Micro-array gene-expression profiling techniques were described in previous reports [7]. In brief, we used 165 primary bladder cancer samples, 23 recurrent non-muscle-invasive tumor tissues, 58 normal-looking bladder mucosa surrounding cancer, and 10 normal bladder mucosa for micro-array analysis. Hierarchical clustering was used to stratify the prognosis-related gene classifiers. The full micro-array data set is available online at http://www.ncbi.nlm.nih.gov/geo/ under the data series accession number GSE13507.

Sixty-two patients with MIBC were selected by micro-array profiling (original cohort). We collected new tumor specimens from 124 MIBC patients with histologically verified urothelial cell carcinoma as a validation cohort. To reduce confounding factors affecting the analyses, and to delineate a more homogeneous study population, any patients diagnosed with a concomitant carcinoma in situ or for whom data collection was incomplete were excluded. All tumors were macro-dissected, typically within 15 minutes of surgical resection. Each bladder cancer specimen was confirmed by pathological analysis of a part of the tissue sample in fresh-frozen sections from cystectomy and transurethral resection (TUR) specimens, then frozen in liquid nitrogen and stored at -80℃ until use. The collection and analysis of all samples was approved by the Institutional Review Board of Chungbuk National University (IRB approval number 2006-01-001), and informed consent was obtained from each subject.

Tumors were staged according to the 2002 tumor-node-metastasis (TNM) classification and the 1973 World Health Organization grading system, respectively [4,16]. All diagnoses were confirmed by pathological analysis of frozen sections from cystectomy and TUR specimens. Patients with localized or locally advanced tumors and good Eastern Cooperative Oncology Group (ECOG) performance status (0 or 1) underwent radical cystectomy and complete pelvic lymph node dissection. Patients who were not eligible for radical cystectomy owing to metastatic disease, poor life expectancy, or poor ECOG performance status (≥2) underwent TUR for histopathological diagnosis. Patients with pT3, pT4, or node-positive disease according to the analysis of radical cystectomy specimens, or with metastatic disease but good performance status, received at least four cycles of cisplatin-based chemotherapy. Each patient was followed and managed according to standard protocols [17]. In this study, progression was defined as local regional recurrence or new distant metastasis in the cystectomized group and a ≥20% increment of mass or new distant metastasis in the noncystectomized group.

One milliliter TRIzol (Invitrogen, Carlsbad, CA, USA) was added to bladder cancer tissue and homogenized in a 5-ml glass tube. The homogenate was transferred to a 1.5-ml tube and was mixed with 200 µl chloroform. After incubation for 5 minutes at 4℃, the homogenate was centrifuged for 13 minutes at 13,000×g at 4℃. The upper aqueous phase was transferred to a clean tube and 500 µl isopropanol was added, followed by incubation for 60 minutes at 4℃. The tube was then centrifuged for 8 minutes at 13,000×g and 4℃. Then, the upper aqueous phase was removed, mixed with 500 µl of 75% ethanol, and centrifuged for 5 minutes at 13,000×g and 4℃. After the upper aqueous layer was discarded, the pellet was dried at room temperature, dissolved with diethylpyrocarbonate-treated water, and stored at -80℃. The quality and integrity of RNA were confirmed by agarose gel electrophoresis and ethidium bromide staining, followed by visual examination under ultraviolet light. The cDNA was then prepared from 1 µg of random priming by using a First-Strand cDNA Synthesis Kit (Amersham Biosciences Europe GmbH, Freiburg, Germany) according to the manufacturer's protocol.

