Mayo Clinic Center Research Articles

Inhibition of the Pentose Phosphate Pathway in Cytotoxic T Lymphocytes Preserves Neuronal Function and Ameliorates Neuroinflammatory Disease

Abstract In diseases such as multiple sclerosis and autoimmune encephalitides, activated CD8+ cytotoxic T cells infiltrate the brain and are found in clonally expanded clusters that outnumber CD4+ cells in progressive stages of disease. At present, the pathophysiological role of these cells is poorly understood and therapeutic strategies for preventing or ameliorating autoinflammatory disease progression are limited. T cells exhibit profound metabolic reprogramming that facilitates the energetic and proliferative demands of effector T cells. Key metabolic pathways in this reprogramming include upregulation of glycolysis and the pentose phosphate pathway (PPP). Here we show that 1) pharmacologic inhibition of the PPP suppresses effector phenotype acquisition and proinflammatory cytokine secretion by CD8+ T cells; 2) engagement of the PPP is enhanced by pharmacologic Nrf2 activation, implicating reactive oxygen species-induced activation as a transcriptional regulator of PPP-dependent immune activation; 3) Nrf2-driven PPP flux promotes the acquisition of specific CD8+ effector phenotypes; 4) limiting the metabolic flux of glucose-derived metabolites through the PPP prevents CD8+ T cell-mediated injury of neurons expressing cognate antigen and preserves their electrophysiological function; and 5) pharmacologic inhibition of the PPP in B6 mice at symptom onset is sufficient to delay and ameliorate motor disability in the MOG EAE preclinical model of MS. Overall, these results underscore the potential of immunometabolic therapies to limit T cell mediated injury in autoinflammatory and autoimmune diseases across organ systems including the prevention of neurologic deficits in patients with T cell driven neurologic disease. This work was supported by the NIH (R01NS115126 to CLH; T32GM065841 to EMG), the Mayo Clinic Graduate School of Biomedical Sciences, the Mayo Clinic Center for MS and Autoimmune Neurology, and generous donations from Eugene and Marcia Applebaum, Donald J. and Frances I. Herdrich, Bob and Loralee Marvin, and Rob and Debbie Mudge.

ST8Sia6 expression in beta cells mitigates onset of autoimmune diabetes in the murine NOD model

Abstract In this study, we harnessed immunomodulatory Siglec-sialic acid interactions to mitigate autoimmune rejection of beta cells in Type 1 Diabetes (T1D). Mice engineered to express the sialyltransferase ST8Sia6 in beta cells (βST mice) were backcrossed to the non-obese diabetic mouse model of T1D and followed for disease; 60% of female littermates spontaneously developed diabetes by 300 days, compared to minimal occurrence (5%) in female βST mice. Histologic analysis of pancreas sections from non-diabetic βST and littermate mice suggested a similar degree of immune infiltration at 4 and 8 weeks of age, but the severity of insulitis was drastically reduced in βST mice beyond 20 weeks of age as compared to littermates. IHC for various immune populations suggested no difference in composition of insulitis at 8 weeks between βST and littermate mice. ELISA for serum anti-insulin antibodies demonstrated no difference in concentration between female βST and littermate mice. After female euglycemic βST and littermate mice at 16 weeks of age were challenged with anti-PD-L1 monoclonal antibodies, both groups resulted in similarly rapid induction of disease, suggesting that ST8Sia6 dependent protection occurs peripherally. Shut-off of ST8Sia6 expression in euglycemic βST mice at 20 weeks of age did not abrogate protection from disease, supporting persistent immune tolerance beyond this time. Flow analysis of islet infiltrating immune cells at 300 days confirmed reduced number of infiltrating immune cells in βST mice. Thus, induced expression of ST8Sia6 has been shown to mitigate autoimmune attack in the NOD mouse and presents a novel path forward for improving the success of curative islet transplant into T1D patients. Supported by funding from the Mayo Clinic (Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic MSTP) and the NIH (5R21AI135858-02, 1F30DK127564). Supported by funding from the Mayo Clinic (Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic MSTP) and the NIH (5R21AI135858-02, 1F30DK127564).

Enhanced anti-tumor immunity in ST8Sia6 knockout mice

Abstract Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells, which recognize specific sialic acid modifications on cell surface glycans. Sialyltransferases are the enzymes that catalyzing the addition of sialic acids onto glycoproteins and glycolipids. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6 on O-linked glycoproteins and functions to inhibit activation of innate immune cells. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered macrophage polarization and increased tumor growth through engagement of ligands generated by ST8Sia6 with Siglec-E. Here, we examine the role of ST8Sia6 on immune cells in regulating anti-tumor immunity. The loss of ST8Sia6 in tumor bearing mice reduced tumor growth, increased survival, and altered the immune response as compared to ST8Sia6 sufficient tumor bearing mice. Using MC38, B16, and B16-OVA flank tumor mouse models, tumor growth was decreased in ST8Sia6 KO mice as compared to C57Bl/6 mice. Within the tumor microenvironment of tumors present in ST8Sia6 KO mice compared to tumors present in C57Bl/6 mice, macrophages and dendritic cells altered their phenotype towards enhanced immune activation. Tregs from tumors in ST8Sia6 KO mice also displayed an increase in RORγt and a reduction in TNFRII, suggesting a possible shift to a less suppressive Treg. These alterations in the immune response to tumors were due to the loss of ST8Sia6 in macrophages and dendritic cells and not T-cells. Thus, ST8Sia6 generates ligands for Siglecs that dampen anti-tumor immunity. Supported by grants from NIH (R01CA243545, 2T32AI007425) and Mayo Clinic Center of Biomedical Discovery

Lessons Learned from Clinical and Translational Science Faculty and Student Survey as COVID-19 Pandemic Continues to Shift Education Online

