Pathogenesis Of Adrenocortical Carcinoma Research Articles

MicroRNAs as Potential Biomarkers in Adrenocortical Cancer: Progress and Challenges

Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis and limited therapeutic options. Over the last decade, pan-genomic analyses of genetic and epigenetic alterations and genome-wide expression profile studies allowed major advances in the understanding of the molecular genetics of ACC. Besides the well-known dysfunctional molecular pathways in adrenocortical tumors, such as the IGF2 pathway, the Wnt pathway, and TP53, high-throughput technologies enabled a more comprehensive genomic characterization of adrenocortical cancer. Integration of expression profile data with exome sequencing, SNP array analysis, methylation, and microRNA (miRNA) profiling led to the identification of subgroups of malignant tumors with distinct molecular alterations and clinical outcomes. miRNAs post-transcriptionally silence their target gene expression either by degrading mRNA or by inhibiting translation. Although our knowledge of the contribution of deregulated miRNAs to the pathogenesis of ACC is still in its infancy, recent studies support their relevance in gene expression alterations in these tumors. Some miRNAs have been shown to carry potential diagnostic and prognostic values, while others may be good candidates for therapeutic interventions. With the emergence of disease-specific blood-borne miRNAs signatures, analyses of small cohorts of patients with ACC suggest that circulating miRNAs represent promising non-invasive biomarkers of malignancy or recurrence. However, some technical challenges still remain, and most of the miRNAs reported in the literature have not yet been validated in sufficiently powered and longitudinal studies. In this review, we discuss the current knowledge regarding the deregulation of tumor-associated and circulating miRNAs in ACC patients, while emphasizing their potential significance in pathogenic pathways in light of recent insights into the role of miRNAs in shaping the tumor microenvironment.

Targeting polo-like kinase 1, a regulator of p53, in the treatment of adrenocortical carcinoma.

BackgroundAdrenocortical carcinoma (ACC) is an aggressive cancer with a 5 year survival rate of 20–30 %. Various factors have been implicated in the pathogenesis of ACC including dysregulation of the G2/M transition and aberrant activity of p53 and MDM2. Polo-like kinase 1 (PLK-1) negatively modulates p53 functioning, promotes MDM2 activity through its phosphorylation, and is involved in the G2/M transition. Gene expression profiling of 44 ACC samples showed that increased expression of PLK-1 in 29 % of ACC. Consequently, we examined PLK-1’s role in the modulation of the p53 signaling pathway in adrenocortical cancer.MethodsWe used siRNA knock down PLK-1 and pharmacological inhibition of PLK-1 and MDM2 ACC cell lines SW-13 and H295R. We examined viability, protein expression, p53 transactivation, and induction of apoptosis.ResultsKnocking down expression of PLK-1 with siRNA or inhibition of PLK-1 by a small molecule inhibitor, BI-2536, resulted in a loss of viability of up to 70 % in the ACC cell lines H295R and SW-13. In xenograft models, BI-2536 demonstrated marked inhibition of growth of SW-13 with less inhibition of H295R. BI-2536 treatment resulted in a decrease in mutant p53 protein in SW-13 cells but had no effect on wild-type p53 protein levels in H295R cells. Additionally, inhibition of PLK-1 restored wild-type p53’s transactivation and apoptotic functions in H295R cells, while these functions of mutant p53 were restored only to a smaller extent. Furthermore, inhibition of MDM2 with nutlin-3 reduced the viability of both the ACC cells and also reactivated wild-type p53′s apoptotic function. Inhibition of PLK-1 sensitized the ACC cell lines to MDM2 inhibition and this dual inhibition resulted in an additive apoptotic response in H295R cells with wild-type p53.ConclusionsThese preclinical studies suggest that targeting p53 through PLK-1 is an attractive chemotherapy strategy warranting further investigation in adrenocortical cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-015-0080-3) contains supplementary material, which is available to authorized users.

