Microarray analysis reveals differential expression of benign and malignant pheochromocytoma

in Endocrine-Related Cancer
Authors:
Jens Waldmann Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Volker Fendrich Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Julia Holler Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Malte Buchholz Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Ernst Heinmöller Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Peter Langer Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Annette Ramaswamy Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Birgit Samans Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Martin K Walz Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Matthias Rothmund Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Detlef K Bartsch Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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Emily P Slater Department of Surgery, Division of Gastroenterology, Department of Pathology, Department of Pathology, Institute of Molecular Biology and Tumor Research, Department of Surgery, University Giessen, Department of Surgery, Center for Minimally Invasive Surgery, Philipps-Universität Marburg, Baldingerstraße, D-35043 Marburg, Germany

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The diagnosis of a malignant pheochromocytoma (PC) can only be established by the presence of distant metastases, but a subset of apparently benign PCs develop metastases. We have employed a microarray analysis to identify a typical gene expression profile which distinguishes malignant from benign PC. Total RNA was isolated from fresh-frozen tissue of five benign and five malignant PCs. The reference consisted of laser microdissected tissue from normal adrenal medulla. After generating Cy3- and Cy5-fluorescently labeled cDNAs, F-chips containing 11 540 spots were hybridized. Data were analyzed with the IMAGENE 3.0 software. Gene expression levels were validated by real-time (RT)-PCR and immunohistochemistry (IHC). The analysis revealed a more than twofold difference in expression between benign and malignant PCs in 132 genes: 19 were up-regulated and 113 were down-regulated. Expression differences of six genes (calsequestrin, NNAT, neurogranin, secreted protein acidic and rich in cysteine (SPARC), EGR2, and MAOB) were confirmed by RT-PCR in 25 PCs. IHC for calsequestrin revealed an overexpression in malignant PCs (7/10 vs 1/10, P=0.03). Comparative analysis by microarray of all ten PCs (benign/malignant) versus normal adrenal medulla revealed a more than twofold expression difference in 455/539 and 491/671 genes respectively. Several of these genes are known to participate on adrenal tumorigenesis, potential tumor suppressor genes, and oncogenes. Comprehensive gene expression analysis of malignant and benign PCs revealed different gene profiles, which could be used to discriminate between malignant and benign PCs. Based on these findings, the strategy for further follow-up and treatment could be modified accordingly.

Abstract

The diagnosis of a malignant pheochromocytoma (PC) can only be established by the presence of distant metastases, but a subset of apparently benign PCs develop metastases. We have employed a microarray analysis to identify a typical gene expression profile which distinguishes malignant from benign PC. Total RNA was isolated from fresh-frozen tissue of five benign and five malignant PCs. The reference consisted of laser microdissected tissue from normal adrenal medulla. After generating Cy3- and Cy5-fluorescently labeled cDNAs, F-chips containing 11 540 spots were hybridized. Data were analyzed with the IMAGENE 3.0 software. Gene expression levels were validated by real-time (RT)-PCR and immunohistochemistry (IHC). The analysis revealed a more than twofold difference in expression between benign and malignant PCs in 132 genes: 19 were up-regulated and 113 were down-regulated. Expression differences of six genes (calsequestrin, NNAT, neurogranin, secreted protein acidic and rich in cysteine (SPARC), EGR2, and MAOB) were confirmed by RT-PCR in 25 PCs. IHC for calsequestrin revealed an overexpression in malignant PCs (7/10 vs 1/10, P=0.03). Comparative analysis by microarray of all ten PCs (benign/malignant) versus normal adrenal medulla revealed a more than twofold expression difference in 455/539 and 491/671 genes respectively. Several of these genes are known to participate on adrenal tumorigenesis, potential tumor suppressor genes, and oncogenes. Comprehensive gene expression analysis of malignant and benign PCs revealed different gene profiles, which could be used to discriminate between malignant and benign PCs. Based on these findings, the strategy for further follow-up and treatment could be modified accordingly.

