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Wnt/β-catenin signaling is activated in the heart after myocardial infarction (MI). This study aims to investigate if β-catenin deletion affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at 8 weeks after MI, associated with reduced scar size and attenuated chamber dilation. qPCR analyses of both myocardial tissues and purified cardiomyocytes demonstrated upregulation of Wnt pathway genes in border and infarct regions after MI, including Wnt ligands (such as Wnt4) and receptors (such as Fzd1 and Fzd2). At 1 week after MI, cardiac sodium channel gene (Scn5a) transcript was reduced in WT but not in KO hearts, consistent with previous studies showing Scn5a inhibition by Wnt/β-catenin signaling. At 8 weeks after MI when Wnt genes have declined, Scn5a returned to near sham levels and K+ channel gene downregulations were not different between WT and KO mice. This study demonstrated that VT susceptibility in the chronic phase after MI is reduced in mice with cardiomyocyte-specific β-catenin deletion primarily through attenuated structural remodeling, but not ion channel gene alterations.

Background Survivors of community-acquired pneumonia (CAP) are at increased risk of cardiovascular disease, cognitive and functional decline, and death, but the mechanisms remain unknown. Research Question Do CAP survivors have evidence of increased inflammatory activity in their lung parenchyma on 2-deoxy-2-[18F]fluoro- d -glucose (18FDG)-PET/CT imaging after clinical resolution of infection? Study Design and Methods We obtained 18FDG-PET/CT scans from 22 CAP survivors during their hospitalization with pneumonia (acute CAP) and 30 to 45 days after hospital discharge (post-CAP). In each set of scans, we assessed the lungs for foci of increased 18FDG uptake by visual interpretation and by total pulmonary glycolytic activity (tPGA), a background-corrected measure of total metabolic activity (as measured by 18FDG uptake). We also measured, post-CAP, the glycolytic activity of CAP survivor lung areas with volumes similar to the areas in 28 matched historical control subjects without pneumonia. Results Overall, 68% of CAP survivors (95% CI, 45%-85%) had distinct residual areas of increased 18FDG uptake in their post-CAP studies. tPGA decreased from 821.5 (SD, 1,140.2) in the acute CAP period to 80.0 (SD, 81.4) in the post-CAP period (P = .006). The tPGA post-CAP was significantly higher than that in lung areas of similar volume in control subjects (80.0 [SD, 81.4] vs –19.4 [SD, 5.9]; P Interpretation An important proportion of CAP survivors have persistent pulmonary foci of increased inflammatory activity beyond resolution of their infection. As inflammation contributes to cardiovascular disease, cognitive decline, functional waning, and mortality risk in the general population, this finding provides a plausible mechanism for the increased morbidity and mortality that have been observed post-CAP.

Background: Wnt signaling plays a critical role in both embryonic cardiogenesis and cardiac remodeling in adult heart disease. We have previously demonstrated that the canonical Wnt/β-catenin pathway inhibits cardiac sodium current, but it remains unclear whether the noncanonical Wnt pathway affects cardiac electrophysiology. Methods and Results: Western blot analysis of ventricular tissues from patients with heart failure (n=6) demonstrated a 2.3x fold increase (p90 : 273±77ms, n=5) as compared to control cells (42±12 ms, n=7, p Conclusion: Wnt5a is increased in the myocardium of patients with heart failure. Viral expression of Wnt5a in rat ventricular tissue increases QT interval and ventricular arrhythmia susceptibility, which is associated with prolongation of action potentials in cardiomyocytes. This may be an important target for future therapies.

This study aimed to evaluate markers of systemic as well as imaging markers of inflammation in the ascending aorta, bone marrow, and spleen measured by 18F-FDG PET/CT, in HIV+ patients at baseline and following therapy with rosuvastatin. Of the 35 HIV+ patients enrolled, 17 were randomized to treatment with 10 mg/day rosuvastatin and 18 to usual care for 6 months. An HIV− control cohort was selected for baseline comparison of serum inflammatory markers and monocyte markers of inflammation. 18F-FDG-PET/CT imaging of bone marrow, spleen, and thoracic aorta was performed in the HIV+ cohort at baseline and 6 months. While CD14++CD16− and CCR2 expressions were reduced, serum levels of IL-7, IL-8, and MCP-1 were elevated in the HIV+ population compared to the controls. There was a significant drop in FDG uptake in the bone marrow (TBRmax), spleen (SUVmax) and thoracic aortic (TBRmax) in the statin-treated group compared to the control group (bone marrow: − 10.3 ± 16.9% versus 5.0 ± 18.9%, p = .0262; spleen: − 9.8 ± 20.3% versus 11.3 ± 28.8%, p = .0497; thoracic aorta: − 19.1 ± 24.2% versus 4.3 ± 15.4%, p = .003). HIV+ patients had significantly markers of systemic inflammation including monocyte activation. Treatment with low-dose rosuvastatin in the HIV+ cohort significantly reduced bone marrow, spleen and thoracic aortic FDG uptake.

