Severe Hepatopathy in National Wilms Tumor Studies 3-5: Prevalence, Clinical Features, and Outcomes After Reintroduction of Chemotherapy.

PURPOSE: The safety of reintroducing chemotherapy in the pediatric renal tumor setting after severe hepatopathy (SH), including sinusoidal obstruction syndrome (SOS), is uncertain. We describe the incidence, severity, outcomes, and impact on subsequent treatment for patients with SH from National Wilms Tumor Study (NWTS) protocols 3-5. PATIENTS AND METHODS: Archived charts for patients enrolled on NWTS 3-5 who met study inclusion criteria for SH by using established hepatopathy grading scales and clinical criteria were reviewed for demographics, tumor characteristics, radio- and chemotherapy details, SH-related dose modifications, and oncologic outcomes. Genomic analysis for candidate polymorphisms associated with SH was performed in 14 patients. RESULTS: Seventy-one of 8,862 patients (0.8%) met study inclusion criteria. The median time from therapy initiation to SH was 51 days (range, 2-293 days). Sixty percent received radiotherapy, and 56% had right-sided tumors. Grade 1-4 thrombocytopenia was noted in 70% at initial occurrence of SH (median 22,000/microliter). Among 69 of 71 children with SH occurring before the end of therapy (EOT) and post-SH treatment information available, chemotherapy was delayed posthepatopathy for 65% (69% of these at a reduced dose), continued without delay for 20% (57% of these at reduced dose), and stopped completely for 15% (4 of 10 of whom died of SH). Overall, 42% of patients with dose reductions achieved full dose by EOT. The five-year post-SH event-free survival for patients who continued therapy was 89% (95% CI, 81 to 98), with no significant differences by whether delay or dose reduction occurred. We identified no SH-associated pharmacogenomic polymorphism. CONCLUSION: The incidence of SH on NWTS 3-5 was low; many had associated severe thrombocytopenia. Careful reintroduction of chemotherapy appeared to be feasible for the majority of patients who developed severe chemotherapy- and/or radiotherapy-induced liver toxicity.

Systematic Review of Pharmacogenetic Factors That Influence High-Dose Methotrexate Pharmacokinetics in Pediatric Malignancies.

Methotrexate (MTX) is a mainstay therapeutic agent administered at high doses for the treatment of pediatric and adult malignancies, such as acute lymphoblastic leukemia, osteosarcoma, and lymphoma. Despite the vast evidence for clinical efficacy, high-dose MTX displays significant inter-individual pharmacokinetic variability. Delayed MTX clearance can lead to prolonged, elevated exposure, causing increased risks for nephrotoxicity, mucositis, seizures, and neutropenia. Numerous pharmacogenetic studies have investigated the effects of several genes and polymorphisms on MTX clearance in an attempt to better understand the pharmacokinetic variability and improve patient outcomes. To date, several genes and polymorphisms that affect MTX clearance have been identified. However, evidence for select genes have conflicting results or lack the necessary replication and validation needed to confirm their effects on MTX clearance. Therefore, we performed a systematic review to identify and then summarize the pharmacogenetic factors that influence high-dose MTX pharmacokinetics in pediatric malignancies. Using the PRISMA guidelines, we analyzed 58 articles and 24 different genes that were associated with transporter pharmacology or the folate transport pathway. We conclude that there is only one gene that reliably demonstrates an effect on MTX pharmacokinetics: SLCO1B1.

Genetic variants associated with methotrexate-induced mucositis in cancer treatment: A systematic review and meta-analysis.

Methotrexate (MTX), an important chemotherapeutic agent, is often accompanied with mucositis. The occurrence and severity are unpredictable and show large interindividual variability. In this study, we review and meta-analyze previously studied genetic variants in relation to MTX-induced mucositis. We conducted a systematic search in Medline and Embase. We included genetic association studies of MTX-induced mucositis in cancer patients. A meta-analysis was conducted for single nucleotide polymorphisms (SNPs) for which at least two studies found a statistically significant association. A total of 34 SNPs were associated with mucositis in at least one study of the 57 included studies. Two of the seven SNPs included in our meta-analysis were statistically significantly associated with mucositis: MTHFR c.677C > T (recessive, grade ≥3 vs grade 0-2, OR 2.53, 95 %CI [1.48-4.32], False Discovery Rate[FDR]-corrected p-value 0.011) and MTRR c.66A > G (overdominant, grade ≥1 vs grade 0, OR 2.08, 95 %CI [1.16-3.73], FDR-corrected p-value 0.042).

