Precision myeloablation with TDM-guided busulfan in pediatric primary immunodeficiencies: a real-world study of 28 patients

Introduction

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for many severe primary immunodeficiency (PID) disorders. However, this population, predominantly infants and young children, presents unique challenges. Physiologic organ immaturity and high prevalence of active opportunistic infections at transplant create a narrow therapeutic window for conditioning (1).

Balancing effective myeloablation against life-threatening regimen-related toxicity (RRT) is the central dilemma. Traditional myeloablative conditioning (MAC) ensures donor engraftment but incurs high rates of sinusoidal obstruction syndrome (SOS), pulmonary toxicity, and severe mucositis. Conversely, reduced-intensity conditioning (RIC) reduces immediate toxicity but increases risks of graft rejection, unstable mixed chimerism, and inadequate immune reconstitution (2,3).

Busulfan (Bu), a key alkylating agent, has a narrow therapeutic index and exhibits high inter-individual pharmacokinetic (PK) variability (4,5). Conventional weight-based dosing often results in unpredictable exposure, leading to either toxicity or graft failure (6,7). Therapeutic drug monitoring (TDM) offers a rational solution by enabling personalized dose adjustments to achieve a target area under the curve (AUC).

We hypothesized that a TDM-guided, personalized Flu/Bu regimen would safely deliver effective myeloablation. This real-world study evaluates the efficacy and safety of this precision medicine approach in a contemporary cohort of pediatric PID patients. We present this article in accordance with the STROBE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0344/rc).

Methods

Study design and participants

This retrospective study was approved by the Institutional Review Board of Beijing Children’s Hospital, which served as the central ethics committee for this multi-center study (No. [2026]-E-040-R). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. We reviewed electronic medical records of 28 consecutive PID patients who underwent first allo-HSCT between March 2022 and September 2025. Inclusion criteria were: (I) age <18 years; (II) diagnosed with PID according to international standard criteria (8); (III) received first allogeneic HSCT (allo-HSCT) regardless of the donor and cell source; (IV) complete remission (CR) of the primary disease prior to transplantation (with no active severe infections and stable organ function); and (V) receipt of TDM-guided Bu/Flu MAC.

Stem cell mobilization and graft sources

Graft source included bone marrow (BM) and granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSCs). PBSCs were used in matched sibling donor (MSD) and matched unrelated donor (MUD) transplantation, while BM combined with PBSCs were used in the haplo-HSCT. Umbilical cord blood (UCB) served as an auxiliary measure to promote engraftment among three patients, as their donors had cryopreserved UCB at birth, and these individuals presented with a significantly elevated risk of transplant failure (e.g., active infection, prior sensitization).

Grafts were infused on day 0 for BM and day 1 for G-CSF mobilized PBSC without T-cell depletion. The target CD34⁺ cell count was above 5×10⁶/kg, but above 4×10⁶/kg was also considered acceptable.

For patients with high-level mixed chimerism (donor chimerism 50–95%) persisting beyond day +60 post-transplant without evidence of graft rejection, we considered additional cellular therapies. Mesenchymal stem cell (MSC) infusions (1–2 ×10⁶/kg per dose, two doses given 7 days apart) were administered to patients with evidence of poor graft function (persistent cytopenias) or ongoing inflammation. Donor lymphocyte infusions (DLIs) (escalating doses from 0.5 to 1.0×10⁵ CD3⁺ cells/kg) were reserved for patients with falling donor chimerism (<80% on two consecutive measurements) without active graft-versus-host disease (GVHD). The decision to administer MSC and/or DLI was made by multidisciplinary team consensus. In this cohort, three patients received these interventions (indicated by asterisks in Table 1).

Conditioning regimen and TDM protocol

All patients received intravenous Bu (q6h for 3 or 4 days) and Flu (150–180 mg/m²). Initial Bu dosing was weight-based per manufacturer recommendations: 1 mg/kg (<9 kg), 1.2 mg/kg (9–16 kg), 1.1 mg/kg (16–23 kg), 0.95 mg/kg (23–34 kg), and 0.8 mg/kg (>34 kg) (9). Blood samples for PK analysis were collected via central line at pre-infusion, 0.5, 1, 2, 2.5, 4, and 6 hours post-first infusion. Bu concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A nonlinear mixed effects (NLME) model (Phoenix 8.0, Certara USA Inc., Princeton, NJ, USA) was used for PK analysis.

