Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer.

We report a comprehensive analysis of 412 muscle-invasive bladder cancers characterized by multiple TCGA analytical platforms. Fifty-eight genes were significantly mutated, and the overall mutational load was associated with APOBEC-signature mutagenesis. Clustering by mutation signature identified a high-mutation subset with 75% 5-year survival. mRNA expression clustering refined prior clustering analyses and identified a poor-survival "neuronal" subtype in which the majority of tumors lacked small cell or neuroendocrine histology. Clustering by mRNA, long non-coding RNA (lncRNA), and miRNA expression converged to identify subsets with differential epithelial-mesenchymal transition status, carcinoma in situ scores, histologic features, and survival. Our analyses identified 5 expression subtypes that may stratify response to different treatments.

Intraosseous hibernoma: clinicopathologic and imaging analysis of 18 cases.

AIMS: Intraosseous hibernomas are rarely reported tumours with brown adipocytic differentiation of unknown aetiology, with only 38 cases documented in the literature. We sought to further characterise the clinicopathologic, imaging and molecular features of these tumours. METHODS AND RESULT: Eighteen cases were identified occurring in eight females and 10 males (median age = 65 years, range = 7-75). Imaging indication was cancer surveillance/staging in 11 patients and clinical concern for a metastasis was raised in 13 patients. The innominate bone (7), sacrum (5), mobile spine (4), humerus (1) and femur (1) were involved. Median tumour size was 1.5 cm (range = 0.8-3.8). Tumours were sclerotic (11), mixed sclerotic and lytic (4) or occult (1). Microscopically, tumours were composed of large polygonal cells with distinct cell membranes, finely vacuolated cytoplasm, central or paracentral small bland nuclei with prominent scalloping. Growth around trabecular bone was observed. Tumour cells were immunoreactive for S100 protein (15/15) and adipophilin (5/5), while negative for keratin AE1/AE3(/PCK26) (0/14) and brachyury (0/2). Chromosomal microarray analysis, performed on four cases, did not show clinically significant copy number variation across the genome or on 11q, the site of AIP and MEN1. CONCLUSION: Analysis of 18 cases of intraosseous hibernoma, to our knowledge, the largest series to date, revealed that these tumours are most often detected in the spine and pelvis of older adults. Tumours were generally small, sclerotic and frequently found incidentally and can raise concern for metastasis. Whether or not these tumours are related to soft tissue hibernomas is uncertain.

Expression Analysis of Same-Patient Metachronous and Synchronous Upper Tract and Bladder Urothelial Carcinoma.

PURPOSE: We compared upper tract urothelial carcinoma (UTUC) and bladder urothelial carcinoma (BUC) in same-patient metachronous UTUC and synchronous UTUC and BUC using next-generation sequencing. MATERIALS AND METHODS: Consecutive untreated same-patient samples of UTUC and BUC were macrodissected from unstained formalin-fixed, paraffin-embedded slides after quality control. Samples were divided into 4 groups: 1) UTUC-metachronous BUC, 2) BUC-metachronous UTUC, 3) synchronous UTUC-BUC, 4) UTUC without BUC. Exclusions were inadequate clinical data or histological tumor purity <30%. Whole transcriptome RNA sequencing was performed. After quality assessment, gene expression clusters using unsupervised hierarchical consensus clustering and correlation with pertinent clinicopathologic variables, a prior RNASeq data set and other published data were performed. RESULTS: RNAseq was performed on 95 samples (UTUC=61, BUC=34) from 40 untreated patients. Unsupervised consensus clustering segregated the tumors into 2 clusters that were enriched with BASE47 basal-like or luminal-like gene expression. Almost two-thirds (61.9%) of Group 2 tumors were basal-like, while the majority of Groups 1, 3, 4 (80.6%, 70.0% and 69.6%, respectively) were luminal-like (p=0.017). Further analyses revealed that the differences in basal-like and luminal-like gene expression were associated with differential fibroblast and immune cell gene expression signatures. In all, 87.5% of metachronous tumors maintained subtype membership. CONCLUSIONS: Gene expression analysis of same-patient metachronous UTUC-BUC suggests that the majority of mUTUC developing after BUC appear more basal-like, while synchronous and initial UTUC tumors appear luminal-like. Metachronous tumors largely maintain molecular subtype membership of the initial tumor regardless of chronologic development or anatomical origin.

