Metrics details Bladder cancer (BlCa) taxonomy has proved its impact in patient outcome and selection for targeted therapies but such transcriptomic-based classification has not yet translated to routine practice epithelial-to-mesenchymal transition (EMT) has shown relevance in acquisition of more aggressive BlCa phenotype We aimed to test the usefulness of the molecular classification as defined by immunohistochemistry (a routinely performed and easy-to-implement technique) in a well-defined BlCa cohort of both non-muscle invasive (NMIBC) and muscle invasive (MIBC) disease we aimed to assess the additional prognostic value of the mesenchymal marker vimentin to the stratification strategy Immunohistochemistry/RT-qPCR for luminal markers GATA3/FOXA1 basal markers KRT5/KRT6A and vimentin were performed mRNA expression levels of the markers positively correlated with immunoexpression scores We found substantial overlapping in immunoexpression of luminal and basal markers basal tumors developed recurrence more frequently NMIBC patients with higher vimentin immunoexpression endured poorer disease-free survival and increased expression was observed from normal bladder-NMIBC-MIBC-metastases The classification has the potential to be implemented in routine but further adjustments in practical scoring should be defined; focusing on additional markers may further refine BlCa molecular taxonomy Patients and controls were enrolled after informed consent This study was approved by the institutional review board (Comissão de Ética para a Saúde) of IPO Porto (CES103-14) In total, 186 samples were available for immunohistochemistry studies: the 126 primary BlCa specimens, plus the 25 normal bladder mucosae and 35 BlCa metastases. Immunohistochemistry methods are described in detail in Additional file 1: Table S1 three micrometer-thick tissue sections from the formalin-fixed and paraffin-embedded samples were ordered and slides were incubated with the primary antibodies for FOXA1 3,3′-diaminobenzidine (Sigma-Aldrich™) was used as chromogen for visualization and slides were counterstained with hematoxylin Appropriate tissue controls were used per run Immunoexpression patterns were evaluated by a dedicated uropathologist Cases were classified using a semi-quantitative scale for both staining intensity (0—no staining; 1—low intensity only barely discernible at 400 × magnification; 2—moderate intensity well appreciated at 400× magnification but faint at 100× magnification; 3—high intensity strong and well appreciated at 40× magnification) and percentage of positive cells (0— < 10%; 1—10–33%; 2—33–67%; 3— > 67%) Results were then combined in a single continuous score (Score S = staining intensity × percentage of positive cells) assigned to each tumor mRNA expression analyses were performed on fresh frozen tissues available for 108 of the patients included in the study (all were run for VIM expression RNA was extracted from tissues using TRIzol® (Invitrogen RNA quantification and purity were assessed in NanoDrop™ Lite Spectophotometer (Cat cDNA synthesis was performed using the RevertAid™ RT Reverse Transcription Kit (Cat The reaction was performed in MyCycler™ Thermal Cycler System (Cat Bio-Rad) using the following conditions: 5 min at 25 °C VIM mRNA expression levels were evaluated using 4.5 µL of diluted cDNA 5 µL of TaqMan® Universal PCR Master Mix No AmpErase® UNG (Applied Biosystems®) and 0.5 µL of TaqMan® Gene Expression Assay two TaqMan® Gene Expression assays were used as internal controls: beta-glucoronidase—GUSB—assay ID Hs99999908 Applied biosystems®; and Hypoxanthine–guanine phosphoribosyltransferase—HPRT1—assay ID Hs01003267 in an ABI 7500 Real Time PCR System (Thermo Fisher) in the following conditions: 2 min at 50 °C followed by enzyme activation for 10 min at 95 °C and 45 cycles which included a denaturation stage at 95 °C for 15 s and an extending stage at 60 °C for 60 s Serial dilutions of cDNA obtained from Human Reference Total RNA (Cat Agilent Technologies®) were used to compute standard curves for each plate All experiments were run in triplicate and two negative controls were included in each plate Relative expression of target genes tested in each sample was determined as: [Gene Expression Level = (Gene Mean Quantity/(HPRT1 & GUSB) Mean Quantity) × 1000] For GATA3, FOXA1, KRT5 and KRT6A genes, transcript levels were also assessed using 2.5 µL of diluted cDNA, 0.25 µL of forward and reverse