Two populations of double minute chromosomes harbor distinct amplicons, the MYC locus at 8q24.2 and a 0.43-Mb region at 14q24.1, in the SW613-S human carcinoma cell line.

High-level amplifications observed in tumor cells are usually indicative of genes involved in oncogenesis. We report here a high resolution characterization of a new amplified region in the SW613-S carcinoma cell line. This cell line contains tumorigenic cells displaying high-level MYC amplification in the form of double minutes (DM(+) cells) and non tumorigenic cells exhibiting low-level MYC amplification in the form of homogeneously staining regions (DM(-) cells). Both cell types were studied at genomic and functional levels. The DM(+) cells display a second amplification, corresponding to the 14q24.1 region, in a distinct population of DMs. The 0.43-Mb amplified and overexpressed region contains the PLEK2, PIGH, ARG2, VTI1B, RDH11, and ZFYVE26 genes. Both amplicons were stably maintained upon in vitro and in vivo propagation. However, the 14q24.1 amplicon was not found in cells with high-level MYC amplification in the form of HSRs, either obtained after spontaneous integration of endogenous DM MYC copies or after transfection of DM(-) cells with a MYC gene expression vector. These HSR-bearing cells are highly tumorigenic. The 14q24.1 amplification may not play a role in malignancy per se but might contribute to maintaining the amplification in the form of DMs.

High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma.

AIMS: Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities. METHODS AND RESULTS: We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-alpha of p16/INK4 and exon 1-beta of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression. CONCLUSIONS: Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.

Genetic heterogeneity in supratentorial and infratentorial primitive neuroectodermal tumours of the central nervous system.

AIMS: Medulloblastoma (MB), a kind of infratentorial primitive neuroectodermal tumour (PNET), is the most frequent malignant brain tumour in childhood. In contrast, supratentorial PNET (sPNET) are very infrequent tumours, but they are histologically similar to MB, although they present a worse clinical outcome. We investigated the differences in genetic abnormalities between sPNET and MB. METHODS AND RESULTS: We analysed 20 central PNET (14 MB and six sPNET) by conventional comparative genomic hybridization (CGH) in order to determine whether a different genetic profile for each tumour exists. Isochromosome 17q was detected in four of the 14 MB cases, but not in any sPNET. Gains at 17q and 7 happened more frequently in MB, and those at 1q in sPNET. Losses at chromosome 10 were detected only in MB, while losses at 16p and 19p happened more frequently in sPNET. A new amplification site, on 4q12, was detected in two MB. CONCLUSIONS: Central PNET are a heterogeneous group of tumours from the genetic point of view. The present and previous data, together with further results from larger series, might contribute to the establishment of specific treatments for supratentorial and infratentorial PNET.

Stromal cells and human malignant neuroblasts derived from bone marrow metastasis may share common karyotypic abnormalities: the case of the IGR-N-91 cell line.

BACKGROUND: Stage IV neuroblastoma is characterized by tumor invasion and metastatic dissemination. Cell lines derived from such neuroblastomas have a high in vitro proliferation capacity. PROCEDURE: We established three neuroblastoma cell lines derived from involved bone marrow of three patients with stage IV neuroblastoma and performed a cytogenetic study. RESULTS: Various culture conditions allowed us to distinguish two cell subpopulations: malignant neuroblasts (Nb-type) and substrate-adherent stromal cells (Str-type). Karyotypic analyses revealed two specific chromosomal abnormalities in diploid malignant IGR-N-331 neuroblasts, der(1)t(1;7)(p22;q11) and der(5)t(5;17)(q35;q21), one unbalanced translocation der(1)t(1;17)(p35;q21)x2 in hyperdiploid malignant IGR-N-337 neuroblasts, and a normal karyotype in both corresponding stromal subpopulations. In contrast, in the IGR-N-91 model, both cell types shared two unbalanced translocations, t(1;4)(q12;p15) and t(2;10)(p14;q11), suggesting that stromal cells and malignant neuroblasts originate from a common stem cell. CONCLUSIONS: Based on our findings, we postulate that genetically modified stromal cells may influence the metastatic potential of malignant neuroblasts.

Mapping of the 7q31 subregion common to the small chromosome 7 derivatives from two sporadic papillary renal cell carcinomas: increased copy number and overexpression of the MET proto-oncogene.

