Merkel cell polyomavirus (MCPyV) is associated to Merkel cell carcinoma (MCC). We studied 113 MCC tumoral skin lesions originating from 97 patients. MCPyV detection was higher in fresh-frozen (FF) biopsies (94%) than in formalin-fixed paraffin-embedded biopsies (39–47%). Mean viral load in FF tumor was of 7.5 copies per cell with a very wide range (0.01–95.4). Nineteen complete sequences of LTAg were obtained, mainly from FF biopsies when the viral load was high. Seventeen showed stop codons, all localized downstream of the pRb protein binding domain. Sequence comparison and phylogenetic analysis showed that all sequences clustered in the large C clade of MCPyV strains. MCPyV integration was demonstrated in 19 out of 27 FF MCC DNA biopsies without evidence of specific host cellular genome integration site. In 13/19 cases, the viral junction was located within the second exon of the LTAg, after the pRB binding domain.

To retrospectively evaluate the incidence of tumour cell contamination of peripheral blood stem cell (PBSC) collections and to correlate these data with the clinical outcome after high-dose chemotherapy (HDCT) with stem cell rescue in patients with a high-risk Ewing tumour. Peripheral blood stem cell collections obtained from 171 patients were analysed. Tumour contamination was assessed by reverse transcriptase–polymerase chain reaction (RT–PCR). The files of 88 patients who underwent HDCT followed by PBSC reinfusion were reviewed in detail, and their outcome compared to the PBSC RT–PCR results. Seven of 88 PBSC collections (8%) contained tumour cells as detected by RT–PCR. Peripheral blood stem cells were collected after a median of five cycles of chemotherapy. No clinical factor predictive of tumour cell contamination of PBSC harvest could be identified. Event-free survival (EFS) and overall survival (OS) of the whole study population were 45.3 % and 51.8 % at 3 years from the date of the graft, respectively. Forty-five patients relapsed with a median time of 15 months after graft, only four of whom had tumour cell contamination of the PBSC harvest. Tumour cell contamination of PBSC collection is rare and does not seem to be associated with a significantly poorer EFS or OS in this high-risk population.

Le cancer du col utérin est le deuxième cancer le plus fréquent chez la femme dans le monde. Les carcinomes infiltrants s'accompagnent d'une intégration génomique cellulaire d'ADN de papillomavirus humain (PVH), en particulier les PVH à haut risque de type 16 et 18. L'oncogenèse du col utérin nécessite la présence des oncoprotéines E6 et E7 du PVH qui induit la prolifération cellulaire par dégradation des protéines TP53 et pRB. Il s'ensuit une instabilité génétique, nécessaire à la transformation maligne. Les conséquences moléculaires de cette instabilité génétique ont été peu caractérisées jusqu'à présent. La technique de puce génome (CGH array, Comparative Genomic Hybridization array) permet de caractériser les altérations du nombre de copies d'ADN d'un prélèvement tumoral. Le principe consiste en une hybridation génomique comparative d'un mélange d'ADN tumoral et d'ADN normal sur de multiples sondes génomiques représentatives de l'ensemble des locus chromosomiques. Nous avons utilisé cette méthodologie pour 37 prélèvements de carcinome du col utérin : 8 lignées cellulaires et 29 carcinomes primitifs infiltrants. Après coupure enzymatique, l'ADN tumoral a été marqué par un fluorochrome (cyanine 5) et l'ADN normal par un autre fluorochrome (cyanine 3). Les deux ADN ont hybridé de façon compétitive sur la puce génome, sur laquelle ont été placés 3 500 clones de BAC (Bacterial Artificial Chromosome). Les niveaux d'intensité de chaque fluorochrome ont été mesurés pour chaque clone. Les pertes et gains d'ADN tumoral ont été déduits du rapport d'intensité des 2 fluorochromes. Les pertes chromosomiques les plus fréquentes étaient observées pour les bras chromosomiques 2q, 3p, 9p, 11q et 16q. Les gains chromosomiques les plus fréquents portaient sur les bras chromosomiques 1q, 3q, 5p, 8q, 16p et 20q. L'analyse détaillée de ces déséquilibres alléliques a permis de délimiter précisément les bornes chromosomiques de ces altérations génomiques. Nous avons également identifié 20 amplifications génomiques présentes dans 14 tumeurs. Quatre locus chromosomiques comportaient des amplifications récurrentes : 11q22.2 contenant les gènes MMP7 et MMP20 (4 cas), 1p21.1 (3 cas), 8q24.21 contenant le gène MYC (2 cas) et 11q13.3 contenant le gène CCND1 (2 cas). Ces résultats, couplés à ceux de l'analyse du transcriptome, permettent de caractériser précisément les anomalies moléculaires des carcinomes du col utérin et d'identifier de nouveaux gènes potentiellement impliqués dans l'oncogenèse du col utérin.

