Identification of a Human Papillomavirus–Associated Oncogenic miRNA Panel in Human Oropharyngeal Squamous Cell Carcinoma Validated by Bioinformatics Analysis of The Cancer Genome Atlas

Abstract

High-risk human papillomavirus (HPV) is a causative agent for an increasing subset of oropharyngeal squamous cell carcinomas (OPSCCs), and current evidence supports these tumors as having identifiable risk factors and improved response to therapy. However, the biochemical and molecular alterations underlying the pathobiology of HPV-associated OPSCC (designated HPV+ OPSCC) remain unclear. Herein, we profile miRNA expression patterns in HPV+ OPSCC to provide a more detailed understanding of pathologic molecular events and to identify biomarkers that may have applicability for early diagnosis, improved staging, and prognostic stratification. Differentially expressed miRNAs were identified in RNA isolated from an initial clinical cohort of HPV+/− OPSCC tumors by quantitative PCR–based miRNA profiling. This oncogenic miRNA panel was validated using miRNA sequencing and clinical data from The Cancer Genome Atlas and miRNA in situ hybridization. The HPV-associated oncogenic miRNA panel has potential utility in diagnosis and disease stratification and in mechanistic elucidation of molecular factors that contribute to OPSCC development, progression, and differential response to therapy. High-risk human papillomavirus (HPV) is a causative agent for an increasing subset of oropharyngeal squamous cell carcinomas (OPSCCs), and current evidence supports these tumors as having identifiable risk factors and improved response to therapy. However, the biochemical and molecular alterations underlying the pathobiology of HPV-associated OPSCC (designated HPV+ OPSCC) remain unclear. Herein, we profile miRNA expression patterns in HPV+ OPSCC to provide a more detailed understanding of pathologic molecular events and to identify biomarkers that may have applicability for early diagnosis, improved staging, and prognostic stratification. Differentially expressed miRNAs were identified in RNA isolated from an initial clinical cohort of HPV+/− OPSCC tumors by quantitative PCR–based miRNA profiling. This oncogenic miRNA panel was validated using miRNA sequencing and clinical data from The Cancer Genome Atlas and miRNA in situ hybridization. The HPV-associated oncogenic miRNA panel has potential utility in diagnosis and disease stratification and in mechanistic elucidation of molecular factors that contribute to OPSCC development, progression, and differential response to therapy. Head and neck squamous cell carcinoma (HNSCC) is a leading cause of cancer death worldwide. High-risk human papillomavirus (HPV), particularly HPV16, is a causative agent for an increasing subset of oropharyngeal SCCs (OPSCCs). Current evidence supports these tumors (designated HPV+ OPSCC) as having unique biology because they have relatively low mutational density, wild-type cellular tumor antigen p53, and p16 protein overexpression.1Miller D.L. Puricelli M.D. Stack M.S. Virology and molecular pathogenesis of HPV (human papillomavirus)-associated oropharyngeal squamous cell carcinoma.Biochem J. 2012; 443: 339-353Crossref PubMed Scopus (52) Google Scholar HPV+ OPSCC is already well established as a distinct clinical entity with identifiable epidemiologic characteristics and risk factors.2Gillison M.L. D'Souza G. Westra W. Sugar E. Xiao W. Begum S. Viscidi R. Distinct risk factor profiles for human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck cancers.J Natl Cancer Inst. 2008; 100: 407-420Crossref PubMed Scopus (1194) Google Scholar However, a detailed understanding of the biology and the translation of that understanding into clinically applicable tools to decrease mortality and to lessen morbidity from treatment is clearly lacking. Although clinical trials assessing differing management strategies for the two diseases are under way, currently, HPV+ patients are treated within the standard of care guidelines for OPSCC, in the same manner as HPV− patients. However, HPV+ tumors of the oropharynx are associated with distinct survival strata because HPV+ patients have prolonged progression-free responses and often, with appropriate surgical management, chemotherapy, and irradiation, experience complete responses that seem durable.3Ang K.K. Harris J. Wheeler