Tumor Biology

, Volume 35, Issue 6, pp 5777–5786 | Cite as

Methylation of tumor suppressor genes in a novel panel predicts clinical outcome in paraffin-embedded bladder tumors

  • Rodrigo García-Baquero
  • Patricia Puerta
  • Manuel Beltran
  • Miguel Alvarez-Mújica
  • Jose Luis Alvarez-Ossorio
  • Marta Sánchez-Carbayo
Research Article


DNA methylation of tumor suppressor genes (TSGs) represents a frequent and early epigenetic event with potential applications for cancer detection and disease evolution. Our aim was to examine the stratification and prognostic biomarker role of the methylation of a novel panel of TSGs in bladder cancer. The methylation status of 18 TSGs was evaluated in bladder cancer cells (n = 14) and paraffin-embedded primary bladder tumors (n = 61), using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Recurrence, progression, and disease-specific survival were analyzed using univariate and multivariate Cox models. PRDM2, HLTF, ID4, DLC1, BNIP3, H2AFX, CACNA1G, TGIF, and CACNA1A were discovered methylated in bladder cancer. The methylation of RUNX3 (p = 0.026), TWIST1 (p = 0.009), SFRP4 (p = 0.002), and CCND2 (p = 0.027) correlated to tumor stage. Univariate analyses indicated prognostic associations for recurrence (DLC1, SFRP5, H2AFX, CACNA1G), progression (DLC1, SFRP5, CACNA1G), disease-specific (PRDM2, DLC1, SFRP5, CACNA1G, and TIMP3), and overall survival (SFRP5 and TIMP3). In multivariate analyses, several TSGs remained as independent prognosticators for recurrence (SFRP5, H2AFX), progression (CACNA1G), and disease-specific survival (SFRP5). Thus, a novel set of TSGs was identified, frequently methylated in bladder cancer cells and tumors. TSG methylation allowed histopathologic and outcome stratification using paraffin-embedded tumors. This is clinically relevant by offering a strategy for the management of patients affected with uroepithelial neoplasias in pathology routine laboratories.


MS-MLPA DNA methylation Bladder cancer Biomarker Tissue 



The authors would like to thank all members of the laboratory of Dr Sánchez-Carbayo at the Tumor Markers Group at the CNIO for constructive suggestions in the preparation of this manuscript, and especially Noreli Franco and Laura Grau for their technical assistance. We would like to thank all the members of our clinical collaborators involved in this study for their support in facilitating the urinary and tumor specimens and the clinical follow-up of the bladder cancer cases analyzed. This study was supported by grants (SAF2009-13035 and SAF2012-40206) from the Spanish Ministry of Science and Innovation and from the Mutua Madrileña 2010 to Dr Sánchez-Carbayo.

Conflicts of interest


Supplementary material

13277_2014_1767_MOESM1_ESM.doc (44 kb)
ESM 1 (DOC 44 kb)


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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Rodrigo García-Baquero
    • 1
    • 2
  • Patricia Puerta
    • 1
  • Manuel Beltran
    • 3
  • Miguel Alvarez-Mújica
    • 4
  • Jose Luis Alvarez-Ossorio
    • 2
  • Marta Sánchez-Carbayo
    • 1
  1. 1.Bladder Cancer Group, Proteomics UnitCIC bioGUNEDerioSpain
  2. 2.Urology DepartmentHospital Puerta del MarCádizSpain
  3. 3.Pathology DepartmentHospital Puerta del MarCádizSpain
  4. 4.Urology DepartmentHospital Central de AsturiasOviedoSpain

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