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BACKGROUND: Several molecular classification systems for bladder cancer have been proposed, but due to differences on how to define molecular subtypes, controversies and misunderstandings have arisen. OBJECTIVE: To discuss different aspects of the molecular classification of bladder cancer and to point to the consequences of using different conceptual approaches. To question some underlying assumptions when defining molecular subtypes. METHODS: To critically reflect on some of the principles and methods used when defining molecular subtypes. RESULTS: Depending on underlying assumptions and aims for the definitions of subtypes, different types of molecular subtypes will be arrived at. CONCLUSION: The underlying assumptions and their consequences must be better clarified when defining molecular subtypes.

The precise classification of tumors into relevant molecular subtypes will facilitate both future research and optimal treatment. Here, the Lund Taxonomy system for molecular classification of urothelial carcinoma was applied to two large and independent cohorts of non-muscle-invasive tumors. Of 752 tumors classified, close to 100% were of the luminal subtypes, 95% urothelial-like (Uro; UroA, UroB, or UroC) and 5% genomically unstable. The obtained subtype structure organized the tumors into groups with specific and coherent gene mutation, genomic, and clinical profiles. The intrasubtype variability in the largest group of tumors, UroA, was caused by infiltration and proliferation, not considered as cancer cell type-defining properties. Within the UroA subtype, a HOXB/late cell-cycle gene expression polarity was found, strongly associated with FGFR3, STAG2, and TP53 mutations, as well as with chromosome 9 losses. Kaplan-Meier analyses identified the genomically unstable subtype as a progression high-risk group, also valid in the subgroup of T1 tumors. Almost all progression events occurred within 12 months in this subtype. Also, a general progression gene signature was derived that identifies high- and low-risk tumors. All findings were demonstrated in two independent cohorts. The Lund Taxonomy system is applicable to both non-muscle- and muscle-invasive tumors and may be a useful biological framework for translational studies.

Background For muscle-invasive bladder cancer (MIBC), no tissue biomarkers are available for clinical use to predict response to neoadjuvant chemotherapy. Objective To investigate how molecular subtypes impact pathological response and survival in patients receiving preoperative cisplatin-based chemotherapy. Design, setting, and participants Classification of a retrospective cohort of 149 patients was performed by tumor transcriptomic profiling and immunostaining. A cohort treated with radical cystectomy alone and public data sets were used for comparison and external validation. Outcome measurements and statistical analysis Complete pathological response in the cystectomy specimen (ypT0N0) and survival were compared in predefined molecular subtypes. Differential gene expression and chemotherapy response were explored beyond molecular subtypes. Results and limitations Patients with genomically unstable (GU) and urothelial-like (Uro) tumors had higher proportions of complete pathological response (16/31 [52%] and 17/54 [31%]), versus five out of 24 (21%) with the basal/squamous (Ba/Sq) subtype following neoadjuvant chemotherapy and radical cystectomy. Molecular subtype was independently associated with improved survival for patients with GU tumors (hazard ratio [HR] 0.29, 95% confidence interval [CI]: 0.11–0.79) and UroC tumors (HR 0.37, 95% CI: 0.14–0.94) compared with Ba/Sq tumors, adjusting for clinical stage. In addition, expression of the gene coding for osteopontin (SPP1) showed a subtype-dependent effect on chemotherapy response. Conclusions Urothelial cancer of the luminal-like (GU and Uro) subtypes is more responsive to cisplatin-based neoadjuvant chemotherapy. A second-generation of subtype-specific biomarkers, for example, SPP1, may be a way forward to develop a more precision-based treatment approach for neoadjuvant chemotherapy in MIBC. Patient summary This study shows that tumor classification by gene expression profiling and molecular subtyping can identify patients who are more likely to benefit from chemotherapy before radical cystectomy for muscle-invasive bladder cancer. Together with other markers for response, molecular subtypes could have a role in selective administration of such chemotherapy.

Supplementary Table S3.

XLS file - 193K, Patient and sample data

PDF file - 51K, Univariate analyses in the whole cohort and in melanomas with a Breslow thickness > 2mm

Purpose: For primary melanomas, tumor thickness, mitotic rate, and ulceration are well-laid cornerstones of prognostication. However, a molecular exposition of melanoma aggressiveness is critically missing. We recently uncovered a four-class structure in metastatic melanoma, which predicts outcome and informs biology. This raises the possibility that a molecular structure exists even in the early stages of melanoma and that molecular determinants could underlie histophenotype and eventual patient outcome.Experimental Design: We subjected 223 archival primary melanomas to a horizontally integrated analysis of RNA expression, oncogenic mutations at 238 lesions, histomorphometry, and survival data.Results: Our previously described four-class structure that was elucidated in metastatic lesions was evident within the expression space of primary melanomas. Because these subclasses converged into two larger prognostic and phenotypic groups, we used the metastatic lesions to develop a binary subtype-based signature capable of distinguishing between “high” and “low” grade forms of the disease. The two-grade signature was subsequently applied to the primary melanomas. Compared with low-grade tumors, high-grade primary melanomas were significantly associated with increased tumor thickness, mitotic rate, ulceration (all P < 0.01), and poorer relapse-free (HR = 4.94; 95% CI, 2.84–8.59), and overall (HR = 3.66; 95% CI, 2.40–5.58) survival. High-grade melanomas exhibited elevated levels of proliferation and BRCA1/DNA damage signaling genes, whereas low-grade lesions harbored higher expression of immune genes. Importantly, the molecular-grade signature was validated in two external gene expression data sets.Conclusions: We provide evidence for a molecular organization within melanomas, which is preserved across all stages of disease. Clin Cancer Res; 18(15); 4026–36. ©2012 AACR.

