Pediatr Transplantation 2015
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Pediatric Transplantation DOI: 10.1111/petr.12538
Hematopoietic stem cell transplant for hyper-IgM syndrome due to CD40L defects: A single-center experience Al-Saud B, Al-Mousa H, Al-Ahmari A, Al-Ghonaium A, Ayas M, Alhissi S, Al-Muhsen S, Al-Seraihy A, Arnaout R, Al-Dhekri H, Hawwari A. (2015) Hematopoietic stem cell transplant for hyper-IgM syndrome due to CD40L defects: A single-center experience. Pediatr Transplant, 00: 1–6. DOI: 10.1111/petr.12538.
Bandar Al-Saud1,2, Hamoud Al-Mousa1,2, Ali Al-Ahmari2,3, Abdulaziz Al-Ghonaium1, Mouhab Ayas3, Safa Alhissi4, Saleh Al-Muhsen1,5, Amal Al-Seraihy3, Rand Arnaout1,2, Hasan Al-Dhekri1 and Abbas Hawwari4
Abstract: HIGMI is a disease with a high risk for morbidity and mortality. HSCT has been shown to be a curative option. This study retrospectively reviewed and analyzed data from five patients who received HSCT at King Faisal Specialist Hospital & Research Centre (KFSH&RC) in Riyadh, Saudi Arabia, between 2005 and 2013. Five patients with HIGMI syndrome underwent HSCT at a median age of 41 months (range, 9–72 months). The median time from diagnosis to transplantation was 30 months (range, 5–58 months). For all five patients, the donors were HLA-identical siblings. In three patients, the conditioning regimen was composed of BU and CY. Fludarabine and melphalan with either ATG or alemtuzumab was used in two patients. For GVHD prophylaxis, cyclosporine was used in two patients, and the combination of cyclosporine and MTX was used in three patients. The survival rate was 100%, with a median follow-up of 69 months (range, 13–100 months). All patients engrafted. Two patients developed acute GVHD. Four patients showed complete immune recovery with positive CD40L expression in activated T cells and discontinued IVIG replacement. HSCT in early stage from an HLA-matched sibling donor is potentially effective at curing the disease.
1
Section of Pediatric Allergy/Immunology, Department of Pediatrics, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia, 2 Colleges of Medicine, Alfaisal University, Riyadh, Saudi Arabia, 3Department of Pediatric Hematology/ Oncology, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia, 4Department of Genetics, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia, 5Department of Pediatrics, College of Medicine, King Saud University, Riyadh, Saudi Arabia Key words: primary immunodeficiency – neutropenia – cryptosporidium – stems cell transplantation Bandar Al-Saud, MD, Section of Allergy and Immunology, Department of Pediatrics, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354 Riyadh 11211, MBC-58 Saudi Arabia Tel.: +96614647272 ext: 39038 Fax: +96614427784 E-mail:
[email protected] Accepted for publication 20 May 2015
Hyper-IgM syndrome (HIGM) is a rare type of primary immunodeficiency disease that was first described in 1961 (1, 2). It is most commonly found in boys and is often characterized by
Abbreviations: ATG, antithymocyte globulin; BM, bone marrow; BMT, bone marrow transplant; BU, busulfan; CsA, cyclosporine A; CSR, class-switch recombination; CY, cyclophosphamide; G-CSF, granulocyte colony-stimulating factor; GVHD, graft-versus-host disease; HIGMI, hyper-IgM syndrome I; HSCT, hematopoietic stem cell transplantation; IVIG, intravenous immunoglobulin; MTX, methotrexate; PCR, polymerase chain reaction; PJP, Pneumocystis jiroveci pneumonia; PMA, phorbol 12-myristate 13-acetate; SCT, stem cell transplantation; SHM, somatic hypermutation; STR, short tandem repeat; VOD, veno-occlusive disease.
