OrganoID: A versatile deep learning platform for tracking and analysis of single-organoid dynamics.

Organoids have immense potential as ex vivo disease models for drug discovery and personalized drug screening. Dynamic changes in individual organoid morphology, number, and size can indicate important drug responses. However, these metrics are difficult and labor-intensive to obtain for high-throughput image datasets. Here, we present OrganoID, a robust image analysis platform that automatically recognizes, labels, and tracks single organoids, pixel-by-pixel, in brightfield and phase-contrast microscopy experiments. The platform was trained on images of pancreatic cancer organoids and validated on separate images of pancreatic, lung, colon, and adenoid cystic carcinoma organoids, which showed excellent agreement with manual measurements of organoid count (95%) and size (97%) without any parameter adjustments. Single-organoid tracking accuracy remained above 89% over a four-day time-lapse microscopy study. Automated single-organoid morphology analysis of a chemotherapy dose-response experiment identified strong dose effect sizes on organoid circularity, solidity, and eccentricity. OrganoID enables straightforward, detailed, and accurate image analysis to accelerate the use of organoids in high-throughput, data-intensive biomedical applications.

Automated microfluidic platform for dynamic and combinatorial drug screening of tumor organoids.

Three-dimensional (3D) cell culture technologies, such as organoids, are physiologically relevant models for basic and clinical applications. Automated microfluidics offers advantages in high-throughput and precision analysis of cells but is not yet compatible with organoids. Here, we present an automated, high-throughput, microfluidic 3D organoid culture and analysis system to facilitate preclinical research and personalized therapies. Our system provides combinatorial and dynamic drug treatments to hundreds of cultures and enables real-time analysis of organoids. We validate our system by performing individual, combinatorial, and sequential drug screens on human-derived pancreatic tumor organoids. We observe significant differences in the response of individual patient-based organoids to drug treatments and find that temporally-modified drug treatments can be more effective than constant-dose monotherapy or combination therapy in vitro. This integrated platform advances organoids models to screen and mirror real patient treatment courses with potential to facilitate treatment decisions for personalized therapy.

Quality of Life in Long-term Survivors of Muscle-Invasive Bladder Cancer.

PURPOSE: Health-related quality of life (QOL) has not been well-studied in survivors of muscle-invasive bladder cancer (MIBC). The present study compared long-term QOL in MIBC patients treated with radical cystectomy (RC) versus bladder-sparing trimodality therapy (TMT). METHODS AND MATERIALS: This cross-sectional bi-institutional study identified 226 patients with nonmetastatic cT2-cT4 MIBC, diagnosed in 1990 to 2011, who were eligible for RC and were disease free for ≥2 years. Six validated QOL instruments were administered: EuroQOL EQ-5D, European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Core Questionnaire and EORTC MIBC module, Expanded Prostate Cancer Index Composite bowel scale, Cancer Treatment and Perception Scale, and Impact of Cancer, version 2. Multivariable analyses of the mean QOL scores were conducted using propensity score matching. RESULTS: The response rate was 77% (n=173). The median follow-up period was 5.6 years. Of the 173 patients, 64 received TMT and 109, RC. The median interval from diagnosis to questionnaire completion was 9 years after TMT and 7 years after RC (P=.009). No significant differences were found in age, gender, comorbidities, tobacco history, performance status, or tumor stage. On multivariable analysis, patients who received TMT had better general QOL by 9.7 points of 100 compared with those who had received RC (P=.001) and higher physical, role, social, emotional, and cognitive functioning by 6.6 to 9.9 points (P≤.04). TMT was associated with better bowel function by 4.5 points (P=.02) and fewer bowel symptoms by 2.7 to 7.1 points (P≤.05). The urinary symptom scores were similar. TMT was associated with better sexual function by 8.7 to 32.1 points (P≤.02) and body image by 14.8 points (P<.001). The patients who underwent TMT reported greater informed decision-making scores by 13.6 points (P=.01) and less concern about the negative effect of cancer by 6.8 points (P=.006). The study limitations included missing baseline QOL data and different follow-up times. CONCLUSIONS: Both TMT and RC result in good long-term QOL outcomes in MIBC survivors, supporting TMT as a good alternative to RC for selected patients. Whether TMT leads to superior QOL requires prospective validation.

