Schimke, Jill M.

e18097 Background: AB160 is a 160 nm nano-immunoconjugate consisting of nab-paclitaxel (NP) nanoparticles non-covalently coated with bevacizumab (BEV) for targeted delivery into tissues expressing high levels of vascular-endothelial growth factor (VEGF). Both taxanes (GOG-0129C, GOG-0126R, GOG-0127V) and BEV (GOG-0229E, AURELIA, GOG-0227C) have demonstrated clinical activity in previously treated metastatic endometrial cancer (EC), ovarian cancer (OC), and cervical cancer (CC), respectively. Methods: A 3+3 phase I trial was conducted in patients with EC, platinum-resistant OC and CC who had prior systemic treatment for metastatic disease to determine the maximum tolerated dose of AB160 administered intravenously on days 1, 8 and 15 of a 28-day cycle. The starting dose level (DL1) was NP at 125 mg/m2 with BEV at 50 mg/m2. There were 2 higher dose levels: DL2 (NP at 150 mg/m2 with BEV at 60 mg/m2) and DL3 (NP at 175 mg/m2 with BEV at 70 mg/m2). Dose limiting toxicities (DLT) included grade (G) 4 neutropenia or anemia, PLT < 25,000, serum creatinine ≥2 times baseline, G2-4 neurologic toxicity or G3-4 non-hematologic toxicities. Disease evaluations were conducted after every 2 treatment cycles using RECIST criteria. Patients were treated until disease progression or intolerability. Samples were collected for pharmacokinetic (PK) studies. Results: Nine women 41 – 74 years of age (median 57) have enrolled (5 with EC and 4 with OC); data are available for the first 8. No DLTs have been observed among the 3 women enrolled on DL1, 3 women on DL2, and 2 women on DL3. All 3 patients on DL3 continue on treatment. The other 6 patients have discontinued due to adverse reactions (3), progression (2), and patient choice (1). The median number of cycles administered is 6 (4-14). The most common severe (G3/4) toxicities include neutropenia (37.5%) and leukopenia (25%). There have been 5 partial responses (62.5%): 1 on DL1 and 2 each on DL2 and DL3. PK evaluation is pending. Conclusions: AB160 therapy is safe and demonstrates promising clinical activity in patients with previously treated metastatic gynecologic malignancies. Further clinical testing is being pursued in this patient population. Clinical trial information: NCT02020707.

e15184 Background: Clinical responses to anti-PD1 immunotherapy in patients with metastatic melanoma (MM) remain challenging to predict. This clinical heterogeneity is also reflected in the tumor-immune microenvironment among patients and within a single tumor lesion. With the emergence of multiplex imaging platforms, defining complex phenotypes at single cell resolution has become possible. Here, we sought to objectively quantify paracrine tumor-immune interactions contributing to the variable clinical responses observed in patients receiving anti-PD1 therapy. Methods: Excisional lymph node (LN) biopsies were obtained from treatment-naïve patients with MM who underwent subsequent anti-PD1 therapy. A single 5µm section of LN tissue was used to assess a 42 analyte panel by multiplex immunofluorescence. From 30 LN samples, 418 fields of view (FOVs) were selected resulting in 14,360 high-resolution images of 4 anatomical subregions: tumor core, tumor-immune interface, tumor infiltrate and adjacent immune stroma. Following image processing, we developed an adaptive classification for tumor-centric cellular neighborhoods (TCCN) to identify and quantify critical paracrine interactions within the tumor-immune microenvironment. Results: Stratification based on responsiveness to anti-PD1 therapy resulted in 4 complete responders (CR) and 12 non-responders (NR) at 12-week follow-up. From 126 FOVs, we defined the cellular composition of 197,865 TCCN across patients based on clinical response and LN subregions. Overall, the percentage of TCCN devoid of any T cells, B cells or macrophages was significantly higher in NR compared to CR irrespective of subregion. However, other markers differentiated TCCN based on subregion. Specifically in CR, tumor core regions were enriched for CD8 T cells, while enrichment for B cells and endothelial cells was demonstrated at the tumor-immune interface. Strikingly, tumor infiltrate regions demonstrate robust immune reactivity with enrichment for M1 polarized macrophages, NK cells and B cells in CR compared to NR. Complete data from the 30 patient cohort across 418 FOVs will be presented. Conclusions: Taken together, this data suggests cellular composition of TCCN across subregions of the LN is dynamic within a single metastatic site. In this small cohort, we introduce a formalized stratification to quantify and classify critical paracrine interactions within the immune-tumor microenvironment with the potential to inform clinical responsiveness to therapy.

