Pathogenesis Of Adrenocortical Tumors Research Articles

Characterizing the Cortex to Improve Clinical Care. Single cell sequencing: lessons for the pathogenesis of adrenocortical tumours

Characterizing the Cortex to Improve Clinical Care. Single cell sequencing: lessons for the pathogenesis of adrenocortical tumours

RNA Sequencing and Somatic Mutation Status of Adrenocortical Tumors: Novel Pathogenetic Insights.

Pathogenesis of autonomous steroid secretion and adrenocortical tumorigenesis remains partially obscure. To investigate the relationship between transcriptome profile and genetic background in a large series of adrenocortical tumors and identify new potential pathogenetic mechanisms. Cross-sectional study. University Hospitals of the European Network for the Study of Adrenal Tumors (ENSAT). We collected snap-frozen tissue from patients with adrenocortical tumors (n = 59) with known genetic background: 26 adenomas with Cushing syndrome (CS- cortisol-producing adenoma [CPA]), 17 adenomas with mild autonomous cortisol secretion (MACS-CPAs), 9 endocrine-inactive adenomas (EIAs), and 7 adrenocortical carcinomas (ACCs). Ribonucleic acid (RNA) sequencing. Gene expression, long noncoding RNA (lncRNA) expression, and gene fusions. Correlation with genetic background defined by targeted Sanger sequencing, targeted panel- or whole-exome sequencing. Transcriptome analysis identified 2 major clusters for adenomas: Cluster 1 (n = 32) mainly consisting of MACS-CPAs with CTNNB1 or without identified driver mutations (46.9% of cases) and 8/9 EIAs; Cluster 2 (n = 18) that comprised CP-CPAs with or without identified driver mutation in 83.3% of cases (including all CS-CPAs with PRKACA mutation). Two CS-CPAs, 1 with CTNNB1 and 1 with GNAS mutation, clustered separately and relatively close to ACC. lncRNA analysis well differentiate adenomas from ACCs. Novel gene fusions were found, including AKAP13-PDE8A in one CS-CPA sample with no driver mutation. MACS-CPAs and EIAs showed a similar transcriptome profile, independently of the genetic background, whereas most CS-CPAs clustered together. Still unrevealed molecular alterations in the cAMP/PKA or Wnt/beta catenin pathways might be involved in the pathogenesis of adrenocortical tumors.

SAT-179 Increased Telomere Length in Adrenocortical Tumors Is Associated with Abnormal Expression of Chromatin Remodelling Factors

Background: The pathogenesis of adrenocortical tumors (ACTs) in the pediatric population is partially known, and few prognostic factors have been identified in this age group. Recently, ATRX and DAXX have been implicated in the pathogenesis and prognosis of a variety of cancers. Their altered function has been shown to affect telomere length through a telomerase-independent mechanism. Objective: To investigate ATRX and DAXX gene expression, ATRX and DAXX protein expression, and telomere length, as well as their clinical significance, in ACT samples from pediatric patients. Methods: The records of 110 pediatric patients with available ACT samples were reviewed. ATRX, DAXX, TERT and TERC gene expression was assessed by qPCR (n = 100 ACTs; n = 12 normal adrenals). ATRX and DAXX protein expression was assessed by IHC (n = 45 ACTs). Telomere length was assessed by qPCR (n = 64 ACTs). For survival analysis, Kaplan-Meier curves were obtained. For association analysis, simple linear regression models were adjusted. Results: Most patients were female (70.9%) and harbored germline TP53 mutations (90.2%). Median age at diagnosis was 21.1 months (2.1 – 199). Younger patients (< 3 years) had better survival (p < 0.01), while those with metastasis at diagnosis and carcinomas (classified by the Wieneke score) had worse survival (p < 0.01). ATRX gene expression was decreased (p < 0.01), while DAXX gene expression was increased (p < 0.01) in ACTs, compared to normal adrenals. ATRX gene expression was even lower in the context of the germline TP53 (R337H) mutation (p < 0.01). TERT expression was not detected in ACTs or normal adrenals, and TERC expression was not altered (p = 0.69). ATRX protein expression was lost in the majority of ACTs (95.6%), while DAXX was lost in a minority (21.1%). There was no association between gene or protein expression and disease-free or overall survival. There was a significant association between decreased ATRX and DAXX gene expression and increased telomere length (p < 0.01 and p = 0.03, respectively). Conclusion: In pediatric ACTs, decreased ATRX and DAXX gene expression was associated with increased telomere length, independently of TERT or TERC expression. In these tumors, ATRX gene expression was decreased and ATRX protein expression was overall lost, while DAXX gene expression was increased and DAXX protein expression was overall retained. No significant association between these alterations and prognosis was found in this cohort. These findings suggest that ATRX and DAXX altered function may be more involved in the pathogenesis of pediatric ACTs than in the prognosis of the affected patients.

