TITLE Heterogeneity in Recent Onset Type 1 Diabetes – A Clinical Trial Perspective Short Title : Heterogeneity in Recent Onset T 1 D

Background: Type 1 Diabetes TrialNet is an NIH-sponsored clinical trial network aimed at altering the disease course of type 1 diabetes. The purpose of this study is to evaluate age-dependent heterogeneity in clinical, metabolic, and immunologic characteristics of individuals with recent-onset type 1 diabetes (T1D), to identify cohorts of interest and to aid in planning of future studies. Methods: 883 individuals with recent onset T1D involved in five TrialNet studies were categorized by age as: ≥ 18, age 12-17, ages 8-12, and age <8. Data was compared with healthy age-matched subjects in the National Health and Nutrition Examination Survey. Results: While only 2.0 % of individuals overall were excluded due to insufficient Cpeptide values (<0.2 pmol/ml), 9.0% of those < age 8 did not meet this entry criteria. Leukopenia was present in 21.2% of individuals and lymphopenia in 11.6%; these frequencies were markedly different than age-matched healthy population. 24.5% of the cohort was overweight or obese. 31.1% of adults and 21.1% of children had neither HLA DR3 nor DR4. Conclusions: The ability of recent onset T1D patients to meet key entry criteria for TrialNet studies, including C-peptide >0.2 pmol/ml, varies by age. Lower C-peptide level requirements for younger participants should be considered in the design of future trials. These data also highlight subgroups of type 1 diabetes patients, such as those with abnormal WBC or who are overweight, which allow for targeted studies of etiopathology and interventions. This article is protected by copyright. All rights reserved. INTRODUCTION Type 1 Diabetes TrialNet is an international consortium of clinical diabetologists and immunologists whose aim is to conduct multiple clinical trials to alter the natural history of the disease; specifically by delaying or stopping beta cell destruction. In these studies, Rituximab[1] and Abatacept[2] both demonstrated improvement in residual insulin secretion in drug as compared to placebo treated individuals, whereas GAD65-alum[3], MMF/DZB[4] and Canakinumab[5] did not. Within all studies and treatment arms however, heterogeneous responses were apparent. For example, we and others have highlighted age as an important variable accounting for some of this heterogeneity, finding significant differences in the disease course in children as compared with adults [6-8]. As a result, future studies may be restricted to narrower age ranges of participants or age category may be used as a stratification variable. With the aim to further dissect heterogeneity in type 1 diabetes, we use combined TrialNet data to evaluate clinical, immunological, and metabolic characteristics of these subjects at study entry according to age. This evaluation should aid in the planning and design of future type 1 diabetes intervention trials. MATERIALS AND METHODS Clinical sites: Studies took place at 15 clinical centers in North America and one in Italy. Protocols and consent documents were approved by the institutional review board or independent ethics committee at each participating clinical center as previously reported and all subjects underwent informed consent and assent prior to participation in any study activities. Study Interventions: The studies were designed to evaluate therapies with an array of mechanisms aimed at immunomodulation to preserve beta cells, including immunosuppressive agents (mycophenolate mofetil [MMF] and daclizumab), a therapy directed at B cells (anti-CD20 rituximab), a therapy directed at antigenspecific tolerance (GAD-alum vaccine), co-stimulation blockade (abatacept), and anti IL1B (canakinumab). Eligibility Criteria. Study eligibility criteria were similar across studies with the exception of age and autoantibodies as described below. Inclusion criteria included Mixed Meal Tolerance Test (MMTT) stimulated peak C-peptide levels of at least 0.2 pmol/ml conducted within 3 weeks to 3 months after diagnosis, and randomization within 100 days of clinical diagnosis. Patients were eligible to participate in the GAD-alum study if they had glutamic acid decarboxylase-65 antibodies (GAD65ab). Eligibility for all other studies required at least one diabetes-related autoantibody: microassayed insulin antibodies (mIAA) [if duration of insulin therapy was less than 7 days]; GAD65ab; insulinoma antigen 2 antibodies (IA-2ab) or islet-cell autoantibodies (ICA). ICA was often measured only when mIAA, GAD65ab, and IA-2ab were negative. In sum, a total of 754 subjects in the five studies underwent testing for all three antibodies (GADab, ICA, and IA-2ab). Znt8 antibodies were only measured in ten otherwise antibody negative subjects in the most recent study testing canakinumab. All trials had age 45 as the upper age limit for eligibility; the lower age limit for eligibility was 8 years for Rituximab and This article is protected by copyright. All rights reserved. MMF/DZB