CANCER PREVENTION RESEARCH | RESEARCH BRIEF A Novel Saliva and Serum miRNA Panel as a Potential Useful Index for Oral Cancer and the Association of miR-21 with Smoking History: a Pilot Study Dimitra P. Vageli, Panagiotis G. Doukas, Rema Shah, Trinithas Boyi, Christina Liu, and Benjamin L. Judson ABSTRACT ◥ Tobacco use is implicated in the carcinogenesis of oral squamous cell carcinoma (OSCC), which is associated with poor survival if not diagnosed early. Identification of novel noninvasive, highly sensitive, and cost-effective diagnostic and risk assessment methods for OSCC would improve early detection. Here, we report a pilot study assessing salivary and serum miRNAs associated with OSCC and stratified by smoking status. Saliva and paired serum samples were collected from 23 patients with OSCC and 21 healthy volunteers, with an equal number of smokers and nonsmokers in each group. Twenty head and neck cancer–related miRNAs were quantified by qPCR (dual-labeled LNA probes) and analyzed by Welch t test (95% confidence interval). Four saliva miRNAs, miR- 21, miR-136, miR-3928, and miR-29B, showed statistically significant overexpression in OSCC versus healthy con- trols (P < 0.05). miR-21 was statistically significantly overexpressed in OSCC smokers versus nonsmokers (P ¼ 0.006). Salivary miR-21, miR-136, and miR-3928, and serum miR-21 and miR-136, showed statistically signifi- cant differential expression in early-stage tumors versus controls (P < 0.05), particularly miR-21 in smokers (P < 0.005). This pilot study provides a novel panel of saliva and serum miRNAs associated with oral cancer. Further validation as a potential useful index of oral cancer, particularly miR-21 in smokers and early-stage OSCC is warranted. Prevention Relevance: Saliva and serum miR-21, miR- 136, miR-3928, and miR-29B, are potentially associated with oral cancer even at an early stage, especially miR-21 in individuals with a smoking history, a further validation in a larger cohort of subjects with premalignant and early malignant lesions need to confirm. Introduction Head and neck cancer not related to human papillomavirus (HPV) is an aggressive malignancy and is more successfully treated if diagnosed early. Early detection of head and neck squamous cell carcinoma (HNSCC) would be improved with the identification of specific biomarkers that have been validated through large-scale studies (1). Most known biomar- kers exhibit limitations in the early diagnosis and prognosis, and tissue specificity, limiting their role in screening strate- gies (2). Small noncoding miRNA molecules, which are often altered during head and neck carcinogenesis (3), can be defined as tissue-specific biomarkers and can have diagnostic and predictive value (4–9). Notably, recent preclinical findings from our team have documented deregulations of specific miRNAs, such as miR-21, associated with early oncogenic events in head and neck carcinogenesis induced by chronic exposure to known risk factors, such as tobacco smoke components (6, 10–14). Although miRNAs can be detected in tissue biopsy, which is an invasive method, miRNAs can also be detected in body fluids, such as serum, plasma, or saliva, using noninvasive collection methods (15–18). In addition, extracellular miRNAs have been proven to exist in a stable cell-free form (18). Therefore, saliva or circulating (serum) miRNAs may be valuable biomarkers in cancer. Although previous studies have presented several miRNA markers detected in saliva or serum with a detection value in HNSCC, the data remain unclear due to different collection methods or miRNA analysis focus, as well as diverse types of head and neck cancer malignancies included in the studies (19, 20). To the best of our knowledge, so far, only a few studies have investigated miRNA markers in paired saliva and serum samples from the same patients with oral cancer of various risk factors, such as tobacco smoking, alcohol, and GERD, compared with healthy controls (21, 22). Such noninvasive and rapid approaches could provide prom- ising diagnostic and prognostic biomarkers with the ability to screen and monitor high-risk patients (23). We hypothesized that there is a specific panel of miRNAs that are altered in saliva and serum of a common HPV- negative HNSCCs, such as oral squamous cell carcinoma The Yale Larynx Laboratory; Department of Surgery (Otolaryngology), Yale School of Medicine, New Haven, Connecticut. Corresponding Author: Dimitra P. Vageli, The Yale Larynx Laboratory, Depart- ment of Surgery (Otolaryngology), Yale School of Medicine, 310 Cedar Street (BML212), New Haven, CT 06510. E-mail: dimitra.vangeli@yale.edu Cancer Prev Res 2023;16:653–60 doi: 10.1158/1940-6207.CAPR-23-0219 Ó2023 American Association for Cancer Research AACRJournals.org | 653 Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/16/12/653/3387092/653.pdf by Yale University user on 21 December 2023