5th International ACC Symposium: Future and Current Therapeutic Trials in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare and complex disease associated with a high mortality rate. Despite intensive translational and clinical research, prognosis remains poor. Over the past decade, a significant effort has been made to develop multinational, collaborative studies to better understand the pathogenesis and clinical features of this rare disease in attempt to improve the therapeutic strategies and patient outcome. The results of both standard and newer treatments are discussed in this review as well as the recent discovery of pathways involved in ACC pathogenesis that provide the rationale to introduce new molecular target therapies. Finally, remaining issues regarding how to improve available therapies in adjuvant setting are raised and addressed.

Future directions in the diagnosis and medical treatment of adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare disease with a poor prognosis. Discrimination between ACCs and adrenocortical adenomas (ACAs) remains challenging, with the current gold standard being the Weiss score, consisting of several histopathological characteristics. However, new markers like Ki67, a marker for proliferation, and the staining of reticulins are promising not only as it comes to identifying malignancy but also as prognostic markers in patients with ACC. Currently, surgery is still the only curative treatment for ACC. Mitotane, an adrenolytic drug, is used in the adjuvant setting and in case of metastatic or advanced disease. Patients with progressive disease are frequently treated with mitotane, alone or in combination with etoposide, doxorubicine and cisplatin. Radiotherapy is indicated in selected cases. The low response rates and high toxicity of the systemic therapies emphasize the need for markers that enable the identification of responders and non-responders. Consequently, research is focusing on predictive factors varying from the expression of DNA repair genes to clinical patient characteristics. Subgroups of ACC with different prognosis have been identified based on transcriptome characteristics. As a conclusion from large molecular studies, ACCs appear to harbor many abnormalities compared to ACAs. Altered pathways driving ACC pathogenesis include the IGF, TP53 and the Wnt signaling pathway, allowing these as new potential targets for medical therapy. However, despite efforts in preclinical and clinical studies investigating efficacy of targeting these pathways, most novel therapies appear to be effective in only a subset of patients with ACC. New treatment concepts are therefore urgently needed.

Integrated genome-wide analysis of genomic changes and gene regulation in human adrenocortical tissue samples.

To gain insight into the pathogenesis of adrenocortical carcinoma (ACC) and whether there is progression from normal-to-adenoma-to-carcinoma, we performed genome-wide gene expression, gene methylation, microRNA expression and comparative genomic hybridization (CGH) analysis in human adrenocortical tissue (normal, adrenocortical adenomas and ACC) samples. A pairwise comparison of normal, adrenocortical adenomas and ACC gene expression profiles with more than four-fold expression differences and an adjusted P-value < 0.05 revealed no major differences in normal versus adrenocortical adenoma whereas there are 808 and 1085, respectively, dysregulated genes between ACC versus adrenocortical adenoma and ACC versus normal. The majority of the dysregulated genes in ACC were downregulated. By integrating the CGH, gene methylation and expression profiles of potential miRNAs with the gene expression of dysregulated genes, we found that there are higher alterations in ACC versus normal compared to ACC versus adrenocortical adenoma. Importantly, we identified several novel molecular pathways that are associated with dysregulated genes and further experimentally validated that oncostatin m signaling induces caspase 3 dependent apoptosis and suppresses cell proliferation. Finally, we propose that there is higher number of genomic changes from normal-to-adenoma-to-carcinoma and identified oncostatin m signaling as a plausible druggable pathway for therapeutics.

MicroRNA-7 as a tumor suppressor and novel therapeutic for adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) has a poor prognosis with significant unmet clinical need due to late diagnosis, high rates of recurrence/metastasis and poor response to conventional treatment. Replacing tumor suppressor microRNAs (miRNAs) offer a novel therapy, however systemic delivery remains challenging. A number of miRNAs have been described to be under-expressed in ACC however it is not known if they form a part of ACC pathogenesis. Here we report that microRNA-7-5p (miR-7) reduces cell proliferation in vitro and induces G1 cell cycle arrest. Systemic miR-7 administration in a targeted, clinically safe delivery vesicle (EGFREDVTM nanocells) reduces ACC xenograft growth originating from both ACC cell lines and primary ACC cells. Mechanistically, miR-7 targets Raf-1 proto-oncogene serine/threonine kinase (RAF1) and mechanistic target of rapamycin (MTOR). Additionally, miR-7 therapy in vivo leads to inhibition of cyclin dependent kinase 1 (CDK1). In patient ACC samples, CDK1 is overexpressed and miR-7 expression inversely related. In summary, miR-7 inhibits multiple oncogenic pathways and reduces ACC growth when systemically delivered using EDVTM nanoparticles. This data is the first study in ACC investigating the possibility of miRNAs replacement as a novel therapy.