X-linked inheritance (3). The X-linked form of HIGM is also known as HIGMI, and CD40 ligand (CD40L) gene defects were found to be responsible for this type of hyper-IgM syndrome (4–8). CD40L is expressed in activated T cells and interacts with CD40 on B cells, leading to immunoglobulin isotype CSR and SHM (9). Therefore, a defect in this mechanism results in markedly decreased serum levels of IgG and IgA and elevated or normal levels of IgM. Moreover, CD40-CD40L interactions are important for Tcell activation and function (10). Patients with HIGMI present with a spectrum of clinical manifestations, including recurrent infections with bacteria, viruses, fungi, and parasites. Patients are more susceptible to opportunistic infections, such as PJP and Cryptosporidium, the latter of 1
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which causes protracted diarrhea and can lead to failure to thrive, sclerosing cholangitis, cirrhosis, and liver failure. Other associated features include severe neutropenia, oral ulcers, autoimmunity, and malignancy (11, 12). Patients with HIGMI are universally managed with prophylactic immunoglobulin replacement and PJP prophylaxis. G-CSF is used in patients with severe neutropenia. Patients with severe infectious illnesses may require prolonged courses of antifungal agents or antibiotics. Patients with protracted diarrhea are tested for Cryptosporidium infection and treated with antibiotics; in addition, these patients may also require aggressive nutritional support or total parenteral nutrition (13). Despite the close follow-up and aggressive management of these patients, morbidity and mortality remain high, and in one series, the survival to 25 yr of age was 20% (11). Stem cell transplant therapy was therefore recently tested as a potential to cure for this disease (14, 15), but only a few single-center studies have been published (16–18). In addition, a European survey was published approximately a decade ago (19), but the available publications are otherwise limited to case reports (20–36). This report represent a single-center experience of HSCT in a cohort of five patients with HIGM I syndrome. Methods Patients This is a retrospective study of five patients with HIGMI who underwent HSCT at our institution between 2005 and 2013 with a median follow-up of 69 months (range, 13– 100 months). Written informed consent to the transplantation was obtained from the parents of each patient, and the study was approved by the institutional research review board. Data of interest were collected from the patients’ medical records. The five patients belonged to three families. Two families (family 2 and family 3) each included two patients (2a, 2b, 3a, and 3b).
The patients’ clinical characteristics are described in Table 1. The median age of diagnosis was 14 months (range, 4–15 months). Patient 1 had no significant manifestations of the disease; however, his brother died in the first year of life from recurrent infections. In contrast, the four other patients showed significant disease manifestations (Table 1). Three patients suffered protracted diarrhea (patients 2a, 2b and 3b), two patients had sclerosing cholangitis (3a and 3b), and one patient was ventilated for a severe pneumonia (2b). Patient 2b also had chronic active hepatitis secondary to maternal transmission of the hepatitis B virus. PJP or Cryptosporidium was not isolated from any of the patients before transplantation. All five patient diagnoses were confirmed by demonstrating the absence of CD40L expression on cells stimulated with a calcium ionophore (ionomycin, 300 ng/ mL; Sigma, St. Louis, MO, USA) and PMA (15 ng/mL; Sigma) as previously described by O’Gorman et al. (37). Data were acquired on a FACS-Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and analyzed using CellQuest software. CD40L expression was analyzed on in vitro-activated CD8-negative cells. All patients received IVIG replacement and PJP prophylaxis before HSCT (Table 1). Three patients required subcutaneous injections of G-CSF before HSCT for persistent severe neutropenia.
Conditioning regimen and GVHD prophylaxis The pretransplant conditioning regimen included the intravenous administration of BU at a dose of 4 mg/kg for four days, from transplant day 10 to day 7 (total dose 16 mg/kg). Three patients were administered intravenous CY at 50 mg/kg on day 5 to day 2 (total dose 200 mg/kg) (Table 2). Two patients received fludarabine at 120 mg/m2 on day 7 to day 3 and melphalan at 140 mg/m2 on day 2. One patient received rabbit ATGs (ATG-Fresenius) at 10 mg/kg daily on day 2 to day +2. One patient received alemtuzumab at 1 mg/kg on day 7 to day 3. All patients received GVHD prophylaxis that consisted of CsA and MTX at standard doses for three patients and CsA alone for two patients (Table 2). In the absence of GVHD, CsA was tapered after 100 days. For all five patients, the grafts were non-manipulated BM from HLA-identical siblings.
Supportive care All patients were isolated in HEPA-filtered or laminar flow facilities during the transplantation. None of the patients
Table 1. Pretransplantation patient status
Patient
Sex
Age at diagnosis (months)
1 2a
Male Male
11 4
2b
Male
15
3a
Male
15
3b
Male
14
*By flow cytometry.