primers (Additional file 2: Table S2) 5 µL of Xpert Fast SYBER Mastermix Blue (GRiSP Research Solutions GUSB was used for normalization and plates were set as described above The run followed the following conditions: 2 min at 95 °C followed by 45 cycles of 5 s at 95 °C and 30 s at 60 °C Data was tabulated using Microsoft Excel 2016 and analyzed and plotted using GraphPad Prism 6 and IBM Statistical Package for Social Sciences (SPSS v24) Percentages were calculated based on the number of cases with available data together with median and interquartile range Mann–Whitney and Kruskal–Wallis tests were used for comparing expression levels among samples p-values were adjusted for multiple comparisons using Dunn’s test Chi square and Fisher exact test were used as necessary for establishing associations between categorical variables Spearman correlation test was used to correlate continuous variables Disease-specific survival (DSS) and disease-free survival (DFS) curves were plotted using Kaplan–Meier statistics and Cox regression models with respective hazard ratios (HR) were computed Statistical significance was set at p < 0.05 there was no significant association between the luminal/basal-like subtype (as defined by immunohistochemistry described above) and the event of metastization (p = 0.933) the “basal-like” cases disclosed disease recurrence in 8/20 cases (40.0%) and the “luminal-like” in a similar proportion of cases (13/31 “basal-like” cancer developed recurrence in 11/34 cases (32.4%) whereas in “luminal-like” this occurred in a lower proportion of patients [only 5/38 cases (13.2%)] Concerning survival analyses, the luminal/basal-like classification did not show significant impact on DSS or DFS, both for NMIBC or MIBC (NMIBC: p = 0.762 and p = 0.625; MIBC: p = 0.346, p = 0.185, respectively). Illustrative examples of immunoexpression patterns for the several markers are depicted in Fig. 1. Immunoexpression of luminal and basal markers in the bladder cancer cohort b FOXA1 strong and diffuse immunoexpression in two bladder cancer specimens d: GATA3 strong and diffuse immunoexpression in two bladder cancer specimens f: CK5/6 strong multifocal immunoexpression in two bladder cancer specimens Correlation between mRNA and protein expression of the several luminal and basal markers in the bladder cancer cohort (both MIBC and NMIBC included) KRT5 (e and f) and KRT6A (g and h) analyses mRNA expression levels are plotted as relative expression levels Red dash and bars represent median and interquartile range The immunoexpression score (intensity × percentage) is plotted in the xx-axis The graphs include n = 83 matched samples (*p < 0.05; ****p < 0.0001) Vimentin transcript and protein levels within the bladder cancer cohort a differential mRNA expression of vimentin between non-muscle (NMIBC) and muscle-invasive (MIBC) bladder cancer normalized to GUSB and HPRT1; b differential protein (immuno)expression of vimentin between NMIBC and MIBC The immunoexpression score (intensity × percentage) is plotted; c immunoexpression of vimentin among normal bladder The immunoexpression score (intensity × percentage) is plotted Correction for multiple comparisons was employed and adjusted p-values are represented (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001) Disease-free survival in non-muscle invasive bladder cancer (NMIBC) patients according to vimentin protein expression Immunoexpression of vimentin in the bladder cancer cohort b: immunoexpression of vimentin in primary bladder cancer specimens one NMIBC (a) and one MIBC (b); c and d: immunoexpression of vimentin in bladder cancer metastases that can be determined in tissue samples upon radical cystectomy and also non-invasively The main aim of our work was to assess the protein expression of these markers and attempt to classify these tumors in a well-defined cohort of BlCa representative of the diagnostic routine of a tertiary cancer center This also substantiates the applicability of the classification We hypothesize that the classification could also be extended to upper urothelial tract carcinomas with 15/57 tumors (26.3%) showing CK5/6 immunoexpression (data not shown) Our work goes further and indicates the clinical potential of VIM as a prognostic marker within luminal vs Limitations of this work include its retrospective nature and the relatively low number of samples with complete clinical information available not all samples in