R. Weber R. Rosenthal D.I. Nguyen-Tân P.F. Westra W.H. Chung C.H. Jordan R.C. Lu C. Kim H. Axelrod R. Silverman C.C. Redmond K.P. Gillison M.L. Human papillomavirus and survival of patients with oropharyngeal cancer.N Engl J Med. 2010; 363: 24-35Crossref PubMed Scopus (4806) Google Scholar Thus, early identification and characterization of this patient cohort is necessary to tailor care to the unique biology of their tumors. As such, characterization of an HPV-driven OPSCC tumor is itself a biomarker that demands management strategies distinct from those associated with traditional HNSCC. Owing to public health efforts regarding smoking cessation, the overall incidence of most HNSCC has decreased in recent years. In contrast, US incidence rates of OPSCC have increased significantly yearly between 1973 and 2004 for base of tongue (1.3%) and tonsillar (0.6%) carcinomas, corresponding to a 225% increase in the population-level incidence of HPV+ OPSCC between 1984 and 2004.4Marur S. D'Souza G. Westra W.H. Forastiere A.A. HPV-associated head and neck cancer: a virus-related cancer epidemic.Lancet Oncol. 2010; 11: 781-789Abstract Full Text Full Text PDF PubMed Scopus (1398) Google Scholar, 5Chaturvedi A.K. Engels E.A. Pfeiffer R.M. Hernandez B.Y. Xiao W. Kim E. Jiang B. Goodman M.T. Sibug-Saber M. Cozen W. Liu L. Lynch C.F. Wentzensen N. Jordan R.C. Altekruse S. Anderson W.F. Rosenberg P.S. Gillison M.L. Human papillomavirus and rising oropharyngeal cancer incidence in the United States.J Clin Oncol. 2011; 29: 4294-4301Crossref PubMed Scopus (2668) Google Scholar Among individuals aged 12 to 69 years, oral HPV infection has an overall prevalence of@∼7%; however, men have a significantly higher prevalence compared with women (10.1% versus 3.6%).6Gillison M.L. Broutian T. Pickard R.K. Tong Z.Y. Xiao W. Kahle L. Graubard B.I. Chaturvedi A.K. Prevalence of oral HPV infection in the United States, 2009-2010.JAMA. 2012; 307: 693-703Crossref PubMed Scopus (825) Google Scholar These numbers are heavily influenced by sexual behavior and smoking history: prevalence seems to be as high as 20% in heavy smokers or individuals with >20 lifetime sexual partners. These trends are globally relevant because OPSCC is increasing in several countries, particularly in younger men.7Chaturvedi A.K. Anderson W.F. Lortet-Tieulent J. Curado M.P. Ferlay J. Franceschi S. Rosenberg P.S. Bray F. Gillison M.L. Worldwide trends in incidence rates for oral cavity and oropharyngeal cancers.J Clin Oncol. 2013; PubMed Scopus Google Scholar cancer incidence is in countries, HPV-associated are to numbers of in the United by HPV+ OPSCC the HPV-associated cancer in the United A.K. Engels E.A. Pfeiffer R.M. Hernandez B.Y. Xiao W. Kim E. Jiang B. Goodman M.T. Sibug-Saber M. Cozen W. Liu L. Lynch C.F. Wentzensen N. Jordan R.C. Altekruse S. Anderson W.F. Rosenberg P.S. Gillison M.L. Human papillomavirus and rising oropharyngeal cancer incidence in the United States.J Clin Oncol. 2011; 29: 4294-4301Crossref PubMed Scopus (2668) Google Scholar strategies for HPV+ OPSCC are and clinically biomarkers that are to the of the to and the and are of and in cancer cell and have patterns the of in tumor and of miRNA miRNA profiles and prognostic in cancer and diseases that may N. D.L. Liu Harris C.C. expression profiles associated with and in 2008; PubMed Scopus Google Scholar, C. M. K.K. biomarkers in for of PubMed Scopus Google Scholar was to profile HPV+ versus HPV− OPSCC to provide a more detailed understanding of pathologic molecular events and to identify biomarkers that may have applicability for early diagnosis, improved staging, and prognostic stratification. identify an oncogenic miRNA panel that the response to an oncogenic HPV infection and this panel in clinical using sequencing and clinical data from The Cancer Genome Atlas and miRNA in situ of human OPSCC This molecular may oropharyngeal tumors with distinct management strategies and mechanistic elucidation of molecular factors that contribute to OPSCC development, progression, and response to therapy. for the initial cohort were from the of surgical to with and tonsillar or base of tongue for was identified by for p16 as a for risk stratification in oropharyngeal squamous cell 2012; PubMed Scopus Google Scholar to the and a of the and was using a with tumor and or in the of were as for p16 we identified from surgical that were for p16 with or the tumor for using the a of cancer were and were RNA the the were using a and a of were an miRNA as a of to the of the RNA quantitative for miRNA was for profiling. of that was The the miRNA in the human miRNA was using the and was using the was with and of and a for the of was to using the unique to the was using the the of was using the using the and was to the and and was to was to the for in a high of and were the of was as using as a for data from the were to the the of the and to that the of the and of and compared with cancer for these M. 