ARTHRITIS & RHEUMATISMVol. 60, No. 7, July 2009, pp 2156–2168DOI 10.1002/art.24637© 2009, American College of Rheumatology

A Multicenter Survey of Rituximab Therapy for RefractoryAntineutrophil Cytoplasmic Antibody–Associated Vasculitis

Rachel B. Jones,1 Alastair J. Ferraro,2 Afzal N. Chaudhry,1 Paul Brogan,3 Alan D. Salama,4

Kenneth G. C. Smith,5 Caroline O. S. Savage,2 and David R. W. Jayne1

Objective. B cell depletion with rituximab hasallowed remissions in relapsing or refractory antineu-trophil cytoplasmic antibody (ANCA)–associated vascu-litis in small studies. The aim of this study was todetermine the efficacy and safety of rituximab forANCA-associated vasculitis in a larger multicenter co-hort. This permitted comparison of rituximab dosingregimens, the value of continuing immunosuppression,and investigation of ANCA and B cell levels as re-treatment biomarkers.

Methods. Retrospective, standardized data collec-tion from 65 sequential patients receiving rituximab forrefractory ANCA-associated vasculitis at 4 centers inthe UK was used.

Results. All patients achieved B cell depletion.Complete remission occurred in 49 of the 65 patients

(75%), partial remission in 15 (23%), and no response in1 (2%). The prednisolone dosage was reduced from 12.5mg/day (median) to 9.0 mg/day at 6 months (P �0.0006). Immunosuppressive therapy was withdrawn in37 of 60 patients (62%). Twenty-eight of 49 patients whoachieved full remission (57%) experienced relapse (me-dian 11.5 months). B cell return preceded relapse in 14of 27 patients (52%). Although ANCA levels fell afterrituximab therapy, relapse was not associated with ANCApositivity or a rise in ANCA levels. Neither the initialrituximab regimen (4 infusions of 375 mg/m2 each given1 week apart or 2 infusions of 1 gm each given 2 weeksapart) nor withdrawal of immunosuppressive therapy(37 of 60 patients [62%]) influenced the timing ofrelapse. Thirty-eight patients received >2 courses ofrituximab, and complete remission was induced ormaintained in 32 of them (84%). IgM levels fell, al-though IgG levels remained stable. Forty-six seriousadverse events occurred, including 2 episodes of late-onset neutropenia, which were attributed to rituximab.

Conclusion. Rituximab was effective remissioninduction therapy for refractory ANCA-associated vas-culitis in this study. There was no difference in efficacybetween the 2 main treatment regimens. Continuingimmunosuppression did not reduce relapses. Relapsesoccurred, but re-treatment was effective and safe. Therewas no clear influence of rituximab on the frequency ofserious adverse events. ANCA and B cell levels lackedsufficient sensitivity to guide the timing of re-treatment.

Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis is a multisystem autoimmune dis-ease characterized by ANCA production and small-vessel inflammation. The ANCA-associated vasculitidescomprise Wegener’s granulomatosis (WG), microscopicpolyangiitis (MPA), and Churg-Strauss syndrome (CSS),which have similar clinical and serologic features andsimilar treatment responses.

Standard immunosuppressive therapies for

Supported by the National Institute for Health ResearchCambridge Biomedical Research Centre. Dr. Ferraro’s work wassupported by an MRC Clinical Research Training Fellowship. Dr.Salama’s work was supported by the National Institute for HealthResearch Imperial College Biomedical Research Centre.

1Rachel B. Jones, BSc, MBBS, MRCP, Afzal N. Chaudhry,BSc, MBBS, PhD, FRCP, David R. W. Jayne, MD, FRCP: Adden-brooke’s Hospital, Cambridge, UK; 2Alastair J. Ferraro, BM, MCh,MRCP, Caroline O. S. Savage, FRCP, FMedSci: University Hospital,Birmingham, UK; 3Paul Brogan, BSc, MBChB, MRCPCH, MSc, PhD:Great Ormond Street Hospital for Children, London, UK; 4Alan D.Salama, MBBS, PhD, FRCP: Hammersmith Hospital, London, UK;5Kenneth G. C. Smith, PhD, FRACP, FRCPA, FRCP, FMedSci:Addenbrooke’s Hospital, and University of Cambridge School ofClinical Medicine, Cambridge, UK.

Dr. Salama has received consulting fees, speaking fees, and/orhonoraria from Roche and Novartis (less than $10,000 each). Dr.Smith has received an honorarium from Roche for attending anAdvisory Board meeting (less than $10,000). Dr. Savage has receivedconsulting fees from Talecris Biotherapeutics and Biovitrum andspeaking fees from Roche (less than $10,000 each). Dr. Jayne hasreceived consulting fees, speaking fees, and/or honoraria from Rocheconcerning rituximab (less than $10,000), as well as research grantsupport from Roche for investigator-initiated studies.

Address correspondence and reprint requests to Rachel B.Jones, BSc, MBBS, MRCP, Vasculitis Office, Box 118, Renal Unit,Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ, UK.E-mail: rbjones@doctors.org.uk.

Submitted for publication July 15, 2008; accepted in revisedform April 1, 2009.

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ANCA-associated systemic vasculitis (AAV), such ascyclophosphamide (CYC) with high-dose glucocorti-coids, achieve initial remission rates of 70–90%. Theremaining patients run a persistent disease course, and50% of those who achieve initial remission will relapsewithin 2 years of diagnosis. Therapy-related toxicities,including sepsis and myelosuppression, remain a majorproblem and contribute to mortality (1). Newer thera-pies are needed.

Rituximab is a chimeric monoclonal antibodythat targets the B cell–specific CD20 calcium channel,resulting in depletion of peripheral B cells, but notplasma cells. The drug was licensed in the US andEurope in 1997 for the treatment of B cell lymphomas(2) and in 2006 for rheumatoid arthritis (RA) (3–6).Preliminary studies have reported benefit with rituximabtreatment in other autoimmune diseases, including sys-temic lupus erythematosus (7–9) and multiple sclerosis(10,11).

