Pathology focused review of morphologic subtypes and molecular variants of urothelial carcinoma with an emphasis on clinical/treatment relevance.

Urothelial carcinoma (UC) has significant morbidity, mortality, and remains the most financially costly carcinoma to manage and treat. This review will cover special morphologic features of UC that may be noted by the pathologist and any subsequent significance in terms of clinical management or treatment considerations as mentioned or recommended in the latest WHO 2022 classification of GU tumors. Many important potentially therapy altering morphologic findings can be consistently identified and reported on routine microscopic examination of hematoxylin and eosin (H&E) stained slides. Furthermore, there has been a rapid advancement of molecular diagnostics and tailored therapies throughout oncology, and we will briefly highlight some of these as they relate to the management of UC. We will actively attempt to limit the discussion of histologic descriptions or pathologic diagnostic criteria of these entities and focus rather on the recognition of their importance/implication for clinicians who must make clinical management decisions based upon these findings. Finally, the importance of open lines of communication with the pathologists who review clinical specimens as well as their practice and reporting methods cannot be overstated.

Molecular profile of bladder cancer progression to clinically aggressive subtypes.

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

Plasmacytoid urothelial carcinoma (UC) are luminal tumors with similar CD8+ Tcell density and PD-L1 protein expression on immune cells as compared to conventional UC.

BACKGROUND: Plasmacytoid urothelial carcinoma (UC) is a rare pathological variant of UC with low chemotherapeutic sensitivity and dismal outcomes. The molecular and immune profiles of such tumors remain poorly investigated. METHODS: Herein, we investigated the phenotypical features of a cohort of plasmacytoid UC (n=32) by comparison to a control group of conventional high-grade UC with matched clinicopathological characteristics (n=30). Histopathological analysis included the following antibodies: p63, GATA3, CK5/6, CK20 and HER2. In addition, the density of intra-tumor CD8+ lymphocytes, and PD-L1 expression in tumor (TC) and immune cells (IC) were evaluated. RESULTS: Plasmacytoid UC expressed GATA3 (97% vs 86% P=0.18), CK20 (59% vs 36% P=0.08) markers and showed a significantly higher rate of HER2 overexpression (2+ and 3+ score: 25% vs 0%, P<0.01) compared to controls. A significantly lower expression of CK5/6 (22% vs 56%, P<0.05) and p63 (41% vs 80%, P<0.05) was observed in plasmacytoid UC compared to controls. The density of tumor-infiltrating CD8+ cells was similar between plasmacytoid and conventional UC (P=0.9). PD-L1 expression on IC was similar compared to conventional UC (P=0.3). CONCLUSIONS: Together, our study demonstrated that plasmacytoid UC belong to the luminal subtype and display a rather inflamed microenvironment similar to conventional UC. These data support the inclusion of plasmacytoid variant of UC in clinical trials evaluating immune checkpoint inhibitors monotherapy or combination immunotherapeutic strategies.

Identification of an IDO1-based immune classifier for survival prediction of upper tract urothelial carcinoma.

The limited response rate of immunotherapy in upper tract urothelial carcinoma (UTUC) might be attributed to additional immunosuppressive mechanisms in vivo. As a promising immune checkpoint target, the expression and prognostic role of indoleamine 2,3-dioxygenase 1 (IDO1) in UTUC remains unknown. In this study, the expression and prognostic value of IDO1 was analyzed in 251 patients from 3 independent cohorts. The least absolute shrinkage and selection operator (LASSO) Cox regression model was used to construct an IDO1-based immune classifier and external validation was performed to further validate the classifier. RNA sequencing and immunofluorescence were used to explore the immune contexture of different risk groups stratified by classifier. We found that high IDO1 expression on tumor cells (TC) indicated a poorer overall survival and disease-free survival in all cohorts. Patients with high expression of IDO1 TC possessed increased infiltration of CD4+ , CD8+ and Foxp3+ T cells. An immune classifier based on intratumoral CD8+ lymphocytes, IDO1 TC, and stromal PD-L1 expression status was developed, with its area under the curves (AUCs) values for overall survival at 5 y being 0.79 (95% confidence interval [CI] 0.65-0.93) in the discovery cohort, 0.75 (95% CI 0.58-0.92) and 0.78 (95% CI 0.65-0.92) in the internal and external validation cohorts, respectively. The high-risk group stratified by the immune classifier was associated with immunosuppressive contexture, accompanied by enhanced CD8+ T cells exhaustion patterns. Our IDO1-based immune classifier can provide a superior accuracy for survival prediction and lead to individual stratification of UTUC immune subtypes.

