoint was the pCR rate. The secondary endpoints included the clinical response rate, disease-free survival, pathologic response rate (defined as pCR or minimal residual invasive disease only in the breast), breast-conserving surgery rate, and safety. Forty-six patients were enrolled. One patient met the exclusion criteria because of the coexistence of another malignant disease; therefore, we evaluated 45 patients in the entire study. One patient experienced rapid disease progression during EC therapy, leaving 44 patients evaluable for nab-PTX treatment. Of the 45 patients, 49% achieved a pCR. The pCR rate was 36% and 71% in those with estrogen receptor-positive and -negative cancer, respectively. Of all the study treatments, the most frequent reason for delay or dose reduction was hematologic toxicity; only 1 patient required a dose reduction for nab-PTX because of peripheral neuropathy. Neoadjuvant therapy using this combination appears to be effective and safe. Copyright © 2015 Elsevier Inc. All rights reserved.

Locally advanced rectal cancer: post-chemoradiotherapy ADC histogram analysis for predicting a complete response.

PubMed

Cho, Seung Hyun; Kim, Gab Chul; Jang, Yun-Jin; Ryeom, Hunkyu; Kim, Hye Jung; Shin, Kyung-Min; Park, Jun Seok; Choi, Gyu-Seog; Kim, See Hyung

2015-09-01

The value of diffusion-weighted imaging (DWI) for reliable differentiation between pathologic complete response (pCR) and residual tumor is still unclear. Recently, a few studies reported that histogram analysis can be helpful to monitor the therapeutic response in various cancer research. To investigate whether post-chemoradiotherapy (CRT) apparent diffusion coefficient (ADC) histogram analysis can be helpful to predict a pCR in locally advanced rectal cancer (LARC). Fifty patients who underwent preoperative CRT followed by surgery were enrolled in this retrospective study, non-pCR (n = 41) and pCR (n = 9), respectively. ADC histogram analysis encompassing the whole tumor was performed on two post-CRT ADC600 and ADC1000 (b factors 0, 600 vs. 0, 1000 s/mm(2)) maps. Mean, minimum, maximum, SD, mode, 10th, 25th, 50th, 75th, 90th percentile ADCs, skewness, and kurtosis were derived. Diagnostic performance for predicting pCR was evaluated and compared. On both maps, 10th and 25th ADCs showed better diagnostic performance than that using mean ADC. Tenth percentile ADCs revealed the best diagnostic performance on both ADC600 (AZ 0.841, sensitivity 100%, specificity 70.7%) and ADC1000 (AZ 0.821, sensitivity 77.8%, specificity 87.8%) maps. In comparison between 10th percentile and mean ADC, the specificity was significantly improved on both ADC600 (70.7% vs. 53.7%; P = 0.031) and ADC1000 (87.8% vs. 73.2%; P = 0.039) maps. Post-CRT ADC histogram analysis is helpful for predicting pCR in LARC, especially, in improving the specificity, compared with mean ADC. © The Foundation Acta Radiologica 2014.

Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer.

PubMed

Gianni, Luca; Zambetti, Milvia; Clark, Kim; Baker, Joffre; Cronin, Maureen; Wu, Jenny; Mariani, Gabriella; Rodriguez, Jaime; Carcangiu, Marialuisa; Watson, Drew; Valagussa, Pinuccia; Rouzier, Roman; Symmans, W Fraser; Ross, Jeffrey S; Hortobagyi, Gabriel N; Pusztai, Lajos; Shak, Steven

2005-10-10

We sought to identify gene expression markers that predict the likelihood of chemotherapy response. We also tested whether chemotherapy response is correlated with the 21-gene Recurrence Score assay that quantifies recurrence risk. Patients with locally advanced breast cancer received neoadjuvant paclitaxel and doxorubicin. RNA was extracted from the pretreatment formalin-fixed paraffin-embedded core biopsies. The expression of 384 genes was quantified using reverse transcriptase polymerase chain reaction and correlated with pathologic complete response (pCR). The performance of genes predicting for pCR was tested in patients from an independent neoadjuvant study where gene expression was obtained using DNA microarrays. Of 89 assessable patients (mean age, 49.9 years; mean tumor size, 6.4 cm), 11 (12%) had a pCR. Eighty-six genes correlated with pCR (unadjusted P < .05); pCR was more likely with higher expression of proliferation-related genes and immune-related genes, and with lower expression of estrogen receptor (ER) -related genes. In 82 independent patients treated with neoadjuvant paclitaxel and doxorubicin, DNA microarray data were available for 79 of the 86 genes. In univariate analysis, 24 genes correlated with pCR with P < .05 (false discovery, four genes) and 32 genes showed correlation with P < .1 (false discovery, eight genes). The Recurrence Score was positively associated with the likelihood of pCR (P = .005), suggesting that the patients who are at greatest recurrence risk are more likely to have chemotherapy benefit. Quantitative expression of ER-related genes, proliferation genes, and immune-related genes are strong predictors of pCR in women with locally advanced breast cancer receiving neoadjuvant anthracyclines and paclitaxel.

Neoadjuvant Chemotherapy versus Chemoradiation Prior to Esophagectomy: Impact on Rate of Complete Pathologic Response and Survival in Esophageal Cancer Patients.

PubMed

Samson, Pamela; Robinson, Clifford; Bradley, Jeffrey; Lockhart, A Craig; Puri, Varun; Broderick, Stephen; Kreisel, Daniel; Krupnick, A Sasha; Patterson, G Alexander; Meyers, Bryan; Crabtree, Traves

2016-12-01

The aim of this study was to evaluate differences in pathologic complete response (pCR) rates and overall survival among patients receiving either neoadjuvant chemotherapy or chemoradiation before esophagectomy for locally advanced esophageal cancer. Patients with esophageal cancer receiving either neoadjuvant chemotherapy or chemoradiation before esophagectomy were identified using the National Cancer Database. Univariate analysis compared patient, tumor, and postoperative outcome characteristics. Logistic regression was performed to identify variables associated with achieving pCR. Kaplan-Meier analysis was performed to compare overall median survival by neoadjuvant therapy type and pCR status. Finally, a Cox proportional hazards model was fitted to identify variables associated with increased mortality hazard. From 2006 to 2012, a total of 916 of 7338 of patients (12.5%) received neoadjuvant chemotherapy whereas 6422 (87.5%) received neoadjuvant chemoradiation. Patients who received neoadjuvant chemoradiation were more likely to achieve a pCR (17.2% versus 6.4%, p < 0.001) and less likely to have positive margins (5.6% versus 11.5%, p < 0.001) than were patients who received neoadjuvant chemotherapy, with no difference in 30- or 90-day mortality. Achieving a pCR was associated with improved overall median survival (59.5 ± 4.0 months versus 30.1 ± 0.76 months for those with persistent disease, p < 0.001). On logistic regression, neoadjuvant chemoradiation therapy was independently associated with achieving a pCR (OR = 2.75, 95% confidence interval: 2.01-3.77, p < 0.001). Despite improvement in the pCR rate with neoadjuvant chemoradiation, neoadjuvant therapy type was not independently associated with long-term survival (hazard ratio = 1.12; 95% confidence interval: 0.97-1.30, p = 0.12). Although neoadjuvant chemoradiation is more successful in downstaging esophageal cancer before esophagectomy, it was not independently prognostic for improved long-term survival. Other factors affecting long-term survival among pathologic complete responders and among patients with persistent disease should be investigated to clarify this association. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Establishing a probabilistic reversal learning test in mice: evidence for the processes mediating reward-stay and punishment-shift behaviour and for their modulation by serotonin.

PubMed

Ineichen, Christian; Sigrist, Hannes; Spinelli, Simona; Lesch, Klaus-Peter; Sautter, Eva; Seifritz, Erich; Pryce, Christopher R

2012-11-01

Valid animal models of psychopathology need to include behavioural readouts informed by human findings. In the probabilistic reversal learning (PRL) task, human subjects are confronted with serial reversal of the contingency between two operant stimuli and reward/punishment and, superimposed on this, a low probability (0.2) of punished correct responses/rewarded incorrect responses. In depression, reward-stay and reversals completed are unaffected but response-shift following punished correct response trials, referred to as negative feedback sensitivity (NFS), is increased. The aims of this study were to: establish an operant spatial PRL test appropriate for mice; obtain evidence for the processes mediating reward-stay and punishment-shift responding; and assess effects thereon of genetically- and pharmacologically-altered serotonin (5-HT) function. The study was conducted with wildtype (WT) and heterozygous mutant (HET) mice from a 5-HT transporter (5-HTT) null mutant strain. Mice were mildly food deprived and reward was sugar pellet and punishment was 5-s time out. Mice exhibited high motivation and adaptive reversal performance. Increased probability of punished correct response (PCR) trials per session (p = 0.1, 0.2 or 0.3) led to monotonic decrease in reward-stay and reversals completed, suggesting accurate reward prediction. NFS differed from chance-level at p PCR = 0.1, suggesting accurate punishment prediction, whereas NFS was at chance-level at p = 0.2-0.3. At p PCR = 0.1, HET mice exhibited lower NFS than WT mice. The 5-HTT blocker escitalopram was studied acutely at p PCR = 0.2: a low dose (0.5-1.5 mg/kg) resulted in decreased NFS, increased reward-stay and increased reversals completed, and similarly in WT and HET mice. This study demonstrates that testing PRL in mice can provide evidence on the regulation of reward and punishment processing that is, albeit within certain limits, of relevance to human emotional-cognitive processing, its dysfunction and treatment. Copyright © 2012 Elsevier Ltd. All rights reserved.

Pathologic complete response and disease-free survival are not surrogate endpoints for 5-year survival in rectal cancer: an analysis of 22 randomized trials.

PubMed

Petrelli, Fausto; Borgonovo, Karen; Cabiddu, Mary; Ghilardi, Mara; Lonati, Veronica; Barni, Sandro

2017-02-01

We performed a literature-based analysis of randomized clinical trials to assess the pathologic complete response (pCR) (ypT0N0 after neoadjuvant therapy) and 3-year disease-free survival (DFS) as potential surrogate endpoints for 5-year overall survival (OS) in rectal cancer treated with neoadjuvant (chemo)radiotherapy (CT)RT. A systematic literature search of PubMed, EMBASE, the Web of Science, SCOPUS, CINAHL, and the Cochrane Library was performed. Treatment effects on 3-year DFS and 5-year OS were expressed as rates of patients alive (%), and those on pCR as differences in pCR rates (∆ pCR% ). A weighted regression analysis was performed at individual- and trial-level to test the association between treatment effects on surrogate (∆ pCR% and ∆ 3yDFS ) and the main clinical outcome (∆ 5yOS ). Twenty-two trials involving 10,050 patients, were included in the analysis. The individual level surrogacy showed that the pCR% and 3-year DFS were poorly correlated with 5-year OS (R=0.52; 95% CI, 0.31-0.91; P=0.002; and R=0.60; 95% CI, 0.36-1; P=0.002). The trial-level surrogacy analysis confirmed that the two treatment effects on surrogates (∆ pCR% and ∆ 3yDFS ) are not strong surrogates for treatment effects on 5-year OS % (R=0.2; 95% CI, -0.29-0.78; P=0.5 and R=0.64; 95% CI, 0.29-1; P=0.06). These findings were confirmed in neoadjuvant CTRT studies but not in phase III trials were 3-year DFS could still represent a valid surrogate. This analysis does not support the use of pCR and 3-year DFS% as appropriate surrogate endpoints for 5-year OS% in patients with rectal cancer treated with neoadjuvant therapy.

Prognostic Value of p16 Status on the Development of a Complete Response in Involved Oropharynx Cancer Neck Nodes After Cisplatin-Based Chemoradiation: A Secondary Analysis of NRG Oncology RTOG 0129

DOE Office of Scientific and Technical Information (OSTI.GOV)

Galloway, Thomas J., E-mail: thomas.galloway@fccc.edu; Zhang, Qiang; Nguyen-Tan, Phuc Felix

Purpose: To determine the relationship between p16 status and the regional response of patients with node-positive oropharynx cancer treated on NRG Oncology RTOG 0129. Methods and Materials: Patients with N1-N3 oropharynx cancer and known p16 status who underwent treatment on RTOG 0129 were analyzed. Pathologic complete response (pCR) rates in patients treated with a postchemoradiation neck dissection (with p16-positive or p16-negative cancer) were compared by Fisher exact test. Patients managed expectantly were compared with those treated with a neck dissection. Results: Ninety-nine (34%) of 292 patients with node-positive oropharynx cancer and known p16 status underwent a posttreatment neck dissection (p16-positive:more » n=69; p16-negative: n=30). The remaining 193 patients with malignant lymphadenopathy at diagnosis were observed. Neck dissection was performed a median of 70 (range, 17-169) days after completion of chemoradiation. Neither the pretreatment nodal stage (P=.71) nor the postradiation, pre-neck dissection clinical/radiographic neck assessment (P=.42) differed by p16 status. A pCR was more common among p16-positive patients (78%) than p16-negative patients (53%, P=.02) and was associated with a reduced incidence of local–regional failure (hazard ratio 0.33, P=.003). On multivariate analysis of local–regional failure, a test for interaction between pCR and p16 status was not significant (P=.37). One-hundred ninety-three (66%) of 292 of initially node-positive patients were managed without a posttreatment neck dissection. Development of a clinical (cCR) was not significantly influenced by p16-status (P=.42). Observed patients with a clinical nodal CR had disease control outcomes similar to those in patients with a pCR neck dissection. Conclusions: Patients with p16-positive tumors had significantly higher pCR and locoregional control rates than those with p16-negative tumors.« less

Prognostic Value of p16 Status on the Development of a Complete Response in Involved Oropharynx Cancer Neck Nodes After Cisplatin-Based Chemoradiation: A Secondary Analysis of NRG Oncology RTOG 0129.

PubMed

Galloway, Thomas J; Zhang, Qiang Ed; Nguyen-Tan, Phuc Felix; Rosenthal, David I; Soulieres, Denis; Fortin, André; Silverman, Craig L; Daly, Megan E; Ridge, John A; Hammond, J Alexander; Le, Quynh-Thu

2016-10-01

To determine the relationship between p16 status and the regional response of patients with node-positive oropharynx cancer treated on NRG Oncology RTOG 0129. Patients with N1-N3 oropharynx cancer and known p16 status who underwent treatment on RTOG 0129 were analyzed. Pathologic complete response (pCR) rates in patients treated with a postchemoradiation neck dissection (with p16-positive or p16-negative cancer) were compared by Fisher exact test. Patients managed expectantly were compared with those treated with a neck dissection. Ninety-nine (34%) of 292 patients with node-positive oropharynx cancer and known p16 status underwent a posttreatment neck dissection (p16-positive: n=69; p16-negative: n=30). The remaining 193 patients with malignant lymphadenopathy at diagnosis were observed. Neck dissection was performed a median of 70 (range, 17-169) days after completion of chemoradiation. Neither the pretreatment nodal stage (P=.71) nor the postradiation, pre-neck dissection clinical/radiographic neck assessment (P=.42) differed by p16 status. A pCR was more common among p16-positive patients (78%) than p16-negative patients (53%, P=.02) and was associated with a reduced incidence of local-regional failure (hazard ratio 0.33, P=.003). On multivariate analysis of local-regional failure, a test for interaction between pCR and p16 status was not significant (P=.37). One-hundred ninety-three (66%) of 292 of initially node-positive patients were managed without a posttreatment neck dissection. Development of a clinical (cCR) was not significantly influenced by p16-status (P=.42). Observed patients with a clinical nodal CR had disease control outcomes similar to those in patients with a pCR neck dissection. Patients with p16-positive tumors had significantly higher pCR and locoregional control rates than those with p16-negative tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

Impact of the Addition of Carboplatin and/or Bevacizumab to Neoadjuvant Once-per-Week Paclitaxel Followed by Dose-Dense Doxorubicin and Cyclophosphamide on Pathologic Complete Response Rates in Stage II to III Triple-Negative Breast Cancer: CALGB 40603 (Alliance)

PubMed Central

Sikov, William M.; Berry, Donald A.; Perou, Charles M.; Singh, Baljit; Cirrincione, Constance T.; Tolaney, Sara M.; Kuzma, Charles S.; Pluard, Timothy J.; Somlo, George; Port, Elisa R.; Golshan, Mehra; Bellon, Jennifer R.; Collyar, Deborah; Hahn, Olwen M.; Carey, Lisa A.; Hudis, Clifford A.; Winer, Eric P.

2015-01-01

Purpose One third of patients with triple-negative breast cancer (TNBC) achieve pathologic complete response (pCR) with standard neoadjuvant chemotherapy (NACT). CALGB 40603 (Alliance), a 2 × 2 factorial, open-label, randomized phase II trial, evaluated the impact of adding carboplatin and/or bevacizumab. Patients and Methods Patients (N = 443) with stage II to III TNBC received paclitaxel 80 mg/m2 once per week (wP) for 12 weeks, followed by doxorubicin plus cyclophosphamide once every 2 weeks (ddAC) for four cycles, and were randomly assigned to concurrent carboplatin (area under curve 6) once every 3 weeks for four cycles and/or bevacizumab 10 mg/kg once every 2 weeks for nine cycles. Effects of adding these agents on pCR breast (ypT0/is), pCR breast/axilla (ypT0/isN0), treatment delivery, and toxicities were analyzed. Results Patients assigned to either carboplatin or bevacizumab were less likely to complete wP and ddAC without skipped doses, dose modification, or early discontinuation resulting from toxicity. Grade ≥ 3 neutropenia and thrombocytopenia were more common with carboplatin, as were hypertension, infection, thromboembolic events, bleeding, and postoperative complications with bevacizumab. Employing one-sided P values, addition of either carboplatin (60% v 44%; P = .0018) or bevacizumab (59% v 48%; P = .0089) significantly increased pCR breast, whereas only carboplatin (54% v 41%; P = .0029) significantly raised pCR breast/axilla. More-than-additive interactions between the two agents could not be demonstrated. Conclusion In stage II to III TNBC, addition of either carboplatin or bevacizumab to NACT increased pCR rates, but whether this will improve relapse-free or overall survival is unknown. Given results from recently reported adjuvant trials, further investigation of bevacizumab in this setting is unlikely, but the role of carboplatin could be evaluated in definitive studies, ideally limited to biologically defined patient subsets most likely to benefit from this agent. PMID:25092775

The role of quantitative estrogen receptor status in predicting tumor response at surgery in breast cancer patients treated with neoadjuvant chemotherapy.

PubMed

Raphael, Jacques; Gandhi, Sonal; Li, Nim; Lu, Fang-I; Trudeau, Maureen

2017-07-01

Estrogen receptor (ER) negative (-) breast cancer (BC) patients have better tumor response rates than ER-positive (+) patients after neoadjuvant chemotherapy (NCT). We conducted a retrospective review using the institutional database "Biomatrix" to assess the value of quantitative ER status in predicting tumor response at surgery and to identify potential predictors of survival outcomes. Univariate followed by multivariable regression analyses were conducted to assess the association between quantitative ER and tumor response assessed as tumor size reduction and pathologic complete response (pCR). Predictors of recurrence-free survival (RFS) were identified using a cox proportional hazards model (CPH). A log-rank test was used to compare RFS between groups if a significant predictor was identified. 304 patients were included with a median follow-up of 43.3 months (Q1-Q3 28.7-61.1) and a mean age of 49.7 years (SD 10.9). Quantitative ER was inversely associated with tumor size reduction and pCR (OR 0.99, 95% CI 0.99-1.00, p = 0.027 and 0.98 95% CI 0.97-0.99, p < 0.0001, respectively). A cut-off of 60 and 80% predicted best the association with tumor size reduction and pCR, respectively. pCR was shown to be an independent predictor of RFS (HR 0.17, 95% CI 0.07-0.43, p = 0.0002) in all patients. At 5 years, 93% of patients with pCR and 72% of patients with residual tumor were recurrence-free, respectively (p = 0.0012). Quantitative ER status is inversely associated with tumor response in BC patients treated with NCT. A cut-off of 60 and 80% predicts best the association with tumor size reduction and pCR, respectively. Therefore, patients with an ER status higher than the cut-off might benefit from a neoadjuvant endocrine therapy approach. Patients with pCR had better survival outcomes independently of their tumor phenotype. Further prospective studies are needed to validate the clinical utility of quantitative ER as a predictive marker of tumor response.

The time course of phase correction: A kinematic investigation of motor adjustment to timing perturbations during sensorimotor synchronization

PubMed Central

Hove, Michael J.; Balasubramaniam, Ramesh; Keller, Peter E.

2014-01-01

Synchronizing movements with a beat requires rapid compensation for timing errors. The phase-correction response (PCR) has been studied extensively in finger tapping by shifting a metronome onset and measuring the adjustment of the following tap time. How the response unfolds during the subsequent tap cycle remains unknown. Using motion capture, we examined finger kinematics during the PCR. Participants tapped with a metronome containing phase perturbations. They tapped in ‘legato’ and ‘staccato’ style at various tempi, which altered the timing of the constituent movement stages (dwell at the surface, extension, flexion). After a phase perturbation, tapping kinematics changed compared to baseline, and the PCR was distributed differently across movement stages. In staccato tapping, the PCR trajectory changed primarily during finger extension across tempi. In legato tapping, at fast tempi the PCR occurred primarily during extension, whereas at slow tempi most phase correction was already completed during dwell. Across conditions, timing adjustments occurred primarily 100-250 ms into the following tap cycle. The change in movement around 100 ms represents the time to integrate information into an already planned movement and the rapidity suggests a subcortical route. PMID:25151103

[Axillary pathologic response after neoadjuvant chemotherapy in locally advanced breast cancer with axillary involvement].

PubMed

Jiménez-Ballvé, A; Serrano-Palacio, A; García-Sáenz, J A; Ortega Candil, A; Salsidua-Arroyo, O; Román-Santamaría, J M; Pelayo Alarcón, A; Fuentes Ferrer, M E; Carreras-Delgado, J L

2015-01-01

To compare axillary involvement (N+) at initial staging in locally advanced breast cancer (LABC) with axillary lymphadenectomy histologic results after neoadjuvant chemotherapy treatment (NeoChemo). Retrospective study between November 2011 and September 2013 of LABC cases treated with neoadjuvant chemotherapy based on docetaxel (associated with trastuzumab in HER2 positive cases and carboplatin/adriamycin in HER2 negative cases). Those clinically or radiologically suspected cases of axillary involvement were histologically confirmed. When there was no suspicion of axillary involvement, sentinel lymph node radioguided biopsy (SLNRB) was performed using intradermal injection of (99m)Tc-nanocolloid albumin prior to neoadjuvant treatment. Axillary lymphadenectomy after NeoChemo was undertaken in all cases with positive axilla. Final pathologic response was classified as complete (pCR) when there was no evidence of tumoral disease and as non-pathologic complete response (no pCR) in the opposite case. A total of 346 patients treated with docetaxel were reviewed, identifying 105 LABC. Axillary involvement at initial staging was detected in 70 (67%) before starting NeoChemo. From these 70, 73% (n=51) were N+ (fine needle biopsy and/or biopsy) and the remaining 19 (27%) were occult N+ detected by SLNRB. Axillary lymphadenectomy detected pCR in 56% (39/70), increasing up to 84% pCR when initial N+ status was reached using SNLB. On the other hand, when N+ was detected using fine needle biopsy/lymph biopsy, pCR was only 45%. More than 50% of women affected by locally advanced breast cancer with tumoral axillary involvement at initial diagnosis present free metastatic axilla after therapeutic neoadjuvant chemotherapy effect. This increases up to almost 90% in case of occult metastatic axilla detected with sentinel node biopsy prior starting neoadjuvant chemotherapy. Copyright © 2014 Elsevier España, S.L.U. and SEMNIM. All rights reserved.

Digital gene expression profiling analysis and its application in the identification of genes associated with improved response to neoadjuvant chemotherapy in breast cancer.

PubMed

Liu, Xiaozhen; Jin, Gan; Qian, Jiacheng; Yang, Hongjian; Tang, Hongchao; Meng, Xuli; Li, Yongfeng

2018-04-23

This study aimed to screen sensitive biomarkers for the efficacy evaluation of neoadjuvant chemotherapy in breast cancer. In this study, Illumina digital gene expression sequencing technology was applied and differentially expressed genes (DEGs) between patients presenting pathological complete response (pCR) and non-pathological complete response (NpCR) were identified. Further, gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were then performed. The genes in significant enriched pathways were finally quantified by quantitative real-time PCR (qRT-PCR) to confirm that they were differentially expressed. Additionally, GSE23988 from Gene Expression Omnibus database was used as the validation dataset to confirm the DEGs. After removing the low-quality reads, 715 DEGs were finally detected. After mapping to KEGG pathways, 10 DEGs belonging to the ubiquitin proteasome pathway (HECTD3, PSMB10, UBD, UBE2C, and UBE2S) and cytokine-cytokine receptor interactions (CCL2, CCR1, CXCL10, CXCL11, and IL2RG) were selected for further analysis. These 10 genes were finally quantified by qRT-PCR to confirm that they were differentially expressed (the log 2 fold changes of selected genes were - 5.34, 7.81, 6.88, 5.74, 3.11, 19.58, 8.73, 8.88, 7.42, and 34.61 for HECTD3, PSMB10, UBD, UBE2C, UBE2S, CCL2, CCR1, CXCL10, CXCL11, and IL2RG, respectively). Moreover, 53 common genes were confirmed by the validation dataset, including downregulated UBE2C and UBE2S. Our results suggested that these 10 genes belonging to these two pathways might be useful as sensitive biomarkers for the efficacy evaluation of neoadjuvant chemotherapy in breast cancer.

[Application of decision curve on evaluation of MRI predictive model for early assessing pathological complete response to neoadjuvant therapy in breast cancer].

PubMed

He, Y J; Li, X T; Fan, Z Q; Li, Y L; Cao, K; Sun, Y S; Ouyang, T

2018-01-23

Objective: To construct a dynamic enhanced MR based predictive model for early assessing pathological complete response (pCR) to neoadjuvant therapy in breast cancer, and to evaluate the clinical benefit of the model by using decision curve. Methods: From December 2005 to December 2007, 170 patients with breast cancer treated with neoadjuvant therapy were identified and their MR images before neoadjuvant therapy and at the end of the first cycle of neoadjuvant therapy were collected. Logistic regression model was used to detect independent factors for predicting pCR and construct the predictive model accordingly, then receiver operating characteristic (ROC) curve and decision curve were used to evaluate the predictive model. Results: ΔArea(max) and Δslope(max) were independent predictive factors for pCR, OR =0.942 (95% CI : 0.918-0.967) and 0.961 (95% CI : 0.940-0.987), respectively. The area under ROC curve (AUC) for the constructed model was 0.886 (95% CI : 0.820-0.951). Decision curve showed that in the range of the threshold probability above 0.4, the predictive model presented increased net benefit as the threshold probability increased. Conclusions: The constructed predictive model for pCR is of potential clinical value, with an AUC>0.85. Meanwhile, decision curve analysis indicates the constructed predictive model has net benefit from 3 to 8 percent in the likely range of probability threshold from 80% to 90%.

Using Flow Characteristics in Three-Dimensional Power Doppler Ultrasound Imaging to Predict Complete Responses in Patients Undergoing Neoadjuvant Chemotherapy.

PubMed

Shia, Wei-Chung; Huang, Yu-Len; Wu, Hwa-Koon; Chen, Dar-Ren

2017-05-01

Strategies are needed for the identification of a poor response to treatment and determination of appropriate chemotherapy strategies for patients in the early stages of neoadjuvant chemotherapy for breast cancer. We hypothesize that power Doppler ultrasound imaging can provide useful information on predicting response to neoadjuvant chemotherapy. The solid directional flow of vessels in breast tumors was used as a marker of pathologic complete responses (pCR) in patients undergoing neoadjuvant chemotherapy. Thirty-one breast cancer patients who received neoadjuvant chemotherapy and had tumors of 2 to 5 cm were recruited. Three-dimensional power Doppler ultrasound with high-definition flow imaging technology was used to acquire the indices of tumor blood flow/volume, and the chemotherapy response prediction was established, followed by support vector machine classification. The accuracy of pCR prediction before the first chemotherapy treatment was 83.87% (area under the ROC curve [AUC] = 0.6957). After the second chemotherapy treatment, the accuracy of was 87.9% (AUC = 0.756). Trend analysis showed that good and poor responders exhibited different trends in vascular flow during chemotherapy. This preliminary study demonstrates the feasibility of using the vascular flow in breast tumors to predict chemotherapeutic efficacy. © 2017 by the American Institute of Ultrasound in Medicine.

Pathological complete response in breast cancer patients receiving anthracycline and taxane-based neoadjuvant chemotherapy: Evaluating the effect of race/ethnicity

PubMed Central

Chavez-MacGregor, Mariana; Litton, Jennifer; Chen, Huiqin; Giordano, Sharon H.; Hudis, Clifford A.; Wolff, Antonio C.; Valero, Vicente; Hortobagyi, Gabriel N.; Bondy, Melissa L.; Gonzalez-Angulo, Ana Maria

2010-01-01

Purpose To evaluate the influence of race/ethnicity and tumor subtype in pathological complete response (pCR) following treatment with neoadjuvant chemotherapy. Methods 2074 patients diagnosed with breast cancer between 1994 and 2008, treated with neoadjuvant anthracycline- and taxane-based chemotherapy, were included. pCR was defined as no residual invasive cancer in the breast and axilla. Kaplan-Meier product-limit was used to calculate survival outcomes. Cox proportional hazards models were fitted to determine the relationship of patient and tumor variables with outcome. Results Median age was 50 years, 14.6% patients were black, 15.2% Hispanic, 64.3% White, and 5.9% other race. There were no differences in pCR rates among race/ethnicity: (12.3% in black, 14.2% in Hispanics, 12.3% in whites and 11.5% in others, p=.788). Lack of pCR, breast cancer subtype, grade 3 tumors, and lymphovascular invasion were associated with worse RFS and OS (p≤.0001). Differences in RFS by race/ethnicity were seen in the patients with hormone receptor-positive disease, p=.007. In multivariate analysis, Hispanics had improved RFS (HR, 95% CI 0.69; 0.49-0.97) and OS (HR, 95% CI 0.63; 0.41-0.97); blacks had a trend to worse outcomes (RFS:HR, 95% CI 1.28; 0.97-1.68, OS:HR, 1.32; 95% CI; 0.97-1.81) when compared to whites. Conclusions In this cohort of patients, race/ethnicity was not significantly associated with pCR rates. In a multivariate analysis we observed improved outcomes in Hispanics and a trend towards worse outcomes in black patients, when compared to whites. Further research is needed to explore the potential differences in biology and outcomes. PMID:20564153

Response evaluation after primary systemic therapy of Her2 positive breast cancer – an observational cross-sectional study.

PubMed

Tőkés, Tímea; Szentmártoni, Gyöngyvér; Torgyík, László; Kajáry, Kornélia; Lengyel, Zsolt; Györke, Tamás; Molnár, Béla Á; Tőkés, Anna-Mária; Kulka, Janina; Dank, Magdolna

2015-04-01

To evaluate (I) trastuzumab-containing primary systemic therapy (PST) in human epidermal growth factor receptor 2 (Her2) overexpressing breast carcinomas.; (II) compare the patients who achieved and those who did not achieve pathological complete remission (pCR), and (III) analyze the accuracy of different clinical-imaging modalities in tumor response monitoring. 188 patients who received PST between 2008 and 2014 were reviewed and 43 Her2 overexpressing breast cancer patients (28 Luminal B/Her2-positive and 15 Her2-positive) were enrolled. 26 patients received mostly taxane-based PST without trastuzumab (Group 1) and 17 patients received trastuzumab-containing PST (Group 2). We compared the concordance between pCR and complete remission (CR) defined by breast-ultrasound, CR defined by standard 18F-fluoro-deoxy-glucose positron emission tomography and computerized tomography (FDG-PET/CT) criteria (Method 1) and CR defined by a novel, breast cancer specific FDG-PET/CT criteria (Method 2). Sensitivity (sens), specificity (spec), and positive (PPV) and negative predictive values (NPV) were calculated. Ten patients (38.5%) in Group 1 and eight (47%) in Group 2 achieved pCR. pCR was significantly more frequent in Her2-positive than in Luminal B/Her2-positive tumors in both Group 1: (P=0.043) and Group 2: (P=0.029). PET/CT evaluated by the breast cancer specific criteria (Method 2) differentiated pCR from non-pCR more accurately in both groups (Group 1: sens=77.8%, spec=%, PPV=100%, NPV=71.4%; Group 2: sens=87.5%, spec=62.5%, PPV=70%, NPV=83.3%) than standard PET/CT criteria (Method 1) (Group 1: sens=22.2% spec=100% PPV=100% NPV=41.7%; in Group 2: sens=37.5%, spec=87.5%, PPV=75% NPV=58.3%) or breast ultrasound (Group 1, sens=83.3% spec=25% PPV=62.5% NPV=50%; Group 2, sens=100% spec=12.5% PPV=41.6% NPV=100%). The benefit of targeted treatment with trastuzumab-containing PST in Her2 overexpressing breast cancer was defined in terms of pCR rate. Luminal B/Her2-positive subtype needs further subdivision to identify patients who would benefit from PST. Combined evaluation of tumor response by our novel, breast cancer specific FDG-PET/CT criteria accurately differentiated pCR from non-pCR patients.

Cetuximab Combined With Induction Oxaliplatin and Capecitabine, Followed by Neoadjuvant Chemoradiation for Locally Advanced Rectal Cancer: SWOG 0713.

PubMed

Leichman, Cynthia Gail; McDonough, Shannon L; Smalley, Stephen R; Billingsley, Kevin G; Lenz, Heinz-Josef; Beldner, Matthew A; Hezel, Aram F; Velasco, Mario R; Guthrie, Katherine A; Blanke, Charles D; Hochster, Howard S

2018-03-01

Neoadjuvant chemoradiation (NCRT) is standard treatment for locally advanced rectal cancer. Pathologic complete response (pCR) has associated with improved survival. In modern phase III trials of NCRT, pCR ranges from 10% to 20%. Cetuximab improves response in KRAS (KRAS proto-oncogene) wild type (wt) metastatic colorectal cancer. S0713 was designed to assess improvement in pCR with additional use of cetuximab with induction chemotherapy and NCRT for locally advanced, KRAS-wt rectal cancer. Patient eligibility: stage II to III biopsy-proven, KRAS-wt rectal adenocarcinoma; no bowel obstruction; adequate hematologic, hepatic and renal function; performance status of 0 to 2. Target enrollment: 80 patients. induction chemotherapy with wCAPOX (weekly capecitabine and oxaliplatin) and cetuximab followed by the same regimen concurrent with radiation (omitting day 15 oxaliplatin). If fewer than 7 pCRs were observed at planned interim analysis after 40 patients received all therapy, the study would close. Eighty eligible patients would provide 90% power given a true pCR rate > 35% at a significance of 0.04. The regimen would lack future interest if pCR probability was ≤ 20%. Between February 2009 and April 2013, 83 patients registered. Four were ineligible and 4 not treated, leaving 75 evaluable for clinical outcomes and toxicity, of whom 65 had surgery. Of 75 patients, 20 had pCR (27%; 95% confidence interval [CI], 17%-38%); 19 (25%) had microscopic cancer; 36 (48%) had minor/no response (including 10 without surgery). Three-year disease-free survival was 73% (95% CI, 63%-83%). Our trial did not meet the pCR target of 35%. Toxicity was generally acceptable. This regimen cannot be recommended outside the clinical trial setting. Copyright © 2017 Elsevier Inc. All rights reserved.

Neoadjuvant Sandwich Treatment With Oxaliplatin and Capecitabine Administered Prior to, Concurrently With, and Following Radiation Therapy in Locally Advanced Rectal Cancer: A Prospective Phase 2 Trial

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Yuan-Hong; Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou; Lin, Jun-Zhong

Purpose: Systemic failure remains the major challenge in management of locally advanced rectal cancer (LARC). To optimize the timing of neoadjuvant treatment and enhance systemic control, we initiated a phase 2 trial to evaluate a new strategy of neoadjuvant sandwich treatment, integrating induction chemotherapy, concurrent chemoradiation therapy, and consolidation chemotherapy. Here, we present preliminary results of this trial, reporting the tumor response, toxicities, and surgical complications. Methods and Materials: Fifty-one patients with LARC were enrolled, among which were two patients who were ineligible because of distant metastases before treatment. Patients were treated first with one cycle of induction chemotherapy consistingmore » of oxaliplatin, 130 mg/m² on day 1, with capecitabine, 1000 mg/m² twice daily for 14 days every 3 weeks (the XELOX regimen), followed by chemoradiation therapy, 50 Gy over 5 weeks, with the modified XELOX regimen (oxaliplatin 100 mg/m²), and then with another cycle of consolidation chemotherapy with the XELOX regimen. Surgery was performed 6 to 8 weeks after completion of radiation therapy. Tumor responses, toxicities, and surgical complications were recorded. Results: All but one patent completed the planned schedule of neoadjuvant sandwich treatment. Neither life-threatening blood count decrease nor febrile neutropenia were observed. Forty-five patents underwent optimal surgery with total mesorectal excision (TME). Four patients refused surgery because of clinically complete response. There was no perioperative mortality in this cohort. Five patients (11.1%) developed postoperative complications. Among the 45 patients who underwent TME, pathologic complete response (pCR), pCR or major regression, and at least moderate regression were achieved in 19 (42.2%), 37 (82.2%), and 44 patients (97.8%), respectively. Conclusions: Preliminary results suggest that the strategy of neoadjuvant sandwich treatment using XELOX regimen as induction, concomitant, and consolidation chemotherapy to the conventional radiation is well tolerated. The strategy is highly effective in terms of pCR and major regression, which warrants further investigation.« less

Evaluation with 3.0-T MR imaging: predicting the pathological response of triple-negative breast cancer treated with anthracycline and taxane neoadjuvant chemotherapy.

PubMed

Kim, Min Jung; Kim, Eun-Kyung; Park, Seho; Moon, Hee Jung; Kim, Seung Il; Park, Byeong-Woo

2015-09-01

Triple-negative breast cancer (TNBC) which expresses neither hormonal receptors nor HER-2 is associated with poor prognosis and shorter survival. Several studies have suggested that TNBC patients attaining pathological complete response (pCR) after neoadjuvant chemotherapy (NAC) show a longer survival than those without pCR. To assess the accuracy of 3.0-T breast magnetic resonance imaging (MRI) in predicting pCR and to evaluate the clinicoradiologic factors affecting the diagnostic accuracy of 3.0-T breast MRI in TNBC patients treated with anthracycline and taxane (ACD). This retrospective study was approved by the institutional review board; patient consent was not required. Between 2009 and 2012, 35 TNBC patients with 3.0-T breast MRI prior to (n = 26) or after (n = 35) NAC were included. MRI findings were reviewed according to pCR to chemotherapy. The diagnostic accuracy of 3.0-T breast MRI for predicting pCR and the clinicoradiological factors affecting MRI accuracy and response to NAC were analyzed. 3.0-T MRI following NAC with ACD accurately predicted pCR in 91.4% of TNBC patients. The residual tumor size between pathology and 3.0-T MRI in non-pCR cases showed a higher correlation in the Ki-67-positive TNBC group (r = 0.947) than in the Ki-67 negative group (r = 0.375) with statistical trends (P = 0.069). Pre-treatment MRI in the non-pCR group compared to the pCR group showed a larger tumor size (P = 0.030) and non-mass presentation (P = 0.015). 3.0-T MRI in TNBC patients following NAC with ACD showed a high accuracy for predicting pCR to NAC. Ki-67 can affect the diagnostic accuracy of 3.0-T MRI for pCR to NAC with ACD in TNBC patients. © The Foundation Acta Radiologica 2014.

Prospective validation of immunological infiltrate for prediction of response to neoadjuvant chemotherapy in HER2-negative breast cancer--a substudy of the neoadjuvant GeparQuinto trial.

PubMed

Issa-Nummer, Yasmin; Darb-Esfahani, Silvia; Loibl, Sibylle; Kunz, Georg; Nekljudova, Valentina; Schrader, Iris; Sinn, Bruno Valentin; Ulmer, Hans-Ullrich; Kronenwett, Ralf; Just, Marianne; Kühn, Thorsten; Diebold, Kurt; Untch, Michael; Holms, Frank; Blohmer, Jens-Uwe; Habeck, Jörg-Olaf; Dietel, Manfred; Overkamp, Friedrich; Krabisch, Petra; von Minckwitz, Gunter; Denkert, Carsten

2013-01-01

We have recently described an increased lymphocytic infiltration rate in breast carcinoma tissue is a significant response predictor for anthracycline/taxane-based neoadjuvant chemotherapy (NACT). The aim of this study was to prospectively validate the tumor-associated lymphocyte infiltrate as predictive marker for response to anthracycline/taxane-based NACT. The immunological infiltrate was prospectively evaluated in a total of 313 core biopsies from HER2 negative patients of the multicenter PREDICT study, a substudy of the neoadjuvant GeparQuinto study. Intratumoral lymphocytes (iTuLy), stromal lymphocytes (strLy) as well as lymphocyte-predominant breast cancer (LPBC) were evaluated by histopathological assessment. Pathological complete response (pCR) rates were analyzed and compared between the defined subgroups using the exact test of Fisher. Patients with lymphocyte-predominant breast cancer (LPBC) had a significantly increased pCR rate of 36.6%, compared to non-LPBC patients (14.3%, p<0.001). LPBC and stromal lymphocytes were significantly independent predictors for pCR in multivariate analysis (LPBC: OR 2.7, p = 0.003, strLy: OR 1.2, p = 0.01). The amount of intratumoral lymphocytes was significantly predictive for pCR in univariate (OR 1.2, p = 0.01) but not in multivariate logistic regression analysis (OR 1.2, p = 0.11). Confirming previous investigations of our group, we have prospectively validated in an independent cohort that an increased immunological infiltrate in breast tumor tissue is predictive for response to anthracycline/taxane-based NACT. Patients with LPBC and increased stromal lymphocyte infiltration have significantly increased pCR rates. The lymphocytic infiltrate is a promising additional parameter for histopathological evaluation of breast cancer core biopsies.

Predictive factors of pathologic complete response of HER2-positive breast cancer after preoperative chemotherapy with trastuzumab: development of a specific predictor and study of its utilities using decision curve analysis.

PubMed

Jankowski, Clémentine; Guiu, S; Cortet, M; Charon-Barra, C; Desmoulins, I; Lorgis, V; Arnould, L; Fumoleau, P; Coudert, B; Rouzier, R; Coutant, C; Reyal, F

2017-01-01

The aim of this study was to assess the Institut Gustave Roussy/M.D. Anderson Cancer Center (IGR/MDACC) nomogram in predicting pathologic complete response (pCR) to preoperative chemotherapy in a cohort of human epidermal growth factor receptor 2 (HER2)-positive tumors treated with preoperative chemotherapy with trastuzumab. We then combine clinical and pathological variables associated with pCR into a new nomogram specific to HER2-positive tumors treated by preoperative chemotherapy with trastuzumab. Data from 270 patients with HER2-positive tumors treated with preoperative chemotherapy with trastuzumab at the Institut Curie and at the Georges François Leclerc Cancer Center were used to assess the IGR/MDACC nomogram and to subsequently develop a new nomogram for pCR based on multivariate logistic regression. Model performance was quantified in terms of calibration and discrimination. We studied the utility of the new nomogram using decision curve analysis. The IGR/MDACC nomogram was not accurate for the prediction of pCR in HER2-positive tumors treated by preoperative chemotherapy with trastuzumab, with poor discrimination (AUC = 0.54, 95% CI 0.51-0.58) and poor calibration (p = 0.01). After uni- and multivariate analysis, a new pCR nomogram was built based on T stage (TNM), hormone receptor status, and Ki67 (%). The model had good discrimination with an area under the curve (AUC) at 0.74 (95% CI 0.70-0.79) and adequate calibration (p = 0.93). By decision curve analysis, the model was shown to be relevant between thresholds of 0.3 and 0.7. To the best of our knowledge, ours is the first nomogram to predict pCR in HER2-positive tumors treated by preoperative chemotherapy with trastuzumab. To ensure generalizability, this model needs to be externally validated.

A 17-molecule set as a predictor of complete response to neoadjuvant chemotherapy with docetaxel, cisplatin, and 5-fluorouracil in esophageal cancer

PubMed Central

Fumoto, Shoichi; Shibata, Tomotaka; Nishiki, Kohei; Tsukamoto, Yoshiyuki; Etoh, Tsuyoshi; Moriyama, Masatsugu; Shiraishi, Norio; Inomata, Masafumi

2017-01-01

Background Recently, neoadjuvant chemotherapy with docetaxel/cisplatin/5-fluorouracil (NAC-DCF) was identified as a novel strong regimen with a high rate of pathological complete response (pCR) in advanced esophageal cancer in Japan. Predicting pCR will contribute to the therapeutic strategy and the prevention of surgical invasion. However, a predictor of pCR after NAC-DCF has not yet been developed. The aim of this study was to identify a novel predictor of pCR in locally advanced esophageal cancer treated with NAC-DCF. Patients and methods A total of 32 patients who received NAC-DCF followed by esophagectomy between June 2013 and March 2016 were enrolled in this study. We divided the patients into the following 2 groups: pCR group (9 cases) and non-pCR group (23 cases), and compared gene expressions between these groups using DNA microarray data and KeyMolnet. Subsequently, a validation study of candidate molecular expression was performed in 7 additional cases. Results Seventeen molecules, including transcription factor E2F, T-cell-specific transcription factor, Src (known as “proto-oncogene tyrosine-protein kinase of sarcoma”), interferon regulatory factor 1, thymidylate synthase, cyclin B, cyclin-dependent kinase (CDK) 4, CDK, caspase-1, vitamin D receptor, histone deacetylase, MAPK/ERK kinase, bcl-2-associated X protein, runt-related transcription factor 1, PR domain zinc finger protein 1, platelet-derived growth factor receptor, and interleukin 1, were identified as candidate molecules. The molecules were mainly associated with pathways, such as transcriptional regulation by SMAD, RB/E2F, and STAT. The validation study indicated that 12 of the 17 molecules (71%) matched the trends of molecular expression. Conclusions A 17-molecule set that predicts pCR after NAC-DCF for locally advanced esophageal cancer was identified. PMID:29136005

Density of CD8+ lymphocytes in biopsy samples combined with the circulating lymphocyte ratio predicts pathologic complete response to chemoradiotherapy for rectal cancer.

PubMed

Xiao, Binyi; Peng, Jianhong; Zhang, Rongxin; Xu, Jing; Wang, Yongchun; Fang, Yujing; Lin, Junzhong; Pan, Zhizhong; Wu, Xiaojun

2017-01-01

The systemic status and local immune status, as determined by the neutrophil-lymphocyte ratio (NLR) or the lymphocyte ratio (LYMR) and tumor-infiltrating lymphocyte (TIL) count, respectively, have been suggested as predictors of the tumor response to neoadjuvant chemoradiotherapy (nCRT) in rectal cancer, although the utility of these measures remains controversial. We aimed to investigate the values of the LYMR, NLR and TIL count and their combinations (TIL-LYMR/TIL-NLR) in predicting pathologic complete response (pCR) after nCRT. Pretreatment biopsy samples and data from the blood tests of 92 patients with rectal cancer who underwent curative resection after nCRT were retrospectively obtained. CD8+ TILs were immunostained using an antibody against CD8. The density of CD8+ TILs was recorded as the number of CD8+ T cells per square millimeter, and the results were classified as either "high" or "low". The LYMR and NLR were calculated using pretreatment blood test data and categorized into either "high" or "low" groups. TIL-LYMR was graded as "low," "mid" or "high" when neither, one or both the CD8+ TIL count and LYMR were "high," respectively. TIL-NLR was graded similarly. The associations between TILs and LYMR, NLR and their combinations (TIL-LYMR/TIL-NLR) were evaluated. pCR was significantly associated with a high LYMR, a low NLR and increased chemotherapy cycles ( P =0.039, P =0.043 and P =0.015, respectively), but not with the CD8+ TIL count or carcinoembryonic antigen (CEA) level ( P =0.100 and P =0.590, respectively). Additionally, 40% of patients with high LYMR and 40.7% with low NLR achieved pCR, whereas only 19.7% with low LYMR and 20.3% with high NLR did so. When the combinations were assessed, TIL-LYMR showed a positive correlation with pCR ( P =0.038), while no association between TIL-NLR and pCR was found ( P =0.916). In multivariate analysis, TIL-LYMR remained an independent predictor of pCR (odds ratio [OR]=1.833, 95% confidence interval [CI]=1.069-3.142, P =0.028). High LYMR, low NLR and high TIL-LYMR at baseline are predictive of pCR to nCRT for patients with rectal cancer. These parameters may help identify pCR patients and provide additional information for therapeutic decision-making.

Impact of neoadjuvant chemotherapy and pathological complete response on eligibility for breast-conserving surgery in patients with early breast cancer: A meta-analysis.

PubMed

Criscitiello, Carmen; Golshan, Mehra; Barry, William T; Viale, Giulia; Wong, Stephanie; Santangelo, Michele; Curigliano, Giuseppe

2018-05-04

We conducted a meta-analysis of randomised trials evaluating pathological complete response (pCR) and surgical outcomes after neoadjuvant systemic therapy (NST) in patients with early breast cancer (EBC). The primary outcome was breast-conserving surgery (BCT) rate. Secondary outcomes were pCR rate and association to BCT. Meta-analyses were performed using random effects models that use inverse-variance weighting for each treatment arm based on evaluable patients. Point estimates are reported with 95% confidence interval (CI), and p < 0.05 was considered statistically significant. Thirty-six studies were identified (N = 12,311 patients). We selected for the analysis 16 of 36 studies reporting both pCR and BCT for at least one treatment arm. Arms per study ranged from one to six; 42 independent units were available to evaluate the association between pCR and BCT. BCT rate ranged 5-76% across arms with an average BCT of 57% (95% CI 52-62%). Significant heterogeneity was observed among the trials (Cochrane Q = 787, p < 0.001, I 2  = 97%). In the meta-regression model, BCT rates were not significantly associated with year of first patient-in (p = 0.89), grade (p = 0.93) and hormone-receptor status (p = 0.39). Clinical N-stage (p = 0.01) and human epidermal growth factor receptor (HER2) status (p = 0.03) were significantly associated with BCT. pCR rate ranged 3-60% across studies. The average pCR across all study arms was 24% (95% CI 19-29%). No association was observed between pCR rate in a study arm and the resulting BCT rate in a univariate model (p = 0.34) nor after adjusting for HER2 and clinical nodal status (p = 0.82). In the subset of 14 multi-arm studies, no significant association was seen between the differences in pCR and BCT between treatment arms (p = 0.27). pCR does not increase BCT in patients receiving NST for EBC. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pathological complete response after neoadjuvant chemotherapy is an independent predictive factor irrespective of simplified breast cancer intrinsic subtypes: a landmark and two-step approach analyses from the EORTC 10994/BIG 1-00 phase III trial.

PubMed

Bonnefoi, H; Litière, S; Piccart, M; MacGrogan, G; Fumoleau, P; Brain, E; Petit, T; Rouanet, P; Jassem, J; Moldovan, C; Bodmer, A; Zaman, K; Cufer, T; Campone, M; Luporsi, E; Malmström, P; Werutsky, G; Bogaerts, J; Bergh, J; Cameron, D A

2014-06-01

Pathological complete response (pCR) following chemotherapy is strongly associated with both breast cancer subtype and long-term survival. Within a phase III neoadjuvant chemotherapy trial, we sought to determine whether the prognostic implications of pCR, TP53 status and treatment arm (taxane versus non-taxane) differed between intrinsic subtypes. Patients were randomized to receive either six cycles of anthracycline-based chemotherapy or three cycles of docetaxel then three cycles of eprirubicin/docetaxel (T-ET). pCR was defined as no evidence of residual invasive cancer (or very few scattered tumour cells) in primary tumour and lymph nodes. We used a simplified intrinsic subtypes classification, as suggested by the 2011 St Gallen consensus. Interactions between pCR, TP53 status, treatment arm and intrinsic subtype on event-free survival (EFS), distant metastasis-free survival (DMFS) and overall survival (OS) were studied using a landmark and a two-step approach multivariate analyses. Sufficient data for pCR analyses were available in 1212 (65%) of 1856 patients randomized. pCR occurred in 222 of 1212 (18%) patients: 37 of 496 (7.5%) luminal A, 22 of 147 (15%) luminal B/HER2 negative, 51 of 230 (22%) luminal B/HER2 positive, 43 of 118 (36%) HER2 positive/non-luminal, 69 of 221(31%) triple negative (TN). The prognostic effect of pCR on EFS did not differ between subtypes and was an independent predictor for better EFS [hazard ratio (HR) = 0.40, P < 0.001 in favour of pCR], DMFS (HR = 0.32, P < 0.001) and OS (HR = 0.32, P < 0.001). Chemotherapy arm was an independent predictor only for EFS (HR = 0.73, P = 0.004 in favour of T-ET). The interaction between TP53, intrinsic subtypes and survival outcomes only approached statistical significance for EFS (P = 0.1). pCR is an independent predictor of favourable clinical outcomes in all molecular subtypes in a two-step multivariate analysis. EORTC 10994/BIG 1-00 Trial registration number NCT00017095. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Association Between Adjuvant Chemotherapy and Overall Survival in Patients With Rectal Cancer and Pathological Complete Response After Neoadjuvant Chemotherapy and Resection.

PubMed

Dossa, Fahima; Acuna, Sergio A; Rickles, Aaron S; Berho, Mariana; Wexner, Steven D; Quereshy, Fayez A; Baxter, Nancy N; Chadi, Sami A

2018-04-19

Although American guidelines recommend use of adjuvant chemotherapy in patients with locally advanced rectal cancer, individuals who achieve a pathological complete response (pCR) following neoadjuvant chemoradiotherapy are less likely to receive adjuvant treatment than incomplete responders. The association and resection of adjuvant chemotherapy with survival in patients with pCR is unclear. To determine whether patients with locally advanced rectal cancer who achieve pCR after neoadjuvant chemoradiation therapy and resection benefit from the administration of adjuvant chemotherapy. This retrospective propensity score-matched cohort study identified patients with locally advanced rectal cancer from the National Cancer Database from 2006 through 2012. We selected patients with nonmetastatic invasive rectal cancer who achieved pCR after neoadjuvant chemoradiation therapy and resection. We matched patients who received adjuvant chemotherapy to patients who did not receive adjuvant treatment in a 1:1 ratio. We separately matched subgroups of patients with node-positive disease before treatment and node-negative disease before treatment to investigate for effect modification by pretreatment nodal status. We compared overall survival between groups using Kaplan-Meier survival methods and Cox proportional hazards models. We identified 2455 patients (mean age, 59.5 years; 59.8% men) with rectal cancer with pCR after neoadjuvant chemoradiation therapy and resection. We matched 667 patients with pCR who received adjuvant chemotherapy and at least 8 weeks of follow-up after surgery to patients with pCR who did not receive adjuvant treatment. Over a median follow-up of 3.1 years (interquartile range, 1.94-4.40 years), patients treated with adjuvant chemotherapy demonstrated better overall survival than those who did not receive adjuvant treatment (hazard ratio, 0.44; 95% CI, 0.28-0.70). When stratified by pretreatment nodal status, only those patients with pretreatment node-positive disease exhibited improved overall survival with administration of adjuvant chemotherapy (hazard ratio, 0.24; 95% CI, 0.10-0.58). The administration of adjuvant chemotherapy in patients with rectal cancer with pCR is associated with improved overall survival, particularly in patients with pretreatment node-positive disease. Although this study suggests a beneficial effect of adjuvant treatment on survival in patients with pCR, these results are limited by the presence of potential unmeasured confounding in this nonrandomized study.

Circulating myeloid-derived suppressor cells increase in patients undergoing neo-adjuvant chemotherapy for breast cancer.

PubMed

Wesolowski, Robert; Duggan, Megan C; Stiff, Andrew; Markowitz, Joseph; Trikha, Prashant; Levine, Kala M; Schoenfield, Lynn; Abdel-Rasoul, Mahmoud; Layman, Rachel; Ramaswamy, Bhuvaneswari; Macrae, Erin R; Lustberg, Maryam B; Reinbolt, Raquel E; Mrozek, Ewa; Byrd, John C; Caligiuri, Michael A; Mace, Thomas A; Carson, William E

2017-11-01

This study sought to evaluate whether myeloid-derived suppressor cells (MDSC) could be affected by chemotherapy and correlate with pathologic complete response (pCR) in breast cancer patients receiving neo-adjuvant chemotherapy. Peripheral blood levels of granulocytic (G-MDSC) and monocytic (M-MDSC) MDSC were measured by flow cytometry prior to cycle 1 and 2 of doxorubicin and cyclophosphamide and 1st and last administration of paclitaxel or paclitaxel/anti-HER2 therapy. Of 24 patients, 11, 6 and 7 patients were triple negative, HER2+ and hormone receptor+, respectively. 45.8% had pCR. Mean M-MDSC% were <1. Mean G-MDSC% and 95% confidence intervals were 0.88 (0.23-1.54), 5.07 (2.45-7.69), 9.32 (4.02-14.61) and 1.97 (0.53-3.41) at draws 1-4. The increase in G-MDSC by draw 3 was significant (p < 0.0001) in all breast cancer types. G-MDSC levels at the last draw were numerically lower in patients with pCR (1.15; 95% CI 0.14-2.16) versus patients with no pCR (2.71; 95% CI 0-5.47). There was no significant rise in G-MDSC from draw 1 to 3 in African American patients, and at draw 3 G-MDSC levels were significantly lower in African Americans versus Caucasians (p < 0.05). It was concluded that G-MDSC% increased during doxorubicin and cyclophosphamide therapy, but did not significantly differ between patients based on pathologic complete response.

Detection of hepatitis C viral sequences in formalin-fixed, paraffin-embedded liver tissue: effect of interferon alpha therapy.

PubMed

Diamond, D A; Davis, G L; Qian, K P; Lau, J Y

1994-03-01

To determine the effect of interferon-alpha (IFN) therapy on hepatitis C virus (HCV) in liver, reverse transcription "nested" polymerase chain reaction (RT-PCR) was applied to detect HCV RNA in formalin-fixed, paraffin-embedded liver biopsy specimens obtained before and at the end of IFN therapy in 42 patients with chronic HCV infection. Results were correlated with the clinical and biochemical outcome in 36 cases. Fifteen patients were nonresponders to IFN; 13 patients had a complete response to IFN but relapsed shortly after IFN was stopped (responders who relapsed); and 8 patients showed a complete and sustained response to IFN therapy (sustained responders). Total RNA was extracted using proteinase K digestion and phenol/chloroform/isoamyl alcohol extraction, and HCV RNA was detected by standard RT-PCR using primers from the highly conserved 5' untranslated region. HCV RNA was detected in 41 of the 42 pretreatment specimens. Of the 36 patients with paired posttreatment samples, HCV RNA was detected in all 15 patients who did not respond to IFN and 9 of 13 who responded to IFN but relapsed shortly after IFN was stopped. In contrast, only one of the eight patients who had a sustained response to IFN therapy had HCV RNA detected by RT-PCR (P < 0.04). These data confirm 1) the feasibility of detecting HCV RNA in formalin-fixed, paraffin-embedded tissue from patients with chronic HCV infection, 2) show that sustained response to IFN is associated with loss of liver HCV RNA at the end of IFN therapy, and 3) offer an explanation for recurrence in patients who relapse.

Impact of implementing an EMR on physical exam documentation by ambulance personnel.

PubMed

Katzer, R; Barton, D J; Adelman, S; Clark, S; Seaman, E L; Hudson, K B

2012-01-01

Georgetown University has a student run Emergency Medical Services (EMS) organization with over 100 emergency medical technicians (EMTs). We set out to determine whether implementing an electronic patient care report (ePCR) system was associated with improved physical exam documentation. This study evaluated documentation of the physical exam on prehospital patient care reports (PCRs). An ePCR system was implemented. ePCR documentation was compared to that of the previously used paper PCRs. This study looked retrospectively at 154 PCRs. 77 were hand written PCRs from before the electronic system. The PCRs involved chief complaints that were primarily respiratory, neurologic, or both. 77 ePCRs of matching chief complaint categories were used for comparison. Each chart was reviewed for completion of certain physical exam findings. The mean percentage of documented components from the ePCRs was compared to that of the hand written PCRs. The null hypothesis was that the absolute increase in the mean was not more than 20 percent. The two exclusion criteria were PCRs completed by study investigators after the design of the project and partially or completely missing PCRs. The absolute increase in mean physical exam component documentation was 36% (95% CI = 29-43%). A weighted kappa of 0.894 showed very good agreement between chart reviewers. This study rejected the null hypothesis that the ePCR system was associated with a mean increase of no more than 20%. It observed increase in physical exam documentation. Limitations of this study included the inability to determine whether documentation of physical exam findings reflected performance of the physical exam, and what components of the ePCR system bundle were responsible for the increase in physical exam component documentation.

Impact of implementing an EMR on physical exam documentation by ambulance personnel

PubMed Central

Katzer, R.; Barton, D.J.; Adelman, S.; Clark, S.; Seaman, E.L.; Hudson, K.B.

2012-01-01

Objectives Georgetown University has a student run Emergency Medical Services (EMS) organization with over 100 emergency medical technicians (EMTs). We set out to determine whether implementing an electronic patient care report (ePCR) system was associated with improved physical exam documentation. Methods This study evaluated documentation of the physical exam on prehospital patient care reports (PCRs). An ePCR system was implemented. ePCR documentation was compared to that of the previously used paper PCRs. This study looked retrospectively at 154 PCRs. 77 were hand written PCRs from before the electronic system. The PCRs involved chief complaints that were primarily respiratory, neurologic, or both. 77 ePCRs of matching chief complaint categories were used for comparison. Each chart was reviewed for completion of certain physical exam findings. The mean percentage of documented components from the ePCRs was compared to that of the hand written PCRs. The null hypothesis was that the absolute increase in the mean was not more than 20 percent. The two exclusion criteria were PCRs completed by study investigators after the design of the project and partially or completely missing PCRs. Results The absolute increase in mean physical exam component documentation was 36% (95% CI = 29–43%). A weighted kappa of 0.894 showed very good agreement between chart reviewers. Conclusions This study rejected the null hypothesis that the ePCR system was associated with a mean increase of no more than 20%. It observed increase in physical exam documentation. Limitations of this study included the inability to determine whether documentation of physical exam findings reflected performance of the physical exam, and what components of the ePCR system bundle were responsible for the increase in physical exam component documentation. PMID:23646077

Pathway level alterations rather than mutations in single genes predict response to HER2-targeted therapies in the neo-ALTTO trial.

PubMed

Shi, W; Jiang, T; Nuciforo, P; Hatzis, C; Holmes, E; Harbeck, N; Sotiriou, C; Peña, L; Loi, S; Rosa, D D; Chia, S; Wardley, A; Ueno, T; Rossari, J; Eidtmann, H; Armour, A; Piccart-Gebhart, M; Rimm, D L; Baselga, J; Pusztai, L

2017-01-01

We performed whole-exome sequencing of pretreatment biopsies and examined whether genome-wide metrics of overall mutational load, clonal heterogeneity or alterations at variant, gene, and pathway levels are associated with treatment response and survival. Two hundred and three biopsies from the NeoALTTO trial were analyzed. Mutations were called with MuTect, and Strelka, using pooled normal DNA. Associations between DNA alterations and outcome were evaluated by logistic and Cox-proportional hazards regression. There were no recurrent single gene mutations significantly associated with pathologic complete response (pCR), except PIK3CA [odds ratio (OR) = 0.42, P = 0.0185]. Mutations in 33 of 714 pathways were significantly associated with response, but different genes were affected in different individuals. PIK3CA was present in 23 of these pathways defining a ‘trastuzumab resistance-network’ of 459 genes. Cases with mutations in this network had low pCR rates to trastuzumab (2/50, 4%) compared with cases with no mutations (9/16, 56%), OR = 0.035; P < 0.001. Mutations in the ‘Regulation of RhoA activity’ pathway were associated with higher pCR rate to lapatinib (OR = 14.8, adjusted P = 0.001), lapatinib + trastuzumab (OR = 3.0, adjusted P = 0.09), and all arms combined (OR = 3.77, adjusted P = 0.02). Patients (n = 124) with mutations in the trastuzumab resistance network but intact RhoA pathway had 2% (1/41) pCR rate with trastuzumab alone (OR = 0.026, P = 0.001) but adding lapatinib increased pCR rate to 45% (17/38, OR = 1.68, P = 0.3). Patients (n = 46) who had no mutations in either gene set had 6% pCR rate (1/15) with lapatinib, but had the highest pCR rate, 52% (8/15) with trastuzumab alone. Mutations in the RhoA pathway are associated with pCR to lapatinib and mutations in a PIK3CA-related network are associated with resistance to trastuzumab. The combined mutation status of these two pathways could define patients with very low response rate to trastuzumab alone that can be augmented by adding lapatinib or substituting trastuzumab with lapatinib.

Effects of neoadjuvant chemotherapy with or without zoledronic acid on pathological response: A meta-analysis of randomised trials.

PubMed

Kroep, J R; Charehbili, A; Coleman, R E; Aft, R L; Hasegawa, Y; Winter, M C; Weilbaecher, K; Akazawa, K; Hinsley, S; Putter, H; Liefers, G J; Nortier, J W R; Kohno, N

2016-02-01

The addition of bisphosphonates to adjuvant therapy improves survival in postmenopausal breast cancer (BC) patients. We report a meta-analysis of four randomised trials of neoadjuvant chemotherapy (CT) +/- zoledronic acid (ZA) in stage II/III BC to investigate the potential for enhancing the pathological response. Individual patient data from four prospective randomised clinical trials reporting the effect of the addition of ZA on the pathological response after neoadjuvant CT were pooled. Primary outcomes were pathological complete response in the breast (pCRb) and in the breast and lymph nodes (pCR). Trial-level and individual patient data meta-analyses were done. Predefined subgroup-analyses were performed for postmenopausal women and patients with triple-negative BC. pCRb and pCR data were available in 735 and 552 patients respectively. In the total study population ZA addition to neoadjuvant CT did not increase pCRb or pCR rates. However, in postmenopausal patients, the addition of ZA resulted in a significant, near doubling of the pCRb rate (10.8% for CT only versus 17.7% with CT+ZA; odds ratio [OR] 2.14, 95% confidence interval [CI] 1.01-4.55) and a non-significant benefit of the pCR rate (7.8% for CT only versus 14.6% with CT+ZA; OR 2.62, 95% CI 0.90-7.62). In patients with triple-negative BC a trend was observed favouring CT+ZA. This meta-analysis shows no impact from the addition of ZA to neoadjuvant CT on pCR. However, as has been seen in the adjuvant setting, the addition of ZA to neoadjuvant CT may augment the effects of CT in postmenopausal patients with BC. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

De-Escalation Strategies in Human Epidermal Growth Factor Receptor 2 (HER2)-Positive Early Breast Cancer (BC): Final Analysis of the West German Study Group Adjuvant Dynamic Marker-Adjusted Personalized Therapy Trial Optimizing Risk Assessment and Therapy Response Prediction in Early BC HER2- and Hormone Receptor-Positive Phase II Randomized Trial-Efficacy, Safety, and Predictive Markers for 12 Weeks of Neoadjuvant Trastuzumab Emtansine With or Without Endocrine Therapy (ET) Versus Trastuzumab Plus ET.

PubMed

Harbeck, Nadia; Gluz, Oleg; Christgen, Matthias; Kates, Ronald Ernest; Braun, Michael; Küemmel, Sherko; Schumacher, Claudia; Potenberg, Jochem; Kraemer, Stefan; Kleine-Tebbe, Anke; Augustin, Doris; Aktas, Bahriye; Forstbauer, Helmut; Tio, Joke; von Schumann, Raquel; Liedtke, Cornelia; Grischke, Eva-Maria; Schumacher, Johannes; Wuerstlein, Rachel; Kreipe, Hans Heinrich; Nitz, Ulrike Anneliese

2017-09-10

Purpose Human epidermal growth factor receptor 2 (HER2)-positive/hormone receptor (HR)-positive breast cancer is a distinct subgroup associated with lower chemotherapy sensitivity and slightly better outcome than HER2-positive/HR-negative disease. Little is known about the efficacy of the combination of endocrine therapy (ET) with trastuzumab or with the potent antibody-cytotoxic, anti-HER2 compound trastuzumab emtansine (T-DM1) with or without ET for this subgroup. The West German Study Group trial, ADAPT (Adjuvant Dynamic Marker-Adjusted Personalized Therapy Trial Optimizing Risk Assessment and Therapy Response Prediction in Early Breast Cancer) compares pathologic complete response (pCR) rates of T-DM1 versus trastuzumab with ET in early HER2-positive/HR-positive breast cancer. Patients and Methods In this prospective, neoadjuvant, phase II trial, 375 patients with early breast cancer with HER2-positive and HR-positive status (n = 463 screened) were randomly assigned to 12 weeks of T-DM1 with or without ET or to trastuzumab with ET. The primary end point was pCR (ypT0/is/ypN0). Early response was assessed in 3-week post-therapeutic core biopsies (proliferation decrease ≥ 30% Ki-67 or cellularity response). Secondary end points included safety and predictive impact of early response on pCR. Adjuvant therapy followed national standards. Results Baseline characteristics were well balanced among the arms. More than 90% of patients completed the therapy per protocol. pCR was observed in 41.0% of patients treated with T-DM1, 41.5% of patients treated with T-DM1 and ET, and 15.1% with trastuzumab and ET ( P < .001). Early responders (67% of patients with assessable response) achieved pCR in 35.7% compared with 19.8% in nonresponders (odds ratio, 2.2; 95% CI, 1.24 to 4.19). T-DM1 was associated with a significantly higher prevalence of grade 1 to 2 toxicities, especially thrombocytopenia, nausea, and elevation of liver enzymes. Overall toxicity was low; seventeen therapy-related severe adverse events (T-DM1 arms v trastuzumab plus ET; 5.3% v 3.1%, respectively) were reported. Conclusion The ADAPT HER2-positive/HR-positive trial demonstrates that neoadjuvant T-DM1 (with or without ET) given for only 12 weeks results in a clinically meaningful pCR rate. Thus, a substantial number of patients are spared the adverse effects of systemic chemotherapy.

RNA Disruption and Drug Response in Breast Cancer Primary Systemic Therapy.

PubMed

Pritzker, Kenneth; Pritzker, Laura; Generali, Daniele; Bottini, Alberto; Cappelletti, Maria Rosa; Guo, Baoqing; Parissenti, Amadeo; Trudeau, Maureen

2015-05-01

As there is now evidence that switching clinical nonresponders early in primary systemic therapy to alternate treatment regimens can enhance survival in some breast cancer patients, the need for a robust intermediate endpoint that can guide treatment response across all tumor subtypes is urgent. Recently, chemotherapy drugs have been shown to induce a decrease in RNA quality in tumor cells from breast cancer biopsies in some patients at midtherapy, and that this has been associated with subsequent achievement of pathological complete response (pCR). The decrease in RNA quality has been shown to be associated with RNA disruption; aberrant RNA bands visualized by RNA electrophoresis have been associated with subsequent tumor cell death. The objectives of these studies are to show that a new assay based on induction of RNA disruption in tumor cells by chemotherapy can stratify at midtherapy, pCR responders from non-pCR responders irrespective of clinical response and to present early evidence that clinically useful RNA disruption can be detected as early as 14 days after initiation of treatment. RNA disruption in tumor cells was quantified by analysis of the RNA electrophoresis banding pattern and expressed as an RNA disruption index (RDI). To develop the RNA disruption assay (RDA), RDI was correlated with clinical outcome (pCR) from the NCIC-CTG MA.22 breast cancer clinical trial (ClinicalTrials.gov NCT00066443). RDA Zones were established by stratifying patients using RDI values into Zone 1, Zone 2, and Zone 3. Zone 3 included seven out of eight pCR responders, whereas Zone 1 contained no pCR responders. An intermediate zone (Zone 2) was established which contained one pCR. Subsequently, to determine early drug response, RNA disruption was examined by RDI after 14 days exposure to trastuzumab, zoledronic acid, or letrozole + cyclophosphamide ± sorafenib therapy. In MA.22, RDA stratified 23 of 85 patients in Zone 1 as pCR nonresponders, 24 patients in Zone 2, an intermediate zone, and 38 patients in Zone 3, pCR responders and non-pCR patients who share RDI comparable to those achieving pCR. In the early response studies, after 14 days exposure to chemotherapy, some RNA disruption as measured by RDI elevation could be detected in 3/12 trastuzumab, 7/15 zoledronic acid, 5/29 letrozole + cyclophosphamide, and 5/23 letrozole + cyclophosphamide + sorafenib patients. RDA is a novel intermediate endpoint that has promise for clinical utility for breast cancers early in response-guided primary systemic therapy. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Association between radiation dose and pathological complete response after preoperative radiochemotherapy in esophageal squamous cell cancer.

PubMed

Ordu, Arif Deniz; Nieder, Carsten; Geinitz, Hans; Scherer, Vera; Kup, Philipp Günther; Schuster, Tibor; Combs, Stephanie E; Fakhrian, Khashayar

2014-12-01

This study was undertaken to examine the impact of radiation dose on pathological complete response (pCR) rates following neoadjuvant radiochemotherapy (N-RCT) for squamous cell esophageal cancer (ESCC). From 1988 to 2011, 218 patients were treated with 30-30.6 Gy (1.8-2 Gy per fraction), 39.6-40 Gy (1.8-2 Gy per fraction) or 44-45 Gy (1.8-2 Gy per fraction) and concomitant cisplatin ± 5-fluorouracil (5-FU), oxaliplatin + 5-FU or 5-FU alone. The most commonly used concomitant chemotherapy was continuous infusion of 5-FU-alone with a dose of 300 mg/m(2)/day during the whole course of treatment (n=111). To eliminate the dispersing effect of potentially different efficacy levels of these drug regimens on pCR, we excluded patients with regimens other than 5-FU-alone. Histomorphological regression grade 1a (0% residual tumor), 1b (<10% residual tumor), 2 (10-50% residual tumor) and 3 (>50% residual tumor) was observed in 26 (23%), 24 (22%), 36 (32%) and 25 (23%) patients, respectively. pCR was observed in 9 out of 71 (13%) patients treated with 30 Gy-30.6 Gy, 13 of 34 (38%) patients treated with 39.6-40 Gy and 4 of 6 (67%) patients treated with 44-45 Gy (p=0.001). Median follow-up time from the start of N-RCT was 191 months (range=2-262 months). The estimated 5-year overall survival (OS) was 33% for the whole cohort. OS at 5 years was 58% for patients with pCR compared to 25% for patients with less favorable response to N-RCT (p=0.009), respectively. The dose of radiation correlates significantly with the likelihood of achieving a pCR in stage II/III squamous cell esophageal cancer patients. Prospective randomized trials are required to definitively evaluate the impact of application of higher radiation doses on efficacy and safety/tolerability in the context of N-RCT on the clinical outcomes. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

FY*X real-time polymerase chain reaction with melting curve analysis associated with a complete one-step real-time FY genotyping.

PubMed

Ansart-Pirenne, H; Martin-Blanc, S; Le Pennec, P-Y; Rouger, P; Cartron, J-P; Tournamille, C

2007-02-01

The Duffy (FY) blood group system is controlled by four major alleles: FY*A and FY*B, the Caucasian common alleles, encoding Fy(a) and Fy(b) antigens; FY*X allele responsible for a poorly expressed Fy(b) antigen, and FY*Fy a silent predominant allele among Black population. Despite the recent development of a real-time fluorescent polymerase chain reaction (PCR) method for FY genotyping FY*X genotyping has not been described by this method. This study focused on the real-time FY*X genotyping development associated with a complete, one-step real-time FY genotyping, based on fluorescence resonance energy transfer (FRET) technology. Seventy-two blood samples from Fy(a+b-) Caucasian blood donors were studied by real-time PCR only. Forty-seven Caucasian and Black individual blood samples, referred to our laboratory, were studied by PCR-RFLP and real-time PCR. For each individual, the result of the genotype was compared to the known phenotype. The FY*X allele frequency calculated in an Fy(a+b-) Caucasian blood donors population was 0.014. With the Caucasian and Black patient samples we found a complete correlation between PCR-RFLP and the real-time PCR method whatever the alleles combination tested. When the known phenotype was not correlated to FY*X genotype, the presence of the Fy(b) antigen was always confirmed by adsorption-elution. The real-time technology method is rapid and accurate for FY genotyping. From now, we are able to detect the FY*X allele in all the alleles combinations studied. Regarding its significant frequency, the detection of the FY*X allele is useful for the correct typing of blood donors and recipients considering the therapeutic use of blood units and the preparation of test red blood cells for antibody screening.

Assessment of early response biomarkers in relation to long‐term survival in patients with HER2‐negative breast cancer receiving neoadjuvant chemotherapy plus bevacizumab: Results from the Phase II PROMIX trial

PubMed Central

Kimbung, Siker; Markholm, Ida; Bjöhle, Judith; Lekberg, Tobias; von Wachenfeldt, Anna; Azavedo, Edward; Saracco, Ariel; Hellström, Mats; Veerla, Srinivas; Paquet, Eric; Bendahl, Pär‐Ola; Fernö, Mårten; Bergh, Jonas; Loman, Niklas

2017-01-01

Pathologic complete response (pCR) is a predictor for favorable outcome after neoadjuvant treatment in early breast cancer. Modulation of gene expression may also provide early readouts of biological activity and prognosis, offering the possibility for timely response‐guided treatment adjustment. The role of early transcriptional changes in predicting response to neoadjuvant chemotherapy plus bevacizumab was investigated. One‐hundred‐and‐fifty patients with large, operable and locally advanced HER2‐negative breast cancer received epirubicin and docetaxel, with the addition of bevacizumab. Patients underwent tumor biopsies at baseline, after Cycle 2 and at the time of surgery. The primary end point, pCR, and its relation with the secondary endpoints event‐free survival (EFS), overall survival (OS) and gene expression profiles, are reported. The pCR rate was 13% (95% CI 8.6–20.2), with significantly more pCRs among triple‐negative [28% (95% CI 14.8–45.4)] than among hormone receptor positive (HR+) tumors [9% (95% CI 4.6–16.3); (OR = 3.9 [CI = 1.5–10.3])]. pCR rates were not associated with EFS or OS. PAM50 subtypes significantly changed after Cycle 2 (p = 0.03) and an index of absolute changes in PAM50 correlations between these time‐points was associated with EFS [HR = 0.62 (CI = 0.3–1.1)]. In univariable analyses, signatures for angiogenesis, proliferation, estrogen receptor signaling, invasion and metastasis, and immune response, measured after Cycle 2, were associated with pCR in HR+ tumors. Evaluation of changes in molecular subtypes and other signatures early in the course of neoadjuvant treatment may be predictive of pCR and EFS. These factors may help guide further treatment and should be considered when designing neoadjuvant trials. PMID:28940389

The short-term outcomes of induction SOX (S-1 + oxaliplatin) ± cetuximab chemotherapy followed by short-course chemoradiotherapy in patients with poor-risk locally advanced rectal cancer.

PubMed

Beppu, Naohito; Yoshie, Hidenori; Kimura, Fumihiko; Aihara, Tsukasa; Doi, Hiroshi; Kamikonya, Norihiko; Matsubara, Nagahide; Tomita, Naohiro; Yanagi, Hidenori; Yamanaka, Naoki

2016-10-01

To evaluate the safety and efficacy of induction SOX (S-1 + oxaliplatin) ± cetuximab chemotherapy followed by short-course chemoradiotherapy and surgery in patients with poor-risk locally advanced rectal cancer. We enrolled eligible patients with poor-risk rectal cancer defined as T3 lower rectal cancer with mesorectal fascia involvement, T4a or T4b tumors or cases with lateral lymph node swelling. The primary endpoint was a pathological complete response (pCR), and the secondary endpoints were the objective response rate (ORR) and the pathological high response rate (Grade 2 plus 3). Twenty eligible patients were enrolled. The majority (75.0 %, 15/20) of the patients completed four cycles of induction chemotherapy, and all patients completed the radiotherapy (25 Gy/10 fractions/5 days). The global rate of Grade 3-4 toxicities was 30.0 % (6/20 patients). The ORRs were 85.0 % (17/20) and 95.0 % (19/20) in the patients who underwent R0 and R1 resection, respectively. The pathological high response rate was 70.0 % (14/20) and the pCR was 10.0 % (2/20). The regimen of induction SOX (S-1 + oxaliplatin) ± cetuximab chemotherapy followed by short-course chemoradiotherapy is safe and is associated with good tumor regression in patients with poor-risk locally advanced rectal cancer.

Impact of treatment characteristics on response of different breast cancer phenotypes: pooled analysis of the German neo-adjuvant chemotherapy trials.

PubMed

von Minckwitz, Gunter; Untch, Michael; Nüesch, Eveline; Loibl, Sibylle; Kaufmann, Manfred; Kümmel, Sherko; Fasching, Peter A; Eiermann, Wolfgang; Blohmer, Jens-Uwe; Costa, Serban Dan; Mehta, Keyur; Hilfrich, Jörn; Jackisch, Christian; Gerber, Bernd; du Bois, Andreas; Huober, Jens; Hanusch, Claus; Konecny, Gottfried; Fett, Werner; Stickeler, Elmar; Harbeck, Nadia; Müller, Volkmar; Jüni, Peter

2011-01-01

Pathological complete response (pCR) to neoadjuvant treatment correlates with outcome in breast cancer. We determined whether characteristics of neoadjuvant therapy are associated with pCR. We used multi-level models, which accounted for heterogeneity in pCR across trials and trial arms, to analyze individual patient data from 3332 women included in 7 German neoadjuvant trials with uniform protocols. PCR was associated with an increase in number of chemotherapy cycles (odds ratio [OR] 1.2 for every two additional cycles; P = 0.009), with higher cumulative anthracycline doses (OR 1.6; P = 0.002), higher cumulative taxane doses (OR 1.6; P = 0.009), and with capecitabine containing regimens (OR 1.62; P = 0.022). Association of pCR with increase in number of cycles appeared more pronounced in hormone receptor (HR)-positive tumors (OR 1.35) than in HR-negative tumors (OR 1.04; P for interaction = 0.046). Effect of anthracycline dose was particularly pronounced in HER2-negative tumors (OR 1.61), compared to HER2-positive tumors (OR 0.83; P for interaction = 0.14). Simultaneous trastuzumab treatment in HER2-positive tumors increased odds of pCR 3.2-fold (P < 0.001). No association of pCR and number of trastuzumab cycles was found (OR 1.20, P = 0.39). Dosing characteristics appear important for successful treatment of breast cancer. Longer treatment, higher cumulative doses of anthracyclines and taxanes, and the addition of capecitabine and trastuzumab are associated with better response. Tailoring according to breast cancer phenotype might be possible: longer treatment in HR-positive tumors, higher cumulative anthracycline doses for HER2-negative tumors, shorter treatment at higher cumulative doses for triple-negative tumors, and limited number of preoperative trastuzumab cycles in HER2-positive tumors.

A gene expression signature of Retinoblastoma loss-of-function predicts resistance to neoadjuvant chemotherapy in ER-positive/HER2-positive breast cancer patients.

PubMed

Risi, Emanuela; Grilli, Andrea; Migliaccio, Ilenia; Biagioni, Chiara; McCartney, Amelia; Guarducci, Cristina; Bonechi, Martina; Benelli, Matteo; Vitale, Stefania; Biganzoli, Laura; Bicciato, Silvio; Di Leo, Angelo; Malorni, Luca

2018-07-01

HER2-positive (HER2+) breast cancers show heterogeneous response to chemotherapy, with the ER-positive (ER+) subgroup deriving less benefit. Loss of retinoblastoma tumor suppressor gene (RB1) function has been suggested as a cardinal feature of breast cancers that are more sensitive to chemotherapy and conversely resistant to CDK4/6 inhibitors. We performed a retrospective analysis exploring RBsig, a gene signature of RB loss, as a potential predictive marker of response to neoadjuvant chemotherapy in ER+/HER2+ breast cancer patients. We selected clinical trials of neoadjuvant chemotherapy ± anti-HER2 therapy in HER2+ breast cancer patients with available information on gene expression data, hormone receptor status, and pathological complete response (pCR) rates. RBsig expression was computed in silico and correlated with pCR. Ten studies fulfilled the inclusion criteria and were included in the analysis (514 patients). Overall, of 211 ER+/HER2+ breast cancer patients, 49 achieved pCR (23%). The pCR rate following chemotherapy ± anti-HER2 drugs in patients with RBsig low expression was significantly lower compared to patients with RBsig high expression (16% vs. 30%, respectively; Fisher's exact test p = 0.015). The area under the ROC curve (AUC) was 0.62 (p = 0.005). In the 303 ER-negative (ER-)/HER2+ patients treated with chemotherapy ± anti-HER2 drugs, the pCR rate was 43%. No correlation was found between RBsig expression and pCR rate in this group. Low expression of RBsig identifies a subset of ER+/HER2+ patients with low pCR rates following neoadjuvant chemotherapy ± anti-HER2 therapy. These patients may potentially be spared chemotherapy in favor of anti-HER2, endocrine therapy, and CDK 4/6 inhibitor combinations.

Predictive value of PET-CT for pathological response in stages II and III breast cancer patients following neoadjuvant chemotherapy with docetaxel.

PubMed

García García-Esquinas, Marta A; Arrazola García, Juan; García-Sáenz, José A; Furió-Bacete, V; Fuentes Ferrer, Manuel E; Ortega Candil, Aída; Cabrera Martín, María N; Carreras Delgado, José L

2014-01-01

To prospectively study the value of PET-CT with fluorine-18 fluorodeoxyglucose (FDG) to predict neoadjuvant chemotherapy (NAC) response of locoregional disease of stages II and III breast cancer patients. A written informed consent and approval were obtained from the Ethics Committee. PET-CT accuracy in the prediction of pathologic complete response (pCR) after NAC was studied in primary tumors and lymph node metastasis in 43 women (mean age: 50 years: range: 27-71 years) with histologically proven breast cancer between December 2009 and January 2011. PET-CT was performed at baseline and after NAC. SUV(max) percentage changes (ΔSUV(max)) were compared with pathology findings at surgery. Receiver-operator characteristic (ROC) analysis was used to discriminate between locoregional pCR and non-pCR. In patients not achieving pCR, it was investigated if ΔSUV(max) could accurately identify the residual cancer burden (RCB) classes: RCB-I (minimal residual disease (MRD)), RCB-II (moderate RD), and RCB-III (extensive RD). pCR was obtained in 11 patients (25.6%). Residual disease was found in 32 patients (74.4%): 16 (37.2%) RCB-I, 15 (35.6%) RCB-II and 2 (4.7%) RCB-III. Sensitivity, specificity, and accuracy to predict pCR were 90.9%, 90.6%, and 90.7%, respectively. Specificity was 94.1% in the identification of a subset of patients who had either pCR or MRD. Accuracy of ΔSUV(max) in the locoregional disease of stages II and III breast cancer patients after NAC is high for the identification of pCR cases. Its specificity is potentially sufficient to identify a subgroup of patients who could be managed with conservative surgery. Copyright © 2013 Elsevier España, S.L. and SEMNIM. All rights reserved.

