Faculty Bibliography

OBJECTIVES/OBJECTIVE:A perception derived from cross-sectional studies of small systemic lupus erythematosus (SLE) cohorts is that there is a marked discrepancy between antinuclear antibody (ANA) assays, which impacts on clinicians' approach to diagnosis and follow-up. We compared three ANA assays in a longitudinal analysis of a large international incident SLE cohort retested regularly and followed for 5 years. METHODS:Demographic, clinical and serological data was from 805 SLE patients at enrolment, year 3 and 5. Two HEp-2 indirect immunofluorescence assays (IFA1, IFA2), an ANA ELISA, and SLE-related autoantibodies were performed in one laboratory. Frequencies of positivity, titres or absorbance units (AU), and IFA patterns were compared using McNemar, Wilcoxon and kappa statistics, respectively. RESULTS:At enrolment, ANA positivity (≥1:80) was 96.1% by IFA1 (median titre 1:1280 (IQR 1:640-1:5120)), 98.3% by IFA2 (1:2560 (IQR 1:640-1:5120)) and 96.6% by ELISA (176.3 AU (IQR 106.4 AU-203.5 AU)). At least one ANA assay was positive for 99.6% of patients at enrolment. At year 5, ANA positivity by IFAs (IFA1 95.2%; IFA2 98.9%) remained high, while there was a decrease in ELISA positivity (91.3%, p<0.001). Overall, there was >91% agreement in ANA positivity at all time points and ≥71% agreement in IFA patterns between IFA1 and IFA2. CONCLUSION/CONCLUSIONS:In recent-onset SLE, three ANA assays demonstrated commutability with a high proportion of positivity and titres or AU. However, over 5 years follow-up, there was modest variation in ANA assay performance. In clinical situations where the SLE diagnosis is being considered, a negative test by either the ELISA or HEp-2 IFA may require reflex testing.

OBJECTIVE:Delayed detection of lupus nephritis associates with worse outcomes. There are conflicting recommendations regarding a threshold level of proteinuria at which biopsy will likely yield actionable management. This study addressed the association of urine protein creatinine ratios (UPCR) with clinical characteristics and investigated the incidence of proliferative and membranous histology in patients with a UPCR between 0.5 and 1. METHODS:275 SLE patients (113 first biopsy, 162 repeat) were enrolled in the multicentre multi-ethnic/racial Accelerating Medicines Partnership across 15 U.S. sites at the time of a clinically indicated renal biopsy. Patients were followed for 1 year. RESULTS:At biopsy, 54 patients had UPCR <1 and 221 had UPCR >1. Independent of UPCR or biopsy number, a majority (92%) of patients had class III, IV, V or mixed histology. Moreover, patients with UPCR <1 and class III, IV, V, or mixed had a median activity index of 4.5 and chronicity index of 3, yet 39% of these patients had an inactive sediment. Neither anti-dsDNA nor low complement distinguished class I or II from III, IV, V, or mixed in patients with UPCR <1. Of 29 patients with baseline UPCR <1 and class III, IV, V or mixed, 23 (79%) had a UPCR <0.5 at one year. CONCLUSION/CONCLUSIONS:In this prospective study three quarters of patients with UPCR <1 had histology showing class III, IV, V or mixed with accompanying activity and chronicity despite an inactive sediment or normal serologies. These data support renal biopsy at thresholds lower than a UPCR of 1.

OBJECTIVE:Previous studies suggest a link between high serum type I interferon (IFN) and lupus nephritis (LN). We determined whether serum IFN activity is associated with subtypes of LN and studied renal tissues and cells to understand the impact of IFN in LN. METHODS:). Podocyte cell line gene expression was measured by real-time PCR. RESULTS:expression was not closely co-localized with pDCs. IFN directly activated podocyte cell lines to induce chemokines and proapoptotic molecules. CONCLUSION/CONCLUSIONS:Systemic high IFN is involved in the pathogenesis of severe LN. We do not find co-localization of pDCs with IFN signature in renal tissue, and instead observe the greatest intensity of IFN signature in glomerular areas, which could suggest a blood source of IFN.

OBJECTIVES/OBJECTIVE:Current treatments are effective only in 30% of lupus nephritis patients emphasizing the need for novel therapeutic strategies. To develop mechanistic hypotheses and explore novel biomarkers, we analyzed the longitudinal urinary proteomic profiles in patients with lupus nephritis undergoing treatment. METHODS:We quantified 1,000 urinary proteins in 30 patients with lupus nephritis at the time of the diagnostic renal biopsy and after 3, 6, and 12 months. The proteins and molecular pathways detected in the urine proteome were then analyzed with respect to baseline clinical features and longitudinal trajectories. The intrarenal expression of candidate biomarkers was evaluated using single cell transcriptomics of renal biopsies from lupus nephritis patients. RESULTS:Our analysis revealed multiple biological pathways including chemotaxis, neutrophil activation, platelet degranulation, and extracellular matrix organization that could be noninvasively quantified and monitored in the urine. We identified 237 urinary biomarkers associated with lupus nephritis as compared to controls without SLE. IL-16, CD163, and TGF-β mirrored intrarenal nephritis activity. Response to treatment was paralleled by a reduction of urinary IL-16, a CD4 ligand with proinflammatory and chemotactic properties. Single cell RNA sequencing independently demonstrated that IL16 is the second most expressed cytokine by most infiltrating immune cells in lupus nephritis kidneys. IL-16 producing cells were found at key sites of kidney injury. CONCLUSION/CONCLUSIONS:Urine proteomics may profoundly change the diagnosis and management of lupus nephritis by noninvasively monitor active intrarenal biological pathways. These findings implicate IL-16 in lupus nephritis pathogenesis designating it as a potentially treatable target and biomarker.