Bu dose adjustments were guided by: (I) first-dose AUC [targeting 900–1,500 µmol·min/L, per Food and Drug Administration (FDA) and European Medicines Agency (EMA) recommendations (10)]; and/or (II) cumulative exposure targets [severe combined immunodeficiency (SCID): 60–70 mg·h/L; non-SCID: 85–95 mg·h/L (11)]; in addition to (III) thepatients’s clinical status during conditioning (e.g., infection, organ function). For conversion, 1 mg·h/L of Bu is approximately equivalent to 60 µmol·min/L (based on molecular weight of 246 g/mol).

GVHD prophylaxis and supportive care

For MSD, prophylaxis comprised cyclosporine A (CsA) and short-course methotrexate (MTX). CsA was administered intravenously at 2.5 mg/kg/day either as a continuous infusion or in divided doses every 12 hours, starting on day −1. MTX was given at 15 mg/m² on day +1, followed by 10 mg/m² on days +3, +6, and +11.

For MUD/haploidentical donors, rabbit anti-thymocyte globulin (ATG, 5–10 mg/kg total) or anti-T lymphocyte globulin (ATLG, 20–30 mg/kg total) was added with CsA, mycophenolate mofetil (MMF), and MTX. CsA serum concentrations were closely monitored every third day to maintain therapeutic levels (100–150 ng/mL).

Phenytoin was administered for seizure prophylaxis. Rituximab (375 mg/m²) was given 24 hours pre-infusion for post-transplant lymphoproliferative disease (PTLD) prophylaxis. Co-trimoxazole was administered orally for Pneumocystis jirovecii pneumonia (PJP) prophylaxis. Intravenous acyclovir (10 mg/kg Q12H) commenced on day 0 as herpes virus prophylaxis. Letermovir prophylaxis (240 mg once daily, or 120 mg once daily with CsA) was initiated from day +1 or +14 until day +100 for patients at high risk of cytomegalovirus (CMV) infection (donor or recipient CMV-seropositive) (12). Before January 2024, letermovir was not routinely available; therefore, these patients received preemptive therapy guided by weekly CMV PCR monitoring. CMV reactivation was defined as a whole-blood CMV DNA level of ≥500 copies/mL on two consecutive tests. Caspofungin was recommended for antifungal prophylaxis. Antimicrobial selection prior to engraftment was guided by the patient’s documented infection history and microbial colonization patterns.

Endpoints and definitions

Primary endpoints: overall survival (OS) and event-free survival (EFS). Events were defined as death or graft failure.

Secondary endpoints:

• Neutrophil engraftment: the first of 3 consecutive days with ANC >0.5×10⁹/L;
• Platelet engraftment: the first of 7 consecutive days with platelets >20×10⁹/L without transfusion;
• Donor chimerism: complete donor chimerism (≥95%), high-level mixed chimerism (50–94%), low-level mixed chimerism (10–49%) or graft failure (10%);
• Regimen-related toxicities: mucositis [Common Terminology Criteria for Adverse Events (CTCAE) v5.0], veno-occlusive disease (VOD)/SOS [European Society for Blood and Marrow Transplantation (EBMT) criteria (13)], transplant-associated thrombotic microangiopathy (TA-TMA) (Cho criteria (14);
• Acute GVHD [Mount Sinai criteria (15)], chronic GVHD [cGVHD; National Institutes of Health (NIH) criteria (16)];
• Transplant-related mortality (TRM): death unrelated to underlying disease;
• Immune reconstitution: lymphocyte subsets by flow cytometry (BD FACSCanto II, BD Biosciences, San Jose, CA, USA) using monoclonal antibodies: CD3-FITC, CD4-PE, CD8-APC, CD19-PE-Cy7 (BD Biosciences).

Immune reconstitution monitoring

Immune recovery was assessed at +3, +6, +9, +12, +18, and +24 months post-transplant. T-cell reconstitution was evaluated by CD3⁺, CD4⁺, and CD8⁺ counts; B-cell recovery by CD19⁺ counts and serum IgG levels; NK-cell recovery by CD56⁺ counts.

Statistical analysis

Continuous variables are presented as median [interquartile range (IQR)] or mean ± standard deviation (SD). Categorical variables are presented as frequencies and percentages. Group comparisons were performed using Fisher’s exact test (categorical) and Mann-Whitney U test (continuous). Survival rates were estimated using the Kaplan-Meier method. Statistical significance was set at P<0.05. Analyses were performed using SPSS (IBM Corp) and R 4.3.3 (R Foundation for Statistical Computing). Graphs were generated using GraphPad Prism 10.0.

