Therapeutic options for patients with metastatic medullary thyroid carcinoma (MTC) are limited due to lack of effective treatments. Thus, there is a need to thoroughly characterize the pathways of molecular pathogenesis and to identify potential targets for therapy in MTC. Since epidermal growth factor receptor (EGFR) seems to play a crucial role for RET activation, a key feature of MTCs, and several promising EGFR/vascular endothelial growth factor receptor 2 (VEGFR2)-targeted drugs have been developed, the present study was designed to investigate whether these proteins are altered in MTCs. We used a well-characterized series of 153 MTCs to evaluate EGFR activation by sequencing and FISH analysis, and to perform EGFR and VEGFR2 immunohistochemistry. EGFR tyrosine kinase domain mutations were not a feature of MTCs; however, EGFR polysomy and a strong EGFR expression were detected in 15 and 13% of the tumors respectively. Interestingly, EGFR was significantly overexpressed in metastases compared with primary tumors (35 vs 9%, PZ0.002). We also studied whether specific RET mutations were associated with EGFR status, and found a decrease in EGFR polysomies (PZ0.006) and a tendency towards lower EGFR expression for the most aggressive RET mutations (918, 883). Concerning VEGFR2, metastasis showed a higher expression than primary tumors (PZ2.8!10 K8 ). In this first study investigating the relationship between EGFR, RET, and VEGFR2 in a large MTC series, we found an activation of EGFR and VEGFR2 in metastasis, using both independent and matched primary/metastasis samples. This suggests that some MTC patients may benefit from existing anti-EGFR/VEFGR2 therapies, although additional preclinical and clinical evidence is needed.

Objective: The purpose of this study was to determine the global patterns of aberrant DNA methylation in thyroid cancer. Research Design and Methods:We have used DNA methylation arrays to determine, for the first time, the genome-wide promoter methylation status of papillary, follicular, medullary, and anaplastic thyroid tumors. Results:We identified 262 and 352 hypermethylated and 13 and 21 hypomethylated genes in differentiated papillary and follicular tumors, respectively. Interestingly, the other tumor types analyzed displayed more hypomethylated genes (280 in anaplastic and 393 in medullary tumors) than aberrantly hypermethylated genes (86 in anaplastic and 131 in medullary tumors). Among the genes indentified, we show that 4 potential tumor suppressor genes (ADAMTS8, HOXB4, ZIC1, and KISS1R) and 4 potential oncogenes (INSL4, DPPA2, TCL1B, and NOTCH4) are frequently regulated by aberrant methylation in primary thyroid tumors. In addition, we show that aberrant promoter hypomethylation-associated overexpression of MAP17 might promote tumor growth in thyroid cancer. Conclusions:Thyroid cancer subtypes present differential promoter methylation signatures, and nondifferentiated subtypes are characterized by aberrant promoter hypomethylation rather than hypermethylation. Additional studies are needed to determine the potential clinical interest of the tumor subtype-specific DNA methylation signatures described herein and the role of aberrant promoter hypomethylation in nondifferentiated thyroid tumors. (J Clin Endocrinol Metab 98: 2811-2821, 2013)

Thyroid cancer is a heterogeneous disease with several subtypes characterized by cytological, histological and genetic alterations, but the involvement of epigenetics is not well understood. Here, we investigated the role of aberrant DNA methylation in the development of well-differentiated thyroid tumors. We performed genome-wide DNA methylation profiling in the largest well-differentiated thyroid tumor series reported to date, comprising 83 primary tumors as well as 8 samples of adjacent normal tissue. The epigenetic profiles were closely related to not only tumor histology but also the underlying driver mutation; we found that follicular tumors had higher levels of methylation, which seemed to accumulate in a progressive manner along the tumorigenic process from adenomas to carcinomas. Furthermore, tumors harboring a BRAF or RAS mutation had a larger number of hypo-or hypermethylation events, respectively. The aberrant methylation of several candidate genes potentially related to thyroid carcinogenesis was validated in an independent series of 52 samples. Furthermore, through the integration of methylation and transcriptional expression data, we identified genes whose expression is associated with the methylation

BackgroundCardiovascular (CV) disease due to atherosclerosis is a major cause of morbidity and mortality in adult patients with diabetes, either type 1 or type 2 diabetes. The aim of the study was to assess the association of the frequency and the burden of subclinical carotid atherosclerotic disease in patients with type 1 diabetes according to the presence and severity of diabetic retinopathy (DR).MethodsA cross-sectional study was conducted in 340 patients with type 1 diabetes (41.5% with DR), and in 304 non-diabetic individuals. All participants were free from previous CV disease and chronic kidney disease (CKD). B-mode carotid ultrasound imaging was performed in all the study subjects. Patients with type 1 diabetes underwent a full eye examination, and DR patients were divided into two groups: mild disease and advanced disease.ResultsIn the group of patients with type 1 diabetes, the percentage of patients with carotid plaques was higher in those with DR compared with those without DR (44.7% vs. 24.1%, p < 0.001). Patients with DR also presented a higher incidence of ≥ 2 carotid plaques (25.5% vs. 11.1%, p < 0.001). Apart from other traditional cardiovascular risk factors, the presence of advanced stages of DR was independently associated with the presence (p = 0.044) and the burden (≥ 2 carotid plaques; p = 0.009) of subclinical carotid atherosclerosis.ConclusionsIn patients with type 1 diabetes without previous CV disease or established CKD, the presence of advanced stages of DR is associated with a higher atherosclerotic burden in the carotid arteries. The presence of DR identifies patients at risk for carotid atherosclerotic disease.Electronic supplementary materialThe online version of this article (10.1186/s12933-018-0706-z) contains supplementary material, which is available to authorized users.

