Stem cell transplantation for autoimmune diseases

Best Practice & Research Clinical Haematology Vol. 14, No. 4, pp. 755±776, 2001

doi:10.1053/beha.2001.0171, available online at http://www.idealibrary.com on

6 Stem cell transplantation for autoimmune diseases A. Gratwohl* J. Passweg I. Gerber

MD

MD MD

A. Tyndall

MD

for the International Stem Cell Project for Autoimmune Diseases Division of Haematology, Department of Internal Medicine, Kantonsspital Basel and Department of Rheumatology, Felix Platter-Spital, University of Basel, Switzerland

Much progress has been made in the ®eld of haemopoietic stem cell transplants (HSCTs) for severe autoimmune disorders. Theoretical considerations, animal data and anecdotal evidence suggested some time ago that intensive immunoablation followed by autologous HSCT could restore normal immune reactivity in patients with severe autoimmune disorders. Based on a concept statement issued in 1995, two European societies, the European League Against Rheumatism (EULAR) and the European Group for Blood and Marrow Transplantation (EBMT) began collecting phase I/II trial data in an international collaborative network. Sucient information from more than 350 patients allows a preliminary assessment with level three evidence. Autologous HSCTs can induce remissions in all disease categories tested so far. Remissions can be transient or durable. HSCTs are associated with signi®cant morbidity and mortality. Treatment-related mortality (TRM) is near 10% at 1 year and is associated with the intensity of the conditioning and the stage of the disease at the time of transplant. Marked interdisease di€erences exist. There are few data available in haematological autoimmune diseases, more in systemic sclerosis (SSc), systemic lupus erythematosus (SLE), juvenile idiopathic arthritis (JIA) and multiple sclerosis (MS). Patient selection has been recognized as a crucial element from the phase I±II trials. Patients with advanced disease, severely compromised organ function or irreversible organ damage should not be considered as candidates for HSCT. Prospective randomized studies should now determine the value of HSCT compared to standard therapy. Such trials are ongoing for patients with systemic sclerosis (ASTIS trial ± Autologous Stem Cell Transplantation International Scleroderma Trial) or are planned for patients with multiple sclerosis (ASTIMS trial ± Autologous Stem Cell Transplantation International Multiple Sclerosis Trial) and rheumatoid arthritis (ASTIRA trial ± Autologous Stem Cell Transplantation International Rheumatoid Arthritis Trial). More phase II data are needed for other indications such as SLE and JIA. Key words: autologous stem cell transplantation; autoimmune disease; EBMT/EULAR; review. *Address for correspondence: Division of Haematology, Department of Internal Medicine, Kantonsspital Basel, CH-4031 Basel, Switzerland. 1521±6926/01/040755‡22 $35.00/00

c 2001 Harcourt Publishers Ltd. *

756 A. Gratwohl et al

Haemopoietic stem cell transplants (HSCTs) are standard therapy for many haematological malignancies, including certain subgroups of lymphoid malignancies and multiple myeloma. Mechanisms of cure are poorly understood but go beyond the traditional thinking of dose escalation and killing of the last tumour cell.1 It is naõÈ ve to think that the same methods used to eradicate `malignant' lymphoid clones could be used to re-educate `aberrant' lymphoid cell populations in severe autoimmune diseases. Pragmatism proves that the concept might be correct even though the mechanisms are not yet understood. Series of patients with severe refractory autoimmune disorders have responded to high-dose chemotherapy and autologous HSCTs2, have softened their thickened skin in systemic sclerosis3, cleared their gadolinium lesions in multiple sclerosis4, come o€ any therapy for their juvenile idiopathic arthritis5 or have normalized their platelet counts in previously refractory immune thrombopenia.6 The challenge lies ahead to prove whether these ®ndings are not mere chance events, that they remain durable in a substantial proportion of patients and that the positive gain is not o€set by toxicity or mortality. The last 5 years have shown that HSCTs are feasible and are associated with response and risk factors for excessive toxicity that have been identi®ed.2 The expectation is that within the next 5 years prospective randomized studies will clarify the value of HSCT in selected patients with some autoimmune disorders compared to standard current therapy. Hopefully, by careful trial design, methodological errors such as those made during the introduction of HSCTs in the treatment of breast cancer7 will not be repeated.

THE CONCEPT OF HSCT IN SEVERE AUTOIMMUNE DISEASES Recognition of a close similarity between the phenomena of chronic graft-versus-host disease (GVHD) and some autoimmune disorders such as systemic sclerosis, SjoÈgren's syndrome or primary biliary cirrhosis have fostered the concept that haematopoietic stem cells are involved in the pathogenesis of severe autoimmune disorders.8±10 These ®ndings form the basis for the postulate that a disease which is provoked by aberrant stem cells or their subsequent immune competent progeny could be restored by profound immunosuppression and `re-setting', supported by HSCT. Theoretical considerations encouraged by animal experiments and observations from patients with haematological malignancies and concomitant autoimmune disease followed the same direction. Results from recent phase I/II studies con®rm the concept to be correct at least in part. Animal data Experience with animal studies has been reviewed recently.11 It includes models of genetically induced autoimmune diseases as well as models with genetic predisposition but environmental or external induction of autoimmunity.12±16 Experimental models show that HSCT can treat established disease as well as prevent development of genetically driven diseases. Most animal data are based on allogeneic HSCTs. A few autologous HSCT experiments form the basis for the development of the clinical projects. Of key importance were observations in rats with Freund's adjuvant-induced arthritis. Autologous HSCTs could abrogate established arthritis and protect from rechallenge. These data did, indeed, suggest that tolerance could be re-established by autologous HSCT (Figure 1).17 So far, animal models have been fundamental in

Stem cell transplantation for autoimmune diseases 757

understanding pathogenetic mechanisms and in proving principles. In contrast, they have failed to predict the role of conditioning regimens. For example, the need for total body irradiation in certain disease conditions11 has not been substantiated so far by clinical experience. Concomitant disease Multiple patients have been reported with HSCT for haematological malignancies or aplastic anaemia and concomitant autoimmune disorders. They have been extensively reviewed.18±33 Reports include autologous and, more frequently, allogeneic HSCTs, and they cover the broad spectrum of autoimmune disorders. They are summarized in Tables 1 and 2. Overall, there have been complete, long-lasting responses, and transient improvements or responses, but relapses with the underlying disease. In addition, a few relapses with donor-type cells were observed as well as recurrence of auto-antibodies without recurrence of the disease.29 It remains a matter of debate whether such cure of autoimmune disease by allogeneic HSCT is solely the consequence of full chimerism and restoration of normal haematopoetic function or whether it depends on a graft-versus-autoimmune e€ect. Similarily, it remains controversial whether autologous HSCT is invariably associated with a high relapse rate and whether or not this relapse is in¯uenced by graft manipulation. In parallel, it is accepted today that autoimmune diseases can be transferred by allogeneic HSCT. This relates to most autoimmune disorders, and donors and recipients need to be informed accordingly. The presence of an autoimmune disease in a potential donor need not be an exclusion criterion.34±36 The severity of the recipient's disease and Table 1. Outcome of concomitant autoimmune disease by HSCT (coincidental autoimmune disease and allogeneic HSC transplantation). Disease for which transplant performed AD present SAA SAA SAA SAA SAA SAA SAA AML CML AML ALL CML Various

