Clinical implications of molecular subtyping in bladder cancer.

PURPOSE OF REVIEW: The purpose of this review is to examine and evaluate similarities and differences in bladder cancer expression subtypes and to understand the clinical implications of the molecular subtyping. RECENT FINDINGS: Four independent classification systems have been described, and there are broad similarities among the subtyping callers. Two major subtypes have been identified, that is, luminal and basal, with underlying subcategories based on various distinct characteristics. Luminal tumors generally bear a better prognosis and increased survival than basal tumors, although there is subtle variation in prognosis among the different subtypes within the luminal and basal classifications. Clinical subtyping is now commercially available, although there are limitations to its generalizability and application. SUMMARY: Expression subtyping is a new method to personalize bladder cancer management. However, there is probably not sufficient evidence to incorporate use into current standards-of-care. Validation cohorts with clinically meaningful outcomes may further establish the clinical relevance of molecular subtyping of bladder cancer. Additionally, genetic alterations in bladder cancer may 'color' the interpretation of individual tumors beyond the expression subtype to truly personalize care for bladder cancer.

Urine Biopsy as Dynamic Biomarker to Enhance Clinical Staging of Bladder Cancer in Radical Cystectomy Candidates.

PURPOSE: There is significant interest in identifying complete responders to neoadjuvant chemotherapy (NAC) before radical cystectomy (RC) to potentially avoid removal of a pathologically benign bladder. However, clinical restaging after NAC is highly inaccurate. The objective of this study was to develop a next-generation sequencing-based molecular assay using urine to enhance clinical staging of patients with bladder cancer. METHODS: Urine samples from 20 and 44 patients with bladder cancer undergoing RC were prospectively collected for retrospective analysis for molecular correlate analysis from two clinical trials, respectively. The first cohort was used to benchmark the assay, and the second was used to determine the performance characteristics of the test as it correlates to responder status as measured by pathologic examination. RESULTS: First, to benchmark the assay, known mutations identified in the tissue (MT) of patients from the Accelerated Methotrexate, Vinblastine, Doxorubicin, Cisplatin trial (ClinicalTrials.gov identifier: NCT01611662, n = 16) and a cohort from University of California-San Francisco (n = 4) were cross referenced against mutation profiles from urine (MU). We then determined the correlation between MU persistence and residual disease in pre-RC urine samples from a second prospective clinical trial (The pT0 trial; ClinicalTrials.gov identifier: NCT02968732). Residual MU status correlated strongly with residual disease status (pT0 trial; n = 44; P = .0092) when MU from urine supernatant and urine pellet were assessed separately and analyzed in tandem. The sensitivity, specificity, PPV, and NPV were 91%, 50%, 86%, and 63% respectively, with an overall accuracy of 82% for this second cohort. CONCLUSION: MU are representative of MT and thus can be used to enhance clinical staging of urothelial carcinoma. Urine biopsy may be used as a reliable tool that can be further developed to identify complete response to NAC in anticipation of safe RC avoidance.

Synthesis, characterization, and biological activity of a triphenylphosphonium-containing imidazolium salt against select bladder cancer cell lines.

Imidazolium salts have shown great promise as anticancer materials. A new imidazolium salt (TPP1), with a triphenylphosphonium substituent, has been synthesized and evaluated for in vitro and in vivo cytotoxicity against bladder cancer. TPP1 was determined to have a GI50 ranging from 200 to 250 µM over a period of 1 h and the ability to effectively inhibit bladder cancer. TPP1 induces apoptosis, and it appears to act as a direct mitochondrial toxin. TPP1 was applied intravesically to a bladder cancer mouse model based on the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Cancer selectivity of TPP1 was demonstrated, as BBN-induced tumors exhibited apoptosis but normal adjacent urothelium did not. These results suggest that TPP1 may be a promising intravesical agent for the treatment of bladder cancer.

Urine Biopsy-Liquid Gold for Molecular Detection and Surveillance of Bladder Cancer.