The Wnt/β-catenin signaling regulates ion channel gene expressions in cardiomyocytes. Because Wnt/β-catenin signaling is activated in myocardial infarction (MI), this study aims to investigate if β-catenin inhibition affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at week-8 after MI, associated with attenuated structural remodeling (reduced scar size and attenuated left ventricle dilation) as compared to WT. However, at the subacute phase (week-1) and chronic phase (week-8) after MI, Wnt/β-catenin signaling activation was found in non-cardiomyocytes, but not in cardiomyocytes. Downregulations of Scn5a (encoding Nav1.5) and Gja1 (encoding Cx43) were found at week-1 but not at week-8, while downregulations of K+ channel genes were present at both week-1 and -8. Consistent with no activation of Wnt/β-catenin pathway in cardiomyocytes at week-1 and -8, these alterations in ion channel/transporter genes were not different between KO and WT mice. This study demonstrated that mice with cardiomyocyte-specific β-catenin deletion have reduced VT susceptibility after MI which is caused by attenuated structural remodeling, instead of alterations in ion channel gene expressions.

Background: Both inherited arrhythmogenic diseases (such as Brugada Syndrome, BrS) and heart failure are associated with reduced voltage-gated Na+ current (lNa) which promotes lethal arrhythmias and sudden deaths. We have shown that WNT/β-catenin signaling, which is active in heart disease, inhibits lNa in rat cardiomyocytes. But whether WNT/β-catenin signaling also regulates lNa in human cardiomyocytes and represents a novel therapeutic target is unknown. Objectives: To investigate if Wnt/β-catenin signaling inhibits lNa in healthy human cardiomyocytes, and to test if inhibition of β-catenin can rescue lNa in BrS patient cardiomyocytes. Methods: Cardiomyocytes were differentiated from human induced pluripotent stem cells (iPSC-CMs) that were derived from healthy volunteers or BrS patients. CHIR-99021 was used to activate Wnt pathway and shRNA was used to knockdown β-catenin. lNa and action potentials were recorded using whole-cell patch-clamp technique. Results: In healthy iPSC-CMs, activation of Wnt/β-catenin signaling reduced peak lNa and action potential upstroke, which were associated with reduced lNa channel protein (nav1.5) and transcript (SCN5A). In BrS iPSC-CMs, peak lNa is only 16% of that in healthy iPSC-CMs. shRNA inhibition of β-catenin in BrS iPSC-CMs led to a 4.4 fold increase in Nav1.5 without affecting cardiac Ca2+ channel. Accordingly, β-catenin inhibition in BrS iPSC-CMs increased peak lNa by 4.9-fold to 78% of the levels in healthy cells, offsetting the fundamental genetic defect. Conclusions: This study demonstrated Wnt/β-catenin-mediated inhibition of human cardiac lNa and, using Brugada Syndrome as an example, demonstrated that β-catenin inhibition is potentially a new therapeutic strategy to rescue lNa in human heart disease.

Introduction: Cardiovascular disease (CVD) is a major source of morbidity and mortality in HIV+ patients, which is thought to be in part due to a state of persistent systemic inflammation that results in accelerated atherosclerosis. Hypothesis: We hypothesized that inflammation, as measured by 18F-fluorodeoxyglucose (18F-FDG) uptake measured by 18F-FDG positron emission tomography/computed tomography (18F-FDG-PET/CT) in the bone marrow, spleen and thoracic aorta would improve in HIV+ patients following therapy with rosuvastatin compared to HIV+ patients not receiving a statin. Methods: HIV+ patients with a moderate Framingham risk score were enrolled in the study and randomized to treatment with rosuvastatin or usual treatment for 6 months. Patients were matched for age, sex, smoking status, Framingham risk score, duration of anti-retroviral therapy (ART) and type of ART. Fasting blood was collected and 18F-FDG-PET/CT imaging of bone marrow, spleen, and thoracic aorta was performed at baseline and 6 months. Results: Thirty-five HIV+ patients were enrolled in the study; 17 were randomized to treatment with rosuvastatin and 18 were randomized to the control group. There was a significant drop in the 18F-FDG bone marrow, spleen and thoracic aortic uptake in the statin-treated group compared to the control group (bone marrow: -10.3±16.9% versus 5.0±18.9%, p=0.0262; spleen: -9.8±20.3% versus 11.3±28.8%, p=0.0497; thoracic aorta: -8.6±24.5% versus 12.6±29.5%, p=0.0343). 18F-FDG changes over the study period are shown in Figure 1. Conclusions: The present study observed a significant decrease in 18F-FDG-PET uptake in the bone marrow, spleen, and thoracic aorta following treatment with rosuvastatin for 6 months in HIV+ patients. These data suggest a wide-range anti-inflammatory effect of rosuvastatin in HIV+ individuals with well-controlled infection on ART, ultimately resulting in decreased inflammatory activity in the arterial walls of these patients.

