p16INK4a Immunohistochemistry Research Articles

Human papillomavirus-driven head and neck cancers in Japan during 2008-2009 and 2018-2019: The BROADEN study.

There is limited understanding of epidemiology and time trends of human papilloma virus (HPV)-driven head and neck cancers (HNC) in Japan, especially outside of the oropharynx. To assess HPV-driven HNC, a non-interventional study (BROADEN) of HNC patients diagnosed in 2008-2009 and 2018-2019 was conducted in Japan. Adult patients with oropharyngeal, nasopharyngeal, laryngeal, hypopharyngeal or oral cavity cancers were included in this study. HPV was centrally tested using p16INK4a immunohistochemistry, HPV-DNA PCR and HPV E6*I mRNA. HPV attributability required positivity in at least two tests (p16INK4a immunohistochemistry, HPV-DNA PCR, HPV E6*I mRNA) in the oropharynx, and HPV-DNA and HPV E6*I mRNA positivity for non-oropharynx sites. Nineteen hospitals included a total of 1108 patients, of whom 981 had valid samples. Men accounted for 82% of HNC diagnoses. Patients in the earlier cohort were younger and included a higher percentage of smokers. There was an increasing trend of HPV-driven oropharyngeal cancer over the last decade, from 44.2% to 51.7%. HPV attribution in nasopharyngeal cancers was 3.2% in 2008-2009 and 7.5% in 2018-2019; and 4.4% and 0% for larynx respectively. In total, 95.2% of HPV-driven HNC were attributed to HPV genotypes included in the 9-valent HPV vaccine being HPV16 the most prominent genotype. These results suggest that an epidemiologic shift is happening in Japan, with a decrease in smoking and alcohol use and an increase in HPV-driven HNC. The increasing trend of HPV-driven HNC in Japan highlights the need for preventive strategies to mitigate the rise of HPV-driven HNC.

The importance of combined HPV and CINtec® PLUS genotyping testing for p16 in women with cervical squamous cell carcinoma.

Immunohistochemistry (IHC) for p16INK4A (p16) is a reliable surrogate test for the presence of a high-risk, potentially transformative human papillomavirus (HPV) infection in precursor and malignant lesions of the cervix. The purpose of this study was to evaluate changes in cervical cells caused by persistent HPV infection, by IHC (p16 protein) by comparison with HPV genotyping. The study included female patients aged between 26 and 57 years who presented to a public hospital, with complaints related to the genital area, namely vaginal bleeding and dyspareunia. After selecting the patients, samples were subjected to cytological testing and IHC for p16 and for the determination of HPV types. The relationship between HPV status and p16 status was statistically significant (p=0.0001), of the 41 patients, 53.7% were HPV positive, respectively 56.1% were p16 positive, the agreement relationship between the two indicators was very high (weighted kappa: 0.951). The clinical performance of CINtec® PLUS triage for p16 shows a high positive predictive value (PPV) and a high negative predictive value (NPV) of 95.7% and 100%, respectively, as regards HPV. The p16 marker (CINtec® PLUS) can be used as a prognostic biomarker and provides clinical usefulness through increased sensitivity (Se) and specificity (Sp) in the triage of women at risk of developing precancerous lesions, compared to cytology that is based on morphology, but has a rather low Se and high Sp, while HPV testing is very sensitive but slightly more specific.

Detection of serum biomarkers of HPV-16 driven oropharynx and oral cavity cancer in Brazil

BackgroundHPV-16 driven oropharynx/oral cavity squamous cell carcinomas prevalence varies globally. We evaluated the presence of HPV-16 ctDNA and HPV-16 E6 antibodies in samples obtained from participants treated at the Instituto do Cancer do Estado de Sao Paulo, ICESP, and from whom tumoral HPV DNA, HPV-16 E6*I mRNA, and p16INK4a status was also accessed. MethodsHPV was genotyped by PCR-hybridization. All HPV DNA positive and ∼10 % HPV DNA negative cases underwent p16INK4a immunohistochemistry and E6*I RNA testing using a multiplex bead based protocol. HPV-16 ctDNA and anti-E6 antibodies were assessed by ddPCR (digital droplet PCR) and multiplex serology, respectively. ResultsThe prevalence of HPV-16 in oropharynx carcinoma (OPC) cases was low (8.7 %) when considering solely HPV-16 DNA detection, and even lower (5.2 %) when taken into consideration the concomitant detection of HPV-16 E6*I RNA and/or p16INK4 (HPV-16 attributable fraction - AF). None of the oral cavity cancer (OCC) cases were detected with HPV-16 DNA. HPV-16 ctDNA was more commonly detected than HPV-16 E6 antibodies (29.8 % versus 10.6 %). Both serum biomarkers attained 100 % sensitivity of detecting HPV-16 AF OPC, however the specificity of the HPV-16 anti-E6 biomarker was higher compared to ctDNA (93.2 % versus 75.0 %). Finally, when both HPV-16 ctDNA and anti-E6 biomarkers were considered together, the sensitivity and specificity for HPV-16 OPC detection was 100 % and about 70 %, respectively, independently of analyzing HPV-16 DNA positive or HPV-16 AF tumors. ConclusionsOur findings corroborate that serum biomarkers are highly sensitive and specific biomarkers for detection of HPV-associated OPC.

