ABSTRACT
Genetic lesions of IKZF1 are frequent events and well-established markers of adverse risk in acute lymphoblastic leukemia. However, their function in the pathophysiology and impact on patient outcome in acute myeloid leukemia (AML) remains elusive. In a multicenter cohort of 1606 newly diagnosed and intensively treated adult AML patients, we found IKZF1 alterations in 45 cases with a mutational hotspot at N159S. AML with mutated IKZF1 was associated with alterations in RUNX1, GATA2, KRAS, KIT, SF3B1, and ETV6, while alterations of NPM1, TET2, FLT3-ITD, and normal karyotypes were less frequent. The clinical phenotype of IKZF1-mutated AML was dominated by anemia and thrombocytopenia. In both univariable and multivariable analyses adjusting for age, de novo and secondary AML, and ELN2022 risk categories, we found mutated IKZF1 to be an independent marker of adverse risk regarding complete remission rate, event-free, relapse-free, and overall survival. The deleterious effects of mutated IKZF1 also prevailed in patients who underwent allogeneic hematopoietic stem cell transplantation (n = 519) in both univariable and multivariable models. These dismal outcomes are only partially explained by the hotspot mutation N159S. Our findings suggest a role for IKZF1 mutation status in AML risk modeling.
Subject(s)
Hematopoietic Stem Cell Transplantation , Leukemia, Myeloid, Acute , Adult , Humans , Nucleophosmin , Mutation , Transcription Factors/genetics , Leukemia, Myeloid, Acute/pathology , fms-Like Tyrosine Kinase 3/genetics , Prognosis , Ikaros Transcription Factor/geneticsABSTRACT
Tandem-duplication mutations of the UBTF gene (UBTF-TDs) coding for the upstream binding transcription factor have recently been described in pediatric patients with acute myeloid leukemia (AML) and were found to be associated with particular genetics (trisomy 8 (+8), FLT3-internal tandem duplications (FLT3-ITD), WT1-mutations) and inferior outcome. Due to limited knowledge on UBTF-TDs in adult AML, we screened 4247 newly diagnosed adult AML and higher-risk myelodysplastic syndrome (MDS) patients using high-resolution fragment analysis. UBTF-TDs were overall rare (n = 52/4247; 1.2%), but significantly enriched in younger patients (median age 41 years) and associated with MDS-related morphology as well as significantly lower hemoglobin and platelet levels. Patients with UBTF-TDs had significantly higher rates of +8 (34% vs. 9%), WT1 (52% vs. 7%) and FLT3-ITD (50% vs. 20.8%) co-mutations, whereas UBTF-TDs were mutually exclusive with several class-defining lesions such as mutant NPM1, in-frame CEBPAbZIP mutations as well as t(8;21). Based on the high-variant allele frequency found and the fact that all relapsed patients analyzed (n = 5) retained the UBTF-TD mutation, UBTF-TDs represent early clonal events and are stable over the disease course. In univariate analysis, UBTF-TDs did not represent a significant factor for overall or relapse-free survival in the entire cohort. However, in patients under 50 years of age, who represent the majority of UBTF-mutant patients, UBTF-TDs were an independent prognostic factor for inferior event-free (EFS), relapse-free (RFS) and overall survival (OS), which was confirmed by multivariable analyses including established risk factors such as age and ELN2022 genetic risk groups (EFS [HR: 2.20; 95% CI 1.52-3.17, p < 0.001], RFS [HR: 1.59; 95% CI 1.02-2.46, p = 0.039] and OS [HR: 1.64; 95% CI 1.08-2.49, p = 0.020]). In summary, UBTF-TDs appear to represent a novel class-defining lesion not only in pediatric AML but also younger adults and are associated with myelodysplasia and inferior outcome in these patients.
