Wouters, B.J.

Risk stratification of intermediate-risk acute myeloid leukemia: Integrative analysis of a multitude of gene mutation and gene expression markers (Article)

2011-07-28T00:00:00Z

Numerous molecular markers have been recently discovered as potential prognostic factors in acute myeloid leukemia (AML). It has become of critical importance to thoroughly evaluate their interrelationships and relative prognostic importance. Gene expression profiling was conducted in a well-characterized cohort of 439 AML patients (age < 60 years) to determine expression levels of EVI1, WT1, BCL2, ABCB1, BAALC, FLT3, CD34, INDO, ERG and MN1. A variety of AML-specific mutations were evaluated, that is, FLT3, NPM1, N-RAS, K-RAS, IDH1, IDH2, and CEBPADM/SM(double/single). Univariable survival analysis shows that (1) patients with FLT3ITDmutations have inferior overall survival (OS) and event-free survival (EFS), whereas CEBPADMand NPM1 mutations indicate favorable OS and EFS in intermediate-risk AML, and (2) high transcript levels of BAALC, CD34, MN1, EVl1, and ERG predict inferior OS and EFS. In multivariable survival analysis, CD34, ERG, and CEBPADMremain significant. Using survival tree and regression methodologies, we show that CEBPADM, CD34, and IDH2 mutations are capable of separating the intermediate group into 2 AML subgroups with highly distinctive survival characteristics (OS at 60 months: 51.9% vs 14.9%). The integrated statistical approach demonstrates that from the multitude of biomarkers a greatly condensed subset can be selected for improved stratification of intermediate-risk AML.

Prognostic impact, concurrent genetic mutations, and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients: Further evidence for CEBPA double mutant AML as a distinctive disease entity (Article)

2011-02-24T00:00:00Z

We evaluated concurrent gene mutations, clinical outcome, and gene expression signatures of CCAAT/enhancer binding protein alpha (CEBPA) double mutations (CEBPAdm) versus single mutations (CEBPAsm) in 1182 cytogenetically normal acute myeloid leukemia (AML) patients (16-60 years of age). We identified 151 (12.8%) patients with CEBPA mutations (91 CEBPAdmand 60 CEBPAsm). The incidence of germline mutations was 7% (5 of 71), including 3 C-terminal mutations. CEBPAdmpatients had a lower frequency of concurrent mutations than CEBPAsmpatients (P < .0001). Both, groups were associated with a favorable outcome compared with CEBPAwt(5-year overall survival [OS] 63% and 56% vs 39%; P < .0001 and P = .05, respectively). However, in multivariable analysis only CEBPAdmwas a prognostic factor for favorable OS outcome (hazard ratio [HR] 0.36, P < .0001; event-free survival, HR 0.41, P < .0001; relapse-free survival, HR 0.55, P = .001). Outcome in CEBPAsmis dominated by concurrent NPM1 and/or FLT3 internal tandem duplication mutations. Unsupervised and supervised GEP analyses showed that CEBPAdmAML (n = 42), but not CEBPAsmAML (n = 18), expressed a unique gene signature. A 25-probe set prediction signature for CEBPAdmAML showed 100% sensitivity and specificity. Based on these findings, we propose that CEBPAdmshould be clearly defined from CEBPAsmAML and considered as a separate entity in the classification of AML.

HAT: Hypergeometric Analysis of Tiling-arrays with application to promoter-GeneChip data (Article)

2010-05-21T00:00:00Z

Background: Tiling-arrays are applicable to multiple types of biological research questions. Due to its advantages (high sensitivity, resolution, unbiased), the technology is often employed in genome-wide investigations. A major challenge in the analysis of tiling-array data is to define regions-of-interest, i.e., contiguous probes with increased signal intensity (as a result of hybridization of labeled DNA) in a region. Currently, no standard criteria are available to define these regions-of-interest as there is no single probe intensity cut-off level, different regions-of-interest can contain various numbers of probes, and can vary in genomic width. Furthermore, the chromosomal distance between neighboring probes can vary across the genome among different arrays.Results: We have developed Hypergeometric Analysis of Tiling-arrays (HAT), and first evaluated its performance for tiling-array datasets from a Chromatin Immunoprecipitation study on chip (ChIP-on-chip) for the identification of genome-wide DNA binding profiles of transcription factor Cebpa (used for method comparison). Using this assay, we can refine the detection of regions-of-interest by illustrating that regions detected by HAT are more highly enriched for expected motifs in comparison with an alternative detection method (MAT). Subsequently, data from a retroviral insertional mutagenesis screen were used to examine the performance of HAT among different applications of tiling-array datasets. In both studies, detected regions-of-interest have been validated with (q)PCR.Conclusions: We demonstrate that HAT has increased specificity for analysis of tiling-array data in comparison with the alternative method, and that it accurately detects regions-of-interest in two different applications of tiling-arrays. HAT has several advantages over previous methods: i) as there is no single cut-off level for probe-intensity, HAT can detect regions-of-interest at various thresholds, ii) it can detect regions-of-interest of any size, iii) it is independent of probe-resolution across the genome, and across tiling-array platforms and iv) it employs a single user defined parameter: the significance level. Regions-of-interest are detected by computing the hypergeometric-probability, while controlling the Family Wise Error. Furthermore, the method does not require experimental replicates, common regions-of-interest are indicated, a sequence-of-interest can be examined for every detected region-of-interest, and flanking genes can be reported.