RA RA RA RA RA RA RA Psoriasis Psoriasis Ulcerative colitis Autoimmune hepatitis Multiple sclerosis Hyperthyroidism IDDM SLE, RA Crohn's disease Vasculitis Dermatitis herpetiformis

Outcome of AD Remission Remission Remission Remission Partial Remission Remission Remission Remission Remission Remission Remission Remission No recurrence

Patient outcome

Reference

Died Died Died Well Well Well Well Well Well Well Well Well Alive

Baldwin et al 197720 Baldwin et al 197720 Baldwin et al 197720 Baldwin et al 197720 Jacobs et al 198621 Lowenthal et al 199322 Lowenthal et al 199322 Eedy et al 199023 Yin & Jowitt 199224 Liu Yin & Jowitt 199224 Vento et al 199625 McAllister et al 199726 Nelson et al 199727

SAA ˆ severe aplastic anaemia; AML ˆ acute myeloid leukaemia; CML ˆ chronic myeloid leukaemia: ALL ˆ acute lymphoblastic leukaemia; RA ˆ rheumatoid arthritis; IDDM ˆ insulin dependent diabetes mellitus; SLE ˆ systemic lupus erythematosus.

758 A. Gratwohl et al Table 2. Outcome of concomitant autoimmune disease by HSCT (coincidental autoimmune disease and autologous HSC transplantation). Disease for which transplant performed

AD present

Outcome of AD

Patient outcome

Reference

NHL Ovarian cancer NHL NHL NHL NHL CML NHL NHL NHL AML Plasma cell leukaemia

Myasthenia gravis Thyroiditis myasthenia SLE Atopic dermatitis RA SLE SLE RA RA Psoriasis Psoriasis Psoriasis

Remission Relapse Relapse Relapse Relapse Remission Remission Remission Relapse Relapse Relapse Relapse

Well Alive Died Alive Alive Alive Alive Alive Alive Alive Alive Alive

Salzmann et al 199428 Euler et al 199629 Euler et al 199629 Euler et al 199629 Euler et al 199629 Snowden et al 199730 Meloni et al 199731 Jondeau et al 199732 Cooley et al 199733 Cooley et al 199733 Cooley et al 199733 Cooley et al 199733

NHL ˆ non-Hodgkin's lymphoma. Other abbreviations as in Table 1.

the severity of the respective autoimmune disease need to be balanced against each other. The magnitude of the problem is unknown because there is no systematic review on transmission of autoimmune disease, and reporting is probably in¯uenced by publication bias. Whether HSCT from a donor with any autoimmune disease will invariably induce autoimmune disease in the recipient needs to be addressed in the future. In one case report, transient systemic lupus erythematosus (SLE) serological events without overt disease were observed in a recipient (leukaemia) who received an HSCT from a brother with known SLE.37 Vice versa, it remains open whether the frequently observed autoimmune disease-like phenomena in the recipient always re¯ect graft-versus-host disease. There are at least some reports suggesting that skewed recovery of immune function could be associated with late autoimmune-like phenomena in autologous as well as allogeneic HSCT recipients.38 Potential mechanisms Understanding the mechanisms of cure by HSCT lags behind even more than our understanding of autoimmunity. Eradication of autoreactive cells has been the driving concept of initial attempts of high-dose therapy. It is likely that it represents a toosimplistic model. Additional mechanisms are being discussed in the treatment of malignancies by HSCT, including induction of graft-verus-tumour mechanisms.39,40 Intensive immunosuppression is clearly part of the therapeutic concept. Early data show that patients may have prolonged periods of lymphopenia, especially CD4-penia, following HSCT with highly selected CD34 cells. These periods appear to be associated with increased susceptibility to infectious complications but also with disease response.41 A more optimistic view sees HSCT as a repetition of ontogeny. Intensive immunoablation allows restoration of immunity de novo and, hopefully, restoration of tolerance via thymic re-education of T cells which are derived from the re-infused haematopoetic stem cells. The ®nding of large numbers of T cell receptor excision circles (TREC) positive cells ± for example, cells representing recent thymic emigrants ± in patients with CD34-selected HSCT for autoimmune disorders support such theories.42,43 However, clearly, T cell immune reconstitution is severely impaired even though B cell reconstitution in many ways repeats ontogeny.42 More observations