Prognostic impact of molecular muscle-invasive bladder cancer subtyping approaches and correlations with variant histology in a population-based mono-institutional cystectomy cohort.

PURPOSE: Recently discovered molecular classifications for urothelial bladder cancer appeared to be promising prognostic and predictive biomarkers. The present study was conducted to evaluate the prognostic impact of molecular subtypes assessed by two different methodologies (gene and protein expression), to compare these two approaches and to correlate molecular with histological subtypes in a consecutively collected, mono-institutional muscle-invasive bladder cancer (MIBC) cohort. METHODS: 193 MIBC were pathologically re-evaluated and molecular subtypes were assessed on mRNA (NanoString technology, modified 21-gene-containing MDACC approach) and protein levels (immuno-histochemical [IHC] analysis of CK5, CK14, CD44, CK20, GATA3 and FOXA1). Descriptive statistical methods and uni-/multi-variable survival models were employed to analyze derived data. RESULTS: Neither gene expression nor protein-based subtyping showed significant associations with disease-specific (DSS) or recurrence-free survival (RFS). Agreement between mRNA (reference) and protein-based subtyping amounted 68.6% for basal, 76.1% for luminal and 50.0% for double-negative tumors. Histological subtypes associated with RFS in uni-variable (P = 0.03), but not in multivariable survival analyses. Tumors with variant histology predominantly showed luminal subtypes (gene expression subtyping: 36/55 cases, 65.5%; protein subtyping: 44/55 cases, 80.0%). Squamous differentiation significantly associated with basal subtypes (gene expression subtyping: 44/45 squamous cases, 97.8%; protein subtyping: 36/45 cases, 80.0%). CONCLUSION: In our consecutive cystectomy cohort, neither gene, protein expression-based subtyping, nor histological subtypes associated with DSS or RFS in multi-variably adjusted survival analyses. Application of a limited IHC subtyping marker panel showed high concordance of 83.9% with gene expression-based subtyping, thus underlining the utility for subtyping in pathological routine diagnostics. In addition, histological MIBC subtypes are strong indicators for intrinsic subtypes.

Heterogeneity in NECTIN4 Expression Across Molecular Subtypes of Urothelial Cancer Mediates Sensitivity to Enfortumab Vedotin.

PURPOSE: Enfortumab vedotin (EV) is an antibody-drug conjugate (ADC) targeting NECTIN4 (encoded by the PVRL4/NECTIN4 gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance. EXPERIMENTAL DESIGN: Molecular subtyping and NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1,915 total specimens) were used to assess NECTIN4 expression across molecular subtypes. The outcome of the transcriptomic analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays. RESULTS: NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes. NECTIN4 expression is positively correlated with luminal markers GATA3, FOXA1, and PPARG across all cohorts. NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 leads to EV resistance. CONCLUSIONS: Sensitivity to EV is mediated by expression of NECTIN4, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials.See related commentary by Teo and Rosenberg, p. 4950.

Utility of pT3 substaging in lymph node-negative urothelial carcinoma of the bladder: do pathologic parameters add to prognostic sub-stratification?