Secondary tumors of the bladder: A survival outcome study.

The urinary bladder may be involved by a variety of secondary tumors that originate from other organs. Bladder secondary tumors are rare and may be mistaken as bladder primary tumors because of their overlapping morphologic features. To avoid the diagnostic pitfalls, we analyzed the clinicopathologic features of bladder secondary tumors in a large cohort of patients. Our patient cohort consisted of 45 females and 38 males with a mean age of 58.7 ± 15.4 years (range 10-87 years). The tumors involved the bladder via direct extension from adjacent organs (n = 42) and distant metastasis (n = 41). In females, the majority of secondary tumors originated from the gynecologic tract (n = 25), and other common origins included the colon/rectum (n = 5) and breast (n = 4). In males, the most common origin was the prostate (n = 18), followed by the colon/rectum (n = 4) and kidney (n = 3). 75.9% of the secondary tumors were adenocarcinoma (n = 63), and other common tumor types included sarcoma (n = 6), squamous cell carcinoma (n = 5), melanoma (n = 4), and neuroendocrine carcinoma (n = 3). 67.5% of patients (n = 56) died of the disease with a median overall survival of 23 months from the time of secondary involvement of the bladder. Patients with secondary tumors via direct extension had a median survival time of 20 months, which was not significantly different from that for patients with secondary involvement via distant metastasis (24 months) (p = 0.83). Median survival in cases with prostate primary was 20 months as compared to 23 months for all other tumor types (p = 0.68). The majority of secondary tumors are composed of adenocarcinoma, which highlights the importance of differentiating primary from secondary involvement in bladder adenocarcinoma. Regardless of the origin, bladder secondary tumors are associated with a poor prognosis.

Cardiac paragangliomas: A case series with clinicopathologic features and succinate dehydrogenase B immunostaining.

Cardiac paragangliomas (PGs) are very rare tumors that comprise less than 1% of all cardiac tumors. PGs can occur sporadically, but inherited syndromes may also play a role in the development of PGs. Approximately one-third of PGs are associated with mutations in the succinate dehydrogenase (SDH) complex, specifically SDHB, as part of syndrome-associated PGs or sporadic PGs. SDH mutations have been assessed by SDHB immunohistochemistry, as negative staining indicates a high likelihood of mutation in PGs in other sites, but not in cardiac PGs. This study aims to evaluate the clinical and pathologic characteristic of cardiac PG cases and assess the expression of SDHB by immunohistochemistry. A retrospective chart analysis of 10 patients with cardiac PG was performed to assess the patient age, sex, size, site of the tumor, and clinical symptoms. Histologically the tumors showed the classic pattern of nested tumor cells surrounded by sustentacular cells. Immunohistochemistry for SDHB was performed in five cases. One case showed a complete absence of SDHB immunohistochemical staining and the others showed staining ranging from a weak-to-strong granular cytoplasmic staining pattern. We conclude that SDHB immunostaining is cost-effective in identifying cases with SDH mutation. It is recommended to assess SDH mutation in patients with cardiac PG to predict the aggressive behavior that has been reported by previous studies from PGs of other sites.

Time-to-progression after front-line fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy for chronic lymphocytic leukaemia: a retrospective, multicohort study.

