Faculty Bibliography

Background/Purpose : The heterogeneity of the clinical manifestations of systemic lupus erythematosus (SLE) and lack of a diagnostic test make SLE difficult for epidemiologists to study. The Centers for Disease Control and Prevention (CDC) supported five population-based SLE surveillance registries, using harmonized methodology, to better estimate incidence and prevalence of SLE in diverse areas in the United States (US). Leveraging these data, we performed a meta-analysis to estimate the general prevalence of SLE in the US. Methods : The CDC registries were established in Michigan, Georgia, California, New York and the through the Indian Health Service (IHS). All registries used the 1997 revised ACR classification criteria for SLE as their primary case definition, and the surveillance time periods ranged from 2003 to 2009. Age-standardized prevalence was stratified by sex and race/ethnicity from the state-based registries; the American Indian/Alaska Native (AI/AN) estimate was based on the IHS registry that covered multiple states. For pooling data across the four sites with data on different racial/ethnic groups, we used Cochran ' s Q and I2 statistic to test for heterogeneity across sites. Due to significant heterogeneity, we used a random effects model to calculate pooled prevalence, which allows for more variation across sites. We then extrapolated to the 2017 Census population data according to sex and race-stratified groups, including data from the IHS registry, and summed the stratum-specific estimates to provide a total population estimate of SLE cases in the US. Results : The registries contributed 5,417 classified cases of SLE from a mix of urban and rural areas. From the metaanalysis of the four state-based registries, the overall prevalence was 72.8 (95%CI 65.3, 81.0) per 100,000 population. The prevalence among females was about 9 times higher than males (128.7 vs 14.6). In the meta-analysis, prevalence was highest among black females (230.9, 95%CI 178.2, 299.2), followed by Hispanic females (120.7, 95%CI 84.0, 173.4), white females (84.7, 95%CI 68.4, 104.8) and Asian/Pacific Islander females (84.4, 95%CI 48.3, 147.4). Among males, prevalence followed a similar pattern with the highest rates among black males (26.7, 95%CI 19.6, 36.4) followed by Hispanic males (18.0, 95%CI 15.6, 20.8), Asian/Pacific Islander males (11.2, 95%CI 5.7, 21.9), and white males (8.9, 95%CI 8.0, 10.1). The AI/AN prevalence estimates, which were not included in the meta-analysis, had the highest rates of SLE for both females (271, 95%CI 238, 307) and males (54, 95%CI 36, 77). Applying our sexand race-specific prevalence estimates to the corresponding population denominators from 2017 Census data, we estimated that 198,677 persons (179,186 females and 19,491 males) in the US fulfill ACR SLE classification criteria, Table 1. Conclusion : Using estimates from a coordinated network of population-based SLE registries, a more accurate prevalence estimate for the US was obtained. Our methods did not capture undiagnosed, incomplete, or other forms of lupus such as cutaneous lupus. Other case definitions may yield different results

Background/Purpose : Lupus nephritis (LN) complicates up to 60% of patients with systemic lupus erythematosus (SLE) and carries a high morbidity and mortality. The definitive diagnosis is based on kidney biopsy. This is invasive and not always readily available, thus delaying treatment. Sometimes multiple biopsies are required over the course of the disease. Importantly, while renal pathology is accurate at describing the morphology of renal disease, the underlying biology and molecular pathways are not thoroughly assessed. Urine proteomics is a non-invasive strategy that may provide insights regarding ongoing renal disease. Methods : One thousand proteins were quantified (RayBiotech Kiloplex assay) on a total of 112 longitudinal urine samples from 32 SLE patients with active LN and 7 healthy controls (HC) enrolled in the Accelerating Medicines Partnership (AMP). All patients underwent treatment as directed by their own physicians. Differentially excreted proteins at baseline (SLE vs HC, proliferative vs membranous LN, responders vs non