Cohort profile: COBLAnCE: a French prospective cohort to study prognostic and predictive factors in bladder cancer and to generate real-world data on treatment patterns, resource use and quality of life.

PURPOSE: Bladder cancer is a complex disease with a wide range of outcomes. Clinicopathological factors only partially explain the variability between patients in prognosis and treatment response. There is a need for large cohorts collecting extensive data and biological samples to: (1) investigate gene-environment interactions, pathological/molecular classification and biomarker discovery; and (2) describe treatment patterns, outcomes, resource use and quality of life in a real-world setting. PARTICIPANTS: COBLAnCE (COhort to study BLAdder CancEr) is a French national prospective cohort of patients with bladder cancer recruited between 2012 and 2018 and followed for 6 years. Data on patient and tumour characteristics, treatments, outcomes and biological samples are collected at enrolment and during the follow-up. FINDINGS TO DATE: We describe the cohort at enrolment according to baseline surgery and tumour type. In total, 1800 patients were included: 1114 patients with non-muscle-invasive bladder cancer (NMIBC) and 76 patients with muscle-invasive bladder cancer (MIBC) had transurethral resection of a bladder tumour without cystectomy, and 610 patients with NMIBC or MIBC underwent cystectomy. Most patients had a solitary lesion (56.3%) without basement membrane invasion (71.7% of Ta and/or Tis). Half of the patients with cystectomy were stage ≤T2 and 60% had non-continent diversion. Surgery included local (n=298) or super-extended lymph node dissections (n=11) and prostate removal (n=492). Among women, 16.5% underwent cystectomy and 81.4% anterior pelvectomy. FUTURE PLANS: COBLAnCE will be used for long-term studies of bladder cancer with focus on clinicopathological factors and molecular markers. It will lead to a much-needed improvement in the understanding of the disease. The cohort provides valuable real-world data, enabling researchers to study various research questions, assess routine medical practices and guide medical decision-making.

A Case-Only Genome-Wide Interaction Study of Smoking and Bladder Cancer Risk: Results from the COBLAnCE Cohort.

Bladder cancer (BC) is the 6th most common cancer worldwide, with tobacco smoking considered as its main risk factor. Accumulating evidence has found associations between genetic variants and the risk of BC. Candidate gene-environment interaction studies have suggested interactions between cigarette smoking and NAT2/GSTM1 gene variants. Our objective was to perform a genome-wide association case-only study using the French national prospective COBLAnCE cohort (COhort to study BLAdder CancEr), focusing on smoking behavior. The COBLAnCE cohort comprises 1800 BC patients enrolled between 2012 and 2018. Peripheral blood samples collected at enrolment were genotyped using the Illumina Global Screening Array with a Multi-Disease drop-in panel. Genotyping data (9,719,614 single nucleotide polymorphisms (SNP)) of 1674, 1283, and 1342 patients were analyzed for smoking status, average tobacco consumption, and age at smoking initiation, respectively. A genome-wide association study (GWAS) was conducted adjusting for gender, age, and genetic principal components. The results suggest new candidate loci (4q22.1, 12p13.1, 16p13.3) interacting with smoking behavior for the risk of BC. Our results need to be validated in other case-control or cohort studies.

Automated DNA, RNA, and Protein Extraction from Urine for Biobanking.