Findings from a Malaysian multicentre study on oropharyngeal squamous cell carcinoma

BackgroundIn addition to the conventional aetiologic agents of oropharyngeal squamous cell carcinoma (OPSCC) such as tobacco usage, alcohol consumption and betel quid usage, it has been established that a proportion of OPSCC are driven by persistent oncogenic human papillomavirus (HPV) infections. Currently, there is a lack of data on the burden of HPV- associated OPSCC in Asian countries including Malaysia.MethodsA cross-sectional multicentre study with tissue analysis of Malaysian patients diagnosed with primary OPSCC within a five-year period, from 2015 to 2019 between 01/01/2015 to 31/12/2019 was undertaken. Determination of HPV status was carried out using p16INK4a immunohistochemistry on tissue microarrays constructed from archived formalin-fixed paraffin-embedded tissue.ResultsFrom the cases identified, 184 cases had sufficient tissue material for analysis. Overall, median age at diagnosis was 63.0 years (IQR = 15) and 76.1% of patients were males. In our cohort, 35.3% of patients were Indian, 34.2% were Chinese, 21.2% were Malay and 9.2% were from other ethnicities. The estimated prevalence of HPV-associated OPSCC in our cohort was 31.0% (CI 24.4–38.2%). The median age for the HPV-associated OPSCC sub-group of patients was not significantly lower than the median age of patients with HPV-independent OPSCC. More than half of HPV-associated OPSCC was seen in patients of Chinese ethnicity (54.4%). Patients with HPV-associated OPSCC had a much better overall survival than patients with HPV-independent OPSCC (Log rank test; p < 0.001). Patients with HPV-associated OPSCC with no habit-related risk factors such as smoking, were found to have much better overall survival when compared to all other sub-groups.ConclusionsThe findings from our study suggests that prevalence of HPV-associated OPSCC in Malaysia, though not as high as some developed countries, is however on an upward trend. HPV-associated OPSCC appears to be more frequently encountered in patients of Chinese ethnicity. Conventional risk-factors associated with OPSCC such as smoking, alcohol consumption and betel quid chewing should still be considered when estimating prognosis of patients with HPV-associated OPSCC.

Assessing the burden of HPV-related head and neck cancers in mainland China: protocol of a nationwide, multisite, cross-sectional study

BackgroundPersistent human papillomavirus (HPV) infection is a known cause of a subset of head and neck cancers (HNCs). In the last two decades, the proportion of HNCs attributable to HPV...

TP53 mutation and human papilloma virus status as independent prognostic factors in a Norwegian cohort of vulva squamous cell carcinoma.

Vulva squamous cell carcinoma (VSCC) develops through two separate molecular pathways-one involving high-risk human papilloma virus infection (HPV-associated), and the other without HPV infection (HPV-independent) often involving TP53 mutation. HPV-associated VSCC generally has a better progression-free survival than HPV-independent VSCC. The aim of this study was to determine TP53 mutation status using immunohistochemistry, compare different methods of HPV detection and correlate both with survival in a retrospective cohort of 123 patients with VSCC. Immunohistochemistry for p53, Ki67 and p16INK4A (a surrogate marker for HPV infection) was performed on formalin-fixed paraffin-embedded tissues from a cohort of surgically treated VSCC patients to identify molecular subtypes of VSCC. Presence of HPV infection was detected by HPV DNA PCR and HPV mRNA insitu hybridization (ISH). The Pearson chi-square test and multivariable Cox regression model were used to investigate the association of different parameters with progression-free survival and disease-specific survival (DSS), and Kaplan-Meier curves were used to show the association of different parameters with survival. The results of p53 and p16INK4A immunohistochemistry confirmed three VSCC subtypes associated with different prognosis. The TP53 mutation status was identified as an independent prognostic factor of worse progression-free survival (p = 0.024) after adjustment for FIGO stage. p16INK4A immunohistochemistry, mRNA ISH, and DNA PCR had excellent concordance in terms of HPV detection. According to the multivariable Cox regression model, the presence of hrHPV mRNA correlated significantly with increased progression-free survival (p = 0.040) and DSS (p = 0.045), after adjustment for other confounders. p53 and p16INK4A immunohistochemistry stratify VSCC cohort into three subtypes with TP53mutated patients having the worst prognosis. The detection of hrHPV mRNA by ISH was an independent predictor of increased survival. Thus, the combined detection of p53 and HPV mRNA might improve risk stratification in VSCC.

