THE ROLE OF EZH2 AND ARID1A IN THE DIAGNOSIS OF FLAT UROTHELIAL LESIONS WITH ATYP

Background. Diagnosis of urothelial carcinoma in situ is of great importance because it has prognostic and therapeutic value.We aim to determine the utility of EZH2 and ARID1A as a new tool in the diagnosis of carcinoma in situ.Material and Methods. This retrospective cross-sectional study included Twenty-four specimens of flat urothelial lesions, twenty specimens of CIS, and 10 of normal adjacent urothelium that was taken by cystoscopic resection biopsy procedure. immunohistochemical expression of EZH2 and ARID1A. were evaluated in all studied cases.Results. All normal urothelium specimens showed high nuclear staining for ARID1A and negative nuclear staining for EZH2. High EZH2 expression was observed in 80 % of CIS specimens compared to 20 % of flat urothelial lesions with atypia (p=0.001 ), while high ARID1A expression was observed in 70.8 % of flat urothelial lesions with atypia compared to 25 % of CIS specimens (p=0.001). EZH2 was more accurate and specific in the diagnosis of carcinoma in situ.Conclusion. EZH2 and ARID1A are promising diagnostic markers for urothelial CIS. EZH2 is more accurate and specific than ARID1A in the diagnosis of carcinoma in situ versus other flat urothelial lesions.

PD-L2 Is Constitutively Expressed in Normal and Malignant Urothelium

The use of immune checkpoint blockade, in particular PD-1 and PD-L1 inhibitors, is now commonplace in many clinical settings including the treatment of muscle-invasive bladder cancer (MIBC). Notwithstanding, little information exists regarding the expression of the alternative PD-1 ligand, PD-L2 in urothelial bladder cancer (UBC). We therefore set out to characterise the expression of PD-L2 in comparison to PD-L1. Firstly, we assessed PD-L2 expression by immunohistochemistry and found widespread expression of PD-L2 in UBC, albeit with reduced expression in MIBC. We further investigated these findings using RNA-seq data from a cohort of 575 patients demonstrating that PDCD1LG2 (PD-L2) is widely expressed in UBC and correlated with CD274 (PD-L1). However, in contrast to our immunohistochemistry findings, expression was significantly increased in advanced disease. We have also provided detailed evidence of constitutive PD-L2 expression in normal urothelium and propose a mechanism by which PD-L2 is cleaved from the cell surface in MIBC. These data provide a comprehensive assessment of PD-L2 in UBC, showing PD-L2 is abundant in UBC and, importantly, constitutively present in normal urothelium. These data have implications for future development of immune checkpoint blockade, and also the understanding of the function of the immune system in the normal urinary bladder.

TERT Promoter Mutation Analysis of Whole-Organ Mapping Bladder Cancers

Background: Multifocal occurrence is a main characteristic of urothelial bladder cancer (UBC). Whether urothelial transformation is caused by monoclonal events within the urothelium, or by polyclonal unrelated events resulting in several tumor clones is still under debate. TERT promoter mutations are the most common somatic alteration identified in UBC. In this study, we analyzed different histological tissues from whole-organ mapping bladder cancer specimens to reveal TERT mutational status, as well as to discern how tumors develop. Methods: Up to 23 tissues from nine whole-organ mapping bladder tumor specimens, were tested for TERT promoter mutations including tumor associated normal urothelium, non-invasive urothelial lesions (hyperplasia, dysplasia, metaplasia), carcinoma in situ (CIS) and different areas of muscle invasive bladder cancers (MIBC). The mutational DNA hotspot region within the TERT promoter was analyzed by SNaPshot analysis including three hot spot regions (−57, −124 or −146). Telomere length was measured by the Relative Human Telomere Length Quantification qPCR Assay Kit. Results: TERT promoter mutations were identified in tumor associated normal urothelium as well as non-invasive urothelial lesions, CIS and MIBC. Analysis of separate regions of the MIBC showed 100% concordance of TERT promoter mutations within a respective whole-organ bladder specimen. Polyclonal events were observed in five out of nine whole-organ mapping bladder cancers housing tumor associated normal urothelium, non-invasive urothelial lesions and CIS where different TERT promoter mutations were found compared to MIBC. The remaining four whole-organ mapping bladders were monoclonal for TERT mutations. No significant differences of telomere length were observed. Conclusions: Examining multiple whole-organ mapping bladders we conclude that TERT promoter mutations may be an early step in bladder cancer carcinogenesis as supported by TERT mutations detected in tumor associated normal urothelium as well as non-invasive urothelial lesions. Since mutated TERT promoter regions within non-invasive urothelial lesions are not sufficient alone for the establishment of cancerous growth, this points to the contribution of other gene mutations as a requirement for tumor development.

