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Rheumatology Advance Access originally published online on February 22, 2007
Rheumatology 2007 46(6):952-956; doi:10.1093/rheumatology/kem001
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Long-term effects of combination treatment with fludarabine and low-dose pulse cyclophosphamide in patients with lupus nephritis

G. G. Illei1, C. H. Yarboro1, T. Kuroiwa1, R. Schlimgen1, H. A. Austin2, J. F. Tisdale2, P. Chitkara1, T. Fleisher3, J. H. Klippel1, J. E. Balow2 and D. T. Boumpas1,4

1Office of the Clinical Director, National Institute of Arthritis and Musculoskeletal and Skin Diseases, 2National Institute of Diabetes and Digestive and Kidney Diseases, 3Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA and 4Division of Rheumatology, Clinical Immunology and Allergy, University of Crete Medical School, 711 10 Heraklion, Greece.

Correspondence to: G. G. Illei, MD, Sjögren's Syndrome Clinic, National Institute of Dental and Craniofacial Research, National Institutes of Health, 10 Center Drive, Building 10 Room 9S205, Bethesda, MD 20892, USA. E-mail: illeig{at}mail.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. To determine the safety and efficacy of a short course of fludarabine combined with cyclophoshamide in lupus nephritis.

Methods. A phase I/II open label pilot study. Thirteen patients with active proliferative lupus nephritis received monthly oral boluses of low-dose cyclophoshamide (0.5 gm/m2 on day 1) and subcutaneous fludarabine (30 mg/m2 on days 1–3) for 3–6 cycles. Concomitant prednisone was aggressively tapered from 0.5 mg/kg/day to a low-dose, alternate-day schedule. Patients were followed for at least 24 months after therapy. The primary outcome was the number of patients achieving renal remission defined as stable creatinine, proteinuria <1 gm/day and inactive urine sediment for at least 6 months.

Results. The study was terminated early because of bone marrow toxicity. Eleven patients who received at least three cycles were evaluated for efficacy. Ten patients improved markedly with seven patients achieving complete remission and three patients achieving partial remission. There were three serious haematological adverse events during the treatment with one death due to transfusion-associated graft vs host disease. Profound and prolonged CD4 (mean CD4: 98/µl at 7 months and 251/µl at 12 months) and CD20 lymphocytopenia was noted in most patients. Three patients developed Herpes zoster infections.

Conclusions. A short course of low-dose fludarabine and cyclophoshamide can induce long-lasting remissions in patients with proliferative lupus nephritis, but severe myelosuppression limits its widespread use.

KEY WORDS: systemic lupus erythematosus, chemotherapy, myelosuppression, remission, safety


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Several clinical trials have demonstrated the efficacy of cyclophosphamide in preserving renal function. However, even with a long course (30 months or longer) of therapy, up to 40% of patients fail to achieve renal remission and up to 20% of patients develop end-stage renal disease (ESRD) [1–4].

Cyclophosphamide can be associated with adverse effects which have to be weighed against morbidity occurring as a result of uncontrolled disease and the need for extended therapy. The risk of premature ovarian failure, a common long-term side effect, is dependent on the age of the patient at the initiation of therapy and the cumulative dose of cyclophosphamide [5–7]. Combining cyclophoshamide with drugs that have additive effects but a different side-effect profile may allow the reductuion of the cumulative dose of cyclophosphamide and decrease the risk of amenorrhea without loss of efficacy.

Fludarabine is a halogenated adenosine analog, used in the treatment of haematological malignancies. It induces profound and prolonged immunosuppression by depleting both T- and B-lymphocytes [8] and inhibiting cytokine-induced activation of STAT1 in resting as well as activated lymphocytes [9], which may contribute to the immunosuppressive effect of fludarabine.

In a Phase I study, continuous 7-day infusion of 2-CdA, another adenosine analogue with a similar mechanism of action, led to complete response in 3/7 patients and all 7 had maintained stable renal function during the 1-yr follow-up [10] and 6-month course of fludarabine led to improvement in proteinuria and filtration rate in a pilot study in membranous nephritis [11]. In vitro studies [12] demonstrating a synergistic action of fludarabine and cyclophosphamide were supported by clinical studies in chronic lymphocytic leukaemia (CLL) [13]. Based on these experiences, we conducted a pilot study in lupus nephritis to evaluate the safety and to collect preliminary data about the efficacy of a short course of low-dose cyclophosphamide combined with fludarabine.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Study design
This were single centre, phase I/II, open label pilot study. We intended to treat 15 patients with proliferative lupus nephritis. The Institutional Review Board of the National Institute of Allergy and Infectious Diseases, NIH approved the study. Written informed consent was obtained from all patients.