This is a confirmatory study about usefulness of SDHB and SDHA immunostaining in assessment of SDH mutations in paragangliomas and pheochromocytomas. Paraganglioma/pheochromocytoma syndrome (PGL/PCC syndrome) consists of different entities, associated with germline mutations in five different genes: SDHD, SDHAF2, SDHC, SDHA and SDHB. It has been suggested that negative immunostaining of SDHB can be taken as an indicator of the presence of a mutation in one of the five SDH genes. We have performed SDHB and SDHA immunohistochemical staining in a series of paragangliomas and pheochromocytomas from 64 patients. The patients had been previously checked for mutations in SDHD, SDHC and SDHB, but also for mutation in RET and VHL. All 14 patients with SDH mutations (9 with SDHB and 5 with SDHD mutations) exhibited negative or weak-diffuse SDHB staining pattern in tumour tissue, whereas cells of the 23 RET mutated and 8 VHL mutated tumours showed a positive SDHB immunostaining. Sixteen of the patients that did not exhibit a mutation in any gene showed positive SDHB immunostaining in tumour tissue, while only three of the patients without mutation exhibited negative staining. All patients exhibited positive pattern of SDHA immunostaining. The results confirm the value of SDHB immunohistochemical status in assessment of germline mutations in PGL/PCC syndrome.

Current genetic evidence in mice indicates that SIRT1 has potent tumor suppressor activity in a variety of cancer models, with no evidence yet for SIRT1 oncogenic activity in vivo. We report here that transgenic Sirt1 expression is oncogenic in murine thyroid and prostate carcinogenesis initiated by Pten-deficiency. Based on mRNA expression analyses of pre-tumoral murine thyroids, we find that SIRT1 increases c-MYC transcriptional programs. Moreover, we show higher c-MYC protein levels in murine thyroid cancers from Sirt1 transgenic mice. Similarly, SIRT1 is overexpressed in human thyroid cancers and it is positively correlated with c-MYC protein levels. Finally, we show in cultured thyroid cancer cells that SIRT1 stabilizes c-MYC protein. These results implicate SIRT1 as a new candidate target for the treatment of thyroid carcinomas.

MicroRNA deregulation could be a crucial event in thyroid carcinogenesis. However, current knowledge is based on studies that have used inherently biased methods. Thus, we aimed to define in an unbiased way a list of deregulated microRNAs in well-differentiated thyroid cancer in order to identify diagnostic and prognostic markers. We performed a microRNA deep-sequencing study using the largest well-differentiated thyroid tumor collection reported to date, comprising 127 molecularly characterized tumors with follicular or papillary patterns of growth and available clinical follow-up data, and 17 normal tissue samples. Furthermore, we integrated microRNA and gene expression data for the same tumors to propose targets for the novel molecules identified. Two main microRNA expression profiles were identified: one common for follicular-pattern tumors, and a second for papillary tumors. Follicular tumors showed a notable overexpression of several members of miR-515 family, and downregulation of the novel microRNA miR-1247. Among papillary tumors, top upregulated microRNAs were miR-146b and the miR-221~222 cluster, while miR-1179 was downregulated. BRAF-positive samples displayed extreme downregulation of miR-7 and -204. The identification of the predicted targets for the novel molecules gave insights into the proliferative potential of the transformed follicular cell. Finally, by integrating clinical follow-up information with microRNA expression, we propose a prediction model for disease relapse based on expression of two miRNAs (miR-192 and let-7a) and several other clinicopathological features. This comprehensive study complements the existing knowledge about deregulated microRNAs in the development of well-differentiated thyroid cancer and identifies novel markers associated with recurrence-free survival.

Cyclin D1 (Ccnd1) is a proto-oncogen amplified in many different cancers and nuclear accumulation of Ccnd1 is a characteristic of tumor cells. Ccnd1 activates the transcription of a large set of genes involved in cell cycle progress and proliferation. However, Ccnd1 also targets cytoplasmic proteins involved in the regulation of cell migration and invasion. In this work, we have analyzed by immunohistochemistry the localization of Ccnd1 in endometrial, breast, prostate and colon carcinomas with different types of invasion. The number of cells displaying membranous or cytoplasmic Ccnd1 was significantly higher in peripheral cells than in inner cells in both collective and pushing invasion patterns of endometrial carcinoma, and in collective invasion pattern of colon carcinoma. Also, the cytoplasmic localization of Ccnd1 was higher when tumors infiltrated as single cells, budding or small clusters of cells. To evaluate cytoplasmic function of cyclin D1, we have built a variant (Ccnd1-CAAX) that remains attached to the cell membrane therefore sequestering this cyclin in the cytoplasm. Tumor cells harboring Ccnd1-CAAX showed high levels of invasiveness and metastatic potential compared to those containing the wild type allele of Ccnd1. However, Ccnd1-CAAX expression did not alter proliferative rates of tumor cells. We hypothesize that the role of Ccnd1 in the cytoplasm is mainly associated with the invasive capability of tumor cells. Moreover, we propose that subcellular localization of Ccnd1 is an interesting guideline to measure cancer outcome.