HPV Integration Research Articles

Characteristics of HPV integration in cervical adenocarcinoma and squamous carcinoma

PurposeHPV integration usually occurs in HPV-related cancer, and is the main cause of cancer. But the carcinogenic mechanism of HPV integration is unclear. The study aims to provide a theoretical basis for understanding the pathogenesis of cervical adenocarcinoma (AC) and cervical squamous carcinoma (SCC).MethodsWe used HPV capture sequencing to obtain HPV integration sites in AC and SCC, and analyzed cytobands, distribution of genetic and genomic elements, identified integration hotspot genes, clinicopathological parameters, breakpoints of HPV16 and performed pathway analysis. Then we conducted immunohistochemical (IHC) assay to preliminarily verify the expression of most frequently integrated genes in AC, STARD3 and ERBB2.ResultsThe results revealed that the most frequently observed integrated cytoband was 17q12 in AC and 21p11.2 in SCC, respectively. The breakpoints in both AC and SCC were more tended to occur within gene regions, compared to intergenetic regions. Compared to SCC samples, AC samples had a higher prevalence of genomic elements. In AC, HPV integration has no significantly difference with clinicopathological parameters, but in SCC integration correlated with differentiation (P < 0.05). Breakpoints of HPV in SCC located in LCR more frequently compared to AC, which destroyed the activation of promoter p97. Hotspot genes of HPV integration were STARD3 and ERBB2 in AC, and RNA45S rDNA and MIR3648-1 in SCC, respectively. Meanwhile, we preliminarily proved that the expression of STARD3 and ERBB2, the most frequently integrated genes, would increase after integration.ConclusionThese results suggested that HPV may utilize the powerful hosts’ promoters to express viral oncogenes and overexpression of viral oncogenes plays a significant role in the carcinogenesis of SCC. In AC, HPV integration may affect hosts’ oncogenes, and the dysregulation of oncogenes may primarily contribute to progression of AC.

Whole genome sequencing in high-grade cervical intraepithelial neoplasia patients from different ethnic groups in China.

Cervical cancer (CC) is the fourth most common cancer in women worldwide. It develops through precancerous lesions (cervical intraepithelial neoplasia (CIN), graded from low-grade (CIN1) to high-grade (CIN2-3)). It is well established that precancerous and cancerous cervical lesions are caused by a persistent infection with high-risk types of the human papilloma virus (hrHPV). To have a deeper understanding of the pathogenesis of CIN and CC, we systematically analyzed the landscape of genomic alterations and HPV integration profiles in high-grade CIN2/3. We performed deep whole genome sequencing on exfoliated cervical cells and matched peripheral blood samples from a cohort of 51 Chinese patients (of whom 35 were HPV+) with high-grade CIN from 3 ethnic groups and constructed strict integrated workflow of genomic analysis. In addition, the HPV types and integration breakpoints in the exfoliated cervical cells from these patients were examined. Genomic analysis identified 6 significantly mutated genes (SMGs), including CDKN2A, PIK3CB, FAM20A, RABEP1, TMPRSS2 and SS18L1, in 51 CIN2/3 samples. As none of them had previously been identified as SMGs in the Cancer Genome Atlas cervical squamous cell carcinoma and endocervical adenocarcinoma (TCGA-CESC) cohort, future studies with larger sample size of CINs may be needed to validate our findings. Mutational signature analysis showed that mutational signatures of CINs were dramatically different from CCs, highlighting their different mutational processes and etiologies. Moreover, non-silent somatic mutations were detected in all of the CIN2/3 samples, and 88% of these mutations occurred in genes that also mutated in CCs of TCGA cohort. CIN2 samples had significantly less non-silent mutations than CIN3 samples (P = .0006). Gene ontology and pathway level analysis revealed that functions of mutated genes were significantly associated with tumorigenesis, thus these genes may be involved in the development and progression of CC. HPV integration breakpoints occurred in 28.6% of the CIN2/3 samples with HPV infection. Integrations of common high risk HPV types in CCs, including HPV16, 52, 58 and 68, also occurred in the CIN samples. Our results lay the groundwork for a deeper understanding of the molecular mechanisms underlying the pathogenesis of CC and pave the way for new tools for screening, diagnosis and treatment of cervical precancerous and cancerous lesions.

