Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Fanconi Anemia: Results of a Single Center with a Fludarabine-Based Conditioning Regimen

Sabrine Mekni ^*, Lamia Torjemane ^*, Malek Khayati , Dorra Belloumi , Rimmel Yosra Kanoun , Insaf Ben Yaiche , Rihab Ouerghi , Ines Turki , Saloua Ladeb , Tarek Ben Othman , Nour Ben Abdeljelil

1. Department of Hematology and Transplantation, National Center of Bone Marrow Transplantation, Tunis, Tunisia

* Correspondences: Sabrine Mekni and Lamia Torjemane

Academic Editor: Mobil Akhmedov

Special Issue: Allogeneic Stem Cell Transplantation in Fanconi Anemia and Pediatric Bone Marrow Failure

Received: June 29, 2024 | Accepted: October 31, 2024 | Published: November 11, 2024

OBM Transplantation 2024, Volume 8, Issue 4, doi:10.21926/obm.transplant.2404226

Recommended citation: Mekni S, Torjemane L, Khayati M, Belloumi D, Kanoun RY, Yaiche IB, Ouerghi R, Turki I, Ladeb S, Othman TB, Abdeljelil NB. Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Fanconi Anemia: Results of a Single Center with a Fludarabine-Based Conditioning Regimen. OBM Transplantation 2024; 8(4): 226; doi:10.21926/obm.transplant.2404226.

© 2024 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.

1. Introduction

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous recessive disorder characterized by diverse congenital malformations, progressive pancytopenia, and a predisposition to both hematologic malignancies (myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and solid tumors [1]. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option for bone marrow failure (BMF) and other hematologic complications of FA, the risk of solid tumors is still unchanged [2,3,4]. Over the last three decades, allo-HCT has significantly improved outcomes by optimizing conditioning regimens. Taking into account the high sensitivity of FA patients to DNA alkylating agents, toxicity-related chemotherapy had decreased by reducing the intensity of conditioning regimens by using fludarabine (FLU) and improving Graft versus host disease (GVHD) prophylaxis and supportive care [5,6,7]. The majority of published series described single-center experiences with small patient populations. However, a significant cohort study conducted by the European Group for Blood and Marrow Transplantation Working Party found that patients who were transplanted before the age of ten, before clonal development, from a matched family donor, and following a fludarabine based conditioning had superior outcomes [8]. Our prior findings with patients transplanted before 2005, using low-dose cyclophosphamide and busulfan as conditioning, revealed 18% and 23% rates for early graft failure and acute GVHD, respectively [9]. We switched then to a fludarabine-based conditioning program with rabbit anti-thymocyte globulin (ATG). Our study aimed to describe the short- and long-term results of allo-HSCT in FA patients.

2. Material and Methods

2.1 Patients-Donors and Inclusion Criteria

We enrolled all patients with FA who underwent allo-HSCT from an HLA-identical matched sibling donor at the Tunisian National Center of Stem Cell Transplantation from January 2006 to December 2021. Lymphocyte chromosome breaks increasing after exposure to DNA cross-linking agents confirmed the diagnosis of FA. In all cases, a molecular genetic approach involving polymerase chain reaction (PCR) amplification and reversed blot (RDB) hybridization (LiPA tests from Innogenetics) was utilized to type HLA class II on donors. All patients or legal guardians have provided informed consent for the transplant procedure and using their data for scientific research. The hospital's Institutional Committee on Hematopoietic Stem Cell Transplantation approved the study.

2.2 Data Collection and Definitions

Data were gathered from medical records on clinical characteristics at the time of transplantation (growth retardation, skin hypopigmentation, and congenital malformations…), indications for stem cell transplantation, hematological status before transplant, transplant-related data, and short- and long-term results. The severity of abnormalities was determined based on the six anatomical areas implicated. Malformations were classified as extensive if they involved at least three sites and limited if they involved fewer than three.

2.3 Transplant Procedures

A conditioning regimen based on fludarabine was administered to all patients, comprising a total dose of fludarabine 120 mg/m² and a total dose of cyclophosphamide 40 mg/kg. Five patients received an additional dose of anti-thymocyte globulin (ATG) because they were at high risk of graft failure (presence of specific donor HLA antibodies). Bone marrow was the stem cell source used in all patients. GVHD prophylaxis consisted of methotrexate (MTX 5 mg/m² on days 1, 3, and 6) and cyclosporine A (CsA) at a dosage of 3 mg/kg/day i.v. Starting on day 1. Then, when oral intake was possible, CsA was administered orally at a dosage of 6 mg/kg/day in two divided doses, with a gradual tapering throughout 12 to 18 months or longer if the presence of GVHD. Systemic monitoring of CsA levels was performed to maintain a level between 150 and 300 ng/ml. All patients received supportive care (acyclovir and fluconazole prophylaxis, growth factors, and blood products) according to local procedures [9].

