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PURPOSE: Results are presented of the SIOP study MMT-98 for paediatric metastatic rhabdomyosarcoma (RMS), which evaluated intensive chemotherapy followed by low intensity 'maintenance' chemotherapy in standard risk patients (SRG). For poor risk patients (PRG), the value of a therapeutic window study, sequential high dose monotherapy to achieve a complete response (CR) followed by low dose maintenance chemotherapy was examined. PATIENTS AND METHODS: From November 1998 to 2005, 146 patients aged 6 months to 18 years with metastatic RMS were entered. Forty-five were SRG, i.e. age<10 years and no bone marrow or bone involvement. Treatment was a 6-drug regimen with local therapy of surgery and/or radiotherapy followed by maintenance of 9 courses of vincristine, actinomycin D and cyclophosphamide (VAC). One hundred and one patients were PRG, i.e. >10 years, or with bone marrow or bone metastases. An upfront window study, high dose monotherapy, local treatment and then VAC maintenance therapy were given. RESULTS: With a median follow-up of 1.52 years, the 3-year event-free survival (EFS) and overall survival (OS) for SRG were 54.92% and 62.14%, respectively, whilst for the PRG 16.17% and 23.17%. The corresponding adverse hazard ratio (HR) for the PRG was HR=2.65 (95% CI 1.63-4.31, p-value<0.001) for EFS and HR=2.51 (CI 1.53-4.11, p-value<0.001) for OS. CONCLUSION: SRG patients' EFS and OS were comparable to those of previous studies. For PRG patients there was no improvement in survival. [ABSTRACT FROM AUTHOR]

Background: EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines., Methods: EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts., Results: Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts., Conclusions: Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

Neuroblastoma (NB) originates from neural crest-derived precursors and represents the most common childhood extracranial solid tumour. MicroRNAs (miRNAs), a class of small non-coding RNAs that participate in a wide variety of biological processes by regulating gene expression, appear to play an essential role within the NB context. High-throughput next generation sequencing (NGS) was applied to study the miRNA transcriptome in a cohort of NB tumours with and without MYCN-amplification (MNA and MNnA, respectively) and in dorsal root ganglia (DRG), as a control. Out of the 128 miRNAs differentially expressed in the NB vs. DRG comparison, 47 were expressed at higher levels, while 81 were expressed at lower levels in the NB tumours. We also found that 23 miRNAs were differentially expressed in NB with or without MYCN-amplification, with 17 miRNAs being upregulated and 6 being downregulated in the MNA subtypes. Functional annotation analysis of the target genes of these differentially expressed miRNAs demonstrated that many mRNAs were involved in cancer-related pathways, such as DNA-repair and apoptosis as well as FGFR and EGFR signalling. In particular, we found that miR-628-3p negatively affects MYCN gene expression. Furthermore, we identified a novel miRNA candidate with variable expression in MNA vs. MNnA tumours, whose putative target genes are implicated in the mTOR pathway. The present study provides further insight into the molecular mechanisms that correlate miRNA dysregulation to NB development and progression.

The role of histone deacetylase (HDAC) 4 and 6 in glioblastoma (GBM) radioresistance was investigated. We found that tumor samples from 31 GBM patients, who underwent temozolomide and radiotherapy combined treatment, showed HDAC4 and HDAC6 expression in 93.5% and 96.7% of cases, respectively. Retrospective clinical data analysis demonstrated that high-intensity HDAC4 and/or HDAC6 immunostaining was predictive of poor clinical outcome. In vitro experiments revealed that short hairpin RNA-mediated silencing of HDAC4 or HDAC6 radiosensitized U87MG and U251MG GBM cell lines by promoting DNA double-strand break (DSBs) accumulation and by affecting DSBs repair molecular machinery. We found that HDAC6 knock-down predisposes to radiation therapy-induced U251MG apoptosis- and U87MG autophagy-mediated cell death. HDAC4 silencing promoted radiation therapy-induced senescence, independently by the cellular context. Finally, we showed that p53 WT expression contributed to the radiotherapy lethal effects and that HDAC4 or HDAC6 sustained GBM stem-like radioresistant phenotype. Altogether, these observations suggest that HDAC4 and HDAC6 are guardians of irradiation-induced DNA damages and stemness, thus promoting radioresistance, and may represent potential prognostic markers and therapeutic targets in GBM., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway.Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS.

Background: Rhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype. This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied., Methods: RH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity., Results: In vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival. Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively. The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential., Conclusions: These results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment.

