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Heat shock proteins 60 and 70 are associated with long-term outcome of T1-stage high-grade urothelial tumors of the bladder treated with intravesical Bacillus Calmette-Guerin immunotherapy.

ABSTRACT: OBJECTIVES:High-grade nonmuscle-invasive urothelial tumors of the bladder that fail intravesical Bacillus Calmette-Guérin (BCG) immunotherapy are at the highest risk of progression. Initial evidence links heat shock protein expression levels and outcome of bladder cancer after BCG treatment. We aimed to determine the association between HSP60, 70, and 90 expression levels and long-term outcomes of T1 high-grade (T1HG) urothelial bladder tumors treated with BCG immunotherapy. MATERIALS AND METHODS:Data of 54 consecutive patients with primary T1HG bladder tumors who underwent transurethral resection between 2002 and 2008 and received at least an induction course of BCG were reviewed. Immunohistochemical staining for heat shock protein (HSP)60, 70, and 90 were performed on resected specimens. Study outcomes included disease recurrence and progression. The association between HSP expression levels and outcomes were evaluated with univariable and multivariable Cox proportional hazards models. RESULTS:During a median follow-up of 9.6 years, 25 patients had a disease recurrence and 14 patients a disease progression. Estimated 5-year recurrence and progression-free survival were 59% and 81%, respectively. On multivariable analyses, HSP60 staining >65% was associated with a higher risk for progression (hazard ratio [HR]?=?3.96, 95% confidence interval [CI] 1.35-11.58, P?=?0.012), and HSP70 staining >5% was associated with a decreased risk for progression (HR?=?0.33, 95% CI 0.11-0.98, P?=?0.045), and recurrence (HR?=?0.29, 95% CI 0.13-0.65, P?=?0.003). HSP90 expression was not associated with disease recurrence or progression. Five patients had both a HSP60 staining >65% and a HSP70 staining ?5% all of whom recurred at a median time of 6 months (interquartile range 3, 16) and 80% of whom progressed at a median time of 26 months (interquartile range 5, 60). CONCLUSIONS:HSP60 and 70 cellular expression levels are associated with long-term outcome following BCG treatment of T1HG urothelial bladder tumors. These findings, if further validated, may be used to better stratify the risk of disease recurrence and progression in this group of patients.

SUBMITTER: Mano R

PROVIDER: S-EPMC6289697 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Heat shock proteins 60 and 70 are associated with long-term outcome of T1-stage high-grade urothelial tumors of the bladder treated with intravesical Bacillus Calmette-Guérin immunotherapy.

Mano Roy R Zilber Sofia S Di Natale Renzo G RG Kedar Daniel D Lifshitz David A DA Yossepowitch Ofer O Baniel Jack J Margel David D

Urologic oncology 20181015 12

<h4>Objectives</h4>High-grade nonmuscle-invasive urothelial tumors of the bladder that fail intravesical Bacillus Calmette-Guérin (BCG) immunotherapy are at the highest risk of progression. Initial evidence links heat shock protein expression levels and outcome of bladder cancer after BCG treatment. We aimed to determine the association between HSP60, 70, and 90 expression levels and long-term outcomes of T1 high-grade (T1HG) urothelial bladder tumors treated with BCG immunotherapy.<h4>Materials ...[more]

PMID: 30337218

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Project description:BackgroundIntravesical Bacillus Calmette-Guerin (BCG) is an effective treatment for bladder superficial carcinoma and it is being tested in interstitial cystitis patients, but its precise mechanism of action remains poorly understood. It is not clear whether BCG induces the release of a unique set of cytokines apart from its pro-inflammatory effects. Therefore, we quantified bladder inflammatory responses and alterations in urinary cytokine protein induced by intravesical BCG and compared the results to non-specific pro-inflammatory stimuli (LPS and TNF-alpha). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder.MethodsC57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Morphometric analyses were conducted in bladders isolated from all groups and urine was collected for multiplex analysis of 18 cytokines. In addition, chromatin immune precipitation combined with real-time polymerase chain reaction assay (CHIP/Q-PCR) was used to test whether intravesical BCG would alter bladder cytokine gene expression.ResultsAcute BCG instillation induced edema which was progressively replaced by an inflammatory infiltrate, composed primarily of neutrophils, in response to weekly administrations. Our morphological analysis suggests that these polymorphonuclear neutrophils are of prime importance for the bladder responses to BCG. Overall, the inflammation induced by BCG was higher than LPS or TNF-alpha treatment but the major difference observed was the unique granuloma formation in response to BCG. Among the cytokines measured, this study highlighted the importance of IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-10, IL-17, GM-CSF, KC, and Rantes as discriminators between generalized inflammation and BCG-specific inflammatory responses. CHIP/Q-PCR indicates that acute BCG instillation induced an up-regulation of IL-17A, IL-17B, and IL-17RA, whereas chronic BCG induced IL-17B, IL-17RA, and IL-17RB.ConclusionTo the best of our knowledge, the present work is the first to report that BCG induces an increase in the IL-17 family genes. In addition, BCG induces a unique type of persisting bladder inflammation different from TNF-alpha, LPS, and, most likely, other classical pro-inflammatory stimuli.