Global DNA (hydroxy)methylation is stable over time under several storage conditions and temperatures.

BACKGROUND: Epigenetic markers are often quantified and related to disease in stored samples. While, effects of storage on stability of these markers have not been thoroughly examined. In this longitudinal study, we investigated the influence of storage time, material, temperature, and freeze-thaw cycles on stability of global DNA (hydroxy)methylation. METHODS: EDTA blood was collected from 90 individuals. Blood (n = 30, group 1) and extracted DNA (n = 30, group 2) were stored at 4°C, -20°C and -80°C for 0, 1 (endpoint blood 4°C), 6, 12 or 18 months. Additionally, freeze-thaw cycles of blood and DNA samples (n = 30, group 3) were performed over three days. Global DNA methylation and hydroxymethylation (mean ± SD) were quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with between-run precision of 2.8% (methylation) and 6.3% (hydroxymethylation). Effects on stability were assessed using linear mixed models. RESULTS: global DNA methylation was stable over 18 months in blood at -20°C and -80°C and DNA at 4°C and -80°C. However, at 18 months DNA methylation from DNA stored at -20°C relatively decreased -6.1% compared to baseline. Global DNA hydroxymethylation was more stable in DNA samples compared to blood, independent of temperature (p = 0.0131). Stability of global DNA methylation and hydroxymethylation was not affected up to three freeze - thaw cycles. CONCLUSION: Global DNA methylation and hydroxymethylation stored as blood and DNA can be used for epigenetic studies. The relevance of  small differences occuring during storage depend on the expected effect size and research question.

Clinical Characteristics of Invasive Fungal Infections in Pediatric Oncology Patients With Solid Tumors.

Invasive fungal infections (IFIs) are an important cause of morbidity and mortality in children with cancer. Studies on the clinical characteristics of IFI in children with solid tumors are limited. This Dutch retrospective cohort study reviewed the medical records of 61 children with solid tumors to analyze the clinical characteristics during their full treatment period. Seven IFI episodes were reported in 6/61 patients (10%), all diagnosed with intermediate-risk or high-risk Wilms tumor or neuroblastoma. Larger studies are necessary to reveal the determinants of IFI in this group of patients and the value of fungal prophylaxis.

Changes in intracellular folate metabolism during high-dose methotrexate and Leucovorin rescue therapy in children with acute lymphoblastic leukemia.

BACKGROUND: Methotrexate (MTX) is an important anti-folate agent in pediatric acute lymphoblastic leukemia (ALL) treatment. Folinic acid rescue therapy (Leucovorin) is administered after MTX to reduce toxicity. Previous studies hypothesized that Leucovorin could 'rescue' both normal healthy cells and leukemic blasts from cell death. We assessed whether Leucovorin is able to restore red blood cell folate levels after MTX. METHODS: We prospectively determined erythrocyte folate levels (5-methyltetrahydrofolate (THF) and non-methyl THF) and serum folate levels in 67 children with ALL before start (T0) and after stop (T1) of HD-MTX and Leucovorin courses. RESULTS: Erythrocyte folate levels increased between T0 and T1 (mean ± SD: 416.7 ± 145.5 nmol/L and 641.2 ± 196.3 nmol/L respectively, p<0.001). This was due to an increase in 5-methyl THF levels (mean increase: 217.7 ± 209.5 nmol/L, p<0.001), whereas non-methyl THF levels did not change (median increase: 0.6 nmol/L [-9.9-11.1], p = 0.676). Serum folate levels increased between T0 and T1 (median increase: 29.2 nmol/L [32.9-74.0], p<0.001). Results were not significantly affected by age, sex, ALL immunophenotype and MTHFR c.677C>T genotype. CONCLUSION: Intracellular folate levels accumulate after HD-MTX and Leucovorin therapy in children with ALL, suggesting that Leucovorin restores the intracellular folate pool. Future studies are necessary to assess concomitant lower uptake of MTX.

Frequency and Determinants of Invasive Fungal Infections in Children With Solid and Hematologic Malignancies in a Nonallogeneic Stem Cell Transplantation Setting: A Narrative Review.