Tumour size is the only predictive factor of distant recurrence after pathological complete response to neoadjuvant chemotherapy in patients with large operable or locally advanced breast cancers: a sub-study of EORTC 10994/BIG 1-00 phase III trial.

PubMed

Fei, F; Messina, C; Slaets, L; Chakiba, C; Cameron, D; Bogaerts, J; Bonnefoi, H

2015-02-01

Although achieving a pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) in breast cancer predicts a better outcome, some patients still relapse. The objectives of this study were to describe the types of events in this group of patients and to identify predictive factors for relapse. Patients with large operable or locally advanced breast cancers (T4d tumours were excluded) were randomised to receive either six cycles of anthracycline-based chemotherapy or three cycles of docetaxel followed by three cycles of eprirubicin/docetaxel. pCR was defined as no evidence of residual invasive cancer (or very few scattered tumour cells) in the primary tumour and axillary lymph nodes at surgery. Two Cox regression analyses were performed to identify predictive factors of relapse: one for recurrence-free interval (RFI) and one for distant recurrence-free interval (DRFI). Out of 283 eligible patients who achieved a pCR, 40 (14.1%) and 28 (9.9%) presented an event of interest for the RFI and DRFI analyses, respectively. Five-year RFI, DRFI and overall survival (OS) were 85.3% (95% confidence interval (CI), 80.1-89.3), 89.6% (95% CI, 85.0-92.9) and 91.9% (95% CI, 87.2-94.9), respectively. No predictors for RFI after pCR were identified. For DRFI, tumour size was the only predictor: Hazard ratio (HR) T3 versus T1-2=3.62 (95% CI, 1.66-7.89); HR T4 versus T1-2: HR, 2.80 (95% CI, 0.62-12.64) p=0.0048. In this study, clinical tumour size emerged as the only predictor for DRFI after pCR, with T3 and T4 tumours having an increased risk for distant recurrence compared to T1-2 tumours. Copyright © 2014 Elsevier Ltd. All rights reserved.

Chemosensitivity and Endocrine Sensitivity in Clinical Luminal Breast Cancer Patients in the Prospective Neoadjuvant Breast Registry Symphony Trial (NBRST) Predicted by Molecular Subtyping.

PubMed

Whitworth, Pat; Beitsch, Peter; Mislowsky, Angela; Pellicane, James V; Nash, Charles; Murray, Mary; Lee, Laura A; Dul, Carrie L; Rotkis, Michael; Baron, Paul; Stork-Sloots, Lisette; de Snoo, Femke A; Beatty, Jennifer

2017-03-01

Hormone receptor-positive (HR+) tumors have heterogeneous biology and present a challenge for determining optimal treatment. In the Neoadjuvant Breast Registry Symphony Trial (NBRST) patients were classified according to MammaPrint/BluePrint subtyping to provide insight into the response to neoadjuvant endocrine therapy (NET) or neoadjuvant chemotherapy (NCT). The purpose of this predefined substudy was to compare MammaPrint/BluePrint with conventional 'clinical' immunohistochemistry/fluorescence in situ hybridization (IHC/FISH) subtyping in 'clinical luminal' [HR+/human epidermal growth factor receptor 2-negative (HER2-)] breast cancer patients to predict treatment sensitivity. NBRST IHC/FISH HR+/HER2- breast cancer patients (n = 474) were classified into four molecular subgroups by MammaPrint/BluePrint subtyping: Luminal A, Luminal B, HER2, and Basal type. Pathological complete response (pCR) rates were compared with conventional IHC/FISH subtype. The overall pCR rate for 'clinical luminal' patients to NCT was 11 %; however, 87 of these 474 patients were reclassified as Basal type by BluePrint, with a high pCR rate of 32 %. The MammaPrint index was highly associated with the likelihood of pCR (p < 0.001). Fifty-three patients with BluePrint Luminal tumors received NET with an aromatase inhibitor and 36 (68 %) had a clinical response. With BluePrint subtyping, 18 % of clinical 'luminal' patients are classified in a different subgroup, compared with conventional assessment, and these patients have a significantly higher response rate to NCT compared with BluePrint Luminal patients. MammaPrint/BluePrint subtyping can help allocate effective treatment to appropriate patients. In addition, accurate identification of subtype biology is important in the interpretation of neoadjuvant treatment response since lack of pCR in luminal patients does not portend the worse prognosis associated with residual disease in Basal and HER2 subtypes.

Computational pathology of pre-treatment biopsies identifies lymphocyte density as a predictor of response to neoadjuvant chemotherapy in breast cancer.

PubMed

Ali, H Raza; Dariush, Aliakbar; Provenzano, Elena; Bardwell, Helen; Abraham, Jean E; Iddawela, Mahesh; Vallier, Anne-Laure; Hiller, Louise; Dunn, Janet A; Bowden, Sarah J; Hickish, Tamas; McAdam, Karen; Houston, Stephen; Irwin, Mike J; Pharoah, Paul D P; Brenton, James D; Walton, Nicholas A; Earl, Helena M; Caldas, Carlos

2016-02-16

There is a need to improve prediction of response to chemotherapy in breast cancer in order to improve clinical management and this may be achieved by harnessing computational metrics of tissue pathology. We investigated the association between quantitative image metrics derived from computational analysis of digital pathology slides and response to chemotherapy in women with breast cancer who received neoadjuvant chemotherapy. We digitised tissue sections of both diagnostic and surgical samples of breast tumours from 768 patients enrolled in the Neo-tAnGo randomized controlled trial. We subjected digital images to systematic analysis optimised for detection of single cells. Machine-learning methods were used to classify cells as cancer, stromal or lymphocyte and we computed estimates of absolute numbers, relative fractions and cell densities using these data. Pathological complete response (pCR), a histological indicator of chemotherapy response, was the primary endpoint. Fifteen image metrics were tested for their association with pCR using univariate and multivariate logistic regression. Median lymphocyte density proved most strongly associated with pCR on univariate analysis (OR 4.46, 95 % CI 2.34-8.50, p < 0.0001; observations = 614) and on multivariate analysis (OR 2.42, 95 % CI 1.08-5.40, p = 0.03; observations = 406) after adjustment for clinical factors. Further exploratory analyses revealed that in approximately one quarter of cases there was an increase in lymphocyte density in the tumour removed at surgery compared to diagnostic biopsies. A reduction in lymphocyte density at surgery was strongly associated with pCR (OR 0.28, 95 % CI 0.17-0.47, p < 0.0001; observations = 553). A data-driven analysis of computational pathology reveals lymphocyte density as an independent predictor of pCR. Paradoxically an increase in lymphocyte density, following exposure to chemotherapy, is associated with a lack of pCR. Computational pathology can provide objective, quantitative and reproducible tissue metrics and represents a viable means of outcome prediction in breast cancer. ClinicalTrials.gov NCT00070278 ; 03/10/2003.

Neoadjuvant chemotherapy in breast cancer: prediction of pathologic response with PET/CT and dynamic contrast-enhanced MR imaging--prospective assessment.

PubMed

Tateishi, Ukihide; Miyake, Mototaka; Nagaoka, Tomoaki; Terauchi, Takashi; Kubota, Kazunori; Kinoshita, Takayuki; Daisaki, Hiromitsu; Macapinlac, Homer A

2012-04-01

To clarify whether fluorine 18 ((18)F) fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) and dynamic contrast-enhanced (DCE) magnetic resonance (MR) imaging performed after two cycles of neoadjuvant chemotherapy (NAC) can be used to predict pathologic response in breast cancer. Institutional human research committee approval and written informed consent were obtained. Accuracy after two cycles of NAC for predicting pathologic complete response (pCR) was examined in 142 women (mean age, 57 years: range, 43-72 years) with histologically proved breast cancer between December 2005 and February 2009. Quantitative PET/CT and DCE MR imaging were performed at baseline and after two cycles of NAC. Parameters of PET/CT and of blood flow and microvascular permeability at DCE MR were compared with pathologic response. Patients were also evaluated after NAC by using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 based on DCE MR measurements and European Organization for Research and Treatment of Cancer (EORTC) criteria and PET Response Criteria in Solid Tumors (PERCIST) 1.0 based on PET/CT measurements. Multiple logistic regression analyses were performed to examine continuous variables at PET/CT and DCE MR to predict pCR, and diagnostic accuracies were compared with the McNemar test. Significant decrease from baseline of all parameters at PET/CT and DCE MR was observed after NAC. Therapeutic response was obtained in 24 patients (17%) with pCR and 118 (83%) without pCR. Sensitivity, specificity, and accuracy to predict pCR were 45.5%, 85.5%, and 82.4%, respectively, with RECIST and 70.4%, 95.7%, and 90.8%, respectively, with EORTC and PERCIST. Multiple logistic regression revealed three significant independent predictors of pCR: percentage maximum standardized uptake value (%SUV(max)) (odds ratio [OR], 1.22; 95% confidence interval [CI]: 1.11, 1.34; P < .0001), percentage rate constant (%k(ep)) (OR, 1.07; CI: 1.03, 1.12; P = .002), and percentage area under the time-intensity curve over 90 seconds (%AUC(90)) (OR, 1.04; CI: 1.01, 1.07; P = .048). When diagnostic accuracies are compared, PET/CT is superior to DCE MR for the prediction of pCR (%SUV(max) [90.1%] vs %κ(ep) [83.8%] or %AUC(90) [76.8%]; P < .05). The sensitivities of %SUV(max) (66.7%), %k(ep) (51.7%), and %AUC(90) (50.0%) at (18)F-FDG PET/CT and DCE MR after two cycles of NAC are not acceptable, but the specificities (96.4%, 92.0%, and 95.2%, respectively) are high for stratification of pCR cases in breast cancer. © RSNA, 2012.

Hemoglobin level and XRCC1 polymorphisms to select patients with locally advanced rectal cancer candidate for neoadjuvant chemoradiotherapy with concurrent capecitabine and a platinum salt.

PubMed

Formica, Vincenzo; Benassi, Michaela; Del Vecchio Blanco, Giovanna; Doldo, Elena; Martano, Laura; Portarena, Ilaria; Nardecchia, Antonella; Lucchetti, Jessica; Morelli, Cristina; Giudice, Emilia; Rossi, Piero; Anselmo, Alessandro; Sileri, Pierpaolo; Sica, Giuseppe; Orlandi, Augusto; Santoni, Riccardo; Roselli, Mario

2018-05-02

A platinum salt (oxaliplatin or cisplatin) is widely used to enhance chemoradation (CRT) response. The potential of cisplatin in neoadjuvant CRT for locally advanced rectal cancer (LARC) has not been fully investigated. Consecutive patients with histologically confirmed LARC were treated with standard pelvic radiotherapy and concurrent cisplatin plus capecitabine (CisCape CRT). Surgery and eight cycles of adjuvant FOLFOX4 were offered to all patients after CRT. Common biochemical variables and key germline genetic polymorphisms were analyzed as predictors of pathological complete response (pCR). Fifty-one patients were enrolled. pCR (regression AJCC grade 0) was documented in 7 patients (14%), nearly complete response (AJCC grade 1) in 10 pts. There was a strong association between disease-free survival and AJCC grade (p 0.0047). Grade 3-4 toxicities (mainly diarrhea) was observed in 41% of patients. Among all analyzed variables, baseline hemoglobin (Hb) was significantly associated with AJCC grade 0-1 response (p 0.027). As for the pharmacogenetic analysis, XRCC1 rs25487 polymorphism was significantly associated with AJCC grade 0-1, Odds Ratio 25.8, p 0.049. AJCC grade 0-1 response rate for patients with high Hb and/or XRCC1 rs25487 G/G genotype was as high as 57%. Baseline Hb and XRCC1 polymorphisms are valuable selection criteria for the CisCape CRT regimen, given its otherwise meaningful toxicity.

Addition of zoledronic acid to neoadjuvant chemotherapy does not enhance tumor response in patients with HER2-negative stage II/III breast cancer: the NEOZOTAC trial (BOOG 2010-01).

PubMed

Charehbili, A; van de Ven, S; Smit, V T H B M; Meershoek-Klein Kranenbarg, E; Hamdy, N A T; Putter, H; Heijns, J B; van Warmerdam, L J C; Kessels, L; Dercksen, M; Pepels, M J; Maartense, E; van Laarhoven, H W M; Vriens, B; Wasser, M N; van Leeuwen-Stok, A E; Liefers, G J; van de Velde, C J H; Nortier, J W R; Kroep, J R

2014-05-01

The role of zoledronic acid (ZA) when added to the neoadjuvant treatment of breast cancer (BC) in enhancing the clinical and pathological response of tumors is unclear. The effect of ZA on the antitumor effect of neoadjuvant chemotherapy has not prospectively been studied before. NEOZOTAC is a national, multicenter, randomized study comparing the efficacy of TAC (docetaxel, adriamycin and cyclophosphamide i.v.) followed by granulocyte colony-stimulating factor on day 2 with or without ZA 4 mg i.v. q 3 weeks inpatients withstage II/III, HER2-negative BC. We present data on the pathological complete response (pCR in breast and axilla), on clinical response using MRI, and toxicity. Post hoc subgroup analyses were undertaken to address the predictive value of menopausal status. Addition of ZA to chemotherapy did not improve pCR rates (13.2% for TAC+ZA versus 13.3% for TAC). Postmenopausal women (N = 96) had a numerical benefit from ZA treatment (pCR 14.0% for TAC+ZA versus 8.7% for TAC, P = 0.42). Clinical objective response did not differ between treatment arms (72.9% versus 73.7%). There was no difference in grade III/IV toxicity between treatment arms. Addition of ZA to neoadjuvant chemotherapy did not improve pathological or clinical response to chemotherapy. Further investigations are warranted in postmenopausal women with BC, since this subgroup might benefit from ZA treatment.

Association of PTP1B with Outcomes of Breast Cancer Patients Who Underwent Neoadjuvant Chemotherapy.

PubMed

Rivera Franco, Monica M; Leon Rodriguez, Eucario; Martinez Benitez, Braulio; Villanueva Rodriguez, Luisa G; de la Luz Sevilla Gonzalez, Maria; Armengol Alonso, Alejandra

2016-01-01

PTP1B is involved in the oncogenesis of breast cancer. In addition, neoadjuvant therapy has been widely used in breast cancer; thus, a measurement to assess survival improvement could be pathological complete response (pCR). Our objective was to associate PTP1B overexpression with outcomes of breast cancer patients who underwent neoadjuvant chemotherapy. Forty-six specimens were included. Diagnostic biopsies were immunostained using anti-PTP1B antibody. Expression was categorized as negative (<5%) and overexpression (≥5%). Patients' responses were graded according to the Miller-Payne system. Sixty-three percent of patients overexpressed PTP1B. There was no significant association between PTP1B overexpression and pCR ( P = 0.2). However, when associated with intrinsic subtypes, overexpression was higher in human epidermal growth factor receptor 2-positive-enriched specimens ( P = 0.02). Ten-year progression-free survival showed no differences. Our preliminary results do not show an association between PTP1B over-expression and pCR; however, given the limited sample and heterogeneous treatment in our cohort, this hypothesis cannot be excluded.

Prospective validation of pathologic complete response models in rectal cancer: Transferability and reproducibility.

PubMed

van Soest, Johan; Meldolesi, Elisa; van Stiphout, Ruud; Gatta, Roberto; Damiani, Andrea; Valentini, Vincenzo; Lambin, Philippe; Dekker, Andre

2017-09-01

Multiple models have been developed to predict pathologic complete response (pCR) in locally advanced rectal cancer patients. Unfortunately, validation of these models normally omit the implications of cohort differences on prediction model performance. In this work, we will perform a prospective validation of three pCR models, including information whether this validation will target transferability or reproducibility (cohort differences) of the given models. We applied a novel methodology, the cohort differences model, to predict whether a patient belongs to the training or to the validation cohort. If the cohort differences model performs well, it would suggest a large difference in cohort characteristics meaning we would validate the transferability of the model rather than reproducibility. We tested our method in a prospective validation of three existing models for pCR prediction in 154 patients. Our results showed a large difference between training and validation cohort for one of the three tested models [Area under the Receiver Operating Curve (AUC) cohort differences model: 0.85], signaling the validation leans towards transferability. Two out of three models had a lower AUC for validation (0.66 and 0.58), one model showed a higher AUC in the validation cohort (0.70). We have successfully applied a new methodology in the validation of three prediction models, which allows us to indicate if a validation targeted transferability (large differences between training/validation cohort) or reproducibility (small cohort differences). © 2017 American Association of Physicists in Medicine.

Association between CHEK2 H371Y mutation and response to neoadjuvant chemotherapy in women with breast cancer.

PubMed

Liu, Yin; Xu, Ye; Ouyang, Tao; Li, Jinfeng; Wang, Tianfeng; Fan, Zhaoqing; Fan, Tie; Lin, Benyao; Xie, Yuntao

2015-03-28

Our previous study suggested that the recurrent CHEK2 H371Y mutation is a novel pathogenic mutation that confers an increased risk of breast cancer. The purpose of this study was to investigate whether breast cancer patients with CHEK2 H371Y mutation were more likely to respond to neoadjuvant chemotherapy. We screened a cohort of 2334 Chinese women with operable primary breast cancer who received a neoadjuvant chemotherapy regimen for CHEK2 H371Y germline mutations. Pathologic complete response (pCR) was defined as the absence of tumor cells in the breast after the completion of neoadjuvant chemotherapy. Thirty-nine patients (1.7%) with CHEK2 H371Y germline mutation were identified in this cohort of 2334 patients. CHEK2 H371Y mutation carriers had a significantly higher pCR rate than non-carriers (33.3% versus 19.5%, P = 0.031) in the entire study population, and CHEK2 H371Y mutation-positive status remained an independent favorable predictor of pCR in a multivariate analysis (odds ratio [OR] = 3.01; 95% confidence interval [CI]: 1.34- 6.78, P = 0.008). CHEK2 H371Y carriers had a slightly worse distant recurrence-free survival than non-carriers (adjusted hazard ratio [HR] =1.24, 95% CI: 0.59-2.63). CHEK2 H371Y mutation carriers are more likely to respond to neoadjuvant chemotherapy than are non-carriers.

[Contribution of Leishmania identification using polymerase chain reaction--restriction fragment length polymerase for epidemiological studies of cutaneous leishmaniasis in Tunisia].

PubMed

Bousslimi, N; Ben Abda, I; Ben Mously, R; Siala, E; Harrat, Z; Zallagua, N; Bouratbine, A; Aoun, K

2014-02-01

Three forms of cutaneous leishmaniasis (CL) are endemic in Tunisia. The identification of the causative species is useful to complete epidemiological data and to manage the cases. The aim of this study is to assess PCR-RFLP technique in the identification of Leishmania species responsible of CL in Tunisia and to compare the results of this technique to those of isoenzyme analysis. Sixty-one CL lesions were sampled. Dermal samples were tested by culture on NNN medium and analyzed by PCR-RFLP assay targeting the ITS1 region of ribosomal DNA. Species identification was performed by both iso-enzymatic typing for positive cultures and analysis of restriction profiles after enzymatic digestion by HaeIII of the obtained amplicons. Thirty-eight (62%) samples were positive by culture. The iso-enzymatic typing of 32 isolates identified 3 L. infantum, 23 L. major MON-25 and 6 L. tropica MON-8. Sixty samples were positive by PCR. The PCR-RFLP digestion profiles of the 56 PCR products identified 12 L. infantum, 38 L. major and 6 L. tropica. The results of both techniques were concordant in the 32 strains identified by both techniques. Species identification correlated with the geographical distribution of CL forms endemic in Tunisia. Results of PCR-RFLP revealed highly concordant with those of isoenzyme electrophoresis. Thanks to its simplicity, rapidity and ability to be performed directly on biological samples, this technique appears as an interesting alternative for the identification of Leishmania strains responsible of CL in Tunisia. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Pre-operative assessment of residual disease in locally advanced breast cancer patients: A sequential study by quantitative diffusion weighted MRI as a function of therapy.

PubMed

Agarwal, Khushbu; Sharma, Uma; Sah, Rani G; Mathur, Sandeep; Hari, Smriti; Seenu, Vurthaluru; Parshad, Rajinder; Jagannathan, Naranamangalam R

2017-10-01

The potential of diffusion weighted imaging (DWI) in assessing pathologic response and surgical margins in locally advanced breast cancer patients (n=38) undergoing neoadjuvant chemotherapy was investigated. DWI was performed at pre-therapy (Tp0), after I (Tp1) and III (Tp3) NACT at 1.5T. Apparent diffusion coefficient (ADC) of whole tumor (ADC WT ), solid tumor (ADC ST ), intra-tumoral necrosis (ADC Nec ) was determined. Further, ADC of 6 consecutive shells (5mm thickness each) including tumor margin to outside tumor margins (OM1 to OM5) was calculated and the data analyzed to define surgical margins. Of 38 patients, 6 were pathological complete responders (pCR), 19 partial responders (pPR) and 13 were non-responders (pNR). Significant increase was observed in ADC ST and ADC WT in pCR and pPR following therapy. Pre-therapy ADC was significantly lower in pCR compared to pPR and pNR indicating the heterogeneous nature of tumor which may affect drug perfusion and consequently the response. ADC of outside margins (OM1, OM2, and OM3) was significantly different among pCR, pPR and pNR at Tp3 which may serve as response predictive parameter. Further, at Tp3, ADC of outside margins (OM1, OM2, and OM3) was significantly lower compared to that seen at Tp0 in pCR, indicating the presence of residual disease in these shells. Pre-surgery information may serve as a guide to define cancer free margins and the extent of residual disease which may be useful in planning breast conservation surgery. Copyright © 2017 Elsevier Inc. All rights reserved.

Prognostic Effect of Carcinoma In Situ in Muscle-invasive Urothelial Carcinoma Patients Receiving Neoadjuvant Chemotherapy

PubMed Central

Thomas, Derek E.; Kaimakliotis, Hristos Z.; Rice, Kevin R.; Pereira, Jose A.; Johnston, Paul; Moore, Marietta L.; Reed, Angela; Cregar, Dylan M.; Franklin, Cindy; Loman, Rhoda L.; Koch, Michael O.; Bihrle, Richard; Foster, Richard S.; Masterson, Timothy A.; Gardner, Thomas A.; Sundaram, Chandru P.; Powell, Charles R.; Beck, Stephen D.W.; Grignon, David J.; Cheng, Liang; Albany, Costantine; Hahn, Noah M.

2017-01-01

We performed a single-institution retrospective analysis of 137 patients with muscle-invasive urothelial carcinoma who underwent neoadjuvant chemotherapy and radical cystectomy to assess the prognostic significance of carcinoma in situ (CIS). The pathologic complete response rates were significantly decreased for patients with CIS identified on transurethral resection of the bladder tumor before treatment. The long-term follow-up data from patients with isolated CIS at cystectomy revealed prolonged progression-free and overall survival. Background Carcinoma in situ (CIS) is a poor prognostic finding in urothelial carcinoma. However, its significance in muscle-invasive urothelial carcinoma (MIUC) treated with neoadjuvant chemotherapy (NAC) is uncertain. We assessed the effect of CIS found in pretreatment transurethral resection of bladder tumor (TURBT) biopsies on the pathologic and clinical outcomes. Materials and Methods Subjects with MIUC treated with NAC before cystectomy were identified. The pathologic complete response (pCR) rates stratified by TURBT CIS status were compared. The secondary analyses included tumor response, progression-free survival (PFS), overall survival (OS), and an exploratory post hoc analysis of patients with pathologic CIS only (pTisN0) at cystectomy. Results A total of 137 patients with MIUC were identified. TURBT CIS was noted in 30.7% of the patients. The absence of TURBT CIS was associated with a significantly increased pCR rate (23.2% vs. 9.5%; odds ratio, 4.08; 95% confidence interval, 1.19–13.98; P = .025). Stage pTisN0 disease was observed in 19.0% of the TURBT CIS patients. TURBT CIS status did not significantly affect the PFS or OS outcomes. Post hoc analysis of the pTisN0 patients revealed prolonged median PFS (104.5 vs. 139.9 months; P = .055) and OS (104.5 vs. 152.3 months; P = .091) outcomes similar to those for the pCR patients. Conclusion The absence of CIS on pretreatment TURBT in patients with MIUC undergoing NAC was associated with increased pCR rates, with no observed differences in PFS or OS. Isolated CIS at cystectomy was frequently observed, with lengthy PFS and OS durations similar to those for pCR patients. Further studies aimed at understanding the biology and clinical effect of CIS in MIUC are warranted. PMID:28040424

Standardizing of Pathology in Patients Receiving Neoadjuvant Chemotherapy.

PubMed

Bossuyt, Veerle; Symmans, W Fraser

2016-10-01

The use of neoadjuvant systemic therapy for the treatment of breast cancer patients is increasing. Pathologic response in the form of pathologic complete response (pCR) and grading systems of partial response, such as the residual cancer burden (RCB) system, gives valuable prognostic information for patients and is used as a primary endpoint in clinical trials. The breast cancer and pathology communities are responding with efforts to standardize pathology in patients receiving neoadjuvant chemotherapy. In this review, we summarize the challenges that postneoadjuvant systemic therapy surgical specimens pose and how pathologists and the multidisciplinary team can work together to optimize handling of these specimens. Multidisciplinary communication is essential. A single, standardized approach to macroscopic and microscopic pathologic examination makes it possible to provide reliable response information. This approach employs a map of tissue sections to correlate clinical, gross, microscopic, and imaging findings in order to report the presence of pCR (ypT0 ypN0 and ypT0/is ypN0) versus residual disease, the ypT and ypN stage using the current AJCC/UICC staging system, and the RCB.

Gene expression patterns in formalin-fixed, paraffin-embedded core biopsies predict docetaxel chemosensitivity in breast cancer patients.

PubMed

Chang, Jenny C; Makris, Andreas; Gutierrez, M Carolina; Hilsenbeck, Susan G; Hackett, James R; Jeong, Jennie; Liu, Mei-Lan; Baker, Joffre; Clark-Langone, Kim; Baehner, Frederick L; Sexton, Krsytal; Mohsin, Syed; Gray, Tara; Alvarez, Laura; Chamness, Gary C; Osborne, C Kent; Shak, Steven

2008-03-01

Previously, we had identified gene expression patterns that predicted response to neoadjuvant docetaxel. Other studies have validated that a high Recurrence Score (RS) by the 21-gene RT-PCR assay is predictive of worse prognosis but better response to chemotherapy. We investigated whether tumor expression of these 21 genes and other candidate genes can predict response to docetaxel. Core biopsies from 97 patients were obtained before treatment with neoadjuvant docetaxel (4 cycles, 100 mg/m2 q3 weeks). Three 10-microm FFPE sections were submitted for quantitative RT-PCR assays of 192 genes that were selected from our previous work and the literature. Of the 97 patients, 81 (84%) had sufficient invasive cancer, 80 (82%) had sufficient RNA for QRTPCR assay, and 72 (74%) had clinical response data. Mean age was 48.5 years, and the median tumor size was 6 cm. Clinical complete responses (CR) were observed in 12 (17%), partial responses in 41 (57%), stable disease in 17 (24%), and progressive disease in 2 patients (3%). A significant relationship (P<0.05) between gene expression and CR was observed for 14 genes, including CYBA. CR was associated with lower expression of the ER gene group and higher expression of the proliferation gene group from the 21 gene assay. Of note, CR was more likely with a high RS (P=0.008). We have established molecular profiles of sensitivity to docetaxel. RT-PCR technology provides a potential platform for a predictive test of docetaxel chemosensitivity using small amounts of routinely processed material.

Value of Diffusion-Weighted Magnetic Resonance Imaging for Prediction and Early Assessment of Response to Neoadjuvant Radiochemotherapy in Rectal Cancer: Preliminary Results

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lambrecht, Maarten, E-mail: maarten.lambrecht@uzleuven.be; Vandecaveye, Vincent; De Keyzer, Frederik

2012-02-01

Purpose: To evaluate diffusion-weighted magnetic resonance imaging (DWI) for response prediction before and response assessment during and early after preoperative radiochemotherapy (RCT) for locally advanced rectal cancer (LARC). Methods and Materials: Twenty patients receiving RCT for LARC underwent MRI including DWI before RCT, after 10-15 fractions and 1 to 2 weeks before surgery. Tumor volume and apparent diffusion coefficient (ADC; b-values: 0-1000 s/mm{sup 2}) were determined at all time points. Pretreatment tumor ADC and volume, tumor ADC change ( Increment ADC), and volume change ( Increment V) between pretreatment and follow-up examinations were compared with histopathologic findings after total mesorectalmore » excision (pathologic complete response [pCR] vs. no pCR, ypT0-2 vs. ypT3-4, T-downstaging or not). The discriminatory capability of pretreatment tumor ADC and volume, Increment ADC, and Increment V for the detection of pCR was compared with receiver operating characteristics analysis. Results: Pretreatment ADC was significantly lower in patients with pCR compared with patients without (in mm{sup 2}/s: 0.94 {+-} 0.12 Multiplication-Sign 10{sup -3} vs. 1.19 {+-} 0.22 Multiplication-Sign 10{sup -3}, p = 0.003), yielding a sensitivity of 100% and specificity of 86% for detection of pCR. The volume reduction during and after RCT was significantly higher in patients with pCR compared with patients without (in %: {Delta}V{sub during}: -62 {+-} 16 vs. -33 {+-} 16, respectively, p = 0.015; and {Delta}V{sub post}: -86 {+-} 12 vs. -60 {+-} 21, p = 0.012), yielding a sensitivity of 83% and specificity of 71% for the {Delta}V{sub during} and, respectively, 83% and 86% for the {Delta}V{sub post}. The Increment ADC during ({Delta}ADC{sub during}) and after RCT ({Delta}ADC{sub post}) showed a significantly higher value in patients with pCR compared with patients without (in %: {Delta}ADC{sub during}: 72 {+-} 14 vs. 16 {+-} 12, p = 0.0006; and {Delta}ADC{sub post}: 88 {+-} 35 vs. 26 {+-} 19, p = 0.0011), yielding a sensitivity and specificity of 100% for the {Delta}ADC{sub during} and, respectively, 100% and 93% for the {Delta}ADC{sub post}. Conclusions: These initial findings indicate that DWI, using pretreatment ADC, {Delta}ADC{sub during}, and {Delta}ADC{sub post} may be useful for prediction and early assessment of pathologic response to preoperative RCT of LARC, with higher accuracy than volumetric measurements.« less

Acute Toxicity and Tumor Response in Locally Advanced Rectal Cancer After Preoperative Chemoradiation Therapy With Shortening of the Overall Treatment Time Using Intensity-Modulated Radiation Therapy With Simultaneous Integrated Boost: A Phase 2 Trial

DOE Office of Scientific and Technical Information (OSTI.GOV)

But-Hadzic, Jasna, E-mail: jbut@onko-i.si; Anderluh, Franc; Brecelj, Erik

Background and Purpose: This phase 2 study investigated the efficacy and safety of preoperative intensity modulated radiation therapy with a simultaneous integrated boost (IMRT-SIB) without dose escalation, concomitant with standard capecitabine chemotherapy in locally advanced rectal cancer. Methods and Materials: Between January 2014 and March 2015, 51 patients with operable stage II-III rectal adenocarcinoma received preoperative IMRT with pelvic dose of 41.8 Gy and simultaneously delivered 46.2 Gy to T2/3 and 48.4 Gy to T4 tumor in 22 fractions, concomitant with capecitabine, 825 mg/m{sup 2}/12 hours, including weekends. The primary endpoint was pathologic complete response (pCR). Results: Fifty patients completed preoperative treatment according to themore » protocol, and 47 underwent surgical resection. The sphincter preservation rate for the low rectal tumors was 62%, and the resection margins were free in all but 1 patient. Decrease in tumor and nodal stage was observed in 32 (68%) and 39 (83%) patients, respectively, with pCR achieved in 12 (25.5%) patients. There were only 2 G ≥ 3 acute toxicities, with infectious enterocolitis in 1 patient and dermatitis over the sacral area caused by the bolus effect of the treatment table in the second patient. Conclusions: Preoperative IMRT-SIB without dose escalation is well tolerated, with a low acute toxicity profile, and can achieve a high rate of pCR and downstaging.« less

Acute Toxicity and Tumor Response in Locally Advanced Rectal Cancer After Preoperative Chemoradiation Therapy With Shortening of the Overall Treatment Time Using Intensity-Modulated Radiation Therapy With Simultaneous Integrated Boost: A Phase 2 Trial.

PubMed

But-Hadzic, Jasna; Anderluh, Franc; Brecelj, Erik; Edhemovic, Ibrahim; Secerov-Ermenc, Ajra; Hudej, Rihard; Jeromen, Ana; Kozelj, Miran; Krebs, Bojan; Oblak, Irena; Omejc, Mirko; Vogrin, Andrej; Velenik, Vaneja

2016-12-01

This phase 2 study investigated the efficacy and safety of preoperative intensity modulated radiation therapy with a simultaneous integrated boost (IMRT-SIB) without dose escalation, concomitant with standard capecitabine chemotherapy in locally advanced rectal cancer. Between January 2014 and March 2015, 51 patients with operable stage II-III rectal adenocarcinoma received preoperative IMRT with pelvic dose of 41.8 Gy and simultaneously delivered 46.2 Gy to T2/3 and 48.4 Gy to T4 tumor in 22 fractions, concomitant with capecitabine, 825 mg/m 2 /12 hours, including weekends. The primary endpoint was pathologic complete response (pCR). Fifty patients completed preoperative treatment according to the protocol, and 47 underwent surgical resection. The sphincter preservation rate for the low rectal tumors was 62%, and the resection margins were free in all but 1 patient. Decrease in tumor and nodal stage was observed in 32 (68%) and 39 (83%) patients, respectively, with pCR achieved in 12 (25.5%) patients. There were only 2 G ≥ 3 acute toxicities, with infectious enterocolitis in 1 patient and dermatitis over the sacral area caused by the bolus effect of the treatment table in the second patient. Preoperative IMRT-SIB without dose escalation is well tolerated, with a low acute toxicity profile, and can achieve a high rate of pCR and downstaging. Copyright © 2016 Elsevier Inc. All rights reserved.

Nottingham Clinico-Pathological Response Index (NPRI) after neoadjuvant chemotherapy (Neo-ACT) accurately predicts clinical outcome in locally advanced breast cancer.

PubMed

Abdel-Fatah, Tarek M; Ball, Graham; Lee, Andrew H S; Pinder, Sarah; MacMilan, R Douglas; Cornford, Eleanor; Moseley, Paul M; Silverman, Rafael; Price, James; Latham, Bruce; Palmer, David; Chan, Arlene; Ellis, Ian O; Chan, Stephen Y T

2015-03-01

There is a need to identify more sensitive clinicopathologic criteria to assess the response to neoadjuvant chemotherapy (Neo-ACT) and guide subsequent adjuvant therapy. We performed a clinicopathologic assessment of 426 patients who had completed Neo-ACT for locally advanced breast cancer (LABC) with a median follow-up of 70 months. Patients were divided into a training set treated with anthracycline combination chemotherapy (n = 172); an internal validation set treated with anthracycline and taxane (n = 129); and an external validation set treated with anthracycline with or without taxane (n = 125). A multivariate Cox regression model demonstrated the absence of fibrosis, presence of lymphovascular invasion, increasing number of lymph node metastases, and administration of hormone therapy were significantly associated with short breast cancer-specific survival (BCSS) and disease-free survival (DFS); Ps < 0.01, while reduction of tumor size was associated with DFS (P = 0.022). Nottingham Clinico-Pathological Response Indexes (NPRI) were calculated, and four prognostic groups (NPRI-PG) were identified. Patients in prognostic group 2 (NPRI-PG2) for BCSS (66 of 172; 38.4%) have the same prognosis as those who achieved pathologic complete response (pCR; NPRI-PG1; 15%). Receiver-operating characteristic (ROC) curves indicated that the NPRI outperformed the currently used prognostic factors and adding the NPRI improved their performance as a predictor for both BCSS (area under the curve [AUC], 0.88) and DFS (AUC, 0.87). The NPRI predicts BCSS and DFS, with a higher sensitivity than pCR. The NPRI can also improve the sensitivity and specificity of clinicopathologic response as a study endpoint, for assessing response to Neo-ACT, and can serve as a valuable tool for the discovery of future predictive molecular markers. ©2014 American Association for Cancer Research.

A deep learning classifier for prediction of pathological complete response to neoadjuvant chemotherapy from baseline breast DCE-MRI

NASA Astrophysics Data System (ADS)

Ravichandran, Kavya; Braman, Nathaniel; Janowczyk, Andrew; Madabhushi, Anant

2018-02-01

Neoadjuvant chemotherapy (NAC) is routinely used to treat breast tumors before surgery to reduce tumor size and improve outcome. However, no current clinical or imaging metrics can effectively predict before treatment which NAC recipients will achieve pathological complete response (pCR), the absence of residual invasive disease in the breast or lymph nodes following surgical resection. In this work, we developed and applied a convolu- tional neural network (CNN) to predict pCR from pre-treatment dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) scans on a per-voxel basis. In this study, DCE-MRI data for a total of 166 breast cancer pa- tients from the ISPY1 Clinical Trial were split into a training set of 133 patients and a testing set of 33 patients. A CNN consisting of 6 convolutional blocks was trained over 30 epochs. The pre-contrast and post-contrast DCE-MRI phases were considered in isolation and conjunction. A CNN utilizing a combination of both pre- and post-contrast images best distinguished responders, with an AUC of 0.77; 82% of the patients in the testing set were correctly classified based on their treatment response. Within the testing set, the CNN was able to produce probability heatmaps that visualized tumor regions that most strongly predicted therapeutic response. Multi- variate analysis with prognostic clinical variables (age, largest diameter, hormone receptor and HER2 status), revealed that the network was an independent predictor of response (p=0.05), and that the inclusion of HER2 status could further improve capability to predict response (AUC = 0.85, accuracy = 85%).

Decreased background parenchymal enhancement of the contralateral breast after two cycles of neoadjuvant chemotherapy is associated with tumor response in HER2-positive breast cancer.

PubMed

You, Chao; Gu, Yajia; Peng, Wen; Li, Jianwei; Shen, Xuxia; Liu, Guangyu; Peng, Weijun

2018-07-01

Background Several recent studies have focused on the association between background parenchymal enhancement (BPE) and tumor response to neoadjuvant chemotherapy (NAC), but early prediction of tumor response based on BPE has yet not been investigated. Purpose To retrospectively investigate whether changes in the BPE of the contralateral breast following NAC could help predict tumor response in early stage HER2-positive breast cancer. Material and Methods Data from 71 patients who were diagnosed with unilateral HER2 positive breast cancer and then underwent NAC with trastuzumab before surgery were analyzed retrospectively. Two experienced radiologists independently categorized the patients' levels of BPE of the contralateral breast into four categories (1 = minimal, 2 = mild, 3 = moderate, 4 = marked) at baseline and after the second cycle of NAC. After undergoing surgery, 34 patients achieved pathologic complete response (pCR) and 37 patients had residual disease (non-pCR). The association between BPE and histopathologic tumor response was analyzed. Result The level of BPE was higher in premenopausal than post-menopausal women both at baseline and after the second cycle of NAC ( P < 0.005). A significant reduction in BPE ( P < 0.001) was observed after the second NAC cycle; however, a more obvious decrease in BPE was identified in premenopausal relative to post-menopausal women ( P = 0.041). No significant association was identified between pCR and baseline BPE ( P = 0.287). However, after the second NAC cycle, decreased BPE was significantly associated with pCR ( P = 0.003). Conclusion For HER2-positive patients, changes in BPE may serve as an additional imaging biomarker of treatment response at an early stage.

Histologic, Serologic, and Molecular Analysis of Persistent Ehrlichiosis in a Murine Model

PubMed Central

Olano, Juan P.; Wen, Gary; Feng, Hui-Min; McBride, Jere W.; Walker, David H.

2004-01-01

Human monocytotropic ehrlichiosis caused by Ehrlichia chaffeensis was reported in 1987. An animal model to study acute fatal ehrlichiosis in mice that has been developed closely resembles the fatal form of human monocytotropic ehrlichiosis. However, animal models for persistent infection in the genus Ehrlichia in immunocompetent mice have not been characterized. We report the histopathological progression of Ehrlichia muris infection in immunocompetent mice (AKR and C57BL/6 strains) correlated with their antibody response determined by indirect immunofluorescence and Western immunoblotting, and the distribution and quantity of the ehrlichial load by immunohistochemistry, polymerase chain reaction (PCR), and real-time PCR in lungs, liver, and spleen. Mild to moderate correlation was observed between histopathological grading in these organs and relative ehrlichial loads. The highest ehrlichial loads were present between days 4 and 14 after infection. E. muris was detected in tissues examined up to 150 days after infection by real-time PCR. Analysis of the serological response revealed several immunodominant antigens, including 200-, 180-, 100-, 73/75-, 45-, and 28-kd proteins. In conclusion, we have provided for the first time a complete histopathological, serological, immunohistochemical, and quantitative analysis of an animal model for the study of persistent ehrlichial infection. PMID:15331423

Effectiveness of evaluating tumor vascularization using 3D power Doppler ultrasound with high-definition flow technology in the prediction of the response to neoadjuvant chemotherapy for T2 breast cancer: a preliminary report

NASA Astrophysics Data System (ADS)

Shia, Wei-Chung; Chen, Dar-Ren; Huang, Yu-Len; Wu, Hwa-Koon; Kuo, Shou-Jen

2015-10-01

The aim of this study was to evaluate the effectiveness of advanced ultrasound (US) imaging of vascular flow and morphological features in the prediction of a pathologic complete response (pCR) and a partial response (PR) to neoadjuvant chemotherapy for T2 breast cancer. Twenty-nine consecutive patients with T2 breast cancer treated with six courses of anthracycline-based neoadjuvant chemotherapy were enrolled. Three-dimensional (3D) power Doppler US with high-definition flow (HDF) technology was used to investigate the blood flow in and morphological features of the tumors. Six vascularity quantization features, three morphological features, and two vascular direction features were selected and extracted from the US images. A support vector machine was used to evaluate the changes in vascularity after neoadjuvant chemotherapy, and pCR and PR were predicted on the basis of these changes. The most accurate prediction of pCR was achieved after the first chemotherapy cycle, with an accuracy of 93.1% and a specificity of 85.5%, while that of a PR was achieved after the second cycle, with an accuracy of 79.31% and a specificity of 72.22%. Vascularity data can be useful to predict the effects of neoadjuvant chemotherapy. Determination of changes in vascularity after neoadjuvant chemotherapy using 3D power Doppler US with HDF can generate accurate predictions of the patient response, facilitating early decision-making.

The effect of obesity on pathological complete response and survival in breast cancer patients receiving uncapped doses of neoadjuvant anthracycline-taxane-based chemotherapy.

PubMed

Farr, Alex; Stolz, Myriam; Baumann, Lukas; Bago-Horvath, Zsuzsanna; Oppolzer, Elisabeth; Pfeiler, Georg; Seifert, Michael; Singer, Christian F

2017-06-01

The effect of obesity in breast cancer patients undergoing neoadjuvant chemotherapy (NAC) remains controversial. The aim of this study was to determine the obesity-related effect on pathological complete response (pCR) and survival in women receiving full uncapped doses of NAC. We retrospectively analyzed the data of all consecutive women who underwent anthracycline-taxane-based NAC for primary breast cancer between 2005 and 2015 at the Department of Obstetrics and Gynecology, Medical University of Vienna. Following the WHO criteria, women with a body mass index (BMI) ≥30 kg/m 2 at baseline were considered obese, whereas those with a BMI <30 kg/m 2 were considered non-obese. Those with dose reductions or dose capping were not eligible for study inclusion. Cox regression and logistic regression were performed. The Kaplan-Meier method was used to analyze disease-free, progression-free, and overall survival. The pCR served as the main outcome measure. Among 120 women who received neoadjuvant epirubicin plus cyclophosphamide and docetaxel, 28 (23.3%) were obese and 92 (76.7%) were non-obese. In the multivariate logistic regression model that adjusted for potentially confounding variables, obesity had an independent positive predictive effect on pCR (OR 4.29, 95% CI, 1.42-13.91; p = 0.011), which was significant in the postmenopausal subgroup (OR 4.72, 95% CI, 1.47-15.84; p = 0.01). When comparing non-obese with obese women, we found that obese women experienced longer progression-free survival (HR 0.10, 95% CI, 8.448 × 10 -4 -0.81; p = 0.025). Obese women receiving full uncapped doses of anthracycline-taxane-based NAC have increased pCR and favorable progression-free survival. This could result from increased dose intensity with increased efficacy and toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Diagnosis and Monitoring of Chronic Myeloid Leukemia: Chiang Mai University Experience.

PubMed

Tantiworawit, Adisak; Kongjarern, Supanat; Rattarittamrong, Ekarat; Lekawanvijit, Suree; Bumroongkit, Kanokkan; Boonma, Nonglak; Rattanathammethee, Thanawat; Hantrakool, Sasinee; Chai-Adisaksopha, Chatree; Norasetthada, Lalita

2016-01-01

A diagnosis of chronic myeloid leukemia (CML) is made on discovery of the presence of a Philadelphia (Ph) chromosome. The success of the treatment of this form of leukemia with tyrosine kinase inhibitor (TKI) is monitored by reduction of the Ph chromosome. To compare the role of conventional cytogenetic (CC) methods with a real time quantitative polymerase chain reaction (RQ-PCR) and fluorescence in situ hybridization (FISH) for diagnosis and treatment monitoring of CML patients. The secondary outcome was to analyze the treatment responses to TKI in CML patients. This was a retrospective study of CML patients who attended the Hematology clinic at Chiang Mai University Hospital from 2005-2010. Medical records were reviewed for demographic data, risk score, treatment response and the results of CC methods, FISH and RQ-PCR. One hundred and twenty three cases were included in the study, 57.7% of whom were male with a mean age of 46.9 years. Most of the patients registered as intermediate to high risk on the Sokal score. At diagnosis, 121 patients were tested using the CC method and 118 (95.9%) were identified as positive. Five patients failed to be diagnosed by CC methods but were positive for BCR-ABL1 using the FISH method. Imatinib was the first-line treatment used in 120 patients (97.6%). In most patients (108 out of 122, 88.5%), a complete cytogenetic response (CCyR) was achieved after TKI therapy and in 86 patients (70.5%) CCyR was achieved long term by the CC method. Five out of the 35 analyzed patients in which CCyR was achieved by the CC method had a positive FISH result. Out of the 76 patients in which CCyR was achieved, RQ-PCR classified patients to only CCyR in 17 patients (22.4%) with a deeper major molecular response (MMR) in 4 patients (5.3%) and complete molecular response (CMR) in 55 patients (72.4%). In the case of initial therapy, CCyR was achieved in 95 patients (79.1%) who received imatinib and in both patients who received dasatinib (100%). For the second line treatment, nilotinib were used in 30 patients and in 19 of them (63.3%) CCyR was achieved. In half of the 6 patients (50%) who received dasatinib as second line or third line treatment CCyR was also achieved. CML patients had a good response to TKI treatment. FISH could be useful for diagnosis in cases where CC analysis failed to detect the Ph chromosome. RQ-PCR was helpful in detecting any residual disease and determining the depth of the treatment response at levels greater than the CC methods.

Prospective study of neoadjuvant chemoradiotherapy using intensity-modulated radiotherapy and 5 fluorouracil for locally advanced rectal cancer - toxicities and response assessment.

PubMed

Simson, David K; Mitra, Swarupa; Ahlawat, Parveen; Saxena, Upasna; Sharma, Manoj Kumar; Rawat, Sheh; Singh, Harpreet; Bansal, Babita; Sripathi, Lalitha Kameshwari; Tanwar, Aditi

2018-01-01

The past 2 decades witnessed the strengthening of evidence favoring the role of neoadjuvant chemoradiation (CHRT) in the treatment of locally advanced rectal cancer. The study aims to evaluate the response and acute toxicities to neoadjuvant CHRT using intensity-modulated radiotherapy (IMRT) in the treatment of rectal cancer. Predictive factors to achieve pathological complete response (pCR) were analyzed, as a secondary endpoint. All consecutive patients who underwent IMRT as part of neoadjuvant CHRT in the treatment of rectal cancer between August 2014 and December 2016 at a tertiary cancer care center were accrued for the study. The cohort underwent CHRT with IMRT technique at a dose of 50.4 Gy in 28 fractions concurrent with continuous infusion of 5 fluorouracil during the first and the last 4 days of CHRT. Surgery was performed 6 weeks later and the pathological response to CHRT was noted. Forty-three subjects were accrued for the study. Radiation dermatitis and diarrhea were the only observed grade ≥3 acute toxicities. Sphincter preservation rate (SPR) was 43.3%. pCR was observed in 32.6%. Univariate and multivariate logistic regression showed that carcinoembryonic antigen was the only independent predictive factor to achieve pCR. IMRT as part of neoadjuvant CHRT in the treatment of locally advanced rectal cancer is well tolerated and gives comparable results with respect to earlier studies in terms of pathological response and SPR. Further randomized controlled studies are needed to firmly state that IMRT is superior to 3-dimensional conformal radiotherapy.

Pertuzumab/Trastuzumab/CT Versus Trastuzumab/CT Therapy for HER2+ Breast Cancer: Results from the Prospective Neoadjuvant Breast Registry Symphony Trial (NBRST).

PubMed

Beitsch, Peter; Whitworth, Pat; Baron, Paul; Rotkis, Michael C; Mislowsky, Angela M; Richards, Paul D; Murray, Mary K; Pellicane, James V; Dul, Carrie L; Nash, Charles H; Stork-Sloots, Lisette; de Snoo, Femke; Untch, Sarah; Lee, Laura A

2017-09-01

Pertuzumab became a standard part of neoadjuvant therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancers approximately halfway through Neoadjuvant Breast Registry Symphony Trial (NBRST) enrollment, providing a unique opportunity to determine biologically which clinical HER2+ patients benefit most from dual targeting. As a neoadjuvant phase 4 study, NBRST classifies patients by both conventional and molecular subtyping. Of 308 clinical HER2+ patients enrolled in NBRST between 2011 and 2014 from 62 U.S. institutions, 297 received neoadjuvant chemotherapy (NCT) with HER2-targeted therapy and underwent surgery. This study compared the pathologic complete response (pCR) rate of BluePrint versus clinical subtypes with treatment, specifically differences between trastuzumab (T) treatment and trastuzumab and pertuzumab (T/P) treatment. In this study, 60% of the patients received NCT-T, and 40% received NCT-T/P. The overall pCR rate (ypT0/isN0) was 47%. BluePrint classified 161 tumors (54%) as HER2 type, with a pCR rate of 65%. This was significantly higher than the pCR rate for the 91 HER2+ tumors (31%) classified as luminal (18%) (p = 0.00001) and the 45 tumors (15%) classified as basal (44%) (p = 0.0166). The patients treated with T/P had higher pCR rates than those treated with trastuzumab alone. The difference was most pronounced in the BluePrint luminal patients (8 vs. 31%). The highest pCR was reached by the BluePrint HER2-type patients treated with T/P (76%). The addition of pertuzumab leads to increased pCR rates for all HER2+ patient groups except for the BluePrint basal-type patients. This better response was most pronounced for the BluePrint luminal-type patients.

Tumor response ratio predicts overall survival in breast cancer patients treated with neoadjuvant chemotherapy.

PubMed

Miller, Marian; Ottesen, Rebecca A; Niland, Joyce C; Kruper, Laura; Chen, Steven L; Vito, Courtney

2014-10-01

Neoadjuvant chemotherapy (NAC) is commonly used to treat locally advanced breast cancer. Pathologic complete response (pCR) predicts improved overall survival (OS); however, prognosis of patients with partial response remains unclear. We evaluated whether tumor response ratio (TRR) is a better predictor of OS than current staging methods. Using the National Comprehensive Cancer Network Breast Cancer Outcomes Database, we identified patients with stage I-III breast cancer who had NAC and pretreatment imaging at City of Hope (1997-2010). Patient demographics, tumor characteristics, and OS were analyzed. TRR was calculated as residual in-breast disease divided by size on pre-NAC imaging. Four TRR groups were stratified; TRR 0 (pCR), TRR > 0-0.4 (strong partial response, SPR), TRR > 0.4-1.0 (weak partial response, WPR), or TRR > 1.0 (tumor growth, TG). OS was estimated by the Kaplan-Meier method and tested by the log-rank test. Cox regression was performed to evaluate associations between OS and TRR in a multivariable analysis while controlling for potential confounders. There were 218 eligible patients identified; 59 (27 %) had pCR, 61 (28 %) SPR, 72 (33 %) WPR, and 26 (12 %) TG. Five-year OS decreased continuously with increasing TRR:pCR (90 %), SPR (79 %), WPR (66 %), and TG (60 %). TRR was the only measure that significantly predicted OS (p = 0.0035); pathologic stage (p = 0.23) and pre-NAC clinical tumor stage (cT) (p = 0.87) were not significant. TRR continued to be statistically significant by multivariable analysis (p = 0.016). TRR takes into account both pretreatment and residual disease and more accurately predicts OS than pathologic stage and pre-NAC cT. TRR may be useful to more accurately assess prognosis and OS in breast cancer patients undergoing NAC.

Pathological response to neoadjuvant chemotherapy: a new prognosis tool for the curative management of peritoneal colorectal carcinomatosis.

PubMed

Passot, Guillaume; You, Benoît; Boschetti, Gilles; Fontaine, Juliette; Isaac, Sylvie; Decullier, Evelyne; Maurice, Christele; Vaudoyer, Delphine; Gilly, François-Noël; Cotte, Eddy; Glehen, Olivier

2014-08-01

The primary objective of this study was to determine the incidence rate of pathological complete responses (pCRs) following neoadjuvant systemic chemotherapy for the treatment of peritoneal carcinomatosis (PC) of colorectal origin. The secondary objective was to evaluate whether pathological response assessments predict survival of patients treated with curative intent by complete cytoreductive surgery (CRS). A retrospective review was performed of 115 patients who underwent preoperative irinotecan- or oxaliplatin-based chemotherapy, followed by 124 procedures of complete CRS alone or combined with hyperthermic intraperitoneal chemotherapy (HIPEC). The pathological response was defined as the mean percentage of cancer cells remaining within all specimens. Univariate and multivariate analyses were performed to identify predictors of survival and pathological response outcome. Twelve procedures (9.7 %) resulted in pCRs, defined as no residual cancer cells in all specimens, 25 (20.2 %) resulted in major responses (1 to 49 % residual cancer cells), and 87 (70.1 %) resulted in minor or no responses (>50 % residual cancer cells). The cumulative 5-year survival rates were 75 and 57 % for patients with pCR and major responses, respectively. Using multivariate analysis, pathological response was the only independent predictor of survival (P = 0.01; major response: hazard ratio [HR] = 4.91; minor response: HR = 13.46). No significant predictor of pathological response was identified. Pathological complete response can be achieved with preoperative systemic chemotherapy for patients with PC of colorectal origin. The degree of pathological response can be assessed and represented as a new outcome for prognosis following treatment with curative intent.

Ibrutinib plus Venetoclax for the Treatment of Mantle-Cell Lymphoma.

PubMed

Tam, Constantine S; Anderson, Mary Ann; Pott, Christiane; Agarwal, Rishu; Handunnetti, Sasanka; Hicks, Rodney J; Burbury, Kate; Turner, Gillian; Di Iulio, Juliana; Bressel, Mathias; Westerman, David; Lade, Stephen; Dreyling, Martin; Dawson, Sarah-Jane; Dawson, Mark A; Seymour, John F; Roberts, Andrew W

2018-03-29

Both the BTK inhibitor ibrutinib and the BCL2 inhibitor venetoclax are active as monotherapy in the treatment of mantle-cell lymphoma. Complete response rates of 21% have been observed for each agent when administered as long-term continuous therapy. Preclinical models predict synergy in combination. We conducted a single-group, phase 2 study of daily oral ibrutinib and venetoclax in patients, as compared with historical controls. Patients commenced ibrutinib monotherapy at a dose of 560 mg per day. After 4 weeks, venetoclax was added in stepwise, weekly increasing doses to 400 mg per day. Both drugs were continued until progression or an unacceptable level of adverse events. The primary end point was the rate of complete response at week 16. Minimal residual disease (MRD) was assessed by flow cytometry in bone marrow and by allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) in blood. The study included 24 patients with relapsed or refractory mantle-cell lymphoma (23 patients) or previously untreated mantle-cell lymphoma (1 patient). Patients were 47 to 81 years of age, and the number of previous treatments ranged from none to six. Half the patients had aberrations of TP53, and 75% had a high-risk prognostic score. The complete response rate according to computed tomography at week 16 was 42%, which was higher than the historical result of 9% at this time point with ibrutinib monotherapy (P<0.001). The rate of complete response as assessed by positron-emission tomography was 62% at week 16 and 71% overall. MRD clearance was confirmed by flow cytometry in 67% of the patients and by ASO-PCR in 38%. In a time-to-event analysis, 78% of the patients with a response were estimated to have an ongoing response at 15 months. The tumor lysis syndrome occurred in 2 patients. Common side effects were generally low grade and included diarrhea (in 83% of the patients), fatigue (in 75%), and nausea or vomiting (in 71%). In this study involving historical controls, dual targeting of BTK and BCL2 with ibrutinib and venetoclax was consistent with improved outcomes in patients with mantle-cell lymphoma who had been predicted to have poor outcomes with current therapy. (Funded by Janssen and others; AIM ClinicalTrials.gov number, NCT02471391 .).

Tumor Metabolism and Blood Flow as Assessed by PET Varies by Tumor Subtype in Locally Advanced Breast Cancer

PubMed Central

Specht, Jennifer M.; Kurland, Brenda F.; Montgomery, Susan K.; Dunnwald, Lisa K.; Doot, Robert K.; Gralow, Julie R.; Ellis, Georgina K.; Linden, Hannah M.; Livingston, Robert B.; Allison, Kimberly H.; Schubert, Erin K.; Mankoff, David A.

2010-01-01

Purpose Dynamic PET imaging can identify patterns of breast cancer metabolism and perfusion in patients receiving neoadjuvant chemotherapy (NC) that are predictive of response. This analysis examines tumor metabolism and perfusion by tumor subtype. Experimental Design Tumor subtype was defined by immunohistochemistry (IHC) in 71 patients with LABC undergoing NC. Subtype was defined as luminal (ER/PR positive), triple-negative (TN; ER/PR negative, HER2 negative) and HER2 (ER/PR negative, HER2 over-expressing). Metabolic rate (MRFDG) and blood flow (BF) were calculated from PET imaging prior to NC. Pathologic complete response (pCR) to NC was classified as pCR versus other. Results Twenty-five (35%) of 71 patients had TN tumors, 6 (8%) were HER2 and 40 (56%) were luminal. MRFDG for TN tumors was on average 67% greater than for luminal tumors (95% CI 9% – 156%), and average MRFDG/BF ratio was 53% greater in TN compared to luminal tumors (95% CI 9% – 114%) (p < 0.05 for both). Average blood flow levels did not differ by subtype (p = 0.73). Most luminal tumors showed relatively low MRFDG and BF (and did not achieve pCR); high MRFDG was generally matched with high BF in luminal tumors, and predicted pCR. This was not true in TN tumors. Conclusions The relationship between breast tumor metabolism and perfusion differed by subtype. The high MRFDG/BF ratio that predicts poor response to NC was more common in TN tumors. Metabolism and perfusion measures may identify subsets of tumors susceptible and resistant to NC and may help direct targeted therapy. PMID:20460489

Evaluation of tolerability and efficacy of incorporating carboplatin in neoadjuvant anthracycline and taxane based therapy in a BRCA1 enriched triple-negative breast cancer cohort.

PubMed

Sella, Tal; Gal Yam, Einav Nili; Levanon, Keren; Rotenberg, Tal Shapira; Gadot, Moran; Kuchuk, Iryna; Molho, Rinat Bernstein; Itai, Amit; Modiano, Tami Mekel; Gold, Raya; Kaufman, Bella; Shimon, Shani Paluch

2018-05-22

The addition of carboplatin (Cb) to neoadjuvant chemotherapy in triple-negative breast cancer (TNBC) has been demonstrated to improve pathologic complete response (pCR) at the expense of increased toxicity. We aimed to evaluate the effectiveness and tolerability of dose-dense anthracycline & cyclophosphamide (ddAC) followed by weekly paclitaxel (wT) in combination with weekly Cb. Retrospective data was collected on patients with clinical stage I-III TNBC treated with neoadjuvant ddAC-wTCb (four cycles of ddA 60 mg/m 2 and ddC 600 mg/m 2 every 2 weeks followed by 12 cycles of wT 80 mg/m 2 with Cb AUC 1.5). Indices of tolerability and pCR were evaluated and compared to a historical cohort (n = 76) treated with ddAC-T. A secondary objective was to evaluate the rates of pCR by BRCA status. For 43 eligible patients, mean age was 41.5 years, 51% had clinical stage II disease, 81.4% were clinically node positive and 32.6% carried a deleterious BRCA1 mutation. Only 35% completed all scheduled doses of chemotherapy. Grade 3/4 neutropenia was observed in 42.5% of patients. Overall pCR was 51.2%; 44.8% in BRCA wild-type compared to 64.3% in BRCA-associated TNBC (p = 0.232). pCR rates with ddAC-wTCb were similar to historic institutional rates with ddAC-T (51.2% vs. 51.3%, p = 0.987) and were comparable when stratified by BRCA status. In pooled multivariate analysis, only BRCA status (HR 4.00, 95%CI 1.65-9.75, p = 0.002) was significantly associated with pCR. Neoadjuvant ddAC-wTCb is less tolerable in clinical practice compared to most clinical trials, with a pCR comparable to historic rates using non-platinum regimen. The role of Cb in neoadjuvant chemotherapy for BRCA mutated TNBC remains uncertain. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Differential Contribution of the Innate Immune System to a Good Pathological Response in the Breast and Axillary Lymph Nodes Induced by Neoadjuvant Chemotherapy in Women with Large and Locally Advanced Breast Cancers

PubMed Central

Verma, Chandan; Eremin, Jennifer M.; Cowley, Gerard; Ilyas, Mohammad; Satthaporn, Sukchai; Eremin, Oleg

2017-01-01

The tumour microenvironment consists of malignant cells, stroma, and immune cells. The role of adaptive immunity in inducing a pathological complete response (pCR) in breast cancer with neoadjuvant chemotherapy (NAC) is well studied. The contribution of innate immunity, however, is poorly documented. Breast tumours and axillary lymph nodes (ALNs) from 33 women with large and locally advanced breast cancers (LLABCs) undergoing NAC were immunohistochemically assessed for tumour-infiltrating macrophages (TIMs: M1 and M2), neutrophils (TINs), and dendritic cells (TIDCs) using labelled antibodies and semiquantitative methods. Patients' blood neutrophils (n = 108), DCs (mDC1 and pDC), and their costimulatory molecules (n = 30) were also studied. Pathological results were classified as pCR, good (GPR) or poor (PRR). In breast and metastatic ALNs, high levels of CD163+ TIMs were significantly associated with a pCR. In blood, high levels of neutrophils were significantly associated with pCR in metastatic ALNs, whilst the % of mDC1 and pDC and expression of HLA-DR, mDC1 CD40, and CD83 were significantly reduced. NAC significantly reduced tumour DCs but increased blood DCs. PPRs to NAC had significantly reduced HLA-DR, CD40, and CD86 expression. Our study demonstrated novel findings documenting the differential but important contributions of innate immunity to pCRs in patients with LLABCs undergoing NAC. PMID:28913366

Preoperative Radiotherapy of Advanced Rectal Cancer With Capecitabine and Oxaliplatin With or Without Cetuximab: A Pooled Analysis of Three Prospective Phase I-II Trials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weiss, Christian, E-mail: christian.weiss@kgu.d; Arnold, Dirk; Dellas, Kathrin

2010-10-01

Purpose: A pooled analysis of three prospective trials of preoperative radiochemotherapy (RCT) for rectal cancer by using oxaliplatin and capecitabine with or without cetuximab was performed to evaluate the impact of additional cetuximab on pathologic complete response (pCR) rates and tumor regression (TRG) grades. Methods and Materials: Of 202 patients, 172 patients met the inclusion criteria (primary tumor stage II/III, M0). All patients received concurrent RCT, and 46 patients received additional cetuximab therapy. A correlation of pretreatment clinicopathologic factors and cetuximab treatment with early pCR rates (TRG > 50%) was performed with univariate and multivariate analyses. Toxicity data were recordedmore » for all patients. Results: Of 172 patients, 24 (14%) patients achieved a pCR, and 84 of 172 (71%) patients showed a TRG of >50% in the surgical specimen assessment after preoperative treatment. Age, gender, and T/N stages, as well as localization of the tumor, were not associated with pCR or good TRG. The pCR rate was 16% after preoperative RCT alone and 9% with concurrent cetuximab therapy (p = 0.32). A significantly reduced TRG of >50% was found after RCT with cetuximab compared to RCT alone (p = 0.0035). This was validated by a multivariate analysis with all available clinical factors (p = 0.0037). Acute toxicity and surgical complications were not increased with additional cetuximab. Conclusions: Triple therapy with RCT and cetuximab seems to be feasible, with no unexpected toxicity. Early response assessment (TRG), however, suggests subadditive interaction. A longer follow-up (and finally randomized trials) is needed to draw any firm conclusions with respect to local and distant failure rates.« less

Cyclophosphamide dose intensification may circumvent anthracycline resistance of p53 mutant breast cancers.

PubMed

Lehmann-Che, Jacqueline; André, Fabrice; Desmedt, Christine; Mazouni, Chafika; Giacchetti, Sylvie; Turpin, Elisabeth; Espié, Marc; Plassa, Louis-François; Marty, Michel; Bertheau, Philippe; Sotiriou, Christos; Piccart, Martine; Symmans, W Fraser; Pusztai, Lajos; de Thé, Hugues

2010-01-01

The predictive value of p53 for the efficacy of front-line anthracycline-based chemotherapy regimens has been a matter of significant controversy. Anthracyclines are usually combined with widely different doses of alkylating agents, which may significantly modulate tumor response to these combinations. We analyzed three series of de novo stage II-III breast cancer patients treated front line with anthracycline-based regimens of various cyclophosphamide dose intensities: 65 patients with estrogen receptor (ER)(-) tumors treated with anthracyclines alone (Institut Jules Bordet, Brussels), 51 unselected breast cancer patients treated with intermediate doses of cyclophosphamide (MD Anderson Cancer Center, Houston, TX), and 128 others treated with a dose-dense anthracycline-cyclophosphamide combination (St. Louis, Paris). After chemotherapy and surgery, pathologic complete response (pCR) was evaluated. p53 status was determined by a yeast functional assay on the pretreatment tumor sample. In a multivariate analysis of the pooled results, a lack of ER expression and high-dose cyclophosphamide administration were associated with a higher likelihood of pCR. A sharp statistical interaction was detected between p53 status and cyclophosphamide dose intensity. Indeed, when restricting our analysis to patients with ER(-) tumors, we confirmed that a mutant p53 status was associated with anthracycline resistance, but found that p53 inactivation was required for response to the dose-intense alkylating regimen. The latter allowed very high levels of pCR in triple-negative tumors. Thus, our data strongly suggest that cyclophosphamide dose intensification in ER(-) p53-mutated breast cancer patients could significantly improve their response.

Prospective study of neoadjuvant chemoradiotherapy using intensity-modulated radiotherapy and 5 fluorouracil for locally advanced rectal cancer – toxicities and response assessment

PubMed Central

Simson, David K; Mitra, Swarupa; Ahlawat, Parveen; Saxena, Upasna; Sharma, Manoj Kumar; Rawat, Sheh; Singh, Harpreet; Bansal, Babita; Sripathi, Lalitha Kameshwari; Tanwar, Aditi

2018-01-01

Aims and objectives The past 2 decades witnessed the strengthening of evidence favoring the role of neoadjuvant chemoradiation (CHRT) in the treatment of locally advanced rectal cancer. The study aims to evaluate the response and acute toxicities to neoadjuvant CHRT using intensity-modulated radiotherapy (IMRT) in the treatment of rectal cancer. Predictive factors to achieve pathological complete response (pCR) were analyzed, as a secondary endpoint. Materials and methods All consecutive patients who underwent IMRT as part of neoadjuvant CHRT in the treatment of rectal cancer between August 2014 and December 2016 at a tertiary cancer care center were accrued for the study. The cohort underwent CHRT with IMRT technique at a dose of 50.4 Gy in 28 fractions concurrent with continuous infusion of 5 fluorouracil during the first and the last 4 days of CHRT. Surgery was performed 6 weeks later and the pathological response to CHRT was noted. Results Forty-three subjects were accrued for the study. Radiation dermatitis and diarrhea were the only observed grade ≥3 acute toxicities. Sphincter preservation rate (SPR) was 43.3%. pCR was observed in 32.6%. Univariate and multivariate logistic regression showed that carcinoembryonic antigen was the only independent predictive factor to achieve pCR. Conclusion IMRT as part of neoadjuvant CHRT in the treatment of locally advanced rectal cancer is well tolerated and gives comparable results with respect to earlier studies in terms of pathological response and SPR. Further randomized controlled studies are needed to firmly state that IMRT is superior to 3-dimensional conformal radiotherapy. PMID:29593430

Evaluation of a 30-gene paclitaxel, fluorouracil, doxorubicin and cyclophosphamide chemotherapy response predictor in a multicenter randomized trial in breast cancer

PubMed Central

Tabchy, Adel; Valero, Vicente; Vidaurre, Tatiana; Lluch, Ana; Gomez, Henry; Martin, Miguel; Qi, Yuan; Barajas-Figueroa, Luis Javier; Souchon, Eduardo; Coutant, Charles; Doimi, Franco D; Ibrahim, Nuhad K; Gong, Yun; Hortobagyi, Gabriel N; Hess, Kenneth R; Symmans, W Fraser; Pusztai, Lajos

2010-01-01

Purpose We examined in a prospective, randomized, international clinical trial the performance of a previously defined 30-gene predictor (DLDA-30) of pathologic complete response (pCR) to preoperative weekly paclitaxel and fluorouracil, doxorubicin, cyclophosphamide (T/FAC) chemotherapy, and assessed if DLDA-30 also predicts increased sensitivity to FAC-only chemotherapy. We compared the pCR rates after T/FAC versus FAC×6 preoperative chemotherapy. We also performed an exploratory analysis to identify novel candidate genes that differentially predict response in the two treatment arms. Experimental Design 273 patients were randomly assigned to receive either weekly paclitaxel × 12 followed by FAC × 4 (T/FAC, n=138), or FAC × 6 (n=135) neoadjuvant chemotherapy. All patients underwent a pretreatment FNA biopsy of the tumor for gene expression profiling and treatment response prediction. Results The pCR rates were 19% and 9% in the T/FAC and FAC arms, respectively (p<0.05). In the T/FAC arm, the positive predictive value (PPV) of the genomic predictor was 38% (95%CI:21–56%), the negative predictive value (NPV) 88% (CI:77–95%) and the AUC 0.711. In the FAC arm, the PPV was 9% (CI:1–29%) and the AUC 0.584. This suggests that the genomic predictor may have regimen-specificity. Its performance was similar to a clinical variable-based predictor nomogram. Conclusions Gene expression profiling for prospective response prediction was feasible in this international trial. The 30-gene predictor can identify patients with greater than average sensitivity to T/FAC chemotherapy. However, it captured molecular equivalents of clinical phenotype. Next generation predictive markers will need to be developed separately for different molecular subsets of breast cancers. PMID:20829329

Long-term remission in BCR/ABL-positive AML-M6 patient treated with Imatinib Mesylate.

PubMed

Pompetti, Franca; Spadano, Antonio; Sau, Antonella; Mennucci, Antonio; Russo, Rosa; Catinella, Virginia; Franchi, Paolo Guanciali; Calabrese, Giuseppe; Palka, Giandomenico; Fioritoni, Giuseppe; Iacone, Antonio

2007-04-01

BCR/ABL-positive acute myeloid leukemia (AML) is a rare disease, characterized by a poor prognosis, with resistance to induction chemotherapy and frequent relapses in responsive patients. Here we report a case of BCR/ABL-positive AML-M6 who, after relapse, was treated with Imatinib Mesylate (600 mg/die) and within 4 months achieved a cytogenetic and molecular complete response. After more than 4 years of continuous Imatinib therapy, nested RT-PCR for BCR/ABL is persistently negative. The case reported shows that the response obtained with Imatinib Mesylate in BCR/ABL-positive AML may be long lasting, offering a chance of successful treatment for this poor prognosis group of patients.

The value of forceps biopsy and core needle biopsy in prediction of pathologic complete remission in locally advanced rectal cancer treated with neoadjuvant chemoradiotherapy.

PubMed

Tang, Jing-Hua; An, Xin; Lin, Xi; Gao, Yuan-Hong; Liu, Guo-Chen; Kong, Ling-Heng; Pan, Zhi-Zhong; Ding, Pei-Rong

2015-10-20

Patients with pathological complete remission (pCR) after treated with neoadjuvant chemoradiotherapy (nCRT) have better long-term outcome and may receive conservative treatments in locally advanced rectal cancer (LARC). The study aimed to evaluate the value of forceps biopsy and core needle biopsy in prediction of pCR in LARC treated with nCRT. In total, 120 patients entered this study. Sixty-one consecutive patients received preoperative forceps biopsy during endoscopic examination. Ex vivo core needle biopsy was performed in resected specimens of another 43 consecutive patients. The accuracy for ex vivo core needle biopsy was significantly higher than forceps biopsy (76.7% vs. 36.1%; p < 0.001). The sensitivity for ex vivo core needle biopsy was significantly lower in good responder (TRG 3) than poor responder (TRG ≤ 2) (52.9% vs. 94.1%; p = 0.017). In vivo core needle biopsy was further performed in 16 patients with good response. Eleven patients had residual cancer cells in final resected specimens, among whom 4 (36.4%) patients were biopsy positive. In conclusion, routine forceps biopsy was of limited value in identifying pCR after nCRT. Although core needle biopsy might further identify a subset of patients with residual cancer cells, the accuracy was not substantially increased in good responders.

The value of forceps biopsy and core needle biopsy in prediction of pathologic complete remission in locally advanced rectal cancer treated with neoadjuvant chemoradiotherapy

PubMed Central

Gao, Yuan-Hong; Liu, Guo-Chen; Kong, Ling-Heng; Pan, Zhi-Zhong; Ding, Pei-Rong

2015-01-01

Patients with pathological complete remission (pCR) after treated with neoadjuvant chemoradiotherapy (nCRT) have better long-term outcome and may receive conservative treatments in locally advanced rectal cancer (LARC). The study aimed to evaluate the value of forceps biopsy and core needle biopsy in prediction of pCR in LARC treated with nCRT. In total, 120patients entered this study. Sixty-one consecutive patients received preoperative forceps biopsy during endoscopic examination. Ex vivo core needle biopsy was performed in resected specimens of another 43 consecutive patients. The accuracy for ex vivo core needle biopsy was significantly higher than forceps biopsy (76.7% vs. 36.1%; p < 0.001). The sensitivity for ex vivo core needle biopsy was significantly lower in good responder (TRG 3) than poor responder (TRG ≤ 2) (52.9% vs. 94.1%; p = 0.017). In vivo core needle biopsy was further performed in 16 patients with good response. Eleven patients had residual cancer cells in final resected specimens, among whom 4 (36.4%) patients were biopsy positive. In conclusion, routine forceps biopsy was of limited value in identifying pCR after nCRT. Although core needle biopsy might further identify a subset of patients with residual cancer cells, the accuracy was not substantially increased in good responders. PMID:26416245

Tumor infiltrating lymphocytes in triple negative breast cancer receiving neoadjuvant chemotherapy

PubMed Central

Castaneda, Carlos A; Mittendorf, Elizabeth; Casavilca, Sandro; Wu, Yun; Castillo, Miluska; Arboleda, Patricia; Nunez, Teresa; Guerra, Henry; Barrionuevo, Carlos; Dolores-Cerna, Ketty; Belmar-Lopez, Carolina; Abugattas, Julio; Calderon, Gabriela; De La Cruz, Miguel; Cotrina, Manuel; Dunstan, Jorge; Gomez, Henry L; Vidaurre, Tatiana

2016-01-01

AIM To determine influence of neoadjuvant-chemotherapy (NAC) over tumor-infiltrating-lymphocytes (TIL) in triple-negative-breast-cancer (TNBC). METHODS TILs were evaluated in 98 TNBC cases who came to Instituto Nacional de Enfermedades Neoplasicas from 2005 to 2010. Immunohistochemistry staining for CD3, CD4, CD8 and FOXP3 was performed in tissue microarrays (TMA) sections. Evaluation of H/E in full-face and immunohistochemistry in TMA sections was performed in pre and post-NAC samples. STATA software was used and P value < 0.05 was considered statistically significant. RESULTS Higher TIL evaluated in full-face sections from pre-NAC tumors was associated to pathologic-complete-response (pCR) (P = 0.0251) and outcome (P = 0.0334). TIL evaluated in TMA sections showed low level of agreement with full-face sections (ICC = 0.017-0.20) and was not associated to pCR or outcome. TIL in post-NAC samples were not associated to response or outcome. Post-NAC lesions with pCR had similar TIL levels than those without pCR (P = 0.6331). NAC produced a TIL decrease in full-face sections (P < 0.0001). Percentage of TIL subpopulations was correlated with their absolute counts. Higher counts of CD3, CD4, CD8 and FOXP3 in pre-NAC samples had longer disease-free-survival (DFS). Higher counts of CD3 in pre-NAC samples had longer overall-survival. Higher ratio of CD8/CD4 counts in pre-NAC was associated with pCR. Higher ratio of CD4/FOXP3 counts in pre-NAC was associated with longer DFS. Higher counts of CD4 in post-NAC samples were associated with pCR. CONCLUSION TIL in pre-NAC full-face sections in TNBC are correlated to longer survival. TIL in full-face differ from TMA sections, absolute count and percentage analysis of TIL subpopulation closely related. PMID:27777881

Preoperative Chemoradiation Therapy in Combination With Panitumumab for Patients With Resectable Esophageal Cancer: The PACT Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kordes, Sil, E-mail: s.kordes@amc.uva.nl; Berge Henegouwen, Mark I. van; Hulshof, Maarten C.

Purpose: Preoperative chemoradiation therapy (CRT) has become the standard treatment strategy for patients with resectable esophageal cancer. This multicenter phase 2 study investigated the efficacy of the addition of the epidermal growth factor receptor (EGFR) inhibitor panitumumab to a preoperative CRT regimen with carboplatin, paclitaxel, and radiation therapy in patients with resectable esophageal cancer. Methods and Materials: Patients with resectable cT1N1M0 or cT2-3N0 to -2M0 tumors received preoperative CRT consisting of panitumumab (6 mg/kg) on days 1, 15, and 29, weekly administrations of carboplatin (area under the curve [AUC] = 2), and paclitaxel (50 mg/m{sup 2}) for 5 weeks and concurrent radiation therapy (41.4 Gy in 23more » fractions, 5 days per week), followed by surgery. Primary endpoint was pathologic complete response (pCR) rate. We aimed at a pCR rate of more than 40%. Furthermore, we explored the predictive value of biomarkers (EGFR, HER 2, and P53) for pCR. Results: From January 2010 until December 2011, 90 patients were enrolled. Patients were diagnosed predominantly with adenocarcinoma (AC) (80%), T3 disease (89%), and were node positive (81%). Three patients were not resected due to progressive disease. The primary aim was unmet, with a pCR rate of 22%. Patients with AC and squamous cell carcinoma reached a pCR of 14% and 47%, respectively. R0 resection was achieved in 95% of the patients. Main grade 3 toxicities were rash (12%), fatigue (11%), and nonfebrile neutropenia (11%). None of the biomarkers was predictive for response. Conclusions: The addition of panitumumab to CRT with carboplatin and paclitaxel was safe and well tolerated but could not improve pCR rate to the preset criterion of 40%.« less

Textural features of 18F-FDG PET after two cycles of neoadjuvant chemotherapy can predict pCR in patients with locally advanced breast cancer.

PubMed

Cheng, Lin; Zhang, Jianping; Wang, Yujie; Xu, Xiaoli; Zhang, Yongping; Zhang, Yingjian; Liu, Guangyu; Cheng, Jingyi

2017-08-01

This study was designed to evaluate the utility of textural features for predicting pathological complete response (pCR) to neoadjuvant chemotherapy (NAC). Sixty-one consecutive patients with locally advanced breast cancer underwent 18 F-FDG PET/CT scanning at baseline and after the second course of NAC. Changes to imaging parameters [maximum standardized uptake value (SUV max ), metabolic tumor volume (MTV), total lesion glycolysis (TLG)] and textural features (entropy, coarseness, skewness) between the 2 scans were measured by two independent radiologists. Pathological responses were reviewed by one pathologist, and the significance of the predictive value of each parameter was analyzed using a Chi-squared test. Receiver operating characteristic curve analysis was used to compare the area under the curve (AUC) for each parameter. pCR was observed more often in patients with HER2-positive tumors (22 patients) than in patients with HER2-negative tumors (5 patients) (71.0 vs. 16.7%, p < 0.001). ∆ %SUV max , ∆ %entropy and ∆ %coarseness were significantly useful for differentiating pCR from non-pCR in the HER2-negative group, and the AUCs for these parameters were 0.928, 0.808 and 0.800, respectively (p = 0.003, 0.032 and 0.037). In the HER2-positive group, ∆ %SUV max and ∆ %skewness were moderately useful for predicting pCR, and the respective AUCs were 0.747 and 0.758 (p = 0.033 and 0.026). Although there was no significant difference in the AUCs between groups for these parameters, an additional 3/22 patients in the HER2-positive group with pCR were identified when ∆ %skewness and ∆ %SUV max were considered together (p = 0.031). The absolute values for each parameter before NAC and after 2 cycles cannot predict pCR in our patients. Neither ∆ %MTV nor ∆ %TLG was efficiently predictive of pCR in any group. The early changes in the textural features of 18 F-FDG PET images after two cycles of NAC are predictive of pCR in both HER2-negative and HER2-positive patients; this evidence warrants confirmation by further research.

Effect of age, diet, and tissue type on PCr response to creatine supplementation.

PubMed

Solis, Marina Yazigi; Artioli, Guilherme Giannini; Otaduy, Maria Concepción García; Leite, Claudia da Costa; Arruda, Walquiria; Veiga, Raquel Ramos; Gualano, Bruno

2017-08-01

Creatine/phosphorylcreatine (PCr) responses to creatine supplementation may be modulated by age, diet, and tissue, but studies assessing this possibility are lacking. Therefore we aimed to determine whether PCr responses vary as a function of age, diet, and tissue. Fifteen children, 17 omnivorous and 14 vegetarian adults, and 18 elderly individuals ("elderly") participated in this study. Participants were given placebo and subsequently creatine (0.3 g·kg -1 ·day -1 ) for 7 days in a single-blind fashion. PCr was measured through phosphorus magnetic resonance spectroscopy ( 31 P-MRS) in muscle and brain. Creatine supplementation increased muscle PCr in children ( P < 0.0003) and elderly ( P < 0.001), whereas the increase in omnivores did not reach statistically significant difference ( P = 0.3348). Elderly had greater PCr increases than children and omnivores ( P < 0.0001 for both), whereas children experienced greater PCr increases than omnivores ( P = 0.0022). In relation to diet, vegetarians ( P < 0.0001), but not omnivores, had significant increases in muscle PCr content. Brain PCr content was not affected by creatine supplementation in any group, and delta changes in brain PCr (-0.7 to +3.9%) were inferior to those in muscle PCr content (+10.3 to +27.6%; P < 0.0001 for all comparisons). PCr responses to a standardized creatine protocol (0.3 g·kg -1 ·day -1 for 7 days) may be affected by age, diet, and tissue. Whereas creatine supplementation was able to increase muscle PCr in all groups, although to different extents, brain PCr was shown to be unresponsive overall. These findings demonstrate the need to tailor creatine protocols to optimize creatine/PCr accumulation both in muscle and in brain, enabling a better appreciation of the pleiotropic properties of creatine. NEW & NOTEWORTHY A standardized creatine supplementation protocol (0.3 g·kg -1 ·day -1 for 7 days) effectively increased muscle, but not brain, phosphorylcreatine. Older participants responded better than younger participants whereas vegetarians responded better than omnivores. Responses to supplementation are thus dependent on age, tissue, and diet. This suggests that a single "universal" protocol, originally designed for increasing muscle creatine in young individuals, may lead to heterogeneous muscle responses in different populations or even no responses in tissues other than skeletal muscle. Copyright © 2017 the American Physiological Society.

Effect of Neoadjuvant Radiation Dose on Surgical and Oncological Outcome in Locally Advanced Esophageal Cancer.

PubMed

Van Daele, E; Ceelen, W; Boterberg, T; Varinl, O; Van Nieuwenhove, Y; Putte, D Van de; Geboes, K; Pattyn, P

2015-01-01

Neoadjuvant chemoradiation (CRT) confers a survival benefit in locally advanced esophageal cancer. The optimal dose of radiotherapy remains undefined. From a prospective database, we identified patients who received CRT followed by Ivor Lewis esophagectomy. Surgical complications, pathological response, and oncological outcome were compared between patients who received a radiotherapy (RT) dose of 36 Gy (group1) versus a dose of > 40 Gy (group 1). 147 patients were evaluated: 109 received 36 Gy, while 38 received 41-50Gy. Mean age was 61 ± 9 years (84% male). Median hospital stay was 16 days. Anastomotic leakage occurred in 4.0%. Pulmonary complications occurred in 41.8%, neither being influenced by RT dose. Complete resection (R0) was achieved in 95% (group 1) and 100% (group 2), P = 0.3. Pathological complete response (pCR) was observed in 19% (group 1) and 37% (group 1), P = 0.04. Local recurrence developed in 9% in group 1, and 3% in group 2 (P = 0.3), but regional recurrence developed significantly higher in the low dose group (18% vs 3%, P < 0.001). Metastatic recurrence occurred in 48% in group 1 and 13% in group 1 (P < 0.001). In patients with locally advanced esophageal cancer a higher RT dose does not affect surgical outcome, enhances pCR rate, and reduces the locoregional and metastatic recurrence risk.

