HPV-positive Cases Research Articles

The association of human papillomavirus infection in indian cohort of head-and-neck squamous cell cancer patients and its impact on treatment outcome

Context: Head and Neck Squamous Cell Carcinomas (HNSCCs) patients with HPV-infected tumors have more favorable prognosis, however data is very sparse in Indian literature. Aim: Our study aims to detect p16 (a surrogate for tumor HPV DNA) in HNSCC and its effect on the survival of the patients. Settings and Design: Observational (prospective study). Materials and Methods: This study was conducted amongst 50 cases of HNSCC. All tissue samples for biopsy were subjected to Immunohistochemistry to study p16 expression, a surrogate marker for HPV. The patients were treated by Radiotherapy or concurrent chemo-radiotherapy depending on performance status and stage of disease. Evaluation was done at 3, 6, 12, 18, and 24 months after treatment. Survival analysis was used to check the outcome of Radiotherapy using Kaplan Meyer survival curves and cox proportional hazards model. Results: Majority of patients had Stage III disease (33 patients – 66%). 16 (32%) patients were HPV positive and 34(64%) were negative. Out of the 16 HPV positive cases, majority, 15 (93.75%) cases were associated with oropharyngeal carcinoma. 2 year DFS for HPV positive was 84% compared to 58% in HPV negative patients (P = 0.089) and 2 year overall survival for HPV positive patients was 83% compared to 52% for HPV negative patients (P = 0.03). Conclusions: Our study concluded that 32% of the HNSCC patients were positive for HPV. Patients who were HPV positive had better disease free survival and overall survival.

In UK, rise in oropharyngeal cancers not solely driven by HPV

A recently published study from the United Kingdom demonstrated that the rise in the incidence of oropharyngeal squamous cell carcinoma (OPSCC) was not solely related to an increase in the incidence of human papillomavirus (HPV)- positive disease because the percentage of HPV-positive and HPV-negative cases remained the same during the study period.1 Terry Jones, MD, professor of head and neck surgery in the department of molecular and clinical cancer medicine at the University of Liverpool in the United Kingdom, notes that because multiple factors besides HPV infection can cause OPSCC, the data should be considered by public health officials when developing disease prevention strategies. Among the other factors contributing to OPSCC are smoking and alcohol consumption. OPSCC is the most common form of head and neck cancer, and its incidence has been increasing throughout the developed world since the 1990s, Dr. Jones says. Several studies have suggested that the increase is due to the rise in HPV-related disease, but Dr. Jones and his colleagues wanted to further investigate whether that was the case in the United Kingdom. A large, multidisciplinary team comprised of researchers from major head and neck cancer centers in the United Kingdom determined the HPV status of archival tumor tissue from 1602 patients who had been diagnosed with OPSCC between 2002 and 2011. Each sample was analyzed using 3 validated commercial tests for HPV, and results were obtained for samples from 1474 patients. The prevalence of HPV infection among these samples was 51.8%. When considered by year of disease diagnosis, the percentage of samples testing positive for HPV remained static at approximately 50%. As a result, researchers were surprised to learn that, although the overall incidence of OPSCC rose each year, the percentage of cases attributable to HPV did not change. HPV-negative disease is rising in parallel to HPV-positive OPSCC, Dr. Jones says, adding that this information contrasts with trends reported elsewhere in the developed world. For that reason, he concludes that the underlying causes of the increase in OPSCC incidence must be analyzed in a population-specific manner. He adds that the evidence points to the need for a genderneutral HPV vaccination policy, and that alcohol consumption could be driving the increase in HPV-negative disease, although that hypothesis has yet to be proven. One difference between the United Kingdom and the United States is that, although the magnitude of the increase in rates of HPV-positive OPSCC is very similar between the countries, the percentage of HPV-negative disease declined in the United States whereas it rose in the United Kingdom during the study period. As a result, researchers have concluded that risk factor exposures may vary substantially between the 2 countries.

