hMICL and CD123 in combination with a CD45/CD34/CD117 backbone – a universal marker combination for the detection of minimal residual disease in acute myeloid leukaemia

Real‐time quantitative polymerase chain reaction (qPCR) has been extensively validated for the detection of minimal residual disease (MRD) in acute myeloid leukaemia (AML). Meanwhile, multicolour flow cytometry (MFC) has received less attention because the so‐called leukaemia‐associated immunophenotypes (LAIPs) are generally of lower sensitivity and specificity, and prone to change during therapy. To improve MRD assessment by MFC, we here evaluate the combination of human Myeloid Inhibitory C‐type Lectin (hMICL, also termed C‐type lectin domain family 12, member A, CLEC12A) and CD 123 (also termed interleukin‐3 receptor alpha, IL3RA) in combination with CD34 and CD117 (KIT), as an MRD assay in pre‐clinical and clinical testing in 69 AML patients. Spiking experiments revealed that the assay could detect MRD down to 10^?4 in normal bone marrow with sensitivities equalling those of validated qPCR assays. Moreover, it provided at least one MFC MRD marker in 62/69 patients (90%). High levels of hMICL/CD123 LAIPs at the post‐induction time‐point were a strong prognostic marker for relapse in patients in haematological complete remission (P < 0·001). Finally, in post induction samples, hMICL/CD123 LAIPs were strongly correlated (r = 0·676, P = 0·0008) to applied qPCR targets. We conclude the hMICL/CD123‐based MFC assay is a promising MRD tool in AML.     

The role of multiparameter flow cytometry for disease monitoring in AML

Monitoring of the minimal residual disease (MRD) load has become essential for the choice of post-induction strategies in patients with acute myeloid leukemia (AML). Quantitative real-time and nested PCR guarantee highest sensitivities, but suitable markers for follow-up are available for ～60% of patients only: e.g. for those with reciprocal gene rearrangements or those with NPM1 mutations. On the other hand, for most AML patients, multiparameter flow cytometry (MFC) represents a good option for MRD monitoring. In virtually all AML patients, leukemia-associated immunophenotypes (LAIPs) are detectable with MFC. These can be targeted with high sensitivity ranging up to 10(-4) during the course of disease. Numerous studies demonstrated the prognostic power of the MRD levels determined by MFC at post-induction as well as post-consolidation time points in adults and children considered to be in hematologic remission of AML. The post-consolidation MRD status seems to have more prognostic power than post-induction levels. Thus, MFC can significantly contribute to risk assessment of patients with AML during and after treatment and allows clinicians to consider alternative strategies (e.g. allogeneic hematopoietic stem cell transplantation) earlier. Clinical studies need to focus on a standardization of these approaches to facilitate the translation of MFC-based MRD assessment into therapeutic decisions in patients with AML.     

Monitoring of minimal residual disease in acute myeloid leukemia

Monitoring minimal residual disease (MRD) becomes increasingly important in the risk-adapted management of patients with acute myeloid leukemia (AML). The two most sensitive and quantitative methods for MRD detection are multiparameter flow cytometry (MFC) and real-time polymerase chain reaction (QRT-PCR). Fusion gene-specific PCR in AML is based on the RNA level, and thus in contrast to MFC expression levels rather than cell counts are assessed. For both methods independent prognostic values have been shown. The strong power of MFC has been shown mainly in the assessment of early clearance of the malignant clone. MRD levels in AML with fusion genes have the strongest prognostic power after the end of consolidation therapy. In addition, with QRT-PCR highly predictive initial expression levels can be assessed. With both methods early detection of relapse is possible. So far, validated PCR-based MRD was done with fusion genes that are detectable in only 20-25% of all AML MFC is superior since it is applicable for most AML. However, QRT-PCR is still more sensitive in most cases. Thus, it is desirable to establish new molecular markers for PCR-based studies. Large clinical trials will determine the role and place of immunologic and PCR-based monitoring in the prognostic stratification of patients with AML.     