Diagnostic accuracy of HPV16 early antigen serology for HPV-driven oropharyngeal cancer is independent of age and sex.

A growing proportion of head and neck cancer (HNC), especially oropharyngeal cancer (OPC), is caused by human papillomavirus (HPV). There are several markers for HPV-driven HNC, one being HPV early antigen serology. We aimed to investigate the diagnostic accuracy of HPV serology and its performance across patient characteristics. Data from the VOYAGER consortium was used, which comprises five studies on HNC from North America and Europe. Diagnostic accuracy, that is, sensitivity, specificity, Cohen's kappa and correctly classified proportions of HPV16 E6 serology, was assessed for OPC and other HNC using p16INK4a immunohistochemistry (p16), HPV in situ hybridization (ISH) and HPV PCR as reference methods. Stratified analyses were performed for variables including age, sex, smoking and alcohol use, to test the robustness of diagnostic accuracy. A risk-factor analysis based on serology was conducted, comparing HPV-driven to non-HPV-driven OPC. Overall, HPV serology had a sensitivity of 86.8% (95% CI 85.1-88.3) and specificity of 91.2% (95% CI 88.6-93.4) for HPV-driven OPC using p16 as a reference method. In stratified analyses, diagnostic accuracy remained consistent across sex and different age groups. Sensitivity was lower for heavy smokers (77.7%), OPC without lymph node involvement (74.4%) and the ARCAGE study (66.7%), while specificity decreased for cases with <10 pack-years (72.1%). The risk-factor model included study, year of diagnosis, age, sex, BMI, alcohol use, pack-years, TNM-T and TNM-N stage. HPV serology is a robust biomarker for HPV-driven OPC, and its diagnostic accuracy is independent of age and sex. Future research is suggested on the influence of smoking on HPV antibody levels.

A study on H-score threshold for p16ink4a immunoperoxidase expression in squamous cell tumours of oral cavity.

Validity of various detection methods used are likely contributing factor to this wide variation of prevalence of HPV (0-73%) by using GP5/GP6/MY09/MY11 (L1) primer. PCR is a sensitive method but does not identify transcriptionally active High-risk Human papillomavirus and also does not indicate whether the virus is isolated from malignant tumour cells and non-neoplastic cells. P16ink4a Immunohistochemistry is a highly sensitive and Cost-effective surrogate marker for transcriptionally active high-risk HPV for oral cancer. Objective The aim of the present study was to evaluate the H-SCORE of p16 expression in the surface epithelial tumour sites of a large cohort of squamous cell carcinoma (SCC), severe dysplasia (SD). we sought to determine whether the p16 algorithm is reliable in Oral cavity SCC and severe dysplasia (SD). This study used Immunohistochemistry in archival Formalin-fixed paraffin embedded specimens for assessment of p16 protein expression, cytoplasmic and nuclear staining intensity was categorized based on score (range, 0-3) and presence of tumour cell staining (0-100%). The majority of positive cases had low H-score of p16 staining except 3/161 (1.8%) cases of tongue SCC had positive for p16 with diffuse moderate staining with ≥2 scores. There were no significant differences in the distribution of demographic, exposure and histopathological characteristics between patients with and without P16 expression. The present study demonstrated that p16 expression is a reliable HPV marker in the lateral border of the tongue with tonsil involvement but no other sites of the oral cavity. Further p16 IHC detection is required in large cohort of all sites of tongue squamous cell carcinoma studies to validate the marker of HPV.

