Abstract 1974: Clonal and subclonal mutational landscapes in circulating tumor DNA in metastatic colorectal cancer: An exploratory analysis from the phase III PARADIGM study
Authors: Tsuchihara Katsuya , Yamashita Riu , Yoshino Takayuki , Shitara Kohei , Watanabe Jun , Yasui Hirofumi , Ohori Hisatsugu , Shiozawa Manabu , Yamazaki Kentaro , Oki Eiji , Sato Takeo , Naitoh Takeshi , Komatsu Yoshito , Kato Takeshi , Yamanaka Kazunori , Mori Ikuo , Hihara Masamitsu , Soeda Junpei , Yamanaka Takeharu , Akagi Kiwamu , Ochiai Atsushi , Muro Kei , Velculescu Victor E. , Uetake Hiroyuki 
Published: September 2020

Abstract
Introduction: We performed an exploratory analysis of circulating tumor DNA (ctDNA) in patients (pts) with RAS wild-type (WT) chemotherapy-naïve stage IV metastatic colorectal cancer (mCRC) from the ongoing phase III PARADIGM study (NCT02394834). The clonal and subclonal mutational landscapes of RAS WT pts were analyzed using a mCRC-focused custom ctDNA sequencing panel.
Methods: Randomized pts (1:1) received mFOLFOX6 plus either panitumumab or bevacizumab. Pre-treatment plasma samples (DNA yield &gt;10ng/mL and &gt;10nM) were sequenced using the custom ctDNA panel (PlasmaSELECTTM-R 91, PGDx) to detect mCRC mutations, amplifications, and rearrangements in 90, 26, and 3 genes, respectively, as well as microsatellite instability (MSI), in 250kb targeted regions using stringent quality criteria. Pre-treatment archival tissue samples from 590 pts were analyzed by the Broad Institute Solid Tumor panel, which covered 1,072 genes with 7.3Mb targeted regions.
Results: Between May 29, 2015 and June 8, 2017, 823 pts were enrolled across 197 sites. Plasma samples were collected from 756 pts, with 747 (98.8%) samples meeting the quality criteria. The average total and distinct sequencing coverages were 34,200 and 5,865, respectively, with 49.1% of bases mapping to regions of interest. Mutation frequencies in ctDNA were generally consistent with those observed in RAS-WT TCGA CRC and rectal cancers (n=283), except for TET2 and DNMT3A potentially due to clonal hematopoiesis. Mutations in BRAF, APC, CTNNB1, PTEN, PIK3CA, and MSI-status observed in ctDNA were significantly different between left and right-sided tumors, consistent with known differences in tumor sidedness. In the 747 plasma samples, 4,072 mutations were observed; 1,871 had a mutant allele frequency (MAF) &lt;25% of the highest MAF in each sample (defined as subclonal) and 2,201 had a MAF above this threshold (defined as clonal). Clonal mutations were 77.5% concordant with mutations in corresponding tissue samples. Subclonal mutations showed 17.3% concordance between tissue and plasma, suggesting that tumor heterogeneity may result in subclonal mutations in ctDNA. Although pts with KRAS/NRAS mutations (G12, G13, A59, Q61, K117, A146) in archival FFPE tissue were not eligible for this study, 38 RAS mutations (58% subclonal) were observed in ctDNA of 32 pts, suggesting that sub-populations of tumor cells may have KRAS mutations in these cases. Patient-level subclonality analyses suggested that the fraction of subclonal mutations were higher in pts with metastases in ≥2 organ sites.
Conclusion: The validated ultra-deep plasma sequencing panel was concordant with tissue sequencing and detected tumor heterogeneity. The PARADIGM study will report the efficacy of anti-EGFR/VEGF therapies; post-treatment collection of ctDNA is ongoing. The relationship between therapeutic effects and clonal and subclonal mutational landscapes will be examined in the future. Funded by Takeda Pharmaceutical Company Limited, Tokyo, Japan. ClinicalTrial.gov number: NCT02394834
Citation Format: Katsuya Tsuchihara, Riu Yamashita, Takayuki Yoshino, Kohei Shitara, Jun Watanabe, Hirofumi Yasui, Hisatsugu Ohori, Manabu Shiozawa, Kentaro Yamazaki, Eiji Oki, Takeo Sato, Takeshi Naitoh, Yoshito Komatsu, Takeshi Kato, Kazunori Yamanaka, Ikuo Mori, Masamitsu Hihara, Junpei Soeda, Takeharu Yamanaka, Kiwamu Akagi, Atsushi Ochiai, Kei Muro, Victor E. Velculescu, Hiroyuki Uetake. Clonal and subclonal mutational landscapes in circulating tumor DNA in metastatic colorectal cancer: An exploratory analysis from the phase III PARADIGM study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1974.