Hematopoietic Cell Transplantation Cures Adenosine Deaminase 2 Deficiency: Report on 30 Patients

We conducted an investigator-driven retrospective international non-interventional multicenter study on HCT for DADA2. Invitations to participate were sent to the physicians allied to the DADA2 Foundation, the European Group for Blood and Marrow Transplantation (EBMT), and the European Society for Immunodeficiencies (ESID). We also invited all authors of published single case reports on HCT in DADA2 to participate in the study. Data collection began after the Second Inaugural International Conference on DADA2 hosted by the DADA2 Foundation on November 9, 2018. The study was approved by the Ethics Committee of Leuven University Hospitals (study number S63982). The study was performed in accordance with the principles of the Declaration of Helsinki. The authors assume responsibility for the accuracy and completeness of the data and analyses and for fidelity to the study protocol.

The criteria for patient inclusion in the study were as follows: (1) genetic diagnosis of DADA2 and/or clinical findings consistent with DADA2 and plasma ADA2 activity level in the deficient range and (2) HCT performed with a follow-up time for survivors of at least 3 months after HCT. All participating physicians completed a questionnaire. All patients or their guardians gave written informed consent for data collection. Patients for whom incomplete data were obtained (indication for HCT, age at HCT, total nucleated cell dose or CD34 + stem cell dose, stem cell donor, conditioning regimen, graft-versus-host disease (GvHD) prophylaxis, time to engraftment, graft failure, conditioning for subsequent HCTs, chimerism) were excluded from the study.

Neutrophil engraftment was defined as the first of three consecutive days with a neutrophil count ≥ 0.5 × 10⁹/L and platelet engraftment as the first of seven consecutive days with a platelet count ≥ 20 × 10⁹/L without platelet transfusion in the prior 7 days. Full donor chimerism was defined as ≥ 95% donor cells in myeloid or whole-blood fractions. The type of test was at the discretion of the transplant center. Primary and secondary graft failures (GF) were defined according to EBMT guidelines. Second (or third) HCT was defined as the infusion of hematopoietic progenitor cell containing product, according to CIBMTR, regardless of conditioning regimen. The diagnosis and grading of acute and chronic GvHD were based on international standard criteria [22]. Transplant regimen, GvHD prophylaxis, antimicrobial prophylaxis, and pre-emptive treatment were chosen according to center preferences. Preparative regimens were classified as reduced intensity conditioning (RIC) if the dose of alkylating agents or TBI is reduced by at least 30% from a myeloablative conditioning (MAC) approach. A total dose of treosulfan > 30 g/m² was considered MAC whether or not combined with another alkylator, whereas a total busulfan dose < 8 mg/kg and fludarabine-melphalan regimens were considered RIC.

Kaplan–Meier curves were plotted for overall survival (OS) and GvHD-free relapse-free survival (GRFS), and p values were obtained for Mantel-Cox log-rank tests performed with Graph-Pad Prism Software version 9. Values of p < 0.05 were considered statistically significant. GvHD relapse-free survival was calculated as the time from first HCT until the first occurrence of any of the following events: grades 3–4 aGvHD or moderate/severe cGvHD GF, disease relapse (poor graft function/graft failure with DADA2 disease relapse requiring repeat transplant), or death. HCTs inadvertently using affected donors were excluded. The cumulative incidence of GvHD and GF were also calculated using competing risk analysis, using R, for all HCT procedures, but excluding HCTs from affected donors.

We included 30 DADA2 patients undergoing HCT between 2000 and 2020 in this study. Four other patients were excluded due to incomplete data sets. Patients underwent HCT at 21 different centers from 12 countries in Europe and North America. Twenty of the patients have been reported before [4, 5, 7, 9, 19‐21, 23‐27]. Median age at disease onset was 2.25 years (range: birth to 16 years). Median age at genetic diagnosis was 12 years (range: 2–28 years) (Table 1). DADA2 diagnosis was confirmed at the molecular level in all patients, by demonstrating the presence of biallelic pathogenic ADA2 variants. Plasma ADA2 activity was assessed before HCT in 18 patients and was low in all cases. Twenty-six patients harbored known pathogenic ADA2 mutations. The R169Q variant was the most common mutation, found in 15 patients. Four patients harbored novel mutations, all with combined annotation-dependent depletion (CADD) scores above the mutation significance cutoff (MSC) for this gene; all these variants were private or had a MAF < 10^–6 [28], strongly suggestive of a deleterious effect. Three of these patients were tested for ADA2 enzyme activity, which was found to be low or absent (Table 2). Patient and HCT characteristics are summarized in Table S1 in the Online Supplement.

PRCA was documented in 8/30 patients, isolated neutropenia in 6/30, combined RCA and neutropenia in 5/30, severe aplastic anemia in 1/30, severe lymphopenia in 1/30, anemia and neutropenia in 2/30, autoimmune hemolytic anemia (AIHA) in 2/30, and pancytopenia in 5/30 patients, at presentation. Six patients had a hematological malignancy or myelodysplasia and received HCT as part of the therapeutic approach (P4, P9, P17, P21, P26, and P27). Twenty-nine patients had received at least one immunosuppressive treatment prior to HCT including 13 patients who received at least 3 lines of immunosuppressive medications. Fourteen patients had received anti-TNF agents before HCT, without effect on cytopenias. P6 received single agent etanercept, which failed to reverse neutropenia; a combination of adalimumab, cyclosporine, and low-dose prednisone resulted in normal neutrophil counts for 6 months prior to HCT. Two patients received pre-HCT Interleukin-1 receptor antagonist (anakinra), without amelioration of cytopenias or immune dysregulation. Seven patients received granulocyte colony stimulating factor (G-CSF) for neutropenia, with no response.

