Combination of a Novel Gene Expression Signature with a Clinical Nomogram Improves the Prediction of Survival in High-Risk Bladder Cancer

Urothelial carcinoma of the urinary bladder is the fifth most common cancer in the Western world, and the ninth most frequent cancer worldwide, representing 3% of cancers diagnosed globally (1). Treatment of bladder cancer has not changed significantly in more than 20 years, and outcomes for patients remain suboptimal. Approximately 20% to 30% of newly diagnosed patients present with muscle-invasive disease (stages T2–T4) or metastatic disease, whereas up to a third of patients with initially non–muscle-invasive disease (stages Ta/T1/Tis) later progress to muscle-invasive or metastatic disease (2). Clinical features including stage and grade are strongly associated with outcome and play an important role in determining treatment. For example, the 5-year tumor-specific mortality rates range from less than 5% for low-stage and low-grade disease up to approximately 50% for all muscle-invasive lesions (3, 4). However, even though grade and stage are important predictors of outcome, there remains significant variability in the prognosis of patients with similar characteristics. This highlights the need to identify additional tumor characteristics that predict clinical behavior.

In recent years, there has been a growing interest in the use of gene expression signatures for risk stratification of patients with cancer (5). Multiple groups have produced urothelial cancer gene signatures predictive of a range of tumor characteristics and outcomes, including stage (6–8), molecular subtype (9, 10), likelihood of recurrence and progression of non–muscle-invasive (11–13) and muscle-invasive disease (14), and survival (7). Here, we aimed the following: (i) to explore whether novel cancer gene expression patterns can be identified that stratify patients undergoing radical cystectomy for urothelial cancer by risk of recurrence and death; (ii) to test our novel signatures and all previously developed signatures in all available bladder cancer microarray data sets; and (iii) to investigate the gain in predictive accuracy of a validated clinical nomogram by combination with gene signature information.

We utilized previously unpublished cancer gene expression data from 93 patients undergoing radical cystectomy at Memorial Sloan-Kettering Cancer Center (MSKCC), New York, between 1993 and 2004. Specimens were collected with MSKCC Institutional Review Board approval, and a waiver of authorization to review associated clinical data was obtained from the Board. The clinicopathologic characteristics are summarized in Table 1 (see Supplementary Table S1 for details). In those 15 patients with non–muscle-invasive disease on final pathologic analysis after radical cystectomy, 4 had muscle-invasive disease histologically in tissue obtained at the time of prior transurethral resection (TUR) and the remaining patients had high-risk features (e.g., extensive volume of disease, recurrent, or Bacillus Calmette—Guerin (BCG) refractory disease). Lymph node dissection was done in 77 patients; no patient had metastatic disease at the time of radical cystectomy. Chemotherapy was administered to 3 patients as neoadjuvant, 16 patients as adjuvant, and 19 patients as salvage for recurrent disease. Case selection was restricted to those with frozen specimens with measurable volume of malignancy and adequate percentage of tumor.

Overall survival time was defined as the time from date of radical cystectomy to date of death or last follow-up. Disease-specific survival was defined as the time from radical cystectomy to death attributed to urothelial cancer, when death occurred with known and progressive metastatic disease. If death was recorded in the institutional database without knowledge of a recurrent cancer or with documentation of another malignancy or a nonmalignancy cause, death was attributed to other causes. Recurrence was defined as pelvic if within the pelvis below the aortic bifurcation, distant if there was visceral metastasis or recurrence above the aortic bifurcation, and urothelial if within the remaining urinary tract (renal pelvis, ureter, and urethra). In the analysis of the development of metastases, only pelvic and distance recurrences were included. Primary endpoint in this study was overall survival if not mentioned otherwise.

All patient specimens collected at the time of surgery were processed expeditiously within the Department of Pathology and stored in an institutional biospecimen bank. Frozen bladder tissues were examined by a genitourinary pathologist to identify tumor content, which was microdissected. Ten 50-μm sections were cut, with confirmation of tumor content by pathologic review. RNA was isolated with TRIzol (Invitrogen) and cleaned with the RNeasy Mini Kits (Qiagen) according to manufacturer protocols. Expression profiling was conducted in the MSKCC Genomics Core Facility. In brief, 5 to 10 μg of RNA was transcribed utilizing a T7-oligo dT primer and converted to cDNA (Invitrogen cDNA synthesis kit). Biotinylated aRNA was produced from the cDNA by an in vitro transcription kit (Enzo Diagnostics). Following quality assessment using an Agilent Bioanalyzer, the aRNA was fragmented and hybridized onto Affymetrix U133 Plus 2.0 arrays, containing 54,675 probe sets. Data were uploaded to National Center for Biotechnology Information (NCBI) Geo (15) under accession number GSE31684.