responders) were identified using a linearmodel with moderated t statistic. Response to treatment was defined based on proteinuria at 1 year as complete (< 0.5g/24h) or partial (50% reduction but >0.5/24h). In the longitudinal analysis, a mixed model was employed to identify markers associated with proteinuria. Pathway enrichment analysis was performed using the genes coding for the differentially excreted analytes using Ingenuity Pathway Analysis (IPA) and other publicly available pathway libraries. Results : There were 186 proteins increased in SLE patients (Fig. 1). The most enriched pathway was TNFa (p< 0.001). We found 74 differentially excreted proteins comparing proliferative and pure membranous LN. CD4, MCP-1, MIP-1a, RANTES, IL-16, and IL-7, markers involved in CD4 T cell and monocyte biology, were enriched in proliferative disease. A few targets were exclusively identified in either class (i.e. CD4 in proliferative nephritis). We used a longitudinal model to identify specific urine proteins associated with worse proteinuria as a marker of severity. Proteinu-ria was associated with 105 markers (FDR < 0.05), the strongest association being CD163 (p = 10-9), a phagocyte marker. IPA implicated several pathways involving fibrosis, acute phase response, LPS/IL1, RXR, ICOS signaling and macrophage/fibroblasts (Fig. 2). Next, we identified 27 differentially excreted proteins in non-responders. IPA revealed that tretinoin, GM-CSF, TNF, and IL1 were among the top upstream regulators (Fig. 3). Conclusion : There is an inflammatory signature in the urine of patients with LN implicating monocyte and TNFa pathways. These signatures are associated with proliferative disease, worse proteinuria, and non-response to treatment. Of note, TNFa is involved in LN and has therapeutic potential. In phase 1 of AMP, monocytes were the main urine cell type identified by singe cell RNA sequencing in patients with LN. These results suggest that urine proteomics might identify and infer active pathological mechanisms in LN, paving the way for a more personalized approach to treatment. Further work in Phase 2 of AMP is being pursued to validate and extend these findings

Background/Purpose : The impact of renal injury in lupus nephritis (LN) is widespread with consequences to resident cells in other tissue beds, even non-lesional, non-sun exposed skin. Faithful reflection of a relevant renal tissue pathway in a more readily accessible compartment would allow for less invasive diagnostic alternatives. While ongoing studies are exploiting single cell RNA sequencing to link phenotype to biotype and identify cell specific pathways in the kidney, this study was initiated to address the hypothesis that these pathways may be reflected in uninvolved skin which is more likely to be serially biopsied. Methods : Single cell RNAseq was performed on cell suspensions prepared from ~2 mm punch biopsies of nonlesional, non-sun-exposed skin from the buttocks of 5 healthy controls, 4 SLE patients without LN and 18 SLE patients with proteinuria (with skin biopsies obtained within 24 hrs of the kidney biopsy). Histology revealed Class III ( n=6 ), Class III/V or IV/V mixed ( n=11 ), Class V ( n=1 ), and nephrosclerosis ( n=1 ). Dissociation of cryostored skin biopsies with collagenase and trypsin enzymes was followed by scRNA-seq using the 10x Genomics platform using V2 and V3 reagents. Results : We obtained 8,019 and 17,655 high-quality scRNA-seq profiles from single cell suspensions of control and SLE non-lesional, non-sun-exposed skin, respectively. A graph-based clustering method was applied and identified major clusters of cells as visualized by t-distributed stochastic neighbor embedding (tSNE). Differential gene expression analysis guided by established markers revealed these cell clusters as keratinocyte (KC), one smooth muscle cell cluster (SMC), fibroblast (FB), melanocyte (MEL), vascular endothelial cells (VEC), lymphatic endothelial cells (LEC), macrophages-dendritic cells (MAC-DC), T cells (TC) and sweat gland cells (SGC) (Figure 1A). Ranking cells by abundance, the result of the SLE skin cells was KC >FB >VEC >LEC, SMC, MAC-DC, TC, MEL and SGC. Overall, samples processed using the recent V3 