The Lund taxonomy for bladder cancer classification - from gene expression clustering to cancer cell molecular phenotypes, and back again.

Treatment of bladder cancer patients depends on precise diagnosis. Molecular subtyping by gene expression profiling may contribute substantially to subclassification of bladder cancer. Several classification systems have been proposed. Most of these base their classification on whole biopsy features, and molecular subtypes are therefore often defined by a combination of features from the cancer cells as well as infiltrating noncancer cells. This makes the link to what is seen at the cancer cell level unclear. The aim of the Lund taxonomy (LundTax) has been to align gene expression-level classification with immunohistochemical classification to identify cancer cell phenotypes independent of infiltration and proliferation. A systematic approach was used in which gene expression clusters were validated and adjusted by immunohistochemistry using markers expressed only by the cancer cells. This review provides a rationale for defining molecular subtypes and a step-by-step description of the development of the LundTax with motivations for each modification and extension. As the cancer cell phenotype defined by gene expression profiling corresponds with the immunohistochemistry of cancer cells, the LundTax represents a harmonization of the gene expression and immunohistochemical levels. Furthermore, the classification system is independent of pathological stage and is, thus, applicable to all urothelial carcinomas. A unified classification system relevant for both the molecular biologist and pathologist will facilitate systematization of current treatment practices, as well as the development of new treatments. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A comparison of rule-based and centroid single-sample multiclass predictors for transcriptomic classification.

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.

A quantitative polymerase chain reaction based method for molecular subtype classification of urinary bladder cancer-Stromal gene expressions show higher prognostic values than intrinsic tumor genes.

Transcriptome-based molecular subtypes of muscle-invasive bladder cancer (MIBC) have been shown to be both prognostic and predictive, but are not used in routine clinical practice. We aimed to develop a feasible, reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based method for molecular subtyping. First, we defined a 68-gene set covering tumor intrinsic (luminal, basal, squamous, neuronal, epithelial-to-mesenchymal, in situ carcinoma) and stromal (immune, extracellular matrix, p53-like) signatures. Then, classifier methods with this 68-gene panel were developed in silico and validated on public data sets with available subtype class information (MD Anderson [MDA], The Cancer Genome Atlas [TCGA], Lund, Consensus). Finally, expression of the selected 68 genes was determined in 104 frozen tissue samples of our MIBC cohort by RT-qPCR using the TaqMan Array Card platform and samples were classified by our newly developed classifiers. The prognostic value of each subtype classification system and molecular signature scores were assessed. We found that the reduced marker set combined with the developed classifiers were able to reproduce the TCGA II, MDA, Lund and Consensus subtype classification systems with an overlap of 79%, 76%, 69% and 64%, respectively. Importantly, we could successfully classify 96% (100/104) of our MIBC samples by using RT-qPCR. Neuronal and luminal subtypes and low stromal gene expressions were associated with poor survival. In conclusion, we developed a robust and feasible method for the molecular subtyping according to the TCGA II, MDA, Lund and Consensus classifications. Our results suggest that stromal signatures have a superior prognostic value compared to tumor intrinsic signatures and therefore underline the importance of tumor-stroma interaction during the progression of MIBC.

Complete metabolic response with [18 F]fluorodeoxyglucose-positron emission tomography/computed tomography predicts survival following induction chemotherapy and radical cystectomy in clinically lymph node positive bladder cancer.