Low testosterone has a similar prevalence among men with sexual dysfunction due to either Peyronie's disease or erectile dysfunction and does not correlate with Peyronie's disease severity.

INTRODUCTION: Low testosterone (T) has been suggested as a risk factor for Peyronie's disease (PD) that may correlate with disease severity. Low T is common in men with sexual dysfunction but its role in the pathogenesis of PD remains unclear. AIM: The aim of this study was to compare the prevalence of low T (<300 ng/dL) in patients presenting with PD or erectile dysfunction (ED), as well as disease severity between men with PD and either low T or normal T (≥300 ng/dL). METHODS: Retrospective review of 300 men with either PD or ED was conducted. Men were excluded for combined PD and ED, psychogenic ED, or prior T use. For men with PD, plaque size, degree of curvature, and surgical correction rate were compared. MAIN OUTCOME MEASURES: The main outcome measures were (i) mean T levels in men with PD or ED and (ii) plaque size, degree of curvature, and surgical correction rates among men with PD and either low T or normal T. RESULTS: Eighty-seven men with PD and 98 men with ED were identified. Men with PD had mean total T and free T of 328 ng/dL and 11.5 ng/dL, while men with ED had mean levels of 332 ng/dL and 12.1 ng/dL, respectively (P > 0.05). Of PD men, 52.9% had low T, compared with 45.9% of men with ED (P = 0.35). T levels did not correlate with plaque size or degree of curvature in the PD group (P > 0.05). CONCLUSIONS: Men with sexual dysfunction characterized by either PD or ED had similarly low T levels, and low T did not correlate with PD severity or surgical correction rate. The comparable prevalence of low T in men with PD or ED suggests the high rate of low T in PD men may be related to a common process among men with abnormal erectile physiology and not specifically causative in plaque formation.

Resident involvement and experience do not affect perioperative complications following robotic prostatectomy.

PURPOSE: Most urologic training programs use robotic prostatectomy (RP) as an introduction to teach residents appropriate robotic technique. However, concerns may exist regarding differences in RP outcomes with resident involvement. Our objective was therefore to evaluate whether resident involvement affects complications, operative time, or length of stay (LOS) following RP. METHODS: Using the National Surgical Quality Improvement Program database (2005-2011), we identified patients who underwent RP, stratified them by resident presence or absence during surgery, and compared hospital LOS, operative time, and postoperative complications using bivariable and multivariable analyses. A secondary analysis comparing outcomes of interest across postgraduate year (PGY) levels was also performed. RESULTS: A total of 5,087 patients who underwent RPs were identified, in which residents participated in 56%, during the study period. After controlling for potential confounders, resident present and absent groups were similar in 30-day mortality (0.0 vs. 0.2%, p = 0.08), serious morbidity (1.8 vs. 2.1%, p = 0.33), and overall morbidity (5.1 vs. 5.4%, p = 0.70). While resident involvement did not affect LOS, operative time was longer when residents were present (median 208 vs. 183 min, p < 0.001). Similar findings were noted when assessing individual PGY levels. CONCLUSIONS: Regardless of PGY level, resident involvement in RPs appears safe and does not appear to affect postoperative complications or LOS. While resident involvement in RPs does result in longer operative times, this is necessary for the learning process.

Nutritional predictors of complications following radical cystectomy.

