Abstract 554: Qualification of a p16INK4A immunohistochemistry (IHC) assay for determination of HPV status in archival Squamous Cell Carcinoma of the Head and Neck (SCCHN) formalin fixed paraffin embedded (FFPE) specimens from a randomized phase 3 trial
Authors: Oliner Kelly S. , Ekdahl Mark , Kyshtoobayeva Ainura , Bloom Kenneth , Bach Bruce A. 
Published: August 2012

Abstract
Introduction: HPV positive SCCHN has a distinct set of molecular alterations and a better prognosis in the locally advanced setting. When HPV is integrated into the host cell genome, HPV protein E7 degrades host retinoblastoma protein, an inhibitor of p16INK4A transcription. Thus, in persistent HPV gene expressing tumors, elevated p16INK4A RNA and protein levels serve as a surrogate of HPV infection applicable to FFPE archival tumor specimens. Our aim was to qualify an existing IHC assay for reliable detection of active HPV expression in SCCHN archival FFPE tumor samples from a randomized phase 3 trial in the recurrent/metastatic (R/M) treatment setting. Methods: The CINtec™ p16INK4A Histology Kit is a semi quantitative, immunocytochemical assay for the evaluation of p16INK4A protein expression in FFPE sections. Banked SCCHN specimens were examined for p16INK4A expression to establish repeatability, reproducibility, and long term analyte stability (blocks and slides). Comparative studies were performed with a p16INK4A reverse transcription quantitative polymerase chain reaction (RT qPCR) assay and an HPV in situ hybridization assay to examine intra assay concordance. Results: 101 banked SCCHN FFPE tumor specimens were utilized for the qualification study. 42 of 101 (42%) SCCHN specimens (all sites) and 17 of 41 (41%) oropharyngeal specimens were HPV positive by p16INK4A. Five slides from each of 6 SCCHN specimens ranging in the percentage of cells expressing p16INK4A (positive, low positive, and negative) were tested on a single day to demonstrate inter assay repeatability. Serial sections from 6 tumors were tested on 3 consecutive working days using multiple automated staining instruments run by different technicians to determine intra assay reproducibility. Inter pathologist scoring, blinded to run, was demonstrably consistent. Positive p16INK4A staining was demonstrated in blocks stored up to 19 years. Analyte stability was demonstrated in cut sections prepared 27 months in advance. Comparative assay studies yielded consistent, though not identical, results. These results led to the design and implementation of a detailed imaging scoring guideline. Conclusions: The CINtecTM p16INK4A Histology Kit was qualified for use as a surrogate for HPV infection in SCCHN specimens. This assay was then utilized to test banked specimens from a randomized, phase 3 study of the anti EGFR antibody panitumumab in patients with HPV positive R/M SCCHN tumors. In this study, patients with HPV positive tumors did not benefit from the addition panitumumab to a platinum + 5 fluorouracil chemotherapy regimen whereas there was a significant improvement in overall survival in the HPV negative group (Vermorken et al., European Multidisciplinary Cancer Congress 2011).
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 554. doi:1538-7445.AM2012-554