OBJECTIVE: To determine whether repeated [18 F]fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET-CT) scans can predict increased cancer-specific survival (CSS) after induction chemotherapy followed by radical cystectomy (RC). PATIENTS AND METHODS: Between 2007 and 2018, 86 patients with clinically lymph node (LN)-positive bladder cancer (T1-T4, N1-N3, M0-M1a) were included and underwent a repeated FDG-PET-CT during cisplatin-based induction chemotherapy. The 71 patients that had a response to chemotherapy underwent RC. Response to chemotherapy was evaluated in LNs through repeated FDG-PET-CT and stratified as partial response or complete response using three different methods: maximum standardised uptake value (SUVmax ), adapted Deauville criteria, and total lesion glycolysis (TLG). Progression-free survival (PFS) and CSS were analysed for all three methods by Cox regression analysis. RESULTS: After a median follow-up of 40 months, 15 of the 71 patients who underwent RC had died from bladder cancer. Using SUVmax and the adapted Deauville criteria, multivariable Cox regression analyses adjusting for age, clinical tumour stage and LN stage showed that complete response was associated with increased PFS (hazard ratio [HR] 3.42, 95% confidence interval [CI] 1.20-9.77) and CSS (HR 3.30, 95% CI 1.02-10.65). Using TLG, a complete response was also associated with increased PFS (HR 5.17, 95% CI 1.90-14.04) and CSS (HR 6.32, 95% CI 2.06-19.41). CONCLUSIONS: Complete metabolic response with FDG-PET-CT predicts survival after induction chemotherapy followed by RC in patients with LN-positive bladder cancer and comprises a novel tool in evaluating response to chemotherapy before surgery. This strategy has the potential to tailor treatment in individual patients by identifying significant response to chemotherapy, which motivates the administration of a full course of induction chemotherapy with a higher threshold for suspending treatment due to toxicity and side-effects.

Molecular changes during progression from nonmuscle invasive to advanced urothelial carcinoma.

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.

Molecular profiling in muscle-invasive bladder cancer: more than the sum of its parts.

Bladder cancers are biologically and clinically heterogeneous. Recent large-scale transcriptomic profiling studies focusing on life-threatening muscle-invasive cases have demonstrated a small number of molecularly distinct clusters that largely explain their heterogeneity. Similar to breast cancer, these clusters reflect intrinsic urothelial cell-type differentiation programs, including those with luminal and basal cell characteristics. Also like breast cancer, each cell-based subtype demonstrates a distinct profile with regard to its prognosis and its expression of therapeutic targets. Indeed, a number of studies suggest subtype-specific differential responses to cytotoxic chemotherapy and to therapies that inhibit a number of targets, including growth factors (EGFR, ERBB2, FGFR) and immune checkpoint (PD1, PDL1) inhibitors. Despite burgeoning evidence for important clinical implications, subtyping has yet to enter into routine clinical practice. Here we review the conceptual basis for intrinsic cell subtyping in muscle-invasive bladder cancer and discuss evidence behind proposed clinical uses for subtyping as a prognostic or predictive test. In deliberating barriers to clinical implementation, we review pitfalls associated with transcriptomic profiling and illustrate a simple immunohistochemistry (IHC)-based subtyping algorithm that may serve as a faster, less expensive alternative. Envisioned as a research tool that can easily be translated into routine pathology workflow, IHC-based profiling has the potential to more rapidly establish the utility (or lack thereof) of cell type profiling in clinical practice. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular pathology of the luminal class of urothelial tumors.

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.

FGFR3 mutation increases bladder tumourigenesis by suppressing acute inflammation.

Recent studies of muscle-invasive bladder cancer show that FGFR3 mutations are generally found in a luminal papillary tumour subtype that is characterised by better survival than other molecular subtypes. To better understand the role of FGFR3 in invasive bladder cancer, we examined the process of tumour development induced by the tobacco carcinogen OH-BBN in genetically engineered models that express mutationally activated FGFR3 S249C or FGFR3 K644E in the urothelium. Both occurrence and progression of OH-BBN-driven tumours were increased in the presence of an S249C mutation compared to wild-type control mice. Interestingly, at an early tumour initiation stage, the acute inflammatory response in OH-BBN-treated bladders was suppressed in the presence of an S249C mutation. However, at later stages of tumour progression, increased inflammation was observed in S249C tumours, long after the carcinogen administration had ceased. Early-phase neutrophil depletion using an anti-Ly6G monoclonal antibody resulted in an increased neutrophil-to-lymphocyte ratio at later stages of pathogenesis, indicative of enhanced tumour pathogenesis, which supports the hypothesis that suppression of acute inflammation could play a causative role. Statistical analyses of correlation showed that while initial bladder phenotypes in morphology and inflammation were FGFR3-dependent, increased levels of inflammation were associated with tumour progression at the later stage. This study provides a novel insight into the tumour-promoting effect of FGFR3 mutations via regulation of inflammation at the pre-tumour stage in the bladder. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Discordant molecular subtype classification in the basal-squamous subtype of bladder tumors and matched lymph-node metastases.