Abstract A136: p16INK4a status of primary tumor modulates local immune response in vulvar squamous cell carcinoma

Abstract Background: The p16INK4a is not a surrogate marker for high-risk human papilloma virus genotypes (hr)HPV-DNA) and was suggested to indicate better prognosis of vulvar squamous carcinoma (vSCC). Our recent study confirmed substantial mismatch between p16ink4a and (hr)HPV-status as well as prognostic significance exclusively for p16INK4a-overexpression in cohort of 85 patients. Aim: To compare the infiltration of tumor infiltrating lymphocytes (TILs) and to assess their prognostic significance in cases having tumors with opposed p16INK4a and (hr)HPV-status. Methods: TILs (CD8+, CD4+, FOXP3+, CD68+, CD56+ and GzB+ cells) were calculated in 85 vSCCs with previously defined p16INK4a and (hr)HPV-status. Number of intraepithelial (IE) TIL-subtypes were compared between tumors with different p16INK4a and (hr)HPV-status separately. Survival analyses included the Kaplan-Meier method, log-rank test and Cox proportional hazards model. Results: p16INK4a-negative tumors were infiltrated more intensely by CD8+, CD4+ and GZB+ cells than p16INK4a-positive vSCCs (p = 0.032, p = 0.016 and p = 0.007 respectively). (hr)HPV-status did not impact the infiltration of TILs. Median follow up was 89.20 months (range 1.7-189.5). High (IE)CD4+ infiltrates were prognostic in general cohort and in p16INK4a-positive cases (p = 0.032 and p = 0.039 respectively). High (IE)CD56+ infiltrates were correlated with prognosis in p16INK4a-positive and (hr)HPV-positive cases (p = 0.013 and p = 0.0458 respectively) while high (IE)CD68+ infiltrates were inversely correlated with survival in cases with p16INK4a-negative and (hr)HPV-negative tumors (p = 0.018 and p = 0.0441 respectively). Conclusion: p16INK4a-status modulates local immune surveillance as represented by TILs. Immunologic effects contributing to clinical outcome might depend on p16INK4a-overexpression. Citation Format: Jacek J. Sznurkowski, Anton Zawrocki. p16INK4a status of primary tumor modulates local immune response in vulvar squamous cell carcinoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A136.

Human papillomavirus detection in fine needle aspiration cytology of lymph node metastasis of head and neck squamous cell cancer

BackgroundCurrently, testing on HPV in oropharyngeal squamous cell carcinoma (OPSCC) is performed on histological material. However, in a certain percentage of the cases who present with lymph node metastases no primary tumor can be identified and only fine needle aspiration cytology (FNAC) is available for analysis. ObjectivesPurpose of this study was to assess HPV status on FNAC and to validate it using histological material of the same patients. Study designPatients with cervical metastasis from OPSCC or cancer of an unknown primary tumor (CUP), diagnosed between 2007 and 2012 were included. In 6 of the 47 patients, no primary tumor could be identified. HPV detection and genotyping was performed in both FNAC slides scrapings and formalin fixed paraffin embedded (FFPE) histological material from the same patients, using the HPV SPF10-LiPA25 assay. HPV PCR analysis on FFPE material was considered the reference standard for HPV status of each case. ResultsCompared with HPV negative cases (n=22), significantly more HPV positive cases (n=25) presented initially with cervical metastasis (27% vs 56% respectively; p=0·047). The HPV PCR assay on FNAC material showed a high sensitivity (96%; 95% CI 86.6–97.4) and specificity (100%; 95% CI 85.1–96.7) using the reference standard of HPV PCR analysis on FFPE material of the same patients. ConclusionIn this study, testing on HPV in FNAC of cervical lymph node metastases of SCC is validated. It provides a valuable alternative for testing of HPV on histological material from patients with oropharyngeal squamous cell carcinoma or cancer of an unknown primary tumor.

PD-L1 expression in tonsillar cancer is associated with human papillomavirus positivity and improved survival: implications for anti-PD1 clinical trials.

In this study, we examined PD-L1 expression by immunohistochemistry in 99 patients with tonsillar cancer and known human papillomavirus (HPV) status to assess its clinical significance. We showed that the pattern of PD-L1 expression is strongly related to HPV status. The PD-L1 positivity rate was 83.3% in HPV-positive cases and 56.9% in HPV-negative cases (p < 0.05). Patients with HPV-positive/PD-L1-positive cancer had significantly better event free survival and overall survival compared with patients with HPV-negative/PD-L1-negative cancer. Relative to those patients with HPV-negative/PD-L1-negative disease who had the highest risk of death, patients with HPV-positive/PD-L1-positive cancers had a 2.85 fold lower risk of developing an event (HR 0.35, 95% CI: 0.16–0.79) and a 4.5 fold lower risk of death (HR =0.22, 95% CI: 0.09–0.53). Our findings will help to guide future clinical trial design in immunotherapy based on PD-L1 expression in tonsillar cancer.

