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Find video protocols related to scientific articles indexed in Pubmed.
Revised risk estimation and treatment stratification of low- and intermediate-risk neuroblastoma patients by integrating clinical and molecular prognostic markers.
Clin. Cancer Res.
PUBLISHED: 09-19-2014
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Purpose: To optimize neuroblastoma treatment stratification, we aimed at developing a novel risk estimation system by integrating gene expression-based classification and established prognostic markers. Material and Methods: Gene expression profiles were generated from 709 neuroblastoma specimens using customized 4x44K microarrays. Classification models were built using 75 tumors with contrasting courses of disease. Validation was performed in an independent test set (n=634) by Kaplan-Meier estimates and Cox regression analyses. Results: The best-performing classifier predicted patient outcome with an accuracy of 0.95 (sensitivity 0.93, specificity 0.97) in the validation cohort. The highest potential clinical value of this predictor was observed for current low-risk patients (LR: 5-year EFS 0.84±0.02 vs 0.29±0.10; 5-year OS 0.99±0.01vs 0.76±0.11; both p<0.001) and intermediate-risk patients (IR: 5-year EFS 0.88±0.06 vs 0.41±0.10; 5-year OS 1.0 vs 0.70±0.09; both p<0.001). In multivariate Cox regression models for LR/IR patients the classifier outperformed risk assessment of the current German trial NB2004 (EFS: HR 5.07, 95%-CI 3.20-8.02, OS: HR 25.54, 95%-CI 8.40-77.66; both p<0.001). Based on these findings, we propose to integrate the classifier into a revised risk stratification system for LR/IR patients. According to this system, we identified novel subgroups with poor outcome (5-year EFS 0.19±0.08; 5-year OS 0.59±0.1), for whom we propose intensified treatment, and with beneficial outcome (5-year EFS 0.87±0.05; 5-year OS 1.0), who may benefit from treatment de-escalation. Conclusion: Combination of gene expression-based classification and established prognostic markers improves risk estimation of LR/IR neuroblastoma patients. We propose to implement our revised treatment stratification system in a prospective clinical trial.
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Lack of association between MDM2 promoter SNP309 and clinical outcome in patients with neuroblastoma.
Pediatr Blood Cancer
PUBLISHED: 01-04-2014
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While a polymorphism located within the promoter region of the MDM2 proto-oncogene, SNP309 (T?>?G), has previously been associated with increased risk and aggressiveness of neuroblastoma and other tumor entities, a protective effect has also been reported in certain other cancers. In this study, we evaluated the association of MDM2 SNP309 with outcome in 496 patients with neuroblastoma and its effect on MDM2 expression. No significant difference in overall or event-free survival was observed among patients with neuroblastoma with or without MDM2 SNP309. The presence of SNP309 does not affect MDM2 expression in neuroblastoma.
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Histone chaperone CHAF1A inhibits differentiation and promotes aggressive neuroblastoma.
Cancer Res.
PUBLISHED: 12-12-2013
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Neuroblastoma arises from the embryonal neural crest secondary to a block in differentiation. Long-term patient survival correlates inversely with the extent of differentiation, and treatment with retinoic acid or other pro-differentiation agents improves survival modestly. In this study, we show the histone chaperone and epigenetic regulator CHAF1A functions in maintaining the highly de-differentiated state of this aggressive malignancy. CHAF1A is a subunit of the chromatin modifier CAF1 and it regulates H3K9 tri-methylation of key target genes regulating proliferation, survival and differentiation. Elevated CHAF1A expression strongly correlated with poor prognosis. Conversely, CHAF1A loss-of-function was sufficient to drive neuronal differentiation in vitro and in vivo. Transcriptome analysis of cells lacking CHAF1A revealed repression of oncogenic signaling pathways and a normalization of glycolytic metabolism. Our findings demonstrate that CHAF1A restricts neural crest differentiation and contributes to the pathogenesis of high-risk neuroblastoma.
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Constitutional 11q14-q22 chromosome deletion syndrome in a child with neuroblastoma MYCN single copy.
Eur J Med Genet
PUBLISHED: 03-18-2013
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Constitutional 11q deletion is a chromosome imbalance possibly found in MCA/MR patients analyzed for chromosomal anomalies. Its role in determining the phenotype depends on extension and position of deleted region. Loss of heterozygosity of 11q (region 11q23) is also associated with neuroblastoma, the most frequent extra cranial cancer in children. It represents one of the most frequent cytogenetic abnormalities observed in the tumor of patients with high-risk disease even if germline deletion of 11q in neuroblastoma is rare. Hereby, we describe a 18 months old girl presenting with trigonocephaly and dysmorphic facial features, including hypotelorism, broad depressed nasal bridge, micrognathia, synophrys, epicanthal folds, and with a stage 4 neuroblastoma without MYCN amplification, carrying a germline 11q deletion (11q14.1-q22.3), outside from Jacobsen syndrome and from neuroblastoma 11q critical regions. The role of 11q deletion in determining the clinical phenotype and its association with neuroblastoma development in the patient are discussed.
