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The current standard for molecular profiling of colorectal cancer (CRC) is using resected or biopsied tissue specimens. However, they are limited regarding sampling frequency, representation of tumor heterogeneity, and sampling can expose patients to adverse side effects. The analysis of cell-free DNA (cfDNA) from blood plasma, which is part of a liquid biopsy, is minimally invasive and in principle enables detection of all tumor-specific mutations. Here, we analyzed cfDNA originating from nucleus and mitochondria and investigated their characteristics and mutation status in a cohort of 18 CRC patients and 10 healthy controls using targeted next-generation sequencing (NGS) and digital PCR. Longitudinal analyses of nuclear cfDNA level and size during chemotherapy revealed a decreasing cfDNA content and a shift from short to long fragments, indicating an appropriate therapy response, while shortened cfDNAs and increased cfDNA content corresponded with tumor recurrence. Comparative NGS analysis of nuclear tissue and plasma DNA demonstrated a good patient-level concordance and cfDNA revealed additional variants in three of the cases. Analysis of mitochondrial cfDNA surprisingly revealed a higher plasma copy number in healthy subjects than in CRC patients. These results highlight the potential clinical utility of liquid biopsies in routine diagnostics and surveillance of CRC patients as complementation to tissue biopsies or as an attractive alternative in cases where tissue biopsies are risky or the quantity/quality does not allow testing.

Comparative analysis of nuclear and mitochondrial DNA from tissue and liquid biopsies of colorectal cancer patients