Primitive neuroectodermal tumor (PNET) is part of the Ewing sarcoma family of tumors. The present case reports a primitive neuroectodermal tumor (PNET) of rare location in the bladder in a newborn. It was evaluated with prenatal ultrasound and postnatal tomography that revealed a mass in the posterior wall of the bladder. The patient underwent partial cystectomy with subsequent analysis of the surgical piece removed, the histopathological study indicated a tumor of mesenchymal origin, and immunohistochemical staining confirmed the diagnosis of PNET of the bladder. Satisfactory result and short-term follow-up. / Revisión por pares

The bladder urothelium is a stratified epithelium that interconnects with the ureters to form the urinary outflow tract. A defining feature of the urothelium is its luminal population of uroplakin-expressing Superficial cells that function to create a protective, waterproof barrier. As such, proper differentiation of urothelial cell types during development and regeneration is essential for bladder function. Previous work has demonstrated that the urothelium is derived from a transient endodermal progenitor population, termed P-cells. However, two remaining uncertainties are the timing and regulation of P-cell fate. Here we show through lineage tracing that the specification of P-cells into luminal, uroplakin-expressing cell types is temporally related to the timing of endogenous retinoic acid (RA) signaling. Selective inhibition of RA signaling in P-cells through ShhCre-driven expression of the RaraT403 dominant-negative (RaraDN) mutant receptor redirected cells towards an abnormal K14+ basal fate that underwent Notch-mediated stratification into a keratinizing squamous epithelium that largely resembled epidermis. Transcriptome analysis of mutant P-cells identified aberrant expression of transcriptional regulators that have critical roles in squamous differentiation and epidermal commitment. Interestingly, inhibition of RA signaling in P-cells also resulted in improper connections between the ureters and the bladder through reduced Caspase 9-mediated apoptosis and remodeling of the common nephric duct. These observations demonstrate that RA signaling in bladder urothelial progenitor cells is required not only for proper epithelial differentiation, but also for regulating inter-organ signals that orchestrate morphogenesis of the urinary outflow tract.

Objective: Rare co-existance of disease or pathology Background: The lipid-rich variant is a rare and aggressive type of urothelial carcinoma (UCa), with less than 40 cases reported in the literature. This variant usually presents as an advanced-stage primary tumor. Case Report: We report the case of a 61-year-old man with previous history of T1 high-grade conventional urothelial carcinoma treated with local therapy. The patient later presented with a new 6.5-cm exophytic bladder mass. Histopathological examination revealed a T2 urothelial carcinoma of the lipid-rich variant. Retrospective review of the previous biopsies confirmed conventional high-grade urothelial car0cinoma, but scattered rare individual or small clusters of cells that resemble the lipid-rich variant urothelial carcinoma were also noted. Conclusions: The findings in this case suggest that the differential sensitivity of conventional urothelial carcinoma to local therapy may have allowed the lipid-rich variant to predominate in the recurrence. Pathologists should be aware of the lipid-rich variant of urothelial carcinoma. The prognostic significance of rare lipoblast-like cells among predominantly conventional urothelial carcinoma may requires further study.