Introduction/Objective: DNA, RNA, and proteins are unavoidable human biomarkers. Today, blood remains the commonly used source of biomarkers despite numerous limitations. Therefore, other sources of biomarkers such as urine could be more appropriate for research in the field of bladder cancer. The aim of this study was to set up a new automated procedure for urinary DNA, RNA, and protein extraction and to evaluate their quality and quantity. Materials and Methods: This study was conducted in the setting of the COBLAnCE cohort. Urinary DNA and RNA were extracted using the Maxwell 16 system, and urinary proteins were isolated by precipitation from the supernatant and the cell pellet. The concentration and purity of nucleic acids were determined by spectrophotometry. RNA integrity was determined by the Agilent Bioanalyzer. PCR assays were also used to ensure the quality of DNA and RNA samples. The quality of protein samples obtained was determined by Western blot analysis. Results: PCR experiments performed highlighted that it is possible to use the DNA and RNA samples for amplification, gene expression, or genotyping. However, DNA and RNA recovery from urine was highly variable among patients, with a significant impact of the patient's gender. The samples were highly degraded. Finally, our protocol of protein isolation was effective in extracting urinary supernatant proteins as well as pellet proteins. Discussion: Therefore, urine samples could constitute valuable resources for subsequent investigations in bladder cancer. These samples will allow identifying new easy-access biomarkers for the early detection of cancer, monitoring cancer progression, and assessing response to therapy.

Identification of immunosuppressive factors in retinoblastoma cell secretomes and aqueous humor from patients.

The microenvironment of retinoblastoma, the solid malignancy of the developing retina, is immunosuppressive. To study the interactions between tumor-associated microglia/macrophages (TAMs) and tumor cells in retinoblastomas, we analyzed immunohistochemistry markers in 23 patient samples and characterized 105 secreted cytokines of 11 retinoblastoma cell models in culture. We detected profuse infiltration of CD163+ protumoral M2-like polarized TAMs in eyes enucleated due to cancer progression. Previous treatment of patients increased the number of TAMs but did not affect M2-like polarization. M2-like microglia/macrophages were almost absent in five eyes obtained from children enucleated due to nontumoral causes. CD8+ tumor-infiltrating lymphocytes (TILs) were moderately abundant in tumor eyes and very scarce in nontumoral ones. The expression of the immune checkpoint molecule PD-L1 was absent in 95% of the tumor samples, which is concordant with the finding of FOXP3+ Tregs infiltrating tumors. We confirmed the pathology results using single-cell transcriptome analysis of one tumor. We identified the cytokines extracellular matrix metalloproteinase inducer (EMMPRIN) and macrophage migration inhibitory factor (MIF), both with reported immunosuppressive activity, secreted at high levels in retinoblastoma primary cell cultures. Gene expression analysis of a large retinoblastoma cohort and single-cell transcriptome analysis confirmed that MIF and EMMPRIN were significantly upregulated in retinoblastomas, which led us to quantify both proteins by immunoassays in liquid biopsies (aqueous humor obtained from more than 20 retinoblastoma patients). We found a significant increase in the concentration of MIF and EMMPRIN in cancer patients, compared to 12 noncancer ones. Finally, we showed that macrophages derived from peripheral blood mononuclear cells increased the expression of markers of M2-like polarization upon exposure to retinoblastoma-conditioned medium or recombinant MIF. Overall, our findings suggest that retinoblastoma cell secretions induce the protumoral phenotype of this tumor. Our results might have clinical impact in the fields of biomarkers and treatment. © 2022 The Pathological Society of Great Britain and Ireland.

Multilayer spectrum of intratumoral heterogeneity in basal bladder cancer.

Basal/squamous (Ba/Sq) subtype represents an intrinsic and robust group in the consensus molecular classification of muscle-invasive bladder cancer (MIBC), with poor outcome and controversial chemosensitivity. We aimed to investigate the spectrum of intratumor heterogeneity (ITH) in the Ba/Sq subtype. First, we validated a 29-gene NanoString CodeSet to predict the Ba/Sq subtype for FFPE samples. We identified heterogeneous Ba/Sq tumors in a series of 331 MIBC FFPE samples using dual GATA3/KRT5/6 immunohistochemistry (IHC). Heterogeneous regions with distinct immunostaining patterns were studied separately for gene expression using the 29-gene CodeSet, for mutations by targeted next-generation sequencing, and for copy number alteration (CNA) by microarray hybridization. Among 83 Ba/Sq tumors identified by GATA3/KRT5/6 dual staining, 19 tumors showed heterogeneity at the IHC level. In one third of the 19 cases, regions from the same tumor were classified in different distinct molecular subtypes. The mutational and CNA profiles confirmed the same clonal origin for IHC heterogeneous regions with possible subclonal evolution. Overall, two patterns of intratumoral heterogeneity (ITH) were observed in Ba/Sq tumors: low ITH (regions with distinct immunostaining, but common molecular subtype and shared CNA) or high ITH (regions with distinct immunostaining, molecular subtype, and CNA). These results showed multilayer heterogeneity in Ba/Sq MIBC. In view of personalized medicine, this heterogeneity adds complexity and should be taken into account for sampling procedures used for diagnosis and treatment choice. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A high-risk retinoblastoma subtype with stemness features, dedifferentiated cone states and neuronal/ganglion cell gene expression.

