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 individually-designed 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 ctDNA monitoring but do not provide sufficient information to predict the proteomic composition of tumors.
Highlights
Circulating tumor DNA is released from tumor cells into blood [1,2]
Target sequencing using a HiSeq 2000 System (Illumina, Inc., CA) conducted for 151 genes that have been implicated in a wide range of cancers yielded 254,606,558 sequence reads from 30 primary tumor regions as well as corresponding peripheral blood mononuclear cells (PBMCs)
With 5% variant allele frequencies (VAFs) set as the minimum threshold, 363 single nucleotide variants (SNVs) and 480 insertion and/or deletion mutations (INDELs) were detected as tumor-unique based on the comparison between PBMCs and primary tumors
Summary
Circulating tumor DNA (ctDNA) is released from tumor cells into blood [1,2]. the majority of ctDNA shows very low variant allele frequencies (VAFs). A highly-sensitive method to measure ctDNA is needed for use in applications involving personalized tumor markers to predict relapse/regrowth, evaluate drug therapy efficacy, and confirm disease-free states [3,4,5,6]. Clinical applications using ctDNA are not limited to predicting utility of molecular targeting drugs; instead, the VAF dynamics of ctDNA can be applied for monitoring of tumor burden during the course of standard, targeted or immune-therapy. We proposed a pipeline to track tumor burden in combination with panel sequencing followed by ctDNA monitoring using digital PCR (dPCR) to identify patient-unique mutations [13,14,15]. Whether the mutations identified by this approach represent the complete spectrum of tumor genetic heterogeneity that can be reflected in ctDNA monitoring is unclear
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