Genomic analysis of acute leukemia

Acute leukemia is the commonest childhood cancer and a major cause of morbidity from hematologic malignancies in adults. Acute lymphoblastic leukemia (ALL) is commonest in children, and acute myeloid leukemia (AML) is more frequent in adults. Apart from childhood ALL, the prognosis of acute leukemia is suboptimal, with many patients experiencing relapse, which carries a poor prognosis, or toxicities from nonspecific therapies. Recent years have witnessed great interest in the application of high‐resolution, genome wide approaches to the study of acute leukemia. These studies have identified multiple novel genetic alterations targeting critical cellular pathways that contribute to leukemogenesis, including alterations of genes regulting lymphoid development, tumor suppressors, apoptosis regulators, and oncogenes. These studies have also delineated novel genetic alterations that are associated with prognosis, and have demonstrated substantial evolution in patterns of genetic alterations from diagnosis to relapse, indicating that specific genetic changes determine resistance to therapy in ALL. Overall, fewer recurring alterations have been identified in AML. These studies have demonstrated the power of genome‐wide approaches to identify new lesions in acute leukemia, and suggest that ongoing genomic analyses, including deep resequencing and epigenetic analysis, will continue to yield novel, clinically relevant insights into the pathogenesis of this disease.     

Acquired copy number alterations in adult acute myeloid leukemia genomes

Cytogenetic analysis of acute myeloid leukemia (AML) cells has accelerated the identification of genes important for AML pathogenesis. To complement cytogenetic studies and to identify genes altered in AML genomes, we performed genome-wide copy number analysis with paired normal and tumor DNA obtained from 86 adult patients with de novo AML using 1.85 million feature SNP arrays. Acquired copy number alterations (CNAs) were confirmed using an ultra-dense array comparative genomic hybridization platform. A total of 201 somatic CNAs were found in the 86 AML genomes (mean, 2.34 CNAs per genome), with French-American-British system M6 and M7 genomes containing the most changes (10–29 CNAs per genome). Twenty-four percent of AML patients with normal cytogenetics had CNA, whereas 40% of patients with an abnormal karyotype had additional CNA detected by SNP array, and several CNA regions were recurrent. The mRNA expression levels of 57 genes were significantly altered in 27 of 50 recurrent CNA regions <5 megabases in size. A total of 8 uniparental disomy (UPD) segments were identified in the 86 genomes; 6 of 8 UPD calls occurred in samples with a normal karyotype. Collectively, 34 of 86 AML genomes (40%) contained alterations not found with cytogenetics, and 98% of these regions contained genes. Of 86 genomes, 43 (50%) had no CNA or UPD at this level of resolution. In this study of 86 adult AML genomes, the use of an unbiased high-resolution genomic screen identified many genes not previously implicated in AML that may be relevant for pathogenesis, along with many known oncogenes and tumor suppressor genes.     

DNA copy number alterations mark disease progression in paediatric chronic myeloid leukaemia

Early recognition of children with chronic phase chronic myeloid leukaemia (CML‐CP) at risk for developing a lymphoid blast crisis (LyBC) is desirable, because therapy options in CML‐LyBC are limited. We used Multiplex Ligation‐dependent Probe Amplification to determine whether B‐cell lymphoid leukaemia‐specific copy number alterations (CNAs) (e.g. IKZF1, PAX5, CDKN2A deletions) could be detected in CML‐CP and may be used to predict disease progression to LyBC. CNAs were detected in all patients with CML‐LyBC, but in none of the 77 patients with CML‐CP. Based on this study we conclude that CNAs remain a hallmark of disease progression.     

Evaluation of gene expression signatures predictive of cytogenetic and molecular subtypes of pediatric acute myeloid leukemia

Background

Pediatric acute myeloid leukemia is a heterogeneous disease characterized by non-random genetic aberrations related to outcome. The genetic subtype is currently detected by different diagnostic procedures which differ in success rate and/or specificity.

