Alignment of molecular subtypes across multiple bladder cancer subtyping classifiers.

BACKGROUND: Treatment of patients with muscle-invasive bladder cancer (MIBC) includes cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC). Molecular subtypes have been associated with patient outcomes after NAC and RC, but the reported results have been highly inconsistent. OBJECTIVE: To evaluate the association of molecular subtypes from different classifiers with overall survival (OS) among patients with MIBC who underwent RC. MATERIALS AND METHODS: We analyzed gene expression data generated from transurethral resection of MIBC from a previously assembled and published meta-cohort, NACmeta (N = 601, 247 treated with NAC+RC and 354 RC without NAC), where extended follow-up was available. Molecular subtypes were assigned using the Genomic Subtyping Classifier (GSC), the Consensus Classifier, The Cancer Genome Atlas (TCGA) Classifier, and the Lund Classifier. For survival analysis, inverse probability weighting was used to balance the clinical NAC and non-NAC patient groups. RESULTS: A high consistency in gene expression patterns and nomenclature was observed between luminal-like subtypes, defined as GSC-Luminal, Consensus-Luminal Papillary (LumP), TCGA Luminal-Papillary (LumP) and Lund-UroA, but not for basal-like subtypes such GSC-Basal, Consensus Basal/Squamous, TCGA-Basal/Squamous and Lund-Basal/Squamous. Patients with luminal-like subtypes demonstrated no difference in 3-year OS when treated with or without NAC (P = 0.7 for GSC, P = 0.94 for Consensus, P = 0.87 for TCGA and P = 0.66 for Lund-UroA, respectively). CONCLUSION: Luminal-like molecular subtypes identify a subgroup of MIBC patients who do not appear to benefit from current NAC regimens, even for locally advanced disease. In addition, we were able to illustrate differences in subtyping nomenclature that are not reflected in the underlying biological definition of the subtypes. PATIENT SUMMARY: Muscle-invasive bladder cancer exhibits molecular diversity, and various classifications identify different groups who do not benefit from chemotherapy. On the other hand, there is a high inconsistency in the way cancer groupings are named.

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.

Immune profiles and DNA methylation alterations related with non-muscle-invasive bladder cancer outcomes.

BACKGROUND: Non-muscle-invasive bladder cancer (NMIBC) patients receive frequent monitoring because ≥ 70% will have recurrent disease. However, screening is invasive, expensive, and associated with significant morbidity making bladder cancer the most expensive cancer to treat per capita. There is an urgent need to expand the understanding of markers related to recurrence and survival outcomes of NMIBC. METHODS AND RESULTS: We used the Illumina HumanMethylationEPIC array to measure peripheral blood DNA methylation profiles of NMIBC patients (N = 603) enrolled in a population-based cohort study in New Hampshire and applied cell type deconvolution to estimate immune cell-type proportions. Using Cox proportional hazard models, we identified that increasing CD4T and CD8T cell proportions were associated with a statistically significant decreased hazard of tumor recurrence or death (CD4T: HR = 0.98, 95% CI = 0.97-1.00; CD8T: HR = 0.97, 95% CI = 0.95-1.00), whereas increasing monocyte proportion and methylation-derived neutrophil-to-lymphocyte ratio (mdNLR) were associated with the increased hazard of tumor recurrence or death (monocyte: HR = 1.04, 95% CI = 1.00-1.07; mdNLR: HR = 1.12, 95% CI = 1.04-1.20). Then, using an epigenome-wide association study (EWAS) approach adjusting for age, sex, smoking status, BCG treatment status, and immune cell profiles, we identified 2528 CpGs associated with the hazard of tumor recurrence or death (P < 0.005). Among these CpGs, the 1572 were associated with an increased hazard and were significantly enriched in open sea regions; the 956 remaining CpGs were associated with a decreased hazard and were significantly enriched in enhancer regions and DNase hypersensitive sites. CONCLUSIONS: Our results expand on the knowledge of immune profiles and methylation alteration associated with NMIBC outcomes and represent a first step toward the development of DNA methylation-based biomarkers of tumor recurrence.

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.

Impact of clinical and pathological subtypes of carcinoma in situ (CIS) of the bladder: Lessons learned from long-term follow-up of a series of CIS patients treated with BCG.

