Decker, Paul A.

Abstract Background Glioblastoma (GBM) is an incurable disease with few approved therapeutic interventions. Radiation therapy (RT) and temozolomide (TMZ) remain the standards of care. The efficacy and optimal deployment schedule of the orally bioavailable small-molecule tumor checkpoint controller lisavanbulin alone, and in combination with, standards of care were assessed using a panel of IDH-wildtype GBM patient-derived xenografts. Methods Mice bearing intracranial tumors received lisavanbulin +/−RT +/−TMZ and followed for survival. Lisavanbulin concentrations in plasma and brain were determined by liquid chromatography with tandem mass spectrometry, while flow cytometry was used for cell cycle analysis. Results Lisavanbulin monotherapy showed significant benefit (P < .01) in 9 of 14 PDXs tested (median survival extension 9%-84%) and brain-to-plasma ratios of 1.3 and 1.6 at 2- and 6-hours postdose, respectively, validating previous data suggesting significant exposure in the brain. Prolonged lisavanbulin dosing from RT start until moribund was required for maximal benefit (GBM6: median survival lisavanbulin/RT 90 vs. RT alone 69 days, P = .0001; GBM150: lisavanbulin/RT 143 days vs. RT alone 73 days, P = .06). Similar observations were seen with RT/TMZ combinations (GBM39: RT/TMZ/lisavanbulin 502 days vs. RT/TMZ 249 days, P = .0001; GBM26: RT/TMZ/lisavanbulin 172 days vs. RT/TMZ 121 days, P = .04). Immunohistochemical analyses showed a significant increase in phospho-histone H3 with lisavanbulin treatment (P = .01). Conclusions Lisavanbulin demonstrated excellent brain penetration, significant extension of survival alone or in RT or RT/TMZ combinations, and was associated with mitotic arrest. These data provide a strong clinical rationale for testing lisavanbulin in combination with RT or RT/TMZ in GBM patients.

P-selectin is a cellular adhesion molecule that enhances procoagulant activity and activation of leukocyte integrins. P-selectin has been shown to be crucial in the development of atherosclerosis. Using a stratified random sample of 2406 participants in the Multi-Ethnic Study of Atherosclerosis (MESA) at Exam 2, we investigated the association of soluble P-selectin and cardiovascular disease. The Tobit model was used to investigate the association of P-selectin levels and coronary artery calcium (CAC) and Cox proportional hazards regression was used to assess the association between protein level and incident cardiovascular disease (CVD) events (myocardial infarction, stroke, angina, or other atherosclerotic death). Mean levels of soluble P-selectin differed significantly by ethnicity; 46±18 ng/mL African Americans, 58±20 ng/mL Caucasians, 43±15 ng/mL Chinese, and 61±20 ng/mL Hispanics (p<0.0001). CAC was present in 46% of African Americans, 61% of Caucasians, 52% of Chinese, and 53% of Hispanics. P-selectin levels were significantly associated with CAC in Caucasians (p < 0.001) and African Americans (p = 0.025), see Table. This association was robust to adjustment for traditional risk factors and a significant interaction of P-selectin and ethnicity was observed (p= 0.02). Over an average of 6.5 years of follow-up there were 111 incident CVD events after MESA exam 2, 26 in African Americans, 33 in Caucasians, 16 in Chinese, and 36 in Hispanics. The risk of CVD events increased 34% per standard deviation increase in P-selectin levels only in Caucasians (p = 0.007) and this finding was consistent after adjustment for CVD risk factors (HR = 1.30, p = 0.017). We have shown in this large cohort that mean circulating P-selectin levels differed by ethnicity. Despite similar percentages of subjects with CAC in each ethnic group, P-selectin levels were significantly associated with CAC only in African Americans and Caucasians. Furthermore, in Caucasians only, P-selectin levels were associated with incident CVD events. Association of P-selectin, per SD (20 ng/mL) for the population and cardiovascular disease Outcome African Americans n = 579 Caucasians n = 620 Chinese n = 602 Hispanics n = 605 CAC, Agatston Score Beta (S.E.) P-value Beta (S.E.) P-value Beta (S.E.) P-value Beta (S.E.) P-value Model 1 85.9 (38.4) 0.025 99.4 (28.7) <0.001 8.2 (33.3) 0.81 32.1 (27.3) 0.24 Model 2 79.3 (39.8) 0.046 94.7 (29.1) 0.001 −19.5 (33.6) 0.56 27.8 (27.6) 0.31 CVD Events HR (CI) HR (CI) HR (CI) HR (CI) Model 1 1.08 (0.75, 1.57) 0.67 1.34 (1.08, 1.65) 0.007 1.09 (0.57, 2.08) 0.80 0.99 (0.71, 1.39) 0.97 Model 2 1.10 (0.76, 1.58) 0.61 1.30 (1.05, 1.62) 0.017 0.82 (0.38, 1.78) 0.62 1.00 (0.71, 1.43) 0.98 Model 1 = age and sex ; Model 2 = age, sex, BMI, smoking status, LDL, HDL, triglycerides, hypertension, diabetes

