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G P Tonini

Publications and source records attributed to G P Tonini.

At least 19 recordsLinked to original sources
PubMed2003

Biological and clinical role of p73 in neuroblastoma.

The p73 gene is a p53 homologue localized at 1p36.3, a chromosomal region frequently deleted in neuroblastoma. p73 was originally considered an oncosuppressor gene. However, it was soon realized that its mode of action did not resemble that of a classic anti-oncogene. The recent discovery of N-terminal truncated isoforms, with oncogenic properties, showed that p73 has a 'two in one' structure. Indeed, the full-length variants are strong inducers of apoptosis while the truncated isoforms inhibit the pro-apoptotic activity of p53 and of the full-length p73. This review summarizes some aspects of p73 biology with particular reference to its possible role in neuroblastoma.

Alternative Splicing↗
PubMed2003

Quality assessment of genetic markers used for therapy stratification.

PURPOSE: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

Biomarkers, Tumor↗
PubMed2002

Expression of DeltaNp73 is a molecular marker for adverse outcome in neuroblastoma patients.

The p73 gene is a p53 homologue which induces apoptosis and inhibits cell proliferation. Although p73 maps at 1p36.3 and is frequently deleted in neuroblastoma (NB), it does not act as a classic oncosuppressor gene. In developing sympathetic neurons of mice, p73 is predominantly expressed as a truncated anti-apoptotic isoform (DeltaNp73), which antagonizes both p53 and the full-length p73 protein (TAp73). This suggests that p73 may be part of a complex tumor-control mechanism. To determine the role of DeltaNp73 in NB we analyzed the pattern of expression of this gene in vivo and evaluated the prognostic significance of its expression. Our results indicate that DeltaNp73 expression is associated with reduced apoptosis in a NB tumor tissue. Expression of this variant in NB patients significantly correlates with age at diagnosis and VMA urinary excretion. Moreover it is strongly associated with reduced survival (HR=7.93; P<0.001) and progression-free survival (HR=5.3; P<0.001) and its role in predicting a poorer outcome is independent from age, primary tumor site, stage and MYCN amplification (OS: HR=5.24, P=0.012; PFS: HR=4.36, P=0.005). In conclusion our data seem to indicate that DeltaNp73 is a crucial gene in neuroblastoma pathogenesis.

Apoptosis↗
PubMed2002

Localized unresectable neuroblastoma: results of treatment based on clinical prognostic factors.

BACKGROUND: We previously reported that stage 3 neuroblastoma comprises (i) a low-risk group including all infants (age 0-11 months) as well as older children with non-abdominal primaries, and (ii) a high-risk group made up of children >1 year of age with abdominal primaries. Aggressive chemotherapy was effective only in the latter group. PATIENTS AND TREATMENT: On this basis, in 1990 we designed a new protocol by which all low-risk patients received standard-dose chemotherapy, while the high-risk ones received very aggressive chemotherapy. RESULTS: Between November 1990 and December 1997 a total of 95 eligible and evaluable children were enrolled: 47 were low-risk (35 infants and 12>1 year of age at diagnosis and having non-abdominal primaries), and 48 were high-risk (being >1 year of age and having abdominal primaries). Of the 47 low-risk patients, five relapsed and four subsequently died. The 5-year overall survival (OS) was 91%. Of the 48 patients in the high-risk group, 22 relapsed or progressed, 18 of whom died from their disease and two from toxicity, and one was lost to follow-up. The 5-year OS was 60%. Univariate analysis showed that age, site of primary, risk-group, urine vanillylmandelic excretion, plasma levels of lactate dehydrogenase, ferritin and neurone-specific enolase, and MYCN status correlated with outcome. However, multivariate analysis showed that only MYCN status retained prognostic value. CONCLUSIONS: In low-risk stage 3 neuroblastoma, standard-dose chemotherapy is associated with an excellent chance of being cured. Aggressive chemotherapy is effective for high-risk patients, but results are still unsatisfactory. MYCN gene amplification is a prognostic indicator for most, but not all, treatment failures.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2001

Somatostatin receptor type 2 gene expression in neuroblastoma, measured by competitive RT-PCR, is related to patient survival and to somatostatin receptor imaging by indium -111-pentetreotide.

