Functional Pathway Analysis Using SCNP of FLT3 Receptor Pathway Deregulation in AML Provides Prognostic Information Independent from Mutational Status

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.     

The NPM1 Mutation Type Has No Impact on Survival in Cytogenetically Normal AML

NPM1 mutations represent frequent genetic alterations in patients with acute myeloid leukemia (AML) associated with a favorable prognosis. Different types of NPM1 mutations have been described. The purpose of our study was to evaluate the relevance of different NPM1 mutation types with regard to clinical outcome. Our analyses were based on 349 NPM1-mutated AML patients treated in the AMLCG99 trial. Complete remission rates, overall survival and relapse-free survival were not significantly different between patients with NPM1 type A or rare type mutations. The NPM1 mutation type does not seem to play a role in risk stratification of cytogenetically normal AML.     

Prognostic impact of the simple L-index and absolute lymphocyte count early after allogeneic hematopoietic stem cell transplantation

Background aimsThe L-index, designed as a quantitative parameter to simultaneously assess the duration and severity of lymphopenia, and absolute lymphocyte count (ALC) have a prognostic impact after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, discrepancies have been reported in the impact of ALC, and limited information is currently available on the L-index.MethodsTo search for a better clinical tool, the authors retrospectively compared the simple L-index at 30 days (sL-index(30)), which aims to make the original L-index more compact, and ALC at 30 days (ALC(30)) after allo-HSCT in 217 patients who underwent allo-HSCT at the authors’ institutions.ResultsMedian sL-index(30) was 11 712 (range, 4419–18 511) and median ALC(30) was 404 (range, 0–3754). In a multivariate analysis, higher sL-index(30) was associated with a significantly higher cumulative incidence of relapse (CIR) (hazard ratio [HR], 1.01, 95% confidence interval [CI], 1.00–1.02, P = 0.02 for every increase of 100 in sL-index(30)) as well as non-relapse mortality (NRM) (HR, 1.02, 95% CI, 1.00–1.03, P = 0.01 for every increase of 100 in sL-index(30)). Although higher ALC(30) was associated with significantly lower CIR (HR, 0.94, 95% CI, 0.89–1.00, P = 0.04 for every increase of 100/μL in ALC(30)), it was not extracted as an independent risk factor for NRM (HR, 0.96, 95% CI, 0.88–1.05, P = 0.39). Higher sL-index(30) was associated with a slightly higher rate of grade 3–4 acute graft-versus-host disease (GVHD) (HR, 1.02, 95% CI, 1.00–1.04, P = 0.12 for every increase of 100 in sL-index(30)) but not chronic GVHD (HR, 1.00, 95% CI, 0.99–1.01, P = 0.63). ALC(30) was not associated with rates of grade 3–4 acute GVHD (HR, 1.02, 95% CI, 0.88–1.17, P = 0.81) or chronic GVHD (HR, 1.02, 95% CI, 0.98–1.06, P = 0.34). In a receiver operating characteristic curve, the cutoff values of sL-index(30) and ALC(30) for CIR were 9000 and 500, respectively, and the cutoff value of sL-index(30) for NRM was 12 000.ConclusionssL-index(30) is a promising tool that may be applied to various survival outcomes. A large-scale prospective study is needed to clarify whether medical interventions based on sL-index(30) values will improve the clinical prognosis of patients.     

A Hypertrophic Cardiomyopathy-associated MYBPC3 Mutation Common in Populations of South Asian Descent Causes Contractile Dysfunction

Hypertrophic cardiomyopathy (HCM) results from mutations in genes encoding sarcomeric proteins, most often MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). A recently discovered HCM-associated 25-base pair deletion in MYBPC3 is inherited in millions worldwide. Although this mutation causes changes in the C10 domain of cMyBP-C (cMyBP-C^C10mut), which binds to the light meromyosin (LMM) region of the myosin heavy chain, the underlying molecular mechanism causing HCM is unknown. In this study, adenoviral expression of cMyBP-C^C10mut in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca²⁺ transients, compared with wild-type cMyBP-C expression (cMyBP-C^WT) and controls. Forty-eight hours after infection, 44% of cMyBP-C^WT and 36% of cMyBP-C^C10mut protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-C^C10mut to the C-zone, whereas cMyBP-C^WT mostly showed C-zone staining, suggesting that cMyBP-C^C10mut could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-C^C10mut resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-C^C10mut displayed significantly reduced fractional shortening, sarcomere shortening, and relaxation velocities, apparently caused by defects in sarcomere function, because Ca²⁺ transients were unaffected. Co-sedimentation and protein cross-linking assays confirmed that C10^mut causes the loss of C10 domain interaction with myosin LMM. Protein homology modeling studies showed significant structural perturbation in cMyBP-C^C10mut, providing a potential structural basis for the alteration in its mode of interaction with myosin LMM. Therefore, expression of cMyBP-C^C10mut protein is sufficient to cause contractile dysfunction in vitro.     