Introduction

Pheochromocytomas (PCs) are rare neoplasms of the adrenal medulla, which are derived from chromaffin cells of the neural crest. Extra-adrenal PC or paraganglioma (PG) arise from closely related chromaffin cells in sympathetic or parasympathetic paraganglia. The annual incidence is ∼1/100 000 patient years, and PC causes secondary hypertension in about 1% (Beard et al. 1983, Sinclair et al. 1987, Omura et al. 2004), although a prevalence of 0.05% is reported in autopsy studies (Platts et al. 1995, McNeil et al. 2000, Khorram-Manesh et al. 2004). Sporadic PCs are three times more frequent than the familial form of PC, which is a result of germline mutations affecting cancer susceptibility genes. Germline mutations affect the oncogene RET and the oncosuppressors VHL, SDHB, SDHD, and NF1. These familial PCs represent ∼25% of cases, and are part of the MEN2 syndrome, the PG syndromes 1, 3, and 4, the von Hippel–Lindau disease, and neurofibromatosis (Neumann et al. 2002). In the case of an inherited tumor syndrome, one must consider the higher rate of malignancy (most commonly associated with SDHB mutation), recurrence, and bilateral disease before patients are scheduled for surgery, which emphasizes the need for genetic testing in every patient with PC.

The surgical treatment of unilateral sporadic PC is well established, and laparoscopic adrenalectomy has become safe in the background of an adequate α-blockage. The management of patients with bilateral disease is still unsettled. An indefinite follow-up is suggested by the NIH consensus conference for familial cases, and at least a 10-year follow-up is warranted for sporadic cases as recurrent and malignant diseases occur in about 17% (Pacak et al. 2001, Amar et al. 2005). The major problem regarding the clinical management is the identification of patients with malignant disease before they develop distant metastases (DM) or recurrence as malignant PC is incurable.

The prevalence of malignancy in PC ranges from 2.4 to 26%, depending on the definition of malignancy (Melicow 1977, Proye et al. 1992). Although the study of Proye et al. apparently overestimated the rate of malignancy by defining microscopic venous thrombi and regional invasion as true signs of malignancy. Only 14/91 (15%) patients revealed DM. The highest rate of malignancy is observed in patients with SDHB mutations, and reaches 50% (Gimenez-Roqueplo et al. 2003). However, the histological distinction of benign from malignant PC is not reliable. Malignancy is only proven by the presence of distant or lymph node metastases. Efforts have been made to establish molecular markers for malignancy such as overexpression of HSP90, human telomerase reverse transcriptase, HIF-2α, tenascin, N-cadherin, COX-2, and others. Comparative genomic hybridization experiments in malignant and benign PC revealed losses of 1p, 3q, and 6q, and gains of 9q and 17q. Progression to malignant PC was associated with deletions on 6q and 17p (Dannenberg et al. 2000).

A recent microarray analysis of rat PC compared with normal adrenal medulla revealed numerous differentially expressed genes and ∼60% of these overlap with a human PC database (Elkahloun et al. 2006). Following the study from Giordano who initially assessed gene expression profiles of malignant and benign PC, five other groups have described similar studies (Eisenhofer et al. 2004a,b, Dahia et al. 2005, Brouwers et al. 2006, Thouennon et al. 2007). These clearly identified a set of genes that could be used as multimarkers for malignancy. Over 80% of differentially expressed genes were down-regulated, which led to the hypothesis that the malignant potential is related to a less differentiated expression pattern. Interestingly, Tischler found that PCs in Nf1 knockout mice express genes which are typical for neural progenitor cells (Powers et al. 2007).

Despite these data, the development of malignancy, tumorigenesis, and its underlying molecular basis are poorly understood.

In this study, we compared five benign and five malignant PCs to normal human adrenal medulla by cDNA microarrays to identify differentially expressed genes that discriminate malignant PC from benign PC and to improve our knowledge of underlying molecular mechanisms.