In studies of small numbers of patients, rituximabhas been associated with high rates of remission, often�80%, in patients with vasculitis that had been refrac-tory to standard therapies (9,12–19). Relapses of vascu-litis following rituximab therapy have occurred, althoughefficacy with second and subsequent courses of ritux-imab has been reported (9,13,14). The value of B celland ANCA levels as re-treatment guides in this contexthas not been determined. Rituximab dosing regimenshave varied, with the majority of investigators usingeither 4 infusions of 375 mg/m2 given 1 week apart (the“lymphoma regimen”) or 2 infusions of 1 gm each given2 weeks apart (the “RA” regimen). Until now, outcomesfollowing different dosing regimens in vasculitis patientshave not been compared. In previous studies, the ma-jority of vasculitis patients received additional immuno-suppressive therapies along with the rituximab, and insome studies, these therapies were continued after ri-tuximab to prevent relapse. The safety of withdrawal ofimmunosuppressive therapy at the time of rituximabwithdrawal has not been evaluated. We thus performeda multicenter survey to investigate these areas of con-troversy as well as to provide further safety and efficacydata with rituximab from a larger cohort of vasculitispatients with refractory disease.

PATIENTS AND METHODS

Participating centers and inclusion criteria. Four UKvasculitis centers participated in this study: Addenbrooke’sHospital in Cambridge, the University Hospital in Birming-ham, Great Ormond Street Hospital for Children in London,and the Hammersmith Hospital in London. One investigator

per site completed standardized data sheets for sequentialpatients who met the inclusion criteria. Data collection in-cluded information on demographics, disease activity, andsafety results at the time of each rituximab infusion and every6 months thereafter. For the purpose of the rituximab responseanalysis, patient followup was censored at the time of ritux-imab re-treatment in patients receiving multiple courses. In-cluded in this study were 11 patients from an earlier study (9)and 2 patients whose cases have previously been described(19,20).

Study inclusion required a diagnosis of AAV (WG,MPA, or CSS) (21). Fifty-five of the 65 study patients (85%)had a history of ANCA positivity at some time in their diseasecourse. It was not a requirement that ANCA be present atinitiation of rituximab. All patients received rituximab asoff-label therapy for refractory disease, encompassing thosewhose disease had failed to remit or had relapsed despitestandard immunosuppression, as well as patients in whomstandard therapies were contraindicated (22). Patients treatedwith rituximab as first-line therapy were excluded, as werethose with fewer than 6 months of followup after a firstrituximab course.

Clinical and laboratory assessments. Complete remis-sion was defined as the absence of disease symptoms and signswith a reduction in the steroid dosage (22). Partial remissionwas defined as a �50% reduction in disease activity, asassessed by the Disease Extent Index (DEI) (23). The DEIcomprises 10 organ systems and an additional category forconstitutional symptoms. Organ systems affected by activevasculitis are scored as 2 points and constitutional symptoms as1 point. The maximum score is 21. Relapse was defined as arecurrence of symptoms attributable to vasculitis that war-ranted therapy escalation beyond a temporary increase in thecorticosteroid dosage. Serious adverse events were those re-sulting in hospitalization, intravenous (IV) therapy, life-threatening situations, or death.

B cell depletion was defined as counts that were�0.02 � 109/liter, as measured by fluorescence-activated cellsorting (FACS), and B cell repopulation was defined as countsthat were �0.02 � 109/liter. The ANCA staining pattern(cytoplasmic [cANCA] or perinuclear [pANCA]) was assessedby indirect immunofluorescence, and ANCA specificity formyeloperoxidase (MPO) or proteinase 3 (PR3) was detectedby enzyme-linked immunosorbent assay (ELISA). The upperlimit of normal for the binding levels of both MPO ANCA andPR3 ANCA ranged from 6 units/ml to 25 units/ml betweencenters. The MPO ANCA and PR3 ANCA results wereinterpreted according to local reference ranges.

Disease activity was classified according to the inves-tigators’ global assessment as new disease activity/worsening ofdisease activity, stable persistent disease activity for at least 3months, or remission, as well as according to the DEI (23). TheDEI score was chosen in preference to the BirminghamVasculitis Activity Score (BVAS) (24) in order to minimizeinaccuracies with interpretations of retrospective data.

Patient population. This study included 65 patients, 40from Addenbrooke’s Hospital in Cambridge, 16 from theUniversity Hospital in Birmingham, 5 from the HammersmithHospital in London, and 4 from the Great Ormond StreetHospital in London (Table 1). The median age of the patientsat the first course of rituximab was 47 years (age range 7–77years), 52% of them were male, and the majority had WG

RITUXIMAB THERAPY FOR REFRACTORY ANCA-ASSOCIATED VASCULITIS 2157

Table 1. Characteristics and treatments in the patients with ANCA-associated vasculitis, by rituximab treatment group*

Rituximab protocol

All patients(n � 65)

Two infusions of1 gm each

given 2 weeks apart(n � 32)

Four infusions of375 mg/m2 each

given 1 week apart(n � 26)

Other(n � 7)†

Patient characteristicsAge at first rituximab course, years 49 (16–77) 47 (11–69) 25 (7–56) 47 (7–77)No. (%) male 16 (50) 13 (50) 5 (71) 34 (52)Diagnosis, no. of patients WG in 26, MPA in 4,

CSS in 2WG in 16, MPA in

6, CSS in 3,unclassified in 1

WG in 4, unclassifiedin 3

WG in 46, MPA in 10,CSS in 5, unclassifiedin 4

History of ANCA by diagnosis, no. of patients PR3 in 21, MPO in 2,cANCA in 2,pANCA in 1

PR3 in 11, MPO in5, cANCA in 5,pANCA in 1

PR3 in 5, cANCA in 1,MPO in 1

PR3 in 37, MPO in 8,cANCA in 8, pANCAin 2

Disease duration before rituximab, months 72 (2–288) 84 (10–360) 60 (7–216) 72 (1.5–360)Cumulative CYC dose (oral plus IV), gm 28 (1–150) 17 (0–90) 15 (0–100) 24 (0–150) (2 patients

took no CYC)Previous immunotherapy, no. (%)

