Human Papillomavirus Integration Research Articles

Microhomology-mediated repair machinery and its relationship with HPV-mediated oncogenesis.

Human Papillomaviruses (HPV) are a diverse family of non-enveloped dsDNA viruses that infect the skin and mucosal epithelia. Persistent HPV infections can lead to cancer frequently involving integration of the virus into the host genome, leading to sustained oncogene expression and loss of capsid and genome maintenance proteins. Microhomology-mediated double-strand break repair, a DNA double-stranded breaks repair pathway present in many organisms, was initially thought to be a backup but it's now seen as vital, especially in homologous recombination-deficient contexts. Increasing evidence has identified microhomology (MH) near HPV integration junctions, suggesting MH-mediated repair pathways drive integration. In this comprehensive review, we present a detailed summary of both the mechanisms underlying MH-mediated repair and the evidence for its involvement in HPV integration in cancer. Lastly, we highlight the involvement of these processes in the integration of other DNA viruses and the broader implications on virus lifecycles and host innate immune response.

Navigating therapeutic strategies: HPV classification in head and neck cancer.

The World Health Organisation recognised human papillomavirus (HPV) as the cause of multiple cancers, including head and neck cancers. HPV is a double-stranded DNA virus, and its viral gene expression can be controlled after infection by cellular and viral promoters. In cancer cells, the HPV genome is detected as either integrated into the host genome, episomal (extrachromosomal), or a mixture of integrated and episomal. Viral integration requires the breakage of both viral and host DNA, and the integration rate correlates with the level of DNA damage. Interestingly, patients with HPV-positive head and neck cancers generally have a good prognosis except for a group of patients with fully integrated HPV who show worst clinical outcomes. Those patients present with lowered expression of viral genes and limited infiltration of cytotoxic T cells. An impediment to effective therapy applications in the clinic is the sole testing for HPV positivity without considering the HPV integration status. This review will discuss HPV integration as a potential determinant of response to therapies in head and neck cancers and highlight to the field a novel therapeutic avenue that would reduce the cancer burden and improve patient survival.

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 2297: Longitudinal monitoring of HPV circulating tumor DNA in cervical cancer

Abstract Introduction Detecting circulating tumor DNA (ctDNA) in the blood using minimally invasive techniques known as a "liquid biopsy", is gaining tremendous popularity in oncology research. Integration of human papillomavirus (HPV) DNA and the overexpression of the E6 and E7 oncogenes are crucial steps in the development of cervical cancer. We investigated the potential utility of HPV ctDNA as a biomarker for monitoring treatment outcomes in patients with cervical cancer. Material and Methods We use droplet digital PCR (ddPCR) to measure the amount of HPV16/18 ctDNA in plasma of cervical cancer patients. The HPV standard GP5+/6+ primers were used to determine HPV positivity in our patients. Specific primers were designed to amplify specifically the E6 oncogene of HPV16 and HPV18. Plasmid constructs were made using the PCR-purified products of HeLa and SiHa cell lines which would act as a positive control for the assay. HPV16 and 18 plasmids were serially diluted and then tested in qPCR as a template using different primer sets. Standard curves were made from the recombinant plasmids. This will serve as a positive control and will also provide a correlation between the sensitivity and specificity of qPCR vs ddPCR. Results and Discussions We hypothesize that our dPCR test will be able to detect minute quantities of HPV ctDNA in plasma of cervical cancer patients as ctDNA levels are expected to be decrease as treatment progresses. The assay sensitivity and specificity will be obtained by comparing results to QPCR. The study samples involve a tumor biopsy and a blood sample at the baseline prior to the therapy and serial blood sampling in different stages of treatment and follow-ups. These samples will also serve for treatment monitoring of these patients through ddPCR. Tumor samples are used to confirm the HPV status of patients using HPV-specific GP5+/GP6+ qPCR. We have recruited 75 cervical cancer patients with majority of them being squamous cell carcinoma and stage IIB disease. The qPCR results from tumor biopsy revealed HPV 16 was most prevalent (32/43), followed by HPV18 (6/43) and HPV 33(2/43). Conclusion we will now use Qiagen digital PCR system to understand HPV ctDNA levels in our patient cohort. we believe that our comprehensive sample collection after every treatment milestone and thorough follow-up for upto 2 years after diagnosis will allow us to study the utility of this sensitive test in monitoring disease and relapse. Citation Format: Preetiparna Parida, Krishna Sharan, Shirley Lewis, Rama Rao Damerla. Longitudinal monitoring of HPV circulating tumor DNA in cervical cancer [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 2297.

