Abstract 5715: Circulating tumor DNA in colorectal cancer patients with resectable liver metastases: Preliminary results of the MIRACLE study
Authors: Wullaert Lissa , Jansen Maurice P. , Kraan Jaco , van Rees Jan M. , Beaufort Corine M. , Meyer Yannick M. , Galjart Boris , Nierop Pieter M. , Buisman Florian E. , Höppener Diederik J. , van der Stok Erik P. , Motta Gianmarco , Grünhagen Dirk J. , Verheul Henk M. , Sleijfer Stefan , Wilting Saskia M. , Verhoef Cornelis , Martens John W. 
Published: April 2023

Abstract
Introduction: Recurrence risk after curative surgery for colorectal liver metastases (CRLM) remains high, underlining the need for novel prognostic markers. Liquid biopsies provide promising biomarkers to detect minimal residual disease (MRD) after local treatment. The aim of this study was to determine and compare the characteristics of cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) in liquid biopsies taken before and after surgical treatment of CRLM.
Materials & Methods: Patients with isolated CRLM were recruited before undergoing potentially curative hepatic resection. Only patients who did not receive per-operative chemotherapy were selected. Peripheral blood samples were collected in CellSave preservative tubes at four different time points and processed into plasma within 96 hours after withdrawal. Subsequently, cfDNA was isolated from 4mL plasma, in which the total cfDNA concentration was measured. Mutations characterizing ctDNA were determined before surgery by next generation sequencing (NGS) using a colon-specific targeted panel (Oncomine Colon cfDNA assay, 14 genes). Samples were sequenced at &gt;20.000 average reads depth and variants were called mutations when the variant allele frequency was above their predefined limit of detection. Longitudinal blood samples of ctDNA-positive patients were evaluated by digital PCR (dPCR) using mutation-specific assays, in which samples were considered ctDNA positive when &gt;5 mutant copies were detected.
Results: Blood samples of 231 patients were collected one day before surgery (T0), and were repeated one day (T1), one week (T2), and three weeks (T3) after surgery. The cfDNA yields per mL plasma were higher at T1 and T2 (median 93 ng and 64 ng, respectively) compared to T0 (6 ng, p&lt;0.001) and T3 (12 ng, p&lt;0.001). NGS at T0 was performed for 196 patients and detected ctDNA in 129 patients (66%). Mutations had a median allele frequency of 6.3% (ranging from 0.28 to 86.4%), and were observed in 12 genes, predominantly in TP53 (n=69), KRAS (n=57), APC (n=40), and PIK3CA (n=23). dPCR analyses were performed for 87 patients out of the 129 patients with ctDNA at T0, and confirmed TP53, KRAS, PIK3CA, and BRAF mutations in 75 patients, with average ctDNA loads of 9%, 8%, 18% and 4% for these genes. Longitudinal dPCR analyses were performed in 58 patients of whom subsequent post-surgical samples were available, and revealed that ctDNA was detectable in 22 patients (40%) at T1/T2, and in 21 patients (36%) at T3. Nine patients (16%) had detectable ctDNA at both T1/T2 and T3. Average mutation loads were 0.97% at T1/T2 and 1.79% at T3 (p=0.36).
Discussion/Conclusion: ctDNA is a potential biomarker to detect MRD after curative treatment for CRLM. Our results demonstrate that out of all patients with detectable ctDNA at baseline (before surgery), ctDNA is still present in 36% of patients three weeks after resection.
Citation Format: Lissa Wullaert, Maurice P. Jansen, Jaco Kraan, Jan M. van Rees, Corine M. Beaufort, Yannick M. Meyer, Boris Galjart, Pieter M. Nierop, Florian E. Buisman, Diederik J. Höppener, Erik P. van der Stok, Gianmarco Motta, Dirk J. Grünhagen, Henk M. Verheul, Stefan Sleijfer, Saskia M. Wilting, Cornelis Verhoef, John W. Martens. Circulating tumor DNA in colorectal cancer patients with resectable liver metastases: Preliminary results of the MIRACLE study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5715.