Modulation of metabolism and cytotoxicity of cytosine arabinoside with N-(phosphon)-acetyl-L-aspartate in human leukemic blast cells and cell lines

Cytosine arabinoside (Ara-C) activation to cytosine arabinoside triphosphate (Ara-CTP) and subsequent incorporation into DNA is regulated by the pyrimidine nucleotides UTP, CTP and dCTP. Inhibition of the de novo synthesis of these pyrimidine nucleotides by N-(phosphon)-acetyl-L-aspartate (PALA) may enhance the cytotoxicity of Ara-C. We therefore studied the effect of PALA on Ara-C cytotoxicity and on Ara-CTP accumulation and incorporation into DNA on cell lines and patient samples, fifty micromolar PALA increased the growth inhibitory effect of Ara-C in U937 cells several fold both with pre- and coincubation. Ara-C cytotoxicity was not potentiated by PALA in HL60 cells. However, coincubation with PALA did not enhance Ara-CTP accumulation both in HL60 and U937 cells, nor affect Ara-C incorporation into DNA. Ara-C cytotoxicity to leukemic blast cells from 11 untreated patients with different types of leukemia was only modulated to a small extent by high PALA concentrations in only two cases. Ara-CTP accumulation in leukemic blast cells varied from non-detectable levels to 200 pmol/106 cells. Fifty micromolar PALA enhanced the accumulation of Ara-CTP significantly in only one patient with no apparent effect on UTP and CTP levels. Raising PALA to 500 μM decreased UTP and CTP levels to 50% but had no effect on Ara-CTP levels. In conclusion, modulation by PALA of Ara-C cytotoxicity and metabolism is limited in leukemic cells, both in culture and from patients. This suggests the possibility for selective modulation of other agents by PALA on non-hematological cells

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

Molecular Drug Imaging: 89Zr-Bevacizumab PET in Children with Diffuse Intrinsic Pontine Glioma

Predictive tools for guiding therapy in children with brain tumors are urgently needed. In this first molecular drug imaging study in children, we investigated whether bevacizumab can reach tumors in children with diffuse intrinsic pontine glioma (DIPG) by measuring the tumor uptake of 89Zr-labeled bevacizumab by PET. In addition, we evaluated the safety of the procedure in children and determined the optimal time for imaging. Methods: Patients received 89Zr-bevacizumab (0.1 mg/kg; 0.9 MBq/kg) at least 2 wk after completing radiotherapy. Whole-body PET/CT scans were obtained 1, 72, and 144 h after injection. All patients underwent contrast (gadolinium)-enhanced MRI. The biodistribution of 89Zr-bevacizumab was quantified as SUVs. Results: Seven DIPG patients (4 boys; 6-17 y old) were scanned without anesthesia. No adverse events occurred. Five of 7 primary tumors showed focal 89Zr-bevacizumab uptake (SUVs at 144 h after injection were 1.0-6.7), whereas no significant uptake was seen in the healthy brain. In 1 patient, multiple metastases all showed positive PET results. We observed inter- and intratumoral heterogeneity of uptake, and 89Zr-bevacizumab uptake was present predominantly (in 4/5 patients) within MRI contrast-enhanced areas, although 89Zr-bevacizumab uptake in these areas was variable. Tumor targeting results were quantitatively similar at 72 and 144 h after injection, but tumor-to-blood-pool SUV ratios increased with time after injection (P = 0.045). The mean effective dose per patient was 0.9 mSv/MBq (SD, 0.3 mSv/MBq). Conclusion: 89Zr-bevacizumab PET studies are feasible in children with DIPG. The data suggest considerable heterogeneity in drug delivery among patients and within DIPG tumors and a positive, but not 1:1, correlation between MRI contrast enhancement and 89Zr-bevacizumab uptake. The optimal time for scanning is 144 h after injection. Tumor 89Zr-bevacizumab accumulation assessed by PET scanning may help in the selection of patients with the greatest chance of benefit from bevacizumab treatment

Prediction of Bloodstream Infection in Pediatric Acute Leukemia by Microbiota and Volatile Organic Compounds Analysis

Introduction: Bloodstream infections (BSIs) cause treatment-related mortality in pediatric acute leukemia. We explored the potential of intestinal microbiota and fecal volatile organic compounds (VOCs) analyses to predict BSI. Methods: In this case-control study, fecal samples of pediatric acute leukemia patients were collected. Microbiota composition and fecal VOC profiles of BSI cases and matched non-BSI controls were compared. Results: In total, 6 patients were included, of which 1 developed BSI and 1 neutropenic fever. Both showed reduced microbial diversity and stability of Bacteroidetes. In the BSI case, Pantoea was identified 15 days before BSI. Significant differences in fecal VOC profiles were measured between the case and controls. Conclusion: Microbiota and fecal VOC could serve as biomarkers to predict BSI in pediatric leukemia

