Probes for INS

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.

Abstract
Background:

Although alcohol and tobacco use are known risk factors for development of squamous cell carcinoma in the head and neck, human papillomavirus (HPV) has been increasingly associated with this group of cancers. We describe the case of a married couple who presented with HPV-positive oropharynx squamous cell carcinoma within two months of each other.

Methods:

Tumor biopsies were positive for p16 and high-risk HPV in both patients. Sanger sequencing showed a nearly identical HPV16 strain in both patients. Both patients received chemoradiation, and one  patient also underwent transoral robotic tongue base resection with bilateral neck dissection.

Results:

Both patients showed no evidence of recurrent disease on follow-up PET imaging.

Conclusions:

New head and neck symptoms should be promptly evaluated in the partner of a patient with known HPV-positive oropharynx cancer. This case expands the limited current literature on concurrent presentation of HPV-positive oropharynx squamous cell carcinoma in couples. 

Although human papillomavirus (HPV)-related oropharyngeal carcinomas (HPV-OPCs) are generally regarded as "poorly differentiated," they actually maintain a close resemblance to the lymphoepithelium of the tonsillar crypts from which they arise: they are basaloid, exhibit minimal keratinization, and are often permeated by lymphocytes. In rare cases, the presence of cilia in a primary HPV-OPC and their persistence in lymph node metastasis can confound the distinction between a benign and malignant process. Three cases of ciliated HPV-OPCs were identified from the archives of The Johns Hopkins Head and Neck Pathology consultation service. HPV status was determined using p16 immunohistochemistry and high-risk HPV in situ hybridization. All 3 patients presented with a cystic lymph node metastasis without a known primary carcinoma. One metastasis was originally diagnosed as a branchial cleft cyst only to regionally recur 7 years later. In 2 cases, a primary HPV-OPC was found in the tonsil. The carcinomas exhibited both nonkeratinizing squamous epithelium and cystic/microcystic spaces lined by ciliated columnar cells. Both the squamous and ciliated cells were HPV positive. This report draws attention to a novel variant of HPV-related head and neck cancer that exhibits ciliated columnar cells. This variant challenges prevailing notions that: (1) HPV-OPCs are uniformly poorly differentiated cancers; (2) cilia are an infallible feature of benignancy; and (3) presence of cilia is a reliable criterion for establishing branchial cleft origin when dealing with cystic lesions of the lateral neck.

Human papillomavirus (HPV) is an independent risk factor for development of oral squamous cell carcinoma (OSCC). This study aimed to investigate the role of HPV infection and the trend in percentage of HPV-associated OSCC over a five-year period in northeastern Thailand. In this case-control study, 91 exfoliated oral cell samples and 80 lesion cell samples from OSCC cases and exfoliated oral cells from 100 age/gender-matched controls were collected. HPV infection was investigated by PCR using GP5+/GP6+ primers followed by HPV genotyping using reverse line blot hybridization. Quantitative RT-PCR was used to evaluate HPV oncogene transcription. Temporal trends of HPVinfection were evaluated in archived formalin-fixed paraffin-embedded (FFPE) OSCC tissues using in situ hybridization. HPV DNA was found in 17.5% (14/80) of lesion samples from OSCC cases and 29.7% (27/91) of exfoliated oral cell samples from the same cases. These values were significantly higher than in exfoliated oral cell samples from controls (13%, 13/100). HPV-16 was the genotype most frequently found in OSCC cases (92.8%, 13/14 infected cases). Interestingly, HPV oncogene mRNA expression was detected and correlated with OSCC cases (P < 0.005). Of 146 archived FFPE OSCC samples, 82 (56.2%) were positive for high-risk HPV DNA and 64 (43.8%) cases were positive for HPV E6/E7 mRNA expression. There was a trend of increasing percentage of HPV-associated OSCC from 2005 to 2010. This was especially so for females with well-differentiated tumors in specific tongue sub-sites. We suggest that HPV infection plays an important role in oral carcinogenesis in northeastern Thailand.