Human Papillomavirus Integration Sites Research Articles

Identification of transcriptionally-active human papillomavirus integrants through nanopore sequencing reveals viable targets for gene therapy against cervical cancer.

Integration of the human papillomavirus (HPV) genome into the cellular genome is a key event that leads to constitutive expression of viral oncoprotein E6/E7 and drives the progression of cervical cancer. However, HPV integration patterns differ on a case-by-case basis among related malignancies. Next-generation sequencing technologies still face challenges for interrogating HPV integration sites. In this study, utilizing Nanopore long-read sequencing, we identified 452 and 108 potential integration sites from the cervical cancer cell lines (CaSki and HeLa) and five tissue samples, respectively. Based on long Nanopore chimeric reads, we were able to analyze the methylation status of the HPV long control region (LCR), which controls oncogene E6/E7 expression, and to identify transcriptionally-active integrants among the numerous integrants. As a proof of concept, we identified an active HPV integrant in between RUNX2 and CLIC5 on chromosome 6 in the CaSki cell line, which was supported by ATAC-seq, H3K27Ac ChIP-seq, and RNA-seq analysis. Knockout of the active HPV integrant, by the CRISPR/Cas9 system, dramatically crippled cell proliferation and induced cell senescence. In conclusion, identifying transcriptionally-active HPV integrants with Nanopore sequencing can provide viable targets for gene therapy against HPV-associated cancers.

Abstract 2937: Accurate detection of human papillomavirus breakpoints in cervical cancers using a novel library preparation strategy for nanopore sequencing technology

Abstract Introduction: Integration of human papillomavirus (HPV) DNA into the human genome is considered as a key event in cervical carcinogenesis. Different methods have been employed in exploring HPV integration sites, such as APOT, DIPS-PCR, and NGS. However, these methods have their drawbacks. APOT heavily relies on the quality of the sample due to its dependence on RNA, while DIPS-PCR mostly identifies known sites and has limited capability in uncovering new integration sites. Although the detection sensitivity of virus infection status increased significantly through the Illumina sequencing platform, there were still disadvantages remain for further improvement, including the detection accuracy and the complex integrated genome structure identification, etc. Also, it requires large amounts of sequencing data, and thus is not applicable in clinical usage, which requires fast and accurate results. Therefore, the development of a new method for HPV integration detection with high accuracy and prompt reporting capacity is crucial for future clinical application. Methods: We established a novel library preparation strategy for the detection of HPV integration in the human genome using Oxford Nanopore sequencing. Using this novel technology, Nanopore libraries were prepared from HPV positive cell lines HeLa and SiHa followed by sequencing on the Oxford Nanopore. We also determined HPV integration sites in five patients with cervical cancer patients with HPV positive tumors using Ilumina WGS. HPV integrations and breakpoints in human genome from both Nanopore and Ilumina WGS were extracted using Minimap and ViFi tool respectively. All HPV integration sites from cell lines and patient samples were validated using Sanger sequencing. Results: The novel library preparation approach for detection of HPV integrations with Nanopore sequencing technology were sensitive and efficient in detection of commonly reported HPV breakpoints in HPV positive cell lines HeLa and SiHa. Additionally, we applied this method to clinical samples from five patients subjected to Whole Genome Sequencing, consistently yielding concordant results. Conclusion: Our Nanopore based assay for detecting HPV integrations without the need for WGS demonstrated high efficacy. These data highlight the sensitivity and efficiency of this methodology in detecting HPV integrations events, emphasizing its potential as a reliable diagnostic tool in understanding pathophysiology of HPV-related malignancies and its use as prognostic markers in clinical settings. Citation Format: Preetiparna Parida, Nivedita Mukherjee, Agastya Singh, Ashima Singh, Krishna Sharan, Mahadev Rao, Shirley Lewis, Sabarinathan Radhakrishnan, Rama Rao Damerla. Accurate detection of human papillomavirus breakpoints in cervical cancers using a novel library preparation strategy for nanopore sequencing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2937.