Introduction: Increased levels of hepatocyte growth factor (HGF), active in cell growth, motility, and morphogenesis, are associated with the presence of obesity, poor metabolic health, and cardiovascular disease. Hypothesis: We assessed the hypothesis that higher baseline levels of HGF will be associated with increased risk of diabetes. Methods: We examined the association between HGF and incident diabetes in MESA, including 5395 men and women 45-84 years of age at enrollment (2000-02). Fasting serum HGF was measured at baseline and on a subsample of participants at exam 2 (n = 1915). From 2000-11, incidence of diabetes was ascertained over 4 follow-up examinations, determined by new use of insulin or oral hypoglycemic medication or fasting glucose ≥ 126 mg/dL. Cox regression was used to estimate hazard ratios (HR) for incident diabetes according to 1 standard deviation unit (SDU) of HGF (1 SDU =256 pg/mL), before and after adjustment for age, sex, race/ethnicity, education, study center, smoking status, alcohol consumption, BMI, WC, fasting glucose and insulin, CRP, and IL-6 levels. Similarly, hazard ratios for incident diabetes were estimated according to change in HGF levels from exam 1 to exam 2 in the subsample. Results: At baseline, older age, male sex, current smoking, and higher body mass index (BMI), waist circumference (WC), fasting glucose and insulin, C-reactive protein (CRP) and interleukin-6 (IL-6) levels were all associated with higher levels of HGF, while greater education and physical activity were associated with lower serum HGF. Incidence of diabetes in this analytic sample was 12% (n cases = 670). Per 1 SDU increase in baseline HGF level, unadjusted risk for diabetes increased 1.46 fold (95% CI=1.37, 1.56). After adjustment, diabetes risk per 1 SDU increase in HGF was attenuated but remained significantly increased (HR=1.22; 95% CI=1.12, 1.32). No association was found between change in HGF level between exam 1 and exam 2 and incidence of diabetes. There was no evidence of effect modification by race/ethnicity for either analysis. Conclusion: In conclusion, in this ethnically diverse U.S. adult population, higher levels of serum HGF were independently associated with increased incidence of diabetes.

Background: Given the global burden of peripheral arterial disease (PAD) and the associated morbidity, continuing investigation of potential biomarkers as therapeutic targets for preventing PAD is warranted. The association of P-selectin, an endothelial cell adhesion molecule, with the ankle brachial index (ABI) and PAD is not well established. Thus, we examined the association of circulating P-selectin with prevalent and incident PAD, the ABI, and change in the ABI. Methods: The Multi-Ethnic Study of Atherosclerosis (MESA) is a prospective population-based cohort including 6814 non-Hispanic white, African American, Hispanic and Chinese men and women aged 45-84 at baseline (2000-02). For this analysis, 5700 participants with both P-selectin at Exam 2 (2002-04) and the ABI at Exam 3 (2004-05) were available, with change in ABI from Exam 3 to Exam 5 (2010-12) available on 4276 participants. P-selectin was modeled per standard deviation (SD), while prevalent and incident PAD were defined as ABI ≤ 0.90 after excluding those with ABI>1.4. Linear regression was used for ABI, growth curve models for change in ABI, and logistic regression for prevalent and incident PAD. Results: In models adjusted for demographics, lifestyle factors, comorbidities, blood pressure, lipids, fasting glucose, c-reactive protein (CRP), interleukin-6 (IL-6), D-dimer, and fibrinogen, each SD (13 ng/mL) higher P-selectin was significantly associated with 0.007 lower ABI (95% CI ((-0.011, -0.004)), p<0.001), and an average change in the ABI of -0.006 ((-0.010, -0.003, p<0.001). P-selectin was also significantly associated with a 1.14-fold greater odds of prevalent PAD ((1.00, 1.28), p=0.04), and a 1.30-fold greater odds of incident PAD ((1.10, 1.55), p<0.001). There were no significant interactions of p-selectin by race/ethnicity or gender. Addition of P-selectin to models containing traditional PAD risk factors as well as CRP, IL-6, D-dimer and fibrinogen did not improve the c-statistic or net reclassification for incident PAD. Conclusions: P-selectin appears to be strongly associated with the development of PAD. However, further research is needed in population-based studies to confirm prospective associations of p-selectin with incident PAD and change in the ABI.