Four-fold staining including CD45 gating improves the sensitivity of multiparameter flow cytometric assessment of minimal residual disease in patients with acute myeloid leukemia

Multiparameter flow cytometry (MFC) is capable of quantifying minimal residual disease (MRD) in acute myeloid leukemia (AML). Its broad application, however, is limited by a lack of sensitivity in about 20% of patients. CD45 gating may improve sensitivity. A broad panel of four-fold combinations of monoclonal antibodies including CD45 in each was used to define leukemia-associated aberrant immunophenotypes (LAIP), to define their sensitivities in normal bone marrow samples, and to compare results to data obtained without CD45 gating using triple staining. In 45 patients, a LAIP was defined, 11 normal bone marrow samples were analyzed as controls. The median percentage of LAIP-positive AML cells with and without CD45 gating was 21.95% (range, 3.31-82.52%) and 20.52% (range, 3.22-81.94%). The median percentage of LAIP-positive normal bone marrow cells ranged from 0.01 to 0.42% (median, 0.02%) and 0.02 to 0.58% (median, 0.15%) with and without CD45 gating. The difference of LAIP-positive cells between AML and normal bone marrow samples amounted to a median of 3.08 log (range, 1.22-4.01) and 2.28 log (range, 1.12-3.34) with and without CD45 gating. CD45 gating improves the sensitivity of MFC-based MRD monitoring in AML by 1 log.     

Prognostic impact of minimal residual disease analysis by flow cytometry in patients with acute myeloid leukemia before and after allogeneic hemopoietic stem cell transplantation

Allogeneic stem cell transplantation (allo‐SCT) has become the treatment of choice in patients with intermediate‐risk and high‐risk acute myeloid leukemia (AML). The quality of response to treatment, assessed in terms of detection of minimal residual disease (MRD), has been consistently associated with prognosis and clinical outcome in patients with AML. The aim of the present study was to evaluate the prognostic impact of analyzing MRD in bone marrow using 4‐color multiparametric flow cytometry (MFC) in 29 patients with AML before and after allo‐SCT. Eighteen patients who were shown to be MRD‐negative [≤0.1% leukemia‐associated immunophenotypes (LAIPs)] by MFC at transplantation and underwent allo‐SCT had lower rates of relapse (15% vs. 66%, P = 0.045), better overall 1‐yr survival (83% vs. 52%, P = 0.021) and a lower cumulative incidence of relapse (P = 0.032) than patients who were MRD‐positive (>0.1%). All post‐transplant MRD‐positive patients underwent a therapeutic intervention after transplant (tapering of immunosuppression, donor lymphocyte infusion, or re‐transplant) with the intention of preventing relapse. Disease was controlled and MRD disappeared in five of these patients. Disease recurred in the other seven patients. We can conclude that follow‐up with MFC for the detection of MRD in AML before and after SCT is useful for predicting relapse. In the post‐transplant setting, monitoring of MRD by MFC could be a key preemptive intervention.     

Flow cytometric assessment of CD15+CD117+ cells for the detection of minimal residual disease in adult acute myeloid leukaemia

