Stem cell transplants for patients with X-linked agammaglobulinemia

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Clinical Immunology 107 (2003) 98 –102

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Stem cell transplants for patients with X-linked agammaglobulinemia Vanessa Howard,a Laurie A. Myers,b David A. Williams,c Gary Wheeler,d E. Victoria Turner,e John M. Cunningham,f and Mary Ellen Conleya,g,* a

Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA b Department of Pediatrics, Duke University School of Medicine, Durham, NC 27708, USA c Department of Pediatrics, Cincinnati Children’s Hospital, Cincinnati, OH 45241, USA d Department of Pediatrics, University of Arkansas School of Medicine, Little Rock, AR 72202, USA e Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA f Department of Hematology–Oncology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA g Department of Pediatrics, University of Tennessee College of Medicine, Memphis, TN 38105, USA Received 26 November 2002; accepted with revision 31 January 2003

Abstract Six young patients with X-linked agammaglobulinemia and proven mutations in Btk were treated with cord blood or bone marrow transplants from HLA-matched siblings. Complete blood counts, serum chemistries, serum immunoglobulin concentrations, lymphocyte cell surface markers, and physical findings were evaluated at 3- to 5-day intervals for the first 2 weeks after transplant and then every 3 to 6 months. The first three patients were not given any preparative regimen or antirejection drugs and at 24 to 42 months posttransplant these patients have shown no benefit or harm related to the transplants. The second three patients were not given a preparative regimen but were treated with cyclosporine A (70 days) and mycophenolate mophetil (28 days) after transplant. Two of these patients have developed normal sized, nontender cervical lymph nodes 3 to 12 months after transplant but none of the three patients have shown an increase in serum IgM or an increase in the number of peripheral blood B cells. It is likely that successful engraftment will require more aggressive immunosupressive medications. © 2003 Elsevier Science (USA). All rights reserved. Keywords: B-lymphocytes; Agammaglobulinemia; Hematopoietic stem cell transplantation; Lymphoid tissue; Transplantation conditioning; Graft rejection

Introduction The majority of patients with X-linked agammaglobulinemia (XLA) are recognized to have immunodeficiency when they are hospitalized for a major infection at 1 to 5 years of age [1]. Laboratory studies generally show very low but detectable serum IgG and serum concentrations of IgM and IgA that are less than 20 mg/dl [2,3]. The most distinctive laboratory feature of XLA is the marked paucity or absence of B cells in the peripheral circulation [4,5]. Most patients with XLA do well on standard therapy, which

* Corresponding author. University of Tennessee College of Medicine, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Fax: ⫹1-901-495-3977. E-mail address: [email protected] (M.E. Conley).

consists of gammaglobulin replacement and aggressive use of antibiotics, but about 10% of patients have significant problems despite optimum treatment and chronic lung disease is common in older patients [3,6]. In all patients with XLA, treatment is expensive and burdensome. For patients who are doing well, the cumulative cost of gammaglobulin replacement exceeds $ 1 million by the time the patient is 30 to 40 years of age. XLA is caused by mutations in Btk [7,8], a cytoplasmic tyrosine kinase that is activated by cross-linking of the B cell antigen receptor [9,10]. Studies done in females heterozygous for mutations in Btk have shown that B cell precursors with a normal Btk have a strong selective advantage in proliferation, differentiation, or survival compared with B cell precursors with mutant Btk [11,12]. In an animal model of XLA, irradiated Btk-deficient mice demonstrate

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V. Howard et al. / Clinical Immunology 107 (2003) 98 –102

correction of the B cell defect when only a small fraction of the donor marrow comes from a host with normal Btk [13]. Unirradiated Btk-deficient mice that have not received any immunosuppression can be completely corrected if given 2.5 ⫻ 106 bone marrow cells from a congenic donor with normal Btk [A. S. Porpiglia et al., this issue]. These findings suggest that in patients with XLA, the engraftment of a small number of stem cells or B cell precursors from a donor with normal Btk might provide significant clinical benefit. Under some circumstances, donor cells may remain in a patient’s peripheral blood for prolonged periods of time, even in the absence of immunosuppression. Women who have had male offspring may have circulating CD34⫹ Ychromosome-positive stem cells as long as 20 years after the birth of a male baby [14]. Y chromosome cells can also be found in the circulation of females who have been extensively transfused for sickle cell disease or after major trauma [15,16]. Together with the findings showing the selective advantage of Btk-positive cells, these findings suggest that it may be possible to provide patients with XLA with donor-derived B cells in the absence of the toxic preparative regimen typically used for stem cell transplants.