2.4 End Points and Statistical Analysis

Engraftment was the first of 3 consecutive days with an absolute neutrophil count greater than 0.5 × 10⁹/L without growth factors and unsupported platelets greater than 20 × 10⁹/L. Primary graft failure was diagnosed if neutrophil recovery was not reached by day 30. Secondary graft failure was defined as recurrent pancytopenia with an absolute neutrophil count <0.5 × 10⁹/L without severe GVHD. The GVHD was graded using the Glucksberg revised criteria and the International Bone Marrow Transplant Registry (IBMTR) Severity Index [10,11]. The c GVHD was diagnosed using published criteria [12].

Overall survival (OS) was defined as the time between the date of allo-HSCT and the date of last patient contact. Event-free survival (EFS) was defined as the time between the date of allo-HSCT and the event (graft failure, secondary malignancy, or leukemia transformation). The probability of survival was calculated using the Kaplan-Meier method. The cumulative incidence (CI) of secondary graft failure and aGVHD and c GVHD were estimated by competing risk function [13]. Kaplan-Meier curves were used to calculate the effect of independent categorical variables on survival. Chi-square or Fisher’s exact tests were used to compare categorical variables. P ≤ 0.05 was considered to indicate a significant association. Statistical analysis was performed using IBM SPSS Statistics for Windows, version 25.0.

3. Results

3.1 Study Cohort

Twenty-three patients were included (14 females and 9 males). Characteristics of the patients and transplant procedures are summarized in Table 1.

Table 1 Characteristics of patients and transplant procedures.

The median age at diagnosis was 10 years (range 5–35). The median age at the time of transplant was 11 years (range: 5–39), and the median time from diagnosis to transplant was 8 months (range: 2–147). Twenty-one (91%) patients had malformation syndrome. Kidney and urogenital tract malformations were found in 11 (47%) of patients. Indications of transplant were BMF in 19 (83%) patients and clonal evolution in 3 (13%) patients. Abnormalities were: 46, XX karyotype with t (1;9) (n = 1); 46, XX karyotype with +21 and -7 (n = 1); and 45, XX with -7 (n = 1). Only one patient was transplanted with myelodysplastic syndrome with complex cytogenetic abnormalities. Seventeen patients (74%) were transfused before transplantation with a median of 5 units of blood products (range: 1-22). Before allo-HSCT, the median neutrophil count was 0.71 × 10³ (0.290-20), and the median platelet count was 23 × 10³ (30-1100). The median number of nucleated cell doses infused per kilogram of recipient body weight was 2.93 × 10⁸ (range; 0.8-6.5).

3.2 Short –Term Outcome

Engraftment occurred in all patients. The median time to neutrophil and platelet engraftment were 12 days (range: 10–18) and 14 days (range: 9-21), respectively. Peripheral blood karyotype analysis showed mixed cytogenetic chimerism in 46% of patients.

3.2.1 Regimen-Related Toxicity (RRT)

Mucositis grade 3 requiring morphine occurred in 14 (61%) patients. Liver dysfunction (transient elevation of liver enzymes) occurred in eight patients (35%), mainly after methotrexate administration, without veno-occlusive disease criteria. Acute renal injury grade 0-2, according to the AKIN classification, with increased creatinine less than twice the basal level, occurred in seven patients (30%). Causes of transient acute renal failure were nephrotoxic drugs such as antibiotics or calcineurin inhibitors (n = 6). Two patients developed CsA-induced thrombotic microangiopathy (TMA) and PRESS syndrome, respectively. CsA withdrawal and its replacement by mycophenolate mofetil (MMF) were indicated for the two patients (8.2%). One patient developed acute pancreatitis (n = 1). No other severe toxicities related to cyclophosphamide, such as hemorrhagic cystitis, were observed. There were no deaths related to chemotherapy. The main complications are summarized in Table 2.

Table 2 Complications following allogenic Stem Cell Transplantation.

