MON-514 Detection of RAS Mutations and RET/PTC Fusions in Thyroid Cancer Using Microfluidic Digital PCR

Abstract

Background:The identification of somatic mutations and gene fusions is crucial for guiding therapeutic decisions in patients with thyroid cancer. Microfluidic digital PCR is currently considered as a technique of choice for highly sensitive detection of gene mutations/fusion. We recently demonstrated that dPCR is a useful tool for detection of BRAFV600E and TERT promoter mutations in thyroid tumors.Objectives:This study aimed to determine the analytic and clinical validity of dPCR for detection of RAS mutations and RET/PTC fusions in thyroid cancer tissue.Material and Methods:Thyroid tissues from 75 patients with PTCs (58 classical PTC (CPTC) and 17 follicular variant (FVPTC) were used for DNA and RNA extraction. The rare mutation SNP genotyping assays which were multiplexed for detection of mutant and wild type NRASQ61; as well as RET/PTC1 and RET/PTC3 were synthesized by Thermo Fisher Scientific. Digital PCR was performed using a QuantStudio 3D Digital PCR platform. QuantStudio Software was used for relative and quantitative data analysis.Results:NRASQ61 was detected in 0/58 CPTC and in 6/17 (35%) FVPTC. The ratios of mutant/total varying from 11.7% to 61.5%. Among patients with FVPTC there were no significant associations between the presence of NRASQ61 and patient’s age, sex, multifocal growth, extra-thyroidal invasion and lymph node metastases. The ratios mutant/total correlated with tumor size in patients harboring NRASQ61. In 23 cases, RET/PTC1 and RET/PTC3 transcripts were examined. RET/PTC1 and RET/PTC3 transcripts were detected in 3 and 1 case, respectively. RET/PTCs were detected in CPTC, but not in FVPTC. RET/PTC positive tumors were characterized by multi-focal patterns of growth, presence of extra-thyroidal invasion, and presence of lymph node metastases (4 of 4 cases with RET/PTC). There were not RET/PTCs positive tumors harboring simultaneously anomalies in RAS oncogene.Conclusions:Microfluidic digital PCR allows specific, sensitive and rapid detection of RAS mutations and RET/PTC fusions in thyroid tissue samples. Implementation of dPCR-based assays may facilitate analysis of thyroid tumors and support research in patients with thyroid cancer.