High-risk Human Papillomavirus Status Research Articles

A Prospective Cohort Study of Human Papillomavirus-Driven Oropharyngeal Cancers: Implications for Prognosis and Immunisation

AimsOropharyngeal cancer (OPC) is increasing on a global scale, including the component driven by high-risk human papillomavirus (HR-HPV); contemporary data that provides insight into the prognosis of this disease in addition to the fraction attributable to HR-HPV are essential to inform primary and secondary disease management strategies. Materials and methodsA population-based cohort of 235 patients diagnosed with OPC between 2013 and 2015 in Scotland was assessed for HPV status using molecular genotyping. Associations between HR-HPV status and key clinical and demographic variables were estimated using the Pearson chi-squared test. Rates of overall survival and progression-free survival were estimated and visualised using Kaplan–Meier curves. ResultsHPV DNA (largely HPV 16) was identified in 60% of cases. After adjustment for age, gender, deprivation, smoking, alcohol consumption and tumour stage, patients with HR-HPV-positive OPC had an 89% reduction in the risk of death (hazard ratio = 0.11, 95% confidence interval 0.05–0.25) and an 85% reduction in the risk of disease progression (hazard ratio = 0.15, 95% confidence interval 0.07–0.30). HPV positivity was not associated with age, deprivation or smoking status, whereas those who reported excess alcohol consumption were less likely to be positive for HR-HPV. ConclusionsThe prevalence of HR-HPV-associated OPC is high in Scotland and strongly associated with dramatically improved clinical outcomes, including survival. Demographic/behavioural variables did not reliably predict HPV positivity in this cohort, which underlines the importance of laboratory confirmation. Finally, the dominance of HPV 16 in OPC indicates the significant impact of prophylactic immunisation on this disease.

PD-L1/PD1 Expression, Composition of Tumor-Associated Immune Infiltrate, and HPV Status in Conjunctival Squamous Cell Carcinoma

PurposeConjunctival squamous cell carcinoma (SCC), a type of ocular surface neoplasia, is primarily treated by surgical resection and topical immuno- or chemotherapy. Metastatic disease may be treated with systemic chemo- or immunotherapy, albeit with variable response. The purpose of this study was to determine whether immune checkpoint blockade might be considered in the management of conjunctival SCC.MethodsIn this retrospective study, we evaluated tumor programmed death-ligand 1 (PD-L1) expression, high-risk human papillomavirus (HPV) status, and immunohistochemical expression of cluster of differentiation 3 (CD3), cluster of differentiation 8 (CD8), and programmed death 1 (PD1) in tumor-associated immune infiltrate in a series of 31 conjunctival SCCs.ResultsPD-L1 expression in ≥1% of tumor cells was noted in 14 conjunctival SCCs (47%) and was more prevalent in invasive than in situ SCC and among tumors with higher American Joint Committee on Cancer (AJCC) T category (≥T3 versus ≤T2). The density of CD3-positive T cells was higher in primary than recurrent tumors and higher in invasive than in situ tumors. Density of CD3-positive and CD8-positive T cells was higher in higher AJCC stage tumors. Density of CD8-positive T cells was higher in HPV-positive than HPV-negative tumors. PD-L1 expression correlated with a higher density of CD3-, CD8-, and PD1-positive cells in the tumor-associated immune infiltrate but not with HPV status.ConclusionsOur findings demonstrate that PD-L1 is expressed in almost half of conjunctival SCCs. The density of tumor-associated immune cells correlated with invasive SCC, stage, and HPV status in conjunctival SCC. Our findings support further studies to establish the potential application of immune checkpoint blockade in the management of conjunctival SCC.

Survival outcomes by high-risk human papillomavirus status in nonoropharyngeal head and neck squamous cell carcinomas: A propensity-scored analysis of the National Cancer Data Base.

The prognostic relevance of human papillomavirus (HPV) status in patients with nonoropharyngeal (OPX) squamous cell cancer (SCC) of the head and neck is controversial. In the current study, the authors evaluated the impact of high-risk HPV status on overall survival (OS) in patients with non-OPX SCC using a large database approach. The National Cancer Data Base was queried to identify patients diagnosed from 2004 through 2014 with SCC of the OPX, hypopharynx (HPX), larynx, and oral cavity (OC) with known HPV status. Survival was estimated using Kaplan-Meier methods; distributions were compared using log-rank tests. Propensity score-matching and inverse probability of treatment weighing (IPTW) methods were used; cohorts were matched based on age, sex, Charlson-Deyo score, clinical American Joint Committee on Cancer (AJCC) group stage, treatments received, and anatomic subsite. Propensity analyses were stratified by group stage of disease. A total of 24,740 patients diagnosed from 2010 through 2013 were analyzed: 1085 patients with HPX, 4804 with laryngeal, 4,018 with OC, and 14,833 with OPX SCC. The percentages of HPV-positive cases by disease site were 17.7% for HPX, 11% for larynx, 10.6% for OC, and 62.9% for OPX. HPV status was found to be prognostic in multiple unadjusted and propensity-adjusted non-OPX populations. HPV positivity was associated with superior OS in patients with HPX SCC with a hazard ratio (HR) of 0.61 (P<.001 by IPTW), in patients with AJCC stage III to IVB laryngeal SCC (HR, 0.79; P=.019 by IPTW), and in patients with AJCC stage III to IVB OC SCC (HR, 0.78; P=.03 by IPTW). Positive high-risk HPV status appears to be associated with longer OS in multiple populations of patients with non-OPX head and neck disease (HPX, locally advanced larynx, and OC). If prospectively validated, these findings have implications for risk stratification.

