Cytoplasmic Expression of p16 Is Associated with Carcinoma Breast: It Is Not an Artifact.

Background & Objective: p16 has different roles in the nuclear and cytoplasmic locations. The nuclear localization of the p16 protein explains its role in cell cycle regulation. Cytoplasmic expression was considered an artifact in the initial years, but there is evidence to prove that cytoplasmic localization is real and that p16 has different roles in the nuclear and cytoplasmic locations. We aimed to study the immunoexpression of p16 protein in the nuclear and cytoplasmic locations of the epithelial and stromal compartments of fibroadenoma, invasive breast carcinoma, and a select number of phyllodes tumors. Methods: The study included a total of 107 patients, comprising 51 cases of invasive breast carcinoma, 51 cases of fibroadenoma, 4 cases of benign phyllodes tumors, and 1 case of lobular carcinoma in situ (LCIS). The p16 immunohistochemistry was evaluated for nuclear and cytoplasmic localization in the epithelial and stromal compartments of the tumors. Results: Of the 51 fibroadenoma cases, 23 showed strong nuclear p16 epithelial expression, but no case showed cytoplasmic expression. In 19/51 cases, stromal cells also showed strong p16 nuclear expression. Moderate stromal p16 expression was observed in 3 out of 4 cases of benign phyllodes. Out of the 51 cases of invasive carcinoma, 31 showed moderate to strong nuclear p16 immunopositivity, while 27 cases exhibited cytoplasmic p16 expression. We found a statistically significant correlation between moderate to strong nuclear p16 immunoexpression and the molecular subtype of breast carcinoma. Conclusion: The cytoplasmic localization of p16 immunohistochemistry is not seen in epithelial components of fibroadenoma, while it is seen frequently in breast carcinoma. Nuclear p16 expression has a statistically significant correlation with molecular subtypes of breast carcinoma.

Evaluation of high-risk human papillomavirus in sinonasal papillomas and squamous cell carcinomas.

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.

Penile intraepithelial neoplasia: Distribution of subtypes, HPV genotypes and p16INK4a in 84 international cases.

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.

Human Papillomavirus-Associated Oral Cavity Squamous Cell Carcinoma: An Entity with Distinct Morphologic and Clinical Features.

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.

The utility of high-risk human papillomavirus in situ hybridization in cytology cell block material from cystic head and neck lesions.

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.

Prognostic Value of HPV Infection Assessed by p16 Immunohistochemistry and the Influence of Tobacco Usage in Oropharyngeal Cancers: Real World Scenario.

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.

2020 WHO Classification of Female Genital Tumors.

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 unveiled reality of human papillomavirus as risk factor for oral cavity squamous cell carcinoma.

The prognostic impact of human papillomavirus (HPV) in oropharyngeal cancer is generally acknowledged, and HPV-status is assessed routinely in clinical practice. Paradoxically, while the oral cavity seems the predilection site for productive HPV-infections, figures on HPV-attribution in oral cavity squamous cell carcinoma (OCSCC) differ widely, and prognostic impact is uncertain. Major obstacles are the lack of reproducible assays to detect HPV in nonoropharyngeal cancers, the relatively small cohorts studied and consequently the shortfall of convincing data. In our study, we used a validated, nucleic acid-based workflow to assess HPV-prevalence in a consecutive cohort of 1016 OCSCCs, and investigated its prognostic impact. In parallel, we analyzed p16-immunohistochemistry (p16-IHC) as surrogate marker for transforming HPV-infection and independent prognosticator. All OCSCC-patients diagnosed between 2008 and 2014 at two Dutch university medical centers were included (N = 1069). Formalin-fixed, paraffin-embedded (FFPE)-samples of 1016 OCSCCs could be retrieved. Punch biopsies were taken from the tumor area in the FFPE-blocks and tested for HPV. P16-IHC was performed on 580 OCSCCs, including all HPV-positive tumors. From 940 samples (92.5%), nucleic acids were of sufficient quality for HPV-testing. In total, 21 (2.2%) OCSCCs were HPV DNA-positive. All HPV DNA-positive tumors were E6 mRNA-positive and considered as true HPV-positive. There was no difference in survival between HPV-positive and HPV-negative OCSCCs. In total, 46 of 580 (7.9%) OCSCCs were p16-immunopositive, including all HPV-positive tumors. Survival was comparable in p16-positive and p16-negative OCSCCs. To conclude, HPV-prevalence is very low in OCSCC and neither HPV-status nor p16-status affects outcome. Based on these data, determining HPV-status in OCSCC seems irrelevant for clinical management.

