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Publications and source records attributed to Peter J M Valk.
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Knockdown of the transcription factor PU.1 (encoded by Sfpi1) leads to acute myeloid leukemia (AML) in mice. We examined the transcriptome of preleukemic hematopoietic stem cells (HSCs) in which PU.1 was knocked down (referred to as 'PU.1-knockdown HSCs') to identify transcriptional changes preceding malignant transformation. Transcription factors c-Jun and JunB were among the top-downregulated targets. Restoration of c-Jun expression in preleukemic cells rescued the PU.1 knockdown-initiated myelomonocytic differentiation block. Lentiviral restoration of JunB at the leukemic stage led to loss of leukemic self-renewal capacity and prevented leukemia in NOD-SCID mice into which leukemic PU.1-knockdown cells were transplanted. Examination of human individuals with AML confirmed the correlation between PU.1 and JunB downregulation. These results delineate a transcriptional pattern that precedes leukemic transformation in PU.1-knockdown HSCs and demonstrate that decreased levels of c-Jun and JunB contribute to the development of PU.1 knockdown-induced AML by blocking differentiation and increasing self-renewal. Therefore, examination of disturbed gene expression in HSCs can identify genes whose dysregulation is essential for leukemic stem cell function and that are targets for therapeutic interventions.
BACKGROUND: Accurate interpretation of data obtained by unsupervised analysis of large scale expression profiling studies is currently frequently performed by visually combining sample-gene heatmaps and sample characteristics. This method is not optimal for comparing individual samples or groups of samples. Here, we describe an approach to visually integrate the results of unsupervised and supervised cluster analysis using a correlation plot and additional sample metadata. RESULTS: We have developed a tool called the HeatMapper that provides such visualizations in a dynamic and flexible manner and is available from http://www.erasmusmc.nl/hematologie/heatmapper/. CONCLUSION: The HeatMapper allows an accessible and comprehensive visualization of the results of gene expression profiling and cluster analysis.
BACKGROUND: Intensity values measured by Affymetrix microarrays have to be both normalized, to be able to compare different microarrays by removing non-biological variation, and summarized, generating the final probe set expression values. Various pre-processing techniques, such as dChip, GCRMA, RMA and MAS have been developed for this purpose. This study assesses the effect of applying different pre-processing methods on the results of analyses of large Affymetrix datasets. By focusing on practical applications of microarray-based research, this study provides insight into the relevance of pre-processing procedures to biology-oriented researchers. RESULTS: Using two publicly available datasets, i.e., gene-expression data of 285 patients with Acute Myeloid Leukemia (AML, Affymetrix HG-U133A GeneChip) and 42 samples of tumor tissue of the embryonal central nervous system (CNS, Affymetrix HuGeneFL GeneChip), we tested the effect of the four pre-processing strategies mentioned above, on (1) expression level measurements, (2) detection of differential expression, (3) cluster analysis and (4) classification of samples. In most cases, the effect of pre-processing is relatively small compared to other choices made in an analysis for the AML dataset, but has a more profound effect on the outcome of the CNS dataset. Analyses on individual probe sets, such as testing for differential expression, are affected most; supervised, multivariate analyses such as classification are far less sensitive to pre-processing. CONCLUSION: Using two experimental datasets, we show that the choice of pre-processing method is of relatively minor influence on the final analysis outcome of large microarray studies whereas it can have important effects on the results of a smaller study. The data source (platform, tissue homogeneity, RNA quality) is potentially of bigger importance than the choice of pre-processing method.