IntroductionAs the pandemic continues with new variants emerging, faculty and students require support with education's rapid shift to the virtual space. The Mayo Clinic Center for Clinical and Translational Science curriculum team works closely with faculty to support a smooth transition to offering graduate courses in a virtual learning environment. The aim of the present project was to explore faculty and student perceptions of these remote learning strategies to gain an understanding of the innovations required to improve future educational offerings.MethodsAll faculty and learners involved in nine Clinical and Translational Science courses in spring 2020 were invited to participate in a web-based questionnaire. Quantitative analysis was performed on closed-ended items, including 5-point Likert-scale questions used to assess the range of views. Qualitative free-text responses were independently analyzed for repetitive themes and summarized. Additionally, comparisons of faculty and course evaluations and student grade point averages (GPAs) from the in-person courses and their subsequent virtual course offerings were considered.ResultsSurvey results indicated several positive impacts with moving courses into the virtual environment, including increased accessibility as well as more student-centered education. Learners joining from sites outside of the originating campus were especially grateful for the virtual classroom because they felt newly integrated within classes. Faculty and course evaluations, as well as student GPAs, remained consistent.ConclusionNew COVID-19 variants continue to shift education online, and innovative ideas are required to further improve future virtual course offerings. Increased engagement is warranted, both from faculty to incorporate activities designed specifically for a virtual classroom, and from students to increase participation by activating their microphones and webcams. Greater opportunities for global involvement and connectedness arise. Finally, this project advocates for adequate eLearning staffing to support quality online education as the need for pedagogical and technical provision continues.

Classical and Non-Classical Phenotypes of Erdheim-Chester Disease: Correlating Clinical, Radiographic, and Genotypic Findings

▪Background: Erdheim-Chester disease (ECD) is characterized by multi-organ infiltration of clonal histiocytes bearing activating mutations predominantly in the MAPK pathway. The diagnosis of ECD is clinico-pathologic; histopathologic findings alone are often non-specific. Characteristic pathognomonic finding of ECD is the symmetric osteosclerosis of the distal femur and proximal tibia/fibula, seen in >90% of cases, and referred to herein as classic ECD (C-ECD). There is a paucity of data on the phenotypic and mutational differences between C-ECD and non-classic ECD (NC-ECD). Determining phenotypic patterns may allow for earlier suspicion and diagnosis.Methods: Patients who met the revised ECD criteria proposed by Haroche J et al (Blood 2020;135:1311-1318) and had full body imaging that included the lower legs (18-FDG-PET/CT or CT/bone scan) were included. ECD diagnosis was made when >1 major criteria plus >1 minor criteria were present. Major criteria: 1) symmetric meta-diaphyseal osteosclerosis in legs; 2) “hairy kidneys”; “coated aorta”, right atrial pseudotumor, xanthelasma, exophthalmos; or osteosclerosis of paranasal sinuses. Minor criteria: 1) histologic finding of typical foamy histiocytes (CD68+/CD163+/CD1a-) associated with fibrosis; 2) mutation/gene fusion of BRAF, CSF1R, or MAPK/PI3K pathways. We compared the organ involvement and BRAF V600E mutational status between C-ECD and NC-ECD.Results: A total of 105 patients were included. The median age at diagnosis was 57 years (range, 38-81) and most were males (62%). Majority had 18-FDG-PET/CT (83%) and BRAF V600E testing (65%). The main organ systems involved were skeletal (83%), renal (64%), adrenal (44%), and pulmonary (42%). Central diabetes insipidus (DI), “hairy kidneys”, and “coated aorta” were present in 27 (26%), 54 (51%), and 44 (42%) patients, respectively. Among those tested, BRAF V600E mutation was found in 48/67 (72%) by immunohistochemistry. In our cohort, most patients (n=87. 83%) had C-ECD. NC-ECD had significantly lower number of organs/systems involved compared with C-ECD (median 3 vs 6, p=0.002). C-ECD had significantly higher rates of involvement of paranasal sinuses (51%/7%, p=0.002), DI (26%/0%, p=0.02), and similar rates of lung (44%/43%), cardiac (34%/14%), and skin (14%14%) involvement when compared to NC-ECD. BRAF V600E was significantly more common in C-ECD (88%/30%, p=0.004). Thirty-nine (37%) patients underwent next generation sequencing, of whom 33 (31%) had successful testing. In C-ECD, 3 patients had mutations other than BRAF V600E, these included: NRAS, MAP2K1, and MEF2C-FLT3 fusion. In NC-ECD, 5 patients had mutations other than BRAF V600E, these included: MAP2K1, KRAS, and NF1.Conclusions: Our study suggests distinct differences in clinical presentation and molecular findings exist between C-ECD and NC-ECD. C-ECD has a higher degree of organ involvement and harbor BRAF V600E more frequently than NC-ECD. Further analysis of histopathologic findings and outcomes in this cohort may provide insights into these ECD subsets that can optimize future management of this disease.Figure 1: Sites of involvement of classical Erdheim-Chester Disease (C-ECD) versus non-classical Erdheim-Chester Disease (NC-ECD) [Display omitted] DisclosuresVassallo: Bristol-Myers-Squibb: Research Funding; Sun Pharma.: Research Funding; Pfizer: Research Funding. Tobin: Mayo Clinic Center for MS and Autoimmune Neurology: Research Funding; Mallinckrodt Pharmaceuticals: Research Funding; National Institutes of Health: Research Funding. Bennani: Verastem: Other: Advisory Board; Purdue Pharma: Other: Advisory Board; Vividion: Consultancy, Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board.