Stem cell transplantation for autoimmune diseases 759

and a longer follow-up are needed to ®nd out whether indeed an unbalanced immune system can be `reset' to time zero and whether normal immunity can be restored. THE EBMT/EULAR INTERNATIONAL PROJECT In 1995, EBMT and EULAR initiated a joint international project to evaluate the role of HSCT in the treatment of severe autoimmune disorders.44 A minimum time frame was given to start phase I/II-like studies, to leave enough freedom for individual institutions but to allow standardized evaluation. The concept was based on four main principles: patient selection, mobilization, conditioning and data collection. For safety®rst reasons, priority was given to autologous HSCT. As the main selection criterion, the severity of the patients' illness should be sucient to justify the risks of the procedure, but patients should be still well enough to pro®t from potential success. Mobilization should be restricted to standard procedures, using G-CSF with or without preceeding cyclophosphamide priming. No stipulations were made concerning manipulation of the graft. Conditioning should be restricted to four main standard regimens: cyclophosphamide (cy) + ATG, cyclophosphamide and total body irradiation (TBI) + ATG, busulfan and cyclophosphamide + ATG or BEAM (BCNU, Etoposide, Ara-C, Melphalan) polychemotherapy + ATG. The use of novel conditioning regimens was discouraged.45 All teams were invited to report their patients from mobilization with a minimal data set and to provide follow-up on a regular basis. In response, teams were informed regularly about all new emerging aspects. The network was harmonized with the North American partners; currently, data from the Americas are collected by the IBMTR/ABMTR registry, and data from Europe and Australasia via the EBMT/EULAR International Stem Cell Project in Basel. CURRENT RESULTS Overview As of Spring 2001, about 500 HCSTs have been performed worldwide; about 350 are included in this project and were reported recently (27th Annual Meeting European Group for Blood and Marrow Transplantation and 17th Nurses' Group Meeting, Maastricht, The Netherlands, 2001). They include primarily patients with neurological (n ˆ 101; 32.4%), rheumatological (n ˆ 183; 60.9%) and haematological (n ˆ 18; 5.8%) autoimmune disorders and are summarized in Table 3. Information on these 312 patients (115 male and 197 female) was obtained from 78 teams in 21 countries (see Acknowledgements). There were paediatric and adult patients of from 2 to 65 years of age (median 35), and time from diagnosis to treatment varied from 1 to 28 years (median 7). Bone marrow harvest was used to collect stem cells in 43 patients (14%), mainly in children. Peripheral blood as stem cell source was used in 265 patients (85%), while four patients had both stem cell sources (1%). For stem cell mobilization the following methods were used: G-CSF alone (n ˆ 61; 20%), cyclophosphamide + G-CSF + GMCSF + others (n ˆ 188; 60%), and miscellaneous methods/missing cases (n ˆ 20; 6.4%). No mobilization was performed in the 43 cases with bone marrow as stem cell source. Not all mobilized patients proceeded to HSCT; 302 patients were transplanted, two out of the total of 302 patients twice. Either mobilization was unsuccessful or HSCT was no longer considered appropriate owing to disease progression or improvement.

760 A. Gratwohl et al Table 3. Main indications for HSCT in autoimmune diseases. Number of patients

%

Neurological disorders Multiple sclerosis Myasthenia gravis Polyneuropathy Cerebellar degeneration Amyotrophic lateral sclerosis

101 97 1 1 1 1

32.4 96 1 1 1 1

Rheumatological disorders Systemic sclerosis Rheumatoid arthritis Juvenile idiopathic arthritis Systemic lupus erythematosus Dermatomyositis MCTD BehcËet's disease Psoriatic arthritis Ankylosing spondylitis SjoÈgren's syndrome

183 55 48 39 25 6 3 3 2 1 1

58.7 30 26.2 21.3 13.7 3.3 1.6 1.6 1.1 0.6 0.6

7 3 3 1

2.2 43 43 14

18 9 4 2 2 1

5.8 50 22.2 11.1 11.1 5.6

2 1 1 1

0.6 50 50 0.3

Disease and disease category

Vasculitides Wegener's Cryoglobulinaemia Not classi®ed Haematological Immunocytopenlas Immune thrombopenia Pure red cell aplasia Autoimmune haemolytic anaemia Thrombotic thrombocytopenic anaemia Evans' syndrome Gastrointestinal Enteropathy In¯ammatory bowel disease Mixed expressed autoimmune disease Total

312

100

Data from the International Autoimmune Disease Stem Cell Project, Basel, Switzerland.

Moreover, three patients died following mobilization (all su€ering from systemic sclerosis (SSc)). Mobilization failures are well known in autologous HSCTs. Mobilization deaths, in contrast, are usually not reported to registries and are often not published. There were indeed novel problems encountered during mobilization for patients with autoimmune disorders: some patients with multiple sclerosis had signs of ¯ares, some patients with systemic sclerosis had lethal infectious complications, probably as a consequence of their organ ®brosis and reduced cardiac function, and in two (three internationally) patients with juvenile idiopathic arthritis fatal macrophage activation syndromes were observed.2,46,47 In the 302 patients reported as transplanted a variety of conditioning regimens were used (Table 4). There was preferential use of certain conditioning regimens, based mainly on ®rst use and tradition. BEAM conditioning regimen was the preferred choice for patients with multiple sclerosis, cyclophosphamide and ATG for rheumatoid

Stem cell transplantation for autoimmune diseases 761 Table 4. Conditioning regimens used for patients with HSCT in autoimmune diseases. Conditioning regimen

ND

Cyclophosphamide Cyclophosphamide + ATG + other drugs Cyclophosphamide ‡ TBI + other drugs or ATG Busulfan + cyclophosphamide + ATG + other drugs BEAM + ATG Other/missing

1 16 6 15 57 5

RD

HD

Others

81 51 21 6 6 16

3 10 1 1 0 3

1 0 0 0 0 2

Total TBI ˆ total body irradiation (includes some patients with total lymphoid irradiation). ND ˆ neurological diseases; RD ˆ rheumatological diseases; HD ˆ haematological BEAM ˆ BCNU,VP16, ara-C, melphalan.

Total 86 77 28 22 63 26 302 diseases.

arthritis. The groups are too heterogeneous to permit conclusions to be drawn concerning the ecacy of individual regimens. Nevertheless, responses and failures were reported with all the regimens used. In contrast, there was a report of an association between dose intensity and transplant-related mortality (TRM) based on 290 patients.48 TRM was reported to be 19 + 10% (95 CI) in patients given a highintensity conditioning regimen (de®ned as either cyclophosphamide (Cy) with radiation or busulfan with Cy or other drugs) compared to 8 + 5% and 4 + 4% in patients given an intermediate or low-intensity conditioning regimen. No de®nitive statements can yet be made concerning disease response or disease progression in relation to conditioning or to use of T- or B-cell-reducing approaches. Response and relapses were reported with all regimens used, with or without TBI (total body irradiation), with or without T- or B-cell depletion. Clearly, the earlier statement that relapse will invariably occur with unmanipulated grafts cannot be maintained as such.29 Outcome of transplants is listed by disease categories. Di€erent outcomes were observed in all disease categories. They include mortality or withdrawal before HSCT, mortality during HSCT, complete responses, improvements, stabilization of disease or progression despite HSCT. All response categories were observed in all disease categories (Table 5). Many patients had been reported as case reports, single institutional series or in earlier reviews but are also included in the international project. Results of the combined data are worse than published short series for two main reasons. Multicentre observational data are routinely worse than single-centre pilot studies, and successful data are more readily published than negative ones. Patient selection and publication bias explain this situation. Systemic sclerosis (SSc) Systemic sclerosis comprises a heterogeneous group of disorders with a presumed autoimmune pathogenesis, poor response to available treatment and limited survival once vital organs are a€ected.49 Patients with systemic sclerosis were considered among the ®rst to be treated with HSCT and were indeed among the ®rst to be reported with successful HSCT.50,51 It was soon recognized that patients with systemic sclerosis present speci®c problems during mobilization and during conditioning, with a high procedurerelated mortality, exceeding 10%.3 Today, a clear statement can be made based on a ®rst series of patients studied in detail and published.