Management of cognitive difficulties is a significant unmet need for individuals with stroke. Incorporating multiple functions, including memory aids and communication tools, smartphones have potential to improve everyday cognitive function and independence in daily activities post-stroke. We aimed to investigate patterns of smartphone use, facilitators and barriers to use, and relationships between smartphone use and daily functioning. Twenty-nine participants with stroke and 29 comparison participants with no history of neurological conditions completed measures of smartphone use, objective and subjective cognitive function, mood and community integration. The majority of participants used smartphones, though the proportion of users was lower in the stroke group (62%) than the comparison group (86%). Older participants were less likely to use smartphones. Using apps that support memory was a main benefit of smartphone use post-stroke. In the stroke group, frequent users of memory apps had significantly fewer motor symptoms (d = 1.20), and higher productivity (d = 0.84). Stroke survivors identified difficulty learning how to use smartphones, but only one participant had assistance with this from a clinician. These results suggest that smartphones have potential as assistive technology post-stroke, however, support in using them is essential, particularly for older individuals with motor dysfunction.

Aims: Both inherited arrhythmogenic diseases (such as Brugada Syndrome) and heart failure are associated with reduced voltage-gated Na + current (I Na ) which promotes lethal arrhythmias and sudden deaths. We and others have shown that Wnt/β-catenin signaling (Wnt signaling), which is active in heart disease, inhibits I Na in rat and mouse cardiomyocytes. But whether Wnt signaling regulates I Na in human cardiomyocytes and represents a novel therapeutic target is unknown. This study aims to investigate if Wnt signaling inhibits I Na in human cardiomyocytes, to elucidate the underlying mechanisms, and to test if blocking Wnt signaling can rescue I Na in Brugada Syndrome patient cardiomyocytes. Methods and Results: Cardiomyocytes were differentiated from human induced pluripotent stem cells (hiPSC-CMs) that were derived from healthy volunteers or a Brugada Syndrome patient. Whole-cell patch-clamp technique was used for I Na measurement. Activation of Wnt signaling in healthy hiPSC-CMs led to a 69% reduction in I Na amplitude (peak current at -20 mV: -10.5±2.8 pA/pF, n=9 cells vs. control -33.6±4.1 pA/pF, n=6 cells, pSCN5A mRNA (encoding the pore-forming α subunit of I Na , Na v 1.5). In addition, Wnt signaling also reduced Na v 1.5 glycosylation causing a depolarizing shift of I Na activation curve (a mechanism found in human, but not in rodent, cardiomyocytes). Blocking Wnt signaling in Brugada hiPSC-CMs with shRNA-mediated β-catenin knockdown led to a 13-fold increase in I Na amplitude (pNa, blocking Wnt signaling also upregulated SCN5A mRNA and Na v 1.5 protein, without affecting expression of other cardiac ion channels in Brugada hiPSC-CMs. Conclusions: This study demonstrated Wnt-inhibition of human cardiac I Na and, using Brugada Syndrome as an example, demonstrated that blocking Wnt signaling is a novel therapeutic strategy to rescue I Na in heart disease.