Retinoblastoma is the most frequent intraocular malignancy in children, originating from a maturing cone precursor in the developing retina. Little is known on the molecular basis underlying the biological and clinical behavior of this cancer. Here, using multi-omics data, we demonstrate the existence of two retinoblastoma subtypes. Subtype 1, of earlier onset, includes most of the heritable forms. It harbors few genetic alterations other than the initiating RB1 inactivation and corresponds to differentiated tumors expressing mature cone markers. By contrast, subtype 2 tumors harbor frequent recurrent genetic alterations including MYCN-amplification. They express markers of less differentiated cone together with neuronal/ganglion cell markers with marked inter- and intra-tumor heterogeneity. The cone dedifferentiation in subtype 2 is associated with stemness features including low immune and interferon response, E2F and MYC/MYCN activation and a higher propensity for metastasis. The recognition of these two subtypes, one maintaining a cone-differentiated state, and the other, more aggressive, associated with cone dedifferentiation and expression of neuronal markers, opens up important biological and clinical perspectives for retinoblastomas.

FGFR3 Mutation Status and FGFR3 Expression in a Large Bladder Cancer Cohort Treated by Radical Cystectomy: Implications for Anti-FGFR3 Treatment?†.

Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer (BC). FGFR3 mutations are common in noninvasive BC and associated with favorable BC prognosis. Overexpression was reported in up to 40% of FGFR3 wild-type muscle-invasive BC. We analyzed FGFR3 mutations, FGFR3, and p53 protein expression and assessed their prognostic value in a cohort of 1000 chemotherapy-naive radical cystectomy specimens. FGFR3 mutations were found in 11%, FGFR3 overexpression was found in 28%, and p53 overexpression was found in 69% of tumors. Among FGFR3 mutant tumors, 73% had FGFR3 overexpression versus 22% among FGFR3 wild-type tumors. FGFR3 mutations were significantly associated with lower pT stage, tumor grade, absence of carcinoma in situ, pN0, low-level p53, and longer disease-specific survival (DSS). FGFR3 overexpression was associated only with lower pT stage and tumor grade. Moreover, FGFR3 overexpression was not associated with DSS in patients with FGFR3 wild-type tumors. In conclusion, FGFR3 mutations identified patients with favorable BC at cystectomy. Our results suggest that FGFR3 mutations have a driver role and are functionally distinct from FGFR3 overexpression. Hence, patients with FGFR3 mutations would be more likely to benefit from anti-FGFR3 therapy. Ideally, further research is needed to test this hypothesis. PATIENT SUMMARY: Oncogenic fibroblast growth factor receptor 3 (FGFR3) mutations are very common in bladder cancer. In this report, we found that these FGFR3 mutations were associated with favorable features and prognosis of bladder cancer compared with patients with FGFR3 overexpressed tumors only. As a consequence, patients with FGFR3 mutant tumors would be more likely to benefit from anti-FGFR3 therapy than patients with FGFR3 protein overexpression only.

High Prevalence of a Hotspot of Noncoding Somatic Mutations in Intron 6 of GPR126 in Bladder Cancer.