High-risk human papillomavirus infection in female and subsequent risk of infertility: a population-based cohort study

To study whether infection with high-risk human papillomavirus (HPV), registered both as a single HPV positive test and as HPV persistence, increases the risk of female factor infertility in later reproductive life. Population-based cohort study. Not applicable. A random sample of 11,088 women (20-29years of age at enrollment) tested for cervical HPV at enrollment during 1991-93 and again after 2years. Information on female factor infertility was obtained by linkage to the Danish Infertility Cohort. None. Follow-up for each study participant was the period from the date of enrollment or date of the second visit until diagnosis of female factor infertility (main outcome), conception, death, emigration, disappearance, or end of study period. Data were analyzed by means of a Cox proportional hazards regression model. Hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between HPV status and female factor infertility after adjustment for potentially confounding factors were determined. After relevant exclusions, 10,595 women were eligible for analysis, 1,861 (17.6%) of whom were high-risk HPV positive at the first visit. There was no association between a positive HPV test at first visit (HR = 0.88; 95% CI, 0.75-1.02) or positivity for the same high-risk HPV type at the first and second visit (persistence; HR = 0.97; 95% CI, 0.66-1.44) and subsequent risk of female factor infertility in reproductive life. We found no association between a high-risk HPV infection and risk of female factor infertility, neither for a single HPV positive test nor for a persistent HPV infection.

High-Risk Human Papillomavirus E6/E7 mRNA Is Rarely Detected in Nonanogenital Cutaneous Squamous Cell Carcinoma: An RNA In Situ Hybridization-Based Tissue Microarray Study.

High-risk human papillomavirus (HR-HPV) is known to play an oncogenic role in squamous cell carcinoma (SCC) at certain anatomical sites, namely the uterine cervix, oropharynx, and anogenital skin. However, the association between HR-HPV and nonanogenital cutaneous SCC (CSCC) remains controversial. In this study, we addressed this controversy by performing HR-HPV E6/E7 mRNA in situ hybridization (ISH) on 243 CSCC samples. A cocktail of E6/E7 mRNA ISH probes, recognizing 18 HR-HPV genotypes, was applied to a tissue microarray of paraffin-embedded sections of 154 invasive and 89 in situ CSCC specimens. The anatomical sites of CSCC included the head and neck (n = 100), extremities (n = 100), trunk (n = 25), and anogenitalia (n = 18). We also investigated the correlation between the p16 expression and HR-HPV status by immunohistochemistry. The results of HR-HPV E6/E7 mRNA ISH showed that 5.8% (14/243) of all CSCC samples were positive for HR-HPV, including 66.7% (12/18) of the anogenital and only 0.9% (2/225) of the nonanogenital CSCC samples (P < 0.01). For the detection of diffuse p16 expression by immunohistochemistry, the sensitivity was 100% (14/14 HR-HPV-positive CSCC samples), and the specificity was 72.1% (165/229 HR-HPV-negative specimens). Thus, HR-HPV E6/E7 mRNA was rarely detected in nonanogenital CSCC, making it unlikely that the virus contributes to the pathogenesis of this malignancy. In addition, p16 immunoreactivity has a limited value as a surrogate marker for transcriptionally active HR-HPV in nonanogenital CSCC.

Periodontal status at diagnosis predicts non-disease-specific survival in a geographically defined cohort of patients with oropharynx squamous cell carcinoma

Background: The aim of this study was to examine periodontal status with a time-efficient screening method from a cohort of newly diagnosed oropharynx squamous cell carcinoma (OPSCC) patients and to study to what extent dental disease level predicted survival.Aims/objective: Can measuring level of dental pathology based on a blind investigation of a routine orthopantogram (OPG) obtained during diagnostic workup reveal prognostic information?Materials and methods: We included 97 patients diagnosed between 2003 and 2010. Radiographic alveolar bone loss was measured. At least 4 mm bone loss from cement–enamel junction on at least two teeth was registered as periodontal pathology. The number of missing and filled teeth (MFT), residual roots and apical radiolucencies were noted. Clinical data were determined through hospital patient records.Results: The horizontal bone loss discriminated between hr-HPV(+) versus hr-HPV(−) status, but secondary to age and smoking history at diagnosis. Vertical and horizontal bone loss predicted survival directly, and adjusted by gender, patient, smoking history, TN stage and hr-HPV tumor infection at diagnosis.Conclusions: Degree of periodontal OPG pathology at the time of OPSCC diagnosis to some extent predicted hr-HPV infection, but predicted non-disease-specific long-term survival.Significance: Degree of periodontal OPG pathology at diagnosis predicts prognosis.

