Accurate HPV testing: a requirement for precision medicine for head and neck cancer
2013; Elsevier BV; Volume: 24; Issue: 11 Linguagem: Inglês
10.1093/annonc/mdt417
ISSN1569-8041
Autores Tópico(s)Cancer-related molecular mechanisms research
ResumoHuman papilloma virus-related head and neck cancer (HPV-positive HNC) is clinically and biologically distinct from tobacco-related (HPV-negative) HNC [1.Leemans C.R. Braakhuis B.J.M. Brakenhoff R.H. The molecular biology of head and neck cancer.Nature Publishing Group. 2010; 11: 9-22PubMed Google Scholar, 2.Hayes D.N. Grandis J.R. El-Naggar A.K. The cancer genome atlas: integrated analysis of genome alterations in squamous cell carcinoma of the head and neck.J Clin Oncol. 2013; 31 (suppl; abstr 6009) 2013Crossref Google Scholar]. HPV-positive tumors have a favorable prognosis and respond better to chemotherapy, radiation and chemoradiation [3.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 (4633) Google Scholar, 4.Posner M.R. Lorch J.H. Goloubeva O. et al.Survival and human papillomavirus in oropharynx cancer in TAX 324: a subset analysis from an international phase III trial.Ann Oncol. 2011; 22: 1071-1077Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 5.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 (2112) Google Scholar, 6.Lassen P. Eriksen J.G. Hamilton-Dutoit S. et al.Effect of HPV-associated p16INK4A expression on response to radiotherapy and survival in squamous cell carcinoma of the head and neck.J Clin Oncol. 2009; 27: 1992-1998Crossref PubMed Scopus (508) Google Scholar, 7.Rischin D. Young R.J. Fisher R. et al.Prognostic significance of p16INK4A and human papillomavirus in patients with oropharyngeal cancer treated on TROG 02.02 phase III trial.J Clin Oncol. 2010; 28: 4142-4148Crossref PubMed Scopus (600) Google Scholar]. While currently HPV status does not influence treatment choices, increasingly clinical trials are exploring whether HPV-positive tumors may require less therapy, thus potentially reducing long-term side-effects in a younger patient population. Also, emerging insights into the genetic differences between HPV-positive and HPV-negative HNC may eventually guide treatment choices: e.g. HPV-negative tumors appear to lack epidermal growth factor receptor (EGFR) and other kinase amplifications, and consistently show lower expression levels of EGFR [2.Hayes D.N. Grandis J.R. El-Naggar A.K. The cancer genome atlas: integrated analysis of genome alterations in squamous cell carcinoma of the head and neck.J Clin Oncol. 2013; 31 (suppl; abstr 6009) 2013Crossref Google Scholar, 8.Keck M.K. Zuo Z. Khattri A. et al.Genomic profiling of kinase genes in head and neck squamous cell carcinomas to identify potentially targetable genetic aberrations in FGFR1/2, DDR2, EPHA2, and PIK3CA.J Clin Oncol. 2013; 31 (suppl; abstr 6010)Crossref Google Scholar, 9.Young R.J. Rischin D. Fisher R. et al.Relationship between epidermal growth factor receptor status, p16INK4A, and outcome in head and neck squamous cell carcinoma.Cancer Epidemiol Biomarkers Prev. 2011; 20: 1230-1237Crossref PubMed Scopus (79) Google Scholar, 10.Vokes E.E. Seiwert T.Y. EGFR-directed treatments in SCCHN.Lancet Oncol. 2013; 14: 672-673Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar]. In contrast, HPV-positive tumors have higher rates of PI3K-pathway alterations [11.Seiwert T.Y. Keck M. Zuo Z. et al.Genomic profiling of a clinically annotated cohort of locoregionally advanced head and neck cancers (HNC) treated with definitive chemoradiotherapy.J Clin Oncol. 2012; 30 (suppl; abstr 5517)Crossref Google Scholar, 12.Lui V.W. Hedberg M.L. Li H. et al.Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers.Cancer Discov. 2013; 3: 761-769Crossref PubMed Scopus (430) Google Scholar]. HPV-positive HNC invariably expresses the viral proteins E6 and E7, which are essential for oncogenic transformation: while 83% of HPV-negative tumors harbor TP53 mutations, TP53 is virtually always wild-type in HPV-positive HNC [2.Hayes D.N. Grandis J.R. El-Naggar A.K. The cancer genome atlas: integrated analysis of genome alterations in squamous cell carcinoma of the head and neck.J Clin Oncol. 2013; 31 (suppl; abstr 6009) 2013Crossref Google Scholar, 13.Zuo Z. Keck M. Khattri A. et al.Multimodality determination of HPV status in head and neck cancers (HNC) and development of an HPV signature.J Clin Oncol. 