The analysis of HPV integration sites based on nanopore sequencing and the profiling changes along the course of photodynamic therapy

ObjectiveTo detect the HPV genotype and integration sites in patients with high-risk HPV infection at different stages of photodynamic therapy using nanopore technology and to evaluate the treatment effect.MethodsFour patients with HPV infection were selected and subjected to photodynamic therapy, and cervical exfoliated cell was sampled at before treatment, after three courses of treatment and six courses of treatment, their viral abundance and insertion sites were analyzed by nanopore technology, and pathological examinations were performed before and after treatment. In this study, we developed a novel assay that combined viral sequence enrichment and Nanopore sequencing for identification of HPV genotype and integration sites at once. The assay has obvious advantages over qPCR or NGS-based methods, as it has better sensitivity after viral sequences enrichment and can generate long-reads (kb to Mb) for better detection rate of structure variations, moreover, fast turn-around time for real-time viral sequencing and analysis.ResultsThe pathological grade was reduced in all four patients after photodynamic therapy. Virus has been cleared in two cases after treatment, the virus amount reduced after treatment but not completely cleared in one case, and two type viruses were cleared and one type virus persisted after treatment in the last patient with multiple infection. Viral abundance and the number of integration sites were positively correlated. Gene enrichment analysis showed complete viral clearance in 1 patient and 3 patients required follow-up.ConclusionNanopore sequencing can effectively monitor the abundance of HPV viruses and integration sites to show the presence status of viruses, and combined with the results of gene enrichment analysis, the treatment effect can be dynamically assessed.

Circulating cell-free HPV DNA is a strong marker for disease severity in cervical cancer.

For cervical cancer (CC), circulating cell-free HPV DNA (ccfHPV) may establish disease severity. Furthermore, HPV integration has been correlated to viral load and survival. In this study, pre-treatment plasma from 139 CC cases (50 primary surgery patients, 22 primary surgery + adjuvant oncological therapy patients, and 67 primary oncological therapy patients) was collected (2018-2020). Furthermore, plasma from 25 cervical intraepithelial neoplasia grade 3 patients and 15 healthy women (negative controls) were collected. Two next-generation sequencing (NGS) panels were used to establish ccfHPV presence and human papillomavirus type 16 (HPV16) integration status. ccfHPV was detected in four primary surgery (8.0%), eight primary surgery + adjuvant oncology (36.4%), and 54 primary oncology (80.6%) patients. For primary oncology patients with HPV16-related cancer (n = 37), more ccfHPVneg than ccfHPVpos patients had HPV16 integration (P = 0.04), and in patients with HPV16 integration (n = 13), ccfHPVpos patients had higher disease stages than ccfHPVneg patients (P = 0.05). In summary, ccfHPV presence is related to disease severity and may add to the debated Sedlis criteria used for identifying patients for adjuvant oncological therapy. However, ccfHPV detection is influenced by HPV integration status and disease stage, and these factors need to be considered in ccfHPVneg patients.

784P HPV integration promotes HPV carcinogenesis via remodeling chromatin interactions between universal stripe factors and super-enhancer in HPV-related carcinoma

784P HPV integration promotes HPV carcinogenesis via remodeling chromatin interactions between universal stripe factors and super-enhancer in HPV-related carcinoma

Abstract PO-026: Intra-tumor heterogeneity of HPV integration and its association with focal genomic instability in oropharyngeal cancer

