Abstract A46: Next-generation sequencing of circulating tumor DNA for detecting minimal residual disease and predicting recurrence in colorectal cancer patients
Authors: Chan Hiu Ting , Nagayama Satoshi , Chin Yoon Ming , Otaki Masumi , Hayashi Rie , Kiyotani Kazuma , Fukunaga Yosuke , Ueno Masashi , Nakamura Yusuke , Low Siew-Kee 
Published: June 2020

Abstract
Background: Although the prognosis of colorectal cancer (CRC) has improved in the past decade, a subset of CRC patients may still suffer from relapse due to the progression from minimal residual disease (MRD) after surgical resection. A sensitive and noninvasive method to efficiently detect MRD is essential to further improve clinical prognosis. In this study, we have evaluated the feasibility of circulating tumor DNA (ctDNA) analysis in detecting MRD in CRC patients.
Methods: Plasma samples were prospectively collected from 38 CRC patients, ranging from stage I to IV, who underwent surgical resection. A preoperative blood sample was taken just before surgery and postoperative samples were collected on multiple time-points to monitor the changes of mutation profiles. The extracted nucleic acid from matched surgically resected tumor tissues, peripheral blood cells (PBCs), and plasma were subjected to ultradeep targeted next-generation sequencing (NGS) using Oncomine Pan-Cancer Panel.
Results: Cell-free DNA/RNA (cfDNA/RNA) was extracted from 208 preoperative and postoperative plasma samples with a median input of 20 ng for targeted-NGS (ranged from 8-20 ng). The median of sequencing depth, molecular coverage, and barcode tagging efficiency of the sequencing were 54,000x, 4,642x and 84%, respectively. 74 nonsynonymous mutations were identified from tumor tissues and 64 mutations from the preoperative plasma samples. 41 (64.1%) of the 64 mutations detected in plasma samples were concordantly identified from the tumor tissue DNA. Paired PBCs sequencing identified 11 of the mutations detected from plasma and 4 mutations detected from tumor tissues to be clonal hematopoiesis-related mutations. After the exclusion of clonal hematopoiesis-related mutations, 34 (89.5%) of the 38 patients harbor at least one preoperative mutation either from tumor tissue or plasma to be monitored postoperatively. Fourteen patients were detected with ctDNA mutations from postoperative plasma samples and to date, two of them have developed clinical recurrence. One patient was detected to carry TP53-G245D and KRAS-A146T from both the preoperative plasma and resected-tumor tissue. Both mutations were not detected from plasma during adjuvant chemotherapy. At 4.5 months after the completion of chemotherapy, both mutations were positively detected from plasma with no signs of radiologic or clinical evidence of recurrence. Clinical recurrence was confirmed by PET scan 3 months after the detection of molecular recurrence from ctDNA. Similarly, positive ctDNA mutations also preceded radiologic and clinical evidence of recurrence by 3.5 months in another patient who has developed recurrence.
Conclusions: Our current results indicate that ctDNA analysis allows the detection of MRD in CRC patients. The integration of ctDNA analysis with current standard monitoring guidelines holds great promise in early detection of recurrence to allow clinical intervention to be applied promptly.
Citation Format: Hiu Ting Chan, Satoshi Nagayama, Yoon Ming Chin, Masumi Otaki, Rie Hayashi, Kazuma Kiyotani, Yosuke Fukunaga, Masashi Ueno, Yusuke Nakamura, Siew-Kee Low. Next-generation sequencing of circulating tumor DNA for detecting minimal residual disease and predicting recurrence in colorectal cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A46.