The sinonasal tract is considered a second hotspot for human papillomavirus (HPV)-related tumors in the head and neck, with HPV being identified in up to 62% of squamous cell carcinomas (SCCs) and 38% of papillomas. There is limited data from geographical regions with low prevalence of high-risk (HR)-HPV on the association of HR-HPV in sinonasal neoplasms and on utility of p16 as a surrogate marker. p16 immunohistochemistry, HR-HPV mRNA ISH and quantitative real-time PCR (qPCR) were performed on a retrospective cohort of sinonasal papillomas and SCCs. KRAS mutation analysis was done in oncocytic papillomas. p16 positivity was present in 22/142 cases (15.5%) including eight inverted papillomas, one oncocytic papilloma (OP), and 13 SCC. Among these, mRNA ISH showed HR-HPV in the OP and two SCC, while another SCC was found to harbour HPV18 by qPCR. Two HPV-associated SCCs had foci of OP. mRNA ISH was negative in all p16 negative cases. p16 immunohistochemistry showed 68% concordance with mRNA ISH, and had sensitivity and negative predictive value of 100%; specificity was 67%, and positive predictive value was 14.3%. Association with HR-HPV in sinonasal papillomas and SCC is rare, and may be seen in cases demonstrating oncocytic morphology. p16 immunohistochemistry has low specificity and positive predictive value in low-prevalence populations; thus, reflex direct HR-HPV testing should be performed in p16 immunopositive cases. This two-step approach is viable in resource-limited settings, as the proportion of p16 positive cases is small.

To find if an association could be established between Human Papilloma Virus (HPV) infection and oropharyngeal cancers (OPCs) in a group of patients known to be regular users of tobacco, and to determine the impact of HPV status on clinical outcomes.Case records of 212 patients with AJCC-7 (The American Joint Committee on Cancer 7th edition) stages II-IVB non metastatic squamous cell carcinoma of the oropharynx treated using radical radiotherapy with or without chemotherapy during the years 2015-2018 were retrieved. Formalin-fixed, paraffin-embedded blocks from oropharyngeal biopsies were available for 177 patients and were evaluated for p16 expression by immunohistochemical (IHC) staining. More than 50% nuclear staining with or without cytoplasmic staining was considered HPV+ . The association between tobacco use and HPV, as well as the influence of HPV status on survival outcomes were assessed. p16 expression was found to be positive in 23(13%) patients. Significant association was found between chewable tobacco usage and HPV positivity (p = 0.051). The median follow up was 20.5 months (range: 3-80). 5-year Overall Survival was 43.4% and 29.8% (p = 0.044) in HPV+ and HPV- patients, respectively. Local control was significantly better in HPV+ patients (38.6% vs. 25.3%, p = 0.049). There was also a trend towards improved Disease-free Survival in HPV+ patients (31 months vs. 15 months, p = 0.078). Though less in prevalence among the Indian population, improved outcomes in HPV+ OPC patients and widely available IHC HPV assays signifies the routine implementation of p16 testing in day-to-day clinical practice.

There are few pathologic or molecular studies of penile precancerous lesions, and the majority refers to lesions associated with invasive carcinomas. Penile Intraepithelial Neoplasia (PeIN) is classified in two morphologically and distinctive molecular groups, non-HPV and HPV-related with special subtypes. The primary purpose of this international series was to classify PeIN morphologically, detect HPV genotypes and determine their distribution according to PeIN subtypes. A secondary aim was to evaluate the p16INK4a immunostaining as a possible HPV surrogate for high-risk HPV infection in penile precancerous lesions. Samples consisted of 84 PeIN cases, part of a retrospective cross-sectional analysis of 1095 penile carcinomas designed to estimate the HPV DNA prevalence in penile cancers using PCR and p16INK4a immunostaining. Penile Intraepithelial Neoplasia (PeIN) was classified in HPV-related (basaloid, warty-basaloid, warty, hybrid, and mixed subtypes) and non-HPV-related (differentiated), the former being the most frequent. PeIN subtypes were differentiated (non-HPV-related) and basaloid, warty-basaloid, warty, hybrid and mixed (HPV-related). Basaloid PeIN was the most commonly diagnosed subtype, and HPV16 was the most frequent HPV genotype detected. Warty-basaloid and warty PeIN showed a more heterogeneous genotypic composition. Most HPV genotypes were high-risk but low-risk HPV genotypes were also present in a few cases (4%). A single HPV genotype was detected in 82% of HPV positive cases. In contrast, multiple genotypes were detected in the remaining 18% of cases. The findings in this study support the paradigm that penile in situ neoplasia, like its invasive counterparts, is HPV dependent or independent and has distinctive morphological subtypes readily identified in routine practice. Considering that HPV16 is clearly the predominant type, and that the three available vaccines have HPV16, all of them will be suitable for vaccination programs; the price of the vaccines will be probably the main determinant to choose the vaccine.