Patient selection
Thirteen patients older than 18 yrs and with a diagnosis of SLE according to the 1982 criteria of the American College of Rheumatology were enrolled. Inclusion criteria included a renal biopsy showing proliferative lupus nephritis within 1 year prior to first dose of study drug and (i) >10 RBC/hpf and cellular (RBC, WBC or mixed) casts, or (ii) >10 RBC/hpf and proteinuria >2 g/day, or (iii) proteinuria >3.5 g/day.

Exclusion criteria included severe renal disease (rapidly progressive glomerulonephritis or fibrinoid necrosis and/or cellular crescents affecting >25% of glomeruli); severe impairment of renal function (serum creatinine >2.5 mg/dl or GFR<50 ml/min measured by inulin clearance); treatment with azathioprine, cyclosporine or methotrexate within 4 weeks; prior exposure to substantial dose of cyclophosphamide [>3 pulses (maximum 1 g/m2/pulse) within the last 6 months or since last renal biopsy showing active disease or ≥4 pulses, unless there is a biopsy showing active disease after a period of ≥6 months since last treatment]; pulse therapy with glucocorticoids or any experimental therapy during the 4 weeks before study entry or need at study entry of oral corticosteroids in dosages >0.5 mg/kg/day of prednisone to control extrarenal disease; active or chronic infection; pregnancy, breast-feeding or inadequate birth control; history of cerebrovascular accident, seizures within the last 5 years or chronic neurological disease; history of malignancy other than squamous cell and/or basal carcinoma of the skin and haematological disease (haemoglobin <8 mg/dl, platelets <50 000/µl or WBC <1500/µl).

Following a serious adverse event in patient #4, the exclusion criteria about past cyclophophamide use and abnormal haematological values were changed. Subjects were excluded if they received >3 pulses of cyclophosphamide (maximum 1 g/m2/pulse) within the last 12 months or since last renal biopsy showing active disease or >6 pulses ever. The exclusion criteria for abnormal haematological values were changed to: haemoglobin <8 mg/dl, platelets <100 000/µl or WBC <2500/µl.

Concomitant therapy
All patients received oral prednisone 0.5 mg/kg/day for 4 weeks. Prednisone dose was then tapered by 5 mg every other day each week to 0.25 mg/kg every other day. After month 6, prednisone was tapered at the treating physician's discretion to low-dose alternate-day regimen.

Prophylactic therapy
All patients were vaccinated with pneumococcal, haemophilus influenzae B (HiB), influenza (during influenza season) and tetanus toxoid vaccines. Patients received Pneumocystis carinii and herpes prophylaxis based on their absolute lymphocyte counts.

Study treatment
Patients received monthly cycles of cyclophosphamide 500 mg/m2 orally on day 1 followed by fludarabine 30 mg/m2 subcutaneously (SQ) on days 1–3 for 6 monthly cycles. The dose of fludarabine and/or cyclophosphamide was reduced according to predetermined criteria for severe lymphopenia, leukopenia or thrombocytopenia. The protocol was modified to decrease the number of cycles to 3 after patient #4 developed severe aplastic crises after the third cycle.

Outcomes and data analysis
The primary outcome was the determination of safety (rates of serious adverse events and infections) and tolerability. Secondary outcomes included assessment of the rate of amenorrhea and renal efficacy measures: renal remission was defined as proteinuria <1 gm/day, inactive urine sediment (<10 red blood cells/hpf and no cellular casts in the sediment of a 50 ml urine sample) and stable serum creatinine. Partial response was defined as ≥50% reduction in 24 h proteinuria, if baseline >2 g/day, with persistently active urine sediment; or inactive urine sediment (if active at baseline) with proteinuria <2 g/day if nephrotic at baseline (or <50% of baseline if non-nephrotic). All patients were included in the toxicity analysis. Patients must have completed 3-cycles of therapy to be included in the analysis for efficacy.

The primary and secondary outcomes were determined at 12 months. All patients were regularly followed for at least 18 months after that to establish long-term effects of therapy; over 5 years of follow-up data are available for all but one.