We obtained 6 independent bladder cancer microarray data sets (Table 2; refs. 6, 7, 9, 10, 13, 16) totaling 578 additional patients and encompassing a broad spectrum of stages, grades, and histologic types. Overall survival data were available for the Blaveri and colleagues, Sanchez-Carbayo and colleagues, Lindgren and colleagues, and Kim and colleagues cohorts (6, 7, 9, 13; see Supplementary data for preprocessing details). Disease-specific survival time information was only documented for patients in the cohort from Kim and colleagues. The primary endpoint was, therefore, overall survival in the meta-analysis.

Expression estimates were obtained by the GCRMA algorithm (17).

The gene expression profiles were evaluated for significant correlation with multiple clinical variables and outcomes, including stage, lymph node status, recurrence, and overall survival. For the identification of differentially expressed probe sets, we used the LIMMA method (18). If not stated otherwise, the cutoff value of the adjusted P values [the false discovery rate (FDR); ref. 19] was set to 0.01 and the minimum fold change (FC) to 1.5. The probability of observing n or more differentially expressed genes was estimated by permutation tests. For the external data sets, the lists of differentially expressed genes were downloaded from the Supplementary Material of the respective articles (6, 7, 9, 10, 13, 16) when available, otherwise calculated with LIMMA. When comparing lists of differentially expressed genes with corresponding lists from external data sets, the significance of overlaps was calculated in Bioconductor (GSEABase), using 25,000 permutations.

Classifiers were generated with the Fisher linear discriminant and support vector machines (SVM) and were leave-one-out cross-validated. The R packages MASS and e1071 were used for building the classifiers. We utilized a linear SVM kernel and default, untuned parameters. Receiver operating characteristic (ROC) curves were generated with the ROCR package (20).

For each patient, a risk score was calculated by a Cox proportional hazards model that was fitted using the gene expression profiles of all remaining patient samples (leave-one-out cross-validation). The risk score was defined as the sum of the gene signature expression values, weighted by the Cox regression coefficients (21). Each individual was then classified into low-risk or high-risk categories, based on the median leave-one-out cross-validated risk score: patients were classified as low-risk when their risk score was smaller than the cohort median, otherwise as high-risk. This was done independently for all data sets for which survival information was available. The significance of survival curve differences of cross-validated models was estimated with permutation tests. The process from cross-validation to risk stratification was repeated 500 times with shuffled survival labels. This empirical χ² distribution was then utilized to estimate P values. For noncross-validated models, the log-rank test was used. Multivariate survival prediction models were compared by the C-statistic, an estimator of the model concordance (22) and by likelihood ratio tests. The concordance represents the probability that given 2 random noncensored individuals, the one with the higher risk score has a shorter survival time.

For unsupervised clustering, we used the Ward clustering method and the Pearson correlation distance as implemented in the pvclust R package (23). The significance of a cluster was reported as its bootstrap value, which is the proportion of 10,000 bootstrap samples showing this particular cluster topology. The clustering was applied to the 6 external data sets of bladder cancer samples.

We compiled 49 published gene signatures (Supplementary Table S2) associated with malignancy: 39 from Lauss and colleagues (24) as well as other bladder (11–14) and melanoma signatures (25, 26); the melanoma signatures were included to test whether signatures from other solid tumors would conduct well in bladder cancer. The 49 gene signatures were tested in our data set and the 4 external gene expression data sets with survival information. Associations of gene expression signatures with outcome and other covariates were calculated using global test (27); this test can be used to estimate whether the expression of a group of genes is significantly associated with a particular response variable, for example, stage or survival. Gene signatures were further tested by leave-one-out cross-validation. To avoid problems with highly correlated covariates in multivariate Cox proportional hazards models, expression values were scaled by principal component analysis (PCA). The number of components was chosen so that at least 99% of the expression variance was included in the model, with a maximum of 20 components. The choice of this cutoff value was examined by varying the maximum number of components from 3 to 30 (Supplementary Table S2). The performance of these cross-validated models was reported as the C-statistic of a univariate Cox model with the risk score as covariate.

