Matched Unrelated Bone Marrow Transplantation for Combined Immunodeﬁciency

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Bone Marrow Transplantation (2000) 25, 613–621  2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt Matched unrelated bone marrow transplantation for combined immunodeﬁciency

I Dalal1, B Reid1, J Doyle2, M Freedman2, S Calderwood2, F Saunders2 and CM Roifman1

1Divisions of Immunology/Allergy, Haematology/Oncology, 2Department of Paediatrics, The Infection, Immunity, Injury and Repair Programme, Research Institute, The Hospital for Sick Children and The University of Toronto, Canada

Summary: marrow transplantation (BMT) using marrow from geno- typically identical sibling donors remains the treatment of Bone marrow transplantation (BMT) from siblings is choice for most forms of severe combined immunodefi- the treatment of choice for severe combined immuno- ciency (SCID). Currently, however, only approximately deficiency (SCID). The objective of this study was to 10% of infants with SCID have healthy, HLA-matched sib- evaluate the efficiency of BMT from matched unrelated ling bone marrow donors.3 Attempts to use alternative pro- donors (MUD) in congenital immunodeficiencies when cedures have been less successful. Immunologic reconsti- a sibling donor is unavailable. Sixteen consecutive tution following fetal thymus and/or liver transplantation is patients with SCID (n = 9) and CID (n = 7), were inconsistent; engraftment is achieved in less than one third referred for an unrelated donor search. Acceptable of cases and a survival rate of less than 20% has been donors were found for all patients. Fifteen patients reported.4,5 received busulfan and cyclophosphamide pretransplant T lymphocyte-depleted, haploidentical bone marrow, conditioning. One patient had an early loss of graft and usually of parental origin, is regarded by many transplant was reconditioned using cyclophosphamide and total centers as the treatment of choice when fully matched sib- body irradiation. The graft-versus-host disease (GVHD) ling donors are unavailable. Although survival for all types prophylaxis used was methylprednisolone, cyclosporin of SCID hovers around 50%,6 recent preliminary experi- A with or without methotrexate. Neutrophil ence shows a higher survival rate (70–78%) in selective engraftment was rapid and was achieved in all patients SCID phenotypes mainly including T−B+ SCID7,8 and ADA within a mean of 15.4 days. Only 13 episodes of fever deficiency8 using this method. However, this approach has were recorded shortly after BMT. GVHD of grade II serious limitations, including prolonged time to or more was apparent in 2/9 (22%) of SCID patients engraftment,9–12 and very slow B lymphocyte engraftment, and in 4/7 (57%) of CID patients. Overall survival was which ranges in large series of patients from 40 to 70%.9–13 75% with a mean follow-up of 47.4 months (range 18– Both of these factors contribute to a high rate of infection.6–15 101). Six out of nine SCID patients (67%) and 6/7 (86%) Moreover, engraftment and survival rate of profound T cell of CID patients are alive and well. Eleven patients had immunodefiencies with residual function (CID) using this normal humoral immunity, and cell-mediated immunity method have been poor. as measured by flow cytometry and mitogenic Combined immunodeficiency (CID) refers to a hetero- responses, was intact in all patients. Intradermal can- geneous group of patients, who present with severe recur- dida skin test was positive in 9/10 patients tested. We rent infections and have profound but not absolute conclude that BMT from MUD results in rapid deficiencies of humoral and cell-mediated function. In engraftment and is therefore associated with a low rate recent years, the molecular defects of several CID syn- of infection contributing to the improved survival rate. dromes have been identified, including lymphocyte signal The protocol used is especially favorable for patients transduction elements such as Zap-70, IL-2R␣, CD3,16–18 with combined immunodeficiency. Bone Marrow Trans- as well as ‘atypical’ phenotypes arising from mutations in plantation (2000) 25, 613–621. IL-2R␥19 or ‘partial’ ADA deficiency.20 Experience with Keywords: severe combined immunodeficiency; bone the application of BMT to patients with CID or T cell marrow transplantation; combined immunodeficiency; immunodeficiency with residual function is extremely lim- matched unrelated donors; graft-versus-host disease ited.21 Among patients transplanted for CID with T cell- depleted bone marrow, engraftment rates have been poor, ranging from 14% to 30%, while short-term survival rates reached 47%,15 and long-term survival as low as 10% have The first successful allogeneic bone marrow transplant for been recorded.18 These discouraging results point to a 1,2 lethal immunodeficiency was reported in 1968. Bone pressing need for better transplant alternatives to ensure a full engraftment and reconstitution of immunological function, and a better long-term survival rate. Correspondence: Dr CM Roifman, Division of Immunology/Allergy, The Infection, Immunity, Injury and Repair Program, The Hospital for Sick We prospectively enrolled infants and children with Children, Toronto, ON M5G 1X8, Canada SCID (n = 9) or CID (n = 7) with no sibling donor into a Received 9 August 1999; accepted 7 November 1999 protocol using matched unrelated donors (MUD) in an Matched unrelated BMT for CID I Dalal et al 614 attempt to develop a better alternative to T cell-depleted administered to all patients from the day prior to transplant, haploidentical bone marrow transplantation. We conclude consisted of cyclosporin A (3 mg/kg/day i.v.) or adjusted that BMT from MUD results in rapid engraftment and is to keep a level of 150 ␮g/l and methylprednisolone (2 associated with a low morbidity and improved survival rate mg/kg/day i.v.) for an average of 90 days. Addition of especially in patients with combined immunodeficiency that methotrexate 10 mg/m2 on days 3, 6, 11 and 18 post BMT possess residual function. was added to the protocol of nine patients as previously suggested by other investigators.24 Prednisone at various doses was continued as long as GVHD persisted. All Materials and methods patients were infused with 3–5 × 108 donor bone marrow mononuclear non-depleted cells per/kg of recipient weight. Patients Eleven patients received GM-CSF (5.5 mg/kg/day subcutaneously) and five received G-CSF (5.0 mg/kg/day = Sixteen consecutive patients with SCID (n 9), CID or i.v.) from the day of transplant until the absolute neutrophil T cell immunodeficiency with residual function (n = 7), count was Ͼ1.0 × 109/l for 3 consecutive days. diagnosed in the Division of Immunology at the Hospital for Sick Children (Toronto) between December 1989 and August 1997, who lacked histocompatible siblings or Engraftment and chimerism studies closely matched related donors, were referred to the Canad- Neutrophil engraftment was considered to have occurred on ian Unrelated Bone Marrow Donor Registry for an unre- the first of 3 consecutive days in which the absolute count lated donor search. All donors and recipients were matched exceeded 500 cells per cubic millimeter. Platelet by DNA genotype analysis. Data were collected until Janu- engraftment was considered to have occurred on the first ary 1999, so that all children had a minimum follow-up of day of 7 consecutive days in which the platelet count 12 months after BMT. exceeded 20000 per cubic millimeter without platelet transfusion. HLA typing and donor selection Peripheral blood mononuclear cells were isolated 8–10 weeks after transplant by Ficoll–Hypaque gradient centri- Lymphocytes from all patients were tested for HLA A, B, fugation and were analyzed for lymphocyte subpopulation C and DR typing using standard serological methods. Class engraftment using flow cytometry (Coulter Electronics, II histocompatibility was further analyzed by DNA Hialeah, FL, USA). Where sex difference between donor hybridization with sequence-specific oligonucleotide and recipient existed, DNA was analyzed using an Y- 22 probes. Mixed lymphocyte reactions (MLR) were perfor- specific probe (ZFX/ZFY) to confirm donor lymphocyte med in nine out of 16 recipient–donor pairs as previously engraftment. In the absence of sex difference restriction 23 reported. Patients who received a haploidentical bone length fragment polymorphism (RFLP) analyses were per- marrow and/or showed a MLR reactivity of more than 20% formed by studying genetic polymorphisms which are due were not included in the study. to a variable number of tandem repeats (VNTR) or (CA)n in varying regions of the genome. Blood group phenotyping Transplantation procedures served as an additional engraftment marker. Patients were admitted into private HEPA-filtered rooms on the bone marrow transplant unit. Two patients (P10 and Immune function studies P14) were admitted to the intensive care unit due to respir- Humoral reconstitution was evaluated by measuring serum atory failure caused by Pneumocystis carinii pneumonitis immunoglobulin levels, isohemagglutinin and specific anti- and one of them (P14) received his marrow transfusion in body titers in response to immunization with diphtheria, the intensive care unit. Patients were maintained under tetanus toxoid and polio viruses. Evaluation of specific anti- strict reverse isolation procedures throughout the admission bodies was done at least 6 months after cessation of IVIG until they were discharged from the hospital. treatment. Cell mediated immunity was evaluated by meas- Trimethoprim/sulfametaxozole was used for Pneumocystis uring T cell subpopulations, in vitro response to mitogens carinii prophylaxis. Two patients were switched to pentam- and in vivo response to heat killed intradermal Candida idine (4 mg/kg/dose i.v. every second week) because of albicans skin test (1:100 or 1:10 dilution). All assays were possible drug-related neutropenia. Intravenous immuno- performed by standard methods as previously described. globulin (IVIG) at a dose of 600 mg/kg was infused as required to maintain IgG level above 6 g/l. Acyclovir (1500 2 mg/m /day) was administered i.v. to five patients who Results received bone marrow from cytomegalovirus (CMV) sero- positive donors. All blood products were CMV negative Patient population and characteristics and irradiated and were infused through a 170 ␮g filter. Pretransplant conditioning therapy consisted of busulfan The clinical features of the 16 patients are summarized in given over 4 days in doses of 4 mg/kg/day for patients over Table 1. Twelve of 16 patients were males and the mean 3 years of age (n = 1), or 5 mg/kg/day for patients younger age at diagnosis was 7.4 months (range: birth–46 months). than 3 years (n = 15). This was followed by a 4-day course Patients were classified according to criteria of the World of cyclophosphamide (50 mg/kg/day). GVHD prophylaxis, Health Organization.25 Nine patients (1–9) had typical clini-