Abstract 4334: Insight into the structure of breakage fusion bridge events and chromothripsis in cancer cell lines using long-read sequencing

Abstract Cervical cancer is caused by human papillomavirus (HPV) infection and is the most common cause of cancer death in low-resource countries. We characterized 18 cervical and 4 head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, and cancer driver mutations. This cell line panel represents the major molecular subtypes of cervical cancer. Structural variation analysis revealed recurrent chromosomal alteration and telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alteration in cancer that has not been studied using long-read sequencing. Analysis of the inversion sites revealed staggered ends consistent with exonuclease deletion of the DNA after breakage. Some BFB events are complex, involving interchromosomal or intrachromosomal insertions or rearrangements. In total, there were three different types of BFB events: Type I, II, and III. Type I BFB events have staggered ends but no additional insertion at the fusion site. Type II BFB events have an insertion of DNA from another chromosome at the junction, and Type III has an insertion of sequences from the same chromosome as the BFB. BFB cycles are thought to resolve with the addition of a telomere. However, a search for telomeric sequences did not identify any on chromosomes impacted by BFB. In many cancers, BFB events co-occur with chromothripsis; however, in our cervical cancers, only 1 out of 13 had chromosomal shattering near the BFB event, indicating that the mechanism of chromosome rearrangement may be different in cervical cancers. Five cell lines have a Chr11q BFB event, with YAP1/BIRC2/BIRC3 gene amplification. Analysis of 911 publicly available cervical cancer exomes showed that YAP1 amplification is associated with a 10-year earlier age of diagnosis of cervical cancer (x2, p<0.0001) and is three times more common in African Americans (x2=12, p=0.003). Therefore, cervical cancer patients with YAP1/BIRC2/BIRC3 amplification, especially those of African American ancestry, might benefit from anti-YAP1/BIRC2/BIRC3 therapies. Our panel provides models for all the major HPV types and subtypes of cervical cancer that will aid in developing new therapeutic approaches. In summary, we uncovered novel insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing. We are expanding long-read sequencing to other cancer cell lines to gain knowledge of events associated with BFB. Citation Format: Isabel Rodriguez, Nicole Rossi, Ayse Gokce Kesus, Yi Xie, Tanveer Ahmad, Asher Bryant, Hong Lou, Jesica Godinez Paredes, Rose Milano, Mikhail Kolmogorov, Sonam Tulsyan, Jazmyn Bess, Vera Mukhina, Daria Gaykalova, Laksh Malik, Kimberley J. Billingsley, Cornelis Blauwendraat, Lisa Mirabello, Michael Dean. Insight into the structure of breakage fusion bridge events and chromothripsis in cancer cell lines using long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4334.

Insights into the mechanisms and structure of breakage-fusion-bridge cycles in cervical cancer using long-read sequencing

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

Tumorigenic activation around HPV integrated sites in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) is causally involved in the development of head and neck squamous cell carcinoma (HNSCC). The integration of HPV drives tumorigenesis through expression of oncogenic viral genes as well as genomic alterations in surrounding regions. To elucidate involvement of epigenetic dysregulation in tumorigenesis, we here performed integrated analyses of the epigenome, transcriptome and interactome using ChIP-seq, RNA-seq and Hi-C and 4C-seq for HPV(+) HNSCCs. We analyzed clinical HNSCC using The Cancer Genome Atlas data and found that genes neighboring HPV integration sites were significantly upregulated and were correlated with oncogenic phenotypes in HPV(+) HNSCCs. While we found four HPV integration sites in HPV(+) HNSCC cell line UPCI-SCC-090 through target enrichment sequencing, 4C-seq revealed 0.5 to 40 Mb of HPV-interacting regions (HPVIRs) where host genomic regions interacted with integrated HPV genomes. While 9% of the HPVIRs were amplified and activated epigenetically forming super-enhancers, the remaining non-amplified regions were found to show a significant increase in H3K27ac levels and an upregulation of genes associated with GO terms, for example, Signaling by WNT and Cell Cycle. Among those genes, ITPR3 was significantly upregulated, involving UPCI-SCC-090-specific super-enhancer formation around the ITPR3 promoter and in the 80-kb-downstream region. The knockdown of ITPR3 by siRNA or CRISPR deletions of the distant enhancer region led to a significant suppression of cell proliferation. The epigenetic activation of HPVIRs was also confirmed in other cell lines, UM-SCC-47 and UM-SCC-104. These data indicate that epigenetic activation in HPVIRs contributes, at least partially, to genesis of HPV(+) HNSCC.