BACKGROUND: HPV-associated oral cavity squamous cell carcinoma (SCC) is not well-characterized in the literature, and also has a clinical significance that is poorly understood.
METHODS: We gathered a cohort of oral cavity (OC) SCC with nonkeratinizing morphology, either in the invasive or in situ carcinoma (or both), tested for p16 by immunohistochemistry and high risk HPV E6/E7 mRNA by RTPCR (reference standard for transcriptionally-active high risk HPV) and gathered detailed morphologic and clinicopathologic data.
RESULTS: Thirteen patients from two institutions were proven to be HPV-associated by combined p16 and high risk HPV mRNA positivity. All 13 patients (100%) were males, all were heavy smokers (average 57 pack/year), and most were active drinkers (9/11 or 81.8%). All 13 (100%) involved the tongue and/or floor of mouth. All had nonkeratinizing features, but maturing squamous differentiation varied widely (0-90%; mean 37.3%). Nonkeratinizing areas had high N:C ratios and larger nests, frequently with pushing borders, and minimal (or no) stromal desmoplasia. The carcinoma in situ, when present, was Bowenoid/nonkeratinizing with cells with high N:C ratios, full thickness loss of maturation, and abundant apoptosis and mitosis. HPV was type 16 in 11 patients (84.6%) and type 33 in two (15.4%). Nine patients had treatment data available. These underwent primary surgical resection with tumors ranging from 1.6 to 5.2 cm. Most had bone invasion (6/9-66.7% were T4a tumors), and most (6/9-66.7%) had extensive SCC in situ with all 6 of these patients having final margins positive for in situ carcinoma.
CONCLUSIONS: HPV-associated OCSCC is an uncommon entity that shows certain distinct clinical and pathologic features. Recognition of these features may help pathologic diagnosis and could potentially help guide clinical management.

BACKGROUND: Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV-OPSCC) presents frequently as metastasis in a neck lymph node that may be cystic or necrotic. Fine-needle aspiration (FNA) biopsies are often first-line diagnostic procedures. p16 immunohistochemistry (IHC) is a surrogate marker for high-risk HPV (hrHPV) infection but can be challenging to interpret. This study evaluated the use of hrHPV in situ hybridization (ISH) in cytology cell blocks of cystic neck lesions.
METHODS: Twenty-four FNA cases with cell blocks and surgical correlates were evaluated. p16 IHC and hrHPV ISH were assessed on cell blocks (C-p16 and C-hrHPV ISH), and hrHPV ISH on surgical samples (S-hrHPV ISH). All results were classified as negative, positive, or equivocal.
RESULTS: Two cases were excluded because of insufficient tissue on recut. On the basis of C-hrHPV ISH cases, 12 were positive, 5 were negative, and 5 were equivocal. All 12 positive C-hrHPV ISH cases had concordant S-hrHPV ISH with no false positives. Of the 5 negative C-hrHPV ISH cases, 4 had concordant S-hrHPV ISH, and 1 had a discordant S-hrHPV ISH. Of the 5 equivocal C-hrHPV ISH cases, S-hrHPV ISH were both positive and negative. Fourteen cases were equivocal by C-p16; 9 cases were reliably classified by C-hrHPV ISH (5 positive, 4 negative; 64%).
CONCLUSIONS: C-hrHPV ISH can be reliably used, especially when positive. A negative or equivocal interpretation of C-hrHPV ISH may warrant repeat testing. Compared to C-p16, C-hrHPV ISH is more frequently diagnostic and could be helpful for HPV-OSCC diagnosis and management.

The 2020 WHO classification is focused on the distinction between HPV-associated and HPV-independent squamous cell carcinoma of the lower female genital organs. Differentiating according to HPV association does not replace the process of grading; however, the WHO classification does not recommend any specific grading system. VIN are also differentiated according to whether they are HPV(p16)-associated. HPV-independent adenocarcinoma (AC) of the cervix uteri has an unfavorable prognosis. Immunohistochemical p16 expression is considered to be a surrogate marker for HPV association. HPV-associated AC of the cervix uteri is determined using the prognostically relevant Silva pattern.

The Lower Anogenital Squamous Terminology (LAST) recommendations classify human papillomavirus-associated squamous lesions into low- and high-grade squamous intraepithelial lesions (LSILs/HSILs). Our study aimed to assess interobserver agreement among 6 experienced pathologists in assigning 40 anal lesions previously diagnosed as anal intraepithelial neoplasia 2 (AIN 2) to either HSIL or non-HSIL categories.
Agreement based on photomicrographs of H&E alone or H&E plus p16 immunohistochemistry was calculated using κ coefficients.
Agreement was fair based on H&E alone (κ = 0.42; 95% confidence interval [CI], 0.34-0.52). Adding p16 improved agreement to moderate (κ = 0.55; 95% CI, 0.54-0.62). On final diagnosis, 21 cases (53%) had unanimous diagnoses, and 19 (47%) were divided. When designating p16 results as positive or negative, agreement was excellent (κ = 0.92; 95% CI, 0.83-0.95). Among variables (staining location, extent, and intensity), staining of the basal/parabasal layers was a consistent feature in cases with consensus for positive results (20/20). Of the 67 H&E diagnoses with conflicting p16 results, participants modified 32 (48%), downgrading 23 HSILs and upgrading 9 non-HSILs.
Although p16 increased interobserver agreement, disagreement remained considerable regarding intermediate lesions. p16 expression, particularly if negative, can reduce unwarranted HSIL diagnoses and unnecessary treatment.

