Biomedical subjects

Gian Paolo Tonini

Publications and source records attributed to Gian Paolo Tonini.

At least 19 recordsLinked to original sources

PubMed2007

An interferon-sensitive response element is involved in constitutive caspase-8 gene expression in neuroblastoma cells.

We previously identified a 1.2 Kb DNA element (P-1161/+16), 5' to caspase-8 exon-1, that acts as promoter in caspase-8-positive, but not in caspase-8-negative neuroblastoma (NB) cells. The P-1161/+16 DNA element regulates both constitutive and interferon IFN-gamma-inducible caspase-8 expression. Two GAS (IFN-activated sequence, STAT-1 binding site) and two ISRE (interferon sensitive response element, IRF binding site) were present in P-1161/+16. Deletion studies indicated that elements essential for promoter activity in NB cells were present in a 167 bp region 5' flanking exon-1 (P-151/+16), which contains an ISRE at position -32. The transcription initiation site was mapped by 5' rapid amplification of cDNA end (RACE) at position -20 from caspase-8 cDNA reference sequence. Disruption of the ISRE-32 indicated that it is required for both constitutive and IFN-gamma-inducible caspase-8 expression. IRF-1 and IRF-2 transcription factors bind to the (-151/+16) DNA fragment in vitro. Chromatin immunoprecipitation (ChIP) assays showed that IRF-1 and IRF-2 bind to the DNA region at the 5' of caspase-8 gene in NB cells, which show constitutive expression but not in caspase-8 negative cells. In these last cells, up-regulation of caspase-8 by IFN-gamma was associated to induction of IRF-1 and IRF-2 binding to caspase-8 promoter and increased histone acetylation. Moreover, RNA interference experiments also supported the involvement of IRF-1 and IRF-2 in constitutive caspase-8 expression in NB cells.

Antiviral Agents↗

PubMed2006

Molecularly guided therapy of neuroblastoma: a review of different approaches.

Neuroblastoma (NB) is the most frequent extra-cranial solid tumor and the first cause of lethality in pre-school age children. NB accounts for 9-10% of pediatric tumors and affects more than ten thousand children a year. It originates from the sympathetic nervous system and is characterized by heterogeneous pathological and clinical presentation. Stage 4 NB represents approximately 50% of the cases and shows metastatic dissemination at onset; its prognosis is grim, with 20% of the patients surviving at 5 years from diagnosis in spite of aggressive chemotherapy with autologous hematopoietic stem cell support. Novel therapeutic strategies are urgently needed to improve the prognosis of stage 4 NB patients. Here we review the most promising approaches to NB treatment that have already reached clinical testing or have proved to be successful in preclinical models of the disease. All of these approaches are molecularly guided, since their rational development has benefited from the enormous amount of information on the biology of neuroblastoma gathered through molecular biology and genetics studies. The following topics are reviewed: MYCN oncogene amplification as parameter for therapeutic decision, minimal residual disease, immunotherapy, gene therapy, differentiation and apoptotic therapy, anti-angiogenic therapy, gene expression profiling as tool for generating novel therapeutic approaches. Although several efforts are still needed to reach a significant cure of patients with neuroblastoma, molecularly guided approaches have opened new ways to neuroblastoma treatment and can represent useful models for other cancers of either childhood or adulthood.

Angiogenesis Inhibitors↗

PubMed2005

Oligogenic inheritance in neuroblastoma.

Neuroblastoma (NB), a childhood malignancy affecting neural crest deriving cell lineages, is characterized by great clinical variability and histological heterogeneity. As NB usually occurs as sporadic form, molecular studies were mainly carried out on tumor samples and derived cell lines, leading to the identification of several somatic alterations. Although familial NB is rare, linkage data obtained from different families have provided evidence of linkage to markers mapping to different chromosomal regions, indicating a remarkable genetic heterogeneity of NB. The first evidence of germline mutations in NB pedigrees has been recently reported in the paired-like homeobox 2B (PHOX2B) gene, involved in the development of neural crest deriving cells. Nevertheless, as only a few NB families but not others have been shown to carry PHOX2B mutations, the role of this gene in NB predisposition has still to be clarified. On the basis of the current data, familial NB cannot be modeled as a Mendelian monogenic trait. Instead, an oligogenic mode of inheritance might explain the existence of different NB loci genetically interacting to cause and/or modify the disease-phenotype.

