The significance of HPV status in patients with anal cancer: A comparative technical analysis.

Abstract

425 Background: HPV status is an important prognostic factor for outcome in head and neck cancers, with improved survival noted in HPV-positive vs -negative tumors. However, this effect has not been studied in anal cancer. Challenges exist in defining reliable and methods of detecting the presence of high-risk HPV virus or the expression of HPV-related proteins. Purpose: To determine p16 expression and HPV16 status, in association with HPV subtyping, in pre-treatment anal cancers, and to correlate marker status with clinical outcome. Methods: This retrospective study evaluated anal cancer patients treated between 1992-2005 with definitive RT or CRT at a single institution (Tom Baker Cancer Centre, Calgary, Canada). HPV subtyping, p16 protein expression via conventional immunohistochemistry (IHC) and automated quantitative IHC (AQUA), and HPV16 in-situ hybridization (CISH) was performed in a subset of patient tumors with sufficient pretreatment tumor specimen. The correlation between results using these different techniques and association of HPV markers with clinical outcome was evaluated. Results: 89 patients were identified; M:F 1:2; median age 57 years. Median tumor size was 3.5 cm. All patients were treated with radical external beam RT with curative intent; 81% received concurrent chemotherapy. Clinical CR was observed in 73% of patients at 3 months post-treatment. Median OS was 82 months. Of tumor specimens analyzed for HPV subtype, 78% (28/36) were HPV16. Using conventional IHC (DAB), 80% (28/35) of tumors over-expressed p16 (score 3). HPV16 CISH was positive in 81% (34/42). P16 AQUA™ score correlated with HPV16 subtyping, CISH for HPV16, and p16 DAB. With HPV16 subtyping as the gold standard for HPV16 infection, false positive and false negative rates were 8% and 4% with DAB, and 10% and 13% with CISH. In univariate analysis only p16 AQUA score > 244 (upper 15%) was associated with PFS (p= 0.006-95% CI 8.4 [1.8-38.6]) and OS (p= 0.013-95% CI 4.5 [1.4-15]). Conclusions: p16 expression via quantitative IHC correlates with other methods, including HPV16 subtyping, and appears more reliable for defining HPV16 status than CISH. Only quantitative IHC identified a subset of patients with worse outcome. No significant financial relationships to disclose.