OBJECTIVE: Some attempts have previously been made to stratify patients with CIS for the purpose of risk-adapted clinical management and clinical trial design. In particular, two classification systems have been proposed: clinical classification, comprising primary (P-CIS), concomitant (C-CIS), and secondary (S-CIS) disease, and pathological classification, comprising P-CIS, cTa-CIS, and cT1-CIS. The aim of the present study was to assess the impact of both classifications on BCG response, recurrence-free survival (RFS), progression-free survival (PFS), overall survival (OS), and cancer-specific survival (CSS). PATIENTS AND METHODS: We performed a retrospective analysis of 386 patients with bladder CIS, with or without associated cTa/cT1 disease, treated with BCG instillations between 2008 and 2015. Patients were stratified according to the two classification systems. Cox multivariate regression models were used to assess the impact of these subtypes on BCG response, RFS, PFS, OS, and CSS. We also performed a cumulative meta-analysis according to PRISMA guidelines. RESULTS: The median follow-up was 70.5 months. According to the clinical classification, 34 (8.8%) patients had P-CIS, 81 (21%) S-CIS, and 271 (70.2%) C-CIS. The pathological classification showed 34 (8.8%) patients to have P-CIS, 190 (49.2%) cTa-CIS, and 162 (42%) cT1-CIS. In the overall cohort, BCG response was reported in 296 (76.7%); 159 (41.2%) had recurrence, 55 (14.2%) had progression, and 67 (17.4%) underwent radical cystectomy. Death from any cause was recorded in 135 (35%) and death from urothelial carcinoma in 38 (9.9%). Cox multivariate regression analysis showed that neither clinical classification nor pathological classification is an independent predictive factor for BCG response, RFS, PFS, OS, or CSS after adjusting for confounders. In the pooled meta-analysis, two studies and the present series were included for evidence synthesis, recruiting a total of 941 patients. We found no statistically significant difference across the groups for both classifications with respect to BCG response, RFS, PFS, and CSS. CONCLUSIONS: Currently, the supporting evidence for an impact of clinical classification and pathological classification on oncological outcomes of CIS of the bladder is insufficient to justify their use to guide clinical management or follow-up.

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.

Incidence, mortality and survival of bladder neoplasms in the Czech Republic in the period 1977-2017: a time trend analysis.

BACKGROUND: Bladder neoplasms represent a significant disease burden in the Czech population. This study aimed to perform a complex time trend analysis of incidence, mortality, and survival of 76,505 patients with bladder neoplasms based on the Czech National Cancer Registry for 1977-2017. MATERIALS AND METHODS: The time trends of incidence and mortality were evaluated using the joinpoint regression. The relative survival and Cox proportional hazards model were used for survival analysis. RESULTS: After 2004, a significant annual decrease by 1.9% in the incidence of malignant tumors of the bladder (C67) was observed, accompanied by a sharp annual increase in the incidence of in situ bladder cancer (D090) by 16.9%. For mortality from malignant tumors of the bladder, a significant decrease by 1.4% annually was detected after 1998. The decline in both incidence and mortality was most pronounced in the below-65 years age group and in patients with a localized stage at dia-gnosis. While a significant decline in both incidence and mortality was observed for the first primary malignant tumors of the bladder, both these measures increased for malignant tumors of the bladder as subsequent primary neoplasms. The five-year relative survival of patients with malignant tumors of the bladder increased from 52.1% in 1990-1993 to 62.3% in 2013-2017. However, comparing the periods 2003-2007 and 2013-2017, a decrease has been observed. CONCLUSION: The decrease in the incidence and survival of malignant tumors of the bladder in the recent period is in particular caused by improved detection of in situ bladder cancer and classification changes. Other reasons for the decreasing survival include the increasing age at dia-gnosis, the growing number of subsequent primary neoplasms, and the increasing proportion of smokers among patients.

Ubiquitination-Related Molecular Subtypes and a Novel Prognostic Index for Bladder Cancer Patients.

Objective: To develop and validate ubiquitination-related molecular subtypes and a novel prognostic index using ubiquitination-related genes (URGs) for patients with bladder cancer (BCa). Materials and Methods: We downloaded the clinical data and transcriptome data of BCa from TCGA and GEO database. Consensus clustering analysis was conducted to identify ubiquitination-related molecular subtypes for BCa. Besides, we performed univariate and multivariate Cox regression analysis to develop a novel prognostic URGs-related index for BCa. We conducted internal and external verification in TCGA cohort and GEO cohort, respectively. Furthermore, the associations of ubiquitination-related molecular subtypes and prognostic index with tumor immune environment were also investigated. Results: A total of four ubiquitination-related molecular subtypes of BCa were finally identified. These four molecular subtypes had significantly different clinical characteristics, prognosis, PD-L1 expression level and tumor microenvironment. Besides, we developed a novel prognostic index using six URGs (including HLA-A, TMEM129, UBE2D1, UBE2N, UBE2T and USP5). The difference in OS between high and low-risk group was statistically significant in training cohort, testing cohort, and validating cohort. The area under ROC curve (AUC) for OS prediction was 0.736, 0.723, and 0.683 in training cohort, testing cohort, and validating cohort, respectively. Multivariate survival analysis showed that this index was an independent predictor for OS. This prognostic index was especially suitable for subtype 1 and 3, older, male, high grade, AJCC stage III-IV, stage N0, stage T3-4 BCa patients. Conclusions: This study identified a total of four ubiquitination-related molecular subtypes with significantly different tumor microenvironment, prognosis, clinical characteristics and PD-L1 expression level. Besides, a novel ubiquitination-related prognostic index for BCa patients was developed and successfully verified, which performed well in predicting prognosis of BCa.