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Human papillomavirus and rising oropharyngeal cancer incidence in the United States.J Clin Oncol. 2011; 29: 4294-4301Crossref PubMed Scopus (2668) Google Scholar In contrast, have These trends are A.K. Anderson W.F. Lortet-Tieulent J. Curado M.P. Ferlay J. Franceschi S. Rosenberg P.S. Bray F. Gillison M.L. Worldwide trends in incidence rates for oral cavity and oropharyngeal cancers.J Clin Oncol. 2013; PubMed Scopus Google Scholar Thus, is of to identify miRNAs of an oncogenic HPV infection of the head and as these miRNAs may biomarkers and may provide a more detailed understanding of the molecular pathogenesis of the miRNA profiles of of patients with of the with and of these supports HPV-associated of and of the and of The associated with HPV+ disease is a of cellular by the the cell to of that in with Thus, HPV may miRNAs that as in in and in In to is necessary for the of in OPSCC, and HPV seems to be a in tonsillar S. HPV and tonsillar Clin PubMed Scopus Google Scholar However, to HPV is to the of the cell and in to cell and that is in to that in N. L. J. R. N. S. and human papillomavirus PubMed Scopus Google Scholar miRNAs be to or E. P.F. of human 2013; PubMed Scopus Google Scholar an miRNA to of of these in the same M.P. G. F. E. C. J. W. J. of the tumor as a of factor 2011; PubMed Scopus Google Scholar Thus, cellular for or are for HPV to miRNA have miRNA expression in the of HPV and head and neck with a of of profiles in squamous cell carcinoma of the head and neck cell by human 2011; PubMed Scopus Google Scholar, W. L. B. W. J. B. J. Liu prognostic miRNAs in HPV-associated oropharyngeal Cancer 2013; PubMed Scopus Google Scholar, L. B. R. E. M. L. B. S. C. miRNA of oral and squamous cell a J 2011; PubMed Scopus Google Scholar, G. J. expression for the of oropharyngeal squamous cell 2013; PubMed Scopus Google Scholar, E. L. B. M. M. H. L. L. C. 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B. W. J. B. J. Liu prognostic miRNAs in HPV-associated oropharyngeal Cancer 2013; PubMed Scopus Google Scholar as in HPV+ In the we of and in and however, is expressed relatively low In contrast, and of the are expressed the of these are the of the two in HPV+ OPSCC be because is expression of these miRNAs low may miRNA are and are to of and are of the of the of miRNAs in human with oncogenic in and L. J. R. T. and of the of miRNA 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, for the in and 2008; Full Text Full Text PDF PubMed Scopus Google Scholar and are of the as such, their is to that of and and they are to have C. The of in and J. 2012; PubMed Scopus Google Scholar However, the of are as of with is that of the same with may have of the seem to be for HPV+ from HPV− however, the mechanistic for is HPV+ HNSCC and are by expression of distinct and of cell and P.F. S. in cell in human and human and PubMed Scopus Google Scholar, N. B. Chung C.H. of oropharyngeal carcinomas HPV-associated J 2013; PubMed Scopus Google Scholar and of is with the protein for the in and 2008; Full Text Full Text PDF PubMed Scopus Google Scholar is a and factors may be in and C. E. M. S. A.K. of the and 2013; PubMed Scopus Google Scholar The has and expression with M.P. of and from Google Scholar This is because has as a protein biomarker that between HPV+ and HPV− P.F. of biomarkers that human papillomavirus versus head and neck in a Natl PubMed Scopus Google Scholar Thus, the may be for HPV+ from HPV− HNSCC as relatively increased expression of this is of HPV+ tumors. is evidence that and as with in factor factor and increased cell F. R. M.S. E. Liu M. L. S. H. G. and in 2008; Full Text Full Text PDF PubMed Scopus Google Scholar of with expression is associated with of and and in and