Transplantation (2001) 27, 589–592  2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00 www.nature.com/bmt Paroxysmal nocturnal haemoglobinuria Successful unrelated donor bone marrow transplantation for paroxysmal nocturnal hemoglobinuria

P Woodard1, W Wang2, N Pitts1, E Benaim1, E Horwitz1, J Cunningham1 and L Bowman1

1Division of Bone Marrow Transplantation, 2Division of , Department of Hematology/, St Jude Children’s Research Hospital, Memphis, TN, USA

Summary: sible for inhibition of complement-mediated cell lysis, leads to hemolysis and hemoglobinuria. Interestingly, clonal Paroxysmal nocturnal hemoglobinuria (PNH) is an populations of cells with the PNH genotype and phenotype acquired clonal of hematopoiesis due to a can be detected in normal individuals using very sensitive mutation in the PIG-A gene. Affected patients may dem- flow cytometric and PCR techniques.3 The abnormal hema- onstrate hemolysis or venous , and may topoiesis of PNH may develop after treatment of aplastic develop MDS or aplastic . Successful results may anemia with immunosuppressive drugs or after chemo- be obtained after conditioning and transplantation from therapy for malignancy.4 Hematopoietic cells with the PNH syngeneic or genotypically matched sibling donors. phenotype can be identified in cases of aplastic anemia, Experience with transplantation from matched unre- , or .5,6 lated donors (MUD) is limited to eight patients, with Affected patients may demonstrate hemolysis, venous only one survivor. We report three patients who thrombosis, and the development of myelodysplastic syn- underwent successful MUD BMT for PNH. All three drome or aplastic anemia. Three patients treated with high- patients had severe aplastic anemia (SAA) and PNH at dose erythropoietin and low-dose demon- the time of BMT. Unrelated donors were six-antigen strated improvements in hemoglobin and reduction in trans- HLA-matched (n = 2) or HLA-A mismatched (n = 1). fusion requirements.7 Treatment with the androgen danazol Conditioning consisted of cytarabine, cyclophospham- resulted in improvement in hematocrit and eventual ide, TBI, and ATG. Grafts were T cell-depleted by anti- cessation of transfusions in four of five cases.8 CD6/CD8 ؉ complement. Further GVHD Cases of bone marrow transplantation for patients with prophylaxis consisted of cyclosporine. Patients received aplastic anemia, myelodysplastic syndrome, recurrent sev- ؋ 108 nucleated cells/kg and 1.1–2.1 ؋ 106 ere hemolysis, and Budd–Chiari syndrome have been 1.1–0.7 CD34+ cells/kg. engraftment occurred at 16– reported.9–17 Syngeneic donors with or without pretrans- 21 days. One patient developed grade 1 acute GVHD. plant conditioning have been utilized with variable results. Although all three patients experienced significant The first reported patient underwent unconditioned PBSC transplant-related complications, they ultimately transplantation, which was unsuccessful. A subsequent resolved and all patients are alive and well 30–62 infusion of syngeneic bone marrow led to engraftment and months after BMT. T cell-depleted MUD BMT is an disease correction.17 Champlin et al11 reported four patients effective treatment option for PNH-related MDS and who failed to engraft after unconditioned syngeneic BMT. SAA. Bone Marrow Transplantation (2001) 27, 589–592. All four received successful second transplants after cyclo- Keywords: paroxysmal nocturnal hemoglobinuria; alter- phosphamide with or without TBI. Kolb et al10 reported nate donor BMT; T cell depletion two patients with PNH who underwent syngeneic BMT. One patient with frequent, severe hemolytic episodes underwent unconditioned BMT with no response. A second patient with PNH underwent successful syngeneic BMT Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired after myeloablative conditioning. Myeloablation appears to clonal disease of hematopoiesis due to a mutation in the be necessary for successful engraftment in the majority of 1,2 X-linked PIG-A gene. This mutation results in defective patients with syngeneic donors.9,16 Endo et al16 showed that synthesis of the glycosylphosphatidylinositol (GPI) anchor unconditioned syngeneic transplantation led to a transient necessary for the attachment of many proteins to the cell increase in donor peripheral blood cells with normal surface. Deficiency of one of these proteins, CD59, respon- expression of GPI-anchored proteins. This patient was asymptomatic after transplantation. At 17 months post- BMT, the percentage of donor cells decreased and was Correspondence: Dr P Woodard, Division of Bone Marrow Transplan- tation, Department of Hematology/Oncology, St Jude Children’s Research associated with recurrent hemolysis. The gradual increase Hospital, 332 North Lauderdale Street, Memphis TN 38105–2794, USA in abnormal PNH cells suggested that the abnormal stem