Invasive fungal infections (IFIs) are an important cause of morbidity and mortality in children with cancer. An overview of studies on the frequency and determinants of IFI in pediatric oncology patients in nonallogeneic stem cell transplantation settings is lacking. We performed a literature review in Pubmed and Embase, and included 13 prospective and 23 retrospective studies. The IFI frequency (proven/probable based on EORTC criteria) in nonallogeneic stem cell transplantation pediatric cancer patients ranged between 1.0% and 38.0%, with the highest frequencies reported in hematologic malignancies. The most common fungal species seen in the studied population was Candida, followed by Aspergillus. IFI are not well investigated in solid tumor patients. Significant recurrent determinants from univariate analysis were the diagnosis acute myeloid leukemia, (prolonged) neutropenia and an older age (above 10 years). The only 2 significant determinants based on multivariate analysis were the preceding number of days of broad-spectrum antibiotics (odds ratio, 1.05; 95% confidence interval, 1.02-1.07; P=0.0006) and the number of days of corticosteroids (odds ratio, 1.05; 95% confidence interval, 1.02-1.09; P=0.005), that were both based on a group of acute myeloid leukemia patients only. Future studies are necessary to determine the frequency and determinants of IFI in pediatric oncology including a representative number of solid tumor patients.

The role of genetic polymorphisms in the thymidylate synthase (TYMS) gene in methotrexate-induced oral mucositis in children with acute lymphoblastic leukemia.

OBJECTIVE: Methotrexate (MTX) is an important drug in the treatment of pediatric acute lymphoblastic leukemia (ALL). MTX is cytotoxic as it impairs DNA and RNA synthesis by inhibiting the enzymes dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS). The association between genetic variants within the TYMS gene and MTX-induced toxicity has been studied, but results are inconsistent. We determined the role of three previously described variants within the TYMS gene and MTX-induced oral mucositis in a prospective cohort of Dutch children with ALL and performed a meta-analysis of the previous results. MATERIALS AND METHODS: We analyzed the presence of a 28-base pair tandem repeat (rs34743033; 2R3R), a single nucleotide polymorphism present within the 28-base pair repeat on the 3R allele (rs2853542; 3RG>C) and a 6-base pair deletion (rs15126436; TTAAAG) within the TYMS gene in germline DNA of 117 pediatric patients with ALL. Oral mucositis was defined as grade≥3 according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v.3.0. Data were analyzed for the individual rs34743033 (2R3R) and rs151264360 (6 bp deletion) polymorphisms, whereas rs2853542 (3RG>C) was combined with rs34743033 (2R3R) and analyzed according to predicted expression levels of TYMS: low expression (2R/2R, 2R/3RC and 3RC/3RC), median expression (2R/3RG and 3RC/3RG) and high expression (3RG/3RG). We performed a meta-analysis of the current literature on these polymorphisms in relation to oral mucositis using a fixed effects model. RESULTS: The 2R2R genotype (rs34743033) was not significantly associated with developing MTX-induced oral mucositis compared with the 2R3R/3R3R genotypes, which was confirmed in a meta-analysis [odds ratio (OR): 1.17 (0.62-2.19)]. Patients carrying the low-expression TYMS genotype (2R2R, 2R3RC, 3RC3RC) had an increased odds of developing MTX-induced oral mucositis [OR: 2.42 (0.86-6.80)], which did not reach statistical significance. The 6-bp deletion [rs151264360, OR: 0.79 (0.20-3.19)] was not associated with the development of MTX-induced oral mucositis. CONCLUSION: The TYMS 6-bp deletion and 2R3R polymorphism were not associated with MTX-induced oral mucositis. Validation studies in prospective cohorts are necessary to assess the possible role of the low-expression TYMS genotypes in relation to MTX-induced oral mucositis.

Global methylation in relation to methotrexate-induced oral mucositis in children with acute lymphoblastic leukemia.

