Background: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis.Methods: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol.Results: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P = 0.04). A significant correlation (P = 0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS = 46% vs 75%, P = 0.023; OS = 66.8% vs 100%, P = 0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P = 0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P = 0.018).Conclusions: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

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, p53-72Pwasmore 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. Neoplasia (2012) 14, 634–643 Introduction Neuroblastoma (NB) is a childhood solid tumor that accounts for 8% to 10% of all childhood cancers and for ∼15% of all deaths because of pediatric malignancies [1]. NB arises from neuroectodermal precursor cells of the neural crest, and therefore, tumors can develop anywhere in the sympathetic nervous system [1]. Clinically, NB is remarkably heterogeneous: age at diagnosis [2], clinical stage (based on the International Neuroblastoma Staging System [3]), and tumor histology [4] are the most important variables for predicting disease risk and selecting appropriate therapeutic protocols. Children older than 18 months, with tumor at advanced stage (3 or 4) or with unfavorable histologic findings have an adverse outcome despite intensive multimodal treatments such as high-dose myeloablative chemotherapy followed by rescue with autologous bone marrow [5]. Clinical heterogeneity correlates with several genetic abnormalities whose detection in tumor cells has further improved risk stratification [5]. V-myc myelocytomatosis viral-related oncogene NB-derived (MYCN) oncogene amplification [6], hemizygous deletions of chromosomal region 1p36 [7], and unbalanced gain of 17q regions [8] are the most common genomic aberrations in NB. MYCN amplification occurs in ∼20% of cases and represents the most powerful marker of poor outcome [9]. In contrast to other malignancies, only 2% to 3% of NBs have mutations of the p53 gene [10]. p53 is a tumor suppressor gene that encodes for a nuclear phosphoprotein, which, on activation, regulates many biologic processes such as cell cycle checkpoints, apoptosis, and cellular senescence [11]. Impairment of such processes has important implications for clinical behavior and response to therapy of tumors with nonfunctional p53 [12]. p53 also activates the transcription of the murine double minute (MDM2) gene that encodes for the major negative regulator of p53 [13]. In recent years, there has been an increasing interest in identifying and assessing the frequency of gene variants (polymorphisms) as a tool to predict interindividual cancer risk and response to cancer therapies [14]. A polymorphism is defined as a DNA sequence change that occurs in a significant proportion (>1%) of a large population [15]. The most common type of genetic variation is a single nucleotide polymorphism (SNP). For the p53 gene, an SNP has been identified at codon 72 within exon 4 causing an Arg>Pro substitution [16]. The p53-72R isoform seems to be more potent than the p53-72P isoform in inducing apoptosis, and increased mitochondrial localization and reduced affinity of the p53-72R isoform for the p53 inhibitor iASPP are among the proposed mechanisms that may be responsible for such an effect [17,18]. In this study, we compared the frequency of the p53 codon 72 Arg/ Arg, Arg/Pro, and Pro/Pro genotypes in 288 control subjects and 286 newly diagnosed NBs and correlated these frequencies with clinicalbiologic variables such as age at diagnosis, primary tumor site, clinical stage, and MYCN amplification. We report here that patients with the Pro/Pro genotype, including those with normal MYCN status and advanced disease stages, seem to have a shorter 5-year survival than those with the Arg/Pro or Arg/Arg genotype. The more aggressive disease of patients with NB with the Pro/Pro genotype correlated with lower apoptosis and enhanced survival of cytotoxic drug or ionizing radiation (IR)–treated p53-null LAN-1 cells expressing the p53-72P compared with the p53-72R isoform. By contrast, expression of the p53-72P isoform, alone or in the presence of a low concentration of etoposide, induced an increase in senescent cells. Together, these findings suggest that, although relatively rare, the p53 codon 72 Pro/Pro genotype might identify a subgroup of patients with NB with aggressive disease independently of the status of other markers predictive of poor outcome. Moreover, patients with this genotype may respond less efficiently to treatments that induce DNA 636 Role of p53Arg72Pro Polymorphism in Neuroblastoma Cattelani et al. Neoplasia Vol. 14, No. 7, 2012 damage and increased p53 expression/activity and may benefit from therapeutic protocols that include cytotoxic drugs with p53-independent mechanisms of action. Materials and Methods Subjects Two hundred eighty-six patients with NB (258 Italian patients, most of whom were previously included to assess the frequency of the MDM2 SNP-309 polymorphism [19] and 28 British patients) were selected for the analysis of the p53 codon 72 genotype. Their clinical and biologic characteristics are listed in Table W1. Tumor DNA was analyzed in each patient, whereas peripheral blood DNA was also genotyped in 40 cases to confirm that polymorphism frequency did not reflect somatic mutation. Selection criteria were lack of previous treatments and availability of adequate amount of DNA. Tumor DNA was used because peripheral blood lymphocytes are not always readily attainable from patients with NB. Institutional written informed consent was obtained from parents or legal guardians of patients with NB. The study underwent ethical review and approval according to local institutional guidelines. Controls The p53 codon 72 genotype was also evaluated in peripheral blood DNA of 288 healthy individuals from anonymous blood donors randomly selected during a 5-year period (2000-2005) at several Northern Italy Blood Banks and from control subjects stored at the “Oncology Institute of Veneto,” Padova, Italy. Their age ranged from 25 to 60 years (median, 45 years) and the male-to-female ratio was approximately 46% to 54%. NB Cell Lines DNA DNA from NB-69, NBL-W, NBL-S, BE1N, TR14, NGP, NB1691, and LS NB cell lines was from Dr Tweddle’s laboratory, whereas DNA from RN-GA, SH-EP, SK-, GI-CAN, and SK-NBE2 cell lines was from Dr Raschellà’s laboratory. DNA from LAN-5, HTLA-230, GI-CAN, SH-SY5Y, IMR32, LAN-1, and SK-N-AS cell lines was obtained using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) (Table W2). p53 Codon 72 Genotyping The p53 DNA segment for codon 72 genotype was amplified by polymerase chin reaction (PCR) using 150 ng of DNA and a pair of forward (5′-TTGCCGTCCCAAGCAATGGATGA-3′) and reverse (5′-TCTGGGAAGGGACAGAAGATGAC-3′) primers that generated a 199-bp DNA product. DNA amplification was done as follows: 95°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes for 40 cycles. PCR products were separated by electrophoresis in 1% agarose gel with ethidium bromide, extracted using a PCR purification kit (Roche Diagnostics GmbH, Mannheim, Germany), and each fragment was sequenced from both ends on an ABI PRISM 377 DNA Sequencer using the ABI PRISM Big Dye Terminator (PE Biosystem, Foster City, CA). Thirty tumor and control DNA were independently resequenced to confirm the genotype’s accuracy. Statistical Analysis The frequencies of p53 polymorphism at codon 72 were crosstabulated in patients with NB versus healthy controls using a two-sided Fisher exact test. The frequencies of p53 polymorphism at codon 72 in patients with NB versus known prognostic factors were also crosstabulated using the Fisher exact test to evaluate the significance of the association. Clinical-pathologic variables were categorized as follows: age at diagnosis (<18 vs ≥18 months) [2], primary site (adrenal vs nonadrenal), clinical stage according to the International Neuroblastoma Staging System [3] (stage 1, 2, and 4 S vs 3 and 4), and MYCN status (single copy vs amplified). Five-year overall survival curves on the basis of the p53 genotype at codon 72 in all patients with NB or in subgroups defined on the basis of established prognostic factors were calculated according to Kaplan and Meier [20], and the statistical significance of the differences was assessed using log-rank test. Cox multiple regression analysis [21] was carried out, including clinical stage,MYCN status, age at diagnosis, and p53 polymorphism as covariates. All statistical tests were two-sided, and P < .05 was considered statistically significant. The analyses were carried out using the software package SPSS 11.0 for Windows (SPSS, Inc, Chicago, IL). The Hardy-Weinberg equilibrium was assessed by χ test. Plasmids p53-72R-ER a

