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Summary: Sarcomatoid urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARC and 84 cases of conventional urothelial carcinoma (UC), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs show a distinct mutational landscape, with enrichment of TP53, RB1, and PIK3CA mutations. They are related to the basal molecular subtype of conventional UCs and could be divided into epithelial-basal and more clinically aggressive mesenchymal subsets on the basis of TP63 and its target gene expression levels. Other analyses reveal that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of the EMT network, and nearly half exhibit a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer. : Guo et al. report that sarcomatoid carcinoma of the bladder evolves by the progression of the basal subtype of conventional urothelial carcinoma with the enrichment of mutagenesis signature 1 and mutations of TP53, RB1, and PIK3CA. It is driven by the dysregulation of the EMT network and shows increased immune infiltrate with overexpression of PD-L1. Keywords: bladder cancer, sarcomatoid carcinoma, urothelial carcinoma, epithelial-mesenchymal transformation, genomic expression, chromatin remodeling, microRNA expression, molecular classification, basal subtype, immune phenotype

Genomic profiling studies have demonstrated that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to frontline chemotherapy. We analyzed the mRNA expressions of signature luminal and basal genes in bladder cancer tumor samples from publicly available and MD Anderson Cancer Center cohorts. We developed a quantitative classifier referred to as basal to luminal transition (BLT) score which identified the molecular subtypes of bladder cancer with 80–94% sensitivity and 83–93% specificity. In order to facilitate molecular subtyping of bladder cancer in primary care centers, we analyzed the protein expressions of signature luminal (GATA3) and basal (KRT5/6) markers by immunohistochemistry, which identified molecular subtypes in over 80% of the cases. In conclusion, we provide a tool for assessment of molecular subtypes of bladder cancer in routine clinical practice.

Summary We report a comprehensive molecular analysis of 34 cases of small cell carcinoma (SCC) and 84 cases of conventional urothelial carcinoma (UC), with The Cancer Genome Atlas cohort of 408 conventional UC bladder cancers used as the reference. SCCs showed mutational landscapes characterized by nearly uniform inactivation of TP53 and were dominated by Sanger mutation signature 3 associated with loss of BRCA1/2 function. SCCs were characterized by downregulation of luminal and basal markers and were referred to as double-negative. Transcriptome analyses indicated that SCCs displayed lineage plasticity driven by a urothelial-to-neural phenotypic switch with a dysregulated epithelial-to-mesenchymal transition network. SCCs were depleted of immune cells, and expressed high levels of the immune checkpoint receptor, adenosine receptor A2A (ADORA2A), which is a potent inhibitor of immune infiltration. Our observations have important implications for the prognostication and development of more effective therapies for this lethal bladder cancer variant., Graphical Abstract, Highlights • SCCs show TP53/RB1 loss with mutational signature of BRCA1/2 loss of function • SCCs are driven by neural phenotypic switch with dysregulated EMT network • SCCs show depleted immune phenotype with upregulation of ADORA2A, Biological Sciences; Genomics; Cancer Systems Biology; Cancer; Transcriptomics

Background It has been suggested that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to chemotherapy. We aimed to validate these subtypes in several clinical cohorts and identify signature immunohistochemical markers that would permit simple and cost-effective classification of the disease in primary care centers. Methods We analyzed genomic expression profiles of bladder cancer in three cohorts of fresh frozen tumor samples: MD Anderson (n = 132), Lund (n = 308), and The Cancer Genome Atlas (TCGA) (n = 408) to validate the expression signatures of luminal and basal subtypes and relate them to clinical follow-up data. We also used an MD Anderson cohort of archival bladder tumor samples (n = 89) and a parallel tissue microarray to identify immunohistochemical markers that permitted the molecular classification of bladder cancer. Findings Bladder cancers could be assigned to two candidate intrinsic molecular subtypes referred to here as luminal and basal in all of the datasets analyzed. Luminal tumors were characterized by the expression signature similar to the intermediate/superficial layers of normal urothelium. They showed the upregulation of PPARγ target genes and the enrichment for FGFR3, ELF3, CDKN1A, and TSC1 mutations. In addition, luminal tumors were characterized by the overexpression of E-Cadherin, HER2/3, Rab-25, and Src. Basal tumors showed the expression signature similar to the basal layer of normal urothelium. They showed the upregulation of p63 target genes, the enrichment for TP53 and RB1 mutations, and overexpression of CD49, Cyclin B1, and EGFR. Survival analyses showed that the muscle-invasive basal bladder cancers were more aggressive when compared to luminal cancers. The immunohistochemical expressions of only two markers, luminal (GATA3) and basal (KRT5/6), were sufficient to identify the molecular subtypes of bladder cancer with over 90% accuracy. Interpretation The molecular subtypes of bladder cancer have distinct clinical behaviors and sensitivities to chemotherapy, and a simple two-marker immunohistochemical classifier can be used for prognostic and therapeutic stratification. Funding U.S. National Cancer Institute and National Institute of Health., Highlights • Bladder cancer can be assigned to two intrinsic molecular subtypes referred to as luminal and basal. • Luminal tumors are characterized by the expression signature of intermediate/superficial layers of normal urothelium and they show up-regulation of PARγ target genes with enrichment for FGFR3, ELF3, CDKN1A, and TSC1 mutations. • Basal tumors show the expression signature similar to the basal layer of normal urothelium and they show the up-regulation of p63 target genes with the enrichment of TP53 and RB1 mutations. • Muscle invasive tumors of basal type are more aggressive when compared to luminal cancers. • The immunohistochemical expression of only two markers, luminal (GATA3) and basal (KRT5/6) is sufficient to identify the molecular subtypes of bladder cancer with over 90% accuracy. Our analyses of several large cohorts comprising of a total of 937 bladder cancer tumors samples with annotated clinical data showed that bladder cancers can be classified into molecular subtypes referred to as luminal and basal. Luminal tumors are similar in their gene expression pattern to more differentiated intermediate and superficial layers of normal urothelium. Basal cancers recapitulate the gene expression signature of the less differentiated basal layer of the urothelium. These molecular subtypes have distinct up-regulation of transcription factors that regulate the expression pattern of their target genes and are enriched for specific mutations. Basal tumors which deeply invade the bladder wall are more aggressive when compared to luminal types and they show distinct sensitivities to frontline chemotherapy. The classification of bladder cancer into its intrinsic molecular subtypes can be accomplished with two immunohistochemical markers such as GATA3 for luminal tumors and KRT5/6 for basal tumors which can be performed in primary care centers.