Application of organoid culture from HPV18-positive small cell carcinoma of the uterine cervix for precision medicine.

Small cell carcinoma of the uterine cervix (SCCC) is a rare and highly malignant human papillomavirus (HPV)-associated cancer in which human genes related to the integration site can serve as a target for precision medicine. The aim of our study was to establish a workflow for precision medicine of HPV-associated cancer using patient-derived organoid. Organoid was established from the biopsy of a patient diagnosed with HPV18-positive SCCC. Therapeutic targets were identified by whole exome sequencing (WES) and RNA-seq analysis. Drug sensitivity testing was performed using organoids and organoid-derived mouse xenograft model. WES revealed that both the original tumor and organoid had 19 somatic variants in common, including the KRAS p.G12D pathogenic variant. Meanwhile, RNA-seq revealed that HPV18 was integrated into chromosome 8 at 8q24.21 with increased expression of the proto-oncogene MYC. Drug sensitivity testing revealed that a KRAS pathway inhibitor exerted strong anti-cancer effects on the SCCC organoid compared to a MYC inhibitor, which were also confirmed in the xenograft model. In this study, we confirmed two strategies for identifying therapeutic targets of HPV-derived SCCC, WES for identifying pathogenic variants and RNA sequencing for identifying HPV integration sites. Organoid culture is an effective tool for unveiling the oncogenic process of rare tumors and can be a breakthrough for the development of precision medicine for patients with HPV-positive SCCC.

Cells with stem-like properties are associated with the development of HPV18-positive cervical cancer.

The cellular origins of cervical cancer and the histological differentiation of human papillomavirus (HPV)-infected cells remain unexplained. To gain new insights into the carcinogenesis and histological differentiation of HPV-associated cervical cancer, we focused on cervical cancer with mixed histological types. We conducted genomic and transcriptomic analyses of cervical cancers with mixed histological types. The commonality of the cellular origins of these cancers was inferred using phylogenetic analysis and by assessing the HPV integration sites. Carcinogenesis was estimated by analyzing human gene expression profiles in different histological types. Among 42 cervical cancers with known HPV types, mixed histological types were detected in four cases, and three of them were HPV18-positive. Phylogenetic analysis of these three cases revealed that the different histological types had a common cell of origin. Moreover, the HPV-derived transcriptome and HPV integration sites were common among different histological types, suggesting that HPV integration could occur before differentiation into each histological type. Human gene expression profiles indicated that HPV18-positive cancer retained immunologically cold components with stem cell properties. Mixed cervical cancer has a common cellular origin among different histological types, and progenitor cells with stem-like properties may be associated with the development of HPV18-positive cervical cancer.

HPV Integration Site Mapping: A Rapid Method of Viral Integration Site (VIS) Analysis and Visualization Using Automated Workflows in CLC Microbial Genomics.

Human papillomavirus (HPV) integration within the host genome may contribute to carcinogenesis through various disruptive mechanisms. With next-generation sequencing (NGS), identification of viral and host genomic breakpoints and chimeric sequences are now possible. However, a simple, streamlined bioinformatics workflow has been non-existent until recently. Here, we tested two new, automated workflows in CLC Microbial Genomics, i.e., Viral Hybrid Capture (VHC) Data Analysis and Viral Integration Site (VIS) Identification for software performance and efficiency. The workflows embedded with HPV and human reference genomes were used to analyze a publicly available NGS dataset derived from pre- and cancerous HPV+ cervical cytology of 21 Gabonese women. The VHC and VIS workflow median runtimes were 19 and 7 min per sample, respectively. The VIS dynamic graphical outputs included read mappings, virus-host genomic breakpoints, and virus-host integration circular plots. Key findings, including disrupted and nearby genes, were summarized in an auto-generated report. Overall, the VHC and VIS workflows proved to be a rapid and accurate means of localizing viral-host integration site(s) and identifying disrupted and neighboring human genes. Applying HPV VIS-mapping to pre- or invasive tumors will advance our understanding of viral oncogenesis and facilitate the discovery of prognostic biomarkers and therapeutic targets.

