miR-15a and miR-24-1 as putative prognostic microRNA signatures for pediatric pilocytic astrocytomas and ependymomas.

In the current setting, we attempted to verify and validate miRNA candidates relevant to pediatric primary brain tumor progression and outcome, in order to provide data regarding the identification of novel prognostic biomarkers. Overall, 26 resected brain tumors were studied from children diagnosed with pilocytic astrocytomas (PAs) (n = 19) and ependymomas (EPs) (n = 7). As controls, deceased children who underwent autopsy and were not present with any brain malignancy were used. The experimental approach included microarrays covering 1211 miRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the expression profiles of miR-15a and miR-24-1. The multiparameter analyses were performed with MATLAB. Matching differentially expressed miRNAs were detected in both PAs and EPs, following distinct comparisons with the control cohort; however, in several cases, they exhibited tissue-specific expression profiles. On correlations between miRNA expression and EP progression or outcome, miR-15a and miR-24-1 were found upregulated in EP relapsed and EP deceased cases when compared to EP clinical remission cases and EP survivors, respectively. Taken together, following several distinct associations between miRNA expression and diverse clinical parameters, the current study repeatedly highlighted miR-15a and miR-24-1 as candidate oncogenic molecules associated with inferior prognosis in children diagnosed with ependymoma.

Analysis of somatic hypermutations of immunoglobulin gene rearrangements in childhood acute lymphoblastic leukemia.

The current study investigated the presence, frequency, and status of somatic hypermutations as well as their role in children with B lineage ALL. The obtained sequences were analyzed using IMGT/V-QUEST. Totally, 150 IGH sequences were evaluated; 139 from the 111 patients at the time of diagnosis and 11 from 8/111 patients at the time of relapse. The findings of the current report revealed the presence of somatically mutated V genes in childhood B lineage ALL. A higher frequency of somatic hypermutations was noted in unproductive rearrangements and was generally attributed to nucleotide mutation type, region, and IGHV gene subgroup biases.

Evaluation of antitumor activity of gefitinib in pediatric glioblastoma and neuroblastoma cells.

BACKGROUND: This work was undertaken to investigate the efficacy of gefitinib, an EGFR tyrosine kinase inhibitor, in tumor cell lines of the CNS by studying cell proliferation and phosphorylation of the tyrosine kinase domain of METHODS: The study included neuroblastoma (SHSY5Y) and glioblastoma (A172) cell lines. The MTT cell proliferation assay was performed in order to quantify the cytotoxic effect of gefitinib in A172 and SH-SY5Y cells, whilst ELISA assay was used to assess the effect on the phosphorylation of tyrosine residue 1068 of EGFR. RESULTS: As the concentration of gefitinib increased, MTT conversion into formazan was observed to progressively decrease, confirming the cytotoxic activity of gefitinib. In the ELISA assay for both cell lines investigated, as the dose of gefitinib increased, a gradual decrease in EGFR tyrosine phosphorylation was detected. CONCLUSIONS: The findings of the current study could form the basis of research regarding the use of novel inhibitors in the treatment of solid tumors in pediatric patients and a shift to targeted therapy with higher efficacy and fewer side effects.

Fas and Bcl-2 protein expression in thyrocytes of patients with nodular goiter.

OBJECTIVE: The relative expression of the apoptotic protein Fas and the anti-apoptotic protein Bcl-2 were investigated in thyrocytes from patients with non-toxic nodular goiter (NTG, n=20) and Hashimoto's thyroiditis (HT, n=5), who underwent fine-needle aspiration biopsy for diagnostic reasons. On the basis of the clinical and cytological findings, the patients with NTG were sub-classified into the group of those with colloid nodules (n=9), degenerative nodules (n=6) and adenomatous nodules (n=5). METHODS: Fine-needle biopsy aspirates were examined by immunocytochemistry for Fas and Bcl-2 expression, using specific monoclonal antibodies. For the evaluation of Fas and Bcl-2 immuno-reactivity, an expression index, based on the number of cells with positive staining, was used: grade 1 included samples with positive staining in <20% of cells; grade 2 included samples with 20-50% positive cells; and grade 3 included samples with >50% positive cells. RESULTS: Fas protein expression was generally low (grade 1) in patients with nodular goiter, in contrast to patients with HT, in whom high expression was detected (grade 3). Only in aspirates from degenerative nodules (four out of six), and in which lymphocytes were also present, was Fas expressed at an intermediate level (grade 2). On the other hand, Bcl-2 protein was differentially expressed among the nodule subtypes. It was low in colloid and degenerative nodules (grade 1) but high in adenomatous ones (grades 2 and 3). Bcl-2 expression was also low in patients with HT (grade 1). CONCLUSION: It is concluded that in comparison to HT, where there is up-regulation of Fas and down-regulation of Bcl-2 protein, Fas expression is low in human goiter, indicating low apoptotic activity. The regulation of Bcl-2 protein differs between adenomatous and colloid nodules, suggesting that this protein may play a role in the differentiation of thyroid nodules.

