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XLSX file - 32K, Phosphorylation of downstream intracellular kinases.

Although most children with acute myeloid leukemia (AML) achieve complete remission, the relapse rate is 30% to 40%. Because it is thought that leukemia-initiating cells (LIC) are responsible for AML relapses, targeting these cells might improve outcome. Treatment of pediatric AML blasts with the receptor tyrosine kinase (RTK) inhibitor PTK787/ZK 222584 (PTK/ZK) induces cell death in vitro. However, the role of PTK/ZK inhibition on outgrowth of (pediatric) LICs is unknown. In this study, we cultured CD34+ cells from pediatric patients with AML on MS5 stromal cells in long-term cocultures. In analogy to adult AML, long-term expansion of leukemic cells up to 10 weeks could be generated in 9 of 13 pediatric AMLs. Addition of PTK/ZK to long-term cocultures significantly inhibited leukemic expansion in all samples, ranging from 4% to 80% growth inhibition at week 5 compared with untreated samples. In 75% of the samples, the inhibitory effect was more pronounced at week 10. Proteome profiler array analysis of downstream kinases revealed that PTK/ZK reduced activation of PI3K/Akt kinase signaling. Although main targets of PTK/ZK are VEGF receptors (VEGFR), no effect was seen on outgrowth of LICs when cultured with bevacizumab (monoclonal VEGFA-antibody), specific antibodies against VEGFR2 or VEGFR3, or exposed to stroma-derived VEGFA. These data suggest that the effect of PTK/ZK on LICs is not only dependent on inhibition of VEGFA/VEGFR signaling. Taken together, our data elucidated antileukemic properties of PTK/ZK in long-term expansion cultures, and suggest that targeting multiple RTKs by PTK/ZK might be a potential effective approach in eradicating (pediatric) LICs. Mol Cancer Res; 11(4); 339–48. ©2013 AACR.

XLSX file - 21K, Specific primers for RT-PCR.

PDF file - 85K

PDF file - 147K, Effect of VEGFA-addition, Bevacizumab, specific VEGFR-inhibitor and MK2206 on CD34+ sorted pediatric AML cells.

PDF file - 161K, Effect of PTK787/ZK 222584 and MK2206 on AML cell lines.

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. [Cancer Res 2009;69(14):5987–95]

Supplementary Table 3 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Figure Legend from Repression of Vascular Endothelial Growth Factor Expression by the Runt-Related Transcription Factor 1 in Acute Myeloid Leukemia

Supplementary Figure 3 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Figure 2 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Table 4 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Figure 1 from Repression of Vascular Endothelial Growth Factor Expression by the Runt-Related Transcription Factor 1 in Acute Myeloid Leukemia

Supplementary Figure 1 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Table 2 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Figure 4 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Table 1 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Objective This study investigated the course, predictors, and impact of caregiving stress on the functioning of primary caregivers of children with cancer during the first year after a child's cancer diagnosis. Methods Primary caregivers (N = 95, 100% mother, 86% response rate) of consecutive newly diagnosed paediatric cancer patients (0–18 years) completed measures of caregiving stress, depressive symptoms, anxiety, and self-reported health at diagnosis, and 3, 6, and 12 months thereafter. Results Results indicated a significant decrease in caregiving stress (especially during the first 3 months after diagnosis). Caregiving stress was predicted by single marital status and the ill child being the mother's only child. Multilevel analyses, controlled for socio-demographic and medical covariates, showed that, over time, the decline in caregiving stress was accompanied by a reduction in depressive symptoms and anxiety. The amount of variance explained by caregiving stress was 53% for depressive symptoms, 47% for anxiety, and 3% for self-reported health. Conclusions The present study suggests that caregiving stress is an important factor in understanding parental adjustment to childhood cancer. This offers possibilities for developing interventions aimed at preventing caregiving stress, and strengthening mothers' confidence in their ability to provide good care

ObjectiveThe aim of this study was to examine the longitudinal effects of communication styles on marital satisfaction and distress of parents of children treated for cancer. MethodsMarital dissatisfaction (Maudsley Marital Questionnaire), intimacy, avoidance, destructive and incongruent communication (Communication Skills Inventory) and psychological distress (General Health Questionnaire) were assessed in 115 parents of pediatric cancer patients shortly after diagnosis (T1) and 5years later (T2). ResultsOnly mothers' marital dissatisfaction increased significantly over time. No gender differences in dissatisfaction were found. Mothers had a significantly higher lack of intimacy score than fathers. All T1 communication styles were significantly univariately related to fathers' and mothers' T2 marital dissatisfaction, while not to T2 distress. Mothers' T1 marital dissatisfaction accounted for 67% and fathers' for 12% in the explained variance of T2 dissatisfaction. T1 destructive communication uniquely affected fathers' T2 marital dissatisfaction and T1 avoidant communication that of mothers. ConclusionsFive years after cancer diagnosis in their children, the quality of parents' marital relationships seemed largely unchanged. Parents' use of communication skills at diagnosis appeared to have limited effect on their marital dissatisfaction and no effect on their distress 5years later. While avoidant communication seemed indicative of mothers' marital distress, fathers' seemed affected by destructive communication. Copyright (c) 2014 John Wiley & Sons, Ltd.