Abstract A175: Novel natural withanolides potently inhibit adrenocortical carcinomas in vivo with superior safety over standard cytotoxic therapeutics.

Abstract Background: Current adjuvant strategies for an adrenocortical carcinoma (ACC) using mitotane or multidrug cytotoxics as in the Italian protocol have limited efficacy yet carry significant morbidity and toxicity. We recently demonstrated that novel natural withanolides from the North American Physalis plant potently and selectively inhibit several solid tumors including ACCs in vitro. These anticancer mechanisms through a combination of oxidative stress and heat shock protein client modulation in ACCs specifically target key tumorigenic proteins and pathways in the disease. Therefore, we hypothesize that these novel withanolides will potently inhibit ACC tumor growth in xenograft murine models without the toxicity associated with standard therapies for this disease. Methods: Tumor xenografts were established by subcutaneous injection of 5x106mouse Y1 cells into the flank region of 6-week old female, athymic, Nu/Nu mice. Drug treatment with 5mg/kg/day of withanolides (WA or WGA-TA or WGB-DB), mitotane (300mg.kg/day), or cisplatin (5-7.5 mg/Kg once weekly for 3 weeks) or control was administered ip daily for 21 days once tumors were at least 4mm in diameter. Tumor size, weight and body score as an estimate of gross toxicity were measured thrice weekly. Assessment of apoptosis and modulation of oncogenic pathway proteins was by done by immunoblot analysis. Significance was set for p&amp;lt;0.05 with differences between two or more means determined by two-way ANOVA t test. Results: Daily treatment of Y1 tumors with the novel acetyleated withanolide, WGA-TA, resulted in a two-fold decrease in tumor volume compared to controls (p&amp;lt;0.01) while WA and WGB-DB showed only slight decrease in tumor volume. All the withanolides were well tolerated in vivo without any weight loss or adverse side effects noted during treatment. While response rates for mitotane and cisplatin were similar to WGA-TA (∼2-fold decrease) compared to controls, both of these drugs carried mild to moderate toxicity that was not observed with any of the withanolides, suggesting their superior safety and potent efficacy in this disease. Induction of apoptosis, including PARP cleavage and Caspase 3 activation was measured on Western blot analysis of tumor samples. Evaluation of several key regulatory proteins of ACC pathogenesis, including Akt, mTOR, HEDGEHOG, and heat shock protein 90 client proteins after treatment with withanolides indicated significant down regulation of HSP90 client proteins (phospho-Akt, jagged 1, HSF1 and mTOR downstream regulators p70S6K and 4EBP1) compared to untreated controls, indicating a putative role of HSP90 modulation in the therapeutic efficacy of withanolides in ACC. Conclusion: Natural withanolides are potent novel therapeutics against adrenocortical carcinomas in vivo with improved toxicity compared to standard agents like mitotane and cisplatin. These data support further translational evaluation of WGA-TA toward clinical evaluation in this difficult malignancy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A175. Citation Format: Chitra Subramanian, Patrick T. Grogan, Joseph Bazzill, Rob Gallagher, Hauping Zhang, Barbara Timmermann, Mark S. Cohen. Novel natural withanolides potently inhibit adrenocortical carcinomas in vivo with superior safety over standard cytotoxic therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A175.

CpG Island Methylator Phenotype in Adrenocortical Carcinoma: Fact or Fiction?

CpG Island Methylator Phenotype in Adrenocortical Carcinoma: Fact or Fiction?