Identification of high-risk human papillomavirus and Rb/E2F pathway genomic alterations in mutually exclusive subsets of colorectal neuroendocrine carcinoma

Colorectal neuroendocrine carcinomas, both small cell and large cell types, are highly aggressive tumors with poor prognosis compared with colorectal adenocarcinoma. The molecular drivers of neuroendocrine carcinoma are best defined in small cell lung cancer, which shows near-universal genomic alterations in TP53 and RB1. The genetics of colorectal neuroendocrine carcinoma remain poorly understood; recent studies demonstrated infrequent RB1 alterations and genetics closely resembling colorectal adenocarcinoma. To better define the molecular pathogenesis of colorectal neuroendocrine carcinoma, we performed capture-based next-generation sequencing on 25 cases and evaluated for expression of p53, Rb, p16, and high-risk human papillomavirus (HR-HPV) subtypes using immunohistochemistry, in situ hybridization, and polymerase chain reaction. Rb/E2F pathway dysregulation was identified in nearly all cases (23/25, 92%) and occurred via three distinct mechanisms. First, RB1 genomic alteration was present in 56% (14/25) of cases and was accompanied by Rb protein loss, high p16 expression, and absence of HR-HPV; these cases also had frequent genomic alterations in TP53, the PI3K/Ras and Wnt pathways, as well as in DNA repair genes, with 4/14 cases being hypermutated. Second, 16% (4/25) of cases, all left-sided, had TP53 alteration without RB1 alteration; half of these harbored high-level amplifications in CCNE1 and MYC or MYCN and arose in patients with ulcerative colitis. Finally, 28% (7/25) of cases, all rectal or anal, lacked genomic alterations in RB1 or TP53 but were positive for HR-HPV. Our data demonstrate that Rb/E2F pathway dysregulation is essential in the pathogenesis of colorectal neuroendocrine carcinoma, akin to neuroendocrine carcinomas in other anatomic sites. Moreover, colorectal neuroendocrine carcinomas stratify into three distinct molecular subgroups, which can be differentiated based on Rb protein and HR-HPV status. HR-HPV infection represents a distinct mechanism for Rb and p53 inactivation in cases lacking genomic alterations in either gene. Differential treatment strategies for hypermutated and HPV-driven cases could improve patient outcomes.

Prevalence, survival outcomes, and clinicopathologic factors associated with negative high risk human papillomavirus in surgical specimens of cervical cancer with pretreatment negative DNA genotype test.

The aim of this study was to detect high risk human papillomavirus in cervical cancer with a pretreatment negative high risk human papillomavirus DNA genotype test and to evaluate clinicopathologic characteristics and survival outcomes according to high risk human papillomavirus status. We investigated high risk human papillomavirus status in surgical specimens from 30 cases of cervical cancer using polymerase chain reaction. Polymerase chain reaction primers were set to detect the presence of the common L1 and E7 regions of human papillomavirus types 16, 18, 31, 33, 45, 52, and 58. We analyzed the following clinicopathologic parameters to evaluate their relationships with high risk human papillomavirus status: age, histology, stage, tumor size, invasion depth, lymphovascular invasion, and recurrent status. Among 30 cases with a pretreatment negative DNA genotype test, high risk human papillomavirus was detected in 12 (40.0%), whereas 18 (60.0%) were negatives. Of 12 high risk human papillomavirus positive cases, 10 (33.3%) were positive for the L1 region, 6 (20.0%) of the 7 types were positive for the E7 region, and 4 (13.1%) were positive for both L1 and E7 regions. According to a multiple logistic regression model, tumor size (odds ratio 7.80; 95% confidence interval 1.476 to 41.216; P=0.0097) and stage (odds ratio 7.00; 95% confidence interval 1.293 to 37.910; P=0.0173) were associated with negative high risk human papillomavirus DNA status. Kaplan-Meier survival plots showed that negative high risk human papillomavirus status was associated with worse disease free survival in contrast with positive high risk human papillomavirus status (P=0.0392). Negative high risk human papillomavirus was found in 60% of cervical cancers with a pretreatment negative DNA genotype test. Moreover, the negative high risk human papillomavirus group was associated with worse survival outcome.

Impact of the human papillomavirus status on the development of high-grade cervical intraepithelial neoplasia in women negative for intraepithelial lesions or malignancy at the baseline: A 9-year Swedish nested case-control follow-up study.

