HPV Copy Number Research Articles

Prostaglandin E2 suppresses KCNH1 gene expression and inhibits the proliferation of CaSki cervical cells through its four prostanoid PTGER subtypes

The main risk factor for cervical cancer is the persistent infection of high-risk HPV subtypes, notably HPV16. Another contributing factor is proinflammatory prostaglandin E2 (PGE2), a lipid abundantly found in seminal fluid. PGE2, along with its receptors (PTGER1-4), contributes to cancer development; however, its specific role in the proliferation of cervical cancer models with high HPV16 copy numbers remains unclear. In this study, we investigated the effects of PGE2 on the proliferation of CaSki cells, a cell line with a high HPV16 viral load. Surprisingly, PGE2 inhibited CaSki cell proliferation, while it increased the proliferation of SiHa, HeLa, and C-33 A cervical cancer cells. The effect of PGE2 on CaSki cell proliferation was specific, as estradiol increased cell growth. Furthermore, PGE2 suppressed expression and promoter activity of the cervical tumoral marker KCNH1. To discern the specific role of each receptor in cell proliferation, we generated stable CaSki cell lines overexpressing each receptor alongside control cells with an empty vector. Notably, PGE2 significantly inhibited cell proliferation in all stable transfected CaSki cells, suppressing oncogenic KCNH1 expression and its promoter activity. In conclusion, our findings indicate that PGE2 inhibits the proliferation of CaSki cervical cancer cells with a high HPV16 load, at least in part, by suppressing the expression of the oncogenic KCNH1 gene.

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.

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.

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.

Exosomes Derived from Post-Surgical Drain Fluid of HPV+ Oropharyngeal Cancer Patients Contain HPV DNA and can Induce E6 mRNA Transcription in HPV-Negative Cells

<h3>Purpose/Objective(s)</h3> Human papillomavirus (HPV) infection predisposes to oropharyngeal squamous cell carcinoma (OPC). Exosomes are a component of cell-free DNAs that are actively shed by tumor and immune cells. The role of exosomes in cancer metastasis and in HPV-induced carcinogenesis remains unknown. We hypothesize that exosomes derived from surgical drain fluid (SDF) in HPV+ OPC patients contain HPV viral DNA that can be transmitted directly into HPV-negative cells, resulting in transcription of viral oncogenes. <h3>Materials/Methods</h3> Following surgical neck dissection, SDF samples were collected and processed from 21 HPV+ and 5 HPV- OPC patients (discovery cohort). Exosomes were isolated from SDFs using either the Qiagen exoEasy kit or the SBI SEC EV isolation kit. Exosome structure was characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Immunoblotting was performed to verify exosome-specific biomarkers. Exosomes were pretreated with Dnase (NEB), then exosomal DNA was extracted using a cell-free DNA isolation kit (Qiagen). HPV16 copy number was determined by TaqMan quantitative PCR. Validation was then performed using SDF from a separate cohort of 10 HPV+ OPC patients. To examine transcription of exosome-derived HPV DNA, exosomes were isolated from 3 HPV+ SDF and 1 HPV- SDF. After incubating these exosomes with two HPV- cell lines (SCC22A and SCC25), we isolated total cell line RNA, and performed reverse transcriptase PCR (RT-PCR). <h3>Results</h3> TEM and NTA results revealed rounded structures with a size of 70-150 nm, respectively. Immunoblots detected CD63, CD9, and TSG101 in the protein lysates, indicating the vesicles isolated from SDF were indeed exosomes. In the discovery cohort, HPV16 DNA was detected in SDF exosomes isolated from 81% (n = 17) of patients with HPV+ OPC, while SDF-derived exosomes from all (n=5) HPV- patients had undetectable HPV16 DNA. Importantly, paired comparison of HPV16 levels revealed a significant proportion (46.7±34.4%) of HPV16 cell-free DNA in SDF was contained within exosomes. Results from our validation cohort showed that all the exosome samples (100%) isolated from SDF from 10 HPV+ OPC patients contained HPV DNA, regardless of the exosome isolation method; 24.4±14.4% of HPV16 cell-free DNA was localized in exosomes. Furthermore, an <i>in vitro</i> cell culture assay showed that exosomal HPV16 DNA can undergo uptake by SCC22A and SCC25 cells. RT-PCR of RNA from SCC22A cells incubated with HPV DNA-containing exosomes revealed that exosomal HPV16 DNA can be transcribed into HPV16 E6 mRNA in HPV- recipient cells. <h3>Conclusion</h3> SDF, a novel post-surgical biofluid analyte, is enriched for HPV-containing exosomes from HPV+ OPC patients. Exosomes from these HPV+ OPC patients can transmit viral oncogenes to HPV-negative cells, suggesting a novel mode of virus-mediated spread of disease. Furthermore, exosomal HPV DNA can mediate HPV16 E6 mRNA transcription in HPV- recipient cells, indicating its potential role in OPC tumorigenesis.