Purpose/Objectives: To explore the response shift phenomenon in pediatric patients with cancer and to determine its effects on ratings of health-related quality of life (HRQOL).Design: Retrospective pre- and post-test design.Setting: Pediatric oncology department in the northern part of the Netherlands.Sample: 37 children newly diagnosed with cancer and 80 parents.Methods: The then-test method was used to determine response shift. HRQOL was assessed within two weeks postdiagnosis (pretest) and three months later (post-test) using both child and parent reports of PedsQL and Cantril's ladder. The post-test and then-test were administered concurrently.Main Research Variables: Overall and multidimensional HRQOL.Findings: Scores on Cantril's then-test were lower than the pretest in both child and parent reports, indicating response shift in the assessment of overall HRQOL. Children experienced a greater response shift than parents. No differences were found between the PedsQL then- and pretests.Conclusions: Both child- and parent-report ratings of overall HRQOL were affected by response shift, resulting in an underestimation of the improvement in overall HRQOL between diagnosis and three months postdiagnosis. No response shift was demonstrated in the more specific domains of HRQOL (PedsQL).Implications for Nursing: Knowledge of the response shift phenomenon helps nurses to better interpret the outcomes of HRQOL. The use of the PedsQL instrument is recommended in future studies that aim to demonstrate changes in HRQOL.

Purpose: To perform a systematic literature review for critical evaluation of prevalence and factors contributing to malnutrition in childhood cancer. Methods: A systematic search resulting in 46 suitable articles. Results: Due to lack of uniform criteria and adequate studies, the prevalence rates of malnutrition can only be estimated. Based on strengths and weaknesses of included references, prevalence rates are estimated to be 0-10% for leukemia, 20-50% for neuroblastoma, and 0-30% for other malignancies. Whether energy deficiency or inflammation contributed to malnutrition could not be confirmed because the occurrence of energy deficit (low energy intake, increased metabolic rate) or inflammation (related to cachexia) was not convincing. Also, a relationship between these factors and malnutrition was not studied. Conclusion: Longitudinal studies are needed to determine which children are at risk of malnutrition, and to investigate the impact of energy deficiency and inflammation on the nutritional status and body composition of childhood cancer patients. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Eph/ephrin signaling has bcen implicated in various types of key cancer-enhancing processes, like migration, proliferation, and angiogenesis. In medulloblastoma, invading tumor cells characteristically lead to early recurrence and a decreased prognosis. Based on kinase-activity profiling data published recently, we hypothesized a key role for the Eph/ephrin signaling system in medulloblastoma invasion. In primary medulloblastoma samples, a significantly higher expression of EphB2 and the ligand ephrin-B1 was observed compared with normal cerebellum. Furthermore, medulloblastoma cell lines showed high expression of EphA2, EphB2, and EphB4. Stimulation of medulloblastoma cells with ephrin-B1 resulted in a marked decrease in in vitro cell adhesion and an increase in the invasion capacity of cells expressing high levels of EphB2. The cell lines that showed an ephrin-B1-induced phenotype possessed increased levels of phosphorylated EphB2 and, to a lesser extent, EphB4 after stimulation. Knockdown of EphB2 expression by short hairpin RNA completely abolished ephrin ligand-induced effects on adhesion and migration. Analysis of signal transduction identified p38, Erk, and mTOR as downstream signaling mediators potentially inducing the ephrin-B1 phenotype. In conclusion, the observed deregulation of Eph/ephrin expression in medulloblastoma enhances the invasive phenotype, suggesting a potential role in local tumor cell invasion and the formation of metastases.

BACKGROUND: High vascular endothelial growth factor (VEGFA) levels at the time of diagnosis confer a worse prognosis to multiple malignancies. Our aim was to investigate the role of VEGFA in promoting tumour growth through interaction with its environment.METHODS: HL-60 cells were transduced with VEGFA165 or control vector using retroviral constructs. Control cells (n = 7) or VEGFA165 cells (n = 7) were subcutaneously injected into NOD/SCID mice. Immunohistochemistry of markers for angiogenesis (CD31) and cell proliferation (Ki67) and gene expression profiling of tumours were performed. Paracrine effects were investigated by mouse-specific cytokine arrays.RESULTS: In vivo we observed a twofold increase in tumour weight when VEGFA165 was overexpressed (P = 0.001), combined with increased angiogenesis (P = 0.002) and enhanced tumour cell proliferation (P = 0.001). Gene expression profiling revealed human genes involved in TGF-beta signalling differentially expressed between both tumour groups, that is, TGFBR2 and SMAD5 were lower expressed whereas the inhibitory SMAD7 was higher expressed with VEGFA165. An increased expression of mouse-derived cytokines IFNG and interleukin 7 was found in VEGFA165 tumours, both described to induce SMAD7 expression.CONCLUSION: These results suggest a role for VEGFA-driven tumour growth by TGF-beta signalling inhibition via paracrine mechanisms in vivo, and underscore the importance of stromal interaction in the VEGFA-induced phenotype. British Journal of Cancer (2011) 105, 1856-1863. doi: 10.1038/bjc.2011.460 www.bjcancer.com Published online 1 November 2011 (C) 2011 Cancer Research UK