Efficacy of Cobimetinib in Rosai-Dorfman Disease

▪INTRODUCTIONRosai-Dorfman disease (RDD) is a rare histiocytic disorder associated with histiocytes expressing high level of macrophage colony stimulating factor receptor (c-fms). Mutations in mitogen-activated protein kinase (MAPK) pathway have been reported in RDD, indicating an underlying clonal process in some patients. Aberrant activation of MAPK pathway can occur due to mutations or high c-fms expression. Data on the use of MAPK/extracellular-signal-related kinase (ERK)-inhibitor therapy in RDD are very limited. Cobimetinib is a MEK inhibitor, which is listed in the National Comprehensive Cancer Network Compendium ® for the treatment of RDD. We present our retrospective experience of cobimetinib treatment in a large series of patients with RDD.METHODSFollowing approval of the institutional reviewed board, patients (pts) with RDD who were consecutively seen at Mayo Clinic and treated with cobimetinib between 2013-2021 were included. Response was assessed as described by Goyal et al (Haematologica 2020;105:2). Cobimetinib was given 20-60 mg once a day by mouth for 21 days in a 28-day cycle. Adverse events (AEs) were graded using the National Cancer Institute Common Toxicity Criteria v5.0. Time-to-event analyses were calculated from the time of treatment initiation, while duration of response was calculated from the time of 1 st response. Tumor genomic profile was analyzed using Tempus-xT ® assay (Chicago, IL), which includes targeted DNA sequencing (648 oncogenes) and whole transcriptome sequencing.RESULTSOf the 54 pts with RDD, ten were treated with cobimetinib and included in this study. The median follow-up was 16 months (m) [95% confidence interval (CI): 13-not reached (NR)] and most (n=8; 80%) were females. The median age at the time of treatment was 63 years (range: 36-74). Cobimetinib was used as first line therapy in four pts.Overall response rate was 70%, with 30% and 40% achieving complete (CR) and partial (PR) responses, respectively. One had stable disease (SD) and two had progressive disease (PD) (Table 1). The median progression-free-survival was 6.4 m (95%CI: 4.9-NR) and at 6 and 12 m, 67% and 50% of pts remained free from disease progression or death, respectively (Figure 1). The median duration of response was 7.6 m (95%CI: 3.1-NR). At the time of last follow-up, nine (90%) pts were alive. One pt died due to myelodysplastic syndrome with excessive blasts. She had multiple chemotherapy treatments before initiation of cobimetinib. Cobimetinib was discontinued in this pt due to grade 3 anemia and skin toxicity after 6 m of therapy and had SD as the best response.Median duration of treatment for the entire cohort was 6 m (range: 2-14). In the seven pts who responded, the median duration of treatment was 7 m (range: 2-14). Cobimetinib was discontinued in seven pts: disease progression (n=3; one disease progression after achieving PR and two PD while on therapy), grade 3 left ventricular systolic dysfunction (n=1), grade 3 diarrhea (n=1), grade 3 anemia (n=1), and grade 2 creatinine elevation (n=1). Five (50%) pts were started at a reduced dose of cobimetinib at the discretion of treating physician [20 mg (n=4) and 40mg, (n=1)]. Two pts were started at 60 mg and dose reduced to 20 mg after one cycle of treatment due to grade 2 skin toxicity in one and grade 2 skin toxicity and diarrhea in the other. Both pts achieved a PR but progressed after four and six cycles, respectively. One pt started at 40 mg and reduced to 20 mg due to grade 2 skin toxicity after two cycles achieving a PR. The response was sustained in this pt for at least 9 months, until the time of last follow-up (Table 1).Tissue molecular testing showed five pts with MAPK pathway alterations and all of them responded to therapy (CR=3 and PR=2). Of the other five pts who either did not have a MAPK pathway alteration (n=4) or had test failure (n=1), responses were observed in two (40%) pts, both PR (Table 1).CONCLUSIONOur study suggests that cobimetinib is an effective treatment in RDD, with responses observed in all pts having MAPK pathway alterations. Less than half of pts without a MAPK pathway alteration achieved a response. However, treatment discontinuations due to AEs were common despite dose reductions. Patients who were treated at low doses of cobimetinib also had remarkable clinical response. Further studies are needed in evaluating other MAPK pathway inhibitors with better toxicity profile to improve adherence and outcomes. [Display omitted] DisclosuresTobin: National Institutes of Health: Research Funding; Mayo Clinic Center for MS and Autoimmune Neurology: Research Funding; Mallinckrodt Pharmaceuticals: Research Funding. Bennani: Vividion: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Verastem: Other: Advisory Board; Purdue Pharma: Other: Advisory Board; Kymera: Other: Advisory Board. Vassallo: Pfizer: Research Funding; Bristol-Myers-Squibb: Research Funding; Sun Pharma.: Research Funding.

Peak Absolute Lymphocyte Count Post CAR-T Is Associated with Clinical Response and Survival Outcome in Aggressive Lymphoma

IntroductionChimeric antigen receptor T cell (CAR-T) expansion has been consistently identified as a predictor of clinical response. However, there are no clinically available test to measure CAR-T presence after infusion for the FDA approved CAR-T therapy. We hypothesize that the lymphocyte expansion which can be readily measured as absolute lymphocyte count (ALC) in blood count differential could be a clinically accessible surrogate for CAR-T expansion in patients who receive FDA approved CD19 CAR-T to treat aggressive B-cell non-Hodgkin lymphoma (NHL). We examined the ALC levels in the first 2 weeks after CAR-T infusion and correlation with clinical outcomes in NHL patients who received CAR-T at our institution.MethodsRecords were reviewed for patients who received CAR-T between 6/2016 and 1/2021 at Mayo Clinic, Rochester. CAR+ T cells were identified by flow cytometry using an anti-FMC63 antibody. Receiver operator curve was generated using nominal logistic regression to predict complete response (CR) as best response. Progression-free survival and overall survival were calculated using Kaplan-Meier method and between-group differences were assessed using log-rank test. Continuous variables were compared using wilcoxon text and categorical variables were compared using chi-square test.ResultsAmong the 87 patients who received CAR-T for NHL, majority of the pts received axicabtagene ciloleucel (86, 99%). The highest ALC level (ALC peak) in the first 15 days were identified for all patients and median ALC peak was 0.44 X10 9/L (range, 0 - 2.55x10 9/L). The median time to ALC peak was 10 ±3 days. Increasing ALC peak levels correlated with increased CAR+ T cells in blood measured by flow cytometry (n=16, R=0.63, p=0.0008). Using ROC analysis, an ALC peak level of 0.67 was identified as the cut point for best association with CR (AUC=0.68, p=0.0004). Baseline demographics were similar between the high ALC peak (N=35) and low ALC peak (N=52) groups, as shown in Table 1. There was no difference in the incidence, high grade, or duration of cytokine release syndrome (CRS) and immune effector associated neurotoxicity syndrome (ICANS) between the two groups (Table 2). Patients who received a higher cumulative dose of steroid for management of CRS and ICANS had lower ALC peak. A higher ALC peak was associated with an increased CR rate and durable CR at month 6. Similarly, a higher ALC peak was associated with increased progression-free survival (PFS, 6.4 months vs. 2.7 months, p=0.004) and overall survival (OS, 31 months vs. 12 months, p=0.04), as shown in figure 1.ConclusionGiven the typical CAR-T expansion seen in the first 2 weeks post infusion, ALC peak in the first 15 days is a clinically accessible, reliable surrogate for CAR-T expansion and predicts durable CR and longer PFS, OS.This study was supported in part by the Mayo Clinic Center for Individualized Medicine, Bernard E. and Edith B. Waterman, Henry J. Predolin Foundation and other generous benefactors of Mayo Clinic. [Display omitted] DisclosuresBennani: Verastem: Other: Advisory Board; Purdue Pharma: Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board; Vividion: Other: Advisory Board; Kymera: Other: Advisory Board. Paludo: Karyopharm: Research Funding. Wang: Genentech: Research Funding; Eli Lilly: Membership on an entity's Board of Directors or advisory committees; LOXO Oncology: Membership on an entity's Board of Directors or advisory committees, Research Funding; MorphoSys: Research Funding; InnoCare: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. Lin: Janssen: Consultancy, Research Funding; Legend: Consultancy; Bluebird Bio: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Vineti: Consultancy; Gamida Cell: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Research Funding; Merck: Research Funding; Sorrento: Consultancy; Juno: Consultancy.

