Antibody Profiling: Kinetics with Native Biomarkers for Diagnostic Assay and Drug Developments.

Despite remarkable progress in applied Surface Plasmon Resonance (SPR)-based methods, concise monitoring of kinetic properties for native biomarkers from patient samples is still lacking. Not only are low concentrations of native targets in patient samples, often in the pM range, a limiting and challenging factor, but body fluids as complex matrices furthermore complicate measurements. The here-described method enables the determination of kinetic constants and resulting affinities for native antigens from patients' cerebrospinal fluid (CSF) and sera binding to antibodies. Using a significantly extended target-enrichment step, we modified a common sandwich-assay protocol, based on a primary and secondary antibody. We successfully analyze antibody kinetics of native targets from a variety of origins, with consistent results, independent of their source. Moreover, native neurofilament light chain (NFL) was investigated as an exemplary biomarker. Obtained data reveal antibodies recognizing recombinant NFL with high affinities, while showing no, or only significantly weakened binding to native NFL. The indicated differences for recombinant vs. native material demonstrate another beneficial application. Our assay is highly suitable for gaining valuable insights into characteristics of native biomarkers, thus impacting on the binder development of diagnostic reagents or pharmaceutical drugs.

Compliance With American Urological Association Guidelines for Nonmuscle Invasive Bladder Cancer Remains Poor: Assessing Factors Associated With Noncompliance and Survival in a Rural State.

OBJECTIVE: To identify factors associated with nonmuscle invasive bladder cancer (NMIBC) American Urological Association (AUA) guideline compliance in a rural state, to evaluate compliance rates over time, and to assess the impact of patient and provider rurality on delivery of NMIBC care. METHODS: We identified 847 Iowans in Surveillance, Epidemiology, and End Results-Medicare from 1992 to 2009 with high-grade NMIBC who survived 2 years and were not treated with cystectomy or radiation during this period. Compliance with AUA guidelines was assessed over time and compared to patient demographic, tumor, and treatment institution variables. Impact of rurality was analyzed using Surveillance, Epidemiology, and End Results ZIP code data travel distance of patient to nearest urologist practice location. RESULTS: Overall compliance with AUA guidelines was low (<1%), and did not markedly improve over the study period. In the multivariable model, only care at an academic medical center (OR 11.68, 95% CI 7.07-19.29) and tumor stage (Tis; OR 3.24, 95% CI 1.86-5.63) increased the odds of compliance. Patients living closer (<10 miles) to their urologists underwent more cystoscopies than patients living further (>30 miles) but distance did not affect compliance with other measures. Compliance was not associated with cancer-specific survival. CONCLUSION: Compliance with post-Transurethral Resection of Bladder Tumor (TURBT) NMIBC treatment guidelines has improved but remains suboptimal in our rural state, and is highly associated with treatment at an academic cancer center for reasons that could not be fully explained with these data.

Effects of tumor size and location on survival in upper tract urothelial carcinoma after nephroureterectomy.

INTRODUCTION: Upper Tract Urothelial Carcinoma (UTUC) is a rare disease with few prognostic determinants. We sought to evaluate the impact of tumor size and location on patient survival following nephroureterectomy for UTUC. MATERIALS AND METHODS: Data on 8284 patients treated with radical nephroureterectomy for UTUC in the United States between 1998 and 2011 were analyzed from the National Cancer Data Base. Univariable survivorship curves were generated based on pT stage, pN stage, grade, tumor size, and tumor site (renal pelvis vs. ureter). A Cox proportional hazards model was used to evaluate the effect of age, comorbidity, T stage, lymph node involvement, tumor site, and tumor size on survival. RESULTS: The median follow-up time was 46 months. A majority of the patients were male (55.4%) with a tumor size of ≥3.5 cm (52.0%) and pT stage

Esophageal squamous cell carcinoma invasion is inhibited by Activin A in ACVRIB-positive cells.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a global public health issue, as it is the eighth most common cancer worldwide. The mechanisms behind ESCC invasion and progression are still poorly understood, and warrant further investigation into these processes and their drivers. In recent years, the ligand Activin A has been implicated as a player in the progression of a number of cancers. The objective of this study was to investigate the role of Activin A signaling in ESCC. METHODS: To investigate the role Activin A plays in ESCC biology, tissue microarrays containing 200 cores from 120 ESCC patients were analyzed upon immunofluorescence staining. We utilized three-dimensional organotypic reconstruct cultures of dysplastic and esophageal squamous tumor cells lines, in the context of fibroblast-secreted Activin A, to identify the effects of Activin A on cell invasion and determine protein expression and localization in epithelial and stromal compartments by immunofluorescence. To identify the functional consequences of stromal-derived Activin A on angiogenesis, we performed endothelial tube formation assays. RESULTS: Analysis of ESCC patient samples indicated that patients with high stromal Activin A expression had low epithelial ACVRIB, the Activin type I receptor. We found that overexpression of stromal-derived Activin A inhibited invasion of esophageal dysplastic squamous cells, ECdnT, and TE-2 ESCC cells, both positive for ACVRIB. This inhibition was accompanied by a decrease in expression of the extracellular matrix (ECM) protein fibronectin and podoplanin, which is often expressed at the leading edge during invasion. Endothelial tube formation was disrupted in the presence of conditioned media from fibroblasts overexpressing Activin A. Interestingly, ACVRIB-negative TE-11 cells did not show the prior observed effects in the context of Activin A overexpression, indicating a dependence on the presence of ACVRIB. CONCLUSIONS: We describe the first observation of an inhibitory role for Activin A in ESCC progression that is dependent on the expression of ACVRIB.

Activin a signaling regulates cell invasion and proliferation in esophageal adenocarcinoma.

TGFß signaling has been implicated in the metaplasia from squamous epithelia to Barrett's esophagus and, ultimately, esophageal adenocarcinoma. The role of the family member Activin A in Barrett's tumorigenesis is less well established. As tumorigenesis is influenced by factors in the tumor microenvironment, such as fibroblasts and the extracellular matrix, we aimed to determine if epithelial cell-derived Activin affects initiation and progression differently than Activin signaling stimulation from a mimicked stromal source. Using Barrett's esophagus cells, CPB, and the esophageal adenocarcinoma cell lines OE33 and FLO-1, we showed that Activin reduces colony formation only in CPB cells. Epithelial cell overexpression of Activin increased cell migration and invasion in Boyden chamber assays in CPB and FLO-1 cells, which exhibited mesenchymal features such as the expression of the CD44 standard form, vimentin, and MT1-MMP. When grown in organotypic reconstructs, OE33 cells expressed E-cadherin and Keratin 8. As mesenchymal characteristics have been associated with the acquisition of stem cell-like features, we analyzed the expression and localization of SOX9, showing nuclear localization of SOX9 in esophageal CPB and FLO-1 cells.In conclusion, we show a role for autocrine Activin signaling in the regulation of colony formation, cell migration and invasion in Barrett's tumorigenesis.