This retrospective case–control study investigated the association between the relative expression of eight folate pathway genes and TTR in 181 patients with stage II colon cancer. The aim was to elucidate whether gene expression in tumor and/or mucosa tissue could serve as a prognostic marker for recurrent disease. The selected genes encode folate transporters (RFC-1, PCFT, ABCC3, and MFT), enzymes involved in the polyglutamation of folates (FPGS and GGH), and the folate metabolism (AMT and TYMS). Low tumoral expression of TYMS, emergency surgery, and pT4 tumors were independently associated with worse TTR. Significant differences in the expression levels of all examined genes were observed between tumors and mucosa, and for some genes, expression also varied with tumor location and MSI status.

Increased expression of RFC-1 and PCFT has been reported during folate deficiency (Thakur et al. 2015), whereas folate over-supplementation leads to downregulation of intestinal folate uptake via decreased transcription of RFC-1 and PCFT (Ashokkumar et al. 2007). The results showed significantly lower RFC-1, but higher PCFT expression in mucosa compared to tumors. High PCFT expression was particularly pronounced in mucosa matching poorly differentiated tumors, suggesting a more acidic microenvironment surrounding these tumors. Additionally, RFC-1 expression was higher in the mucosa of right-sided or MSI-H colon cancers compared to left-sided or MSS/MSI-L cases, whereas the expression of both RFC-1 and PCFT was lower in right- than in left-sided tumors. These findings indicate an association between tumor location (i.e., tumor sidedness) and tissue folate status.

The expression of MFT was higher in tumor tissue compared to mucosa, indicating a high demand for mitochondrial folates in tumors. Although high tumoral expression of MFT has been correlated with reduced survival of patients with different types of solid cancers (Santoro et al. 2020), no association with TTR was found in the present study. The expression of AMT was lower in tumors compared to mucosa, suggesting less AMT activity in the mitochondria of tumor tissue. AMT expression was lower in right-sided or MSI-H colon cancer compared to left-sided or MSS/MSI-L cases, while mucosal AMT expression was higher in right- than left-sided colon cancers, again underscoring the importance of tumor sidedness for certain biological processes.

In agreement with previous studies on chemotherapy-naïve stage I–IV CRC (Hinoshita et al. 2000; Hlavata et al. 2012; Thakur et al. 2015), tumor tissue exhibited significantly lower expression of ABCC3 compared to mucosa. There was no difference in tumoral ABCC3 expression based on tumor location; however, higher expression was observed in the mucosa of right-sided or MSI-H colon cancers compared to left-sided or MSS/MSI-L cases. These results suggest that sidedness-related differences in the efflux of folates or other organic anions in the mucosa may contribute to the development of microenvironments that favor different pathways of tumorigenesis.

In the present study, ABCC3 expression was higher in the mucosa of patients with high-grade (G3) tumors compared to those with low-grade (G1/G2) differentiation. Additionally, ABCC3 expression was elevated in pT4 compared to pT3 tumors. However, ABCC3 expression did not correlate with prognosis nor did it impact TTR. These findings suggest that ABCC3 expression may serve as a potential marker for assessing disease severity and tumor progression, particularly in association with more aggressive disease forms, rather than as a predictor of clinical outcomes such as prognosis.

FPGS catalyzes the polyglutamation of intracellular folates, including MeTHF, which facilitates better stabilization of the ternary complex with the TS enzyme (Chen et al. 2017). In contrast, GGH is involved in removal of folate glutamates in the lysosomes, and it is suggested that monoglutamated folates from the lysosomes are released into the cytosol (Odin et al. 2019). In the present study, both FPGS and GGH were expressed at higher levels in tumors compared to mucosa, indicating an increased intracellular folate turnover in tumors, a finding further supported by the high expression of the proliferation marker TYMS in tumor tissue.

The expression of GGH was lower in both tumor and mucosa of right-sided and MSI-H colon cancers compared to left-sided and MSS/MSI-L cases. A lower expression of GGH was also observed in high-grade (G3) tumors compared to low-grade (G1/G2). This suggests that the availability of polyglutamated folates in the cytosol varies with tumor location and differentiation grade. High FPGS expression has been associated with increased chemosensitivity, whereas GGH overexpression is related to drug resistance (Kim 2020; Schneider and Ryan 2006; Sohn et al. 2004). Therefore, information on FPGS, GGH and TYMS expression may be important when selecting a treatment strategy for high-risk stage II patients who are likely to benefit from adjuvant chemotherapy.

Interestingly, low tumoral expression of TYMS was independently associated with worse TTR, whereas MSI-H correlated with a better TTR, but not independently. In agreement with a previous study, higher TYMS expression was observed in MSI-H tumors, compared to MSS/MSI-L (Odin et al. 2007). MSI-H has been associated with right-sided colorectal cancer and an immune-active tumor microenvironment (Hua et al. 2022), suggesting that high tumor TYMS expression and MSI-H may collectively indicate heightened immune activity within the tumor microenvironment. Contradictory results have been reported regarding the relationship between TYMS expression and prognosis in patients with colorectal cancer (Jensen et al. 2008; Lu et al. 2013; Soong et al. 2008). These discrepant results may have multiple explanations. For example, the TYMS gene is autoregulated; that is, a high transcription does not necessarily lead to increased protein expression and/or TS activity (Tai et al. 2004). Furthermore, differences in study cohorts regarding relevant covariates known to be associated with TYMS expression (e.g., MSI, tumor stages, tumor location) may contribute to these inconsistencies.

Patients who underwent surgical removal of colon cancer in 2002–2015 were included in this retrospective case–control study. During that time-period, it was not possible to evaluate some of the clinico-pathological parameters which are used today in the assessment of colon cancer, e.g., tumor budding. However, the current study consisted of a relatively large patient cohort which was well-selected and well-controlled. The investigation of the association between MSI status and gene expression gives the study further strength. In the era of next-generation sequencing (NGS), we opted to use qPCR for gene expression analysis. qPCR is particularly well-suited for focused studies targeting specific genes of interest, in contrast to the broad, exploratory scope of NGS. The targeted qPCR methodology offers faster turnaround times compared to NGS, a critical advantage in clinical settings where timely decision-making is essential. It also provides high sensitivity and specificity, enabling the detection of low-abundance transcripts and subtle changes in gene expression. The study examined the relative expression of the selected genes, but not the corresponding protein expression or enzyme activities. In future studies, it will be valuable to analyze the correlation between TYMS gene and protein expression in tumor tissues and to investigate the possible association between TS activity and TTR. It will also be of interest to evaluate folate-associated gene expression and MSI status in relation to tumor budding and other relevant parameters. To address these questions, a follow-up study of the patient cohort is planned.