Global DNA methylation profile in medullary thyroid cancer patients

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Abstract

Background

Changes in global DNA methylation have been suggested to cause genomic instability leading to increased risk of cancer. The accumulation of epigenetic changes is believed to contribute to tumorigenesis and dedifferentiation, but the effects of such changes in thyroid cancer are still yet defined.

Objective

To evaluate the global DNA methylation levels in thyroid cancer patients.

Methods

Total DNA was extracted from peripheral blood leukocytes of the medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC) patients and the methylation pattern was evaluated using the Imprint Methylated DNA Quantification kit (Sigma-Aldrich).

Results

A total of 42 patients were analyzed (24 MTC, 12 PTC, and 6 controls). For MTC, the mean age was 41 ± 20 years, 54% were women and 12 cases were sporadic. The median calcitonin level at diagnosis was 1692 (637–8865), 65% of the MTC patients had local metastases and 23% distant metastases. For PTC, the median age was 43 ± 15 years, 58% were women and 50% had local metastases. The percentage of overall methylation differed according to the tumor subtype. Patients with MTC had a higher level of DNA methylation when compared to individuals with PTC (35 (24–48) vs. 17 (6.5–20.5); P = 0.002, respectively). Interestingly, among patients with MTC, individuals with the sporadic form of the disease had a higher level of methylation when compared to the hereditary form (25 (16–37) vs. 43 (33–52); P = 0.025, respectively). No association was observed between global methylation levels and clinical and/or oncological characteristics of the disease.

Conclusion

Global methylation levels were higher in MTC as compared to PTC patients. These results suggest the overall DNA methylation profile may be influenced by the histological subtype of thyroid cancer.

Introduction

Thyroid cancer is the most frequent endocrine neoplasia, with an overall estimate of 56,870 new cases by 2017, and its incidence has been increasing in recent decades (SEER, 2017). Thyroid cancers can be classified according to histopathological characteristics as papillary thyroid carcinoma (PTC, >80%), follicular thyroid carcinoma (FTC, 10%), medullary thyroid carcinoma (MTC, 4%) and anaplastic thyroid carcinoma (ATC, <1%). These malignancies arise from different cell types present in the thyroid gland (Davies and Welch, 2014; Pellegriti et al., 2013). Endoderm-derived follicular cells give rise to papillary, follicular, and probably anaplastic carcinomas. While the neuroendocrine-derived, calcitonin-producing C cells are considered precursors (Johansson et al., 2015).

Management options for thyroid cancer include observation, surgery, and pharmacotherapy. Controversy exists regarding the optimal management approaches, since the histologic subtype impacts prognosis of the thyroid cancer. Differentiated tumors generally respond well to treatment, thereby carrying a good prognosis. However, poorly differentiated tumors are typically aggressive and highly metastatic and have a significantly worse prognosis (NCCN, 2017). The origin and characteristics of thyroid cancers are important factors influencing disease progression and response to therapy.

Although genetic mutations have been implicated in the neoplastic process, about 30–50% of the thyroid tumors remain without genetic origin identified. Thus, the search for as yet unidentified genetic changes that might provide with new therapeutic approaches to the thyroid cancer is critical, since there are no effective therapeutic options for the advanced disease (Kapiteijn et al., 2012; NCCN, 2017).

DNA methylation is the most studied and well-understood mechanism of epigenetic regulation. It has been demonstrated that DNA methylation plays an important role in the regulation of gene expression. Indeed, hypo and hypermethylation across the genome contribute to changes in gene expression. Variations in the global DNA methylation levels have been observed in different cancer types and may be a potential source of noninvasive cancer biomarkers, (Esteller, 2008; Feinberg and Tycko, 2004; Witte, Plass, and Gerhauser, 2014).

Recently, epigenetic alterations have been shown to play a role in the development and progression of thyroid cancer. Hypermethylation in the promoter region of thyroid cancer-associated genes (RASSF1, TSHR, PTEN, SLC5A, DAPK, P16, and CDH1) has been associated with an increased risk of thyroid cancer. Methylation profiling stands out as a promising strategy for clinical use in patients with advanced cancer. However, in MTC, which originates from a very small population of parafollicular C-cells of the thyroid gland, few studies have explored methylation profiling of the malignancy (Joo et al., 2017; Khatami et al., 2017).

Here, we aimed to evaluate global DNA methylation percentage (5-mC%) in individuals with thyroid cancer, particularly with MTC, and correlate our findings with other thyroid cancer subtype, clinical and oncological features.

Section snippets

Patients

Patients diagnosed with MTC or PTC attending the Endocrine Division at Hospital de Clínicas de Porto Alegre were enrolled in this study.

The MTC patients underwent a complete clinical examination, laboratory tests (levels of basal calcitonin (Until December 2003, Calcitonin IRMA-DSL7700, Diagnostic Systems Laboratories, Inc., Webster, TX, USA, reference range < 10 pg/ml and, after January 2004, Immulite 2000, Diagnostic Products Corporation, Los Angeles, CA, USA; reference value (VR)

MTC patients

A total of 24 MTC patients were analyzed. The mean age was 41 ± 20 years, 54% were women and 12 cases were sporadic. The median calcitonin level at MTC diagnosis was 1692 (637–8865), 65% of patients had local metastases and 23% distant metastases (Table 1). Of the 12 hereditary cases, 8 harbored the C634R and 4 the M918 T mutation. The percentage of DNA methylation was 35 (24–48). No association was observed between mutational profile and methylation status. Interestingly, the individuals with

Discussion

In this cross-sectional study with thyroid cancer patients, we found that global methylation levels were higher in MTC as compared to PTC patients. Furthermore, sporadic MTC patients presented higher methylation levels compared to those with the inherited form or controls. To the best of our knowledge, this is the first study to specifically examine the association between global DNA methylation and thyroid cancer types.

There is growing evidence that leukocyte DNA methylation status is

Acknowledgements

This work was supported by: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant MCTI/CNPq/CT-Saúde/MS/SCTIE/DECIT; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) scholarship; Fundação de Amparo Pesquisa do Estado do Rio Grande do Sul (FAPERGS), grant PRONEX FAPERGS/CNPq; and Fundo de Incentivo à Pesquisa do Hospital de Clínicas de Porto Alegre (FIPE), project number 15-0160.

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