Global gene expression in papillary thyroid carcinoma: search for diagnostic, prognostic markers and therapeutic targets

• Grant number: 10/09526-7
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: January 01, 2011
• End date: January 31, 2014
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Luiz Paulo Kowalski
• Grantee: Mateus de Camargo Barros Filho
• Host Institution: A C Camargo Cancer Center. Fundação Antonio Prudente (FAP). São Paulo , SP, Brazil

Abstract

Papillary thyroid carcinoma (PTC) is the most frequent malignant endocrine neoplasia, presenting an evident increase in incidence in the last years. Although the behavior of CPT is usually indolent, a subset of patients presents aggressive disease, for which there is no effective therapeutic options. Despite the mutation in BRAF is often described as a poor prognostic factor, to date, no molecular marker is used in clinical routine. Aims. To identify diagnostic, prognostic markers and potential candidates for therapeutic intervention in PTC by global gene expression analysis. Methods. Two hundred and fifty PTC cases treated by total thyroidectomy and radioiodine therapy were included. The BRAFV600E mutation was detected and quantified by pyrosequencing (225 PTC). Microarray experiments for gene expression analysis were conducted on 61 PTC and 13 non-neoplastic thyroids adjacent to the tumor samples (NT). The expression profile of PTC was compared with NT and according to BRAF mutation status, lymph node involvement and clinical outcome. The results were compared with those deposited in public databases. In silico molecular analysis and confirmation of the findings for a subset of genes were also performed. Thirty-nine targets transcripts and five references were selected for confirmation by RT-qPCR in 206 PTC, NT 33 and 27 benign thyroid lesions (BTL). Results. Five hundred and eighty-nine differentially expressed transcripts were identified in PTC compared with NT, in which GABRB2 was ranked as the most frequently deregulated by meta-analysis. The results were interpreted collectively by in silico analysis, showing evidence of a possible loss of sensitivity to thyroxine and altered metabolism of retinoic acid (RA) as a result of EZH2 and histone deacetylases (HDAC) activation. Twenty-eight transcripts were confirmed as differentially expressed by RT-qPCR. The combination of CLDN10, HMGA2 and LAMB3 expression was able to discriminate all PTC from normal/benign samples in a training set (86 PTC, 23 NT and 8 BTL). The diagnostic algorithm was validated in an independent set (120 PTC, 10 NT and 19 BTL) achieving an area under the curve (AUC) of 0.99 with 97.5% sensitivity and 90.3% negative predictive value (NPV). Higher algorithm scores were associated with lymph node involvement. The expression profile was also compared according to BRAF mutation (present in 62.5% of cases) and associated with the presence of extrathyroidal extension. An unsupervised hierarchical clustering analysis revealed that the frequency of BRAFV600E alleles is an important factor in transcriptional variability of PTC. One hundred and eighty-eight differentially expressed genes were identified in tumors harboring the mutation (confirmed by comparison with external databases), highlighting a possible modulation of the neuregulin pathway. Eleven out of 12 transcripts, including ERBB3, were confirmed by RT-qPCR. To search for prognostic markers, groups were compared according to clinical outcome (relapse as outcome) and lymph node status, finding 28 and 32 genes, respectively. Among the 12 genes selected for confirmation, reduced GADD45B expression was associated with better clinical outcome and cervical lymph nodes free of lesions at diagnosis. Conclusions. Together, these results help to understand the biology of PTC, revealing EZH2 and HDAC as potential therapeutic targets. Moreover, the results provide a promising diagnostic tool with prognostic implications. It was evidenced a possible involvement of neuregulin pathway in PTC harboring BRAF mutation and GADD45B as a useful marker to better stratify PTC patients according to the risk of relapse. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

BARROS-FILHO, MATEUS CAMARGO; MARCHI, FABIO ALBUQUERQUE; PINTO, CLOVIS ANTONIO; ROGATTO, SILVIA REGINA; KOWALSKI, LUIZ PAULO. High Diagnostic Accuracy Based on CLDN10, HMGA2, and LAMB3 Transcripts in Papillary Thyroid Carcinoma. JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, v. 100, n. 6, p. E890-E899, JUN 2015. (10/18370-0, 10/09526-7, 08/57887-9)