Probes for P16

Oropharyngeal squamous cell carcinoma is frequently associated with high-risk HPV infection, which confers a good prognosis. Immunohistochemistry for p16 is used as a surrogate for HPV status, but discrepant results are occasionally seen. Here, we report a case with a unique pattern of partial loss of p16.A 63 year old male presented with a base of tongue nonkeratinizing squamous cell carcinoma and a large metastatic neck mass. The primary lesion and multiple regions of the metastatic mass were assessed with p16 immunohistochemistry, RNA in situ hybridization for high-risk HPV, and HPV16 genome sequencing.The primary lesion was p16 negative, and the metastatic neck mass had large, confluent regions that were either strongly p16 positive or entirely p16 negative. All of these regions were positive for high-risk HPV with identical HPV16 genomes.This unusual case illustrates a potential diagnostic pitfall, and it raises important questions regarding molecular mechanisms and prognostic implications of p16 staining in oropharyngeal squamous cell carcinoma.

Sinonasal squamous cell carcinoma (SNSCC) is a rare and aggressive malignancy with poor prognosis. Human papilloma virus (HPV) can induce SNSCC although its incidence and impact on patients' outcomes remains unclear. We performed a retrospective cohort study of patients with SNSCC treated consecutively in a comprehensive cancer center. HPV status was determined with p16 immunohistochemistry followed by RNA in situ hybridization (RNAscope). The incidence, clinical characteristics, and oncologic outcomes of HPV+SNSCC were assessed. P16 prognostic value was evaluated. Fifty-nine patients were included. Eleven (18.6%) SNSCC were p16+ with five (8.4%) doubtful cases. RNAscope was positive in nine cases (15.2%). Patients with HPV+SNSCC were younger (p = 0.0298) with a primary tumor originating mainly in nasal fossa (p < 10-4). Pathologic findings were not different according to HPV status. Among patients who were curatively treated, overall survival was better for HPV+SNSCC (p = 0.022). No prognostic value of p16 expression was reported. Patients with HPV+SNSCC have better oncologic outcomes, probably due to earlier tumor stage with primary location predominantly in the nasal fossa, a more suitable epicenter to perform a surgical resection with clear margins. P16 expression seems not to be a good surrogate of HPV status in SNSCC.

Detecting human papillomavirus (HPV) infection in head and neck squamous cell carcinoma (HNSCC) is clinically relevant, but there is no agreement about the most appropriate methodology. We have studied 64 oropharyngeal carcinomas using p16 immunohistochemistry, HPV DNA in situ hybridisation (ISH) and HPV DNA polymerase chain reaction (PCR) followed by pyrosequencing. We have also evaluated a new assay, RNAscope, designed to detect HPV E6/E7 RNA transcripts. Using a threshold of 70 % labelled tumour cells, 21 cases (32.8 %) were p16 positive. Of these, 19 cases scored positive with at least one HPV detection assay. Sixteen cases were positive by HPV DNA-ISH, and 18 cases were positive using the E6/E7 RNAscope assay. By PCR and pyrosequencing, HPV16 was detected in 15 cases, while one case each harboured HPV33, 35 and 56. All p16-negative cases were negative using these assays. We conclude that p16 expression is a useful surrogate marker for HPV infection in HNSCC with a high negative predictive value and that p16-positive cases should be further evaluated for HPV infection, preferably by PCR followed by type determination. Using RNase digestion experiments, we show that the RNAscope assay is not suitable for the reliable discrimination between E6/E7 RNA transcripts and viral DNA.

Oropharyngeal squamous cell carcinoma (SCC) is strongly associated with human papillomavirus (HPV) infection, which is distinctively different from most other head and neck cancers. However, a robust quantitative reverse transcription PCR (RT-qPCR) method for comprehensive expression profiling of HPV genes in routinely fixed tissues has not been reported. To address this issue, we have established a new real-time RT-PCR method for the expression profiling of the E6 and E7 oncogenes from 13 high-risk HPV types. This method was validated in cervical cancer and by comparison with another HPV RNA detection method (in situ hybridization) in oropharyngeal tumors. In addition, the expression profiles of selected HPV-related human genes were also analyzed. HPV E6 and E7 expression profiles were then analyzed in 150 archived oropharyngeal SCC samples and compared with other variables and with patient outcomes. Our study showed that RT-qPCR and RNA in situ hybridization were 100% concordant in determining HPV status. HPV transcriptional activity was found in most oropharyngeal SCC (81.3%), a prevalence that is higher than in previous studies. Besides HPV16, three other HPV types were also detected, including 33, 35 and 18. Furthermore, HPV and p16 had essentially identical expression signatures, and both HPV and p16 were prognostic biomarkers for the prediction of disease outcome. Thus, p16 mRNA or protein expression signature is a sensitive and specific surrogate marker for HPV transcriptional activity (all genotypes combined).

