Arja ter Elst

Evidence Based Selection of Housekeeping Genes

For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes (reference or internal control genes) is required. It is known that commonly used housekeeping genes (e.g. ACTB, GAPDH, HPRT1, and B2M) vary considerably under different experimental conditions and therefore their use for normalization is limited. We performed a meta-analysis of 13,629 human gene array samples in order to identify the most stable expressed genes. Here we show novel candidate housekeeping genes (e.g. RPS13, RPL27, RPS20 and OAZ1) with enhanced stability among a multitude of different cell types and varying experimental conditions. None of the commonly used housekeeping genes were present in the top 50 of the most stable expressed genes. In addition, using 2,543 diverse mouse gene array samples we were able to confirm the enhanced stability of the candidate novel housekeeping genes in another mammalian species. Therefore, the identified novel candidate housekeeping genes seem to be the most appropriate choice for normalizing gene expression data.

Determination of TP53 Mutation Is More Relevant Than Microsatellite Instability Status for the Prediction of Disease-Free Survival in Adjuvant-Treated Stage III Colon Cancer Patients

PURPOSE Microsatellite instability (MSI), TP53 mutation, and KRAS mutation status have been reported as prognostic factors in colon cancer. Most studies, however, have included heterogeneous groups of patients with respect to cancer stage. We determined the prognostic relevance of high-frequency MSI (MSI-H), TP53 mutations, and KRAS mutations in a well-defined group of patients with stage III colon cancer (N = 391), randomly assigned for adjuvant treatment with fluorouracil-based chemotherapy. METHODS Three hundred ninety-one tumor specimens were available. MSI was determined in 273 specimens, and mutation analyses of TP53 and KRAS were performed in 220 and 205 specimens, respectively. RESULTS In a univariate analysis, MSI-H (44 of 273; 16%) was associated with a longer disease-free survival (DFS; P = .038), but in a multivariate model adjusting for nodal involvement, histology, invasion, and grade of tumor, the association of MSI status with DFS did no longer reach statistical significance, though the risk estimate for microsatellite stability versus MSI-H tumors did not change much. Mutant TP53, found in 116 (53%) of 220 tumors, was associated with a shorter DFS, both in univariate (P = .009) and multivariate analyses (P = .018), whereas KRAS mutations (58 of 205; 28%) did not show any prognostic significance. CONCLUSION Both mutant TP53 and MSI-H seem to be prognostic indicators for disease-free survival, but only TP53 retains statistical significance after adjusting for clinical heterogeneity. Thus, in adjuvantly treated patients with stage III colon cancer, presence or absence of a TP53 mutation should be considered as a better predictor for DFS than MSI status.

Bone marrow-derived cells and tumor growth: Contribution of bone marrow-derived cells to tumor micro-environments with special focus on mesenchymal stem cells

Research has provided evidence that tumor growth depends on the interaction of tumor cells with stromal cells, as already suggested in 1889 by Paget. Experimental and clinical studies have revealed that tumor stromal cells can be derived from bone marrow (BM)-derived progenitor cells, such as mesenchymal stem cells (MSCs), which can be mobilized into the circulation and incorporate into tumor micro-environments. Many observations indicate that, in the tumor micro-environment, MSCs have several tumor growth promoting functions, including expression of growth factors, promotion of tumor vessel formation and creation of tumor stem cell niches. This review will discuss the currently known tumor growth promoting BM-derived cells and focus on the role of MSCs in modulating tumor micro-environments. In addition, we will discuss the potential of inhibiting BM-derived cells and their utilization as cellular vehicles for selective delivery of cancer therapeutics as additional strategies in the treatment of cancer.

Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Progression in pediatric brain tumor growth is thought to be the net result of signaling through various protein kinase-mediated networks driving cell proliferation. Defining new targets for treatment of human malignancies, without a priori knowledge on aberrant cell signaling activity, remains exceedingly complicated. Here, we introduce kinome profiling using flow-through peptide microarrays as a new concept for target discovery. Comprehensive tyrosine kinase activity profiles were identified in 29 pediatric brain tumors using the PamChip kinome profiling system. Previously reported activity of epidermal growth factor receptor, c-Met, and vascular endothelial growth factor receptor in pediatric brain tumors could be appreciated in our array results. Peptides corresponding with phosphorylation consensus sequences for Src family kinases showed remarkably high levels of phosphorylation compared with normal tissue types. Src activity was confirmed applying Phos-Tag SDS-PAGE. Furthermore, the Src family kinase inhibitors PP1 and dasatinib induced substantial tumor cell death in nine pediatric brain tumor cell lines but not in control cell lines. Thus, this study describes a new high-throughput technique to generate clinically relevant tyrosine kinase activity profiles as has been shown here for pediatric brain tumors. In the era of a rapidly increasing number of small-molecule inhibitors, this approach will enable us to rapidly identify new potential targets in a broad range of human malignancies.

High VEGFC expression is associated with unique gene expression profiles and predicts adverse prognosis in pediatric and adult acute myeloid leukemia

High VEGFC mRNA expression of acute myeloid leukemia (AML) blasts is related to increased in vitro and in vivo drug resistance. Prognostic significance of VEGFC on long-term outcome and its associated gene expression profiles remain to be defined. We studied effect of VEGFC on treatment outcome and investigated gene expression profiles associated with VEGFC using microarray data of 525 adult and 100 pediatric patients with AML. High VEGFC expression appeared strongly associated with reduced complete remission rate (P = .004), reduced overall and event-free survival (OS and EFS) in adult AML (P = .002 and P < .001, respectively). Multivariable analysis established high VEGFC as prognostic indicator independent of cytogenetic risk, FLT3-ITD, NPM1, CEBPA, age, and white blood cell count (P = .038 for OS; P = .006 for EFS). Also, in pediatric AML high VEGFC was related to reduced OS (P = .041). A unique series of differentially expressed genes was identified that distinguished AML with high VEGFC from AML with low VEGFC, that is, 331 up-regulated genes (representative of proliferation, vascular endothelial growth factor receptor activity, signal transduction) and 44 down-regulated genes (eg, related to apoptosis) consistent with a role in enhanced chemoresistance. In conclusion, high VEGFC predicts adverse long-term prognosis and provides prognostic information in addition to well-known prognostic factors.

Analysis of a new homozygous deletion in the tumor suppressor region at 3p12.3 reveals two novel intronic noncoding RNA genes

Homozygous deletions or loss of heterozygosity (LOH) at human chromosome band 3p12 are consistent features of lung and other malignancies, suggesting the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene has been cloned thus far from the overlapping region deleted in lung and breast cancer cell lines U2020, NCI H2198, and HCC38. It is DUTT1 (Deleted in U Twenty Twenty), also known as ROBO1, FLJ21882, and SAX3, according to HUGO. DUTT1, the human ortholog of the fly gene ROBO, has homology with NCAM proteins. Extensive analyses of DUTT1 in lung cancer have not revealed any mutations, suggesting that another gene(s) at this location could be of importance in lung cancer initiation and progression. Here, we report the discovery of a new, small, homozygous deletion in the small cell lung cancer (SCLC) cell line GLC20, nested in the overlapping, critical region. The deletion was delineated using several polymorphic markers and three overlapping P1 phage clones. Fiber‐FISH experiments revealed the deletion was approximately 130 kb. Comparative genomic sequence analysis uncovered short sequence elements highly conserved among mammalian genomes and the chicken genome. The discovery of two EST clusters within the deleted region led to the isolation of two noncoding RNA (ncRNA) genes. These were subsequently found differentially expressed in various tumors when compared to their normal tissues. The ncRNA and other highly conserved sequence elements in the deleted region may represent miRNA targets of importance in cancer initiation or progression. Published 2006 Wiley‐Liss, Inc.

Mesenchymal Stem Cells Contribute to Tumor Cell Proliferation by Direct Cell-Cell Contact Interactions

ABSTRACT Bone marrow (BM)-derived mesenchymal stem cells (MSCs) have been implicated in tumor progression, making MSCs important targets for anti-cancer strategies. In this study, we show that MSCs promote tumor growth in vivo in a lymphoma xenograft model. We show that MSCs provide direct cell–cell contact interactions and, to a lesser extend, soluble factors that promote tumor cell proliferation and survival in vitro. PTK787/ZK 222584 reduces tumor growth-promoting effects of MSCs both in vitro and in vivo. Our results address the importance of targeting the MSCs for future anti-cancer strategies.

