Pairwise analysis of plasma cell-free DNA before and after palliative second-line paclitaxel plus ramucirumab treatment in patients with metastatic gastric cancer

Abstract

Background: This study compared plasma cell-free DNA (cfDNA) and tumor tissue DNA (ttDNA) to explore the clinical applicability of cfDNA in patients with metastatic gastric cancer (mGC) receiving palliative second-line paclitaxel + ramucirumab treatment.

Methods: Targeted sequencing of 106 genes was conducted using germline DNA and cfDNA at baseline (baseline-cfDNA) and progressive disease (PD-cfDNA). The results were compared with those of ttDNA-based cancer panel data.

Results: Of 76 consecutive patients, 46 (27 males; median age 57.5 [range, 32-73] years) who had all three samples were included. Combined analysis of ttDNA and baseline-cfDNA revealed that TP53 (58.7%) was the most frequently mutated gene, followed by CDH1 (26.1%), KRAS (21.7%), and APC (13.0%). For these genes, the sensitivity and positive predictive value of baseline-cfDNA over ttDNA were 71.8% and 51.9%, respectively. When baseline-cfDNA and PD-cfDNA results were combined, 34 patients (73.9%) were found to have additional mutations compared with ttDNA results alone. PD-cfDNA analysis revealed 14 novel pathogenic mutations in ten patients (21.7%). At baseline, patients with a high circulating tumor DNA fraction concentration showed a significantly shorter progression-free survival (PFS) (P = 0.016) in univariable and multivariable analyses. High maximal variant allele frequency (VAF) (P = 0.022), high sum of VAF (P = 0.028), and high TP53 VAF (P = 0.022) were associated with worse PFS in univariable analysis.

Conclusions: Although cfDNA alone cannot replace ttDNA entirely, cfDNA analysis revealed additional mutations. Notably, PD-cfDNA analysis revealed novel pathogenic mutations that emerged during treatment. Moreover, the baseline circulating tumor DNA fraction concentration and VAF values were associated with longer PFS.

Keywords: Anti-cancer therapy; Cell-free DNA; Circulating tumor DNA; Clonal evolution; Gastric cancer.

Fig. 1

Oncoplot of 46 patients with gastric cancer. For each gene, each patient had two boxes denoting different sample types: ttDNA (upper box) and cfDNA at baseline and PD (lower box). Vertical bar plots at the top and right show the number of mutations with different colors representing different sample types. SNV single nucleotide variant, CNV copy number variation, PD progressive disease, MSI microsatellite instability, MSS microsatellite stable, MSI-H microsatellite instability-high, VAF variant allele frequency, cfDNA cell-free DNA, ttDNA tumor tissue DNA

Fig. 2

Clonal evolution dynamics before and after the treatment. A Variant allele frequency values of ttDNA, baseline-cfDNA, and PD-cfDNA were visualized in eight patients harboring single nucleotide variants and small indels: GC025, GC050, GC058, GC070, GC085, GC099, GC116, and GC177. B Copy number values of ttDNA, baseline-cfDNA, and PD-cfDNA were visualized in three patients harboring copy number variations: GC047, GC081, and GC116. PD progressive disease, ttDNA tumor tissue DNA, cfDNA cell-free DNA

Fig. 3

PFS according to DNA concentration and VAF. A Patients with a high cfDNA concentration (> 17.27 ng/µL) tended to have shorter PFS (P = 0.0502). B Patients with a high ctDNA concentration (> 4.38 ng/µL) at 110–160 bp had significantly shorter PFS (P = 0.016). C Among patients with any mutations in the baseline-cfDNA (N = 36), those with higher maximal VAF values (> 0.1045) demonstrated significantly worse PFS (P = 0.022). D Patients with a higher sum of VAF values (> 0.2071) had significantly shorter PFS (P = 0.028). E Among patients harboring TP53 mutations (N = 24), those with high TP53 VAF values (> 0.1014) showed significantly worse PFS (P = 0.022). PD progressive disease, cfDNA cell-free DNA, PFS progression-free survival