Aims: Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR-derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR-2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1-3 mRNA expression was assessed. Methods: VEGFR-2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser-microdissected blood vessels. Results: Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR-2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a 13.6-fold average enrichment of VEGFR-2 expression in the laser-microdissected endothelium compared to whole tumour. Also the expression of VEGFR-1 and -3 was highly enriched in the endothelium fraction with an average fold-enrichment of 16.5 and 50.8 respectively. Conclusions: Phosphorylated VEGFR-2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1-3 mRNA expression. This suggests a crucial role for VEGF/VEGFR-induced angiogenesis in the progression and maintenance of these tumours.

Objective This prospective 5-year longitudinal study examined the use of coping styles of fathers and mothers of pediatric cancer patients over time and the prospective effects of coping on distress. Methods Psychological distress (General Health Questionnaire) and the use of seven coping styles (Utrecht Coping List: active problem focussing, palliative and passive reaction patterns, avoidance, social support seeking, expression of emotions, and comforting cognition) were assessed in 115 parents shortly after diagnosis, 6 and 12-months, and 5-years later. Results At diagnosis, parents' use of coping styles did not differ from the norm population except more frequent use of support seeking. No significant change over time was found in a palliative reaction pattern. Support seeking declined and emotional expression increased linearly, whereas use of the remaining coping styles decreased, followed by an increase. At 5-years, parents' use differed from the norm population only in less use of expression of emotions and comforting cognitions. Initial coping use significantly predicted fathers' future distress at 6 and 12-months but not at 5-years. This was not found for mothers. Changes in coping were significantly associated with both parents' changes in distress only during the first year. Increased passive reaction pattern and social support seeking were the risk factors for mothers. Increased avoidance, a passive reaction pattern, expression of emotions, and decreased active problem focussing formed the risk factors for fathers. Conclusion Findings illustrate that coping seems to be a situation-specific process and that coping predictors vary as a function of parents' gender. Copyright (c) 2011 John Wiley & Sons, Ltd.

Background Acute 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. Methods Vessel 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. Results High 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. Conclusion Different 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.

Fijlstra M, Rings EH, Verkade HJ, van Dijk TH, Kamps WA, Tissing WJ. Lactose maldigestion during methotrexate-induced gastrointestinal mucositis in a rat model. Am J Physiol Gastrointest Liver Physiol 300: G283-G291, 2011. First published November 18, 2010; doi:10.1152/ajpgi.00462.2010.-Patients with chemotherapy-induced gastrointestinal mucositis suffer from anorexia, diarrhea, and stomach pain, often causing weight loss and malnutrition. When the intestinal function during mucositis would be known, a rational feeding strategy might improve the nutritional state, accelerate recuperation, and increase survival of mucositis patients. We developed a methotrexate (MTX)-induced mucositis rat model to study nutrient digestion and absorption. To determine lactose digestion and absorption of its derivative glucose during mucositis, we injected Wistar rats intravenously with MTX (60 mg/kg) or 0.9% NaCl (controls). Four days later, we orally administered trace amounts of [1-C-13] lactose and [U-C-13] glucose and quantified the appearance of labeled glucose in the blood for 3 h. Finally, we determined plasma citrulline level and harvested the small intestine to assess histology, myeloperoxidase level, glycohydrolase activity, immunohistochemical protein, and mRNA expression. MTX-treated rats showed profound villus atrophy and epithelial damage. During the experimental period, the absorption of lactose-derived [1-C-13] glucose was 4.2-fold decreased in MTX-treated rats compared with controls (P

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.

Background. The Currently used National Cancer Institute (NCI) adverse events criteria for mucosal barrier injury (MBI) are insufficient for use in children. We searched for objective, easily measurable indicators for MBI in children with cancer. Purpose. In children with acute myeloid leukemia, various MBI-related clinical and laboratory tests were investigated, reflecting clinical severity (NCI symptomatic adverse events criteria (gold standard), daily gut score (DGS)), inflammation (plasma and fecal interleukin-8 (IL-8), fecal calprotectin), enterocytic loss (plasma citrulline, ratio fecal human DNA/total DNA) and intestinal permeability (sugar absorption tests). Results. Intestinal MBI as detected by the NCI adverse events criteria was found in 55% of chemotherapy cycles, correlating well with the continuous DGS (n = 55, rho = 0.581; P

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. [Cancer Res 2009;69(14):5987–95]

