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

Abstract

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.

Keywords

MS-MLPA DNA methylation Bladder cancer Biomarker Tissue 

Supplementary material

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

References

  1. 1.
    Wolff EM, Liang G, Jones PA. Mechanisms of disease: genetic and epigenetic alterations that drive bladder cancer. Nat Clin Pract Urol. 2005;2:502–10.CrossRefPubMedGoogle Scholar
  2. 2.
    Sanchez-Carbayo M. Hypermethylation in bladder cancer: biological pathways and translational applications. Tumor Biol. 2005;33:347–61.CrossRefGoogle Scholar
  3. 3.
    Esteller M. CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene. 2002;21:5427–40.CrossRefPubMedGoogle Scholar
  4. 4.
    Sánchez-Carbayo M. Urine epigenomics: a promising path for bladder cancer diagnostics. Exp Rev Mol Diagn. 2012;5:429–32.CrossRefGoogle Scholar
  5. 5.
    Aleman A, Adrien L, Lopez-Serra L, Cordon-Cardo C, Esteller M, Belbin TJ, et al. Identification of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays. Br J Cancer. 2008;98:466–73.PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Nygren AO, Ameziane N, Duarte HM, Vijzelaar RN, Waisfisz Q, Hess CJ, et al. Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res. 2005;33:e128.PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Serizawa RR, Ralfkiaer U, Dahl C, Lam GW, Hansen AB, Steven K, et al. Custom-designed MLPA using multiple short synthetic probes: application to methylation analysis of five promoter CpG islands in tumor and urine specimens from patients with bladder cancer. J Mol Diagn. 2010;12:402–8.PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Cabello MJ, Grau L, Franco N, Orenes E, Alvarez M, Blanca A, et al. Multiplexed methylation profiles of TSGs in bladder cancer. J Mol Diagn. 2011;13:29–40.PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Zuiverloon TC, Beukers W, van der Keur KA, Munoz JR, Bangma CH, Lingsma HF, et al. A methylation assay for the detection of non-muscle-invasive bladder cancer (NMIBC) recurrences in voided urine. BJU Int. 2012;109:941–8.CrossRefPubMedGoogle Scholar
  10. 10.
    Agundez M, Grau L, Palou J, Algaba F, Villavicencio H, Sanchez-Carbayo M. Evaluation of the methylation status of tumour suppressor genes for predicting bacillus Calmette-Guérin response in patients with T1G3 high-risk bladder tumours. Eur Urol. 2011;60:131–40.CrossRefPubMedGoogle Scholar
  11. 11.
    Castro M, Grau L, Puerta P, Gimenez L, Venditti J, Quadrelli S, et al. Multiplexed methylation profiles of tumor suppressor genes and clinical outcome in lung cancer. J Transl Med. 2010;8:86.PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    García-Baquero R, Puerta P, Beltran M, Alvarez M, Sacristan R, Alvarez-Ossorio JL, et al. Methylation of a novel panel of tumor suppressor genes in urine moves forward noninvasive diagnosis and prognosis of bladder cancer: a 2-center prospective study. J Urol. 2013;190:723–30.CrossRefPubMedGoogle Scholar
  13. 13.
    Kirkali Z, Chan T, Manoharan M, Algaba F, Busch C, Cheng L, et al. Bladder cancer: epidemiology, staging and grading, and diagnosis. Urology. 2005;66:4–34.CrossRefPubMedGoogle Scholar
  14. 14.
    Kim WJ, Kim EJ, Jeong P, Quan C, Kim J, Li QL, et al. RUNX3 inactivation by point mutations and aberrant DNA methylation in bladder tumors. Cancer Res. 2005;65:9347–54.CrossRefPubMedGoogle Scholar
  15. 15.
    Yu J, Zhu T, Wang Z, Zhang H, Qian Z, Xu H, et al. A novel set of DNA methylation makers in urine sediments for sensitive/specific detection of bladder cancer. Clin Cancer Res. 2007;13:7296–304.CrossRefPubMedGoogle Scholar
  16. 16.