Vascular endothelial growth factor signaling in acute myeloid leukemia

This review is designed to provide an overview of the current literature concerning vascular endothelial growth factor signaling (VEGF) in acute myeloid leukemia (AML). Aberrant VEGF signaling operates in the bone marrow of AML patients and is related to a poor prognosis. The altered signaling pathway demonstrated to interfere in several autocrine and paracrine signaling pathways. VEGF signaling promotes autocrine AML blast cell proliferation, survival, and chemotherapy resistance. In addition, VEGF signaling can mediate paracrine vascular endothelial cell-controlled angiogenesis in AML. Both effects presumably explain the association of high VEGF levels and poor therapeutic outcome. More recently, researches focusing on bone marrow stem cell niches demonstrate a role for VEGF signaling in the preservation of several cell types within these niches. The bone marrow niches are proposed to be a protective microenvironment for AML cells that could be responsible for relapses in AML patients. This implies the need of sophisticated VEGF-targeted therapeutics in AML therapy strategies. This review highlights our current understanding of aberrant VEGF signaling in AML, appoints the interference of VEGF signaling in the AML-associated microenvironment, and reflects the novelty of current VEGF-targeted therapeutics used in clinical trails for the treatment of AML.

Endogenous Vascular Endothelial Growth Factor-C Expression Is Associated with Decreased Drug Responsiveness in Childhood Acute Myeloid Leukemia

Purpose: We hypothesized that downstream effects of endogenous vascular endothelial growth factor (VEGF)/VEGF receptor signaling on acute myelogenous leukemia (AML) cell survival resulted in increased in vitro cellular drug resistance and a longer time to kill most leukemic cells in vivo upon drug exposure. Experimental Design: In primary AML cells from pediatric patients, VEGFA and VEGFC mRNA expression and in vitro cellular resistance to nine cytotoxic drugs were studied. As in vivo equivalents for in vitro drug resistance, in vivo AML blast reduction upon drug exposure, measured as blast cell reduction on day 15 in the bone marrow and as time in days from diagnosis to complete remission (CR) were used. Results: Increased endogenous VEGFC levels significantly correlated with increased in vitro resistance for six typical AML drugs in primary AML cells from pediatric patients. Patients with >5% blasts on day 15 showed a 12.9-fold increase in the median VEGFC level compared with patients with ≤5% blasts (P = 0.002). Time to reach CR was studied using linear regression analysis with VEGFC, age at diagnosis, sex, treatment protocol, FAB type, cytogenetic risk profile, and WBC counts as variables. There was a significant positive independent association between VEGFC levels and time to CR (b = 6.02, SE = 1.58, P ≤ 0.0001, n = 72). Conclusions: These results suggest for the first time that higher endogenous VEGFC levels of AML cells are related to decreased in vitro and in vivo drug responsiveness.

High acute myeloid leukemia derived VEGFA levels are associated with a specific vascular morphology in the leukemic bone marrow.

BackgroundAcute Myeloid Leukemia (AML) bone marrow biopsies at diagnosis display enhanced angiogenesis and increased VEGFA expression. In a xenograft mouse model it was described that availability of free VEGFA versus bound VEGFA is related to different vascular morphology. In this study we investigate the relationship between vascular morphology within AML bone marrow biopsies and AML derived VEGFA levels.MethodsVessel count and surface area (Chalkley count) were calculated in AML bone marrow biopsies at diagnosis (n = 32), at remission (n = 8) and Normal Bone Marrow (n = 32) using immunohistochemical staining for FVIII, CD31, CTIV, SMA and VEGFA. VEGFA protein levels were measured.ResultsHigh vessel count was associated with an immature vessel status. Combining vessel count and Chalkley count different vessel morphology patterns were quantified within AML bone marrow biopsies. Three different subgroups could be distinguished. The subgroup (37.5% of the samples) exhibiting a high vessel count and vessels with predominantly large lumen (normal Chalkley count) was associated with high secreted VEGFA protein levels.ConclusionDifferent vasculature patterns are seen in AML bone marrow biopsies, defined by combining number and size of vessel. These quantified morphology patterns, combined with VEGFA levels, might be of value in the success of VEGF/VEGFR-signaling interference approaches.