Background: Rhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children. MicroRNAs (miRNAs) represent a class of short, non-coding, regulatory RNAs which play a critical role in different cellular processes. Altered miRNA levels have been reported in human cancers, including RMS., Methods: Using deep sequencing technology, a total of 685 miRNAs were investigated in a group of alveolar RMSs (ARMSs), embryonal RMSs (ERMSs) as well as in normal skeletal muscle (NSM). Q-PCR, MTT, cytofluorimetry, migration assay, western blot and immunofluorescence experiments were carried out to determine the role of miR-378a-3p in cancer cell growth, apoptosis, migration and differentiation. Bioinformatics pipelines were used for miRNA target prediction and clustering analysis., Results: Ninety-seven miRNAs were significantly deregulated in ARMS and ERMS when compared to NSM. MiR-378 family members were dramatically decreased in RMS tumour tissue and cell lines. Interestingly, members of the miR-378 family presented as a possible target the insulin-like growth factor receptor 1 (IGF1R), a key signalling molecule in RMS. MiR-378a-3p over-expression in an RMS-derived cell line suppressed IGF1R expression and affected phosphorylated-Akt protein levels. Ectopic expression of miR-378a-3p caused significant changes in apoptosis, cell migration, cytoskeleton organization as well as a modulation of the muscular markers MyoD1, MyoR, desmin and MyHC. In addition, DNA demethylation by 5-aza-2'-deoxycytidine (5-aza-dC) was able to up-regulate miR-378a-3p levels with a concomitant induction of apoptosis, decrease in cell viability and cell cycle arrest in G2-phase. Cells treated with 5-aza-dC clearly changed their morphology and expressed moderate levels of MyHC., Conclusions: MiR-378a-3p may function as a tumour suppressor in RMS and the restoration of its expression would be of therapeutic benefit in RMS. Furthermore, the role of epigenetic modifications in RMS deserves further investigations.

Wilms' tumor (WT) is a common childhood renal cancer. A 25-year single center UK experience is reported. During 1985-2010, 97 children underwent immediate nephrectomy or delayed resection of tumor after chemotherapy. Survival, morbidity, and late effects following treatment are described. Tumor distribution was: Stage I, 25.7% (n = 25); Stage II, 24.7% (n = 24); Stage III, 26.8% (n = 26); Stage IV, 17.5% (n = 17); and Stage V, 5.2% (n = 5). Immediate nephrectomy was performed in 39% (n = 38) patients with elective delayed resection in 61% (n = 59) cases. Ten patients had cavotomy to excise tumor involving vena cava territory. Two cases required cardiopulmonary bypass. Tumor rupture was recorded in eight (8.5%) total operated cases-after immediate (n = 5/37), 13.5% vs delayed nephrectomy-(n = 3/57), 5.2%; X(2) P = .154. From 2001 onwards, one case of tumor rupture was recorded at this center after the universal adoption of UKW3 and SIOP guidelines advocating preoperative chemotherapy and delayed nephrectomy for all WT. Three treatment-related deaths occurred-hepatic veno-occlusive disease (n = 2) with actinomycin D and a single WT fatality due to vascular injury. Overall survival was 84.5% (82/97 cases). Two patients developed "late malignancies" -thyroid cancer and a basal cell carcinoma. This study demonstrates excellent survival for WT comparable with national outcomes and international cooperative studies. Adverse events with chemotherapy and surgery, including "late onset," second malignancies deserve special consideration.

Childhood neuroblastic tumors are characterized by heterogeneous clinical courses, ranging from benign ganglioneuroma (GN) to highly lethal neuroblastoma (NB). Although a refined prognostic evaluation and risk stratification of each tumor patient is becoming increasingly essential to personalize treatment options, currently only few biomolecular markers (essentially MYCN amplification, chromosome 11q status and DNA ploidy) are validated for this purpose in neuroblastic tumors. Here we report that Galectin-3 (Gal-3), a β-galactoside-binding lectin involved in multiple biological functions that has already acquired diagnostic relevance in specific clinical settings, is variably expressed in most differentiated and less aggressive neuroblastic tumors, such as GN and ganglioneuroblastoma, as well as in a subset of NB cases. Gal-3 expression is associated with the INPC histopathological categorization (P<0.001) and Shimada favorable phenotype (P=0.001), but not with other prognostically relevant features. Importantly, Gal-3 expression was associated with a better 5-year overall survival (P=0.003), and with improved cumulative survival in patient subsets at worse prognosis, such as older age at diagnosis, advanced stages or NB histopathological classification. In vitro, Gal-3 expression and nuclear accumulation accompanied retinoic acid-induced cell differentiation in NB cell lines. Forced Gal-3 overexpression increased phenotypic differentiation and substrate adherence, while inhibiting proliferation. Altogether, these findings suggest that Gal-3 is a biologically relevant player for neuroblastic tumors, whose determination by conventional immunohistochemistry might be used for outcome assessment and patient's risk stratification in the clinical setting.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. β-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/β-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed β-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of β-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including β-catenin, glycogen synthase kinase-3β, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of β-catenin, stabilization of the active cytosolic form and nuclear translocation of β-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/β-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target.

Background: The adverse prognostic impact of elevated levels of circulating Vascular Endothelial Growth Factor (VEGF) is described in several malignancies. However, no information is available in childhood rhabdomyosarcoma (RMS). In the present study, serum VEGF-A (sVEGF-A) was measured at diagnosis in a series of patients with RMS., Procedure: sVEGF-A was assessed retrospectively in 17 newly diagnosed RMS patients. sVEGF-A concentrations were determined by quantitative enzyme-linked immunoabsorbent ELISA kit and their possible associations with age at diagnosis, gender, histology, primary site, primary size, Intergroup Rhabdomyosarcoma Study (IRS) post-surgical group, and outcome were investigated., Results: sVEGF-A median value in patients with RMS was significantly higher than in controls: 499.0 pg/ml, range: 2,648.0 versus 301.5 pg/ml, range: 716.0 (P = 0.013). Although not statistically significant probably due to the limited number of patients, sVEGF-A median levels resulted higher in unfavorable primary sites (277.0 vs. 539.0 pg/ml; P = 0.31), and advanced groups (390.0 vs. 715.0; P = 0.29). Patients with shorter 5-year overall survival (OS) and 5-year progression-free survival (PFS) times also had higher sVEGF-A levels, although again the difference was not statistically significant (P = 0.18 and P = 0.22, respectively)., Conclusions: Circulating VEGF is significantly increased in pediatric patients with newly diagnosed RMS. Further studies in larger series of RMS patients are needed to understand whether measurements of circulating VEGF might have a role in assessing prognosis and modulating treatment., (Copyright © 2012 Wiley Periodicals, Inc.)

