DETERMINING THE INflAMMATORY RESPONSE IN ORAL SQUAMOUS CELL CARCINOMA BY SALIVA ANALYSIS

Objectives Oral squamous cell carcinoma (OSCC) often shows a pronounced inflammatory infiltrate and there is accumulating evidence that this inflammatory infiltrate plays an active role in tumor development and progression. Analyses of saliva may provide a non-invasive approach to study the inflammatory response in OSCC. Our aim is to develop a protocol for collection and analysis of saliva in OSCC patients and to use it to characterize both the inflammatory cell profile and cytokine profile in the saliva of patients across all stages of OSCC. Methods 42 patients undergoing treatment for OSCC at the Toronto Odette Cancer Centre (stages I to IV), 25 healthy patients, and 9 patients with periodontitis were enrolled. Saliva samples were obtained by rinsing with 3 ml of saline for 30 sec. The samples were kept on ice and stabilized with protease inhibitor until filtration using a 20 μm membrane filter. Cell pellets were separated by centrifugation and supernatants were analyzed using a BioFLex 30-Plex inflammatory panel (BioRad) and Luminex® detection technology. Cell pellets were fixed in 4% PFA and analyzed using multichannel flow cytometry. The fluorescent markers included CD45, CD66b, CD3, CD4, CD8, CD25, CD56, CD68, CD138, Siglec8, PD1 and PDL1. Findings Distinctive, reproducible changes were observed in salivary cytokines and inflammatory cell profile of patients with OSCC compared to control and periodontal disease patients. Using our protocol, we were able to describe specific patterns of inflammation for oral cancer, including altered CD4/CD8 ratio and marked increase in IL-1b, IL-6 and TNF-a. Conclusion We created a reproducible protocol to collect saliva and perform high-throughput analysis of inflammatory profile of saliva. This technology can be used to develop non-invasive, early detection/prognostic tests for OSCC, new adjuvant therapies and new techniques to monitor response to treatment.