Genetic alterations in cfDNA of benign and malignant thyroid nodules based on amplicon-based next-generation sequencing

Circulating cell-free DNA (cfDNA) serves as a biomarker in multiple malignant diseases. However, controversy still surrounds the role of cfDNA detection in the diagnosis and monitoring of papillary thyroid carcinoma (PTC). This study set out to identify the role of cfDNA detection in distinguishing between benign and malignant thyroid nodules.

We found that BRAFV600E mutation was a credible disease-related mutation in PTC; however, it could not be detected in cfDNA. Moreover, there was a large difference in mutation gene distribution between benign and malignant thyroid nodules.

Tissue, blood cell, and plasma samples were collected from 10 patients with benign nodules and 10 patients with malignant nodules. The DNA isolated from these samples was subject to PCR-based amplification using primers designed for 50 proto-oncogenes and tumor suppressor genes. PCR products were sequenced using Illumina technology, and the mutations were detected with varScan among sequencing data for each sample and comparative analysis was carried out.

Through amplicon sequencing, we found one non-synonymous somatic mutation in the benign nodules and three in the malignant nodules. Among these four mutations, BRAFV600E mutation was detected in the tissue samples of 8 out of the 10 PTC patients, but it was not detected in the benign nodules. However, no BRAFV600E mutation was detected in cfDNA. Further differential analysis of cfDNA indicated that some genes had more mutations in benign patients than in malignant patients, such as MET and IDH, and some genes had more mutations in malignant patients, such as PIK3CA and EZH2. Conclusions: We found that BRAFV600E mutation was a credible disease-related mutation in PTC; however, it could not be detected in cfDNA. Moreover, there was a large difference in mutation gene distribution between benign and malignant thyroid nodules.