Analysis of the p16INK4 and TP53 Tumor Suppressor Genes in Bone Sarcoma Pediatric Patients

Recent data suggest that deletion of p16INK4 and mutation of TP53 are among the most common genetic events in the development of human cancer, since the codified proteins act as brakes of the abnormal cell cycle. As the molecular events leading to the development of pediatric bone sarcomas remain unclear, we analyzed 75 osteosarcoma and Ewing sarcoma samples from 43 pediatric patients to search for alterations at the TP53 or p16INK4 tumor suppressor genes. By means of PCR-DGGE (polymerase chain reaction and denaturing gradient gel electrophoresis) we detected TP53 point mutations in 18.6% of the tumor samples, but no constitutional mutations. In the analysis of p16INK4, 7% of the samples harbored deletions of the gene but no point mutations were detected by SSCP (single strand conformation polymorphism) analysis, just the polymorphism Ala-->Thr at codon 148. These data support the hypothesis that TP53 alterations may play a role in the development of pediatric bone tumors and that the primary mechanism of inactivation of p16INK4 seems to be homozygous deletion rather than point mutation

El ensayo de micronúcleos como medida de inestabilidad genética inducida por agentes genotóxicos

Human genetic integrity is compromised by the intense industrial activity, which emphasizes the importance to determine an "acceptable" genetic damage level and to carry out routine genotoxicity assays in the populations at risk. Micronuclei are cytoplasmatic bodies of nuclear origin which correspond to genetic material that is not correctly incorporated in the daughter cells in the cellular division; they reflect the existence of chromosomal aberrations and are originated by chromosomal breaks, replication errors followed by cellular division of the DNA and/or exposure to genotoxic agents. There are several factors able to modify the number of micronuclei present in a given cell, among them are age, gender, vitamins, medical treatments, daily exposure to genotoxic agents, etc. The cytogenetic assay for the detection of micronuclei (CBMN: cytokinesis-block micronucleus) is based on the use of a chemical agent, cytochalasin-B, which is able to block cytocinesis but allowing the nuclear division, therefore yielding binucleated and monodivided cells. The micronuclei scoring is performed on 1000 binucleated cells and the starting sample may vary, although most studies are performed on peripheral blood lymphocytes. The micronuclei assay is considered a practical, universally validated and technically feasible protocol which is useful to evaluate the genetic instability induced by genotoxic agent

Cost-effectiveness analysis of tropisetron vs. chlorpromazine-dexamethasone in the control of acute emesis induced by highly emetogenic chemotherapy in children

To perform a cost-effectiveness analysis (CEA) between a standard antiemetic regimen-chlorpromazine + dexamethasone (CPM-DEX)- and a 5-HT3 receptor antagonist-tropisetron (TROP)--in the control of acute emesis induced by highly emetogenic chemotherapy in children, considering two analytic perspectives: hospital and patients. METHODS: The CEA was performed by constructing a decision tree, for both analytic perspectives, of the possible outcomes of treatment with TROP (single 0.2 mg/kg i.v.) or CPM (5-15 mg i.v. infusion for 3 doses) plus DEX (2 mg/m2 i.v. bolus i.v. x2). The patients were stratified by age in two groups (2-12 and 13-17). To estimate the probability of each endpoint at the decision tree we have taken as a base a trial developed in the Department of Pediatrics. Direct medical cost of primary therapy, failure, complications and side effects were included in the cost calculations. RESULTS: From patients' analytic perspective, TROP was more cost-effective than CPM-DEX for both groups of patients. Discrepancy between both analytic perspectives in 13-17 year-old patient's group was resolved in favour of the option chosen from the patients' analytic perspective (TROP). Sensitivity analysis showed the reliability of the results. CONCLUSIONS: 1. TROP was more cost-effective than CPM-DEX. 2. Taking into account the patients' analytic perspective is essential when we compare antiemetics pharmacoeconomically. 3. It seems necessary to increase the effectiveness of TROP in pediatric patients receiving highly emetogenic chemotherapy with strategies such as the addition of a steroid