Bone Marrow Transplantation Matched unrelated BMT for CID I Dalal et al 615 Table 1 Clinical features of patients with immunodeﬁciency

Patient Immunodeﬁciency Age at diagnosis Sex Pre-BMT features (months)

1 SCID T−B+ 9 F Pneumonia, FTT 2 SCID T−B+ 5 M Pneumonia, FTT 3 SCID T−B− 1 M None 4 SCID T−B+ 5 M Pneumonitis 5 SCID T−B+ 4 M mouth ulcers, FTT 6 SCID T−B+ 3 M FTT 7 SCID T−B+ 10 M Candida esophagitis, FTT 8 SCID T−B+ 4 M PCP, FTT 9 SCID T−B+ birth M none 10 CID (Cartilage hair hypoplasia) (T+B+) 6 F PCP, failed haploidentical T cell-depleted BMT 11a CID (T+B+) 46 M Severe varicella, malabsorption, recurrent otitis media 12a CID (T+B+) 1 F none 13 CID (T+B+) 6 M PCP, FTT 14 Omenn’s syndrome (T+B+)5. M PCP, mechanical ventilation, exfoliative dermatitis 15 Omenn’s syndrome (T+B+) 6 F Exfoliative dermatitis, pneumonitis, FTT 16 Omenn’s syndrome (T+B+)6. M PCP, exfoliative dermatitis aSiblings. PCP = Pneumocystis carinii pneumonitis; FTT = failure to thrive.

