Background - 5-fluorouracil (5-FU) in combination with the folate leucovorin (LV) has formed the backbone of chemotherapy for advanced colorectal cancer for several decades. A number of genes encode proteins that participate in transportation of LV into the cells, as well as in subsequent metabolic action. We previously reported that high tumoral expression of genes involved in folate transport, polyglutamation, and metabolism was associated with decreased risk of recurrent disease in patients with stage III colorectal cancer treated with 5-FU + LV (FLV) alone, or in combination with oxaliplatin (FLOX) according to the Nordic bolus regimen. The aim of the present study was to determine the association between expression of the folate-associated genes ABCC3, MTHFD2, SLC19A1, SLC25A32, SLC46A1, and TYMS and outcome of patients with metastatic colorectal cancer subjected to palliative chemotherapy.

Patients and Methods - A total of 290 patients treated with FLV (n = 113), FLOX (n = 102) or FLV + irinotecan (FLIRI, n = 75) were included. Relative gene expression (ΔCt) was determined in primary tumors by quantitative PCR and analyzed in relation to clinical benefit, based on RECIST criteria and 3-year progression-free survival (PFS). Analyses were conducted on the whole study group, and on subgroups based on tumor stage at primary surgery (subgroup 1, stage I-III; subgroup 2, stage IV). An ANOVA test was used to assess the relationship between expression and clinical benefit. A multivariate Cox proportional hazard model was applied to assess potential associations between genetic markers, clinical variables and PFS. A Stepwise model selection was used to identify a minimal set of variables associated with PFS.

Results - Low expression of TYMS and MTHFD2, and high expression of ABCC3 was significantly associated with a clinical benefit in the whole group (p<0.0001, p=0.017, and p=0.028, respectively). The association between TYMS expression and clinical benefit was seen in both sub-groups, whereas ABCC3 expression was significant in subgroup 2 (p=0.041). Multivariate models showed that low TYMS and high SLC25A32 expression in subgroup 1 and high ABCC3 expression in subgroup 2 correlated significantly with better PFS (Hazard Ratio (HR) = 0.75 (95% CI = 0.57-1.0), HR = 2.21 (95% CI = 1.37-3.6), and HR = 1.34 (95% CI = 1.08 -1.7), respectively).

Conclusion - Expression of TYMS, the target enzyme of 5-FU, was strongly associated with clinical benefit in the whole group, whereas expression of TYMS and the folate transporters SLC25A32, and ABCC3 was associated with PFS in the subgroups (stage I-III and stage IV), respectively. The prospective global phase III study AGENT is presently conducted on patients with advanced colorectal cancer, to determine whether expression of these genes can predict response to 5-FU-based chemotherapy that includes LV or the novel folate arfolitixorin.

Citation Format: Yvonne Wettergren, Elisabeth Odin, Göran Carlsson, Pushpa Saksena, Anders Edsjö, Alessandro Di Cara, Roger Tell, Bengt Gustavsson. Tumoral expression of folate-associated genes is associated with progression-free survival of patients with advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 346.