Spiera, Robert

ObjectiveTo discover biomarkers involved in the pathophysiology of antineutrophil cytoplasmic antibody–associated vasculitis (AAV) and to determine whether low‐density granulocytes (LDGs) contribute to gene expression signatures in AAV.MethodsThe source of clinical data and linked biologic specimens was a randomized controlled treatment trial in AAV. RNA sequencing of whole blood from patients with AAV was performed during active disease at the baseline visit and during remission 6 months later. Gene expression was compared between patients who met versus those who did not meet the primary trial outcome of clinical remission at 6 months (responders versus nonresponders). Measurement of neutrophil‐related gene expression was confirmed in peripheral blood mononuclear cells (PBMCs) to validate the findings in whole blood. A negative‐selection strategy isolated LDGs from PBMC fractions.ResultsDifferential expression between responders (n = 77) and nonresponders (n = 35) was detected in 2,346 transcripts at the baseline visit (P < 0.05). Unsupervised hierarchical clustering demonstrated a cluster of granulocyte‐related genes, including myeloperoxidase (MPO) and proteinase 3 (PR3). A granulocyte multigene composite score was significantly higher in nonresponders than in responders (P < 0.01) and during active disease than during remission (P < 0.01). This signature strongly overlapped an LDG signature identified previously in lupus (false discovery rate by gene set enrichment analysis <0.01). Transcription of PR3 measured in PBMCs was associated with active disease and treatment response (P < 0.01). LDGs isolated from patients with AAV spontaneously formed neutrophil extracellular traps containing PR3 and MPO.ConclusionIn AAV, increased expression of a granulocyte gene signature is associated with disease activity and decreased response to treatment. The source of this signature is likely LDGs, a potentially pathogenic cell type in AAV.

ObjectiveNonsevere relapses are more common than severe relapses in antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV), but their clinical course and treatment outcomes remain largely unexamined. We undertook this study to analyze the outcomes of patients with nonsevere relapses in the Rituximab in ANCA‐Associated Vasculitis (RAVE) trial who were treated with prednisone according to a prespecified protocol.MethodsRAVE was a randomized, double‐blind, placebo‐controlled trial comparing rituximab (RTX) to cyclophosphamide (CYC) followed by azathioprine (AZA) for induction of remission. Patients who experienced nonsevere relapses between months 1 and 18 were treated with a prednisone increase without a concomitant change in their nonglucocorticoid immunosuppressants, followed by a taper.ResultsForty‐four patients with a first nonsevere relapse were analyzed. In comparison to the 71 patients who maintained relapse‐free remission over 18 months, these patients were more likely to have proteinase 3–ANCAs, diagnoses of granulomatosis with polyangiitis (Wegener's), and a history of relapsing disease at baseline. A prednisone increase led to remission in 35 patients (80%). However, only 13 patients (30%) were able to maintain second remissions through the followup period (mean 12.5 months); 31 patients (70%) had a second disease relapse, 14 of them with severe disease. The mean time to second relapse was 9.4 months (4.7 months in the group treated with RTX versus 13.7 months in the group treated with CYC/AZA; P < 0.01). Patients who experienced nonsevere relapses received more glucocorticoids than those who maintained remission (6.7 grams versus 3.8 grams; P < 0.01).ConclusionTreatment of nonsevere relapses in AAV with an increase in glucocorticoids is effective in restoring temporary remission in the majority of patients, but recurrent relapses within a relatively short interval remain common. Alternative treatment approaches are needed for this important subset of patients.

ObjectiveCD5+ B cells have been conceptualized as a possible surrogate for Breg cells. The aim of the present study was to determine the utility of CD5+ B cells as biomarkers in antineutrophil cytoplasmic antibody–associated vasculitis (AAV).MethodsThe absolute and relative numbers (percentages) of CD5+ B cells (explanatory variables) were measured longitudinally during 18 months in 197 patients randomized to receive either rituximab (RTX) or cyclophosphamide (CYC) followed by azathioprine (AZA) for the treatment of AAV (Rituximab in ANCA‐Associated Vasculitis [RAVE] trial). Outcome variables included disease activity (status of active disease versus complete remission), responsiveness to induction therapy, disease relapse, disease severity, and, in RTX‐treated patients, relapse‐free survival according to the percentage of CD5+ B cells detected upon B cell repopulation.ResultsCD5+ B cell numbers were comparable between the treatment groups at baseline. After an initial decline, absolute CD5+ B cell numbers progressively increased in patients in the RTX treatment arm, but remained low in CYC/AZA‐treated patients. In both groups, the percentage of CD5+ B cells increased during remission induction and slowly declined thereafter. During relapse, the percentage of CD5+ B cells correlated inversely with disease activity in RTX‐treated patients, but not in patients who received CYC/AZA. No significant association was observed between the numbers of CD5+ B cells and induction treatment failure or disease severity. The dynamics of the CD5+ B cell compartment did not anticipate disease relapse. Following B cell repopulation, the percentage of CD5+ B cells was not predictive of time to flare in RTX‐treated patients.ConclusionThe percentage of peripheral CD5+ B cells might reflect disease activity in RTX‐treated patients. However, sole staining for CD5 as a putative surrogate marker for Breg cells did not identify a subpopulation of B cells with clear potential for meaningful clinical use. Adequate phenotyping of Breg cells is required to further explore the value of these cells as biomarkers in AAV.

