Gene Expression Profiling of Childhood Adrenocortical Tumors

2007;67:600-608. Cancer Res Alina Nico West, Geoffrey A. Neale, Stanley Pounds, et al. TumorsGene Expression Profiling of Childhood Adrenocortical

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Gene Expression Profiling of Childhood Adrenocortical Tumors

Alina Nico West,1Geoffrey A. Neale,

2Stanley Pounds,

4Bonald C. Figueredo,

8

Carlos Rodriguez Galindo,5Mara Albonei D. Pianovski,

9Antonio G. Oliveira Filho,

10

David Malkin,7Enzo Lalli,

11Raul Ribeiro,

3,5and Gerard P. Zambetti

6

1Interdisciplinary Science Program, University of Tennessee Health Science Center; 2Hartwell Center for Biotechnology; 3InternationalOutreach Program; and the Departments of 4Biostatistics, 5Oncology, and 6Biochemistry, St. Jude Children’s Research Hospital,Memphis, Tennessee; 7Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada;8Center for Molecular Genetics and Cancer Research in Children and 9Division of Pediatric Hematology and Oncology,Erasto Gaertner Hospital, and Department of Pediatrics, Universidade Federal do Parana, Curitiba, Brazil; 10Institutode Pesquisa e Ensino Boldrini, Campinas, Brazil; and 11Institut de Pharmacologie Moleculaire et Cellulaire CentreNational de la Recherche Scientifique Unite Mixte de Recherche 6097, Valbonne, France

Abstract

Pediatric adrenocortical tumors (ACT) are rare and often fatalmalignancies; little is known regarding their etiology andbiology. To provide additional insight into the nature of ACT,we determined the gene expression profiles of 24 pediatrictumors ( five adenomas, 18 carcinomas, and one undeter-mined) and seven normal adrenal glands. Distinct patternsof gene expression, validated by quantitative real-time PCRand Western blot analysis, were identified that distinguishnormal adrenal cortex from tumor. Differences in geneexpression were also identified between adrenocortical ade-nomas and carcinomas. In addition, pediatric adrenocorticalcarcinomas were found to share similar patterns of geneexpression when compared with those published for adultACT. This study represents the first microarray analysis ofchildhood ACT. Our findings lay the groundwork for establish-ing gene expression profiles that may aid in the diagnosisand prognosis of pediatric ACT, and in the identification ofsignaling pathways that contribute to this disease. [Cancer Res2007;67(2):600–8]

Introduction

Pediatric adrenocortical tumors (ACT) are rare malignanciesoccurring at a rate of 0.3 to 0.4 annual cases per million childrenunder the age of 18 years (1, 2). Signs and symptoms of ACTinclude virilization, acne, deep voice, facial hair, muscle weakness,facial hyperemia, hypertension, and other signs of Cushingsyndrome. The tumor size and weight, disease staging, and selectedhistologic criteria have been used to classify ACT as eithercarcinoma or adenoma. Large tumors (>200 g), and locally invasiveor metastatic tumors, have been associated with poor outcome.However, in many cases, clinical and pathologic features fail toidentify patients with localized disease that eventually relapse.Current therapy for pediatric ACT primarily relies on surgicalresection of the tumor, although mitotane (a DDT-relatedcompound)—with or without DNA-damaging agents—has beenused with some success (3). The overall 5-year disease-free survival

is 50%; however, patients with stage IV disease have less than a 10%chance of long-term survival (2).

The adrenal cortex synthesizes essential steroids (e.g., glucocor-ticoids, androgens, and mineralocorticoids) that regulate diversebiological processes such as blood pressure, glucose metabolism,immune surveillance, and sexual development (4, 5). Duringgestation, the cortex is subdivided into the outer-definitive andinner-fetal zones, which contribute to the maintenance of normalpregnancy through the production of dihydroepiandrosteronesulfate. As this function is no longer required after birth, theadrenal gland rapidly loses 50% of its volume within the first2 weeks due to massive apoptosis. Subsequently, the adrenal cortexundergoes significant tissue remodeling and develops into threedefined regions: outer zona glomerulosa, middle zona fasciculata,and inner zona reticularis. The zona glomerulosa is primarilyresponsible for the production of aldosterone, whereas the zonafasciculata and zona reticularis produce corticosteroids andandrogens, respectively. Various genetic abnormalities, eitheracquired or inherited (see below), promote ACT developmentduring childhood or late adulthood (6).