Sex as an Independent Variable in the Measurement of Satiation: A Retrospective Cohort Study

Background: Satiation is a key component of food intake regulation as it brings an eating episode to an end. The effect of sex on satiation measurement has not been characterized. Methods: This retrospective cohort study aimed to investigate the effect of sex on satiation measurement in adult participants across all body mass indices (BMI). We measured satiation with a nutrient-drink test to assess volume to fullness (VTF) and maximum tolerated volume (MTV), and/or an ad libitum meal test to assess calories consumed to fullness (CTF). We performed univariate and multiple regression analyses to estimate the contribution of sex to VTF, MTV, and CTF, compared to other biological variables, such as age, weight, height, BMI, waist-to-hip circumference (W/H), and lean mass percentage (LM%), that are known to affect these parameters. Findings: We included 959 participants (mean age 39 [SD 12] years; 70·7% female, and BMI 33 kg/m2 [8]). Females had higher BMI, W/H, and LM%. VTF, MTV, and CTF were lower in females: 704 [323] vs. 783 [328] mL, p=0·001; 1226 [384] vs. 1419 [410] mL, p<0·001; and 871 [291] vs. 1086 [326] kcal, p<0·001; respectively. Sex was a strong and independent predictor of VTF, MTF and CTF: parameter estimate [PE]=-80·8, p=0·006; PE=-124·2, p=0·0007; and PE=-110, p=0·001; respectively. Interpretation: Sex has a strong effect on satiation measured by VTF, MTV, and CTF, even after adjusting for other biological factors known to affect these parameters. Females seem to integrate intra-meal inhibition signals to consume fewer calories unrelated to body size or composition. Funding Information: Dr. Acosta is supported by NIH (NIH K23-DK114460), Mayo Clinic Center for Individualized Medicine – Gerstner Career Development Award. Dr. Camilleri receives funding related to obesity from the National Institutes of Health (NIH RO1-DK67071). Dr. Kapoor is supported by the NIA grant U54 AG044170. Declaration of Interests: Dr. Acosta is a stockholder in Gila Therapeutics, Phenomix Sciences; he serves as a consultant for Rhythm Pharmaceuticals, General Mills. Dr. Camilleri is a stockholder in Phenomix Sciences and Enterin and serves as a consultant to Takeda, Allergan, Rhythm, Kallyope, and Arena with compensation to his employer, Mayo Clinic. Dr. Kapoor serves as a consultant for Mithra and Astellas Pharmaceuticals. She is also a consultant for the company Womaness. The rest of the authors have nothing to disclose. Ethics Approval Statement: This retrospective cohort study was approved by the Institutional Review Board. All participants signed informed consent before enrollment in the original studies, and they consented to the use of their data, medical records, and samples for research.

CT-387: Prognostic Role of Lymphocyte to Monocyte Ratio in Pts Treated with CAR-T for Aggressive Lymphoma

Background Low absolute lymphocyte to monocyte ratio (ALC/AMC) predicts decreased survival in aggressive lymphoma (NHL) patients (pts) receiving chemotherapy and ASCT. We report the clinical significance of ALC/AMC and associated cellular phenotype changes in pts receiving chimeric antigen receptor T-cell (CAR-T) therapy. Methods We conducted a retrospective chart review of all pts who received axicabtagene ciloleucel for NHL at Mayo Clinic, Rochester, between 6/2016 and 12/2020. ALC and AMC were obtained from a clinical lab before lymphodepletion chemotherapy (LD). The receiver operator characteristic curve was generated using nominal logistic regression on ALC/AMC to predict complete remission (CR). Survivals were calculated using the Kaplan–Meier method. Blood immune phenotypes were assayed by multiparametric flow cytometry. Results Low ALC/AMC (≤0.8, n=29) prior to LD was associated with a lower CR rate (AUC=0.68, p=0.0004). Baseline characteristics were similar between the low and high ALC/AMC groups (>0.8, n=52), except for the use of prior ASCT and bridging therapy. The low ALC/AMC group also had higher CRP on the day of CAR-T infusion and was associated with shorter EFS and OS (EFS: 2.6 vs 6.4 months, p≤0.0001; OS: 5.3 months vs not reached, p=0.0006). Prognostic association remained statistically significant on multivariate analysis, which included prior ASCT, bridging therapy, and CRP. Compared to the high ALC/AMC group, the low ALC/AMC group had decreased CD4 Tem and increased CD16+CCR2+ monocytes (n=26). In addition, when comparing patients who achieved CR initially and relapsed versus those who maintained durable CR for 6 months or longer, there was a difference in these phenotypes, as well as the number of CD27+CD45RA+CD62L+ CD8 T cells and PD-1+TIGIT+ T cells. Conclusion ALC/AMC is a clinically accessible test that is strongly associated with CAR-T response and survival. Immune characterization revealed that the biological effect is not just associated with cell numbers. Phenotypes, such as monocytes with inflammatory capacity and T-cell subsets, may further distinguish between primary refractory disease, early relapse, and durable response. These data could inform future therapeutic strategies to improve clinical outcomes with CAR-T. This study was supported in part by the Mayo Clinic Center for Individualized Medicine and Henry J. Predolin Foundation.

Does Reduction Mammaplasty for Treatment of Macromastia Play a Role in the Functionality and Performance of Female Athletes?

