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Identification of Biomarkers of Adrenocortical Carcinoma Using Genomewide Gene Expression Profiling

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Identification of Biomarkers of Adrenocortical Carcinoma Using Genomewide Gene Expression Profiling PAPER Identification of Biomarkers of Adrenocortical Carcinoma Using Genomewide Gene Expression Profiling Gustavo G. Fernandez-Ranvier, MD; Julie Weng, BS; Ru-Fang Yeh, PhD; Elham Khanafshar, MD; Insoo Suh, MD; Christopher Barker, PhD; Quan Yang Duh, MD; Orlo H. Clark, MD; Electron Kebebew, MD Hypothesis: The gene expression profiles of benign and Results: We found 37 genes differentially expressed by malignant adrenocortical tumors are different. 8-fold higher or lower. Fifteen genes were downregu- lated and 22 were upregulated in adrenocortical carci- Design: Genomewide gene expression profiling and vali- noma. Of the 37 genes, 29 differentially expressed by dation. microarray correlated with the gene expression levels by quantitative RT-PCR (PՅ.01). Of the 37 genes vali- Setting: Tertiary medical center. dated by RT-PCR, 22 were significantly differentially expressed between benign and malignant adrenocorti- Patients: Eighty-five patients with benign adrenocorti- cal tumors (PϽ.05). Five of these 22 genes had an AUC cal tumors (n=74) and adrenocortical carcinoma (n=11). of 0.80 or greater (the AUC for IL13RA2 was 0.90; HTR2B, 0.87; CCNB2, 0.86; RARRES2, 0.86; and Intervention: Real-time quantitative reverse transcrip- tion–polymerase chain reaction (RT-PCR) in 89 adre- SLC16A9, 0.80), indicating high diagnostic accuracy for nocortical tissue samples (11 malignant and 78 be- distinguishing benign from malignant adrenocortical nign). The criteria for differentially expressed genes tumors. between benign and malignant adrenocortical tumors were a false discovery rate of less than 5% and an adjusted Conclusion: We identified 37 genes that are dysregu- PϽ.01. Genes differentially expressed by 8-fold higher lated in adrenocortical carcinoma, and several of the dif- or lower were validated by RT-PCR. ferentially expressed genes have excellent diagnostic ac- curacy for distinguishing benign from malignant Main Outcome Measures: The diagnostic accuracy adrenocortical tumors. of differentially expressed genes as determined by the area under the receiver operating characteristic curve (AUC). Arch Surg. 2008;143(9):841-846 DRENOCORTICAL TUMORS vasion or metastatic disease. However, are common, with a preva- most adrenocortical tumors are local- lence of approximately 4% ized, and there are no clinically reliable cri- in the US population. They teria to distinguish benign from malig- are discovered as a result nant localized adrenocortical tumors. Aof hormonal hypersecretion (Cushing syn- Consequently, sometimes patients are mis- drome, primary hyperaldosteronism, and diagnosed as having benign tumors based other conditions), because of local symp- on histologic examination but later de- Author Affiliations: toms (back or abdominal pain), and, most velop aggressive recurrent disease, even af- Departments of Surgery frequently, on abdominal imaging for ter initial complete resection.5 On the other (Drs Fernandez-Ranvier, Suh, another clinical indication (adrenal hand, many patients with adrenal inci- Duh, Clark, and Kebebew and incidentaloma).1,2 In contrast, adrenocor- dentaloma are subjected to adrenalec- Ms Weng), Epidemiology and tical carcinoma is rare, with an annual in- tomy based on the risk of adrenocortical Biostatistics (Dr Yeh), and cidence of 2 cases per million persons per carcinoma as estimated by tumor size, but Pathology (Dr Khanafshar), year, and it accounts for 0.2% of cancer histologic examination and long-term fol- J. David Gladstone Institutes deaths.3,4 Five-year survival for patients low-up show their tumors to be benign.6 Genomics Core Laboratory with adrenocortical carcinoma varies from Because of the clinical limitations of re- (Dr Barker), and UCSF Helen 3 Diller Family Comprehensive 32% to 45%. liably distinguishing between benign and Cancer Center (Drs Clark and It is relatively easy to distinguish be- malignant adrenocortical tumors, and to Kebebew), University of nign from malignant adrenocortical tu- gain some biological insight into the patho- California, San Francisco. mors when there is gross locoregional in- genesis of adrenocortical carcinoma, sev- (REPRINTED) ARCH SURG/ VOL 143 (NO. 9), SEP 2008 WWW.ARCHSURG.COM 841 ©2008 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 ing to confirm tissue diagnosis and type (adrenal cortex and me- Table 1. Clinical and Pathologic Features of Patients With dulla). Total RNA was extracted from homogenized frozen tis- Benign Adrenocortical Tumors or Adrenocortical Carcinoma sue using TRIzol reagent (Invitrogen, Carlsbad, California) and was purified using the RNeasy Mini Kit (Qiagen, Valencia, Cali- Benign