OROPHARYNGEAL SQUAMOUS CELL CARCINOMA: A SERIES OF 152 CASES FROM RIO DE JANEIRO

<h3>Objectives</h3> The aim of the present study was to evaluate the clinicopathologic features of oropharyngeal squamous cell carcinoma (OPSCC) in a population from Rio de Janeiro, Brazil. <h3>Study Design</h3> One hundred fifty-two patients were diagnosed with primary OPSCC in an oral pathology laboratory between 2014 and 2020. Demographic and clinical data were retrospectively collected from the records and all cases were microscopically classified as keratinizing, nonkeratinizing, or nonkeratinizing with maturation. Immunohistochemistry for p16INK4a was performed in 46 cases (30.2%) and the positive ones were submitted to DNA in situ hybridization (ISH) for human papillomavirus (HPV). <h3>Results</h3> There were 29 females and 123 males, with a mean age of 61 years (range, 39-89). Most patients regularly consumed tobacco (69%) and alcohol (50.7%) and lived in low socioeconomic status areas. Most tumors measured more than 2 cm and were located mainly in the soft palate (48%), followed by unspecified oropharynx, tonsil, and base of the tongue. The 3 histologic subtypes were similarly distributed among the cases. Five cases were positive for p16INK4a (10.9%), and of these, 4 were positive for HPV by DNA ISH (8.7%), most of them nonkeratinizing. <h3>Conclusions</h3> HPV prevalence is low in OPSCC cases from this Brazilian population.

Prevaccine Human Papillomavirus Status in Invasive and Intraepithelial Lesions of the Vulva in New Zealand Women.

The human papillomavirus (HPV) vaccine, introduced in New Zealand (NZ) in 2008, is predicted to substantially lower the incidence of HPV-associated precancers and cancers. The aim of this study is to estimate the proportion of vulvar intraepithelial neoplasia (VIN) lesions and invasive vulvar squamous cell carcinomas (SCCV) attributable to HPV in NZ women treated by the Auckland Regional Gynecological Oncology Service, covering an estimated 50% of the NZ population. Polymerase chain reaction and reverse hybridization were used to analyze retrospective histologically proven SCCV from 1990 to 2007 and VIN lesions from 2000 to 2007 for HPV content and genotype in a collaborative study with the Catalan Institute of Oncology. Immunohistochemistry for p16INK4a was performed on SCCV, which were attributed to HPV if both tested positive. Polymerase chain reaction testing for HPV content and genotype was performed on 66 VIN lesions (all high-grade squamous intraepithelial lesions) and 189 SCCV. In addition, p16 immunohistochemistry was performed on 168 of the 189 SCCV (88.9%) tested for HPV-DNA. Overall, 61 SCCV cases (36.3%) were attributed to HPV (HPV+/p16+), and 89 SCCV cases (53%) were considered to have developed independently of HPV (HPV-/p16-). Known high-risk HPV genotypes were present in 96.8% of HPV-DNA-positive vulvar high-grade squamous intraepithelial lesions and 98.4% of HPV-attributable SCCV. Human papillomavirus 16 represented the most common genotype in both. Overall, the HPV vaccine is likely to substantially alter the profile of SCCV in our region. The results provide a baseline assessment of the HPV status of vulvar neoplasia before the introduction of the HPV vaccine.

Concordance of p16INK4a and E6*I mRNA among HPV-DNA-Positive Oropharyngeal, Laryngeal, and Oral Cavity Carcinomas from the ICO International Study.