Role of Endocrine Gland-Derived Vascular Endothelial Growth Factor (EG-VEGF) and Its Receptors in Adrenocortical Tumors.

Angiogenesis is essential for tumor growth and metastasis. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is an angiogenic factor predominantly expressed in steroidogenic organs like the adrenal gland, ovary, testes, and placenta. EG-VEGF has antiapoptotic, mitogenic, and chemoattractive properties mediated via the two G protein-coupled receptors prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2). We investigated the expression of EG-VEGF and its receptors in a large number of normal adrenal glands (NAG), adrenocortical adenomas (ACA), and carcinomas (ACC) using real-time PCR (NAG, n = 12; ACA, n = 24; and ACC, n = 30) and immunohistochemistry (NAG, n = 9; ACA, n = 23; and ACC, n = 163) and evaluated its impact on patients’ survival. EG-VEGF, PKR1, and PKR2 mRNA and protein are expressed in NAG and the vast majority of ACA and ACC samples. The mean EG-VEGF mRNA expression was significantly lower in ACC (606.5 ± 77.1 copies) compared to NAG (4,043 ± 1,111) and cortisol-producing adenomas (CPA) (4,433 ± 2,378) (p < 0.01 and p < 0.05, respectively). However, cytoplasmic and nuclear EG-VEGF protein expression was either significantly higher or similar in ACC (H score 2.4 ± 0.05, p < 0.05 and 1.7 ± 0.08, n.s., respectively) compared to NAG (1.8 ± 0.14 and 1.7 ± 0.2). Nuclear protein expression of either EG-VEGF or PKR1 or both is predictive for a higher mortality compared to patients without nuclear expression (hazard ratio (HR) = 5.15; 95 % confidence interval (CI) = 1.24–21.36, n = 100, p = 0.02 independent of age, sex, and tumor stage). These findings suggest that EG-VEGF and its receptor PKR1 might play a role in the pathogenesis of adrenocortical tumors and could serve as prognostic markers for this rare malignant disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s12672-015-0236-z) contains supplementary material, which is available to authorized users.

PRKACA: the catalytic subunit of protein kinase A and adrenocortical tumors.

Cyclic-AMP (cAMP)-dependent protein kinase (PKA) is the main effector of cAMP signaling in all tissues. Inactivating mutations of the PRKAR1A gene, coding for the type 1A regulatory subunit of PKA, are responsible for Carney complex and primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A inactivation and PKA dysregulation have been implicated in various types of adrenocortical pathologies associated with ACTH-independent Cushing syndrome (AICS) from PPNAD to adrenocortical adenomas and cancer, and other forms of bilateral adrenocortical hyperplasias (BAH). More recently, mutations of PRKACA, the gene coding for the catalytic subunit C alpha (Cα), were also identified in the pathogenesis of adrenocortical tumors. PRKACA copy number gain was found in the germline of several patients with cortisol-producing BAH, whereas the somatic Leu206Arg (c.617A>C) recurrent PRKACA mutation was found in as many as half of all adrenocortical adenomas associated with AICS. In vitro analysis demonstrated that this mutation led to constitutive Cα activity, unregulated by its main partners, the PKA regulatory subunits. In this review, we summarize the current understanding of the involvement of PRKACA in adrenocortical tumorigenesis, and our understanding of PKA's role in adrenocortical lesions. We also discuss potential therapeutic advances that can be made through targeting of PRKACA and the PKA pathway.

Role of steroidogenic factor-1 in the pathogenesis of adrenocortical tumours

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Single Nucleotide Polymorphism Array Profiling of Adrenocortical Tumors - Evidence for an Adenoma Carcinoma Sequence?

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 beningn 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.