PURPOSE: The value of bladder cancer (BC) substaging into macroscopic (pT3b) and microscopic (pT3a) perivesical fat extension in lymph node (Ln)-negative patients is controversially discussed and limited evidence for prognostic relevance of additional histopathological factors in pT3 BC exists. We evaluated the prognostic value of pT3 substaging and established pathological and clinical parameters with focus on tumor invasive front (TIF) and tumor size. METHODS: Specimens of 52 patients treated with radical cystectomy (RC) for pT3 a/b muscle-invasive BC were reviewed and re-evaluated by a pathologist specialized in uropathology. Clinical variables and standard histopathologic characteristics were assessed including TIF and tumor size. Their value as prognosticators for overall survival (OS) and recurrence-free survival (RFS) was evaluated. RESULTS: Mean age of patients was 67.55 years. Tumors were staged pT3a in 28 patients (53.8%) and pT3b in 24 (46.8%). Median OS was 34.51 months. Median tumor size was 3.2 cm, median TIF was 11.0 mm. Differences in OS between pT3a and pT3b were not significant (p = 0.45). Carcinoma in situ (CIS) and lymphovascular invasion (LVI) were significantly associated with pT3b tumors. Univariate analysis could not identify pathological prognosticators like TIF or tumor size for OS and RFS (p for all > 0.05). CONCLUSION: No significant differences in OS or RFS were observed comparing Ln-negative pT3 BC following radical cystectomy. Additional pathologic variables like TIF could not be identified as prognosticator. Relevance of pT3 BC substaging needs reevaluation in larger prospective cohorts.

Does multidetector computed tomographic urography (MDCTU) T staging classification correspond with pathologic T staging in upper tract urothelial carcinoma?

PURPOSE: Multidetector computed tomographic urography (MDCTU) is not yet sufficient to be used in the clinical staging of upper tract urothelial carcinoma (UTUC). This study aimed to compare the diagnostic accuracy of MDCTU T stage classification and pathologic T staging for UTUC. METHODS: We retrospectively evaluated 125 patients with UTUC who underwent preoperative MDCTU. A single radiologist classified the MDCTU pattern of the tumors as either low or advanced T stage for localized or locally advanced tumors, respectively. The diagnostic values of MDCTU for locally advanced tumors and the kappa agreement between MDCTU and pathologic T stage were investigated. RESULTS: Among 85 pathologic low T stage (Ta-T2) tumors, 71 low T stage tumors were correctly detected by MDCTU, while 30 out of 40 advanced T stage (T3-T4) tumors were correctly diagnosed by MDCTU. MDCTU led to under-staging in 8% (10/125) tumors and over-staging in 11.2% (14/125) tumors. Therefore, the overall accuracy of MDCTU in the diagnosis of low and advanced T stage tumors was 80.8% (101/125 patients). The sensitivity for advanced T stage tumors was 75% (30/40), the specificity was 83.5% (71/85), and the positive and negative predictive values were 68.1% (30/44) and 87.6% (71/81), respectively. The kappa agreement value between the MDCTU T stage and pathologic T stage was 0.57 (95% confidence interval (CI) 0.42-0.72), which was statistically significant (P = 0.001). CONCLUSION: MDCTU T stage classification may be relatively accurate for the detection and staging of UTUC correspondence with a pathologic stage.

The value and limitations of urothelial bladder carcinoma molecular classifications to predict oncological outcomes and cancer treatment response: A systematic review and meta-analysis.