Pediatric ACT is frequently reported in families with Li-Fraumenisyndrome and Li-Fraumeni–like syndrome, which are usuallyassociated with TP53 tumor-suppressor germ line mutations(7, 8). The most frequently observed tumors in Li-Fraumenisyndrome include soft tissue sarcomas, osteosarcomas, breastcarcinomas, brain tumors, and adrenocortical carcinomas. Indeed,it has been proposed that pediatric ACT is almost diagnostic of agerm line TP53 mutation (9), but clearly alternative factors cancontribute to this tumor type (e.g., Beckwith-Wiedemannsyndrome, Carney’s complex, and multiple endocrine neoplasiatype I; ref. 10). Beckwith-Wiedemann syndrome is characterizedby the overgrowth of tissues and organs, including the adrenalgland. Beckwith-Wiedemann syndrome is usually sporadic; how-ever, it also occurs as a familial autosomal dominant form linkedto the loss of imprinting at the insulin-like growth factor-II (IGF-II)locus on chromosome 11p15.5, resulting in the overproduction ofIGF-II (6). The underlying genetic events responsible for theBeckwith-Wiedemann syndrome phenotype are complex, withmultiple genes (e.g., KCNQ1 and CDKN1C) being implicated in itsetiology (11, 12).

The cooperating factors and signaling pathways that promotethe development of childhood ACT are not well defined. Animalstudies implicate inhibin-a, a glycoprotein with homology totransforming growth factor-h, as a suppressor of ACT development(13). Deletion of inhibin-a by gene targeting in gonadectomizedmice causes fully penetrant ACT by 4 to 5 weeks of age. Consistentwith the mouse model, mutation of inhibin-a with loss of

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

A.N. West and G.A. Neale contributed equally to this work.Requests for reprints: Gerard P. Zambetti, Department of Biochemistry, St. Jude

Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Phone: 901-495-3429; Fax: 901-525-8025; E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-06-3767

Cancer Res 2007; 67: (2). January 15, 2007 600 www.aacrjournals.org

Research Article



heterozygosity at chromosome 2q33 was commonly observed inhuman pediatric ACT (13). Comparative genomic hybridizationanalysis of pediatric ACT also showed recurrent chromosomalalterations, such as the amplification of chromosome 9q34 (14).Localized within this region is the nuclear orphan receptorsteroidogenic factor-1 (SF1, NR5A1), which is required for normaladrenal gland development. Subsequent studies showed that SF1 isamplified and overexpressed in f90% of pediatric ACT (15, 16).Similarly, both pediatric and adult ACT express elevated levels ofIGF-II (17, 18).

Due to the rarity of pediatric ACT, it becomes necessary toconsolidate resources to maximize efforts in studying this diseasein a comprehensive and thorough manner. We therefore estab-lished an International Pediatric Adrenocortical Tumor Registryand Bank at St. Jude Children’s Research Hospital.12 More than 250subjects have enrolled in the registry component since 1990 (theadrenal tissue bank has been in existence since 2000). To identifykey factors and signaling pathways that may be involved inadrenocortical tumorigenesis, we conducted an Affymetrix geneexpression profiling analysis of pediatric ACT. As we report here,distinct expression signatures have been identified that discrimi-nate between normal adrenal cortex and ACT. In addition, ourretrospective analyses identified profiles that may aid in thedifferential diagnosis of adenoma from carcinoma. Insight into thecell type of origin that gives rise to ACT has also been generated.Our findings provide the basis for identifying signaling pathwaysthat are corrupted during adrenocortical tumorigenesis, with thegoal of establishing new therapeutic targets that could be exploitedin treating this often fatal disease.

Materials and Methods

Institutional review board approval. The institutional review board

(IRB) of St. Jude and the ethics committees of the Hospital de Clinicas of the

Federal University of Parana, Hospital Erasto Gaertner, and the CentroInfantil de Investigacoes Hematologicas Dr. Domingos A. Boldrini approved

the genetic analysis of pediatric normal adrenal cortex and ACT. Informed

consent was obtained for each subject.Total RNA preparation. Tissue samples were classified according to

established histopathologic criteria and verified by two independent

pathologists. Total RNA was isolated from 50 to 100 mg of pediatric ACT

using the RNeasy RNA Midi-Prep kit (Qiagen, Valencia, CA). Tumors wereprepared in a 4jC cold room, sliced into fine pieces using a sterile scalpel,

and homogenized with 18- and 19-gauge needles in lysis Buffer RLT

(Qiagen) containing h-mercaptoethanol. Total RNA was isolated by the

Animal Tissues protocol following the manufacturer’s recommendations.The RNA was resuspended in diethyl pyrocarbonate–treated water,

quantified by UV absorbance at 260/280 nm, and stored at �80jC.cDNA amplification and real-time PCR analysis. cDNA was generated

from 1 Ag total RNA using the iScript cDNA amplification kit according to

the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA). cDNA

was diluted 1:2 using sterile double-distilled water before real-time PCR

analysis. The following genes were amplified by real-time PCR using theiQSybrGreen PCR amplification mix (Bio-Rad Laboratories; according to the

manufacturer’s instructions) and 400 ng per primer: IGF-II, type II 3b-hydroxysteroid dehydrogenase (HSD3B2), fibroblast growth factor receptor-4