Numerous pangenomic studies identified protein-coding genes and signaling pathways involved in bladder carcinogenesis. However, noncoding somatic alterations remain unexplored. A recent study revealed a mutational hotspot in intron 6 of GPR126 gene in 2.7% of a large breast cancer series. As GPR126 is highly expressed in bladder tissues, we investigated here the prevalence and the prognostic significance of these mutations in bladder cancer. We analyzed a cohort of 103 bladder cancers including 44 nonmuscle-invasive bladder cancers (NMIBC) and 59 muscle-invasive bladder cancers (MIBC). GPR126 mutations were analyzed by high-resolution melting and Sanger sequencing, and GPR126 expression levels were assessed using real-time quantitative RT-PCR. In NMIBC, somatic GPR126 noncoding mutations occurred in 47.7% of samples and were negatively associated with GPR126 mRNA levels. GPR126 mutations had higher frequencies in nonsmoker patients and were associated with a prior history of NMIBC. GPR126 overexpression was detected in 70.5% of samples. GPR126 mutation and overexpression status were not associated with outcome. In MIBC, somatic GPR126 mutations occurred in 44.1% of samples. Mutations were more frequent in females. GPR126 overexpression was detected in 27.1% of the sample. A trend toward significance was observed between GPR126 overexpression and better outcome. We identified the second most frequent mutational hotspot after TERT promoter (∼70%) in bladder cancer, with a mutation rate of approximately 50%. IMPLICATIONS: The GPR126 intronic mutational hotspot could be a promising clinical biomarker candidate to monitor tumor burden using circulating tumor DNA in bladder cancer.

Reappraisal of HER2 status in the spectrum of advanced urothelial carcinoma: a need of guidelines for treatment eligibility.

Although human epidermal growth factor receptor 2 (HER2) may represent a therapeutic target, its evaluation in urothelial carcinoma of the bladder does not rely on a standardized scoring system by immunohistochemistry or fluorescent in situ hybridization (FISH), as reflected by various methodology in the literature and clinical trials. Our aim was to improve and standardize HER2 amplification detection in bladder cancer. We assessed immunohistochemical criteria derived from 2013 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAPs) guidelines for breast cancer and investigated intratumoral heterogeneity in a retrospective multicentric cohort of 188 patients with locally advanced urothelial carcinoma of the bladder. Immunohistochemistry was performed on 178 primary tumors and 126 lymph node metastases, eligible cases (moderate/strong, complete/incomplete membrane staining) were assessed by FISH. HER2 overexpression was more frequent with 2013 ASCO/CAP than 2007 ASCO/CAP guidelines (p < 0.0001). The rate of positive HER2 FISH was similar between primary tumor and lymph node metastases (8%). Among positive FISH cases, 48% were associated with moderate/strong incomplete membrane staining that were not scored eligible for FISH by 2007 ASCO/CAP criteria. Among 3+ immunohistochemistry score cases, 67% were associated with HER2-positive FISH. Concordance between primary tumors and matched lymph node metastases was moderate for immunohistochemistry (κ = 0.54 (CI 95%, 0.41-0.67)) and FISH (κ = 0.50 (CI 95%, 0.20-0.79)). HER2-positive FISH was more frequent in micropapillary carcinomas (12%) and carcinoma with squamous differentiation (11%) than in pure conventional carcinoma (6%). Intratumoral heterogeneity for HER2 immunohistochemistry was observed in 7% primary tumor and 6% lymph node metastases; 24% positive HER2 FISH presented intratumoral heterogeneity. Our study suggests that HER2 evaluation should include an immunohistochemistry screening step with eligibility for FISH including incomplete/complete and moderate/strong membrane staining. Spatial or temporal intratumoral heterogeneity prompts to perform evaluation on both tumor and lymph node, and for each histological variant observed.

Evidence of estrone-sulfate uptake modification in young and middle-aged rat prostate.