Cadmium induces apoptosis differentially on immune system cell lines.

We investigate the role of cadmium-induced apoptosis in the immune system, studying the apoptotic effect of Cd2+ in three human cell lines, the T-cell line CCRF-CEM, the B-cell line Raji and the lymphoblastoid cell line Molt-3. Cd2+ was found to be dose-dependently toxic for these cell lines, after 18 h incubation. The 50% lethal dose (LD50) for CCRF-CEM was 25 +/- 20 microM, for Molt-3 was 22.5 +/- 2.4 microM, and for Raji was 13.5 +/- 2.2 microM. DNA electrophoresis and quantitation of apoptosis after 18 h incubation with different Cd2+ concentrations was carried out. In CCRF-CEM cells, apoptosis was detected at 10 microM, reaching a maximum at 30 microM. In Molt-3, apoptosis was detected at 10 microM, increased thereafter and a plateau effect was observed from 30 to 50 microM Cd2+. In Raji, apoptosis was detected at 5 microM, while a plateau effect was observed from 20 to 30 microM Cd2+. The above results indicated that Raji cells were more sensitive to cadmium compared to both CCRF-CEM and Molt-3 cells, suggesting a differential Cd2+-induced apoptotic effect, which may disturb the immune system normal growth and development.

Metallothionein expression prevents apoptosis: a study with antisense phosphorothioate oligodeoxynucleotides in a human T cell line.

Human metallothioneins (hMTs), are low molecular weight cysteine-rich proteins that constitute the majority of intracellular protein thiols. Their transcription is regulated by metals, glucocorticoids and cytokines, and in certain tissues it is a highly specialized phenomenon. Although their physiological function is not entirely understood, hMTs induction has been observed to be associated with protection from heavy metal toxicity and cellular resistance to cytotoxic anticancer drugs. However, the main problem in the investigation of the physiological function of hMTs is the absence of any known specific inhibitor, as well as the fact that many genes constitute the hMTs family. As the identification of genes preventing apoptosis is of great interest, we attempted to examine the role of hMTs in the apoptotic process by inhibiting their expression in the immature T cell line CCRF-CEM with antisense sequence-specific phosphorothioate oligodeoxynucleotides (ODNs). In the experimental procedure the cells were activated and cultured in medium containing 20% FBS instead of 10%, during maintenance. We found that the inhibition of hMTs synthesis, induced by the incubation of the cells for 24 hours with ODNs, stimulated the apoptotic process, as confirmed by the characteristic morphological alterations and DNA fragmentation. Quantitative analysis of apoptosis has shown that inhibition of hMTs expression results in a dose-dependent and ODNs sequence-specific induction of apoptosis. Immunocyto-chemical detection of hMTs followed by Tunel assay showed that all the Tunel positive cells were hMTs negative, suggesting that hMTs expression prevents apoptosis. As hMTs induction is rapid and transient in response to stress and/or environmental stimuli, these results indicate that hMTs constitute a cellular protective mechanism, neutralizing external apoptotic signals.

Study of minimal residual disease in B-cell lineage acute lymphoblastic leukemia of childhood, using clone-specific probes.

In B-cell lineage acute lymphoblastic leukemia (B-ALL), the clonal rearrangements of the immunoglobulin heavy chain gene locus (IgH), can be used as a molecular marker for the detection of minimal residual disease (MRD). Patients in complete remission may still harbor leukemic cells undetectable by conventional methods such as light-microscopic examination, immunophenotyping and cytogenetics. 30 children with B-ALL were screened at diagnosis by polymerase chain reaction (PCR) for their IgH gene repertoire. 7/30 patients were extensively studied using patient-specific oligonucleotide probes derived from the sequence analysis of bone marrow (BM) samples at diagnosis. 210 PCR products from follow-up BM samples corresponding to these 7 patients were hybridized with the appropriate clone-specific probe in order to detect MRD with high sensitivity and specificity. All the patients were in morphological remission during and after therapy. 25/30 patients were PCR positive at diagnosis. 4/7 patients who were examined for MRD had detectable disease in various periods after diagnosis. Molecular signs of residual cells can persist for a long time during and after therapy. Long term follow-up of MRD could determine the period of therapy and predict relapse, indicating therapeutic interventions.