Abstract Human papillomavirus (HPV) integration has been considered as one of the driver factors for cancer progression, but its pattern and role in carcinogenesis is still unclear. Whole genome sequencing data of 14 Japanese HPV16+ OPSCC and 13 HPV+ OPSCC from Pan-Cancer Analysis of Whole Genomes were used to identify integrations and somatic mutations. The phylogenetic analysis of HPV16 revealed that in tumors of 14 Japanese subjects, the A4 variant was the most common (8/14, 57.1%), followed by the A5 variant (2/14, 14.3%), which is considered unique to HPV16 in East and Southeast Asian, and the A2/A3/D2/D3 variants (1/14, 7.1%), showing a significant difference from the distribution of the HPV16 sublineages in Europe and the United States, where the A1 variant accounts for half. A total of 250 integration breakpoints (BPs) in 20 of the 27 tumors (74.1%) were detected. We estimated the cancer cell fraction of integration BPs and found that HPV integration also shows intra-tumor heterogeneity, indicating that nearly half of the integration events occurred after carcinogenesis. Clonal BPs were more likely to occur in the E1 (P = 0.036), confirming that the disruption of the E1 gene may result in abnormal negative regulation of the E6/E7 oncogenes and promote OPSCC carcinogenesis. Subclonal BPs were less likely to occur in the E6 (P = 0.045), suggesting that the E6 may also play an important role in the process of cancer evolution after carcinogenesis. Four states of the HPV genome were identified: (1) episomal-only (7/27, 25.9%), (2) integrated-only (4/27, 14.8%), (3) clonally-mixed (episomal + clonally integrated) (11/27, 40.7%), and (4) subclonally-mixed (episomal + subclonally integrated) (5/27, 18.5%). Since nearly half of these cancers have developed with just episomal copies of HPV, integration itself is not essential for carcinogenesis and can occur during and after carcinogenesis. Interestingly, the E6/E7 were conserved in all four cancers with integrated-only HPV, while the E2 and E1 were disrupted or deleted. This confirms that the constitutive expression of E6/E7 is essential for the carcinogenesis of HPV+ OPSCC. These results suggest that some integrations may be drivers of carcinogenesis with disruption of E1/E2, as in these four cancers, and others may arise randomly due to genomic instability, as in the cancers with the E6/E7 deleted integrants. The number of BPs of structural variations (SVs) occurred in the overlapping 100 kb regions flanking integration BPs (ITG regions) was positively correlated with the number of integration BPs (Spearman’s rank correlation ρ = 0.78; Permutation test P = 9.9 × 10−5). The genomic instability that causes clonal integrations during carcinogenesis is considered to cause clonal SVs, mainly large duplications, and subclonal integrations and SVs such as large deletions. Clonal SVs, mainly large duplications, were also observed in the regions where only subclonal integrations occurred, suggesting that large duplications occur before integrations. Citation Format: Noah Sasa, Hirotaka Eguchi, Hidenori Tanaka, Takahito Fukusumi, Motoyuki Suzuki, Yukinori Takenaka, Yukinori Okada, Hidenori Inohara. Intra-tumor heterogeneity of HPV integration and its association with focal genomic instability in oropharyngeal cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-026.

Human papillomavirus integration transforms chromatin to drive oncogenesis

BackgroundHuman papillomavirus (HPV) drives almost all cervical cancers and up to 70% of head and neck cancers. Frequent integration into the host genome occurs predominantly in tumorigenic types of HPV. We hypothesize that changes in chromatin state at the location of integration can result in changes in gene expression that contribute to the tumorigenicity of HPV.ResultsWe find that viral integration events often occur along with changes in chromatin state and expression of genes near the integration site. We investigate whether introduction of new transcription factor binding sites due to HPV integration could invoke these changes. Some regions within the HPV genome, particularly the position of a conserved CTCF binding site, show enriched chromatin accessibility signal. ChIP-seq reveals that the conserved CTCF binding site within the HPV genome binds CTCF in 4 HPV+ cancer cell lines. Significant changes in CTCF binding pattern and increases in chromatin accessibility occur exclusively within 100 kbp of HPV integration sites. The chromatin changes co-occur with out-sized changes in transcription and alternative splicing of local genes. Analysis of The Cancer Genome Atlas (TCGA) HPV+ tumors indicates that HPV integration upregulates genes which have significantly higher essentiality scores compared to randomly selected upregulated genes from the same tumors.ConclusionsOur results suggest that introduction of a new CTCF binding site due to HPV integration reorganizes chromatin state and upregulates genes essential for tumor viability in some HPV+ tumors. These findings emphasize a newly recognized role of HPV integration in oncogenesis.

HPV integration as a prognostic biomarker for metastatic anal cancer: A next-generation sequencing ctDNA-based approach.