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) have been shown to favor tumor growth, suggesting the relevance of pharmaceutical inhibition of MSCs for the treatment of malignancies. We tested the effect of PTK787/ZK 222584 (PTK) on the outgrowth of MSCs from human bone marrow-derived mononuclear cells (MNCs) and the migration and tube formation capacity of MSCs in vitro. PTK dose-dependently inhibited the outgrowth of BM-MSCs from BM-MNCs (LC50 1.12 microM PTK), while hematopoietic colony formation (HCF) was only slightly hampered (13 +/- 19% at 1 microM PTK, and stable at approximately 50% at higher concentrations of PTK). Addition of 10 microM PTK inhibited proliferation of MSCs by 74 +/- 6.6% compared to control (p < 0.0001) and increased apoptosis of MSCs by 63 +/- 7.7% (p < 0.01). In addition, upon addition of PTK, BM-MSCs showed impaired tube formation as well as reduced migration (52 +/- 19%, p = 0.006) compared to control. Pepchip array analysis revealed that PTK effectively inhibits activity of kinases involved in cell cycling (WEE1 and several cyclin dependent kinases), and migratory processes (including Rho kinase). In conclusion, we show that PTK impairs outgrowth, proliferation, migration and tube formation of human BM-MSCs. In addition, we show the usability of Pepchip array analysis as a powerful tool for kinase activity profiling in functional studies since the effect of PTK on the kinome profile of MSCs corresponds with the observed functional effects of PTK on proliferation and migration. Inhibition of BM-MSCs and their contribution to tumor growth may be an additional strategy for treatment of cancer in the future.

Summary Background Genetic subtypes of acute lymphoblastic leukaemia (ALL) are used to determine risk and treatment in children. 25% of precursor B-ALL cases are genetically unclassified and have intermediate prognosis. We aimed to use a genome-wide study to improve prognostic classification of ALL in children. Methods We constructed a classifier based on gene expression in 190 children with newly diagnosed ALL (German Cooperative ALL [COALL] discovery cohort) by use of double-loop cross-validation and validated this in an independent cohort of 107 newly diagnosed patients (Dutch Childhood Oncology Group [DCOG] independent validation cohort). Hierarchical cluster analysis with classifying gene-probe sets revealed a new ALL subtype, the underlying genetic abnormalities of which were characterised by comparative genomic hybridisation-arrays and molecular cytogenetics. Findings Our classifier predicted ALL subtype with a median accuracy of 90·0% (IQR 88·3–91·7) in the discovery cohort and correctly identified 94 of 107 patients (accuracy 87·9%) in the independent validation cohort. Without our classifier, 44 children in the COALL cohort and 33 children in the DCOG cohort would have been classified as B-other. However, hierarchical clustering showed that many of these genetically unclassified cases clustered with BCR–ABL1 -positive cases: 30 (19%) of 154 children with precursor B-ALL in the COALL cohort and 14 (15%) of 92 children with precursor B-ALL in the DCOG cohort had this BCR–ABL1 -like disease. In the COALL cohort, these patients had unfavourable outcome (5-year disease-free survival 59·5%, 95% CI 37·1–81·9) compared with patients with other precursor B-ALL (84·4%, 76·8–92·1%; p=0·012), a prognosis similar to that of patients with BCR–ABL1 -positive ALL (51·9%, 23·1–80·6%). In the DCOG cohort, the prognosis of BCR–ABL1 -like disease (57·1%, 31·2–83·1%) was worse than that of other precursor B-ALL (79·2%, 70·2–88·3%; p=0.026), and similar to that of BCR–ABL1 -positive ALL (32·5%, 2·3–62·7%). 36 (82%) of the patients with BCR–ABL1 -like disease had deletions in genes involved in B-cell development, including IKZF1, TCF3, EBF1, PAX5, and VPREB1 ; only nine (36%) of 25 patients with B-other ALL had deletions in these genes (p=0·0002). Compared with other precursor B-ALL cells, BCR–ABL1 -like cells were 73 times more resistant to L-asparaginase (p=0·001) and 1·6 times more resistant to daunorubicin (p=0·017), but toxicity of prednisolone and vincristine did not differ. Interpretation New treatment strategies are needed to improve outcome for this newly identified high-risk subtype of ALL. Funding Dutch Cancer Society, Sophia Foundation for Medical Research, Paediatric Oncology Foundation Rotterdam, Centre of Medical Systems Biology of the Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research, American National Institute of Health, American National Cancer Institute, and American Lebanese Syrian Associated Charities.

We investigated the psychological functioning of parents of children suffering from pediatric cancer using a prospective design over a five-year time period. Parents of children diagnosed with cancer participated at diagnosis (T1), six months (T2), twelve months (T3), and five years later (T4, n = 115). Repeated measures ANOVAs were calculated for the three measures of psychological distress (GHQ, SCL-90 and STAI-S) to examine changes over time and gender differences. Independent T-tests were computed to examine differences between the mean scores of the parents at T4 and the norm groups. The effects of health status and earlier levels of distress on T4 functioning were examined using ANOVA and partial correlational analysis. Results showed that levels of reported distress, psychoneurotic symptoms and state anxiety significantly decreased across time to normal levels at T4 except on the GHQ. A significantly higher percentage of parents (27%) than in the norm group (15%) showed clinically elevated scores on the GHQ. Mothers had higher scores than fathers only on state anxiety. Parents of relapsed children reported higher anxiety levels than parents of surviving and deceased children. Psychological functioning at T1 was significantly related to functioning at T4. These results support the conclusion that although parental distress decreases with time, a significant number of parents still suffer from clinical distress after five years. Parents of relapsed children are at risk for long-term psychological problems as are those with higher levels of psychosomatic complaints at diagnosis. Copyright (c) 2005 John Wiley & Sons, Ltd.

