Integrative Analysis of Multiplex Interphase FISH Copy Number with Whole Exome Sequencing Identifies Tumor Evolution in Stage II Colon Cancer

Abstract Colon cancer ranks as the second leading cause of cancer-related death worldwide. Although standard therapies are highly effective in stage II lymph-node negative colon cancers, about 10-15% of these patients show disease relapse within the next 5-year period after intended curative surgery. Extensive prior research has highlighted the role of intratumor heterogeneity, arising from genomic instability, as a leading contributor to metastasis in colon cancer. In the current study, we pioneered the integration of Multiplex Interphase FISH (miFISH) and Whole Exome Sequencing (WES) data aiming to depict intratumor heterogeneity and delineate the landscape of genetic aberrations that underlie the early metastatic potential in stage II colon cancer patients. To this aim, we have showcased the genome-wide copy-number alterations and single nucleotide mutations derived from WES analysis of samples obtained from primary stage II colon tumors and their patient-matched liver metastases of nine patients using multi-region sampling. miFISH was employed for the concurrent quantification of copy-numbers for nine genes relevant to colon cancer and a centromeric control probe in intact tumor nuclei derived from archival patient material allowing us to establish ploidy baselines and gain and loss patterns for the genes analyzed. Our study was revealing of several copy number alterations, such as gains of chromosomes 1q, 7, 8q, 13q and 20, and losses affecting chromosomes 17p and 18, which were confirmed by miFISH. Based on our SNV data, most frequently mutated genes were APC, TP53, KRAS. We were able to show several mutations in genes belonging to specific pathways most commonly altered in colorectal cancer, such as APC, TCF7L2, AXIN2, FBXW7 in WNT signaling, SMAD4 in TGF-ß signaling, PIK3CA and PTEN in PI3K signaling and KRAS in RTK-RAS signaling pathways. In nearly all cases, subclones that led to metastasis were already present in the primary cancer, indicating a direct progression from the primary to metastatic disease. However, the divergence from the primary tumor to metastasis enabled us to uncover several potential genetic aberrations present in the metastasis but not observed in the primary tumor. Moreover, in one of our cases, despite harboring APC and TP53 mutations in both the primary and metastatic tumor, unique mutations for these genes along with several distinct mutations in the metastasis were identified. These observations suggest that the metastasis might not be related to the primary tumor analyzed and may have arisen from an independent lesion. In conclusion, applying miFISH and WES to decipher the mutational and copy number landscape in metastatic stage II colon cancer provided comprehensive insights into the complex and heterogenous nature of primary colon cancer and the clonal evolution leading to subsequent liver metastases. Citation Format: Sanam Yaghoubi, Kerstin Heselmeyer-Haddad, Ivan Archilla, Carolina Parra, Darawalee Wangsa, Giancarlo Castellano, Jack Zhu, Diba Yaghoubi, Sara Lahoz, Veronica Pablo-Fontecha3, Daniela Hirsch, Wei-Dong Chen, Thomas Ried, Miriam Cuatrecasas, Paul S. Meltzer, Jordi Camps. Integrative analysis of multiplex interphase FISH copy number with whole exome sequencing identifies tumor evolution in Stage II colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4360.