Mutational Signature and Integrative Genomic Analysis of Human Papillomavirus-Associated Penile Squamous Cell Carcinomas from Latin American Patients.

Simple SummaryDNA sequencing has been crucial to comprehending cancer mutational patterns, leading to the identification of driver genes and altered signaling pathways. Thus, identifying new pathogenic variants and their impact on tumor onset, progression, and treatment response has fueled tumor biology research. Here, we present novel findings addressing the first whole-exome sequencing (WES) of human papillomavirus (HPV)-associated penile squamous cell carcinoma (PSCC) from Latin Americans and its association with pathogenesis. We also compared the molecular profile of the tumors to that of three previous studies from populations with different genetic and socioeconomic backgrounds, the majority of which was HPV-negative. We describe the most altered genes and the main pathogenic variants found in the Latin Americans, ten of which are exclusive to our study sample. The data allowed us to identify molecular pathways and druggable targets with potential treatment value for this still-neglected HPV-associated carcinoma.High-throughput DNA sequencing has allowed for the identification of genomic alterations and their impact on tumor development, progression, and therapeutic responses. In PSCC, for which the incidence has progressively increased worldwide, there are still limited data on the molecular mechanisms involved in the disease pathogenesis. In this study, we characterized the mutational signature of 30 human papillomavirus (HPV)-associated PSCC cases from Latin Americans, using whole-exome sequencing. Copy number variations (CNVs) were also identified and compared to previous array-generated data. Enrichment analyses were performed to reveal disrupted pathways and to identify alterations mapped to HPV integration sites (HPVis) and miRNA–mRNA hybridization regions. Among the most frequently mutated genes were NOTCH1, TERT, TTN, FAT1, TP53, CDKN2A, RYR2, CASP8, FBXW7, HMCN2, and ITGA8. Of note, 92% of these altered genes were localized at HPVis. We also found mutations in ten novel genes (KMT2C, SMARCA4, PTPRB, AJUBA, CR1, KMT2D, NBEA, FAM135B, GTF2I, and CIC), thus increasing our understanding of the potential HPV-disrupted pathways. Therefore, our study reveals innovative targets with potential therapeutic benefits for HPV-associated PSCCs. The CNV analysis by sequencing (CNV-seq) revealed five cancer-associated genes as the most frequent with gains (NOTCH1, MYC, NUMA1, PLAG1, and RAD21), while 30% of the tumors showed SMARCA4 with loss. Additionally, four cancer-associated genes (CARD11, CSMD3, KDR, and TLX3) carried untranslated regions (UTRs) variants, which may impact gene regulation by affecting the miRNAs hybridization regions. Altogether, these data contribute to the characterization of the mutational spectrum and its impact on cellular signaling pathways in PSCC, thus reinforcing the pivotal role of HPV infection in the molecular pathogenesis of these tumors.

Human papilloma virus integration sites and genomic signatures in head and neck squamous cell carcinoma.

A prevalence of around 26% of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) has been previously reported. HPV induced oncogenesis mainly involving E6 and E7 viral oncoproteins. In some cases, HPV viral DNA has been detected to integrate with the host genome and possibly contributes to carcinogenesis by affecting the gene expression. We retrospectively assessed HPV integration sites and signatures in 80 HPV positive patients with HNSCC, by using a double capture‐HPV method followed by next‐generation Sequencing. We detected HPV16 in 90% of the analyzed cohort and confirmed five previously described mechanistic signatures of HPV integration [episomal (EPI), integrated in a truncated form revealing two HPV‐chromosomal junctions colinear (2J‐COL) or nonlinear (2J‐NL), multiple hybrid junctions clustering in a single chromosomal region (MJ‐CL) or scattered over different chromosomal regions (MJ‐SC) of the human genome]. Our results suggested that HPV remained episomal in 38.8% of the cases or was integrated/mixed in the remaining 61.2% of patients with HNSCC. We showed a lack of association of HPV genomic signatures to tumour and patient characteristics, as well as patient survival. Similar to other HPV associated cancers, low HPV copy number was associated with worse prognosis. We identified 267 HPV‐human junctions scattered on most chromosomes. Remarkably, we observed four recurrent integration regions: PDL1/PDL2/PLGRKT (8.2%), MYC/PVT1 (6.1%), MACROD2 (4.1%) and KLF5/KLF12 regions (4.1%). We detected the overexpression of PDL1 and MYC upon integration by gene expression analysis. In conclusion, we identified recurrent targeting of several cancer genes such as PDL1 and MYC upon HPV integration, suggesting a role of altered gene expression by HPV integration during HNSCC carcinogenesis.

