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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.

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.

Motivation Gene expression-based multiclass prediction, such as tumor subtyping, is a non-trivial bioinformatic problem. Most classifier methods operate by comparing expression levels relative to other samples. Methods that base predictions on the expression pattern within a sample have been proposed as an alternative. As these methods are invariant to the cohort composition and can be applied to a sample in isolation, they can collectively be termed single sample predictors (SSP). Such predictors could potentially be used for preprocessing-free classification of new samples and be built to function across different expression platforms where proper batch and dataset normalization is challenging. Here, we evaluate the behavior of several multiclass SSPs based on binary gene-pair rules (k-Top Scoring Pairs, Absolute Intrinsic Molecular Subtyping and a new Random Forest approach) and compare them to centroids built with centered or raw expression values, with the criteria that an optimal predictor should have high accuracy, overcome differences in tumor purity, be robust across expression platforms and provide an informative prediction output score. Results We found that gene-pair-based SSPs showed excellent performance on many expression-based classification tasks. The three methods differed in prediction score output, handling of tied scores and behavior in low purity samples. The k-Top Scoring Pairs and Random Forest approach both achieved high classification accuracy while providing an informative prediction score. Although gene-pair-based SSPs have been touted as being cross-platform compatible (through training on mixed platform data), out-of-the-box compatibility with a new dataset remains a potential issue that warrants cohort-to-cohort verification. Availability and implementation Our R package ‘multiclassPairs’ (https://cran.r-project.org/package=multiclassPairs) (https://doi.org/10.1093/bioinformatics/btab088) is freely available and enables easy training, prediction, and visualization using the gene-pair rule-based Random Forest SSP method and provides additional multiclass functionalities to the switchBox k-Top-Scoring Pairs package. Supplementary information Supplementary data are available at Bioinformatics online.

We used the fact that patients with non-muscle invasive bladder tumors show local recurrences and multiple tumors to study re-initiation of tumor growth from the same urothelium. By extensive genomic analyses we show that tumors from the same patient are clonal. We show that gross genomic chromosomal aberrations may be detected in one tumor, only to be undetected in a recurrent tumor. By analyses of incompatible changes i.e., genomic alterations that cannot be reversed, we show that almost all tumors from a single patient may show such changes, thus the tumors cannot have originated from each other. As recurring tumors share both genomic alterations and driver gene mutations, these must have been present in the urothelium in periods with no tumor growth. We present a model that includes a growing and evolving field of urothelial cells that occasionally, and locally, produce bursts of cellular growth leading to overt tumors.

Molecular changes occurring during invasion and clinical progression of cancer are difficult to study longitudinally in patient‐derived material. A unique feature of urothelial bladder cancer (UBC) is that patients frequently develop multiple nonmuscle invasive tumors, some of which may eventually progress to invade the muscle of the bladder wall. Here, we use a cohort of 73 patients that experienced a total of 357 UBC diagnoses to study the stability or change in detected molecular alterations during cancer progression. The tumors were subtyped by gene expression profiling and analyzed for hotspot mutations in FGFR3, PIK3CA and TERT, the most frequent early driver mutations in this tumor type. TP53 alterations, frequent in advanced UBC, were inferred from p53 staining pattern, and potential genomic alterations were inferred by gene expression patterns at regions harboring frequent copy number alterations. We show that early driver mutations were largely preserved in UBC recurrences. Changes in FGFR3, PIK3CA or TERT mutation status were not linked to changes in molecular subtype and aggressive behavior. Instead, changes into a more aggressive molecular subtype seem to be associated with p53 alterations. We analyze changes in gene expression from primary tumors, to recurrences and progression tumors, and identify two modes of progression: Patients for whom progression is preceded by or coincides with a radical subtype shift, and patients who progress without any systematic molecular changes. For the latter group of patients, progression may be either stochastic or depending on factors already present at primary tumor initiation., What's new? Molecular changes occurring during invasion and clinical progression of cancer are difficult to study longitudinally in patient‐derived material. A unique feature of urothelial bladder cancer is that patients frequently develop multiple nonmuscle invasive tumors, some of which may eventually progress to invade the muscle of the bladder wall. Here, the authors perform multi‐level longitudinal analyses on patients with progression from non‐muscle invasive to advanced disease and describe novel modes of progression related to shifts in molecular profiles. Combined with the theory of field cancerization, these results identify limitations in predicting clinical progression based on molecular data from non‐muscle invasive tumors.