SUMMARYThe sarcomatoid variant of urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARCs and 84 cases of conventional urothelial carcinomas (UCs), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs showed a distinct mutational landscape with enrichment ofTP53, RB1, and PIK3CAmutations. They were related to the basal molecular subtype of conventional UCs and could be divided into epithelial/basal and more clinically aggressive mesenchymal subsets based on TP63 and its target genes expression levels. Other analyses revealed that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of cell cycle and EMT networks, and nearly half exhibited a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer.

Background Bladder cancer is among the common human malignancies that show a heavy mutational load and copy number variations of numerous chromosomes, which makes them a target for diagnostic explorations. Objective We aimed to design a multicolor fluorescence in situ hybridization (FISH) test referred to as the quartet test for the detection of bladder cancer in urine. Design, setting, and participants We performed genome-wide copy number variation analysis on cohorts from the University of Texas MD Anderson Cancer Center ( n =40) and The Cancer Genome Atlas ( n =129), and identified the most frequently amplified chromosomal regions. These data were used to select four of the amplified regions to design a multicolor FISH test, referred to as the quartet test. Assay validation was performed on urine samples from 98 patients with bladder cancer: 56 with low-grade papillary, 42 with high-grade invasive disease, and 48 benign controls. Intervention The quartet test can be used in clinical practice for noninvasive detection of bladder cancer. Outcome measurements and statistical analysis We initially analyzed samples using a fraction of abnormal cell scores and then by the quantitative score, which included not only the proportion of cells with abnormal copy numbers, but also the proportion of cells with numbers of altered copies and degree of amplification. We used receiver operator characteristic (ROC) curves to identify cutoff values for the scores at which performances of sensitivity and specificity were maximized. Results and limitations The copy number status assessed by probes detected in voided urine reflected the amplification status of the primary tumor. An ROC curve summarizing the proportion of assayed cells with any abnormal copy numbers gave specificity of 93.8% and sensitivity of 78.6% using the proportion of cells with abnormal copy numbers. The quantitative score giving extra weight to cells with multiple simultaneous amplifications provided 95.8% specificity and 76.8% sensitivity. Both percentage of abnormal cells and quantitative scores were highly effective for assessing the grade of the tumor. The full spectrum of potential clinical applications was not explored in the current study, and further validation studies are needed. Conclusions The quartet test shows promising specificity and sensitivity results, but it requires validation on a larger multi-institutional cohort of samples. Patient summary The quartet test can be used for noninvasive detection of bladder cancer in voided urine. It can also be used to assess the grade of the tumor and tumor recurrence as well as post-treatment effects.

We developed a whole organ mapping approach that combines microscopic histological assessment of the entire mucosal membrane of the bladder with comprehensive genomic profiling. We used the approach to study the locoregional molecular changes in a human bladder containing multi-focal cancer. Uniform and widespread DNA methylation changes were identified in almost the entire mucosa representing the initial field effect. The field effect was associated with sub-clonal low allele frequency mutations and a small numer of uniform DNA copy number alterations that were typically restricted to small areas. An inactivating founder mutation in the pro-apoptotic and RNA splicing gene, ACIN1, was identified in normal appearing mucosa and expanded clonally with an additional 21 mutations in the development of in situ precursor lesions, and progression to carcinoma. The patterns of mutations and copy number changes in carcinoma in situ and several foci of carcinoma were almost identical, confirming their clonal origins. Importantly, the pathways affected by the CNVs and DNA mutations, including the Kras pathway, appeared to be preceded by the field changes in DNA methylation, suggesting that the DNA-based changes reinforced mechanisms that had already been initiated by methylation. Overall, the results demonstrate that field changes in DNA methylation can serve as the initiators of carcinogenesis in the bladder.

