Folates provoke cellular efflux and drug resistance of substrates of the multidrug resistance protein 1 (MRP1)

Cellular folate concentration was earlier reported to be a critical factor in the activity and expression of the multidrug resistance protein MRP1 (ABCC1). Since MRP1 mediates resistance to a variety of therapeutic drugs, we investigated whether the cellular folate concentration inf

Effect of polymorphisms in folate-related genes on in vitro methotrexate sensitivity in pediatric acute lymphoblastic leukemia

We studied whether common polymorphisms in genes involved in folate metabolism affect methotrexate (MTX) sensitivity. Ex vivo MTX sensitivity of lymphoblasts obtained from pediatric patients with acute lymphoblastic leukemia (ALL; n = 157) was determined by the in situ thymidylate synthase inhibition assay after either continuous (21 hours; TSI(50, cont)) or short-term (3 hours; TSI(50, short)) MTX exposure. DNA was isolated from lymphoblasts obtained from cytospin slides. Polymorphisms in methylenetetrahydrofolate reductase (MTHFR 677C>T, MTHFR 1298A>C), methionine synthase (MTR 2756A>G), methionine synthase reductase (MTRR 66A>G), methylenetetrahydrofolate dehydrogenase (MTHFD1 1958G>A), serine hydroxymethyl transferase (SHMT1 1420C>T), thymidylate synthase (TS 2R3R), and the reduced folate carrier (RFC 80G>A) were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or real-time PCR. Patients with the MTHFR 1298AC variant or the MTRR 66 G-allele showed decreased in vitro MTX sensitivity measured under both test conditions. SHMT1 1420TT homozygotes only showed decreased MTX sensitivity in the TSI(50, cont). In conclusion, polymorphisms in the folate-related genes MTHFR, MTRR, and SHMT1 are related to MTX resistance in pediatric patients with ALL

The neurovascular unit in diffuse intrinsic pontine gliomas

Aims: Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a median overall survival of eleven months. Lack of chemotherapy efficacy may be related to an intact blood-brain barrier (BBB). In this study we aim to investigate the neurovascular unit (NVU) in DIPG patients. Methods: DIPG biopsy (n = 4) and autopsy samples (n = 6) and age-matched healthy pons samples (n = 20) were immunohistochemically investigated for plasma protein extravasation, and the expression of tight junction proteins claudin-5 and zonula occludens-1 (ZO-1), basement membrane component laminin, pericyte marker PDGFR-β, and efflux transporters P-gp and BCRP. The mean vascular density and diameter were also assessed. Results: DIPGs show a heterogeneity in cell morphology and evidence of BBB leakage. Both in tumor biopsy and autopsy samples, expression of claudin-5, ZO-1, laminin, PDGFR-β and P-gp was reduced compared to healthy pontine tissues. In DIPG autopsy samples, vascular density was lower compared to healthy pons. The density of small vessels (<10 µm) was significantly lower (P<0.001), whereas the density of large vessels (≥10 µm) did not differ between groups (P = 0.404). The median vascular diameter was not significantly different: 6.21 µm in DIPG autopsy samples (range 2.25-94.85 µm), and 6.26 µm in controls (range 1.17-264.77 µm). Conclusion: Our study demonstrates evidence of structural changes in the NVU in DIPG patients, both in biopsy and autopsy samples, as well as a reduced vascular density in end-stage disease. Adding such a biological perspective may help to better direct future treatment choices for DIPG patients

PARP inhibition sensitizes childhood high grade glioma, medulloblastoma and ependymoma to radiation

Poly ADP-ribose polymerase (PARP) is a protein involved in single strand break repair. Recently, PARP inhibitors have shown considerable promise in the treatment of several cancers, both in monotherapy and in combination with cytotoxic agents. Synthetic lethal action of PARP inhibitors has been observed in tumors with mutations in double strand break repair pathways. In addition, PARP inhibition potentially enhances sensitivity of tumor cells to DNA damaging agents, including radiotherapy. Aim of this study is to determine the radiosensitizing properties of the PARP inhibitor Olaparib in childhood medulloblastoma, ependymoma and high grade glioma (HGG). Increased PARP1 expression was observed in medulloblastoma, ependymoma and HGG, as compared to non-neoplastic brain tissue. Pediatric high grade glioma, medulloblastoma and ependymoma gene expression profiling revealed that high PARP1 expression is associated with poor prognosis. Cell growth inhibition assays with Olaparib resulted in differential sensitivity, with IC50 values ranging from 1.4 to 8.4 μM, irrespective of tumor type and PARP1 protein expression. Sensitization to radiation was observed in medulloblastoma, ependymoma and HGG cell lines with subcytotoxic concentrations of Olaparib, which coincided with persistence of double strand breaks. Combining PARP inhibitors with radiotherapy in clinical studies in childhood high grade brain tumors may improve therapeutic outcome

Use of granulocyte colony-stimulating factor and risk of relapse in pediatric patients treated for acute myeloid leukemia according to NOPHO-AML 2004 and DB AML-01