Retroviral insertion mutagenesis is considered a powerful tool to identify cancer genes in mice, but its significance for human cancer has remained elusive. Moreover, it has recently been debated whether common virus integrations are always a hallmark of tumor cells and contribute to the oncogenic process. Acute myeloid leukemia (AML) is a heterogeneous disease with a variable response to treatment. Recurrent cytogenetic defects and acquired mutations in regulatory genes are associated with AML subtypes and prognosis. Recently, gene expression profiling (GEP) has been applied to further risk stratify AML. Here, we show that mouse leukemia genes identified by retroviral insertion mutagenesis are more frequently differentially expressed in distinct subclasses of adult and pediatric AML than randomly selected genes or genes located more distantly from a virus integration site. The candidate proto-oncogenes showing discriminative expression in primary AML could be placed in regulatory networks mainly involved in signal transduction and transcriptional control. Our data support the validity of retroviral insertion mutagenesis in mice for human disease and indicate that combining these murine screens for potential proto-oncogenes with GEP in human AML may help to identify critical disease genes and novel pathogenetic networks in leukemia.
BACKGROUND: Increased CD38 expression by leukemic cells has been suggested as an adverse prognostic factor in B-CLL. Several approaches have been proposed to quantify its level of expression by flow cytometry. METHODS: We compared the use of (i) the percentage of CD38 positive cells, (ii) CD38 antibodies bound per cell (ABC), and (iii) a semi-quantitative method based on the shape of the CD38 histogram, within a cohort of 78 B-CLL patients. RESULTS: A decreased overall survival was seen with >30% CD38 positivity among B-CLL cells, with CD38 ABC >100, and with bimodal or unimodal, strongly positive CD38 histograms. However, patients with unimodal weakly positive CD38 histograms also showed a significantly reduced survival as did patients with intermediate proportions (i.e. 5-30%) of CD38+ cells. Furthermore, within the group with <5% CD38 positivity among their B-CLL cells, 84% of patients showed prognostically favourable mutated IGVH gene segments and 100% had low ZAP70 gene expression. For 5-30% CD38 positivity, these proportions were 50 and 83%, while for >30% CD38 positivity, these proportion were only 28 and 56%, respectively. CONCLUSIONS: We found a simple method of quantitation of CD38 expression (i.e., >5% CD38 positivity among B-CLL cells) to be sufficient to identify patients with an unfavourable prognosis. The level of CD38 expression as defined with this method correlated well with the IGVH mutation status and ZAP70 gene expression.
BACKGROUND AND OBJECTIVES: The mutational status of the immunoglobulin heavy chain variable region genes (IGVH) is a strong indicator of prognosis in B-cell chronic lymphocytic leukaemia (CLL). Since the determination of the IGVH mutation status is very labor-intensive, alternative prognostically relevant markers would facilitate CLL diagnostics. DESIGN AND METHODS: Ten genes were selected from previously published gene expression profiling studies based on their differential expression in IGVH mutated versus unmutated cases of CLL, and tested with real-time quantitative polymerase chain reaction (RQ-PCR) in unpurified samples from 130 CLL patients. To ascertain potential contaminating effects by normal hematopoietic cells, the expression levels of the selected genes were determined in normal monocytes, B cells, T cells, NK cells and granulocytes. RESULTS: The selected genes, i.e., ZAP70, LPL, SPG20, ADAM29, NRIP1, AKAP12, DMD, SEPT10, TPM2 and CLECSF2, showed prognostic significance. In multivariate logistic regression analysis expression levels of LPL, ZAP70, ADAM29 and SEPT10 were the most predictive for IGVH mutational status. In univariate analysis the expression of LPL was the best predictor. For survival, expression of LPL was the strongest prognostic factor. In combination with the three cytogenetic markers associated with a poor prognosis, i.e., deletions 17p13, 11q22 and trisomy 12, expression of LPL and IGVH mutational status performed equally well with regard to their predictive value for survival, both being more predictive than ZAP70. INTERPRETATION AND CONCLUSIONS: This study demonstrates that LPL expression is a predictor for survival in CLL, and for this purpose is as good as IGVH mutational status and more reliable than ZAP70 expression when tested in unpurified CLL samples.
