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Introduction Human papilloma virus (HPV) associated vulval squamous cell carcinoma (VSCC) has better prognosis than HPV-independent VSCC. Ancillary tests are necessary as morphology alone is unreliable in differentiating these. Methods We analysed 18 VSCC cases by comparing their morphology with three ancillary tests performed to identify the HPV status. Results Basaloid/warty type VSCC considered to be HPV-associated; accounted for 6/18 cases (33.3%). Of these, 5 cases (83.3%) showed block p16 positivity and 3 cases (50%) had a positive HPV genotyping result. Keratinising VSCC considered to be HPV-independent; accounted for 11/18 cases (61.1%). Amongst these, 3 cases (27.3%) showed block 16 positivity and 2 cases (18.2%) had positive HPV genotyping results. In one case, the VSCC was too small to assess the morphology. Usual type VIN considered to be HPV-associated, was seen in 11/18 cases (61.1%). Of these, 8 cases (72.7%) showed block p16 positivity and 6 cases (54.5%) had a positive HPV genotyping result. Differentiated VIN considered to be HPV-independent, was seen in 4/18 cases (22.2%). Amongst these, 0 cases (0%) showed block p16 positivity and 0 cases (0%) had a positive HPV genotyping result. HPV genotyping detected high risk HPV in 6/18 cases (33.3%) and all these cases showed block p16 positivity. In 3/9 cases (33.3%), with block p16 positivity, genotyping did not reveal any HPV. HPV immunohistochemistry was negative in all cases. Conclusion Our study illustrates that in the absence of a gold standard test for HPV, p16 immunohistochemistry and HPV genotyping are complimentary ancillary tests in VSCC.

Typically, HPV-related cancers are sexually transmitted, however, the natural history of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) is unclear. HPV16 transmission has been reported previously between five couples with OPSCC. We report the clinico-pathological features of a further four couples with HPV-related OPSCC and compare them with the published cases.We identified four couples in long-term heterosexual relationships that all had HPV-related OPSCC. The couples were treated at three UK hospitals and presented between 2009 and 2015. HPV tests included p16 immunohistochemistry, high-risk HPV DNA in-situ hybridisation and Roche Cobas HPV test. DNA sequencing was used to determine the HPV variant.The four couples represented2% of patients with HPV-related OPSCC at the three contributing hospitals (8 of 457 consecutive patients). The couples' tumours all contained HPV16. The mean age was 63 years old (range 52-72 years). The interval between the index cancer and the partner's cancer was 16, 24, 26 and 64 months respectively. The majority of patients had Stage I disease (UICC TNM8). Six of eight patients are disease free, one patient is alive with disease and there was one death from loco-regional recurrence.This report highlights the occurrence of HPV-related OPSCC in heterosexual couples and raises the possibility of transmission of HPV16. Despite increasing prevalence of HPV-related OPSCC and increased awareness of the disease, there is a paucity of couples with the disease, suggesting either under-reporting or that the development of OPSCC following HPV transmission between couples is a rare event.

BACKGROUND Hormonal changes in pregnancy are known to alter melanocytic lesions, with some nevi noted to have increased mitotic figures and increased Ki-67 proliferation index. Additionally, cytomorphologic changes have also been noted, referred to as superficial micronodules of pregnancy. These changes may alarm the pathologist for malignancy, particularly nevoid melanoma. Immunohistochemistry for p16 has been recently utilized to distinguish benign nevi from melanoma. We assessed the use of p16 immunohistochemistry for distinguishing melanocytic nevi of pregnant patients from nevoid melanomas. METHODS Fourteen nevomelanocytic lesions were obtained from pregnant or postpartum patients along with 20 nevoid melanomas for comparison. Immunohistochemistry with p16 was performed on each melanocytic lesion. The percentage of nuclear p16 staining of dermal melanocytes was grouped on a scale of 25% to 50%, and >50%. RESULTS The majority of nevi from pregnant patients (81%) showed staining of >5% for p16. In contrast, the majority of nevoid melanomas (65%) had staining of

There are at least two different etio-pathogenic pathways for the development of vulvar squamous cell carcinoma (VSCC): one associated with infection by human papillomavirus (HPV) and another independent of HPV. We aimed to describe the histological characteristics of HPV-associated and HPV-independent tumors and to determine the best strategy to identify HPV in VSCC. A single paraffin block was available for review from a series of 1594 VSCCs. In all cases HPV DNA detection was analyzed using the SPF10PCR/DEIA/LiPA25 system and p16 immunohistochemistry (IHC). A tumor was considered as unquestionably HPV-associated if both HPV DNA and p16 IHC were positive. A tumor was considered indisputably HPV-independent if both HPV DNA and p16 IHC were negative. Two groups of tumors were classified as non-conclusive: 1) HPV DNA+/p16-; and 2) HPV DNA-/p16+. WHO typing and a thorough histological evaluation were conducted in all cases. 441 tumors were HPV DNA+ with 367 cases (23.0%) being HPV DNA+/p16+. These HPV DNA+/p16+ tumors were more frequently basaloid or warty (49.8%), but 36.5% were of the keratinizing type. 1153 tumors were HPV DNA-, with 1060 cases (66.5%) being HPV DNA-/p16-. These HPV DNA-/p16- tumors were mostly keratinizing (81.2%) but were occasionally basaloid or warty (5.2%). The features of HPV DNA-/p16+ cases (n=93) were similar to those of the HPV-associated VSCC, and HPV DNA+/p16- (n=74) cases had a more diverse profile, although they were more similar to HPV-independent tumors. Several histological characteristics were more frequently associated with HPV-related VSCC (koilocytotic-like change, necrosis, moderate to marked pleomorphism, invasive front in nests; p