有核红细胞数在白血病患者危险度分层评估中的意义

摘要 目的：探讨有核红细胞数(nucleated red blood cell count,NRBCs)在白血病患者危险度分层评估中的意义。方法：选择2016年2月到2019年7月在厦门大学附属成功医院(本院)诊治的急性髓系白血病(Acute myeloid leukemia,AML)患者120例,检测其NRBCs并进行危险度分层,回顾分析患者的临床资料并与其NRBCs进行相关性分析。结果：120例患者中,危险度分层为低危40例,中危60例,高危20例。低危组和中高危组的患者年龄、性别、核仁磷酸蛋白(nucleophosmin,NPM1)突变、骨髓原始细胞等对比差异无统计学意义(P>0.05),其外周血原始细胞、FMS 样酪氨酸激酶-3(FMS-like tyrosinekinase 3,FLT3)突变、急性生理和慢性健康状况Ⅱ(acute physiology andchron ic health evaluationⅡ,APACHEⅡ)评分、白细胞（white blood cell,WBC）、血红蛋白（hemoglobin,Hb）、血小板（platelet,PLT） 、白蛋白（albumin,ALB）与丙氨酸氨基转移酶（alanine aminotransferase,ALT）值等对比差异有统计学意义(P<0.05)。低危组的NRBCs为3.94±0.29个,显著低于中高危组(11.87±2.11个,P=0.000)。Pearson相关分析显示危险度分层与NRBCs、外周血原始细胞、APACHEⅡ评分、FLT3突变、PLT有显著相关性(r=0.823、0.566、0.494、0.578、0.781,P<0.05)。logistic回归分析显示NRBCs、外周血原始细胞、APACHEⅡ评分、FLT3突变、PLT为影响急性髓系白血病患者危险度分层的主要因素(P<0.05)。结论：不同危险度分层的白血病患者的NRBCs具有显著差异,其与患者的病理特征显著相关,也是影响患者危险度分层的主要因素。     

Effect of FLT3 Ligand on Survival and Disease Phenotype in Murine Models Harboring a FLT3 Internal Tandem Duplication Mutation