Bladder cancer is a common and highly heterogeneous malignancy with a relatively poor outcome. Patient-derived tumor organoid cultures have emerged as a preclinical model with improved biomimicity. However, the impact of the different methods being used in the composition and dynamics of the models remains unknown. This study aims to systematically review the literature regarding patient-derived organoid models for normal and cancer tissue of the bladder, and their current and potential future applications for tumor biology studies and drug testing. A PRISMA-compliant systematic review of the PubMED, Embase, Web of Sciences, and Scopus databases was performed. The results were analyzed based on the methodologies, comparison with primary tumors, functional analysis, and chemotherapy and immunotherapy testing. The literature search identified 536 articles, 24 of which met the inclusion criteria. Bladder cancer organoid models have been increasingly used for tumor biology studies and drug screening. Despite the heterogeneity between methods, organoids and primary tissues showed high genetic and phenotypic concordance. Organoid sensitivity to chemotherapy matched the response in patient-derived xenograft (PDX) models and predicted response based on clinical and mutation data. Advances in bioengineering technology, such as microfluidic devices, bioprinters, and imaging, are likely to further standardize and expand the use of organoids.

There are no established biomarkers to guide patient selection for neoadjuvant chemotherapy prior to radical cystectomy for muscle-invasive bladder cancer. Recent studies suggest that molecular subtype classification holds promise for predicting chemotherapy response and/or survival benefit in this setting. Here, we summarize and discuss the scientific literature examining transcriptomic or panel-based molecular subtyping applied to neoadjuvant chemotherapy-treated patient cohorts. We find that there is not sufficient evidence to conclude that the basal subtype of muscle-invasive bladder cancer responds well to chemotherapy, since only a minority of studies support this conclusion. More evidence indicates that luminal-like subtypes may have the most improved outcomes after neoadjuvant chemotherapy. There are also conflicting data concerning the association between biopsy stromal content and response. Subtypes indicative of high stromal infiltration responded well in some studies and poorly in others. Uncertainties when interpreting the current literature include a lack of reporting both response and survival outcomes and the inherent risk of bias in retrospective study designs. Taken together, available studies suggest a role for molecular subtyping in stratifying patients for receiving neoadjuvant chemotherapy. The precise classification system that best captures such a predictive effect, and the exact subtypes for which other treatment options are more beneficial remains to be established, preferably in prospective studies.

