Low Impact of Clonal Hematopoiesis on the Determination of RAS Mutations by Cell-Free DNA Testing in Routine Clinical Diagnostics

Targeted sequencing of circulating cell-free DNA (cfDNA) is used in routine clinical diagnostics for the identification of predictive biomarkers in cancer patients in an advanced stage. The presence of KRAS mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) might represent a confounding factor. We used an amplicon-based targeted sequencing panel, covering selected regions of 52 genes, for circulating cell-free total nucleic acid (cfTNA) analysis of 495 plasma samples from cancer patients. The cfDNA test failed in 4 cases, while circulating cell-free RNA (cfRNA) sequencing was invalid in 48 cases. In the 491 samples successfully tested on cfDNA, at least one genomic alteration was found in 222 cases (45.21%). We identified 316 single nucleotide variants (SNVs) in 21 genes. The most frequently mutated gene was TP53 (74 variants), followed by KRAS (71), EGFR (56), PIK3CA (33) and BRAF (19). Copy number variations (CNVs) were detected in 36 cases, while sequencing of cfRNA revealed 6 alterations. Analysis with droplet digital PCR (ddPCR) of peripheral blood leukocyte (PBL)-derived genomic DNA did not identify any KRAS mutations in 39 cases that showed KRAS mutations at cfDNA analysis. These findings suggest that the incidence of CHIP-associated KRAS mutations is relatively rare in routine clinical diagnostics.