INTRODUCTION: Bladder cancer (BC) is highly prevalent. It presents as either non-muscle invasive or muscle-invasive disease. The prognosis of muscle invasive disease is poor, with a 5-year survival rate of less than 50%. Treatment approaches for both types of BC have not advanced much in the last few years and new therapies are needed to overcome the large burden of BC. Recently, a large effort has been undertaken to classify BC into molecular subtypes. These analyses have revealed significant alterations in epigenetic modifiers in BC. A previous study from our group revealed that SH3GL2, a negative regulator of receptor tyrosine kinase (RTK) signaling, was lost with high frequency in BC, leading to increased growth of tumor cells in-vitro and in-vivo. Conversely, forced expression of SH3GL2 in BC cell lines attenuated oncogenic behaviors including growth and migration. In addition to genomic deletion, SH3GL2 is subject to methylation-induced silencing, a key epigenetic mechanism. OBJECTIVE: Epigenetic mechanisms of gene regulation are known to be perturbed in BC. The objectives of this study were to investigate methylation of the SH3GL2 promoter and to test whether agents that promote Deoxyribonucleic acid (DNA) demethylation could be used to re-express SH3GL2 thereby restoring regulation of RTK signaling. METHODS: Methylation of a specific CpG island in the SH3GL2 promoter was analyzed using methylation-specific Polymerase Chain Reaction (PCR) in a panel of BC cell lines with known SH3GL2 messenger Ribonucleic Acid (mRNA) status. Selected BC cell lines were treated with a variety of demethylating agents at different doses and for different times to evoke the re-expression of silenced SH3GL2. Demethylation inhibitors were combined with the histone deacetylase inhibitor, trichostatin A (TSA), to determine whether further re-expression could be achieved. RESULTS: The SH3GL2 promoter displayed differing extents of promoter methylation among cell lines examined. In RT4 cells, the only cell line with detectable expression of SH3GL2 mRNA and protein, the promoter was completely unmethylated. In contrast, T24 and 253J cells displayed significant promoter methylation with little to no SH3GL2 mRNA expressed, consistent with methylation-induced silencing. Treatment of T24 and 253J with 5-Aza-2’-deoxycytidine (5-Aza-dC, 20 M), a DNA methyltransferase (DNMT) inhibitor increased gene expression but this was not dose- or time-dependent. Two additional DNMT inhibitors, Zebularine and RG-108 were also tested. A much higher dosage of Zebularine was required to trigger activation (500 M) while RG-108 was unable to trigger gene reactivation at all. Combination treatment with 5-Aza-dC and TSA further increased SH3GL2 expression compared to either agent alone. These results suggest that DNA methyltransferase inhibition is an effective treatment to re-express SH3GL2 in cells with SH3GL2 promoter silencing. CONCLUSION: The present study shows silencing of SH3GL2 in a variety of BC cell lines as a consequence of DNA promoter hypermethylation. Treatment with demethylating agents was able to increase gene expression. Based on prior findings showing attenuation of tumor cell growth and migration with forced expression of SH3GL2, DNA methyltransferase inhibition represents an effective strategy to re-express SH3GL2 in BC and normalize tumor cell behavior. / 2020-07-03T00:00:00Z

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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Bladder cancer is currently the 6th most common cancer in the United States, resulting in 17,000 deaths annually. Clinically, bladder cancers are mostly urothelial carcinoma, classified as either non-muscle invasive bladder cancer (NMIBC) or muscle-invasive bladder cancer (MIBC), with the latter having a 5-year survival rate of merely 50%. With recent advances in next-generation sequencing, several international consortia have elucidated molecular subtypes of MIBC. The two major subtypes of MIBCs are basal and luminal; the basal subtype frequently exhibits hallmarks of squamous differentiation and highly expressed basal markers (CD44, KRT14, KRT6A, KRT6B). Tumors of the luminal subtype have papillary morphology and highly express differentiation-associated luminal markers (e.g., KRT20, PPARG, UPKs, and FOXA1). Notably, the transcription factor Peroxisome Proliferator Activated Receptor Gamma (PPARG) gene is frequently amplified in luminal MIBC. And recurrent activating mutations have been reported for its obligatory functional partner Retinoic X Receptor (RXR). In addition, the basal subtype is immune-infiltrated and is postulated as more likely to respond to immunotherapies. In contrast, the luminal subtype is immune-cold. Despite these advances in recent years, the molecular driver of subtype determination, specifically in luminal MIBC, remains poorly understood. Furthermore, subtype-specific targeted therapy for MIBCs is still in its infancy. Our previous work determined that Pparg activation can drive luminal tumor formation. We generated a novel Krt5CreERT2; VP16;Pparg transgenic mouse model, where Pparg expression is constitutively active in basal urothelial cells upon tamoxifen induction. During homeostasis, constitutive Pparg promoted luminal differentiation and cell cycle exit in basal cells but did not produce tumors. However, increased Pparg signaling in activated basal cells following 1-month exposure to bladder-specific carcinogen BBN produced luminal tumors. These tumors are similar both in morphology and molecular markers to human luminal MIBCs. The resulting VP16;Pparg luminal tumors have reduced Nf-kb expression and are immune cold compared to basal tumors. These findings suggest that Pparg is a driver of luminal tumor formation and a suppressor of immune infiltration in bladder cancer. In Chapter 2 of the thesis, I focus on the therapeutic potential of activating Pparg in basal MIBC. We treated mice bearing BBN-induced, Pparg-negative basal tumors with synthetic Pparg ligand - Rosiglitazone (Rosi) and Mek1/2 inhibitor Trametinib (Tram), both of which have been shown to induce Pparg signaling in vitro and in vivo. The combined RosiTram treatment induced apoptosis and significantly reduced tumor burden. The post-treatment urothelium appeared similar in morphology to a healthy urothelium. RosiTram treatment also restored normal urothelial differentiation and generated resident cell types (e.g., superficial cells, intermediate cells, Keratin5+ (K5), basal cells, and Keratin14+ (K14) basal cells) that are normally seen in a healthy urothelium. In contrast, basal tumors are almost entirely composed of K14-Basal cells. Mechanistically, RosiTram treatment partially restores differentiation through retinoic acid signaling and Ezh2 inhibition. Together, our study established targeted transcriptionally and epigenetically reprogramming as a promising differentiation therapeutic approach for basal bladder tumors.

