Study on developmental-behavioural pediatrics training experiences of pediatricians and pediatric trainees working in Nepal.

BACKGROUND: There are growing concerns about developmental, behavioural, social and emotional wellbeing of children throughout the world. A huge gap exists between the instructions provided on medical disciplines and on areas of Developmental-Behavioural Pediatrics (DBP) during pediatric training in our region. OBJECTIVE: This study aims to evaluate the Developmental-Behavioural pediatrics (DBP) training experiences of pediatricians and pediatric trainees during their post graduate training in pediatrics. METHODS: Questionnaires were sent to the pediatricians / trainees and data was analyzed using SPSS software. RESULT: More than 56 % of respondents were satisfied in 10 out of 11 medical disciplines whereas more than 56% were unsatisfied in 10 out of 11 developmental behavioural pediatrics disciplines. This study has demonstrated that between 50-60 % of pediatricians did not see the cases of ADHD, learning difficulty and family dysfunction for a period of three months prior to the survey which are relatively common conditions. Even in post graduate training nearly 66 % of pediatricians were unsatisfied with their instructions in DB pediatrics. CONCLUSION: Pediatricians working in Nepal have difficulties in addressing the issues of developmental pediatrics, raising a question about the need to review the curriculum of post graduate training in Pediatrics.

Genome complexity in acute lymphoblastic leukemia is revealed by array-based comparative genomic hybridization.

Chromosomal abnormalities are important for the classification and risk stratification of patients with acute lymphoblastic leukemia (ALL). However, approximately 30% of childhood and 50% of adult patients lack abnormalities with clinical relevance. Here, we describe the use of array-based comparative genomic hybridization (aCGH) to identify copy number alterations (CNA) in 58 ALL patients. CNA were identified in 83% of cases, and most frequently involved chromosomes 21 (n=42), 9 (n=21), 6 (n=16), 12 (n=11), 15 (n=11), 8 (n=10) and 17 (n=10). Deletions of 6q (del(6q)) were heterogeneous in size, in agreement with previous data, demonstrating the sensitivity of aCGH to measure CNA. Although 9p deletions showed considerable variability in both the extent and location, all encompassed the CDKN2A locus. Six patients showed del(12p), with a common region encompassing the ETV6 gene. Complex CNA were observed involving chromosomes 6 (n=2), 15 (n=2) and 21 (n=11) with multiple regions of loss and gain along each chromosome. Chromosome 21 CNA shared a common region of gain, with associated subtelomeric deletions. Other recurrent findings included dim(13q), dim(16q) and enh(17q). This is the first report of genome-wide detection of CNA in ALL patients using aCGH, and it has demonstrated a higher level of karyotype complexity than anticipated from conventional cytogenetic analysis.

The cloning, mapping and expression of a novel gene, BRL, related to the AF10 leukaemia gene.

The MLL gene is reciprocally translocated with one of a number of different partner genes in a proportion of human acute leukaemias. The precise mechanism of oncogenic transformation is unclear since most of the partner genes encode unrelated proteins. However, two partner genes, AF10 and AF17 are related through the presence of a cysteine rich region and a leucine zipper. The identification of other proteins with these structures will aid our understanding of their role in normal and leukaemic cells. We report the cloning of a novel human gene (BRL) which encodes a protein containing a cysteine rich region related to that of AF10 and AF17 and is overall most closely related to the previously known protein BR140. BRL maps to chromosome 22q13 and shows high levels of expression in testis and several cell lines. The deduced protein sequence also contains a bromodomain, four potential LXXLL motifs and four predicted nuclear localization signals. A monoclonal antibody raised to a BRL peptide sequence confirmed its widespread expression as a 120 Kd protein and demonstrated localization to the nucleus within spermatocytes.

Expression pattern and cellular distribution of the murine homologue of AF10.

We have cloned Af10, the murine homologue of the MLL partner gene AF10. The predicted open reading frame of Af10 contains 1069 aa which are 90% identical to those of AF10. Af10 contains an N-terminal cysteine-rich region with a LAP/PHD finger, a leucine zipper domain and a glutamine-rich region at the C-terminus, features also found in the human proteins AF10 and AF17. A single 5. 5-kb transcript was detected in murine tissues with the highest level of expression in the testes. A polyclonal antibody raised to the cysteine-rich region of AF10 was able to identify a double band of 140 kDa on Western analysis in mouse testicular extracts. After subcellular separation Af10 was identified in both the nuclear and cytoplasmic extracts, again as a double band of 140 kDa in size. In situ hybridisation studies were performed with sense and antisense digoxigenin-labelled oligonucleotides. High levels of expression were noted in postmeiotic germ cells, especially in spermatids from around stage VI to stage VIII. High levels of expression were also seen in the white matter of the cerebellum, extending into the granular layer. The expression in differentiated rather than in proliferating cells suggests that the role of Af10 may lie in the suppression of proliferation rather than in differentiation. Since the LAP/PHD finger domains are lost in the MLL-AF10 fusion, arguably such a function could be carried out by this domain.