Over the last decades, significant advances have been made in the knowledge and treatment of acute myeloid leukemia (AML). The WHO has recognized this new information by incorporating into its classification morphologic, immunophenotypic, genetic, and clinical features in an attempt to define biologically and clinically relevant entities. Nevertheless, well-defined cytogenetic subgroups exhibit considerable heterogeneity, and in many AML subtypes the pathogenic event is still not known. A classification system based on the underlying molecular pathogenetic abnormalities would be ideal, but such detailed knowledge is not yet available. Novel approaches in genomics, such as surveying the expression levels of thousands of genes in parallel using DNA microarray technology, open possibilities to further refine the studies on AML. Today, gene expression profiling in AML is becoming well established and has already been proven to be valuable in diagnosing different cytogenetic subtypes, discovering novel AML subclasses, and predicting clinical outcome. Recently, gene expression profiling studies in AML showed a remarkable level of concordance in findings, which may ultimately lead to an increasingly refined molecular taxonomy. While many challenges remain to be overcome, a combination of gene expression profiling with other microarray-based applications, high-throughput mutational analyses and proteomic approaches will not only significantly contribute to the classification and therapeutic decision making of AML, but also give important insights into the true pathobiologic nature of this type of leukemia.
Mutations in nucleophosmin NPM1 are the most frequent acquired molecular abnormalities in acute myeloid leukemia (AML). We determined the NPM1 mutation status in a clinically and molecularly well-characterized patient cohort of 275 patients with newly diagnosed AML by denaturing high-performance liquid chromatography (dHPLC). We show that NPM1 mutations are significantly underrepresented in patients younger than 35 years. NPM1 mutations positively correlate with AML with high white blood cell counts, normal karyotypes, and fms-like tyrosine kinase-3 gene (FLT3) internal tandem duplication (ITD) mutations. NPM1 mutations associate inversely with the occurrence of CCAAT/enhancer-binding protein-alpha (CEBPA) and NRAS mutations. With respect to gene expression profiling, we show that AML cases with an NPM1 mutation cluster in specific subtypes of AML with previously established gene expression signatures, are highly associated with a homeobox gene-specific expression signature, and can be predicted with high accuracy. We demonstrate that patients with intermediate cytogenetic risk AML without FLT3 ITD mutations but with NPM1 mutations have a significantly better overall survival (OS) and event-free survival (EFS) than those without NPM1 mutations. Finally, in multivariable analysis NPM1 mutations express independent favorable prognostic value with regard to OS, EFS, and disease-free survival (DFS).
The AML1-ETO fusion protein, generated by the t(8;21) in acute myeloid leukemia (AML), exerts dominant-negative functions and a variety of gains of function, including a positive effect on the growth of primary human CD34+ hematopoietic stem/progenitor cells. We now show that AML1-ETO expression up-regulates the level of TRKA mRNA and protein in these cells and that AML1-ETO-expressing CD34+ hematopoietic cells grown in the presence of five early-acting hematopoietic cytokines further proliferate in response to nerve growth factor (NGF). These cells also show a unique response to NGF and IL-3; namely, they expand in liquid culture. To determine the biological relevance of our findings, we analyzed 262 primary AML patient samples using real-time RT-PCR and found that t(8;21)-positive AML samples express significantly higher levels of TRKA mRNA than other subtypes of AML. NGF, which is normally expressed by bone marrow stromal cells, could provide important proliferative or survival signals to AML1-ETO-expressing leukemic or preleukemic cells, and the NGF/TRKA signaling pathway may be a suitable target for therapeutic approaches to AML.
PURPOSE OF REVIEW: This review deals with the emerging promises of gene expression profiling (GEP) and the currently accumulating knowledge about the classification and the discovery of novel disease entities in clinical acute myeloid leukemia (AML). RECENT FINDINGS: Gene expression profiling studies in AML have shown that known and novel classes of disease can be recognized by unsupervised analyses. Prognostically informative molecular signatures can be deduced. Supervised analyses show that particular clinically relevant subsets of AML can be predicted with high accuracy with minimal sets of genes. SUMMARY: The AML GEP studies published to date show a remarkable level of concordance in findings, especially for similar GEP platforms. This confirms the robustness of the methodology and the promise for future applicability of GEP in clinical diagnostics. For the time being, certain technical hurdles remain to be overcome. These relate, for instance, to the conversion of data between different GEP platforms, the effect of differences between various statistical clustering methods, and the still incomplete understanding of the effect of biologic (eg, morphology) and genetic factors on the expression signature. GEP analyses, perhaps in combination with high-throughput mutation analysis and proteomic approaches, may ultimately result in the establishment of a comprehensive diagnostic approach that will yield a key to the precise pathobiologic nature of AML.
