To assess the clinical effects of rituximab therapy in patients with antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV).
To assess the clinical effects of rituximab therapy in patients with antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV).
The study group comprised 11 patients who had active AAV despite receiving maximally tolerated doses of cyclophosphamide or had contraindications for cyclophosphamide use. All patients had ANCA reactive against proteinase 3. The patients received rituximab infusions and glucocorticoids to induce remission. Three patients also received plasma exchange. No other immunosuppressive agents were used. Glucocorticoids were tapered as soon as control of disease activity was achieved. Disease activity was monitored using the Birmingham Vasculitis Activity Score, modified for Wegener's granulomatosis.
Rituximab therapy was well tolerated by all patients, and adverse events were rare. Following the rituximab infusions, circulating B lymphocytes became undetectable, and ANCA titers decreased significantly. Remission was achieved in all patients and was maintained while B lymphocytes were absent.
The ability to achieve stable remissions with rituximab in patients with AAV refractory to conventional therapy suggests that B lymphocyte depletion may be a safe, effective, mechanism-based treatment modality for treatment of patients with these conditions.
Wegener's granulomatosis (WG) and microscopic polyangiitis are potentially fatal forms of systemic vasculitis associated with antineutrophil cytoplasmic antibodies (ANCA). Cyclophosphamide remains the most effective treatment for ANCA-associated vasculitis (AAV) with severe organ- or life-threatening disease presentations (1). Selective suppression of immunoglobulin secretion by human B lymphocytes is thought to be an important mechanism explaining the efficacy of cyclophosphamide in AAV (2). Unfortunately, in ∼10% of patients the disease remains refractory to conventional therapy, and these patients experience severe, dose-limiting side effects of cyclophosphamide (1). Few treatment options are available for such patients.
Rituximab, a chimeric monoclonal antibody, induces selective depletion of B lymphocytes by targeting the CD20 surface antigen. Rituximab is an established treatment of non-Hodgkin's lymphoma, and interest in its use for autoimmune disease is growing (3). Anticipating that B lymphocyte depletion with rituximab, and a potential downstream effect on plasma cells, would result in the resolution of vasculitic symptoms and disappearance of ANCA and allow the tapering of glucocorticoids without relapse, we used rituximab to treat 11 patients with severe AAV refractory to conventional therapy, on a compassionate-use basis.
The study group comprised 11 consecutive patients who received treatment for severe AAV refractory to conventional therapy between January 2000 and September 2002. The review of these cases was approved by the Institutional Review Board. Patients were defined as having “refractory disease” if they had active disease that had not been controlled by the maximally tolerated cyclophosphamide dose given in conjunction with glucocorticoids (n = 8) or if treatment with cyclophosphamide was contraindicated because of previous drug-induced cystitis (n = 1) or profound and prolonged cytopenias (n = 2).
Clinical activity was assessed using a disease-specific activity index, the Birmingham Vasculitis Activity Score modification for WG (BVAS/WG) (4). BVAS/WG scores were determined by consensus between 2 physician-investigators (KK and US). “Severe disease” was defined as disease that posed an immediate threat to the patient's life or the function of a critical individual organ (items classified as “major” on the BVAS/WG instrument  and generally deemed to require therapy with cyclophosphamide). Remission was defined as a BVAS/WG score of 0 (4).
The clinical characteristics of the patients are summarized in Table 1. The median age of the patients (6 men and 5 women) was 31 years (range 14–71 years). The mean (±SD) disease duration was 57.8 ± 48.5 months. Ten patients had WG, and 1 had microscopic polyangiitis. All patients had active, severe disease, with BVAS/WG scores ranging from 3 to 11. Four patients had alveolar hemorrhage, which was documented by bronchoscopy with bronchoalveolar lavage in 3. (The fourth patient had hemoptysis, a decline in the hemoglobin concentration, and the rapid onset of ground-glass infiltrates on computed tomography of the chest in the absence of infection.) Five patients had active renal disease, which was documented by red blood cell (RBC) casts on urine microscopy; 3 of these patients had active glomerulonephritis confirmed by renal biopsy.