To quantify the expression levels of FAM70B, real-time PCR amplification was performed with a Rotor Gene 6000 instrument (Corbett Research, Mortlake, Australia). Real-time PCR assays using SYBR Premix EX Taq (Takara Bio Inc., Otsu, Japan) were carried out in micro-reaction tubes (Corbett Research, Mortlake, Australia). The primers used for amplifying FAM70B (220 bp) were 5'-CCC TCG CCC GCC TAC TAT-3' and 5'-GCT GGG CGG GGT TGT AGA-3'. The PCR reaction was performed in a final volume of 10 µl, consisting of 5 µl of 2x SYBR premix EX Taq buffer, 0.5 µl of each 5'- and 3'- primer (10 pmol/µl), and 1 µl of the sample cDNA. The product was purified with a QIAquick Extraction kit (QIAGEN, Hilden, Germany), quantified with a spectrometer (Perkin Elmer MBA2000, Fremont, CA, USA), and sequenced with an automated laser fluorescence sequencer (ABI PRISM 3100 Genetic Analyzer, Foster City, CA, USA). The known concentration of the product was 10-fold serially diluted from 100 pg/µl to 0.1 pg/µl. The dilution series of PCR products were used for establishing the standard curve of real-time PCR. The real-time PCR conditions were 1 cycle at 96℃ for 20 seconds, followed by 40 cycles of 3 seconds at 96℃ for denaturation, 15 seconds at 60℃ for annealing, and 15 seconds at 72℃ for extension. The melting program was performed at 72 to 95℃ with a heating rate of 1℃ per 45 seconds. Spectral data were captured and analyzed by using Rotor-Gene Real-Time Analysis Software 6.0 Build 14 (Corbett Research, Mortlake, Australia). All samples were run in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was applied as an endogenous RNA reference gene. Gene expression was normalized to the expression of GAPDH.

To normalize the highly skewed distribution of FAM70B mRNA expression, the data were examined as the natural log. Patients were classified as having a high expression of FAM70B or low expression of FAM70B, with the median (115.8×10⁴ copies/µl) of the mRNA expression as the cutoff value. The Kaplan-Meier method was used to estimate cancer-specific survival, and differences were assessed by using log-rank statistics. The prognostic value of FAM70B expression was analyzed by using univariate and multivariate Cox proportional hazard regression models. Statistical analyses were performed by using SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA), and p-values of less than 0.05 were considered statistically significant.

In the original cohort, FAM70B was associated with higher risk for cancer-specific death (hazard ratio [HR], 33.675; p<0.001), cancer progression (HR, 30.82; p=0.007), and overall survival (HR, 20.903; p=0.002) in patients with MIBC. Kaplan-Meier survival curves showing the effect of FAM70B expression on progression-free survival (log-rank test, p=0.005), cancer-specific survival (log-rank test, p=0.013), and overall survival (log-rank test, p=0.008) are shown in Fig. 1A, 1B, and 1C, respectively.

The baseline characteristics of the newly enrolled patients are listed in Table 1. Mean patient age was 65.2 years (range, 38 to 87 years) and mean±standard deviation follow-up was 37.8±45.9 months (median, 16.9; range, 2.1 to 193.1 months). A total 70 of 124 patients (56.5%) underwent radical cystectomy for MIBC. Other patients (43.5%) underwent TUR or biopsy for histopathological diagnosis. Systemic chemotherapy was performed in 56 patients (45.2%) after intervention. During the follow-up, 63 of 124 patients (50.8%) with MIBC experience progression and 61 (49.2%) died of their bladder tumor. Progression-free survival and cancer-specific survival were 32.9 months (median, 13.2 months) and 37.7 months (median, 16.9 months), respectively.

To create more homogeneous patient groups, patients were categorized according to chemotherapy and radical cystectomy status. In the patient group who underwent radical cystectomy, Kaplan-Meier estimates revealed a significant association between FAM70B expression and cancer-specific survival (log-rank test, p=0.013) (Fig. 3A). Similar results were identified in the chemotherapy group (log-rank test, p=0.013) (Fig. 4A). However, in the patient group who had not undergone radical cystectomy and had not received chemotherapy, FAM70B expression did not predict cancer-specific survival (Figs. 3B, 4B). Also, FAM70B expression successfully predicted progression-free survival and overall survival in MIBC patients who underwent radical cystectomy (log-rank test, p=0.036, p=0.005, respectively). In the chemotherapy group, FAM70B expression was associated with progression-free survival (log-rank test, p=0.042; data not shown).

Kaplan-Meier estimates revealed that FAM70B expression successfully predicted cancer-specific survival in the subjects with localized or locally advanced tumors (log-rank test, p=0.016) (Fig. 5A). In contrast, FAM70B expression was not associated with the cancer-specific survival in patients with tumor stage T4 or ≥N1 or M1 (log-rank test, p=0.372) (Fig. 5B).