Ultrasensitive Detection of BRAF Mutations in Circulating Tumor DNA of Patients With Metastatic Thyroid Cancer

Background: Liquid biopsy is a promising technology that can offer various advantages for molecular testing over tissue-based approaches. Most studies trying to address the utility of liquid biopsy in thyroid cancer have failed so far to achieve satisfactory rates of detection of relevant mutations. In this study, we examined a newly developed approach for ultrasensitive detection of oncogenic mutations in thyroid cancer using BRAF mutation as a proof-of-concept. In an exploratory analysis, we also correlated our findings with clinical outcomes and with levels of standard of care biomarkers. Methods: We included a group of patients with metastatic thyroid carcinoma. Cell free DNA (cfDNA) was isolated from an average of 2 ml of plasma and from matched formaldehyde fixed paraffin tissue blocks (FFPB) that were obtained from prior surgery. Extracted DNA was subject to preamplification of mutant copies using Q5 High-Fidelity PCR kit. Digital droplet PCR was performed on pre-amplified purified DNA where BRAF mutated allele frequencies (AF) were measured using BioRad ddPCR Qx200. Results: Thirty-three patients were included in our study with a median age at diagnosis of 62. Our method achieved a sensitivity of detection of 47.6% and a specificity of 80%. Mutant BRAF V600E was detected in cfDNA of 54.5% of patients (n=18) compared to 80.8% in isolated DNA from matched FFPB. Median overall survival (OS) was shorter in patients with wild type (WT) BRAF in both ctDNA and tissue (127m vs 218m, p=0.015; 116m vs 223m, p=0.004). Thyroglobulin (Tg) levels did not correlate with BRAF mutations either quantitatively or qualitatively. In the papillary thyroid carcinoma-classic variant cohort (n=20), however, patients with cfDNA mutant BRAF were more likely to have elevated Tg (90.9% versus 44.4% respectively, p=0.05). Conclusions: Our study provided a proof of concept for a newly developed technique to provide high sensitivity of mutation detection in thyroid cancer. The achieved sensitivity of detection is the highest to date using liquid biopsy in this tumor type. While we addressed only BRAF mutations in our study, the same approach can potentially be used for other mutations as well, likely changing the paradigm for use of liquid biopsy in thyroid cancer.