|Patient/age/sex||Duration of AAV, months||Treatment failure or contraindication||Failing therapy at time of rituximab||Active organ involvement at time of rituximab||BVAS/WG score, baseline/6 mos.||Followup, months||No. of rituximab courses||Comments on current status|
|1/71/M||67||CYC, AZA, MTX, MMF||MMF, 500 mg bid; pred., 40 mg qd||Kidney, joints||5/0||42||3||Stable remission with persistent B cell suppression†|
|2/69/F||2||CYC, MTX||CYC, 100 mg qd; pred., 60 mg qd||Lung, kidney, joints||8/0||19||1||Stable remission; clinical tolerance restored|
|3/23/M||78||CYC, MTX, MMF||CYC, 50 mg qd; pred., 30 mg qd||Lung, joints, skin||11/0||16||3||Stable remission with persistent B cell suppression|
|4/65/F||39||CYC, MTX, AZA||pred., 60 mg qd||ENT, eye||5/0||16||2||Stable remission, ANCA positive|
|5/50/M||62||CYC, MTX||CYC, 0.5 gm/m2/month; pred., 30 mg qd||ENT, lung, kidney, eye||10/0||17||1||Stable remission, clinical tolerance restored|
|6/31/M||142||CYC||MTX, 25 mg/week; pred., 80 mg qd||Lung, joints||5/0||14||1||Stable remission, clinical tolerance restored|
|7/62/F||18||CYC, MTX||CYC, 50 mg qd||Lung, kidney||7/0||12||2||Stable remission with persistent B cell suppression|
|8/29/F||142||CYC, MTX, MMF||CYC, 0.5 gm/m2/month; pred., 20 mg qd||Eye, joints||3/0||10||1||Stable remission; now receiving routine posttransplantation immunosuppression only‡|
|9/17/M||9||CYC, MTX||CYC, 100 mg qd; pred., 40 mg qd||Kidney, skin||7/0||10||2||Stable remission, ANCA positive|
|10/23/M||57||CYC, AZA||CYC, 150 mg qd; pred., 50 mg qd||ENT, lung||4/1||10||2||Stable remission with persistent B cell suppression|
|11/14/F||20||CYC, MTX||CYC, 50 mg qd||ENT, lung||3/0||10||1||Stable remission with persistent B cell suppression|
Glomerular filtration rates were estimated using the Cockroft-Gault equation. B cells were measured by fluorescence-activated cell sorting (FACS) analysis using CD19 as marker, with a lower detection limit of 1 cell per microliter. ANCA were determined at the Mayo Clinic Rochester Clinical Immunology laboratory using previously described procedures (5). The current procedure for ANCA testing, which was used in most of the patients in this study, is to perform proteinase 3 ANCA (PR3 ANCA)–specific direct enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays together. Before implementation of this practice early in the course of this study, patients for whom classic ANCA (cANCA) immunofluorescence testing had been performed had PR3 ANCA reactivity confirmed by capture ELISA (5). In our laboratory, cANCA titer determinations have an interobserver and intraobserver variability of ±1 dilution. Each titer is determined by 2 independent readers, and in case of disagreement, by a third reader. The average is reported as the final titer. The laboratory personnel were blinded to patients' clinical diagnoses or status.
The treatment protocol included rituximab (4 weekly doses of 375 mg/m2) and prednisone (up to 1 mg/kg/day), which was tapered once disease activity improved. In 8 patients, prednisone was preceded by intravenous methylprednisolone (1 gm/day for 3 days). Three patients with renal disease also received plasma exchange before receiving rituximab: patient 2 had three 3-liter replacements with fresh frozen plasma, patient 5 had six 4.5-liter replacements with albumin solution, and patient 9 had three 5-liter replacements with 1:4 fresh frozen plasma and albumin.