Patients and methods

Patients and tumors

All ten patients with PC included in this study underwent adrenalectomy in the Department of Surgery, University of Marburg between 1998 and 2006. The tumor tissue was frozen in liquid nitrogen 10–20 min after surgical removal, and stored in our collection of fresh-frozen adrenal tissue.

This study was performed according to the local ethics committee guidelines, and all patients gave their informed consent in written form.

For the purpose of this study, we chose five benign PCs and five malignant PCs. We only used sporadic benign PCs where a germline mutation had been excluded by mutation analysis (see below). Malignant PC tumor tissue was only available in five cases. The diagnosis of a malignant PC was based on the presence of DM. Frozen tumor samples were formalin-fixed, embedded in paraffin, and sections were evaluated with hematoxylin and eosin (HE) staining to determine the diagnosis and the neoplastic cellularity. To exclude contamination with normal adrenal medulla or cortical tissue, only tumor samples with a neoplasticity of at least 90% were subjected to further analysis.

In total, we analyzed 36 different primary PC samples: 10 in the microarray analysis, 25 by real-time (RT)-PCR, and 20 by immunohistochemistry (IHC). Adrenal tumors were classified as PCs, and extradrenal tumors were classified as PG.

Normal control

Normal adrenal medulla that served as the normal control consisted of 60 fresh-frozen sections from ten different patients who underwent adrenal surgery for reasons other than PC (five Conn's syndrome and five Cushing's syndrome). They were chosen randomly from our tissue bank that contains over 200 frozen samples of adrenal tumors and normal adrenal tissue. From ten slides of each normal adrenal tissue, we collected the adrenal medulla cells by laser microdissection (LMD) using a P.A.L.M. MicroBeam (Bernried, Germany).

Laser microdissection

Owing to the irregular distribution of adrenal medulla and cortex in an adrenal specimen, we performed a LMD to prevent cortical contamination in the normal medulla control. We stained six frozen sections (10-μm thickness) of each normal adrenal tissue in methylene blue (1:10) in DEPC-H2O under RNAse-free conditions. Staining was followed by LMD with a P.A.L.M. Microlaser System. Tissue fragments were collected in the adhesive lid of a microfuge tube. Immediately following LMD, tissue was removed from the lid and immersed in 100-μl lysis buffer (Macherey Nagel, Düren, Germany; Heinmoller et al. 2003, Niyaz et al. 2005).

RNA extraction

Total RNA from frozen tumor samples and from the LMD normal controls was isolated by homogenization of tissue in lysis buffer and purification by DNAse treatment. RNA was recovered using the RNA Kit NucleoSpin (Machery Nagel) according to the manufacturer. RNA integrity was ensured by RT-PCR of chromogranin A (CgA) and β-actin with the Qiagen one-step RT-PCR Kit by the standard protocol. CgA was used to assure the medullar origin of the normal control, as it is expressed in adrenal medulla and PC, but not expressed in adrenal cortex. Primer sequences for CgA (GenBank accession number: BT006869) and β-actin (Genebank accession M10277) were as follows:

  • CgA: forward 5′-GAGTGGGAGGACTCCAAACG-3′ reverse 5′-CCACTTTCTCCAGCTCTGCC-3′ (Amplicon: 340 bp)

  • β-actin: forward 5′-GATGATGATATCGCCGCGCTCGTCGTC-3′ reverse 5′-GTGCCTCAGGGCAGCGGAACCGCTCA-3′ (Amplicon: 778 bp)

After visualization of the product from the one step RT-PCR, only samples which showed expression of both were included in the further analysis.

Amplification of RNA

Two micrograms of each tumor RNA and 2 μg of the pooled normal adrenal medulla RNA (10×200 ng) were amplified with the Message Amp aRNA Kit. Amino allyl cDNA was synthesized with 2 μg aRNA, and then labeled and purified with the CyScribe Post-Labeling Kit (Amersham Biosciences). The amino allyl-modified cDNA was chemically labeled with CyDye NHS esters. The coupling reactions of amino allyl-modified cDNA were performed separately with Cy3 and Cy5. The labeled probes were purified with Qiagen spin columns and combined before hybridization. Tumors were labeled with Cy3 and normal adrenal medulla with Cy5.