Background Supportive-care use of granulocyte colony-stimulating factor (G-CSF) in pediatric acute myeloid leukemia (AML) remains controversial due to a theoretical increased risk of relapse and limited impact on neutropenic complications. We describe the use of G-CSF in patients treated according to NOPHO-AML 2004 and DB AML-01 and investigated associations with relapse. Procedure Patients diagnosed with de novo AML completing the first week of therapy and not treated with hematopoietic stem cell transplantation in the first complete remission were included (n = 367). Information on G-CSF treatment after each course (yes/no) was registered prospectively in the study database and detailed information was gathered retrospectively from each center. Descriptive statistics were used to describe G-CSF use and Cox regression to assess the association between G-CSF and risk of relapse. Results G-CSF as supportive care was given to 128 (35%) patients after 268 (39%) courses, with a large variation between centers (0-93%). The use decreased with time-the country-adjusted odds ratio was 0.8/diagnostic year (95% confidence interval [CI] 0.7-0.9). The median daily dose was 5 mu g/kg (range 3-12 mu g/kg) and the median cumulative dose was 75 mu g/kg (range 7-1460 mu g/kg). Filgrastim was used in 82% of G-CSF administrations and infection was the indication in 44% of G-CSF administrations. G-CSF was associated with increased risk of relapse-the adjusted hazard ratio was 1.5 (95% CI 1.1-2.2). Conclusions G-CSF as supportive care was used in a third of patients, and use decreased with time. Our results indicate that the use of G-CSF may be associated with an increased risk of relapse.Peer reviewe

Causes of early death and treatment-related death in newly diagnosed pediatric acute myeloid leukemia:Recent experiences of the Dutch Childhood Oncology Group

Background: With the current more effective treatment regimens for pediatric acute myeloid leukemia (AML), research on early death (ED), treatment-related mortality (TRM), and toxicity becomes increasingly important. The aim of this study was to give an overview of the frequency, clinical features, and risk factors associated with ED and TRM in first complete remission (CR1) during the last three consecutive treatment protocols of the Dutch Childhood Oncology Group (DCOG) between 1998 and 2014. Methods: Incidence and risk factors associated with ED and TRM in CR1 were retrospectively studied in 245 patients treated according to the Dutch ANLL-97/AML-12 (n = 118), AML-15 (n = 60), or DB AML-01 (n = 67) protocols. Results: The incidence of ED was, respectively, 5.1%, 6.7%, and 3.0% excluding deaths before treatment (P = NS), and 7.4%, 11.1%, and 4.4% including deaths before the onset of treatment. Severe underweight at initial diagnosis was significantly associated with more frequent ED. When relapse was included as a competing risk, cumulative incidence of death in CR1 were 5.9%, 5.0%, and 4.6% for ANLL97, AML15, and DB01, respectively (P = NS). The most important cause of TRM included infectious and SCT-related complications. Conclusion: We report relatively stable rates of ED and TRM in CR1 in the latest completed DCOG protocols for newly diagnosed AML patients. The most important causes of TRM were SCT- or infection-related, warranting further evaluation and awareness

Attenuated AMPA Receptor Expression Allows Glioblastoma Cell Survival in Glutamate-Rich Environment

Background: Glioblastoma multiforme (GBM) cells secrete large amounts of glutamate that can trigger AMPA-type glutamate receptors (AMPARs). This commonly results in Na+ and Ca2+-permeability and thereby in excitotoxic cell death of the surrounding neurons. Here we investigated how the GBM cells themselves survive in a glutamate-rich environment. Methods and Findings: In silico analysis of published reports shows down-regulation of all ionotropic glutamate receptors in GBM as compared to normal brain. In vitro, in all GBM samples tested, mRNA expression of AMPAR subunit GluR1, 2 and 4 was relatively low compared to adult and fetal total brain mRNA and adult cerebellum mRNA. These findings were in line with primary GBM samples, in which protein expression patterns were down-regulated as compared to the normal tissue. Furthermore, mislocalized expression of these receptors was found. Sequence analysis of GluR2 RNA in primary and established GBM cell lines showed that the GluR2 subunit was found to be partly unedited. Conclusions: Together with the lack of functional effect of AMPAR inhibition by NBQX our results suggest that down-regulation and afunctionality of AMPARs, enable GBM cells to survive in a high glutamate environment without going into excitotoxic cell death themselves. It can be speculated that specific AMPA receptor inhibitors may protect normal neurons against the high glutamate microenvironment of GBM tumor

Harnessing gene expression profiles for the identification of ex vivo drug response genes in pediatric acute myeloid leukemia