which immunohistochemistry was performed had fresh-frozen material available for performing transcript analyses although immunohistochemistry may be subjected to inter-observer variability allowing for evaluating morphology simultaneously and perceiving details related to tumor heterogeneity this work also extends the molecular classification to NMIBC which should be further explored in the future we show that BlCa molecular classification has the potential to be effectively translated to the diagnostic routine but effort must be made to consistently define the tumor categories acknowledged by transcriptomic studies using routine techniques with the ultimate goal of maintaining the same clinically meaningful input expression of EMT markers may be useful for predicting relapse and adjusting therapeutic strategy in which it provided useful prognostic information and dictated survival outcome Adjunctive markers to the molecular classification merit attention as they might further improve BlCa molecular taxonomy The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries Bladder cancer incidence and mortality: a global overview and recent trends Economic burden of bladder cancer across the European Union WHO Classification of Tumours of the Urinary System and Male Genital Organs (4th Edition) Novel therapeutic targets in advanced urothelial carcinoma MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Immunotherapy: a new treatment paradigm in bladder cancer A consensus molecular classification of muscle-invasive bladder cancer Molecular subtypes of urothelial bladder cancer: results from a meta-cohort analysis of 2411 tumors Molecular subtypes of bladder cancer: academic exercise or clinical relevance Molecular subtypes of non-muscle invasive bladder cancer Patient-derived non-muscular invasive bladder cancer Xenografts of main molecular subtypes of the tumor for Anti-Pd-l1 treatment assessment Identifying distinct classes of bladder carcinoma using microarrays Bladder cancer outcome and subtype classification by gene expression Combined gene expression and genomic profiling define two intrinsic molecular subtypes of urothelial carcinoma and gene signatures for molecular grading and outcome A molecular taxonomy for urothelial carcinoma A validation and extended description of the Lund taxonomy for urothelial carcinoma using the TCGA cohort Comprehensive molecular characterization of urothelial bladder carcinoma Molecular subtypes and response to immunotherapy in bladder cancer patients Identification of distinct basal and luminal subtypes of muscle-invasive bladder cancer with different sensitivities to frontline chemotherapy Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm Impact of molecular subtypes in muscle-invasive bladder cancer on predicting response and survival after neoadjuvant chemotherapy Bladder cancer: two molecular subtypes identified Comprehensive molecular characterization of muscle-invasive bladder cancer Epigenetic mechanisms influencing epithelial to mesenchymal transition in bladder cancer Epithelial-to-mesenchymal transition: event and core associates in bladder cancer Prognostic role of epithelial-mesenchymal transition markers “E-Cadherin Role of epithelial-to-mesenchymal transition (EMT) in drug sensitivity and metastasis in bladder cancer Epithelial-To-mesenchymal transition and its correlation with clinicopathologic features in patients with urothelial carcinoma of the bladder Expression profile of epithelial-mesenchymal transition markers in non-muscle-invasive urothelial carcinoma of the bladder: correlation with intravesical recurrence following transurethral resection Update on the management of non-muscle invasive bladder cancer A multiplex test assessing MiR663ame and VIMme in urine accurately discriminates bladder cancer from inflammatory conditions Urinary TERT promoter mutations as non-invasive biomarkers for the comprehensive detection of urothelial cancer EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2016 Immunotherapy in urothelial cancer: recent results and future perspectives Targeting the immune system and epigenetic landscape of urological tumors Liquid biopsy biomarkers in bladder cancer: a current need for patient diagnosis and monitoring Liquid biopsies in the management of bladder cancer: next-generation biomarkers for diagnosis New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer Epithelial-mesenchymal transitions: twist