ObjectiveDisease relapses are frequent in antineutrophil cytoplasmic antibody–associated vasculitis (AAV). This study was undertaken to evaluate outcomes in patients with AAV who are re‐treated with rituximab (RTX) and prednisone for severe disease relapses.MethodsThe Rituximab in AAV trial was a randomized, double‐blind, placebo‐controlled trial comparing the rates of remission induction among patients treated with RTX (n = 99) and patients treated with cyclophosphamide (CYC) followed by azathioprine (AZA) (n = 98). Prednisone was tapered to discontinuation after 5.5 months. After remission was achieved, patients who experienced a severe disease relapse between months 6 and 18 were eligible to receive RTX and prednisone on an open‐label basis according to a prespecified protocol. Investigators remained blinded with regard to the original treatment assignment.ResultsTwenty‐six patients received RTX for disease relapse after remission had initially been achieved with their originally assigned treatment. Fifteen of these patients were initially randomized to receive RTX and 11 to receive CYC/AZA. Thirteen (87%) of the patients originally assigned to receive RTX and 10 (91%) originally assigned to receive CYC/AZA achieved remission again with open‐label RTX (an overall percentage of 88%). In half of the patients treated with open‐label RTX, prednisone could be discontinued entirely. Patients in this cohort experienced fewer adverse events compared to the overall study population (4.7 adverse events per patient‐year versus 11.8 adverse events per patient‐year).ConclusionRe‐treatment of AAV relapses with RTX and glucocorticoids appears to be a safe and effective strategy, regardless of previous treatment.

ObjectiveTo 1) describe the distribution of patient global assessment (PtGA) scores of disease activity in patients with granulomatosis with polyangiitis (GPA; Wegener's), 2) explore the discordance between PtGA scores and physician global assessment (PhGA) scores of disease activity, and 3) explore whether PtGA scores during disease remission are associated with future disease relapse.MethodsData from the Wegener's Granulomatosis Etanercept Trial (WGET) were used. PtGA and PhGA scores were assessed on 100‐mm visual analog scales (VAS). Presence of active disease was determined using the Birmingham Vasculitis Activity Score for WG (BVAS/WG), and remission was defined as a BVAS/WG score of 0. Disease relapse was defined as a BVAS/WG score of >0 after remission had been achieved. Discordance between PtGA and PhGA scores was defined as a difference of ≥20 points between the two measures. Mixed linear models were used in longitudinal analysis of PtGA scores.ResultsData were obtained from 180 patients in the WGET cohort, seen at a total of 1,719 study visits. The mean ± SD PtGA and PhGA disease activity scores (on 100‐mm VAS) at baseline were 64.2 ± 27.4 and 55.5 ± 23.4, respectively. PtGA–PhGA discordance occurred in 53% of patients at baseline, and this was inversely associated with newly diagnosed disease (as opposed to relapsing disease) at baseline (odds ratio 0.37, 95% confidence interval [95% CI] 0.20–0.68) but not with age, sex, or presence of renal or pulmonary disease. Patients were in disease remission during 62% of the study visits. The mean PtGA score during visits immediately prior to relapse was 4.52 points higher (95% CI 0.66–8.4) than that at other remission visits (P = 0.03).ConclusionPtGA–PhGA discordance is common in GPA. A rise in the PtGA disease activity score during times defined by physicians as periods of remission is associated with subsequent occurrence of disease relapse. These findings support the addition of PtGA as an outcome measure for GPA.