Immunotherapy is revolutionizing cancer treatment, but success is limited to a fraction of patients. Tumor immunosurveillance and immunotherapy relies on presentation of tumor-associated antigens by conventional dendritic cells type 1 (cDC1). However, tumors develop mechanisms to avoid immune recognition such as downregulation of antigen presentation and exclusion of cDC1. We have previously demonstrated that enforced expression of the transcription factors PU.1, IRF8 and BATF3 (PIB) imposes the lineage conversion of fibroblasts to cDC1 by direct cell reprogramming. Here, we hypothesize that PIB reprograms cancer cells directly into functional tumor-antigen presenting cells (tumor-APCs) with enhanced immunogenicity. First, we show that enforced expression of PIB in a wide range of murine and human cancer cells from different origins is sufficient to induce surface expression of hematopoietic and DC-lineage specific markers (CD45 and Clec9a). Moreover, reprogramming restored the expression of antigen presentation complexes (MHC-I and MHC-II) and activated the expression of the co-stimulatory molecules CD40, CD80 and CD86, required for productive T cell activation. Transcriptomic analysis using mRNA-sequencing showed that PIB imposes a global cDC1 gene signature and an antigen presentation program in tumor cells as early as day 3 of reprogramming, overriding the original cancer cell program. Furthermore, Assay for Transposase-Accessible Chromatin (ATAC) sequencing analysis revealed that PIB-mediated cDC1 reprogramming elicited rapid epigenetic remodeling followed by gradual rewiring of transcriptional program and stabilization of cDC1 identity. Functionally, tumor-APCs present endogenous antigens on MHC-I, prime naïve CD8+ T and become prone to CD8+ T cell mediated killing. Tumor-APCs secrete pro-inflammatory cytokines (IL-12) and chemoattractants (CXCL10), uptake and process exogenous antigens, phagocyte dead cells, and cross-present exogenous antigens to activate naïve T-cells. In addition, reprogrammed tumor cells harboring TP53, KRAS and PTEN mutations downregulated proliferation and showed impaired tumorigenicity in vitro and in vivo. Importantly, we show that intra-tumoral injection of reprogrammed tumor-APCs elicited tumour growth control in vivo alongside increasing infiltration of CD8+ T and NK cells in B16-OVA tumors. Finally, we showed that our approach can be employed to convert primary cancer cells derived from melanoma, lung, breast, pancreatic, urothelial, and head and neck carcinomas as well as cancer associated fibroblasts. In summary, we provide evidence for the direct reprogramming of tumor cells into immunogenic cDC1-like cells, with restored antigen presentation capacity and the ability to reinstate anti-tumor immunity. Our approach elicits the immune system against cancer and counteract major tumor evasion mechanisms including tumor heterogeneity and impaired antigen presentation, laying the foundation for developing immunotherapeutic strategies based on the cellular reprogramming of human cancer cells. Citation Format: Alexandra G. Ferreira, Olga Zimmermannova, Ervin Ascic, Ilia Kurochkin, Diego Soto-Cabrera, Ariane Eceiza, Hreinn Benonisson, Inês Caiado, Rita Silvério-Alves, Fábio F. Rosa, Cristiana F. Pires, David Gomez-Jimenez, Carina Bernardo, Monika Bauden, Roland Anderson, Mattias Höglund, Kenichi Miharada, Yukio Nakamura, Lennart Greiff, Malin Lindstedt, Carlos-Filipe Pereira. Restoring tumor immunogenicity with dendritic cell reprogramming [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A40.

