Molecular diagnostics in the treatment of childhood acute lymphoblastic leukemia

Somatically acquired genetic alterations play an important role in the pathogenesis of acute lymphoblastic leukemia. The molecular analysis of these alterations has increased our understanding of the mechanisms of leukemogenesis. In addition, this information has led to improvements in our abilities to predict treatment response and to deliver the optimal intensity of treatment to individual patients. For example, the prognosis for patients with acute lymphoblastic leukemia whose leukemic cells express the TEL-AML1 fusion is favorable when they are treated on modem chemotherapy protocols, whereas patients whose leukemic lymphoblasts contain the MLL-AF4 or the BCR-ABL fusion sometimes require allogeneic hematopoietic stem cell transplantation for cure. Molecular techniques are also used to detect minimal residual disease and genetic polymorphisms that are important in optimizing drug therapy.     

Nested PCR detection of WT1 expression in the peripheral blood in childhood acute leukemia

Detection of minimal residual disease (MRD) in childhood leukemia is not possible by cytomorphology or Southern blotting due to their low sensitivity. On the other hand, the use of DNA markers and PCR amplification is helpful in a smaller proportion of leukemia cases (20-30%). Since childhood leukemia is characterized by WT1 gene expression in the majority of cases,monitoring of WT1 expression in the peripheral blood was suggested to be a method of choice to detect MRD. We have studied 22 newly diagnosed childhood acute leukemias and 17 cases in remission. As controls, 19 patients with non-leukemic diseases were included. The majority of our acute leukemia cases (80%) were proved to be WT1 expressors using a highly sensitive nested PCR technique. Ten WT1 + cases have been monitored for a year throughout the inicial therapy phase, using peripheral blood tests. We observed that in 20% of the follow-up cases MRD was suggested which was not detectable by any other methods. It is our intention to introduce this new molecular technique into the clinical management of childhood acute leukemia.     

Genomic DNA of leukemic patients: target for clinical diagnosis of MLL rearrangements

Genomic DNA is the optimal resource to analyze questions concerning genetic changes that are related to oncogenesis. This article tries to summarize recent efforts to analyze chromosomal changes that trigger the development of human acute myeloid and lymphoblastic leukemias. The aim of this study was to establish an universal method that enables the identification and characterization of chromosomal translocations of the human MLL gene at the genomic nucleotide level. Chromosomal translocations of the MLL gene are the result of illegitimate recombination events in hematopoietic stem or precursor cells, strictly associated with the onset of highly malignant leukemic diseases. The applied technology was able to identify specific fusion alleles that were generated by chromosomal translocations, chromosomal deletions, chromosomal inversions and partial tandem duplications. Moreover, it allowed us to investigate even highly complex genetic changes by applying systematic breakpoint analyses. On the basis of these analyses, patient-specific molecular markers were established that turned out to be a very good source for monitoring minimal residual disease (MRD). MRD analyses control the efficiency and efficacy of current treatment protocols and have become a very sensitive molecular tool to monitor therapeutic success or failure in individual leukemia patients.     

The association between microRNA polymorphisms and the risk of childhood acute lymphoblastic leukemia: A meta-analysis

The aim of this meta-analysis was to determine the relationship between microRNA polymorphisms and the risk of childhood acute lymphoblastic leukemia comprehensively. PubMed, EMBASE, Scopus, Web of Science, the Cochrane Library, Global Index Medicus, Clinicaltrials.gov, ProQuest, and Open Grey databases were used to find relevant papers. Using the STATA 16.0 and CMA 3.0 software, the significance of relationships between microRNA polymorphisms and childhood acute lymphoblastic leukemia risk was evaluated using odds ratios (ORs) and 95 % confidence intervals (95 % CIs) for five genetic models. The results of the meta-analysis showed that there was no significant association between the polymorphism of miR-146a rs2910164 and childhood acute lymphoblastic leukemia risk in different genetic models. Also, in the sensitivity analysis, removing Xue's study from the analysis indicated that both the homozygote and recessive models are significantly affected. Additionally, there was a statistically significant relationship between the polymorphisms of pri-miR-34b/c rs4938723 (in the homozygote and recessive models) and miR-612 rs12803915 (in the allele and dominant models) and childhood acute lymphoblastic leukemia risk. These findings suggest that the rs4938723 and rs12803915 polymorphisms may have a role in the development of childhood acute lymphoblastic leukemia.     

