HPV Integration Research Articles

HPV16 integration regulates ferroptosis resistance via the c-Myc/miR-142-5p/HOXA5/SLC7A11 axis during cervical carcinogenesis

BackgroundFerroptosis, a newly identified form of regulated cell death triggered by small molecules or specific conditions, plays a significant role in virus-associated carcinogenesis. However, whether tumours arising after high-risk HPV integration are associated with ferroptosis is unexplored and remains enigmatic.MethodsHigh-risk HPV16 integration was analysed by high­throughput viral integration detection (HIVID). Ferroptosis was induced by erastin, and the levels of ferroptosis were assessed through the measurement of lipid-reactive oxygen species (ROS), malondialdehyde (MDA), intracellular Fe2+ level and transmission electron microscopy (TEM). Additionally, clinical cervical specimens and an in vivo xenograft model were utilized for the study.ResultsExpression of HPV16 integration hot spot c-Myc negatively correlates with ferroptosis during the progression of cervical squamous cell carcinoma (CSCC). Further investigation revealed that the upregulated oncogene miR-142-5p in HPV16-integrated CSCC cells served as a critical downstream effector of c-Myc in its target network. Inhibiting miR-142-5p significantly decreased the ferroptosis-suppressing effect mediated by c-Myc. Through a combination of computational and experimental approaches, HOXA5 was identified as a key downstream target gene of miR-142-5p. Overexpression of miR-142-5p suppressed HOXA5 expression, leading to decreased accumulation of intracellular Fe2+ and lipid peroxides (ROS and MDA). HOXA5 increased the sensitivity of CSCC cells to erastin-induced ferroptosis via transcriptional downregulation of SLC7A11, a negative regulator of ferroptosis. Importantly, c-Myc knockdown increased the anti-tumour activity of erastin by promoting ferroptosis both in vitro and in vivo.ConclusionsCollectively, these data indicate that HPV16 integration hot spot c-Myc plays a novel and indispensable role in ferroptosis resistance by regulating the miR-142-5p/HOXA5/SLC7A11 signalling axis and suggest a potential therapeutic approach for HPV16 integration-related CSCC.

Risk assessment and triage strategy of cervical cancer primary screening on HPV integration status: 5-year follow-up of a prospective cohort study

ObjectiveWe investigated the relation man papillomavirus (HPV) integration status and the immediate risk of cervical intraepithelial neoplasia (CIN), as well as the triage strategy based on HPV integration test. Methods4086 women aged 20 to 65 years in China were enrolled in 2015 for a prospective, population-based, clinical observational study to evaluate the triage performance of HPV integration. Cervical exfoliated cells were collected for HPV testing and cytologic test. If high-risk HPV was positive, HPV integration test was performed at baseline, 2-year and 5-year follow-up. ResultsAt baseline, HPV integration was positively correlated with the severity of cervical pathology, ranging from 5.0% (15/301) in normal diagnosis, 6.9% (4/58) in CIN1, 31.0% (9/29) in CIN2, 70% (14/20) in CIN3, and 100% (2/2) in cervical cancer (P < 0.001). Compared with cytology, HPV integration exhibits comparable sensitivity and negative predictive value for the diagnosis of CIN3+, higher specificity (92.8% [90.2%–95.4%] vs. 75.5% [71.2%–79.8%], P < 0.001) and higher positive predictive value (36.4% [22.1%–50.6%] vs. 15.2% [8.5%–21.8%], P < 0.001). HPV integration testing strategy yielded a significantly lower colposcopy referral rate than cytology strategy (10.7% [44/410] vs. 27.3% [112/410], P < 0.001). The HPV integration-negative group exhibited the lowest immediate risk for CIN3+ (1.6%) and accounted for the largest proportion of the total population (89.3%), when compared with the normal cytology group (risk, 1.7%; proportion, 72.7%). ConclusionAs a key molecular basis for the development of cervical cancer, HPV integration might be a promising triage strategy for HPV-positive patients.

LAMP-based electrochemical platform for monitoring HPV genome integration at the mRNA level associated with higher risk of cervical cancer progression.

Human papillomaviruses (HPVs) represent a diverse group of double-stranded DNA viruses associated with various types of cancers, notably cervical cancer. High-risk types of HPVs exhibit their oncogenic potential through the integration of their DNA into the host genome. This integration event contributes significantly to genomic instability and the progression of malignancy. However, traditional detection methods, such as immunohistochemistry or PCR-based assays, face inherent challenges, and thus alternative tools are being developed to fasten and simplify the analysis. Our study introduces an innovative biosensing platform that combines loop-mediated amplification with electrochemical (EC) analysis for the specific detection of HPV16 integration. By targeting key elements like the E7 mRNA, a central player in HPV integration, and the E2 viral gene transcript lost upon integration, we show clear distinction between episomal and integrated forms of HPV16. Our EC data confirmed higher E7 expression in HPV16-positive cell lines having integrated forms of viral genome, while E2 expression was diminished in cells with fully integrated genomes. Moreover, we revealed distinct expression patterns in cervical tissue of patients, correlating well with digital droplet PCR, qRT-PCR, or immunohistochemical staining. Our platform thus offers insights into HPV integration in clinical samples and facilitates further advancements in cervical cancer research and diagnostics.

HPV-16 E6 mutation and viral integration related host DNA methylation implicate the development and progression of cervical cancer

Background HPV-16 infection and viral-host integration are the most important risk factors for cervical cancer (CC). The aim of this study is to develop a new molecular strategy integrated both the viral and host genome variations identifying and monitoring CC. Method A total of 312 methylation and 538 RNA-seq datasets were collected from public databases to identify differentially methylated and expressed genes. HPV associated virus integration sites (VISs) were analysed using the ViMIC database. From September 2020 to August 2021, the 70 HPV-16 positive cases retrospectively collected from multi-centre cohorts were subjected to HPV-16 E6 deep sequencing and PCR-based host gene (ASTN1, DLX1, ITGA4, RXFP3, SOX17, ZNF671) methylation detection. RNAseq and expression validation (NNF671) were performed in C-33A cell line harbouring HPV D32E. Lasso and logistic regression algorithm were used to construct the CC diagnostic model. Results A positive correlation was observed between the average methylation level of CC patients and their pathological features including tumour stage (p = 0.0077) and HPV subtype (p < 0.001). ZNF671 was identified as a CC-specific methylation marker, with an impressive 93% sensitivity. Both HPV-16 D32E mutation and integration of HPV-16 down-regulated the ZNF671 expression. Finally, a CC diagnostic nomogram was developed by integrating ZNF671 methylation level and HPV E6 mutation feature, yielding an exceptional AUC of 0.997 (95% CI: 0.934–1.000). Conclusions Our study demonstrated HPV viral mutations are closely related to host gene epigenetic alterations in CC. Integration of the viral and host genetic information might be a new promising strategy for CC screening.

HPV integration: a precise biomarker for detection of residual/recurrent disease after treatment of CIN2-3

BackgroundThis study aimed to investigate whether persistent human papillomavirus integration at the same loci (PHISL) before and after treatment can predict recurrent/residual disease in women with CIN2-3.MethodsA total of 151 CIN2-3 women treated with conization between August 2020 and September 2021 were included. To investigate the precision of HPV integration, we further analyzed HPV integration-positive patients. Sensitivity, specificity, positive and negative predictive values (PPV and NPV, respectively), and the Youden index for predicting recurrence/residual disease were calculated.ResultsAmong the 151 enrolled CIN2-3 women, 56 were HPV integration-positive and 95 had HPV integration-negative results. Six (10.7%) experienced recurrence among 56 HPV integration-positive patients, which was more than those in HPV integration-negative patients (one patient, 1.1%). In the 56 HPV integration-positive patients, 12 had positive HPV results after treatment, seven had PHISL, and two had positive cone margin. Among the seven patients who tested with PHISL, six (85.7%) had residual/recurrent disease. PHISL was a prominent predictor of persistent/recurrent disease. The HPV test, the HPV integration test, and PHISL all had a sensitivity of 100% and a NPV of 100% for residual/recurrent disease. PHISL showed better specificity (98.0% vs. 82.0%, p = 0.005) and PPV (85.7% vs. 40.0%, p = 0.001) than the HPV test for predicting recurrence.ConclusionsThe HPV-integration-positive CIN2-3 women had much higher relapse rates than HPV-integration-negative CIN2-3 women. The findings indicate that PHISL derived from preoperative and postoperative HPV integration tests may be a precise biomarker for the identification of residual/recurrent CIN 2/3.

