Faculty Bibliography

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.

Background Classification and treatment decisions in lupus nephritis (LN) are largely based on renal histology. Single-cell RNA sequencing (scRNAseq) analysis may accurately differentiate types of renal involvement at the transcriptomic level, and better inform treatment decisions and prognosis. Methods scRNAseq was performed on kidney and non-lesional skin tissue collected from 20 SLE patients undergoing a clinically indicated renal biopsy. Cell types were determined using principal component analysis and t-distributed stochastic neighbor embedding (tSNE) plotting, resulting in the definitive identification of keratinocytes, tubular cells, mesangial cells, fibroblasts, endothelial cells, and leukocytes. Results LN patients expressed upregulated IFN response genes in both their tubular cells and keratinocytes. This IFN response signature in tubular cells predicted poor response to therapy 6 months post-biopsy. Tubular cells of non-responder patients also expressed upregulated extracellular matrix proteins and fibrotic markers (figure 1A and 1B). Using logistic regression analysis, a 4-gene tubular fibrosis score was created and able to predict response to treatment with an area under curve of 0.9 (figure 1C). Keratinocytes of non-responders also upregulated certain extracellular matrix genes and this response was not observed in peripheral blood mononuclear cells. Differential expression analysis between histology classes indicated an upregulation of IFN and TNF signaling in the tubular cells of patients with proliferative LN compared with membranous. Conclusions scRNAseq from 2-10 mm of renal biopsy tissue in SLE can differentiate between the different classes of LN, and provide important insights into potential pathogenic mechanisms. Further, changes in the skin of LN patients can provide a useful source of biomarkers and may reflect important information concerning concurrent kidney pathological events

Background The impact of renal injury in lupus nephritis 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. Single-cell transcriptional states as performed in this study may provide a framework for understanding how in vivo biological function emerges from complex cell ensembles, thus allowing for a clearer understanding of potential mutual pathways. Methods Patients with proteinuria and known ISN/RPS Class and controls were recruited to discovery 1 and 2 cohorts. Single cell RNAseq was performed on cell suspensions prepared from ~2 mm punch biopsies of non-lesional non sun-exposed skin from the buttocks. The libraries were prepared on the Fluidigm C1 platform (discovery 1) and 10X Genomics platform (discovery 2) along with Illumina HiSeq 2500 sequencing. Results Sorting based on COL1A1, COL1A2, COL3A1, MFAP5 and MFAP4 expression yielded 12 fibroblasts from 3 patients. The 1 Class II subject yielded 5 single-cell transcriptomes. The other 2 subjects (1 Class IV,V; 1 Class III,V) yielded 7 single-cell transcriptomes. 22 transcriptomes were derived from 3 controls. The aggregate data were used to determine the top upregulated genes in cases versus controls, most of which belonged to the interferon-stimulated gene category and the extracellular matrix category (DAVID databases). Fewer cells were obtained using Fluidigm C1 (36 single-cell) than 10X Genomics (7280 single-cell). For the latter, the major biopsy classes were represented (Class III, III/IV, III/V, V and no LN). We applied graph-based clustering and identified 12 major clusters of cells from the patient skin as visualized by t-distributed stochastic neighbor embedding (t-SNE; figure 1). Differential gene expression analysis guided by established lineage markers revealed three keratinocyte clusters (KC1- KC3), two fibroblast clusters (FB1, FB2), smooth muscle cells (SMC), two endothelial cell clusters (VEC, LEC), melanocytes (MEL), sweat gland cells (SG), macrophages/dendritic cells (MAC-DC) and T cells (TC). Ranked by abundance, patient skin exhibited KC>FB>EC>MAC-DC>SMC>TC>SG> MEL. Conclusions Single-cell RNAseq is both feasible and informative in cell-specific transcriptome analysis of fresh non-lesional non-sun exposed LN skin biopsies. 10X genomics significantly increases cell numbers and facilitates identification of major cell clusters compared to Fluidigm C1. The expression of fibroblasts and genes reflective of fibrotic and interferonrelated pathways support the application of this approach to study readily accessible tissue for biomarkers related to disease progression. (Figure Presented)