The Role of MiR-124 in Bladder Cancer – A Review of the Literature

MicroRNAs (miRNAs) are a group of non-coding RNA molecules that have an important role in modulating the expression of genes involved in regulating cellular functions. A growing number of studies suggest the abnormal expression of microRNAs in different types of cancer cells. MiRNA-124 is a microRNA that is down-regulated in many types of cancer cells, including bladder cancer. Our objective is to provide a review of the key publications that studied the effect of miR-124 on bladder cancer. This review focus on the targets and different pathways of miR-124 that were identified in various studies and differences between their expressions in normal urothelium and tumor tissues. We also include data regarding urinary methylations levels of miR-124 and their role in bladder cancer diagnosis and prognosis. Subsequently, we establish future perspectives of miR-124 research and its promising role in bladder cancer.

FGFR3 – a Central Player in Bladder Cancer Pathogenesis?

The identification of mutations in FGFR3 in bladder tumors in 1999 led to major interest in this receptor and during the subsequent 20 years much has been learnt about the mutational profiles found in bladder cancer, the phenotypes associated with these and the potential of this mutated protein as a target for therapy. Based on mutational and expression data, it is estimated that >80% of non-muscle-invasive bladder cancers (NMIBC) and ∼40% of muscle-invasive bladder cancers (MIBC) have upregulated FGFR3 signalling, and these frequencies are likely to be even higher if alternative splicing of the receptor, expression of ligands and changes in regulatory mechanisms are taken into account. Major efforts by the pharmaceutical industry have led to development of a range of agents targeting FGFR3 and other FGF receptors. Several of these have entered clinical trials, and some have presented very encouraging early results in advanced bladder cancer. Recent reviews have summarised the drugs and related clinical trials in this area. This review will summarise what is known about the effects of FGFR3 and its mutant forms in normal urothelium and bladder tumors, will suggest when and how this protein contributes to urothelial cancer pathogenesis and will highlight areas that may benefit from further study.

Combined lectin- and immuno-histochemistry (CLIH) for applications in cell biology and cancer diagnosis: Analysis of human urothelial carcinomas

Lectin histochemistry (LHC) and immunohistochemistry (IHC), which demonstrate the composition and localisation of sugar residues and proteins in cell membranes, respectively, are generally used separately. Using these two methods, we previously demonstrated that malignant transformation of urothelial cells results in the alterations of protein glycosylation and reduced expression of urothelium-specific integral membrane proteins uroplakins (UPs). However, the correlation between these changes was not studied yet. To evaluate this correlation, we developed innovative method, which we named combined lectin- and immuno- histochemistry (CLIH). We used human biopsies of 6 normal urothelia and 9 papillary urothelial carcinomas, i.e. 3 papillary urothelial neoplasms of low malignant potential (PUNLMP), 3 non-invasive papillary urothelial carcinomas of low grade (pTa, l.g.), and 3 invasive papillary urothelial carcinomas of high grade (pT1, h.g.). We tested five different protocols (numbered 1-5) of CLIH on paraffin and cryo-semithin sections and compared them with LHC and IHC performed separately. Additionally, we carried out western and lectin blotting with antibodies against UPs and lectins Amaranthus caudatus agglutinin (ACA), Datura stramonium agglutinin (DSA), and jacalin, respectively. We showed that incubation with primary antibodies first, followed by the mixture of secondary antibodies and lectins is the most efficient CLIH method (protocol number 5). Additionally, 300 nm thick cryo-semithin sections enabled better resolution of co-localisation between sugar residues and proteins than 5 µm thick paraffin sections. In the normal urothelium, CLIH showed co-localisation of lectins ACA and jacalin with UPs in the apical plasma membrane (PM) of superficial umbrella cells. In papillary urothelial carcinomas, all three lectins (ACA, DSA and jacalin) labelled regions of apical PM, where they occasionally co-localised with UPs. Western and lectin blotting confirmed the differences between normal urothelium and papillary urothelial carcinomas. Our results show that CLIH, when used with various sets of lectins and antigens, is a useful, quick, and reliable method that could be applied for basic cell biology research as well as detailed subtyping of human urothelial carcinomas.