(FGFR4), NURR1, NGF1-B , and nephroblastoma overexpressed (NOV ).Ubiquitin was also amplified as a loading control. Each normal adrenal

and tumor sample was amplified in triplicate via separate PCR conditions

and compared with ubiquitin expression levels using the DDC t method (19).

Primer sequences and PCR conditions are described in SupplementaryTable S1.

Western blot analysis. Protein was isolated from normal adrenal cortex

and tumor tissues by homogenization in T-PER lysis buffer (Pierce

Chemical, Rockford, IL) containing a protease inhibitor cocktail (RocheDiagnostics Corporation, Indianapolis, IN). Total protein (50 Ag) was

analyzed by SDS-PAGE using the Novex NuPAGE system (Invitrogen,

Carlsbad, CA). Proteins were separated by electrophoresis and transferred

to 0.45-Am nitrocellulose membranes. Membranes were blocked in TBS-Tbuffer [10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.1% Tween 20]

containing 5% nonfat milk and probed with the following primary

antibodies: goat polyclonal anti-human IGF-II (1:500; Sigma-Aldrich

Chemical, St. Louis, MO), rabbit polyclonal anti-human HSD3B2 (1:500;gift from Dr. C. Richard Parker Jr., University of Alabama, Birmingham,

AL), and mouse monoclonal anti-human actin (1:2000; Sigma-Aldrich

Chemical). Membranes were washed with TBS-T and hybridized with thefollowing horseradish peroxidase–linked antibodies diluted in TBS-T

containing 5% nonfat milk: rabbit anti-goat (1:1,000; Calbiochem, San

Diego, CA), donkey anti-rabbit (1:3,000; Amersham Biosciences, Piscataway,

NJ), and sheep anti-mouse (1:2,000; Amersham Biosciences). The mem-branes were washed with TBS-T and developed using Supersignal West

Dura chemiluminescence reagent (Pierce Chemical) according to the

manufacturer’s protocol.

Microarray analysis. The Affymetrix U133A GeneChip was used tocollect expression data for 22,215 probe sets on each of 31 samples

(18 adrenocortical carcinomas, five adenomas, one undetermined ACT,

and seven normal adrenal cortex). Microarray analysis was done in theHartwell Center Affymetrix core laboratory at St. Jude. High-quality RNA,

confirmed by UV spectrophotometry and an Agilent 2100 Bioanalyzer, was

processed according to the Affymetrix one-cycle labeling protocol.13 In

brief, 5 to 10 Ag total RNA was annealed to an oligo-dT(24)-T7 primer toinitiate cDNA synthesis. Purified double-stranded cDNA was used as a

template to synthesize biotin-labeled cRNA using T7 RNA polymerase.

Labeled cRNA (20 Ag) was fragmented, added to a mixture containing

blocking agents and array controls, and hybridized overnight at 45jC to thegene chip array. Following hybridization, arrays were stringently washed,

stained with streptavidin-conjugated phycoerythrin, and scanned using an

Affymetrix GeneChip Scanner 3000. Relative expression signals for eachgene was calculated using the Affymetrix GCOS software (version 1.4) using

the global normalization method where the 2% trimmed mean signal was

set to a target value of 500.

Statistical analysis. Microarray signals were summarized and normal-ized using Affymetrix GCOX software as described above. No probe set was

excluded before subsequent statistical analysis because filtering has been

found to be of questionable value (20). The Wilcoxon rank-sum test was

used to compare median expression between normal and tumor tissues ineach probe set (21). Likewise, the rank-sum test was used to compare the

median expression level of each probe set between adrenocortical

adenomas and carcinomas. To account for multiple testing in each of

these analyses, we used a robust method to estimate the false discovery rate(22). These analyses were implemented using S-plus software, version 6.2

for Windows (Microsoft).14 The robust false discovery rate method was

implemented using our freely available routines.15

To compare expression profiles in our pediatric ACT samples with data

in other reports (23, 24), U133A probe sets were matched by either Genbank

accession ID (23) or by the Affymetrix ‘‘best match’’ criteria (24). Fold-

change point estimates were computed by exponentiation of the differenceof means of log-transformed signals. This estimate of fold change can be

interpreted as an estimate of the ratio median expression levels of the two

groups. The t distribution was used to compute 95% confidence intervals

for the difference of means of log signals; these intervals were transformed

12 www.stjude.org.

