Probes for P16

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).

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.

Viruses are well known drivers of several human malignancies. A causative factor for oral cavity squamous cell carcinoma (OSCC) in patients with limited exposure to traditional risk factors, including tobacco use, is yet to be identified. Our study aimed to comprehensively evaluate the role of viral drivers in OSCC patients with low cumulative exposure to traditional risk factors. Patients under 50 years of age with OSCC, defined using strict anatomic criteria were selected for WGS. The WGS data was interrogated using viral detection tools (Kraken 2 and BLASTN), together examining >700,000 viruses. The findings were further verified using tissue microarrays of OSCC samples using both immunohistochemistry and RNA in situ hybridisation (ISH). 28 patients underwent WGS and comprehensive viral profiling. One 49-year-old male patient with OSCC of the hard palate demonstrated HPV35 integration. 657 cases of OSCC were then evaluated for the presence of HPV integration through immunohistochemistry for p16 and HPV RNA ISH. HPV integration was seen in 8 (1.2%) patients, all middle-aged men with predominant floor of mouth involvement. In summary, a wide-ranging interrogation of >700,000 viruses using OSCC WGS data showed HPV integration in a minority of male OSCC patients and did not carry any prognostic significance.

Background Oropharyngeal squamous cell carcinoma (OPSCC) incidence is rising worldwide, especially human papillomavirus (HPV)-associated disease. Historically, high levels of protein kinase CK2 were linked with poor outcomes in head and neck squamous cell carcinoma (HNSCC), without consideration of HPV status. This retrospective study examined tumor CK2α protein expression levels and related clinical outcomes in a cohort of Veteran OPSCC patient tumors which were determined to be predominantly HPV(+). Methods Patients at the Minneapolis VA Health Care System with newly diagnosed primary OPSCC from January 2005 to December 2015 were identified. A total of 119 OPSCC patient tumors were stained for CK2α, p16 and Ki-67 proteins and E6/E7 RNA. CK2α protein levels in tumors and correlations with HPV status and Ki-67 index were assessed. Overall survival (OS) analysis was performed stratified by CK2α protein score and separately by HPV status, followed by Cox regression controlling for smoking status. To strengthen the limited HPV(−) data, survival analysis for HPV(−) HNSCC patients in the publicly available The Cancer Genome Atlas (TCGA) PanCancer RNA-seq dataset was determined for CSNK2A1. Results The patients in the study population were all male and had a predominant history of tobacco and alcohol use. This cohort comprised 84 HPV(+) and 35 HPV(−) tumors. CK2α levels were higher in HPV(+) tumors compared to HPV(−) tumors. Higher CK2α scores positively correlated with higher Ki-67 index. OS improved with increasing CK2α score and separately OS was significantly better for those with HPV(+) as opposed to HPV(−) OPSCC. Both remained significant after controlling for smoking status. High CSNK2A1 mRNA levels from TCGA data associated with worse patient survival in HPV(−) HNSCC. Conclusions High CK2α protein levels are detected in HPV(+) OPSCC tumors and demonstrate an unexpected association with improved survival in a strongly HPV(+) OPSCC cohort. Worse survival outcomes for high CSNK2A1 mRNA levels in HPV(−) HNSCC are consistent with historical data. Given these surprising findings and the rising incidence of HPV(+) OPSCC, further study is needed to understand the biological roles of CK2 in HPV(+) and HPV(−) HNSCC and the potential utility for therapeutic targeting of CK2 in these two disease states.