The p53 gene is rarely mutated in neuroblastoma, but codon 72 polymorphism that modulates its proapoptotic activity might influence cancer risk and clinical outcome. We investigated whether this polymorphism affects neuroblastoma risk and disease outcome and assessed the biologic effects of the p53-72R and p53-72P isoforms in p53-null cells. Comparison of 288 healthy subjects and 286 neuroblastoma patients revealed that the p53-72 polymorphism had no significant impact on the risk of developing neuroblastoma; however, patients with the Pro/Pro genotype had a shorter survival than those with the Arg/Arg or the Arg/Pro genotypes even in the stage 3 and 4 subgroup without MYCN amplification. By Cox regression analysis, the p53 Pro/Pro genotype seems to be an independent marker of poor prognosis (hazard ratio = 2.74; 95% confidence interval = 1.14-6.55, P = .014) together with clinical stage, MYCN status, and age at diagnosis. In vitro, p53-72P was less effective than p53-72R in inducing apoptosis and inhibiting survival of p53-null LAN-1 cells treated with etoposide, topotecan, or ionizing radiation but not taxol. By contrast, p53-72P was more effective in promoting p21-dependent accelerated senescence, alone or in the presence of etoposide. Thus, the p53-72 Pro/Pro genotype might be a marker of poor outcome independent of MYCN amplification, possibly improving risk stratification. Moreover, the lower apoptosis and the enhanced accelerated senescence by the p53-72P isoform in response to DNA damage suggest that patients with neuroblastoma with the p53-72 Pro/Pro genotype may benefit from therapeutic protocols that do not rely only on cytotoxic drugs that function, in part, through p53 activation.

Purpose: The role of surgery in the management of neuroblastoma yields conflicting reports. We report a 20-year experience from a UK centre in the context of evolving cancer therapies for neuroblastoma., Methods: Hospital records of 91 neuroblastoma patients from 1985 to 2005 were studied. Patient demographics, data from operating notes and tumour biology (MYCN status) where available were analysed., Results: Surgery consisted of primary resection or delayed operation following tumour biopsy/chemotherapy. Overall survival was 100% for stage 1(n = 3), 90% for stage 2 (n = 10), 46% for stage 3 (n = 13), 13% for stage 4 (n = 55) and 56% for stage 4S disease (n = 9). During the eras 1985-1994 versus 1995-2005, survival for stage 3 lesions was 25% and 80% (P = 0.04) with marginal increase in survival observed in stage 4 disease (12% vs. 22%, P = 0.083). Delayed tumour resection was not performed in 20 (36%) stage 4 patients due to progressive disease. Complete tumour resection was achieved in 62% of stage 3-4 patients during 1995-2005 compared to 38% in 1985-1994. The extent of surgical resection (complete vs. partial) showed no significant differences in overall survival or relapse rates. Postoperative morbidity occurred in 15.7% of cases emphasising technical challenges in resection of neuroblastoma. No child with MYCN amplification survived versus 59% survival in non-amplified cases (P = 0.012)., Conclusions: While complete tumour resection may be desirable in advanced neuroblastoma (stage 3-4) these findings suggest that the radicality of operation is not significantly associated with better overall survival/relapse. Improving outcomes in the 1995-2005 era for patients with stage 3-4 tumours complements the introduction of new high dose-intensive chemotherapy regimens and other adjuvant therapies for this enigmatic disease., (Copyright © 2011 Wiley Periodicals, Inc.)

MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53(S46) kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53(S46) phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53(S46) phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma., (©2010 AACR.)

Aim: Activity and toxiciy of gefitinib in combination with topotecan and cyclophosphamide (CPA) were evaluated in a case-series of relapsed neuroblastoma (NB) patients. The in vitro activity of the combination was also assessed., Procedure: Gefitinib (250 mg/day), topotecan (0.8 mg/m(2)/day), and CPA (50 mg/m(2)/day) (GTC) were administered orally for 14 consecutive days out of 28 days. Antitumor activity of gefitinib as single agent and in combination with either topotecan or CPA was assessed in a panel of NB cell lines., Results: Ninety-two courses were given in 10 patients. Grade 4 neutropenia was observed in 7/92 courses (8%) and grade 4 thrombocytopenia in 8/92 (9%). Two patients had a grade 2 liver toxicity, four a grade 1/2 skin toxicity, and two a grade 1/2 diarrhea. Dose reduction of topotecan and/or CPA was required in eight patients. After four courses, three patients were in partial response (PR) and four with a stable disease (SD), while three experienced a progressive disease (PD). Time to progression (TTP) was 9 months (range, 1-27). After a median follow-up of 16 months (range 5-54), seven patients are died of disease (DOD) and three alive with disease (AWD). All but one patient discontinued oral chemotherapy because of a PD, whilst one patient stopped chemotherapy after 27 months with a SD. In vitro, gefitinib was synergistic with topotecan and additive with CPA., Conclusion: The GTC combination was well tolerated and the TTP was encouraging. These promising results, also supported by in vitro evidence, should be further confirmed in a phase II study., (Copyright 2009 Wiley-Liss, Inc.)

