Probes for HPV

Abstract

Background

Accurate identification of HPV-driven oropharyngeal cancer (OPC) is a major issue and none of the current diagnostic approaches is ideal. An in situ hybridization (ISH) assay that detects high-risk HPV E6/E7 mRNA, called the RNAscope HPV-test, has been recently developed. Studies have suggested that this assay may become a standard to define HPV-status.

Methods

To further assess this test, we compared its performance against the strategies that are used in routine clinical practice: p16 immunohistochemistry (IHC) as a single test and algorithms combining p16-IHC with HPV-DNA identification by PCR (algorithm-1) or ISH (algorithm-2).

Results

105 OPC specimens were analyzed. The prevalence of HPV-positive samples varied considerably: 67% for p16-IHC, 54% for algorithm-1, 61% for algorithm-2 and 59% for the RNAscope HPV-test. Discrepancies between the RNAscope HPV-test and p16-IHC, algorithm-1 and 2 were noted in respectively 13.3%, 13.1%, and 8.6%.

The 4 diagnostic strategies were able to identify 2 groups with different prognosis according to HPV-status, as expected. However, the greater survival differential was observed with the RNAscope HPV-test [HR: 0.19, 95% confidence interval (CI), 0.07–0.51, p = 0.001] closely followed by algorithm-1 (HR: 0.23, 95% CI, 0.08–0.66, p = 0.006) and algorithm-2 (HR: 0.26, 95% CI, 0.1–0.65, p = 0.004). In contrast, a weaker association was found when p16-IHC was used as a single test (HR: 0.33, 95% CI, 0.13–0.81, p = 0.02).

Conclusions

Our findings suggest that the RNAscope HPV-test and p16-based algorithms perform better that p16 alone to identify OPC that are truly driven by HPV-infection. The RNAscope HPV-test has the advantage of being a single test.

Abstract

BACKGROUND:

There is increasing evidence that high-risk human papillomavirus plays significant role in oropharyngeal cancer; however, there is lack of knowledge on the interplay between the virus and its downstream related molecules and their possible prognostic values. The objectives of the study are to better understand the interplay of the HR-HPV and its associated downstream molecules and to evaluate potential biomarkers for patient outcomes.

METHODS:

We conducted a retrospective study with available formalin-fixed, paraffin-embedded tissue from 244 oropharyngeal cancer patients that received curative radiotherapy or concurrent chemoradiotherapy from 2000 to 2008. In addition to chart review, we performed HPV DNA and RNA in situ hybridization and immunohistochemistry for p53, the retinoblastoma protein, p16, and cyclin D1 analysis. Cox-proportional hazard and Kaplan-Meier survival analysis were used to determine the prognostic markers for clinical outcomes.

RESULTS:

Patients averaged 57.3±9.4 year-old and were mostly males (76.2%) and ever-smokers (76.2%). All patients received curative radiotherapy and 44.3% received concurrent chemoradiotherapy. We detected the human papillomavirus in 77.9% of study patients. Ever-smokers, more advanced tumor stage, and receiving radiotherapy only had poorer 5-year overall survival, disease-specific survival, and loco-regional recurrence. Cases with positive human papillomavirus and p53 overexpression had poorer disease-specific survival. Cases without human papillomavirus, but cyclin D1 overexpression, was associated with poorer 5-year overall survival.

CONCLUSIONS:

Our data suggests that additional p53 and cyclin D1 testing may benefit oropharyngeal cancer patients with known human papillomavirus status.

Human Papillomavirus (HPV) is known as causative for squamous cell carcinoma (SCC) of the oropharynx, but is also not infrequently found in carcinomas of the sinonasal tract. Recently, a subset of these carcinomas was recognized to harbour HPV33 and have a significant morphological overlap with adenoid cystic carcinoma (ACC), a rare and aggressive carcinoma originating in the minor salivary glands. Termed HPV-related carcinoma with ACC-like features, only 9 cases have been reported. To clarify the occurrence of these tumours we screened a large material for presence of HPV-related ACC-like carcinoma. The identified tumours were characterized immunohistochemically and with fluorescence in situ hybridization and clinicopathologic information for all cases is presented.