Adenovirus infection after pediatric bone marrow transplantation: is treatment always necessary?

BACKGROUND: Adenovirus infections are associated with significant rates of morbidity and mortality among children after bone marrow transplantation (BMT). Many transplantation units use molecular virological methods, such as polymerase chain reaction (PCR), for surveillance for adenovirus infection and give preemptive antiviral therapy to children with evidence of disseminated adenovirus infection. This treatment strategy has never been evaluated in clinical trials. METHODS: We retrospectively tested blood samples obtained from a cohort of children who had undergone BMT before the introduction of regular weekly surveillance for adenovirus infection. A total of 273 samples collected from 26 patients between May 1998 and June 2002 were tested for adenovirus infection by quantitative PCR. Virus load was quantified for each sample yielding positive test results, and the clinical notes and virological records of each child were reviewed. RESULTS: Evidence of adenovirus infection was found in 11 children (42%), 7 of whom had not previously had positive test results. Receipt of T cell-depleted transplants was associated with a significantly higher incidence of adenovirus infection during the posttransplantation period. The 2 children who died from adenovirus disease developed infection within 2 weeks after transplantation, and both had very low absolute lymphocyte counts at the time of diagnosis. Seven of 11 children with blood samples that were found to be positive for adenovirus by PCR cleared the virus without antiviral therapy. CONCLUSIONS: Surveillance for adenovirus by PCR is better than symptomatic testing for detecting adenovirus infection. Antiviral therapy may not be necessary for all children who develop adenovirus viremia after BMT.

Biochemical analyses of the AF10 protein: the extended LAP/PHD-finger mediates oligomerisation.

Leukaemogenesis correlates with alterations in chromatin structure brought about by the gain or loss of interactive domains from regulatory factors that are disrupted by chromosomal translocations. The gene MLL, a target of such translocation events, forms chimaeric fusion products with a variety of partner genes. While MLL appears to be involved in chromatin-mediated gene regulation, the functions of its partner genes are largely speculative. We report the biochemical analysis of the MLL partner gene AF10 and its possible role in leukaemogenesis. AF10 has been reported to be re-arranged with genes other than MLL leading to the same phenotype, a myeloid leukaemia. We have identified a novel protein-protein interaction motif in the AF10 protein comprising the extended LAP/PHD-finger. This domain mediates homo-oligomerisation of recombinant AF10 and is conserved in several proteins, including MLL itself. AF10 binds cruciform DNA via a specific interaction with an AT-hook motif and is localised to the nucleus by a defined bipartite nuclear localisation signal in the N-terminal region.

Primary extra-thoracic T-cell non-Hodgkin's lymphoma of childhood. The United Kingdom Children's Cancer Study Group.

Between 60-80% of non-Hodgkin's lymphoma (NHL) of T-cell lineage in children, present with primary mediastinal disease. On current therapy, this and nodal low-stage disease are associated with a relatively favourable outcome. In order to evaluate whether the primary site of disease is a prognostic factor, we examined the clinical characteristics and outcome of 36 children with primary extrathoracic T-cell NHL diagnosed during a 7-year period by the United Kingdom Children's Cancer Study Group. Eight, eight, 11 and nine children had stage I, II, III and IV disease, respectively. The primary site of disease was nodal in 22, skin and subcutaneous tissue in five, abdominal in three, bone in three and at other sites in three. Eighteen (50%) had lymphoblastic, ten (28%) large cell anaplastic (LCA) and seven (19%) pleomorphic large or medium cell (PLC) tumours. In one child the histology was inconclusive. All children with lymphoblastic, three with LCA, three with PLC disease and one with unknown histology were treated on intensive, sustained and continuous leukaemia protocols. Ten, seven with LCA and three with PLC, were treated on regimens of short duration pulsed therapy and one child with PLC with radiotherapy only. The three-year event-free survival (EFS) was 78% for the group as a whole. Those with stage IV lymphoblastic disease had a significantly worse prognosis (p < 0.0005). Primary site of disease, gender, therapeutic regimen and histology were not predictive for survival. In primary extrathoracic T-cell NHL with lymphoblastic histology, leukaemia-like therapy is recommended. For non-lymphoblastic disease, in particular the Ki-1 positive tumours, evidence suggests that short duration pulsed therapy may be optimal.

Identification and molecular characterisation of a CALM-AF10 fusion in acute megakaryoblastic leukaemia.