BACKGROUND: Fludarabine, cyclophosphamide, and rituximab (FCR) has become a gold-standard chemoimmunotherapy regimen for patients with chronic lymphocytic leukaemia. However, the question remains of how to treat treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia. We therefore aimed to develop and validate a gene expression signature to identify which of these patients are likely to achieve durable remissions with FCR chemoimmunotherapy. METHODS: We did a retrospective cohort study in two cohorts of treatment-naive patients (aged ≥18 years) with chronic lymphocytic leukaemia. The discovery and training cohort consisted of peripheral blood samples collected from patients treated at the University of Texas MD Anderson Cancer Center (Houston, TX, USA), who fulfilled the diagnostic criteria of the International Workshop on Chronic Lymphocytic Leukemia, had received at least three cycles of FCR chemoimmunotherapy, and had been treated between Oct 10, 2000, and Oct 26, 2006 (ie, the MDACC cohort). We did transcriptional profiling on samples obtained from the MDACC cohort to identify genes associated with time to progression. We did univariate Cox proportional hazards analyses and used significant genes to cluster IGHV-unmutated samples into two groups (intermediate prognosis and unfavourable prognosis). After using cross-validation to assess robustness, we applied the Lasso method to standardise the gene expression values to find a minimum gene signature. We validated this signature in an external cohort of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia enrolled on the CLL8 trial of the German Chronic Lymphocytic Leukaemia Study Group who were treated between July 21, 2003, and April 4, 2006 (ie, the CLL8 cohort). FINDINGS: The MDACC cohort consisted of 101 patients and the CLL8 cohort consisted of 109 patients. Using the MDACC cohort, we identified and developed a 17-gene expression signature that distinguished IGHV-unmutated patients who were likely to achieve a long-term remission following front-line FCR chemoimmunotherapy from those who might benefit from alternative front-line regimens (hazard ratio 3·83, 95% CI 1·94-7·59; p<0·0001). We validated this gene signature in the CLL8 cohort; patients with an unfavourable prognosis versus those with an intermediate prognosis had a cause-specific hazard ratio of 1·90 (95% CI 1·18-3·06; p=0·008). Median time to progression was 39 months (IQR 22-69) for those with an unfavourable prognosis compared with 59 months (28-84) for those with an intermediate prognosis. INTERPRETATION: We have developed a robust, reproducible 17-gene signature that identifies a subset of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia who might substantially benefit from treatment with FCR chemoimmunotherapy. We recommend testing the value of this gene signature in a prospective study that compares FCR treatment with newer alternative therapies as part of a randomised clinical trial. FUNDING: Chronic Lymphocytic Leukaemia Global Research Foundation and the National Institutes of Health/National Cancer Institute.

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma.

BACKGROUND: Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist. METHODS: We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis. RESULTS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH). CONCLUSIONS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).

Clinical risk stratification in patients with surgically resectable micropapillary bladder cancer.

OBJECTIVE: To analyse survival in patients with clinically localised, surgically resectable micropapillary bladder cancer (MPBC) undergoing radical cystectomy (RC) with and without neoadjuvant chemotherapy (NAC) and develop risk strata based on outcome data. PATIENTS AND METHODS: A review of our database identified 103 patients with surgically resectable (≤cT4acN0 cM0) MPBC who underwent RC. Survival estimates were calculated using Kaplan-Meier method and compared using log-rank tests. Classification and regression tree (CART) analysis was performed to identify risk groups for survival. RESULTS: For the entire cohort, estimated 5-year overall survival and disease-specific survival (DSS) rates were 52% and 58%, respectively. CART analysis identified three risk subgroups: low-risk: cT1, no hydronephrosis; high-risk: ≥cT2, no hydronephrosis; and highest-risk: cTany with tumour-associated hydronephrosis. The 5-year DSS for the low-, high-, and highest-risk groups were 92%, 51%, and 17%, respectively (P < 0.001). Patients down-staged at RC

Next-generation sequencing identifies high frequency of mutations in potentially clinically actionable genes in sebaceous carcinoma.