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Epigenetic Silencing of DKK3 in Medulloblastoma.
Int J Mol Sci
PUBLISHED: 02-06-2013
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Medulloblastoma (MB) is a malignant pediatric brain tumor arising in the cerebellum consisting of four distinct subgroups: WNT, SHH, Group 3 and Group 4, which exhibit different molecular phenotypes. We studied the expression of Dickkopf (DKK) 1-4 family genes, inhibitors of the Wnt signaling cascade, in MB by screening 355 expression profiles derived from four independent datasets. Upregulation of DKK1, DKK2 and DKK4 mRNA was observed in the WNT subgroup, whereas DKK3 was downregulated in 80% MBs across subgroups with respect to the normal cerebellum (p < 0.001). Since copy number aberrations targeting the DKK3 locus (11p15.3) are rare events, we hypothesized that epigenetic factors could play a role in DKK3 regulation. Accordingly, we studied 77 miRNAs predicting to repress DKK3; however, no significant inverse correlation between miRNA/mRNA expression was observed. Moreover, the low methylation levels in the DKK3 promoters (median: 3%, 5% and 5% for promoter 1, 2 and 3, respectively) excluded the downregulation of gene expression by methylation. On the other hand, the treatment of MB cells with Trichostatin A (TSA), a potent inhibitor of histone deacetylases (HDAC), was able to restore both DKK3 mRNA and protein. In conclusion, DKK3 downregulation across all MB subgroups may be due to epigenetic mechanisms, in particular, through chromatin condensation.
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A p53 Drug Response Signature Identifies Prognostic Genes in High-Risk Neuroblastoma.
PLoS ONE
PUBLISHED: 01-01-2013
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Chemotherapy induces apoptosis and tumor regression primarily through activation of p53-mediated transcription. Neuroblastoma is a p53 wild type malignancy at diagnosis and repression of p53 signaling plays an important role in its pathogenesis. Recently developed small molecule inhibitors of the MDM2-p53 interaction are able to overcome this repression and potently activate p53 dependent apoptosis in malignancies with intact p53 downstream signaling. We used the small molecule MDM2 inhibitor, Nutlin-3a, to determine the p53 drug response signature in neuroblastoma cells. In addition to p53 mediated apoptotic signatures, GSEA and pathway analysis identified a set of p53-repressed genes that were reciprocally over-expressed in neuroblastoma patients with the worst overall outcome in multiple clinical cohorts. Multifactorial regression analysis identified a subset of four genes (CHAF1A, RRM2, MCM3, and MCM6) whose expression together strongly predicted overall and event-free survival (p<0.0001). The expression of these four genes was then validated by quantitative PCR in a large independent clinical cohort. Our findings further support the concept that oncogene-driven transcriptional networks opposing p53 activation are essential for the aggressive behavior and poor response to therapy of high-risk neuroblastoma.
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Impact of interleukin-6 -174 g>c gene promoter polymorphism on neuroblastoma.
PLoS ONE
PUBLISHED: 01-01-2013
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Common variants in DNA may predispose to onset and progression of neuroblastoma (NB). The genotype GG of single nucleotide polymorphism (SNP) rs1800795 (-174 G>C) in interleukin (IL)-6 promoter has been associated with lower survival of high-risk NB.
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Inhibition of N-linked glycosylation impairs ALK phosphorylation and disrupts pro-survival signaling in neuroblastoma cell lines.
BMC Cancer
PUBLISHED: 06-21-2011
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The Anaplastic Lymphoma Kinase (ALK) is an orphan receptor tyrosine kinase, which undergoes post-translational N-linked glycosylation. The catalytic domain of ALK was originally identified in the t(2;5) translocation that produces the unglycosylated oncogenic protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL). Recently, both germline and somatic activating missense mutations of ALK have been identified in neuroblastoma (NB), a pediatric cancer arising from neural crest cells. Moreover, we previously reported that ALK expression is significantly upregulated in advanced/metastatic NB. We hypothesized that ALK function may depend on N-linked glycosylation and that disruption of this post-translational modification would impair ALK activation, regardless the presence of either gene mutations or overexpression.
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"DNA-Dressed NAnopore" for complementary sequence detection.
Biosens Bioelectron
PUBLISHED: 06-18-2011
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Single molecule electrical sensing with nanopores is a rapidly developing field with potential revolutionary effects on bioanalytics and diagnostics. The recent success of this technology is in the simplicity of its working principle, which exploits the conductance modulations induced by the electrophoretic translocation of molecules through a nanometric channel. Initially proposed as fast and powerful tools for molecular stochastic sensing, nanopores find now application in a range of different domains, thanks to the possibility of finely tuning their surface properties, thus introducing artificial binding and recognition sites. Here we show the results of DNA translocation and hybridization experiments at the single molecule level by a novel class of selective biosensor devices that we call "DNA-Dressed NAnopore" (DNA(2)), based on solid state nanopore with large initial dimensions, resized and activated by functionalization with DNA molecules. The presented data demonstrate the ability of the DNA(2) to selectively detect complementary target sequences, that is to distinguish between molecules depending on their affinity to the functionalization. The DNA(2) can thus constitute the basis for the design of integrable parallel devices for mutation DNA analysis, diagnostics and bioanalytic investigations.
