Succinate Dehydrogenase B Mutations And EPAS1 Overexpression Predict Invasion And Metastasis Of Pheochromocytomas Or Paragangliomas

Objective:Pheochromocytoma (PHEO) and paraganglioma (PGL) are tumors of adrenal glands medulla and ganglions of abdomen, thorax, head and neck. Genetic mutations account for approximately 30%of functional (secrete catecholamines) and nonfunctional cases. In addition to RET, VHL and NF-1, genes encoding succinate dehydrogenase complex subunit B (SDHB), SDHC, SDHD and SDHAF2 are recognized as susceptibility genes for PHEO and PGL. Recently, approximately 15% PHEOs and PGLs caused by genetic mutations of SDHB, SDHC and SDHD were established as hereditary pheochromocytoma paraganglioma syndrome (HPPS). The aim of this study was to analyze relationship between invasion& metastasis and SDHB mutations& EPAS1 overexpression in PHEOs and PGLs, and to evaluate clinical parameters as predictors of invasion and metastasis, such as germline mutation, IGF-1, MIB-1 and EPAS1.Material and methods:1. Forty-three PHEOs/PGLs patients were analyzed for germline mutations:SDH genes, VHL and RET. Results were correlated to clinical characteristics including gender, age, tumor localization and multifocality. The surgical approach was evaluated in terms of tumor origin, sequelae and subsequent evolution. DNA analysis was carried out for SDHB (1q36.1- 1q35, exonsl-8), SDHC (1q21, exons1-5), SDHD (11q23, exonsl-4), SDHAF2 (11ql2.2,1exons1-4), RET (1Oq11.2, exonslO, 11,13,14&15, and 16) and VHL (3p25.3, exons1-3) genes. Techniques used for the analysis were direct sequence analysis. Promoter region methylation of the succinate dehydrogenase was detected in PH EO/PGL peripheral blood samples.2. We performed immunohistochemistry for SDHB, EPAS1, VEGF-1R, CgA and MIB-1 on 64 tumors with PHEO or PGL, known SDH mutation status partly. We defined positive as granular cytoplasmic staining (a mitochondrial pattern), high expression(+++) as above 100 positive staining cells per high magnification (>50%), weak diffuse as a cytoplasmic blush lacking definite granularity, and negative as completely absent staining in the presence of an internal positive control.3. In light of earlier studies showing that the activation of the insulin-like growth factor I receptor (IGF-IR) exerts a strong neuroprotective effect, we investigated the ability of IGF-I to induce PC-12 cells differention and proliferation. PC 12 cells exposed to recombinant human insulin-liked growth factor (IGF)-1, observe proliferation and differentiation effection of IGF-1 stimulate PC 12 rat pheochromocytoma cells and modulation of catecholamine-releasing.Results:1. Germinal mutations were found in 17 patients (17.5%,17/97). Most frequent were mutations in RET proto-oncogene (8.24%,8/97), seven mutation in SDHB genes (7.2%,7/97), followed by VHL gene (2.06%,2/97). A comparison of some of the clinical features of both groups (with and without mutation) showed statistically significant differences.2. EPAS1 showed weak diffuse or negative staining in normal adrenal medulla. Moderate positive expression in benign pheochromocytoma/paraganglioma and high positive expression in malignant paraganglioma.15 of 17 tumors with known mutations of VHL, RET, and SDHB showed positive staining. One VHL associated tumor showed weak diffuse staining. Of 46 tumors without germline mutations,13 showed positive staining. One paraganglioma with no known SDH mutation but clinical features suggesting familial disease was negative, and one showed weak diffuse staining. Overexpression of EPAS1 was related to CgA, MIB-1, VEGF-1R and CD34.3. Treatment of PC 12 cells with recombinant human IGF-1 synergistically increased the efficiency of proliferation outgrowth, in a dose-and time-dependent manner by activating different signaling pathways compared with treatment of norepinephrine (NE) group.Conclusions:The results of our study show that genetic predisposition is frequent in chromaffin tissue tumors, which indicates that DNA analysis is necessary in every case, also because of possible atypical clinical presentation. We conclude that approximately 7.2%of all pheochromocytomas or paragangliomas are associated with germline SDH mutation and that immunohistochemistry can be used to triage genetic testing. Evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). Pathogcnesis and Mechanism of Invasion and metastasis of PHEO/PGL were related to activation of CD34,VEGF-1R,IGF and TGF. IGF may be associated with the modulation network of proliferation and differentiation of the pHEO/PGL tumor cells. Complex regulation involving multiple factors and multiple synthetic and/or degradative steps are implicated in this process needs further studies.