Integrative analysis of SF-1 transcription factor dosage impact on genome-wide binding and gene expression regulation

Steroidogenic Factor-1 (SF-1) is a nuclear receptor that has a pivotal role in the development of adrenal glands and gonads and in the control of steroid hormone production, being also implicated in the pathogenesis of adrenocortical tumors. We have analyzed the mechanisms how SF-1 controls gene expression in adrenocortical cells and showed that it regulates different categories of genes according to its dosage. Significant correlations exist between the localization of SF-1-binding sites in chromatin under different dosage conditions and dosage-dependent regulation of gene expression. Our study revealed unexpected functional interactions between SF-1 and Neuron-Restrictive Silencer Factor/RE1-Silencing Transcription Factor (NRSF/REST), which was first characterized as a repressor of neuronal gene expression in non-neuronal tissues, in the regulation of gene expression in steroidogenic cells. When overexpressed, SF-1 reshapes the repertoire of NRSF/REST—regulated genes, relieving repression of key steroidogenic genes. These data show that NRSF/REST has a novel function in regulating gene expression in steroidogenic cells and suggest that it may have a broad role in regulating tissue-specific gene expression programs.

Expression of sfrp1 and activation of the Wnt pathway in the adrenal glands of healthy ferrets and neutered ferrets with hyperadrenocorticism

Gonadectomy induces the pathogenesis of luteinising hormone receptor positive, androgen and oestrogen producing tumours in the adrenal cortex of ferrets. In mice, the castration-dependent appearance of adrenocortical tumours has been attributed to loss of expression of the tumour suppressor gene Secreted Frizzled Related Protein 1 (sfrp1), a dominant inhibitor of the Wnt pathway, which controls cell proliferation and ‘cell faith’ decisions. This study investigated whether sfrp1 and the Wnt pathway play a similar role in the pathogenesis of hyperadrenocorticism in ferrets. The expression of sfrp1 and three target genes of the Wnt pathway (c-myc, axin2 and cyclinD1) in seven adrenal glands from healthy ferrets and in 13 adrenocortical tumours were studied by quantitative real-time PCR. Nuclear β-catenin staining was assessed by immunohistochemistry. Sfrp1 mRNA expression was up-regulated and axin2 and cyclinD1 were down-regulated in the tumour group in comparison with the control group. Decreased nuclear β-catenin staining supported the decrease in active Wnt signalling in adrenocortical tumours in ferrets. Therefore, it is unlikely that the involvement of sfrp1 and the Wnt pathway in the pathogenesis of adrenocortical tumours in ferrets is similar to that described in mice.

MiR-195 and miR-483-5p Identified as Predictors of Poor Prognosis in Adrenocortical Cancer

PURPOSE: Adrenocortical adenomas are common, whereas adrenocortical carcinomas are rare. Discriminating between benign and malignant adrenocortical tumors using conventional histology can be difficult. In addition, adrenocortical carcinomas generally have poor prognosis and limited treatment options. MicroRNAs are short noncoding RNAs that are involved in regulation of gene transcription. EXPERIMENTAL DESIGN: To identify microRNAs involved in the pathogenesis of adrenocortical tumors, expression profiling of microRNAs was done on a cohort of 22 adrenocortical carcinomas, 27 adrenocortical adenomas, and 6 normal adrenal cortices. RESULTS: Twenty-three microRNAs were found to be significantly differentially expressed between adrenocortical carcinomas and adrenocortical adenomas. miR-335 and miR-195 were significantly downregulated in adrenocortical carcinomas compared with adrenocortical adenomas. This result was further validated in an external cohort of six adrenocortical carcinomas and four adrenocortical adenomas. Using Kaplan-Meier analysis, downregulation of miR-195 and upregulation of miR-483-5p in adrenocortical carcinomas were significantly associated with poorer disease-specific survival. CONCLUSIONS: These findings indicate that deregulation of microRNAs is a recurring event in human adrenocortical carcinomas and that aberrant expression of miR-195 and miR-483-5p identifies a subset of poorer prognosis adrenocortical carcinomas. (Clin Cancer Res 2009;15(24):7684-92).

ES08 MICRORNA MICROARRAY ANALYSIS OF HUMAN ADRENOCORTICAL TUMOURS

Purpose: A dilemma in the management of adrenal tumours is differentiating between benign and malignant tumours. Adrenocortical adenomas (ACAs) are common while adrenocortical carcinomas (ACCs) are rare. ACCs also have poor prognosis with limited treatment options. MicroRNAs (miRs) are non‐coding, 20–22 nucleotide RNAs which have been implicated in tumorigenesis. The aim of this study was to use microarray analysis to identify miRs which play a role in the pathogenesis of ACCs and have the ability to discriminate between ACCs and ACAs as well as to serve as therapeutic targets.Methodology: RNA from 6 normal adrenal cortices, 27 ACAs and 22 ACCs were hybridised to Exiqon mirCURYTM LNA Arrays version 10.0.Results: 14 miRs were significantly upregulated while 9 were significantly downregulated between ACCs and ACAs. Several miRs which have previously been reported to be associated with other cancers and act as regulators of genes such as EGFR, PTEN and RAS were found in our group of significantly differentially expressed miRs. One of the significantly downregulated miRs is located in a 10.4 megabase common minimal region of loss on 17p13 which we have previously delineated in ACCs. In addition, one of the 14 significantly upregulated miRs was located in an intron of IGF2, a gene frequently overexpressed in ACCs compared to ACAs.Conclusions: In this study, we found miRs which were significantly differentially expressed between ACCs, ACAs and normal adrenal cortex, indicating that miRs are involved in the pathogenesis of adrenocortical tumours and have the potential to serve as diagnostic markers and therapeutic targets.