Knowledge of the HPV status biases cytotechnologists' interpretation of Pap tests originally diagnosed as negative for intraepithelial lesion or malignancy.

Because of cervical cancer screening recommendations and forthcoming first-line human papillomavirus (HPV) screening, many Papanicolaou (Pap) tests will be performed in patients with known concurrent HPV results. This study was designed to evaluate whether knowledge of the HPV status affects cytotechnologists' interpretation of Pap tests. A retrospective search of cervical screening Pap tests with known concurrent HPV results provided 250 ThinPrep Pap tests, which were chosen to reflect an atypical rate similar to the rate of the Cleveland Clinic's normal practice. Fifty percent of negative for intraepithelial lesion or malignancy (NILM) and atypical squamous cells of undetermined significance (ASCUS) cases were from patients with positive HPV results. Slides were re-evaluated twice by 8 cytotechnologists blinded to the diagnosis and study purpose. The HPV status was provided for 50% of the cases in the first phase; after a washout period, knowledge of the HPV status for each case was reversed in the second phase. Follow-up information was collected from the medical record. In both phases, there was a significant bias for HPV-positive NILM cases to be upgraded to ASCUS or worse when the HPV-positive status was provided (P < .001). When the HPV status was withheld, there was no difference in upgrading NILM cases (phase 1, P = .69; phase 2, P = .066). A combined analysis showed a significant bias in referral to the pathologist when the HPV-positive status was provided rather than withheld (P < .001). Follow-up data revealed no significant effect of bias when the HPV-positive status was provided between patient groups with benign, low-grade, or high-grade follow-up. A known HPV-positive status biases cytotechnologists' interpretation of Pap tests, and this results in a higher rate of upgrading to ASCUS or worse; however, it does not improve sensitivity for disease detection. Cancer Cytopathol 2017;125:60-69. © 2016 American Cancer Society.

Identification of genetic variation in the lncRNA HOTAIR associated with HPV16-related cervical cancer pathogenesis.

Previously, over-expression of the long noncoding RNA (lncRNA) HOTAIR has been found to be associated with the invasive and metastatic capacities of several epithelial cancers, including cervical cancer (CaCx). Here, we aimed at identifying functionally relevant genetic variants that may be employed to differentiate between clinically distinct CaCx subtypes, i.e., those exhibiting high HOTAIR levels and molecular signatures of metastasis and those lacking such signatures in the presence of low HOTAIR expression levels. Genomic DNA isolated from various cervical tissue samples (characterized by histopathology and HPV status) was used for HOTAIR amplicon sequencing, followed by validation of the findings by Sanger sequencing. The impact of the genetic variants found on the secondary structure of HOTAIR and the concomitant alterations in miRNA binding sites were determined through in silico analysis, followed by miRNA expression analysis by quantitative real-time PCR and confirmation of miRNA binding using a luciferase reporter assay. We found that rs2366152C was over-represented [ORage-adjusted = 2.58 (1.23-5.57); p = 0.014] in low HOTAIR expressing HPV positive CaCx cases compared to HPV negative controls. This genetic variant showed the propensity of a secondary structure alteration and gain of a miR-22 binding site in HOTAIR, which was found to be concordant with miR-22 over-expression in low HOTAIR CaCx cases compared to controls. We found that miR-22 expression negatively correlated with HOTAIR and E7 expression in HPV16 positive cases and in an E7 transfected HPV negative CaCx-derived cell line (C33A), but was not altered in high HOTAIR cases compared to controls. Reduced luciferase activity of a HOTAIR rs2366152C expression plasmid in C33A cells through miR-22 co-transfection confirmed the ability of miR-22 to specifically target rs2366152C-harbouring HOTAIR lncRNA in CaCx cells, ultimately leading to its down-regulation. Our data indicate that rs2366152C not only has the potential to serve as a marker for singling out CaCx cases lacking metastatic molecular signatures, but also to explain the functional inactivation of HOTAIR in these cases, including the mechanism of its down-regulation.

Prevalence of human papillomavirus types, viral load and physical status of HPV16 in head and neck squamous cell carcinoma from the South Swedish Health Care Region.

