Prognostic value of folate-associated gene expression in stage II colon cancer

Abstract

Purpose: Prognostic variability in stage II colon cancer underscores the need for better risk stratification. Analyzing folate-associated gene expression in stage II colon cancer could provide researchers and clinicians with deeper insights into tumor biology and potentially aid in identifying early prognostic and/or predictive biomarkers.

Methods: Patients with stage II colon cancer and recurrence (n = 48) were matched to patients with a 5 year recurrence-free follow-up (n = 133). Gene expression of ABCC3, AMT, FPGS, GGH, MFT, PCFT, RFC-1, and TYMS was analyzed in tumor tissue and matching colon mucosa using qPCR and evaluated in relation to time to recurrence (TTR), as well as to demographic and clinicopathological variables.

Results: Independent of other covariates, TYMS expression in tumors, pT4 stage, and emergency surgery were associated with TTR. There were significant differences in expression levels of all examined genes between tumor and mucosa. ABCC3, GGH, and RFC-1 expression levels differed in mucosa between microsatellite instability-high (MSI-H) compared to microsatellite stable/microsatellite instability-low (MSS/MSI-L) tumors, whereas tumoral expression of AMT, GGH, and TYMS differed between MSI-H and MSS/MSI-L tumors. Depending on tumor location, the expression of ABCC3, AMT, GGH, and RFC-1 in mucosa, as well as the tumoral expression of AMT, GGH, PCFT and RFC-1 differed.

Conclusion: Low tumoral expression of TYMS was associated with worse TTR, independent of MSI status, pT stage, and emergency surgery. The indication of a better outcome for patients with MSI-H status and high tumoral TYMS expression might be of particular interest in the stratification of patients for immunotherapy.

Keywords: Biomarker; Cancer; Colorectal Neoplams; Folic acid; Prognosis; Thymidylate synthase.

Fig. 1

Simplified overview of the folate mediated metabolism, polyglutamation and transport in eukaryotic cells. Polyglutamated folate is converted to monoglutamated folate extracellularly before transport into the cell through the transporter PCFT or RFC-1, depending on pH. MFT transports folates into the mitochondrial matrix. The ATP-dependent protein ABCC3 transports monoglutamated folate amongst other substrates out of the cell. Intracellularly, monoglutamated folate is partly converted into polyglutamated folate by FPGS, whereas GGH hydrolyzes polyglutamated folates to monoglutamated forms. AMT participates in the glycine cleavage system within the mitochondria and is responsible for the formation of 5,10-methyleneTHF. This co-factor forms a ternary complex with dUMP and the TS enzyme, encoded by the TYMS gene. Abbreviations: ATP Adenosine triphosphate, dTMP Deoxythymidine monophosphate, dUMP Deoxyuridine monophosphate, H Hydrogen, OP Organic phosphate, THF Tetrahydrofolate

Fig. 2

Comparison of gene expression levels in non-cancerous mucosa (M) and tumors (T) obtained at primary surgery from patients with stage II colon cancer. Expression data could be analysed in each of the non-cancerous mucosa samples (n = 181), and in 179/181 tumor samples, except for PCFT (n = 178). Expression levels are presented as violin plots with median values and interquartile ranges depicted as horizontal lines. The scale on the y-axis has been reversed to simplify interpretation as a high ΔCt value represents low gene expression and vice versa. Statistical significance by asterisks, ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05

Fig. 3

Gene expression levels in a non-cancerous mucosa and b tumors dichotomized by tumoral MSI status (MSS or MSI). The MSS and MSI-L cases were combined into a single group labelled as MSS, while the MSI-H cases were categorized as the MSI group. Expression levels are presented as violin plots with median values and interquartile ranges depicted as horizontal lines. The scale on the y-axis has been reversed to simplify interpretation as a high ΔCt value represents low gene expression and vice versa. Statistical significance by asterisks, ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05

Fig. 4

Gene expression levels in a non-cancerous mucosa and b tumors dichotomized by tumor location (right (R) or left (L) side of colon. Expression levels are presented as violin plots with median values and interquartile ranges depicted as horizontal lines. The scale on the y-axis has been reversed to simplify interpretation as a high ΔCt value represents low gene expression and vice versa. Statistical significance by asterisks, ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05

Fig. 5

Kaplan–Meier curve showing the probability of time to recurrence (TTR) in stage II colon cancer patients dichotomized by high and low tumoral TYMS gene expression