Subject(s)
Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Adult , Humans , Child , Nuclear Proteins/genetics , Nucleophosmin , Leukemia, Myeloid, Acute/genetics , Mutation , Myelodysplastic Syndromes/genetics , Prognosis , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
Minimal residual disease (MRD) detection is a strong predictor for survival and relapse in acute myeloid leukemia (AML). MRD can be either determined by molecular assessment strategies or via multiparameter flow cytometry. The degree of bone marrow (BM) dilution with peripheral blood (PB) increases with aspiration volume causing consecutive underestimation of the residual AML blast amount. In order to prevent false-negative MRD results, we developed Cinderella, a simple automated method for one-tube simultaneous measurement of hemodilution in BM samples and MRD level. The explainable artificial intelligence (XAI) Cinderella was trained and validated with the digital raw data of a flow cytometric "8-color" AML-MRD antibody panel in 126 BM and 23 PB samples from 35 patients. Cinderella predicted PB dilution with high accordance compared to the results of the Holdrinet formula (Pearson's correlation coefficient r = 0.94, R2 = 0.89, p < 0.001). Unlike conventional neuronal networks Cinderella calculated the distributions of 12 different cell populations that were assigned to true hematopoietic counterparts as a human in the loop (HIL) approach. Besides characteristic BM cells such as myelocytes and myeloid progenitor cells the XAI identified discriminating populations, which were not specific for BM or PB (e.g., T cell/NK cell subpopulations and CD45 negative cells) and considered their frequency differences. Thus, Cinderella represents a HIL-XAI algorithm capable to calculate the degree of hemodilution in BM samples with an AML MRD immunophenotype panel. It is explicable, transparent, and paves a simple way to prevent false negative MRD reports.
Subject(s)
Bone Marrow , Leukemia, Myeloid, Acute , Humans , Neoplasm, Residual/diagnosis , Artificial Intelligence , HemodilutionABSTRACT
BACKGROUND: The bone marrow blast count is central to the diagnosis and monitoring of myelodysplastic syndromes (MDS). It is an independent risk factor for worse prognosis whether based on the morphology blast count or the flow cytometry (FC) myeloid progenitor (MyP) count. It is a principal population in FC MDS analysis also because once defined; it provides significant contributions to the overall FC MDS score. METHODS: We elected to investigate inter-analyst agreement for the most fundamental parameter of the FC MDS diagnostic score: the MyP count. A common gating strategy was agreed and used by seven cytometrists for blind analysis of 34 routine bone marrows sent for MDS work-up. Additionally, we compared the results with a computational approach. RESULTS: Concordance was excellent: Intraclass correlation was 0.993 whether measuring %MyP of total cells or CD45+ cells, and no significant difference was observed between files from different centers or for samples with abnormal MyP phenotypes. Computational and manual results were similar. Applying the common strategy to individual laboratories' control cohorts produced similar MyP reference ranges across centers. CONCLUSION: The FC MyP count offers a reliable diagnostic and prognostic measurement in MDS. The use of manual and computational approaches side by side may allow for optimizing both strategies. Considering its known prognostic power, the MyP count could be considered a useful and reliable addition to existing prognostic scoring systems.
Subject(s)
Myelodysplastic Syndromes , Humans , Flow Cytometry/methods , Myelodysplastic Syndromes/diagnosis , Myelodysplastic Syndromes/genetics , Bone Marrow , Bone Marrow Cells , Myeloid Progenitor CellsABSTRACT
Functional impairment of the bone marrow (BM) niche has been suggested as a major reason for prolonged cytopenia and secondary graft failure after allogeneic hematopoietic cell transplantation (alloHCT). Because mesenchymal stromal cells (MSCs) serve as multipotent progenitors for several niche components in the BM, they might play a key role in this process. We used collagenase digested trephine biopsies to directly quantify MSCs in 73 patients before (n = 18) and/or after alloHCT (n = 65). For the first time, we demonstrate that acute graft-versus-host disease (aGvHD, n = 39) is associated with a significant decrease in MSC numbers. MSC reduction can be observed even before the clinical onset of aGvHD (n = 10). Assessing MSCs instantly after biopsy collection revealed phenotypic and functional differences depending on the occurrence of aGvHD. These differences vanished during ex vivo expansion. The MSC endotypes observed revealed an enhanced population of donor-derived classical dendritic cells type 1 and alloreactive T cells as the causing agent for compartmental inflammation and MSC damage before clinical onset of aGvHD was ascertained. In conclusion, MSCs endotypes may constitute a predisposing conductor of alloreactivity after alloHCT preceding the clinical diagnosis of aGvHD.
