HPV status, like politics, is local—evaluating p16 staining and a new staging system in a Dutch cohort of oropharynx cancer
Authors: R.K. Orosco, J.A. Califano
Journal: Annals of Oncology (2018)

HPV-negative and HPV-positive OPSCC are two fundamentally distinct diseases with different mechanisms of tumorigenesis, presentation, prognosis, and response to treatment [1.Ang K.K. Harris J. Wheeler R. et al.Human papillomavirus and survival of patients with oropharyngeal cancer.N Engl J Med. 2010; 363: 24-35Crossref PubMed Scopus (3977) Google Scholar, 2.Castellsague X. Alemany L. Quer M. et al.HPV involvement in head and neck cancers: comprehensive assessment of biomarkers in 3680 patients.J Natl Cancer Inst. 2016; 108: djv403.Crossref PubMed Scopus (355) Google Scholar, 3.Fakhry C. Westra W.H. Li S. et al.Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial.J Natl Cancer Inst. 2008; 100: 261-269Crossref PubMed Scopus (1931) Google Scholar, 4.Vokes E.E. Agrawal N. Seiwert T.Y. HPV-associated head and neck cancer.J Natl Cancer Inst. 2015; 107: djv344.Crossref PubMed Scopus (96) Google Scholar]. In the landmark study by Ang et al. based on the US patients, p16 immunohistochemistry (IHC) and HPV in situ hybridization and other DNA-based testing were highly concordant in defining a favorable prognosis, HPV-related subtype of OPSCC. Reflecting this and other subsequent studies, the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual defines separate staging systems for HPV-negative and HPV-positive OPSCC, with p16 IHC as an acceptable standard for defining HPV-related OPSCC [5.Burd E.M. Human papillomavirus laboratory testing: the changing paradigm.Clin Microbiol Rev. 2016; 29: 291-319Crossref PubMed Scopus (71) Google Scholar]. In this issue of Annals of Oncology, Nauta et al. [6.Nauta I.H. Rietbergen M.M. van Bokhoven A.A.J.D. et al.Evaluation of the eighth TNM classification on p16-positive oropharyngeal squamous cell carcinomas in the Netherlands and the importance of additional HPV DNA testing.Ann Oncol. 2018; 29: 1273-1279Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar] evaluate the new AJCC 8th edition staging system and explore our current methodology for classifying HPV-related tumors in a cohort of 1204 patients with OPSCC from two Dutch medical centers. Tumor samples underwent p16 IHC, and positive specimens underwent additional high-risk HPV-DNA testing via PCR amplification. Reassuringly, AJCC 8th edition staging for HPV-related OPSCC based on p16 IHC exhibited superior prognostic power over 7th edition staging. More interestingly, the authors identified and characterized a subset of patients (p16-positive/HPV-DNA-negative) that may be incorrectly classified as having HPV-related cancers. The clinical, demographic, and oncologic behavior of this group was closer to that of p16-negative (non-HPV-related) patients than to their p16-positive/HPV-DNA-positive counterparts, including a worse overall survival. Of note, these p16-positive/HPV-DNA-negative patients were more likely to be treated with radiation alone compared with chemoradiation, lending a note of caution to interpretation, although no difference in survival was noted in this subgroup based on treatment. By stratifying patients based on p16 and HPV-DNA status, the authors highlight critical definitions within OPSCC subpopulation with implications for treatment and clinical trial design. Based solely on p16-status, p16-positive/HPV-DNA-negative patients may spuriously be labeled, and incorrectly staged, as HPV-associated OPSCC, noting misclassification in 12% of Dutch patients in their study [6.Nauta I.H. Rietbergen M.M. van Bokhoven A.A.J.D. et al.Evaluation of the eighth TNM classification on p16-positive oropharyngeal squamous cell carcinomas in the Netherlands and the importance of additional HPV DNA testing.Ann Oncol. 