Oral Oncol. Apr; 50(4):306–310.
Poling JS, Ma XJ, Bui S, Luo Y, Li R, Koch WM, Westra WH (2014).
PMID: 24485566 | DOI: 10.1016/j.oraloncology.2014.01.006.
OBJECTIVES:
The human papillomavirus (HPV) is an important cause of some head and neck squamous cell carcinomas (HNSCCs), but its role in cancer of the lateral tongue is debatable. Suspicion of HPV causation is heightened when these lateral tongue carcinomas arise in patients that are young and/or have never smoked. The purpose of this study was to determine the incidence of transcriptionally active high risk HPV in these tumors, with a particular emphasis on non-smoking patients who are often presumed to have HPV-positive tumors.
METHODS:
We evaluated 78 HNSCCs of the lateral tongue for the presence of HPV using p16 immunohistochemistry and an RNA in situ hybridization assay targeting HPV E6/E7 mRNA. The study population was enriched for patients without traditional risk factors such as smoking and drinking.
RESULTS:
P16 overexpression was detected in 9 (11.5%) of 78 cases, but HPV E6/E7 mRNA transcripts were detected in only 1 (1.3%) case (positive predictive value of p16 staining for the presence of transcriptionally active HPV=0.12). HPV mRNA transcripts were not detected in any patient under 40 (n=11), or in patients who had never smoked (n=44), had quit smoking (n=15), and/or were only light consumers of alcohol (n=57).
CONCLUSIONS:
HPV is not detected in the vast majority of lateral tongue carcinomas. In light of the observation that HPV plays little if any role in the development of these cancers, routine HPV testing is unwarranted , even for patients without traditional risk factors. P16 staining is not a reliable marker for the presence of transcriptionally active HPV at this particular anatomic site.
HDAC1/2 control proliferation and survival in adult epidermis and pre-basal cell carcinoma via p16 and p53
The Journal of investigative dermatology
Zhu, X;Leboeuf, M;Liu, F;Grachtchouk, M;Seykora, JT;Morrisey, EE;Dlugosz, AA;Millar, SE;
PMID: 34284046 | DOI: 10.1016/j.jid.2021.05.026
HDAC inhibitors show therapeutic promise for skin malignancies; however, the roles of specific HDACs in adult epidermal homeostasis and disease are poorly understood. We find that homozygous epidermal co-deletion of Hdac1 and Hdac2 in adult mouse epidermis causes reduced basal cell proliferation, apoptosis, inappropriate differentiation, and eventual loss of Hdac1/2-null keratinocytes. Hdac1/2 deficient epidermis displays elevated acetylated p53 and increased expression of the senescence gene p16. Loss of p53 partially restores basal proliferation, whereas p16 deletion promotes long-term survival of Hdac1/2-null keratinocytes. In activated GLI2-driven pre-basal cell carcinoma, Hdac1/2 deletion dramatically reduces proliferation and increases apoptosis, and knockout of either p53 or p16 partially rescues both proliferation and basal cell viability. Topical application of the HDAC inhibitor Romidepsin to normal epidermis or GLI2ΔN-driven lesions produces similar defects to genetic Hdac1/2 deletion, and these are partially rescued by loss of p16. These data reveal essential roles for HDAC1/2 in maintaining proliferation and survival of adult epidermal and basal cell carcinoma progenitors and suggest efficacy of therapeutic HDAC1/2 inhibition will depend in part on the mutational status of p53 and p16.
Dreyer JH, Hauck F, Oliveira-Silva M, Barros MH, Niedobitek G. (2013).
PMID: 23503925 | DOI: 10.1007/s00428-013-1393-5.
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.
Annals of Diagnostic Pathology (2018)
Nicol AF, de Andrade CV, Gomes SC, Brusadelli MG, Lodin HM, Wells SI, Nuovo GJ.