762 A. Gratwohl et al Table 5. Main outcome of HSCT for autoimmune diseases according to disease category.a Disease group and number of patients Patients reported

MS

Patients with HSCT not available/too early treatment related mortality after mobilization after transplantation disease mortality response/better response, then progression stable disease no response/progression/worse

SSc

RA

97 9

55 8

48 2

± 5 2

3 4 4

33 3 28 17

27 9 ± ±

JIA

SLE

ITP

39 1

25 ±

9 ±

± 1 ±

± 5 1

± 4 ±

± 2 ±

7 24 1 13

20 10 ± 2

13 7 ± 1

2 1 3 1

aData

from the International Autoimmune Disease Stem Cell Project, Basel, Switzerland. MS ˆ multiple sclerosis; SSc ˆ systemic sclerosis; RA ˆ rheumatoid arthritis; JIA ˆ juvenile idiopathic arthritis; SLE ˆ systemic lupus erythematodes; ITP ˆ immune thrombopenia.

Some 41 patients, 37 with predominantly di€use skin form and four with limited sclerosis, between 12 and 58 years of age (median 41 years), were treated with HSCT. All were considered to have severe disease, 76% had lung involvement with 570% forced vital capacity (FVC) in half of the patients, pulmonary artery hypertension in seven and renal involvement in ®ve patients. Skin score was 450% of maximum in two-thirds of the patients. Some 37 patients had Raynaud's phenomenon, 21 oesophageal and four gastrointestinal involvement.

100 conditioning: cyclo + 4Gy PSA vs Syn BMT reimmunization

% arthritic score

80

60

40

20

0

2°60mg/kg Cyclo + 4 Gy + PSA BMT (n=9) 2°60mg/kg Cyclo + 4 Gy + Syn BMT (n=10)

0

5

imm.BMT

10

15

20

weeks after immunization

reimm.

Figure 1. Cure of arthritis in rats by autologous HSCT: adjuvant arthritis in Bu€alo rats. Reprinted with permission: Knaan-Shanzer S et al.17 PSA ˆ Pseudoautologous; BMT ˆ bone marrow transplantation.

Stem cell transplantation for autoimmune diseases 763

Change in Skin Score

0.8 0.6 0.4 †

0.2 0

†

–0.2 †

–0.4 –0.6 –0.8

–30

0 30 90 180 Time (Days after transplant)

385

730

Figure 2. Change in skin score. The serial skin score data are presented for the 37 patients with di€use scleroderma. The proportional change from baseline measurements was calculated for each patient at each available time point. Two horizontal lines are marked, which represent changes of 25%. The x axis is not drawn to scale. Data obtained before administration of priming regimen are shown at ÿ30 days (although the temporal relation to conditioning was variable). A vertical line is drawn to show the timing of conditioning treatment. {ˆpatient death, but is only shown for deaths beyond 90 days. Reprinted with permission: Binks et al.3

All but one had peripheral blood stem cell grafts, obtained after mobilization with cyclophosphamide and G-CSF or G-CSF alone. Seven conditioning regimens were used. The majoritiy, 86%, used cyclophosphamide + ATG + TBI. In all but three cases CD34 cell selection was used. The results are striking. A prolonged improvement in skin score of 425% was observed in 70% of the evaluable patients (Figure 2). Lung function tended to be stabilized. Only one of the ®ve patients with renal involvement showed progression of deterioration, and pulmonary artery hypertension was stabilized in all patients. Many patients reported subjective improvement in wellbeing. Disease progression was reported in 19%, and three patients died of progressive disease. Seven patients died as a consequence of the procedure, bringing the transplant-related mortality to 17%. Three of these patients died before HSCT from the mobilization procedure ± two cardiac and one septic event. Four patients died after HSCT, two from interstitial pneumonitis, one from di€use alveolar haemorrhage and one from CNS bleeding. The cumulative probability of survival at 1 year was 73% (58±88, 95% CI). Autopsy in one case showed cardiac SSc changes as cause of death, despite careful cardiac assessment before enrolment.47 An update at the EBMT Meeting in Maastricht with 55 patients con®rmed the response rates but currently shows a markedly reduced transplant-related mortality of about 10%, indicating a learning curve with better patient selection. Consensus at the Basel Meeting in October 2000, as well as at later meetings, was that patient selection should be restricted to patients without severe pulmonary or cardiac failure (Table 6) and that a prospective randomized trial should now prove the concept (see below).2 Multiple sclerosis Immunosuppression or immunomodulation has been the mainstay of treatment for patients with multiple sclerosis, the most common neurological autoimmune disease.

*Capable of response

Full®llment of ARA-criteria for RA 18±55 years old Disease duration 4 2 and 5 15 years Active disease de®ned as 5 six swollen joints*, 5 six tender joints, 5 one of early morning sti€ness, ESR 4 28 mm/hour or CRP 4 20 mg/dl . Lack of response to MTX (25 mg/week) or lack of response to LEF (20 mg OD) if intolerant of MTX . Failure of 5 four DMARDs (including MTX and/ or LEF), combination therapy and anti-TNF (with no other e€ective treatment options available) . Informed consent

. . . .

Rheumatoid arthritis (RA)

EDSS ˆ expanded disability status scale (0 ˆ no disability, 10 ˆ dead from disease); mir ˆ magnetic resonance imaging; DLCO ˆ di€usion lung capacity in oxygen; FVC ˆ forced vital capacity; LVEF ˆ left ventricular ejection fraction; ARA ˆ American Rheumatism Association; MTX ˆ methotrexate; LEF ˆ Le¯umonid; DMARDS ˆ disease modifying anti-rheumatic drugs; TNF ˆ tumor necrosis factor.

. . .

. . .

. Full®llment of ARA-criteria for SSc . 16±60 years old . Di€use scleroderma 4 4 years plus Rodnan skin score 5 15 plus major organ involvement such as: (a) respiratory involvement: DLCO and/or (F)VC 4 70% (of predicted) plus evidence of interstitional lung disease; (b) renal involvement: hypertension, persistent urinalysis abnormalities, microangiopathic anaemia, new renal insuciency; (c) cardiac involvement: reversible congestive heart failure; LVEF 5 45%, signi®cant atrial or ventricular rhythm disturbances, pericardial e€usion . Informed consent

. . . .