Many of the mutations contributing to leukemogenesis in acute myeloid leukemia have been identified. A common activating mutation is an internal tandem duplication (ITD) mutation in the FLT3 gene that is found in approximately 25% of patients and confers a poor prognosis. FLT3 inhibitors have been developed and have some efficacy, but patients often relapse. Levels of FLT3 ligand (FL) are significantly elevated in patients during chemotherapy and may be an important component contributing to relapse. We used a mouse model to investigate the possible effect of FL expression on leukemogenesis involving FLT3-ITD mutations in an in vivo system. FLT3^ITD/ITD FL^−/− (knockout) mice had a statistically significant increase in survival compared with FLT3^ITD/ITD FL^+/+ (wildtype) mice, most of which developed a fatal myeloproliferative neoplasm. These findings suggest that FL levels may have prognostic significance in human patients. We also studied the effect of FL expression on survival in a FLT3-ITD NUP98–HOX13 (NHD13) fusion mouse model. These mice develop an aggressive leukemia with short latency. We asked whether FL expression played a similar role in this context. The NUP98-HOX13 FLT3^ITD/wt FL^−/− mice did not have a survival advantage, compared with NUP98-HOX13 FLT3^ITD/wt FL^+/+ mice (normal FL levels). The loss of the survival advantage of the FL knockout group in the NUP98–HOX13 model suggests that adding a second mutation changes the effect of FL expression in the context of more aggressive disease.Abbreviations: AML, acute myeloid leukemia; FL, FLT3 ligand; FLT3, FMS-like tyrosine kinase 3; ITD, internal tandem duplication; MPN, myeloproliferative neoplasmFMS-like tyrosine kinase 3 (FLT3) is normally activated by binding of its ligand (FL) to 2 FLT3 molecules, causing them to dimerize, autophosphorylate, and activate downstream targets.^20,26,31 Although FL expression is relatively ubiquitous, the FLT3 receptor is found predominantly on hematopoietic cells and has an important role in hematopoiesis.^6,13,24 Several mutations in the FLT3 gene can lead to constitutive activation that occurs independent of ligand binding and leads to activation of downstream targets; these mutations typically are found in patients with acute myeloid leukemia (AML). The most common mutation described in AML is an internal tandem duplication (ITD) that occurs in the juxtamembrane domain of FLT3. The ITD mutations vary in length,^17,25 but these forms all constitutively activate FLT3 kinase activity to result in autophosphorylation and phosphorylation of its downstream targets.^4,14,28,32 The ITD mutation is seen in approximately 25% of adult AML cases and is associated with a poor prognosis.^18,19,23Despite the fact that FTL3-ITD is constitutively activated, some evidence indicates that FL may continue to play a role in FLT3 signaling and affect AML prognosis.³⁵ Elevated plasma levels of FL have been reported in patients that have undergone chemotherapy.^2,30 In addition, elevated levels of FL have been shown to increase the amount of FLT3 inhibitor needed to reduce the levels of phosphorylated FLT3-ITD in a cell line (Molm14) model.^8,21,34 When a lentivirus was used to introduce a FLT3-ITD mutation into mouse embryonic fibroblast cells from FL-knockout mice, the addition of FL to the culture media resulted in an increase in the level of phosphorylated FLT3, further supporting the idea that FL may play a role in FLT3-ITD–associated AML.³³ These previous models have all used cell lines, cultured cells, and plasma from patient samples to address the potential importance of FL expression in cases where an ITD mutation is present.Here we use primary hematopoietic cells from a combination of genetically engineered mouse models to investigate the role of FLT3 and FL in the pathogenesis of AML. The first model is a FLT3-ITD knockin mouse model with an 18-bp insertion in the juxtamembrane domain of FLT3 that was generated and characterized by our lab. This mouse model consistently and predictably develops myeloproliferative neoplasia (MPN) with moderately elevated WBC counts, splenomegaly, and myeloid expansion in the bone marrow, as evidenced by histopathologic changes and increased granulocytic/ monocytic fractions by flow cytometry.¹¹ A small percentage (7%; 9 of 129) of the FLT3-ITD homozygous (FLT3^ITD/ITD) mice spontaneously developed fully transformed leukemia.¹⁰ The second mouse model uses transgenic expression of a Nup98–Hox13 fusion (NHD13) that is expressed primarily in hematopoietic tissues. Mice that carry this mutation typically develop a myelodysplastic syndrome that often progresses to acute leukemia after a long lag time.¹² When these mice were bred to our FLT3-ITD mice, the resulting double-mutant Nup98–Hox13 (NHD13) FLT3^wt/ITD mice predominantly developed an AML with minimal differentiation and demonstrated a markedly shorter latency to disease. Interestingly, a subset of mice display loss of heterozygosity of the wildtype Flt3 allele in the bone marrow⁷ as occurs in a fraction of human FLT3-ITD AML patients.^22,29 The third model is a FL-knockout mouse model that was developed at Immunex (Seattle, WA) and is currently commercially available. These mice have the majority of the FL extracellular domain coding region disrupted by insertion of a PKG–Neo cassette. These mice demonstrated reduced cellularity in the bone marrow and an overall reduction in hematopoietic precursors, especially of the myeloid and lymphoid lineages.¹⁶To examine the effect of FL expression on disease conferred by a FLT3-ITD mutation, we used 2 genetically engineered mouse models: the first is the model of MPN generated by the FLT3^ITD/ITD mutation alone. The second was a leukemia model that is generated by the combination of a FLT3^wt/ITD together with a NHD13 mutation. Into both of these models, we bred mice that were either wildtype for FL or that had FL knocked out. We then characterized survival and disease phenotype data from each cohort to ascertain the effect of FL expression on MPN and AML generated by FLT3-ITD expression.     

Expression of Nucleophosmin/NPM1 correlates with migration and invasiveness of colon cancer cells

Background

We aimed to examine the expression level of Nucleophosmin (NPM1) protein in colon cancer tissues and to investigate the potential role of NPM1 in the regulation of cell migration and invasiveness.