    Wolff EM, Liang G, Cortez CC, Tsai YC, Castelao JE, Cortessis VK, et al. RUNX3 methylation reveals that bladder tumors are older in patients with a history of smoking. Cancer Res. 2008;68:6208–14.PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Kim EJ, Kim YJ, Jeong P, Ha YS, Bae SC, Kim WJ. Methylation of the RUNX3 promoter as a potential prognostic marker for bladder tumor. J Urol. 2008;108:1141–5.CrossRefGoogle Scholar
  18. 18.
    Renard I, Joniau S, van Cleynenbreugel B, Collette C, Naômé C, Vlassenbroeck I, et al. Identification and validation of the methylated TWIST1 and NID2 genes through real-time methylation-specific polymerase chain reaction assays for the noninvasive detection of primary bladder cancer in urine samples. Eur Urol. 2010;58:96–104.CrossRefPubMedGoogle Scholar
  19. 19.
    Suzuki M, Shigematsu H, Shames DS, Sunaga N, Takahashi T, Shivapurkar N, et al. DNA methylation-associated inactivation of TGFbeta-related genes DRM/Gremlin, RUNX3, and HPP1 in human cancers. Br J Cancer. 2005;93:1029–37.PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Dhawan D, Hamdy FC, Rehman I, Patterson J, Cross SS, Feeley KM, et al. Evidence for the early onset of aberrant promoter methylation in urothelial carcinoma. J Pathol. 2006;209:336–43.CrossRefPubMedGoogle Scholar
  21. 21.
    Hoque MO, Begum S, Brait M, Jeronimo C, Zahurak M, Ostrow KL, et al. TIMP3 promoter methylation is an independent prognostic factor for bladder cancer. J Urol. 2008;179:743–7.PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Jarmalaite S, Andrekute R, Scesnaite A, Suziedelis K, Husgafvel-Pursiainen K, Jankevicius F. Promoter hypermethylation in tumour suppressor genes and response to interleukin-2 treatment in bladder cancer: a pilot study. J Cancer Res Clin Oncol. 2010;136:847–54.CrossRefPubMedGoogle Scholar
  23. 23.
    Yates DR, Rehman I, Abbod MF, Meuth M, Cross SS, Linkens DA, et al. Promoter hypermethylation identifies progression risk in bladder cancer. Clin Cancer Res. 2007;13:2046–53.CrossRefPubMedGoogle Scholar
  24. 24.
    Shigematsu H, Suzuki M, Takahashi T, Miyajima K, Toyooka S, Shivapurkar N, et al. Aberrant methylation of HIN-1 (high in normal-1) is a frequent event in many human malignancies. Int J Cancer. 2005;113:600–4.CrossRefPubMedGoogle Scholar
  25. 25.
    Wiklund ED, Bramsen JB, Hulf T, Dyrskjøt L, Ramanathan R, Hansen TB, et al. Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer. Int J Cancer. 2010;6:1327–34.Google Scholar
  26. 26.
    Marsit CJ, Karagas MR, Andrew A, Liu M, Danaee H, Schned AR, et al. Epigenetic inactivation of SFRP genes and TP53 alteration act jointly as markers of invasive bladder cancer. Cancer Res. 2005;65:7081–5.CrossRefPubMedGoogle Scholar
  27. 27.
    Urakami S, Shiina H, Enokida H, Kawakami T, Kawamoto K, Hirata H, et al. Combination analysis of hypermethylated Wnt-antagonist family genes as a novel epigenetic biomarker panel for bladder cancer detection. Clin Cancer Res. 2006;12:2109–16.CrossRefPubMedGoogle Scholar
  28. 28.
    Pu RT, Laitala LE, Clark DP. Methylation profiling of urothelial carcinoma in bladder biopsy and urine. Acta Cytol. 2006;50:499–506.CrossRefPubMedGoogle Scholar
  29. 29.
    Brait M, Begum S, Carvalho AL, Dasgupta S, Vettore AL, Czerniak B, et al. Aberrant promoter methylation of multiple genes during pathogenesis of bladder cancer. Cancer Epidemiol Biomarkers Prev. 2008;10:2786–94.CrossRefGoogle Scholar
  30. 30.
    Friedrich MG, Chandrasoma S, Siegmund KD, Weisenberger DJ, Cheng JC, Toma MI, et al. Prognostic relevance of methylation markers in patients with non-muscle invasive bladder carcinoma. Eur J Cancer. 2005;41:2769–78.CrossRefPubMedGoogle Scholar

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

Personalised recommendations