Summary: We used whole-organ mapping to study the locoregional molecular changes in a human bladder containing multifocal cancer. Widespread DNA methylation changes were identified in the entire mucosa, representing the initial field effect. The field effect was associated with subclonal low-allele frequency mutations and a small number of DNA copy alterations. A founder mutation in the RNA splicing gene, ACIN1, was identified in normal mucosa and expanded clonally with an additional 21 mutations in progression to carcinoma. The patterns of mutations and copy number changes in carcinoma in situ and foci of carcinoma were almost identical, confirming their clonal origins. The pathways affected by the DNA copy alterations and mutations, including the Kras pathway, were preceded by the field changes in DNA methylation, suggesting that they reinforced mechanisms that had already been initiated by methylation. The results demonstrate that DNA methylation can serve as the initiator of bladder carcinogenesis. : Majewski et al. report that methylation changes suppressing innate immunity and dysregulating Ras-related pathways initiate bladder carcinogenesis. Keywords: Whole-organ map, bladder cancer, DNA methylation, DNA copy alterations, founder mutation, field effect, clonal origins, clonal expansion, urothelial carcinoma, gene signature

Bladder Cancer is one of the most frequent human cancer which develops on two tracks, papillary and non-papillary that correspond to clinically different forms of the disease. Most bladder cancers are chemically induced with tobacco smoking being the leading risk factor. Recent advances of bladder cancer research for which our laboratory has significantly contributed have enhanced the understanding of the origin of bladder cancer from urothelial progenitor cells via field effects along papillary/luminal non-papillary/basal pathways. Here we present the contribution to the development of novel, both diagnostic and therapeutic, biomarkers using genomic high throughput technologies. The analysis of hits associated with growth advantage of preneoplastic lesions allowed us to identify six chromosomal regions mapping 3q22, 5q22.2-5q23.3, 9q22.12, 10q26.1, 13q14 and 17p13 that may be critical for the development of bladder cancer and contain a distinct class of genes referred to us forerunner (FR) genes involved in the clonal expansion of precursor conditions. We concentrated our efforts on one of these regions; which contain a model tumor suppressor RB1. We used high-resolution whole-organ mapping with SNPs, which facilitated the identification of five positional candidate FR genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11) mapping contiguously to RB1. We hypothesized that identification of novel forerunner genes and the investigation in early occult phases of human bladder preneoplasia may not only provide important mechanistic clues to the development of human cancer but also identify a novel class of early detection markers capable of detecting the clinically and microscopically occult phases of human cancer development. We used the whole-organ histologic and genetic mapping strategy coupled with next generation whole exome sequencing using complemented with genotyping using Illumina HumanOmni2.5_8 chips and whole genome methylation analyses to define the molecular alterations that were associated with the progression of muscle-invasive bladder cancer in one surgical specimen. We identified a total of 18 structurally significant mutations, three of which appeared to be clear "driver" mutations that had been annotated previously. Alterations in chromatin hyper- and hypomethylation predominated in all of the regions that contained normal-appearing urothelium or low-grade dysplasia. In total, 31 genes were hypermethylated and 26 genes were hypomethylated. Copy number alterations were numerous (n=242) in areas that contained high-grade dysplasia suggesting that genomic instability proceeded the emergence of driver mutations in what ultimately emerged as the dominant tumor subclone. Chromosomal gains (n=216) vastly outnumbered losses (n=26).

Recent progress in skeletal molecular biology has led to the clarification of the transcriptional mechanisms of chondroblastic and osteoblastic lineage differentiation. Three master transcription factors-Sox9, Runx2, and Osterix-were shown to play an essential role in determining the skeletal progenitor cells' fate. The present study evaluates the expression of these factors in 4 types of benign bone tumors-chondromyxoid fibroma, chondroblastoma, osteoid osteoma, and osteoblastoma-using immunohistochemistry and tissue microarrays. Osteoid osteoma and osteoblastoma showed strong nuclear expression of Osterix and Runx2. In contrast, only a few chondroblastomas showed positive nuclear expression of Osterix. Strong nuclear expression of Sox9 was detected in all chondroblastomas, whereas nearly half of the osteoblastomas showed focal weak cytoplasmic expression of Sox9.

Expression of various members of the ErbB family (epidermal growth factor receptor/ErbB-1, ErbB-2, ErbB-3 and ErbB-4) is associated with disease stage and survival in patients with urothelial carcinoma. We examined the correlation of ErbB family receptor expression with the progression of urothelial carcinoma and survival.A urothelial carcinoma tissue array was constructed from 248 archival paraffin blocks and quality control studies were ascertained. The tissue microarray was stained for epidermal growth factor receptor, ErbB-2, ErbB-3 and ErbB-4, and analyzed using an automated reader. Patient data included grade, stage, growth pattern, recurrence and survival.Kaplan-Meier estimates of 5-year overall and recurrence-free survival were 58% and 27%, respectively. Patients with high grade, invasive or nonpapillary disease had a worse prognosis than patients with low grade, superficial or papillary disease (p0.0001). High epidermal growth factor receptor or low ErbB-4 expression was associated with nonpapillary, high grade and invasive tumors as well as with significantly shorter recurrence-free and overall survival (p0.002, 0.028 and 0.047, respectively). Levels of ErbB-2 and ErbB-3 expression were not associated with overall or recurrence-free survival.The expression profiles of ErbB-4 and epidermal growth factor receptor are prognostic in urothelial carcinoma. They may help in selecting patients at high risk with bladder cancer for more aggressive therapy.

