Detection of minimal residual disease in acute leukemia by flow cytometry.

Patients with acute leukemia in clinical remission may still have up to 10(10) residual malignant cells (the upper limit of detection by standard morphologic techniques). Sensitive techniques to detect minimal residual disease (MRD) may allow better estimates of the leukemia burden and help the selection of appropriate therapeutic strategies. Flow cytometry and polymerase chain reaction have emerged as the most promising methods for detecting submicrospopic levels of leukemia. Flowcytometric detection of MRD is based on the identification of immunophenotypic combinations expressed on leukemic cells but not on normal hematopoietic cells. It affords the detection of one leukemic cell among 10,000 normal bone marrow cells, and can be currently applied to at least two thirds of all patients with acute leukemia. Prospective studies in large series of patients have demonstrated a strong correlation between MRD levels during clinical remission and treatment outcome. Therefore, MRD assays can be reliably used to assess early response to treatment and predict relapse. In this review, we discuss methodologic aspects and clinical results of flowcytometric detection of MRD in patients with acute leukemia.     

CD304 is preferentially expressed on a subset of B-lineage acute lymphoblastic leukemia and represents a novel marker for minimal residual disease detection by flow cytometry

Minimal residual disease (MRD) has emerged as a major prognostic factor for monitoring patients with B-lineage acute lymphoblastic leukemia (B-ALL). The quantification of MRD by flow cytometry (FC) is based on the identification of a leukemia-associated phenotype (LAP). Because phenotypic switch is common during treatment, multiple LAPs must be available and used for MRD detection over time. We evaluated the potential usefulness of CD304 as a new marker for monitoring MRD. CD304 was expressed in 48% of B-ALL (24/50) with discriminative fluorescence intensity compared with CD304-negative normal B-cell precursors (n = 15). The sensitivity of CD304-based MRD detection reached 10(-4), as with some of established LAPs. The stability of CD304 expression evaluated during therapy and at relapse confirms the usefulness of this marker for MRD quantification. Finally, CD304 was repeatedly expressed in patients with TEL-AML1 gene rearrangement, which warrants further investigation on its potential relevance as a prognosis marker or therapeutic target.     

Treatment outcomes for childhood acute lymphoblastic leukemia in low-middle income country before minimal residual disease risk stratification

BackgroundOutcome of childhood acute lymphoblastic leukemia (ALL) in low- and middle-income countries is lagging in many aspects including diagnosis, risk stratification, access to treatment and supportive care.Objectiveto report the outcome of childhood ALL at Ain Shams University Children’s Hospitals with the use of risk-based protocols before the implementation of minimal residual disease technology and to evaluate the use of double delayed intensification (DDI) in standard risk patients.MethodsTwo hundred and twenty patients with ALL diagnosed between January 2005 and December 2014 were included in the study. Patients were treated according to a modified CCG 1991 and 1961 for standard and high risk respectively. Patients were stratified into three risk groups: standard risk (SR), high-risk standard arm (HR-SA), and high-risk augmented arm (HR-AA).ResultsAmong the whole cohort, the 10-year event-free survival (EFS) and overall survival (OS) were 78.1% and 84.3% respectively. Patients with Pre-B immunophenotype (IPT) had significantly better outcome than T-cell IPT (EFS 82.0% versus 58.6%, p < 0.001; OS 86.9% versus 69%, p = 0.003 for Pre-B and T-cell respectively). Among the SR group, patients treated with single delayed intensification (SDI) had comparable EFS and OS rates when compared to patients treated with DDI with EFS 82.4% versus 87.5%, p = 0.825 and OS 88.2% versus 93.5%, p = 0.638 for SDI and DDI groups, respectively.ConclusionThe use of risk-based protocol with simple laboratory techniques resulted in acceptable survival outcome in resource limited settings. The use of double delayed intensification showed no survival advantage in patients with standard risk.     

Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease.

The aim of this study was to investigate to which extent acute leukemias could be monitored for residual disease by using atypical antigen combinations as leukemia-related markers. Atypical antigenic features were determined by double color flow cytometry and included coexpression of lymphoid and myeloid related antigens, unphysiological coexpression of immature and mature antigens, and lack of an antigen that is normally expressed during maturation. Atypical immunophenotypes were detected in 35 of 68 patients with AML (51.5%) and 15 of 24 patients with ALL (62.5%). When 12 patients with leukemia-associated markers were again analyzed at relapse, the relevant antigen combinations were retained in 11 of them. The sensitivity of this two color flow cytometric assay as determined in dilution experiments was 1 in 10(3) to 10(4) cells. Follow-up studies of bone marrow samples revealed that, after induction chemotherapy cells with leukemia-associated markers were detectable in several patients at a frequency of 0.5 to 4%, but only patients in whom the cells with atypical antigens never disappeared suffered from relapse. In contrast, patients who became negative for the atypical cells remained in complete remission (median remission duration after the first negative bone marrow assessment by flow cytometry 52 weeks, range 20-102). We conclude that atypical antigen combinations, which are present in a meaningful number of acute leukemias, are a valuable means of monitoring acute leukemia patients during follow-up. This flow cytometric approach can complement other strategies to get a more accurate definition of remission in acute leukemia.     

