Caroline Moraes Beltrami

Integrated data analysis reveals potential drivers and pathways disrupted by DNA methylation in papillary thyroid carcinomas

BackgroundPapillary thyroid carcinoma (PTC) is a common endocrine neoplasm with a recent increase in incidence in many countries. Although PTC has been explored by gene expression and DNA methylation studies, the regulatory mechanisms of the methylation on the gene expression was poorly clarified. In this study, DNA methylation profile (Illumina HumanMethylation 450K) of 41 PTC paired with non-neoplastic adjacent tissues (NT) was carried out to identify and contribute to the elucidation of the role of novel genic and intergenic regions beyond those described in the promoter and CpG islands (CGI). An integrative and cross-validation analysis were performed aiming to identify molecular drivers and pathways that are PTC-related.ResultsThe comparisons between PTC and NT revealed 4995 methylated probes (88% hypomethylated in PTC) and 1446 differentially expressed transcripts cross-validated by the The Cancer Genome Atlas data. The majority of these probes was found in non-promoters regions, distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes (mainly in the promoter and body regions, respectively) with negative and positive correlation, respectively. Genes showing negative correlation underlined FGF and retinoic acid signaling as critical canonical pathways disrupted by DNA methylation in PTC. BRAF mutation was detected in 68% (28 of 41) of the tumors, which presented a higher level of demethylation (95% hypomethylated probes) compared with BRAF wild-type tumors. A similar integrative analysis uncovered 40 of 254 differentially expressed genes, which are potentially regulated by DNA methylation in BRAFV600E-positive tumors. The methylation and expression pattern of six selected genes (ERBB3, FGF1, FGFR2, GABRB2, HMGA2, and RDH5) were confirmed as altered by pyrosequencing and RT-qPCR.ConclusionsDNA methylation loss in non-promoter, poor CGI and enhancer-enriched regions was a significant event in PTC, especially in tumors harboring BRAFV600E. In addition to the promoter region, gene body and 3’UTR methylation have also the potential to influence the gene expression levels (both, repressing and inducing). The integrative analysis revealed genes potentially regulated by DNA methylation pointing out potential drivers and biomarkers related to PTC development.

Prognostic Classifier Based on Genome-Wide DNA Methylation Profiling in Well-Differentiated Thyroid Tumors

Context: Even though the majority of well‐differentiated thyroid carcinoma (WDTC) is indolent, a number of cases display an aggressive behavior. Cumulative evidence suggests that the deregulation of DNA methylation has the potential to point out molecular markers associated with worse prognosis. Objective: To identify a prognostic epigenetic signature in thyroid cancer. Design: Genome‐wide DNA methylation assays (450k platform, Illumina) were performed in a cohort of 50 nonneoplastic thyroid tissues (NTs), 17 benign thyroid lesions (BTLs), and 74 thyroid carcinomas (60 papillary, 8 follicular, 2 Hürthle cell, 1 poorly differentiated, and 3 anaplastic). A prognostic classifier for WDTC was developed via diagonal linear discriminant analysis. The results were compared with The Cancer Genome Atlas (TCGA) database. Results: A specific epigenetic profile was detected according to each histological subtype. BTLs and follicular carcinomas showed a greater number of methylated CpG in comparison with NTs, whereas hypomethylation was predominant in papillary and undifferentiated carcinomas. A prognostic classifier based on 21 DNA methylation probes was able to predict poor outcome in patients with WDTC (sensitivity 63%, specificity 92% for internal data; sensitivity 64%, specificity 88% for TCGA data). High‐risk score based on the classifier was considered an independent factor of poor outcome (Cox regression, P < 0.001). Conclusions: The methylation profile of thyroid lesions exhibited a specific signature according to the histological subtype. A meaningful algorithm composed of 21 probes was capable of predicting the recurrence in WDTC.