Anaplastic large cell lymphomas (ALCLs) can be subdivided into two subgroups on the basis of their expression of the ALK protein. ALK protein expression leads to activation of signal transducer and activator of transcription (STAT) 3, which is more commonly expressed in ALK-positive than in ALK-negative tumours. Activated STAT3 leads to the induction of several genes such as Mcl-1, Bcl-2 and Bcl-X(L), and tissue inhibitor of metalloproteinase (TIMP)-1. In this study, we analysed TIMP-1 expression in five ALCL cell lines and 11 tumours by quantitative RT-PCR and immunohistochemistry. We identified high-level TIMP-1 expression by RT-PCR in three ALK-positive ALCL-derived cell lines and in all ALK-positive ALCLs, whereas ALK-negative ALCLs generally demonstrated a lower level of TIMP-1 expression. Concordant with these results, we observed TIMP-1 immunostaining in all ALK-positive ALCLs and in only two of six ALK-negative ALCLs. No relationship was observed between the levels of ALK and TIMP-1 expression in the ALK-positive tumours. STAT3 expression levels were similar in all ALCL samples. Double staining with either CD30 or CD68 demonstrated that TIMP-1 expression was restricted to macrophages in the majority of TIMP-1-positive tumours. Expression of the TIMP-1 substrate MMP-2 was more prominent in ALK-negative tumours, while MMP-9 levels were low in all cases. Expression levels of IL-6 and TGF-beta1, which are cytokines known to induce TIMP-1, were higher in ALK-negative ALCLs and moderate in ALK-positive tumours. No clear relationship was observed between IL-10 expression and ALK positivity. Overall, no correlation was seen in ALCLs between the expression of TIMP-1 and that of cytokines that induce TIMP-1. Lack of TIMP-1 expression in the tumour cells of ALK-positive ALCLs argues against a direct role for ALK-induced activation of STAT3 in the regulation of TIMP-1 expression in ALCL.

Purpose: Recombinant human insulin-like growth factor I (rhIGF-I) has been reported to ameliorate vincristine-induced neuropathy, the dose-limiting side effect of this antimitotic anticancer drug. However, rhIGF-I also might have adverse effects, as has been shown in vitro, where it stimulates growth of cancer cells and protects them from cytotoxicity of anticancer drugs. The influence of rhIGF-I on the cytotoxicity of vincristine has not yet been studied. Furthermore, studies performed have been done under serum-free conditions, which are far from physiological. Methods: We studied the influence of rhIGF-I on the growth of two rhabdomyosarcoma cell lines (Rh30 and Rh1) and on the antitumor effects of vincristine, cisplatin, etoposide, doxorubicin, and topotecan under serum-free and serum-containing conditions. To extend the in vitro data, we grew Rh30 cells as xenografts in mice and determined the effects of vincristine, rhIGF-I or their combination on tumor growth. Results: In vitro, both cell lines demonstrated a functional type I IGF receptor, as shown by the rapid activation of ribosomal p70 S6 kinase after stimulation with rhIGF-I. Under serum-free conditions, rhIGF-I stimulated growth of both cell lines. Exposure to cytotoxic drugs with and without rhIGF-I resulted in higher cell numbers in cultures exposed to rhIGF-I. However, relative to the appropriate control, fractional growth inhibition and or cell kill of the cytotoxic drugs was identical with and without rhIGF-I. Under serum-containing conditions, rhIGF-I had no effect on cell growth or drug cytotoxicity. In vivo we did not find a significant influence of rhIGF-I on HxRh30 cell growth, or on the antitumor activity of vincristine. Conclusions: These studies show that rhIGF-I has no adverse effects on human rhabdomyosarcoma growth or on the antitumor effect of cytotoxic drugs under serum-containing conditions in vitro or in tumor-bearing mice. Potentially, therefore, rhIGF-I may ameliorate vincristine-induced neuropathy without adversely influencing tumor growth or vincristine cytotoxicity in children.

This study investigated the course, predictors, and impact of caregiving stress on the functioning of primary caregivers of children with cancer during the first year after a child's cancer diagnosis.Primary caregivers (N = 95, 100% mother, 86% response rate) of consecutive newly diagnosed paediatric cancer patients (0-18 years) completed measures of caregiving stress, depressive symptoms, anxiety, and self-reported health at diagnosis, and 3, 6, and 12 months thereafter.Results indicated a significant decrease in caregiving stress (especially during the first 3 months after diagnosis). Caregiving stress was predicted by single marital status and the ill child being the mother's only child. Multilevel analyses, controlled for socio-demographic and medical covariates, showed that, over time, the decline in caregiving stress was accompanied by a reduction in depressive symptoms and anxiety. The amount of variance explained by caregiving stress was 53% for depressive symptoms, 47% for anxiety, and 3% for self-reported health.The present study suggests that caregiving stress is an important factor in understanding parental adjustment to childhood cancer. This offers possibilities for developing interventions aimed at preventing caregiving stress, and strengthening mothers' confidence in their ability to provide good care.

BACKGROUND & AIMS: Under- and overnutrition are linked to adverse outcomes during and after childhood cancer treatment. Therefore, understanding the timing of weight loss and weight gain and their contributory factors is essential for improving outcomes. We aimed to determine in which period of treatment changes in nutritional status occurred and which factors contributed to these changes.METHODS: A prospective cohort study of 133 newly diagnosed cancer patients with hematological, solid, and brain malignancies was performed. Anthropometric data and related factors were assessed at 0, 3, 6 and 12 months after diagnosis.RESULTS: Despite initial weight loss at the beginning of treatment in patients with hematological and solid malignancies, body mass index (BMI) and fat mass (FM) increased within 3 months with 0.13 SDS (P < 0.001) and 0.05 SDS (P = 0.021) respectively. Increase continued during the following months and resulted in a doubling of the number of overnourished patients. Fat free mass (FFM), which was already low at diagnosis, remained low. During the entire study period about 17% of the patients were undernourished on the basis of low FFM. Tube feeding and diminished activity level were related to increases in BMI and %FM respectively. No relationship was found between energy intake or corticosteroids and increase in BMI or %FM.CONCLUSIONS: BMI and FM increased during and after the period of intensive treatment, while FFM remained low. Improvement of nutritional status might be accomplished by increasing physical activity from the early phase of treatment.

BackgroundPI3K/AKT pathway mutations are found in T-cell acute lymphoblastic leukemia, but their overall impact and associations with other genetic aberrations is unknown. PTEN mutations have been proposed as secondary mutations that follow NOTCH1-activating mutations and cause cellular resistance to gamma-secretase inhibitors.Design and MethodsThe impact of PTEN, PI3K and AKT aberrations was studied in a genetically well-characterized pediatric T-cell leukemia patient cohort (n=146) treated on DCOG or COALL protocols.ResultsPTEN and AKT E17K aberrations were detected in 13% and 2% of patients, respectively. Defective PTEN-splicing was identified in incidental cases. Patients without PTEN protein but lacking exon-, splice-, promoter mutations or promoter hypermethylation were present. PTEN/AKT mutations were especially abundant in TAL- or LMO-rearranged leukemia but nearly absent in TLX3-rearranged patients (P=0.03), the opposite to that observed for NOTCH1-activating mutations. Most PTEN/AKT mutant patients either lacked NOTCH1-activating mutations (P=0.006) or had weak NOTCH1-activating mutations (P=0.011), and consequently expressed low intracellular NOTCH1, cMYC and MUSASHI levels. T-cell leukemia patients without PTEN/AKT and NOTCH1-activating mutations fared well, with a cumulative incidence of relapse of only 8% versus 35% for PTEN/AKT and/or NOTCH1-activated patients (P=0.005).ConclusionsPI3K/AKT pathway aberrations are present in 18% of pediatric T-cell acute lymphoblastic leukemia patients. Absence of strong NOTCH1-activating mutations in these cases may explain cellular insensitivity to gamma-secretase inhibitors.

Little is known about the etiology of childhood acute lymphoblastic leukemia (ALL). The presence of atopic disease has been shown to protect against developing childhood ALL. The aim of this study was to examine whether single nucleotide polymorphisms (SNPs) in innate immunity genes previously associated with atopic disease, can elucidate the inverse association between childhood ALL and atopic disease. We studied 525 children, including 192 with childhood ALL, 149 with atopic disease and 184 healthy control subjects. We compared genotype distributions of 29 SNPs in genes of TLR2, TLR4, TLR6, TLR9, TLR10 and CD14 between the three groups and corrected for multiple testing. The genotype distributions of two SNPs in the TLR6 gene, rs5743798 and rs6531666, differed significantly between children with ALL, children with atopic disease and control subjects. Particularly in children with atopic eczema, risk alleles for atopic disease were observed more often than in control subjects, and less often in children with ALL than in control subjects. These findings support the immune surveillance hypothesis as an explanation for the protective association of atopic disease on childhood ALL. Further investigation is warranted to examine in more detail the role of innate immunity in the development of childhood ALL.

Item does not contain fulltext To date, the biology of acute leukemia has been unclear, and defining new therapeutic targets without prior knowledge remains complicated. The use of high-throughput techniques would enable us to learn more about the biology of the disease, and make it possible to directly assess a broader range of therapeutic targets. In this study we have identified comprehensive tyrosine kinase activity profiles in leukemia samples using the PamChip(R) kinase activity profiling system. Strikingly, 31% (44/120) of the detected peptides were active in all three groups of leukemia samples. The recently reported activity of platelet-derived growth factor receptor (PDGFR) and neurotrophic tyrosine kinase receptors (NTRK1 and NTRK2) in leukemia could be appreciated in our array results. In addition, high levels of peptide phosphorylation were demonstrated for peptides related to macrophage stimulating 1 receptor (MST1R). A provisional signal transduction scheme of the common active peptides was constructed and used to specifically select an inhibitor for leukemic blast cell survival assays. As expected, a dose-dependent decrease in leukemic blast cell survival was achieved for all leukemia samples. Our data demonstrate that kinase activity profiling in leukemic samples is feasible and provides novel insights into the pathogenesis of leukemia. This approach can be used for the rapid discovery of potential drug targets.