Introduction Neutropenic fever following high-dose chemotherapy and autologous stem cell transplantation (ASCT) is a common (incidence 63-100%) and potentially life-threatening complication. Recommended time to antibiotic (TTA) administration is within 1 hr of fever onset with delays associated with significant morbidity, prolonged hospitalization, and mortality. Standard of care guidelines emphasize patient self-monitoring for fever, with instructions to seek immediate medical attention if body temperature (temp) reaches 100.4°F or higher. In this study, we evaluated if a novel wearable, continuous temp monitor, tPatch, could reliably estimate core body temp and detect fever in an outpatient setting following ASCT. Additionally, we gathered preliminary data to explore early detection and prediction of clinically relevant temp rise in this clinical setting. Methods Patients (N = 86) with hematologic malignancies (62% multiple myeloma) who underwent high-dose chemotherapy followed by ASCT at Mayo Clinic, MN were prospectively enrolled between June 2018 and March 2019. Patients (82% male) wore an axilla-placed tPatch continuously for 7 days in an outpatient setting during the post ASCT period and were asked to record self-measured oral temp in 3-4 hr intervals daily using a standardized thermometer after appropriate training . Patients followed standard of care procedures with daily clinic assessment of temp, blood counts, and vital signs. An optional patient questionnaire was given at end-of-study. A model was trained using both patient- and clinic-assessed oral temp measures to estimate core temp from 2 sensors on the tPatch device. Core temp estimates and trends were then compared to patient- and clinic-assessed measurements. Fever was defined as a temperature ≥100.4°F for at least 1 hr. Results When compared to all oral temp reads, the tPatch estimated core temp within 0.03 ± 0.7℉. Among the 86 patients, clinic-assessed fever incidence was 29.4% while tPatch-assessed incidence was 58.8%. Using all clinic-recorded temp readings as "ground truth," the sensitivity and specificity of the tPatch algorithm in detecting fevers were 88% and 86%, respectively, while patient self fever detection sensitivity was 62% and specificity 93%. With "fever episode" defined as a temp ≥100.4°F for at least 1 hr, tPatch detected 9.6 times the number of fever episodes vs. clinic reads. The average lead time of tPatch detection of clinic-recorded fevers was 3.7 hours. In 25% of all intervals between clinic temp readings, either tPatch or patients detected at least 1 fever episode. The tPatch was well-tolerated, the only adverse events reported were grade 1 skin irritation and discomfort in 4 (5%) patients. Of 65 patients who completed the survey, 95% reported the tPatch as "quite" or "somewhat" comfortable and 94% stated no difficulty in using the tPatch. Exploration of tPatch temp trends over various time intervals for use in fever prediction is ongoing. Conclusions Patient self-monitoring of temp has low sensitivity and is not feasible for long intervals of time (e.g., overnight). Continuous temperature monitoring by a wearable device overcomes these challenges and has the potential to improve early detection and consequently shorten time to antibiotic initiation. A follow-up randomized study is planned to assess the clinical benefits of continuous temp monitoring through patient and clinician alerts triggering early clinical intervention for febrile neutropenia. Figure Disclosures Vera-Aguilera: Verily Life Sciences: Research Funding. Haji-Abolhassani:Verily Life Sciences: Employment. Kulig:Verily Life Sciences: Employment. Heitz:Verily Life Sciences: Employment. Paludo:Verily Life Sciences: Research Funding; Verily Life Sciences: Research Funding; Celgene: Research Funding; Celgene: Research Funding. Ghoreyshi:Verily Life Sciences: Employment. Scheevel:Verily Life Sciences: Research Funding. Schimke:Verily Life Sciences: Research Funding. Markovic:Verily Life Sciences: Research Funding.

