Faculty Bibliography

Background/Purpose : Osteoporosis and bone fractures are a frequent cause of morbidity in systemic lupus erythematosus (SLE), and are felt to be related both to disease activity and glucocorticoid (GC) exposure. Dual energy X-ray absorptiometry (DEXA) is the standard tool to assess bone density, but it does not measure bone quality or strength and is not a robust predictor of fractures in SLE. Clinical 3T MRI scans have been shown to assess information about bone not captured by DEXA. This study aims to evaluate factors associated with bone structure measured by DEXA and MRI in an SLE cohort. Methods : DEXAs were performed on 31 women with SLE and 3T MRI of the non-dominant hip were performed on 29 of these cases. Results were associated with multiple demographic, clinical and laboratory measures. MRI parameters measured included trabecular plate width (PW), trabecular plate to rod ratio (PRR), plate volume fraction (PVF), rod volume fraction (RVF), trabecular bone thickness (Tb.Th), trabecular spacing (Tb.Sp) and trabecular network area (TNA). DEXA BMD was measured, and osteoporosis (OP) was defined as hip, spine or femoral neck Z score < -2.0 in premenopausal women, and T score < -2.5 in others, and low bone density (LBD) as Z score < -2.0 in premenopausal women and T score < -1.0 in others. Results : By DEXA, 8/31 (25.8%) had OP and 12 (38.7%) had LBD. History of lymphopenia (75.0% vs. 31.8%, p=0.049) and lower concurrent HCQ dose (340 vs. 400 mg, p=0.006) associated with DEXA OP, while older age (48.3 vs. 36.3 y, p=0.024) associated with LBD. Higher ESR was inversely correlated with favorable bone structure (PW r(22) = -.49, p=0.025, PRR rs = -.51, p=0.018, PVF rs = -.51, p=0.018, RVF rs = .51, p=0.018, Tb.Th rs = -.58, p=0.005, Tb.Sp rs = .44, p=0.046, TNA rs = -.50, p=0.022). Higher CRP was likewise inversely correlated with favorable bone structure (PW r(20) = -.61, p=0.004, PRR rs = -.57, p=0.009, PVF rs = -.57, p=0.009, RVF rs =.57, p=0.009, Tb.Th rs = -.56, p=.011, Tb.Sp rs =.67, p=0.001, TNA rs = -.64, p=0.002). A history of lupus nephritis was associated with unfavorable bone structure (PW 705.3 vs. 833.3 mum, p=0.048, PRR 6.6 vs. 8.1, p=0.024, PVF 0.83 vs. 0.89, p=0.024, RVF 0.17 vs. 0.11, p=0.024, Tb.Th 178.1 vs. 193.4 mm, p=0.012, Tb.Sp 358.6 vs. 296.5 mm, p=0.056, TNA 0.41 vs. 0.54 (1/mm), p=0.009). ESR, CRP and history of lupus nephritis were not significantly associated with DEXA hip BMD, OP or LBD. MRI parameters for favorable bone structure were inversely correlated with DEXA hip BMD (PW r(28) = -.47, p=0.011, Tb.Th rs = -.53, p=0.003) and BMI (PW r(28) = -.54, p=0.003, TbTh rs = -.72, p< 0.001, TNA rs = -.44, p=0.017). Conclusion : Higher ESR and CRP and a history of lupus nephritis associated with MRI parameters of unfavorable bone structure, but did not associate with DEXA abnormalities in SLE patients. MRI may be a more sensitive tool than DEXA to measure inflammatory effects on bone and potentially cumulative dose of steroid exposure. There were inverse correlations of MRI parameters with traditional osteoporosis risk factors and BMD measures on DEXA, and it is possible that each tool evaluates different aspects of bone health. Further evaluation of MRI screening for fracture risk in SLE and GC exposed individuals is warranted to better quantify risk and guide treatment