Full®llment of Poser's criteria for MS 18±50 years old Disease duration 4 1 year Relapse-remitting, secondary-progressive, progressive relapsing course MS-speci®c brain-MRI-®ndings (Barkhof criteria) EDSS between 3.5 and 6.5 Disability progression sustained for 6 months in previous 2 years of: (a) 4 1.5 points if entry EDSS 3.0±5.0, (b) 4 1.0 point if entry EDSS 4 5.5 Clinical or MRI activity in the last year Unsatisfactory response Informed consent

Systemic sclerosis (SSc)

Multiple sclerosis (MS)

Table 6. Inclusion criteria for future trials.

764 A. Gratwohl et al

Stem cell transplantation for autoimmune diseases 765

Components of myelin are discussed as the target and in®ltration of T-cells, while macrophages and in¯ammatory cells form the basis for focal neurological dysfunction and demyelination.52 The course of disease is highly variable from benign (10%) to rapidly malignant (1±3%). Multiple sclerosis (MS) typically presents as a relapsing remitting disease (80%) with a secondary progressive phase several years later. About one-®fth of patients show primary progressive forms of disease from onset.53,54 Multiple sclerosis not only a€ects quality of life but is also associated with high morbidity and costs; it is also associated with a signi®cant reduction of life compared to age-matched healthy individuals.55,56 Many animal models of experimental allergic encephalomyelitis have shown that both allogeneic and autologous HSCTs can induce remissions and cures.15,57,58 Several case reports of concomitant disease have con®rmed that it is possible to achieve prolonged remissions of the disease in humans by HSCT in selected situations. Two groups reported the feasibility of HSCT in this disease, using either a polychemotherapy-based regimen, BEAM or a TBI-based regimen.59,60 Today, data on nearly 100 patients show that HSCTs can stabilize progressive MS ± possibly better than any other available therapy.61 Nevertheless, comparative studies are required.62 Evidence comes from a recent analysis of the international project based on 85 patients, aged 20±58 years (39 years median), including 52 female and 33 male patients with progressive MS who were treated in 20 centres.61 The majority su€ered from primary progressive (19 patients), relapsing progressive (13 patients) or secondary progressive (48 patients) disease. Disease was severe, with a median expanded disability status scale (EDSS) score of 6.5 (4.5±8.5) and disease progression by at least one EDSS point in the preceding 12 months in 78 of 82 evaluable patients. The stem cell source was bone marrow in six and peripheral blood in 79 patients. Mobilization was performed with cyclophosphamide and G-CSF/GM-CSF in 80%. BEAM + ATG was used in 54 patients (63%), cyclophosphamide + ATG + TBI in 15 patients (18%), and busulfan and cyclophosphamide + ATG in 15 (18%). Stem cell manipulation to remove T cells was performed in 52 patients. Neurological improvement by 51 EDSS point was reported in 18 (21%) patients. Con®rmed progression-free survival was 74% (+12%) at 3 years. It was lower in patients with primary progressive MS (59 + 27%) than in non-primary progressive MS (79 + 12%). Toxicity was considerable. Neurological deterioration occurred in 21 patients during the transplant. It was transient in most but associated with MS progression in ®ve patients, two of whom died. In total, there were seven deaths, ®ve due to toxicity and infectious complications and two due to disease progression. Treatment-related mortality at 3 years was 8%. Neurological deterioration was reported in another series as well during mobilization.63 It remains open whether this was due to a direct e€ect of G-CSF or to the consequence of indirect cytokine release.64,65 Most remarkable are the observations of an ongoing Italian multicentre study with regular MRI scans during follow-up. In all except one of 10 patients studied, all gadolinium-enhanced lesions disappeared upon follow-up.4 The opinion of many experts in the ®eld of MS is that current data show feasibility and promising results, as was anticipated by the Milan consensus group.66 It is now the role for prospective randomized trials to prove superiority compared to standard treatment. Entry criteria have been de®ned, and a prospective international phase III trial is in preparation (ASTIMS trial).

766 A. Gratwohl et al

Rheumatoid arthritis (RA) Data on a retrospective analysis of 70 rheumatoid arthritis (RA) patients were provided during an international meeting in Basel (Switzerland) in October, 2000.2 HSCT was well tolerated: only one death was reported among 70 patients (none in the cyclophosphamide-only conditioning regimens). Most patients responded, with ACR responses similar to those seen after anti-TNF-a treatment. However, about 50% relapsed, most having synovitis ¯ares. Many relapses responded well to standard treatment with a single disease-modifying antirheumatic drug (DMARD: methotrexate, le¯unomide or cyclosporin)±drugs which had failed before transplant. However, in a small number of patients it was observed that cyclosporin-A may have a negative impact on T cell immune reconstitution after transplant for naõÈ ve T cell re-population and thymic function; it is therefore not recommended as the ®rst DMARD for maintenance after transplant. Data are in line with other published small series or case reports.67±69 Dose intensity might correlate with response.70 No one regimen was clearly better than another. Some patients did not proceed to HSCT after they were found to have improved signi®cantly after 4 g cyclophosphamide as mobilization. One case of seronegative RA received a syngeneic HSCT from his identical twin brother and did well.71 Consensus was reached on the need and feasibility for an HSCT randomized controlled trial.2,72 The trial's name is ASTIRA (Autologous Stem Cell Transplantation International in RA) and the inclusion/exclusion criteria are listed in Table 6. The trial design is shown in Figure 3(c). Systemic lupus erythematosus (SLE) SLE is a multi-organ disease that especially a€ects the skin and the blood vessels.73 Disease activity is often re¯ected in a rise in autoantibody production, in particular the anti-double-stranded-DNA-antibody and by a fall in serum complement levels. Its pathogenesis remains unclear but an autoimmune component is generally accepted. Standard treatment for severe disease consists of intensive immunosuppression with pulse dose cyclophosphamide. Responses to HSCT in patients with concomitant SLE and a haematological malignancy have been reported early and formed the basis for several attempts with HSCT as the basic treatment.29 A successful response in ®ve patients was recently reported74; all had advanced aggressive disease. In addition, single case reports have been encouraging.5 In the EBMT/EULAR database a total of 25 SLE patients are registered. A detailed analysis has been performed on 22 patients (A. Marmont on the behalf of the EBMT on Autoimmune Disease Working Party, 27th Annual Meeting European Group for Blood and Marrow Transplantation and 17th Nurses Group Meeting, Maastricht, The Netherlands, 2001). They represented a highly heterogenous group. The female-to-male ratio was 6.3, the group including 19 females and three males. The median age was 27 (15±52 years range). The median duration of disease was 7 years; systemic lupus erythematodes activity index (SLEDAI) scores pre-transplant were mostly high, reaching from 6 to 38, with a medium value of 18 (maximum score 105). Before mobilization there was evidence of autoimmune activity with anti-dsDNS-antibodies strongly positive in 89% patients. The main clinical features before transplantation were arthralgias (90%) and renal involvement (85%) followed by CNS