Metallothionein expression prevents apoptosis. II: Evaluation of the role of metallothionein expression on the chemotherapy-induced apoptosis during the treatment of acute leukemia.

Metallothioneins (MT) are low molecular weight cysteine-rich proteins, present in a wide variety of eukaryotes. Although their physiological function is not entirely understood, recently it was found that in vitro human MTs (hMTs) expression prevents apoptosis. In the present study, the apoptosis preventing effect of hMTs is evaluated in vivo, in order to correlate the apoptotic effect of chemotherapy during the treatment of acute leukemia with the expression of hMTs. The expression of hMTs was studied immunocytochemically in bone marrow smears and peripheral blood cytocentrifugations of 47 children with acute leukemia at diagnosis and during treatment. Apoptosis was quantitatively studied in peripheral blood samples during the induction therapy. Eighteen cases were found to be positive for hMTs expression at diagnosis and the mean apoptosis curve of these cases showed maximal effect on the second day of treatment, the apoptotic action of chemotherapy being completed on the tenth day. The mean apoptosis curve of the hMTs negative cases (29 cases) showed maximal effect on the first day of treatment and the apoptotic action of chemotherapy was completed on the sixth day. When considering the day on which the maximal apoptotic effect appeared and the day on which the apoptotic action of treatment was completed, the results indicated retardation of the chemotherapy-induced apoptosis dependent on hMTs expression, as a result of resistance to treatment. Furthermore, the study of hMTs expression during treatment, showed that although the apoptotic action of chemotherapy eliminates blast cells, a cell population positive for hMTs survived and increased during treatment, since they were able to escape apoptotic cell death. These findings, indicated that in vivo, hMTs constitute a cellular protective mechanism preventing chemotherapy-induced apoptosis, thus regulating the response of patients to treatment.

Study of apoptosis in peripheral blood of patients with acute lymphoblastic leukemia during induction therapy.

In acute lymphoblastic leukemia (ALL) the apoptosis of blast cells in peripheral blood (PB) and bone marrow before and/or during treatment, is of great interest. As the morphological changes during apoptosis provide the most reliable markers, in the present study we utilized a nuclear stain based on ethidium bromide (EtBr) for the rapid qualitative and quantitative measurement of circulating apoptotic cells directly in PB suspensions without fractionation. By using a fluorescent microscope the apoptotic cells appeared clearly visible, making the estimation of their percentage straightforward. We studied apoptosis before and during the onset of chemotherapy in PB from 16 children with ALL at diagnosis, and one upon relapse. In the cases studied at diagnosis the circulating apoptotic cells were found in variable percentages after 24 hours of treatment. Maximal apoptosis was observed after 24 hours of treatment in five cases and after 48 hours in two cases. After 96 hours of treatment the cases studied at diagnosis could be divided into three groups: those with a) negligible apoptotic cells, b) between 8% and 12% apoptotic cells and c) a high percentage of apoptotic cells (more than 20%). The relapsed case was characterized by P-glycoprotein positive blast cells, and circulating apoptotic cells which remained very low at all time points. Thus, it is possible to evaluate the response to treatment by studying apoptosis directly in peripheral blood. Therefore, the maximum apoptotic effect and the percentage of circulating apoptotic cells at the different time intervals must be considered.

Gingival overgrowth as the initial paraneoplastic manifestation of Hodgkin's lymphoma in a child. A case report.

BACKGROUND: The purpose of this paper is to present the first case of gingival overgrowth, premature root resorption, and alveolar bone loss, which preceded the diagnosis of a stage IVB Hodgkin's lymphoma (HL) in a 9-year-old boy. METHODS: The child presented complaining of gingival pain which first appeared 3 months prior. Clinical examination revealed inflamed, hyperplastic gingivae, while x-ray showed premature root resorption and alveolar bone loss. Medical work-up was significant for cervical lymphadenopathy. Gingival biopsy, followed by lymph node resection, was performed twice. RESULTS: Histological examination of both gingival biopsies disclosed a mixed inflammatory infiltrate, while classical Hodgkin's lymphoma of the nodular sclerosis type was diagnosed from the second lymph node biopsy. Chemotherapy was instituted with mustard-vincristine-procarbazine-prednizone and adriamycine-bleomycine-vinblastine-dacarbazine. Remission of the lymphoma was observed with concomitant regression of the gingival overgrowth. CONCLUSIONS: The inflammatory gingival overgrowth, premature root resorption of deciduous teeth, and alveolar bone loss in this case, in conjunction with the regression of gingival overgrowth which followed the completion of chemotherapy, are strongly indicative of a paraneoplastic manifestation of HL. The postulated mechanism for the development of the manifestation is the constitutive activation of the transcription factor NF-kB. The gingival inflammatory reaction was probably further aggravated by the bacterial-stimulated cytokine secretion released by monocytes.

