Citations

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly lethal hepatobiliary neoplasm with limited therapeutic options. Thus, new therapeutic strategies are needed to advance our treatment armamentarium for this devastating disease. MicroRNAs represent a new class of endogenous nucleic acids regulating development, cell death, and carcinogenesis. Recent studies have indicated that the miR-106b∼25 cluster, which is upregulated in cancer, has potential antiapoptotic properties. Meanwhile, we have demonstrated that Hedgehog (Hh) signaling is activated in CCA and also has a strong antiapoptotic effect. Based on this information, we posited that Hh signaling may regulate mir-106b∼25 expression. Due to the dependence of CCA on antiapoptotic signals, we conducted a study to determine the role of miR-106b∼25 in CCA apoptosis and unveil its regulatory mechanisms. Experimental procedures: Immortalized benign and malignant human cholangiocyte cell lines were used for the experiments. MicroRNA expression was analyzed using real-time quantitative RT-PCR (qRT-PCR) assays. The effect of the microRNAs was studied using Lipofectamine-based transfection with precursor microRNAs or locked-nucleic acid antagonists (LNAs) to respectively potentiate or decrease microRNAs expression. Functional apoptosis studies employed DAPI staining with fluorescent microscopic analysis of nuclear morphology, or biochemical measurement of caspase-3/7 activity. Western blotting and luciferase assays were conducted to identify potential messenger RNA targets. Results: Expression of miR-106b∼25 was enhanced in malignant vs. benign cholangiocyte cell lines. Treatment with the known antagonist of Hh signaling, cyclopamine, resulted in downregulation of the cluster. Because CCA cells express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in vivo, we next examined TRAIL-induced apoptosis in CCA cells. TRAIL-mediated apoptosis was attenuated by transfection with miR-25 and enhanced by LNAs. Computational analysis identified the TRAIL death receptor 4 (DR4) as a potential target of this cluster. Indeed, we observed downregulation of DR4 cellular protein levels during overexpression of miR-106b∼25 and upregulation of DR4 upon cluster inhibition with LNAs. The 3’untranslated region (UTR) of the DR4 mRNA was cloned into a luciferase reporter, and the potential direct regulation of DR4 by miR-106b∼25 examined. Indeed, the regulation of DR4 by miR-106b∼25 was confirmed by this luciferase assay demonstrating a direct inhibitory effect of the miR-106b∼25 polycistron on the DR4 3’UTR. Conclusions: The miR-106b∼25 cluster is overexpressed in malignant cholangiocyte cell lines, likely by a Hh signaling pathway. miR-106b∼25 inhibits apoptosis, at least in part, via downregulation of DR4. LNA targeted inhibition of miR-106b∼25 sensitizes CCA cells to apoptosis and may represent a new therapeutic strategy for this cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 199. doi:10.1158/1538-7445.AM2011-199

Background Atrial fibrillation ( AF ) is a common sequela of hypertrophic cardiomyopathy ( HCM ), but evidence on its prevalence, risk factors, and effect on mortality is sparse. We sought to evaluate the prevalence of AF , identify clinical and echocardiographic correlates, and assess its effect on mortality in a large high‐risk HCM population. Methods and Results We identified HCM patients who underwent evaluation at our institution from 1975 to 2012. AF was defined by known history (either chronic or paroxysmal), electrocardiogram, or Holter monitoring at index visit. We examined clinical and echocardiographic variables in association with AF . The effect of AF on overall and cause‐specific mortality was evaluated with multivariate Cox proportional hazards models. Of 3673 patients with HCM , 650 (18%) had AF. Patients with AF were older and more symptomatic ( P <0.001). AF was less common among patients with obstructive HCM phenotype and was associated with larger left atria, higher E/e’ ratios, and worse cardiopulmonary exercise tolerance (all P values<0.001). During median (interquartile range) follow‐up of 4.1 (0.2 to 10) years, 1069 (29%) patients died. Patients with AF had worse survival compared to those without AF ( P <0.001). In multivariate analysis adjusted for established risk factors of mortality in HCM , the hazard ratio (95% confidence interval) for the effect of AF on overall mortality was 1.48 (1.27 to 1.71). AF did not have an effect on sudden or nonsudden cardiac death. Conclusions In this large referral HCM population, approximately 1 in 5 patients had AF . AF was a strong predictor of mortality, even after adjustment for established risk factors.

Abstract Background: Because of the faltering sensitivity and/or specificity, urine-based assays currently have a limited role in the management of patients with bladder cancer. The aim of this study was to externally validate our previously reported protein biomarker panel from multiple sites in the United States and Europe. Methods: This multicenter external validation study included a total of 320 subjects (bladder cancer = 183). The 10 biomarkers (IL8, MMP9, MMP10, SERPINA1, VEGFA, ANG, CA9, APOE, SDC1, and SERPINE1) were measured using commercial ELISA assays in an external laboratory. The diagnostic performance of the biomarker panel was assessed using receiver operator curves (ROC) and descriptive statistical values. Results: Utilizing the combination of all 10 biomarkers, the area under the ROC for the diagnostic panel was noted to be 0.847 (95% confidence interval, 0.796–0.899), outperforming any single biomarker. The multiplex assay at optimal cutoff value achieved an overall sensitivity of 0.79, specificity of 0.79, positive prediction value of 0.73, and negative prediction value of 0.84 for bladder cancer classification. Sensitivity values of the diagnostic panel for high-grade bladder cancer, low-grade bladder cancer, muscle invasive bladder cancer, and non-muscle invasive bladder cancer were 0.81, 0.90, 0.95, and 0.77, respectively. Conclusions: Urinary levels of the biomarker panel enabled discrimination of patients with bladder cancer and controls, and the levels of biomarker subsets were associated with advancing tumor grade and stage. Impact: If proven to be reliable, urinary diagnostic biomarker assays can detect bladder cancer in a timely manner such that the patient can expect improvements in overall survival and quality of life. Cancer Epidemiol Biomarkers Prev; 23(9); 1804–12. ©2014 AACR.