Conventional Chemotherapy and Emerging Targeted Therapy for Advanced Adrenocortical Carcinoma

Adrenocortical carcinoma (ACC) is a rare but typically aggressive malignancy. Radical surgery remains the potentially curative option. However, about one third of patients initially present with distant metastases. Regarding to chemotherapy, mitotane alone or in combination with cytotoxic drugs should be the first selection. Meanwhile, a phase lll clinical trial of etoposide, doxorubicin, cisplatin plus mitotane or streptozotocin plus mitotane is currently undergoing worldwide. The study on molecular pathogenesis of ACC is progressing. A lot of targeted therapies are also enrolled in preclinical investigations and clinical trials, including small-molecule tyrosine kinase inhibitors, antiangiogenic compounds. This article introduced the conventional chemotherapy, newly developed targeted therapy for advanced ACC.

Lower expression of ATM and gene deletion is more frequent in adrenocortical carcinomas than adrenocortical adenomas

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy accounting for approximately 0.02-0.2% of all cancer deaths. The molecular pathogenesis of ACC has been the hot topic of recent reviews but it is still poorly understood. It is imperative to have a better understanding on the pathophysiology of ACC so as to establish precise diagnosis and effective treatment. This study aims to identify the molecular markers between ACCs and adrenocortical adenomas (ACAs). With MLPA, we checked on 10 ACA and 9 ACC tissue samples. The MLPA results showed deletion on chromosomes 18q, 11q, 11p, and 13q and duplication on chromosomes 3q, 4q, 6p, and 19p. There was a significant difference in the number of aberration copies of the ataxia telangiectasia-mutated (ATM) gene located on chromosome 11q22-q23 between ACCs and ACAs. Five out of 9 (56%) ACC specimens had deletion of ATM (P=0.011). RT-PCR result then demonstrated that ATM mRNA level is lower in ACCs than in ACAs (P<0.001). In addition, immunohistochemistry (IHC) study of the 19 ACA and 18 ACC samples confirmed lower expression of ATM protein in ACCs than in ACAs (P<0.001). The study demonstrated that ATM expression was diminished in ACC than in ACA, suggesting an important role of ATM in the tumorigenesis of ACC.

Adrenocortical Carcinoma: Current Therapeutic State-of-the-Art.

Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy that generally conveys a poor prognosis. Currently, surgical resection is considered the lone curative treatment modality. In addition, the low prevalence of ACC has limited effective clinical trial design to develop evidence-based approaches to ACC therapy. The proper role of radio- and chemotherapy treatment for ACC is still being defined. Similarly, the molecular pathogenesis of ACC remains to be fully characterized. Despite these challenges, progress has been made in several areas. After years of refinement, an internationally accepted staging system has been defined. International collaborations have facilitated increasingly robust clinical trials, especially regarding agent choice and patient selection for chemotherapeutics. Genetic array data and molecular profiling have identified new potential targets for rational drug design as well as potential tumor markers and predictors of therapeutic response. However, these advances have not yet been translated into a large outcomes benefit for ACC patients. In this paper, we summarize established therapy for ACC and highlight recent findings in the field that are impacting clinical practice.

Is There a Role of Targeted Agents in the Management of Adrenocortical Cancers?

Background. Adrenocortical carcinoma (ACC) is a rare and aggressive tumor arising from the adrenal cortex with an incidence of one to two cases per million within the general US population. Recent developments in the understanding of the pathogenesis of ACC have led to multiple clinical trials involving targeted agents in the management of ACC. Patients and Methods. We report two cases of refractory adrenocortical cancer (cisplatin, adriamycin, etoposide, and mitotane) who were treated with targeted agents such as erlotinib and sutent, respectively. A total of 2 women with adrenocortical cancer were reviewed and followed for a median time of 6 months. Radiological response, duration of response and toxicities were evaluated. Results. In both cases, the targeted agents were able to control the disease for a short duration, but due to the deterioration in performance status and fatigue the agents were discontinued. Conclusion. The current observations emphasize the need for better targeted treatment modalities and strategies for the management of this fatal disease.

