Immunohistochemical assays for bladder cancer molecular subtyping: Optimizing parsimony and performance using Lund taxonomy

Transcriptomic and proteomic profiling reliably classifies bladder cancers into luminal and basal molecular subtypes. Based on their prognostic and predictive associations, these subtypes may improve clinical management of bladder cancers. However, the complexity of published subtyping algorithms has limited their translation into practice. Here we optimize and validate compact subtyping algorithms based on the Lund taxonomy. We reanalyzed immunohistochemistry (IHC) expression data of muscle-invasive bladder cancer samples from Lund 2017 (n=193) and 2012 (n=76) cohorts. We characterized and quantified IHC expression patterns, and determined the simplest, most accurate decision tree models to identify subtypes. We tested the utility of a previously published algorithm using routine antibody assays commonly available in surgical pathology laboratories (GATA3, KRT5 and p16) to identify basal/luminal subtypes and to distinguish between luminal subtypes, Urothelial-Like (Uro) and Genomically Unstable (GU). We determined the dominant decision tree classifiers using four-fold cross-validation with separate uniformly distributed train (75%) and validation (25%) sets. Using the three-antibody algorithm resulted in 86-95% accuracy across training and validation sets for identifying basal/luminal subtypes, and 67-86% accuracy for basal/Uro/GU subtypes. Although antibody assays for KRT14 and RB1 are not routinely used in pathology practice, these features achieved the simplest and most accurate models to identify basal/luminal and Uro/GU/basal subtypes, achieving 93-96% and 85-86% accuracies, respectively. When translated to a more complex model using eight antibody assays, accuracy was comparable to simplified models, with 86% (train) and 82% (validation). We conclude that a simple immunohistochemical classifier can accurately identify luminal (Uro, GU) and basal subtypes and pave the way for clinical implementation.

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Longitudinal Follow‐up and Performance Validation of a mRNA‐based Urine Test (Xpert® Bladder Cancer Monitor) for surveillance in Non‐Muscle Invasive Bladder Cancer Patients

BJU International ◽

10.1111/bju.15418 ◽

2021 ◽

Author(s):

Barrett Cowan ◽

Eric Klein ◽

Ken Jansz ◽

Karl Westenfelder ◽

Timothy Bradford ◽

...

Keyword(s):

Bladder Cancer ◽

Cancer Patients ◽

Invasive Bladder Cancer ◽

Urine Test ◽

Muscle Invasive Bladder Cancer ◽

And Performance ◽

Muscle Invasive ◽

Performance Validation

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Gene expression profiling in bladder cancer to identify potential therapeutic targets.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.4518 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 4518-4518

Author(s):

Syed A. Hussain ◽

Daniel H. Palmer ◽

Wing Kin Syn ◽

Joseph J Sacco ◽

Bryony Lloyd ◽

...

Keyword(s):

Gene Expression ◽

Bladder Cancer ◽

Hierarchical Clustering ◽

Expression Profiles ◽

Expression Patterns ◽

Invasive Cancer ◽

Classical Complement Pathway ◽

Grade 3 ◽

Muscle Invasive

4518 Background: Characterization of gene expression patterns in bladder cancer (BC) allows the identification of pathways involved in its pathogenesis, and may stimulate the development of novel therapies targeting these pathways. Methods: Between 2004 and 2005, cystoscopic bladder biopsies were obtained from 19 patients and 11 controls. These were subjected to whole transcript-based microarray analysis. Unsupervised hierarchical clustering was used to identify samples with similar expression profiles. Results: Hierarchical clustering defined signatures, which differentiated between cancer and normal, muscle-invasive or non-muscle invasive cancer and normal, g1 and g3. Pathways associated with cell cycle and proliferations were markedly upregulated in muscle-invasive and grade 3 cancers. Genes associated with the classical complement pathway were downregulated in non-muscle invasive cancer. Osteopontin was markedly overexpressed in invasive cancer as compared to normal tissue. Conclusions: This study contributes to a growing body of work on gene expression signatures in BC. The data support an important role for osteopontin in BC, and identify several pathways worthy of further investigation. [Table: see text]

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Smoking status and PD-L1 mRNA-expression as a predictor of response to neoadjuvant chemotherapy in patients diagnosed with muscle invasive bladder cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.6_suppl.530 ◽

2020 ◽

Vol 38 (6_suppl) ◽

pp. 530-530

Author(s):

Moritz Reike ◽

Hendrik Juette ◽

Ralph Wirtz ◽

Philipp Erben ◽

Karl Tully ◽

...