Female↗

PubMed2005

Application of microarray-based technology to neuroblastoma.

In the past decade, microarray technology has become a major tool for high-throughput comprehensive analysis of gene expression, genotyping and re-sequencing applications. High-throughput microarrays are used for expression profiling analyses with the aims of gene or pathway discovery, tumor subclassification or relapse risk assessment. The introduction of microarray CGH provides a powerful tool to precisely detect and quantify genomic aberrations and map these directly onto the human genome. This review summarizes the current status of the application of microarray technology to neuroblastoma research.

Humans↗

PubMed2005

Distinct CpG methylation profiles characterize different clinical groups of neuroblastic tumors.

The hypermethylation of CpG islands within gene promoter regions is an epigenetic phenomenon that is often, but not always, associated with the transcriptional silencing of downstream genes and contributes to carcinogenesis. We have determined the pattern of methylation of several genes involved in distinct biological pathways, including cell proliferation and apoptosis, in neuroblastoma and in the nonmalignant ganglioneuroma. The purpose of this work was to search for epigenetic signatures that could be associated with defined clinical and biological parameters and that, in prospective, could identify specific risk categories among the patients. We have analysed 31 malignant neuroblastoma with or without MYCN amplification and 13 benign ganglioneuroma and we have observed dramatic differences in the methylation pattern of five genes (CASP8, 14.3.3sigma, DeltaN-p73, RASSF1A and DCR2) between these tumors indicating that this phenomenon is not tissue-specific and can be considered as cancer-dependent. Furthermore, the methylation pattern of 14.3.3sigma, RASSF1A and of an intragenic segment of CASP8 was significantly different between MYCN amplified and single copy neuroblastoma suggesting a specific role of epigenetic alterations in aggressive neuroblastoma.

14-3-3 Proteins↗

PubMed2005

PHOX2B mutations and genetic predisposition to neuroblastoma.

Neuroblastoma (NB) is a childhood malignancy originating from neural crest cells, which seldom occurs in association with other neurocristopathies. Owing to the rarity of familial NB cases, only a few linkage data are available and no mutations in candidate genes have been demonstrated up till now. Germline mutations in a small proportion of NB patients have been recently reported in the paired-like homeobox 2B (PHOX2B) gene, suggesting its role in NB predisposition. On the basis of this indication, we screened three Italian families with recurrence of NB and one family with occurrence of ganglioneuroblastoma and isolated Hirschsprung disease for PHOX2B defects. Our analysis did not show any mutation, excluding PHOX2B as the NB susceptibility gene in the families we analysed. Our findings combined with those derived from other PHOX2B mutation screenings and from genome-wide linkage analysis support a remarkable genetic heterogeneity of NB and suggest an oligogenic model of disease transmission. Furthermore, as PHOX2B mutations were mainly observed in some NB families with multifocal and syndromic NB, features that are missing in the families we have studied, we suggest they represent second-site modifications responsible for a specific phenotype rather than causal mutations of a major locus.

Female↗

PubMed2005

Clinical and molecular evidence for c-kit receptor as a therapeutic target in neuroblastic tumors.

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.

Adolescent↗

PubMed2005

Genome analysis and gene expression profiling of neuroblastoma and ganglioneuroblastoma reveal differences between neuroblastic and Schwannian stromal cells.

Neuroblastic tumours are a group of paediatric cancers with marked morphological heterogeneity. Neuroblastoma (Schwannian stroma-poor) (NB-SP) is composed of undifferentiated neuroblasts. Ganglioneuroblastoma intermixed (Schwannian stroma-rich) (GNBi-SR) is predominantly composed of Schwannian stromal (SS) and neuroblastic (Nb) cells. There are contrasting reports suggesting that SS cells are non-neoplastic. In the present study, laser capture microdissection (LCM) was employed to isolate SS and Nb cells. Chromosome 1p36 deletion and MYCN gene amplification were found to be associated in two out of seven NB-SPs, whereas no abnormalities were observed in five GNBi-SRs. In some cases, loss of heterozygosity (LOH) at 1p36 loci was detected in Nb cells but not in the bulk tumour by LCM; furthermore, LOH was also identified in both SS and tumour tissue of a GNBi-SR. DNA gain and loss studied by comparative genomic hybridization were observed at several chromosome regions in NB-SP but in few regions of GNBi-SR. Finally, gene expression profiles studied using an oligo-microarray technique displayed two distinct signatures: in the first, 32 genes were expressed in NB-SP and in the second, 14 genes were expressed in GNBi-SR. The results show that NB-SP is composed of different morphologically indistinguishable malignant cell clones harbouring cryptic mutations that are detectable only after LCM. The degree of DNA imbalance is higher in NB-SP than in GNBi-SR. However, when the analysis of chromosome 1p36 is performed at the level of microdissection, LOH is also observed in SS cells. These data provide supportive evidence that SS cells have a less aggressive phenotype and play a role in tumour maturation.

Chromosomes, Human, Pair 1↗

PubMed2005

Glutathione S-transferase polymorphisms and susceptibility to neuroblastoma.

There is evidence to suggest that polymorphic variations in the glutathione S-transferase (GSTs) are associated with cancer susceptibility. The GST supergene family includes several genes with well characterized polymorphisms. Approximately 50% of the Caucasian population is homozygous for deletions in GSTM1 and approximately 20% are homozygous for deletions in GSTT1. Deletions lead to an absence of the protein, thus resulting in conjugation deficiency of mutagenic electrophiles to glutathione. The GSTP1 gene displays a polymorphism at codon 105 resulting in an Ile to Val substitution, which alters the enzymatic activity of the protein, and this has been suggested as a putative high-risk genotype in various cancers. In the present study, we investigated the relationship between GSTs polymorphism and the susceptibility to neuroblastoma, comparing GSTs genotypes of 256 children with neuroblastoma with those of 392 normal control subjects. No significant differences of allele frequencies were found between patients and controls. Within the neuroblastoma group, we further investigated whether any particular GSTs genotype was correlated with clinical and biological characteristics at diagnosis, but no association was detected. Our data do not support an important effect of GSTs genotype on neuroblastoma susceptibility.

Genetic Predisposition to Disease↗

PubMed2004

Detection of neuroblastoma cells in bone marrow and peripheral blood by different techniques: accuracy and relationship with clinical features of patients.

PURPOSE: Detection of metastatic tumor cells in bone marrow (BM) and peripheral blood (PB) of children with neuroblastoma is crucial for prognosis and planning of therapy. Aims of this large descriptive repeated survey were to evaluate the diagnostic accuracy of different techniques in diagnostic samples obtained at several disease course time points and to correlate positive results with patient clinical features and outcome. EXPERIMENTAL DESIGN: BM aspirates, trephine biopsies, PB, and peripheral blood stem cell (PBSC) samples from Italian children with neuroblastoma were analyzed by morphological and histologic techniques, as well as by immunocytochemistry (IC) for disialoganglioside GD(2) and reverse transcription-PCRs (RT-PCRs) for tyrosine hydroxylase (TH) and pgp9.5 genes. The diagnostic odd ratio (DOR) was used to measure the accuracy of the different techniques. RESULTS: A total of 2,247 evaluations were done on 561 BM, 265 PB, and 69 PBSC samples from 247 patients. IC showed the best accuracy. Whereas TH RT-PCR accuracy was satisfactory, that of pgp9.5 was very low. Positive results obtained by IC in BM and PB samples at diagnosis from stage 1, 2, and 3 patients correlated with unfavourable outcome. No correlation was found between positive results obtained by IC or TH RT-PCR in BM, PB, and PBSC samples from stage 4 patients and their outcome. CONCLUSIONS: Because of its elevated diagnostic accuracy, IC may represent a useful adjunct to conventional morphological techniques, especially in view of its potential prognostic role in patients with localized disease. Longitudinal multicenter studies are warranted to definitely establish the clinical usefulness of TH RT-PCR.

Adolescent↗

PubMed2004

Detection of MYCN amplification and chromosome 1p36 loss in neuroblastoma by cDNA microarray comparative genomic hybridization.

BACKGROUND: In the last decade, microarray technology has been extensively used to evaluate gene expression profiles and genome imbalances. We have developed a microarray-based comparative genomic hybridization (CGH) approach to identify MYCN gene amplification and 1p36 chromosome loss, two markers of tumor aggressiveness in neuroblastoma. AIM: The aim was to use microarray CGH technology to detect the two major prognostic markers for neuroblastoma, MYCN amplification and 1p36 chromosome deletion, in neuroblastoma patients and, therefore, confirm the usefulness of this approach in this cancer. METHODS: DNA was purified from 16 tumors containing at least 90% malignant neuroblasts and collected at the onset of disease. Pooled fluorescent-labeled reference and neuroblastoma tumor genomic DNA was hybridized to epoxide-coated glass slides on laboratory-made complementary DNA microarray. The microarray contained cDNA mapped at the 1p36.33-36.1 chromosomal region and MYCN gene. cDNA from the 2q33-q34 and 12p13 chromosomes was used as a control and Arabidopsis thaliana DNA was spotted to control unspecific hybridization. Fluorescence in situ hybridization analysis was also performed to validate results from the microarray CGH. RESULTS: Both MYCN amplification and 1p36 chromosome deletion were detected by microarray CGH. The sensitivity and specificity for 1p36 loss detection were 66.7% and 90.0%, respectively. The method had a sensitivity of 66.7% and specificity of 90.9% to detect MYCN amplification. DISCUSSION: Our results demonstrated that the microarray CGH can be efficiently applied to study DNA gain and loss of specific chromosome regions.

Cell Nucleus↗

PubMed2003

Familial neuroblastoma: a complex heritable disease.

Genomic surveys carried out over the years have revealed a great heterogeneity in the pattern of genetic aberrations occurring in neuroblastoma (NB) cells. Studies on familial NB could lead to the discovery of susceptibility genes and their contribution to the development of the disease. The two-hit hypothesis is considered the most consistent model of inherited predisposition to NB, but an oligogenic inheritance governed by a major gene should be also taken into account. The rarity of familial clustering of NB cases and the difficulty to recruit informative families has not allowed the identification of the disease gene until now. In fact, linkage analysis has suggested more than one region candidate to harboring NB predisposing gene(s). These findings indicate that genetic heterogeneity might be the molecular basis of the disorder. Given the complexity of the disease additional studies are required to elucidate the genetic determination of NB.

Chromosomes, Human↗

PubMed2003

Genetic and epigenetic alterations in neuroblastoma.

Neuroblastoma is one of the most common solid tumors of childhood. Despite the most recent advances in combined therapy, the overall survival of patients with disseminated disease has not improved in the last 20 years. On the contrary, the increased knowledge of the genetic and molecular abnormalities that characterize neuroblastoma allowed the identification of several important prognostic factors and possible pathways of neuroblastoma development. Moreover, several other structural and numerical non-random chromosome abnormalities were recognized by comparative genomic hybridization and their role is actively investigated. More recently, it has become evident also that epigenetic factors may alter the pattern of expression of multiple genes whose role in neuroblastoma begins to be understood. It is thus likely that a combination of events that include gene translocation and rearrangement and the altered methylation of crucial genes, contribute to neuroblastoma development and progression and are likely responsible for the clinical heterogeneity of this tumor.

Chromosome Aberrations↗

PubMed2003

Homozygous inactivation of NF1 gene in a patient with familial NF1 and disseminated neuroblastoma.

Neurofibromatosis type 1 (NF1) patients are susceptible to tumor development. In the present study we describe a child with NF1 and disseminated neuroblastoma whose death resulted from disease progression. The mother had café-au-lait spots suggesting a familial NF1. Neuroblastoma cells showed MYCN amplification and chromosome 1p36 deletion, common features associated with tumor progression in this malignancy. The NF1 gene displayed a germline T --> C transition of intron 14 in both the proband and mother DNA. This mutation, not yet previously described, occurs in a splicing donor site and produces a new mRNA variant observed together with normal NF1 mRNA. Furthermore, the SSCP analysis of the NF1 gene in tumor cells showed a somatic deletion encompassing the intron 26 and 27b of the paternal NF1 allele. Hence, neuroblastoma cells displayed both somatic and germline mutation of the NF1 gene. Our data suggest that, although rare, neuroblastoma in patients with NF1 may display homozygous gene inactivation.

Bone Marrow Neoplasms↗

PubMed2003

Neuroblastic tumors associated with opsoclonus-myoclonus syndrome: histological, immunohistochemical and molecular features of 15 Italian cases.

The aim of this study was to investigate the histological, immunohistochemical and molecular features of a series of children with neuroblastic tumors (NTs) and opsoclonus-myoclonus syndrome (OMS). Of 1187 children (age 0-15 years) with previously untreated NTs registered between 1979 and 1995, 15 (1.3%) had OMS at presentation. The majority of patients showed favorable biological characteristics, such as lack of amplification of the neuroblastoma-associated avian myelocytomatosis homolog MYCN oncogene and aneuploid nuclear DNA content. Tumor histology was reviewed according to the International Neuroblastoma Pathology Classification. Histology of the 15 cases of NTs with OMS was ganglioneuroblastoma, intermixed, in 10 patients; ganglioneuroma, maturing, in 1; and neuroblastoma in 4. Of 15 tumors, 12 (10 ganglioneuroblastomas, 2 neuroblastomas) showed abundant interstitial or perivascular lymphoid infiltrates, the latter often organized in secondary lymphoid follicles. The three remaining cases had only minimal infiltrates. A review of 91 cases of age- and stage-matched neuroblastic tumors not associated with OMS tested as controls showed that the degree of lymphoid infiltration was significantly lower than that detected in OMS-related tumors. Furthermore, lymphoid follicles were always present in the latter tumors, whereas they were detected only in a few ganglioneuroma, intermixed tumors from the control group. In conclusion, ganglioneuroblastoma, intermixed subtype, lack of MYCN amplification, aneuploid DNA content and presence of lymphoid infiltrates may contribute to favorable prognosis in NTs associated with OMS.

Adolescent↗

PubMed2003

Disseminated neuroblastoma in children older than one year at diagnosis: comparable results with three consecutive high-dose protocols adopted by the Italian Co-Operative Group for Neuroblastoma.

PURPOSE: To compare the outcomes associated with modifications in three consecutive protocols employed by the Italian Co-Operative Group for Neuroblastoma (ICGNB) in disseminated neuroblastoma. PATIENTS AND METHODS: Between January 1985 and November 1997, a total of 359 children aged 1 to 15 years with newly diagnosed stage 4 neuroblastoma were enrolled in three consecutive protocols. Compared with ICGNB-85, the ICGNB-89 protocol contained two more chemotherapy cycles, and some drugs were given at greater doses, whereas in the ICGNB-92 protocol, the induction phase included a chelating agent, and individual cycles contained four drugs instead of two. RESULTS: A total of 330 of 359 evaluable children were included in this analysis; 106 children were treated with ICGNB-85, 65 children were treated with ICGNB-89, and 159 children were treated with ICGNB-92 protocols. Radical resection of primary tumor was carried out in 59.4%, 50.8%, and 57.9% of the patients, respectively. Major tumor response after induction therapy was achieved in 66.7%, 69.2%, and 68.6% of the patients, respectively. A total of 218 of 232 patients received consolidation therapy consisting of conventional chemotherapy in 65 patients and of high-dose chemotherapy in 153 patients. Disease recurrence or progression occurred in 82.1%, 69.2%, and 74.8% of the patients, respectively. Therapy-related deaths occurred in 1.9%, 12.3%, and 6.9% of the patients, respectively. Five-year overall survival (OS) for the three studies was 26%, 23%, and 28%, and event-free survival (EFS) was 19%, 17%, and 17%, respectively. CONCLUSION: The therapeutic modifications adopted in the ICGNB-89 and ICGNB-92 protocols were not associated with a significant improvement in response rate or in the 5-year OS and EFS as compared with the ICGNB-85 protocol. Attempts at intensifying chemotherapy were associated with greater toxicity.

Adolescent↗

PubMed2002

Weak linkage at 4p16 to predisposition for human neuroblastoma.

The most frequent genetic alterations described in neuroblastoma (NB) are amplification of MYCN oncogene and deletion of chromosome 1p, although somatic deletions have been demonstrated at other chromosomal intervals. Since loss of heterozygosity (LOH) at distal 4p has been observed in about 20-29% of neuroblastomas, we have evaluated deletions in 41 Italian NB samples by LOH analysis at loci mapping to 4p as follows: pter-D4S2936-D4S412-D4S2957-D4S432-D4S3023-D4S431-cen. Our analysis showed allele losses in eight out of 41 samples (19.5%) and allowed the identification of a smallest region of overlapping deletion (SRO) of 3.0 cM, delimited by D4S412 and D4S3023. Two of these tumors with 4p LOH are from patients belonging to a family with recurrent NB. Interestingly the genotyping of this family revealed an identical haplotype that includes the nonrecombinant loci D4S412, D4S2957 and D4S432 shared by all affected children and demonstrated that this haplotype is retained in the two tumors carrying somatic deletions from patients of this family. Furthermore linkage analysis was performed in two NB families and yielded an overall lod-score of 3.0 in the interval including the haplotype. This provides a confirmatory indication that the region delimited by D4S2936 and D4S3023, which also includes the new defined SRO, may harbor NB predisposing gene/s.

Child↗

PubMed2002

Two regions of deletion in 9p22- p24 in neuroblastoma are frequently observed in favorable tumors.

Neuroblastoma is a tumor of infancy that presents several chromosomal abnormalities. Nonrandom deletion of chromosome arm 9p has been identified in primary neuroblastoma suggesting the presence of a tumor suppressor gene located on this chromosome. In previous work, we showed that CDKN2A and CDKN2B genes, mapped at 9p21, were not deleted in neuroblastoma cells. In the present article, we refine the deleted region of 9p using polymerase chain reaction-based analysis of highly polymorphic simple sequence repeats and a two color fluorescence in situ hybridization technique on interphase nuclei. We analyzed 71 primary tumors of patients at the onset of the disease. We found loss of heterozygosity (LOH) in 16 of 71 (23%) cases; the frequency of LOH for 9p was higher (28%) in favorable stages 1, 2, and 4s than in unfavorable stages 3 and 4 (14%). Our results identify two regions of frequent allelic loss: the first at the locus D9S1849 and the second at the locus D9S157. These regions appear to be distant from CDKN2A and CDKN2B loci suggesting that other genes may be involved in 9p deletion. Finally, our data show that 9p deletion is more frequent in tumors of patients with a favorable prognosis, indicating that deleted genes may not be crucial for tumor progression.

Alleles↗