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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 examined whether: (1) the goals of adolescents with cancer at 3 months post-diagnosis (T1) and healthy peers differed in terms of content, valuation, and abstraction level, (2) the content, valuation and abstraction level of the goals of the adolescents with cancer differed between 3 and 12 months post-diagnosis (T2). Methods: Thirty-three adolescents with cancer and 66 matched controls completed the Personal Project Analysis Inventory. After nine months, the adolescents with cancer completed the measure again. Results: Compared to controls, adolescents with cancer at 3 months post-diagnosis (T1) reported more intrinsic than extrinsic goals, appraised intrinsic goals as more important than extrinsic goals and reported more concrete goals. Within the adolescents with cancer, the content, valuation and abstraction level of the goals did not differ between T1 and T2. Conclusions: Adolescents recently diagnosed with cancer set different types of goals than healthy peers and continue to set these types of goals until one year post-diagnosis. Future research can help determine how the personal goals of adolescents with cancer develop in the long term and to what extent personal goal setting during cancer influences the attainment of age-graded developmental tasks and well-being

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.

Social competence, i.e. appropriate or effective social functioning, is an important determinant of quality of life. Social competence consists of social skills, social performance and social adjustment. The current paper reviews social skills, in particular emotion recognition performance and its relationship with social adjustment in children with brain disorders. In this review, normal development and the neuro-anatomical correlates of emotion recognition in both healthy children and adults and in various groups of children with brain disorders, will be discussed. A systematic literature search conducted on PubMed, yielded nine papers. Emotion recognition tasks were categorized on the basis of task design and emotional categories to ensure optimal comparison across studies before an explorative meta-analysis was conducted. This meta-analytic review suggests that children with brain disorders show impaired emotion recognition, with the recognition of sad and fearful expressions being most impaired. Performance did not seem to be related to derivative measures of social adjustment. Despite the limited number of studies on a variety of brain disorders and control groups, outcomes were quite consistent across analyses and corresponded largely with the existing literature on development of emotion recognition in typically developing children. More longitudinal prospective studies on emotion recognition are needed to gain insight into recovery and subsequent development of children with distinct brain disorders. This will aid development, selection and implementation of interventions for improvement of social competence and quality of life in children with a brain disorder.

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.

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.

PURPOSE Infections are a major cause of morbidity and mortality in pediatric cancer patients. The aim of this study was to establish the microbiological spectrum and the susceptibility patterns of bacteremia-causing bacteria in pediatric cancer patients with febrile neutropenia in relation to the use of prophylactic and empirical antibiotics. METHODS We analyzed positive blood cultures of pediatric cancer patients presenting with febrile neutropenia between 2004 and 2011 in Groningen and Amsterdam (the Netherlands) and in Bern (Switzerland), using different antibiotic prophylactic and empirical regimens. RESULTS A total of 156 patients with 202 bacteremias, due to 248 bacteria species, were enrolled. The majority (73%) of bacteremias were caused by Gram-positive bacteria. Gram-negative bacteria, especially Pseudomonas aeruginosa, were observed significantly more often in Bern, where no fluoroquinolone prophylaxis was used. Ciprofloxacin-resistant bacteria were cultured more often from patients who did receive ciprofloxacin prophylaxis, compared to the patients who did not (57 versus 11%, p = 0.044). CONCLUSIONS Gram-positive bacteria predominated in this study. We showed that the use of prophylactic antibiotics in pediatric cancer patients was associated with increased resistance rates, which needs further study. The strategy for empiric antimicrobial therapy for febrile neutropenia should be adapted to local antibiotic resistance patterns.