Recombinant Human Erythropoietin for the Treatment of Anemia in Children With Solid Malignant Tumors

Cancer is often associated with chronic anemia which frequently requires blood transfusions. This study was performed to assess the efficacy and safety of r-HuEPO therapy in children with cancer. PATIENTS AND METHODS: Twenty-five patients under 18 years of age with solid malignant tumors were treated with 150 U/kg/day of r-HuEPO 5 times weekly for 12 weeks. Response was defined as an increase of the baseline hemoglobin level by at least 2 g/dl. r-HuEPO patients were compared to 25 matched historical controls. RESULTS: Response was achieved in 72% of r-HuEPO patients. Hemoglobin level increased from 9.8 +/- 0.6 g/dl at baseline to 12.4 +/- 1.7 g/dl at the end of treatment in the r-HuEPO group and increased from 9.5 +/- 0.7 g/dl to 9.6 +/- 1.4 g/dl in the control group (P < .001, Student's t-test). Only 16% of patients receiving r-HuEPO required blood transfusions vs 96% of control patients (P < .001, Student's t-test), with mean units of blood transfused per patient being 0.35 in the r-HuEPO group and 3.56 in controls (P < .001, Student's t-test). There was a statistically significance improvement in Karnofsky's index in r-HuEPO patients. No adverse reaction related to r-HuEPO therapy was observed. CONCLUSIONS: r-HuEPO is a safe and effective means of increasing hemoglobin level and reducing blood requirements in children with solid malignant tumors receiving chemotherapy

Screening of the human tumor necrosis factor-alpha (TNF-α) gene promoter polymorphisms by PCR–DGGE analysis

We have designed a new PCR-DGGE technique that enables detection of base changes in the TNF-alpha gene promoter. Screening of 130 samples from Spanish children has shown that this technique accurately detects the altered band patterns induced by the presence of the polymorphisms at positions -376, -308, -238 and -163 of the promoter sequence. Although further analysis are needed to fully characterise the alterations detected, we believe that this PCR-DGGE technique is a rapid and sensitive first approach to the genetic characterisation of the TNF-alpha promote

Estudio de la densidad mineral ósea mediante osteosonografía en niños y adolescentes sanos: valores de normalidad

Osteoporosis is a frequent health problem in adults. Optimization of bone mass acquisition during childhood and adolescence may play a major role in the prevention of this disease. Osteosonography is a recent technique for measuring bone mineralization without exposing the patient to radiation. OBJECTIVES: To measure bone mineral density using osteosonography in healthy Spanish Caucasian children and adolescents in order to determine normal values. METHODS: We performed a cross sectional study of 829 healthy child and adolescent volunteers (360 girls and 469 boys) randomly selected from the urban area of Pamplona in Navarre (Spain). Ages ranged from 6 to 18 years. ADBM Sonic 1200 ultrasound densitometer from IGEA was used. Daily calcium dietary intake and amount of physical activity were recorded. RESULTS: Cross sectional standards for Ad-SOS are presented. Ad-SOS did not significantly change between the ages of 6 and 9 years in girls or until the age of 10 years in boys. From the ages of 10 to 14 years, Ad-SOS values were higher in girls than in boys. After the age of 14 years, no significant differences were found. No correlation was found between calcium dietary intake, amount of physical exercise or bone mineralization values. CONCLUSIONS: Measurement of Ad-SOS by osteosonography is an easy, fast and inexpensive method for measuring bone mineral density in children and adolescents without exposing them to radiation. It can be used in the pediatric population to detect early alterations in bone mineralization