Stem cell transplantation for autoimmune diseases 767

(a) ASTIS Trial Autologous Stem cell Transplantation International Scleroderma Trial ENTRY HSCT

CONTROL

Mobilization - Cy 4 g/m2 Conditioning–Cy 200 mg/kg, ATG 7.5 mg/kg CD 34 selection

12 monthly pulse Cy 750 mg/m2 Rescue immunosuppression

(b) ASTIMS Trial

Autologous Stem cell Transplantation International Multiple SclerosisTrial

ENTRY CONTROL

HSCT Mobilization - Cy 4g, G-CSF 5mg Conditioning - BEAM + ATG unselected graft

3-monthly Mitoxanthrone 12 mg/m2

(c) ASTIRA Trial

Autologous Stem cell Transplantation International Rheumatoid Arthritis Trial

ENTRY Mobilization - Cy 4 g/m2 harvest and store HSCT Conditioning - Cy 200 mg/kg Unpurged graft product

CONTROL Continued best standard

Figure 3. Trial design of prospective randomized studies comparing HSCT with conventional therapy. (a) ASTIS trial; (b) ASTIMS trial; (c) ASTIRA trial.

a€ection (40%) and positive ACA/LA (anticardiolipin-antibodies/Lupus anticoagulant) (40%). Less often reported were symptoms such as serositis (18%), vasculitis/ thrombosis and myositis (9% each); Evans' syndrome and autoimmunethrombopenia occurred in 5%. Bone marrow was used as a source of stem cells in six patients (27%), peripheral blood in 16 (73%). Mobilization was performed using cyclophosphamide in doses from 2 to 4 g/m2, CD34‡ selection being carried out in 13 cases (59%). A multitude of di€erent conditioning regimens was used: 200 mg cyclophosphamide/kg body weight + ATG (nine patients), BEAM (four patients); cyclophosphamide plus thiopeta or methylprednisolone (three patients each), VP-melphalan (two patients) and

768 A. Gratwohl et al

cyclophosphamide plus total body irradiation (TBI) with 400 cGy (one patient). Four patients were considered to have died through treatment-related causes; one died because of gastrointestinal bleeding and DIC zero days after HSCT, and one died 11 days after SCT due to graft and multi-organ failure. Two young patients (age 16) died, one of thrombotic thrombocytopenic purpura (TTP), the other from sepsis and multiorgan failure, 65 and 22 days respectively post-SCT. Seventeen patients have been followed-up, another currently being too early for follow-up. Out of these 17 followup cases, ®ve are now o€ therapy (30%), while four need the same treatment as before SCT (24%); in eight patients (47%) new immunosuppressive therapy was needed in addition to corticosteroids. These data show an overall survival of 82% and a high TRM. There was no disease-free survival after transplant, although a signi®cant improvement was seen in SLEDAI scores (median value of 3 at 100 days after SCT) and in antibody production. Results were intensively discussed at the EBMT meeting in Maastricht (March, 2001) and at the 6th International Lupus Conference in Barcelona (March, 2001). The conclusion of the evaluated phase I/II studies is that HSCT has a signi®cant therapeutic e€ect in otherwise treatment-resistant patients. It was associated with a high TRM, emphasizing the importance of patient selection and strict interdisciplinary collaboration. There was a trend to progressive serological and, ®nally, clinical relapse as the antibody production rises slowly but continuously after HSCT.75 Many patients who relapsed responded to medication which had been ine€ective pre-transplant. More phase II data are required, especially with respect to patient selection, before phase III randomized controlled trials can be planned. Juvenile idiopathic arthritis (JIA) Juvenile idiopathic arthritis (JIA) can present a major clinical problem in its severe refractory form and lends itself to more aggressive therapy.76 Most of the reported cases of HSCT for JIA were for the systemic, polyarticular type (Still's disease).46 Fifteen of these were transplanted in two centres in The Netherlands: Utrecht and Leiden (reported by N. Wul€raat). In this cohort eight full and two partial remissions were recorded, while two deaths occurred through macrophage activation syndrome. Internationally a further 10 complete remissions were noted as well as another death through macrophage activation syndrome. Protocols were modi®ed accordingly to avoid transplantation during a phase of signi®cant systemic activity, which should be controlled with corticosteroid treatment. The impact of the modi®ed protocol using bolus prednisolone to control systemic disease before HSCT has not yet been assessed, but as there have been no further such deaths it may reduce the TRM to below the current 15%, making a comparative trial feasible. Over the next 12 months the ®nal JIA protocol will be worked on and reported (N. Wul€raat). There was consensus on a potential role for HSCT in this disease.2,77 Immune thrombopenia Immune thrombopenia (ITP) is considered as autoimmune disease. Antibodies against platelet-speci®c surface receptors form the basis for immune destruction of circulating platelets and corresponding increased megacaryocyte production.78 A ®rst report of successful HSCT was published in 19976, presenting two patients with refractory ITP responding to HSCT with normalization of their platelet counts. Conditioning was with cyclophosphamide and unmanipulated cells were used. Two case reports shortly