Women’s sports are on the rise, with an increase in enthusiasts, viewers, and, most importantly, participants. Female athletics has made remarkable strides, gaining traction with media coverage and with professional sports associations implementing increased salaries to their athletes. With the women’s sports world ascending, the number of female athletes finding a passion in sport is swiftly growing. From 2017 to 2018, a total of 216,378 female athletes competed in the National Collegiate Athletic Association (NCAA), making up 44 percent of the student-athlete body nationwide. Women’s teams tallied 54 percent of NCAA teams in the country 1. However, with an increase in female participants in athletics, this indubitably opens the discussion of macromastia and its consequences within this population. Macromastia is an issue for many women and is associated with a variety of complaints, such as musculoskeletal pain,2 as well as psychosocial effects, including depression and decreased self-esteem.3 Macromastia and its effects are well known to female athletes. This may subsequently discourage women from participating in sports. For those who decide to pursue sports regardless of their symptomatology due to macromastia, important questions arise. Do female athletes with macromastia experience a reduction in functionality and performance compared with female athletes without macromastia? If so, what role can reduction mammoplasty play in this population? Reduction mammaplasty has been shown to ameliorate symptoms related to macromastia.2,4 Studies have evaluated the relief of symptoms in macromastia patients following reduction mammaplasty. Many studies report improvement in discomfort and pain postoperatively.2 One study focused on pulmonary function before and after reduction mammaplasty. Maximum voluntary ventilation, peak expiratory flow rate, and inspiratory capacity were significantly improved postoperatively. In addition, per analysis of preoperative and postoperative patient questionnaires, subjective findings, such as shortness of breath, were noted to improve.4 With this, it is important to evaluate whether reduction mammaplasty in female athletes would result in improved objective performance within distinct sports. Specific training tests could be administered before and after reduction mammaplasty, such as sprint, jump, agility, and endurance tests, to assess for statistically significant differences in the functionality and performance of the athlete. Macromastia can affect an athlete at any level of sport; however, it is worth noting its effects on elite-level female athletes. A 2018 study reported that elite-level female athletes encounter contact breast injuries appreciably more often in those athletes with larger breasts or greater body mass index, as well as in contact sports. It found that 21 percent of participants who sustained an injury to the breast perceived that it had a negative impact on their athletic performance. Many of these athletes reported altering their physical movements to avoid breast injury, subsequently limiting their physical activity during performance.5 In summary, macromastia is an issue for many women and reduction mammaplasty has proven benefits.2,4 Female athletes are a group that deserves further research to assess whether reduction mammaplasty for treatment of macromastia plays a role in functionality and performance in athletics. If studies were to support this hypothesis, we could see a significant change in the landscape of women’s sports and competition. DISCLOSURE The authors have no financial interest or conflicts of interest to declare in relation to the content of this article. ACKNOWLEDGMENTS This study was supported in part by the Mayo Clinic Center for Individualized Medicine and the Plastic Surgery Foundation.

Abstract P57: Risk Factors for Incidentally Discovered Silent Brain Infarcts and White Matter Disease in a Real World Cohort Identified by Artificial Intelligence

Introduction: Silent brain infarcts (SBIs) and white matter disease (WMD) are highly prevalent and associated with increased risk of ischemic stroke in patients with traditional stroke risk factors (RFs) in prospective cohort studies. Their frequency and associations with stroke RFs have not been well described in real world populations. Methods: This was a cross-sectional study of patients age ≥ 50 in the Kaiser Permanente-Southern California (KPSC) health system between 2009-2019 with a head CT or MRI for non-stroke indications and no history of ischemic stroke, transient ischemic attack, or dementia. A natural language processing (NLP) algorithm developed at Mayo Clinic and Tufts Medical Center was applied to the KPSC EHR to identify individuals with reported SBIs or WMD. Multivariable Poisson regression with robust error variance was used to estimate risk ratios of demographics, stroke RFs (from the Framingham Stroke Risk Score), and scan modality on the presence of SBIs or WMD. Results: Among 262,875 individuals, the NLP identified 13,154 (5.0%) with SBIs and 78,330 (29.8%) with WMD. Stroke RFs were highly prevalent in this cohort. The majority underwent CTs (74.8%) instead of MRIs as their initial neuroimaging. After adjustment for demographics and RFs, advanced age demonstrated a strong association with increased risk of SBIs and WMD (table). MRI was associated with a reduced risk of reported SBIs (ARR: 0.87, 95% CI 0.83-0.91) and an increased risk of reported WMD (ARR 2.86, 95% CI 2.83-2.90). Despite being prevalent, traditional stroke RFs had weak associations with increased risk of SBIs or increased risk of WMD. Conclusions: Advanced age is strongly associated with incidentally discovered SBIs and WMD on neuroimaging studies obtained in routine care. The development of SBIs and WMD may not be fully attributable to traditional stroke RFs.

95233 Analysis of 5'UTR Variation in Rare Disease Patients Reveals Variants of Potential Disease Relevance

ABSTRACT IMPACT: This work sheds diagnostic insight on patients with idiopathic rare disease and has the potential to further their care and treatment as a result. OBJECTIVES/GOALS: Correct diagnosis is imperative to treating patients with idiopathic, suspected genetic conditions, yet sequencing approaches leave up to 70% of these patients undiagnosed. We sought to improve diagnosis rates for a cohort patients referred for sequencing by characterizing deleterious variants within the 5’UTR. METHODS/STUDY POPULATION: We retrospectively analyzed whole exome sequencing (WES) data from 472 unsolved rare disease patients within the Mayo Clinic Center for Individualized Medicine to identify variants within the 5’UTR that affect the presence of upstream open reading frames (uORFs). uORFs are short regions (typically 30bp - 600bp) that typically influence downstream gene translation by sequestering ribosomes. We specifically searched for variants with the potential to disrupt existing uORFs or introduce new uORFs within the 5’UTR, and developed a pipeline to annotate these variants with information including GnomAD allele frequency and gene loss of function intolerance (pLI) score. To aid in variant interpretation, we applied two deep learning tools to predict variant impacts on transcript ribosome load (TITER and FramePool). RESULTS/ANTICIPATED RESULTS: Our pipeline identified a median of 21 variants per patient that were predicted to have a deleterious impact on the translational efficiency of protein coding transcripts, primarily by introducing new start codons within the 5’UTR or by altering the Kozak consensus of existing start codons. A median of 10 of these variants occur upstream of haploinsufficient genes with an existing disease association. We also identified a subset of variants that are predicted to introduce translationally active N-terminal extensions to protein coding transcripts, with the potential to disrupt protein localization and processing. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work demonstrates that analysis of 5’UTR variants can be incorporated into existing WES pipelines, and identifies a group of variants with potential significance to patient disease. Further experimental evidence is necessary to ascertain the pathogenicity of these variants.