13 http://www.affymetrix.com/support/technical/manual/expression_manual.affx.14 www.splus.com.15 http://www.stjuderesearch.org/depts/biostats/robustfdr/index.html.

Microarray Analysis of Pediatric Adrenocortical Tumors

www.aacrjournals.org 601 Cancer Res 2007; 67: (2). January 15, 2007



into confidence intervals for fold changes by exponentiation. The fold-change confidence intervals are not adjusted for multiple testing.

As measures of how fold changes observed in our study correlated withfold changes observed in other studies, we computed the number of probesets with a directional agreement (i.e., the fold-change estimates from thetwo studies were in the same direction) and Kendall et al.’s method (25)with the two sets of fold changes as input. We used a permutation methodto assess the statistical significance of the observed values of thesemeasures of agreement. The permutation assessment was done bycomputing the fold changes on 1,000 data sets, derived by randomlyreassigning group labels in our data set to the expression profiles in ourdata set, and then computing the agreement statistics. We counted thenumber of permuted data sets in which stronger values of the agreementstatistics were observed to obtain the P values.

Estimates of overall and relapse-free survival were computed usingthe Kaplan-Meier method with SEs determined using the method of Petoet al. (26). Overall survival was defined as the duration from date ofdiagnosis to date of death with those living at last follow-up consideredcensored. Relapse-free survival was defined as the duration from date ofdiagnosis to date of relapse or death with those alive and relapse-free at lastfollow-up censored.

Results

Clinical information. Pediatric adrenocortical adenoma and

carcinoma patients were enrolled on the International Pediatric

Adrenocortical Tumor Registry and Bank protocol. Tumor speci-

mens were harvested during surgery and snap frozen in liquid

nitrogen to preserve tissue integrity. Data have been compiled

for eight males and 15 females between 0 and 16 years of age.

Table 1 summarizes the primary clinical information for each

subject (excluding sample Unk1 with ACT of undetermined

histology), including stage of the disease, tumor class, sex, age,

relapse-free survival, and overall survival. Details regarding clinical

features and treatment were also collected.All subjects presented with virilization. Eleven patients had signs

and symptoms consistent with an increased secretion of gluco-

corticoids (Cushing syndrome) and eight patients were hyperten-

sive at presentation. Normal adrenal glands were obtained with IRB

approval as discarded tissue from seven cases of Wilms’ tumor.

These patients, whose age ranged from 2 to 6 years, had not

Table 1. Clinical data of 24 pediatric adrenocortical cancer patients

Sample Sample ID Sex Age* (y) Tumor ornormal

Histologic type(adenoma or ACC)

Tumor stage Virilization Cushingsyndrome

Outcome Treatment

1 ACC1 M 8 T ACC 4 Y N CR SC2 ACA1 F 12 T Adenoma — Y Y CCR S

3 ACC2 F 5 T ACC 2 Y N DD SC

4 ACA2 F <1 T Adenoma — Y Y CCR S

5 ACC3 M 2 T ACC 3 Y N DD SC6 ACC4 F 2 T ACC 1 Y N CCR S

7 Unk1 Unk Unk T Unk Unk Unk Unk Unk Unk

8 ACC5 M 4 T ACC 2 Y N CR SC9 ACC6 F 13 T ACC 4 Y Y Deceased SC

10 ACA3 F 3 T Adenoma — Y N CCR S

11 ACA4 F 2 T Adenoma — Y Y CCR S

12 ACA5 F 4 T Adenoma — Y N CCR S13 ACC7 F 11 T ACC 2 Y Y CR SC

14 ACC8 M 9 T ACC 2 Y Y DD SC

15 ACC9 M 2 T ACC 1 Y Y DD SC

16 ACC10 F <1 T ACC 2 Y Y CCR S17 ACC11 M 2 T ACC 1 Y N CCR S

18 ACC12 M 12 T ACC 3 Y Y CCR SC

19 ACC13 M 4 T ACC 3 Y Y CR SC

20 ACC14 F 10 T ACC 3 Y N CR SC21 ACC15 F 3 T ACC 1 Y Y CR SC

22 ACC16 F 6 T ACC 2 Y N SC

23 ACC17 F 15 T ACC 4 Y N DD SC24 ACC18 F 3 T ACC 3 Y N CCR SC

25 Nor001 Unk Unk N NA NA NA NA NA NA


27 Nor006 Unk Unk N NA NA NA NA NA NA28 Nor007 Unk Unk N NA NA NA NA NA NA




NOTE: Adenomas were not staged based on standard ACT staging criteria (2).