3525 Background: Anal cancer is an HPV-associated malignancy with increasing prevalence in the United States. Immune checkpoint blockade demonstrates limited efficacy in patients with metastatic squamous cell cancer of the anal canal (mSCCA), and no prognostic or predictive biomarkers have demonstrated clinical utility in this population. Available circulating tumor DNA (ctDNA) assays have utilized ddPCR methodologies to report only the most common HPV types (HPV-16 and HPV-18) and HPV copy numbers. To extend the utility of an HPV ctDNA assay, we created a novel next-generation sequencing HPV ctDNA assay to analyze HPV integration as a prognostic biomarker for patients with mSCCA. Methods: Using an IRB-approved protocol, ctDNA isolated from the plasma of patients with mSCCA was sequenced using a 1.4Mb hybrid-capture target-enrichment panel covering the whole genome sequences of all 193 HPV types. HPV type, HPV copy number, and HPV integration status/locus were determined using a bioinformatic pipeline developed in-house. A Fisher's exact test was used to compare radiographic and ctDNA changes in response to systemic treatment. Unpaired t-tests were used to compare demographics between patients with HPV integration (I) or no integration (NI) status in the ctDNA. Median survival was estimated (Kaplan Meier) and compared according to integration status using a log-rank test. Results: 68 plasma samples from 27 patients with mSCCA were analyzed. While HPV-16 was detected in ctDNA of all 27 patients, five patients had additional oncogenic HPV types [HPV-18 (2), HPV-45 (2), HPV-73 (1)]. Radiographic changes in metastatic disease volume were concordant with HPV copy number changes in the ctDNA (odds ratio 12.2, 95% confidence interval (CI) 2.0-75; p = .007). HPV integration events were detected in the ctDNA for 16 (59%) patients (median 2, range 1-7 unique events per patient). For the 23 patients with mSCCA who received anti-PD1 immunotherapy (15 I, 8 NI), there were no differences in age (60 vs 59 years, p = .88) or prior lines of treatment (3.0 vs 3.5, p = .72). There was a trend towards improved median progression-free survival for NI ctDNA status (5.8 vs 3.2 months, hazard ratio (HR) 1.8, 95% CI .71-4.5). Relative to NI status, I status was associated with worse overall survival rates (OS) (22.5 vs 44.9 months, HR 5.0, 95% CI 1.5-17, p = .005). Conclusions: Detection of HPV integration status for all HPV types is feasible using a ctDNA-based approach for patients with SCCA. HPV integration was prognostic for worse OS in patients with mSCCA. Identification of HPV integration as a novel prognostic biomarker for survival outcomes in mSCCA warrants further evaluation in larger clinical trials.

Polymorphisms in the Nonhomologous End-joining DNA Repair Pathway are Associated with HPV Integration in Cervical Dysplasia.

HPV integration in premalignant lesions and is thought to be an important driver of carcinogenesis. However, it is unclear what factors promote integration. The use of targeted genotyping among women presenting with cervical dysplasia has the potential to be an effective tool in assessing the likelihood of progression to cancer.

PIBF1 regulates multiple gene expression via impeding long-range chromatin interaction to drive the malignant transformation of HPV16 integration epithelial cells

IntroductionHuman papillomavirus (HPV) integration can induce gene expression dysregulation by destroying higher-order chromatin structure in cervical cancer. ObjectivesWe established a 13q22 site-specific HPV16 gene knock-in cell model to interrogate the changes in chromatin structure at the initial stages of host cell malignant transformation. MethodsWe designed a CRISPR-Cas9 system with sgRNA targeting 13q22 site and constructed the HPV16 gene donor. Cells were cotransfected, screened, and fluorescence sorted. The whole genome sequencing (WGS) was used to confirm the precise HPV16 gene integration site. Western blot and qRT-PCR were used to measure gene expression. In vitro and in vivo analysis were performed to estimate the tumorigenic potential of the HPV16 knock-in cell model. Combined Hi-C, chromatin immunoprecipitation and RNA sequencing analyses revealed correlations between chromatin structure and gene expression. We performed a coimmunoprecipitation assay with anti-PIBF1 antibody to identify endogenous interacting proteins. In vivo analysis was used to determine the role of PIBF1 in the tumor growth of cervical cancer cells. ResultsWe successfully established a 13q22 site-specific HPV16 gene knock-in cell model. We found that HPV integration promoted cell proliferation, invasion and stratified growth in vitro, and monoclonal proliferation in vivo. HPV integration divided the affected topologically associated domain (TAD) into two smaller domains, and the progesterone-induced blocking factor 1 (PIBF1) gene near the integration site was upregulated, although PIBF1 was not enriched at the domain boundary by CUT-Tag signal analysis. Moreover, PIBF1 was found to interact with the cohesin complex off chromatin to reduce contact domain formation by disrupting the cohesin ring-shaped structure, causing dysregulation of tumorigenesis-related genes. Xenograft experiments determined the role of PIBF1 in the proliferation in cervical cancer cells. ConclusionWe highlight that PIBF1, a potential chromatin structure regulatory protein, is activated by HPV integration, which provides new insights into HPV integration-driven cervical carcinogenesis.