AIM: To evaluate the predictive value of molecular subtypes on oncological outcomes and response to cancer treatment in patients with urothelial bladder carcinoma (UBC). MATERIALS AND METHODS: A literature search using PubMed, Scopus, and Cochrane Library was conducted on April 2020 to identify relevant studies according to the preferred reporting items for systematic review and meta-analysis guidelines. The pooled overall survival (OS), cancer-specific survival (CSS), and progression-free survival were calculated using a fixed or random effects model. RESULTS: We identified 66 studies (including 21,447 molecular subtype records) evaluating the impact of molecular classification on oncologic outcomes in patients with UBC. We found significant association of different molecular subtypes with OS, CSS, progression-free survival, recurrence-free survival, and response to treatment. Totally, 11 studies were included in the meta-analysis. Basal group and NE-like subtypes were associated with worse OS (pooled HR: 1.78, 95%CI: 1.49-2.12, and pooled HR: 2.67, 95%CI: 1.08-6.60, respectively) in patients with muscle invasive bladder cancer. Luminal group was also associated with worse CSS (pooled HR of 3.67, 95%CI: 2.19-6.14). CONCLUSIONS: Based on these data, UBC molecular classifications are significant predictors of oncological outcomes and identify patients who are most likely to benefit from intensified or different therapies. The optimal consensus on molecular classification remains to be verified in well-designed prospective studies to allow precise prognostic and predictive value assessment.

Nested Variant of Urothelial Carcinoma Is a Luminal Bladder Tumor With Distinct Coexpression of the Basal Marker Cytokeratin 5/6.

OBJECTIVES: The nested variant of urothelial carcinoma (NVUC) is a rare bladder tumor that may possess a luminal molecular phenotype. We sought to determine whether a small immunohistochemical (IHC) panel using common surrogates for molecular phenotypes would reliably classify a cohort of pure NVUC cases. METHODS: IHC staining with a panel composed of markers for basal subtypes (CK5/6, CK14) and luminal subtypes (FOXA1, GATA3) was performed on pure small NVUC cases (n = 23) and 5 large NVUC cases (n = 5). Scoring of IHC stains was performed semiquantitatively. Individual cases were analyzed using previously reported IHC-based surrogates for molecular subtype. RESULTS: The phenotype of NVUC was classified as luminal from 60.1% (FOXA1+/CK5/6-) to 100% (GATA3+/CK14-) of cases using composite phenotypes. No cases possessed a basal or squamous cell carcinoma-like phenotype. The majority of small NVUC cases (69.5%) showed subset CK5/6 expression distinctly localized to the basal layers of tumor cell nests. Intratumoral heterogeneity was also noted in CK5/6 (21.7% of small NVUC cases) but no other markers. CONCLUSIONS: NVUC appears to express markers of both basal and luminal bladder tumors. Definitive gene expression profiling may be valuable to further characterize this unique histologic variant.

Urothelial carcinoma: variant histology, molecular subtyping, and immunophenotyping significant for treatment outcomes.

Although urothelial carcinoma (UC) has been recognised as a homogenous disease entity until recently, it exhibits widely diverse histological variants. Recent studies have revealed that some histological variants may serve as markers of very high risk for advanced cancers and poor prognoses. Certain histological variants can generate a pathological T stage, which may result in unnecessary surgery. Though platinum based chemotherapy is the standard treatment, the use of immune checkpoint inhibitors (ICIs) for UC treatment has become a major trend in oncology. UCs showing specific histological variants have responded exceptionally well to chemotherapy and ICIs. Currently, molecular studies base molecular classification on gene expression profile signatures in order to make diagnoses or predict responses to chemotherapies and ICIs. Notably, some histological variants correlate with specific molecular subtypes. The usefulness of immunophenotyping for classification purposes was recognised only recently. Immunophenotypes are classified into three categories according to lymphocyte distribution in or around the cancer cell nest: desert, excluded, and inflamed. This immunophenotyping has been increasingly shown to be of value in predicting the response to ICIs. This review describes the morphological characteristics of histological variants as well as the advantages and limitations in determining them, with particular reference to clinical benefits. Subsequently, we describe the concept of molecular classification and immunophenotypes, and their morphological features, which are easily interpreted and amenable to daily practice via hematoxylin and eosin staining. We also consider the clinical advantages, limitations, and issues encountered while using these in routine clinical practice.

Comparison of urine cytology diagnostic reports before and after the implementation of the Paris System classification system in China.