The t(10;11)(p13;q14-21) is a non-random translocation described in acute lymphoblastic and myeloid leukaemias. It results in the fusion of the gene CALM, which encodes a clathrin assembly protein, on 11q14 to the gene AF10, a putative transcription factor on 10p13. Here we describe for the first time, the occurrence of a CALM-AF10 fusion in a case of acute megakaryoblastic leukaemia. Fluorescence in situ hybridisation and reverse transcriptase polymerase chain reaction were used to confirm the presence of a CALM-AF10 fusion. A novel splice variant of CALM missing nt 1927-2091 was also detected. Though CALM is a cytoplasmic protein, the chimaeric fusion product is able to localise to both the nucleus and cytoplasm. Analysis of the fusion variants suggests, however, that the critical fusion product is likely to be cytoplasmic and contain the interactive leucine zipper of AF10.

Is there a danger in delaying radiotherapy in childhood medulloblastoma?

Approximately 45-50% of children with medulloblastoma are cured by conventional surgery and radiotherapy, but survivors may face severe late neuropsychological toxicity. Studies showing good partial responses to platinum-based chemotherapy in relapsed patients and the theoretical possibility of a therapeutic window immediately after surgery have prompted neoadjuvant treatment studies which are ongoing. However, the absolute benefit of chemotherapy for the treatment of medulloblastoma in childhood is, as yet, not proven. There is a danger that chemotherapy may simply delay radiotherapy, and in so doing reduce the radiological impact of this known effective treatment. We report four children with medulloblastoma presenting consecutively to this unit over a 6-month period, whose management was problematic because of either failure to respond to neoadjuvant chemotherapy or their very young age. These cases are discussed in the light of the current literature and future treatment strategies that must seek to improve the therapeutic ratio of multimodality therapy.

Detection of rabies virus in different tissues of experimentally infected mice at preclinical and postclinical stages of the disease.

Rabies fixed virus (CVS) was passaged 10 times in mice by intramuscular (im) route followed by experimental inoculation of the titrated virus in 4 groups of mice with the dose of 0.1 ml of 1000 mouse (LD50 0.03 ml) using intracerebral (ic), intravenous (iv), intramuscular (im), intraocular (io), and intranasal (in) routes respectively. No marked variation in clinical signs due to variation of routes could be detected. Involvement of brain with io route could be detected even in preclinical stage. Although the virus could be detected in the postclinical stage in all the tissues under study (brain, skin, salivary gland and corneal impression), with io and ic routes spread of the virus was observed in comparatively higher concentrations.

An odyssey in search of a cure: the evolution of treatment of childhood acute lymphoblastic leukemia in the United Kingdom.

This review charts the evolution of therapy for childhood acute lymphoblastic leukaemia (ALL) in the United Kingdom. The present chemotherapeutic regimen is the result of experience gained from carefully planned randomised cooperative studies carried out during the last two decades. In common with the experience of the West German and American groups, the best results have been in those treated with post remission intensification blocks. With current chemotherapy protocols, almost 70% of children with ALL in U.K. can be cured but there may be a medical cost of such a cure, in terms of both acute and long term toxicity. This was especially true when central nervous system (CNS) therapy with cranial irradiation was used. Therefore present regimens are examining chemotherapeutic options for CNS disease control and the efficacy of additional post remission intensification. Failure of chemotherapy is most often seen in those children with a presenting white cell count of more than 50 x 10(9)/l, very young children and/or the presence of certain chromosomal rearrangements (e.g. t4: 11, t9: 22). At present the optimum therapeutic option for such high risk patients and for the majority of those in second remission, is an allogenic bone marrow transplant if an HLA-matched sibling is available. Modern day therapy is both complicated and costly and will be beyond the resources available for most children with ALL in developing countries. A significant decrease in worldwide mortality due to ALL will only occur if either the disease can be prevented or a simpler cure devised.

An audit of the activities of the paediatric oncology unit in Edinburgh, 1982-1991.

The activities of the paediatric oncology unit at the Royal Hospital for Sick Children, Edinburgh during 1982-1991 were audited. There were 254 new patients of whom 240 had a malignancy, seven had Langerhans cell histiocytosis and seven had non malignant tumours. Of the 254,131 (52%), 78 (31%), 14 (65), were normally resident within the Lothian, Fife and Borders regions respectively. Therefore, although the unit is a part of the Lothian Health Board, it is in practice, the referral centre for S.E. Scotland. A majority (92%) of patients were treated according to national or international protocols during the last five years and of the 240 children diagnosed to have cancer, 158 (66%) are at present in clinical remission. These figures compare favourably with national averages for protocol compliance and survival. During these ten years, an additional 69 children with cancer from S.E. Scotland were treated at other centres. Therefore, while the unit justifies its existence it has yet to realise its full potential.

