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.

Genetic variation and bone mineral density in long-term adult survivors of childhood cancer.

PURPOSE: Despite similarities in upfront treatment of childhood cancer, not every adult survivor of childhood cancer (CCS) has an impaired bone mineral density (BMD). No data are available on the role of genetic variation on impairment of BMD in CCS. METHODS: This cross-sectional single-center cohort study included 334 adult CCSs (median follow-up time after cessation of treatment: 15 years; median age at follow-up: 26 years). Total body BMD (BMDTB ) and lumbar spine BMD (BMDLS ) were measured by dual x-ray absorptiometry. We selected 12 candidate single-nucleotide polymorphisms (SNPs) in 11 genes (COL1A1, TNFSF11, TNFRSF11, TNRFSA11B, VDR, ESR1, WLS, LRP5, MTHFR, MTRR, IL-6). RESULTS: Multivariate analyses revealed that lower BMD was associated with lower weight and height at follow-up, male sex, and previously administered radiotherapy. Survivors with the homozygous minor allele (GG) genotype of rs2504063 (ESR1: estrogen receptor type 1) had a lower BMDTB values (-1.16 vs. -0.82; P = 0.01) than those with the AG/AA genotype; however, BMDLS was not different. Carriers of two minor alleles (GG) of rs599083 (LRP5: low-density lipoprotein receptor) revealed lower BMDTB (-1.20 vs. -0.78; P = 0.02) and lower BMDLS (-0.95 vs. -0.46; P = 0.01) values than those with the TT/TG genotype. CONCLUSION: CCSs who are carriers of candidate SNPs in the ESR1 or LRP5 genes seem to have an impaired bone mass at an early adult age. Information on genetic variation, in addition to patient- and treatment-related factors, may be helpful in identifying survivors who are at risk for low bone density after childhood cancer treatment.

Genetic Biomarkers to Identify the Risk of Osteonecrosis in Children with Acute Lymphoblastic Leukemia.

Osteonecrosis is a disabling complication of treatment for pediatric acute lymphoblastic leukemia, and much effort has been made to predict which patients are prone to develop this disease. Multiple clinical and genetic factors have already been identified as being associated with osteonecrosis; however, a prediction model that combines pretreatment genetic biomarkers and clinical factors has not yet been designed. Such a prediction model can only be developed with continuing international collaborations and research efforts, including large genome-wide association studies.

Aggravated bone density decline following symptomatic osteonecrosis in children with acute lymphoblastic leukemia.

Osteonecrosis and decline of bone density are serious side effects during and after treatment of childhood acute lymphoblastic leukemia. It is unknown whether osteonecrosis and low bone density occur together in the same patients, or whether these two osteogenic side-effects can mutually influence each other's development. Bone density and the incidence of symptomatic osteonecrosis were prospectively assessed in a national cohort of 466 patients with acute lymphoblastic leukemia (4-18 years of age) who were treated according to the dexamethasone-based Dutch Child Oncology Group-ALL9 protocol. Bone mineral density of the lumbar spine (BMDLS) (n=466) and of the total body (BMDTB) (n=106) was measured by dual X-ray absorptiometry. Bone density was expressed as age- and gender-matched standard deviation scores. Thirty patients (6.4%) suffered from symptomatic osteonecrosis. At baseline, BMDLS and BMDTB did not differ between patients who did or did not develop osteonecrosis. At cessation of treatment, patients with osteonecrosis had lower mean BMDLS and BMDTB than patients without osteonecrosis (respectively, with osteonecrosis: -2.16 versus without osteonecrosis: -1.21, P<0.01 and with osteonecrosis: -1.73 versus without osteonecrosis: -0.57, P<0.01). Multivariate linear models showed that patients with osteonecrosis had steeper BMDLS and BMDTB declines during follow-up than patients without osteonecrosis (interaction group time, P<0.01 and P<0.01). We conclude that bone density status at the diagnosis of acute lymphoblastic leukemia does not seem to influence the occurrence of symptomatic osteonecrosis. Bone density declines from the time that osteonecrosis is diagnosed; this suggests that the already existing decrease in bone density during acute lymphoblastic leukemia therapy is further aggravated by factors such as restriction of weight-bearing activities and destruction of bone architecture due to osteonecrosis. Osteonecrosis can, therefore, be considered a risk factor for low bone density in children with acute lymphoblastic leukemia.

The negative impact of being underweight and weight loss on survival of children with acute lymphoblastic leukemia.

Body mass index and change in body mass index during treatment may influence treatment outcome of pediatric patients with acute lymphoblastic leukemia. However, previous studies in pediatric acute lymphoblastic leukemia reported contradictory results. We prospectively collected data on body composition from a cohort of newly diagnosed Dutch pediatric patients with acute lymphoblastic leukemia (n=762, age 2-17 years). Patients were treated from 1997-2004 and the median follow-up was 9 years (range, 0-10). Body mass index at diagnosis was expressed as age- and gender-matched standard deviation scores and on the basis of these scores the patients were categorized as being underweight, of normal weight or overweight. Multivariate analyses showed that patients who were underweight (8%) had a higher risk of relapse [hazard ratio: 1.88, 95% confidence interval (1.13-3.13)], but similar overall survival and event-free survival as patients who had a normal weight or who were overweight. Patients with loss of body mass index during the first 32 weeks of treatment had a similar risk of relapse and event-free survival, but decreased overall survival [hazard ratio: 2.10, 95% confidence interval (1.14-3.87)] compared to patients without a loss of body mass index. In addition, dual X-ray absorptiometry scans were performed in a nested, single-center cohort. Data from these scans revealed that a loss of body mass consisted mainly of a loss of lean body mass, while there was a gain in the percentage of fat. In conclusion, being underweight at diagnosis is a risk factor for relapse, and a decrease in body mass index early during treatment is associated with decreased survival. In addition, loss of body mass during treatment seems to consist mainly of a loss of lean body mass. This study was approved by the Medical Ethical Committee in 1996 (trial number NTR460/SNWLK-ALL-9).