Abbreviations: Unk, unknown; ACC, adrenocortical carcinoma; NA, not applicable; CR, complete remission; CCR, continuous complete remission; DD,

died from disease; Deceased, died from unknown causes; S, surgical resection only; SC, surgical resection plus chemotherapy.*Ages were rounded to the nearest full year.

Cancer Research




received chemotherapy before surgery, thus avoiding complicationsof chemotherapeutic effects. Normal adrenal cortex was subse-quently isolated by an American Board–certified pathologist andprocessed as described in the Materials and Methods.

Gene expression profiling distinguishes ACT from normaladrenal tissue. Gene expression profiles for the ACT and normaladrenal cortex samples were generated using the Affymetrix U133Agene chip, which recognizes 14,500 genes using 22,215 probe sets.We estimate that at least 33% of the probe sets on the array aredifferentially expressed between tumor and normal tissues; for1,019 of the probe sets, we detected differences that weresignificant at P = 0.001 (see Supplementary Table S2). Furthermore,we estimate that 1.5% or fewer of the 1,019 detected differences arefalse discoveries. Hierarchical clustering analysis was used tovisualize the variability between ACT and normal cortex (Fig. 1).

Validation of the gene expression data set. Among the 1,019significant probe sets, we identified 25 with the greatest and leastratios of median expression in tumor samples to that of normalsamples (Table 2). The median expression of FGFR4 in the ACTsamples was 21 times that of the normal samples (95% confidenceinterval, 11.4–38.8) and represents the highest induced gene withinthe group. Previous studies implicate FGFR4 in breast cancerprogression and other tumors (27).

The median expression of IGF-II in ACT samples was 18 timesthat of the normal samples (95% confidence interval, 7.8–42.7).Overexpression of IGF-II in the ACT samples was subsequentlyverified at the RNA and protein level by quantitative real-time PCR(qRT-PCR) assay and Western blot analysis, respectively (Fig. 2Aand B). The qRT-PCR assay revealed higher expression values in theACT samples than that determined by microarray analysis, mostlikely due to the larger dynamic range of the RT-PCR assay. IGF-IIprotein levels were also significantly higher in the tumors than inthe normal adrenal cortex samples and correlated with changes inmRNA expression. However, the 7.5-kDa mature form of IGF-II wasselectively expressed in the normal adrenal tissue, whereas multipleproforms of IGF-II, including the prominent 20-kDa form of theprotein, were overexpressed in the ACT samples. These results areconcordant with previous biochemical and microarray analyses ofadult and pediatric ACT (18, 24, 28, 29). In addition, the expressionof NOV, a member of the CCN gene family of secretory proteins thatplays a role in cell adhesion, was significantly lower in the ACTsamples compared with normal adrenal tissue (Table 2; data notshown). Loss of NOV expression in the pediatric ACT samples isalso in agreement with those reported in adult ACT studies byMartinerie et al. (30), further corroborating our data set.

Cell origin of pediatric ACT. HSD3B2, a steroidogenic enzymeresponsible for the conversion of pregnenolone to progesterone inthe synthesis of glucocorticoids, mineralocorticoids, and andro-gens, is expressed at programmed times during adrenal develop-ment ( for review, see ref. 31). During late embryogenesis, HSD3B2is preferentially expressed in the adrenocortical definitive zone butnot the fetal zone. After birth, HSD3B2 expression is restrictedlargely to the zona glomerulosa and zona fasciculata. Microarrayanalyses showed that the median expression of HSD3B2 inpediatric ACT samples is f40-fold less than that of normalcontrols. This finding was confirmed by qRT-PCR and Western blotanalysis (Fig. 2C and D).

Moreover, the expression of NURR1 (NR4A2) and NGF1-B(NR4A1), transcriptional regulators of HSD3B2 gene expression(32), were concomitantly lower in the ACT samples (Table 2; anddata not shown). The expression of KCNQ1 , which encodes a

voltage-dependent potassium channel, was also lower (f85-fold)in the pediatric ACT samples than in normal adrenal cortex(Table 2). Murine Kcnq1 is preferentially expressed in the corticalzona glomerulosa (33), but not in the adrenal medulla. Takentogether, these results suggest that pediatric ACT may arise fromeither the fetal zone or the more developmentally mature zonareticularis or zona fasciculata.