Association of current molecular subtypes in urothelial carcinoma with patterns of muscularis propria invasion.

Urothelial carcinoma is subdivided into luminal (L), basal (B), and p53-wild-type (WT) molecular subtypes, with basal and p53-WT groups showing more aggressive course and poor treatment response, respectively. The literature on molecular subtypes of UC includes a mixture of different stages. We investigated the molecular profile and outcome of pure cohort of muscle invasive bladder carcinoma (MIBC) considering two distinct patterns of muscularis propria (MP) invasion. Forty-three cystectomies harboring stage pT2 were retrospectively identified in 18 years. MP invasion was subclassified into patterns 1 (tumor encasing intact detrusor muscle bundles) and 2 (tumor dissecting/replacing detrusor muscle). Using IHC, B/L phenotypes, p53, and Ki67 were assessed, and survival data was collected. Pattern 1 invasion was noted in 16 (37%) and pattern 2 in 27 (63%), with mean age of pattern 1 being 10 years younger. B/L phenotypes were successfully determined in 83.7%; 48.8% and 34.8% revealed L and B phenotypes, respectively (indeterminate phenotype in 16.4%). Pattern 1 was associated with L phenotype (GATA3 and HER-2 expressions: p = 0.02 & p = 0.04, respectively). Ki67 ≥ 5/10HPF was noted in pattern 2 and B phenotype (p = 0.03). B phenotype showed association with p53-WT (p = 0.007). In median follow-up of 60.7 months, 63.6% of pattern 1 cases were alive without disease compared to 32% of pattern 2 (not significant). A panel of CK20 and GATA3 for luminal and CK5/6 and CK14 for basal subtypes can provide reliable molecular classification in UC. Also, morphology of MIBC can predict the molecular phenotype and the behavior of the UC.

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.

Nuclear hypochromasia: Shedding light on the "lightness" of high-grade urothelial carcinoma.

BACKGROUND: The Paris system (TPS) has provided a standardized classification for reporting urinary cytology, specifically high-grade urothelial carcinoma (HGUC). While hyperchromasia is well described in HGUC, there exists little data on nuclear hypochromasia in HGUC. Our focus was to investigate the incidence of hypochromasia in HGUC and if it should become a criterion for HGUC. DESIGN: All cases of HGUC at our institution for a 3-year interval (2017-2019) using TPS were reviewed. Each case had a single ThinPrep slide and concurrent biopsy or resection specimen to confirm the diagnosis within 30 days. The presence of hypochromasia was evaluated, and cases with hypochromasia were stratified based on the tumor cell percentage. Cases with hypochromasia in 5% or greater of the tumor cells were considered "positively identified" for hypochromasia. RESULTS: We reviewed 117 cases of HGUC and identified nuclear hypochromasia in 12 cases (10.2%) within 5% or greater of the tumor cells. These 12 cases were further assessed based on if tumor cells showed hypochromasia in 5%-49% of the sample, or greater than 50% of the sample. Hypochromasia in 5%-49% of tumor cells was present in 8/117 cases (6.8%); whereas in 50% or greater samples 4/117 cases (3.4%) showed hypochromasia. No cases were identified where hypochromasia was noted in less than 5% of the tumor cells. CONCLUSION: TPS and use of hyperchromasia as a feature of HGUC is affirmed. However, hypochromasia, while not diagnostic in isolation, is present in a sub-set of patients with HGUC (10.2%) and should be considered as a variance noted in the nuclei of HGUC.

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.

Expression of Nectin-4 in Bladder Urothelial Carcinoma, in Morphologic Variants, and Nonurothelial Histotypes.