Abstract A13: Gene body methylation could regulate the expression of genes associated to papillary thyroid carcinomas

Background: Papillary thyroid carcinoma (PTC) is the histological subtype more frequently reported among the endocrine malignancies. Deregulation of DNA methylation has been extensively reported in cancer with hypermethylation usually found in promoter and hypomethylation in nonpromoter regions. The methylation profile of PTC revealed altered genome regions; however, its impact on the gene expression remains poorly understood. DNA methylation profile in PTC patients was performed, aiming to investigate the role of novel genic and intergenic regions on the expression of genes. Patients and Methods: The methylation profile of 41 patients diagnosed with PTC was evaluated using the Methylation 450 Human Infinium®BeadChip platform (Illumina). The data were normalized and analyzed using SVA, watermelon, and LIMMA packages. Delta beta of 0.15 and adjusted p-value Results: Methylation analysis revealed 4,995 differentially methylated probes. A global hypomethylation was found in PTC samples (88% hypomethylated probes) mostly in nonpromoter regions (especially in gene body), distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes with negative and positive correlation, respectively. Genes showing negative correlation highlighted FGF and retinoic acid signaling as crucial pathways disrupted by DNA methylation in PTC. The selected genes were confirmed as altered in both methylation and expression levels. Interestingly, four of them (ERBB3, FGF1, GABRB2, and HMGA2) were mapped in gene body, suggesting that methylation in this region regulates the gene expression. Conclusion: Loss of methylation was extensively observed in PTC, especially in nonpromoter, poor CGI, and enhancer-enriched regions. Gene body and promoter regions have the potential to influence the gene expression levels (both repressing and inducing). Genes potentially regulated by DNA methylation were identified from the integrative and cross-validation analyses. ERBB3, FGF1, FGFR2, GABRB2, HMGA2, and RDH5 biomarkers are regulated by methylation in PTC samples. Citation Format: Caroline Moraes Beltrami, Mateus Camargo Barros-Filho, Mariana Bisarro dos Reis, Fabio Albuquerque Marchi, Hellen Kuasne, Skirant Ambatipudi, Zdenko Herceg, Luiz Paulo Kowalski, Silvia Regina Rogatto. Gene body methylation could regulate the expression of genes associated to papillary thyroid carcinomas [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; Sao Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A13.

Abstract 4416: MiRNAs genes are regulated by methylation in papillary thyroid carcinomas

Background: Papillary thyroid cancer (PTC) is the most common thyroid malignancy. DNA methylation associated with microRNAs (miRNA) regulation is an effective epigenetic mechanism for controlling gene expression. This mechanism is poorly explored in PTC. Patients and Methods: To investigate miRNAs genes regulated by methylation, global DNA methylation (Infinium® Human Methylation450 BeadChip, Illumina) analysis was performed in 50 matched PTC and normal tissues (NT) and compared with those deposited in The Cancer Genome Atlas (TCGA) (515 PTC and 56 NT). An integrative analysis was performed in 510 cases evaluated by whole methylation and miRNA expression analysis (TCGA). Using these findings, three miRNAs were investigated by RT-qPCR and pyrosequencing in PTC (N=103), normal thyroid tissues (NT, N=40) and benign thyroid lesions (BTL, N=32). In addition, seven target genes of these miRNAs were evaluated by RT-qPCR (44PTC, 30BTL and 28NT). Functional assays using three PTC cell lines (TPC1, K1 and BCPAP) were used to investigate the role of methylation in the miRNAs expression. Results: We found 50 differentially methylated probes of which 42 (27 microRNA genes) were confirmed in the TCGA database. The miRNA expression data from TCGA presented 67 differentially expressed miRNAs in PTC. The integrative analysis revealed three miRNAs (hsa-miR-146b-5p, hsa-miR-146b-3p and hsa-miR-21-5p) as candidates to be regulated by methylation. A significant hypomethylation pattern in PTC compared to NT and BTL (thyroid benign lesions) was found for these three miRNAs. Increased expression levels of hsa-miR-21-5p and hsa-miR-146b-5p were detected in PTC compared to NT and BTL. Combining these two miRNAs expression values, 45/47 PTC were distinguished from 66/67 non-malignant tissues (96% sensitivity and 99% specificity). Similarly, the association of methylation and expression data distinguished 41/45 PTC from 55/60 non-malignant tissues (91% sensitivity and 92% specificity) for MIR21 gene and 45/47 PTC from 61/61 NT/BTL (96% sensitivity and 100% specificity) for MIR146B gene. Seven target genes (MPPED2, STXBP5L, MRO, FHL1, FLRT1, DOK6 and MOB3B) of these miRNAs were significantly down regulated in PTC compared to NT and BTL. The BRAFV600E mutation was significantly associated to hypomethylation and overexpression of hsa-miR-21-5p and hsa-miR-146b-5p. Methylation and expression data in these three cell lines revealed that MIR146B is putatively regulated by methylation. Conclusion: We provide evidences that MIR21 and MIR146B genes are regulated by methylation having control in the expression of target genes associated with PTC. These miRNAs have a strong potential to be used as diagnostic markers in the clinical practice. Citation Format: Isabella Maria D. Ortiz, Mateus de Camargo Barros-Filho, Mariana B. dos Reis, Caroline Moraes Beltrami, Fabio Albuquerque Marchi, Hellen Kuasne, Clovis Pinto, Luiz Kowalski, Silvia Rogatto. MiRNAs genes are regulated by methylation in papillary thyroid carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4416.