The causal relation between high-risk human papillomavirus (HPV) and cervical cancer and its precursor lesions has led to the use of sensitive HPV molecular tests for screening. This study examined the impact of the baseline HPV status on the future risk of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) among women with cytology negative for intraepithelial lesions or malignancy (NILM). This was a nested case-control study including women with NILM baseline cytology participating in the Swedish cervical screening program in 2005-2007. Ninety-six cases of CIN2+ and 5 age-matched controls per case were identified through the National Cervical Screening Registry by follow-up through 2014. Baseline liquid-based cytology samples were tested for HPV. Conditional logistic regression analysis was used to calculate odds ratios (ORs) with confidence intervals (CIs). The risk of future high-grade cervical intraepithelial neoplasia (CIN) was strongly associated with the baseline HPV status. For women younger than 30 years, HPV-16/18 showed a significant association with future risk for CIN2+ (OR, 9.44; 95% CI, 3.37-26.4). Other HPV types were not significantly associated with future CIN2+ in these younger women. For women 30 years old or older, both HPV-16/18 and other HPV subtypes conferred a significant risk. The presence of HPV-16/18 among women with NILM cytology is associated with an elevated future risk of high-grade CIN. HPV types other than HPV-16/18 seem to have a greater impact on women 30 years old or older than younger women. Women with NILM cytology and HPV-16/18 need specific follow-up management within screening.

Importance of Colposcopy Impression in the Early Diagnosis of Posthysterectomy Vaginal Cancer.

The aim of the study was to investigate the value of cytology, high-risk human papillomavirus (hrHPV) status and colposcopy in the early diagnosis of vaginal cancer after hysterectomy. A retrospective study was performed in the Obstetrics and Gynecology Hospital of Fudan University. Posthysterectomy patients who were diagnosed with vaginal high-grade intraepithelial lesion (HSIL) by colposcopy-directed biopsy with colposcopy impression of extensive HSIL or suspicion of cancer and underwent upper or total vaginectomy from January 2009 to December 2017 were included. Eighty-six posthysterectomy vaginal HSIL patients were included. Available abnormal cytology and positive hrHPV were observed in 90.7% (49/54) and 96.2% (51/53) of the patients, respectively. A total of 18.6% (16/86) of the patients were diagnosed with squamous cell cancer by vaginectomy, and the average interval between hysterectomy and vaginectomy was 3.5 years. Among them, 62.5% (10/16) cancers occurred after hysterectomy for cervical cancer, 31.2% (5/16) after hysterectomy for cervical precancer, and 6.3% (1/16) after hysterectomy for myoma. An indication for hysterectomy (cervical cancer vs HSIL, odds ratio = 7.2, 95% CI = 1.9-28.0, p = .004) and colposcopy impression of vaginal cancer (vaginal cancer vs HSIL, odds ratio = 5.9, 95% CI = 1.3-26.8, p = .021) were high-risk factors of cancer confirmed by vaginectomy in colposcopy-directed biopsy vaginal intraepithelial neoplasia 2/3 posthysterectomy in multiple logistic regression analysis. Colposcopy is pivotal in the evaluation of abnormal cytology/hrHPV tests in follow-up of cervical cancer patients after hysterectomy and decision-making for vaginectomy in detecting early cancer.

Screening for persistent high-risk HPV infections may be a valuable screening method for young women; A retrospective cohort study

IntroductionScreening of young women is often discouraged because of the high risk of unnecessary diagnostics or overtreatment. Multiple countries therefore use cytology instead of high risk human papillomavirus (hrHPV)-testing as screening method for young women because of the limited specificity of hrHPV-testing. The objective of this study was to investigate how hrHPV screening before the age of 30, can be used to reduce the future prevalence of high-grade cervical lesions in young women.MethodsWe retrospectively analyzed follow-up data from a cohort study on HPV prevalence in unscreened Dutch women aged 18–29 years. Women performed multiple self-collected cervico-vaginal samples for HPV detection and genotyping. At least one valid cervical pathology result was obtained from 1,018 women. Women were categorized as hrHPV negative, cleared- or persistent hrHPV infection. Anonymized follow-up data for each group was obtained. Composite outcome measures were defined as; normal, low-grade squamous intraepithelial lesion (LSIL) or high-grade squamous intraepithelial lesion (HSIL). The association between prior hrHPV status and cytology and histology outcome was analyzed.ResultsAfter exclusion, a pathology result was registered for 962 women. The prevalence of HSIL was 19.3% in women with a persistent HPV infection at a younger age. This is significantly higher (p<0,001) compared with the HSIL prevalence of 1.5% in HPV-negative women, and 3.1% (n = 8) in women who cleared the hrHPV infection in the past.ConclusionWomen with a persistent hrHPV infection in their 20s, show an increased prevalence of HSIL lesions in their early 30s. Screening for persistent hrHPV infections, instead of cytology screening before the age of 30, can be used to reduce the future prevalence of cervical cancer in young women.