Incidence of human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is rising in several countries. Intriguingly, large variations of HPV16 viral load and different proportions of the physical viral status among HNSCC have been reported. We analysed fresh biopsies of 275 HNSCC patients from the South Swedish Health Care Region for HPV types with modified general primer PCR and Luminex. Seventy-eight HPV16-positive HNSCC cases were further investigated for viral DNA load and physical status using quantitative PCR for HPV E2 and E7 genes. Presence of intact E2 gene, as a surrogate marker for episomal HPV, was investigated with conventional PCR. Fifteen different HPV types were detected in HNSCC cases and HPV16 was present in 74 % of the HPV-positive cases. HPV was detected in 65 % (92/141) and 11 % (15/134) of oropharyngeal and non-oropharyngeal carcinomas, respectively (P<0.0001). HPV was detected in 73 % (75/103) of tonsillar carcinomas. The median load of HPV16 was 13 copies cell-1 (range 0.003-1080). Among HPV16-positive patients with oropharyngeal carcinoma, metastases to regional lymph nodes were observed in 100 % (17/17) and 68 % (40/58) for those with <1 HPV16 copy cell-1 and >1 HPV16 copy cell-1, respectively (P=0.007). Among HPV16 cases, purely integrated HPV16 was found in 6 %, whereas entirely episomal and mixed virus was detected in 51 and 42 % of cases, respectively. Conclusively, HPV16 viral DNA load demonstrated a large diversity among HNSCCs. Although integration of HPV16 is common (48 %), the episomal HPV16 is salient (93 %) among HPV16 HNSCCs. In addition, low amount of HPV16 was associated with lymph node metastases among oropharyngeal carcinomas.

Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2.

We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.

The variable clinicopathological categories and role of human papillomavirus in cervical adenocarcinoma: A hospital based nation-wide multi-center retrospective study across China.

We investigated HPV in adenocarcinoma presenting and managed as cervical adenocarcinoma (CADC) at seven major representative regional cancer centres across China. From 1,051 CADC cases diagnosed locally in 2005-2010, 881 had available paraffin embedded tissue. Initial review excluded 154 cases as other diagnoses or inappropriate specimens. In 718 eligible cases consensus panel pathology diagnosis was made using an algorithm incorporating p16 and progesterone receptor immunohistochemistry (IHC). Classification of cervical adenocarcinoma categories was subject to substantial pathological disagreement. High-risk human papillomaviruses (HR-HPV) DNA was studied by the sensitive SPF10 PCR-DEIA-LiPA25 version 1 for L1 genes and type-specific HR-HPV E6/7 gene PCR's. HR-HPV prevalence in whole tissue samples in eligible tested CADC was 74.5%: 100.0% in neuro-endocrine carcinoma (NEC), 82.2% in classical cervical adenocarcinoma (ADC-CX), 40.0% in adenocarcinoma-not otherwise specified (ADC-NOS) and 33.3% in endometrioid adenocarcinoma (ADC-ENDO). Higher mean age at diagnosis correlated with histological categories showing low HPV prevalence (Linear regression: β= -13.794, p < 0.001). HPV-16 and 18 were associated with early development of CADC and a lower mean age correlated with carcinogenic risk of associated HPV (β = -0.1829, p < 0.001). HPV-16 or HPV-18 was found in 88.2% of all HPV positive cases including multiple-infections. HPV-18 was the commonest HPV type in NEC (58.3%), ASC (40.2%) and ADC-CX (40.9%). The proportion of HPV-unrelated CADC and in different final histological categories varied geographically and by age. Although HPV negativity was predominantly associated with special categories of CADC, some HPV-negative usual adenocarcinomas indistinguishable by adjudicated microscopic diagnosis from ADC-CX were found and varied in frequency across China.

Human papillomavirus genotypes distribution in cervical cancer cases in Gabon.