Adrenocortical Adrenocortical fornia). We used 1 µg of total RNA for amplification and label- Tumor Carcinoma ing with a kit (MessageAmp aRNA Kit; Ambion Inc, Foster City, Feature (n=74) (n=11) California). Labeled and fragmented complementary RNA, 12 µg, Sex, No. (%)a was hybridized to a gene chip (Affymetrix Human Genome U133 Male 24 (34) 2 (29) Plus 2.0 GeneChip; Affymetrix Inc, Santa Clara, California) for Female 47 (66) 5 (71) 16 hours at 45°C. The gene chip arrays were stained and washed Male to female ratio 1:1.88 1:2.50 (Affymetrix Fluidics Station 400; Affymetrix Inc), according to Age at initial operation, 49.6 (11.8) [18-72] 49.7 (21.8) [18-77] the manufacturer’s protocol. The probe intensities were mea- mean (SD) [range], y sured using an argon laser confocal scanner (GeneArray Scan- Tumor size, mean 3.2 (2.0-6.5) 9.9 (3.5-20.0) ner; Hewlett-Packard, Palo Alto, California). (range), cm Functioning, No. 54 (24 with Cushing 7 (4 with virilizing syndrome and disease and REAL-TIME QUANTITATIVE RT-PCR 30 with 3 with Cushing hyperaldosteronism) syndrome) Genes differentially expressed in the microarray experiments Nonfunctioning, No. 20 4 were validated by means of real-time quantitative TaqMan (Ap- Follow-up, mean 25.7 (1-120) 25.6 (1-63) plied Biosystems, Foster City) RT-PCR in individual samples. (range), mo The same stock of total RNA used for the gene array experi- ments was used for the real-time quantitative RT-PCR. Total aSome patients had multiple tumor samples. RNA, 125 ng/µL, was reverse transcribed using the RT script complementary DNA synthesis kit (USB Corp, Cleveland, Ohio). eral investigators have used genomewide gene expres- Real-time quantitative PCR was used to measure messenger RNA sion profiling. Some of these studies7-10 have identified expression levels relative to glyceraldehyde-3-phosphate de- hydrogenase messenger RNA expression. The gene expres- candidate diagnostic biomarkers of adrenocortical car- ⌬ ϫ cinoma and molecular profiles that distinguish between sion level is as follows: Ct=−2 (Ct of the gene of interest−Ct of glyceraldehyde-3-phosphate dehydrogenase), where Ct is the benign and malignant adrenocortical tumors. However, PCR cycle threshold. The PCR primers and probes for the genes the specific candidate genes found in these studies have were purchased from Applied Biosystems (Assay-on-Demand been discordant, possibly as a result of different ap- Kit). All the PCR experiments were performed in a final vol- proaches to data analysis. In addition, some studies have ume of 20 µL, with 1 µL of complementary DNA template on not validated microarray data using quantitative meth- a detection system (ABI PRISM 7900 Sequence Detection Sys- ods. Most important, to our knowledge, a formal analy- tem; Applied Biosystems). The PCR thermal cycler condition sis of the diagnostic accuracy of the proposed biomark- was 95°C for 12 minutes, followed by 40 cycles at 95°C for ers of adrenocortical carcinoma has not been performed. 15 seconds and 60°C for 1 minute. We, therefore, analyzed the genomewide expression pro- file of benign vs malignant adrenocortical tumors to iden- DATA ANALYSIS AND STATISTICAL ANALYSIS tify candidate markers. We then validated these mark- ers using real-time quantitative reverse transcription– Raw microarray data were analyzed using the Affy package polymerase chain reaction (RT-PCR) and evaluated their (Affymetrix GeneChip Operating software; Affymetrix Inc) in diagnostic accuracy. the free statistical environment R/Bioconductor to generate an intensity value in log2 scale for each probe set using the ro- bust multiarray average method with default variables.11-13 For METHODS the class comparison (benign vs malignant), we used the limma package in R/Bioconductor to calculate the moderated t statis- TISSUE SAMPLES tics and the associated P values and the log posterior odds ra- tio (B statistic) that a gene is differentially expressed vs not dif- A total of 89 human adrenocortical tissue samples were ob- ferentially expressed.14 The P values were adjusted for multiple tained at surgical resection and were immediately snap frozen testing by controlling for the false discovery rate using the Ben- and stored at −80°C. Patient and tumor characteristics are sum- jamini-Hochberg method.15 marized in Table 1. Each tumor sample was reviewed again To evaluate the accuracy of candidate markers to distin- and was confirmed to be adrenocortical tissue. Approval for this guish benign from malignant adrenocortical tumors, we deter- study was obtained from the Committee on Human Research mined the area under the receiver operating characteristic curve at the University of California, San Francisco. (AUC). A stepwise regression analysis was used to determine Adrenocortical carcinoma was defined when gross local in- the AUC for the combination of markers. vasion or
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