Objective: Although Japan is a non-endemic area with nasopharyngeal carcinoma (NPC), the proportion of WHO type I NPC in Japan are different from that in non-endemic areas such as North America and Europe. Recently, it is said that not only Epstein-Barr virus (EBV) but also human papillomavirus (HPV) has an influence on NPC in non-endemic areas. The aim of this study is to clarify the influence of HPV on NPC in Japan. Methods: Paraffin-embedded tumor specimens were available for 59 patients with NPC diagnosed between 1996 and 2015. We detected the virus status by p16 immunohistochemistry, HPV PCR, and in situ hybridization for Epstein-Barr virus (EBV)-encoded RNA. Kaplan-Meier curves were used to compare the overall survival by viral status. Results: Among the 59 patients, 49 (83%) were EBV-positive/HPV-negative, 2 (3%) were EBV-positive/HPV-positive, and 8 (16%) were EBV-negative/HPV-negative. All HPV-positive NPCs were co-infected with EBV. There were no significant differences between the overall survival in the three groups (p=0.111). Conclusion: In Japan, HPV was detected in a few patients with NPC, and we suggest that HPV has no influence on NPC carcinogenesis in this population. © 2016 Elsevier Ireland Ltd., Embargo period 12 months

Visual interpretation of cervical biopsies is subjective and variable, generally showing fair to moderate inter‐reader agreement in distinguishing high from low grade cervical intraepithelial neoplasia (CIN). We investigated the performance of two objective p16 quantitative tests in comparison with visual assessment: (i) p16‐mRNA assay and (ii) digital analysis of sections stained for p16 protein. The primary analysis considered 232 high‐risk human papilloma virus positive (HPV+) samples from diagnostic cervical specimens. A p16 RT‐qPCR (p16‐mRNA assay) was run on mRNA extracted from formalin‐fixed paraffin‐embedded sections. Two p16 immunohistochemistry (IHC) readings, a visual read by a histopathologist (Visual IHC) and a digital read of a high‐resolution scan (Digital IHC), were done on adjacent sections. The worst reviewed CIN grade (agreed by at least two histopathologists) from up to two biopsies and a loop excision was taken, with CIN2/3 as the primary endpoint. Visual IHC attained a specificity of 70% (95%CI 61–77) for 85% (95%CI 77–91%) sensitivity. The four‐point Visual IHC staining area under the curve (AUC) was 0.77 (95%CI 0.71–0.82), compared with 0.71 (95%CI 0.64–0.77) for p16‐mRNA and 0.67 (95%CI 0.60–0.74) for Digital IHC. Spearman rank‐order correlations were: visual to p16‐mRNA 0.41, visual to digital 0.49 and p16‐mRNA to digital: 0.22. The addition of p16‐mRNA assay to visual reading of p16 IHC improved the AUC from 0.77 to 0.84 (p = 0.0049). p16‐mRNA testing may be complementary to visual IHC p16 staining for a more accurate diagnosis of CIN, or perhaps a substitute in locations with a lack of skilled pathologists., What's new? Visual examination of cervical tissue obtained by biopsy provides information on morphological changes associated with human papillomavirus (HPV) infection and transformation to precancerous cervical intraepithelial neoplasia 2/3 (CIN2/3). The accuracy of CIN2/3 diagnosis by visual interpretation, however, could be improved through the use of complementary objective tests. This study describes two such tests: quantitative p16 mRNA analysis and digital analysis of immunohistochemical p16‐protein staining. CIN2/3 diagnostic accuracy benefitted especially from the addition of p16‐mRNA testing. Both new approaches could prove useful in locations lacking trained pathologists and could help avoid overtreatment in HPV‐positive women, many of whom recover spontaneously.

Introduction: p16 or p16/INK4a is a Cyclin Dependant Kinase Inhibitor 2 (CDKN2) and is upregulated in HPV-related lesions. Therefore, p16 can be considered an indirect marker for altered HR-HPV and growth cycle transformation. Aim: To study p16 Immunohistochemistry (IHC) in spectrum of Cervical Intraepithelial Neoplasia (CIN) and access the possible utility of p16 IHC in differentiating Low grade Squamous Intraepithelial Lesions (CIN1/L-SIL) from High Grade Lesions (CIN2,3/H-SIL). Materials and Methods: Retrospective Hospital Based study of 50 consecutive biopsies with CIN changes was conducted from January 2016 to March 2017. Routine Hematoxylin and Eosin (H&E) and IHC with p16 was performed. p16 IHC was scored according to a scoring system by a previous study. HPV status wherever present was correlated. Statistical analysis was done using SPSS 18.0 software and Fisher’s-exact test was used for comparison between p16 IHC and CIN. Association of p16 IHC and HPV DNA was calculated using the Phi co-efficient test. Results: The age range of CIN cases was 24-50 years and mean age was 35.9 years. The percentage of p16 positive cells, the intensity of IHC reaction, cellular reaction pattern and p16 IHC positivity, all increased with increasing grades of dysplasia. The use of p16 was statistically significant to differentiate between CIN1/L-SIL and CIN2, 3/H-SIL but not between CIN2 and CIN3. In p16 IHC intracellular pattern, weak cytoplasmic positivity of p16 was seen in L-SIL while strong cytoplasmic positivity was seen in H-SIL cases. High risk HPV positivity increased with increase in grade of dysplasia. Conclusion: p16 can be used as an adjunct to histopathology and will definitely improve reporting of grades of CIN.

Human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (SCC) is a well-known cause and prognostic indicator, and the utility of p16 as a surrogate marker for HPV status has been established. P16 and its relationship with HPV have not been defined in sinonasal malignancy nor has a link with outcomes been established. Patients with sinonasal SCC from 2011 to 2017 were identified from our pathology database. P16 immunohistochemistry and HPV RNA in situ hybridization were performed on tissue specimens. Forty-seven patients were included. Disease-free survival for p16+ patients was significantly higher than p16- patients (P = .043). Fewer HPV+ patients died (P = .052) or experienced recurrence (P = .0437). Odds ratio between p16 and HPV status was 14.19 (95% CI: 1.72, 442.03). Our findings demonstrate improved survival in both the p16+ and HPV+ groups and a positive association between p16 and HPV. There may be similar potential for modifying classification for HPV+ sinonasal SCC.