Molecular cytogenetic analysis of several sporadic papillary renal cell carcinomas and of their xenografts in immunodeficient mice had previously allowed us to delimit a minimal overrepresented region of chromosome 7 shared by all of them to band 7q31. We have refined the location of the overlapping region to the junction of the subbands 7q31.2 and 7q31.3 by reverse painting with two differently labelled probes prepared from the small chromosome 7 derivatives microdissected from the cells of two distinct tumours. This small region was shown to contain the MET proto-oncogene, present at three to four copies per cell as determined by Southern blot analysis. The increased copy number of the MET gene was found to be associated with its overexpression at the mRNA level. However, no change in MET copy number or expression level was observed in the cells from two xenografted tumours serially transplanted into immunodeficient mice, as compared to those from the corresponding initial tumours. Our results indicate that expression of the MET proto-oncogene above a critical threshold is required for the maintenance of the tumorigenic phenotype of at least some papillary renal cell carcinomas, but does not further increase during tumour progression.

Gain of chromosome arm 17q and adverse outcome in patients with neuroblastoma.

BACKGROUND: Gain of genetic material from chromosome arm 17q (gain of segment 17q21-qter) is the most frequent cytogenetic abnormality of neuroblastoma cells. This gain has been associated with advanced disease, patients who are > or =1 year old, deletion of chromosome arm 1p, and amplification of the N-myc oncogene, all of which predict an adverse outcome. We investigated these associations and evaluated the prognostic importance of the status of chromosome 17. METHODS: We compiled molecular cytogenetic analyses of chromosome 17 in primary neuroblastomas in 313 patients at six European centers. Clinical and survival information were collected, along with data on 1p, N-myc, and ploidy. RESULTS: Unbalanced gain of segment 17q21-qter was found in 53.7 percent of the tumors, whereas the chromosome was normal in 46.3 percent. The gain of 17q was characteristic of advanced tumors and of tumors in children > or =1 year of age and was strongly associated with the deletion of 1p and amplification of N-myc. No tumor showed amplification of N-myc in the absence of either deletion of 1p or gain of 17q. Gain of 17q was a significant predictive factor for adverse outcome in univariate analysis. Among the patients with this abnormality, overall survival at five years was 30.6 percent (95 percent confidence interval, 21 to 40 percent), as compared with 86.0 percent (95 percent confidence interval, 78 to 91 percent) among those with normal 17q status. in multivariate analysis, gain of 17q was the most powerful prognostic factor, followed by the presence of stage 4 disease and deletion of 1p (hazard ratios, 3.4, 2.3, and 1.9, respectively). CONCLUSIONS: Gain of chromosome segment 17q21-qter is an important prognostic factor in children with neuroblastoma.

Comparative genomic hybridization analysis of sporadic neuroendocrine tumors of the digestive system.

Little information is available on the molecular mechanisms underlying neuroendocrine tumorigenesis. To obtain an overview of the genomic imbalances characterizing these tumors, we studied 20 benign or malignant sporadic endocrine gastroenteropancreatic tumors by comparative genomic hybridization. Chromosomal imbalances were found in all tumors. Gains of chromosomal material were more frequent than losses. The most frequent gains were of chromosomes and chromosome arms 5 (55%), 14 (55%), 17q (55%), and 7 (50%). Losses were most frequent from 11q (30%) and 16p (30%). Gains of chromosome 5 did not occur in nonmetastatic tumors, whereas losses of 9p were observed exclusively in intestinal tumors. In addition, we found two high-level amplifications, of 17q11-21 and 19q13. A complementary FISH analysis revealed that the gain in 17q11-21 included amplification of the protooncogene HER2/neu. As in multiple endocrine neoplasia type-1-associated tumors, deletions of chromosome band 11q13 appear to be involved in the development of sporadic digestive tract neuroendocrine tumors, but our results suggest that other chromosomal regions are also involved.

Mapping of the tyrosine kinase receptors trkA (NTRK1), trkB (NTRK2) and trkC(NTRK3) to human chromosomes 1q22, 9q22 and 15q25 by fluorescence in situ hybridization.

trk (NTRK) genes encode tyrosine kinase transmembrane receptors that are stimulated by neurotrophins, and are responsible for the transduction of signals controlling neuropoiesis and neuron survival in the central and peripheral nervous system, trkA gene has earlier been assigned to three different loci on chromosome 1. To resolve these conflicting results, and confirm the localization of trkB and trkC, probes specific to each of these related genes were constructed and used in fluorescent in situ hybridization on human metaphase cells. Our results indicate that trkA, trkB and trkC are located in chromosome bands 1q22, 9q22 and 15q25, respectively.

Recurrent chromosomal abnormalities in hepatocellular carcinoma detected by comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to evaluate and map genomic aberrations in 50 hepatocellular carcinomas (HCCs) from patients chronically infected with hepatitis B virus (HBV). CGH clearly detected nonrandom genomic imbalances. Losses were most prevalent on chromosome regions 4q (70%), 8p (65%), 16q (54%), 17p (51%), 13q and 6q (37% each), and lp (30%). The most frequent gains occurred on 8q (60%), 1q (58%), and 6p and 17q (33% each). In a few cases, sequence amplifications were detected that were mapped to bands 11q12, 12p11, 14q12, and 19q13.1. This study represents the first analysis of primary liver cancers by CGH, and it confirms the presence of previously known chromosomal aberrations in HCC and highlights new quantitative abnormalities and sequence amplifications. These findings should lead to the characterization of new loci involved in liver cancer pathogenesis.