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Role of CXCL13-CXCR5 crosstalk between malignant neuroblastoma cells and Schwannian stromal cells in neuroblastic tumors.
Mol. Cancer Res.
PUBLISHED: 06-03-2011
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Neuroblastoma is a stroma-poor (SP) aggressive pediatric cancer belonging to neuroblastic tumors, also including ganglioneuroblastoma and ganglioneuroma, two stroma-rich (SR) less aggressive tumors. Our previous gene-expression profiling analysis showed a different CXCL13 mRNA expression between SP and SR tumors. Therefore, we studied 13 SP and 13 SR tumors by reverse transcription quantitative real-time PCR (RT-qPCR) and we found that CXCR5b was more expressed in SP than in SR and CXCL13 was predominantly expressed in SR tumors. Then, we isolated neuroblastic and Schwannian stromal cells by laser capture microdissection and we found that malignant neuroblasts express CXCR5b mRNA, whereas Schwannian stromal cells express CXCL13. Immunohistochemistry confirmed that stroma expresses CXCL13 but not CXCR5. To better understand the role of CXCL13 and CXCR5 in neuroblastic tumors we studied 11 neuroblastoma cell lines and we detected a heterogeneous expression of CXCL13 and CXCR5b. Interestingly, we found that only CXCR5b splice variant was expressed in both tumors and neuroblastoma lines, whereas CXCR5a was never detected. Moreover, we found that neuroblastoma cells expressing CXCR5 receptor migrate toward a source of recombinant CXCL13. Lastly, neuroblastoma cells induced to glial cell differentiation expressed CXCL13 mRNA and protein. The chemokine released in the culture medium was able to stimulate chemotaxis of LA1-5S neuroblastoma cells. Collectively, our data suggest that CXCL13 produced by stromal cells may contribute to the generation of an environment in which the malignant neuroblasts are retained, thus limiting the possible development of metastases in patients with SR tumor.
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Bone marrow of neuroblastoma patients shows downregulation of CXCL12 expression and presence of IFN signature.
Pediatr Blood Cancer
PUBLISHED: 05-25-2011
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At diagnosis, children with neuroblastoma (NB) present with either localized or metastatic disease. Since the mechanisms responsible for BM invasion are not well known, we investigated the transcriptome of resident BM cells from NB patients as compared to healthy children.
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Chromosomal aberrations and aneuploidy in oral potentially malignant lesions: distinctive features for tongue.
BMC Cancer
PUBLISHED: 05-23-2011
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The mucosae of the oral cavity are different at the histological level but appear all equally exposed to common genotoxic agents. As a result of this exposure, changes in the mucosal epithelia may develop giving rise to Oral Potentially Malignant Lesions (OPMLs), which with time may in turn progress to Oral Squamous Cell Carcinomas (OSCCs). Therefore, much effort should be devoted to identify features able to predict the likeliness of progression associated with an OPML. Such features may be helpful in assisting the clinician to establish both appropriate therapies and follow-up schedules. Here, we report a pilot study that compared the occurrence of DNA aneuploidy and chromosomal copy number aberrations (CNAs) in the OPMLs from different oral anatomical subsites.
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Gene expression profiling identifies eleven DNA repair genes down-regulated during mouse neural crest cell migration.
Int. J. Dev. Biol.
PUBLISHED: 03-23-2011
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Neural Crest Cells (NCCs) are transient multipotent migratory cells that derive from the embryonic neural crest which is itself derived from the margin of the neural tube. DNA repair genes are expressed in the early stages of mammalian development to reduce possible replication errors and genotoxic damage. Some birth defects and cancers are due to inappropriate or defective DNA repair machinery, indicating that the proper functioning of DNA repair genes in the early stages of fetal development is essential for maintaining DNA integrity. We performed a genome-wide expression analysis combining laser capture microdissection (LCM) and high-density oligo-microarray of murine NCCs at pre-migratory embryonic days 8.5 (E8.5), and at E13.5, as well as on neural crest-derived cells from the adrenal medulla at postnatal day 90. We found 11 genes involved in DNA repair activity (response to DNA damage stimulus, DNA damage checkpoint, base-excision repair, mismatch repair), over-expressed in the early stages of mouse embryo development. Expression of these 11 genes was very low or undetectable in the differentiated adrenal medulla of the adult mouse. Amongst the 11 genes, 6 had not been previously reported as being over-expressed during mouse embryonic development. High expression of DNA repair genes in enriched NCCs during early embryonic development may contribute to maintaining DNA integrity whilst failure of some of these genes may be associated with the onset of genetic disease and cancer. Our model of enriched murine NCCs and neural crest-derived cells can be used to elucidate the key roles of genes during normal embryonic development and in cancer pathogenesis.
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Loss of 10q26.1-q26.3 in association with 7q34-q36.3 gain or 17q24.3-q25.3 gain predict poor outcome in pediatric medulloblastoma.