PDF file - 425K, Clusters of genes with coordinated expression across samples

PDF file - 79K, Significant canonical pathways (p

Purpose: Chromosomal instability (CIN) is believed to have an important role in the pathogenesis of urothelial cancer (UC). The aim of this study was to evaluate whether disturbances of mitotic segregation contribute to CIN in UC, if these processes have any effect on the course of disease, and how deregulation of these mechanisms affects tumor cell growth.Experimental Design: We developed molecular cytogenetic methods to classify mitotic segregation abnormalities in a panel of UC cell lines. Mitotic instabilities were then scored in biopsies from 52 UC patients and compared with the outcome of tumor disease. Finally, UC cells were exposed in vitro to a telomerase inhibitor to assess how this affects mitotic stability and cell proliferation.Results: Three distinct chromosome segregation abnormalities were identified: (a) telomere dysfunction, which triggers structural rearrangements and loss of chromosomes through anaphase bridging; (b) sister chromatid nondisjunction, which generates discrete chromosomal copy number variations; and (c) supernumerary centrosomes, which cause dramatic shifts in chromosome copy number through multipolar cell division. Chromosome segregation errors were already present in preinvasive tumors and a high rate mitotic instability was an independent predictor of poor survival. However, induction of even higher levels of the same segregation abnormalities in UC cells by telomerase inhibition in vitro led to reduced tumor cell proliferation and clonogenic survival.Conclusion: Several distinct chromosome segregation errors contribute to CIN in UC, and the rate of such mitotic errors has a significant effect on the clinical course. Efficient tumor cell proliferation may depend on the tight endogenous control of these processes.

Purpose: To evaluate performances of published gene signatures for the assessment of urothelial carcinoma.Experimental Design: We evaluated 28 published gene signatures designed for diagnostic and prognostic purposes of urothelial cancer. The investigated signatures include eight signatures for stage, five for grade, four for progression, and six for survival. We used two algorithms for classification, nearest centroid classification and support vector machine, and Cox regression to evaluate signature performance in four independent data sets.Results: The overlap of genes among the signatures was low, ranging from 11% among stage signatures to 0.6% among survival signatures. The published signatures predicted muscle-invasive and high-grade tumors with accuracies in the range of 70% to 90%. The performance for a given signature varied considerably with the validation data set used, and interestingly, some of the best performing signatures were not designed for the tested classification problem. In addition, several nonbladder-derived gene signatures performed equally well. Large randomly selected gene signatures performed better than the published signatures, and by systematically increasing signature size, we show that signatures with >150 genes are needed to obtain robust performance in independent validation data sets. None of the published survival signatures performed better than random assignments when applied to independent validation data.Conclusion: We conclude that gene expression signatures with >150 genes predict muscle-invasive growth and high-grade tumors with robust accuracies. Special considerations have to be taken when designing gene signatures for outcome in bladder cancer. Clin Cancer Res; 16(17); 4421–33. ©2010 AACR.

PDF file - 91K, Flow chart describing the analysis

Supplementary Figure S1 from Distinct Mitotic Segregation Errors Mediate Chromosomal Instability in Aggressive Urothelial Cancers

PDF file - 392K, Expression of cell adhesion genes

Supplementary Tables S1-S2 from Distinct Mitotic Segregation Errors Mediate Chromosomal Instability in Aggressive Urothelial Cancers

PDF file - 48K, Multi�class SAM analysis revealed 503 probes which were found to significantly differ between the four subtypes in GSE22155 at FDR=0

PDF file - 95K, The expression values for these genes were averaged across GSE22155 samples in the two classes thereby providing us with two distinct centroids

PDF file - 116K, Among the set of 36 BRAF-mutated cases, high-grade tumors showed worse survival than low-grade lesions