The role of microRNA deregulation in the pathogenesis of adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is an aggressive tumor showing frequent metastatic spread and poor survival. Although recent genome-wide studies of ACC have contributed to our understanding of the disease, major challenges remain for both diagnostic and prognostic assessments. The aim of this study was to identify specific microRNAs (miRNAs) associated with malignancy and survival of ACC patients. miRNA expression profiles were determined in a series of ACC, adenoma, and normal cortices using microarray. A subset of miRNAs showed distinct expression patterns in the ACC compared with adrenal cortices and adenomas. Among others, miR-483-3p, miR-483-5p, miR-210, and miR-21 were found overexpressed, while miR-195, miR-497, and miR-1974 were underexpressed in ACC. Inhibition of miR-483-3p or miR-483-5p and overexpression of miR-195 or miR-497 reduced cell proliferation in human NCI-H295R ACC cells. In addition, downregulation of miR-483-3p, but not miR-483-5p, and increased expression of miR-195 or miR-497 led to significant induction of cell death. Protein expression of p53 upregulated modulator of apoptosis (PUMA), a potential target of miR-483-3p, was significantly decreased in ACC, and inversely correlated with miR-483-3p expression. In addition, high expression of miR-503, miR-1202, and miR-1275 were found significantly associated with shorter overall survival among patients with ACC (P values: 0.006, 0.005, and 0.042 respectively). In summary, we identified additional miRNAs associated with ACC, elucidated the functional role of four miRNAs in the pathogenesis of ACC cells, demonstrated the potential involvement of the pro-apoptotic factor PUMA (a miR-483-3p target) in adrenocortical tumors, and found novel miRNAs associated with survival in ACC.

Cushing's Syndrome in an Infant Secondary to Malignant Adrenocortical Tumors with Somatic Mutation of Beta-Catenin

A role of beta-catenin (CTNNB1) in the molecular pathogenesis of adrenocortical carcinoma (ACC) has been suspected in adult ACC and pediatric pigmented nodular adrenocortical disease, but it has never been reported in pediatric ACC. We present the case of a 4-month-old Thai infant who had Cushing's syndrome secondary to bilateral adrenal tumors with hepatic metastasis. The child was successfully treated with a bilateral adrenalectomy and wedge resection of the liver nodule. Histopathology revealed bilateral adrenocortical tumors with different histologic grades; the right tumor had a higher score, according to modified Weiss criteria. On molecular study, a deletion mutation of beta-catenin involving codons 44 to 45 was detected in the right adrenal tumor. The same mutation was found in peripheral blood before treatment; this mutation disappeared after tumor removal. The left tumor harbored wild-type beta-catenin. Immunohistochemistry showed nuclear accumulation of beta-catenin on the right adrenal tumor and the metastatic nodule. In summary, we present evidence that supports the role of the Wnt-signaling pathway in the carcinogenesis of pediatric adrenocortical carcinoma.

Safety, tolerability, and pharmacokinetics of the anti-IGF-1R monoclonal antibody figitumumab in patients with refractory adrenocortical carcinoma

Insulin-like growth factor 1 receptor signaling through upregulation of the stimulatory ligand IGF-II has been implicated in the pathogenesis of adrenocortical carcinoma. As there is a paucity of effective therapies, this dose expansion cohort of a phase 1 study was undertaken to determine the safety, tolerability, pharmacokinetics, and effects on endocrine markers of figitumumab in patients with adrenocortical carcinoma. Figitumumab was administered on day 1 of each 21-day cycle at the maximal feasible dose (20 mg/kg) to a cohort of patients with metastatic, refractory adrenocortical carcinoma. Serum glucose, insulin, and growth hormone were measured pre-study, at cycle 4 and study end. Pharmacokinetic evaluation was performed during cycles 1 and 4. Fourteen patients with adrenocortical carcinoma received 50 cycles of figitumumab at the 20 mg/kg. Treatment-related toxicities were generally mild and included hyperglycemia, nausea, fatigue, and anorexia. Single episodes of grade 4 hyperuricemia, proteinuria, and elevated gamma-glutamyltransferase were observed. Pharmacokinetics of figitumumab was comparable to patients with solid tumors other than adrenocortical carcinoma. Treatment with figitumumab increased serum insulin and growth hormone levels. Eight of 14 patients (57%) had stable disease. The side effect profile and pharmacokinetics of figitumumab were similar in patients with adrenocortical carcinoma in comparison to patients with other solid tumors. While hyperglycemia was the most common adverse event, no clear patterns predicting severity were observed. The majority of patients receiving protocol therapy with single agent figitumumab experienced stability of disease, warranting further evaluation.