| DOI: 10.1016/j.anndiagpath.2018.12.001
Importin-β, exportin-5, p16, Ki-67, Mcl1, PDL1, and cFLIP are each over-expressed in the majority of CIN 1 lesions. These biomarkers, plus HPV E6/E7 RNA, were analyzed in carcinoma-in-situ (CIS), microinvasive, and squamous cell carcinoma (SCC) of the uterine cervix and cervical carcinoma cell lines. Only p16 and Ki-67 continued to be over-expressed in CIS, with a concomitant marked increase in E6/E7 RNA. There was a highly significant increase in PDL1 expression and decrease in Ki-67 (each p < 0.001) in microinvasive cancer compared to CIS whereas p16 and E6/E7 remained stable. As the lesion progressed to SCC, p16 and E6/E7 RNA remained strongly overexpressed with a concomitant over expression of importin-β and Ki67. HPV positive Caski cells showed significant elevations of p16, importin-β, exportin-5 and PDL1 compared to the HPV negative cervical cancer cell line C33A, consistent with viral induction of these biomarkers. The data suggest that PDL1 may be a useful biomarker to differentiate CIS from microinvasive cancer and, thus, anti-PDL1 therapy may inhibit the progression of CIS to the invasive stage.
Craig SG, Anderson LA, Schache AG, Moran M, Graham L, Currie K, Rooney K, Robinson M, Upile NS, Brooker R, Mesri M, Bingham V, McQuaid S, Jones T, McCance DJ, Salto-Tellez M, McDade SS and James JA
PMID: 30890775 | DOI: 10.1038/s41416-019-0414-9
BACKGROUND: TNM8 staging for oropharyngeal squamous cell carcinomas (OPSCC) surrogates p16 immunohistochemistry for HPV testing. Patients with p16+ OPSCC may lack HPV aetiology. Here, we evaluate the suitability of TNM8 staging for guiding prognosis in such patients. METHODS: HPV status was ascertained using p16 immunohistochemistry and high-risk HPV RNA and DNA in situ hybridisation. Survival by stage in a cohort of OPSCC patients was evaluated using TNM7/TNM8 staging. Survival of p16+/HPV- patients was compared to p16 status. RESULTS: TNM8 staging was found to improve on TNM7 (log rank p = 0.0190 for TNM8 compared with p = 0.0530 for TNM7) in p16+ patients. Patients who tested p16+ but were HPV- (n = 20) had significantly reduced five-year survival (33%) compared to p16+ patients (77%) but not p16- patients (35%). Cancer stage was reduced in 95% of p16+/HPV- patients despite having a mortality rate twice (HR 2.66 [95% CI: 1.37-5.15]) that of p16+/HPV+ patients under new TNM8 staging criteria. CONCLUSION: Given the significantly poorer survival of p16+/HPV- OPSCCs, these data provide compelling evidence for use of an HPV-specific test for staging classification. This has particular relevance in light of potential treatment de-escalation that could expose these patients to inappropriately reduced treatment intensity as treatment algorithms evolve.
High-risk human papillomavirus and ZEB1 in ocular adnexal sebaceous carcinoma
Journal of cutaneous pathology
Moore, RF;Zhang, XR;Allison, DB;Rooper, LM;Campbell, AA;Eberhart, CG;
PMID: 33745190 | DOI: 10.1111/cup.13987
Ocular adnexal (OA) sebaceous carcinoma is an aggressive malignancy. Oncologic drivers of ocular sebaceous carcinoma are incompletely understood. A retrospective search of our pathology archives for OA sebaceous carcinoma identified 18 primary resection specimens. Immunohistochemistry for p16 and ZEB1 and RNA in situ hybridization for high-risk human papillomavirus (HPV) subtypes were performed. High-risk HPV was demonstrated in 2/11 (18%) cases. p16 overexpression was observed in 10/11 (91%). No association between gender, age at presentation, tumor location, intraepithelial spread, tumor size, and T stage was observed between HPV-driven and nonviral cases. High expression of ZEB1 was observed in the intraepithelial component of 4/14 (28%) cases and in the subepithelial component of 1/13 (7%) cases. ZEB1 overexpression was not associated with HPV-status, T stage, or tumor size. As previously described by others, our findings suggest that a subset of OA sebaceous carcinomas may arise via an HPV-dependent pathway. However, unlike high-risk HPV-driven carcinomas of the oropharynx, we did not identify an association between HPV-status and prognostic features. Furthermore, p16 expression was not a useful surrogate marker for HPV-driven disease. ZEB1 overexpression is not associated with HPV in our cohort of ocular sebaceous carcinoma.
Roopera LM, Gandhib M, Bishop JA, Westraa WH
PMID: - | DOI: 10.1016/j.oraloncology.2016.02.008
Objectives
Evaluation of human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (OPSCC) has become increasingly important for prognostication and clinical trial enrollment. This assessment is confounded in OPSCCs that are p16 positive by immunohistochemistry (IHC) but HPV negative by DNA in situ hybridization (DISH). This study evaluates whether E6/E7 mRNA in situ hybridization (RISH) can detect transcriptionally active HPV in these problematic cases.