Design and Methods

We examined the potential of gene expression profiles to classify pediatric acute myeloid leukemia. Gene expression microarray data of 237 children with acute myeloid leukemia were collected and a double-loop cross validation approach was used to generate a subtype-predictive gene expression profile in the discovery cohort (n=157) which was then tested for its true predictive value in the independent validation cohort (n=80). The classifier consisted of 75 probe sets, representing the top 15 discriminating probe sets for MLL-rearranged, t(8;21)(q22;q22), inv(16)(p13q22), t(15;17)(q21;q22) and t(7;12)(q36;p13)-positive acute myeloid leukemia.

Results

These cytogenetic subtypes represent approximately 40% of cases of pediatric acute myeloid leukemia and were predicted with 92% and 99% accuracy in the discovery and independent validation cohort, respectively. However, for NPM1, CEBPA, MLL(-PTD), FLT3(-ITD), KIT, PTPN11 and N/K-RAS gene expression signatures had limited predictive value. This may be caused by a limited frequency of these mutations and by underlying cytogenetics. This latter is exemplified by the fact that different gene expression signatures were discovered for FLT3-ITD in patients with normal cytogenetics and in those with t(15;17)(q21;q22)-positive acute myeloid leukemia, which pointed to HOXB-upregulation being specific for FLT3-ITD⁺ cytogenetically normal acute myeloid leukemia.

Conclusions

In conclusion, gene expression profiling correctly predicted the most prevalent cytogenetic subtypes of pediatric acute myeloid leukemia with high accuracy. In clinical practice, this gene expression signature may replace multiple diagnostic tests for approximately 40% of pediatric acute myeloid leukemia cases whereas only for the remaining cases (predicted as ‘acute myeloid leukemia-other’) are additional tests indicated. Moreover, the discriminative genes reveal new insights into the biology of acute myeloid leukemia subtypes that warrants follow-up as potential targets for new therapies.     

Annular promyelocytes in acute myeloid leukemia

Summary Granulocytes with ring-shaped nuclei (annular granulocytes; ring granulocytes) are normal bone marrow constituents in rodents. Studies in man have shown a small number of these cells in cases of myeloproliferative disease. Myelocytes and metamyelocytes have also been described. Similar to rodents and some other animal species, the annular promyelocyte also exists in humans. The significance of these very rare cells in human haemopoesis becomes an interesting question.     

Integrated analysis of homozygous deletions, focal amplifications, and sequence alterations in breast and colorectal cancers

We have performed a genome-wide analysis of copy number changes in breast and colorectal tumors using approaches that can reliably detect homozygous deletions and amplifications. We found that the number of genes altered by major copy number changes, deletion of all copies or amplification to at least 12 copies per cell, averaged 17 per tumor. We have integrated these data with previous mutation analyses of the Reference Sequence genes in these same tumor types and have identified genes and cellular pathways affected by both copy number changes and point alterations. Pathways enriched for genetic alterations included those controlling cell adhesion, intracellular signaling, DNA topological change, and cell cycle control. These analyses provide an integrated view of copy number and sequencing alterations on a genome-wide scale and identify genes and pathways that could prove useful for cancer diagnosis and therapy.     

Novel monoclonal antibody-based therapies for acute myeloid leukemia

There has been long-standing interest in using monoclonal antibodies to improve outcomes of people with acute myeloid leukemia (AML). While several candidate therapeutics have failed at various stages of clinical testing, improved survival of some patients receiving the CD33 antibody-drug conjugate gemtuzumab ozogamicin has provided first evidence that monoclonal antibodies have a role in the armamentarium against AML. Over the last several years, work to improve the success of monoclonal antibody-based therapies in AML has focused on the identification and exploration of new antigen targets as much as on the development of novel treatment formats such as use of unconjugated engineered monoclonal antibodies and conjugated antibodies, delivering highly potent small molecule drugs or radionuclides to AML cells. Here, we will provide a brief overview of current efforts with such investigational monoclonal antibody-based therapeutics.     