2018; 29: 1273-1279Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar]. This Dutch study highlights the imperfect performance of p16 status for categorizing OPSCC patients as having HPV-related disease in specific populations. The false-positive rate for p16 staining was nearly 19% in early-work by Ang et al. [1.Ang K.K. Harris J. Wheeler R. et al.Human papillomavirus and survival of patients with oropharyngeal cancer.N Engl J Med. 2010; 363: 24-35Crossref PubMed Scopus (3977) Google Scholar], and the present study cites a 5%–20% rate across several others. Although lower, the false-negative rate for p16 staining is not insignificant [1.Ang K.K. Harris J. Wheeler R. et al.Human papillomavirus and survival of patients with oropharyngeal cancer.N Engl J Med. 2010; 363: 24-35Crossref PubMed Scopus (3977) Google Scholar, 2.Castellsague X. Alemany L. Quer M. et al.HPV involvement in head and neck cancers: comprehensive assessment of biomarkers in 3680 patients.J Natl Cancer Inst. 2016; 108: djv403.Crossref PubMed Scopus (355) Google Scholar]. A recent systematic review and meta-analysis of the diagnostic accuracy of p16 IHC in OPSCCs analyzed 24 studies and concluded p16INK4a IHC has a high sensitivity, but modest specificity to classify HPV status when used as a single test. When both p16 IHC and HPV-DNA PCR testing were combined, there was significantly higher specificity with preservation of high sensitivity [7.Prigge E.S. Arbyn M. von Knebel Doeberitz M. Reuschenbach M. Diagnostic accuracy of p16(INK4a) immunohistochemistry in oropharyngeal squamous cell carcinomas: a systematic review and meta-analysis.Int J Cancer. 2017; 140: 1186-1198Crossref PubMed Scopus (108) Google Scholar]. This test performance depends on disease prevalence, and the attributable fraction of HPV-related OPSCC cases has been shown to vary by geography, cancer site, and gender [2.Castellsague X. Alemany L. Quer M. et al.HPV involvement in head and neck cancers: comprehensive assessment of biomarkers in 3680 patients.J Natl Cancer Inst. 2016; 108: djv403.Crossref PubMed Scopus (355) Google Scholar]. Therefore, the utility of p16 status as a surrogate marker for HPV will vary according to the prevalence of HPV-related disease in particular population, which can range from low single digits Brazil to as high as 60% in the US populations [8.Anantharaman D. Abedi-Ardekani B. Beachler D.C. et al.Geographic heterogeneity in the prevalence of human papillomavirus in head and neck cancer.Int J Cancer. 2017; 140: 1968-1975Crossref PubMed Scopus (69) Google Scholar]. Issues with the determination of true HPV status have broad implications for the use of p16 for cancer staging, treatment selection, and clinical trials design and eligibility. P16-positive patients who are HPV-DNA-negative, but untested, may potentially ‘contaminate’ the pool of favorable prognosis HPV-related patients. These patients may also be inappropriately considered for treatment de-intensification protocols. Furthermore, p16-positive/HPV-DNA-negative patients assigned to clinical trials designed for HPV-positive tumors may interfere with therapeutic effects and lead to flawed conclusions. These data indicate that HPV determination, clinical trials eligibility, staging, treatment algorithms, and interpretation of clinical trials data based on p16 status alone may be dependent on the prevalence of HPV-related OPSCC within a specific population as well as other factors. Current assays for HPV DNA on formalin-fixed paraffin-embedded OPSCC include an array of IHC, DNA in situ hybridization, PCR, and sequencing for a variety of HPV strains, protein products, and associated markers, including p16. Ideally, HPV genotype in OPSCC tumors would be determined by consensus assays that are validated across populations with facile standardized measures. HPV-DNA studies and genotyping have been integrated into cervical cancer screening and diagnosis algorithms [5.Burd E.M. Human papillomavirus laboratory testing: the changing paradigm.Clin Microbiol Rev. 2016; 29: 291-319Crossref PubMed Scopus (71) Google Scholar], but there are several barriers to adopting these practices in the management of OPSCC including: cost, need to validate the assays in diverse populations, and limitations imposed by formalin-fixed paraffin-embedded specimens as opposed to pap smear swabs. The findings by Nauta et al. [6.Nauta I.H. Rietbergen M.M. van Bokhoven A.A.J.D. et al.Evaluation of the eighth TNM classification on p16-positive oropharyngeal squamous cell carcinomas in the Netherlands and the importance of additional HPV DNA testing.Ann Oncol. 2018; 29: 1273-1279Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar] indicate that there is a need for increased sophistication in our determination of HPV status in the OPSCC population. Additional work is needed to improve our understanding of p16 and HPV test performance in other populations, as well as improve standardization of determination of HPV status in OPSCC across these diverse populations, so that diagnosis, treatment, trial enrollment, and interpretation of these data truly reflect a cohort of OPSCC patients defined by HPV-related disease. None declared.