The signatures were optimized by stepwise selection, an iterative procedure which serially removes and adds probe sets from a pool of candidates. The procedure was terminated when adding, removing, or replacing a probe set did not further significantly improve the mean leave-one-out cross-validated area under receiver operating curve (AUC; for the stage and subtype signatures) or C-statistic (for the survival signature) over all data sets (see Supplemental data for details). Thus all data sets were used for training and validation. In total, over a million promising signatures were cross-validated in all data sets.

We calculated the recurrence-free survival probabilities in our patients using the postoperative nomogram developed by the International Bladder Cancer Nomogram Consortium (IBCNC; ref. 28). This nomogram, developed from data of more than 9,000 patients, includes age, sex, time from diagnosis to surgery, pathologic tumor stage and grade, tumor histologic subtype, and regional lymph note status. The contribution of our newly curated gene signature for overall survival to the predictive ability of the nomogram was then evaluated. Multivariate modeling with nomogram information applied the leave-one-out cross-validated gene signature risk score as a second covariate. Thus 2 Cox models were used: the first model estimated the risk as described for the Kaplan–Meier analysis using the gene signature alone; the second model combined the nomogram and the signature score. The right endpoint of this second model was set to be recurrence-free survival. Concordance was estimated with the C-statistics approach (22) for a univariate model with the nomogram alone and then for a multivariate model with the addition of the survival gene signature. Prediction models were compared by the likelihood ratio test.

In Fig. 1, we provide an overview of the analyses conducted in this study.

When comparing non–muscle-invasive with muscle-invasive samples in our data set, we found 636 significantly differentially expressed probe sets, which significantly overlapped with the corresponding lists of genes in the 6 independent studies (P < 0.0001, Supplementary Tables S3A, S4, and S5, Supplementary Fig. S1). Fibronectin 1 (FN1), a member of the integrin signaling pathway and several other members or close interaction partners of this pathway were upregulated in muscle-invasive as compared with non–muscle-invasive tumors in most data sets we investigated; these genes include ACTN1, COL1A2, COL3A1, COL5A2, COL6A3, COL11A1, COL16A1, FBN1, FLNA, LUM, TGFBI, and TNC. Furthermore, we found that members of the TGF-β signaling pathway were present in most lists of differentially expressed genes; for example, SMAD3, SMAD6, and BMP7 were overexpressed in non–muscle-invasive as compared with muscle-invasive tumors, whereas INHBA, NNMT, and POSTN were overexpressed in muscle-invasive samples. To further characterize the association of gene expression with pathologic stage, we created a classifier based on consistently differentially expressed genes among all data sets. The optimized classifier (Supplementary Table S6) identified muscle-invasive tumors with a mean accuracy of 89.0% (88.4% SVM, Supplementary Fig. S3) over all data sets.

We then compared the gene expression patterns of patients who developed metastasis (pelvic or distant recurrence) and died of bladder cancer with those of patients who did not. When considering samples of all stages, we identified no significantly differentially expressed genes. However, when restricting this comparison to patients with pathologically organ-confined muscle-invasive (pT2N0) tumors (17 patients, 5 of whom developed metastasis), we identified 53 differentially expressed genes with FDR less than 10% in our data set (P < 0.06, Supplementary Table S3B). In the external data sets, the number of pT2N0 tumors with later metastasis was too small for the identification of significantly differentially expressed genes in this comparison. Among those genes that were differentially expressed in patients who developed metastasis and died were PERP (a TP53 apoptosis factor), ATXN10 (an activator of the Ras-MAPK pathway; ref. 24); and PCM1 (associated with papillary thyroid cancer, chronic myeloid leukemia, and myeloproliferative disorders; ref. 25).

We then evaluated 49 published gene signatures (Supplementary Table S2) alongside our classifiers described earlier for correlation with clinical features in our data set and with the 4 independent gene expression data sets with available survival information (6, 7, 9, 13). Because of the smaller number of genes on the Lindgren and colleagues and Blaveri and colleagues cDNA microarrays, only fractions of these signatures' genes could be mapped. We observed overfitting of published survival signatures so that most signatures achieved significance, in terms of survival information, only in the data sets used to identify them (Table 3). Applying previously published signatures to our cohort, we found no significant association between survival and any signatures. Nevertheless, when we analyzed all genes from all survival and progression signatures in a univariate fashion, several markers that achieved low P values in multiple data sets emerged; for example BST2, HLA-G, ICAM1, and LIMCH1 from the Smith and colleagues signature (14); DGCR2, DSC3, and SCEL from Kim and colleagues (13); UBE2C, VCAM1, and WNT5B from Blaveri and colleagues (6); APOBEC3B from Lindgren and colleagues (9); and ATXN10, C17orf39, and INPP4B from Sanchez-Carbayo and colleagues (7; Supplementary Table S2). Especially striking are mitogen—activated protein (MAP) kinases, with different members present in various signatures (7, 9, 11, 14, 29).

