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Quantitative Analysis of Methylation of Genomic Loci in Early-Stage Rectal Cancer Predicts Distant Recurrence

Michiel F.G. de Maat, Cornelis J.H. van de Velde, Martijn P.J. van der Werff, Hein Putter, Naoyuki Umetani, Elma Meershoek Klein-Kranenbarg, Roderick R. Turner, J. Han J.M. van Krieken, Anton Bilchik, Rob A.E.M. Tollenaar, Dave S.B. Hoon

From the Department of Molecular Oncology, John Wayne Cancer Institute; Department of Surgical Pathology, Saint John's Health Center, Santa Monica, CA; Department of Surgery, Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden; and Department of Pathology, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands

Corresponding author: Dave S.B. Hoon, PhD, Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404; e-mail: hoon{at}jwci.org

Purpose: There are no accurate prognostic biomarkers specific for rectal cancer. Epigenetic aberrations, in the form of DNA methylation, accumulate early during rectal tumor formation. In a preliminary study, we investigated absolute quantitative methylation changes associated with tumor progression of rectal tissue at multiple genomic methylated-in-tumor (MINT) loci sequences. We then explored in a different clinical patient group whether these epigenetic changes could be correlated with clinical outcome.

Patients and Methods: Absolute quantitative assessment of methylated alleles was used to assay methylation changes at MINT 1, 2, 3, 12, 17, 25, and 31 in sets of normal, adenomatous, and malignant tissues from 46 patients with rectal cancer. Methylation levels of these biomarkers were then assessed in operative specimens of 251 patients who underwent total mesorectal excision (TME) without neoadjuvant radiotherapy in a multicenter clinical trial.

Results: Methylation at MINT 2, 3, and 31 increased 11-fold (P = .005), 15-fold (P < .001), and two-fold (P = .02), respectively, during adenomatous transformation in normal rectal epithelium. Unsupervised grouping analyses of quantitative MINT methylation data of TME trial patients demonstrated two prognostic subclasses. In multivariate analysis of node-negative patients, this subclassification was the only predictor for distant recurrence (hazard ratio [HR], 4.17; 95% CI, 1.72 to 10.10; P = .002), cancer-specific survival (HR, 3.74; 95% CI, 1.4 to 9.43; P = .003), and overall survival (HR, 2.68; 95% CI, 1.41 to 5.11; P = .005).

Conclusion: Methylation levels of specific MINT loci can be used as prognostic variables in patients with American Joint Committee on Cancer stage I and II rectal cancer. Quantitative epigenetic classification of rectal cancer merits evaluation as a stratification factor for adjuvant treatment in early disease.

Supported by the Ruth and Martin H. Weil Fund, the Gonda Foundation, and Coates Laboratories.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.