Keyword(s):

Bladder Cancer ◽

Neoadjuvant Chemotherapy ◽

Mrna Expression ◽

Smoking Status ◽

Expression Patterns ◽

Invasive Bladder Cancer ◽

Study Cohort ◽

Muscle Invasive Bladder Cancer ◽

Former Smokers ◽

Muscle Invasive

530 Background: Patients with muscle-invasive bladder cancer (MIBC) that underwent neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC) show improved overall survival. Patients with a pathological complete response (pCR) usually have the best prognosis. In the literature, impaired response to immune checkpoint therapy has been reported in active smokers. The aim of our study was to examine the association of smoking status with pCR at RC after NAC. Moreover, we investigated the interaction of smoking status and Programmed Death Ligand 1 (PD-L1) mRNA expression at transurethral resection (TUR) and pCR prediction at RC after NAC. Methods: Clinical Data and formalin fixed paraffin embedded tumor tissue samples from TUR and RC of 49 patients with MIBC were retrospectively analyzed. Using RT-PCR PD-L1 mRNA expression was measured in 40-∆Ct values and normalized against the control gene CALM2. Smoking status was defined as never, former and active. After NAC, RC was performed and the specimens were evaluated for pCR, defined as ypT0N0M0. Statistical analyses comprised nonparametric and chi2 testing, partition models and spearman correlation analyses. Results: The study cohort had a median age of 63 years and consisted of 38/49 (78%) males. Regarding smoking status 11/49 (22%) were never, 17/49 (35%) were former and 21/49 (43%) were active smokers. After NAC, 17/49 patients (35%) had a pCR. Never/former smokers did not show a higher rate of pCR compared to active smokers (43%vs.24%, p=0.16). Comparing smoking status (never/former vs. active smokers) within the subgroup showing high PD-L1 expression (≥32.1∆Ct), a higher rate of pCR was found in never/former smokers (58% vs. 25%, p=0.047). Conclusions: Never and former smokers with MIBC that show high PD-L1 mRNA expression patterns are more likely to show pCR at RC after NAC. Smoking cessation is important for the management of MIBC patients undergoing NAC and RC.

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Clinical outcomes of muscle invasive bladder Cancer according to the BASQ classification

BMC Cancer ◽

10.1186/s12885-019-6042-1 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 4

Author(s):

Hyeong Dong Yuk ◽

Chang Wook Jeong ◽

Cheol Kwak ◽

Hyeon Hoe Kim ◽

Kyung Chul Moon ◽

...

Keyword(s):

Bladder Cancer ◽

Classification System ◽

Expression Patterns ◽

Treatment Strategies ◽

Invasive Bladder Cancer ◽

Molecular Characteristics ◽

Muscle Invasive Bladder Cancer ◽

Cancer Specific Survival ◽

Muscle Invasive

Abstract Background We evaluated the clinical efficacy and prognosis of muscle-invasive bladder cancer according to the basal/squamous-like (BASQ) classification system based on immunohistochemical staining [CK5/6(+), CK14(+), GATA3(−), and FOXA1(−)]. Methods One hundred patients diagnosed with muscle-invasive bladder cancer (cT2-4 N0-3 M0) were included in the study. All patients underwent radical cystectomy after transurethral removal of bladder tumor. Immunostaining was performed for CK5/6, CK14, FOXA1, and GATA3 antibodies on tissue microarray slides, and expression patterns were quantitatively analyzed using a scanning program. Results The median follow-up time was 77.4 (interquartile range: 39–120.9) months. The mean age of the patients was 65.1 ± 11.2 years. FOXA1 or CK14 expression greater than 1% was respectively positively and negatively correlated with overall survival (OS; p = 0.011 and p = 0.042, respectively), cancer-specific survival (CSS; p = 0.050 for both), and recurrence-free survival (RFS; p = 0.018 and p = 0.040, respectively). For CK5/6+ and GATA3- or FOXA1- expression, 10% CK5/6+ cells were negatively correlated with OS (p = 0.032 and p = 0.039, respectively) and with RFS in combination with FOXA1- only (p = 0.050). Conclusions In this study, CK14 expression was associated with a poor prognosis. The new classification system of bladder cancer based on molecular characteristics is expected to helpful tool for the establishment of personalized treatment strategies and associated prediction of therapeutic responses.

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Muscle invasive bladder cancer (MIBC) demonstrates neoadjuvant cisplatin-based chemotherapy (NAC) related changes in molecular subtype and immune infiltration.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.7_suppl.443 ◽

2019 ◽

Vol 37 (7_suppl) ◽

pp. 443-443

Author(s):

Samuel Aaron Funt ◽

Alexander Solovyov ◽

Bishoy Morris Faltas ◽

Gopa Iyer ◽

Mariel Elena Boyd ◽

...