Cancer Lett.
PUBLISHED: 02-24-2011
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Medulloblastoma (MB) is the most common malignant brain tumor of childhood. We have investigated for novel chromosomal imbalances and prognostic markers of pediatric MB. Forty MBs out of 64, were analyzed using high resolution prometaphase comparative genomic hybridization. Chromosome 10q26.1-q26.3 loss combined with 17q24.3-q25.3 gain and/or 7q34-q36.3 gain in tumors predicted poor patients survival. A minimal deleted region of 14.12cM at 10q26.1-q26.3 was refined by LOH analysis. We propose a new prognostic marker for pediatric MB patient risk stratification based on the presence of 10q26.1-q26.3 loss plus 17q24.3-q25.3 gain and/or 7q34-q36.3 gain associations.
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Chromosome 9q and 16q loss identified by genome-wide pooled-analysis are associated with tumor aggressiveness in patients with classic medulloblastoma.
OMICS
PUBLISHED: 02-24-2011
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Medulloblastoma (MB) is one of the most aggressive pediatric brain tumor. We report genome-wide pooled-analysis of classic MB variant of patients over 3 years of age at diagnosis. We combined array comparative genomic hybridization (aCGH) results from experimental analysis (31 cases) with two public databases (55 cases) in a final evaluation of 86 MBs. The most common chromosome structural aberrations were gains of 17q (45.3%), 1q (22.1%), and losses of 8p (15.1%), 10q (19.8%), 17p (37.2%), and 16q (16.3%). Isochromome (17q) was observed in 29.1% MBs. A significant association between poor patients survival and losses of 9q (p?
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A multilocus technique for risk evaluation of patients with neuroblastoma.
Clin. Cancer Res.
PUBLISHED: 02-18-2011
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Precise and comprehensive analysis of neuroblastoma genetics is essential for accurate risk evaluation and only pangenomic/multilocus approaches fulfill the present-day requirements. We present the establishment and validation of the PCR-based multiplex ligation-dependent probe amplification (MLPA) technique for neuroblastoma.
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Simultaneous tumors: acute myeloid leukemia infiltrating mediastinal ganglioneuroblastoma.
Pediatr Blood Cancer
PUBLISHED: 01-22-2011
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We report the case of a child presenting with concurrent thoracic ganglioneuroblastoma and acute myeloid leukemia. The peculiarity was the close relation between the two tumors with the latter infiltrating the former one. Histological and genomic studies indicate the different clonal origins of the malignancies in the patient, and we hypothesized that GNB and AML developed independently. Our observation suggests that in patients with more than one tumor, though discovered at different times, one neoplasm is not always secondary.
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Multiple target molecular monitoring of bone marrow and peripheral blood samples from patients with localized neuroblastoma and healthy donors.
Pediatr Blood Cancer
PUBLISHED: 01-19-2011
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Multiple target molecular monitoring of minimal residual disease in neuroblastoma (NB) patients may increase sensitivity and overcome tumor heterogeneity. However, multiple target analysis is costly and time consuming, thus improvement with respect to single target monitoring needs to be achieved.
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Research trends for early cancer biomarker detection in Italy: an integrated program in oncology (PIO) survey.
Tumori
PUBLISHED: 12-28-2010
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In 2007, an Italian Research Network proposed to the Ministry of Health a concerted action aimed at developing a specific pathway for the analytical and clinical validation of new biomarkers for early cancer diagnosis. The action, funded by the Italian Ministry of Health within the Integrated Program in Oncology (PIO) and coordinated by the National Cancer Institute of Bari, started in 2008 involving 37 national research teams.
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A novel splice variant of the human dicer gene is expressed in neuroblastoma cells.
FEBS Lett.
PUBLISHED: 05-07-2010
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Dicer is a ribonuclease playing a key role in the biogenesis of microRNAs and small interfering RNAs. Here we report the identification of a novel splice variant of human dicer gene, the first one bearing a modified coding sequence. It encodes a truncated protein, t-Dicer that lacks the dsRNA-binding domain and is defective in one of the two RNase III catalytic centers. The splice variant was found in neuroblastoma cells and in cells induced to neuronal differentiation, whereas it was not detectable in other cell lines or in normal tissues. Interestingly, it occurred in primary neuroblastic tumors, mainly in stroma poor neuroblastomas.
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MDM2 SNP309 genotype is associated with ferritin and LDH serum levels in children with stage 4 neuroblastoma.
Pediatr Blood Cancer
PUBLISHED: 03-17-2010
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MDM2 SNP309, characterised by a T-to-G substitution in the MDM2 promoter, is associated with higher gene expression compared to wild type and was recently found to be a negative prognostic factor for patients with stage 4 neuroblastoma (NB), but not for children with localised disease. This polymorphism was not associated with any clinical or genetic tumour characteristics, including MYCN amplification and 1p chromosome deletion.
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Transcribed-ultra conserved region expression profiling from low-input total RNA.