MicroRNAs are a class of sophisticated regulators of gene expression, acting as post-transcriptional inhibitors that recognize their target mRNAs through base pairing with short regions along the 3'UTRs. Several microRNAs are tissue specific, suggesting a specialized role in tissue differentiation or maintenance, and quite a few are critically involved in tumorigenesis. We studied miR-128, a brain-enriched microRNA, in retinoic acid-differentiated neuroblastoma cells, and we found that this microRNA is up-regulated in treated cells, where it down-modulates the expression of two proteins involved in the migratory potential of neural cells: Reelin and DCX. Consistently, miR-128 ectopic overexpression suppressed Reelin and DCX, whereas the LNA antisense-mediated miR-128 knockdown caused the two proteins to increase. Ectopic miR-128 overexpression reduced neuroblastoma cell motility and invasiveness, and impaired cell growth. Finally, the analysis of a small series of primary human neuroblastomas showed an association between high levels of miR-128 expression and favorable features, such as favorable Shimada category or very young age at diagnosis. Thus, we provide evidence for a role for miR-128 in the molecular events modulating neuroblastoma progression and aggressiveness.

Rhabdomyosarcoma (RMS) is a common childhood solid tumor, resulting from dysregulation of the skeletal myogenesis program. Two major histological subtypes occur in childhood RMS, embryonal and alveolar. While chromosomal rearrangements account for the majority of alveolar tumors, the genetic defects underlying the pathogenesis of embryonal RMS remain largely undetermined. A few studies performed on small series of embryonal tumors suggest that dysregulation of RAS function may be relevant to disease pathogenesis. To explore further the biological and clinical relevance of mutations with perturbing consequences on RAS signaling in embryonal RMS, we investigated the prevalence of PTPN11, HRAS, KRAS, NRAS, BRAF, MEK1, and MEK2 mutations in a relatively large cohort of primary tumors. While HRAS and KRAS were found to be rarely mutated, we identified somatic NRAS lesions in 20% of cases. All mutations were missense and affected codon 61, with the introduction of a positive charged amino acid residue representing the most common event. PTPN11 was found mutated in one tumor specimen, confirming that somatic defects in this gene are relatively uncommon in RMS, while no mutation was observed in BRAF and MEK genes. Although no clear association of mutations with any clinical variable was observed, comparison of the outcome between mutation-positive and mutation-negative cases indicated a trend for a higher percentage of patients exhibiting a better outcome in the former. Our findings provide evidence that dysregulation of RAS signaling is a major event contributing to embryonal RMS pathogenesis., (Copyright 2009 Wiley-Liss, Inc.)

Purpose: The CXC chemokine receptor-4 (CXCR4)/stromal-derived factor-1 and c-Met/hepatocyte growth factor axes promote the metastatic potential of rhabdomyosarcoma cell lines in experimental models, but no data are available on their role in rhabdomyosarcoma tumors. The expressions of CXCR4 and c-Met were evaluated in primary tumors and isolated tumor cells in marrow, and were correlated with clinicopathologic variables and survival., Experimental Design: Forty patients with recently diagnosed rhabdomyosarcoma were retrospectively enrolled. CXCR4 and c-Met expression was investigated in primary tumors by immunohistochemistry, in isolated marrow-infiltrating tumor cells using double-label immunocytology. Results were expressed as the mean percentage of immunostained tumor cells., Results: CXCR4 and c-Met were expressed in >/=5% of tumor cells from 40 of 40 tumors, with 14 of 40 cases showing >/=50% of immunostained tumor cells (high expression). High CXCR4 expression correlated with alveolar histology (P = 0.006), unfavorable primary site (P = 0.009), advanced group (P < 0.001), marrow involvement (P = 0.007), and shorter overall survival and event-free survival (P < 0.001); high c-Met expression correlated with alveolar histology (P = 0.005), advanced group (P = 0.04), and marrow involvement (P = 0.02). In patients with a positive diagnosis for isolated tumor cells in marrow (n = 16), a significant enrichment in the percentage of CXCR4-positive (P = 0.001) and c-Met-positive (P = 0.003) tumor cells was shown in marrow aspirates compared with the corresponding primary tumors., Conclusions: CXCR4 and c-Met are widely expressed in both rhabdomyosarcoma subtypes and, at higher levels, in isolated marrow-infiltrating tumor cells. High levels of expression are associated with unfavorable clinical features, tumor marrow involvement and, only for CXCR4, poor outcome. In rhabdomyosarcoma, CXCR4 and c-Met represent novel exploitable targets for disease-directed therapy.