studies, 6 years for canakinumab and abatacept studies and 3 years for the GAD-alum trial. Exclusion criteria included complicating medical issues, active infection, positive PPD, serologic evidence of HIV, hepatitis B or hepatitis C infection, history of immunodeficiency or lymphopenia, or chronic use of steroids or other immunosuppressive agents. EBV and CMV serology was measured in all 5 studies along with EBV PCR to rule out active infection in all studies with the exception of the GAD-alum trial. Study Assessments: Similar, but not identical information was obtained during all trials. For example, in some studies, HLA typing was performed on all screened subjects, while in others HLA was done only on randomized subjects. Methods: Samples were sent to a central laboratory for measurements of HbA1c, C-peptide, glucose, autoantibodies, chemistries, viral serology and PCR as previously published. CBCs were determined at the center’s local clinical laboratory. All local clinical laboratories at US sites were CLIA certified, Canadian sites were certified by Ontario Medical Association Laboratory Quality Management ProgramLaboratory Services; similar certification was obtained at the Italian site. Values outside normal ranges for either the central laboratory or local laboratory measures were graded according to Common Terminology Criteria for Adverse Events (CTCAE) criteria. While children were enrolled in all of these studies, the age of children eligible for trials may be limited in some studies due to regulatory or ethical considerations. Frequently, considerations for enrollment of children involve consideration of emotional and intellectual maturation stages as children vs adults (< or ≥ age 18); teenagers ages 12-17; children who are considered developmentally mature enough to understand study participation (age 8-12) and younger children (<age 8). These age categories were thus applied to the data presented. For comparison of TrialNet type 1 diabetes participants with healthy subjects, CBC data from 2009-2010 National Health and Nutrition Examination Survey (NHANES) study participants, ages 3-45, was used (n=5172). Age and gender-specified cutoffs for normal values available in the 2009-2010 NHANES study documentation[9] were applied to WBC, PMN and lymphocyte counts. Analysis: Categorical variables were compared among age groups by Pearson’s chi-square test or Fisher Exact test when cell sizes were insufficient. Continuous variables were summarized either by mean and standard deviation or median and inter-quartile range. ANOVA F-tests were done to determine significant differences among age groups in mean values. The association of age with level of WBC, polymorphonuclear neutrophils (PMN) and lymphocyte counts was tested using the multivariable logistic regression model. Time from diagnosis, HbA1c, autoantibody status and c-peptide level were included in the model to adjust for possible confounding factors. Tests of significance were two-tailed. Probability (p) value < 0.05 was considered to be statistically significant. Statistical analyses were performed with SAS Version 9.2 (Cary, NC). This article is protected by copyright. All rights reserved. RESULTS A total of 883 subjects were screened and 541 (61%) patients enrolled in one of the five intervention trials. The mean age of patients who presented for screening was 17.1 (±9.0) years, with a range from 3.5 to 46 years; the age distribution favored younger subjects (Figure 1). Descriptive characteristics of the patients screened for the intervention studies by age category are presented in Table 1. 90.5% of research participants were white and 8.8% were Hispanic or Latino. They were more often male (58.9%), particularly among older subjects, and were screened a mean of 54 days and a median of 56 days from type 1 diabetes diagnosis. While the information was not available on almost a third of participants, 24.7% of the participants reported having other family members with type 1 diabetes and 5.3% report other autoimmune disease themselves or within the family. The proportions of overweight and obese individuals were different by age category (p=0.045). While the mean BMI for adults was within the normal range, 24.5% of all enrolled subjects were classified as overweight or obese by BMI or BMIZ criteria including 43 obese children. The mean HbA1c was 7.09% and this did not differ significantly by age category. Stimulated C-peptide values increased across age categories whether measured by AUC or peak value during MMTT. However, there was no significant relationship between age and HbA1c or age and C-peptide when considered as continuous variables (data not shown). When considering only those subjects who were tested for GAD65, IA-2ab, and ICA, adults were less likely ICA or IA-2ab positive then the cohorts under age 18, yet little differences were found with prevalence of antibodies between the three younger cohorts. Too few subjects were assessed for ZnT8 antibodies (N=10) to evaluate the effect of age on rate o