Molecular subtypes of muscle-invasive bladder tumors have emerged as a promising research tool with potential to stratify patients for neoadjuvant treatment. Prior to radical cystectomy, the utility of molecular classification and biomarkers depend on concordance between tissue from transurethrally resected specimens and disseminated disease. We assess the concordance of molecular subtypes and a large number of potential biomarkers in 67 pairs of muscle-invasive bladder tumors and synchronous lymph-node metastases. Tissue cores were stained for 29 immunohistochemistry markers and immunohistochemistry-based molecular subtype classification was performed. Molecular subtype was determined by mRNA profiling for 57 bladder tumors and 28 matched lymph-node metastases. Full section immunohistochemistry was performed to assess intra-tumor subtype heterogeneity in discordant cases, and exome sequencing was performed for 20 sample pairs. Discordant subtype classification between the bladder tumor and lymph-node metastasis was generally rare (12/67, 18%), but most (7/12, 58%) involved the Basal/Squamous-like subtype. Discordant Basal/Squamous-like tumors showed either Urothelial-like or Genomically Unstable, luminal-like phenotype in the lymph-node metastasis. Full section immunohistochemistry revealed intra-tumor subtype heterogeneity for six discordant cases including four involving the Basal/Squamous-like subtype. Subtype concordance for non- Basal/Squamous-like tumors was 91%. RNA-based classification agreed with immunohistochemistry classification but quantitative assessment is necessary to avoid false detection of subtype shifts. Most high confidence cancer mutations were shared between samples (n = 93, 78%), and bladder tumor private mutations (n = 20, 17%) were more frequent than those private to the lymph-node metastasis (n = 7, 6%). We conclude that bladder tumors and lymph-node metastases have overall similar molecular subtype, biomarker expression, and cancer mutations. The main exception was tumors of the Basal/Squamous-like subtype where most cases showed discordant classification, some with evidence of intra-tumor heterogeneity. The data are of relevance for neoadjuvant treatment stratification and raises questions on the dynamics of molecular subtypes during bladder cancer progression.

Molecular classification of urothelial carcinoma: global mRNA classification versus tumour-cell phenotype classification.

Global mRNA expression analysis is efficient for phenotypic profiling of tumours, and has been used to define molecular subtypes for almost every major tumour type. A key limitation is that most tumours are communities of both tumour and non-tumour cells. This problem is particularly pertinent for analysis of advanced invasive tumours, which are known to induce major changes and responses in both the tumour and the surrounding tissue. To identify bladder cancer tumour-cell phenotypes and compare classification by tumour-cell phenotype with classification by global gene expression analysis, we analysed 307 advanced bladder cancers (cystectomized) both by genome gene expression analysis and by immunohistochemistry with antibodies for 28 proteins. According to systematic analysis of gene and protein expression data, focusing on key molecular processes, we describe five tumour-cell phenotypes of advanced urothelial carcinoma: urothelial-like, genomically unstable, basal/SCC-like, mesenchymal-like, and small-cell/neuroendocrine-like. We provide molecular pathological definitions for each subtype. Tumours expressing urothelial differentiation factors show inconsistent and abnormal protein expression of terminal differentiation markers, suggesting pseudo-differentiation. Cancers with different tumour-cell phenotypes may co-cluster (converge), and cases with identical tumour-cell phenotypes may cluster apart (diverge), in global mRNA analyses. This divergence/convergence suggests that broad global commonalities related to the invasive process may exist between muscle-invasive tumours regardless of specific tumour-cell phenotype. Hence, there is a systematic disagreement in subtype classification determined by global mRNA profiling and by immunohistochemical profiling at the tumour-cell level. We suggest that a combination of molecular pathology (tumour-cell phenotype) and global mRNA profiling (context) is required for adequate subtype classification of muscle-invasive bladder cancer. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Toward a molecular pathologic classification of urothelial carcinoma.