We used human bladder cancer as a model system and the whole-organ histologic and genetic mapping strategy to identify clonal genetic hits associated with growth advantage, tracking the evolution of bladder cancer from intraurothelial precursor lesions. Six putative chromosomal regions critical for clonal expansion of intraurothelial neoplasia and development of bladder cancer were identified by using this approach. Focusing on one of the regions, which includes the model tumor suppressor RB1 , we performed allelotyping of single-nucleotide polymorphic sites and identified a 1.34-Mb segment around RB1 characterized by a loss of polymorphism associated with the initial expansion of in situ neoplasia. This segment contains several positional candidate genes referred to by us as forerunner genes that may contribute to such expansion. We subsequently concentrated our efforts on the two neighbor genes flanking RB1 , namely ITM2B and CHC1L , as well as P2RY5 , which is located inside RB1 . Here, we report that ITM2B and P2RY5 modulated cell survival and were silenced by methylation or point mutations, respectively, and thus by functional loss may contribute to the growth advantage of neoplasia. We also show that homozygous inactivation of P2RY5 was antecedent to the loss of RB1 during tumor development, and that nucleotide substitutions in P2RY5 represent a cancer predisposing factor.

Our goal was to perform studies on the specificity and antimelanoma mechanism of a novel bis-anthracycline, WP760. WP760 initially identified in the NCI 160 screen as anti-melanoma.The methyl thiazolyl tetrazolium reduction (MTT) assay was used to test tumor cell growth inhibition; confocal microscopy to view WP760 intracellular distribution; flow cytometry for cell-cycle arrest and apoptosis; and Western blotting was employed to identify and compare quantities and kinetics of cell growth related molecule levels.WP760 induced G(2)/M-phase cell-cycle arrest and apoptosis in melanoma cell lines and short-term melanoma explants established from clinical specimens in a time and concentration dependent manner at nM concentrations. In contrast, effects on fibroblasts and A549 lung cancer cells required higher concentrations, suggesting that WP760 possesses selectivity for melanoma. Molecular studies indicated that WP760 induced p53 stabilization, checkpoint kinase 2 and p27(Kip1) protein upregulation, and activation of caspase-3. Endogenous nitric oxide (NO) production has been implicated in the chemoresistance of melanoma; WP760 caused inhibition of the inducible nitric oxide synthase (iNOS) protein as well as inhibition of phosphorylation of ERK, known to drive the iNOS pathway. Based on WP760 localization into mitochondria, and caspase-3 inhibitor block the killing of WP760, the intrinsic pathway of apoptosis appears to have been activated.Our results indicate that WP760 affects a critical and unique set of growth regulatory effects in melanoma, and is a promising candidate for further preclinical studies.

BACKGROUND Trastuzumab (Herceptin; Genentech, South San Francisco, CA) is a humanized anti-ErbB-2 monoclonal antibody that has demonstrated antitumor function, especially in combination with other chemotherapies such as paclitaxel (Taxol; Bristol Myers-Squibb, Princeton, NJ), in patients with tumors that overexpress ErbB-2. Because the repeated administration of low-dose chemotherapy, such as paclitaxel, endorsed an antiangiogenic effect in vitro, and because trastuzumab was shown to inhibit angiogenesis in tumor xenografts, the authors investigated whether ErbB-2-mediated angiogenic responses would be inhibited more effectively by the combined treatment of paclitaxel plus trastuzumab. METHODS Tumor xenografts were established in 37 severe combined immunodeficiency mice by injecting the mammary fat pad with ErbB-2-overexpressing human breast carcinoma cells. Mice then were treated with an immunoglobulin (IgG) control, trastuzumab, paclitaxel, or a combination treatment of trastuzumab plus paclitaxel. Tumorigenicity, lung metastasis, and tumor microvessel density (MVD) were evaluated. Vascular endothelial growth factor (VEGF) secretion, endothelial cell migration after treatment, and the status of phosphorylated Akt were evaluated in vitro to determine mechanisms underlying the inhibition of ErbB-2-induced angiogenesis. RESULTS Mice treated with the trastuzumab plus paclitaxel combination exhibited significantly reduced mean tumor volumes compared with mice treated with the IgG control (419.5 mm3 vs. 786.6 mm3, P < 0.0001). Mice treated with trastuzumab had a mean tumor volume of 543.9 mm3, and mice treated with paclitaxel had a mean tumor volume of 574.9 mm3. Tumors from the trastuzumab-plus-paclitaxel group also had significantly decreased mean MVD compared with the control (30 ± 8 MVD vs. 44 ± 12 MVD, P < 0.05). The trastuzumab group had tumors with a MVD of 35 ± 7, similar to the paclitaxel-treated group (35 ± 9). Forty-four percent of the mice in the trastuzumab-plus-paclitaxel group had metastases to the lungs compared with 50%, 63%, and 75% of the mice in the paclitaxel, trastuzumab, and control groups, respectively. In vitro, the ErbB-2-overexpressing cells treated with combined trastuzumab plus paclitaxel secreted less VEGF than the cells treated with trastuzumab, paclitaxel, or control (185.9 pg/mL vs. 233.2 pg/mL, 261.3 pg/mL, and 286.4 pg/mL, respectively). In addition, the conditioned media from the combination group stimulated less mean endothelial cell migration (31.0 cells vs. 47.0 cells, 39.2 cells, and 67.5 cells, respectively). Furthermore, Akt phosphorylation contributed to VEGF up-regulation and Akt phosphorylation was reduced more effectively by combined trastuzumab plus paclitaxel treatment compared with the other treatments. CONCLUSIONS Combined trastuzumab plus paclitaxel treatment more effectively inhibited ErbB-2-mediated angiogenesis than either treatment alone, which resulted in more pronounced tumoricidal effects. This effect may be mediated via the reduction of phosphorylated Akt. Cancer 2003;98:1377–85. © 2003 American Cancer Society. DOI 10.1002/cncr.11656

The equilibrium binding and association kinetics of the fos-jun dimer (basic and leucine zipper domain) to the AP-1 DNA were studied using a quantitative assay. The basic-region and leucine zipper (bZip) domain of c-fos was expressed as a fusion protein with glutathione S-transferase, and it was bound to glutathione-agarose. The GST-fused fos bZip region was allowed to form a heterodimer with the bZip domain of c-jun, to which radiolabeled AP-1 nucleotides were added. After thorough washing, the gel-bound radioactivity was counted. The binding and dissociation rate constants (k(1) and k-(1)) of the fos-jun dimer and DNA could be obtained from a time-course experiment. The association binding constant (K(1)) was determined using both a thermodynamic equation and kinetic parameters. Nordihydroguaiaretic acid (NDGA), momordin I, natural product inhibitors of the fos-jun/DNA complex formation, was applied to this jun-GST-fused fos system and it was found to decrease the apparent equilibrium binding of dimer and DNA. The thermodynamic constant of dimer and inhibitor binding was also determined.

Background: The idea that microscopically recognizable dysplasia and carcinoma in situ act as precursor conditions for invasive cancer is the prevailing view of how many common epithelial malignancies develop. However, new evidence suggests that these are not the initiating events of carcinogenesis but represent the evolution of pre-existing occult disease, caused by the field effects of carcinogens and/or chronic inflammation, which shows minimal phenotypic deviation from normal tissue. These effects may form large plaques in the mucosa of the affected organ but the mechanisms driving their developments are unknown. Design: We developed a strategy that combines histologic and genetic mapping that permits interrogation of the chronology of genetic changes associated with cancer development on a whole-organ scale. By using this approach, we analyzed the sequence of genetic alterations contiguous to the tumor suppressor RB1 and identified a set of alternative target genes that we term “forerunner” (FR) genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11) whose silencing was associated with development of clonal plaque-like mucosal field effects initiating bladder carcinogenesis. We focused on two model FR genes, LPAR6 and CAB39L, and validated their involvement in the development of mucosal field effects by in vitro mechanistic studies and in vivo models. Results: We provide evidence that silencing of prototypic FR genes by hypermethylation (LPAR6 and CAB39L) and less frequently by mutations (LPAR6) is associated with the initial expansion of intraurothelial neoplasia and sets the stage for subsequent events of carcinogenesis. We also found that LPAR6 and CAB39L control cell survival and proliferation consistent with the loss of function being contributory to tumorigenesis. We validated their involvement in three large independent cohorts of bladder cancer, which were profiled according to a novel molecular taxonomy and show that they distinctively contribute to the development of luminal (LPAR6) and basal (CAB39L) subtypes. The in vitro studies using cell lines and their ability to form urospheres showed that these genes contribute to carcinogenesis by altering the transition from basal to luminal phenotypes. Consistent with these observations the BBN induced cancers in Lpar6−/− mice were of luminal type while the cancers induced in Lpar6+/+ mice were of basal type. Conclusion: Loss of prototypic FR genes contributes to the development of mucosal field effects initiating bladder carcinogenesis by altering the basal to luminal urothelial transition program. Citation Format: Sangkyou Lee, Jolanta Bondaruk, Sooyoung Lee, June G. Lee, Tadeusz Majewski, Woonyoung Choi, Charles Guo, Colin Dinney, Li Zhang, Keith Baggerly, Richard Behringer, David McConkey, Bogdan Czerniak. Forerunner genes contribute to bladder carcinogenesis by altering the basal to luminal urothelial transition program. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5172.

Urothelial cancer has served as one of the most important sources of information about the mutational events that underlie the development of human solid maligancies. Although “field effects” that affect the entire bladder mucosa appear to initiate disease, tumors develop along two distinct biological “tracks” that present vastly different challenges for clinical management. Recent whole genome methodologies have facilitated even more rapid progress in the identification of the molecular mechanisms involved in bladder cancer initiation and progression. Specifically, whole organ mapping combined with high resolution, high throughput SNP analyses have identified a novel class of candidate tumor suppressors (“forerunner genes”) that localize near more familiar tumor suppressors but are disrupted at an earlier stage of cancer development. Furthermore, whole genome comparative genomic hybridization (CGH) and mRNA expression profiling have demonstrated that the two major subtypes of urothelial cancer (papillary/superficial and non-papillary/muscle-invasive) are truly distinct molecular entities, and in recent work our group has discovered that muscle-invasive tumors express molecular markers characteristic of a developmental process known as “epithelial-to-mesenchymal transition” (EMT). Emerging evidence indicates that urothelial cancers contain subpopulations of tumor-initiating cells (“cancer stem cells”) but the phenotypes of these cells in different tumors are heterogeneous, raising questions about whether or not the two major subtypes of cancer share a common precursor. This review will provide an overview of these new insights and discuss priorities for future investigation.

Chromosome missegregation and the resulting aneuploidy is a common change in neoplasia. The Aurora kinase A (AURKA) gene, which encodes a key regulator of mitosis, is frequently amplified and/or overexpressed in cancer cells, and the level of AURKA amplification is associated with the level of aneuploidy. We examined whether AURKA gene amplification is a biomarker for the detection of bladder cancer.The effect of ectopic expression of Aurora kinase A (AURKA) using an adenoviral vector in simian virus 40-immortalized urothelial cells (SV-HUC) on centrosome multiplication and chromosome copy number was measured in vitro by immunofluorescence and fluorescence in situ hybridization (FISH), respectively. The FISH test was also used to examine AURKA gene copy number in exfoliated cells in voided urine samples from 23 patients with bladder cancer and 7 healthy control subjects (training set), generating a model for bladder cancer detection that was subsequently validated in an independent set of voided urine samples from 100 bladder cancer patients and 148 control subjects (92 healthy individuals and 56 patients with benign urologic disorders). An AURKA gene score (the proportion of cells with three or more AURKA signals) was used to produce receiver operating characteristic (ROC) curves and to calculate the specificity and sensitivity of the AURKA FISH test. Differences between mean AURKA scores in different pathogenetic groups of bladder cancer stratified according to histological grade and stage were tested by unpaired Mann-Whitney t tests or one-way Wilcoxon tests. All statistical tests were two-sided.Forced overexpression of AURKA in urothelial cells induced amplification of centrosomes, chromosome missegregation, and aneuploidy, and natural overexpression was detectable in in situ lesions from patients with bladder cancer. The FISH test for the AURKA gene copy number performed on the validation set yielded a specificity of 96.6% (95% confidence interval [CI] = 92.3% to 98.5%) and sensitivity of 87% (95% CI = 79.0% to 92.2%) and an area under the ROC curve of 0.939 (95% CI = 0.906 to 0.971; P.001).Overexpression of AURKA can cause aneuploidy in urothelial cells, and the AURKA gene copy number is a promising biomarker for detection of bladder cancer.

The search for the genomic sequences involved in human cancers can be greatly facilitated by maps of genomic imbalances identifying the involved chromosomal regions, particularly those that participate in the development of occult preneoplastic conditions that progress to clinically aggressive invasive cancer. The integration of such regions with human genome sequence variation may provide valuable clues about their overall structure and gene content. By extension, such knowledge may help us understand the underlying genetic components involved in the initiation and progression of these cancers. We describe the development of a genome-wide map of human bladder cancer that tracks its progression from in situ precursor conditions to invasive disease. Testing for allelic losses using a genome-wide panel of 787 microsatellite markers was performed on multiple DNA samples, extracted from the entire mucosal surface of the bladder and corresponding to normal urothelium, in situ preneoplastic lesions, and invasive carcinoma. Using this approach, we matched the clonal allelic losses in distinct chromosomal regions to specific phases of bladder neoplasia and produced a detailed genetic map of bladder cancer development. These analyses revealed three major waves of genetic changes associated with growth advantages of successive clones and reflecting a stepwise conversion of normal urothelial cells into cancer cells. The genetic changes map to six regions at 3q22–q24, 5q22–q31, 9q21–q22, 10q26, 13q14, and 17p13, which may represent critical hits driving the development of bladder cancer. Finally, we performed high-resolution mapping using single nucleotide polymorphism markers within one region on chromosome 13q14, containing the model tumor suppressor gene RB1, and defined a minimal deleted region associated with clonal expansion of in situ neoplasia. These analyses provided new insights on the involvement of several non-coding sequences mapping to the region and identified novel target genes, termed forerunner (FR) genes, involved in early phases of cancer development.

Context.—Sarcomas are rare, numerous in type, and often difficult to definitively classify. Work in the last 2 decades has revealed that a significant subset of sarcomas are associated with specific chromosomal translocations producing chimeric (fusion) genes that play a role in the sarcomas' biology and are helpful in their differential diagnosis.Objective.—To briefly review the sarcomas associated with specific translocations presenting Ewing sarcoma and synovial sarcoma as archetypes and to further explain how cytogenetic and molecular biologic approaches are being used in the diagnosis of sarcomas.Data Sources.—This work is based on a selected review of the relevant medical and scientific literature and our extensive experience with molecular testing in sarcomas.Conclusions.—In addition to, and complementing, the traditional diagnostic methods of examination of hematoxylin-eosin stained slides, immunohistochemistry, and sound clinical-pathologic correlation, additional cytogenetic and molecular biologic methods are being increasingly utilized and relied on in sarcoma pathology. These methods include chromosomal karyotyping, fluorescence in-situ hybridization, spectral karyotyping, and polymerase chain reaction– based methods for demonstrating specific chromosomal translocations and fusion genes. Understanding the basis of these methods and their application is critical to better provide accurate and validated specific diagnoses of sarcomas.

We developed a strategy that combines histologic and genetic mapping that permits interrogation of the chronology of genetic changes associated with cancer development on a whole-organ scale. By using this approach, we analyzed the sequence of genetic alterations contiguous to the tumor suppressor RB1 and identified a set of alternative target genes that we term “forerunner” (FR) genes whose silencing was associated with development of clonal plaque-like mucosal field effects initiating bladder carcinogenesis. Expression and methylation studies identified five candidate FR genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11). In vitro mechanistic studies demonstrated that three of these genes (ITM2B, LPAR6 and ARL11) control cell survival and proliferation consistent with their loss of function being contributory to tumorigenesis, and in vivo genetic ablation of Lpar6 in a mouse model resulted in activation of basal/stem cell transcriptional programs involving STAT3 and NFκB causing proliferative expansion of the basal layer of the urothelium. Whole genome analyses of the field effects in a representative whole-organ map revealed a sequential accumulation of alterations in RHO/RAC/Cdc42 pathways that control invasion and cell motility. These studies identify FR genetic alterations in tissue fields as one of the earliest events in carcinogenesis and provide comprehensive description of field cancerization in a primary human tumor. The results have important implications for the development of novel detection markers and chemoprevention therapeutic strategies. Citation Format: Bogdan A. Czerniak, Sangkyou Lee, Jolanta Bondaruk, Sooyong Lee, Tadeusz Majewski, Shizhen Zhang, Li Shen, Yuexin Liu, Charles Guo, Colin Dinney, H. Barton Grossman, Wei Zhang, Menashe Bar-Eli, Keith Baggerly, Richard Behringer, David McConkey, David McConkey, Margaret Knowles, Woonbok Chung, Jean-Pierre Issa. Mechanism of field cancerization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5256. doi:10.1158/1538-7445.AM2014-5256

