Dataset Information

Detection of Human Papillomavirus Integration in Brain Metastases from Oropharyngeal Tumors by Targeted Sequencing.

ABSTRACT: Human papillomavirus (HPV) positive and negative head and neck squamous cell carcinoma (HNSCC) are known to have differential phenotypes, including the incidence and location of metastases. HPV positive (HPV+) HNSCC are more likely to metastasize to distant sites, such as the lung, brain, and skin. Among these locations, metastasis to the brain is a rare event, and little is known about specific risk factors for this phenotype. In this report, we describe two patients who developed brain metastases from HNSCC. Both patient tumors had p16INK4a overexpression, suggesting these tumors were HPV+. This was confirmed after PCR, in situ hybridization, and mass spectrometry detected the presence of HPV type 16 (HPV16) DNA, RNA and protein. To further characterize the presence of HPV16, we used a target enrichment strategy on tumor DNA and RNA to isolate the viral sequences from the brain metastases. Analysis by targeted next generation sequencing revealed that both tumors had the HPV genome integrated into the host genome at known hotspots, 8q24.21 and 14q24.1. Applying a similar target enrichment strategy to a larger cohort of HPV+ HNSCC brain metastases could help to identify biomarkers that can predict metastasis and/or identify novel therapeutic options.

SUBMITTER: McEllin B

PROVIDER: S-EPMC8402651 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Detection of Human Papillomavirus Integration in Brain Metastases from Oropharyngeal Tumors by Targeted Sequencing.

McEllin Brian B Searle Brian C BC DePledge Lisa L Sun George G Cobbs Charles C Karimi Mohsen M

Viruses 20210803 8

Human papillomavirus (HPV) positive and negative head and neck squamous cell carcinoma (HNSCC) are known to have differential phenotypes, including the incidence and location of metastases. HPV positive (HPV+) HNSCC are more likely to metastasize to distant sites, such as the lung, brain, and skin. Among these locations, metastasis to the brain is a rare event, and little is known about specific risk factors for this phenotype. In this report, we describe two patients who developed brain metasta ...[more]

PMID: 34452401

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Project description:IntroductionIn North America and in most European countries, Human Papillomavirus (HPV) is responsible for over 70% of oropharyngeal squamous cell carcinomas. The burden of OPSCC, in high-income countries, has been steadily increasing over the past 20 years. As a result, in the USA and in the UK, the burden of HPV-related oropharyngeal squamous cell carcinoma in men has now surpassed that of cervical cancer in women. However, the oncogenic impact of high-risk HPV integration in oropharyngeal squamous cell carcinomas hasn't been extensively studied. The present study aimed to explore the patterns of HPV integration in oropharyngeal squamous cell carcinomas and to assess the feasibility and reliability of long-read sequencing technology in detecting viral integration events in oropharyngeal head and neck cancers.MethodsA cohort of eight HPV-positive OPSCC pre-treatment patient tumors (four males and four females), were selected. All patients received a p16INK4A positive OPSCC diagnosis and were treated at the McGill University Health Centre, a quaternary center in Montreal. A minimum of 20mg of tumor tissue was used for DNA extraction. Extracted DNA was subjected to Nanopore long-read sequencing to detect and analyze for the presence of high-risk HPV sequences. PCR and Sanger sequencing experiments were performed to confirm Nanopore long-read sequencing readings.ResultsNanopore long-read sequencing showed that seven out of eight patient samples displayed either integrated or episomal high-risk HPV sequences. Out of these seven samples, four displayed verifiable integration events upon bioinformatic analysis. Integration confirmation experiments were designed for all four samples using PCR-based methods. Sanger sequencing was also performed. Four distinct HPV integration patterns were identified: concatemer chromosomal integration in a single chromosome, bi-chromosomal concatemer integration, single chromosome complete integration and bi-chromosomal complete integration. HPV concatemer integration also proved more common than full HPV integration events.Conclusion and relevanceLong-read sequencing technologies can be effectively used to assess HPV integration patterns in OPSCC tumors. Clinically, more research should be conducted on the prognostication value of high-risk HPV integration in OPSCC tumors using long-read sequencing technology.