Integration and viral oncogene expression of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma and gastric cancer.

Persistent high-risk human papillomavirus (HR-HPV) infections cause cervical cancer and a fraction of head and neck cancer. To investigate whether HR-HPV infection might be also involved in the development of gastric cancer (GC), we developed a platform utilizing a rolling circle amplification (RCA)-based nested L1 polymerase chain reactionwith Sanger sequencing to genotype the HPV DNA in cancer tissues of 361 GC and 89 oropharyngeal squamous cell carcinomas (OPSCC). HPV transcriptional activity was determined by E6/E7 mRNA expression and a 3' rapid amplification of cDNA ends was performed to identify HPV integration and expression of virus-host fusion transcripts. Ten of 361 GC, 2of 89 OPSCC, and 1of 22 normal adjacent tissues were HPV L1 DNA-positive. Five of the 10 HPV-positive GC were genotyped as HPV16 by sequencing and 1of 2 GC with RCA/nested HPV16 E6/E7 DNA detection exhibited HPV16 E6/E7 mRNA. Two OPSCC displayed HPV16 L1 DNA and E6/E7 mRNA, of which 1OPSCC tissue showed virus-host RNA fusion transcripts from an intron region of KIAA0825 gene. Together, our data reveal viral oncogene expression and/or integration in GC and OPSCC and a possible etiology role of HPV infections in gastric carcinogenesis.

Abstract LB252: HPV integration events are heterogenous, clonally selected and associated with spatially distinct transcriptomic profiles in aggressive HPV positive oropharyngeal squamous cell carcinoma

Abstract HPV positive Oropharyngeal Squamous Cell Carcinoma (HPV positive OPSCC) is rising in incidence and, for patients that recur, survival is extremely poor. Recent technological advances have led to a better understanding of patterns of HPV integration in primary tumors but have not yet tested the evolution of these events in deadly forms of the disease. Therefore, we performed multi-region sequencing analysis on a cohort of 34 recurrent and metastatic patients with HPV positive OPSCC (29% survival rate) plus one recurrent HPV positive model pair, UPCI-90 and UPCI-152. We then used our recently developed capture-based sequencing HPV integration caller and assembler pipeline, SearcHPV, to identify patterns of viral integration into the host genome and Oxford Nanopore long-read whole genome sequencing to validate the integration events in selected cases. In total, 1591 HPV integrations were called and assembled. Unexpectedly, our analysis revealed that HPV integration events are heterogenous as multiple independent integration events were observed in different spatial regions of the same tumor. Further, when comparing independent and anatomically distinct regions of recurrence from the same patient, independent integration events were also observed. Heterogeneity of integration structures was found to include variation of local HPV copy number, distribution of the specific HPV integration sites within the structures as well as distinct characteristics of rearrangement breakpoints (human-HPV, HPV-HPV, human-human). Given this observation, we then evaluated the hypothesis that HPV integration events could be clonally selected by comparing the integration profiles from a cohort of matched primary and recurrent tumors. In this cohort, 64 integration events (11 exact same; 53 clustered in the same genes) were maintained throughout tumor progression to recurrence, while other integration events were lost, supporting a model of clonal evolution with driver and passenger integrations. We then explored this further using one recurrent HPV positive model pair, UPCI-90 and UPCI-152, using long-read whole genome sequencing which validated the heterogeneity and clonal selection of HPV integration events. Finally, to test if differences in HPV integration content in spatially distinct regions could drive spatially distinct phenotypic differences, we performed multi-region RNA-seq analysis on 8 HPV16 positive OPSCCs, which demonstrated spatial differences in HPV16 E7 expression, that were validated with RNAscope assays, as well as statistically significant spatial enrichment of independent oncogenic gene sets. Collectively, we report evidence showing that HPV integration events are heterogenous, clonally selected during progression and associated with distinct transcriptomic profiles. Based on this data, we now propose a new working model of disease pathogenesis in which heterogeneity and clonal evolution of HPV integration events serve as a critical driver of the disease process. Citation Format: Wenjin Gu, Apurva Bhangale, Collin V. Brummel, Elizabeth Gensterblum-Miller, Matthew E. Spector, Ryan E. Mills, Chad Brenner. HPV integration events are heterogenous, clonally selected and associated with spatially distinct transcriptomic profiles in aggressive HPV positive oropharyngeal 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 LB252.

