EACR25-2209

Performance of a novel NGS-based technology for mutation profiling in liquid biopsies derived from metastatic colorectal cancer patients

A. Ganesan1,2, A. Korkiakoski3,4, S. Adamusová3,4, E. Andersson5, J. Laine3,4,6, S. Kytölä5, P. Osterlund7,8,9, P. Nummela1,2, M. Tamminen3,4, A. Ristimäki1,2
1University of Helsinki, Applied Tumor Genomics Research Program, Research Programs Unit, Helsinki, Finland
2University of Helsinki and Helsinki University Hospital, Department of Pathology, HUS Diagnostic Center, Helsinki, Finland
3Genomill Health Inc, Turku, Finland
4University of Turku, Department of Biology, Turku, Finland
5Helsinki University Hospital and University of Helsinki, Department of Genetics, HUS Diagnostic Center, Helsinki, Finland
6Turku University Hospital, Department of Oncology, Turku, Finland
7Helsinki University Hospital and University of Helsinki, Department of Oncology, Comprehensive Cancer Center, Helsinki, Finland
8Tampere University Hospital and University of Tampere, Department of Oncology,, Tampere, Finland
9Karolinska University Hospital and Karolinska Institute, Department of Gastrointestinal Oncology, Stockholm, Sweden
Introduction:

Colorectal cancer (CRC) is the most prevalent gastrointestinal malignancy and the second leading cause of cancer-related deaths. Symptoms of CRC often manifest only in the advanced stages. Circulating tumor DNA (ctDNA), tumor-released DNA in the bloodstream, may display cancer-related mutations originating from all metastatic lesions of the patient. Liquid biopsy in forms of ctDNA analysis provides a non-invasive method for monitoring treatment response, detecting minimal residual disease (MRD), and identifying resistance-associated mutations. In this study, we used serial plasma samples from metastatic colorectal cancer (mCRC) patients to assess a novel next generation sequencing (NGS)-based Bridge Capture technology for mutation profiling and MRD detection. We also evaluated the scalability of the technology using synthetic DNA targets.

Material and method:

This study included 80 plasma samples collected from ten mCRC patients participating in the AXOAXI phase II trial. Written informed consent was obtained from all patients. Each patient had a codon 12 or 13 KRAS mutation in the primary tumor specimen, enabling a tumor-informed approach to ctDNA analysis. Cell-free DNA (cfDNA) was extracted from 2 ml plasma using QiaSymphony DSP Circulating DNA Kit (QIAGEN). The performance of a novel Bridge Capture™ technology was compared to those of droplet digital PCR (ddPCR; Bio-Rad) and Ion AmpliSeq™ Cancer Hotspot Panel v2 (Thermo Fisher Scientific).

Result and discussion:

Bridge Capture 282-probe panel and ddPCR detected KRAS mutation in 35 of the 80 ctDNA samples (44%), while 33 samples (41%) were negative with both methods. Twelve samples showed discrepant results, with eight samples being positive with Bridge Capture but negative with ddPCR and four samples being positive with ddPCR but negative with Bridge Capture. The KRAS variant allele frequency (VAF) values showed a very strong correlation (Spearman rs = 0.86). In a subset of ten samples analyzed with NGS-based AmpliSeq, both Bridge Capture and AmpliSeq identified 15 CRC driver mutations with strong correlation in VAF values (rs = 0.74). Bridge Capture further detected several additional oncogenic mutations. Scalability of Bridge Capture was confirmed using an expanded 851-probe panel and synthetic DNA targets, with dilutions from 0.03% to 1% VAF showing a very strong linear correlation between the observed and expected total VAF signals for all probes (Pearson r = 0.99).

Conclusion:

This study demonstrates the potential of Bridge Capture to improve mutation profiling and MRD detection in mCRC plasma samples. It shows substantial agreement with other technologies in clinical sample validation, and the VAF values strongly correlate with those from ddPCR and the NGS-based Ion AmpliSeq. The high scalability of the probe panel enables comprehensive ctDNA analysis. Further validation in larger and diverse cohorts is necessary to establish its clinical utility.