ObjectiveTo evaluate the reasons that complete remission is not achieved or maintained with original treatment in some patients with antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV) treated with rituximab (RTX) or with cyclophosphamide/azathioprine (CYC/AZA).MethodsThe Rituximab in AAV trial was a randomized, double‐blind, placebo‐controlled trial comparing the rate of remission induction among patients treated with RTX (n = 99) and patients treated with CYC followed by AZA (n = 98). Glucocorticoids were tapered over a period of 5 months. The primary outcome measure was lack of disease activity without glucocorticoid treatment at 6 months. To determine the most important reason for failure to achieve the primary outcome, 7 hierarchical categories of reasons were defined retrospectively (uncontrolled disease, adverse event leading to therapy discontinuation, severe flare, limited flare, Birmingham Vasculitis Activity Score for Wegener's Granulomatosis >0, prednisone treatment at any dosage, and other).ResultsAlthough remission (lack of disease activity) was achieved in 170 of the 197 patients (86%) in the first 6 months, the primary outcome measure was not achieved in 42%. There were 3 deaths. Twenty‐four percent of the patients failed to achieve the primary end point due to active disease: 10 (5%) experienced uncontrolled disease in the first month and 37 (19%) experienced flares after initial improvement. In the majority of such patients, treatment with blinded crossover or according to best medical judgment led to disease control. Ninety‐one percent of patients who had uncontrolled disease or experienced a severe flare had proteinase 3 (PR3)–ANCA. When patients with uncontrolled disease were excluded from analysis, those who were PR3‐ANCA positive were found to experience fewer flares when treated with RTX compared to CYC/AZA (8 of 59 [14%] versus 20 of 62 [32%]; P = 0.02). Neither ANCA titers nor B cell counts predicted disease flare.ConclusionCurrent treatment regimens are largely successful in controlling AAV, but in approximately one‐fourth of patients, active disease persists or recurs in the first 6 months despite treatment. PR3‐ANCA positivity is a risk factor for recurrence or persistence of severe disease. ANCA titers and B cell detectability are poor predictors of both disease relapse and disease quiescence in the first 6 months.

ObjectiveTo identify genetic determinants of granulomatosis with polyangiitis (Wegener's) (GPA).MethodsWe carried out a genome‐wide association study (GWAS) of 492 GPA cases and 1,506 healthy controls (white subjects of European descent), followed by replication analysis of the most strongly associated signals in an independent cohort of 528 GPA cases and 1,228 controls.ResultsGenome‐wide significant associations were identified in 32 single‐nucleotide polymorphic (SNP) markers across the HLA region, the majority of which were located in the HLA–DPB1 and HLA–DPA1 genes encoding the class II major histocompatibility complex (MHC) DPβ chain 1 and DPα chain 1 proteins, respectively. Peak association signals in these 2 genes, emanating from SNPs rs9277554 (for DPβ chain 1) and rs9277341 (DPα chain 1) were strongly replicated in an independent cohort (in the combined analysis of the initial cohort and the replication cohort, P = 1.92 × 10−50 and 2.18 × 10−39, respectively). Imputation of classic HLA alleles and conditional analyses revealed that the SNP association signal was fully accounted for by the classic HLA–DPB1*04 allele. An independent single SNP, rs26595, near SEMA6A (the gene for semaphorin 6A) on chromosome 5, was also associated with GPA, reaching genome‐wide significance in a combined analysis of the GWAS and replication cohorts (P = 2.09 × 10−8).ConclusionWe identified the SEMA6A and HLA–DP loci as significant contributors to risk for GPA, with the HLA–DPB1*04 allele almost completely accounting for the MHC association. These two associations confirm the critical role of immunogenetic factors in the development of GPA.

Granulomatosis with polyangiitis (Wegener's) is a rare autoimmune neutrophil-mediated vasculitis that can cause renal disease and mucosal manifestations. Antineutrophil cytoplasmic antibodies (ANCA) are present in many patients, vary in level over time, and induce neutrophil activation through engagement with Fc receptors (FcRs). Given roles for FcRs in ANCA-mediated neutrophil activation and IgA antibodies in mucosal immunity, we hypothesized that FcR genetics and previously unappreciated IgA ANCA affect clinical presentation. We assembled a total of 673 patients and 413 controls from two multicenter cohorts, performed ELISA and immunofluorescence assays to determine IgA and IgG ANCA positivity, and used Illumina, TaqMan, or Pyrosequencing to genotype eight haplotype-tagging SNPs in the IgA FcR ( FCAR ) and to determine NA1/NA2 genotype of FCGR3B, the most prevalent neutrophil IgG FcR. We evaluated neutrophil activation by measuring degranulation marker CD11b with flow cytometry or neutrophil extracellcular trap formation with confocal microscopy. Functional polymorphisms in FCGR3B and FCAR differed between patient groups stratified by renal involvement. IgA ANCA were found in ∼30% of patients and were less common in patients with severe renal disease. Neutrophil stimulation by IgA or IgG ANCA led to degranulation and neutrophil extracellcular trap formation in a FcR allele-specific manner (IgA:FCAR P = 0.008; IgG:FCGR3B P = 0.003). When stimulated with IgA and IgG ANCA together, IgG ANCA induced neutrophil activation was reduced ( P = 0.0001). FcR genotypes, IgA ANCA, and IgG ANCA are potential prognostic and therapeutic targets for understanding the pathogenesis and presentation of granulomatosis with polyangiitis (Wegener's).