There is little information available regarding immunophenotypic monitoring of minimal residual disease (MRD) in acute myeloid leukaemia (AML). We investigated leukaemic cells co‐expressing CD15 and CD117 (CD15+CD117+) in 72 adult AML cases at diagnosis. In 22 cases (31%) with various AML subtypes, more than 5% of leukaemic cells showed the CD15+CD117+ phenotype (range 5.22–55.48%). These 22 cases were younger and had a higher complete remission (CR) rate than the other AML cases, but the CD15+CD117+ cell percentage at diagnosis showed no correlation with the CR duration among the 72 cases. The CD15+CD117+ cell percentage showed a range of 0.00–0.08% in bone marrow cells from 10 haematologically normal subjects. We also investigated CD15+CD117+ cells in sequential bone marrow samples from 17 AML patients who achieved CR and who had had more than 5% CD15+CD117+ leukaemic cells at diagnosis. Because the CD15+CD117+ cell percentage varied among these AML cases, we calculated the percentage of MRD {MRD% = [CD15+CD117+ cells (%) in each sequential marrow sample] ÷ [CD15+CD117+ cells (%) at diagnosis of the corresponding case] × 100}. A high MRD% after 10 months of CR was significantly associated with a short CR duration (P = 0.0004), whereas continuation of a well‐reduced MRD% was associated with a long CR duration. The leukaemic cells conserved the CD15+CD117+ phenotype in all of the eight cases who relapsed. Flow cytometric monitoring of CD15+CD117+ cells is simple and can be applied to a substantial fraction of AML cases. This monitoring may be useful for predicting relapse of adult AML.     

The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells

In CD34(+) acute myeloid leukemia (AML), the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously, we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population, containing the CD34(+)CD38(-)CLL-1(+) cells, does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission, both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future.     

Different organization of von Willebrand factor oligomers in type-2A and -2B von Willebrand disease variants: effects of DDAVP infusion and protease inhibitors

The existence of leukemic-associated phenotypes has been suggested to be a valuable tool for the detection of minimal residual disease (MRD) in AML patients, as they would allow to distinguish leukemic blast cells from normal hematopoietic progenitors. The present study was designed to analyze in which proportion of AML patients the immunological detection of MRD is feasible, based on the presence of aberrant phenotypes that allow the distinction of leukemic from normal cells. For this purpose we have prospectively investigated the blast cells from 40 AML patients at diagnosis with a large panel of MoAb in double and triple staining combinations analyzed at flow cytometry, in order to detect aberrant phenotypes on blast cells (lineage infidelity, antigenic overexpression, and asynchronous antigenic expression, as well as aberrant lightscatter pattern). In the analysis of the 40 AML cases more than one blast cell subset, distinguished by its different antigenic expression, was detected in 85% of the patients: five different phenotypic blast cell subsets were observed in six cases, four in 13 patients, three subsets in three cases, and two in 12 patients; only six cases showed a homogeneous phenotypical blast cell population. Twenty-nine of the 40 AML cases analyzed (73%) showed the existence of at least one aberrant phenotype: in 15 cases the myeloid blast cells co-expressed lymphoid-associated antigens (CD2, CD5, CD7, and/or CD19) - lineage infidelity -; asynchronous antigen expression was detected in 25 patients (CD34+CD56+, CD34+CD11b+, CD34+CD14+, CD117+CD15+, CD33-CD13+, CD13-CD15+, HLADR+CD15+++, HLADR-CD14+CD11b+ CD4+); seven cases displayed antigen overexpression (CD13, CD33, CD15, or CD14); and in 13 patients leukemic cells had an abnormal FSC/SSC distribution according to their phenotype. These results suggest that immunological methods for the detection of MRD based on the existence of aberrant phenotypes could be used in the majority of AML patients.     

Predictive role of minimal residual disease and log clearance in acute myeloid leukemia: a comparison between multiparameter flow cytometry and Wilm’s tumor 1 levels

In acute myeloid leukemia (AML), the detection of minimal residual disease (MRD) as well as the degree of log clearance similarly identifies patients with poor prognosis. No comparison was provided between the two approaches in order to identify the best one to monitor follow-up patients. In this study, MRD and clearance were assessed by both multiparameter flow cytometry (MFC) and WT1 expression at different time points on 45 AML patients achieving complete remission. Our results by WT1 expression showed that log clearance lower than 1.96 after induction predicted the recurrence better than MRD higher than 77.0 copies WT1/10⁴ ABL. Conversely, on MFC, MRD higher than 0.2 % after consolidation was more predictive than log clearance below 2.64. At univariate and multivariate analysis, positive MRD values and log clearance below the optimal cutoffs were associated with a shorter disease-free survival (DFS). At the univariate analysis, positive MRD values were also associated with overall survival (OS). Therefore, post-induction log clearance by WT1 and post-consolidation MRD by MFC represented the most informative approaches to identify the relapse. At the optimal timing of assessment, positive MRD and log-clearance values lower than calculated thresholds similarly predicted an adverse prognosis in AML.     

Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia

Quantification of minimal residual disease (MRD) reveals significant prognostic information in patients treated for acute myeloid leukemia (AML). The application of multiparameter flow cytometry (MFC) for MRD assessment has resulted in significant prognostic information in selected cases in previous analyses. We analyzed MRD in unselected patients with AML in complete remission (CR) after induction (n = 58) and consolidation (n = 62) therapies. By using a comprehensive panel of monoclonal antibodies we identified at least one leukemia-associated aberrant immunophenotype (LAIP) in each patient. The degree of reduction between diagnosis and CR in LAIP-positive cells (log difference [LD]) as a continuous variable was significantly related to relapse-free survival (RFS) both after induction (P = .0001) and consolidation (P = .000 08) therapies, respectively. The LD determined after consolidation therapy was the only parameter related to overall survival (OS) (P = .005). Separation of patients based on the 75th percentile of LD after consolidation therapy resulted in groups with highly different RFS (83.3% versus 25.7%, P = .0034) and OS (87.5% versus 51.4%, P = .0507) at 2 years. Multivariate analysis identified LD as an independent prognostic factor for RFS at both checkpoints. MFC-based quantification of MRD reveals important prognostic information in unselected patients with AML in addition to cytogenetics and should be further evaluated and used in clinical trials.     

CD34+ cell subpopulations detected by 8-color flow cytometry in bone marrow and in peripheral blood stem cell collections: application for MRD detection in leukemia patients

Fast development in polychromatic flow cytometry (PFC) makes it possible to study CD34+ cells with two scatter and eight fluorescence parameters. Minimal residual disease (MRD) is determined as persistence of leukemic cells at submicroscopic levels in bone marrow (BM) of patients in complete remission. MRD can be present in collections of hematopoietic stem cell from blood (HSC-B). Using PFC, we have defined patterns of antigen expression in CD34+ cell subpopulations in BM and applied them as templates in MRD analysis. Twelve BM samples from hospital control (HC) patients with no signs of hematological malignancy were studied using five 8-color monoclonal antibody combinations detecting subsets of CD34+ cells. These patterns have been used as templates to determine levels of MRD in HSC-B collections from six AML patients. Several subsets of CD34+ precursor cells were found to be present at very low frequencies (<10⁻⁴) in BM and/or HSC-B collections. All six HSC-B collections from AML patients showed MRD by 8-color technique and only three by previously applied 3-color method. The 8-color technique showed promising results in efficient detection of different CD34+ subpopulations of HSC-B and in MRD quantification. Monitoring of MRD should become a part of quality control of HSC-B collections.     

Oral azacitidine modulates the bone marrow microenvironment in patients with acute myeloid leukaemia in remission: A subanalysis from the QUAZAR AML-001 trial

Oral azacitidine (Oral-AZA) maintenance therapy improved relapse-free (RFS) and overall survival (OS) significantly versus placebo for AML patients in remission after intensive chemotherapy (IC) in the phase 3 QUAZAR AML-001 study. Immune profiling was performed on the bone marrow (BM) at remission and on-treatment in a subset of patients with the aim of identifying prognostic immune features and evaluating associations of on-treatment immune effects by Oral-AZA with clinical outcomes. Post-IC, increased levels of lymphocytes, monocytes, T cells and CD34 + CD117+ BM cells were prognostically favourable for RFS. CD3+ T-cell counts were significantly prognostic for RFS in both treatment arms. At baseline, high expression of the PD-L1 checkpoint marker was identified on a subset of CD34 + CD117+ BM cells; many of which were PD-L2+. High co-expression of T-cell exhaustion markers PD-1 and TIM-3 was associated with inferior outcomes. Oral-AZA augmented T-cell numbers during early treatment, increased CD4+:CD8+ ratios and reversed T-cell exhaustion. Unsupervised clustering analysis identified two patient subsets defined by T-cell content and expression of T-cell exhaustion markers that were enriched for MRD negativity. These results indicate that Oral-AZA modulates T-cell activity in the maintenance setting of AML, and these immune-mediated responses are associated with clinical outcomes.     