Abstract 4352: A high rate of episomal HPV16 is present in head and neck squamous cell carcinoma tumors by long-read whole genome sequencing

Abstract Persistent Human Papillomavirus (HPV) infection causes an increased risk of Head and Neck Squamous Cell Carcinoma (HNSCC). However, a comprehensive understanding of the intricate molecular events within virus-positive tumors remains a critical knowledge gap. Therefore, we used long-read sequencing to study the intricacies of HPV integration events within the genomic landscape of HPV+ HNSCC tumors. A total of 16 tissue biopsies, including 10 tumors and 6 matched adjacent normal tissues from histopathologically confirmed HNSCC subjects were sequenced by LSK114 chemistry of Oxford Nanopore Technology with an average coverage of 30X and 10X, respectively. The predicted mitochondrial haplotype from Mitomaster confirmed that the tumor/normal pair was from the same individual. The HPV integration status was determined by aligning fastq_pass files at the Cancer Genomics Cloud and viewing the breakpoints in the Integrated Genome Viewer. HPV lineage and sub-lineage classification was determined using ClustalOmega. We detected HPV16 in 8/10 tumors, with sublineages A1 and A2 accounting for 38% (3/8) and D3 for 25% (2/8). One tumor sample had a very low HPV load with only one HPV16 sequence read and low detection by PCR. Interestingly, we found that 5/8 HPV positive tumors had episomal HPV16 with different states. So, two tumors had intact HPV episomes, two more had either a small deletion or an insertion in the episomal HPV, and the last one had a rearranged episomal multimer (Table 1). The remaining three tumors had integrated HPV16: one with type 1 integrations at 11q12.13 and two others with type 2 integrations at chr1q42.12 and chr14q21.1. Our results indicate that episomal HPV16 is more prevalent than integrated HPV16 in primary HPV+ HNSCC samples. These findings provide new insights into the molecular mechanisms and consequences of HPV infection in HNSCC. Table 1: HPV characterization in HNSCC studied tissue biopsies. Patient ID Race HPV status HPV sublineages Predicted Haplotype HPV pattern #PID_1 Caucasian HPV16 + A2 T2b (T2b) Type 2 Integration (chr1q42.12) #PID_2 - HPV- - I1a (I1a1b) - #PID_3 Caucasian HPV16 + A1 H1q (H1q) Episomal (rearranged multimer) PID_4 African American HPV16 + A1 - Episomal (deletion of 636bp from 4892 to 5528) #PID_5 - HPV16 + A2 H1 (H1+16189) Type 1 Integration (complex chr11q12.13) *#PID_6 Caucasian HPV16 + - T2b (T2b) - PID_7 Caucasian HPV16 + D3 - Episomal #PID_8 African American HPV16 + D3 L1c (L1c3a) Type 2 Integration (chr14q21.1) PID_9 African American HPV16 + A2 - Episomal (insertion of 257bp from 7310-7567) PID_10 Caucasian HPV16 + A1 - Episomal *Very weakly HPV16 positive; #Tumor-normal pair Citation Format: Sonam Tulsyan, Vera Mukhina, Isabel Rodriguez, Subhendu R. Choudhury, Erin Allor, Kyle Hatte, Michael Dean, Daria A. Gaykalova. A high rate of episomal HPV16 is present in head and neck squamous cell carcinoma tumors by long-read whole genome 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 4352.

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.