Statistical analysis
All P-values are two-sided. Averages are expressed as the mean and standard deviation. Changes in the absolute values and percentage change from baseline at different time-points were compared by repeated measures analysis of variance (ANOVA). Adjustments for multiple comparisons were made using Scheffe's method. All statistical analyses were done with the Statview V.5 statistical software package (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Patient characteristics
Thirteen patients entered the protocol. A significant number of patients exhibited features that have been associated with poor renal outcomes such as male gender (3 patients), African American ethnicity (5 patients) and diffuse proliferative (7 patients) or mixed membranous and proliferative glomerulonephritis (4 patients). The majority of patients (10/13) had nephrotic range proteinuria; 5 had anti-dsDNA antibodies and 7 of the 13 had low C3 concentrations. Only two patients received more than six cycles of cyclophosphamide pulses before entering the study.

Adverse events
Clinically significant adverse events are summarized in Table 1. There were three serious haematological adverse events during the treatment period. Patient #4 developed aplastic crisis with neutropenic fever 1 week after receiving the third cycle. She eventually died of a transfusion-associated graft versus host-like disease (TA-GVHD), as reported earlier [14]. Following this episode of aplastic crisis, the protocol was revised and the number of treatment cycles was reduced from six to three. Two other patients experienced Grade 4 neutropenia; neither of these was associated with any clinical complications and both patients recovered spontaneously (Table 1). Bone marrow biopsy was normal in both patients. However, based on the unexpected rate and severity of bone marrow suppression, the study was terminated early. Both of these patients along with a third patient had intermittent leucopenia during their long-term follow-up, with no clinical sequalae.


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  		TABLE 1. Clinical course of patients during and after the protocol

 
Two patients had immune mediated thrombocytopenia and three patients had Herpes zoster infections; two of them had history of herpes infections and the third patient developed it 1 week after the first study treatment. No other serious infections were observed.

Renal outcome
A total of 11 patients who received at least three cycles were evaluated for efficacy (Table 1 and Fig. 1).


Figure 1
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  		FIG. 1. Renal and immunological changes. Proteinuria decreased promptly (A) and most patients achieved sustained partial renal response by 6 months and complete flare-free response by 18 months (B). Lymphodepletion was achieved promptly with slow recovery of B cells and activated CD4+ T cells (CD4+ CD25+) (C and D).

 
Proteinuria
Proteinuria improved rapidly and substantially in all patients (Fig. 1A). By 6 months, mean proteinuria decreased to 2.1 gram/day from 5.6 gram/day at baseline with all but two patients having at least 50% improvement. At 12 months, mean proteinuria was 0.9 gm/day, all patients had more than 70% improvement, with 7/11 patients having <1 gram/day protein excretion.

Remissions
By 6 months nine patients achieved at least partial response (Fig. 1B) despite aggressive tapering of steroids (mean daily dose ± SD: 7.9 ± 1.9 mg vs 27.5 ± 9.6 mg at baseline). At 12 months, four patients were in remission; all other patients met criteria for partial response. Three of those with partial remission were treated with additional immunosuppressive therapy for ongoing renal disease. Two of these patients had biopsies after the protocol; both showed mild focal proliferative disease. One patient was started on mycophenolate mofetil and is in remission on this treatment; the other patient became pregnant soon after the biopsy and was treated with moderate dose prednisone during her pregnancy. One patient had mixed membranous and focal proliferative disease and continued to have significant proteinuria. She was started on cyclosporine first and then switched to mycophenolate mofetil. Ultimately, she failed both and progressed to ESRD. Seven patients continued to maintain or advance to complete renal response; at 30 months 7/11 patients were in remission and remained in renal remission throughout the follow-up (68–80 months).

Extrarenal lupus
Extra-renal manifestations of lupus improved rapidly, despite aggressive tapering of prednisone. One patient with a history of recurrent uveitis had a few episodes that responded well to topical treatment and a new onset rash 1 month after the last cycle; this responded well to hydroxychloroquine. Two patients had thrombocytopenia more than 18 months after the treatment; one of them required splenectomy, the other was treated with immunosuppressants. Another patient experienced an episode of pleuritis and rashes more than a year after completing treatment.

Amenorrhoea
None of the patients developed amenorrhoea during the study. Three patients had a total of five uncomplicated pregnancies with term deliveries.