Recurrent integration of human papillomavirus genomes at transcriptional regulatory hubs

Oncogenic human papillomavirus (HPV) genomes are often integrated into host chromosomes in HPV-associated cancers. HPV genomes are integrated either as a single copy or as tandem repeats of viral DNA interspersed with, or without, host DNA. Integration occurs frequently in common fragile sites susceptible to tandem repeat formation and the flanking or interspersed host DNA often contains transcriptional enhancer elements. When co-amplified with the viral genome, these enhancers can form super-enhancer-like elements that drive high viral oncogene expression. Here we compiled highly curated datasets of HPV integration sites in cervical (CESC) and head and neck squamous cell carcinoma (HNSCC) cancers, and assessed the number of breakpoints, viral transcriptional activity, and host genome copy number at each insertion site. Tumors frequently contained multiple distinct HPV integration sites but often only one “driver” site that expressed viral RNA. As common fragile sites and active enhancer elements are cell-type-specific, we mapped these regions in cervical cell lines using FANCD2 and Brd4/H3K27ac ChIP-seq, respectively. Large enhancer clusters, or super-enhancers, were also defined using the Brd4/H3K27ac ChIP-seq dataset. HPV integration breakpoints were enriched at both FANCD2-associated fragile sites and enhancer-rich regions, and frequently showed adjacent focal DNA amplification in CESC samples. We identified recurrent integration “hotspots” that were enriched for super-enhancers, some of which function as regulatory hubs for cell-identity genes. We propose that during persistent infection, extrachromosomal HPV minichromosomes associate with these transcriptional epicenters and accidental integration could promote viral oncogene expression and carcinogenesis.

Detection of HPV and Human Chromosome Sites by Dual-Color Fluorescence In Situ Hybridization Reveals Recurrent HPV Integration Sites and Heterogeneity in Cervical Cancer.

Human papillomavirus (HPV) integration in the human genome is suggested to be an important cause of cervical cancer. With the development of sequencing technologies, an increasing number of integration “hotspots” have been identified. However, this HPV integration information was derived from analysis of whole cervical cancer tissue, and we know very little about the integration in different cancer cell subgroups or individual cancer cells. This study optimized the preparation of probes and provided a dual-color fluorescence in situ hybridization (FISH) method to detect HPV integration sites in paraffin-embedded cervical cancer samples. We used both HPV probes and site-specific probes: 3p14 (FHIT), 8q24 (MYC), 13q22 (KLF5/KLF12), 3q28 (TP63), and 5p15 (TERT). We detected HPV signals in 75 of the 96 cases of cervical cancer; 62 cases showed punctate signals, and 13 cases showed diffuse punctate signals. We identified 3p14 as a high-frequency HPV integration site in 4 cervical cancer cases. HPV integration at 8p14 occurred in 2 cases of cervical cancer. In the same cervical cancer tissue of sample No.1321, two distinct subgroups of cells were observed based on the HPV probe but showed no difference in cell and nucleus morphology. Our study provides a new method to investigate the frequent HPV integration sites in cervical cancer and reports the heterogeneity within cervical cancer from the perspective of HPV integration.

Multi-omics mapping of human papillomavirus integration sites illuminates novel cervical cancer target genes.