Background: p16 immunohistochemistry is widely used to diagnose human papillomavirus (HPV)-related squamous neoplasms of cervix, anogenital, head, and neck tissues. The incidence of these HPV-related squamous neoplasms is markedly increased in the HIV-infected population. Ocular surface squamous neoplasia (OSSN) is also more common in HIV-infected patients. However, the expression pattern of p16 in OSSN among HIV-infected patients is unclear. Here, we examined the expression of p16 in OSSN surgical excisions collected from a large HIV-infected cohort from ­Mozambique. Methods: OSSN surgical tissue specimens were collected from 75 Mozambican patients. Formalin-fixed, paraffin-embedded tissue blocks from these OSSNs were sectioned, stained with hematoxylin and eosin (H&E), and p16 expression by immunohistochemistry. H&E slides were reviewed to determine if OSSNs were noninvasive conjunctival intraepithelial neoplasms or invasive squamous cell carcinomas (SCC). Cases were classified as p16 positive or negative based on diffuse nuclear and cytoplasmic expression of p16 in neoplastic cells. Results: p16 positivity was found in a minority of OSSN cases (14/75). p16 positivity was significantly associated with the invasive SCC type of OSSN in HIV-infected patients (p value of 0.026). Conclusions: The majority of OSSNs in our HIV-infected cohort do not express p16. However, those cases that are p16-positive are significantly more likely to be the invasive SCC form of OSSN. We propose that p16 expression may identify more aggressive OSSNs in HIV-infected populations.

Objective Limited data on the prevalence of human papilloma virus (HPV)–positive oropharyngeal squamous cell carcinoma (OPSCC) in Southern Africa exist. The aim of the current study was to determine the prevalence of HPV-positive OPSCC at a major referral public hospital (Tygerberg Hospital) in the Western Cape region, South Africa. Methods Sequential surgical samples of 266 cases of OPSCC diagnosed over a 10-year period (2007-2017) were selected for evaluation and relevant patient characteristics were documented. p16 immunohistochemistry was performed as a screening test. All p16-positive cases were further evaluated for HR-HPV using a novel DNA polymerase chain reaction technology (BD Onclarity HPV assay). Results Of 266 cases, 14% were positive for p16 (n = 36). Of 36 p16-positive cases, 23 were negative and 13 were positive for HR-HPV (5%) when evaluated by PCR. p16 was found to have a positive predictive value of 36.1%. HPV subtypes were HPV-16 (n = 10), HPV-18 (n = 1), HPV-52 (n = 1), and HPV-31 (n = 1). One case was positive for HPV-16 and HPV-31. HPV-positive OPSCC occurred in 10 men and 3 women with a mean age of 51 years (range, 33-72 years). Most HPV-positive OPSCC were nonkeratinizing (n = 10) or partially keratinizing (n = 1). In contrast, HPV-negative OPSCC were predominantly keratinizing (n = 218). Conclusions The presence of HR-HPV in 5% of cases suggests that HR-HPV as a minor etiologic agent in OPSCC at this institution. Because of its suboptimal positive predictive value (36.1%), p16 immunohistochemistry alone is insufficient to confirm an HPV-positive OPSCC at this center and, when positive, HPV-specific testing must be performed by one of the available platforms.

Background This study examined the prognostic significance of human papillomavirus (HPV) in patients with oropharyngeal and oral cavity squamous cell carcinoma (SCC). Methods Tissue microarrays were constructed from oropharyngeal and oral cavity SCC (n = 143). The presence of functional HPV in tumour was determined by combined assessments of p16 immunohistochemistry and HPV in situ hybridisation. Results Oropharyngeal SCC patients presented with more advanced disease in comparison with oral cavity SCC patients (P = 0.001). HPV is present in 60% and 61% of oropharyngeal and oral cavity SCC patients, respectively. HPV-positive oropharyngeal SCC patients with advanced TNM stages displayed better overall and disease-free survival outcomes than HPV-negative patients (P = 0.022 and 0.046, respectively). Such survival differences were not observed in oral cavity SCC. Conclusions HPV is common in both oropharyngeal and oral cavity SCC and is associated with better survival outcome in oropharyngeal SCC but not in oral cavity SCC patients.

Human papillomavirus (HPV), especially HPV16 genotype, is associated with oropharyngeal squamous cell carcinoma (OPSCC). We aim to determine the prevalence and characterize the high-risk (HR)-HPV genotypes in head and neck SCC (HNSCC) in a South-East Asian multi-ethnic society in Singapore and examine its prognostic significance. 159 HNSCC archival tissue samples were retrieved and tumour DNA was screened for 18 HR-HPV genotypes using a PCR-based assay (Qiagen, digene HPV genotyping RH test). P16 protein overexpression was identified using immunohistochemistry (IHC). Statistical correlation between clinical outcomes were performed between HPV-positive and negative HNSCC patients. Six HR-HPVs (HPV16, 18, 31, 45, 56, 68) were detected in 90.6% of HNSCC; and 79.9% had multiple HPV genotypes detected. HPV31 and HPV45 were the most prevalent (79.2% and 87.4%, respectively); and HPV16 was predominantly found in OPSCC (p < 0.001). HPV-DNA PCR assay yielded a high sensitivity (96%) but low specificity (11%) when compared to p16 immunohistochemistry as the reference standard. P16-positive HNSCC was predominantly observed in OPSCC (73.7%; p = 0.005); and p16-positive OPSCC exhibited improved overall survival compared to p16-negative OPSCC (p = 0.022). Similarly, smoking and alcohol consumption were poor prognostic factors of overall survival (p = 0.007; p = 0.01) in OPSCC patients. HR-HPVs were identified in 90.6% of HNSCC patients using the HPV-DNA PCR assay. This test had a poor specificity when compared to p16 IHC; making it an unreliable detection technique in selecting patients for radiation dose de-escalation treatment protocol. P16-positive tumor was predominantly found in the oropharynx these patients demonstrated better overall survival than those with p16-negative OPSCC.

