A. Valent

Monoallelically Expressed Gene Related to p53 at 1p36, a Region Frequently Deleted in Neuroblastoma and Other Human Cancers

We describe a gene encoding p73, a protein that shares considerable homology with the tumor suppressor p53. p73 maps to 1p36, a region frequently deleted in neuroblastoma and other tumors and thought to contain multiple tumor suppressor genes. Our analysis of neuroblastoma cell lines with 1p and p73 loss of heterozygosity failed to detect coding sequence mutations in remaining p73 alleles. However, the demonstration that p73 is monoallelically expressed supports the notion that it is a candidate gene in neuroblastoma. p73 also has the potential to activate p53 target genes and to interact with p53. We propose that the disregulation of p73 contributes to tumorigenesis and that p53-related proteins operate in a network of developmental and cell cycle controls.

Apoptosis regulation in tetraploid cancer cells

Tetraploidy can result in cancer‐associated aneuploidy. As shown here, freshly generated tetraploid cells arising due to mitotic slippage or failed cytokinesis are prone to undergo Bax‐dependent mitochondrial membrane permeabilization and subsequent apoptosis. Knockout of Bax or overexpression of Bcl‐2 facilitated the survival of tetraploid cells at least as efficiently as the p53 or p21 knockout. When tetraploid cells were derived from diploid p53 and Bax‐proficient precursors, such cells exhibited an enhanced transcription of p53 target genes. Tetraploid cells exhibited an enhanced rate of spontaneous apoptosis that could be suppressed by inhibition of p53 or by knockdown of proapoptotic p53 target genes such as BBC3/Puma, GADD45A and ferredoxin reductase. Unexpectedly, tetraploid cells were more resistant to DNA damaging agents (cisplatin, oxaliplatin and camptothecin) than their diploid counterparts, and this difference disappeared upon inhibition of p53 or knockdown of p53‐inducible ribonucleotide reductase. Tetraploid cells were also more resistant against UVC and γ‐irradiation. These data indicate the existence of p53‐dependent alterations in apoptosis regulation in tetraploid cells.

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.

P73 functionally replaces p53 in Adriamycin‐treated, p53‐deficient breast cancer cells

p53‐related genes, p73 and p63, encode 2 classes of proteins, TA‐p73/p63 and ΔN‐p73/p63. TA‐p73/p63 demonstrate p53‐like properties including gene transactivation and cell death promotion, whereas ΔN‐p73/p63 lack these p53‐like functions. Although p53‐deficient cancer cells are often less responsive to chemotherapy, they are not completely drug resistant, suggesting that other apoptotic pathways are at work. Here, we compared for the first time to our knowledge p73 and p63 activation in various breast cancer (BC) cell lines after Adriamycin (ADR) treatment, an agent considered as mandatory in breast cancer chemotherapy. Our study was carried out using 1 p53‐proficient BC cell line (MCF7 cells) and 3 BC cell lines deficient in p53 response (MCF7/ADRIGR, MDA‐MB157 and T47D) after ADR‐induced genotoxic stress. We report that in cells with no p53 response after ADR treatment, TAp73, but not TAp63 or ΔN‐p73/p63, may replace p53 in triggering not only apoptosis but also cell cycle arrest or DNA repair effectors such as p21, GADD45, 14‐3‐3σ and p53R2. We also demonstrate that TAp73 siRNA inhibits the accumulation of TAp73 in response to ADR treatment in MDA‐MB157 cells and confers protection against ADR. ADR‐induced downregulation of the ΔNp73 isoform in the T47D cell line with nonfunctional mutant p53 further supports anti‐apoptotic function of the isoform antagonistic to both p53 and TA‐p73/p63. Exogenous TAp73 and ΔNp73 overexpression in p53‐response‐deficient cell lines further confirms these results. cDNA microarray techniques demonstrated that the cellular response induced by p73 during ADR treatment could involve specific genes.

Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification.

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.

Diagnosis of HER2 gene amplification in breast carcinoma

Amplification of the HER2 gene, mapping to 17q21.1, is present in about 20 % of breast carcinomas. Amplification leads to an overexpression of the protein that made it possible to develop a targeted therapy by the monoclonal antibody trastuzumab (Herceptin). A good response to the treatment requires a stringent assessment of the gene status in tumours; only patients whose tumour shows a high expression of the protein or an amplification of the gene are eligible. Cases with intermediate level expression are checked by in situ hybridization, mainly by FISH, to identify amplifications in this subset of tumours. Results are sometimes difficult to interpret due to the frequent aneuploidy of the tumours. Moreover, copy number cut-offs of the gene for defining an amplification are variable according to the studies. A tumour is considered now as amplified when showing more than six HER2 copies per nucleus, or a ratio HER2 to centromere 17 greater than 2.2. The phenomenon of HER2 amplification in breast cancers is discussed in this paper, and distinguished from gene overrepresentation. It is recommended that tumours showing six to seven copies of HER2 are assessed with a kit including the centromere 17. Clusters of signals are characteristic of amplifications. The process designed for the assessment of HER2 is a model of strategies that will be used for the evaluation of markers involved in future targeted therapies.