OBJECTIVE: In 2013, The Paris System for Reporting Urinary Cytology (TPS) was developed as a uniform practical urine cytology system that could be applied worldwide. Here, we investigated the effectiveness of TPS diagnostic approach compared with that of the traditional urine cytological diagnosis method used in China. METHODS: Based on the diagnostic criteria of TPS, 412 urine samples from 143 patients with histological follow-up data were retrospectively analysed, and the diagnoses were compared with the original cytological diagnoses. RESULTS: In total, 110 patients were histologically diagnosed with high-grade urothelial carcinoma (HGUC), and 33 patients were diagnosed with low-grade urothelial neoplasia. Based on the traditional urine cytological analysis method, 50 patients (34.9%) were diagnosed as negative, 48 patients (33.6%) were diagnosed as having atypical urothelial cells, and 45 patients (31.5%) were diagnosed as positive. After reclassification using TPS, urine samples from 11 cases (7.7%) were categorised as unsatisfactory, 34 cases (23.8%) were negative, 21 cases (14.7%) were categorised as having atypical urothelial cells, 12 cases (8.4%) were diagnosed as suspicious for HGUC, 59 cases (41.2%) were diagnosed with HGUC, and six cases (4.2%) were reclassified as having low-grade urothelial neoplasia. Thus, after implementing TPS criteria, the sensitivity for positive malignancy diagnoses (HGUC alone) increased from 38.2% to 50.9%, while the specificity of the diagnosis was barely changed. CONCLUSIONS: The Paris System for Reporting Urinary Cytology greatly contributes to the standardisation of urine cytology reports and significantly improves the diagnostic sensitivity for HGUC.

What Is the Significance of Variant Histology in Urothelial Carcinoma?

CONTEXT: Urothelial carcinoma can exhibit a wide range of variant morphologies. Many variants present diagnostic challenges and carry clinical implications that inform prognosis and treatment decisions. OBJECTIVE: To provide an overview of the diagnostic, therapeutic, and prognostic significance of histological variants of urothelial carcinoma. EVIDENCE ACQUISITION: A PubMed/MEDLINE-based literature search was conducted using the key terms "urothelial carcinoma", "variant histology", "nested", "micropapillary", "microcystic", "sarcomatoid", "squamous differentiation", "glandular differentiation", "clear cell", "plasmacytoid", "lymphoepithelioma-like carcinoma", "squamous cell carcinoma", "small cell carcinoma", "adenocarcinoma", "radiotherapy", "neoadjuvant chemotherapy", and "adjuvant chemotherapy". EVIDENCE SYNTHESIS: The incidence of variant histology is increasing due to improved recognition. Nonetheless, diagnosis can pose challenges due to sampling limitations and interobserver variability. Although associated with advanced disease at presentation, survival outcomes for most variants do not differ significantly compared with pure urothelial carcinoma of the same stage. Controversy exists regarding optimal management due to the low quality of available evidence. For most cases, radical cystectomy with pelvic lymph node dissection (with neoadjuvant chemotherapy when appropriate) represents the standard of care. Small cell carcinoma and lymphoepithelioma-like carcinoma appear to be particularly chemosensitive. CONCLUSIONS: Accurate identification of variant histological subtypes is an important part of risk stratification, as these variants exhibit aggressive biological behaviour. Variant histology tumours are associated with advanced disease at presentation, which must be considered when counselling patients regarding survival outcomes. Optimal management remains to be defined but in most cases; neoadjuvant chemotherapy and radical cystectomy with pelvic lymph node dissection remains the mainstay of treatment. PATIENT SUMMARY: It is important to recognise histological variants of urothelial carcinoma as they indicate aggressive disease. When compared with patients with pure urothelial carcinoma of the same disease stage, survival does not appear to be significantly worse. In most cases, patients with invasive variant histology should be treated with neoadjuvant chemotherapy and radical cystectomy. Take Home Messages Accurate identification of variant histology is important as it exhibits aggressive biological behaviour and affects treatment. Although associated with advanced disease at presentation, with appropriate treatment, survival outcomes are not significantly different compared with pure urothelial carcinoma of the same stage.