Objective The aim of this study was to determine if there is a different p16 expression pattern between colposcope-directed and random (colposcope-undetectable) biopsies of cervical intraepithelial neoplasia (CIN2) and CIN3. Methods Cervical biopsies that were positive for CIN2 or CIN3 were selected from a database of samples acquired during a large population-based clinical trial in Guangdong Province in China (Shenzhen Cervical Cancer Screening Study II). Blocks were recut, reread, and then immunostained for p16. Biopsies were categorized as either colposcope-directed or random biopsies. Diffuse staining was considered p16 positive, whereas focal or no staining was considered p16 negative. Differences were determined by the Fisher exact test. Results Among the patients with CIN3, there were 232 individual biopsies of CIN3. Sixty were randomly collected, and 172 were colposcopy directed. p16 positivity for the colposcope-directed and random biopsies was 97.7% and 91.7%, respectively (p = 0.052). Like the CIN3 biopsies, colposcope-directed and random CIN2 samples expressed p16 similarly (86.8% [46/53] and 82.6% [19/23], p = .73, respectively). Conclusions Based on our data, even small colposcope-undetectable biopsies of CIN3 are significant. Random biopsies of CIN2 or CIN3 demonstrate similar p16 positivity as visible lesions and therefore might be expected to have a similar natural history.

Background The Xpert® HPV Assay (Cepheid®, Sunnyvale, USA) is a rapid, cartridge-based HPV test validated for use on cervical cytology samples. However, there is an increasing demand for HPV annotation of formalin fixed paraffin embedded (FFPE) material. Objectives The aim of this study was to determine the suitability of the Xpert HPV assay for the detection of nucleic acid (NA) derived from FFPE samples. Study design A total of 88, 10 μm sections derived from FFPE tissue blocks were assessed, 74 originated from oropharyngeal squamous cell carcinomas (OPSCC) and 14 from a range of other sites. All had previously been tested with a sensitive Luminex® based assay with a component also tested with p16 immunohistochemistry (IHC). NA was extracted from samples using the easyMag® platform and after dilution was added directly to the Xpert cartridge. Agreement between assays was assessed. Results Overall agreement between the assays was 92%; with a Kappa for HR-HPV detection of 0.833 (95% CI 0.725–0.953). In the 50 samples that had been annotated for p16 status overall agreement between the Xpert assay and the p16 IHC was 90%. Conclusions These data indicate that FFPE material is amenable to HPV detection by the Xpert assay. To our knowledge, this is the first study to interrogate the use of the Xpert® HPV assay for this application.

CDKN2A (p16) expression is a well-established prognostic marker and its loss is a common molecular feature in tobacco/alcohol-associated head and neck squamous cell carcinoma (HNSCC). Over the past two decades evidence has also suggested that high-risk human papilloma virus (HR-HPV) infection, which results in overexpression of p16, is implicated in the development of some HNSCC. HNSCC is the sixth most common cancer worldwide and the seventh in Jamaica. Mortality rates for HNSCC in the Caribbean is among the highest in the world. This study will for the first time examine the molecular features of HNSCC as it relates to p16 expression and HPV in patients from Jamaica. There were no previous studies of this kind conducted in the English-speaking Caribbean. Methods: This retrospective cohort study was conducted using archival formalin-fixed paraffin-embedded tissue from patients diagnosed with HNSCC at the Department of Pathology, University of the West Indies, and the National Public Health Laboratory from 2001-2010. Clinical information was extracted for all patients. P16 immunohistochemistry was performed using P16INK4A clone E6H4 mouse anti-human protein and identification of HR-HPV DNA was done by standard PCR using MY09/MY11 consensus primers of the L1 HR-HPV gene. Results: The study population consisted of 326 HNSCC cases with ages ranging from 18 to 92 years (mean, 65 yrs. ± 15 SD). There were 254 males, 63 females and 9 of unknown sex with M: F ratio of 4:1. The majority of tumors were either moderately differentiated 147/326 (45%) or were poorly differentiated 55/326 (17%). All cases were tested for p16, and the majority 255/326 (78.22%) were negative and 71/326 (21.8%) were positive. All p16+ cases along with a subset of 60 randomly selected p16- cases were tested for HR-HPV by PCR. There were nine p16+ cases which were also positive for HR-HPV DNA. HPV 16 was detected in all cases, one case had HPV16 and 18, and another had HPV 16, 18, 39, 68 and 73. None of the sixty p16- cases were positive for HR-HPV. The nine patients that were HR-HPV+ and p16+ had a mean age of 61 yrs. ± 15 SD (range 39-83 years) with a M: F ratio of 8:1. All patients had moderately differentiated tumors, and the majority (78%) of these was from non-oropharyngeal sites with predominance in the larynx. Conclusion: In Jamaica, the prevalence of HPV-related HNSCC appears to be low with the majority of patients showing no expression of p16 and diagnosed in older individuals. Based on what is known in the literature, our study results suggest that in Jamaica most patients are diagnosed with alcohol and tobacco driven disease. The role of HPV in HNSCC warrants further investigation in this population. Citation Format: Sharon R Harrison, Carlos Tl Escoffery, Sheray Chin, Norma D McFarlane-Anderson, Marvin E Reid, Camille C Ragin. Characterization of head and neck squamous cell carcinoma from Jamaican patients using p16 immunohistochemistry and HPV polymerase chain reaction in archival formalin-fixed, paraffin-embedded tissues [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A071.