Integration of human papillomavirus (HPV) into the host genome is a dominant feature of invasive cervical cancer (ICC), yet the tumorigenicity of cis genomic changes at integration sites remains largely understudied. Combining multi-omics data from The Cancer Genome Atlas with patient-matched long-read sequencing of HPV integration sites, we developed a strategy for using HPV integration events to identify and prioritise novel candidate ICC target genes (integration-detected genes (IDGs)). Four IDGs were then chosen for in vitro functional studies employing small interfering RNA-mediated knockdown in cell migration, proliferation and colony formation assays. PacBio data revealed 267 unique human-HPV breakpoints comprising 87 total integration events in eight tumours. Candidate IDGs were filtered based on the following criteria: (1) proximity to integration site, (2) clonal representation of integration event, (3) tumour-specific expression (Z-score) and (4) association with ICC survival. Four candidates prioritised based on their unknown function in ICC (BNC1, RSBN1, USP36 and TAOK3) exhibited oncogenic properties in cervical cancer cell lines. Further, annotation of integration events provided clues regarding potential mechanisms underlying altered IDG expression in both integrated and non-integrated ICC tumours. HPV integration events can guide the identification of novel IDGs for further study in cervical carcinogenesis and as putative therapeutic targets.

Circulating HPV DNA as a Marker for Early Detection of Relapse in Patients with Cervical Cancer.

Almost all cervical cancers are caused by human papillomavirus (HPV) and patients with advanced stage are at high risk for relapse. Circulating HPV DNA (HPV ctDNA) may serve as a residual tumor marker at the end of chemoradiation or to predict relapse during the follow-up period. We analyzed serum samples from 94 HPV16- or HPV18-related CCs from the BioRAIDs prospective cohort. Samples were collected before and after treatment and during an 18-month follow-up period. Using digital droplet PCR (ddPCR), we assessed the relevance of circulating HPV E7 gene as a marker for residual disease compared to HPV integration site and PIK3CA mutations. Finally, the prognostic impact of circulating HPV E7 gene was assessed with its prediction value of relapse. HPV E7 gene was the most sensitive tumor marker, superior to both HPV integration sites and PIK3CA mutations in serum. Circulating HPV DNA (HPV ctDNA) was detected in 63% (59/94) of patients, before treatment. HPV ctDNA detection in serum sample was associated with high FIGO stage (P = 0.02) and para-aortic lymph node involvement (P = 0.01). The level of HPV ctDNA was positively correlated with HPV copy number in the tumor (R = 0.39, P < 0.001). Complete clearance of HPV ctDNA by the end of treatment was significantly associated with a longer PFS (P < 0.0001). Patients with persistent HPV ctDNA in serum relapsed with a median time of 10 months (range, 2-15) from HPV ctDNA detection. HPV ctDNA detection is a useful marker to predict relapse in cervical cancer.See related commentary by Wentzensen and Clarke, p. 5733.

SearcHPV: A novel approach to identify and assemble human papillomavirus-host genomic integration events in cancer.

Human papillomavirus (HPV) is a well-established driver of malignant transformation at a number of sites, including head and neck, cervical, vulvar, anorectal, and penile squamous cell carcinomas; however, the impact of HPV integration into the host human genome on this process remains largely unresolved. This is due to the technical challenge of identifying HPV integration sites, which includes limitations of existing informatics approaches to discovering viral-host breakpoints from low-read-coverage sequencing data. To overcome this limitation, the authors developed SearcHPV, a new HPV detection pipeline based on targeted capture technology, and applied the algorithm to targeted capture data. They performed an integrated analysis of SearcHPV-defined breakpoints with genome-wide linked-read sequencing to identify potential HPV-related structural variations. Through an analysis of HPV+ models, the authors showed that SearcHPV detected HPV-host integration sites with a higher sensitivity and specificity than 2 other commonly used HPV detection callers. SearcHPV uncovered HPV integration sites adjacent to known cancer-related genes, including TP63, MYC, and TRAF2, and near regions of large structural variation. The authors further validated the junction contig assembly feature of SearcHPV, which helped to accurately identify viral-host junction breakpoint sequences. They found that viral integration occurred through a variety of DNA repair mechanisms, including nonhomologous end joining, alternative end joining, and microhomology-mediated repair. In summary, SearcHPV is a new optimized tool for the accurate detection of HPV-human integration sites from targeted capture DNA sequencing data.

DeepHPV: a deep learning model to predict human papillomavirus integration sites.