2013; 31 (suppl; abstr 6008)Crossref Google Scholar], where it is inactivated at the protein level by E6 [14.McLaughlin-Drubin M.E. Munger K. Oncogenic activities of human papillomaviruses.Virus Res. 2009; 143: 195-208Crossref PubMed Scopus (232) Google Scholar]. In HPV-negative tumors, the cell cycle is activated by loss of the tumor suppressor p16, as well as frequent cyclin D1 amplification. Still p16 loss is not universal and an estimated 10%–15% of HPV-negative tumors express p16 [2.Hayes D.N. Grandis J.R. El-Naggar A.K. The cancer genome atlas: integrated analysis of genome alterations in squamous cell carcinoma of the head and neck.J Clin Oncol. 2013; 31 (suppl; abstr 6009) 2013Crossref Google Scholar, 13.Zuo Z. Keck M. Khattri A. et al.Multimodality determination of HPV status in head and neck cancers (HNC) and development of an HPV signature.J Clin Oncol. 2013; 31 (suppl; abstr 6008)Crossref Google Scholar]. In contrast, HPV-positive tumors typically do not show p16 loss, as the cell cycle is activated via E7, which inhibits the cell-cycle regulator pRB, bypassing p16 [14.McLaughlin-Drubin M.E. Munger K. Oncogenic activities of human papillomaviruses.Virus Res. 2009; 143: 195-208Crossref PubMed Scopus (232) Google Scholar]. HPV-positive HNC arises likely over a period of years by viral persistence, immune escape and accumulation of additional genetic aberrations [14.McLaughlin-Drubin M.E. Munger K. Oncogenic activities of human papillomaviruses.Virus Res. 2009; 143: 195-208Crossref PubMed Scopus (232) Google Scholar]. Commonly used HPV tests rely either on the detection of HPV genetic material or on the expression of the p16 protein. Given the ease of using standard formalin-fixed paraffin-embedded diagnostic material, p16 detection by immunohistochemistry (IHC), or HPV in-situ hybridization (ISH), which hybridizes probes for detection to high-risk HPV genetic material (most commonly HPV DNA), are the dominant HPV tests in clinical practice. Both p16 IHC and HPV ISH are relatively easy to implement since they utilize standard formalin-fixed paraffin embedded (FFPE) diagnostic material. Both tests perform well, especially for oropharyngeal primary tumors, where the pretest probability for HPV positivity is high and both are powerful prognostic biomarkers [3.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 (4633) Google Scholar, 4.Posner M.R. Lorch J.H. Goloubeva O. et al.Survival and human papillomavirus in oropharynx cancer in TAX 324: a subset analysis from an international phase III trial.Ann Oncol. 2011; 22: 1071-1077Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 5.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 (2112) Google Scholar, 6.Lassen P. Eriksen J.G. Hamilton-Dutoit S. et al.Effect of HPV-associated p16INK4A expression on response to radiotherapy and survival in squamous cell carcinoma of the head and neck.J Clin Oncol. 2009; 27: 1992-1998Crossref PubMed Scopus (508) Google Scholar, 7.Rischin D. Young R.J. Fisher R. et al.Prognostic significance of p16INK4A and human papillomavirus in patients with oropharyngeal cancer treated on TROG 02.02 phase III trial.J Clin Oncol. 2010; 28: 4142-4148Crossref PubMed Scopus (600) Google Scholar]. The problem with p16 IHC is the suboptimal specificity and in particular, a 3.8%–7.3% false-positive rate; the problem with HPV ISH is the low sensitivity with a 13%–41% false-negative rate [15.Thavaraj S. Stokes A. Guerra E. et al.Evaluation of human papillomavirus testing for squamous cell carcinoma of the tonsil in clinical practice.J Clin Pathol. 2011; 64: 308-312Crossref PubMed Scopus (115) Google Scholar, 16.Schlecht N.F. Brandwein-Gensler M. Nuovo G.J. et al.A comparison of clinically utilized human papillomavirus detection methods in head and neck cancer.Mod Pathol. 2011; 24: 1295-1305Crossref PubMed Scopus (161) Google Scholar]. As we move closer toward new HPV-specific treatment paradigms, there are two pivotal questions that have to be answered:(i)Is HPV status the only prognostic biomarker (and driver of biologic behavior)- or does p16 expression status potentially add additional information –, e.g. in HPV-negative, p16-positive HNC?