Methotrexate Pharmacokinetics and Survival in Osteosarcomat

The aim of this study was to analyze the relationship between exposure to high-dose methotrexate (HDMTX) and tumor response in terms of survival in children with osteosarcoma. PROCEDURE: This study included 44 patients (479 courses) who received a median dose of 5.92 g/m2 of MTX (interquartile range (IQR) 2.37 g/m2) in a 4-hr infusion. The mean area under the concentration-time curve (AUC) estimated by parametric methods (non-parametric expectation maximization, NPEM), and the mean concentration at the end of the infusion were considered to be the exposure parameters. Tumor response was recorded as disease-free survival (DFS), overall survival (OS), and histologic tumor response. The relationship between MTX exposure and survival parameters was analyzed by Cox regression. RESULTS: The group of 11 patients who were the least exposed to MTX (AUC <2,400 micromol/L hr) presented a high DFS, probably due to the shorter interval of time between MTX courses that led to a higher dose density. In patients with AUC >2,400 micromol/L hr, an increase in the AUC was related to an increase in the DFS. Significant differences were observed in the DFS between patients whose mean AUC was below or above 4,000 micromol/L hr (P=0.024), such that 4,000 micromol/L hr was considered as the minimum AUC to be aimed at for future patients. CONCLUSIONS: Dose density seems to be an important factor in osteosarcoma response, but this must be confirmed in further studies. In order to improve the response to osteosarcoma in children, it is recommended that the dose of MTX to be increased such as to obtain an AUC higher than 4,000 micromol/L hr

Methotrexate in Pediatric Osteosarcoma: Response and Toxicity in Relation to Genetic Polymorphisms and Dihydrofolate Reductase and Reduced Folate Carrier 1 Expression

To determine the influence of the genotype and the level of expression of different enzymes involved in folate metabolism on the response to and toxicity of high-dose methotrexate treatment in pediatric osteosarcomas. STUDY DESIGN: DHFR and Reduced folate carrier 1 (RFC1) semiquantitative expression was analyzed in 34 primary and metastatic osteosarcoma tissues by real-time polymerase chain reaction. The following polymorphisms were also analyzed in peripheral blood from 96 children with osteosarcoma and 110 control subjects: C677T, A1298C (MTHFR), G80A (RFC1), A2756G (MTR), C1420T (SHMT), the 28bp-repeat polymorphism, and 1494del6 of the TYMS gene. Treatment toxicity was scored after each cycle according to criteria from the World Health Organization. RESULTS: DHFR and RFC1 expression was lower in initial osteosarcoma biopsy specimens than in metastases (P = .024 and P = .041, respectively). RFC1 expression was moderately decreased in samples with poor histologic response to preoperative treatment (P = .053). Patients with osteosarcoma with G3/G4 hematologic toxicity were more frequently TT than CT/CC for C677T/MTHFR (P = .023) and GG for A2756G/MTR (P = .048 and P = .057 for gastrointestinal and hematologic toxicity, respectively). CONCLUSIONS: The role of C677T/MTHFR and A2756G/MTR on chemotherapy-induced toxicity should be further investigated in pediatric osteosarcomas receiving high-dose methotrexate. Altered expression of DHFR and RFC1 is a feasible mechanism by which osteosarcoma cells become resistant to methotrexate

Mutational Activation of ras Genes is Absent in Pediatric Osteosarcoma

Activation of ras oncogenes is found in human cancers; overall it is observed in 15% of all neoplasms. The purpose of this study was to assess the extent of involvement of ras oncogenes in osteosarcoma. Tumor samples from a series of 49 pediatric patients diagnosed with osteosarcoma and treated at our institution were evaluated. Paraffin-embedded tumor samples from diagnostic biopsies, from tumor en bloc resection tissue after neoadjuvant chemotherapy, and samples from metastases were examined in search of point mutations in H, K, and N-ras genes at codons 12 and 61 by means of polymerase chain reaction (PCR), slot-blotting, and radioactive labeled specific DNA probes. A total of 92 archival samples were studied. No point mutations activating these genes were found. These findings suggest that the activation by point mutations at codons 12 and 61 of the H, K, and N-ras genes does not play a role in the pathogenesis of human osteosarcoma. Since no point mutations in codons 12 and 61 were detected, it was not possible to establish any correlation between the ras genes and clinical or histologic finding