2511 Background: CD73 is implicated in tumor resistance to checkpoint immunotherapy (CPI) and plays a critical role in adenosine-mediated immune suppression. Uliledlimab, a differentiated CD73 antibody, inhibits the adenosine pathway in a non-competitive and unique intra-dimer binding mode. Uliledlimab suppresses tumor growth when combined with a PD-(L)1 inhibitor in multiple pre-clinical models. Methods: This 3+3 dose-escalation phase 1 study (NCT03835949) evaluated safety, tolerability, PK, PD and preliminary efficacy in cancer patients. Uliledlimab was administered intravenously at doses of 5, 10 or 15 mg/kg weekly (QW) or 15 or 20 mg/kg every 3 weeks (Q3W) alone in the first cycle and in combination with atezolizumab (1,200 mg Q3W) starting on week 4. Soluble CD73 in serum and CD73 receptor occupancy (RO) in circulating CD19+ B cells were measured. Expression of PD-L1, CD73 and A2A receptor was analyzed in baseline tumor specimens (n = 14). Tumor responses were assessed by RECIST/iRECIST. Results: As of 17 January 2021, 20 patients with advanced solid tumors were enrolled (M:F 8:12; mean age = 64; median prior regimens = 3 (range 1-9)). Uliledlimab was well-tolerated with no dose limiting toxicity. The most common treatment-related adverse events were first dose infusion related reactions (65%, n = 13) most commonly comprising chills/rigors, nausea, and vomiting (Grade 1 or 2) that resolved in subsequent infusions. PK appears linear at doses ≥ 10 mg/kg and modelling indicated a mean derived effective half-life of ̃19 days. Soluble CD73 was undetectable and complete RO was achieved in all patients after the first dose at ≥ 10 mg/kg. Anti-drug antibody was detected in 3/20 patients (15%). Among 13 efficacy-evaluable patients dosed at ≥ 10 mg/kg, complete response (CR = 1) and partial response (PR = 2) were observed in 3 patients (ORR = 23%) together with 3 stable disease (SD) patients (DCR = 46%). One PD-(L)1 inhibitor naïve patient with clear cell ovarian cancer achieved CR at 10 mg/kg QW and remains on study after 12 months. Two patients with NSCLC dosed at 15 mg/kg QW and 20 mg/kg Q3W, respectively, achieved PR. One patient failed nivolumab and the other received no prior PD-(L)1 inhibitor treatment. CD73 was expressed on 78% (mean) of malignant cells from archival tumor specimens in responders compared to 23% in non-responders. Conclusions: Uliledlimab is safe and well tolerated up to 20 mg/kg Q3W and 15 mg/kg QW. Full saturation of circulating and cell-bound CD73 was achieved at doses ≥ 10 mg/kg. Uliledlimab exhibited evidence of clinical activity in both PD-(L)1 treatment naïve and refractory cancer patients with high archival tumor expression of CD73. The results of this phase 1 study encourage further clinical investigation to evaluate the efficacy of uliledlimab in the treatment of solid tumors. Clinical trial information: NCT03835949.

Background: Triple negative breast cancer (TNBC) is a heterogeneous disease with several molecular subtypes: basal-like1 (BL1), basal-like 2 (BL-2), mesenchymal(M), mesenchymal-stem-like(MSL), immune-modulatory(IM) and unclassified (UNC) Molecular evolution of TNBC through chemotherapy selection pressure is well recognized but poorly understood. This study was carried out to perform paired genomic analysis of TNBC comparing primary breast cancer with recurrent/refractory disease. Here we report the result of the first10 paired tissue analysis. Methods: 49 paired specimens were identified through an IRB-approved protocol via COH biorepository search (2002- 2015). miRNA and mRNA profiling of 22 samples were performed. The miRNA libraries were prepared and sequenced on Hiseq2500. Sequences were aligned to hg19 genome and miRNA expression levels were counted by in house built R scripts. Go and pathway annotation was performed using DAVID online tool. Affymetrix human Genechip 2.0st was used for mRNA expression profiling. Raw data were normalized and processed using Expression Console, and linear regression was performed using Limma to identify the differentially expressed genes between primary and recurrent/refractory TNBCs. Result: Through mRNA profiling, we identified several unique gene expression patterns comparing the paired TNBC. Significant mRNA expression alterations were observed in: cell cycle, DNA repair and adhesion. Using Vanderbilt TNBC sub-classification tool, we have identified “phenotype shift” between primary and recurrent TNBCs. Of the 8 paired specimen analyzed, 3 paired tissue remain in the same subclass (2 in IM, 1 in M). Phenotype shift observed in: 1 from BL1 to BL2, 1 from BL2 to BL1, 1 UNC to IM; 1 MSL to UNC; 1 from M to UNC. 15 up regulated and 13 down regulated miRNAs were identified. Most significantly differentially expressed miRNA (with more than 4 fold expression changes, P-value < 0.001) included: miR-206, miR-203, miR-144, miR-16-2, miR-15b, and miR-20b (un-regulated) and miR-10b, miR-125b and let-7c(down-regulated). These miRNA genes are involved in regulation of hormonal receptor signaling, cell cycle, proliferation and metastases. Statistically significant differentially expressed miRNAs identified from our TNBC patient cohort will be further validated using RT-PCR. Conclusion: A number of mRNA gene pathways and miRNAs showed differential expression between paired recurrent and primary TNBC tumor specimen. The underlying biology driven the phenotype shift is being studied. Further analysis to include a total of 49 paired TNBCs is currently underway. Contact information: Yuan Yuan MD PhD, Email: yuyuan@coh.org. Citation Format: Yuan Y, Li A, Yost S, Yuan Y-C, Chu P, Warden C, Wang J, Liu Z, Liang Y, Goldstein L, Wu X. Genomic analysis of molecular discordance of paired primary and recurrent triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-26.