Identification of complement C3f‐desArg and its derivative for acute leukemia diagnosis and minimal residual disease assessment

Therapeutic conditions for acute leukemia (AL) mainly rely on diagnosis and detection of minimal residual disease (MRD). However, no serum biomarker has been available for clinicians to make diagnosis of AL and assessment of MRD. In this study, we performed bead fractionation/MALDI‐TOF‐MS analysis on sera from patients with AL. Support vector machine algorithm was used to obtain diagnostic model that discriminated proteomic spectra of patients with AL from that of controls. Twenty‐six features with p<0.00001 had optimal discriminatory performance, with 97% sensitivity and 100% specificity. Statistical analysis revealed that two peptides with m/z 1778 and 1865 were gradually decreased in their relative intensities with increase of remission degree. Moreover, the peptide with m/z 1865 was also found to be correlated with AL types. With FT‐ICR‐MS detection, both the peptides were identified as fragments of complement C3f. Linear regression analysis showed that the combined use of them could discriminate PML/RARα positive M3 from molecular remission M3. Two fragments of complement C3f were significantly correlated with MRD levels and could be used for clinical practice in MRD assessment.     

Detection of minimal disease in acute leukemia using flow cytometry: studies in a rat model for human acute leukemia

The study was made using a rat model for human acute myelocytic leukemia (BNML), which shows striking similarities with human acute myelocytic leukemia (AML). A monoclonal antibody (MCA-Rm124), raised against BNML cells, allowed the recognition of the leukemic cell fraction. The discriminative capacity of the monoclonal antibody is based on differences in labeling intensities between normal and leukemic cells. After i.v. cellular transfer of leukemia, the growth of the leukemic cell population in the bone marrow, the liver, and the spleen was monitored using MCA-Rm124 and flow cytometry. For the bone marrow and the liver, a clonogenic assay for leukemic cells was used to quantify the cell content in these organs. A good correlation was found between the bioassay-derived data and the flow-cytometry-derived data. The doubling times of the leukemic cell population were not equal for the two organs studied, indicating that a number of different processes contribute to the net cell production per organ. Apart from their application in the detection of residual leukemia, monoclonal antibodies might be employed in the analysis of the growth kinetics of the "invisible" leukemic cell population.     

A dysfunctional Mo1 glycoprotein is present on a subline of the KG1 acute myelogenous leukemia cell line

Mo1 is a glycoprotein heterodimer found on the surface of phagocytic cells. By use of monoclonal antibodies directed against various epitopes of the 155 kd alpha-chain of Mo1, two distinct functions of this glycoprotein have been identified. Mo1 serves as the receptor (CR3) for iC3b, one of the breakdown products of the third component of complement, and in addition appears to be involved in promoting cell-cell adhesion of granulocytes. In studies performed with a subline of the acute myelogenous leukemia tumor cell line KG1, we found cells from this subline (KG1m) incapable of iC3b-mediated binding despite these cells having surface Mo1. Five distinct epitopes on Mo1 identifiable on normal cells by a panel of anti-Mo1 alpha-chain monoclonal antibodies (including OKM10, thought to be identical to or close to the iC3b binding site) were equally present on KG1m by indirect immunofluorescence. The electrophoretic mobilities of both the alpha- and beta-chains of Mo1 derived from KG1m were identical to those isolated from normal granulocytes. To determine whether altered receptor mobility or decreased surface density of Mo1 was responsible for the lack of Mo1-related functions, binding to EiC3b of isolated Mo1 derived from KG1m lysate was measured. KG1m-derived Mo1 did not bind to EiC3b when compared with normal granulocyte-derived Mo1, suggesting that the lack of iC3b binding is not secondary to decreased surface receptor number or mobility. This subline of KG1 provides an excellent model for the study of the relationship between surface receptor structure and function.     