Abstract 3829: DNA methylation profile in papillary thyroid cancer according to BRAF (V600E) mutation

Background: Thyroid cancer is an important health burden and the endocrine neoplasia with highest incidence in worldwide. Papillary thyroid cancer (PTC), the most common thyroid malignancy, is frequently associated with BRAF V600E mutation. This mutation occurs in around 45% of PTC cases and it has been associated with aberrant gene methylation. This finding suggests an association between genetic and epigenetic alterations in tumor development. The goal of this study was to evaluate the methylation profile in PTC samples according to BRAF mutation. Patients and Method: Forty-two PTC were included in this study (28 positive for BRAF V600E mutation). Normal and tumoral samples were obtained from patients submitted to total thyroidectomy and radioiodine therapy. The pyrosequencing method was used to detect and quantify the BRAF V600E mutation in tumor samples. The methylation analysis was performed by microarray platform Methylation 450 Human Infinium®BeadChip (Illumina), according to the manufacturer9s recommendations. The data were normalized and analyzed using SVA, wateRmelon and LIMMA package. It was considered only probes with Delta-Beta (β) = 0.1 and p value ≤ 0.001. Integrative analyses were performed using expression data generated from previous analysis. The findings were compared with 56 samples from The Cancer Genome Atlas (TCGA). Genes enrichment to biological pathways were performed using Ingenuity Pathway Analysis software. Results: It was identified 404 differentially methylated probes (291 hypomethylated and 113 hypermethylated) of which 195 were validated by TCGA. After the integrative analysis with expression data, nine TOP genes (INF2, TACSTD2, RAB15, TM7SF4, GJB3, CDSN, BHLHE41, GTF2IRD1, CXXC5) were selected with hypomethylation and overexpression. Three of them, BHLHE41, CDSN and INF2, were enriched to cancer and endocrine system disorders pathways (P = 4,47×10−2), highlighting the relevance of these genes in PTC Conclusion: This study point out putative drivers in PTC according to BRAF mutation revealing epigenetic mechanisms that contributes with cell proliferation and aggressiveness in thyroid carcinoma. Citation Format: Caroline Moraes Beltrami, Mariana Bisarro dos Reis, Mateus Camargo Barros-Filho, Fabio Albuquerque Marchi, Hellen Kuasne, Srikant Ambatipudi, Zdenko Herceg, Luiz Paulo Kowalski, Silvia Regina Rogatto. DNA methylation profile in papillary thyroid cancer according to BRAF (V600E) mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3829. doi:10.1158/1538-7445.AM2015-3829