Purpose: MDM2 is a key negative regulator of p53 activity, and a single nucleotide polymorphism (SNP309, T>G change; rs 2279744) in its promoter increases the affinity for the transcription factor SP1, enhancing MDM2 expression. We carried out a pilot study to investigate the effect of this polymorphism on development and behavior of neuroblastoma, an extracranial pediatric tumor with unfrequent genetic inactivation of p53., Experimental Design: We genotyped the MDM2-SNP309 alleles of tumor DNA from 239 neuroblastoma patients and peripheral blood DNA from 237 controls. In 40 of 239 neuroblastomas, the MDM2-SNP309 alleles were also genotyped in peripheral blood DNA. Data were analyzed by two-sided Fisher's exact test, log-rank test, and Kaplan-Meier statistics. Where appropriate, data are reported with 95% confidence intervals (CI)., Results: The frequency of both the T/G and G/G genotypes or the G/G or T/G genotype only was higher in neuroblastoma DNA samples than in controls: 60.3% (95% CI, 54.1-66.5) versus 47.3% (95% CI, 40.9-53.6), 30.4% (95% CI, 22.4-37.8) versus 15.0% (95% CI, 9.2-20.7), and 52.0% (95% CI, 45.0-59.9) versus 41.9% (95% CI, 35.3-48.5), respectively; Two-Sided Fisher's Exact Test P values were 0.006, 0.003, and 0.048, respectively; Odds ratios were 1.69 (95% CI, 1.18-2.43), 2.45 (95% CI, 1.37-4.39) and 1.51 (95% CI, 1.02-2.22), respectively. A significant association (P = 0.016) between heterozygous (T/G)/homozygous (G/G) genotypes at SNP309 and advanced clinical stages was also shown. Homozygous/heterozygous SNP309 variant carriers had a shorter 5-year overall survival than patients with the wild-type allele (P = 0.046; log-rank test). A shorter overall survival in patients with heterozygous/homozygous SNP309 was also observed in the subgroups with age at diagnosis >1 year and adrenal primary tumor (P = 0.024 and P = 0.014, respectively)., Conclusions: Data from this pilot study suggest that the MDM2 G/G and T/G-SNP309 alleles are markers of increased predisposition to tumor development and disease aggressiveness in neuroblastoma. However, additional studies with larger patient cohorts are required for a definitive assessment of the clinical relevance of these data.

Background: Increased expression of Eph receptors and their ephrin ligands has been implicated in promoting angiogenesis and tumour progression in several malignancies. Here the expression of mRNA for ephrin-B and EphB receptors in rhabdomyosarcoma (RMS) cell lines and primary tumours was investigated., Materials and Methods: Expression of mRNA for ephrin-B and EphB receptors in RMS cell lines and primary tumours was measured by real-time RT-PCR and compared with the expression in normal striated muscle., Results: A dysregulation of both ligands and receptors was found in all cell lines. In embryonal tumours, overexpression of ephrin-B1 correlated with overexpression of EphB1 (r = 0.97, p < 0.01) and EphB3 (r = 0.94, p < 0.05); overexpression of ephrin-B2 correlated with overexpression of EphB1 (r = 0.94, p < 0.05), EphB2 (r = 0.88, p < 0.01) and EphB4 (r = 0.76, p < 0.01). In alveolar tumours, no similar correlations were found. A correlation between EphB2 and EphB4 receptors was demonstrated in both tumour types, being positive in embryonal cases (r = 0.81, p < 0.01) and negative in alveolar (r = -1.00, p < 0.01)., Conclusion: A global up-regulation of ephrin-B and EphB receptors in RMS tumours was found. The correlation between EphB2 and EphB4 receptors suggests a possible role for ephrin-B and EphB receptors in RMS development.

Permanent alopecia can occur following treatment for pediatric malignant disease, especially cranial irradiation, resulting in identity and self-image problems. This late effect is usually addressed through external cosmesis and psychological adjustment. Surgical options are less commonly utilized. The experience of reconstructive procedures in patients at RLC NHS Trust, Alder Hey with alopecia is presented. Four patients had scalp tissue expansion and one had hair transplantation. The reconstructive options available are discussed as well as the potential opportunities and difficulties in this population. Post-radiotherapy alopecia can be successfully addressed by reconstructive surgery, and should be considered more often in this population., ((c) 2007 Wiley-Liss, Inc.)

High levels of PDGFR expression in primary rhabdomyosarcoma (RMS) have been associated with disease progression. To date however, there are no reports on the activity of imatinib mesylate, a selective PDGFR inhibitor, in RMS preclinical models. A panel of 5 RMS cell lines was used to investigate the expression of PDGFRalpha and PDGFRbeta, c-Kit and the multidrug transporter ABCG2 (also inhibited by imatinib). In vitro and in vivo experiments were performed using RD (embryonal) and RH30 (alveolar) cell lines to determine the efficacy of imatinib as single agent and in combination with topotecan (TPT). PDGFRbeta was significantly expressed in all cell lines, with the highest levels in RD, while PDGFR alpha and ABCG2 were significantly expressed only in RH30 and RMZ-RC2. c-Kit was not detected. PDGFRbeta signaling was active in RD but not in RH30, whilst PDGFRalpha signaling was not active in either cell lines. Significant ABCG2-mediated extrusion of Hoechst 33342 was demonstrated in RH30 but not in RD, and was inhibited by imatinib and the specific ABCG2 inhibitor Ko143. In vitro, imatinib was not active as a single agent at therapeutic concentrations, but significantly potentiated TPT antitumor activity in both cell lines. In vivo experiments using tumor xenografts confirmed the synergistic interaction in both cell lines. These results suggest that at least 2 different mechanisms--inhibition of ABCG2 and/or PDGFRbeta--are involved in the synergistic interaction between imatinib and TPT, and support the use of this combination for the treatment of high-risk RMS patients., (Copyright 2006 Wiley-Liss, Inc.)