Human papillomavirus (HPV) integrating into human genome is the main cause of cervical carcinogenesis. HPV integration selection preference shows strong dependence on local genomic environment. Due to this theory, it is possible to predict HPV integration sites. However, a published bioinformatic tool is not available to date. Thus, we developed an attention-based deep learning model DeepHPV to predict HPV integration sites by learning environment features automatically. In total, 3608 known HPV integration sites were applied to train the model, and 584 reviewed HPV integration sites were used as the testing dataset. DeepHPV showed an area under the receiver-operating characteristic (AUROC) of 0.6336 and an area under the precision recall (AUPR) of 0.5670. Adding RepeatMasker and TCGA Pan Cancer peaks improved the model performance to 0.8464 and 0.8501 in AUROC and 0.7985 and 0.8106 in AUPR, respectively. Next, we tested these trained models on independent database VISDB and found the model adding TCGA Pan Cancer performed better (AUROC: 0.7175, AUPR: 0.6284) than the model adding RepeatMasker peaks (AUROC: 0.6102, AUPR: 0.5577). Moreover, we introduced attention mechanism in DeepHPV and enriched the transcription factor binding sites including BHLHA15, CHR, COUP-TFII, DMRTA2, E2A, HIC1, INR, NPAS, Nr5a2, RARa, SCL, Snail1, Sox10, Sox3, Sox4, Sox6, STAT6, Tbet, Tbx5, TEAD, Tgif2, ZNF189, ZNF416 near attention intensive sites. Together, DeepHPV is a robust and explainable deep learning model, providing new insights into HPV integration preference and mechanism. Availability: DeepHPV is available as an open-source software and can be downloaded from https://github.com/JiuxingLiang/DeepHPV.git, Contact: huzheng1998@163.com, liangjiuxing@m.scnu.edu.cn, lizheyzy@163.com.

HPV Detection in Head and Neck Squamous Cell Carcinomas: What Is the Issue?

Besides classic tobacco and alcohol risk factors, human papillomavirus (HPV) plays a role in the development of a subset of head and neck squamous cell carcinomas (HNSCCs), and notably oropharynx squamous cell carcinomas (OPSCCs). HPV-induced OPSCCs have a different biological behavior and a better prognosis compared to non-HPV-induced OPSCCs and the eighth-edition TNM classification now separates these two entities. Therefore, determining the HPV status of patients with OPSCC is now essential for treatment, prognosis, and development of clinical trials. In this review, after reminding essential steps of HPV implication in the cell cycle, we describe the existing tools that are currently feasible in routine practice according to facilities available in health structures, with their benefits and drawbacks: HPV PCR, E6/E7 mRNA RT-PCR, E6/E7 mRNA in situ hybridization, HPV DNA in situ hybridization, and P16 immunochemistry. Besides these traditional HPV detection tools, novel diagnostic approaches are being evaluated for HPV-induced OPSCC “ultrastaging.” E6 humoral response and ddPCR-detecting HPVct DNA are two techniques performed on blood and are therefore non-invasive. Baseline E6 humoral levels could have a prognostic value, and HPVct DNA could be helpful for HPV OPSCC recurrence monitoring. At last, next-generation sequencing (NGS)-based “capture HPV” is a technique feasible on biopsies and circulating DNA material. It helps characterize HPV integration status and sites, and it could define prognostic subgroups in HPV-induced OPSCC. These novel precision detection tools could be further integrated in the care of patients with HPV-induced OPSCC.

Accurate Detection of HPV Integration Sites in Cervical Cancer Samples Using the Nanopore MinION Sequencer Without Error Correction.