Loss of Heterozygosity of 17p13, With Possible Involvement of ACADVL and ALOX15B, in the Pathogenesis of Adrenocortical Tumors

To determine the minimal common region of loss on 17p13 in a cohort of adrenocortical carcinomas (ACCs) (defined by a Weiss score > or =3) and adrenocortical adenomas (ACAs) (defined by a Weiss score <3) and subsequently to assess 3 genes in this region that could be involved in adrenocortical tumorigenesis. Loss of heterozygosity (LOH) of 17p13 has been demonstrated to occur more frequently in ACCs compared with ACAs. Using 12 microsatellite markers across 17p13, LOH analysis was performed on 37 paired blood and adrenocortical tumor samples (23 ACC and 14 ACA samples) to determine the minimal common region of loss for ACCs and ACAs. From this minimal region of loss, 3 genes were selected for quantitative real time reverse transcription polymerase chain reaction analysis on 20 ACCs and 30 ACAs. LOH at 17p13 was found in 74% of ACCs compared with 14% of ACAs. There was a 10.4-Mb common minimal region of loss in ACCs whereas no minimal region of loss in ACAs could be demonstrated. Expression of Acyl coenzyme-A dehydrogenase very long chain (ACADVL) and Arachidonate 15-lipoxygenase second type (ALOX15B) was significantly down-regulated in ACCs compared with ACAs whereas there was no difference in expression of Potassium channel tetramerization domain containing 11 (KCTD11) in ACCs and ACAs. We demonstrated a minimal common region of loss of 10.4-Mb on 17p13 in ACCs and within this region, we found that ACADVL and ALOX15B expression are good discriminators between ACCs and ACAs.

ES07 MICROARRAY GENE EXPRESSION ANALYSIS OF HUMAN ADRENOCORTICAL TUMOURS

Introduction Adrenal tumours are common, occurring in 7% of patients over the age of 50. Adrenocortical carcinomas, however, are rare, with an incidence of two per million population per year. The management of adrenocortical tumours is complex, compounded by the difficulty in discriminating benign from malignant tumours using conventional histology. A molecular marker which could reliably distinguish between the two groups would be valuable in patient management.Objectives The aim of this study was to identify molecular markers which will discriminate between adrenocortical carcinomas and adenomas using microarray gene expression analysis.Methods This study used RNA from 6 normal adrenal cortices, 16 adrenocortical adenomas and 12 carcinomas. Only samples with an RNA integrity number of 7.5 or greater were used. The samples were hybridised to Affymetrix HGU133plus2.0 genechips. Data analysis was performed with Partek and affylmgui softwares.Results Using a cutoff of B &gt; 2 and M &gt; 2 or &lt;−2, 217 genes were found to be significantly differentially expressed between adrenocortical adenomas and carcinomas. Of these genes, 120 were unpregulated while 97 were downregulated. Seven of these genes have been selected for validation studies with real time reverse transcription polymerase chain reaction.Conclusion In this study, we found 217 genes which were significantly differentially expressed between adrenocortical adenomas and carcinomas. With validation and further studies, these genes will provide further insight into the pathogenesis of adrenocortical tumours as well as possibly proving to be reliable discriminators between adrenocortical adenomas and carcinomas.

Mutational Analysis of PRKAR1A and Gsα in Sporadic Adrenocortical Tumors

Little is known about the pathogenesis of adrenocortical tumors. The cAMP-dependent pathway is physiologically activated by ACTH in adrenocortical cells and different components of this cascade may be altered in some functioning adrenocortical tumors. Recently, mutations of the gene encoding the PKA type 1 A regulatory subunit (R1 A), PRKAR1A, associated with loss of heterozygosity (LOH) at PRKAR1A locus, have been demonstrated in primary pigmented nodular adrenocortical disease (PPNAD), either isolated or associated with Carney complex. Moreover, activating mutations of the Gs(alpha) gene (the gsp oncogene) have also been found in a small number of adrenocortical cortisol-secreting adenomas. Aim of this study was to investigate the presence of such genetic alterations on a series of 10 ACTH-independent Cushing syndrome due to non-PPNAD adrenocortical adenomas. The coding sequence of PRKAR1A, evaluated by PCR and direct sequencing analysis, revealed the absence of mutations while heterozygosity for at least 1 polymorphism excluded LOH in most tumors. In one single adenoma gsp mutation was detected. In conclusion, we provide additional evidence that the only mutational changes able to activate the cAMP pathway so far identified, i.e. PRKAR1A mutations and gsp oncogene, are a rare event in adrenocortical tumors.