Purpose: Signaling through insulin-like growth factor I receptor (IGF-IR) is important for growth and survival of many tumor types. Neuroblastoma is sensitive to IGF., Experimental Design: We assessed the ability of NVP-AEW541, a recently developed small molecule that selectively inhibits IGF-IR activity, for neuroblastoma growth effects in vitro and in vivo. Our data showed that, in a panel of 10 neuroblastoma cell lines positive for IGF-IR expression, NVP-AEW541 inhibited in vitro proliferation in a submicromolar/micromolar (0.4-6.8) range of concentrations., Results: As expected, NVP-AEW541 inhibited IGF-II-mediated stimulation of IGF-IR and Akt. In addition to growth inhibition, the drug also induced apoptosis in vitro. Oral administration of NVP-AEW541 (50 mg/kg twice daily) inhibited tumor growth of neuroblastoma xenografts in nude mice. Analysis of tumors from the drug-treated animals revealed a marked apoptotic pattern and a decrease in microvascularization compared with controls. Interestingly, quantitative real-time PCR detected both in vitro and in vivo a significant down-regulation of mRNA for vascular endothelial growth factor (VEGF) caused by NVP-AEW541. In addition, in Matrigel-coated chambers and in severe combined immunodeficient mice tail vein injected with neuroblastoma cells, tumor invasiveness was significantly reduced by this agent. Analysis of IGF-IR expression in a series of 43 neuroblastoma primary tumors revealed IGF-IR positivity in 86% of cases., Conclusions: Taken together, these data indicate that NVP-AEW541 can be considered as a novel promising candidate for treatment of neuroblastoma patients.

Background: Sacrococcygeal teratoma (SCT) is uncommon (1:35,000-1:40,000 newborns). We report a 25-year single-center experience with a focus on late effects., Methods: Surgical and tumor registries identified patients with SCT between 1977 and 2001. Perinatal data, associated anomalies, operative findings, histology, and survival were recorded. Continence was assessed clinically. Urodynamics and anorectal manometry were performed as indicated., Results: Thirty-three patients (28 females) were treated for SCT. Before 1988, 2 of 18 were diagnosed antenatally compared with 8 of 15 between 1988 and 2001. Ten babies were delivered by cesarean birth. Seven children presented after the neonatal period. Surgery comprised tumor excision with coccygectomy. Histology was benign in 26 (79%), malignant in 6 (18%), and immature in a single patient. Presentation beyond the newborn period was associated with malignant disease and poorer outcome. Overall survival was 94%. Neuropathic bladder or bowel disturbance was identified in 7 of 20 patients on long-term follow-up., Conclusions: Antenatal diagnosis of SCT appears to be increasing. Parental counseling should include the continence problems that may follow removal of even benign tumors. Resection by surgical oncologists and reconstruction by colorectal specialists may improve function. Follow-up by oncologists, surgeons, and urologists remains an important part of SCT management.

Multiple defects in apoptotic pathways have been described in peripheral neuroblastic tumours (NTs). Mitosis-karyorrhexis index (MKI) is a reliable morphological marker identifying favourable and unfavourable NTs. The extent to which apoptotic processes contribute to determine the clinical significance of MKI is still undefined. Apoptosis was investigated in a series of 110 peripheral NTs by comparing MKI to immunohistochemical and molecular apoptotic features. High MKI was found in 55 out of 110 NTs (50%) and was associated with advanced stage (P = 0.007), neuroblastoma (NB) histological category (P = 0.024), MYCN amplification (P < 0.001), and poor outcome (P = 0.011). Overall survival probability was 45% in patients with high MKI compared to 73% in patients with low MKI. In the same 110 NTs, the expression of Bcl-2, Bcl-XL, Bax and Mcl-1 was studied by immunohistochemistry, but no significant associations were found with clinicohistological features. Microarray analysis of apoptotic genes was performed in 40 out of 110 representative tumours. No significant association was found between the expression of apoptotic genes and MKI or clinicohistological features. Proliferative activity was assessed in 60 out of 110 representative tumours using Ki67 immunostaining, but no significant correlations with MKI or clinicobiological features were found. In NTs, the combination of apoptosis and proliferation as expressed by MKI is a significant prognostic parameter, although neither of them is per se indicative of the clinicobiological behaviour and outcome.

The PTPN11 gene encodes SHP-2, a widely expressed cytoplasmic protein tyrosine phosphatase functioning as a signaling transducer. Germ-line PTPN11 mutations cause Noonan syndrome (NS), a developmental disorder characterized by an increased risk of malignancies. Recently, a novel class of activating mutations in PTPN11 has been documented as a somatic event in a heterogeneous group of leukemias. Because of the relatively higher prevalence of certain solid tumors in children with NS and the positive modulatory function of SHP-2 in RAS signaling, a wider role for activating PTPN11 mutations in cancer has been hypothesized. Here, we screened a number of solid tumors, including those documented in NS or in which deregulated RAS signaling occurs at significant frequency, for PTPN11 mutations. No disease-associated mutation was identified in rhabdomyosarcoma (n = 13), neuroblastoma (n = 32), melanoma (n = 50), thyroid (n = 85), and colon (n = 48) tumors; a novel missense change, promoting an increased basal phosphatase activity of SHP-2, was observed in one glioma specimen. Our data document that deregulated SHP-2 function does not represent a major molecular event in pediatric and adult tumors, further supporting our previous evidence indicating that the oncogenic role of PTPN11 mutations is cell-context specific.

