Clonal diversity in KRAS mutant colorectal adenocarcinoma under treatment: Monitoring of cfDNA using reverse hybridization and DNA sequencing platforms

Biological heterogeneity is a key feature of malignancies that significantly contributes to disease progression and therapy resistance. Residual/relapsed tumor foci may represent genetically divergent subclones, which remain uncovered as repeated and multiple tumor sampling is usually limited. The analysis of circulating free DNA (cfDNA) from the peripheral blood plasma (also called a liquid biopsy, LB) is a new achievement that provides an effective tool for follow-up monitoring of cancer-related genetic status. The present study highlights the phenomenon of mutational variability observed in patients with metastatic KRAS mutant colorectal cancer (mCRC) during treatment with bevacizumab in combination in a longitudinal fashion.

The prospective study included 490 mCRC patients evaluated between 2020 and 2022 in our institution. Out of the 211 KRAS mutant cases (43.06%) 12 tumors were identified with multiple KRAS gene variants (5.68%). Detailed follow-up investigations were possible in 3 of these patients including the genotyping of the primary and available metastatic tumors, and the peripheral blood cfDNA. cfDNA was collected from three different time points before and between cycles of combined treatment with bevacizumab chemotherapy. KRAS gene variants were identified using reverse-hybridization strips, and next-generation sequencing (NGS), and confirmed by conventional Sanger sequencing.

Interestingly, surgery and multiple treatment cycles reorganized the mutational profiles in the selected cases. The effect of the treatments resulted either in the overrepresentation of one of the pre-existing gene variants or in the appearance of new KRAS variants absent in the primary sample, according to the plasma cfDNA findings. Besides the KRAS variants demonstrated by targeted analysis, NGS mutational profiling identified some additional pathogenic variants from the cfDNA samples (including NRAS and MET alterations).

In conclusion, plasma cfDNA sampling enables the monitoring of mutational heterogeneity and subclonal dynamics of the actual metastatic tumor mass in mCRC. The pattern of molecular profile potentially reflects a differential drug response determining further progression.