British Journal o!Haematology, 1995,89,79-82

The effect of cyclosporine on haematological parameters in patients with paroxysmal nocturnal haemoglobinuria

HARMEN VAN KAMP, GUSTAAF W. VAN IMHOFF, JOOST TH. M. DE WOLF, JAN W. SMIT,· M. RUUD HALlB

AND EDO VBLLENGA Department 0/ Haematology, and ·Central Laboratory 0/ Clinical Haematology, University 0/ Groningen, The Netherlands

Received 20 July 1994; accepted/or publication 16 September 1994

Summary. Four patients with paroxysmal nocturnal haemoglobinuria (PNH) were treated with cyclosporine. The treatment with cyclosporine was based on the hypothesis that immune-mediated bone-marrow damage is the common pathogenetic mechanism of aplasia and PNH, with lack of GPI-linked ligands for an immune attack (i.e. LFA-3, CD58) rendering PNH cens a growth advantage over other bone marrow cells. In the !irst patient, presenting with a mixed AA/PNH syndrome, a gradual recovery from aplasia was seen after prolonged treatment with cyclosporine. In a second patient, with a mixed AA/PNH syndrome, no haematological improvement was noted during cyclosporine administration, but this patient became transfuslon­independent with increasing neutrophil and platelet counts after a course of ATG in combination with androgen therapy. Both these patients showed an increment in the

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired disorder of haemopoiesis characterized by recur­rent episodes of intravascular haemolysis due to an Increased sensitivity for complement-mediated lysis of red blood cells (Rotoll & Luzzatto, 1989). The pathophysiological basis of the disorder has been identified as the inability of the affected cens to synthesize a glycosylphosphatidyllnositol (GPI) moiety necessary to bind several proteins, among which are complement-regulatory proteins, to the cell surface (Rosse, 1990; Mahoney et aI, 1992; Takeda et aI, 1993). PNH is closely associated with acquired aplastic anaemia (AA) (Lewis & Dacie, 1967; Tichelli et al, 1988, 1992; De Planque et al, 1989) with marrow failure as the primary phenomenon and the degree of emergence of the PNH clone determining the clinical appearance as pure aplaSia, the so-called mixed AAjPNH syndrome or typical

Correspondence: Dr H. van Kamp, Department of Internal Medicine, Division of Haematology, Unlverslty of Gronlngen, P.O. Box 30.001, 9700 RB Groningen. The Netherlands.

79

proportion of neutrophils with normal expression of GPI­linked proteins concurrently with the Improvement of haematological characteristics. In the two other patients, presenting with typical PNH, cyclosporine treatment did not result in any change in haematologlcal characteristics, nor in PNH parameters. No Significant change in haemolytic parameters was seen In any of the patients.

It is concluded that immunosuppressive therapy may be of benefit in patients with a mixed AA/PNH syndrome. This effect became apparent after prolonged treatment with cyclosporine In one patient, and after a subsequent course of ATG with concomitant androgen therapy in another.

Keywords: cyclosporine, PNH, aplasia, haemolysis, GPI­linked proteins.

PNH (Rotoll & Luzzatto, 1989). Immune mechanisms suppressing normal haemopolesis contribute to the pathogenesis of aplasia (Camitta et aI, 1982). Lack of some GPI-llnked proteins, such as lymphocyte function-associated antigen 3 (LFA-3. CD58), which is the ligand for the T--cell glycoprotein CD2, may explain why PNH cells escape from a cytotOXiC attack and have a growth advantage when compared to their unaffected counterparts In an aplastic marrow (Selvaraj et al, 1987; Young, 1992).

Immunosuppressive therapy with antlthymocyte globulin (ATG) as a single agent or in combination with cortico­steroids or androgens is an established treatment for patients with AA (Champlin et al, 1985; Young et al, 1988). The efficacy of immunosuppression with oral cyclosporine in AA patients has also been demonstrated (Stryckmans et al, 1984; Frickhofen et al, 1991). It is still unknown, however, whether PNH patients will also benefit from immuno­suppression with restoration of normal haemopolesis and extinction of the PNH clone.

We report a patient with a mixed AA/PNH syndrome

80 Harmen van Kamp et al treated with cyclosporine for a prolonged period, and also the results of the treatment with cyclosporine of three subsequent PNH patients who were treated according to a study protocol designed to assess the therapeutic effect of oral cyclosporlne on the degree of Intravascular haemolysis during a 3-month period.