Objective: The cytomorphological changes associated with atrophic cellular pattern (ACP) in cervical cytology smears may mimic high-grade squamous intraepithelial lesion (HSIL). Due to this, there may be higher chances of cytomorphological overinterpretation in cases with ACP. Estrogen therapy (ET) (topical or systemic) would reverse the changes related to atrophy and repeat Pap smear after ET should correct the false positives. This approach would minimize the unindicated invasive interventions. However, performing immediate biopsies following “higher than low-grade squamous intraepithelial lesion (LSIL) (atypical squamous cells-cannot exclude HSIL, low-grade squamous intraepithelial lesions-cannot exclude HSIL, and HSIL) interpretations” in such cases, is a general trend. Pap smears with “higher than LSIL interpretations” in association with ACP over a period of 10 years were selected. Materials and Methods: A total of 657,871 cases over 10 years were reviewed, of which 188 Pap smears interpreted as higher than LSIL interpretations with ACP were selected randomly for this study. Result: Of these 188 cases, 67 underwent biopsies which were reviewed and compared with 67 biopsies performed for “higher than LSIL interpretation” cases without ACP. The follow-up biopsy material was reviewed including elective p16 immunohistochemistry with other clinical details including high-risk HPV test results as indicated. Conclusion: The findings demonstrated that Pap smears with ACP have higher false positives due to tendency for cytomorphologic overinterpretation as compared to non-ACP group.

Current human papillomavirus (HPV) detection methods in oropharyngeal squamous cell carcinoma (OPSCC) have varying sensitivity and specificity. We aimed to compare different HPV-detection methods against the test used in clinical practice, ie, p16 immunohistochemistry (IHC) and to evaluate whether another HPV-detection test additional to p16 IHC would be worthwhile in OPSCC specimens. The study cohort comprised 357 consecutive OPSCC patients during two time periods: 2000-2009 and 2012-2016. From tumor tissue slides, HPV mRNA via in situ hybridization (ISH), HPV DNA via ISH and HPV DNA via polymerase chain reaction (PCR) were detected. The results of these methods were compared with p16 IHC results. Additionally, clinicopathological factors were compared with the methods studied. The sensitivity of HPV mRNA ISH, HPV DNA ISH and HPV DNA PCR were 93.4%, 86.3%, and 83.5%, respectively. The corresponding specificity was 92.4%, 95.3%, and 89.1%, respectively. The negative predictive value for p16 IHC was highest (89.0%) when using mRNA ISH, and followed by DNA ISH (83.5%). ISH for high-risk HPV E6/E7 mRNA was found to be a highly specific and sensitive method for detecting HPV in OPSCC. As p16 protein may be overexpressed due to HPV-independent mechanisms, all p16 IHC positive OPSCCs should be considered for retesting using mRNA ISH in order to verify transcriptionally active HPV. This is especially critical when considering de-escalated treatment approaches for patients with HPV-positive tumors and still maintaining favorable outcomes for this subgroup of patients. (C) 2019 Elsevier Inc. All rights reserved.

The aim of this study was to identify the association between human papilloma virus (HPV) infection and ocular surface squamous neoplasia (OSSN) using p16 immunohistochemistry (IHC) and deoxyribonucleic acid (DNA) chip test. Thirty-eight patients who underwent surgical excision of OSSN were retrospectively studied using tissue samples. The IHC was performed to assess the expression of p16 and DNA chip test was used to detect 24 HPV serotypes. Among the 38 OSSN samples, 32 cases (84.2%) were histopathologically categorized as pre-invasive type and 6 cases (15.8%) as invasive type. The IHC for p16 showed strong positivity in 12 cases (31.6%), whereas it was negative in 26 cases (68.4%). On the other hand, only one case (2.6%) of invasive OSSN was positive for the HPV16 serotype, as assessed by DNA chip test. In OSSN, p16 expression was positive in approximately 1/3rd of the cases, whereas the majority of the 24 HPV serotypes were negative for p16. Our findings suggest that only a weak association exists between HPV infection and OSSN.

Context.— 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. Objective.— 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. Design.— 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. Results.— 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. Conclusions.— 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.

HPV testing is a better alternative for cervical cancer screening, but additional procedures are required for triage of HPV positive women. HPV encoded oncoproteins E6 and E7, as the main effectors of HPV carcinogenicity represent promising triage alternatives. To evaluate performance of the test, we included 155 women from a screening study and 59 from the same referral population attending colposcopy and with precancerous lesions. All were HPV-tested with HC2 and genotyped with LiPA, and cervical swabs were tested for HPV16/18 E6 oncoproteins. Histologic specimens were reviewed and adjudicated using p16 immunohistochemistry and 55 women had confirmed histologic HSIL, 31 (56.3%) associated with HPV 16/18, 23 with other HPV types and one HPV negative. Sensitivity and specificity were estimated with histologic HSIL/cancer as gold standard. E6 oncoprotein was detectable in all but one HSIL and in all cancers where HPV16/18 DNA was detected, but in none of the cases associated with other HPV types or HPV negatives. Among the few HPV16/18 DNA positive subjects initially without HSIL (n = 4) who were E6 oncoprotein positive, precancer was detected during follow-up in 2 out of 3 with available information. Estimated sensitivity for HPV16/18-related HSIL+ was 96.8% (95%CI = 83.8-99.8) and for all HSIL+ regardless of HPV type it was 56.4% (95%CI = 43.3-68.6). Specificity was 97.5% (95%CI = 93.7-99.0). E6 oncoprotein proved as a highly sensitive and specific marker for detection of HPV16/18-related HSIL lesions in this Honduran population with limited previous screening and may be useful as a triage method in screening programs, particularly in low income countries.

Background At times, distinguishing Bowen disease (BD) and benign seborrheic keratosis (SK) is histologically challenging, especially when SK shows clonal features (clonal seborrheic keratosis [CSK]). While p16 is often reported as positive in BD and negative in SK, p16 expression in CSK is rarely studied. Here we investigate p16 immunohistochemistry in CSK, SK, and BD. Methods p16 immunohistochemistry with pattern of expression was noted for 14 CSK, 12 SK, and 18 BD. The degree of inflammation among lesions with respect to p16 expression was also noted. Results When examining p16 staining in clonal nests of CSK, 57% showed diffuse or patchy/diffuse positivity, 21% showed patchy positivity, and 21% showed clusters of single positive cells. 67% of BD showed diffuse positivity, 11% showed patchy/diffuse positivity, 17% showed patchy positivity, and 6% were negative. 25% of SK showed focal areas of patchy to full thickness positivity, 25% showed moderate number of positive single cells with or without patchy staining, and 50% showed negative/scattered single cell positivity. Conclusion Our findings support that p16 positivity limited to clonal nests in CSK is normal. p16 positivity in clonal nests of CSK in isolation without concurrent atypical histologic features should not be used to support a diagnosis of BD.