A Scalable Framework for Telehealth: The Mayo Clinic Center for Connected Care Response to the COVID-19 Pandemic.

Background: The Mayo Clinic Center for Connected Care has an established organizational framework for telehealth care delivery. It provides patients, consumers, care teams, and referring providers access to clinical knowledge through technologies and integrated practice models. Central to the framework are teams that support product management and operational functions. They work together across the asynchronous, synchronous video telemedicine, remote patient monitoring (RPM), and mobile core service lines.Methods: The organizational framework of the Center for Connected Care and Mayo Clinic telehealth response to the COVID-19 pandemic is described. Barriers to telehealth delivery that were addressed by the public health emergency are also reported. This report was deemed exempt from full review by the Mayo Clinic IRB.Results: After declaration of the COVID-19 pandemic, there was rapid growth in established telehealth offerings, including patient online services account creation, secure messaging, inpatient eConsults, express care online utilization, and video visits to home. Census for the RPM program for patients with chronic conditions remained stable; however, its framework was rapidly adapted to develop and implement a COVID-19 RPM service. In addition to this, other new telehealth and virtual care services were created to support the unique needs of patients with COVID-19 symptoms or disease and the health care workforce, including a digital COVID-19 self-assessment tool and video telemedicine solutions for ambulances, emergency departments, intensive care units, and designated medical–surgical units.Conclusion: Rapid growth, adoption, and sustainability of telehealth services through the COVID-19 pandemic were made possible by a scalable framework for telehealth and alignment of regulatory and reimbursement models.

Integrating Genomic Screening into Primary Care: Provider Experiences Caring for Latino Patients at a Community-Based Health Center.

Introduction:Minority communities have had limited access to advances in genomic medicine. Mayo Clinic and Mountain Park Health Center, a Federally Qualified Health Center in Phoenix, Arizona, partnered to assess the feasibility of offering genomic screening to Latino patients receiving care at a community-based health center. We examined primary care provider (PCP) experiences reporting genomic screening results and integrating those results into patient care.Methods:We conducted open-ended, semi-structured interviews with PCPs and other members of the health care team charged with supporting patients who received positive genomic screening results. Interviews were recorded, transcribed, and analyzed thematically.Results:Of the 500 patients who pursued genomic screening, 10 received results indicating a genetic variant that warranted clinical management. PCPs felt genomic screening was valuable to patients and their families, and that genomic research should strive to include underrepresented minorities. Providers identified multiple challenges integrating genomic sequencing into patient care, including difficulties maintaining patient contact over time; arranging follow-up medical care; and managing results in an environment with limited genetics expertise. Providers also reflected on the ethics of offering genomic sequencing to patients who may not be able to pursue diagnostic testing or follow-up care due to financial constraints.Conclusions:Our results highlight the potential benefits and challenges of bringing advances in precision medicine to community-based health centers serving under-resourced populations. By proactively considering patient support needs, and identifying financial assistance programs and patient-referral mechanisms to support patients who may need specialized medical care, PCPs and other health care providers can help to ensure that precision medicine lives up to its full potential as a tool for improving patient care.

The Adjustable Intragastric Balloon for Treatment of Obesity: Results of a Multicenter, Randomized Controlled Trial

Background: Intragastric balloons are anatomy preserving, minimally invasive therapies for obesity. Enhanced tolerance and durability inform clinical adoption. We investigated the safety and efficacy of an adjustable intragastric balloon (aIGB) with prolonged dwelling time in adults with obesity.Methods: Patients were randomized to the Spatz3 (Spatz FGIA, Great Neck, NY) aIGB with lifestyle intervention versus lifestyle intervention alone (control) for 32 weeks. The aIGB volume could be increased to facilitate weight loss or decreased for tolerability. Co-primary endpoints included mean total body weight loss (TBL) and responder rate (≥ 5% TBL) at 32 weeks. Weight loss maintenance (WLM) was reported at 14 months. Improvement in obesity-related comorbidities was reported at 32 weeks. Data were analyzed in a multiple imputed intention to treat (miITT) population. Adverse events were recorded throughout the study duration.Findings: 288 adults with obesity were randomized to aIGB (n = 187) or control (n = 101) arms. Mean TBL was 15‧0% [95% CI 13‧9%, 16‧1%] in the aIGB group vs 3‧3% [95% CI 2‧0%,4‧6%] in controls (p Interpretation: The adjustable intragastric balloon induced significant weight loss that was maintained in most patients. Adjustability of volume significantly improved safety and tolerance highlighting advantages of this therapy for clinical practice.Trial Registration: This study was registered with clinicaltrial.gov -NCT02812160.Funding: SPATZ MedicalDeclaration of Interest: BKA reports consultant roles with Endogenex, Endo-TAGSS, Metamodix, and BFKW; consultant and grant/research support from USGI, Cairn Diagnostics, Aspire Bariatrics, Boston Scientific; Speaker roles with Olympus, Johnson and Johnson; speaker and grant/research support from Medtronic, Endogastric solutions; and research support from Apollo Endosurgery, and Spatz Medical. CGC reports consultant roles with Olympus; consultant roles and payment or honoraria Apollo Endosurgery, Boston Scientific; payment or honoraria from Abbive; participation in a Data Safety Monitoring Board and consultant roles with ERBE, BFKW Inc, and Nitinotes Surgical. VP reports consultancy fees from Obalon Therapeutics Inc. PJ reports grants from Boston Scientific; licenses to Endosim; consulting fees from Endogastric solutions, ERBE; support for meetings from USGI medical; consulting fees and research support from GI dynamics; research support from Apollo Endosurgery, and Fractyl. AA reports grants from NIH-NIDDK, American Neurogastroenterology and Motility Society, Mayo Clinic Center for Individualized Medicine; Stockholder of Gila Therapeutics, and Phenomix Sciences; personal fees from Rhythm Pharmaceuticals, General Mills, Gila Therapeutics; he reports patent PCT. US 62/589,915 licensed to Phenomix Sciences. ACS reports institutional research grants from Boston Scientific, Enterasense, Endogenex; consulting fees from Olympus; consulting fees and research grants from Endo-TAGSS, and Apollo Endosurgery; Participation in Data Safety Monitoring Board with GI Dynamics, and ERBE. MR reports consultant roles with Boston Scientific, Fujifilm, Medtronic, Enterasense, and GI Windows; Consultant and research support from Cook Medical. DM reports financial support from SPATZ medical for statistical analysis for this trial. CCT reports institutional research grants from Aspire Bariatrics, ERBE, SPATZ; Royalties from Endosim, Enterasense; Consulting fees and research grants from Apollo Endosurgery, Boston Scientific, Covidien/ Medtronic, Fractyl, Fujifilm, GI Dynamics, Lumendi, Olympus, and USGI; Payment or honoraria from Boston Scientific and Olympus; Patents licensed to Boston Scientific, GI Windows, Enterasense, EnVision Endoscopy, Endosim; Participation in Data Safety Monitoring Board with GI Dynamics, Fractyl, USGI; Leadership or fiduciary role in other board with Enterasense, EnVision Endoscopy, GI Windows, ASGE Governing board, BlueFlame Healthcare Venture Fund; Stockholder in GI Windows, Enterasense, EnVision Endoscopy; has received equipment on loan from Fujifilm, Olympus, and Boston Scientific. All other authors report no conflict of interests.Ethical Approval: It was conducted under a US Food and Drug Administration (FDA) Investigational Device Exemption (IDE G160061), approved on 29-Jul-2016, in compliance with 21 CFR812