Comparison between adult and pediatric ACT. Giordanoet al. (24) recently identified differences in gene expression pat-terns between adult ACT and normal tissue using the Affymetrixhuman U95A gene chip. Independently, Rainey et al. (23) comparedthe gene expression profiles of normal human fetal and adultadrenal cortex using a cDNA microarray approach. To our know-ledge, there have been no published studies to date compar-ing adult and pediatric ACT gene expression in a comprehensivemanner.

To compare expression profiles across studies, we queried ourmicroarray data set for the genes identified as significantly changedin the other two studies. We then used expression values relative tonormal tissues within each study (log2 ratio) to compare geneprofiles across studies. These analyses showed that the mostsignificant differences identified in the comparison between adultadrenal tumors and normal adult adrenal cortex (24) wereremarkably similar to our findings comparing childhood ACT(adenoma and carcinoma) to normal cortex (s = 0.56, P = 0.001;Fig. 3). Moreover, the observed direction of association was the

Figure 1. Heat map and hierarchical clustering analysis comparing pediatricACT and normal cortex. Relative expression signals of 1,019 unique probe sets:red, overexpressed; green, underexpressed. Differentially expressed geneswere significant at P = 0.001. N, normal; ACC, adrenocortical carcinoma;Ad, adenoma; U , unknown. Bar , SD from the mean.





same for 147 of 153 probe sets in our study corresponding to theirreported fold changes (P < 0.001).

Expression of IGF-II and HSD3B2 was dysregulated in a similarmanner in both adult and childhood ACT; however, the degreeof IGF-II expression seems to be greater in the adult tumors(200- versus 20-fold; Supplementary Table S3), possibly due to

the relatively lower levels of IGF-II in the normal adult adrenalcortex (23, 24). There was also a remarkable correlation in geneexpression profiles between normal fetal adrenal tissue (23)and pediatric ACT (s = 0.34, P = 0.022; Fig. 3). Additionally,the direction of association agreed for 99 of 127 probe setscorresponding to genes for which they report fold changes

Table 2. Dysregulated genes in pediatric ACT

Probe Set ID Gene symbol Ratio of medians 95% Low 95% High

Increased

211237_s_at FGFR4 21.1 11.4 38.8

204597_x_at STC1 19.1 8.2 44.4210881_s_at IGF-II 18.3 7.9 42.7

202410_x_at IGF-II 16.0 6.2 41.4

203213_at CDC2 13.8 5.7 33.6219918_s_at ASPM 13.4 4.2 43.5

204285_s_at PMAIP1 13.1 5.9 29.1

213562_s_at SQLE 12.9 6.2 27.1

205345_at BARD1 12.8 8.6 18.9218755_at KIF20A 12.0 5.6 25.7

204056_s_at MVK 10.6 4.1 27.4

218009_s_at PRC1 10.4 4.2 25.9

220091_at SLC2A6 10.2 5.3 19.7207414_s_at PCSK6 10.0 3.7 26.9

218585_s_at RAMP 9.6 3.7 24.6

203828_s_at NK4 9.3 4.2 20.4

204641_at NEK2 8.6 3.3 22.5201292_at TOP2A 8.6 3.2 23.0

201890_at RRM2 8.6 3.0 24.8

201291_s_at TOP2A 8.6 2.5 29.3213126_at MED8 8.2 3.8 18.0

203708_at PDE4B 8.0 3.0 21.4

209218_at SQLE 8.0 4.1 15.3

202779_s_at UBE2S 7.9 2.3 27.5213479_at NPTX2 7.8 4.4 13.9

Decreased

204487_s_at KCNQ1 85.5 49.2 148.6

206294_at HSD3B2 41.1 16.1 104.7204621_s_at NR4A2 26.9 11.5 63.0

214630_at CYP11B2 26.2 11.9 57.9

214321_at NOV 24.4 10.8 55.3209613_s_at ADH1B 21.6 10.9 42.8

216248_s_at NR4A2 19.2 8.5 43.6

204501_at NOV 19.1 9.6 38.2

211959_at IGFBP5 18.2 11.3 29.5203523_at LSP1 17.9 10.5 30.5

208606_s_at WNT4 17.3 8.3 36.1

213764_s_at MFAP5 15.7 4.3 56.6

204622_x_at NR4A2 15.5 7.8 31.1209496_at RARRES2 14.3 6.7 30.2

202768_at FOSB 14.0 4.8 41.2

217767_at C3 13.7 7.2 26.2211217_s_at KCNQ1 13.6 6.4 29.1

202994_s_at FBLN1 13.2 6.9 25.3

203131_at PDGFRA 13.0 7.9 21.6

205969_at AADAC 12.2 5.4 27.8203798_s_at VSNL1 12.0 6.0 23.8

203424_s_at IGFBP5 11.1 6.6 18.5

204457_s_at GAS1 11.0 5.3 22.9

213994_s_at SPON1 10.8 3.9 30.0211896_s_at DCN 10.8 6.2 18.8

Cancer Research




(P = 0.006; Supplementary Table S4). These results indicate thatboth adult and pediatric ACT resemble fetal tissue with respect togene expression patterns. Furthermore, our findings suggest thatadult and childhood ACT may select for common genetic andbiochemical alterations and may be more physiologically relatedthan previously considered.