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thereafter reported unsuccessful outcome and persisting disease, despite a similar approach and the additional use of highly selected CD34 cells.79,80 A phase I/II study was performed and recently presented by the National Institutes of Health in the USA.81 Twelve patients with advanced ITP, refractory to steroids, intravenous immunoglobulins and splenectomy were treated in a standardized protocol, using cyclophosphamide and ATG and selected CD34 cells. All patients tolerated the procedure with no major bleeding complications during the aplastic phase, even though multiple platelet transfusions were necessary. Three of the 12 patients responded completely and recovered normal platelet counts with an observation time exceeding 1 year in all. Two patients showed severe complications; one patient was found to su€er from lung cancer, and one patient had a cerebral Aspergillus infection. These data are compatible with the results from the International project where two of nine patients showed a clear response to HSCT. No information is yet available on potential factors in¯uencing response. No international consensus has yet been reached on the topic. Overall, and together with the data from the International project, refractory ITP can respond to HSCT. Probably, in a prospective randomized study, HSCT should be compared to splenectomy in patients refractory to steroids and intravenous immunoglobulins. Miscellaneous There are several case reports on HSCT in gastrointestinal autoimmune disease, in vasculitides, such as cryoglobulinaemias or BehcËet's disease, or other autoimmune disease categories. Successful responses have been described in refractory BehcËet's disease.82 No information is available on unsuccessful, unpublished or non-reported cases. Too little information is available to give any recommendations for treatment ± except that all such patients should be reported to the data registries and that further phase I/II data are required.83 PROSPECTIVE RANDOMIZED STUDIES There is now sucient information from phase I/II trials that HSCT can induce lasting remissions in patients with severe autoimmune disorders. Remissions can be obtained with a variety of conditioning regimens and with bone marrow and peripheral blood stem cells using technologies which are standard in HSCT for other disease categories. Results are obtained at the expense of considerable morbidity and mortality, of the same order of magnitude as observed in HSCTs for advanced disease in malignancies. There is no more than level III evidence about the usefulness of this approach, and there are no studies so far to prove superiority over conventional approaches. It is therefore the primary target of the International project to conduct the appropriate prospective randomized studies while still continuing to collect the observational data and to continue the phase I/II-like trials in those disease categories with less information. The decision to proceed is based on several considerations (Table 7). In the ideal situation, enough information is available from phase I/II trials; inherent mortality of the disease justi®es the risk of the procedure; prognostic factors of the disease are known to de®ne patients at high risk for disease progression; HSCT morbidity and mortality is acceptably low; the risk of disease progression after HSCT is low; and little or no alternative conventional therapy is available. Currently, such criteria are suciently met for systemic sclerosis, multiple sclerosis and rheumatoid arthritis.

770 A. Gratwohl et al Table 7. Considerations for planned trials of HSCT in autoimmune diseases. Inherent Prognostic HSCT Relapse Alternative mortality factors known mortality after HSCT therapy available Systemic sclerosis Rheumatoid arthritis Systemic lupus erythematosus Multiple sclerosis

‡‡ ‡ ‡ ‡ ‡

‡‡‡ ‡‡ ‡ ‡‡‡

ÿ ÿ ÿÿÿ ‡

ÿ ÿÿÿ ÿÿ ÿ

ÿ ÿÿÿ ÿÿÿ ÿ

Factors in favour (‡) or against (ÿ) HSCT.

ASTIS (Autologous Stem Cell Transplantation International Scleroderma Trial) This trial will compare patients with severe systemic sclerosis, treated with autologous HSCT or monthly pulse cyclophosphamide. Inclusion criteria are listed in Table 6 and the trial design is illustrated in Figure 3(a). Mobilization will be performed with cyclophosphamide 2  2 g/m2 followed by G-CSF. Cells can be CD34-selected by two di€erent methods, and conditioning will consist of cyclophosphamide 200 mg/kg and rabbit ATG. Attention will be given to patient selection in order to exclude patients with too-advanced disease. It is anticipated that 200 patients will be included. The primary endpoint will be the time to occurrence of an event, an event being de®ned as death or major organ failure. Details of the trial can be obtained from the study administration oce on the webpage http://www.astistrial.com. Patient recruitment for the trial began in March, 2001. ASTIMS (Autologous Stem Cell Transplantation International Multiple Sclerosis Trial) This trial will compare patients with severe progressive multiple sclerosis, treated with autologous HSCT or 3-monthly mitoxantrone. Inclusion criteria are listed in Table 6, and the trial design in illustrated in Figure 3(a). Mobilization will be performed with cyclophosphamide 2  2 g/m2 followed by G-CSF. Cells will not be CD34-selected, and conditioning will consist of high-dose combination chemotherapy BEAM followed by rabbit ATG. Attention will be given to patient selection in order to exclude patients with too-advanced disease. The number of patients to be included, as well as the primary and secondary endpoints, are currently still being discussed. Details of the trial can be obtained from the study administration oce or on the study website: http://www.astimstrial.com. ASTIRA (Autologous Stem Cell Transplantation International Rheumatoid Arthritis Trial) This trial will assess the value of HSCT in patients with severe refractory rheumatoid arthritis. The design of the study is currently being ®nalized. It will most likely include an approach with inclusion of all registered patients and mobilization with cyclophosphamide in all patients. Successfully mobilized patients will then be randomized to immediate HSCT or continued `best available' treatment. This approach is proposed because high rates of response are expected from the high-dose cyclophosphamide alone

Stem cell transplantation for autoimmune diseases 771

used for mobilization. Conditioning will most likely consist of cyclophosphamide 200 mg/kg body weight without ATG, and there will be no CD34 selection. The number of patients to be included, as well as the primary and secondary endpoints, are currently still being discussed. Details of the trial can be obtained from the study administration oce or on the study website: http://www.astiratrial.com. DISCUSSION Substantial evidence has accumulated over the past 5 years since the introduction of the joint International EBMT/EULAR project. Clinical data, obtained from a substantial number of teams all over the world in local or national phase I/II-like trials, con®rm the postulated previous theoretical concepts. Observations from patients with HSCT for haematological diseases and concomitant autoimmune disease can be validated by results from HSCT in patients with isolated autoimmune disease. Animal models have an important but limited role concerning clinical e€ects. International co-operation based on voluntary participation and rapid exchange of information is possible.2 Taken together, current results show that HSCT can be performed in patients with severe autoimmune diseases of any type. Standard techniques as used routinely in conventional indications for HSCT can be employed and, with a few exceptions, the same problems are encountered. HSCT can induce long-lasting remissions in patients' subgroups. It is associated with substantial morbidity and mortality. It does not provide a simplistic solution for all patients, and failures as well as relapses ± despite initial response ± must be anticipated. Based on adequate numbers, sucient consensus has been reached to initiate prospective controlled comparative trials in de®ned disease categories: systemic sclerosis, multiple sclerosis and rheumatoid arthritis. Too few data are available for all other indications, and ongoing phase I/II trial data need to be gathered and analysed. Hopefully, early results from the prospective phase III trials will by then ease analysis and decision making. Not unexpectedly, results have also illustrated many problems related to, and well known in, the ®eld of HSCT. As in leukaemia and lymphoma treatment, the main indications for HSCT at the current time, the mechanisms of cure or disease modi®cation, are still poorly understood. Concepts of dose intensi®cation alone are too simplistic. This might explain why no `best conditioning regimen' has been identi®ed and why animal models fail to be of help in this regard. Similarly, as after years of discussion on the role of `purging' in leukaemia and lymphoma treatment, no clues hint at the value of CD34 selection compared to unselected bone marrow or peripheral blood stem cell transplantation.84 It is possible that these questions can never be answered satisfactorily. Still, consensus has been reached among the main investigators in the ®eld that, as a matter of high priority, the basic conceptual question ± HSCT versus conventional treatment ± could and should be answered. It is timely to perform the appropriate trials before too-optimistic views forbid randomized trials with the same arguments used a decade ago, when early results suggested a marked bene®t of HSCT in breast cancer. Similarly, observations from patients scheduled and waiting for HSCT have shown that the inherent mortality of the disease(s) in discussion is not negligible. Several patients died while waiting for HSCT because of logistic or ®nancial reasons (in one centre six out of nine SSc patients, D. Furst, personal communication). The planned and ongoing studies should soon increase our evidence above level III. Last but not least, the concepts behind HSCT in these diseases have already stimulated and enriched the discussions on the pathogenesis of autoimmune disease in

772 A. Gratwohl et al

general. What are the initiating factors? What are the perpetuating factors, and what are the criteria triggering progression or slowing it down? It is the documented wish of the trial co-ordinators to link basic immunological questions to the ongoing or planned trials. Eventually, results of these trials could not only give information on the value of these therapeutic approaches but also on the potential for immune reconstitution41 and correction of aberrant T or B cells. Acknowledgements The participating centres and their collaborators are acknowledged. The participating centres are: Austria: AKH, Vienna. Australia: St Vincent's Hospital, Sydney; Sir Charles Gairdner Hospital, Perth; Royal Hospital, Perth; Royal Hobart Hospital, Tasmania; Alfred Hospital, Melbourne. Belgium: Erasmus Hospital, Brussels; Brussels; Children University Hospital, Brussels; CHU, LieÁge; University Hospital Gasthuisberg, Leuven. China: Drum Tower Hospital, Nanjing. Czech Republic: Charles University Medical School, Prague; Charles University Hospital, Pilsen. France: HoÃpital Jean Minjoz BesancËon; HoÃpital St Antoine, Paris; HoÃpital Necker, Paris; HoÃpital Purpan, Toulouse; HoÃpital St Louis, Paris; HoÃpital Avicennes, Bobigny; Centre HoÃpitalier Universitaire, Clermont Ferrant; HoÃpital Claude Huriez, Lille; HoÃpital Universitaire de Hautepierre, Strasbourg. Germany: Klinikum

Practice points . some animal models demonstrate tolerance induction and long-term remission following autologous stem cell therapy . some patients with coincidental autoimmune diseases and malignancies show long-term remission of the autoimmune disease following HSCT performed for the malignancy . relapses can occur following autologous and allogeneic transplants, even despite documented full chimeric states . patient selection is critical to avoid undue transplant-related mortality . autologous transplantation is preferred to allogeneic transplantation due to lower toxicity . all transplanted patients should be in an approved clinical trial with central data registration . substantial clinical responses have been observed in all disease categories. Treatment-related mortality is 8±10%, e.g. comparable to that observed in patients with advanced malignancies . low treatment-related mortality with RA probably re¯ects less vital organ involvement pre-transplant . sustained skin improvement is seen after transplantation in many SSc patients with few early relapses . stabilization of lung function can be observed following HSCT . phase I and II study experience forms the basis for phase III randomized controlled trials. The same techniques used in HSCT can be used for other indications . international centralized data collection ensured rapid utilization of the data . only through prospective multicentre randomized controlled studies will the role of HSCT in autoimmune disease treatment be established

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Research agenda . comprehensive standardized data collection on all patients (mobilized/ transplanted) with either the EBMT (non-Americas cases) or the IBMTR (for the Americas cases) . patients su€ering from SSc, RA and MS should be transplanted only within the context of a prospective randomized controlled trial . other autoimmune diseases require further phase I and II data using a restricted number of protocols to established randomized trial regimens . standardized and co-ordinated immune reconstitution and function measurement pre-and post-transplant is essential to understand and improve ASCT as a treatment for autoimmune diseases Albert-Ludwig, Freiburg; Medizinische UniversitaÈtsklinik, TuÈbingen; UniversitaÈtsklinikum, Dresden; Medizinische Hochschule, Hannover; Charite Hospital, Berlin; UniversitaÈtsklinik, MuÈnster; University of Heidelberg; University of Jena; DKD KMT Zentrum, Wiesbaden. Hungary: Postgraduate Medical School, Miskolc. Finland: Kuopio University Hospital; Turku University. Greece: George Papanicolaou Hospital, Thessaloniki. Israel: Rambam Medical Center, Haifa; Hadassah University Hospital, Jerusalem. Italy: Ospedale San Martino, Genova; Policlinico San Matteo, Pavia; UniversitaÁ di Palermo; UniversitaÁ `La Sapienza', Roma; UniversitaÁ Tor Vergata, Roma; Ospedale San Camillo, Roma; Ospedale pediatrico Burlo Garofolo, Trieste; Ospedale pediatrico `A.Meyer', Firenze; Ospedale di Careggi, Firenze; Ospedale St Anna, Ferrara; Ospedali Binacchi, Cagliari; Ospedale San Ra€aele, Milano. Japan: Osaka Medical Center, The Netherlands: University Hospital for Children, Utrecht; Akademish Ziekenhuis, Leiden; University Hospital, Nijmegen; University hospital, Rotterdam. Poland: K. Marcinkowski University, Poznan. Russia: Institute of Clinical Immunology, Novosibirsk. Slovakia: University Hospital, Bratislava. Spain: SAS Jerez; Hospital Xeral-Calde, Lugo; Hospital Infantil La Paz, Madrid; Clinica Puerta de Hierro, Madrid; Hospital G.U. Gregorio Maranon, Madrid; Carlos Haya, Malaga; Hospital Universitaire Virgen del Rocio, Sevilla; Hospital Clinic, Barcelona; Hospital Vall d'Hebron, Barcelona; Hospital San Dureta, Palma de Mallorca: Sweden; Sahlgrenska University Hospital, GoÈteborg. Switzerland: Kantonsspital Basel. UK: Queen Elizabeth Hospital, Birmingham; University of Leeds; Royal Free Hospital, London; St George's Hospital, London; University College, London; Royal Victoria In®rmary, Newcastle, Nottingham. Special thanks to Dr Chiara Bocelli-Tyndall for data collection and data management. The International project is supported by unrestricted contributions from AMGEN, Sangstat, Nexell, Milteny Corporations, and it is supported by grants from the Swiss National Research Foundation and from the Horten Foundation.

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