Abstract 1353: The clinical utility of ctDNA in colorectal cancer validated by multiregional sequencing and protein analysis
Authors: Yaegashi Mizunori , Iwaya Takeshi , Fujita Masashi , Ju Zhenlin , Siwak Doris , Sato Kei , Endo Fumitaka , Sugimoto Ryo , Sugai Tamotsu , Liotta Lance , Lu Yiling , Mills Gordon , Nakagawa Hidewaki , Nishizuka Satoshi 
Published: August 2019

Abstract
Background : Circulating tumor DNA (ctDNA) is considered to be a new class of tumor-specific personalized biomarkers. Since the variant allele frequency (VAF) of ctDNA is very low (0.01-1%), it is more important to quantify tumor-specific mutations using specific probes than to perform exploratory analyses. A molecular targeting drug is often selected by mutations, and it has not been verified whether the mutations reflect the level of proteins as the drug target molecule.
Purpose: To clarify: (a) the utility of ctDNA from tumors with genetic heterogeneity; (b) the ability of early detection for relapse; and (c) the validity of protein level prediction by gene mutation.
Methods: Tumor DNA was extracted from three regions of 14 Stage III/IV surgically-resected colorectal tumors. Plasma DNA was also extracted from pre-/post-operative as well as follow-up blood samples every three months. The multi-regional sequencing analysis was conducted using a next generation sequencer (NGS) with a panel of 151 genes and phylogenetic trees were subsequently generated. Of the detected non-synonymous mutations, ctDNA was monitored using digital PCR (dPCR) for which primers and probes were designed by the Hypercool Primer & Probe™ technology to facilitate a specific PCR with short (~70bp) DNA fragments. A reverse phase protein array (RPPA) was used to quantitate 294 proteins in cancer tissues for three regions.
Results: The average number of mutations per tumor and per region was nine and six, respectively. The average number of founder mutations (i.e., mutations found in all three regions) and non-founder mutations was three and seven, respectively. Phylogenetic analysis revealed that mutations with high VAF tended to emerge at the trunk of the evolutional tree. Interestingly, when non-founder mutations in the regions that had not exhibited the mutation by NGS were detected using dPCR in 5 of 13 regions. mutations were still detected in 5 of 13 regions (38.5%). The VAFs of founder mutations were significantly higher than those of non-founder mutations. In pre-operative plasma, ctDNA was detected in 10 of 12 patients (83.3%) with low VAF (range; 0.028-13.3%). Founder mutations in ctDNA were detected in 8 of 10 patients with low VAF (range; 0.04-13.3%). In each patient, founder mutations appeared to exhibit higher VAFs in ctDNA than those of non-founder mutations. Importantly, the VAF of ctDNA started to increase 6 months before the relapse was diagnosed by CT scan. Among 42 gene-protein matched pairs, the level of phosphorylated proteins that could be molecular targets did not seem to be predicted by the coding gene mutation.
Conclusions: To select mutations for ctDNA monitoring, founder mutations with high VAF in a tumor are mandatory. Since mutations in colorectal cancer do not seem to predict the protein level, the proper clinical utility of ctDNA is a tumor marker over the entire course of colorectal cancer therapy.
Citation Format: Mizunori Yaegashi, Takeshi Iwaya, Masashi Fujita, Zhenlin Ju, Doris Siwak, Kei Sato, Fumitaka Endo, Ryo Sugimoto, Tamotsu Sugai, Lance Liotta, Yiling Lu, Gordon Mills, Hidewaki Nakagawa, Satoshi Nishizuka. The clinical utility of ctDNA in colorectal cancer validated by multiregional sequencing and protein analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1353.