Low‐grade squamous intraepithelial lesion, cannot rule out high‐grade lesion: Diagnosis, histological outcomes and human papillomavirus results

The 2014 Bethesda System for Reporting Cervical Cytology classifies squamous intraepithelial lesions (SILs) of cervix into two main categories: low-grade SIL (LSIL) and high-grade SIL (HSIL). In some clinical practices, the LSIL cannot rule out high-grade lesion (LROH) interpretive category is used in cases with LSIL and findings that may raise the possibility of HSIL. Our purpose is to assess follow-up histopathology and high-risk human papillomavirus (hrHPV) results in patients with LROH, in comparison with LSIL, atypical squamous cells, cannot rule out HSIL (ASC-H), and HSIL in our institution. Cervical Papanicolaou tests with LROH, LSIL, ASC-H and HSIL interpretation, surgical follow-up, and hrHPV status were retrieved from the computer database from May 2014 to December 2016. Of 109963 total Papanicolaou tests, LROH comprised 0.3%, LSIL 3.1%, ASC-H 0.2% and HSIL 0.4%. Only 3272 cases with surgical diagnoses were included in the study. The most common histological outcome for ASC-H was cervical intraepithelial neoplasia (CIN)2/3 (32.6%); LSIL was CIN 1 (45.7%); LROH was CIN 1 (46.7%) and HSIL was CIN 2/3 (64.4%). For LROH and LSIL, 31.1% and 7.5% respectively, had CIN 2/3. Approximately 79% of cases were hrHPV positive. Of LROH cases with surgical follow-up, 86.9% tested hrHPV positive, accounting for the second most common positive group after HSIL (92.6%). In our study cohort, LROH interpretation is associated with a higher number of CIN 2 or higher lesions on follow-up compared to patients with LSIL (P<0.0001), and is associated with a significant percentage of positive other hrHPV, supporting LROH as a useful diagnostic category that triggers appropriate follow-up in affected women.

High-risk human papillomavirus status and prognosis in invasive cervical cancer: A nationwide cohort study.

BackgroundHigh-risk human papillomavirus (hrHPV) infection is established as the major cause of invasive cervical cancer (ICC). However, whether hrHPV status in the tumor is associated with subsequent prognosis of ICC is controversial. We aim to evaluate the association between tumor hrHPV status and ICC prognosis using national registers and comprehensive human papillomavirus (HPV) genotyping.Methods and findingsIn this nationwide population-based cohort study, we identified all ICC diagnosed in Sweden during the years 2002–2011 (4,254 confirmed cases), requested all archival formalin-fixed paraffin-embedded blocks, and performed HPV genotyping. Twenty out of 25 pathology biobanks agreed to the study, yielding a total of 2,845 confirmed cases with valid HPV results. Cases were prospectively followed up from date of cancer diagnosis to 31 December 2015, migration from Sweden, or death, whichever occurred first. The main exposure was tumor hrHPV status classified as hrHPV-positive and hrHPV-negative. The primary outcome was all-cause mortality by 31 December 2015. Five-year relative survival ratios (RSRs) were calculated, and excess hazard ratios (EHRs) with 95% confidence intervals (CIs) were estimated using Poisson regression, adjusting for education, time since cancer diagnosis, and clinical factors including age at cancer diagnosis and International Federation of Gynecology and Obstetrics (FIGO) stage. Of the 2,845 included cases, hrHPV was detected in 2,293 (80.6%), and we observed 1,131 (39.8%) deaths during an average of 6.2 years follow-up. The majority of ICC cases were diagnosed at age 30–59 years (57.5%) and classified as stage IB (40.7%). hrHPV positivity was significantly associated with screen-detected tumors, young age, high education level, and early stage at diagnosis (p < 0.001). The 5-year RSR compared to the general female population was 0.74 (95% CI 0.72–0.76) for hrHPV-positive cases and 0.54 (95% CI 0.50–0.59) for hrHPV-negative cases, yielding a crude EHR of 0.45 (95% CI 0.38–0.52) and an adjusted EHR of 0.61 (95% CI 0.52–0.71). Risk of all-cause mortality as measured by EHR was consistently and statistically significantly lower for cases with hrHPV-positive tumors for each age group above 29 years and each FIGO stage above IA. The difference in prognosis by hrHPV status was highly robust, regardless of the clinical, histological, and educational characteristics of the cases. The main limitation was that, except for education, we were not able to adjust for lifestyle factors or other unmeasured confounders.ConclusionsIn this study, women with hrHPV-positive cervical tumors had a substantially better prognosis than women with hrHPV-negative tumors. hrHPV appears to be a biomarker for better prognosis in cervical cancer independent of age, FIGO stage, and histological type, extending information from already established prognostic factors. The underlying biological mechanisms relating lack of detectable tumor hrHPV to considerably worse prognosis are not known and should be further investigated.

Incorporation of Cervista Human Papillomavirus 16/18 Assay Into Algorithms for Classifying Human Papillomavirus Status in Formalin-Fixed, Paraffin-Embedded Head and Neck Squamous Carcinoma Specimens.