Human papillomavirus DNA resides in surgical drain fluid exosomes from HPV+ oropharyngeal squamous cell carcinoma patients and can be spread to neighboring HPV-negative cells.

e18050 Background: Human papillomavirus (HPV) is the primary driver of oropharyngeal squamous cell carcinoma (OPC) in the US. Previous studies have investigated the role of plasma and saliva-based cell-free HPV DNA in OPC, yet the pathophysiological impact of exosomes in HPV+ OPC remains unknown. Exosomes are nanometer-sized membrane vesicles of endocytic origin carrying DNA, RNA and proteins, and have been shown to exist in blood, plasma, and in urine. We hypothesize that serosanguinous fluid drained from the postoperative bed, which we call “surgical drain fluid” (SDF) also contains exosomes. We further hypothesize that exosomes derived from SDF in HPV+ OPC patients contain HPV DNA that can be directly transmitted into HPV-negative cells. Methods: Twenty-six 5-mL SDF samples were collected following surgical neck dissections directly from the surgical drain; 21 from HPV+ and 5 from HPV- OPC patients. Exosomes were isolated from SDF using the Qiagen exoEasy kit. Exosome structure was characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Western blotting (WB) targeted known exosomal surface markers. Exosomes were pretreated with DNase (New England Biolabs) to cleave the surface DNA, then exosomal DNA was extracted using a cell-free DNA isolation kit (Sigma). TaqMan real-time PCR was employed to detect HPV16 copy numbers. Lastly, exosomes isolated from 3 HPV+ patients’ SDF and 1 HPV- patient’s SDF were incubated with HPV- cell lines, SCC22A and SCC25, in order to query exosome-mediated HPV transmission. Results: TEM identified exosomes as round/oval-shaped vesicles measuring between 70 and 150 nm. NTA enumerated an average of 1.5 x 1011 particles per mL of SDF. WB further confirmed exosomal identity with strong CD63, CD9, and TSG101 signal detected. Among SDF exosomes from HPV+ OPC, 81% (n = 17) had detectable HPV16 DNA by real-time PCR, while all HPV- SDF exosomes (n = 5) showed undetectable HPV16. Paired comparison of HPV16 levels within exosomes to total HPV16 in SDF revealed an average of 90% of HPV16 in SDF was contained within exosomes. In an in vitro assay, SCC22A and SCC25 cells treated with HPV16 + exosomes became real-time PCR-positive for HPV16, while cells treated with HPV16- exosomes remained HPV16 negative. Conclusions: This study demonstrates that SDF, a novel post-surgical biofluid analyte, is enriched for HPV-containing exosomes in HPV+ OPC patients. We also found that exosomes from HPV+ OPC patients can transmit HPV to HPV-negative cells, suggesting a novel mode of virus-mediated spread of disease.

