Moving Closer to a Prognostic DNA Methylation Signature in Colon Cancer

Clinical Cancer CCR Translations Research Commentary on Yi et al., p. 1535

F. Javier Carmona1 and Manel Esteller1,2,3

Although large panels of genes undergoing aberrant CpG island methylation in colorectal cancer have been identified, reliable predictors for clinical management in this tumor type remain elusive. A new DNA methylation signature affecting the extracellular matrix (ECM) pathway has been identified with potential prognostic value in colon cancer. Clin Cancer Res; 17(6); 1215–7. Ó2011 AACR.

In this issue of Clinical Cancer Research, Yi and colleagues methylation biomarkers lies in their stability in the face of (1) report a widespread epigenetic inactivation affecting subtle variations in the cell environment, which can never- extracellular matrix (ECM) pathway genes in colon cancer. theless deeply affect transcription profiles, and in the In addition, the authors have tested this signature as a availability of sensitive, simple, and cost-effective techni- prognostic indicator for individualizing stage II colon ques to detect them. Importantly, gene hypermethylation is cancer patients at high risk of recurrence, who would frequently associated with gene inactivation, and, given its benefit from adjuvant treatments (1). plasticity, it is a modification that has the potential to be An aberrant DNA methylation profile has been recog- reverted by epigenetic drugs. Thus, aberrant DNA methyla- nized as a common hallmark in all types of human neo- tion has important implications for cancer research and plasia (2). DNA methylation is an essential process for the management. However, despite all the efforts made, clini- maintenance of homeostatic equilibrium in normal cells, cally relevant biomarkers valuable in the management of and alterations in normal DNA methylation patterns in cancer patients remain scarce, with the exception of MGMT association with human disease affect almost all cellular and GSTP1 hypermethylation in gliomas and prostate pathways. Disruption of normal epigenetic patterns occurs carcinomas, respectively (2). from early stages of tumor development and accumulates In this issue of Clinical Cancer Research, Yi and collea- throughout cancer progression. Research in cancer epige- gues (1) have adopted an integrative approach whereby netics has, so far, yielded a significant number of candidate they combine genomic data about gene mutations genes whose methylation status is informative of cancer cell reported in colon and breast cancer (8) with epigenomic behavior. Specific DNA methylation profiles have been analyses to identify the pathways inactivated in colon associated with particular types of cancer, and biomarkers cancer. As a result, the authors have found a series of exist with the potential to anticipate tumor formation and genes undergoing epigenetic inactivation that result in the to predict metastatic progression or the risk of recurrence impairment of the ECM pathway (1). This pathway seems after surgical removal. Colon cancer, in particular, is a well- to be altered in the majority of colon cancers and prob- established model for DNA methylation studies (3). In ably facilitates cancer cell dissemination, thereby making colorectal tumorigenesis, the promoter CpG island methy- it a poor prognosis factor. Alterations in ECM remodeling, lation-associated silencing of 2 genes, the mismatch repair as a major constituent of the microenvironment, have gene hMLH1 (4) and the 06-methylguanine-DNA methyl- important implications for tumor biology and progres- transferase gene MGMT (5), leads to 2 different mutator sion, providing a permissive milieu for cancer cell growth pathways characterized by insertion and/or deletion in and dissemination (9). The results provided by the many downstream targets and point transition mutations authors include a set of 29 genes featuring cancer-specific in K-ras and p53 (6, 7), respectively. The value of DNA methylation, in other words, genes that are unmethylated in normal colon tissue and that are susceptible to genomic mutations that are enriched in ECM components. Authors' Affiliations: 1Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL); 2Department of Phy- These genes seem to be more often epigenetically inacti- siological Sciences II, School of Medicine, University of Barcelona; 3Insti- vated than genetically disrupted. Interestingly, 19 of these tucio Catalana de Recerca i Estudis Avancats¸ (ICREA), Barcelona, genes display frequent DNA hypermethylation in color- Catalonia, Spain ectal cancer, underlining the relevance of ECM impair- Corresponding Author: Manel Esteller, Cancer Epigenetics and Biology Program (PEBC), 3rd Floor, Hospital Duran i Reynals, Avda. Gran Via de ment in cancer progression. L'Hospitalet 199–203, 08907 L'Hospitalet, Barcelona, Catalonia, Spain. Promoter DNA methylation of selected genes (IGFBP3, Phone: 34-93-2607253; Fax: 34-93-2607219; E-mail: [email protected] EVL, CD109, NRCAM, NTNG, and FLNC) was assessed in doi: 10.1158/1078-0432.CCR-10-3446 relation to survival in 3 cohorts of colon cancer patients. Ó2011 American Association for Cancer Research. The authors report an overall greater risk of mortality in