Prediction of Bloodstream Infection in Pediatric Acute Leukemia by Microbiota and Volatile Organic Compounds Analysis

Introduction: Bloodstream infections (BSIs) cause treatment-related mortality in pediatric acute leukemia. We explored the potential of intestinal microbiota and fecal volatile organic compounds (VOCs) analyses to predict BSI. Methods: In this case-control study, fecal samples of pediatric acute leukemia patients were collected. Microbiota composition and fecal VOC profiles of BSI cases and matched non-BSI controls were compared. Results: In total, 6 patients were included, of which 1 developed BSI and 1 neutropenic fever. Both showed reduced microbial diversity and stability of Bacteroidetes. In the BSI case, Pantoea was identified 15 days before BSI. Significant differences in fecal VOC profiles were measured between the case and controls. Conclusion: Microbiota and fecal VOC could serve as biomarkers to predict BSI in pediatric leukemia

18 F-FDG PET standard uptake values of the normal pons in children: Establishing a reference value for diffuse intrinsic pontine glioma

Background: Positron emission tomography (PET) scanning with [18 F]fluorodeoxyglucose (18 F-FDG) is a useful diagnostic and prediction tool in brain tumors, but its value in childhood diffuse intrinsic pontine glioma (DIPG) is still unclear. For interpretation of 18 F-FDG PET results in DIPG, uptake values of the normal pons of children of increasing ages are mandatory. The aim of this study was to determine 18 F-FDG standard uptake value ratios (SUVr) of the normal pons and to compare these to those of DIPG. Methods: We studied 36 subjects with a normal, non-affected pons (aged 5 to 23 years) and 6 patients with DIPG (aged 4 to 17 years) who underwent 18 F-FDG PET scanning. Magnetic resonance imaging (MRI) was co-registered to define the regions of interest. SUVr and SUVrmax for the pons/cerebellum (SUVrp/c) and the pons/occipital lobe (SUVrp/o) were calculated. Independent-samples t tests and Mann-Whitney U tests were used to compare the mean SUVr and Pearson's test for correlations. Results: For the normal pons, mean SUVrp/c and SUVrp/o were 0.65 (±0.054) and 0.51 (±0.056), respectively. No significant correlations were found between the SUVr of the normal pons and sex, age, nor pontine volume. A modest but statistically significant correlation was found between SUVr and post-injection time acquisition timing. For DIPG, mean SUVrp/c and SUVrp/o were 0.74 (±0.20) and 0.65 (±0.30), respectively, while mean SUVrp(max)/c and SUVrp(max)/o were 1.95 (±0.48) and 1.81 (±0.20), respectively. Conclusion: The SUVr of the unaffected pons are strikingly constant between children, irrespective of sex and age, and can therefore be well used as a reference value for 18 F-FDG PET studies in DIPG. © 2014 Jansen et al.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

RNA-based FLT3-ITD allelic ratio is associated with outcome and ex vivo response to FLT3 inhibitors in pediatric AML

Controversy exists whether internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-internal tandem duplication [ITD]) allelic ratio (AR) and/or length of the ITD should be taken into account for risk stratification of pediatric acute myeloid leukemia (AML) and whether it should be measured on RNA or DNA. Moreover, the ITD status may be of relevance for selecting patients eligible for FLT3 inhibitors. Here, we included 172 pediatric AML patients, of whom 36 (21%) harbored FLT3-ITD as determined on both RNA and DNA. Although there was a good correlation between both parameters AR(spearman) = 0.62 (95% confidence interval, 0.22-0.87) and ITDlength(spearman) = 0.98 (95% confidence interval, 0.90-1.00), only AR >= 0.5 and length >= 48 base pairs (bps) based on RNA measurements were significantly associated with overall survival (AR: P-logrank = .008; ITDlength: P-logrank = .011). In large ITDs (>156 bp on DNA) a remarkable 90-bp difference exists between DNA and RNA, including intron 14, which is spliced out in RNA. Ex vivo exposure (n = 30) to FLT3 inhibitors, in particular to the FLT3-specific inhibitor gilteritinib, showed that colony-forming capacity was significantly more reduced in FLT3-ITD-AR >= 0.5 compared with ITD-AR-low and ITD2 patient samples (P <.001). RNA-based FLT3-ITD measurements are recommended for risk stratification, and the relevance of AR regarding eligibility for FLT3-targeted therapy warrants further study