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 to the development of MPNST. A selected set (n=11) of cell cycle components and proliferation markers (n=2) as well as the neurofibromin protein were analyzed by immunohistochemistry on a TMA with tissue cores from 106 MPNSTs and 3 neurofibromas. The thirteen 5um parallel sections, hybridized with the individual antibodies, were each scanned with high resolution before visual scoring was done from the digital images. The expression data revealed that topoisomerase IIa (TOP2A) and Ki-67 are indeed expressed in most MPNSTs. Fewer tumors than expected had positive score of tumor protein 53 (TP53). Generally, expression of cyclins and CDKs were common among MPNSTs, leading to phosphorylated retinoblastoma (RB1) and G1/S transition. The cyclin-CDK-complex inhibitors were on the other side absent from most MPNSTs, confirming the suspicion of lost control of the cell cycle checkpoint at G1-phase.
MPNST is a highly aggressive cancer disease for which no standard therapy, except surgery, exists.A molecular classification of these tumors in combination with the clinical and pathological evaluations may aid in improved treatment of these patients. The current study has added new knowledge to the understanding of the molecular biology of MPNST.