3.2.2 Infections

Febrile neutropenia occurred in all transplants. Bacteremia was documented in only three (13%) patients, and it was gram-positive infections (n = 3). Cytomegalovirus reactivation occurred in four (17%) patients, of whom one patient had underlying severe GVHD and was on systemic corticosteroids. Two patients (9%) developed probable pulmonary aspergillosis. Two patients developed an EBV infection.

3.2.3 Graft-Versus-Host Disease

Grade 2-3 aGVHD occurred in three (13%) patients. The CI of aGVHD was 17% (Figure 1). It was refractory in one patient, leading to his death. In univariate analysis, the occurrence of aGVHD was independent of age (<10 or ≥10 years) (p = 0.7), major ABO incompatibility (p = 0.33), conditioning including ATG or not (p = 0.12), and infection (p = 0.6). Acute GVHD was significantly associated with sex mismatch of donor-recipient (p = 0.018). The CI of c GVHD was 32% at 10 years (Figure 2). It was mainly mild (n = 5) and moderate in only one patient, respectively. None of the factors studied for the occurrence of a GVHD was associated with the occurrence of cGVHD.

Figure 1 Cumulative incidence of acute GVHD.

Figure 2 Cumulative incidence of chronic GVHD.

3.3 Long-Term Outcome

3.3.1 Graft Failure

Two patients (one with initially BMF and one with MDS) developed late secondary graft failure, 3 and 5 years after transplant, respectively. The CI of secondary graft failure was 10.5% at 10 years (Figure 3). Graft failure was not associated with age (p = 0.3), sex mismatch (p = 0.45), ABO major incompatibility (p = 0.2), conditioning regimen (p = 0.45), clonal abnormalities (p = 0.37), aGVHD and c GVHD (p = 0.5 and 0.55, respectively).

Figure 3 Cumulative incidence of Graft failure.

3.3.2 Clonal Evolution

Clonal evolution to acute myeloid leukemia (AML) occurred in two (8.6%) patients after 32 and 69 months, respectively.

3.3.3 Late Renal Complications

One patient, without initial renal malformations, developed late renal complications, including nephrotic syndrome, and died of renal failure. The main complications were summarized in Table 2.

3.4 Survival Outcomes

After a median follow-up of 120 months (range; 3-242), 19 patients were alive, and 4 patients died. Causes of death were infection associated with refractory GVHD (n = 1), graft failure (n = 2), and renal failure (n = 1). The 10-year overall survival (OS) and event-free survival (EFS) were 80% and 75%, respectively (Figure 4 and Figure 5). In univariate analysis for OS and EFS, clinical abnormalities, time from diagnosis to transplant, conditioning regimen, a GVHD, or c GVHD were not significant predictors. There was a trend towards a better EFS in patients aged <10 years at transplant (100% vs. 61%, respectively; p = 0.06) (Figure 6 and Figure 7).

Figure 4 The 10-year Overall survival.

Figure 5 The 10-year Event free survival.

Figure 6 Overall survival according to age.

Figure 7 Event-free survival according to age.

4. Discussion

The particular characteristics of our cohort were the presence of malformation syndrome in young children with FA, as well as 47% of renal and/or urinary tract abnormalities.

The main indications for transplantation were bone marrow failure (n = 19), Clonal hematopoiesis with myelodysplastic feature (n = 1), or cytogenetic abnormalities (n = 3). All transplants were done from HLA-matched related donors with bone marrow. A fludarabine-based conditioning regimen resulted in long-term engraftment with mild toxicity in all patients. This regimen improved outcomes in patients with FA, as observed in prior studies [14,15,16,17,18]. The most extensive multicenter study by the European Society for Blood and Marrow Transplantation (EBMT), including 795 patients transplanted with FA, reported better outcomes for patients transplanted before the age of 10 years, before clonal evolution (i.e., myelodysplastic syndrome or acute myeloid leukemia), from a matched family donor, after a conditioning regimen without irradiation but including fludarabine [8].

