Biology Contribution
Human Papillomavirus (HPV) Infection in Squamous Cell Carcinomas Arising From the Oropharynx: Detection of HPV DNA and p16 Immunohistochemistry as Diagnostic and Prognostic Indicators—A Pilot Study

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Purpose

Human papillomavirus (HPV) 16 infection is associated with oropharyngeal carcinogenesis and is likely the cause of the reported increase in disease incidence. We evaluated the prevalence of HPV infection and the reliability of different diagnostic tools using primary tumor samples from a cohort of 50 patients.

Methods and Materials

Formalin-fixed paraffin-embedded (FFPE) tumor samples were collected from all 50 consecutive primary oropharyngeal SCC patients who were enrolled in the study; fresh tumor samples were available in 22 cases. NucliSENS EasyQ HPVv1 was used for RNA, and Digene Hybrid Capture-2(HC2) was used for DNA detection. p16 Expression was evaluated by immunohistochemistry in FPPE specimens.

Results

Based on the DNA detection assay on FFPE samples, the frequency of high-risk HPV infection was 32%. The agreement rate between HPV RNA and HPV DNA detection in fresh samples was 100%. The agreement rate between p16 immunohistochemistry (IHC) and the detection of HPV DNA in the FFPE samples was fair but not excellent (κ = 0.618). HPV DNA detection was highly significant, as measured by disease-specific survival and determined using a Wilcoxon test (P=.001). p16 IHC also exhibited a prognostic value but with a lower statistical significance (P=.0475). The detection of HPV DNA, but not p16 IHC, was also significantly correlated with locoregional control (P=.0461).

Conclusion

Diagnostic methods based on the detection of HPV nucleic acids appear to be more reliable and objective because they do not require reading by a trained histopathologist. Furthermore, the detection of HPV DNA exhibits an improved correlation with survival, and therefore appears definitely more reliable than p16 IHC for routine use in clinical practice.

Introduction

More than 50,000 cases of head and neck cancer, predominantly squamous cell carcinomas (HNSCC), occurred in the United States in 2012, resulting in approximately 13,000 deaths (1). HNSCC grossly accounts for 3.5% of all malignant tumors in the United States (1) and is more prevalent in other parts of the world, such as India, Southeast Asia, and Brazil (2). Well-established risk factors for the development of HNSCC include cigarette smoking and alcohol abuse 3, 4. Another emerging risk factor for oropharyngeal SCC (OPSCC) is infection with high-risk (HR) human papillomavirus (HPV) 5, 6, the role of which, in carcinogenesis of the uterine cervix, has been extensively studied (4), although its relevance for carcinogenesis in other head and neck sites (if any) remains unclear 5, 6, 7. The emergence of HR HPV infection underlies a marked increase in the incidence of OPSCC 8, 9, particularly in young patients (10). A general decrease in the incidence of SCC in other head and neck mucosal sites has been observed in Western countries because of successful campaigns for primary prevention, particularly against cigarette smoke 1, 11.

The significance of HPV infection in nonoropharyngeal head and neck sites might previously have been overestimated 12, 13 because of the use of p16 expression as a diagnostic tool, based on a uterine cervix model 14, 15. Importantly, we recently demonstrated that p16 immunohistochemistry (IHC) is unreliable for diagnosing HPV infections of head and neck sites outside the oropharynx (6). The primary aim of this study was to evaluate, on formalin-fixed paraffin-embedded (FFPE) samples, which are by far the most easily available in daily clinical practice, the reliability of p16 IHC and of HPV-DNA detection in the diagnosis of HPV infection in OPSCC, and their clinical and prognostic rsignificance.

Section snippets

Patient characteristics

Patients with oropharyngeal neoplasms are evaluated at the same institution by a multidisciplinary head and neck tumor board, which provides therapeutic recommendations following histological diagnosis and staging according to TNM classification (16). Samples were collected from 50 patients affected by primary, previously untreated, advanced oropharyngeal SCC between March 2009 and December 2011. The patients were enrolled in the study, which had previously been approved by the ethical

Results

Patient and tumor characteristics are shown in Table 1. All patients enrolled in this study were available for follow-up.

The most frequent subsite from which the SCCs originated was the tonsil (68%), followed by the base of the tongue (22%). We observed a marked prevalence of stage IV cases (76%). More than 85% of the patients in our study cohort presented with clinically positive lymph nodes at diagnosis.

The specific genotype of HPV was evaluated in fresh samples and consistently found to be

Discussion

The results of this study confirm the relevance of HPV infection as a risk factor for OPSCC 5, 6, 22. The infection rate in this group of patients is consistent with previous reports on OPSCC from Europe 23, 24, 25, which generally estimate the HR HPV prevalence between 25% and 50%, also taking account of the markedly reduced prevalence in southern Europe over that in other areas such as Scandinavia (26). Nevertheless, the prevalence of disease in this cohort was lower than that observed in our

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    This study was funded exclusively by research grant D1.1 from the Università Cattolica del Sacro Cuore awarded in 2010 to professor Giovanni Delogu.

    Conflict of interest: none.

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