Table 2 Pre-BMT humoral and cell-mediated immunity

Patient Cell mediated immunity Humoral immunity Flow cytometry analysis of patients by lymphocytes Response to mitogens Speciﬁc Lymphocyte subsets % (% of control) Immunoglobulins antibodies

ALC CD2 CD3 CD4 CD8 CD19 CD56 PHA STA Anti IgGb IgAb IgMb Tetanusc Poliod CD3

1 1.10 9.00 0.10 2.00 0.40 89.00 7.00 0.00 161.00 0.00 0.00 0.00 0.00 Ͻ0.01 Ͻ1:8 2a 0.50 58.00 42.00 18.00 12.00 41.00 24.00 0.00 0.00 0.00 0.00 0.00 0.00 Ͻ0.01 Ͻ1:8 3 0.90 65.00 0.00 1.00 0.10 0.20 31.00 0.00 0.00 0.00 0.00 0.00 0.00 Ͻ0.01 Ͻ1:8 4 0.86 7.00 0.10 1.60 2.60 87.00 1.00 0.00 194.00 0.00 0.90 0.10 0.03 Ͻ0.01 Ͻ1:8 5 1.50 8.00 0.20 0.00 0.10 82.00 14.00 0.00 235.00 0.00 0.50 0.00 0.00 Ͻ0.01 Ͻ1:8 6 2.54 1.00 1.00 0.00 0.00 99.00 0.00 0.00 55.00 ND 1.10 Ͻ0.1 00.30 ND ND 7 1.24 6.00 3.00 0.40 2.00 94.00 2.00 0.00 6.00 ND Ͻ0.1 Ͻ0.1 Ͻ0.1 Ͻ0.01 Ͻ1:8 8a 5.92 67.90 67.00 4.10 13.40 43.00 0.16 0.00 50.00 ND 0.02 0.21 ND ND ND 9 1.83 44.00 0.00 0.00 0.00 27.00 46.00 0.00 177.00 ND 8.80 Ͻ0.1 Ͻ0.1 ND ND 10 0.70 30.00 14.00 11.00 2.00 62.00 22.00 7.00 19.00 21.00 4.10 0.10 0.30 Ͻ0.01 0.06 11 1.90 48.00 37.00 8.00 21.00 48.00 17.00 26.00 20.00 28.00 0.70 0.40 0.27 NA NA 12 1.87 37.00 33.00 16.00 14.00 38.00 29.00 39.00 50.00 27.00 2.90 0.30 0.60 1.32 1.26 13 2.50 74.00 64.00 40.00 19.00 19.00 9.00 50.00 132.00 101.00 2.72 1.14 0.29 Ͻ0.01 Ͻ1:8 14 66.50 95.00 92.00 3.00 35.00 0.30 0.50 7.00 30.00 11.00 0.00 0.00 0.00 Ͻ0.01 Ͻ1:8 15 4.75 90.50 74.00 22.00 16.00 4.00 14.40 8.00 72.00 ND 4.00 0.60 0.40 Ͻ0.01 Ͻ1:8 16 7.46 47.00 42.90 9.50 11.80 49.70 4.40 0.00 ND ND 2.28 Ͻ0.15 0.20 Ͻ0.01 Ͻ1:8

Neg = negative; NA = not available; ALC = absolute lymphocyte count in 109/l. aMaternal engraftment. bLevels of immunoglobulins are expressed in grams/l. cProtective level of tetanus antibodies Ͻ0.01 IU/ml. dProtective level of polio antibodies Ͻ1:16. cal and laboratory findings of SCID (Tables 1 and 2). How- (P12) was diagnosed immediately after birth because of the ever, seven other patients (10–16) demonstrated residual T positive family history. Assessment of her immune system cell function and therefore were diagnosed as CID (Table at the age of 3 months revealed an abnormal subset of T 2). cells and a moderately decreased proliferative response to Patients 14–16 were diagnosed with Omenn’s syndrome, mitogens. Soon after, she demonstrated a gradual deterio- and patient 14 has been described in detail elsewhere.26 ration of T cell function. However, she was able to maintain Patients 11 and 12 were siblings and although the molecular normal levels of immunoglobulins and specific antibody defect of their disease remains unknown, they demonstrated production. Patient 13 had normal in vitro immune function the evolution of a late onset T cell immunodeficiency. Case and even a normal thymic morphology. Diagnosis of pro- 11 was diagnosed at the age of almost 4 years, but his sister found immunodeficiency was established only after he