Abstract 1212: Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma

Abstract Human papillomavirus (HPV) infections are currently associated with more than 70% of oropharyngeal squamous cell carcinomas (OPSCCs). HPV-positive OPSCCs display a distinct biologic behavior and treatment response and thus are now considered a distinct entity than HPV-negative OPSCCs. However, the underlying process of HPV-related OPSCC carcinogenesis is not entirely clear. HPV as a driver of cervical carcinogenesis is well-characterized with identification of a canonical mechanism dependent on HPV DNA integration, loss of HPV E2 gene expression, and increased expression of HPV E6 and E7 oncogenes with no active infection present in malignant lesions. However, whole genome sequencing found no HPV DNA integration in ~25% of HPV-associated OPSCC, suggesting an alternative mechanism of HPV carcinogenesis. Expression of all HPV genes, including self-assembling capsid proteins, in a substantial portion of HPV+ OPSCC, led us to hypothesize that tumors with episomal HPV are capable of transferring HPV genes to surrounding cells and that HPV gene transfer contributes to carcinogenesis in this subset of OPSCC. Our data support this hypothesis, showing that growth media from HPV+ OPSCC cell lines, as well as isolates from HPV+ tumors or nodal metastases, effectively transferred functional HPV genes to cells lacking HPV. The HPV DNA Transferring Agent (HDTA) derived from HPV+ OPSCC contained HPV L1 capsid protein and intact HPV genome protected from DNAase degradation. Initial characterization revealed that anti-L1 antibody blocked gene transfer and that exposure of cells to the HDTA initiated a robust innate immune response in recipient cells. Negative staining of HDTA isolate from metastatic lymph nodes revealed many pleomorphic, electron-dense particles with diameter ranging between 20 and 80nM, with approximately 40% of particles being between 50 and 60nM, consistent with the size of HPV. We propose that development and/or maintenance of OPSCCs depend on continuous production of HDTA capable of transferring HPV genes into neighboring cells. This paradigm of tumorigenesis/maintenance in OPSCC is most likely to apply to cancers lacking HPV integration, but may also apply to tumors with integration, since recent data suggested that integrated HPV may excise from the genome to recreate a transient episomal like structure, even containing cellular DNA. Our findings offer novel insights into oropharyngeal carcinogenesis and suggest targeting HDTA as a new therapeutic strategy for HPV-positive OPSCCs. Citation Format: Jason Tasoulas, Wendell Gray Yarbrough, Natalia Issaeva. Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1212.

Abstract 6164: NF-κB and NRF2 signaling pathways affect prognosis in HPV-associated head and neck cancer

Abstract Incidence of HPV associated head and neck squamous cell carcinoma (HNSCC) is on the rise, displaying a significantly favorable prognosis and overall better survival as compared to HPV negative HNSCC. Intense radiotherapy is the primary medical care leading to treatment associated morbidity in patients, thereby generating a need for de-intensification strategies. Biomarkers that can identify patients with less aggressive tumors as candidates for de-escalation therapy would revolutionize treatment. We applied an unbiased approach (weighted gene correlation network analysis (WGCNA)) that allows detection of autocorrelated gene sets on 3 independent cohorts of HPV+ HNSCC and created 22 consensus transcriptional modules by selecting genes that grouped together in WGCNA analyses from all 3 cohorts. Interestingly, only 1 module intrinsically divided HPV+ HNSCC into 2 subtypes, displaying different mutational profiles, mutational signatures, HPV gene expression patterns, HPV integration status, and patient survival. Gene set enrichment analysis revealed that this module was enriched in NF-kB genes and strongly associated with NF-κB signaling, confirming our prior findings that defined HPV+ HNSCC subtypes by presence or absence of NF-kB regulators, TRAF3 or CYLD defects and by the NF-κB activity classifier. Our studies show that NF- κB -driven intrinsic tumor characteristics contribute to increased sensitivity of NF- κB active HPV+ head and neck tumors to radiation, providing patients survival benefits. Indeed, TRAF3 or CYLD deletion activated NF-kB and dramatically increased radiation sensitivity of HPV+ head and neck cancer cells. In line with our RNA seq analysis, we found that activation of NF-κB via TRAF3/CYLD deletion significantly correlated with marked downregulation of NRF2 activity and reduced nuclear localization in in HPV+ HNSCC cells. Interestingly, TRAF3 CRISPR KO cells had lower NRF2 protein levels that were restored by MG132 treatment, indicating an involvement of KEAP1/CUL3 mediated proteasomal degradation of NRF2. In summary, our data unveils a unique relation between NF-κB pathway and radiosensitivity in HPV+ HNSCC. The distinct biological characteristics that separate patients into two subclasses associated with favorable prognosis could serve as a biomarker for personalized therapy. Citation Format: Aditi Kothari, Travis Parke Schrank, Wendell Gray Yarbrough, Natalia Isaeva. NF-κB and NRF2 signaling pathways affect prognosis in HPV-associated head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6164.