Integrative analysis of type-I and type-II aberrations underscores the genetic heterogeneity of pediatric acute myeloid leukemia

Background

Several studies of pediatric acute myeloid leukemia have described the various type-I or type-II aberrations and their relationship with clinical outcome. However, there has been no recent comprehensive overview of these genetic aberrations in one large pediatric acute myeloid leukemia cohort.

Design and Methods

We studied the different genetic aberrations, their associations and their impact on prognosis in a large pediatric acute myeloid leukemia series (n=506). Karyotypes were studied, and hotspot regions of NPM1, CEPBA, MLL, WT1, FLT3, N-RAS, K-RAS, PTPN11 and KIT were screened for mutations of available samples. The mutational status of all type-I and type-II aberrations was available in 330 and 263 cases, respectively. Survival analysis was performed in a subset (n=385) treated on consecutive acute myeloid leukemia Berlin-Frankfurt-Munster Study Group and Dutch Childhood Oncology Group treatment protocols.

Results

Genetic aberrations were associated with specific clinical characteristics, e.g. significantly higher diagnostic white blood cell counts in MLL-rearranged, WT1-mutated and FLT3-ITD-positive acute myeloid leukemia. Furthermore, there was a significant difference in the distribution of these aberrations between children below and above the age of two years. Non-random associations, e.g. KIT mutations with core-binding factor acute myeloid leukemia, and FLT3-ITD with t(15;17)(q22;q21), NPM1- and WT1-mutated acute myeloid leukemia, respectively, were observed. Multivariate analysis revealed a ‘favorable karyotype’, i.e. t(15;17)(q22;q21), t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22). NPM1 and CEBPA double mutations were independent factors for favorable event-free survival. WT1 mutations combined with FLT3-ITD showed the worst outcome for 5-year overall survival (22±14%) and 5-year event-free survival (20±13%), although it was not an independent factor in multivariate analysis.

Conclusions

Integrative analysis of type-I and type-II aberrations provides an insight into the frequencies, non-random associations and prognostic impact of the various aberrations, reflecting the heterogeneity of pediatric acute myeloid leukemia. These aberrations are likely to guide the stratification of pediatric acute myeloid leukemia and may direct the development of targeted therapies.     

Outpatient consolidation chemotherapy in pediatric acute myeloid leukemia: a retrospective analysis

Abstract

Background: To assess the outcomes of outpatient high dose cytosine arabinoside consolidation cycles in pediatric acute myeloid leukemia (AML) patients in comparison to inpatient treatment.

Methods: We retrospectively analyzed 90 cycles of AML consolidation given to 30 patients between July 2003 and July 2007.

Results: Median age was 8 years (range 1·5–15) and 22/30 (73·3%) were males. Sixty-nine of 90 (76·7%) cycles were given on an ambulatory basis; readmission occurred in 25/69 (36·2%) and there was one death. The outpatient cycles in comparison to the inpatient cycles required significantly fewer invasive blood investigations (p<0·001) but had comparable number of blood products administered as supportive therapy. There was no significant difference in the frequency of granulocyte colony stimulating factor usage and recovery time of absolute neutrophil count and platelet count. The incidence of febrile neutropenia though was comparable in the groups (72·5% outpatient versus 76·2% inpatient), but the duration (p=0·003) and subsequent therapeutic antifungal usage (p=0·001) was significantly more in inpatient administered cycles. Second line antibiotics were needed in 16/50 (32%) outpatient episodes of febrile neutropenia in contrast to 10/16 (72·5%) episodes of febrile neutropenia in inpatient courses (p=0·030).

Conclusions: Outpatient AML consolidation therapy is safe and feasible in children. It appears to result in less frequent invasive blood studies; shorter duration of febrile neutropenia and consequently less antimicrobial and antifungal usage as compared to inpatient consolidation cycles. To our knowledge, this report is the first of its kind looking specifically at outpatient consolidation chemotherapy in AML.     