Detection of Locoregional Minimal Residual Disease Immediately After Surgery Through Multi-Omic Analysis of Tumor-Associated Cell-Free DNA in Surgical Drain Fluid

<h3>Purpose/Objective(s)</h3> Liquid biopsy markers of minimal residual disease (MRD) after cancer treatment have been investigated for detection of recurrence, but detection of locoregional MRD using plasma remains a challenge. Our laboratory examined the use of liquid biopsy technology in surgical drain fluid (SDF) immediately after surgery. Here, we: 1) validate the detection of tumor-associated cell-free DNA (cfDNA) in SDF and 2) demonstrate the utility of SDF cfDNA for clinical decision-making in two cancer types. We hypothesize that tumor-associated cfDNA in SDF provides an early, objective measure of locoregional MRD that can guide adjuvant therapy. <h3>Materials/Methods</h3> We collected tumor, SDF, and plasma samples from patients undergoing surgery for HPV(+) oropharyngeal squamous cell carcinoma (OPSCC) and thyroid cancer. TaqMan qPCR assays were designed for detection of HPV DNA and BRAF V600 mutations in HPV (+) OPSCC and thyroid cancer, respectively. HPV(+) OPSCC PCR results were validated using next-generation HPV capture sequencing in a subset of patients. <h3>Results</h3> 62 HPV (+) OPSCC and 13 thyroid carcinoma cases were included. We correlated SDF HPV cfDNA with pathology and showed a dose-dependence between HPV copy number, node positivity, and extranodal extension (ENE). In patients with ENE, locoregional MRD detection rate was 93% in SDF versus 53% in plasma (p=0.035, Fisher's). We validated results using HPV capture sequencing in a subset (n=6) and noted 100% concordance between PCR data and HPV subtyping in SDF. We then measured actionable mutations in SDF and associations with locoregional recurrence. We determined BRAF V600 mutant status in 13 papillary and anaplastic thyroid carcinomas and in paired cfDNA from SDF. In three known BRAF V600 cases, two patients with extrathyroidal extension were SDF mutant positive; one recurred locoregionally while the other only recently had surgery. The SDF mutant negative patient is disease-free at 9 months. <h3>Conclusion</h3> We present the use of a novel multi-omic platform to quantify tumor-associated cfDNA in surgical drain fluid as an immediate postoperative marker of locoregional MRD. Our data suggest tumor-associated cfDNA in surgical drain fluid is highly correlated with aggressive pathologic features in two cancer types, and could enable more accurate determination of adjuvant therapy and prognosis. When combined with plasma tumor-associated cfDNA, this may enable locoregional and distant molecular restaging after surgery. Future clinical applications may include identifying candidates for adjuvant therapy de-escalation after surgery in HPV+ OPSCC; in thyroid cancer, this assay could assist in radioactive iodine therapy planning or introduce targeted therapies into the adjuvant setting.

Performance of oral HPV DNA, oral HPV mRNA and circulating tumor HPV DNA in the detection of HPV-related oropharyngeal cancer and cancer of unknown primary.

A biomarker that is useful for the detection of human papillomavirus (HPV)‐related oropharyngeal cancer (OPC) and cancer of unknown primary (CUP) is indispensable. We evaluated the diagnostic performance of HPV DNA and mRNA in oral gargle samples and circulating tumor HPV16 DNA (ctHPV16DNA) in blood samples. Oral HPV DNA and mRNA were analyzed using commercially available HPV assays of the GENOSEARCH HPV31 and Aptima, respectively. ctHPV16DNA was analyzed using in‐house droplet digital polymerase chain reaction. Seventy‐four patients with OPC and eight patients with CUP were included. The sensitivity and specificity of oral HPV DNA, oral HPV mRNA, and ctHPV16DNA were 82% (95% confidence interval [CI] = 66‐92) and 100% (95% CI = 88‐100), 85% (95% CI = 69‐94) and 94% (95% CI = 73‐100), and 93% (95% CI = 81‐99) and 97% (95% CI = 84‐100), respectively, for HPV16‐related OPC, while those were 20% (95% CI = 1‐72) and 100% (95% CI = 3‐100), 0% (95% CI = 0‐52) and 100% (95% CI = 3‐100), and 100% (95% CI = 54‐100) and 100% (95% CI = 16‐100), respectively, for HPV16‐related CUP. The sensitivity of ctHPV16DNA for HPV16‐related OPC was higher than that of oral biomarkers, though the difference was not statistically significant. ctHPV16DNA remarkably correlated with the anatomic extent of disease, total metabolic tumor volume and HPV16 copy number per tumor genome in patients with HPV16‐related OPC/CUP, whereas oral biomarkers did not. In conclusion, ctHPV16DNA is a potentially promising biomarker for HPV16‐related OPC, while further studies are required for HPV16‐related CUP.