Results

Patient and transplant characteristics

A total of 28 patients (19 male, 9 female) with a median age of 1.7 (IQR, 0.6–4.1) years were included. Diagnoses were SCID (n=11, 39.3%) and non-SCID PID (n=17, 60.7%). Pre-transplant pulmonary infections were present in 67.8% of patients (other detailed infections, please refer to Table 2). Donors were MSD (21.5%), MUD (35.7%), mMUD (28.6%), or haploidentical (14.3%). The median CD34⁺ cell dose was 10.55 (IQR, 9.79–13.48) ×10⁶/kg. Detailed demographics are shown in Tables 1,2.

Bu PK and TDM

Only 11/28 patients (39.3%) achieved the target AUC after the first dose, confirming the high inter-individual PK variability [coefficient of variation (CV) 36.8%] and the critical need for TDM (Figures 1,2, Table 3).

• SCID cohort (target 60–70 mg·h/L): the initial dose AUC for Bu spanned 9.0 to 19.9 mg·h/L (equivalent to 540.2 to 1,192.37 µmol·min/L; median 844 µmol·min/L). Median cumulative AUC was 62.31 (IQR 55.6–80.09) mg·h/L (CV 26.3%). Five of 11 patients (45.5%) were within the target range after the first dose. Among the six patients requiring dose modification, three required dose escalation due to subtherapeutic exposure, one required dose reduction for supratherapeutic levels, and two remained subtherapeutic despite escalation but were not further adjusted due to clinical stablility and full-match donor status.
• Non-SCID cohort (target 85–95 mg·h/L): the initial dose AUC varied widely from 9.75 to 23.93 mg·h/L (equivalent to 585.2 to 1,436 µmol·min/L; median 928.3 µmol·min/L). Median cumulative AUC was 77.49 (IQR, 60.64–88.93) mg·h/L (CV 40.18%). Only 6 of 17 patients (35.3%) achieved the target after the first dose. Eleven patients required dose adjustments: eight required escalation, two required reduction, and one required a 24-hour treatment pause due to bacterial pneumonia and sepsis. Notably, two patients with persistently subtherapeutic exposure despite maximal dose escalation (to 1.3 mg/kg) maintained stable engraftment with full donor chimerism, suggesting that donor type (MUD) may compensate for slightly lower Bu exposure.

Figure 1 Busulfan dose adjustment guided by TDM in SCID patients (target cumulative AUC: 60–70 mg·h/L). Each colored form represents an individual patient. Green round circle indicates the initial cumulative AUC after the first weight-based dose. Red square indicates the final cumulative AUC after TDM-guided dose adjustment (if any). (A) The overlap of the red square and green circle indicates that no dose adjustment is required. The shaded horizontal band indicates the target AUC range (60–70 mg·h/L). AUC values were converted to mg·h/L using the conversion factor 1 mg·h/L =60 μmol·min/L. Detailed PK parameters for each patient are provided in Table S1. AUC, area under the curve; PK, pharmacokinetic; SCID, severe combined immunodeficiency; TDM, therapeutic drug monitoring.

Figure 2 Busulfan dose adjustment guided by TDM in non-SCID patients (target cumulative AUC: 85–95 mg·h/L). Each colored form represents an individual patient. Green round circle indicates the initial cumulative AUC after the first weight-based dose. Red square indicates the final cumulative AUC after TDM-guided dose adjustment (if any). (A) The overlap of the red square and green circle indicates that no dose adjustment is required. The shaded horizontal band indicates the target AUC range (85–95 mg·h/L). AUC values were converted to mg·h/L using the conversion factor 1 mg·h/L=60 μmol·min/L. Detailed PK parameters for each patient are provided in Table S2. AUC, area under the curve; PK, pharmacokinetic; SCID, severe combined immunodeficiency; TDM, therapeutic drug monitoring.

Engraftment and chimerism

Median time to neutrophil and platelet engraftment was 12.4±3.2 and 11.4±4.3 days, respectively. During this period, patients received a median of 4.5 red blood cell transfusions (IQR, 2–7.5) and 2 platelet transfusions (IQR, 1–4.0). Notably, one child achieved sustained platelet recovery (20×10⁹/L) without requiring transfusion support.

At 6 months post-HSCT, 85.7% (24/28) patients achieved complete donor chimerism (median 99.54%, IQR, 97.7–99.76%). Among these, complete donor chimerism was observed in 81.8% (9/11) of SCID patients and 88.2% (15/17) of non-SCID patients (P=0.64). One case (3.6%) of non-SCID patients experienced fatal graft failure. Three patients with initial high-level mixed chimerism (85–95%) improved after MSC infusions and/or DLIs. At last follow-up, two of these three patients maintained stable mixed chimerism (80%). Chimerism kinetics are shown in Figure 3.

