Automated Extraction of FFPE Tissue for High-Risk HPV Testing

The testing for high-risk human papillomavirus (HR-HPV) in head and neck squamous cell carcinomas is important for both prognostication and clinical management. Several testing platforms are available for HR-HPV; however, effective alternative automated approaches are needed.

High-risk human papillomavirus (HR-HPV) is now a well-established etiologic agent in a subset of head and neck squamous cell carcinomas (HNSCCs) that has an incidence on the rise, particularly with respect to oropharyngeal squamous cell carcinoma (OPSCC), which demonstrates an association with transcriptionally active virus in up to 80% of tumors.


Pathologists from Massachusetts General Hospital (Boston, MA, USA) collected from 62 archival specimens of formalin-fixed, paraffin-embedded (FFPE) samples of HNSCC. Overall OPSCC represented slightly more than half (55%) of the HNSCCs tested. These were prepared using the automated system by deparaffinization and dehydration followed by tissue lysis. Corresponding FFPE samples were evaluated for HR-HPV by in situ hybridization (ISH) and p16 by immunohistochemistry. Discrepant cases were adjudicated by polymerase chain reaction (PCR).

The scientists used the automated Roche cobas 4800 System Platform (Roche Diagnostics, Risch-Rotkreuz, Switzerland), which consists of an automated instrument for sample preparation and DNA extraction followed by and coupled to a thermocycler and analyzer for performance of real-time PCR and subsequent detection of target DNA sequences. Immunohistochemical expression of p16 was evaluated by deparaffinizing 5µm FFPE sections of HNSCCs and performing antigen retrieval using the Leica Bond protocol (Leica Biosystems, Buffalo Grove, IL, USA).

The investigators found 32/62 (52%) of formalin-fixed, paraffin-embedded tumors were positive for HR-HPV by cobas. There were 28/32 (88%) of cases were the HPV 16 subtype and 12% (4 /32) were other HR-HPV subtypes. Corresponding testing with ISH was concordant in 92% (57 /62). Compared with the adjudication PCR standard, there were three false-positive cases by cobas. For the OPSCC, cobas had positive and negative predictive values of 100%, whereas for non-OPSCC, the positive predictive value was 57% and the negative predictive value was 100%.

The authors concluded that concordance in HNSCC HR-HPV status between cobas and ISH was more than 90%. The cobas demonstrated a sensitivity of 100% and a specificity of 91% for detection of HR-HPV. They said the advantages favoring cobas included its automation, cost efficiency, objective results, and ease of performance. The study was published in an early online edition of the journal Archives of Pathology & Laboratory Medicine.

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Massachusetts General Hospital
Roche Diagnostics
Leica Biosystems