Abstract 2078: Mitochondrial DNA copy number correlates with trifluridine sensitivity of human tumor cell lines

Abstract

Abstract Background: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. FTD, an antineoplastic thymidine analogue, is incorporated into the genomic DNA of tumor cells. However no detailed analysis of the relation between mitochondrial DNA (mtDNA) in human tumors and FTD’s cytotoxic effects has been reported yet. In this study, we analyzed mtDNA copy number of 23 human cancer cell lines and its relation with FTD sensitivity. Method: Twenty-three human cancer cell lines, representing the nine main tumor types, were tested for sensitivity to FTD by WST-8 assay. The cell lines included lung (NCI-H226, NCI-H23), colon (COLO 205, HCT 116, HCT-15, SW620), pancreas (PANC-1), breast (Hs 578T, MCF7, MDA-MB-231, T-47D), melanoma (Malme-3M, SK-MEL-28, SK-MEL-5), prostate (DU 145, PC-3), renal (786-O, A-498), leukemia (CCRF-CEM, HL-60, MOLT-4), and ovary (OVCAR-3, SK-OV-3). Genomic DNA was purified using a silica membrane spin column-based centrifugation procedure. The copy number of mtDNA was quantitated by array-based digital PCR and real-time PCR. Furthermore, the mtDNA heterogeneity of each cell line was assessed by deep-sequencing as follows: a DNA panel targeting 222 amplicons covering the entire mitochondrial genome was used for NGS library preparation; NGS libraries were subjected to cluster generation in a flow cell and paired-end sequencing for 300 cycles; and finally, the resulting fastq files were uploaded to mtDNA-server for mapping and heteroplasmy evaluation. Results: The IC50s of FTD ranged from 0.6 uM to 52 uM (Median: 5.6 uM). While cell lines were arbitrarily classified as FTD-high- and FTD-low sensitive cell lines according to their respective IC50 values at median, mtDNA copy numbers of FTD-high sensitive cell lines were significantly lower than those of FTD-low sensitive cell lines (p = 0.01). Median heteroplasmy level and counts were 0.057 and 137, respectively. Interestingly, significant negative correlation was found between mtDNA copy number and heteroplasmy level. (Spearman’s Rho = -0.46, p= 0.02). While 6 out of 23 cell lines were microsatellite instable, MSI status also significantly correlated with mtDNA copy number, FTD-sensitivity, and heteroplasmy level (p< 0.05). Conclusions: Integrated analysis of mtDNA copy number and mutational profiling may be useful to predict FTD sensitivity of cancer cells. Citation Format: Takashi Kobunai, Kazuaki Matsuoka, Mamoru Nukatsuka, Hiroshi Tsukihara, Teiji Takechi. Mitochondrial DNA copy number correlates with trifluridine sensitivity of human tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2078.