Impact of detectable measurable residual disease on umbilical cord blood transplantation

The impact of measurable residual disease (MRD) on cord blood transplantation (CBT) outcomes has remained debated. To address this issue, we assessed the impact of measurable MRD at CBT on outcomes in large cohort of patients with acute leukemia. Inclusion criteria included adult patients with acute myeloid (AML) or acute lymphoblastic leukemia (ALL), CBT as first allo-HCT in first or second complete remission (CR) at transplantation, and known MRD status at the time of CBT. Data from 506 patients were included in the analysis. Among them, 317 patients had AML and 189 had ALL. Positive MRD was reported in 169 (33%) patients while the remaining 337 patients were MRD negative at CBT. At 2 years, relapse incidence was 18% in patients with MRD negativity vs 33% in those with MRD positivity at transplantation (P < .001). Two-year leukemia-free survival (LFS) and overall survival (OS) were 57% and 60%, respectively, in MRD negative patients, vs 38% (P < .001) and 48% (P = .004), respectively, in those with MRD positivity. There was no interaction between the impact of MRD on OS and LFS and diagnosis (ie, ALL vs AML), single or double CBT, and reduced-intensity or myeloablative conditioning. On multivariate analysis, MRD positivity was associated with a higher risk of relapse (HR = 1.8, P = .003), comparable non-relapse mortality (P = .44), worse LFS (HR = 1.4, P = .008) and a trend towards worse OS (HR = 1.3, P = .065). In conclusion, these data suggest that novel strategies that are aiming to achieve MRD negativity at CBT are needed for leukemic patients with positive MRD pre-CBT.     

C-kit receptor tyrosine kinase (CD117) expression and its positive predictive value for the diagnosis of Thai adult acute myeloid leukemia

We examined the expression of c-kit receptor tyrosine kinase in 195 Thai adult patients with acute leukemia and determined its specificity and predictive values for the diagnosis of adult acute myeloid leukemia (AML). CD117 was used to detect c-kit expression on CD45 and side-scatter-gated blast cells by flow cytometry. Of 163 AML cases, 67% expressed CD117. None of acute lymphoid leukemia (ALL) had CD117 expression, except one case of T-ALL. The majority of AML patients carrying t(8;21), inv(16), and t(15;17) had high CD117 expression. High proportion of AML cases without c-kit expressed monocytic markers. Significant associations between CD117 and CD34 (P<0.001), CD13 (P=0.006), CD7 (P=0.034), and CD19 (P<0.001) were found in AML cases. The calculated specificity of CD117 for the diagnosis of AML was 0.97, which was higher than CD13 (0.78) and CD33 (0.75) but comparable to MPO (0.97). The positive predictive value (PPV) of CD117 for AML was 0.99, with the negative predictive value of 0.35. In conclusion, the majority of Thai adult AML cases expressed c-kit. C-kit is infrequently expressed in ALL and appeared to be specific for AML with high PPV. Future targeting therapy using c-kit as a therapeutic target should benefit the majority of Thai AML patients who had high c-kit expression.     