Materials and methods Patients This study was approved by the Institutional Review Board of St. Jude Children’s Research Hospital. The families of patients who were referred for genetic testing were informed of the study if their son appeared to be eligible. The study was described to the family on at least two separate occasions and informed consent was signed immediately prior to entry into the study. Patients were admitted to the hospital for hydration the night before the stem cell infusion. According to standard stem cell procedures, the patients were premedicated with antihistamines and hydrocortisone prior to the stem cell infusion and the infusion was given over a period of 25 to 150 min. The patients were discharged the day after the stem cell transplant and they were seen in the outpatient department three times at 3- to 5-day intervals for the first 15 days after the transplant. In accordance with the protocol, the patients are seen in the outpatient department at St. Jude Children’s Research Hospital at 4, 8, and 12 months after the transplant and then at 6-month intervals for 5 years after the transplant. Laboratory studies Btk mutation detection was performed as previously described [17]. To evaluate expression of Btk protein, peripheral blood mononuclear cells were separated by Ficoll–Hypaque centrifugation and permeabilized with Orthopermafix; 5 ⫻ 105 cells were incubated at room temper-

99

ature with 50 ␮l of rabbit immunoglobulin plus 25 ␮l of a 1:100 dilution of mouse monoclonal anti-Btk (clone 48-2H, kindly provided by Satoshi Tsukada [18]). The cells were washed and then stained with FITC-labeled goat antimouse antibody (Southern Biotechnology, Birmingham AL). Cells within the monocyte gate, as determined by forward and side scatter, were analyzed for Btk protein expression. Other laboratory studies were performed according to standard procedures.

Results The eligibility criteria for the XLA stem cell transplant study were designed to focus on patients who were thought to be most likely to engraft with minimal toxic side effects. The study was limited to patients under 6 years of age who had an HLA-matched sibling less than 8 years of age or an HLA-matched sibling cord blood available which could provide at least 107 cells/kg to the patient. Young patients and donors were selected because the normal number of B cell precursors decreases with age [5,19] and because there is a lower risk of graft vs host disease in young patients who receive stem cells from a young donor [20,21]. All of the patients were well at the time of transplant, all had less than 1% CD19⫹ B cells in the peripheral circulation, and all had proven mutations in Btk (Table 1). The patients were maintained on their routine intravenous gammaglobulin therapy throughout the transplant period. None of the patients were receiving additional medications. The first three patients (Patients 1, 2, and 3) were treated without a preparative regimen and without graft vs host or antirejection therapy. Patient 1 received a cord blood transplant and Patients 2 and 3 were given unmanipulated bone marrow from an older, HLA-matched sibling (Table 2). In the posttransplant period, the patients were examined in the outpatient department at 3- to 5-day intervals for the first 2 weeks. None of the patients developed any changes in clinical status; all remained healthy without malaise, rashes, or diarrhea. Blood studies were done to determine if there were a transient increase in serum IgM or circulating B cells provided by mature B cells from the donor. To monitor for signs of graft vs host disease or rejection, we analyzed serum chemistries and the percentage of activated T cells in the peripheral circulation, as measured by coexpression of CD3 and DR. None of the patients demonstrated any consistent changes in the complete blood count with differential, serum chemistries including liver function tests, serum immunoglobulins, or lymphocyte cell surface markers. Specifically, none of the patients had greater than 2% DR⫹ CD3⫹ cells at any time, nor did any of the patients demonstrate an increase in serum IgM or number of CD19⫹ cells. Blood studies were repeated 4, 8, 16, 32, and 52 weeks after transplant and the patients were examined by us at 16, 32, and 52 weeks posttransplant. None of the patients demon-

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Table 1 Pretransplant evaluation of XLA patients Patient No. 1 Age at diagnosis (in months) Age at transplant (in months) Serum Igs (mg/dL) IgM IgG IgA Percentage of CD19⫹ B cells in circulation Btk proteina Mutation in Btk a

2 41 59

19 1130 ⬍7 0.1 Btk⫺ C506F

14 52 15 727 ⬍7 0.03 Btk⫺ Y223X

Table 2 Characteristics of donor cells Patient No.

Type of HSCTa

Gender of donor

Age of donor in years

Total number of cells infused/kg

1 2 3 4 5 6

CBb BMc BM CB BM BM

M M F M F F

— 7 4 — 5 3

0.37 ⫻ 108 3.87 ⫻ 108 4.2 ⫻ 108 0.41 ⫻ 108 3.93 ⫻ 108 1.02 ⫻ 108

HSCT, hematopoietic stem cell transplant. CB, umbilical cord blood. c BM, unmanipulated bone marrow. b

17 32 10 492 ⬍7 0.53 Btk⫺ W588X

4 40 65 ⬍4 296 ⬍6 0.12 Btk⫺ Intron 6 ⫹5 G3T

5 27 39 ⬍4 927 ⬍6 0.22 Btk⫺ R641C

6 45 61 6 1530 ⬍6 0.01 Btk⫺ Intron 18 ⫺2 A3G

Indirect immunofluourescence was used to stain Btk in monocytes.