Microarray analysis

The combined labeled samples were hybridized to the cDNA microarray for 16 h at 55 °C and washed at a stringency of 0.1× SSC/0.1% SDS and 0.1× SSC. The microarray contains 11 551 DNA spots from the human cDNA library ‘Human Sequence-Verified cDNA UniGene Gene Sets gf200, gf201u, and gf202’ (Invitrogen). Each experiment was performed as a sandwich hybridization using two arrays. cDNA microarrays were analyzed using Scan Array Express and Software from Perkin Elmer (Waltham, MA, USA).

To account for spot differences, the background corrected ratio of the two channels was calculated and then log2-transformed. Raw data were standardized to balance intensities to both dyes and to achieve comparable expression levels across experiments. A spatial and intensity-dependent standardization (Yang et al. 2002) was used to correct for inherent bias on each chip (the lowest scatter plot). The chips also contain standardized controls, which were employed to normalize and thus control for bias selected differences.

Mean log ratios were calculated from replicates, as each spot was measured twice due to the sandwich hybridization. Spots were excluded if replicates differed more than the maximum of threefold and 75% of the calculated average log ratio, or if the background intensity was higher than the signal intensity.

Differentially expressed genes were selected based on an absolute value of the t-statistic of 1.96 and a fold-change difference of at least 2. Prior to the cluster analysis, the expression profile of each gene was centered by subtracting the mean observed value. We performed an average linkage hierarchic clustering for genes as well as for chips with the Euclidean distance metric as implemented in the program Genesis (Sturn et al. 2002). Expression data and gene annotations were stored in Array Express (http://www.ebi.ac.uk/arrayexpress/) (accession number E-MEXP-2137), which complies with minimal information about a microarray experiment (MIAME) guidelines.

RT-PCR

The mRNA was reverse transcribed into cDNA with oligo-dT primers using the Superscript 1st Strand System for RT-PCR (Invitrogen) at 42 °C for 50 min. All PCRs were carried out on a 7500 RT-PCR System (Applied Biosystems, Foster City, CA, USA) over 40 cycles, with denaturation for 15 s at 95 °C and combined annealing/extension at 60 °C for 1 min. Following an activation step at 95 °C for 10 min, determination of early growth response factor 1 and 2 (EGR 1 and 2), calsequestrin, monoaminooxidase B (MAOB), SPOCK 2, neurogranin, and neuronatin (NNAT) mRNA expression was performed over 40 cycles with 15 s of denaturation at 95 °C and annealing/extension/data acquisition at 60 °C for 60 s using the Power SYBR Green PCR kit (Applied Biosystems). Primer sequences were as follows:

  • EGR1: forward 5′-TGGGCAAGAACACCATGATG-3′ reverse 5′-AGTTTCTCCAGAGCTGGGTTGT-3′

  • EGR2: forward 5′-CCCTAAAATGGTGAATCAGAGCAT-3′ reverse 5′-CGTTCCTCCCATCACATTGC-3′

  • Calsequestrin: forward 5′-CTGCTAGTGAGCCTTCCCATTT-3′ reverse 5′-TATTGTTGCCCTGGCCAATT-3′

  • MAOB: forward 5′-GAGCCACAATAAGCCACTGGTAT-3′ reverse 5′-AGGGAGTGAAGGAGGATAATTGG-3′

  • SPOCK2: forward 5′-TGGATTTCCGTCTGTAGATTTAACTG-3′ reverse 5′-GGATGACTCACGGGACTCTTG-3′

  • NNAT: forward 5′-AGCGGATCTCGGCAAACC-3′ reverse 5′-AGCCGATGATGAGCAGTTCAG-3′

Relative fold mRNA expression levels were determined using the 2**(−ΔΔCt) method (Livak & Schmittgen 2001) with ribosomal protein, large, P0 (RPLPO) as housekeeping control. RPLP0 primer sequences were as follows: forward 5′-TGGGCAAGAACACCATGATG-3′ and reverse 5′-AGTTTCTCCAGAGCTGGGTTGT-3′.

Immunohistochemistry

For immunostaining, formalin-fixed and paraffin embedded archival tumor samples and corresponding normal tissues were stained as previously described (Waldmann et al. 2008). Briefly, slides from archived ACCs were heated to 60 °C for 1 h, deparaffinized using xylene, and hydrated by a graded series of ethanol washes. Antigen retrieval was accomplished by microwave heating in 10 mM sodium citrate buffer, pH 6.0 for 10 min. For IHC, endogenous peroxidase activity was quenched by a 10-min incubation in 3% H2O2. Nonspecific binding was blocked with 10% serum for 1 h. Sections were then probed with a primary antibody against calsequestrin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a working dilution of 1:100 overnight at 4 °C. For IHC, bound antibodies were detected using the avidin–biotin complex (ABC) peroxidase method (ABC Elite Kit, Vector Labs, Burlingame, CA, USA). Final staining was developed with the Sigma FAST DAB peroxidase substrate kit (Sigma). As a positive control, we used murine heart tissue with every batch, as suggested by the manufacturer.

Statistical analysis

For analyzing proportions, the Mann–Whitney U test was applied. In addition, a discriminant analysis (leave-one-out) was performed to estimate the diagnostic value of RT data. P values <0.05 were considered to be statistically significant. Data were analyzed using SPSS (Chicago, IL, USA), version 14.0 for Microsoft Windows.

Results

Patients

The clinical data of five patients with benign PC and five patients with malignant PC (with DM) are shown in Table 1. For all benign PCs, a hereditary origin was excluded by prior DHPLC analyses of the RET, VHL, SDHB, and SDHD genes.

Table 1

Clinical data of PC which were used for microarray, RT-PCR, and IHC

Patient IDHistologyAge (years)Size (mm)SyndromeKi 67 (%)DM
P1Malignant PC4450None1y LN
P2Malignant PC4295None1y LN
P3Malignant PC65130None5y LN
P4Malignant PC7850None3y LN
P5Malignant PC3020MEN2B1y LN
P6Benign PC5435NoneNDn
P7Benign PC2033None5n
P8Benign PC4795None1n
P9Benign PC5350None1n
P10Benign PC3545None1n
P11Benign PC2135MEN2A<1n
P12Benign PC7160None<1n
P13Benign PC4875None<1n
P14Benign PC4840None1n
P15Benign PC6060None<1n
P16Benign PC4140None1n
P17Benign PC41100None2n
P18Benign PC4640MEN2A<1n
P19Malignant PG3330None10y LG/LN
P20Benign PC4380NoneNDn
P21Benign PC3840MEN2A<1n
P22Benign PC4245MEN2A<1n
P23Benign PC4360NoneNDn
P24Benign PC5570None<1n
P25Benign PG4740None1n
P26Benign PC3470None<1n
P27Benign PC7840None<1n
P28Benign PC6275None<1n
P29Benign PC4830None<1n
P30Benign PC4635None<1n
P31Malignant PG39130PGS (SDHB)5y LM
P32Benign PC4795None<1n
P33Malignant PC2045PGS (SDHB)3y BN, P
P34Malignant PC5775None<1y BN
P35Malignant PC8230None1y LM
P36Malignant PG3720PGS (SDHD)15y HM

DM, distant metastases; PC, pheochromocytoma; PG, paraganglioma; n, no; y, yes; PGS, paraganglioma syndrome; LM, liver metastases; LN, lymph node metastases; LG, lung metastases; BN, bone metastases; P, peritoneum; HM, heart metastases; RT, real-time PCR; IHC, immunohistochemistry. For microarray n=10: P1–P10; for RT n=25: P1–P25; and for IHC n=20: P1, P3–5, P19, P31, P33–36 versus P9, P12, P15, P24, P26–30, P32.

