Assessment of Circularized HPV16 E7 RNA, GLUT1, and PD-L1 in Anal Squamous Cell Carcinoma

BACKGROUND: Anal squamous cell carcinoma (ASCC) is a rare, deadly malignancy caused by high-risk human papillomaviruses in up to 90% of cases and continues to be treated by cytotoxic therapy established 40 years ago. There is a dearth of reliable biomarkers for ASCC. The prognostic implication of programmed death-ligand 1 (PD-L1) expression remains controversial while other biomarkers, like glucose-1-transporter (GLUT1) expression levels, have not been examined in the setting of ASCC. More recently, covalently closed circular RNAs has been (circRNA) expression has been shown to be widespread in cancers, and circRNAs have been proposed to be potential biomarkers. In previous studies, we discovered a novel circular E7 RNA expressed by HPV16 (circE7), which has not been assessed as a potential biomarker in any diseases. OBJECTIVE: We hypothesize that human papillomavirus infection status, increased GLUT-1 expression, increased PD-L1 expression, and HPV E7 RNA expression will serve as biomarkers for higher mortality in patients with anal squamous cell carcinoma. METHODS: A retrospective, translational case series was conducted on twenty-two subjects to analyze PD-L1, GLUT1, HPV-ISH, and HPV circE7 in relation to the clinical features and overall survival of patients with anal squamous cell carcinoma. To supplement understanding of the HPV circE7 biomarker, bioinformatic analyses of RNA-Seq data from the Cancer Genome Atlas was performed on 875 subjects with HPV-driven head and neck cancer and cervical cancer. RESULTS: Improved overall survival could be predicted histologically by pure basaloid architecture (p=0.013), PD-L1 expression (p=0.08), HPV-ISH positivity (p<0.001), but not GLUT1 expression. Quantitative RT-PCR of archived tumors revealed that high levels of circE7 in ASCC were predictive of improved overall survival (p=0.023). Bioinformatic analyses suggested that the presence of high amounts of circE7 correlated to improved survival in 875 subjects (p=0.074). CONCLUSION: Glut-1 overexpression was ubiquitous among all anal squamous cell carcinoma cases but was not predictive of survival. This study adds to the growing evidence of PD-L1 expression correlation to improved survival in ASCC. CircE7 levels correlate with improved survival in anal squamous cell carcinoma but larger, prospective studies are necessary to confirm the potential role of circE7 as a biomarker.