strated any signs of benefit or harm from the transplant (Table 3). Because it was felt that the failure of the first three patients to show any signs of engraftment was most likely caused by rejection due to minor histocompatibility differences, the second group of three patients (Patients 4, 5, and 6) were treated with antirejection medications in the posttransplant period. Three days before transplant, the patients were started on oral cyclosporine A with a goal of achieving a serum level of 200 ng/ml. The cyclosporine was maintained for 70 days and then tapered over a period of 6 weeks. Approximately 12 h after the transplant, the patients were started on mycophenolate mofetil (MMF) and this drug was continued for 28 days. The patients were evaluated in the same manner as the first three patients. The patients were allowed to go to preschool or kindergarten while they were receiving immunosuppressive medications. Patients 4 and 6 developed one or more, nontender, 1-cm anterior cervical lymph nodes approximately 3 and 12 months after transplant, respectively; these lymph nodes were not associated with acute infections or changes in serum immunoglobulins or B cell numbers. In Patient 4, the lymph nodes have not changed significantly over the last 12 months. There were no other signs of benefit or harm related to the transplant in the second group of patients.

a

3

Discussion The six stem cell transplants reported in this paper were performed as a pilot study to determine the least toxic regimen that could allow improved antibody production in patients with X-linked agammaglobulinemia. The strong selective advantage of B cell precursors with normal Btk and the fact that donor cells sometimes persist for long periods of time in patients who have not received immunosuppressive medications left open the possibility that partial or complete B cell correction could be seen in patients with XLA who had not received any preparative regimen before transplant. Our results indicate that it is unlikely that patients with XLA who are not given any pretreatment or antirejection therapy will show clinical benefit from HLAmatched sibling stem cell transplants. It is more difficult to interpret the findings in the three patients with XLA who were not given any preparative regimen but who were given antirejection medications for 70 days posttransplant, in part because these transplants were performed more recently and insufficient time has passed to allow complete assessment. Our studies in xid mice, an animal model of XLA, show that improved antibody production can be seen in mice that have received relatively small numbers of donor bone marrow cells and no preparative regimen; however, improved B cell function sometimes took more than 20 weeks to develop [13]. There are no comparable studies in the human that would allow us to estimate the time required to detect some antibody production in the patients. Two of the patients who received antirejection medications have developed normal sized, nontender, cervical lymph nodes. These lymph nodes were not associated with infection or an inflammatory reaction. Although it is possible that they represent a sign of graft vs host disease, we think it is more likely that the nodes are a subtle indicator of some B cell engraftment. Our murine studies indicate that improved serum IgG may be the most sensitive indicator of improved B cell function [A.S. Porpiglia et al., this issue]. Because the patients in this study were maintained on therapeutic doses

V. Howard et al. / Clinical Immunology 107 (2003) 98 –102

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Table 3 Most recent posttransplant evaluation Patient No. 1 Months posttransplant Serum Igs (mg/dL) IgM IgG IgA Percentage of CD19⫹B cells in circulation Development of normal sized cervical nodes

2

3

4

5

6

42

36

24

18

18

12

⬍4 286 11 ⬍1% —

⬍4 1050 ⬍6 ⬍1% —

⬍5 337 ⬍6 ⬍1% —

⬍4 721 ⬍6 ⬍1% ⫹

4 687 ⬍6 ⬍1% —

18 820 ⬍6 ⬍1% ⫹

of IgG replacement, measurement of serum IgG would not be an adequate reflection of endogenous production of IgG. However, it is likely that a more aggressive approach to transplant will be required to provide significant clinical benefit for the patients. Because patients with XLA generally do well when treated with gammaglobulin replacement, it is important to carefully evaluate the risk/benefit ratio when proposing a treatment that might cure the disorder. Preclinical studies indicate that gene therapy approaches, using current retroviral vectors, are unlikely to be successful [22]. However, bone marrow transplants in mice with defects in B cell development suggest that it may not be necessary to make “space” in the bone marrow to achieve donor B cell engraftment [A. S. Porpiglia et al., this issue]. This finding implies that minimally toxic preparative regimens may be sufficient to permit long-term B cell engraftment in patients with XLA. Nonablative stem cell transplants have been used for patients with malignancies and immunodeficiencies [23– 26]. However, the goal in these studies has been to achieve complete donor chimerism of the hematopoietic systems and the preparative regimens for these transplants have included myelotoxic agents. Although many of these transplants have been successful, they have been associated with a substantial risk of graft versus host disease and infection. It is harder to justify these risks in patients with XLA. In XLA, it is unlikely that full chimerism, in which the host hematopoietic system is replaced by donor cells, would be required to provide clinical benefit. Further, the selective advantage of Btk normal cells versus Btk mutant cells suggests that extensive donor engraftment may be unnecessary. Immunosuppressive or immunomodulatory treatments that allow the patient to develop tolerance to donor cells may be sufficient to allow donor engraftment with an acceptable risk/benefit ratio.

Acknowledgments We appreciate the willingness of the patients and their families to participate in these research studies. We thank Judy Rush for secretarial assistance. These studies were supported by Grant AI25129 from the National Institute of

Health, National Cancer Institute Grant P30 CA21765, the Assisi Foundation, American Lebanese Syrian Associated Charities, and by funds from the Federal Express Chair of Excellence.

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