Long-read sequencing reveals the structural complexity of genomic integration of HPV DNA in cervical cancer cell lines

BackgroundCervical cancer (CC) causes more than 311,000 deaths annually worldwide. The integration of human papillomavirus (HPV) is a crucial genetic event that contributes to cervical carcinogenesis. Despite HPV DNA integration is known to disrupt the genomic architecture of both the host and viral genomes in CC, the complexity of this process remains largely unexplored.ResultsIn this study, we conducted whole-genome sequencing (WGS) at 55-65X coverage utilizing the PacBio long-read sequencing platform in SiHa and HeLa cells, followed by comprehensive analyses of the sequence data to elucidate the complexity of HPV integration. Firstly, our results demonstrated that PacBio long-read sequencing effectively identifies HPV integration breakpoints with comparable accuracy to targeted-capture Next-generation sequencing (NGS) methods. Secondly, we constructed detailed models of complex integrated genome structures that included both the HPV genome and nearby regions of the human genome by utilizing PacBio long-read WGS. Thirdly, our sequencing results revealed the occurrence of a wide variety of genome-wide structural variations (SVs) in SiHa and HeLa cells. Additionally, our analysis further revealed a potential correlation between changes in gene expression levels and SVs on chromosome 13 in the genome of SiHa cells.ConclusionsUsing PacBio long-read sequencing, we have successfully constructed complex models illustrating HPV integrated genome structures in SiHa and HeLa cells. This accomplishment serves as a compelling demonstration of the valuable capabilities of long-read sequencing in detecting and characterizing HPV genomic integration structures within human cells. Furthermore, these findings offer critical insights into the complex process of HPV16 and HPV18 integration and their potential contribution to the development of cervical cancer.

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.

Mapping the landscape of HPV integration and characterising virus and host genome interactions in HPV-positive oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV) integration into the host genome is an important factor in HPV(+)OPSCC carcinogenesis, in conjunction with HPV oncoproteins E6/E7. However, a well-studied investigation about virus-host interaction still needs to be completed. Our objective is to characterise HPV integration to investigate potential mechanisms of tumourigenesis independent of E6/E7 oncoproteins. High-throughput viral integration detection was performed on 109 HPV(+)OPSCC tumours with relevant clinicopathological information. Of these tumours, 38 tumours underwent targeted gene sequencing, 29 underwent whole exome sequencing and 26 underwent RNA sequencing. HPV integration was detected in 94% of tumours (with a mean integration count of 337). Tumours occurring at the tonsil/oropharyngeal wall that exhibit higher PD-L1 expression demonstrated increased integration sites (p=.024). HPV exhibited a propensity for integration at genomic sites located within specific fragile sites (FRA19A) or genes associated with functional roles such as cell proliferation and differentiation (PTEN, AR), immune evasion (CD274) and glycoprotein biosynthesis process (FUT8). The viral oncogenes E2, E4, E6 and E7 tended to remain intact. HPV fragments displayed enrichment within host copy number variation (CNV) regions. However, insertions into genes related to altered homologous recombination repair were infrequent. Genes with integration had distinct expression levels. Fifty-nine genes whose expression level was affected by viral integration were identified, for example, EPHB1, which was reported to be involved in cellular protein metabolic process. HPV can promote oncogenesis through recurrent integration into functional host genome regions, leading to subsequent genomic aberrations and gene expression disruption. This study characterises viral integrations and virus-host interactions, enhancing our understanding of HPV-related carcinogenesis mechanisms.

The significance of human papillomavirus integration in carcinogenesis and the development of specific diagnostics and countermeasures

Human papillomavirus (HPV) infection is associated with various tumors, notably in the cervix, oropharyngeal region, and anus. As the disease progresses, integration of the viral genome into the host genome is often observed, yet the means of integration and its intrinsic relationship to carcinogenesis remain unclear. To address this gap, novel sequencing technologies have been developed to enhance the accuracy, intuitiveness, and cost-effectiveness of identifying integration breakpoints. HPV genome integration is thought to induce imbalances or dysfunctions in gene expression, epigenetic changes, chromosomal translocation, and genetic instability. The precise mechanisms underlying the changes in gene expression caused by genome integration offer avenues for tumor risk prediction and prognosis assessment. Personalized precision medicine, grounded in the integration patterns unique to each patient, holds promise for cancer treatment. This review summarizes and discusses the current state of knowledge regarding the mechanisms, carcinogenesis, detection and analysis, and clinical significance of HPV integration. It synthesizes existing information to offer a comprehensive overview, potentially enhancing our understanding of HPV-related tumorigenesis and aiding in the development of more effective diagnostic and therapeutic strategies.

Multiple HPV integration mode in the cell lines based on long-reads sequencing.