Extracting hypertrophic cardiomyopathy features from cardiac magnetic resonance reports by natural language processing

Abstract Objective Determine if information regarding hypertrophic cardiomyopathy (HCM) can be accurately retrieved from cardiac magnetic resonance (CMR) reports using natural language processing (NLP). Background CMR imaging is used for diagnosis and risk stratification of HCM. Manual annotation of information from CMR is time-consuming. NLP is an artificial intelligence method for automating extraction of information from narrative text. Methods We identified 200 HCM patients who had CMR reports from 1998 to 2018. These patients were randomly allocated into training (100 patients with 185 CMR reports) and testing sets (100 patients with 206 reports). An NLP system with 2 tiers was developed; the first extracted information regarding HCM diagnosis while second extracted categorical or numeric concepts for HCM classification. NLP performance was compared with gold-standard manual annotation. Results NLP algorithms achieved very high performance across all concepts with mean positive predictive value (PPV) = 0.96. An outlier was the performance for abstracting the presence of an apical pouch from CMR reports, which had noticeably lower PPV= 0.78 which be attributed to the low number of cases with this finding. Conclusions The algorithms developed can be translated to clinical decision support systems to increase efficiency and contribute to improved quality of care. Funding Acknowledgement Type of funding source: Other. Main funding source(s): Study supported by the National Heart, Lung and Blood Institute of National Institutes of Health (K01HL124045), the Mayo Clinic Center for Clinical and Translational Science (CCaTS), and the Mayo Clinic K2R award. Content is solely the responsibility of authors and does not necessarily represent official views of the National Institutes of Health.

Anti-CD19 chimeric antigen receptor T-cell therapy in acute lymphocytic leukaemia: a systematic review and meta-analysis

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has shown remarkable activity in patients with refractory or relapsed acute lymphocytic leukaemia. Various anti-CD19 CAR T-cell constructs have been trialled and responses vary widely among different studies. We aimed to systematically analyse the outcomes of patients with acute lymphocytic leukaemia treated with anti-CD19 CAR T cells and identify factors associated with differences in outcomes. We did a systematic review and meta-analysis of published and unpublished clinical trials that reported data on the outcomes of adult or paediatric patients that were treated with anti-CD19 CAR T cells for relapsed or refractory B-cell acute lymphocytic leukaemia, reported between Jan 1, 2012, and April 14, 2020. Studies with two patients or fewer were excluded and summary data were extracted from the reports. The primary outcome was the number of patients who had complete remission at any time after anti-CD19 CAR T-cell infusion. This study is not registered in PROSPERO. From 1160 studies, we identified 40 potentially appropriate studies, 35 (88%) of which met the eligibility criteria and were included in the final analysis (n=953 patients). The pooled complete remission was 80% (95% CI 75·5-84·8) and heterogeneity between studies was moderate (I2=56·96%). In the prespecified subgroup analyses, 195 (75% [95% CI 66·9-82·9, I2=35·22%]) of 263 patients in adult studies and 242 (81% [72·9-87·2, I2=54·45%]) of 346 patients in paediatric studies achieved complete remission, p=0·24. The pooled complete remission did not significantly differ with anti-CD19 CAR T-cell construct type or single-chain variable fragment clone, but was higher with autologous T-cell origin (727 [83%, 78·5-86·5, I2=44·34%] of 901 patients), compared with allogeneic T-cell origin (29 [55%, 30·6-79·0, I2=62·64%] of 52 patients; p=0·018). 242 (26% [95% CI 18·5-34·1]) of 854 patients developed grade 3 or worse cytokine release syndrome and 97 (12% [6·6-19·2]) of 532 developed grade 3 or worse neurotoxicity. There was no difference in the proportion of patients who achieved complete remission or who had cytokine release syndrome or neurotoxicity between different anti-CD19 CAR T-cell constructs. The risk of bias was assessed as low in 17 studies and moderate in 18 studies. The high response rates after anti-CD19 CAR T-cell therapy can be used to guide the use of therapy in patients with relapsed or refractory acute lymphocytic leukaemia. Comparison studies are required to further determine differences in efficacy between different anti-CD19 CAR T-cell constructs in the setting of relapsed or refractory acute lymphocytic leukaemia. National Cancer Institute, National Comprehensive Cancer Network, Mayo Clinic K2R Research Pipeline, and Mayo Clinic Center for Individualized Medicine.

Epidemiology of autosomal-dominant polycystic liver disease in Olmsted county.