PURPOSE: To determine the impact of preoperative nutritional status on the development of surgical complications following cystectomy using the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP). METHODS: We performed a retrospective review of the NSQIP 2005-2012 Participant Use Data Files. ACS-NSQIP collects data on 135 variables, including pre- and intraoperative data and 30-day postoperative complications and mortality on all major surgical procedures at participating institutions. Preoperative albumin (<3.5 or >3.5 g/dl), weight loss 6 months before surgery (>10 %), and body mass index (BMI) were identified as nutritional variables within the database. The overall complication rate was calculated, and predictors of complications were identified using multivariable logistic regression models. RESULTS: A total of 1,213 patients underwent cystectomy for bladder cancer between 2005 and 2012. The overall 30-day complication rate was 55.1 % (n = 668). While 14.7 % (n = 102) had a preoperative albumin <3.5 g/dL, 3.4 % had >10 % weight loss in the 6 months prior to surgery and the mean BMI was 28 kg/m(2). After controlling for age, sex, medical comorbidities, medical resident involvement, operation year, operative time, and prior operation, only albumin <3.5 g/dl was a significant predictor of experiencing a postoperative complication (p = 0.03). This remained significant when albumin was evaluated as a continuous variable (p = 0.02). CONCLUSIONS: Poor nutritional status measured by serum albumin is predictive of an increased rate of surgical complications following radical cystectomy. This finding supports the importance of preoperative nutritional status in this population and highlights the need for the development of effective nutritional interventions in the preoperative setting.

Metabolic syndrome as a predictor for postoperative complications after urologic surgery.

OBJECTIVE: To elucidate whether metabolic syndrome (MS) has an effect on outcomes after nephrectomy, prostatectomy, or cystectomy. METHODS: Using the American College of Surgeons National Surgical Quality Improvement Program's database, patients undergoing cystectomy, nephrectomy, or prostatectomy between 2005 and 2011 were reviewed to assess for the presence of MS and a variety of perioperative complications. RESULTS: The overall complication rate for cystectomy, nephrectomy, and prostatectomy was 52.4%, 20.2%, and 8.7%, respectively. On multivariate analysis controlling for age, sex, body mass index, cardiac comorbidity, functional status, surgical approach (prostatectomy and nephrectomy), and surgery within 30 days, MS was not associated with perioperative complications in patients undergoing cystectomy (odds ratio [OR], 0.760; 95% confidence interval [CI], 0.476-1.213). On multivariate analysis, the presence of MS was a significant predictor of perioperative complications after radical nephrectomy (adjusted OR, 1.489; 95% CI, 1.146-1.934). With regards to prostatectomy, MS was not a significant predictor of complications (OR, 1.065; 95% CI, 0.739-1.535). CONCLUSION: Patients in this cohort with MS undergoing cystectomy or prostatectomy did not experience a higher rate of complications compared with patients without MS, although patients with MS undergoing nephrectomy had a higher complication rate. It may be warranted to preoperatively counsel patients with MS undergoing nephrectomy that complication rates may be higher.

Sarcopenia as a predictor of complications and survival following radical cystectomy.

PURPOSE: Patients undergoing radical cystectomy face substantial but highly variable risks of major complications. Risk stratification may be enhanced by objective measures such as sarcopenia. Sarcopenia (loss of skeletal muscle mass) has emerged as a novel biomarker associated with adverse outcomes in many clinical contexts relevant to cystectomy. Based on these data we hypothesized that sarcopenia would be associated with increased 30-day major complications and mortality after radical cystectomy for bladder cancer. MATERIALS AND METHODS: We performed a retrospective study of patients treated with radical cystectomy at our institution from 2008 to 2011. Sarcopenia was assessed by measuring cross-sectional area of the psoas muscle (total psoas area) on preoperative computerized tomography. Cutoff points were developed and evaluated using ROC curves to determine predictive ability in men and women for outcomes of major complications and survival. RESULTS: Of 224 patients with bladder cancer 200 underwent preoperative computerized tomography within 1 month of surgery. Total psoas area was calculated with a mean score of 712 and 571 cm2/m2 in men and women, respectively. A clear association was noted between major complications and lower total psoas area in women using a cutoff of 523 cm2/m2 to define sarcopenia (AUC 0.70). Sarcopenia was not significantly associated with complications in men. There was a nonsignificant trend of sarcopenia with worse 2-year survival. CONCLUSIONS: Sarcopenia in women was a predictor of major complications after radical cystectomy. Further research confirming sarcopenia as a useful predictor of complications would support the development of targeted interventions to mitigate the untoward effects of sarcopenia before cancer surgery.