Accurate determination of human papilloma virus (HPV) status is critical when identifying patients with oropharyngeal squamous cell carcinoma (OPSCC) who may be candidates for de-escalation trials. In this study we investigated whether local p16 screening, by immunohistochemistry (IHC), has high positive predictive value (PPV) for HPV status in a good prognosis HPV positive OPSCC (HPVOPSCC) population treated on a clinical trial.
Patients enrolled on the TROG 12.01 randomised trial for good prognosis HPVOPSCC were randomised based on local p16 IHC testing but subsequently had central p16 IHC and HPV RNA in situ hybridisation (HPV RNA ISH) testing. Correlations between the local and central p16 and central HPV RNA ISH were studied. The main outcome was the positive predictive value (PPV) of local pathology laboratory testing of p16.
176/182 patients had samples available for central testing. 172/176 were evaluable for central testing of p16, and all were confirmed to be p16 positive (172/172, 100%, 95% CI = [97.9%, 100%]). Similarly, 100% of those evaluable for HPV RNA ISH (155/155, 100%, 95% CI = [97.6%, 100%]) were confirmed HPV positive, indicating p16 overexpression driven by transcriptionally active HPV and a PPV of 100% for local p16 testing.
Our results validate the suitability of local pathology laboratory p16 testing alone, in populations with a high attributable fraction of OPSCC due to HPV, to screen and enrol low risk HPVOPSCC patients onto de-intensification trials. This obviates the need for upfront more complex and expensive HPV assays and/or central laboratory testing.

Evidence from current studies show that squamous cell carcinomas at oral and oropharyngeal sites are distinct and unique, with their own separate etiopathogenesis, treatment, and prognosis. The aim of this work is to correlate p16 immunohistochemical expression with histomorphological features suggestive of HPV infection in oral and oropharyngeal squamous cell carcinoma. A total of 50 consecutive biopsy cases of oral squamous cell carcinoma (OSCC) and 50 consecutive biopsy cases of oropharyngeal squamous cell carcinoma (OPSCC) were evaluated for features suggestive of HPV infection like focal basaloid appearance, nests, and lobules of tumor cells with pushing borders, absence of stromal reaction, central necrosis, focal lymphoepithelial morphology, presence of koilocytes, and non-keratinizing or hybrid morphology. Immunostaining was performed using p16 monoclonal antibody (clone mouse 16P04). Only cases showing a moderate (2+) to high intensity (3+) staining in more than 75% cells were taken as p16 immunopositive. The histological features were correlated with p16 immunopositivity. A total of 18/50 (36%) cases of oral squamous cell carcinoma and 27/50 (54%) cases of oropharyngeal squamous cell carcinoma were p16 immunopositive. On statistical analysis, only nests/lobules with pushing borders were found to have a significant correlation with p16 immunopositivity (P value = 0.0012) for OSCC cases. For OPSCC cases, four histological features namely nests and lobules with pushing borders (P value = 0.0001), focal basaloid appearance (P value = 0.0041), lymphoepithelial morphology (P value = 0.0029), and non-keratinizing/hybrid morphology (P value = 0.0141) had a significant correlation with p16 immunopositivity. Histomorphological features are more helpful in predicting p16 immunopositivity in OPSCC than OSCC.

The lungs are a common site of metastasis of head and neck (H&N) squamous cell carcinomas (SCC). This study attempts to define p16 immunoexpression and presence of HPV in primary SCC of the lung and determine their usefulness in discriminating between primary lung SCC and metastasis from HPV-associated oropharyngeal primary. Pathology archives were searched for patients with SCC of the lung without SCC elsewhere. Tissue microarray was constructed and immunohistochemistry performed using anti-p40 and anti-p16 antibodies. All cases were tested for HPV viral proteins E6/E7 by RNA in situ hybridization (ISH) and available positive cases for HPV DNA by polymerase chain reaction (PCR). Eight of 25 (32%) showed cytoplasmic and nuclear expression of p16: 2 (8%) strong and 2 (8%) moderate in > 70% of tumor cells; 1 (4%) strong, 1 (4%) moderate, and 1 (4%) weak in 50-70% of tumor cells; 1 (4%) weak in < 50% of tumor cells. E6/E7 mRNA ISH was negative in all cases. Seven of 8 (87.5%) p16-expressing cases were available for testing by HPV PCR; all were negative for HPV DNA. A retrospective control group of 12 patients with possible SCC metastatic to lung was also identified; high-risk HPV DNA was present in 3, confirming metastasis. p16 expression in lung SCC is not uncommon and may not discriminate between primary pulmonary SCC and metastasis from HPV-associated oropharyngeal primary. Confirmatory HPV testing (high risk HPV DNA or E6/E7 mRNA) is recommended to differentiate metastasis from oropharyngeal primary from two separate primaries.