The antibody-drug conjugate enfortumab-vedotin acts by targeting nectin-4, a protein that is nearly ubiquitously expressed in conventional urothelial cancer. However, expression of nectin-4 in morphologic variants of urothelial carcinoma and nonurothelial histotypes was unknown. Immunohistochemistry for nectin-4 using was performed on 169 patients including 83 with nonmuscle invasive bladder cancer and 86 patients with muscle invasive bladder cancer. Staining was scored for intensity (0 to 3) and extent (% positive cells) using the histological score system, where >15 was considered positive. Overall, 72/83 (87%) samples of nonmuscle invasive urothelial carcinoma were positive, including 29/30 (97%) noninvasive papillary urothelial carcinomas, 7/8 (87.5%) carcinomas in situ, 36/45 (80%) papillary urothelial carcinomas invading the lamina propria. Overall, 50/86 muscle invasive tumors were positive, including 15/22 (68.2%) urothelial carcinomas, 7/10 (70%) squamous cell carcinomas, 3/11 (28%) micropapillary tumors, 4/6 (66%) adenocarcinomas, 2/4 (50%) nested carcinomas, 5/8 (63%) plasmacytoid, 1/10 (10%) sarcomatoid carcinomas, and 0/15 (0%) small cell carcinomas. Whole transcriptome RNA sequencing revealed that compared with conventional urothelial carcinomas, most sarcomatoid carcinomas and all but 2 small cell carcinomas expressed very low levels of nectin-4 mRNA but expressed significant levels of either trop2 or ERBB2, which are the molecular targets of 2 other antibody-drug conjugates-sacituzumab gavitecan (trop2) or trastuzumab deruxtecan (ERBB2/HER2). In summary, our study demonstrates that there is heterogeneity of expression of nectin-4 in morphologic variants of urothelial cancer and nonurothelial histotypes, and suggests that testing expression of nectin-4 should be considered in morphologic variants or nonurothelial histotypes found to have lower expression.

Construction of Decision Trees Based on Gene Expression Omnibus Data to Classify Bladder Cancer and Its Subtypes.

BACKGROUND Bladder cancer is a malignant tumor of the genitourinary system. Different subtypes of bladder cancer have different treatment methods and prognoses. Therefore, identifying hub genes affecting other genes is of great significance for the treatment of bladder cancer. MATERIAL AND METHODS We obtained expression profiles from the GSE13507 and GSE77952 datasets from the Gene Expression Omnibus database. First, principal component analysis was used to identify the difference in gene expression in different types of tissues. Differential expression analysis was used to find the differentially expressed genes between normal and tumor tissues, and between tumors with and without muscle infiltration. Further, based on differentially expressed genes, we constructed 2 decision trees for differentiating between tumor and normal tissues, and between muscle-infiltrating and non-muscle-infiltrating tumor tissues. A receiver operating characteristic curve was used to evaluate the prediction effect of the decision trees. RESULTS FAM107A and C8orf4 showed significantly lower expression in bladder cancer tissues than in normal tissues. Regarding muscle infiltration, CTHRC1 showed lower expression and HMGCS2 showed higher expression in non-muscle-infiltrating samples than in those with muscle infiltration. We constructed 2 decision trees for differentiating between tumor and normal tissue, and between tissues with and without muscle infiltration. Both decision trees showed good prediction results. CONCLUSIONS These newly discovered hub genes will be helpful in understanding the occurrence and development of different subtypes of bladder cancer, and will provide new therapeutic targets and biomarkers for bladder cancer.

Comprehensive Gene Expression Analyses of Immunohistochemically Defined Subgroups of Muscle-Invasive Urinary Bladder Urothelial Carcinoma.

A number of urinary bladder urothelial carcinoma (UB UC) mRNA-based classification systems have been reported. It also has been observed that treatment response and prognosis are different for each molecular subtype. In this study, cytokeratin (CK)5/6 and CK20 immunohistochemistry (IHC) were performed, and IHC-based subgroup classification was applied. UB UC was classified into CK5/6 single-positive (SP), CK20 SP, double-positive (DP) and double-negative (DN) subgroups, and transcriptional analysis was performed. The results of gene ontology (GO) terms and functional analysis using differentially expressed genes indicate that, CK5/6 SP and DP subgroups were enriched in cell migration, immune activation, interleukin 6-Janus kinase-signal transducer and activator of transcription 3 (IL6-JAK-STAT3) signaling pathway and tumor necrosis factor-α signaling via the nuclear factor-κB (NF-κB) signaling pathway signature gene. In addition, compared with the other subgroups, the DN subgroup showed inhibited cell movement, cell migration, and cell activation. Furthermore, in survival analysis, the CK5/6 SP subgroup was significantly associated with poor progression-free survival (p = 0.008). The results of our study indicate that the CK5/6 positive subgroup exhibited high gene expression signature related to aggressive behavior and exhibited worse clinical outcome.