Roadmap for the development of the University of North Carolina at Chapel Hill Genitourinary OncoLogy Database--UNC GOLD.

BACKGROUND: The management of genitourinary malignancies requires a multidisciplinary care team composed of urologists, medical oncologists, and radiation oncologists. A genitourinary (GU) oncology clinical database is an invaluable resource for patient care and research. Although electronic medical records provide a single web-based record used for clinical care, billing, and scheduling, information is typically stored in a discipline-specific manner and data extraction is often not applicable to a research setting. A GU oncology database may be used for the development of multidisciplinary treatment plans, analysis of disease-specific practice patterns, and identification of patients for research studies. Despite the potential utility, there are many important considerations that must be addressed when developing and implementing a discipline-specific database. METHODS AND MATERIALS: The creation of the GU oncology database including prostate, bladder, and kidney cancers with the identification of necessary variables was facilitated by meetings of stakeholders in medical oncology, urology, and radiation oncology at the University of North Carolina (UNC) at Chapel Hill with a template data dictionary provided by the Department of Urologic Surgery at Vanderbilt University Medical Center. Utilizing Research Electronic Data Capture (REDCap, version 4.14.5), the UNC Genitourinary OncoLogy Database (UNC GOLD) was designed and implemented. RESULTS: The process of designing and implementing a discipline-specific clinical database requires many important considerations. The primary consideration is determining the relationship between the database and the Institutional Review Board (IRB) given the potential applications for both clinical and research uses. Several other necessary steps include ensuring information technology security and federal regulation compliance; determination of a core complete dataset; creation of standard operating procedures; standardizing entry of free text fields; use of data exports, queries, and de-identification strategies; inclusion of individual investigators' data; and strategies for prioritizing specific projects and data entry. CONCLUSIONS: A discipline-specific database requires a buy-in from all stakeholders, meticulous development, and data entry resources to generate a unique platform for housing information that may be used for clinical care and research with IRB approval. The steps and issues identified in the development of UNC GOLD provide a process map for others interested in developing a GU oncology database.

Neoadjuvant chemotherapy for bladder cancer does not increase risk of perioperative morbidity.

OBJECTIVE: To determine whether neoadjuvant chemotherapy (NAC) is a predictor of postoperative complications, length of stay (LOS), or operating time after radical cystectomy (RC) for bladder cancer. PATIENTS AND METHODS: A retrospective review of the American College of Surgeons National Surgical Quality Improvement Program (NSQIP) database was performed to identify patients receiving NAC before RC from 2005 to 2011. Bivariable and multivariable analyses were used to determine whether NAC was associated with 30-day perioperative outcomes, e.g. complications, LOS, and operating time. RESULTS: Of the 878 patients who underwent RC for bladder cancer in our study, 78 (8.9%) received NAC. Excluding those patients who were ineligible for NAC due to renal insufficiency, 78/642 (12.1%) received NAC. In all, 457 of the 878 patients (52.1%) undergoing RC had at least one complication ≤30 days of RC, including 43 of 78 patients (55.1%) who received NAC and 414 of 800 patients (51.8%) who did not (P = 0.58). On multivariable logistic regression, NAC was not a predictor of complications (P = 0.87), re-operation (P = 0.16), wound infection (P = 0.32), or wound dehiscence (P = 0.32). Using multiple linear regression, NAC was not a predictor of increased operating time (P = 0.24), and patients undergoing NAC had a decreased LOS (P = 0.02). CONCLUSIONS: Our study is the first large multi-institutional analysis specifically comparing complications after RC with and without NAC. Using a nationally validated, prospectively maintained database specifically designed to measure perioperative outcomes, we found no increase in perioperative complications or surgical morbidity with NAC. Considering these findings and the well-established overall survival benefit over surgery alone, efforts are needed to improve the uptake of NAC.