(ii)Which HPV tests are most accurate- and provide sufficient accuracy to enable us to confidently make treatment decisions? Arguably, the biggest contribution Rietbergen et al's article in this issue of Annals of Oncology [17.Rietbergen M.M. Brakenhoff R.H. Bloemena E. et al.Human papillomavirus detection and comorbidity: critical issues in selection of patients with oropharyngeal cancer for treatment de-escalation trials.Ann Oncol. 2013; 24: 2740-2745Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar] provides is a clear answer to the first question: HPV status is the most important prognostic biomarker, while p16 status is only an approximation for HPV, and not an independent prognostic factor. Rietbergen et al show that in patients with oropharyngeal HNC 3.8% of p16-positive tumors are in reality HPV negative and this is consistent with a report by Jordan et al. reporting a false-positive rate of 7.3% [18.Jordan R.C. Lingen M.W. Perez-Ordonez B. et al.Validation of methods for oropharyngeal cancer HPV status determination in US cooperative group trials.Am J Surg Pathol. 2012; 36: 945-954Crossref PubMed Scopus (305) Google Scholar]. The authors demonstrate that such tumors retain the clinical and prognostic characteristics of HPV-negative tumors. Furthermore, a recent report by the Radiation Therapy Oncology Group (RTOG) indicated that HPV status (as measured by p16 IHC and HPV ISH) is also prognostic outside the oropharynx [19.Chung C.H. Zhang Q. Kong C. et al.P16 expression as a human papillomavirus (HPV)-independent prognostic biomarker in non-oropharyngeal squamous cell carcinoma (non-OPSCC).J Clin Oncol. 2013; 31 (suppl; abstr 6010)Crossref Google Scholar], where only a smaller percentage (∼5%) of tumors is HPV positive [19.Chung C.H. Zhang Q. Kong C. et al.P16 expression as a human papillomavirus (HPV)-independent prognostic biomarker in non-oropharyngeal squamous cell carcinoma (non-OPSCC).J Clin Oncol. 2013; 31 (suppl; abstr 6010)Crossref Google Scholar, 20.Lingen M.W. Xiao W. Schmidt A. et al.Low etiologic fraction for high-risk human papillomavirus in oral cavity squamous cell carcinomas.Oral Oncol. 2012; : 1-8PubMed Google Scholar]. This report also suggested that p16-positive/ISH-negative patients have an 'intermediate' prognosis. How can this at first contradictory appearing result be reconciled with the current report by Rietbergen et al., where p16-positive, HPV-negative tumors have no better prognosis than p16/HPV double-negative tumors? The answer is likely related to the significantly higher sensitivity of PCR (>98%) compared with ISH (∼57–89%) [13.Zuo Z. Keck M. Khattri A. et al.Multimodality determination of HPV status in head and neck cancers (HNC) and development of an HPV signature.J Clin Oncol. 2013; 31 (suppl; abstr 6008)Crossref Google Scholar, 15.Thavaraj S. Stokes A. Guerra E. et al.Evaluation of human papillomavirus testing for squamous cell carcinoma of the tonsil in clinical practice.J Clin Pathol. 2011; 64: 308-312Crossref PubMed Scopus (115) Google Scholar, 16.Schlecht N.F. Brandwein-Gensler M. Nuovo G.J. et al.A comparison of clinically utilized human papillomavirus detection methods in head and neck cancer.Mod Pathol. 2011; 24: 1295-1305Crossref PubMed Scopus (161) Google Scholar]. It is a prime example of why the use of an insufficiently accurate biomarker hampers interpretation of study results and could potentially negatively affect our ability to make appropriate treatment decisions. Along the same line of argument, Ang et al suggested that the improved predictive power of p16 over ISH in the RTOG0129 study is explained by HPV-positive tumors that are missed by ISH due to the lower sensitivity of the assay [3.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 (4633) Google Scholar, 18.Jordan R.C. Lingen M.W. Perez-Ordonez B. et al.Validation of methods for oropharyngeal cancer HPV status determination in US cooperative group trials.Am J Surg Pathol. 2012; 36: 945-954Crossref PubMed Scopus (305) Google Scholar, 21.Ukpo O.C. Flanagan J.J. Ma X.-J. et al.High-risk human papillomavirus E6/E7 mRNA detection by a novel in-situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma.Am J Surg Pathol. 2011; 35: 1343-1350Crossref PubMed Scopus (241) Google Scholar]. Rietbergen et al used a more rigorous PCR-based methodology combined with p16 IHC, which enabled them to isolate p16 IHC's lack of prognostic implications in patients with HPV-negative tumors [17.Rietbergen M.M. Brakenhoff R.H. Bloemena E. et al.Human papillomavirus detection and comorbidity: critical issues in selection of patients with oropharyngeal cancer for treatment de-escalation trials.Ann Oncol. 