Differences between pediatric adrenocortical carcinomaand adenoma. There are no definitive tests to predict ACTmalignant potential. Tumor size is one of the most consistentprognostic indicators in children with completely resected ACT (1),although it is not uncommon for patients with small tumors toexperience relapses. We therefore compared gene expression

profiles of ACT that were classified by histologic criteria as eitheradenoma or carcinoma to identify changes that may distinguishbetween these risk groups.

For 52 probe sets, we detected differences in expression betweenadrenocortical adenomas and carcinomas that were significantat the P = 0.001 level (Fig. 4; Supplementary Table S5). We estimatethat 56% or more of the detected differences represent truediscoveries. Among this set was a consistent and marked decreasein the expression of major histocompatibility class II genes.Specifically, the median expressions of HLA-DRB1, HLA-DPB1, HLA-DRA , andHLA-DPA1 mRNA levels were 6- to 8-fold lower in pediatricadrenocortical carcinomas than in adenomas. Similar findings

Figure 2. Dysregulation of IGF-II andHSD3B2 gene expression in pediatricadrenocortical carcinoma. A, IGF-II mRNAlevels are significantly higher in tumorscompared with normal tissue. Bars , SD.B, IGF-II protein is overexpressed inadrenal tumors but is incompletelyprocessed. C, HSD3B2 transcripts aremarkedly lower in adrenal tumors than innormal tissue. Bars , SD. D, HSD3B2protein levels are reduced in tumorscompared with normal tissue.





have been recently reported by Bornstein and coworkers (34, 35) in astudy of adult ACT. HLA-class II expression may therefore serve as amarker for distinguishing between adrenocortical carcinoma andadenoma.

Discussion

We have established the first pediatric ACT gene expressionprofile database. Analysis of the ACT panel revealed a markedincrease in FGFR4 and IGF-II expression, and a sharp decrease inKCNQ1, CDKN1C , and HSD3B2 gene expression in the ACT samplescompared with normal adrenal cortex. In support of these results,qRT-PCR and Western blot analyses confirmed the differentialexpression of several of these factors (Fig. 2; and data not shown).Giordano et al. (24) also detected a similar pattern of IGF-II,KCNQ1 , and CDKN1C expression in adult adrenocortical carci-

nomas. All three of these genes are localized to an imprinted locuson chromosome 11p15, with IGF-II being normally expressed fromthe paternal allele and CDKN1C and KCNQ1 being expressed onlyfrom the maternal allele ( for review, see refs. 11, 12). The coupleddysregulation of IGF-II, CDKN1C , and KCNQ1 (Table 2) implies animprinting defect, similar to what has been observed in Beckwith-Wiedemann syndrome (11, 12).

Overexpression of IGF-II in pediatric ACT was anticipatedbased on previously published reports (Fig. 2A and B ; Table 2;refs. 18, 24, 28, 36). However, the finding that the majority ofthe tumors grossly overexpress immature forms of IGF-II wassurprising, but not unprecedented based on adult ACT studies (29).Pro-IGF-II must be posttranslationally modified by glycosyla-tion and proteolytic cleavage before its mature, active 7.5-kDaform is secreted (37). Here, we have detected, in the ACT samples,IGF-II proteins ranging from 14 to 22 kDa, but not the 7.5-kDaform, which was readily evident in normal adrenal cortex tissue.It is generally considered that the overexpression of IGF-II inACT provides a growth advantage that drives tumorigenesis.Consistent with this hypothesis, transgenic mice engineered toexpress high levels of IGF-II develop adrenal hyperplasia (38) andrecombinant IGF-II stimulates human fetal adrenocortical cellproliferation in culture (39). Because the IGF-I receptor isconcomitantly up-regulated in the pediatric tumors analyzed here(Supplementary Table S2), it is reasonable to speculate that IGF-IImay also play a role in pediatric adrenocortical tumorigenesisand therefore serve as a drug target. However, further consider-ation must be given as to whether these adrenal tumors secretean active form of IGF-II that contributes to the growth and survivalof these cells.