Emergence and scattering of multiple neurofibromatosis (NF1)-related sequences during hominoid evolution suggest a process of pericentromeric interchromosomal transposition.

Type 1 neurofibromatosis (NF1) gene encodes for a member of the GTPase activating protein family and is considered to be a tumor suppressor gene. Its very high rate of de novo mutation in humans led us to study a specific feature of this gene: the presence of numerous NF1-related sequences. According to our results, the human genome contains at least 11 NF1-related sequences, nine of which are scattered near centromeric sequences of seven different chromosomes. These NF1-related sequences, whose extent is quite varied according to loci, are unprocessed copies of the NF1 gene, and bear numerous mutations. A phylogenetic analysis of the six largest sequences indicates that they are all derived from a common ancestor, which would have appeared 22-33 million years ago, and was subsequently duplicated several times during hominoid evolution. The most recent duplication and interchromosomal transposition occurred in the last million years suggesting that the process could still be ongoing. Intriguing similarities between the evolution of alpha-satellite DNA and NF1-related sequences suggest the involvement of a common genetic mechanism for the generation and pericentric spreading of these NF1 partial copies.

Additional copies of a 25 Mb chromosomal region originating from 17q23.1-17qter are present in 90% of high-grade neuroblastomas.

Neuroblastoma shows remarkable heterogeneity, ranging from spontaneous regression to progression toward highly malignant tumors. In search of genetic abnormalities that could explain this variability, we have characterized neuroblastoma tumors by using multiple fluorescent hybridizations. Our results indicate that chromosome 17 is rearranged very frequently in the form of unbalanced translocations with numerous chromosomal partners, all leading to the presence of supernumerary copies of a 25 Mb chromosomal region originating from 17q23.1-qter. Additional 17q material was detected in more than 90% of untreated high-grade neuroblastomas and, along with 1p36 deletion, should represent the most frequent genetic abnormality of neuroblastoma observed until now.

Assignment of the NTRK4 (trkE) gene to chromosome 6p21.

Reverse transcriptase-polymerase chain reactions using foetal brain RNA with reverse and forward primers of the first, second and third NTRK4 region allowed us to obtain three amplified NTRK4 fragments. The specificity of amplified fragments was checked by digestion with restriction endonucleases AvrII, HindIII and PspII for the first, second and third regions, respectively. Each restriction site was specific for each amplified fragment. The fragment of the NTRK4 first region was also sequenced and the sequence determined was identical to the human NTRK4 sequence. The three amplified fragments were cloned in pBS. For the Southern technique, plasmid pBS-NTRK4a (with an insert of 1052 bp) detected a human 9-kb HindIII sequence which was localised unambiguously on chromosome 6. For fluorescence in situ hybridisation, the three plasmids, pBS-NTRK4a, pBS-NTRK4b (insert 924 bp) and pBS-NTRK4c (insert 1114 bp) were pooled and used as a probe. This NTRK4 probe was localised on 6p21. Of 50 metaphases analysed, 49 contained twin spot signals on both sister chromatids.

Mapping of 22 YACs on human chromosomes by fish using yeast DNA Alu-PCR products for competition.

Chromosomal assignment and analysis of chimerism of 22 YACs was performed by FISH. Probes were obtained by PCR amplification of the human YAC inserts with Alu primers. Maximum amplification of various inter-Alu elements was obtained when the primer annealing temperature was below the optimal temperature needed for high specificity. In these conditions, yeast DNA contributed to the amplification of various Alu-PCR products and, since strong competition was required for the suppression of all Alu sequences, yeast Alu-PCR products fulfilled this purpose efficiently.

MDM2 amplification in a primary alveolar rhabdomyosarcoma displaying a t(2;13)(q35;q14).

This report describes a case of rhabdomyosarcoma associated with a 2;13 translocation and multiple double minute chromosomes. The origin of the amplified DNA was identified using comparative genomic hybridization, which pinpointed a unique spot at 12q13-->q14. Band 12q13 has been shown to contain several genes that are occasionally amplified in other sarcomas. Fluorescene in situ hybridization to tumor metaphases with probes specific for this region indicated that the double minutes contained the MDM2 gene but not the CDK4 gene. MDM2 amplification was further quantified by Southern hybridization, which showed a mean value of 25 copies per haploid genome. This is the first example of MDM2 amplification in a rhabdomyosarcoma.