Diagnostic de l’amplification du gène HER2 dans les cancers du sein: Le point de vue génétique

Amplification of the HER2 gene, mapping to 17q21.1, is present in about 20 % of breast carcinomas. Amplification leads to an overexpression of the protein that made it possible to develop a targeted therapy by the monoclonal antibody trastuzumab (Herceptin). A good response to the treatment requires a stringent assessment of the gene status in tumours; only patients whose tumour shows a high expression of the protein or an amplification of the gene are eligible. Cases with intermediate level expression are checked by in situ hybridization, mainly by FISH, to identify amplifications in this subset of tumours. Results are sometimes difficult to interpret due to the frequent aneuploidy of the tumours. Moreover, copy number cut-offs of the gene for defining an amplification are variable according to the studies. A tumour is considered now as amplified when showing more than six HER2 copies per nucleus, or a ratio HER2 to centromere 17 greater than 2.2. The phenomenon of HER2 amplification in breast cancers is discussed in this paper, and distinguished from gene overrepresentation. It is recommended that tumours showing six to seven copies of HER2 are assessed with a kit including the centromere 17. Clusters of signals are characteristic of amplifications. The process designed for the assessment of HER2 is a model of strategies that will be used for the evaluation of markers involved in future targeted therapies.

Diagnostic de l’amplification du gène HER2 dans les cancers du sein

Amplification of the HER2 gene, mapping to 17q21.1, is present in about 20 % of breast carcinomas. Amplification leads to an overexpression of the protein that made it possible to develop a targeted therapy by the monoclonal antibody trastuzumab (Herceptin). A good response to the treatment requires a stringent assessment of the gene status in tumours; only patients whose tumour shows a high expression of the protein or an amplification of the gene are eligible. Cases with intermediate level expression are checked by in situ hybridization, mainly by FISH, to identify amplifications in this subset of tumours. Results are sometimes difficult to interpret due to the frequent aneuploidy of the tumours. Moreover, copy number cut-offs of the gene for defining an amplification are variable according to the studies. A tumour is considered now as amplified when showing more than six HER2 copies per nucleus, or a ratio HER2 to centromere 17 greater than 2.2. The phenomenon of HER2 amplification in breast cancers is discussed in this paper, and distinguished from gene overrepresentation. It is recommended that tumours showing six to seven copies of HER2 are assessed with a kit including the centromere 17. Clusters of signals are characteristic of amplifications. The process designed for the assessment of HER2 is a model of strategies that will be used for the evaluation of markers involved in future targeted therapies.

Diagnostic de l’amplification du gène HER2 dans les cancers du sein: Le point de vue génétiqueDiagnosis of HER2 gene amplification in breast carcinoma: The genetic point of view

Amplification of the HER2 gene, mapping to 17q21.1, is present in about 20 % of breast carcinomas. Amplification leads to an overexpression of the protein that made it possible to develop a targeted therapy by the monoclonal antibody trastuzumab (Herceptin). A good response to the treatment requires a stringent assessment of the gene status in tumours; only patients whose tumour shows a high expression of the protein or an amplification of the gene are eligible. Cases with intermediate level expression are checked by in situ hybridization, mainly by FISH, to identify amplifications in this subset of tumours. Results are sometimes difficult to interpret due to the frequent aneuploidy of the tumours. Moreover, copy number cut-offs of the gene for defining an amplification are variable according to the studies. A tumour is considered now as amplified when showing more than six HER2 copies per nucleus, or a ratio HER2 to centromere 17 greater than 2.2. The phenomenon of HER2 amplification in breast cancers is discussed in this paper, and distinguished from gene overrepresentation. It is recommended that tumours showing six to seven copies of HER2 are assessed with a kit including the centromere 17. Clusters of signals are characteristic of amplifications. The process designed for the assessment of HER2 is a model of strategies that will be used for the evaluation of markers involved in future targeted therapies.

SFCE-05 – Cancérologie, hématologie, immunologie – Classification génomique dans le neuroblastome : utilité pour la prise en charge thérapeutique

Objectifs Le neuroblastome (NB) est un cancer pediatrique avec une grande heterogeneite clinique. Des nombreuses alterations genetiques recurrentes ont ete decrites : une amplification de MYCN associee a un pronostic pauvre, ainsi que des variations de la ploidie ou des alterations chromosomiques segmentaires (deletions du chromosome 1p, 3p, 4p, 11q ; gain du chromosome 2p, 17q) dont l’importance pronostique reste a etre determinee. Afin d’etudier l’association de ces alterations genetiques entre elles et leur impact pronostique, nous avons entrepris une analyse en CGH-array d’une grande serie de NB. Methodes 389 echantillons de NB ont ete analyses en hybridation genomique comparative (CGH), sur une puce d’ADN a BAC/PAC avec une resolution moyenne de 1Mb. Resultats L’analyse du profil genomique permet de distinguer 2 differents types d’instabilite genetique : une instabilite numerique et une instabilite segmentaire. L’instabilite numerique est caracterisee par une variation en nombre de chromosomes entiers sans alterations segmentaires (n = 162). Elle est associee a une survie sans progression et une survie globale excellente. L’instabilite segmentaire se caracterise par des translocations chromosomiques desequilibrees. Elle a ete observee dans des tumeurs sans (n = 45) ou avec (n = 97) variations numeriques, ou en association avec une amplification de MYCN (n = 67). Ce type genomique est associe a un risque eleve de rechute (p Conclusion Dans le NB, un profil genomique de type segmentaire est le marqueur pronostique le plus fort. Ceci souligne l’importance du mecanisme a l’origine des translocations desequilibrees dans l’oncogenese du NB. Le typage genomique devra donc etre pris en compte pour l’attribution a un groupe de risque et la stratification therapeutique. Le NB est le premier modele de l’utilite d’une classification genomique pour une meilleure prise en charge therapeutique.