Analysis of selected genes in neuroendocrine tumours: insulinomas and phaeochromocytomas.

Insulinomas and phaeochromocytomas are neuroendocrine tumours that may be either sporadic or manifestation of a familial cancer syndromes and are both derived from the neural crest. In the present study, gene components of different signalling pathways were investigated in sporadic human insulinomas and phaeochromocytomas to identify the responsible candidates. Ret and k-ras were tested for activating point mutations, and NF1, p53, BRCA1, nm23-H1, SDHB and SDHD for loss of heterozygosity (LOH). Twenty-two sporadic insulinomas and 15 phaeochromocytomas were analysed by the polymerase chain reaction using restriction fragment length polymorphism or dinucleotide repeat polymorphism methods. The results of our analysis demonstrate that the most frequent changes were point mutations of k-ras: 23% of insulinomas and 62% of phaeochromocytomas harboured k-ras mutations. The analysis also showed two phaeochromocytomas with point mutations of the ret oncogene. Only one insulinoma showed LOH of NF1, and another showed LOH of p53. Allelic loss of BRCA1 was detected in two insulinomas, and of nm23-H1 in another insulinoma. Allelic losses of the SDHB gene were present in two phaeochromocytoma and one insulinoma cases and allelic losses of SDHD were present in one phaeochromocytoma case. The changes observed in phaeochromocytomas were more homogenous and confined to k-ras and ret oncogenes, whereas insulinomas showed more heterogenic situation. Our findings may contribute to a better understanding of the genetic profile of neuroendocrine tumours.

The expression and role of insulin-like growth factor II in malignant hemangiopericytomas.

Hemangiopericytoma is a rare soft tissue tumor originating from contractile pericapillary pericytes. To address the issue of molecular genetic events that participate in genesis and progression of hemangiopericytoma we analyzed insulin-like growth factor (IGF) II and IGF I receptor in 29 tumors collected from a human tumor bank network. Seven of these tumors were associated with severe hypoglycemia; six were retroperitoneal and one was located in the leg. Of 22 tumors tested 12 (54.5%) exhibited IGF II mRNA, while almost 90% (17 of 19) of hemangiopericytomas exhibited IGF I receptor mRNA. Sera from some patients whose tumors expressed IGF II mRNA contained elevated levels of IGF II. Removal of the tumor eliminated most of the IGF II immunoreactivity from the sera. The potential role of IGF II as a growth-promoting factor was examined on three malignant primary hemangiopericytoma cell cultures. Extracellular addition of IGF II significantly enhanced cell proliferation in a dose-dependent manner. Antisense oligodeoxynucleotides that specifically inhibit IGF II mRNA, at a concentration of 40 or 80 micrograms/ml, inhibited the growth of hemangiopericytoma cells significantly, by 40%. Simultaneous administration of antisense deoxyoligonucleotides to both IGF II and IGF I receptor inhibited tumor cell proliferation by even 80%. Our data suggest that tumor cells produce IGF II, and that this in turn stimulates their proliferation by autocrine mechanisms.

Molecular pathology of hemangiopericytomas accompanied by severe hypoglycemia: oncogenes, tumor-suppressor genes and the insulin-like growth factor family.

Relatively little is known about molecular genetic events that participate in the genesis and progression of hemangiopericytoma. In this study, we describe two cases of hemangiopericytoma accompanied by severe hypoglycemia. Tumor cells from patient 1 exhibited insulin-growth factor I (IGF I) and insulin-like growth factor I receptor (IGF IR) mRNA transcripts. Tumor cells from patient 2 exhibited IGF II, IGF IR and IGF binding proteins 1-3 mRNA. Serum from patient 2 contained IGF II, mostly in a large molecular form ("big" IGF II); the IGF II level did not change after the tumor removal. The presence of IGF IR in tumor cells was confirmed by immunoprecipitation with antibodies that recognize human IGF IR subunit (visualized as a 460-kDa band). The hemangiopericytoma cells derived from patient 1 expressed 210000 IGF I receptors/cell. Specific binding of IGF I to the tumor cell membrane fraction was higher in tissue from patient 1, while the tissue of patient 2 showed relatively low IGF I binding. In contrast, IGF II binding was much higher in tissue from patient 2. Both tumor tissues showed positive immunostaining for c-Jun; one tumor showed strong immunostaining for c-Myc, H-Ras and p53, while the other exhibited strong reaction with H-Ras antibodies only. No loss of the heterozygosity at the genes APC, NFI and nm23-H1 loci in tumor tissue obtained from patient 1 was found. In effect, our results suggest multiple molecular genetic changes in hemangiopericytoma -- activation of some oncogenes and the IGF growth factor family. IGF ligands together with IGF IR could be responsible for hypoglycemia and perhaps the transformed phenotype.

Multiple genetic alterations in malignant metastatic insulinomas.

Proto-oncogenes, growth factors/receptors, and tumour suppressor genes were analysed in malignant metastatic insulinomas. Normal pancreas showed only a moderate immunoreaction for c-myc proto-oncogene and a strong reaction for insulin. Benign insulinomas were slightly or moderately positive for transforming growth factor alpha (TGF alpha), weakly positive for epidermal growth factor receptor (EGF-R), and strongly positive for c-myc and insulin. In malignant insulinomas, besides a strong immunoreaction for c-myc and TGF alpha, activation of c-K-ras and overexpression of p53 protein were found. Insulin reaction was moderate or strong. Three out of six malignant insulinomas displayed a c-K-ras point mutation at codon 12. All mutations were guanine to cytosine transversion, resulting in amino acid substitution, glycine to arginine. Mutations were present in metastatic insulinomas only. Patients with mutated c-K-ras oncogene had overexpression of p53 protein as well as c-myc and TGF alpha overexpression. Our results support the view that malignant progression is a consequence of more than one genetic lesion and suggest that activation of myc, TGF alpha an ras genes plays a role in a multistep process of tumour progression, perhaps serving as an initiating event.