Unrelated Umbilical Cord Blood Transplantation For Children With Hereditary Leukodystrophy: Single Center Experience


 Background

Hereditary leukodystrophies are diverse metabolic diseases caused by gene mutations that result in the abnormal development or degeneration of myelin. Transplantation of matched sibling donor umbilical cord blood (UCB) can slow progression and prolong survival in some cases. For patients without siblings, however, UCB transplantation from unrelated donors may be the only option.
Methods

This retrospective study assessed unrelated UCB transplantation (UCBT) efficacy following busulfan- and cyclophosphamide-based myeloablative chemotherapy.
Results

The study cohort included 12 pediatric patients (ten males), nine with adrenoleukodystrophy (ALD) and three with globoid cell leukodystrophy (GLD), treated between April 2015 and March 2020. All received HLA-matched or partially mismatched UCBT. Median age at UCBT was 7.2 years (range, [0.8–12.9 years]). There were no cases of graft rejection. Median neutrophil engraftment time was 20 days [12–33 days] and median platelet engraftment time was 29 days [14–65 days]. Median follow-up was 28 months [1–73 months], and overall survival rate was 83.3% (10/12). Seven patients had higher Loes scores post-transplantation, and two patients died of infection. Four patients with rapid neurological deterioration pre-UCBT exhibited worse neurological symptoms post-UCBT. In contrast, four patients with stable neurological symptoms pre-UCBT demonstrated symptom stability post-UCBT, and two with no neurological symptoms pre-UCBT were also symptom-free post-UCBT. Further, lipid profiles of surviving ALD patients were improved post-treatment.
Conclusions

Hereditary leukodystrophy patients with mild neurological symptoms can benefit from UCBT, while UCBT cannot reverse advanced disease.


Introduction
Hereditary leukodystrophies are a rare group of inherited metabolic diseases caused by mutations in genes encoding metabolic enzymes or factors leading to abnormal development or diffuse damage to the myelin sheath. Adrenoleukodystrophy (ALD) is an X-linked βoxidation disorder of very-long-chain fatty acids (VLCFAs) caused by ABCD1 gene mutation [1]. More than 500 unique mutations in ABCD1 associated with ALD have been identi ed [2]. The abnormal accumulation of metabolic substrates in the brain and adrenal cortex leads to progressive demyelination and adrenal cortex dysfunction [3]. Neurological symptoms include audiovisual de cits, mental retardation, cognitive impairments, behavioral abnormalities, and neuropsychiatric disorders. There are eight ALD subtypes, of which cerebral ALD in childhood accounts for about 30% of all cases and has the most severe clinical manifestations. After the onset of cerebral ALD, patients may exhibit disability and dementia followed by death in a few months to years [4]. Globoid cell leukodystrophy (GLD) is an autosomal recessive genetic disease caused by GALC mutations that cause demyelination through lack of galactocerebrosidase activity and ensuing accumulation of β-galactosides and derivatives. More than 270 different mutations in GALC related to GLD have been cataloged in the Human Gene Mutation Database [5]. Globoid cell leukodystrophy includes four clinical subtypes, early infantile phenotype, late infantile phenotype, juvenile phenotype, and adult phenotype. Patients with the early infantile phenotype are often younger than 6 months old and present with agitation, convulsion, audiovisual de cits, and feeding di culties; further, the disease progresses rapidly, and median survival is only two years [6]. Alternatively, patients with the later-onset types present with dyskinesia, visual impairment, mental decline, and seizures [7].
In developed countries, newborn screening enables early diagnosis and intervention [8,9]. In developing countries like China, however, newborn screening does not include hereditary leukodystrophies. Further, early diagnosis is di cult due to the insidious onset, diversity, and non-speci city of symptoms. Dietary control and glucocorticoid administration may alleviate some symptoms, but these measures do not improve neurological de cits. Alternatively, allogeneic hematopoietic stem cell transplantation (allo-HSCT) can slow the progression of neuropathy and promote the long-term survival of children with cerebral ALD and GLD [8, [10][11][12]. However, many hereditary leukodystrophy patients lack a matched sibling donor (MSD) of hematopoietic stem cells. Therefore, it is critical that patients without an MSD nd a suitable unrelated donor as soon as possible. Unrelated umbilical cord blood stem cells (UCB) which are advantageous due to easy isolation and low HLA compatibility requirements provide an alternative source of hematopoietic stem cells.
As hereditary leukodystrophies are relatively rare, there are few case series on the e cacy of unrelated umbilical cord blood stem cell transplantation (UCBT). Here we report the outcomes of twelve consecutive patients receiving unrelated UCBT at a single center to identify the most promising candidates.