Early detection and screening for urinary tract illnesses is a complex and widespread process which has implications for both preventative care, diagnosis, and treatment monitoring. In this paper, we investigate the use of Raman spectroscopy (RS) for the analysis of urine, a complex biological solution, for the detection of bladder cancer (BCa) and hematuria. Raman spectroscopy is a rapid, low cost, non-destructive analysis method with wide-ranging applicability due to the holistic data capturing nature of the scanning technique. Each Raman scan can be considered a 'snapshot' of the molecular makeup of the sample, and, through proper applications of algorithmic transformation and statistical analysis, many types of assessments can be performed on each sample. In this paper we address creating and utilizing a data pipeline for the purposes of analyzing and characterizing potential samples with hematuria and BCa. The algorithmic transformations utilized include baselining using either the Goldindec or ISREA methods, and intensity normalization. The statistical analysis methods utilized include principal component analysis (PCA), discriminant analysis of principal components (DAPC), analysis of variance (ANOVA), pairwise ANOVA, leave-one-out cross-validation (LOOCV), and partial least squares regression (PLSR). These components of the data pipeline serve to output qualitative or quantitative data, depending on the application. The Rametrix toolbox encompasses the tools required to transform and assess Raman spectra with PCA and DAPC. Using the Rametrix toolbox as well as ANOVA, pairwise ANOVA, and LOOCV, we were able to significantly detect the presence of bladder cancer in a specimen with 80% accuracy. Using the Rametrix toolbox, ANOVA, pairwise ANOVA, LOOCV, and PLSR, we were able to classify samples as pure urine, micro-, or macrohematuria with a greater than 91% accuracy, and quantify the amount of blood in the sample with a high correlation (R-squared value of 0.92). In combination, this style of data pipeline is shown to rapidly and accurately test for multiple symptoms or diseases using similar methodologies. / Master of Science / In the United States, over 37 million people live with chronic kidney disease, over 81 thousand new cases of bladder cancer will be diagnosed, and over 17 thousand people will die from bladder cancer. These serious renal and urinary tract illnesses require urinalysis as a major component of detection, diagnosis, and monitoring of the diseases. This level of required testing has significant costs, both in labor and financial impact. Reduction in both the labor and consumable reagent costs associated with urinalysis would serve to improve the ability for the healthcare system provide the necessary testing for these patients, and reduce the risk of shortages in both reagents and staff. We present a new analysis method, termed 'data pipeline', which would take data from a spectrographic data collection method, Raman Spectroscopy (RS), and generate useable output in the form of classification and quantification. These outputs are highly desired for urinalysis, as urine collection is largely the least invasive testing method related to the urinary tract. As we have shown, the RametrixTM toolbox, an algorithmic package of mathematical methods for assessing spectra, is the backbone of a data pipeline capable of detecting both hematuria, an early warning symptom of many urinary tract illness, and bladder cancer, a notably difficult to detect disease, with high accuracy. This method of analysis is non-destructive of the samples, requires no reagents or single use dipsticks, avoids subjective color assessment, and provides rapid results in a repeatable, potentially automatable manner. We investigate a critical component of this process, the baselining method, in order to further examine and refine the methodology by comparing the accuracy and statistical quality of the results with different baseline methods. It is our goal to implement this methodology with the best component processes, in order to achieve a highly robust, accurate tool for assisting in urinalysis testing.

Yes / Background: Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. Methods: We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. Results: Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. Conclusions: Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.