Methylation of IGF2 regulatory regions to diagnose adrenocortical carcinomas

Abstract

Adrenocortical carcinoma (ACC) is a rare malignancy with a poor prognosis. Discrimination of ACCs from adrenocortical adenomas (ACAs) is challenging on both imaging and histopathological grounds. High IGF2 expression is associated with malignancy, but shows large variability. In this study, we investigate whether specific methylation patterns of IGF2 regulatory regions could serve as a valuable biomarker in distinguishing ACCs from ACAs. Pyrosequencing was used to analyse methylation percentages in DMR0, DMR2, imprinting control region (ICR) (consisting of CTCF3 and CTCF6) and the H19 promoter. Expression of IGF2 and H19 mRNA was assessed by realtime quantitative PCR. Analyses were performed in 24 ACCs, 14 ACAs and 11 normal adrenals. Using receiver operating characteristic (ROC) analysis, we evaluated which regions showed the best predictive value for diagnosis of ACC and determined the diagnostic accuracy of these regions. In ACCs, the DMR0, CTCF3, CTCF6 and the H19 promoter were positively correlated with IGF2 mRNA expression (P < 0.05). Methylation in the most discriminating regions distinguished ACCs from ACAs with a sensitivity of 96%, specificity of 100% and an area under the curve (AUC) of 0.997 ± 0.005. Our findings were validated in an independent cohort of 9 ACCs and 13 ACAs, resulting in a sensitivity of 89% and a specificity of 92%. Thus, methylation patterns of IGF2 regulatory regions can discriminate ACCs from ACAs with high diagnostic accuracy. This proposed test may become the first objective diagnostic tool to assess malignancy in adrenal tumours and facilitate the choice of therapeutic strategies in this group of patients.

European Society of Endocrinology Clinical Practice Guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European Network for the Study of Adrenal Tumors

“…We included 17 publications (37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53) that contributed data to either the diagnosis of ACC in the context of adrenal vs non-adrenal distinction (4 studies) or in the context of benign vs malignant adrenocortical tumor distinction (15 studies) (two of them contributing to both subquestions (40,45)). Details of studies are shown in Appendix 1 (in all samples, diagnosis based on histological examination).…”

MDR1 inhibition increases sensitivity to doxorubicin and etoposide in adrenocortical cancer

“…MDR1 mRNA expression was measured in cell lines and adrenocortical tissues. RNA isolation, cDNA synthesis and RT-PCR were performed as previously described, but using other primers (Supplementary Table 1, see section on supplementary data given at the end of this article; Sigma-Aldrich) (Creemers et al 2016b). Three housekeeping genes were used to normalize mRNA levels using the Vandesompele method: hypoxanthineguanine phosphoribosyl transferase 1 (HPRT1; Sigma-Aldrich), Beta-actin (B-actin; Thermo Fisher Scientific) and glucuronidase beta (GUSB; Thermo Fisher Scientific) (Vandesompele et al 2002).…”

Insulin-Like Growth Factor 2 in Physiology, Cancer, and Cancer Treatment

“…In mice, it has been shown that 5-azacytidine, which inhibits DNA methylation, causes hypomethylation of the ICR in the IGF2 gene (Figure 3). Since the maternal ICR in IGF2 gene is not methylated and as a consequence of 5-azacytidine treatment, ICR on the paternal IGF2 becomes hypomethylated and IGF2 expression from the paternal gene would be strongly inhibited [149]. In line with this, 5azacytidine treatment inhibited rhabdomyosarcoma cell growth through repression of IGF2 expression and re-expression of H19 in vitro [150].…”