The integration of human papillomavirus (HPV) is closely related to the occurrence of cervical cancer. However, little is known about the complete state of HPV integration into the host genome. In this study, three HPV-positive cell lines, HeLa, SiHa, and CaSki, were subjected to NANOPORE long-read sequencing to detect HPV integration. Analysis of viral integration patterns using independently developed software (HPV-TSD) yielded multiple complete integration patterns for the three HPV cell lines. We found distinct differences between the integration patterns of HPV18 and HPV16. Furthermore, the integration characteristics of the viruses were significantly different, even though they all belonged to HPV16 integration. The HPV integration in the CaSki cells was relatively complex. The HPV18 integration status in HeLa cells was the dominant, whereas the percentage of integrated HPV 16 in SiHa and CaSki cells was significantly lower. In addition, the virus sequences in the HeLa cells were incomplete and existed in an integrated state. We also identified a large number of tandem repeats in HPV16 and HPV18 integration. Our study not only clarified the feasibility of high-throughput long-read sequencing in the study of HPV integration, but also explored a variety of HPV integration models, and confirmed that viral integration is an important form of HPV in cell lines. Elucidating HPV integration patterns will provide critical guidance for developing a detection algorithm for HPV integration, as well as the application of virus integration in clinical practice and drug research and development.

Cervical Cancer Stages, Human Papillomavirus Integration, and Malignant Genetic Mutations: Integrative Analysis of Datasets from Four Different Cohorts

Cervical cancer represents a significant global health concern, stemming from persistent infections with high-risk types of human papillomavirus (HPV). The understanding of cervical cancer’s clinical correlates, risk factors, molecular mechanisms, stages, and associated genetic mutations is important for early detection and improved treatment strategies. Through integrated analysis of clinical and molecular datasets, this study aims to identify key factors that are overlapping and distinct across four cohorts of different races and regions. Here, datasets from four distinct cohorts of patients from Uganda (N = 212), the United States of America (USA) (N = 228), China (N = 106), and Venezuela (N = 858) were examined to comprehensively explore the relationships between cervical cancer stages, HPV types (clades), productive HPV integration, and malignant genetic mutations. Cohort-specific findings included the occurrence frequencies of cervical cancer stages and grades. The majority of patients from the USA and China were diagnosed with stages I and II, while those from Uganda were diagnosed with stages II and III, reflecting levels of awareness and the availability of HPV vaccines and screening services. Conversely, cervical cancer and its stages were positively correlated with HPV types (clades), HPV integration, and risk-factor habits across the cohorts. Our findings indicate that the more common squamous cervical cancer can be potentially due to productive HPV16 (clade 9) integration. At the molecular level, pathways related to HPV infection, cancer-related conditions, and viral carcinogenesis were among the most significant pathways associated with mutated genes in cervical cancer (across cohorts). These findings collectively corroborate the prominent role of HPV infection and integration leading to genetic mutation and hence to the development of cervical cancer and its stages across patients of distinct races and regions.

Whole exome sequencing identifies common mutational landscape of cervix and endometrium small cell neuroendocrine carcinoma.

Primary small cell neuroendocrine carcinomas of the cervix and endometrium are rare gynecological malignancies with limited treatment options. This study aimed to improve the understanding of the carcinogenesis process and identify potential therapeutic targets for these two tumor types by constructing the mutational landscape at the whole exome level. Primary tumor tissues and their matched blood samples were obtained from 10 patients with small cell cervical neuroendocrine carcinoma (NECC) and five patients with small cell endometrial neuroendocrine carcinoma (NECE). Whole exome sequencing was performed to construct the somatic mutation profiles. Mutational signature and recurrent mutated gene analysis were used to identify tumor subtypes and common carcinogenesis processes. Based on the burden of different mutational signatures, the NECCs in this work can be divided into two subtypes, including the mismatch repair deficiency like (dMMR-like) type (4/10) and the high spontaneous deamination type (6/10). Components of the PI3K/AKT signaling and RAS signaling were exclusively mutated in these two subtypes, respectively. The integration of human papillomavirus made a limited contribution to tumorigenesis in NECC (20%). The dysfunction of the mismatch repair system and microsatellite instability are the major features of NECE. PI3K/AKT, JAK/STAT signaling, and chromatin remodeling activity were the common mutated pathways in NECE. PIK3CA, WNK2, and KMT2B underwent mutations in both the dMMR-like subtype of NECC (50% - 75%) and in NECE (60% - 80%) specimens, while exhibiting infrequent mutational occurrences in publicly available data pertaining to neuroendocrine carcinomas of the lung or bladder (< 10%). We identified the two subtypes of NECC with distinct mutated pathways and potential therapy targets. The dMMR-like type NECC and NECE may share a similar carcinogenesis process that include dysfunction of PI3K/AKT signaling, cell cycle, antiapoptotic processes, and chromatin remodeling activity.

