Abstract 26: Exosomal microRNA as salivary biomarkers of head and neck squamous cell carcinoma

Head and neck cancer is the 10th most common cancer overall and 5th most common among men in the United States, the vast majority of which are squamous cell carcinoma (HNSCC). Despite approximately two-thirds of cases being diagnosed at an advanced stage, the only routine screening approach currently in widespread use is visual inspection and palpation, which is provided by dentists and clinicians on an opportunistic basis, lacks sensitivity, and varies according to the skill of the clinician. This underscores the urgent and compelling need for establishment of novel and effective biomarkers to facilitate early detection. Exosomes may offer a new avenue for discovery and development of novel biomarkers of HNSCC. These nanosized (40-150 nm), membrane-encapsulated vesicles are released by both normal and malignant cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including microRNA (miRNA), which can affect the phenotype of recipient cells. Importantly, exosomes are present in essentially all extracellular biofluids, including saliva, and thus can readily be collected by noninvasive means. Through previous cell culture work, we observed striking differences in exosomal miRNA secreted by HNSCC cells relative to healthy oral epithelial cells, with a high degree of overlap in exosomal miRNA profiles across HNSCC cell lines. Although salivary exosomes have been theorized to be a potentially powerful biomarker source for HNSCC, there has been an extreme paucity of work conducted in this arena. To begin to address this question, we conducted a small pilot study in which we collected 2-mL whole-saliva samples from 5 HNSCC cases (collected prior to initiation of treatment) and 5 cancer-free controls. Exosomes were isolated from each saliva sample via differential ultracentrifugation, and total RNA was extracted for miRNA-sequencing (miRNA-seq), which was performed by the University of Cincinnati Genomics, Epigenomics & Sequencing Core (GESC). The number of reads for each sample during miRNA-seq ranged from 1.6 million to 27.4 million reads (median = 12.4 million). After aligning the reads to all human miRNA sequences catalogued in miRBase, there were a total of 1,334 mature miRNA transcripts detected across samples, with 307 transcripts (12%) detected solely in salivary exosomes from cases. Moreover, despite the small sample size of the pilot study, we observed significant differential exosomal secretion of miR-10b-5p (p = 0.006), with transcripts present at relatively high levels in salivary exosomes from 3 of the 5 cases but none detected in those from the 5 controls. We also preliminarily assessed the translational potential of 8 miRNA transcripts that were solely and universally expressed by our HNSCC cell lines (i.e. not secreted by healthy oral epithelial cells) in our previous cell culture work using the salivary exosome miRNA-seq data for the 5 cases and 5 controls. All 8 transcripts were detected in salivary exosomes obtained from the 5 HNSCC cases. In particular, miR-486-5p and miR-486-3p showed considerable promise, with 40% of cases expressing drastically higher levels of these transcripts relative to controls. When either miR-486-5p or miR-486-3p was combined with miR-10b-5p, the substantial separation of these markers could clearly distinguish 80% (4/5) of the HNSCC cases from controls. These findings demonstrate the potential of salivary miRNA as biomarkers of HNSCC and highlight the necessity of further workup and validation in full-scale studies involving human saliva. Citation Format: Scott M. Langevin, Damaris Kuhnell, Xiang Zhang, Trisha M. Wise-Draper, Keith A. Casper. Exosomal microRNA as salivary biomarkers of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 26.