Incident hypertension varies across races/ethnicities. Mechanisms implicated in the pathogenesis are endothelial dysfunction with reduction of nitric oxide and alterations of the Renin-Angiotensin-Aldosterone System (RAAS). Stromal Derived Factor 1 (SDF1) and Transforming Growth Factor β1 (TGFβ1) are essential in these processes. SDF1 is a chemotactic protein that regulates migration of endothelial progenitor cells from the bone marrow to the arterial wall to repair endothelial damage; TGFβ1 promotes extra-cellular matrix deposition within arterial vessels, inhibits nitric oxide production and stimulates renin release. In order to investigate the association of circulating levels of these proteins with incident hypertension across different ethnicities, an ethnic stratified random sample of 2,880 participants from the Multi-Ethnic Study of Atherosclerosis (MESA) was used. Of those, 988 participants had available plasma samples and were without hypertension, defined as blood pressure ≥ 140/90 mmHg, or current use of antihypertensive medications, at baseline. During a median follow up of 8.5 years, 388 new cases of hypertension (101 in non-Hispanic whites, 94 in Chinese, 101 in African Americans and 92 in Hispanics) were observed. Time to hypertension incidence was estimated using Cox proportional hazards regression. Hazard Ratios were adjusted for age, sex, diabetes, LDL, HDL, triglycerides, BMI, smoking status and current alcohol use. The race/ethnicity by protein interaction p-values were 0.055 and 0.066 for TGFβ1 and SDF1 respectively, suggesting heterogeneity across races/ethnicities. In Chinese, a difference of one standard deviation of plasma TGFβ1 was associated with a 22% increase in incident hypertension (HR (95% CI) 1.22 (1.02, 1.47), p-value=0.03); and a difference of one standard deviation of plasma SDF1 was associated with a 31% decrease in incident hypertension (HR (95% CI) 0.69 (0.52, 0.91), p-value 0.008). No significant association was found between these proteins and incident hypertension among non-Hispanic whites, African or Hispanic Americans. In conclusion, soluble markers of endothelial integrity and of RAAS activation were associated with the incidence of hypertension among Chinese Americans. These results suggest heterogeneity in the pathophysiology of hypertension according to race/ethnicity.

L-selectin is an adhesion molecule constitutively expressed on leukocytes and mediating their interaction with the endothelium. As a member of the selectin family, it plays a crucial role in leukocyte adhesion to and migration through the endothelial wall during inflammation. A protective role of L-selectin in early atherosclerosis was demonstrated in animal models. Previous studies assessing the relationship of this protein with cardiovascular disease were inconsistent. Lower serum L-selectin levels were found in patients with coronary artery disease (CHD) and peripheral artery disease (PAD) compared to controls. In contrast, plasma levels of L-selectin were increased in patients with CHD and PAD. These inconsistencies might be due to an interaction of soluble L-selectin with clotting factors or anticoagulants present in plasma but not in serum. Also, the small number and Caucasian predominance of patients included in previous studies represent limitations that need to be addressed using a rigorous, population-based approach. Herein, we seek to investigate the association of serum L-selectin with subclinical outcomes in a large, ethnically heterogeneous population. In a randomly selected subset of 720 subjects for each of the four ethnicities included in the Multi-Ethnic Study of Atherosclerosis (MESA), serum samples were available for 2403 individuals (579 African Americans, 620 Caucasians, 600 Chinese and 604 Hispanics). Serum L-selectin levels and intima-media thickness (IMT) were measured at exam 2 and CAC was measured at exam 2 or 3 during the cohort follow up. Regression analysis and the Tobit model were used to assess the association of L-selectin levels with IMT and CAC respectively. The mean age was 63±10 years and 47% were men. The mean±SD protein levels were significantly different across ethnicities (867±192 ng/mL in African Americans, 956±215 ng/mL in Caucasians, 834±174 ng/mL in Chinese and 904±195 ng/mL in Hispanics; p<0.0001). In the univariate analysis, age was inversely correlated to the protein levels; in addition, levels were lower in males, regardless of ethnicity and in smokers, except among Hispanics. L-selectin was associated with modestly lower CAC only among Chinese (p=0.030), the association persisted after adjustment for traditional risk factors (p=0.046). No significant association was demontrated between the protein levels and IMT. In conclusion, in this large diverse population, we found that serum L-selectin was modestly associated to measures of subclinical atherosclerosis in Chinese but not in African, European or Hispanic Americans.