Sebaceous carcinoma (SC) is a rare but aggressive malignancy with frequent recurrence and metastases. Surgery is the mainstay of therapy, but effective systemic therapies are lacking because the molecular alterations driving SC remain poorly understood. To identify these, we performed whole-exome next-generation sequencing of 409 cancer-associated genes on 27 SCs (18 primary/locally recurrent ocular, 5 paired metastatic ocular, and 4 primary extraocular) from 20 patients. In ocular SC, we identified 139 non-synonymous somatic mutations (median/lesion 3; range 0-23). Twenty-five of 139 mutations (18%) occurred in potentially clinically actionable genes in 6 of 16 patients. The most common mutations were mutations in TP53 (n = 9), RB1 (n = 6), PIK3CA (n = 2), PTEN (n = 2), ERBB2 (n = 2), and NF1 (n = 2). TP53 and RB1 mutations were restricted to ocular SC and correlated with aberrant TP53 and RB protein expression. Systematic pathway analyses demonstrated convergence of these mutations to activation of the PI3K signalling cascade, and PI3K pathway activation was confirmed in tumours with PTEN and/or PIK3CA mutations. Considerable inter-tumoural heterogeneity was observed between paired primary and metastatic ocular SCs. In primary extraocular SC, we identified 77 non-synonymous somatic mutations (median/lesion 22.5; range 3-29). This overall higher mutational load was attributed to a microsatellite instability phenotype in three of four patients and somatically acquired mutations in mismatch repair genes in two of four patients. Eighteen of 77 mutations (23%) were in potentially clinically actionable genes in three of four patients, including BTK, FGFR2, PDGFRB, HRAS, and NF1 mutations. Identification of potentially clinically actionable mutations in 9 of 20 SC patients (45%) underscores the importance of next-generation sequencing to expand the spectrum of genotype-matched targeted therapies. Frequent activation of PI3K signalling pathways provides a strong rationale for application of mTOR inhibitors in the management of this disease. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Intratumoral morphologic and molecular heterogeneity of rhabdoid renal cell carcinoma: challenges for personalized therapy.

Rhabdoid histology in clear-cell renal cell carcinoma is associated with a poor prognosis. The prognosis of patients with clear-cell renal cell carcinoma may also be influenced by molecular alterations. The aim of this study was to evaluate the association between histologic features and salient molecular changes in rhabdoid clear-cell renal cell carcinoma. We macrodissected the rhabdoid and clear-cell epithelioid components from 12 cases of rhabdoid clear-cell renal cell carcinoma. We assessed cancer-related mutations from eight cases using a clinical next-generation exome-sequencing platform. The transcriptome of rhabdoid clear-cell renal cell carcinoma (n=8) and non-rhabdoid clear-cell renal cell carcinoma (n=37) was assessed by RNA-seq and gene expression microarray. VHL (63%) showed identical mutations in all regions from the same tumor. BAP1 (38%) and PBRM1 (13%) mutations were identified in the rhabdoid but not in the epithelioid component and were mutually exclusive in 3/3 cases and 1 case, respectively. SETD2 (63%) mutations were discordant between different histologic regions in 2/5 cases, with mutations called only in the epithelioid and rhabdoid components, respectively. The transcriptome of rhabdoid clear-cell renal cell carcinoma was distinct from advanced-stage and high-grade clear-cell renal cell carcinoma. The diverse histologic components of rhabdoid clear-cell renal cell carcinoma, however, showed a similar transcriptomic program, including a similar prognostic gene expression signature. Rhabdoid clear-cell renal cell carcinoma is transcriptomically distinct and shows a high rate of SETD2 and BAP1 mutations and a low rate of PBRM1 mutations. Driver mutations in clear-cell renal cell carcinoma are often discordant across different morphologic regions, whereas the gene expression program is relatively stable. Molecular profiling of clear-cell renal cell carcinoma may improve by assessing for gene expression and sampling tumor foci from different histologic regions.

Clinical outcomes of cT1 micropapillary bladder cancer.

PURPOSE: While many urologists recommend radical cystectomy for micropapillary bladder cancer invading the lamina propria (cT1), contradictory small reports exist on the efficacy of conservative management with intravesical bacillus Calmette-Guérin for this disease. We report our updated experience in what to our knowledge is the largest series of patients with cT1 micropapillary bladder cancer. MATERIALS AND METHODS: An institutional review board approved review of our cancer database identified 283 patients with micropapillary bladder cancer, including 72 staged with cT1N0M0 disease at diagnosis and initiation of therapy. Survival analysis was performed using the Kaplan-Meier estimator and compared using the log rank test. RESULTS: In this cohort of 72 patients 40 received primary intravesical bacillus Calmette-Guérin and 26 underwent up-front radical cystectomy. Of patients who received bacillus Calmette-Guérin 75%, 45% and 35% experienced disease recurrence, progression and lymph node metastasis, respectively. Patients treated with up-front cystectomy had improved survival compared to patients treated with primary bacillus Calmette-Guérin (5-year disease specific survival 100% vs 60% p = 0.006) and patients who underwent delayed cystectomy after recurrence (5-year disease specific survival 62%, p = 0.015). Prognosis was especially poor in patients who waited for progression before undergoing radical cystectomy with an estimated 5-year disease specific survival of only 24% and a median survival of 35 months. In patients treated with up-front cystectomy pathological up-staging was found in 27%, including 20% with lymph node metastasis. CONCLUSIONS: While certain patients with T1 micropapillary bladder cancer may respond to intravesical bacillus Calmette-Guérin, survival is improved in those who undergo early radical cystectomy. Further molecular studies are needed to identify subsets of patients in whom the bladder can be safely spared.

