Analysis of mutational and proteomic heterogeneity of gastric cancer suggests an effective pipeline to monitor post-treatment tumor burden using circulating tumor DNA

Abstract

Circulating tumor DNA (ctDNA) is released from tumor cells into blood in advanced cancer patients. Although gene mutations in individual tumors can be diverse and heterogenous, ctDNA has the potential to provide comprehensive biomarker information. Here, we performed multi-region sampling (three sites) per resected specimen from 10 gastric cancer patients followed by targeted sequencing and proteomic profiling using reverse-phase protein arrays. A total of 126 non-synonymous mutations were identified from 30 samples from 10 tumors. Of these, 16 (12.7%) were present in all three regions and were designated as founder mutations. Variant allele frequencies (VAFs) of founder mutations were significantly higher than those of non-founder mutations. Phylogenetic analysis also demonstrated a good concordance between founder and truncal mutations, defined as mutations shared by all simulated clones at the trunk of the tumor phylogenetic tree. These findings led us to prioritize founder mutations for quantitative ctDNA monitoring by digital PCR with individuallydesigned primer/probe sets. In preoperative plasma, the average ctDNA VAF of founder mutations was significantly higher than that of non-founder mutations (p = 0.039). Proteomic heterogeneity was present across the tumor regions both within and between patients independent of mutational status. Our results suggest that, in practice, mutations having high VAF identified without multi-regional sequencing may be immediately useful for quantitative

Impact of sensitive circulating tumor DNA monitoring on CT scan intervals during postoperative colorectal cancer surveillance

“…31 We previously demonstrated that, in contrast to next-generation sequencing (NGS), frequent ctDNA monitoring by dPCR enabled early relapse prediction, treatment efficacy evaluation, and disease-free corroboration in the management of gastrointestinal cancers, including CRC. [32][33][34][35] Here, we examined results of ctDNA assays from over 1,500 plasma samples and found that ctDNA monitoring by dPCR can provide early relapse detection and disease-free corroboration at molecular level during postoperative CRC surveillance as compared to conventional CTS and CEA monitoring.…”

“…The dPCR assay for quantitative monitoring of ctDNA levels was performed as described previously. [32][33][34][35] Briefly, specific primers and probes labeled for wild-type and mutant alleles were specifically designed for each mutation identified in a primary tumor, using Hypercool Primer & Probe™ technology (Nihon Gene Research Laboratories, Sendai, Japan). For frequently recurring missense mutations, commercially available primer/probe sets were used (Thermo Fisher Scientific, Waltham, MA and Quantdetect, Inc, Tokyo, Japan).…”

“…35,37 Monitoring ctDNA levels using dPCR The dPCR assay for quantitative monitoring of ctDNA levels was performed as described previously. [34][35][36][37] Briefly, primers and probes labeled for wild-type and mutant alleles were specifically designed for each mutation identified in a primary tumor. Between 1 and 4 mutations per tumor that had a VAF >10% in primary tumors were prioritized for dPCR analysis.…”

“…Our previous studies also demonstrated that tumor-informed ctDNA monitoring has the clinical validity in terms of early relapse prediction, treatment efficacy evaluation, and non-relapse corroboration in management of several types of gastrointestinal cancers, including CRC. [34][35][36][37] In the present study, we further investigated whether ctDNA monitoring by dPCR can alter intervals of CT testing during postoperative intensive surveillance without loss of critical therapeutic opportunities for clinical relapse.…”

Reply

“…We found that most "clonal" mutations exhibit higher variant allele frequencies (VAFs) compared to "subclonal" mutations in gastric and colorectal cancer, which offers opportunities for detection in ctDNA. 3,4 Multiregional sequencing of ESCCs also revealed that "clonal" mutations had high VAFs in all cases. 5 In our study, the clinical validity of ctDNA monitoring was confirmed in 91% (31/34) of ESCC patients, using fewer than 3 mutations per patient.…”