The purpose of this study was to explore patient perceptions of primary care providers and their offices relative to their physician’s philosophy (medical degree [MD] vs doctorate in osteopathic medicine [DO]), specialty (internal medicine vs family medicine), US region, and gender (male vs female). Using the Healthgrades website, the average satisfaction rating for the physician, office parameters, and wait time were collected and analyzed for 1267 physicians. We found female doctors tended to have lower ratings in the Midwest, and staff friendliness of female physicians were rated lower in the northwest. In the northeast, male and female MDs were rated more highly than DOs. Wait times varied regionally, with northeast and northwest regions having the shortest wait times. Overall satisfaction was generally high for most physicians. Regional differences in perception of a physician based on gender or degree may have roots in local culture, including proximity to a DO school, comfort with female physicians, and expectations for waiting times.

Introduction: Unplanned 30 day readmission prediction models exist for general cancer population1. However, the models may not perform well for hematology patients as their readmission risk may be associated with a variety of population specific factors including labs and diagnoses that are not typically used in models for general population2. Including all lab and diagnostic elements in predictive models may not be practical due to large number of variables relative to number of observations. Embeddings, originated in the field of natural language processing, have the potential to represent medical concepts in low dimensional spaces3. In this study, we developed a machine learning model utilizing clinical embedding representations of ICD and LOINC codes to predict readmission risk with prospective validation. Methods: This is a single institutional study examining inpatient 30 day unplanned readmissions between Jan 2013 and Dec 2016 (n = 16361 total, n = 5685 in hematology). The overall readmission rate was 18% (24% for hematology). We used gradient boosted trees models (lightGBM) including several baseline factors: gender, age, length of stay, service line, number of admissions within 6 months, primary insurance, discharge disposition, ICU admission, year of admission, and average pain score at rest. In addition, we utilized publicly available clinical embeddings3 to generate 300 dimensional representations for ICD9 and LOINC codes in the patients' Electronic Medical Records. We considered diagnoses (ICD9s) within individual admission or within 6 months prior to admission, and lab tests (LOINCs) ordered during the admission. We used ten-fold cross-validation to identify optimal model hyper-parameters. We used records between Jan 2017 and Dec 2017 for prospective validation. To eliminate potential data leakage, we only considered new patients (n = 3785 total, n = 1424 in hematology), resulting in 17% overall readmission rate (22% for hematology). Results: In the prospective validation, the Area Under Receiver Operating Characteristic Curve (AUC) using baseline factors were 0.73 (average precision “AP” = 0.352) and 0.667 (AP = 0.347) for the overall and hematology populations, respectively. The AUC for hematology was notably lower. With the inclusion of ICD9 and LOINC embeddings, AUCs of 0.775 (AP = 0.44) and 0.722 (AP = 0.4) were obtained for overall and hematology populations, corresponding to 6% and 8% AUC improvements, respectively. Conclusions: This study found that readmission prediction performance for hematology patients is lower than the overall cancer population, and utilizing clinical embeddings improves the prediction performance. Given the higher readmission rates for hematology patients, this model shows potential to improve patient care and reduce associated costs, by predicting and preventing readmissions. 1 J Surg Oncol. 2018; 117:1113-1118. 2 JAMA Network Open. 2019;2(7):e196476. 3 AMIA Jt Summits Transl Sci Proc. 2016;41-50. Citation Format: Chi Wah Wong, Chen Chen, Lorenzo A. Rossi, Jerry Wang, Monga Abila, Janet Munu, Zahra Eftekhari. Improving 30 day readmission prediction for hematological cancer patients via clinical embeddings [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2089.

2040 Background: Existing models typically predict unplanned 30-day readmission for cancer patients at discharge1. Performing prediction dynamically during hospital stay may allow earlier intervention for high risk patients. In addition, readmission risk may be associated with the outcome of a variety of labs and diagnoses. Models including all those elements may not be practical due to large number of variables relative to number of samples. Embeddings have the potential to represent medical concepts in low dimensional spaces2. In this study, we developed a machine learning model utilizing embedding representations of ICD and LOINC codes to dynamically predict readmission risk. Methods: This is a single institutional study examining inpatient 30-day unplanned readmissions from Jan 2013 to Dec 2016 (n = 16361 total, n = 5685 in hematology). The readmission rate was 18% (24% for hematology). We used gradient boosted trees models with 10-fold cross validation and included baseline factors that are typically available shortly after admission: gender, age, service, admission count within 6 months, insurance, emergency admission, admission year, allogeneic or autologous stem cell transplant (hematology only). For dynamic factors, we randomly selected a timepoint (TP, median = 2.4 days) during each visit. We utilized publicly available clinical embeddings2 to generate 300 dimensional representations for ICD9s and LOINCs in the patients’ Electronic Medical Records. We considered diagnoses (ICD9) between 6 months prior to admission and TP, and lab tests (LOINC) ordered between admission time and TP. We used records from Jan 2017 to Dec 2017 for prospective validation (n = 3785 total, n = 1424 in hematology), with 17% readmission rate (22% for hematology). Results: Prospective validation Area Under Receiver Operating Characteristic Curve (AUC) using baseline factors were 0.72 (average precision “AP” = 0.33) and 0.65 (AP = 0.32) for overall and hematology populations, respectively. By including dynamic factors, we obtained AUCs of 0.74 (AP = 0.4) and 0.7 (AP = 0.39) for overall and hematology populations, corresponding to 3% and 8% AUC (21% and 22% AP) improvements, respectively. Conclusions: We found that dynamic readmission prediction utilizing clinical embeddings improves the prediction performance comparing with using baseline factors only. The model shows potential to improve patient care and reduce costs by predicting and preventing readmissions when the patient is still in the hospital. 1 J Surg Oncol 2018; 117:1113-1118. 2 AMIA Jt Summits Transl Sci Proc. 2016;41-50.