Background Autoantibody and laboratory testing is essential for SLE diagnosis. ANA by indirect immunofluorescence (ANA IIF) remains the gold standard to screen for lupus and studies demonstrate preclinical phase during which autoantibodies accumulate. Prevalence of ANA IIF alone without more specific autoantibodies and the accompanying clinical phenotype of these patients uncertain. Methods Queried 602 patients in the NYU Lupus Registry with 4 or more ACR or SLICC criteria as adjudicated by the authors for prevalence of ANA IIF, dsDNA, Sm, Ro, La, RNP, aPL, C3, C4. Compared clinical features of isolated ANA (ANA IIF alone) with the ANA IIF plus one or more lupus associated abnormalities (ANA IIF +). Results 590/602 (98%) ANA IIF positive. 548/590 (93%) patients at least one of associated tests compared to only 42/ 590 (7%) ANA IIF alone. SLE nephritis significantly more prevalent in ANA IIF+254/548 (46%), compared to 13/42 (31%) recorded with renal criteria ANA IIF alone. ANA IIF +, 158/254 (62%) biopsy proven nephritis (LN), rather than relying on proteinuria for diagnosis, compared to 5/13 (38%) of ANA IIF alone biopsy proven LN. Remaining 8 ANA IIF alone, uPCR exceeded 0.5 g in 1 of 44 (2%) encounters. Low incidence of proteinuria explained by complete renal response or prior proteinuria misconstrued as evidence of LN. In comparison, uPCR >0.5 g was present in 694 of 1157 (60%) encounters in ANA IIF +, casting doubt on validity of LN diagnosis in 8 ANA IIF alone without biopsy. Leucopenia, lymphocytopenia, AITP, AIHA statistically less ANA IIF alone compared to ANA IIF + ; 24% vs 36%, 29% vs 40%, 7% vs 15% and 0% vs 7%, respectively. 42 patients with ANA IIF alone prevalence of potentially misattributed (e.g. not result of IMID) clinical criteria such as photosensitivity (64%) and malar rash (60%) greater compared to ANA IIF +, 38% and 44%, respectively. Prevalence oral ulcers, DLE, arthritis, serositis, seizures and psychosis equivalent in both. Conclusions ANA IIF alone rare and patients infrequently develop nephritis, leucopenia, lymphocytopenia, AITP, AIHA. In patients ANA IIF alone attribution of ACR/SLICC clinical criteria needs to be point of emphasis and unless biopsy proven, alternative explanation for proteinuria should be considered. Data argues inclusion criteria for clinical trials, rather than allowing ANA IIF alone or dsDNA, may need to require ANA IIF and at least one of the following (dsDNA, Sm, Ro, La, aPL, or hypocomplementemia) to avoid enrolling patients that do not have SLE

Objective/UNASSIGNED:A common problem in clinical trials is missing data due to participant dropout and loss to follow-up, an issue which continues to receive considerable attention in the clinical research community. Our objective was to examine and compare current and alternative methods for handling missing data in SLE trials with a particular focus on multiple imputation, a flexible technique that has been applied in different disease settings but not to address missing data in the primary outcome of an SLE trial. Methods/UNASSIGNED:Data on 279 patients with SLE randomised to standard of care (SoC) and also receiving mycophenolate mofetil (MMF), azathioprine or methotrexate were obtained from the Lupus Foundation of America-Collective Data Analysis Initiative Database. Complete case analysis (CC), last observation carried forward (LOCF), non-responder imputation (NRI) and multiple imputation (MI) were applied to handle missing data in an analysis to assess differences in SLE Responder Index-5 (SRI-5) response rates at 52 weeks between patients on SoC treated with MMF versus other immunosuppressants (non-MMF). Results/UNASSIGNED:The rates of missing data were 32% in the MMF and 23% in the non-MMF groups. As expected, the NRI missing data approach yielded the lowest estimated response rates. The smallest and least significant estimates of differences between groups were observed with LOCF, and precision was lowest with the CC method. Estimated between-group differences were magnified with the MI approach, and imputing SRI-5 directly versus deriving SRI-5 after separately imputing its individual components yielded similar results. Conclusion/UNASSIGNED:The potential advantages of applying MI to address missing data in an SLE trial include reduced bias when estimating treatment effects, and measures of precision that properly reflect uncertainty in the imputations. However, results can vary depending on the imputation model used, and the underlying assumptions should be plausible. Sensitivity analysis should be conducted to demonstrate robustness of results, especially when missing data proportions are high.