Interestingly, basic FGF-2 (bFGF-2) suppresses the processingof IGF-II in human ACT cells, thereby blocking its secretion,resulting in a marked accumulation of intracellular IGF-II (40).Consistent with the high levels of partially processed IGF-II pro-tein in the adrenal tumors, FGFR1 and FGFR4 , both of whichcan be activated by bFGF-2, were found by microarray analysisto be significantly up-regulated in the ACT samples (Table 2;Supplementary Table S2). Moreover, because bFGF is a potentangiogenic factor and is mitogenic for fetal adrenal cortex cells(41, 42), the inhibition of the FGFR signaling pathway mayrepresent a rational approach in developing new treatments forpediatric ACT. In support of this concept, 17 of the most signifi-cant genes dysregulated in pediatric ACT (Fig. 1; SupplementaryTable S2) function within the mitogen-activated protein kinasepathway, including NRAS , an immediate downstream target ofFGFR signaling.

The finding that the expression of KCNQ1, HSD3B2 , and itscorresponding transcriptional regulators NURR1 and NGF1B ismarkedly lower in pediatric ACT compared with normal adrenalcortex supports the thesis that the tumors originate from eitherthe fetal zone during embryogenesis or the developing zonafasciculata or zona reticularis during the first few years of life.At the very least, the pediatric adrenal tumors share biochemicalcharacteristics of these compartments. Because normal adult tissueis significantly different from the fetal adrenal cortex (23), theremarkable and somewhat unexpected similarity between adultand pediatric ACT implies (Fig. 3; Supplementary Table S3) theexistence of an adrenal stem cell that may become corrupted togive rise to the developing tumor. Alternatively, the tumors,whether adult or pediatric, may undergo dedifferentiation as theydevelop (43).

Figure 3. Comparisons of pediatric ACT gene expression profiles with thoseobserved in adult ACT (top panel ) and fetal adrenal cortex (bottom panel ).

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In the present study, patterns of gene expression have been iden-tified that distinguish adrenocortical carcinomas from adenomas,which is often difficult to assess by standard histopathologicapproaches. Interestingly, two adrenocortical carcinoma cases,which have not yet relapsed, segregated with the adrenocorticaladenoma group (Fig. 4; Supplementary Table S5 and Fig. S1),underscoring the limitations of the histologic criteria to predicttumor malignant potential. Future prospective studies shoulddetermine the usefulness of gene expression analysis in theclassification and prognosis of pediatric ACT.

Significant changes in the expression profiles between adreno-cortical adenomas and carcinomas included the MHC class IIgenes, which are largely restricted to hematopoietic lineages.Interestingly, the adrenocortical reticular zone also expresses MHCclass II antigens after 4 years of age (34, 35). Based on the age of thepatients diagnosed with adrenocortical adenoma, it is reasonableto speculate that the relatively high MHC class II expressionreflects an infiltration of immune cells that limits tumor potential(B.F.; data not shown). Conversely, the association of low MHCclass II expression in the carcinomas may represent a mechanismto evade immune surveillance, which could contribute to itsmalignant phenotype (35).

Little is known regarding the pathways and factors that promotepediatric ACT and there is no proven therapy for this raremalignancy other than surgery. Our findings identify potentiallyimportant components that may contribute to adrenocorticaltumorigenesis. However, the establishment of genetically engi-neered mice, primary tissue culture cell lines, and/or human ACTxenografts will be required to explore new potential targets, such asFGFR4, IGF-II , and other dysregulated genes identified here. Onlythrough these efforts can advancements in the treatment ofpediatric ACT be made.

Acknowledgments

Received 10/12/2006; accepted 11/14/2006.Grant support: NIH/National Cancer Institute grants CA63230 and CA71907 (G.P.

Zambetti), Fondation pour la Recherche Medicale (E. Lalli), L’Association pour laRecherche sur le Cancer (E. Lalli), Cancer Center Core grant CA21765, National CancerInstitute of Canada with funds from the Canadian Cancer Society, and the AmericanLebanese Syrian Associated Charities.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Robert Lorsbach for his assistance in processing tissue samples, theHartwell Center for the Affymetrix U133A GeneChip and biostatistical analyses, Dr. C.Richard Parker, Jr., for his generosity in providing the antihuman HSD3B2 probe, andDr. Donald D. Samulack for his editorial assistance.

Figure 4. Heat map of differentially expressedgenes comparing pediatric adrenocorticalcarcinomas and adenomas. Median expressionvalues calculated by the Wilcoxon rank-sumtest generated data for 52 unique probe setsbetween adenoma and carcinoma significant atP = 0.001. U, unknown. Red, overexpressed;green, underexpressed. Bar , SD from themean.





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Gene Expression Profiling of Childhood Adrenocortical Tumors

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