Background: In neuroblastoma (NB), the presence of segmental chromosome alterations (SCAs) is associated with a higher risk of relapse. Methods: In order to analyse the role of SCAs in infants with localised unresectable/disseminated NB without MYCN amplification, we have performed an array CGH analysis of tumours from infants enroled in the prospective European INES trials. Results: Tumour samples from 218 out of 300 enroled patients could be analysed. Segmental chromosome alterations were observed in 11%, 20% and 59% of infants enroled in trials INES99.1 (localised unresectable NB), INES99.2 (stage 4s) and INES99.3 (stage 4) ( P <0.0001). Progression-free survival was poorer in patients whose tumours harboured SCA, in the whole population and in trials INES99.1 and INES99.2, in the absence of clinical symptoms (log-rank test, P =0.0001, P =0.04 and P =0.0003, respectively). In multivariate analysis, a SCA genomic profile was the strongest predictor of poorer progression-free survival. Conclusion: In infants with stage 4s MYCN- non-amplified NB, a SCA genomic profile identifies patients who will require upfront treatment even in the absence of other clinical indication for therapy, whereas in infants with localised unresectable NB, a genomic profile characterised by the absence of SCA identifies patients in whom treatment reduction might be possible. These findings will be implemented in a future international trial.

Circumvention of the p53 checkpoint in neuroblastoma (NB) might arise from increased expression of its main negative regulator MDM2. The SNP309, a T-to-G substitution in the MDM2 promoter, was associated with higher levels of MDM2 mRNA and protein, with consequent attenuation of the p53 pathway. The association between MDM2 SNP309 and disease progression and survival was evaluated in a cohort of 142 children with stage 4 NB. The SNP309 GG patients had a worse overall survival and a worse survival after relapse than the TT ones, whereas the heterozygotes showed an intermediate behaviour (p=0.043 and p=0.049, respectively, log-rank test for trend). No evident association between SNP309 and event free survival was found. The lack of association between SNP309 and MYCN status indicates that MDM2 SNP309 may be a new independent prognostic factor for stage 4 NB.