Human papillomavirus (HPV) DNA in situ hybridization (ISH) assay and p16 immunohistochemistry (IHC) are used to determine high-risk HPV status in formalin-fixed, paraffin-embedded (FFPE) tissues in oropharyngeal squamous cell carcinoma (SCC). Although high sensitivity and specificity for HPV can be obtained by combined p16 IHC and HPV DNA ISH, the occasional discrepancy between these assays has prompted evaluation of Cervista HPV assays in FFPE tissue from patients with oropharyngeal SCC. To compare the efficacy of Cervista HPV 16/18 and Cervista HPV HR assay to that of HPV DNA ISH assay and p16 IHC in FFPE tissue in head and neck squamous cell carcinoma of oropharyngeal origin. Archived FFPE tissue from 84 patients with SCC of oropharyngeal origin and available HPV DNA ISH and p16 IHC test results were tested with the Cervista HPV 16/18 assay and further verified by polymerase chain reaction (PCR)-based HPV16/18 genotyping tests in cases with discrepancy. Of the 84 specimens, 75% (63 of 84) were positive and 16% (13 of 84) had discrepant or equivocal findings by p16 IHC and HPV DNA ISH testing. Use of Cervista HPV assays, either to clarify discrepant/equivocal findings or as confirmation after initial p16 IHC/HPV DNA ISH tests, identified 81% (68 of 84) of HPV-positive cases without equivocal HPV results. Five of 13 cases with discrepancy or equivocal HPV DNA ISH results tested positive for HPV16 or HPV18 by Cervista HPV 16/18 assay, which was further confirmed by PCR-based HPV 16/18 genotyping. The Cervista HPV assays are a reasonable alternative to HPV DNA ISH in determining HPV status in FFPE tissue specimens from patients with oropharyngeal SCC.

Survival Rates for Patients With Barrett High-grade Dysplasia and Esophageal Adenocarcinoma With or Without Human Papillomavirus Infection

High-risk human papillomavirus (HPV) has been associated with Barrett dysplasia and esophageal adenocarcinoma. Nevertheless, the prognostic significance of esophageal tumor HPV status is unknown. To determine the association between HPV infection and related biomarkers in high-grade dysplasia or esophageal adenocarcinoma and survival. Retrospective case-control study. The hypothesis was that HPV-associated esophageal tumors would show a favorable prognosis (as in viral-positive head and neck cancers). Pretreatment biopsies were used for HPV DNA determination via polymerase chain reaction, in situ hybridization for E6 and E7 messenger RNA (mRNA), and immunohistochemistry for the proteins p16INK4A and p53. Sequencing of TP53 was also undertaken. The study took place at secondary and tertiary referral centers, with 151 patients assessed for eligibility and 9 excluded. The study period was from December 1, 2002, to November 28, 2017. Disease-free survival (DFS) and overall survival (OS). Among 142 patients with high-grade dysplasia or esophageal adenocarcinoma (126 [88.7%] male; mean [SD] age, 66.0 [12.1] years; 142 [100%] white), 37 were HPV positive and 105 were HPV negative. Patients who were HPV positive mostly had high p16INK4A expression, low p53 expression, and wild-type TP53. There were more Tis, T1, and T2 tumors in HPV-positive patients compared with HPV-negative patients (75.7% vs 54.3%; difference, 21.4%; 95% CI, 4.6%-38.2%; P = .02). Mean DFS was superior in the HPV-positive group (40.3 vs 24.1 months; difference, 16.2 months; 95% CI, 5.7-26.8; P = .003) as was OS (43.7 vs 29.8 months; difference, 13.9 months; 95% CI, 3.6-24.3; P = .009). Recurrence or progression was reduced in the HPV-positive cohort (24.3% vs 58.1%; difference, -33.8%; 95% CI, -50.5% to -17.0%; P < .001) as was distant metastasis (8.1% vs 27.6%; difference, -19.5%; 95% CI, -31.8% to -7.2%; P = .02) and death from esophageal adenocarcinoma (13.5% vs 36.2%; difference, -22.7%; 95% CI, -37.0% to -8.3%; P = .01). Positive results for HPV and transcriptionally active virus were both associated with a superior DFS (hazard ratio [HR], 0.33; 95% CI, 0.16-0.67; P = .002 and HR, 0.44; 95% CI, 0.22-0.88; P = .02, respectively [log-rank test]). Positivity for E6 and E7 mRNA, high p16INK4A expression, and low p53 expression were not associated with improved DFS. On multivariate analysis, superior DFS was demonstrated for HPV (HR, 0.39; 95% CI, 0.18-0.85; P = .02), biologically active virus (HR, 0.36; 95% CI, 0.15-0.86; P = .02), E6 and E7 mRNA (HR, 0.36; 95% CI, 0.14-0.96; P = .04), and high p16 expression (HR, 0.49; 95% CI, 0.27-0.89; P = .02). Barrett high-grade dysplasia and esophageal adenocarcinoma in patients who are positive for HPV are distinct biological entities with a favorable prognosis compared with viral-negative esophageal tumors. Confirmation of these findings in larger cohorts with more advanced disease could present an opportunity for treatment de-escalation in the hope of reducing toxic effects without deleteriously affecting survival.

Expression of human papillomavirus oncoproteins E6 and E7 inhibits invadopodia activity but promotes cell migration in HPV-positive head and neck squamous cell carcinoma cells.