BMC Genomics
PUBLISHED: 01-21-2010
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Ultra Conserved Regions (UCRs) are a class of 481 noncoding sequences located in both intra- and inter-genic regions of the genome. The recent findings that they are significantly altered in adult chronic lymphocytic leukemias, carcinomas, and pediatric neuroblastomas lead to the hypothesis that UCRs may play a role in tumorigenesis.
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The Integrated Oncology Program of the Italian Ministry of Health. Analytical and clinical validation of new biomarkers for early diagnosis: network, resources, methodology, quality control, and data analysis.
Int. J. Biol. Markers
PUBLISHED: 09-30-2009
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In 2007, an Italian cancer research group proposed a specific concerted action aimed at the "analytical and clinica validation of new biomarkers for early diagnosis: Network, resources, methodology, quality control, and data analysis." The proposal united 37 national operative units involved in different biomarker studies and it created a strong coordinative body with the necessary expertise in methodologies, statistical analysis, quality control, and biological resources to perform ad hoc validation studies for new biomarkers of early cancer diagnosis. The action, financed by the Italian Ministry of Health within the Integrated Oncology Program (PIO) coordinated by NCI-Istituto Tumori Bari, started in 2007 and activated 7 projects, each of which focused on disease-specific biomarker studies. Overall, the 37 participating units proposed studies on 50 biomarkers, including analytical and clinical validation procedures. Clusters of units were specifically involved in research of early-detection biomarkers for cancers of the lung, digestive tract, prostate/bladder, and nervous system, as well as female cancers. Furthermore, a cluster involved in biomarkers for bioimaging and infection-related cancers was created. The first investigators meeting, "Analytical and clinical validation of new biomarkers for early diagnosis," was held on 9 September 2008 in Bari. During this meeting, methodological aspects, scientific programs and preliminary results were presented and discussed.
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Mutation-independent anaplastic lymphoma kinase overexpression in poor prognosis neuroblastoma patients.
Cancer Res.
PUBLISHED: 09-01-2009
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Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALK(high) 12.8%, ALK(low) 73%, P = 0.0035; cell death: ALK(high) 56.4%, ALK(low) 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs.
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Transcribed-Ultra Conserved Region expression is associated with outcome in high-risk neuroblastoma.
BMC Cancer
PUBLISHED: 08-14-2009
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Neuroblastoma is the most common, pediatric, extra-cranial, malignant solid tumor. Despite multimodal therapeutic protocols, outcome for children with a high-risk clinical phenotype remains poor, with long-term survival still less than 40%. Hereby, we evaluated the potential of non-coding RNA expression to predict outcome in high-risk, stage 4 neuroblastoma.
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MDM2 SNP309 genotype influences survival of metastatic but not of localized neuroblastoma.
Pediatr Blood Cancer
PUBLISHED: 06-16-2009
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MDM2 is a major negative regulator of p53 function and is directly regulated by MYCN in neuroblastoma (NB) cells. MDM2 SNP309, a T-to-G substitution in the MDM2 promoter associated with higher gene expression compared to wild-type, may attenuate the p53 pathway in NB, in which p53 mutations are rare. We investigated its impact on NB development and survival in relation with major clinical and biological characteristics.
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c.1810C>T polymorphism of NTRK1 gene is associated with reduced survival in neuroblastoma patients.
BMC Cancer
PUBLISHED: 05-15-2009
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TrkA (encoded by NTRK1 gene), the high-affinity tyrosine kinase receptor for neurotrophins, is involved in neural crest cell differentiation. Its expression has been reported to be associated with a favourable prognosis in neuroblastoma. Therefore, the entire coding sequence of NTRK1 gene has been analysed in order to identify mutations and/or polymorphisms which may alter TrkA receptor expression.
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Risk of malignant pleural mesothelioma and polymorphisms in genes involved in the genome stability and xenobiotics metabolism.
Mutat. Res.
PUBLISHED: 03-16-2009
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Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer mostly attributable to asbestos exposure. Many polymorphic genes encoding for xenobiotic and oxidative metabolism enzymes (XME) or involved in genome stability (GS) can modulate individual MPM risk in exposed populations. An association study was carried out in a case-control setting including 119 MPM patients and two groups of referent subjects (104 with and 695 without documented asbestos exposure). Forty-eight polymorphisms in XME genes and 75 in GS-genes were evaluated. Statistical analysis revealed some significant associations of studied polymorphisms with MPM risk, but most of them disappeared after applying Bonferroni correction (new threshold for statistical significance: p=4.07 x 10(-4)). On the other hand, the nucleotidic change 282C>T within NAT2 held the statistical significance (OR=3.54; 95% CI 1.75-7.16; p=0.0002), reinforcing existing evidences that describe genetic polymorphisms of NAT2 possibly involved in the etiology of the MPM.
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Translational research in hormone-related cancer.
Ann. N. Y. Acad. Sci.
