Clinical application of circulating tumor DNA (ctDNA) in patients with metastatic gastric cancer.
Authors: Hong Moonki , Lee Kwang Seob , Park Sejung , Kim Kyoo Hyun , Kim Hyunwook , Che Jingmin , Kwon Woo Sun , Lee Choong-kun , Jung Minkyu , Chung Hyun Cheol Cheol , Shin Saeam , Lee Seung-Tae , Choi Jong Rak , Rha Sun Young 
Published: January 2023

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Background: Circulating tumor DNA (ctDNA) is a minimally invasive approach, effective for the real-time follow-up of cancer. However, the clinical significance in gastric cancer is not yet determined. Therefore, we investigated both ctDNA and tissue biopsy NGS at diagnosis of stage IV gastric cancer patients to evaluate the relationship of ctDNA with clinical parameters and concordance of ctDNA and tissue NGS. Methods: All tissue samples were sequenced with the targeted panel sequencing – CancerMaster Panel V2 (in-house 522 gene panel, Kwon et al., ASCO GI 2021) or TruSight Oncology (TSO) 500 (illumina, Inc. USA). 20mL of whole blood in DxTube(Dxome, Korea) was used for library preparation using DxSeq ctDNA TMB500 for Illumina Platform Kit (Dxome, Korea) according to the manufacturer’s instructions. For each sample, PBMC was sequenced as germline paired controls using the identical panel, and library kits targeting an average depth of >3,000x. ctDNA analysis using TMB500 panel reports total ctDNA amount, genomic alterations including copy number variations, tumor mutational burden (ctTMB) and MSI status (Lee et al, Clinical Chemistry accepted). Her-2, EBV, dMMR status in the tissues were performed in standard manner. Results: ctDNA were analysed since 2 samples were not qualified. 27 chromosomal instable (CIN) type and 46 Genome stable (GS) type based on pathologic evaluation were identified. Higher ctDNA were detected in CIN group than GS group (17.8 vs. 6.8, p<0.01) and ctTMB was higher in EBV or CIN than GS group (12.8 vs. 9.1, p=0.02). Patients with high CEA, LDH or systemic metastases including liver, bone, lung, or distant lymph node showed higher ctTMB than those without such factors (14.7 vs. 8.5, p<0.01, 12 vs. 8.8, p<0.01, and 12.5 vs. 7.8, p<0.01, respectively). ctDNA amount was higher in LDH high and systemic metastases patients (31.3 vs. 6.9, p<0.01 and 12.8 vs 6.3, p<0.01, respectively). Among 53 patients with paired ctDNA and tissue NGS, 49 (92.4%) and 47 (88.7%) had detectable ctDNA alterations and tissue alterations respectively, with concordance of 95.9%. Tissue TMB was higher than ctTMB (14.8 vs. 8.0, p<0.01), but the two showed positive correlation (Pearson c. 0.34, p=0.01). Druggable targets (TP53 mutation, KRAS, ERBB2, FGFR, MET or RICTOR amplification) were more frequently detected by ctDNA than tissue NGS. In 25 patients with TP53 mutation in both tissue and ctDNA, TP53 genetic alterations were identical except 1 patient. When compared with ERBB2 IHC, specificity was 93.6% in ctDNA, 97.9% in tissue NGS and sensitivity of both were 50%. Patients with false positive of ERBB2 in ctDNA showed lesser copy number than those with true positive in ctDNA. Conclusions: Our study showed a potential role of ctDNA in advanced gastric cancer, suggesting ctDNA’s sensitivity in detecting various druggable target genes. Investigation to reveal relationship between ctDNA and clinical outcomes is ongoing.