Objective: To investigate the preferences of children with cancer, their parents, and survivors of childhood cancer regarding medical communication with child patients and variables associated with these preferences.Methods: Preferences regarding health-care provider empathy in consultations, and children's involvement in information exchange and medical decision making were investigated by means of vignettes. Vignettes are brief descriptions of hypothetical situations, in which important factors are systematically varied following an experimental design. In total, 1440 vignettes were evaluated by 34 children with cancer (aged 8-16), 59 parents, and 51 survivors (aged 8-16 at diagnosis, currently aged 10-30). Recruitment of participants took place in three Dutch university-based pediatric oncology centers. Data were analyzed by multilevel analyses.Results: Patients, parents, and survivors indicated the importance of health-care providers' empathy in 81% of the described situations. In most situations (70%), the three respondent groups preferred information about illness and treatment to be given to patients and parents simultaneously. Preferences regarding the amount of information provided to patients varied. The preference whether or not to shield patients from information was mainly associated with patients' age and emotionality. In most situations (71%), the three respondent groups preferred children to participate in medical decision making. This preference was mainly associated with patients' age.Conclusions: To be able to adapt communication to parents' and patients' preferences, health-care providers should repeatedly assess the preferences of both groups. Future studies should investigate how health-care providers balance their communication between the sometimes conflicting preferences of patients and parents. Copyright (C) 2010 John Wiley & Sons, Ltd.

To evaluate the adverse late effects of ALL treatment on cognitive functions and brain morphology; to integrate the results of a neuropsychological and neuroradiological study.Cranial magnetic resonance imaging (MRI) and neuropsychological assessments (NA) were performed in 35 children treated for acute lymphoblastic leukemia (ALL) with cranial irradiation (CI) and intrathecal and intravenous methotrexate. Patients were under the age of 7 years (MD: 3.5 years) at diagnosis; median follow-up at MRI and NA was 8 years since diagnosis.MRI's were classified as definitely abnormal in 51% and as probably abnormal in another 17% of the patients. White matter damage was most frequently seen. MRI abnormalities were not related to CI dose or age at diagnosis. Patients showed significantly lower scores, compared to the norm group on measures of intelligence, verbal auditory memory, visual motor integration, and fine motor functioning. Lower scores significantly correlated with higher CI dose (25-32 Gy compared to 18-20 Gy) and younger age at diagnosis (4.0 years compared toor = 4.0 years). Forty percent of the patients had to be referred to schools for learning disabled.ALL treatment, including CI and MTX, at a young age is associated with persistent cognitive impairment and MRI abnormalities. However, no correlation was found between MRI results and neuropsychological or academic performance.

This prospective 5-year longitudinal study examined the use of coping styles of fathers and mothers of pediatric cancer patients over time and the prospective effects of coping on distress.Psychological distress (General Health Questionnaire) and the use of seven coping styles (Utrecht Coping List: active problem focussing, palliative and passive reaction patterns, avoidance, social support seeking, expression of emotions, and comforting cognition) were assessed in 115 parents shortly after diagnosis, 6 and 12 months, and 5 years later.At diagnosis, parents' use of coping styles did not differ from the norm population except more frequent use of support seeking. No significant change over time was found in a palliative reaction pattern. Support seeking declined and emotional expression increased linearly, whereas use of the remaining coping styles decreased, followed by an increase. At 5 years, parents' use differed from the norm population only in less use of expression of emotions and comforting cognitions. Initial coping use significantly predicted fathers' future distress at 6 and 12 months but not at 5 years. This was not found for mothers. Changes in coping were significantly associated with both parents' changes in distress only during the first year. Increased passive reaction pattern and social support seeking were the risk factors for mothers. Increased avoidance, a passive reaction pattern, expression of emotions, and decreased active problem focussing formed the risk factors for fathers.Findings illustrate that coping seems to be a situation-specific process and that coping predictors vary as a function of parents' gender.

From 1991 until 2004 children with acute lymphoblastic leukemia (ALL) in the Netherlands were treated according to protocols ALL-8 and ALL-9 which were based on different principles. An earlier study showed that the outcome of adolescents highly differed on these protocols.In this retrospective study, we analyzed whether the outcome of older children 10-15 years of age at diagnosis differed between the Berlin-Frankfurt-Münster (BFM)-based ALL-8 regimen and the ALL-9 regimen. Two hundred fifty-four older children who were treated according to protocol ALL-8 (n = 82) or ALL-9 (n = 172) were included in the analysis.A higher 5-year event-free survival (EFS) rate was found for patients treated according to ALL-8 compared to ALL-9 (79 ± 5% vs. 65 ± 4%, P = 0.02). Patient characteristics did not differ except for a slightly higher age in ALL-8. Therefore, additional analyses were done including only patients who were 12-15 years of age. In this age group there was also a difference in the 5-year EFS (82 ± 5% vs. 61 ± 5%, P = 0.00) as well as in the 5-year overall survival rate; 89 ± 4% compared to 68 ± 5%, respectively (P = 0.01). Major difference between protocols was the use of a consolidation and reinduction/intensification course and higher cumulative doses of asparaginase, methotrexate, and anthracyclines in ALL-8.Children 10-15 years of age have been undertreated with the ALL-9 regimen and benefit by intensive treatment components as used in ALL-8. We recommend using BFM-based protocols for these older children with ALL.