Deletion of chromosome arm 1p is one of the most frequent genetic alterations in neuroblastoma. However, using conventional comparative genomic hybridization, we have observed amplifications on 1p in 2 neuroblastoma tumors at bands 1p34.2 and 1p36.3, respectively. Using a medium‐resolution genomic array containing 178 PACs/BACs from 1p and then 2 high‐resolution arrays containing contigs of overlapping PACs/BACs from the amplified regions, we could precisely map and delineate both amplicons. The 1p34.2 amplicon appeared as a homogeneous amplification unit, whereas the 1p36.3 amplicon had a more complex structure, with 2 noncontiguous, highly amplified regions and several moderate amplification units. In this case, fluorescence in situ hybridization analysis confirmed the amplification of several clones and indicated that the 2 highest amplification units corresponded to 2 populations of double minute chromosomes, one of which also contained the MYCN locus. This is the first report of 1p amplifications in primary neuroblastomas. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat/index.html. © 2004 Wiley‐Liss, Inc.

PURPOSE The presence of metastasis is a major prognostic factor in Ewing tumor (ET). The relapse pattern of patients with localized tumors has long indicated that cases with disseminated ET cells escape detection at diagnosis. ET cells are characterized by specific gene fusions that can be detected with high sensitivity and specificity by reverse transcriptase polymerase chain reaction (RT-PCR). PATIENTS AND METHODS RT-PCR targeting EWS-FLI-1 or EWS-ERG transcripts was used to search for occult tumor cells in peripheral blood (PB) and bone marrow (BM) at diagnosis in 172 patients with ET, and the prognostic significance of this parameter was assessed. RESULTS As we suggested previously in a smaller series of patients, RT-PCR positivity of the BM was correlated with a high risk of adverse outcome in the overall study population (P =.007). More interestingly, among patients with otherwise localized tumors, BM micrometastasis also predicted significantly poorer disease-free survival rates (P =.043). The presence of circulating tumor cells (CTC) was more frequently observed in patients with large tumors (P =.006). CTC were associated with a poor outcome among patients with clinically localized disease (P =.045). Patients with clinically localized disease and peripheral occult tumor cells as evidenced by BM and/or PB RT-PCR positivity had axial or proximal tumors and experienced relapses at a systemic rather than at a local level. CONCLUSION Patients with localized ET and BM micrometastasis or CTC are comparable to patients with metastases in terms of the localization of the primary tumor, outcome, and relapse pattern.

The t(X;18) translocation is known to be a useful marker for the diagnosis of synovial sarcoma. In this study, the authors describe a new real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method to detect SYT/SSX fusion transcripts using paraffin-embedded and frozen tumor specimens. A series of 38 soft tissue sarcomas were analyzed. Diagnosis was based on clinical, histologic, and immunohistochemical examination. The fusion transcripts were detected in 16 of 17 synovial sarcoma samples (the 17th sample was not suitable for molecular analysis). No t(X;18)-fusion transcript was PCR-amplified in the 21 nonsynovial sarcoma mesenchymal tumors. Therefore, real-time PCR amplification appears to be a powerful, rapid, specific, and sensitive technique that can be used routinely to diagnose the synovial sarcoma t(X;18) translocation. In addition, the t(X;18) can be detected not only on frozen but also on paraffin-embedded tumor samples.

The occurrence of secondary chromosome changes is frequent in Ewing tumors, in particular trisomies for chromosomes 8 and 12, and unbalanced (1;16) translocations leading to gains of 1q and losses of 16q. The prognostic value of these secondary aberrations has not been statistically demonstrated. We report here a CGH analysis of a series of 43 primary tumors corresponding to 21 localized and 22 metastatic tumors. For five of them, a sufficient amount of DNA for the CGH analysis was available from the frozen samples. For 19 samples, a preliminary step of DOP-PCR amplification of the DNA was necessary. For the last 19 tumors, DNA was obtained after DOP-PCR amplification of small amount of DNA contaminating the RNA. As a whole, the main chromosome imbalances previously described, such as trisomies for 1q, 8, and 12, were observed. It is noteworthy that the mean number of imbalances was more frequent in localized versus metastatic tumors. Gain of 1q was more frequent in metastatic than in localized tumors. Nevertheless, these two results do not reach statistical significance. Conversely, a statistically significant excess of copy number of chromosome 2 was observed in non-metastatic tumors, suggesting that this imbalance, which has never been previously reported, could be associated with more favorable tumor behavior.

