Malignant peripheral nerve sheath tumors : amplicon search by FISH and protein expression profiling using tissue micro arrays

• Main Author: Brekke, Helge Roar
• Format: Dissertation
• Language: Norwegian

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant inherited disorders, and carriers are at greatly increased risk of developing malignant peripheral nerve sheath tumours (MPNST). The NF1 patients are carriers of a germline mutation in the NF1 gene, which encodes the protein neurofibromin. This protein is in normal cells responsible for the deactivation of RAS through GTP-hydrolysis and inactivation of the gene will lead to hyperstimulation of the MAP-kinase pathway. However, inactivations of both NF1 alleles are also found in the benign precursor lesions, neurofibromas, showing that additional genetic events are necessary for malignant transformation. MPNSTs usually have complex karyotypes showing numerous chromosomal aberrations. We have previously shown recurrent copy number gains and losses, including frequent loss of 9p and 13q, and gain of 17q. The target gene for the 9p losses is the CDKN2A and for the 17q gain there is evidence for TOP2A as one target gene. The target gene(s) for the 13q losses remains unknown, although the RB1 is a possible candidate. In the present study we have analyzed topoisomerase II á, TOP2A and other chromosome 17 probes in a series of MPNSTs and neurofibromas (n=32) by metaphase- and inter-phase fluorescence in situ hybridization, FISH. Excess of TOP2A signals, relative to the centromere signals of the same chromosome, was found in 12 tumors. This support that gene amplification of TOP2A partly explain the previously observed increased expression of TOP2A in this type of tumors. The fact that TOP2A is the molecular target of several well-established chemotherapeutic agents, and that no consensus for therapy, except surgery, exists, underline the importance of these findings. In search for an additional and more distal amplicon at 17q we first analyzed distal loci by interphase FISH and, indeed we found amplification in four MPNST with normal copy number of the 17 centromere and TOP2A. By a global genomic approach, so called COBRA-FISH, we thereafter identified four MPNST with chromosomal breakpoints involving 17q. This pinpointed the region of interest for further FISH studies using BAC probes in order to identify the gene(s) targets. The second part of this study focused on in situ expression taking advantage of tissue microarray (TMA) as a tool for analyses of a large clinical series. Previous studies have suggested that some central cell cycle components, in particular deficient p16, contribute