The leukemia and lymphoma disease locus Evi12 was mapped to the noncoding region of a novel gene, Gnn (named for Grp94 neighboring nucleotidase), that is located immediately upstream of the Grp94/Tra1 gene on mouse chromosome 10. The Gnn gene is conserved in mice and humans. Expression of fusion constructs between GFP and Gnn cDNA isoforms in HEK-293 cells showed that Gnn proteins are located mainly in the cytoplasm. Immunoblotting experiments demonstrated the presence of multiple Gnn protein isoforms in most organs, with the lowest levels of expression of the protein detected in bone marrow and spleen. The Evi12-containing leukemia cell line NFS107 showed high levels of expression of a approximately 150-kDa Gnn isoform (Gnn107) that was not observed in control cell lines. Overexpression may be due to the viral insertion in Evi12. The Gnn107 protein is probably encoded by a Gnn cDNA isoform that is expressed exclusively in NFS107 cells and that includes sequences of TU12B1-TY, a putative protein with homology to 5'-nucleotidase enzymes. Interestingly, using Affymetrix gene expression data of a cohort of 285 patients with acute myeloid leukemia (AML), we found that GNN/TU12B1-TY expression was specifically increased in two AML clusters. One cluster consisted of all AML patients with a t(8;21) translocation, and the second cluster consisted of AML patients with a normal karyotype carrying a FLT3 internal tandem duplication. These findings suggest that we identified a novel proto-oncogene that may be causally linked to certain types of human leukemia.
Hematopoietic stem cells (HSCs) sustain blood formation throughout life. Pathways regulating maintenance of adult HSCs are largely unknown. Here we report that the Ets-related transcription factor Tel/Etv6, the product of a locus frequently involved in translocations in leukemia, is a selective regulator of HSC survival. Following inactivation of Tel/Etv6, HSCs are lost in the adult bone marrow but their progeny are unaffected and transiently sustain blood formation. Accordingly, absence of Tel/Etv6 after lineage commitment is ostensibly without consequence except for unexpected impairment of maturation of megakaryocytes. Thus, we establish Tel/Etv6 as a selective and essential regulator of postembryonic HSCs.
BACKGROUND: In patients with acute myeloid leukemia (AML) a combination of methods must be used to classify the disease, make therapeutic decisions, and determine the prognosis. However, this combined approach provides correct therapeutic and prognostic information in only 50 percent of cases. METHODS: We determined the gene-expression profiles in samples of peripheral blood or bone marrow from 285 patients with AML using Affymetrix U133A GeneChips containing approximately 13,000 unique genes or expression-signature tags. Data analyses were carried out with Omniviz, significance analysis of microarrays, and prediction analysis of microarrays software. Statistical analyses were performed to determine the prognostic significance of cases of AML with specific molecular signatures. RESULTS: Unsupervised cluster analyses identified 16 groups of patients with AML on the basis of molecular signatures. We identified the genes that defined these clusters and determined the minimal numbers of genes needed to identify prognostically important clusters with a high degree of accuracy. The clustering was driven by the presence of chromosomal lesions (e.g., t(8;21), t(15;17), and inv(16)), particular genetic mutations (CEBPA), and abnormal oncogene expression (EVI1). We identified several novel clusters, some consisting of specimens with normal karyotypes. A unique cluster with a distinctive gene-expression signature included cases of AML with a poor treatment outcome. CONCLUSIONS: Gene-expression profiling allows a comprehensive classification of AML that includes previously identified genetically defined subgroups and a novel cluster with an adverse prognosis.