single cell reagent kit showed higher genes and transcript captures compared to V2. However, these samples also captured more mitochondrial transcripts (Figure 1B). An analysis of gene expression changes in KC, SMC, and VSC from the LN patients versus controls demonstrated overexpression of interferon stimulated genes. However, the degree of interferon response varied in these cell types with KCs (basal KC, p=0.00312 and hair follicle KC, p=0.000012) showing the highest response followed by VECs (p=0.0043) and SMCs (p=0.0068). In addition to the interferon response signature, VECs from the LN patients also showed upregulation of MHC-II genes such as HLA-DRB5 and HLA-DRB1, suggesting increased antigen presentation capacity (Figure 1C). Conclusion : scRNA-seq identifies major skin cell types and further clustering identifies rarer cell populations. KCs, SMCs, and VECs from the skin of LN patients reveal diverse IFN response states and additionally VECs also show higher antigen presentation potential. The V3 upgrade of 10x Genomics single cell reagents capture more genes and UMIs per cell, but also higher mitochondrial content compared to the V2 version

Background/Purpose : Pregnancy counseling of all anti-Ro positive women includes advice regarding the development of congenital heart block (CHB), albeit the risk is only 2% for primigravida women or those with previously unaffected offspring. Despite this low risk, the prevailing surveillance recommendation is weekly echocardiography. While evidence from basic research laboratories support that high titers of antibodies confer clinically meaningful risk, unfortunately the majority of commercial laboratories use the BioPlex assay, which provides positive and negative values with limited information on actual levels because the sera or plasma are not diluted past a specified cutoffgiven cost (e.g. values of anti-Ro inclusive of Ro52 or Ro60 by laboratories such as Quest or LabCorp provide positive as 1-8 or > 8 units with no further information). The present study was initiated to assess whether the Bio-Plex assay used by many commercial laboratories provides adequate stratification of risk for counseling regarding management. Methods : The study group comprised healthy non-pregnant donors (N = 9), healthy pregnant donors (N = 62), women testing positive for anti-Ro by commercial BioPlex but without CHB children (N = 60 SLE and 2 SS), and women with CHB children (N = 83). Anti-Ro60 reactivity was assessed using native antigen and anti-Ro52 using recombinant protein. Sera were applied to coated microtiter plates at serial dilutions ranging from 1:1000 -1:50,000 for 1h at RT and run in duplicate. Tested samples were multiplied by the dilution factor which gives an OD in the range of 0.3-0.8. Results were considered positive at 123 ELISA units (EU) for Ro60 and 215 EU for Ro52 as this represented the mean +3 SD of the values obtained for healthy control sera. Results : Of the 83 CHB mothers tested, 74 had titers of Ro60 and Ro52 > 1000 EU, in 1 anti-Ro60 was > 1000 EU and anti-52 Ro between 215 -1000, in 3 anti-Ro52 was > 1000 EU and anti-Ro60 between 300 -1000, and 1 mother had anti-Ro60 > 1000 EU and was negative for anti-Ro52. Albeit all positive, the sera from 4 CHB mothers obtained 15 years after the birth of the affected child were < 1000 EU for both anti-Ro60 and Ro52. With these results setting thresholds ( > 1000 EU in either Ro60 or Ro52 for CHB risk), we assessed patients testing positive for anti-Ro based on the BioPlex assay. Of 42 patients with values of > 8 on BioPlex testing, 14 had titers > 1000 EU for both anti-Ro60 and Ro52, 7 had anti-Ro60 > 1000 EU, and 8 had anti-Ro52 > 1000 EU. Thus, 13 of 42 (25%) with commercial Ro > 8 did not meet the threshold EU for CHB risk. Of 20 patients considered positive for anti-Ro by BioPlex with values between 1-8, none had levels of either anti-Ro60 or Ro52 at 1000 EU. No patient or healthy control testing negative by the BioPlex assay was positive for CHB risk in our ELISA. Conclusion : These data suggest that commercial testing using the BioPlex assay may fall short of stratifying risk for CHB. Women with positive values < 8 are not likely at risk, obviating the cost and burden of weekly fetal echo surveillance. Moreover, even those considered high titer on commercial testing may be at low risk supporting the need for more quantitative commercial testing than is currently available. (Figure Presented)