Bone Marrow Transplantation Matched unrelated BMT for CID I Dalal et al 616 failed to reject a skin graft. Recently the molecular defect GVHD. Blood group phenotyping was switched to donor has been identified as a novel mutation in the common ␥ type in the nine patients who had differences in blood group chain of the IL2 receptor.19 before transplant (Table 4). Nine patients (56%) had a pulmonary infection before RFLP studies were performed in 14 cases, 8 to 10 weeks transplant, which is considered a bad prognostic factor,9 post BMT. All 14 cases demonstrated virtually 100% and three of them were very high-risk transplants. Patient engraftment of donor cells, without any evidence of 14 received a bone marrow infusion during his admission residual recipient cells; including the patient who had pre- to the intensive care unit due to severe pneumocystic carinii viously failed a haploidentical T cell-depleted BMT. In one pneumonitis requiring prolonged mechanical ventilation. other patient karyotyping analysis showed 100% female Prior to her MUD transplant patient 10 failed to engraft a donor cells in peripheral blood. haploidentical T cell-depleted BMT and reconstituted her own marrow (Table 1). Patient 16 had a rapid engraftment Humoral immunity with an HLA-identical MUD donor, but lost the graft within 4 weeks. He was re-conditioned with a cyclophos- Evaluation of humoral immune reconstitution is shown in phamide and irradiation protocol and was re-transplanted Table 5. Serum IgM and IgA normalized in all patients using a harvest obtained from the same donor. The mean within a mean of 3.5 and 6 months, respectively, except age at transplantation was 15.2 months (range, 2–59) for patient 5 whose IgA was still Ͻ0.1 g/l when he died 11 (Table 3). months after transplant. Eleven of 12 alive patients reached normal levels of IgG within 4 to 17 months (average 8.2 Donor selection months). At the present time, patient 15 has not yet normalized her IgG levels 18 months post BMT. Eleven Acceptable donors were found for all patients within a patients have been immunized with four doses of DPT vac- mean of 4.7 months (range, 1–13). Thirteen patients had cine. Levels of specific antibodies were measured 1 to 2 phenotypically identical donors while the rest had donors weeks after the second and the third immunizations, as well mismatched at one locus: A, B or C (one each). MLR was as at 6-month intervals. Patients began producing protective performed in nine cases. None of the MLRs demonstrated levels of specific antibodies rapidly, within 5 months of more than 7% reactivity in the direction of donor cells BMT. All immunized patients developed and sustained pro- responding against irradiated recipient cells (Table 3). tective levels of antibodies against tetanus toxoid and polio viruses. Hematological engraftment Cell-mediated immunity Neutrophil engraftment was achieved in all patients at a mean of 15.4 days (range, 11–22), including the patients Total lymphocyte counts and subsets of T cells, studied by who failed previous BMTs. Engraftment of platelets was flow cytometry, 1 to 2 months after BMT showed evidence achieved in 13 out of 16 patients with a mean of 16 days of engraftment in all patients (Table 6). Four patients dem- (range, 7–60). Patients 6 and 12 remained platelet depen- onstrated proliferative response to mitogen stimulation dent until they died from GVHD post BMT. Patient 7 had within 2 months after BMT. Most recent evaluation in 10 delayed engraftment of platelets until day 237, due to patients showed that all have normal absolute lymphocyte

Table 3 Characteristics of unrelated BMT in 16 patients with immunodeﬁciency

Patient Donor BMT HLA match MLR GVHD Febrile GVHD Post BMT Outcome Age, Sex administered R/D prophylaxisa episodes (grade) (months) (months)

1 36, F 16 Identical ND 1 1 1 72 A&W 2 47, M 12 Identical ND 1 1 1 68 A&W 3 31, M 7 A28/A31 2% 2 1 1 56 A&W 4 48, M 14 Cw3, 5/Cw2, 4 Ͻ1% 2 1 1 53 A&W 5 34, F 6.5 Identical ND 2 0 1 — Died; BM aplasia 6 34, M 4.5 Identical 5% 2 0 3 — Died; GVHD 7 34, M 15 Identical 3% 2 1 1 31 A&W 8 46, F 9 Identical ND 1 1 3 — Died; GVHD 9 25, M 2 Identical ND 1 1 1 18 A&W 10 36, M 23 B57/B37 2% 2 0 1 52 A&W 11 33, F 59 Identical Ͻ1% 2 1 3 52 A&W 12 33, F 24 Identical Ͻ1% 2 1 3 — Died; GVHD 13 36, M 11.5 Identical Ͻ1% 2 1 2 43 A&W 14 37, F 10.5 Identical 7% 1 1 2 101 A&W 15 37, F 12 Identical ND 1 1 1 20 A&W 16 38, M 16 Identical ND 1 1 1 6 A&W

R/D = Recipient/Donor; MLR = mixed lymphocyte reaction; Bu/Cy = Busulfan/cyclophosphamide; A&W = alive and well. aMethylprednisolone + cyclosporin A; 2, methylprednisolone + cyclosporin A + methotrexate.

Bone Marrow Transplantation Matched unrelated BMT for CID I Dalal et al 617 Table 4 Engraftment

Patient WBC Neutrophils Platelets Red cells Blood type PBL Karyotype (days) (days) (days) (days) pre/post RFLP T cellsa transplant (% donor)

11219127A+/O+ ND ND 214172825B−/A− 100 3 9 11 26 23 A+/O+ 100 4 9 11 18 24 A+/A+ 100 512131110O+/O+ 100 6 16 18 Remained plt Remained red cell O+ 100 dependent dependent 7 17 19 237 229 B−/A+ 100 8 8 11 10 5 O+/A+ 100 9 11 12 7 14 A+/A+ 100 10 15 16 12 21 O+/O+ 100 11 14 16 60 41 O+/A+ 100 12 20 22 Remained plt Remained red cell O+/A+ 100 dependent dependent 13 12 14 11 11 O−/B+ 100 14 11 13 12 46 A+/O+ 100% F 15 15 16 38 17 AB+/A+ 100 16b 18 19 30 25 O+/O+ 100

PBL = peripheral blood lymphocytes; RFLP = restriction length fragment polymorphosis. aT cell karyotyping performed on PHA stimulated lymphocytes. bPost 2nd transplant.