BackgroundCervical cancer is a real public health problem in African countries. The relation between HPV and cervical cancer is well established. However, it is known that the distribution of HPV genotypes differ geographically and this may influence the effectiveness of the three available vaccines, which among other HPV genotypes targets the genotypes 16 and 18 that cause about 70 % of cervical cancers cases. The objective of this study was to identify for the first time the HPV genotypes distribution in cervical cancer specimens obtained from Gabonese women.MethodsA total of 105 cervical samples including 93 formalin-fixed paraffin embedded tissues collected between 2007 and 2013 and 12 fresh biopsies collected in August 2013 were investigated. The presence of HPV DNA was analyzed by nested PCR with primers MY09/11 and GP5+/6+ followed by sequencing for HPV genotyping.ResultsAmplification of the housekeeping gene (β-globin) with PCO4/GH20 primers was successful for 91.4 % (96/105) of the cervical cancer samples and HPV DNA was detected in all the 96 samples. Five different HPV genotypes were identified. HPV 16 [58.3 %; 95 % IC: 48.44–68.16] was the most common genotype followed by HPV 33 [25.0 %; 95 % IC: 16.34–33.66], HPV 18 [8.4 %; 95 % IC: 2.86–13.94], HPV 70 [7.3 %; 95 % IC: 2.1–12.5] and HPV 31 [1.1 %; 95 % IC: −0.986–3.186]. HPV 16 was also the most prevalent in all histological malignant lesions. It was found in 56.6 % of squamous cervical carcinoma and 69.2 % of adenocarcinoma. Concerning the HPV positive adenocarcinoma cases, HPV 18 was identified in 7.7 % (1/13).ConclusionThese findings show the predominance of HPV 16 in cervical cancer cases among Gabonese women. However, HPV33 is more prevalent than HPV18. Our study suggests that HPV vaccines may be effective at reducing the burden of cervical cancer in Gabon.

Higher prevalence and gene amplification of HPV16 in oropharynx as compared to oral cavity.

ABSTRACTObjective The objective of this study was to clarify differences regarding HPV16 infection and gene amplification between the oral cavity and oropharynx in healthy individuals.Material and Methods The subjects were 94 healthy asymptomatic individuals (41 males, 53 females; mean age 58.6 years, range 16-97 years) who visited the Department of Oral and Maxillofacial Reconstructive Surgery of the Hiroshima University Hospital from 2014 to 2015. Oral epithelial cells were collected from oral rinse and pharynx gargle samples and placed in saline. The human endogenous retrovirus gene ERV3-1 was used as a reference to estimate the number of human cells in each sample. DNA samples were extracted from approximately 10,000 human cells and tested for HPV16 DNA by PCR using a type-specific primer. Similarly, we analyzed the HPV16 viral copy number in HPV16-positive cases using real-time PCR to examine genomic amplification.Results The percentage of HPV16-positive cases was higher in the gargle (28.7%) as compared to the rinse (16.0%) samples. In the oral rinse samples, males (26.8%) showed a significantly higher rate of HPV16 than females (7.5%) (P=0.021). Importantly, in older subjects (aged 60-89 years), gargle samples showed a significantly higher rate of HPV16 (33.3%) than oral rinse samples (13.7%) (P=0.034). The average number of viral copies was approximately 8 times higher in the gargle than in the oral rinse samples (0.16±0.27 vs. 1.35±1.26 copy numbers per cell), a significant difference (P<0.001).Conclusion Our findings suggest that the oropharynx is more susceptible to HPV16 infection as compared to the oral cavity, while HPV16 gene amplification is also more commonly found in the oropharynx.

Abstract 3440: Time and regional incidence trends of human papillomavirus-associated oropharyngeal cancer in Ontario, Canada

