MON-563 Quantitative Assessment Of BRAF Mutations In Thyroid Cancer Using Droplet Digital PCR.

Abstract

Background:Identification of somatic mutations is crucial for guiding therapeutic decisions in patients with thyroid cancer. Droplet digital PCR (ddPCR) is a sensitive and particularly useful method of nucleic acid detection for quantification of gene mutations. This study aimed to determine the analytic and clinical validity of BRAF ddPCR mutational testing in thyroid cancer tissue. Material and Methods: Initially we examined a training cohort of 30 patients with thyroid cancer that had been previously tested for BRAF mutation with Sanger sequencing and included 15 BRAF positive and 15 BRAF negative subjects. A second group included 65 samples from 18 patients with thyroid cancer. Samples were obtained from central and invasive areas of tumors, normal thyroid tissue and lymph node metastasis. The DNA was extracted using Thermo Fisher Scientific King Fisher Duo Prime Purification System, and DNA concentration was measured by NanoDrop. Digital PCR was performed using a QuantStudio 3D Digital PCR platform. QuantStudio Software was used for relative and quantitative data analysis. Results: In the training set, BRAF mutations were detected in 15/15 BRAF-positive thyroid cancers and in 0/15 BRAF- negative tumors. Analysis of tissue samples from the second group of patients demonstrated detection of wild type BRAF alleles in normal thyroid as well as in thyroid cancer tissue. In contrast, BRAFV600 mutant alleles were detected only in thyroid cancer tissue samples (13/18 (72%) examined patients). In cancer samples, the ratios of mutant/total BRAF alleles ranged from 1.47% to 42.8% (average 24.6%). Quantification of BRAF mutant alleles did not reveal differences between male and female patients or patient age. The ratios of mutant/total BRAF did not correlate with tumor size, and were not different in central and invasive areas of the thyroid tumors. Metastases at the time of surgery were detected in 2/5 patients with BRAF-negative tumors, and in 6/13 patients with BRAF-positive tumors. The ratios of mutant/total BRAF alleles were higher in cancers presenting with metastases at the time of surgery (average 26.6%) compared to tumors without metastases (average 19.6%). However, these differences were not statistically significant. The quantifications of mutant BRAF alleles in metastatic lesions showed that the ratios of mutant/total BRAF were similar to corresponding primary tumors in 3 cases, and were decreased in 3 cases. Conclusion: Overall, droplet digital PCR allows specific, sensitive and rapid detection of BRAF mutations in thyroid tissue samples. Quantitative evaluation of BRAF mutations offers an opportunity for additional assessment of prognosis and potential response to treatment with tyrosine kinase inhibitors. Further research on implementation of ddPCR-based assays of thyroid tumors may provide important insights to the management of patients with thyroid cancer.