METHODS AND RESULTS:

Forty-seven candidate cases were screened for presence of HPV. Six cases were identified and genotyped as HPV types 33, 35 and 56. All six cases had areas of dysplastic mucosal lining and showed remarkable heterogeneous morphologies. MYB, MYBL1, and NFIB genes were intact and, interestingly, staining for MYB protein was largely negative in contrast to what was found in ACC. One patient experienced a local recurrence 11 years after initial treatment and the remaining five patients were alive without evidence of disease.

CONCLUSION:

We report six new cases of HPV-related ACC-like carcinoma and found that, although in a small material, the prognosis for these patients seems more favourable than for ACC. For the distinction between ACC and HPV-related ACC-like carcinoma, p16, MYB immunohistochemistry, or investigation of MYB, MYBL1, and NFIB gene status are valuable. This article is protected by copyright. All rights reserved.

Abstract

BACKGROUND:

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) is increasing among older adults. It is unknown whether these trends can be explained by human papillomavirus (HPV) and whether HPV-related tumors remain associated with an improved prognosis among older patients.

METHODS:

In a retrospective study of OPSCCs diagnosed from 1995 to 2013 at 2 National Comprehensive Cancer Network-designated cancer centers, p16 immunohistochemistry and in situ hybridization (ISH) for HPV-16, high-risk DNA, and/or E6/E7 RNA were performed. The median age at diagnosis was compared by p16 and ISH tumor status. Trends in age were analyzed with nonparametric trends. Survival was analyzed with the Kaplan-Meier method and Cox proportional hazards models.

RESULTS:

Among 239 patients, 144 (60%) were p16-positive. During 1998-2013, the median age increased among p16-positive patients (Ptrend = .01) but not among p16-negative patients (Ptrend = .71). The median age of p16-positive patients increased from 53 years (interquartile range [IQR] in 1995-2000, 45-65 years) to 58 years (IQR for 2001-2013, 53-64 years). Among patients ≥ 65 years old, the proportion of OPSCCs that were p16-positive increased from 41% during 1995-2000 to 75% during 2007-2013 (Ptrend = .04). Among all age groups, including older patients, a p16-positive tumor status conferred improved overall survival in comparison with a p16-negative status.

CONCLUSIONS:

The median age at diagnosis for HPV-related OPSCC is increasing as the proportion of OPSCCs caused by HPV rises among older adults. The favorable survival conferred by an HPV-positive tumor status persists in older adults. Cancer 2018. © 2018 American Cancer Society.

The performance characteristics of neuroendocrine-specific and squamous-specific immunohistochemical markers in head and neck squamous cell carcinomas (SCC), in particular in oropharyngeal tumors in this era of human papillomavirus (HPV)-induced cases, are not well-established. The differential diagnosis for poorly differentiated SCCs, for nonkeratinizing oropharyngeal SCCs, and for other specific SCC variants such as basaloid SCC and undifferentiated (or lymphoepithelial-like) carcinomas includes neuroendocrine carcinomas. Given that neuroendocrine carcinomas of the head and neck are aggressive regardless of HPV status, separating them from SCC is critically important. In this study, we examined the neuroendocrine markers CD56, synaptophysin, and chromogranin-A along with the squamous markers p40 and cytokeratin 5/6 in a large tissue microarray cohort of oral, oropharyngeal, laryngeal, and hypopharyngeal SCCs with known HPV results by RNA in situ hybridization for the oropharyngeal tumors. Results were stratified by site and specific SCC variant. The neuroendocrine stains were rarely expressed in SCC (<1% overall) with CD56 the least, and chromogranin-A the most, specific markers. Further, p40 and cytokeratin 5/6 were very consistently expressed in all head and neck SCC (>98% overall), including very strong, consistent staining in oropharyngeal HPV-related nonkeratinizing SCC. Undifferentiated (or lymphoepithelial-like) carcinomas of the oropharynx are more frequently p40 or cytokeratin 5/6 negative or show only weak or focal expression. In summary, markers of neuroendocrine and squamous differentiation show very high specificity and sensitivity, respectively, across the different types of head and neck SCC.