www.aacrjournals.org 1215

Normal colon epithelium (Unmethylated CpG islands of tumor suppressor genes)

Tumor formation

Primary colon cancer (Hypermethylation-associated inactivation of MGMT, hMLH1, p16INK4a, p14ARF…)

Prognostic signature based on methylation status of selected Figure 1. CpG island ECM pathway genes hypermethylation in colon cancer and the proposed DNA Hypermethylation of ECM • Poor prognosis methylation signature for pathway genes • High risk of recurrence prognosis. IGFBP3 EVL CD109 NRCAM Transcriptional repression NTNG FLNC Unmethylated ECM • Good prognosis pathway genes • Low risk of recurrence

Transcriptional activation

relation to the DNA hypermethylation of individual genes independent series of patients. Another interesting report or of combinations of them (Fig. 1). Of special relevance described LINE-1 hypomethylation levels to be of prog- is the signature composed of IGFBP3, EVL, FLNC, and nostic significance (10). Nevertheless, to date, few reports CD109, whose CpG island methylation is associated with have provided conclusive results on the prognosis of colon an 8-fold increase in mortality risk relative to that of cancer patients, and this is the first one to encompass both patients with no DNA methylation of these genes. The genomic and epigenomic events. Major translational impli- results were similar in 1 of the 2 validation cohorts sur- cations arise from this study because it has the potential to veyed (John Hopkins University and the Netherlands enable stage II colon cancer patients to be categorized and Cohort Study on Diet and Cancer), most likely because those benefitting from adjuvant therapy to be selected (1). of diverse clinico-pathologic and environmental factors Overall, this is a valuable report that proves the signifi- influencing the populations examined. However, indivi- cance of a prognostic signature with potential clinical dual hypermethylation of CD109 kept its previous trend, applicability. This and other studies of outcome prediction and EVL retained its prognostic significance in both signatures should be reproduced by independent labora- cohorts. The signature proposed here has obvious potential tories, and the findings confirmed by a wide range of to be of prognostic significance at a clinical level. This techniques, and then borne in mind in the design of further finding should be confirmed in prospective studies. studies. In addition to these analyses, it would also be Furthermore, the authors describe an association interesting to investigate quantitative variation at the level between DNA methylation of 2 of the 6 selected ECM of CpG island methylation in specific genes, because this genes (IGFBP3 and CD109) and a high risk of recurrence might yield important clues about tumor progression. (Fig. 1). This finding was tested on a homogeneous cohort High-throughput, unbiased strategies aiming to comple- (John Hopkins University) and should be confirmed in ment currently available data are likely to provide