LMO2与T细胞白血病

Lmo2基因是LMO(LIM-only)家族的成员之一。作为一个原癌基因,Lmo2的染色体异位t(11;14)(p13;q11)或t(7;11)(q35;p13)与T细胞急性淋巴细胞白血病密切相关。LMO2是细胞中介导转录因子复合物形成的重要接头分子。现对LMO2的分子结构及其在正常和白血病细胞中的调控作用机制的差异作重点介绍。在此基础上还讨论了LMO2成为逆转录病毒介导的基因治疗X染色体连锁的严重联合免疫缺陷综合征过程中成为病毒插入靶位点的可能原因。     

Minimal residual disease in leukemia: studies in an animal model for acute myelocytic leukemia (BNML)

The possibilities for studying minimal residual disease (MRD) in human acute myelocytic leukemia (AML) are limited. Animal models are, therefore, indispensable for gaining insight into the characteristics of leukemia growth during the MRD phase. Studies were done to compare AML to acute myelocytic leukemia in the Brown Norway rat (BNML). The BNML model exhibited a high degree of similarity to human AML with regard to its general growth characteristics, its cell kinetic parameters, its biophysical parameters and its response to chemotherapy. This implied that studies of the BNML model have predictive value for clinical application. In the BNML model a number of independent methods are available to quantify the number of leukemic cells, i.e., indirectly by means of various bioassays or directly by using monoclonal antibody labeling and flow cytometry. Studies of the BNML model in relation to the understanding of various aspects of MRD in leukemia are discussed in this concise review. Insight has been obtained with regard to the kinetics of MRD; the efficacy of certain treatment modalities, e.g., cytostatic drug treatment with or without total body irradiation to eradicate MRD; the efficacy of various methods for eliminating residual leukemic cells from autologous marrow grafts; the emergence of drug resistance during MRD; and the progression of residual disease during the remission phase ultimately leading to a relapse and the implications of these observations for staging leukemia patients during the phase of MRD.     

mTOR Inhibition by Everolimus in Childhood Acute Lymphoblastic Leukemia Induces Caspase-Independent Cell Death

Increasingly, anti-cancer medications are being reported to induce cell death mechanisms other than apoptosis. Activating alternate death mechanisms introduces the potential to kill cells that have defects in their apoptotic machinery, as is commonly observed in cancer cells, including in hematological malignancies. We, and others, have previously reported that the mTOR inhibitor everolimus has pre-clinical efficacy and induces caspase-independent cell death in acute lymphoblastic leukemia cells. Furthermore, everolimus is currently in clinical trial for acute lymphoblastic leukemia. Here we characterize the death mechanism activated by everolimus in acute lymphoblastic leukemia cells. We find that cell death is caspase-independent and lacks the morphology associated with apoptosis. Although mitochondrial depolarization is an early event, permeabilization of the outer mitochondrial membrane only occurs after cell death has occurred. While morphological and biochemical evidence shows that autophagy is clearly present it is not responsible for the observed cell death. There are a number of features consistent with paraptosis including morphology, caspase-independence, and the requirement for new protein synthesis. However in contrast to some reports of paraptosis, the activation of JNK signaling was not required for everolimus-induced cell death. Overall in acute lymphoblastic leukemia cells everolimus induces a cell death that resembles paraptosis.     

Molecular and cellular bases of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the overproduction of granulocytes, which leads to high white blood cell counts and splenomegaly in patients. Based on clinical symptoms and laboratory findings, CML is classified into three clinical phases, often starting with a chronic phase, progressing to an accelerated phase and ultimately ending in a terminal phase called blast crisis. Blast crisis phase of CML is clinically similar to an acute leukemia; in particular, B-cell acute lymphoblastic leukemia (B-ALL) is a severe form of acute leukemia in blast crisis, and there is no effective therapy for it yet. CML is induced by the BCR-ABL oncogene, whose gene product is a BCR-ABL tyrosine kinase. Currently, inhibition of BCR-ABL kinase activity by its kinase inhibitor such as imatinib mesylate (Gleevec) is a major therapeutic strategy for CML. However, the inability of BCR-ABL kinase inhibitors to completely kill leukemia stem cells (LSCs) indicates that these kinase inhibitors are unlikely to cure CML. In addition, drug resistance due to the development of BCR-ABL mutations occurs before and during treatment of CML with kinase inhibitors. A critical issue to resolve this problem is to fully understand the biology of LSCs, and to identify key genes that play significant roles in survival and self-renewal of LSCs. In this review, we will focus on LSCs in CML by summarizing and discussing available experimental results, including the original studies from our own laboratory.     

Prognostic value of the reaction to steroids in the treatment of acute lymphoblastic leukemia in children]

Relationship between the result of therapy in 48 cases of the acute lymphoblastic leukemia in childhood and character of response to corticosteroids, classified according to BMF group, has been assessed. Follow up period ranged from 13 to 75 months (mean 36 months, median 39 months). In was found, that the probability of survival free from any events, probability of complete remission persistence, and probability of survival after diagnosis have been statistically significantly higher in the group of patients with positive response to corticosteroids in comparison with patients non-responding to these agents. However, there was no significant difference in the number of recurrencies with the involvement of CNS. Authors share the opinion that their results confirm an opinion of Riehm et al. that the response to corticosteroids is of prognostic value in the acute lymphoblastic leukemia in childhood.     

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.     

