Disease monitoring by analysis of circulating cell-free DNA in metastatic colorectal cancer patients

Introduction: Colorectal cancer (CRC) is characterized by a high level of genetic heterogeneity. Changes in the genetic profile induced by chemotherapy affect treatment results. Acquired resistance of tumors is defined as a result of clonal evolution and clonal selection under systemic chemotherapy. Repeated tumor tissue biopsies are difficult to obtain and cannot be easily used for dynamic monitoring of therapy response or failure due to marked tumor heterogeneity. Promising data for circulating cell-free DNA (cfDNA) as a tool for studying tumor evolution were recently published.

Aims and methods: In this study we analyzed cfDNA from 27 patients with metastatic CRC during 1^st, 2^nd and 3^rd line palliative chemotherapy. By droplet digital PCR we analyzed the total amount of cfDNA and performed targeted genotyping of cfDNA and tracking of clonal evolution of the most frequent KRAS mutations (G12A, G12C, G12D, G12R, G12S, G12V, G13C, G13D, Q61R, A146T and A59T). We did correlation studies between cfDNA results and established tools (RECIST 1.1 and CEA).

Results: cfDNA was detectable in all patients of the study cohort. In 82.2% cfDNA amount correlated with course of disease, as monitored by CT scans every 8 weeks. In 85.7% cfDNA amount correlated with CEA levels. Of note, the changes in cfDNA by non-invasive analysis were detectable weeks prior to confirmed therapy failure by CT scans. The results of targeted genotyping indicate that there is evidence also for KRAS screening in the blood prior to initiating of a treatment with anti-EGFR antibodies. Tissue analysis alone underlies the single biopsy bias and tumors are wrongly classified as RAS wild-type. Our results provide the rationale for repeated KRAS testing under EGFR blockade, because mutated KRAS fragments occurred under anti-EGFR therapy with impact of treatment results. Interestingly, we also could show that mutated KRAS clones in the blood disappeared under chemotherapy and EGFR blockade and re-occurred at the time point of disease progression.

Conclusion: The analysis of cfDNA in combination with targeted genotyping shows promising results for non-invasive disease monitoring and has the potential to overcome the restrictions of tumor tissue genotyping.