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: 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 : Autoantibody production precedes SLE or SS by years, including anti-Ro. Anti-Ro + mothers of children with congenital heart block (CHB) are a unique population at risk for pathologic autoimmunity, as many are asymptomatic (Asym/UAS) and become aware of autoantibodies due to fetal disease and yet have a 10-year progression rate to SS/SLE of 20%-30%. We hypothesized that variation in the oral microbiome correlates with transition to SLE or SS and pathogenicity involves sequence homology between Ro60 and bacterial von Willebrand factor type A domain protein (vWFA).
Method(s): The oral microbiome of 25 anti-Ro + mothers of CHB children (Asym/UAS, N=9; SS/SLE, N=16) and 7 healthy controls (HC) were processed using 16S ribosomal RNA sequencing. Analysis of variance methods compared the centered log ratio transformed relative abundances for 1) HC vs. anti-Ro + mothers, and 2) assuming an ordering of severity from HC < Asym/UAS < SS/SLE. To adjust for multiple comparisons, a taxonomic stepdown method coupled with false discovery rate (FDR) was used. The Basic Local Alignment Search Tool evaluated homology of Ro60 at aa 371-381 and peptides of vWFA. Results : Sequencing 16S rRNA identified microorganisms from 2 kingdoms, 16 phyla, 25 classes, 41 orders, 70 families, 164 genera, and 166 species. The Shannon Index (H) revealed that for each taxonomic level except species, there were significant reductions in diversity in the anti-Ro + mothers relative to HC (P <= 0.05). There were global differences in the microbiota of these mothers relative to HC (perMANOVA P=0.00049). The phylum Actinobacteria was more abundant in the anti-Ro + mothers vs HC (P FDR =0.0231). Within Actinobacteria , the class Coriobacteriia and subsequent lower taxonomic levels down to Atopobium parvulum , all exhibited increases in relative abundance in the anti-Ro + mothers compared to HC. There was a significant reduction in the relative abundance as clinical severity increased within one of the most frequent phyla, Proteobacteria (P FDR =0.030; mean+/-SD; HC 0.24+/-0.07; Asym/UAS 0.19+/-0.12; SS/SLE 0.11+/-0.08). The difference in the relative abundances between Asym/UAS and SS/SLE within Proteobacteria was significant (P=0.042). Within Proteobacteria , the common class Betaproteobacteria also showed reduced relative abundance with increasing clinical severity (P FDR =0.0037; HC 0.11+/-0.04; Asym/UAS 0.072+/-0.07; SS/ SLE 0.031+/-0.04). These ordered differences were maintained down the taxonomic hierarchy to the genus ( Lautropia , P FDR =0.0072) and species within this genus ( L. mirabilis , P FDR =0.012). Next, sequences of vWFA secreted by these taxa were evaluated. For a comparison of Ro60 T cell epitope, FLLAVDVSASMNQ, the vWFA, VLVVFDNSSSMTA vWFA of A. parvulum was not a fit due to the aromatic and polar aa at positions 5 and 9, respectively. In contrast, the vWFA of L. mirabilis , LLLLLDVSGSMAG, was identical at 7 of the first 11 aa. Conclusion : These data provide evidence that the microbiome differs along a clinical spectrum of autoimmunity. In part, the data refiect a path involving depletion of L. mirabilis , which is secondary to a pathologic role of anti-Ro along with an expansion of A. parvulum , an opportunistic taxon

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 : 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 : Early treatment initiation in rheumatoid arthritis (RA) is fundamental to avoid chronic joint destruction and disability. Despite remarkable advances in RA therapeutics, oral methotrexate (MTX) remains the anchor drug and mainstay of treatment worldwide. However, MTX bioavailability has a wide inter-individual variability and >50% of patients with moderate or severe RA show no or suboptimal improvement in their symptoms in response to MTX. The reasons for these disparities in treatment response remain unclear. Prior studies have shown that the biotransformation of MTX is altered in germ-free and microbiome-depleted mice, prompting us to hypothesize that inter-individual differences in the human gut microbiome could impact drug bioavailability and thus clinical efficacy. We sought to determine differences in the microbiome of drug-naive, new onset RA (NORA) patients that could predict response to MTX therapy. Methods : We enrolled 27 drug-naive, NORA patients priori to MTX initiation (test cohort), and classified them as either MTX-responders (MTX-R; 39% of the cohort) or non-responders (MTX-NR; 61%) based on a stringent definition of clinical response (delta improvement of DAS28 >1.8 by month 4). We performed 16S rRNA gene and Shotgun Metagenomic sequencing on the baseline gut microbiomes of these NORA patients and confirmed the results in an independent validation cohort (n=31). NMR and LC-MS were performed in ex vivo incubations to measure the capacity of each NORA microbiome to metabolize MTX. Results : Our analysis revealed significant associations between the abundance of gut bacterial taxa and future MTX response. Patients that responded to therapy had significantly lower microbial diversity (p< 0.05). A significant difference in overall gut microbial community structure was also observed between groups (Bray-Curtis distance; PERMANOVA < 0.05). At the class level, we observed statistically higher abundance of Clostridia and lower abundance of Bacteroidia in MTX-NR (p< 0.05; q< 0.2). Furthermore, the baseline metagenome separated most MTX-R from MTX-NR (PCoA; PERMANOVA p< 0.05). We identified 8 microbial modules and 23 pathways, whose abundance significantly differed between groups (p< 0.05, q< 0.2), including genes related with purine and MTX metabolism, indicating a major difference in metabolic and biosynthetic potential between the microbiome of MTX-R and MTX-NR patients. Machine learning techniques were applied to this metagenomic data, resulting in a robust model based on bacterial gene abundance that accurately predicted response to MTX in an independent cohort. Finally, MTX available levels remaining after ex vivo incubation with distal gut samples from pre-treatment RA patients significantly correlated with the magnitude of future clinical response, suggesting a direct effect of the gut microbiome on MTX bioavailability and response to therapy. Conclusion : Together, these results provide the first step towards predicting response to oral MTX in NORA patients and support the utility of the gut microbiome as a prognostic tool and perhaps even as a target for manipulation in the treatment of rheumatic and autoimmune disease

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