Patients
All hereditary leukodystrophy patients treated by unrelated UCBT from April 2015 to March 2020 at the Children's Hospital of Fudan University were included in this study. Diagnosis was based on clinical manifestations, enzyme detection, and neuroimaging examinations, and then con rmed by gene sequencing. All patients were younger than 18 years old and none had an MSD. All guardians provided written informed consent before transplantation and the study was approved by the ethics committee of the Children's Hospital of Fudan University (2016-162).

Chimeric Monitoring and Engraftment De nition
Donor-recipient chimerism was tested using the short tandem repeat technique at 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, and 1 year after transplantation. More than 95% donor-derived cells was de ned as complete donor chimerism. An absolute neutrophil count > 0.5 × 10 9 /L for 3 consecutive days was de ned as neutrophil engraftment, and platelet count > 20 × 10 9 /L for 7 consecutive days without platelet transfusion was de ned as platelet engraftment.

Neurological Function Scores, Performance Status, and Loes Scores
Each patient was assigned a Neurologic Function Score (NFS) pre-and post-UCBT based on evaluations of vision, hearing, communication, swallowing, urinary and fecal control, movement, and the presence of afebrile convulsions [13]. Performance status (PS) was scored by the Lansky standard [14]. In addition, a Loes score of cranial MRI lesion severity was assigned by a senior radiologist [15,16].

Supportive Treatment
All patients were cared for in an independent laminar ow ward before neutrophil engraftment. Ganciclovir (10 mg/kg/day, from the beginning of conditioning to day -1 pre-UCBT) and acyclovir (750 mg/m 2 /day, from day 0 to day +270 post-UCBT) were used to prevent virus infection, caspofungin (from the beginning of conditioning to neutrophil engraftment) and voriconazole (from neutrophil engraftment to day +180) were used to prevent fungal infection, and sulfamethoxazole (25 mg/kg/day 2 days per week, from neutrophil engraftment to 6 months after immunosuppressant discontinuation) was used to prevent Pneumocystis carinii infection. Patients received intravenous immunoglobulin (500 mg/kg/dose) every 2 weeks starting on day +1 post-UCBT and continuing until B-lymphocyte count surpassed 200/μL.