Rodent skeletal muscle mitochondrial DNA has been shown to be a potential site of oxidative damage during aging. Caloric restriction (CR) is reported to reduce oxidative stress and prolong life expectancy in rodents. Gene expression profiling and measurement of mitochondrial ATP production capacity were performed in skeletal muscle of male rats after feeding them either a control diet or calorie-restricted diet (60% of control diet) for 36 wk to determine the potential mechanism of the beneficial effects of CR. CR enhanced the transcripts of genes involved in reactive oxygen free radical scavenging function, tissue development, and energy metabolism while decreasing expression of those genes involved in signal transduction, stress response, and structural and contractile proteins. Real-time PCR measurments confirmed the changes in transcript levels of cytochrome- c oxidase III, superoxide dismutase (SOD)1, and SOD2 that were noted by the microarray approach. Mitochondrial ATP production and citrate synthase were unaltered by the dietary changes. We conclude that CR alters transcript levels of several genes in skeletal muscle and that mitochondrial function in skeletal muscle remains unaltered by the dietary intervention. Alterations in transcripts of many genes involved in reactive oxygen scavenging function may contribute to the increase in longevity reported with CR.

High-fat diets are reported to increase oxidative stress in a variety of tissues, whereas antioxidant supplementation prevents many diseases attributed to high-fat diet. Rodent skeletal muscle mitochondrial DNA has been shown to be a potential site of oxidative damage. We hypothesized that the effects of a high-fat diet on skeletal muscle DNA functions would be attenuated or partially reversed by antioxidant supplementation. Gene expression profiling and measurement of mitochondrial ATP production capacity were performed in skeletal muscle from male rats after feeding one of three diets (control, high-fat diet with or without antioxidants) for 36 wk. The high-fat diet altered transcript levels of 18 genes of 800 surveyed compared with the control-fed rats. Alterations included reduced expression of genes involved in free-radical scavenging and tissue development and increased expression of stress response and signal transduction genes. The magnitude of these alterations due to high-fat diet was reduced by antioxidant supplementation. Real-time PCR measurements confirmed the changes in transcript levels of cytochrome c oxidase subunit III and superoxide dismutase-1 and -2 noted by microarray approach. Mitochondrial ATP production was unaltered by dietary changes or antioxidant supplemention. It is concluded that the high-fat diet increases the transcription of genes involved in stress response but reduces those of free-radical scavenger enzymes, resulting in reduced DNA repair/metabolism (increased DNA damage). Antioxidants partially prevent these changes. Mitochondrial functions in skeletal muscle remain unaltered by the dietary intervention due to many adaptive changes in gene transcription.

Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Aktα and GYS3β phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3β when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) enhance insulin sensitivity and glucose homeostasis in rodent models of insulin resistance. These beneficial effects have been linked with anti-inflammatory properties, but emerging data suggest that the mechanisms may also converge on mitochondria. We evaluated the influence of dietary n-3 PUFAs on mitochondrial physiology and muscle lipid metabolites in the context of high-fat diet (HFD) in mice. Mice were fed control diets (10% fat), HFD (60% fat), or HFD with fish oil (HFD+FO, 3.4% kcal from n-3 PUFAs) for 10 wk. Body mass and fat mass increased similarly in HFD and HFD+FO, but n-3 PUFAs attenuated the glucose intolerance that developed with HFD and increased expression of genes that regulate glucose metabolism in skeletal muscle. Despite similar muscle triglyceride levels in HFD and HFD+FO, long-chain acyl-CoAs and ceramides were lower in the presence of fish oil. Mitochondrial abundance and oxidative capacity were similarly increased in HFD and HFD+FO compared with controls. Hydrogen peroxide production was similarly elevated in HFD and HFD+FO in isolated mitochondria but not in permeabilized muscle fibers, likely due to increased activity and expression of catalase. These results support a hypothesis that n-3 PUFAs protect glucose tolerance, in part by preventing the accumulation of bioactive lipid mediators that interfere with insulin action. Furthermore, the respiratory function of skeletal muscle mitochondria does not appear to be a major factor in sphingolipid accumulation, glucose intolerance, or the protective effects of n-3 PUFAs.