Subject(s)
Graft vs Host Disease , Hematopoietic Stem Cell Transplantation , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Humans , Graft vs Host Disease/diagnosis , Bone Marrow/pathology , Mesenchymal Stem Cell Transplantation/adverse effects , Hematopoietic Stem Cell Transplantation/adverse effects , Mesenchymal Stem Cells/metabolismABSTRACT
Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.Luspatercept has high clinical activity in patients with transfusion-dependent lower-risk myelodysplastic syndromes (LR-MDS) and ring sideroblasts (RS) relapsed or refractory to erythropoietin. We report long-term luspatercept safety and efficacy in 108 patients with LR-MDS in the PACE-MDS study, including 44 non-RS and 34 non-transfusion-dependent or previously untreated patients. The primary end point was safety. Secondary end points included rates of hematologic improvement (HI) erythroid (HI-E), HI neutrophil, and HI platelet. Exploratory end points included erythropoiesis biomarker quantitation and mutation data. Median duration of luspatercept exposure was 315 days (range, 21-1,934 days). No new safety signals emerged. HI-E was observed in 53.7% of patients, including 36.4% of non-RS and 70.6% of non-transfusion-dependent patients. HI neutrophil and HI platelet were observed in 33.3% and 9.5% of patients, respectively. An almost three-fold increase in bone marrow late to early progenitor cell ratio accompanied HI-E response, irrespective of RS status. Lower baseline erythropoietin levels in non-RS patients (69.6 v 623.3 IU/L; P = .0077) and higher late to early erythroid progenitor cell ratio (10.44 v 4.48; P = .0106) in RS patients were associated with HI-E. This study highlights luspatercept's effects across LR-MDS subtypes, including untreated MDS-RS, serving as a platform for future trials.
Subject(s)
Anemia , Erythropoietin , Myelodysplastic Syndromes , Humans , Myelodysplastic Syndromes/complications , Myelodysplastic Syndromes/drug therapy , Activin Receptors, Type II/therapeutic use , Anemia/chemically induced , Anemia/drug therapyABSTRACT
Measurable residual disease (MRD) detected by multiparametric flow cytometry (MFC) is associated with unfavorable outcome in patients with AML. A simple, broadly applicable eight-color panel was implemented and analyzed utilizing a hierarchical gating strategy with fixed gates to develop a clear-cut LAIP-based DfN approach. In total, 32 subpopulations with aberrant phenotypes with/without expression of markers of immaturity were monitored in 246 AML patients after completion of induction chemotherapy. Reference values were established utilizing 90 leukemia-free controls. Overall, 73% of patients achieved a response by cytomorphology. In responders, the overall survival was shorter for MRDpos patients (HR 3.8, p = 0.006). Overall survival of MRDneg non-responders was comparable to MRDneg responders. The inter-rater-reliability for MRD detection was high with a Krippendorffs α of 0.860. The mean time requirement for MRD analyses at follow-up was very short with 04:31 minutes. The proposed one-tube MFC approach for detection of MRD allows a high level of standardization leading to a promising inter-observer-reliability with a fast turnover. MRD defined by this strategy provides relevant prognostic information and establishes aberrancies outside of cell populations with markers of immaturity as an independent risk feature. Our results imply that this strategy may provide the base for multicentric immunophenotypic MRD assessment.