Influence of TLR4 and TLR9 polymorphisms and haplotypes on multiple hrHPV infections and HPV16 copy number in cervical cancer and cervicitis

Genetic polymorphism in pathogen recognition receptors tends to influence infection, disease susceptibility, and progression. We analyzed the association of TLR4 and TLR9 gene polymorphisms with multiple hrHPV infections and HPV16 copy number in cervicitis and cervical cancer. A total of 440 cervical cancer, cervicitis, and healthy individuals were studied using PCR-based assays. Student t-test, chi-square test, Welch's t-test, and Fisher's Exact test were utilized to evaluate the association of HPV infection with polymorphisms. Haploview and FAMHAP were used to analyze haplotype association with HPV infection and viral load. Study results revealed HPV45 infection as the most common one in cervical cancer after HPV16, and one-fourth HPV positive cervical cancer patients possessed multiple HPV infections. Mean HPV16 copy number of 264.4 ± 58.7 and 2.1 ± 3.3 copies/cell was detected in cervical cancer and cervicitis, respectively. TLR4 rs10759931 was protective against multiple hrHPV infections. TLR4 haplotype ACAC was associated with an increased risk of multiple hrHPV infections. TLR9 SNPs rs187084, rs352140, and rs352139 were associated with decreased risk of high HPV16 copy number. Augmentation of efforts for the multivalent HPV vaccination in India is suggested. The analyzed polymorphisms were shown to modulate hrHPV co-infections and HPV16 viral load that warrants further analysis.

STAT1 expression and HPV16 viral load predict cervical lesion progression.

Cervical cancer is the fourth leading cause of cancer-associated mortality worldwide. However, its underlying molecular mechanisms are unclear. It is important to explore these mechanisms in order to identify novel diagnostic and prognostic biomarkers. The present study determined the association between STAT1 and human papillomavirus (HPV)16 in cervical lesions. STAT1 expression was detected by immunohistochemistry. Quantitative PCR was used to detect HPV16 viral load and STAT1 expression in cervical lesions. The potential associations among STAT1 expression, HPV16 viral load and the severity of cervical lesions in patients were analyzed using receiver operating characteristic (ROC) curves. The Cancer Genome Atlas database was used to analyze STAT1 expression and survival. High STAT1 expression was observed in 10.71 (3/28), 41.18 (14/34), 53.06 (26/49) and 90.00% (27/30) of normal tissue, low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL) and cervical squamous cell carcinoma samples, respectively. The HPV16 copy number gradually increased with the progression of cervical lesions, with the highest copy number observed in cervical cancer samples. In addition, STAT1 expression was positively correlated with HPV16 viral load. Furthermore, ROC curve analysis demonstrated that the combination of STAT1 expression and HPV16 viral load was able to differentiate between LSIL/HSIL and cervical cancer samples. Bioinformatics analysis revealed that STAT1 expression was associated with improved survival in cervical cancer. Additionally, STAT1 expression was positively associated with the progression of cervical lesions, and HPV16 viral load may affect STAT1 expression. Overall, these findings indicate that STAT1 may be an indicator of the status of cervical lesions.

Rational genomic optimization of DNA detection for human papillomavirus type 16 in head and neck squamous cell carcinoma.

