Prognostic Role of Survivin and Macrophage Infiltration Quantified on Protein and mRNA Level in Molecular Subtypes Determined by RT-qPCR of KRT5, KRT20, and ERBB2 in Muscle-Invasive Bladder Cancer Treated by Adjuvant Chemotherapy

Objectives: Bladder cancer is a heterogeneous malignancy. Therefore, it is difficult to find single predictive markers. Moreover, most studies focus on either the immunohistochemical or molecular assessment of tumor tissues by next-generation sequencing (NGS) or PCR, while a combination of immunohistochemistry (IHC) and PCR for tumor marker assessment might have the strongest impact to predict outcome and select optimal therapies in real-world application. We investigated the role of proliferation survivin/BIRC5 and macrophage infiltration (CD68, MAC387, CLEVER-1) on the basis of molecular subtypes of bladder cancer (KRT5, KRT20, ERBB2) to predict outcomes of adjuvant treated muscle-invasive bladder cancer patients with regard to progression-free survival (PFS) and disease-specific survival (DSS). Materials and Methods: We used tissue microarrays (TMA) from n = 50 patients (38 males, 12 female) with muscle-invasive bladder cancer. All patients had been treated with radical cystectomy followed by adjuvant triple chemotherapy. Median follow-up time was 60.5 months. CD68, CLEVER-1, MAC387, and survivin protein were detected by immunostaining and subsequent visual inspection. BIRC5, KRT5, KRT20, ERBB2, and CD68 mRNAs were detected by standardized RT-qPCR after tissue dot RNA extraction using a novel stamp technology. All these markers were evaluated in three different centers of excellence. Results: Nuclear staining rather than cytoplasmic staining of survivin predicted DSS as a single marker with high levels of survivin being associated with better PFS and DSS upon adjuvant chemotherapy (p = 0.0138 and p = 0.001, respectively). These results were validated by the quantitation of BIRC5 mRNA by PCR (p = 0.0004 and p = 0.0508, respectively). Interestingly, nuclear staining of survivin protein was positively associated with BIRC5 mRNA, while cytoplasmic staining was inversely related, indicating that the translocation of survivin protein into the nucleus occurred at a discrete, higher level of its mRNA. Combining survivin/BIRC5 levels based on molecular subtype being assessed by KRT20 expression improved the predictive value, with tumors having low survivin/BIRC5 and KRT20 mRNA levels having the best survival (75% vs. 20% vs. 10% 5-year DSS, p = 0.0005), and these values were independent of grading, node status, and tumor stage in multivariate analysis (p = 0.0167). Macrophage infiltration dominated in basal tumors and was inversely related with the luminal subtype marker gene expression. The presence of macrophages in survivin-positive or ERBB2-positive tumors was associated with worse DSS. Conclusions: For muscle-invasive bladder cancer patients, the proliferative activity as determined by the nuclear staining of survivin or RT-qPCR on the basis of molecular subtype characteristics outperforms single marker detections and single technology approaches. Infiltration by macrophages detected by IHC or PCR is associated with worse outcome in defined subsets of tumors. The limitations of this study are the retrospective nature and the limited number of patients. However, the number of molecular markers has been restricted and based on predefined assumptions, which resulted in the dissection of muscle-invasive disease into tumor–biological axes of high prognostic relevance, which warrant further investigation and validation.</p

Identifying the Molecular Mechanisms Contributing to Progression, Metastasis, and Death in Low-grade Non-muscle-invasive Bladder Cancer: A Case Report

Transitional cell carcinoma of the bladder is a common malignancy with an estimated 549 393 new cases occurring in 2018 alone. Both non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) show high recurrence and progression rates, and therefore impose a great burden on patients and health care systems. Current risk stratification and therapy strategies are predominantly based on clinical and histopathological findings for tumor stage and grade. The chemoresistance and metastasis of low-grade tumors suggest an incomplete understanding of disease mechanisms, despite numerous studies on differentiating molecular subtypes of bladder cancer to identify tumor drivers and potential therapeutic targets. We present a highly unusual course for a low-grade bladder tumor leading to metastasis and death, for which we used postmortem histopathological and molecular analyses to evaluate targetable alterations in key signaling pathways driving the underlying tumor biology. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of European Association of Urology

Prognostic Role of Survivin and Macrophage Infiltration Quantified on Protein and mRNA Level in Molecular Subtypes Determined by RT-qPCR of KRT5, KRT20, and ERBB2 in Muscle-Invasive Bladder Cancer Treated by Adjuvant Chemotherapy

