Stone, John H.

BACKGROUND Baseline expression of FCRL5 , a marker of naive and memory B cells, was shown to predict response to rituximab (RTX) in rheumatoid arthritis. This study investigated baseline expression of FCRL5 as a potential biomarker of clinical response to RTX in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). METHODS A previously validated quantitative PCR–based (qPCR-based) platform was used to assess FCRL5 expression in patients with GPA/MPA (RAVE trial, NCT00104299). RESULTS Baseline FCRL5 expression was significantly higher in patients achieving complete remission (CR) at 6, 12, and 18 months, independent of other clinical and serological variables, among those randomized to RTX but not cyclophosphamide-azathioprine (CYC/AZA). Patients with baseline FCRL5 expression ≥ 0.01 expression units (termed FCRL5 hi ) exhibited significantly higher CR rates at 6, 12, and 18 months as compared with FCRL5 lo subjects (84% versus 57% [ P = 0.016], 68% versus 40% [ P = 0.02], and 68% versus 29% [ P = 0.0009], respectively). CONCLUSION Our data taken together suggest that FCRL5 is a biomarker of B cell lineage associated with increased achievement and maintenance of complete remission among patients treated with RTX and warrant further investigation in a prospective manner. FUNDING The analysis for this study was funded by Genentech Inc.

Objective To identify risk alleles relevant to the causal and biologic mechanisms of antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV). Methods A genome‐wide association study and subsequent replication study were conducted in a total cohort of 1,986 cases of AAV (patients with granulomatosis with polyangiitis [Wegener's] [GPA] or microscopic polyangiitis [MPA]) and 4,723 healthy controls. Meta‐analysis of these data sets and functional annotation of identified risk loci were performed, and candidate disease variants with unknown functional effects were investigated for their impact on gene expression and/or protein function. Results Among the genome‐wide significant associations identified, the largest effect on risk of AAV came from the single‐nucleotide polymorphism variants rs141530233 and rs1042169 at the HLA–DPB1 locus (odds ratio [OR] 2.99 and OR 2.82, respectively) which, together with a third variant, rs386699872, constitute a triallelic risk haplotype associated with reduced expression of the HLA–DPB1 gene and HLA–DP protein in B cells and monocytes and with increased frequency of complementary proteinase 3 (PR3)–reactive T cells relative to that in carriers of the protective haplotype. Significant associations were also observed at the SERPINA1 and PTPN22 loci, the peak signals arising from functionally relevant missense variants, and at PRTN3 , in which the top‐scoring variant correlated with increased PRTN3 expression in neutrophils. Effects of individual loci on AAV risk differed between patients with GPA and those with MPA or between patients with PR3‐ANCAs and those with myeloperoxidase‐ANCAs, but the collective population attributable fraction for these variants was substantive, at 77%. Conclusion This study reveals the association of susceptibility to GPA and MPA with functional gene variants that explain much of the genetic etiology of AAV, could influence and possibly be predictors of the clinical presentation, and appear to alter immune cell proteins and responses likely to be key factors in the pathogenesis of AAV.

