Carcinoma of the adrenal cortex accounts for between 0.02 and 0.2% of all cancer deaths. It is one of the least common but most lethal endocrine malignancies. Surgical removal offers the only current potential for cure. Unfortunately, adrenocortical cancers (ACC) have undergone metastatic spread in approximately 40-70% of patients by the time of diagnosis. This late diagnosis and a lack of effective systemic treatments result in a relatively poor overall 5-year survival rate of 20 to 35%. Newer and more effective therapeutic drugs against ACC are clearly needed. The modern molecular technologies developed during the era of the sequencing of the human genome project offer the potential to analyze DNA alterations and the expression changes of thousands of genes in human tumor tissue. Along with affording knowledge of the oncogenic processes in ACC, increased disease specific genetic knowledge leads to the identification of novel targets for therapeutic agents.

The objectives of this study are to make use of genomic technologies at TGen for the molecular analysis of adrenocortical carcinoma (ACC) tissue collected by investigators at collaborating clinical centers. The TGen facility has expertise in a variety of genomic technologies including DNA copy assessment by array-based comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) assessment, expression profiling on multiple platforms, mutation detection and analysis, immunohistochemistry, and tissue microarrays (TMAs).

Current Projects

1. Patient Samples: The TGen ACC tissue bank has received frozen tumor tissue for this study from collaborating institutions around the world. The collection process will continue throughout the research project with tumors contributed from scientific institutes as well as through participation in the ATAC Fund tumor donation process. Samples will be collected prospectively by surgeons, from patients undergoing medically indicated procedures at their participating hospitals. No patients will be subjected to collection procedures specifically for this protocol nor will additional tissue be removed that would not have been removed for medically indicated reasons.

2. Expression Profiling: RNA from experimental samples is extracted, amplified, labeled, and hybridized to either spotted microarrays or fabricated oligonucleotide microarrays. Using these technologies, the amount of fluorescence can be monitored at each feature, enabling the simultaneous quantitation of the tens of thousands of different RNA transcripts, representing gene activity (expression analysis). Agilent microarrays utilize the hybridization of control and experimental samples to a single array while Affymetrix GeneChip microarrays assay one individual sample per chip. Both platforms result in the determination of gene regulation differences in experimental and control samples, leading to a picture of aberrantly expressed genes within the tumor tissue.

3. Comparative Genomic Hybridization (CGH) and Single Nucleotide Polymorphism (SNP) Analysis: DNA will be extracted from tissue samples for CGH analysis and SNP analysis. CGH is a molecular cytogenetic method of screening a tumor for genetic changes. The alterations are classified as DNA gains and losses and reveal a characteristic pattern that includes mutations at chromosomal and sub chromosomal levels. New CGH technologies employ an Agilent array-based assay requiring minute quantities of DNA able to evaluate elements across the human genome. DNA genotyping and copy number changes will be assessed through Affymetrix SNP chip arrays containing elements assaying 100,000 SNPs across the genome, highlighting areas of genomic variation possibly linked to tumor presence and progression.

4. Tissue Sections and Tissue Microarrays (TMA): Very thin tissue sections will be cut from the donor block and after staining, the tumor will be examined using a microscope to assess the tumors’ appearance. Regions of interest from each tumor block will be utilized for the creation of a tissue microarray (TMA). TGen scientists construct TMAs by extracting a small paraffin core from the tumor block and then insert this core into a composite recipient TMA block. Each block will consist of three tissue cores from each tumor to enable a thorough sample of all areas of the tumor.

5. Immunohistochemistry (IHC): Antibodies or antisera are effective histological tools for identifying patterns of antigen distribution within a tissue sample. An antibody (or mixture of antibodies) that binds to a specific protein or other antigen is tagged with a fluorescent chemical or an enzyme that can convert a substrate to a visible dye. The tagged antibody is incubated with the tissue and after washing unbound antibody away, the bound antibody distribution is revealed by fluorescence microscopy or incubation with a chromogenic substrate. This type of analysis confirms the findings of increased or diminished expression seen on the gene expression microarray.

6. Drosophila Chemical Synthetic Lethal: In collaboration with the University of Arizona and the Harvard Drosophila RNAi Screening Center, genes sensitizing cells to mitotane treatment will be determined through knockout using interfering RNAs (RNAi). The model Drosophila system will be utilized.

7. In vitro and in vivo Testing: Potential therapeutic targets identified through genomic analyses are immediately translated into cell culture and xenograft mice trials using both approved drugs and compounds in various stages of development.

8. Statistical Design: TGen has access to a variety of sophisticated statistical tools which have been shown viable in previous work. These statistical tools will be utilized to analyze data created through the multiple techniques employed in the ACC research project. One of notable interest is a statistical method to link gene expression microarray data to the results of the microarray CGH and SNP analysis. To aid in this type of analysis, investigators have access to the TGen-ASU supercomputer, ranked in the top 15 of computers dedicated to biomedical research.

9. Data Sharing: Data produced from samples in this study will have all patient identifier information removed after resulting data is returned to the submitting physician (in the case of prospective submission where the patient has requested results from their tumor sample). De-identified data from Agilent expression profiling, Affymetrix expression profiling, Agilent 44K CGH, and Affymetrix 100K SNP are available by clicking here.

Collaborations

Investigators from Arizona, Minnesota, Canada, Australia and Germany have contributed tumor samples to this ongoing project. The TGen ACC project has formed a collaboration with the Critical Pathway Institute (C-Path) to develop patient oriented web-based data collection. C-Path is working with the FDA’s Division on Orphan Diseases to develop methods to speed drug development and approval for the treatment of patients with rare diseases, such as adrenocortical cancer. The ACC research team at TGen is eager to work with all investigators on efforts to improve treatments for affected patients.

Staff:

Kimberly J Bussey, PhD, Associate Investigator
Michael J Demeure, MD, Senior Investigator
Erica Dastrup, Research Associate

Presentations:

Watch the Advancing Treatments for Adrenocortical Carcinoma video
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