Fatalities from organophosphate (OP) insecticide result from both occupational and deliberate exposure; significantly impacting human health. Like nerve agents, insecticides are neurotoxins which target and inhibit acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with a pyridinium aldoxime e.g. pralidoxime, to reactivate the OP-inhibited AChE. However, commonly used oximes inefficiently cross the bloodbrain barrier and are rapidly cleared and their benefit is debated. Recent findings have demonstrated the ability of a novel zwitterionic, centrally acting, brain penetrating oxime (RS194B) to reverse severe symptoms and rapidly reactivate sarin-inhibited AChE in macaques, but it has not been tested following OP pesticide poisoning. In the present study, the symptoms following a lethal dose of inhaled paraoxon (100 ug/kg), were shown to mimic those in insecticide poisoned individuals and were also rapidly reversed in macaques by post-exposure IM administration of 80 mg/kg of RS194B. This occurred with a concomitant reactivation of AChE to 40–100% in < 1 hr and BChE (40% in 8 h). These findings will be used to develop a macaque model with RS194 B as a post-exposure treatment for insecticide poisoning and generate efficacy data for approval under the FDA Animal rule.

The Rehabilitation Act Amendments of 1992 mandate that state and federal public vocational rehabilitation extend efforts to further include “traditionally underserved” populations. Section 2 1 of the Rehabilitation Act Amendments specifies the Asian Pacific American population as an “underserved group, and other data indicate that they are the fastest growing minority population in the United States. Yet, current data related to rehabilitation and disability at the national level are unavailable for Asian Pacific Americans. This article uses data taken from the Hawaii vocational rehabilitation program to illustrate the need for Asian Pacific American data to be reported, not only in aggregate form, but also for each ethnic group within the aggregate. It proposes a policy change for the way data are gathered and collected for the Asian Pacific American population.