Correlation of cyclin D1, HER2, and AMACR expressions with histologic grade in bladder urothelial carcinomas.

BACKGROUND AND OBJECTIVE: Bladder cancer is the ninth most common type of cancer worldwide. We aimed to investigate the relationship between tumor grade, lamina propria invasion, muscularis propria invasion, and lymphovascular invasion and human epidermal growth factor receptor 2 (HER-2), cyclin D1, and alpha-methyl-CoA racemase (AMACR) expressions in bladder cancer. MATERIALS AND METHODS: The study included patients who underwent complete TURBT. In total, 72 cases of bladder cancer diagnosed by two pathologists were selected. AMACR, HER-2, cyclin D1 expressions were detected immunohistochemically. RESULTS: The study population comprised 80% (57) males and 20% (15) females (mean age, 68 years). Further, 35 cases were noninvasive and 37 invasive urothelial carcinoma and 38 patients had low-grade tumor and 34 high-grade tumor. Intense immunostaining was observed with cyclin D1 for 75% tumors, AMACR for 39%, and HER-2 for 86%. High expressions of cyclin D1 and AMACR were observed in high-grade tumors (P < 0.05 and P < 0.005, respectively). High expression of HER-2 (2 and 3 positive) was found both at low- and high-grade tumors (84% and 88%, respectively). CONCLUSION: Cyclin D1, AMACR expressions were found to be significant predictive factors of high-grade tumors. High Her-2 expression in patients with bladder carcinoma may indicate that they are potential targets for treatment. These markers may be important in determining prognosis of tumors and may be valuable for guiding treatment options.

Defining muscle-invasive bladder cancer immunotypes by introducing tumor mutation burden, CD8+ T cells, and molecular subtypes.

Immunotherapy, especially anti-PD-1, is becoming a pillar of modern muscle-invasive bladder cancer (MIBC) treatment. However, the objective response rates (ORR) are relatively low due to the lack of precise biomarkers to select patients. Herein, the molecular subtype, tumor mutation burden (TMB), and CD8+ T cells were calculated by the gene expression and mutation profiles of MIBC patients. MIBC immunotypes were constructed using clustering analysis based on tumor mutation burden, CD8+ T cells, and molecular subtypes. Mutated genes, enriched functional KEGG pathways and GO terms, and co-expressed network-specific hub genes have been identified. We demonstrated that ORR of immunotype A patients identified by molecular subtype, CD8+ T cells, and TMB is about 36% predictable. PIK3CA, RB1, FGFR3, KMT2C, MACF1, RYR2, and EP300 are differentially mutated among three immunotypes. Pathways such as ECM-receptor interaction, PI3K-Akt signaling pathway, and TGF-beta signaling pathway are top-ranked in enrichment analysis. Low expression of ACTA2 was associated with the MIBC survival benefit. The current study constructs a model that could identify suitable MIBC patients for immunotherapy, and it is an important step forward to the personalized treatment of bladder cancers.

Should "suspicious for high-grade urothelial carcinoma" and "positive for high-grade urothelial carcinoma" remain separate categories?

BACKGROUND: The Paris System (TPS) for Reporting Urinary Cytology aims to standardize urine cytology reporting. Per TPS, the diagnosis of "suspicious for high-grade urothelial carcinoma (SHGUC)" is applied in cases that have few urothelial cells with severe atypia but are quantitatively insufficient for a diagnosis of "high-grade urothelial carcinoma (HGUC)." In our study, we compared the diagnostic accuracy and risk of malignancy (ROM) of these 2 categories to assess whether they could be combined in clinical practice to perhaps improve overall interobserver variability. METHODS: All urine specimens with a diagnosis of either SHGUC or HGUC from January 2016 to July 2019 were retrieved from the pathology database of 2 large academic institutions. Only cases with follow-up biopsies within 6 months were included. RESULTS: One hundred eighty-nine cases met the study criteria. Of these, 122 had a cytologic diagnosis of SHGUC, and 67 had a diagnosis of HGUC. Ninety-five (78%) cases from the SHGUC group and 64 (96%) cases from the HGUC group had biopsy-proven HGUC. The majority of cases with discordance had a history of treatment with either intravesical bacillus Calmette-Guérin or mitomycin. The difference in the rate of biopsy-proven HGUC between the SHGUC category and the HGUC category (95/122 vs 64/67, respectively) was statistically significant (P < .001). CONCLUSIONS: The difference in ROM between SHGUC and HGUC was statistically significant in our study cohort. Intravesical chemotherapy was frequently observed in negative biopsy cases in both groups. Our preliminary findings suggest that the 2 TPS categories should remain separate.

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.