High plasma exposure to estrogens is often associated with prostate cancer. Reducing this phenomenon may present therapeutic benefits. The involvement of estrone sulphate (E1S), the most abundant circulating estrogen in men, has been partially studied in this age-related pathology. To investigate the consequences of plasma E1S overload on blood and prostate sex steroid levels and inflammatory tissue responses, young and middle-aged male rats were treated with E1S with or without steroid sulfatase (STS) inhibitor STX64 for 21 consecutive days. A plasma and prostate tissue steroid profile was determined. STS activity, mRNA expression of E1S organic anion transporting polypeptides (slco1a2, slco2b1, slco4a1) and pro-inflammatory cytokines (Il1-beta, Il6, TNF-alpha) were evaluated in prostate tissue according to age and treatment group. A significant correlation between plasma and prostate steroid levels related to hormone treatment was observed in all rat age groups. However, while the E1S level in prostate tissue increased in middle-aged treated rats (p<0.0001), no significant variation was observed in young treated rats. The protective effect of STX64 during E1S infusion was observed by the maintenance of low free estrogen concentrations in both plasma and tissue. However, this protection was not associated with mRNA expression stability of pro-inflammatory cytokines in older rat prostate. These results suggest that E1S uptake in rat prostate cells increases during aging. Therefore, if a similar phenomenon existed in men, preventively reducing the STS activity could be of interest to limit uptake of estrogens in prostate when high E1S plasma level is assayed.

Lipidosterolic extract of serenoa repens modulates the expression of inflammation related-genes in benign prostatic hyperplasia epithelial and stromal cells.

Despite the high prevalence of histological Benign Prostatic Hypeplasia (BPH) in elderly men, little is known regarding the molecular mechanisms and networks underlying the development and progression of the disease. Here, we explored the effects of a phytotherapeutic agent, Lipidosterolic extract of the dwarf palm plant Serenoa repens (LSESr), on the mRNA gene expression profiles of two representative models of BPH, BPH1 cell line and primary stromal cells derived from BPH. Treatment of these cells with LSESr significantly altered gene expression patterns as assessed by comparative gene expression profiling on gene chip arrays. The expression changes were manifested three hours following in vitro administration of LSESr, suggesting a rapid action for this compound. Among the genes most consistently affected by LSESr treatment, we found numerous genes that were categorized as part of proliferative, apoptotic, and inflammatory pathways. Validation studies using quantitative real-time PCR confirmed the deregulation of genes known to exhibit key roles in these biological processes including IL1B, IL1A, CXCL6, IL1R1, PTGS2, ALOX5, GAS1, PHLDA1, IL6, IL8, NFkBIZ, NFKB1, TFRC, JUN, CDKN1B, and ERBB3. Subsequent analyses also indicated that LSESr treatment can impede the stimulatory effects of certain proinflammatory cytokines such as IL6, IL17, and IL15 in these cells. These results suggest that LSESr may be useful to treat BPH that manifest inflammation characteristics. This also supports a role for inflammation in BPH presumably by mediating the balance between apoptosis and proliferation.

Androgens regulate Hedgehog signalling and proliferation in androgen-dependent prostate cells.

Prostate cancer (PCa) is androgen sensitive in its development and progression to metastatic disease. Hedgehog (Hh) pathway activation is important in the initiation and growth of various carcinomas including PCa. We and others have observed aberrations of Hh pathway during the progression of PCa to the castration-resistant state. The involvement of androgen signalling in Hh pathway activation, however, remains largely elusive. Here we investigate the direct role of androgen signalling on Hh pathway. We examined the effect of Dihydrosterone (DHT), antiandrogen, bicalutamide, and Hh pathway inhibitor, KAAD-cyclopamine in four human prostate cell lines (two cancerous: LNCaP, VCaP, and two normal: PNT2 and PNT2-ARm which harbours a mutant version of androgen receptor (AR) that is commonly found in LNCaP). Cell proliferation as well as Hh pathway members (SHH, IHH, DHH, GLI, PTCH) mRNA expression levels were assessed. We showed that KAAD-cyclopamine decreased cell proliferation of DHT-stimulated LNCaP, VCaP and PNT2-ARm cells. SHH expression was found to be downregulated by DHT in all AR posititve cells. The negative effect of DHT on SHH expression was counteracted when cells were treated by bicalutamide. Importantly, KAAD-cyclopamine treatment seemed to inhibit AR activity. Moreover, bicalutamide as well as KAAD-cyclopamine treatments induced GLI and PTCH expression in VCaP and PNT2-ARm. Our results suggest that Hh pathway activity can be regulated by androgen signalling. Specifically, we show that the DHT-induced inhibition of Hh pathway is AR dependent. The mutual interaction between these two pathways might be important in the regulation of cell proliferation in PCa.