BACKGROUND: We previously reported that human neuroblastoma cell lines and primary neuroblastoma tumors expressed a variable amount of mRNA for type 2 somatostatin (sst2) receptor gene. We also found that high level of sst2 expression were positively related to patient survival. PROCEDURE: We studied retrospectively 49 primary neuroblastomas. To detect and measure sst2 mRNA expression we developed a quantitative RT-PCR based on competitive PCR. When possible the number of MYCN copies was also measured with competitive PCR. RESULTS;. We found that the lowest level of sst2 mRNA was detected in advanced stages of neuroblastomas (stage IV) when compared with the other stages (P< 0.005). Patients with high levels of sst2 expression (>7 x 10(7) molecules/microg RNA) had a cumulative survival better than those with low sst2 expression (P < 0.0005). This predictive independent value of sst2 (P= 0.005) is retained after stratification for N-myc amplification. Finally we verified that the ex vivo sst2 gene expression in tumor samples was positively related (P < 0.01) to the in vivo semiquantitative determination of sst2 protein, assessed by 111In-pentetreotide imaging. CONCLUSIONS: Our data indicate that the measurement of sst2 mRNA measurement could represent a relevant tool in the prediction of neuroblastoma outcome, independently from MYCN amplification.

Biomarkers, Tumor↗
PubMed2001

Exclusion of candidate genes and chromosomal regions in familial neuroblastoma.

Two families with recurrence of neuroblastoma one Italian and one British with three and two affected children respectively were genotyped using polymorphic markers on chromosome 1 spanning the p32-p36 region frequently deleted in neuroblastoma tumor cells. Linkage to this region was excluded by haplotype inspection and negative lod scores. Furthermore, the exclusion of genes involved in neurocristopathies sometimes associated with neuroblastoma was carried out by typing the Italian family with polymorphic markers located in or near the corresponding genes. Finally, linkage analysis in the two families showed negative lod scores for markers spanning the 16p12-13 chromosomal region where a locus for familial neuroblastoma has been recently mapped. Our findings indicate that different genes are involved in the pathogenesis of familial neuroblastoma.

Chromosome Mapping↗
PubMed2000

p27Kip1 accumulation is associated with retinoic-induced neuroblastoma differentiation: evidence of a decreased proteasome-dependent degradation.

Development of human neuroblastoma is due to an arrest in the differentiation program of neural crest sympathoadrenal progenitor cells. However, neuroblastomas, as well as their derived cell lines, maintain the potentiality of terminal differentiation. We investigated the molecular mechanisms by which retinoic acid, a molecule introduced in clinical trials for chemotherapy, induces differentiation in neuroblastoma cell lines. Our findings demonstrate that the retinoic acid-dependent growth arrest of LAN-5 neuroblastoma cell line is associated to a very large accumulation (>tenfold) of p27Kip1 protein, a cyclin-dependent kinase inhibitor; the protein binds and inhibits cyclin-dependent kinase 2, 4 and 6 activities, thus hampering pRb and p107 phosphorylation. p27Kip1 build-up was observable as an early phenomenon (12 - 24 h) after retinoic exposure and resulted in a time-dependent accumulation of high quantities of a free p27Kip1 form. Furthermore, retinoic treatment causes an increase of cyclin-dependent kinase 5 level and activity; however, immunoprecipitation studies proved the absence of interaction with p27kip1. No noticeable variation of other components of G1 phase cell cycle engine was observed. Pulse-chase experiments showed a remarkable elongation of p27Kip1 half-life in retinoic-treated LAN-5, while no enhancement of p27Kip1 gene expression and of the translational efficiency of its messenger RNA were demonstrated. In vivo degradation of p27Kip1 was sensitive to two highly specific proteasome inhibitors, LLnL and lactacystin, while the calpain inhibitor II ALLM and the cysteine protease inhibitor E64 did not modify the level of the protein. LLnL treatment caused a very rapid (2 h) build-up of the Cdk inhibitor content and the accumulation of higher molecular weight anti-p27Kip1 immunoreactive bands, which probably represent ubiquitinated forms of the protein. Finally, in vitro experiments demonstrated that extracts prepared from retinoic-treated LAN-5 cells degraded recombinant p27Kip1 at a rate remarkably slower than the untreated cells. Our results indicate that retinoic acid strongly increases p27Kip1 levels by down-regulating the ubiquitin-proteasome p27Kip1 degrading pathway.

Cell Cycle↗
PubMed2000

Reduced expression of transforming growth factor-beta receptor type III in high stage neuroblastomas.