PUBLISHED: 03-03-2009
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Hormones are implicated in various types of cancer, however, several aspects of steroid activity on carcinogenesis remain elusive. Recent progress has made it possible for us to directly study the biological mechanisms of disease development, including hormone-cancer relationships, from numerous viewpoints, from numerous viewpoints, including genome abnormalities. One tool is comparative genomic hybridization array (aCGH). Furthermore, it is possible to identify the so-called "cancer signature" by gene expression profiling, which provides new information about the role of steroids on carcinogenesis. DNA mutations and gene expression abnormalities may be associated with hormone-related cancer. The recent discovery of microRNA provides new opportunities for understanding the fine regulation of gene expression in cancer cells, and the role of microRNA in the relationship between hormones and cancer. From these experimental models we should be able to rapidly develop translation-to-treatment protocols. The final goal should be to design specific labs on a microchip for prognosis and therapy of individual patients. While in clinical research there is renewed attention to stratification of patients, especially those at high risk.
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Characterisation and validation of insertions and deletions in 173 patient exomes.
PLoS ONE
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Recent advances in genomics technologies have spurred unprecedented efforts in genome and exome re-sequencing aiming to unravel the genetic component of rare and complex disorders. While in rare disorders this allowed the identification of novel causal genes, the missing heritability paradox in complex diseases remains so far elusive. Despite rapid advances of next-generation sequencing, both the technology and the analysis of the data it produces are in its infancy. At present there is abundant knowledge pertaining to the role of rare single nucleotide variants (SNVs) in rare disorders and of common SNVs in common disorders. Although the 1,000 genome project has clearly highlighted the prevalence of rare variants and more complex variants (e.g. insertions, deletions), their role in disease is as yet far from elucidated.We set out to analyse the properties of sequence variants identified in a comprehensive collection of exome re-sequencing studies performed on samples from patients affected by a broad range of complex and rare diseases (N = 173). Given the known potential for Loss of Function (LoF) variants to be false positive, we performed an extensive validation of the common, rare and private LoF variants identified, which indicated that most of the private and rare variants identified were indeed true, while common novel variants had a significantly higher false positive rate. Our results indicated a strong enrichment of very low-frequency insertion/deletion variants, so far under-investigated, which might be difficult to capture with low coverage and imputation approaches and for which most of study designs would be under-powered. These insertions and deletions might play a significant role in disease genetics, contributing specifically to the underlining rare and private variation predicted to be discovered through next generation sequencing.
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Replication of GWAS-identified neuroblastoma risk loci strengthens the role of BARD1 and affirms the cumulative effect of genetic variations on disease susceptibility.
Carcinogenesis
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Several neuroblastoma (NB) susceptibility loci have been identified within LINC00340, BARD1, LMO1, DUSP12, HSD17B12, DDX4, IL31RA, HACE1 and LIN28B by genome-wide association (GWA) studies including European American individuals. To validate and comprehensively evaluate the impact of the identified NB variants on disease risk and phenotype, we analyzed 16 single nucleotide polymorphisms (SNPs) in an Italian population (370 cases and 809 controls). We assessed their regulatory activity on gene expression in lymphoblastoid (LCLs) and NB cell lines. We evaluated the cumulative effect of the independent loci on NB risk and high-risk phenotype development in Italian and European American (1627 cases and 2575 controls) populations. All NB susceptibility genes replicated in the Italian dataset except for DDX4 and IL31RA, and the most significant SNP was rs6435862 in BARD1 (P = 8.4 × 10(-15)). BARD1 showed an additional and independent SNP association (rs7585356). This variant influenced BARD1 mRNA expression in LCLs and NB cell lines. No evidence of epistasis among the NB-associated variants was detected, whereas a cumulative effect of risk variants on NB risk (European Americans: P (trend) = 6.9 × 10(-30), Italians: P (trend) = 8.55 × 10(13)) and development of high-risk phenotype (European Americans: P (trend) = 6.9 × 10(-13), Italians: P (trend) = 2.2 × 10(-1)) was observed in a dose-dependent manner. These results provide further evidence that the risk loci identified in GWA studies contribute to NB susceptibility in distinct populations and strengthen the role of BARD1 as major genetic contributor to NB risk. This study shows that even in the absence of interaction the combination of several low-penetrance alleles has potential to distinguish subgroups of patients at different risks of developing NB.
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High genomic instability predicts survival in metastatic high-risk neuroblastoma.
Neoplasia
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We aimed to identify novel molecular prognostic markers to better predict relapse risk estimate for children with high-risk (HR) metastatic neuroblastoma (NB). We performed genome- and/or transcriptome-wide analyses of 129 stage 4 HR NBs. Children older than 1 year of age were categorized as "short survivors" (dead of disease within 5 years from diagnosis) and "long survivors" (alive with an overall survival time ? 5 years). We reported that patients with less than three segmental copy number aberrations in their tumor represent a molecularly defined subgroup with a high survival probability within the current HR group of patients. The complex genomic pattern is a prognostic marker independent of NB-associated chromosomal aberrations, i.e., MYCN amplification, 1p and 11q losses, and 17q gain. Integrative analysis of genomic and expression signatures demonstrated that fatal outcome is mainly associated with loss of cell cycle control and deregulation of Rho guanosine triphosphates (GTPases) functioning in neuritogenesis. Tumors with MYCN amplification show a lower chromosome instability compared to MYCN single-copy NBs (P = .0008), dominated by 17q gain and 1p loss. Moreover, our results suggest that the MYCN amplification mainly drives disruption of neuronal differentiation and reduction of cell adhesion process involved in tumor invasion and metastasis. Further validation studies are warranted to establish this as a risk stratification for patients.
