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.

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.

Non-Hodgkin's lymphomas and myeloid disorders: deletions associated with t(2;5) and t(3;5) detected by FISH.

The nucleophosmin (NPM) gene is involved in two recurrent translocations in hematological malignancies: t(2;5) (p23;q35) in anaplastic large cell lymphoma (ALCL) and t(3;5)(q25.1;q34-35) in myelodysplasia and acute non-lymphocytic leukemia (ANLL). Using eight YACs encompassing the 5q34-q35 region, we could easily detect these two translocations. In both types of translocation, probable unexpected deletions were also discovered downstream of the breakpoint at 5q35.

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.

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.