Project description:Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression in the bladder target organ beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following chronic intravesical BCG therapy and to compare the results to non-specific pro inflammatory stimuli (LPS and TNF-alpha). For this purpose, C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Seven days after the last instillation, the urothelium along with the submucosa was removed from detrusor muscle and the RNA was extracted from both layers for cDNA array experiments. Microarray results were normalized by a robust regression analysis and only genes with an expression above a conditional threshold of 0.001 (3SD above background) were selected for analysis. Next, genes presenting a 3-fold ratio in regard to the control group were entered in Ingenuity Pathway Analysis (IPA) for a comparative analysis in order to determine genes specifically regulated by BCG, TNF-alpha, and LPS. In addition, the transcriptome was precipitated with an antibody against RNA polymerase II and real-time polymerase chain reaction assay (Q-PCR) was used to confirm some of the BCG-specific transcripts.Molecular networks of treatment-specific genes generated several hypotheses regarding the mode of action of BCG. BCG-specific genes involved small GTPases and BCG-specific networks overlapped with the following canonical signaling pathways: axonal guidance, B cell receptor, aryl hydrocarbon receptor, IL-6, PPAR, Wnt/beta-catenin, and cAMP. In addition, a specific detrusor network expressed a high degree of overlap with the development of the lymphatic system. Interestingly, TNF-alpha-specific networks overlapped with the following canonical signaling pathways: PPAR, death receptor, and apoptosis. Finally, LPS-specific networks overlapped with the LPS/IL-1 mediated inhibition of RXR. Because NF-kappaB occupied a central position in several networks, we further determined whether this transcription factor was part of the responses to BCG. Electrophoretic mobility shift assays confirmed the participation of NF-kappaB in the mouse bladder responses to BCG. In addition, BCG treatment of a human urothelial cancer cell line (J82) also increased the binding activity of NF-kappaB, as determined by precipitation of the chromatin by a NF-kappaB-p65 antibody and Q-PCR of genes bearing a NF-kappaB consensus sequence. Next, we tested the hypothesis of whether small GTPases such as LRG-47 are involved in the uptake of BCG by the bladder urothelium.As expected, BCG treatment induces the transcription of genes belonging to common pro-inflammatory networks. However, BCG also induces unique genes belonging to molecular networks involved in axonal guidance and lymphatic system development within the bladder target organ. In addition, NF-kappaB seems to play a predominant role in the bladder responses to BCG therapy. Finally, in intact urothelium, BCG-GFP internalizes in LRG-47-positive vesicles. These results provide a molecular framework for the further study of the involvement of immune and nervous systems in the bladder responses to BCG therapy.

Project description:IntroductionIntravesical therapy (IVT), including Bacillus Calmette-Guérin (BCG), is the standard of care for high grade (HG) non-muscle invasive bladder cancer (NMIBC). Despite the use of IVT, many patients recur after treatment. The bladder microbiome and its role in disease processes has recently risen to prominence. We aim to characterize changes that occur in the bladder microbiome over the course of intravesical therapy and assess whether these changes correlate with outcomes in patients with NMIBC.MethodsPatients with NMIBC undergoing induction BCG or intravesical therapy were prospectively enrolled from January 2019 to March 2020. Patients with clinical T2 or greater pathology or active urinary tract infection at enrollment were excluded. Twenty-nine patients had catheterized (bladder) urine samples collected prior to induction intravesical therapy and prior to each IVT instillation. Twenty-seven received BCG while 2 received intravesical gemcitabine. Bacteria were identified using 16S ribosomal RNA gene sequencing. Bladder microbiome changes were evaluated and differences between patients who recurred and patients who did not recur after IVT were investigated.ResultsAcross the 29 patients analyzed, bacterial richness decreased significantly following intravesical therapy (Richness, P=0.01). Evenness and overall diversity did not change significantly (Pielou, P=0.62; Shannon, P=0.13). Patients who experienced recurrence had a higher relative abundance of Aerococcus in their urine (P<0.01), while those who did not recur had significantly more Ureaplasma (P=0.01) and Escherichia/Shigella species (P=0.05). Patients with decreased levels of alpha diversity were more likely to fall within the non-recurrence cohort.ConclusionIVT for NMIBC appears to change the urinary microbiome by decreasing richness while not altering evenness or overall diversity. The presence of Aerococcus species may be predictive of a poor cancer response to IVT, while the presence of Ureaplasma and Escherichia/Shigella may predict a favorable response to IVT. Further studies are warranted to elucidate and confirm the significance of changes in the bladder microbiome.

Dataset Information

Heat shock proteins 60 and 70 are associated with long-term outcome of T1-stage high-grade urothelial tumors of the bladder treated with intravesical Bacillus Calmette-Guerin immunotherapy.

Publications

Heat shock proteins 60 and 70 are associated with long-term outcome of T1-stage high-grade urothelial tumors of the bladder treated with intravesical Bacillus Calmette-Guérin immunotherapy.

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