Data collection was censored on June 30, 2003. The initial treatment outcome of the index case at 18 months has been reported previously (6). Comparisons were performed using JMP software, version 5.0 (SAS Institute, Cary, NC). Student's paired t-tests were used when similar measurements were obtained in the same patient at different time intervals. For nonparametric data (BVAS/WG, ANCA), comparisons were made using a Wilcoxon's/Kruksal-Wallis test. Data are presented as the mean ± SD or median with interquartile ranges. P values less than 0.05 were considered significant.
Following rituximab therapy, peripheral blood B lymphocytes became undetectable in all patients. Disease remission was induced in all patients (Figure 1A), and glucocorticoids were tapered successfully in all (median dose 0 mg [range 0–1.5 mg]). Ten patients achieved BVAS/WG scores of 0 within 6 months. One patient (patient 10) had persistent lung nodules but no evidence of active disease in other organs. These nodules were given a score of 1 point (persistent BVAS/WG item) for as long as they were shrinking (4 months). Once no further change occurred (8 months), they were considered to be damage rather than active disease, and the BVAS/WG score was 0.
Six months after the initiation of rituximab therapy, significant reductions in the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) level were observed. The mean (±SD) ESR fell from 48.9 ± 48 mm/hour to 19 ± 22 mm/hour (n = 7; P < 0.02), and the mean (±SD) CRP level fell from 9.1 ± 9.5 mg/dl to 1.1 ± 1 mg/dl (n = 5; P < 0.002).
All patients were cANCA/PR3 ANCA positive before and at the time of rituximab infusion, with a mean ±SD of 4.1 ± 3 measurements preceding the first infusion. The baseline measurements shown in Figure 1B were obtained 5.3 ± 7 days before the start of infusion. Titers of cANCA decreased significantly in all patients, with 8 of the 11 patients becoming ANCA negative (Figure 1B).
For the 5 patients with active renal disease, the calculated mean ± SD creatinine clearance was 34 ± 25 ml/minute at baseline and 46 ± 33 ml/minute at 6 months (P = 0.48). Serial serum creatinine determinations for the individual patients are shown in Figure 1D. Two patients who were dialysis dependent before the initiation of rituximab also received plasma exchange. Both were able to discontinue dialysis. In another patient with renal involvement, the serum creatinine level increased from 1.3 gm/dl to 2.8 gm/dl within the first 3 weeks of therapy but then stabilized. Renal function in the other 2 patients with active renal disease remained unchanged during the period of observation. The microscopic hematuria with RBC casts resolved by 6 months in all 5 patients, and protein concentrations of spot urine samples fell from a mean ± SD of 120 ± 66 mg/dl to 56.8 ± 50 mg/dl (P = 0.008).
Following completion of glucocorticoid tapering, remission was maintained in every patient as long as B lymphocytes were undetectable. B lymphocytes returned in 9 patients during the followup period (4–12 months after infusion). Three of these 9 patients remained ANCA negative and experienced disease in remission (5, 8, and 10 months after return of B lymphocytes). Two of the 9 patients experienced a relapse after discontinuation of glucocorticoid therapy (at 7 and 12 months following rituximab treatment, respectively). Both relapses were associated with normal B lymphocyte counts and elevations in ANCA titers. Remissions were restored in both patients by prompt reinstitution of the rituximab/glucocorticoid regimen. Three of the 9 patients whose B lymphocytes recovered after treatment were re-treated preemptively with rituximab without glucocorticoids for rising ANCA titers (Figure 1C). In all 3 patients, disease remained in remission during followup. Finally, 1 patient who never became ANCA negative (albeit her baseline titers declined markedly) was re-treated preemptively when her ANCA titer rose and had no disease flare during the observation period.
