Page 81 - Haematologica May 2020

Extensive mixed chimerism analysis in sickle cell disease post HSCT
Introduction
Sickle cell disease (SCD) is a severe, monogenic disease associated with high mortality and morbidity rates.1 Together with β-thalassemia, SCD constitutes the world’s most prevalent inherited disorder.2,3 Allogeneic human leukocyte antigen (HLA)-matched hematopoietic stem cell transplantation (HSCT) is the only curative treatment. Non-transplanted patients with SCD have a significantly shortened life expectancy, and experience disease-related complications throughout their lives.4–6 With the aim of improving care for patients with SCD, non-myeloablative, reduced-intensity conditioning regi- mens and haploidentical transplants are now being inves- tigated.7–13 Furthermore, recent advances in gene therapy offer new perspectives for the treatment of this serious disease.14,15 However, the curative level of engraftment by genetically modified cells has yet to be determined.
Following HSCT, SCD patients may develop mixed chimerism (MC), i.e. the co-existence of host- and donor- derived cells, which can nevertheless result in the clinical control of the disease.16–21 MC is favored by the increasing use of non-myeloablative reduced-intensity conditioning regimens9–11 and high doses of antithymocyte globulin.22 The minimum level of donor chimerism required to reverse the clinical symptoms of SCD is still subject to debate.21,23 Some literature data show that a donor white blood cell (WBC) percentage as low as 11% is enough to provide clinically adequate disease control20 probably because the healthy cells have a survival advantage over SCD erythroid cells.24 This advantage is also observed in beta-thalassemia.25,26 However, donor chimerism at differ- ent stages of hematopoietic differentiation/development has yet to be analyzed in detail in a large cohort of SCD patients. Furthermore, donor chimerism (typically quanti- fied as the proportion of donor-derived total circulating WBC) might be a poor indicator of the clinical outcome in patients with MC.
We therefore decided to perform an extensive analysis of donor chimerism in different cell lineage populations among a cohort of SCD patients having a mixed chimerism defined in the present study as host cells >0.05% after a full myeloablative conditioning regimen and then genoidentical HSCT. Our objective was to study the hematopoietic reconstitution after HSCT in SCD patients and determine the engraftment threshold for sta- ble disease control. To this end, we performed a multilin- eage analysis of donor chimerism concomitantly in highly purified peripheral blood myeloid and lymphoid lineages, in erythroid and granulomonocytic progenitors/precur- sors, and mature RBC in a large cohort of SCD patients with MC at last follow up.
Our present results may have implications not only for allogeneic HSCT but also for gene therapy trials based on the autologous transplantation of genetically modified CD34+ cells.
Methods
Between May 1990 and December 2013, 119 patients with SCD (registered at the Paris region’s Pediatric Reference Center for SCD (Créteil, France)) underwent HLA-matched allogeneic HSCT at various transplantation centers. These patients are part of the French cohort previously published.4,22 Patients with symp-
tomatic SCD (genotype: S/S or S/ 0) and an HLA-identical sibling donor (hemoglobin [Hb] genotype: AA, AS, A/ 0 or A/D-Punjab) underwent HSCT. The myeloablative conditioning regimen con- sisted of busulfan, cyclophosphamide (total dose: 200 mg/kg), and rabbit anti-thymocyte globulin (total dose: 20 mg/kg). The total dose of intravenous busulfan was adjusted to the recipient’s body weight: 12.8 mg/kg for >34 kg, 15.2 mg/kg for 23-34 kg, 17.6 mg/kg for 16-23 kg, and 19.2 mg/kg for 9-16 kg.
The main inclusion criteria for the present study were the development of MC for total WBC, at least 12 months of follow- up, and regular monitoring at the Reference Center. Post-HSCT blood samples were collected as part of routine care at last follow up. The patients’ medical records were analyzed retrospectively.
Sorting of hematopoietic subpopulations
Cells were stained with specific, directly labeled monoclonal antibodies, according to the manufacturer’s instructions. Chimerism was analyzed only when the population purity was ≥90%.
Clonogenic assay and DNA extraction
Erythroid burst-forming-units (BFU-E) and granulocyte- macrophage colony-forming-units (CFU-GM) progenitors/pre- cursors were grown in semisolid methylcellulose medium with or without supplemented erythropoietin.
Hemoglobin fraction analysis
Values for Hb fractions HbS, HbF, and HbA were determined by cation-exchange high-performance liquid chromatography (HPLC).
Chimerism analysis in mature lymphoid and myeloid populations and in progenitors/precursors
Chimerism was determined in sorted, mature myeloid and lymphoid populations and concomitantly in BFU-E and CFU- GM. Analysis was performed in the Molecular Hematology Laboratory at Henri Mondor Hospital (Creteil, France). Chimerism was first analyzed using quantitative real-time PCR assays for indel genomic polymorphisms (KimerDx kit, GenDX, Netherlands), using a method adapted from a previous publica- tion27 and by PCR-STR when the level was above 10%. Mixed chimerism was defined as a recipient cell percentage above 0.05%. Patients were divided into three groups according to the level of donor chimerism. The 70% and 95% cutoffs were cho- sen on the basis of published data22 and according to the limit usually employed in clinics, respectively. The donor chimerism in peripheral mature RBC was obtained by calculating the post- HSCT proportion of donor HbA.
Statistical analyses
Statistical analysis was performed with ad hoc routines imple- mented in R software (http://www.R-project.org). The data are pre- sented as proportions for categorical data and as median, interquartile range and range for quantitative data. Quantitative variables were compared with the non-parametric Wilcoxon tests and proportions with the Fisher’s exact tests or the chi- squared tests, as appropriate. Correlations between continuous variables were calculated using the non-parametric Spearman’s rank correlation test. Wilcoxon signed-rank test for paired data was used to compare donor chimerism levels in RBC relative to BFU-E, CFU-GM and CD15+ cells. A P-value of 0.05 was consid- ered statistically significant for all analyses. Two-sided tests were used in all analyses. Please see the Online Supplementary Materials and Methods for a more detailed description of the methods used.
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