Introduction: Despite many advances in our understanding of breast cancer biology over the last decades, there are no therapies that can effectively prevent or treat metastatic cancer. During the growth of the primary tumor mass and its dissemination through the body during metastasis, cancer cells are exposed to a host of mechanical environments generated by the local matrix compliance, pressure and tension from tumor mass expansion, and fluidic shear stresses from interstitial and vascular fluid flow. These biophysical forces are emerging as powerful regulators of cancer growth, quiescence and metastasis; however, our understanding of the mechanisms of the biomechanical regulation of tumor biology remains very limited. The overall goal was to identify the role of mechanical forces in regulating key steps in tumor metastasis and progression. Results: We recently developed a mesofluidic system that allows the high throughput study of tumor cell interactions under flow. This device allows us to simulate the adhesion of cancer cells to endothelial cells under physiological flow conditions to model this step of the metastatic cascade and to apply shear forces to cells in a 96-well format. In addition, we have designed a system to apply mechanical stretch to cells in a high throughput format (576 wells simultaneously). Using these two systems, we examined the interplay between mechanical cues and the propensity of breast cancer cells to metastasize and undergo epithelial-to-mesenchymal transition (EMT). Mechanical strain increases circulating tumor cell adhesion to endothelial cells and extracellular matrix (ECM). We applied cyclic mechanical strain (5% maximal strain) to MDA-MB-231 and MCF-7 cancer cells for 24 hours and then performed adhesion assays of the cancer cells to activated and non-activated endothelial cells (ECS) and purified ECM proteins. We found that cyclic strain increased cancer cell adhesion to activated ECs in comparison to non-strained cells. In addition cancer cells exposed to mechanical strain adhered more to collagen I, laminin, and vitronectin, while they adhered less to collagen II and fibronectin in comparison to non-strained control cells. To determine which integrins were involved in the strain-induced change in adhesion, we treated the cells with a library of integrin inhibitors while applying strain for 24 hours. Cilengitide, P11, ATN-161, Bio 1211, and RGDS peptides reduced the adhesion of cancer cells back to the level of non-strained cancer cells, indicating the role of αvβ3, αvβ5, α5β1, and α4β1 integrins in the biophysical regulation of circulating tumor cell adhesion. Cyclic strain alters the ability of breast cancer cells to invade through an endothelial monolayer. We next used the high throughput system to apply multiple levels of strain to the cancer cell lines (0, 2.5, 5, 7.5, 10, 12.5, 15 and 17.5% strain). We then trypsinized the cells and measured their invasion in a Transwell assay with a confluent layer of endothelial cells cultured on a porous membrane. We found that the mid-level strains ranging from 7.5-15% strain decreased the invasiveness of the MDA-MB-231 cells compared to the non-strained control cells. For the MCF-7 cells, mechanical strain of 5% or higher led to decreased invasion through the endothelial layer. Mechanical forces alter signaling through the TGF-β and Yap/Taz pathways as well the expression of markers of epithelial-to-mesenchymal transition (EMT). We applied cyclic strain at multiple levels (2.5-17.5% strain) to breast cancer cells for 24 hours then immunostained for various signaling proteins. In MDA-MB-231 cells, we saw a significant decrease in the intensity of Smad2/3 and nuclear phospho-Smad2/3 at mid-range strains (5-12.5% strain) as well as an increase in the nuclear localization of Yap/Taz using fluorescent immunostaining. At high levels of strain (15 and 17.5% strain), there was increased nuclear p-Smad2/3 intensity. Consistent with these findings, we found increased Smad activity in the cells using a luciferase reporter assay. An analysis of EMT markers using PCR, western blotting and immunostaining showed a reduction in mesenchymal markers including α−SMA, Slug, ZEB, fibronectin and vimentin with mid-range mechanical strain levels. Conclusion: Together, our studies demonstrate that mechanical forces can profoundly alter the propensity of cancer cells to adhere and invade during metastasis. Moreover, the response to mechanical strain is dependent on the magnitude of the strain and cancer cell type. These effects are mediated, in part, through integrin interactions and the differential regulation of the TGF-β and Hippo pathways by biophysical forces. Citation Format: Adrianne Spencer, Jason Lee, Katerina Lee, Darshil Choksi, Jerry Wang, Christopher Spruell, Aaron Baker. Biophysical Regulation of Breast Cancer Metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A38.

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies.

Introduction: Hypogonadism and impaired fertility remain critical problems in transfusion dependent thalassemia (TDT) patients who develop iron overload. Pituitary iron toxicity and resultant gland shrinkage were shown to occur in these patients, starting even during their first decade of life. While MRI screening of iron load in the heart has been linked with cardiac function and has a direct effect on iron chelation practices, little is known on pituitary iron and anterior pituitary volume, as they relate to hormone secretion and reproductive potential. We investigated such an association in order to better understand the development of hypogonadism and infertility and its pathogenesis. Methods: A total of38TDTpatients, age 12-45 y, 20 females and 18 males were enrolled in this study. Relative pituitary iron (R2) and anterior volume was assessed by MRI. These MRI parameters were transformed to Z-scores and compared to published age- and sex-matched normal subjects. Laboratory assessment of gonadal function and predictors of fertility status was performed in patients grouped into adolescents (12-18 y), young adults (19-29 y) and adults (>29 y). LH, FSH were obtained in both genders. In females, Anti Mullerian Hormone (AMH, a predictor of ovarian antral follicle count) was tested, while in males inhibin B (a hormone correlating with sperm count) was obtained; both are a measure of fertility potential. Results: Among the 9 adolescent patients (12-18 y), 3 had delayed puberty. In this cohort, the anterior pituitary iron was moderately increased compared to normal (Z = 2.2 ) and it did not result in a decrease in the gland size (Z = 0.04), while in patients 19-29 years old, a higher R2 with reduced gland size (Z = 5.6 and -2.4, respectively) was noted. These patients had an associated high liver (LIC) and cardiac iron (T2*). Pituitary findings were similar in patients older than 29y (see table). Overall, pituitary iron did not statistically correlate with liver and cardiac iron, though iron has increased with age in all these organs. Pituitary volume, but not iron, correlated with LH level in patients >19 y (r=0.4, P=0.03). AMH levels in women decreased with age in the 3 groups: 5.8±5, 3.1±2.4 and 1.2±0.6 ng/mL, respectively. Inhibin B, in men, decreased as well (124± 60; 115±70; 85±50 pg/mL, respectively) Abstract 1048. Table 1Age (y)nanterior pit. volume (mm3)Z score (pit. vol.)R2 (s-1)Z score(R2)LIC(mg/g dry wt)cardiac T2*(ms)LH(U/L)FSH(U/L)16 ± 19512 ± 1570.0413.1±1.62.210.4 ± 7.030 ± 106.2±8.04.5±1.625 ± 318358 ± 127-2.416.4±2.85.622.8 ± 13.022 ± 104.4±3.04.0±3.034 ± 411392 ± 180-2.216.0±2.84.512.0 ± 5.520 ± 102.6±3.04.0±4.0 Normal range: LH (U/L): 2-10 (m), < 30 (f) ; FSH (U/L): 1.4-21 (m), 0.9-15 (f); AMH (ng/mL): 2.1-6 (f); Inhibin B (pg/mL): 25-325 (m). Discussion: These results suggest a significant reduction in pituitary size along with increased iron load in patients in their 20’s affecting reproductive potential, while there is less impact of pituitary iron on hormone secretion in the younger patients. Retrospective analysis of the patients 19-29 years old showed that the majority did not have delayed puberty or primary amenorrhea in their second decade of life, suggesting that their pituitary studies may have been similar to the younger age group in this study. The decline in pituitary size and function as patients’ age could represent decreased or inconsistent adherence to iron chelation, but may also be a result of prolonged and constant oxidative stress damage from labile plasma iron. Efforts to target this age group for closer screening, including monitoring pituitary gland size, along with intensified chelation, could help prevent the gland shrinkage and result in a higher fertility potential. Disclosures Paley: Novartis Pharma: Employment.

