Multiplexed digital PCR for detection of HPV in tissue samples from oropharyngeal cancer patients.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e17552-e17552 ◽  
Author(s):  
Sunil Kumar ◽  
Bhishamjit S. Chera ◽  
Brian Beaty ◽  
David Marron ◽  
Stuart Jefferys ◽  
...  
Keyword(s):  
False Positive ◽  
Oropharyngeal Cancer ◽  
Copy Number ◽  
False Positive Rate ◽  
Diagnostic Testing ◽  
Digital Pcr ◽  
Tissue Samples ◽  
Diploid Genome ◽  
Hpv Dna ◽  
Cellular Genes

e17552 Background: p16 immunohistochemistry (IHC) is a commonly used method for identifying HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). However, p16 overexpression in a minority of HPV negative OPSCC can give rise to false positive results. In contrast, HPV nucleic acid testing lacks adequate sensitivity for routine diagnostic testing. The goal of this study was to investigate whether a multiplexed digital PCR assay can detect and quantify HPV DNA in p16 positive OPSCC treated with definitive chemo-radiotherapy (CRT). Methods: Formalin-fixed paraffin embedded (FFPE) residual diagnostic biopsy specimens were collected from 57 patients. Macrodissection was performed to ensure tumor cellularity > 70%. Extracted genomic DNA was analyzed by next generation sequencing (NGS) using a hybrid capture assay (UNCSeq) targeting > 200 cellular genes and HPV 16/18 genomes. We also designed, validated, and implemented an multiplexed droplet digital PCR (dPCR) assay to detect and quantify HPV DNA (strains 16, 18, 31, 33 and 35) in tissue samples, relative to a genomic control locus (chromosome 6). Results: HPV strain 16 DNA was identified in 50 patients (87.7%), whereas 5 patients (8.8%) had HPV DNA from an alternative high-risk strain (18/31/33/35). HPV DNA was undetectable in 2 patients, indicating a false positive rate of 3.5% for p16 IHC testing in this cohort. HPV DNA copy number per diploid genome equivalent varied significantly across samples, with a median value of 19.7 (range 0.23-1712). A significant correlation was observed between the copies of HPV detected by dPCR and NGS (R2 = 0.5853, p < 0.0001). Evidence for HPV integration was detected by NGS in 23 out of 56 evaluable tumors (42%). There was a trend towards a higher prevalence of HPV integration in tumors with less than 10 HPV copies per diploid genome relative to cancers with > / = 10 HPV copy number (63% versus 34%, p = 0.07). Conclusions: Multiplexed digital PCR demonstrates excellent sensitivity for detection and typing of HPV DNA in diagnostic FFPE specimens from patients with p16 positive oropharyngeal cancer. HPV copy number varies significantly across samples, with a possible association with HPV integration status. Future investigations of potential correlation between HPV copy number, integration status, and clinical outcomes are warranted. Clinical trial information: NCT02281955, NCT03077243.

scholarly journals Copy Number and Prevalence of Porcine Endogenous Retroviruses (PERVs) in German Wild Boars

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 419
Author(s):  
Luise Krüger ◽  
Milena Stillfried ◽  
Carolin Prinz ◽  
Vanessa Schröder ◽  
Lena Katharina Neubert ◽  
...  
Keyword(s):  
Wild Boar ◽  
Copy Number ◽  
Digital Pcr ◽  
Endogenous Retroviruses ◽  
Wild Boars ◽  
Domestic Pigs ◽  
Copy Numbers ◽  
Cellular Genes ◽  
Porcine Endogenous Retroviruses ◽  
Different Populations

Porcine endogenous retroviruses (PERVs) are integrated in the genome of pigs and are transmitted like cellular genes from parents to the offspring. Whereas PERV-A and PERV-B are present in all pigs, PERV-C was found to be in many, but not all pigs. When PERV-C is present, recombination with PERV-A may happen and the PERV-A/C recombinants are characterized by a high replication rate. Until now, nothing has been known about the copy number of PERVs in wild boars and little is known about the prevalence of the phylogenetically youngest PERV-C in ancient wild boars. Here we investigated for the first time the copy number of PERVs in different populations of wild boars in and around Berlin using droplet digital PCR. Copy numbers between 3 and 69 per genome have been measured. A lower number but a higher variability was found compared to domestic pigs, including minipigs reported earlier (Fiebig et al., Xenotransplantation, 2018). The wild boar populations differed genetically and had been isolated during the existence of the Berlin wall. Despite this, the variations in copy number were larger in a single population compared to the differences between the populations. PERV-C was found in all 92 analyzed animals. Differences in the copy number of PERV in different organs of a single wild boar indicate that PERVs are also active in wild boars, replicating and infecting new cells as has been shown in domestic pigs.

scholarly journals Comparative analysis of HER2 copy number between plasma and tissue samples in gastric cancer using droplet digital PCR

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Boram Kim ◽  
Soo Kyung Nam ◽  
Soo Hyun Seo ◽  
Kyoung Un Park ◽  
Sang-Hoon Ahn ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Comparative Analysis ◽  
Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Tissue Samples

scholarly journals Author Correction: Comparative analysis of HER2 copy number between plasma and tissue samples in gastric cancer using droplet digital PCR

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Boram Kim ◽  
Soo Kyung Nam ◽  
Soo Hyun Seo ◽  
Kyoung Un Park ◽  
Sang-Hoon Ahn ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Comparative Analysis ◽  
Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Tissue Samples

scholarly journals Detection of High-Risk Human Papillomavirus in Oral Cavity Squamous Cell Carcinoma Using Multiple Analytes and Their Role in Patient Survival

2018 ◽  
pp. 1-33 ◽  
Author(s):  
Vinayak Palve ◽  
Jamir Bagwan ◽  
Neeraja M. Krishnan ◽  
Manisha Pareek ◽  
Udita Chandola ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Human Papillomavirus ◽  
Oral Cavity ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Copy Number ◽  
Patient Survival ◽  
Digital Pcr ◽  
Hpv Dna ◽  
Multiple Analytes

Purpose Accurate detection of human papillomavirus (HPV) in oral cavity squamous cell carcinoma (OSCC) is essential to understanding the role of HPV in disease prognosis and management of patients. We used different analytes and methods to understand the true prevalence of HPV in a cohort of patients with OSCC with different molecular backgrounds, and we correlated HPV data with patient survival. Methods We integrated data from multiple analytes (HPV DNA, HPV RNA, and p16), assays (immunohistochemistry, polymerase chain reaction [PCR], quantitative PCR [qPCR], and digital PCR), and molecular changes (somatic mutations and DNA methylation) from 153 patients with OSCC to correlate p16 expression, HPV DNA, and HPV RNA with HPV incidence and patient survival. Results High prevalence (33% to 58%) of HPV16/18 DNA did not correlate with the presence of transcriptionally active viral genomes (15%) in tumors. Eighteen percent of the tumors were p16 positive and only 6% were both HPV DNA and HPV RNA positive. Most tumors with relatively high copy number HPV DNA and/or HPV RNA, but not with HPV DNA alone (irrespective of copy number), were wild-type for TP53 and CASP8 genes. In our study, p16 protein, HPV DNA, and HPV RNA, either alone or in combination, did not correlate with patient survival. Nine HPV-associated genes stratified the virus-positive from the virus-negative tumor group with high confidence ( P < .008) when HPV DNA copy number and/or HPV RNA were considered to define HPV positivity, and not HPV DNA alone, irrespective of copy number ( P < .2). Conclusion In OSCC, the presence of both HPV RNA and p16 is rare. HPV DNA alone is not an accurate measure of HPV positivity and therefore may not be informative. HPV DNA, HPV RNA, and p16 do not correlate with patients’ outcome.

scholarly journals Glutathione Peroxidase (GPx) and Superoxide Dismutase (SOD) in Oropharyngeal Cancer Associated with EBV and HPV Coinfection

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1008
Author(s):  
Małgorzata Strycharz-Dudziak ◽  
Sylwia Fołtyn ◽  
Jakub Dworzański ◽  
Małgorzata Kiełczykowska ◽  
Maria Malm ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Glutathione Peroxidase ◽  
Oropharyngeal Cancer ◽  
Hpv Infection ◽  
Cancer Tissue ◽  
Upper Aerodigestive Tract ◽  
Sod Activity ◽  
Tissue Samples ◽  
Hpv Dna

Recent reports have pointed to the link between persistent inflammation, oxidative stress, and carcinogenesis; however most of the studies concerning the role of viruses in head and neck cancer (HNC) are focused mainly on one type of virus. Our present study aimed to study the relationship between Epstein–Barr virus/human papilloma virus (EBV/HPV) coinfection and glutathione peroxidase (GPx) and superoxide dismutase (SOD) level in oropharyngeal cancer. Fresh-frozen tumor tissue samples were collected from 128 patients with oropharyngeal cancer infected with EBV or HPV or with EBV/HPV coinfection. After DNA extraction, EBV and HPV DNA was detected using a polymerase chain reaction (PCR) assay. GPx and SOD activity was determined in homogenates of cancer tissue using diagnostic kits produced by Randox Laboratories. Both GPx and SOD activity was statistically lower in patients with EBV/HPV coinfection than in a single EBV or HPV infection. Analysis of GPx and SOD activity in relation to histological grading and tumor, node (TN) classification revealed that in poorly-differentiated tumors, the level of antioxidant enzymes was lower compared with well-differentiated lesions and in cases with greater tumor dimensions and lymph-node involvement, both GPx and SOD activity was decreased. Further studies are necessary to clarify the influence of interplay between EBV, HPV, and oxidative stress on malignant transformation of upper aerodigestive tract epithelial cells.