Received 4 October 2000; accepted 18 December 2000 cells might have a survival advantage. Doukas et al9 Successful unrelated donor BMT for PNH P Woodard et al 590 reported successful transplantation of a 24-year-old patient Table 1 Patient characteristics with multiple venous thromboses with syngeneic bone mar- row after busulfan and cylcophosphamide conditioning. Age (years) 13, 15 and 20 This patient engrafted and remains free of PNH red cells Original Dx (n) SAA 2 over 11 years after transplantation. Mild to Moderate AA 1 Several reports describe matched sibling BMT for aplas- Time to PNH Dx (years) 1.5, 8 and 10.3 tic anemia, recurrent thromboses, or refractory hemolysis Time from PNH Dx to BMT (months) 3, 17 and 30 associated with PNH.12,13 Bemba et al13 reported 16 Blood exposures from original diagnosis patients transplanted after conditioning with matched sib- until BMT RBC (units) 8, 17 and 29 ling donors. Six of these had PNH after severe aplastic ane- (units) 3, 10 and 192 mia while 10 had de novo PNH. Conditioning consisted Prior therapy before BMT (n) of (150 mg/kg) and thoraco-abdominal ATG 3 (2 with multiple courses) irradiation in 15 and cyclophosphamide and 8 Gy TBI in Corticosteroids 2 Cyclosporine 1 one patient. There were nine survivors, with seven deaths Oxymetholone 1 due to and/or GVHD. In a report by Kawahara et al12 six patients were transplanted with matched sibling donors for aplastic complications (n = 4), anemia and hem- = = olysis (n 1), and hemolysis and thromboses (n 1) due Severe aplastic anemia to PNH. All six patients were long-term survivors. A recent report by Raiola et al18 describes successful MSD BMT in The original diagnosis of severe aplastic anemia occurred seven patients with PNH. Saso et al14 summarized 57 at ages 3 and 13 years, respectively, and resulted in treat- patients reported to the International Bone Marrow Trans- ment with equine anti-thymocyte globulin for two courses. plant Registry (IBMTR). This experience included 48 One of these patients additionally received four courses of matched sibling donors, six unrelated donors, and one hap- corticosteroids, three courses of cyclosporine, and two loidentical BMT. Patients were treated with varying con- courses of oxymetholone. The diagnosis of PNH occurred ditioning regimens. Thirty-seven of 48 (77%) evaluable 11 years after treatment with multiple immunosuppressive MSD recipients had sustained donor engraftment. 34% agents in one patient, while PNH occurred 1.5 years after developed уacute GVHD, while 33% developed chronic treatment with ATG in the second patient. The diagnosis GVHD. Overall survival was 56% for the MSD cohort at of PNH was followed by the development of MDS 10 and a median of 44 months after BMT. Causes of death 21 months later, respectively. included graft failure, interstitial pneumonitis, infection and GVHD. Strikingly, only 1/7 alternate donor BMT recipients Moderate aplastic anemia survived. Causes of death for alternate donor recipients included graft failure (n = 2), interstitial pneumonitis One patient was diagnosed with moderate AA at age 4 (n = 1), organ failure (n = 1), chronic GVHD (n = 1), and years. This patient initially received no treatment, but was new malignancy (n = 1). The overall reported experience treated with equine ATG and hydrocortisone after evolu- for alternate donor BMT for PNH is limited to eight ation to severe aplastic anemia and PNH 8 years later. patients with only one survivor. Our series reports three patients undergoing matched unrelated donor allogeneic Specifics of BMT BMT for PNH. All three patients experienced side-effects that are often associated with transplantation of marrow Patient and transplant specifics are given in Table 2. The from matched unrelated donors, yet all are survivors at a median time from PNH diagnosis to BMT was 17 months median of 3 years after BMT. (range, 3–30 months). Patients received a median of 17 units of pRBCs (range, 8–29 units) and 10 units of platelets (3–192 units) before BMT. Typing was performed by sero- Materials and methods logic techniques for class 1 and high-resolution DNA typing for class 2. Three unrelated donor recipients received grafts matched at six antigens (n = 2) or with a mismatch Patients Patient characteristics are listed in Table 1. Three patients with PNH and myelodysplastic syndrome (MDS) (n = 2) or Table 2 Transplant characteristics severe aplastic anemia (SAA) (n = 1) received BMT Unrelated donor match grade (n) between 1995 and 1998 at St Jude Children’s Research 6/6 2 Hospital. Diagnosis of MDS was made by review of bone 5/6 1 marrow aspirates using the FAB classification.19 Diagnosis Cell dose of PNH was originally made by sucrose hemolysis and TNC/kg 0.7, 1.0 and 1.1 ϫ 108 ϫ 6 Ham’s test in two patients and by flow cytometry to CD55 CD34/kg 1.1, 2.0 and 2.1 10 ANC Ͼ500 (days) 15, 16 and 21 and CD59 in one patient. Two patients had an original diag- Acute GVHD (n)1 nosis of severe aplastic anemia, while one patient had mod- Chronic GVHD (n)0 erate aplastic anemia. Transplant-related toxicity was Follow-up (months) 35 (19–59) graded by the Bearman criteria.20