Evaluation of cancer testis antigen (CT10, PRAME) and MHC I expression in high-grade urothelial carcinoma of the bladder.

Immunotherapeutic strategies are increasingly used in the treatment of a number of malignancies including high-grade urothelial carcinoma (HGUC) of the bladder. Because of this, detailed and accurate assessment of the tumor immune microenvironment is paramount. In this study, we aimed to correlate the composition of the tumor immune microenvironment with oncologic outcome and the expression of two cancer testis antigens (CTAs), CT10 and PRAME, potential cancer vaccine targets, as well as major histocompatibility complex I (MHC I), a molecule associated with tumor immune escape and resistance to immunotherapy. Triplicate tissue microarrays (TMAs) were constructed using 207 cases of HGUC of the bladder. Oncologic outcome data was gathered for each case. Consecutive sections from the TMA blocks were stained with CD3, CD4, CD8, FOXP3, PD1, PD-L1, CT10, PRAME, and MHC I. Twenty-one percent and 15% of cases expressed CT10 and PRAME, respectively. Eighty-eight percent of cases showed absent or decreased MHC I expression. CT10-expressing tumors showed a significantly worse disease specific survival (p = 0.007, hazard ratio 2.245, confidence interval 1.223-4.122). CT10, PRAME, and MHC I expression significantly correlated with other some immune parameters. CT10 and PRAME are expressed in a subset of HGUC and CTA and MHC I expression correlate with a number of important immune parameters. Together, these findings highlight the potential for exploring novel immune therapeutic strategies in HGUC. Additional studies evaluating the clinical relevance of these findings are warranted.

Micropapillary urothelial carcinoma of urinary bladder displays immunophenotypic features of luminal and p53-like subtypes and is not a variant of adenocarcinoma.

OBJECTIVES: Micropapillary urothelial carcinoma of the urinary bladder (MPUC) is a rare variant of urothelial carcinoma which has aggressive clinical characteristics. The objective is to investigate the molecular subtypes of MPUC and the impact to the clinical outcome and determine whether MPUC represents a variant of adenocarcinoma. MATERIALS AND METHODS: We evaluated surrogate immunohistochemical markers of luminal, basal, and p53-like subtypes and correlated with prognosis and the expression of markers related to bladder adenocarcinoma and glandular differentiation in 56 cases of MPUC (10 cases of transurethral resection and 46 cases of radical cystectomy). Biomarker expression in co-existing conventional urothelial carcinoma was also analyzed. Cox regression analysis was performed to study the impact of molecular subtype on the clinical outcome. RESULTS: Thirty-four cases (61%) met criteria for the luminal subtype. Twenty-two cases (39%) displayed a p53-like subtype. In contrast, 40/56 (71%) cases of coexisting conventional urothelial carcinoma were classified as luminal subtype and 16/56 (29%) cases were designated as p53-like subtype. There was no significant survival difference between luminal subtype and p53-like subtype. CDX2, villin, and cadherin 17 were negative in all cases. MUC1 was strongly and diffusely expressed in the stroma-facing surface of MPUC tumor cells in all the cases. CONCLUSIONS: Our findings suggest that MPUC possesses characteristics of luminal and p53-like subtypes, and does not harbor phenotypic features of the basal subtype. There is no significant difference in the prognosis between luminal and p53-like subtype MPUC. MPUC is not a variant of adenocarcinoma and does not represent a form of glandular differentiation, in contrast to other organ sites.

Concordance among four commercially available, validated programmed cell death ligand-1 assays in urothelial carcinoma.

