Severe congenital neutropenia (SCN) defines a rare, heterogeneous group of disorders of neutrophil maturation. Prevalence estimates range from 1-9 cases per million individuals (1). More than thirty genes have been identified in which variants impair neutrophil differentiation, though this is not a universal finding (2–4). The most common of these are heterozygous ELANE mutations, accounting for approximately 50-60% of cases of SCN (5, 6). ELANE encodes neutrophil elastase, a serine protease enzyme contained in neutrophil azurophilic granules that hydrolyzes cell surface proteins and is released during the neutrophil activation process (6–8). Approximately two hundred distinct ELANE mutations have been identified, and the correlation of specific mutations to SCN phenotype are beginning to be understood (9, 10).

SCN typically presents with multiple severe infections in infancy and early childhood. These include omphalitis, pneumonia, bacteremia, abscess, and otitis media. Patients also frequently experience aphthous stomatitis and periodontitis. Historically, SCN had an approximate mortality rate of 50% in the first year of life due to sepsis, with a subsequent mortality of 6-7% per year. Since the advent of G-CSF therapy, patients with SCN have experienced a significant improvement in average lifespan and the mortality rate has been reduced to 0.9% per year (11). Patients with ELANE-mediated SCN on long-term G-CSF, however, have a cumulative risk of 22% for progression to MDS/leukemia after 15 years of treatment (11). Despite this leukemogenic risk, G-CSF is the initial treatment of choice, with a goal of achieving ANCs exceeding 1.0x10⁹/L (12). Approximately 10% of patients are unable to maintain this ANC and remain at risk for morbidity and mortality due to sepsis (13). Allogeneic stem cell transplant (AlloSCT) is the only curative treatment option for SCN. Definitive reasons to pursue HSCT include failure to maintain appropriate ANC with G-CSF or progression to MDS/leukemia. The cumulative risk of malignancy with G-CSF raises questions regarding the advisability of pre-emptive transplant for patients with high G-CSF requirements, recurrent intractable infections, or at some centers high-risk ELANE variants (9). In cases where a MSD is available, transplant is often considered. The appropriateness of pre-emptive alloSCT for patients with only alternative donor options is less clear (14, 15).

Given the rarity of SCN, there remains a paucity of data on the most appropriate conditioning regimen. To date, most patients who have had alloSCT for SCN have received myeloablative conditioning with busulfan and cyclophosphamide (Bu/Cy), and a small minority have received varied other regimens (16). This Bu/Cy myeloablative regimen carries numerous well-documented risks, including increased risk of veno-occlusive disease, pulmonary toxicity, infertility, and secondary malignancy (17). Busulfan and fludarabine has been proposed in this patient population as an alternative conditioning that can mitigate the severe side effects associated with cyclophosphamide (18–21). Our institutional experience supports this regimen, and we currently have an open phase I clinical trial to assess the efficacy of a busulfan/fludarabine-based conditioning regimen in bone marrow failure (BMF) patients. We present five pediatric patients with SCN transplanted using a conditioning regimen of busulfan, fludarabine, and ATG.

ELANE-related SCN is a rare disorder of neutrophil maturation that carries a high risk of life-threatening infections. It requires lifelong G-CSF therapy to mitigate this risk, but G-CSF may cause severe long-term side effects, including significant osteopenia, splenomegaly, and risk of myeloid malignancies. AlloSCT is the only currently available curative therapy for SCN. The discussion to consider alloSCT becomes more acute in patients who develop clonal evolution involving somatic RUNX1 mutations or cytogenetic abnormalities and many centers will consider using best available alternative donor in these cases. Thus, the question of which patients should consider pre-emptive transplant in the absence of myeloid malignancies is much debated. Most centers consider patients who require high standing doses of G-CSF (≥8 mcg/kg/day) and who have a suitable available MSD to be excellent candidates for pre-emptive transplant, but whether this approach should be offered to patients with lower daily G-CSF requirements or for those whose options are limited to alternative donor transplant is less certain.

There are no defined standards of care for conditioning in alloSCT for ELANE-related SCN. Busulfan and cyclophosphamide is the most common regimen used in published cases (16), but poses significant risks from dual alkylating agents. We present 5 patients that received MSD-BMT using a reduced toxicity busulfan and fludarabine conditioning regimen for ELANE-mutant SCN without myeloid malignancy. None of the patients in this study developed significant lung, liver, or kidney toxicity, and overall survival was 100%. This strategy also did not compromise efficacy. All patients exhibited event-free survival, time to neutrophil and platelet engraftment and total donor chimerism comparable to busulfan and cyclophosphamide-based regimens (21–23). Our patients exhibited low rates of GVHD with no cases requiring ongoing systemic therapy. Viral reactivation occurred in a minority of patients and resolved without end-organ damage in all cases.

MSD-BMT with myeloablative but reduced-toxicity conditioning for ELANE-mutant SCN provides excellent outcomes with minimal toxicity. Additional reduced-toxicity regimens could also be considered, including treosulfan/fludarabine. Treosulfan has demonstrated comparable efficacy and a favorable toxicity profile compared to busulfan (24–26), and may represent an opportunity to further optimize outcomes. However, the currently enrolling clinical trial BMT-CTN 1904 examining a treosulfan-based conditioning regimen for patients with bone marrow failure excludes patients with SCN due to a paucity of data using similar regimens including busulfan/fludarabine. As our study demonstrates excellent engraftment for a busulfan/fludarabine/ATG regimen, we theorize that treosulfan might perform similarly and should be tested in patients with SCN.

Our study is limited in scope by the small size and lack of control group receiving traditional busulfan/cyclophosphamide regimens for comparison; however, comparisons to cases in the literature are favorable. These pilot data suggest that this busulfan and fludarabine-based approach should be studied in multicenter clinical trials, where any patient with ELANE-related SCN who has a MSD and daily G-CSF requirement would be eligible.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Children’s Hospital of Philadelphia Institutional Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

JO: Writing – original draft, Writing – review & editing, Conceptualization, Methodology. NG: Writing – original draft, Writing – review & editing, Data curation, Formal analysis. KV: Investigation, Writing – review & editing, Resources, Project administration. CE: Conceptualization, Resources, Supervision, Writing – review & editing. LW: Conceptualization, Resources, Writing – review & editing. JW: Supervision, Writing – review & editing. TO: Conceptualization, Supervision, Writing – original draft, Writing – review & editing, Methodology.