Detection Of Human Papillomavirus By Chromogenic In Situ Hybridization And Expression Of P16 Protein In Malignant Lung Lesions: A Histomolecular Analysis

Abstract

Lung cancer is the most prevalent and lethal malignant neoplasm worldwide, presenting high incidence and mortality rates. New studies have investigated the association between the Human Papillomavirus (HPV) and pulmonary carcinogenesis, suggesting that this virus may act as an additional risk factor for lung cancer, promoting the overexpression of protein p16(INK4a), a tumor suppressor and marker of cell cycle dysregulation, in malignant lung cells. However, the etiological relationship between HPV and lung cancer remains unclear. In this sense, the aim of this study was to detect and genotype HPV in lung malignant biopsy samples by nested-PCR, Chromogenic in situ Hybridization (CISH) and Multiplex-PCR with Reverse Line Blot Hybridization (PCR-RDB), correlating its presence with the immunohistochemical (IHC) expression of p16. 62 formalin-fixed paraffin-embedded biopsy samples were analyzed, especially adenocarcinomas and squamous cell carcinomas. 16.12% of samples were HPV-16 positive, while only 4.83% were HPV-18 positive. PCR-RDB identified the presence of HPV-16 E6 protein in 62.86% of cases. Positive expression of p16 by IHC was identified in 65.62% of lung biopsy samples. CISH assays of HPV-positive lung tumor samples were positive in 20% of cases. No significant differences were identified between comparisons by Mann-Whitney test. Significant differences were observed in Chi-Square and Fisher’s analysis for HPV detection and p16 expression. In the Spearman’s test significant correlations were observed, among others, between tumor stage and lesion (p = 0. 018), tumor stage and detection of HPV-16 E6 (p = 0.016), p16 expression and detection of HPV-16 E6 (p = 0.010) and between HPV detection and p16 expression (p = 0.030). For the diagnosis and differentiation of malignant lung lesions (SCCs or adenocarcinomas), p16 obtained excellent sensitivity (100%) and reasonable specificity (50%). Estimates for NPV were 100% and for PPV 82.10%. For the diagnosis and differentiation of HPV-positive tumors, p16 showed good sensitivity (67.74%) and excellent specificity (83.87%), with NPV corresponding to 55.32% and PPV to 89.82%. Our results reinforce the importance of HPV detection and genotyping, as well as p16 analysis as a complementary biomarker to the diagnosis of lung carcinomas and HPV-associated lesions. New studies, especially longitudinal and prospective ones, may improve p16 analysis by RT-qPCR or NGS, generating more accurate and robust results. HPV analysis should also include the evaluation of its oncogenes expression by quantitative methods, such as RT-qPCR, ensuring more accurate diagnoses and prognoses and a more assertive therapeutic approach.