BackgroundThe rapid increase in the incidence of head and neck squamous cell carcinoma (HNSCC) is caused by high‐risk human papillomavirus (HPV) infections. The HPV oncogenes E6 and E7 promote carcinogenesis by disrupting signaling pathways that control survival and proliferation. Although these cancers are often diagnosed with metastases, the mechanisms that regulate their dissemination are unknown.AimsThe aim of this study was to determine whether the HPV‐16 E6 and E7 oncogenes affected the invasive and migratory properties of HNSCC cells which promote their spread and metastasis.Methods and resultsInvasiveness was determined using invadopodia assays which allow for quantitation of extracellular matrix (ECM) degradation by invadopodia which are proteolytic membrane protrusions that facilitate invasion. Using cell lines and genetic manipulations, we found that HPV inhibited invadopodia activity in aggressive cell lines which was mediated by the E6 and E7 oncogenes. Given these findings, we also tested whether HPV caused differences in the migratory ability of HNSCC cells using Transwell assays. In contrast to our invadopodia results, we found no correlation between HPV status and cell migration; however, blocking the expression of the E6 and E7 oncoproteins in a HPV‐positive (HPV+) HNSCC cell line resulted in decreased migration.ConclusionsOur data suggest that the E6 and E7 oncoproteins are negative regulators of invadopodia activity but may promote migration in HPV+ HNSCC cells. Despite the need for ECM proteolysis to penetrate most tissues, the unique structure of the head and neck tissues in which these cancers arise may facilitate the spread of migratory cancer cells without significant proteolytic ability.

Prognostic model based on magnetic resonance imaging, whole-tumour apparent diffusion coefficient values and HPV genotyping for stage IB-IV cervical cancer patients following chemoradiotherapy.

To develop and validate a prognostic model of integrating whole-tumour apparent diffusion coefficient (ADC) from pretreatment diffusion-weighted (DW) magnetic resonance (MR) imaging with human papillomavirus (HPV) genotyping in predicting the overall survival (OS) and disease-free survival (DFS) for women with stage IB-IV cervical cancer following concurrent chemoradiotherapy (CCRT). We retrospectively analysed three prospectively collected cohorts comprising 300 patients with stage IB-IV cervical cancer treated with CCRT in 2007-2014 and filtered 134 female patients who underwent MR imaging at 3.0 T for final analysis (age, 24-92 years; median, 54 years). Univariate and multivariate Cox regression analyses were used to evaluate the whole-tumour ADC histogram parameters, HPV genotyping and relevant clinical variables in predicting OS and DFS. The dataset was randomly split into training (n = 88) and testing (n = 46) datasets for construction and independent bootstrap validation of the models. The median follow-up time for surviving patients was 69 months (range, 9-126 months). Non-squamous cell type, ADC10 <0.77 × 10-3 mm2/s, T3-4, M1 stage and high-risk HPV status were selected to generate a model, in which the OS and DFS for the low, intermediate and high-risk groups were significantly stratified (p < 0.0001). The prognostic model improved the prediction significantly compared with the International Federation of Gynaecology and Obstetrics (FIGO) stage for both the training and independent testing datasets (p < 0.0001). The prognostic model based on integrated clinical and imaging data could be a useful clinical biomarker to predict OS and DFS in patients with stage IB-IV cervical cancer treated with CCRT. • ADC 10 is the best prognostic factor among ADC parameters in cervical cancer treated with CCRT • A novel prognostic model was built based on histology, ADC 10 , T and M stage and HPV status • The prognostic model outperforms FIGO stage in the survival prediction.

Methylation of the hsa-miR-124, SOX1, TERT, and LMX1A genes as biomarkers for precursor lesions in cervical cancer

ObjectivesThe methylation profile of genes in precursor lesions in cervical cancer was characterized to improve screening techniques for high-grade intraepithelial neoplasia. MethodsA total of 447 cervical cytology samples obtained from women who underwent colposcopy were examined. The cases were distributed as follows: (1) cervices without cervical intraepithelial neoplasia (CIN; n = 152); (2) cervices with a CIN grade of 1 (CIN 1; n = 147); and (3) cervices with a CIN grade of 2 or 3 (CIN 2/3; n = 148). The methylation pattern for a panel of 15 genes was analysed by quantitative methylation-specific PCR (qMSP) and compared between the groups (≤CIN 1 vs. CIN 2+). ResultsIn the validation set, seven genes presented significantly different methylation profiles according to diagnosis, namely, DAPK1 (p = 0.001), EPB41L3 (p = 0.001), HIC1 (p = 0.028), hsa-miR-124-2 (p = 0.001), LMX1A (p = 0.001), SOX1 (p = 0.001), and TERT (p = 0.001). Six genes showed a significant increase in the frequency of methylation in the presence of hr-HPV, namely, DAPK1 (p = 0.001), EPB41L3 (p = 0.001), hsa-miR-124-2 (p = 0.001), LMX1A (p = 0.001), SOX1 (p = 0.001), and TERT (p = 0.001). The methylation of the hsa-miR-124 gene showed sensitivity and specificity (86.7% and 61.3%, respectively) similar to that of the HPV test (91.3% and 50.0%, respectively). The independent factors associated with the diagnosis of CIN 2+ and the methylation of the hsa-miR-124-2 (OR = 5.1), SOX1 (OR = 2.8), TERT (OR = 2.2), and LMX1A (OR = 2.0) genes were a positive test for hr-HPV (odds ratio [OR] = 5.5). ConclusionsHypermethylation of the hsa-miR-124-2, SOX1, TERT, and LMX1A genes may be a promising biomarker for precursor lesions in cervical cancer regardless of the hr-HPV status.