Background/Purpose : Based on encouraging bench to bedside results including experimental evidence supporting Toll-like receptor signaling in the pathogenesis of CHB, a case control study demonstrating CHB risk reduction in hydroxychoroquine (HCQ) exposed fetuses of anti-Ro positive SLE women, and a historical cohort study supporting a reduction in recurrence rate, an open label single arm Phase 2 clinical trial was initiated to evaluate whether HCQ reduces the CHB recurrence rate (pi) below the historical recurrence rate of 18%. Methods : A two-stage trial design (N=19 first stage; N=54 second stage) using Simon's optimal approach was employed to allow for early stopping due to absence of treatment efficacy. The null hypothesis, H 0 :pi <= 18%, would be rejected and HCQ considered efficacious at the end of the trial if <= 5 of 54 mothers with anti-Ro and a previous CHB child had a subsequent child with 2 nd or 3 rd degree block (primary outcome). The protocol required HCQ initiation or maintenance at 400mg by 10 wks gestation. Mothers underwent serial echocardiograms, with bloods drawn each trimester and delivery for cord blood to measure antibody and HCQ levels. Results : Sixty five mothers (all with previous CHB child and anti-Ro52 or Ro60 > 1,000 EU; 47.9% with anti-La; 71.4% White; 47.6% SLE and/or SS; 42.9% started HCQ solely for CHB prevention; 41% prior CHB child died, 3.2% had > 1 CHB child) signed consent. Ten were considered screen failures (2 miscarriages < 12 wks, 7 wherein dating of conception placed HCQ initiation at > 10 wks, 1 given dexamethasone (dex) 1mg at 10 wks) and 1 was lost to follow up before delivery leaving 54 pregnancies evaluable with serial fetal echos and birth or one yr EKG or echo results known. In Stage I, 2/19 fetuses had CHB, and the study proceeded to Stage II. By intention to treat analysis, 4/54 pregnancies resulted in CHB (7.4%; p = 0.02 for H 0 ), all at 19-20 wks. Three presented with 2 nd degree block, one reverted to NSR at birth following dex and two progressed to 3 rd degree despite dex and IVIG (one electively terminated). One presenting with 1 st degree was treated with dex prophylactically (eliminating this case from evaluating HCQ exposure alone), progressed to 2 nd but reverted to NSR at birth. At 2 yrs, the 2 in NSR had intermittent 2 nd degree on Holter monitor. In 8 mothers potentially confounding medications, IVIG and/or dex, were prescribed after enrollment for lupus flare, cardiac concerns apart from advanced block (APCs, echo brightness, 1 st degree block), and/or physician decision to consider additional prophylaxis. To evaluate HCQ alone, 9 additional mothers were enrolled, one whose fetus developed 3 rd degree block at 19 wks. Including only pregnancies exposed to HCQ alone prior to confirmed 2 nd or 3 rd degree block, 4/54 developed CHB (7.4%; p = 0.02). In total 5/63 pregnancies (7.9%) resulted in advanced block. HCQ levels in the second trimester confirmed a 98% adherence rate. Anti-Ro levels remained > 1,000 EU (considered vulnerable for CHB) throughout pregnancy. No CHB developed in any of the 7 mothers screened out because of low dose or delayed start of HCQ. Conclusion : These prospective data from a single-arm clinical trial support that HCQ significantly reduces the recurrence of CHB below the historical rate

Background/Purpose : Null mutations in DNASE1L3 cause severe familial SLE with prominent anti-DNA antibodies (Abs), suggesting that DNASE1L3 is a key driver of tolerance to DNA. Indeed, DNASE1L3-deficient mice rapidly develop anti-chromatin and anti-DNA Abs followed by renal involvement. DNASE1L3 is a secreted DNase that has the preferential capacity to digest DNA within nucleosomes and/or encapsulated by membranes. Our previous studies suggest that chromatin carried by circulating apoptotic microparticles (MP) is an important physiological substrate of DNASE1L3. Accordingly, the present study was initiated to address the hypothesis that dysfunction of