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M.M. Cuadrado et al.
function. Supportive care including growth factors and blood products are routinely administered but the latter can be associated with allo-immunization and transfu- sion-related iron overload. Other approaches, including the use of thrombopoietin-receptor agonists, are currently being investigated in early phase clinical trials. Second allogeneic SCT or infusions of unmanipulated peripheral blood stem cells are other options but are associated with a high risk of GvHD and non-relapse mortality.6 Larocca et al.7 reported on the use of CD34+-selected stem cell infu- sions from the original donor without conditioning for correction of poor graft function based on the premise that the risk of GvHD would be low. Clinical outcomes were favorable when compared to those of historical cohorts of patients who were given either no treatment or unmanip- ulated bone marrow/peripheral blood stem cells without pre-conditioning. Several recent series of patients admin- istered CD34+-selected stem cell infusions have also shown promising results with an improvement of graft function reported in 72-81% of patients (Online Supplementary Table S1).8-11 Although these studies were very important in establishing the principle of CD34+- selected cells in the management of poor graft function, the small number of patients and heterogeneity, in terms of definitions of poor graft function or response, have made it difficult to predict which patients will benefit most from this treatment. Some studies excluded patients with GvHD, with active infection or use of myelosuppres- sive drugs, although in practice it is often difficult to deter- mine the relative effect of such factors on the graft. Furthermore, all the studies to date have excluded patients with significant mixed chimerism, a group with increasing prevalence given the frequent use of reduced intensity conditioning and T-cell depletion. Thus, there is a need to identify predictors of response in clinically relevant popu- lations of patients to ensure both suitable resource alloca- tion and appropriate requests for repeat donor harvesting.
Here we report the outcome and analysis of predictors of recovery in 62 consecutive patients with poor graft func- tion who were treated with donor CD34+-selected infusion without conditioning. While the majority of patients had complete or partial recovery, cytomegalovirus (CMV) seropositivity, donor-recipient sex mismatching and active infection were all associated with inferior outcomes. Thus, our findings demonstrate the overall feasibility of the approach but also indicate that new strategies are still required in some groups of patients.
Methods
Definitions
Engraftment was defined as the first of 3 consecutive days when the absolute neutrophil count was ≥0.5x109/L and the absolute platelet count was ≥20x109/L with or without the administration of granulocyte colony-stimulating factor and without transfusion. Primary poor graft function was defined by: (i) failure to ever achieve count recovery in at least one lineage (neutrophils ≥0.5x109/L, platelets ≥20 x109/L and hemoglobin ≥8 g/dL in the absence of transfusion) after transplantation; (ii) a hypoplastic bone marrow; (iii) the absence of relapse; and (iv) the presence of donor cells as detected by peripheral blood chimerism studies. Secondary poor graft function was defined as for primary poor graft function with the exception that blood counts fell in at least one lineage after the initial achievement of
engraftment. Recovery was categorized as complete or partial. Complete recovery was defined as a hematological improvement in all three cell lineages (hemoglobin ≥8 g/dL, platelets ≥30x109/L and neutrophils ≥1.5x109/L) without the need for transfusion or growth factor support. Partial recovery was defined as a hemato- logic improvement in one or two lineages. Acute GvHD after CD34+-selected infusion was defined according to the criteria of Glucksberg et al.,12 and chronic GvHD was defined as mild, mod- erate or severe, following the National Institutes of Health con- sensus criteria.13 Active infection was identified using the surro- gate of parenteral antimicrobial therapy at the time of CD34+- selected infusion.
Patients
Between 1999-2018, 1996 allogeneic SCT were performed at University College Hospital and Royal Free Hospital in London, UK (Table 1). Seventy patients who received CD34+-selected infusions were identified; eight patients were excluded from the analysis because of disease relapse. This research project was considered by the NHS Health Research Authority as a non- Research Ethics Committee study and was conducted in line with the harmonized UK-wide edition of the Governance Arrangements for Research Ethics Committees (GAfREC) 2018 and the UK Policy Framework for Health and Social Care Research (2017).
Chimerism analysis
Chimerism was analyzed by fluorescence in situ hybridization using the XX/XY dual color probe in whole blood or by lineage- specific chimerism using polymerase chain reaction analysis of informative minisatellite regions (short tandem repeat loci), as previously described,14 within 60 days prior to CD34+-selected infusion. This information was available for 87% of patients. Mixed chimerism of individual cell fractions was defined as the co-existence of donor and recipient DNA with the detection limit being 1-5% according to the individual short tandem repeat marker and the combination of homozygosity versus heterozy- gosity for each marker between donor and patient. Full donor chimerism was defined as the absence of detectable donor DNA in the relevant cell fraction using these sensitivity thresholds.
CD34+ stem cell selection
CD34+ cells were selected from peripheral blood stem cells that had been mobilized into the periphery by granulocyte colony- stimulating factor using the CliniMACS CD34 enrichment sys- tem (Miltenyi Biotec GmbH, Germany)15 (Online Supplementary Comment 1). The CD34+-selected cells were infused within 24 h of selection and without cryopreservation. In six patients with mixed chimerism, a fixed dose of T cells (median CD3+ dose of 1x106, range 1x106 - 1x108) was administered at the time of the CD34+-selected cell infusion.
Statistical analysis
Recovery was compared between categorical variables using the c2 test or Fisher exact test as appropriate, and between con- tinuous variables using the Mann-Whitney U test. Variables for which significant differences were found in univariate analyses were entered into a logistic regression analysis with a forward stepping procedure to find the best model. Probabilities of overall survival were calculated using the Kaplan-Meier method and groups compared with the log-rank test. Probabilities of recovery were estimated using the cumulative incidence procedure, and groups compared using Gray's test. SPSS version 24.0 (IBM SPSS Statistics for Windows, version 24.0. IBM Corp, Armonk, NY, USA) and R version 3.4.2 16 were used for all analyses.
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