OBJECTIVE:To evaluate seroreactivity and disease flares after COVID-19 vaccination in a multi-ethnic/racial cohort of patients with systemic lupus erythematosus (SLE). METHODS:90 SLE patients and 20 healthy controls receiving a complete COVID-19 vaccine regimen were included. IgG seroreactivity to the SARS-CoV-2 spike receptor-binding domain (RBD) and SARS-CoV-2 microneutralization were used to evaluate B cell responses; IFN-γ production to assess T cell responses was measured by ELISpot. Disease activity was measured by the hybrid SLE disease activity index (SLEDAI) and flares were assigned by the SELENA/SLEDAI flare index. RESULTS:Overall, fully vaccinated SLE patients produced significantly lower IgG antibodies against SARS-CoV-2 spike RBD than controls. Twenty-six SLE patients (28.8%) generated an IgG response below that of the lowest control (<100 units/ml). In logistic regression analyses, the use of any immunosuppressant or prednisone and a normal anti-dsDNA level prior to vaccination associated with decreased vaccine responses. IgG seroreactivity to the SARS-CoV-2 Spike RBD strongly correlated with the SARS-CoV-2 microneutralization titers and antigen-specific IFN-γ production determined by ELISpot. In a subset of patients with poor antibody responses, IFN-γ production was likewise diminished. Pre-/post-vaccination SLEDAI scores were similar. Only 11.4% of patients had a post-vaccination flare; 1.3% were severe. CONCLUSION/CONCLUSIONS:In a multi-ethnic/racial study of SLE patients 29% had a low response to the COVID-19 vaccine which was associated with being on immunosuppression. Reassuringly, disease flares were rare. While minimal protective levels remain unknown, these data suggest protocol development is needed to assess efficacy of booster vaccination.

Background Patients with Systemic Lupus Erythematosus (SLE) represent a unique population at risk for COVID-19 due to underlying immune abnormalities and regular use of immunosuppressant medications. This study was initiated to evaluate for the presence of SARS-CoV-2 IgG antibodies in SLE patients with and without prior COVID-19-related symptoms or COVID-19 RT PCR testing. Methods A total of 329 patients with SLE from two cohorts, one serially monitored for COVID-19 in Spring 2020 (the Web-based Assesment of Autoimmune, Immune-Mediated and Rheumatic Patients (WARCOV) and one undergoing routine surveillance (NYU Lupus Cohort) were tested for SARS-CoV-2 IgG via commercially available immunoassays processed through hospital or outpatient laboratories between April 29, 2020 and February 9, 2021. Results Overall, 16% of 329 patients had a reactive SARSCoV- 2 IgG antibody test. Seropositive patients were more likely to be Hispanic. Other demographic variables, lupus-specific factors and immunosuppressant use were not associated with reactivity. Of the 29 patients with prior RT-PCR confirmed COVID-19, 83% developed an antibody response despite 62% being on immunosuppressants. Six percent of patients who had symptoms suspicious for COVID-19 but negative concurrent RT-PCR testing developed an antibody response. Twenty-three percent of patients who had COVID- 19-related symptoms but no RT-PCR testing and 5% of patients who had no symptoms of COVID-19 developed an antibody response. Among patients initially SARS-CoV-2 IgG positive, the majority maintained reactivity serially. In COVID- 19-confirmed patients high percentages had antibody positivity beyond 30 weeks from disease onset, 88% up to 10 weeks, 83% up to 20 weeks, and 80% up to 30 weeks. Conclusions Most patients with SLE and confirmed COVID- 19 were able to produce a serologic response despite use of a variety of immunosuppressants. These findings provide reassurances regarding the efficacy of humoral immunity and possible reinfection protection in patients with SLE