Subject(s)
Leukemia, Myeloid, Acute , Flow Cytometry , Humans , Immunophenotyping , Neoplasm, Residual , Prognosis , Reproducibility of ResultsABSTRACT
Three different Flow Cytometry datasets consisting of diagnostic samples of either peripheral blood (pB) or bone marrow (BM) from patients without any sign of bone marrow disease at two different health care centers are provided. In Flow Cytometry, each cell rapidly passes through a laser beam one by one, and two light scatter, and eight surface parameters of more than 100.000 cells are measured per sample of each patient. The technology swiftly characterizes cells of the immune system at the single-cell level based on antigens presented on the cell surface that are targeted by a set of fluorochrome-conjugated antibodies. The first dataset consists of N=14 sample files measured in Marburg and the second dataset of N=44 data files measured in Dresden, of which half are BM samples and half are pB samples. The third dataset contains N=25 healthy bone marrow samples and N=25 leukemia bone marrow samples measured in Marburg. The data has been scaled to log between zero and six and used to identify cell populations that are simultaneously meaningful to the clinician and relevant to the distinction of pB vs BM, and BM vs leukemia. Explainable artificial intelligence methods should distinguish these samples and provide meaningful explanations for the classification without taking more than several hours to compute their results. The data described in this article are available in Mendeley Data [1].
ABSTRACT
In patients who have undergone allogeneic hematopoietic cell transplantation (HCT), myeloid mixed donor chimerism (MC) is a risk factor for disease relapse. In contrast, several studies found favorable outcome in patients with lymphoid MC. Thus far, most studies evaluating MC focused on a short-term follow-up period. Here, we report the first case series of long-term survivors with MC. We screened 1,346 patients having undergone HCT for myeloid neoplasms at our center from 1996 to 2016; 443 patients with data on total peripheral blood mononuclear cells (PBMC)/CD4+/CD34+ short tandem repeat (STR) donor chimerism (DC) and follow-up ≥24 months post-HCT were included. We identified 10 patients with long-term MC (PBMC DC <95% at ≥12 months post-HCT). Median follow-up was 11 years. All patients had received combined ex vivo/in vivo T cell-depleted (TCD) peripheral blood stem cells; none experienced ≥grade 2 acute graft-versus-host disease (GVHD). The mean total PBMC, CD4+, and CD34+ DC of all patients were 95.88%, 85.84%, and 90.15%, respectively. Reduced-intensity conditioning (RIC) was associated with a trend to lower mean total DC. Of note, two patients who experienced relapse had lower CD34+ DC but higher CD4+ DC as compared with patients in continuous remission. Bone marrow evaluation revealed increased CD4+/FOXP3+ cells in patients with MC, which might indicate expansion of regulatory T cells (Tregs). Our results support known predictive factors associated with MC such as RIC and TCD, promote the value of CD34+ MC as a potential predictor of relapse, highlight the potential association of CD4+ MC with reduced risk of GVHD, and indicate a possible role of Tregs in the maintenance of immune tolerance post-HCT.
ABSTRACT
The bone marrow microenvironment (BMME) plays a key role in the pathophysiology of myelodysplastic syndromes (MDS), clonal blood disorders affecting the differentiation, and maturation of hematopoietic stem and progenitor cells (HSPCs). In lower-risk MDS patients, ineffective late-stage erythropoiesis can be restored by luspatercept, an activin receptor type IIB ligand trap. Here, we investigated whether luspatercept can modulate the functional properties of mesenchymal stromal cells (MSCs) as key components of the BMME. Luspatercept treatment inhibited Smad2/3 phosphorylation in both healthy and MDS MSCs and reversed disease-associated alterations in SDF-1 secretion. Pre-treatment of MDS MSCs with luspatercept restored the subsequent clonogenic potential of co-cultured HSPCs and increased both their stromal-adherence and their expression of both CXCR4 and ß3 integrin. Luspatercept pre-treatment of MSCs also increased the subsequent homing of co-cultured HSPCs in zebrafish embryos. MSCs derived from patients who had received luspatercept treatment had an increased capacity to maintain the colony forming potential of normal but not MDS HSPCs. These data provide the first evidence that luspatercept impacts the BMME directly, leading to a selective restoration of the ineffective hematopoiesis that is a hallmark of MDS.