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Towards a turning point of neuroblastoma therapy.
Cancer Lett.
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In 1983, the MYCN oncogene amplification was discovered in neuroblastoma cells and few years later its prognostic role was clearly demonstrated. The first European study, in which MYCN gene status is taken as prognostic factor for therapeutic decision, was released by SIOPEN and GPOH in 1995. Afterward, other trials were released by SIOPEN, GPOH, COG, and JNBSG in which MYCN gene amplification was employed as prognostic risk factor. However, since MYCN is abnormal in about 20% of tumors and is a reliable prognostic marker for only some subgroups of patients, additional chromosomal abnormalities have been introduced for clinical decisions: 1p deletions/imbalances (GPOH), 11q deletions/imbalances (COG), and structural copy number aberrations (SIOPEN). MYCN gene status and chromosome aberrations improved patients risk evaluation and helped to develop tailored therapy for diverse subgroups of patients. Unfortunately, high risk patients still have an unfavorable prognosis and are the major challenge for oncologists. In the last decade, the advent of genome-wide analysis and the next generation sequencing technique have given the opportunity to deeply investigate the genome of neuroblastoma to identify both candidate genes associated with tumor progression and druggable target genes. ALK gene is an actual candidate for the therapy with small molecule inhibitors, but others as mTOR, Aurora, and TRK, are suitable targets for subgroups of high risk patients. The huge amount of clinical and biological information collected in the last 20 years suggests that it is time to turn on for new and more personalized therapies of neuroblastoma.
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The p53 codon 72 Pro/Pro genotype identifies poor-prognosis neuroblastoma patients: correlation with reduced apoptosis and enhanced senescence by the p53-72P isoform.
Neoplasia
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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.
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Dissecting the genomic complexity underlying medulloblastoma.
Nature
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Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.
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Identification of ALK germline mutation (3605delG) in pediatric anaplastic medulloblastoma.
J. Hum. Genet.
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The anaplastic lymphoma kinase (ALK) gene has been found either rearranged or mutated in several neoplasms such as anaplastic large-cell lymphoma, non-small-cell lung cancer, neuroblastoma and anaplastic thyroid cancer. Medulloblastoma (MB) is an embryonic pediatric cancer arising from nervous system, a tissue in which ALK is expressed during embryonic development. We performed an ALK mutation screening in 52 MBs and we found a novel heterozygous germline deletion of a single base in exon 23 (3605delG) in a case with marked anaplasia. This G deletion results in a frameshift mutation producing a premature stop codon in exon 25 of ALK tyrosine kinase domain. We also screened three human MB cell lines without finding any mutation of ALK gene. Quantitative expression analysis of 16 out of 52 samples showed overexpression of ALK mRNA in three MBs. In the present study, we report the first mutation of ALK found in MB. Moreover, a deletion of ALK gene producing a stop codon has not been detected in human tumors up to now. Further investigations are now required to elucidate whether the truncated form of ALK may have a role in signal transduction.
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Multiple endocrine neoplasias type 2B and RET proto-oncogene.
Ital J Pediatr
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Multiple Endocrine Neoplasia type 2B (MEN 2B) is an autosomal dominant complex oncologic neurocristopathy including medullary thyroid carcinoma, pheochromocytoma, gastrointestinal disorders, marphanoid face, and mucosal multiple ganglioneuromas. Medullary thyroid carcinoma is the major cause of mortality in MEN 2B syndrome, and it often appears during the first years of life. RET proto-oncogene germline activating mutations are causative for MEN 2B. The 95% of MEN 2B patients are associated with a point mutation in exon 16 (M918/T). A second point mutation at codon 883 has been found in 2%-3% of MEN 2B cases. RET proto-oncogene is also involved in different neoplastic and not neoplastic neurocristopathies. Other RET mutations cause MEN 2A syndrome, familial medullary thyroid carcinoma, or Hirschsprungs disease. RET gene expression is also involved in Neuroblastoma. The main diagnosis standards are the acetylcholinesterase study of rectal mucosa and the molecular analysis of RET. In our protocol the rectal biopsy is, therefore, the first approach. RET mutation detection offers the possibility to diagnose MEN 2B predisposition at a pre-clinical stage in familial cases, and to perform an early total prophylactic thyroidectomy. The surgical treatment of MEN 2B is total thyroidectomy with cervical limphadenectomy of the central compartment of the neck. When possible, this intervention should be performed with prophylactic aim before 1 year of age in patients with molecular genetic diagnosis. Recent advances into the mechanisms of RET proto-oncogene signaling and pathways of RET signal transduction in the development of MEN 2 and MTC will allow new treatment possibilities.