DNASE1L3 and its downstream consequences contribute to the development of sporadic human SLE. Methods : Reactivity to DNASE1L3-sensitive antigens on MP was measured by incubating plasma samples with control or DNASE1L3-treated MP and analyzing IgG binding to MP by flow cytometry. DNASE1L3 activity was measured based on its preferential capacity to digest complex DNA substrates with the readout expressed as the fraction of activity measured in healthy control subjects. The DNA load of MP was measured by qPCR of genomic DNA purified from the MP and MP-depleted fractions of plasma. In considering a spectrum from benign to clinical autoimmunity, subjects included those with active biopsy proven lupus nephritis as well as those with anti-Ro antibodies absent SLE. Results : IgG-binding to MP was assessed in 116 SLE patients, 45 anti-Ro Ab-positive mothers whose children have neonatal lupus (17 asymptomatic or having an undifferentiated autoimmune syndrome, 2 SLE, 12 SS, 14 SLE/SS) and 16 healthy controls. IgG-reactivity to MP was not detected in any healthy controls. In contrast, 36% of SLE patients showed IgG binding to MP reversed by DNASE1L3 pretreatment (DNASE1L3-senstive reactivity). Only two of these patients were heterozygous for the known hypomorphic DNASE1L3 (R206C) variant. DNASE1L3-sensitive reactivity correlated with anti-dsDNA Abs (p< .0001) but did not overlap, indicating that reactivity is directed to more complex DNA. DNASE1L3-sensitive reactivity correlated with active proteinuria (p=.0003), low complement levels (p=.01) and overall SLEDAI (p< .0001). Among 45 anti-Ro-positive mothers, all were unreactive except the only 2 SLE mothers that developed lupus nephritis. The DNA load of MP and DNASE1L3 activity were assessed in a subset of SLE patients (n=40). Of these, patients with proteinuria showed lower plasma DNASE1L3 activity (p=.034) than those without proteinuria (Fig. 1). Moreover, increased partitioning of plasma DNA into MP correlated with reduced DNASE1L3 activity, and both parameters strongly correlated with DNASE1L3-sensitive IgG binding to MPs (p< 0.0001) (Fig. 2). Conclusion : The activity of plasma DNASE1L3 is reduced in a significant fraction of SLE patients with renal disorder unrelated to a genetic explanation and is associated with DNA accumulation in MP targeted by Abs. Collectively, these data suggest that digestion of circulating chromatin by DNASE1L3 is a fundamental mechanism of tolerance to DNA which when disrupted may lead to sporadic SLE, particularly lupus nephritis

Background/Purpose: Both plasmacytoid dendritic cells (pDCs) and switched memory B cells (SMBCs) are considered to be key effector cells in systemic lupus erythematosus. It seems likely that within these classical cell lineages, additional diversity of function will exist that will contribute to disease pathogenesis. To explore this question, we performed single-cell RNA sequencing in pDCs and SMBCs from SLE patients and controls to assess gene expression patterns and cellular sub-groupings within these lineages. Methods : pDCs and SMBCs from SLE patients (n=10) and Healthy controls (n=5) were purified by magnetic separation. For deep sequencing, we used the Fluidigm C1 HT system with 800 capture site chips to capture single cells. Single cell capture was verified by direct visualization using the Array Scan system, allowing us to remove empty wells and wells with multiple cells. After quality control and adaptor trimming, the data was analyzed using SeqGeq software. pDCs and SMBCs were clustered using UMAP and pseudo-time analysis was performed using the Monocle program. Type I IFN activity in SLE plasma was measured using reporter cell assay. Results : A total of 2774 pDCs and 2578 SMBCs from SLE and healthy controls passed the quality control and were used for further analysis. In pDCs, we observed unique clusters for patients with high interferon, low interferon, and controls, indicating that the IFN response is a major determinant of overall gene expression patterns in SLE patient pDCs. IFN signature in pDCs correlated with circulating type I IFN activity in the SLE patients measured at the same time. Other genes upregulated in pDCs included the type I interferon regulator AXL and MACC1. The SMBCs were heterogeneous in patients and controls, and in contrast to the pDCs, the overall clustering pattern was independent of the IFN score. SMBC clusters were predominantly defined by genes indicating cellular activation or proliferation such as HLA-DRs and CREB1, or genes associated with nucleic acid processing such as DNASE1 and SNORD3B-1. Conclusion : We find distinct clusters of cells defined transcriptionally within the pDC and SMBC lineages, and the transcripts which define these subgroups differ between cell lineages. Type I IFN induced transcripts are important to pDC diversity, while in SMBCs transcripts related to cellular activation and nucleic acid processing are critical markers of transcriptional heterogeneity

Background/Purpose : Given the heterogeneity of systemic lupus erythematosus (SLE), the effect of any intervention is expected to vary. The ability to identify those most and least likely to benefit from a treatment would improve the interpretability of trial outcomes and advance medical care. Conventional subgroup analyses suffer from low power, can encompass only a few variables at a time, and require a priori specification of cut-points for continuous variables. We explored the utility of a machine learning-based algorithm for discovering in a SLE trial the subgroups in which adding experimental therapy to standard of care considerably enhances or diminishes response compared to placebo (PBO). Methods : A two-step virtual twin (VT) method was applied to combined data from the BLISS-52 (N=865) and BLISS-76 (N=819) trials. A random forest algorithm was first used to estimate for each patient, given baseline characteristics, the probabilities of SRI-4 response to belimumab and PBO. A regression tree was then constructed to partition the study population into distinct subgroups and identify those in which the estimated difference in these response probabilities is much greater or smaller than the treatment effect in the overall population. Two separate VT analyses were conducted of the 10 mg/kg and 1 mg/kg belimumab doses compared to PBO. Cross-validation was used to assess the method ' s performance. Results : In the combined BLISS trials, response rates to the primary endpoint (SRI-4) were 51% in those receiving 10 mg/kg belimumab, 46% (1 mg/kg), and 39% (PBO). VT analysis of 10 mg/kg vs. PBO found a 23% belimumab response advantage over PBO in patients with SLEDAI >= 7 & steroid dose >= 4 mg/d & low C4 & no BILAG A at baseline , vs 12% in the total population. In contrast, the estimated response difference in those entering with SLEDAI < 7 & normal C4 was 5% lower on 10 mg/kg than PBO. In analysis of 1 mg/kg vs. PBO, two subgroups showed enhanced belimumab effect: SLEDAI >= 8 & steroid dose >= 19 mg/d and SLEDAI >= 8 & steroid dose < 19 mg/d & BLyS >= 1.9 ng/mL ; average estimated between-treatment response differences were 18% and 14%, respectively, compared to 7% in the overall population. But in patients with SLEDAI < 8 & steroid dose < 16 mg/d & age < 43 , the 1 mg/kg belimumab response rate was estimated to be 7% lower. Cross-validation indicated the accuracy of the VT method to identify subgroups exceeded 70%. Conclusion : Enhanced belimumab response was associated with low C4 and higher disease activity, steroid dose, and BLyS levels, as in prior studies. However, the VT method identified alternative cutpoints for continuous variables and additional features predicting non-response. SLEDAI >= 7 or 8 was most predictive of response to treatment. Thus, lower response difference is identified in patients who are potentially too ill (BILAG A severity) or not ill enough (minimal disease criteria) to benefit from adding belimumab. The 1 mg/kg belimumab effect was enhanced only in those on high baseline steroid doses. The VT and other machine learning techniques are promising for subgroup discovery in SLE trials as more sophisticated biomarkers, especially potent but less common indicators, become available