Keyword(s):

Bladder Cancer ◽

Bladder Tumor ◽

Molecular Subtypes ◽

Molecular Subtype ◽

Expression Patterns ◽

The Cancer Genome Atlas ◽

Rna Expression ◽

Cancer Therapies ◽

Primary Bladder ◽

Muscle Invasive

443 Background: Defining the role of MIBC molecular subtypes and immune expression in determining clinical outcomes is an area of active investigation. However, changes in these transcriptomic profiles pre- and post-NAC have not been well characterized. Methods: This retrospective study reviewed 53 pts with MIBC treated with NAC, of whom 12 pts without complete pathological response had both pre- and post-NAC samples of sufficient quality. Post-NAC staging was > = pT2 in 11 pts and pT1 in 1 pt. We performed RNA expression analysis of matched pre-NAC transurethral resection of bladder tumor specimens and post-treatment radical cystectomy primary bladder tumor specimens. We used a customized NanoString panel incorporating previously reported immune signatures (Ayers, JCI 2017; O’Donnell, ASCO 2017) and additional genes to assign basal ( CD14, CD44, PDGFC, KRT14, KRT5) and luminal ( GATA3, PPARG, SHH, CD24, FOXA1, WNT7B, ERBB2) molecular subtypes. Results: We first classified the bladder cancer cohort of The Cancer Genome Atlas into basal and luminal subtypes using the BASE47 signature (Damrauer, PNAS 2014) and the NanoString panel and there was good agreement (Rand Index = 0.72). We then assigned subtypes using the NanoString panel on matched pre- and post-NAC samples and found marked subtype shift (Table). We identified two robust clusters of samples according to immune expression with a 3-fold change of immune expression between them (FDR = 0.0008). We found that 4 pts switched from the low to the high cluster, while 2 switched from the high to the low cluster after NAC (Table). Conclusions: MIBC molecular subtype membership is dynamic and is influenced by NAC. NAC can induce both enhanced and suppressed immune activity. These findings have implications on future studies exploring the predictive value of RNA expression patterns for bladder cancer therapies as well as post-NAC immunotherapy. [Table: see text]

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BioXmark® liquid fiducial markers for image-guided radiotherapy in muscle invasive bladder cancer: a safety and performance trial

British Journal of Radiology ◽

10.1259/bjr.20200241 ◽

2020 ◽

Vol 93 (1111) ◽

pp. 20200241

Author(s):

Mischa de Ridder ◽

Lara C Gerbrandy ◽

Theo M de Reijke ◽

Karel A Hinnen ◽

Maarten C.C.M. Hulshof

Keyword(s):

Bladder Cancer ◽

Learning Curve ◽

Fiducial Marker ◽

Invasive Bladder Cancer ◽

The Novel ◽

Muscle Invasive Bladder Cancer ◽

Image Guided Radiotherapy ◽

Promising Tool ◽

And Performance ◽

Muscle Invasive

Objective: This study evaluated the performance of the novel liquid fiducial marker (BioXmark®) in IGRT for bladder cancer. Methods: 20 patients with muscle invasive bladder cancer were entered in this prospective, single center, Phase I-II study. The novel BioXmark® liquid markers were injected around the tumor using a flexible cystoscopy. Visibility and stability of the markers were evaluated on planning-CT and CBCT. Prospectively defined threshold for success was set at a visibility of 75%. Results: In total, 76 markers were implanted in 20 patients. Of those, 60 (79% 95% CI ± 9%) were visible on CT scan. Due to the learning curve of the technique, the visibility improved in the last 75% of patients (86% visibility) compared to the first 25% of patients with 58% visibility. Concerning stability of the BioXmark® marker, all visible markers after CT acquisition were still detectable at the last CBCT without displacement. In 15/20 (75%) of the patients, three or more markers were visible on CT. No BioXmark® related adverse events were reported. Conclusion: The success rate of this novel fiducial marker was 79%, which is above the prospectively defined threshold rate. A distinct learning curve of the injection of the liquid marker was seen over the study period. The marker showed sustained visibility and positional stability during treatment phases and also appears to be safe and easy to inject. Advances in knowledge: This novel liquid BioXmark® marker seems to be a very promising tool in daily-adaptive IGRT for bladder preserving chemoradiotherapy in muscle invasive bladder cancer.

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Expression patterns and prognostic role of transketolase-like 1 in muscle-invasive bladder cancer

World Journal of Urology ◽

10.1007/s00345-014-1473-4 ◽

2015 ◽

Vol 33 (10) ◽

pp. 1403-1409 ◽

Cited By ~ 5

Author(s):

Mauro Semilia ◽

Jörg Hennenlotter ◽

Carlo Pavone ◽

Teresa Bischoff ◽

Ursula Kühs ◽

...