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

Purpose In this study, we evaluated C-reactive protein (CRP), interleukin (IL)-8, procalcitonin (PCT), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as predictors for bacterial infection in febrile neutropenia, plus their usefulness in febrile neutropenia during chemotherapy-induced gastrointestinal mucositis. Methods Plasma was obtained from pediatric oncology patients at presentation with febrile neutropenia (n = 43) and 24–48 h later (n = 17). The patients were classified as having or not having a bacterial infection. Plasma was also obtained of patients in the absence and in the presence of mucositis (n = 26). Results At presentation with febrile neutropenia, median IL-8 and PCT levels were significantly increased in patients with a bacterial infection, in contrast to CRP and sTREM-1. IL-8 was the most sensitive marker for the early detection of bacterial infection, in combination with clinical parameters or PCT the sensitivity reached 100%. After 24–48 h, only PCT was significantly elevated during bacterial infection. IL-8 levels were significantly increased during mucositis. Mucositis did not cause considerable changes in PCT levels. Conclusions IL-8 is the most useful marker for the early detection of bacterial infections, compared with CRP, PCT, and sTREM-1. IL-8 in combination with clinical parameters or PCT might be even more useful. Gastrointestinal mucositis alone does not affect PCT levels, in contrast to IL-8 levels, and therefore, PCT might be more useful for the detection of bacterial infections during mucositis than IL-8.

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.

Infections are a major cause of morbidity and mortality in children with acute lymphoblastic leukemia (ALL). Susceptibility to infections increases as the neutrophil count decreases. Despite identical treatment patients vary considerably in the number of neutropenic episodes. Toll-like receptor 4 (TLR4) has been shown to have a role in inhibiting apoptosis of neutrophils. Therefore, we hypothesized that polymorphisms in the TLR4 gene may influence the number of chemotherapy-induced neutropenic episodes. Eight single-nucleotide polymorphisms (SNPs) of the TLR4 gene were determined in 194 children aged 0-17 years, who were diagnosed with ALL. We compared the genotype distributions of the SNPs with the frequency of neutropenic episodes during treatment with chemotherapeutic regimens. The number of neutropenic episodes varied from 0 to 17, with a median of four neutropenic episodes. Four SNPs in the TLR4 gene (rs10759931, rs11536889, rs1927911 and rs6478317) were associated with an increased risk of developing chemotherapy-induced neutropenia, each sustaining correction for multiple testing. Further studies are required to elucidate whether pediatric patients with ALL with the particular SNPs in the TLR4 gene also experience more infections and would benefit from prophylactic antibiotic treatment, by a reduction of morbidity and mortality due to infections.

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.

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.

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]

Background. Normally, humans are protected against infections by their anaerobic intestinal microorganisms providing colonization resistance. In immunocompromised patients, the endogenous intestinal gram-positive and gram-negative pathogens often cause infectious complications. Therefore, we analyzed the effect of chemotherapy treatment and antimicrobial prophylaxis on intestinal bacterial populations (microbiota) among pediatric patients with acute myeloid leukemia who are prone to intestinal mucositis and infections.Methods. During 36 chemotherapy cycles, fecal samples were collected from pediatric patients with acute myeloid leukemia. Fecal bacterial populations were analyzed by polymerase chain reaction denaturing gradient gel electrophoresis fingerprinting. Fluorescent in situ hybridization analysis with specific bacterial oligonucleotide probes was used to quantify the fecal bacteria.Results. During chemotherapy treatment, the total number of bacteria in fecal samples was 109 per gram of dry weight feces, which was 100-fold lower than than in healthy control samples. Fluorescent in situ hybridization analysis showed that this decrease was the result of an up to 10,000-fold decrease in anaerobic bacteria, partly compensated for by a 100-fold increase in potentially pathogenic enterococci. Additional experiments showed that both prophylactic and therapeutic use of antibiotics could not sufficiently explain the tremendous changes in intestinal microbial composition. In vitro tests showed a direct bacteriostatic effect of chemotherapeutics.Conclusions. Patients with acute myeloid leukemia treated with chemotherapy and prophylactic antibiotics are unable to maintain colonization resistance because of a decrease in anaerobic bacteria and an increase in potentially pathogenic aerobic enterococci. We hypothesize that this disturbance in the balance between anaerobic and aerobic bacteria will further increase the risk of gram-positive aerobic infections among immunocompromised patients with cancer.

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.