Immunological effects
Lymphodepletion was achieved promptly in all patients. CD4+ T cells recovered more slowly than CD8+ T cells and B cells. The delayed recovery was most pronounced in activated T cells expressing the IL-2 receptor (CD4+ CD25+); this, however, was not associated with an increase in infectious complications (Fig. 1).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
In this pilot study, a limited course of subcutaneous fludarabine in combination with low-dose oral pulse cyclophosphamide was associated with an unexpectedly high rate of haematological side effects. Despite some encouraging preliminary efficacy data the risk-benefit assessment of this treatment in its current format is unfavourable.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We would like to thank the nurses of the rheumatology outpatient and inpatient units of the NIH Clinical Center for their devoted care of the patients in this study.

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Austin HA, Klippel JH, Balow JE, et al. Therapy of lupus nephritis. Controlled trial of prednisone and cytotoxic drugs. N Engl J Med (1986) 314:614–9.[Abstract]
  2. Boumpas DT, Austin HA, Vaughn EM, et al. Controlled trial of pulse methylprednisolone versus two regimens of pulse cyclophosphamide in severe lupus nephritis. Lancet (1992) 340:741–5.[CrossRef][Web of Science][Medline]
  3. Gourley MF, Austin HA, Scott D, et al. Methylprednisolone and cyclophosphamide, alone or in combination, in patients with lupus nephritis. A randomized, controlled trial. Ann Intern Med (1996) 125:549–57.[Abstract/Free Full Text]
  4. Illei GG, Austin HA, Crane M, et al. Combination therapy with pulse cyclophosphamide plus pulse methylprednisolone improves long-term renal outcome without adding toxicity in patients with lupus nephritis. Ann Intern Med (2001) 135:248–57.[Abstract/Free Full Text]
  5. Belmont HM, Storch M, Buyon J, Abramson S. New York University/Hospital for Joint Diseases experience with intravenous cyclophosphamide treatment: efficacy in steroid unresponsive lupus nephritis. Lupus (1995) 4:104–8.[Abstract/Free Full Text]
  6. Boumpas DT, Austin HA, Vaughan EM, Yarboro CH, Klippel JH, Balow JE. Risk for sustained amenorrhea in patients with systemic lupus erythematosus receiving intermittent pulse cyclophosphamide therapy. Ann Intern Med (1993) 119:366–9.[Abstract/Free Full Text]
  7. Langevitz P, Klein L, Pras M, Many A. The effect of cyclophosphamide pulses on fertility in patients with lupus nephritis. Am J Reprod Immunol (1992) 28:157–8.[Medline]
  8. Plunkett W, Gandhi V, Cheson B, Plunkett W, Keating M. Cellular pharmacology, mechanisms of action and strategies for combination therapy. In: Nucleoside Analogs in Cancer Therapy 1—Cheson B, Keating M, Plunkett W, eds. (1997) New York: Marcel Dekker. 1–35.
  9. Frank DA, Mahajan S, Ritz J. Fludarabine-induced immunosuppression is associated with inhibition of STAT1 signaling. Nat Med (1999) 5:444–7.[CrossRef][Web of Science][Medline]
  10. Davis JC Jr, Austin H, Boumpas D, et al. A pilot study of 2-chloro-2'-deoxyadenosine in the treatment of systemic lupus erythematosus-associated glomerulonephritis. Arthritis Rheum (1998) 41:335–43.[CrossRef][Web of Science][Medline]
  11. Boumpas DT, Tassiulas IO, Fleisher TA, et al. A pilot study of low-dose fludarabine in membranous nephropathy refractory to therapy. Clin Nephrol (1999) 52:67–75.[Web of Science][Medline]
  12. Bellosillo B, Villamor N, Colomer D, Pons G, Montserrat E, Gil J. In vitro evaluation of fludarabine in combination with cyclophosphamide and/or mitoxantrone in B-cell chronic lymphocytic leukemia. Blood (1999) 94:2836–43.[Abstract/Free Full Text]
  13. O'Brien S, Kantarjian H, Beran M. Fludarabine (FAMP) and cyclophosphamide therapy in chronic lymphocytic leukemia (CLL). Ann Oncol (1997) 7:34.
  14. Leitman SF, Tisdale JF, Bolan CD, et al. Transfusion-associated GVHD after fludarabine therapy in a patient with systemic lupus erythematosus. Transfusion (2003) 43:1667–71.[CrossRef][Web of Science][Medline]
Submitted 24 May 2006; revised version accepted 3 January 2007.
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