Marc C. Levesque

Rituximab in the Treatment of Refractory Adult and Juvenile Dermatomyositis and Adult Polymyositis: A Randomized, Placebo-phase Trial

OBJECTIVE To assess the safety and efficacy of rituximab in a randomized, double-blind, placebo-phase trial in adult and pediatric myositis patients. METHODS Adults with refractory polymyositis (PM) and adults and children with refractory dermatomyositis (DM) were enrolled. Entry criteria included muscle weakness and ≥2 additional abnormal values on core set measures (CSMs) for adults. Juvenile DM patients required ≥3 abnormal CSMs, with or without muscle weakness. Patients were randomized to receive either rituximab early or rituximab late, and glucocorticoid or immunosuppressive therapy was allowed at study entry. The primary end point compared the time to achieve the International Myositis Assessment and Clinical Studies Group preliminary definition of improvement (DOI) between the 2 groups. The secondary end points were the time to achieve ≥20% improvement in muscle strength and the proportions of patients in the early and late rituximab groups achieving the DOI at week 8. RESULTS Among 200 randomized patients (76 with PM, 76 with DM, and 48 with juvenile DM), 195 showed no difference in the time to achieving the DOI between the rituximab late (n = 102) and rituximab early (n = 93) groups (P = 0.74 by log rank test), with a median time to achieving a DOI of 20.2 weeks and 20.0 weeks, respectively. The secondary end points also did not significantly differ between the 2 treatment groups. However, 161 (83%) of the randomized patients met the DOI, and individual CSMs improved in both groups throughout the 44-week trial. CONCLUSION Although there were no significant differences in the 2 treatment arms for the primary and secondary end points, 83% of adult and juvenile myositis patients with refractory disease met the DOI. The role of B cell-depleting therapies in myositis warrants further study, with consideration for a different trial design.

Patients with Idiopathic Pulmonary Fibrosis with Antibodies to Heat Shock Protein 70 Have Poor Prognoses

RATIONALE Diverse autoantibodies are present in most patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that specific autoantibodies may associate with IPF manifestations. OBJECTIVES To identify clinically relevant, antigen-specific immune responses in patients with IPF. METHODS Autoantibodies were detected by immunoblots and ELISA. Intrapulmonary immune processes were evaluated by immunohistochemistry. Anti-heat shock protein 70 (HSP70) IgG was isolated from plasma by immunoaffinity. Flow cytometry was used for leukocyte functional studies. MEASUREMENTS AND MAIN RESULTS HSP70 was identified as a potential IPF autoantigen in discovery assays. Anti-HSP70 IgG autoantibodies were detected by immunoblots in 3% of 60 control subjects versus 25% of a cross-sectional IPF cohort (n = 122) (P = 0.0004), one-half the patients with IPF who died (P = 0.008), and 70% of those with acute exacerbations (P = 0.0005). Anti-HSP70 autoantibodies in patients with IPF were significantly associated with HLA allele biases, greater subsequent FVC reductions (P = 0.0004), and lesser 1-year survival (40 ± 10% vs. 80 ± 5%; hazard ratio = 4.2; 95% confidence interval, 2.0-8.6; P < 0.0001). HSP70 protein, antigen-antibody complexes, and complement were prevalent in IPF lungs. HSP70 protein was an autoantigen for IPF CD4 T cells, inducing lymphocyte proliferation (P = 0.004) and IL-4 production (P = 0.01). IPF anti-HSP70 autoantibodies activated monocytes (P = 0.009) and increased monocyte IL-8 production (P = 0.049). ELISA confirmed the association between anti-HSP70 autoreactivity and IPF outcome. Anti-HSP70 autoantibodies were also found in patients with other interstitial lung diseases but were not associated with their clinical progression. CONCLUSIONS Patients with IPF with anti-HSP70 autoantibodies have more near-term lung function deterioration and mortality. These findings suggest antigen-specific immunoassays could provide useful clinical information in individual patients with IPF and may have implications for understanding IPF progression.

Predictors of Clinical Improvement in Rituximab-Treated Refractory Adult and Juvenile Dermatomyositis and Adult Polymyositis

To identify the clinical and laboratory predictors of clinical improvement in a cohort of myositis patients treated with rituximab.

Single cell analysis reveals oligoclonality among “low count” monoclonal B cell lymphocytosis

Monoclonal B-cell lymphocytosis (MBL) is a preclinical hematologic syndrome characterized by small accumulations of CD5+ B lymphocytes. Most MBL share phenotypic characteristics with chronic lymphocytic leukemia (CLL). Although some MBL progress to CLL, most MBL have apparently limited potential for progression to CLL, particularly those MBL with normal absolute B-cell counts (‘low-count’ MBL). Most CLL are monoclonal and it is not known whether MBL are monoclonal or oligoclonal; this is important because it is unclear whether MBL represent indolent CLL or represent a distinct premalignant precursor before the development of CLL. We used flow cytometry analysis and sorting to determine immunophenotypic characteristics, clonality and molecular features of MBL from familial CLL kindreds. Single-cell analysis indicated four of six low-count MBL consisted of two or more unrelated clones; the other two MBL were monoclonal. 87% of low-count MBL clones had mutated immunoglobulin genes, and no immunoglobulin heavy-chain rearrangements of VH family 1 were observed. Some MBL were diversified, clonally related populations with evidence of antigen drive. We conclude that although low-count MBL share many phenotypic characteristics with CLL, many MBL are oligoclonal. This supports a model for step-wise development of MBL into CLL.