Abstract Macrophage inflammatory protein (MIP-1α), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1α have been well documented, the in vivo biological activities of MIP-1α in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1α and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10–24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1α stimulation. These studies substantiate the biological effects of MIP-1α on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1α to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.

Abstract Background: Up to 70% of patients with non–muscle-invasive bladder cancer (NMIBC) experience disease recurrence, making it one of the most prevalent cancers in the United States. The purpose of this study was to test the performance of a multiplex urinary biomarker assay for the monitoring of voided urine for recurrent bladder cancer. Methods: This retrospective, multicenter study included a total of 125 subjects with a history of bladder cancer. Voided urine specimens were collected before procedure from these subjects (53 with confirmed tumor recurrence and 72 with confirmed non-tumor recurrence) for analysis. A prediction rule generated from the performance characteristics of 10 single biomarkers (IL8, MMP9, MMP10, SERPINA1, VEGFA, ANG, CA9, APOE, SERPINE1, and SDC1) was measured using ELISA. The diagnostic performance of the biomarker panel was assessed using receiver operator curves (ROC) and descriptive statistical values (e.g., sensitivity and specificity). Results: The combination of all 10 biomarkers outperformed any single biomarker with a calculated AUROC for the diagnostic panel of 0.904 [95% confidence interval (CI), 0.853–0.956]. The multiplex assay achieved an overall sensitivity of 79% and specificity of 88% for recurrent bladder cancer and significantly outperformed the Urovysion cytogenetic assay (sensitivity 42%, specificity 94%) and voided urinary cytology (sensitivity 33%, specificity 90%). Conclusions: A diagnostic panel of 10 urinary biomarkers that accurately detects primary bladder cancer also performs well for the detection of recurrent bladder cancer. Impact: The identification of a reliable urine-based surveillance and detection assay would be of benefit to both patients and the healthcare system. Cancer Epidemiol Biomarkers Prev; 23(7); 1340–5. ©2014 AACR.

PurposePrevious research suggests that school-age children with minimal hearing loss (CMHL) are at risk for a variety of psychoeducational problems. However, CMHL are a heterogeneous group, and the profile of at-risk children is unknown. Data regarding the characteristics of early school-age CMHL are needed to extend previous findings and determine potential risk factors associated with psychoeducational difficulties.MethodPsychoeducational outcomes were evaluated at baseline and longitudinally in age-matched groups of 27 CMHL (ages 4–10 years) and 26 children with normal hearing (CNH) using assessments of language, reading, behavior, speech recognition in noise, and cognition. Additional analyses were used to identify demographic characteristics among CMHL that are associated with psychoeducational difficulties.ResultsAt the earliest age tested, CMHL had greater teacher-rated attention difficulties in the classroom than CNH. Differences in the rate of psychoeducational development were not observed between groups. Among CMHL, psychoeducational difficulties were associated with delays in identification of hearing loss and low maternal education.ConclusionsClassroom attention abilities should be monitored for early school-age CMHL. Late-identified CMHL and CMHL with low maternal education levels may be in particular need of academic and social support. Continued efforts for early identification of CMHL should be made to improve outcomes for these children.

Abstract The canonical function of plasminogen activator inhibitor-1 (PAI-1/SERPINE1) is as an inhibitor of urokinase-type plasminogen activator for blood clot maintenance, but it is now also considered a pleiotropic factor that can exert diverse cellular and tumorigenic effects. However, the mechanism controlling its pleiotropic effects is far from being understood. To elucidate the tumorigenic role of PAI-1, we tested the effects of PAI-1 after manipulation of its expression or through the use of a small-molecule inhibitor, tiplaxtinin. Downregulation of PAI-1 significantly reduced cellular proliferation through an inability to progress from the G0–G1 phase of the cell cycle. Accordingly, overexpression of PAI-1 augmented proliferation by encouraging S-phase entry. Biochemically, cell-cycle arrest was associated with the depletion of the G1-phase transition complexes, cyclin D3/cdk4/6 and cyclin E/cdk2, in parallel with the upregulation of the cell-cycle inhibitors p53, p21Cip1/Waf1, and p27Kip1. PAI-1 depletion significantly decreased the tumor size of urothelial T24 and UM-UC-14 xenografts, and overexpression of PAI-1 substantially increased the tumor size of HeLa xenografts. Finally, immunohistochemical analysis of human bladder and cervical tumor tissue microarrays revealed increased expression of PAI-1 in cancerous tissue, specifically in aggressive tumors, supporting the relevance of this molecule in human tumor biology. Implications: Targeting PAI-1 has beneficial antitumoral effects and should be further investigated clinically. Mol Cancer Res; 12(3); 322–34. ©2014 AACR.

A 16‐year‐old high school football player presented with 4 months of anterior knee pain and small, mobile, prepatellar “lumps” after falling onto an opponent's cleat. He reported knee pain primarily during knee flexion and direct pressure during squatting and kneeling. Knee radiographs were unremarkable. Ultrasonography revealed multiple, freely mobile, subcutaneous nodules of variable size and echogenicity in the prepatellar region. Analysis of magnetic resonance imaging suggested possible fat necrosis but was nondiagnostic. The patient opted for surgical exploration, at which time multiple, opalescent subcutaneous nodules were removed. Pathology was consistent with encapsulated fat necrosis. After surgery, his symptoms resolved, and he returned to sports without restrictions.