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The CCAAT/enhancer binding protein alpha is an essential transcription factor for granulocytic differentiation. Recent studies reported N- and C-terminal CEBPA mutations in approximately 7% of acute myeloid leukaemia (AML) patients. C-terminal mutations are usually in-frame and occur in the basic-leucine zipper (bZIP) domain, resulting in deficient DNA binding. Using a rapid PCR approach, we screened for bZIP mutations and determined the prognostic value of these mutations in a cohort of 277 de novo AMLs. In addition, we set out to quantify CEBPA mRNA levels by 'real-time' PCR using TaqMan technology. In-frame insertions were observed in 12 (4.3%) cases. All cases with mutations carried an intermediate-risk karyotype and all but one belonged to M1 or M2 FAB class. Further sequence analysis revealed that CEBPA C-terminal mutations are associated with frameshift mutations in the N-terminus of CEBPA. These two mutations were always found in different alleles. Event-free survival (EFS) and overall survival (OS) of patients with CEBPA mutations were significantly increased (P=0.02 and 0.03, respectively) in comparison to the patients lacking these mutations. Mutations were associated with a significantly reduced hazard ratio for death (OS: HR=0.35, P=0.04) and failure (EFS: no CR, death in CR or relapse, HR=0.37, P=0.03). This favourable hazard ratio was maintained after adjustment for cytogenetic risk, FLT3-ITD and CEBPA expression levels in multivariable analysis. In contrast, low CEBPA expression in AML with intermediate-risk karyotype (n=6) seemed to be associated with poor prognosis (not significant). By including this newly developed PCR assay, we define a subgroup of good-risk patients within the heterogeneous intermediate-risk group of AML.
DNA from 110 adult de novo acute myeloid leukaemia (AML) patients exhibiting either inv(16) (n = 63) or t(8;21) (n = 47) was screened for mutations in the c-KIT (exon 8 and Asp816) and FLT3 (ITD and Asp835) genes. c-KIT exon 8 mutations were found in 15/63 (23.8%) inv(16) patients and 1/47 (2.1%) t(8;21) patients. c-KIT Asp816 mutations were present in 5/63 (7.9%) inv(16) AML and 5/47 (10.6%) t(8;21) AML. FLT3 mutations were identified in five patients (7.9%) with inv(16) and three patients (5.6%) with t(8;21) AML. All mutations were mutually exclusive; 40% of inv(16) AML patients possessed either a c-KIT or FLT3 mutation. c-KIT exon 8 mutations were shown to be a significant factor adversely affecting relapse rate.
The identification of common virus integration sites (cVIS) in retrovirally induced tumors in mice provides a powerful strategy to isolate novel transforming genes. Applying virus LTR-specific inverse-PCR and RT-PCR combined with automated sequencing on CasBr-M Murine Leukemia Virus (MuLV) induced myeloid leukemias, 126 virus integration sites were cloned. Using locus- and LTR-specific primers, a nested-PCR/Southern-blotting procedure was developed on genomic DNA from a large panel of MuLV-induced leukemias, to analyse whether a particular virus insertion represented a cVIS. In fact 39 out of 41 integrations analysed this way appeared to represent a common virus integration. We recognized six previously cloned cVISs, i.e. Evi1, Hoxa7, c-Myb, Cb2/Evi11, Evi12, and His1 and 33 novel common insertions, designated Cas-Br Virus Integration Site (Casvis). Among this group we found integrations in or near genes encoding nuclear proteins, e.g. Dnmt-2, Nm23-M2, Ctbp1 or Erg, within receptor genes, e.g. Cb2 or mrc1, novel putative signaling or transporter genes, the ringfinger-protein gene Mid1 and a panel of genes encoding novel proteins with unknown function. The finding that 39 out of 41 integrations analysed represented a cVIS, suggests that the majority of the other virus insertions that were not yet analysed by the PCR/Southern-blotting method are located in a cVIS as well and may therefore also harbor novel disease genes.