Erg and AP-1 as determinants of glucocorticoid response in acute lymphoblastic leukemia.

Glucocorticoids (GCs) are among the most widely prescribed medications in clinical practice. The beneficial effects of GCs in acute lymphoblastic leukemia (ALL) are based on their ability to induce apoptosis, but the underlying transcriptional mechanisms remain poorly defined. Computational modeling has enormous potential in the understanding of biological processes such as apoptosis and the discovery of novel regulatory mechanisms. We here present an integrated analysis of gene expression kinetic profiles using microarrays from GC sensitive and resistant ALL cell lines and patients, including newly generated and previously published data sets available from the Gene Expression Omnibus. By applying time-series clustering analysis in the sensitive ALL CEM-C7-14 cells, we identified 358 differentially regulated genes that we classified into 15 kinetic profiles. We identified GC response element (GRE) sequences in 33 of the upregulated known or potential GC receptor (GR) targets. Comparative study of sensitive and resistant ALL showed distinct gene expression patterns and indicated unexpected similarities between sensitivity-restored and resistant ALL. We found that activator protein 1 (AP-1), Ets related gene (Erg) and GR pathways were differentially regulated in sensitive and resistant ALL. Erg protein levels were substantially higher in CEM-C1-15-resistant cells, c-Jun was significantly induced in sensitive cells, whereas c-Fos was expressed at low levels in both. c-Jun was recruited on the AP-1 site on the Bim promoter, whereas a transient Erg occupancy on the GR promoter was detected. Inhibition of Erg and activation of GR lead to increased apoptosis in both sensitive and resistant ALL. These novel findings significantly advance our understanding of GC sensitivity and can be used to improve therapy of leukemia.

5T4 oncofetal antigen is expressed in high risk of relapse childhood pre-B acute lymphoblastic leukemia and is associated with a more invasive and chemotactic phenotype.

Although the overall prognosis in childhood acute lymphoblastic leukemia (ALL) is good, outcome after relapse is poor. Recurrence is frequently characterized by the occurrence of disease at extramedullary sites, such as the central nervous system and testes. Subpopulations of blasts able to migrate to such areas may have a survival advantage and give rise to disease recurrence. Gene expression profiling of 85 diagnostic pre-B-ALL bone marrow samples revealed higher 5T4 oncofetal antigen transcript levels in cytogenetic high-risk subgroups of patients (P<0.001). Flow cytometric analysis determined that bone marrow from relapse patients have a significantly higher percentage of 5T4-positive leukemic blasts than healthy donors (P=0.005). The high-risk Sup-B15 pre-B-ALL line showed heterogeneity in 5T4 expression, and the derived, 5T4(+) (Sup5T4) and 5T4(-) (Sup) subline cells, displayed differential spread to the omentum and ovaries following intraperitoneal inoculation of immunocompromised mice. Consistent with this, Sup5T4 compared with Sup cells show increased invasion in vitro concordant with increased LFA-1 and VLA-4 integrin expression, adhesion to extracellular matrix and secretion of matrix metalloproteases (MMP-2/-9). We also show that 5T4-positive Sup-B15 cells are susceptible to 5T4-specific superantigen antibody-dependent cellular toxicity providing support for targeted immunotherapy in high-risk pre-B-ALL.

Temporal changes in the incidence and pattern of central nervous system relapses in children with acute lymphoblastic leukaemia treated on four consecutive Medical Research Council trials, 1985-2001.

Despite the success of contemporary treatment protocols in childhood acute lymphoblastic leukaemia (ALL), relapse within the central nervous system (CNS) remains a challenge. To better understand this phenomenon, we have analysed the changes in incidence and pattern of CNS relapses in 5564 children enrolled in four successive Medical Research Council-ALL trials between 1985 and 2001. Changes in the incidence and pattern of CNS relapses were examined and the relationship with patient characteristics was assessed. The factors affecting outcome after relapse were determined. Overall, relapses declined by 49%. Decreases occurred primarily in non-CNS and combined relapses with a progressive shift towards later (> or =30 months from diagnosis) relapses (P<0.0001). Although isolated CNS relapses declined, the proportional incidence and timing of relapse remained unchanged. Age and presenting white blood cell (WBC) count were risk factors for CNS relapse. On multivariate analysis, the time to relapse and the trial period influenced outcomes after relapse. Relapse trends differed within biological subtypes. In ETV6-RUNX1 ALL, relapse patterns mirrored overall trends whereas in high hyperdiploidy (HH) ALL, these seem to have plateaued over the latter two trial periods. Intensive systemic and intrathecal chemotherapy have decreased the overall CNS relapse rates and changed the patterns of recurrence. The heterogeneity of therapeutic response in the biological subtypes suggests room for further optimization using currently available chemotherapy.