Identification of novel somatic cell-free DNA variants by next-generation sequencing in breast cancer patients

Objectives: Breast cancer is a heterogeneous disease affecting women worldwide and is one of the leading causes of mortality in India. Sampling bias due to tumor heterogeneity and invasive nature of biopsies necessitate noninvasive methods for comprehensive tumor profiling. Circulating cell-free DNA presents a complete mutation profile of the tumor, enabling the non-invasive monitoring of disease in real-time. This study aimed to identify tumor-specific variants in cfDNA with potential applications in the liquid-biopsy based testing of breast cancer. Material and Methods: Next-generation sequencing was performed for cell-free DNA, lymphocyte DNA, and tumor DNA from 21 breast cancer patients. Variant calling was performed using Torrent Suite Server v.5.0 and somatic variants were annotated using web-based tools. Pathogenic variants detected in cell-free DNA and tumor DNA of three patients were validated by Sanger sequencing. Results: Fifty-nine somatic variants were detected in the cell-free DNA of 10 breast cancer patients. Hotspot variants were detected in PIK3CA, TP53, and KRAS genes. In addition, previously unreported missense variants in ABL1 and PIK3CA genes were predicted to be pathogenic and potential driver mutations. Several frameshift indels were detected in two triple negative breast cancer patients. Conclusion: Sequencing of cell-free DNA from breast cancer patients identified somatic variants including several potentially pathogenic variants which have not been reported previously. These variants may have potential applications as non-invasive biomarkers for breast cancer.