With recent advancements in a non-invasive approach to cancer diagnosis and surveillance, the term "liquid biopsy" has gained traction but is currently limited by technological challenges in identifying and isolating circulating tumor cells (CTCs), proteins, cell-free DNA (cfDNA), or other nucleic acids. Tumor tissue biopsy, especially in genitourinary (GU) system is sometimes inadequate and requires invasive surgical options, especially for upper tract urothelial cancer. Urine can prove to be "liquid gold" since it may be a more abundant source of tumor-derived material without the background noise; however, urine DNA (uDNA) may be associated with low mutant allele fraction (MAF). Molecular detection of mutations in uDNA requires a sensitive and accurate method of analysis that allows a high depth of sequencing while minimizing artifacts. Several sequencing approaches to address this hurdle using enhanced library preparation techniques such as Tagged amplicon deep sequencing (TAm-Seq), Safe-SeqS, FAST-SeqS, and CAPP-Seq approaches have been developed. Urine biopsy utilizing next-generation sequencing (NGS) can prove useful at all stages of urologic malignancy care, where urine can be collected to aid in clinical decision making through the identification of commonly known mutations, and potentially reduce or avoid all forms of invasive procedures.

Interfacially Engineered Pyridinium Pseudogemini Surfactants as Versatile and Efficient Supramolecular Delivery Systems for DNA, siRNA, and mRNA.

This article presents the synthesis, self-assembly, and biological activity as transfection agents for pDNA, siRNA, and mRNA of novel pyridinium pseudogemini surfactants, interfacially engineered from the most efficient gemini surfactants and lipids generated in our amphiphile research program. Formulation of novel amphiphiles in water revealed supramolecular properties very similar to those of gemini surfactants, despite their lipidlike charge/mass ratio. This dual character was found also to enhance endosomal escape and significantly increase the transfection efficiency. We were also successful in identifying the parameters governing the efficient delivery of pDNA, siRNA, and mRNA, drawing valuable structure-activity and structure-property relationships for each nucleic acid type, and establishing DNA/siRNA/mRNA comparisons. Several supramolecular complexes identified in this study proved to be extremely efficient nucleic acid delivery systems, displaying excellent serum stability and tissue penetration in three-dimensional organoids.

Expression and purification of bioactive high-purity recombinant mouse CXCL14 in Escherichia coli.

Mouse CXCL14/BRAK is a monocyte-selective chemokine which is expressed in almost all normal tissues. A flood of reports on its new functions of tumor suppression and fat metabolism modulation has left CXCL14 a potential therapeutic candidate for these diseases. Therefore, a simple accessible method is on demand for large-scale production of recombinant mouse CXCL14 protein for in vivo animal studies. Here, we introduce an efficient method for large-scale production of recombinant mouse CXCL14, by which an 18-mg protein is produced from 2-L Escherichia coli culture with good bioactivity and low level of endotoxin.

Neutralization of chemokine CXCL14 (BRAK) expression reduces CCl4 induced liver injury and steatosis in mice.

Using mouse gene expression microarray analysis, we earlier obtained dynamic profiles of whole genome expression in the CCl(4)-induced liver injury mouse model. CXCL14 expression was increased in the liver injury phase and returned to normal after liver regeneration suggesting its involvement in the liver injury or regeneration regulation. The role of CXCL14 in liver injury was investigated. The dynamic of CXCL14 transcription was analyzed in CCl(4)-induced mouse liver damage by qRT-PCR. Plasmid mediated CXCL14 overexpression and antibody neutralization of endogenous CXCL14 were used to demonstrate its effects and mechanisms on CCl(4)-induced liver injury and acute liver failure. We showed that CXCL14 expression was immediately upregulated post CCl(4) injection with a dose-dependent response. CXCL14 over-expression aggravated CCl(4)-induced liver injuries, evidenced by enhanced acidophilic change and necrosis of hepatocyte, increased fat deposition in hepatocytes (P<0.01), and inhibited hepatocyte proliferation (P<0.01). On the contrary, anti-CXCL14 antibody treatment reduced the severity of CCL4-induced liver injuries Significant reductions in hepatic necrosis area (P<0.05), the liver fat deposition (P<0.01), and the lipid peroxidation measured by serum MDA (P<0.05) were observed. Importantly, the antibody treatment reduced the mouse mortality caused by CCl4-induced liver failure (P<0.05). The data suggest that CXCL14 and its receptor present potential targets for the treatment of liver diseases.