Methods

Immunohistochemical assay was performed to examine the expression pattern of NPM1 in 31 groups of colonic carcinoma samples, including colon tumors, adjacent normal tissues, and matched metastatic lymph nodes from the same patients. Small interfering RNA technique and exogenous expression of wild type NPM1 methods were used to further verify the function of NPM1.

Results

High-expression of NPM1 correlates with lymph node metastasis (P = 0.0003) and poor survival rate of human colon cancer patients (P = 0.017). SiRNA-mediated reduction of NPM1 was also shown to inhibit the migration and invasiveness of metastatic colon cancer HCT116 cell line. In addition, the exogenous expression of NPM1 in HT29 cells, a NPM1 low expression and low invasive colon cancer cell line, enhanced cell migration and invasiveness along with increased cell proliferation.

Conclusions

The current study uncovered the critical role of NPM1 in the regulation of colon cancer cells migration and invasion, and NPM1 may serve as a potential marker for the prognosis of colon cancer patients.     

NPM1 Deletion Is Associated with Gross Chromosomal Rearrangements in Leukemia

Background

NPM1 gene at chromosome 5q35 is involved in recurrent translocations in leukemia and lymphoma. It also undergoes mutations in 60% of adult acute myeloid leukemia (AML) cases with normal karyotype. The incidence and significance of NPM1 deletion in human leukemia have not been elucidated.

Methodology and Principal Findings

Bone marrow samples from 145 patients with myelodysplastic syndromes (MDS) and AML were included in this study. Cytogenetically 43 cases had isolated 5q-, 84 cases had 5q- plus other changes and 18 cases had complex karyotype without 5q deletion. FISH and direct sequencing investigated the NPM1 gene. NPM1 deletion was an uncommon event in the “5q- syndrome” but occurred in over 40% of cases with high risk MDS/AML with complex karyotypes and 5q loss. It originated from large 5q chromosome deletions. Simultaneous exon 12 mutations were never found. NPM1 gene status was related to the pattern of complex cytogenetic aberrations. NPM1 haploinsufficiency was significantly associated with monosomies (p<0.001) and gross chromosomal rearrangements, i.e., markers, rings, and double minutes (p<0.001), while NPM1 disomy was associated with structural changes (p = 0.013). Interestingly, in complex karyotypes with 5q- TP53 deletion and/or mutations are not specifically associated with NPM1 deletion.

Conclusions and Significance

NPM1/5q35 deletion is a consistent event in MDS/AML with a 5q-/-5 in complex karyotypes. NPM1 deletion and NPM1 exon 12 mutations appear to be mutually exclusive and are associated with two distinct cytogenetic subsets of MDS and AML.     

Methylenetetrahydrofolate reductase gene polymorphisms contribute to acute myeloid leukemia and chronic myeloid leukemia susceptibilities: Evidence from meta-analyses

PurposeThe expression of methylenetetrahydrofolate reductase (MTHFR) is associated with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Most studies have linked the common functional C677T and A1298C polymorphisms of the MTHFR gene and susceptibility to AML and CML, but the results were not consistent. The aim of the present study was to derive a more precise estimation of the relationship.MethodsMeta-analyses assessing the association of MTHFR C677T and A1298C variations with AML and CML were conducted. Eligible articles were identified from the PubMed and EMBASE databases. All statistical analyses were conducted using Review Manager Software.Results10 and 10 studies were included in the meta-analysis about the role of C677T polymorphism on the AML and CML risks, respectively; 6 and 4 studies were included about the role of A1298C polymorphism on the AML and CML risks, respectively. Overall, both the C677T and A1298C polymorphisms were significantly associated with CML risk under the recessive model (P = 0.04, OR = 1.35, 95% CI = 1.02–1.79 for C677T and P = 0.003, OR = 2.17, 95% CI = 1.29–3.63 for A1298C). In addition, the risk of CML was higher in 1298CC genotype carriers than in 1298AA genotype carriers (P = 0.004, OR = 2.17, 95% = 1.28–3.69). Conversely, the overall data failed to indicate a significant association of C677T or A1298C polymorphisms with AML risk under any model.ConclusionsThe findings provide evidence that C677T and A1298C polymorphisms are risk factors for CML risk.     

