Integrated genomic analysis of nodular tissue in macronodular adrenocortical hyperplasia: progression of tumorigenesis in a disorder associated with multiple benign lesions

Abstract

Context: Massive macronodular adrenocortical disease or ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a clinically and genetically heterogeneous disorder.

Objective and design: Whole-genome expression profiling and oligonucleotide array comparative genomic hybridization changes were analyzed in samples of different nodules from the same patients with AIMAH. Quantitative RT-PCR and staining were employed to validate the mRNA array data.

Results: Chromosomal gains were more frequent in larger nodules when compared with smaller nodules from the same patients. Among the 50 most overexpressed genes, 50% had a chromosomal locus that was amplified in the comparative genomic hybridization data. Although the list of most over- and underexpressed genes was similar between the nodules of different size, the gene set enrichment analysis identified different pathways associated with AIMAH that corresponded to the size; the smaller nodules were mainly enriched for metabolic pathways, whereas p53 signaling and cancer genes were enriched in larger nodules. Confirmatory studies demonstrated that BCL2, E2F1, EGF, c-KIT, MYB, PRKCA, and CTNNB1 were overexpressed in the larger nodules at messenger and/or protein levels. Chromosomal enrichment analysis showed that chromosomes 20q13 and 14q23 might be involved in progression of AIMAH from smaller to larger tumors.

Conclusion: Integrated transcriptomic and genomic data for AIMAH provides supporting evidence to the hypothesis that larger adrenal lesions, in the context of this chronic, polyclonal hyperplasia, accumulate an increased number of genomic and, subsequently, transcript abnormalities. The latter shows that the disease appears to start with mainly tissue metabolic derangements, as suggested by the study of the smaller nodules, but larger lesions showed aberrant expression of oncogenic pathways.

Fig. 1.

AIMAH patient 1. A, Macroscopic appearance of the left adrenal gland and a summary of chromosomal aberrations; B, all chromosomes with identified gains and losses; C, from two nodules of different size: red bars and points (*) to the right of the chromosome ideogram indicate a gain, whereas green bars and points (*) to the left indicate loss of genetic material.

Fig. 2.

AIMAH patient 2. A, Macroscopic appearance of the right adrenal in patient 2, showing macronodules and a summary of chromosomal aberrations; B, all chromosomes with indentified gains and losses; C, from two nodules of different size: red bars and points (*) to the right of the chromosome ideogram indicate a gain, whereas green bars and points (*) to the left indicate loss of genetic material.

Fig. 3.

A, Heatmap visualization of gene expression data displaying differentially expressed genes in normal adrenals and AIMAH nodules from patient 2; B and C, functional analysis of whole-genome transcriptome profiling of nodule 1 (small) and nodule 7 (large) compared with normal adrenal tissue. The array functional analysis was performed using DAVID Bioinformatics Resources 2008, National Institute of Allergy and Infectious Diseases, NIH (http://david.abcc.ncifcrf.gov/home.jsp).

Fig. 4.

A, A quantitative RT-PCR array including 84 genes involved in oncogenesis demonstrated that key oncogenes were overexpressed in the larger nodules when compared with smaller AIMAH nodules; B, a small and large nodule from patient 2, stained by hematoxylin and eosin (magnification, ×5): BCL-2 and c-KIT were highly expressed in the large nodule but not as much in the smaller nodule (magnification, ×10); C, strong CTNNB1 staining in a large nodule and weak staining in the small nodule (magnification, ×10).

Fig. 5.

A, Basal and cAMP-stimulated PKA activity in AIMAH nodules of different size from the same subject; C–D, Cα (B) and Cβ (C) PKA catalytic subunits and PRKX (D) staining was weaker in a large nodule compared with the small nodule from the same patient (magnification, ×5).