OBJECTIVE The main objective of this study was to analyze the presence of human papillomavirus (HPV) by polymerase chain reaction (PCR) in invasive squamous cell carcinomas of the oral cavity. MATERIALS AND METHODS A selection was made of 155 cases of squamous cell carcinoma of the oral cavity treated at the University Hospital of Geneva. HPV detection was performed at the Laboratory of Molecular Pathology using a PCR technique followed by in situ hybridization of the viral DNA. Sections were studied for the immunohistochemical expression of P16INK4a . RESULTS The presence of HPV-DNA was found in 3.3% of the cases (95% CI: 1.3%-7.5%). The only HPV genotype found was HR-HPV 16. In contrast, 7.7% (95% CI: 1.3%-7.5%) of the cases showed an overexpression of the P16INK4a . We found no significant differences in age, sex, or tobacco and alcohol consumption in relation to the P16INK4a or HPV positivity. CONCLUSIONS Human papillomavirus is rarely associated with squamous cell carcinomas of the oral cavity. Our results suggest that an overexpression of the P16INK4a protein, even in the absence of the virus, is linked to an increase in the survival of the patient.

Context.—Making an accurate diagnosis for melanocytic lesions has always been challenging for pathologists, especially when dealing with difficult-to-diagnose cases. Misdiagnosis of melanoma and melanocytic lesions in general has tremendous medical-legal implications, often leading to unnecessary and excessive use of adjunctive tests. Although molecular testing is of much interest and there is great support for its development, currently, for most melanocytic lesions, immunohistochemical studies remain the most practical method for assistance in the routine diagnosis of melanocytic lesions for the average pathologist.Objectives.—To review the practical use of p16 immunohistochemistry for evaluating melanocytic lesions, particularly for differentiating benign from malignant tumors, and to perform a meta-analysis of primary studies evaluating p16 immunohistochemistry in melanocytic lesions.Data Sources.—A PubMed database search for literature reporting melanocytic lesions and p16 immunohistochemistry was performed. Essential information from each study (number of samples, antibody used, collection dates, overall p16 immunohistochemistry results, and general method of interpretation) was tabulated and analyzed. Examples of representative cases showing p16 immunostaining pattern are also illustrated.Conclusions.—Incorporation of p16 immunohistochemistry for the diagnosis of melanocytic lesions is of limited use, especially for the purpose of differentiating benign from malignant lesions. Evaluation of multiple studies reveals a wide range of results. However, there appears to be some value for the use of p16 in distinguishing nodal nevi from metastatic melanoma within nodes. The method of interpretation (nuclear versus cytoplasmic staining) also appears to give differing results, as studies considering only nuclear staining appeared to show more consistent results from study to study.

Background Nasopharyngeal carcinoma (NPC) is related to Epstein‐Barr virus (EBV) in endemic areas; however, the role of viruses in nonendemic countries is unclear. Our nationwide study investigated the prevalence and prognostic significance of EBV and human papillomaviruses (HPVs) in Finnish NPC tumors. Methods We analyzed samples from 150 patients diagnosed between 1990 and 2009. Viral status was determined using EBV and HPV RNA in situ hybridizations, and p16 immunohistochemistry. Patient and treatment characteristics were obtained from patient records. Results In our white patient cohort, 93 of 150 (62%) patients were EBV‐positive and 21/150 (14%) patients were HPV‐positive with no coinfections. Thirty‐six (24%) tumors were negative for both viruses. The 5‐year disease‐specific survival for patients with EBV‐positive, HPV‐positive, and EBV/HPV‐negative tumors was 69%, 63%, and 39%, respectively. In multivariable‐adjusted analysis, overall survival was better among patients with EBV‐positive (P = .005) and HPV‐positive (P = .03) tumors compared to patients with EBV/HPV‐negative tumors. Conclusions In our low‐incidence population, EBV and HPV are important prognostic factors for NPC.

Context.—Testing for high-risk human papillomavirus (HR-HPV) in head and neck squamous cell carcinomas (HNSCCs) is important for both prognostication and clinical management. Several testing platforms are available for HR-HPV; however, effective alternative automated approaches are needed. Objective.—To assess the performance of the automated Roche cobas 4800 HPV real-time polymerase chain reaction-based system on formalin-fixed, paraffin-embedded HNSCC specimens and compare results with standard methods of in situ hybridization (ISH) and p16 immunohistochemistry. Design.—Formalin-fixed, paraffin-embedded samples of HNSCC were collected from archival specimens in the Department of Pathology, Massachusetts General Hospital (Boston), and prepared using the automated system by deparaffinization and dehydration followed by tissue lysis. Samples were integrated into routine cervical cytology testing runs by cobas. Corresponding formalin-fixed, paraffin-embedded samples were evaluated for HR-HPV by ISH and p16 by immunohistochemistry. Discrepant cases were adjudicated by polymerase chain reaction. Results.—Sixty-two HNSCC samples were analyzed using the automated cobas system, ISH, and immunohistochemistry. Fifty-two percent (n = 32 of 62) of formalin-fixed, paraffin-embedded tumors were positive for HR-HPV by cobas. Eighty-eight percent (n = 28 of 32) of cases were the HPV 16 subtype and 12% (n = 4 of 32) were other HR-HPV subtypes. Corresponding testing with ISH was concordant in 92% (n = 57 of 62) of cases. Compared with the adjudication polymerase chain reaction standard, there were 3 false-positive cases by cobas. Conclusions.—Concordance in HNSCC HR-HPV status between cobas and ISH was more than 90%. The cobas demonstrated a sensitivity of 100% and a specificity of 91% for detection of HR-HPV. Advantages favoring cobas include its automation, cost efficiency, objective results, and ease of performance.