Insights into the role of genetic alterations in adrenocortical tumorigenesis

Whereas benign adrenocortical tumors are frequent in the population, adrenocortical carcinoma (ACC) is a rare cancer. Significant advances in the understanding of the pathogenesis of sporadic ACCs have been possible through the study of hereditary syndromes responsible for ACCs. The genetic alterations involved in these syndromes have also been found in sporadic ACCs. Several specific genes have been shown to be altered in sporadic ACCs. Despite these progresses, the underlying sequence(s) of events remains to be elucidated. Progressive transformation of a normal tissue into a benign tumor and ultimately into a carcinoma occurs via accumulation of genetic and epigenetic alterations. Likewise, a multistage model has been proposed for the adrenal tumor development. This review summarizes the molecular alterations likely involved in the multistage tumorigenesis and describes a mouse model which allows us to evaluate the effect of individual genes or combination of genes in the development of adrenocortical tumors.

Candidate Diagnostic Markers and Tumor Suppressor Genes for Adrenocortical Carcinoma by Expression Profile of Genes on Chromosome 11q13

The most common genetic change observed in adrenocortical carcinoma is loss of heterozygozity on chromosome 11q13. As genes on this chromosome may be important in the pathogenesis of adrenocortical carcinoma, we compared their expression profile between benign and malignant adrenocortical tissue. We used the Affymetrix GeneChip (U133 plus 2.0) array in 54 adrenocortical tumors (11 carcinoma and 43 benign). Differential gene expression was defined as a twofold higher or lower gene expression level (p<0.05). Differentially expressed genes on microarray analysis were validated by real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The area under the receiver operating characteristic (ROC) curve (AUC) was used to determined the diagnostic accuracy of the differently expressed genes for distinguishing benign from malignant tumors. We found 25 of the 314 genes on chromosome 11q13 to be differentially expressed between adrenocortical carcinoma and benign adrenocortical tumor. All 25 were downregulated in adrenocortical carcinoma by 2-fold to 4.8-fold; 21 were validated to be differentially expressed by RT-PCR (Pearson's coefficient>0.5). Six genes (SERPING1, MRPL48, TM7SF2, DDB1, NDUSF8, PRDX5) validated by RT-PCR were significantly differentially expressed between benign and malignant adrenocortical tumors (p<0.05) with an overall accuracy of 89% for SERPING1, 91% for MRPL48, 87% for TM7SF2, 88% for DDB1, 91% for NDUFS8, and 89% for PRDX5. The AUC was 0.89 for the combination of SERPING1, MRPL48, TM7SF2, DDB1, and NDUFS8. We have identified 25 genes located on chromosome 11q13 that are downregulated in adrenocortical carcinoma and may be candidate tumor suppressor genes. Six of these genes were good diagnostic markers for distinguishing adrenocortical carcinoma from adenoma.

The molecular genetics of adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy defined by a heterogeneous clinical presentation, dismal prognosis, and lack of effective therapeutic regimens. The incidence of ACC ranges from 0.5 to 2 cases per million people per year, accounting for 0.02% of all reported cancers [1]. Unfortunately, most patients present with metastatic disease which reduces the 5 year survival rate to less than 10% [2]. The resultant limited clinical experience has hampered significant research interest, public awareness and overall support from a limited pool of funding agencies. This review aims to summarize emerging genetic and molecular events implicated in the pathogenesis of ACC that can serve to energize current and future efforts to provide effective diagnostic and therapeutic approaches to this deadly cancer.