Viridans group streptococci (VGS) are a well-known cause of infections in immunocompromised patients, accounting for severe morbidity and mortality. Streptococcus mitis group species ( Streptococcus mitis , Streptococcus pneumoniae , Streptococcus oralis ) are among the VGS most often encountered in clinical practice. Identifying the portal of entry for S. mitis group strains is crucial for interventions preventing bacterial translocation. Unfortunately, tracking the source of S. mitis group strains is dependent on a combination of extremely laborious and time-consuming cultivation and molecular techniques (enterobacterial repetitive intergenic consensus-PCR [ERIC-PCR]). To simplify this procedure, a PCR analysis with newly designed primers targeting the household gene glucose kinase ( gki ) was used in combination with denaturing gradient gel electrophoresis (DGGE). This gki -PCR-DGGE technique proved to be specific for S. mitis group strains. Moreover, these strains could be detected in samples comprised of highly diverse microbiota, without prior cultivation. To study the feasibility of this new approach, a pilot study was performed. This confirmed that the source of S. mitis group bacteremia in pediatric patients with acute myeloid leukemia could be tracked back to the throat in five out of six episodes of bacteremia, despite the fact that throat samples are polymicrobial samples containing multiple S. mitis group strains. In contrast, using the classical combination of cultivation techniques and ERIC-PCR, we could detect these strains in only two out of six cases, showing the superiority of the newly developed technique. The new gki -PCR-DGGE technique can track the source of S. mitis group strains in polymicrobial samples without prior cultivation. Therefore, it is a valuable tool in future epidemiological studies.

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.

Background: In this national multicentre study, we examined the safety of reducing antibiotics in selected paediatric cancer patients with febrile neutropenia.Methods: Patients with signs of a bacterial infection and/or abnormal vital signs indicating sepsis were considered high risk and received antibiotic therapy. Remaining patients were allocated to low-or medium risk, depending on their interleukin-8 level. Low-risk patients did not receive any antibiotics and were discharged from the hospital after having been afebrile for 12 h. Medium-risk patients were re-evaluated after 72 h of antibiotic treatment and, in selected patients, antibiotics were stopped.Results: Two hundred thirty-three febrile neutropenic episodes in 141 paediatric cancer patients were included in the study. Sixty-four episodes were classified high risk (28%), 122 medium risk (52%), and 47 (20%) low risk. In the medium-risk group, antibiotics were stopped after 72 h in 50 in 122 episodes (41%). Median duration of antibiotic treatment and hospital admission was significantly lower in low-and medium-risk episodes with early discharge. No failures were observed in the medium-risk group with early discharge. In the low-risk group, six failures were observed (12.8%), due to coagulase-negative staphylococci-positive blood cultures and recurrent fever.Conclusion: We showed that it is safe to shorten antibiotic treatment to 72 h in selected medium-risk patients with febrile neutropenia, regardless of the neutrophil count. The safety of withholding antibiotics in selected low-risk paediatric cancer patients with febrile neutropenia requires further investigation, using more suitable definitions for safety. Reduction in hospital admissions allows children with cancer more time at home and consequently improves their quality of life. (C) 2015 Elsevier Ltd. All rights reserved.

In children with cancer a well-known risk factor for cardiotoxicity is a high cumulative dose of anthracyclines, but little is known about cardiac function in low-dose anthracycline-treated survivors. Also, it is unclear if a safe anthracycline-dose exists at all.Cardiac function was assessed in 23 long-term ALL-survivors with a median follow-up of 22 years (range 19.5-24.5) post-treatment. Age at diagnosis and current age were 5.0 (2.0-14.0) and 29.0 (24.0-39.0) years. All 23 survivors were treated according to DCLSG protocol ALL-5, including 18-25 Gy cranial irradiation. Thirteen of them received 4 x 25 mg/m(2) daunorubicin by randomization. Cardiac evaluation included blood pressure measurement, echocardiography, and (24 h-) electrocardiogram. Results were compared with an earlier assessment at median 12 years post-treatment.None of the survivors had cardiac abnormalities. Cardiac status of daunorubicin-treated survivors showed no deterioration compared with the previous assessment in 1995. CONCLUSION AND IMPLICATION FOR CANCER SURVIVORS: After prolonged follow-up (more than 20 years post-treatment), ALL-survivors treated with low dose daunorubicin had no clinical relevant deterioration of cardiac function.