CD33‐targeting extracellular vesicles deliver antisense oligonucleotides against FLT3‐ITD and miR‐125b for specific treatment of acute myeloid leukaemia

IntroductionAcute Myeloid Leukaemia (AML) is the most common blood cancer in adults. Although 2 out of 3 AML patients go into total remission after chemotherapies and targeted therapies, the disease recurs in 60%–65% of younger adult patients within 3 years after diagnosis with a dramatically decreased survival rate. Therapeutic oligonucleotides are promising treatments under development for AML as they can be designed to silence oncogenes with high specificity and flexibility. However, there are not many well validated approaches for safely and efficiently delivering oligonucleotide drugs. This issue could be resolved by utilizing a new generation of delivery vehicles such as extracellular vesicles (EVs).MethodsIn this study, we harness red blood cell‐derived EVs (RBCEVs) and engineer them via exogenous drug loading and surface functionalization to develop an efficient drug delivery system for AML. Particularly, EVs are designed to target CD33, a common surface marker with elevated expression in AML cells via the conjugation of a CD33‐binding monoclonal antibody onto the EV surface.ResultsThe conjugation of RBCEVs with the CD33‐binding antibody significantly increases the uptake of RBCEVs by CD33‐positive AML cells, but not by CD33‐negative cells. We also load CD33‐targeting RBCEVs with antisense oligonucleotides (ASOs) targeting FLT3‐ITD or miR‐125b, 2 common oncogenes in AML, and demonstrate that the engineered EVs improve leukaemia suppression in in vitro and in vivo models of AML.ConclusionTargeted RBCEVs represent an innovative, efficient, and versatile delivery platform for therapeutic ASOs and can expedite the clinical translation of oligonucleotide drugs for AML treatments by overcoming current obstacles in oligonucleotide delivery.

In this study, we harness red blood cell‐derived EVs (RBCEVs) and engineer them with surface functionalization and exogenous drug loading to develop an efficient drug delivery system for AML. Anti‐CD33 antibody was conjugated to RBCEVs using an enzymatic method combined with the streptavidin‐biotin system. We load the antibody conjugated RBCEVs with ASOs targeting FLT3‐ITD or miR‐125b, 2 common oncogenes in AML, and demonstrate that the treatment with engineered EVs improve leukaemia suppression both in vitro and in vivo.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

FLT3 and NPM-1 mutations in a cohort of acute promyelocytic leukemia patients from India

Background:

Acute promyelocytic leukemia (APL) with t (15;17) is a distinct category of acute myeloid leukemia (AML) and is reported to show better response to anthracyclin based chemotherapy. A favorable overall prognosis over other subtypes of AML has been reported for APL patients but still about 15% patients relapse.

Methods:

This study evaluated the presence of Famus like tyrosine kinase-3 (FLT3) and nucleophosmin-1 (NPM1) gene mutations in a cohort of 40 APL patients. Bone marrow/peripheral blood samples from patients at the time of diagnosis and follow-up were processed for immunophenotyping, cytogenetic markers and isolation of DNA and RNA. Samples were screened for the presence of mutations in FLT3 and NPM1 genes using polymerase chain reaction followed by sequencing.

Results:

Frequency of FLT3/internal tandem duplication and FLT3/tyrosine kinase domain was found to be 25% and 7% respectively. We observed a high frequency of NPM1 mutation (45%) in the present population of APL patients.     

Hemolysis and hyperhomocysteinemia caused by cobalamin deficiency: three case reports and review of the literature

Background

Molecular characterisation of normal karyotype acute myeloid leukemia (NK-AML) allows prognostic stratification and potentially can alter treatment choices and pathways. Approximately 45–60% of patients with NK-AML carry NPM1 gene mutations and are associated with a favourable clinical outcome when FLT3-internal tandem duplications (ITD) are absent. High resolution melting (HRM) is a novel screening method that enables rapid identification of mutation positive DNA samples.

Results

We developed HRM assays to detect NPM1 mutations and FLT3-ITD and tested diagnostic samples from 44 NK-AML patients. Eight were NPM1 mutation positive only, 4 were both NPM1 mutation and FLT3-ITD positive and 4 were FLT3-ITD positive only. A novel point mutation Y572C (c.1715A>G) in exon 14 of FLT3 was also detected. In the group with de novo NK-AML, 40% (12/29) were NPM1 mutation positive whereas NPM1 mutations were observed in 20% (3/15) of secondary NK-AML cases. Sequencing was performed and demonstrated 100% concordance with the HRM results.