[18F]fluoro-2-deoxyglucose-positron emission tomography for the assessment of histopathological response after preoperative chemoradiotherapy in advanced oral squamous cell carcinoma.

PubMed

Shimomura, Hiroyuki; Sasahira, Tomonori; Yamanaka, Yasutsugu; Kurihara, Miyako; Imai, Yuichiro; Tamaki, Shigehiro; Yamakawa, Nobuhiro; Shirone, Norihisa; Hasegawa, Masatoshi; Kuniyasu, Hiroki; Kirita, Tadaaki

2015-04-01

[(18)F]fluoro-2-deoxyglucose-positron emission tomography (FDG-PET) is widely used to evaluate tumor metabolic activity. The aim of this study was to evaluate the usefulness of FDG-PET in assessing the histopathological response to preoperative concurrent chemoradiotherapy (CRT) in patients with oral squamous cell carcinoma (OSCC). Forty-five patients with resectable advanced OSCC who had received preoperative CRT followed by tumor ablative surgery between January 2004 and December 2011 were included in the study. All patients underwent FDG-PET before and after preoperative CRT. The maximum standardized uptake value (SUVmax) before (pre-SUV) and after preoperative CRT (post-SUV) and the SUVmax reduction rate (ΔSUV %) were used to evaluate the response to preoperative CRT. Correlations among SUVmax, histopathological response, and expression of cancer antigen Ki-67 and hypoxia-inducible factor-1α (HIF-1α) were analyzed. Preoperative CRT significantly reduced intratumoral FDG uptake (P < 0.001). The pre-SUV and post-SUV were significantly lower in patients with a pathological complete response (pCR) than in those with a non-pCR (pre-SUV P = 0.037; post-SUV P = 0.001). ΔSUV % was higher in patients with pCR than in those with non-pCR (P = 0.029). The pre-SUV was significantly correlated with Ki-67 and HIF-1α expression in pretreatment biopsy specimens (Ki-67 P = 0.046, R = 0.292; HIF-1α P = 0.007, R = 0.385). The expression of both Ki-67 and HIF-1α was significantly lower in patients with pCR than in those with non-pCR (Ki-67 P < 0.001; HIF-1α P < 0.001). Low pre-SUV and post-SUV and high ΔSUV % may predict a good histopathological response to preoperative CRT. Ki-67 and HIF-1α expression in pretreatment biopsy specimens were predictors of histopathological response to preoperative CRT.

Recommendations for standardized pathological characterization of residual disease for neoadjuvant clinical trials of breast cancer by the BIG-NABCG collaboration

PubMed Central

Bossuyt, V.; Provenzano, E.; Symmans, W. F.; Boughey, J. C.; Coles, C.; Curigliano, G.; Dixon, J. M.; Esserman, L. J.; Fastner, G.; Kuehn, T.; Peintinger, F.; von Minckwitz, G.; White, J.; Yang, W.; Badve, S.; Denkert, C.; MacGrogan, G.; Penault-Llorca, F.; Viale, G.; Cameron, D.; Earl, Helena; Alba, Emilio; Lluch, Ana; Albanell, Joan; Amos, Keith; Biernat, Wojciech; Bonnefoi, Hervé; Buzdar, Aman; Cane, Paul; Pinder, Sarah; Carson, Lesley; Dickson-Witmer, Diana; Gong, Gyungyub; Green, Jimmy; Hsu, Chih-Yi; Tseng, Ling-Ming; Kroep, Judith; Leitch, A. Marilyn; Sarode, Venetia; Mamounas, Eleftherios; Marcom, Paul Kelly; Nuciforo, Paolo; Paik, Soonmyung; Peg, Vicente; Peston, David; Pierga, Jean-Yves; Quintela-Fandino, Miguel; Salgado, Roberto; Sikov, William; Thomas, Jeremy; Unzeitig, Gary; Wesseling, Jelle

2015-01-01

Neoadjuvant systemic therapy (NAST) provides the unique opportunity to assess response to treatment after months rather than years of follow-up. However, significant variability exists in methods of pathologic assessment of response to NAST, and thus its interpretation for subsequent clinical decisions. Our international multidisciplinary working group was convened by the Breast International Group-North American Breast Cancer Group (BIG-NABCG) collaboration and tasked to recommend practical methods for standardized evaluation of the post-NAST surgical breast cancer specimen for clinical trials that promote accurate and reliable designation of pathologic complete response (pCR) and meaningful characterization of residual disease. Recommendations include multidisciplinary communication; clinical marking of the tumor site (clips); and radiologic, photographic, or pictorial imaging of the sliced specimen, to map the tissue sections and reconcile macroscopic and microscopic findings. The information required to define pCR (ypT0/is ypN0 or ypT0 yp N0), residual ypT and ypN stage using the current AJCC/UICC system, and the Residual Cancer Burden system were recommended for quantification of residual disease in clinical trials. PMID:26019189

Planned Pregnancy in a Chronic Myeloid Leukemia Patient in Molecular Remission

PubMed Central

Pavlovsky, Carolina; Giere, Isabel; Van Thillo, Germán

2012-01-01

Excellent response rates and a good quality of life have been observed since the introduction of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) treatment. Consequently, some challenges began to appear in CML women in child-bearing age wishing to become pregnant. Currently, many women around the world are in stable major/complete molecular response MMR/CMR (MMR: <0.1% BCR-ABL/ABL and CMR: undetectable BCR-ABL mRNA by RQ-PCR transcript levels on the international scale). The condition of stable MMR/CMR is linked to a long-term virtual absence of progression to the accelerated and blastic phase and to the possibility of stopping the TKI treatment with the maintenance of a condition of CMR in a proportion of cases. Imatinib teratogenic and prescribing information prohibits the use of it during pregnancy. We describe the case of a 36-year-old female patient with CML in chronic phase who stopped imatinib after 2 years in major molecular response (MMR) to plan a pregnancy. Molecular monitoring by RQ-PCR was performed quarterly. She achieved a safe pregnancy and delivery maintaining an optimal molecular response throughout the pregnancy. Isolated literature reports have been described, but no formal advice has been described at present time. PMID:22928126

Implications of Pathologic Complete Response Beyond Mediastinal Nodal Clearance With High-Dose Neoadjuvant Chemoradiation Therapy in Locally Advanced, Non-Small Cell Lung Cancer.

PubMed

Vyfhuis, Melissa A L; Burrows, Whitney M; Bhooshan, Neha; Suntharalingam, Mohan; Donahue, James M; Feliciano, Josephine; Badiyan, Shahed; Nichols, Elizabeth M; Edelman, Martin J; Carr, Shamus R; Friedberg, Joseph; Henry, Gavin; Stewart, Shelby; Sachdeva, Ashutosh; Pickering, Edward M; Simone, Charles B; Feigenberg, Steven J; Mohindra, Pranshu

2018-06-01

To determine, in a retrospective analysis of a large cohort of stage III non-small cell lung cancer patients treated with curative intent at our institution, whether having a pathologic complete response (pCR) influenced overall survival (OS) or freedom from recurrence (FFR) in patients who underwent definitive (≥60 Gy) neoadjuvant doses of chemoradiation (CRT). At our institution, 355 patients with locally advanced non-small cell lung cancer were treated with curative intent with definitive CRT (January 2000-December 2013), of whom 111 underwent mediastinal reassessment for possible surgical resection. Ultimately 88 patients received trimodality therapy. Chi-squared analysis was used to compare categorical variables. The Kaplan-Meier analysis was performed to estimate OS and FFR, with Cox regression used to determine the absolute hazards. Using high-dose neoadjuvant CRT, we observed a mediastinal nodal clearance (MNC) rate of 74% (82 of 111 patients) and pCR rate of 48% (37 of 77 patients). With a median follow-up of 34.2 months (range, 3-177 months), MNC resulted in improved OS and FFR on both univariate (OS: hazard ratio [HR] 0.455, 95% confidence interval [CI] 0.272-0.763, P = .004; FFR: HR 0.426, 95% CI 0.250-0.726, P = .002) and multivariate analysis (OS: HR 0.460, 95% CI 0.239-0.699, P = .001; FFR: HR 0.455, 95% CI 0.266-0.778, P = .004). However, pCR did not independently impact OS (P = .918) or FFR (P = .474). Mediastinal nodal clearance after CRT continues to be predictive of improved survival for patients undergoing trimodality therapy. However, a pCR at both the primary and mediastinum did not further improve survival outcomes. Future therapies should focus on improving MNC to encourage more frequent use of surgery and might justify use of preoperative CRT over chemotherapy alone. Copyright © 2018 Elsevier Inc. All rights reserved.

Weekly paclitaxel and concurrent pazopanib following doxorubicin and cyclophosphamide as neoadjuvant therapy for HER-negative locally advanced breast cancer: NSABP Foundation FB-6, a phase II study.

PubMed

Tan, A R; Johannes, H; Rastogi, P; Jacobs, S A; Robidoux, A; Flynn, P J; Thirlwell, M P; Fehrenbacher, L; Stella, P J; Goel, R; Julian, T B; Provencher, L; Bury, M J; Bhatt, K; Geyer, C E; Swain, S M; Mamounas, E P; Wolmark, N

2015-01-01

This multicenter single-arm phase II study evaluated the addition of pazopanib to concurrent weekly paclitaxel following doxorubicin and cyclophosphamide as neoadjuvant therapy in human epidermal growth factor receptor (HER2)-negative locally advanced breast cancer (LABC). Patients with HER2-negative stage III breast cancer were treated with doxorubicin 60 mg/m(2) and cyclophosphamide 600 mg/m(2) for four cycles every 3 weeks followed by weekly paclitaxel 80 mg/m(2) on days 1, 8, and 15 every 28 days for four cycles concurrently with pazopanib 800 mg orally daily prior to surgery. Post-operatively, pazopanib was given daily for 6 months. The primary endpoint was pathologic complete response (pCR) in the breast and lymph nodes. Between July 2009 and March 2011, 101 patients with stage IIIA-C HER2-negative breast cancer were enrolled. The pCR rate in evaluable patients who initiated paclitaxel and pazopanib was 17 % (16/93). The pCR rate was 9 % (6/67) in hormone receptor-positive tumors and 38 % (10/26) in triple-negative tumors. Pre-operative pazopanib was completed in only 39 % of patients. The most frequent grade 3 and 4 adverse events during paclitaxel and pazopanib were neutropenia (27 %), diarrhea (5 %), ALT and AST elevations (each 5 %), and hypertension (5 %). Although the pCR rate of paclitaxel and pazopanib following AC chemotherapy given as neoadjuvant therapy in women with LABC met the pre-specified criteria for activity, there was substantial toxicity, which led to a high discontinuation rate of pazopanib. The combination does not appear to warrant further evaluation in the neoadjuvant setting for breast cancer.

Enrichment of individual KIR2DL4 sequences from genomic DNA using long-template PCR and allele-specific hybridization to magnetic bead-bound oligonucleotide probes.

PubMed

Roberts, C H; Turino, C; Madrigal, J A; Marsh, S G E

2007-06-01

DNA enrichment by allele-specific hybridization (DEASH) was used as a means to isolate individual alleles of the killer cell immunoglobulin-like receptor (KIR2DL4) gene from heterozygous genomic DNA. Using long-template polymerase chain reaction (LT-PCR), the complete KIR2DL4 gene was amplified from a cell line that had previously been characterized for its KIR gene content by PCR using sequence-specific primers (PCR-SSP). The whole gene amplicons were sequenced and we identified two heterozygous positions in accordance with the predictions of the PCR-SSP. The amplicons were then hybridized to allele-specific, biotinylated oligonucleotide probes and through binding to streptavidin-coated beads, the targeted alleles were enriched. A second PCR amplified only the exonic regions of the enriched allele, and these were then sequenced in full. We show DEASH to be capable of enriching single alleles from a heterozygous PCR product, and through sequencing the enriched DNA, we are able to produce complete coding sequences of the KIR2DL4 alleles in accordance with the typing predicted by PCR-SSP.

Neoadjuvant oral vs. infusional chemoradiotherapy on locally advanced rectal cancer: Prognostic factors.

PubMed

Conde, Sofia; Borrego, Margarida; Teixeira, Tânia; Teixeira, Rubina; Sá, Anabela; Soares, Paula

2012-01-01

To evaluate the prognostic factors and impact on survival of neoadjuvant oral and infusional chemoradiotherapy in patients with locally advanced rectal cancer. There is still no definitive consensus about the prognostic factors and the impact of neoadjuvant chemoradiotherapy on survival. Some studies have pointed to an improvement in overall survival (OS) and progression-free survival (PFS) in patients with tumor downstaging (TD) and nodal downstaging (ND). A set of 159 patients with LARC were treated preoperatively. Group A - 112 patients underwent concomitant oral chemoradiotherapy: capecitabine or UFT + folinic acid. Group B - 47 patients submitted to concomitant chemoradiation with 5-FU in continuous infusion. 63.6% of patients were submitted to adjuvant chemotherapy. pathologic complete response (pCR) - 18.7%; TD - 55.1%; ND - 76%; loco-regional response - 74.8%. Group B: pCR - 11.4%; TD - 50%; ND - 55.8%; LRR - 54.5%. The loco-regional control was 95.6%. There was no difference in survival between both groups. Those with loco-regional response had better PFS. Tumor and nodal downstaging, loco-regional response and a normal CEA level turned out to be important prognostic factors in locally advanced rectal cancer. Nodal downstaging and loco-regional response were higher in Group A. Those with tumor downstaging and loco-regional response from Group A had better OS. Adjuvant chemotherapy had no impact on survival except in those patients with loco-regional response who achieved a higher PFS.

Phase II randomized clinical trial evaluating neoadjuvant chemotherapy regimens with weekly paclitaxel or eribulin followed by doxorubicin and cyclophosphamide in women with locally advanced HER2-negative breast cancer: NSABP Foundation Study FB-9.

PubMed

Abraham, Jame; Robidoux, André; Tan, Antoinette R; Limentani, Steven; Sturtz, Keren; Shalaby, Ibrahim; Alcorn, Hope; Buyse, Marc E; Wolmark, Norman; Jacobs, Samuel A

2015-07-01

Locally advanced breast cancer (LABC) is a good setting in which to monitor response to neoadjuvant chemotherapy, to downsize the tumor (which facilitates breast-conserving surgery), and to test newer agents in untreated patients. Eribulin (E) has shown activity in patients who have undergone previous taxane, anthracycline, and capecitabine treatment. We aimed to evaluate the neoadjuvant use of E followed by doxorubicin and cyclophosphamide (AC) in patients with HER2-negative LABC, using as a control a randomized group of women who received weekly paclitaxel (WP). Fifty women with LABC were accrued January-August 2013. Patients were randomized (1:2) to receive either WP (N = 19) for 12 treatments or E (N = 31) every 3 weeks for 4 cycles followed by AC every 3 weeks for 4 cycles before surgery. 17/19 patients who took WP and 25/30 who took E completed all cycles. Patients were evaluated by clinical examination and breast MRI at baseline and after completion of E or WP. Surgical pCR in breast and lymph nodes was determined by a local pathologist following chemotherapy. Forty-nine patients received ≥1 dose of neoadjuvant chemotherapy and are included in this analysis. Forty-eight underwent surgery; one had disease that was inoperable (on E) and is included as no-pCR patient. 17/19 of these patients who took WP completed 12 doses; 28/30 on E completed 4 cycles. Six discontinued treatment on WP, E, or AC. Both treatments were well tolerated. pCR on WP = 5/19(26 %) and on E = 5/30(17 %). Both regimens were equally well tolerated with no unexpected toxicities. pCR did not suggest higher activity with E than with other standard regimens in these LABC patients.

Predictions of the pathological response to neoadjuvant chemotherapy in patients with primary breast cancer using a data mining technique.

PubMed

Takada, M; Sugimoto, M; Ohno, S; Kuroi, K; Sato, N; Bando, H; Masuda, N; Iwata, H; Kondo, M; Sasano, H; Chow, L W C; Inamoto, T; Naito, Y; Tomita, M; Toi, M

2012-07-01

Nomogram, a standard technique that utilizes multiple characteristics to predict efficacy of treatment and likelihood of a specific status of an individual patient, has been used for prediction of response to neoadjuvant chemotherapy (NAC) in breast cancer patients. The aim of this study was to develop a novel computational technique to predict the pathological complete response (pCR) to NAC in primary breast cancer patients. A mathematical model using alternating decision trees, an epigone of decision tree, was developed using 28 clinicopathological variables that were retrospectively collected from patients treated with NAC (n = 150), and validated using an independent dataset from a randomized controlled trial (n = 173). The model selected 15 variables to predict the pCR with yielding area under the receiver operating characteristics curve (AUC) values of 0.766 [95 % confidence interval (CI)], 0.671-0.861, P value < 0.0001) in cross-validation using training dataset and 0.787 (95 % CI 0.716-0.858, P value < 0.0001) in the validation dataset. Among three subtypes of breast cancer, the luminal subgroup showed the best discrimination (AUC = 0.779, 95 % CI 0.641-0.917, P value = 0.0059). The developed model (AUC = 0.805, 95 % CI 0.716-0.894, P value < 0.0001) outperformed multivariate logistic regression (AUC = 0.754, 95 % CI 0.651-0.858, P value = 0.00019) of validation datasets without missing values (n = 127). Several analyses, e.g. bootstrap analysis, revealed that the developed model was insensitive to missing values and also tolerant to distribution bias among the datasets. Our model based on clinicopathological variables showed high predictive ability for pCR. This model might improve the prediction of the response to NAC in primary breast cancer patients.

Neoadjuvant therapy in the treatment of breast cancer

PubMed Central

Teshome, Mediget; Hunt, Kelly K.

2014-01-01

Synopsis Neoadjuvant systemic therapy in the treatment of breast cancer was initially employed for patients with inoperable disease. Over the past several decades this treatment approach has proved beneficial in many other patients including those with early-stage, operable breast cancer. Several randomized prospective studies have shown comparable survival rates when compared with adjuvant systemic therapy. Additionally, neoadjuvant chemotherapy can decrease the tumor burden facilitating breast conservation in selected patients without significant increases in local recurrence. Response to therapy has proven to be a strong predictor of outcome, with patients achieving pathologic complete response (pCR) demonstrating improved survival compared with those achieving less than a pCR. Furthermore, molecular subtype analysis has shown improved response following neoadjuvant chemotherapy in certain tumor types providing patients with the most aggressive subtypes a chance at cure with targeted therapies. In particular, targeting the HER2-positive subtype with trastuzumab and other HER2-directed therapies has markedly improved the outcome in these patients. Conversely, the early recognition of poor responders is important in limiting the toxicity of ineffective therapy and altering management. Neoadjuvant endocrine therapy in postmenopausal women with hormone receptor-positive tumors consistently decreases tumor size improving rates of breast conservation. Aromatase inhibitors have demonstrated superiority to tamoxifen with improved response and favorable toxicity profiles. Imaging modalities have shown promise in predicting patients with pCR, however they have not yet eliminated the need for surgical intervention. Less invasive surgical strategies such as breast conserving surgery and sentinel lymph node dissection have been shown to be safe following neoadjuvant chemotherapy in selected patients. A multidisciplinary approach with primary systemic therapy when indicated, improves the likelihood for breast conservation, provides a window into tumor biology and predicts patient outcomes. PMID:24882348

Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients.

PubMed

Yung, Tony K F; Chan, K C Allen; Mok, Tony S K; Tong, Joanna; To, Ka-Fai; Lo, Y M Dennis

2009-03-15

We aim to develop a digital PCR-based method for the quantitative detection of the two common epidermal growth factor receptor (EGFR) mutations (in-frame deletion at exon 19 and L858R at exon 21) in the plasma and tumor tissues of patients suffering from non-small cell lung cancers. These two mutations account for >85% of clinically important EGFR mutations associated with responsiveness to tyrosine kinase inhibitors. DNA samples were analyzed using a microfluidics system that simultaneously performed 9,180 PCRs at nanoliter scale. A single-mutant DNA molecule in a clinical specimen could be detected and the quantities of mutant and wild-type sequences were precisely determined. Exon 19 deletion and L858R mutation were detectable in 6 (17%) and 9 (26%) of 35 pretreatment plasma samples, respectively. When compared with the sequencing results of the tumor samples, the sensitivity and specificity of plasma EGFR mutation analysis were 92% and 100%, respectively. The plasma concentration of the mutant sequences correlated well with the clinical response. Decreased concentration was observed in all patients with partial or complete clinical remission, whereas persistence of mutation was observed in a patient with cancer progression. In one patient, tyrosine kinase inhibitor was stopped after an initial response and the tumor-associated EGFR mutation reemerged 4 weeks after stopping treatment. The sensitive detection and accurate quantification of low abundance EGFR mutations in tumor tissues and plasma by microfluidics digital PCR would be useful for predicting treatment response, monitoring disease progression and early detection of treatment failure associated with acquired drug resistance.

Assessment of an RNA interference screen-derived mitotic and ceramide pathway metagene as a predictor of response to neoadjuvant paclitaxel for primary triple-negative breast cancer: a retrospective analysis of five clinical trials.

PubMed

Juul, Nicolai; Szallasi, Zoltan; Eklund, Aron C; Li, Qiyuan; Burrell, Rebecca A; Gerlinger, Marco; Valero, Vicente; Andreopoulou, Eleni; Esteva, Francisco J; Symmans, W Fraser; Desmedt, Christine; Haibe-Kains, Benjamin; Sotiriou, Christos; Pusztai, Lajos; Swanton, Charles

2010-04-01

Addition of taxanes to preoperative chemotherapy in breast cancer increases the proportion of patients who have a pathological complete response (pCR). However, a substantial proportion of patients do not respond, and the prognosis is particularly poor for patients with oestrogen-receptor (ER)/progesterone-receptor (PR)/human epidermal growth factor receptor 2 (HER2; ERBB2)-negative (triple-negative) disease who do not achieve a pCR. Reliable identification of such patients is the first step in determining who might benefit from alternative treatment regimens in clinical trials. We previously identified genes involved in mitosis or ceramide metabolism that influenced sensitivity to paclitaxel, with an RNA interference (RNAi) screen in three cancer cell lines, including a triple-negative breast-cancer cell line. Here, we assess these genes as a predictor of pCR to paclitaxel combination chemotherapy in triple-negative breast cancer. We derived a paclitaxel response metagene based on mitotic and ceramide genes identified by functional genomics studies. We used area under the curve (AUC) analysis and multivariate logistic regression to retrospectively assess the metagene in six cohorts of patients with triple-negative breast cancer treated with neoadjuvant chemotherapy; two cohorts treated with paclitaxel (n=27, 30) and four treated without paclitaxel (n=88, 28, 48, 39). The metagene was associated with pCR in paclitaxel-treated cohorts (AUC 0.79 [95% CI 0.53-0.93], 0.72 [0.48-0.90]) but not in non-paclitaxel treated cohorts (0.53 [0.31-0.77], 0.59 [0.22-0.82], 0.53 [0.36-0.71], 0.64 [0.43-0.81]). In multivariate logistic regression, the metagene was associated with pCR (OR 19.92, 2.62-151.57; p=0.0039) with paclitaxel-containing chemotherapy. The paclitaxel response metagene shows promise as a paclitaxel-specific predictor of pCR in patients with triple-negative breast cancer. The metagene is suitable for development into a reverse transcription-PCR assay, for which clinically relevant thresholds could be established in randomised clinical trials. These results highlight the potential for functional genomics to accelerate development of drug-specific predictive biomarkers without the need for training clinical trial cohorts. UK Medical Research Council; Cancer Research UK; the National Institute for Health Research (UK); the Danish Council for Independent Research-Medical Sciences (FSS); Breast Cancer Research Foundation (New York); Fondation Luxembourgeoise contre le Cancer; the Fonds National de la Recherche Scientifique; Brussels Region (IRSIB-IP, Life Sciences 2007) and Walloon Region (Biowin-Keymarker); Sally Pearson Breast Cancer Fund; and the European Commission. 2010 Elsevier Ltd. All rights reserved.

Assessing treatment response in triple-negative breast cancer from quantitative image analysis in perfusion magnetic resonance imaging.

PubMed

Banerjee, Imon; Malladi, Sadhika; Lee, Daniela; Depeursinge, Adrien; Telli, Melinda; Lipson, Jafi; Golden, Daniel; Rubin, Daniel L

2018-01-01

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is sensitive but not specific to determining treatment response in early stage triple-negative breast cancer (TNBC) patients. We propose an efficient computerized technique for assessing treatment response, specifically the residual tumor (RT) status and pathological complete response (pCR), in response to neoadjuvant chemotherapy. The proposed approach is based on Riesz wavelet analysis of pharmacokinetic maps derived from noninvasive DCE-MRI scans, obtained before and after treatment. We compared the performance of Riesz features with the traditional gray level co-occurrence matrices and a comprehensive characterization of the lesion that includes a wide range of quantitative features (e.g., shape and boundary). We investigated a set of predictive models ([Formula: see text]) incorporating distinct combinations of quantitative characterizations and statistical models at different time points of the treatment and some area under the receiver operating characteristic curve (AUC) values we reported are above 0.8. The most efficient models are based on first-order statistics and Riesz wavelets, which predicted RT with an AUC value of 0.85 and pCR with an AUC value of 0.83, improving results reported in a previous study by [Formula: see text]. Our findings suggest that Riesz texture analysis of TNBC lesions can be considered a potential framework for optimizing TNBC patient care.

The Lcn2-engineered HEK-293 cells show senescence under stressful condition

PubMed Central

Bahmani, Bahareh; Amiri, Fatemeh; Mohammadi Roushandeh, Amaneh; Bahadori, Marzie; Harati, Mozhgan Dehghan; Habibi Roudkenar, Mehryar

2015-01-01

Objective(s): Lipocalin2 (Lcn2) gene is highly expressed in response to various types of cellular stresses. The precise role of Lcn2 has not been fully understood yet. However, it plays a key role in controlling vital cellular processes such as proliferation, apoptosis and metabolism. Recently it was shown that Lcn2 decreases senescence and increases proliferation of mesenchymal stem cells (MSC) with finite life span under either normal or oxidative stress conditions. However, Lcn2 effects on immortal cell line with infinite proliferation are not defined completely. Materials and Materials and Methods: HEK-293 cells were transfected with recombinant pcDNA3.1 containing Lcn2 fragment (pcDNA3.1-Lcn2). Expression of lipocalin2 in transfected cells was evaluated by RT-PCR, real time RT-PCR, and ELISA. Different cell groups were treated with H2O2 and WST-1 assay was performed to determine their proliferation rate. Senescence was studied by β-galactosidase and gimsa staining methods as well as evaluation of the expression of senescence-related genes by real time RT-PCR. Results: Lcn2 increased cell proliferation under normal culture condition, while the proliferation slightly decreased under oxidative stress. This decrease was further found to be attributed to senescence. Conclusion: Our findings indicated that under harmful conditions, Lcn2 gene is responsible for the regulation of cell survival through senescence. PMID:26124931

Early results of multicenter phase II trial of perioperative oxaliplatin and capecitabine without radiotherapy for high-risk rectal cancer: CORONA I study.

PubMed

Kamiya, T; Uehara, K; Nakayama, G; Ishigure, K; Kobayashi, S; Hiramatsu, K; Nakayama, H; Yamashita, K; Sakamoto, E; Tojima, Y; Kawai, S; Kodera, Y; Nagino, M

2016-06-01

Perioperative introduction of developed chemotherapy into the treatment strategy for locally advanced rectal cancer (LARC) may be a promising option. However, the most prevalent treatment for high-risk LARC remains preoperative chemoradiotherapy (CRT) in Western countries. A phase II trial was undertaken to evaluate safety and efficacy of perioperative XELOX without radiotherapy (RT) for patients with high-risk LARC. Patients received 4 cycles of XELOX before and after surgery, respectively. Primary endpoint was disease-free survival. We enrolled 41 patients between June 2012 and April 2014. The completion rate of the preoperative XELOX was 90.3%. Twenty-nine patients (70.7%) could start postoperative XELOX, 15 of these patients (51.7%) completed 4 cycles. Allergic reaction to oxaliplatin was experienced by 5 patients (17.2%) during postoperative XELOX. One patient received additional RT after preoperative XELOX. Consequently, the remaining 40 patients underwent primary resection. Major complications occurred in 6 of 40 patients (15.0%). Pathological complete response (pCR) rate was 12.2%, and good tumor regression was exhibited in 31.7%. N down-staging (cN+ to ypN0) and T down-staging were detected in 56.7% and 52.5%, respectively. Clinical T4 tumor was a predictor of poor pathological response (p < 0.001). We could show the favorable pCR rate after preoperative XELOX alone. However, the T and N down-staging rate was likely to be insufficient. When tumor regression is essential for curative resection, the use of preoperative CRT is likely to be recommended. For patients with massive LN metastasis, the additional Bev to NAC might be a promising option. Copyright © 2016 Elsevier Ltd. All rights reserved.

Heat shock fusion protein-based immunotherapy for treatment of cervical intraepithelial neoplasia III

PubMed Central

Einstein, Mark H.; Kadish, Anna S.; Burk, Robert D.; Kim, Mimi Y.; Wadler, Scott; Streicher, Howard; Goldberg, Gary L.; Runowicz, Carolyn D.

2007-01-01

Objectives SGN-00101 (HspE7, Nventa, San Diego, CA) is a novel therapeutic vaccine consisting of a fusion protein containing an M. bovis BCG heat shock protein (Hsp65) covalently linked to the entire sequence of HPV 16 E7. This trial was designed to evaluate the efficacy and toxicities of HspE7 in women with CIN III. Methods HIV (−) women with biopsy-proven CIN III were eligible. Two cohorts were accrued; one cohort to establish efficacy and a second cohort with a longer follow-up period to improve the precision of the trial to estimate response rates. Each patient underwent 3 monthly subcutaneous vaccinations with 500 µg of HspE7 followed by monthly colposcopic follow-up for 1 month in cohort 1 and an extended observation period (2 months) in cohort 2. All patients then underwent a LEEP or cone biopsy of the cervix. A complete pathologic response (pCR) was defined as no evidence of CIN or CIN I (only HPV changes). A partial response (PR) was defined as colposcopic lesion regression of >50% in size. Cervicovaginal lavage samples were obtained at each visit for HPV typing using MY09/ MY11 HPV PCR. Results Seventy-two patients were registered and screened, of whom 64 were eligible. Fifty-eight patients completed the trial and were evaluable (31 in cohort 1, 27 in cohort 2). There were no significant epidemiologic or HPV type differences between the 2 cohorts so responses were combined for analysis. Of the 58 evaluable patients, 13 (22.5%) had a pCR; 32 (55%) had a PR and 11 (19%) had stable disease. Two (3.5%) patients in cohort 2 had microinvasive disease and were defined as progressive disease. Thirty-three of 58 (57%) of the patients were infected with HPV 16 prior to vaccination or in subsequent visits. There was no significant difference in regression in women infected with HPV 16 compared to those without HPV 16 infection (88% vs. 70%; p=0.12). Women who had a previous LEEP or ablation for CIN were 2.7 times more likely to have a complete response compared to patients without previous treatment, although the difference was not statistically significant (95% CI for rate ratio: 0.95–6.19, p=0.10). At a cellular level, there was a significant association between local inflammation and response; lower grade of lesional inflammation correlated with a response to HspE7 (p=0.04 using Wilcoxon rank sum test). Conclusions HspE7 appeared to demonstrate activity in women with CIN III and met a priori assumptions for efficacy; however, it is unclear whether this response was due to natural regression rather than treatment effect. HspE7, which targets the HPV 16 E7 oncoprotein, had efficacy in patients infected with HPV types other than 16, suggesting cross-reactivity. A larger randomized, controlled trial is needed to better define efficacy and to identify subsets of women most likely to benefit from immunotherapy. PMID:17586030

The Expression of Checkpoint and DNA Repair Genes in Head and Neck Cancer as Possible Predictive Factors.

PubMed

Rusz, Orsolya; Pál, Margit; Szilágyi, Éva; Rovó, László; Varga, Zoltán; Tomisa, Bernadett; Fábián, Gabriella; Kovács, Levente; Nagy, Olga; Mózes, Petra; Reisz, Zita; Tiszlavicz, László; Deák, Péter; Kahán, Zsuzsanna

2017-04-01

DNA damage response failure may influence the efficacy of DNA-damaging treatments. We determined the expression of 16 genes involved in distinct DNA damage response pathways, in association with the response to standard therapy. Twenty patients with locoregionally advanced, squamous cell head and neck carcinoma were enrolled. The treatment included induction chemotherapy (iChT) with docetaxel, cisplatin and 5-fluorouracil followed by concomitant chemoradiotherapy (ChRT) or radiotherapy (RT) alone. The volumetric metabolic therapeutic response was determined by [18F]FDG-PET/CT. In the tumor and matched normal tissues collected before treatment, the gene expressions were examined via the quantitative real-time polymerase chain reaction (qRT-PCR). The down-regulation of TP53 was apparently associated with a poor response to iChT, its up-regulation with complete regression in 2 cases. 7 cases with down-regulated REV1 expression showed complete regression after ChRT/RT, while 1 case with REV1 overexpression was resistant to RT. The overexpression of WRN was an independent predictor of tumor relapse. Our results suggest that an altered expression of REV1 predicts sensitivity to RT, while WRN overexpression is an unfavorable prognostic factor.

The Role of Neoadjuvant Trials in Drug Development for Solid Tumors.

PubMed

Funt, Samuel A; Chapman, Paul B

2016-05-15

The relatively low success rate of phase II oncology trials in predicting success of novel drugs in phase III trials and in gaining regulatory approval may be due to reliance on the endpoint of response rate defined by the RECIST. The neoadjuvant treatment paradigm allows the antitumor activity of a novel therapy to be determined on a pathologic basis at the time of surgery instead of by RECIST, which was not developed to guide clinical decision making or correlate with long-term outcomes. Indeed, the FDA endorsed pathologic complete response (pCR) as a surrogate for overall survival (OS) in early-stage breast cancer and granted accelerated approval to pertuzumab based on this endpoint. We propose that pCR is a biologically rational method of determining treatment effect that may be more likely to predict OS. We discuss some advantages of the neoadjuvant trial design, review the use of neoadjuvant therapy as standards of care, and consider the neoadjuvant platform as a method for drug development. Clin Cancer Res; 22(10); 2323-8. ©2016 AACR. ©2016 American Association for Cancer Research.

Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies.

PubMed

Azuara, Daniel; Santos, Cristina; Lopez-Doriga, Adriana; Grasselli, Julieta; Nadal, Marga; Sanjuan, Xavier; Marin, Fátima; Vidal, Joana; Montal, Robert; Moreno, Victor; Bellosillo, Beatriz; Argiles, Guillem; Elez, Elena; Dienstmann, Rodrigo; Montagut, Clara; Tabernero, Josep; Capellá, Gabriel; Salazar, Ramon

2016-05-01

The clinical significance of low-frequent RAS pathway-mutated alleles and the optimal sensitivity cutoff value in the prediction of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients remains controversial. We aimed to evaluate the added value of genotyping an extended RAS panel using a robust nanofluidic digital PCR (dPCR) approach. A panel of 34 hotspots, including RAS (KRAS and NRAS exons 2/3/4) and BRAF (V600E), was analyzed in tumor FFPE samples from 102 mCRC patients treated with anti-EGFR therapy. dPCR was compared with conventional quantitative PCR (qPCR). Response rates, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutated allele fraction. Tumor response evaluations were not available in 9 patients and were excluded for response rate analysis. Twenty-two percent of patients were positive for one mutation with qPCR (mutated alleles ranged from 2.1% to 66.6%). Analysis by dPCR increased the number of positive patients to 47%. Mutated alleles for patients only detected by dPCR ranged from 0.04% to 10.8%. An inverse correlation between the fraction of mutated alleles and radiologic response was observed. ROC analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value for all combinations of RAS and BRAF analysis. In addition, this threshold also optimized prediction both PFS and OS. We conclude that mutation testing using an extended gene panel, including RAS and BRAF with a threshold of 1% improved prediction of response to anti-EGFR therapy. Mol Cancer Ther; 15(5); 1106-12. ©2016 AACR. ©2016 American Association for Cancer Research.

COLD-PCR Technologies in the Area of Personalized Medicine: Methodology and Applications.

PubMed

Mauger, Florence; How-Kit, Alexandre; Tost, Jörg

2017-06-01

Somatic mutations bear great promise for use as biomarkers for personalized medicine, but are often present only in low abundance in biological material and are therefore difficult to detect. Many assays for mutation analysis in cancer-related genes (hotspots) have been developed to improve diagnosis, prognosis, prediction of drug resistance, and monitoring of the response to treatment. Two major approaches have been developed: mutation-specific amplification methods and methods that enrich and detect mutations without prior knowledge on the exact location and identity of the mutation. CO-amplification at Lower Denaturation temperature Polymerase Chain Reaction (COLD-PCR) methods such as full-, fast-, ice- (improved and complete enrichment), enhanced-ice, and temperature-tolerant COLD-PCR make use of a critical temperature in the polymerase chain reaction to selectively denature wild-type-mutant heteroduplexes, allowing the enrichment of rare mutations. Mutations can subsequently be identified using a variety of laboratory technologies such as high-resolution melting, digital polymerase chain reaction, pyrosequencing, Sanger sequencing, or next-generation sequencing. COLD-PCR methods are sensitive, specific, and accurate if appropriately optimized and have a short time to results. A large variety of clinical samples (tumor DNA, circulating cell-free DNA, circulating cell-free fetal DNA, and circulating tumor cells) have been studied using COLD-PCR in many different applications including the detection of genetic changes in cancer and infectious diseases, non-invasive prenatal diagnosis, detection of microorganisms, or DNA methylation analysis. In this review, we describe in detail the different COLD-PCR approaches, highlighting their specificities, advantages, and inconveniences and demonstrating their use in different fields of biological and biomedical research.

Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

DOEpatents

Wong, Kwong-Kwok

2000-01-01

The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

Commentary on "Prognostic effect of carcinoma in situ in muscle-invasive urothelial carcinoma patients receiving neoadjuvant chemotherapy."

PubMed

Thomas, D E; Kaimakliotis, H Z; Rice, K R; Pereira, J A; Johnston, P; Moore, M L; Reed, A; Cregar, D M; Franklin, C; Loman, R L; Koch, M O; Bihrle, R; Foster, R S; Masterson, T A; Gardner, T A; Sundaram, C P; Powell, C R; Beck, Sdw; Grignon, D J; Cheng, L; Albany, C; Hahn, N M

2018-07-01

Carcinoma in situ (CIS) is a poor prognostic finding in urothelial carcinoma. However, its significance in muscle-invasive urothelial carcinoma (MIUC) treated with neoadjuvant chemotherapy (NAC) is uncertain. We assessed the effect of CIS found in pretreatment transurethral resection of bladder tumor (TURBT) biopsies on the pathologic and clinical outcomes. Subjects with MIUC treated with NAC before cystectomy were identified. The pathologic complete response (pCR) rates stratified by TURBT CIS status were compared. The secondary analyses included tumor response, progression-free survival (PFS), overall survival (OS), and an exploratory post hoc analysis of patients with pathologic CIS only (pTisN0) at cystectomy. A total of 137 patients with MIUC were identified. TURBT CIS was noted in 30.7% of the patients. The absence of TURBT CIS was associated with a significantly increased pCR rate (23.2% vs. 9.5%; odds ratio = 4.08; 95% CI: 1.19-13.98; P = 0.025). Stage pTisN0 disease was observed in 19.0% of the TURBT CIS patients. TURBT CIS status did not significantly affect the PFS or OS outcomes. Post hoc analysis of the pTisN0 patients revealed prolonged median PFS (104.5 vs. 139.9 months; P = 0.055) and OS (104.5 vs. 152.3 months; P = 0.091) outcomes similar to those for the pCR patients. The absence of CIS on pretreatment TURBT in patients with MIUC undergoing NAC was associated with increased pCR rates, with no observed differences in PFS or OS. Isolated CIS at cystectomy was frequently observed, with lengthy PFS and OS durations similar to those for pCR patients. Further studies aimed at understanding the biology and clinical effect of CIS in MIUC are warranted. Copyright © 2018 Elsevier Inc. All rights reserved.

Prognostic Effect of Carcinoma In Situ in Muscle-invasive Urothelial Carcinoma Patients Receiving Neoadjuvant Chemotherapy.

PubMed

Thomas, Derek E; Kaimakliotis, Hristos Z; Rice, Kevin R; Pereira, Jose A; Johnston, Paul; Moore, Marietta L; Reed, Angela; Cregar, Dylan M; Franklin, Cindy; Loman, Rhoda L; Koch, Michael O; Bihrle, Richard; Foster, Richard S; Masterson, Timothy A; Gardner, Thomas A; Sundaram, Chandru P; Powell, Charles R; Beck, Stephen D W; Grignon, David J; Cheng, Liang; Albany, Costantine; Hahn, Noah M

2017-08-01

Carcinoma in situ (CIS) is a poor prognostic finding in urothelial carcinoma. However, its significance in muscle-invasive urothelial carcinoma (MIUC) treated with neoadjuvant chemotherapy (NAC) is uncertain. We assessed the effect of CIS found in pretreatment transurethral resection of bladder tumor (TURBT) biopsies on the pathologic and clinical outcomes. Subjects with MIUC treated with NAC before cystectomy were identified. The pathologic complete response (pCR) rates stratified by TURBT CIS status were compared. The secondary analyses included tumor response, progression-free survival (PFS), overall survival (OS), and an exploratory post hoc analysis of patients with pathologic CIS only (pTisN0) at cystectomy. A total of 137 patients with MIUC were identified. TURBT CIS was noted in 30.7% of the patients. The absence of TURBT CIS was associated with a significantly increased pCR rate (23.2% vs. 9.5%; odds ratio, 4.08; 95% confidence interval, 1.19-13.98; P = .025). Stage pTisN0 disease was observed in 19.0% of the TURBT CIS patients. TURBT CIS status did not significantly affect the PFS or OS outcomes. Post hoc analysis of the pTisN0 patients revealed prolonged median PFS (104.5 vs. 139.9 months; P = .055) and OS (104.5 vs. 152.3 months; P = .091) outcomes similar to those for the pCR patients. The absence of CIS on pretreatment TURBT in patients with MIUC undergoing NAC was associated with increased pCR rates, with no observed differences in PFS or OS. Isolated CIS at cystectomy was frequently observed, with lengthy PFS and OS durations similar to those for pCR patients. Further studies aimed at understanding the biology and clinical effect of CIS in MIUC are warranted. Copyright © 2016 Elsevier Inc. All rights reserved.

A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.

PubMed

Junqueira, Bruna Rayane Teodoro; Nicolini, Cícero; Lucinda, Natalia; Orílio, Anelise Franco; Nagata, Tatsuya

2014-03-01

Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants. Copyright © 2013 Elsevier B.V. All rights reserved.

The Impact of Residual Disease After Preoperative Systemic Therapy on Clinical Outcomes in Patients with Inflammatory Breast Cancer.

PubMed

Nakhlis, Faina; Regan, Meredith M; Warren, Laura E; Bellon, Jennifer R; Hirshfield-Bartek, Judith; Duggan, Margaret M; Dominici, Laura S; Golshan, Mehra; Jacene, Heather A; Yeh, Eren D; Mullaney, Erin E; Overmoyer, Beth

2017-09-01

Inflammatory breast cancer (IBC) is a rare and aggressive disease treated with multimodality therapy: preoperative systemic therapy (PST) followed by modified radical mastectomy (MRM), chest wall and regional nodal radiotherapy, and adjuvant biologic therapy and/or endocrine therapy when appropriate. In non-IBC, the degree of pathologic response to PST has been shown to correlate with time to recurrence (TTR) and overall survival (OS). We sought to determine if pathologic response correlates with oncologic outcomes of IBC patients. Following review of IBC patients' records (1997-2014), we identified 258 stage III IBC patients; 181 received PST followed by MRM and radiotherapy and were subsequently analyzed. Pathologic complete response (pCR) to PST, hormone receptor and human epidermal growth factor receptor 2 (HER2) status, grade, and histology were evaluated as predictors of TTR and OS by Cox model. Overall, 95/181 (52%) patients experienced recurrence; 93/95 (98%) were distant metastases (median TTR 3.2 years). Seventy-three patients (40%) died (median OS 6.9 years). pCR was associated with improved TTR (hazard ratio [HR] 0.20, 95% confidence interval [CI] 0.09-0.46, p < 0.01, univariate; HR 0.17, 95% CI 0.07-0.41, p < 0.0001, multivariate) and improved OS (HR 0.26, 95% CI 0.11-0.65, p < 0.01, univariate). In patients with pCR, grade III (HR 1.91, 95% CI 1.16-3.13, p = 0.01), and triple-negative phenotype (HR 3.54, 95% CI 1.79-6.98, p = 0.0003) were associated with shorter TTR, while residual ductal carcinoma in situ was not (HR 0.85, 95% CI 0.53-1.35, p = 0.48, multivariate). In stage III IBC, pCR was associated with prognosis, further influenced by grade, hormone receptor, and HER2 status. Investigating mechanisms that contribute to better response to PST could help improve oncologic outcomes in IBC.

Patient-Specific Circulating Tumor DNA Detection during Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer.

PubMed

Riva, Francesca; Bidard, Francois-Clement; Houy, Alexandre; Saliou, Adrien; Madic, Jordan; Rampanou, Aurore; Hego, Caroline; Milder, Maud; Cottu, Paul; Sablin, Marie-Paule; Vincent-Salomon, Anne; Lantz, Olivier; Stern, Marc-Henri; Proudhon, Charlotte; Pierga, Jean-Yves

2017-03-01

In nonmetastatic triple-negative breast cancer (TNBC) patients, we investigated whether circulating tumor DNA (ctDNA) detection can reflect the tumor response to neoadjuvant chemotherapy (NCT) and detect minimal residual disease after surgery. Ten milliliters of plasma were collected at 4 time points: before NCT; after 1 cycle; before surgery; after surgery. Customized droplet digital PCR (ddPCR) assays were used to track tumor protein p53 ( TP53 ) mutations previously characterized in tumor tissue by massively parallel sequencing (MPS). Forty-six patients with nonmetastatic TNBC were enrolled. TP53 mutations were identified in 40 of them. Customized ddPCR probes were validated for 38 patients, with excellent correlation with MPS ( r = 0.99), specificity (≥2 droplets/assay), and sensitivity (at least 0.1%). At baseline, ctDNA was detected in 27/36 patients (75%). Its detection was associated with mitotic index ( P = 0.003), tumor grade ( P = 0.003), and stage ( P = 0.03). During treatment, we observed a drop of ctDNA levels in all patients but 1. No patient had detectable ctDNA after surgery. The patient with rising ctDNA levels experienced tumor progression during NCT. Pathological complete response (16/38 patients) was not correlated with ctDNA detection at any time point. ctDNA positivity after 1 cycle of NCT was correlated with shorter disease-free ( P < 0.001) and overall ( P = 0.006) survival. Customized ctDNA detection by ddPCR achieved a 75% detection rate at baseline. During NCT, ctDNA levels decreased quickly and minimal residual disease was not detected after surgery. However, a slow decrease of ctDNA level during NCT was strongly associated with shorter survival. © 2016 American Association for Clinical Chemistry.

Clinical applicability and prognostic significance of molecular response assessed by fluorescent-PCR of immunoglobulin genes in multiple myeloma. Results from a GEM/PETHEMA study.

PubMed

Martinez-Lopez, Joaquin; Fernández-Redondo, Elena; García-Sánz, Ramón; Montalbán, María Angeles; Martínez-Sánchez, Pilar; Pavia, Bruno; Mateos, María Victoria; Rosiñol, Laura; Martín, Marisa; Ayala, Rosa; Martínez, Rafael; Blanchard, María Jesus; Alegre, Adrian; Besalduch, Joan; Bargay, Joan; Hernandez, Miguel T; Sarasquete, María Eugenia; Sanchez-Godoy, Pedro; Fernández, Manuela; Blade, Joan; San Miguel, Jesús F; Lahuerta, Juan Jose

2013-12-01

Minimal residual disease monitoring is becoming increasingly important in multiple myeloma (MM), but multiparameter flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) techniques are not routinely available. This study investigated the prognostic influence of achieving molecular response assessed by fluorescent-PCR (F-PCR) in 130 newly diagnosed MM patients from Grupo Español Multidisciplinar de Melanoma (GEM)2000/GEM05 trials (NCT00560053, NCT00443235, NCT00464217) who achieved almost very good partial response after induction therapy. As a reference, we used the results observed with simultaneous MFC. F-PCR at diagnosis was performed on DNA using three different multiplex PCRs: IGH D-J, IGK V-J and KDE rearrangements. The applicability of F-PCR was 91·5%. After induction therapy, 64 patients achieved molecular response and 66 non-molecular response; median progression-free survival (PFS) was 61 versus 36 months, respectively (P = 0·001). Median overall survival (OS) was not reached (NR) in molecular response patients (5-year survival: 75%) versus 66 months in the non-molecular response group (P = 0·03). The corresponding PFS and OS values for patients with immunophenotypic versus non-immunophenotypic response were 67 versus 42 months (P = 0·005) and NR (5-year survival: 95%) versus 69 months (P = 0·004), respectively. F-PCR analysis is a rapid, affordable, and easily performable technique that, in some circumstances, may be a valid approach for minimal residual disease investigations in MM. © 2013 John Wiley & Sons Ltd.

Recommendations for standardized pathological characterization of residual disease for neoadjuvant clinical trials of breast cancer by the BIG-NABCG collaboration.

PubMed

Bossuyt, V; Provenzano, E; Symmans, W F; Boughey, J C; Coles, C; Curigliano, G; Dixon, J M; Esserman, L J; Fastner, G; Kuehn, T; Peintinger, F; von Minckwitz, G; White, J; Yang, W; Badve, S; Denkert, C; MacGrogan, G; Penault-Llorca, F; Viale, G; Cameron, D

2015-07-01

Neoadjuvant systemic therapy (NAST) provides the unique opportunity to assess response to treatment after months rather than years of follow-up. However, significant variability exists in methods of pathologic assessment of response to NAST, and thus its interpretation for subsequent clinical decisions. Our international multidisciplinary working group was convened by the Breast International Group-North American Breast Cancer Group (BIG-NABCG) collaboration and tasked to recommend practical methods for standardized evaluation of the post-NAST surgical breast cancer specimen for clinical trials that promote accurate and reliable designation of pathologic complete response (pCR) and meaningful characterization of residual disease. Recommendations include multidisciplinary communication; clinical marking of the tumor site (clips); and radiologic, photographic, or pictorial imaging of the sliced specimen, to map the tissue sections and reconcile macroscopic and microscopic findings. The information required to define pCR (ypT0/is ypN0 or ypT0 yp N0), residual ypT and ypN stage using the current AJCC/UICC system, and the Residual Cancer Burden system were recommended for quantification of residual disease in clinical trials. © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Laser micro-dissection and qPCR for identifying specific HPV types responsible for malignancy in penile lesions.

PubMed

Lebelo, Ramokone L; Thys, Sofie; Benoy, Ina; Depuydt, Christophe E; Bogers, John-Paul; Bida, Meshack N; Mphahlele, M Jeffrey

2015-10-01

The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections. © 2015 Wiley Periodicals, Inc.

Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing.

PubMed

Banjo, Masaya; Iguchi, Atsushi; Seto, Kazuko; Kikuchi, Taisei; Harada, Tetsuya; Scheutz, Flemming; Iyoda, Sunao

2018-06-01

In Escherichia coli , more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system, E. coli H-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes, fliC and its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. The E. coli H-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenic E. coli . Copyright © 2018 American Society for Microbiology.

SU-F-R-48: Early Prediction of Pathological Response of Locally Advanced Rectal Cancer Using Perfusion CT:A Prospective Clinical Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nie, K; Yue, N; Jabbour, S

Purpose: To prospectively evaluate the tumor vascularity assessed by perfusion CT for prediction of chemo-radiation treatment (CRT) response in locally advanced rectal cancer (LARC). Methods: Eighteen consecutive patients (61.9±8.8 years, from March–June 2015) diagnosed with LARC who underwent 6–8 weeks CRT followed by surgery were included. The pre-treatment perfusion CT was acquired after a 5s delay of contrast agent injection for 45s with 1s interval. A total of 7-cm craniocaudal range covered the tumor region with 3-mm slice thickness. The effective radiation dose is around 15mSv, which is about 1.5 the conventional abdomen/pelvis CT dose. The parametric map of bloodmore » flow (BF), blood volume (BV), mean transit time (MTT), permeability (PMB), and maximum intensity map (MIP) were obtained from commercial software (Syngo-CT 2011A, Siemens). An experienced radiation oncologist outlined the tumor based on the pre-operative MR and pathologic residual region, but was blinded with regards to pathological tumor stage. The perfusion parameters were compared to histopathological response quantified by tumor regression grade as good-responder (GR, TRG 0-1) vs. non-good responder (non-GR). Furthermore, the predictive value for pathological complete response (pCR) was also investigated. Results: Both BV (p=0.02) and MTT (P=0.02) was significantly higher and permeambility was lower (p=0.004) in the good responders. The BF was higher in GR group but not statistically significant. Regarding the discrimination of pCR vs non-pCR, the BF was higher in the pCR group (p=0.08) but none of those parameters showed statistically significant differences. Conclusion: BV and MTT can discriminate patients with a favorable response from those that fail to respond well, potentially selecting high-risk patients with resistant tumors that may benefit from an aggressive preoperative treatment approach. However, future studies with more patient data are needed to verify the prognostic value of perfusion CT especially for pCR prediction. This work is supported by the National High-tech R&D program for Young Scientists by the Ministry of Science and Technology of China (Grant No. 2015AA020917), Natural Science Foundation of China (NSFC Grant No. 81201091).« less

A single tube PCR assay for simultaneous amplification of HSV-1/-2, VZV, CMV, HHV-6A/-6B, and EBV DNAs in cerebrospinal fluid from patients with virus-related neurological diseases.

PubMed

Yamamoto, T; Nakamura, Y

2000-10-01

Cerebrospinal fluid (CSF) specimens from 27 patients with encephalitis, meningitis, and other neurological diseases were studied for the presence of herpes simplex virus types 1 and 2 (HSV-1/-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), human herpesviruses 6A and 6B (HHV-6A/-6B) and Epstein-Barr virus (EBV) DNA using the polymerase chain reaction (PCR) method. The DNAs were amplified using two sets of consensus primer pairs in a single tube, bringing simultaneous amplification of the herpesviruses. The PCR products were analyzed by agarose gel electrophoresis, and Southern blot hybridization with virus-type specific probes, thus allowing discrimination between the different types of herpesviruses to be made. Each virus-specific probe was highly specific for identifying the PCR product. Thirty CSF specimens from 13 patients with encephalitis and 10 specimens from 10 patients with meningitis, respectively, were examined using this method. Eight patients with encephalitis and six with meningitis were positive for different herpesviruses, including patients with coinfections (HSV-1/-2 and VZV, VZV and CMV). Among four CSF specimens from four patients with other neurological disorders, dual amplification of CMV and EBV was present. Since identification of the types of herpesviruses in this system requires a very small amount of CSF, and is completed with one PCR, it is useful for routine diagnosis of herpesvirus infections in diagnostic laboratories. The viruses responsible for central nervous system infection are easily detected with various coinfection and serial patterns of herpesviruses, by this consensus primer-based PCR method. This may give an insight into the relationship between virus-related neurological diseases (VRNDS) and herpesvirus infections.

Antibacterial activity and mechanism of action of ε-poly-L-lysine.

PubMed

Ye, Ruosong; Xu, Hengyi; Wan, Cuixiang; Peng, Shanshan; Wang, Lijun; Xu, Hong; Aguilar, Zoraida P; Xiong, Yonghua; Zeng, Zheling; Wei, Hua

2013-09-13

ε-Poly-L-lysine (ε-PL)(2) is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS)(3) levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR)(4) indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response)(5) regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence. Copyright © 2013. Published by Elsevier Inc.

Prognostic value of tumour regression grade in locally advanced rectal cancer: a systematic review and meta-analysis.

PubMed

Kong, J C; Guerra, G R; Warrier, S K; Lynch, A Craig; Michael, M; Ngan, S Y; Phillips, W; Ramsay, G; Heriot, A G

2018-03-27

The current standard of care for locally advanced rectal cancer involves neoadjuvant chemoradiotherapy (CRT) followed by total mesorectal excision. There is a spectrum of response to neoadjuvant therapy; however, the prognostic value of tumour regression grade (TRG) in predicting disease-free survival (DFS) or overall survival (OS) is inconsistent in the literature. This study was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A systematic search was undertaken using Ovid MEDLINE, Embase and Google Scholar. Inclusion criteria were Stage II and III locally advanced rectal cancer treated with long-course CRT followed by radical surgery. The aim of the meta-analysis was to assess the prognostic implication of each TRG for rectal cancer following neoadjuvant CRT. Long-term prognosis was assessed. The main outcome measures were DFS and OS. A random effects model was performed to pool the hazard ratio (HR) from all included studies. There were 4875 patients from 17 studies, with 775 (15.9%) attaining a pathological complete response (pCR) and 719 (29.9%) with no response. A significant association with OS was identified from a pooled-estimated HR for pCR (HR = 0.47, P = 0.002) and nonresponding tumours (HR = 2.97; P < 0.001). Previously known tumour characteristics, such as ypN, lymphovascular invasion and perineural invasion, were also significantly associated with DFS and OS, with estimated pooled HRs of 2.2, 1.4 and 2.3, respectively. In conclusion, the degree of TRG was of prognostic value in predicting long-term outcomes. The current challenge is the development of a high-validity tests to predict pCR. Colorectal Disease © 2018 The Association of Coloproctology of Great Britain and Ireland.

TGFbeta1 (Leu10Pro), p53 (Arg72Pro) can predict for increased risk for breast cancer in south Indian women and TGFbeta1 Pro (Leu10Pro) allele predicts response to neo-adjuvant chemo-radiotherapy.

PubMed

Rajkumar, Thangarajan; Samson, Mani; Rama, Ranganathan; Sridevi, Veluswami; Mahji, Urmila; Swaminathan, Rajaraman; Nancy, Nirmala K

2008-11-01

The breast cancer incidence has been increasing in the south Indian women. A case (n=250)-control (n=500) study was undertaken to investigate the role of Single Nucleotide Polymorphisms (SNP's) in GSTM1 (Present/Null); GSTP1 (Ile105Val), p53 (Arg72Pro), TGFbeta1 (Leu10Pro), c-erbB2 (Ile655Val), and GSTT1 (Null/Present) in breast cancer. In addition, the value of the SNP's in predicting primary tumor's pathologic response following neo-adjuvant chemo-radiotherapy was assessed. Genotyping was done using PCR (GSTM1, GSTT1), Taqman Allelic discrimination assay (GSTP1, c-erbB2) and PCR-CTPP (p53 and TGFbeta1). None of the gene SNP's studied were associated with a statistically significant increased risk for the breast cancer. However, combined analysis of the SNP's showed that p53 (Arg/Arg and Arg/Pro) with TGFbeta1 (Pro/Pro and Leu/Pro) were associated with greater than 2 fold increased risk for breast cancer in Univariate (P=0.01) and Multivariate (P=0.003) analysis. There was no statistically significant association for the GST family members with the breast cancer risk. TGFbeta1 (Pro/Pro) allele was found to predict complete pathologic response in the primary tumour following neo-adjuvant chemo-radiotherapy (OR=6.53 and 10.53 in Univariate and Multivariate analysis respectively) (P=0.004) and was independent of stage. This study suggests that SNP's can help predict breast cancer risk in south Indian women and that TGFbeta1 (Pro/Pro) allele is associated with a better pCR in the primary tumour.

Use of sequential endorectal US to predict the tumor response of preoperative chemoradiotherapy in rectal cancer.

PubMed

Li, Ning; Dou, Lizhou; Zhang, Yueming; Jin, Jing; Wang, Guiqi; Xiao, Qin; Li, Yexiong; Wang, Xin; Ren, Hua; Fang, Hui; Wang, Weihu; Wang, Shulian; Liu, Yueping; Song, Yongwen

2017-03-01

Accurate prediction of the response to preoperative chemoradiotherapy (CRT) potentially assists in the individualized selection of treatment. Endorectal US (ERUS) is widely used for the pretreatment staging of rectal cancer, but its use for preoperatively predicting the effects of CRT is not well evaluated because of the inflammation, necrosis, and fibrosis induced by CRT. This study assessed the value of sequential ERUS in predicting the efficacy of preoperative CRT for locally advanced rectal cancer. Forty-one patients with clinical stage II/III rectal adenocarcinoma were enrolled prospectively. Radiotherapy was delivered to the pelvis with concurrent chemotherapy of capecitabine and oxaliplatin. Total mesorectal excision was performed 6 to 8 weeks later. EUS measurements of primary tumor maximum diameter were performed before (ERUS1), during (ERUS2), and 6 to 8 weeks after (ERUS3) CRT, and the ratios of these were calculated. Correlations between ERUS values, tumor regression grade (TRG), T down-staging rate, and pathologic complete response (pCR) rate were assessed, and survival was analyzed. There was no significant correlation between ERUS2/ERUS1 and TRG. The value of ERUS3/ERUS1 correlated with pCR rate and TRG but not T down-staging rate. An ERUS3 value of 6.3 mm and ERUS3/ERUS1 of 52% were used as the cut-off for predicting pCR, and patients were divided into good and poor prognosis groups. Although not statistically significant, 3-year recurrence and survival rates of the good prognosis group were better than those of the poor prognosis group. Sequential ERUS may predict therapeutic efficacy of preoperative CRT for locally advanced rectal cancer. (Clinical trial registration number: NCT01582750.). Copyright © 2017 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

cDNA-AFLP analysis reveals differential gene expression in response to salt stress in foxtail millet (Setaria italica L.).

PubMed

Jayaraman, Ananthi; Puranik, Swati; Rai, Neeraj Kumar; Vidapu, Sudhakar; Sahu, Pranav Pankaj; Lata, Charu; Prasad, Manoj

2008-11-01

Plant growth and productivity are affected by various abiotic stresses such as heat, drought, cold, salinity, etc. The mechanism of salt tolerance is one of the most important subjects in plant science as salt stress decreases worldwide agricultural production. In our present study we used cDNA-AFLP technique to compare gene expression profiles of a salt tolerant and a salt-sensitive cultivar of foxtail millet (Seteria italica) in response to salt stress to identify early responsive differentially expressed transcripts accumulated upon salt stress and validate the obtained result through quantitative real-time PCR (qRT-PCR). The expression profile was compared between a salt tolerant (Prasad) and susceptible variety (Lepakshi) of foxtail millet in both control condition (L0 and P0) and after 1 h (L1 and P1) of salt stress. We identified 90 transcript-derived fragments (TDFs) that are differentially expressed, out of which 86 TDFs were classified on the basis of their either complete presence or absence (qualitative variants) and 4 on differential expression pattern levels (quantitative variants) in the two varieties. Finally, we identified 27 non-redundant differentially expressed cDNAs that are unique to salt tolerant variety which represent different groups of genes involved in metabolism, cellular transport, cell signaling, transcriptional regulation, mRNA splicing, seed development and storage, etc. The expression patterns of seven out of nine such genes showed a significant increase of differential expression in tolerant variety after 1 h of salt stress in comparison to salt-sensitive variety as analyzed by qRT-PCR. The direct and indirect relationship of identified TDFs with salinity tolerance mechanism is discussed.

Enzymatic and viability RT-qPCR assays for evaluation of enterovirus, hepatitis A virus and norovirus inactivation: Implications for public health risk assessment.

PubMed

Monteiro, S; Santos, R

2018-04-01

To assess the potential of a viability dye and an enzymatic reverse transcription quantitative PCR (RT-qPCR) pretreatment to discriminate between infectious and noninfectious enteric viruses. Enterovirus (EntV), norovirus (NoV) GII.4 and hepatitis A virus (HAV) were inactivated at 95°C for 10 min, and four methods were used to compare the efficiency of inactivation: (i) cell culture plaque assay for HAV and EntV, (ii) RT-qPCR alone, (iii) RT-qPCR assay preceded by RNase treatment, and (iv) pretreatment with a viability dye (reagent D (RD)) followed by RT-qPCR. In addition, heat-inactivated NoV was treated with RD coupled with surfactants to increase the efficiency of the viability dye. No treatment was able to completely discriminate infectious from noninfectious viruses. RD-RT-qPCR reduced more efficiently the detection of noninfectious viruses with little to no removal observed with RNase. RD-RT-qPCR method was the closest to cell culture assay. The combination of surfactants and RD did not show relevant improvements on the removal of inactivated viruses signal compared with viability RT-qPCR, with the exception of Triton X-100. The use of surfactant/RD-RT-qPCR, although not being able to completely remove the signal from noninfectious viral particles, yielded a better estimation of viral infectivity. Surfactant/RD-RT-qPCR may be an advantageous tool for a better detection of infectious viruses with potential significant impact in the risk assessment of the presence of enteric viruses. © 2017 The Society for Applied Microbiology.

Pentostatin and Rituximab Therapy for Previously Untreated B-CLL

PubMed Central

Kay, Neil E.; Wu, Wenting; Kabat, Brian; LaPlant, Betsy; Lin, Thomas S.; Byrd, John C.; Jelinek, Diane F.; Grever, Michael R.; Zent, Clive S.; Call, Timothy G.; Shanafelt, Tait D.

2010-01-01

We have shown that the combination of pentostatin (P), cyclophosphamide (C) and rituximab (R) achieves an overall response (OR) rate >90% with more than 40% complete responses (CR) in patients with untreated CLL. To evaluate if the tolerability of this regimen could be enhanced without sacrificing efficacy, we conducted a phase II trial of P and R without cyclophosphamide, using a higher P dose (4 mg/m2). Among the 33 patients enrolled, 82% were male, median age was 65 (9 patients ≥70 years) and 64% were Rai stage III-IV. The OR rate was 76% with 9 CR (27%), 5 nPR, and 11 PRs. At the time of this analysis, 29/33 patients are still alive and the median follow up for patients still alive is 14 months (range: 1-34.8 months). Four (12%) patients experienced grade 3 or higher hematologic toxicity and 5 (15%) experienced grade 3 or higher non-hematologic toxicity. Comparison of this trial to our previous PCR trial showed that patients treated with PCR had a higher OR rate (91% vs. 76%) and CR rate (41% vs. 27%) compared to patients treated with PR. Median treatment-free survival for all accrued patients was notably longer in PCR treated patients compared to PR (30 vs. 16 months). These findings suggest that increasing the dose of the purine nucleoside analogue does not eliminate the need for cyclophosphamide in chemoimmunotherapy for treatment of CLL. PMID:20187101

Pandemic preparedness in Hawaii: a multicenter verification of real-time RT-PCR for the direct detection of influenza virus types A and B.

PubMed

Whelen, A Christian; Bankowski, Matthew J; Furuya, Glenn; Honda, Stacey; Ueki, Robert; Chan, Amelia; Higa, Karen; Kumashiro, Diane; Moore, Nathaniel; Lee, Roland; Koyamatsu, Terrie; Effler, Paul V

2010-01-01

We integrated multicenter, real-time (RTi) reverse transcription polymerase chain reaction (RT-PCR) screening into a statewide laboratory algorithm for influenza surveillance and response. Each of three sites developed its own testing strategy and was challenged with one randomized and blinded panel of 50 specimens previously tested for respiratory viruses. Following testing, each participating laboratory reported its results to the Hawaii State Department of Health, State Laboratories Division for evaluation and possible discrepant analysis. Two of three laboratories reported a 100% sensitivity and specificity, resulting in a 100% positive predictive value and a 100% negative predictive value (NPV) for influenza type A. The third laboratory showed a 71% sensitivity for influenza type A (83% NPV) with 100% specificity. All three laboratories were 100% sensitive and specific for the detection of influenza type B. Discrepant analysis indicated that the lack of sensitivity experienced by the third laboratory may have been due to the analyte-specific reagent probe used by that laboratory. Use of a newer version of the product with a secondary panel of 20 specimens resulted in a sensitivity and specificity of 100%. All three laboratories successfully verified their ability to conduct clinical testing for influenza using diverse nucleic acid extraction and RTi RT-PCR platforms. Successful completion of the verification by all collaborating laboratories paved the way for the integration of those facilities into a statewide laboratory algorithm for influenza surveillance and response.

Standardized Index of Shape (DCE-MRI) and Standardized Uptake Value (PET/CT): Two quantitative approaches to discriminate chemo-radiotherapy locally advanced rectal cancer responders under a functional profile

PubMed Central

Petrillo, Antonella; Fusco, Roberta; Petrillo, Mario; Granata, Vincenza; Delrio, Paolo; Bianco, Francesco; Pecori, Biagio; Botti, Gerardo; Tatangelo, Fabiana; Caracò, Corradina; Aloj, Luigi; Avallone, Antonio; Lastoria, Secondo

2017-01-01

Purpose To investigate dynamic contrast enhanced-MRI (DCE-MRI) in the preoperative chemo-radiotherapy (CRT) assessment for locally advanced rectal cancer (LARC) compared to18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT). Methods 75 consecutive patients with LARC were enrolled in a prospective study. DCE-MRI analysis was performed measuring SIS: linear combination of percentage change (Δ) of maximum signal difference (MSD) and wash-out slope (WOS). 18F-FDG PET/CT analysis was performed using SUV maximum (SUVmax). Tumor regression grade (TRG) were estimated after surgery. Non-parametric tests, receiver operating characteristic were evaluated. Results 55 patients (TRG1-2) were classified as responders while 20 subjects as non responders. ΔSIS reached sensitivity of 93%, specificity of 80% and accuracy of 89% (cut-off 6%) to differentiate responders by non responders, sensitivity of 93%, specificity of 69% and accuracy of 79% (cut-off 30%) to identify pathological complete response (pCR). Therapy assessment via ΔSUVmax reached sensitivity of 67%, specificity of 75% and accuracy of 70% (cut-off 60%) to differentiate responders by non responders and sensitivity of 80%, specificity of 31% and accuracy of 51% (cut-off 44%) to identify pCR. Conclusions CRT response assessment by DCE-MRI analysis shows a higher predictive ability than 18F-FDG PET/CT in LARC patients allowing to better discriminate significant and pCR. PMID:28042958

Standardized Index of Shape (DCE-MRI) and Standardized Uptake Value (PET/CT): Two quantitative approaches to discriminate chemo-radiotherapy locally advanced rectal cancer responders under a functional profile.

PubMed

Petrillo, Antonella; Fusco, Roberta; Petrillo, Mario; Granata, Vincenza; Delrio, Paolo; Bianco, Francesco; Pecori, Biagio; Botti, Gerardo; Tatangelo, Fabiana; Caracò, Corradina; Aloj, Luigi; Avallone, Antonio; Lastoria, Secondo

2017-01-31

To investigate dynamic contrast enhanced-MRI (DCE-MRI) in the preoperative chemo-radiotherapy (CRT) assessment for locally advanced rectal cancer (LARC) compared to18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT). 75 consecutive patients with LARC were enrolled in a prospective study. DCE-MRI analysis was performed measuring SIS: linear combination of percentage change (Δ) of maximum signal difference (MSD) and wash-out slope (WOS). 18F-FDG PET/CT analysis was performed using SUV maximum (SUVmax). Tumor regression grade (TRG) were estimated after surgery. Non-parametric tests, receiver operating characteristic were evaluated. 55 patients (TRG1-2) were classified as responders while 20 subjects as non responders. ΔSIS reached sensitivity of 93%, specificity of 80% and accuracy of 89% (cut-off 6%) to differentiate responders by non responders, sensitivity of 93%, specificity of 69% and accuracy of 79% (cut-off 30%) to identify pathological complete response (pCR). Therapy assessment via ΔSUVmax reached sensitivity of 67%, specificity of 75% and accuracy of 70% (cut-off 60%) to differentiate responders by non responders and sensitivity of 80%, specificity of 31% and accuracy of 51% (cut-off 44%) to identify pCR. CRT response assessment by DCE-MRI analysis shows a higher predictive ability than 18F-FDG PET/CT in LARC patients allowing to better discriminate significant and pCR.

Circulating tumour cells and pathological complete response: independent prognostic factors in inflammatory breast cancer in a pooled analysis of two multicentre phase II trials (BEVERLY-1 and -2) of neoadjuvant chemotherapy combined with bevacizumab.

PubMed

Pierga, J-Y; Bidard, F-C; Autret, A; Petit, T; Andre, F; Dalenc, F; Levy, C; Ferrero, J-M; Romieu, G; Bonneterre, J; Lerebours, F; Bachelot, T; Kerbrat, P; Campone, M; Eymard, J-C; Mouret-Reynier, M-A; Gligorov, J; Hardy-Bessard, A-C; Lortholary, A; Soulie, P; Boher, J-M; Proudhon, C; Charafe-Jaufret, E; Lemonnier, J; Bertucci, F; Viens, P

2017-01-01

We present a pooled analysis of predictive and prognostic values of circulating tumour cells (CTC) and circulating endothelial cells (CEC) in two prospective trials of patients with inflammatory breast cancer (IBC) treated with neoadjuvant chemotherapy combined with neoadjuvant and adjuvant bevacizumab. Nonmetastatic T4d patients were enrolled in two phase II multicentre trials, evaluating bevacizumab in combination with sequential neoadjuvant chemotherapy of four cycles of FEC followed by four cycles of docetaxel in HER2-negative tumour (BEVERLY-1) or docetaxel and trastuzumab in HER2-positive tumour (BEVERLY-2). CTC and CEC were detected in 7.5 and 4 ml of blood, respectively, with the CellSearch System. From October 2008 to September 2010, 152 patients were included and 137 were evaluable for CTC and CEC. At baseline, 55 patients had detectable CTC (39%). After four cycles of chemotherapy, a dramatic drop in CTC to a rate of 9% was observed (P < 0.01). Pathological complete response (pCR) rate was 40%. No correlation was found between CTC or CEC levels and pCR rate. Median follow-up was 43 months. CTC detection (≥1 CTC/7.5 ml) at baseline was associated with shorter 3-year disease-free survival (39% versus 70% for patients without CTC, P < 0.01, HR 2.80) and shorter 3-year overall survival (OS) (P < 0.01). In multivariate analysis, independent prognostic parameters for shorter survival were absence of hormonal receptors, no pCR and CTC detection at baseline. CEC level at baseline or variations during treatment had no prognostic value. In this pooled analysis of two prospective trials in nonmetastatic IBC, detection rate of CTC was 39% with a strong and independent prognostic value for survival. Combination of pCR after neoadjuvant treatment with no CTC detection at baseline isolated a subgroup of IBC with excellent OS (94% 3-year OS), suggesting that CTC count could be part of IBC stratification in prospective trials. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Anti-HER2 CD4(+) T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer.

PubMed

Datta, Jashodeep; Berk, Erik; Xu, Shuwen; Fitzpatrick, Elizabeth; Rosemblit, Cinthia; Lowenfeld, Lea; Goodman, Noah; Lewis, David A; Zhang, Paul J; Fisher, Carla; Roses, Robert E; DeMichele, Angela; Czerniecki, Brian J

2015-05-23

A progressive loss of circulating anti-human epidermal growth factor receptor-2/neu (HER2) CD4(+) T-helper type 1 (Th1) immune responses is observed in HER2(pos)-invasive breast cancer (IBC) patients relative to healthy controls. Pathologic complete response (pCR) following neoadjuvant trastuzumab and chemotherapy (T + C) is associated with decreased recurrence and improved prognosis. We examined differences in anti-HER2 Th1 responses between pCR and non-pCR patients to identify modifiable immune correlates to pathologic response following neoadjuvant T + C. Anti-HER2 Th1 responses in 87 HER2(pos)-IBC patients were examined using peripheral blood mononuclear cells pulsed with 6 HER2-derived class II peptides via IFN-γ ELISPOT. Th1 response metrics were anti-HER2 responsivity, repertoire (number of reactive peptides), and cumulative response across 6 peptides (spot-forming cells [SFC]/10(6) cells). Anti-HER2 Th1 responses of non-pCR patients (n = 4) receiving adjuvant HER2-pulsed type 1-polarized dendritic cell (DC1) vaccination were analyzed pre- and post-immunization. Depressed anti-HER2 Th1 responses observed in treatment-naïve HER2(pos)-IBC patients (n = 22) did not improve globally in T + C-treated HER2(pos)-IBC patients (n = 65). Compared with adjuvant T + C receipt, neoadjuvant T + C - utilized in 61.5 % - was associated with higher anti-HER2 Th1 repertoire (p = 0.048). While pCR (n = 16) and non-pCR (n = 24) patients did not differ substantially in demographic/clinical characteristics, pCR patients demonstrated dramatically higher anti-HER2 Th1 responsivity (94 % vs. 33 %, p = 0.0002), repertoire (3.3 vs. 0.3 peptides, p < 0.0001), and cumulative response (148.2 vs. 22.4 SFC/10(6), p < 0.0001) versus non-pCR patients. After controlling for potential confounders, anti-HER2 Th1 responsivity remained independently associated with pathologic response (odds ratio 8.82, p = 0.016). This IFN-γ(+) immune disparity was mediated by anti-HER2 CD4(+)T-bet(+)IFN-γ(+) (i.e., Th1) - not CD4(+)GATA-3(+)IFN-γ(+) (i.e., Th2) - phenotypes, and not attributable to non-pCR patients' immune incompetence, host-level T-cell anergy, or increased immunosuppressive populations. In recruited non-pCR patients, anti-HER2 Th1 repertoire (3.7 vs. 0.5, p = 0.014) and cumulative response (192.3 vs. 33.9 SFC/10(6), p = 0.014) improved significantly following HER2-pulsed DC1 vaccination. Anti-HER2 CD4(+) Th1 response is a novel immune correlate to pathologic response following neoadjuvant T + C. In non-pCR patients, depressed Th1 responses are not immunologically "fixed" and can be restored with HER2-directed Th1 immune interventions. In such high-risk patients, combining HER2-targeted therapies with strategies to boost anti-HER2 Th1 immunity may improve outcomes and mitigate recurrence.

Gravity Persistent Signal 1 (GPS1) reveals novel cytochrome P450s involved in gravitropism.

PubMed

Withers, John C; Shipp, Matthew J; Rupasinghe, Sanjeewa G; Sukumar, Poornima; Schuler, Mary A; Muday, Gloria K; Wyatt, Sarah E

2013-01-01

Gravity is an important environmental factor that affects growth and development of plants. In response to changes in gravity, directional growth occurs along the major axes and lateral branches of both shoots and roots. The gravity persistent signal (gps) mutants of Arabidopsis thaliana were previously identified as having an altered response to gravity when reoriented relative to the gravity vector in the cold, with the gps1 mutant exhibiting a complete loss of tropic response under these conditions. Thermal asymmetric interlaced (TAIL) PCR was used to identify the gene defective in gps1. Gene expression data, molecular modeling and computational substrate dockings, quantitative RT-PCR analyses, reporter gene fusions, and physiological analyses of knockout mutants were used to characterize the genes identified. Cloning of the gene defective in gps1 and genetic complementation revealed that GPS1 encodes CYP705A22, a cytochrome P450 monooxygenase (P450). CYP705A5, a closely related family member, was identified as expressed specifically in roots in response to gravistimulation, and a mutation affecting its expression resulted in a delayed gravity response, increased flavonol levels, and decreased basipetal auxin transport. Molecular modeling coupled with in silico substrate docking and diphenylboric acid 2-aminoethyl ester (DBPA) staining indicated that these P450s are involved in biosynthesis of flavonoids potentially involved in auxin transport. The characterization of two novel P450s (CYP705A22 and CYP705A5) and their role in the gravity response has offered new insights into the regulation of the genetic and physiological controls of plant gravitropism.

Positive regulation of humoral and innate immune responses induced by inactivated Avian Influenza Virus vaccine in broiler chickens.

PubMed

Abdallah, Fatma; Hassanin, Ola

2015-12-01

Avian Influenza (AI) vaccines are widely used for mammals and birds in a trial to eliminate the Avian Influenza virus (AIV) infection from the world. However and up till now the virus is still existed via modulation of its antigenic structure to evade the pressure of host immune responses. For a complete understanding of the immune responses following AI vaccination in chickens, the modulations of the chickens humoral immune responses and interferon-alpha signaling pathway, as a fundamental part of the innate immune responses, were investigated. In our study, we measured the humoral immune response using hemagglutination-inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) tests. In addition, chicken interferon-alpha pathway components was measured at RNA levels using Quantitative Real-time PCR (qRT-PCR) following one dose of inactivated H5N1 influenza vaccine at 14 days of age. In this study, the protective levels of humoral antibody responses were observed at 14, 21 and 28 days following immunization with inactivated (Re-1/H5N1) AI vaccine. In the chicken spleen cells, up regulation in the chicken interferon-alpha pathway components (MX1 & IRF7) was existed as early as 48 h post vaccination and remained until 28 days post vaccination at the endogenous state. However, after the recall with ex-vivo stimulation, the up regulation was more pronounced in the transcriptional factor (IRF7) compared to the antiviral gene (MX1) at 28 days post vaccination. So far, from our results it appears that the inactivated H5N1 vaccine can trigger the chicken interferon-alpha signaling pathway as well as it can elicit protective humoral antibody responses.

Novel in Vitro Modification of Bone for an Allograft with Improved Toughness Osteoconductivity

DTIC Science & Technology

2015-06-01

osteocalcin, Runx2, and col1a1 by RT-PCR. Spectrophotometry and fluorescence microscopy were used to quantify AGEs. 2. KEYWORDS Fracture toughness, R...markers (alkaline phosphatase, osteocalcin, RUNX2 and COL1A1 ) Completed Task 10 Data analysis, publications, reports Completed Task 1. Retrieval...FEMALE 25 Task 9. Measure expression of molecular markers of mineralization, osteocalcin, RUNX2 and COL1A1 using quantitative RT-PCR with specific

Complete Genome Sequence of Diaphorina citri-associated C virus, a Novel Putative RNA Virus of the Asian Citrus Psyllid, Diaphorina citri.

PubMed

Nouri, Shahideh; Salem, Nidà; Falk, Bryce W

2016-07-21

We present here the complete nucleotide sequence and genome organization of a novel putative RNA virus identified in field populations of the Asian citrus psyllid, Diaphorina citri, through sequencing of the transcriptome followed by reverse transcription-PCR (RT-PCR). We tentatively named this virus Diaphorina citri-associated C virus (DcACV). DcACV is an unclassified positive-sense RNA virus. Copyright © 2016 Nouri et al.

A simplified strategy for studying the etiology of viral diseases: Apple stem grooving virus as a case study.

PubMed

Dhir, Sunny; Walia, Yashika; Zaidi, A A; Hallan, Vipin

2015-03-01

A simple method to amplify infective, complete genomes of single stranded RNA viruses by long distance PCR (LD PCR) from woody plant tissues is described in detail. The present protocol eliminates partial purification of viral particles and the amplification is achieved in three steps: (i) easy preparation of template RNA by incorporating a pre processing step before loading onto the column (ii) reverse transcription by AMV or Superscript reverse transcriptase and (iii) amplification of cDNA by LD PCR using LA or Protoscript Taq DNA polymerase. Incorporation of a preprocessing step helped to isolate consistent quality RNA from recalcitrant woody tissues such as apple, which was critical for efficient amplification of the complete genomes of Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV). Complete genome of ASGV was cloned under T7 RNA polymerase promoter and was confirmed to be infectious through transcript inoculation producing symptoms similar to the wild type virus. This is the first report for the largest RNA virus genome amplified by PCR from total nucleic acid extracts of woody plant tissues. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of complete genome sequence of the spring viremia of carp virus isolated from common carp (Cyprinus carpio) in China.

PubMed

Teng, Y; Liu, H; Lv, J Q; Fan, W H; Zhang, Q Y; Qin, Q W

2007-01-01

The complete genome of spring viraemia of carp virus (SVCV) strain A-1 isolated from cultured common carp (Cyprinus carpio) in China was sequenced and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed and the DNA was sequenced. It showed that the entire genome of SVCV A-1 consists of 11,100 nucleotide base pairs, the predicted size of the viral RNA of rhabdoviruses. However, the additional insertions in bp 4633-4676 and bp 4684-4724 of SVCV A-1 were different from the other two published SVCV complete genomes. Five open reading frames (ORFs) of SVCV A-1 were identified and further confirmed by RT-PCR and DNA sequencing of their respective RT-PCR products. The 5 structural proteins encoded by the viral RNA were ordered 3'-N-P-M-G-L-5'. This is the first report of a complete genome sequence of SVCV isolated from cultured carp in China. Phylogenetic analysis indicates that SVCV A-1 is closely related to the members of the genus Vesiculovirus, family Rhabdoviridae.

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

PubMed Central

Espy, M. J.; Uhl, J. R.; Sloan, L. M.; Buckwalter, S. P.; Jones, M. F.; Vetter, E. A.; Yao, J. D. C.; Wengenack, N. L.; Rosenblatt, J. E.; Cockerill, F. R.; Smith, T. F.

2006-01-01

Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory. PMID:16418529

Molecular approaches to Taenia asiatica.

PubMed

Jeon, Hyeong-Kyu; Eom, Keeseon S

2013-02-01

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.

Obesity is an independent prognostic factor of decreased pathological complete response to neoadjuvant chemotherapy in breast cancer patients.