Classifying Anal Intraepithelial Neoplasia 2 Based on LAST Recommendations.

OBJECTIVES: 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. METHODS: Agreement based on photomicrographs of H&E alone or H&E plus p16 immunohistochemistry was calculated using κ coefficients. RESULTS: 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. CONCLUSIONS: Although p16 increased interobserver agreement, disagreement remained considerable regarding intermediate lesions. p16 expression, particularly if negative, can reduce unwarranted HSIL diagnoses and unnecessary treatment.

Validation of local p16 testing for determination of human papilloma virus status eligibility on a low risk oropharyngeal cancer trial - A Trans-Tasman Radiation Oncology Group study.

OBJECTIVE: 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. METHODS AND MATERIALS: 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. RESULTS: 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. CONCLUSIONS: 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.

Are Histomorphological Features Predictive of p16 Immunopositivity Different for Oral and Oropharyngeal Squamous Cell Carcinoma?

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.

Metastatic HPV-Associated Oropharyngeal Versus Primary Pulmonary Squamous Cell Carcinoma: is p16 Immunostain Useful?

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.

p16 Expression Correlates with Invasive Ocular Surface Squamous Neoplasms in HIV-Infected Mozambicans.

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.

P16 and human papillomavirus in sinonasal squamous cell carcinoma.

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.

Inclusion of an E7 DNA Amplification Test Improves the Robustness of Human Papillomavirus-Associated Oropharyngeal Squamous Cell Carcinoma Diagnosis.

BACKGROUND: The rise in human papillomavirus (HPV) infection rates over the last few decades in the USA has contributed to a significant increase in the overall incidence of patients diagnosed with squamous cell carcinoma of the head and neck. These head and neck carcinomas develop in the oropharynx, with more than 90% of them caused by infection with high-risk HPV type 16. Patients diagnosed with HPV-induced oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis and treatment response than those diagnosed with head and neck cancers caused by alcohol consumption and tobacco use. To identify patients with HPV-positive OPSCC, new guidelines recommend positive staining of oropharyngeal tissues for p16 INK4a (p16) by immunohistochemistry (IHC). Herein we discuss the testing algorithm that was adopted to address discrepant results between p16 IHC and a DNA in situ hybridization (ISH) test used routinely to diagnose HPV-positive OPSCC patients. METHODS: A DNA polymerase chain reaction (PCR) test that amplifies HPV16 and HPV18 E7 was developed to aid in the diagnosis of HPV-positive OPSCC in a subset of patients. Specimens from these patients stained positive for p16 by an IHC test, but negative for high-risk HPV by a commercial DNA ISH test. Moreover, these results did not match the histopathological characteristics of the specimens, nor the clinical presentations of the patients. RESULTS: Of 21 patients' specimens that were tested for p16 by IHC, 11 specimens showed concordant results with the high-risk HPV 16/18 DNA ISH test. Whereas, in eight p16 IHC positive specimens, HPV viral DNA was not detected by HPV16/18 DNA ISH, and two specimens were not tested by DNA ISH. When these eight p16 IHC positive specimens with discrepant p16 IHC and DNA ISH results were further tested by DNA PCR, six specimens showed concordance with p16 IHC with positive results for HPV16 E7, while two specimens were negative for HPV16 E7 by DNA PCR. All tested specimens were negative for HPV18 E7 by DNA PCR. Thus, the addition of the HPV16 and HPV18 E7 DNA PCR test identified a significant number of false negative test results by the HPV16/18 DNA ISH test and likely several false positive results by p16 IHC. CONCLUSIONS: Inclusion of an HPV16 E7 DNA PCR test improved the robustness of HPV-associated OPSCC diagnosis in patients with discrepant results from p16 IHC staining and a DNA ISH test, and identified patients for proper management with less misclassification.

Current Status of p16 Immunohistochemistry and HPV Testing in Fine Needle Aspiration Specimens of the Head and Neck.

Human papilloma virus (HPV)-related squamous cell carcinoma (SCC) is biologically unique and has a better prognosis than conventional SCC of the head and neck. p16 immunohistochemistry emerged as a valuable surrogate marker for HPV in oropharyngeal SCC. The criteria for a positive p16 result in tissue specimens are well established. However, there is no consensus regarding interpreting p16 staining in cell blocks and other cytology specimens. This review discusses the current evidence on p16 testing in cytology specimens and also highlights other methods for HPV testing, including DNA and RNA in situ hybridization, as well as other molecular HPV tests.