Purpose: Identifying intraepithelial sebaceous carcinoma cells in small periocular biopsies can be difficult, particularly in the conjunctiva. The goal of this study was to evaluate p53 and p16 immunohistochemistry as potential markers of intraepithelial sebaceous carcinoma. Procedures: A total of 25 tumors, including 4 recurrent lesions, were stained for p16 and p53, with intensity scored as negative, weak, moderate or strong. Results: Expression of p16 was detected in intraepithelial sebaceous carcinoma cells in 24 of the 25 cases (96%), with only 1 case showing weak immunoreactivity. Intraepithelial p53 immunoreactivity was present in 17 of 25 tumors (68%), but was weak in 3 cases. Expression levels remained relatively stable in primary and recurrent tumors, but varied in a few cases between intraepithelial and subepithelial sites. Conclusions: Intraepithelial sebaceous carcinomas stained for p53 and p16 demonstrated moderate to strong immunoreactivity in 100% of cases for at least one of these proteins, suggesting that together they are useful markers for determining the extent of tumor spread. Of the two, p16 was immunoreactive in more cases than p53.

Background This study provides Australian data on the characteristics of human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) over the last 2 decades. Methods The HPV status of 515 patients with oropharyngeal SCC diagnosed between 1987 and 2010 was determined by HPV E6-targeted multiplex real time polymerase chain reaction assay (PCR) and p16 immunohistochemistry. Results The HPV positivity rate increased from 20.2% (1987-1995) to 63.5% (2006-2010). Among HPV-positive oropharyngeal SCC over the study period, the median age increased from 55.4 years to 59.8 years (p = .004) and there was a trend of an increasing proportion of never smokers (19.2% to 34.0%). The use of radiation therapy (RT) in patients with HPV-positive oropharyngeal cancer increased from 26.9% to 68.1% (p = .007) and we also observed a trend of improved outcomes. Conclusion Our data show a rising prevalence of HPV-positive oropharyngeal SCC in Australia over the last 2 decades. These patients with HPV-positive oropharyngeal SCC are now presenting at an older age and about one third have never smoked.

Introduction: Diagnostic reproducibility and determination of prognostic factors in cervical intraepithelial neoplasias grades 1 and 2 are still relevant problems in the daily practice of gynecological histopathology. Objective: To correlate the value of morphological reclassification and of p16 immunoexpression in cervical intraepithelial neoplasias grades 1 and 2 with clinical outcome. Materials and methods: Sixty-six patients were included (34 with cervical intraepithelial neoplasia grade 1, and 32 with grade 2); an immunohistochemical study with p16 and reclassification according to the Lower Anogenital Squamous Terminology (LAST) Consensus and by the alternative proposal of Herfs and Crum were done; unfavorable outcome was defined as a subsequent histologic diagnosis of cervical intraepithelial neoplasia grade 3 or invasive squamous cell carcinoma. Results: We observed superior performance of the alternative morphological classification (p = 0.002) to determine unfavorable outcome. We also detected superior performance of p16 in the same determination (p = 0.002). Conclusion: The use of an alternative morphological classification is promising; in the context of the use of immunohistochemical antibodies as biomarkers, p16 showed good sensitivity and negative predictive value in the determination of cases in which the outcome was unfavorable. RESUMO Introdução: A reproducibilidade diagnóstica e a determinação de fatores prognósticos em neoplasias intraepiteliais cervicais graus 1 e 2 ainda são problemas relevantes na prática diária da histopatologia ginecológica. Objetivo: Correlacionar o valor da reclassificação morfológica e da imunoexpressão do marcador p16 em neoplasias intraepiteliais cervicais graus 1 e 2 com o desfecho clínico. Materiais e métodos: Incluídas 66 pacientes (34 com neoplasia intraepitelial cervical grau 1 e 32 com grau 2); realizou-se estudo imuno-histoquímico com p16 e reclassificação segundo o Consenso Lower Anogenital Squamous Terminology (LAST) e a proposta alternativa de Herfs e Crum; desfecho desfavorável foi definido como diagnóstico histológico subsequente de neoplasia intraepitelial cervical grau 3 ou carcinoma de células escamosas invasivo. Resultados: Observamos performance superior da classificação morfológica alternativa (p = 0,002) para determinação de desfecho desfavorável. Também detectamos performance superior do marcador p16 na mesma determinação (p = 0,002). Conclusão: A utilização de uma classificação morfológica alternativa é promissora. No âmbito da utilização dos anticorpos imuno-histoquímicos como biomarcadores, o p16 apresentou boa sensibilidade e valor preditivo negativo na determinação dos casos em que o desfecho foi desfavorável.

p16 immunohistochemistry is recommended by the CAP-ASCCP Lower Anogenital Squamous Terminology (LAST) Standardization Project for human papillomavirus associated Lesions as an adjunct to morphologic assessment in the diagnosis of high-grade squamous intraepithelial lesion. This study evaluates the performance of different p16 clones as compared with E6H4 (CINtec) in detecting high-grade squamous intraepithelial lesion. The 54 high-quality articles addressing the performance of p16 identified by work group 4 of the LAST Project were evaluated for: specific p16 clone, scoring method, number of cases, anatomic site, and histologic diagnoses. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for each clone. Two-proportion z tests (pooled) were used to evaluate significance. In total, 32 of the 54 studies met the inclusion criteria. The most commonly used clone was E6H4 (17 studies, 3507 cases) with smaller numbers (1-4) of studies evaluating the following: 16P04, JC8, 16P07, G175-405, K5334, K5336, and 7962. p16 clones 16P04 and JC8 performed better than E6H4 with 16P04 exhibiting statistically significant higher sensitivity (94% vs. 87% for E6H4), specificity (94% vs. 81%), and positive predictive value (96% vs. 69%) while JC8 exhibited higher specificity (91% vs. 81%) and positive predictive value (88% vs. 69%). 16P07 performed similarly to E6H4 and the other 4 clones did not perform as well as E6H4. p16 clones 16P04, JC8, and 16P07 clones perform as well or better than the widely used p16 clone E6H4 (CINtec). However, further studies are indicated to determine the reproducibility of these findings and the impact of interlaboratory variation on test performance.