C. E. M. S. A.K. of the and 2013; PubMed Scopus Google Scholar In expression of factor and of factor A.K. F. E. G. B. S. M. M. H. F. J. The of the in 2010; Full Text Full Text PDF PubMed Scopus Google Scholar expression of and in was M. S. with to 2011; PubMed Scopus Google Scholar leading to the that in the of high expression may the for the numbers of for These the of in miRNA oncogenic and the that the current of and in OPSCC tumors supports the oncogenic by the and In to the of is in HPV+ has to potential of to cancer for an and L. J. H. E. J. F. S. F. F. J. a and cancer 2010; PubMed Scopus Google Scholar is in patients with HPV+ OPSCC, and increased in HPV+ to HPV− OPSCC were in the and W. L. B. W. J. B. J. Liu prognostic miRNAs in HPV-associated oropharyngeal Cancer 2013; PubMed Scopus Google Scholar, G. J. expression for the of oropharyngeal squamous cell 2013; PubMed Scopus Google Scholar we was a between and expression in OPSCC using and of OPSCC cell of HPV however, between and was of is with in with early expression as an prognostic for and S. L. M. N. H. and factor expression in and head and neck squamous cell 2008; PubMed Scopus Google Scholar recent that the expression of is in OPSCC, with between and or or of of expression and or patient in oropharyngeal squamous cell a 2012; PubMed Scopus Google Scholar that tumors from the oropharynx high and T. E. M. C. C. B. T. G. profile of head and neck squamous cell carcinomas p16 high Oncol. 2013; PubMed Scopus Google Scholar these that high in HPV+ OPSCC is that be may the in HPV+ OPSCC, as is evidence that is into or may as a tumor G. Jiang J. J. C. N. cell and by the J. 2012; PubMed Scopus Google Scholar, C.H. of in 2011; PubMed Scopus Google Scholar expression may F. M. M. M. L. N. M. and of in human and to Natl PubMed Scopus Google Scholar In is evidence that may be in and F. J. S. Liu W. Xiao the expression of and in human carcinoma 2013; PubMed Scopus Google Scholar and response are lacking. the biology associated with response in HPV+ OPSCC is of and clinically relevant as these tumors are for with cell death or cell death and cell S. S. Westra W.H. B. H. L. for a of the in of HPV-associated head and neck squamous cell 2013; PubMed Scopus Google Scholar has that most HPV+ OPSCC tumors cell death the of this and to patient survival may be S. S. Westra W.H. B. H. L. for a of the in of HPV-associated head and neck squamous cell 2013; PubMed Scopus Google Scholar, expression for in human oropharyngeal squamous cell 2013; PubMed Scopus Google Scholar Thus, in of of in HPV+ OPSCC, expression and the of of and has in HPV+ OPSCC and L. B. R. E. M. L. B. S. C. miRNA of oral and squamous cell a J 2011; PubMed Scopus Google Scholar, E. L. B. M. M. H. L. L. C. The of miRNAs in human between head and neck cancer and J 2012; PubMed Scopus Google Scholar were in an of and a potential mechanistic to early of the HPV Human expression to 2013; PubMed Scopus Google Scholar The patient that and were in patient and expression of miRNAs the an and for the data supports an of The of is in the of a of and has to be a Human expression to 2013; PubMed Scopus Google Scholar a in the with a in of this miRNA in compared with that seems to the of expression of a in with a of in These in data be as having because miRNA profiles in HNSCC and cancer an HPV-associated of and L. B. R. E. M. L. B. S. C. miRNA of oral and squamous cell a J 2011; PubMed Scopus Google Scholar these data that HPV may to for and that this miRNA in human tumors. In this we as a miRNA in HPV HPV+ HNSCC tumors in the of the of the These tumors are from in as such, most patients with However, to patients with HPV− OPSCC, HPV+ is with a to increase in overall K.K. Harris J. Wheeler R. Weber R. Rosenthal D.I. Nguyen-Tân P.F. Westra W.H. Chung C.H. Jordan R.C. Lu C. Kim H. Axelrod R. Silverman C.C. Redmond K.P. Gillison M.L. Human papillomavirus and survival of patients with oropharyngeal cancer.N Engl J Med. 2010; 363: 24-35Crossref PubMed Scopus (4806) Google Scholar that the is HPV therapy. The HPV-associated oncogenic miRNA panel identified may be into a that contribute to early disease stratification to in oropharyngeal tumors with distinct management the oncogenic miRNA panel the mechanistic elucidation of molecular factors that contribute to OPSCC development, progression, and response to therapy.