The recombinant humanized anti-ErbB2/HER2 monoclonal antibody Herceptin (Trastuzumab) has been shown to significantly enhance the tumoricidaleffects of antitumor drugs such as paclitaxel (Taxol) in patients with ErbB2-overexpressing breast cancers. Here, we investigated the molecular mechanisms by which Herceptin enhances the antitumor effects of Taxol. Because activation of p34(Cdc2) is required for Taxol-induced apoptosis and because overexpression of ErbB2 blocks Taxol-induced apoptosis by inhibiting p34(Cdc2) activation, we studied the effect of Herceptin treatment on p34(Cdc2) kinase activation and apoptosis in Taxol-treated human breast carcinoma cell lines MDA-MB-435, SKBr3, MDA-MB-453, and 435.eB, which is an ErbB2 transfectant of MDA-MB-435. Herceptin treatment down-regulated ErbB2, reduced the inhibitory phosphorylation of Cdc2 on Tyr-15, and down-regulated the expression of p21(Cip1), a Cdc2 inhibitor. Herceptin plus Taxol treatment led to higher levels of p34(Cdc2) kinase activity and apoptosis in ErbB2-overexpressing breast cancer cells, which is likely attributable to inhibition of Cdc2-Tyr-15 phosphorylation and p21(Cip1) expression. Because significant dephosphorylation of Cdc2-Tyr-15 and down-regulation of p21(Cip1) occur at least 24 h after Herceptin treatment, we investigated whether 24 h Herceptin pretreatment will render ErbB2-overexpressing breast cancer cells more sensitive to Taxol-induced apoptosis compared with the simultaneous treatment of Herceptin plus Taxol. Indeed, Herceptin pretreatment increased Taxol-induced apoptosis and cytotoxicity in vitro and more effectively inhibited the growth of tumor xenografts with enhanced in vivo apoptosis. Thus, Herceptin treatment of ErbB2-overexpressing cells can inhibit ErbB2-mediated Cdc2-Tyr-15 phosphorylation and p21(Cip1) up-regulation, which allows effective p34(Cdc2) activation and induction of apoptosis upon Taxol treatment. Herceptin pretreatment renders ErbB2-overexpressing breast cancers more susceptible to Taxol-induced cell death, which may have important clinical therapeutic implications.

Our prior studies have suggested that genes mapping around the tumor suppressor RB1 termed forerunner (FR) genes were silenced by methylation or mutations in in situ phases of urothelial carcinogenesis and were potentially driving the development of clonal mucosal field effects. In order to verify these correlative observations, we have developed a mouse ablation model of a candidate FR gene, P2RY5 mapping within intron 17 of RB1 and encoding G protein receptor. In order to facilitate a germline ablation of P2RY5, a knock-out construct was generated to delete the P2RY5 reading frame. The construct was inserted into mouse embryonic stem cells and the gene was ablated by mating the animals with CMV-cre mice. The effect of P2RY5 deletion on cell proliferation was assessed on cultured fibroblasts derived from embryos with P2RY5 ablation (P2RY5-/-) and from control embryos (P2RY5 +/+). The effect of P2RY5 ablation in the urothelium was assessed on bladders of P2RY5-/- and P2RY5 +/+ mice. The bladders were fixed in formalin and the proliferation status in the urothelium was assessed by immunohistochemical and immunoflourescent staining for Ki67and PCNA. The status of the urothelium was assessed by immunohistochemical and immunoflourescent staining for cytokeratins 5, 14, and 18. In addition, the effect of silencing P2RY5 on global gene expression patterns was evaluated on mouse embryonic fibroblasts using Illumina mouse cDNA expression microarrays. P2RY5-/- embryonic fibroblasts showed dramatically increased proliferation and colony formation rates as compared to P2RY5 +/+ cells. cDNA micro analysis shows that silencing P2RY5 activated several important oncogenic pathways including NFκB and STAT3. Similarly, P2RY5-/- urothelium contained nearly three times more proliferating cells as compared to P2RY5 +/+ urothelium. The increased proliferation rate was associated with the striking expansion of the peribasal layer as documented by staining for cytokeratin 5 and 14. In the P2RY5 +/+ urothelium the cytokeratin 5 and 14 positive cells formed a sparse single basal layer. In contrast, in the P2RY5-/- urothelium, these cells formed irregular mounds overcrowded with peribasal cells. Taken together, our observations show that the silencing of P2RY5 increases the growth rate of mouse embryonic fibroblasts via the activation of several oncogenic pathways and causes an increase in the proliferation rate in the urothelium resulting in urothelial hyperplasia recapitulating incipient events of urothelial carcinogenisis. Citation Format: Bogdan A. Czerniak, Sangkyou Lee, Ying Wang, Jolanta Bondaruk, Taddeusz Majewski, Woongyoung Choi, David McConkey, Richard R. Behringer. Germline ablation of candidate forerunner gene, P2RY5, in a mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 307. doi:10.1158/1538-7445.AM2013-307

The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5'-untranslated regions (UTRs) that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5'-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an "in-frame cDNA library." We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11), cellular retinoic acid binding protein 2 (CRABP2), and phosphoglycerate mutase 1 (PGAM1). Thus, the in-frame cDNA library without 5'-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems.