Table 5 Humoral immune reconstitution after unrelated BMT

Patient IgG IgA IgM Tetanus Polio (months post BMT)

1 (7) 8.5 0.7 2.6 0.47 1:512 (32) 9.3 1.1 1.7 0.83 Ͼ1:64 2 (4) 5.2 Ͻ0.01 0.5 Ͻ0.01 neg (58) 5.4 0.3 0.9 3.33 Ͼ1:64 3 (5) 3.7 0.2 Ͻ0.1 0.11 ND (42) 15.6 1.7 1.2 0.42 Ͻ1:64 4 (6) 5.5 0.1 0.4 0.11 1:512 (48) 9.4 0.9 1.3 0.15 Ͼ1:64 5 (2) IVIG Ͻ0.1 0.2 ND ND (6) 3.0 Ͻ0.1 0.2 ND ND 6 (1) IVIG 0.1 0.8 ND ND 7 (6) IVIG 0.1 0.2 ND ND (32) 6.2 0.4 0.7 0.85 Ͼ1:64 8 (5) 4.5 0.5 0.4 ND ND (12) IVIG NA NA ND ND 9 (6) IVIG Ͻ0.1 0.2 ND Ͼ1:64 (20) 5.1 Ͻ0.2 0.5 1.06 ND 10 (6) 9.0 1.8 4.0 0.17 Ͼ1:64 (45) 8.1 1.8 1.1 0.28 Ͼ1:64 11 (6) IVIG 0.1 0.4 ND ND (53) 5.6 1.8 1.9 0.18 Ͼ1:64 12 (3) IVIG 0.8 3.4 ND ND (6) IVIG 8.1 13.0 ND ND 13 (2) IVIG 0.1 0.16 ND ND (17) 3.5 0.3 0.8 Ͼ1.05 Ͼ1:64 14 (6) 3.69 Ͻ0.01 0.32 0.02 ND (99) 9.1 1.3 0.7 0.59 Ͼ1:64 15 (8) IVIG 0.1 0.4 ND ND (18) 2.4 0.7 1.5 Ͼ2.00 Ͼ1:64 16 (3) IVIG 0.1 0.1 ND ND (12) 4.2 0.5 0.8 0.6 Ͼ1:64

Bone Marrow Transplantation Matched unrelated BMT for CID I Dalal et al 618 Table 6 Cell-mediated reconstitution after unrelated BMT

Patient ALC CD2 % CD3 % CD4 % CD8 % CD56 % PHA % of STA Candida (months) control skin test

1 (2) 1.00 80 35 13 18 52 5 25 pos (32) 2.64 75 52 19 40 6 36 97 2 (2) 0.70 46 28 6 8 33 11 31 neg (58) 2.73 78 74 38 23 4.5 112 349 3 (2) 0.70 66 57 26 30 9 24 34 pos (42) 3.53 83 76 39 26 16 90 133 4 (2) 0.65 58 40 31 8 22 ND ND pos (48) 4.30 68 61.6 33 19 8.5 142 100 5 (2) 0.71 62 45 33 25 21 ND ND ND (6) 0.92 77 42 25 39 38 ND ND 6 (1) 0.30 94 68 48 16 30 ND ND ND 7 (1) 0.22 87 56 44 9 40 ND ND pos (32) 2.15 69 66 46 14 4 121 106 8 (1) 0.53 89 76 33 35 12 ND ND ND (5) 0.46 84 40 24 14 55 ND ND 9 (1) 0.33 87 78 54 13 20 ND ND pos (20) 1.04 80 75 30 36 9 164 48 10 (2) 0.90 83 53 34 14 31 ND ND pos (45) 2.60 72.6 62 22 28 12 69 277 11 (2) 0.30 80 67 16 47 19 ND ND pos (53) 3.90 92 66 41 19 3.9 61 166 12 (2) 0.15 91 64 21 58 30 ND ND ND (5) 0.26 92 68 28 28 32 ND ND 13 (2) 0.32 80 61 29 29 7 ND ND pos (17) 1.5 72 63 33 24 14 133 331 14 (2) 1.20 96 73 35 35 31 21 42 pos (99) 2.61 72 63 33 24 14 76 95 15 (1) 0.46 99 98 65 23 2 ND ND ND (18) 1.43 73 53 27 20 27 ND ND 16 (1) 0.30 92 79 20 44 21 ND ND ND (3) 0.60 93 55 10 33 55 ND ND