Bone Marrow Transplantation Successful unrelated donor BMT for PNH P Woodard et al 591 at the HLA-A locus (n = 1). Pre-transplant conditioning ence suggests a relatively high rate of graft failure with the consisted of Ara-C 18 g/m2 in six divided doses adminis- use of non-conditioned transplants. Endo et al16 reported tered every 12 h, cyclophosphamide 45 mg/kg/day ϫ 2 transient correction of clinical manifestations of PNH asso- days, fractionated TB1 (14 Gy), Mesna 45 mg/kg/day in ciated with an increase in normal donor cells with normal five divided doses, and ATG 30 mg/kg/day ϫ 3 days. T cell GPI-anchored proteins in a patient with a history of recur- depletion was performed by treatment of grafts with anti- rent hemoglobinuria and chest pain. The patient remained CD6/CD8 antibodies ϩ complement.21 Cyclosporine was asymptomatic for 13 months, but subsequently experienced administered post-BMT as GVHD prophylaxis. Donor recurrent symptoms and progressive dominance of hemato- chimerism studies were performed by variable nucleotide poiesis by GPI-anchored protein-deficient stem cells. This tandem repeats (VNTR). report suggested that abnormal PNH cells appeared to have Patients received a median of 1.1 ϫ 108 total nucleated a proliferative advantage over normal syngeneic donor cells/kg (range, 0.74–1.1 ϫ 108/kg). A median of 2.0 ϫ 106 cells. ϩ CD34 cells/kg were infused (range, 1.1–2.1 ϫ 106/kg). Bemba et al13 reported 16 patients with PNH trans- The median time to recovery of ANC Ͼ500 cells/mm3 was planted with matched sibling donors over a 20-year period 16 days (range, 15–21 days). One patient experienced tran- at the Hospital Saint-Louis. Six patients developed PNH sient grade I acute GVHD of the skin. No patient developed after therapy for aplastic anemia while 10 had de novo chronic GVHD. PNH. Of the 10 de novo cases, six had prior therapy with Three patients experienced side-effects that were typical androgens or anti-lymphocyte globulin. Four had evidence for unrelated donor transplantation. One had two significant of alloimmunization. All patients engrafted after receiving complications, hemorrhagic cystitis and hemolytic-uremic pretransplant conditioning followed by a median of syndrome (HUS). Grade III hemorrhagic cystitis occurred 2.6 ϫ 108 nucleated cells/kg. Grade II–III acute GVHD 2–3 months post BMT and required cystoscopy and bladder occurred in eight patients, while three developed chronic irrigation. Urine cultures for bacterial and viral pathogens GVHD. The 5-year survival rate was 58%, with seven were negative. Cytology specimens from the cystoscopy deaths from GVHD and/or infection. GVHD morbidity and were negative for viral changes. This patient also developed mortality was relatively high, perhaps related to single transient HUS responsive to erythropoietin. A second agent prophylaxis for GVHD. patient developed grade I veno-occlusive disease (VOD) of Saso et al14 reported the outcome of 57 patients reported the liver with a maximum total bilirubin of 3.2 mg/dl. One to the International Bone Marrow Transplant Registry patient developed three significant toxicities, including (IBMTR). Patients were transplanted between 1978 and Epstein–Barr virus lymphoproliferative disease (EBV- 1995. Forty-nine patients were transfused pre-BMT with a LPD), severe hemorrhagic cystitis, and transient congestive median of 40 units (range, 1–300). Conditioning regimens heart failure (CHF). This patient has been described in were variable but most often included busulfan/ detail previously.22 Bulky cervical and nasopharyngeal cyclophosphamide or cyclophosphamide/irradiation. Two lymphomatous disease developed 5 months post BMT. The patients received syngeneic BMT while 48 received HLA- inflammatory response associated with infiltration of EBV- matched sibling donor BMT and seven received alternate specific cytotoxic T-lymphocytes (CTLs) resulted in airway obstruction and mucosal sloughing, requiring tracheostomy. donor (six matched unrelated donor, one phenotypically During this acute life-threatening illness, this patient also matched family member) BMT. Donor engraftment experienced grade III hemorrhagic cystitis requiring tem- occurred in only 77%. Both recipients of syngeneic BMT porary suprapubic catheter placement. EBV DNA was iso- had successful transplants, while 58% of matched sibling lated from cystoscopy washings. During the episode of donor BMT recipients were alive at 2 years. Only one of hemorrhagic cystitis, the patient developed transient grade seven alternate