Project description:In-depth information regarding the DFCI OncoPanel sequencing panel has been described previously. Briefly, sequencing is performed using an Illumina HiSeq 2500 system (RRID:SCR_016383) with 2×100 paired-end reads. Samples must meet an average 50X coverage and minimum of 30X coverage for 80% of targets for analysis. For all samples, a board-certified molecular pathologist evaluates alterations and writes interpretations of the results. The three generations of OncoPanel use Mutect (RRID:SCR_000559) and GATK (RRID:SCR_001876) to interrogate possible alterations in the complete exonic DNA sequences of 275, 309, and 447 cancer-related genes, respectively, including substitutions, insertions, and deletions. Because tumor tissues are tested without a paired normal from individual patients, additional informatics steps are taken to identify common single nucleotide polymorphisms (SNPs). Any SNP present at >0.1% in Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP) (RRID:SCR_012761), or present in dbSNP (RRID:SCR_002338) is filtered; however, variants also present in at least twice in COSMIC (RRID:SCR_002260) are rescued for manual review. Variants that appear two or more times in a panel of 150 normal samples sequenced in-house and are not present in COSMIC are also filtered. For copy number alteration (CNA) analysis, the workflow employs an in-house algorithm (RobustCNV) to cover exonic regions of targeted genes as well as select intronic regions. The depth of coverage of these regions is determined by counting the number of reads aligning within defined genomic intervals and then normalized against a panel of normals and corrected for GC bias. The copy number for a segmented genomic interval is calculated as a log2 ratio of the depth of coverage of this sample compared to a panel of normal (non-cancer) samples that are run on the same plate. In parallel (samples not included), additional cases were previously sequenced via the MSK-IMPACT platform. To ensure maximum overlap between the platforms, only single nucleotide variants (SNVs), dinucleotide/oligonucleotide variants (DNVs/ONVs), insertions/deletions (indels), high copy gains (amplifications), and homozygous deletions from 182 genes shared between all versions of both platforms were evaluated in our analyses involving samples from both platforms. To filter pathogenic and passenger mutations, missense, truncating (nonsense, frameshift, and splice site), and in-frame indel mutations from the 182 genes overlapping between the DFCI OncoPanel and MSK-IMPACT panels were subjected to analysis in the Cancer Genome Interpreter (CGI) suite (RRID:SCR_023752) with cancer type set to “Skin Cutaneous Melanoma (SKCM)”. Known pathogenic mutations, defined as those listed as pathogenic in ClinVar (RRID:SCR_006169), OncoKB (RRID:SCR_014782), and/or CGI databases, were included in downstream analyses. Mutations which had not been previously annotated but were predicted to be pathogenic mutations by CGI were also included. Missense SNV mutations called passengers in CGI were subjected to another round of analysis in the ChasmPlus suite with cancer type set to SKCM. Missense mutations called pathogenic via this approach (p < 0.05; FDR Q < 0.3) were salvaged and included in further analyses. All other mutations were excluded. TERT promoter mutations were evaluated via FATHMM-MKL. Mutations called pathogenic via this tool were included in downstream analyses. Clinical data for these cases is available in a supplementary file from the associated publication. OF NOTE, THE RAW SEQUENCING DATA FROM THESE SAMPLES CANNOT BE MADE PUBLICLY AVAILABLE BECAUSE THE RESEARCH PARTICIPANT CONSENT DOES NOT INCLUDE AUTHORIZATION TO SHARE IDENTIFIABLE DATA.