As reported in multiple other studies (6, 7, 9), bladder tumors cluster in 2 very distinct molecular subtypes. We reproduced this finding in all analyzed data sets (Supplementary Fig. S4), and further, found significant differences in survival between the 2 subtypes (Supplementary Fig. S5). The expression differences between the subtypes were remarkable and overlapped significantly across data sets (P < 0.00004, Supplementary Table S5). This allowed a robust classification of tumors by subtype with a novel 19-gene signature (Supplementary Table S7, Supplementary Fig. S6). The very heterogeneous landscape of muscle-invasive bladder tumors motivated us to evaluate the performance of all signatures in the 2 subtypes separately. Two progression signatures, one developed for bladder (6) and one for breast cancer (30), were significantly associated with survival in our cohort when applied to one of the 2 subtypes (Supplementary Fig. S7). By analyzing primary and recurrent tumors from the Kim and colleagues cohort (13), we found evidence that tumors can progress from one molecular subtype to the other (Supplementary Table S8).

We then curated a new 20-gene overall survival signature for muscle-invasive tumors (Table 4) by stepwise optimization and tested its predictive accuracy in our data set and the 4 external data sets with available survival information. A leave-one-out cross-validated multivariate Cox proportional hazards model based on this signature classified tumors into 2 equally sized risk groups with significantly different survival in all data sets (Fig. 2A–E). As an example, with the model applied to the data set by Kim and colleagues (13), there was a 5-fold increase in the risk of death in those classified as high-risk [HR: 5.05; 95% confidence interval (CI), 2.26–11.3; P < 0.008] compared with those classified as low risk. See Supplementary Figs. S8 and S9 for details of the signature. Neoadjuvant chemotherapy, administered to 3 patients in our cohort, had no impact on the prediction accuracies (Supplementary Table S9). We next compared the prediction accuracies of the signature with pathologic stage and grade in all cohorts and found highly significant improvements of model concordance and likelihood in all data sets (mean ΔC-statistic: 0.14; 95% CI, 0.01–0.27; likelihood ratio test, P < 0.001; Fig. 2F, Supplementary Table S10B).

We then incorporated the new signature developed for muscle-invasive tumors (Table 4) into the IBCNC nomogram (28), which was designed to predict 5-year recurrence-free survival after radical cystectomy. Necessary data points for the nomogram were not available in the external data sets. When tested in our muscle-invasive patient cohort, a multivariate Cox model predicting recurrence-free survival with both nomogram and gene signature score (C-statistic: 0.66; 95% CI, 0.56–0.76) was a significant improvement (ΔC-statistic: 0.08; 95% CI, −0.04–0.20; P < 0.005, likelihood ratio test) over a model based on the nomogram score alone (C-statistic: 0.58; 95% CI, 0.43–0.72). As the signature was designed for overall survival due to the lack of recurrence dates in the independent data, the prediction accuracies increased profoundly in a model predicting overall survival (ΔC-statistic: 0.15; 95% CI, 0.07–0.23; Supplementary Table S10C–D).

This study shows the utility of adding molecular information to an existing prognostic tool in patients with bladder cancer who undergo radical cystectomy. Gene expression data from a new cohort of patients with bladder cancer was analyzed and compared with gene expression data from multiple published studies. A robust expression signature was identified that improved the predictive outcome of a well-accepted nomogram and was independently predictive of survival in all cohorts where survival data were available. These findings are particularly striking given the relative homogeneity of the population analyzed, as our cohort, having all undergone the most aggressive surgical therapy, is highly selected for being at high risk of death from disease.