Molecular subtypes of urothelial carcinoma may be divided into luminal and nonluminal tumors. Nonluminal tumors are composed of cases with basal/squamous‐like or small cell/neuroendocrine features, with a consensus on the molecular characteristics of the respective subtype. In contrast, luminal tumors are more disparate with three to five suggested subtypes and with definitions that do not always cohere. To resolve some of these disparities we assembled a cohort of 344 luminal tumors classified as urothelial‐like (Uro), with the subtypes UroA, UroAp, UroB, and UroC, or genomically unstable (GU) according to the LundTax system. Cases were systematically analyzed by immunohistochemistry using antibodies for proteins representing important biological processes or cellular states: KRT5, EGFR, and CDH3 for the integrity of a basal cell layer; CCNB1, Ki67, and FOXM1 for proliferation; FGFR3 and ERBB2 for receptor tyrosine kinase status; CCND1, CDKN2A(p16), RB1, and E2F3 for cell cycle regulation; PPARG, GATA3, and TP63 for the differentiation regulatory system; and KRT20 and UPK3 for the differentiation readout. We show that Uro tumors form one, albeit heterogenous, group characterized by FGFR3, CCND1, and RB1 expression, but low or absence of CDKN2A(p16) and ERBB2 expression. The opposite expression pattern is observed in GU cases. Furthermore, Uro tumors are distinguished from GU tumors by showing a high RB1/p16 expression ratio. Class defining characteristics were independent of pathological stage and growth pattern, and thus intrinsic. In Uro tumors, proliferation was limited to a well‐defined single layer of basal‐like cells in UroA tumors but occurred throughout the tumor parenchyma, independent of the basal layer, in the more progressed UroAp and UroC tumors. A similar change in proliferation topology was not observed in GU. We conclude that luminal urothelial carcinomas consist, at the molecular pathology level, of two major subtypes, the larger heterogenous Uro and the biologically distinct GU subtype. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cancer progression entails close crosstalk between tumor cells and immune system. Recent advances in understanding mutual tumor-immune interactions translated into the development of cancer immunotherapy, changing the cancer treatment paradigm. Nevertheless, despite achieving long-term effect even in advanced malignancies, only a minor subset of patients responds to immunotherapy. The activation of the immune system, as well as good response to the immunotherapy strongly depends on tumor immunogenicity and effective presentation of tumor-associated antigens by conventional dendritic cells type 1 (cDC1). However, downregulation of antigen-presentation machinery and cDC1 exclusion represent major mechanisms of cancer immune evasion, leading to immunotherapy failure. Previously, we identified a combination of 3 transcription factors, PU.1, IRF8, and BATF3 (PIB) that instructed direct reprogramming of fibroblasts into cDC1-like cells endowed with the ability to present and cross-present antigens. Here, we hypothesize that cDC1-direct reprogramming employing PIB transcription factors can impose antigen-presentation directly in human tumor cells, generating functional tumor-antigen presenting cells (tumor-APCs) able to prime antigen-specific T cell responses. Here, we used a large panel of human cell lines (>30) derived from leukemia and multiple solid tumors including glioblastoma, melanoma, breast, pancreatic, ovarian, prostate, head and neck, and lung adenocarcinoma and evaluated dendritic cell reprogramming efficiency. Enforced expression of PIB induced a cDC1-phenotype in tumor cells, as demonstrated by an emerging CD45+HLA-DR+ population and the activation of the cDC1-markers CD11c, CD141, and CLEC9A. Induced cells showed global transcriptional reprogramming towards cDC1 fate imposing a tumor-APC signature in cancer cells at varying efficiencies ranging between 0.2±0.1% to 94.5±7.6% according to the tumor cell of origin. PIB induces rapid transcriptomic and epigenetic rewiring of cancer cells towards cDC1-fate, activating antigen presentation and processing machinery. This reprogramming process is asynchronous and leads to the generation of intermediate populations with active tumor-APC gene expression signatures. Furthermore, tumor-APCs upregulated expression of HLA-ABC and the co-stimulatory molecules CD40, CD80, and CD86, suggesting the acquired ability to prime immune-effectors. Functionally, tumor-APCs secrete pro-inflammatory cytokines including IL12p70, TNFα, CXCL10, and IL-29 upon TLR3/4 stimulation with Poly:IC and LPS, are competent in phagocytosis of dead cells and proteins, and can activate antigen-specific CD8+ T-cells. Importantly, cDC1-cell fate adoption was accompanied by downregulation of proliferation and diminished tumorigenicity, as evidenced by the loss of anchorage-independent growth. Importantly, cDC1-reprogramming was efficient in primary tissues obtained from head and neck, breast, urothelial and pancreatic carcinoma, as well as melanoma patients. Notably, PIB-mediated cDC1 reprogramming of tumor cells is substantially augmented when combined with a histone deacetylase inhibitor, valproic acid. In summary, PIB expression imposes cDC1 fate and function in a broad range of human cancer cells suggesting a capacity to elicit anti-tumor responses by orchestrating innate and adaptive immunity. Our strategy combines cDC1 antigen-processing and presenting abilities with the endogenous generation of tumor-antigens. This approach merges cell fate reprogramming with cancer immunotherapy, paving the way for the development of novel gene therapy for cancer. Disclosures No relevant conflicts of interest to declare.

Introduction To describe the genetic relationship between the primary and recurrent bladder tumors and to identify their origins, different models such as the field cancerization, seeding, and intraepithelial migration have been proposed. The aim of this study was to investigate the genetic relationship in syn- and metachronous bladder tumors using different types of data, and to highlight supportive evidence for the proposed models. Methods The exophytic part of primary and recurrence urothelial tumors were collected by cold-cup biopsy from patients undergoing transurethral resection at the Lund University Hospital, Sweden, between 2001 and 2007. In total, 49 tumors from 18 patients and their 18 matched normal samples obtained from peripheral blood were collected and hybridized on HumanCNV370-Duo Genotyping BeadChips (Illumina Inc.). Gene expression data were generated using Illumina HumanHT-12 Expression BeadChip. Tumors were classified according the Lund classification system. For 27 tumor samples and matched normal blood samples from 10 patients, a custom target captured panel was used to sequence 1697 cancer genes. Results We used the fact that patients with non-muscle invasive bladder tumors show frequent local recurrences and often have multiple tumors to study re-initiation of tumor growth from the same urothelium. By extensive genomic analyses we show that tumors from the same patient are clonal. We show that gross genomic chromosomal aberrations may be present then absent in a recurrent tumor. By analyses of incompatible changes i.e., genomic alterations that cannot be reversed, we show that almost all tumors from a single patient may show such changes, thus the tumors cannot have originated from each other. Conclusions As recurring tumors share both genomic alterations and driver gene mutations these must have been present in the urothelium in periods with no overt tumor growth. The presence of similar mutational and CN profiles combined with incompatible events suggests that the primary and the recurrent tumors have a shared ancestry but are not originating from each other. This supports a model that includes a growing and evolving field of urothelial cells that occasionally, and locally, produce bursts of cellular growth leading to overt tumors.