Clinical Conditions Before Transplantation
Twelve patients with hereditary leukodystrophy were treated by UCBT. Clinical and demographic characteristics of these patients (p1-p12) are summarized in Table 1. Nine (p1-p9 in Table 1) were diagnosed with cerebral ALD and harbored unique maternally inherited ABCD1 mutations (mainly point mutations in exons 1 and 3), and the remaining three (p10-p12) were diagnosed with GLD and harbored inherited GALC gene mutations. The median onset age was 7 years (range, about 3 years to 11.9 years). Ten patients (all except p3 with ALD and p11 with GLD) had neurological symptoms before transplantation, eleven patients (all except p11) had abnormal brain white matter signals on cranial MRI before transplantation, and seven patients (p1, p3, and p5-p9) had adrenal cortex dysfunction and were receiving glucocorticoid replacement therapy. Serum VLCFAs were elevated in all ALD patients and leukocyte galactocerebrosidase activity was reduced in all GLD patients (see Table 1 for details). mg/kg). Rabbit anti-human thymocyte immunoglobulin (ATG, 5 mg/kg) was used in ten patients. Oral tacrolimus (FK506) was administered starting 4 days before transplantation to prevent graft versus host disease (GvHD). The target concentration of FK506 was 5-10 ng/mL. In addition to FK506, mycophenolate mofetil (MMF) was administered to the patient (p5) receiving UCB with 7/10 HLA matching on the rst day post-transplantation. The median age at UCBT was 7.2 years (range, 0.8 to 12.9 years) and median body weight at UCBT was 24.3 kg (range, 8.5 to 38 kg). See Table 2 for details of each procedure.  Table 3 for details of post-UBCT complications and treatments.  (20) AIHA, autoimmune hemolytic anemia; CDC, complete donor chimerism; # Nervous system asymptomatic Comparison of Neurologic Function Score, Performance Status, and Loes Score Pre-UCBT and Post-UCBT Neurologic function, PS, and Loes scores were evaluated before and regularly after transplantation. Eight patients with ALD (all except p3) and two patients with GLD (all except p11) demonstrated neurological impairments of varying severity before transplantation (NFS range, 1-17 points), and two of these patients (both with ALD) died after UCBT. Eleven patients (all except p2) were followed-up for at least three months after transplantation, and all showed varying degrees of neurologic symptom aggravation. In patient 6, neurologic symptoms deteriorated markedly after transplantation (6 points before transplantation to 9 points three months after transplantation to 24 points six months after transplantation) due to severe autoimmune hemolytic anemia, and there was no signi cant recovery during follow-up. Among the remaining seven patients (p1, p3, p4, p6-p9), the NFS of p1 was similar to pre-UBCT baseline at 5 years follow-up (Figure 1 for details). All other ALD cases demonstrated different degrees of brain damage before transplantation as measured by Loes scoring of magnetic resonance images, and most exhibited further increases in brain lesion severity after UCBT with stabilization by nine months post-treatment.
Two female patients (p10 and p11) with GLD were siblings. The neurological symptoms of the elder sibling (p10) progressed slowly before and after transplantation as evidenced by stable NFS and PS. Similarly, the younger sibling (p11) had no imaging lesions or nervous system involvement before or after UCBT. Another male (p12) with GLD was also neurologically stable before and after UCBT, although the Loes score increased slightly after transplantation. The changes in Loes scores for each patient throughout the treatment period are shown in Figure 2. Finally, the PS of six patients (p3, p7, p8, p10, p11, and p12) was stable after UCBT, while all other patients demonstrated different degrees of PS deterioration (Table 4). The serum VLCFA concentrations of most surviving ALD patients (except p4) were measured regularly following treatment. Both absolute C26:0 concentration and the C26:0/C22:0 ratio were signi cantly reduced one year post-UCBT (Figure 3), indicating partial restoration of ABCD1 activity.