Abstract Background: Despite the increasing incidence of human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) in the Western World, there are little data describing the change in HPV incidence within Ontario, Canada. We therefore assessed HPV-positivity in a retrospective study of 612 OPC patient samples. Methods: Diagnostic formalin-fixed and paraffin embedded OPC patient samples belonging to two cohorts were retrieved from institutions across Ontario, Canada over two time periods: 1998-1999 (n = 245) and 2003-2004 (n = 367). In all samples, HPV status was assessed initially using p16 immunohistochemistry (IHC). For p16 negative and equivocal staining samples, HPV in situ hybridization (ISH; using an HPV subtype 16/18 probe) was performed as the final indicator for HPV positivity. HPV-positivity rate was then compared over time and geographic region. Results: Overall, p16-positivity was observed in 52.3% (320 of 612) of the OPC patients. For the p16-negative (n = 246) and p16-equivocal (n = 46) samples, 25.3% (n = 74) were positive by HPV16/18 ISH. The HPV infection incidence for the 1998-1999 cohort was 54.3% (133 of 245 samples; 95% CI: 53.9(47.4-60.2)). The HPV infection incidence increased significantly in the 2003-2004 cohort to 71.1% (261 of 367 samples; 95% CI: 71.4(66.5-76.0)) (p&amp;lt;0.0001). When divided based on geographical region, HPV incidence also increased significantly over time (Northern Ontario by 49%, p = 0.04; Eastern Ontario by 23%, p = 0.009; Southern Ontario by 17%, p = 0.009; Central Ontario by 12%, p = 0.05). There were no significant differences between urban and rural areas over the two cohort periods (urban p = 0.8; rural, p = 0.6). Conclusions: HPV-associated OPC increased in incidence in Ontario, Canada, over a 6-year period from 1998 to 2004, which appeared to be applicable across the entire province, regardless of urban or rural regions. A composite HPV detection method using p16 in combination with HPV ISH identified an additional 25% of HPV-positive OPC cases. Citation Format: Wei Shi, Stephen F. Hall, Katrina Rey-McIntyre, Rebecca Griffiths, Wei Xu, Jie Su, Jeff Bruce, Shaohui Huang, Brian O’Sullivan, Kenneth W Yip, Fei-Fei Liu. Time and regional incidence trends of human papillomavirus-associated oropharyngeal cancer in Ontario, Canada. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3440.

Effect of human papillomavirus 16 oncoproteins on oncostatin M upregulation in oral squamous cell carcinoma.

Human papillomavirus (HPV) infection modulates several host cytokines contributing to cancer development. Oncostatin M (OSM), an IL-6 family cytokine, acts to promote cell senescence and inhibit growth. Its dysregulation promotes cell survival, cell proliferation and metastasis in various malignancies. The effect of HPV on OSM dysregulation has not been investigated. To elucidate this, immunohistochemistry was used on formalin-fixed, paraffin-embedded oral squamous cell carcinoma (OSCC) tissues: HPV-positive (50) and HPV-negative (50) cases. Immortalized human cervical keratinocytes expressing HPV16E6 (HCK1T, Tet-On system) were used to demonstrate the role of HPV16E6 in OSM expression. In addition, a vector containing HPV16E6/E7 was transiently transfected into oral cancer cell lines. Cell viability, cell-cycle progression and cell migration were evaluated using flow cytometry and a wound healing assay, respectively. The results showed various intensities of OSM expression in OSCC. Interestingly, the median percentages of strongly stained cells were significantly higher in HPV-positive OSCCs than in HPV-negative OSCCs. To explore the role of HPV oncoproteins on OSM expression, the expression of HPV16E6 in the HCK1T Tet-On condition was induced by doxycycline and HPV16E6 was found to significantly upregulate levels of OSM mRNA and protein, with concomitant upregulation of c-Myc. In addition, the levels of OSM mRNA and protein in E6/E7 transiently transfected oral cancer cells also gradually increased in a time-dependent manner and these transfected cells showed greater viability and higher migration rates and cell-cycle progression than controls. This result demonstrates that HPV16 oncoproteins upregulate OSM and play an important role to promote OSCC development.

Colposcopists’ experiences of HPV Test of Cure for the follow up of cervical intra-epithelial neoplasia

ObjectiveTo survey lead colposcopists in England to explore their views on the recently introduced HPV Test of Cure (TOC) following treatment for cervical intra-epithelial neoplasia (CIN) and to determine the extent to which it has impacted their clinical practice and affected their patients. MethodsAn online survey was sent to lead colposcopists across England. Questions were asked focusing on the clinicians’ confidence in the ability of TOC to guide follow up in various clinical scenarios and how the implementation of TOC had changed patient management. ResultsThere was a 50% (N=88) response rate. 90% of respondents indicated they were happy with the new procedure. In the follow-up questions, 20% indicated they were uncomfortable with the procedure when it was applied to women who were CIN2+ with incomplete excision at the endocervical margin. Open-ended questions elicited positive aspects of TOC including reduced follow-up, increased reassurance for patients and clinicians and a faster return to the call–recall system. Negative observations included concerns around HPV positive cases, possible false negatives and anxiety in those women who were originally subject to the pre-TOC guidelines and were now returned to call–recall “earlier” than originally indicated to them. 11% of respondents also indicated they work around the new guidelines to some extent. ConclusionAlthough clinicians are on the whole positive towards the introduction of TOC, concerns were raised which centre primarily around those patients with CIN2+ combined with positive endocervical margins, issues related to HPV positive cases and the possibility of a false negative HPV result. The possibility of patient anxiety due to return to routine screening earlier than originally expected was also identified as a concern.