Pulse pressure (PP), defined as the difference between systolic and diastolic blood pressure, is considered a marker of large artery stiffness and has been shown to independently predict cardiovascular disease. Differences among ethnicities with regard to PP have been previously described, yet the underlying biological mechanisms are poorly understood. In addition, biomarkers of inflammation and matrix remodeling have been shown to be involved in the development of arterial stiffness. The aim of this study was to investigate the role of adhesion pathway components in the development of arterial stiffness measured by PP among four ethnicities. E-cadherin, HGF, L-selectin, P-selectin, 6-Ckine, MMP1, MMP2, RANTES, SDF1, SLPI, TGFβ1, TIMP2 and VCAM were measured in a random sample of 2402 participants (579 African Americans, 619 Caucasians, 600 Chinese and 604 Hispanics) in the Multi-Ethnic Study of Atherosclerosis (MESA) study at Exam 2. PP was measured concurrently and its association with each adhesion protein level was assessed via linear regression in each ethnicity. All analyses were adjusted for age, sex, and traditional cardiovascular risk factors. Mean PP was significantly different among ethnicities: 55.9±18.0 mmHg in African Americans, 52.1± 16.1 mmHg in Caucasians, 51.4±16.4 mmHg in Chinese, and 55.0±17.5 in Hispanics, p<0.001. MMP2 was positively associated with PP among African Americans (β=2.3 (0.6), p<0.001) and Caucasians (β=1.7 (0.5), p=0.001); TIMP2 among Caucasians (β=1.5 (0.5), p=0.002) and to a lesser extent among African Americans (β=1.2 (0.6) p=0.050) and Chinese (β=1.3 (0.6), p=0.034). SDF1 was associated to PP in African Americans (β=1.4 (0.6), p=0.017) and in Chinese (β=1.1 (0.5), p=0.042); finally an association existed between TGFβ1 and PP in C (β=1.1 (0.5), p=0.025). None of the studied proteins was associated with PP in Hispanics. In conclusion, components of the adhesion pathway were correlated to PP in this large multi-ethnic cohort, possibly giving new insight on the pathophysiology of arterial stiffness. In addition, heterogeneity by ethnicity suggests different mechanisms in the development of arterial stiffness.

Abstract Background Appropriately designed preclinical patient-derived xenograft (PDX) experiments are important to accurately inform human clinical trials. There is little experimental design guidance regarding choosing the number of PDX lines to study, and the number of mice within each PDX line. Methods Retrospective data from IDH-wildtype glioblastoma preclinical experiments evaluating a uniform regimen of fractionated radiation (RT), temozolomide (TMZ) chemotherapy, and concurrent RT/TMZ across 27 PDX lines were used to evaluate experimental designs and empirically estimate statistical power for ANOVA and Cox regression. Results Increasing the number of PDX lines resulted in more precise and reproducible estimates of effect size. To achieve 80% statistical power using ANOVA, experiments using a single PDX line required subsampling of 6 mice per PDX for each treatment group to detect a difference in survival of 135 days, and 9 mice per PDX to detect a difference of 100 days. Alternatively, a design that used 10 PDX lines had greater than 80% power to detect a difference of 135 days with a single mouse per PDX per treatment group, a difference of 100 days with 2 mice per PDX per treatment, and 35 days with more than 10 mice per PDX per treatment. Power for Cox regression was slightly smaller than ANOVA for very small experiments regardless of effect size and slightly higher than ANOVA for detecting a smaller effect size of 35 days difference in survival for moderate-to-large experiments. Conclusions Experimental designs using few mice across many PDX lines can provide robust results and account for inter-tumor variability.

Abstract Background Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important. Methods PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH, and phosphoproteomics. Results Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment. Conclusions Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.

Background: Prevention strategies for Alzheimer’s disease and Alzheimer’s disease related dementias (AD/ADRDs) are urgently needed for reducing incidence. Intra-patient variability in lipid levels is a potentially modifiable risk factor for incident AD/ADRD. Although regular lipid measurements are a part of common clinical practice and longitudinal data routinely available in electronic health records (EHR), research examining this association between AD/ADRD and lipid variability across multiple lipid types remains scarce. Methods: All residents living in Olmsted County, MN on 1/1/2006 age ≥60 years without an AD/ADRD diagnosis were identified using Centers for Medicare & Medicaid Services diagnostic codes. Persons with ≥3 lipid measurements (total cholesterol or triglycerides) in the 5 years prior to index date were retained. Lipid variability was quantified using variability independent of the mean (VIM). Models were adjusted for traditional risk factors. Associations between lipid variability quintiles and incident AD/ADRD were assessed using Cox proportional hazards regression. Multiple imputation was used for missing covariates. Participants were followed through 2018 for incident AD/ADRD. Results: The final analysis included data on 11,551 participants with total cholesterol and 11,502 participants with triglycerides. Participants had a mean age of 71 (range 60-99) years, and were primarily white (96%). Females (54%) were also slightly more frequent than males. Median follow up was 12.9 years (range: 0-13). Participants in the highest quintile of variability for total cholesterol and triglycerides had a 20% increased risk of incident AD/ADRD. Similar results were found in the subset with complete covariate data. Conclusion: In a large EHR derived cohort, persons in the highest quintile of lipid variation had an increased risk of incident AD/ADRD. Further studies to identify the mechanisms behind this risk factor and replication of these results across a more diverse population are needed.