Introduction Large efforts have been made to divide bladder cancer into prognostic or predictive subtypes by unsupervised or semi-supervised clustering of mRNA data. To validate and make use of subtypes, classifiers must be constructed that accurately predicts subtypes in new samples. Variations of nearest centroid classification is commonly used, where each class is represented as a vector of mean fold changes of selected genes. The similarity to these vectors is then used to predict the class of a new sample. Using relative fold change values means that classification becomes related to both the composition of the training cohort and the cohort the new sample originates from. Without thorough data preprocessing, this can result in major classification errors. Optimally, we want to classify a new sample in isolation in a manner robust to preprocessing, sample purity, and mRNA measurement technology. Methods We evaluated single sample classification methods based on using sets of gene-pair rules (e.g. Gene A>Gene B indicates Subtype X), using the R packages AIMS and SwitchBox, and a custom iterative RandomForest (RF) approach, as well as the single sample centroid approach used by Kamoun et. el. These were tested on Lund, TCGA, and IMvigor210 data. Results Rule-based and centroid based single sample classifiers were able to achieve similar accuracy in predicting LundTaxonomy subtypes in the datasets. The single sample centroid classifier based on raw expression values suffered from poor separation between predictions (delta between best and second-best prediction), which becomes worse as more genes were included. Rule-based methods, particularly RF, had much more distinct subtype calls. The custom RF approach outperformed the published AIMS and SwitchBox methods. Conclusions Each single sample classifier method had strengths and limitations. The RF based approach emerged as a promising new method, being robust even when using very few genes, while also allowing for evaluation of class stability and putative incorrect reference labels during training. The main challenge is how to select which gene-rules to use in the classifier. An R package (“multiclassPairs”) for building SwitchBox and RandomForest classifier models is currently being prepared.

Schistosomiasis is the most important helminthiasis worldwide in terms of morbidity and mortality. Most of the infections occurs in Africa, which about two thirds are caused by Schistosoma haematobium. The infection with S. haematobium is considered carcinogenic leading to squamous cell carcinoma (SCC) and urothelial carcinoma of the urinary bladder. Additionally, it is responsible for female genital schistosomiasis leading to infertility and higher risk of human immunodeficiency virus (HIV) transmission. Remarkably, a recent outbreak in Corsica (France) drew attention to its potential re-mergence in Southern Europe. Thus far, little is known related to host-parasite interactions that trigger carcinogenesis. However, recent studies have opened new avenues to understand mechanisms on how the parasite infection can lead cancer and other associated pathologies. Here, we present a historical perspective of schistosomiasis, and review the infection-associated pathologies and studies on host–parasite interactions that unveil tentative mechanisms underlying schistosomiasis-associated carcinogenesis.