Materials and methods
Eighty-two head and neck squamous cell carcinoma cases that had previously undergone p16 IHC and HPV DISH were evaluated with two RISH platforms and a second-generation DISH probe. The study included 21 p16+/DISH+ concordant cases, 19 p16−/DISH− concordant cases, and 42 p16+/DISH− discordant cases.
Results
RISH identified E6/E7 mRNA in 37 (88%) p16+/DISH− cases, 21 (100%) p16+/DISH+ cases, and 0 (0%) p16−/DISH− cases. RISH signals were clearly visible at low to medium magnification in 97% of positive cases, facilitating almost-perfect inter-observer reproducibility. The performance of the manual and automated RISH platforms were equivalent (kappa = 0.915). Only 29% of carcinomas that demonstrated E6/E7 mRNA transcriptional activity were positive using the 2nd generation DISH probe.
Conclusions
HPV RISH is a highly sensitive and specific platform that can clarify the HPV status of those perplexing OPSCCs that are p16 positive by IHC but HPV negative by DISH. Moreover, it is easy to interpret, readily adaptable to the clinical laboratory, and provides direct evidence of HPV transcriptional activity. E6/E7 RISH should be considered as a first-line platform for determination of HPV status in OPSCCs.
Augustin J, Mandavit M, Outh-Gauer S, Grard O, Gasne C, Lépine C, Mirghani H, Hans S, Bonfils P, Denize T, Bruneval P, Bishop JA, Fontugne J, Péré H, Tartour E, Badoual C.
PMID: - | DOI: 10.1038/s41379-018-0090-y
HPV-related and HPV-unrelated oropharyngeal squamous cell carcinomas are two distinct entities according to the Union for International Cancer Control, with a better prognosis conferred to HPV-related oropharyngeal squamous cell carcinomas. However, variable clinical outcomes are observed among patients with p16 positive oropharyngeal squamous cell carcinoma, which is a surrogate marker of HPV infection. We aimed to investigate the prognostic value of RNA CISH against E6 and E7 transcripts (HPV RNA CISH) to predict such variability. We retrospectively included 50 histologically confirmed p16 positive oropharyngeal squamous cell carcinomas (p16 positive immunostaining was defined by a strong staining in 70% or more of tumor cells). HPV RNA CISH staining was assessed semi-quantitatively to define two scores: RNA CISH “low” and RNA CISH “high”. Negative HPV RNA CISH cases were scored as RNA CISH “low”. This series contained 29 RNA CISH low cases (58%) and 21 RNA CISH high cases (42%). Clinical and pathologic baseline characteristics were similar between the two groups. RNA CISH high staining was associated with a better overall survival in both univariate and multivariate analyses (p = 0.033 and p = 0.042, respectively). Other recorded parameters had no prognostic value. In conclusion, HPV RNA CISH might be an independent prognostic marker in p16 positive oropharyngeal squamous cell carcinomas and might help guide therapeutics.
Chahoud, J;Zacharias, NM;Pham, R;Qiao, W;Guo, M;Lu, X;Alaniz, A;Segarra, L;Martinez-Ferrer, M;Gleber-Netto, FO;Pickering, CR;Rao, P;Pettaway, CA;
PMID: 36551510 | DOI: 10.3390/cancers14246024
Penile Squamous Cell Carcinoma (PSCC) is associated with high-risk human papillomavirus (HR-HPV). The immunohistochemical (IHC) test for p16INK4a (p16) is highly correlated with HR-HPV expression in other SCCs. To investigate whether the expression of p16 IHC or HR-HPV is associated with survival in PSCC, we conducted a single institution analysis of 143 patients with a diagnosis of PSCC and, available tissue were tested for p16 IHC staining patterns, histological subtype, tumor grade, and lymphovascular invasion (LVI) by an experienced pathologist. HR-HPV status using the Cobas PCR Assay or the RNAScope high-risk HPV in situ hybridization kit were also assessed. Patient characteristics were summarized using descriptive statistics of clinico-pathologic variables. Kaplan-Meier was used to estimate median overall survival (OS), cancer specific survival (CSS) and correlated with HPV, p16, and other study variables. Patients with p16+ tumors had a significantly longer median CSS in comparison to the p16- group (p = 0.004), with respective 5-year CSS probability of 88% (95% CI; 0.84, 1) versus 58% (95% CI; 0.55, 0.76; p = 0.004). HPV status did not predict survival outcomes. Multivariable analysis with respect to OS and CSS, showed that p16+ status was associated with a lower risk of death (HR = 0.36, 95%CI; 0.20-0.67, p = 0.001), and improved CSS (HR = 0.20, 95% CI; 0.07-0.54, p = 0.002) after adjusting for covariates. In conclusion, tumor p16 status via IHC was an easy to perform independent prognostic factor for OS and CSS that correlates with HR-HPV expression.
