Pre-Operative Decitabine in Colon Cancer Patients: Analyses on WNT Target Methylation and Expression

Abstract

DNA hypermethylation is common in colon cancer. Previously, we have shown that methylation of WNT target genes predicts poor prognosis in stage II colon cancer. The primary objective of this study was to assess whether pre-operative treatment with decitabine can decrease methylation and increase the expression of WNT target genes APCDD1, AXIN2 and DKK1 in colon cancer patients. A clinical study was conducted, investigating these potential effects of decitabine in colon cancer patients (DECO). Patients were treated two times with 25 mg/m² decitabine before surgery. Methylation and expression of LINE1 and WNT target genes (primary outcome) and expression of endogenous retroviral genes (secondary outcome) were analysed in pre- and post-treatment tumour samples using pyrosequencing and rt-PCR. Ten patients were treated with decitabine and eighteen patients were used as controls. Decitabine treatment only marginally decreased LINE1 methylation. More importantly, no differences in methylation or expression of WNT target or endogenous retroviral genes were observed. Due to the lack of an effect on primary and secondary outcomes, the study was prematurely closed. In conclusion, pre-operative treatment with decitabine is safe, but with the current dosing, the primary objective, increased WNT target gene expression, cannot be achieved.

Keywords: DNA methylation; clinical translation study; colon cancer; decitabine.

Figure 1

(A) Flow chart of DECO study. In blue squares, daily routine steps in clinical care are shown; in green squares, additional steps for DECO study are shown; (B) Baseline characteristics of decitabine-treated patients included in DECO study. Indicated stage is pathological staging after resection was done. Initial staging (for inclusion) was performed based on CT scan; (C) Timeline for DECO study. Red arrow shows the moment of inclusion. Blood tests before inclusion were part of standard of care. * For the patients with fresh frozen material, an extra endoscopy was performed to obtain freshly frozen biopsies as pre-treatment sample. For FFPE patients, pre-treatment samples were obtained from the diagnostic endoscopy performed for clinical purposes. IC = informed consent, ND = not detected, OS = overall survival.

Figure 2

(A) Methylation of LINE1 in the control group (n = 15) and in the treated group (n = 10) (before and after treatment with 25 mg/m² decitabine two times) measured by pyrosequencing. In both cohorts, FF tumour samples and FFPE samples were included. Open symbols and dotted lines represent FF samples, and closed symbols and lines represent FFPE samples. In the treated cohort, two technical replicates per time point were averaged and two biological replicates (two different samples from the same tumour) were used. For patient 6 to patient 10, no biological replicate was available for the pre-treatment sample. For statistical analyses, for pre-treatment and post-treatment samples, the average of all measurements was used. A paired t-test revealed no significant difference in the control cohort (p = 0.9718). For the treated cohort, a significant (p = 0.0075) difference was shown; (B) Methylation of WNT target genes before and after treatment with decitabine in colon cancer patients measured by pyrosequencing (n = 5); (C) Expression of LINE1 and WNT target genes after treatment with decitabine measured by quantitative rt-PCR in fresh-frozen samples (n = 5). Values are the average of two samples (both for pre- and post-treatment samples), except for patient 3, where only one post-treatment sample was available.

Figure 3

(A) Representable Ki67 staining of biopsy and resection material of the tumour from one patient. The scale bar represents 100 µm; (B) Percentage of Ki67 positive cells compared to total cells from nine treated patients. A representative area of the tumour block was used for quantification. No significant difference between pre- and post-treatment samples (p = 0.7618) was observed using a paired t-test.

Figure 4

Expression of ERVL and interferon genes DDX58 and OASL in pre- and post-treatment samples (n = 2 per patients except from patient 3) from patients treated with decitabine. Only fresh-frozen samples were used.