The p63 protein isoform ΔNp63α inhibits epithelial-mesenchymal transition in human bladder cancer cells: role of MIR-205.

Epithelial-mesenchymal transition (EMT) is a physiological process that plays important roles in tumor metastasis, "stemness," and drug resistance. EMT is typically characterized by the loss of the epithelial marker E-cadherin and increased expression of EMT-associated transcriptional repressors, including ZEB1 and ZEB2. The miR-200 family and miR-205 prevent EMT through suppression of ZEB1/2. p53 has been implicated in the regulation of miR-200c, but the mechanisms controlling miR-205 expression remain elusive. Here we report that the p53 family member and p63 isoform, ΔNp63α, promotes miR-205 transcription and controls EMT in human bladder cancer cells. ΔNp63α, E-cadherin and miR-205 were coexpressed in a panel of bladder cancer cell lines (n = 28) and a cohort of primary bladder tumors (n = 98). Stable knockdown of ΔNp63α in the "epithelial" bladder cancer cell line UM-UC6 decreased the expression of miR-205 and induced the expression of ZEB1/2, effects that were reversed by expression of exogenous miR-205. Conversely, overexpression of ΔNp63α in the "mesenchymal" bladder cancer cell line UM-UC3 induced miR-205 and suppressed ZEB1/2. ΔNp63α knockdown reduced the expression of the primary and mature forms of miR-205 and the miR-205 "host" gene (miR-205HG) and decreased binding of RNA Pol II to the miR-205HG promoter, inhibiting miR-205HG transcription. Finally, high miR-205 expression was associated with adverse clinical outcomes in bladder cancer patients. Together, our data demonstrate that ΔNp63α-mediated expression of miR-205 contributes to the regulation of EMT in bladder cancer cells and identify miR-205 as a molecular marker of the lethal subset of human bladder cancers.

Refining patient selection for neoadjuvant chemotherapy before radical cystectomy.

PURPOSE: We evaluated the survival of patients with muscle invasive bladder cancer undergoing radical cystectomy without neoadjuvant chemotherapy to confirm the utility of existing clinical tools to identify low risk patients who could be treated with radical cystectomy alone and a high risk group most likely to benefit from neoadjuvant chemotherapy. MATERIALS AND METHODS: We identified patients with muscle invasive bladder cancer who underwent radical cystectomy without neoadjuvant chemotherapy at our institution between 2000 and 2010. Patients were considered high risk based on the clinical presence of hydroureteronephrosis, cT3b-T4a disease, and/or histological evidence of lymphovascular invasion, micropapillary or neuroendocrine features on transurethral resection. We evaluated survival (disease specific, progression-free and overall) and rate of pathological up staging. An independent cohort of patients from another institution was used to confirm our findings. RESULTS: We identified 98 high risk and 199 low risk patients eligible for analysis. High risk patients exhibited decreased 5-year overall survival (47.0% vs 64.8%) and decreased disease specific (64.3% vs 83.5%) and progression-free (62.0% vs 84.1%) survival probabilities compared to low risk patients (p <0.001). Survival outcomes were confirmed in the validation subset. On final pathology 49.2% of low risk patients had disease up staged. CONCLUSIONS: The 5-year disease specific survival of low risk patients was greater than 80%, supporting the distinction of high risk and low risk muscle invasive bladder cancer. The presence of high risk features identifies patients with a poor prognosis who are most likely to benefit from neoadjuvant chemotherapy, while many of those with low risk disease can undergo surgery up front with good expectations and avoid chemotherapy associated toxicity.