Simple SummaryThe utility of a diagnostic algorithm for the detection of HPV-driven oral cavity (OCC), oropharyngeal (OPC), and laryngeal (LC) carcinomas using HPV-DNA testing followed by p16INK4a immunohistochemistry, taking E6*I mRNA detection as the reference standard, was assessed in HPV-DNA-positive formalin-fixed paraffin-embedded samples from 29 countries. The concordance of p16INK4a and E6*I mRNA among 78, 257, and 51 HPV-DNA-positive OCC, OPC, and LC, respectively, was moderate to substantial in OCC and OPC but only fair in LC. A different p16INK4a expression pattern was observed in those cases HPV-DNA-positive for types other than HPV16, as compared to HPV16-positive cases. We concluded that the diagnostic algorithm of HPV-DNA testing followed by p16INK4a immunohistochemistry might be helpful in the diagnosis of HPV-driven OCC and OPC, but not LC. Our study provides new insights into the use HPV-DNA, p16INK4a, and HPV-E6*I mRNA for diagnosing an HPV-driven head and neck carcinoma.Background: Tests or test algorithms for diagnosing HPV-driven oral cavity and laryngeal head and neck carcinomas (HNC) have not been yet validated, and the differences among oral cavity and laryngeal sites have not been comprehensively evaluated. We aimed to assess the utility of a diagnostic algorithm for the detection of HPV-driven oral cavity (OCC), oropharyngeal (OPC) and laryngeal (LC) carcinomas using HPV-DNA testing followed by p16INK4a immunohistochemistry, taking E6*I mRNA detection as the reference standard. Methods: Formalin-fixed paraffin-embedded OCC, OPC, and LC carcinomas were collected from pathology archives in 29 countries. All samples were subjected to histopathological evaluation, DNA quality control, and HPV-DNA detection. All HPV-DNA-positive samples (including 78 OCC, 257 OPC, and 51 LC out of 3680 HNC with valid HPV-DNA results) were also tested for p16INK4a immunohistochemistry and E6*I mRNA. Three different cutoffs of nuclear and cytoplasmic staining were evaluated for p16INK4a: (a) >25%, (b) >50%, and (c) ≥70%. The concordance of p16INK4a and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases was assessed. Results: A total of 78 OCC, 257 OPC, and 51 LC were HPV-DNA-positive and further tested for p16INK4a and E6*I mRNA. The percentage of concordance between p16INK4a (cutoff ≥ 70%) and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases was 79.5% (95% CI 69.9–89.1%), 82.1% (95% CI 77.2–87.0%), and 56.9% (95% CI 42.3–71.4%), respectively. A p16INK4a cutoff of >50% improved the concordance although the improvement was not statistically significant. For most anatomical locations and p16INK4a cutoffs, the percentage of discordant cases was higher for HPV16- than HPV-non16-positive cases. Conclusions: The diagnostic algorithm of HPV-DNA testing followed by p16INK4a immunohistochemistry might be helpful in the diagnosis of HPV-driven OCC and OPC, but not LC. A different p16INK4a expression pattern was observed in those cases HPV-DNA-positive for types other than HPV16, as compared to HPV16-positive cases. Our study provides new insights into the use HPV-DNA, p16INK4a, and HPV-E6*I mRNA for diagnosing an HPV-driven HNC, including the optimal HPV test or p16INK4a cutoffs to be used. More studies are warranted to clarify the role of p16INK4a and HPV status in both OPC and non-OPC HNC.

Dysplastic Lesions of the Gingiva: Prevalence and Risk Factors Including Human Papillomavirus (HPV) Infection

<h3>Background</h3> Verrucous gingival lesions are common within the spectrum of proliferative verrucous leukoplakia (PVL), and the gingiva is not an unusual location for oral carcinoma. However, non-PVL associated dysplastic gingival lesions appear to be less common and may not progress directly to malignancy. In addition, it appears HPV likely plays a limited etiologic role in any form of dysplasia of the gingiva. <h3>Materials and Methods</h3> Following IRB approval, all "dysplasia" cases between 2009-2019 were identified from the archives of the University of Florida Oral Pathology Biopsy Service. Exclusion criteria included verrucous lesions, lesions from multiple sites compatible with PVL, and cases diagnosed as mild atypia/dysplasia. The remaining gingival dysplasia cases were evaluated for age, gender, contributory history, clinical appearance/impression, symptoms, duration, and p16INK4a immunohistochemistry (IHC) results. <h3>Results</h3> A total of 72 biopsies were included in our database from 54 patients. The mean age was 70.3 years (range of 27 - 90), with a female: male ratio of 1.4:1. 69.4% (n=50) were located on the posterior gingiva and 62.5% (n=45) the mandibular gingiva. Reported contributing histories included smoking (n=9, 12.5%), previous oral dysplasia (n=9, 12.5%), or oral cavity malignancy (n=24, 33.3%). The two most common clinical impressions were leukoplakia (n=16, 16.7%) and carcinoma (n=18, 25%). Twenty-five of the 72 cases (34.7%) were symptomatic with pain, swelling and/or bleeding. The most recent biopsy from 32 of the patients was low-grade dysplasia, 34 had high-grade dysplasia, and 6 koilocytic dysplasia. Only 4 of 6 koilocytic dysplasia cases were positive for p16, with no low-grade or high-grade lesions demonstrating positivity. <h3>Conclusion</h3> This study confirms that p16 expression is rarely present in dysplastic lesions of the gingiva, unless koilocytic dysplasia is also present.