Transforming growth factor beta (TGF-beta) is a powerful inhibitor of cell proliferation and a potent inducer of differentiation. Resistance to TGF-beta action is a characteristic of many malignancies and has been attributed to alterations of TGF-beta receptors as well as disturbance of downstream transduction pathways. To analyse the TGF-beta response in neuroblastoma, the expression of TGF-beta1 and TGF-beta type I, II and III receptor genes was investigated in 61 cancer samples by means of reverse transcription polymerase chain reaction. The specimens analysed belong to different stages, namely nine samples of stage 1, ten of stage 2, nine of stage 3 and 28 of stage 4. Moreover, five samples were of stage 4S, which represents a tumour form undergoing spontaneous regression. The results obtained show that TGF-beta1 and TGF-beta type I and II receptor genes appear to be almost equally expressed in neuroblastomas of all stages. Conversely, TGF-beta type III receptor gene expression, which is required for an efficacious TGF-beta binding and function, is strongly reduced exclusively in neuroblastomas of stages 3 and 4. These findings were directly confirmed by immunohistochemical analyses of ten neuroblastoma specimens. Our results suggest the occurrence of an altered TGF-beta response in advanced neuroblastomas which might be an important mechanism for escaping growth control and for developing invasiveness. Moreover, our findings allow the proposal of a novel mechanism, namely down-regulation of TGF-beta type III receptor gene expression, to avoid TGF-beta inhibitory activity.

Cell Division↗
PubMed2000

Numerical and structural aberrations in advanced neuroblastoma tumours by CGH analysis; survival correlates with chromosome 17 status.

Rapid tumour progression in neuroblastoma is associated with MYCN amplification, deletion of the short arm of chromosome 1 and gain of 17q. However, patients with advanced disease without MYCN amplification and/or 1p deletion have a very poor outcome too, which suggests other genetic defects may predict an unfavourable prognosis. We employed CGH to study 22 tumours of patients at stages 3 and 4 over one year of age (6 and 16 cases respectively). Patients were divided in groups (A) long-term survivors and (B) short-term survivors. CGH showed a total of 226 chromosome imbalances (110 in group A and 116 in group B). The neuroblastoma cells of long-term survivors showed a preponderance of numerical aberrations (54%vs 43%); particularly gains of entire chromosomes 1 (P< 0.03), 7 (P< 0.04) and 19 (P< 0.05). An extra copy of 17 was detected in 6/8 (75%) samples of group A and only 1/14 (7%) samples of group B (P< 0.002). Conversely, tumours of patients who died from disease progression displayed a higher frequency of structural abnormalities (43%vs 35%), including loss of 1p, 9p, 11q, 15q and 18q and gain of 12q, although the difference was not significant (P = 0.24). Unbalanced gain of 17q was detected in 8/14 (57%) tumours of group B and only 1/8 (13%) tumours of group A (P< 0.05). The peculiar genetic difference observed in the tumours of long and short-term survivors may have prognostic relevance.

Child↗
PubMed2000

Quantitative determination of sst2 gene expression in neuroblastoma tumor predicts patient outcome.

Neuroblastoma (NB) is the most common pediatric neuroendocrine tumor, and it is characterized by a quite variable clinical course. We previously found a great variability in the expression of somatostatin receptor type 2 (sst2) in several human NB cell lines and primary tumors. In this report we investigated whether expression of sst2 is somehow related to clinical outcome. We performed a retrospective study on 54 patients with a maximum follow-up of 100 months. The concentration of specific messenger ribonucleic acid (mRNA) for sst2 was measured by competitive RT-PCR and validated, in a small subset of samples, by quantitative imaging of gene (in situ hybridization) and protein (immunohistochemistry) expression. We found that sst2 mRNA was variably expressed in all NB tumors (range, 2.5 x 10(5) to 8 x 10(9) molecules/microg RNA) with a relevant reduction in the more advanced stage (P < 0.01). Analysis of Kaplan-Meier curves indicated that sst2 expression is positively related to the overall (P < 0.0001) and event-free (P < 0.0001) survival. Expression of sst2 was negatively related to tumor stage (P < 0.02) and MYCN amplification (P < 0.001), a poor prognostic factor. However, the prognostic information derived from sst2 is apparently independent from MYCN amplification, as assessed by stratifying sst2 values according to MYCN. In addition, the expression of sst2 was the only significant prognostic factor (P < 0.02) when it was included in a multivariate model containing other well known prognostic factors such as age, stage, and MYCN amplification. Hence, we propose that sst2 expression represents a new prognostic marker for NB. The main clinical value of a quantitative measure of sst2 lies in its ability to detect patients at low risk, independently from other prognostic factor, including MYCN amplification.

Brain Neoplasms↗
PubMed1999

Stage-independent expression and genetic analysis of tp73 in neuroblastoma.

The tp73 gene, a tp53 homologue, has been sub-regionally mapped at 1p36.3, a chromosomal region frequently deleted in neuroblastoma. Due to its chromosomal localization and to the mono-allelic expression observed in some neuroblastoma cell lines, it was proposed that tp73 might be involved in the pathogenesis of neuroblastoma. Functional assays have demonstrated that tp73 can inhibit cell proliferation and induce apoptosis. The role of this gene in tumorigenesis, however, is still unclear. We analyzed tp73 expression in 95 sporadic neuroblastoma samples by RT-PCR and we detected the tp73 transcript in 46 cases (48.4%), without significant correlation with age, clinical stage or 3-year overall survival. A genetic polymorphism in the 2nd exon of tp73 was utilized to identify the transcribed allele in tumor-cell samples. Expression from only one of the tp73 alleles was found in 13 out of 16 heterozygous tumors, while in 3 samples both alleles were present. Genotype analysis of 73 patients and 150 controls showed a significant deviation (p = 0.0308) from the Hardy-Weinberg equilibrium for a tp73 allele only among neuroblastoma patients. The absence of correlation between tp73 expression and clinical stage, age and survival suggests that this gene does not play an essential function in the clinical course of the disease. However, the distribution of genomic tp73 alleles in patients indicates that a role of this gene in the development of neuroblastoma cannot be completely ruled out. Int. J. Cancer (Pred. Oncol.) 84:365-369, 1999.

Alleles↗
PubMed1999

Expression of B-myb in neuroblastoma tumors is a poor prognostic factor independent from MYCN amplification.

The transcription factors of the Myb family are expressed in several tissues and play an important role in cell proliferation, differentiation, and survival In this study, the expression of A-myb, B-myb, and c-myb was investigated in a group of 64 neuroblastomas at different dinical stages by a sensitive reverse transcription-PCR tchnique and correlated with patients' survival. All of the myb genes were frequently expressed in neuroblastoma tumors. Interestingly, the expression of B-myb, which was detected in 33 cases, was associated with an increased risk of death (P = 0.027 in a univariate analysis), whereas there was no correlation with A-myb and c-myb expression. In addition, in a multivariate Cox regression analysis that included myb gene expression, MYCN status, age at diagnosis, and tumor staging, MYCN amplification and B-myb expression were independently associated to an increased risk (P < 0.01 and P = 0.015, respectively). In overall survival curves obtained by stratifying the neuroblastoma cases on the basis of MYCN status and B-myb expression, the group of patients without MYCN amplification and positive for B-myb expression had worse survival probability than that without MYCN amplification and nonexpressing B-myb (P < 0.01). In summary, these findings provide the first demonstration that B-myb expression can be a useful prognostic marker in human neuroblastoma. Moreover, B-myb expression has a prognostic value complementary to MYCN amplification and can identify a group of high-risk patients that would not be predicted on the basis of the MYCN status only.

Cell Cycle Proteins↗
PubMed1999

Neuroblastoma in two siblings supports the role of 1p36 deletion in tumor development.

Familial neuroblastoma occurs rarely. We studied a family with three children; one of them has a disseminated (stage 4) and another has a localized (stage 2) neuroblastoma. We observed subtelomeric locus D1Z2 (1p36) deletion in both tumors by using double-color fluorescence in situ hybridization. The MYNC gene was found in single copy in both tumors. Loss of heterozygosity (LOH) and restriction fragment length polymorphism analyses were performed by using DNA from frozen tumor cells and from microdissected tumor areas excised from paraffin-embedded sections. We detected somatic LOH at locus D1S468 (1p36) in a tumor-cell population with a trisomy 1 of the stage-2 patient. Neuroblastoma cells of the stage-4 patient were diploid and showed allelic loss at the following loci: D1S172, D1S80, D1S94, D1S243, D1S468, D1S214, D1S241, and D1S164. Haplotype study showed that the siblings inherited the same paternal 1p36-->pter chromosome region by homologous recombination and that, in the two tumors, arm 1p of different chromosomes of maternal origin was damaged. Our results suggest that the siblings inherited the predisposition to neuroblastoma associated with paternal 1p36 region and that tumors developed as a consequence of somatic loss of the maternal 1p36 allele.

Abdominal Neoplasms↗
PubMed1999

Detection of DNA polymorphisms and point mutations of high-affinity nerve growth factor receptor (TrkA) in human neuroblastoma.

We have studied nerve growth factor (NGF) receptors in 63 neuroblastoma tissues. We found DNA polymorphisms of TrkA consisting in C-->T transitions in tyrosine kinase domain at 6773, 7232 and 7301 nucleotides. Both C/T alleles were detected in tumors belonging to patients at stages 1, 2, 3 and 4 but not in 4S that expresses only the allele with C-->T. Furthermore, we detected a GT transversion resulting in a Gly-->Val substitution in a stage 4 and a stage 4S samples. We report the first evidence of TrkA polymorphism and point mutations in neuroblastoma.

Humans↗