Overcoming minimal residual disease using intensified conditioning with medium-dose etoposide,cyclophosphamide and total body irradiation in allogeneic stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia in adults

Background aimsAn intensified conditioning regimen incorporating medium-dose etoposide (VP16) is an option for patients with acute lymphoblastic leukemia (ALL). However, the prognostic impacts of the addition of VP16 to cyclophosphamide (CY) and total body irradiation (TBI) in patients with Philadelphia chromosome-positive (Ph+) ALL with regard to minimal residual disease (MRD) status have not been elucidated.MethodsThe authors retrospectively compared the outcomes of patients with Ph+ ALL who underwent allogeneic transplantation following VP16/CY/TBI (n = 101) and CY/TBI (n = 563).ResultsAt 4 years, the VP16/CY/TBI group exhibited significantly better disease-free survival (DFS) (72.6% versus 61.7%, P = 0.027) and relapse rate (11.5% versus 21.1%, P = 0.020) and similar non-relapse mortality (16.0% versus 17.2%, P = 0.70). In subgroup analyses, the beneficial effects of the addition of VP16 on DFS were more evident in patients with positive MRD status (71.2% versus 48.4% at 4 years, P = 0.022) than those with negative MRD status (72.8% versus 66.7% at 4 years, P = 0.24). Although MRD positivity was significantly associated with worse DFS in patients who received CY/TBI (48.4% versus 66.7%, P < 0.001), this was not the case in those who received VP16/CY/TBI (71.2% versus 72.8%, P = 0.86).ConclusionsThis study demonstrated the benefits of the addition of VP16 in Ph+ ALL patients, especially those with positive MRD status. VP16/CY/TBI could be a potential strategy to overcome the survival risk of MRD positivity.     

Interaction between integrin alpha(5) and fibronectin is required for metastasis of B16F10 melanoma cells

In this study, we report the role of integrin alpha(5) in promoting melanoma metastasis. The alpha(5) expression was remarkably elevated in highly metastatic B16F10 melanoma cells compared to lowly metastatic B16F1 cells, whereas no significant changes were detected in those of integrin alpha(4), alpha(v), and beta(1) subunits. Neutralization of alpha(5) with anti-alpha(5) antibody significantly suppressed the potential of B16F10 cells for pulmonary metastasis in mice and inhibited cell adhesion or spreading to fibronectin in vitro. Furthermore, loss of the interaction between alpha(5) and fibronectin diminished cell survival and induced apoptosis in B16F10 cells. Above results provide clear evidence that integrin alpha(5) is positively correlated with melanoma metastasis and might be an anti-melanoma target.     

Critical period for a teratogenic VLA-4 antagonist: Developmental effects and comparison of embryo drug concentrations of teratogenic and non-teratogenic VLA-4 antagonists

BACKGROUND: Integrins such as VLA-4 (Very late antigen 4, integrin alpha4beta1) play key roles in cell-cell interactions that are critical for development. Homozygous null knockouts of the VLA-4 alpha4-subunit or VCAM-1 (VLA-4 cell surface ligand) in mice result in failure of the allantois and chorion to fuse leading to interrupted placentation and cardiac development and embryo lethality. Embryo-fetal studies of three VLA-4 antagonists, IVL745, IVL984, and HMR1031 [Crofts et al., Birth Defects Res B 71:55-68 (this issue), 2004] with exposure on gestation days (GD) 6-17 (rat), 6-18 (rabbit) or 6-15 (mouse) showed that only IVL984 treatment resulted in embryo lethality and cardiac defects. Objectives of the current study were to determine the critical period for inducing IVL984-related embryo-fetal effects, and to test the hypothesis that these effects were due to higher embryo drug concentrations. METHODS: IVL984 was administered at 40 mg/kg/day to pregnant rats on GD 4 and 5, GD 6 and 7, GD 8 and 9, GD 10 and 11, or GD 12 and 13. Animals were euthanized on GD 21 and uteri and fetuses were examined. A treatment period of GD 10-12 was selected for subsequent toxicokinetic (TK) studies in which IVL984, HMR1031, or IVL745 was administered to pregnant rats and rabbits. On GD 12, maternal plasma, extra-embryonic tissue (placenta and amniotic fluid), and embryonic tissue were collected and analyzed for drug concentrations. RESULTS: In the IVL984 critical period study in pregnant rats, treatment on GD 10 and 11 resulted in increased post-implantation loss, skeletal variations, and spiral septal defects similar to those observed in standard embryo-fetal development studies with treatment throughout organogenesis. There were no embryo-fetal effects after treatment on GD 4 and 5, GD 6 and 7, or GD 8 and 9. There was a single aorta malformation after treatment on GD 12 and 13. In the TK studies, IVL745, HMR1031, and IVL984 were all detectable in embryonic tissue and there was no evidence for accumulation. Rat and rabbit embryo exposures (AUC or dose-adjusted AUC) on GD 12 could not explain the observed teratology (IVL984相似文献   