BACKGROUND: Children with acute lymphoblastic leukemia (ALL) often suffer from toxicity of chemotherapeutic drugs such as Methotrexate (MTX). Previously, we reported that 20% of patients receiving high-dose MTX developed oral mucositis. MTX inhibits folate metabolism, which is essential for DNA methylation. We hypothesize that MTX inhibits DNA methylation, which results into adverse effects. We studied DNA methylation markers during high-dose methotrexate treatment in pediatric acute lymphoblastic leukemia (ALL) in relation to developing oral mucositis. MATERIALS & METHODS: S-Adenosyl-Methionine (SAM) and S-Adenosyl-Homocysteine (SAH) levels and LINE1 DNA methylation were measured prospectively before and after high-dose methotrexate (HD-MTX 4 x 5g/m2) therapy in 82 children with ALL. Methotrexate-induced oral mucositis was registered prospectively. Oral mucositis (grade ≥ 3 National Cancer Institute Criteria) was used as clinical endpoint. RESULTS: SAM levels decreased significantly during methotrexate therapy (-16.1 nmol/L (-144.0 -+46.0), p<0.001), while SAH levels and the SAM:SAH ratio did not change significantly. LINE1 DNA methylation (+1.4% (-1.1 -+6.5), p<0.001) increased during therapy. SAM and SAH levels were not correlated to LINE1 DNA methylation status. No association was found between DNA methylation markers and developing oral mucositis. CONCLUSIONS: This was the first study that assessed DNA methylation in relation to MTX-induced oral mucositis in children with ALL. Although global methylation markers did change during methotrexate therapy, methylation status was not associated with developing oral mucositis.

The miR-1206 microRNA variant is associated with methotrexate-induced oral mucositis in pediatric acute lymphoblastic leukemia.

Five-year survival rates of pediatric acute lymphoblastic leukemia (ALL) have reached 90% in the developed countries. However, toxicity because of methotrexate (MTX) occurs frequently. Variety in the occurrence of toxicity is partly determined by single nucleotide polymorphisms (SNPs) in coding regions. Recently, five SNPs in non-coding pre-microRNAs and microRNA processing (miRNA) genes were identified in association with MTX-induced oral mucositis. This study aimed to replicate the association of these miRNA variants in relation to MTX-induced oral mucositis in a prospective childhood ALL cohort. Three out of five SNPs with a minor allele frequency more than 0.15 [CCR4-NOT transcription complex (CNOT4) rs3812265, miR-1206 rs2114358, miR-2053 rs10505168] were analyzed in 117 pediatric ALL patients treated with 5 g/m MTX (DCOG ALL-10). Oral mucositis was defined as grade more than or equal to 3 according to the National Cancer Institute criteria. rs2114358 in miR-1206 was associated with oral mucositis [odds ratio (OR): 3.6; 95% confidence interval (CI): 1.1-11.5], whereas we did not confirm the association of CNOT4 rs3812265 (OR: 0.69; 95% CI: 0.27-1.80) and miR-2053 rs10505168 (OR: 2.50; 95% CI: 0.76-8.24). Our results replicate the association between rs2114358 in miR-1206 and MTX-induced oral mucositis in childhood ALL. Genetic variation in miR-1206 has potential as a novel biomarker to predict MTX-induced toxicity.

Neuro-imaging findings in infants with congenital cytomegalovirus infection: relation to trimester of infection.

BACKGROUND: Congenital cytomegalovirus (cCMV) infection early in pregnancy may result in major disabilities. Cerebral abnormalities detected using cranial ultrasound (cUS) and magnetic resonance imaging (MRI) have been related to neurological sequelae. OBJECTIVE: To evaluate the additional value of MRI and assess the relationship between time of infection during pregnancy and outcome in infants with cCMV infection. METHODS AND STUDY DESIGN: Demographic and clinical data were collected in infants with cCMV infection (1992-2013). Trimester of infection, neuro-imaging results and outcome were reviewed. Cerebral abnormalities were categorized into none, mild (lenticulostriate vasculopathy, germinolytic cysts, high signal intensity on T2-weighted images) and severe (migrational disorder, ventriculomegaly, cerebellar hypoplasia). Results were statistically analysed. RESULTS: Thirty-six infants were eligible for analysis. cUS was performed in all and cranial MRI in 20 infants. Migrational disorders were only diagnosed using MRI (p < 0.01). In 17 infants trimester of infection was ascertained. Seven out of 10 infants infected during the first trimester had severe abnormalities on cUS (5 confirmed on MRI) and adverse sequelae; 3 had no/mild abnormalities on cUS/MRI and normal outcome. Two out of 3 infants infected during the second trimester with no/mild abnormalities on cUS/MRI had normal outcome; 1 with mild cUS and MRI abnormalities developed sensorineural hearing loss. Four infants infected during the third trimester with no/mild abnormalities on cUS/MRI had normal outcome. CONCLUSION: Infants with a first trimester cCMV infection are most at risk of severe cerebral abnormalities and neurological sequelae. MRI, and not cUS, enables an early diagnosis of migrational disorders, which can improve prediction of outcome.