2
Clinical presentation Failure to thrive, neutropenia Protracted diarrhea, neutropenia, G6PD deficiency Pneumonia, ventilated, protracted diarrhea, chronic active hepatitis B, Failure to thrive, neutropenia, G6PD deficiency Recurrent upper respiratory tract infections, sclerosing cholangitis, failure to thrive, neutropenia Recurrent upper respiratory tract infections, protracted diarrhea, sclerosing cholangitis, failure to thrive, neutropenia
Isolated organisms
Treatment
CD 40L expression*
Bacrim/IVIG Pentamidine/IVIG
Absent Absent
Hepatitis B infection
Pentamidine /IVIG/ Lamivudine
Absent
Pseudomonas aeruginosa (cellulitis), salmonella (stool) Salmonella (stool)
Bacrim/IVIG
Absent
Bacrim/IVIG
Absent
received anticryptosporidial prophylaxis or treatment at the time of transplantation. All blood products were irradiated and leukocyte-filtered. All patients received acyclovir prophylaxis at a dose of 500 mg/m2/dose intravenous every eight h for 28 days starting 3 pre-BMT if the donor was CMV positive. Engraftment was defined as an ANC >0.5 9 109/L and a platelet count of >20 000 9 106/L for three consecutive days. Chimerism was analyzed using STR. CD40L expression was assessed through flow cytometry using the procedure described above.
Cryptosporidium, adenovirus gastroenteritis Adenovirus gastroenteritis, herpes zoster, renal impairment related to medication CsA
12
22
Yes (Skin, Gut, Liver)
DNA sequencing
FLU, fludarabine; MEL, melphalan; aGVHD, acute graft-versus-host disease.
3.17 72 3b
58
HLA-id sibling/BM
8.35
No 16 13 CsA 6.8 HLA-id sibling/BM 61 3a
46
FLU/MEL/ Alemtuzumab FLU/MEL/ATG
9.44
CMV, pansinusitis Yes (Gut) 21 16 CsA/MTX 6.03 NA HLA-id sibling/BM BU/CY 26 2b
11
18.25 9 2a
5
BU/CY
HLA-id sibling/BM
5.99
CsA/MTX
20
31
No
Rhinovirus A
Alive and well (1 yr 1 m) Alive and well (4 yr 10 m) Alive and well (5 yr 9 m) Alive and not cured (6 yr 5 m) Alive and well (8 yr 4 m) No No 16 16 CsA/MTX 6.99 36.3 HLA-id sibling/BM 41 1
30
BU/CY
Type of transplant Patient
Time from diagnosis to HSCT (months) Age at HSCT (months)
Table 2. Patients’ transplantation data
Conditioning regimen
Nucleated cells (108/kg)
CD34+ (106/kg)
GVHD prophylaxis
Neutrophil engraftment (day)
Platelet engraftment (day)
aGVHD
Infection and complications
Outcome (years post-HSCT)
HSCT for hyper-IgM syndrome I
Genomic DNA was extracted from whole blood samples obtained from patients and their families using a Gentra Puregene Blood Extraction kit (Qiagen, Valencia, CA, USA). Promoter, exons, and both 50 and 30 untranslated regions from the CD40L gene were amplified by PCR using HotStarTaq DNA Polymerase (Qiagen). Primers were designed from within the intron regions to span the splice sites and the exon/intron boundaries. In addition, M13 sequences were attached to the 50 end of each primer to enable forward and reverse sequencing. Amplifications were performed using touchdown PCR. The extension temperatures ranged from 68 to 55 °C (primer sequences are available and will be provided upon request). PCR products were purified using an AgencourtRAMPureR XP PCR Purification Kit (Beckman Coulter, Beverly, MA, USA) and sequenced with a BigDyeR Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the M13 Forward primer. After sequencing, the DNA was purified using an AgencourtRCleanSeqR Kit (Beckman Coulter) and processed with a 3730XI DNA Analyzer (Applied Biosystems). All kits were used in accordance with the manufacturers’ instructions. Sequencing data were analyzed for mutations using SeqMan II software (DNA Star Inc, Madison, WI, USA). Samples with mutations were confirmed by sequencing the reverse DNA strand using the M13 Reverse primer. Once a mutation was found, DNA from the patient’s siblings and parents, if available, were sequenced to determine the heterozygote (carrier) status of each family member. To rule out the possibility that the mutations detected were unreported normal variations within the Saudi population, 250 DNA samples from normal individuals from the blood bank of the King Faisal Specialist Hospital & Research Centre were sequenced.
Statistical analysis For this study, all statistical analyses were performed using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). Descriptive statistics for continuous variables are reported as the median, and categorical variables are summarized as frequencies and percentages.