Infusion-related adverse events were rare. One patient experienced mild angioedema following the first infusion, which was followed by polyarthritis after the fourth infusion. The polyarthritis was attributed to serum sickness and responded to short-term glucocorticoid treatment. Another patient described dizziness with the first infusion but tolerated subsequent infusions well. Other adverse events are included in Table 2, along with comments regarding their potential attribution. All 7 patients who had immunoglobulin determinations after their rituximab infusions had reduced IgM levels (mean ± SD nadir 14.3 ± 13.5 mg/dl; normal 60–300 mg/dl) and mildly reduced IgG levels (mean ± SD nadir 429.3 ± 145 mg/dl; normal 600–1,500 mg/dl).
|1||Presumed bacterial bronchitis (2 episodes)||Prompt response to oral antibiotics|
|3||Viral upper respiratory tract infection with conjunctivitis||Spontaneous resolution|
|4||Lower extremity petechiae (days 3–6 postinfusion)||Spontaneous resolution|
|5||Dizziness (during first infusion); MRSA sepsis with vertebral osteomyelitis following dialysis catheter placement||MRSA olecranon bursitis prior to rituximab treatment|
|6||Multiple exacerbations of respiratory infection||Known bronchiectasis; no change in frequency of infections before or after therapy|
|7||Thrombocytopenia (platelet count 43,000/mm3)||Timing and duration suggest that the cause was dapsone|
|8||Hypertension (during third infusion)||Volume-sensitive hemodialysis patient with similar prior episodes|
|11||Mild angioedema (during first infusion); polyarthritis following fourth infusion||Responded to a short burst in steroid dose|
The only proven therapy for severe AAV—cyclophosphamide combined with glucocorticoids—is associated with substantial morbidity and many potentially dose-limiting adverse effects (7, 8). Many of the deaths that occur during the treatment of AAV are related to therapy (1, 7, 8). Furthermore, a subset of patients fail to achieve stable remissions even with the maximally tolerated doses of cyclophosphamide (7, 8). Consequently, safe and effective alternatives to cyclophosphamide are needed for the treatment of AAV. This report of successful remission induction using rituximab in 11 patients with disease refractory to conventional treatment has several important implications.
First, the fact that in all patients disease remained in remission during the period of B lymphocyte depletion supports a central pathogenic role for B lymphocytes in AAV (2, 9). B lymphocytes may exert their pathogenic effects in AAV via several mechanisms, including costimulation, antigen presentation, and autoantibody production.
Second, the observation that disease remained in remission after restoration of B lymphocyte counts for as long as ANCA remained undetectable provides additional evidence for the importance of ANCA in disease pathophysiology. Prospective clinical studies have shown that the persistence or recurrence of ANCA in patients who have achieved clinical remission is a predictor of relapse (10). Many in vitro studies designed to investigate the pathogenic role of ANCA have identified a variety of proinflammatory effects of ANCA, and a direct pathogenic role of ANCA in vivo has now been documented in mice (11). Consequently, therapeutic interventions aimed at elimination of ANCA or at suppression of ANCA production are appealing. Compared with cyclophosphamide, rituximab may contribute to this goal in a more selective manner.
Third, our results suggest that either B lymphocytes or short-lived plasma cells (or both) rather than long-lived plasma cells are the cellular source of ANCA. ANCA titers declined in all of our patients and became negative in most. Long-lived plasma cells are not affected by anti-CD20 therapy. Consequently, as in other autoimmune diseases (12), B lymphocytes and short-lived plasma cells (which are dependent on antigen-specific B lymphocyte progenitors) seem to be primarily responsible for autoantibody production. However, our patients who had the highest initial ANCA titers did not become ANCA negative and subsequently had rises in the ANCA titer when B lymphocytes returned. Thus, for reasons that remain to be investigated formally, a single course of rituximab may not eliminate all ANCA-producing cells or their precursors in some patients.