Inferring early genetic progression in cancers with unobtainable premalignant disease

Analysis of premalignant tissue has identified the typical order of somatic events leading to invasive tumors in several cancer types. For other cancers, premalignant tissue is unobtainable, leaving genetic progression unknown. Here, we demonstrate how to infer progression from exome sequencing of primary tumors. Our computational method, PhylogicNDT, recapitulated the previous experimentally determined genetic progression of human papillomavirus-negative (HPV–) head and neck squamous cell carcinoma (HNSCC). We then evaluated HPV+ HNSCC, which lacks premalignant tissue, and uncovered its previously unknown progression, identifying early drivers. We converted relative timing estimates of driver mutations and HPV integration to years before diagnosis based on a clock-like mutational signature. We associated the timing of transitions to aneuploidy with increased intratumor genetic heterogeneity and shorter overall survival. Our approach can establish previously unknown early genetic progression of cancers with unobtainable premalignant tissue, supporting development of experimental models and methods for early detection, interception and prognostication.

Intratumoral Heterogeneity and Clonal Evolution Induced by HPV Integration.

Long-read sequencing of HPV-positive cancers revealed "heterocateny," a previously unreported form of genomic structural variation characterized by heterogeneous, interrelated, and repetitive genomic rearrangements within a tumor. Heterocateny is driven by unstable concatemerized HPV genomes, which facilitate capture, rearrangement, and amplification of host DNA, and promotes intratumoral heterogeneity and clonal evolution. See related commentary by McBride and White, p. 814. This article is highlighted in the In This Issue feature, p. 799.

Effects of CpG sites methylation modification of HPV16 integration essential gene on the proliferation of cervical cancer cells.

The mechanism of methylation of HPV CpG sites in the occurrence and prognosis of cervical carcinogenesis remains unclear. We investigated the effects of demethylation of the CpG sites of E2 and E6, essential genes of HPV16 integration, on cervical cancer cell expression, integration, and proliferation. HPV16-positive (Caski) cells were treated with different concentrations of the demethylation compound 5-aza-dc (0, 5, 10, 20μmol/l) in vitro. After the intervention, the methylation statuses of HPV16 E2 and E6 were detected by TBS, the expression levels of E2 and E6 mRNA and protein were detected by real-time PCR and western blot, cell proliferation activity was detected by CCK8, and cell cycle and apoptosis were determined by FCM. GraphPad Prism version 8.4.2 and R version 4.2.3 were used for relevant data analyses. The methylation levels of HPV16 E2 and E6 CpG sites decreased gradually with increasing 5-aza-dc intervention concentrations. With decreasing E2 and E6 methylation rates, E2 expression increased, the E2/E6 ratio increased, E6 expression decreased, and the growth inhibition rate of Caski cells increased. E2 and E6 expression were negatively and positively correlated with their degrees of methylation respectively, while the E2/E6 mRNA to protein ratio was negatively correlated with the methylation degrees of E2 and E6. Demethylation can be used as a prospective treatment to affect HPV expression and persistent infection, providing a new theoretical basis for the clinical treatment of viral infections.

