Abstract 2229: Molecular profile of histological and mutational heterogeneity of adenocarcinoma of the stomach in tumor burden monitoring using circulating tumor DNA
Authors: Sasaki Noriyuki , Iwaya Takeshi , Chiba Takehiro , Fujita Masashi , Endo Fumitaka , Yaegashi Mizunori , Sugimoto Ryo , Sugai Tamotsu , Siwak Doris , Liotta Lance , Lu Yilling , Mills Gordon , Nakagawa Hidewaki , Nishizuka Satashi S. 
Published: August 2019

Abstract
PURPOSE: This study aimed to perform molecular profiling of the heterogeneous histological and mutational background of primary adenocarcinoma of the stomach for pre- and post-operational tumor burden monitoring using circulating tumor DNA (ctDNA).
METHODS: Ten patients with gastric adenocarcinoma who underwent surgical resection for clinical Stage IB or higher were included. Multi-region samples (three sites) per resected specimen were used for cancer cellulality estimation, a 151-gene ClearSeq panel sequencing on Next Generation Sequencer (NGS), and proteomic profiling with 294 proteins using reverse-phase protein arrays (RPPAs). The ctDNA level was evaluated by digital PCR (dPCR) using 25 originally-designed sets with the Hypercool Primer & ProbeTM technology based on mutations of individual tumors. With the median follow-up of 808 days, a total of 187 serial plasma samples were examined for ctDNA.
RESULTS: A total of 103 mutations were detected in 30 regions from 10 tumors. Twenty founder mutations (i.e., mutations found in all three regions) were observed in eight tumors whereas two tumors had only non-founder mutations. Twenty-three and 60 non-founder mutations were detected in two regions and one region per tumor, respectively. Variant allele frequencies (VAFs) of founder mutations were higher than those of non-founder mutations (31.2% vs 14.5%; p &lt; 0.01) in primary tumors. With sample regions in which mutations were not detected in trio by NGS, non-founder mutations could be detected by dPCR at low VAFs (ranging from 0.02 to 2.2%) in 95% (19/20) regions. In preoperative patient plasma, ctDNA was detected in 30% (3/10) patients (with a mean VAF of 0.59%). The rate of preoperative ctDNA detection was lower than that of esophageal squamous cell cancer (24/26, 92.3%) and colorectal cancer (10/12, 83.3%) patients. With respect to the tumor stromal effect to ctDNA, no ctDNA was detected in three cases of scirrhous type cancers (0/3, 0%), whereas three of six cases of the other stromal types (3/6, 50%) showed detectable ctDNA (VAF&gt;0.03%). In two relapsed cases with peritoneal dissemination, the elevation of ctDNA was not apparent even at the time of diagnosis of the relapse by CT scan. Among 42 gene-protein matched pairs, the level of proteins did not seem to predict the coding gene mutation. However, tumors with TP53 mutations had significantly higher levels of p53 than those with the wild-type, which was likely due to protein stabilization (p = 0.0004).
CONCLUSIONS: Detecting ctDNA in gastric cancer patients may not be as feasible as in other gastrointestinal cancer patients likely due to the heterogeneous histological and mutational background. If applicable, founder mutations are the most suitable marker for detection and monitoring ctDNA. Mutations in the primary tumor itself did not appear to predict protein levels except for TP53.
Citation Format: Noriyuki Sasaki, Takeshi Iwaya, Takehiro Chiba, Masashi Fujita, Fumitaka Endo, Mizunori Yaegashi, Ryo Sugimoto, Tamotsu Sugai, Doris Siwak, Lance Liotta, Yilling Lu, Gordon Mills, Hidewaki Nakagawa, Satashi S. Nishizuka. Molecular profile of histological and mutational heterogeneity of adenocarcinoma of the stomach in tumor burden monitoring using circulating tumor DNA [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 2229.