The Dutch Childhood Oncology Group (DCOG) has used two treatment strategies for children with acute lymphoblastic leukemia (ALL) based on Pinkel's St Jude Total Therapy or the Berlin-Frankfurt-Munster (BFM) backbone. In four successive protocols, 1734 children were treated. Studies ALL-6 and ALL-9 followed the Total Therapy approach; cranial irradiation was replaced by medium-dose methotrexate infusions and prolonged triple intrathecal therapy; dexamethasone was used instead of prednisone. Studies ALL-7 and ALL-8 had a BFM backbone, including more intensive remission induction, early reinduction and maintenance therapy without vincristine and prednisone pulses. The 5-year event-free survival and overall survival increased from 65.4 to 80.6% (P100 x 109/l, and T-lineage ALL have prognostic impact. We conclude that the chemotherapy-only approach in children with ALL in Total Therapy-based strategies and BFM-backbone treatment does not jeopardize survival and preserves cognitive functioning. This experience is implemented in the current DCOG-ALL-10 study using a BFM backbone and minimal residual disease-based stratification. Leukemia (2010) 24, 309-319; doi: 10.1038/leu.2009.258; published online 17 December 2009

Increased levels of circulating VEGF-A have been demonstrated in patients with non-Hodgkin lymphoma (NHL) and are associated with progressive disease and poor clinical outcome. We investigated the role of VEGF-A in lymphoma tumour growth on a molecular level in order to identify the mechanism of VEGF-A-promoted tumour growth and to identify the potential targets for therapy. We used a model in which Daudi (human Burkitt lymphoma) tumour cells were transduced with VEGF-A165 or an empty vector (negative control) and subcutaneously injected in NOD/SCID mice. The weight of tumours over-expressing VEGF-A was increased 4-fold compared to that of control tumours (p

The currently used National Cancer Institute (NCI) adverse events criteria for mucosal barrier injury (MBI) are insufficient for use in children. We searched for objective, easily measurable indicators for MBI in children with cancer.In children with acute myeloid leukemia, various MBI-related clinical and laboratory tests were investigated, reflecting clinical severity (NCI symptomatic adverse events criteria (gold standard), daily gut score (DGS)), inflammation (plasma and fecal interleukin-8 (IL-8), fecal calprotectin), enterocytic loss (plasma citrulline, ratio fecal human DNA/total DNA) and intestinal permeability (sugar absorption tests).Intestinal MBI as detected by the NCI adverse events criteria was found in 55% of chemotherapy cycles, correlating well with the continuous DGS (n = 55, rho = 0.581; P0.001). Intestinal cell loss as measured by the ratio fecal human DNA/total DNA and plasma citrulline correlated well with both NCI criteria (n = 61, rho = 0.357, P = 0.005 resp. n = 58, rho = -0.482; P0.001) and DGS (n = 54, rho = 0.352, P = 0.009 resp. n = 55, rho = -0.625; P0.001). Plasma IL-8 correlated strongly to plasma citrulline (n = 46, rho = -0.627; P0.001).MBI was reflected by parameters indicating inflammation (IL-8) and cell loss (plasma citrulline, ratio fecal human DNA/total DNA). We conclude that plasma citrulline might be a good parameter for MBI. Further studies are needed to show whether plasma citrulline can be used as a marker for MBI in future research.

Contains fulltext : 81501.pdf (Publisher’s version ) (Open Access) BACKGROUND: The purpose of this article is to describe and evaluate the methodology of online focus group discussions within the setting of paediatric oncology. METHODS: Qualitative study consisting of separate moderated asynchronous online discussion groups with 7 paediatric cancer patients (aged 8-17), 11 parents, and 18 survivors of childhood cancer (aged 8-17 at diagnosis). RESULTS: All three participant groups could be actively engaged over a one-week period. Respondents highly valued the flexibility and convenience of logging in at their own time and place to join the discussion. Adolescent patients and survivors emphasized that the anonymity experienced made them feel comfortable to express their views in detail. The findings indicate a strong preference for online group discussions across all participant groups. CONCLUSION: The findings show that online focus group methodology is a feasible tool for collecting qualitative data within the setting of paediatric oncology, and may offer new opportunities to collect data in other hard-to-include populations. The evaluations seem to indicate that the online group discussions have given participants an opportunity to articulate their experiences and views in a way they might not have done in a traditional group discussion. 9 p.