One important contribution of this study is the clinical validation of published gene signatures. To be clinically relevant, new survival markers must (i) stratify patients in groups with significantly different survival; (ii) deliver survival information that is not already included in established clinically used predictors such as grade and stage; and (iii) increase the accuracies of existing prediction models to an extent that warrants the cost and effort of obtaining biomarker status (31). Previously published gene signatures (Supplementary Table S2), however, did poorly in our patient cohort; no gene signature provided more survival information than would be expected by chance. This finding can partly be explained by cross-platform issues like incomplete mapping of genes to available probe sets or probe sets targeting different exons. Another reason is a likely overfitting of published signatures due to rather small training data sets for the very heterogeneous group of muscle-invasive bladder cancers. To circumvent these limitations, we optimized the survival signatures for the subgroup of muscle-invasive tumors, and showed that gene expression clearly stratifies patients into 2 groups, denoted as low-risk and high-risk. The new signature further segregated by risk 305 patients with muscle-invasive tumors in the external data sets containing gene expression and survival information. Interestingly, 4 genes in our muscle-invasive survival signature, APOBEC3B, DGCR2, PRMT1, SLC1A5, are located on chromosomes 22 and 19, in neighboring chromosome bands of single-nucleotide polymorphisms (SNP) recently described as associated with an increased risk of developing bladder cancer (32). In addition to our signature, several other genes (Supplementary Table S2) significantly correlated with survival in multiple independent cohorts. These genes identified are potential therapeutic targets and worthy of further investigation to understand their role in bladder cancer. For example, inhibitors of MAP2K1 (i.e., MEK1), which is part of our overall survival signature (Table 4), are currently being evaluated in several clinical trials and our results provide a rationale for this in bladder cancer. Our screen for potential biomarkers in multiple data sets thus also has important applications in gene selection for technologies such as NanoString, which can target only a limited number of genes.

To place our signature in the context of currently known clinical data, we applied the survival signature to a robust nomogram that predicts outcome after radical cystectomy (28). This postoperative nomogram was shown to have a significantly better predictive accuracy (concordance index, 0.75) than standard tumor-node-metastasis (TNM; concordance index, 0.68; P < 0.001) or pathologic (concordance index, 0.62; P < 0.001) subgroupings. When this model was applied to our patient cohort with the addition of our newly developed survival signature, we observed statistically significant improvements in the prediction of risk of death as compared with the use of the nomogram only. As the signature reflects unique molecular features associated with risk, its addition to the nomogram thus provides more insights into the biologic behavior of a malignancy of patient, and leads to improved prognostication as compared with the use of the nomogram alone. A more accurate prediction of progression may help select the higher-risk patients for additional treatment, such as adjuvant systemic therapy after surgery. External validation of these findings is ongoing.

In addition, our analysis led to the identification of 53 genes significantly associated with the development of metastasis and death of patients with pathologically organ-confined muscle-invasive disease (pT2N0). This list of genes establishes a molecular signature of less favorable outcome, which may help stratify patients in this identically staged subgroup of bladder tumors to more or less intensive multimodal therapy. Patients with this stage of disease are typically followed expectantly, but evaluation of gene expression data or testing for downstream products of expression may identify those patients at higher risk for progression who may benefit from systemic therapy after radical surgery. The investigation of this list in a prospective collection of similar cystectomy samples is essential. An interesting additional opportunity for evaluation would be among patients with muscle-invasive disease at the time of TUR—by characterizing a greater risk of aggressive behavior, we may more carefully select patients for chemotherapy prior to radical surgery as well, to improve our survival outcomes using multimodal therapy.

We found that FN1 was significantly overexpressed in muscle-invasive tumors as compared with non–muscle-invasive samples in all data sets we analyzed. The role of FN1 in cancer invasion and metastasis is still poorly understood, but its increased expression might be the result of an enhanced recruitment of cancer-associated fibroblasts (33, 34). The consistency across data sets suggests that the abundant differential expression of FN1 and other extracellular matrix (ECM) genes is unlikely due to stromal contamination. Our stage classifier (Supplementary Table S6) could identify patients as having muscle-invasive disease with a mean accuracy of 89%, which is within the range of previously published classifiers (6, 24). However, our stage classifier was validated in more data sets and consists of fewer genes than the published classifiers, which offer advantages for its potential clinical use. Furthermore, the accuracy rate of 89% does not imply a false classification rate of 11% as some tumors might have been incorrectly classified histologically. Urothelial cancer of the bladder is exceptionally difficult to stage accurately prior to cystectomy, given technical limitations of endoscopic resection and imaging. Significant rates of upstaging from time of TUR to radical cystectomy have been reported, ranging from 31% to 61% (35–37). In our patient cohort, more than 80% of the tumors were determined to have higher pathologic stage at the time of cystectomy, when compared with the preoperative TUR specimens, with many tumors found to be locally advanced (>pT2) when clinically staged as organ confined (≤pT2).

