Statement of findings
Autoimmune diseases that are resistant to conventional treatment cause severe morbidity and even mortality. In the present study we demonstrate that complete remissions can be achieved in refractory polychondritis and systemic lupus erythematosus (SLE), even at advanced stage, with the use of autologous stem-cell transplantation (SCT). Remissions persisted after reconstitution of the immune system. In the treatment of advanced systemic sclerosis (SSc), stable disease may be achieved with autologous SCT.
Keywords:autologous stem-cell transplantation; polychondritis; refractory autoimmune disease; systemic lupus erythematosus; systemic sclerosis
Patients with persistently active autoimmune diseases are considered to be candidates for autologous SCT. We performed a phase 1/2 study in a limited number of patients who were refractory to conventional immunosuppressive treatment. Following a period of uncontrolled disease activity for at least 6 months, autologous SCT was performed, after in vivo immunoablation and ex vivo depletion of mononuclear cells.
To investigate feasibility, toxicity and efficacy of the treatment, and the incidence of emergent infections.
Seven patients (aged between 23 and 48 years) were included in the single-centre trial: one had relapsing polychondritis, three had treatment-refractory SLE and three patients had SSc. Stem-cell mobilization was achieved by treatment with moderate-dose cyclophosphamide (2 g/m2; in terms of myelotoxic side effects or myelosuppression) and granulocyte colony-stimulating factor (G-CSF). CD34- cells of the leukapheresis products were removed by high-gradient magnetic cell sorting. After stem-cell collection, immunoablation was performed with high-dose cyclophosphamide (200 mg/kg body weight) and antithymocyte globulin (ATG; 90 mg/kg body weight). Autologous SCT was followed by reconstitution of the immune system, which was monitored by six-parameter flow cytometry and standard serology. The trial fulfilled the European League Against Rheumatism (EULAR) and the European Group for Blood and Marrow Transplantation (EBMT) guidelines for blood and bone marrow stem-cell transplants in autoimmune disease.
Among the seven patients studied, the patient with relapsing polychondritis and the patients with SLE were successfully treated and remained in complete remission during a follow up of 10-21 months. Remission persisted despite reconstitution of the immune system, resulting in high numbers of effector-/memory-type T-helper lymphocytes and increasing populations in the naïve T-cell compartment. Before autologous SCT, one of the patients with SLE had a long-lasting secondary antiphospholipid syndrome, with high anticardiolipin antibodies and thromboembolic events. After autologous SCT the antiphospholipid antibodies became negative, and no thrombosis occurred during follow up. Two of the patients with SSc were unaffected by treatment with autologous SCT for 6 or 13 months. The other patient with SSc died 2 days after autologous SCT because of cardiac failure.
During stem-cell mobilization with G-CSF, flares of autoimmune disease were seen in the patient with polychondritis and in one patient with SLE. The strategy utilized for depletion of CD34- cells led to a reduction by 4.5-5 log of contaminating CD3+ cells in the transplant. T-cell add-back was required in the patient with polychondritis and in one patient with SLE to provide a dose of 1×104 CD3+ cells/kg body weight for the transplant.
In vivo immunoablation in combination with autologous SCT after ex vivo depletion of CD34- cells can block the autoimmune process in relapsing polychondritis or SLE without incidence of severe infections. The remissions were achieved in patients with advanced disease that was refractory to previous intensive immunosuppressive therapy. The present results do not indicate that large-scale contamination of the stem-cell transplant with autoreactive cells after selection for CD34+cells occurred. After the preparative regimen, the application of G-CSF was avoided, because induction of flares of the autoimmune disease were noticed during the mobilization of stem cells. In SSc patients, distinct remissions were not observable after autologous SCT; the serological and clinical status did not improve. Follow-up periods of more than 12 months may be required to identify successful treatment with autologous SCT in SSc patients. Among the various autoimmune diseases the efficacy of autologous SCT appears to be dependent on the underlying pathophysiology. The results of the present phase 1/2 study suggest that patients with advanced stage SSc should not be treated with autologous SCT, until the reasons for the lack of response and the possible mortality due to cardiac complications are identified. The observation of flares of autoimmune disease after application of G-CSF emphasizes the need for critical evaluation of the role of G-CSF in immunoablative regimens.