Next-generation sequencing-defined minimal residual disease before stem cell transplantation predicts acute myeloid leukemia relapse

In acute myeloid leukemia (AML), the assessment of post-treatment minimal residual disease (MRD) may inform a more effective management approach. We investigated the prognostic utility of next-generation sequencing (NGS)-based MRD detection undertaken before hematopoietic stem cell transplantation (HSCT). Forty-two AML subjects underwent serial disease monitoring both by standard methods, and a targeted 42-gene NGS assay, able to detect leukemia-specific mutant alleles (with >0.5% VAF) (mean 5.1 samples per subject). The prognostic relevance of any persisting diagnostic mutation before transplant (≤27 days) was assessed during 22.1 months (median) of post-transplant follow-up. The sensitivity of the NGS assay (27 MRD-positive subjects) exceeded that of the non-molecular methods (morphology, FISH, and flow cytometry) (11 positive subjects). Only one of the 13 subjects who relapsed after HSCT was NGS MRD-negative (92% assay sensitivity). The cumulative incidence of post-transplant leukemic relapse was significantly higher in the pre-transplant NGS MRD-positive (vs MRD-negative) subjects (P = .014). After adjusting for TP53 mutation and transplant conditioning regimen, NGS MRD-positivity retained independent prognostic significance for leukemic relapse (subdistribution hazard ratio = 7.3; P = .05). The pre-transplant NGS MRD-positive subjects also had significantly shortened progression-free survival (P = .038), and marginally shortened overall survival (P = .068). In patients with AML undergoing HSCT, the pre-transplant persistence of NGS-defined MRD imparts a significant, sensitive, strong, and independent increased risk for subsequent leukemic relapse and death. Given that NGS can simultaneously detect multiple leukemia-associated mutations, it can be used in the majority of AML patients to monitor disease burdens and inform treatment decisions.     

Upregulation of CD200R1 in lineage-negative leukemic cells is characteristic of AML1-ETO-positive leukemia in mice

Activating mutations of c-Kit are frequently found in acute myeloid leukemia (AML) patients harboring t(8;21) chromosomal translocation generating a fusion protein AML1-ETO. Here we show that an active mutant of c-Kit cooperates with AML1-ETO to induce AML in mouse bone marrow transplantation models. Leukemic cells expressing AML1-ETO with c-Kit^D814V were serially transplantable. Transplantation experiments indicated that lineage^?c-Kit⁺Sca-1⁺ (KSL) leukemic cells, but not lineage⁺ leukemic cells, were enriched for leukemia stem cells (LSCs). Comparison of gene expression profiles between KSL leukemic and normal cells delineated that CD200R1 was highly expressed in KSL leukemic cells as compared with KSL normal cells. Upregulation of CD200R1 was verified in lineage^? leukemic cells, but not in lineage⁺ leukemic cells. CD200R1 expression in the lineage^? leukemic cells was not correlated with the frequency of LSCs, indicating that CD200R1 is not a useful marker for LSCs in these models. Interestingly, CD200R1 was upregulated in KSL cells transduced with AML1-ETO, but not with other leukemogenic mutants, including c-Kit^D814V, AML1^D171N, and AML1^S291fsX300. Consistently, upregulation of CD200R1 in lineage^? leukemic cells was observed only in the BM of mice suffering from AML1-ETO-positive leukemia. In conclusion, AML1-ETO upregulated CD200R1 in lineage^? cells, which was characteristic of AML1-ETO-positive leukemia in mice.     

Detection of measurable residual disease may better predict outcomes than mutations based on next-generation sequencing in acute myeloid leukaemia with biallelic mutations of CEBPA