Gene expression profiling of minimally differentiated acute myeloid leukemia: M0 is a distinct entity subdivided by RUNX1 mutation status (Article)

2009-11-19T00:00:00Z

Minimally differentiated acute myeloid leukemia (AML-M0) is defined by immature morphology and expression of early hematologic markers. By gene expression profiling (GEP) and subsequent unsupervised analysis of 35 AML-M0 samples and 253 previously reported AML cases, we demonstrate that AML-M0 cases express a unique signature that is largely separated from other molecular subtypes. Hematologic transcription regulators such as CEBPA, CEBPD, and ETV6, and the differentiation associated gene MPO appeared strongly down-regulated, in line with the primitive state of this leukemia. AML-M0 frequently carries loss-of-function RUNX1 mutation. Unsupervised analyses revealed a subdivision between AML-M0 cases with and without RUNX1 mutations. RUNX1 mutant AML-M0 samples showed a distinct up-regulation of B cell-related genes such as members of the B-cell receptor complex, transcription regulators RUNX3, ETS2, IRF8, or PRDM1, and major histocompatibility complex class II genes. Importantly, prediction with high accuracy of the AML-M0 subtype and prediction of patients carrying RUNX1 mutation within this subtype were possible based on the expression level of only a few transcripts. We propose that RUNX1 mutations in this AML subgroup cause lineage infidelity, leading to aberrant coexpression of myeloid and B-lymphoid genes. Furthermore, our results imply that AML-M0, although originally determined by morphology, constitutes a leukemia subgroup.

Genomic and Epigenomic Studies of Acute Myeloid Leukemia with CEBPA Abnormalities (Doctoral Thesis)

2009-05-20T00:00:00Z

Since the introduction of molecular genetics, our knowledge about the abnormalities underlying human AML has increased tremendously. This is well reflected in the evolution that classification of the disease has undergone, from cytomorphology (according the FAB system, introduced in the 1970s) to the recognition of entities of AML defined by gene mutations (WHO classification 2008). The main goal of this thesis was to further progress the molecular dissection of AML. Special attention throughout this thesis was given to abnormalities affecting CEBPA, the gene encoding the transcription factor CCAAT/enhancer binding protein alpha, one of the master regulators of normal myeloid differentiation. To address the research questions posed in the various studies, several genome-wide research techniques were applied, with a central role for GEP. In chapter 2, we reviewed the use of genome-wide GEP in AML and described some of the common research questions that investigators have addressed using this technique, i.e. those related to class discovery, class prediction, outcome prediction and dissection of pathobiology. We also discussed limitations of the technology and we described promising future developments. Chapter 3 focused on the diagnostic potential of GEP-based class prediction of cytogenetically and genetically defined AML subgroups. These analyses indicated, in agreement with previous observations, that the chromosomal defects t(8;21), t(15;17) and inv(16) can be predicted with high accuracy, but that many other abnormalities do not result in distinctive classifiers. For CEBPA mutations, we observed two groups of AML cases: one that could be well predicted, and one that could not. This latter finding was studied in further detail in chapter 9. Together, these results confirmed that GEP can be applied in a d! iagnostic setting for AML, albeit at the moment only for a selected and defined number of subtypes. In chapters 4-6, we investigated the molecular characteristics and pathobiology of a novel subset of acute leukemia. Chapter 4 described, through mouse modeling, a novel mechanism of inhibition of C/EBPalpha causing AML, i.e. elevated expression of Trib2. Trib2 was shown to bind C/EBPalpha protein, inducing its degradation. Analysis of our previously established gene expression data base of human AML samples highlighted an association of high TRIB2 expression with a particular cluster characterized by CEBPA mutations. In contrast to the other cases in this cluster, the 6 leukemias that appeared particularly associated with high TRIB2 levels did not exhibit CEBPA mutations. Additional experiments, described in chapters 5 and 6, uncovered distinctive characteristics of these specific leukemias, including, most notably, epigenetic silencing of CEBPA expression and an overall DNA methylation profile characterized by hypermethylation. These molecular features were accompanied by! an immature, mixed myeloid/T-lymphoid immunophenotype. In chapters 7 through 9, we studied AMLs with mutations in the CEBPA gene. In chapter 7, it was found that an insertion in the second transactivation domain of C/EBPalpha (HP196-197ins) is a common polymorphism and not an AML-specific mutation. Chapter 8 described that homozygosity of CEBPA mutations is frequently associated with copy number neutral loss-of-heterozygosity of the chromosomal region that includes CEBPA, presumably through mitotic recombination. Chapter 9 presented previously unrecognized heterogeneity of CEBPA mutant AML. While it was already known that there are AMLs in which both CEBPA alleles are affected as well as AMLs with only a single heterozygous aberration, it was not known whether the reported prognostic impact of CEBPA mutations, i.e. favorable, was equal in both groups. Our analyses suggested that only AML patients with two mutations in the CEBPA gene, presumably affecting both alleles, should be considered to have favorable risk. In support of th! e prognostic differences, single and double mutant specimens also strikingly differed in genome-wide transcriptional profiles. In the final chapter, chapter 10, we identified putative novel target genes of C/EBPalpha in a myeloid cell line model using ChIP-chip. We performed similar experiments with a variant C/EBPalpha protein that carries a C-terminal insertion mutation in the bZIP region resembling human AML. Our preliminary analysis of these data demonstrated impaired DNA binding of the mutant, providing possible leads to an understanding of the mechanism involved in human leukemogenesis by the mutant protein.

Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome (Article)

2009-03-26T00:00:00Z

Mutations in CCAAT/enhancer binding protein α (CEBPA) are seen in 5% to 14% of acute myeloid leukemia (AML) and have been associated with a favorable clinical outcome. Most AMLs with CEBPA mutations simultaneously carry 2 mutations (CEBPAdouble-mut), usually biallelic, whereas single heterozygous mutations (CEBPAsingle-mut) are less frequently seen. Using denaturing high-performance liquid chromatography and nucleotide sequencing, we identified among a cohort of 598 newly diagnosed AMLs a subset of 41 CEBPA mutant cases (28 CEBPAdouble-mut and 13 CEBPA single-mut cases) CEBPAdouble-mut associated with a unique gene expression profile as well as favorable overall and event-free survival, retained in multi-variable analysis that included cytoge-netic risk, FZT3-ITD and NPM1 mutation, white blood cell count, and age. In contrast, CEBPA single-mut AMLs did not express a discriminating signature and could not be distinguished from wild-type cases as regards clinical outcome. These results demonstrate significant underlying heterogeneity within CEBPA mutation-positive AML with prognostic relevance.

Genome-wide epigenetic analysis delineates a biologically distinct immature acute leukemia with myeloid/T-lymphoid features (Article)

2009-03-19T00:00:00Z

Acute myeloid leukemia is a heterogeneous disease from the molecular and biologic standpoints, and even patients with a specific gene expression profile may present clinical and molecular heterogeneity. We studied the epigenetic profiles of a cohort of patients who shared a common gene expression profile but differed in that only half of them harbored mutations of the CEBPA locus, whereas the rest presented with silencing of this gene and coexpression of certain T-cell markers. DNA methylation studies revealed that these 2 groups of patients could be readily segregated in an unsupervised fashion based on their DNA methylation profiles alone. Furthermore, CEBPA silencing was associated with the presence of an aberrant DNA hypermethylation signature, which was not present in the CEBPA mutant group. This aberrant hypermethylation occurred more frequently at sites within CpG islands. CEBPA-silenced leukemias also displayed marked hypermethylation compared with normal CD34+ hematopoietic cells, whereas CEBPA mutant cases showed only mild changes in DNA methylation compared with these normal progenitors. Biologically, CEBPA-silenced leukemias presented with a decreased response to myeloid growth factors in vitro.

Response: CEBPA promoter hypermethylation in a subset of myeloid/T-lymphoid leukemias with a distinct gene expression profile (Article)

2009-02-19T00:00:00Z

A decade of genome-wide gene expression profiling in acute myeloid leukemia: Flashback and prospects (Article)

2009-01-08T00:00:00Z

The past decade has shown a marked increase in the use of high-throughput assays in clinical research into human cancer, including acute myeloid leukemia (AML). In particular, genome-wide gene expression profiling (GEP) using DNA microarrays has been extensively used for improved understanding of the diagnosis, prognosis, and pathobiology of this heterogeneous disease. This review discusses the progress that has been made, places the technologic limitations in perspective, and highlights promising future avenues.