scholarly journals High-risk human papillomavirus in oral cavity squamous cell carcinoma

2016 ◽  
Author(s):  
Vinayak Palve ◽  
Jamir Bagwan ◽  
Neeraja M Krishnan ◽  
Manisha Pareek ◽  
Udita Chandola ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Human Papillomavirus ◽  
Oral Cavity ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Copy Number ◽  
Patient Survival ◽  
Digital Pcr ◽  
Assay Sensitivity ◽  
Hpv Dna

ABSTRACTPurposeThe prevalence of human papillomavirus (HPV) in oral cavity squamous cell carcinoma (OSCC) varies significantly based on assay sensitivity and patient geography. Accurate detection is essential to understand the role of HPV in disease prognosis and management of patients with OSCC.MethodsWe generated and integrated data from multiple analytes (HPV DNA, HPV RNA, and p16), assays (immunohistochemistry, PCR, qPCR and digital PCR) and molecular changes (somatic mutations and DNA methylation) from 153 OSCC patients to correlate p16 expression, HPV DNA, and HPV RNA with HPV incidence and patient survival.ResultsHigh prevalence (33-58%) of HPV16/18 DNA did not correlate with the presence of transcriptionally active viral genomes (15%) in tumors. Eighteen percent of the tumors were p16 positive. and only 6% were both HPV DNA and RNA positive. Most tumors with relatively high-copy HPV DNA, and/or HPV RNA, but not with HPV DNA alone (irrespective of copy number), were wild-type for TP53 and CASP8 genes. In our study, p16 protein, HPV DNA and HPV RNA, either alone or in combinations, did not correlate with patient survival. Nine HPV-associated genes stratified the virus +ve from the –ve tumor group with high confidence (p<0.008) when HPV DNA copy number and/or HPV RNA were considered to define HPV positivity and not HPV DNA alone irrespective of their copy number (p < 0.2).ConclusionsIn OSCC, the presence of both HPV RNA and p16 are rare. HPV DNA alone is not an accurate measure of HPV positivity and therefore not informative. Moreover, HPV DNA, RNA or p16 don’t correlate with outcome.

scholarly journals HPV Status as Prognostic Biomarker in Head and Neck Cancer—Which Method Fits the Best for Outcome Prediction?

Cancers ◽  
2021 ◽  
Vol 13 (18) ◽  
pp. 4730
Author(s):  
Jan Philipp Kühn ◽  
Wendelin Schmid ◽  
Sandrina Körner ◽  
Florian Bochen ◽  
Silke Wemmert ◽  
...  
Keyword(s):  
Head And Neck Cancer ◽  
Head And Neck ◽  
Neck Cancer ◽  
Oropharyngeal Cancer ◽  
Direct Detection ◽  
Multivariable Analysis ◽  
Tissue Samples ◽  
Hpv Dna ◽  
Hpv Status ◽  
Dna Pcr

The incidence of human papillomavirus (HPV)-related head and neck cancer (HNSCC) is rising globally, presenting challenges for optimized clinical management. To date, it remains unclear which biomarker best reflects HPV-driven carcinogenesis, a process that is associated with better therapeutic response and outcome compared to tobacco/alcohol-induced cancers. Six potential HPV surrogate biomarkers were analyzed using FFPE tissue samples from 153 HNSCC patients (n = 78 oropharyngeal cancer (OPSCC), n = 35 laryngeal cancer, n = 23 hypopharyngeal cancer, n = 17 oral cavity cancer): p16, CyclinD1, pRb, dual immunohistochemical staining of p16 and Ki67, HPV-DNA-PCR, and HPV-DNA-in situ hybridization (ISH). Biomarkers were analyzed for correlation with one another, tumor subsite, and patient survival. P16-IHC alone showed the best performance for discriminating between good (high expression) vs poor outcome (low expression; p = 0.0030) in OPSCC patients. Additionally, HPV-DNA-ISH (p = 0.0039), HPV-DNA-PCR (p = 0.0113), and p16-Ki67 dual stain (p = 0.0047) were significantly associated with prognosis in uni- and multivariable analysis for oropharyngeal cancer. In the non-OPSCC group, however, none of the aforementioned surrogate markers was prognostic. Taken together, P16-IHC as a single biomarker displays the best diagnostic accuracy for prognosis stratification in OPSCC patients with a direct detection of HPV-DNA by PCR or ISH as well as p16-Ki67 dual stain as potential alternatives.

