MicroRNAS as Novel Classifiers of Premalignant Progression within an Oral Cancer Field

BACKGROUND: Oral Squamous Cell Carcinoma (OSCC) is one of the most commonly diagnosed malignancies worldwide, with a dismal 5-year survival rate of ~50% that has not changed for decades.  An improved understanding of the molecular basis of OSCC, particularly the alterations that govern disease initiation and progression, is essential for developing novel strategies for diagnosis, predicting prognosis and establishing effective therapeutics.  To date, the role of microRNAs (miRNAs), a class of short, non-coding, single stranded RNAs that negatively regulate gene expression, in oral tumorigenesis – and oral premalignant lesions (OPLs) specifically, is largely unknown.  Thus, the objective of this study is to identify miRNAs that are deregulated in early OPLs and to select those with the greatest potential as future biomarkers. METHODS: MiRNA expression is tissue-specific and can be influenced by a number of extrinsic factors, such as smoking and diet.  To remove variation due to timing differences in sampling, we analyzed histologically different OPL and normal tissue biopsies taken at the same time from a single, contiguous field in a patient’s mouth.  For each of the 10 patient cases, we obtained microdissected tissues from normal, dysplastic and carcinoma in situ lesions.  Diseased regions in the oral cavity were detected by a hand-held Fluorescence Visualization (FV) device capable of delineating occult disease proximal to oral tumors in real-time.  This device uses a blue/violet light (400-460 nm) to scan oral tissues.  While normal tissues re-emit this light as pale green, abnormal tissues show loss of such autofluorescence and appear dark brown.  Total RNA was isolated from each microdissected specimen and profiled for the expression of 742 human miRNAs using miRCURY LNA™ Universal RT microRNA PCR.  Data were normalized based on the manufacturer guidelines and a minimum of 2-fold expression change relative to the matched normal was used to define candidate miRNAs. RESULTS: We have identified several candidate miRNAs that were differentially expressed at the earliest stages of oral cancer development.  The most frequently up-regulated and down-regulated miRNAs were miR-21 and miR-375, respectively.  Along with our other candidates, these miRNAs are widely mentioned in the literature and are suggested to play crucial roles in the oncogenic pathways of multiple malignancies, including those of the head and neck.  We are currently validating these findings in a larger cohort of patient samples through in situ hybridization experiments. CONCLUSIONS: Molecular characterization of intralesional progression of the altered oral field allows for the delineation of key genetic events driving oral cancer progression.  Ultimately, our data will aid in the development of a novel genomic platform for risk assessment, diagnosis and novel targeted therapies, leading to improved disease management.