13P HPV integration status conversion and risk stratification by HPV integration levels in HPV integration-positive women: A 1-year follow-up

13P HPV integration status conversion and risk stratification by HPV integration levels in HPV integration-positive women: A 1-year follow-up

Multi-feature next-generation liquid biopsy for diagnosis and prognosis in HPV-associated head and neck cancer.

6064 Background: Human papillomavirus-associated head and neck squamous cell carcinoma (HPV+HNSCC) releases circulating tumor HPV DNA (ctHPVDNA) into the blood which is a highly accurate biomarker of disease status. Existing approaches based on droplet digital PCR (ddPCR) have limitations in sensitivity, genotype coverage, and do not annotate additional prognostic genomic features. In this study, we tested the diagnostic and prognostic performance of a custom HPV whole-genome next-generation sequencing (NGS) liquid biopsy, termed HPV-DeepSeek, at the time of diagnosis with HPV+HNSCC, compared to ctHPVDNA ddPCR, HPV serology, and clinical work-up. Methods: 304 participants (152 untreated HPV+HNSCC patients, 152 population-level controls) were prospectively enrolled in this single-center prospective cohort study. Plasma samples were analyzed using HPV-DeepSeek, a multiplexed serology assay, single-plex ddPCR, and a 5-genotype multiplexed ddPCR assay. We tested the hypothesis that HPV-DeepSeek would have the highest diagnostic accuracy. We secondarily examined the accuracy of HPV-DeepSeek for prognostic feature annotation including high-risk HPV16 single nucleotide polymorphisms (SNPs), integration events, and PIK3CA mutations. Results: Of 152 cases, 114 (75%) were AJCC stage 1 and 138/152 (91%) were oropharynx primary site cancers. The sensitivity (98.7%), specificity (98.7%), and diagnostic accuracy (0.974) of HPV-DeepSeek was superior (P&lt;0.001) to singleplex ddPCR (94.2%, 98.6%, 0.928), multiplex ddPCR (90.6%, 96.3%, 0.869), HPV serology (86.4%, 96.3%, 0.827), and first attempt clinical diagnostic biopsy (78% sensitive). Utilizing HPV-DeepSeek, 8 different genotypes were detected. 4 patients were found to have multi-genotype infections. 137/152 cases were classified as HPV16, with A1 the most common sub-lineage (90/137). 21 of 137 HPV+HNSCC HPV16 cases (15%) displayed high risk SNPs. Head-to-head comparison of HPV SNP genotyping by HPV-DeepSeek, with the gold-standard tissue-based HPV whole genome sequencing assay, demonstrated 89% concordance in tissue and 85% in blood. The internal concordance rate for HPV-DeepSeek between tissue and blood was 91%. 48 of 152 cases (32%) had at least one viral integration event detected and 36 of 152 cases (24%) had a PIK3CA mutation detected by HPV-DeepSeek. Fragmentomic analysis demonstrated a mean ctHPVDNA fragment size of 148bp for ctHPVDNA versus 167bp for human cell free (cf)DNA from HPV+HNSCC patients and 168bp for control patient cfDNA. Baseline ctHPVDNA quantity was strongly correlated with viral integration status, T stage and N stage (P&lt;0.001). Conclusions: HPV-DeepSeek has improved diagnostic accuracy relative to ddPCR, HPV serology and clinical work-up at the time of diagnosis and demonstrates detection of prognostic features including high-risk HPV16 SNPs, viral integration events, and PIK3CA mutations.

Multiomics sequencing and immune microenvironment characteristics define three subtypes of small cell neuroendocrine carcinoma of the cervix.

Small cell cervical carcinoma (SCCC) is the most common neuroendocrine tumor in the female genital tract, with an unfavorable prognosis and lacking an evidence-based therapeutic approach. Until now, the distinct subtypes and immune characteristics of SCCC combined with genome and transcriptome have not been described. We performed genomic (n = 18), HPV integration (n = 18), and transcriptomic sequencing (n = 19) of SCCC samples. We assessed differences in immune characteristics between SCCC and conventional cervical cancer, and other small cell neuroendocrine carcinomas, through bioinformatics analysis and immunohistochemical assays. We stratified SCCC patients through non-negative matrix factorization and described the characteristics of these distinct types. We further validated it using multiplex immunofluorescence (n = 77) and investigated its clinical prognostic effect. We confirmed a high frequency of PIK3CA and TP53 alterations and HPV18 integrations in SCCC. SCCC and other small cell carcinoma had similar expression signatures and immune cell infiltration patterns. Comparing patients with SCCC to those with conventional cervical cancer, the former presented immune excluded or 'desert' infiltration. The number of CD8+ cells in the invasion margin of SCCC patients predicted favorable clinical outcomes. We identified three transcriptome subtypes: an inflamed phenotype with high-level expression of genes related to the MHC-II complex (CD74) and IFN-α/β (SCCC-I), and two neuroendocrine subtypes with high-level expression of ASCL1 or NEUROD1, respectively. Combined with multiple technologies, we found that the neuroendocrine groups had more TP53 mutations and SCCC-I had more PIK3CA mutations. Multiplex immunofluorescence validated these subtypes and SCCC-I was an independent prognostic factor of overall survival. These results provide insights into SCCC tumor heterogeneity and potential therapies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer.

Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.

Abstract 2280: HPV integration identifies emerging oncogenes and carcinogenic mechanisms, potentially indicating tumor recurrences

Abstract HPV integration events in Head and neck squamous cell carcinomas (HNSCC) are strongly associated with carcinogenesis and tumor progression. Previous studies have elucidated the mechanisms and structures of HPV-human fusion events, pinpointing potential integration “hotspots” (i.e. genes with nearby HPV integration in recurrent patients). However, none has comprehensively investigated the effects of HPV integration on both HPV and human gene expression, nor assessed the clinical implications of these bidirectional expression changes. In this study, we conducted bulk RNA sequencing on 67 Formalin-Fixed Paraffin-Embedded Oropharyngeal Squamous Cell Carcinoma (OPSCC) tumor samples from the University of Michigan hospital, and analyzed them in conjunction with bulk and single cell RNA-seq from another &amp;gt;200 HPV(+) HNSCC patients from four previous studies. Among the 117 samples confidently identified as HPV integration positive, we recaptured all previously known integration “hotspot” genes as well as several novel ones that have altered expression in the integrated samples. Exploring the impact of HPV integration on the expression of human and HPV genes, we found that HPV integration in HNSCC is strongly associated with keratinization, immune response, and the development of HPV(+) HNSCC subtypes. Upon HPV integration, expression of non-oncogenic HPV genes is often lost. We hypothesized that the expression ratio of these potentially HPV immunogenic genes (E1-E5, L1,L2) to the HPV oncogenic genes E6 and E7 (termed the ImmunOnco Score) would predict clinical outcomes. Survival analysis of 70 University of Michigan patients showed low ImmunOnco Score is associated with tumor recurrence. Citation Format: Shiting Li, Shaomiao Xia, Maria Lawas, Tingting Qin, Bailey Grab, Nisha D'Silva, Laura Rozek, Maureen Sartor. HPV integration identifies emerging oncogenes and carcinogenic mechanisms, potentially indicating tumor recurrences [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 2280.

Abstract 3491: Tumor subtype classification of HPV-associated head and neck cancers is central to key clinically relevant variables

Abstract Cancer types are typically categorized according to the cell of origin, but within these groupings there exists vast heterogeneity. Thus, subtypes based on molecular features are often defined that have clinical utility as prognostic biomarkers, aid in selection of therapeutic strategies, or are associated with treatment response. Head and neck cancer (HNC) is the seventh most common cancer globally and projected to have 54,540 new cases in the U.S. in 2023. Within the US, HPV(+) HNC is now more prevalent than HPV(+) cervical cancer, and it is expected to continue to rise. Although there is wide morphologic and epigenetic diversity within HPV(+) HNC, tumor subtyping is not yet widely used for this cancer population, largely because of HPV(+) HNC’s unique tumorigenesis process. Unlike other cancers that are driven by specific genetic signatures, HNC is driven by viral gene mechanisms. Given the large degree of heterogeneity among HPV(+) HNC cases and the lack of key mutations to define subtypes, the classification of HPV(+) HNSC subtypes requires a more sophisticated approach. In this study, we introduce a robust, ensemble machine learning (ML) classifier for subtyping HPV(+) HNC that was trained on multiple cohorts and gene feature sets from RNA-seq data, and rigorously tested to ensure high reproducibility. The results classify HPV(+) HNC into the two main recognized subtypes: IMU (immune strong) and KRT(highly keratinized). Using a cohort of 227 patients, we show that the IMU/KRT classification is highly correlated with key clinically relevant variables in HNC. Of 41 molecular, clinical, and epidemiologic variables tested, 24 significantly associated with subtype. The IMU subtype was significantly associated with CD8 T-cells (p-value = 4.21 × 10−9), Dendritic cells (p-value = 1.65 × 10−8), B-cells (p-value = 2.20E−8), and epithelial to mesenchymal transition (p-value = 0.00013146). The KRT subtype is significantly associated with keratinization (p-value = 7.11 × 10−9), HPV integration (p-value = 3.14 × 10−6), radiation resistance (p-value = 0.00307), female sex (p-value = 0.00641), and high T stage (p-value = 0.0324). Genetic, epigenetic, and HPV gene-related variables were also among those significantly associated with subtype. HPV(+) HNC subtypes have been shown to be associated with survival, with KRT-like patients having worse clinical outcomes than IMU. Given the different carcinogenic processes underlying IMU and KRT tumors, our ensemble ML subtype classifier web tool will help inform future studies of HPV(+) HNC. Future work to assess how HPV(+) subtypes can be incorporated into precision treatment strategies is well-motivated by our findings. Citation Format: Bailey F. Garb, Shiting Li, Tingting Qin, Elizabeth Lopez, Sarah Soppe, Snehal Patil, Laura Rozek, Nisha D'Silva, Maureen Sartor. Tumor subtype classification of HPV-associated head and neck cancers is central to key clinically relevant variables [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 3491.

Abstract 1769: Pan-cancer genomic characterization of human papillomavirus associated tumors reveals patterns of somatic alteration that associate with virus status and anatomic site

Abstract Human papilloma virus (HPV) infection causes over 600,000 human cancers yearly and accounts for nearly all cervical cancers, increasing rates of head and neck squamous cell carcinomas (HNSCC), and many anogenital cancers - all with varying clinical outcomes due to a lack of personalized care. While recent integrative genomic studies have described molecular features of individual cancer types, few studies have compared genomic changes between HPV(+) and HPV(-) cancers across anatomic sites. Here, we conducted the first pan-cancer genomic analysis of HPV-associated tumors across multiple anatomic tumor types using whole exome and transcriptome data from The Cancer Genome Atlas (TCGA) cohorts of cervical (n=254) and HNSCCs (n=514), and targeted exome sequencing of 800 cancer genes plus full length HPV16/18 genomes in a clinical cohort of squamous tumors from the head and neck (n=458), cervix (n=78), vulva (n=23), anal canal (n=5), and vagina (n=2). Somatic variant calling and filtering, followed by an integrative pathway analysis of commonly altered targets, defined the catalog of somatic mutations and copy number alterations (CNAs) that drive HPV(+) and HPV(-) tumorigenesis. Sequencing reads from viral RNA or DNA determined HPV status, and HPV type, genome structure, integration events, and viral load were characterized in a subset of samples via de novo assembly of viral aligned reads followed by copy number analysis, breakpoint identification, and the calling of structural variants. Overall HPV positivity was 50% (668 out of 1334 total), with HPV16 accounting for 96%, 89%, and 60% of all HPV(+) anogenital, head and neck, and cervical tumors respectively. Significant differences in somatic mutation frequency between HPV(+) and HPV(-) tumors were observed in the full cohort, as well as in analyses stratified by anatomic site. Interestingly, we noticed recurrently mutated “hotspots” attributable to increased APOBEC-mutagenesis in HPV(+) samples across anatomic sites (PIK3CA:E545K, FGFR3:S249C, EP300:D1399N), while hotspot mutations likely caused by tobacco smoking predominate HPV(-) HNSCC (PIK3CA:H1047R/L, CDKN2A:R80*, TP53:R282W). Focal and arm level CNAs were distinctive, including gains of 11q22 in HPV(+) cervical and HPV(-) HNSCC, and losses of 11q22 in HPV(+) HNSCC. Biological pathways commonly altered include epithelial differentiation, cell death, innate immunity, growth factor/kinase signaling, and cell cycle control. In summary, pan-cancer genomic analysis revealed distinct patterns of somatic alteration of conserved biological pathways that associate with HPV status and anatomic site. These findings improve our understanding of tumor biology unique to HPV-associated cancers and may lead to novel treatment and classification strategies to improve patient outcomes in the clinical setting. Citation Format: Jeremiah Ray Holt, Paul Little, Heejoon Jo, Xiaobei Zhao, Hyo Young Choi, Vonn Walter, Benjamin Wahle, Jose P. Zevallos, Angela Mazul, Katherine A. Hoadley, Michele Hayward, David N. Hayes. Pan-cancer genomic characterization of human papillomavirus associated tumors reveals patterns of somatic alteration that associate with virus status and anatomic site [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 1769.

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.

Identification of novel genomic hotspots and tumor-relevant genes via comprehensive analysis of HPV integration in Chinese patients of cervical cancer.

Cervical cancer accounts for 10-15% of cancer-related mortality among women globally. Infection with high-risk human papillomavirus (HPV) types constitutes a significant etiological factor in the development of cervical carcinoma. The integration of HPV DNA into the host genome is considered a pivotal event in cervical carcinogenesis. Nevertheless, the precise mechanisms underlying HPV integration and its role in promoting cancer progression remain inadequately understood. Therefore, this study aims to identify potential common denominators at HPV DNA integration sites and to analyze the adjacent cellular sequences. We conducted whole-genome sequencing on 13 primary cervical cancer samples, employing the chromosomal coordinates of 537 breakpoints to assess the statistical overrepresentation of integration sites in relation to various chromatin features. Our analysis, which encompassed all chromosomes, identified several integration hotspots within the human genome, notably at 14q32.2, 10p15, and 2q37. Additionally, our findings indicated a preferential integration of HPV DNA into intragenic and gene-dense regions of human chromosomes. A substantial number of host cellular genes impacted by the integration sites were associated with cancer, including IKZF2, IL26, AHRR, and PDCD6. Furthermore, the cellular genes targeted by integration were enriched in tumor-related terms and pathways, as demonstrated by gene ontology and KEGG analysis. In conclusion, these findings enhance our understanding of HPV integration sites and provide deeper insights into the molecular mechanisms underlying the pathogenesis of cervical carcinoma.

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.

Preliminary study of HPV integration status on the occurrence and development of vaginal intraepithelial neoplasia.

The aim of this study was to investigate how the integration status of HPV in the vaginal epithelium affects the development of vaginal intraepithelial neoplasia (VaIN). Twenty-four vaginal tissues were collected before applying high-throughput viral integration detection (HIVID), medical records of them were documented, including age, thin-prep cytologic test (TCT) and HPV test results, colposcopic biopsy pathology, and other clinical data, such as history of total hysterectomy for cervical lesions, whether they were infected with HPV16/18 with a follow-up span of 2 years. We summarized the distribution of HPV integration on the host chromosome and HPV type, as well as the hotspot integration gene and its role in the development of VaIN. In this study, 24 cases suffered from VaIN were involved. HPV integration was detected in 11 cases; furthermore, we discovered HPV 16 and 73, chromosome 1 and 2 possessed most HPV integration sites while EMBP1, CLO5A1, EHF, ELF5 as dominate hot spots. Taken clinical outcome into account, we found a significant difference between HPV integration occurrence and VaIN (p = 0.011). (1) This study found a statistical difference between HPV integration and the occurrence of VaIN; (2) HPV integration may provide a new clinical predictor for VaIN and facilitate risk assessment and stratified management of high-risk patients.

Cis-regulatory effect of HPV integration is constrained by host chromatin architecture in cervical cancers.

Human papillomavirus (HPV) infections are the primary drivers of cervical cancers, and often HPV DNA gets integrated into the host genome. Although the oncogenic impact of HPV encoded genes is relatively well known, the cis-regulatory effect of integrated HPV DNA on host chromatin structure and gene regulation remains less understood. We investigated genome-wide patterns of HPV integrations and associated host gene expression changes in the context of host chromatin states and topologically associating domains (TADs). HPV integrations were significantly enriched in active chromatin regions and depleted in inactive ones. Interestingly, regardless of chromatin state, genomic regions flanking HPV integrations showed transcriptional upregulation. Nevertheless, upregulation (both local and long-range) was mostly confined to TADs with integration, but not affecting adjacent TADs. Few TADs showed recurrent integrations associated with overexpression of oncogenes within them (e.g. MYC, PVT1, TP63 and ERBB2) regardless of proximity. Hi-C and 4C-seq analyses in cervical cancer cell line (HeLa) demonstrated chromatin looping interactions between integrated HPV and MYC/PVT1 regions (~ 500 kb apart), leading to allele-specific overexpression. Based on these, we propose HPV integrations can trigger multimodal oncogenic activation to promote cancer progression.

Abstract B004: Molecular crosstalk between NF-κB and NRF2 signaling affects prognosis in HPV-associated head and neck cancer

Abstract The incidence of head and neck squamous cell carcinoma (HNSCC) is on the rise and is expected to increase by 50% by 2030. Around half of the patient population affected are diagnosed when the tumor is at a locally advanced stage. These patients are usually treated with intense radiotherapy, leading to severe side-effects. Precision medicine would revolutionize the treatment of head and neck cancer by accurately identifying patients that are at low risk of recurrence, reduce treatment related side-effects and aid in overall improvement in quality of life of patients after treatment. This generates the need to develop biomarkers to identify patients with less aggressive tumors as candidates for treatment de-escalation. 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. We hypothesized that NF-kB-driven intrinsic tumor characteristics contribute to increased sensitivity of NF-kB active HPV+ head and neck tumors to radiation, providing patients survival benefits. Indeed, TRAF3 or CYLD deletion dramatically increased radiation sensitivity of HPV+ head and neck cancer cells. As we began to investigate mechanisms of radiation sensitization associated with TRAF3 and CYLD deletion, we found that activation of NF-κB significantly correlated with marked downregulation of nuclear factor erythroid 2-related factor 2 (NRF2) activity in tumors from 3 independent cohorts, as well as in HPV+ HNSCC cells that harbor constitutively active NF-kB due to deletion of TRAF3 or CYLD. 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 showcases an inverse correlation between NF-kB and NRF2 pathways in HPV+ HNSCC. Currently, there are no prognostic biomarkers to distinguish patients who require aggressive therapy versus those that are appropriate for reduced intensity treatment. Identification of markers distinguishing the 2 subtypes of HPV+ HNSCC (high NF-kB and low NRF2 activity) may serve as prognostic biomarkers to help clinicians with therapeutic decisions. Citation Format: Aditi Kothari, Travis Schrank, Wendell Yarbrough, Natalia Isaeva. Molecular crosstalk between NF-κB and NRF2 signaling affects prognosis in HPV-associated head and neck cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B004.

Abstract A132: Pathological analysis of a newly established immunocompetent mice with full Hpv16 genome integration

Abstract Human papillomaviruses (HPV) are associated with both benign and malignant epithelial lesions. High-risk HPV subtype HPV 16 is the causative agent of cervical and the majority of oropharyngeal squamous cell carcinomas. Despite the continuous increase in the disease burden, mechanisms and pathways of HPV16-induced cancer growth are not fully understood, thus leading to suboptimal treatment. In last decades, several transgenic mouse models were developed, helped tremendously to study HPV-induced carcinogenesis. However, all existent HPV transgenic mice were targeted early genes expression driven by an artificial promoter and did not encompass the functionality of the whole HPV genome, including the upstream regulatory region (URR) that contains HPV promoters, regulatory elements, and a replication origin, limited our ability to investigate HPV-associated malignant transformation and the role of host factors in this process. Therefore, we generated HPV16 transgenic mouse by using CRISPR/Cas9-mediated genome editing to insert the full-length HPV16 genome into the Rosa26 locus. Two founder mice, with single HPV copy and with tandem HPV integration, were used to establish mouse colonies. Interestingly, regardless of HPV copy number, ~50% of the HPV knock-in mice exhibited early death during the first 2 weeks after birth, while survived pups were significantly smaller than the same age wild-type mice, and had severe hair loss. Intriguingly, the growth retardant and hair loss phenotype in the HPV16 knock-in mice disappeared in ~4 weeks, most likely indicating that viral genes compromise specific stages of mouse development. We evaluated the expression of HPV genes during development, in different mouse organs, and in established fibroblasts and epithelial cell cultures. Our newly established HPV16 knock-in mouse model provides viral gene expression from natural viral promoters offering an essential basis to explore the role of cellular factors in HPV-driven pathologies. Citation Format: Xue Li, Aditi Kothari, Hina Rehmani, Wendell Yarbrough, Natalia Isaeva. Pathological analysis of a newly established immunocompetent mice with full Hpv16 genome integration [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A132.

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.