Purpose: Even though urothelial cancer is the fourth most common tumor type among males, progress in treatment has been scarce. A problem in day-to-day clinical practice is that precise assessment of individual tumors is still fairly uncertain; consequently efforts have been undertaken to complement tumor evaluation with molecular biomarkers. An extension of this approach would be to base tumor classification primarily on molecular features. Here, we present a molecular taxonomy for urothelial carcinoma based on integrated genomics.Experimental Design: We use gene expression profiles from 308 tumor cases to define five major urothelial carcinoma subtypes: urobasal A, genomically unstable, urobasal B, squamous cell carcinoma like, and an infiltrated class of tumors. Tumor subtypes were validated in three independent publically available data sets. The expression of 11 key genes was validated at the protein level by immunohistochemistry.Results: The subtypes show distinct clinical outcomes and differ with respect to expression of cell-cycle genes, receptor tyrosine kinases particularly FGFR3, ERBB2, and EGFR, cytokeratins, and cell adhesion genes, as well as with respect to FGFR3, PIK3CA, and TP53 mutation frequency. The molecular subtypes cut across pathologic classification, and class-defining gene signatures show coordinated expression irrespective of pathologic stage and grade, suggesting the molecular phenotypes as intrinsic properties of the tumors. Available data indicate that susceptibility to specific drugs is more likely to be associated with the molecular stratification than with pathologic classification.Conclusions: We anticipate that the molecular taxonomy will be useful in future clinical investigations. Clin Cancer Res; 18(12); 3377–86. ©2012 AACR.

XLS file - 22K, Summary of patient and sample data

PDF file - 1.8MB, Validation of tumor clusters in external data sets

PDF file - 95K

PDF file - 199K

Supplementary Figures S1-S4.

Supplementary Table 3 from Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

Supplementary Table 8 from Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

Supplementary Table 6 from Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

Supplementary Figure Legends 1-6 from Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

Supplementary Table 2 from Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

Supplementary Table 7 from Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

Supplementary Table 5 from Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

Supplementary Table 2 from Genome-Wide Array-Based Comparative Genomic Hybridization Reveals Multiple Amplification Targets and Novel Homozygous Deletions in Pancreatic Carcinoma Cell Lines

Supplementary Table 1 from Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

Pancreatic carcinomas display highly complex chromosomal abnormalities, including many structural and numerical aberrations. There is ample evidence indicating that some of these abnormalities, such as recurrent amplifications and homozygous deletions, contribute to tumorigenesis by altering expression levels of critical oncogenes and tumor suppressor genes. To increase the understanding of gene copy number changes in pancreatic carcinomas and to identify key amplification/deletion targets, we applied genome-wide array-based comparative genomic hybridization to 31 pancreatic carcinoma cell lines. Two different microarrays were used, one containing 3,565 fluorescence in situ hybridization-verified bacterial artificial chromosome clones and one containing 25,468 cDNA clones representing 17,494 UniGene clusters. Overall, the analyses revealed a high genomic complexity, with several copy number changes detected in each case. Specifically, 60 amplicons at 32 different locations were identified, most frequently located within 8q (8 cases), 12p (7 cases), 7q (5 cases), 18q (5 cases), 19q (5 cases), 6p (4 cases), and 8p (4 cases). Amplifications of 8q and 12p were mainly clustered at 8q23–24 and 12p11–12, respectively, whereas amplifications on other chromosome arms were more dispersed. Furthermore, our analyses identified several novel homozygously deleted segments located to 9p24, 9p21, 9q32, 10p12, 10q22, 12q24, and 18q23. The individual complexity and aberration patterns varied substantially among cases, i.e., some cell lines were characterized mainly by high-level amplifications, whereas others showed primarily whole-arm imbalances and homozygous deletions. The described amplification and deletion targets are likely to contain genes important in pancreatic tumorigenesis.

Supplementary Table 4 from Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

Supplementary Figures 1-6 from Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

Several groups have during past years produced molecular classification schemes for bladder cancer. Even though no consensus on how to define a subtype exists, one approach has been to base definitions on how tumours cluster according to their mRNA expression profiles. In many cases, obtained profiles, and thus class defining features, are affected by signals from non-tumour cells within the biopsy. To overcome this issue, we combined gene expression analyses with analyses of the actual tumour cells by extensive immunohistochemistry (IHC). By this approach we were able to define tumour cell phenotypes i.e., subtypes defined by features of the tumour cells only, and adjust mRNA-based algorithms accordingly. In the present investigation we address the non-luminal Basal/Squamous-like (Ba/Sq) and Small cell/Neuroendocrine-like (Sc/NE) categories of tumours defined by mRNA-based classification. We make use of IHC data for 15 proteins, all known to be instrumental for defining molecular subtypes of urothelial carcinoma. We show that the UroB type of tumours, frequently grouped together with Ba/Sq, are different from the Ba/Sq entity at several essential features and is a derivative of Urothelial-like tumours (Uro). We show that the Sc/NE tumours are similar to but represents extreme versions of Genomically Unstable (GU) tumours. We apply clustering to 423 cases representing all subtypes using IHC data for 14 proteins and show that the obtained grouping conforms well with the mRNA-based classification. This work describes in detail the molecular pathology of non-luminal RNA-based bladder cancer subtypes and highlight similarities/dissimilarities suggestive of origin.