We have studied a series of 20 primary retinoblastomas by karyotypic analysis and comparative genomic hybridization (CGH), to perform an exhaustive evaluation of chromosome imbalances in this tumor. In addition, 4 tumors were studied by CGH only. On the whole, CGH results were largely in agreement with those of karyotypic analysis and with known cytogenetic data. The most frequent imbalances were +6p (13/24 cases), +1q (12/24), -16/-16q (11/24), and +2p (9/24). Recurrent high-level amplifications were observed in 2p23-25 and 1q21. Amplification of 2p23-25, present in 4 cases among which 3 showed double-minute chromosomes, was related to MYCN amplification, as demonstrated by FISH and PCR. No evident correlation was found in this small series between any of the imbalances identified and either the differentiation or the histoprognostic risk. (C) 2000 Wiley-Liss, Inc.

Parmi les crises épileptiques du nourrisson, on a isolé, ces dernières années, un ensemble syndromique appelé convulsions familiales bénignes du nourrisson. Les principales caractéristiques cliniques et évolutives sont: nourrisson de moins de 1 an, salves de crises épileptiques brèves, de type partiel, complexe, avec généralisation rapide, excellent pronostic et incidence familiale très élevée. Ce syndrome apparaît hétérogène sur le plan génétique.Benign familial infantile convulsion is a syndrome recently identified among the epileptic seizures of infancy. The main characteristics are: occurrence before one year of age, brief epileptic bursts of partial type seizures with secondary generalization, excellent prognosis with normal mental and motor development, high familial incidence. This syndrome appears genetically heterogeneous.

Malignant rhabdoid tumors are rare and aggressive neoplasms of childhood, occurring in the kidney or in various extrarenal locations. Most cytogenetic studies of these tumors have shown the frequent involvement of chromosome 22, including translocations and/or deletions, with a critical region for a rhabdoid tumor gene mapping to chromosome segment 22q11, close to BCR. We report a case of an extrarenal rhabdoid tumor with a t(1;22)(p36;q11.2) that was associated with deletions of chromosomes 1 and 22. We have performed fluorescence in situ hybridization to bracket the translocation breakpoints on both chromosomes and microsatellite analysis to establish the deletion of chromosome 22 more precisely. The chromosome 22 translocation breakpoint is localized close to BCR, in the region covered by the overlapping YACs 446B5 and 361D9, and it is associated with a proximal hemizygous deletion of approximatively 2 Mb. On chromosome 1, the translocation breakpoint maps to a 25 cM region, proximal to D1Z2 and distal to PND, and is also associated with an estimated deletion of 8 Mb. Moreover, microsatellite analysis has demonstrated a homozygous deletion of chromosome 22 for three contiguous loci, immediately distal to BCR. This result suggests that a tumor suppressor gene involved in rhabdoid tumor oncogenesis could be localized in this region of chromosome 22.

The t(11;22) and t(21;22) translocations that are specific for Ewing's tumor are responsible for production of chimeric transcripts between the EWS gene on chromosome 22 and the FLI-1 or ERG gene on chromosome 11 or 21, The development of molecular biology methods capable of detecting these composites have added to the diagnosis of Ewing's sarcoma. The specific marker can be identified in sites other than the primary, providing the opportunity for studying incipient lesions, Ewing's sarcoma cells were looked for in blood and/or bone marrow specimens from 46 patients in whom a specific composite had been detected in the primary. At diagnosis, 16 of 44 patients had tumor cells in their bloodstream and 14 of 31 had micrometastases in their bone marrow, Presence of tumor cells in the bone marrow, but not in the blood-stream, was associated with a greater likelihood of severe metastatic disease. In all the patients who were tested before and after chemotherapy, the blood tests reverted to negative after chemotherapy, and this effect was not influenced by the quality of the response of the primary to the treatment. In contrast, patients with positive bone marrow tests after chemotherapy were more likely to have a poor response of their primary. Bone marrow testing for Ewing's cells at diagnosis and during therapy may add substantially to the staging and follow-up of patients with Ewing's sarcoma.