Molecular profile of bladder cancer progression to clinically aggressive subtypes.

Bladder cancer is a histologically and clinically heterogenous disease. Most bladder cancers are urothelial carcinomas, which frequently develop distinct histological subtypes. Several urothelial carcinoma histological subtypes, such as micropapillary, plasmacytoid, small-cell carcinoma and sarcomatoid, show highly aggressive behaviour and pose unique challenges in diagnosis and treatment. Comprehensive genomic characterizations of the urothelial carcinoma subtypes have revealed that they probably arise from a precursor subset of conventional urothelial carcinomas that belong to different molecular subtypes - micropapillary and plasmacytoid subtypes develop along the luminal pathway, whereas small-cell and sarcomatoid subtypes evolve along the basal pathway. The subtypes exhibit distinct genomic alterations, but in most cases their biological properties seem to be primarily determined by specific gene expression profiles, including epithelial-mesenchymal transition, urothelial-to-neural lineage plasticity, and immune infiltration with distinct upregulation of immune regulatory genes. These breakthrough studies have transformed our view of bladder cancer histological subtype biology, generated new hypotheses for therapy and chemoresistance, and facilitated the discovery of new therapeutic targets.

Plasmacytoid urothelial carcinoma of the urinary bladder-A clinicopathological and molecular analysis of 52 cases.

Plasmacytoid urothelial carcinoma (UC) is a rare histologic subtype of bladder cancer that is associated with an aggressive clinical behavior. We analyzed the clinicopathologic and molecular features of plasmacytoid UC in 52 patients from a single institute. The patients included 44 men and 8 women, with a mean age of 64 years (range, 41-91 years). All bladder cancers were high-grade UC, and plasmacytoid component accounted for a mean of 47% of bladder tumors (range, 5-100%). Distinct gene mutations were found in most plasmacytoid UCs (n = 49); the most common mutations were TP53 (n = 30), followed by TERT (n = 20), and CDH1 (n = 18). Copy number analysis was performed in 34 patients, and 13 of them showed copy number variations. Expression of HER2 was analyzed in 18 patients by immunohistochemistry, and 3 of them showed HER2 overexpression, which was confirmed by fluorescence in situ hybridization analysis. Thirty-two patients died of disease in a median of 15 months (range, 1-45 months). No individual gene mutations were significantly associated with clinical outcome, but mutations in the mammalian target of rapamycin (mTOR) pathway, including PICK3CA and PIK3R1 mutations, were associated with a significantly shorter survival duration (p < 0.05). Plasmacytoid UC is an aggressive histologic subtype that demonstrates frequent somatic gene mutations and CNVs, which may underlie its oncogenesis and progression. Gene mutations of the mTOR pathway are associated with poor outcome in a subset of patients with plasmacytoid UC.

Inferring Bladder Cancer Evolution from Mucosal field Effects by Whole-Organ Spatial Mutational, Proteomic, and Metabolomic Mapping.

Multi-platform mutational, proteomic, and metabolomic spatial mapping was used on the whole-organ scale to identify the molecular evolution of bladder cancer from mucosal field effects. We identified complex proteomic and metabolomic dysregulations in microscopically normal areas of bladder mucosa adjacent to dysplasia and carcinoma in situ. The mutational landscape developed in a background of complex defects of protein homeostasis which included dysregulated nucleocytoplasmic transport, splicesome, ribosome biogenesis, and peroxisome. These changes were combined with altered urothelial differentiation which involved lipid metabolism and protein degradations controlled by PPAR. The complex alterations of proteome were accompanied by dysregulation of gluco-lipid energy-related metabolism. The analysis of mutational landscape identified three types of mutations based on their geographic distribution and variant allele frequencies. The most common were low frequency α mutations restricted to individual mucosal samples. The two other groups of mutations were associated with clonal expansion. The first of this group referred to as ß mutations occurred at low frequencies across the mucosa. The second of this group called γ mutations increased in frequency with disease progression. Modeling of the mutations revealed that carcinogenesis may span nearly 30 years and can be divided into dormant and progressive phases. The α mutations developed gradually in the dormant phase. The progressive phase lasted approximately five years and was signified by the advent of ß mutations, but it was driven by γ mutations which developed during the last 2-3 years of disease progression to invasive cancer. Our study indicates that the understanding of complex alterations involving mucosal microenvironment initiating bladder carcinogenesis can be inferred from the multi-platform whole-organ mapping.