Figure 3 Donor chimerism kinetics after transplantation in (A) SCID and (B) non-SCID patients. Chimerism was assessed at months +1, +3, +6, +9, +12, and +24 post-transplant. CDC defined as ≥95% donor cells; HLMC as 50–94%; LLMC as 10–49%; graft failure as <10%. Three patients who received MSC and/or DLI are indicated by asterisks. CDC, complete donor chimerism; DLI, donor lymphocyte infusion; HLMC, high-level mixed chimerism; LLMC, low-level mixed chimerism; MSC, mesenchymal stem cell; SCID, severe combined immunodeficiency.

Toxicity and GVHD

Grade 3–4 mucositis occurred in only one patient (3.5%). One patient (3.5%) was diagnosed with definite VOD, which resolved with defibrotide. Three additional patients had clinical VOD-like features that resolved with defibrotide prophylactic therapy. No grade 3–4 acute GVHD occurred. Grade 1–2 aGVHD occurred in 10 patients (35.7%), with comparable rates between SCID (4/11, 36.4%) and non-SCID (6/17, 35.3%) patients. The median time to onset of aGVHD was day +35 post-transplant (range: day +18 to +62). cGVHD developed in 7 of 27 evaluable patients (25.9%). The median time to cGVHD onset was month +6 post-transplant (range: month +4 to +11). According to NIH consensus criteria (15), the severity was mild in one patient (3.7%), moderate in 5 (18.5%), and severe in 1 (3.7%). Organ involvement included lung (n=6, 85.7% of cGVHD cases), skin (n=2, 28.5%), oral mucosa (n=2, 28.6%), and liver (n=1, 14.3%) (Table 4). Notably, the incidence of cGVHD was higher in patients receiving PBSC grafts (6/21, 28.5%) compared to those receiving BM-containing grafts (1/7, 0%; P=0.64), although this did not reach statistical significance due to small sample size.

Infections and complications

CMV reactivation occurred in 11 patients (39.3%), all of whom were in the high-risk serostatus group. Among these, 6 patients had not received letermovir prophylaxis (transplanted prior to 2024 or letermovir unavailable). The reactivation rate was lower in the letermovir group compared to the no-letermovir group [3/12 (25.0%) vs. 8/16 (50%)], although this difference was not statistically significant (P=0.25), likely due to small sample size. No patient developed CMV disease. Epstein-Barr virus (EBV) reactivation occurred in 3 patients (11.1%) without progression to PTLD. BK virus-associated hemorrhagic cystitis (grade I) occurred in 2 patients (7.1%). New bacterial, fungal, or viral co-infections were identified in six patients, including one adenovirus case. Autoimmune hemolytic anemia (AIHA) and TA-TMA occurred in 3 (11.1%) and 2 (7.1%) patients, respectively.

Immune reconstitution

Robust immune reconstitution was observed in both cohorts (Figure 4).

Figure 4 XXXXXXXXXXXXX. (A) T-cell reconstitution after TDM-guided BuFlu HSCT CD4⁺ and CD8⁺ T-cell counts over 24 months post-transplant in SCID patients (n=11) and non-SCID patients (n=17). Shaded areas represent IQRs. Dashed horizontal line indicates CD4⁺ count of 500 cells/µL, a threshold associated with reduced infection risk. (B,C) B-cell and immunoglobulin reconstitution after TDM-guided BuFlu (C) CD19⁺ B-cell counts in SCID and non-SCID patients over 24 months. (D) Serum IgG levels. All patients received monthly IVIG infusions during the first-year post-transplant (shaded area). The dashed line indicates the lower limit of normal for age (500 mg/dL). IVIG was discontinued in all surviving patients by 12 months. Data are presented as median (IQR). Bu, busulfan; Flu, fludarabine; HSCT, hematopoietic stem cell transplantation; IQR, interquartile range; IVIG, intravenous immunoglobulin; SCID, severe combined immunodeficiency; TDM, therapeutic drug monitoring.