Development of a quantitative method for the study of apoptosis in peripheral blood.

In vitro and ex vivo many methods are effective for the quantitation of apoptotic cells. These methods cannot be applied for the study of apoptosis in peripheral blood (PB) samples, because of the disappearance of circulating apoptotic cells and the heterogeneity of cell populations. In the present study we describe a nuclear chromatin staining method using ethidium bromide (EtBr), by which we were able to identify and quantify morphologically apoptotic cells in an early stage of the apoptotic process, before their disappearance. The application of the EtBr nuclear stain in CCRF-CEM cells incubated with epirubicin, provides a reliable and reproducible estimation of apoptosis compared with other assays. The main advantage of this method is that it permits the study of four cellular subpopulations by their morphological characteristics (normal cells, early apoptotic, late apoptotic and cells which died from necrosis). Furthermore, we utilized the EtBr nuclear stain to identify and quantify the circulating apoptotic cells directly in the PB of patients with acute lymphoblastic leukemia during multidrug chemotherapy. Circulating apoptotic cells were detectable after 24 hours treatment followed by the decrease of peripheral blood mononuclear cells. Thus staining of nuclei with EtBr provides a rapid and useful method for the quantitative study of apoptotic cells in somatic fluids during clinical trials.

Inferior vena cava and right atrial thrombosis in children with nephroblastoma: diagnostic and therapeutic problems.

BACKGROUND: The neoplastic thrombus in Wilms' tumor rarely can extend in to the inferior vena cava or to the right atrium. The neoplastic thrombus usually is diagnosed concurrently with the tumor, although in some cases the diagnosis of the thrombus may precede the diagnosis of nephroblastoma. METHODS: Among 90 children with Wilms' tumor who were treated in the authors' unit, 4 had extensive tumor thrombosis of the inferior vena cava or the right atrium. One of these patients was found with a life-threatening thrombosis of the inferior vena cava and the right atrium, which was treated surgically; in this case, the diagnosis of nephroblastoma was made postoperatively. As for the 3 remaining patients the diagnosis of neoplastic thrombosis and Wilms' tumor was made simultaneously. RESULTS: In the first case, the patient underwent surgical excision of the thrombus with cardiopulmonary bypass and a short period of hypothermic cardiopulmonary arrest. In the other 3 cases the thrombus resolved with chemotherapy only. CONCLUSIONS: Surgical excision of extensive neoplastic thrombosis is suggested in the case of life-threatening thrombosis even with cardiopulmonary bypass. Chemotherapy is suggested in cases lacking clinical symptoms of thrombosis.

Frequency of FLT3 mutations in childhood acute lymphoblastic leukemia.

FLT3 mutations are occasionally observed in acute lymphoblastic leukemia (ALL). These most frequently manifest as internal tandem duplications (ITD) and activation loop (AL) mutations. This study investigated the incidence of FLT3 mutations in 86 pediatric patients diagnosed with ALL and the co-presence of common RAS mutations. A 2.3% (2/86) FLT3/AL mutation rate in terms of total ALL cases and a 22% (2/9) incidence in hyperdiploid cases was observed. This is in accordance to previous studies indicating a higher incidence in the patient subgroup associated with hyperdiploidy.

Rituximab in the treatment of high responding inhibitors in severe haemophilia A.

The development of antibodies to factor VIII (FVIII) in severely affected haemophilia A patients is a serious complication associated with increased morbidity and mortality. Bypassing agents are used to treat acute bleeding episodes; however, elimination of the inhibitors can only be achieved with immune tolerance therapy (ITT) in 60-80% of cases. High responding (HR) inhibitors are more likely to respond to ITT if the titre is decreased to <5 BU over time or in selected cases after the administration of immunosuppressive drugs, plasmapheresis or immunoabsorption, techniques difficult to apply in children. Anti-CD20 (rituximab), a monoclonal antibody, was given as an alternative treatment in two haemophilic children with HR inhibitors and impaired quality of life, due to recurrent haemarthrosis. Rituximab was given at the dose of 375 mg m(-2), once weekly for four consecutive weeks. Both patients showed a partial response to rituximab reducing the inhibitor titre to <5 BU, thus facilitating ITT initiation; however, only the older patient eradicated the inhibitor within 21 days after application of ITT. The second patient, despite depletion of B cells, did not respond to ITT. No long-term side effects have been observed in both patients for a follow-up period of 20 and 18 months respectively. In conclusion, rituximab appears to be an alternative effective therapy to rapidly reduce or eliminate the inhibitor in selected cases of severely affected haemophiliacs before further proceeding to ITT. However, the dose and appropriate schedule, as well as long-term side effects need further investigation.

History of pediatric hematology and oncology in Greece.

The history of pediatric hematology and oncology in ancient and modern Greece is reviewed. Ancient Greek literature concerning cancer starts with Hippocrates, is enriched by Galen during the 2nd century AD, and ends with the end of the Byzantine period. Hematology and oncology in modern Greece were adopted as fields of special interest by a few Greek pediatricians. Their work constituted the basis for and the start of pediatric hematology and oncology, which has followed the advances of science ever since.

Dihydrofolate reductase activity in primary brain tumors in children.

PURPOSE: Dihydrofolate reductase is an enzyme involved in cell proliferation and differentiation processes. A cytochemical method was used to detect and quantitate this enzyme at the cellular level in brain tumors in children. MATERIAL AND METHODS: Twenty-six children, aged 1-12 years, with primary brain tumors were studied, eight with medulloblastoma, 10 with glioma, and eight with ependymoma or other tumors. The cytochemical technique was applied on touch preparations performed in the operating room form biopsy specimens. RESULTS: Enzyme activity was apparent as cytoplasmic granules sometimes overlying the nucleus of tumor cells. CONCLUSIONS: Activity of dihydrofolate reductase in the children with medulloblastomas and high-grade gliomas was higher than that reported in leukemic blast cells. In the other brain tumors, low grade gliomas, and ependymomas, the enzyme activity was weaker.

Action of intermediate doses of methotrexate on dihydrofolate reductase in malignant diseases.

This report describes the effect of intermediate methotrexate (MTX) doses on dihydrofolate reductase (DHFR) activity in vivo in the leukocytes of 16 children with malignant diseases. The authors used a cytochemical technique, and the enzyme was studied in intact cells. The treatment protocols included MTX 500 mg-2 g/m2 weekly with leucovorin rescue. The above doses of MTX partially inhibit DHFR. The reduction of enzyme activity was observed in leukocytes within 24 h after MTX infusion, and it was more obvious in the polymorphonucleas and the monocytes. Complete inhibition of enzyme activity was not observed. These results do not agree with those of previous reports using biochemical techniques, which showed that small amounts of MTX inhibit DHFR activity. Even the large doses of MTX used in this study do not completely inhibit enzyme activity. It would be worthwhile to test the effect of even larger doses of MTX to find out if DHFR activity is inhibited.

Activity of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase in primary brain tumors in children.

The activity of the enzymes 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase has been studied cytochemically in children's primary brain tumors. These enzymes play a significant role in purine biosynthesis. Thirty children, aged 1-12 years, were studied, 12 with medulloblastoma, 14 with glioma grade I-IV, and 4 with ependymoma. The activity of the enzymes was apparent as cytoplasmic granules that sometimes overlie the nucleus of the tumor cells. This coincidence showed that different types of brain tumors exhibit different degrees of enzymic activity, which in some cases correlated positively with the malignant potential of the tumor. Approximately one third of the cases were negative for any activity of these enzymes. The intensity of the staining of 5,10-methenyl tetrahydrofolate cyclohydrolase activity was actually higher than that of 5-formyl tetrahydrofolate cyctodehydrase. The clinical or prognostic significance of these findings remains to be clarified, but we believe that cylochemistry provides a sensitive technique for the detection, localization, and description of these enzymes in brain tumor cells. A clear understanding of the mode of action of these enzymes may contribute to devising novel therapeutic strategies.

Leucocyte glucose-6-phosphate dehydrogenase (g-6-pd) activity in g-6-pd deficient subjects.

The activity of glucose-6-phosphate dehydrogenase (G-6-PD) in leucocytes was studied in the following group of Greek people. Group 1: 43 male children and 16 male students with mean values of enzyme activity of 27.7 +/- 16.6 units and 24.6 +/- 5.6 units, respectively. Group 2: 15 G-6-PD deficient male children who had never experienced an acute haemolytic episode with a mean value of 10.8 +/- 4.6 units. Group 3: 19 G-6-PD deficient male children during favism and 3 months after the haemolytic crisis with mean values of 8 +/- 4 units and 9.2 +/- 1.9 units, respectively. Group 4: 19 mothers of children from group 3 who by definition were carriers of G-6-PD deficiency had a mean value of 18.2 +/- 8.2 units. The difference between means for group 1 and groups 2, 3 and 4 is highly significant (P < 0.001). Therefore the enzymatic defect in Greek people is not limited to the erythrocytes but can be also demonstrated in leucocytes.