count (ALC), absolute CD3+ and CD4+ cells. Seven out of fungal or viral infection. All patients recovered after 10 have a normal number of CD8+ cell counts whereas administration of antibiotic therapy. patients 4, 7 and 11 have slightly lower then normal counts. Proliferative responses of T cells to stimulation with differ- Graft-versus-host disease ent mitogens were within normal limits in all surviving patients, with the exception of patients 15 and 16 who have GVHD of grade II or more was clinically apparent in six not been tested yet due to continued treatment with GVHD patients. In the SCID population, two out of nine (22%) prophylaxis. In vivo evaluation of cell-mediated immunity, developed grade III GVHD and subsequently died from this assessed by intradermal candida skin test, showed positive complication (Table 3). The mean age of transplant for results in nine out of 10 patients tested (Table 5). Patients SCID patients was 6.25 months (range 4.5–6.5 months). 1, 2, 4 and 14 have experienced an uneventful varicella However, in the CID group four out of seven (57%) infection after BMT. developed GVHD of grade II or more, possibly due to their advanced age (mean 26.3) as compared with SCID patients Infections (Table 3). GVHD was reversed in three out of four in CID patients. Patients 11 and 12 were siblings who received During hospitalization a total of 13 episodes of fever were marrow from the same donor at the ages of 59 and 24 recorded for all patients. They all occurred within a few months, respectively. Patient 11 developed grade II GVHD days after BMT, while patients were severely neutropenic. that was reversed after a pulse of methylprednisolone (20 In ﬁve episodes bacterial agents were recovered from blood mg/kg/day) administered over 3 consecutive days. Patient cultures including Enterobacter cloacae, gram-negative 12 did not have any symptoms of GVHD for more than 2 bacilli, Pseudomonas aeruginosa and Staphylococcus coag- months post BMT. She developed a full blown, three organ ulase negative. Blood cultures obtained from patients dur- involvement GVHD several days after GVHD prophylaxis ing the rest of the febrile episodes failed to detect bacterial, was switched from i.v. to equivalent doses of oral medi-

Bone Marrow Transplantation Matched unrelated BMT for CID I Dalal et al 619 cation resulting in a sharp decline in cyclosporin A serum once switched from i.v. to oral immunosuppression, less levels. A pulse of high-dose steroids was administered but than 10 weeks post BMT. had to be discontinued due to a massive gastrointestinal bleed. She responded partially to repeated courses of anti- thymocyte globulin (ATG). A trial of anti-IL-2R antibody Discussion was unsuccessful and she subsequently died 7 months post BMT. Only three patients (1, 9, 15) developed mild chronic We report a series of 16 patients with SCID (n = 9) and GVHD of the skin. In two cases it subsequently resolved CID (n = 7), who received an unmodified BMT from after treatment with topical steroids. Only patient 15 still matched unrelated donors. This procedure is currently one requires low doses of cyclosporin A and steroids to control of two preferred approaches in cases where a suitable donor her chronic GVHD. could not be identified in the patient’s family. Only a small number of reports, which include a relatively limited num- Survival and cause of death ber of cases, have studied the option of MUD BMT for SCID and CID patients.27,28 Two main obstacles appear to Seventy-five percent of patients remain alive and well 6 to be associated with using MUD transplants. First, donor 101 months post BMT (mean 47.4 months). Six out of nine availability through the bone marrow registries, is a parti- (67%) SCID patients and six of seven (86%) CID patients cular concern for smaller or non-participating ethnic are alive. The actuarial survival rate for our group of groups. Furthermore, there may be a waiting period until patients was 74% as shown in Figure 1. Patients 5, 6, 8 the bone marrow is harvested, shipped and finally adminis- and 12 developed GVHD and subsequently died 13, 2.5, tered to the patient. It appears that these concerns have 12 and 7 months post BMT, respectively (Table 3). Patient gradually been addressed, and although they are not yet 5 developed bone marrow aplasia 1 year post BMT presum- optimal, great progress has already been achieved. We have ably due to GVHD and died from fulminant Streptococcus been able to identify a suitable donor through the ever- viridans sepsis. Patient 8 developed progressive liver growing International Registry for all our patients, and GVHD and succumbed to hepatic failure. In both patients waiting time has been reduced from months to sometimes 5 and 8 GVHD surfaced when the dose of corticosteroid weeks. Further improvement is likely to occur in light of prophylaxis was tapered rapidly (Ͼ5 mg per week) and massive enrollment in BMT registries, improved records of aggressive immunosuppression was not introduced HLA determination and enhanced international communi- promptly. Patients 6 and 12 developed intractable GVHD cation. The second major obstacle to declaring this mode of treatment ideal, is the consistent complication, GVHD. All a our patients developed GVHD in spite of treatment with 1 cyclosporin A and corticosteroids, with or without the addition of methotrexate. Although in most patients, this 0.8 complication was transient and limited to the skin, in some it ultimately contributed directly or indirectly to patients’ 0.6 deaths. In two patients, switching prophylaxis to oral administration 6 to 8 weeks post BMT resulted in a sharp 0.4 drop in cyclosporin A level and presumably triggered fatal GVHD. A third patient developed hepatic failure when 0.2 GVHD prophylaxis was reduced, while the fourth patient developed bone marrow aplasia following a rapid reduction 0 of immune suppression 5 to 6 months post transplantation. These results clearly highlight the critical importance of b maintaining adequate cyclosporin A levels during the first 1 3 months post BMT as previously suggested,29 even if no signs of GVHD are apparent. Furthermore, frequent moni- 0.8 toring and strict follow-up appears extremely important when immunosuppression is being reduced, even months 0.6 after bone marrow transplantation. Such follow-up cannot be left to unqualified medical staff and preferably should 0.4 be performed by an experienced bone marrow transplantation facility. 0.2 The second therapeutic approach currently adopted by many centers throughout the world is the use of modified 0 (depleted) haploidentical bone marrow, which is extracted 0 20 40 60 80 100 120 from a family member. Although this approach uses an Time (months) immediately available resource, it is plagued with multiple Figure 1 Survival of MUD BMT recipients. (a) Overall survival; (b) problems regardless of the method used. Engraftment is solid line, SCID patients; dotted line, CID patients. Survival was better slow, exposing patients to fatal infections over long periods among CID patients as compared with SCID patients. of time.30,31 Furthermore, greater than half of survivors