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Cyto-morphologic evaluation of bone marrow in infants with disseminated neuroblastoma.
J. Pediatr. Hematol. Oncol.
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We studied the prevalence and degree of tumor cell infiltration (TCI) in bone marrow (BM) aspirates of 89 infants with stage 4/4 S neuroblastoma and correlated them with MYCN gene status and patient outcome. TCI was scored 0, +, ++, and +++, the last corresponding to an infiltration greater than 10%. TCI 0 was more frequent in stage 4 than in stage 4 S. TCI + and ++ were equally represented. TCI +++ was found only in 9 patients, all with typical stage 4 features (bone or lung involvement). Overall survival was not significantly influenced by the presence and degree of TCI.
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Segmental chromosome aberrations converge on overexpression of mitotic spindle regulatory genes in high-risk neuroblastoma.
Genes Chromosomes Cancer
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Integration of genome-wide profiles of DNA copy number alterations (CNAs) and gene expression variations (GEVs) could provide combined power to the identification of driver genes and gene networks in tumors. Here we merge matched genome and transcriptome microarray analyses from neuroblastoma samples to derive correlation patterns of CNAs and GEVs, irrespective of their genomic location. Neuroblastoma correlation patterns are strongly asymmetrical, being on average 10 CNAs linked to 1 GEV, and show the widespread prevalence of long range covariance. Functional enrichment and network analysis of the genes covarying with CNAs consistently point to a major cell function, the regulation of mitotic spindle assembly. Moreover, elevated expression of 14 key genes promoting this function is strongly associated to high-risk neuroblastomas with 1p loss and MYCN amplification in a set of 410 tumor samples (P < 0.00001). Independent CNA/GEV profiling on neuroblastoma cell lines shows that increased levels of expression of these genes are linked to 1p loss. By this approach, we reveal a convergence of clustered neuroblastoma CNAs toward increased expression of a group of prognostic and functionally cooperating genes. We therefore propose gain of function of the spindle assembly machinery as a lesion potentially offering new targets for therapy of high-risk neuroblastoma.
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The metallophosphodiesterase Mpped2 impairs tumorigenesis in neuroblastoma.
Cell Cycle
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Through microarray analyses, we identified the Mpped2 gene as differentially expressed in two neuroblastoma cell lines induced to differentiation with all-trans retinoic acid. Mpped2 codes for a new metallophosphodiesterase protein, the expression of which inhibits cell proliferation and soft agar colony formation in SH -SY5Y cells. This inhibition is concomitant to an increased proportion of the cells in G0/G1 phase and enhanced caspase 3 activation, effects not seen for the other phosphodiesterases. A Mpped2-null mutation (H67R) abrogates these functions, which indicates that the biochemical activity of Mpped2 is advantageous for cancer suppression. Expression analyses in the "Los Angeles" and "Essen" neuroblastoma gene-array data sets show that increased expression of Mpped2 is associated with good patient prognosis according to Kaplan-Meier analyses. Tumorigenic assays in mice show that overexpression of Mpped2 improves survival rate, substantially impairs tumor growth and induces neuronal differentiation. Altogether, these data show that Mpped2 expression impairs neuroblastoma tumorigenesis, and they establish a basis for future therapeutic applications.
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Bone marrow-infiltrating human neuroblastoma cells express high levels of calprotectin and HLA-G proteins.
PLoS ONE
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Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5 y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.
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Age-dependent accumulation of genomic aberrations and deregulation of cell cycle and telomerase genes in metastatic neuroblastoma.
Int. J. Cancer
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About 50% of children with neuroblastoma (NB) show a metastatic disease and have a poor prognosis. However, disease progression is greatly variable and depends on patients age and MYCN oncogene amplification. To investigate the role of patients age in tumor aggressiveness, we performed array-CGH and gene expression profiles of three groups (G) of metastatic NBs: G1, stage 4S patients and MYCN single copy (MYCN-) tumors; G2, stage 4 patients, ? 18 months of age, MYCN- tumors and favorable outcome and G3, Stage 4 patients, ? 19 months with unfavorable outcome. G1 was characterized by numerical aberrations prevalently; on the contrary, all G3 tumors had structural rearrangements, whereas G2 showed an intermediate pattern. The average of numerical alterations decreased significantly from G1 to G2 to G3 (p < 0.01). Contrarily, the number of structural aberrations increased from G1 to G2 to G3 (p < 2.35 E-05). Noteworthy, G3/MYCN- NBs were characterized by several complex intrachromosome rearrangements. Expression analysis of the three groups showed significant differences in genes of Rho and Ras signaling pathways, development and adhesion, cell cycle regulation and telomerase activity. Accumulation of structural alterations increased with patients age and was associated with a more aggressive disease. Abnormal expression of genes involved in cell cycle and telomerase in G3 may be responsible for the genomic instability in this cohort of patients. The higher DNA instability observed in G3/MYCN- NBs than in MYCN-amplified G3 may also explain why patients ? 19 months have a poor outcome independently by MYCN status.
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