The unsupervised clustering produced 2 strikingly different groups of patients, who were not clearly distinguishable by many clinical variables such as the presence of lymph node metastases or recurrence status. Gene expression showed large differences between the 2 groups (Supplementary Table S3C) with highly significant overlaps in all data sets (Supplementary Table S5), allowing a robust identification of cluster membership by a 19-gene signature (Supplementary Table S7). Lindgren and colleagues (9) similarly identified 2 distinct molecular subtypes (denoted as MS1 and MS2) by analyzing 144 tumor samples obtained by transurethral bladder biopsy, representing a wide range of tumor stage and grade. Their investigation, which included whole-genome and specific mutational analyses, identified a greater number of genomic alterations in the MS2 group, containing nearly all of the muscle-invasive samples. The genomic instability in the MS2 group was also associated with expression of genes involved in cell-cycle pathways and cellular transformation (9), all indicative of more aggressive behavior and worse prognosis. Consistent with their results, we found a progression from MS1 to MS2 to be more likely than a progression from MS2 to MS1 when analyzing 23 recurrent tissue samples from Kim and colleagues (13). While only stage had a significant association with subtype, other covariates not available in this study, for example response to treatments, could be tested for association in prospective data. Not surprisingly, survival signatures often showed very different prediction accuracies in the 2 subtypes. This heterogeneity of bladder cancer tumors thus points to the necessity of large training data to establish robust signatures. Interestingly, from all 49 considered previously published signatures, a breast cancer progression signature (30) showed the highest association with overall survival in the MS2 group of our patient cohort (Supplementary Fig. S7A). This finding suggests that optimizing thoroughly validated signatures designed for other solid tumors might yield more robust signatures than developing new signatures from scratch, in particular when only small training data sets are available. The identification of different molecular signatures for subtype suggests that these subtypes, which are strongly associated with invasion status, may be a building block for future work to noninvasively determine the aggressiveness of tumors; for example, these changes in expression may be detectable in urine.

Our data set represents a high-risk population of patients with bladder cancer who underwent radical cystectomy at a high-volume academic center. The sample size is large for a single-center report of microarray analysis and comprises, to our knowledge, the largest number of high-risk patients with bladder cancer of all published microarray data sets. One limitation of our findings is, however, a possible element of overfitting, resulting from the use of the same data in testing and validation steps, although this concern is mitigated through the use of statistical methods such as cross-validation. This points to the imperative for validation in independent data of any gene signature associated with survival. Such a validation is necessary because signatures established from single, moderately sized data sets often did poorly in external data due to the relatively high probability that genes correlate with survival by chance. However, extending the sample size with independent data, as carried out here, can minimize this problem. For example, a gene found to be predictive in one data set could arise due to chance, but if one gene is predictive with consistent expression in multiple data sets, it is more likely to be a robust survival marker.

A considerable strength of our investigation is its comprehensive effort to combine the majority of published data on gene expression in bladder cancer, all arrayed on different platforms, in a meaningful way. With this study, we have shown that meta-analyses across platforms are feasible. Especially in rare cancers or subtypes, the increase in sample size might often outweigh the disadvantages of meta-analyses, such as the heterogeneity of patient cohorts, the different treatments before and after surgery, and the decrease in potential markers when focusing on genes present on all platforms. Nonetheless, large independent patients cohorts are needed for further validation and optimization (i.e., for other endpoints such as recurrence-free survival or for particular microarray platforms) of the prognostic signatures, before warranting a clinical trial (38). The results of this study strongly motivate such sample collection for urothelial tumors.

Our gene expression analysis identifies novel signatures predictive of tumor stage, progression among patients with organ-confined disease, molecular subtypes of tumors, and overall survival. We further incorporated 6 published gene expression data sets for a large-scale comparison and validation of predictive signatures and found a significant increase in predictive accuracy of a postoperative nomogram by addition of our gene signature. Multiple genes identified across several sets have emerged as highly promising candidates for further investigation. The identification of patients at higher risk for progression or death will provide additional rationale for multimodality management, using chemotherapy and surgery, in an effort to improve survival.

No potential conflicts of interest were disclosed.

The authors thank Parantu Shah, Dhruv Sharma, Levi Waldron, Marta Sanchez-Carbayo, Mithat Gönen, Samir Amin, Yi Li, and the Michor laboratory.

This work was supported from the NCI initiative to found Physical Science Oncology Centers (U54CA143798; M. Riester, F. Michor) and an NRSA training grant T32-CA82088 from the NIH (J.M. Taylor).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.