Genome Methylation In Papillary Thyroid Cancer

Caroline BELTRAMI, AC Camargo Cancer Center, São Paulo, SP, Brazil, Brazil
REIS M. 2 , BARROS FILHO M. 1 , MARCHI F. 1 , KUASNE H. 1,2 , AMBATIPUDI S. 3 , HERCEG Z. 3 , KOWALSKI L. 4 , ROGATTO S. 1,2,5

1 CIPE - AC Camargo Cancer Center, São Paulo, SP, Brazil
2 Dept of Urology, Faculty of Medicine, UNESP, Botucatu, SP, Brazil
3 Epigenetics Group, International Agency for Research on Cancer (IARC), Lyon, France
4 Dept of Head and Neck Surgery, AC Camargo Cancer Center, São Paulo, SP, Brazil
5 Dept of Clinical Genetics, Vejle Hospital, University of Odense, Denmark

Purpose: Papillary thyroid carcinoma (PTC), a usually indolent disorder, is the most frequent thyroid cancer. However, a number of patients present disease progression, for which there are no effective therapeutic options. In this study, the methylation profile was integrated with the large-scale expression gene data of PTC and surrounding normal tissue (NT) aiming to identify putative molecular drivers.
Methods: Forty-one PTC obtained from patients treated with total thyroidectomy and radioiodine therapy were included in this study. The BRAF V600E mutation was evaluated by pyrosequencing method. The microarray platform Methylation 450 Human Infinium®BeadChip (Illumina) was performed in all 41 paired samples. The data were analyzed with wateRmelon package. Thirty-four PTC evaluated by gene expression arrays (Sure Print G3 8x60K; Agilent Technologies) were integrated with the methylome data. Six genes were selected to be confirmed using pyrosequencing and qRT-PCR in 94 PTC and 50 NT samples. The findings were submitted to in silico pathway analysis (IPA and KOBAS).
Results: The BRAF genotyping analysis resulted in 28 cases BRAF V600E. A total of 6,070 probes differentially methylated were identified in PTC samples: 5,425 CpG sites hypomethylated and 645 hypermethylated (p<0.05 and |Δβ|=0.15). An unsupervised hierarchical clustering analysis revealed one cluster with cases presenting poor prognosis (recurrence, male sex, tumors >1cm and extrathyroidal extension). Integrative analysis between expression and methylation data revealed 214 genes with significant negative correlation (p<0.05). We suggested that ERBB3, FGF1, FGFR2, GABRB2, HMGA2 and RDH5 genes are associated with PTC development being regulated by methylation. In silico analysis indicated the involvement of FGF signaling pathway associated to PTC.
Conclusion: These findings suggest that methylation is one mechanism involved in the regulation of the altered gene expression in PTC. Putative molecular drivers were identified that can be useful in the clinical practice. 
Financial Support: FAPESP 2008/57887–9 and CNPq 573589/08–9.