Bone Marrow Transplantation Matched unrelated BMT for CID I Dalal et al 620 remain with humoral immunodeficiency for years and at Acknowledgements least 30%6,32 to 65%8 never show B cell engraftment. In addition, acute and chronic GVHD remains a major concern We would like to thank Drs Fred Rosen and Trudy Small for in up to 50% of patients.10,33,34 their valuable comments, and also Penny Sotiropoulos and Brenda Long-term survival rates in multiple European centers Lynch for their assistance with manuscript preparation. This work using T cell-depleted bone marrow transplants appears to has been supported by the Donald and Audrey Campbell Chair in be below 50%, as reported in 1998.6 Recently, more pre- Immunology (CMR) and the Medical Research Council of liminary and relatively short-term experience reported by Canada (CMR). the same group suggests much improved outcome mainly in a selective group of T−B+ SCID (70%), but remained fairly low for the B−T+ SCID group.7 The best results of T References cell-depleted BMT for mismatched transplants were recently reported by Buckley et al.8 This single centre 1 Gatti RA, Meeuwissen HJ, Aller HD et al. Immunological experience shows that although B cell engraftment is poor, reconstitution of sex-linked lymphopenic immunological at 40%, survival rate was at 78%. Again, most patients had deficiency. Lancet 1968; 2: 1366–1369. either ␥c deficiency or ADA deficiency. Although a group 2 Bach FH, Albertini RJ, Anderson JL et al. Bone marrow transplantation in a patient with the Wiscott–Aldrich syndrome. of other autosomal recessive SCIDs are mentioned, it − + Lancet 1968; 2: 1364–1366. remains unclear whether B T SCIDs, who appear to have 3 Levinski RJ, Davies EG, Butler M, Abai A. Problems of mis- a lower survival rate using this method, were included in matched bone marrow transplantation for severe combined this survey. Consistent with these results, only one of three immunodeficiency after soybean lectin fractionation. Birth Def who have not survived in our SCID series had a mutation 1983; 19: 147–150. in ␥c. Overall, these results suggest that some SCID vari- 4 O’Reilly RJ, Pollack MS, Kapoor N. Fetal liver transplan- ants such as T−B+ and ADA deficiency may have a better tation in man and animals. In: Gale RP (ed). Recent Advances outcome using haploidentical T cell-depleted BMT. In con- in Bone Marrow Transplantation. Alan R Liss: New York, trast, the experience using T cell-depleted bone marrow for 1983, pp 799–830. CID was universally low (19–47% survival rate).21,35 We 5 Touraine JL, Laplace S, Rezzoug F et al. The place of fetal liver transplantation in the treatment of inborn errors of metab- show here that a MUD BMT performed on patients with olism. J Inher Metab Dis 1991; 14: 619–626. residual immune function such as CID and Omenn’s syn- + + 6 Haddad E, Landais P, Friedrich W et al. Long-term immune drome (Rag gene normal, B T ) has a relatively high (85%) reconstitution and outcome after HLA-non-identical T cell- success rate. Further, 5/6 patients with Wiscott–Aldrich depleted bone marrow transplantation for severe combined syndrome have successfully engrafted with MUD trans- immunodeficiency: a European retrospective study of 116 plants in our unit (manuscript in preparation). There is no patients. Blood 1998; 19: 3646–3653. clear explanation for this major difference in the success 7 Bertrand Y, Landais P, Friedrich W et al. Influence of severe rate between the MUD and depleted haploidentical BMT combined immunodeficiency phenotype on the outcome of for CID transplantation. One possible explanation is the HLA non-identical, T-cell-depleted bone marrow transplan- increased risk for overwhelming infection in this patient tation. J Pediatr 1999; 134: 740–748. 8 Buckley RH, Schiff SE, Schiff RI et al. Hematopoietic stem- population due to pre-transplant cytoreduction, skin lesions cell transplantation for the treatment of severe combined and/or delayed engraftment and therefore increased immunodeficiency. New Engl J Med 1999; 340: 508–516. microbial colonization and morbidity. Rapid engraftment, 9 Fischer A, Landais P, Friedrich W et al. European experience and therefore very low rates of infection achieved by MUD of bone marrow transplantation for severe combined immuno- transplantation, may be the critical factor for the increased deficiency. Lancet 1990; 336: 850–854. survival of these patients. 10 O’Reilly RJ, Keever CA, Small TN et al. The use of HLA- Together, these data promote the idea that adhering to non-identical T cell-depleted marrow transplants for correc- one method of BMT for immunodeficiency may not lead to tion of severe combined immunodeficiency disease. Immuno- further significant improvement. Rather, selecting the most def Rev 1989; 1: 273–309. successful modality of therapy for the various subgroups 11 Buckley RH, Schiff SE, Sampson HA et al. Development of immunity in human severe primary T-cell deficiency follow- of patients with immunndeficiency seems more plausible. − + ing haploidentical bone marrow stem cell transplantation. J Thus, patients with T B SCID and ADA deficiency might Immunol 1986; 136: 2398–2407. benefit from haploidentical depleted marrow, while patients 12 Dror Y, Gallagher R, Wara DW et al. Immune reconstitution with CID and Omenn’s syndrome may be best served by in severe combined immunodeficiency disease after lectin- a MUD transplant. treated, T cell-depleted haplocompatible bone marrow transplantation. Blood 1993; 81: 2021–2030. 13 Friedrich W, Goldmann SF, Ebell W et al. Severe combined Note added in proof immunodeficiency; treatment by bone marrow transplantation Since the completion of data collection, patient 15 in 15 infants using HLA-haploidentical donors. Eur J Pediatr developed multiple exacerbations of GVHD, involving 1985; 144: 125–130. 14 Rumelhart SL, Trigg ME, Horowitz SD et al. Monoclonal mainly the skin and liver and eventually resulting in graft antibody T cell-depleted HLA-haploidentical bone marrow failure. The patient’s parents admittedly discontinued transplantation for Wiskott–Aldrich syndrome. Blood 1990; prednisone treatment against medical advice, probably con- 75: 1031–1035. tributing to this outcome. Rescue with cord blood transplan- 15 Fischer A, Friedrich W, Fasth A et al. Reduction of graft fail- tation failed and the patient died of multi-organ failure. ure by a monoclonal antibody (anti-LFA-1 CD11a) after HLA

