Evaluation of Human Papilloma Virus Diagnostic Testing in Oropharyngeal Squamous Cell Carcinoma: Sensitivity, Specificity, and Prognostic Discrimination

Abstract

Purpose: Human papillomavirus-16 (HPV16) is the causative agent in a biologically distinct subset of oropharyngeal squamous cell carcinoma (OPSCC) with highly favorable prognosis. In clinical trials, HPV16 status is an essential inclusion or stratification parameter, highlighting the importance of accurate testing.Experimental Design: Fixed and fresh-frozen tissue from 108 OPSCC cases were subject to eight possible assay/assay combinations: p16 immunohistochemistry (p16 IHC); in situ hybridization for highrisk HPV (HR HPV ISH); quantitative PCR (qPCR) for both viral E6 RNA (RNA qPCR) and DNA (DNA qPCR); and combinations of the above.Results: HPV16-positive OPSCC presented in younger patients (mean 7.5 years younger, P ¼ 0.003) who smoked less than HPV-negative patients (P ¼ 0.007). The proportion of HPV16-positive cases increased from 15% to 57% (P ¼ 0.001) between 1988 and 2009. A combination of p16 IHC/DNA qPCR showed acceptable sensitivity (97%) and specificity (94%) compared with the RNA qPCR "gold standard", as well as being the best discriminator of favorable outcome (overall survival P ¼ 0.002). p16 IHC/HR HPV ISH also had acceptable specificity (90%) but the substantial reduction in its sensitivity (88%) impacted upon its prognostic value (P ¼ 0.02). p16 IHC, HR HPV ISH, or DNA qPCR was not sufficiently specific to recommend in clinical trials when used in isolation.Conclusions: Caution must be exercised in applying HPV16 diagnostic tests because of significant disparities in accuracy and prognostic value in previously published techniques.

p16 Immunohistochemistry As a Standalone Test for Risk Stratification in Oropharyngeal Squamous Cell Carcinoma

“…There are many tests that reflect (or at least correlate with) biologically or transcriptionally-active HPV, including DNA in situ hybridization (ISH) [2,5] (Fig. 1a), RT-PCR for E6 and E7 mRNA [6][7][8], and more recently RNA ISH [9] (Fig. 1b).…”

“…Many advocate p16 IHC as a screening test which, when positive, is to be followed by an HPV specific test such as DNA ISH, PCR, or both [1,7,10,11]. There is an emerging view in oropharyngeal SCC, however, that the high correlation with detectable HPV RNA [9] and the strong risk stratification for patient survival provided by p16 IHC make it a suitable single marker for defining a patient's tumor as biologically and clinically favorable for treatment and counseling.…”

“…Most of the studies are retrospective, but several prospective studies have been performed and analyzed as well [2,3,5,9,12]. There is very high correlation between the HPVspecific tests (of all types) [7,9] and also between p16 immunohistochemistry and HPV-specific testing, particularly with the newer HPV RNA tests (Table 1) [2,3,5,[8][9][10][11][13][14][15]. In a pooled analysis by Robinson et al [1] of 496 patients from several studies utilizing DNA-based HPV testing, the authors reported that only 5% of cases were p16 positive/HPV negative and that 8% were p16 negative/HPV positive.…”

“…In a pooled analysis by Robinson et al [1] of 496 patients from several studies utilizing DNA-based HPV testing, the authors reported that only 5% of cases were p16 positive/HPV negative and that 8% were p16 negative/HPV positive. More recent studies have utilized HPV E6/E7 mRNA testing and have found [7][8][9] only 1-7% of p16 positive tumors to be HPV RNA negative, and only 2-7% of p16 negative tumors to be HPV RNA positive. From all of this data, it is widely acknowledged that p16 overexpression is a very sensitive marker for transcriptionally-active HPV.…”

“…The argument being that ''a significant minority of p16 positive oropharyngeal SCC cases lack the virus, with p16 being overexpressed by other mechanisms [16]. '' It has been suggested that screening patients' tumors for p16 expression be performed first, and if positive, followed by an HPV-specific test, such as DNA ISH or DNA PCR (or both), for practical purposes [1,7,11]. The rationale is that this provides direct evidence that HPV is in the tumor and that, based on p16 overexpression, the HPV is transcriptionally-active.…”