Identification of patients with a poor prognosis for non-metastatic rhabdomyosarcoma (RMS) remains a clinical challenge. Prospective analysis for the presence of disseminated RMS cells in bone marrow at diagnosis, using immunocytochemistry, with MyoD1 and myogenin as markers, was carried out. Thirty-seven patients treated on RMS88 and RMS96 Italian protocols underwent staging investigations, and in addition marrow examination for occult tumour cells. All patients had negative marrow involvement using cytomorphology, but 10/37 were positive with immunostaining. With a median follow-up of 46 months (range, 12-115), 7 patients had died and 30 were disease-free. Overall survival probability was 92% in patients with no occult marrow infiltration, 47% with occult marrow infiltration (P=0.001); event-free survival probability was 89% in the former and 50% in the latter (P=0.01). Disseminated tumour cells are indicative of disease spread and are significantly linked to recurrence at distant sites and poorer outcome. Marrow examination at diagnosis using immunocytochemistry may be an additional tool to modulate treatment.

Wilms' tumour commonly presents with an abdominal mass and gross haematuria. Here, we present the novel application of paediatric renal arterial embolisation to control life-threatening haematuria in Wilms' tumour.

Purpose: Clinicobiological characteristics of neuroblastic tumor (NT) expressing c-kit tyrosine kinase receptor and/or its ligand, stem cell factor (SCF), are debated. This study aimed at investigating the clinicobiological features of primary NTs expressing c-kit and/or SCF in order to define the clinical relevance of selective therapeutic targeting., Experimental Design: c-Kit and SCF expression was studied in 168 NTs using immunohistochemistry and in 106 of 168 using Northern blot. Quantitative determination of c-kit expression in 54 additional NTs was also done using real-time reverse transcription-PCR. Correlations between c-kit and SCF expression and clinicobiological features were analyzed using chi2 test, univariate, and multivariate regression analyses., Results: c-Kit protein was detected in 21 of 168 NTs (13%) and its mRNA in 23 of 106 NTs (22%). SCF protein was shown in 30 of 106 NTs (28%) and its mRNA in 33 of 106 NTs (31%). No mutations in exon 11 of c-kit gene were identified. By univariate analysis, c-kit and SCF expression correlated with advanced stage, MYCN amplification, and 1p36 allelic loss. Cox simple regression analysis showed that overall survival probability was 17% in the c-kit-positive subset versus 68% in the negative (P < 0.001), 43% in the SCF-positive subset versus 78% in the negative (P < 0.001). When using real-time reverse transcription-PCR, significant levels of c-kit mRNA were found in 35 of 54 NTs (65%), but the correlations with clinicobiological features were no longer documented., Conclusions: c-Kit expression can be detected in the majority of primary NTs. High levels of expression are preferentially found in tumors with unfavorable clinicobiological variables. c-Kit may represent a useful therapeutic target in a subset of otherwise untreatable NTs.

Signaling by the transcription factor nuclear factor kappa B (NF-kappaB) involves its release from inhibitor kappa B (IkappaB) in the cytosol, followed by translocation into the nucleus. NF-kappaB regulation of IkappaBalpha transcription represents a delayed negative feedback loop that drives oscillations in NF-kappaB translocation. Single-cell time-lapse imaging and computational modeling of NF-kappaB (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased IkappaBalpha transcription. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF-kappaB signaling may thus depend on number, period, and amplitude of oscillations.

The overall survival of childhood rhabdomyosarcoma has improved dramatically over the past 10 years. Early diagnosis and appropriate referral to a specialised centre leading to an accurate and timely diagnosis reflects on overall outcome. Recent molecular studies have identified different biological subtypes resulting in the recognition of poorer subgroups and allowing more appropriate treatment to be administered. Clinical trials remain the cornerstone to further improve outcome, now carried out on an international basis.

Aim: Orbital rhabdomyosarcoma is the most common primary malignant orbital tumour in children and has a good prognosis. The purpose of this paper was to review the imaging and consequent treatment of patients with localized orbital rhabdomyosarcoma from around the U.K., Materials and Methods: Patients were identified through the U.K. Children's Cancer Study Group (UKCCSG) database. Investigations and therapy were dictated by the Malignant Mesenchymal Tumour '89 (MMT89) protocol. Imaging and radiological reports of 16 patients from 12 centres were reviewed. The number of patients receiving radiotherapy, timing of radiotherapy, and adherence to treatment protocols were assessed., Results: Local radiologists' reports and imaging techniques varied between sequential examinations and centres. The imaging was adequate for management. No reports quoted measurements of the tumours. Treatment protocols were not always followed rigidly with regard to a residual mass at day 80 post-diagnosis. However, the protocol was not explicit for all outcomes. Fifteen out of 16 patients eventually received radiotherapy., Conclusion: There is no standardization of imaging between centres. The presence or absence of a post-therapeutic residue should be stated in the radiology report. Further investigation is needed to differentiate between fibrosis and recurrent tumour. Radiotherapy for residual mass at day 80 is probably more important than standardizing radiological technique.