T cells

In SCID patients, median CD4⁺ T-cell counts demonstrated a consistent increase over time post-transplant: 127.5 (IQR, 34.05–418.5) cells/µL at 3 months, 537 (IQR, 87–860) cells/µL at 6 months, 616.5 (IQR, 113.3–1,498) cells/µL at 9 months, 1,148 (IQR, 205–2,477) cells/µL at 12 months, 1,150 (IQR, 299.8–2,881) cells/µL at 18 months, and 2,366 (IQR, 780–2,881) cells/µL by 24 months; corresponding CD8⁺ T-cell counts also showed robust recovery, rising from 293.5 (IQR, 64.75–2,973) cells/µL at 3 months to 722 (IQR, 338.5–7,888) cells/µL at 6 months, 859 (IQR, 158.3–3,900) cells/µL at 9 months, 1,226 (IQR, 333.8–4,510) cells/µL at 12 months, 1,350 (IQR, 457.5–3,625) cells/µL at 18 months, and reaching 1,695 (IQR, 607.5–3, 126) cells/µL by 24 months. In non-SCID patients, median CD4⁺ T-cell counts showed a gradual increase over time: 158 (IQR, 81–232) cells/µL at 3 months, 347 (IQR, 187.5–474.5) cells/µL at 6 months, 465.5 (IQR, 292–649.3) cells/µL at 9 months, 543 (IQR, 398.8–775.8) cells/µL at 12 months, 712.5 (IQR, 613–930) cells/µL at 18 months, and 812 (IQR, 600.3–942) cells/µL by 24 months post-transplant. In contrast, CD8⁺ T-cell counts followed a distinct trajectory: 760.5 (IQR, 379.3–1,118) cells/µL at 3 months, rising to 974 (IQR, 669.8–3,134) cells/µL at 6 months, 1, 147 (IQR, 710.8–2,798) cells/µL at 9 months, 1,352 (IQR, 940.3–1,939) cells/µL at 12 months, 1,441 (IQR, 798–1,818) cells/µL at 18 months, and reaching 1,648 (IQR 623.3–1,648) cells/µL by 24 months.

B cells

In SCID patients, median CD19⁺ B-cell counts at 3, 6, 9, 12, 18, and 24 months post-transplant were 0, 131 (IQR, 35–356.5), 150.5 (IQR, 55.25–336), 235 (IQR, 108.5–874.8), 373 (IQR, 210.5–867), and 559 (IQR, 370.5–1,189) cells/µL, respectively. In non-SCID patients, corresponding B-cell counts were 9 (IQR, 0.75–85), 123.5 (IQR, 13.5–285.3), 273.5 (IQR, 127.3–450.5), 335 (IQR, 89.5–582), 423.5 (IQR, 278–627.8), and 655 (IQR, 375.5–765.5) cells/µL. Of note, one child with AIHA received rituximab at nine months, resulting in a B-cell count of 0 cells/µL at that time point.

Immunoglobulin

All patients received monthly immunoglobulin infusions during the first year post-transplant. In SCID patients, median IgG levels at 3, 6, 9, 12, 18, and 24 months were 8.73 (IQR, 5.97–16.0), 6.51 (IQR, 4.685–13.43), 6.58 (IQR, 4.075–10.70), 6.465 (IQR, 4.83–8.95), 7.57 (IQR, 5.64–10.73), and 8.09 (IQR, 6.09–9.17) g/L, respectively. In non-SCID patients, corresponding values were 9.96 (IQR, 8.233–11.35), 7.15 (IQR, 6.44–10.2), 8.86 (IQR, 5.4–10.43), 7.97 (IQR, 6.21–10.97), 7.87 (IQR, 5.90–11.85), and 7.13 (IQR, 5.30–10.74) g/L. Critically, all surviving patients achieved intravenous immunoglobulin (IVIG) independence within 1 year post-transplant, indicating functional B-cell recovery (Figure 4).

Survival

With a median follow-up of 829.5 (IQR, 376–1,046) days, the 2-year OS and EFS were 96.4% (Figure 5). One patient died of graft failure complicated by sepsis and multi-organ failure. All other patients are alive and disease-free.

Figure 5 Kaplan-Meier estimates of OS in SCID and non-SCID cohorts. Parentheses indicate number of events/cohort size. OS, overall survival; SCID, severe combined immunodeficiency.

Exploratory analysis: Bu exposure and immune reconstitution

To explore whether higher Bu exposure delays immune recovery, we compared CD4⁺ T-cell reconstitution between patients with cumulative AUC above vs. below the median (78.5 mg·h/L). At 6 months post-transplant, median CD4⁺ counts were similar between the high-exposure and low-exposure groups (219.5 vs. 276 cells/µL, P=0.53), suggesting that within the narrow therapeutic window achieved through TDM, Bu exposure does not significantly impact early T-cell recovery.

Discussion

In this real-world cohort of 28 pediatric PID patients undergoing first allo-HSCT, a TDM-guided, personalized Bu/Flu conditioning regimen was associated with favorable outcomes, including 2-year OS of 96.4%. While these results compare favorably with historical reports of both traditional MAC and RIC regimens (17,18), the absence of a concurrent control group in our study warrants cautious interpretation. While the concept of TDM-guided Bu dosing has been established in larger trials, real-world data focusing exclusively on contemporary pediatric PID patients—particularly those with active infections or organ dysfunction—remain limited. Our study provides granular immune reconstitution data and validates a risk-adapted AUC stratification strategy in this vulnerable population.