Estrogen-like metabolites have been identified in S. haematobium, the helminth parasite that causes urogenital schistosomiasis (UGS) and in patients´ blood and urine during UGS. Estrogen receptor (ER) activation is enriched in the luminal molecular subtype bladder cancer (BlaCa). To date, the significance of ER to these diseases remains elusive. We evaluated ERα and ERβ expression in UGS-related BlaCa (n = 27), UGS-related non-malignant lesions (n = 35), and noninfected BlaCa (n = 80). We investigated the potential of ERα to recognize S. haematobium-derived metabolites by docking and molecular dynamics simulations and studied ERα modulation in vitro using 3 BlaCa cell lines, T24, 5637 and HT1376. ERα was expressed in tumor and stromal cells in approximately 20% noninfected cases and in 30% of UGS-related BlaCa, predominantly in the epithelial cells. Overall, ERα expression was associated with features of tumor aggressiveness such as high proliferation and p53 positive expression. ERα expression correlated with presence of schistosome eggs. ERβ was widely expressed in both cohorts but weaker in UGS-related cases. molecular dynamics simulations of the 4 most abundant S. haematobium-derived metabolites revealed that smaller metabolites have comparable affinity for the ERα active state than 17β-estradiol, while the larger metabolites present higher affinity. Our in vitro findings suggested that ERα activation promotes proliferation in ERα expressing BlaCa cells and that this can be reverted with anti-estrogenic therapy. In summary, we report differential ER expression between UGS-related BlaCa and noninfected BlaCa and provide evidence supporting a role of active ERα during UGS and UGS-induced carcinogenesis. Highlights: Estrogen receptor (ERs) are expressed in both urogenital schistosomiasis and bladder cancer; ERα was associated with aggressive tumor features and presence of schistosome eggs; In silico analysis shows an agonistic putative action of S. haematobium-derived metabolites for ERα; ERα induced proliferation in bladder cancer cells can be reverted by anti-estrogens; Evidence suggests that activation of ERα could play a role in urogenital schistosomiasis and urogenital schistosomiasis-induced carcinogenesis. PJB acknowledges support from award R01CA164719 from the National Cancer Institute (NCI), National Institutes of Health (NIH), USA. info:eu-repo/semantics/publishedVersion

Patient-derived xenograft (PDX ) tumors are models developed by direct transplant of human tumors into immune-compromised hosts such as nude mice. These models retain the histological and genetic characteristics of the primary tumor and are considered a valuable platform for translational cancer research. This chapter describes the methodology to establish and propagate bladder cancer PDX model.

The lipidome of the red seaweed Gracilaria sp., cultivated on land-based integrated multitrophic aquaculture (IMTA) system, was assessed for the first time using hydrophilic interaction liquid chromatography-mass spectrometry and tandem mass spectrometry (HILIC–MS and MS/MS). One hundred and forty-seven molecular species were identified in the lipidome of the Gracilaria genus and distributed between the glycolipids classes monogalactosyl diacylglyceride (MGDG), digalactosyl diacylglyceride (DGDG), sulfoquinovosyl monoacylglyceride (SQMG), sulfoquinovosyl diacylglyceride (SQDG), the phospholipids phosphatidylcholine (PC), lyso-PC, phosphatidylglycerol (PG), lyso-PG, phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatic acid (PA), inositolphosphoceramide (IPC), and betaine lipids monoacylglyceryl- and diacylglyceryl-N,N,N-trimethyl homoserine (MGTS and DGTS). Antiproliferative and anti-inflammatory effects promoted by lipid extract of Gracilaria sp. were evaluated by monitoring cell viability in human cancer lines and by using murine macrophages, respectively. The lipid extract decreased cell viability of human T-47D breast cancer cells and of 5637 human bladder cancer cells (estimated half-maximal inhibitory concentration (IC50) of 12.2 μg/mL and 12.9 μg/mL, respectively) and inhibited the production of nitric oxide (NO) evoked by the Toll-like receptor 4 agonist lipopolysaccharide (LPS) on the macrophage cell line RAW 264.7 (35% inhibition at a concentration of 100 μg/mL). These findings contribute to increase the ranking in the value-chain of Gracilaria sp. biomass cultivated under controlled conditions on IMTA systems.

Several genomic regions are frequently altered and associated with the type, stage and progression of urinary bladder cancer (UBC). We present the characterization of 5637, T24 and HT1376 UBC cell lines by karyotyping, fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and multiplex ligation-dependent probe amplification (MLPA) analysis. Some cytogenetic anomalies present in UBC were found in the three cell lines, such as chromosome 20 aneuploidy and the loss of 9p21. Some gene loci losses (e.g. CDKN2A) and gains (e.g. HRAS, BCL2L1 and PTPN1) were coincident across all cell lines. Although some significant heterogeneity and complexity were detected between them, their genomic profiles exhibited a similar pattern to UBC. We suggest that 5637 and HT1376 represent the E2F3/RB1 pathway due to amplification of 6p22.3, concomitant with loss of one copy of RB1 and mutation of the remaining copy. The HT1376 presented a 10q deletion involving PTEN region and no alteration of PIK3CA region which, in combination with the inactivation of TP53, bears more invasive and metastatic properties than 5637. The T24 belongs to the alternative pathway of FGFR3/CCND1 by presenting mutated HRAS and over-represented CCND1. These cell lines cover the more frequent subtypes of UBC and are reliable models that can be used, as a group, in preclinical studies.