Prognostic value of high-risk human papillomavirus DNA and p16INK4a immunohistochemistry in patients with anal cancer: An individual patient data meta-analysis

BackgroundHigh-risk human papillomavirus (hrHPV) types represent the aetiological agents in a major proportion of anal squamous cell carcinomas (ASCC). Several studies have suggested a prognostic relevance of HPV-related markers, particularly hrHPV DNA and p16INK4a (p16) protein expression, in patients with ASCC. However, broader evaluation of these prognostic marker candidates has been hampered by small cohort sizes and heterogeneous survival data among the individual studies. We conducted an individual patient data (IPD) meta-analysis to determine the prognostic value of hrHPV DNA and p16 in patients with ASCC while controlling for major clinical and tumour covariates. Patients and methodsA systematic literature search was conducted to identify all published studies analysing p16 alone or in combination with hrHPV DNA and reporting survival data in patients with ASCC. Clinical and tumour-related IPD were requested from authors of potentially eligible studies. Survival analyses were performed with a proportional hazard Cox model stratified by study and adjusted for relevant covariates. The study-specific hazard ratios (HRs) for the exposures were pooled using a random-effects model. Kaplan-Meier curves from different studies were pooled per exposure group and weighted by the study's total sample size. ResultsSeven studies providing IPD from 693 patients with ASCC could be included in the meta-analysis. Seventy-six percent of patients were p16+/hrHPV DNA+, whereas 11% were negative for both markers. A discordant marker status was observed in 13% of cases. Patients with p16+/hrHPV DNA+ ASCC showed significantly superior overall survival (OS) compared with patients with p16-/hrHPV DNA- tumours (pooled adjusted HR = 0.26 [95% confidence interval {CI}, 0.14–0.50]) with pooled three-year OS rates of 86% (95% CI, 82–90%) versus 39% (95% CI, 24–54%). Patients with discordant p16 and hrHPV DNA status showed intermediate three-year OS rates (75% [95% CI, 56–86%] for p16+/hrHPV DNA- and 55% [95% CI, 35–71%] for p16-/hrHPV DNA+ ASCC). ConclusionThis first IPD meta-analysis controlling for confounding variables shows that patients with p16+/hrHPV DNA+ ASCC have a significantly better survival than patients with p16-/hrHPV DNA- tumours.

Localization and characterization of human papillomavirus-16 in oral squamous cell carcinoma.

The role of Human papillomavirus (HPV) in the oral squamous cell carcinoma (OSCC) has not been completely elucidated. The purpose of the present study was to investigate the prevalence and localization of HPV-16 virus in OSCC and to correlate HPV-16 positivity and p16INK4A expression with the clinical and pathological features of OSCC. The archives of Oral Pathology at the University of Florida, College of Dentistry were accessed for demographic, clinical, histopathological data, and slides of 114 OSCC patients. HPV-16 positivity of OSCC was evaluated by p16INK4A immunohistochemistry (IHC) and HPV-16 E6/E7mRNA by in situ hybridization (ISH). Out of 114 consecutive pathological slides of OSCC, 16samples (14%) showed positivity for p16INK4A by IHC and 14samples (12%) were positive for HPV-16 E6/E7mRNA ISH and the Positivity showed a significant correlation with the patients' age, alcohol consumption, and the degree of OSSC differentiation. The hard palate showed the highest positivity of p16INK4A IHC and HPV-16mRNA ISH (38%, 36% respectively). HPV-16 is a significant factor in oral carcinogenesis. We recommend using p16INK4A as a surrogate marker for HPV detection in OSCC, which can be complemented by RNA ISH for the identification of HPV subtypes.

A phase II trial of abemaciclib in patients with p16ink4a negative, relapsed mesothelioma.