Evaluation of plasma insulin-like growth factor binding protein-2 as a marker for adrenocortical tumors.

Recent studies have pointed to the role of the IGF system in the pathogenesis of adrenocortical tumors, and it was shown recently that malignant adrenocortical tumors exhibit a high insulin-like growth factor binding protein (IGFBP)-2 content. Circulating markers specific for adrenocortical carcinoma are needed and the aim of this study was to evaluate plasma IGFBP-2 as a marker for these malignant tumors. Plasma IGFBP-2 was determined in 51 patients referred to our institutions for adrenocortical tumors. Fifteen patients were in complete remission (group 1), eight patients had preoperative localized tumors (group 2) and 28 patients had metastatic tumors (group 3). Thirty-six healthy volunteers constituted a control group. There was no significant difference in plasma IGFBP-2 concentration between healthy controls and patients with complete remission or localized tumors. In contrast, patients with metastatic disease had significantly higher IGFBP-2 plasma levels than the control group (P<0.001). IGFBP-2 levels in patients with metastatic disease were inversely correlated with survival (R2=0.308; P=0.0026). In patients with localized tumors, there was no correlation between plasma IGFBP-2 concentration and tumor size or histological features. Analysis of individual IGFBP-2 concentrations showed that five patients (17.8%) with metastatic tumors had normal IGFBP-2 levels and two patients (13.3%) in complete remission had high plasma IGFBP-2 levels. The influence of nutrition, hormone secretion and treatment on IGFBP-2 levels was examined. Nutritional status was evaluated by determining IGF-I levels and was found to be normal in 16 patients (61.5%) with high IGFBP-2 levels, suggesting that malnutrition was not responsible for the high IGFBP-2 concentrations in these patients. IGFBP-2 levels did not differ significantly according to tumor secretion or mitotane treatment. In a follow-up study, plasma IGFBP-2 concentration remained stable in patients with complete remission or stabilized disease and was a late marker of tumor progression in patients with progressive metastatic disease. These results indicate that plasma IGFBP-2 is elevated in patients with malignant adrenocortical tumors and that the major factor affecting IGFBP-2 levels in these patients is tumor stage. However, plasma IGFBP-2 was less sensitive than expected for a tumor marker, which may limit its value in the diagnosis and follow-up of adrenocortical carcinoma.

Structural and functional abnormalities at 11p15 are associated with the malignant phenotype in sporadic adrenocortical tumors: study on a series of 82 tumors.

Abnormalities of the 11p15 region with overexpression of the normally imprinted insulin-like growth factor II (IGF-II) gene have been implicated in the pathogenesis of adrenocortical tumors. We evaluated the frequency and distribution of 11p15 loss of heterozygosity (LOH) and IGF-II gene overexpression in a series of 82 sporadic adrenocortical tumors, screened for pathological functional imprinting of the 11p15 region in tumors not exhibiting LOH and evaluated the expression of H19 gene in these tumors. Abnormalities of the 11p15 region as LOH (loss of the maternal allele and duplication of the paternal allele) and/or IGF-II gene overexpression are frequent features of the malignant state and were found in 27 of 29 (93.1%) of the malignant tumors and in only 3 of 35 (8.6%) of the benign tumors. Tumors without abnormality of the 11p15 region (mainly benign tumors) did not exhibit pathological functional imprinting. In tumors with mosaicism for 11p15 LOH, biallelic expression of the IGF-II gene was constant in the tumor cell contingent not undergoing LOH. Abrogation of H19 expression correlated with the loss of the maternal allele (LOH or pathological imprinting), but did not always correlate with overexpression of the IGF-II gene. These data indicate the involvement of dysregulation of the 11p15 region in late steps of adrenocortical tumorigenesis and provide us with new molecular markers for a better diagnostic and prognostic evaluation of adrenocortical tumors.

Recent advances in the pathogenesis of adrenocortical tumours.

Recent advances in the pathogenesis of adrenocortical tumours.