Class III beta-tubulin expression predicts prostate tumor aggressiveness and patient response to docetaxel-based chemotherapy.

Expression of class III ß-tubulin (ßIII-tubulin) correlates with tumor progression and resistance to taxane-based therapies for several human malignancies, but its use as a biomarker of tumor behavior in prostate cancer (PCa) remains largely unexplored. Here, we describe ßIII-tubulin immunohistochemical staining patterns of prostate tumors obtained from a broad spectrum of PCa patients, some of whom subsequently received docetaxel therapy for castration-resistant PCa (CRPC). Elevated ßIII-tubulin expression was significantly associated with tumor aggressiveness in PCa patients with presumed localized disease, as it was found to be an independent marker of biochemical recurrence after treatment. Additionally, ßIII-tubulin expression in tumor cells was an independent predictor of lower overall survival for patients receiving docetaxel-based chemotherapy for CRPC. Manipulation of ßIII-tubulin expression in human PCa cell lines using a human ßIII-tubulin expression vector or ßIII-tubulin small interfering RNA altered cell survival in response to docetaxel treatment in a manner that supports a role for ßIII-tubulin expression as a mediator of PCa cell resistance to docetaxel therapy. Our findings suggest a role for ßIII-tubulin as candidate theranostic biomarker to predict the response to docetaxel-based chemotherapy as well as to target for treatment of docetaxel-resistant CRPC.

Inflammation in benign prostatic hyperplasia: a 282 patients' immunohistochemical analysis.

INTRODUCTION AND OBJECTIVES: Prostatic inflammation could be a key component in prostate enlargement and benign prostatic hyperplasia (BPH) progression. Our aim was to characterize inflammatory cells infiltrate within BPH tissue and to correlate inflammation and clinical data. MATERIAL AND METHODS: Inflammation was profiled on three clinical outcome tissue microarrays (TMAs), including 282 patients treated by surgery for a complicated and/or symptomatic BPH. Inflammation score was defined by combining six cytological parameters and five markers on immunohistochemistry (IHC). Cytological parameters were lymphocytes, macrophages, and polynuclears leukocytes infiltrates, and three glandular aspect modifications: glandular atrophy, glandular destruction, and tissue fibrosis. IHC markers were CD3, CD4, and CD8 decorating T-lymphocytes, CD20 decorating B-lymphocytes, and CD163 decorating macrophages. RESULTS: The majority of patients had inflammatory cells infiltrating BPH tissues: 81% had T-lymphocytes markers (CD3), 52% had B-lymphocytes markers (CD20), and 82% had macrophages markers (CD163). IPSS score (21 vs. 12; P = 0.02) and prostate volume (77 cm(3) vs. 62 cm(3); P = 0.002) were significantly higher in patients with high-grade prostatic inflammation. CONCLUSION: We characterized inflammatory cells infiltrate in a large cohort of surgically treated BPH specimens. The role of inflammation in BPH development was highlighted by the strong correlation between histological inflammation, IPSS, and prostate volume. Prostate enlargement due to chronic inflammatory process may progressively conduce to BPH progression. Therefore, inflammation is a therapeutic target for BPH.