Loss of LPAR6 and CAB39L dysregulates the basal-to-luminal urothelial differentiation program, contributing to bladder carcinogenesis.

We describe a strategy that combines histologic and molecular mapping that permits interrogation of the chronology of changes associated with cancer development on a whole-organ scale. Using this approach, we present the sequence of alterations around RB1 in the development of bladder cancer. We show that RB1 is not involved in initial expansion of the preneoplastic clone. Instead, we found a set of contiguous genes that we term "forerunner" genes whose silencing is associated with the development of plaque-like field effects initiating carcinogenesis. Specifically, we identified five candidate forerunner genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11) mapping near RB1. Two of these genes, LPAR6 and CAB39L, are preferentially downregulated in the luminal and basal subtypes of bladder cancer, respectively. Their loss of function dysregulates urothelial differentiation, sensitizing the urothelium to N-butyl-N-(4-hydroxybutyl)nitrosamine-induced cancers, which recapitulate the luminal and basal subtypes of human bladder cancer.

Genome architecture marked by retrotransposons modulates predisposition to DNA methylation in cancer.

Epigenetic silencing plays an important role in cancer development. An attractive hypothesis is that local DNA features may participate in differential predisposition to gene hypermethylation. We found that, compared with methylation-resistant genes, methylation-prone genes have a lower frequency of SINE and LINE retrotransposons near their transcription start site. In several large testing sets, this distribution was highly predictive of promoter methylation. Genome-wide analysis showed that 22% of human genes were predicted to be methylation-prone in cancer; these tended to be genes that are down-regulated in cancer and that function in developmental processes. Moreover, retrotransposon distribution marks a larger fraction of methylation-prone genes compared to Polycomb group protein (PcG) marking in embryonic stem cells; indeed, PcG marking and our predictive model based on retrotransposon frequency appear to be correlated but also complementary. In summary, our data indicate that retrotransposon elements, which are widespread in our genome, are strongly associated with gene promoter DNA methylation in cancer and may in fact play a role in influencing epigenetic regulation in normal and abnormal physiological states.

Plasmacytoid urothelial carcinoma, a chemosensitive cancer with poor prognosis, and peritoneal carcinomatosis.

PURPOSE: Plasmacytoid urothelial carcinoma is a rare variant histology with poorly defined clinical behavior. We report clinical outcome information on patients with predominant plasmacytoid urothelial carcinoma. MATERIALS AND METHODS: We retrospectively analyzed treatments and outcomes in patients with predominant plasmacytoid urothelial carcinoma seen at our institution from 1990 through 2010. The Kaplan-Meier method was used to calculate overall and progression-free survival. RESULTS: We identified 31 patients with a median age of 63.5 years, of whom 83.3% were male. TNM stage was cT1N0 in 4 patients, cT2N0 in 7, cT3b-4aN0 in 5 and cT4b, N+ or M+ in 15. Median overall survival was 17.7 months (stage I-III vs IV 45.8 vs 13.3). Five of the 16 patients with potentially surgically resectable plasmacytoid urothelial carcinoma (pT4aN0M0 or less) received neoadjuvant chemotherapy, 10 underwent initial surgery and 1 was treated only with transurethral resection of bladder tumor. Despite pathological down staging in 80% of the patients who received neoadjuvant chemotherapy, relapses were common. There was no survival difference between patients treated with neoadjuvant chemotherapy or initial surgery, although 7 received adjuvant chemotherapy. Surgical up staging with positive margins was also common for surgery alone. The most common site of recurrence was in the peritoneum (19 of 23 patients) with relapses even in those with a pathological complete response at surgery. In patients who presented with metastatic disease and were treated with chemotherapy median survival was 12.6 months. CONCLUSIONS: Plasmacytoid urothelial carcinoma is an aggressive subset with overall poor outcomes. Although down staging is seen with neoadjuvant chemotherapy, there are few long-term survivors. There is a strong predilection for recurrence along the peritoneal lining.