Infection due to Schistosoma haematobium is carcinogenic. However, the cellular and molecular mechanisms underlying urogenital schistosomiasis (UGS)-induced carcinogenesis have not been well defined. Conceptually, early molecular detection of this phenomenon, through non-invasive procedures, seems feasible and is desirable. Previous analysis of urine collected during UGS suggests that estrogen metabolites, including depurinating adducts, may be useful for this purpose. Here, a new direction was pursued: the identification of molecular pathways and potential biomarkers in S. haematobium-induced bladder cancer by analyzing the proteome profiling of urine samples from UGS patients. GeLC-MS/MS followed by protein-protein interaction analysis indicated oxidative stress and immune defense systems responsible for microbicide activity are the most representative clusters in UGS patients. Proteins involved in immunity, negative regulation of endopeptidase activity, and inflammation were more prevalent in UGS patients with bladder cancer, whereas proteins with roles in renal system process, sensory perception, and gas and oxygen transport were more abundant in subjects with urothelial carcinoma not associated with UGS. These findings highlighted a Th2-type immune response induced by S. haematobium, which seems to be further modulated by tumorigenesis, resulting in high-grade bladder cancer characterized by an inflammatory response and complement activation alternative pathway. These findings established a starting point for the development of multimarker strategies for the early detection of UGS-induced bladder cancer. This work was supported by the Portuguese Foundation for Science and Technology (FCT), European Union, QREN, FEDER, and COMPETE for funding the QOPNA; by iBiMED research unit (project PEst-C/QUI/UI0062/2013, UID/BIM/04501/2013, UID/IC/00051/2013, and COST action BM1305) and PhD fellowship SFRH/BD/80855/2011 (CB); and by the Portuguese Mass Spectrometry Network (RNEM). info:eu-repo/semantics/publishedVersion

Introduction To describe the origin and the genetic relationship between primary bladder tumours and their recurrences, different models were proposed, such as, field cancerization, seeding, and intraepithelial migration. This study aims to investigate the genetic relationship in syn- and metachronous bladder tumours using different types of data, and to evaluate the proposed models. Material and methods The exophytic part of primary and recurrent urothelial tumours were collected by cold-cup biopsies from patients undergoing transurethral resection at the Lund University Hospital, Sweden, between 2001 and 2007. In total, 51 tumours from 19 patients and their 19 matched peripheral blood samples were collected and hybridised on Illumina HumanCNV370-Duo Genotyping array. Copy number data was extracted from GenomeStudio 2 software, and normalised using transformed quantile normalisation (tQN). Mirrored B allele frequency values were segmented using BAFsegmentation. Mutation calling was performed using targeted cancer gene sequencing panel. Gene expression data was generated using Illumina HumanHT-12 expression array. Results and discussions A manual inspection of all copy number alterations (CNAs) profiles was performed. Normal cells content, tumour ploidy, and sub clonality were calculated, and the relationship between the primary and recurrent tumours were estimated. Preliminary results show that CNAs are early and stable events during the tumour evolution. Part of the syn- and metachronous bladder tumours shared similar mutational and CNAs profiles. However, even with similar profiles, some events such as loss of heterozygosity in the primary tumours, were not observed in the recurrent tumours, constituting incompatible events. Samples with mutations in TP53 or amplification in MDM2 showed higher number of break points in the genome. A further analysis will be performed using the available CNAs, expression, and mutational data. Conclusion The genetic relationship in syn- and metachronous bladder tumours was investigated using different data types providing a better understanding of bladder cancer evolution. The presence of similar mutational and CNAs profiles combined with incompatible events suggests that the primary and the recurrent tumours were generated separately from a common ancestor. These findings highlight a tumour evolution scenario of a clonal cancerized field.