Background & AimsIsolated autosomal-dominant polycystic liver disease (ADPLD) is generally considered a rare disease. However, the frequency of truncating mutations to ADPLD genes in large, population sequencing databases is 1:496. With the increasing use of abdominal imaging, incidental detection of hepatic cysts and ADPLD has become more frequent. The present study was performed to ascertain the incidence and point prevalence of ADPLD in Olmsted County, MN, USA, and how these are impacted by the increasing utilisation of abdominal imaging.MethodsThe Rochester Epidemiology Project and radiology databases of Mayo Clinic and Olmsted Medical Center were searched to identify all subjects meeting diagnostic criteria for definite, likely, or possible ADPLD. Annual incidence rates were calculated using incident cases during 1980–2016 as numerator, and age- and sex-specific estimates of the population of Olmsted County as denominator. Point prevalence was calculated using prevalence cases as numerator, and age- and sex-specific estimates of the population of Olmsted County on 1 January 2010 as denominator.ResultsThe incidence rate and point prevalence of combined definite and likely ADPLD were 1.01 per 100,000 person-years and 9.5 per 100,000 population, respectively. Only 15 of 35 definite and likely incident ADPLD cases had received a diagnostic code, and only 8 had clinically significant hepatomegaly. The incidence rates were much higher when adding possible cases, mainly identified through radiology databases, particularly in recent years and in older patients because of the increased utilisation of imaging studies.ConclusionsClinically significant isolated ADPLD is a rare disease with a prevalence <1:10,000 population. The overall prevalence of ADPLD, however, to a large extent not clinically significant, is likely much higher and closer to the reported genetic prevalence.Lay summaryIsolated autosomal-dominant polycystic liver disease (ADPLD) is generally considered a rare disease. However, we demonstrate that it is a relatively common disease, which is rarely (<1:10,000 population) clinically significant.

0817 Abnormal REM Sleep Atonia Control In Patients With Chronic Post-traumatic Stress Disorder

Abstract Introduction Post-traumatic stress disorder (PTSD) is characterized by persistent mental and emotional stress following one or more significant physical or psychological traumatic incidents earlier in life. Vivid recall of the events, including traumatic nightmares, and prominent sleep disturbance are usual in PTSD. Previous studies have suggested that PTSD may share some clinical features with idiopathic REM sleep behavior disorder (iRBD) including altered REM sleep without atonia (RSWA) levels. Our group has previously found evidence for altered RSWA control in patients with psychiatric disease, including a pilot sample of PTSD patients with iRBD. We aimed to comparatively analyze RSWA levels between patients with PTSD, PTSD and RBD (PTSD+RBD), iRBD, and controls. Methods We selected 18 PTSD, 18 PTSD+RBD, 15 iRBD, and 51 healthy control patients matched for age and sex from the Mayo Clinic Center for Sleep Medicine’s polysomnography database for RSWA quantification. RSWA amounts in the submentalis (SM) and anterior tibialis (AT) were quantitatively analyzed as a percentage of REM sleep duration, in accordance with previously published methods. Non-parametric analyses were performed to compare RSWA, patient demographics, and PSG data across groups. Significance was set at p &amp;lt; 0.016. Results Patients with PTSD had significantly higher RSWA than controls in all RSWA density measures (p &amp;lt; 0.016 for all). All measures of RSWA, excluding average SM duration, were significantly greater in PTSD+RBD patients compared with controls (p &amp;lt; 0.016 for all). Within the PTSD group, patients on antidepressants did not have significantly higher RSWA in any of the measures. PTSD+RBD patients had significantly higher SM Phasic, AT Any, SM+AT Any, and Tonic RSWA measures than PTSD patients (p &amp;lt; 0.016 for all). Conclusion PTSD patients have significantly higher RSWA than controls, with PTSD+RBD patients having higher RSWA levels than PTSD patients. These data provide the first evidence for abnormal RSWA control in patients with chronic PTSD. This provides evidence of a unique biology in PTSD that could imply a future risk for neurodegenerative disease in PTSD similar to RBD patients. Further prospective studying will need to be performed on patients with PTSD to understand the unique biology. Support

OR02-02 Possible Age and Time-Of-Day Differences in the Hypothalamo-Pituitary-Testicular, and Adrenal, Response to Total Overnight Sleep Restriction

Introduction: In young men, sleep restriction decreases testosterone and increases afternoon cortisol, leading to anabolic-catabolic imbalance, insulin resistance and metabolic, neurocognitive, reproductive, and other adverse effects. Age-related differences in the hypothalamo-pituitary-testicular/adrenal response to sleep restriction could expose older individuals to greater or lesser risk, but this possibility has not been previously studied. Subjects and Methods: Thirty-five healthy young and older men aged 18-30y (n=17) and 60-80y (n=18), underwent blood sampling in the Mayo Clinic Center for Clinical and Translational Science every 10 minutes for 24 hours from 6PM-6PM under two conditions in random order spaced at least 3 weeks apart: awake (no sleep) or sleep (from 10PM to 6AM). Blood was assayed for LH, testosterone (T) and cortisol (F), and then analyzed by automated mathematical deconvolution and with cross approximate entropy statistics to determine hormone secretion and hormone synchrony, respectively. Statistical significance was construed by repeated measures ANOVA using a full factorial model that included age, sleep and the interaction. Results: Sleep deprivation had multiple effects on 24-hour (6PM-6PM) Te secretion with significant reductions in mean concentrations, basal, total and pulsatile secretion, and pulse frequency (each P<0.05), in the absence of detectable changes in LH. These effects were most apparent in older men and differed according to age for some parameters: pulsatile Te secretion (P=0.03) and T pulse frequency (P=0.02). Time-of-day analyses revealed that sleep restriction significantly reduced Te in the morning (6AM-9AM) and afternoon (3PM-6PM), reduced LH in the morning, and increased F in the afternoon, particularly in older men. Cross-approximate entropy statistics showed that sleep restriction enforced greater LH-Te and Te-LH joint synchrony in the morning (P<0.05 for each), but not in the afternoon. Conclusion: Sleep restriction decreases morning LH secretion, morning and afternoon Te secretion, and increases afternoon F secretion, especially in older men. This combination of findings could plausibly cause metabolic and reproductive ill-health when accumulated over decades of life, and may explain how chronic sleep loss contributes to metabolic and reproductive diseases that are more prevalent in older men. These preliminary data also suggest a time-of-day dependent uncoupling of the regulatory control of the testicular axis, and of cortisol secretion. Direct verification by interventions that manipulate hormones during the morning and late afternoon in appropriately matched cohorts of young and older men are now required.