Subject(s)
Activin Receptors, Type II/pharmacology , Chemokine CXCL12/metabolism , Hematopoiesis , Hematopoietic Stem Cells/drug effects , Immunoglobulin Fc Fragments/pharmacology , Mesenchymal Stem Cells/drug effects , Myelodysplastic Syndromes/drug therapy , Recombinant Fusion Proteins/pharmacology , Adult , Aged , Animals , Case-Control Studies , Chemokine CXCL12/genetics , Hematinics/pharmacology , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Humans , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Middle Aged , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/pathology , Smad2 Protein/genetics , Smad2 Protein/metabolism , Tumor Cells, Cultured , ZebrafishSubject(s)
Antibodies, Monoclonal/administration & dosage , Azacitidine/adverse effects , Drug Resistance, Neoplasm/drug effects , Drug-Related Side Effects and Adverse Reactions/diagnosis , Interleukin-3 Receptor alpha Subunit/antagonists & inhibitors , Myelodysplastic Syndromes/drug therapy , Aged , Aged, 80 and over , Antibodies, Monoclonal/adverse effects , Antibody-Dependent Cell Cytotoxicity , Antimetabolites, Antineoplastic/adverse effects , Drug-Related Side Effects and Adverse Reactions/etiology , Female , Follow-Up Studies , Humans , Interleukin-3 Receptor alpha Subunit/immunology , Male , Myelodysplastic Syndromes/immunology , Myelodysplastic Syndromes/pathology , Prognosis , Survival RateABSTRACT
Isolation of mesenchymal stromal cells (MSCs) from pretreated, hematologic patients is challenging. Especially after allogeneic hematopoietic cell transplantation (HCT), standard protocols using bone marrow aspirates fail to reliably recover sufficient cell numbers. Because MSCs are considered to contribute to processes that mainly affect the outcome after transplantation, such as an efficient lymphohematopoietic recovery, extent of graft-versus-host disease as well as the occurrence of leukemic relapse, it is of great clinical relevance to investigate MSC function in this context. Previous studies showed that MSCs can be isolated by collagenase digestion of large bone fragments of hematologically healthy patients undergoing hip replacement or knee surgeries. We have now further developed this procedure for the isolation of MSCs from hematologic patients after allogeneic HCT by using trephine biopsy specimens obtained during routine examinations. Comparison of aspirates and trephine biopsy specimens from patients after allogeneic HCT revealed a significantly higher frequency of clonogenic MSCs (colony-forming unit-fibroblast [CFU-F]) in trephine biopsy specimens (mean, 289.8 ± standard deviation 322.5 CFU-F colonies/1 × 106 total nucleated cells versus 4.2 ± 9.9; P < 0.0001). Subsequent expansion of functional MSCs isolated from trephine biopsy specimen was more robust and led to a significantly higher yield compared with control samples expanded from aspirates (median, 1.6 × 106; range, 0-2.3 × 107 P0 MSCs versus 5.4 × 104; range, 0-8.9 × 106; P < 0.0001). Using trephine biopsy specimens as MSC source facilitates the investigation of various clinical questions.
Subject(s)
Bone Marrow Cells/cytology , Hematopoietic Stem Cell Transplantation/methods , Leukemia/therapy , Mesenchymal Stem Cells/cytology , Adult , Aged , Biopsy , Bone Marrow , Collagenases/pharmacology , Female , Graft vs Host Disease/pathology , Humans , Male , Middle Aged , Tumor Cells, Cultured , Young AdultABSTRACT
Adoptive transfer of T regulatory cells (Treg) has been successfully exploited in the context of graft-versus-host disease, transplantation, and autoimmune disease. For the majority of applications, clinical administration of Treg requires laborious ex vivo expansion and typically involves open handling for culture feeds and repetitive sampling. Here we show results from our approach to translate manual Treg manufacturing to the fully closed automated CliniMACS Prodigy® system reducing contamination risk, hands-on time, and quality variation from human intervention. Polyclonal Treg were isolated from total nucleated cells obtained through leukapheresis of healthy donors by CD8+ cell depletion and subsequent CD25high enrichment. Treg were expanded with the CliniMACS Prodigy® device using clinical-grade cell culture medium, rapamycin, IL-2, and αCD3/αCD28 beads for 13-14 days. We successfully integrated expansion bead removal and final formulation into the automated procedure, finalizing the process with a ready to use product for bedside transfusion. Automated Treg expansion was conducted in parallel to an established manual manufacturing process using G-Rex cell culture flasks. We could prove similar expansion kinetics leading to a cell yield of up to 2.12 × 109 cells with the CliniMACS Prodigy® and comparable product phenotype of >90% CD4+CD25highCD127lowFOXP3+ cells that had similar in vitro immunosuppressive function. Efficiency of expansion bead depletion was comparable to the CliniMACS® Plus system and the final ready-to-infuse product had phenotype stability and high vitality after overnight storage. We anticipate this newly developed closed system expansion approach to be a starting point for the development of enhanced throughput clinical scale Treg manufacture, and for safe automated generation of antigen-specific Treg grafted with a chimeric antigen receptor (CAR Treg).
Subject(s)
Batch Cell Culture Techniques , Immunotherapy, Adoptive , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Automation , Biomarkers , Cell Separation , Cell- and Tissue-Based Therapy , Humans , Immunophenotyping , Immunotherapy, Adoptive/methods , T-Lymphocytes, Regulatory/cytologyABSTRACT
Alloreactivity or opportunistic infections following allogeneic stem cell transplantation are difficult to predict and contribute to post-transplantation mortality. How these immune reactions result in changes to the T-cell receptor repertoire remains largely unknown. Using next-generation sequencing, the T-cell receptor alpha (TRα) repertoire of naïve and memory CD8+ T cells from 25 patients who had received different forms of allogeneic transplantation was analyzed. In parallel, reconstitution of the CD8+/CD4+ T-cell subsets was mapped using flow cytometry. When comparing the influence of anti-T-cell therapy, a delay in the reconstitution of the naïve CD8+ T-cell repertoire was observed in patients who received in vivo T-cell depletion using antithymocyte globulin or post-transplantation cyclophosphamide in case of haploidentical transplantation. Sequencing of the TRα identified a repertoire consisting of more dominant clonotypes (>1% of reads) in these patients at 6 and 18 months post transplantation. When comparing donor and recipient, approximately 50% and approximately 80% of the donors' memory repertoire were later retrieved in the naïve and memory CD8+ T-cell receptor repertoire of the recipients, respectively. Although there was a remarkable expansion of single clones observed in the recipients' memory CD8+ TRα repertoire, no clear association between graft-versus-host disease or cytomegalovirus infection and T-cell receptor diversity was identified. A lower TRα diversity was observed in recipients of a cytomegalovirus-seropositive donor (P=0.014). These findings suggest that CD8+ T-cell reconstitution in transplanted patients is influenced by the use of T-cell depletion or immunosuppression and the donor repertoire.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , CD8-Positive T-Lymphocytes/immunology , Female , Graft vs Host Disease/etiology , Hematopoietic Stem Cell Transplantation , High-Throughput Nucleotide Sequencing , Humans , Immunologic Memory , Lymphocyte Depletion , Male , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Transplantation, HomologousABSTRACT