P56: Expression profile of genes on chromosome 11q13 identify candidate markers of adrenocortical carcinoma and possible tumor suppressor genes

Introduction: The molecular mechanism of adrenocortical carcinogenesis is poorly understood and distinguishing benign from malignant tumors is difficult. Several studies suggest that there is a significantly higher frequency of loss of heterozygosity (LOH) in chromosome 11q13 that is associated with adrenocortical carcinoma in 75 to 100% of cases. The Menin tumor suppressor gene at this chromosomal locus has been studied but found not be involved. These findings suggest that other genes on Chromosome 11q13 may play an important role in the pathogenesis of adrenocortical carcinoma. Therefore, in this study, we studied the expression profile of genes located on Chromosome 11q13 to identify candidate genes that are dysregulated in adrenocortical carcinoma. Material and methods: We used the Affymetrix GeneChip (U133 plus 2.0) array in 45 adrenocortical tumors (9 carcinoma and 36 adenoma). Clinical and histopathologic diagnosis of unequivocal adrenocortical carcinoma and adenoma was confirmed in all cases. The criteria used for differential gene expression between adrenocortical carcinomas and adenomas were 1) 2-fold higher or lower gene expression level, and 2) P < 0.01 by t-test statistics. We validated the differentially expressed genes by real time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Results: We found that 13 of the 317 genes on Chromosome 11q13 were differentially expressed between adrenocortical carcinoma and adenoma. All the genes were downregulated in adrenocortical carcinoma by 2 to 4.5-fold. Eleven of the thirteen genes differentially expressed by cDNA microarray were validated to be differentially expressed by RT-PCR (Pearson’s coefficient > 0.5). Four (FOLR2, DNAJC4, SERPING1, ALDH3B1) of the 13 genes were significantly differentially expressed in adrenocortical adenomas from carcinomas: FOLR2 gene had a sensitivity of 88.8% and specificity of 80.5% (p< 0.01), DNAJC4 gene had a sensitivity of 77.7% and specificity of 97.2% (p< 0.01), SERPING1 gene had a sensitivity of 66.6% and specificity of 97.2% (p< 0.01), and ALDH3B1 gene had a sensitivity of 77.7% and specificity of 94.4% (p = 0.04). Conclusions: We have identified 11 genes located on Chromosome 11q13 that are downregulated and may be candidate tumor suppressor genes in adrenocortical carcinoma. Moreover, 4 of these genes may be good diagnostic markers of adrenocortical carcinoma.

Gene expression profiling of adrenal cortical tumors by cDNA macroarray analysis. Results of a preliminary study

Adrenocortical carcinoma (ACC) are highly malignant tumors with poor prognosis. To verify if it is possible to assess their differential gene expression by a cDNA macroarray analysis using RNA extracted from paraffin sections, we analyzed two different cohorts of adrenal cortical adenoma (ACA) and ACC. Paraffin sections of seven ACC and seven ACA were analyzed. Transcriptional profiles were generated by commercially available c-DNA arrays testing 82 genes. Hybridization signals were quantified by densitometry and the intensity signal was compared for each gene between ACA and ACC cohorts. RNA was successfully extracted in only four out of 14 cases. Four genes displayed a significantly different expression (ACC/ACA ratio > 1.5 or < 0.6). Heat shock protein 60 (HSP-60) (ratio > 2), Ciclin D1 and topoisomerase I (ratio > 1.5) were overexpressed in the ACC cohort, while jun proto-oncogene was down-regulated. cDNA macroarray analysis from paraffin sections of adrenal tumors is feasible, despite with a low success rate. The different expression of HSP-60, Ciclin D1, jun proto-oncogene and topoisomerase I indicates that these genes may play a role in ACC pathogenesis and could represent potential diagnostic/prognostic/therapeutic target markers. Larger series of patients are necessary to confirm the biologic, diagnostic, prognostic and therapeutic implications of these findings.