Expression of nectin-4 in bladder cancer with variant histology.

546 Background: The antibody-drug conjugate enfortumab vedotin is poised to change the bladder cancer (BC) treatment landscape by targeting Nectin-4, near ubiquitously expressed in urothelial cancer (UC). Less is known about this and other targets in BC with pure or mixed variant histology (VH). Methods: Immunohistochemistry (IHC) was performed on a Ventana Discovery Autostainer (Roche Diagnostics) using an ultraView DAB detection kit (Roche Diagnostics) and a Nectin-4 polyclonal antibody (1:100 dilution; Abcam, Cambridge, UK). The intensity and extent of Nectin-4 expression was determined by the histochemical scoring (H-score) used in preclinical testing, defined as the sum of the products of the staining intensity (score of 0–3) x % of cells (0–100) stained at a given intensity. Specimens were assessed by H score as: negative (0–14), weak (15–99), moderate (100–199), and strong (200–300). Results: Forty UC and VH BC were evaluated for Nectin-4 expression by IHC: 15 small cell (SCBC) (8 pure SCBC, 6 mixed SCBC/UC, 1 SCBC/CIS), 8 carcinosarcomas (CS) (7 pure CS, 1 HGUC/sarcomatoid features), and 17 pure HGUCs. Normal urothelium and stroma were negative. Eight of 8 (100%) pure SCBC were negative for Nectin-4. Six of 7 (85.3%) mixed SCBC+HGUC/CIS had weak staining and 1/7 (14.7%) had moderate staining in the urothelial components (comp) while 7/7 (100%) of the SCBC comp were negative. Seven of 7 (100%) pure CS were negative and 1/1 (100%) mixed CS+HGUC showed weak staining in the HGUC comp while the sarcomatoid comp was negative. Expression in UC was: 1/17 (5.9%) strong, 3/17 (17.6%) moderate, 10/17 (58.8%) weak, and 3/17 (17.6%) negative. Gene expression profiling confirmed Nectin-4 was downregulated in VH compared to UC samples, as was ERBB2 and Trop2. Conclusions: There is heterogeneity of expression of Nectin-4 and other targets in BC with VH compared to UC. This may have therapeutic implications, and highlights need for additional research in VH.[Table: see text]

Expression of p53 and p21 in bladder carcinoma of Iraqi patients

Bladder cancer is an abnormal cellular multiplication without control arising from the urinary bladder urothelium. Cells cycle is a strictly controlled process regulated by protein complexes composed of cyclins and cyclin-dependent kinases and also by several tumor suppressor gene protein products acting at the Go/G1 checkpoint of the cell-cycle. This study was designed to evaluate the frequency of p53 and p21 expression as biomarkers to monitor the disease and compare the results with those of others. Seventy-three tumors with different stages and grades were collected from patients with bladder carcinoma and ten normal bladder biopsies from forensic autopsy. Sections were immunohistochemically stained for p53 and p21. Results revealed that p53 positive immune reactivity was observed in (57.5%) which were exclusively nuclear and no immune reactivity detectable in (42.5%) of tumors, while all normal tissues expressed negative patterns. Positive immune staining of p21 was recorded in (69.5%) and negative p21 immune staining was recorded in 22 (31.1%), as well as ten normal urothelium. Statistically p53 and p21 over-expression was significantly related to stage and grade of tumors (P<0.05).