High-valency Anti-CD99 Antibodies Toward the Treatment of T Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that currently requires intensive chemotherapy. While childhood T-ALL is associated with high cure rates, adult T-ALL is not, and both are associated with significant short- and long-term morbidities. Thus, less toxic and effective strategies to treat T-ALL are needed. CD99 is overexpressed on T-ALL blasts at diagnosis and at relapse. Although targeting CD99 with cytotoxic antibodies has been proposed, the molecular features required for their activity are undefined. We identified human antibodies that selectively bound to the extracellular domain of human CD99, and the most potent clone, 10A1, shared an epitope with a previously described cytotoxic IgM antibody. We engineered clone 10A1 in bivalent, trivalent, tetravalent, and dodecavalent formats. Increasing the antibody valency beyond two had no effects on binding to T-ALL cells. In contrast, a valency of ≥3 was required for cytotoxicity, suggesting a mechanism of action in which an antibody clusters ≥3 CD99 molecules to induce cytotoxicity. We developed a human IgG-based tetravalent version of 10A1 that exhibited cytotoxic activity to T-ALL cells but not to healthy peripheral blood cells. The crystal structure of the 10A1 Fab in complex with a CD99 fragment revealed that the antibody primarily recognizes a proline-rich motif (PRM) of CD99 in a manner reminiscent of SH3-PRM interactions. This work further validates CD99 as a promising therapeutic target in T-ALL and defines a pathway toward the development of a selective therapy against T-ALL.     

Cancer Pharmacogenomics May Require Both Qualitative and Quantitative Approaches

Resistance to chemotherapy is a major cause of mortality in patients receiving treatment for most types of cancer, and overcoming drug resistance has become an important focus of current research. A major clinical challenge is the fact that most anticancer drugs have a narrow therapeutic range, that is, their effective dose is relatively close to that associated with substantial toxicity. Significant advances have been achieved in event-free survival of patients with many types of cancer (most dramatically childhood acute lymphoblastic leukemia, ALL) through a better understanding of the pathobiology of human cancers, the cellular mechanisms of cancer chemotherapy, and the determinants of inter-individual differences in drug effects and treatment response. It is anticipated that expanding our knowledge of these areas will lead to the development of new anticancer agents and to more effective use of existing cancer chemotherapy. Pharmacogenomics research aims to elucidate the genetics determinants of drug efficacy and toxicity. Results of recent studies indicate that both qualitative and quantitative genomic analyses may be required for precise pharmacogenomic characterization of some types of human cancer.     

Gene expression signatures and ex vivo drug sensitivity profiles in children with acute lymphoblastic leukemia

Introduction  

Causes of treatment failure in acute lymphoblastic leukemia (ALL) are still poorly understood. Microarray technology gives new possibilities for the analysis of the biology of leukemias. We hypothesize that drug sensitivity in pediatric ALL is driven by specific molecular mechanisms that correlate with gene expression profiles assessed by microarray analysis.     

Genetically engineered mouse models of human B-cell precursor leukemias

B-cell precursor acute lymphoblastic leukemias (pB-ALLs) are the most frequent type of malignancies of the childhood, and also affect an important proportion of adult patients. In spite of their apparent homogeneity, pB-ALL comprises a group of diseases very different both clinically and pathologically, and with very diverse outcomes as a consequence of their biology, and underlying molecular alterations. Their understanding (as a prerequisite for their cure) will require a sustained multidisciplinary effort from professionals coming from many different fields. Among all the available tools for pB-ALL research, the use of animal models stands, as of today, as the most powerful approach, not only for the understanding of the origin and evolution of the disease, but also for the development of new therapies. In this review we go over the most relevant (historically, technically or biologically) genetically engineered mouse models (GEMMs) of human pB-ALLs that have been generated over the last 20 years. Our final aim is to outline the most relevant guidelines that should be followed to generate an “ideal” animal model that could become a standard for the study of human pB-ALL leukemia, and which could be shared among research groups and drug development companies in order to unify criteria for studies like drug testing, analysis of the influence of environmental risk factors, or studying the role of both low-penetrance mutations and cancer susceptibility alterations.     

Stages of B-lymphocyte differentiation in acute lymphoblastic leukemia

Blasts phenotype was determined in 61 children with the acute lymphoblastic leukemia. Non-T-cell acute lymphoblastic leukemia was diagnosed in 51 children. Stages of blasts differentiation were determined with the aid of monoclonal antibodies set using alkaline phosphatase-anti-alkaline phosphatase technique. Blasts in 50 patients belonged to B subpopulation confirmed by the presence of panB CD19 and CD22 antigens. Common antigen was seen in 76.5% of the examined patients with non-T-cell acute lymphoblastic leukemia. Cases of non-T-cell acute lymphoblastic leukemia were divided into 8 subgroups depending on the antigens of B-cells differentiation. An identification of pre-B subgroups of the acute lymphoblastic leukemia indicates heterogenicity of the acute lymphoblastic leukemias in childhood and enables their classification into groups corresponding to the early stages of lymphoblasts maturation.     

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.     

A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies.