[Ionizing radiation received by patients with osteosarcoma during intra-arterial chemotherapy treatment]. / Radiación ionizante recibida por pruebas de radiología intervencionista durante el tratamiento con quimioterapia intraarterial en pacientes afectos de osteosarcoma.

BACKGROUND: Osteosarcoma paediatric patients are usually treated with intra-arterial chemotherapy (QTia) which is admi-nistered directly to the tumour. This procedure exposes patients to ionizing radiation. Paediatric patients are especially sensitive to this exposure. METHODS: The total amount of ionizing radiation received from QTia administration was quantified in a group of 16 osteo-sarcoma paediatric patients from the Clínica Universidad de Navarra. RESULTS: The median of the total radiation received per patient was 33.4 Gy·cm2 (IQR: 43.33 Gy·cm2), and the median number of procedures performed per subject was 10 (IQR: 6.5). CONCLUSIONS: The study highlights the importance of quantifying the radiation received by a group of children and adoles-cents affected by osteosarcoma during treatment with QTia. Long-term side effects of this radiation should be considered in pae-diatric patients. Currently, there are no previous studies that provide data of the amount of ionizing radiation received through this procedure.

[The cytogenetic assay as a measure of genetic instability induced by genotoxic agents]. / 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 agents.

Differential polymerase chain reaction: a technical comparison of three methods for the detection of CDK4 gene amplification in glioblastomas.

Among the different techniques used to detect oncogene amplification in tumor DNA, Southern blot and differential PCR have been the most frequently used. We report on a technical comparison of three different methods to detect gene amplification by differential PCR: ethidium bromide staining, silver staining (both after standard differential PCR), and fluorescent differential PCR. We explored the relative densitometric measure of a 119 bp fragment of the CDK4 gene versus an 82 bp fragment of the IFNG gene. In total agreement with previous studies carried out by Southern blot and differential PCR by other authors, we were able to detect CDK4 amplification in 3 of the 21 glioblastomas (14%), but only by the fluorescent differential PCR method. In conclusion, fluorescent differential PCR is more sensitive than standard differential PCR for detection oncogene amplification in tumor DNAs.

Megestrol acetate therapy for anorexia and weight loss in children with malignant solid tumours.

BACKGROUND: Malnutrition is very frequent in childhood cancer. Its main cause is inadequate intake for energy demands owing to lack of appetite. Megestrol acetate is a synthetic progestin that has been used for reversing anorexia in adult cancer. OBJECTIVE: To assess megestrol acetate efficacy and side-effects in treating anorexia in childhood cancer. METHODS: Thirty-five children with solid tumours were receiving antitumour therapy. Nutritional assessment was by anthropometry. Megestrol acetate efficacy was assessed by evaluating grade of appetite, energy intake and well-being. Side-effects were evaluated by means of clinical history, physical examination, lipid profile, coagulation tests and cortisol rhythm. RESULTS: When compared to baseline all the anthropometric measurements increased (P < 0.05) from the first month of megestrol acetate therapy, as well as appetite and energy intake. No significant side-effects were found. CONCLUSION: Megestrol acetate therapy is a powerful appetite stimulant which led to weight gain, composed of both fat mass and fat-free mass. Megestrol acetate is well tolerated, with few and mild side-effects. If megestrol acetate therapy is started at the onset of anorexia, the use of more expensive, invasive and complicated techniques of nutritional support may be avoided.

Molecular features in a biphenotypic small cell sarcoma with neuroectodermal and muscle differentiation.

We report a case of a 13-year-old girl with soft tissue sarcoma of the hand, which showed muscle and neuroectodermal immunophenotypes. Molecular studies were performed on RNA collected from fine-needle aspiration (FNA) cytology and peripheral blood samples by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis. This biphenotypic tumor showed simultaneous expression of EWS-FLI1 and PAX3-FKHR transcripts, specific of Ewing family tumors and alveolar rhabdomyosarcoma, respectively. Although childhood sarcomas with simultaneous muscle and neural differentiation have been described to have EWS-FLI1 transcripts, there are no reports of tumors with both transcripts. Cytological specimens are a good source of RNA for molecular studies.

Ewing family tumors: potential prognostic value of reverse-transcriptase polymerase chain reaction detection of minimal residual disease in peripheral blood samples.

In more than 95% of patients, the Ewing family of tumors (ET) has chimeric transcripts caused by fusion of the EWS gene to either FLI1 or ERG. The presence of specific EWS-FLI1 or EWS-ERG transcripts in peripheral blood (PB) samples of patients being treated for ET was prospectively evaluated, and these data were correlated to their clinical status. The authors studied 113 PB samples from 28 patients with ET. Treatment included chemotherapy, radiotherapy, and surgical excision of tumor after induction therapy. PB samples were taken prospectively at least 2 weeks after resection of tumor. Nested reverse-transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot was performed in all samples. Resected tumors were reviewed for the degree of response to chemotherapy and volume. Seventy-seven PB samples from 28 patients had EWS-FLI1/ERG transcripts. In 11 patients, PB samples became negative with treatment, and, in 5 of them, the samples remained negative throughout the study. Samples taken during progression were always positive and, in 4 patients, became positive before progression was clinically evident. All patients with transcripts other than EWS-FLI1 type 1 (n = 3) died from tumor progression. This is a sensitive assay to monitor circulating tumor cells in Ewing tumors. The preliminary data suggest that progression is preceded by positive samples and may be related to specific transcript types.

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.

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 findings.

Clinical and molecular features of Ewing sarcoma in a patient with triple-X syndrome.

A case of Ewing sarcoma in a 16-year-old girl with 47 XXXc karyotype is reported.

Nonclonal chromosomal aberrations induced by anti-tumoral regimens in childhood cancer: relationship with cancer-related genes and fragile sites.

Cytogenetic studies were performed on 80 pediatric cancer patients to observe the chromosomal damage, both quantitative and qualitative, induced by chemotherapy. Peripheral blood lymphocytes (PBL) (n = 127) were obtained at diagnosis, during treatment, at remission, and at relapse, and chromosome analysis performed utilizing G-banding standard procedures. The results show a significant increase in the number of altered karyotypes (P = 0.03) in the samples during treatment, returning to values that were similar to those at diagnosis at 2-year remission. Most of the chromosomal aberrations (CA) detected during the chemotherapy regimens were nonclonal, unbalanced (75%), and involved chromosomes 1, 3, 5, 6, 11, 12, 16, and 17 most frequently. There was also a marked increase of CA in samples at relapse with very similar features (type and distribution) to those detected during treatment. There was a good correlation between the chromosomal breakpoints in our series and fragile sites (58%), oncogene (75%), and tumor suppressor gene (33%) loci described in the literature. The results obtained suggest that cytostatic drugs induce a transient increase in chromosome fragility occurring at several cancer-associated breakpoints.

Lack of gene amplification as a mechanism of CDK4 activation in human neuroblastoma.

Neuroblastoma is the most frequent solid tumor in childhood. We have analysed 48 neuroblastomas of different stages and degrees of cellular differentiation for CDK4 amplification by a fluorescent differential PCR assay. We explored the relative densitometric measure of a 119 bp fragment of the CDK4 gene versus an 82 bp fragment of the IFNG gene. We were not able to detect any case of CDK4 gene amplification in the neuroblastomas. In conclusion, CDK4 activation does not seem to be relevant to the development of neuroblastomas, at least through gene amplification.

Screening of the human tumor necrosis factor-alpha (TNF-alpha) 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 promoter.

Transient posterior encephalopathy induced by chemotherapy in children.

The cases of three children, 16, 12, and 12 years of age, who suffered sudden confusional state and cortical blindness lasting 12 to 30 minutes while under treatment with high-dose methotrexate, cyclophosphamide, and dactinomycin for a lower limb osteosarcoma are reported. Transient neuropsychologic deficits arose after the acute phase of treatment: left hemispatial neglect and constructive apraxia (Patient 1); constructive apraxia (Patient 2); and constructive apraxia and alexia without aphasia (Patient 3). The three patients recovered completely from all their deficits within the time frame of 3 hours to 2 weeks. Arterial hypertension and hypomagnesemia were found during the acute phase in all patients. In Patients 2 and 3, magnetic resonance imaging revealed increased parieto-occipital T(2) signal involving gray and white matter. In Patients 1 and 2, HmPAO-SPECT revealed parieto-occipital hypoperfusion that resolved a few days later. The alterations detected by neuroimaging were concurrent with the appearance and disappearance of the clinical symptoms. Such transient acute episodes have been named occipital-parietal encephalopathy. On the basis of our clinical, laboratory, and neuroimaging findings, an explanation for the origin of this syndrome, a migrainelike mechanism, triggered by chemotherapy-induced hypomagnesemia, is proposed.

Osteosarcoma: correlation between radiological and histological changes after intra-arterial chemotherapy.

The statistical correlation between three different radiological methods (conventional radiography, computed tomography and angiography) and tumor necrosis (TN) of the resected specimen have been studied in a series of 31 patients diagnosed with osteosarcoma (OS). They were treated with a multidisciplinary approach including intraarterial and intravenous chemotherapy followed by limb salvage procedures, plus intraoperative radiotherapy and adjuvant chemotherapy. A clear statistical correlation has been obtained between TN and angiography (p = 0.02) and between TN and two specific radiological signs: 'tumoral stain and neovascularity' (p = 0.02) and 'peritumoral fat planes' (p = 0.05). Conventional radiography, computed tomography and other radiological signs studied (nutrient vessel, soft tissue mass and central peripheral calcifications) did not show any significant correlation with TN. These results seem to suggest that angiography is a method to evaluate TN preoperatively and also to define the efficacy of neoadjuvant chemotherapy in OS.

[Role of conservative surgery in the multidisciplinary treatment of Ewing's sarcoma in childhood]. / Papel de la cirugía conservadora en el tratamiento multidisciplinario del sarcoma de Ewing en la infancia.

BACKGROUND: In order to cure Ewing's sarcoma it is necessary to have an approach which considers the radical local control on the sites of macroscopic disease, along with the systemic control of micrometastases. On the present study the experience of the authors in analyzed, remarking the role of cytoreduction surgery on curability. METHODS: From January 1982 to August 1991, 24 patients with the mean age 13 years, 14 boys and 10 girls, previously untreated and with a pathology proven diagnosis have been treated by the authors. The treatment protocol included: alternating chemotherapy with cyclophosphamide, adriamycin, methotrexate, bleomycin, actinomycin D and vincristine; administered simultaneously with preoperative external radiation with a volume that completely included the affected bone and surrounding soft tissues for a total dose of 45 Gy/4.5 weeks. After a resting period of 4 weeks, resection of the involved bone and adjacent healthy bone was performed, followed by a single dose of 10-15 Gy of intraoperative radiotherapy to the tumor bed. Subsequently a custom prostheses or allograft was implanted. RESULTS: Twenty patients had localized disease and 4 had metastatic disease at diagnosis. In 16 cases the tumor was in extremities, 5 axial, and 3 extraskeletical. In 15 patients surgery with limb sparing techniques was performed, 8 had en block resection and one amputation (calcaneous location). At the time of this report 21 patient are alive (87%). Four had disease progression, of this 3 had died (12%). The actuarial disease free survival rate is 80% +/- 9% with a follow-up of 104 months, being the mean survival time of 85.5 months. CONCLUSIONS: The cytoreduction surgery included into a multidisciplinary approach permits to achieve a high rate of cure in Ewing's sarcoma. The toxicity of the program can be considered acceptable.

[Vascular malformations as syndromic markers]. / Malformaciones vasculares como marcadores sindrómicos.

Vascular malformations are static lesions, generally present at the moment of birth, formed by displasic vessels that grow in proportion to the growth of the child. They show normal cell replacements that constitute genuine mistakes of morphogenesis. The absence of regression of these malformations implies that they remain throughout the lifetime. The terminology describing this type of lesions gave rise to confusion in the medical literature until the International Society for the Study of Vascular Anomalies adopted a classification based on the dominant vessel of the malformation in 1996. This classification distinguishes between simple and complex forms depending on whether they affect one or several types of vessel. Vascular malformations can show themselves as isolated lesions or can be associated with other lesions, constituting the guide sign or being the principal marker of some syndromic complexes. This paper describes the principal signs and symptoms of those syndromes in which a vascular malformation is the key that raises suspicion about the existence of other associated lesions.