Purpose 1. To assess health-related quality of life (HRQoL) of adult long term (up to 20 years) and very long term (>20 years) childhood cancer survivors, compared to the HRQoL of an age matched Dutch population sample. 2. To evaluate the impact of cancer-related adverse late effects on the functional, psychological and social health of childhood cancer survivors. Method The RAND-36 was used to assess HRQoL in all adult (⩾18 years) survivors who had attended the long-term follow-up clinic since 1995. The survivors were divided into two groups based on the length of follow-up: Group LF (long term follow-up, follow-up ⩽20 years, n = 129) and Group VLF (very long-term follow-up, follow-up >20 years, n = 184). Data on diagnosis, treatment and complications were obtained from medical records. Late effects were graded using the CTCAEv3. Results The RAND-36 was completed by 313 (86.2%) out of 363 eligible patients. Except for higher scores on the subscale Bodily pain, LF patients did not differ significantly on the RAND-36 subscales from the population sample; VLF patients had significant lower scores on the subscales Physical functioning ( P = 0.003), Social functioning, Vitality and General health perception ( P P Conclusion Childhood cancer survivors who were diagnosed more than 20 years ago have lower scores on the RAND 36, and have significantly more severe late effects than those diagnosed more recently. Patients with longer follow-up are more likely to become lost to follow-up. Time has come to establish new models of care for adult childhood cancer survivors, which are more flexible and appropriate to the needs of adult childhood cancer survivors.

intellectual functioning (verbal, performance and full-scale IQ) in 43 children treated for acute lymphoblastic leukaemia (ALL) with chemotherapy-only was evaluated in a nationwide, prospective, sibling-controlled study. Intellectual assessment was performed at diagnosis and repeated shortly after cessation of 2 years treatment, including intrathecal and systemic chemotherapy. Using hierarchical regression analysis, patients' and siblings' (n = 27) scores were longitudinally analysed and compared to assess possible changes and differences over time. At both assessments, before and after treatment, patients showed average scores on intelligence tests compared to population norms. Longitudinal analysis and cross-sectional comparisons revealed no significant differences between patients and controls. Young patients showed a small relative decline, albeit not significant, on performance-IQ compared to healthy siblings. Despite intensive and potentially neurotoxic treatment, no evident negative effects on intelligence were found. However, it cannot be precluded that younger patients are at risk for a small decline in PIQ. (c) 2006 Elsevier Ltd. All rights reserved.

Langerhans cell histiocytosis (LCH) is a neoplastic disorder that results in clonal proliferation of cells with a Langerhans cell (LQ phenotype. The pathogenesis of LCH is still poorly understood. In the present study, serial analysis of gene expression (SAGE) was applied to LCs generated from umbilical cord blood CD34+ progenitor cells to identify LC-specific genes and the expression of these genes in LCH was investigated. Besides the expression of several genes known to be highly expressed in LCs and LCH such as CD1a, LYZ, and CD207, high expression of genes not previously reported to be expressed in LCs, such as GSN, MMP12, CCL17, and CCL22, was also identified. Further analysis of these genes by quantitative RT-PCR revealed high expression of FSCN1 and GSN in all 12 LCH cases analysed; of CD207, MMP12, CCL22, and CD1a in the majority of these cases; and CCL17 in three of the 12 cases. Immunohistochemistry confirmed protein expression in the majority of cases. The expression of MMP12 was most abundant in multi-system LCH, which is the LCH type with the worst prognosis. This suggests that expression of MMP12 may play a role in the progression of LCH. These data reveal new insight into the pathology of LCH and provide new starting points for further investigation of this clonal proliferative disorder. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.