Prevalence and distribution of 15 high-risk human papillomavirus types in squamous cell carcinoma of the scrotum

Which subtype(s) of high-risk human papillomavirus (hrHPV) are involved in squamous cell carcinoma (SCC) of the scrotum is unknown. Twenty-seven cases of SCC of the scrotum were retrieved, and all 15 subtypes of hrHPV and their viral loads were assessed using multiplex real-time polymerase chain reaction. The results were correlated with the histopathologic features, p16 expression, and in situ hybridization for hrHPV. hrHPV was identified in 18 (67%) of 27 of the cases, including HPV16 (n=8), HPV35 (n=7), HPV31 (n=5), HPV59 (n=5), HPV33 (n=3), HPV18 (n=2), HPV51 (n=2), HPV39 (n=1), HPV56 (n=1), and HPV82 (n=1). Of the 18 cases, 10 (56%) were infected by multiple hrHPV subtypes. In situ carcinomas had higher viral loads than invasive (50M versus 2M in average). The average age of HPV-positive and -negative cases was similar, 55 and 51, respectively. Of 11 cases of invasive carcinoma, 5 (45%) were positive for hrHPV versus 13 of 16 (81%) of in situ carcinomas. The highest proportion of hrHPV-positive cases was seen in basaloid type (7/7; 100%) and warty type (4/4; 100%), followed by usual type (7/16; 44%). Of 18 of the HPV-positive cases, 9 (50%) were also positive for p16 by immunohistochemistry and 6 of 18 (33%) were positive by in situ hybridization. Similar to SCC of the vulva and penis, the most frequently HPV-positive tumors are basaloid and warty types. However, a proportion of SCC usual type are also positive for hrHPV. Our results show that 8 (44%) of 18 of cases are associated with hrHPV subtypes other than 16 and 18. Additionally, 7 (70%) of 10 of hrHPV16/18-positive cases are coinfected with other subtypes.

A beneficial tumor microenvironment in oropharyngeal squamous cell carcinoma is characterized by a high T cell and low IL-17(+) cell frequency.

Patients with HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis than patients with non-HPV-induced OPSCC. The role of the immune response in this phenomenon is yet unclear. We studied the number of T cells, regulatory T cells (Tregs), T helper 17 (Th17) cells and IL-17+ non-T cells (mainly granulocytes) in matched HPV-positive and HPV-negative OPSCC cases (n = 162). Furthermore, the production of IFN-γ and IL-17 by tumor-infiltrating T cells was analyzed. The number of tumor-infiltrating T cells and Tregs was higher in HPV-positive than HPV-negative OPSCC (p < 0.0001). In contrast, HPV-negative OPSCC contained significantly higher numbers of IL-17+ non-T cells (p < 0.0001). Although a high number of intra-tumoral T cells showed a trend toward improved survival of all OPSCC patients, their prognostic effect in patients with a low number of intra-tumoral IL-17+ non-T cells was significant with regard to disease-specific (p = 0.033) and disease-free survival (p = 0.012). This suggests that a high frequency of IL-17+ non-T cells was related to a poor immune response, which was further supported by the observation that a high number of T cells was correlated with improved disease-free survival in the HPV-positive OPSCC (p = 0.008). In addition, we detected a minor Th17 cell population. However, T cells obtained from HPV-positive OPSCC produced significantly more IL-17 than those from HPV-negative tumors (p = 0.006). The improved prognosis of HPV-positive OPSCC is thus correlated with higher numbers of tumor-infiltrating T cells, more active Th17 cells and lower numbers of IL-17+ non-T cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-016-1805-x) contains supplementary material, which is available to authorized users.

Evidence of disrupted high-risk human papillomavirus DNA in morphologically normal cervices of older women.