Novel treatment strategies are of paramount importance to improve clinical outcomes in pediatric AML. Since chemotherapy is likely to remain the cornerstone of curative treatment of AML, insights in the molecular mechanisms that determine its cytotoxic effects could aid further treatment optimization. To assess which genes and pathways are implicated in tumor drug resistance, we correlated ex vivo drug response data to genome-wide gene expression profiles of 73 primary pediatric AML samples obtained at initial diagnosis. Ex vivo response of primary AML blasts towards cytarabine (Ara C), daunorubicin (DNR), etoposide (VP16), and cladribine (2-CdA) was associated with the expression of 101, 345, 206, and 599 genes, respectively (p < 0.001, FDR 0.004–0.416). Microarray based expression of multiple genes was technically validated using qRT-PCR for a selection of genes. Moreover, expression levels of BRE, HIF1A, and CLEC7A were confirmed to be significantly (p < 0.05) associated with ex vivo drug response in an independent set of 48 primary pediatric AML patients. We present unique data that addresses transcriptomic analyses of the mechanisms underlying ex vivo drug response of primary tumor samples. Our data suggest that distinct gene expression profiles are associated with ex vivo drug response, and may confer a priori drug resistance in leukemic cells. The described associations represent a fundament for the development of interventions to overcome drug resistance in AML, and maximize the benefits of current chemotherapy for sensitive patients

Associations between pretherapeutic body mass index, outcome, and cytogenetic abnormalities in pediatric acute myeloid leukemia

Background Associations between body mass index (BMI), outcome, and leukemia-related factors in children with acute myeloid leukemia (AML) remain unclear. We investigated associations between pretherapeutic BMI, cytogenetic abnormalities, and outcome in a large multinational cohort of children with AML. Methods We included patients, age 2-17 years, diagnosed with de novo AML from the five Nordic countries (2004-2016), Hong Kong (2007-2016), the Netherlands and Belgium (2010-2016), and Canada and USA (1995-2012). BMI standard deviations score for age and sex was calculated and categorized according to the World Health Organization. Cumulative incidence functions, Kaplan-Meier estimator, Cox regression, and logistic regression were used to investigate associations. Results In total, 867 patients were included. The median age was 10 years (range 2-17 years). At diagnosis, 32 (4%) were underweight, 632 (73%) were healthy weight, 127 (15%) were overweight, and 76 (9%) were obese. There was no difference in relapse risk, treatment-related mortality or overall mortality across BMI groups. The frequency of t(8;21) and inv(16) increased with increasing BMI. For obese patients, the sex, age, and country adjusted odds ratio of having t(8;21) or inv(16) were 1.9 (95% confidence interval (CI) 1.1-3.4) and 2.8 (95% CI 1.3-5.8), respectively, compared to healthy weight patients. Conclusions This study did not confirm previous reports of associations between overweight and increased treatment-related or overall mortality in children. Obesity was associated with a higher frequency of t(8;21) and inv(16). AML cytogenetics appear to differ by BMI status.Peer reviewe

Clinical, radiologic, pathologic, and molecular characteristics of long-term survivors of diffuse intrinsic pontine glioma (DIPG): a collaborative report from the International and European Society for Pediatric Oncology DIPG registries

Purpose Diffuse intrinsic pontine glioma (DIPG) is a brainstem malignancy with a median survival of &lt; 1 year. The International and European Society for Pediatric Oncology DIPG Registries collaborated to compare clinical, radiologic, and histomolecular characteristics between short-term survivors (STSs) and long-term survivors (LTSs). Materials and Methods Data abstracted from registry databases included patients from North America, Australia, Germany, Austria, Switzerland, the Netherlands, Italy, France, the United Kingdom, and Croatia. Results Among 1,130 pediatric and young adults with radiographically confirmed DIPG, 122 (11%) were excluded. Of the 1,008 remaining patients, 101 (10%) were LTSs (survival ≥ 2 years). Median survival time was 11 months (interquartile range, 7.5 to 16 months), and 1-, 2-, 3-, 4-, and 5-year survival rates were 42.3% (95% CI, 38.1% to 44.1%), 9.6% (95% CI, 7.8% to 11.3%), 4.3% (95% CI, 3.2% to 5.8%), 3.2% (95% CI, 2.4% to 4.6%), and 2.2% (95% CI, 1.4% to 3.4%), respectively. LTSs, compared with STSs, more commonly presented at age &lt; 3 or &gt; 10 years (11% v 3% and 33% v 23%, respectively; P &lt; .001) and with longer symptom duration ( P &lt; .001). STSs, compared with LTSs, more commonly presented with cranial nerve palsy (83% v 73%, respectively; P = .008), ring enhancement (38% v 23%, respectively; P = .007), necrosis (42% v 26%, respectively; P = .009), and extrapontine extension (92% v 86%, respectively; P = .04). LTSs more commonly received systemic therapy at diagnosis (88% v 75% for STSs; P = .005). Biopsies and autopsies were performed in 299 patients (30%) and 77 patients (10%), respectively; 181 tumors (48%) were molecularly characterized. LTSs were more likely to harbor a HIST1H3B mutation (odds ratio, 1.28; 95% CI, 1.1 to 1.5; P = .002). Conclusion We report clinical, radiologic, and molecular factors that correlate with survival in children and young adults with DIPG, which are important for risk stratification in future clinical trials