Circulating Tumor DNA as a Preoperative Marker of Recurrence in Patients with Peritoneal Metastases of Colorectal Cancer: A Clinical Feasibility Study

Abstract

Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy (CRS-HIPEC) may be curative for colorectal cancer patients with peritoneal metastases (PMs) but it has a high rate of morbidity. Accurate preoperative patient selection is therefore imperative, but is constrained by the limitations of current imaging techniques. In this pilot study, we explored the feasibility of circulating tumor (ct) DNA analysis to select patients for CRS-HIPEC. Thirty patients eligible for CRS-HIPEC provided blood samples preoperatively and during follow-up if the procedure was completed. Targeted Next-Generation Sequencing (NGS) of DNA from PMs was used to identify bespoke mutations that were subsequently tested in corresponding plasma cell-free (cf) DNA samples using droplet digital (dd) PCR. CtDNA was detected preoperatively in cfDNA samples from 33% of patients and was associated with a reduced disease-free survival (DFS) after CRS-HIPEC (median 6.0 months vs median not reached, p = 0.016). This association could indicate the presence of undiagnosed systemic metastases or an increased metastatic potential of the tumors. We demonstrate the feasibility of ctDNA to serve as a preoperative marker of recurrence in patients with PMs of colorectal cancer using a highly sensitive technique. A more appropriate treatment for patients with preoperative ctDNA detection may be systemic chemotherapy in addition to, or instead of, CRS-HIPEC.

Keywords: CRS-HIPEC; circulating tumor DNA; colorectal cancer; droplet digital PCR; liquid biopsy; peritoneal metastases.

Figure 1

Flowchart of patient classification after initial inclusion in the study. The reasons for patient exclusion prior to intraoperative assessment are described. After the intraoperative assessment, patients either underwent Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy (CRS-HIPEC) or received an open–close procedure. PMs: Peritoneal Metastases.

Figure 2

(A) An overview of the genes in which a somatic mutation was identified in peritoneal metastases (PMs) and tested for in the corresponding preoperative cfDNA sample. A black square indicates that the mutation was identified in both PMs and cfDNA; a grey square indicates a negative result for the corresponding mutations in the cfDNA. All cfDNA samples were tested using the KRAS G12/13 screening kit. (B) Comparison of the relative proportion of mutations detected in PMs and preoperative cfDNA samples. (C) Concentration of cfDNA samples. A black dot denotes that ctDNA was detected; lines indicate the median and the interquartile range. HRM-sequencing: High-Resolution Melting assay followed by Sanger sequencing.

Figure 3

Survival analysis of patients who received CRS-HIPEC. (A) All patients (n = 24). (B) All patients except those diagnosed with preoperative liver metastases (n = 22). DFS: Disease-free Survival.

Figure 4

Tissue-guided ctDNA analysis of plasma samples taken from 14/24 patients during follow-up after CRS-HIPEC. Ten patients did not provide a sample. Patients are categorized by the location of metastases before or during CRS-HIPEC and the location of recurrence(s) during follow-up. The blue color is to highlight a positive result.