The high inter-individual variability in Bu PKs observed in our cohort (CV 36.8%)—with only 39.3% of patients achieving target AUC after the first dose—underscores the inadequacy of empiric weight-based dosing. This finding aligns with previous reports (7,10) and confirms that TDM is not optional but mandatory for optimizing outcomes. By individualizing doses, we successfully avoided both supratherapeutic peaks (which increase SOS risk) and subtherapeutic valleys (which increase graft failure risk), achieving a narrow distribution of final AUC values around the prespecified targets (Figures 1,2). A key innovation of our protocol is the stratification of target AUC by disease type. The lower target for SCID patients (60–70 mg·h/L) reflects their unique biology: the absence of functional T-cells reduces rejection risk, allowing for dose reduction to protect vulnerable organs, particularly in young infants with active infections. Conversely, the higher target for non-SCID patients (85–95 mg·h/L) was chosen to overcome the barrier of intact innate immunity and ensure robust myeloid engraftment. This is particularly critical in disorders such as chronic granulomatous disease (CGD) and familial hemophagocytic lymphohistiocytosis (HLH), where mixed chimerism below 20–30% may not provide adequate disease control (19,20).

Our AUC targets align with previously published recommendations. In a cohort of 562 patients, Bognàr et al. (2024) showed that a Bu AUC of 70–90 mg·h/L (depending on disease indication) was associated with optimal outcomes. For SCID, HLH-related, and neutrophil disorders, an AUC of 70–90 mg·h/L (optimal 80 mg·h/L) yielded the highest 2-year EFS (87.9%) and donor chimerism (>90%), significantly outperforming lower (<70%) or higher (>90%) exposures. For CID, although an optimal EFS range was not clearly defined, an AUC >70 mg·h/L improved donor chimerism, while an AUC <50 mg·h/L markedly increased graft failure risk. More recently, the EBMT/European Society for Immunodeficiencies (ESID) guidelines (11) recommended risk-adapted Bu exposure: lower AUC (60–70 mg·h/L) for SCID and low rejection risk disorders, and higher AUC (80–95 mg·h/L) for conditions requiring full myeloid engraftment (e.g., CGD, HLH). Our study provides real-world validation of these guideline recommendations.

The low incidence of severe RRT (definite VOD 3.6%, grade 3–4 mucositis 3.6%) compares favorably with historical MAC regimens (21,22) and is attributable to two key modifications: (I) replacement of cyclophosphamide with fludarabine (Flu), which avoids glutathione-pathway hepatic toxicity and reduces endothelial damage (23,24); and (II) TDM-guided avoidance of supratherapeutic Bu exposure, which is directly linked to SOS risk (2,7).

While the absence of grade 3–4 aGVHD is encouraging, the 25.9% incidence of cGVHD—predominantly lung-limited—warrants attention. This may reflect the predominant use of PBSC grafts (85% of patients), which are associated with higher cGVHD risk compared to BM (25). The pulmonary predominance (85.7% of cGVHD cases) underscores the need for long-term pulmonary function monitoring in this cohort.

The 39.3% CMV reactivation rate, while comparable to other Flu/ATG-based regimens (26), highlights the profound T-cell suppression induced by this conditioning approach. Notably, no patient developed CMV disease, reflecting vigilant monitoring and preemptive therapy. The numerically lower reactivation rate in patients receiving letermovir prophylaxis (25% vs. 50%, P=0.253), although not statistically significant due to small sample size, supports the incorporation of universal letermovir prophylaxis in future protocols (27).

Robust immune reconstitution was observed in both cohorts, with all surviving patients achieving IVIG independence by 12 months post-transplant. This functional cure—the ultimate goal of HSCT for PID—demonstrates that TDM-guided Bu/Flu conditioning preserves thymic function and supports durable B-cell engraftment. The exploratory analysis suggesting no significant delay in CD4⁺ recovery with higher exposure (within the target range) provides reassurance that achieving the higher target in non-SCID patients does not compromise immune reconstitution.