Tissue Fixation Conditions for p16 Immunohistochemistry and Human Papillomavirus RNA In Situ Hybridization in Oropharyngeal Squamous Cell Carcinoma.

Human papillomavirus (HPV) has become a critical prognostic biomarker in oropharyngeal squamous cell carcinoma (OPSCC). While retrospective studies suggest that p16 immunohistochemistry and even HPV RNA in situ hybridization work well on tissues and tumors from a variety of labs and various fixation conditions, no formal study of fixation conditions has been performed to date. We took surgically resected specimens from three p16 and HPV RNA in situ hybridization positive OPSCC patients, divided their fresh tumors into small pieces, and varied the time to formalin fixation as 1, 3, 6, 24, and 48 h. Tumors were either held moistened at room temperature or were refrigerated. After fixation and processing, routine hematoxylin and eosin slides were generated and p16 immunohistochemistry and RNA in situ hybridization performed. All three tumors were nonkeratinizing and had strong and diffuse p16 expression at immediate fixation, which, surprisingly, remained positive for all fixation times and conditions and despite significant degeneration at the later points for two of the patients while for one, the nuclear signal dropped out of most cells at early and mid time points, particularly at room temperature, causing false negatives. HPV RNA in situ hybridization stayed positive in all specimens up to 48 h of cold ischemic time refrigerated and even at room temperature, except for overtly autolyzed tumor regions. These findings help to establish that, at least for standard nonkeratinizing, p16 and HPV RNA strongly positive OPSCC patients, and using the most common tests in clinical practice, relatively lenient time to fixation may be acceptable if it cannot be avoided. However, for some patients, p16 immunohistochemistry may be sensitive to signal loss with autolysis. HPV RNA in situ hybridization, in particular, seems remarkably resistant to pretest cold ischemic times.

Relationship between p16 expression and prognosis in different anatomic subsites of OSCC.

BACKGROUND: p16 has often been found to be overexpressed in patients oral squamous cell carcinoma (OSCC), but its prognostic value between anatomic subsites is still unclear. OBJECTIVE: The aim of this study was to investigate the diagnostic and prognostic values of p16 in OSCC originating from tongue, gingiva or buccal mucosa. METHODS: A total of 147 OSCC patients with tumors arising from the tongue, gingiva or buccal mucosa were enrolled in this study. p16 expression was detected using immunohistochemistry (IHC), and the presence of HPV16 was determined by real-time PCR in p16 positive patients. The correlation of p16 expression with the clinical parameters was evaluated. RESULTS: Only one p16 positive patient with a cut off value of 25% and 75% was HPV16 positive. Although overall survival (OS), recurrence free survival (RFS) and metastasis free survival (MFS) had no significant differences between the p16 positive and negative patients, p16 negative patients (cut off value 25%) had more RFS in the buccal mucosa cancer (p= 0.03) than the p16-positive patients. CONCLUSIONS: The prevalence of HPV16 in Chinese OSCC patients was low. p16 overexpression decoupled from HPV infection was not a prognostic marker for OSCC patients except for patients with the buccal mucosa cancer.

Low prevalence of HPV-induced oral squamous cell carcinoma in Geneva, Switzerland.

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.

Smoking and papillomavirus DNA in patients with p16-positive N3 oropharyngeal squamous cell carcinoma.

BACKGROUND: We investigated the survival of patients with a p16-positive N3 oropharyngeal squamous cell carcinoma (OPSCC) and the prognostic significance of patient, tumor, and treatment characteristics. METHODS: We retrospectively reviewed the data of patients treated at our Cancer Center for a p16-positive N3 OPSCC between 2003 and 2016. End points were overall survival (OS) and progression-free survival (PFS). RESULTS: A total of 29 patients were included. The 5-year OS and PFS were 67.5% and 59.1%, respectively. Smoking history above 10 pack-years and the absence of human papillomavirus DNA were associated with worse OS (P = .02 and P = .03, respectively) and PFS (P = .02 and P = .02, respectively). Induction chemotherapy or radical neck dissection were not associated with different treatment outcomes. CONCLUSION: Patients with an N3 p16-positive oropharyngeal cancer in our series had a 5-year OS rate of 67.5%. Smoking history and viral DNA were prognostic factors associated with survival.