The fact that in 3 patients ANCA did not return after reconstitution of B lymphocytes supports the hypothesis that rituximab therapy may result in restoration of tolerance to ANCA target antigens in AAV. If anti-CD20 therapy effectively eliminates B lymphocytes, there is a chance that the entire B cell lineage is restored once B lymphocyte production resumes. Antigens presented to immature lymphocytes tend to trigger tolerance by inducing anergy or elimination (13). In contrast, when antigen is presented only after the cells have matured and migrated to the periphery, cellular activation is the typical result (13). Although conventional immunosuppression failed to suppress ANCA production in our 11 patients, it does induce long-term ANCA negativity in some patients. The ability of rituximab to restore immune tolerance requires formal study.
Our study has several limitations, most of which are related to the fact that it was not a prospective clinical trial. The potentially confounding effects of concomitant therapies require consideration. The use of high-dose glucocorticoids in all patients undoubtedly facilitated the rapid induction of remission. However, most patients had active disease despite receiving significant doses of oral glucocorticoids and other immunosuppressants before receiving rituximab infusions. B cell depletion, in contrast, enabled induction and maintenance of remission despite subsequent tapering of glucocorticoids. This goal had not been achieved in these patients with any other regimen previously used. High-dose glucocorticoid therapy may also have contributed to the more precipitous fall in ANCA titers observed after initial treatment compared with that observed in the 3 patients who received rituximab alone preemptively. Other possible explanations for this observation include differences in sampling frequency and the fact that measured ANCA titers are the result of the balance between ANCA production and consumption. At the time of active systemic inflammation, ANCA consumption may result from ANCA binding to target antigen expressed on the surface of activated neutrophils and monocytes or released from these cells. In contrast, during remission, only the inhibition of ANCA production by rituximab may be a factor, and the rate of ANCA decline is a reflection of the normal half-life of IgG.
Plasma exchange may also have contributed to remission induction in the 3 patients who received this therapy concomitantly. It is remarkable, however, that none of the patients experienced disease flare following glucocorticoid withdrawal. The overall responses of the 5 patients with active renal disease to this rituximab/glucocorticoid regimen is very encouraging, particularly in view of the inability of other treatment approaches to control the disease safely. Further prospective studies of rituximab in patients with renal vasculitis associated with ANCA are indicated.
The lack of frequent B lymphocyte and ANCA determinations at predefined intervals in all patients does not allow us to state with certainty that the recurrences of ANCA or increases in ANCA titers were always preceded by the recurrence of B lymphocytes. However, the recurrence of ANCA or increases in ANCA titers were not detected in the absence of reconstituted B lymphocyte counts, and B lymphocyte reconstitution preceding the recurrence of ANCA or elevations of ANCA titers was documented in 3 cases. Furthermore, although FACS analysis of peripheral blood B lymphocytes represents the only practical way to measure these cells in humans, their numbers may inadequately reflect the presence of pathogenically significant tissue B lymphocytes.
Finally, the occurrence of human antichimeric antibody was not measured in our patients, because the assay is available only from the manufacturer of rituximab and because, to date, no evidence has been published suggesting that human antichimeric antibody formation is an important clinical phenomenon in patients treated with the doses of rituximab that we used. In contrast to what is observed with the use of other chimeric monoclonal antibodies (e.g., infliximab), the mechanism of action of rituximab itself might be expected to prevent formation of human antichimeric antibody. Nevertheless, this issue deserves further study.
Two smaller series of patients with AAV refractory to conventional treatment who appeared to benefit from rituximab were described recently (14, 15). In both series, rituximab was used in conjunction with other immunosuppressive agents, including intravenous cyclophosphamide, mycophenolate mofetil, azathioprine, or leflunomide. In contrast, in our patients all immunosuppressive agents other than glucocorticoids were discontinued before the institution of rituximab therapy.
In summary, our ability to achieve stable remissions using rituximab in patients with AAV refractory to conventional therapy suggests that B lymphocyte depletion may be a safe, effective, mechanism-based treatment modality for AAV. A randomized, double-blinded trial of this medication is now under way.