8558 Background: Genetically stratified therapy for malignant mesothelioma (MM) is lacking. MMs frequently harbour loss of chromosome 9p21.3 locus (CDKN2A) associated with shorter survival. CDKN2A encodes the tumour suppressor p16ink4a, an endogenous suppressor of CDK4 and CDK6. Genetic reconstitution of p16ink4a into CDKN2A suppresses MM in preclinical models, underpinning the rationale for targeting of CDK4/6 in p16ink4a negative MM. We therefore developed a multi-centre molecularly stratified phase IIa trial to test this hypothesis as arm 2 of the Mesothelioma Stratified Therapy umbrella trial (NCT03654833, MiST2). Methods: Patients with histologically confirmed MM (pleural or peritoneal) were molecularly screened by immunohistochemistry for p16ink4a, BAP1, BRCA1, and PDL1 (Dako 22C3). Patients with p16ink4a negative MM were eligible. Key inclusion factors: histological confirmation of MM with an available archival tissue block, ECOG performance status 0-1, prior platinum-based 1st line chemotherapy (any line allowed), evidence of disease progression with measurable disease by CT (RECIST 1.1), and adequate haematological/organ function. Patients received Abemaciclib (Ab) 200mg bd po daily in q21 day cycles. Primary endpoint was disease control rate at 12 weeks (DCR12w). The null hypothesis was rejected if ≥ 11 patients had disease control (A’Hern design). Secondary endpoints: DCR at 24 weeks (DCR24w), best objective response rate and toxicity (NCI CTCAE 4.03). Patients could undergo an optional re-biopsy upon disease progression. Results: Between November 2019 and March 2020, 26 patients with p16ink4a negative MM received at least one dose of Ab. The median age of pts was 67 (range, 64-74) years, 89% were male, 80.8% epithelioid, 84.6% ECOG PS1, &gt; 1 prior systemic therapy 62%. All patients received at least one cycle with 27% completing 6 cycles. No dose reductions occurred in 53.9% of pts. DCR12w: 54% (95% confidence limit (CI), 36% – 71%), DCR24w: 23% (95%CI, 9% – 44%). Best responses (within 24w): partial - 15% (95%CI, 4- 35%), one occurring after 18 weeks; stable disease - 53.8% (33.4 – 73.4%); progression - 7.7% (0.9 – 25.1%). Adverse events (any cause): ≥ grade 3 toxicities affected 5.7% of pts. Conclusions: MiST2 met its primary endpoint, warranting further clinical evaluation of Ab. Whole exome sequencing of the cohort is ongoing to explore genomic correlates of de novo and acquired resistance. Clinical trial information: NCT03654833.

Different patterns of p16INK4a immunohistochemical expression and their biological implications in laryngeal squamous cell carcinoma.

Introduction: p16INK4a immunohistochemistry (IHC) is widely used to facilitate the diagnosis of human papillomavirus (HPV)-associated neoplasia, when ≥70% of cells show strong nuclear and cytoplasmic positivity. In this study, we aim to compare partial expression patterns that do not fulfill the above criteria and seek biological implications in laryngeal squamous cell carcinoma (LSCC). Materials and Methods: p16INK4a IHC staining was conducted on representative sections of archived tissue from 88 LSCCs. Immunoreactivity was described based on four parameters: intracellular localization of immunostaining, intensity of immunostaining, distribution pattern and percentage of positive cells. Results: Six patterns of p16INK4a immunoexpression were observed and defined as: strong diffuse (strong immunostaining, expression in cytoplasm and nucleus in >70% of tumor cells), weak diffuse (moderate or weak immunostaining, expression in cytoplasm in >70% of tumor cells), marginal (strong cytoplasmic immunostaining, limited to the periphery of tumor islets), strong scattered (strong immunostaining, expression in cytoplasm and nucleus in <50% of tumor cells), weak scattered (moderate or weak immunostaining, expression in cytoplasm in <50% of tumor cells), negative (no expression). The pN stage of the patients was associated with p16INK4a immunoexpression patterns, the marginal pattern was only found in the pN0-Nx stages, while the weak diffuse pattern was more frequently observed in pN2-N3 stages. Conclusions: Partial immunostaining with architecturally distinct p16INK4a immunoexpression patterns may prove significant in stratifying characteristic clinicopathological subgroups among LSCC. Our observations may support the hypothesis that p16INK4a has different roles in different subcellular locations, with tumorigenic molecular pathways unrelated to HPV infection.

DNA methylation markers for cancer risk prediction of vulvar intraepithelial neoplasia.

