Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel

Abstract

BackgroundCirculating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile.MethodologyDNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor.ResultsA total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease.ConclusionsThis panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.

Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma

“…This sample and three other samples only showed mutations in loci not covered by the KRAS Screening Multiplex Kit used for ddPCR ( KRAS codon 61, BRAF and TP53 ). Nonetheless, we observed good agreement between ddPCR and NGS, which was also reported in other studies …”

A high-throughput assay for quantitative measurement of PCR errors

“…3, we detected high-frequency errors at a similar rate for all polymerases in 20 cycle PCR reactions in two independent experiments. These recurrent errors can reach a frequency of >0.1%, putting them in the sensitivity range of state-of-art assays for circulating tumor DNA detection 22 . Replicate experiments 23 are unfeasible in this scenario, and therefore appropriate PCR error models and other techniques such as the UMI approach should be used instead for ultra-deep sequencing.

Figure 3Recurrent high-frequency errors in two separate experiments.

…”

Role of circulating free DNA in colorectal cancer

“…The selection of the proper mutation markers to be monitored in cfDNA is as yet unresolved. Sato et al[29] monitored preoperative and postoperative cfDNA levels based on a panel of 50 genes, using ddPCR; only markers with an allele frequency above 0.1% in plasma DNA correlated with the clinical course. Interestingly, cfDNA has also been shown to be useful in the early detection of relapse after metastasectomy of liver metastases, significantly outperforming both CEA and imaging in one study[30].…”