Predictive value of human papillomavirus (HPV) 16 in situ hybridization (ISH) and p16 immunohistochemistry (IHC) for HPV oncogene expression in paraffin-embedded oropharynx squamous cell carcinoma (OPSCC).

Abstract

5512 Background: Tumor HPV status is a strong, independent predictor of OPSCC survival and increasingly used for therapeutic decision making. Methods for classification are variable and not standardized. Here we validate our methods for HPV classification used in past and ongoing cooperative group trials (ECOG and RTOG). Methods: Consecutive OPSCC diagnosed from 2000-2009 were obtained from four referral centers. Tumor DNA and RNA were extracted and categorized as evaluable or inevaluable by DNA detection (ERV3) and mRNA expression (RPLPO) of control genes by quantitative (q) PCR. Presence of 18 high-risk HPV types was detected by consensus primer PCR and line-blot hybridization (Inno-Lipa) followed by viral load measurement and HPV oncogene expression (E6/7) analysis by HPV type-specific q RT-PCR. P16 expression was detected by IHC and HPV16 by ISH and sensitivity (S), specificity (SP), positive (PPV) and negative predictive value (NPV) were compared to gold standard HPVE6/7 oncogene expression. Assay interpretation was performed by three masked pathologists. Inter-assay and inter-rater agreement was evaluated by kappa statistic. Sample size provided 80% power for Cohen’s kappa of 0.8 or higher among three raters. Results: 235/237 OPSCC were evaluable, 183 (78%) HPV DNA-positive, and 158 (67%) HPVE6/7 expression positive (16, 92%; 18, 3%; 33, 3%; 35, 1%; 58, 1%) with median viral load of 15.7 copies/cell, E6/7 expression of 210 copies/1,000 RPLPO, p16 expression 46.7 vs. 2.9 copies/1000 RPLPO for HPVE6/7-negative, p<0.001. S/SP/PPV/NPV of HPV16 ISH for HPV16E6/7 was 91/91/94/86 and for p16 for HPVE6/7 was 97/82/92/93 and for combined testing was 84/97/99/75. P16 IHC and HPV16 ISH had very good agreement (kappa=0.64) with HPVE6/7 expression detected in 99%/63%/50%/3% of ++/+-/-+/-- cases, respectively. Inter-rater agreement was excellent for p16 (kappa=0.95-.98) and HPV16 ISH (kappa.0.83-.91). Conclusions: HPV16 ISH and p16 IHC have excellent assay performance. However, if results are used for therapy “deintensification” among HPV-positive cases, combined testing provides higher SP and PPV.