scholarly journals SMN1 and SMN2 Copy Number Determination by droplet digital PCR (ddPCR) based on Extreme Value Theory for Threshold Estimation

2017 ◽  
Author(s):  
Mohammad Niknazar ◽  
Michael Jansen ◽  
Oscar Puig
Keyword(s):  
Extreme Value Theory ◽  
Copy Number ◽  
Muscular Atrophy ◽  
Diagnostic Testing ◽  
Digital Pcr ◽  
Homozygous Deletion ◽  
Value Theory ◽  
Extreme Value ◽  
Threshold Estimation ◽  
Copy Number Determination

AbstractSpinal muscular atrophy (SMA) is the most frequent genetic cause of infantile death. Homozygous deletion screening of survival of motor neuron (SMN1) represents the first tier in diagnostic testing. In this work, we adopted and optimized a method to increase the accuracy of SMN1 and SMN2 copy number determination on ddPCR platform. This method, which makes no assumption about the distribution of the fluorescence readouts, was shown to significantly increase accuracy and outperform QuantaSoft software on problematic SMN1 samples.Method SummaryIn order to increase the accuracy of SMN1 and SMN2 copy number determination by ddPCR, we adopted and optimized a method based on extreme value theory for threshold estimation. Our method increases total accuracy and improves resolution of problematic samples.

scholarly journals Same-species Contamination Detection With Variant Calling Information From Next-generation Sequencing

2021 ◽  
Author(s):  
Tao Jiang ◽  
Martin Buchkovich ◽  
Alison Motsinger-Reif
Keyword(s):  
Quality Control ◽  
Next Generation Sequencing ◽  
False Positive ◽  
Copy Number ◽  
False Positive Rate ◽  
Variant Calling ◽  
Degraded Samples ◽  
Svm Model ◽  
Positive Rate ◽  
Generation Sequencing

Abstract Background: Same-species contamination detection is an important quality control step in genetic data analysis. Due to a scarcity of methods to detect and correct for this quality control issue, same-species contamination is more difficult to detect than cross-species contamination. We introduce a novel machine learning algorithm to detect same-species contamination in next-generation sequencing (NGS) data using a support vector machine (SVM) model. Our approach uniquely detects contamination using variant calling information stored in variant call format (VCF) files for DNA or RNA. Importantly, it can differentiate between same-species contamination and mixtures of tumor and normal cells.In the first stage, a change-point detection method is used to identify copy number variations (CNVs) and copy number aberrations (CNAs) for filtering. Next, single nucleotide polymorphism (SNP) data is used to test for same-species contamination using an SVM model. Based on the assumption that alternative allele frequencies in NGS follow the beta-binomial distribution, the deviation parameter ρ is estimated by the maximum likelihood method. All features of a radial basis function (RBF) kernel SVM are generated using publicly available or private training data. Results: We demonstrate our approach in simulation experiments. The datasets combine, in silico, exome sequencing data of DNA from two lymphoblastoid cell lines (NA12878 and NA10855). We generate VCF files using variants identified in these data and then evaluate the power and false-positive rate of our approach. Our approach can detect contamination levels as low as 5% with a reasonable false-positive rate. Results in real data have sensitivity above 99.99% and specificity of 90.24%, even in the presence of degraded samples with similar features as contaminated samples. We provide an R software implementation of our approach.Conclusions: Our approach addresses the gap in methods to test for same-species contamination in NGS. Due to its high sensitivity for degraded samples and tumor-normal samples, it represents an important tool that can be applied within the quality control process. Additionally, the user-friendly software has the unique ability to conduct quality control using the VCF format.

scholarly journals ClinSV: Clinical grade structural and copy number variant detection from whole genome sequencing data

2020 ◽  
Author(s):  
Andre E Minoche ◽  
Ben Lundie ◽  
Greg B Peters ◽  
Thomas Ohnesorg ◽  
Mark Pinese ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
False Positive ◽  
Copy Number ◽  
False Positive Rate ◽  
Copy Number Variants ◽  
Copy Number Variant ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
Sequencing Data

AbstractWhole genome sequencing (WGS) has the potential to outperform clinical microarrays for the detection of structural variants (SV) including copy number variants (CNVs), but has been challenged by high false positive rates. Here we present ClinSV, a WGS based SV integration, annotation, prioritisation and visualisation method, which identified 99.8% of pathogenic ClinVar CNVs >10kb and 11/11 pathogenic variants from matched microarrays. The false positive rate was low (1.5–4.5%) and reproducibility high (95–99%). In clinical practice, ClinSV identified reportable variants in 22 of 485 patients (4.7%) of which 35–63% were not detectable by current clinical microarray designs.

Sign in / Sign up

Export Citation Format

Share Document