Several limitations must be acknowledged. First, the retrospective, single-center design with a modest sample size (n=28) limits generalizability and precludes definitive causal inference. Second, the absence of a concurrent control group prevents direct comparison with non-TDM strategies; comparisons with historical data should be interpreted cautiously (Table 5). Third, the median follow-up of 829 days, while adequate for assessing early outcomes, is insufficient to evaluate late effects such as pulmonary sequelae of cGVHD or gonadal function. Fourth, the heterogeneity of graft sources (PBSC predominant) and ATG exposure may have confounded GVHD and immune reconstitution analyses. Finally, the optimal AUC target may vary by specific PID genotype and donor type, warranting further refinement in a larger multicenter cohort. While subgroup analyses by donor type and GVHD prophylaxis did not reveal significant outcome differences, these findings are exploratory and limited by small sample sizes. Sixth, despite systematic chart review, some data on prior non-pulmonary infections and daily organ function trajectories were incomplete due to the retrospective design, potentially introducing bias.

Conclusions

TDM-guided, personalized Bu/Flu conditioning achieves “precision myeloablation” within a therapeutic window, enabling excellent engraftment, favorable safety, and preserved immune reconstitution in pediatric PID patients. This approach successfully extends curative transplantation to high-risk PID patients—including infants, those with active infections, or impaired organ function—who might otherwise be ineligible for traditional MAC, while overcoming suboptimal engraftment associated with RIC regimens. Prospective multicenter studies with larger cohorts and standardized protocols are warranted to validate these findings.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0344/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0344/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0344/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0344/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This retrospective study was approved by the Ethics Committee of Beijing Children’s Hospital (No. [2026]-E-040-R) for all participating centers and was conducted in accordance with the Declaration of Helsink. The requirement for informed consent was waived by both ethics committees due to the retrospective nature of the study, which involved analysis of de-identified medical records and posed minimal risk to participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Squires JE, Raghu VK, Mazariegos GV. Optimizing the pediatric transplant candidate. Curr Opin Organ Transplant 2024;29:43-9. [Crossref] [PubMed]
2. Bartelink IH, Lalmohamed A, van Reij EM, et al. Association of busulfan exposure with survival and toxicity after haemopoietic cell transplantation in children and young adults: a multicentre, retrospective cohort analysis. Lancet Haematol 2016;3:e526-36. [Crossref] [PubMed]
3. Apsel Winger B, Shukla P, Kharbanda S, et al. The Relationship Between Busulfan Exposure and Achievement of Sustained Donor Myeloid Chimerism in Patients with Non-Malignant Disorders. Transplant Cell Ther 2021;27:258.e1-6.
4. Takahashi T, Jaber MM, Brown SJ, et al. Population Pharmacokinetic Model of Intravenous Busulfan in Hematopoietic Cell Transplantation: Systematic Review and Comparative Simulations. Clin Pharmacokinet 2023;62:955-68. [Crossref] [PubMed]
5. Lee JW, Kang HJ, Lee SH, et al. Highly variable pharmacokinetics of once-daily intravenous busulfan when combined with fludarabine in pediatric patients: phase I clinical study for determination of optimal once-daily busulfan dose using pharmacokinetic modeling. Biol Blood Marrow Transplant 2012;18:944-50. [Crossref] [PubMed]
6. Combarel D, Tran J, Delahousse J, et al. Individualizing busulfan dose in specific populations and evaluating the risk of pharmacokinetic drug-drug interactions. Expert Opin Drug Metab Toxicol 2023;19:75-90. [Crossref] [PubMed]
7. Andersson BS, Thall PF, Valdez BC, et al. Fludarabine with pharmacokinetically guided IV busulfan is superior to fixed-dose delivery in pretransplant conditioning of AML/MDS patients. Bone Marrow Transplant 2017;52:580-7. [Crossref] [PubMed]
8. Poli MC, Aksentijevich I, Bousfiha AA, et al. Human inborn errors of immunity: 2024 update on the classification from the International Union of Immunological Societies Expert Committee. J Hum Immun 2025;1:e20250003. [Crossref] [PubMed]