Background Prior studies of SLE clusters based on autoantibodies have utilized cross-sectional data from single centers. We applied clustering techniques to longitudinal and comprehensive autoantibody data from a large multinational, multiethnic inception cohort of well characterized SLE patients to identify clusters associated with disease outcomes. Methods We used demographic, clinical, and serological data at enrolment and follow-up visits years 3 and 5 from 805 patients who fulfilled the 1997 Updated ACR SLE criteria and were enrolled within 15 months of diagnosis. For each visit, ANA, dsDNA, Sm, U1-RNP, SSA/Ro60, SSB/La, Ro52/ TRIM21, histones, ribosomal P, Jo-1, centromere B, PCNA, anti-DFS70, lupus anticoagulant (LAC), IgG and IgM for anticardiolipin, anti-b2GP1, and aPS/PT, and IgG anti-b2GP1 D1 were performed at a single lab (except LAC). K-means clustering algorithm on principal component analysis (10 dimensions) transformed longitudinal ANA/autoantibody profiles was used. We compared cluster demographic/clinical outcomes, including longitudinal disease activity (total and adjusted mean SLEDAI- 2K), SLICC/ACR damage index and organ-specific domains, SLE therapies, and survival, using one-way ANOVA test and a Benjamini-Hochberg correction with false discovery rate alpha=0.05. Results were visualized using t-distributed stochastic neighbor embedding. Results Four unique patient clusters were identified (table 1). Cluster 1, characterized by high frequency of anti-Sm and anti-RNP over time, was the youngest group at disease onset with a high proportion of subjects of Asian and African ancestry. At year 5, they had the highest disease activity, were more likely to have active hematologic and mucocutaneous involvement, and to be on/exposed to immunosuppressants/ biologics. Cluster 2, the largest cluster, had low frequency of anti-dsDNA, were oldest at disease onset, and at year 5, had the lowest disease activity, and were least likely to have nephritis and be on/exposed to immunosuppressants/biologics. Cluster 3 had the highest frequency of antiphospholipid antibodies over time, were more likely to be of European ancestry, have an elevated BMI, be former smokers, and by year 5, to have nephritis, neuropsychiatric involvement, including strokes and seizures (SLICC/ACR damage index). Cluster 4 was characterized by anti-SSA/Ro60, SSB/La, Ro52/TRIM21, histone antibodies, and low complements at year 5. Overall, survival of the 805 subjects was 94% at 5 years, and none of the clusters predicted survival. Conclusions Four SLE patient clusters associated with disease activity, organ involvement, and treatment were identified in this analysis of longitudinal ANA/autoantibody profiles in relation to SLE outcomes, suggesting these subsets might be identifiable based on extended autoantibody profiles early in disease and carry prognostic information

Background/Purpose: The Accelerating Medicines Partnership (AMP) Lupus Network was established with the goal of applying novel technologies to the interrogation of blood and tissue samples from patients with lupus nephritis (LN). In contrast to global LN clinical trials, the AMP LN cohort affords an opportunity to generate outcome data representative of a US multicenter multi-ethnic real-world experience. In this analysis, the AMP clinical dataset was investigated to determine the percentages of patients who attained prespecified definitions of partial or complete responses at 52 weeks. In addition, incorporation of response rates at weeks 12 and 26 to the analysis provided longitudinal patterns of response to standard of care.
Method(s): Patients with LN who were undergoing kidney biopsies as part of standard of care were eligible to enroll in the AMP LN study. Response definitions were only applied to cases whose baseline spot urine protein/creatinine (UPCR) ratios were > 1.0. Complete response (CR) required: 1) UPCR < 0.5; and 2) normal creatinine (< 1.3 mg/dL) or, if abnormal at baseline, < 125% of baseline; and 3) prednisone < 10 mg/day at the time of the study visit. Partial response required: 1) >50% reduction in UPCR without meeting UPCR criterion for CR; and 2) normal creatinine (< 1.3 mg/dL) or, if abnormal, < 125% of baseline; and 3) prednisone dose < 15 mg/day at the time of the study visit. Patients who did not achieve a CR or PR at the specific timepoints were considered non-responders (NR). Only patients with renal biopsies that demonstrated ISN/RPS classes III, IV, V or combined III or IV with V and data available at all four timepoints (baseline, weeks 12, 26 and 52) were included in this analysis. Cross-sectional and longitudinal analyses of responses were performed, and heat maps were generated to graphically display response patterns.
Result(s): Data on 121 patients with LN enrolled in AMP were included in this analysis. Cross-sectional response rates at 52 weeks were: CR: 28.1%; PR: 23.1%; NR: 48.8% (Table 1). Response rates at weeks 12 and 26 are additionally displayed in Table 1, and Figure 1 is a heat map demonstrating longitudinal responses of our patients. All patients were considered NR at baseline. Only 7.4% of patients had week 12 CR responses sustained through week 52, whereas 19% had attained PR or CR at all 3 visits. An additional 14.9% achieved a PR or CR at 26 weeks which was sustained at 52 weeks. Overall, 36.4% of patients were NR at all time points.
Conclusion(s): Clinical data from the AMP Lupus Network revealed rates of 52-week CR and PR that were consistent with placebo response data from recently conducted LN trials. Low sustained CR rates not only underscore the need for more efficacious therapies but highlight how critically important it is to understand the molecular pathways that are associated with response and non-response. (Figure Presented)