The pathogenesis of squamous cell neoplasms arising in the lacrimal drainage system is poorly understood, and the underlying genomic drivers for disease development remain unexplored. We aimed to investigate the genomic aberrations in carcinomas arising in the LDS and correlate the findings to human papillomavirus (HPV) status. The HPV analysis was performed using HPV DNA PCR, HPV E6/E7 mRNA in-situ hybridization, and p16 immunohistochemistry. The genomic characterization was performed by targeted DNA sequencing of 523 cancer-relevant genes. Patients with LDS papilloma (n = 17) and LDS carcinoma (n = 15) were included. There was a male predominance (68%) and a median age at diagnosis of 46.0 years (range 27.5-65.5 years) in patients with papilloma and 63.8 years (range 34.0-87.2 years) in patients with carcinoma. Transcriptional activity of the HPV E6/E7 oncogenes was detected in the whole tumor thickness in 12/15 (80%) papillomas (HPV6, 11, 16) and 10/15 (67%) squamous cell carcinomas (SCC) (HPV11: 3/15 (20%) and HPV16: 7/15 (47%)). Pathogenic variants in PIK3CA, FGFR3, AKT1, and PIK3R1, wildtype TP53, p16 overexpression, and deregulated high-risk E6/E7 transcription characterized the HPV16-positive SCC. The deregulated pattern of HPV E6/E7 expression, correlating with HPV DNA presence and p16 positivity, supports a causal role of HPV in a subset of LDS papillomas and carcinomas. The viral and molecular profile of LDS SCC resembles that of other HPV-driven SCC.

Objective: To assess the clinical features and treatment outcomes in patients with primary ovarian squamous cell carcinoma (POSCC). Methods: Fifteen patients with primary ovarian squamous cell carcinoma diagnosed from January 2009 to December 2018 in Cancer Hospital of the University of Chinese Academy of Sciences were collected. The expression of p16, hMLH1, hMSH2, hMSH6 and PMS2 in POSCC was detected by immunohistochemistry, and the status of high-risk human papillomavirus (HPV) by RNAscope test. Results: Squamous cell carcinoma with different degrees of differentiation was found in 15 cases, including three cases with high differentiation and 12 cases with medium to low differentiation. There were four cases with in situ squamous cell carcinoma, four cases with teratoma, one case with endometrial carcinoma/atypical hyperplasia, and one case with endometriosis. p16 was expressed in five cases (5/15), indicating coexisting high-risk HPV infection. There was no high-risk HPV infection in the remaining 10 cases, and p16 staining was negative. There was no deficient mismatch repair protein in all cases. The overall survival time (P=0.038) and progression free survival (P=0.045) of patients with high-risk HPV infection were longer than those without HPV infection. Conclusions: POSCC is more commonly noted in postmenopausal women and often occurs unilaterally. Elevated serological indexes CA125 and SCC are the most common finding. Morphologically, the tumors show variable degrees of differentiation, but the current data suggest that the degree of differentiation cannot be used as an independent prognostic index. High-risk HPV infection may be associated with the occurrence of POSCC, and that the prognosis of POSCC patients with HPV infection is better than that of patients without infection.

Although both the 2014 and 2020 World Health Organization (WHO) criteria require unequivocal glandular and squamous differentiation for a diagnosis of cervical adenosquamous carcinoma (ASC), in practice, ASC diagnoses are often made in tumors that lack unequivocal squamous and/or glandular differentiation. Considering the ambiguous etiologic, morphologic, and clinical features and outcomes associated with ASCs, we sought to redefine these tumors. We reviewed slides from 59 initially diagnosed ASCs (including glassy cell carcinoma and related lesions) to confirm an ASC diagnosis only in the presence of unequivocal malignant glandular and squamous differentiation. Select cases underwent immunohistochemical profiling as well as human papillomavirus (HPV) testing by in situ hybridization. Of the 59 cases originally classified as ASCs, 34 retained their ASC diagnosis, 9 were reclassified as pure invasive stratified mucin-producing carcinomas, 10 as invasive stratified mucin-producing carcinomas with other components (such as HPV-associated mucinous, usual-type, or ASCs), and 4 as HPV-associated usual or mucinous adenocarcinomas with benign-appearing squamous metaplasia. Two glassy adenocarcinomas were reclassified as poorly differentiated HPV-associated carcinomas based on morphology and immunophenotype. There were no significant immunophenotypic differences between ASCs and pure invasive stratified mucin-producing carcinomas with regard to HPV and other markers including p16 expression. Although limited by a small sample size, survival outcomes seemed to be similar between all groups. ASCs should be diagnosed only in the presence of unequivocal malignant glandular and squamous differentiation. The 2 putative glassy cell carcinomas studied did not meet our criteria for ASC and categorizing them as such should be reconsidered.