Determination of high‐risk HPV status of head and neck squamous cell carcinoma using the Roche cobas HPV test on cytologic specimens and acellular supernatant fluid

Abstract

BackgroundHigh‐risk human papillomavirus–positive (hrHPV+) oropharyngeal squamous cell carcinomas comprise a subset of head and neck squamous cell carcinomas (HNSCCs) with a distinct biology and prognosis. Commonly, the diagnosis of HNSCC is rendered on fine‐needle aspiration (FNA). Because cell blocks may be insufficient for determining HPV status using microscopy‐based techniques, the ability of liquid‐based assays was examined in the current study.MethodsThe performance of the Roche cobas 4800 platform was evaluated on the FNA material from the cell pellet and corresponding cell‐free supernatant fluid specimens of primary and metastatic HNSCCs. These results were compared with the p16 immunostain result from the histologic material obtained from the same patient. Discrepant cases were adjudicated using hrHPV RNA in situ hybridization.ResultsA total of 41 samples (23 primary tumors and 18 lymph node metastases) were acquired from 34 patients with HNSCC. Primary tumors included the oropharynx (20 samples), oral cavity (13 samples), larynx (3 samples), and skin (3 samples). In 2 cases, a primary tumor could not be identified. Twenty‐three samples (56%) were found to be p16 positive by immunohistochemistry. Twenty‐two samples were found to be positive on cobas hrHPV testing from both cell pellet and cell‐free supernatant fluid. Two cell‐free supernatant fluid specimens yielded indeterminate cobas results. At the time additional hrHPV RNA in situ hybridization analysis was performed, one cobas‐positive cell pellet was deemed to be a false‐positive result. The sensitivity of the cobas assay was 100% for pellet material and cell‐free supernatant fluid, with specificities of 94.7% and 100%, respectively.Conclusionscobas hrHPV testing of HNSCC specimens demonstrated high concordance with p16 immunohistochemistry on the corresponding cell block and/or tissue specimen. Using the cell‐free supernatant fluid in this platform could provide accurate HPV results while conserving material for cytomorphologic analyses.

Liquid phase human papillomavirus genotype analysis of aspirated metastatic head and neck squamous cell carcinoma: Fine needle aspiration supernatant is a rich source of tumor DNA that can increase the diagnostic yield

“…Two prior studies have found that the FNA supernatant holds promise as a potential substrate for determining HPV in patients with head and neck cancer 14,22 . Likewise, we found that even the FNA leftover wash component that is generally discarded as useless waste actually represents a rich resource of DNA for HPV analysis.…”

Diagnostic Accuracy of HPV Detection in Patients with Oropharyngeal Squamous Cell Carcinomas: A Systematic Review and Meta-Analysis

“…Nine studies evaluated HPV testing in formalin-fixed paraffin-embedded (FFPE) tissue [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ], and five studies evaluated HPV detection in fine needle aspiration (FNA) [ 30 , 31 , 32 , 33 , 34 ]. Reference methods varied between studies; seven studies used p16 IHC as reference [ 24 , 25 , 27 , 30 , 33 , 34 ], three studies used HPV RNA as reference, where one study detected HPV RNA by ISH [ 22 ] and the other two studies detected HPV RNA by PCR [ 23 , 26 ].…”

“…Nine studies evaluated HPV testing in formalin-fixed paraffin-embedded (FFPE) tissue [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ], and five studies evaluated HPV detection in fine needle aspiration (FNA) [ 30 , 31 , 32 , 33 , 34 ]. Reference methods varied between studies; seven studies used p16 IHC as reference [ 24 , 25 , 27 , 30 , 33 , 34 ], three studies used HPV RNA as reference, where one study detected HPV RNA by ISH [ 22 ] and the other two studies detected HPV RNA by PCR [ 23 , 26 ]. Another three studies used HPV DNA by PCR [ 28 , 29 , 32 ] as reference and finally, two studies combined two detection methods for reference; p16 IHC combined with HPV DNA PCR [ 21 ] and p16 IHC combined with HPV DNA PCR and/or HPV E6 seropositivity [ 31 ], respectively ( Table 1 ).…”

Prevalence and concordance of oral HPV infections with cervical HPV infections in women referred to colposcopy with abnormal cytology

“…Data from a validation study on oral cytobrushes and FFPE samples indicate that the Cobas assay is highly sensitive for detecting low copy numbers of viral DNA and displays high repeatability and reproducibility for detection of HPV in oral samples 17 . A clinical evaluation of the Cobas assay also demonstrated comparable performance to the gold standard p16 immunohistochemistry assay for detection of HPV in cytological preparations from head and neck cancer patients 18 …”