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

Abstract

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.

Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments

“…Nucleosome occupancy/ ‘footprints’ of cfDNAs may be correlated with the expression of various target genes such as cancer drivers and providing critical information of its tissue of origin [ 205 , 206 ]. More recently, studies have shown a positive correlation between a decrease in nuclear cfDNA levels and a transition to longer fragments, and an improved chemotherapeutic response in patients with CRC [ 207 ]. Conversely, increased and shorter nuclear cfDNA content correlated with tumor recurrence [ 207 ].…”

“…More recently, studies have shown a positive correlation between a decrease in nuclear cfDNA levels and a transition to longer fragments, and an improved chemotherapeutic response in patients with CRC [ 207 ]. Conversely, increased and shorter nuclear cfDNA content correlated with tumor recurrence [ 207 ]. cfDNA analysis, thus, offers promising breakthroughs in non-invasive liquid biopsy technologies [ 100 ].…”

Extracellular Nucleic Acids in the Diagnosis and Progression of Colorectal Cancer

“…Several features of the mitochondrial genome can be analyzed, such as mtDNA mutations, mtDNA copy number alterations, heteroplasmy, or cf-mtDNA fragment length distribution. Since mtDNA exhibits characteristics distinct from the nuclear genome, including high copy numbers, high mutation frequencies, and heteroplasmy, it may be considered in some novel applications of liquid biopsies [ 98 ]. Recently, some papers have become available on the putative role of cf-mtDNA in CRC screening and follow-up ( Table 4 ).…”

“…Haupts et al reported a higher cf-mtDNA copy number in the plasma of healthy subjects compared to CRC patients [ 98 ]. Copy number of mitochondrially encoded NADH ubiquinone oxidoreductase core subunit 1 ( MT-ND1 ) in blood plasma has been suggested as a marker of early CRC [ 102 ].…”

Follow‐up of intraocular retinoblastoma through the quantitative analysis of conserved nuclear DNA sequences in aqueous humor from patients

“…A single normal cell contains only two copies of a nuclear DNA in the nucleus, whereas there are thousands of copies of mitochondrial DNA [ 10 ]. In some types of cancer, such as colorectal and papillary thyroid carcinomas, the proportion of mitochondrial and nuclear DNA circulating in plasma samples is different compared to that of patients without cancer [ 11 , 12 ]. The mitochondrial‐to‐nuclear DNA ratio in the blood of healthy individuals is up to four times higher than in cancer patients [ 11 , 12 ].…”