During the carcinogenesis of cervical cancer, the DNA of human papillomavirus (HPV) is frequently integrated into the human genome, which might be a biomarker for the early diagnosis of cervical cancer. Although the detection sensitivity of virus infection status increased significantly through the Illumina sequencing platform, there were still disadvantages remain for further improvement, including the detection accuracy and the complex integrated genome structure identification, etc. Nanopore sequencing has been proven to be a fast yet accurate technique of detecting pathogens in clinical samples with significant longer sequencing length. However, the identification of virus integration sites, especially HPV integration sites was seldom carried out by using nanopore platform. In this study, we evaluated the feasibility of identifying HPV integration sites by nanopore sequencer. Specifically, we re-sequenced the integration sites of a previously published sample by both nanopore and Illumina sequencing. After analyzing the results, three points of conclusions were drawn: first, 13 out of 19 previously published integration sites were found from all three datasets (i.e., nanopore, Illumina, and the published data), indicating a high overlap rate and comparability among the three platforms; second, our pipeline of nanopore and Illumina data identified 66 unique integration sites compared with previous published paper with 13 of them being verified by Sanger sequencing, indicating the higher integration sites detection sensitivity of our results compared with published data; third, we established a pipeline which could be used in HPV integration site detection by nanopore sequencing data without doing error correction analysis. In summary, a new nanopore data analysis method was tested and proved to be reliable in integration sites detection compared with methods of existing Illumina data analysis pipeline with less sequencing data required. It provides a solid evidence and tool to support the potential application of nanopore in virus status identification.

Simultaneous detection and comprehensive analysis of HPV and microbiome status of a cervical liquid-based cytology sample using Nanopore MinION sequencing

Human papillomavirus (HPV) is a major pathogen that causes cervical cancer and many other related diseases. HPV infection related cervical microbiome could be an induce factor of cervical cancer. However, it is uncommon to find a single test on the market that can simultaneously provide information on both HPV and the microbiome. Herein, a novel method was developed in this study to simultaneously detect HPV infection and microbiota composition promptly and accurately. It provides a new and simple way to detect vaginal pathogen situation and also provide valuable information for clinical diagnose. This approach combined multiplex PCR, which targeted both HPV16 E6E7 and full-length 16S rRNA, and Nanopore sequencing to generate enough information to understand the vagina condition of patients. One HPV positive liquid-based cytology (LBC) sample was sequenced and analyzed. After comparing with Illumina sequencing, the results from Nanopore showed a similar microbiome composition. An instant sequencing evaluation showed that 15 min sequencing is enough to identify the top 10 most abundant bacteria. Moreover, two HPV integration sites were identified and verified by Sanger sequencing. This approach has many potential applications in pathogen detection and can potentially aid in providing a more rapid clinical diagnosis.

Discovering HPV16 and HPV18 infection status and cancer gene variations in cervical cancer patient specimens by using Next-generation sequencing.

e13040 Background: Human papillomavirus (HPV) infection has been reported as an important cause of cervical cancer for many years. HPV16 and HPV18 are the two most prevalent types in cervical cancer5. Integration of HPV DNA is a crucial genetic event in cervical carcinogenesis. Therefore, it is important to know the HPV integration hot spots in human genome to predict the cervical cancer development status of patients. Although many studies have addressed the HPV integration sites in human genome, it is still a technique barrier to precisely determine the HPV integration sites of each patient, especially at early disease development stage with limited integration sites existing. Methods: To solve this problem, we first designed HPV probes to capture HPV integrated sequences of genome using next generation sequencing (NGS). And then we analyzed the integration sites by two methods with the NGS results, HPV Detector. We applied the study on 17 patient samples, 15 of them were found HPV16 positive and 2 of them were found HPV18 positive. Among them 16 were detected with HPV integration sites. Results: The results indicated that different samples showed different numbers of integration sites with limited overlapped positions in genome. The integrated positions of HPV16/18 located in all the genes of the virus, but mostly distributed in gene E1, L1 and L2. We identified integration sites HPV16 E6/GATC， E6/WWTR1， L2/DGKB, E1/EHHADH, ect. Some of the integrated sites in human genome are belong to intergenic region. We also used cancer gene panel, which cover 143 cancer related genes exon regions, to identify the mutation sites of each samples. We summed up a highly occurred mutated cancer gene list, which include ALK, NF1, PIK3R1, ROS1, TSC1, TSC2, ect. Most of them are either cancer driver genes or have been found highly related to cervical cancer. Further study is still carrying on. Conclusions: In conclusion, our HPV probe could successfully detect HPV16 and HPV18 in patient samples and enrich HPV integration sites in human genome of cervical samples significantly. It could provide detailed information for cervical cancer discovery and early detection.