Blockade of miR-150 maturation by MLL-fusion/MYC/LIN-28 is required for MLL-associated leukemia.

Expression of microRNAs (miRNAs) is under stringent regulation at both transcriptional and posttranscriptional levels. Disturbance at either level could cause dysregulation of miRNAs. Here, we show that MLL fusion proteins negatively regulate production of miR-150, an miRNA widely repressed in acute leukemia, by blocking miR-150 precursors from being processed to mature miRNAs through MYC/LIN28 functional axis. Forced expression of miR-150 dramatically inhibited leukemic cell growth and delayed MLL-fusion-mediated leukemogenesis, likely through targeting FLT3 and MYB and thereby interfering with the HOXA9/MEIS1/FLT3/MYB signaling network, which in turn caused downregulation of MYC/LIN28. Collectively, we revealed a MLL-fusion/MYC/LIN28⊣miR-150⊣FLT3/MYB/HOXA9/MEIS1 signaling circuit underlying the pathogenesis of leukemia, where miR-150 functions as a pivotal gatekeeper and its repression is required for leukemogenesis.

In vitro-in vivo translation of lipid nanoparticles for hepatocellular siRNA delivery.

A significant challenge in the development of clinically viable siRNA delivery systems is a lack of in vitro-in vivo translatability: many delivery vehicles that are initially promising in cell culture do not retain efficacy in animals. Despite its importance, little information exists on the predictive nature of in vitro methodologies, most likely due to the cost and time associated with generating in vitro-in vivo data sets. Recently, high-throughput techniques have been developed that have allowed the examination of hundreds of lipid nanoparticle formulations for transfection efficiency in multiple experimental systems. The large resulting data set has allowed the development of correlations between in vitro and characterization data and in vivo efficacy for hepatocellular delivery vehicles. Consistency of formulation technique and the type of cell used for in vitro experiments was found to significantly affect correlations, with primary hepatocytes and HeLa cells yielding the most predictive data. Interestingly, in vitro data acquired using HeLa cells were more predictive of in vivo performance than mouse hepatoma Hepa1-6 cells. Of the characterization parameters, only siRNA entrapment efficiency was partially predictive of in vivo silencing potential, while zeta-potential and, surprisingly, nanoparticle size (when <300 nm) as measured by dynamic light scattering were not. These data provide guiding principles in the development of clinically viable siRNA delivery materials and have the potential to reduce experimental costs while improving the translation of materials into animals.

The association between self-reported tooth loss and cognitive function in the REasons for Geographic And Racial Differences in Stroke study: an assessment of potential pathways.

BACKGROUND: Several mechanisms may associate tooth loss and related oral inflammation with cognitive impairment. The authors studied the relationship between tooth loss and cognitive function. METHODS: The REasons for Geographic And Racial Differences in Stroke study is a national longitudinal study of more than 30,000 African American and white adults 45 years or older. Data for tooth loss, cognitive function and potential confounding variables were available for 9,853 participants at the time of analysis. The authors used incremental linear regression modeling to investigate the cross-sectional association between self-reported tooth loss and cognitive function. RESULTS: In unadjusted analysis (mean learning followed by recall; α level of significance of .05), the loss of six to 16 teeth and the loss of more than 16 teeth were associated with poorer cognitive function compared with the loss of no teeth. Attenuated associations persisted after the authors adjusted for demographic and systemic risk factors. The full model, which was adjusted for socioeconomic status (SES), revealed no association between tooth loss and cognitive function. CONCLUSION: Tooth loss may be associated with cognitive function; however, this association is mediated by age and SES. CLINICAL IMPLICATIONS: Tooth loss due to periodontal disease may be a marker for low SES, and the interplay of these factors with advanced age may confer risk of having poorer cognitive function. Further studies are needed to clarify these associations.

Screening for inhibitors of the SOD1 gene promoter: pyrimethamine does not reduce SOD1 levels in cell and animal models.

