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Supplementary Tables S1-S2 from Global and Regional CpG Methylation in Pheochromocytomas and Abdominal Paragangliomas: Association to Malignant Behavior

Purpose: This study aims to quantitatively assess promoter and global methylation changes in pheochromocytomas and abdominal paragangliomas and its relation to tumor phenotypes.Experimental Design: A panel of 53 primary tumors (42 benign, 11 malignant) was analyzed by quantitative bisulfite pyrosequencing. Based on methylation levels in the tumor suppressor genes, p16INK4A, CDH1, DCR2, RARB, RASSF1A, NORE1A, TP73, APC, DAPK1, p14ARF, and PTEN, a CpG island methylator phenotype (CIMP) was defined as concerted hypermethylation in three or more genes. Mean Z scores for the hypermethylated promoters were calculated to characterize overall promoter methylation. Global DNA methylation was quantified for LINE-1 promoter sequences and by using luminescent methylation analysis.Results: Five primary tumors (9.4%) exhibited a CIMP phenotype, four of which were malignant paragangliomas. CIMP was significantly associated with malignant behavior (P = 0.005) and younger age at presentation (P < 0.007) but did not result from BRAF V600E mutation. Global hypomethylation of LINE-1 elements was observed in tumors compared with normal adrenal samples (P < 0.02).Conclusion: We here describe the identification of CIMP in abdominal paragangliomas and a strong association of this phenotype with malignant behavior, as well as young age at presentation. The findings raise a prospective for potential benefits of epigenetically acting drugs for a subgroup of young abdominal paraganglioma patients with adverse prognosis.

Background: Medullary thyroid carcinoma (MTC) originates from parafollicular C cells of the thyroid gland, and are associated with activating constitutional or somatic mutations in the RET proto-oncogene. The common somatic mutation of codon 918 is associated with a less favorable clinical outcome. The catalytic component of telomerase, human telomerase reverse transcriptase (TERT) has been found to be reactivated in most human tumors. Objective: In this study, we aimed to investigate telomerase involvement in relation the RET mutational status in MTC. Design: Our study includes 43 cases of MTC from the Karolinska University Hospital. RET mutations were identified by sequencing of the hot spots in exons 10,_11-,_15-, and 16. Expression of the TERT gene, telomerase activity and telomere length were quantified. Result: Among the 43 cases, 18 carried a RET point mutation in exon15 or exon16 (Group I), 5/43 had a point mutation in exon 10 or 11 (Group II), and 20 were wild-type (Group III). Telomerase activity and TERT expression were closely associated. The TERT gene was expressed in 13/18 cases in Group I, in 0/5 in Group II and in 10/20 of Group III. The median telomerase activity was higher in Group I as compared to Groups II and III. Group I tumors showed the shortest relative telomere length. With regard to follow-up, cases with high telomerase activity and TERT gene expression in Group I had less favorable outcome. Conclusion: The findings suggest that TERT/telomerase activity may contribute to aggressive MTC in addition to, or as a consequence of, RET codon 918 mutations. The very low telomerase activity and TERT expression revealed in several of the MTC cases suggest the involvement of alternative mechanisms for telomere stabilization in the MTC entity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1141. doi:1538-7445.AM2012-1141

Background: Tumors arising from the adrenal cortex (ACT) can be hormonally hyperfunctioning or silent. This functional phenotype, as well as their size, is of clinical importance. MicroRNAs are a class of small non-coding RNAs that negatively regulates gene expression. There is increasing evidence for their significant role in tumorigenesis. Their relevance on the functional phenotype and size of these tumors has not been previously documented. Aim: To characterize differential expression patterns of microRNAs regarding the increasing size and the functional phenotypes of ACT. Methods: Seventy frozen tissue samples were included: 16 aldosterone-producing adenomas (APA), 14 cortisol producing adenomas (CPA), 14 non-hyperfunctioning adenomas (NhFA), 19 adrenocortical carcinomas (ACC) and 7 normal adrenal cortices as controls. Global microRNA expression profiles were determined using the Agilent Human microRNA microarray, and the results were verified by quantitative real-time PCR. Results: We observed distinct microRNA expression patterns among different ACT phenotypes: majority of the APA was clustered separately from NhFA and CPA, and the ACC was independently clustered apart from all benign tumors. A larger number of miRNAs were differentially expressed between APA and CPA, while a smaller subset of differently expressed microRNAs was found between functional and non-hyperfunctional adenomas. Among the benign tumors, we found miR-21 expression was significantly correlated with increasing tumor size. Conclusion: Deregulation of microRNA expression is associated with the functional phenotype and size in ACT, and these microRNAs may have implication related to the development of adrenocortical tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4940. doi:10.1158/1538-7445.AM2011-4940