Conclusion

HRM is a rapid and efficient method of screening NK-AML samples for both novel and known NPM1 and FLT3 mutations. NPM1 mutations can be observed in both primary and secondary NK-AML cases.     

Advantages of peripheral blood stem cells from unrelated donors versus bone marrow transplants in outcomes of adult acute myeloid leukemia patients

Background aimsIn allogeneic stem cell transplantation, unrelated donors are chosen in cases where appropriate related donors are not available. Peripheral blood stem cells (PBSCs) are more often selected as a graft source than bone marrow (BM). However, the prognostic benefits of PBSCs versus BM transplants from unrelated donors have not been carefully examined in patients with acute myeloid leukemia (AML). This study compared outcomes of adult AML patients who underwent unrelated PBSC and BM transplantation, evaluating post-transplant complications, including engraftment, graft-versus-host disease (GVHD) and infections, and determined subgroups of patients who are most likely to benefit from unrelated PBSCs compared with BM transplants.MethodsThe authors analyzed 2962 adult AML patients who underwent unrelated PBSC or BM transplants between 2011 and 2018 (221 PBSC and 2741 BM) using the Japanese nationwide registry database, in which graft source selection is not skewed toward PBSCs.ResultsIn 49.7% of patients, disease status at transplantation was first complete remission (CR1). In 57.1% of cases, HLA-matched donors were selected. Myeloablative conditioning was performed in 75.1% of cases, and anti-thymocyte globulin (ATG) was added to conditioning in 10.5%. Multivariate analyses showed a trend toward favorable non-relapse mortality (NRM) in PBSC recipients compared with BM recipients (hazard ratio [HR], 0.731, P = 0.096), whereas overall survival (OS) (HR, 0.959, P = 0.230) and disease-free survival (DFS) (HR, 0.868, P = 0.221) were comparable between PBSC and BM recipients. Although the rate of chronic GVHD (cGVHD) was significantly higher in PBSC patients (HR, 1.367, P = 0.016), NRM was not increased, mainly as a result of significantly reduced risk of bacterial infections (HR, 0.618, P = 0.010), reflecting more prompt engraftments in PBSC recipients. Subgroup analyses revealed that PBSC transplantation was advantageous in patients transplanted at CR1 and in those without ATG use. PBSC recipients experienced significantly better OS and/or DFS compared with BM recipients in this patient group.ConclusionsThe authors' results confirmed the overall safety of unrelated PBSC transplantation for adult AML patients and suggested an advantage of PBSCs, especially for those in CR1. Further optimization of the prophylactic strategy for cGVHD is required to improve the overall outcome in transplantation from unrelated PBSC donors.     

High-Dose Cytarabine in Acute Myeloid Leukemia Treatment: A Systematic Review and Meta-Analysis

The optimal dose, scheme, and clinical setting for Ara-C in acute myeloid leukemia (AML) treatment remain uncertain. In this study, we performed a meta-analysis to systematically assess the impact of high-dose cytarabine (HDAC) on AML therapy during the induction and consolidation stages. Twenty-two trials with a total of 5,945 de novo AML patients were included in the meta-analysis. Only patients less than 60 year-old were included in the study. Using HDAC in induction therapy was beneficial for RFS (HR = 0.57; 95% CI, 0.35–0.93; P = 0.02) but not so for CR rate (HR = 1.01; 95% CI, 0.93–1.09; P = 0.88) and OS (HR = 0.83; 95% CI, 0.66–1.03; P = 0.1). In consolidation therapy, HDAC showed significant RFS benefits (HR = 0.67; 95% CI, 0.49–0.9; P = 0.008) especially for the favorable-risk group (HR = 0.38; 95% CI, 0.21–0.69; P = 0.001) compared with SDAC (standard dose cytarabine), although no OS advantage was observed (HR = 0.84; 95% CI, 0.55–1.27; P = 0.41). HDAC treatment seemed less effective than auto-BMT/allo-BMT treatment (HR = 1.66, 95% CI, 1.3–2.14; P<0.0001) with similar OS. HDAC treatment led to lower relapse rate in induction and consolidation therapy than SDAC treatment, especially for the favorable-risk group. Auto-BMT/allo-BMT was more beneficial in prolonging RFS than HDAC.     

miR-15a-5p and miR-21-5p contribute to chemoresistance in cytogenetically normal acute myeloid leukaemia by targeting PDCD4, ARL2 and BTG2