2013; 24: 2740-2745Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar]. They demonstrate that accurate HPV-testing matters. In conclusion, in response to the first question, there are no convincing data at this point to suggest that p16 in the absence of HPV (if determined accurately) is meaningful. Further studies of the role of p16 as an independent prognostic marker may still be of interest, if not to confirm the results by Rietbergen et al, but must include accurate HPV testing in order to be interpretable. With respect to the second question regarding optimal HPV testing, for oropharynx tumors the reports by Rietbergen et al. and Jordan et al.[18.Jordan R.C. Lingen M.W. Perez-Ordonez B. et al.Validation of methods for oropharyngeal cancer HPV status determination in US cooperative group trials.Am J Surg Pathol. 2012; 36: 945-954Crossref PubMed Scopus (305) Google Scholar] indicate that between 3.8% and 7.3% of p16 IHC-positive oropharynx cancers are in fact HPV negative. In order to make treatment decisions, e.g. in the curative-intent setting, this is not acceptable: in a worst case scenario 1 in 13 patients would be misclassified, i.e. a high-risk tumor would be assessed as low risk, and with the prospect of de-escalation would potentially receive insufficient therapy. For tumors arising outside the oropharynx, p16IHC performs poorly, and should not be used by itself: in the oral cavity, the positive predictive value for p16 is only 41.3%, meaning 6 out of 10 times a positive p16 IHC result will misclassify the tumor [20.Lingen M.W. Xiao W. Schmidt A. et al.Low etiologic fraction for high-risk human papillomavirus in oral cavity squamous cell carcinomas.Oral Oncol. 2012; : 1-8PubMed Google Scholar]. Similarly, Chung et al. reported a poor correlation of p16 IHC and HPV ISH in oral cavity tumors, and poor-to-moderate correlation for hypopharyngeal and laryngeal tumors [19.Chung C.H. Zhang Q. Kong C. et al.P16 expression as a human papillomavirus (HPV)-independent prognostic biomarker in non-oropharyngeal squamous cell carcinoma (non-OPSCC).J Clin Oncol. 2013; 31 (suppl; abstr 6010)Crossref Google Scholar]. This is in particular important as HPV status is also prognostically important in non-oropharynx tumors [19.Chung C.H. Zhang Q. Kong C. et al.P16 expression as a human papillomavirus (HPV)-independent prognostic biomarker in non-oropharyngeal squamous cell carcinoma (non-OPSCC).J Clin Oncol. 2013; 31 (suppl; abstr 6010)Crossref Google Scholar], and given the similar HPV-driven biology as documented in the Cancer Genome Atlas Project [2.Hayes D.N. Grandis J.R. El-Naggar A.K. The cancer genome atlas: integrated analysis of genome alterations in squamous cell carcinoma of the head and neck.J Clin Oncol. 2013; 31 (suppl; abstr 6009) 2013Crossref Google Scholar], may also be candidates for de-escalation approaches in the future. Both p16 IHC and HPV PCR show excellent sensitivity but have lower than ideal specificity. Still when combined as a composite assay (as employed by Rietbergen et al.[17.Rietbergen M.M. Brakenhoff R.H. Bloemena E. et al.Human papillomavirus detection and comorbidity: critical issues in selection of patients with oropharyngeal cancer for treatment de-escalation trials.Ann Oncol. 2013; 24: 2740-2745Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar]), accurate HPV testing is achievable: by combining both assays, sensitivity remains high, but specificity increases markedly given non-overlapping limitations, i.e. p16 IHC-positive/HPV-negative tumors versus detection of HPV not involved in the oncogenic process (HPV-bystander).Unfortunately, Rietbergen et al. in their study did not employ a true gold standard (i.e. E6/E7 mRNA detection [18.Jordan R.C. Lingen M.W. Perez-Ordonez B. et al.Validation of methods for oropharyngeal cancer HPV status determination in US cooperative group trials.Am J Surg Pathol. 2012; 36: 945-954Crossref PubMed Scopus (305) Google Scholar], or consensus HPV testing [13.Zuo Z. Keck M. Khattri A. et al.Multimodality determination of HPV status in head and neck cancers (HNC) and development of an HPV signature.J Clin Oncol. 