Cross-sectional analysis of circulating tumor DNA in primary colorectal cancer at surgery and during post-surgery follow-up by liquid biopsy

Abstract

Background: Liquid biopsy (LB) in early-stage, non-metastatic colorectal cancer (CRC) must be sensitive enough to detect extremely low circulating tumor DNA (ctDNA) levels. This challenge has been seldom and non-systematically investigated. Methods: Next generation sequencing (NGS) and digital PCR (dPCR) were combined to test tumor DNAs (tDNAs) and paired ctDNAs collected at surgery from 39 patients, 12 of whom were also monitored during the immediate post-surgery follow up. Patients treated for metastatic disease (n = 14) were included as controls. Results: NGS and dPCR concordantly (100% agreement) called at least one single nucleotide variant (SNV) in 34 tDNAs, estimated differences in allelic frequencies being negligible (±1.4%). However, despite dPCR testing, SNVs were only detectable in 15/34 (44.1%) ctDNAs from patients at surgery, as opposed to 14/14 (100%) metastatic patients. This was likely due to striking differences (average 10 times, up to 500) in ctDNA levels between groups. NGS revealed blood-only SNVs, suggesting spatial heterogeneity since pre-surgery disease stages, and raising the combined NGS/dPCR sensitivity to 58.8%. ctDNA levels at surgery correlated with neither tumor size, stage, grade, or nodal status, nor with variant abundance in paired tDNA. LB sensitivity reached 63.6% when ctDNA was combined with CEA. Finally, persistence and absence of ctDNA on the first conventional (month 3) post-surgery follow-up were associated with fast relapse and a disease-free status in 3 and 7 patients, respectively. Conclusions: A simple clinical NGS/dPCR/CEA combination effectively addresses the LB challenge in a fraction of non-metastatic CRC patients.

TMPRSS2, a SARS-CoV-2 internalization protease is downregulated in head and neck cancer patients

“…All DNA samples were then quantified by a Qubit Fluorometer (Termofisher Scientific, Waltham, Massachusetts, USA) using a Qubit® dsDNA HS Assay Kit. Library preparation was performed on 10 ng DNA by the Ion AmpliSeq Library Kit 2.0 (Termofisher Scientific) and the Colon and Lung Panel (Life Technologies) was used to sequencing the entire coding regions of TP53, as previously described [ 16 ]. Briefly, the prepared libraries were sequenced on Ion S5 Sequencer using an Ion 540 Chip and an Ion 540 kit–Chef (all Thermo Fisher Scientific) with configuration 500 flows covering the 200 bp library read length.…”

The Promise of Circulating Tumor DNA (ctDNA) in the Management of Early-Stage Colon Cancer: A Critical Review

“…A retrospective analysis of the IDEA-France data also reported that the detection of ctDNA postoperatively is an independent adverse prognostic marker for cancer recurrence (adjusted HR,1.85; p < 0.001) in stage III patients treated with oxaliplatin-based ACT [ 49 ]. A similar study by Allegretti et al [ 50 ] reported that persistence and absence of ctDNA at the time of first post-operative (3 month) follow-up were associated with fast relapse and a disease-free status in three and seven patients, respectively. In addition, this study reported improved sensitivity (58.8% to 63.6%) when ctDNA result was analyzed in combination with serum CEA level.…”

Direct plasmonic detection of circulating RAS mutated DNA in colorectal cancer patients

“…Spiking experiments prompted us to apply the NESPRI assay to the direct detection of KRAS mutated ctDNA present in the blood of metastatic CRC patients. These specimens were preliminarily validated by NGS and digital PCR (dPCR) 28 , carried a unique RAS mutation per sample and displayed different variant allele frequencies (VAF, i.e., the percentage of mutated reads) and mutated copies per ml (Table 4). We applied the already described NESPRI assay operating under continuous flow conditions so that kinetic curves useful to check the correct outcome of each step of the assay were also recorded during the experiments.…”

“…Data here reported displays the clinical validity of the NESPRI assay and its ability to discriminate between normal and RAS mutated genotype into separate groups (CRC and control group). These were shown to be associated with different treatment indications and clinical outcomes 28 . The assay has been shown to exhibit analytical validity also in testing liquid biopsies from CRC patients.…”