Anti-TNF� 11 (34) 8 (31) 5 (71) 24 (38)IVIG 6 (19) 10 (38) 1 (14) 17 (26)Azathioprine 26 (81) 17 (65) 6 (86) 49 (77)Methotrexate 10 (31) 4 (15) 4 (57) 18 (28)Mycophenolate mofetil 23 (72) 19 (73) 5 (71) 47 (74)Other 10 (31) 13 (50) 2 (29) 25 (38)Total no. of previous therapies/patient 4 (2–8) 4 (2–8) 4 (2–6) 4 (2–8)

No. of previous relapses/patient 3 (1–16) 3 (2–11) 6 (5–7) 3 (1–16)ANCA status at study entry by diagnosis, no. of patients PR3 in 13, MPO in 1,

cANCA in 1PR3 in 8, MPO in 5,

cANCA in 1PR3 in 4 PR3 in 25, MPO in 6,

cANCA in 2, negativein 32

Total followup after rituximab, months 21 (3–39) 17 (3–52) 21 (12–40) 20 (3–55)No. of organs with active disease at first rituximab course 2 (1–4) 2 (1–6) 3 (1–4) 2 (1–6)Indication for rituximab, no. (%)

New/worse disease 23 (71) 26 (100) 6 (86) 55 (85)Persistent disease 9 (28) 0 (0) 1 (14) 10 (15)

BSA, m2 1.85 (1.57–2.61) 1.87 (0.89–2.38) – –Total rituximab dose adjusted for BSA at the first

rituximab course, mg1,081 (766–1,273) 1,500 (1,500–1,500) – –

TreatmentsImmunotherapy before the first rituximab course, no. (%)‡

Mycophenolate mofetil 12 (38) 15 (58) 1 (14) 28; 2 gm/day (0.5–3gm/day)

Azathioprine 3 (9) 2 (8) 2 (29) 7; 75 mg/day (12.5–150mg/day)

IV pulse CYC 6 (19) 0 0 6Oral CYC 3 (9) 0 2 (29) 5; 150 mg/day (25–200

mg/day)Deoxyspergualin 2 (6) 2 (8) 0 4Methotrexate 2 (6) 1 (4) 0 3; 20 mg/day (17.5–25

mg/day)Plasma exchange 1 (3) 0 (0) 0 1ECMS 0 1 (4) 0 1; 2.16 gm/dayAlemtuzumab 0 1 (4) 0 1; 11 months before

rituximabCombination of 2 or 3 therapies 1 (3) 2 (8) 1 (14) 4None 2 (6) 2 (8) 1 (14) 5Oral corticosteroid dosage, mg/day§ 12.5 (0–60) 12.5 (0–60) 20 (0–60) 12.5 (0–60)

Rituximab protocols, no. of patients2 infusions of 1 gm each given 2 weeks apart 32 0 0 32 (total dose 2 gm)4 infusions of 375 mg/m2 each given 1 week apart 0 26 0 26 (total dose �3 gm)2 infusions of 750 mg/m2 each given 2 weeks apart 0 0 3 3 (total dose �3 gm)3 infusions of 1 gm each given 1 week apart 0 0 1 1 (total dose 3 gm)1 infusion of 3.2 gm 0 0 3 3 (total dose 3.2 gm)

Additional therapy with the first rituximab course, no. (%)CYC‡ 12 (38) 12 (46) 4 (57) 28; 0.9 gm (0.2–1.85 gm)IV corticosteroids/5 days 1 mg/kg oral¶ 10 (31) 1 (4) 6 (86) 17None 17 (53) 13 (50) 0 22

* Except where indicated otherwise, values are the median (range). ANCA � antineutrophil cytoplasmic antibody; WG � Wegener’s granulomatosis; MPA �microscopic polyangiitis; CSS � Churg-Strauss syndrome; PR3 � proteinase 3; MPO � myeloperoxidase; cANCA � cytoplasmic ANCA; pANCA � perinuclear ANCA;CYC � cyclophosphamide; anti-TNF� � anti–tumor necrosis factor �; IVIG � intravenous immunoglobulin; BSA � body surface area; ECMS � enteric-coatedmycophenolate sodium.† Other rituximab protocols were as follows: 2 infusions of 750 mg/m2 each given 2 weeks apart; 3 infusions of 1 gm each given 1 week apart; and 1 infusion of 3.2 gm.‡ Values for the entire study group represent the number of patients; median dosage (range).§ Values are the median (range).¶ Patients received either intravenous (IV) corticosteroids (1–3 gm total) or high-dose daily oral corticosteroids (1 mg/kg for 5 days).

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(71%). The median disease duration prior to rituximab therapywas 72 months (range 1.5–360 months). The frequency of newor worse disease activity was 85%, and 15% had persistentdisease activity (Table 1).

At the time of the first rituximab course, a median of 2organs (range 1–6) showed active disease. The frequency ofinitial organ activity is illustrated in Figure 1A. The relativelyhigh rate of endobronchial disease and retroorbital granulomareflected the most common diagnosis (WG) and the complextreatment-resistant nature of this patient cohort. Just prior torituximab administration, 92% were receiving continuous ther-apy with immunosuppressive agents (Figure 1B).

Five patients (8%) were not taking immunosuppressivetherapy at the time of the first rituximab course, and all ofthem had active disease. Four of these 5 patients could nottolerate other immunosuppressive therapies: 2 of them hadWG, and immunosuppressive therapy was withdrawn 1 monthprior to the first rituximab course because of recurrent chestinfections, and the other 2 patients had prolonged recurrentleukopenia. One patient was not receiving immunosuppressive

agents during recovery from chemotherapy for non-Hodgkin’slymphoma.

Treatment protocol and followup. Rituximab was ad-ministered to 26 patients as 4 doses of 375 mg/m2 each given 1week apart, as used to treat lymphoma, and to 32 patients as 2doses of 1 gm each given 2 weeks apart, as used to treat RA.In 7 patients, other dosing regimens were used for the firstrituximab course.