ObjectiveThe Rituximab in ANCA‐Associated Vasculitis (RAVE) trial compared rituximab to cyclophosphamide as induction therapy for the treatment of antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis. We undertook the current study to determine whether known single‐nucleotide polymorphisms (SNPs) for Fcγ receptors (FcγR) or cytochrome P450 (CYP) enzymes were associated with the response to treatment with rituximab and cyclophosphamide, respectively.MethodsFunctional SNPs for FcγR (FcγRIIa 519G>A, FcγRIIb 695T>C, FcγRIIIa 559T>G) and CYP enzymes (CYP2B6 1459C>T, CYP2C19 681G>A) were analyzed by direct sequencing of polymerase chain reaction–amplified genomic DNA. Each SNP was tested as a predictor of complete remission at 6 months or remission with continued prednisone administration using logistic regression and including the covariates of baseline Birmingham Vasculitis Activity Score for Wegener's Granulomatosis, ANCA type, and new versus relapsing disease. The associations of these SNPs with the secondary outcomes of time to complete remission, time to relapse, or time to B cell reconstitution were analyzed by Cox proportional hazard tests.ResultsNo significant associations were identified between complete remission and any FcγR genotype in the rituximab group or any CYP genotype in the cyclophosphamide group. However, when the treatment groups were combined, an association was found between the 519AA genotype of FcγRIIa and complete remission (P = 0.01). The 519AA genotype predicted complete remission (P = 0.006) and a shorter time to complete remission (P < 0.001).ConclusionThe finding that the homozygous FcγRIIa 519AA variant was associated with complete response and a shorter time to complete response in the RAVE trial, independent of treatment type, implies that FcγRIIa may be broadly involved in disease pathogenesis and response to therapy.

Objective S100A8/A9 (calprotectin) has shown promise as a biomarker for predicting relapse in antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV). This study was undertaken to investigate serum S100A8/A9 level as a biomarker for predicting future relapse in a large cohort of patients with severe AAV. Methods Serum levels of S100A8/A9 were measured at baseline and months 1, 2, and 6 following treatment initiation in 144 patients in the Rituximab in ANCA‐Associated Vasculitis trial (cyclophosphamide/azathioprine versus rituximab [RTX] for induction of remission) in whom complete remission was attained. Results Patients were divided into 4 groups: proteinase 3 (PR3)–ANCA with relapse (n = 37), PR3‐ANCA without relapse (n = 56), myeloperoxidase (MPO)–ANCA with relapse (n = 6), and MPO‐ANCA without relapse (n = 45). Serum S100A8/A9 level decreased in all groups during the first 6 months of treatment. The percentage reduction from baseline to month 2 was significantly different between patients who experienced a relapse and those who did not in the PR3‐ANCA group ( P = 0.046). A significantly higher risk of relapse was associated with an increase in S100A8/A9 level between baseline and month 2 ( P = 0.0043) and baseline and month 6 ( P = 0.0029). Subgroup analysis demonstrated that patients treated with RTX who had increased levels of S100A8/A9 were at greatest risk of future relapse ( P = 0.028). Conclusion An increase in serum S100A8/A9 level by month 2 or 6 compared to baseline identifies a subgroup of PR3‐ANCA patients treated with RTX who are at higher risk of relapse by 18 months. Since RTX is increasingly used for remission induction in PR3‐ANCA–positive patients experiencing a relapse, S100A8/A9 level may assist in identifying those patients requiring more intensive or prolonged treatment.

ObjectiveTo examine the relationship of antineutrophil cytoplasmic antibody (ANCA) type and ANCA‐associated vasculitis (AAV) diagnosis with demographic features, disease manifestations, and clinical outcomes. We focused on patients who account for the differences between ANCA type and disease type classifications: anti‐myeloperoxidase (MPO)–ANCA–positive and ANCA‐negative patients with granulomatosis with polyangiitis (Wegener's) (GPA).MethodsWe performed a pooled analysis of the Wegener's Granulomatosis Etanercept Trial and the Rituximab in ANCA‐Associated Vasculitis trial comparing patients with MPO‐ANCA–positive GPA and patients with ANCA‐negative GPA to patients with proteinase 3 (PR3)–ANCA–positive GPA and patients with MPO‐ANCA–positive microscopic polyangiitis (MPA).ResultsOf the 365 patients analyzed, 273 (75%) had PR3‐ANCA–positive GPA, 33 (9%) had MPO‐ANCA–positive GPA, 15 (4%) had ANCA‐negative GPA, and 44 (12%) had MPO‐ANCA–positive MPA. MPO‐ANCA–positive GPA patients were younger at diagnosis compared to MPO‐ANCA–positive MPA patients (53 versus 61 years; P = 0.02). Their disease manifestations and rates of relapse were similar to those of PR3‐ANCA–positive GPA patients. Relapse was more frequent in MPO‐ANCA–positive GPA patients than in patients with MPO‐ANCA–positive MPA at trial entry as well as at 12 and 18 months. ANCA‐negative patients with GPA had lower Birmingham Vasculitis Activity Score for Wegener's Granulomatosis scores at trial entry than PR3‐ANCA–positive patients with GPA (4.5 versus 7.7; P < 0.01), primarily because of a lower prevalence of renal involvement.ConclusionWe were unable to demonstrate important clinical differences between MPO‐ANCA–positive and PR3‐ANCA–positive patients with GPA. The risk of relapse was associated more closely with disease type than with ANCA type in this patient cohort. These findings deserve consideration in the assessment of relapse risk in patients with AAV.