We recently defined molecular subtypes of urothelial carcinomas according to whole genome gene expression. Herein we describe molecular pathologic characterization of the subtypes using 20 genes and IHC of 237 tumors. In addition to differences in expression levels, the subtypes show important differences in stratification of protein expression. The selected genes included biological features central to bladder cancer biology, eg, cell cycle activity, cellular architecture, cell-cell interactions, and key receptor tyrosine kinases. We show that the urobasal (Uro) A subtype shares features with normal urothelium such as keratin 5 (KRT5), P-cadherin (P-Cad), and epidermal growth factor receptor (EGFR) expression confined to basal cells, and cell cycle activity (CCNB1) restricted to the tumor-stroma interface. In contrast, the squamous cell cancer-like (SCCL) subtype uniformly expresses KRT5, P-Cad, EGFR, KRT14, and cell cycle genes throughout the tumor parenchyma. The genomically unstable subtype shows proliferation throughout the tumor parenchyma and high ERBB2 and E-Cad expression but absence of KRT5, P-Cad, and EGFR expression. UroB tumors demonstrate features shared by both UroA and SCCL subtypes. A major transition in tumor progression seems to be loss of dependency of stromal interaction for proliferation. We present a simple IHC/histology-based classifier that is easy to implement as a standard pathologic evaluation to differentiate the three major subtypes: urobasal, genomically unstable, and SCCL. These three major subtypes exhibit important prognostic differences.

Distinct Infiltration of T Cell Populations in Bladder Cancer Molecular Subtypes.

Bladder cancer is a heterogenous disease, and molecular subtyping is a promising method to capture this variability. Currently, the immune compartment in relation to subtypes is poorly characterized. Here, we analyzed the immune compartment in bladder tumors and normal bladder urothelium with a focus on T cell subpopulations using flow cytometry and RNA sequencing. The results were investigated in relation to tumor invasiveness (NMIBC/MIBC) and molecular subtypes according to the Lund Taxonomy system. Whereas the NMIBC/MIBC differed in the overall immune infiltration only, the molecular subtypes differed both in terms of immune infiltration and immune compartment compositions. The Basal/Squamous (Ba/Sq) and genomically unstable (GU) tumors displayed increased immune infiltration compared to urothelial-like (Uro) tumors. Additionally, the GU tumors had a higher proportion of regulatory T cells within the immune compartment compared to Uro tumors. Furthermore, sequencing showed higher levels of exhaustion in CD8+ T cells from GU tumors compared to both Uro tumors and the control. Although no such difference was detected at the transcriptomic level in Uro tumors compared to the controls, CD8+ T cells in Uro tumors showed higher expression of several exhaustion markers at the protein level. Taken together, our findings indicate that depending on the molecular subtype, different immunotherapeutic interventions might be warranted.

Tumor Biomarkers for Bacillus Calmette-Guérin Response: What We Get Is Not What We Want.

Studies to date on biomarkers predictive of response to bacillus Calmette-Guérin (BCG) treatment for non-muscle-invasive bladder cancer have only identified markers with prognostic potential. There is an urgent need for larger study cohorts and for control arms comprising BCG-untreated patients to identify biomarkers with true ability to predict BCG response in classifying this patient population.

UROSCAN and UROSCANSEQ: a large-scale multicenter effort towards translation of molecular bladder cancer subtypes into clinical practice - from biobank to RNA-sequencing in real time.

BACKGROUND: Bladder cancer is molecularly one of the most heterogenous malignancies characterized by equally heterogenous clinical outcomes. Standard morphological assessment with pathology and added immunohistochemical analyses is unable to fully address the heterogeneity, but up to now treatment decisions have been made based on such information only. Bladder cancer molecular subtypes will likely provide means for a more personalized bladder cancer care. METHODS: To facilitate further development of bladder cancer molecular subtypes and clinical translation, the UROSCAN-biobank was initiated in 2013 to achieve systematic biobanking of preoperative blood and fresh frozen tumor tissue in a population-based setting. In a second phase, we established in 2018 a parallel logistic pipeline for molecular profiling by RNA-sequencing, to develop and validate clinical implementation of molecular subtyping and actionable molecular target identification in real-time. RESULTS: Until June 2021, 1825 individuals were included in the UROSCAN-biobank, of which 1650 (90%) had primary bladder cancer, 127 (7%) recurrent tumors, and 48 (3%) unknown tumor status. In 159 patients, multiple tumors were sampled, and metachronous tumors were collected in 83 patients. Between 2016 and 2020 the UROSCAN-biobanking included 1122/2999 (37%) of all primary bladder cancer patients in the Southern Healthcare Region. Until June 2021, the corresponding numbers subjected to RNA-sequencing and molecular subtyping was 605 (UROSCANSEQ), of which 52 (9%) samples were not sequenced due to inadequate RNA-quality (n = 47) or technical failure/lost sample (n = 5). CONCLUSIONS: The UROSCAN-biobanking and UROSCANSEQ-infrastructure for molecular subtyping by real-time RNA-sequencing represents, to our knowledge, the largest effort of evaluating population-wide molecular classification of bladder cancer.

Urodrill - a novel MRI-guided endoscopic biopsy technique to sample and molecularly classify muscle-invasive bladder cancer without fractionating the specimen during transurethral resection.

The current diagnostic pathway for patients with muscle-invasive bladder cancer (MIBC), which involves with computed tomography urography, cystoscopy, and transurethral resection of the bladder (TURB) to histologically confirm MIBC, delays definitive treatment. The Vesical Imaging-Reporting and Data System (VI-RADS) has been suggested for MIBC identification using magnetic resonance imaging (MRI), but a recent randomized trial reported misclassification in one-third of patients. We investigated a new endoscopic biopsy device (Urodrill) for histological confirmation of MIBC and assessment of molecular subtype by gene expression in patients with VI-RADS 4 and 5 lesions on MRI. In ten patients, Urodrill biopsies were guided by MR images to the muscle-invasive portion of the tumor via a flexible cystoscope under general anesthesia. During the same session, conventional TURB was subsequently performed. A Urodrill sample was successfully obtained in nine of ten patients. MIBC was verified in six of nine patients, and seven of nine samples contained detrusor muscle. In seven of eight patients for whom a Urodrill biopsy sample was subjected to RNA sequencing, single-sample molecular classification according to the Lund taxonomy was feasible. No complications related to the biopsy device occurred. A randomized trial comparing this new diagnostic pathway for patients with VI-RADS 4 and 5 lesions and the current standard (TURB) is warranted. Patient summary: We report on a novel biopsy device for patients with muscle-invasive bladder cancer that facilitates histology analysis and molecular characterization of tumor samples.

Predictive value of molecular subtypes and APOBEC3G for adjuvant chemotherapy in urothelial bladder cancer.

OBJECTIVE: Although targeted approaches have become available in second- and third-line settings, platinum-based chemotherapy remains the standard first-line treatment for advanced muscle-invasive bladder cancer (MIBC). Therefore, the prediction of platinum resistance is of utmost clinical importance. METHODS: In this study, we established a routine compatible method for the molecular classification of MIBC samples according to various classification systems and applied this method to evaluate the impact of subtypes on survival after adjuvant chemotherapy. This retrospective study included 191 patients with advanced MIBC (pT≥3 or pN+) who underwent radical cystectomy, with or without adjuvant chemotherapy. A 48-gene panel and classifier rule set were established to determine molecular subtypes according to TCGA, MDA, LundTax, and Consensus classifications. Additionally, 12 single platinum-predictive candidate genes were assessed. The results were correlated with patients' clinicopathological and follow-up data and were validated using independent data sets. RESULTS: Our final evaluation of 159 patients demonstrated better survival in the luminal groups for those who received chemotherapy compared with those who did not. In contrast, no such differences were observed in basal subtypes. The use of chemotherapy was associated with better survival in patients with high APOBEC3G expression (p < 0.002). This association was confirmed using an independent data set of patients who received neoadjuvant platinum therapy. CONCLUSIONS: The proposed method robustly replicates the most commonly used transcriptome-based subtype classifications from paraffin-embedded tissue samples. The luminal, but not basal, molecular subtypes had the greatest benefit from adjuvant platinum therapy. We identified and validated APOBEC3G as a novel predictive marker for platinum-treated patients.

Patient-Derived Bladder Cancer Organoid Models in Tumor Biology and Drug Testing: A Systematic Review.

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.

Molecular pathology of the non-luminal Ba/Sq-like and Sc/NE-like classes of urothelial tumours: An integrated immunohistochemical analysis.

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.

Molecular Subtypes as a Basis for Stratified Use of Neoadjuvant Chemotherapy for Muscle-Invasive Bladder Cancer-A Narrative Review.

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.