BACKGROUND: Antibodies targeting the programmed cell death-1 (PD-1)/PD-ligand 1 (PD-1/PD-L1) checkpoint have shown promising clinical activity in patients with advanced urothelial carcinoma (UC). Expression of PD-L1 in UC tumors has been investigated using different antibody clones, staining protocols, and scoring algorithms. The aim was to establish the extent of concordance among PD-L1 immunohistochemistry (IHC) assays. METHODS: Tumor biopsy samples (N = 335) were assessed using four commercially available PD-L1 assays: VENTANA SP263, VENTANA SP142, PD-L1 IHC 28-8 pharmDx, and PD-L1 IHC 22C3 pharmDx. PD-L1 analytical staining and classification concordance, including agreement between clinically relevant scoring algorithms, were investigated using overall/positive/negative percentage agreement (OPA/PPA/NPA). RESULTS: Good analytical correlation was observed among the VENTANA SP263, PD-L1 IHC 22C3 pharmDx, and PD-L1 IHC 28-8 pharmDx assays for tumor cell (TC) and immune cell (IC) PD-L1 staining with Spearman rank coefficients of 0.92-0.93 for TCs and 0.88-0.91 for ICs. However, concordance (preset criterion: ≥85%) between patient PD-L1 status when applying the TC or ICICArea ≥ 25% (VENTANA SP263) cutoff was only achieved for PD-L1 IHC 22C3 pharmDx versus VENTANA SP263 (OPA 92.2%, PPA 86.4%, NPA 95.4%). Differences were observed between patient populations with UC tumors classified as PD-L1 high versus PD-L1 low/negative using combined positive score (CPS) ≥1, CPS ≥10, IC ≥5%, and TC/IC ≥25%. CONCLUSIONS: The VENTANA SP263 and PD-L1 IHC 22C3 pharmDx assays are analytically similar in UC. When the different PD-L1 assays were combined with their specified clinical scoring algorithms, differences were seen in patient classification driven by substantial differences in scoring approaches.

Distinct genetic alterations and luminal molecular subtype in nested variant of urothelial carcinoma.

AIMS: Nested variant of urothelial carcinoma (NVUC) is rare, and only a few small series exist. Molecular characteristics and the classifying marker profile as well as therapeutic targets of this specific variant are mostly unknown. The aim of this study was to characterise NVUC at the molecular level in one of the largest cohorts to date. In addition, we applied an immunohistochemical marker panel in order to define the molecular subtype. METHODS AND RESULTS: Sixty NVUC cases were collected from different departments. TERT promoter mutation analysis was carried out in all samples using SNaPshot analysis. Targeted sequencing of 48 cancer-related genes by next-generation sequencing (NGS) analysis was performed in a subset of 26 cases. Immunohistochemical markers CD44, CK5, CK14, EGFR, p63, FOXA1, GATA3, CD24 and CK20 were used to elucidate the molecular subtype. A total of 62.5% of NVUC cases harboured a mutation of the TERT promoter. Additionally, TP53, JAK3 and CTNNB1 were among the most frequently mutated genes identified by NGS analysis. Subtyping revealed that all NVUC express luminal markers such as CD24, FOXA1, GATA3 and CK20. CONCLUSIONS: In summary, NVUC belong to the luminal molecular subtype. Moreover, a subset of NVUC seems to be characterised by mutations of the Wnt and inflammatory pathways, including JAK3 mutations, indicating a different biological background compared to conventional urothelial bladder cancer.

Molecular and histopathology directed therapy for advanced bladder cancer.