In addition to the rituximab, 28 patients received IVCYC (median total dose 0.9 gm [range 0.2–1.85 gm]); another8 patients had stopped taking CYC just prior to initiation ofrituximab. A total of 17 patients received additional high-doseoral or IV glucocorticoids at the time of rituximab. Noadditional therapy was administered to 22 patients.

The median followup after rituximab was for 20months (range 3–55 months). All patients had at least 6months of followup, except for 1 patient who died at 3 monthsof followup. The median followup after the first rituximabcourse, with censoring at the time of the second rituximabcourse, was for 14 months (range 3–52 months).

Figure 1. Characteristics of the 65 patients with antineutrophil cytoplasmic antibody–associated vasculitis refractory totreatment and response to therapy with rituximab. A, Proportion of patients with specific organ system activity at the firstrituximab course. ENT � ear, nose, throat; LN � lung nodules; LO � lung other (pulmonary fibrosis and asthma); EB �endobronchial; ROG � retroorbital granuloma; PNS � peripheral nervous system; GIT � gastrointestinal tract; LH �lung hemorrhage; other � muscle. B, Proportion of patients receiving specific immunosuppressive agents prior to thefirst rituximab course. MMF � mycophenolate mofetil; AZA � azathioprine; IV � intravenous; CYC � cyclo-phosphamide; PO � oral; DSG � deoxyspergualin; MTX � methotrexate; PEX � plasma exchange; ECMS �enteric-coated mycophenolate sodium; CAM � Campath 1H (Bayer, Bury St. Edmonds, UK; alemtuzumab); multiple �2 or more concomitant therapies. C, Proportion of patients with specific disease activity states at 0, 6, and 12 monthsfollowing the first rituximab course. D, Disease Extent Index at 0, 6, and 12 months following the first rituximab courseand at the last followup visit. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines insidethe boxes represent the median. Lines outside the boxes represent the 10th and the 90th percentiles. Circles indicateoutliers. E, Daily oral dose of prednisolone at 0, 6, and 12 months following the first rituximab course and currently.Values are the mean and SD. F, Proportion of patients receiving concomitant therapy with immunosuppressive agentsat 0, 6, and 12 months following the first rituximab course and currently.

RITUXIMAB THERAPY FOR REFRACTORY ANCA-ASSOCIATED VASCULITIS 2159

After the first rituximab course, some investigatorsadministered a second course of rituximab if relapse occurred,whereas others administered a second course on a preemptivebasis, either at a set time point (e.g., every 6 months) orfollowing B cell restoration. One investigator did not admin-ister a second course of rituximab, but switched to othertherapies if relapse occurred. Of the 38 patients (59%) whoreceived a second rituximab course, the rituximab was givenpreemptively in 6, after relapse in 27, and to further improvedisease control after either partial remission (4 patients) orpersistently active disease (1 patient) following the first ritux-imab course in 5 patients.

The results after the first rituximab course, with fol-lowup censored at the time of the second rituximab course,provided the most information with regard to the timing of thereturn of B cells, increases in ANCA levels, and relapse ofdisease. Results following multiple additional courses of ritux-imab were informative with regard to long-term changes inimmunoglobulin levels and severe adverse events.

Statistical analysis. Statistical analysis was performedusing SPSS version 11 (SPSS, Chicago, IL) and GraphPadPrism version 4 (GraphPad Software, San Diego, CA) softwarepackages. Changes in variables, including the DEI and theprednisolone dosage, were compared by Mann-Whitney Utest. Relapses were analyzed using Kaplan-Meier survivalanalysis, with log rank analysis for significance. P values lessthan 0.05 were considered significant for all statistical tests.

RESULTS

Efficacy of rituximab. B cell depletion and remis-sion. Depletion of B cells (�0.02 � 109/liter) wasachieved in all patients after the first rituximab course. Bcell repopulation occurred in 28 of 64 patients (44%) ata median of 11 months (range 6–37 months) after thefirst rituximab course (B cell followup data not availablein 1 patient). Of the remaining 36 patients, 19 were givena second rituximab course before the return of B cells,and 17 remained persistently B cell depleted at a medianfollowup of 15 months (range 3–52 months). After asecond rituximab course in 38 patients, B cell depletiondid not occur in 1 of them.

At the time of the first course of rituximab, allpatients had active disease. Fifty-five of the 65 patients(85%) had new or worse disease activity, and theremaining 10 patients (15%) had stable, persistent dis-ease activity for at least 3 months. The median DEIscore at the time of the first rituximab course was 4(range 2–11) in patients classified as having new orworse disease and 2 (range 2–6) in patients with persis-tent disease activity. Figures 1C and D show the changesin disease activity over time. The DEI fell from a medianof 4 (range 2–11) to a median of 0 (range 0–4) at 6months (P � 0.0001) and remained low at 12 months(median 0 [range 0–7]; P � 0.0001).

Complete remission occurred in 49 of the 65patients (75%), with a further 15 patients (23%) expe-riencing partial remission. One patient with a retroor-bital granuloma failed to respond to rituximab therapy.This patient had previously failed to respond to CYC(�100 gm), azathioprine (AZA), and mycophenolatemofetil (MMF). The median time to remission was 2months (range 1–5 months). Remission was accompa-nied by a decrease in the prednisolone dosage from amedian of 12.5 mg/day (range 0–60 mg/day) initially to amedian of 9.0 mg/day (range 0–80 mg/day) at 6 months(P � 0.006) and a median of 7.5 mg/day (range 0–20mg/day) at 12 months (P � 0.001). Figure 1E shows thechanges in the mean prednisolone dosage over time.

Of the 60 patients who were receiving immuno-suppressive agents just prior to the first rituximabcourse, the immunosuppressive therapy was withdrawnin 37 of them (62%) (Figure 1F). Treatments that werewithdrawn were MMF in 16 patients, CYC in 8, deoxy-spergualin in 4, AZA in 2, methotrexate (MTX) in 2,enteric-coated mycophenolate sodium (ECMS) in 1,plasma exchange in 1, alemtuzumab in 1, infliximab pluscyclosporine in 1, and MMF plus cyclosporine in 1. In afurther 4 patients, the dosage of immunosuppressivetherapy was reduced (AZA in 1, MMF in 2, and MTXin 1). In 3 patients, the CYC was switched to anotheragent (MMF in 2 and AZA in 1). In 2 patients, part oftheir combination therapy was reduced (CYC withdrawnwith dapsone continued in 1, and etanercept and MMF

Figure 2. Rituximab courses, disease remission, and disease relapse in65 patients with antineutrophil cytoplasmic antibody–associated vas-culitis refractory to treatment. Values are the total number of patientsreceiving each treatment course (number treated for active disease/number treated preemptively for relapse) and the number (%) ofpatients. One patient received 7 courses of rituximab and was still inremission at the last followup visit.

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withdrawn with IV immunoglobulin [IVIG] continued in1). Ten patients continued taking MMF and 4 continuedtaking AZA, with unchanged dosages.

Fifteen patients received only 1 course of ritux-imab and remained relapse-free thereafter. The medianfollowup in these patients was 21 months (range 3–52months).

Relapse and re-treatment. Relapse occurred in 28of the 49 patients who experienced complete remissioninitially (57%). The median time to relapse was 11.5months (range 4–37 months). No differences in diseasesubtype, initial ANCA status (positive versus negative orMPO versus PR3 ANCA), or organ system involvementwere noted between patients who experienced relapseand patients who did not.

Thirty-eight patients received a second course ofrituximab. Of these 38 patients, 27 were treated fordisease relapse, 6 were treated preemptively to avoidrelapse, either following B cell restoration or at a settime point (e.g., every 6 months) while they were still Bcell depleted, and 5 were treated to further improvedisease control because of either partial remission (n �4) or persistently active disease (n � 1). After the secondcourse of rituximab, 84% experienced a second com-plete remission or maintained remission. The mediantime to remission after a second rituximab course was1.5 months (range 1–4 months). Three or more ritux-imab courses were administered to 20 patients, with 1patient receiving a total of 7 courses. Figure 2 shows therituximab re-treatment, response, and relapse patterns.

Figure 3. Predictors of remission and relapse in patients with antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis refractory to treatment. A and B, Relationship between complete remission in 49 patients(B cell data unavailable in 1), relapse, and B cell repopulation (�0.02 � 109/liter). Shown are the duration offollowup, the timing of B cell return, and the timing of relapse in 25 patients with complete remission and Bcell repopulation occurring during followup (14 with relapse at the time of or after B cell return, 3 with relapsebefore B cell return, and 8 without relapse) (A), as well as the duration of followup and the timing of relapsein 23 patients with complete remission who remained B cell depleted throughout followup (10 with relapse, and13 without relapse) (B). C, Relapse-free survival curves in 63 patients who were positive (n � 33) or negative(n � 30) for ANCA at the time of the first rituximab course. D, Levels of proteinase 3 (PR3) ANCA andmyeloperoxidase (MPO) ANCA binding at 0, 6, and 12 months following the first rituximab course andcurrently in the 31 patients who were initially positive for PR3 ANCA or MPO ANCA by enzyme-linkedimmunosorbent assay (ELISA). Values are the median, with 25th and 75th percentiles. Horizontal line showsthe lowest value for the upper limit of normal (6 units/ml).

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In 2006, 1 of the study centers started routinepreemptive re-treatment with 1 gm of rituximab given in1 infusion every 6 months. Fifteen patients received atleast 1 preemptive course of rituximab (maximum 3courses). Following initiation of the preemptive re-treatment practice, no relapses occurred (median fol-lowup 11 months [range 5–23 months]).

Predictors of remission and relapse. B cell de-pletion after the initial rituximab course was followed byB cell return at a median of 11 months in 28 patients, 25of whom experienced complete remission. Of the 28patients who had a relapse following the initial completeremission, 14 had a relapse at the time of or after B cellreturn, 3 had a relapse before B cell return, and 10 hada relapse but remained persistently B cell depleted forthe remainder of followup (B cell data missing in 1patient who had a relapse). Therefore, 48% of patients(13 of 27) had a relapse before B cell repopulation, and

32% of patients (8 of 25) in whom B cells returnedfollowing an initial complete remission did not have arelapse. Figures 3A and B illustrate the relationshipbetween relapse and B cell return in individual patientsachieving complete remission after the initial course ofrituximab. Twenty-five patients experienced restorationof B cells (Figure 3A), and 23 experienced continueddepletion of B cells (Figure 3B).

At the time of initial treatment, 33 patients wereANCA positive (25 had PR3 ANCA, 6 had MPOANCA, and 2 had cANCA alone), and 32 were ANCAnegative. The initial median DEI score was lower inANCA-negative patients (3.8 versus 4.5 in ANCA-positive patients; P � 0.03). The remission and re-lapse rates were similar in the ANCA-positive andANCA-negative groups (Figure 3C). The mean levels ofMPO ANCA and PR3 ANCA binding, as determined byELISA, were decreased at 6 months after the first

Figure 4. Factors influencing remission and relapse in patients with antineutrophil cytoplasmic antibody–associated vasculitis refractory to treatment. A, B cell depletion survival curves following the firstrituximab course, by rituximab treatment protocol (2 main protocols used): 4 infusions of 375 mg/m2 eachgiven 1 week apart (n � 26) and 2 infusions of 1 gm each given 2 weeks apart (n � 30; B cell dataunavailable in 1 patient). B, Relapse-free survival curves following the first rituximab course, by rituximabtreatment protocol (2 main protocols used): 4 infusions of 375 mg/m2 each given 1 week apart (n � 26)or 2 infusions of 1 gm each given 2 weeks apart (n � 31). C, Relapse-free survival curves following the firstrituximab course in patients who did (n � 10) and those who did not (n � 33) continue immunosuppres-sive therapy. Patients in whom an initial remission did not occur and those who were not receivingimmunosuppressive agents at the time of the first rituximab course were excluded from the analysis.

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rituximab course (P � 0.001) (Figure 3D). Twenty-eight patients experienced a disease relapse after aninitial complete remission; 12 of them were ANCA-positive at the time of rituximab treatment, as de-termined by ELISA. Of these 12 patients, 2 showed aclear increase in ANCA levels before relapse, as deter-mined by ELISA. The majority of patients thereforerelapsed without a major change in the ANCA-bindinglevel.

Factors influencing remission and relapse. Thepredominant rituximab protocols used were 4 infusionsof 375 mg/m2 each given 1 week apart (the lymphomaregimen; n � 26) and 2 infusions of 1 gm each given 2weeks apart (the RA regimen; n � 32). Both regimensinduced depletion of peripheral blood B cells in allpatients and resulted in similar rates of remission (81%and 75%, respectively). Neither the duration of B celldepletion nor the duration of disease remission wasinfluenced by the different dosing regimens (Figures 4Aand B). More of the patients who received the 2 1-gminfusions of rituximab were treated with a course ofCYC just prior to the rituximab course. Other factorsthat may have influenced outcome, including additional

CYC with the rituximab, the average daily corticosteroiddose, withdrawal of immunosuppressive agents, the ini-tial DEI scores, and the disease subtype, were similar inthe 2 main treatment protocol groups (Table 1).

In 60 patients who were receiving immunosup-pressive agents just prior to the first rituximab course,62% (37 patients) had concomitant immunosuppressivetherapy withdrawn following the initial rituximab course(MMF in 16, CYC in 8, deoxyspergualin in 4, AZA in 2,MTX in 2, ECMS in 1, plasma exchange in 1, alemtu-zumab in 1, and combination therapy with infliximabplus cyclosporine in 1 and MMF plus cyclosporine in 1).The withdrawal of immunosuppressive therapy occurredin 32 of the 60 patients by 6 months and in 37 of the 60patients by 12 months. No significant difference in timeto relapse was seen in patients in whom immunosuppres-sive agents were withdrawn as compared with patients inwhom immunosuppressive agents were continued (P �0.79) (Figure 4C). Median steroid dosages were similarbetween these 2 patient groups, as were disease demo-graphics and changes in the DEI score.

Rituximab safety. Rituximab was well tolerated,and there were no severe infusion reactions. During an

Table 2. Serious adverse events occurring in the 65 study patients*

Event category, serious adverse eventNo. (%) of

patientsNo. ofevents

No. of monthssince first

rituximab course

No. of rituximabcourses before the

event

Disease activityEndobronchial disease 5 (7.5) 6 1, 4, 4, 9, 38, 47 1, 1, 1, 1, 2, 3Asthma 2 (3.0) 6 2, 2, 6, 7, 9, 11 1, 1, 1, 1, 1, 1Peripheral neuropathy 1 (1.5) 1 4 1ENT disease 2 (3.0) 2 4, 6 1, 1Pulmonary fibrosis, respiratory failure, and death 1 (1.5) 1 8 1Retroorbital granuloma 1 (1.5) 1 27 2

InfectionPneumonia (organism not specified) 7 (11.0) 10 0.5, 1, 2, 4, 5, 5, 9, 14, 24, 49 1, 1, 1, 1, 1, 1, 2, 2, 1, 2Aspergillus lung infection 1 (1.5) 1 8 2Pseudomonas lung infection 1 (1.5) 1 9 2Line sepsis 1 (1.5) 1 3 1Sepsis 2 (3.0) 2 2, 10 1, 2Cellulitis 1 (1.5) 1 20 2

OtherPulmonary embolism 1 (1.5) 1 24 1Sudden unexplained death 1 (1.5) 1 3 1Addisonian crisis 2 (3.0) 2 5, 27 1, 1Hip surgery 1 (1.5) 1 5 1Aseptic meningitis post-IVIG therapy 1 (1.5) 1 6 1Aortic valve replacement 1 (1.5) 1 8 1Neutropenia 2 (3.0) 2 14, 18 2, 2Failure of B cells to deplete 1 (1.5) 1 19 2Ankle surgery 1 (1.5) 1 24 1Eye surgery 1 (1.5) 1 42 3

* Serious adverse events were those that resulted in hospitalization, intravenous therapy, a life-threatening situation, or death. A total of 46 seriousadverse events occurred in 25 patients. Some patients experienced more than 1 serious adverse event. ENT � ear, nose, throat; IVIG � intravenousimmunoglobulin.

RITUXIMAB THERAPY FOR REFRACTORY ANCA-ASSOCIATED VASCULITIS 2163

overall total of 1,548 months of followup, 45 seriousadverse events occurred in 25 patients (Table 2). Sixteenserious infections occurred a median of 8 months afterthe first rituximab course (range 0.5–49 months). Sev-enteen events, including 1 death, were related to activevasculitis. In 2 patients, neutropenia (�0.5 � 109/liter)occurred at 3 months and 5 months, respectively, afterthe second rituximab course. Both episodes were shortlived, and there was spontaneous recovery of the neu-trophil count, without sepsis. Although concomitantmedications might have been responsible (trimethoprim/sulfamethoxazole and AZA in one patient and cyclo-sporine plus amlodipine in the other), rituximab wasconsidered the likely cause. With the exception of neu-tropenia, no other serious adverse event appeared to bedirectly attributable to rituximab (Table 2).

Two deaths occurred. One sudden unexplaineddeath occurred 3 months following a rituximab course ina 59-year-old patient whose disease was in remission. Apostmortem examination was not performed. The sec-ond death occurred 8 months after a rituximab course ina 7-year-old patient. This boy had severe pulmonaryfibrosis and MPO ANCA positivity (with unclassifiedAAV) prior to treatment with rituximab. The cause ofdeath was believed to be disease-related and not theconsequence of rituximab per se.

IgM levels fell from a median of 0.8 gm/liter(range 0.2–3.3 gm/liter) at 0 months to a median of 0.52gm/liter (range 0.1–1.7 gm/liter) at 6 months (P � 0.002),with a further nonsignificant decline continuing over theremainder of followup (0.50 gm/liter [range 0.09–1.5gm/liter] at the last followup; normal range 0.4–2.2gm/liter). The median IgG level was 7.9 gm/liter (range1.5–28.9 gm/liter) initially and 8.0 gm/liter (range 3.7–17.4 gm/liter) at the last followup (P � 0.65) (normalrange 6–13 gm/liter).

DISCUSSION

B cell depletion with rituximab therapy was asso-ciated with complete remission in 75% of adults andchildren with refractory AAV. Remissions were sus-tained after a single rituximab course, although 58% ofthese patients subsequently experienced a relapse. Mul-tiple courses of rituximab effectively induced furtherremissions following relapses, and re-treatment on apreemptive basis resulted in sustained remissions. Theseresults are consistent with those of previous studies(9,12–20). This is the largest series reported to date, andit pools the results from 4 UK centers. All patients had

active disease at the time rituximab was initiated, andprevious disease courses were characterized by poorlycontrolled disease, multiple relapses, and treatment withmultiple immunosuppressive agents.

Some centers followed the practice of temporaryuse of IV CYC or corticosteroids at the time of ritux-imab therapy to aid early disease control. Given thatcomplete remissions occur an average of 2 months afterthe rituximab course, this approach seems reasonable inpatients with rapidly progressive disease. It is unlikelythat this temporary increase in immunosuppressiveagents would account for the sustained remissions inpatients with active disease who were already receivingcontinuous therapy with immunosuppressive agents.Furthermore, a significant number of patients had sus-tained remissions without additional therapies.

A positive outcome bias may occur in retrospec-tive studies, and this possibility was minimized by includ-ing in our study all eligible patients who were treatedwith rituximab during the study period and by includingdata from 4 independent centers. The overall responseto rituximab in this patient series was superior to thatseen with alternative therapies in similar cohorts ofpatients with refractory vasculitis. In a retrospectiveseries of patients treated with MMF, a reduction indisease activity occurred in 86% (19 of 22); however, fewachieved complete remission, and 47% experienced arelapse by 14 months, which required other therapies(25). In a placebo-controlled trial, 82% of patients (14 of17) responded to IVIG therapy; however, the responsewas not maintained beyond 3 months (26). In a long-term followup of 71 patients treated with alemtuzumab,85% responded; however, relapses occurred in 72% at amedian of 9.2 months (27). Thus, the quality andduration of responses following rituximab treatmentappear to be superior to those following alternativetherapies for refractory disease.

One patient with a retroorbital granuloma failedto respond to rituximab. This patient had a long diseasecourse and was exposed to a high cumulative dose ofCYC. Aries et al (28) reported improvement in only 3 oftheir 8 patients who had disease manifestations andchronicity similar to those in our patient. On the otherhand, our results show that rituximab is effective therapyfor granulomatous manifestations, such as retroorbitalgranuloma and endobronchial disease, in the majority ofpatients. These differences may be partly due to therituximab dosing regimen used by Aries et al (an infu-sion of 375 gm/m2 given every fourth week), as well asthe longer followup in our series of patients, whichallowed for sufficient time to observe the maximumbenefit of rituximab therapy.

2164 JONES ET AL

The earlier use of rituximab in our patient serieswas associated with continued concomitant immunosup-pression. After observing sustained remission, someinvestigators reduced the dosage and then withdrew theconcomitant therapies, and our results showed that therates of relapse were not higher in patients in whomthese treatments were withdrawn. A selection bias inpatients from whom immunosuppressive therapy waswithdrawn is likely, with investigators preferring to with-draw therapies when the perceived risk of relapse is low.However, in 1 center (Addenbrooke’s Hospital; 40 pa-tients), the decision to withdraw immunosuppressivetherapy was dictated by local policy and not solely by thecharacteristics of the patients. In the majority of patientsin this study, the steroid dosages were tapered, but thetreatment was not withdrawn. The continuation of low-dose corticosteroids may have protected patients fromexperiencing a relapse.

No dose-ranging studies have been performed invasculitis patients to ascertain the optimum rituximabdosage, and previous small, single-center studies havebeen unable to compare different dosing regimens. Thepharmacokinetics and pharmacodynamics of rituximabare variable and are influenced by factors such aspolymorphisms in Fc� receptor IIIa (29), proteinuria,and human antichimeric antibodies (30). The rituximabdosing regimen used to treat lymphoma (4 infusions of375 mg/m2 each given 1 week apart) results in an averagetotal dose of 2.6 gm in a person weighing 70 kg. In astudy of 203 lymphoma patients, the mean maximumserum concentration (Cmax) following the fourth ritux-imab infusion was reported to be 486 �g/ml (31). Incomparison, the dosing regimen used to treat RA (2infusions of 1 gm each given 2 weeks apart) results in alower total dose of rituximab (2 gm) that is administeredover a shorter time period, and a mean Cmax of 370�g/ml following the second infusion has been reported(31). The latter regimen is more attractive in terms ofpatient convenience, cost, and ease of administration.An alternative rituximab regimen of 2 infusions of 750mg/m2 given 2 weeks apart was used in the pediatricpatients in our study; this regimen provided the samecumulative dose as used in lymphoma patients, with lessinconvenience to the patient.

Pharmacokinetic modeling in RA suggested thata body surface area–calculated dose would not improvethe predictability of drug exposure (32), and clinicaltrials in RA have found that 2 infusions of 1 gm given 2weeks apart is both effective (5,6) and safe with repeateduse (33). The more extensive organ involvement thatusually occurs in vasculitis as compared with RA raisesthe question of whether 2 infusions of 1 gm given 2

weeks apart would allow sufficient tissue penetration forB cell depletion from lesional tissue, perhaps allowingsustained remissions. The results from our series ofpatients showed no difference in the duration of B celldepletion or the therapeutic effect between the 2 maintreatment regimens (2 infusions of 1 gm each given 2weeks apart and 4 infusions of 375 mg/m2 each given 1week apart), which suggests that the dosing regimen inwhich 2 1-gm infusions are given 2 weeks apart may besufficient in AAV.