PubMed

Karatas, Fatih; Erdem, Gokmen Umut; Sahin, Suleyman; Aytekin, Aydin; Yuce, Deniz; Sever, Ali R; Babacan, Taner; Ates, Ozturk; Ozisik, Yavuz; Altundag, Kadri

2017-04-01

The relation between higher body mass index (BMI) and pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) in breast cancer (BC) is a controversial issue according to the data of Western and Asian patients. The aim of this study is to evaluate BMI and pCR to NAC and discuss the importance of pCR outcomes in Turkish BC patients as a bridging country between Europe and Asia. Of the 4423 BC patients diagnosed between the years 1994 and 2015 in Hacettepe University Cancer Institute, 295 female patients with stage II and III BC were enrolled in the study. Three different group divisions were done according to patients' BMI as normal or underweight (N/U) patients (BMI <25 kg/m 2 ), overweight (OW) patients (BMI = 25-29.9 kg/m 2 ) and obese (OB) patients (BMI ≥30 kg/m 2 ). BC subtypes were defined as luminal-like (ER/PR-positive and HER2-negative), HER2/luminal (ER/PR-positive and HER2-positive), HER2-type (ER/PR-negative and HER2-positive), and triple-negative (TNBC; ER/PR- and HER2-negative). The analysis of overall survival (OS) and recurrence-free survival (RFS) was performed according to Kaplan-Meier method. The Log-rank test was used to compare the subgroup analysis and logistic regression analysis to determine the independent prognostic factors. In this study, a total number of 93 (31.5%) patients were N/U, 107 (36.3%) patients were OW and 95 (32.2%) patients were OB. Among groups, except for the age, no baseline clinicopathological differences were found. In 70 (23.7%) patients, pCR was achieved. pCR rates in N/U, OW and OB were 31.2%, 22.4%, and 17.9% respectively, showing a considerable trend towards significance (P = 0.09 in chi-square test). In the multivariate logistic regression analysis, obesity was an independent adverse prognostic feature on pCR to NAC compared to N/U patients (OR, 0.34; 95% CI, 0.13 to 0.85, P = 0.02). The recurrence rates were slightly increased with the increase of BMI (N/U = 24.7%, OW = 29.0% and OB = 40%; P = 0.06 respectively). Median RFS was significantly higher in N/U group compared to OB patients (150 vs. 76 months respectively, P = 0.03) and was also higher in pCR group compared to non-pCR patients (151 vs. 77 months P = 0.004). Median OS was significantly higher in N/U patients compared to OB patients (N/U = not reached, OW = 211 and OB = 114 months; P = 0.01) and was also higher in pCR group compared to non-pCR patients (not reached vs. 211 months P = 0.04). In Cox regression analysis; pCR, histopathological grade and TNBC were found as independent prognostic factors on OS (HR, 0.29; 95% CI, 0.11 to 0.79, P = 0.015, HR, 2.09; 95% CI, 1.14 to 3.83, P = 0.017, HR, 1.95; 95% CI, 1.01 to 3.77, P = 0.046, respectively). It was observed that obesity was an important independent prognostic factor which has an adverse effect on pCR. Moreover it causes decreasing RFS and OS in BC patients who had received NAC. The probability of inefficient treatment in obese patients should be considered. Copyright © 2016 Elsevier Ltd. All rights reserved.

Application of PCR for detection of toxigenic Corynebacterium diphtheriae strains isolated during the Russian diphtheria epidemic, 1990 through 1994.

PubMed Central

Mikhailovich, V M; Melnikov, V G; Mazurova, I K; Wachsmuth, I K; Wenger, J D; Wharton, M; Nakao, H; Popovic, T

1995-01-01

A total of 250 Corynebacterium diphtheriae isolates from clinical cases and carriers in Russia were assayed by PCR directed at the A subunit of the diphtheria toxin gene to distinguish toxigenic from nontoxigenic strains; 170 strains were positive as indicated by the presence of the 248-bp amplicon. The results of this PCR assay were in complete concordance with those of the standard immunoprecipitation assay (Elek), and the PCR assay is a useful tool for rapid identification in clinical laboratories. PMID:8576378

Value of molecular monitoring during the treatment of chronic myeloid leukemia: a Cancer and Leukemia Group B study.

PubMed

Stock, W; Westbrook, C A; Peterson, B; Arthur, D C; Szatrowski, T P; Silver, R T; Sher, D A; Wu, D; Le Beau, M M; Schiffer, C A; Bloomfield, C D

1997-01-01

Disappearance of the Philadelphia chromosome during treatment for chronic myeloid leukemia (CML) has become an important therapeutic end point. To determine the additional value of molecular monitoring during treatment for CML, we performed a prospective, sequential analysis using quantitative Southern blot monitoring of BCR gene rearrangements of blood and marrow samples from Cancer and Leukemia Group B (CALGB) study 8761. Sixty-four previously untreated adults with chronic-phase CML who were enrolled onto CALGB 8761, a molecular-monitoring companion study to a treatment study for adults with chronic-phase CML (CALGB 9013). Treatment consisted of repetitive cycles of interferon alfa and low-dose subcutaneous cytarabine. Blood and marrow Southern blot quantitation of BCR gene rearrangements was compared with marrow cytogenetic analysis before the initiation of treatment and of specified points during therapy. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was performed to detect residual disease in patients who achieved a complete response by Southern blot or cytogenetic analysis. Quantitative molecular monitoring by Southern blot analysis of blood samples was found to be equivalent to marrow monitoring at all time points. Twelve of 62 (19%) follow-up samples studied by Southern blot analysis had a complete loss of BCR gene rearrangement in matched marrow and blood specimens. Southern blot monitoring of blood samples was also found to be highly correlated to marrow cytogenetic evaluation at all points, although there were four discordant cases in which Southern blot analysis of blood showed no BCR gene rearrangement, yet demonstrated from 12% to 20% Philadelphia chromosome-positive metaphase cells in the marrow. RT-PCR analysis detected residual disease in five of six patients in whom no malignant cells were detected using Southern blot or cytogenetic analyses. Quantitative Southern blot analysis of blood samples may be substituted for bone marrow to monitor the response to therapy in CML and results in the need for fewer bone marrow examinations. To avoid overestimating the degree of response, marrow cytogenetic analysis should be performed when patients achieve a complete response by Southern blot monitoring. This approach provides a rational, cost-effective strategy to monitor the effect of treatment of individual patients, as well as to analyze large clinical trials in CML.

RandomizEd controlled trial for pre-operAtive dose-escaLation BOOST in locally advanced rectal cancer (RECTAL BOOST study): study protocol for a randomized controlled trial.

PubMed

Burbach, J P Maarten; Verkooijen, Helena M; Intven, Martijn; Kleijnen, Jean-Paul J E; Bosman, Mirjam E; Raaymakers, Bas W; van Grevenstein, Wilhelmina M U; Koopman, Miriam; Seravalli, Enrica; van Asselen, Bram; Reerink, Onne

2015-02-22

Treatment for locally advanced rectal cancer (LARC) consists of chemoradiation therapy (CRT) and surgery. Approximately 15% of patients show a pathological complete response (pCR). Increased pCR-rates can be achieved through dose escalation, thereby increasing the number patients eligible for organ-preservation to improve quality of life (QoL). A randomized comparison of 65 versus 50Gy with external-beam radiation alone has not yet been performed. This trial investigates pCR rate, clinical response, toxicity, QoL and (disease-free) survival in LARC patients treated with 65Gy (boost + chemoradiation) compared with 50Gy standard chemoradiation (sCRT). This study follows the 'cohort multiple randomized controlled trial' (cmRCT) design: rectal cancer patients are included in a prospective cohort that registers clinical baseline, follow-up, survival and QoL data. At enrollment, patients are asked consent to offer them experimental interventions in the future. Eligible patients-histologically confirmed LARC (T3NxM0 <1 mm from mesorectal fascia, T4NxM0 or TxN2M0) located ≤10 cm from the anorectal transition who provided consent for experimental intervention offers-form a subcohort (n = 120). From this subcohort, a random sample is offered the boost prior to sCRT (n = 60), which they may accept or refuse. Informed consent is signed only after acceptance of the boost. Non-selected patients in the subcohort (n = 60) undergo sCRT alone and are not notified that they participate in the control arm until the trial is completed. sCRT consists of 50Gy (25 × 2Gy) with concomitant capecitabine. The boost (without chemotherapy) is given prior to sCRT and consists of 15 Gy (5 × 3Gy) delivered to the gross tumor volume (GTV). The primary endpoint is pCR (TRG 1). Secondary endpoints include acute grade 3-4 toxicity, good pathologic response (TRG 1-2), clinical response, surgical complications, QoL and (disease-free) survival. Data is analyzed by intention to treat. The boost is delivered prior to sCRT so that GTV adjustment for tumor shrinkage during sCRT is not necessary. Small margins also aim to limit irradiation of healthy tissue. The cmRCT design provides opportunity to overcome common shortcomings of classic RCTs, such as slow recruitment, disappointment-bias in control arm patients and poor generalizability. The Netherlands Trials Register NL46051.041.13. Registered 22 August 2013. ClinicalTrials.gov NCT01951521 . Registered 18 September 2013.

Lymphocyte density determined by computational pathology validated as a predictor of response to neoadjuvant chemotherapy in breast cancer: secondary analysis of the ARTemis trial.

PubMed

Ali, H R; Dariush, A; Thomas, J; Provenzano, E; Dunn, J; Hiller, L; Vallier, A-L; Abraham, J; Piper, T; Bartlett, J M S; Cameron, D A; Hayward, L; Brenton, J D; Pharoah, P D P; Irwin, M J; Walton, N A; Earl, H M; Caldas, C

2017-08-01

We have previously shown lymphocyte density, measured using computational pathology, is associated with pathological complete response (pCR) in breast cancer. The clinical validity of this finding in independent studies, among patients receiving different chemotherapy, is unknown. The ARTemis trial randomly assigned 800 women with early stage breast cancer between May 2009 and January 2013 to three cycles of docetaxel, followed by three cycles of fluorouracil, epirubicin and cyclophosphamide once every 21 days with or without four cycles of bevacizumab. The primary endpoint was pCR (absence of invasive cancer in the breast and lymph nodes). We quantified lymphocyte density within haematoxylin and eosin (H&E) whole slide images using our previously described computational pathology approach: for every detected lymphocyte the average distance to the nearest 50 lymphocytes was calculated and the density derived from this statistic. We analyzed both pre-treatment biopsies and post-treatment surgical samples of the tumour bed. Of the 781 patients originally included in the primary endpoint analysis of the trial, 609 (78%) were included for baseline lymphocyte density analyses and a subset of 383 (49% of 781) for analyses of change in lymphocyte density. The main reason for loss of patients was the availability of digitized whole slide images. Pre-treatment lymphocyte density modelled as a continuous variable was associated with pCR on univariate analysis (odds ratio [OR], 2.92; 95% CI, 1.78-4.85; P < 0.001) and after adjustment for clinical covariates (OR, 2.13; 95% CI, 1.24-3.67; P = 0.006). Increased pre- to post-treatment lymphocyte density showed an independent inverse association with pCR (adjusted OR, 0.1; 95% CI, 0.033-0.31; P < 0.001). Lymphocyte density in pre-treatment biopsies was validated as an independent predictor of pCR in breast cancer. Computational pathology is emerging as a viable and objective means of identifying predictive biomarkers for cancer patients. NCT01093235. © The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents

PubMed Central

Proctor, Michael; Flaherty, Patrick; Jordan, Michael I; Arkin, Adam P; Davis, Ronald W; Nislow, Corey; Giaever, Guri

2005-01-01

The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups. PMID:16121259

A multicenter study of viable PCR using propidium monoazide to detect Legionella in water samples.

PubMed

Scaturro, Maria; Fontana, Stefano; Dell'eva, Italo; Helfer, Fabrizia; Marchio, Michele; Stefanetti, Maria Vittoria; Cavallaro, Mario; Miglietta, Marilena; Montagna, Maria Teresa; De Giglio, Osvalda; Cuna, Teresa; Chetti, Leonarda; Sabattini, Maria Antonietta Bucci; Carlotti, Michela; Viggiani, Mariagabriella; Stenico, Alberta; Romanin, Elisa; Bonanni, Emma; Ottaviano, Claudio; Franzin, Laura; Avanzini, Claudio; Demarie, Valerio; Corbella, Marta; Cambieri, Patrizia; Marone, Piero; Rota, Maria Cristina; Bella, Antonino; Ricci, Maria Luisa

2016-07-01

Legionella quantification in environmental samples is overestimated by qPCR. Combination with a viable dye, such as Propidium monoazide (PMA), could make qPCR (named then vPCR) very reliable. In this multicentre study 717 artificial water samples, spiked with fixed concentrations of Legionella and interfering bacterial flora, were analysed by qPCR, vPCR and culture and data were compared by statistical analysis. A heat-treatment at 55 °C for 10 minutes was also performed to obtain viable and not-viable bacteria. When data of vPCR were compared with those of culture and qPCR, statistical analysis showed significant differences (P < 0.001). However, although the heat-treatment caused an abatement of CFU/mL ≤1 to 1 log10 unit, the comparison between untreated and heat-treated samples analysed by vPCR highlighted non-significant differences (P > 0.05). Overall this study provided a good experimental reproducibility of vPCR but also highlighted limits of PMA in the discriminating capability of dead and live bacteria, making vPCR not completely reliable. Copyright © 2016 Elsevier Inc. All rights reserved.

Phase II, randomized study of concomitant chemoradiotherapy followed by surgery and adjuvant capecitabine plus oxaliplatin (CAPOX) compared with induction CAPOX followed by concomitant chemoradiotherapy and surgery in magnetic resonance imaging-defined, locally advanced rectal cancer: Grupo cancer de recto 3 study.

PubMed

Fernández-Martos, Carlos; Pericay, Carles; Aparicio, Jorge; Salud, Antonieta; Safont, Mariajose; Massuti, Bertomeu; Vera, Ruth; Escudero, Pilar; Maurel, Joan; Marcuello, Eugenio; Mengual, Jose Luis; Saigi, Eugenio; Estevan, Rafael; Mira, Moises; Polo, Sonia; Hernandez, Ana; Gallen, Manuel; Arias, Fernando; Serra, Javier; Alonso, Vicente

2010-02-10

PURPOSE The optimal therapeutic sequence of the adjuvant chemotherapy component of preoperative chemoradiotherapy (CRT) for patients with locally advanced rectal cancer is controversial. Induction chemotherapy before preoperative CRT may be associated with better efficacy and compliance. PATIENTS AND METHODS A total of 108 patients with locally advanced rectal cancer were randomly assigned to arm A-preoperative CRT with capecitabine, oxaliplatin, and concurrent radiation followed by surgery and four cycles of postoperative adjuvant capecitabine and oxaliplatin (CAPOX)-or arm B-induction CAPOX followed by CRT and surgery. The primary end point was pathologic complete response rate (pCR). Results On an intention-to-treat basis, the pCR for arms A and B were 13.5% (95% CI, 5.6% to 25.8%) and 14.3% (95% CI, 6.4% to 26.2%), respectively. There were no statistically significant differences in other end points, including downstaging, tumor regression, and R0 resection. Overall, chemotherapy treatment exposure was higher in arm B than in arm A for both oxaliplatin (P < .0001) and capecitabine (P < .0001). During CRT, grades 3 to 4 adverse events were similar in both arms but were significantly higher in arm A during postoperative adjuvant CT than with induction CT in arm B. There were three deaths in each arm during the treatment period. CONCLUSION Compared with postoperative adjuvant CAPOX, induction CAPOX before CRT had similar pCR and complete resection rates. It did achieve more favorable compliance and toxicity profiles. On the basis of these findings, a phase III study to definitively test the induction strategy is warranted.

A Clinical and Epidemiological Investigation of the First Reported Human Infection With the Zoonotic Parasite Trypanosoma evansi in Southeast Asia

PubMed Central

Van Vinh Chau, Nguyen; Buu Chau, Le; Desquesnes, Marc; Herder, Stephane; Phu Huong Lan, Nguyen; Campbell, James I.; Van Cuong, Nguyen; Yimming, Benjarat; Chalermwong, Piangjai; Jittapalapong, Sathaporn; Ramon Franco, Jose; Tri Tue, Ngo; Rabaa, Maia A.; Carrique-Mas, Juan; Pham Thi Thanh, Tam; Tran Vu Thieu, Nga; Berto, Alessandra; Thi Hoa, Ngo; Van Minh Hoang, Nguyen; Canh Tu, Nguyen; Khac Chuyen, Nguyen; Wills, Bridget; Tinh Hien, Tran; Thwaites, Guy E.; Yacoub, Sophie; Baker, Stephen

2016-01-01

Background. Trypanosoma is a genus of unicellular parasitic flagellate protozoa. Trypanosoma brucei species and Trypanosoma cruzi are the major agents of human trypanosomiasis; other Trypanosoma species can cause human disease, but are rare. In March 2015, a 38-year-old woman presented to a healthcare facility in southern Vietnam with fever, headache, and arthralgia. Microscopic examination of blood revealed infection with Trypanosoma. Methods. Microscopic observation, polymerase chain reaction (PCR) amplification of blood samples, and serological testing were performed to identify the infecting species. The patient's blood was screened for the trypanocidal protein apolipoprotein L1 (APOL1), and a field investigation was performed to identify the zoonotic source. Results. PCR amplification and serological testing identified the infecting species as Trypanosoma evansi. Despite relapsing 6 weeks after completing amphotericin B therapy, the patient made a complete recovery after 5 weeks of suramin. The patient was found to have 2 wild-type APOL1 alleles and a normal serum APOL1 concentration. After responsive animal sampling in the presumed location of exposure, cattle and/or buffalo were determined to be the most likely source of the infection, with 14 of 30 (47%) animal blood samples testing PCR positive for T. evansi. Conclusions. We report the first laboratory-confirmed case of T. evansi in a previously healthy individual without APOL1 deficiency, potentially contracted via a wound while butchering raw beef, and successfully treated with suramin. A linked epidemiological investigation revealed widespread and previously unidentified burden of T. evansi in local cattle, highlighting the need for surveillance of this infection in animals and the possibility of further human cases. PMID:26908809

Extramural vascular invasion and response to neoadjuvant chemoradiotherapy in rectal cancer: Influence of the CpG island methylator phenotype

PubMed Central

Williamson, Jeremy Stuart; Jones, Huw Geraint; Williams, Namor; Griffiths, Anthony Paul; Jenkins, Gareth; Beynon, John; Harris, Dean Anthony

2017-01-01

AIM To identify whether CpG island methylator phenotype (CIMP) is predictive of response to neoadjuvant chemoradiotherapy (NACRT) and outcomes in rectal cancer. METHODS Patients undergoing NACRT and surgical resection for rectal cancer in a tertiary referral centre between 2002-2011 were identified. Pre-treatment tumour biopsies were analysed for CIMP status (high, intermediate or low) using methylation specific PCR. KRAS and BRAF status were also determined using pyrosequencing analysis. Clinical information was extracted from case records and cancer services databases. Response to radiotherapy was measured by tumour regression scores determined upon histological examination of the resected specimen. The relationship between these molecular features, response to NACRT and oncological outcomes were analysed. RESULTS There were 160 patients analysed with a median follow-up time of 46.4 mo. Twenty-one (13%) patients demonstrated high levels of CIMP methylation (CIMP-H) and this was significantly associated with increased risk of extramural vascular invasion (EMVI) compared with CIMP-L [8/21 (38%) vs 15/99 (15%), P = 0.028]. CIMP status was not related to tumour regression after radiotherapy or survival, however EMVI was significantly associated with adverse survival (P < 0.001). Intermediate CIMP status was significantly associated with KRAS mutation (P = 0.01). There were 14 (9%) patients with a pathological complete response (pCR) compared to 116 (73%) patients having no or minimal regression after neoadjuvant chemoradiotherapy. Those patients with pCR had median survival of 106 mo compared to 65.8 mo with minimal regression, although this was not statistically significant (P = 0.26). Binary logistic regression analysis of the relationship between EMVI and other prognostic features revealed, EMVI positivity was associated with poor overall survival, advanced “T” stage and CIMP-H but not nodal status, age, sex, KRAS mutation status and presence of local or systemic recurrence. CONCLUSION We report a novel association of pre-treatment characterisation of CIMP-H with EMVI status which has prognostic implications and is not readily detectable on pre-treatment histological examination. PMID:28567185

Extramural vascular invasion and response to neoadjuvant chemoradiotherapy in rectal cancer: Influence of the CpG island methylator phenotype.

PubMed

Williamson, Jeremy Stuart; Jones, Huw Geraint; Williams, Namor; Griffiths, Anthony Paul; Jenkins, Gareth; Beynon, John; Harris, Dean Anthony

2017-05-15

To identify whether CpG island methylator phenotype (CIMP) is predictive of response to neoadjuvant chemoradiotherapy (NACRT) and outcomes in rectal cancer. Patients undergoing NACRT and surgical resection for rectal cancer in a tertiary referral centre between 2002-2011 were identified. Pre-treatment tumour biopsies were analysed for CIMP status (high, intermediate or low) using methylation specific PCR. KRAS and BRAF status were also determined using pyrosequencing analysis. Clinical information was extracted from case records and cancer services databases. Response to radiotherapy was measured by tumour regression scores determined upon histological examination of the resected specimen. The relationship between these molecular features, response to NACRT and oncological outcomes were analysed. There were 160 patients analysed with a median follow-up time of 46.4 mo. Twenty-one (13%) patients demonstrated high levels of CIMP methylation (CIMP-H) and this was significantly associated with increased risk of extramural vascular invasion (EMVI) compared with CIMP-L [8/21 (38%) vs 15/99 (15%), P = 0.028]. CIMP status was not related to tumour regression after radiotherapy or survival, however EMVI was significantly associated with adverse survival ( P < 0.001). Intermediate CIMP status was significantly associated with KRAS mutation ( P = 0.01). There were 14 (9%) patients with a pathological complete response (pCR) compared to 116 (73%) patients having no or minimal regression after neoadjuvant chemoradiotherapy. Those patients with pCR had median survival of 106 mo compared to 65.8 mo with minimal regression, although this was not statistically significant ( P = 0.26). Binary logistic regression analysis of the relationship between EMVI and other prognostic features revealed, EMVI positivity was associated with poor overall survival, advanced "T" stage and CIMP-H but not nodal status, age, sex, KRAS mutation status and presence of local or systemic recurrence. We report a novel association of pre-treatment characterisation of CIMP-H with EMVI status which has prognostic implications and is not readily detectable on pre-treatment histological examination.

Simultaneous VENTANA IHC and RT-PCR testing of ALK status in Chinese non-small cell lung cancer patients and response to crizotinib.

PubMed

Xu, Chun-Wei; Wang, Wen-Xian; Chen, Yan-Ping; Chen, Yu; Liu, Wei; Zhong, Li-Hua; Chen, Fang-Fang; Zhuang, Wu; Song, Zheng-Bo; Chen, Xiao-Hui; Huang, Yun-Jian; Guan, Yan-Fang; Yi, Xin; Lv, Tang-Feng; Zhu, Wei-Feng; Lu, Jian-Ping; Wang, Xiao-Jiang; Shi, Yi; Lin, Xian-Dong; Chen, Gang; Song, Yong

2018-04-11

ALK rearrangement-advanced NSCLC patients respond to crizotinib. ALK rearrangement is currently determined with RT-PCR. VENTANA IHC is a standard method to identify ALK protein overexpression in NSCLC; however, VENTANA IHC has rarely been used to determine the response to crizotinib in Chinese patients with NSCLC and ALK overexpression. To better clarify the clinical implication of VENTANA IHC to detect ALK rearrangements, we conducted this study to analyze VENTANA IHC and RT-PCR in a large cohort of Chinese patients with NSCLC undergoing screening for ALK rearrangements. A total of 1720 patients with NSCLC who had ALK rearrangements detected by VENTANA IHC and/or RT-PCR were included in this analysis. We compared the efficacy and survival of ALK-positive patients detected by VENTANA IHC and RT-PCR. We used NGS to identify patients in whom the two methods were inconsistent. Among 1720 patients, 187 (10.87%) were shown to be ALK-positive by VENTANA IHC and/or RT-PCR, and 66 received crizotinib treatment. We identified 10.27% (172/1674) of patients as ALK-positive by the VENTANA IHC method, and 12.73% (41/322) of patients had ALK rearrangements by the RT-PCR method. Twenty-nine of 276 (10.51%) ALK-positive patients were simultaneously analyzed using VENTANA IHC and RT-PCR. The overall response rates were 65.90% (29/44) by VENTANA IHC and 55.88% (19/34) by RT-PCR. The disease control rates were 86.36% (38/44) by VENTANA IHC and 76.47% (26/34) by RT-PCR. In contrast, the median progression-free survival for VENTANA IHC and RT-PCR was 8.5 and 9.2 months, respectively. The VENTANA IHC and RT-PCR results obtained for 6 of 17 ALK-positive patients were inconsistent based on NGS; specifically, 4 patients had EML4-ALK fusions, 2 patients had non EML4-ALK fusions, 1 patient had a KCL1-ALK fusion, and one patient had a FBXO36-ALK fusion. VENTANA IHC is a reliable and rapid screening tool used in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy. VENTANA IHC has moderate sensitivity and a slightly higher association with response to therapy with ALK inhibitors, and some VENTANA IHC-positive, but RT-PCR-negative cases may benefit from crizotinib.

Identification of ATM mutations using extended RT-PCR and restriction endonuclease fingerprinting, and elucidation of the repertoire of A-T mutations in Israel.

PubMed

Gilad, S; Khosravi, R; Harnik, R; Ziv, Y; Shkedy, D; Galanty, Y; Frydman, M; Levi, J; Sanal, O; Chessa, L; Smeets, D; Shiloh, Y; Bar-Shira, A

1998-01-01

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, and radiation sensitivity. The responsible gene, ATM, has an extensive genomic structure and encodes a large transcript with a 9.2 kb open reading frame (ORF). A-T mutations are extremely variable and most of them are private. We streamlined a high throughput protocol for the search for ATM mutations. The entire ATM ORF is amplified in a single RT-PCR step requiring a minimal amount of RNA. The product can serve for numerous nested PCRs in which overlapping portions of the ORF are further amplified and subjected to restriction endonuclease fingerprinting (REF) analysis. Splicing errors are readily detectable during the initial amplification of each portion. Using this protocol, we identified 5 novel A-T mutations and completed the elucidation of the molecular basis of A-T in the Israeli population.

Chemoradiation With Concomitant Boosts Followed by Radical Surgery in Locally Advanced Cervical Cancer: Long-term Results of the ROMA-2 Prospective Phase 2 Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ferrandina, Gabriella, E-mail: gabriella.ferrandina@libero.it; Gambacorta, Antonietta; Gallotta, Valerio

Purpose: This prospective, phase 2 study aimed at assessing the efficacy of accelerated fractionation radiation therapy by concomitant boosts (CBs) associated with chemoradiation therapy (CRT) of the whole pelvis, in improving the rate of pathological complete response (pCR) to treatment in patients with International Federation of Gynaecology and Obstetrics (FIGO) stage IB2-IVA locally advanced cervical cancer. Methods and Materials: Neoadjuvant CRT included conformal irradiation of the whole pelvis with a total dose of 39.6 Gy (1.8 cGy/fraction, 22 fractions), plus additional irradiation of primary tumor and parametria with 10.8 Gy administered with CBs (0.9 cGy/fraction, 12 fractions, every other day). Concomitant chemotherapy included cisplatinmore » (20 mg/m{sup 2}, days 1-4 and 26-30 of treatment), and capecitabine (1300 mg/m{sup 2}/daily, orally) during the first 2 and the last 2 weeks of treatment. Radical hysterectomy plus pelvic with or without aortic lymphadenectomy was performed within 6 to 8 weeks from CRT. Toxicity was recorded according to Radiation Therapy Oncology Group toxicity criteria and Chassagne grading system. Based on the Simon design, 103 cases were required, and the regimen would be considered active if >45 pCR were registered (α error = 0.05; β error = 0.1). Results: pCR was documented in 51 cases (50.5%), and the regimen was considered active, according to the planned statistical assumptions. At median follow-up of 36 months (range: 7-85 months), the 3-year local failure rate was 7%, whereas the 3-year disease-free and overall survival rates were 73.0% and 86.1%, respectively. Grade 3 leukopenia and neutropenia were reported in only 1 and 2 cases, respectively. Gastrointestinal toxicity was always grade 1 or 2. Conclusions: Addition of CBs in the accelerated fractionation modality to the whole pelvis chemoradiation followed by radical surgery results in a high rate of pathologically assessed complete response to CRT and a very encouraging local control rate, with acceptable toxicity.« less

Molecular Approaches to Taenia asiatica

PubMed Central

Jeon, Hyeong-Kyu

2013-01-01

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms. PMID:23467738

The impact of medical insurance coverage and molecular monitoring frequency on outcomes in chronic myeloid leukemia: real-world evidence in China.

PubMed

Sheng, Guangying; Chen, Suning; Zhang, Ri; Miao, Miao; Wu, Depei; Tan, Seng Chuen; Liu, Chao; Xiong, Tengbin

2017-04-01

Imatinib (Glivec) has been covered by critical disease insurance for treatment of chronic myeloid leukemia (CML) in Jiangsu province of China since 2013. Further, free molecular monitoring has been provided to patients at top clinical centers as part of a pilot study that has changed the local treatment pattern and outcomes of patients with CML. This study evaluates the impact of medical insurance coverage and the molecular monitoring frequency on outcomes of patients with CML treated at a central hospital in Jiangsu, China, according to patient-level data. The study investigated 335 CML patients receiving medical treatment in a central hospital between January 1, 2011 and December 31, 2014. Demographic and clinical characteristics were extracted from the patients' clinical records. Univariate and multivariate analyses using the logistic regression model were performed to identify the differences in outcomes of major molecular response (MMR) or complete cytogenetic response (CCyR) between patients who were insured vs uninsured, or between patients with frequency of PCR monitoring ≤2 times vs ≥3 times per year. Both the achievement of MMR (BCR-ABL IS ≤0.1%) (50.4% vs 37.5%) and CCyR (80.7% vs 62.8%) at 12 months have shown significant differences that favored patients with insurance coverage of imatinib, while there was no significant difference in the outcome of BCR-ABL IS ≤1% between insured and non-insured groups (56.0% vs 51.3%) at 6 months. The long-term results at 24 months demonstrated that there was a statistically significant difference in MMR rates between the group with 3 or more PCR monitoring tests per year and the group of patients with 2 or less PCR tests per year (76.9% vs 52.2%). The study findings suggest that CML patients benefit from insurance coverage of imatinib and higher frequency (≥3) of regularly scheduled molecular monitoring PCR in China.

Phase 2 Trial of De-intensified Chemoradiation Therapy for Favorable-Risk Human Papillomavirus–Associated Oropharyngeal Squamous Cell Carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chera, Bhishamjit S., E-mail: bchera@med.unc.edu; Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina; Amdur, Robert J.

Purpose: To perform a prospective, multi-institutional, phase 2 study of a substantial decrease in concurrent chemoradiation therapy (CRT) intensity as primary treatment for favorable-risk, human papillomavirus–associated oropharyngeal squamous cell carcinoma. Methods and Materials: The major inclusion criteria were: (1) T0 to T3, N0 to N2c, M0; (2) human papillomavirus or p16 positive; and (3) minimal/remote smoking history. Treatment was limited to 60 Gy intensity modulated radiation therapy with concurrent weekly intravenous cisplatinum (30 mg/m{sup 2}). The primary study endpoint was pathologic complete response (pCR) rate based on required biopsy of the primary site and dissection of pretreatment positive lymph node regions, regardless ofmore » radiographic response. Power computations were performed for the null hypothesis that the pCR rate is 87% and n=40, resulting in a type 1 error of 14.2%. Secondary endpoint measures included physician-reported toxicity (Common Toxicity Terminology for Adverse Events, CTCAE), patient-reported symptoms (PRO-CTCAE), and modified barium swallow studies. Results: The study population was 43 patients. The pCR rate was 86% (37 of 43). The incidence of CTCAE grade 3/4 toxicity and PRO-CTCAE severe/very severe symptoms was as follows: mucositis 34%/45%, general pain 5%/48%, nausea 18%/52%, vomiting 5%/34%, dysphagia 39%/55%, and xerostomia 2%/75%. Grade 3/4 hematologic toxicities were 11%. Thirty-nine percent of patients required a feeding tube for a median of 15 weeks (range, 5-22 weeks). There were no significant differences in modified barium swallow studies before and after CRT. Conclusions: The pCR rate with decreased intensity of therapy with 60 Gy of IMRT and weekly low-dose cisplatinum is very high in favorable-risk oropharyngeal squamous cell carcinoma, with evidence of decreased toxicity compared with standard therapies. (ClinicalTrials.gov) ID: (NCT01530997).« less

Chemoradiotherapy, with adjuvant surgery for local control, confers a durable survival advantage in adenocarcinoma and squamous cell carcinoma of the oesophagus.

PubMed

Bass, G A; Furlong, H; O'Sullivan, K E; Hennessy, T P J; Walsh, T N

2014-04-01

Oesophageal cancer usually presents with systemic disease, necessitating systemic therapy. Neo-adjuvant chemoradiotherapy improves short-term survival, but its long-term impact is disputed because of limited accrual, treatment-protocol heterogeneity and a short follow-up of randomised trials. Long-term results of two simultaneous randomised controlled trials (RCTs) comparing neo-adjuvant chemo-radiotherapy and surgery (MMT) with surgical monotherapy were examined, and the response of adenocarcinoma (AC) and squamous cell carcinoma (SCC) to identical regimens compared. Between 1990 and 1997, two RCTs were undertaken on 211 patients. Patients with AC (n=113) or SCC (n=98) were separately-randomised to identical protocols of MMT or surgical monotherapy. 211 patients were followed to 206 months; 104 patients were randomised to MMT (58 AC and 46 SCC, respectively) and 107 to surgery. MMT provided a significant survival-advantage over surgical monotherapy for AC (P=0.004), SCC (P=0.01). There was a 54% relative risk-reduction in lymph-node metastasis following MMT, compared with surgery (64% versus 29%, P<0.001). MMT produced a pathologic complete response (pCR) in 25% and 31% of AC and SCC, respectively. Survival advantage accrued to MMT, pCR and node-negative patients: AC pCR versus surgical monotherapy (P=0.001); residual disease following MMT versus surgical monotherapy (P=0.008); SCC pCR versus surgical monotherapy (P=0.033). A survival advantage for MMT persisted long-term in AC and was replicated in SCC. MMT produced loco-regional tumour down-staging to extinction in 25-31% of patients, potentially permitting personalised treatment in this cohort that avoids the morbidity and mortality associated with resection. Node-negative patients with residual localised disease following MMT had a survival advantage over node-negative patients following surgery alone, supporting a systemic effect on micro-metastatic disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

DcR3 binds to ovarian cancer via heparan sulfate proteoglycans and modulates tumor cells response to platinum with corresponding alteration in the expression of BRCA1

PubMed Central

2012-01-01

Background Overcoming platinum resistance is a major obstacle in the treatment of Epithelial Ovarian Cancer (EOC). In our previous work Decoy Receptor 3 (DcR3) was found to be related to platinum resistance. The major objective of this work was to define the cellular interaction of DcR3 with EOC and to explore its effects on platinum responsiveness. Methods We studied cell lines and primary cultures for the expression of and the cells ability to bind DcR3. Cells were cultured with DcR3 and then exposed to platinum. Cell viability was determined by MTT assay. Finally, the cells molecular response to DcR3 was studied using real time RT-PCR based differential expression arrays, standard RT-PCR, and Western blot. Results High DcR3 in the peritoneal cavity of women with EOC is associated with significantly shorter time to first recurrence after platinum based therapy (p = 0.02). None-malignant cells contribute DcR3 in the peritoneal cavity. The cell lines studied do not secrete DcR3; however they all bind exogenous DcR3 to their surface implying that they can be effected by DcR3 from other sources. DcR3s protein binding partners are minimally expressed or negative, however, all cells expressed the DcR3 binding Heparan Sulfate Proteoglycans (HSPGs) Syndecans-2, and CD44v3. DcR3 binding was inhibited by heparin and heparinase. After DcR3 exposure both SKOV-3 and OVCAR-3 became more resistant to platinum with 15% more cells surviving at high doses. On the contrary CaOV3 became more sensitive to platinum with 20–25% more cell death. PCR array analysis showed increase expression of BRCA1 mRNA in SKOV-3 and OVCAR-3 and decreased BRCA1 expression in CaOV-3 after exposure to DcR3. This was confirmed by gene specific real time PCR and Western blot analysis. Conclusions Non-malignant cells contribute to the high levels of DcR3 in ovarian cancer. DcR3 binds readily to EOC cells via HSPGs and alter their responsiveness to platinum chemotherapy. The paradoxical responses seen were related to the expression pattern of HSPGs available on the cells surface to interact with. Although the mechanism behind this is not completely known alterations in DNA repair pathways including the expression of BRCA1 appear to be involved. PMID:22583667

Adaptive Randomization of Neratinib in Early Breast Cancer

PubMed Central

Park, John W.; Liu, Minetta C.; Yee, Douglas; Yau, Christina; van 't Veer, Laura J.; Symmans, W. Fraser; Paoloni, Melissa; Perlmutter, Jane; Hylton, Nola M.; Hogarth, Michael; DeMichele, Angela; Buxton, Meredith B.; Chien, A. Jo; Wallace, Anne M.; Boughey, Judy C.; Haddad, Tufia C.; Chui, Stephen Y.; Kemmer, Kathleen A.; Kaplan, Henry G.; Liu, Minetta C.; Isaacs, Claudine; Nanda, Rita; Tripathy, Debasish; Albain, Kathy S.; Edmiston, Kirsten K.; Elias, Anthony D.; Northfelt, Donald W.; Pusztai, Lajos; Moulder, Stacy L.; Lang, Julie E.; Viscusi, Rebecca K.; Euhus, David M.; Haley, Barbara B.; Khan, Qamar J.; Wood, William C.; Melisko, Michelle; Schwab, Richard; Lyandres, Julia; Davis, Sarah E.; Hirst, Gillian L.; Sanil, Ashish; Esserman, Laura J.; Berry, Donald A.

2017-01-01

Background I-SPY2, a standing, multicenter, adaptive phase 2 neoadjuvant trial ongoing in high-risk clinical stage II/III breast cancer, is designed to evaluate multiple, novel experimental agents added to standard chemotherapy for their ability to improve the rate of pathologic complete response (pCR). Experimental therapies are compared against a common control arm. We report efficacy for the tyrosine kinase inhibitor neratinib. Methods Eligible women had ≥2.5 cm stage II/III breast cancer, categorized into 8 biomarker subtypes based on HER2, hormone-receptor status (HR), and MammaPrint. Neratinib was evaluated for 10 signatures (prospectively defined subtype combinations), with primary endpoint pCR. MR volume changes inform likelihood of pCR for each patient prior to surgery. Adaptive assignment to experimental arms within disease subtype was based on current Bayesian probabilities of superiority over control. Accrual to experimental arm stop at any time for futility or graduation within a particular signature based on Bayesian predictive probability of success in a confirmatory trial. The maximum sample size in any experimental arm is 120 patients, Results With 115 patients and 78 concurrently randomized controls, neratinib graduated in the HER2+/HR− signature, with mean pCR rate 56% (95% PI: 37 to 73%) vs 33% for controls (11 to 54%). Final predictive probability of success, updated when all pathology data were available, was 79%. Conclusion Adaptive, multi-armed trials can efficiently identify responding tumor subtypes. Neratinib added to standard therapy is highly likely to improve pCR rates in HER2+/HR2212; breast cancer. Confirmation in I-SPY 3, a phase 3 neoadjuvant registration trial, is planned. PMID:27406346

Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay.

PubMed

Corey, Lawrence; Huang, Meei-Li; Selke, Stacy; Wald, Anna

2005-07-01

While the clinical manifestations of HSV-1 and -2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV-1 and -2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR-based assay to accurately distinguish HSV-1 from HSV-2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV-1 and -2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV-1 and -2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV-1 and -2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques. Copyright (c) 2005 Wiley-Liss, Inc.

[Rapid identification of meningitis due to bacterial pathogens].

PubMed

Ubukata, Kimiko

2013-01-01

We constructed a new real-time PCR method to detect causative pathogens in cerebrospinal fluid (CSF) from patient due to bacterial meningitis. The eight pathogens targeted in the PCR are Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus agalactiae, Staphylococcus aurues, Neisseria meningitides, Listeria monocytogenes, Esherichia coli, and Mycoplasma pneumoniae. The total time from DNA extraction from CSF to PCR analysis was 1.5 hour. The pathogens were detected in 72% of the CSF samples (n=115) by real-time PCR, but in only 48% by culture, although the microorganisms were completely concordant. The detection rate of pathogens with PCR was significantly better than that with cultures in patients with antibiotic administration.In conclusion, detection with real-time PCR is useful for rapidly identifying the causative pathogens of meningitis and for examining the clinical course of chemotherapy.

FPA-FTIR Microspectroscopy for Monitoring Chemotherapy Efficacy in Triple-Negative Breast Cancer

NASA Astrophysics Data System (ADS)

Zawlik, Izabela; Kaznowska, Ewa; Cebulski, Jozef; Kolodziej, Magdalena; Depciuch, Joanna; Vongsvivut, Jitraporn; Cholewa, Marian

2016-11-01

Triple-negative breast cancer is the most aggressive breast cancer subtype with limited treatment options and a poor prognosis. Approximately 70% of triple-negative breast cancer patients fail to achieve a pathologic complete response (pCR) after chemotherapy due to the lack of targeted therapies for this subtype. We report here the development of a focal-plane-array Fourier transform infrared (FPA-FTIR) microspectroscopic technique combined with principal component analysis (PCA) for monitoring chemotherapy effects in triple-negative breast cancer patients. The PCA results obtained using the FPA-FTIR spectral data collected from the same patients before and after the chemotherapy revealed discriminatory features that were consistent with the pathologic and clinical responses to chemotherapy, indicating the potential of the technique as a monitoring tool for observing chemotherapy efficacy.

Metronomic chemotherapy for non-metastatic triple negative breast cancer: Selection is the key

PubMed Central

Rabanal, Connie; Ruiz, Rossana; Neciosup, Silvia; Gomez, Henry

2017-01-01

Triple negative breast cancer (TNBC) accounts for 15%-20% of all breast cancer, and is still defined as what it is not. Currently, TNBC is the only type of breast cancer for which there are no approved targeted therapies and maximum tolerated dose chemotherapy with taxanes and anthracycline-containing regimens is still the standard of care in both the neoadjuvant and adjuvant settings. In the last years, metronomic chemotherapy (MC) is being explored as an alternative to improve outcomes in TNBC. In the neoadjuvant setting, purely metronomic and hybrid approaches have been developed with the objective of increasing complete pathologic response (pCR) and prolonging disease free survival. These regimens proved to be very effective achieving pCR rates between 47%-60%, but at the cost of great toxicity. In the adjuvant setting, MC is used to intensify adjuvant chemotherapy and, more promisingly, as maintenance therapy for high-risk patients, especially those with no pCR after neoadjuvant chemotherapy. Considering the dismal prognosis of TNBC, any strategy that potentially improves outcomes, specially being the oral agents broadly available and inexpensive, should be considered and certainly warrants further exploration. Finally, the benefit of MC needs to be validated in properly designed clinical trials were the selection of the population is the key. PMID:29291168

Interim heterogeneity changes measured using entropy texture features on T2-weighted MRI at 3.0 T are associated with pathological response to neoadjuvant chemotherapy in primary breast cancer.

PubMed

Henderson, Shelley; Purdie, Colin; Michie, Caroline; Evans, Andrew; Lerski, Richard; Johnston, Marilyn; Vinnicombe, Sarah; Thompson, Alastair M

2017-11-01

To investigate whether interim changes in hetereogeneity (measured using entropy features) on MRI were associated with pathological residual cancer burden (RCB) at final surgery in patients receiving neoadjuvant chemotherapy (NAC) for primary breast cancer. This was a retrospective study of 88 consenting women (age: 30-79 years). Scanning was performed on a 3.0 T MRI scanner prior to NAC (baseline) and after 2-3 cycles of treatment (interim). Entropy was derived from the grey-level co-occurrence matrix, on slice-matched baseline/interim T2-weighted images. Response, assessed using RCB score on surgically resected specimens, was compared statistically with entropy/heterogeneity changes and ROC analysis performed. Association of pCR within each tumour immunophenotype was evaluated. Mean entropy percent differences between examinations, by response category, were: pCR: 32.8%, RCB-I: 10.5%, RCB-II: 9.7% and RCB-III: 3.0%. Association of ultimate pCR with coarse entropy changes between baseline/interim MRI across all lesions yielded 85.2% accuracy (area under ROC curve: 0.845). Excellent sensitivity/specificity was obtained for pCR prediction within each immunophenotype: ER+: 100%/100%; HER2+: 83.3%/95.7%, TNBC: 87.5%/80.0%. Lesion T2 heterogeneity changes are associated with response to NAC using RCB scores, particularly for pCR, and can be useful across all immunophenotypes with good diagnostic accuracy. • Texture analysis provides a means of measuring lesion heterogeneity on MRI images. • Heterogeneity changes between baseline/interim MRI can be linked with ultimate pathological response. • Heterogeneity changes give good diagnostic accuracy of pCR response across all immunophenotypes. • Percentage reduction in heterogeneity is associated with pCR with good accuracy and NPV.

[Application of droplet digital PCR technology for genetic testing and prenatal diagnosis of spinal muscular atrophy].

PubMed

Zou, Yang; Xu, Peiwen; Li, Jie; Huang, Sexin; Gao, Ming; Kang, Ranran; Gao, Xuan; Gao, Yuan

2016-10-01

To explore the clinical application of droplet digital PCR (ddPCR) for genetic testing and prenatal diagnosis of spinal muscular atrophy (SMA) with deletion of SMN1 gene exon 7. A total of 138 clinical samples, including 121 peripheral blood, 13 amniotic fluid, 2 umbilical cord blood and 2 chorionic villi from 56 SMA families, were tested by both ddPCR and multiplex ligation-dependent probe amplification (MLPA). Results of the two approaches were analyzed with commercial software QuantaSoft (ddPCR) and Coffalyser (MLPA), respectively. Among the 138 cases, 25 had two copies, 84 had one copy, and 29 had null copy of exon 7 of the SMN1 gene. The results of ddPCR and MLPA were completely consistent. As a rapid, precise and economically efficient method, ddPCR will provide a new choice for genetic testing of SMA.

Molecular Surveillance of True Nontypeable Haemophilus influenzae: An Evaluation of PCR Screening Assays

PubMed Central

Binks, Michael J.; Temple, Beth; Kirkham, Lea-Ann; Wiertsema, Selma P.; Dunne, Eileen M.; Richmond, Peter C.; Marsh, Robyn L.; Leach, Amanda J.; Smith-Vaughan, Heidi C.

2012-01-01

Background Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Methodology/Principal Findings Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Conclusions/Significance Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease. PMID:22470516

Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays.

PubMed

Binks, Michael J; Temple, Beth; Kirkham, Lea-Ann; Wiertsema, Selma P; Dunne, Eileen M; Richmond, Peter C; Marsh, Robyn L; Leach, Amanda J; Smith-Vaughan, Heidi C

2012-01-01

Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.

High-intensity interval training increases in vivo oxidative capacity with no effect on P(i)→ATP rate in resting human muscle.

PubMed

Larsen, Ryan G; Befroy, Douglas E; Kent-Braun, Jane A

2013-03-01

Mitochondrial ATP production is vital for meeting cellular energy demand at rest and during periods of high ATP turnover. We hypothesized that high-intensity interval training (HIT) would increase ATP flux in resting muscle (VPi→ATP) in response to a single bout of exercise, whereas changes in the capacity for oxidative ATP production (Vmax) would require repeated bouts. Eight untrained men (27 ± 4 yr; peak oxygen uptake = 36 ± 4 ml·kg(-1)·min(-1)) performed six sessions of HIT (4-6 × 30-s bouts of all-out cycling with 4-min recovery). After standardized meals and a 10-h fast, VPi→ATP and Vmax of the vastus lateralis muscle were measured using phosphorus magnetic resonance spectroscopy at 4 Tesla. Measurements were obtained at baseline, 15 h after the first training session, and 15 h after completion of the sixth session. VPi→ATP was determined from the unidirectional flux between Pi and ATP, using the saturation transfer technique. The rate of phosphocreatine recovery (kPCr) following a maximal contraction was used to calculate Vmax. While kPCr and Vmax were unchanged after a single session of HIT, completion of six training sessions resulted in a ∼14% increase in muscle oxidative capacity (P ≤ 0.004). In contrast, neither a single nor six training sessions altered VPi→ATP (P = 0.74). This novel analysis of resting and maximal high-energy phosphate kinetics in vivo in response to HIT provides evidence that distinct aspects of human skeletal muscle metabolism respond differently to this type of training.

Breath-to-breath hypercapnic response in neonatal rats: temperature dependency of the chemoreflexes and potential implications for breathing stability.

PubMed

Cummings, Kevin J; Frappell, Peter B

2009-07-01

The breathing of newborns is destabilized by warm temperatures. We hypothesized that in unanesthetized, intact newborn rats, body temperature (T(B)) influences the peripheral chemoreflex response (PCR response) to hypercapnia. To test this, we delivered square-wave challenges of 8% CO(2) in air to postnatal day 4-5 (P4-P5) rats held at a T(B) of 30 degrees C (Cold group, n = 11), 33 degrees C (Cool group, n = 10), and 35 degrees C thermoneutral zone group [thermoneutral zone (TNZ) group, n = 11], while measuring ventilation (Ve) directly with a pneumotach and mask. Cool animals were challenged with 8% CO(2) balanced in either air or hyperoxia (n = 10) to identify the PCR response. Breath-to-breath analysis was performed on 30 room air breaths and every breath of the 1-min CO(2) challenge. As expected, warmer T(B) was associated with an unstable breathing pattern in room air: TNZ animals had a coefficient of variation in Ve (Ve CV%) that was double that of animals held at cooler T(B) (P < 0.001). Hyperoxia markedly suppressed the hypercapnic ventilatory response over the first 10 breaths (or approximately 4 s), suggesting that this domain is dominated by the PCR response. The PCR response (P = 0.03) and total response (P = 0.04) were significantly greater in TNZ animals compared with hypothermic animals. The total response had a significant, negative relationship with Vco(2) (R(2) = 0.53; P < 0.001). Breathing stability was positively related to the total response (R(2) = 0.36; P < 0.001) and to a lesser extent, the PCR response (R(2) = 0.19; P = 0.01) and was negatively related to Vco(2) (R(2) = 0.34; P < 0.001). ANCOVA confirmed a significant effect of T(B) alone on breathing stability (P < 0.01), with no independent effects of Vco(2) (P = 0.41), the PCR response (P = 0.82), or the total Ve response (P = 0.08). Our data suggest that in early postnatal life, the chemoreflex responses to CO(2) are highly influenced by T(B), and while related to breathing stability, are not predictors of stability after accounting for the independent effect of T(B).

Contrast-enhanced spectral mammography in neoadjuvant chemotherapy monitoring: a comparison with breast magnetic resonance imaging.

PubMed

Iotti, Valentina; Ravaioli, Sara; Vacondio, Rita; Coriani, Chiara; Caffarri, Sabrina; Sghedoni, Roberto; Nitrosi, Andrea; Ragazzi, Moira; Gasparini, Elisa; Masini, Cristina; Bisagni, Giancarlo; Falco, Giuseppe; Ferrari, Guglielmo; Braglia, Luca; Del Prato, Alberto; Malavolti, Ivana; Ginocchi, Vladimiro; Pattacini, Pierpaolo

2017-09-11

Neoadjuvant-chemotherapy (NAC) is considered the standard treatment for locally advanced breast carcinomas. Accurate assessment of disease response is fundamental to increase the chances of successful breast-conserving surgery and to avoid local recurrence. The purpose of this study was to compare contrast-enhanced spectral mammography (CESM) and contrast-enhanced-MRI (MRI) in the evaluation of tumor response to NAC. This prospective study was approved by the institutional review board and written informed consent was obtained. Fifty-four consenting women with breast cancer and indication of NAC were consecutively enrolled between October 2012 and December 2014. Patients underwent both CESM and MRI before, during and after NAC. MRI was performed first, followed by CESM within 3 days. Response to therapy was evaluated for each patient, comparing the size of the residual lesion measured on CESM and MRI performed after NAC to the pathological response on surgical specimens (gold standard), independently of and blinded to the results of the other test. The agreement between measurements was evaluated using Lin's coefficient. The agreement between measurements using CESM and MRI was tested at each step of the study, before, during and after NAC. And last of all, the variation in the largest dimension of the tumor on CESM and MRI was assessed according to the parameters set in RECIST 1.1 criteria, focusing on pathological complete response (pCR). A total of 46 patients (85%) completed the study. CESM predicted pCR better than MRI (Lin's coefficient 0.81 and 0.59, respectively). Both methods tend to underestimate the real extent of residual tumor (mean 4.1mm in CESM, 7.5mm in MRI). The agreement between measurements using CESM and MRI was 0.96, 0.94 and 0.76 before, during and after NAC respectively. The distinction between responders and non-responders with CESM and MRI was identical for 45/46 patients. In the assessment of CR, sensitivity and specificity were 100% and 84%, respectively, for CESM, and 87% and 60% for MRI. CESM and MRI lesion size measurements were highly correlated. CESM seems at least as reliable as MRI in assessing the response to NAC, and may be an alternative if MRI is contraindicated or its availability is limited.

Factors influencing polymerase chain reaction outcomes in patients with clinically suspected ocular tuberculosis.

PubMed

Balne, Praveen Kumar; Modi, Rohit Ramesh; Choudhury, Nuzhat; Mohan, Neha; Barik, Manas Ranjan; Padhi, Tapas Ranjan; Sharma, Savitri; Panigrahi, Satya Ranjan; Basu, Soumyava

2014-03-25

Polymerase chain reaction (PCR) assay can be a useful method for definitive diagnosis in paucibacillary infections such as ocular tuberculosis (TB). In this study, we have evaluated factors affecting PCR outcomes in patients with clinically suspected ocular TB. Patients with clinically suspected ocular TB were investigated by PCR of aqueous or vitreous samples. Three control groups were also tested: group 1 included culture-proven non-tuberculous endophthalmitis, group 2 culture-negative non-tuberculous endophthalmitis, and group 3 patients undergoing surgery for uncomplicated cataract. PCR targeted one or more of following targets: IS6110, MPB64, and protein b genes of Mycobacterium tuberculosis complex. Multiple regression analysis (5% level of significance) was done to evaluate the associations between positive PCR outcome and laterality of disease, tuberculin skin test (TST)/interferon-gamma release assay (IGRA), chest radiography, and type of sample (aqueous or vitreous). The main outcome measures were positive PCR by one or more gene targets, and factors influencing positive PCR outcomes. All 114 samples were tested for MPB64, 110 for protein b, and 88 for IS6110. MPB64 was positive in 70.2% (n = 80) of tested samples, protein b in 40.0% (n = 44), and IS6110 in only 9.1% (n = 8). DNA sequencing of amplicons from four randomly chosen PCR reactions showed homology for M. tuberculosis complex. Of the 80 PCR-positive patients, 71 completed a full course of antitubercular therapy, of which 65 patients (91.5%) had complete resolution of inflammation at final follow-up. Among controls, 12.5% (3 out of 24) in group 1 and 18.7% (6 out of 32) in group 2 also tested positive by PCR. No PCR-positive outcome was observed in control group 3 (n = 25). Multiple regression analysis revealed significant association of positive PCR outcome with bilateral presentation, but not with a positive TST/IGRA, chest radiography, or type of sample (aqueous/vitreous) used. Careful selection of gene targets can yield high PCR positivity in clinically suspected ocular TB. Bilateral disease presentation but not any evidence of latent systemic TB influences PCR outcomes. False-positive results may be seen in ocular inflammation unrelated to ocular TB.

HannaH phase III randomised study: Association of total pathological complete response with event-free survival in HER2-positive early breast cancer treated with neoadjuvant-adjuvant trastuzumab after 2 years of treatment-free follow-up.

PubMed

Jackisch, Christian; Hegg, Roberto; Stroyakovskiy, Daniil; Ahn, Jin-Seok; Melichar, Bohuslav; Chen, Shin-Cheh; Kim, Sung-Bae; Lichinitser, Mikhail; Starosławska, Elżbieta; Kunz, Georg; Falcon, Silvia; Chen, Shou-Tung; Crepelle-Fléchais, Aulde; Heinzmann, Dominik; Shing, Mona; Pivot, Xavier

2016-07-01

In the phase III, open-label, randomised HannaH study, fixed-dose neoadjuvant-adjuvant subcutaneous trastuzumab for human epidermal growth factor receptor 2 (HER2)-positive early breast cancer was non-inferior to standard weight-based intravenous infusion in terms of serum trough concentration and pathological complete response (pCR). Evidence suggests that pCR, particularly total pCR (tpCR), is likely to predict clinical benefit. We report associations between tpCR and event-free survival (EFS) from HannaH (the largest population from a single study of patients presenting with newly diagnosed HER2-positive breast cancer treated with neoadjuvant-adjuvant trastuzumab to date) plus long-term efficacy and safety. Eligible patients received four cycles of neoadjuvant docetaxel followed by four cycles of fluorouracil/epirubicin/cyclophosphamide administered concurrently with 3-weekly subcutaneous (600 mg fixed dose) or intravenous trastuzumab (8 mg/kg loading, 6 mg/kg maintenance doses). Post-surgery, patients received adjuvant trastuzumab as randomised to complete 1 year of standard treatment. In exploratory analyses, we used Cox regression to assess associations between tpCR and EFS. EFS rates per subgroup were estimated using the Kaplan-Meier method. Three-year EFS rates were 76% for subcutaneous and 73% for intravenous trastuzumab (unstratified hazard ratio [HR] 0.95, 95% confidence interval [CI] 0.69-1.30; intention-to-treat population). Three-year overall survival rates were 92% for subcutaneous and 90% for intravenous trastuzumab (unstratified HR 0.76, 95% CI 0.44-1.32). tpCR was associated with a reduced risk of an EFS event: subcutaneous arm HR 0.38 (95% CI 0.22-0.65); intravenous arm HR 0.32 (95% CI 0.18-0.60). Results were similar for subgroups, including oestrogen receptor status. The few additional adverse events occurring during treatment-free follow-up were balanced between arms. Long-term efficacy supports the established non-inferiority of subcutaneous trastuzumab, and its safety profile remains consistent with the known intravenous profile. In each of HannaH's treatment arms, tpCR was associated with improved EFS, adding to evidence that tpCR is associated with clinical benefit in HER2-positive early breast cancer. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

DNA Repair Biomarkers Predict Response to Neoadjuvant Chemoradiotherapy in Esophageal Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alexander, Brian M., E-mail: bmalexander@lroc.harvard.edu; Wang Xiaozhe; Niemierko, Andrzej

2012-05-01

Purpose: The addition of neoadjuvant chemoradiotherapy prior to surgical resection for esophageal cancer has improved clinical outcomes in some trials. Pathologic complete response (pCR) following neoadjuvant therapy is associated with better clinical outcome in these patients, but only 22% to 40% of patients achieve pCR. Because both chemotherapy and radiotherapy act by inducing DNA damage, we analyzed proteins selected from multiple DNA repair pathways, using quantitative immunohistochemistry coupled with a digital pathology platform, as possible biomarkers of treatment response and clinical outcome. Methods and Materials: We identified 79 patients diagnosed with esophageal cancer between October 1994 and September 2002, withmore » biopsy tissue available, who underwent neoadjuvant chemoradiotherapy prior to surgery at the Massachusetts General Hospital and used their archived, formalin-fixed, paraffin-embedded biopsy samples to create tissue microarrays (TMA). TMA sections were stained using antibodies against proteins in various DNA repair pathways including XPF, FANCD2, PAR, MLH1, PARP1, and phosphorylated MAPKAP kinase 2 (pMK2). Stained TMA slides were evaluated using machine-based image analysis, and scoring incorporated both the intensity and the quantity of positive tumor nuclei. Biomarker scores and clinical data were assessed for correlations with clinical outcome. Results: Higher scores for MLH1 (p = 0.018) and lower scores for FANCD2 (p = 0.037) were associated with pathologic response to neoadjuvant chemoradiation on multivariable analysis. Staining of MLH1, PARP1, XPF, and PAR was associated with recurrence-free survival, and staining of PARP1 and FANCD2 was associated with overall survival on multivariable analysis. Conclusions: DNA repair proteins analyzed by immunohistochemistry may be useful as predictive markers for response to neoadjuvant chemoradiotherapy in patients with esophageal cancer. These results are hypothesis generating and need confirmation in an independent data set.« less

The effect of classical swine fever virus NS5A and NS5A mutants on oxidative stress and inflammatory response in swine testicular cells.

PubMed

Dong, Wang; Lv, Huifang; Wang, Yifan; Li, Xiaomeng; Li, Cheng; Wang, Lu; Wang, Chengbao; Guo, Kangkang; Zhang, Yanming

2017-06-01

Infection with classical swine fever virus (CSFV) results in highly significant economic losses; this infection is characterized by being highly contagious and accompanied by hyperthermia and systemic bleeding. Oxidative stress (OS) plays a critical role in the pathological process of viral infection. The function of the nonstructural protein 5A (NS5A) in the pathogenesis of CSFV has not been completely understood. Here, OS and the inflammatory response were studied with NS5A and substitution mutants in swine testicular (ST) cells. ST cell lines stably expressing CSFV NS5A or substitution mutants were established. Reactive oxygen species (ROS) production, antioxidant protein expression and inflammatory response were analyzed by quantitative real-time PCR (qRT-PCR), ELISA and flow cytometry analysis. The results showed that CSFV NS5A did not increase ROS production or the antioxidant protein (Trx, HO-1 and PRDX-6) expression in ST cells. However, NS5A inhibited cyclooxygenase-2 (COX-2) expression, a pro-inflammatory protein related to OS. Further studies have shown that NS5A mutants S15A and S92A increased ROS production and inhibited antioxidant protein expression. S15A, S81A and T274A affected the inflammatory response. This study suggested that CSFV NS5A did not induce OS, and amino acids Ser15 and Ser92 of CSFV NS5A were essential for inhibiting OS. Additionally, Ser15, Ser81 and Thr274 played important roles in the inflammatory response in ST cells. These observations provided insight into the function of CSFV NS5A and the mechanism of CSFV persistent infection in ST cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular cloning and preliminary function study of iron responsive element binding protein 1 gene from cypermethrin-resistant Culex pipiens pallens

PubMed Central

2011-01-01

Background Insecticide resistance jeopardizes the control of mosquito populations and mosquito-borne disease control, which creates a major public health concern. Two-dimensional electrophoresis identified one protein segment with high sequence homology to part of Aedes aegypti iron-responsive element binding protein (IRE-BP). Method RT-PCR and RACE (rapid amplification of cDNA end) were used to clone a cDNA encoding full length IRE-BP 1. Real-time quantitative RT-PCR was used to evaluate the transcriptional level changes in the Cr-IRE strain Aedes aegypti compared to the susceptible strain of Cx. pipiens pallens. The expression profile of the gene was established in the mosquito life cycle. Methyl tritiated thymidine (3H-TdR) was used to observe the cypermethrin resistance changes in C6/36 cells containing the stably transfected IRE-BP 1 gene of Cx. pipiens pallens. Results The complete sequence of iron responsive element binding protein 1 (IRE-BP 1) has been cloned from the cypermethrin-resistant strain of Culex pipiens pallens (Cr-IRE strain). Quantitative RT-PCR analysis indicated that the IRE-BP 1 transcription level was 6.7 times higher in the Cr-IRE strain than in the susceptible strain of 4th instar larvae. The IRE-BP 1 expression was also found to be consistently higher throughout the life cycle of the Cr-IRE strain. A protein of predicted size 109.4 kDa has been detected by Western blotting in IRE-BP 1-transfected mosquito C6/36 cells. These IRE-BP 1-transfected cells also showed enhanced cypermethrin resistance compared to null-transfected or plasmid vector-transfected cells as determined by 3H-TdR incorporation. Conclusion IRE-BP 1 is expressed at higher levels in the Cr-IRE strain, and may confer some insecticide resistance in Cx. pipiens pallens. PMID:22075242

MicroRNA-16 Alleviates Inflammatory Pain by Targeting Ras-Related Protein 23 (RAB23) and Inhibiting p38 MAPK Activation.

PubMed

Chen, Wenjin; Guo, Shengdong; Wang, Shenggang

2016-10-22

BACKGROUND The purpose of our study was to determine the functional role of microRNA (miR)-16 in chronic inflammatory pain and to disclose its underlying molecular mechanism. MATERIAL AND METHODS Inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) to Wistar rats. The pWPXL-miR-16, PcDNA3.1- Ras-related protein (RAB23), and/or SB203580 were delivered intrathecally to the rats. Behavioral tests were detected at 0 h, 4 h, 1 d, 4 d, 7 d, and 14 d after CFA injection. After behavioral tests, L4-L6 dorsal spinal cord were obtained and the levels of miR-16, RAB23, and phosphorylation of p38 (p-p38) were evaluated by quantitative real-time PCR (qRT-PCR). In addition, luciferase reporter assay was performed to explore whether RAB23 was a target of miR-16, and qRT-PCR and Western blotting were used to confirm the regulation between RAB23 and miR-16. RESULTS The level of miR-16 was significantly decreased in the CFA-induced inflammatory pain. Intrathecal injection of miR-16 alleviates pain response and raised pain threshold. The level of RAB23 was significantly increased in the pain model, and intrathecal injection of RAB23 aggravated pain response. Luciferase reporter assay confirmed that RAB23 was a direct target of miR-16, and RAB23 was negatively regulated by miR-16. In addition, we found that simultaneous administration of SB203580 and miR-16 further alleviates pain response compared to only administration of miR-16. CONCLUSIONS Our findings suggest that miR-16 relieves chronic inflammatory pain by targeting RAB23 and inhibiting p38 MAPK activation.

Selective elimination of breast cancer surgery in exceptional responders: historical perspective and current trials.

PubMed

van la Parra, Raquel F D; Kuerer, Henry M

2016-03-08

With improvements in chemotherapy regimens, targeted therapies, and our fundamental understanding of the relationship of tumor subtype and pathologic complete response (pCR), there has been dramatic improvement in pCR rates in the past decade, especially among triple-negative and human epidermal growth factor receptor 2-positive breast cancers. Rates of pCR in these groups of patients can be in the 60 % range and thus question the paradigm for the necessity of breast and nodal surgery in all cases, particularly when the patient will be receiving adjuvant local therapy with radiotherapy. Current practice for patients who respond well to neoadjuvant chemotherapy (NCT) is often to proceed with the same breast and axillary procedures as would have been offered women who had not received NCT, regardless of the apparent clinical response. Given these high response rates in defined subgroups among exceptional responders it is appropriate to question whether surgery is now a redundant procedure in their overall management. Further, definitive radiation without surgical resection with or without systemic therapy has been proven effective for several other malignant disease sites including some stages of esophageal, anal, laryngeal, prostate, cervical, and lung carcinoma. The main impediments for potential elimination of surgery have been the fact that prior and current standard and functional breast imaging methods are incapable of accurate prediction of residual disease and that integrating percutaneous biopsy of the breast primary and nodes following NCT may circumvent this issue. This article highlights historical attempts at omission of surgery following NCT in an earlier era, the current status of breast and nodal imaging to predict residual carcinoma, and ongoing and planned trials designed to identify appropriate patients who might be selected for clinical trials designed to test the safety of selected elimination of breast cancer surgery in percutaneous image-guided biopsy-proven exceptional responders to NCT.

A PCR Based Microbial Monitoring Alternative Method of Detection and Identification of Microbes Aboard ISS

NASA Technical Reports Server (NTRS)

Khodadad, Christina; Oubre, Cherie; Castro, Victoria; Flint, Stephanie; Ott, Mark; Roman, Monserrate; Wheeler, Ray; Melendez, Orlando

2017-01-01

Previous research has shown that microorganisms and potential human pathogens have been detected on the International Space Station (ISS) with additional introduction of new microflora occurring with every exchange of crew or addition of equipment and supplies. These microbes are readily transferred between crew and subsystems (i.e. ECLSS, environmental control and life support systems). As this can be detrimental to astronaut health and optimal performance of ISS systems, monitoring of systems such as ECLSS to include identification of microbial contaminants could prevent adverse effects on human health and life support systems. Current monitoring on ISS is laborious and utilizes culture based methods followed by sample return to Earth for complete analysis. Future, long-distance spaceflight missions will require real-time monitoring capabilities that enable efficient and rapid assessments of the microbial environment allowing for expedited decisions and more targeted response to cope with anomalies. Polymerase chain reaction (PCR), a molecular microbial monitoring method was chosen and numerous PCR instruments investigated for their potential to perform in microgravity conditions. Using ISS as a test bed for PCR verification in microgravity will enable NASA to assess whether molecular based microbiological sensors may be components of reliable, closed-loop life support and habitation systems in spacecraft, enhancing infrastructure capabilities through increased efficiency, reliability, and time savings by enabling sample analysis on orbit. NASA selected the Water Monitoring Suite as one of the rapid spaceflight hardware demonstration activities utilizing a streamlined process to minimize the time required to fly experimental flight hardware. The RAZOR EX (BioFire Defense, Salt Lake City, UT) system was part of the water monitoring suite and is a commercial off-the-shelf (COTS) real-time PCR instrument designed for field work. The RAZOR EX was originally designed for Department of Defense (DoD) under a small business innovative research (SBIR) grant and is ruggedized, compact and provides a rapid, sample to answer in less than an hour. PCR assays using a fluorescent probe were optimized and spiked with known concentrations of DNA (Pseudomonas aeruginosa) ranging from 0.002 to 20 ng. PCR reagents were lyophilized and configured in customized pouches and tested for flight readiness. Three types of water were used to rehydrate the reagents and demonstrate the fidelity of the PCR reaction in microgravity. Molecular grade deionized water served as a control while filtered and unfiltered ISS potable water served to test for chemical or biological inhibitors. All three types were compared to parallel ground test results. Nine tests were run on ISS (3 of each water type) and the critical threshold cycle (Ct) was compared to parallel ground tests completed at Kennedy Space Center, FL and Johnson Space Center, TX. All concentrations of Pseudomonas aeruginosa DNA were detected. A comparison of the Ct produced in real time PCR indicated similarity between flight and ground samples. There appeared to be no significant difference between flight or ground PCR reactions or between any of the three water types. This testing demonstrated the ability to perform molecular testing during spaceflight operations with similar sensitivity. It will allow for future ground development of molecular protocols and minimize the need for spaceflight testing. Future testing will include development of additional targets including environmental and health related organisms.

Evaluation of two real time PCR assays for the detection of bacterial DNA in amniotic fluid.

PubMed

Girón de Velasco-Sada, Patricia; Falces-Romero, Iker; Quiles-Melero, Inmaculada; García-Perea, Adela; Mingorance, Jesús

2018-01-01

The aim of this study was to evaluate two non-commercial Real-Time PCR assays for the detection of microorganisms in amniotic fluid followed by identification by pyrosequencing. We collected 126 amniotic fluids from 2010 to 2015 for the evaluation of two Real-Time PCR assays for detection of bacterial DNA in amniotic fluid (16S Universal PCR and Ureaplasma spp. specific PCR). The method was developed in the Department of Microbiology of the University Hospital La Paz. Thirty-seven samples (29.3%) were positive by PCR/pyrosequencing and/or culture, 4 of them were mixed cultures with Ureaplasma urealyticum. The Universal 16S Real-Time PCR was compared with the standard culture (81.8% sensitivity, 97.4% specificity, 75% positive predictive value, 98% negative predictive value). The Ureaplasma spp. specific Real-Time PCR was compared with the Ureaplasma/Mycoplasma specific culture (92.3% sensitivity, 89.4% specificity, 50% positive predictive value, 99% negative predictive value) with statistically significant difference (p=0.005). Ureaplasma spp. PCR shows a rapid response time (5h from DNA extraction until pyrosequencing) when comparing with culture (48h). So, the response time of bacteriological diagnosis in suspected chorioamnionitis is reduced. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification.

PubMed Central

Coleman, S S; Melanson, D M; Biosca, E G; Oliver, J D

1996-01-01

DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed. In addition, PCR amplification of V. vulnificus from oysters seeded with biotype 1 cells was demonstrated. By the methods described, V. vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells. It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters. V. vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin. The protocol used for detection of V. vulnificus cells in eels required less than 5 h to complete. Optimum MgCl2 concentrations for the PCR of V. vulnificus from oysters and eels were different, although the same primer pair was used for both. This is the first report on the detection of cells of V. vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen. PMID:8919800

Quantifying environmental DNA signals for aquatic invasive species across multiple detection platforms.

PubMed

Nathan, Lucas M; Simmons, Megan; Wegleitner, Benjamin J; Jerde, Christopher L; Mahon, Andrew R

2014-11-04

The use of molecular surveillance techniques has become popular among aquatic researchers and managers due to the improved sensitivity and efficiency compared to traditional sampling methods. Rapid expansion in the use of environmental DNA (eDNA), paired with the advancement of molecular technologies, has resulted in new detection platforms and techniques. In this study we present a comparison of three eDNA surveillance platforms: traditional polymerase chain reaction (PCR), quantitative PCR (qPCR), and digital droplet PCR (ddPCR) in which water samples were collected over a 24 h time period from mesocosm experiments containing a population gradient of invasive species densities. All platforms reliably detected the presence of DNA, even at low target organism densities within the first hour. The two quantitative platforms (qPCR and ddPCR) produced similar estimates of DNA concentrations. The analyses completed with ddPCR was faster from sample collection through analyses and cost approximately half the expenditure of qPCR. Although a new platform for eDNA surveillance of aquatic species, ddPCR was consistent with more commonly used qPCR and a cost-effective means of estimating DNA concentrations. Use of ddPCR by researchers and managers should be considered in future eDNA surveillance applications.

The efficacy of rituximab plus Hyper-CVAD regimen in mantle cell lymphoma is independent of FCgammaRIIIa and FCgammaRIIa polymorphisms.

PubMed

Galimberti, S; Palumbo, G A; Caracciolo, F; Benedetti, E; Pelosini, M; Brizzi, S; Ciabatti, E; Fazzi, R; Stelitano, C; Quintana, G; Conte, E; Tibullo, D; Di Raimondo, F; Petrini, M

2007-06-01

Mantle cell lymphoma (MCL) accounts for 3-10% of all non-Hodgkin's lymphomas, with median overall survival not exceeding 3-4 years. Rituximab in combination with the Hyper-CVAD regimen appears the most promising regimen; thus, we adopted it as a first-line treatment strategy in a series of 24 patients. In addition to evaluation of clinical success of the regimen, we investigated a possible role of polymorphism in IgG Fc receptors, FCgammaRIIIa and FCgammaRIIa. The frequencies of FCgammaRIIIa-158 were as follows: V/V=4/24 (17%); V/F=16/24 (66%); F/F=4/24 (17%). Those of the FCgammaRIIa-131 polymorphism were H/H=11/24 (46%), H/R=9/24 (37%), R/R=4/24 (17%). The overall response rate was 62.5%, with 33% of complete responses (CRs) after four cycles of R-Hyper-CVAD. Two-year progression-free survival (PFS) was 78% for 158V/V patients vs 75% for cases carrying phenylalanine (p=0.88). When the FCgammaRIIa polymorphism was assessed, the 2-year PFS was 82% for 131H/H patients vs 75% for those carrying arginine (p=0.26). Eighty-three percent of cases achieved Polymerase Chain Reaction (PCR)-negativity: the progression rate was significantly influenced by the minimal residual disease clearance, with 12% progression in the subgroup of PCR-negative cases versus 67% progression in PCR-positive cases (p=0.008). The achievement of PCRnegativity was not significantly influenced by FCgammaR polymorphisms. Results confirm that rituximab plus Hyper-CVAD is an effective regimen for the induction of prolonged remission in patients with aggressive MCL and suggest that rituximab efficacy is independent of the FCgammaR polymorphisms.

Genetic variants in VEGF pathway genes in neoadjuvant breast cancer patients receiving bevacizumab: Results from the randomized phase III GeparQuinto study.

PubMed

Hein, Alexander; Lambrechts, Diether; von Minckwitz, Gunter; Häberle, Lothar; Eidtmann, Holger; Tesch, Hans; Untch, Michael; Hilfrich, Jörn; Schem, Christian; Rezai, Mahdi; Gerber, Bernd; Dan Costa, Serban; Blohmer, Jens-Uwe; Schwedler, Kathrin; Kittel, Kornelia; Fehm, Tanja; Kunz, Georg; Beckmann, Matthias W; Ekici, Arif B; Hanusch, Claus; Huober, Jens; Liedtke, Cornelia; Mau, Christine; Moisse, Matthieu; Müller, Volkmar; Nekljudova, Valentina; Peuteman, Gilian; Rack, Brigitte; Rübner, Matthias; Van Brussel, Thomas; Wang, Liewei; Weinshilboum, Richard M; Loibl, Sibylle; Fasching, Peter A

2015-12-15

Studies assessing the effect of bevacizumab (BEV) on breast cancer (BC) outcome have shown different effects on progression-free and overall survival, suggesting that a subgroup of patients may benefit from this treatment. Unfortunately, no biomarkers exist to identify these patients. Here, we investigate whether single nucleotide polymorphisms (SNPs) in VEGF pathway genes correlate with pathological complete response (pCR) in the neoadjuvant GeparQuinto trial. HER2-negative patients were randomized into treatment arms receiving either BEV combined with standard chemotherapy or chemotherapy alone. In a pre-planned biomarker study, DNA was collected from 729 and 724 patients, respectively from both treatment arms, and genotyped for 125 SNPs. Logistic regression assessed interaction between individual SNPs and both treatment arms to predict pCR. Five SNPs may be associated with a better response to BEV, but none of them remained significant after correction for multiple testing. The two SNPs most strongly associated, rs833058 and rs699947, were located upstream of the VEGF-A promoter. Odds ratios for the homozygous common, heterozygous and homozygous rare rs833058 genotypes were 2.36 (95% CI, 1.49-3.75), 1.20 (95% CI, 0.88-1.64) and 0.61 (95% CI, 0.34-1.12). Notably, some SNPs in VEGF-A exhibited a more pronounced effect in the triple-negative subgroup. Several SNPs in VEGF-A may be associated with improved pCR when receiving BEV in the neoadjuvant setting. Although none of the observed effects survived correction for multiple testing, our observations are consistent with previous studies on BEV efficacy in BC. Further research is warranted to clarify the predictive value of these markers. © 2015 UICC.

Nanoparticle albumin-bound paclitaxel as neoadjuvant chemotherapy of breast cancer: a systematic review and meta-analysis.

PubMed

Zong, Yu; Wu, Jiayi; Shen, Kunwei

2017-03-07

The value of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) in neoadjuvant systemic therapy for breast cancer remains uncertain. Both electronic databases and proceedings of oncologic meetings were included in systematic literature search. Pooled rates of pathological complete response (pCR), odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using fixed-effect or random-effect model to determine the effect of neoadjuvant nab-paclitaxel. Twenty-one studies with 2357 patients were included, 3 of which were randomized clinical trials. The aggregate pCR(ypT0/is ypN0) rate was 32% (95% CI 25-38%) in unselected breast cancer patients and variated in different subtypes. Within randomized clinical trials, the probability of achieving pCR was significantly higher in the nab-paclitaxel group than in the conventional taxanes group (OR = 1.383, 95%CI 1.141-1.676, p = 0.001). For non-hematological toxic effect, any grade and grade 3-4 peripheral sensory neuropathy occurred more frequently with nab-paclitaxel compared to paclitaxel (any grade, OR = 2.090, 95%CI 1.016-4.302, p = 0.045; grade3-4, OR = 3.766, 95%CI 2.324-6.100, p < 0.001). Hypersensitivity was more common with paclitaxel than nab-paclitaxel at any grade and grade 3-4. nab-paclitaxel is an effective cytotoxic drug in neoadjuvant treatment of breast cancer, especially for aggressive tumors in terms of pCR. Exchange of nab-paclitaxel for conventional taxanes could significantly improve pCR rate with reasonable toxicities.

Benefit of the addition of hormone therapy to neoadjuvant anthracycline-based chemotherapy for breast cancer: comparison of predicted and observed pCR.

PubMed

Generali, Daniele; Corona, Silvia Paola; Pusztai, Lajos; Rouzier, Roman; Allevi, Giovanni; Aguggini, Sergio; Milani, Manuela; Strina, Carla; Frati, Albane

2018-03-01

Neoadjuvant hormonal therapy is generally considered a valid option for hormone receptor positive breast cancer (BC) patients who are unfit for chemotherapy or surgery. Whilst numerous studies analyzed efficacy of neoadjuvant chemotherapy (CT) or endocrine therapy (HT) alone in hormone receptor positive patients, there is a lack of research looking at the usefulness of a preoperative combinatorial approach of CT and HT in this patient subgroup. Using a predictive model previously described in the literature, developed to analyze the probability of benefit from preoperative chemotherapy, we were able to compare pathological complete response (pCR) rates expected with the use of CT alone with the pCR rates reported in a population of 192 patients treated with the combination of tamoxifen plus anthracycline-based CT at Cremona Hospital between 2003 and 2006. Even with a relatively small patient population, this approach provided insightful information for the selection of hormone receptor positive BC patients most likely to benefit from the use of preoperative HT and CT in combination. Whilst no statistically significant benefit was obtained with the addition of tamoxifen to neoadjuvant chemotherapy in the entire population, or in any of the molecular stratification subgroups, the analysis of the calibration curve showed that a combinatorial approach may improve pCR in patients with luminal B tumors. More specific trials should be designed to confirm our initial results. To the best of our knowledge, this is the first report investigating the efficacy of the combination of CT and HT in the neoadjuvant treatment of hormone receptor positive BC.

Organ Preservation in Rectal Adenocarcinoma: a phase II randomized controlled trial evaluating 3-year disease-free survival in patients with locally advanced rectal cancer treated with chemoradiation plus induction or consolidation chemotherapy, and total mesorectal excision or nonoperative management.

PubMed

Smith, J Joshua; Chow, Oliver S; Gollub, Marc J; Nash, Garrett M; Temple, Larissa K; Weiser, Martin R; Guillem, José G; Paty, Philip B; Avila, Karin; Garcia-Aguilar, Julio

2015-10-23

Treatment of patients with non-metastatic, locally advanced rectal cancer (LARC) includes pre-operative chemoradiation, total mesorectal excision (TME) and post-operative adjuvant chemotherapy. This trimodality treatment provides local tumor control in most patients; but almost one-third ultimately die from distant metastasis. Most survivors experience significant impairment in quality of life (QoL), due primarily to removal of the rectum. A current challenge lies in identifying patients who could safely undergo rectal preservation without sacrificing survival benefit and QoL. This multi-institutional, phase II study investigates the efficacy of total neoadjuvant therapy (TNT) and selective non-operative management (NOM) in LARC. Patients with MRI-staged Stage II or III rectal cancer amenable to TME will be randomized to receive FOLFOX/CAPEOX: a) before induction neoadjuvant chemotherapy (INCT); or b) after consolidation neoadjuvant chemotherapy (CNCT), with 5-FU or capecitabine-based chemoradiation. Patients in both arms will be re-staged after completing all neoadjuvant therapy. Those with residual tumor at the primary site will undergo TME. Patients with clinical complete response (cCR) will receive non-operative management (NOM). NOM patients will be followed every 3 months for 2 years, and every 6 months thereafter. TME patients will be followed according to NCCN guidelines. All will be followed for at least 5 years from the date of surgery or--in patients treated with NOM--the last day of treatment. The studies published thus far on the safety of NOM in LARC have compared survival between select groups of patients with a cCR after NOM, to patients with a pathologic complete response (pCR) after TME. The current study compares 3-year disease-free survival (DFS) in an entire population of patients with LARC, including those with cCR and those with pCR. We will compare the two arms of the study with respect to organ preservation at 3 years, treatment compliance, adverse events and surgical complications. We will measure QoL in both groups. We will analyze molecular indications that may lead to more individually tailored treatments in the future. This will be the first NOM trial utilizing a regression schema for response assessment in a prospective fashion. NCT02008656.

Establishing a novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure for the direct detection of gene doping.

PubMed

Beiter, Thomas; Zimmermann, Martina; Fragasso, Annunziata; Armeanu, Sorin; Lauer, Ulrich M; Bitzer, Michael; Su, Hua; Young, William L; Niess, Andreas M; Simon, Perikles

2008-01-01

So far, the abuse of gene transfer technology in sport, so-called gene doping, is undetectable. However, recent studies in somatic gene therapy indicate that long-term presence of transgenic DNA (tDNA) following various gene transfer protocols can be found in DNA isolated from whole blood using conventional PCR protocols. Application of these protocols for the direct detection of gene doping would require almost complete knowledge about the sequence of the genetic information that has been transferred. Here, we develop and describe the novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure that overcomes this difficulty. Apart from the interesting perspectives that this spiPCR procedure offers in the fight against gene doping, this technology could also be of interest in biodistribution and biosafety studies for gene therapeutic applications.

Evaluation of propidium monoazide real-time PCR for enumeration of probiotic lactobacilli microencapsulated in calcium alginate beads.

PubMed

Oketič, K; Matijašić, B Bogovič; Obermajer, T; Radulović, Z; Lević, S; Mirković, N; Nedović, V

2015-01-01

The aim of the study was to evaluate real-time PCR coupled with propidium monoazide (PMA) treatment for enumeration of microencapsulated probiotic lactobacilli microencapsulated in calcium alginate beads. Lactobacillus gasseri K7 (CCM 7710) and Lactobacillus delbrueckii subsp. bulgaricus (CCM 7712) were analysed by plate counting and PMA real-time PCR during storage at 4 °C for 90 days. PMA was effective in preventing PCR amplification of the target sequences of DNA released from heat-compromised bacteria. The values obtained by real-time PCR of non-treated samples were in general higher than those obtained by real-time PCR of PMA-treated samples or by plate counting, indicating the presence of sub-lethally injured cells. This study shows that plate count could not be completely replaced by culture independent method PMA real-time PCR for enumeration of probiotics, but may rather complement the well-established plate counting, providing useful information about the ratio of compromised bacteria in the samples.

A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies.

PubMed

Bach, H-J; Jessen, I; Schloter, M; Munch, J C

2003-01-01

Real-time TaqMan-PCR assays were developed for detection, differentiation and absolute quantification of the pathogenic subspecies of Clavibacter michiganensis (Cm) in one single PCR run. The designed primer pair, targeting intergenic sequences of the rRNA operon (ITS) common in all subspecies, was suitable for the amplification of the expected 223-nt DNA fragments of all subspecies. Closely related bacteria were completely discriminated, except of Rathayibacter iranicus, from which weak PCR product bands appeared on agarose gel after 35 PCR cycles. Sufficient specificity of PCR detection was reached by introduction of the additional subspecies specific probes used in TaqMan-PCR. Only Cm species were detected and there was clear differentiation among the subspecies C. michiganensis sepedonicus (Cms), C. michiganensis michiganensis (Cmm), C. michiganensis nebraskensis (Cmn), C. michiganensis insidiosus (Cmi) and C. michiganensis tessellarius (Cmt). The TaqMan assays were optimized to enable a simultaneous quantification of each subspecies. Validity is shown by comparison with cell counts.

Longitudinal monitoring of bottlenose dolphins leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Longitudinal monitoring of bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Self-Reported Student Confidence in Troubleshooting Ability Increases after Completion of an Inquiry-Based PCR Practical

ERIC Educational Resources Information Center

Cook, Anthony L.; Snow, Elizabeth T.; Binns, Henrica; Cook, Peta S.

2015-01-01

Inquiry-based learning (IBL) activities are complementary to the processes of laboratory discovery, as both are focused on producing new findings through research and inquiry. Here, we describe the results of student surveys taken pre- and postpractical to an IBL undergraduate practical on PCR. Our analysis focuses primarily student perceptions of…

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Polymerase chain reaction for screening clinical isolates of corynebacteria for the production of diphtheria toxin.

