Background: Human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma (SCCA) has distinct pathophysiologic differences from HPV negative SCCA. Inhibition of tumor suppressor genes p53 and RB by HPV proteins E6 and E7 has been extensively studied. More recently, the senior author has identified novel mechanisms by which E6 and E7 contribute to genomic instability. Alteration of the homologous recombination (HR) pathways by these proteins inhibits double stranded DNA break (DSB) repair. Increased expression of E6 and E7 in cell culture models has been shown to increase the abundance of proteins involved in HR and has been shown to increase HR foci. Activation of these foci, however, has been shown to occur during G1, when HR cannot occur due to lack of a sister chromatid template. Furthermore, HR proteins have been shown to have impaired ability to properly localize to DSB’s. These changes in DSB repair mechanisms create opportunities for genomic integration of viral DNA and subsequent neoplastic changes. The purpose of the present study is to compare expression of HR proteins in HPV positive and negative SCCA through immunohistochemistry.
Methods: A tissue microarray (TMA) was created from twenty-seven HPV positive and nine HPV negative oropharyngeal SCCA surgical specimens. TMA slides were stained with antibodies to two HR proteins, presently termed AH2017.1 and AH2017.2. Immunohistochemical analysis was performed by two independent pathologists. Staining intensity and percentage of nuclear staining was measured, from which a composite score was derived. Composite scores were compared between groups using a Mann-Whitney U test.
Results: Differential staining was identified between groups for both AH2017.1 and AH2017.2. The mean composite scores for HPV positive and negative groups for AH2017.1 were 1.04 and 0.63 respectively, a difference which trended towards statistical significance (p=0.07). AH2017.2 mean composite scores were 2.06 for the HPV positive group and 0.76 for the HPV negative group, which was statistically significant (p=0.002). Inter-rater reliability between pathologists was measured and found to be excellent (Intraclass correlation coefficient 0.901).
Conclusion: Our study demonstrates differential staining in HPV positive and negative SCCA for the DNA repair genes currently termed AH2017.1 and AH2017.2 using a TMA, a high throughput technique for immunohistochemical analysis. This validates data from cell culture models that demonstrate overexpression of HR genes and further differentiates the pathophysiology of HPV positive and negative SCCA. Abrogation of DNA repair pathways represent possible targets for novel targeted molecular therapies in HPV positive tumors.