BACKGROUND: Monitoring of measurable residual disease (MRD) in patients with advanced myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML) who achieve a morphological complete remission can predict haematological relapse. In this prospective study, we aimed to determine whether MRD-guided pre-emptive treatment with azacitidine could prevent relapse in these patients. METHODS: The relapse prevention with azacitidine (RELAZA2) study is an open-label, multicentre, phase 2 trial done at nine university health centres in Germany. Patients aged 18 years or older with advanced MDS or AML, who had achieved a complete remission after conventional chemotherapy or allogeneic haemopoietic stem-cell transplantation, were prospectively screened for MRD during 24 months from baseline by either quantitative PCR for mutant NPM1, leukaemia-specific fusion genes (DEK-NUP214, RUNX1-RUNX1T1, CBFb-MYH11), or analysis of donor-chimaerism in flow cytometry-sorted CD34-positive cells in patients who received allogeneic haemopoietic stem-cell transplantation. MRD-positive patients in confirmed complete remission received azacitidine 75 mg/m2 per day subcutaneously on days 1-7 of a 29-day cycle for 24 cycles. After six cycles, MRD status was reassessed and patients with major responses (MRD negativity) were eligible for a treatment de-escalation. The primary endpoint was the proportion of patients who were relapse-free and alive 6 months after the start of pre-emptive treatment. Analyses were done per protocol. This trial is registered with ClincialTrials.gov, number NCT01462578, and finished recruitment on Aug 21, 2018. FINDINGS: Between Oct 10, 2011, and Aug 20, 2015, we screened 198 patients with advanced MDS (n=26) or AML (n=172), of whom 60 (30%) developed MRD during the 24-month screening period and 53 (88%) were eligible to start study treatment. 6 months after initiation of azacitidine, 31 (58%, 95% CI 44-72) of 53 patients were relapse-free and alive (p<0·0001; one-sided binomial test for null hypothesis pexp≤0·3). With a median follow-up of 13 months (IQR 8·5-22·8) after the start of MRD-guided treatment, relapse-free survival at 12 months was 46% (95% CI 32-59) in the 53 patients who were MRD-positive and received azacitidine. In MRD-negative patients, 12-month relapse-free survival was 88% (95% CI 82-94; hazard ratio 6·6 [95% CI 3·7-11·8], p<0·0001). The most common (grade 3-4) adverse event was neutropenia, occurring in 45 (85%) of 53 patients. One patient with neutropenia died because of an infection considered possibly related to study treatment. INTERPRETATION: Pre-emptive therapy with azacitidine can prevent or substantially delay haematological relapse in MRD-positive patients with MDS or AML who are at high risk of relapse. Our study also suggests that continuous MRD negativity during regular MRD monitoring might be prognostic for patient outcomes. FUNDING: Celgene Pharma, José Carreras Leukaemia Foundation, National Center for Tumor Diseases (NCT), and German Cancer Consortium (DKTK) Foundation.
Subject(s)
Antimetabolites, Antineoplastic/administration & dosage , Azacitidine/administration & dosage , Leukemia, Myeloid, Acute/drug therapy , Myelodysplastic Syndromes/drug therapy , Aged , Antimetabolites, Antineoplastic/adverse effects , Azacitidine/adverse effects , Biomarkers, Tumor/genetics , Biomarkers, Tumor/immunology , Drug Administration Schedule , Female , Germany , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Male , Middle Aged , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/immunology , Neoplasm, Residual , Nucleophosmin , Progression-Free Survival , Prospective Studies , Recurrence , Risk Assessment , Risk Factors , Time FactorsABSTRACT
This study was conducted to characterize and compare peripheral blood stem cell grafts from healthy donors who underwent granulocyte colony-stimulating factor (G-CSF) mobilization and subsequently received 1 dose of plerixafor after insufficient stem cell yields were achieved at the first apheresis. Aliquots from 35 donors were collected from the first apheresis after mobilization with G-CSF alone and from the second apheresis after additional plerixafor administration. Samples were freshly analyzed for cellular subsets by 8-color flow cytometry. Leukapheresis samples mobilized with additional plerixafor showed a significant increase of total nucleated cells, including B cells, CD4+ and CD8+ T cells, and CD34+ hematopoietic stem and progenitor cells. Absolute numbers of plasmacytoid dendritic cells were also significantly increased, whereas no changes were detected for myeloid dendritic cells. Furthermore, absolute numbers of regulatory T cells increased, with naive CD45RA+ regulatory T cells showing the highest rise. Finally, strikingly higher numbers of myeloid-derived suppressor cells were detected in the plerixafor and G-CSF-mobilized graft. The mobilization of peripheral stem cells in healthy donors with G-CSF and plerixafor led to a significant difference in cellular graft composition compared with G-CSF alone. The clinical impact of the different cell composition for the graft recipient warrants further clinical investigation.