Cytarabine and daunorubicin are old drugs commonly used in the treatment of acute myeloid leukaemia (AML). Refractory or relapsed disease because of chemotherapy resistance is a major issue. microRNAs (miRNAs) were incriminated in resistance. This study aimed to identify miRNAs involved in chemoresistance in AML patients and to define their target genes. We focused on cytogenetically normal AML patients with wild-type NPM1 without FLT3-ITD as the treatment of this subset of patients with intermediate-risk cytogenetics is not well established. We analysed baseline AML samples by small RNA sequencing and compared the profile of chemoresistant to chemosensitive AML patients. Among the miRNAs significantly overexpressed in chemoresistant patients, we revealed miR-15a-5p and miR-21-5p as miRNAs with a major role in chemoresistance in AML. We showed that miR-15a-5p and miR-21-5p overexpression decreased apoptosis induced by cytarabine and/or daunorubicin. PDCD4, ARL2 and BTG2 genes were found to be targeted by miR-15a-5p, as well as PDCD4 and BTG2 by miR-21-5p. Inhibition experiments of the three target genes reproduced the functional effect of both miRNAs on chemosensitivity. Our study demonstrates that miR-15a-5p and miR-21-5p are overexpressed in a subgroup of chemoresistant AML patients. Both miRNAs induce chemoresistance by targeting three pro-apoptotic genes PDCD4, ARL2 and BTG2.     

A novel predictive algorithm to personalize autologous T-cell harvest for chimeric antigen receptor T-cell manufacture

Background aimsThe most widely accepted starting materials for chimeric antigen receptor T-cell manufacture are autologous CD3+ T cells obtained via the process of leukapheresis, also known as T-cell harvest. As this treatment modality gains momentum and apheresis units struggle to meet demand for harvest slots, strategies to streamline this critical step are warranted.MethodsThis retrospective review of 262 T-cell harvests, with a control cohort of healthy donors, analyzed the parameters impacting CD3+ T-cell yield in adults with B-cell malignancies. The overall aim was to design a novel predictive algorithm to guide the required processed blood volume (PBV) (L) on the apheresis machine to achieve a specific CD3+ target yield.ResultsFactors associated with CD3+ T-cell yield on multivariate analysis included peripheral blood CD3+ count (natural log, ×10⁹/L), hematocrit (HCT) and PBV with coefficients of 0.86 (95% confidence interval [CI], 0.80–0.92, P < 0.001), 1.30 (95% CI, 0.51–2.08, P = 0.001) and 0.09 (95% CI, 0.07–0.11, P < 0.001), respectively. The authors’ model, incorporating CD3+ cell count, HCT and PBV (L), with an adjusted R² of 0.87 and root-mean-square error of 0.26 in the training dataset, was highly predictive of CD3+ cell yield in the testing dataset. An online application to estimate PBV using this algorithm can be accessed at https://cd3yield.shinyapps.io/cd3yield/.ConclusionsThe authors propose a transferrable model that incorporates clinical and laboratory variables accessible pre-harvest for use across the field of T-cell therapy. Pending further validation, such a model may be used to generate an individual leukapheresis plan and streamline the process of cell harvest, a well-recognized bottleneck in the industry.     

Impact of Chemotherapy Delay on Overall Survival for AML with IDH1/2 Mutations: A Study in Adult Chinese Patients

The effect of time from diagnosis to treatment (TDT) on overall survival of patients with acute myeloid leukemia (AML) remains obscure. Furthermore, whether chemotherapy delay impacts overall survival (OS) of patients with a special molecular subtype has not been investigated. Here, we enrolled 364 cases of AML to assess the effect of TDT on OS by fractional polynomial regression in the context of clinical parameters and genes of FLT3ITD, NPM1, CEBPA, DNMT3a, and IDH1/2 mutations. Results of the current study show IDH1/2 mutations are associated with older age, M0 morphology, an intermediate cytogenetic risk group, and NPM1 mutations. TDT associates with OS for AML patients in a nonlinear pattern with a J shape. Moreover, adverse effect of delayed treatment on OS was observed in patients with IDH1/2 mutations, but not in those with IDH1/2 wildtype. Therefore, initiating chemotherapy as soon as possible after diagnosis might be a potential strategy to improve OS in AML patients with IDH1/2 mutations.