PATIENTS AND METHODS

Patients. Patient 1 developed PNH after successful ATG treatment of AA 7'5 years previously. He showed a gradual relapse of aplasia and now presented with a mixed AA/PNH syndrome. The other patients were selected with a history of PNH confirmed by laboratory features of Increased Intra­vascular haemolysis, a positive acid Ham and sucrose lysis test, and a decreased expression of GPI-linked proteins (CDI6, CD24, CD67) on neutrophils (Van der Schoot et aI, 1990). Other criteria for eligibility Included a haemoglobin level <lOg/d!, and normal renal (creatinine <1·25x the upper limit of normal) function. Patient characteristics prior to treatment are given In Table I. The study protocol was approved by the Medical Ethical Committee of the University Hospital Gronlngen. Informed consent was obtained from the patients before start of treatment.

Treatment design. Cyclosporlne (Sandimmune, Sandoz, Basel, Switzerland) was given twice daily in a total oral dose of 5 mg/kg body weight. Doses were adjusted to reach whole-blood-through levels of 200-300 J.Lg/1 and had to be adapted, when the creatinine level rose up to 1· 5 x the upper limit of normal. Patient 1 was treated for a prolonged period. Treatment according to the study protocol was confined to a 3-month period. Medication was given on an outpatient basis. Folic acid (5 mg/d) was used concurrently by patients 1, 3 and 4. Supportive care was given as medically indicated.

Study parameters. A medical history was taken and a physical examination was performed prior to drug administration and at every visit scheduled at 3-week intervals. Haemoglobin level and haematocrit, reticulocyte count, white blood cell count and differential, and a platelet count was performed every 3 weeks, and also serum creatinine and urinalysis. Parameters of Intravascular haemolysis, including bilirubin, LDH and haptoglobin levels, were also monitored at every visit. An acid Ham and sucrose lysis test was done before the start of drug administration. The expression of GPI-Ilnked proteins on the neutrophils was analysed on a fluorescence-activated cell sorter FACScan (Becton Dickinson, Mountain View, Calif., U.S.A.) using the fluorescein isothiocyanate (FITC) con­jugated monoclonal antibody anti-CD16 (Becton Dickinson), and the monoclonal antibodies anti-CD24 (Boehringer, Mannheim, Germany) and anti-CD67 (Central Laboratory of the Dutch Red Cross, Amsterdam, The Netherlands) In conjunction with FITC-Iabelled rabbit anti-mouse immuno­globulin antisera (Dako, Glostrup, Denmark). In patient 1, GPI-linked protein expreSSion was measured 6 weeks after the start of treatment and after 13 months. In the study patients, GPI-linked protein expression was determined prior to treatment, after 6 weeks, and at the end of treatment. Prior to treatment. a bone marrow smear and a biopsy

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CycIosporine in Patients with PNH 81 Table D. Effect of cyc1osporine on PNH parameters.-

Before treatment After 6 weeks of treatment After 1 S weeks of treatment At follow-upt

CD16 CD24 CD67 CD16 CD24 CD67 CD16 CD24 CD67 CD16 CD24 CD67 Patient (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%)

1 N.D. N.D. N.D. 32 26 29 N.D. N.D. N.D. S4 S3 54 2 71 76 77 80 N.D. 7S 75 72 73 81/93 86/95 N.D./95 3 N.D. 2 1 2 2 2 3 S 2 N.D. N.D. N.D. 4 5 3 3 3 3 3 2 5 4 5 4 2

-Data of expression of CPI-Iinked proteins (CD16. CD24. CD67) on neutrophils are given. t Patient 1. after 13 months of continuous treatment; patient 2. before and after ATG treatment. respectively 6 and 9 months after the end of

cyc1osporine treatment; patient 4. after 9· 5 months of continuous treatment.

specimen were analysed In all patients. Bone marrow analysis was repeated in the study patients.

RESULTS

Four patients with PNH were treated with cyclosporine. Patients 1 and 2 presented with a mixed AA/PNH syndrome and a hypocellular bone marrow. Patients 3 and 4 had typical PNH with a hypercellular bone marrow showing predominant erythropoiesis. Patient 1 was treated continu­ously with cyclosporine, with a total treatment duration of 17 months at the time of writing. This patient showed a gradual Increase in Hb level from 5'6 gldl at the start of treatment to 10'0 gldl 17 months later. The number of neutrophils rose from 0'7 to 2·3 x 10911. The platelet count Increased from 21 x 109/1 after a nadir of 13 x 109/1 at 3 weeks to 70 x 109 /l after 17 months oC treatment. In the other patients treatment was given as indicated by the study protocol. The administration of cyclosporine was continued for another 9 months In patient 4. No effect on haemato­logical parameters was seen in any of these patients. However, after the 3-month study period, patient 2 was treated with androgens and 6 months later he also received a course of ATG. This patient became transfusion-independent 2 months after ATG treatment and blood counts are still Improving. 5 months after ATG treatment the Hb level is now 12'8 g/dl. the neutrophil count increased from 0'5 to 1·4 x 109/1, and the platelet count rose from 6 x 109/1 to 39 X 10911. No significant Improvement of parameters of Intravascular haemolysis was seen in any of the four patients.