PubMed

Pallen, M J; Hay, A J; Puckey, L H; Efstratiou, A

1994-04-01

To assess the performance of the polymerase chain reaction (PCR) when used to screen rapidly large numbers of corynebacteria for toxin production; and to determine the incidence of false positive PCR results with non-toxigenic Corynebacterium diphtheriae isolates. Eighty seven recent British isolates of corynebacteria were assayed by PCR. All isolates were assayed from both blood and tellurite agar within a five day period. Thirty three non-toxigenic isolates of C diphtheriae from six countries were also tested by PCR and by the Elek immunodiffusion assay. There was complete concordance between the results of PCR and traditional methods on the recent British isolates, with one exception: an Elek positive "C ulcerans" isolate, which was PCR positive from tellurite but not from blood agar. One of the thirty three (3%) non-toxigenic isolates of C diphtheriae was PCR positive. These results suggest that PCR compares favourably with traditional methods for the detection of toxigenic corynebacteria and that it represents a powerful new tool in the diagnosis of an old disease.

Polymerase chain reaction for screening clinical isolates of corynebacteria for the production of diphtheria toxin.

PubMed Central

Pallen, M J; Hay, A J; Puckey, L H; Efstratiou, A

1994-01-01

AIMS--To assess the performance of the polymerase chain reaction (PCR) when used to screen rapidly large numbers of corynebacteria for toxin production; and to determine the incidence of false positive PCR results with non-toxigenic Corynebacterium diphtheriae isolates. METHODS--Eighty seven recent British isolates of corynebacteria were assayed by PCR. All isolates were assayed from both blood and tellurite agar within a five day period. Thirty three non-toxigenic isolates of C diphtheriae from six countries were also tested by PCR and by the Elek immunodiffusion assay. RESULTS--There was complete concordance between the results of PCR and traditional methods on the recent British isolates, with one exception: an Elek positive "C ulcerans" isolate, which was PCR positive from tellurite but not from blood agar. One of the thirty three (3%) non-toxigenic isolates of C diphtheriae was PCR positive. CONCLUSIONS--These results suggest that PCR compares favourably with traditional methods for the detection of toxigenic corynebacteria and that it represents a powerful new tool in the diagnosis of an old disease. Images PMID:8027375

Fingerprinting and quantification of GMOs in the agro-food sector.

PubMed

Taverniers, I; Van Bockstaele, E; De Loose, M

2003-01-01

Most strategies for analyzing GMOs in plants and derived food and feed products, are based on the polymerase chain reaction (PCR) technique. In conventional PCR methods, a 'known' sequence between two specific primers is amplified. To the contrary, with the 'anchor PCR' technique, unknown sequences adjacent to a known sequence, can be amplified. Because T-DNA/plant border sequences are being amplified, anchor PCR is the perfect tool for unique identification of transgenes, including non-authorized GMOs. In this work, anchor PCR was applied to characterize the 'transgene locus' and to clarify the complete molecular structure of at least six different commercial transgenic plants. Based on sequences of T-DNA/plant border junctions, obtained by anchor PCR, event specific primers were developed. The junction fragments, together with endogeneous reference gene targets, were cloned in plasmids. The latter were then used as event specific calibrators in real-time PCR, a new technique for the accurate relative quantification of GMOs. We demonstrate here the importance of anchor PCR for identification and the usefulness of plasmid DNA calibrators in quantification strategies for GMOs, throughout the agro-food sector.

[Application of rapid PCR to authenticate medicinal snakes].

PubMed

Chen, Kang; Jiang, Chao; Yuan, Yuan; Huang, Lu-Qi; Li, Man

2014-10-01

To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.

Development of a Novel and Rapid Fully Automated Genetic Testing System.

PubMed

Uehara, Masayuki

2016-01-01

We have developed a rapid genetic testing system integrating nucleic acid extraction, purification, amplification, and detection in a single cartridge. The system performs real-time polymerase chain reaction (PCR) after nucleic acid purification in a fully automated manner. RNase P, a housekeeping gene, was purified from human nasal epithelial cells using silica-coated magnetic beads and subjected to real-time PCR using a novel droplet-real-time-PCR machine. The process was completed within 13 min. This system will be widely applicable for research and diagnostic uses.

No Clinical or Molecular Evidence of Plasmodium falciparum Resistance to Artesunate–Mefloquine in Northwestern Brazil

PubMed Central

Ladeia-Andrade, Simone; de Melo, Gladson Naber P.; de Souza-Lima, Rita de Cássia; Salla, Laís C.; Bastos, Melissa S.; Rodrigues, Priscila T.; Luz, Francisco das Chagas O.; Ferreira, Marcelo U.

2016-01-01

We evaluated the clinical efficacy of artesunate–mefloquine (ASMQ) fixed-dose combination to treat uncomplicated malaria in Juruá Valley, the main Plasmodium falciparum transmission hotspot in Brazil. Between November 2010 and February 2013, we enrolled 162 patients aged 4–73 years, with fever or a history of fever, and a single-species P. falciparum infection confirmed by microscopy and polymerase chain reaction (PCR). All 154 patients who completed the 42-day follow-up presented an adequate clinical and parasitologic response. ASMQ was well tolerated and no adverse event caused treatment interruption. Gametocytes were detected in 46.3% patients; 35.2% had gametocytes at enrollment, whereas others developed patent gametocytemia 1–14 days after starting ASMQ. By day 3 of treatment, all subjects had cleared asexual parasitemia, but parasite DNA remained PCR detectable in 37.6% of them. Day-3 PCR positivity was associated with prolonged gametocyte carriage. We found no molecular evidence of resistance to either MQ (pfmdr1 gene amplification) or AS (mutations in selected kelch13 gene domains known to be associated with AS resistance) in the local P. falciparum population. These results strongly support the use of ASMQ as a first-line regimen to treat uncomplicated P. falciparum malaria in northwestern Brazil, but underscore the need for gametocytocidal drugs to reduce the transmission potential of ASMQ-treated patients (ClinicalTrials.gov number NCT01144702). PMID:27068396

Lab-on-a-chip enabled HLA diagnostic: combined sample preparation and real time PCR for HLA-B57 diagnosis

NASA Astrophysics Data System (ADS)

Gärtner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Schattschneider, Sebastian; Frank, Rainer; Willems, Andreas

2015-05-01

The diverse human HLA (human leukocyte antigen) system is responsible for antigen presentation and recognition. It is essential for the immune system to maintain a stable defense line, but also is also involved in autoimmunity as well as metabolic disease. HLA-haplotype (HLA-B27), for instance, is associated with inflammatory diseases such as Bechterew's disease. The administration of the HIV drug Abacavir in combination with another HLA-haplotype (HLAB57) is associated with severe hypersensitivity reactions. Accordingly, the HLA status has to be monitored for diagnosis or prior to start of therapy. Along this line, a miniaturized microfluidic platform has been developed allowing performing the complete analytical process from "sample-in" to "answer-out" in a point-of-care environment. The main steps of the analytical cascade inside the integrated system are blood cell lysis and DNA isolation, DNA purification, real-time PCR and quantitative monitoring of the rise of a fluorescent signal appearing during the PCR based sequence amplification. All bio-analytical steps were intended to be performed inside one chip and will be actuated, controlled and monitored by a matching device. This report will show that all required processes are established and tested and all device components work well and interact with the functional modules on the chips in a harmonized fashion.

COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing.

PubMed

Castellanos-Rizaldos, Elena; Milbury, Coren A; Guha, Minakshi; Makrigiorgos, G Mike

2014-01-01

Detection of low-level mutations is important for cancer biomarker and therapy targets discovery, but reliable detection remains a technical challenge. The newly developed method of CO-amplification at Lower Denaturation temperature PCR (COLD-PCR) helps to circumvent this issue. This PCR-based technology preferentially enriches minor known or unknown variants present in samples with a high background of wild type DNA which often hampers the accurate identification of these minority alleles. This is a simple process that consists of lowering the temperature at the denaturation step during the PCR-cycling protocol (critical denaturation temperature, T c) and inducing DNA heteroduplexing during an intermediate step. COLD-PCR in its simplest forms does not need additional reagents or specific instrumentation and thus, can easily replace conventional PCR and at the same time improve the mutation detection sensitivity limit of downstream technologies. COLD-PCR can be applied in two basic formats: fast-COLD-PCR that can enrich T m-reducing mutations and full-COLD-PCR that can enrich all mutations, though it requires an intermediate cross-hybridization step that lengthens the thermocycling program. An improved version of full-COLD-PCR (improved and complete enrichment, ice-COLD-PCR) has also been described. Finally, most recently, we developed yet another form of COLD-PCR, temperature-tolerant-COLD-PCR, which gradually increases the denaturation temperature during the COLD-PCR reaction, enriching diverse targets using a single cycling program. This report describes practical considerations for application of fast-, full-, ice-, and temperature-tolerant-COLD-PCR for enrichment of mutations prior to downstream screening.

Molecular and Pharmacological Determinants of the Therapeutic Response to Artemether-Lumefantrine in Multidrug-Resistant Plasmodium falciparum Malaria

PubMed Central

Price, Ric N.; Uhlemann, Anne-Catrin; van Vugt, Michele; Al Brockman; Hutagalung, Robert; Nair, Shalini; Nash, Denae; Singhasivanon, Pratap; Anderson, Tim J. C.; Krishna, Sanjeev; White, Nicholas J.; Nosten, François

2015-01-01

Background Our study examined the relative contributions of host, pharmacokinetic, and parasitological factors in determining the therapeutic response to artemether-lumefantrine (AL). Methods On the northwest border of Thailand, patients with uncomplicated Plasmodium falciparum malaria were enrolled in prospective studies of AL treatment (4- or 6-dose regimens) and followed up for 42 days. Plasma lumefantrine concentrations were measured by high performance liquid chromatography; malaria parasite pfmdr1 copy number was quantified using a real-time polymerase chain reaction assay (PCR), and in vitro drug susceptibility was tested. Results All treatments resulted in a rapid clinical response and were well tolerated. PCR-corrected failure rates at day 42 were 13% (95% confidence interval [CI], 9.6%–17%) for the 4-dose regimen and 3.2% (95% CI, 1.8%–4.6%) for the 6-dose regimen. Increased pfmdr1 copy number was associated with a 2-fold (95% CI, 1.8–2.4-fold) increase in lumefantrine inhibitory concentration50 (P = .001) and an adjusted hazard ratio for risk of treatment failure following completion of a 4-dose regimen, but not a 6-dose regimen, of 4.0 (95% CI, 1.4–11; P = .008). Patients who had lumefantrine levels below 175 ng/mL on day 7 were more likely to experience recrudescence by day 42 (adjusted hazard ratio, 17; 95% CI, 5.5–53), allowing prediction of treatment failure with 75% sensitivity and 84% specificity. The 6-dose regimen ensured that therapeutic levels were achieved in 91% of treated patients. Conclusions The lumefantrine plasma concentration profile is the main determinant of efficacy of artemether-lumefantrine. Amplification in pfmdr1 determines lumefantrine susceptibility and, therefore, treatment responses when plasma lumefantrine levels are subtherapeutic. PMID:16652314

Molecular and pharmacological determinants of the therapeutic response to artemether-lumefantrine in multidrug-resistant Plasmodium falciparum malaria.

PubMed

Price, Ric N; Uhlemann, Anne-Catrin; van Vugt, Michele; Brockman, Al; Hutagalung, Robert; Nair, Shalini; Nash, Denae; Singhasivanon, Pratap; Anderson, Tim J C; Krishna, Sanjeev; White, Nicholas J; Nosten, François

2006-06-01

Our study examined the relative contributions of host, pharmacokinetic, and parasitological factors in determining the therapeutic response to artemether-lumefantrine (AL). On the northwest border of Thailand, patients with uncomplicated Plasmodium falciparum malaria were enrolled in prospective studies of AL treatment (4- or 6-dose regimens) and followed up for 42 days. Plasma lumefantrine concentrations were measured by high performance liquid chromatography; malaria parasite pfmdr1 copy number was quantified using a real-time polymerase chain reaction assay (PCR), and in vitro drug susceptibility was tested. All treatments resulted in a rapid clinical response and were well tolerated. PCR-corrected failure rates at day 42 were 13% (95% confidence interval [CI], 9.6%-17%) for the 4-dose regimen and 3.2% (95% CI, 1.8%-4.6%) for the 6-dose regimen. Increased pfmdr1 copy number was associated with a 2-fold (95% CI, 1.8-2.4-fold) increase in lumefantrine inhibitory concentration(50) (P=.001) and an adjusted hazard ratio for risk of treatment failure following completion of a 4-dose regimen, but not a 6-dose regimen, of 4.0 (95% CI, 1.4-11; P=.008). Patients who had lumefantrine levels below 175 ng/mL on day 7 were more likely to experience recrudescence by day 42 (adjusted hazard ratio, 17; 95% CI, 5.5-53), allowing prediction of treatment failure with 75% sensitivity and 84% specificity. The 6-dose regimen ensured that therapeutic levels were achieved in 91% of treated patients. The lumefantrine plasma concentration profile is the main determinant of efficacy of artemether-lumefantrine. Amplification in pfmdr1 determines lumefantrine susceptibility and, therefore, treatment responses when plasma lumefantrine levels are subtherapeutic.

[Complete genome sequencing and sequence analysis of BCG Tice].

PubMed

Wang, Zhiming; Pan, Yuanlong; Wu, Jun; Zhu, Baoli

2012-10-04

The objective of this study is to obtain the complete genome sequence of Bacillus Calmette-Guerin Tice (BCG Tice), in order to provide more information about the molecular biology of BCG Tice and design more reasonable vaccines to prevent tuberculosis. We assembled the data from high-throughput sequencing with SOAPdenovo software, with many contigs and scaffolds obtained. There are many sequence gaps and physical gaps remained as a result of regional low coverage and low quality. We designed primers at the end of contigs and performed PCR amplification in order to link these contigs and scaffolds. With various enzymes to perform PCR amplification, adjustment of PCR reaction conditions, and combined with clone construction to sequence, all the gaps were finished. We obtained the complete genome sequence of BCG Tice and submitted it to GenBank of National Center for Biotechnology Information (NCBI). The genome of BCG Tice is 4334064 base pairs in length, with GC content 65.65%. The problems and strategies during the finishing step of BCG Tice sequencing are illuminated here, with the hope of affording some experience to those who are involved in the finishing step of genome sequencing. The microarray data were verified by our results.

Cytomegalovirus induced sensorineural hearing loss with persistent cochlear inflammation in neonatal mice

PubMed Central

Schachtele, Scott J.; Mutnal, Manohar B.; Schleiss, Mark R.; Lokensgard, James R.

2011-01-01

Congenital cytomegalovirus (CMV) infection is the leading cause of sensorineural hearing loss (SNHL) in children. During murine (M)CMV-induced encephalitis, the immune response is important for both the control of viral dissemination and the clearance of virus from the brain. While the importance of CMV-induced SNHL has been described, the mechanisms surrounding its pathogenesis and the role of inflammatory responses remain unclear. This study presents a neonatal mouse model of profound SNHL in which MCMV preferentially infected both cochlear perilymphatic epithelial cells and spiral ganglion neurons. Interestingly, MCMV infection induced cochlear hair cell death by 21 days post-infection, despite a clear lack of direct infection of hair cells and the complete clearance of the virus from the cochlea by 14 dpi. Flow cytometric, immunohistochemical, and quantitative PCR analysis of MCMV-infected cochlea revealed a robust and chronic inflammatory response, including a prolonged increase in reactive oxygen species production by infiltrating macrophages. These data support a pivotal role for inflammation during MCMV-induced SNHL. PMID:21416394

Cytomegalovirus-induced sensorineural hearing loss with persistent cochlear inflammation in neonatal mice.

PubMed

Schachtele, Scott J; Mutnal, Manohar B; Schleiss, Mark R; Lokensgard, James R

2011-06-01

Congenital cytomegalovirus (CMV) infection is the leading cause of sensorineural hearing loss (SNHL) in children. During murine (M)CMV-induced encephalitis, the immune response is important for both the control of viral dissemination and the clearance of virus from the brain. While the importance of CMV-induced SNHL has been described, the mechanisms surrounding its pathogenesis and the role of inflammatory responses remain unclear. This study presents a neonatal mouse model of profound SNHL in which MCMV preferentially infected both cochlear perilymphatic epithelial cells and spiral ganglion neurons. Interestingly, MCMV infection induced cochlear hair cell death by 21 days post-infection, despite a clear lack of direct infection of hair cells and the complete clearance of the virus from the cochlea by 14 dpi. Flow cytometric, immunohistochemical, and quantitative PCR analysis of MCMV-infected cochlea revealed a robust and chronic inflammatory response, including a prolonged increase in reactive oxygen species production by infiltrating macrophages. These data support a pivotal role for inflammation during MCMV-induced SNHL.

Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

PubMed

Greenspoon, S A; Sykes, K L V; Ban, J D; Pollard, A; Baisden, M; Farr, M; Graham, N; Collins, B L; Green, M M; Christenson, C C

2006-12-20

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.

Molecular and clinical analyses of Helicobacter pylori colonization in inflamed dental pulp.

PubMed

Nomura, Ryota; Ogaya, Yuko; Matayoshi, Saaya; Morita, Yumiko; Nakano, Kazuhiko

2018-04-16

Recently, dental pulp has been considered a possible source of infection of Helicobacter pylori (H. pylori) in children. We previously developed a novel PCR system for H. pylori detection with high specificity and sensitivity using primer sets constructed based on the complete genome information for 48 H. pylori strains. This PCR system showed high sensitivity with a detection limit of 1-10 cells when serial dilutions of H. pylori genomic DNA were used as templates. However, the detection limit was lower (10 2 -10 3 cells) when H. pylori bacterial DNA was detected from inflamed pulp specimens. Thus, we further refined the system using a nested PCR method, which was much more sensitive than the previous single PCR method. In addition, we examined the distribution and virulence of H. pylori in inflamed pulp tissue. Nested PCR system was constructed using primer sets designed from the complete genome information of 48 H. pylori strains. The detection limit of the nested PCR system was 1-10 cells using both H. pylori genomic DNA and bacterial DNA isolated from inflamed pulp specimens. Next, distribution of H. pylori was examined using 131 inflamed pulp specimens with the nested PCR system. In addition, association between the detection of H. pylori and clinical information regarding endodontic-infected teeth were investigated. Furthermore, adhesion property of H. pylori strains to human dental fibroblast cells was examined. H. pylori was present in 38.9% of inflamed pulp specimens using the nested PCR system. H. pylori was shown to be predominantly detected in primary teeth rather than permanent teeth. In addition, samplings of the inflamed pulp were performed twice from the same teeth at 1- or 2-week intervals, which revealed that H. pylori was detected in most specimens in both samplings. Furthermore, H. pylori strains showed adhesion property to human dental fibroblast cells. Our results suggest that H. pylori colonizes inflamed pulp in approximately 40% of all cases through adhesion to human dental fibroblast cells.

Rolling circle amplification of metazoan mitochondrialgenomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simison, W. Brian; Lindberg, D.R.; Boore, J.L.

2005-07-31

Here we report the successful use of rolling circle amplification (RCA) for the amplification of complete metazoan mt genomes to make a product that is amenable to high-throughput genome sequencing techniques. The benefits of RCA over PCR are many and with further development and refinement of RCA, the sequencing of organellar genomics will require far less time and effort than current long PCR approaches.

An advanced telerobotic system for shuttle payload changeout room processing applications

NASA Technical Reports Server (NTRS)

Sklar, M.; Wegerif, D.

1989-01-01

To potentially alleviate the inherent difficulties in the ground processing of the Space Shuttle and its associated payloads, a teleoperated, semi-autonomous robotic processing system for the Payload Changeout Room (PCR) is now in the conceptual stages. The complete PCR robotic system as currently conceived is described and critical design issues and the required technologies are discussed.

An evaluation of direct PCR amplification

PubMed Central

Hall, Daniel E.; Roy, Reena

2014-01-01

Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837

Random Amplification and Pyrosequencing for Identification of Novel Viral Genome Sequences

PubMed Central

Hang, Jun; Forshey, Brett M.; Kochel, Tadeusz J.; Li, Tao; Solórzano, Víctor Fiestas; Halsey, Eric S.; Kuschner, Robert A.

2012-01-01

ssRNA viruses have high levels of genomic divergence, which can lead to difficulty in genomic characterization of new viruses using traditional PCR amplification and sequencing methods. In this study, random reverse transcription, anchored random PCR amplification, and high-throughput pyrosequencing were used to identify orthobunyavirus sequences from total RNA extracted from viral cultures of acute febrile illness specimens. Draft genome sequence for the orthobunyavirus L segment was assembled and sequentially extended using de novo assembly contigs from pyrosequencing reads and orthobunyavirus sequences in GenBank as guidance. Accuracy and continuous coverage were achieved by mapping all reads to the L segment draft sequence. Subsequently, RT-PCR and Sanger sequencing were used to complete the genome sequence. The complete L segment was found to be 6936 bases in length, encoding a 2248-aa putative RNA polymerase. The identified L segment was distinct from previously published South American orthobunyaviruses, sharing 63% and 54% identity at the nucleotide and amino acid level, respectively, with the complete Oropouche virus L segment and 73% and 81% identity at the nucleotide and amino acid level, respectively, with a partial Caraparu virus L segment. The result demonstrated the effectiveness of a sequence-independent amplification and next-generation sequencing approach for obtaining complete viral genomes from total nucleic acid extracts and its use in pathogen discovery. PMID:22468136

Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma

PubMed Central

Lahuerta, Juan J.; Pepin, François; González, Marcos; Barrio, Santiago; Ayala, Rosa; Puig, Noemí; Montalban, María A.; Paiva, Bruno; Weng, Li; Jiménez, Cristina; Sopena, María; Moorhead, Martin; Cedena, Teresa; Rapado, Immaculada; Mateos, María Victoria; Rosiñol, Laura; Oriol, Albert; Blanchard, María J.; Martínez, Rafael; Bladé, Joan; San Miguel, Jesús; Faham, Malek; García-Sanz, Ramón

2014-01-01

We assessed the prognostic value of minimal residual disease (MRD) detection in multiple myeloma (MM) patients using a sequencing-based platform in bone marrow samples from 133 MM patients in at least very good partial response (VGPR) after front-line therapy. Deep sequencing was carried out in patients in whom a high-frequency myeloma clone was identified and MRD was assessed using the IGH-VDJH, IGH-DJH, and IGK assays. The results were contrasted with those of multiparametric flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). The applicability of deep sequencing was 91%. Concordance between sequencing and MFC and ASO-PCR was 83% and 85%, respectively. Patients who were MRD– by sequencing had a significantly longer time to tumor progression (TTP) (median 80 vs 31 months; P < .0001) and overall survival (median not reached vs 81 months; P = .02), compared with patients who were MRD+. When stratifying patients by different levels of MRD, the respective TTP medians were: MRD ≥10−3 27 months, MRD 10−3 to 10−5 48 months, and MRD <10−5 80 months (P = .003 to .0001). Ninety-two percent of VGPR patients were MRD+. In complete response patients, the TTP remained significantly longer for MRD– compared with MRD+ patients (131 vs 35 months; P = .0009). PMID:24646471

Eradication of Borrelia burgdorferi infection in primary marginal zone B-cell lymphoma of the skin.

PubMed

Roggero, E; Zucca, E; Mainetti, C; Bertoni, F; Valsangiacomo, C; Pedrinis, E; Borisch, B; Piffaretti, J C; Cavalli, F; Isaacson, P G

2000-02-01

Primary cutaneous B-cell lymphomas have been associated with Borrelia burgdorferi, the spirochete responsible for Lyme disease. Recently, cutaneous marginal zone B-cell lymphoma has been proposed as a distinct clinical-pathological entity. We report a case of primary cutaneous marginal zone lymphoma, associated with B burgdorferi infection. Polymerase chain reaction (PCR) amplification of the third complementarity determining region (CDR3) of the immunoglobulin heavy chain gene showed the presence of a monoclonal lymphoproliferation, therefore strengthening the histological diagnosis of a malignant process. B burgdorfer-specific hbb gene sequences were detected by PCR in the lymphoma tissue at diagnosis but not after antibiotic treatment. A nearly complete clinical and histological regression was observed after B burgdorferi eradication, with immunohistochemistry studies showing disappearance of plasma cell differentiation and a marked decline in the number of CD3+ T cells and Ki-67+ cells. Our case confirms the link between B burgdorferi and some cutaneous lymphomas. The disappearance of the microorganism accompanied by the unequivocal decrease of most indicators of active T- and B-cell immune response strongly supported a pathogenetic role for B burgdorferi in sustaining an antigen-driven development and growth of this cutaneous marginal zone lymphoma. Antibiotic therapy (analogous to Helicobacter pylori infection in gastric MALT lymphoma) might be helpful with the aim of averting or at least deferring the indication for more aggressive treatment.

Prognostic value of sequencing-based minimal residual disease detection in patients with multiple myeloma who underwent autologous stem-cell transplantation.

PubMed

Takamatsu, H; Takezako, N; Zheng, J; Moorhead, M; Carlton, V E H; Kong, K A; Murata, R; Ito, S; Miyamoto, T; Yokoyama, K; Matsue, K; Sato, T; Kurokawa, T; Yagi, H; Terasaki, Y; Ohata, K; Matsumoto, M; Yoshida, T; Faham, M; Nakao, S

2017-10-01

Most patients with multiple myeloma (MM) are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper response are required. We retrospectively analyzed 125 patients with MM who underwent high-dose melphalan plus autologous stem-cell transplantation (ASCT) to detect MRD in autograft/bone marrow (BM) cells using a next-generation sequencing (NGS)-based method and allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR). NGS-based method was applicable to 90% and this method had at least one to two logs greater sensitivity compared to ASO-PCR. MRD negative by NGS [MRDNGS(-)] (defined as <10-6) in post-ASCT BM cases (n = 26) showed a significantly better progression-free survival (PFS) (96% at 4 years, P < 0.001) and overall survival (OS) (100% at 4 years, P =0.04) than MRDNGS(+) in post-ASCT BM cases (n = 25). When restricting the analysis to the 39 complete response cases, patients who were MRDNGS(-) (n = 24) showed a significantly better PFS than those that were MRDNGS(+) (n = 15) (P =0.02). Moreover, MRDNGS(-) in post-ASCT BM cases (n = 12) showed significantly a better PFS than MRDNGS(+) cases (n = 7) where MRD was not detected by ASO-PCR (P = 0.001). Patients whose autografts were negative by NGS-based MRD assessment (<10-7) (n = 19) had 92% PFS and 100% OS at 4 years post-ASCT. Conversely, the NGS-based MRD positive patients who received post-ASCT treatment using novel agents (n = 49) had a significantly better PFS (P = 0.001) and tended to have a better OS (P= 0.214) than those that were untreated (n = 33). Low level MRD detected by NGS-based platform but not ASO-PCR has significant prognostic value when assessing either the autograft product or BM cells post-ASCT. © The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

Molecular Technique to Understand Deep Microbial Diversity

NASA Technical Reports Server (NTRS)

Vaishampayan, Parag A.; Venkateswaran, Kasthuri J.

2012-01-01

Current sequencing-based and DNA microarray techniques to study microbial diversity are based on an initial PCR (polymerase chain reaction) amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of the minor template appears to be suppressed by the exponential amplification of the more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck has overlooked the presence of the less abundant minority population, and underestimated their role in the ecosystem maintenance. To generate PCR amplicons for subsequent diversity analysis, bacterial l6S rRNA genes are amplified by PCR using universal primers. Two distinct PCR regimes are employed in parallel: one using normal and the other using biotinlabeled universal primers. PCR products obtained with biotin-labeled primers are mixed with streptavidin-labeled magnetic beads and selectively captured in the presence of a magnetic field. Less-abundant DNA templates that fail to amplify in this first round of PCR amplification are subjected to a second round of PCR using normal universal primers. These PCR products are then subjected to downstream diversity analyses such as conventional cloning and sequencing. A second round of PCR amplified the minority population and completed the deep diversity picture of the environmental sample.

Clinical and economic impact of the introduction of a nucleic acid amplification assay for Clostridium difficile.

PubMed

Guinta, Margaret M; Bunnell, Kristen; Harrington, Amanda; Bleasdale, Susan; Danziger, Larry; Wenzler, Eric

2017-12-04

The clinical outcomes and cost implications of a diagnostic shift from an EIA- to PCR-based assay for Clostridium difficile infection (CDI) have not been completely described in the literature. The impact of the PCR-based assay on the incidence and duration of CDI therapy was compared to the EIA assay for patients with a negative CDI diagnostic result. Secondary clinical and economic outcomes were also evaluated. Independent predictors of receipt of antibiotic therapy were assessed via logistic regression. 141 EIA and 140 PCR patients were included. Significantly more patients were started or continued on anti-CDI antibiotic therapy after a known negative assay result in the EIA group (26 patients vs. 8 patients, P = 0.002). Duration of antibiotic therapy after a known negative result was significantly shorter in the PCR group (1 vs. 4 days, P = 0.029) and a 23% reduction in the number of tests obtained per patient was observed (1.41 ± 0.86 vs. 1.82 ± 1.35, P = 0.007). The over fourfold difference in per-test cost of the EIA assay ($8.33 vs. $42.86, P < 0.0001) was offset by the overall medication costs required for the increased treatment in the EIA group ($546.60 vs. $188.96, P = 0.191). Utilization of the EIA-based CDI assay was associated with increased odds of CDI treatment after a negative test (aOR 4.71, 95% CI 1.93-11.46, P = 0.001). The transition from an EIA to PCR-based assay for diagnosing CDI resulted in a significant decrease in the number of patients treated and the duration of treatment in response to a negative test result. This significant decrease in treatment resulted in decreased costs offsetting the utilization of a more expensive molecular test for patients with a negative CDI diagnostic result.

Biological nutrient removal with limited organic matter using a novel anaerobic–anoxic/oxic multi-phased activated sludge process

PubMed Central

Naseer, Rusul; Abualhail, Saad; Xiwu, Lu

2012-01-01

An anaerobic–anoxic/oxic (A2/O) multi-phased biological process called “phased isolation tank step feed technology (PITSF)” was developed to force the oscillation of organic and nutrient concentrations in process reactors. PITSF can be operated safely with a limited carbon source in terms of low carbon requirements and aeration costs whereas NAR was achieved over 95% in the last aerobic zone through a combination of short HRT and low DO levels. PCR assay was used for XAB quantification to correlate XAB numbers with nutrient removal. PCR assays showed, high NAR was achieved at XAB population 5.2 × 108 cells/g MLVSS in response to complete and partial nitrification process. It was exhibited that low DO with short HRT promoted XAB growth. Simultaneous nitrification and denitrification (SND) via nitrate were observed obviously, SND rate was between 69–72%, at a low DO level of 0.5 mg/l in the first aerobic tank during main phases and the removal efficiency of TN, NH4+-N, COD, TP was 84.7 .97, 88.3 and 96% respectively. The removal efficiencies of TN, NH4+-N, and TP at low C/N ratio and DO level were 84.2, 98.5 and 96.9% respectively which were approximately equal to the complete nitrification–denitrification with the addition of external carbon sources at a normal DO level of (1.5–2.5 mg/l). PMID:23961214

Cost-effectiveness of a modified two-step algorithm using a combined glutamate dehydrogenase/toxin enzyme immunoassay and real-time PCR for the diagnosis of Clostridium difficile infection.

PubMed

Vasoo, Shawn; Stevens, Jane; Portillo, Lena; Barza, Ruby; Schejbal, Debra; Wu, May May; Chancey, Christina; Singh, Kamaljit

2014-02-01

The analytical performance and cost-effectiveness of the Wampole Toxin A/B EIA, the C. Diff. Quik Chek Complete (CdQCC) (a combined glutamate dehydrogenase antigen/toxin enzyme immunoassay), two RT-PCR assays (Progastro Cd and BD GeneOhm) and a modified two-step algorithm using the CdQCC reflexed to RT-PCR for indeterminate results were compared. The sensitivity of the Wampole Toxin A/B EIA, CdQCC (GDH antigen), BD GeneOhm and Progastro Cd RT-PCR were 85.4%, 95.8%, 100% and 93.8%, respectively. The algorithm provided rapid results for 86% of specimens and the remaining indeterminate results were resolved by RT-PCR, offering the best balance of sensitivity and cost savings per test (algorithm ∼US$13.50/test versus upfront RT-PCR ∼US$26.00/test). Copyright © 2012. Published by Elsevier B.V.

Selection of reliable reference genes for RT-qPCR studies in Octopus vulgaris paralarvae during development and immune-stimulation.

PubMed

García-Fernández, P; Castellanos-Martínez, S; Iglesias, J; Otero, J J; Gestal, C

2016-07-01

The common octopus, Octopus vulgaris is a new candidate species for aquaculture. However, rearing of octopus paralarvae is hampered by high mortality and poor growth rates that impede its entire culture. The study of genes involved in the octopus development and immune response capability could help to understand the key of paralarvae survival and thus, to complete the octopus life cycle. Quantitative real-time PCR (RT-qPCR) is the most frequently tool used to quantify the gene expression because of specificity and sensitivity. However, reliability of RT-qPCR requires the selection of appropriate normalization genes whose expression must be stable across the different experimental conditions of the study. Hence, the aim of the present work is to evaluate the stability of six candidate genes: β-actin (ACT), elongation factor 1-α (EF), ubiquitin (UBI), β-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GADPH) and ribosomal RNA 18 (18S) in order to select the best reference gene. The stability of gene expression was analyzed using geNorm, NormFinder and Bestkeeper, in octopus paralarvae of seven developmental stages (embryo, paralarvae of 0, 10, 15, 20, 30 and 34days) and paralarvae of 20days after challenge with Vibrio lentus and Vibrio splendidus. The results were validated by measuring the expression of PGRP, a stimuli-specific gene. Our results showed UBI, EF and 18S as the most suitable reference genes during development of octopus paralarvae, and UBI, ACT and 18S for bacterial infection. These results provide a basis for further studies exploring molecular mechanism of their development and innate immune defense. Copyright © 2016 Elsevier Inc. All rights reserved.

Long-term results with oral fluoropyrimidines and oxaliplatin-based preoperative chemoradiotherapy in patients with resectable rectal cancer. A single-institution experience.

PubMed

Díaz Beveridge, Robert; Aparicio, Jorge; Tormo, Alejandro; Estevan, Rafael; Artes, Josefina; Giménez, Alejandra; Segura, Ángel; Roldán, Susana; Palasí, Rosana; Ramos, David

2012-06-01

Neoadjuvant 5-FU-based chemoradiotherapy in resectable rectal cancer (RC) is a standard of treatment. The use of oral fluoropyrimidines and new agents such as oxaliplatin may improve efficacy and tolerance. Between 1999 and 2009, 126 RC patients with T3-T4 and/or N+ disease were given three successive protocols: UFT (32), UFT-oxaliplatin (75) and capecitabine-oxaliplatin (19), alongside 45 Gy of radiotherapy; with surgery 4-6 weeks after. Adjuvant treatment was given in all patients. The primary objective was pathologic complete response (pCR). Preoperative therapy was well tolerated, with no toxic deaths and a 15% grade 3-4 toxicity rate. Eighty-five percent of patients received the full chemotherapy dose, 56% had an abdominoperineal resection, 6% reinterventions and 57% received the full adjuvant chemotherapy planned. The pCR rate was 13%. The downstaging rate was 80%; 8% had progression of disease. The relapse rate was 20%, with local relapse in 6%. By 5 years of followup, 92% of relapses had occurred. Median follow-up was 73 months, 5- and 10-year disease-free survival rates were 75% and 50%, and 5- and 10-year overall survival rates were 79% and 66% respectively. There was no benefit from the use of oxaliplatin regarding survival or pCR rates. Older patients had worse long-term outcomes. Neoadjuvant chemoradiotherapy with oral fluoropyrimidines and oxaliplatin is feasible and well tolerated. The risk of early progression is low. However, there was no added benefit with the use of oxaliplatin. There were no relapses in patients with pCR. The role of adjuvant chemotherapy is unclear.

Characterization of miRNAs in response to short-term waterlogging in three inbred lines of Zea mays

USDA-ARS?s Scientific Manuscript database

To characterize the involvement of miRNAs and their targets in response to short-term hypoxia conditions, a quantitative real time PCR (qRT-PCR) assay was used to quantify the expression of the 24 candidate mature miRNA signatures (22 known and 2 novel mature miRNAs, representing 66 miRNA loci) and ...

Molecular detection of Toxoplasma gondii in water samples from Scotland and a comparison between the 529bp real-time PCR and ITS1 nested PCR.

PubMed

Wells, Beth; Shaw, Hannah; Innocent, Giles; Guido, Stefano; Hotchkiss, Emily; Parigi, Maria; Opsteegh, Marieke; Green, James; Gillespie, Simon; Innes, Elisabeth A; Katzer, Frank

2015-12-15

Waterborne transmission of Toxoplasma gondii is a potential public health risk and there are currently no agreed optimised methods for the recovery, processing and detection of T. gondii oocysts in water samples. In this study modified methods of T. gondii oocyst recovery and DNA extraction were applied to 1427 samples collected from 147 public water supplies throughout Scotland. T. gondii DNA was detected, using real time PCR (qPCR) targeting the 529bp repeat element, in 8.79% of interpretable samples (124 out of 1411 samples). The samples which were positive for T. gondii DNA originated from a third of the sampled water sources. The samples which were positive by qPCR and some of the negative samples were reanalysed using ITS1 nested PCR (nPCR) and results compared. The 529bp qPCR was the more sensitive technique and a full analysis of assay performance, by Bayesian analysis using a Markov Chain Monte Carlo method, was completed which demonstrated the efficacy of this method for the detection of T. gondii in water samples. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Evaluation of chemotherapy response in patients with advanced head and neck cancer using [F-18]fluorodeoxyglucose positron emission tomography.

PubMed

Lowe, V J; Dunphy, F R; Varvares, M; Kim, H; Wittry, M; Dunphy, C H; Dunleavy, T; McDonough, E; Minster, J; Fletcher, J W; Boyd, J H

1997-12-01

[F-18]Fluorodeoxyglucose (FDG)-positron emission tomography (PET) can measure the metabolic activity of tissues; FDG-PET may be able to predict response to chemotherapy by identifying changes in tumor metabolism. Measurement of response to treatment may help improve survival in the management of advanced head and neck cancer. We evaluated this particular use of FDG-PET in patients participating in a neoadjuvant organ-preservation protocol using taxol and carboplatin and compared pathologic response after chemotherapy with changes in tumor metabolism measured by FDG-PET. Serial FDG-PET studies (n = 56) were performed in patients (n = 28) with stage III/IV head and neck cancer participating in a neoadjuvant organ-preservation protocol. The FDG-PET studies were performed before and after chemotherapy. All patients had tissue biopsies before and after chemotherapy. Patients were classified as pathologic complete response (PCR) or residual disease (RD) based on tissue biopsies. Visual analysis of PET scans was performed to identify patients with complete response by PET, and these findings were compared with pathology results. Metabolic changes were also evaluated using standardized uptake ratios (SUR) of FDG. The sensitivity and specificity of PET for residual cancer after therapy was 90% (19/21) and 83% (5/6), respectively. Two patients had initially negative biopsies and positive PET studies for persistent disease. Pathology review and rebiospy led to confirmation of the PET results in these cases, giving a sensitivity of 90% for initial tissue biopsy. In this preliminary analysis, FDG-PET was accurate in classifying response to chemotherapy in most patients. Fluorodeoxyglucose-PET may identify residual viable tumor when it is otherwise undetectable.

Evaluation of Commercial Cell Preparations as Sources of Calibration Standards for Real-Time qPCR Analysis of Enterococci in Recreational Waters

EPA Science Inventory

In response to the Beach Act, the U.S. EPA has developed a quantitative PCR (qPCR) method for enterococci that meets requirements for rapid, risk-based water quality assessments of recreational waters. Widespread implementation of this method will require that different laborator...

Mycophenolate mofetil prevents the delayed T cell response after pilocarpine-induced status epilepticus in mice

PubMed Central

Engelmann, Robby; Sellmann, Tina; Köhling, Rüdiger; Müller-Hilke, Brigitte

2017-01-01

Growing clinical and laboratory evidence corroborates a role for the immune system in the pathophysiology of epilepsy. In order to delineate the immune response following pilocarpine-induced status epilepticus (SE) in the mouse, we monitored the kinetics of leukocyte presence in the hippocampus over the period of four weeks. SE was induced following a ramping protocol of pilocarpine injection into 4–5 weeks old C57BL/6 mice. Brains were removed at days 1–4, 14 or 28 after SE, and the hippocampi were analyzed via flow cytometry, via quantitative reverse transcriptase PCR (qRT-PCR) and via immunohistochemistry. Epileptogenesis was confirmed by Timm staining of mossy fiber sprouting in the inner molecular layer of the dentate gyrus. The flow cytometry data revealed a biphasic immune response following pilocarpine-induced SE with a transient increase in activated CD11b+ and F4/80+ macrophages within the first four days replaced by an increase in CD3+ T-lymphocytes around day 28. This delayed T cell response was confirmed via qRT-PCR and via immunohistochemistry. In addition, qRT-PCR data could show that the delayed T cell response was associated with an increased CD8/CD4 ratio indicating a cytotoxic T cell response after SE. Intriguingly, early intervention with mycophenolate mofetil administration on days 0–3 after SE prevented this delayed T cell response. These results show an orchestrated immunological sequela and provide evidence that the delayed T cell response is sensitive to early immunomodulatory intervention. PMID:29182639

Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site

PubMed Central

Watzinger, Franz; Hörth, Elfriede; Lion, Thomas

2001-01-01

Despite the recent introduction of real-time PCR methods, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. Here we describe a shifted restriction-site competitive PCR (SRS-cPCR) assay based on a modified type of competitor. The competitor fragments are designed to contain a recognition site for a restriction endonuclease that is also present in the target sequence to be quantified, but in a different position. Upon completion of the PCR, the amplicons are digested in the same tube with a single restriction enzyme, without the need to purify PCR products. The generated competitor- and target-specific restriction fragments display different sizes, and can be readily separated by electrophoresis and quantified by image analysis. Suboptimal digestion affects competitor- and target-derived amplicons to the same extent, thus eliminating the problem of incorrect quantification as a result of incomplete digestion of PCR products. We have established optimized conditions for a panel of 20 common restriction endonucleases permitting efficient digestion in PCR buffer. It is possible, therefore, to find a suitable restriction site for competitive PCR in virtually any sequence of interest. The assay presented is inexpensive, widely applicable, and permits reliable and accurate quantification of nucleic acid targets. PMID:11376164

Molecular Identification of Bacteria from Aseptically Loose Implants

PubMed Central

Kobayashi, Naomi; Procop, Gary W.; Krebs, Viktor; Kobayashi, Hideo

2008-01-01

Polymerase chain reaction (PCR) assays have been used to detect bacteria adherent to failed orthopaedic implants, but some PCR assays have had problems with probable false-positive results. We used a combination of a Staphylococcus species-specific PCR and a universal PCR followed by DNA sequencing to identify bacteria on implants retrieved from 52 patients (92 implants) at revision arthroplasty. We addressed two questions in this study: (1) Is this method able to show the existence of bacterial DNA on presumed aseptic loosed implants?; and (2) What proportion of presumed aseptic or culture-negative implants was positive for bacterial DNA by PCR? Fourteen implants (15%) were believed infected, whereas 74 implants (85%) were believed aseptic. Each implant was sonicated and the resulting solution was submitted for dual real-time PCR assay and culture. All implants believed aseptically loose were culture-negative, but nine of the 74 (12%) had bacterial DNA by PCR; two (2.7%) were PCR-positive and also showed histologic findings suggestive of infection. Uniquely developed PCR and bacterial sequencing assays showed bacterial DNA on 12% of implants removed for presumed aseptic loosening. Additional studies are needed to determine the clinical importance of bacterial DNA detected by PCR but not by conventional culture. Level of Evidence: Level III, diagnostic study. See the Guidelines for Authors for a complete description of levels of evidence. PMID:18438724

Rapid detection of Porcine circovirus 2 by recombinase polymerase amplification.

PubMed

Wang, Jianchang; Wang, Jinfeng; Liu, Libing; Li, Ruiwen; Yuan, Wanzhe

2016-09-01

Porcine circovirus-associated disease, caused primarily by Porcine circovirus 2 (PCV-2), has become endemic in many pig-producing countries and has resulted in significant economic losses to the swine industry worldwide. Tests for PCV-2 infection include PCR, nested PCR, competitive PCR, and real-time PCR (rtPCR). Recombinase polymerase amplification (RPA) has emerged as an isothermal gene amplification technology for the molecular detection of infectious disease agents. RPA is performed at a constant temperature and therefore can be carried out in a water bath. In addition, RPA is completed in ~30 min, much faster than PCR, which usually takes >60 min. We developed a RPA-based method for the detection of PCV-2. The detection limit of RPA was 10(2) copies of PCV-2 genomic DNA. RPA showed the same sensitivity as rtPCR but was 10 times more sensitive than conventional PCR. Successful amplification of PCV-2 DNA, but not other viral templates, demonstrated high specificity of the RPA assay. This method was also validated using clinical samples. The results showed that the RPA assay had a diagnostic agreement rate of 93.7% with conventional PCR and 100% with rtPCR. These findings suggest that the RPA assay is a simple, rapid, and cost-effective method for PCV-2 detection, which could be potentially applied in clinical diagnosis and field surveillance of PCV-2 infection. © 2016 The Author(s).

Droplet Digital PCR for Minimal Residual Disease Detection in Mature Lymphoproliferative Disorders.

PubMed

Drandi, Daniela; Ferrero, Simone; Ladetto, Marco

2018-01-01

Minimal residual disease (MRD) detection has a powerful prognostic relevance for response evaluation and prediction of relapse in hematological malignancies. Real-time quantitative PCR (qPCR) has become the settled and standardized method for MRD assessment in lymphoid disorders. However, qPCR is a relative quantification approach, since it requires a reference standard curve. Droplet digital TM PCR (ddPCR TM ) allows a reliable absolute tumor burden quantification withdrawing the need for preparing, for each experiment, a tumor-specific standard curve. We have recently shown that ddPCR has a good concordance with qPCR and could be a feasible and reliable tool for MRD monitoring in mature lymphoproliferative disorders. In this chapter we describe the experimental workflow, from the detection of the clonal molecular marker to the MRD monitoring by ddPCR, in patients affected by multiple myeloma, mantle cell lymphoma and follicular lymphoma. However, standardization programs among different laboratories are needed in order to ensure the reliability and reproducibility of ddPCR-based MRD results.

Creation of a bovine herpes virus 1 (BoHV-1) quantitative particle standard by transmission electron microscopy and comparison with established standards for use in real-time PCR.

PubMed

Hoferer, Marc; Braun, Anne; Sting, Reinhard

2017-07-01

Standards are pivotal for pathogen quantification by real-time PCR (qPCR); however, the creation of a complete and universally applicable virus particle standard is challenging. In the present study a procedure based on purification of bovine herpes virus type 1 (BoHV-1) and subsequent quantification by transmission electron microscopy (TEM) is described. Accompanying quantitative quality controls of the TEM preparation procedure using qPCR yielded recovery rates of more than 95% of the BoHV-1 virus particles on the grid used for virus counting, which was attributed to pre-treatment of the grid with 5% bovine albumin. To compare the value of the new virus particle standard for use in qPCR, virus counter based quantification and established pure DNA standards represented by a plasmid and an oligonucleotide were included. It could be shown that the numbers of virus particles, plasmid and oligonucleotide equivalents were within one log10 range determined on the basis of standard curves indicating that different approaches provide comparable quantitative values. However, only virus particles represent a complete, universally applicable quantitative virus standard that meets the high requirements of an RNA and DNA virus gold standard. In contrast, standards based on pure DNA have to be considered as sub-standard due to limited applications. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

The molecular epidemiological study of bovine leukemia virus infection in Myanmar cattle.

PubMed

Polat, Meripet; Moe, Hla Hla; Shimogiri, Takeshi; Moe, Kyaw Kyaw; Takeshima, Shin-Nosuke; Aida, Yoko

2017-02-01

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide and affects both health status and productivity. However, no studies have examined the distribution of BLV in Myanmar, and the genetic characteristics of Myanmar BLV strains are unknown. Therefore, the aim of this study was to detect BLV infection in Myanmar and examine genetic variability. Blood samples were obtained from 66 cattle from different farms in four townships of the Nay Pyi Taw Union Territory of central Myanmar. BLV provirus was detected by nested PCR and real-time PCR targeting BLV long terminal repeats. Results were confirmed by nested PCR targeting the BLV env-gp51 gene and real-time PCR targeting the BLV tax gene. Out of 66 samples, six (9.1 %) were positive for BLV provirus. A phylogenetic tree, constructed using five distinct partial and complete env-gp51 sequences from BLV strains isolated from three different townships, indicated that Myanmar strains were genotype-10. A phylogenetic tree constructed from whole genome sequences obtained by sequencing cloned, overlapping PCR products from two Myanmar strains confirmed the existence of genotype-10 in Myanmar. Comparative analysis of complete genome sequences identified genotype-10-specific amino acid substitutions in both structural and non-structural genes, thereby distinguishing genotype-10 strains from other known genotypes. This study provides information regarding BLV infection levels in Myanmar and confirms that genotype-10 is circulating in Myanmar.

Allele-Specific PCR for Determination of IL28B Genotype

PubMed Central

Cook, Linda; Diem, Kurt; Kim, Woo; Scott, John D.

2012-01-01

The IL28B genotype is a critical determinant of interferon response in patients infected with hepatitis C virus genotype 1. We describe an allele-specific PCR assay for the IL28B genotype. The assay is simple and robust, uses commonly available real-time PCR instrumentation, and is well suited for clinical laboratories offering IL28B genotyping. PMID:23052312

A Phase 3, Double-Blind, Randomized Study of Arterolane Maleate-Piperaquine Phosphate vs Artemether-Lumefantrine for Falciparum Malaria in Adolescent and Adult Patients in Asia and Africa.

PubMed

Toure, Offianan Andre; Valecha, Neena; Tshefu, Antoinette K; Thompson, Ricardo; Krudsood, Srivicha; Gaye, Oumar; Rao, Bappanaidu Hoigegudde Krishnamurthy; Sagara, Issaka; Bose, Tarit Kumar; Mohanty, Sanjib; Rao, Ballamudi Srinivas; Anvikar, Anupkumar R; Mwapasa, Victor; Noedl, Harald; Arora, Sudershan; Roy, Arjun; Iyer, Sunil S; Sharma, Pradeep; Saha, Nilanjan; Jalali, Rajinder K; Tiacoh, Landry; Enosse, Sonia; Tangpukdee, Noppadon; Kokolomami, Jack; Ndiaye, Jean-Louis; Rao, Deepak; Yumva, Ntamabyaliro Nsengi; Sidibe, Bouran; Mohanty, Rajesh; Jha, A C; Nyirenda, Mulinda; Starzengruber, Peter; Swoboda, Paul

2016-04-15

Artemisinins, which are derived from plants, are subject to risk of supply interruption due to climatic changes. Consequently, an effort to identify a new synthetic antimalarial was initiated. A fixed-dose combination of arterolane maleate (AM), a new synthetic trioxolane, with piperaquine phosphate (PQP), a long half-life bisquinoline, was evaluated in patients with uncomplicatedPlasmodium falciparummalaria. In this multicenter, randomized, double-blind, comparative, parallel-group trial, 1072 patients aged 12-65 years withP. falciparummonoinfection received either AM-PQP (714 patients) once daily or artemether-lumefantrine (A-L; 358 patients) twice daily for 3 days. All patients were followed up until day 42. Of the 714 patients in the AM-PQP group, 638 (89.4%) completed the study; of the 358 patients in the A-L group, 301(84.1%) completed the study. In both groups, the polymerase chain reaction corrected adequate clinical and parasitological response (PCR-corrected ACPR) on day 28 in intent-to-treat (ITT) and per-protocol (PP) populations was 92.86% and 92.46% and 99.25% and 99.07%, respectively. The corresponding figures on day 42 in the ITT and PP populations were 90.48% and 91.34%, respectively. After adjusting for survival ITT, the PCR-corrected ACPR on day 42 was >98% in both groups. The overall incidence of adverse events was comparable. AM-PQP showed comparable efficacy and safety to A-L in the treatment of uncomplicatedP. falciparummalaria in adolescent and adult patients. AM-PQP demonstrated high clinical and parasitological response rates as well as rapid parasite clearance. India. CTRI/2009/091/000101. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Utility of Real-Time PCR for Detection of Exserohilum rostratum in Body and Tissue Fluids during the Multistate Outbreak of Fungal Meningitis and Other Infections

PubMed Central

Gade, Lalitha; Grgurich, Dale E.; Kerkering, Thomas M.; Brandt, Mary E.

2014-01-01

Exserohilum rostratum was the major cause of the multistate outbreak of fungal meningitis linked to contaminated injections of methylprednisolone acetate produced by the New England Compounding Center. Previously, we developed a fungal DNA extraction procedure and broad-range and E. rostratum-specific PCR assays and confirmed the presence of fungal DNA in 28% of the case patients. Here, we report the development and validation of a TaqMan real-time PCR assay for the detection of E. rostratum in body fluids, which we used to confirm infections in 57 additional case patients, bringing the total number of case patients with PCR results positive for E. rostratum to 171 (37% of the 461 case patients with available specimens). Compared to fungal culture and the previous PCR assays, this real-time PCR assay was more sensitive. Of the 139 identical specimens from case patients tested by all three methods, 19 (14%) were positive by culture, 41 (29%) were positive by the conventional PCR assay, and 65 (47%) were positive by the real-time PCR assay. We also compared the utility of the real-time PCR assay with that of the previously described beta-d-glucan (BDG) detection assay for monitoring response to treatment in case patients with serially collected CSF. Only the incident CSF specimens from most of the case patients were positive by real-time PCR, while most of the subsequently collected specimens were negative, confirming our previous observations that the BDG assay was more appropriate than the real-time PCR assay for monitoring the response to treatment. Our results also demonstrate that the real-time PCR assay is extremely susceptible to contamination and its results should be used only in conjunction with clinical and epidemiological data. PMID:25520443

Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective

PubMed Central

Nombela, Ivan; Carrion, Aurora; Puente-Marin, Sara; Chico, Verónica; Mercado, Luis; Perez, Luis; Coll, Julio; Ortega-Villaizan, Maria del Mar

2017-01-01

Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection  have not been studied yet. Methods: Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results: IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions: Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs. PMID:29333244

Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective.

PubMed

Nombela, Ivan; Carrion, Aurora; Puente-Marin, Sara; Chico, Verónica; Mercado, Luis; Perez, Luis; Coll, Julio; Ortega-Villaizan, Maria Del Mar

2017-01-01

Background : Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection  have not been studied yet. Methods : Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results : IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1 , pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions : Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs.

A Randomized Phase 2 Study of Neoadjuvant Chemoradiaton Therapy With 5-Fluorouracil/Leucovorin or Irinotecan/S-1 in Patients With Locally Advanced Rectal Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Minkyu; Shin, Sang Joon; Koom, Woong Sub

2015-12-01

Purpose: The purpose of this study was to evaluate the rate of pathologic complete response (pCR) in patients with locally advanced rectal cancer (LARC) treated with neoadjuvant chemoradiation therapy (CRT) with leucovorin (FL) versus irinotecan/S-1 (IS). Methods and Materials: Patients with resectable LARC (clinical stage T3/4, lymph node positive, or both) were randomly assigned to receive preoperative radiation (45-50.4 Gy in 25 to 28 daily fractions) and concomitant chemotherapy either with a bolus injection of FL (400 mg/m{sup 2}/day 5-fluorouracil and 20 mg/m{sup 2}/day leucovorin) for 3 consecutive days every 4 weeks for 2 cycles (FL group) or with 40 mg/m{sup 2} irinotecan on daysmore » 1, 8, 15, 22, and 29, and 35 mg/m{sup 2} S-1 twice on the day of irradiation (IS group). Curative surgery was performed approximately 4 to 8 weeks after the completion of CRT. The postoperative chemotherapy regimen was FL with a primary endpoint of a pCR rate evaluation. Results: One hundred forty-two eligible patients were randomly assigned, and the median follow-up duration was 43.8 months (95% confidence interval, 40.8-46.8 months). One hundred thirty-three patients (93.7%) of 142 underwent total mesorectal excision; pCR was achieved in 11 (16.7%) of 66 patients in the FL group and 17 (25.8%) of 67 patients in the IS group (P=.246). When good responders were defined as patients with Mandard grades 1 and 2, the rate of good responders was significantly higher in the IS group than in the FL group (54.6% vs 36.4%, respectively, P=.036). The preoperative rates of grade 3 and 4 toxicities were higher in the IS group (7.0%) than in the FL group (1.4%, P=.095). The 3-year disease-free survival was not significantly different between the 2 groups (79.7% vs 76.6%, respectively, P=.896). Conclusions: IS-based preoperative CRT did not increase pCR rate, but it did increase acute toxicities compared with standard 5-FU treatment. Therefore, further investigation is needed.« less

Clinicopathological predictors of long-term benefit in breast cancer treated with neoadjuvant chemotherapy

PubMed Central

Galvez, Marco; Castaneda, Carlos A; Sanchez, Joselyn; Castillo, Miluska; Rebaza, Lia Pamela; Calderon, Gabriela; Cruz, Miguel De La; Cotrina, Jose Manuel; Abugattas, Julio; Dunstan, Jorge; Guerra, Henry; Mejia, Omar; Gomez, Henry L

2018-01-01

AIM To investigate the survival impact of clinicopathological factors, including pathological complete response (pCR) and tumor-infiltrating lymphocytes (sTIL) levels according to subtypes, in breast cancer (BC) patients who received neo-adjuvant chemotherapy (NAC). METHODS We evaluated 435 BC patients who presented and received NAC at the Instituto Nacional de Enfermedades Neoplasicas from 2003 to 2014. sTIL was analyzed as the proportion of tumor stroma occupied by lymphocytes, and was prospectively evaluated on hematoxylin and eosin-stained sections of the preNAC core biopsy. pCR was considered in the absence of infiltrating cancer cells in primary tumor and axillary lymph nodes. Analysis of statistical association between clinical pathological features, sTIL, pCR and survival were carried out using SPSSvs19. RESULTS Median age was 49 years (range 24-84 years) and the most frequent clinical stage was IIIB (58.3%). Luminal A, Luminal B, HER2-enriched and (triple-negative) TN phenotype was found in 24.6%, 37.9%, 17.7% and 19.8%, respectively. pCR was observed in 11% and median percentage of sTIL was 40% (2%-95%) in the whole population. pCR was associated to Ct1-2 (P = 0.045) and to high sTIL (P = 0.029) in the whole population. There was a slight trend towards significance for sTIL (P = 0.054) in Luminal A. sTIL was associated with grade III (P < 0.001), no-Luminal A subtype (P < 0.001), RE-negative (P < 0.001), PgR-negative (P < 0.001), HER2-positive (P = 0.002) and pCR (P = 0.029) in the whole population. Longer disease-free survival was associated with grade I-II (P = 0.006), cN0 (P < 0.001), clinical stage II (P = 0.004), ER-positive (P < 0.001), PgR-positive (P < 0.001), luminal A (P < 0.001) and pCR (P = 0.002). Longer disease-free survival was associated with grade I-II in Luminal A (P < 0.001), N0-1 in Luminal A (P = 0.045) and TNBC (P = 0.01), clinical stage II in Luminal A (P = 0.003) and TNBC (P = 0.038), and pCR in TNBC (P < 0.001). Longer overall survival was associated with grade I-II (P < 0.001), ER-positive (P < 0.001), PgR-positive (P < 0.001), Luminal A (P < 0.001), cN0 (P = 0.002) and pCR (P = 0.002) in the whole population. Overall survival was associated with clinical stage II (P = 0.017) in Luminal A, older age (P = 0.042) in Luminal B, and pCR in TNBC (P = 0.005). CONCLUSION Predictive and prognostic values of clinicopathological features, like pCR and sTIL, differ depending on the evaluated molecular subtype PMID:29651385

Modelling in vivo creatine/phosphocreatine in vitro reveals divergent adaptations in human muscle mitochondrial respiratory control by ADP after acute and chronic exercise.

PubMed

Ydfors, Mia; Hughes, Meghan C; Laham, Robert; Schlattner, Uwe; Norrbom, Jessica; Perry, Christopher G R

2016-06-01

Mitochondrial respiratory sensitivity to ADP is thought to influence muscle fitness and is partly regulated by cytosolic-mitochondrial diffusion of ADP or phosphate shuttling via creatine/phosphocreatine (Cr/PCr) through mitochondrial creatine kinase (mtCK). Previous measurements of respiration in vitro with Cr (saturate mtCK) or without (ADP/ATP diffusion) show mixed responses of ADP sensitivity following acute exercise vs. less sensitivity after chronic exercise. In human muscle, modelling in vivo 'exercising' [Cr:PCr] during in vitro assessments revealed novel responses to exercise that differ from detections with or without Cr (±Cr). Acute exercise increased ADP sensitivity when measured without Cr but had no effect ±Cr or with +Cr:PCr, whereas chronic exercise increased sensitivity ±Cr but lowered sensitivity with +Cr:PCr despite increased markers of mitochondrial oxidative capacity. Controlling in vivo conditions during in vitro respiratory assessments reveals responses to exercise that differ from typical ±Cr comparisons and challenges our understanding of how exercise improves metabolic control in human muscle. Mitochondrial respiratory control by ADP (Kmapp ) is viewed as a critical regulator of muscle energy homeostasis. However, acute exercise increases, decreases or has no effect on Kmapp in human muscle, whereas chronic exercise surprisingly decreases sensitivity despite greater mitochondrial content. We hypothesized that modelling in vivo mitochondrial creatine kinase (mtCK)-dependent phosphate-shuttling conditions in vitro would reveal increased sensitivity (lower Kmapp ) after acute and chronic exercise. The Kmapp was determined in vitro with 20 mm Cr (+Cr), 0 mm Cr (-Cr) or 'in vivo exercising' 20 mm Cr/2.4 mm PCr (Cr:PCr) on vastus lateralis biopsies sampled from 11 men before, immediately after and 3 h after exercise on the first, fifth and ninth sessions over 3 weeks. Dynamic responses to acute exercise occurred throughout training, whereby the first session did not change Kmapp with in vivo Cr:PCr despite increases in -Cr. The fifth session decreased sensitivity with Cr:PCr or +Cr despite no change in -Cr. Chronic exercise increased sensitivity ±Cr in association with increased electron transport chain content (+33-62% complexes I-V), supporting classic proposals that link increased sensitivity to oxidative capacity. However, in vivo Cr:PCr reveals a perplexing decreased sensitivity, contrasting the increases seen ±Cr. Functional responses occurred without changes in fibre type or proteins regulating mitochondrial-cytosolic energy exchange (mtCK, VDAC and ANT). Despite the dynamic responses seen with ±Cr, modelling in vivo phosphate-shuttling conditions in vitro reveals that ADP sensitivity is unchanged after high-intensity exercise and is decreased after training. These findings challenge our understanding of how exercise regulates skeletal muscle energy homeostasis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions

USGS Publications Warehouse

Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Shively, Dawn A.; Nevers, Meredith B.

2010-01-01

Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100 ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) (R = 0.650, P N = 32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher (P R = 0.565, P N = 32). Culturable E. coli and enterococci densities were significantly correlated (R = 0.682, P N = 32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R2 = 0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity × wave height (adjusted R2 = 0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results

Characterization of a new apple luteovirus identified by high-throughput sequencing.

PubMed

Liu, Huawei; Wu, Liping; Nikolaeva, Ekaterina; Peter, Kari; Liu, Zongrang; Mollov, Dimitre; Cao, Mengji; Li, Ruhui

2018-05-15

'Rapid Apple Decline' (RAD) is a newly emerging problem of young, dwarf apple trees in the Northeastern USA. The affected trees show trunk necrosis, cracking and canker before collapse in summer. In this study, we discovered and characterized a new luteovirus from apple trees in RAD-affected orchards using high-throughput sequencing (HTS) technology and subsequent Sanger sequencing. Illumina NextSeq sequencing was applied to total RNAs prepared from three diseased apple trees. Sequence reads were de novo assembled, and contigs were annotated by BLASTx. RT-PCR and 5'/3' RACE sequencing were used to obtain the complete genome of a new virus. RT-PCR was used to detect the virus. Three common apple viruses and a new luteovirus were identified from the diseased trees by HTS and RT-PCR. Sequence analyses of the complete genome of the new virus show that it is a new species of the genus Luteovirus in the family Luteoviridae. The virus is graft transmissible and detected by RT-PCR in apple trees in a couple of orchards. A new luteovirus and/or three known viruses were found to be associated with RAD. Molecular characterization of the new luteovirus provides important information for further investigation of its distribution and etiological role.

Complete Genome Sequence of a Putative Densovirus of the Asian Citrus Psyllid, Diaphorina citri.

PubMed

Nigg, Jared C; Nouri, Shahideh; Falk, Bryce W

2016-07-28

Here, we report the complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri Diaphorina citri densovirus (DcDNV) was originally identified through metagenomics, and here, we obtained the complete nucleotide sequence using PCR-based approaches. Phylogenetic analysis places DcDNV between viruses of the Ambidensovirus and Iteradensovirus genera. Copyright © 2016 Nigg et al.

Biochemical pathways and enhanced degradation of di-n-octyl phthalate (DOP) in sequencing batch reactor (SBR) by Arthrobacter sp. SLG-4 and Rhodococcus sp. SLG-6 isolated from activated sludge.

PubMed

Zhang, Ke; Liu, Yihao; Chen, Qiang; Luo, Hongbing; Zhu, Zhanyuan; Chen, Wei; Chen, Jia; Mo, You

2018-04-01

Two bacterial strains designated as Arthrobacter sp. SLG-4 and Rhodococcus sp. SLG-6, capable of utilizing di-n-octyl phthalate (DOP) as sole source of carbon and energy, were isolated from activated sludge. The analysis of DOP degradation intermediates indicated Arthrobacter sp. SLG-4 could completely degrade DOP. Whereas DOP could not be mineralized by Rhodococcus sp. SLG-6 and the final metabolic product was phthalic acid (PA). The proposed DOP degradation pathway by Arthrobacter sp. SLG-4 was that strain SLG-4 initially transformed DOP to PA via de-esterification pathway, and then PA was metabolized to protocatechuate acid and eventually converted to tricarboxylic acid (TCA) cycle through meta-cleavage pathway. Accordingly, Phthalate 3,4-dioxygenase genes (phtA) responsible for PA degradation were successfully detected in Arthrobacter sp. SLG-4 by real-time quantitative PCR (q-PCR). q-PCR analysis demonstrated that the quantity of phthalate 3,4-dioxygenase was positively correlated to DOP degradation in SBRs. Bioaugmentation by inoculating DOP-degrading bacteria effectively shortened the start-up of SBRs and significantly enhanced DOP degradation in bioreactors. More than 91% of DOP (500 mg L -1 ) was removed in SBR bioaugmented with bacterial consortium, which was double of the control SBR. This study suggests bioaugmentation is an effective and feasible technique for DOP bioremediation in practical engineering.

Preoperative cetuximab, irinotecan, cisplatin, and radiation therapy for patients with locally advanced esophageal cancer.

PubMed

Lee, Michael S; Mamon, Harvey J; Hong, Theodore S; Choi, Noah C; Fidias, Panagiotis M; Kwak, Eunice L; Meyerhardt, Jeffrey A; Ryan, David P; Bueno, Raphael; Donahue, Dean M; Jaklitsch, Michael T; Lanuti, Michael; Rattner, David W; Fuchs, Charles S; Enzinger, Peter C

2013-01-01

To determine the efficacy and toxicity of weekly neoadjuvant cetuximab combined with irinotecan, cisplatin, and radiation therapy in patients with locally advanced esophageal or gastroesophageal junction cancer. Patients with stage IIA-IVA esophageal or gastroesophageal junction cancer were enrolled in a Simon's two-stage phase II study. Patients received weekly cetuximab on weeks 0-8 and irinotecan and cisplatin on weeks 1, 2, 4, and 5, with concurrent radiotherapy (50.4 Gy on weeks 1-6), followed by surgical resection. In the first stage, 17 patients were enrolled, 16 of whom had adenocarcinoma. Because of a low pathologic complete response (pCR) rate in this cohort, the trial was discontinued for patients with adenocarcinoma but squamous cell carcinoma patients continued to be enrolled; two additional patients were enrolled before the study was closed as a result of poor accrual. Of the 19 patients enrolled, 18 patients proceeded to surgery, and 16 patients underwent an R0 resection. Three patients (16%) had a pCR. The median progression-free survival interval was 10 months, and the median overall survival duration was 31 months. Severe neutropenia occurred in 47% of patients, and severe diarrhea occurred in 47% of patients. One patient died preoperatively from sepsis, and one patient died prior to hospital discharge following surgical resection. This schedule of cetuximab in combination with irinotecan, cisplatin, and radiation therapy was toxic and did not achieve a sufficient pCR rate in patients with localized esophageal adenocarcinoma to undergo further evaluation.

New mutations of DAX-1 genes in two Japanese patients with X-linked congenital adrenal hypoplasia and hypogonadotropic hypogonadism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanase, Toshihiko; Takayanagi, Ryoichi; Oba, Koichi

Congenital adrenal hypoplasia, an X-linked disorder, is characterized by primary adrenal insufficiency and frequent association with hypogonadotropic hypogonadism. The X-chromosome gene DAX-1 has been most recently identified and shown to be responsible for this disorder. We analyzed the DAX-1 genes of two unrelated Japanese patients with congenital adrenal hypoplasia and hypogonadotropic hypogonadism by using PCR amplification of genomic DNA and its complete exonic sequencing. In a family containing several affected individuals, the proband male patient had a stop codon (TGA) in place of tryptophan (TGG) at amino acid position 171. As expected, his mother was a heterozygous carrier for themore » mutation, whereas his father and unaffected brother did not carry this mutation. In another male patient with noncontributory family history, sequencing revealed a 1-bp (T) deletion at amino acid position 280, leading to a frame shift and, subsequently a premature stop codon at amino acid position 371. The presence of this mutation in the patients` genome was further confirmed by digestion of genomic PCR product with MspI created by this mutation. Family studies using MspI digestion of genomic PCR products revealed that neither parent of this individual carried the mutation. These results clearly indicate that congenital adrenal hypoplasia and hypogonadotropic hypogonadism result from not only inherited but also de novo mutation in the DAX-1 gene. 31 refs., 4 figs., 2 tabs.« less

Antibacterial activity and mechanism of action of ε-poly-L-lysine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Ruosong; Xu, Hengyi; Wan, Cuixiang

Highlights: •Antibacterial activity and mechanism of ε-PL against E. coli O157:H7 was investigated. •Critical inhibitory factors toward the growth of E. coli O157:H7 by ε-PL was analyzed. •Cell membrane integrity and cell morphology of E. coli O157:H7 was affected by ε-PL. •A positive correlation between reactive oxygen species levels and ε-PL concentration in E. coli O157:H7 cells. •ε-PL induced the expression of different genes related to oxidative/redox stress, SOS response, virulence. -- Abstract: ε-Poly-L-lysine (ε-PL) is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level hasmore » not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS) levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR) indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response) regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence.« less

Use of 16S-23S rRNA spacer-region (SR)-PCR for identification of intestinal clostridia.

PubMed

Song, Yuli; Liu, Chengxu; Molitoris, Denise; Tomzynski, Thomas J; Mc Teague, Maureen; Read, Erik; Finegold, Sydney M

2002-12-01

The suitability of a species identification technique based on PCR analysis of 16S-23S rRNA spacer region (SR) polymorphism for human intestinal Clostridium species was evaluated. This SR-PCR based technique is highly reproducible and successfully differentiated the strains tested, which included 17 ATCC type strains of Clostridium and 152 human stool Clostridium isolates, at the species or intraspecies level. Ninety-eight of 152 stool isolates, including C. bifermentans, C. butyricum, C. cadaveris, C. orbiscindens, C. paraputrificum, C. pefringens, C. ramosum, C. scindens, C. spiroforme, C. symbiosum and C. tertium, were identified to species level by SR-PCR patterns that were identical to those of their corresponding ATCC type strains. The other 54 stool isolates distributed among ten SR-PCR patterns that are unique and possibly represent ten novel Clostridium species or subspecies. The species identification obtained by SR-PCR pattern analysis completely agreed with that obtained by 16S rRNA sequencing, and led to identification that clearly differed from that obtained by cellular fatty acid analysis for 23/152 strains (15%). These results indicate that SR-PCR provides an accurate and rapid molecular method for the identification of human intestinal Clostridium species.

PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines

USGS Publications Warehouse

Jarvi, Susan I.; Schultz, Jeffrey J.; Atkinson, Carter T.

2002-01-01

Several polymerase chain reaction (PCR)-based methods have recently been developed for diagnosing malarial infections in both birds and reptiles, but a critical evaluation of their sensitivity in experimentally-infected hosts has not been done. This study compares the sensitivity of several PCR-based methods for diagnosing avian malaria (Plasmodium relictum) in captive Hawaiian honeycreepers using microscopy and a recently developed immunoblotting technique. Sequential blood samples were collected over periods of up to 4.4 yr after experimental infection and rechallenge to determine both the duration and detectability of chronic infections. Two new nested PCR approaches for detecting circulating parasites based on P. relictum 18S rRNA genes and the thrombospondin-related anonymous protein (TRAP) gene are described. The blood smear and the PCR tests were less sensitive than serological methods for detecting chronic malarial infections. Individually, none of the diagnostic methods was 100% accurate in detecting subpatent infections, although serological methods were significantly more sensitive (97%) than either nested PCR (61–84%) or microscopy (27%). Circulating parasites in chronically infected birds either disappear completely from circulation or to drop to intensities below detectability by nested PCR. Thus, the use of PCR as a sole means of detection of circulating parasites may significantly underestimate true prevalence.

Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

PubMed

Taira, Chiaki; Matsuda, Kazuyuki; Takeichi, Naoya; Furukawa, Satomi; Sugano, Mitsutoshi; Uehara, Takeshi; Okumura, Nobuo; Honda, Takayuki

2018-01-01

ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care. © 2017 Wiley Periodicals, Inc.

Data on DNA gel sample load, gel electrophoresis, PCR and cost analysis.

PubMed

Kuhn, Ramona; Böllmann, Jörg; Krahl, Kathrin; Bryant, Isaac Mbir; Martienssen, Marion

2018-02-01

The data presented in this article provide supporting information to the related research article "Comparison of ten different DNA extraction procedures with respect to their suitability for environmental samples" (Kuhn et al., 2017) [1]. In that article, we compared the suitability of ten selected DNA extraction methods based on DNA quality, purity, quantity and applicability to universal PCR. Here we provide the data on the specific DNA gel sample load, all unreported gel images of crude DNA and PCR results, and the complete cost analysis for all tested extraction procedures and in addition two commercial DNA extraction kits for soil and water.

One-step cross-genogroup multiplex RT-qPCR with an internal control system for the detection of infectious pancreatic necrosis virus (IPNV).

PubMed

Hoferer, Marc; Braun, Anne; Skrypski, Julia; Bock, Sabine; Thalheim, Sabine; Sting, Reinhard

2017-09-01

Infectious pancreatic necrosis virus (IPNV) causes great losses in fish hatcheries world-wide. The detection of IPNV can be challenging in certain circumstances, particularly due to low viral load and the genetic variability of this RNA virus. For the first time, this project created a quantitative triplex real-time reverse transcription PCR (RT-qPCR), including an endogenous control system, for specific, sensitive and rapid detection of IPNV in routine diagnostics. Multiple sequence alignment of 46 nucleotide sequences of the segment A genome obtained from the NCBI database allowed the design of two RT-qPCR systems covering the IPNV genogroup 1 and genogroups 2-5, respectively. The completed triplex RT-qPCR including a salmonid-specific endogenous control showed high specificity and an analytical sensitivity of 20-40 oligonucleotide copies. Testing of dilution series of virus-loaded cell culture suspensions proved equality of the triplex RT-qPCR with virus detection in cell culture and a higher sensitivity than conventional RT-PCR in field samples. In comparative studies of a total of 77 field samples tested, 51 showed identical positive and 19 identical negative results in cell culture and the triplex RT-qPCR. However, seven other samples yielded positive results in the triplex RT-qPCR, but negative results in cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of individual powdery mildew fungi infecting leaves and direct detection of gene expression by single conidium polymerase chain reaction.

PubMed

Matsuda, Yoshinori; Sameshima, Takeshi; Moriura, Nobuyuki; Inoue, Kanako; Nonomura, Teruo; Kakutani, Koji; Nishimura, Hiroaki; Kusakari, Shin-Ichi; Takamatsu, Susumu; Toyoda, Hideyoshi

2005-10-01

ABSTRACT Greenhouse-grown tomato seedlings were inoculated naturally with two genera of powdery mildew conidia forming appressorial germ tubes that could not be differentiated by length alone. For direct identification, single germinated conidia were removed from leaves by means of a glass pipette linked to the manipulator of a high-fidelity digital microscope. This microscope enabled in vivo observation of the fungi without leaf decoloration or fungal staining. The isolated conidia were subjected to PCR amplification of the 5.8S rDNA and its adjacent internal transcribed spacer sequences followed by nested PCR to attain sensitivity high enough to amplify target nucleotide sequences (PCR/nested PCR). Target sequences from the conidia were completely coincident with those of the pathogen Oidium neolycopersici or Erysiphe trifolii (syn. Microsphaera trifolii), which is nonpathogenic on tomato. Using RT-PCR/nested PCR or multiplex RT-PCR/nested PCR, it was possible to amplify transcripts expressed in single conidia. Conidia at pre- and postgermination stages were removed individually from tomato leaves, and two powdery mildew genes were monitored. The results indicated that the beta-tubulin homolog TUB2-ol was expressed at pre- and postgermination stages and the cutinase homolog CUT1-ol was only expressed postgermination. Combining digital microscopic micromanipulation and two-step PCR amplification is thus useful for investigation of individual propagules on the surface of plants.

Assessment of African Swine Fever Diagnostic Techniques as a Response to the Epidemic Outbreaks in Eastern European Union Countries: How To Improve Surveillance and Control Programs

PubMed Central

Nieto, R.; Soler, A.; Pelayo, V.; Fernández-Pinero, J.; Markowska-Daniel, I.; Pridotkas, G.; Nurmoja, I.; Granta, R.; Simón, A.; Pérez, C.; Martín, E.; Fernández-Pacheco, P.; Arias, M.

2015-01-01

This study represents a complete comparative analysis of the most widely used African swine fever (ASF) diagnostic techniques in the European Union (EU) using field and experimental samples from animals infected with genotype II ASF virus (ASFV) isolates circulating in Europe. To detect ASFV, three different PCRs were evaluated in parallel using 785 field and experimental samples. The results showed almost perfect agreement between the Universal ProbeLibrary (UPL-PCR) and the real-time (κ = 0.94 [95% confidence interval {CI}, 0.91 to 0.97]) and conventional (κ = 0.88 [95% CI, 0.83 to 0.92]) World Organisation for Animal Health (OIE)-prescribed PCRs. The UPL-PCR had greater diagnostic sensitivity for detecting survivors and allows earlier detection of the disease. Compared to the commercial antigen enzyme-linked immunosorbent assay (ELISA), good-to-moderate agreement (κ = 0.67 [95% CI, 0.58 to 0.76]) was obtained, with a sensitivity of 77.2% in the commercial test. For ASF antibody detection, five serological methods were tested, including three commercial ELISAs, the OIE-ELISA, and the confirmatory immunoperoxidase test (IPT). Greater sensitivity was obtained with the IPT than with the ELISAs, since the IPT was able to detect ASF antibodies at an earlier point in the serological response, when few antibodies are present. The analysis of the exudate tissues from dead wild boars showed that IPT might be a useful serological tool for determining whether or not animals had been exposed to virus infection, regardless of whether antibodies were present. In conclusion, the UPL-PCR in combination with the IPT was the most trustworthy method for detecting ASF during the epidemic outbreaks affecting EU countries in 2014. The use of the most appropriate diagnostic tools is critical when implementing effective control programs. PMID:26041901

Assessment of African Swine Fever Diagnostic Techniques as a Response to the Epidemic Outbreaks in Eastern European Union Countries: How To Improve Surveillance and Control Programs.

PubMed

Gallardo, C; Nieto, R; Soler, A; Pelayo, V; Fernández-Pinero, J; Markowska-Daniel, I; Pridotkas, G; Nurmoja, I; Granta, R; Simón, A; Pérez, C; Martín, E; Fernández-Pacheco, P; Arias, M

2015-08-01

This study represents a complete comparative analysis of the most widely used African swine fever (ASF) diagnostic techniques in the European Union (EU) using field and experimental samples from animals infected with genotype II ASF virus (ASFV) isolates circulating in Europe. To detect ASFV, three different PCRs were evaluated in parallel using 785 field and experimental samples. The results showed almost perfect agreement between the Universal ProbeLibrary (UPL-PCR) and the real-time (κ = 0.94 [95% confidence interval {CI}, 0.91 to 0.97]) and conventional (κ = 0.88 [95% CI, 0.83 to 0.92]) World Organisation for Animal Health (OIE)-prescribed PCRs. The UPL-PCR had greater diagnostic sensitivity for detecting survivors and allows earlier detection of the disease. Compared to the commercial antigen enzyme-linked immunosorbent assay (ELISA), good-to-moderate agreement (κ = 0.67 [95% CI, 0.58 to 0.76]) was obtained, with a sensitivity of 77.2% in the commercial test. For ASF antibody detection, five serological methods were tested, including three commercial ELISAs, the OIE-ELISA, and the confirmatory immunoperoxidase test (IPT). Greater sensitivity was obtained with the IPT than with the ELISAs, since the IPT was able to detect ASF antibodies at an earlier point in the serological response, when few antibodies are present. The analysis of the exudate tissues from dead wild boars showed that IPT might be a useful serological tool for determining whether or not animals had been exposed to virus infection, regardless of whether antibodies were present. In conclusion, the UPL-PCR in combination with the IPT was the most trustworthy method for detecting ASF during the epidemic outbreaks affecting EU countries in 2014. The use of the most appropriate diagnostic tools is critical when implementing effective control programs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity

NASA Technical Reports Server (NTRS)

Orihuela, C. J.; Mills, J.; Robb, C. W.; Wilson, C. J.; Watson, D. A.; Niesel, D. W.

2001-01-01

Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

Dynamics of communities of bacteria and ammonia-oxidizing microorganisms in response to simazine attenuation in agricultural soil.

PubMed

Wan, Rui; Wang, Zhao; Xie, Shuguang

2014-02-15

Autochthonous microbiota plays a crucial role in natural attenuation of s-triazine herbicides in agricultural soil. Soil microcosm study was carried out to investigate the shift in the structures of soil autochthonous microbial communities and the potential degraders associated with natural simazine attenuation. The relative abundance of soil autochthonous degraders and the structures of microbial communities were assessed using quantitative PCR (q-PCR) and terminal restriction fragment length polymorphism (TRFLP), respectively. Phylogenetic composition of bacterial community was also characterized using clone library analysis. Soil autochthonous microbiota could almost completely clean up simazine (100 mg kg(-1)) in 10 days after herbicide application, indicating a strong self-remediation potential of agricultural soil. A significant increase in the proportion of s-triazine-degrading atzC gene was found in 6 days after simazine amendment. Simazine application could alter the community structures of total bacteria and ammonia-oxidizing archaea (AOA) and bacteria (AOB). AOA were more responsive to simazine application compared to AOB and bacteria. Actinobacteria, Alphaproteobacteria and Gammaproteobacteria were the dominant bacterial groups either at the initial stage after simazine amendment or at the end stage of herbicide biodegradation, but Actinobacteria predominated at the middle stage of biodegradation. Microorganisms from several bacterial genera might be involved in simazine biodegradation. This work could add some new insights on the bioremediation of herbicides contaminated agricultural soils. Copyright © 2013 Elsevier B.V. All rights reserved.

Primer development to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus.

PubMed

Agustiningsih, Agustiningsih; Trimarsanto, Hidayat; Setiawaty, Vivi; Artika, I Made; Muljono, David Handojo

2016-08-30

Influenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009. An optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested. The developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.

Transient downregulation of microRNA-206 protects alkali burn injury in mouse cornea by regulating connexin 43

PubMed Central

Li, Xiaoyan; Zhou, Huanfen; Tang, Weiqiang; Guo, Qing; Zhang, Yan

2015-01-01

Purpose: Chemical burn in cornea may cause permanent visual problem or complete blindness. In the present study, we investigated the role of microRNA 206 (miR-206) in relieving chemical burn in mouse cornea. Method: An alkali burn model was established in C57BL/6 mice to induce chemical corneal injury. Within 72 hours, the transient inflammatory responses in alkali-treated corneas were measured by opacity and corneal neovascularization (CNV) levels, and the gene expression profile of miR-206 was measured by quantitative real-time PCR (qPCR). Inhibitory oligonucleotides of miR-206, miR-206-I, were intrastromally injected into alkali-burned corneas. The possible protective effects of down-regulating miR-206 were assessed by both in vivo measurements of inflammatory responses and in vitro histochemical examinations of corneal epithelium sections. The possible binding of miR-206 on its molecular target, connexin43 (Cx43), was assessed by luciferase reporter (LR) and western blot (WB) assays. Cx43 was silenced by siRNA to examine its effect on regulating miR-206 modulation in alkali-burned cornea. Results: Opacity and CNV levels, along with gene expression of miR-206, were all transiently elevated within 72 hours of alkali-burned mouse cornea. Intrastromal injection of miR-206-I into alkali-burned cornea down-regulated miR-206 and ameliorated inflammatory responses both in vivo and in vitro. LR and WB assays confirmed that Cx43 was directly targeted by miR-206 in mouse cornea. Genetic silencing of Cx43 reversed the protective effect of miR-206 down-regulation in alkali-burned cornea. Conclusion: miR-206, associated with Cx43, is a novel molecular modulator in alkali burn in mouse cornea. PMID:26045777

Antimicrobial peptides and pro-inflammatory cytokines are differentially regulated across epidermal layers following bacterial stimuli.