Context: Oral and pharyngeal cancer, grouped together, is the sixth most common cancer in the world. In the past few years, human papillomavirus (HPV) infection has been suggested as a risk factor for oral cancer apart from traditional risk factors such as smoking, tobacco, and alcohol consumption. Aims: The aim of this study was to determine HPV status of the tumors using polymerase chain reaction (HPV-DNA PCR) and p16 immunostaining and to correlate p16 overexpression as an indicator of HPV-associated oral dysplasia and carcinoma. Settings and Design: A prospective study was conducted in fifty cases of suspected oral cancer. Materials and Methods: PCR Amplification of extracted HPV-DNA was done for HPV-DNA status in fresh tissue of suspected oral cancer cases. Histomorphological features of the cases were analyzed, and p16 immunohistochemistry was performed on the same specimen after making paraffin blocks to study p16 overexpression. Statistical Analysis Used: Chi-square test was used to analyze the differences between discrete variables. Results: 5/6 (83.3%) HPV-DNA-positive cases were positive for p16 expression, whereas 26/44 (59.09%) p16-positive cases which were negative for HPV-DNA. Sensitivity and specificity of p16 as a surrogate marker for HPV-DNA were found to be 83.3% and 40%, respectively. Conclusions: p16 immunostaining is a good first-line assay for eliminating HPV-negative cases from additional analysis, but other causes of p16 overexpression in oral tumorigenesis related to tobacco consumption in keratinizing squamous cell carcinoma needs to be explored further.

Oropharyngeal squamous cell carcinomas (OPSCCs) are traditionally caused by smoking and excessive alcohol consumption. However, in the last decades high-risk human papillomavirus (HPV) infections play an increasingly important role in tumorigenesis. HPV-driven OPSCCs are known to have a more favorable prognosis, which has led to important and marked changes in the recently released TNM-8. In this 8th edition, OPSCCs are divided based on p16 immunostaining, with p16 overexpression as surrogate marker for the presence of HPV. The aims of this study are to evaluate TNM-8 on a Dutch consecutive cohort of patients with p16-positive OPSCC and to determine the relevance of additional HPV DNA testing.All OPSCC patients without distant metastases at diagnosis and treated with curative intent at VU University Medical Center (2000-2015) and Erasmus Medical Center (2000-2006) were included (N = 1204). HPV status was determined by p16 immunostaining followed by HPV DNA PCR on the p16-immunopositive cases. We compared TNM-7 and TNM-8 using the Harrell's C index.In total, 388 of 1204 (32.2%) patients were p16-immunopositive. In these patients, TNM-8 had a markedly better predictive prognostic power than TNM-7 (Harrell's C index 0.63 versus 0.53). Of the 388 p16-positive OPSCCs, 48 tumors (12.4%) were HPV DNA-negative. This subgroup had distinct demographic, clinical and morphologic characteristics and showed a significantly worse five-year overall survival compared with the HPV DNA-positive tumors (P 0.001).TNM-8 has a better predictive prognostic power than TNM-7 in patients with p16-positive OPSCC. However, within p16-positive OPSCCs, there is an HPV DNA-negative subgroup with distinct features and a worse overall survival, indicating the importance to perform additional HPV DNA testing when predicting prognosis and particularly for selecting patients for de-intensified treatment regimens.

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

Oral squamous cell carcinomas (OSCCs) are common neoplasms of dogs and are of unknown cause. Whereas papillomaviruses (PVs) are an established cause of human OSCCs, few studies have investigated canine OSCCs for a PV aetiology. In humans, a PV aetiology can be determined by detecting PV DNA and PV-induced increased p16(CDKN2A) protein (p16) within the OSCC. In this study, PCR, using four different primer sets and p16 immunohistochemistry, was used to evaluate 28 canine OSCCs for a possible PV aetiology. None of the primers amplified PV DNA from any of the OSCCs although four neoplasms contained intense p16 immunostaining. Intense p16 immunostaining would indicate a PV aetiology in a human OSCC but the absence of PV DNA suggests that the increase in p16 was not due to PV infection. Overall the results indicated that PVs are not a significant cause of canine OSCCs.

High-risk human papillomavirus (HPV) is both causative and prognostic in the majority of oropharyngeal squamous cell carcinomas (OPSCCs). The aim of this study was to evaluate for transcriptionally-active HPV in matched primary OPSCCs and their distant metastases given the implications of HPV status for diagnosis and treatment. Twenty matched pairs of primary OPSCC and their distant metastases were retrieved from departmental files. Two study pathologists reviewed all cases to confirm the diagnoses and to evaluate histologic features. Real-time PCR (RT-PCR) for detection of E6/E7 mRNA for all major high-risk HPV types and p16 immunohistochemistry were performed. Distant metastases were to lung (70 %), bone (20 %), non-regional lymph nodes (5 %) and pericardium (5 %). Histologically, 15 primary tumors were nonkeratinizing, 3 nonkeratinizing with maturation, one basaloid, and one keratinizing. Seventeen (85 %) of the metastases had the same histologic type as the primary tumor. All 20 matched pairs were concordant for HPV status by RT-PCR and for p16 expression with 19 of 20 cases positive for high risk HPV and one negative. HPV types were concordant in all cases. These findings show that the distant metastases from HPV-related primary OPSCCs uniformly retain transcriptionally-active HPV and p16 overexpression. They also retain similar morphology. This argues that HPV status can be utilized to differentiate metastatic OPSCC from separate, new, primary squamous cell carcinomas in other organs, and that therapies specifically targeting HPV or virus-related proteins in patients with distant metastases can be utilized.