e16013 Background: Epidemiological and molecular findings suggest that estrogen may play a role in bladder carcinogenesis and in urogenital schistosomiasis (UGS). Methods: A comprehensive analysis of expression of estrogen receptors (ER) in urothelial carcinomas (UC), n = 80 from non-infected patients and UGS-related bladder carcinoma (BlaCa) n = 27 was undertaken. I n vitro, we evaluated the effect of estradiol (E2) and anti-estrogens on the survival of human BlaCa cells. In addition, molecular dynamics simulations are ongoing to assess the ability of estradiol-like metabolites produced by the Schistosoma haematobiumto bind ERs. Results: Tumor cell expression of ERα was found in 17.5% of the non-infected cases, and was associated with proliferative tumors (p = 0.004, ES = 0.326) and lower overall survival. Expression of ERα was also found in normal urothelium adjacent to the tumor and in tumor stromal cells, particularly in T2 and late stage tumors (p = 0.034, ES = 0.498and p = 0.037, ES = 0.357, respectively). In UGS-related BlaCa, 22% of tumor cells were ERα positive. Presence of parasite eggs was associated with higher ERα expression in cancer cells and adjacent urothelium (p = 0.038, ES = 0.399 and p = 0.020, ES = 0.600, respectively). ERβ was found in 91% of non-infected UC and 78% of the UGS-related tumors. ERβ was equally expressed across different tumor stages and grade but more intense in tumor cells than in urothelium of normal appearance and in non-infected than in UGS-related cases. In the latter group, staining intensity was increased in women and in samples with parasite eggs. Using 3 human BlaCa cell lines, HT1374, T24 and 5637, we observed that 5637 cells express ERα and can be stimulated by E2 and inhibited by the anti-estrogens (4OH-tamoxifen and ICI 182 780). T24 cells expressed ERα at very low levels and did not respond to E2, but were inhibited by anti-estrogens; HT1376 do not express ERα. ERβ mRNA was not detected in any of the cell lines. Conclusions: Our results document an association between expression of ERα and aggressive features. The potential of expression of ERs as a prognostic tool and as therapeutic target should be further investigated especially for tumors with aggressive profile and during UGS-related carcinogenesis.

Patient-derived tumor xenografts (PDTXs) are said to accurately reflect the heterogeneity of human tumors. In the case of human bladder cancer, few studies are available featuring these models. The best methodology to develop and the real value of the model remain unclear. This systematic review aims to elucidate the best methodology to establish and use PDTXs to study the characteristics and behavior of human bladder tumors. The value and potential application of these models are also addressed. A comprehensive literature search was performed to identify published studies using xenograft models directly established from human bladder cancer samples into mice. A total of 12 studies were included in the final analysis. All studies differed in design; the reported take rate varied between 11% and 80%, with the implantation via dorsal incision and with matrigel obtaining the higher take rate. Advanced stage and high-grade tumors were associated with increased take rate. Xenografts preserved the original tumor identity in the establishment phase and after serial passages. Although some studies suggest a correlation between engraftment success and clinical prognosis, evidence about the association between the response of xenografts to treatment and the clinical response of the tumor of origin is still missing. All methodological approaches resulted in the establishment of tumor xenografts with preservation of the original tumor identity but variable take rate. The time needed to establish the model and propagate xenografts to a number suitable for drug testing is the main limitation of the model, along with the success rate and lack of consistency in the early passages. Comparison between tumor response in mice and clinical outcome remains to be assessed.

More than 70% of muscle invasive bladder cancers (MIBC) express the cell-surface antigen sialyl-Tn (sTn) that promotes motility and invasive potential of tumor cells. Effective drug testing models to optimize therapy against these tumors are warranted.Fragments of sTn-positive MIBC were subcutaneously engrafted into nude mice and expanded until the third passage. Histology and immunoexpression of tumor markers (p53, p63, Ki-67, CK20, sTn) were studied in order to evaluate tumor phenotype maintenance.Tumor take rate was low in the first passage (1/9) but increased and became consistent, therefore suitable for drug testing, in the third passage (13/13). Histology and immunoexpression patterns were similar between primary tumors and xenografts. However, p53 and ki-67 levels increased with passages suggesting a selection of more proliferative clones. STn expression, even though decreased, was preserved in xenografts.We describe the first patient-derived sTn-positive xenograft model to be used for drug testing and identification of prognostic biomarkers.