1216 Clin Cancer Res; 17(6) March 15, 2011 Clinical Cancer Research

meaningful biomarkers that increase the discriminatory cancer patients are expected to provide sensitive biomar- power of the available predictive signatures. Molecular kers for rapid, unaggressive, and cost-effective assessment markers to predict recurrence in stage II colon cancer of risk of recurrence or metastatic development. patients are available in the literature based on DNA The past decade has witnessed major advances in our methylation (10), gene expression (11), or microRNA knowledge of the clinical and molecular aspects of colon profiling (12). Nevertheless, no prognostic signature so cancer. However, the gaps remaining in our understanding of farhasbeentransferredtotheclinic,andstandardsfor cancer are large and significant. Further efforts need to be clinical management are still based on clinico-pathologic madetodesigninformativepanelsofbiomarkersthatare able parameters (node involvement, tumor infiltration, etc.) to anticipate the malignant phenotype effectively. The and limited molecular clues, MSI microsatellite instability authors whose work is discussed here have designed an being the best example. Furthermore, many of the current integrative approach that provides encouraging data and screening guidelines entail invasive protocols that are places 2 clinically relevant issues, the prediction of cancer traumatic for the patient. Predictive imprints need to cellbehaviorand patient stratification, firmlyin the spotlight. be validated in extensive prospective studies in order to develop (epi) genetic-based prediction models to help guide the selection of adjuvant treatments for patients. Disclosure of Potential Conflicts of Interest The signature reported here should follow similar stan- dardized pipelines to prove its clinical usefulness. No potential conflicts of interest were disclosed. Furthermore, ongoing trials evaluating available signa- Received January 1, 2011; accepted January 24, 2011; published online tures or single indicators in stool and blood from colon March 16, 2011.

References 1. Yi JM, Dhir M, Van Neste L, Downing SR, Jana Jeschke J, Glockner S, guanine-DNA methyltransferase is associated with the presence of G: et al. Genomic and epigenomic integration identifies a prognostic C to A:T transition mutations in p53 in human colorectal tumorigen- signature in colon cancer. Clin Can Res 2011;17:1535–45. esis. Cancer Res 2001;61:4689–92. 2. Esteller M. Epigenetics in cancer. N Engl J Med 2008;358:1148–59. 8. Wood LD, Parsons DW, Jones S, Lin J, Sjoblom€ T, Leary RJ, et al. The 3. Carmona FJ, Esteller M. Epigenomics of human colon cancer. Mutat genomic landscapes of human breast and colorectal cancers. Res 2010;693:53–60. Science 2007;318:1108–13. 4. Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JP, et al. 9. Barkan D, Green JE, Chambers AF. Extracellular matrix: a gatekeeper Incidence and functional consequences of hMLH1 promoter hyper- in the transition from dormancy to metastatic growth. Eur J Cancer methylation in colorectal carcinoma. Proc Natl Acad Sci U S A 2010;46:1181–8. 1998;95:6870–5. 10. Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Chan AT, Schernham- 5. Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG. Inactiva- mer ES, et al. A cohort study of tumoral LINE-1 hypomethylation tion of the DNA repair gene O6-methylguanine-DNA methyltransfer- and prognosis in colon cancer. J Natl Cancer Inst 2008;100: ase by promoter hypermethylation is a common event in primary 1734–8. human neoplasia. Cancer Res 1999;59:793–7. 11. Jorissen RN, Gibbs P, Christie M, Prakash S, Lipton L, Desai J, et al. 6. Esteller M, Toyota M, Sanchez-Cespedes M, Capella G, Peinado MA, Metastasis-Associated Gene Expression Changes Predict Poor Out- Watkins DN, et al. Inactivation of the DNA repair gene O6-methylgua- comes in Patients with Dukes Stage B and C Colorectal Cancer. Clin nine-DNA methyltransferase by promoter hypermethylation is asso- Cancer Res 2009;15:7642–51. ciated with G to A mutations in K-ras in colorectal tumorigenesis. 12. Schepeler T, Reinert JT, Ostenfeld MS, Christensen LL, Silahtaroglu Cancer Res 2000;60:2368–71. AN, Dyrskjt L, et al. Diagnostic and prognostic microRNAs in stage II 7. Esteller M, Risques RA, Toyota M, Capella G, Moreno V, Peinado MA, colon cancer. Cancer Res 2008;68:6416–24. et al. Promoter hypermethylation of the DNA repair gene O(6)-methyl-

www.aacrjournals.org Clin Cancer Res; 17(6) March 15, 2011 1217

F. Javier Carmona and Manel Esteller

Clin Cancer Res 2011;17:1215-1217.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/17/6/1215

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2011/03/11/17.6.1215.DC1

Cited articles This article cites 12 articles, 8 of which you can access for free at: http://clincancerres.aacrjournals.org/content/17/6/1215.full#ref-list-1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/17/6/1215. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.