Subject(s)
Anti-HIV Agents/therapeutic use , Granulocyte Colony-Stimulating Factor/therapeutic use , Hematopoietic Stem Cell Mobilization/methods , Heterocyclic Compounds/therapeutic use , Leukapheresis/methods , Peripheral Blood Stem Cell Transplantation/methods , Peripheral Blood Stem Cells/metabolism , Transplants/transplantation , Anti-HIV Agents/pharmacology , Benzylamines , Cyclams , Female , Granulocyte Colony-Stimulating Factor/pharmacology , Healthy Volunteers , Heterocyclic Compounds/pharmacology , Humans , Male , Tissue DonorsSubject(s)
Blood Donors , High-Throughput Nucleotide Sequencing , Receptors, Antigen, T-Cell/genetics , Adult , Humans , MaleABSTRACT
INTRODUCTION: Acute myeloid leukemia (AML) rarely involves the central nervous system (CNS). Little is known about the clinical course in adult AML patients since most studies examined pediatric patients. Therefore, this study analyzed the data of patients treated in three prospective trials of the "Study Alliance Leukemia" (SAL) study group for CNS involvement. METHODS: In all, 3,261 AML patients included in the prospective AML96, AML2003, and AML60+ trials of the SAL study group were analyzed. Symptomatic patients underwent cerebrospinal fluid (CSF) puncture and CNS involvement was diagnosed depending on morphology and/or flow cytometry of the CSF. Cytogenetic, molecular, clinical, and laboratory parameters were analyzed in order to identify risk factors. RESULTS: A total of 55 patients had proven symptomatic CNS involvement. Significantly more patients revealed CNS involvement at relapse (34 patients, 2.9%) compared with first diagnosis (21 patients, 0.6%), p<0.001. CNS involvement at initial diagnosis had a significantly higher frequency in patients with complex aberrant karyotypes, high serum lactate dehydrogenase activity, French-American-British M5 subtype, FLT3-internal tandem duplication (ITD) mutations alone, and co-occurrence of a FLT3-ITD and NPM1 mutation. Furthermore, AML patients with CNS involvement at diagnosis had an inferior outcome compared with patients without CNS involvement even if treated with intrathecal chemotherapy with an overall survival of 11% versus 30% at 5 years, p=0.004. CONCLUSION: This study analyzed the largest data set of adult AML patients with proven CNS involvement reported so far. The data demonstrated very low prevalence of CNS involvement at initial diagnosis in adult patients with AML, and described new risk factors. In patients with risk factors, intense diagnostic and treatment strategies should be employed in the future.
ABSTRACT
Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment approach for patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). Graft versus leukemia (GVL) effects, which are exerted by donor T cells directed against leukemic-associated antigens (LAAs), are considered to play a crucial role in disease eradication. Although the expansion of cytotoxic T lymphocytes (CTLs) specific for cytomegalovirus (CMV) in response to an infection has been shown in multiple studies, data on CTLs mediating GVL effects are limited. To evaluate a potential increase or decrease of T lymphocytes specific for LAAs in the setting of allogeneic HSCT, we monitored leukemia-specific CD8+ T cells throughout the first year after HSCT in 18 patients using streptamer technology. A broad panel of promising LAAs was selected: Wilms tumor protein, proteinase 3, receptor for hyaluronan acid-mediated motility, apoptosis regulator Bcl-2, survivin, nucleophosmin, and fibromodulin. T cells specifically directed against AML- or CLL-associated antigens were found at very low frequencies in peripheral blood. Substantial frequencies of LAA-specific T cells could not be measured at any time point by flow cytometry. In contrast, abundant CMV-pp65-specific T cells were detected in CMV-seropositive patient-recipient pairs and an increase prompted by CMV infection could be demonstrated. In conclusion, T lymphocytes with specificities for the aforementioned LAAs can only be detected in minimal quantities in the early phase after allogeneic HSCT.