AbstractContext:Both training and normal body mass index are associated with high insulin sensitivity, but the mechanism may be different.Objective:The aim of the study was to examine whether lean trained humans may be protected from acute free fatty acid (FFA)-induced insulin resistance compared with lean sedentary humans.Design and Setting:We conducted an interventional trial using either a 6-h lipid (20% Intralipid at 90 ml/h) or glycerol (2.25 g/100 ml at 90 ml/h) infusion along with a concurrent hyperinsulinemic-euglycemic clamp and serial muscle biopsies (0, 120, 360 min) at a clinical research unit at the University of Minnesota.Patients or Participants:The study included lean endurance-trained (n = 14) and sedentary (n = 14) individuals matched for age, gender, and body mass index.Main Outcome Measures:We measured the decline in glucose infusion rate (GIR) during the hyperinsulinemic-euglycemic clamp.Results:The trained group had higher baseline mitochondrial DNA copy number, mRNA of cytochrome C oxidase subunit 3, and insulin sensitivity (as measured by GIR) compared with the sedentary group. When FFA was acutely elevated to the upper physiological range (0.6–0.7 mEq/liter) by lipid infusion, the GIR in both activity groups declined similarly compared with their respective glycerol controls, although insulin signaling, as measured by Ser 473 pAKT/AKT, remained comparable. Specific to the trained group, the stimulatory effect of hyperinsulinemia on mitochondrial mRNA levels during the glycerol infusion was absent during the lipid infusion.Conclusions:Elevated FFA had similar effects in reducing insulin sensitivity in trained and sedentary humans. In trained participants, this decline was associated with alterations in the skeletal muscle mitochondrial mRNA response to hyperinsulinemia.

AbstractContext:Hyperandrogenism and oxidative stress are related in polycystic ovary syndrome (PCOS), but it is unknown whether hyperandrogenemia can activate oxidative stress.Objective:The purpose of this study was to determine the effect of oral androgen administration on fasting and glucose-stimulated leukocytic reactive oxygen species (ROS) generation, reduced nicotinamide adenine dinucleotide phosphate oxidase p47phox subunit gene expression, and plasma thiobarbituric acid-reactive substances (TBARS) in lean healthy reproductive-age women.Participants, Design, and Setting:Sixteen lean healthy ovulatory reproductive-age women were treated with 130 mg dehydroepiandrosterone (DHEA) or placebo (n = 8 each) for 5 d in this randomized, controlled, double-blind study that was performed at an an academic medical center.Main Outcome Measures:Leukocytic ROS generation, p47phox gene expression, and plasma TBARS were quantified in the fasting state and 2 h after glucose ingestion, before and after treatment.Results:Before treatment, subjects receiving DHEA or placebo exhibited no differences in androgens or any prooxidant markers while fasting and after glucose ingestion. Compared with placebo, DHEA administration raised levels of testosterone, androstenedione, and DHEA-sulfate, increased the percent change in glucose-challenged p47phox RNA content, and increased the percent change in fasting and glucose-challenged ROS generation from mononuclear cells and polymorphonuclear cells, p47phox protein content, and plasma TBARS.Conclusion:Elevation of circulating androgens comparable to what is present in PCOS increases leukocytic ROS generation, p47phox gene expression, and plasma TBARS to promote oxidative stress in lean healthy reproductive-age women. Thus, hyperandrogenemia activates and sensitizes leukocytes to glucose in this population.