Keyword(s):

Bladder Cancer ◽

Expression Patterns ◽

Invasive Bladder Cancer ◽

Prognostic Role ◽

Muscle Invasive Bladder Cancer ◽

Muscle Invasive

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Identification of Biomarkers for Esophageal Squamous Cell Carcinoma Using Feature Selection and Decision Tree Methods

The Scientific World JOURNAL ◽

10.1155/2013/782031 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 16

Author(s):

Chun-Wei Tung ◽

Ming-Tsang Wu ◽

Yu-Kuei Chen ◽

Chun-Chieh Wu ◽

Wei-Chung Chen ◽

...

Keyword(s):

Feature Selection ◽

Decision Tree ◽

Squamous Cell ◽

Expression Profiles ◽

Expression Patterns ◽

Cell Cancer ◽

Design Decision ◽

Discrimination Ability ◽

Normal Tissues ◽

Tree Models

Esophageal squamous cell cancer (ESCC) is one of the most common fatal human cancers. The identification of biomarkers for early detection could be a promising strategy to decrease mortality. Previous studies utilized microarray techniques to identify more than one hundred genes; however, it is desirable to identify a small set of biomarkers for clinical use. This study proposes a sequential forward feature selection algorithm to design decision tree models for discriminating ESCC from normal tissues. Two potential biomarkers of RUVBL1 and CNIH were identified and validated based on two public available microarray datasets. To test the discrimination ability of the two biomarkers, 17 pairs of expression profiles of ESCC and normal tissues from Taiwanese male patients were measured by using microarray techniques. The classification accuracies of the two biomarkers in all three datasets were higher than 90%. Interpretable decision tree models were constructed to analyze expression patterns of the two biomarkers. RUVBL1 was consistently overexpressed in all three datasets, although we found inconsistent CNIH expression possibly affected by the diverse major risk factors for ESCC across different areas.

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Association of KRT20 and KRT5 with response to neoadjuvant chemotherapy in patients diagnosed with muscle invasive bladder cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.6_suppl.562 ◽

2020 ◽

Vol 38 (6_suppl) ◽

pp. 562-562

Author(s):

Florian Roghmann ◽

Moritz Reike ◽

Ralph Wirtz ◽

Maximilian Kriegmair ◽

Philipp Erben ◽

...

Keyword(s):

Bladder Cancer ◽

Neoadjuvant Chemotherapy ◽

Mrna Expression ◽

Expression Patterns ◽

High Risk Group ◽

Invasive Bladder Cancer ◽

Study Cohort ◽

Muscle Invasive Bladder Cancer ◽

Tissue Samples ◽

Muscle Invasive

562 Background: Patients with muscle-invasive bladder cancer (MIBC) that underwent neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC) show improved overall survival. Those with a pathological complete response (pCR) usually have the best prognosis. In the literature, improved response to NAC has been associated with basal tumor characteristics in MIBC so far. The aim of the present study was to examine the association of luminal (KRT20) and basal (KRT5) mRNA expression patterns at transurethral resection (TUR) with pCR at RC after NAC in a contemporary cohort of consecutive MIBC patients. Methods: Clinical Data and formalin fixed paraffin embedded tumor tissue samples from TUR and RC of 49 patients with MIBC were retrospectively analyzed. Using RT-PCR KRT20 and KRT5 mRNA expression were measured in 40-∆Ct values and normalized against the control gene CALM2. Statistical analyses comprised nonparametric and chi2 testing, partition models and spearman correlation analyses. Results: The study cohort had a median age of 63 years and consisted of 38/49 (78%) males. After NAC, 17/49 (35%) patients had cPR. Using partition models, we found that patients with high-KRT20 (≥39.5 ∆Ct) had a higher chance of pCR (57% vs. 26%, p=0.04). Using a cutoff for KRT5 at <38.1 ∆Ct within the subgroup of patients with low-KRT20 (<39.5 ∆Ct, n=35), we found poorest response among low-KRT20/low-KRT5 compared to low-KRT20/high-KRT5 and high-KRT20 (13% vs. 37% vs. 57%, p=0.29), respectively. For low-KRT20/low-KRT5, low-KRT20/high-KRT5 and high-KRT20 median KRT5 was 34.8 vs. 39.5 vs. 34.1 ∆Ct ( p=0.001) and median KRT20 was 37.9 vs. 32.9 vs. 40.1 ∆Ct,( p=0.001), respectively. Conclusions: Patients with MIBC showing high expression of KRT20 were more likely to show pCR at RC after NAC. Moreover, we were able to identify a high risk group of patients with lowKRT20/lowKRT5 that was less likely to achieve pCR at RC after NAC. Our findings are contradicting previous studies and need further verification in larger cohorts. However, our results might be useful for treatment stratification in MIBC patients.

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