We aimed to use genomic data for optimizing polymerase chain reaction (PCR) primer/probe sets for detection of human papillomavirus (HPV)-16 in body fluids of patients with HPV-related head and neck squamous cell carcinoma (HPV-HNSCC). We used genomic HPV-HNSCC sequencing data from a single institutional and a TCGA cohort. Optimized primer/probe sets were designed and tested for analytical performance in CaSki HPV-16 genome and confirmed in salivary rinse samples from patients with HPV-HNSCC. The highest read density was observed between E5 and L2 regions. The E1 region contained a region that was universally present. Among candidate PCR primer/probe sets created, six reliably detected 30 HPV-16 copy number. In a CLIA certified laboratory setting, the combination of two novel primer/probe with E7 sets improved performance in salivary rinse samples with a sensitivity of 96% and specificity of 100%. PCR-based detection of HPV-16 DNA in HPV-HNSCC can be improved using rational genomic design.

Multiplexed digital PCR for detection of HPV in tissue samples from oropharyngeal cancer patients.

e17552 Background: p16 immunohistochemistry (IHC) is a commonly used method for identifying HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). However, p16 overexpression in a minority of HPV negative OPSCC can give rise to false positive results. In contrast, HPV nucleic acid testing lacks adequate sensitivity for routine diagnostic testing. The goal of this study was to investigate whether a multiplexed digital PCR assay can detect and quantify HPV DNA in p16 positive OPSCC treated with definitive chemo-radiotherapy (CRT). Methods: Formalin-fixed paraffin embedded (FFPE) residual diagnostic biopsy specimens were collected from 57 patients. Macrodissection was performed to ensure tumor cellularity &gt; 70%. Extracted genomic DNA was analyzed by next generation sequencing (NGS) using a hybrid capture assay (UNCSeq) targeting &gt; 200 cellular genes and HPV 16/18 genomes. We also designed, validated, and implemented an multiplexed droplet digital PCR (dPCR) assay to detect and quantify HPV DNA (strains 16, 18, 31, 33 and 35) in tissue samples, relative to a genomic control locus (chromosome 6). Results: HPV strain 16 DNA was identified in 50 patients (87.7%), whereas 5 patients (8.8%) had HPV DNA from an alternative high-risk strain (18/31/33/35). HPV DNA was undetectable in 2 patients, indicating a false positive rate of 3.5% for p16 IHC testing in this cohort. HPV DNA copy number per diploid genome equivalent varied significantly across samples, with a median value of 19.7 (range 0.23-1712). A significant correlation was observed between the copies of HPV detected by dPCR and NGS (R2 = 0.5853, p &lt; 0.0001). Evidence for HPV integration was detected by NGS in 23 out of 56 evaluable tumors (42%). There was a trend towards a higher prevalence of HPV integration in tumors with less than 10 HPV copies per diploid genome relative to cancers with &gt; / = 10 HPV copy number (63% versus 34%, p = 0.07). Conclusions: Multiplexed digital PCR demonstrates excellent sensitivity for detection and typing of HPV DNA in diagnostic FFPE specimens from patients with p16 positive oropharyngeal cancer. HPV copy number varies significantly across samples, with a possible association with HPV integration status. Future investigations of potential correlation between HPV copy number, integration status, and clinical outcomes are warranted. Clinical trial information: NCT02281955, NCT03077243.

Human Papillomavirus-16 DNA Quantitation Differentiates High-Grade Anal Neoplasia