Objective/UNASSIGNED:Epidemiological data for primary discoid lupus erythematosus (pDLE) remain limited, particularly for racial/ethnic populations in the USA. The Manhattan Lupus Surveillance Program (MLSP) is a population-based retrospective registry of cases with SLE and related diseases including pDLE in Manhattan and was used to provide estimates of the prevalence and incidence of pDLE across major racial/ethnic populations. Methods/UNASSIGNED:MLSP cases were identified from rheumatologists, hospitals and population databases. Two case definitions were used for pDLE: the primary case definition which was any physician diagnosis found in the chart and a secondary case definition which was limited to cases diagnosed by a rheumatologist and/or dermatologist. Rates among Manhattan residents were age-adjusted, and capture-recapture analyses were conducted to assess case under-ascertainment. Results/UNASSIGNED:Based on the primary definition, age-adjusted overall prevalence and incidence rates of pDLE among Manhattan residents were 6.5 and 0.8 per 100 000 person-years, which increased to 9.0 and 1.3 after capture-recapture adjustment. Prevalence and incidence rates were approximately two and six times higher, respectively, among women compared with men (p<0.0001). Higher prevalence was also found among non-Latino blacks (23.5) and Latinos (8.2) compared with non-Latino whites (1.8) and non-Latino Asians (0.6) (p<0.0001). Incidence was highest among non-Latino blacks (2.4) compared with all other racial/ethnic groups. Similar relationships were observed for the secondary case definition. Conclusion/UNASSIGNED:Data from the MLSP provide epidemiological estimates for pDLE among the major racial/ethnic populations in the USA and reveal disparities in pDLE prevalence and incidence by sex and race/ethnicity among Manhattan residents.

OBJECTIVE:Cutaneous neonatal lupus (cNL) occurs in possibly 5%-16% of anti-Ro±anti-La antibody-exposed infants. Data suggest in utero exposure to hydroxychloroquine (HCQ) may prevent cardiac NL. The aim was to assess whether in utero exposure to HCQ decreases the risk of cNL and/or delays onset. METHODS:A multicentre case-control study was performed with 122 cNL cases and 434 controls born to women with a rheumatological disease who had documentation of maternal anti-Ro±anti-La antibodies at pregnancy and confirmation of medication use and the child's outcome. A secondary analysis was performed on 262 cNL cases, irrespective of maternal diagnosis, to determine if HCQ delayed time to cNL onset. RESULTS:Twenty (16%) cNL cases were exposed to HCQ compared with 146 (34%) controls (OR 0.4 (95% CI 0.2 to 0.6); p<0.01). Exposure to HCQ was associated with a reduced risk of cNL; exposure to anti-La antibody and female gender were associated with an increased risk of cNL. Exposure to HCQ remained significantly associated with a reduced cNL risk in the analyses limited to mothers with systemic lupus erythematosus and those who developed rash ≤1 month. When analysing all 262 cNL cases, HCQ-exposed infants were older (6.0 (95% CI 5.7 to 6.3) weeks) at cNL onset versus HCQ-non-exposed infants (4.4 (95% CI 3.9 to 5.0) weeks), but the difference was not statistically significant (p=0.21). CONCLUSION/CONCLUSIONS:Exposure to HCQ was associated with a reduced risk of cNL. Among cNL cases, those exposed to HCQ tend to have later onset of rash. Both findings suggest a protective effect of HCQ on cNL.

Non-bacterial thrombotic endocarditis in antiphospholipid syndrome presents a management dilemma. Large mobile valvular lesions pose an increased risk of stroke and arterial embolization. However, surgical excision or valve replacement in such patients carries high morbidity and mortality, while anticoagulation alone has limited data. We describe two patients with antiphospholipid syndrome presenting with neurologic events and large non-bacterial aortic valve vegetations. Both patients were successfully managed with anticoagulation and demonstrated rapid dissolution of lesions without evidence of recurrent embolic events. We provide a literature review describing additional cases managed with anticoagulation with dissolution of valvular lesions over time. Our cases further support the efficacy and safety of anticoagulation in patients with antiphospholipid syndrome and non-bacterial thrombotic endocarditis in the context of arterial embolization.