PubMed

Percoco, Giuseppe; Merle, Chloé; Jaouen, Thomas; Ramdani, Yasmina; Bénard, Magalie; Hillion, Mélanie; Mijouin, Lily; Lati, Elian; Feuilloley, Marc; Lefeuvre, Luc; Driouich, Azeddine; Follet-Gueye, Marie-Laure

2013-12-01

The skin is a natural barrier between the body and the environment and is colonised by a large number of microorganisms. Here, we report a complete analysis of the response of human skin explants to microbial stimuli. Using this ex vivo model, we analysed at both the gene and protein level the response of epidermal cells to Staphylococcus epidermidis (S. epidermidis) and Pseudomonas fluorescens (P. fluorescens), which are present in the cutaneous microbiota. We showed that both bacterial species affect the structure of skin explants without penetrating the living epidermis. We showed by real-time quantitative polymerase chain reaction (qPCR) that S. epidermidis and P. fluorescens increased the levels of transcripts that encode antimicrobial peptides (AMPs), including human β defensin (hBD)2 and hBD3, and the pro-inflammatory cytokines interleukin (IL)-1α and (IL)-1-β, as well as IL-6. In addition, we analysed the effects of bacterial stimuli on the expression profiles of genes related to innate immunity and the inflammatory response across the epidermal layers, using laser capture microdissection (LCM) coupled to qPCR. We showed that AMP transcripts were principally upregulated in suprabasal keratinocytes. Conversely, the expression of pro-inflammatory cytokines was upregulated in the lower epidermis. These findings were confirmed by protein localisation using specific antibodies coupled to optical or electron microscopy. This work underscores the potential value of further studies that use LCM on human skin explants model to study the roles and effects of the epidermal microbiota on human skin physiology. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Systematic Analysis of Sequences and Expression Patterns of Drought-Responsive Members of the HD-Zip Gene Family in Maize

PubMed Central

Zhao, Yang; Zhou, Yuqiong; Jiang, Haiyang; Li, Xiaoyu; Gan, Defang; Peng, Xiaojian; Zhu, Suwen; Cheng, Beijiu

2011-01-01

Background Members of the homeodomain-leucine zipper (HD-Zip) gene family encode transcription factors that are unique to plants and have diverse functions in plant growth and development such as various stress responses, organ formation and vascular development. Although systematic characterization of this family has been carried out in Arabidopsis and rice, little is known about HD-Zip genes in maize (Zea mays L.). Methods and Findings In this study, we described the identification and structural characterization of HD-Zip genes in the maize genome. A complete set of 55 HD-Zip genes (Zmhdz1-55) were identified in the maize genome using Blast search tools and categorized into four classes (HD-Zip I-IV) based on phylogeny. Chromosomal location of these genes revealed that they are distributed unevenly across all 10 chromosomes. Segmental duplication contributed largely to the expansion of the maize HD-ZIP gene family, while tandem duplication was only responsible for the amplification of the HD-Zip II genes. Furthermore, most of the maize HD-Zip I genes were found to contain an overabundance of stress-related cis-elements in their promoter sequences. The expression levels of the 17 HD-Zip I genes under drought stress were also investigated by quantitative real-time PCR (qRT-PCR). All of the 17 maize HD-ZIP I genes were found to be regulated by drought stress, and the duplicated genes within a sister pair exhibited the similar expression patterns, suggesting their conserved functions during the process of evolution. Conclusions Our results reveal a comprehensive overview of the maize HD-Zip gene family and provide the first step towards the selection of Zmhdz genes for cloning and functional research to uncover their roles in maize growth and development. PMID:22164299

Systematic analysis of sequences and expression patterns of drought-responsive members of the HD-Zip gene family in maize.

PubMed

Zhao, Yang; Zhou, Yuqiong; Jiang, Haiyang; Li, Xiaoyu; Gan, Defang; Peng, Xiaojian; Zhu, Suwen; Cheng, Beijiu

2011-01-01

Members of the homeodomain-leucine zipper (HD-Zip) gene family encode transcription factors that are unique to plants and have diverse functions in plant growth and development such as various stress responses, organ formation and vascular development. Although systematic characterization of this family has been carried out in Arabidopsis and rice, little is known about HD-Zip genes in maize (Zea mays L.). In this study, we described the identification and structural characterization of HD-Zip genes in the maize genome. A complete set of 55 HD-Zip genes (Zmhdz1-55) were identified in the maize genome using Blast search tools and categorized into four classes (HD-Zip I-IV) based on phylogeny. Chromosomal location of these genes revealed that they are distributed unevenly across all 10 chromosomes. Segmental duplication contributed largely to the expansion of the maize HD-ZIP gene family, while tandem duplication was only responsible for the amplification of the HD-Zip II genes. Furthermore, most of the maize HD-Zip I genes were found to contain an overabundance of stress-related cis-elements in their promoter sequences. The expression levels of the 17 HD-Zip I genes under drought stress were also investigated by quantitative real-time PCR (qRT-PCR). All of the 17 maize HD-ZIP I genes were found to be regulated by drought stress, and the duplicated genes within a sister pair exhibited the similar expression patterns, suggesting their conserved functions during the process of evolution. Our results reveal a comprehensive overview of the maize HD-Zip gene family and provide the first step towards the selection of Zmhdz genes for cloning and functional research to uncover their roles in maize growth and development.

Evaluation of a spot-on imidacloprid-moxidectin formulation (Advocate®) for the treatment of naturally occurring esophageal spirocercosis in dogs: a double-blinded, placebo-controlled study.

PubMed

Segev, Gilad; Rojas, Alicia; Lavy, Eran; Yaffe, Marganit; Aroch, Itamar; Baneth, Gad

2018-03-05

Dogs are the definitive hosts of Spirocerca lupi. Spirocercosis is treated by prolonged avermectin administration by injection or daily oral doses. In this prospective, double-blinded, placebo-controlled, clinical trial, the efficacy of imidacloprid and moxidectin spot-on formulation (Advocate®) was compared to injectable doramectin (Dectomax®). Dogs diagnosed with benign esophageal spirocercosis were divided randomly into doramectin (400 μg/kg IM) or moxidectin and imidacloprid spot-on (2.5-6.25 mg/kg and 10-25 mg/kg, respectively) groups and treated weekly for 12 consecutive weeks. Dogs were followed for 20 weeks by physical examination, owners' questionnaire, blood work, fecal floatation, PCR and endoscopy. All the doramectin group dogs (n = 10) completed the treatment and follow-up, and the disease had completely resolved in all by week 12. Of the Advocate® group (n = 10), four had complete resolution at week 12, four had partial resolution, one dog did not respond to treatment, and one dog was switched to the doramectin protocol on week 5 due to persistent severe clinical signs. PCR analysis was more sensitive in detecting S. lupi eggs compared to fecal floatation. Discrepancies were detected on 22 occasions, of which on 20 occasions, the PCR was positive while fecal floatation was negative, and only on two occasions the PCR results were negative while fecal flotation was positive. The present results indicate that weekly Advocate® spot-on administration may be effective for treating benign esophageal spirocercosis, but is less effective than the currently used injectable doramectin therapy at the dose and duration used herein.

Pretreatment to avoid positive RT-PCR results with inactivated viruses.

PubMed

Nuanualsuwan, Suphachai; Cliver, Dean O

2002-07-01

Enteric viruses that are important causes of human disease must often be detected by reverse transcription-polymerase chain reaction (RT-PCR), a method that commonly yields positive results with samples that contain only inactivated virus. This study was intended to develop a pretreatment for samples, so that inactivated viruses would not be detected by the RT-PCR procedure. Model viruses were human hepatitis A virus, vaccine poliovirus 1 and feline calicivirus as a surrogate for the Norwalk-like viruses. Each virus was inactivated (from an initial titer of approximately 10(3) PFU/ml) by ultraviolet light, hypochlorite or heating at 72 degrees C. Inactivated viruses, that were treated with proteinase K and ribonuclease for 30 min at 37 degrees C before RT-PCR, gave a negative result, which is to say that no amplicon was detected after the reaction was completed. This antecedent to the RT-PCR method may be applicable to other types of viruses, to viruses inactivated in other ways and to other molecular methods of virus detection.

Targeting GPR30 in Abiraterone and MDV3100 Resistant Prostate Cancer

DTIC Science & Technology

2017-12-01

ID Labs, London, ON, Canada) following the manufacturer’s protocols. Quantitative real- time PCR Total RNA was treated with RNase-free DNase (Qiagen...99-gene panel for confirmation based on a literature search showing their relatedness to cell-mediated immune responses. Quantitative real- time PCR...mouse neutrophils (Geiser et al. 1993, Schaider et al. 2003), we analyzed murine neutrophil-related cytokine genes using quantitative real- time PCR

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics.

PubMed

Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

2014-02-20

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics

NASA Astrophysics Data System (ADS)

Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

2014-02-01

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

Survey of diagnostic and typing capacity for Clostridium difficile infection in Europe, 2011 and 2014.

PubMed

van Dorp, Sofie M; Notermans, Daan W; Alblas, Jeroen; Gastmeier, Petra; Mentula, Silja; Nagy, Elisabeth; Spigaglia, Patrizia; Ivanova, Katiusha; Fitzpatrick, Fidelma; Barbut, Frédéric; Morris, Trefor; Wilcox, Mark H; Kinross, Pete; Suetens, Carl; Kuijper, Ed J

2016-07-21

Suboptimal laboratory diagnostics for Clostridium difficile infection (CDI) impedes its surveillance and control across Europe. We evaluated changes in local laboratory CDI diagnostics and changes in national diagnostic and typing capacity for CDI during the European C. difficile Infection Surveillance Network (ECDIS-Net) project, through cross-sectional surveys in 33 European countries in 2011 and 2014. In 2011, 126 (61%) of a convenience sample of 206 laboratories in 31 countries completed a survey on local diagnostics. In 2014, 84 (67%) of these 126 laboratories in 26 countries completed a follow-up survey. Among laboratories that participated in both surveys, use of CDI diagnostics deemed 'optimal' or 'acceptable' increased from 19% to 46% and from 10% to 15%, respectively (p  < 0.001). The survey of national capacity was completed by national coordinators of 31 and 32 countries in 2011 and 2014, respectively. Capacity for any C. difficile typing method increased from 22/31 countries in 2011 to 26/32 countries in 2014; for PCR ribotyping from 20/31 countries to 23/32 countries, and specifically for capillary PCR ribotyping from 7/31 countries to 16/32 countries. While our study indicates improved diagnostic capability and national capacity for capillary PCR ribotyping across European laboratories between 2011 and 2014, increased use of 'optimal' diagnostics should be promoted. This article is copyright of The Authors, 2016.

MRPrimerW: a tool for rapid design of valid high-quality primers for multiple target qPCR experiments

PubMed Central

Kim, Hyerin; Kang, NaNa; An, KyuHyeon; Koo, JaeHyung; Kim, Min-Soo

2016-01-01

Design of high-quality primers for multiple target sequences is essential for qPCR experiments, but is challenging due to the need to consider both homology tests on off-target sequences and the same stringent filtering constraints on the primers. Existing web servers for primer design have major drawbacks, including requiring the use of BLAST-like tools for homology tests, lack of support for ranking of primers, TaqMan probes and simultaneous design of primers against multiple targets. Due to the large-scale computational overhead, the few web servers supporting homology tests use heuristic approaches or perform homology tests within a limited scope. Here, we describe the MRPrimerW, which performs complete homology testing, supports batch design of primers for multi-target qPCR experiments, supports design of TaqMan probes and ranks the resulting primers to return the top-1 best primers to the user. To ensure high accuracy, we adopted the core algorithm of a previously reported MapReduce-based method, MRPrimer, but completely redesigned it to allow users to receive query results quickly in a web interface, without requiring a MapReduce cluster or a long computation. MRPrimerW provides primer design services and a complete set of 341 963 135 in silico validated primers covering 99% of human and mouse genes. Free access: http://MRPrimerW.com. PMID:27154272

Chronic myelogenous leukemia in eastern Pennsylvania: an assessment of registry reporting.

PubMed

Mertz, Kristen J; Buchanich, Jeanine M; Washington, Terri L; Irvin-Barnwell, Elizabeth A; Woytowitz, Donald V; Smith, Roy E

2015-01-01

Chronic myelogenous leukemia (CML) has been reportable to the Pennsylvania Cancer Registry (PCR) since the 1980s, but the completeness of reporting is unknown. This study assessed CML reporting in eastern Pennsylvania where a cluster of another myeloproliferative neoplasm was previously identified. Cases were identified from 2 sources: 1) PCR case reports for residents of Carbon, Luzerne, or Schuylkill County with International Classification of Diseases for Oncology, Third Edition (ICD-O-3) codes 9875 (CML, BCR-ABL+), 9863 (CML, NOS), and 9860 (myeloid leukemia) and date of diagnosis 2001-2009, and 2) review of billing records at hematology practices. Participants were interviewed and their medical records were reviewed by board-certified hematologists. PCR reports included 99 cases coded 9875 or 9863 and 9 cases coded 9860; 2 additional cases were identified by review of billing records. Of the 110 identified cases, 93 were mailed consent forms, 23 consented, and 12 medical records were reviewed. Hematologists confirmed 11 of 12 reviewed cases as CML cases; all 11 confirmed cases were BCR/ABL positive, but only 1 was coded as positive (code 9875). Very few unreported CML cases were identified, suggesting relatively complete reporting to the PCR. Cases reviewed were accurately diagnosed, but ICD-0-3 coding often did not reflect BCR-ABL-positive tests. Cancer registry abstracters should look for these test results and code accordingly.

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.

PubMed Central

Kox, L F; Noordhoek, G T; Kunakorn, M; Mulder, S; Sterrenburg, M; Kolk, A H

1996-01-01

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization. PMID:8862568

Development of a rapid method to quantify Salmonella Typhimurium using a combination of MPN with qPCR and a shortened time incubation.

PubMed

Kim, Sun Ae; Park, Si Hong; Lee, Sang In; Ricke, Steven C

2017-08-01

A novel method was developed for the specific quantification of S. Typhimurium using a most-probable-number (MPN) combined with qPCR and a shortened incubation time (MPN-qPCR-SIT). For S. Typhimurium enumeration, dilutions of samples were transferred into three wells on a microtiter plate and the plate was incubated for 4 h. The S. Typhimurium presence in the wells was identified using a qPCR and populations were determined based on an MPN calculation. The R 2 between the MPN-qPCR-SIT and conventional MPN exhibited a high level of correlation (0.9335-0.9752), suggesting that the MPN-qPCR-SIT offers a reliable alternative method for S. Typhimurium quantification. Although plating and qPCR were limited in their ability to detect low levels of S. Typhimurium (e.g. 0.18 log MPN/ml), these levels could be successfully detected with the MPN-qPCR-SIT. Chicken breast samples inoculated with S. Typhimurium were incubated at 0, 4, and 24 h and incubated samples were subjected to microbiome analysis. Levels of Salmonella and Enterobacteriaceae increased significantly with incubation time. The obvious benefits of the MPN-qPCR-SIT are: 1) a further confirmation step is not required, 2) the detection limit is as low as conventional MPN, but 3) is more rapid, requiring approximately 7 h to simultaneously complete quantification. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of a high-speed real-time PCR system for rapid and precise nucleotide recognition

NASA Astrophysics Data System (ADS)

Terazono, Hideyuki; Takei, Hiroyuki; Hattori, Akihiro; Yasuda, Kenji

2010-04-01

Polymerase chain reaction (PCR) is a common method used to create copies of a specific target region of a DNA sequence and to produce large quantities of DNA. A few DNA molecules, which act as templates, are rapidly amplified by PCR into many billions of copies. PCR is a key technology in genome-based biological analysis, revolutionizing many life science fields such as medical diagnostics, food safety monitoring, and countermeasures against bioterrorism. Thus, many applications have been developed with the thermal cycling. For these PCR applications, one of the most important key factors is reduction in the data acquisition time. To reduce the acquisition time, it is necessary to decrease the temperature transition time between the high and low ends as much as possible. We have developed a novel rapid real-time PCR system based on rapid exchange of media maintained at different temperatures. This system consists of two thermal reservoirs and a reaction chamber for PCR observation. The temperature transition was achieved within 0.3 sec, and good thermal stability was achieved during thermal cycling with rapid exchange of circulating media. This system allows rigorous optimization of the temperatures required for each stage of the PCR processes. Resulting amplicons were confirmed by electrophoresis. Using the system, rapid DNA amplification was accomplished within 3.5 min, including initial heating and complete 50 PCR cycles. It clearly shows that the device could allow us faster temperature switching than the conventional conduction-based heating systems based on Peltier heating/cooling.

A survey of tools for the analysis of quantitative PCR (qPCR) data.

PubMed

Pabinger, Stephan; Rödiger, Stefan; Kriegner, Albert; Vierlinger, Klemens; Weinhäusel, Andreas

2014-09-01

Real-time quantitative polymerase-chain-reaction (qPCR) is a standard technique in most laboratories used for various applications in basic research. Analysis of qPCR data is a crucial part of the entire experiment, which has led to the development of a plethora of methods. The released tools either cover specific parts of the workflow or provide complete analysis solutions. Here, we surveyed 27 open-access software packages and tools for the analysis of qPCR data. The survey includes 8 Microsoft Windows, 5 web-based, 9 R-based and 5 tools from other platforms. Reviewed packages and tools support the analysis of different qPCR applications, such as RNA quantification, DNA methylation, genotyping, identification of copy number variations, and digital PCR. We report an overview of the functionality, features and specific requirements of the individual software tools, such as data exchange formats, availability of a graphical user interface, included procedures for graphical data presentation, and offered statistical methods. In addition, we provide an overview about quantification strategies, and report various applications of qPCR. Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

PubMed Central

Chang, Shy-Shin; Hsu, Hsung-Ling; Cheng, Ju-Chien; Tseng, Ching-Ping

2011-01-01

Background Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. Methodology/Principal Findings We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Conclusions/Significance Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories. PMID:21637859

Survival after neoadjuvant chemotherapy with or without bevacizumab or everolimus for HER2-negative primary breast cancer (GBG 44-GeparQuinto)†.

PubMed

von Minckwitz, G; Loibl, S; Untch, M; Eidtmann, H; Rezai, M; Fasching, P A; Tesch, H; Eggemann, H; Schrader, I; Kittel, K; Hanusch, C; Huober, J; Solbach, C; Jackisch, C; Kunz, G; Blohmer, J U; Hauschild, M; Fehm, T; Nekljudova, V; Gerber, B

2014-12-01

The GeparQuinto study showed that adding bevacizumab to 24 weeks of anthracycline-taxane-based neoadjuvant chemotherapy increases pathological complete response (pCR) rates overall and specifically in patients with triple-negative breast cancer (TNBC). No difference in pCR rate was observed for adding everolimus to paclitaxel in nonearly responding patients. Here, we present disease-free (DFS) and overall survival (OS) analyses. Patients (n = 1948) with HER2-negative tumors of a median tumor size of 4 cm were randomly assigned to neoadjuvant treatment with epirubicin/cyclophosphamide followed by docetaxel (EC-T) with or without eight infusions of bevacizumab every 3 weeks before surgery. Patients without clinical response to EC ± Bevacizumab were randomized to 12 weekly cycles paclitaxel with or without everolimus 5 mg/day. To detect a hazard ratio (HR) of 0.75 (α = 0.05, β = 0.8) 379 events had to be observed in the bevacizumab arms. With a median follow-up of 3.8 years, 3-year DFS was 80.8% and 3-year OS was 89.7%. Outcome was not different for patients receiving bevacizumab (HR 1.03; P = 0.784 for DFS and HR 0.974; P = 0.842 for OS) compared with patients receiving chemotherapy alone. Patients with TNBC similarly showed no improvement in DFS (HR = 0.99; P = 0.941) and OS (HR = 1.02; P = 0.891) when treated with bevacizumab. No other predefined subgroup (HR+/HER2-; locally advanced (cT4 or cN3) or not; cT1-3 or cT4; pCR or not) showed a significant benefit. No difference in DFS (HR 0.997; P = 0.987) and OS (HR 1.11; P = 0.658) was observed for nonearly responding patients receiving paclitaxel with or without everolimus overall as well as in subgroups. Long-term results, in opposite to the results of pCR, do not support the neoadjuvant use of bevacizumab in addition to an anthracycline-taxane-based chemotherapy or everolimus in addition to paclitaxel for nonearly responding patients. NCT 00567554, www.clinicaltrials.gov. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Detection of Francisella tularensis within infected mouse tissues by using a hand-held PCR thermocycler.

PubMed

Emanuel, Peter A; Bell, Ryan; Dang, Jessica L; McClanahan, Rebecca; David, John C; Burgess, Robert J; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

2003-02-01

The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5' hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.

Detection of Francisella tularensis within Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler

PubMed Central

Emanuel, Peter A.; Bell, Ryan; Dang, Jessica L.; McClanahan, Rebecca; David, John C.; Burgess, Robert J.; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

2003-01-01

The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5′ hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field. PMID:12574268

Dramatic Increase in the Signal and Sensitivity of Detection via Self-Assembly of Branched DNA

PubMed Central

Kim, Kyung-Tae; Chae, Chi-Bom

2011-01-01

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA. PMID:21870112

Identification of genus Acinetobacter: Standardization of in-house PCR and its comparison with conventional phenotypic methods.

PubMed

Kulkarni, Sughosh S; Madalgi, Radhika; Ajantha, Ganavalli S; Kulkarni, Raghavendra D

2017-01-01

Acinetobacter is grouped under nonfermenting Gram-negative bacilli. It is increasingly isolated from pathological samples. The ability of this genus to acquire drug resistance and spread in the hospital settings is posing a grave problem in healthcare. Specific treatment protocols are advocated for Acinetobacter infections. Hence, rapid identification and drug susceptibility profiling are critical in the management of these infections. To standardize an in-house polymerase chain reaction (PCR) for identification of genus Acinetobacter and to compare PCR with two protocols for its phenotypic identification. A total of 96 clinical isolates of Acinetobacter were included in the study. An in-house PCR for genus level identification of Acinetobacter was standardized. All the isolates were phenotypically identified by two protocols. The results of PCR and phenotypic identification protocols were compared. The in-house PCR standardized was highly sensitive and specific for the genus Acinetobacter . There was 100% agreement between the phenotypic and molecular identification of the genus. The preliminary identification tests routinely used in clinical laboratories were also in complete agreement with phenotypic and molecular identification. The in-house PCR for genus level identification is specific and sensitive. However, it may not be essential for routine identification as the preliminary phenotypic identification tests used in the clinical laboratory reliably identify the genus Acinetobacter .

Rapid and sensitive detection of mink circovirus by recombinase polymerase amplification.

PubMed

Ge, Junwei; Shi, Yunjia; Cui, Xingyang; Gu, Shanshan; Zhao, Lili; Chen, Hongyan

2018-06-01

To date, the pathogenic role of mink circovirus (MiCV) remains unclear, and its prevalence and economic importance are unknown. Therefore, a rapid and sensitive molecular diagnosis is necessary for disease management and epidemiological surveillance. However, only PCR methods can identify MiCV infection at present. In this study, we developed a nested PCR and established a novel recombinase polymerase amplification (RPA) assay for MiCV detection. Sensitivity analysis showed that the detection limit of nested PCR and RPA assay was 10 1 copies/reaction, and these methods were more sensitive than conventional PCR, which has a detection limit of 10 5 copies/reaction. The RPA assay had no cross-reactivity with other related viral pathogens, and amplification was completed in less than 20 min with a simple device. Further assessment of clinical samples showed that the two assays were accurate in identifying positive and negative conventional PCR samples. The detection rate of MiCV by the RPA assay in clinical samples was 38.09%, which was 97% consistent with that by the nested PCR. The developed nested PCR is a highly sensitive tool for practical use, and the RPA assay is a simple, sensitive, and potential alternative method for rapid and accurate MiCV diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Acetyl group availability influences phosphocreatine degradation even during intense muscle contraction

PubMed Central

Timmons, James A; Constantin-Teodosiu, Dumitru; Poucher, Simon M; Greenhaff, Paul L

2004-01-01

We previously established that activation of the pyruvate dehydrogenase complex (PDC) using dichloroacetate (DCA) reduced the reliance on substrate-level phosphorylation (SLP) at the onset of exercise, with normal and reduced blood flow. PDC activation also reduced fatigue development during contraction with reduced blood flow. Since these observations, several studies have re-evaluated our observations. One study demonstrated a performance benefit without a reduction in SLP, raising a question mark over PDC's role in the regulation of ATP regeneration and our interpretation of fatigue mechanisms. Using a model of muscle contraction similar to the conflicting study (i.e. tetanic rather than twitch stimulation), we re-examined this question. Using canine skeletal muscle, one group was infused with saline while the other was pretreated with 300 mg (kg body mass)−1 DCA. Muscle biopsies were taken at rest, peak tension (1 min) and after 6 min of tetanic electrical stimulation (75 ms on−925 ms off per second) and blood flow was limited to 25% of normal values observed during contraction. DCA reduced phosphocreatine (PCr) degradation by 40% during the first minute of contraction, but did not prevent the almost complete depletion of PCr stores at 6 min, while muscle fatigue did not differ between the two groups. During intermittent tetanic stimulation PCr degradation was 75% greater than with our previous 3 Hz twitch contraction protocol, despite a similar rate of oxygen consumption at 6 min. Thus, in the present study enhanced acetyl group availability altered the time course of PCr utilization but did not prevent the decline towards depletion. Consistent with our earlier conclusions, DCA pretreatment reduces muscle fatigue only when SLP is attenuated. The present study and our met-analysis indicates that enhanced acetyl group availability results in a readily measurable reduction in SLP when the initial rate of PCr utilization is ∼1 mmol (kg dry mass)−1 s−1 or less (depending on intrinsic mitochondrial capacity). When measured early during an uninterrupted period of muscle contraction, acetyl group availability is likely to influence SLP under any condition where mitochondria are responsible for a significant proportion of ATP regeneration. PMID:15498812

Faster and economical screening for vancomycin-resistant enterococci by sequential use of chromogenic agar and real-time polymerase chain reaction.

PubMed

Tan, Thean Yen; Jiang, Boran; Ng, Lily Siew Yong

2017-08-01

Screening for vancomycin-resistant enterococci (VRE) by culture takes days to generate results, while polymerase chain reaction (PCR) testing directly from clinical specimens lacks specificity. The aims of this study were to develop a real-time PCR to detect and identify Enterococcus faecium, Enterococcus faecalis, and vanA and vanB genes, and to evaluate the impact of this PCR on test-reporting times when performing it directly from suspect VRE isolates present on screening chromogenic media. The tetraplex PCR primers were designed to amplify E. faecium, E. faecalis, and vanA and vanB genes, with melt-curve analysis of PCR products. Following analytical and clinical validation of the molecular assay, PCR testing was performed for target colonies present on VRE chromogenic media. PCR results were evaluated against conventional phenotypic identification and susceptibility testing, with the time to result being monitored for both modalities. A total of 519 colonies from clinical specimens were tested concurrently by real-time PCR and phenotypic methods. In all, 223 isolates were identified with phenotypic vancomycin resistance (vanA, n = 108; vanB, n = 105; non-vanA/vanB = 10), with complete agreement between PCR and phenotypic testing for vancomycin-resistant E. faecium and E. faecalis. The majority (88.6%) of PCR results were reported, on average, 24.8 hours earlier than those of phenotypic testing, with 68% reduction in total costs. The use of culture on selective media, followed by direct colony PCR confirmation allows faster and economical VRE screening. Copyright © 2015. Published by Elsevier B.V.

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C).

PubMed

Fong, Wai-Ying; Ho, Chi-Chun; Poon, Wing-Tat

2017-05-12

Thiopurine intolerance and treatment-related toxicity, such as fatal myelosuppression, is related to non-function genetic variants encoding thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Genetic testing of the common variants NUDT15:NM_018283.2:c.415C>T (Arg139Cys, dbSNP rs116855232 T allele) and TPMT: NM_000367.4:c.719A>G (TPMT*3C, dbSNP rs1142345 G allele) in East Asians including Chinese can potentially prevent treatment-related complications. Two complementary genotyping approaches, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length morphism (PCR-RFLP) analysis were evaluated using conventional PCR and Sanger sequencing genotyping as the gold standard. Sixty patient samples were tested, revealing seven patients (11.7%) heterozygous for NUDT15 c.415C>T, one patient homozygous for the variant and one patient heterozygous for the TPMT*3C non-function allele. No patient was found to harbor both variants. In total, nine out of 60 (15%) patients tested had genotypic evidence of thiopurine intolerance, which may require dosage adjustment or alternative medication should they be started on azathioprine, mercaptopurine or thioguanine. The two newly developed assays were more efficient and showed complete concordance (60/60, 100%) compared to the Sanger sequencing results. Accurate and cost-effective genotyping assays by real-time PCR-HRM and PCR-RFLP for NUDT15 c.415C>T and TPMT*3C were successfully developed. Further studies may establish their roles in genotype-informed clinical decision-making in the prevention of morbidity and mortality due to thiopurine intolerance.

Effect of long interval between hyperthermochemoradiation therapy and surgery for rectal cancer on apoptosis, proliferation and tumor response.

PubMed

Kato, Toshihide; Fujii, Takaaki; Ide, Munenori; Takada, Takahiro; Sutoh, Toshinaga; Morita, Hiroki; Yajima, Reina; Yamaguchi, Satoru; Tsutsumi, Soichi; Asao, Takayuki; Oyama, Tetsunari; Kuwano, Hiroyuki

2014-06-01

Neoadjuvant chemoradiotherapy is commonly used to improve the local control and resectability of locally advanced rectal cancer, with surgery performed after an interval of a number of weeks. We have been conducting a clinical trial of preoperative chemoradiotherapy in combination with regional hyperthermia (hyperthermo-chemoradiation therapy; HCRT) for locally advanced rectal cancer. In the current study we assessed the effect of a longer (>10 weeks) interval after neoadjuvant HCRT on pathological response, oncological outcome and especially on apoptosis, proliferation and p53 expression in patients with rectal cancer. Forty-eight patients with proven rectal adenocarcinoma who underwent HCRT followed by surgery were identified for inclusion in this study. Patients were divided into two groups according to the interval between HCRT and surgery, ≤ 10 weeks (short-interval group) and >10 weeks (long-interval group). Patients in the long-interval group had a significantly higher rate of pathological complete response (pCR) (43.5% vs. 16.0%) than patients of the short-interval group. Patients of the long-interval group had a significantly higher rate of down-staging of T-stage (78.3% vs. 36.0%) and relatively higher rate of that of N-stage (52.2% vs. 36.0%) than patients of the short-interval group. Furthermore, apoptosis in the long-interval group was relatively higher compared to that of the short-interval group, without a significant difference in the Ki-67 proliferative index and expression of p53 in the primary tumor. In conclusion, we demonstrated that a longer interval after HCRT (>10 weeks) seemed to result in a better chance of a pCR, a result confirmed by the trends in tumor response markers, including apoptosis, proliferation and p53 expression. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Self-reported student confidence in troubleshooting ability increases after completion of an inquiry-based PCR practical.

PubMed

Cook, Anthony L; Snow, Elizabeth T; Binns, Henrica; Cook, Peta S

2015-01-01

Inquiry-based learning (IBL) activities are complementary to the processes of laboratory discovery, as both are focused on producing new findings through research and inquiry. Here, we describe the results of student surveys taken pre- and postpractical to an IBL undergraduate practical on PCR. Our analysis focuses primarily student perceptions of knowledge acquisition and their ability to troubleshoot problems. The survey results demonstrate significant self-reported gains in knowledge related to DNA structure and PCR, and an increase in confidence with "troubleshooting problems during scientific experiments." We conclude that the IBL-based approach that combines PCR primer design with wet laboratory experimentation using student-designed primers, provides students a sense of confidence by imparting workplace and research skills that are integral to diverse forms and applications of laboratory practices. © 2015 The International Union of Biochemistry and Molecular Biology.

Correlation of transforming growth factor-β messenger RNA (TGF-β mRNA) expression with cellular immunoassays in Triamcinolone-treated captive hybrid striped bass

USGS Publications Warehouse

Harms, Craig A.; Ottinger, Christopher A.; Kennedy-Stoskopf, S.

2000-01-01

Assessing fish immune status with molecular markers has been hampered by a lack of specific reagents. A quantitative polymerase chain reaction (PCR) method (reverse transcription quantitative–competitive PCR, RT-qcPCR) for measuring transforming growth factor-β (TGF-β) transcription from a broad range of teleost fish has recently been developed. The quantitative PCR now permits monitoring production of this important immunosuppressive cytokine in response to immunomodulating agents and conditions. We examined anterior kidney and spleen mononuclear cells from hybrid striped bass (female striped bass Morone saxatilis× male white bass M. chrysops) for production of TGF-β messenger RNA (mRNA) in response to administration of the synthetic glucocorticoid triamcinolone. We also compared TGF-β transcription with anterior kidney macrophage bactericidal activity and splenic lymphocyte blastogenesis. Anterior kidney mononuclear cell TGF-β mRNA levels decreased, whereas bactericidal activity increased. Spleen TGF-β mRNA levels did not change significantly, and splenic lymphocyte pokeweed mitogen stimulation index increased in triamcinolone-treated fish. Since triamcinolone is used therapeutically as a suppressive immunomodulator, the enhanced immune functions indicated by the cellular immunoassays were unexpected; however, the inverse response of TGF-β production and macrophage bactericidal activity was consistent with the known relationship between TGF-β and macrophage activation in mammals. Induced immunomodulation in hybrid striped bass was detectable by both traditional cellular immunoassays and the new RT-qcPCR for TGF-β.

[Iditification of five imported cases of Plasmodium ovale wallikeri infection in Zhejiang Province].

PubMed

Zhang, Ling-ling; Ruan, Wei; Chen, Hua-liang; Lu, Qiao-yi; Yao, Li-nong

2014-10-01

To identify and analyze Plasmodium ovale wallikeri in 5 imported malaria cases, who were detected positive by microscopy and negative by conventional PCR. Epidemiological information and blood samples were collected from the five patients. The detection was conducted by microscopy, Rapid Diagnostic Test (RDT) and nested PCR with Plasmodium genus-specific, species-specific and Plasmodium ovale wallikeri-specific primers. The amplified products were sequenced and Blast analysis was performed on line in NCBI. The five patients returned from Africa, and all had a history of malaria. They were microscopically positive for Plasmodium sp., and two cases showed Pan positive RDT result. All blood samples were negative for four Plasmodium spp. by conventional nested PCR, but positive by nested PCR with Plasmodium ovale wallikeri-specific primers. Blast analysis showed that the amplified sequences of the five cases had complete homology with P. ovale wallikeri clone RSH10 18S ribosomal RNA gene (Accession No. KF219561.1). The five cases which classified as positive by microscopy while negative by conventional PCR have been confirmed as Plasmodium ovale wallikeri infection by nested PCR with P. ovale wallikeri-specific primers.

Polymerase chain reaction for detection of invasive Shigella flexneri in food.

PubMed

Lampel, K A; Jagow, J A; Trucksess, M; Hill, W E

1990-06-01

The polymerase chain reaction (PCR) was used to amplify a 760-base-pair (bp) fragment with the 220-kbp invasive plasmids of enteroinvasive Escherichia coli, Shigella flexneri, Shigella dysenteriae, Shigella boydii, and Shigella sonnei as templates. This PCR product was easily detected by agarose gel electrophoresis. A 210-bp AccI-PstI fragment lying within the amplified region was used as a probe in Southern hybridization blots and showed that the PCR-generated product was derived from the invasive plasmid. The application of PCR as a rapid method to detect enteroinvasive bacteria in foods was tested by inoculating lettuce with 10(4) S. flexneri cells per g in shigella broth base. Plasmid DNA was isolated from cultures of inoculated and uninoculated lettuce in broth after 0, 4, and 24 h of incubation. With the PCR, the 760-bp fragment was generated only from lettuce inoculated with S. flexneri, as shown by gel electrophoresis and confirmed both by Southern blotting and by nucleotide sequencing of the amplified region. Because the isolation of plasmid DNA, the performance of PCR, and gel electrophoresis all can be completed in 6 to 7 h, invasive enteric bacteria can be detected in less than 1 day.

Phase 1 Study of Preoperative Chemoradiation Therapy With Temozolomide and Capecitabine in Patients With Locally Advanced Rectal Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Jae Ho; Hong, Yong Sang; Park, Yangsoon

Purpose: Preoperative chemoradiation therapy (CRT) with capecitabine is a standard treatment strategy in patients with locally advanced rectal cancer (LARC). Temozolomide improves the survival of patients with glioblastoma with hypermethylated O{sup 6}-methylguanine DNA methyltransferase (MGMT); MGMT hypermethylation is one of the colorectal carcinogenesis pathways. We aimed to determine the dose-limiting toxicity (DLT) and recommended dose (RD) of temolozomide in combination with capecitabine-based preoperative CRT for LARC. Methods and Materials: Radiation therapy was delivered with 45 Gy/25 daily fractions with coned-down boost of 5.4 Gy/3 fractions. Concurrent chemotherapy comprised fixed and escalated doses of capecitabine and temozolomide, respectively. The MGMT hypermethylation was evaluatedmore » in pretreatment tumor samples. This trial is registered with (ClinicalTrials.gov) with the number (NCT01781403). Results: Twenty-two patients with LARC of cT3-4N0 or cT{sub any}N1-2 were accrued. Dose level 3 was chosen as the RD because DLT was noticeably absent in 10 patients treated up to dose level 3. An additional 12 patients were recruited in this group. Grade III adverse events were noted, and pathologic complete response (pCR) was observed in 7 patients (31.8%); MGMT hypermethylation was detected in 16. The pCR rate was 37.5% and 16.7% in the hypermethylated and unmethylated MGMT groups, respectively (P=.616). Conclusions: There was a tendency toward higher pCR rates in patients with hypermethylated MGMT. Future randomized studies are therefore warranted.« less

Efficacy and safety analysis of trastuzumab and paclitaxel based regimen plus carboplatin or epirubicin as neoadjuvant therapy for clinical stage II-III, HER2-positive breast cancer patients: a phase 2, open-label, multicenter, randomized trial

PubMed Central

Yang, Wentao; Xu, Binghe; Huang, Tao; Yang, Hongjian; Zheng, Hong; Wang, Yongsheng; Song, Erwei; Zhang, Jin; Cui, Shude; Pang, Da; Tang, Lili; Lei, Yutao; Geng, Cuizhi; Shao, Zhiming

2015-01-01

This trial was designed to compare the efficacy and safety between epirubicin (E) and carboplatin (C) in combination with paclitaxel (P) and trastuzumab (H) in neoadjuvant setting. In 13 Chinese cancer centers, 100 patients with HER2-positive, locally advanced breast cancer were 1:1 randomized to receive medication as follows: trastuzumab and paclitaxel weekly combined with carboplatin weekly for PCH group, or epirubicin every 3 weeks for PEH group. Patients were given 4 to 6 cycles of chemotherapy. The primary endpoint was pathologic complete response (pCR) rate, which was no significant difference in PCH and PEH regimen (39.1% vs. 48.8%; p=0.365). However, PEH regimen achieved higher pCR in luminal-B (HER2-poitive) subgroup (55.0% vs. 24.0%; p = 0.033), but not in ERBB2+ subgroup (42.9% vs. 57.1%; p = 0.355). PEH regimen showed a favorable efficacy in PIK3CA mutated subgroup (69.2% vs.23.5%, p=0.012). No significant difference was observed in the subgroup analysis of TP53 mutation status, PTEN expression, FCGR2A SNP and FCGR3A SNP. Both regimens as neoadjuvant chemotherapy achieve similar efficacy and safety. PEH might improve pCR rate, especially in the luminal-B subtype and PIK3CA mutation subtype. PEH is feasible and less likely to increase the incidence of acute cardiac events compared to PCH. PMID:26084292

BCR-ABL1 expression, RT-qPCR and treatment decisions in chronic myeloid leukaemia.

PubMed

Latham, Susan; Bartley, Paul A; Budgen, Bradley; Ross, David M; Hughes, Elizabeth; Branford, Susan; White, Deborah; Hughes, Timothy P; Morley, Alexander A

2016-09-01

RT-qPCR is used to quantify minimal residual disease (MRD) in chronic myeloid leukaemia (CML) in order to make decisions on treatment, but its results depend on the level of BCR-ABL1 expression as well as leukaemic cell number. The aims of the study were to quantify inter-individual differences in expression level, to determine the relationship between expression level and response to treatment, and to investigate the effect of expression level on interpretation of the RT-qPCR result. BCR-ABL1 expression was studied in 248 samples from 65 patients with CML by determining the difference between MRD quantified by RT-qPCR and DNA-qPCR. The results were analysed statistically and by simple indicative modelling. Inter-individual levels of expression approximated a normal distribution with an SD of 0.36 log. Expression at diagnosis correlated with expression during treatment. Response to treatment, as measured by the number of leukaemic cells after 3, 6 or 12 months of treatment, was not related to the level of expression. Indicative modelling suggested that interpretation of RT-qPCR results in relation to treatment guidelines could be affected by variation in expression when MRD was around 10% at 3 months and by both expression variation and Poisson variation when MRD was around or below the limit of detection of RT-qPCR. Variation between individuals in expression of BCR-ABL1 can materially affect interpretation of the RT-qPCR when this test is used to make decisions on treatment. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Decreased frontal lobe phosphocreatine levels in methamphetamine users

PubMed Central

Sung, Young-Hoon; Yurgelun-Todd, Deborah A.; Shi, Xian-Feng; Kondo, Douglas G.; Lundberg, Kelly J.; McGlade, Erin C.; Hellem, Tracy L.; Huber, Rebekah S.; Fiedler, Kristen K.; Harrell, Renee E.; Nickerson, Bethany R.; Kim, Seong-Eun; Jeong, Eun-Kee; Renshaw, Perry F.

2012-01-01

BACKGROUND Mitochondria-related mechanisms have been suggested to mediate methamphetamine (METH) toxicity. However, changes in brain energetics associated with highenergy phosphate metabolism have not been investigated in METH users. Phosphorus-31 (31P) magnetic resonance spectroscopy (MRS) was used to evaluate changes in mitochondrial high energy phosphates, including phosphocreatine (PCr) and β-nucleoside triphosphate (β-NTP, primarily ATP in brain) levels. We hypothesized that METH users would have decreased high-energy PCr levels in the frontal gray matter. METHODS Study participants consisted of 51 METH (age=32.8±6.7) and 23 healthy comparison (age=31.1±7.5) subjects. High-energy phosphate metabolite levels were compared between the groups and potential gender differences were explored. RESULTS METH users had lower ratios of PCr to total pool of exchangeable phosphate (PCr/TPP) in the frontal lobe as compared to the healthy subjects (p=0.001). The lower PCr levels in METH subjects were significantly associated with lifetime amount of METH use (p=0.003). A sub-analysis for gender differences revealed that female METH users, who had lower daily amounts (1.1±1.0 gram) of METH use than males (1.4±1.7 gram), had significantly lower PCr/TPP ratios than male METH users, controlling for the amount of METH use (p=0.02). CONCLUSIONS The present findings suggest that METH compromises frontal lobe high-energy phosphate metabolism in a dose-responsive manner. Our findings also suggest that the abnormality in frontal lobe high-energy phosphate metabolism might be more prominent in female than in male METH users. This is significant as decreased PCr levels have been associated with depressive symptoms, and poor responses to antidepressant treatment have been reported in those with decreased PCr levels. PMID:23084413

Complete mitochondrial genome of freshwater shark Wallago attu (Bloch & Schneider) from Indus River Sindh, Pakistan.

PubMed

Laghari, Muhammad Younis; Lashari, Punhal; Xu, Peng; Zhao, Zixia; Jiang, Li; Narejo, Naeem Tariq; Xin, Baoping; Sun, Xiaowen; Zhang, Yan

2016-01-01

Complete mitochondrial genome of fresh water giant catfish, Wallago attu, was isolated by LA PCR (TakaRa LAtaq, Dalian, China); and sequenced by Sanger's method to obtain the complete mitochondrial genome. The complete mitogenome was 15,639 bp in length and contains 13 typical vertebrate protein-coding genes, 2 rRNA and 22 tRNA genes. The whole genome base composition was estimated to be 31.17% A, 28.15% C, 15.55% G and 25.12% T. The complete mitochondrial genome of catfish, W. attu, provides the fundamental tools for genetic breeding.

Cell type-specific suppression of mechanosensitive genes by audible sound stimulation.

PubMed

Kumeta, Masahiro; Takahashi, Daiji; Takeyasu, Kunio; Yoshimura, Shige H

2018-01-01

Audible sound is a ubiquitous environmental factor in nature that transmits oscillatory compressional pressure through the substances. To investigate the property of the sound as a mechanical stimulus for cells, an experimental system was set up using 94.0 dB sound which transmits approximately 10 mPa pressure to the cultured cells. Based on research on mechanotransduction and ultrasound effects on cells, gene responses to the audible sound stimulation were analyzed by varying several sound parameters: frequency, wave form, composition, and exposure time. Real-time quantitative PCR analyses revealed a distinct suppressive effect for several mechanosensitive and ultrasound-sensitive genes that were triggered by sounds. The effect was clearly observed in a wave form- and pressure level-specific manner, rather than the frequency, and persisted for several hours. At least two mechanisms are likely to be involved in this sound response: transcriptional control and RNA degradation. ST2 stromal cells and C2C12 myoblasts exhibited a robust response, whereas NIH3T3 cells were partially and NB2a neuroblastoma cells were completely insensitive, suggesting a cell type-specific response to sound. These findings reveal a cell-level systematic response to audible sound and uncover novel relationships between life and sound.

Cell type-specific suppression of mechanosensitive genes by audible sound stimulation

PubMed Central

Takahashi, Daiji; Takeyasu, Kunio; Yoshimura, Shige H.

2018-01-01

Audible sound is a ubiquitous environmental factor in nature that transmits oscillatory compressional pressure through the substances. To investigate the property of the sound as a mechanical stimulus for cells, an experimental system was set up using 94.0 dB sound which transmits approximately 10 mPa pressure to the cultured cells. Based on research on mechanotransduction and ultrasound effects on cells, gene responses to the audible sound stimulation were analyzed by varying several sound parameters: frequency, wave form, composition, and exposure time. Real-time quantitative PCR analyses revealed a distinct suppressive effect for several mechanosensitive and ultrasound-sensitive genes that were triggered by sounds. The effect was clearly observed in a wave form- and pressure level-specific manner, rather than the frequency, and persisted for several hours. At least two mechanisms are likely to be involved in this sound response: transcriptional control and RNA degradation. ST2 stromal cells and C2C12 myoblasts exhibited a robust response, whereas NIH3T3 cells were partially and NB2a neuroblastoma cells were completely insensitive, suggesting a cell type-specific response to sound. These findings reveal a cell-level systematic response to audible sound and uncover novel relationships between life and sound. PMID:29385174

Response of tobacco to the Pseudomonas syringae pv. Tomato DC3000 is mainly dependent on salicylic acid signaling pathway.

PubMed

Liu, Yang; Wang, Li; Cai, Guohua; Jiang, Shanshan; Sun, Liping; Li, Dequan

2013-07-01

Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) was the first pathogen to be demonstrated to infect Arabidopsis and to cause disease symptoms in the laboratory setting. However, the defense response to Pst DC3000 was unclear in tobacco. In this report, the expression profiles of twelve defense response-related genes were analyzed after treatment with salicylic acid (SA), jasmonic acid (JA), and pathogen Pst DC3000 by qRT-PCR. According to our results, it could be presented that the genes primarily induced by SA were also induced to higher levels after Pst DC3000 infection. SA accumulation could be induced to a higher level than that of JA after Pst DC3000 infection. In addition, SA could result in hypersensitive response (HR), which did not completely depend on accumulation of reactive oxygen species. These results indicated that tobacco mainly depended on SA signaling pathway rather than on JA signaling pathway in response to Pst DC3000. Further study demonstrated that JA could significantly inhibit the accumulation of SA and the generation of the HR induced by Pst DC3000. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

The Molecular Basis of the Response to Radiation

DTIC Science & Technology

1999-07-01

S. cerevisiae and S. pombe ). After PCR amplification of human cDNA libraries as described below, PCR products are analyzed on 4% NuSieve agarose...media lacking uracil as well as counterselected against on media containing uracil and 0.1% 5-fluoroorotic acid (5FOA). Induction of the LacZ gene...evolutionarily distant species (S. cerevisiae andS. pombe ) to develop degenerate PCR based primers. For example, a fission yeast homolog of RAD9 named rhp9 was

Type-Specific Detection of 30 Oncogenic Human Papillomaviruses by Genotyping both E6 and L1 Genes

PubMed Central

Peng, Junping; Gao, Lei; Guo, Junhua; Wang, Ting; Wang, Ling; Yao, Qing; Zhu, Haijun

2013-01-01

Human papillomavirus (HPV) is the principal cause of invasive cervical cancer and benign genital lesions. There are currently 30 HPV types linked to cervical cancer. HPV infection also leads to other types of cancer. We developed a 61-plex analysis of these 30 HPV types by examining two genes, E6 and L1, using MassARRAY matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (PCR-MS). Two hundred samples from homosexual males (HM) were screened by PCR-MS and MY09/MY11 primer set-mediated PCR (MY-PCR) followed by sequencing. One hundred thirty-five formalin-fixed, paraffin-embedded (FFPE) cervical cancer samples were also analyzed by PCR-MS, and results were compared to those of the commercially available GenoArray (GA) assay. One or more HPV types were identified in 64.5% (129/200) of the samples from HM. Comprising all 30 HPV types, PCR-MS detected 51.9% (67/129) of samples with multiple HPV types, whereas MY-PCR detected only one single HPV type in these samples. All PCR-MS results were confirmed by MY-PCR. In the cervical cancer samples, PCR-MS and GA detected 97% (131/135) and 90.4% (122/135) of HPV-positive samples, respectively. PCR-MS and GA results were fully concordant for 122 positive and 4 negative samples. The sequencing results for the 9 samples that tested negative by GA were completely concordant with the positive PCR-MS results. Multiple HPV types were identified in 25.2% (34/135) and 55.6% (75/135) of the cervical cancer samples by GA and PCR-MS, respectively, and results were confirmed by sequencing. The new assay allows the genotyping of >1,000 samples per day. It provides a good alternative to current methods, especially for large-scale investigations of multiple HPV infections and degraded FFPE samples. PMID:23152557

Capsular typing of Streptococcus agalactiae (Lancefield group B streptococci) from fish using multiplex PCR and serotyping

USDA-ARS?s Scientific Manuscript database

Streptococcus spp. including Streptococcus agalactiae (Lancefield group B streptococci) are considered emerging pathogens responsible for approximately $1 billion USD in annual losses to the global tilapia (Oreochromis sp.) aquaculture industry. This study evaluated a published multiplex PCR capsul...

Evaluation of the performance of C. DIFF QUIK CHEK COMPLETE and its usefulness in a hospital setting with a high prevalence of Clostridium difficile infection.

PubMed

Chung, Hae-Sun; Lee, Miae

2017-01-01

Rapid and accurate diagnosis of Clostridium difficile infection (CDI) is crucial for patient care, infection control, and efficient surveillance. We evaluated C. DIFF QUIK CHEK COMPLETE (QCC; TechLab), which detects glutamate dehydrogenase (GDH) antigen (QCC-Ag) and toxin A/B (QCC-Tox) simultaneously, and compared it to the laboratory diagnostics for CDI currently in use in a tertiary hospital setting with a high prevalence of CDI. QCC, RIDASCREEN C. difficile toxin A/B assay (Toxin EIA; R-Biopharm AG), chromID C. difficile agar (bioMérieux) culture (ChromID culture), and Xpert C. difficile PCR assay (Xpert PCR; Cepheid) were performed according to the manufacturers' instructions. Performances of the assays were compared against that of Xpert PCR as a reference. Of the 231 loose stool specimens, 83 (35.9%) were positive by Xpert PCR. The sensitivity, specificity, and positive and negative predictive values were 97.6%, 93.9%, 90.0%, and 98.6%, respectively, for QCC-Ag and 55.4%, 100%, 100%, and 80.0%, respectively, for QCC-Tox. The median threshold cycle values of the QCC-Tox(+) specimens were lower than those of the QCC-Tox(-) specimens. Results of QCC as an initial screening test were confirmed in 81.0% (187/231) of samples; these specimens did not require further testing. QCC is a rapid, easy, and cost-effective method that would be a useful first-line screening assay for laboratory diagnosis of CDI in a tertiary hospital with a high prevalence of CDI. A two-step algorithm using QCC as an initial screening tool, followed by Xpert PCR as a confirmatory test, is a practical and cost-effective approach. Copyright © 2016 American Federation for Medical Research.

Decreased frontal N-acetylaspartate levels in adolescents concurrently using both methamphetamine and marijuana.

PubMed

Sung, Young-Hoon; Carey, Paul D; Stein, Dan J; Ferrett, Helen L; Spottiswoode, Bruce S; Renshaw, Perry F; Yurgelun-Todd, Deborah A

2013-06-01

The potential neurochemical toxicity associated with methamphetamine (MA) or marijuana (MJ) use on the developing adolescent brain is unclear, particularly with regard to individuals with concomitant use of MA and MJ (MA+MJ). In this study, proton magnetic resonance spectroscopy (MRS) was utilized to measure in vivo brain N-acetylaspartate plus N-acetylaspartyl glutamate (tNAA, an indicator of intact neuronal integrity) levels. Three adolescent groups from Cape Town, South Africa completed MRS scans as well as clinical measures including a drug use history. Subjects included (1) nine MA (age=15.7±1.37), (2) eight MA+MJ (age=16.2±1.16) using adolescents and (3) ten healthy controls (age=16.8±0.62). Single voxel spectra were acquired from midfrontal gray matter using a point-resolved spectroscopy sequence (PRESS). The MRS data were post-processed in the fully automated approach for quantitation of metabolite ratios to phosphocreatine plus creatine (PCr+Cr). A significant reduction in frontal tNAA/PCr+Cr ratios was seen in the MA+MJ group compared to the healthy controls (p=0.01, by 7.2%) and to the MA group (p=0.04, by 6.9%). Significant relationships were also observed between decreased tNAA/PCr+Cr ratios and drug use history of MA or MJ (total cumulative lifetime dose, age of onset, and duration of MA and MJ exposure) only in the MA+MJ group (all p<0.05). These findings suggest that in adolescents, concomitant heavy MA+MJ use may contribute to altered brain metabolites in frontal gray matter. The significant associations between the abnormal tNAA/PCr+Cr ratios and the drug use history suggest that MA+MJ abuse may induce neurotoxicity in a dose-responsive manner in adolescent brain. Copyright © 2013 Elsevier B.V. All rights reserved.

Novel high-speed droplet-allele specific-polymerase chain reaction: application in the rapid genotyping of single nucleotide polymorphisms.

PubMed

Taira, Chiaki; Matsuda, Kazuyuki; Yamaguchi, Akemi; Sueki, Akane; Koeda, Hiroshi; Takagi, Fumio; Kobayashi, Yukihiro; Sugano, Mitsutoshi; Honda, Takayuki

2013-09-23

Single nucleotide alterations such as single nucleotide polymorphisms (SNP) and single nucleotide mutations are associated with responses to drugs and predisposition to several diseases, and they contribute to the pathogenesis of malignancies. We developed a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) with our droplet-PCR machine (droplet-AS-PCR). Using 8 SNP loci, we evaluated the specificity and sensitivity of droplet-AS-PCR. Buccal cells were pretreated with proteinase K and subjected directly to the droplet-AS-PCR without DNA extraction. The genotypes determined using the droplet-AS-PCR were then compared with those obtained by direct sequencing. Specific PCR amplifications for the 8 SNP loci were detected, and the detection limit of the droplet-AS-PCR was found to be 0.1-5.0% by dilution experiments. Droplet-AS-PCR provided specific amplification when using buccal cells, and all the genotypes determined within 9 min were consistent with those obtained by direct sequencing. Our novel droplet-AS-PCR assay enabled high-speed amplification retaining specificity and sensitivity and provided ultra-rapid genotyping. Crude samples such as buccal cells were available for the droplet-AS-PCR assay, resulting in the reduction of the total analysis time. Droplet-AS-PCR may therefore be useful for genotyping or the detection of single nucleotide alterations. Copyright © 2013 Elsevier B.V. All rights reserved.

[Isolation of ABA-regulated genes in Oryza sativa through fluorescent differential display PCR (FDD-PCR)].

PubMed

Xu, Shou Ling; Shen, Si Shi; Xu, Zhi Hong; Xue, Hong Wei

2002-12-01

Abscisic acid (ABA) was critical in plant seed development and response to environmental factors such as stress situations. To study the possible ABA related signaling transduction pathways, we tried to isolate the ABA-regulated genes through fluorescent differential display PCR (FDD-PCR) technology using rice seedling as materials (treated with ABA for 2, 4, 8 and 12h). In the 17 fragments isolated, 14 and 3 clones were up-and down-regulated respectively. Sequence analyses revealed that the encoded proteins were involved in photosynthesis (7 fragments), signal transduction (1 fragments), transcription (2 fragments), metabolism and resistance (6 fragments), and unknown protein (1 fragments). 3 clones, encoding putative alpha/beta hydrolase fold, putative vacuolar H+ -ATPase B subunit, putative tyrosine phosphatase, were confirmed to be regulated under ABA treatment by RT-PCR and northern blot analysis. FDD-PCR and possible functional mechanisms of ABA were discussed.

Galactomannan enzyme immunoassay and quantitative Real Time PCR as tools to evaluate the exposure and response in a rat model of aspergillosis after posaconazole prophylaxis.

PubMed

Cendejas-Bueno, Emilio; Forastiero, Agustina; Ruiz, Isabel; Mellado, Emilia; Buitrago, María José; Gavaldà, Joan; Gomez-Lopez, Alicia

2016-11-01

A steroid-immunosuppressed rat model of invasive pulmonary aspergillosis was use to examine the usefulness of galactomannan enzyme immunoassay (GM) and quantitative real time PCR (RT-PCR) in evaluating the association between response and exposure after a high dose of prophylactic posaconazole. Two different strains of Aspergillus fumigatus with different in vitro posaconazole susceptibility were used. Serum concentrations demonstrated similar posaconazole exposure for all treated animals. However, response to posaconazole relied on the in vitro susceptibility of the infecting strain. After prophylaxis, galactomannan index and fungal burden only decreased in those animals infected with the most susceptible strain. This study demonstrated that both biomarkers may be useful tools for predicting efficacy of antifungal compounds in prophylaxis. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Yeast microbiota associated with spontaneous sourdough fermentations in the production of traditional wheat sourdough breads of the Abruzzo region (Italy).

PubMed

Valmorri, Sara; Tofalo, Rosanna; Settanni, Luca; Corsetti, Aldo; Suzzi, Giovanna

2010-02-01

The aims of this study were to describe the yeast community of 20 sourdoughs collected from central Italy and to characterize the sourdoughs based on chemical properties. A polyphasic approach consisting of traditional culture-based tests (spore-forming and physiological tests) and molecular techniques (PCR-RFLP, RAPD-PCR, PCR-DGGE) and chemical analysis (total acidity, acids, and sugar contents), was utilized to describe the yeast population and to investigate the chemical composition of the doughs. PCR-RFLP analysis identified 85% of the isolates as Saccharomyces cerevisiae, with the other dominant species being Candida milleri (11%), Candida krusei (2.5%), and Torulaspora delbrueckii (1%). RAPD-PCR analysis, performed with primers M13 and LA1, highlighted intraspecific polymorphism among the S. cerevisiae strains. The diversity of the sourdoughs from the Abruzzo region is reflected in the chemical composition, yeast species, and strain polymorphism. Our approach using a combination of phenotypic and genotypic methods identified the yeast species in the 20 sourdough samples and provided a complete overview of the yeast populations found in sourdoughs from the Abruzzo region.

Development of homologous viral internal controls for use in RT-PCR assays of waterborne enteric viruses.

PubMed

Parshionikar, Sandhya U; Cashdollar, Jennifer; Fout, G Shay

2004-10-01

Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides a means to rapidly detect low levels of these viruses, but it is sensitive to inhibitors that are present in water samples. Inhibitors of RT-PCR are concentrated along with viruses during sample processing. While procedures have been developed to remove inhibitors, none of them completely remove all inhibitors from all types of water matrices. This problem requires that adequate controls be used to distinguish true from potentially false-negative results. To address this problem, we have developed homologous viral internal controls for hepatitis A virus (HAV), poliovirus, Norwalk virus and rotavirus. These internal controls can be used in RT-PCR assays for the detection of the above viruses by competitive amplification, thereby allowing the detection of false negatives in processed water samples. The internal controls developed in this study were successfully tested with virus-seeded environmental water sample concentrates.

Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells.

PubMed

Greve, Tine; Clasen-Linde, Erik; Andersen, Morten T; Andersen, Mette K; Sørensen, Stine D; Rosendahl, Mikkel; Ralfkiaer, Elisabeth; Andersen, Claus Yding

2012-11-22

Some women suffering from leukemia require bone marrow transplantation to be cured. Bone marrow transplantation is associated with a high risk of sterility, and some patients are offered fertility preservation by cryopreservation of the ovarian cortex. Transplantation of the ovarian cortex to women cured of leukemia who became menopausal is currently not performed because of the risk of introducing the disease. In this study, individual pieces of ovarian cortex intended for reimplantation from 25 patients with leukemia were transplanted to each of 25 nude mice for 20 weeks. The ovarian cortex was examined before and after transplantation by histology and immunohistochemistry, and RT-quantitative PCR (in the 7 patients with a known marker). Seventeen patients had the ovarian cortex retrieved when they were in complete remission. Before transplantation, 4 of 7 pieces (2 from patients in complete remission) of ovarian cortex had a positive RT-quantitative PCR. After transplantation, none of the mice revealed any sign of disease, neither in the pieces of ovarian cortex transplanted nor in any of the murine organs evaluated. Thus, the ovaries from patients in complete remission do not appear to contain viable malignant cells contrasting ovarian tissue retrieved before treatment.

SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.

PubMed

Niba, Emma Tabe Eko; Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Lai, Poh San; Bouike, Yoshihiro; Nishio, Hisahide; Shinohara, Masakazu

2017-12-18

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA. To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA. DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus. The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP. Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first-tier diagnostic method of SMA.

Sustained high-throughput polymerase chain reaction diagnostics during the European epidemic of Bluetongue virus serotype 8.

PubMed

van Rijn, Piet A; Heutink, René G; Boonstra, Jan; Kramps, Hans A; van Gennip, René G P

2012-05-01

A real-time reverse transcription polymerase chain reaction assay (PCR test) based on genome segment 10 of Bluetongue virus (BTV) was developed. The PCR test consists of robotized viral RNA isolation from blood samples and an all-in-one method including initial denaturation of genomic double-stranded RNA, reverse transcription polymerase chain reaction (RT-PCR), and real-time detection and analysis. Reference strains of the 24 recognized BTV serotypes, isolates from different years, and geographic origins were detected. Other orbiviruses such as African horse sickness virus, Epizootic hemorrhagic disease virus, and Equine encephalosis virus were not detected. Experimentally infected animals were PCR positive from 2 days postinoculation, which was earlier than fever, other clinical signs, or seroconversion. The diagnostic sensitivity and specificity were very close to or even 100%. The PCR test played a key role in the detection of BTV serotype 8 in August 2006 in The Netherlands. The outbreak in a completely naive ruminant population allowed for further evaluation of the PCR test with field samples. In 2006, the correlation between enzyme-linked immunosorbent assay and PCR results was estimated to be 95%. In the following years, the PCR test was used for diagnosis of diseased animals, for testing of healthy animals for trade purposes, and for detection of BTV RNA in different species of the insect vector, Culicoides. In the autumn of 2008, BTV serotype 6 unexpectedly emerged in northwest Europe and was also detected with the PCR test developed in the current study. The performance in routine use over 5 years has been recorded and evaluated.

Introduction on Using the FastPCR Software and the Related Java Web Tools for PCR and Oligonucleotide Assembly and Analysis.

PubMed

Kalendar, Ruslan; Tselykh, Timofey V; Khassenov, Bekbolat; Ramanculov, Erlan M

2017-01-01

This chapter introduces the FastPCR software as an integrated tool environment for PCR primer and probe design, which predicts properties of oligonucleotides based on experimental studies of the PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations. These include the standard PCR as well as the multiplex, long-distance, inverse, real-time, group-specific, unique, overlap extension PCR for multi-fragments assembling cloning and loop-mediated isothermal amplification (LAMP). It also contains a built-in program to design oligonucleotide sets both for long sequence assembly by ligase chain reaction and for design of amplicons that tile across a region(s) of interest. The software calculates the melting temperature for the standard and degenerate oligonucleotides including locked nucleic acid (LNA) and other modifications. It also provides analyses for a set of primers with the prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity as well as a primer dilution and resuspension calculator. The program consists of various bioinformatical tools for analysis of sequences with the GC or AT skew, CG% and GA% content, and the purine-pyrimidine skew. It also analyzes the linguistic sequence complexity and performs generation of random DNA sequence as well as restriction endonucleases analysis. The program allows to find or create restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It performs efficient and complete detection of various repeat types with visual display. The FastPCR software allows the sequence file batch processing that is essential for automation. The program is available for download at http://primerdigital.com/fastpcr.html , and its online version is located at http://primerdigital.com/tools/pcr.html .

Ultrasensitive Quantification of Hepatitis B Virus A1762T/G1764A Mutant by a SimpleProbe PCR Using a Wild-Type-Selective PCR Blocker and a Primer-Blocker-Probe Partial-Overlap Approach ▿

PubMed Central

Nie, Hui; Evans, Alison A.; London, W. Thomas; Block, Timothy M.; Ren, Xiangdong David

2011-01-01

Hepatitis B virus (HBV) carrying the A1762T/G1764A double mutation in the basal core promoter (BCP) region is associated with HBe antigen seroconversion and increased risk of liver cirrhosis and hepatocellular carcinoma (HCC). Quantification of the mutant viruses may help in predicting the risk of HCC. However, the viral genome tends to have nucleotide polymorphism, which makes it difficult to design hybridization-based assays including real-time PCR. Ultrasensitive quantification of the mutant viruses at the early developmental stage is even more challenging, as the mutant is masked by excessive amounts of the wild-type (WT) viruses. In this study, we developed a selective inhibitory PCR (siPCR) using a locked nucleic acid-based PCR blocker to selectively inhibit the amplification of the WT viral DNA but not the mutant DNA. At the end of siPCR, the proportion of the mutant could be increased by about 10,000-fold, making the mutant more readily detectable by downstream applications such as real-time PCR and DNA sequencing. We also describe a primer-probe partial overlap approach which significantly simplified the melting curve patterns and minimized the influence of viral genome polymorphism on assay accuracy. Analysis of 62 patient samples showed a complete match of the melting curve patterns with the sequencing results. More than 97% of HBV BCP sequences in the GenBank database can be correctly identified by the melting curve analysis. The combination of siPCR and the SimpleProbe real-time PCR enabled mutant quantification in the presence of a 100,000-fold excess of the WT DNA. PMID:21562108

Comparison of Three Different Hepatitis C Virus Genotyping Methods: 5'NCR PCR-RFLP, Core Type-Specific PCR, and NS5b Sequencing in a Tertiary Care Hospital in South India.

PubMed

Daniel, Hubert D-J; David, Joel; Raghuraman, Sukanya; Gnanamony, Manu; Chandy, George M; Sridharan, Gopalan; Abraham, Priya

2017-05-01

Based on genetic heterogeneity, hepatitis C virus (HCV) is classified into seven major genotypes and 64 subtypes. In spite of the sequence heterogeneity, all genotypes share an identical complement of colinear genes within the large open reading frame. The genetic interrelationships between these genes are consistent among genotypes. Due to this property, complete sequencing of the HCV genome is not required. HCV genotypes along with subtypes are critical for planning antiviral therapy. Certain genotypes are also associated with higher progression to liver cirrhosis. In this study, 100 blood samples were collected from individuals who came for routine HCV genotype identification. These samples were used for the comparison of two different genotyping methods (5'NCR PCR-RFLP and HCV core type-specific PCR) with NS5b sequencing. Of the 100 samples genotyped using 5'NCR PCR-RFLP and HCV core type-specific PCR, 90% (κ = 0.913, P < 0.00) and 96% (κ = 0.794, P < 0.00) correlated with NS5b sequencing, respectively. Sixty percent and 75% of discordant samples by 5'NCR PCR-RFLP and HCV core type-specific PCR, respectively, belonged to genotype 6. All the HCV genotype 1 subtypes were classified accurately by both the methods. This study shows that the 5'NCR-based PCR-RFLP and the HCV core type-specific PCR-based assays correctly identified HCV genotypes except genotype 6 from this region. Direct sequencing of the HCV core region was able to identify all the genotype 6 from this region and serves as an alternative to NS5b sequencing. © 2016 Wiley Periodicals, Inc.

Development of a fast PCR protocol enabling rapid generation of AmpFℓSTR® Identifiler® profiles for genotyping of human DNA

PubMed Central

2012-01-01

Background Traditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. The purpose of this study was to develop and validate a fast PCR protocol that enabled amplification of the 16 loci targeted by the AmpFℓSTR® Identifiler® primer set, allowing decreased cycling times. Methods Fast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR™ HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach. Results The total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA. Conclusions The fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations. PMID:22394458

Development of a fast PCR protocol enabling rapid generation of AmpFℓSTR® Identifiler® profiles for genotyping of human DNA.

PubMed

Foster, Amanda; Laurin, Nancy

2012-03-06

Traditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. The purpose of this study was to develop and validate a fast PCR protocol that enabled amplification of the 16 loci targeted by the AmpFℓSTR® Identifiler® primer set, allowing decreased cycling times. Fast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR™ HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach. The total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA. The fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations.

Determinants of short and long term outcomes in patients undergoing immediate breast reconstruction following neoadjuvant chemotherapy.

PubMed

Wengler, Craig A; Valente, Stephanie A; Al-Hilli, Zahraa; Woody, Neil M; Muntean, Julia H; Abraham, Jame; Tendulkar, Rahul D; Djohan, Risal; O'Rourke, Colin; Crowe, Joseph P; Grobmyer, Stephen R

2017-12-01

We evaluated oncologic outcomes and complications of skin-sparing mastectomy (SSM) and nipple-sparing mastectomy (NSM) with immediate reconstruction (IR) after neoadjuvant chemotherapy (NAC) in patients with early-stage and locally advanced breast cancer (BC). BC patients from 2000 to 2014 treated with NAC followed by SSM/NSM and IR were reviewed. Patient demographics, tumor characteristics, NAC response, complications, and recurrence were analyzed. Two hundred sixty-nine patients with 280 BCs were treated with NAC followed by SSM (94%) or NSM (6%) with IR. Median age was 47 (26-72) years with a median follow-up of 45 months. Pathologic complete response (pCR) was noted in 49 (17.5%) cases. Overall 30-day complication rate was 13.2%. Variables associated with complications included BMI (P < 0.0001), tobacco use (P = 0.015), and adjuvant radiation (P = 0.025). Local-regional recurrence was 3.2% and metastatic recurrence was 13.2%. Variables predicting recurrence risk were pre-NAC tumor size (P < 0.001), residual tumor size (P = 0.002), Grade III (P = 0.002), HER-2 negative (P = 0.025), pre-NAC nodal disease (P = 0.05), and lack of pCR (P = 0.045). Following NAC, risk factors for complications in patients undergoing SSM/NSM with IR are high BMI, smoking, and adjuvant XRT. SSM/NSM following NAC is associated with excellent local control. These data support expanding the indications for NSM/SSM to include patients receiving NAC. © 2017 Wiley Periodicals, Inc.

Visual format for detection of Mycobacterium tuberculosis and M. bovis in clinical samples using molecular beacons.

PubMed

Kumar, Parameet; Nath, Kapili; Rath, Bimba; Sen, Manas K; Vishalakshi, Potharuju; Chauhan, Devender S; Katoch, Vishwa M; Singh, Sarman; Tyagi, Sanjay; Sreenivas, Vishnubhatla; Prasad, Hanumanthappa K

2009-09-01

A real-time polymerase chain reaction (PCR) assay for the direct identification of Mycobacterium tuberculosis and M. bovis using molecular beacons was developed. The assay was modified for use in regular thermal cyclers. Molecular beacons that were specific for M. tuberculosis (Tb-B) and M. bovis (Bo-B) were designed. The fluorescence of the target PCR product-molecular beacon probe complex was detected visually using a transilluminator. The results were then compared with those of conventional multiplex PCR (CM-PCR) assays and biochemical identification. The detection limit of Tb-B and Bo-B beacons was 500 fg and 50 fg by the visual format and real-time PCR assay, respectively, compared with 5 pg by CM-PCR assay. Pulmonary and extrapulmonary samples were examined. The agreement between culture and the two assays was very good in sputum samples and fair in extrapulmonary samples. The agreement between clinical diagnoses with the two assays was moderate in extrapulmonary samples. There was very good agreement between CM-PCR and visual format assays for all samples used in the study. Concordance in the identification of isolates by the visual, CM-PCR assay, and biochemical identification was seen. Hence, the use of molecular beacon detection of M. tuberculosis and M. bovis in clinical samples is feasible by setting up two asymmetric PCRs concurrently. The assay is sensitive, specific, simple to interpret, and takes less than 3 hours to complete.

Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

PubMed Central

Lucero, David E.; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W.; Peña, Reynaldo; Morrissey, Leslie A.; Rizzo, Donna M.; Stevens, Lori

2014-01-01

Background In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. Methodology/Principal Findings We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). Conclusions/Significance We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors. PMID:25474154

Visual Format for Detection of Mycobacterium tuberculosis and M. bovis in Clinical Samples Using Molecular Beacons

PubMed Central

Kumar, Parameet; Nath, Kapili; Rath, Bimba; Sen, Manas K.; Vishalakshi, Potharuju; Chauhan, Devender S.; Katoch, Vishwa M.; Singh, Sarman; Tyagi, Sanjay; Sreenivas, Vishnubhatla; Prasad, Hanumanthappa K.

2009-01-01

A real-time polymerase chain reaction (PCR) assay for the direct identification of Mycobacterium tuberculosis and M. bovis using molecular beacons was developed. The assay was modified for use in regular thermal cyclers. Molecular beacons that were specific for M. tuberculosis (Tb-B) and M. bovis (Bo-B) were designed. The fluorescence of the target PCR product-molecular beacon probe complex was detected visually using a transilluminator. The results were then compared with those of conventional multiplex PCR (CM-PCR) assays and biochemical identification. The detection limit of Tb-B and Bo-B beacons was 500 fg and 50 fg by the visual format and real-time PCR assay, respectively, compared with 5 pg by CM-PCR assay. Pulmonary and extrapulmonary samples were examined. The agreement between culture and the two assays was very good in sputum samples and fair in extrapulmonary samples. The agreement between clinical diagnoses with the two assays was moderate in extrapulmonary samples. There was very good agreement between CM-PCR and visual format assays for all samples used in the study. Concordance in the identification of isolates by the visual, CM-PCR assay, and biochemical identification was seen. Hence, the use of molecular beacon detection of M. tuberculosis and M. bovis in clinical samples is feasible by setting up two asymmetric PCRs concurrently. The assay is sensitive, specific, simple to interpret, and takes less than 3 hours to complete. PMID:19661384

A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

PubMed

Saingamsook, Jassada; Saeung, Atiporn; Yanola, Jintana; Lumjuan, Nongkran; Walton, Catherine; Somboon, Pradya

2017-10-10

Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale. A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR). The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory. Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

Authentication of an endangered herb Changium smyrnioides from different producing areas based on rDNA ITS sequences and allele-specific PCR.

PubMed

Sun, Xiaoqin; Wei, Yanglian; Qin, Minjian; Guo, Qiaosheng; Guo, Jianlin; Zhou, Yifeng; Hang, Yueyu

2012-03-01

The rDNA ITS region of 18 samples of Changium smyrnioides from 7 areas and of 2 samples of Chuanminshen violaceum were sequenced and analyzed. The amplified ITS region of the samples, including a partial sequence of ITS1 and complete sequences of 5.8S and ITS2, had a total length of 555 bp. After complete alignment, there were 49 variable sites, of which 45 were informative, when gaps were treated as missing data. Samples of C. smyrnioides from different locations could be identified exactly based on the variable sites. The maximum parsimony (MP) and neighbor joining (NJ) tree constructed from the ITS sequences based on Kumar's two-parameter model showed that the genetic distances of the C. smyrnioides samples from different locations were not always related to their geographical distances. A specific primer set for Allele-specific PCR authentication of C. violaceum from Jurong of Jiangsu was designed based on the SNP in the ITS sequence alignment. C. violaceum from the major genuine producing area in Jurong of Jiangsu could be identified exactly and quickly by Allele-specific PCR.

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics

PubMed Central

Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

2014-01-01

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas. PMID:24553130

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

PubMed Central

Ghosh, Prakash; Khan, Md. Anik Ashfaq; Duthie, Malcolm S.; Vallur, Aarthy C.; Picone, Alessandro; Howard, Randall F.; Reed, Steven G.

2017-01-01

Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites in VL, PKDL and RVL patients, as well as being capable of assessing response to treatment. As such, this real time PCR assay represents an important contribution in efforts to eliminate VL. PMID:28957391

Identification of the WRKY gene family and functional analysis of two genes in Caragana intermedia.

PubMed

Wan, Yongqing; Mao, Mingzhu; Wan, Dongli; Yang, Qi; Yang, Feiyun; Mandlaa; Li, Guojing; Wang, Ruigang

2018-02-09

WRKY transcription factors, one of the largest families of transcriptional regulators in plants, play important roles in plant development and various stress responses. The WRKYs of Caragana intermedia are still not well characterized, although many WRKYs have been identified in various plant species. We identified 53 CiWRKY genes from C. intermedia transcriptome data, 28 of which exhibited complete open reading frames (ORFs). These CiWRKYs were divided into three groups via phylogenetic analysis according to their WRKY domains and zinc finger motifs. Conserved domain analysis showed that the CiWRKY proteins contain a highly conserved WRKYGQK motif and two variant motifs (WRKYGKK and WKKYEEK). The subcellular localization of CiWRKY26 and CiWRKY28-1 indicated that these two proteins localized exclusively to nuclei, supporting their role as transcription factors. The expression patterns of the 28 CiWRKYs with complete ORFs were examined through quantitative real-time PCR (qRT-PCR) in various tissues and under different abiotic stresses (drought, cold, salt, high-pH and abscisic acid (ABA)). The results showed that each CiWRKY responded to at least one stress treatment. Furthermore, overexpression of CiWRKY75-1 and CiWRKY40-4 in Arabidopsis thaliana suppressed the drought stress tolerance of the plants and delayed leaf senescence, respectively. Fifty-three CiWRKY genes from the C. intermedia transcriptome were identified and divided into three groups via phylogenetic analysis. The expression patterns of the 28 CiWRKYs under different abiotic stresses suggested that each CiWRKY responded to at least one stress treatment. Overexpression of CiWRKY75-1 and CiWRKY40-4 suppressed the drought stress tolerance of Arabidopsis and delayed leaf senescence, respectively. These results provide a basis for the molecular mechanism through which CiWRKYs mediate stress tolerance.

MRPrimerW: a tool for rapid design of valid high-quality primers for multiple target qPCR experiments.

PubMed

Kim, Hyerin; Kang, NaNa; An, KyuHyeon; Koo, JaeHyung; Kim, Min-Soo

2016-07-08

Design of high-quality primers for multiple target sequences is essential for qPCR experiments, but is challenging due to the need to consider both homology tests on off-target sequences and the same stringent filtering constraints on the primers. Existing web servers for primer design have major drawbacks, including requiring the use of BLAST-like tools for homology tests, lack of support for ranking of primers, TaqMan probes and simultaneous design of primers against multiple targets. Due to the large-scale computational overhead, the few web servers supporting homology tests use heuristic approaches or perform homology tests within a limited scope. Here, we describe the MRPrimerW, which performs complete homology testing, supports batch design of primers for multi-target qPCR experiments, supports design of TaqMan probes and ranks the resulting primers to return the top-1 best primers to the user. To ensure high accuracy, we adopted the core algorithm of a previously reported MapReduce-based method, MRPrimer, but completely redesigned it to allow users to receive query results quickly in a web interface, without requiring a MapReduce cluster or a long computation. MRPrimerW provides primer design services and a complete set of 341 963 135 in silico validated primers covering 99% of human and mouse genes. Free access: http://MRPrimerW.com. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Chronic myeloid leukaemia with extreme thrombocytosis.

PubMed

Verma, Shailendra Prasad; Subbiah, Arunkumar; Jacob, Sajini Elizabeth; Basu, Debdatta

2015-08-19

We report two cases of chronic myeloid leukaemia (CML) with extreme thrombocytosis. The first patient was a 65-year-old man who presented with prolonged history of upper abdominal discomfort, anorexia and two episodes of recent gum bleeds without fever or other bleeding manifestations. He was a chronic smoker with no other comorbidities. Examination revealed moderate hepatosplenomegaly. On investigation, he was found to have extreme thrombocytosis (3,500,000/mm(3)) and leucocytosis with moderate anaemia. In view of the leucocytosis, he was investigated for CML and found to be positive for BCR-ABL by reverse transcription PCR (RT-PCR). He received imatinib 400 mg/day and achieved complete haematological response at the end of 3 months. The second patient was a 7-year-old boy who presented with fever, cough and cold of 2-week duration. Examination revealed mild hepatomegaly with palpable spleen tip. Haemogram and peripheral smear revealed moderate leucocytosis with extreme thrombocytosis (2,800,000/mm(3)). On evaluation, he was found to be BCR-ABL positive and responded well to imatinib treatment. In both these cases, massive thrombocytosis was an unusual presentation of a well-known entity, namely, CML. This degree of thrombocytosis is usually seen only in essential thrombocytosis. 2015 BMJ Publishing Group Ltd.

Coexistence of SHV-4- and TEM-24-Producing Enterobacter aerogenes Strains before a Large Outbreak of TEM-24-Producing Strains in a French Hospital

PubMed Central

Mammeri, H.; Laurans, G.; Eveillard, M.; Castelain, S.; Eb, F.

2001-01-01

In 1996, a monitoring program was initiated at the teaching hospital of Amiens, France, and carried out for 3 years. All extended-spectrum β-lactamase (ESBL)-producing Enterobacter aerogenes isolates recovered from clinical specimens were collected for investigation of their epidemiological relatedness by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and determination of the type of ESBL harbored by isoelectric focusing and DNA sequencing. Molecular typing revealed the endemic coexistence, during the first 2 years, of two clones expressing, respectively, SHV-4 and TEM-24 ESBLs, while an outbreak of the TEM-24-producing strain raged in the hospital during the third year, causing the infection or colonization of 165 patients. Furthermore, this strain was identified as the prevalent clone responsible for outbreaks in many French hospitals since 1996. This study shows that TEM-24-producing E. aerogenes is an epidemic clone that is well established in the hospital's ecology and able to spread throughout wards. The management of the outbreak at the teaching hospital of Amiens, which included the reinforcement of infection control measures, failed to obtain complete eradication of the clone, which has become an endemic pathogen. PMID:11376055

Evaluating the addition of oxaliplatin to single agent fluoropyrimidine in the treatment of locally advanced rectal cancer: a systematic review and meta-analysis.

PubMed

Thavaneswaran, Subotheni; Kok, Peey Sei; Price, Timothy

2017-10-01

Multimodality treatment of patients with locally advanced rectal cancer (LARC) has significantly improved local disease control, however the unaltered overall survival (OS) implicates an inability to further control micrometastases, providing rationale for intensified systemic treatment. A systematic review was conducted to evaluate the efficacy and toxicity of adding oxaliplatin to a fluoropyrimidine (intervention) compared with fluoropyrimidine alone (control) in the treatment of LARC. We searched CENTRAL, Medline Ovid, PubMed and EMBASE databases. Randomised trials comparing the intervention and control delivered either pre- or post-operatively were included. Seven trials involving 4444 patients were identified; five studies evaluated the intervention vs control preoperatively; one study peri-operatively; and one, post-operatively. There was no significant difference in OS with oxaliplatin addition, HR 0.89, 95% CI, 0.75 to 1.06. There was however an improvement in disease free survival, 3-year local and distant recurrence rates (RR) favouring oxaliplatin. Preoperative oxaliplatin improved pathological complete response (pCR), but with a greater toxicity and reduced compliance with radiation. There is no OS benefit with oxaliplatin, despite improved pCR, local and distant RR. Before drawing definitive conclusions, longer follow-up in included trials and availability of published data from other eligible studies, including the induction setting, are needed.

Predicting response before initiation of neoadjuvant chemotherapy in breast cancer using new methods for the analysis of dynamic contrast enhanced MRI (DCE MRI) data

NASA Astrophysics Data System (ADS)

DeGrandchamp, Joseph B.; Whisenant, Jennifer G.; Arlinghaus, Lori R.; Abramson, V. G.; Yankeelov, Thomas E.; Cárdenas-Rodríguez, Julio

2016-03-01

The pharmacokinetic parameters derived from dynamic contrast enhanced (DCE) MRI have shown promise as biomarkers for tumor response to therapy. However, standard methods of analyzing DCE MRI data (Tofts model) require high temporal resolution, high signal-to-noise ratio (SNR), and the Arterial Input Function (AIF). Such models produce reliable biomarkers of response only when a therapy has a large effect on the parameters. We recently reported a method that solves the limitations, the Linear Reference Region Model (LRRM). Similar to other reference region models, the LRRM needs no AIF. Additionally, the LRRM is more accurate and precise than standard methods at low SNR and slow temporal resolution, suggesting LRRM-derived biomarkers could be better predictors. Here, the LRRM, Non-linear Reference Region Model (NRRM), Linear Tofts model (LTM), and Non-linear Tofts Model (NLTM) were used to estimate the RKtrans between muscle and tumor (or the Ktrans for Tofts) and the tumor kep,TOI for 39 breast cancer patients who received neoadjuvant chemotherapy (NAC). These parameters and the receptor statuses of each patient were used to construct cross-validated predictive models to classify patients as complete pathological responders (pCR) or non-complete pathological responders (non-pCR) to NAC. Model performance was evaluated using area under the ROC curve (AUC). The AUC for receptor status alone was 0.62, while the best performance using predictors from the LRRM, NRRM, LTM, and NLTM were AUCs of 0.79, 0.55, 0.60, and 0.59 respectively. This suggests that the LRRM can be used to predict response to NAC in breast cancer.

Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients

PubMed Central

Xu, Yuanxin; Theobald, Valerie; Sung, Crystal; DePalma, Kathleen; Atwater, Laura; Seiger, Keirsten; Perricone, Michael A; Richards, Susan M

2008-01-01

Background HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis. Methods Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126–35(27L), IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127–35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established. Results The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods. Conclusion Our results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses. PMID:18945350

Intracellular angiotensin II directly induces in vitro transcription of TGF-β1, MCP-1 and NHE-3 mRNAs in isolated rat renal cortical nuclei via activation of nuclear AT1 receptors

PubMed Central

Li, Xiao C.; Zhuo, Jia L.