Prediction of molecular subtypes in acute myeloid leukemia based on gene expression profiling (Article)

2009-01-01T00:00:00Z

We examined the gene expression profiles of two independent cohorts of patients with acute myeloid leukemia [n=247 and n=214 (younger than or equal to 60 years)] to study the applicability of gene expression profiling as a single assay in prediction of acute myeloid leukemia-specific molecular subtypes. The favorable cytogenetic acute myeloid leukemia subtypes, i.e., acute myeloid leukemia with t(8;21), t(15;17) or inv(16), were predicted with maximum accuracy (positive and negative predictive value: 100%). Mutations in NPM1 and CEBPA were predicted less accurately (positive predictive value: 66% and 100%, and negative predictive value: 99% and 97% respectively). Various other characteristic molecular acute myeloid leukemia subtypes, i.e., mutant FLT3 and RAS, abnormalities involving 11q23, -5/5q-, -7/7q-, abnormalities involving 3q (abn3q) and t(9;22), could not be correctly predicted using gene expression profiling. In conclusion, gene expression profiling allows accurate prediction of certain acute myeloid leukemia subtypes, e.g. those characterized by expression of chimeric transcription factors. However, detection of mutations affecting signaling molecules and numerical abnormalities still requires alternative molecular methods.

Gene expression profiling for improved dissection of acute leukemia: A recently identified immature myeloid/T-lymphoid subgroup as an example (Article)

2008-05-01T00:00:00Z

In this concise overview, we discuss recent findings concerning a distinct subgroup of acute myeloid/T-lymphoid leukemia. We describe how we identified these leukemias in multiple cohorts of acute myeloid leukemia (AML) using a combination of gene expression profiling and additional analytic approaches, and how we obtained insight in possible mechanisms leading to their phenotype.

Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1 (Article)

2007-11-15T00:00:00Z

Gene expression profiling of acute myeloid leukemia (AML) allows the discovery of previously unrecognized molecular entities. Here, we identified a specific subgroup of AML, defined by an expression profile resembling that of AMLs with mutations in the myeloid transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), while lacking such mutations. We found that in these leukemias, the CEBPA gene was silenced, which was associated with frequent promoter hypermethylation. The leukemias phenotypically showed aberrant expression of T-cell genes, of which CD7 was most consistent. We identified 2 mechanisms that may contribute to this phenotype. First, absence of Cebpa led to up-regulation of specific T-cell transcripts (ie, Cd7 and Lck) in hematopoietic stem cells isolated from conditional Cebpa knockout mice. Second, the enhanced expression of TRIB2, which we identify here as a direct target of the T-cell commitment factor NOTCH1, suggested aberrantly activated Notch signaling. Putatively activating NOTCH1 mutations were found in several specimens of the newly identified subgroup, while a large set of control AMLs was mutation negative. A gene expression prediction signature allowed the detection of similar cases of leukemia in independent series of AML.

Segmental uniparental disomy as a recurrent mechanism for homozygous CEBPA mutations in acute myeloid leukemia [13] (Article)

2007-11-01T00:00:00Z

Text-derived concept profiles support assessment of DNA microarray data for acute myeloid leukemia and for androgen receptor stimulation (Article)

2007-02-19T00:00:00Z

Background: High-throughput experiments, such as with DNA microarrays, typically result in hundreds of genes potentially relevant to the process under study, rendering the interpretation of these experiments problematic. Here, we propose and evaluate an approach to find functional associations between large numbers of genes and other biomedical concepts from free-text literature. For each gene, a profile of related concepts is constructed that summarizes the context in which the gene is mentioned in literature. We assign a weight to each concept in the profile based on a likelihood ratio measure. Gene concept profiles can then be clustered to find related genes and other concepts. Results: The experimental validation was done in two steps. We first applied our method on a controlled test set. After this proved to be successful the datasets from two DNA microarray experiments were analyzed in the same way and the results were evaluated by domain experts. The first dataset was a gene-expression profile that characterizes the cancer cells of a group of acute myeloid leukemia patients. For this group of patients the biological background of the cancer cells is largely unknown. Using our methodology we found an association of these cells to monocytes, which agreed with other experimental evidence. The second data set consisted of differentially expressed genes following androgen receptor stimulation in a prostate cancer cell line. Based on the analysis we put forward a hypothesis about the biological processes induced in these studied cells: secretory lysosomes are involved in the production of prostatic fluid and their development and/or secretion are androgen-regulated processes. Conclusion: Our method can be used to analyze DNA microarray datasets based on information explicitly and implicitly available in the literature. We provide a publicly available tool, dubbed Anni, for this purpose.

A recurrent in-frame insertion in a CEBPA transactivation domain is a polymorphism rather than a mutation that does not affect gene expression profiling-based clustering of AML [5] (Article)

2007-01-01T00:00:00Z