Background: Due to their higher rates of anal dysplasia/cancer, human immunodeficiency virus (HIV)-positive individuals are recommended to undergo anal dysplasia screening, which consists of anal cytology (AC) and high resolution anoscopy (HRA) with anal biopsy (AB) after abnormal AC result. However, AC variability limits its usefulness. Our objective was to evaluate human papillomavirus (HPV)-16 DNA quantitation as part of the screening algorithm. Methods: HPV-16 was detected in AC specimens from 75 HIV-positive participants using quantitative real-time polymerase chain reaction. AB results were available from 18/44 patients who had abnormal AC. Statistical tests included Mann-Whitney U, Kruskal-Wallis, receiver operating characteristic (ROC) analysis and Kappa coefficient tests. Results: HPV-16 copy numbers differed significantly across AC (p = 0.001) and AB grades (p = 0.009). HPV-16 ≥ 65 copies/cell predicted high-grade AB (p = 0.04). Using this cut-off in comparison to AB, it had better specificity (1.00) than AC (0.75) and specificity (0.77) than qualitative HPV-16 detection (0.38). Also, the Kappa coefficient of the cut-off (κ = 0.649) was higher than AC (κ = 0.557) and qualitative HPV-16 detection (κ = 0.258) to AB. Conclusion: Higher HPV-16 copy numbers corresponded to higher AC and AB grades, suggesting the importance of HPV burden on disease stage. Furthermore, HPV-16 ≥ 65 copies/cell distinguished high-grade disease and demonstrated better sensitivity, specificity, and agreement with AB than AC or qualitative HPV-16 detection. These results support the potential use of HPV quantitation in conjunction with AC in anal dysplasia screening.

Increased high mobility group A 2 expression promotes transition of cervical intraepithelial neoplasm into cervical cancer.

Integration of the high risk human papillomavirus (HR-HPV) genome into host chromatin is an important step in cervical carcinogenesis. We identified HR-HPV integration sites within the human genome through detection of integrated papillomavirus sequences-PCR and assessed the role of high mobility group A 2 (HMGA2) in cervical carcinogenesis in clinical samples and cell lines. HPV integration sites were analyzed in 40 cervical cancer samples, while copy number variation and protein expression were assessed in 19 normal cervixes, 49 cervical intraepithelial neoplasia (CIN), and 52 cervical cancer samples. Overall, 25 HR-HPV integrating loci were detected in 24 cervical samples; HMGA2 was the only recurring integration site. Both HPV copy number and HMGA2 protein expression were higher in cervical cancer than CIN samples. Area under the curve (AUC) values for HMGA2 expression and HPV copy number were 0.910 (95% CI: 0.844–0.976) and 0.848 (95% CI: 0.772–0.923), respectively. Expression of Bcl-2 and Caspase 3 can indicate the cell proliferation and apoptosis. Transfection of HMGA2 siRNA decreased HMGA2 mRNA and protein expression, Bcl-2 expression, inhibited cell proliferation, and increased Caspase 3 expression and apoptosis in SiHa, CaSki and S12 cervical cancer cells. HMGA2 overexpression had the opposite effects. These results suggest that elevated HMGA2 expression is associated with transformation of CIN into cervical cancer and that HMGA2 might be a useful biomarker for assessing the risk of cervical lesion progression.

Reduction in the copy number and expression level of the recurrent human papillomavirus integration gene fragile histidine triad (FHIT) predicts the transition of cervical lesions.

Cervical cancer is the second most common cancer and the third leading cause of cancer death in females worldwide, especially in developing countries. High risk human papillomavirus (HR-HPV) infection causes cervical cancer and precancerous cervical intraepithelial neoplasia (CIN). Integration of the HR-HPV genome into the host chromatin is an important step in cervical carcinogenesis. The detection of integrated papillomavirus sequences-PCR (DIPS-PCR) allowed us to explore HPV integration in the human genome and to determine the pattern of this integration. We performed DIPS-PCR for 4 cell lines including 3 cervical cancer cell lines and 40 tissue samples. Overall, 32 HR-HPV integration loci were detected in the clinical samples and the HeLa and SiHa cell lines. Among all the integration loci, we identified three recurrent integration loci: 3p14.2 (3 samples), 13q22.1 (2 samples and a SiHa cell line) and 8q24 (1 sample and a HeLa cell line). To further explore the effect of HR-HPV integration in the 3p14.2 locus, we used fluorescence in situ hybridization (FISH) to determine the copy number of the 3p14.2 locus and immunohistochemistry (IHC) to determine the protein expression levels of the related FHIT gene in the clinical samples. Both the 3p14.2 locus copy number and FHIT protein expression levels showed significant decreases when CIN transitioned to cervical cancer. HPV copy number was also evaluated in these clinical samples, and the copy number of HPV increased significantly between CIN and cervical cancer samples. Finally, we employed receiver operating characteristic curve (ROC curve) analysis to evaluate the potential of all these indexes in distinguishing CIN and cervical cancer, and the HPV copy number, FHIT copy number and FHIT protein expression levels have good diagnostic efficiencies.

Physical state and copy numbers of HPV16 in oral asymptomatic infections that persisted or cleared during the 6-year follow-up.

Persistent human papillomavirus (HPV) infection is a key event in HPV-induced carcinogenesis. As part of the prospective Finnish Family HPV Study, we analysed the physical state and viral copy numbers of HPV16 in asymptomatic oral infections that either persisted or cleared during the 6-year follow-up. The persister group comprised 14 women and 7 men with 51 and 21 HPV16-positive brush samples. The clearance group included 41 women and 13 men, with 64 and 24 samples, respectively. Physical state and viral DNA load were assessed by using quantitative PCR for HPV16 E2 and E6 genes. E2/E6 ratio was calculated and HPV16 was classified as episomal, mixed or integrated with values of 0.93-1.08, <0.93 and 0, respectively. In both genders, the physical state of HPV16 was significantly different between the cases and controls (P<0.001). HPV16 was episomal in all men and 66 % (27/41) of women who cleared their infection. HPV16 was mixed and/or integrated in71 % and 57 %of the women and men persisters, respectively. The mean HPV16 copy number per 50 ng genomic DNA was nearly 5.5-fold higher in the women than in the men clearance group (P=0.011). Only in men, HPV16 copy numbers were higher in persisters than in the clearance group (P=0.039). To conclude, in both genders, persistent oral HPV16 infections were associated with the mixed or integrated form of HPV16, while in the clearance groups, episomal HPV16 predominated. This indicates that HPV16 integration is a common event even in asymptomatic oral infections, which might predispose the infected subjects to progressive disease.

Daily self-sampling for high-risk human papillomavirus (HR-HPV) testing

BackgroundSelf-sampling for HPV as part of primary screening is a well-tolerated method for women not attending organized Pap smear screening and could increase coverage of cervical cancer screening. ObjectiveTo investigate if the prevalence of HR-HPV varies from day to day in infected women and if one single sample is reliable for detecting an ongoing infection. Study designThis is a prospective cohort study on 12 premenopausal and 13 postmenopausal women performing daily self-sampling for HR-HPV testing. They were all HR-HPV-positive 1–3 months ago. Postmenopausal women were sampled for 28 days and premenopausal women sampled during bleeding-free days in one menstrual cycle. A possible difference in viral load between the estrogen-dominated proliferative phase and the progesterone-dominated secretory phase was analyzed. Results and conclusionsConsistent results throughout the sampling period were observed for 19 women, with either a daily presence of HPV (14 women) or no HPV at all during the sampling period (5 women). Of 607 samples from 25 women, 596 were consistently positive or negative for HPV during the sampling period and 11 were inconsistent (2%). There was no difference in HPV copy number between the estrogen dominated proliferative or progesterone dominated secretory menstrual cycle phases.The major finding was a high degree of consistency concerning HR-HPV positivity and negativity of HR-HPV in vaginal fluid during a sustained period of daily self-sampling. It does not appear to matter whether the sample is collected in the proliferative or secretory phase.

Biologic predictors of serologic responses to HPV in oropharyngeal cancer: The HOTSPOT study

We hypothesized that viral and host factors impact the serologic responses to HPV early antigens in HPV-positive oropharyngeal cancer (HPVOPC). We conducted a multicenter study to measure HPV16-specific IgG among patients with HPVOPC, their long-term sexual partners, and healthy volunteers. Risk factor surveys and rinse and gargle specimens were collected. Peripheral blood samples at diagnosis were evaluated for IgG Abs to HPV16 antigens using a programmable ELISA assay. Predictors for HPV16 serologic responses were evaluated using univariate and multivariable linear regression. 116 patients with HPVOPC, 43 partners, and 81 healthy volunteers were enrolled and had baseline sera for analysis. Cases were primarily male (90%), with a median age of 56 years. Abs to E1, E2, E6 or E7 antigens were detected more often in HPVOPC compared with volunteers or partner sera (p<0.0001). HPV16 Abs to at least one early protein (E1, E2, E4, E5, E6, or E7) were detected in the sera of 90.6% of cases, 0% of partners and 7.4% of healthy volunteers. Gender, race, sexual behavior, and viral integration were not associated with antibody response. Younger age and higher oral HPV16 copy number were associated with higher HPV16 E6 and NE2 antibody levels. HPV16 seroreactivity is commonly detected among patients with HPVOPC at diagnosis, but not among partners or healthy volunteers. Seroreactivity among cases are correlated with viral load and stage and not with other demographic or behavioral factors. Positive HPV16 serology was strongly associated with HPV 16 oropharyngeal cancer.

Importance of Human Papillomavirus Integration in Malignant Transformation of Inverted Papilloma

Objectives: This study investigated the role of human papillomavirus (HPV) in malignant transformation of paranasal inverted papilloma (IP). Methods: HPV presence and viral load and physical status of HPV-16 were examined by polymerase chain reaction-based methods using fresh frozen samples or paraffin-embedded samples obtained from 17 patients with IP (IP group), 5 with IP and squamous cell carcinoma (IP+SCC group), 16 with SCC (SCC group), and 67 with chronic inflammatory lesions (inflammatory group). Results: The presence of the HPV genome was detected in 29.4%, 40.0%, 25.0%, and 6.0% of patients in the IP, IP+SCC, SCC, and inflammatory groups, respectively. The IP group showed significantly higher HPV-positive rates than the inflammatory group. All types of HPV detected were high-risk HPV, especially HPV-16. The relative HPV-16 copy numbers varied from 2.5 to 7953 per 50 ng genomic DNA. Viral load was higher in the IP+SCC group than in the inflammatory group. In the IP group, no significant relationship was found between HPV-16 viral load and clinical characteristics. All patients with IP+SCC and SCC showed integration of HPV-16. Conclusions: High viral load and integration of HPV have an important role in malignant lesion in association with IP.

Oral Human Papillomavirus (HPV) Infection in HPV-Positive Patients With Oropharyngeal Cancer and Their Partners

To better understand oral human papillomavirus (HPV) infection and cancer risk among long-term sexual partners of patients with HPV-positive oropharyngeal cancer (HPV-OPC). An oral rinse sample, risk factor survey, cancer history, and oral examination (partners only) were collected from patients with HPV-OPC and their partners. Oral rinse samples were evaluated for 36 types of HPV DNA using PGMY 09/11 primers and line-blot hybridization and HPV16 copy number using quantitative polymerase chain reaction. Oral HPV prevalence was compared with infection among those age 45 to 65 years using National Health and Nutrition Examination Survey (NHANES) 2009-2010. A total of 164 patients with HPV-OPC and 93 of their partners were enrolled. Patients were primarily men (90%), were never-smokers (51%), and had performed oral sex (97%), with a median age of 56 years; they had a high prevalence of oncogenic oral HPV DNA (61%) and oral HPV16 DNA (54%) at enrollment. Female partners had comparable oncogenic oral HPV prevalence compared with members of the general population of the same age (1.2% v 1.3%). Among the six male partners, no oncogenic oral HPV infections were detected. No precancers or cancers were identified during partner oral cancer screening examinations. However, a history of cervical disease was reported by nine partners (10.3%) and two female patients (11.8%), and three patients (2.0%) reported a previous partner who developed invasive cervical cancer. Oral HPV16 DNA is commonly detected among patients with HPV-OPC at diagnosis, but not among their partners. Partners of patients with HPV-OPC do not seem to have elevated oral HPV infection compared with the general population.