Rituximab Therapy for Primary Sjögren’s Syndrome: An Open-Label Clinical Trial and Mechanistic Analysis

OBJECTIVE To study the safety and clinical efficacy of rituximab therapy for primary Sjögrens syndrome, as well as to investigate its mechanisms. METHODS Patients with primary Sjögrens syndrome were enrolled in an open-label trial, were given rituximab (1 gm) infusions on days 1 and 15, and were monitored through week 52. The primary end point was safety, with secondary end points evaluating clinical and biologic efficacy. Blood was obtained for enumeration of lymphocyte subsets, measurement of serum autoantibody and BAFF levels, and analysis of gene expression. RESULTS Twelve female patients with primary Sjögrens syndrome were administered rituximab. They had a median age of 51 years (range 34-69 years) and a median disease duration of 8.0 years (range 2-18 years). We observed no unexpected toxicities from the rituximab therapy. Modest improvements were observed at week 26 in patient-reported symptoms of fatigue and oral dryness, with no significant improvement in the objective measures of lacrimal and salivary gland function. The recovery of blood B cells following the nadir from rituximab therapy was characterized by a predominance of transitional B cells and a lack of memory B cells. While blood B cell depletion was associated with an increase in serum BAFF levels, no significant changes were observed in the levels of serum anti-Ro/SSA, anti-La/SSB, and anti-type 3 muscarinic acetylcholine receptor autoantibodies or in the blood interferon signature. CONCLUSION In patients with primary Sjögrens syndrome, a single treatment course of rituximab was not associated with any unexpected toxicities and led to only modest clinical benefits despite effective depletion of blood B cells.

Plasma B Lymphocyte Stimulator and B Cell Differentiation in Idiopathic Pulmonary Fibrosis Patients

We hypothesized B cells are involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a progressive, restrictive lung disease that is refractory to glucocorticoids and other nonspecific therapies, and almost invariably lethal. Accordingly, we sought to identify clinically associated B cell–related abnormalities in these patients. Phenotypes of circulating B cells were characterized by flow cytometry. Intrapulmonary processes were evaluated by immunohistochemistry. Plasma B lymphocyte stimulating factor (BLyS) was assayed by ELISA. Circulating B cells of IPF subjects were more Ag differentiated, with greater plasmablast proportions (3.1 ± 0.8%) than in normal controls (1.3 ± 0.3%) (p < 0.03), and the extent of this differentiation correlated with IPF patient lung volumes (r = 0.44, p < 0.03). CD20+ B cell aggregates, diffuse parenchymal and perivascular immune complexes, and complement depositions were all prevalent in IPF lungs, but much less prominent or absent in normal lungs. Plasma concentrations of BLyS, an obligate factor for B cell survival and differentiation, were significantly greater (p < 0.0001) in 110 IPF (2.05 ± 0.05 ng/ml) than among 53 normal (1.40 ± 0.04 ng/ml) and 90 chronic obstructive pulmonary disease subjects (1.59 ± 0.05 ng/ml). BLyS levels were uniquely correlated among IPF patients with pulmonary artery pressures (r = 0.58, p < 0.0001). The 25% of IPF subjects with the greatest BLyS values also had diminished 1-y survival (46 ± 11%), compared with those with lesser BLyS concentrations (81 ± 5%) (hazard ratio = 4.0, 95% confidence interval = 1.8–8.7, p = 0.0002). Abnormalities of B cells and BLyS are common in IPF patients, and highly associated with disease manifestations and patient outcomes. These findings have implications regarding IPF pathogenesis and illuminate the potential for novel treatment regimens that specifically target B cells in patients with this lung disease.

Immunophenotypic and gene expression analysis of monoclonal B-cell lymphocytosis shows biologic characteristics associated with good prognosis CLL

Monoclonal B-cell lymphocytosis (MBL) is a hematologic condition wherein small B-cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and ‘CLL-like’ MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. In all, 91 unique MBL clones were detected: 73 CLL-like MBL (CD5+CD20dimsIgdim), 11 atypical MBL (CD5+CD20+sIg+) and 7 CD5neg MBL (CD5negCD20+sIgneg). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70 and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on seven CLL-like MBL, and showed activation of B-cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.

The effect of targeted rheumatoid arthritis therapies on anti-citrullinated protein autoantibody levels and B cell responses.