Human papillomavirus (HPV) oncogenic activity is the result of viral oncogene E6 and E7 expression in infected cells. Oncogene expression analysis is however not part of the routine diagnostic evaluation of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) since it requires fresh tumor tissue. We compared the diagnostic accuracy of several methods commonly employed for HPV characterization in OPSCC with the results of the newly available HPV E6/E7 mRNA in situ hybridization (ISH) on formalin-fixed, paraffin-embedded biopsy samples, in order to establish if the latter should be introduced in the diagnostic routine to increase accuracy when fresh tissue is not available. p16 immunostain, DNA ISH for high risk (HR) HPV genotypes, SPF LiPA amplification and genotyping, and HPV16 E6 amplification were performed on 41 consecutive OPSCC samples. Twenty (48,7%) cases were positive by mRNA ISH; sensitivity and specificity were 100% and 90% for p16, 90% and 100% for DNA ISH, 70% and 76% for SPF10 LiPA, 90% and 76% for E6 amplification. A diagnostic algorithm considering p16 immunostain as first step followed by either HR HPV DNA ISH or HPV16 E6 amplification in p16-positive cases correctly characterized 90% of mRNA-positive and all mRNA-negative cases; combining the 3 tests correctly identified all cases. While no stand-alone test was sufficiently accurate for classifying HPV-associated OPSCC, the high sensitivity and specificity of the established combination of p16 immunostain, DNA ISH and HPV16 DNA amplification suggests that the introduction of labour- and cost-intensive mRNA ISH, is not necessary in the diagnostic routine of oropharyngeal tumors.
International Journal of Cancer, 132(4), 882–890.
Gao G, Chernock RD, Gay HA, Thorstad WL, Zhang TR, Wang H, Ma XJ, Luo Y, Lewis JS Jr, Wang X (2013).
PMID: 22821242 | DOI: 10.1002/ijc.27739.
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 American journal of surgical pathology, 37(9):1349–1356.
Mehrad M, Carpenter DH, Chernock RD, Wang H, Ma XJ, Luo Y, Luo J, Lewis JS Jr, El-Mofty SK. (2013).
PMID: 23797720 | DOI: 10.1097/PAS.0b013e318290427d.
A relationship between human papillomavirus (HPV) infection and papillary squamous cell carcinoma (PSCC) has been suggested. However, to date, no studies have thoroughly and directly evaluated for transcriptional activity of the virus or the clinicopathologic significance of HPV-positive PSCC. Forty-eight cases of PSCC were retrieved from our surgical pathology database and were reviewed by 4 study pathologists, with tumors defined as SCC with a significant component of papillary growth in the tumor. Immunohistochemical analysis for p16 and p53 was performed. Overexpression of p16 was used as a surrogate marker of transcriptionally active HPV. Transcriptional activity was also directly evaluated using RNA in situ hybridization to detect high-risk HPV E6/E7 mRNA. Clinical follow-up data were obtained by chart review. Seven cases were located in the oral cavity, 19 in the oropharynx, and 22 in the larynx. Two morphologic types of PSCC were identified: keratinizing type, in which the epithelial cells showed a maturation trend with minimal surface parakeratin, and nonkeratinizing type, in which the papillae were completely covered by immature basaloid cells. Transcriptionally active HPV was present in 23 of 43 (53.4%) tumors. The majority of tumors harboring transcriptionally active HPV arose in the oropharynx, showed nonkeratinizing morphology, were p16 positive, and p53 negative. Transcriptionally active HPV was also present in many laryngeal and oral cavity PSCCs. Overall survival, disease-specific survival, and disease-free survival were favorable and did not significantly differ by anatomic subsite. However, HPV-related tumors showed a trend toward better survival.