Discussion
Treatment of hereditary leukodystrophy is limited by a lack of effective drugs. Diet adjustment and glucocorticoids may alleviate some symptoms, but cannot improve neuropathy, which is the predominant cause of functional impairment and frequently an indirect cause of death from infection. Currently, hematopoietic stem cell transplantation is the only way to prevent the progression of neuropathy and prolong survival. Cerebral type ALD and GLD in childhood are both indications for HSCT [11], while ADL patients with genetic diagnosis but mild or absent symptoms should instead receive regular MRI and neurological examinations to assess myelination status. Once cerebral manifestations occur, transplantation should be conducted as early as possible to prevent further progression and improve prognosis [17].
The bene ts of HSCT for GLD are attributed to "cross correction", a process in which the GALC enzyme secreted by donor cells binds to surface receptors on recipient host cells and is accumulated by pinocytosis, thereby compensating for the enzymatic de cit. If GALC activity is su cient, demyelination and neuropathy may be prevented [18]. In contrast, the therapeutic mechanisms of HSCT for ALD are still unclear. It has been proposed that monocytes from the donor can cross the blood-brain barrier and differentiate into microglia, and that these microglia help restore ABCD1 activity [19].
The neuropathy associated of childhood cerebral ALD and GLD progresses rapidly, so timely transplantation is critical. The European Society for Blood and Marrow Transplantation recommends related donors as the rst choice for stem cell transplantation, followed by unrelated donors with at least 4/6 site matching. UCB is easy to obtain and rich in stem cells [18]. However, the failure rate of UCB engraftment is higher than that of bone marrow or peripheral blood stem cells, so myeloablative conditioning should be used prior to transplantation. In this study, twelve patients were treated with BU/CY-based myeloablative conditioning and all demonstrated complete donor cell chimerism two weeks after transplantation that remained stable during follow-up.
Ten patients with hereditary leukodystrophy had nervous system symptoms before transplantation. However, the median time from onset to UCBT was 6 months (2 to 58 months), which may explain the continued deterioration in some patients after treatment. At present, hereditary leukodystrophies are not part of neonatal screening in China. In most patients, the initial symptoms are non-speci c, so diagnosis is often delayed, resulting in progression of neuropathy before transplantation. In our group of ALD patients, both Loes and NFS scores indicated substantial structural and functional neurological impairment before transplantation. Although neurological function was generally more stable following transplantation, pretreatment damage was largely irreversibly, underscoring the urgency of diagnostic con rmation and treatment.
In previous reports, the overall survival rates of cerebral ALD and GLD following UCBT ranged from 59-69% and 43-100%, respectively, and analysis of prognostic factors showed that early-stage disease (no neurological symptoms or mild symptoms), fewer and less extensive brain imaging lesions, and su cient stem cells were all predictive of better prognosis [11,13,20,21]. For GLD patients, transplantation reduced the risk of death by 45% [22]. Asymptomatic GLD newborns can obtain 100% engraftment and 100% survival through UCBT. After transplantation, the myelin gradually forms and skills development gradually improves [21].
GvHD and infection are common complications following transplantation. Compared to stem cells from bone marrow or peripheral blood, the immunogenicity of UCB is lower, so the incidence of severe GvHD after UCBT is relatively reduced (only about 20-35% in previous reports) [11]. In the current study, there were no cases of chronic or severe GvHD, only four cases of acute grade II GvHD responsive to glucocorticoid therapy. Children with hereditary leukodystrophy may have weak cough re exes and urinary incontinence due to neuropathy as well as poor immune function after pretreatment, which in combination can increase the risks of pulmonary and urinary tract infections. Indeed, previous studies have found that severe infection associated with disease progression after transplantation is the main cause of death among children with hereditary leukodystrophy [11]. In the present study, six of twelve patients developed pulmonary infection after transplantation, and two died, while another four developed CMV viremia. Fortunately, these CMV viremia cases were cured by ganciclovir or foscarnet treatment. In addition, the two case of urinary tract infection and the two cases of hemorrhagic cystitis were improved by antibiotic treatment or support therapy. Therefore, clinical outcome following UCBT for hereditary leukodystrophy may be improved by more intensive nursing care to prevent pulmonary infection.
Multiple factors can affect the prognosis of neurological function in hereditary leukodystrophy patients after transplantation, including disease severity before transplantation, donor chimerism level after transplantation, GvHD severity, and other transplantation related complications, especially pulmonary infection. At present, most studies on prognosis have focused on survival rate, while few studies have conducted longitudinal assessment of nerve function [23]. Peters and colleagues found that only 16% of patients with neurological de cits before transplantation demonstrated improvement after transplantation, while 56% of patients without neurological de cits before transplantation had no neurological de cits following transplantation [24]. Van den Broek and colleagues found that 50% of hereditary leukodystrophy patients with a mild decline in functional status score before transplantation remained stable after transplantation, while 50% exhibited neurological deterioration after transplantation [11]. In such cases, disease progression may occur due to enzyme insu ciency before substantial engraftment.
We also found that lesion severity does not necessarily predict post-treatment outcome. Rather, neurological function may be the predominant predictive factor. Patient 7 had a relatively high Loes score before transplantation, while neurological function score was relatively low (NFS = 1) with no audiovisual dysfunction, and this patient obtained the greatest bene t after transplantation (NFS = 1). Thus, audiovisual function before transplantation may be a good predictor of prognosis.

Conclusions
Unrelated UCB transplantation is more effective for hereditary leukodystrophy patients with mild or no neurological symptoms, so early diagnosis and timely treatment prior to substantial progression are critical. For these patients lacking MSD, unrelated UCBT is safe and effective. In contrast, the potential value of UCBT for children with rapid disease progression and severe neurological impairment may be limited, so parental expectations should be carefully managed. Figure 1 Time course of Neurologic Function Score changes for all patients.

Figure 2
Time course of Loes score changes for all patients. In some cases, there was no post-UCBT data because of early death (p2) or because the parent (of p4) refused MRI examination after transplantation.