Current clinical and histological classifications are unable to determine the risk of vulvar squamous cell carcinoma (VSCC) in high‐grade vulvar intraepithelial neoplasia (VIN), making prognostic biomarkers highly needed. We studied host‐cell DNA methylation markers in high‐grade squamous intraepithelial lesion (HSIL) and differentiated VIN (dVIN) without VSCC, in HSIL and dVIN adjacent to VSCC and in human papillomavirus (HPV) positive and negative VSCC, relative to control vulvar tissues. A series of 192 formalin‐fixed paraffin‐embedded vulvar samples, including VSCC (n = 58), VIN adjacent to VSCC (n = 30), VIN without VSCC during follow‐up (n = 41) and normal vulvar tissues (n = 63), were tested for 12 DNA methylation markers with quantitative multiplex methylation‐specific PCR (qMSP). HPV status was determined by p16INK4A immunohistochemistry and high‐risk HPV PCR analysis. Logistic regression analyses were used to determine methylation patterns and methylation marker performance for VIN and VSCC detection. Methylation markers showed significantly higher methylation levels with increasing severity of disease. VIN adjacent to VSCC showed a similar methylation‐high pattern as VSCC, while VIN without VSCC displayed a heterogeneous methylation pattern. Vulvar carcinogenesis is associated with increased DNA methylation. Higher DNA methylation levels in VIN seem to reflect higher cancer risk, emphasizing the high potential of DNA methylation biomarkers in the diagnostic workup of VIN. As a next step, longitudinal studies are needed to verify the prognostic value of methylation biomarkers as a clinical tool for stratification of cancer risk in women with VIN.

Cellular senescence evaluated by P16INK4a immunohistochemistry is a prevalent phenomenon in advanced calcific aortic valve disease

BackgroundFibrosis, calcification, and ossification are histopathologic hallmarks of calcific aortic valve disease (CAVD), a leading cause of morbidity and mortality in the aging population. Cellular senescence contributes to a functional decay in chronic diseases by intensifying tissue remodeling and impairing tissue regeneration. We evaluated the expression of P16INK4A and P53 as surrogate markers of senescence in CAVD. MethodsAortic valves from 27 individuals with severe CAVD requiring aortic valve replacement were selected for routine histologic processing. Immunohistochemical expression of P16INK4A and P53 was quantified using computerized image analysis on fields matching compartments with varying degrees of tissue remodeling. ResultsAll aortic valves demonstrated P16INK4A and P53-positive cells. The percentage of P16INK4A -positive cells, but not of P53, was higher in areas of calcification and/or ossification (57.21%±26.31, n=40) and severe fibrosis (54.79%±27.19, n=25) than in areas with minimal to mild tissue remodeling (13.69% ± 11.88, n=16, P<.0001). P16INK4A expression was observed in interstitial valve cells within all compartments proportional to the degree of fibrosis and did not correlate with age, severity of aortic stenosis, or P53 expression. Multiple linear regression analysis by backward elimination revealed P16INK4A expression was lower among statin users (P<.01). ConclusionsP16INK4A- expression is ubiquitous in calcified aortic valves and correlates with severity of tissue remodeling, suggesting a role of cellular senescence in the progression of CAVD. Further research is needed to identify possible treatment modalities as disease modifying agents for CAVD.

HPV58 E7 Protein Expression Profile in Cervical Cancer and CIN with Immunohistochemistry.

Background: The persistent infection of high-risk human papillomavirus (HR-HPV) is one of the most common causes of cervical cancer worldwide, and HPV type 58 (HPV58) is the third most common HPV type in eastern Asia. The E7 oncoprotein is constitutively expressed in HPV58-associated cervical cancer cells and plays a key role during tumorigenesis. This study aimed to assess the HPV58 E7 protein expression in the tissues of cervical cancer and cervical intraepithelial neoplasia (CIN).Methods: A total of 67 HPV58-positive cervical samples were collected, including 25 cervical cancer samples and 42 CIN samples. All the tissues were examined by HPV58 E7, p16INK4a and Ki67 immunohistochemistry (IHC). At last, we analyzed their association with clinical and pathological variables.Results: HPV58 E7 expression was detected in 96% of the HPV58 DNA-positive cervical cancer tissues and 85.7% of HPV58-positive CIN tissues. 65 samples of cervical cancer and CIN tissues had p16-positive staining, while 59 samples were Ki-67 positive.Conclusions: HPV58 E7 protein is highly expressed in both cervical cancer and CIN tissues. HPV58 E7 IHC could be sensitive and specific for evaluating HPV-driven cervical cancer and pre-cancerous lesions, in combination with p16 and Ki-67 IHC.

Epidemiological aspects of preinvasive cervical lesions and the role of p16INK4a immunohistochemistry in their diagnosis: Experience from a tertiary hospital in South-east Nigeria

Epidemiological aspects of preinvasive cervical lesions and the role of p16INK4a immunohistochemistry in their diagnosis: Experience from a tertiary hospital in South-east Nigeria