Long-read sequencing of oropharyngeal squamous cell carcinoma tumors reveal diverse patterns of high-risk Human Papillomavirus integration.

In 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. A 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. Nanopore 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. Long-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.

Introduction of E-Score: A gene expression score to characterize immunogenic versus oncogenic HPV expression in HNSCC.

6057 Background: Human Papillomavirus (HPV) integration in the human genome occurs in a majority of HPV(+) head and neck squamous cell carcinomas (HNSCC), with direct and indirect effects on host gene expression and tumorigenesis. Although previous studies showed that HPV integration in HNSCC is overrepresented in genes often somatically mutated in head and neck, lung, and urogenital cancers, reports are mixed for whether patients with HPV integration have worse clinical outcomes. These disparate results may be due to differences in how integration is defined. Integration can often lead to partial loss of the viral genome (e.g. HPV genes E1, E2, or E5) with retention of the oncogenes E6 and E7, which led to a low E2/E7 expression ratio being used as a biomarker for integration. Conversely, others have used the direct detection of DNA or RNA HPV-human breakpoints to identify tumors harboring integrated HPV. HPV integration and the retention or deletion of HPV genes can dramatically change the expression of HPV genes, and we hypothesize it also affects the host immune response. Hypothesis: Loss of non-oncogenic HPV genes relative to E6 and E7 levels reduces immunogenicity, leading to weakened immune cell infiltration and worse clinical outcomes. Methods: Bulk RNA sequencing data from HPV(+) HNSCC tumors (n = 136) was used to characterize the differences in retention or loss of HPV genes and calculate the E-Score as the sum of the logCPM of HPV genes E1, E2, E4, and E5 relative to E7. Cell type deconvolution was performed using ComBat to correct for batch effects, and CIBERSORTx to quantify cell type proportions within the tumor samples. Kaplan-Meier estimates of recurrence and overall survival (OS) were calculated, and Cox proportional hazards tests with covariates were used to test for association with recurrence and OS. To test the correlation between immune cell type inferred proportions and E-Score, T-Tests were performed between samples with high and low E-scores. Results: Low E-Score was significantly associated with recurrence (p = 0.012) but not with OS (p = 0.39) in the multivariate model accounting for age, tumor stage (AJCC8), smoking status, drinking status, BMI, marital status, and molecular tumor subtype (defined as IMU or KRT). For integration positive samples (n = 66), higher E-Score, suggesting lower rates of HPV gene loss, was correlated with higher percentages of dendritic cells (p = 0.044), macrophages (p = 0.021), and CD8+ T Cells (p = 0.024), but not CD4+ T cells (p = 0.14). This significance did not hold when looking at integration negative samples. Conclusions: These results suggest that HPV+ HNSCC patients with a loss of E1, E2, E4 and E5 tend to have less immune cell infiltration and are more likely to have recurrence. We conclude that E-Score is a potential prognostic biomarker of recurrence in HPV+ HNSCC and potential predictive biomarker for immunotherapy.

Integration of human papillomavirus associated anal cancer screening into HIV care and treatment program in Pakistan: perceptions of policymakers, managers, and care providers

BackgroundThe incidence of anal cancer, largely associated with anal human papillomavirus (HPV) infection, is increasing among men who have sex with men (MSM), and transgender women living with or without HIV. Screening for anal cancer to detect anal precancerous lesions in high-risk groups is an important opportunity for prevention but still lacking in many low-and-middle-income countries. The aim of this study was to explore the readiness of Pakistan’s healthcare system to integrate anal cancer and HPV screening into a national HIV program, as perceived by policymakers, health managers, and healthcare providers.DesignThis qualitative study using key-informant interviews with participants influence in policy making, implementation and advocacy from public and private sector were conducted between March 2021 to August 2021 in Karachi Pakistan.MethodsKey informants were purposely selected from different domains of the healthcare system responsible for the target group of interest, MSM and transgender-women in general and people living with HIV in particular. A total of 18 key informants, at different levels of seniority were recruited from governmental and non-governmental organizations, high-level infectious disease healthcare managers, and United Nations Program representatives. Qualitative content analysis was used to identify the manifest and latent themes, based on socioecological framework.ResultsThe results were grouped into five major themes; (1) The policy context and priorities, (2) Health systems factors, (3) Community environment, (4) Healthcare setting & providers and (5) Individual-level obstacles. The policy actors expressed their concerns about their limited voice in country’s health and health related priority setting. Informants reported a lack of political will and suggested that government should bring a change in the paradigm of healthcare service delivery from reactive to proactive approach. Although, participants unanimously favored integration of HPV preventive services into existing HIV program, they also identified several service delivery barriers including trained workforce shortage, limited capacity of information technology, lack of supplies needed for screening, lack of financing, and lack of services that could meet key-populations needs. Participants also predicted other implementation challenges such as stigma, social victimization, and systemic discrimination against at-risk groups at healthcare facilities.ConclusionAlthough policy makers and health providers in Pakistan saw a clear need to scale-up and integrate anal cancer screening for key populations, the feasibility of this is dependent on political will, financing, anti-stigma and discrimination interventions and health system efficiency.

A cross-sectional study of human papillomavirus genotype distribution and integration status in penile cancer among Chinese population

Human papillomavirus (HPV) has been recognized as an important risk factor in penile cancer. This study aimed to investigate the HPV subtypes and integration status in Chinese patients. Samples were collected from 103 penile cancer patients aged 24–90 years between 2013 and 2019. We found that HPV infection rate was 72.8%, with 28.0% integration. The aging patients were more susceptible to HPV (p = 0.009). HPV16 was the most frequent subtype observed (52/75) and exhibited the highest frequency of integration events, with 11 out of 30 single infection cases showing integration positive. The HPV integrations sites in the viral genome were not randomly distributed, the breakpoints were enriched in the E1 gene (p = 0.006) but relatively scarce in L1, E6 and E7. Our research might provide some clues how HPV leads to the progression of penile cancer.

Multi-omics data reveals novel impacts of human papillomavirus integration on the epigenomic and transcriptomic signatures of cervical tumorigenesis.

Integration of human papilloma virus (HPV) DNA into the human genome may progressively contribute to cervical carcinogenesis. To explore how HPV integration affects gene expression by altering DNA methylation during carcinogenesis, we analyzed a multiomics dataset for cervical cancer. We obtained multiomics data by HPV-capture sequencing, RNA sequencing, and Whole Genome Bisulfite Sequencing from 50 patients with cervical cancer. We detected 985 and 485 HPV-integration sites in matched tumor and adjacent paratumor tissues. Of these, LINC00486 (n = 19), LINC02425 (n = 11), LLPH (n = 11), PROS1 (n = 5), KLF5 (n = 4), LINC00392 (n = 3), MIR205HG (n = 3) and NRG1 (n = 3) were identified as high-frequency HPV-integrated genes, including five novel recurrent genes. Patients at clinical stage II had the highest number of HPV integrations. E6 and E7 genes of HPV16 but not HPV18 showed significantly fewer breakpoints than random distribution. HPV integrations occurring in exons were associated with altered gene expression in tumor tissues but not in paratumor tissues. A list of HPV-integrated genes regulated at transcriptomic or epigenetic level was reported. We also carefully checked the candidate genes with regulation pattern correlated in both levels. HPV fragments integrated at MIR205HG mainly came from the L1 gene of HPV16. RNA expression of PROS1 was downregulated when HPV integrated in its upstream region. RNA expression of MIR205HG was elevated when HPV integrated into its enhancer. The promoter methylation levels of PROS1 and MIR205HG were all negatively correlated with their gene expressions. Further experimental validations proved that upregulation of MIR205HG could promote the proliferative and migrative abilities of cervical cancer cells. Our data provides a new atlas for epigenetic and transcriptomic regulations regarding HPV integrations in cervical cancer genome. We demonstrate that HPV integration may affect gene expression by altering methylation levels of MIR205HG and PROS1. Our study provides novel biological and clinical insights into HPV-induced cervical cancer.