The Prognostic Value of Immune Factors in the Tumor Microenvironment of Penile Squamous Cell Carcinoma.

The host’s immune system plays a pivotal role in many tumor types, including squamous cell carcinomas (SCCs). We aim to identify immunological prognosticators for lymph node metastases (LNM) and disease-specific survival (DSS) in penile SCC. For this retrospective observational cohort study, penile SCC patients (n = 213) treated in the Netherlands Cancer Institute, were selected if sufficient formalin-fixed, paraffin-embedded tumor material was available. Analysis included previously described high-risk human papilloma virus (hrHPV) status, immunohistochemical scores for classical and non-classical human leukocyte antigen (HLA) class I, programmed death ligand-1 (PD-L1) expression, and novel data on tumor-infiltrating macrophages and cytotoxic an regulatory T-cells. Clinicopathological characteristics and extended follow-up were also included. Regression analyses investigated relationships of the immune parameters with LNM and DSS. In the total cohort, diffuse PD-L1 tumor-cell expression, CD163+ macrophage infiltration, non-classical HLA class I upregulation, and low stromal CD8+ T-cell infiltration were all associated with LNM. In the multivariable model, only tumor PD-L1 expression remained a significant predictor for LNM (odds ratio (OR) 2.8, p = 0.05). hrHPV negativity and diffuse PD-L1 tumor-cell expression were significantly associated with poor DSS and remained so upon correction for clinical parameters [hazard ratio (HR) 9.7, p < 0.01 and HR 2.8, p = 0.03]. The only immune factor with different expression in HPV+ and HPV− tumors was PD-L1, with higher PD-L1 expression in the latter (p = 0.03). In the HPV− cohort (n = 158), LNM were associated with diffuse PD-L1 tumor-cell expression, high intratumoral CD163+ macrophage infiltration, and low number of stromal CD8+ T-cells. The first two parameters were also linked to DSS. In the multivariable regression model, diffuse PD-L1 expression remained significantly unfavorable for DSS (HR 5.0, p < 0.01). These results emphasize the complexity of the tumor microenvironment in penile cancer and point toward several possible immunotherapy targets. Here described immune factors can aid risk-stratification and should be evaluated in clinical immunotherapy studies to ultimately lead to patient tailored treatment.

Ezrin\u2013Radixin\u2013Moesin Binding Phosphoprotein 50: A Potential Novel Biomarker in Human Papilloma Virus-Associated Head and Neck Squamous Cell Carcinomas

High-risk human papilloma virus (HR-HPV) has increasingly been associated with head and neck squamous cell carcinoma (HNSCC), in particular oropharyngeal cancers. Ezrin–Radixin–Moesin Binding Phosphoprotein 50 (EBP50), a putative tumour suppressor, localises to the plasma membrane in suprabasal epithelium and to the cytoplasm in proliferative basal layers, and is a target for degradation by the HR-HPV E6 oncoprotein. The aim of this study was to investigate EBP50 protein expression patterns in HNSCC in a large Scottish cohort to determine if there was a correlation with HPV status and clinical outcomes. EBP50 expression patterns were assessed in 156 HNSCC including oropharyngeal (37.8%), laryngeal (24%), oral (19%) and other sites (18.5%), which were genotyped for presence of HR-HPV. HNSCC were generally negative for membranous EBP50. EBP50 expression was either cytoplasmic/absent, being ‘predominantly cytoplasmic’ in 76 (49%), ‘weak/negligible cytoplasmic’ in 44 (28%), ‘strongly cytoplasmic’ in 5 (3%), ‘heterogeneous’ in 26 (17%) and ‘other’ in 5 (3%) samples. Forty tumours (25%) were positive for HPV DNA, predominantly HR-HPV 16, and 44 (28%) were p16 positive. The majority of tumours (71%) with ‘weak/negligible cytoplasmic’ EBP50 expression originated in the oropharynx were more likely to have positive neck nodes, overexpression of p16 and positive tumour HR-HPV status (P < 0.001). Differences in EBP50 levels between oropharyngeal and non-oropharyngeal tumours may be linked to degradation of EBP50 by HR-HPV, and loss of EBP50 may therefore be a surrogate biomarker for HR-HPV infection in oropharyngeal tumours.