in development and metastasis Differential expression of E-cadherin and P-cadherin in pT3 prostate cancer: correlation with clinical and pathological features E-cadherin clone 36 nuclear staining dictates adverse disease outcome in lobular breast cancer patients Epithelial to mesenchymal transition in renal cell carcinoma: implications for cancer therapy Epithelial-mesenchymal transition in gastric cancer Dysregulation of EMT drives the progression to clinically aggressive sarcomatoid bladder cancer Characteristics of bladder transitional cell carcinoma with E-cadherin and N-cadherin double-negative expression Cell adhesion and urothelial bladder cancer: the role of cadherin switching and related phenomena Prognostic significance of the epithelial-to-mesenchymal transition markers e-cadherin Overexpression of vimentin: role in the invasive phenotype in an androgen-independent model of prostate cancer Vimentin and epithelial-mesenchymal transition in human breast cancer–observations in vitro and in vivo E-cadherin and Par6 expression in epithelial and stromal compartment in non-small-cell lung cancer Identification and prognostic significance of an epithelial-mesenchymal transition expression profile in human bladder tumors Epithelial mesenchymal transition in urothelial carcinoma: twist in the tale Sarcomatoid urothelial carcinoma of the bladder: analysis of 28 cases with emphasis on clinicopathologic features and markers of epithelial-to-mesenchymal transition Download references This research was funded by Research Center of Portuguese Institute of Porto (CI-IPOP-27-2016) JL and SM-R are recipients of fellowships from FCT - Fundação para a Ciência e Tecnologia (SFRH/BD/132751/2017 and SFRH/BD/112673/2015 Catarina Guimarães-Teixeira shared first authorship Cancer Biology and Epigenetics Group IPO Porto Research Center (GEBC CI-IPOP) Portuguese Oncology Institute of Porto (IPO Porto) & Porto Comprehensive Cancer Center (P.CCC) Portuguese Oncology Institute of Porto (IPOP) Department of Pathology and Molecular Immunology Institute of Biomedical Sciences Abel Salazar SM-R and CG-T performed molecular analyses and wrote the manuscript JL collected the clinical data and reviewed histological specimens IC prepared the histological sections for immunohistochemistry CJ and RH supervised the work and revised the manuscript All authors read and approved the final manuscript This study was approved by the ethics committee of Portuguese Oncology Institute of Porto (Comissão de Ética para a Saúde-CES103-14) All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards The authors declare that they have no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations  S1: Immunoexpression of neuroendocrine markers in a bladder cancer specimen negative for CK5/6 A: CD56; B: Synaptophysin; C: Chromogranin unless otherwise stated in a credit line to the data Download citation DOI: https://doi.org/10.1186/s12967-020-02475-w Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article (ANS - Manique) – Fr Jeronimo Rocha Monteiro passed away on August 31st in the city of Manique 61 spent in the Salesian religious life and 50 as a priest he served the World Federation of Don Bosco Past Pupils and visited many countries in the East Asia-Oceania region to the province of Portugal and to the World Federation of Past Pupils Fr Cereda expressed great appreciation for the strength with which Fr Jeronimo Rocha Monteiro carried out his work Fr Monteiro made his first religious profession in the Salesian Congregation in Manique on August 16 In his life Fr Monteiro held positions as director advisor and vicar of various Salesian communities was director of "Edições Salesianas" of Portugal (2001-2002) Provincial Delegate for the Salesian Family since 2008 he had brilliantly held the role of World Delegate of the World Confederation of Past Pupils wished to express his thoughts on social networks "Today I learned the news that Fr Jeronimo Rocha Monteiro has returned to the House of the Father," he wrote "He has always been friendly and very close to the whole Salesian Family and It is right to remember his poems and his musical compositions always ready to put himself at the service of others and published after his visit to East Timor on the Salesian Bulletin Its title is "Timor": "Breath of a sea of new people welcome people rich in soul and limitless in their generosity." 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