Objectives: Bladder cancer is a heterogeneous malignancy. Therefore, it is difficult to find single predictive markers. Moreover, most studies focus on either the immunohistochemical or molecular assessment of tumor tissues by next-generation sequencing (NGS) or PCR, while a combination of immunohistochemistry (IHC) and PCR for tumor marker assessment might have the strongest impact to predict outcome and select optimal therapies in real-world application. We investigated the role of proliferation survivin/BIRC5 and macrophage infiltration (CD68, MAC387, CLEVER-1) on the basis of molecular subtypes of bladder cancer (KRT5, KRT20, ERBB2) to predict outcomes of adjuvant treated muscle-invasive bladder cancer patients with regard to progression-free survival (PFS) and disease-specific survival (DSS). Materials and Methods: We used tissue microarrays (TMA) from n = 50 patients (38 males, 12 female) with muscle-invasive bladder cancer. All patients had been treated with radical cystectomy followed by adjuvant triple chemotherapy. Median follow-up time was 60.5 months. CD68, CLEVER-1, MAC387, and survivin protein were detected by immunostaining and subsequent visual inspection. BIRC5, KRT5, KRT20, ERBB2, and CD68 mRNAs were detected by standardized RT-qPCR after tissue dot RNA extraction using a novel stamp technology. All these markers were evaluated in three different centers of excellence. Results: Nuclear staining rather than cytoplasmic staining of survivin predicted DSS as a single marker with high levels of survivin being associated with better PFS and DSS upon adjuvant chemotherapy (p = 0.0138 and p = 0.001, respectively). These results were validated by the quantitation of BIRC5 mRNA by PCR (p = 0.0004 and p = 0.0508, respectively). Interestingly, nuclear staining of survivin protein was positively associated with BIRC5 mRNA, while cytoplasmic staining was inversely related, indicating that the translocation of survivin protein into the nucleus occurred at a discrete, higher level of its mRNA. Combining survivin/BIRC5 levels based on molecular subtype being assessed by KRT20 expression improved the predictive value, with tumors having low survivin/BIRC5 and KRT20 mRNA levels having the best survival (75% vs. 20% vs. 10% 5-year DSS, p = 0.0005), and these values were independent of grading, node status, and tumor stage in multivariate analysis (p = 0.0167). Macrophage infiltration dominated in basal tumors and was inversely related with the luminal subtype marker gene expression. The presence of macrophages in survivin-positive or ERBB2-positive tumors was associated with worse DSS. Conclusions: For muscle-invasive bladder cancer patients, the proliferative activity as determined by the nuclear staining of survivin or RT-qPCR on the basis of molecular subtype characteristics outperforms single marker detections and single technology approaches. Infiltration by macrophages detected by IHC or PCR is associated with worse outcome in defined subsets of tumors. The limitations of this study are the retrospective nature and the limited number of patients. However, the number of molecular markers has been restricted and based on predefined assumptions, which resulted in the dissection of muscle-invasive disease into tumor&ndash;biological axes of high prognostic relevance, which warrant further investigation and validation

Prediction of Response to Cisplatin-Based Neoadjuvant Chemotherapy of Muscle-Invasive Bladder Cancer Patients by Molecular Subtyping including KRT and FGFR Target Gene Assessment

Patients with muscle-invasive urothelial carcinoma achieving pathological complete response (pCR) upon neoadjuvant chemotherapy (NAC) have improved prognosis. Molecular subtypes of bladder cancer differ markedly regarding sensitivity to cisplatin-based chemotherapy and harbor FGFR treatment targets to various content. The objective of the present study was to evaluate whether preoperative assessment of molecular subtype as well as FGFR target gene expression is predictive for therapeutic outcome&mdash;rate of ypT0 status&mdash;to justify subsequent prospective validation within the &ldquo;BladderBRIDGister&rdquo;. Formalin-fixed paraffin-embedded (FFPE) tissue specimens from transurethral bladder tumor resections (TUR) prior to neoadjuvant chemotherapy and corresponding radical cystectomy samples after chemotherapy of 36 patients were retrospectively collected. RNA from FFPE tissues were extracted by commercial kits, Relative gene expression of subtyping markers (e.g., KRT5, KRT20) and target genes (FGFR1, FGFR3) was analyzed by standardized RT-qPCR systems (STRATIFYER Molecular Pathology GmbH, Cologne). Spearman correlation, Kruskal&ndash;Wallis, Mann&ndash;Whitney and sensitivity/specificity tests were performed by JMP 9.0.0 (SAS software). The neoadjuvant cohort consisted of 36 patients (median age: 69, male 83% vs. female 17%) with 92% of patients being node-negative during radical cystectomy after 1 to 4 cycles of NAC. When comparing pretreatment with post-treatment samples, the median expression of KRT20 dropped most significantly from DCT 37.38 to 30.65, which compares with a 128-fold decrease. The reduction in gene expression was modest for other luminal marker genes (GATA3 6.8-fold, ERBB2 6.3-fold). In contrast, FGFR1 mRNA expression increased from 33.28 to 35.88 (~6.8-fold increase). Spearman correlation revealed positive association of pretreatment KRT20 mRNA levels with achieving pCR (r = 0.3072: p = 0.0684), whereas pretreatment FGFR1 mRNA was associated with resistance to chemotherapy (r = &minus;0.6418: p &lt; 0.0001). Hierarchical clustering identified luminal tumors of high KRT20 mRNA expression being associated with high pCR rate (10/16; 63%), while the double-negative subgroup with high FGFR1 expression did not respond with pCR (0/9; 0%). Molecular subtyping distinguishes patients with high probability of response from tumors as resistant to neoadjuvant chemotherapy. Targeting FGFR1 in less-differentiated bladder cancer subgroups may sensitize tumors for adopted treatments or subsequent chemotherapy