9. Nguyen L, Fuller D, Lennon S, et al. I.V. busulfan in pediatrics: a novel dosing to improve safety/efficacy for hematopoietic progenitor cell transplantation recipients. Bone Marrow Transplant 2004;33:979-87.
10. Palmer J, McCune JS, Perales MA, et al. Personalizing Busulfan-Based Conditioning: Considerations from the American Society for Blood and Marrow Transplantation Practice Guidelines Committee. Biol Blood Marrow Transplant 2016;22:1915-25. [Crossref] [PubMed]
11. Lankester AC, Albert MH, Booth C, et al. EBMT/ESID inborn errors working party guidelines for hematopoietic stem cell transplantation for inborn errors of immunity. Bone Marrow Transplant 2021;56:2052-62. [Crossref] [PubMed]
12. Aldalati AY, Hussein AM, Balbaa E, et al. Efficacy and Safety of Letermovir for Cytomegalovirus Prophylaxis in Pediatric Hematopoietic Stem Cell Transplantation Recipients: A Systematic Review, Meta-Analysis, and Meta-Regression. Transpl Infect Dis 2025;27:e70006. [Crossref] [PubMed]
13. Ichikawa H, Yakushijin K, Kurata K, et al. Utility of the refined EBMT diagnostic and severity criteria 2023 for sinusoidal obstruction syndrome/veno-occlusive disease. Bone Marrow Transplant 2024;59:518-25. [Crossref] [PubMed]
14. Cho BS, Yahng SA, Lee SE, et al. Validation of recently proposed consensus criteria for thrombotic microangiopathy after allogeneic hematopoietic stem-cell transplantation. Transplantation 2010;90:918-26. [Crossref] [PubMed]
15. Harris AC, Young R, Devine S, et al. International, Multicenter Standardization of Acute Graft-versus-Host Disease Clinical Data Collection: A Report from the Mount Sinai Acute GVHD International Consortium. Biol Blood Marrow Transplant 2016;22:4-10. [Crossref] [PubMed]
16. Filipovich AH, Weisdorf D, Pavletic S, et al. National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 2005;11:945-56.
17. Harris AC, Boelens JJ, Ahn KW, et al. Comparison of pediatric allogeneic transplant outcomes using myeloablative busulfan with cyclophosphamide or fludarabine. Blood Adv 2018;2:1198-206. [Crossref] [PubMed]
18. Pfeiffer T, Murray L, Gao F, et al. Alemtuzumab, Fludarabine, Melphalan, and Thiotepa Conditioning for Transplantation in Inborn Errors of Immunity. Transplant Cell Ther 2026;32:470.e1-10.
19. Bognàr T, Garcia-Rosa M, Lalmohamed A, et al. Association of busulfan exposure and outcomes after HCT for patients with an inborn error of immunity. Blood Adv 2024;8:5137-45. [Crossref] [PubMed]
20. Ju HY, Kang HJ, Hong CR, et al. Targeted busulfan and fludarabine-based conditioning for bone marrow transplantation in chronic granulomatous disease. Korean J Pediatr 2016;59:S57-9. [Crossref] [PubMed]
21. El-Serafi I, Remberger M, Ringdèn O, et al. Reduced Risk of Sinusoidal Obstruction Syndrome of the Liver after Busulfan-Cyclophosphamide Conditioning Prior to Allogeneic Hematopoietic Stem Cell Transplantation. Clin Transl Sci 2020;13:293-300. [Crossref] [PubMed]
22. N BA. Busulfan-cyclophosphamide vs fludarabine-busulfan for allogeneic transplant in acute myeloid leukemia. Future Oncol 2025;21:1887-94. [Crossref] [PubMed]
23. Hassan Z, Hellström-Lindberg E, Alsadi S, et al. The effect of modulation of glutathione cellular content on busulphan-induced cytotoxicity on hematopoietic cells in vitro and in vivo. Bone Marrow Transplant 2002;30:141-7. [Crossref] [PubMed]
24. Yuan J, Sun N, Feng X, et al. Optimization of Busulfan Dosing Regimen in Pediatric Patients Using a Population Pharmacokinetic Model Incorporating GST Mutations. Pharmgenomics Pers Med 2021;14:253-68. [Crossref] [PubMed]
25. Alkhunaizi M, Patel B, Bueno L, et al. Risk Factors for Bronchiolitis Obliterans Syndrome after Initial Detection of Pulmonary Impairment after Hematopoietic Cell Transplantation. Transplant Cell Ther 2023;29:204.e1-7.
26. Ljungman P, de la Camara R, Robin C, et al. Guidelines for the management of cytomegalovirus infection in patients with haematological malignancies and after stem cell transplantation from the 2017 European Conference on Infections in Leukaemia (ECIL 7). Lancet Infect Dis 2019;19:e260-72. [Crossref] [PubMed]
27. Chemaly RF, Ullmann AJ, Stoelben S, et al. Letermovir for cytomegalovirus prophylaxis in hematopoietic-cell transplantation. N Engl J Med 2014;370:1781-9. [Crossref] [PubMed]
28. Chandra S, Chandrakasan S, Dávila Saldaña BJ, et al. Experience with a Reduced Toxicity Allogeneic Transplant Regimen for Non-CGD Primary Immune Deficiencies Requiring Myeloablation. J Clin Immunol 2021;41:89-98. [Crossref] [PubMed]