Project description:ImportanceHuman papillomavirus-related oropharyngeal carcinoma (HPV-OPC) is increasing in incidence in the United States. Although HPV-OPC has favorable prognosis, 10% to 25% of HPV-OPCs recur. Detection of human papillomavirus (HPV) DNA in oral rinses is associated with HPV-OPC, but its potential as a prognostic biomarker is unclear.ObjectiveTo determine whether HPV DNA detection in oral rinses after treatment for HPV-OPC is associated with recurrence and survival.Design, setting, and participantsProspective cohort study of patients with incident HPV-OPC diagnosed from 2009 to 2013 at 4 academic tertiary referral cancer centers in the United States. Oral rinse samples were collected at diagnosis and after treatment (9, 12, 18, and 24 months after diagnosis), and evaluated for HPV DNA. Among an initial cohort of 157 participants with incident HPV-OPC treated with curative intent, 124 had 1 or more posttreatment oral rinses available and were included in this study.Main outcomes and measuresDisease-free survival (DFS) and overall survival (OS) were estimated by the Kaplan-Meier method, and the association of HPV DNA detection in oral rinses with survival was evaluated using Cox regression analysis.ResultsOral HPV type 16 (HPV16) DNA was common at diagnosis (67 of 124 participants [54%]). In contrast, oral HPV16 DNA was detected in only 6 participants after treatment (5%), including 5 with HPV16 DNA also detected at diagnosis (persistent oral HPV16 DNA). Two-year DFS and OS were 92% (95% CI, 94%-100%) and 98% (95% CI, 93%-99%). Persistent oral HPV16 DNA was associated with worse DFS (hazard ratio, 29.7 [95% CI, 9.0-98.2]) and OS (hazard ratio, 23.5 [95% CI, 4.7-116.9]). All 5 participants with persistent oral HPV16 DNA developed recurrent disease, 3 with local disease involvement. In contrast, just 9 of 119 participants (8%) without persistent oral HPV16 DNA developed recurrent disease, only 1 (11%) with local disease involvement. Median (range) time from earliest posttreatment oral HPV16 DNA detection to recurrence was 7.0 (3.7-10.9) months.Conclusions and relevanceHuman papillomavirus type 16 DNA in oral rinses is common at diagnosis but rare after treatment for HPV-OPC. Our data suggest that, although infrequent, persistent HPV16 DNA in posttreatment oral rinses is associated with poor prognosis and is a potential tool for long-term tumor surveillance, perhaps more so for local recurrence.

Project description:BackgroundNew ultrasensitive methods for detecting residual disease after surgery are needed in human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OPSCC).MethodsTo determine whether the clearance kinetics of circulating tumor human papillomavirus DNA (ctHPVDNA) is associated with postoperative disease status, a prospective observational study was conducted in 33 patients with HPV+OPSCC undergoing surgery. Blood was collected before surgery, postoperative days 1 (POD 1), 7, and 30 and with follow-up. A subcohort of 12 patients underwent frequent blood collections in the first 24 hours after surgery to define early clearance kinetics. Plasma was run on custom droplet digital polymerase chain reaction (ddPCR) assays for HPV genotypes 16, 18, 33, 35, and 45.ResultsIn patients without pathologic risk factors for recurrence who were observed after surgery, ctHPVDNA rapidly decreased to <1 copy/mL by POD 1 (n = 8/8). In patients with risk factors for macroscopic residual disease, ctHPVDNA was markedly elevated on POD 1 (>350 copies/mL) and remained elevated until adjuvant treatment (n = 3/3). Patients with intermediate POD 1 ctHPVDNA levels (1.2-58.4 copies/mL) all possessed pathologic risk factors for microscopic residual disease (n = 9/9). POD 1 ctHPVDNA levels were higher in patients with known adverse pathologic risk factors such as extranodal extension >1 mm (P = .0481) and with increasing lymph nodes involved (P = .0453) and were further associated with adjuvant treatment received (P = .0076). One of 33 patients had a recurrence that was detected by ctHPVDNA 2 months earlier than clinical detection.ConclusionsPOD 1 ctHPVDNA levels are associated with the risk of residual disease in patients with HPV+OPSCC undergoing curative intent surgery and thus could be used as a personalized biomarker for selecting adjuvant treatment in the future.Lay summaryHuman papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OPSCC) is increasing at epidemic proportions and is commonly treated with surgery. This report describes results from a study examining the clearance kinetics of circulating tumor HPV DNA (circulating tumor human papillomavirus DNA [ctHPVDNA]) following surgical treatment of HPV+OPSCC. We found that ctHPVDNA levels 1 day after surgery are associated with the risk of residual disease in patients with HPV+OPSCC and thus could be used as a personalized biomarker for selecting adjuvant treatment in the future. These findings are the first to demonstrate the potential utility of ctHPVDNA in patients with HPV+OPSCC undergoing surgery.

Dataset Information

Detection of Human Papillomavirus Integration in Brain Metastases from Oropharyngeal Tumors by Targeted Sequencing.

Publications

Detection of Human Papillomavirus Integration in Brain Metastases from Oropharyngeal Tumors by Targeted Sequencing.

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