2008-01-01

The present study tested the hypothesis that intracellular angiotensin II (Ang II) directly induces transcriptional effects by stimulating AT1 receptors in the nucleus of rat renal cortical cells. Intact nuclei were freshly isolated from the rat renal cortex and transcriptional responses to Ang II were studied using in vitro RNA transcription assays and semi-quantitative RT-PCR. High power phase contrast micrographs showed that isolated nuclei were encircled by an intact nuclear envelop, stained strongly by the DNA marker DAPI, but not by the membrane or endosomal markers. FITC-labeled Ang II and [125I]-Val5-Ang II binding confirmed the presence of Ang II receptors in the nuclei with a predominance of AT1 receptors. RT-PCR showed that AT1a mRNA expression was 3-fold greater than AT1b receptor mRNAs in these nuclei. In freshly isolated nuclei, Ang II increased in vitro [α-32P]CTP incorporation in a concentration manner, and the effect was confirmed by autoradiography and RNA electrophoresis. Ang II markedly increased in vitro transcription of mRNAs for transforming growth factor-β1 by 143% (p < 0.01), macrophage chemoattractant protein-1 by 89% (p < 0.01), and the sodium and hydrogen exchanger-3 by 110% (p < 0.01). These transcriptional effects of Ang II on the nuclei were completely blocked by the AT1 receptor antagonist losartan (p < 0.01). By contrast, Ang II had no effects on transcription of angiotensinogne and GAPDH mRNAs. Since these transcriptional effects of Ang II in isolated nuclei were induced by Ang II in the absence of cell surface receptor-mediated signaling and completely blocked by losartan, we concluded that Ang II may directly stimulate nuclear AT1a receptors to induce transcriptional responses that are associated with tubular epithelial sodium transport, cellular growth and hypertrophy, and proinflammatory cytokines. PMID:18256274

Complete mitochondrial genome of the Freshwater Catfish Rita rita (Siluriformes, Bagridae).

PubMed

Lashari, Punhal; Laghari, Muhammad Younis; Xu, Peng; Zhao, Zixia; Jiang, Li; Narejo, Naeem Tariq; Deng, Yulin; Sun, Xiaowen; Zhang, Yan

2015-01-01

The complete mitochondrial genome of Catfish, Rita rita, was isolated by LA PCR (TakaRa LAtaq, Dalian, China); and sequenced by Sanger's method to obtain the complete mitochondrial genome, which is listed Critically Endangered and Red Listed species. The complete mitogenome was 16,449 bp in length and contains 13 typical vertebrate protein-coding genes, 2 rRNA and 22 tRNA genes. The whole genome base composition was estimated to be 33.40% A, 27.43% C, 14.26% G and 24.89% T. The complete mitochondrial genome of catfish, Rita rita provides the basis for genetic breeding and conservation studies.

Tapping in synchrony with a perturbed metronome: the phase correction response to small and large phase shifts as a function of tempo.

PubMed

Repp, Bruno H

2011-01-01

When tapping is paced by an auditory sequence containing small phase shift (PS) perturbations, the phase correction response (PCR) of the tap following a PS increases with the baseline interonset interval (IOI), leading eventually to overcorrection (B. H. Repp, 2008). Experiment 1 shows that this holds even for fixed-size PSs that become imperceptible as the IOI increases (here, from 400 to 1200 ms). Earlier research has also shown (but only for IOI=500 ms) that the PCR is proportionally smaller for large than for small PSs (B. H. Repp, 2002a, 2002b). Experiment 2 introduced large PSs and found smaller PCRs than in Experiment 1, at all of the same IOIs. In Experiments 3A and 3B, the author investigated whether the change in slope of the sigmoid function relating PCR and PS magnitudes occurs at a fixed absolute or relative PS magnitude across different IOIs (600, 1000, 1400 ms). The results suggest no clear answer; the exact shape of the function may depend on the range of PSs used in an experiment. Experiment 4 examined the PCR in the IOI range from 1000 to 2000 ms and found overcorrection throughout, but with the PCR increasing much more gradually than in Experiment 1. These results provide important new information about the phase correction process and pose challenges for models of sensorimotor synchronization, which presently cannot explain nonlinear PCR functions and overcorrection. Copyright © Taylor & Francis Group, LLC

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples.

PubMed

Zuo, Zhuang; Chen, Su S; Chandra, Pranil K; Galbincea, John M; Soape, Matthew; Doan, Steven; Barkoh, Bedia A; Koeppen, Hartmut; Medeiros, L Jeffrey; Luthra, Rajyalakshmi

2009-08-01

KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 80 degrees C (vs 94 degrees C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by >4.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffin-embedded tissue.

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

PubMed

Bassler, H A; Flood, S J; Livak, K J; Marmaro, J; Knorr, R; Batt, C A

1995-10-01

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.

Complete genome sequence of a natural Escherichia coli O145:H11 isolate that belongs to Phylo-group A

USDA-ARS?s Scientific Manuscript database

Escherichia coli O145:H11 strain RM14721 was originally isolated from wildlife feces near a leafy greens-growing region in Yuma, Arizona. This strain was initially positive in stx1; however, in the subsequent cultures, stx1 was not detected by PCR. Here we report the complete genome sequence and ann...

Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples

EPA Science Inventory

Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protectio...

qPCR for Second Year Undergraduates: A Short, Structured Inquiry to Illustrate Differential Gene Expression

ERIC Educational Resources Information Center

McCauslin, Christine Seitz; Gunn, Kathryn Elaine; Pirone, Dana; Staiger, Jennifer

2015-01-01

We describe a structured inquiry laboratory exercise that examines transcriptional regulation of the "NOS2" gene under conditions that simulate the inflammatory response in macrophages. Using quantitative PCR and the comparative C[subscript T] method, students are able determine whether transcriptional activation of "NOS2"…

Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR

USDA-ARS?s Scientific Manuscript database

The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as internal reference for data nor...

PCR detection of potato cyst nematode.

PubMed

Reid, Alex

2009-01-01

Potato cyst nematode (PCN) is responsible for losses in potato production totalling millions of euros every year in the EC. It is important for growers to know which species is present in their land as this determines its subsequent use. The two species Globodera pallida and Globodera rostochiensis can be differentiated using an allele-specific PCR.

Monitoring the Single-Cell Stress Response of the Diatom Thalassiosira pseudonana by Quantitative Real-Time Reverse Transcription-PCR

PubMed Central

Shi, Xu; Gao, Weimin; Chao, Shih-hui

2013-01-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition. PMID:23315741

Monitoring the single-cell stress response of the diatom Thalassiosira pseudonana by quantitative real-time reverse transcription-PCR.

PubMed

Shi, Xu; Gao, Weimin; Chao, Shih-hui; Zhang, Weiwen; Meldrum, Deirdre R

2013-03-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

Treatment of adult chronic indeterminate Chagas disease with benznidazole and three E1224 dosing regimens: a proof-of-concept, randomised, placebo-controlled trial.

PubMed

Torrico, Faustino; Gascon, Joaquim; Ortiz, Lourdes; Alonso-Vega, Cristina; Pinazo, María-Jesús; Schijman, Alejandro; Almeida, Igor C; Alves, Fabiana; Strub-Wourgaft, Nathalie; Ribeiro, Isabela

2018-04-01

Chagas disease is a major neglected vector-borne disease. In this study, we investigated the safety and efficacy of three oral E1224 (a water-soluble ravuconazole prodrug) regimens and benznidazole versus placebo in adult chronic indeterminate Chagas disease. In this proof-of-concept, double-blind, randomised phase 2 clinical trial, we recruited adults (18-50 years) with confirmed diagnosis of Trypanosoma cruzi infection from two outpatient units in Bolivia. Patients were randomised with a computer-generated randomisation list, which was stratified by centre and used a block size of ten. Patients were randomly assigned (1:1:1:1:1) to five oral treatment groups: high-dose E1224 (duration 8 weeks, total dose 4000 mg), low-dose E1224 (8 weeks, 2000 mg), short-dose E1224 (4 weeks + 4 weeks placebo, 2400 mg), benznidazole (60 days, 5 mg/kg per day), or placebo (8 weeks, E1224-matched tablets). Double-blinding was limited to the E1224 and placebo arms, and assessors were masked to all treatment allocations. The primary efficacy endpoint was parasitological response to E1224 at the end of treatment, assessed by PCR. The secondary efficacy endpoints were parasitological response to benznidazole at end of treatment, assessed by PCR; sustainability of parasitological response until 12 months; parasite clearance and changes in parasite load; incidence of conversion to negative response in conventional and non-conventional (antigen trypomastigote chemiluminescent ELISA [AT CL-ELISA]) serological response; changes in levels of biomarkers; and complete response. The primary analysis population consisted of all randomised patients by their assigned treatment arms. This trial is registered with ClinicalTrials.gov, number NCT01489228. Between July 19, 2011, and July 26, 2012, we screened 560 participants with confirmed Chagas disease, of whom 231 were enrolled and assigned to high-dose E1224 (n=45), low-dose E1224 (n=48), short-dose E1224 (n=46), benznidazole (n=45), or placebo (n=47). Parasite clearance was observed with E1224 during the treatment phase, but no sustained response was seen with low-dose and short-dose regimens, whereas 13 patients (29%, 95% CI 16·4-44·3) had sustained response with the high-dose regimen compared with four (9%, 2·4-20·4) in the placebo group (p<0·0001). Benznidazole had a rapid and sustained effect on parasite clearance, with 37 patients (82%, 67·9-92·0) with sustained response at 12-month follow-up. After 1 week of treatment, mean quantitative PCR repeated measurements showed a significant reduction in parasite load in all treatment arms versus placebo. Parasite levels in the low-dose and short-dose E1224 groups gradually returned to placebo levels. Both treatments were well tolerated. Reversible, dose-dependent liver enzyme increases were seen with E1224 and benznidazole. 187 (81%) participants developed treatment-emergent adverse events and six (3%) developed treatment-emergent serious adverse events. Treatment-emergent adverse events were headaches, nausea, pruritus, peripheral neuropathy, and hypersensitivity. E1224 is the first new chemical entity developed for Chagas disease in decades. E1224 displayed a transient, suppressive effect on parasite clearance, whereas benznidazole showed early and sustained efficacy until 12 months of follow-up. Despite PCR limitations, our results support increased diagnosis and access to benznidazole standard regimen, and provide a development roadmap for novel benznidazole regimens in monotherapy and in combinations with E1224. Drugs for Neglected Diseases initiative. Copyright © 2018 Elsevier Ltd. All rights reserved.

A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples.

PubMed

Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Guillier, Laurent; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

2015-05-18

Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Complete mitochondrial genome of the desert toad-headed agama, Phrynocephalus przewalskii (Reptilia, Squamata, Agamidae), a novel gene organization in vertebrate mtDNA.

PubMed

Li, Donghai; Song, Sen; Chen, Tao; Zhang, Caihong; Chang, Cheng

2015-01-01

The mitogenome of the desert toad-headed agama, Phrynocephalus przewalskii, was amplified using polymerase chain reaction (PCR), long-and-accurate PCR and directly sequenced by primer walking. The complete mitogenome was 16,892 bp in size and contained 13 protein-coding, 23 tRNA, and 2 rRNA genes, and 1 control region. The mitogenome of the P. przewalskii was similar to those of other Phrynocephalus sand lizards in gene arrangement and composition, except that tRNA-Phe and tRNA-Pro were exchanged and tRNA-Phe had two copies. The control region comprised three parts, one between tRNA-Thr and tRNA-Phe, a second between the two tRNA-Phe copies, and a third between tRNA-Pro and 12S RNA. The overall nucleotide composition of the H-strand was 36.3% A, 26.7% T, 12.5% G, 24.6% C. The complete mitogenome of P. przewalskii will contribute to understanding the evolution of the genus Phrynocephalus and the family Agamidae.

Quantitative microbiological study of human carious dentine by culture and real-time PCR: association of anaerobes with histopathological changes in chronic pulpitis.

PubMed

Martin, F Elizabeth; Nadkarni, Mangala A; Jacques, Nicholas A; Hunter, Neil

2002-05-01

The bacteria found in carious dentine were correlated with the tissue response of the dental pulps of 65 teeth extracted from patients with advanced caries and pulpitis. Standardized homogenates of carious dentine were plated onto selective and nonselective media under anaerobic and microaerophilic conditions. In addition, real-time PCR was used to quantify the recovery of anaerobic bacteria. Primers and fluorogenic probes were designed to detect the total anaerobic microbial load, the genera Prevotella and Fusobacterium, and the species Prevotella melaninogenica, Porphyromonas endodontalis, Porphyromonas gingivalis, and Micromonas (formerly Peptostreptococcus) micros. The pulpal pathology was categorized according to the cellular response and degenerative changes. Analysis of cultured bacteria showed a predominance of gram-positive microorganisms, particularly lactobacilli. Gram-negative bacteria were also present in significant numbers with Prevotella spp., the most numerous anaerobic group cultured. Real-time PCR analysis indicated a greater microbial load than that determined by colony counting. The total number of anaerobes detected was 41-fold greater by real-time PCR than by colony counting, while the numbers of Prevotella and Fusobacterium spp. detected were 82- and 2.4-fold greater by real-time PCR than by colony counting, respectively. Real-time PCR also identified M. micros, P. endodontalis, and P. gingivalis in 71, 60, and 52% of carious samples, respectively. Correlation matrices of the real-time PCR data revealed significant positive associations between M. micros and P. endodontalis detection and inflammatory degeneration of pulpal tissues. These anaerobes have been strongly implicated in endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of high levels of these bacteria in carious lesions may be indicative of irreversible pulpal pathology.

Transcriptional expression of type-I interferon response genes and stability of housekeeping genes in the human endometrium and endometriosis.

PubMed

Vestergaard, Anna L; Knudsen, Ulla B; Munk, Torben; Rosbach, Hanne; Martensen, Pia M

2011-04-01

Endometriosis is a painful chronic female disease defined by the presence of endometrial tissue implants in ectopic (Ec) locations. The pathogenesis is much debated, and type-I interferons (IFNs) could be involved. The expression of genes of the type-I IFN response were profiled by a specific PCR array of RNA obtained from Ec and eutopic (Eu) endometrium collected from nine endometriosis patients and nine healthy control women. Transcriptional expression levels of selected IFN-regulated and housekeeping genes (HKGs) were investigated by real-time quantitative reverse transcriptase PCR (qRT-PCR). Stably expressed HKGs for valid normalization of transcriptional studies of endometrium and endometriosis have not yet been published. Here, seven HKGs were evaluated for stability using the GeNorm and NormFinder software. A normalization factor based on HMBS, TBP and YWHAZ expression was suitable for normalization of qRT-PCR studies of Eu versus Ec endometrium. In the endometrial cell lines HEC1A, HEC1B, Ishikawa and RL95-2, HMBS and HPRT1 were the most stably expressed. The IFN-specific PCR array indicated significantly different expression of the genes BST2, COL16A1, HOXB2 and ISG20 between the endometrial tissue types. However, by correctly normalized qRT-PCR, levels of BST2, COL16A1 and the highly type-I IFN-stimulated genes ISG12A and 6-16 displayed insignificant variations. Conversely, HOXB2 and ISG20 transcriptions were significantly reduced in endometriosis lesions compared with endometrium from endometriosis patients and healthy controls. In conclusion, appropriate HKGs for normalization of qRT-PCR studies of endometrium and endometriosis have been identified here. Abolished expression of ISG20 and HOX genes could be important in endometriosis.

The clinical potential of Enhanced-ice-COLD-PCR.

PubMed

Tost, Jörg

2016-01-01

Enhanced-ice-COLD-PCR (E-ice-COLD-PCR) is a novel assay format that allows for the efficient enrichment and sensitive detection of all mutations in a region of interest using a chemically modified blocking oligonucleotide, which impedes the amplification of wild-type sequences. The assay is compatible with DNA extracted from tissue and cell-free circulating DNA. The main features of E-ice-COLD-PCR are the simplicity of the setup and the optimization of the assay, the use of standard laboratory equipment and the very short time to results (~4 h including DNA extraction, enrichment and sequence-based identification of mutations). E-ice-COLD-PCR is therefore a highly promising technology for a number of basic research as well as clinical applications including detection of clinically relevant mutated subclones and monitoring of treatment response or disease recurrence.

Detection of methicillin-resistant and methicillin-susceptible Staphylococcus aureus colonization of healthy military personnel by traditional culture, PCR, and mass spectrometry.

PubMed

Shaw, Ashley G; Vento, Todd J; Mende, Katrin; Kreft, Rachael E; Ehrlich, Garth D; Wenke, Joseph C; Spirk, Tracy; Landrum, Michael L; Zera, Wendy; Cheatle, Kristelle A; Guymon, Charles; Calvano, Tatjana P; Rini, Elizabeth A; Tully, Charla C; Beckius, Miriam L; Murray, Clinton K

2013-10-01

Methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus colonization is associated with increased rates of infection. Rapid and reliable detection methods are needed to identify colonization of nares and extra-nare sites, particularly given recent reports of oropharynx-only colonization. Detection methods for MRSA/MSSA colonization include culture, PCR, and novel methods such as PCR/electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). We evaluated 101 healthy military members for S. aureus colonization in the nares, oropharynx, axilla, and groin, using CHROMagar S. aureus medium and Xpert SA Nasal Complete PCR for MRSA/MSSA detection. The same subjects were screened in the nares, oropharynx, and groin using PCR/ESI-TOF-MS. By culture, 3 subjects were MRSA-colonized (all oropharynx) and 34 subjects were MSSA-colonized (all 4 sites). PCR detected oropharyngeal MRSA in 2 subjects, which correlated with culture findings. By PCR, 47 subjects were MSSA-colonized (all 4 sites); however, 43 axillary samples were invalid, 39 of which were associated with deodorant/anti-perspirant use (93%, p < 0.01). By PCR/ESI-TOF-MS, 4 subjects were MRSA-colonized, 2 in the nares and 2 in the oropharynx; however, neither of these correlated with positive MRSA cultures. Twenty-eight subjects had MSSA by PCR/ESI-TOF-MS, and 41 were found to have possible MRSA (S. aureus with mecA and coagulase-negative Staphylococcus (CoNS)). The overall 3% MRSA colonization rate is consistent with historical reports, but the oropharynx-only colonization supports more recent findings. In addition, the use of deodorant/anti-perspirant invalidated axillary PCR samples, limiting its utility. Defining MRSA positivity by PCR/ESI-TOF-MS is complicated by co-colonization of S. aureus with CoNS, which can also carry mecA.

Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction

PubMed Central

Kulkarni, Raghavendra D.; Mishra, Mukti Nath; Mohanraj, Jeevanandam; Chandrasekhar, Arun; Ajantha, G. S.; Kulkani, Sheetal; Bhat, Shama

2018-01-01

BACKGROUND: Nosocomial infections are often caused by multidrug-resistant bacteria and the incidence is increasing. Acinetobacter, a Gram-negative bacillus, is commonly associated with the use of intravascular catheterization and airway intubation. Polymerase chain reaction (PCR) for identification of Acinetobacter baumannii from samples has been standardized that use conventional wet-reagent mix. We have designed and optimized a dry-reagent mix for identification of Acinetobacter species by PCR. The dry-reagent mix can be stored at room temperature, has less chances of contamination, and thus can be used at point-of-care diagnosis. AIM AND OBJECTIVE: The present work was focused on comparing the sensitivity and specificity of dry-reagent PCR mix over conventional wet-reagent PCR mix for identification of Acinetobacter species. MATERIALS AND METHODS: Conventional wet-reagent mix based and dry-reagent mix based PCR were carried out for the DNA isolated from Acinetobacter species. The latter was also applied directly on bacterial growth without prior DNA extraction process. Equal numbers of bacterial isolates other than Acinetobacter species were also subjected to identification by the same protocols for determining the sensitivity and specificity of the test. RESULTS: The Acinetobacter species showed amplification of the target rpoB gene and the band was observed at 397 bp. The dry-reagent PCR mix results matched completely with the conventional wet-reagent PCR mix assay. All the non-Acinetobacter isolates were negative for the PCR. This indicates that the test is highly specific. The dry-reagent mix also contained an enzyme resistant to PCR inhibitors and capable of amplifying DNA directly from cells. CONCLUSION: Performance of dry-reagent PCR mix without the need for DNA extraction and preparation of a PCR mix proved to be more sensitive and reduce the handling error, minimizes the time, manual work, and skilled labor. PMID:29403209

Long-PCR based next generation sequencing of the whole mitochondrial genome of the peacock skate Pavoraja nitida (Elasmobranchii: Arhynchobatidae).

PubMed

Yang, Lei; Naylor, Gavin J P

2016-01-01

We determined the complete mitochondrial genome sequence (16,760 bp) of the peacock skate Pavoraja nitida using a long-PCR based next generation sequencing method. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement. Primers, protocols, and procedures used to obtain this mitogenome are provided. We anticipate that this approach will facilitate rapid collection of mitogenome sequences for studies on phylogenetic relationships, population genetics, and conservation of cartilaginous fishes.

Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome.

PubMed

Badri, Amine; Stefani, Franck O P; Lachance, Geneviève; Roy-Arcand, Line; Beaudet, Denis; Vialle, Agathe; Hijri, Mohamed

2016-10-01

Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172 ± 304 SE (n = 6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.

Development of a direct PCR assay to detect Taenia multiceps eggs isolated from dog feces.

PubMed

Wang, Ning; Wang, Yu; Ye, Qinghua; Yang, Yingdong; Wan, Jie; Guo, Cheng; Zhan, Jiafei; Gu, Xiaobin; Lai, Weimin; Xie, Yue; Peng, Xuerong; Yang, Guangyou

2018-02-15

Taenia multiceps is a tapeworm that leads to the death of livestock, resulting in major economic losses worldwide. The adult stage of this parasite invades the small intestine of dogs and other canids. In the present study, we developed a direct PCR assay to detect T. multiceps eggs isolated from dog feces to help curb further outbreaks. The genomic DNA was rapidly released using a lysis buffer and the PCR reaction was developed to amplify a 433-bp fragment of the T. multiceps mitochondrial gene encoding NADH dehydrogenase subunit 5 (nad5) from eggs isolated from dog feces. The procedure could be completed within 3 h, including flotation. The sensitivity of the assay was determined by detecting DNA from defined numbers of eggs, and the specificity was determined by detecting DNA from other intestinal tapeworm and roundworm species that commonly infect dogs. In addition, 14 taeniid-positive fecal samples determined by the flotation technique were collected and further evaluated by the regular PCR and our direct PCR. The results showed that the direct PCR developed herein was sensitive enough to detect the DNA from as few as 10 T. multiceps eggs and that no cross-reactions with other tapeworm and roundworm were observed, suggesting its high sensitivity and specificity for T. multiceps detection. Moreover, 14 taeniid-positive samples were screened by the regular PCR and direct PCR, with detection rates of 78.6% and 85.7%, respectively. In conclusion, the direct PCR assay developed in the present study has high sensitivity and specificity to identify T. multiceps eggs isolated from dog feces and therefore could represent an invaluable tool to identify T. multiceps outbreaks and would contribute to future clinical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients.

PubMed

Caliendo, A M; St George, K; Kao, S Y; Allega, J; Tan, B H; LaFontaine, R; Bui, L; Rinaldo, C R

2000-06-01

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.

Comparison of Quantitative Cytomegalovirus (CMV) PCR in Plasma and CMV Antigenemia Assay: Clinical Utility of the Prototype AMPLICOR CMV MONITOR Test in Transplant Recipients

PubMed Central

Caliendo, Angela M.; St. George, Kirsten; Kao, Shaw-Yi; Allega, Jessica; Tan, Ban-Hock; LaFontaine, Robert; Bui, Larry; Rinaldo, Charles R.

2000-01-01

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients. PMID:10834964

RNAi-induced silencing of embryonic tryptophan oxygenase in the Pyralid moth, Plodia interpunctella

PubMed Central

Fabrick, Jeffrey A.; Kanost, Michael R.; Baker, James E.

2004-01-01

Gene silencing through the introduction of double-stranded RNA (RNA interference, RNAi) provides a powerful tool for the elucidation of gene function in many systems, including those where genomics and proteomics are incomplete. The use of RNAi technology for gene silencing in Lepidoptera has lacked significant attention compared to other systems. To demonstrate that RNAi can be utilized in the lepidopteran, Plodia interpunctella, we cloned a cDNA for tryptophan oxygenase, and showed that silencing of tryptophan oxygenase through RNAi during embryonic development resulted in loss of eye-color pigmentation. The complete amino acid sequence of Plodia tryptophan oxygenase can be accessed through NCBI Protein Database under NCBI Accession # AY427951. Abbreviation RNAi RNA interference PCR polymerase chain reaction RT-PCR reverse transcription-PCR PMID:15861231

Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias.

PubMed

Clarke, Laurence J; Soubrier, Julien; Weyrich, Laura S; Cooper, Alan

2014-11-01

Studies of insect assemblages are suited to the simultaneous DNA-based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR-amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR-amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR-amplified the blend using five primer sets, targeting either COI or 16S, with high-throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers. © 2014 John Wiley & Sons Ltd.

Developmental and genetic toxicity of stannous chloride in mouse dams and fetuses.

PubMed

El-Makawy, Aida I; Girgis, Shenouda M; Khalil, Wagdy K B

2008-12-08

Humans are exposed to stannous chloride (SnCl2) present in packaged food, soft drinks, biocides, dentifrices, etc. Health effects in children exposed to tin and tin compounds have not been investigated yet. Therefore, we evaluated the possible teratogenic and genotoxic effects of SnCl2 in pregnant female mice and their fetuses. Teratogenic effects including morphological malformation of the fetus and its skeleton were observed. Exposures to environmental stressors including toxic chemicals that have the potential of modulating the immune system can often be linked to ecologically relevant endpoints, such as reduced resistance to disease. Therefore, the semi-quantitative reverse-transcription PCR (RT-PCR) assay was used to evaluate the expression of immune-response genes in the liver of SnCl2-treated dams and their fetuses. Bone-marrow cells of dams and fetuses were investigated for the presence of aberrant chromosomes. Three oral doses of SnCl2 (2, 10 and 20 mg/kg bw) were tested. The results of the teratological study show that SnCl2 induced a significant decrease in the number of living fetuses and a significant increase in the number of post-implantation losses. The high dose of SnCl2 induced complete post-implantation loss. Furthermore, SnCl2 caused reduction in the ossification of the fetal skeleton. The RT-PCR assay showed that the immune-response genes GARP and SIMP were not expressed in the liver of dams and fetuses in the controls or in the group treated with SnCl2 at 2 mg/kg bw. However, the expression of these genes was up-regulated in the groups treated with the other doses of SnCl2. Regarding the chromosome analysis, SnCl2 induced a dose-dependent increase in the frequency of individual and total chromosomal aberrations (P

Seroprevalence of occult hepatitis B among Egyptian paediatric hepatitis C cancer patients.

PubMed

Raouf, H E; Yassin, A S; Megahed, S A; Ashour, M S; Mansour, T M

2015-02-01

Occult hepatitis B infection is characterized by the presence of hepatitis B virus (HBV) DNA in the serum in the absence of hepatitis B surface antigen (HBsAg). Prevalence of hepatitis C virus (HCV) infections in Egypt is among the highest in the world. In this study, we aim at analysing the rates of occult HBV infections among HCV paediatric cancer patients in Egypt. The prevalence of occult HBV was assessed in two groups of paediatric cancer patients (HCV positive and HCV negative), in addition to a third group of paediatric noncancer patients, which was used as a general control. All groups were negative for HBsAg and positive for HCV antibody. HBV DNA was detected by nested PCR and real-time PCR. HCV was detected by real-time PCR. Sequencing was carried out in order to determine HBV genotypes to all HBV patients as well as to detect any mutation that might be responsible for the occult phenotype. Occult hepatitis B infection was observed in neither the non-HCV paediatric cancer patients nor the paediatric noncancer patients but was found in 31% of the HCV-positive paediatric cancer patients. All the detected HBV patients belonged to HBV genotype D, and mutations were found in the surface genome of HBV leading to occult HBV. Occult HBV infection seems to be relatively frequent in HCV-positive paediatric cancer patients, indicating that HBsAg negativity is not sufficient to completely exclude HBV infection. These findings emphasize the importance of considering occult HBV infection in HCV-positive paediatric cancer patients especially in endemic areas as Egypt. © 2014 John Wiley & Sons Ltd.

Highly personalized detection of minimal Ewing sarcoma disease burden from plasma tumor DNA.

PubMed

Hayashi, Masanori; Chu, David; Meyer, Christian F; Llosa, Nicolas J; McCarty, Gregory; Morris, Carol D; Levin, Adam S; Wolinsky, Jean-Paul; Albert, Catherine M; Steppan, Diana A; Park, Ben Ho; Loeb, David M

2016-10-01

Even though virtually all patients with Ewing sarcoma achieve a radiographic complete response, up to 30% of patients who present with localized disease and up to 90% of those who present with metastases experience a metastatic disease recurrence, highlighting the inability to identify patients with residual disease at the end of therapy. Up to 95% of Ewing sarcomas carry a driving EWS-ETS translocation that has an intronic breakpoint that is specific to each tumor, and the authors developed a system to quantitatively detect the specific breakpoint DNA fragment in patient plasma. The authors used a long-range multiplex polymerase chain reaction (PCR) technique to identify tumor-specific EWS-ETS breakpoints in Ewing sarcoma cell lines, patient-derived xenografts, and patient tumors, and this sequence was used to design tumor-specific primer sets to detect plasma tumor DNA (ptDNA) by droplet digital PCR in xenograft-bearing mice and patients. Tumor-specific breakpoint DNA fragments were detected in the plasma of xenograft-bearing mice, and the signal correlated with tumor burden during primary tumor growth, after surgical resection, and at the time of metastatic disease recurrence. Furthermore, the authors were able to detect the specific breakpoint in plasma DNA obtained from 3 patients with Ewing sarcoma and in 2 patients the authors were able to detect ptDNA when there was radiographically undetectable disease present. The use of droplet digital PCR to detect tumor-specific EWS-ETS fusion gene breakpoint ptDNA fragments can be developed into a highly personalized biomarker of disease recurrence that can be optimized in animal studies for ultimate use in patients. Cancer 2016;122:3015-3023. © 2016 American Cancer Society. © 2016 American Cancer Society.

Outbreak of H3N2 influenza at a US military base in Djibouti during the H1N1 pandemic of 2009.

PubMed

Cosby, Michael T; Pimentel, Guillermo; Nevin, Remington L; Fouad Ahmed, Salwa; Klena, John D; Amir, Ehab; Younan, Mary; Browning, Robert; Sebeny, Peter J

2013-01-01

Influenza pandemics have significant operational impact on deployed military personnel working in areas throughout the world. The US Department of Defense global influenza-like illness (ILI) surveillance network serves an important role in establishing baseline trends and can be leveraged to respond to outbreaks of respiratory illness. We identified and characterized an operationally unique outbreak of H3N2 influenza at Camp Lemonnier, Djibouti occurring simultaneously with the H1N1 pandemic of 2009 [A(H1N1)pdm09]. Enhanced surveillance for ILI was conducted at Camp Lemonnier in response to local reports of a possible outbreak during the A(H1N1)pdm09 pandemic. Samples were collected from consenting patients presenting with ILI (utilizing a modified case definition) and who completed a case report form. Samples were cultured and analyzed using standard real-time reverse transcriptase PCR (rt-RT-PCR) methodology and sequenced genetic material was phylogenetically compared to other published strains. rt-RT-PCR and DNA sequencing revealed that 25 (78%) of the 32 clinical samples collected were seasonal H3N2 and only 2 (6%) were A(H1N1)pdm09 influenza. The highest incidence of H3N2 occurred during the month of May and 80% of these were active duty military personnel. Phylogenetic analysis revealed that sequenced H3N2 strains were genetically similar to 2009 strains from the United States of America, Australia, and South east Asia. This outbreak highlights challenges in the investigation of influenza among deployed military populations and corroborates the public health importance of maintaining surveillance systems for ILI that can be enhanced locally when needed.

Development of a PCR-based marker utilizing a deletion mutation in the dihydroflavonol 4-reductase (DFR) gene responsible for the lack of anthocyanin production in yellow onions (Allium cepa).

PubMed

Kim, Sunggil; Yoo, Kil Sun; Pike, Leonard M

2005-02-01

Bulb color in onions (Allium cepa) is an important trait, but the mechanism of color inheritance is poorly understood at the molecular level. A previous study showed that inactivation of the dihydroflavonol 4-reductase (DFR) gene at the transcriptional level resulted in a lack of anthocyanin production in yellow onions. The objectives of the present study were the identification of the critical mutations in the DFR gene (DFR-A) and the development of a PCR-based marker for allelic selection. We report the isolation of two additional DFR homologs (DFR-B and DFR-C). No unique sequences were identified in either DFR homolog, even in the untranslated region (UTR). Both genes shared more than 95% nucleotide sequence identity with the DFR-A gene. To obtain a unique sequence from each gene, we isolated the promoter regions. Sequences of the DFR-A and DFR-B promoters differed completely from one another, except for an approximately 100-bp sequence adjacent to the 5'UTR. It was possible to specifically amplify only the DFR-A gene using primers designed to anneal to the unique promoter region. The sequences of yellow and red DFR-A alleles were the same except for a single base-pair change in the promoter and an approximately 800-bp deletion within the 3' region of the yellow DFR-A allele. This deletion was used to develop a co-dominant PCR-based marker that segregated perfectly with color phenotypes in the F2 population. These results indicate that a deletion mutation in the yellow DFR-A gene results in the lack of anthocyanin production in yellow onions.

Outbreak of H3N2 Influenza at a US Military Base in Djibouti during the H1N1 Pandemic of 2009

PubMed Central

Cosby, Michael T.; Pimentel, Guillermo; Nevin, Remington L.; Fouad Ahmed, Salwa; Klena, John D.; Amir, Ehab; Younan, Mary; Browning, Robert; Sebeny, Peter J.

2013-01-01

Background Influenza pandemics have significant operational impact on deployed military personnel working in areas throughout the world. The US Department of Defense global influenza-like illness (ILI) surveillance network serves an important role in establishing baseline trends and can be leveraged to respond to outbreaks of respiratory illness. Objective We identified and characterized an operationally unique outbreak of H3N2 influenza at Camp Lemonnier, Djibouti occurring simultaneously with the H1N1 pandemic of 2009 [A(H1N1)pdm09]. Methods Enhanced surveillance for ILI was conducted at Camp Lemonnier in response to local reports of a possible outbreak during the A(H1N1)pdm09 pandemic. Samples were collected from consenting patients presenting with ILI (utilizing a modified case definition) and who completed a case report form. Samples were cultured and analyzed using standard real-time reverse transcriptase PCR (rt-RT-PCR) methodology and sequenced genetic material was phylogenetically compared to other published strains. Results rt-RT-PCR and DNA sequencing revealed that 25 (78%) of the 32 clinical samples collected were seasonal H3N2 and only 2 (6%) were A(H1N1)pdm09 influenza. The highest incidence of H3N2 occurred during the month of May and 80% of these were active duty military personnel. Phylogenetic analysis revealed that sequenced H3N2 strains were genetically similar to 2009 strains from the United States of America, Australia, and South east Asia. Conclusions This outbreak highlights challenges in the investigation of influenza among deployed military populations and corroborates the public health importance of maintaining surveillance systems for ILI that can be enhanced locally when needed. PMID:24339995

Evaluation of nifurtimox treatment of chronic Chagas disease by means of several parasitological methods.

PubMed

Muñoz, Catalina; Zulantay, Inés; Apt, Werner; Ortiz, Sylvia; Schijman, Alejandro G; Bisio, Margarita; Ferrada, Valentina; Herrera, Cinthya; Martínez, Gabriela; Solari, Aldo

2013-09-01

Currently, evaluation of drug efficacy for Chagas disease remains a controversial issue with no consensus. In this work, we evaluated the parasitological efficacy of Nifurtimox treatment in 21 women with chronic Chagas disease from an area of endemicity in Chile who were treated according to current protocols. Under pre- and posttherapy conditions, blood (B) samples and xenodiagnosis (XD) samples from these patients were subjected to analysis by real-time PCR targeting the nuclear satellite DNA of Trypanosoma cruzi (Sat DNA PCR-B, Sat DNA PCR-XD) and by PCR targeting the minicircle of kinetoplast DNA of T. cruzi (kDNA PCR-B, kDNA PCR-XD) and by T. cruzi genotyping using hybridization minicircle tests in blood and fecal samples of Triatoma infestans feed by XD. In pretherapy, kDNA PCR-B and kDNA PCR-XD detected T. cruzi in 12 (57%) and 18 (86%) cases, respectively, whereas Sat DNA quantitative PCR-B (qPCR-B) and Sat DNA qPCR-XD were positive in 18 cases (86%) each. Regarding T. cruzi genotype analysis, it was possible to observe in pretherapy the combination of TcI, TcII, and TcV lineages, including mixtures of T. cruzi strains in most of the cases. At 13 months posttherapy, T. cruzi DNA was detectable in 6 cases (29.6%) and 4 cases (19.1%) by means of Sat DNA PCR-XD and kDNA PCR-XD, respectively, indicating treatment failure with recovery of live parasites refractory to chemotherapy. In 3 cases, it was possible to identify persistence of the baseline genotypes. The remaining 15 baseline PCR-positive cases gave negative results by all molecular and parasitological methods at 13 months posttreatment, suggesting parasite response. Within this follow-up period, kDNA PCR-XD and Sat DNA qPCR-XD proved to be more sensitive tools for the parasitological evaluation of the efficacy of Nifurtimox treatment than the corresponding PCR methods performed directly from blood samples.

Evaluation of Nifurtimox Treatment of Chronic Chagas Disease by Means of Several Parasitological Methods

PubMed Central

Muñoz, Catalina; Zulantay, Inés; Apt, Werner; Ortiz, Sylvia; Schijman, Alejandro G.; Bisio, Margarita; Ferrada, Valentina; Herrera, Cinthya; Martínez, Gabriela

2013-01-01

Currently, evaluation of drug efficacy for Chagas disease remains a controversial issue with no consensus. In this work, we evaluated the parasitological efficacy of Nifurtimox treatment in 21 women with chronic Chagas disease from an area of endemicity in Chile who were treated according to current protocols. Under pre- and posttherapy conditions, blood (B) samples and xenodiagnosis (XD) samples from these patients were subjected to analysis by real-time PCR targeting the nuclear satellite DNA of Trypanosoma cruzi (Sat DNA PCR-B, Sat DNA PCR-XD) and by PCR targeting the minicircle of kinetoplast DNA of T. cruzi (kDNA PCR-B, kDNA PCR-XD) and by T. cruzi genotyping using hybridization minicircle tests in blood and fecal samples of Triatoma infestans feed by XD. In pretherapy, kDNA PCR-B and kDNA PCR-XD detected T. cruzi in 12 (57%) and 18 (86%) cases, respectively, whereas Sat DNA quantitative PCR-B (qPCR-B) and Sat DNA qPCR-XD were positive in 18 cases (86%) each. Regarding T. cruzi genotype analysis, it was possible to observe in pretherapy the combination of TcI, TcII, and TcV lineages, including mixtures of T. cruzi strains in most of the cases. At 13 months posttherapy, T. cruzi DNA was detectable in 6 cases (29.6%) and 4 cases (19.1%) by means of Sat DNA PCR-XD and kDNA PCR-XD, respectively, indicating treatment failure with recovery of live parasites refractory to chemotherapy. In 3 cases, it was possible to identify persistence of the baseline genotypes. The remaining 15 baseline PCR-positive cases gave negative results by all molecular and parasitological methods at 13 months posttreatment, suggesting parasite response. Within this follow-up period, kDNA PCR-XD and Sat DNA qPCR-XD proved to be more sensitive tools for the parasitological evaluation of the efficacy of Nifurtimox treatment than the corresponding PCR methods performed directly from blood samples. PMID:23836179

Complete mitochondrial genome and taxonomic revision of Cardiodactylus muiri Otte, 2007 (Gryllidae: Eneopterinae: Lebinthini).

PubMed

Dong, Jiajia; Vicente, Natallia; Chintauan-Marquier, Ioana C; Ramadi, Cahyo; Dettai, Agnès; Robillard, Tony

2017-05-15

In the present study, we report the high-coverage complete mitochondrial genome (mitogenome) of the cricket Cardiodactylus muiri Otte, 2007. The mitogenome was sequenced using a long-PCR approach on an Ion Torrent Personal Genome Machine (PGM) for next generation sequencing technology. The total length of the amplified mitogenome is 16,328 bp, representing 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one noncoding region (D-loop region). The new sets of long-PCR primers reported here are invaluable resources for future comparative evolutionary genomic studies in Orthopteran insects. The new mitogenome sequence is compared with published cricket mitogenomes. In the taxonomic part, we present new records for the species and describe life-history traits, habitat and male calling song of the species; based on observation of new material, the species Cardiodactylus buru Gorochov & Robillard, 2014 is synonymized under C. muiri.

[Inverse PCR amplification of the complete major capsid protein gene of lymphocystis disease virus isolated from Rachycentron canadum and the phylogenetic analysis of the virus].

PubMed

Fu, Xiao-Zhe; Shi, Cun-Bin; Li, Ning-Qiu; Pan, Hou-Jun; Chang, Ou-Qin; Wu, Shu-Qin

2007-09-01

The major capsid protein of lymphocystis disease virus isolated from Rachycentron canadum (LCDV-rc) was amplified and analysed. The 457bp DNA core fragment was amplified with the degenerate primers designed according to the conserved sequences of MCP gene of iridoviruses, then the flaking sequences adjacent to the core region were amplified by inverse PCR, and the complete sequence was obtained by combining all of them. The open reading frame of the gene is 1380bp in length, encoding a putative protein of 459 aa with molecular weight 51.12 kD and pI 6.87. Constructing the phylogenetic tree for comparing the MCP amino acid of iridoviruses, the results indicated that LCDV-rc is most homologous to the other Lymphocystis viruses and all of them constitute a branch. Accordingly LCDV-rc is identified as Lymphocystivirus.

Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples

PubMed Central

Bushell, Claire A.; Grant, Paul R.; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M.; Žel, Jana; Milavec, Mojca; Foy, Carole A.; Nastouli, Eleni; Garson, Jeremy A.; Huggett, Jim F.

2015-01-01

Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening. PMID:26659206

Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

PubMed

Whale, Alexandra S; Bushell, Claire A; Grant, Paul R; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M; Žel, Jana; Milavec, Mojca; Foy, Carole A; Nastouli, Eleni; Garson, Jeremy A; Huggett, Jim F

2016-02-01

Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening. Copyright © 2016 Whale et al.

Molecular characterization of African orthobunyaviruses.

PubMed

Yandoko, E Nakouné; Gribaldo, S; Finance, C; Le Faou, A; Rihn, B H

2007-06-01

The genus Orthobunyavirus is composed of segmented, negative-sense RNA viruses that are responsible for mild to severe human diseases. To date, no molecular studies of bunyaviruses in the genus Orthobunyavirus from central Africa have been reported, and their classification relies on serological testing. Four new primer pairs for RT-PCR amplification and sequencing of the complete genomic small (S) RNA segments of 10 orthobunyaviruses isolated from the Central African Republic and pertaining to five different serogroups have been designed and evaluated. Phylogenetic analysis showed that these 10 viruses belong to the Bunyamwera serogroup. The S segment sequences differ from those of the Bunyamwera virus reference strain by 5-15 % at the nucleotide level, and both overlapping reading frames, encoding the nucleocapsid (N) and non-structural (NS) proteins, were evident in sequenced genomes. This study should improve diagnosis and surveillance of African bunyaviruses.

STAT3 Mediates Nilotinib Response in KIT-Altered Melanoma: A Phase II Multicenter Trial of the French Skin Cancer Network.

PubMed

Delyon, Julie; Chevret, Sylvie; Jouary, Thomas; Dalac, Sophie; Dalle, Stephane; Guillot, Bernard; Arnault, Jean-Philippe; Avril, Marie-Françoise; Bedane, Christophe; Bens, Guido; Pham-Ledard, Anne; Mansard, Sandrine; Grange, Florent; Machet, Laurent; Meyer, Nicolas; Legoupil, Delphine; Saiag, Philippe; Idir, Zakia; Renault, Victor; Deleuze, Jean-François; Hindie, Elif; Battistella, Maxime; Dumaz, Nicolas; Mourah, Samia; Lebbe, Celeste

2018-01-01

Mutated oncogenic KIT is a therapeutic target in melanoma. We conducted a multicenter phase II trial on the KIT inhibitor nilotinib in patients with unresectable melanoma harboring KIT alteration. The primary endpoint was the response rate (complete response or partial response following Response Evaluation Criteria in Solid Tumors criteria) at 6 months. Pharmacodynamic studies using KIT sequencing, qPCR array, and immunostaining of downstream KIT effectors were performed during treatment. Twenty-five patients were included and received 400 mg oral nilotinib twice daily. At 6 months, nilotinib induced tumor response in four patients. The best overall response rate was 20% and the disease control rate was 56%, limited to patients harboring exon 11 or 13 mutations. Four patients exhibited durable response, including three persisting (3.6 and 2.8 years for two patients with stage IIIC and 2.5 years for one with IVM1b melanoma). A reduction in signal transducer and activator of transcription (STAT) 3 phosphorylation and its effectors (BCL-2, MCL-1) in tumors during follow-up was significantly associated with clinical response. In the KIT-mutated melanoma cell line M230, nilotinib reduced STAT3 signaling and STAT inhibitors were as efficient as KIT inhibitors in reducing cell proliferation. Our study evidences a significant association between STAT3 inhibition and response to nilotinib, and provides a rationale for future research assessing STAT inhibitors in KIT-mutated melanoma. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Virtual PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, S N; Clague, D S; Vandersall, J A

2006-02-23

The polymerase chain reaction (PCR) stands among the keystone technologies for analysis of biological sequence data. PCR is used to amplify DNA, to generate many copies from as little as a single template. This is essential, for example, in processing forensic DNA samples, pathogen detection in clinical or biothreat surveillance applications, and medical genotyping for diagnosis and treatment of disease. It is used in virtually every laboratory doing molecular, cellular, genetic, ecologic, forensic, or medical research. Despite its ubiquity, we lack the precise predictive capability that would enable detailed optimization of PCR reaction dynamics. In this LDRD, we proposed tomore » develop Virtual PCR (VPCR) software, a computational method to model the kinetic, thermodynamic, and biological processes of PCR reactions. Given a successful completion, these tools will allow us to predict both the sequences and concentrations of all species that are amplified during PCR. The ability to answer the following questions will allow us both to optimize the PCR process and interpret the PCR results: What products are amplified when sequence mixtures are present, containing multiple, closely related targets and multiplexed primers, which may hybridize with sequence mismatches? What are the effects of time, temperature, and DNA concentrations on the concentrations of products? A better understanding of these issues will improve the design and interpretation of PCR reactions. The status of the VPCR project after 1.5 years of funding is consistent with the goals of the overall project which was scoped for 3 years of funding. At half way through the projected timeline of the project we have an early beta version of the VPCR code. We have begun investigating means to improve the robustness of the code, performed preliminary experiments to test the code and begun drafting manuscripts for publication. Although an experimental protocol for testing the code was developed, the preliminary experiments were tainted by contaminated products received from the manufacturer. Much knowledge has been gained in the development of the code thus far, but without final debugging, increasing its robustness and verifying it against experimental results, the papers which we have drafted to share our findings still require the final data necessary for publication. The following sections summarize our final progress on VPCR as it stands after 1.5 years of effort on an ambitious project scoped for a 3 year period. We have additional details of the methods than are provided here, but would like to have legal protection in place before releasing them. The result of this project, a suite of programs that predict PCR products as a function of reaction conditions and sequences, will be used to address outstanding questions in pathogen detection and forensics at LLNL. VPCR should enable scientists to optimize PCR protocols in terms of time, temperature, ion concentration, and primer sequences and concentrations, and to estimate products and error rates in advance of performing experiments. Our proposed capabilities are well ahead of all currently available technologies, which do not model non-equilibrium kinetics, polymerase extension, or predict multiple or undesired PCR products. We are currently seeking DHS funding to complete the project, at which time licensing opportunities will be explored, an updated patent application will be prepared, and a publication will be submitted. A provisional and a full patent application have already been filed (1).« less

An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research

PubMed Central

Yan, Xu; Bishop, David J.

2018-01-01

Gene expression analysis by quantitative PCR in skeletal muscle is routine in exercise studies. The reproducibility and reliability of the data fundamentally depend on how the experiments are performed and interpreted. Despite the popularity of the assay, there is a considerable variation in experimental protocols and data analyses from different laboratories, and there is a lack of consistency of proper quality control steps throughout the assay. In this study, we present a number of experiments on various steps of quantitative PCR workflow, and demonstrate how to perform a quantitative PCR experiment with human skeletal muscle samples in an exercise study. We also tested some common mistakes in performing qPCR. Interestingly, we found that mishandling of muscle for a short time span (10 mins) before RNA extraction did not affect RNA quality, and isolated total RNA was preserved for up to one week at room temperature. Demonstrated by our data, use of unstable reference genes lead to substantial differences in the final results. Alternatively, cDNA content can be used for data normalisation; however, complete removal of RNA from cDNA samples is essential for obtaining accurate cDNA content. PMID:29746477

PCR synthesis of double stranded DNA labeled with 5-bromouridine. A step towards finding a bromonucleoside for clinical trials.

PubMed

Michalska, Barbara; Sobolewski, Ireneusz; Polska, Katarzyna; Zielonka, Justyna; Zylicz-Stachula, Agnieszka; Skowron, Piotr; Rak, Janusz

2011-12-05

Incorporation of 5-bromouridine (5BrdU) into DNA makes it sensitive to UV and ionizing radiation, which opens up a prospective route for the clinical usage of 5-bromouridine and other halonucleosides. In the present work the polymerase chain reaction (PCR) protocol, which enables a long DNA fragment (resembling DNA synthesized in the cell in the presence of halonucleosides) to be completely substituted with 5BrdU, was optimized. Using HPLC coupled to enzymatic digestion, it was demonstrated that the actual amounts of native nucleosides and 5BrdU correspond very well to those calculated from the sequence of PCR products. The synthesized DNA is photosensitive to photons of 300nm. HPLC analysis demonstrated that the photolysis of labeled PCR products leads to a significant decrease in the 5BrdU signal and the simultaneous occurrence of a uridine peak. Agarose and polyacrylamide gel electrophoresis suggest that single strand breaks and cross-links are formed as a result of UV irradiation. The PCR protocol described in the current paper may be employed for labeling DNA not only with BrdU but also with other halonucleosides. Copyright © 2011 Elsevier B.V. All rights reserved.

Quantitative PCR monitoring of the effect of azoxystrobin treatments on Mycosphaerella graminicola epidemics in the field.

PubMed

Rohel, Eric A; Laurent, Paul; Fraaije, Bart A; Cavelier, Nadine; Hollomon, Derek W

2002-03-01

Quantitative PCR and visual monitoring of Mycosphaerella graminicola epidemics were performed to investigate the effect of curative and preventative applications of azoxystrobin in wheat field crops. A non-systemic protectant and a systemic curative fungicide, chlorothalonil and epoxiconazole, respectively, were used as references. PCR diagnosis detected leaf infection by M graminicola 3 weeks before symptom appearance, thereby allowing a clear distinction between curative and preventative treatments. When applied 1 week after the beginning of infection, azoxystrobin curative activity was intermediate between chlorothalonil (low effect) and epoxiconazole. When applied preventatively, none of the fungicides completely prevented leaf infection. There was some indication that azoxystrobin preventative treatments may delay fungal DNA increase more than epoxiconazole at the beginning of leaf infection. Both curative and preventative treatments increased the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Azoxystrobin only slightly decreased the speed of necrotic area increase compared with epoxiconazole. Hence, azoxystrobin activity toward M graminicola mainly resides in lengthening the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Information generated in this way is useful for optimal positioning of azoxystrobin treatments on M graminicola.

Performance of four HRP-2/pLDH combination rapid diagnostic tests and field microscopy as screening tests for malaria in pregnancy in Indonesia: a cross-sectional study.

PubMed

Ahmed, Rukhsana; Levy, Elvira I; Maratina, Sylvia S; de Jong, Judith J; Asih, Puji B S; Rozi, Ismail E; Hawley, William; Syafruddin, Din; ter Kuile, Feiko

2015-10-29

Malaria in pregnancy poses a major public health problem in Indonesia with an estimated six million pregnancies at risk of Plasmodium falciparum or Plasmodium vivax malaria annually. In 2010, Indonesia introduced a screen and treat policy for the control of malaria in pregnancy at first antenatal visit using microscopy or rapid diagnostic tests (RDTs). A diagnostic study was conducted in Sumba, Indonesia to compare the performance of four different RDTs in predominately asymptomatic pregnant women under field condition. Women were screened for malaria at antenatal visits using field microscopy and four HRP-2/pLDH combination RDTs (Carestart™, First-Response(®), Parascreen(®) and SD-Bioline(®)). The test results were compared with expert microscopy and nested PCR. End user experience of the RDTs in the field was assessed by questionnaire. Overall 950 were recruited and 98.7 % were asymptomatic. The prevalence of malaria was 3.0-3.4 % by RDTs, and 3.6, 5.0 and 6.6 % by field microscopy, expert microscopy and PCR, respectively. The geometric-mean parasite density was low (P. falciparum = 418, P. vivax = 147 parasites/µL). Compared with PCR, the overall sensitivity of the RDTs and field microscopy to detect any species was 24.6-31.1 %; specificities were >98.4 %. Relative to PCR, First-Response(®) had the best diagnostic accuracy (any species): sensitivity = 31.1 %, specificity = 98.9 % and diagnostic odds ratio = 39.0 (DOR). The DOR values for Carestart™, Parascreen(®), SD-Bioline(®), and field microscopy were 23.4, 23.7, 23.5 and 29.2, respectively. The sensitivity of Pan-pLDH bands to detect PCR confirmed P. vivax mono-infection were 8.6-13.0 %. The sensitivity of the HRP-2 band alone to detect PCR confirmed P. falciparum was 10.3-17.9 %. Pan-pLDH detected P. falciparum cases undetected by the HRP-2 band resulting in a better test performance when both bands were combined. First Response(®) was preferred by end-users for the overall practicality. The diagnostic accuracy to detect malaria among mostly asymptomatic pregnant women and perceived ease of use was slightly better with First-Response(®), but overall, differences between the four RDTs were small and performance comparable to field microscopy. Combination RDTs are a suitable alternative to field microscopy to screen for malaria in pregnancy in rural Indonesia. The clinical relevance of low density malaria infections detected by PCR, but undetected by RDTs or microscopy needs to be determined.

DEVELOPMENT OF AN ASTROVIRUS RT-PCR DETECTION ASSAY FOR USE WITH CONVENTIONAL, REAL-TIME, AND INTEGRATED CELL CULTURE/RT-PCR

EPA Science Inventory

Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed an...

Development of allele-specific primer PCR for a swine TLR2 SNP and comparison of the frequency among several pig breeds of Japan and the Czech Republic.

PubMed

Muneta, Yoshihiro; Minagawa, Yu; Kusumoto, Masahiro; Shinkai, Hiroki; Uenishi, Hirohide; Splichal, Igor

2012-05-01

In the present study, we have developed an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 2 (TLR2) (C406G), which is related to the prevalence of pneumonia caused by Mycoplasma hyopneumoniae. We also compared the allele frequency among several pig breeds of Japan and the Czech Republic. Allele-specific primers were constructed by introducing 1-base mismatch sequence before the SNP site. The swine TLR2 C406G mutation was successfully determined by the ASP-PCR using genomic DNA samples in Japan as previously genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from pig blood obtained from 110 pigs from 7 different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR were completely matched with the results from the sequencing method. The allele frequency of the swine TLR2 C406G mutation was 27.5% in the Czech Republic and 3.6% in Japan. The C406G mutation was only found in the Landrace breed in Japan, and was almost exclusively found in the Landrace breed in the Czech Republic as well. These results indicated the usefulness of ASP-PCR for detecting a specific SNP for swine TLR2.

Descriptive Biomarkers for Assessing Breast Cancer Risk

DTIC Science & Technology

2010-10-01

and we are making significant progress on Tasks 6 and 7. We completed methylation analyses of three genes (RASSF1, SFRP1 and GSTP1 ) on all samples...promoter hypermethylation; RASSF1, GSTP1 , SFRP1 12 karcaro@nre.umass.edu Arcaro, Kathleen F Annual Report...methylation analysis by pyrosequencing. PCR amplification and pyrosequencing has been completed for three genes, RASSF1, SFRP1 and GSTP1 and have

Actuation method and apparatus, micropump, and PCR enhancement method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ullakko, Kari; Mullner, Peter; Hampikian, Greg

An actuation apparatus includes at least one magnetic shape memory (MSM) element containing a material configured to expand and/or contract in response to exposure to a magnetic field. Among other things, the MSM element may be configured to pump fluid through a micropump by expanding and/or contracting in response to the magnetic field. The magnetic field may rotate about an axis of rotation and exhibit a distribution having a component substantially perpendicular to the axis of rotation. Further, the magnetic field distribution may include at least two components substantially orthogonal to one another lying in one or more planes perpendicularmore » to the axis of rotation. The at least one MSM element may contain nickel, manganese, and gallium. A polymerase chain reaction (PCR) may be enhanced by contacting a PCR reagent and DNA material with the MSM element.« less

Phase II trial of short-term neoadjuvant docetaxel and complete androgen blockade in high-risk prostate cancer

PubMed Central

Mellado, B; Font, A; Alcaraz, A; Aparicio, L A; Veiga, F J G; Areal, J; Gallardo, E; Hannaoui, N; Lorenzo, J R M; Sousa, A; Fernandez, P L; Gascon, P

2009-01-01

Background: The low probability of curing high-risk prostate cancer (PC) with local therapy suggests the need to study modality of therapeutic approaches. To this end, a prospective phase II trial of neoadjuvant docetaxel (D) and complete androgen blockade (CAB) was carried out in high-risk PC patients. The primary end point was to detect at least 10% of pCRs after chemohormonal treatment. Methods: Patients with T1c–T2 clinical stage with prostate-specific antigen (PSA) >20 ng ml−1 and/or Gleason score ⩾7 (4+3) and T3 were included. Treatment consisted of three cycles of D 36 mg m−2 on days 1, 8 and 15 every 28 days concomitant with CAB, followed by radical prostatectomy (RP). Results: A total of 57 patients were included. Clinical stage was T1c, 11 patients (19.3%); T2, 30 (52.6%) and T3, 16 (28%) patients. Gleason score was ⩾7 (4+3) in 44 (77%) patients and PSA >20 ng ml−1 in 15 (26%) patients. Treatment was well tolerated with 51 (89.9%) patients completing neoadjuvant therapy together with RP. The rate of pCR was 6% (three patients). Three (6%) additional patients had microscopic residual tumour (near pCR) in prostate specimen. With a median follow-up of 35 months, 18 (31.6%) patients presented PSA relapse. Conclusion: Short-term neoadjuvant D and CAB induced a 6% pCR rate, which is close to what would be expected with ADT alone. The combination was generally well tolerated. PMID:19755998

Nine year trends of dengue virus infection in Mumbai, Western India.

PubMed

Shastri, Jayanthi; Williamson, Manita; Vaidya, Nilima; Agrawal, Sachee; Shrivastav, Om

2017-01-01

Dengue virus (DENV) causes a wide range of diseases in humans, from acute febrile illness Dengue fever (DF) to life-threatening Dengue hemorrhagic fever (DHF) or Dengue shock syndrome (DSS). Factors believed to be responsible for spread of Dengue virus infection include explosive population growth, unplanned urban overpopulation with inadequate public health systems, poor standing water and vector control, climate changes and increased international recreational, business, military travel to endemic areas. All of these factors must be addressed to control the spread of Dengue and other mosquito-borne infections. The detection of Dengue virus RNA by reverse transcriptase PCR (RT-PCR) in human serum or plasma samples is highly indicative of acute Dengue fever. Moreover, the method is able to identify the Dengue virus serotype by demonstrating defined sequence homologies in the viral genomic RNA. During the nine year period of this study analysis, 6767 strongly suspected cases were tested by RT-PCR. 1685 (24.9%) were Dengue PCR positive and confirmed as Dengue cases. Observations on the seasonality were based on the nine year's data as the intensity of sampling was at its maximum during monsoon season. Dengue typing was done on 100 positive samples after storage of Dengue RNA at - 80°C. Dengue serotypes were detected in 69 samples of which Dengue 2 was most predominant. 576 samples were processed for NS1 antigen and PCR simultaneously. 19/576 were positive (3.3 %) for NS1 as well as by PCR. 23/576 samples were negative for NS1 antigen, but were positive by RT-PCR. The remaining 534 samples which were negative for NS1 antigen were also negative by Dengue RT-PCR. In this study we sought to standardize rapid, sensitive, and specific fluorogenic probe-based RT-PCR assay to screen and serotype a representative range of Dengue viruses that are found in and around Mumbai. Qualitative Dengue virus TaqMan assays could have tremendous utility for the epidemiological investigation of Dengue illness and especially for the study of the viremic response with candidate live-attenuated dengue virus vaccines.

Evaluation of clinical pathology parameters in fecal PCR-positive or PCR-negative goats for Johne's disease.

PubMed

Bonelli, Francesca; Fratini, F; Turchi, B; Cantile, C; Ebani, V V; Colombani, G; Galiero, A; Sgorbini, M

2017-10-01

Johne's disease (JD) is an economically important infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This study evaluated the differences in various hematological and biochemical parameters between healthy goats and goats with JD. Forty goats were chosen randomly from a herd endemic for JD. A complete physical examination was performed. Blood and fresh fecal samples were collected from each goat. A complete blood cell (CBC) count and a protein electrophoresis were performed. Polymerase chain reaction (PCR) on fecal samples was performed in order to divide goats into two groups: group A "positive PCR on feces"; and group B "control (negative)." A Student's t test was performed for each parameter to verify differences between groups A vs B. Twenty goats were included in each group. Clinical signs likely related to JD were found in the history of 4/40 (10%) goats, while 36/40 (90%) goats were reported to be asymptomatic. CBC and electrophoresis values were within reference intervals in both groups. No differences were found for CBC parameters between the two groups. Values for alpha 1, beta, gamma globulins, and total protein (TP) were statistically higher in group A vs those in group B, while those for albumin and albumin/globulin (A/G) ratio were lower. An increase in TP, hypoalbuminemia, and hypergammaglobulinemia has been reported in group A, while no abnormalities were found concerning CBC. JD-positive goats seem to show earlier clinical pathological alternations than clinical signs. Protein electrophoresis may help the diagnosis of JD in asymptomatic goat herds, acting as an economical screening method.

Effects of Minocycline on Urine Albumin, Interleukin-6, and Osteoprotegerin in Patients with Diabetic Nephropathy: A Randomized Controlled Pilot Trial

PubMed Central

Wang, Ying; Tong, Lili; Pak, Youngju; Andalibi, Ali; LaPage, Janine A.; Adler, Sharon G.

2016-01-01

Background We tested minocycline as an anti-proteinuric adjunct to renin-angiotensin-aldosterone system inhibitors (RAASi) in diabetic nephropathy (DN) and measured urinary biomarkers to evaluate minocycline’s biological effects. Methods Design: Prospective, single center, randomized, placebo-controlled, intention-to-treat pilot trial. Inclusion. Type 2 diabetes/DN; Baseline creatinine clearance > 30 mL/min; proteinuria ≥ 1.0 g/day; Age ≥30 years; BP <150/95 mm Hg; intolerant of/at maximum RAASi dose. Protocol. 3-wk screening; Baseline randomization; Urine and blood measures at months 1, 2, 4, and Month 6 study completion. Urine interleukin-6 (IL-6) and osteoprotegerin were measured in a subset. Primary outcome. Natural log of urine protein/creatinine (ln U P:Cr) ratio at Month 6 vs Baseline. Results 30 patients completed the study. The 15% decline in U P: Cr in minocycline patients (6 month P:Cr ÷ Baseline P:Cr, 0.85 vs. 0.92) was not significant (p = 0.27). Creatinine clearance did not differ in the 2 groups. Urine IL-6:Cr (p = 0.03) and osteoprotegerin/Cr (p = 0.046) decrements were significant. Minocycline modified the relationship between urine IL-6 and proteinuria, suggesting a protective biological effect. Conclusions Although the decline in U P:Cr in minocycline patients was not statistically significant, the significant differences in urine IL-6 and osteoprotegerin suggest that minocycline may confer cytoprotection in patients with DN, providing a rationale for further study. Trial Registration Clinicaltrials.gov NCT01779089 PMID:27019421

Complete genome sequences of two divergent isolates of strawberry crinkle virus coinfecting a single strawberry plant.

PubMed

Koloniuk, Igor; Fránová, Jana; Sarkisova, Tatiana; Přibylová, Jaroslava

2018-05-04

Strawberry crinkle disease is one of the major diseases that threatens strawberry production. Although the biological properties of the agent, strawberry crinkle virus (SCV), have been thoroughly investigated, its complete genome sequence has never been published. Existing RT-PCR-based detection relies on a partial sequence of the L protein gene, presumably the least expressed viral gene. Here, we present complete sequences of two divergent SCV isolates co-infecting a single plant, Fragaria x ananassa cv. Čačanská raná.

ALK-rearranged pulmonary adenocarcinoma in Thai Patients: From diagnosis to treatment efficacy.

PubMed

Incharoen, Pimpin; Reungwetwattana, Thanyanan; Saowapa, Sakditad; Kamprerasart, Kaettipong; Pangpunyakulchai, Duangjai; Arsa, Lalida; Jinawath, Artit

2016-05-03

Anaplastic lymphoma kinase (ALK) gene rearrangement is detected in 3% to 13% of non-small cell lung carcinoma patients, and these patients benefit from ALK inhibitors. The aim of this study was to determine the prevalence, the clinical and histological characteristics and the treatment outcomes of ALK-rearranged lung adenocarcinoma using immunohistochemistry (IHC) IHC, reverse transcription polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) methodologies. A total of 268 pulmonary adenocarcinoma patients were screened for ALK expression by ALK IHC, which was confirmed by FISH and/or RT-PCR for ALK gene rearrangement. The treatment outcomes of ALK-rearranged patients were retrospectively reviewed. ALK gene rearrangement was identified in 26 cases (9.7%) with no EGFR co-mutation, and it showed significant associations with younger age, female sex and non-smoker status (p < 0.05). A cribriform growth pattern was identified as the dominant histologic feature, and a solid signet ring cell component was focally present in a minority of the cases. Among 12 ALK-rearranged patients with conventional treatment, seven cases in the early stage of disease were cured and alive, and five patients in the late stage of the disease progressed and died, with a median overall survival (OS) at 14 months. Of the 14 patients receiving crizotinib, all of them had clinical benefit from crizotinib treatment, with one patient having a complete response (CR), 12 patients having a partial response (PR) and one patient having stable disease (SD). On the cutoff date, six of 14 patients were continuing crizotinib treatment with a median time of response of 7.5 (3-13) months, while eight patients had disease progression, and five of them died with a median OS at 8 months. ALK gene rearrangement tended to occur in younger, non-smoking, female patients. ALK IHC is a reliable screening method to detect ALK gene rearrangement. Crizotinib therapy provided treatment benefit in ALK-rearranged adenocarcinoma patients especially in advanced stages of the disease.

A single digital droplet PCR assay to detect multiple KIT exon 11 mutations in tumor and plasma from patients with gastrointestinal stromal tumors.

PubMed

Boonstra, Pieter A; Ter Elst, Arja; Tibbesma, Marco; Bosman, Lisette J; Mathijssen, Ron; Atrafi, Florence; van Coevorden, Frits; Steeghs, Neeltje; Farag, Sheima; Gelderblom, Hans; van der Graaf, Winette T A; Desar, Ingrid M E; Maier, Jacqueline; Overbosch, Jelle; Suurmeijer, Albert J H; Gietema, Jourik; Schuuring, Ed; Reyners, Anna K L

2018-03-02

Gastrointestinal stromal tumors (GISTs) are characterized by oncogenic KIT mutations that cluster in two exon 11 hotspots. The aim of this study was to develop a single, sensitive, quantitative digital droplet PCR (ddPCR) assay for the detection of common exon 11 mutations in both GIST tumor tissue and in circulating tumor DNA (ctDNA) isolated from GIST patients' plasma. A ddPCR assay was designed using two probes that cover both hotspots. Available archival FFPE tumor tissue from 27 consecutive patients with known KIT exon 11 mutations and 9 randomly selected patients without exon 11 mutations were tested. Plasma samples were prospectively collected in a multicenter bio-databank from December 2014. ctDNA was analyzed of 22 patients with an exon 11 mutation and a baseline plasma sample. The ddPCR assay detected the exon 11 mutation in 21 of 22 tumors with exon 11 mutations covered by the assay. Mutations in ctDNA were detected at baseline in 13 of 14 metastasized patients, but in only 1 of 8 patients with localized disease. In serial plasma samples from 11 patients with metastasized GIST, a decrease in mutant droplets was detected during treatment. According to RECIST 1.1, 10 patients had radiological treatment response and one patient stable disease. A single ddPCR assay for the detection of multiple exon 11 mutations in ctDNA is a feasible, promising tool for monitoring treatment response in patients with metastasized GIST and should be further evaluated in a larger cohort.

Influence of Medical Student Debt on the Decision to Pursue Careers in Primary Care.

PubMed

Gil, Joseph A; Waryasz, Gregory R; Liu, Dorothy; Daniels, Alan H

2016-07-01

To determine if medical student debt has an effect on medical student specialty choice. A cross-sectional survey was distributed to students at 12 medical schools across the United States to assess the effect of debt on specialty choice. In total, 415 students responded to the survey; 98 medical students reported that they were pursuing a primary care residency (PCR) and 250 reported that they were pursing a non-primary care residency (NPCR). There was no significant difference in average student loan debt anticipated by medical students pursing PCR and NPCR ($142,217 vs $150,784; P>0.46). Medical students pursuing a PCR reported lower estimated salaries on average than medical students pursuing NPCR ($137,711 vs $241,804; p<0.01). Of the surveyed students, 62% of students who are pursuing PCR and 77% of the students who are pursuing a NPCR would not have pursued medicine as a career if residents were responsible for paying tuition. This study revealed no significant difference between the student debt of medical students pursuing PCR compared to those who are pursuing a NPCR. However, a large majority of medical students would not pursue a career in medicine if faced with the responsibility of paying tuition for residency. [Full article available at http://rimed.org/rimedicaljournal-2016-07.asp, free with no login].

Identification of mycoparasitism-related genes against the phytopathogen Sclerotinia sclerotiorum through transcriptome and expression profile analysis in Trichoderma harzianum.

PubMed

Steindorff, Andrei Stecca; Ramada, Marcelo Henrique Soller; Coelho, Alexandre Siqueira Guedes; Miller, Robert Neil Gerard; Pappas, Georgios Joannis; Ulhoa, Cirano José; Noronha, Eliane Ferreira

2014-03-18

The species of T. harzianum are well known for their biocontrol activity against plant pathogens. However, few studies have been conducted to further our understanding of its role as a biological control agent against S. sclerotiorum, a pathogen involved in several crop diseases around the world. In this study, we have used RNA-seq and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum gene expression during growth on cell wall of S. sclerotiorum (SSCW) or glucose. RT-qPCR was also used to examine genes potentially involved in biocontrol, during confrontation between T. harzianum and S. sclerotiorum. Data obtained from six RNA-seq libraries were aligned onto the T. harzianum CBS 226.95 reference genome and compared after annotation using the Blast2GO suite. A total of 297 differentially expressed genes were found in mycelia grown for 12, 24 and 36 h under the two different conditions: supplemented with glucose or SSCW. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on SSCW or glucose. We identified various genes of biotechnological value encoding proteins with functions such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. To validate the expression profile, RT-qPCR was performed using 20 randomly chosen genes. RT-qPCR expression profiles were in complete agreement with the RNA-Seq data for 17 of the genes evaluated. The other three showed differences at one or two growth times. During the confrontation assay, some genes were up-regulated during and after contact, as shown in the presence of SSCW which is commonly used as a model to mimic this interaction. The present study is the first initiative to use RNA-seq for identification of differentially expressed genes in T. harzianum strain TR274, in response to the phytopathogenic fungus S. sclerotiorum. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against S.sclerotiorum. The RNA-seq data presented will facilitate improvement of the annotation of gene models in the draft T. harzianum genome and provide important information regarding the transcriptome during this interaction.

Quantitative Detection and Resolution of BRAF V600 Status in Colorectal Cancer Using Droplet Digital PCR and a Novel Wild-Type Negative Assay.

PubMed

Bidshahri, Roza; Attali, Dean; Fakhfakh, Kareem; McNeil, Kelly; Karsan, Aly; Won, Jennifer R; Wolber, Robert; Bryan, Jennifer; Hughesman, Curtis; Haynes, Charles

2016-03-01

A need exists for robust and cost-effective assays to detect a single or small set of actionable point mutations, or a complete set of clinically informative mutant alleles. Knowledge of these mutations can be used to alert the clinician to a rare mutation that might necessitate more aggressive clinical monitoring or a personalized course of treatment. An example is BRAF, a (proto)oncogene susceptible to either common or rare mutations in codon V600 and adjacent codons. We report a diagnostic technology that leverages the unique capabilities of droplet digital PCR to achieve not only accurate and sensitive detection of BRAF(V600E) but also all known somatic point mutations within the BRAF V600 codon. The simple and inexpensive two-well droplet digital PCR assay uses a chimeric locked nucleic acid/DNA probe against wild-type BRAF and a novel wild-type-negative screening paradigm. The assay shows complete diagnostic accuracy when applied to formalin-fixed, paraffin-embedded tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy

PubMed Central

Di Fiore, F; Blanchard, F; Charbonnier, F; Le Pessot, F; Lamy, A; Galais, M P; Bastit, L; Killian, A; Sesboüé, R; Tuech, J J; Queuniet, A M; Paillot, B; Sabourin, J C; Michot, F; Michel, P; Frebourg, T

2007-01-01

The predictive value of KRAS mutation in metastatic colorectal cancer (MCRC) patients treated with cetuximab plus chemotherapy has recently been suggested. In our study, 59 patients with a chemotherapy-refractory MCRC treated with cetuximab plus chemotherapy were included and clinical response was evaluated according to response evaluation criteria in solid tumours (RECIST). Tumours were screened for KRAS mutations using first direct sequencing, then two sensitive methods based on SNaPshot and PCR-ligase chain reaction (LCR) assays. Clinical response was evaluated according to gene mutations using the Fisher exact test. Times to progression (TTP) were calculated using the Kaplan–Meier method and compared with log-rank test. A KRAS mutation was detected in 22 out of 59 tumours and, in six cases, was missed by sequencing analysis but detected using the SNaPshot and PCR-LCR assays. Remarkably, no KRAS mutation was found in the 12 patients with clinical response. KRAS mutation was associated with disease progression (P=0.0005) and TTP was significantly decreased in mutated KRAS patients (3 vs 5.5 months, P=0.015). Our study confirms that KRAS mutation is highly predictive of a non-response to cetuximab plus chemotherapy in MCRC and highlights the need to use sensitive molecular methods, such as SNaPshot or PCR-LCR assays, to ensure an efficient mutation detection. PMID:17375050

Multiple-endpoints gene alteration-based (MEGA) assay: A toxicogenomics approach for water quality assessment of wastewater effluents.

PubMed

Fukushima, Toshikazu; Hara-Yamamura, Hiroe; Nakashima, Koji; Tan, Lea Chua; Okabe, Satoshi

2017-12-01

Wastewater effluents contain a significant number of toxic contaminants, which, even at low concentrations, display a wide variety of toxic actions. In this study, we developed a multiple-endpoints gene alteration-based (MEGA) assay, a real-time PCR-based transcriptomic analysis, to assess the water quality of wastewater effluents for human health risk assessment and management. Twenty-one genes from the human hepatoblastoma cell line (HepG2), covering the basic health-relevant stress responses such as response to xenobiotics, genotoxicity, and cytotoxicity, were selected and incorporated into the MEGA assay. The genes related to the p53-mediated DNA damage response and cytochrome P450 were selected as markers for genotoxicity and response to xenobiotics, respectively. Additionally, the genes that were dose-dependently regulated by exposure to the wastewater effluents were chosen as markers for cytotoxicity. The alterations in the expression of an individual gene, induced by exposure to the wastewater effluents, were evaluated by real-time PCR and the results were validated by genotoxicity (e.g., comet assay) and cell-based cytotoxicity tests. In summary, the MEGA assay is a real-time PCR-based assay that targets cellular responses to contaminants present in wastewater effluents at the transcriptional level; it is rapid, cost-effective, and high-throughput and can thus complement any chemical analysis for water quality assessment and management. Copyright © 2017 Elsevier Ltd. All rights reserved.

TaqMan 5′-Nuclease Human Immunodeficiency Virus Type 1 PCR Assay with Phage-Packaged Competitive Internal Control for High-Throughput Blood Donor Screening

PubMed Central

Drosten, C.; Seifried, E.; Roth, W. K.

2001-01-01

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5′-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5′-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5′-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a ≥95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1. PMID:11724836

Early diagnosis and monitoring of mucormycosis by detection of circulating DNA in serum: retrospective analysis of 44 cases collected through the French Surveillance Network of Invasive Fungal Infections (RESSIF).

PubMed

Millon, L; Herbrecht, R; Grenouillet, F; Morio, F; Alanio, A; Letscher-Bru, V; Cassaing, S; Chouaki, T; Kauffmann-Lacroix, C; Poirier, P; Toubas, D; Augereau, O; Rocchi, S; Garcia-Hermoso, D; Bretagne, S

2016-09-01

The main objective of this study was to assess the diagnostic performance of a set of three Mucorales quantitative PCR assays in a retrospective multicentre study. Mucormycosis cases were recorded thanks to the French prospective surveillance programme (RESSIF network). The day of sampling of the first histological or mycological positive specimen was defined as day 0 (D0). Detection of circulating DNA was performed on frozen serum samples collected from D-30 to D30, using quantitative PCR assays targeting Rhizomucor, Lichtheimia, Mucor/Rhizopus. Forty-four patients diagnosed with probable (n = 19) or proven (n = 25) mucormycosis were included. Thirty-six of the 44 patients (81%) had at least one PCR-positive serum. The first PCR-positive sample was observed 9 days (range 0-28 days) before diagnosis was made using mycological criteria and at least 2 days (range 0-24 days) before imaging. The identifications provided with the quantitative PCR assays were all concordant with culture and/or PCR-based identification of the causal species. Survival rate at D84 was significantly higher for patients with an initially positive PCR that became negative after treatment initiation than for patients whose PCR remained positive (48% and 4%, respectively; p <10 -6 ). The median time for complete negativity of PCR was 7 days (range 3-19 days) after initiation of l-AmB treatment. Despite some limitations due to the retrospective design of the study, we showed that Mucorales quantitative PCR could not only confirm the mucormycosis diagnosis when other mycological arguments were present but could also anticipate this diagnosis. Quantification of DNA loads may also be a useful adjunct to treatment monitoring. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Genome-Wide Investigation of WRKY Transcription Factors Involved in Terminal Drought Stress Response in Common Bean

PubMed Central

Wu, Jing; Chen, Jibao; Wang, Lanfen; Wang, Shumin

2017-01-01

WRKY transcription factor plays a key role in drought stress. However, the characteristics of the WRKY gene family in the common bean (Phaseolus vulgaris L.) are unknown. In this study, we identified 88 complete WRKY proteins from the draft genome sequence of the “G19833” common bean. The predicted genes were non-randomly distributed in all chromosomes. Basic information, amino acid motifs, phylogenetic tree and the expression patterns of PvWRKY genes were analyzed, and the proteins were classified into groups 1, 2, and 3. Group 2 was further divided into five subgroups: 2a, 2b, 2c, 2d, and 2e. Finally, we detected 19 WRKY genes that were responsive to drought stress using qRT-PCR; 11 were down-regulated, and 8 were up-regulated under drought stress. This study comprehensively examines WRKY proteins in the common bean, a model food legume, and it provides a foundation for the functional characterization of the WRKY family and opportunities for understanding the mechanisms of drought stress tolerance in this plant. PMID:28386267

Genome-Wide Investigation of WRKY Transcription Factors Involved in Terminal Drought Stress Response in Common Bean.

PubMed

Wu, Jing; Chen, Jibao; Wang, Lanfen; Wang, Shumin

2017-01-01

WRKY transcription factor plays a key role in drought stress. However, the characteristics of the WRKY gene family in the common bean ( Phaseolus vulgaris L.) are unknown. In this study, we identified 88 complete WRKY proteins from the draft genome sequence of the "G19833" common bean. The predicted genes were non-randomly distributed in all chromosomes. Basic information, amino acid motifs, phylogenetic tree and the expression patterns of PvWRKY genes were analyzed, and the proteins were classified into groups 1, 2, and 3. Group 2 was further divided into five subgroups: 2a, 2b, 2c, 2d, and 2e. Finally, we detected 19 WRKY genes that were responsive to drought stress using qRT-PCR; 11 were down-regulated, and 8 were up-regulated under drought stress. This study comprehensively examines WRKY proteins in the common bean, a model food legume, and it provides a foundation for the functional characterization of the WRKY family and opportunities for understanding the mechanisms of drought stress tolerance in this plant.

[Serum levels of HCV-RNA determined by branched DNA (bDNA) probe assay in chronic hepatitis C: method and clinical significance on interferon (IFN) therapy].

PubMed

Osada, T; Iwabuchi, S; Takatori, M; Murayama, M; Iino, S

1994-07-01

Recently serum levels of HCV RNA (s-HCV RNA) in chronic hepatitis C has been regarded as one of an important indicator for the activity of disease and outcome of IFN therapy. Quantitative analysis by competitive RT-PCR, however, requires special skills and is too expensive for clinical use. We attempted to determine serial sample of s-HCV RNA and genotypes in 117 cases treated with IFN using bDNA probe assay which has lately been developed by Chiron corporation. Cases with complete response to IFN therapy showed 62% (49/79) in lower than 10(6) levels, 27% (8/30) in 10(6)-10(7) and only 5% (2/41) in higher than 10(7). Genotype III, IV had higher response than type II on the same level of s-HCV RNA. These data indicates that determination of s-HCV levels by bDNA assay may be useful for prediction of the outcome of IFN therapy and making adequate schedule of the therapy in each cases.

miR156 modulates rhizosphere acidification in response to phosphate limitation in Arabidopsis.

PubMed

Lei, Kai Jian; Lin, Ya Ming; An, Guo Yong

2016-03-01

Rhizosphere acidification is a general response to Pi deficiency, especially in dicotyledonous plants. However, the signaling pathway underlying this process is still unclear. Here, we demonstrate that miR156 is induced in the shoots and roots of wild type Arabidopsis plants during Pi starvation. The rhizosphere acidification capacity was increased in 35S:MIR156 (miR156 overexpression) plants, but was completely inhibited in 35S:MIM156 (target mimicry) plants. Both 35S:MIR156 and 35S:MIM156 plants showed altered proton efflux and H(+)-ATPase activity. In addition, significant up-regulation of H(+)-ATPase activity in 35S:MIR156 roots coupled with increased citric acid and malic acid exudates was observed. qRT-PCR results showed that most H(+)-ATPase and PPCK gene transcript levels were decreased in 35S:MIM156 plants, which may account for the decreased H(+)-ATPase activity in 35S:MIM156 plants. MiR156 also affect the root architecture system. Collectively, our results suggest that miR156 regulates the process of rhizosphere acidification in plants.

Identification of differentially expressed proteins of Arthrospira (Spirulina) plantensis-YZ under salt-stress conditions by proteomics and qRT-PCR analysis

PubMed Central

2013-01-01

Arthrospira (Spirulina) platensis as a representative species of cyanobacteria has been recognized and used worldwide as a source of protein in the food, which possesses some unusual and valuable physiological characteristics, such as alkali and salt tolerance. Based on complete genome sequencing of Arthrospira (Spirulina) plantensis-YZ, we compared the protein expression profiles of this organism under different salt-stress conditions (i.e. 0.02 M, 0.5 M and 1.0 M NaCl, respectively), using 2-D electrophoresis and peptide mass fingerprinting, and retrieved 141 proteins showing significantly differential expression in response to salt-stress. Of the 141 proteins, 114 Arthrospira (Spirulina) plantensis-YZ proteins were found with significant homology to those found in Arthrospira (76 proteins in Arthrospira platensis str. Paraca and 38 in Arthrospira maxima CS-328). The remaining 27 proteins belong to other bacteria. Subsequently, we determined the transcriptional level of 29 genes in vivo in response to NaCl treatments and verified them by qRT-PCR. We found that 12 genes keep consistency at both transcription and protein levels, and transcription of all of them but one were up-regulated. We classified the 141 differentially expressed proteins into 18 types of function categories using COG database, and linked them to their respective KEGG metabolism pathways. These proteins are involved in 31 metabolism pathways, such as photosynthesis, glucose metabolism, cysteine and methionine metabolism, lysine synthesis, fatty acid metabolism, glutathione metabolism. Additionally, the SRPs, heat shock protein and ABC transporter proteins were identified, which probably render Arthrospira (Spirulina) plantensis’s resistance against high salt stress. PMID:23363438

Exploring valid internal-control genes in Porphyra yezoensis (Bangiaceae) during stress response conditions

NASA Astrophysics Data System (ADS)

Wang, Wenlei; Wu, Xiaojie; Wang, Chao; Jia, Zhaojun; He, Linwen; Wei, Yifan; Niu, Jianfeng; Wang, Guangce

2014-07-01

To screen the stable expression genes related to the stress (strong light, dehydration and temperature shock) we applied Absolute real-time PCR technology to determine the transcription numbers of the selected test genes in P orphyra yezoensis, which has been regarded as a potential model species responding the stress conditions in the intertidal. Absolute real-time PCR technology was applied to determine the transcription numbers of the selected test genes in P orphyra yezoensis, which has been regarded as a potential model species in stress responding. According to the results of photosynthesis parameters, we observed that Y(II) and F v/ F m were significantly affected when stress was imposed on the thalli of P orphyra yezoensis, but underwent almost completely recovered under normal conditions, which were collected for the following experiments. Then three samples, which were treated with different grade stresses combined with salinity, irradiation and temperature, were collected. The transcription numbers of seven constitutive expression genes in above samples were determined after RNA extraction and cDNA synthesis. Finally, a general insight into the selection of internal control genes during stress response was obtained. We found that there were no obvious effects in terms of salinity stress (at salinity 90) on transcription of most genes used in the study. The 18S ribosomal RNA gene had the highest expression level, varying remarkably among different tested groups. RPS8 expression showed a high irregular variance between samples. GAPDH presented comparatively stable expression and could thus be selected as the internal control. EF-1α showed stable expression during the series of multiple-stress tests. Our research provided available references for the selection of internal control genes for transcripts determination of P. yezoensis.