The apparent benefit of second and subsequentrituximab courses observed in our study suggests a roleof rituximab re-treatment in AAV. Preemptive re-treatment with rituximab in adult patients may be pref-erable to re-treatment following relapse, although datarelating to this issue in children are not provided by thisstudy. Given the variable timing of relapses, it would beideal to target the therapy to the individual patient toavoid unnecessary treatments and costs. We evaluatedthe role of ANCA and B cell levels as predictors ofrelapse and re-treatment. ANCA was present in justover one-half of the patients at the time of initialtreatment, consistent with ANCA positivity in othercohorts of patients with refractory AAV (34,35). Levelsof MPO and PR3 ANCA fell following rituximab treat-ment; however, clinical remissions occurred to the sameextent in both ANCA-positive and ANCA-negative pa-tients, and relapses were preceded by an increase inANCA levels in only a few patients. From these data, wecan conclude that ANCA was not sufficiently sensitive toguide the timing of re-treatment.

Similar to the findings in RA patients treatedwith rituximab (33), the repopulation of B cells in AAVpatients was not a reliable marker of relapse. Just underone-half of the relapses occurred before B cell return,and 32% of the patients whose B cells returned did notexperience a relapse. Therefore, repopulation of B cellsin the peripheral blood is not sufficiently reliable toguide re-treatment alone. Possible explanations for re-lapse in the absence of apparent peripheral blood B cellsare that our FACS analysis did not detect B cell levelsthat were �0.02 �109/liter. A more sensitive analyticalmethod may have shown closer correlations with diseaseactivity. In addition, rituximab results in the reduction ofCD20-positive B cells in tissues as well as in blood(36,37), which may not be complete in tissues (38,39).Certainly, tissue B cell repopulation can occur prior todetectable return to the peripheral blood (20). Further-more, variability in the phenotype of the repopulating Bcells has been demonstrated following rituximab therapyin patients with systemic lupus erythematosus (40), and

RITUXIMAB THERAPY FOR REFRACTORY ANCA-ASSOCIATED VASCULITIS 2165

this may contribute to the variable duration of clinicaleffect.

Rituximab was well tolerated, and there were nosevere infusion reactions. The frequency of seriousadverse events was similar to that seen in other studiesof vasculitis patients and appeared to largely reflect theinfluence of concurrent or previous immunosuppressivetherapy, underlying disease, and corticosteroid therapy.A review of 5 trials of rituximab treatment in patientswith non-Hodgkin’s lymphoma found no additional riskof infection with rituximab and chemotherapy versuschemotherapy alone (41); however, it remains unclearwhether rituximab increases the risk of infection inpatients with vasculitis. Two patients experienced tran-sient neutropenia. Rituximab-associated late-onset neu-tropenia has been described not only in association withthe use of high-dose immunosuppressive therapy inlymphoma patients (42), but also in association withconcomitant low-dose immunosuppressive therapy inpatients with pemphigus vulgaris (as in our patients)(43). Arrest of promyelocyte maturation has been dem-onstrated in late-onset neutropenia following rituximabtherapy (42); however, the mechanism is unclear. Theneutropenia in our patients resolved spontaneously,without the use of granulocyte colony-stimulating factorand without sequelae from infection. Two deaths oc-curred. In neither case was a causal association betweenrituximab and death found.

A significant fall in IgM levels occurred by 6months. IgG levels were maintained within the normalrange. These results are consistent with short-livedplasma cells making a greater contribution to serum IgMthan to serum IgG; conversely, most of the IgG that ispresent in serum is produced by long-lived bone marrowplasma cells (44). Rates of serious infection were notincreased with prolonged followup or after successivecourses of rituximab. Similar decreases in IgM levelshave been reported with multidose rituximab regimensin RA (33), and this may complicate long-term therapyin some patients. Multiple courses of rituximab in thiscohort appeared to be safe, although it is important toemphasize that patients who received multiple coursestended to be in remission, receiving little or no concom-itant immunosuppressive therapy, and taking low dosesof corticosteroids and were therefore generally at alower risk of infection than other vasculitis cohorts. Inorder to minimize the risk of underreporting of infec-tions, only data concerning serious infections were re-corded. It remains to be seen whether more prolongeduse of rituximab will ultimately result in significantdeclines in immunoglobulin levels, as may be expected if

a necessary replenishment of long-lived plasma cells isprevented.

In conclusion, our findings add to previous evi-dence of the high level of efficacy of rituximab therapy inpatients with refractory AAV. This treatment shouldnow be considered for patients with this presentation.While an additional course(s) of rituximab is also effec-tive, the value of a re-treatment protocol in preventingdisease flares remains under investigation. Immunosup-pressive therapy can be withdrawn and steroid dosagestapered in patients achieving complete clinical remis-sion, without jeopardizing the relapse risk, as long aspreemptive re-treatment is considered as a long-termstrategy. The optimum timing of a subsequent course ofrituximab is unclear, and better disease biomarkers areneeded.

ACKNOWLEDGMENTS

We thank all of the nurses and physicians involved inthe care of the patients included in this study.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Jones had full access to all of thedata in the study and takes responsibility for the integrity of the dataand the accuracy of the data analysis.Study conception and design. Jones, Brogan, Savage, Jayne.Acquisition of data. Jones, Ferraro, Chaudhry, Brogan, Salama, Smith,Jayne.Analysis and interpretation of data. Jones, Chaudhry, Brogan, Savage,Jayne.

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DOI 10.1002/art.24604

Clinical Image: Cerebral amyloid angiopathy

The patient, a 73-year-old woman, was admitted to our hospital with a 2-month history of recurrent intracranial hemorrhage.Systemic lupus erythematosus (SLE) had been diagnosed 6 years previously. She did not have thrombocytopenia, was negative forantiphospholipid antibody, and had not received anticoagulant treatment. Computed tomography (CT) of the brain showed anintracerebral hematoma over the subcortical white matter region of the right temporal lobe, a typical area of cerebral amyloidangiopathy. Results of histologic analysis, as shown here, were consistent with the CT findings. Cerebral amyloid angiopathy, a rarereported complication of lupus, should be considered in SLE patients with recurrent intracranial hemorrhages.

Chien-Hsueh Tung, MDChih-En Tseng, MDNing-Sheng Lai, MD, PhDBuddhist Tzu Chi General HospitalDalin, Chia-yi, Taiwan

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