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. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

The expression of the N-methyl-D-aspartate (NMDA) receptor subunit NR2B/epsilon2 (GRIN2B) in the human adult brain was assayed by in situ hybridisation, by using a specific cRNA probe. The full length GRIN2B cDNA was cloned and sequenced. It showed a 90% nucleotide conservation when compared to the rodent homologue. GRIN2B gene is expressed at high levels in the fronto-parieto-temporal cortex and hippocampus pyramidal cells and, at a lower extent, in the basal ganglia (amygdala and striatum). The cerebellar granule cells does not show any mRNA expression. The non-ubiquitous anatomical distribution of the GRIN2B mRNA in the central nervous system suggests that the gene could be involved in specific functions pertaining to the expressing cell groups.

Cytogenetic techniques were used to study the tissue involved in neural tube defects. Eighteen patients have been evaluated and no specific alterations have been detected. We conclude that, whatever are the mechanisms that lead to neural tube defect, their origins must be searched for at the molecular level.

We examined for the presence of 17p11.2 deletion, by Southern blotting and fluorescent in situ hybridization, 3 cases with progressive sensory-motor polyneuropathy and diffuse tomaculous changes at sural nerve biopsy. We demonstrated in all the cases the 17p11.2 deletion, previously reported in hereditary neuropathy with pressure palsy, an inherited disorder of the peripheral nervous system with similar pathologic changes but a different clinical phenotype. The molecular study of the 17p11.2 region should be considered as a non invasive method for differential diagnosis in selected cases of progressive polyneuropathy.

We report the chromosomal characteristics of a recurrent pineal non-seminomatous germ cell tumor in a 16-year-old male patient. This non-seminomatous tumor had the following components: embryonal carcinoma, teratoma, yolk sac tumor, and trophoblastic giant cells. Chromosome analysis showed a near-triploid karyotype (64 chromosomes), including two copies of an isochromosome 12p. This latter finding could be confirmed using 12p-specific competitive in situ hybridization techniques applied to cultured cells (T2219-P6 cell line) derived from the tumor. The present findings are in keeping with the hypothesis that isochromosome 12p formation is associated with the development of malignant extragonadal germ cell tumors.

The 50% survival time for low grade astrocytomas is 50 months and for high grade astrocytomas it is 13 months, underlining the need for new therapies. Several reports show that in vivo histamine antagonists cause retardation of tumour growth in some animal models and prolonged survival in cancer patients. Therefore we have tested the growth modulating effects of histamine and histamine antagonists on human glioma cultures. Twelve freshly excised human gliomas were cultured and tested for their in vitro sensitivity to histamine and histamine antagonists. Four continuous glioma cell lines were used to confirm the glioma-specificity of the effects observed in the primary cell lines. In low serum concentration (0 or 1%) the growth of 5/9 primary glioma-derived cultures could be stimulated with 0.2 mM histamine, and in 4/5 cases with 0.2 microM histamine. One mM of the histamine H2-receptor antagonist cimetidine could inhibit the growth of 4/5 primary glioma cultures when tested in 1% human AB serum, and of 6/13 cases when tested in 1% FCS. Lower concentrations (down to 1 microM) were less effective. The histamine H1-receptor antagonist pyrilamine gave variable results. The specificity of the effects is indicated by the absence of a generalised toxic effect, by the observation that the antagonist-induced inhibition could be reversed with histamine, and by the correlation of the obtained cimetidine-induced growth inhibition with the maximal growth rate of the primary cell lines in 10% FCS. The observed cimetidine-induced inhibition of the in vitro proliferation of gliomas suggests that cimetidine is a relevant candidate for the in vivo growth inhibition of these tumours.

In 420 Philadelphia positive (Ph+) chronic myeloid leukemia (CML) patients karyotyped at diagnosis in our laboratory, 26 Ph variants (6.2%) were observed. Twelve of them are reported. Five cases are “simple” variants without detectable involvement of band 9q34, and seven are “complex,” since a third chromosomal band is involved in the Ph formation. Two translocations [t(7;22)(q36;q11) and t(9;22;12)(q34;q11;q11)] are reported for the first time. Six cases were characterized molecularly, and bcr-abl rearrangement was demonstrated, confirming involvement of 9q34 band also in the cases in which chromosomes 9 appear cytogenically normal. Chimeric mRNAs in which M-BCR exon 3 is joined to abl exon 2 (type b3-a2) were detected in four of six cases; one case showed a DNA breakpoint in zone III, which may also give rise to the same transcript. In one case, mRNA junction was b2-a2. The frequency of the b3-a2 junction occurs more frequently in CML patients with a Ph variant than in patients with the standard translocation, suggesting a preferential correlation between this type of transcript and the involvement of other chromosomes in Ph formation.