Acute myeloid leukaemia (AML) patients with biallelic mutations of CEBPA (bi CEBPA) have a 30–50% relapse rate. This study established the value of mutations based on next-generation sequencing (NGS) and multiparameter flow cytometric measurable residual disease (MFC-MRD) detection and compared the outcomes. From 2014 to 2018, 124 newly diagnosed bi CEBPA AML patients were treated. The median age was 37·5 (16–69) years. The 3-year cumulative incidence of relapse (CIR), relapse-free survival (RFS) and overall survival (OS) were 33·0%, 64·7% and 84·3%, respectively. Patients without additional mutations and with GATA2 mutations were defined as ‘NGS low risk’, which was the only favourable independent factor for CIR and RFS of pretreatment parameters. Patients with sustained positive MRD after two consolidation cycles and MRD negative losses at any time were defined as ‘MRD high risk’, which was the only poor independent factor for CIR, RFS and OS, including pretreatment and post-treatment parameters. In CR2 and non-remission patients who underwent allo-HSCT, superior OS was achieved. We conclude that NGS low risk was a favourable factor in the analysis of pretreatment parameters. MRD risk stratification was an independent prognostic factor in pretreatment and post-treatment parameters. Relapsed patients still have a favourable outcome followed by allo-HSCT.     

Minimal residual disease status determined by multiparametric flow cytometry pretransplantation predicts the outcome of patients with ALL receiving unmanipulated haploidentical allografts

This study evaluated the effects of pretransplantation minimal residual disease (pre-MRD) on outcomes of patients with acute lymphoblastic leukemia (ALL) who underwent unmanipulated haploidentical stem cell transplantation (haplo-SCT). A retrospective study including 543 patients with ALL was performed. MRD was determined using multiparametric flow cytometry. Both in the entire cohort of patients and in subgroup cases with T-ALL or B-ALL, patients with positive pre-MRD had a higher incidence of relapse (CIR) than those with negative pre-MRD in MSDT settings (P < 0.01 for all). Landmark analysis at 6 months showed that MRD positivity was significantly and independently associated with inferior rates of relapse (HR, 1.908; P = 0.007), leukemia-free survival (LFS) (HR, 1.559; P = 0.038), and OS (HR, 1.545; P = 0.049). The levels of pre-MRD according to a logarithmic scale were also associated with leukemia relapse, LFS, and OS, except that cases with MRD <0.01% experienced comparable CIR and LFS to those with negative pre-MRD. A risk score for CIR was developed using the variables pre-MRD, disease status, and immunophenotype of ALL. The CIR was 14%, 26%, and 59% for subjects with scores of 0, 1, and 2-3, respectively (P < 0.001). Three-year LFS was 75%, 64%, and 42%, respectively (P < 0.001). Multivariate analysis confirmed the association of the risk score with CIR and LFS. The results indicate that positive pre-MRD, except for low level one (MRD < 0.01%), is associated with poor outcomes in patients with ALL who underwent unmanipulated haplo-SCT.     

Molecular predictors of immunophenotypic measurable residual disease clearance in acute myeloid leukemia

Measurable residual disease (MRD) is a powerful prognostic factor in acute myeloid leukemia (AML). However, pre-treatment molecular predictors of immunophenotypic MRD clearance remain unclear. We analyzed a dataset of 211 patients with pre-treatment next-generation sequencing who received induction chemotherapy and had MRD assessed by serial immunophenotypic monitoring after induction, subsequent therapy, and allogeneic stem cell transplant (allo-SCT). Induction chemotherapy led to MRD− remission, MRD+ remission, and persistent disease in 35%, 27%, and 38% of patients, respectively. With subsequent therapy, 34% of patients with MRD+ and 26% of patients with persistent disease converted to MRD-. Mutations in CEBPA, NRAS, KRAS, and NPM1 predicted high rates of MRD− remission, while mutations in TP53, SF3B1, ASXL1, and RUNX1 and karyotypic abnormalities including inv (3), monosomy 5 or 7 predicted low rates of MRD− remission. Patients with fewer individual clones were more likely to achieve MRD− remission. Among 132 patients who underwent allo-SCT, outcomes were favorable whether patients achieved early MRD− after induction or later MRD− after subsequent therapy prior to allo-SCT. As MRD conversion with chemotherapy prior to allo-SCT is rarely achieved in patients with specific baseline mutational patterns and high clone numbers, upfront inclusion of these patients into clinical trials should be considered.