We are pursuing a unique strategy to identify early events in bladder cancer development by the genome-wide analysis of somatic changes in tissues from resected bladders and characterized pathologically as urothelial carcinoma. We then extend this analysis into precursor intraurothelial lesions and normal tissue. Here we report on the construction of a high resolution whole-organ histologic and genetic map of bladder cancer development that tracks its progression from in situ precursor conditions to invasive clinically aggressive cancer using total genome and exome sequencing and complemented with HumanOmni 2.5-8 v1 SNP-based Illumina genotyping, combined with CpG island methylation and cDNA microarray platforms derived from 12 whole-organ cystectomy specimens with invasive urothelial carcinoma. This approach provided a high resolution integrated map of structural genome, epigenome, and transcriptome alterations that parallel the development of bladder cancer from early field effects to invasive disease. By analyzing genome-wide patterns of structural genomic, epigenomic, and transcriptome changes, we found three major waves of alterations associated with growth advantage of successive clones reflecting a stepwise conversion of normal urothelial cells into cancer cells. This approach is significant for the identification of specific chromosomal regions as well as epigenetically and transcriptionally modified gene ontologies involved in early clonal expansion of intraurothelial neoplasia and their clonal evolution to carcinoma in situ progressing to invasive cancer. These studies suggest involvement of specific gene networks in early clonal expansion of intraurothelial neoplasia antecedent to microscopically recognizable precursor lesions such as dysplasia and carcinoma in situ. This map represents a prerequisite for specific studies on the mechanisms of incipient phases of bladder cancer development and should have a significant impact on the understanding of human carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5066. doi:1538-7445.AM2012-5066

Genomic maps that track the development of human cancer from its in situ precursor conditions to invasive disease can facilitate the search for genes and genomic sequences involved in the development of cancer. The identification of those chromosomal regions and their positional target genes as well as non-coding sequences that provide growth advantage for the incipient preneoplastic conditions can provide novel insights on molecular mechanisms of cancer development. Such an approach is also critical for the identification of novel biomarkers as well as targets for preventive and therapeutic strategies. We report on the construction of a high resolution whole-organ histologic and genetic map of bladder cancer development that tracks its progression from in situ precursor conditions to invasive clinically aggressive cancer using exome deep sequencing and 1M SNP-based Illumina genotyping combined with CpG island methylation and cDNA microarray technology platforms. This approach provided a high resolution integrated pattern of structural genome, epigenome, and transcriptome alterations that parallel the development of bladder cancer from early field effects to invasive disease. By analyzing genome-wide patterns of structural genomic (copy loss or gain), epigenomic, and transcriptome changes, we found three major waves of alterations associated with growth advantage of successive clones reflecting a stepwise conversion of normal urothelial cells into cancer cells. In particular, this approach is significant for the identification of specific chromosomal regions as well as epigenetically and transcriptionally modified gene ontologies involved in early clonal expansion of intraurothelial neoplasia and their clonal evolution to carcinoma in situ progressing to invasive cancer. These studies suggest involvement of ras/raf gene network in early clonal expansion of intraurothelial neoplasia. This map represents a prerequisite for specific studies on the mechanisms of incipient phases of bladder cancer development and should have a significant impact on the understanding of human carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4837. doi:10.1158/1538-7445.AM2011-4837

Our previously reported whole-organ genome-wide mapping studies of bladder cancer provided evidence that a new class of genes, referred to as forerunner (FR) genes (ITM2B, P2RY5, GPR38 and ARL11), located near known tumor suppressors such as RB1, may drive early clonal expansion of neoplasia. (S Lee, et al. PNAS 104: 13732-13737, 2007; T Majewski et al. Lab Inv 88: 694-712, 2008) These studies suggested three major steps for the involvement of the RB1 locus in tumor development. In the first step, one allele of FR gene and RB1 is inactivated by large deletions. In the second step, homozygous inactivation of the FR genes is accomplished by hypermethylation or less frequently by missence mutations. Homozygous inactivation of FR genes is associated with the clonal expansion of in situ lesions referred to as low-grade intraurothelial neoplasia. In the final, third step, the contiguous tumor suppressor, such as RB1, is inactivated most commonly by a mutation. This step is associated with clonal evolution into a transformed phenotype with microscopic features of carcinoma in situ progressing to invasive cancer. Recently, we concentrated our efforts on the ARL11 candidate FR gene, which encodes ADP-ribosylation factor-like 11 protein, a ras-related member of the small GTPases, that functions as GDP/GTP nucleotide interswitch for signal transduction. We have shown that it has been silenced by hypermethylation in approximately 40% of urothelial carcinomas of the bladder and the silencing was associated with in situ expansion of intraurothelial neoplasia. Moreover, loss of ARL11 expression was almost mutually exclusive with the FGFR3 mutations ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3065.