Pancreatic tumours are rare childhood neoplasms. Inflammatory myofibrohistiocytic tumours (IMTs) represent an uncommon but distinct pathological subgroup that creates diagnostic and therapeutic dilemmas. We report a case of IMT arising from the body and tail of the pancreas in an 8-year-old girl presenting with a mass and abdominal pain. A locally aggressive tumour with no evidence of distant metastasis was encountered at laparotomy and resected. Pathologically, the tumour revealed a mixed inflammatory cell infiltrate with myofibrohistiocytic proliferation. These features can resemble a sarcoma. A review of the literature is provided which emphasises the clinical features, pathological findings, and management strategies for these unusual tumours. Complete surgical excision, aided by radiological surveillance, appears to offer the best guidelines for definitive management.

Purpose: To determine the maximum-tolerated dose (MTD) of cyclophosphamide (CTX) when administered over 2 consecutive days followed by hematopoetic stem-cell rescue given as two sequential courses to children with glioblastoma multiforme, poor-prognosis pontine gliomas, and other recurrent CNS tumors., Patients and Methods: Two identical doses of CTX were administered 24 hours apart to 14 children and followed by hematopoetic stem-cell rescue. This treatment was repeated immediately following hematologic recovery. The starting dose of CTX was 2.5 g/m2/d with increments of 0.5 g/m2/d. CTX pharmacokinetics and metabolism were measured during 22 courses of treatment. Toxicity and tumor response were recorded., Results: There were two toxic deaths at the dose level of 4 g/m2/d. These were not clearly related to cardiac toxicity and may have been due to generalized capillary leak syndrome. Thus, the MTD of CTX was 3.5 g/m2/ d. There were six complete responses (CRs) (46%; (95% confidence interval [CI], 19% to 73%) and four partial responses (PRs) (31%; 95% CI, 6% to 56%), and one patient achieved stable disease. All children with intracranial primitive neuroectodermal tumors (PNETs) improved following CTX. The median duration of tumor response was 6 months (range, 4 to 29) and only one patient remains disease-free following CTX alone. Overall survival is 21% (95% CI, 13% to 29%) at a median follow-up time of 27 months (range, 12 to 34)., Conclusion: The MTD of CTX when followed by hematopoetic stem-cell rescue is 3.5 g/m2 administered on each of 2 consecutive days. This treatment was tolerable in children with poor-prognosis brain tumors and produced complete responses in children with recurrent PNETs.

This is the fourth official document of the SIOP Working Committee on psychosocial issues in pediatric oncology constituted in 1991. This document develops another topic discussed and approved by the SIOP Committee: "communication of the diagnosis" is addressed to the pediatric oncology community as guidelines that could be followed. The highly stressful nature of the diagnostic period must be acknowledged, and communication involving the staff and all family members should cover both medical and psychosocial issues. A well-planned and extensive initial session should be followed by continuing discussions. The goal is a knowledgeable family that can talk openly with its members and with the staff.

To assess the clinical efficacy of ticarcillin, with clavulanic acid, and gentamicin, we conducted a prospective one year study of febrile episodes in neutropenic children. Seventy-five episodes were evaluated in 42 children. The response rate was 32% during persistent neutropenia, whilst another third of episodes responded with neutrophil recovery. Positive blood cultures occurred in 21 episodes and 20 of 24 micro-organisms belonged to the 'community' flora, i.e. organisms carried by healthy people (Streptococcus pneumoniae, Staphylococcus aureus, Branhamella catarrhalis and Escherichia coli). The route of pathogenesis was endogenous in 76% of the patients. There was a substantial superinfection-related morbidity (14%) and mortality (7%), related to emergence of resistance during and after parenteral antibiotic administration. The poor clinical response, combined with emergence of resistance, lead to the conclusion that this combination is of limited value as a first line regimen for neutropenic patients.

Two infants presented with extensive interstitial emphysema of the neck as a result of non-accidental trauma to the pharynx. The clinical presentation, diagnosis, and management of this unusual form of child abuse is discussed.

Twelve totally implantable subcutaneous central venous catheter systems were used in paediatric oncology patients. Complications were few--namely, blockage and four infective episodes, all of which were successfully treated. Infective rate was 0.189 episodes per 100 days' use, which is substantially lower than that reported with other central venous catheters.

Pulmonary disease in the 'histiocytosis syndromes' is not uncommon. Isolated pulmonary histiocytosis, however, is rarely diagnosed. We describe three patients with this condition, with ages ranging from 3 weeks to 9 1/2 years, in whom there was no evidence of disease in any other organ. Their presentation, treatment, and clinical progress over three years of follow up are discussed.

A cross sectional study was carried out in children receiving treatment for acute lymphoblastic leukaemia to determine the prevalence of trimethoprim resistant organisms in their gut flora and to compare this with a control population. There was a significantly higher prevalence of trimethoprim resistant bacteria in the study group (61%) compared with controls (14%). A longitudinal study showed that emergence of these organisms was intermittent during treatment.