IgG levels were low in 12/27 tested patients, IgA levels were low in 13/26, and IgM levels were low in 15/27 tested patients. Recurrent infections were reported in 17 of the 30 patients, mostly viral infections (in 14/17). Herpesvirus infections predominated, with three patients suffering from recurrent herpes zoster, one having protracted CMV infection, one with severe chicken pox, one with recurrent cutaneous HSV-1, two with HHV6 viremia, and four with EBV viremia (2 transient, 1 chronic, and 1 in the context of lymphoproliferative disease). Warts (n = 4), and mollusca contagiosum (n = 4) were also reported. Immunoglobulin substitution treatment was administered to 15 of the 30 patients before HCT, and splenomegaly was reported in 23/30 patients. Fifteen of 30 patients had reported vasculitis prior to HCT (Table 1): 9 had livedo racemosa, 3 had polyarteritis nodosa (PAN) (P6 with livedo, P7 with ICH and livedo, P11 isolated), three patients had ischemic stroke (P2, 26, 27), and four had intracranial hemorrhage (P7,10, 27, P12).

The indications for HCT were cytopenia with or without immunodeficiency and/or lymphoproliferation or malignancy (Table 3). The median age at HCT was 9 years (range: 2–28). Six of the 30 patients had received HCT before the description of DADA2 in 2014. Two patients (P16, P29) were inadvertently transplanted using affected siblings as donors and received salvage second HCT from unrelated donors. A total of 38 HCTs were performed for 30 patients. Twenty patients received MAC (P12 with two subsequent HCTs), eight received RIC, and two received NMA conditioning for the final curative transplant (Tables 3 and S1). The most commonly used regimen (in 11 patients) was treosulfan/fludarabine ± thiotepa with antithymocyte globulin (ATG) or alemtuzumab. Serotherapy was used in 25/30 patients: ATG in 10 (rabbit ATG in all except horse ATG in P6 and P18) and alemtuzumab in 15 patients. The source of the stem cells for the final transplant was peripheral blood (PB) for 10 and bone marrow (BM) for 20 patients. Donors were HLA-matched related (n = 4), HLA-haploidentical sibling (n = 2), 10/10 HLA-matched unrelated (n = 16), and 9/10 HLA-mismatched unrelated (n = 8) for the final transplant (Tables 3 and S1). Two of the six related donors carried heterozygous ADA2 mutations (P3, P22), three were healthy (P1, P2, P21), and one donor was of unknown status (P4).

Median engraftment was d + 20 for neutrophils and d + 23 for platelets. In 25/28 patients receiving grafts from unaffected donors, full donor chimerism was achieved by d + 30. Overall survival at 2 years was 97%, with a median follow-up of survivors of 2 years (range: 0.5–16), accounting for 1545 patient-months post-HCT (Fig. 1A). P24 passed away 2 months post-HCT due to respiratory failure secondary to parainfluenza pneumonia despite full donor chimerism and treatment with steroids and etanercept for the suspicion of immune reconstitution inflammatory syndrome. Viral reactivation occurred in 17/30 patients (56%). Adenovirus, CMV, and BK were most frequent involved, each observed in six patients. GRFS was 73% at 2 years, with all events occurring within the first year post-HCT. Of those transplanted with affected donors (n = 2), P16 had primary graft failure requiring salvage HCT, and P29 had obtained near-full donor chimerism but failed erythroid line engraftment and remained with PRCA. Three patients experienced graft failure. P18 required two and P19 one subsequent HCT for secondary GF. The latter two patients were found to have aggregates of CD8 + T cells in their BM with low donor T cell chimerism (9% and 0%) (Fig. 1B, Fig. 2B). P28 required a second HCT likely due to low stem cell dose (1.4 × 10⁶/kg). P12 originally received MAC, but subsequently required two unconditioned HCTs due to drop in whole blood donor chimerism to 30% and new-onset RCA and agranulocytosis unresponsive to G-CSF. Cumulative incidence of aGvHD grades 2–4 was 20% at 1 year. Moderate-severe chronic GvHD developed in 2/30 patients (7%) at 1 year (Fig. 2A). P1, P19, and P24 developed sinusoidal obstruction syndrome (SOS), which responded to fluid restriction and diuresis in P1 and P19 and to defibrotide in P24. All three patients with SOS received either high-dose cyclophosphamide or myeloablative busulfan.

HCT cured the hematological phenotype in all patients, as confirmed at the most recent follow-up visit (Fig. 1C). No central vascular events were reported after engraftment (Fig. 1C). ADA2 plasma enzyme activity normalized in 16/17 patients tested post-HCT (Table 2). This normalization occurred as early as d + 12, coinciding with the reappearance of monocytes in the PB, as demonstrated by the prospective monitoring of plasma ADA2 enzyme activity in one patient [29]. At last follow-up, 29 patients were still alive, and 28 of these patients displayed full donor chimerism. Donor chimerism fell to 55% in P25 but remained stable over several months, with no evidence of disease. Transient hematological autoimmunity post-HCT was reported in three patients: ITP in two and AIHA in one. This autoimmunity responded to various treatment regimens (intravenous immunoglobulins (IVIG)/steroids/sirolimus/rituximab/bortezomib/romiplostim). Five patients are still on IVIG (one less than a year after HCT, one for mild bronchiectasis, and three post-rituximab treatment).