Three Epigenetic Biomarkers, GDF15, TMEFF2, and VIM, Accurately Predict Bladder Cancer from DNA-Based Analyses of Urine Samples
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Published OnlineFirst October 25, 2010; DOI: 10.1158/1078-0432.CCR-10-1312 Clinical Cancer Imaging, Diagnosis, Prognosis Research Three Epigenetic Biomarkers, GDF15, TMEFF2, and VIM, Accurately Predict Bladder Cancer from DNA-Based Analyses of Urine Samples Vera L. Costa1,2,3,4, Rui Henrique1,5,6, Stine A. Danielsen3,4, Sara Duarte-Pereira1,2, Mette Eknaes3,4, Rolf I. Skotheim3,4, Angelo^ Rodrigues5, Jose S. Magalhaes~ 7, Jorge Oliveira7, Ragnhild A. Lothe3,4,8, Manuel R. Teixeira2,4,6,9, Carmen Jeronimo 1,2,6, and Guro E. Lind3,4 Abstract Purpose: To identify a panel of epigenetic biomarkers for accurate bladder cancer (BlCa) detection in urine sediments. Experimental Design: Gene expression microarray analysis of BlCa cell lines treated with 5-aza-20- deoxycytidine and trichostatin A as well as 26 tissue samples was used to identify a list of novel methylation candidates for BlCa. Methylation levels of candidate genes were quantified in 4 BlCa cell lines, 50 BlCa tissues, 20 normal bladder mucosas (NBM), and urine sediments from 51 BlCa patients and 20 healthy donors, 19 renal cancer patients, and 20 prostate cancer patients. Receiver operator characteristic curve analysis was used to assess the diagnostic performance of the gene panel. Results: GDF15, HSPA2, TMEFF2, and VIM were identified as epigenetic biomarkers for BlCa. The methylation levels were significantly higher in BlCa tissues than in NBM (P < 0.001) and the cancer specificity was retained in urine sediments (P < 0.001). A methylation panel comprising GDF15, TMEFF2, and VIM correctly identified BlCa tissues with 100% sensitivity and specificity. In urine samples, the panel achieved a sensitivity of 94% and specificity of 100% and an area under the curve of 0.975. The gene panel could discriminate BlCa from both healthy individuals and renal or prostate cancer patients (sensitivity, 94%; specificity, 90%). Conclusions: Byusingagenome-wideapproach,wehaveidentifiedabiomarkerpanelthatallowsforearly and accurate noninvasive detection of BlCa using urine samples. Clin Cancer Res; 16(23); 5842–51. Ó2010 AACR. Bladdercancer(BlCa)isone of the leading causes of the second most common genitourinary malignancy, cancer-related morbidity and mortality. Global estimates with about 71,000 new cases and more than 14,000 for 2002 indicate that approximately 357,000 BlCa cases deaths in 2009 (2). The incidence of BlCa increases with were diagnosed and that approximately 145,000 patients age, with an average age at the time of diagnosis in succumbed to the disease (1). In the United States, BlCa is the 60s, and it is 3 times more common in men than in women (3). Although several risk factors (e.g., smoking habits and exposure to carcinogens) have been identified, effective strategies for early detection are still 1 2 Authors' Affiliations: Cancer Epigenetics Group, and Department of not available (4). Genetics, Portuguese Oncology Institute – Porto, Porto, Portugal; 3Cancer Prevention, Institute for Cancer Research, The Norwegian Radium Hospi- The present gold standard strategy for BlCa diagnosis is tal, Oslo University Hospital, Oslo, Norway; 4Centre for Cancer Biomedi- noninvasive, voided urine cytology, followed by cysto- cine, University of Oslo, Oslo, Norway; 5Department of Pathology, Portuguese Oncology Institute – Porto, Porto, Portugal; 6Department of scopic examination. However, both methods have low Pathology and Molecular Immunology, Institute of Biomedical Sciences sensitivity, especially for low-grade tumors (3). Several 7 Abel Salazar, University of Porto, Porto, Portugal; Department of Urology, BlCa markers were recently revised by Vrooman and Witjes Portuguese Oncology Institute – Porto, Porto, Portugal; 8Faculty of Med- icine, University of Oslo, Norway; and 9Cancer Genetics Group, Research (5), but they have concluded that the analysis of those Center of the Portuguese Oncology Institute – Porto, Porto, Portugal markers is costly and did not improve diagnostics com- Note: Supplementary data for this article are available at Clinical Cancer pared with routine urinary cytology. Additional tests have Research Online (http://clincancerres.aacrjournals.org/). been developed during the last few years; however, their C. Jeronimo and G. E. Lind contributed equally to this study. clinical usefulness has yet to be established using large Corresponding Author: Carmen Jeronimo, Cancer Epigenetics Group, clinical trials (5, 6). Research Center of the Portuguese Oncology Institute – Porto, Rua Urothelial carcinoma comprises the most common form Dr. Antonio Bernardino de Almeida, 4200 Porto, Portugal. Phone: þ351962447005;Fax: þ351225084016; E-mail: carmenjeronimo@ipoporto. of BlCa, 70% of which present as papillary nonmuscle- min-saude.pt; or Guro E. Lind, Centre for Cancer Biomedicine, University of invasive tumors, although as many as 50% to 70% of these Oslo, 0310 Oslo, Norway. E-mail: [email protected] tumors (pTa and pT1, classified according to AJCC/UICC; doi: 10.1158/1078-0432.CCR-10-1312 ref. 7) will recur and approximately 10% to 20% will Ó2010 American Association for Cancer Research. progress to invasive disease (3). To predict which patients 5842 Clin Cancer Res; 16(23) December 1, 2010 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst October 25, 2010; DOI: 10.1158/1078-0432.CCR-10-1312 Methylation Markers for Bladder Cancer 100 mg/mL of streptomycin; Gibco), in a humidified atmo- Translational Relevance sphere of 5% CO2 at 37 C. Our study identified a 3-gene panel of epigenetic All BlCa cell lines were subjected to treatment with a biomarkers that accurately detect bladder cancer (BlCa) combination of the demethylating drug 5-aza- 0 m in urine sediments and discriminate it from prostate 2 deoxycytidine (1 mol/L for 72 hours) and the histone m cancer and renal epithelial tumors. This panel might be deacetylase inhibitor trichostatin A (0.5 mol/L added in easily applied for routine screening of BlCa in voided the last 12 hours). In parallel, the same cell lines were urine samples. cultured without treatment for 72 hours and were har- vested before confluency. Patients and tumor sample collection will progress from superficial to invasive disease remains a The 50 BlCa samples that were included in this study challenge. Patients with a diagnosis of early-stage BlCa were obtained from a consecutive series of patients diag- undergo frequent monitoring, currently based on cysto- nosed and treated between 2005 and 2006 at the Portu- scopy and cytology, resulting in BlCa becoming one of the guese Oncology Institute – Porto, Porto, Portugal. Tumor most costly cancer diseases to manage (8). The develop- tissues were collected after transurethral resection or ment of innovative, noninvasive tests for early detection of radical cystectomy. A small tumor sample was immedi- BlCa is essential to lower the morbidity and mortality ately snap-frozen, stored at À80C, and subsequently cut associated with BlCa, and such tests will also be economic- in cryostat for DNA and RNA extraction. The bulk mate- ally beneficial. rial was routinely processed for routine pathologic exam- The alteration of DNA methylation pattern in CpG ination allowing for tumor classification and WHO/ISUP islands is emerging as a key event in transcriptionally grading (13, 14). An independent set of 20 NBMs from repressed regions of the genome. Aberrant promoter BlCa-free individuals (prostate cancer patients submitted methylation at several gene loci has been reported for to radical prostatectomy) was used as controls. Relevant BlCa, and some of them have been shown to be asso- clinical data was collected from patient’s clinical records ciated with cancer development, stage, recurrence, pro- (see Supplementary Table S1). Patients were enrolled gression, and survival (9). Because of the high prevalence following informed consent. These studies were approved of cancer-related gene promoter hypermethylation, sev- by the institutional review board of the Portuguese eral BlCa epigenetic biomarkers have been identified in Oncology Institute – Porto. urine for the purposes of disease detection and monitor- ing (10). Although most of these markers are promising from exploratory studies, many of them fail to show Urine sample collection and processing comparable results in independent studies and a great Morning voided urine samples (1 sample per patient) majority of them are reported in various cancers and thus were collected from 51 patients with a diagnosis of BlCa are not specific for BlCa (11). Consequently, there is a and treated between 2004 and 2006 at the Portuguese need to identify improved epigenetic biomarkers, which Oncology Institute – Porto, from 19 patients with renal might enable a more reliable and accurate detection of the cell tumor and from 20 patients with prostate cancer. disease as well as for predicting the prognosis of BlCa Controls were randomly chosen among healthy donors patients. with no personal or family history of cancer. Relevant We have used a genome-wide strategy (12) to identify demographic data are provided in Supplementary novel epigenetic biomarkers in BlCa and followed a strict Table S2. Patients and controls were enrolled after pipeline to pinpoint and validate those most suitable for informed consent. Urine storage and processing conditions noninvasive, urine-based diagnostic testing. were standardized: each sample was immediately centri- fuged at 4,000 rpm for 10 minutes;