Rheumatoid arthritis (RA) is a complex inflammatory disorder associated with synovitis and joint destruction that affects an estimated 1·3 million Americans and causes significant morbidity, a reduced life‐span and lost work productivity. The use of biological therapies for the treatment of RA is costly, and the selection of therapies is still largely empirical and not guided by the underlying biological features of the disease in individual patients. The synovitis associated with RA is characterized by an influx of B and T cells, macrophages and neutrophils and the expansion of fibroblast‐like synoviocytes, which form pannus and lead to cartilage and bone destruction. RA is associated with synovial production of rheumatoid factor (RF) and anti‐citrullinated protein autoantibodies (ACPA) and with the production of inflammatory cytokines, including interleukin (IL)‐1, IL‐6, IL‐17 and tumour necrosis factor (TNF)‐α, which are targets for RA therapeutics. Recent ideas about the pathogenesis of RA emphasize a genetic predisposition to develop RA, a preclinical phase of disease that is associated with the production of ACPA and the development of symptomatic disease following inflammatory initiating events that are associated with expression of citrullinated epitopes in the joints of patients. However, we still have a limited understanding of the cytokine and intracellular pathways that regulate ACPA levels. In humans, therapy with biological agents affords a unique opportunity to better understand the cytokine and signalling pathways regulating ACPA levels and the impact of ACPA level changes on disease activity. In this study we summarize the effect of RA therapies on ACPA levels and B cell responses.

Do patients with juvenile idiopathic arthritis in clinical remission have evidence of persistent inflammation on 3T magnetic resonance imaging

Up to 90% of adults with rheumatoid arthritis (RA) in clinical remission have persistent synovitis and/or bone marrow lesions (BMLs) on magnetic resonance imaging (MRI). MRI findings in patients with juvenile idiopathic arthritis (JIA) in clinical remission have not been described. We utilized 3T MRI with contrast enhancement to examine JIA patients with hand and/or wrist involvement who were in clinical remission and compared them with a cohort of adult RA patients.

Anti-Cyclic Citrullinated Peptide Testing for the Diagnosis of Rheumatoid Arthritis and the Quest for Improved Sensitivity and Predictive Value

In this issue of Arthritis & Rheumatism, van der Linden and colleagues (1) present comparative studies of different anti–cyclic citrullinated peptide (anti-CCP) tests in patients from the Leiden Early Arthritis Clinic. These studies are important for selecting the most clinically useful anti-CCP tests and for better understanding the role of autoantibodies to citrullinated epitopes in the pathogenesis of rheumatoid arthritis (RA). The use of the term anti-CCP to introduce this topic reminds us of the important and dominant role that this term has taken on in our rheumatology lexicon. The term anti-CCP is often used to refer to not only antibodies against CCP epitopes but also antibodies directed to noncyclic citrullinated protein (or peptide) epitopes (ACPAs). For most practicing rheumatologists, placing an order for an anti-CCP test usually means that results will be generated using a so-called second-generation antiCCP test (anti–CCP-2) that employs a proprietary citrullinated cyclic peptide. The patent for the anti–CCP-2 test is held by the Netherlands Technology Foundation, and exclusive global rights to the proprietary CCP or peptides used in all anti–CCP-2 tests are held by AxisShield (Dundee, UK) and Euro-Diagnostica (Arnhem, The Netherlands). The anti–CCP-2 test is somewhat unusual among laboratory tests, because its derivation was based on empiricism and molding of the assay to achieve an RA diagnostic test with the highest sensitivity and specificity. The development of the anti-CCP test has been somewhat contrary to our usual reductionist tendencies for the establishment of other clinical tests, in which specificity typically requires pathophysiologic knowledge of the disease process and a focus on an explicit antigen or other marker of the disease process. To understand how the anti-CCP test was developed, it is valuable to understand its origins. The antiCCP test had its origins in studies performed more than 40 years ago, when serum autoantibodies that bound to perinuclear structures and to keratin were identified in RA patients (2,3). In 1998, Schellekens and colleagues identified citrulline as an important component of epitopes for RA autoantibodies that react with tissue fillagrin (4). This same group of investigators developed a first-generation anti-CCP test (anti–CCP-1) using a cyclic derivative of a citrullinated fillagrin peptide (5). In contrast to linear peptides, cyclic peptides are more conformationally constrained, and this was the rationale for using cyclic derivatives of citrullinated peptides in the original anti–CCP-1 assay to improve the sensitivity of the assay. The first-generation anti–CCP-1 test had a sensitivity for RA that was lower than the sensitivity of the rheumatoid factor (RF) test but had greater specificity than the RF test (5,6). In 2002, a second-generation anti-CCP test (anti– CCP-2) was developed by screening CCPs using RA patient sera and selecting the cyclic peptides with the highest sensitivity and specificity for a diagnosis of RA. The anti–CCP-2 test retains the high specificity (95%) of the anti–CCP-1 test and has a higher sensitivity than the anti–CCP-1 test, with a sensitivity for RA that is