An increment of the percentage of neutrophlls expressing CD16, CD24 and CD67 was seen in patient 1 after prolonged treatment with cyclosporine. In patient 2 a relative high percentage of neutrophils with normal expression of these GPI-linked proteins was found prior to study entry. No change was noted during cyclosporine administration; however, normal values were found 3 months after ATG treatment. In the other two patients no increase of the proportion of neutrophils that normally expressed GPI­linked proteins was seen. Data of expression of GPI-linked proteins are presented In Table II. The acid Ham and sucrose

lysis test remained positive after 3 months of treatment when tested in patients I, 3 and 4.

The treatment with cyclosporine was well tolerated. However, a slight impairment of renal function was noted in patients 1 and 2.

DISCUSSION

The treatment of PNH patients with cyclosporine as described In this report was based on the hypothesis that immune-mediated bone-marrow damage is the common pathogenetic mechanism of aplasia and PNH, with lack of GPI-linked ligands for an immune attack (I.e. LFA-3, CD58) giving PNH cells a growth advantage over other bone marrow cells (Young, 1992). In one of the two patients with a mixed AA/PNH syndrome a gradual recovery from aplasia was seen after prolonged treatment with cyclosporine. In the other patient with a mixed AAjPNH syndrome no haemato­logical Improvement was noted during cyclosporine admin­istration, but this patient became transfUSion-independent with concomitant improvement of neutrophil and platelet counts after a course of ATG In combination with androgen therapy. Both patients showed an Increment in the proportion of neutrophils with normal expression of GPI­linked proteins concurrently with the improvement of haematological characteristics. In the two other patients. presenting with typical PNH. cyclosporine treatment did not result In any change In haematological characteristics, nor in PNH parameters. No significant change In haemolytic parameters was seen in any of the patients.

The development of PNH is one of the late complications of immunosuppressive therapy for AA. In a survey of the Severe Aplastic Anaemia Working Party of the European Cooperative Group for Bone Marrow Transplantation the development of PNH was reported in 19/209 long-term survivors after immunosuppressive therapy (De Planque et ai, 1989). Tichelli et aI (1992) describe the development ofPNH in 17/117 patients treated with ATG for severe AA. These data raise the question whether Immunosuppressive therapy may even facilitate the outgrowth of the PNH clone Instead of suppressing its proliferation. and therefore the immuno­suppressive treatment of aplasia associated with PNH has

82 Harmen van Kamp et al remained controversial. However. the first two patients described In this report showed haematologlcal recovery after Immunosuppressive treatment and concurrently an Increase In the expression of GPI-linked proteins on their neutrophils. Also. Kusmlnsky et a1 (1988) have reported a patient with a mixed AA/PNH syndrome in whom therapy with ATG resulted In recovery from aplasia and disappear­ance of the PNH abnormality. In contrast. Nakao et a1 (1992) described a patient with a mixed AA/PNH syndrome who demonstrated aggravation of intravascular haemolysis during cyclosporine treatment. Such an expansion of the PNH clone was not seen In any of our patients. and we believe that immunosuppressive treatment can be given safely to patients with a mixed AA/PNH syndrome to treat the underlying bone marrow aplasia provided that the follow-up is done carefully.

The hypothesis that PNH cells escape the bone marrow damage of aplasia by the lack of GPI-linked ligands for an Immune attack (I.e. LFA-3. CD58) (Young. 1992) is partly supported by this report. The patients with a mixed AA/PNH syndrome showed a beneficial effect of Immunosuppressive therapy with improvement of their blood counts. This effect became apparent after prolonged treatment with cyclospor­Ine or after a subsequent course of ATG with concurrent androgen therapy. No effect was seen In the other patients presenting with typical PNH. However. the prevalence of PNH is low. and therefore we could only study a very limited number of patients. To provide definite answers. more patients have to be treated.

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