Identification of trans-acting factors regulating SamDC expression in Oryza sativa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basu, Supratim, E-mail: supratim_genetics@yahoo.co.in; Division of Plant Biology, Bose Institute, Kolkata; Roychoudhury, Aryadeep

2014-03-07

Highlights: • Identification of cis elements responsible for SamDC expression by in silico analysis. • qPCR analysis of SamDC expression to abiotic and biotic stress treatments. • Detection of SamDC regulators using identified cis-elements as probe by EMSA. • Southwestern Blot analysis to predict the size of the trans-acting factors. - Abstract: Abiotic stress affects the growth and productivity of crop plants; to cope with the adverse environmental conditions, plants have developed efficient defense machinery comprising of antioxidants like phenolics and flavonoids, and osmolytes like polyamines. SamDC is a key enzyme in the polyamine biosynthesis pathway in plants. In ourmore » present communication we have done in silico analysis of the promoter region of SamDC to look for the presence of different cis-regulatory elements contributing to its expression. Based on the presence of different cis-regulatory elements we completed comparative analysis of SamDC gene expression in rice lamina of IR-29 and Nonabokra by qPCR in response to the abiotic stress treatments of salinity, drought, cold and the biotic stress treatments of ABA and light. Additionally, to explore the role of the cis-regulatory elements in regulating the expression of SamDC gene in plants we comparatively analyzed the binding of rice nuclear proteins prepared from IR-29 and Nonabokra undergoing various stress treatments. The intensity of the complex formed was low and inducible in IR-29 in contrast to Nonabokra. Southwestern blot analysis helped in predicting the size of the trans-acting factors binding to these cis-elements. To our knowledge this is the first report on the comprehensive analysis of SamDC gene expression in rice and identification of the trans-acting factors regulating its expression.« less

Phase II Trial of Preoperative Irinotecan-Cisplatin Followed by Concurrent Irinotecan-Cisplatin and Radiotherapy for Resectable Locally Advanced Gastric and Esophagogastric Junction Adenocarcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivera, Fernando, E-mail: oncrhf@humv.e; Galan, Maica; Tabernero, Josep

Purpose: To determine in a Phase II trial whether preoperative irinotecan-cisplatin (IC) followed by concurrent IC therapy and radiotherapy (IC/RT) improved outcome in patients with resectable, locally advanced gastric adenocarcinoma (GC) or esophagogastric junction cancer (EGJC). Patients and Methods: Patients with resectable Stage II-IV, M0 GC or EGJC made up the study population. The primary endpoint was pathologic complete response (pCR). Two courses of IC (irinotecan, 65mg/m{sup 2}; cisplatin, 30mg/m{sup 2} on Days 1 and 8 every 21 days) were given. Patients without progression then received IC/RT, consisting of daily radiotherapy (45Gy) with concurrent IC (irinotecan, 65mg/m{sup 2}; cisplatin, 30mg/m{supmore » 2} on Days 1, 8, 15, and 22). Surgical resection was performed, if feasible, 5-8 weeks after the end of radiotherapy. Results: Twenty-three patients were included in the study: 10 with EGJC and 13 with GC. Two patients (9%) achieved pCR. The incidences of Grade 3-4 toxicities were as follows: IC: neutropenia 35% (febrile 13%), anemia 22%, diarrhea 22%, emesis 8%; IC/RT: neutropenia 52% (febrile 5%), asthenia 19%, anemia 9%, emesis 9%, diarrhea 5%, cardiotoxicity 5%. No patients died during IC or IC/RT. R0 resection was achieved in 15 patients (65%). Median survival was 14.5 months, and the actuarial 2-year survival rate was 35%. Conclusions: Preoperative IC followed by IC/RT resulted in moderate response and resection rates with mild toxicity in patients with GC and EGJC.« less

The suppression of tomato defence response genes upon potato cyst nematode infection indicates a key regulatory role of miRNAs.

PubMed

Święcicka, Magdalena; Skowron, Waldemar; Cieszyński, Piotr; Dąbrowska-Bronk, Joanna; Matuszkiewicz, Mateusz; Filipecki, Marcin; Koter, Marek Daniel

2017-04-01

Potato cyst nematode Globodera rostochiensis is an obligate parasite of solanaceous plants, triggering metabolic and morphological changes in roots which may result in substantial crop yield losses. Previously, we used the cDNA-AFLP to study the transcriptional dynamics in nematode infected tomato roots. Now, we present the rescreening of already published, upregulated transcript-derived fragment dataset using the most current tomato transcriptome sequences. Our reanalysis allowed to add 54 novel genes to 135, already found as upregulated in tomato roots upon G. rostochiensis infection (in total - 189). We also created completely new catalogue of downregulated sequences leading to the discovery of 76 novel genes. Functional classification of candidates showed that the 'wound, stress and defence response' category was enriched in the downregulated genes. We confirmed the transcriptional dynamics of six genes by qRT-PCR. To place our results in a broader context, we compared the tomato data with Arabidopsis thaliana, revealing similar proportions of upregulated and downregulated genes as well as similar enrichment of defence related transcripts in the downregulated group. Since transcript suppression is quite common in plant-nematode interactions, we assessed the possibility of miRNA-mediated inverse correlation on several tomato sequences belonging to NB-LRR and receptor-like kinase families. The qRT-PCR of miRNAs and putative target transcripts showed an opposite expression pattern in 9 cases. These results together with in silico analyses of potential miRNA targeting to the full repertoire of tomato R-genes show that miRNA mediated gene suppression may be a key regulatory mechanism during nematode parasitism. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

NMR studies on /sup 15/N-labeled creatine (CR), creatinine (CRN), phosphocreatine (PCR), and phosphocreatinine (PCRN), and on barriers to rotation in creatine kinase-bound creatine in the enzymatic reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kenyon, G.L.; Reddick, R.E.

1986-05-01

Recently, the authors have synthesized /sup 15/N-2-Cr, /sup 15/N-3-Crn, /sup 15/N-2-Crn, /sup 15/N-3-PCrn, /sup 15/N-3-PCr, and /sup 15/N-2-PCr. /sup 1/H, /sup 15/N, /sup 31/P NMR data show that Crn protonates exclusively at the non-methylated ring nitrogen, confirm that PCrn is phosphorylated at the exocyclic nitrogen, and demonstrate that the /sup 31/P-/sup 15/N one-bond coupling constant in /sup 15/N-3-PCr is 18 Hz, not 3 Hz as previously reported by Brindle, K.M., Porteous, R. and Radda, G.K.. The authors have found that creatine kinase is capable of catalyzing the /sup 14/N//sup 15/N positional isotope exchange of 3-/sup 15/N-PCr in the presence ofmore » MgADP, but not in its absence. Further, the exchange does not take place when labeled PCr is resynthesized exclusively from the ternary complex E X Cr X MgATP as opposed to either E X Cr or free Cr. This suggests that the enzyme both imparts an additional rotational barrier to creatine in the complex and catalyzes the transfer of phosphoryl group with essentially complete regiospecificity.« less

[Rapid diagnosis of the most common fetal aneuploidies with the QF-PCR method--a study of 100 cases].

PubMed

Łaczmańska, Izabela; Gil, Justyna; Stembalska, Agnieszka; Makowska, Izabela; Kozłowska, Joanna; Skiba, Paweł; Czemarmazowicz, Halina; Pesz, Karolina; Slęzak, Ryszard; Smigiel, Robert; Jakubiak, Aleksandra; Doraczyńska-Kowalik, Anna; Sąsiadek, Maria M

2015-09-01

The aim of the study was to assess whether commercial kit QF-PCR can be used as the only method for rapic prenatal dia gnosis of chromosomes 13, 18, 21, X and Y aneuploidies, omitting cell culture and complete cyt6genetik analysis of fetal chromosomes. DNA from amniocytes (94 cases) and trophoblast cells (6 cases) was analyzed witt QF-PCR according to the manufacturer's protocol. The obtained products were separated using ABI 310 Genetic Analyzer and the resulting data were analyzed using GeneMarker software. The results of QF-PCR were obtained in 95 out of 100 cases (95%). Abnormalities were found in 28 casea (29.5%). All these results were confirmed in subsequent cytogenetic analysis. Normal results were obtained in 62 patients (70.5%). However in that group, we found three chromosomal aberrations other than those analyzed b3 QF-PCR. Additionally two abnormal and three normal karyotypes were found in patients with inconclusive QF-POF results. QF-PCR is a fast and reliable tool for chromosomal aneuploidy analysis and can be used as the only method without a full analysis of the karyotype, but only in cases of suspected fetal 13, 18, 21 trisomy or numerica aberrations of X chromosome. In other cases, fetal karyotype analysis from cells obtained after cell culture should be offered to the patient.

Detection and typing of low-risk human papillomavirus genotypes HPV 6, HPV 11, HPV 42, HPV 43 and HPV 44 by polymerase chain reaction and restriction fragment length polymorphism.

PubMed

Maver, Polona J; Poljak, Mario; Seme, Katja; Kocjan, Bostjan J

2010-10-01

A novel PCR-restriction fragment length polymorphism assay (PCR-RFLP) was developed for sensitive detection and reliable differentiation of five low-risk human papillomavirus (lr-HPV) genotypes: HPV 6, HPV 11, HPV 42, HPV 43 and HPV 44, as well as differentiation of prototypic and non-prototypic HPV 6 genomic variants. The assay is based on the amplification of a 320-bp fragment of the HPV E1 gene and subsequent analysis of PCR-products with BsaJI and HinFI. Testing on plasmid standards showed that PCR-RFLP enabled simple and reliable identification and differentiation of five targeted lr-HPV genotypes and could detect reproducibly down to 10 copies of viral genome equivalents per PCR. The PCR-RFLP showed almost complete agreement with previously obtained genotyping results on 42 HPV-DNA negative samples and 223 HPV-DNA positive samples (45 HPV 6, 34 HPV 11, 35 HPV 42, 10 HPV 43, 24 HPV 44 positive samples and 75 samples containing 28 non-targeted HPV genotypes). The novel assay is simple and robust, does not require any sophisticated equipment and can be of great value for epidemiological studies, particularly in settings in which financial resources are limited. Copyright (c) 2010 Elsevier B.V. All rights reserved.

[Disappearance of residual disease confirmed by RT-PCR following induction chemotherapy in two hypoplastic leukemia patients with t(8;21)].

PubMed

Sawada, M; Tsurumi, H; Yamada, T; Hara, T; Oyama, M; Moriwaki, H

1999-04-01

Reverse transcriptase-polymerase chain reaction (RT-PCR) methods often detect the AML1/MTG8 fusion transcript even in acute myelogenous leukemia (AML) patients with t(8;21) who have been in long-term remission. We encountered 2 hypoplastic leukemia patients with t(8;21) who achieved cytogenetic remission with short-term conventional chemotherapy. Patient 1 was a 42-year-old woman. Chromosomal analysis detected t(8;21) (q22;q22) and PCR analysis (35 cycles PCR amplification; detection limit 1 x 10(-5) cells) detected the AML1/MTG8 fusion transcript. Complete remission was obtained with 1 course of chemotherapy consisting of low-dose cytarabine (20 mg x 14 days) and etoposide (50 mg x 14 days). After 2 courses of consolidation chemotherapy consisting of conventional-dose cytarabine and mitoxantrone, the RT-PCR findings were negative for the AML1/MTG8 fusion transcript. Patient 2 was a 67-year-old man. Cytogenetic analysis detected t(8;21) (q22;q22), and was positive for the AML1/MTG8 fusion transcript. After 2 courses of induction chemotherapy comprising low-dose cytarabine (20 mg x 14 days) and etoposide (50 mg x 14 days), and 3 courses of conventional consolidation chemotherapy, RT-PCR analysis confirmed the disappearance of the AML1/MTG8 fusion transcript.

Designing, optimization and validation of tetra-primer ARMS PCR protocol for genotyping mutations in caprine Fec genes

PubMed Central

Ahlawat, Sonika; Sharma, Rekha; Maitra, A.; Roy, Manoranjan; Tantia, M.S.

2014-01-01

New, quick, and inexpensive methods for genotyping novel caprine Fec gene polymorphisms through tetra-primer ARMS PCR were developed in the present investigation. Single nucleotide polymorphism (SNP) genotyping needs to be attempted to establish association between the identified mutations and traits of economic importance. In the current study, we have successfully genotyped three new SNPs identified in caprine fecundity genes viz. T(-242)C (BMPR1B), G1189A (GDF9) and G735A (BMP15). Tetra-primer ARMS PCR protocol was optimized and validated for these SNPs with short turn-around time and costs. The optimized techniques were tested on 158 random samples of Black Bengal goat breed. Samples with known genotypes for the described genes, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the tetra-primer ARMS PCR assays and the sequencing results. These results highlight the ability of tetra-primer ARMS PCR in genotyping of mutations in Fec genes. Any associated SNP could be used to accelerate the improvement of goat reproductive traits by identifying high prolific animals at an early stage of life. Our results provide direct evidence that tetra-primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of mutations in caprine Fec genes. PMID:25606428

Detection of intestinal protozoa in paediatric patients with gastrointestinal symptoms by multiplex real-time PCR.

PubMed

Maas, L; Dorigo-Zetsma, J W; de Groot, C J; Bouter, S; Plötz, F B; van Ewijk, B E

2014-06-01

The performance of a multiplex real-time PCR for the detection of Blastocystis, Dientamoeba fragilis, Giardia lamblia, Cryptosporidium species and Entamoeba species in faecal samples was evaluated in an observational prospective study. Paediatric patients (0-18 years) presenting with gastrointestinal symptoms and suspected of having enteroparasitic disease were included. A questionnaire on gastrointestinal symptoms and the chosen treatment was completed at the start of the study and after 6 weeks. Of 163 paediatric patients (mean age, 7.8 years), 114 (70%) had a PCR-positive faecal sample. D. fragilis was detected most frequently, in 101 patients, followed by Blastocystis in 49. In faecal samples of 47 patients, more than one protozoan was detected, mainly the combination of D. fragilis and Blastocystis. Reported gastrointestinal symptoms were abdominal pain (78%), nausea (30%), and altered bowel habits (28%). Eighty-nine of the PCR-positive patients were treated with antibiotics. A significant reduction in abdominal pain was observed both in treated and in untreated patients. This study demonstrated that multiplex real-time PCR detects a high percentage of intestinal protozoa in paediatric patients with gastrointestinal symptoms. However, interpretation and determination of the clinical relevance of a positive PCR result in this population are still difficult. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Development and Evaluation of a Rapid and Sensitive EBOV-RPA Test for Rapid Diagnosis of Ebola Virus Disease.

PubMed

Yang, Mingjuan; Ke, Yuehua; Wang, Xuesong; Ren, Hang; Liu, Wei; Lu, Huijun; Zhang, Wenyi; Liu, Shiwei; Chang, Guohui; Tian, Shuguang; Wang, Lihua; Huang, Liuyu; Liu, Chao; Yang, Ruifu; Chen, Zeliang

2016-06-01

Confirming Ebola virus disease (EVD), a deadly infectious disease, requires real-time RT-PCR, which takes up to a few hours to yield results. Therefore, a rapid diagnostic assay is imperative for EVD diagnosis. A rapid nucleic acid test based on recombinase polymerase amplification (EBOV-RPA) was developed to specifically detect the 2014 outbreak strains. The EBOV-RPA assay was evaluated by testing samples from suspected EVD patients in parallel with RT-PCR. An EBOV-RPA, which could be completed in 20 min, was successfully developed. Of 271 patients who tested positive for Ebola virus by RT-PCR, 264 (sensitivity: 97%, 95% CI: 95.5-99.3%) were positive by EBOV-RPA; 101 of 104 patients (specificity: 97%, 95% CI: 93.9-100%) who tested negative by RT-PCR were also negative by EBOV-RPA. The sensitivity values for samples with a Ct value of <34, which accounted for 95.59% of the samples, was 100%. Discordant samples positive by RT-PCR but negative by EBOV-RPA had significantly high Ct values. Results of external quality assessment samples with EBOV-RPA were 100%, consistent with those of RT-PCR. The EBOV-RPA assay showed 97% sensitivity and 97% specificity for all EVD samples tested, making it a rapid and sensitive test for EVD diagnosis.

Simplified Pan-species Real-time PCR-based Detection of Plasmodium Spp. in Blood Smear.

PubMed

Hassanpour, Gholamreza; Mirhendi, Hossein; Mohebali, Mehdi; Raeisi, Ahmad; Zeraati, Hojjat; Keshavarz, Hossein

2016-01-01

We aimed to quicken and simplify the detection of Plasmodium in blood samples by developing and testing a pan- Plasmodium real-time PCR for accurate screening of individuals suspected of malaria. A single primer/probe set for pan-species Plasmodium -specific real time PCR targeting a conserved region of the small subunit 18S ribosomal DNA was designed and evaluated for rapid diagnosis and screening of malaria infections using dried blood smears. FTA cards were used for rapid and simple DNA extraction. The primers and probes showed a positive response with the DNA extracted from bloods infected with P. falciparum and P. vivax but not with DNA extracted from various smears from uninfected blood samples. Seven positive cases positive by both microscopy and nested PCR were found among 280 blood samples taken from in South and Southeast Iran. Five samples were identified as positive for P. vivax and two as positive for P. falciparum . All positive samples were positive by real-time PCR. Furthermore, all 38-blood samples positive by microscopy were positive by real-time PCR. No microscopy-negative samples were positive by real-time PCR. By using a simple FTA card for DNA extraction and by application of the real-time PCR developed in this study, sensitivity similar to nested-PCR and microscopy was achieved. This format simplifies the detection of Plasmodium in large numbers of samples.

Molecular cloning of a defense-response-related cytochrome P450 gene from tobacco.

PubMed

Takemoto, D; Hayashi, M; Doke, N; Nishimura, M; Kawakita, K

1999-12-01

Plant defenses against pathogen attack involve a series of inducible responses that contribute to resistance. Tobacco leaves injected with HWC (hyphal wall components prepared from Phytophthora infestans) elicitor showed typical defense responses, including the induction of localized necrosis and the accumulation of pathogenesis-related proteins. In order to elucidate the molecular mechanisms by which plant defense systems are activated, we screened tobacco plants for genes differentially expressed in response to HWC. We performed differential screening by RT-PCR with random primers and obtained PCR products specific to HWC-treated leaf RNA. Northern hybridization using the PCR products as probes confirmed that one transcript was actually induced by HWC treatment. As the deduced amino acid sequence of this clone showed the highest degree of similarity to elicitor-induced soybean cytochrome P450 CYP82A4, it was designated CYP82E1. The expression of CYP82E1 was strongly induced in tobacco by the soybean pathogen Pseudomonas syringae pv. glycinea (nonpathogenic on tobacco), but it was activated only slightly and in a delayed fashion by the tobacco pathogen P. syringae pv. tabaci (pathogenic on tobacco), implying that the product of CYP82E1 may be involved in disease resistance in tobacco.

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment.

PubMed

García-Mena, Jaime; Cano-Ramirez, Claudia; Garibay-Orijel, Claudio; Ramirez-Canseco, Sergio; Poggi-Varaldo, Héctor M

2005-06-01

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 microg-1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.

Dataset of proinflammatory cytokine and cytokine receptor gene expression in rainbow trout (Oncorhynchus mykiss) measured using a novel GeXP multiplex, RT-PCR assay

USDA-ARS?s Scientific Manuscript database

A GeXP multiplex, RT-PCR assay was developed and optimized that simultaneously measures expression of a suite of immune-relevant genes in rainbow trout (Oncorhynchus mykiss), concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. The dataset ...

Hypoxia decreases creatine uptake in cardiomyocytes, while creatine supplementation enhances HIF activation.

PubMed

Santacruz, Lucia; Arciniegas, Antonio Jose Luis; Darrabie, Marcus; Mantilla, Jose G; Baron, Rebecca M; Bowles, Dawn E; Mishra, Rajashree; Jacobs, Danny O

2017-08-01

Creatine (Cr), phosphocreatine (PCr), and creatine kinases (CK) comprise an energy shuttle linking ATP production in mitochondria with cellular consumption sites. Myocytes cannot synthesize Cr: these cells depend on uptake across the cell membrane by a specialized creatine transporter (CrT) to maintain intracellular Cr levels. Hypoxia interferes with energy metabolism, including the activity of the creatine energy shuttle, and therefore affects intracellular ATP and PCr levels. Here, we report that exposing cultured cardiomyocytes to low oxygen levels rapidly diminishes Cr transport by decreasing V max and K m Pharmacological activation of AMP-activated kinase (AMPK) abrogated the reduction in Cr transport caused by hypoxia. Cr supplementation increases ATP and PCr content in cardiomyocytes subjected to hypoxia, while also significantly augmenting the cellular adaptive response to hypoxia mediated by HIF-1 activation. Our results indicate that: (1) hypoxia reduces Cr transport in cardiomyocytes in culture, (2) the cytoprotective effects of Cr supplementation are related to enhanced adaptive physiological responses to hypoxia mediated by HIF-1, and (3) Cr supplementation increases the cellular ATP and PCr content in RNCMs exposed to hypoxia. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Characterization of Betula platyphylla gene transcripts associated with early development of male inflorescence.

PubMed

Xing, Lei; Liu, Xue-Mei

2012-02-01

Birch (Betula platyphylla), an eminent tree species in Northeast and Inner Mongolia of China, has been widely used in architecture, furniture, and paper making in recent years. In order to retrieve genes involved in early development of B. platyphylla male inflorescence, RNA populations extracted from early and late developmental stage were analyzed by cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique. Following amplification of 256 pairs of primer combinations, ~7000 fragments were generated, of which 350 transcripts expressing more in early stage than late. Of 350 specific transcripts, 198 clear and reproducible electrophoresis bands were retrieved and sequenced successfully, 74 of them (37%) showing significant homologies to known genes after GO annotation. Majority of the predicted gene products were involved in metabolism (24.56%), cellular process (27.19%), response to stimulus (11.4%) and cell growth (8.7%). Transcripts ME56, ME108, ME206 and ME310, representing metabolism, cellular process, response to stimulus and cell growth, respectively, were selected for further study to validate cDNA-AFLP expression patterns via RT-PCR and qRT-PCR analysis. RT-PCR and qRT-PCR expression pattern results were consistent with cDNA-AFLP analysis results.

Elevated ground-level O(3) changes the diversity of anoxygenic purple phototrophic bacteria in paddy field.

PubMed

Feng, Youzhi; Lin, Xiangui; Yu, Yongchang; Zhu, Jianguo

2011-11-01

The knowledge of the impact of elevated ground-level O(3) below ground the agro-ecosystem is limited. A field experiment in China Ozone Free-Air Concentration Enrichment (FACE-O(3)) facility on a rice-wheat rotation system was carried out to investigate responses of anoxygenic phototrophic purple bacteria (AnPPB) to elevated ground-level O(3). AnPPB community structures and sizes in paddy soil were monitored by molecular approaches including PCR-DGGE and real-time quantitative PCR based upon the pufM gene on three typical rice growth stages. Repetitive sequence-based PCR (rep-PCR) in combination with culture-reliant method was conducted to reveal changes in genotypic diversity. Elevated ground-level O(3) statistically reduce AnPPB abundance and percentage in total bacterial community in flooded rice soil via decreasing their genotypic diversity and metabolic versatility. Concomitantly, their community composition changed after rice anthesis stage under elevated ground-level O(3). Our results from AnPPB potential responses imply that continuously elevated ground-level O(3) in the future would eventually harm the health of paddy ecosystem through negative effect on soil microorganisms.

The use of ELISA, nPCR and qPCR for diagnosis of ocular toxoplasmosis in experimentally infected pigs.

PubMed

Garcia, João Luis; Burrells, Alison; Bartley, Paul M; Bartley, Kathryn; Innes, Elisabeth A; Katzer, Frank

2017-12-01

In the present study we experimentally infected pigs with T. gondii tachyzoites, bradyzoites and oocysts in order to evaluate IgG-ELISA, nested-PCR, and qPCR for diagnosis of ocular infection. Eighteen pigs were divided into four groups: G1 (infected with 10 3 tissue cysts of the M4 strain (type II) at day 28, n=5), G2 (infected with 10 3 oocysts of the M4 strain at day 28, n=5), G3 (infected with tachyzoites of S48 strain (type 1) at day 0, n=5), and G4 (uninfected unchallenged, control group n=3). At day 70 of the experiment all animals were culled, and serum, aqueous humor (AH) and vitreous humor (VH) samples were collected to perform indirect ELISA, and PCR (nPCR, and qPCR). By ELISA nine pigs (60%) out of 15 were positive in VH samples, and seven out of 15 (46%) were positive in AH samples. Both molecular techniques used here, nPCR and qPCR, were able to detect <50fg of T. gondii tachyzoite DNA. The nPCR and qPCR detected six (7/15, 47%) and two (2/15, 13.3%) positive animals respectively. Antibody responses were detected in serum and in AH and VH from the eye, suggesting that pigs may be an animal that could be used as a model to further our understanding of diagnosis of human ocular infection with T. gondii. Copyright © 2017 Elsevier Ltd. All rights reserved.

Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing.

PubMed

Borgmästars, Emmy; Jazi, Mehrdad Mousavi; Persson, Sofia; Jansson, Linda; Rådström, Peter; Simonsson, Magnus; Hedman, Johannes; Eriksson, Ronnie

2017-12-01

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with C q shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.

Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer.

PubMed

Ma, Yanlin; Deng, Yang; Xu, Zhenbo; Liu, Junyan; Dong, Jianjun; Yin, Hua; Yu, Junhong; Chang, Zongming; Wang, Dongfeng

The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30μg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0μg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3.

PubMed

Sharma, Deepa K; Nalavade, Uma P; Deshpande, Jagadish M

2015-10-01

The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre. No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus. rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.

Evaluation of Legionella real-time PCR against traditional culture for routine and public health testing of water samples.

PubMed

Collins, S; Stevenson, D; Walker, J; Bennett, A

2017-06-01

To evaluate the usefulness of Legionella qPCR alongside traditional culture for enumeration of Legionella from water samples as part of both routine and public health investigation testing. Routine water samples (n = 2002) and samples from public health investigations (n = 215) were analysed by culture and qPCR for Legionella spp., Legionella pneumophila and L. pneumophila sg-1. A negative qPCR result was highly predictive of a negative culture result for all water systems (negative predictive values, NPV from 97·4 to 100%). Positive predictive values (PPV) were lower (0-50%). Results for qPCR were generally larger than culture with average log 10 differences of 1·1 for Legionella spp. and 1·2 for L. pneumophila. Alert and action levels of 1000 and 10 000 GU per litre, respectively, are proposed for Legionella qPCR for hot and cold water systems (HCWS). The use of qPCR significantly reduced the time to results for public health investigations by rapidly identifying potential sources and ruling out others, thus enabling a more rapid and efficient response. The high NPV of qPCR supports its use to rapidly screen out negative samples without culture. Alert and action levels for Legionella qPCR for HCWS are proposed. Quantitative PCR will be a valuable tool for both routine and public health testing. This study generated comparative data of >2000 water samples by qPCR and culture. Action and alert levels have been recommended that could enable duty holders to interpret qPCR results to facilitate timely Legionella control and public health protection. © 2017 Crown copyright. Journal of Applied Microbiology © 2017 The Society for Applied Microbiology.

Nodavirus Colonizes and Replicates in the Testis of Gilthead Seabream and European Sea Bass Modulating Its Immune and Reproductive Functions

PubMed Central

Valero, Yulema; Arizcun, Marta; Esteban, M. Ángeles; Bandín, Isabel; Olveira, José G.; Patel, Sonal; Cuesta, Alberto; Chaves-Pozo, Elena

2015-01-01

Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17β-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases. PMID:26691348

The complete title: The effect of interleukin-28B rs12979860 polymorphism on the therapeutic response of Moroccan patients with chronic hepatitis C.

PubMed

Nadia, Kandoussi; Hicham, Elannaz; Reda, Tagajdid Mohamed; Nadia, Touil; Elarbi, Bouaiti; Saâd, Elkabbaj; Mimoun, Zouhdi; Saâd, Mrani

2015-08-15

There is increasing evidence for the effect of rs12979860 IL28B polymorphism in response to the standard treatment PEG-IFN/RBV (i.e. combination of pegylated interferon and ribavirin) in chronic hepatitis C virus (HCV) infection. The present study aimed to determine the impact of IL28B associations in interferon responsiveness in 187 Moroccan patients with chronic HCV infection. HCV RNA levels were measured with a real-time RT-PCR assay and treatment efficacy was assessed by sustained virological response (SVR) and patients were classified as responders or non-responders. IL28B rs12979860 polymorphism genotyping was achieved by PCR-HRM technique. The results demonstrated that SVR was achieved in 102 patients (55%); while 69 were non-responders (37%) and 16 relapsed (8%). Genotype 1 was the predominant HCV genotype detected in 112 patients followed by genotype 2 in 56 patients. The genotype CC was observed in 42 cases (25%); CT in 69 (41%) and TT in 57 (34%) demonstrating a C allele frequency of 46%. The SVR was observed in 32 patients with genotype CC accounting for 76%. The frequencies of rs12979860 CC type in infected individuals with HCV genotype 1 were 47% and 12% respectively in SVR and non-SVR groups. A highly statistically significant association between this SNP and SVR was found (p<0.001). Using multivariate logistic regression analysis, CC genotype was an independent factor for SVR. In the group of patients infected with genotype 2, SVR rate was 79%. The frequency of rs12979860 CC type in SVR group (n=4) was 9% and rs12979860 non-CC genotype was highly associated with SVR (p=0.001). This finding adds evidence that genotyping for the IL-28B rs12979860 SNP can be a good parameter for the prediction of treatment success in patients with chronic hepatitis C before initiation of antiviral therapy in Morocco. Copyright © 2015. Published by Elsevier B.V.

Oxidative stress provokes distinct transcriptional responses in the stress-tolerant atr7 and stress-sensitive loh2 Arabidopsis thaliana mutants as revealed by multi-parallel quantitative real-time PCR analysis of ROS marker and antioxidant genes.

PubMed

Mehterov, Nikolay; Balazadeh, Salma; Hille, Jacques; Toneva, Valentina; Mueller-Roeber, Bernd; Gechev, Tsanko

2012-10-01

The Arabidopsis thaliana atr7 mutant is tolerant to oxidative stress induced by paraquat (PQ) or the catalase inhibitor aminotriazole (AT), while its original background loh2 and wild-type plants are sensitive. Both, AT and PQ, which stimulate the intracellular formation of H₂O₂ or superoxide anions, respectively, trigger cell death in loh2 but do not lead to visible damage in atr7. To study gene expression during oxidative stress and ROS-induced programmed cell death, two platforms for multi-parallel quantitative real-time PCR (qRT-PCR) analysis of 217 antioxidant and 180 ROS marker genes were employed. The qRT-PCR analyses revealed AT- and PQ-induced expression of many ROS-responsive genes mainly in loh2, confirming that an oxidative burst plays a role in the activation of the cell death in this mutant. Some of the genes were specifically regulated by either AT or PQ, serving as markers for particular types of ROS. Genes significantly induced by both AT and PQ in loh2 included transcription factors (ANAC042/JUB1, ANAC102, DREB19, HSFA2, RRTF1, ZAT10, ZAT12, ethylene-responsive factors), signaling compounds, ferritins, alternative oxidases, and antioxidant enzymes. Many of these genes were upregulated in atr7 compared to loh2 under non-stress conditions at the first time point, indicating that higher basal levels of ROS and higher antioxidant capacity in atr7 are responsible for the enhanced tolerance to oxidative stress and suggesting a possible tolerance against multiple stresses of this mutant. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Assessing the reliability of microscopy and rapid diagnostic tests in malaria diagnosis in areas with varying parasite density among older children and adult patients in Nigeria.

PubMed

Ayogu, E E; Ukwe, C V; Nna, E O

2016-01-01

Current malaria control strategies are based on early diagnosis and appropriate treatment of malaria cases. The study aimed at comparing the performance of blood film microscopy and rapid diagnostic test (RDT) in Plasmodium falciparum detection in patients ≥6 years of age. A total of 154 consecutive pyretic patients aged 6-62 years were enrolled, sampled, and tested for malaria using RDT (first response) and microscopy by Giemsa staining. Genomic DNA was extracted after saponin hemolysis and nested polymerase chain reaction (PCR) was used to detect Plasmodium falciparum. The endpoints were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Of the 154 patients, 80 (51.9%) had fever of ≥37.5°C. 106 (68.8%) were positive by First response® , 132 (85.7%) by microscopy, and 121 (78.6%) by PCR. The sensitivity, specificity, PPV, and NPV of first response compared to microscopic method were 82.2%, 100.0%, 100.0%, and 34.3%, respectively, while it was 75.4%, 75.0%, 95.3%, and 31.2%, respectively, when compared to PCR. The sensitivity, specificity, PPV, and NPV of the microscopic method compared to PCR were 92.3%, 50.0%, 90.91%, and 54.5%, respectively. There was a significant difference in the performance of RDT and film microscopy methods (P ≤ 0.05). Microscopy performed better and is more reliable than first response (RDT) in areas with low parasite density among patients ≥6 years of age. Rapid diagnostic tests could be useful in aareas with high parasite density as an alternative to smear microscopy.

Multiplex reverse transcription polymerase chain reaction to study the expression of virulence and stress response genes in Staphylococcus aureus.

PubMed

Shrihari, Rohinishree Yadahalli; Singh, Negi Pradeep

2012-02-01

Staphylococcus aureus survives well in different stress conditions. The ability of this organism to adapt to various stresses is the result of a complex regulatory response, which is attributed to regulation of multiple genes. The aims of the present study were (1) to develop a multiplex PCR for the detection of genes which are involved in stress adaptation (asp23, dnaK, and groEL); alternative sigma factor (sigB) and virulence determination (entB and spa) and (2) to study the expression of these genes during stress conditions for S. aureus culture collection strains (FRI 722 and ATCC 6538) and S. aureus food isolates at mRNA level using multiplex reverse transcription polymerase chain reaction (RT-PCR). During heat shock treatment groEL, dnaK, asp23, sodA, entB, spa, and sigB genes were up regulated up to 2.58, 2.07, 2.76, 2.55, 3.55, 2.71, and 2.62- folds, respectively, whereas in acid shock treatment, sodA and groEL were up regulated; dnaK was downregulated; and entB and sigB genes were not expressed in food isolates. Multiplex PCR assay standardized in this study offers an inexpensive alternative to uniplex PCR for detection of various virulence and stress response genes. This study is relevant to rapid and accurate detection of potential pathogenic S. aureus in foods. © 2012 Institute of Food Technologists®

Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

PubMed Central

Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

2015-01-01

Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients. PMID:26111048

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells.

PubMed

Kostyuk, Svetlana; Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

2015-01-01

Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose-derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

2015-01-01

Background. Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose—derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis. PMID:26273425

Ki67 Proliferation Index as a Tool for Chemotherapy Decisions During and After Neoadjuvant Aromatase Inhibitor Treatment of Breast Cancer: Results From the American College of Surgeons Oncology Group Z1031 Trial (Alliance).

PubMed

Ellis, Matthew J; Suman, Vera J; Hoog, Jeremy; Goncalves, Rodrigo; Sanati, Souzan; Creighton, Chad J; DeSchryver, Katherine; Crouch, Erika; Brink, Amy; Watson, Mark; Luo, Jingqin; Tao, Yu; Barnes, Michael; Dowsett, Mitchell; Budd, G Thomas; Winer, Eric; Silverman, Paula; Esserman, Laura; Carey, Lisa; Ma, Cynthia X; Unzeitig, Gary; Pluard, Timothy; Whitworth, Pat; Babiera, Gildy; Guenther, J Michael; Dayao, Zoneddy; Ota, David; Leitch, Marilyn; Olson, John A; Allred, D Craig; Hunt, Kelly

2017-04-01

Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) -positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was > 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was > 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of > 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred > 2) versus PEPI > 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI > 0 (recurrence hazard ratio [PEPI = 0 v PEPI > 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A Phase III Study; clinical trial information: NCT01953588).

Clostridium difficile ribotypes in humans and animals in Brazil

PubMed Central

Silva, Rodrigo Otávio Silveira; Rupnik, Maja; Diniz, Amanda Nádia; Vilela, Eduardo Garcia; Lobato, Francisco Carlos Faria

2015-01-01

Clostridium difficile is an emerging enteropathogen responsible for pseudomembranous colitis in humans and diarrhoea in several domestic and wild animal species. Despite its known importance, there are few studies aboutC. difficile polymerase chain reaction (PCR) ribotypes in Brazil and the actual knowledge is restricted to studies on human isolates. The aim of the study was therefore to compare C. difficileribotypes isolated from humans and animals in Brazil. Seventy-six C. difficile strains isolated from humans (n = 25), dogs (n = 23), piglets (n = 12), foals (n = 7), calves (n = 7), one cat, and one manned wolf were distributed into 24 different PCR ribotypes. Among toxigenic strains, PCR ribotypes 014/020 and 106 were the most common, accounting for 14 (18.4%) and eight (10.5%) samples, respectively. Fourteen different PCR ribotypes were detected among human isolates, nine of them have also been identified in at least one animal species. PCR ribotype 027 was not detected, whereas 078 were found only in foals. This data suggests a high diversity of PCR ribotypes in humans and animals in Brazil and support the discussion of C. difficile as a zoonotic pathogen. PMID:26676318

Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics.

PubMed

Geng, Tao; Mathies, Richard A

2015-01-01

The presence of PCR inhibitors in forensic and other biological samples reduces the amplification efficiency, sometimes resulting in complete PCR failure. Here we demonstrate a high-performance digital agarose droplet microfluidics technique for single-cell and single-molecule forensic short tandem repeat (STR) typing of samples contaminated with high concentrations of PCR inhibitors. In our multifaceted strategy, the mitigation of inhibitory effects is achieved by the efficient removal of inhibitors from the porous agarose microgel droplets carrying the DNA template through washing and by the significant dilution of targets and remaining inhibitors to the stochastic limit within the ultralow nL volume droplet reactors. Compared to conventional tube-based bulk PCR, our technique shows enhanced (20 ×, 10 ×, and 16 ×) tolerance of urea, tannic acid, and humic acid, respectively, in STR typing of GM09948 human lymphoid cells. STR profiling of single cells is not affected by small soluble molecules like urea and tannic acid because of their effective elimination from the agarose droplets; however, higher molecular weight humic acid still partially inhibits single-cell PCR when the concentration is higher than 200 ng/μL. Nevertheless, the full STR profile of 9948 male genomic DNA contaminated with 500 ng/μL humic acid was generated by pooling and amplifying beads carrying single-molecule 9948 DNA PCR products in a single secondary reaction. This superior performance suggests that our digital agarose droplet microfluidics technology is a promising approach for analyzing low-abundance DNA targets in the presence of inhibitors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Improving molecular tools for global surveillance of measles virus.

PubMed

Bankamp, Bettina; Byrd-Leotis, Lauren A; Lopareva, Elena N; Woo, Gibson K S; Liu, Chunyu; Jee, Youngmee; Ahmed, Hinda; Lim, Wilina W; Ramamurty, Nalini; Mulders, Mick N; Featherstone, David; Bellini, William J; Rota, Paul A

2013-09-01

The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures. Published by Elsevier B.V.

Droplet digital PCR analysis of NOTCH1 gene mutations in chronic lymphocytic leukemia.

PubMed

Minervini, Angela; Francesco Minervini, Crescenzio; Anelli, Luisa; Zagaria, Antonella; Casieri, Paola; Coccaro, Nicoletta; Cumbo, Cosimo; Tota, Giuseppina; Impera, Luciana; Orsini, Paola; Brunetti, Claudia; Giordano, Annamaria; Specchia, Giorgina; Albano, Francesco

2016-12-27

In chronic lymphocytic leukemia (CLL), NOTCH1 gene mutations (NOTCH1mut) have been associated with adverse prognostic features but the independence of these as a prognostic factor is still controversial. In our study we validated a c.7541-7542delCT NOTCH1 mutation assay based on droplet digital PCR (ddPCR); we also analyzed the NOTCH1mut allelic burden, expressed as fractional abundance (FA), in 88 CLL patients at diagnosis to assess its prognostic role and made a longitudinal ddPCR analysis in 10 cases harboring NOTCH1mut to verify the FA variation over time. Our data revealed that with the ddPCR approach the incidence of NOTCH1mut in CLL was much higher (53.4%) than expected. However, longitudinal ddPCR analysis of CLL cases showed a statistically significant reduction of the NOTCH1mut FA detected at diagnosis after treatment (median FA 11.67 % vs 0.09 %, respectively, p = 0.01); the same difference, in terms of NOTCH1mut FA, was observed in the relapsed cases compared to the NOTCH1mut allelic fraction observed in patients in complete or partial remission (median FA 4.75% vs 0.43%, respectively, p = 0.007). Our study demonstrated a much higher incidence of NOTCH1mut in CLL than has previously been reported, and showed that the NOTCH1mut allelic burden evaluation by ddPCR might identify patients in need of a closer clinical follow-up during the "watch and wait" interval and after standard chemotherapy.

Improving molecular tools for global surveillance of measles virus⋆

PubMed Central

Bankamp, Bettina; Byrd-Leotis, Lauren A.; Lopareva, Elena N.; Woo, Gibson K.S.; Liu, Chunyu; Jee, Youngmee; Ahmed, Hinda; Lim, Wilina W.; Ramamurty, Nalini; Mulders, Mick N.; Featherstone, David; Bellini, William J.; Rota, Paul A.

2017-01-01

Background The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. Objectives The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. Study design A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. Results A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. Conclusions These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures. PMID:23806666

Comparative studies of mitochondrial proteomics reveal an intimate protein network of male sterility in wheat (Triticum aestivum L.)

PubMed Central

Wang, Shuping; Zhang, Gaisheng; Zhang, Yingxin; Song, Qilu; Chen, Zheng; Wang, Junsheng; Guo, Jialin; Niu, Na; Wang, Junwei; Ma, Shoucai

2015-01-01

Plant male sterility has often been associated with mitochondrial dysfunction; however, the mechanism in wheat (Triticum aestivum L.) has not been elucidated. This study set out to probe the mechanism of physiological male sterility (PHYMS) induced by the chemical hybridizing agent (CHA)-SQ-1, and cytoplasmic male sterility (CMS) of wheat at the proteomic level. A total of 71 differentially expressed mitochondrial proteins were found to be involved in pollen abortion and further identified by MALDI-TOF/TOF MS (matrix-assisted laser desorption/ionization-time of fight/time of flight mass spectrometry). These proteins were implicated in different cellular responses and metabolic processes, with obvious functional tendencies toward the tricarboxylic acid cycle, the mitochondrial electron transport chain, protein synthesis and degradation, oxidation stress, the cell division cycle, and epigenetics. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways involved in anther abortion and pollen defects. Accordingly, a mitochondria-mediated male sterility protein network in wheat is proposed; this network was further confirmed by physiological data, RT-PCR (real-time PCR), and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. The results provide intriguing insights into the metabolic pathway of anther abortion induced by CHA-SQ-1 and also give useful clues to identify the crucial proteins of PHYMS and CMS in wheat. PMID:26136264

Molecular cloning and expression analysis of the gene encoding proline dehydrogenase from Jatropha curcas L.

PubMed

Wang, Haibo; Ao, Pingxing; Yang, Shuanglong; Zou, Zhurong; Wang, Shasha; Gong, Ming

2015-03-01

Proline dehydrogenase (ProDH) (EC 1.5.99.8) is a key enzyme in the catabolism of proline. The enzyme JcProDH and its complementary DNA (cDNA) were isolated from Jatropha curcas L., an important woody oil plant used as a raw material for biodiesels. It has been classified as a member of the Pro_dh superfamily based on multiple sequence alignment, phylogenetic characterization, and its role in proline catabolism. Its cDNA is 1674 bp in length with a complete open reading frame of 1485 bp, which encodes a polypeptide chain of 494 amino acids with a predicted molecular mass of 54 kD and a pI of 8.27. Phylogenetic analysis indicated that JcProDH showed high similarity with ProDH from other plants. Reverse transcription PCR (RT-PCR) analysis revealed that JcProDH was especially abundant in the seeds and flowers but scarcely present in the stems, roots, and leaves. In addition, the expression of JcProDH increased in leaves experiencing environmental stress such as cold (5 °C), heat (42 °C), salt (300 mM), and drought (30 % PEG6000). The JcProDH protein was successfully expressed in the yeast strain INVSc1 and showed high enzyme activity in proline catabolism. This result confirmed that the JcProDH gene negatively participated in the stress response.

Idiotype vaccination in patients with myeloma reduced circulating myeloma cells (CMC).

PubMed

Abdalla, A O; Kokhaei, P; Hansson, L; Mellstedt, H; Osterborg, A

2008-06-01

Circulating myeloma cells (CMC), exhibiting the same immunoglobulin heavy-chain gene rearrangements as the plasma cells, are part of the myeloma clone. In this study, we evaluated the effect of idiotype (Id) vaccination on CMC. Eleven patients were immunized with the autologous Id in combinations with granulocyte-macrophage colony-stimulating factor and interleukin 12, and followed for CMC by quantitative real-time allele-specific PCR. Id-specific T cells were monitored by proliferation assay, enzyme-linked immunospot (interferon-gamma) assay, and quantitative real-time PCR for cytokines. Regulatory T (T(reg)) cells were analyzed by flow cytometry. CMC were detected in 9 of 11 patients at start of vaccination. In four patients, CMC declined and two had a complete molecular remission. Further two patients had stable levels of CMC during follow-up, while in three patients CMC progressively increased. Six patients had a vaccine-induced Id-specific T-cell response. A significant correlation was observed between reduced/stable levels of CMC and the Id-specific T cells (P < 0.02). The frequency of T(reg) cells was decreased in immune responders, but increased in immune nonresponders (P < 0.05). No significant change in the serum M-protein concentration was, however, observed in any patient. Id vaccination reduced CMC, which correlated with vaccine-induced Id-specific T cells. Further studies are warranted to analyze the clinical significance of CMC and clinical effects of Id vaccination.

Clinical value of polymerase chain reaction in the diagnosis of joint tuberculosis by detecting the DNA of Mycobacterium tuberculosis.

PubMed

Sun, Yong-sheng; Lou, Si-quan; Wen, Jian-min; Lv, Wei-xin; Jiao, Chang-geng; Yang, Su-min; Xu, Hai-bin

2011-02-01

To assess the clinical value of polymerase chain reaction (PCR) in the diagnosis and differential diagnosis of joint tuberculosis (TB). PCR was used blindly to detect the DNA of Mycobacterium tuberculosis (M.TB) in five specimens of M.TB, 5 of BCG, and 10 of other bacteria. Then, M. TB in 98 samples from patients with joint TB and 100 samples from patients with non-tubercular joint disorders were detected by PCR, acid-fast staining and culture,. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PCR were calculated. The χ2 test was used for statistical analysis of the frequency of various factors. At the same time, some problems with PCR were also systematically analyzed. (1) In the "standard samples", both M. TB and BCG showed positive while other bacteria were negative. (2) In 98 cases from patients with joint TB, 81 were positive by PCR, 6 by acid-fast staining, and 17 by culture. In 100 cases from patients with non-tuberculous joint disorders, 9 were positive by PCR, and none by either acid-fast staining or culture. Sensitivity, specificity, accuracy, positive and negative predictive value of PCR were 82.65% (81/98), 91.00% (91/100), 86.87% (172/198), 90.00% (81/90) and 84.26% (91/108), respectively. (3) The positive rates for PCR, acid-fast staining and culture in detection of M. TB were 82.65% (81/98), 6.12% (6/98), and 17.34% (17/98), respectively. There were statistically significant differences between the three methods (P < 0.001). (4) The process of PCR is automatic, and can be completed within 3 to 6 hours, whereas 4 to 8 weeks are required for the conventional culture of M. TB. PCR is a sensitive, specific, rapid, simple and minimally invasive method for detection of M. TB in samples from joint TB, and can play an important role in early and rapid diagnosis and differential diagnosis of joint TB. But it also has some limitations, such as false positivity and false negativity. © 2011 Tianjin Hospital and Blackwell Publishing Asia Pty Ltd.

Day-to-day variability in spot urine protein-creatinine ratio measurements.

PubMed

Naresh, Chetana N; Hayen, Andrew; Craig, Jonathan C; Chadban, Steven J

2012-10-01

Accurate measurement of proteinuria is important in the diagnosis and management of chronic kidney disease (CKD). The reference standard test, 24-hour urinary protein excretion, is inconvenient and vulnerable to collection errors. Spot urine protein-creatinine ratio (PCR) is a convenient alternative and is in widespread use. However, day-to-day variability in PCR measurements has not been evaluated. Prospective cohort study of day-to-day variability in spot urine PCR measurement. Clinically stable outpatients with CKD (n = 145) attending a university hospital CKD clinic in Australia between July 2007 and April 2010. Spot urine PCR. Spot PCR variability was assessed and repeatability limits were determined using fractional polynomials. Spot PCRs were measured from urine samples collected at 9:00 am on consecutive days and 24-hour urinary protein excretion was collected concurrently. Paired results were analyzed from 145 patients: median age, 56 years; 59% men; and median 24-hour urinary protein excretion, 0.7 (range, 0.06-35.7) g/d. Day-to-day variability was substantial and increased in absolute terms, but decreased in relative terms with increasing baseline PCR. For patients with a low baseline PCR (20 mg/mmol [177 mg/g]), a change greater than ±160% (repeatability limits, 0-52 mg/mmol [0-460 mg/g]) is required to indicate a real change in proteinuria status with 95% certainty, whereas for those with a high baseline PCR (200 mg/mmol [1,768 mg/g]), a change of ±50% (decrease to <100 mg/mmol [<884 mg/g] or increase to >300 mg/mmol [>2,652 mg/g]) represents significant change. These study results need to be replicated in other ethnic groups. Changes in PCR observed in patients with CKD, ranging from complete resolution to doubling of PCR values, could be due to inherent biological variation and may not indicate a change in disease status. This should be borne in mind when using PCR in the diagnosis and management of CKD. Copyright © 2012 National Kidney Foundation, Inc. All rights reserved.

rpoB Gene Sequencing for Identification of Corynebacterium Species

PubMed Central

Khamis, Atieh; Raoult, Didier; La Scola, Bernard

2004-01-01

The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species. PMID:15364970

Genetic characterization of avian metapneumovirus subtype C isolated from pheasants in a live bird market.

PubMed

Lee, Eun ho; Song, Min-Suk; Shin, Jin-Young; Lee, Young-Min; Kim, Chul-Joong; Lee, Young Sik; Kim, Hyunggee; Choi, Young Ki

2007-09-01

Complete nucleotide sequences of two avian metapneumoviruses (aMPV), designated PL-1 and PL-2, were isolated from pheasants, revealing novel sequences of the first aMPV to be fully sequenced in Korea. The complete genome of both PL-1 and PL-2 was composed of 13,170 nucleotides. Phylogenetic analysis revealed that PL-1 belonged to aMPV subtype C, sharing higher homology in deduced amino acid sequence identities with hMPV, rather than with aMPV subtypes A and B. Replication of PL-1 in experimentally re-infected pheasants was confirmed by reverse transcription (RT)-polymerase chain reaction (PCR). Chickens and mice were experimentally inoculated with PL-1 to test the replication potential of PL-1 in other species. Although one specimen from the nasal turbinates of an inoculated chicken showed a slight trace of viral replication at 3 days post-infection (dpi), all of the infected mice were negative for aMPV by RT-PCR throughout the experiment, suggesting that PL-1 does not readily infect mammals. This is the first report of the isolation and complete genomic sequence of aMPV subtype C originating from pheasants.

Differential expression of alpha 2 macroglobulin in response to dietylstilbestrol and in ovarian carcinomas in chickens

PubMed Central

2011-01-01

Background Alpha 2 macroglobulin (A2M; also known as ovostatin), a homotetrameric protein with four disulfide-linked subunits, has the unique feature of inactivating/inhibiting most known proteases including serine-, threonine-, cysteine-, aspartic- and metalloproteases. In chickens, A2M has been identified and characterized biochemically, but little is known of its functional role(s) in the oviduct, hormonal regulation of expression or its expression in ovarian carcinomas in chickens. Therefore, we investigated estrogen regulation of A2M gene expression during development of the chicken oviduct, and its expression in normal and cancerous ovaries from chickens. Methods To determine tissue-specific expression of A2M in chickens, we collected various organs from male and female chickens and performed RT-PCR analyses. To examine A2M gene expression in the oviduct of 1-week-old female chicks that received a subcutaneous implant of 15 mg DES in the abdominal region for 20 days, we performed RT-PCR, qPCR and in situ hybridization analyses using cDNAs from control- (n = 5) and DES-treated oviducts (n = 5), and then each segment of the oviduct from DES-treated chicks. To determine if A2M is a biomarker of ovarian cancer in hens, we collected cancerous (n = 10) ovaries from a total of 136 chickens which had completely stopped egg-laying and performed RT-PCR and in situ hybridization analyses. Results We found that A2M is most abundant in the chicken oviduct, specifically luminal (LE) and glandular epithelia (GE), but it was not detected in any other tissues of either sex. We then determined that DES (dietylstilbestrol, a synthetic nonsteroidal estrogen) increased A2M mRNA only in LE and GE of the oviduct of chicks. Further, expression of A2M was most abundant in GE of endometrioid adenocarcinoma of cancerous, but not normal ovaries of hens. Conclusions Collectively, results of the present study indicate that A2M is novel estrogen-stimulated gene expressed in LE and GE of the chicken oviduct and may be used for monitoring effects of therapies for ovarian cancer in laying hens. PMID:21978460

A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control consortium

PubMed Central

2014-01-01

We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the United States Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed, for these and qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings. PMID:25150838

Detection of viable but non-culturable Escherichia coli O157:H7 by PCR in combination with propidium monoazide.

PubMed

Zhong, Junliang; Zhao, Xihong

2018-01-01

The aim of this study was to evaluate the applicability of the conventional PCR detection method combined with propidium monoazide (PMA) treatment for the detection of viable but non-culturable (VBNC) state Escherichia coli O157:H7 in ground beef meatballs. Under low temperature, E. coli O157:H7 cells were induced into the VBNC state in ground beef meatballs at - 20 °C after 152 days. The optimal PMA concentration of 5 µg/mL was obtained in beef meatball samples, which could completely inhibit the DNA amplification on dead cells (10 6  cells/mL) but with no inhibition on viable cells. The established PMA-PCR assay revealed that the VBNC counts exceeded 10 7  CFU/mL in artificial contamination beef samples, which could be used for semi-quantitative detection of VBNC cells in beef meatball samples. This study indicated that the PMA-PCR assay might be a potential method for detection of VBNC state E . coli O157:H7 cells in food products.

Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci.

PubMed

Stevens, Aaron J; Taylor, Millie G; Pearce, Frederick Grant; Kennedy, Martin A

2017-03-10

Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1 , BLCAP , DNMT1 , PLAGL1 , KCNQ1 , and GRB10 These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations. Copyright © 2017 Stevens et al.

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.

PubMed

Goossens, Dirk; Moens, Lotte N; Nelis, Eva; Lenaerts, An-Sofie; Glassee, Wim; Kalbe, Andreas; Frey, Bruno; Kopal, Guido; De Jonghe, Peter; De Rijk, Peter; Del-Favero, Jurgen

2009-03-01

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics. 2008 Wiley-Liss, Inc.

A rapid real-time PCR method to differentiate between mottled skate (Beringraja pulchra) and other skate and ray species.

PubMed

Kim, Mi-Ra; Kwon, Kisung; Jung, Yoo-Kyung; Kang, Tae Sun

2018-07-30

Skates and rays are commercially important fish in South Korea, and among them, Beringraja pulchra has the highest economic value. However, the similar morphological traits among skates and rays are often exploited for seafood fraud. Here, we designed both Beringraja pulchra-specific and skate-universal primer sets, capable of detecting short sequences in the cytochrome oxidase subunit I gene, and developed highly sensitive and reliable quantitative real-time PCR (qPCR) assays to differentiate between Beringraja pulchra and other skate and ray species. AΔCq method based on differences in the amplification efficiency was developed, validated, and then used to confirm the presence of Beringraja pulchra in twenty-six commercial skate products. The averageΔCq value obtained for other skate species (18.94 ± 3.46) was significantly higher than that of Beringraja pulchra (1.18 ± 0.15). For on-site applications, we developed an ultra-fast qPCR assay, allowing for completion of the entire analytical procedure within 30 min. Copyright © 2018 Elsevier Ltd. All rights reserved.

Neoadjuvant chemoradiation therapy and pathological complete response in rectal cancer

PubMed Central

Ferrari, Linda; Fichera, Alessandro

2015-01-01

The management of rectal cancer has evolved significantly in the last few decades. Significant improvements in local disease control were achieved in the 1990s, with the introduction of total mesorectal excision and neoadjuvant radiotherapy. Level 1 evidence has shown that, with neoadjuvant chemoradiation therapy (CRT) the rates of local recurrence can be lower than 6% and, as a result, neoadjuvant CRT currently represents the accepted standard of care. This approach has led to reliable tumor down-staging, with 15–27% patients with a pathological complete response (pCR)—defined as no residual cancer found on histological examination of the specimen. Patients who achieve pCR after CRT have better long-term outcomes, less risk of developing local or distal recurrence and improved survival. For all these reasons, sphincter-preserving procedures or organ-preserving options have been suggested, such as local excision of residual tumor or the omission of surgery altogether. Although local recurrence rate has been stable at 5–6% with this multidisciplinary management method, distal recurrence rates for locally-advanced rectal cancers remain in excess of 25% and represent the main cause of death in these patients. For this reason, more recent trials have been looking at the administration of full-dose systemic chemotherapy in the neoadjuvant setting (in order to offer early treatment of disseminated micrometastases, thus improving control of systemic disease) and selective use of radiotherapy only in non-responders or for low rectal tumors smaller than 5 cm. PMID:26290512

Novel dehydrins lacking complete K-segments in Pinaceae. The exception rather than the rule

PubMed Central

Perdiguero, Pedro; Collada, Carmen; Soto, Álvaro

2014-01-01

Dehydrins are thought to play an essential role in the plant response, acclimation and tolerance to different abiotic stresses, such as cold and drought. These proteins contain conserved and repeated segments in their amino acid sequence, used for their classification. Thus, dehydrins from angiosperms present different repetitions of the segments Y, S, and K, while gymnosperm dehydrins show A, E, S, and K segments. The only fragment present in all the dehydrins described to date is the K-segment. Different works suggest the K-segment is involved in key protective functions during dehydration stress, mainly stabilizing membranes. In this work, we describe for the first time two Pinus pinaster proteins with truncated K-segments and a third one completely lacking K-segments, but whose sequence homology leads us to consider them still as dehydrins. qRT-PCR expression analysis show a significant induction of these dehydrins during a severe and prolonged drought stress. By in silico analysis we confirmed the presence of these dehydrins in other Pinaceae species, breaking the convention regarding the compulsory presence of K-segments in these proteins. The way of action of these unusual dehydrins remains unrevealed. PMID:25520734

The Complete Genome Sequence of the Plant Growth-Promoting Bacterium Pseudomonas sp. UW4

PubMed Central

Duan, Jin; Jiang, Wei; Cheng, Zhenyu; Heikkila, John J.; Glick, Bernard R.

2013-01-01

The plant growth-promoting bacterium (PGPB) Pseudomonas sp. UW4, previously isolated from the rhizosphere of common reeds growing on the campus of the University of Waterloo, promotes plant growth in the presence of different environmental stresses, such as flooding, high concentrations of salt, cold, heavy metals, drought and phytopathogens. In this work, the genome sequence of UW4 was obtained by pyrosequencing and the gaps between the contigs were closed by directed PCR. The P. sp. UW4 genome contains a single circular chromosome that is 6,183,388 bp with a 60.05% G+C content. The bacterial genome contains 5,423 predicted protein-coding sequences that occupy 87.2% of the genome. Nineteen genomic islands (GIs) were predicted and thirty one complete putative insertion sequences were identified. Genes potentially involved in plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, trehalose production, siderophore production, acetoin synthesis, and phosphate solubilization were determined. Moreover, genes that contribute to the environmental fitness of UW4 were also observed including genes responsible for heavy metal resistance such as nickel, copper, cadmium, zinc, molybdate, cobalt, arsenate, and chromate. Whole-genome comparison with other completely sequenced Pseudomonas strains and phylogeny of four concatenated “housekeeping” genes (16S rRNA, gyrB, rpoB and rpoD) of 128 Pseudomonas strains revealed that UW4 belongs to the fluorescens group, jessenii subgroup. PMID:23516524

Differential effects of antifungal agents on expression of genes related to formation of Candida albicans biofilms.

PubMed

Chatzimoschou, Athanasios; Simitsopoulou, Maria; Antachopoulos, Charalampos; Walsh, Thomas J; Roilides, Emmanuel

2016-01-01

The purpose of this study was to analyse specific molecular mechanisms involved in the intrinsic resistance of C. albicans biofilms to antifungals. We investigated the transcriptional profile of three genes (BGL2, SUN41, ECE1) involved in Candida cell wall formation in response to voriconazole or anidulafungin after the production of intermediate and mature biofilms. C. albicans M61, a well-documented biofilm producer strain, was used for the development of intermediate (12 h and 18 h) and completely mature biofilms (48 h). After exposure of cells from each biofilm growth mode to voriconazole (128 and 512 mg l(-1)) or anidulafungin (0.25 and 1 mg l(-1)) for 12-24 h, total RNA samples extracted from biofilm cells were analysed by RT-PCR. The voriconazole and anidulafungin biofilm MIC was 512 and 0.5 mg l(-1) respectively. Anidulafungin caused significant up-regulation of SUN41 (3.7-9.3-fold) and BGL2 (2.2-2.8 fold) in intermediately mature biofilms; whereas, voriconazole increased gene expression in completely mature biofilms (SUN41 2.3-fold, BGL2 2.1-fold). Gene expression was primarily down-regulated by voriconazole in intermediately, but not completely mature biofilms. Both antifungals caused down-regulation of ECE1 in intermediately mature biofilms. © 2015 Blackwell Verlag GmbH.

Transcription factors WRKY11 and WRKY17 are involved in abiotic stress responses in Arabidopsis.

PubMed

Ali, Muhammad Amjad; Azeem, Farrukh; Nawaz, Muhammad Amjad; Acet, Tuba; Abbas, Amjad; Imran, Qari Muhammad; Shah, Kausar Hussain; Rehman, Hafiz Mamoon; Chung, Gyuhwa; Yang, Seung Hwan; Bohlmann, Holger

2018-04-17

Plant WRKY transcription factors play a vital role in abiotic stress tolerance and regulation of plant defense responses. This study examined AtWRKY11 and AtWRKY17 expression under ABA, salt, and osmotic stress at different developmental stages in Arabidopsis. We used reverse transcriptase PCR, quantitative real-time PCR, and promoter:GUS lines to analyze expression. Both genes were upregulated in response to abiotic stress. Next, we applied the same stressors to seedlings of T-DNA insertion wrky11 and 17 knock-out mutants (single and double). Under stress, the mutants exhibited slower germination and compromised root growth compared with the wild type. In most cases, double-mutant seedlings were more affected than single mutants. These results suggest that wrky11 and wrky17 are not strictly limited to plant defense responses but are also involved in conferring stress tolerance. Copyright © 2018 Elsevier GmbH. All rights reserved.

Identification of reference genes for RT-qPCR in ovine mammary tissue during late pregnancy and lactation and in response to maternal nutritional programming.

PubMed

Paten, A M; Pain, S J; Peterson, S W; Blair, H T; Kenyon, P R; Dearden, P K; Duncan, E J

2014-08-01

The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland. Copyright © 2014 the American Physiological Society.

Longitudinal Analysis of Vaginal Microbiome Dynamics in Women with Recurrent Bacterial Vaginosis: Recognition of the Conversion Process

PubMed Central

Lambert, Janet A.; John, Susan; Sobel, Jack D.; Akins, Robert A.

2013-01-01

Bacterial vaginosis (BV) affects ∼30% of women of reproductive age, has a high rate of recurrence, and is associated with miscarriage, preterm birth, and increased risk of acquiring other sexually transmitted infections, including HIV-1. Little is known of the daily changes in the vaginal bacterial composition as it progresses from treatment to recurrence, or whether any of these might be useful in its prediction or an understanding of its causes. We used phylogenetic branch-inclusive quantitative PCR (PB-qPCR) and Lactobacillus blocked/unblocked qPCR (Lb-qPCR) to characterize longitudinal changes in the vaginal microbiota in sequential vaginal self-swabs from five women with recurrent BV, from diagnosis through remission to recurrence. Both patients with acute BV samples dominated by G. vaginalis recurred during the study with similar profiles, whereas the three patients with acute BV samples dominated by other anaerobes did not recur or recurred to an intermediate Nugent score. L. iners dominated remission phases, with intermittent days of abnormal microbial profiles typically associated with menses. The exception was a newly discovered phenomenon, a sustained period of abnormal profiles, termed conversion, which preceded symptomatic acute BV. Species known to have antagonistic activity towards Lactobacillus were detected in pre-conversion samples, possibly contributing to the decline in Lactobacillus. Lb-qPCR scores define two categories of response in the initial post-treatment visit samples; scores <5 may correspond with poor response to treatment or rapid recurrence, whereas scores >8 may predict delayed or no recurrence. Amsel criteria or Nugent scores did not have this potential predictive capability. Larger studies are warranted to evaluate the prognostic potential of detecting conversion and poor Lb-qPCR scores at the post-treatment visit of recurrent BV patients. PMID:24376552

Accurate clinical genetic testing for autoinflammatory diseases using the next-generation sequencing platform MiSeq.

PubMed

Nakayama, Manabu; Oda, Hirotsugu; Nakagawa, Kenji; Yasumi, Takahiro; Kawai, Tomoki; Izawa, Kazushi; Nishikomori, Ryuta; Heike, Toshio; Ohara, Osamu

2017-03-01

Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program-which detects and avoids common SNPs in gene-specific PCR primers-we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.

An Analysis of Thaumarchaeota Populations from the Northern Gulf of Mexico

PubMed Central

Tolar, Bradley B.; King, Gary M.; Hollibaugh, James T.

2013-01-01

We sampled Thaumarchaeota populations in the northern Gulf of Mexico, including shelf waters under the Mississippi River outflow plume that are subject to recurrent hypoxia. Data from this study allowed us to: (1) test the hypothesis that Thaumarchaeota would be abundant in this region; (2) assess phylogenetic composition of these populations for comparison with other regions; (3) compare the efficacy of quantitative PCR (qPCR) based on primers for 16S rRNA genes (rrs) with primers for genes in the ammonia oxidation (amoA) and carbon fixation (accA, hcd) pathways; (4) compare distributions obtained by qPCR with the relative abundance of Thaumarchaeota rrs in pyrosequenced libraries; (5) compare Thaumarchaeota distributions with environmental variables to help us elucidate the factors responsible for the distributions; (6) compare the distribution of Thaumarchaeota with Nitrite-Oxidizing Bacteria (NOB) to gain insight into the coupling between ammonia and nitrite oxidation. We found up to 108 copies L−1 of Thaumarchaeota rrs in our samples (up to 40% of prokaryotes) by qPCR, with maximum abundance in slope waters at 200–800 m. Thaumarchaeota rrs were also abundant in pyrosequenced libraries and their relative abundance correlated well with values determined by qPCR (r2 = 0.82). Thaumarchaeota populations were strongly stratified by depth. Canonical correspondence analysis using a suite of environmental variables explained 92% of the variance in qPCR-estimated gene abundances. Thaumarchaeota rrs abundance was correlated with salinity and depth, while accA abundance correlated with fluorescence and pH. Correlations of Archaeal amoA abundance with environmental variables were primer-dependent, suggesting differential responses of sub-populations to environmental variables. Bacterial amoA was at the limit of qPCR detection in most samples. NOB and Euryarchaeota rrs were found in the pyrosequenced libraries; NOB distribution was correlated with that of Thaumarchaeota (r2 = 0.49). PMID:23577005

Auto-antibodies and Autoimmune Disease during Treatment of Children with Chronic Hepatitis C

PubMed Central

Molleston, Jean P.; Mellman, William; Narkewicz, Michael R.; Balistreri, William F.; Gonzalez-Peralta, Regino P.; Jonas, Maureen M.; Lobritto, Steven J.; Mohan, Parvathi; Murray, Karen F.; Njoku, Dolores; Rosenthal, Philip; Barton, Bruce A.; Talor, Monica V.; Cheng, Irene; Schwarz, Kathleen B.; Haber, Barbara A.

2012-01-01

Objectives Auto-antibodies were studied in a well-characterized cohort of children with chronic hepatitis C (CHC) during treatment with PEG-IFN and ribavirin to assess the relationship to treatment and development of autoimmune disease. Methods 114 children (5–17 years), previously screened for the presence of high titer autoantibodies, were randomized to Peg-IFN with or without ribavirin. Anti-nuclear (ANA), anti-liver-kidney-microsomal (LKM), anti-thyroglobulin (TG), anti-thyroid peroxidase (TPO), insulin (IA2), anti-glutamic acid decarboxylase (GAD) antibodies were measured after trial completion using frozen sera. Results At baseline,19% had auto-antibodies: ANA (8%), LKM (4%), and GAD (4%). At 24 and 72 weeks (24 weeks after treatment completion), 23% and 26% had auto-antibodies (p=0.50, 0.48 compared to baseline). One child developed diabetes and two hypothyroidism during treatment; none developed autoimmune hepatitis. At 24 weeks, the incidence of flu-like symptoms, gastrointestinal symptoms, and headaches were 42%, 8% and 19% in those with auto-antibodies vs. 52%, 17%, and 26% in those without (p=0.18, 0.36, and 0.20, respectively). In children with negative HCV PCR at 24 weeks, there was no difference in the rate of early virologic response /sustained virologic response respectively in those with auto-antibodies 76%/69%, vs 58%/65% in those without (p=0.48). Conclusions Despite screening, we found autoantibodies commonly at baseline, during treatment for CHC and after. The presence of antibodies did not correlate with viral response, side effects, or autoimmune hepatitis. Neither screening nor archived samples assayed for thyroid and diabetes-related antibodies identified the 3 subjects who developed overt autoimmune disease, diabetes (1) and hypothyroidism (2). PMID:23439301

Markers of hepatic regeneration associated with surgical attenuation of congenital portosystemic shunts in dogs.

PubMed

Tivers, Michael S; Lipscomb, Victoria J; Smith, Kenneth C; Wheeler-Jones, Caroline P D; House, Arthur K

2014-05-01

Dogs with congenital portosystemic shunts (CPSS) have liver hypoplasia and hepatic insufficiency. Surgical CPSS attenuation results in liver growth associated with clinical improvement. The mechanism of this hepatic response is unknown, although liver regeneration is suspected. This study investigated whether markers of liver regeneration were associated with CPSS attenuation. Dogs treated with CPSS attenuation were prospectively recruited. Residual liver tissue was collected for gene expression analysis (seven genes) from 24 CPSS dogs that tolerated complete attenuation, 25 dogs that tolerated partial attenuation and seven control dogs. Relative gene expression was measured using quantitative polymerase chain reaction (qPCR). Blood samples were collected before, 24 h and 48 h post-surgery from 36 CPSS dogs and from 10 control dogs. Serum hepatocyte growth factor (HGF) concentration was measured using a canine specific enzyme-linked immunosorbent assay (ELISA). HGF mRNA expression was significantly decreased in CPSS compared with control dogs (P = 0.046). There were significant increases in HGF (P = 0.050) and methionine adenosyltransferase 2 A (MAT2A; P = 0.002) mRNA expression following partial CPSS attenuation. Dogs with complete attenuation had significantly greater MAT2A (P = 0.024) mRNA expression compared with dogs with partial attenuation. Serum HGF concentration significantly increased 24 h following CPSS attenuation (P < 0.001). Hepatic mRNA expression of two markers of hepatocyte proliferation (HGF and MAT2A) was associated with the response to surgery in dogs with CPSS, and serum HGF significantly increased following surgery, suggesting hepatocyte proliferation. These findings support the concept that hepatic regeneration is important in the hepatic response to CPSS surgery. Copyright © 2014 Elsevier Ltd. All rights reserved.

PubMed Central

Taran, Florin-Andrei; Schneeweiss, Andreas; Lux, Michael P.; Janni, Wolfgang; Hartkopf, Andreas D.; Nabieva, Naiba; Overkamp, Friedrich; Kolberg, Hans-Christian; Hadji, Peyman; Tesch, Hans; Wöckel, Achim; Ettl, Johannes; Lüftner, Diana; Wallwiener, Markus; Müller, Volkmar; Beckmann, Matthias W.; Belleville, Erik; Wallwiener, Diethelm; Brucker, Sara Y.; Fasching, Peter A.; Fehm, Tanja N.; Schütz, Florian

2018-01-01

This summary provides an overview of how new therapies or new aspects of established therapies relate to the latest findings. Neoadjuvant therapy, local therapy, new aspects of systemic therapy, and prognostic and predictive factors are presented. In the neoadjuvant setting, the association between pathological complete response (pCR) and prognosis is still of interest as is the identification of new molecular predictors for new therapies such as CDK4/6 inhibitors. As regards surgical treatment, the target is still to reduce the aggressiveness of surgery. To achieve this, a better understanding particularly of ductal carcinoma in situ is required. With regard to systemic therapy, more data on the best combinations and therapy sequences for existing therapies is available. Finally, the use of prognostic and predictive factors may help to avoid overtreatment and ensure that patients only receive therapies which have been shown to be effective for their specific condition and have fewer side effects. PMID:29576629

Interferon-α Based Individualized Treatment of a High Risk Chronic Myelogenous Leukemia Patient Harboring T315I Mutation.

PubMed

Zeng, Yunxin; Zhang, Jingwen; Li, Xiaoqing; Zhang, Ling; Liu, Jiajun

2018-06-01

T315I mutation is the most common BCR-ABL mutation and confers resistance to all the first and second generation BCR-ABL tyrosine kinases, including nilotinib and dasatinib. We report a high risk chronic myelogenous leukemia (CML) patient harboring the T315I mutation treated by Interferon-α (INF-α) solo and subsequently combined with dasatinib. Hematological investigation, bone marrow cytology inspection, chromosomal analysis (G-banding), and real-time quantitative polymerase chain reaction (RQ-PCR) were performed on a 47-year-old male patient. After 8 months IFN-α monotherapy, the patient lost the T315I mutation but acquired a new F359V mutation. After 2 months on dasatinib and INF-α treatment, the patient achieved complete hematologic response (CHR). IFN-α based combination therapy could be a viable treatment option for CML patients harboring T315I BCR-ABL mutation.

Isolation and characterization of StERF transcription factor genes from potato (Solanum tuberosum L.).

PubMed

Wang, Zemin; Zhang, Ning; Zhou, Xiangyan; Fan, Qiang; Si, Huaijun; Wang, Di

2015-04-01

Ethylene response factor (ERF) is a major subfamily of the AP2/ERF family and plays significant roles in the regulation of abiotic- and biotic-stress responses. ERF proteins can interact with the GCC-box cis-element and then initiate a transcriptional cascade activating downstream ethylene response and enhancing plant stress tolerance. In this research, we cloned five StERF genes from potato (Solanum tuberosum L.). The expressional analysis of StERF genes revealed that they showed tissue- or organ-specific expression patterns and the expression levels in leaf, stem, root, flower, and tuber were different. The assays of quantitative real-time polymerase chain reaction (qRT-PCR) and the reverse transcription-PCR (RT-PCR) showed that the expression of five StERF genes was regulated by ethephon, methyl jasmonate (MeJA), salt and drought stress. The result from the yeast one-hybrid experiment showed that five StERFs had trans-activation activity and could specifically bind to the GCC-box cis-elements. The StERFs responded to abiotic factors and hormones suggested that they possibly had diverse roles in stress and hormone regulation of potato. Copyright © 2015 Académie des sciences. Published by Elsevier SAS. All rights reserved.

COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma.

PubMed

Galbiati, Silvia; Monguzzi, Alessandra; Damin, Francesco; Soriani, Nadia; Passiu, Marianna; Castellani, Carlo; Natacci, Federica; Curcio, Cristina; Seia, Manuela; Lalatta, Faustina; Chiari, Marcella; Ferrari, Maurizio; Cremonesi, Laura

2016-07-01

Until now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through the detection of a paternally inherited disease allele in cases where maternal and paternal mutated alleles differ. Conditions for non-invasive identification of fetal paternally inherited mutations in maternal plasma were developed by two independent approaches: coamplification at lower denaturation temperature-PCR (COLD-PCR) and highly sensitive microarrays. Assays were designed for identifying 14 mutations, 7 causing β-thalassaemia and 7 cystic fibrosis. In total, 87 non-invasive prenatal diagnoses were performed by COLD-PCR in 75 couples at risk for β-thalassaemia and 12 for cystic fibrosis. First, to identify the more appropriate methodology for the analysis of minority mutated fetal alleles in maternal plasma, both fast and full COLD-PCR protocols were developed for the most common Italian β-thalassaemia Cd39 and IVSI.110 mutations. In 5 out of 31 samples, no enrichment was obtained with the fast protocol, while full COLD-PCR provided the correct fetal genotypes. Thus, full COLD-PCR protocols were developed for all the remaining mutations and all analyses confirmed the fetal genotypes obtained by invasive prenatal diagnosis. Microarray analysis was performed on 40 samples from 28 couples at risk for β-thalassaemia and 12 for cystic fibrosis. Results were in complete concordance with those obtained by both COLD-PCR and invasive procedures. COLD-PCR and microarray approaches are not expensive, simple to handle, fast and can be easily set up in specialised clinical laboratories where prenatal diagnosis is routinely performed. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture.

PubMed

Hiitiö, Heidi; Riva, Rauna; Autio, Tiina; Pohjanvirta, Tarja; Holopainen, Jani; Pyörälä, Satu; Pelkonen, Sinikka

2015-05-01

Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85.7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83.0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97.0 and 95.8% compared with BC. For Staphylococcus spp., the corresponding figures were 86.7 and 75.4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37.0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.

The effect of ethnicity on the performance of protein-creatinine ratio in the prediction of significant proteinuria in pregnancies at risk of or with established hypertension: an implementation audit and cost implications.

PubMed

Bhatti, Sadia; Cordina, Mark; Penna, Leonie; Sherwood, Roy; Dew, Tracy; Kametas, Nikos A

2018-05-01

The replacement of 24-h urine collection by protein-creatinine ratio (PCR) for the diagnosis of preeclampsia has been recently recommended. However, the literature is conflicting and there are concerns about the impact of demographic characteristics on the performance of PCR. This was an implementation audit of the introduction of PCR in a London Tertiary obstetric unit. The performance of PCR in the prediction of proteinuria ≥300 mg/day was assessed in 476 women with suspected preeclampsia who completed a 24-h urine collection and an untimed urine sample for PCR calculation. Multivariate logistic regression was used to assess the independent predictors of significant proteinuria. In a pregnant population, ethnicity and PCR are the main predictors of ≥300 mg proteinuria in a 24-h urine collection. A PCR cut-off of 30 mg/mmol would have incorrectly classified as non-proteinuric, 41.4% and 22.9% of black and non-black women, respectively. Sensitivity of 100% is achieved at cut-offs of 8.67 and 20.56 mg/mmol for black and non-black women, respectively. Applying these levels as a screening tool to inform the need to perform a 24-h urine collection in 1000 women, would lead to a financial saving of €2911 in non-black women and to an additional cost of €3269 in black women. Our data suggest that a move from screening for proteinuria with a 24-h urine collection to screening with urine PCR is not appropriate for black populations. However, the move may lead to cost-saving if used in the white population with a PCR cut-off of 20.5. © 2018 Nordic Federation of Societies of Obstetrics and Gynecology.

Molecular assessment of microbiota structure and dynamics along mixed olive oil and winery wastewaters biotreatment.

PubMed

Eusébio, Ana; Tacão, Marta; Chaves, Sandra; Tenreiro, Rogério; Almeida-Vara, Elsa

2011-07-01

The major parcel of the degradation occurring along wastewater biotreatments is performed either by the native microbiota or by added microbial inocula. The main aim of this study was to apply two fingerprinting methods, temperature gradient gel electrophoresis (TGGE) and length heterogeneity-PCR (LH-PCR) analysis of 16S rRNA gene fragments, in order to assess the microbiota structure and dynamics during mixed olive oil and winery wastewaters aerobic biotreatment performed in a jet-loop reactor (JLR). Sequence homology analysis showed the presence of bacterial genera Gluconacetobacter, Klebsiella, Lactobacillus, Novosphingobium, Pseudomonas, Prevotella, Ralstonia, Sphingobium and Sphingomonas affiliated with five main phylogenetic groups: alpha-, beta- and gamma-Proteobacteria, Firmicutes and Bacteroidetes. LH-PCR analysis distinguished eight predominant DNA fragments correlated with the samples showing highest performance (COD removal rates of 67 up to 75%). Cluster analysis of both TGGE and LH-PCR fingerprinting profiles established five main clusters, with similarity coefficients higher than 79% (TGGE) and 62% (LH-PCR), and related with hydraulic retention time, indicating that this was the main factor responsible for the shifts in the microbiota structure. Canonical correspondence analysis revealed that changes observed on temperature and O(2) level were also responsible for shifts in microbiota composition. Community level metabolic profile analysis was used to test metabolic activities in samples. Integrated data revealed that the microbiota structure corresponds to bacterial groups with high degradative potential and good suitability for this type of effluents biotreatments.

Comparative analysis of detection methods for congenital cytomegalovirus infection in a Guinea pig model.

PubMed

Park, Albert H; Mann, David; Error, Marc E; Miller, Matthew; Firpo, Matthew A; Wang, Yong; Alder, Stephen C; Schleiss, Mark R

2013-01-01

To assess the validity of the guinea pig as a model for congenital cytomegalovirus (CMV) infection by comparing the effectiveness of detecting the virus by real-time polymerase chain reaction (PCR) in blood, urine, and saliva. Case-control study. Academic research. Eleven pregnant Hartley guinea pigs. Blood, urine, and saliva samples were collected from guinea pig pups delivered from pregnant dams inoculated with guinea pig CMV. These samples were then evaluated for the presence of guinea pig CMV by real-time PCR assuming 100% transmission. Thirty-one pups delivered from 9 inoculated pregnant dams and 8 uninfected control pups underwent testing for guinea pig CMV and for auditory brainstem response hearing loss. Repeated-measures analysis of variance demonstrated no statistically significantly lower weight for the infected pups compared with the noninfected control pups. Six infected pups demonstrated auditory brainstem response hearing loss. The sensitivity and specificity of the real-time PCR assay on saliva samples were 74.2% and 100.0%, respectively. The sensitivity of the real-time PCR on blood and urine samples was significantly lower than that on saliva samples. Real-time PCR assays of blood, urine, and saliva revealed that saliva samples show high sensitivity and specificity for detecting congenital CMV infection in guinea pigs. This finding is consistent with recent screening studies in human newborns. The guinea pig may be a good animal model in which to compare different diagnostic assays for congenital CMV infection.

A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World

PubMed Central

Gilligan, Todd M.; Tembrock, Luke R.; Farris, Roxanne E.; Barr, Norman B.; van der Straten, Marja J.; van de Vossenberg, Bart T. L. H.; Metz-Verschure, Eveline

2015-01-01

The Old World bollworm, Helicoverpa armigera (Hübner), and the corn earworm, H. zea (Boddie), are two of the most important agricultural pests in the world. Diagnosing these two species is difficult—adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP) analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion) to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2) amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae. PMID:26558366

Inactivation of human norovirus and Tulane virus in simple media and fresh whole strawberries by ionizing radiation.

PubMed

DiCaprio, Erin; Phantkankum, Nuttapong; Culbertson, Doug; Ma, Yuanmei; Hughes, John H; Kingsley, David; Uribe, Roberto M; Li, Jianrong

2016-09-02

Human norovirus (NoV) is a major cause of fresh produce-associated outbreaks and human NoV in irrigation water can potentially lead to viral internalization in fresh produce. Therefore, there is a need to develop novel intervention strategies to target internalized viral pathogens while maintaining fresh produce quality. In this study electron beam (E-beam) and gamma radiation were evaluated for efficacy against a human NoV GII.4 strain and Tulane virus (TV). Virus survival following ionizing radiation treatments was determined using direct quantitative reverse transcriptase PCR (RT-qPCR), the porcine gastric mucin magnetic bead (PGM-MB) binding assay followed by RT-qPCR, and plaque assay. In simple media, a high dose of E-beam treatment was required to completely abolish the receptor binding ability of human NoV (35.3kGy) and TV (19.5-24.1kGy), as assessed using the PGM-MB binding assay. Both human NoV and TV were more susceptible to gamma irradiation than E-beam, requiring 22.4kGy to achieve complete inactivation. In whole strawberries, no human NoV or TV RNA was detected following 28.7kGy of E-beam treatment using the PGM-MB binding assay. Overall, human NoV and TV are highly resistant to ionizing radiation and therefore the technology may not be suitable to eliminate viruses in fresh produce at the currently approved levels. In addition, the PGM-MB binding assay is an improved method to detect viral infectivity compared to direct RT-qPCR. Copyright © 2016. Published by Elsevier B.V.

Comparison of culture, polymerase chain reaction, and fluorescent in situ hybridization for detection of Brachyspira hyodysenteriae and "Brachyspira hampsonii" in pig feces.

PubMed

Wilberts, Bailey L; Warneke, Hallie L; Bower, Leslie P; Kinyon, Joann M; Burrough, Eric R

2015-01-01

Swine dysentery is characterized by mucohemorrhagic diarrhea and can occur following infection by Brachyspira hyodysenteriae or "Brachyspira hampsonii ". A definitive diagnosis is often based on the isolation of strongly beta-hemolytic spirochetes from selective culture or by the application of species-specific polymerase chain reaction (PCR) assays directly to feces. While culture is highly sensitive, it typically requires 6 or more days to complete, and PCR, although rapid, can be limited by fecal inhibition. Fluorescent in situ hybridization (FISH) has been described in formalin-fixed tissues; however, completion requires approximately 2 days. Because of the time constraints of available assays, a same-day FISH assay was developed to detect B. hyodysenteriae and "B. hampsonii " in pig feces using previously described oligonucleotide probes Hyo1210 and Hamp1210 for B. hyodysenteriae and "B. hampsonii", respectively. In situ hybridization was simultaneously compared with culture and PCR on feces spiked with progressive dilutions of spirochetes to determine the threshold of detection for each assay at 0 and 48 hr. The PCR assay on fresh feces and FISH on formalin-fixed feces had similar levels of detection. Culture was the most sensitive method, detecting the target spirochetes at least 2 log-dilutions less when compared to other assays 48 hr after sample preparation. Fluorescent in situ hybridization also effectively detected both target species in formalin-fixed feces from inoculated pigs as part of a previous experiment. Accordingly, FISH on formalin-fixed feces from clinically affected pigs can provide same-day identification and preliminary speciation of spirochetes associated with swine dysentery in North America. © 2014 The Author(s).