New approaches in acute myeloid leukemia

Today, treatment of patients with acute myeloid leukemia (AML) remains predominantly a "one-fits-all" approach with intensive cytarabine-based chemotherapy as the mainstay, but we are finally beginning to reap the rewards of decades of basic, translational, and clinical research. Developing individualized, "targeted" therapy for each AML patient based on unique molecular features of disease remains a daunting goal, yet one that we can now begin to envision. Hypothesis-based study designs--from preclinical/laboratory experiments to phase 1 and subsequent efficacy trials--provide the foundation for advances in the diagnosis, risk stratification, and treatment of patients with AML. The following is an outline of several key areas of ongoing AML research.     

When the good go bad: Mutant NPM1 in acute myeloid leukemia

Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein that performs diverse biological functions including molecular chaperoning, ribosome biogenesis, DNA repair, and genome stability. Acute myeloid leukemia (AML) is a heterogeneous disease, more than half of the AML cases exhibit normal karyotype (NK). Approximately 50–60 percent of patients with NK-AML carry NPM1 mutations which are characterized by cytoplasmic dislocation of the NPM1 protein. In AML, mutant NPM1 (NPM1c+) acts in a dominant negative fashion and also blocks the differentiation of myeloid cells through gain-of-function for the AML phenotype. Currently, there is limited knowledge on the gain-of-function mechanism of mutant NPM1. Here, we review the known mechanisms of mutant NPM1 in the pathogenesis of AML. We describe genetic abnormalities, the clinical significance of exon-12 mutations in the NPM1 gene, and chromosomal translocations including the recently discovered NPM1-TYK2, and NPM1-HAUS1. Also, we outline the possible therapeutic interventions for the treatment of AML by targeting NPM1. Overall, the review will summarize present knowledge on mutant NPM1 origin, pathogenesis, and therapy in AML.     

实时定量RT-PCR检测急性早幼粒细胞白血病PML/RARα融合基因的临床意义

目的:探讨实时定量RT-PCR方法检测急性早幼粒细胞白血病(APL) PML/RARα融合基因的临床意义.方法:采用实时定量RT-PCR方法,用Taqman探针检测32例初治APL患者的PML/RARα融合基因3种异构体表达水平,并对治疗过程中的20例患者融合基因表达水平进行动态观察. 结果:①实时定量RT-PCR的敏感度为101拷贝数/μl,标准品日间差异及日内差异平均变异系数均<5%;②32例PML/RARα融合基因阳性的初治APL患者中,21例为PML/RARα融合基因长型异构体,11例为PML/RARα融合基因短型异构体;③32例初治患者PML/RARα融合基因表达量中位数和±s分别为1.44%,(1.29±1.46)%.比较异构体长型及短型标准拷贝数(NCN)中位数和±s分别为1.40%,(1.46±1.18)%和1.28%,(1.39±1.51)%,差异无统计学意义P<0.05;④20例治疗后APL患者PML/RARα融合基因表达水平随着临床治疗而改变.结论:①建立了检测APL微小残留病的高敏感性、高特异性的实时定量RT-PCR方法;②32例初治APL患者PML/RARα融合基因长型及短型异构体mRNA NCN差异无统计学意义;③PML-RARα融合基因表达水平的变化与临床疾病发展相一致,有助于疗效评价、微小残留病的检测和预后判断.     

The role of Clofarabine in the treatment of adults with acute myeloid leukemia

The therapeutic scenario available for adult patients with acute myeloid leukemia (AML) has shown only partial progresses over the last few years. This is especially true for refractory and relapsed AML whose outcome is still extremely disappointing. In this context Clofarabine has offered new promising perspectives within first and second line protocols. This review will firstly describe the initial development in monotherapy, considering then the different potential combination strategies which include both polichemotherapeutic regimens and less conventional approaches with new generation drugs. The potential use of Clofarabine as induction treatment for patients candidate to stem cell transplantation and within conditioning regimens will be finally evaluated.