Refractory autoimmune diseases cause a high degree of morbidity and even mortality, although they are not considered to be malignant diseases. During treatment with conventional and experimental immunosuppression, patients can experience treatment-related morbidity without significant gain in quality of life. Autologous SCT is a novel experimental approach for treating patients with refractory autoimmune diseases . Worldwide, 74 patients with severe autoimmune disease have thus far been treated in 22 centres . Of these 74 patients, 38 received autologous SCT for treatment of rheumatic autoimmune diseases.
In the present study one patient with therapy-resistent polychondritis, three patients with advanced SLE and three patients with SSc qualified for an aggressive experimental therapy. After stem-cell mobilization all patients were treated with a rigorous immunosuppressive regimen including cyclophosphamide and ATG to achieve in vivo depletion of T cells and other mononuclear cells. The preparative regimen was followed by autologous SCT of CD34+ cells after an effective ex vivo depletion of mononuclear cells by high-gradient magnetic cell sorting in order to exclude contamination of the transplant with CD34- cells. The present phase 1/2 trial was aimed at investigating the toxicity of this protocol and the incidence of infections. In addition, the efficacy of autologous SCT with respect to clinical and serological remissions and their duration was evaluated.
Patients and methods
All patients had long-lasting histories of severe and progressive disease without any signs of improvement under conventional immunosuppressive treatment. Inclusion criteria were defined as persistently active disease with poor prognosis and inadequate response to standard protocols (glucocorticoids and at least two different regimens of immunosuppressive drugs, such as intravenous cyclophosphamide 800-1000 mg/application). Furthermore, the patients needed to have adequate function of all major organs in order to tolerate conditioning and transplantation. The exclusion criteria were infections and uncontrolled arrhythmia or congestive heart failure. Further exclusion criteria were as follows: ejection fraction below 50% determined by echocardiogram; lung function test (LFT; transfer factor for carbon monoxide [TLCO] <45%); glomerular filtration rate below 40ml/min or serum creatinine greater than 2.0 mg/dl; hyperalimentation; and age greater than 59 years. The patients were included in the trial only after written consent had been obtained. The present study on autologous SCT for refractory autoimmune diseases was approved by the state ethics committee.
A 41-year-old female was admitted with relapsing polychondritis, which was first diagnosed in 1985. The disease was manifested by severe arthralgias, costosternal pain, vasculitis, scleritis, saddle nose and tracheal involvement; the patient had also sustained a life-threatening episode of pyoderma gangrenosum. Despite continuous and intensive conventional therapy for several years, no remission was achieved. During disease progression there was a risk of developing a tracheo-oesophageal fistula. The previous therapy regimens had included intravenous Ig, high-dose methylprednisolone, methotrexate, anti-CD4 antibody and intravenous cyclophosphamide (cumulative dose 6.0 g/m2 per month) with concomitant application of steroids. At admission, the daily dose of methylprednisolone was 30 mg. Her Karnofsky score was 60%.
A 27-year-old female was diagnosed as having severe SLE at the age of 16 years. During the course of disease, erythema, arthralgia, myalgia, abdominal vasculitis, polyserositis, nephrotic syndrome and pericardial effusions had been observed. Despite consecutive treatments with high-dose methylprednisolone, hydroxychloroquine, azathioprine, intravenous cyclophosphamide (cumulative dose 2.8 g/m2 per month), cyclosporine A, mycophenolate mofetile and daily doses of prednisolone of at least 30mg, the disease activity remained uncontrolled for 1.5 years before stem-cell therapy. The patient had been hospitalized for the 15 months before autologous SCT. Her Karnofsky score was 40% and her European Consensus on Lupus Activity measurement (ECLAM) score was 6.5. This patient had serum antibodies against double-stranded DNA (Table 1); she fulfilled the classification criteria of the American College of Rheumatology .
A 48-year-old female had had severe SLE since 1993. The disease manifested as polyserositis, arthralgias, peripheral neuropathy, nephrotic syndrome, pericardial effusions and ventricular tachycardia (the latter was treated with propanolol). Treatment had included high-dose methylprednisolone, hydroxychloroquine, azathioprine, methotrexate, intravenous Ig, monthly intravenous cyclophosphamide (cumulative dose 2.7 g/m2 per month) and mycophenolate mofetile. At admission, the patient was under treatment with prednisolone (20 mg/day) and oral morphium sulphate (120 mg/day). Her Karnofsky score was 60% and her ECLAM score was 6. This patient had serum antibodies against double-stranded DNA (Table 1); she fulfilled the classification criteria of the American College of Rheumatology .
A 37-year-old male had been diagnosed with SLE in 1989, with a nephrotic syndrome and oral lesions, erythema, arthralgias, and cardiac and pulmonary involvement. Despite treatment with prednisolone, azathioprine, intravenous cyclophosphamide (cumulative dose 7.3 g/m2) and high-dose methylprednisolone, the nephrotic syndrome (histology indicated lupus nephritis of World Health Organization grade IV) and other manifestations had not improved, and the ventricular arrhythmia (multiple couplets, one triplet, multiple bigemini) remained uncontrolled. At admission, the dose of prednisolone was 100 mg/day. His Karnofsky score was 70% and his ECLAM score was 10. This patient had serum antibodies against double-stranded DNA (Table 1); he fulfilled the classification criteria of the American College of Rheumatology .
A 23-year-old female was first diagnosed as having diffuse SSc at age 12 years. During the course of disease, microstomia, xerostomia, arthralgias, dysphagia, cutaneous necrosis with Raynaud's phenomenon and the onset of lung fibrosis (by high-resolution computed tomography [HRCT] scan; LFTs - total lung capacity [TLC] 72.6%, residual volume [as percentage of TLC] 127%, single breath (SB) TLCO 61.8%) had occurred. Progression of disease was observed under consecutive treatment periods with D-penicillamine, prednisolone, azathioprine, cyclosporine A, oral cyclophosphamide for 12 months (cumulative dose 3.8 g/m2) and dapsone. Treatment at admission was only symptomatic and without steroids. Her Karnofsky score was 60% and her skin score was 19.
A 25-year-old male was diagnosed with diffuse SSc in 1995 with microstomia, arthralgias, dysphagia, cutaneous necrosis with Raynaud's phenomenon, and onset of lung fibrosis (by HRCT scan; LFTs - TLC 93.2%, residual volume [in percentage of TLC] 159%, TLCO-SB 86.2 %). His finger mobility was severely limited, and he had lost 10 kg in weight since 1997. Treatment had included prednisolone, azathioprine and symptomatic therapy. At admission, the daily dose of prednisolone was 5 mg. His Karnofsky score was 60%, and his skin score was 30. In this patient steroids were applied due to the rapid progression of the disease.
A 45-year-old female had diffuse SSc that was first diagnosed in 1996. During the preceding 6 months she had lost 13 kg in weight, presumably due to oesophageal involement. Further manifestations were microstomia, xerostomia, arthralgias, Raynaud's phenomenon, cutaneous necrosis, intermittent tachyarrhythmia and the onset of lung fibrosis (by HRCT scan; LFT - TLC 71.1%, residual volume [in percentage of TLC] 182%, steady-state (SS) TLCO 47.3%), but she had normal echocardiography (ejection fraction 60%). Despite pretreatment with prednisolone, methotrexate, mycophenolate mofetile, azathioprine and one course of intravenous cyclophosphamide (cumulative dose 0.7 g/m2), progression of disease continued. During hospitalization before autologous SCT, the patient was treated with prednisolone 30 mg/day. Her Karnofsky score was 40% and her skin score was 32. In this patient steroids were applied due to the rapid progression of the disease.
Specific antibodies and cytometry
Disease-related autoantibodies in SLE and SSc were analyzed at admission and regularly during follow up. Monolayers of Hep-2 cells (Bios GmbH, Gräfelfing/Munich, Germany) were used to detect antinuclear antibodies (ANAs) by indirect immunofluorescence. Anti-double-stranded DNA antibodies were identified by indirect immunofluorescence on Crithidia luciliae and by enzyme-linked immunosorbent assay as previously described . Autoantibodies against extractable nuclear antigens (Sm, U1RNP, Ro/SS-A, La/SS-B, Scl-70, Jo-1, centromere) and anticardiolipin antibodies were analyzed using enzyme-linked immunosorbent assay (IMTEC Immundiagnostika GmbH, Zepernick, Germany).
Antibodies conjugated to phycoerythrin, fluorescein or biotin, and conjugated to peridinin-chlorophyll protein were obtained from Becton Dickinson (Heidelberg, Germany) and Pharmingen (Hamburg, Germany). For cytometry, anti-CD45RO (clone UCHL-1) was coupled to Cy5 (Amersham, Braunschweig, Germany), according to the manufacturer's instructions. Cell staining and flow cytometry were performed using standard protocols on freshly prepared peripheral blood mononuclear cells. The cells were analyzed using a dual-laser, six-parameter FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany); the data were evaluated using commercial software (Becton Dickinson). UCHL-1 (anti-CD45RO) was a generous gift from Imperial Cancer Research Technology (London, UK).
Stem-cell mobilization and collection
In all patients mobilization of stem cells was achieved with cyclophosphamide at 2 g/m2. After 5 days, G-CSF (10 μg/kg body weight) was administered daily, until harvest of CD34+ cells. Leukapheresis was performed when the leucocyte numbers had reached 4.0×109/l. If required, leukapheresis (Cobe Spectra; Cobe BCT, Lake-wood, CO, USA) was repeated until a minimum number of 4×106CD34+ cells/kg body weight had been collected for the transplants.
Engineering of transplants
Removal of CD34- leucocytes from the stem-cell transplant was performed by selection for CD34+ cells through high-gradient magnetic cell sorting, using a CliniMacs™ device (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) [5,6,7]. If required, CD3+ cells from the CD34-fraction were additionally supplied to the purified CD34+cells to transplant a minimum of 1.0×104/kg body weight CD3+cells. Until transplantation the CD34+ cell suspensions were cryopreserved with 5vol% dimethyl sulphoxide.
Preparative regimen and autologous stem-cell transplantation
The preparative regimen consisted of 200 mg cyclophosphamide/kg body weight (days -5 to -2) and ATG (rabbit; obtained from Fresenius, Bad Homburg, Germany) 90 mg/kg body weight (days -4 to -2) . During ATG treatment 500mg methylprednisone was administered twice a day. The median time interval between cyclophosphamide for mobilization of stem cells and autologous SCT was 38 days (range 29-61 days). Supportive care was provided, according to standard protocols for allogeneic bone marrow transplantation, including isolation of the patient and prophylaxis against infection. Substitution of Igs (10 g every other week) was applied to avoid hypoimmunoglobinaemia, and was ended in all patients after 6 months.
Evaluation of response
The function of the organs involved was monitored by technical examinations. Apart from the the clinical course, serological parameters were evaluated (ie ANAs, anti-double-stranded DNA, Scl-70 and other extractable nuclear antigens). Activity of SLE was determined by the ECLAM score . For SSc the skin score was used . Therapeutic response was defined as 50% improvement in clinical and serological parameters. Complete remission was defined as normalization without clinical symptoms of disease. The trial fulfilled the EBMT/EULAR guidelines for blood and bone marrow stem-cell transplants in auto-immune diseases .
Table 1. Clinical outcome and treatment-related morbidity of patients with polychondritis or SLE in complete remission
Table 2. Reconstitution of the immune system
Table 3. Clinical course and treatment-related morbidity in patients with SSc not responding to autologous SCT