donor recipients survived the procedure. A II congestive heart failure (CHF) requiring treatment with substantial proportion of patients, 34% and 33%, respect- digoxin. The etiology of the CHF was presumed to be fluid ively, developed grade II–IV acute GVHD or chronic = overload. The EBV-LPD completely resolved with two GVHD. Deaths occurred due to graft failure (n 7), inter- = = treatment courses of EBV-specific cytotoxic T-lympho- stitial pneumonitis (IP) (n 4), GVHD (n 3), infection cytes. These complications resolved completely in all three (n = 3), adult respiratory distress syndrome (n = 2), and patients. Chimerism analyses done after transplantation hemorrhage (n = 1). In alternate donors, there were six revealed 100% donor-derived hematopoiesis in bone mar- deaths from graft failure (n = 2), IP (n = 1), organ failure row samples from all three patients at multiple time points (n = 1), chronic GVHD (n = 1), and malignancy (n = 1). after BMT. No patient had a period of mixed hematopoietic In our series of three heavily pre-treated, heavily trans- chimerism. All patients are alive and well at a median fol- fused patients, full donor engraftment was achieved. low-up of 3 years (range, 2.5–5.1 years), with no evidence Reports of BMT with unrelated and haploidentical donors of recurrence of aplastic anemia, MDS, or PNH. have shown successful engraftment is achievable despite large numbers of prior transfusions.23,24 In a report by Dav- ies et al23 primary engraftment occurred in six of seven Discussion patients despite extensive red cell transfusions and refractoriness prior to BMT. Tzeng et al24 reported success- Correction of the hematologic manifestations of PNH by ful engraftment in six consecutive patients despite a median transplantation of syngeneic and genotypically matched of 13 units of red blood cells and a median of 39 units of bone marrow has been previously demonstrated. Experi- platelets received before BMT. Full donor engraftment was

Bone Marrow Transplantation Successful unrelated donor BMT for PNH P Woodard et al 592 achieved in our three patients despite extensive red blood 8 Harrington WJS, Kolodny L, Horstman LL et al. Danazol for cell and platelet exposure before BMT. paroxysmal nocturnal haemoglobinuria. Am J Hematol 1997; Although the mortality rate was high in the previous 54: 149–154. report of alternative donor BMT for patients with PNH, the 9 Doukas MA, Fleming D, Jennings D. Identical twin marrow three patients in this report have had successful outcomes transplantation for venous thrombosis in paroxysmal nocturnal hemoglobinuria; long-term complete remission as assessed by after BMT. We have previously reported equivalent out- flow cytometry. Bone Marrow Transplant 1998; 22: 717–721. come of BMT with MSD or matched unrelated donors for 10 Kolb HJ, Holler E, Bender-Gotze C et al. Myeloablative con- 20 children with leukemia. After in vitro T cell depletion ditioning for marrow transplantation in myelodysplastic syn- of unrelated donor grafts with anti-CD6/CD8 monoclonal dromes and paroxysmal nocturnal haemoglobinuria. Bone antibodies ϩ complement followed by cyclosporine pro- Marrow Transplant 1989; 4: 29–34. phylaxis, one patient experienced transient grade 1 acute 11 Champlin RE, Feig SA, Sparkes RS, Gale RP. Bone marrow GVHD and none developed chronic GVHD. These three transplantation from identical twins in the treatment of aplastic patients experienced other transplant-related complications anemia: implication for the pathogenesis of the disease. Br J including hemorrhagic cystitis, hemolytic-uremic syn- Haematol 1984; 56: 455–463. drome, and EBV-LPD. Despite the BMT-related compli- 12 Kawahara K, Witherspoon RP, Storb R. Marrow transplan- cations requiring therapeutic intervention, our patients have tation for paroxysmal noctumal hemoglobinuria. Am J Hema- tol 1992; 39: 283–288. recovered uneventfully and remain free of aplastic anemia 13 Bemba M, Guradiola P, Garderet LU et al. Bone marrow and MDS after 30–62 months of follow-up. These results transplantation for paroxysmal nocturnal haemoglobinuria. Br support the cautious use of T cell-depleted unrelated-donor J Haematol 1999; 105: 366–368. BMT for patients with PNH-related disease processes, such 14 Saso R, Marsh J, Cevreska L et al. Bone marrow transplants as SAA or MDS. In this series, the use of extensive blood for paroxysmal nocturnal haemoglobinuria. Br J Haematol products before BMT was not associated with alloimmuni- 1999; 104: 392–396. zation or graft failure. 15 Graham ML, Rosse WF, Halperin EC et al. Resolution of Future strategies should include more immunosuppres- Budd–Chiari syndrome following bone marrow transplan- sive, less myelobalative conditioning to lessen toxicity. tation for paroxysmal nocturnal haemoglobinuria. 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