Abstract 734: Improved detection of salivary glands’ RNA markers in saliva samples
Authors: Schubert Adrian D. , Izumchenko Evgeny , Wysocki Piotr T. , Sidransky David , Brait Mariana 
Published: July 2017

Abstract
RNA based liquid biopsy in saliva could be part of the diagnostic process and surveillance in patients with Salivary Gland Tumors. There is no established approach for saliva markers in salivary gland tumors. These tumors occur approximately in one out of 100,000 adults per year in the USA. We aim to develop a method to identify RNA shed specifically from the salivary glands.The two most common salivary gland malignancies harbor frequent gene fusions, which may be candidate RNA based markers. In mucoepidermoid carcinoma, the MECT1-MAML2 fusion. In adenoid cystic carcinoma, either a MYB-NFIB or MYBL1-NFIB fusion. We identified collection and processing methods to be tested on healthy individuals’ saliva samples. We compared three saliva stimulation methods: Chewing Gum, Tabasco and Vitamin C powder with unstimulated collected saliva; and also utilized different processing and RNA extraction procedures: Trizol based: QIAzol method (Qiagen), RNEasy saliva protecting reagent (Qiagen), OrageneRNA (DNA Genotek), and mirVana Kit (ThermoFisher). We evaluated the RNA´s quantity and quality with Spectrophotometer and Agilent’s 2100 Bio analyzer. We identified two salivary gland specifically highly expressed genes (HTN3 and CA6) to be analyzed by quantitative RT-PCR. We have collected and processed 221 samples and analysis is ongoing. Bio analyzer showed that RNA from the unstimulated collected saliva has a higher concentration than stimulated saliva. Unstimulated saliva is expected to contain mucosa cells, bacteria and rests of food, therefore those could also be RNA sources. When saliva’s RNA is protected by either of the buffers tested, the RIN scores (RNA Integrity Number) obtained by Bio analyzer are higher. Based on our preliminary data, stimulation with either Tabasco, Chewing Gum or Vitamin C followed by RNA stabilization with the OrageneRNA kit and extraction with Trizol resulted in the highest quality and specificity of the RNA for its salivary gland origin. Unstimulated collection followed by the same processing generates a greater RNA yield. This saliva RNA is likely contaminated with RNA form squamous cells, bacteria and food residues, which may decrease the sensitivity of the specific gene expression of HTN3 and CA6. Once established how to best collect and protect saliva for the extraction of nucleic acids originated from the salivary glands, we anticipate accelerating the path to clinically highly demanded kits for the surveillance of salivary gland cancer patients. We plan to prospectively collect samples from Salivary gland tumor patients with the chosen method and evaluate tumor related markers. The detection of tumor markers in bodily fluids will open a new avenue for the diagnosis and the clinical management of patients with this type of tumor. Moreover, our work creates the possibility of a liquid biopsy-based detection of several disease-specific alterations identified in different types of salivary gland tumors.
Citation Format: Adrian D. Schubert, Evgeny Izumchenko, Piotr T. Wysocki, David Sidransky, Mariana Brait. Improved detection of salivary glands’ RNA markers in saliva samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 734. doi:10.1158/1538-7445.AM2017-734