[Analysis of the involvement of the tumour suppressor genes TP53, p16INK4, p21WAF1, RB1 and the drugs metabolizing enzymes in the development of bone tumours in children]. / Análisis de la implicación de los genes de supresión tumoral TP53, p16INK4, p21WAF1, RB1 y de las enzimas metabolizadoras de drogas en el desarrollo de tumores óseos en niños.

BACKGROUND: Several tumor suppressor genes such as p16INK4, TP53, RB1 y p21WAF1 are involved in cell cycle regulation in response to DNA damage and belong to the complex pathway that regulates cell proliferation and/or differentiation. We have investigated the presence of mutations in those genes and polymorphisms of Drug Metabolizing Enzymes that could be involved in the development of pediatric bone tumors or in their outcome. MATERIALS AND METHODS: By means of PCR-based techniques, we have analyzed the presence of variations in the coding sequence of p16INK4, TP53, RB1 y p21WAF1 and of the Drug Metabolizing Enzymes in a group of 82 osteosarcomas and 47 Ewing's sarcomas as well as in a control group of 115 healthy children. RESULTS: We detected mutations of the TP53 gene in about 25% of the samples analyzed, most frequently in association with tumors of poor prognosis or reduced survival. The p16INK4 gene was homozygously deleted in 18% of the osteosarcomas, also associated with poor prognosis and unfavourable histologic subtypes; RB1 was altered in 21% of the osteosarcomas. We did not detect relevant associations between polymorphisms of the Drug Metabolizing Enzymes or mutation of the p21WAF1 and development of pediatric bone tumors. CONCLUSIONS: Alteration of TP53, p16INK4 and p21WAF1 seems to be involved in the development of pediatric bone tumors and to be an unfavourable prognostic factor in this type of tumors.

[Second tumors in pediatric oncologic patients. Report of 5 cases]. / Segundos tumores en pacientes oncológicos pediátricos. A propósito de cinco observaciones.

The dramatic progress observed in the survival of children treated for cancer in the last two decades due to the use of aggressive chemotherapy and radiotherapy has brought an increased incidence of second malignant tumors. Five clinical cases of second malignant neoplasms after a period of six months to seventeen years after diagnosis are presented. The second tumors observed were: one patient with malignant fibrous histiocytoma of the orbit after treatment bilateral retinoblastoma; one patient with multifocal osteosarcoma after cerebelli medullo-blastoma; one patient with Ewing's sarcoma of the fibula after neuroblastoma of the adrenal gland; one case of carcinoma of the thyroid gland after osteosarcoma of the femur and one patient with acute lymphoblastic leukemia after been treated of osteosarcoma of the femur. The genetic, immunologic and therapeutic risk factors are reviewed and analyzed.

[Characteristics and value of chromosome aberrations induced by antitumor treatments in pediatric patients with cancer]. / Características y valor de las aberraciones cromosómicas inducidas por los tratamientos antitumorales en pacientes pediátricos con cáncer.

Cytogenetic studies were performed on 80 pediatric cancer patients to test the chromosomal damage induced by the chemotherapy treatments. G-banded karyotypes were performed on peripheral blood lymphocytes (PBL) (n = 127) obtained at diagnosis, during treatment, at remission and at relapse. We detected a significant increase in the number of altered karyotypes in the samples during treatment, lowering to similar values to those at diagnosis at two-year remission. Most of the chromosomal aberrations (CA) detected during chemotherapy were unbalanced (75%) and affected most frequently chromosomes 1, 3, 5, 6, 11, 12, 16 and 17. There was also a marked increase of CA in samples at relapse, with similar features (type and distribution) to those detected during treatment. There was an outstanding correlation between the chromosomal breakpoints in our series and fragile sites (58%), oncogene (75%) and tumour suppressor gene (33%) loci described in the literature. The results obtained suggest that the cytostatic drugs induce a transient increase in chromosome fragility that focuses to several cancer-associated breakpoints.

[Lymphatic metastases of osteosarcomas]. / Metástasis ganglionares de osteosarcomas.

Osteosarcoma lymph node metastasis are uncommon. This paper shows two patients having osteoblastic osteosarcoma with loco-regional lymph node involvement. In the first case two inguinal and pelvic adenomegalies were found to have tumor metastasis two years and a half after initial diagnosis in a control radiological study. Currently the patient is alive three months after the lymphadenectomy. In the second case, several inguinal high density tumoral nodules were identified during the workup of primary tumor.