Human BDH2, an anti-apoptosis factor,is a novel poor prognostic factor for de novo cytogenetically normal acute myeloid leukemia

Background

The relevance of recurrent molecular abnormalities in cytogenetically normal (CN) acute myeloid leukemia (AML) was recently acknowledged by the inclusion of molecular markers such as NPM1, FLT3, and CEBPA as a complement to cytogenetic information within both the World Health Organization and the European Leukemia Net classifications. Mitochondrial metabolism is different in cancer and normal cells. A novel cytosolic type 2-hydroxybutyrate dehydrogenase, BDH2, originally named DHRS6, plays a physiological role in the cytosolic utilization of ketone bodies, which can subsequently enter mitochondria and the tricarboxylic acid cycle. Moreover, BDH2 catalyzes the production of 2, 3-DHBA during enterobactin biosynthesis and participates in 24p3 (LCN2)-mediated iron transport and apoptosis.

Results

We observed that BDH2 expression is an independent poor prognostic factor for CN-AML, with an anti-apoptotic role. Patients with high BDH2 expression have relatively shorter overall survival (P = 0.007) and a low complete response rate (P = 0.032). BDH2-knockdown (BDH2-KD) in THP1 and HL60 cells increased the apoptosis rate under reactive oxygen species stimulation. Decrease inducible survivin, a member of the inhibitors of apoptosis family, but not members of the Bcl-2 family, induced apoptosis via a caspase-3-independent pathway upon BDH2-KD.

Conclusions

BDH2 is a novel independent poor prognostic marker for CN-AML, with the role of anti-apoptosis, through surviving.     

Interaction of culture-derived macrophages with the fibroblast-binding domain of fibronectin is a necessary but inefficient signal for fibronectin enhancement of CR1-mediated phagocytosis

Human plasma fibronectin (Fn) enhances ingestion of opsonized particles through its interaction with phagocytic cells. To better characterize the site or sites on Fn responsible for this effect, we subjected Fn to limited proteolytic cleavage by chymotrypsin and used affinity and gel filtration chromatography to isolate a 110,000 dalton cell-binding fragment, a 60,000 dalton fragment that bound both heparin and gelatin, and 50,000 and 45,000 dalton fragments that bound to gelatin but not heparin. The cell-binding fragment mediated adhesion and spreading of fibroblasts on glass slides, whereas the heparin-gelatin and gelatin-binding fragments failed to cause fibroblast spreading. At high concentrations, the cell-binding fragment doubled phagocytosis of C4b-coated sheep erythrocytes by human monocyte-derived macrophages, whereas equal concentrations of the other fragments had minimal enhancing effect on phagocytosis. Interestingly, the effect of the cell-binding fragment on CR1-mediated phagocytosis was always less than the effect of intact Fn, even when a 40-fold higher molar concentration of the cell-binding fragment was used. Fab of a monoclonal anti-Fn, HFn 7.1, which recognizes the 110,000 dalton cell-binding fragment of Fn and inhibits fibroblast binding, blocked enhancement of CR1-mediated phagocytosis by intact Fn. Fab of Fn 8, a monoclonal anti-Fn that binds the heparin-gelatin-binding fragment, failed to inhibit the Fn effect. These data suggest that interaction of the macrophage with the cell-binding domain of Fn is critical for the Fn effect on CR1-mediated phagocytosis. In addition, there may be other domains of the Fn molecule that have a role in augmenting the Fn-phagocyte interaction.     

Inhibition of MEK/ERK signaling induces apoptosis of acute myelogenous leukemia cells via inhibition of eukaryotic initiation factor 4E-binding protein 1 and down-regulation of Mcl-1

We previously showed that the MEK inhibitor AZD6244 induced apoptosis in acute myelogenous leukemia (AML) HL60 cells. However, the mechanisms of AZD6244 to induce apoptosis remain to be fully elucidated. This study found that exposure of HL60 cells to AZD6244 down-regulated the levels of phosphor (p)-4E-binding protein 1 (4E-BP1), a substrate of mammalian target of rapamycin complex 1 (mTORC1), and anti-apoptotic protein Mcl-1. On the other hand, exposure of EOL-1 and MOLM13 cells to AZD6244 failed to induce apoptosis and levels of p-4E-BP1 and Mcl-1 were not down-regulated in these cells. These observations prompted us to hypothesize that down-regulation od 4E-BP1 and Mcl-1 might play an important role in AZD6244-mediated apoptosis. As expected, down-regulation of 4E-BP1 by an siRNA sensitized EOL-1 cells to AZD6244-mediated apoptosis in parallel with down-regulation of Mcl-1. Moreover, we found that blockade of mTORC1 by RAD001 synergistically enhanced the action of AZD6244 in leukemia cells.