Bone Marrow Transplantation Matched unrelated BMT for CID I Dalal et al 621 nonidentical bone marrow transplantation in children with CD4− CD8− T cell subpopulation expressing high levels of IL- immunodeficiencies, osteopetrosis and Fanconi’s anemia: a 5 in Omenn’s syndrome. Clin Exp Immunol 1994; 95: 14–21. European group for immunodeficiency/European Group for 27 Filipovich AH, Shapiro RS, Ramsay NKC et al. Unrelated Bone Marrow Transplantation report. Blood 1991; 77: 249– donor bone marrow transplantation for correction of lethal 256. congenital immunodeficiencies. Blood 1992; 80: 270–276. 16 Arpaia E, Shahar M, Dadi H et al. Defective T cell receptor 28 Kernan NA, Bartsch G, Ash RC et al. Analysis of 462 trans- signaling and CD8+ thymic selection in humans lacking Zap- plantation from unrelated donors facilitated by the national 70 kinase. Cell 1994; 76: 947–958. marrow donor program. New Engl J Med 1993; 328: 593–602. 17 Sharfe N, Dadi HK, Shahar M et al. Human immune disorder 29 Fischer A, Durandy A, de Villartay JP et al. HLA-haploident- arising from mutation of the ␣ chain of the interleukin-2 ical bone marrow transplantation for severe combined immun- receptor. Proc Natl Acad Sci USA 1997; 94: 3168–3171. deficiency using E rosette fractionation and cyclosporin. Blood 18 Alarcon B, Regueiro JR, Arnaiz-Villena A et al. Familial 1986; 67: 444–449. defect in the surface of expression of the T-cell receptor CD3 30 Buckley RH, Schiff SE, Schiff RI et al. Haploidentical bone complex. New Engl J Med 1988; 319: 1203–1208. marrow transplantation in human severe combined immuno- 19 Sharfe N, Shahar M, Roifman CM. An interleukin-2 receptor deficiency. Semin Hematol 1993; 30 (Suppl. 4): 92–104. ␥ chain mutation with normal thymus morphology. J Clin 31 Van Leeuwen JEM, van Tol MJF, Joosten AM et al. Relation- Invest 1997; 100: 3036–3043. ship between patterns of engraftment in peripheral blood and 20 Hirschhorn R. Overview of biochemical abnormalities and immune reconstitution after allogeneic bone marrow transplantation for (severe) combined immunodeficiency. Blood molecular genetics of adenosine deaminase deficiency. Pediatr 1994; 94: 3936–3947. Res 1993; 3 (Suppl. ): S35–S41. 32 Wijnaendts L, LeDeist F, Griscelli C et al. Development of 21 Fischer A, Griscelli C, Friedrich W et al. Bone marrow trans- immunologic functions after bone marrow transplantation in plantation for immunodeficiencies and osteopetrosis: Euro- 33 patients with severe combined immunodeficiency. Blood pean survey: 1968–1985. Lancet 1986; 2: 1080–1083. 1989; 74: 2212–2219. 22 Petersdorf EW, Longton GM, Anasetti G et al. The signifi- 33 Hayward AR, Murphy S, Githens J et al. Failure of a pan- cance of HLA-DRB1 matching on clinical outcome after reactive anti-T cell antibody, OKT3, to prevent graft-versus- HLA-A, B, DR, identical unrelated donor marrow transplan- host disease in severe combined immunodeficiency. J Pediatr tation. Blood 1995; 86: 1606–1613. 1982; 100: 665–668. 23 Hansen JA, Clift RA, Thomas ED et al. Histocompatibility 34 Morgan G, Linen DC, Knott LT et al. Successsful haploident- and marrow transplantation. Transplant Proc 1979; 11: ical mismatched bone marrow transplantation in severe com- 1924–1929. bined immunodeficiency: T cell removal using CAMPATH-1 24 Storb R, Deeg HJ, Whitehead J et al. Methotrexate and cyclos- monoclonal antibody and E-rosetting. Br J Haematol 1986; porin compared with cyclosporin alone for prophylaxis of 62: 421–430. acute graft-versus-host disease after marrow transplantation 35 Fischer A, Landais W, Friedrich W et al. Bone marrow trans- for leukemia. New Engl J Med 1986; 314: 729–735. plantation (BMT) in Europe for primary immunodeficiencies 25 Primary immunodeficiency diseases. Report of a WHO scien- other than severe combined immunodeficiency: a report from tific group. Clin Exp Immunol 1995; 99: 1–24. the European Group for BMT and the European Group for 26 Melamed A, Cohen A, Roifman CM. Expansion of CD3+ Immunodeficiency. Blood 1994; 83: 1149–1154.

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