High-risk human papillomavirus (HR-HPV) causes nearly 100% of cervical carcinoma. However, it remains unclear whether HPV can establish a latent infection, one which may be responsible for the second peak in incidence of cervical carcinoma seen in older women. Therefore, using Ventana in situ hybridisation (ISH), quantitative PCR assays and biomarkers of productive and transforming viral infection, we set out to provide the first robust estimate of the prevalence and characteristics of HPV genomes in FFPE tissue from the cervices of 99 women undergoing hysterectomy for reasons unrelated to epithelial abnormality. Our ISH assay detected HR-HPV in 42% of our study population. The majority of ISH positive samples also tested HPV16 positive using sensitive PCR based assays and were more likely to have a history of preceding cytological abnormality. Analysis of subsets of this population revealed HR-HPV to be transcriptionally inactive as there was no evidence of a productive or transforming infection. Critically, the E2 gene was always disrupted in those HPV16 positive cases which were assessed. These findings point to a reservoir of transcriptionally silent, disrupted HPV16 DNA in morphologically normal cervices, re-expression of which could explain the increase in incidence of cervical cancer observed in later life.

Human papillomavirus genotypes distribution among Gabonese women with normal cytology and cervical abnormalities.

BackgroundCervical cancer is one of the most common tumors affecting women with a disproportionate mortality occurring in developing countries. Despite the high prevalence of cervical cancer and cervical neoplasia in Gabon, few studies have been performed to evaluate the prevalence and determinants of HPV infection in this country. The aim of this study was to determine the HPV prevalence and distribution in a population of Gabonese women with normal cytology and cervical abnormalities.MethodsA total of 200 cervical samples collected in the “Departement d’Anatomie et de Cytologie Pathologiques” of the “Faculté de Medecine et des Sciences de la Santé” in Libreville, Gabonwere analyzed. Cytological status was classified according to Bethesda 2001. Nested polymerase chain reaction (PCR) using consensus degenerate PCR primers (MY09/11 and GP5+/6+) was performed for the detection of HPV DNA and HPV typing was done by DNA sequencing.ResultsCytological analysis showed that 87 % of women had normal cytology (n = 174/200). Among the 26 women with cytological abnormalities, predominance (61.5 %; 16/26) of low grade squamous intraepithelial lesion (LSIL) was found and no cervical cancer case was detected. Overall, HPV DNA was detected in 60 % of women (120/200). With respect to the cytological status, HPV DNA was found in 57.5 % of women with normal cervix and 76.9 % of women with abnormal cytology. HPV genotyping was performed on 114 HPV positive cases and revealed the presence of 11 distinct genotypes: 16, 18, 33, 31, 56, 6, 66, 70, 35, 45 and 81. The high risk type HPV 16 was the most common genotype found in all cytological categories. Six HPV positive samples could not be typed by DNA sequencing, probably due to multiple HPV infection. Evaluation of possible risk factors showed that HPV infection was related positively with number of sexual partners (≥3, OR = 2.3; 95 % CI, 1.3–4.3), history of sexually transmitted infection (Chlamydia, OR = 1.9; 95 % CI, 1.01–3.4) and marital status (single, OR = 2.0; 95 % CI, 1.1–3.5).ConclusionThe prevalence of HPV infection among Gabonese women is high. Our findings highlight the need to set up a national program to fight cervical cancer, combining Pap smear test and HPV testing, to improve cervical cancer prevention in Gabon.

High Prevalence of Human Papillomavirus in Colorectal Cancer in Hispanics: A Case-Control Study.

The role of Human Papillomavirus (HPV) in colorectal carcinogenesis remains elusive. Based on the high incidence of HPV-associated malignancies among Puerto Rican Hispanics, this study aimed to assess the prevalence of HPV infection and viral integration in colorectal tissues in order to evaluate its putative role in colorectal cancer (CRC). In this case-control study, the prevalence of HPV infection in CRC (cases n = 45) and normal colon mucosa from cancer-free subjects (controls n = 36) was assessed by a nested PCR strategy. HPV-16 genotyping was performed in HPV-positive tissues and the physical status of the HPV-16 genome was determined by E2 detection. HPV was detected in 19 of 45 (42.2%) CRC cases (mean age 61.1 ± 10.7 years, 24 males) and in 1 of 36 (2.8%) controls (mean age 60.9 ± 9.6 years, 24 males) with an OR = 25.58 (95% CI 3.21 to 203.49). HPV-16 was detected in 63.2% of the HPV-positive colorectal tumors; genome integration was observed in all HPV-16 positive cases. This is the first report showing the high prevalence of HPV infections in Caribbean Hispanic colorectal tumors. Despite evidence of HPV integration into the host genome, further mechanistic analysis examining HPV oncoprotein expression and the putative role of these oncoproteins in colorectal carcinogenesis is warranted.