Single nucleotide polymorphism array profiling of adrenocortical tumors--evidence for an adenoma carcinoma sequence?

Abstract

Adrenocortical tumors consist of benign adenomas and highly malignant carcinomas with a still incompletely understood pathogenesis. A total of 46 adrenocortical tumors (24 adenomas and 22 carcinomas) were investigated aiming to identify novel genes involved in adrenocortical tumorigenesis. High-resolution single nucleotide polymorphism arrays (Affymetrix) were used to detect copy number alterations (CNAs) and copy neutral losses of heterozygosity (cnLOH). Genomic clustering showed good separation between adenomas and carcinomas, with best partition including only chromosome 5, which was highly amplified in 17/22 malignant tumors. The malignant tumors had more relevant genomic aberrations than benign tumors, such as a higher median number of recurrent CNA (2631 vs 94), CNAs >100 Kb (62.5 vs 7) and CN losses (72.5 vs 5.5), and a higher percentage of samples with cnLOH (91% vs 29%). Within the carcinoma cohort, a precise genetic pattern (i.e. large gains at chr 5, 7, 12, and 19, and losses at chr 1, 2, 13, 17, and 22) was associated with a better prognosis (overall survival: 72.2 vs 35.4 months, P=0.063). Interestingly, >70% of gains frequent in benign were also present in malignant tumors. Notch signaling was the most frequently involved pathway in both tumor entities. Finally, a CN gain at imprinted "IGF2" locus chr 11p15.5 appeared to be an early alteration in a multi-step tumor progression, followed by the loss of one or two alleles, associated with increased IGF2 expression, only in carcinomas. Our study serves as database for the identification of genes and pathways, such as Notch signaling, which could be involved in the pathogenesis of adrenocortical tumors. Using these data, we postulate an adenoma-carcinoma sequence for these tumors.

Figure 1. Copy number amplifications in chromosome 5 are specific for malignant adrenocortical tumors.

A) Unsupervised euclidean distance genomic clustering (complete linkage) in 46 adrenocortical tumor samples (defined as sample ID). Best separation between adenoma (ACA, n=24, white) and carcinoma (ACC, n=22, black) obtained analyzing only the chromosome 5. B) Validation with FISH analysis of the copy number alterations detected with SNP array in chromosome 5. Two representative adrenocortical carcinomas (ACC, samples ID 456C and 1023C) with gains/amplifications at SNP array analysis validated by FISH (probe FISH_5q/5p-Probe: CSF1R/D5S23, D5S721). Gain of the red signal (specific probe) with respect to the green signal (centromer-specific reference probe) in more than 50% of cells. In black the ACC patients affected by ACC and in white the patients affected by ACA.

Figure 2. Whole-genome copy number alterations (CNA) in adrenocortical tumors.

A) Genome-wide distribution of CNA in each adrenocortical tumor according to the genomic segmentation algorithm (Partek GS). ACA=adenoma (n=24, total number of CNA=3603). ACC=carcinoma (n=22, total number of CNA=5017). B) Genome-wide distribution of recurrent CNA in adrenocortical tumors. Minimal overlapping regions in at least 4 samples (MORs, frequency >15%). ACA=adenoma (n=24, total number of CNA=283). ACC=carcinoma (n=22, total number of CNA=3993). CN gains are represented in red and CN losses in blue.

Figure 3. Whole-genome loss of heterozygosity (LOH) events in adrenocortical tumors.

A) Genome-wide distribution of LOH events in each adrenocortical tumor according to the hidden markov model (Partek GS). ACA=adenoma (n=24, total number of LOH=23). ACC=carcinoma (n=22, total number of LOH=2439). B) Genome-wide distribution of copy neutral LOH identified by merged analysis between copy number alteration and LOH analysis.

Figure 4. Correlation between the total number of LOH events and tumor size.

All the evaluated adrenocortical tumors (n=46) were included in the analysis by linear regression analysis. White triangles = adenomas (n=24), black triangles = carcinomas (n=22).

Figure 5. Schematic representation of the genetic alterations detected by SNP array analysis in adrenocortical tumors.

In the upper panel, the percentage of samples affected by different alterations is reported. Interestingly, the simultaneous presence of numerous LOH and large CNA was observed only in malignant tumors (n=22). In the lower panel, some relevant specific alterations in candidate genes and pathway are reported. In particular, some alterations were observed in all tumors (i.e. isolated small CN gains), while others were found only in carcinomas. CNA=copy number alterations, LOH=loss of heterozygosity) detected by SNP array analysis in adrenocortical adenomas (n=24) and carcinomas (n=22).

Figure 6. Pathway analysis for genes frequently affected by copy number alterations in both adenomas and carcinomas.

A) List of the most significantly altered pathways according to the GeneGo analysis (Meta Core Analytical suite) including all the genes (n=696 annotated genes) with copy number alterations observed in at least 4 samples (recurrent CNA) in both adenomas and carcinomas. The P values are expresses as logarithmic scale. B) Graphical representation of the Notch signaling pathway map (gene ontology enrichment, P=2.913 ^e-4). C) Graphical representation of the WNT5 signaling pathway (gene ontology enrichment, P=3.256 ^e-3). Genes with gains are underlined in red and genes with losses in blue. Detailed legend is available at http://pathwaymaps.com/pdf/MC_legend.pdf.

Figure 7. Relationship between genomic alterations at chr 11p15.5 and IGF2 gene expression in adrenocortical tumors.

A) Relative mRNA expression levels evaluated by qRT-PCR for IGF2 gene according to the copy number status observed with SNP array analysis in normal adrenals (n=16), adenomas (n=18) and carcinomas (n=14). The gene actin beta was used as a loading control (reference gene). Data expressed as logarithmic scale. P=0.0003 by one-way ANOVA. B) Schematic representation of the genetic alterations at the 11p15.5 imprinted locus and their impact on IGF2 expression levels. In particular, we firstly report frequent copy number gains in adenomas, leading to a normal IGF2 expression, and we confirm frequent copy neutral LOH events in carcinoma, leading to an increased IGF2 expression, suggesting a progressive multi-step genetic derangement in this locus. We also demonstrate a further allele loss in carcinoma (LOH + CN loss) without effects on IGF2 expression.

Figure 8. Relationship between genomic alterations and clinical outcome in malignant adrenocortical tumors.

A) Unsupervised euclidean distance genomic clustering (complete linkage) in 22 carcinomas. Best cluster obtained including a specific combination of chromosomes (i.e. chr 1, 2, 4, 5, 7, 12, 13, 14, 17, 18, 20, 22, and X). B) Relationship between the genetic clusters and the clinical parameters: tumor size and Weiss score (Mann-Withney test), ENSAT tumor stage (Fisher’s test) and overall survival (Kaplan-Meyer curve and log-rank test). In black the patients with a worst prognosis (overall survival < 24 months), in grey the patients with an intermediate prognosis (overall survival 24-48 months), and in white the patients with a good prognosis (overall survival > 48 months or still alive at the last follow up).