Continuous improvement in the life span of TM patients allows patients to address critical issues of specific organ function. Among these hypogonadotrophic-hypogonadism (HH) and infertility are common complications, having a significant adverse impact on quality of life (QOL). While pregnancies are reported in TM women, paternity is less common in TM men, with significant gaps in our understanding of the underlying pathogenesis of male reduced fertility. Growing evidence indicates that oxidative stress negatively affects sperm quality and function, playing a central role in certain types of male infertility. In addition, abnormal low trace elements, in particular zinc, are important causes for infertility. We hypothesized that deleterious effects of iron overload impair sperm quality beyond that of iron deposition in the pituitary and the resultant HH state, and thus aimed to evaluate potential mechanisms for infertility in TM men: Systemic and pituitary iron burden (liver iron concentration; LIC, pituitary iron by MRI, ferritin), reproductive hormones (LH, FSH) and semen fluid were measured in 7 TM men (median age 26, range 21-30 years). Semen fluid parameters included volume, sperm count, motility, DNA damage determined by DNA Fragmentation Index (DFI), concentration of glutathione (GSH), a major cellular antioxidant which plays a central role in the defense against oxidative stress, and that of pertinent metal/trace elements (iron, zinc, copper). Results were compared to normal reference and Pearson correlation analysis was performed to identify markers that best predict fertility potential and highlight pathogenesis.Sperm and semen plasma analysisLab resultsImagingPtVol. mlCount Mill/mlMotility %DFI %Zn mg/LFe mg/LGSH µmol/LLH/FSH mIU/mLFerritin ng/mLLICMRI R213.51787513141.41.189.54.9/7.96685.413.5± 0.222.500-54.42.732.20.3/0.9369625.216.4± 0.731.400-6.72.530.880.0/0.0428822.816.8± 1.442.314659.7163.02.218.661.6/2.710926.614.5± 0.951.51137---7.52.8/3.419248.4-64.594758.6120.407.54-37752016.7± 0.671.800-80.22.341.060.07/0.330002019.5± 1.2nl>2>20>50 Semen analysis showed that 5/7 (70%) had either low sperm count or complete azoospermia, and 4/7 exhibited reduced sperm motility (57%). The three patients with a measurable sperm count did not exhibit increased DNA fragmentation (normal DFI). Overall, patients had low seminal plasma GSH concentration, indicating increased oxidative stress. Seminal plasma had elevated iron concentration in 5/6 patients; zinc levels were low in 4/6, while copper was undetectable in all patients (nl 0.064 mg/L). Patients with highest pituitary iron (2,3,7) had the lowest LH/FSH levels and azoospermia, but pituitary iron (R2) did not correlate with LH/FSH, with sperm or systemic iron measures. Seminal fluid GSH (p=0.001), iron (p=0.06), and zinc (p=0.02) inversely correlated with sperm motility. Systemic iron status, as measured by LIC and ferritin, was inversely correlated with GSH (p=0.02; 0.04, respectively) and with zinc (p=0.04; 0.04, respectively). Our data suggest that increased free iron, iron-induced oxidative damage and low trace elements are associated with impaired semen integrity and may explain low fertility potential in TM men, beyond the expected effect of pituitary iron toxicity and HH. These abnormalities, however, did not result in sperm DNA damage, in a small analyzed sample size. Oxidative and metal profiles in seminal fluid may be a valuable tool to improve the reproductive evaluation of TM men. Anti-oxidant treatment will likely have an important role in increasing their reproductive potential. Disclosures: No relevant conflicts of interest to declare.