title: Methylation of WNT target genes AXIN2 and DKK1 as robust biomarkers for recurrence prediction in stage II colon cancer.
abstract: Stage II colon cancer (CC) still remains a clinical challenge with patient stratification for adjuvant therapy (AT) largely relying on clinical parameters. Prognostic biomarkers are urgently needed for better stratification. Previously, we have shown that WNT target genes AXIN2, DKK1, APCDD1, ASCL2 and LGR5 are silenced by DNA methylation and could serve as prognostic markers in stage II CC patients using methylation-specific PCR. Here, we have extended our discovery cohort AMC90-AJCC-II (N=65) and methylation was analyzed by quantitative pyrosequencing. Subsequently, we validated the results in an independent EPICOLON1 CC cohort (N=79). Methylation of WNT target genes is negatively correlated to mRNA expression. A combination of AXIN2 and DKK1 methylation significantly predicted recurrences in univariate (area under the curve (AUC)=0.83, confidence interval (CI): 0.72-0.94, P<0.0001) analysis in stage II microsatellite stable (MSS) CC patients. This two marker combination showed an AUC of 0.80 (CI: 0.68-0.91, P<0.0001) in the EPICOLON1 validation cohort. Multivariate analysis in the Academic Medical Center (AMC) cohort revealed that both WNT target gene methylation and consensus molecular subtype 4 (CMS4) are significantly associated with poor recurrence-free survival (hazard ratio (HR)methylation: 3.84, 95% CI: 1.14-12.43; HRCMS4: 3.73, 95% CI: 1.22-11.48). CMS4 subtype tumors with WNT target methylation showed worse prognosis. Combining WNT target gene methylation and CMS4 subtype lead to an AUC of 0.89 (0.791-0.982, P<0.0001) for recurrence prediction. Notably, we observed that methylation of DKK1 is high in BRAF mutant and CIMP (CpG island methylator phenotype)-positive cancers, whereas AXIN2 methylation appears to be associated with CMS4. Methylation of AXIN2 and DKK1 were found to be robust markers for recurrence prediction in stage II MSS CC patients. Further validation of these findings in a randomized and prospective manner could pave a way to identify poor prognosis patients of stage II CC for AT.
authors.affiliation: Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands.|Cancer Genomics Center, Amsterdam, The Netherlands.|Center for Gastrointestinal Research and Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Scott and White Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA.
authors.forename: R
authors.initials: R
authors.lastname: Kandimalla
authors.affiliation: Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands.|Cancer Genomics Center, Amsterdam, The Netherlands.
authors.forename: J F
authors.initials: JF
authors.lastname: Linnekamp
authors.affiliation: Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands.|Cancer Genomics Center, Amsterdam, The Netherlands.
authors.forename: S
authors.initials: S
authors.lastname: van Hooff
authors.affiliation: Institut de Malaties Digestives i Metabòliques, CIBERehd, Hospital Clínic, Barcelona, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Castells
authors.affiliation: University of Yale, New Haven, CT, USA.
authors.forename: X
authors.initials: X
authors.lastname: Llor
authors.affiliation: Gastroenterology Department, Hospital del Mar, Barcelona, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Andreu
authors.affiliation: Servicio de Medicina Digestiva, Hospital General Universitario de Alicante, Instituto de Investigación Sanitaria ISABIAL, Alicante, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Jover
authors.affiliation: Center for Gastrointestinal Research and Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Scott and White Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, Dallas, TX, USA.
authors.forename: A
authors.initials: A
authors.lastname: Goel
authors.affiliation: Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands.|Cancer Genomics Center, Amsterdam, The Netherlands.
authors.forename: J P
authors.initials: JP
authors.lastname: Medema