Abstract Therapeutic monoclonal immunoglobulins (mAbs) are used to treat patients with a wide range of disorders including autoimmune diseases. As pharmaceutical companies bring more fully humanized therapeutic mAb drugs to the healthcare market analytical platforms that perform therapeutic drug monitoring (TDM) without relying on mAb specific reagents will be needed. In this study we demonstrate that liquid-chromatography?mass spectrometry (LC?MS) can be used to perform TDM of mAbs in the same manner as smaller nonbiologic drugs. The assay uses commercially available reagents combined with heavy and light chain disulfide bond reduction followed by light chain analysis by microflow-LC?electrospray ionization-quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF MS). Quantification is performed using the peak areas from multiply charged mAb light chain ions using an in-house developed software package developed for TDM of mAbs. The data presented here demonstrate the ability of an LC?MS assay to quantify a therapeutic mAb in a large cohort of patients in a clinical trial. The ability to quantify any mAb in serum via the reduced light chain without the need for reagents specific for each mAb demonstrates the unique capabilities of LC?MS. This fact, coupled with the ability to phenotype a patient?s polyclonal repertoire in the same analysis further shows the potential of this approach to mAb analysis.

Objective Relapse following remission is common in antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV), particularly with ANCAs directed at proteinase 3 (PR3). This study was undertaken to evaluate the association of an increase in PR3‐ANCA level with subsequent relapse. Methods Data from the Rituximab versus Cyclophosphamide for ANCA‐Associated Vasculitis (RAVE) trial were used. Starting from the time of achieving complete remission, serial measurements by direct and capture enzyme‐linked immunosorbent assays (ELISAs) were analyzed in 93 patients with PR3‐ANCA, using Cox proportional hazards regression. Results An increase in PR3‐ANCA level was identified in 58 of 93 subjects (62.4%) by direct ELISA and in 59 of 93 (63.4%) by capture ELISA. Relapses occurred in 55 of 93 subjects (59.1%), with 25 and 21 occurring within 1 year after an increase by direct ELISA and capture ELISA, respectively. An increase by direct ELISA was associated with subsequent severe relapses (hazard ratio [HR] 4.57; P < 0.001), particularly in patients presenting with renal involvement (HR 7.94; P < 0.001) and alveolar hemorrhage (HR 24.19; P < 0.001). Both assays identified increased risk for severe relapse in the rituximab group (HR 5.80; P = 0.002 for direct ELISA and HR 4.54; P = 0.007 for capture ELISA) but not the cyclophosphamide/azathioprine group ( P = 0.103 and P = 0.197, respectively). Conclusion The association of an increase in PR3‐ANCA level with the risk of subsequent relapse is partially affected by the PR3‐ANCA detection methodology, disease phenotype, and remission induction treatment. An increase in PR3‐ANCA level during complete remission conveys an increased risk of relapse, particularly severe relapse, among patients with renal involvement or alveolar hemorrhage and those treated with rituximab. Serial measurements of PR3‐ANCA may be informative in this subset of patients, but the risk of relapse must be weighed carefully against the risks associated with therapy.