Background/Purpose : Compared to Caucasian, African-American ethnicity is associated with a higher risk of developing systemic lupus erythematosus, lupus nephritis, high-risk histological features, resistance to treatment, and mortality. In phase 1 of the Accelerating Medicines Partnership (AMP), we used single-cell genomics to identify ethnicity associated features. Methods : Single cell RNA sequencing was performed on renal biopsies obtained for clinical purpose; one pipeline applying CEL-Seq2 in a leukocyte enriched sample and the other Fluidigm C1 800 in an agnostic approach to dissociated renal cells. Differential abundance of cell populations was determined using a logistic mixed model. Then, the differential expression profile was determined for each cell cluster and interpreted using pathway enrichment analysis. Results : Samples from 19 African-American and 20 Caucasian patients were obtained. We identified 30 cell clusters. Type I and II interferon inducible genes were upregulated in most cell populations. A cluster of T cells with exceptionally high interferon signature was found to be increased in African-Americans (OR 4.8). Macrophages and DC4-like dendritic cells were instead less abundant (OR 0.3). In African-Americans, type I and II interferon response pathways were enriched in several cell types including T cells, B cells, plasma cells, and activated monocytes. The majority of the differentially expressed genes was specifically inducible by type II interferon. In addition, while there was no local expression of type I interferons, interferon gamma was abundantly expressed by infiltrating NK and CD8 T cells. Conclusion : African-American patients with lupus nephritis have a stronger interferon response pathway activation, especially type II. Our findings suggest an intrinsic biological factor underlying the outcome gap and highlight the role of interferon gamma in lupus nephritis, implicating this pathway as a potential therapeutic target in SLE. Further work in Phase 2 of AMP is being pursued to validate and extend these findings

Background/Purpose : The EULAR/ACR 2019 Classification Criteria for SLE have been validated in an international cohort of 696 SLE patients and 574 non-SLE patients with a sensitivity of 96.1% and a specificity of 93.4%. We comparatively evaluated the performance characteristics of the SLE classification systems in subsets of the validation cohort with regard to gender, race/ethnicity, and disease duration. Methods : 21 SLE expert centers from 16 countries submitted up to 100 SLE cases and 100 SLE mimicking controls each, using a standardized form without knowledge of the new criteria system to form the validation cohort. Cases and control diagnosis (SLE or not SLE) were independently verified by 3 SLE experts. The EULAR/ACR 2019 classifi-cation criteria validation cohort consisted of female (n=1,098) and male (n=172) patients; Asian (n=118), Black (n=68), Hispanic (n=124) and White (n=941) patients; and patients with an SLE duration of less than 1 year (n=34), 1-3 years (n=196), 3-5 years (n=157), and 5 or more years (n=879). Sensitivity and specificity with 95% confidence intervals (CI) were estimated for the EULAR-ACR 2019 criteria, the SLICC 2012 criteria and the ACR 1997 criteria. Results : As shown in Table 1, most of the point estimates for sensitivity and specificity in subsets lay within the 95% confidence intervals of the sensitivity and specificity of the EULAR/ACR 2019 criteria validation. In particular, sensitivity and specificity for all race/ethnicity groups were within the confidence intervals or even higher. Formally, the sensitivity was slightly lower for male patients, corresponding to a higher specificity, but the male 95% confidence intervals (0.86-0.98 for sensitivity, 0.90-0.99 for specificity) overlapped with those of the full cohort. Sensitivity appeared independent of disease duration at least from year 1 on, with all 95% confidence intervals overlapping (for the first year after diagnosis 0.52-1.00 for sensitivity, 0.69-0.97 for specificity). Conclusion : The point estimates of sensitivity and specificity suggest that the EULAR/ACR 2019 SLE classification criteria perform well in diverse race/ethnicity groups, in men and in early disease. These results now need to be independently validated in larger groups of African American/Black, Asian, and Hispanic patients, male patients and in early disease