Sequential gene expression analysis of cervical malignant transformation identifies RFC4 as a novel diagnostic and prognostic biomarker

BackgroundCervical squamous cell carcinoma (SCC) is known to arise through increasingly higher-grade squamous intraepithelial lesions (SILs) or cervical intraepithelial neoplasias (CINs). This study aimed to describe sequential molecular changes and identify biomarkers in cervical malignant transformation.MethodsMultidimensional data from five publicly available microarray and TCGA-CESC datasets were analyzed. Immunohistochemistry was carried out on 354 cervical tissues (42 normal, 62 CIN1, 26 CIN2, 47 CIN3, and 177 SCC) to determine the potential diagnostic and prognostic value of identified biomarkers.ResultsWe demonstrated that normal epithelium and SILs presented higher molecular homogeneity than SCC. Genes in the region (e.g., 3q, 12q13) with copy number alteration or HPV integration were more likely to lose or gain expression. The IL-17 signaling pathway was enriched throughout disease progression with downregulation of IL17C and decreased Th17 cells at late stage. Furthermore, we identified AURKA, TOP2A, RFC4, and CEP55 as potential causative genes gradually upregulated during the normal-SILs-SCC transition. For detecting high-grade SIL (HSIL), TOP2A and RFC4 showed balanced sensitivity (both 88.2%) and specificity (87.1 and 90.1%), with high AUC (0.88 and 0.89). They had equivalent diagnostic performance alone to the combination of p16INK4a and Ki-67. Meanwhile, increased expression of RFC4 significantly and independently predicted favorable outcomes in multi-institutional cohorts of SCC patients.ConclusionsOur comprehensive study of gene expression profiling has identified dysregulated genes and biological processes during cervical carcinogenesis. RFC4 is proposed as a novel surrogate biomarker for determining HSIL and HSIL+, and an independent prognostic biomarker for SCC.

Characteristics of Vaginal Microbiome in Reproductive-Age Females with HPV Infection in Xinjiang, China.

We investigated the characteristics of vaginal microbiome in reproductive-age females with HPV infection in Xinjiang, China. A total of 135 females of reproductive age were enrolled. There were 43 healthy HPV-negative females in control group (N group), 58 HPV-positive females in nonlesion group (P1 group), and 34 HPV-positive females in low-grade squamous intraepithelial lesion group (P2 group). DNA was extracted from the vaginal secretions, and V3-V4 regions of bacterial 16S rDNA were amplified and sequenced by NovaSeq. QIIME2 and R software were used to perform diversity analysis of bacteria. PICRUSt2 was used to predict the function of the vaginal microbiota. Lactobacillus was the main genus of vaginal microbiota in asymptomatic reproductive-age females with or without HPV in Xinjiang. The diversity of vaginal microbiota in the P1 group was significantly higher than that in the N group, and the proportion of Gardnerella increased significantly. The vaginal microbiota structure of the P2 group was different from the N group, characterized by the decrease of Lactobacillus crispatus and the increase of Shuttleworthia. The function of the inordinate microbiome may play a role in accelerating HPV replication and integration. The structure of vaginal microbiota alters under persistent HPV infection in asymptomatic females of reproductive age in Xinjiang. The Gardnerella increase is associated with increased susceptibility to HPV infection, and Lactobacillus iners predominance and Shuttleworthia presence may be a signature of HPV infection with low-grade squamous intraepithelial lesion.

Viral Integration Plays a Minor Role in the Development and Prognostication of Oral Squamous Cell Carcinoma.

Viruses are well known drivers of several human malignancies. A causative factor for oral cavity squamous cell carcinoma (OSCC) in patients with limited exposure to traditional risk factors, including tobacco use, is yet to be identified. Our study aimed to comprehensively evaluate the role of viral drivers in OSCC patients with low cumulative exposure to traditional risk factors. Patients under 50 years of age with OSCC, defined using strict anatomic criteria were selected for WGS. The WGS data was interrogated using viral detection tools (Kraken 2 and BLASTN), together examining >700,000 viruses. The findings were further verified using tissue microarrays of OSCC samples using both immunohistochemistry and RNA in situ hybridisation (ISH). 28 patients underwent WGS and comprehensive viral profiling. One 49-year-old male patient with OSCC of the hard palate demonstrated HPV35 integration. 657 cases of OSCC were then evaluated for the presence of HPV integration through immunohistochemistry for p16 and HPV RNA ISH. HPV integration was seen in 8 (1.2%) patients, all middle-aged men with predominant floor of mouth involvement. In summary, a wide-ranging interrogation of >700,000 viruses using OSCC WGS data showed HPV integration in a minority of male OSCC patients and did not carry any prognostic significance.