Our study added ATG to the conditioning regimen to reduce the risk of primary graft failure in heavily transfused patients with anti-HLA immunization (n = 5). The role of ATG, as in vivo T cell depletion, was to improve engraftment and reduce the incidence of acute and chronic GVHD both in the setting of matched family donors (MRD) as well as in matched unrelated donors (MUD) [7,15,19,20]. Our study omitted busulfan (BU) from the conditioning regimen. The Consensus of German Transplant Centers (CGTC) on HSCT in FA patients also recommended omitting busulfan for low-risk patients defined as cytopenia without evidence of leukemia or cytogenetic aberrations and with an HLA identical donor (i.e., ≥9/10 MSD, MRD, or MUD). However, BU is recommended to facilitate engraftment in patients with mismatched/haplo-identical allografts (<9/10) and as an anti-leukemic agent in patients with bone marrow clonal aberrations and/or MDS/AML. The intravenous formulation is preferred due to its more consistent blood levels after pharmacological monitoring [21].

In our study, the CI of a GVHD and mortality related to severe forms was 17% and 4%, respectively. A lower prevalence of severe GVHD in patients who received the fludarabine-based conditioning was reported [2,15,20]. The cumulative incidence of cGVHD was 32% but lower (13%) in our other studies [8,15,21,22]. These different results may be explained by the heterogeneity of the patient population and the use of heterogeneous strategies for GVHD prophylaxis. ATG(s) and alemtuzumab have also been applied as in vivo T cell-depleting agents in FA allo-HSCT to boost engraftment and reduce the incidence and severity of acute and chronic GVHD [23]. To reduce the risk of GVHD, the CGTC recommended in vivo T cell depletion in all FA patients, even those transplanted from an MSD. Alemtuzumab is superior to ATG in FA patients because of its better GVHD prophylactic effect. CSA (day −1 until day +100, then taper) is still employed for GVHD prophylaxis. MMF is added to CSA for unrelated donors only. The continuation or tapering of CSA and MMF depends on the presence or absence of GVHD [21]. In our study, the historical use of methotrexate in GVHD prophylaxis seems to maintain inevitable toxicity (liver, kidneys, and mucosa) without tangible benefits. Thus, GVHD prophylaxis needs to be adapted to avoid an additional risk of interacting with the primary biological defect of FA (i.e., DNA repair processes).

The long-term outcome of our cohort was still affected by other complications, such as clonal evolution with AML (n = 2). FA patients have a markedly higher risk of malignancy, particularly squamous cell carcinomas of the aero esophageal and anogenital tracts, in addition to MDS and AML [3,4,24]. GVHD may contribute to epithelioid malignancies. The use of peripheral blood stem cells was strongly associated with secondary cancer. This result is important enough to consider marrow as this population's recommended source of stem cells. Older age was identified by itself as an independent risk factor for secondary cancer [8]. Two patients developed impure nephrotic syndrome (NS) after allo-HSCT with graft failure (n = 1), cGVHD (n = 1), and rapid evolution to renal failure. NS is a rare manifestation of GVHD. Monitoring renal function and kidney biopsy are essential tools for obtaining information about renal pathology and guiding specific treatment [25,26].

In our study, the 10-year overall survival (OS) and event-free survival (EFS) were 80% and 75%, respectively. Age ≥10 years at the time of transplant seems to be a negative predictor for EFS. Similar results were reported by single-center and multicenter studies with fludarabine-based conditioning for FA patients [6,8,17,27] but for a shorter follow-up period. Bernard et al. reported an OS of 85.4% with matched-related donors in FA [28]. Also, in a large cohort of 813 patients transplanted for Fanconi anemia, the 5-year OS and EFS were 83% and 78%, respectively, and age ≥ 10 years was an independent factor for worse EFS [29]. Other factors were negative predictors for OS: indication for transplant (BMF vs. MDS/AML), HLA mismatch, conditioning including irradiation, year of transplant, and ex vivo T cell depletion [22].

Our study has limitations: a small sample size of patients, which could be attributed to the disease's rarity. Furthermore, because this is a retrospective research, we had several missing documented data points for some patients. However, the main strength of our study was the long-term follow-up. We conclude with three critical points. First, flu-based protocols are strongly recommended for allogeneic transplantation for FA, which reduces graft failure incidence and severity of GVHD and improves OS with an acceptable toxicity profile. Second, adding ATG, as in vivo T cell depletion, may reduce acute and chronic GVHD incidence and severity. Third and finally, both conditioning regimens and GVHD prophylaxis need to be adapted to the risk factors of each patient taking into account their toxicity and benefits.

Author Contributions

Dr Mekni and Dr Torjemane: writing and editing. Dr Khayati: data collection. All other authors: reviewing and editing.

Competing Interests

The authors have declared that no competing interests exist.