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene are detected in 20% of familial and 3% of sporadic amyotrophic lateral sclerosis (ALS) cases. Although mutant SOD1 is known to induce motor neuron death via multiple adverse acquired functions, its exact pathogenic mechanism is not well defined. SOD1 toxicity is dose dependent; levels of mutant SOD1 protein in transgenic mice determine disease susceptibility, onset and rate of progression. We therefore sought to identify small molecules that reduce SOD1 levels by inhibiting the SOD1 promoter. We tested pyrimethamine (previously reported to suppress SOD1 expression), several compounds currently in trials in human and murine ALS, and a set of 1040 FDA-approved compounds. In a PC12 cell-based assay, no compounds reduced SOD1 promoter activity without concomitant cytotoxicity. Additionally, pyrimethamine failed to repress levels of SOD1 protein in HeLa cells or homogenates of liver, spinal cord and brain of wild-type mice. Thirty-four compounds (including riluzole, ceftriaxone, minocyclin, PBA, lithium, acetylcysteine) in human and mouse ALS trials and an additional set of 1040 FDA-approved compounds also showed no effect on SOD1 promoter activity. This present study thus failed to identify small molecule inhibitors of SOD1 gene expression.

IGF-1:tetanus toxin fragment C fusion protein improves delivery of IGF-1 to spinal cord but fails to prolong survival of ALS mice.

To improve delivery of human insulin-like growth factor-1 (hIGF-1) to brain and spinal cord, we generated a soluble IGF-1:tetanus toxin fragment C fusion protein (IGF-1:TTC) as a secreted product from insect cells. IGF-1:TTC exhibited IGF-1 and TTC activity in vitro; it increased levels of immunoreactive phosphoAkt in treated MCF-7 cells and bound to immobilized ganglioside GT1b. In mice, the fusion protein underwent retrograde transport by spinal cord motor neurons following intramuscular injection, and exhibited both TTC- and IGF-1 activity in the CNS following intrathecal infusion. Analogous to the case with TTC, intrathecal infusion of the fusion protein resulted in substantial levels of IGF-1:TTC in spinal cord tissue extracts. Tissue concentrations of hIGF-1 in lumbar spinal cords of mice infused with IGF-1:TTC were estimated to be approximately 500-fold higher than those in mice treated with unmodified recombinant hIGF-1 (rhIGF-1). Like rhIGF-1, infusion of IGF-1:TTC reduced levels of IGF-1 receptor immunoreactivity in the same extracts. Despite raising levels of exogenous hIGF-1 in spinal cord, intramuscular- or intrathecal administration of IGF-1:TTC had no significant effect on disease progression or survival of high-expressing SOD1(G93A) transgenic mice. IGF-1:TTC may prove to be neuroprotective in other animal models of CNS disease or injury known to be responsive to unmodified IGF-1.

Recombinant GDNF: tetanus toxin fragment C fusion protein produced from insect cells.

Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFRalpha-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

A small molecule inhibitor for phosphatase and tensin homologue deleted on chromosome 10 (PTEN).

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a phosphoinositide 3-phosphatase, is an important regulator of insulin-dependent signaling. The loss or impairment of PTEN results in an antidiabetic impact, which led to the suggestion that PTEN could be an important target for drugs against type II diabetes. Here we report the design and validation of a small- molecule inhibitor of PTEN. Compared with other cysteine-based phosphatases, PTEN has a much wider active site cleft enabling it to bind the PtdIns(3,4,5)P3 substrate. We have exploited this feature in the design of vanadate scaffolds complexed to a range of different organic ligands, some of which show potent inhibitory activity. A vanadyl complexed to hydroxypicolinic acid was found to be a highly potent and specific inhibitor of PTEN that increases cellular PtdIns(3,4,5)P3 levels, phosphorylation of Akt, and glucose uptake in adipocytes at nanomolar concentrations. The findings presented here demonstrate the applicability of a novel and specific chemical inhibitor against PTEN in research and drug development.