A Tsp509I variant in exon 13 of the neurofibromatosis type 1 (NF1) gene allows the identification of both alleles at the mRNA level.

We describe here a new sequence variant occurring in the coding region of the neurofibromatosis (NF1) gene (exon 13). This exonic polymorphism can be directly investigated by simple restriction enzyme digestion of RT-PCR (reverse transcription-polymerase chain reaction) products, making it a powerful tool for examining allele-specific mRNA expression levels.

Neurofibromatosis 1 (NF1) mRNAs expressed in the central nervous system are differentially spliced in the 5' part of the gene.

The neurofibromatosis 1 gene seems to play essential roles at several different stages of life. During embryogenesis, it is involved in cardiac development while in the adult, neurofibromin (the corresponding protein) is mainly expressed in the nervous system, and therein, essentially in neurons, non-myelinating Schwann cells and oligodendrocytes. In addition, the NF1 gene is considered a tumor suppressor gene, since mutations have been associated with the occurrence of benign and malignant tumors in neuralcrest-derived tissues. Using reverse transcription-polymerase chain reaction (RT-PCR) analyses with primers located in exons 7 and 13, we have identified evidence of alternative splicing in this region of the NF1 gene. Cloning and sequencing of cDNA allowed the characterization of an isoform bearing an extra 30 bp sequence between exons 9 and 10a, leading to the insertion of 10 amino acids between residues 420 and 421 of neurofibromin. The insertion is conserved in the mouse. Examination of the pattern of expression of this isoform demonstrated a high level of expression in the central nervous system and an absence of expression in all the other normal tissues tested including peripheral nervous tissues derived from the neural crest. Analysis of brain tumors indicated a reduced expression of the alternative exon in medulloblastomas and oligodendrogliomas. The results presented here are consistent with tissue-specific expression of this alternative exon which we propose to call exon 9br.

A new hybrid cell line containing only human chromosome 15 selected through fluorescence in situ hybridization and characterized by Alu-PCR amplification of the human DNA.

Human Cot1 DNA directly labeled with dUTP-fluorochromes (FITC, Rhodamin) and used as a probe, allowed rapid detection of one or a few human chromosomes in human-hamster hybrid cells by in situ hybridization (FISH). A hybrid cell line containing only a human acrocentric chromosome was isolated (CH35B2D). The DNA from this hybrid was used for PCR amplification with a single Alu (A33) primer. After agarose gel electrophoresis, a continuum of intense bands of between 400-700 bp was observed with A33-PCR products. No amplified product was visible with hamster DNA. FISH on normal human metaphases of biotinylated Alu-PCR products obtained with Alu A33 and compétition with human Cot1 DNA showed decoration, with high specificity for chromosome 15. It was identified after R banding obtained with PI or DAPI in an antifade adjusted to pH11 with NaOH. Under the applied conditions, the Alu (A33) products are expected to be useful for characterization, by specific decoration of chromosome 15 aberrations in pathological cells. CH35B2D could be employed for functional studies of genes located on chromosome 15.

Tissue-specific alternative splicing of neurofibromatosis 1 (NF1) mRNA.

The neurofibromatosis 1 gene NF1 appears to play a crucial role in regulating the proliferation of cells of neural crest origin. The NF1 gene is a 300 kbp gene, encoding a complex pattern of mRNA related to the presence or absence of two alternative splices. The first splice, in the centre of the coding region of the gene, results in the addition of 63 bp in the GAP-related domain. The second splice located 4203 bp downstream, near the 3' terminus of the coding region of the gene, consists of a 54 bp insert. RT-PCR analysis demonstrates that the most prevalent splice variant in human tissues is the one which contains the GAP-related splice and omits the 3' terminal splice. It is also the form expressed in the peripheral nerve, adrenal medulla, benign NF1 neurofibromas and NF1 neurosarcomas. Conversely, a few organs (brain, muscle) exhibit extensive alternative splicing leading to the co-expression of four distinct transcripts. The reproducibility of the relative levels of each of the splice types in the different organs indicates a tissue-specific splicing pattern of the NF1 gene.

Localization of 15 cosmids on human chromosome 22 by fluorescence in situ hybridization.

Fluorescence in situ hybridization has been used in a cytogenetic analysis to map 15 cosmids on human chromosome 22. Thirteen cosmids were localized on the long arm of chromosome 22 (22q) while two other probes displayed a hybridization signal on 22p and the short arm of the acrocentric chromosomes of groups D and G. The regional assignment of these new chromosome markers will improve the mapping of chromosome 22; they can be used to detect numerical and structural aberrations of this chromosome involved in numerous pathologies.