Human papilloma virus presence and its physical status in primary pterygium

Pterygium is one of the most frequent pathologies in ophthalmology, and is a benign, overgrowth of fibrovascular tissue, often with a wing-like appearance, from the conjunctiva over the cornea. It is composed of an epithelium and highly vascular, sub-epithelial, loose connective tissue. There is much debate surround the pathogenesis of pterygium and a number of theories have been put forward including genetic instability, cellular proliferation, inflammatory influence, and degeneration of connective tissue, angiogenesis, aberrant apoptosis and viral infection. At present, the involvement of human papillomavirus (HPV) in the genesis of pterygium is controversial, as have reported that HPV is present in 58% of cases, while others have failed to detect HPV in pterygium. In this study, we evaluated the presence and viral genotype of HPV DNA in pterygia and healthy conjunctiva sample, and virus integration into the cellular genome.Forty primary pterygia samples and 12 healthy conjunctiva samples were analyzed to HPV DNA presence by polymerase chain reaction, using MY09/MY11 primers of HPV-L1 gene. Viral genotype was identified by DNA sequence analysis of this amplicon. HPV integration into the cellular genome was analyzed by western blot detecting HPV-L1 capsid protein.Presence of HPV was observed in 19 of the 40 pterygia samples. In contrast, healthy conjunctiva samples were negative. To determine virus type, sequence analyses were performed. Interestingly, 11 out of the 19-pterygium samples were identified as HPV-11 type, meanwhile, the remaining 8 pterygium samples were identified as HPV-18. HPV-L1 capsid protein were found only in 3 out of the 10 samples studied.In conclusion, our study identified the presence of HPV DNA exclusively in pterygium samples and described HPV-11 and -18 genotypes. Our results suggest that HPV may be involved in the pathogenesis of pterygium. On the other hand, the expression of the L1-HPV protein suggests viral integration into the cellular genome.

Abstract LB153: Inhibiting BRD4 as a proposed alternative therapeutic strategy for HPV16-associated head and neck squamous cell carcinoma

Abstract Integration of Human Papilloma Virus (HPV) 16 into human genomes is an integral event for cancer progression in Head and Neck Squamous Cell Carcinoma (HNSCC). There is a need to differentiate treatment for HPV-16 integrated HNSCC from other subtypes. We hypothesize that by inhibiting Bromodomain Extra Terminal (BET) protein BRD4, a transcriptional co-regulator of host and viral genes can advance treatment in patients with the integrated subtype of HPV16 associated HNSCC. Through small molecule inhibition and knockdown of BRD4 in seven model HPV-16 integrated cell lines, we demonstrated that BRD4 down-regulates the expression of viral genes E6, E7, independent of viral E2 type of integration. Given that E6 expression is mediated by BRD4, E6-p53 interactions was abrogated as revealed through increased expression of p53. Furthermore, BRD4 inhibition caused downregulation of c-Myc. This result also suggests the function of BRD4 in the regulation of proteins in cell cycle regulation, including E2F1 and p21. BRD4 inhibition was also shown to delay DNA damage response (DDR) by reducing the expression of RAD51 in 6 out of 7 cell lines. RNA-sequencing results give further insight regarding the downregulation of genes involved in DDR – RAD51, BRCA1 and BRCA2 in all cell lines. RNA-sequencing data also gives us an understanding of the implication of BRD4-inhibition in other genes involved in cell cycle regulation. Knockdown of BRD4 done through small hairpin RNA (shRNA) constructs phenocopied the effects shown through inhibition of BRD4. Additionally, treating the above-mentioned cell lines with BRD4 inhibitor (JQ1) followed by radiation showed further downregulation of viral oncogenes E6, and E7. This suggests that combining radiation and JQ1 treatment can be a viable combination treatment. De-escalating radiation treatment with BRD4 inhibition has potential to be an effective combination treatment for HPV16 integrated HNSCC. Citation Format: Dhruthi Suresh, Chitrasen Mohanty, Albert R. Wang, Christina Kendziorski, Gopal Iyer. Inhibiting BRD4 as a proposed alternative therapeutic strategy for HPV16-associated head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB153.