Results
Five patients with HIGMI syndrome underwent HSCT at a median age of 41 months (range, 9–72 months). The patients were treated at a median of 30 months post-diagnosis (range, 5–58 months). The median dose of CD34-positive cells was 6.99 9 106/kg (range, 5.99– 9.44 9 106/kg) per kg of recipient body weight (Table 2). All patients had initial engraftment, as 3
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shown in Table 2. The median time to neutrophil recovery was 16 days (range, 12–20 days). The median time to an unsupported platelet count was 21 days (range, 16–31 days) (Table 2). One patient developed acute GVHD grade 2 in the gut only, and a second patient developed acute GVHD grade 2 in the skin, gut, and liver. No patients developed chronic GVHD or VOD. One patient developed a CMV infection, which was resolved with specific therapy. Cryptosporidium was isolated from one patient post-SCT (Table 2). The median post-transplant chimerism rates for donor percentage of lymphocytes and myeloid cells were 79% (range, 4–100%) and 72% (range, 0–100%), respectively. The median CD40L expression in activated T cells was 49% (range 1–58%) (Table 3). Four patients discontinued IVIG replacement. Six month after discontinuing IVIG replacement, three patients showed a positive specific vaccination response (Table 3). The overall survival and cure rates were 100% and 80%, respectively, at a median follow-up of 69 months (range, 13–100 months). One patient (3a) is not cured due to graft failure. We only tested two patients (Pt 1 and Pt 2a) for mutations in the CD40L gene because DNA samples were not available for the other three patients. Patient 1 has a 2-bp deletion (Fig. S1a) in Exon 5 (c.719_720delAT), resulting in a frame shift and stop codon shifted three amino acids downstream (N240SfsX3). This mutation results in a truncated protein. Patient 2a has a mutation in the splice site (Fig. S2a) in the second bp immediately after the end of Exon 1 (c.156+2 T>A). This mutation prevents Exon 1 from splicing to any subsequent exons and thus prevents the formation of a functional protein. Alternatively, Intron 1, which has a stop codon within the splice site (gta aga tga), may be retained (Fig. S2a). Patient 2b is expected to have the same mutation as his brother (Pt 2a). Both mutations were novel and
were predicted to cause disease development using the Mutation Tasting (Figs. S1b and S2b) website (http://www.mutationtaster.org/). Discussion
HIGMI syndrome is a combined form of primary immunodeficiency. The standard care for patients has been IVIG replacement, PJP prophylaxis, and a close follow-up and management of infections and related complications. Nevertheless, patients show a high risk for morbidity and mortality (11). Therefore, HSCT has become a viable option for curing this disease, especially if the patient has a full-match donor. In this study, five patients with HIGMI syndrome underwent HSCT and showed a survival rate of 100% with a complete cure (positive expression of CD40L and discontinued use of IVIG) in four of five patients (80%). Patient 3a had engrafted initially with a chimeric study for both granulocytes and lymphocytes of 21% and 18% donor cells, respectively, at six months post-HSCT, but subsequently, he lost his graft. Nevertheless, he remained clinically well. The failure of the graft is probably related to the use of reduced intensity conditioning. A second HSCT in this patient is considered to be a high risk; this option was discussed with the parents. In the meantime, patient will continue to be under monitoring with close follow-ups in the clinic. A previous Japanese study reported rates of 71% survival and 57% complete cure (16), and a Newcastle study reported rates of 50% and 25%, respectively, for survival and complete cure (18). The improved outcome in our patients may be due to their younger age at transplantation. Our study showed a median age of 41 months, whereas the Newcastle and Japanese studies reported median ages of 69 and 180 months, respectively. Moreover, the European survey of 38 patients from eight centers published in 2004 by Gennery et al.
Table 3. Immune reconstitution profile post-HSCT Donor chimerism* Patient
Time postHSCT (months)
M%
1 2a 2b 3a 3b
9 40 64 75 94
100 56 75 0 72
L%
IgG (mg/dL)
IgA (mg/dL)
IgM (mg/dL)
Immunoglobulin substitution
Specific vaccination response
CD40L expression on activated CD4+ T cells %†
In vitro T-cell proliferation‡
100 79 59 4 90
1130 700 1210 1240 1040
115 106 243 A Mutation: Electrograms of the family members sequenced containing the splice site mutation as indicated. (b) The output of the “Mutation Tasting” for P2a predicting program is shown to indicate the effect of the mutation as disease causing. Table S1 Summary and outcomes of transplantation in the published studies to date.