PURPOSE Gene fusions that result from the chromosome translocations observed in Ewing's tumor (ET) provide tumor-specific markers that can be used to detect the presence of tumor cells in peripheral blood (PB), bone marrow (BM), and stem cell collection (SCC). These markers were used to evaluate, at diagnosis, a series of 67 ET patients. PATIENTS AND METHODS RNA was extracted from nucleated cells from PB and BM and a nested reverse-transcriptase polymerase chain reaction (RT-PCR) was performed to search for EWS-FLI-1 or EWS-ERG fusion transcripts that resulted from the t(11;22) or t(21;22) translocations, respectively. RESULTS At diagnosis, 16 of 62 (26%) patients had circulating tumor cells. This was not correlated with any clinical parameter. In contrast, Ewing's cells were detected by RT-PCR in BM in 14 of 43 (33%) patients and were associated with the presence of clinically detectable metastases and a statistically significant unfavorable outcome in univariate analysis. There was no correlation between the RT-PCR results in PB and in BM. CONCLUSION These results suggested that the monitoring of BM but not of PB by RT-PCR might constitute an important criterion for the staging, at diagnosis, of patients with ET. Further studies should appreciate the relationship or independence of this marker toward other classical prognostic factors in ET, particularly to the presence of clinically detectable metastases.

Les translocations t(11;22) et t(21;22), specifiques des tumeurs d'Ewing, entrainent la synthese de transcrits chimeriques entre le gene EWS sur le chromosome 22, et les genes FLI-1 ou ERG sur les chromosomes 11 ou 21. Le developpement d'approches moleculaires permettant la detection de ces fusions a debouche sur une nouvelle methode diagnostique de ces tumeurs. Ces techniques permettent egalement d'isoler ce marqueur specifique dans des sites extra-tumoraux et donnent ainsi la possibilite d'etudier la maladie minime chez des patients atteints de tumeur d'Ewing. Nous avons recherche la presence de cellules tumorales au niveau sanguin et/ou medullaire chez 46 patients pour lesquels une fusion specifique etait detectee au niveau de la tumeur primitive. Au diagnostic, 16 sur 44 patients presentaient des cellules tumorales circulantes et 14 sur 31 des micrometastases medullaires. Contrairement a la presence de cellules tumorales dans le sang, l'atteinte medullaire semble plus frequemment concerner les formes graves, metastatiques de la maladie. Lorsque les analyses ont pu etre effectuees avant et apres chimiotherapie, les prelevements de sang ont ete negatives quelle que soit la qualite de la reponse a la chimiotherapie appreciee au niveau du site primitif, suggerant que le marqueur sanguin est peu sensible pour apprecier la reponse au traitement. Par contre, les prelevements de moelle, positifs apres chimiotherapie, etaient associes a une mauvaise qualite de la reponse therapeutique au niveau de la tumeur primitive. L'analyse de la moelle au diagnostic et son suivi regulier sous traitement pourraient etre des parametres importants du staging initial et de la surveillance des patients atteints de tumeur d'Ewing.

Les tumeurs malignes des tissus mous constituent un groupe très hétérogène composé de plus de 50 entités différentes dérivées soit du mésenchyme, soit du neuroectoderme. Les difficultés diagnostiques concernent, d'une part, l'établissement d'un diagnostic de malignité et, d'autre part, la détermination précise du type de sarcome. Le pronostic est en grande partie déterminé par l'extension locale et à distance de la maladie. L'étude du génotype des cellules tumorales a permis de mettre en évidence des altérations génétiques. Ces altérations, fréquemment des translocations chromosomiques, constituent des marqueurs spécifiques à certains sous-groupes tumoraux et présentent ainsi un intérêt diagnostique. Ces marqueurs peuvent être détectés par la cytogénétique, l'hybridation in situ ou la biologie moléculaire. La spécificité de ces altérations pour les cellules tumorales et la possibilité de les mettre en évidence par la technique sensible de réaction en chaîne à la polymérase (polymerase chain reaction [PCR]) ont permis de développer des tests visant à rechercher les cellules tumorales au niveau de sites potentiellement métastatiques.

Malignant tumors of soft tissue constitute a very heterogeneous group of tumors which is composed of more than 50 different entities derived from either the mesenchyme or the neuroectoderm. Diagnostic problems can be linked to the difficulty to firmly establish the diagnosis of malignancy and to the precise determination of the type of sarcoma. The prognostic is largely dependent on local and distant extension of the cancer. The study of the genotype of tumor cells has enabled the detection and characterization of genetic alterations. These alterations, frequently chromosome translocations, are specific markers for subgroups of tumors and can thus be of clinical relevance. These markers can be detected by cytogenetic, in situ hybridization or molecular biology techniques. The specificity of these alterations for tumor cells and the possibility to detect them with the highly sensitive polymerase chain reaction (PCR) technique have enabled development of tests aimed at the detection of tumor cells within potential metastatic sites.