Bladder cancer is a heterogeneous group of tumours with at least 40 histological subgroups. Patients with localized disease can be cured with surgical resection or radiotherapy, but such curative options are limited in the setting of recurrent disease or distant spread, in which case systemic therapy is used to control disease and palliate symptoms. Cytotoxic chemotherapy has been the mainstay of treatment for advanced bladder cancer, but high-quality evidence is lacking to inform the management of rare subgroups that are often excluded from studies. Advances in molecular pathology, the development of targeted therapies and the resurgence of immunotherapy have led to the reclassification of bladder cancer subgroups and rigorous efforts to define predictive biomarkers for cancer therapies. In this Review, we present the current evidence for the management of conventional, variant and divergent urothelial cancer subtypes, as well as non-urothelial bladder cancers, and discuss how the integration of genomic, transcriptomic and proteomic characterization of bladder cancer could guide future therapies.

Predicting outcomes in non-muscle invasive (Ta/T1) bladder cancer: the role of molecular grade based on luminal/basal phenotype.

Bladder cancer tumors can be divided into two molecular subtypes referred to as luminal or basal. Each subtype may react differently to current chemotherapy or immunotherapy. Likewise, the technology required for comprehensive molecular analysis is expensive and not yet applicable for routine clinical diagnostics. Therefore, it has been suggested that the immunohistochemical expressions of only two markers, luminal (CK20+, CK5/6-) and basal (CK5/6+, CK20-), is sufficient to identify the molecular subtypes of bladder cancer. This would represent a molecular grade that could be used in daily practice. Molecular classification is done using immunohistochemistry to assess luminal-basal phenotype based on tissular expression of CK20 and CK5/6 as surrogate for luminal or basal subtypes, respectively. A series of 147 non-muscle-invasive bladder carcinoma cases was selected, and the tumors were divided into four subgroups based on the presence of CK20 and/or CK5/6, that is, null (CK20-, CK5/6-), mixed (CK20+, CK5/6+), basal (CK20-, CK5/6+), and luminal (CK20+, CK5/6-) categories. Survival analysis was estimated using the Kaplan-Meier method and the log-rank test. Hazard ratios were calculated by Cox multivariate analysis. The molecular grade included cases with null (n = 89), mixed (n = 6), basal (n = 20), and luminal (n = 32) phenotypes with differences in recurrence-free, progression-free and cancer-specific survival associated with molecular-grade categories in patients with low- or high-grade Ta, or high-grade T1 tumors. The multivariate analysis identified the luminal phenotype as a predictor of more aggressive neoplasms. Our findings provide a rationale to investigate luminal and basal subtypes of bladder cancer using two gene expression signatures as surrogate markers and show that non-muscle-invasive bladder carcinoma can be stratified into biologically and clinically different subgroups by using an immunohistochemical classifier.

Biological molecular layer classification of muscle-invasive bladder cancer opens new treatment opportunities.

BACKGROUND: Muscle-invasive bladder tumors are associated with a high risk of relapse and metastasis even after neoadjuvant chemotherapy and radical cystectomy. Therefore, further therapeutic options are needed and molecular characterization of the disease may help to identify new targets. The aim of this study was to characterize muscle-invasive bladder tumors at the molecular level using computational analyses. METHODS: The TCGA cohort of muscle-invasive bladder cancer patients was used to describe these tumors. Probabilistic graphical models, layer analyses based on sparse k-means coupled with Consensus Cluster, and Flux Balance Analysis were applied to characterize muscle-invasive bladder tumors at a functional level. RESULTS: Luminal and Basal groups were identified, and an immune molecular layer with independent value was also described. Luminal tumors showed decreased activity in the nodes of epidermis development and extracellular matrix, and increased activity in the node of steroid metabolism leading to a higher expression of the androgen receptor. This fact points to the androgen receptor as a therapeutic target in this group. Basal tumors were highly proliferative according to Flux Balance Analysis, which makes these tumors good candidates for neoadjuvant chemotherapy. The Immune-high group showed a higher degree of expression of immune biomarkers, suggesting that this group may benefit from immune therapy. CONCLUSIONS: Our approach, based on layer analyses, established a Luminal group candidate for therapy with androgen receptor inhibitors, a proliferative Basal group which seems to be a good candidate for chemotherapy, and an immune-high group candidate for immunotherapy.