2013; 31 (suppl; abstr 6008)Crossref Google Scholar]). Still their methods are rigorous and sufficiently accurate enabling them to isolate the lack of prognostic implications of p16 IHC-positive/HPV-negative status. Combined p16 IHC/HPV PCR is feasible in many larger pathology laboratories that also do molecular testing, and should be considered when accurate HPV determination is required in order to make treatment decisions. The combination of p16 IHC with HPV ISH in contrast is less helpful as the results are essentially limited (and driven) by the lower sensitivity (and high specificity) of HPV ISH [15.Thavaraj S. Stokes A. Guerra E. et al.Evaluation of human papillomavirus testing for squamous cell carcinoma of the tonsil in clinical practice.J Clin Pathol. 2011; 64: 308-312Crossref PubMed Scopus (115) Google Scholar, 16.Schlecht N.F. Brandwein-Gensler M. Nuovo G.J. et al.A comparison of clinically utilized human papillomavirus detection methods in head and neck cancer.Mod Pathol. 2011; 24: 1295-1305Crossref PubMed Scopus (161) Google Scholar]. Other promising accurate HPV tests are feasible, including more accurate HPV PCRs based on E6/E7 detection as reported by our group recently [13.Zuo Z. Keck M. Khattri A. et al.Multimodality determination of HPV status in head and neck cancers (HNC) and development of an HPV signature.J Clin Oncol. 2013; 31 (suppl; abstr 6008)Crossref Google Scholar], consensus HPV testing [13.Zuo Z. Keck M. Khattri A. et al.Multimodality determination of HPV status in head and neck cancers (HNC) and development of an HPV signature.J Clin Oncol. 2013; 31 (suppl; abstr 6008)Crossref Google Scholar], or alternative HPV mRNA detection methods, e.g. by massively parallel sequencing (RNA-Seq) [2.Hayes D.N. Grandis J.R. El-Naggar A.K. The cancer genome atlas: integrated analysis of genome alterations in squamous cell carcinoma of the head and neck.J Clin Oncol. 2013; 31 (suppl; abstr 6009) 2013Crossref Google Scholar], or RNA-based ISH detection [21.Ukpo O.C. Flanagan J.J. Ma X.-J. et al.High-risk human papillomavirus E6/E7 mRNA detection by a novel in-situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma.Am J Surg Pathol. 2011; 35: 1343-1350Crossref PubMed Scopus (241) Google Scholar]. Even in the presence of accurate HPV testing, we need to be cognizant that additional risk factors modify the risk profile. Ang et al. [3.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 (4633) Google Scholar] and the study by Rietbergen et al in this issue of Annals of Oncology demonstrate that use of tobacco products has negative prognostic implications in patients with HPV-positive tumors. We know that some HPV-positive tumors behave more aggressively, and tobacco history is one way to identify such higher risk patients that should arguably not be candidates for future de-intensification approaches [3.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 (4633) Google Scholar]. This may be related to overlapping biologic processes between some HPV-positive and HPV-negative tumors [13.Zuo Z. Keck M. Khattri A. et al.Multimodality determination of HPV status in head and neck cancers (HNC) and development of an HPV signature.J Clin Oncol. 2013; 31 (suppl; abstr 6008)Crossref Google Scholar]. Furthermore, Rietbergen et al. demonstrate that comorbidities are an additional important risk modifier in HPV-positive HNC. This is the third important finding of this study, although the implications are less clear: should patients with comorbidities not be candidates for de-intensification in the future, or would these patients be the ones benefitting from lower treatment intensity? In conclusion, Rietbergen et al. demonstrate that HPV status and not p16 IHC is the dominant biomarker for HNC patients. Accurate HPV testing will allow us to better risk-stratify patients in conjunction with smoking history, comorbidities and potentially additional molecular markers. Accurate HPV testing can be implemented by combined p16 IHC/HPV PCR testing or a number of the newer HPV detection methods. Unfortunately, neither p16 IHC nor HPV ISH alone or in combination is suitable as a biomarker for our efforts to de-escalate treatment even when limited to tumors arising in the oropharynx. Accurate HPV testing is a required first step to enable us to deliver precision medicine for our patients with HNC in both the curative and palliative settings in the near future. The author has declared no conflicts of interest.
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