We report on the cytogenetic and molecular analysis of a malignant melanoma of the soft parts (MMSP). A t(12;22)(q13;q12) was found as the only structural chromosomal change, and this provides additional support for the important role of this translocation in MMSP development. Molecular analysis revealed in frame fusion between exon 10 of the Ewing's sarcoma oncogene (EWS) and codon 110 of ATF-1. In previously analyzed MMSPs, junctions were observed between EWS exon 8 and ATF-1 codon 65. The present data thus indicate that, as in Ewing's sarcoma, different fusion proteins can occur in MMSP. The presence of the EWS/ATF-1 fusion gene in the tumor cells was demonstrated by dual color fluorescence in situ hybridization on interphase nuclei. Our data provide additional support for the specific association of the t(12;22) and the resulting EWS/ATF-1 gene fusion in MMSP. This particular genetic aberration, therefore, serves as a strong diagnostic marker for MMSP. We conclude that detection of the t(12;22) by cytogenetic or molecular analysis is useful in establishing or confirming the diagnosis of MMSP.

The magnitude of N-myc amplification (NMA) influences the treatment strategy of localized neuroblastomas, Reliable assays are therefore needed for all types of tumor samples, The aim of this comparative study of 119 tumor samples was to determine whether a polymerase chain reaction (PCR)-based assay could replace the current dot blot assay as a routine and reliable means of determining NMA, The 2 assays exhibited comparable sensitivity and were completely concordant for samples containing at least 20% neuroblastoma cells, In their present state, both assays remain semi-quantitative since an absolute quantification of the N-myc copy number in clinical samples is limited by uncertainty about the amplification level of reference cell lines and by the estimation of the proportion of malignant cells, However, PCR offers several advantages over dot blotting, such as feasibility on minute samples, simplicity, standardization, rapidity and cost effectiveness. (C) 1997 Wiley-Liss, Inc.

As a result of chromosome translocations, the EWS gene is fused to a variety of transcription factors in human solid tumors. Up to now, gene fusions of EWS with 6 different partners have been described. In all fusions presently reported the entire N-terminal domain of EWS (NTD-EWS) composed of 265 amino acids encoded by the first 7 exons of EWS was always included in the chimeric proteins, suggesting that the integrity of this domain was mandatory for the oncogenic property of the fusion proteins. We report the molecular characterization of a Ewing tumor demonstrating a reciprocal t(21;22)(q22;q12) translocation. No EWS/ERG fusion transcript could be detected with previously reported RT-PCR primers. However, Southern-blot experiments demonstrated that the EWS gene was disrupted within a 2-kb PstI genomic fragment including exon 7. PCR amplification and sequence of the translocation junction fragments indicated that the breakpoint was localized within exon 7 of EWS. The resulting fusion gene encoded a chimeric protein in which a truncated NTD-EWS was linked, in frame, to the ETS DNA-binding domain of ERG. This observation indicates that, to avoid false negative results, RT-PCR-based diagnosis of tumors with EWS fusion transcripts should now include the search for such rare variants. It also suggests that the amino-terminal portion of the NTD-EWS, but not its carboxy terminal part, might be fundamental for the oncogenicity of the chimeric proteins. (C) 1996 Wiley-Liss, Inc.

Neuroblastoma is characterized by a wide variability of its clinical course, and considerable effort has been made to identify factors determining outcome in this disease. In a series of 82 patients from a single institution, we have investigated the prognostic impact of multiple clinical, biological and genetic parameters. Univariate testing showed that advanced stage of disease, abdominal localization of the primary tumor, elevated urinary dopamine levels, N‐myc amplification (NMA) and loss of heterozygosity of chromosome 1p (LOH 1p) were related to a poor outcome. Most of these parameters were strong indicators of treatment failure in children younger than 12 months of age but none of them, apart from stage, had a significant prognostic impact in patients older than 12 months at diagnosis. Interestingly, the shorter survival time associated with the presence of 1p deletions or NMA appears to be more strongly linked to a poorer outcome after relapse or progression than to a shorter progression‐free interval. Although different types of LOH 1p have been described in neuroblastoma and may be associated with different biological features, as suggested by a different pattern of catecholamine secretion, tumors with LOH 1p present an aggressive clinical behavior, regardless of the type of LOH 1p. In this study, LOH 1p is an indicator of poor prognosis and identifies a larger population at risk than NMA alone. © 1996 Wiley‐Liss, Inc.