We conducted a gene expression profile comparing five benign and five malignant PCs to normal adrenal medulla. After background subtraction and data normalization, the ratios were averaged for all tumors.

Differentially expressed genes in benign and malignant PC compared with normal adrenal medulla

In the analysis, we found 455 spots in benign PC and 539 spots in malignant PC that were up-regulated more than twofold, which represent 344 and 389 unique genes respectively. A similar number of cDNA probes were down-regulated in benign (491) and malignant (674) PCs, which correspond to 411 and 572 unique genes respectively.

Among these, we identified several genes which are known to play a role in the tumorigenesis of adrenal tumors (e.g. IGF2, RGS, CDK 5, and ubiquitin).

One of the main goals of microarray analysis is to identify potential tumor suppressor genes (TSGs), which led us to focus on clearly down-regulated genes. Interestingly, MGST1 and GSTA 1,3,4 were down-regulated 62/52- and 131/84-fold in both malignant/benign PC respectively. Other potential TSGs were NPY1R, MAX, IGFB6, and Ezrin.

Genes up-regulated more than twofold and which potentially promote tumorigenesis were identified in malignant and benign PCs, and are enlisted in Table 2. One of the most interesting candidates was small ubiquitin-like modifier (SUMO1), which is known to be involved in the by SUMOylation of several TSGs and transcription factors, such as p53, c-jun, and NF-κB.

Table 2

Gene sets which show different expressions in both benign and malignant pheochromocytoma (PC) compared with normal adrenal medulla

Gene symbolsGene title/definitionGene IDChromosomal locationFold changeDirection
MGST1Microsomal glutathione S-transferase 1425712p12.3-p12.161.98/131.73Down
GSTA3Glutathione S-transferase α329406p12.152.33/83.61Down
GPIGlucose phosphate isomerase282119q13.124.69/55.37Down
MAXMYC associated factor X414914q2318.72/27.15Down
FDX1Ferredoxin 1223011q2217.39/27.15Down
NPY1RNeuropeptide Y receptor Y148864q31.3-q32 14.89/25.70Down
NUPR1Nuclear protein tranascriptional regul. 12647116p11.212.07/15.92Down
MAOAMonoamine oxidase A4128Xp11.35.05/13.48Down
LAMA2Laminin, α239086q22-q237.69/9.00Down
MT1MMetallothionein 1M449916q136.87/10.20Down
GSTA4Glutathione S-transferase α429416p12.16.31/8.74Down
IGFBP6IGF-binding protein 6348912q137.05/8.30Down
VIL2Villin 2 (ezrin)74306q25.2-q266.32/7.40Down
GSTT1Glutathione S-transferase θ 1295222q11.238.22/6.99Down
DNASE1L3Deoxyribonuclease I-like 317763p21.1-p14.37.45/6.34Down
NGFRNerve growth factor receptor480417q21-q223.55/5.08Down
EGFREpidermal growth factor receptor19567p123.17/4.51Down
SP100SP100 nuclear antigen66722q37.12.40/4.15Down
CTNNB1Catenin (cadherin-associated protein)14993p213.25/3.30Down
SUMO1SMT3 suppressor of mif two 3 homolog 173412q3314.31/9.99Up
IGF2IGF2 (somatomedin A)348111p15.512.90/9.65Up
RGS5Regulator of G-protein signaling 584901q23.16.45/4.52Up
CDK5R2Cyclin-dependent kinase 5, regulatory subunit 2 (p39)89419q334.63/3.81Up
UBE2D3Ubiquitin-conjugating enzyme E2D 3 (UBC4/5 homolog, yeast)73234q243.86/3.58Up
MUM1Melanoma associated antigen (mutated) 18493919p13.33.74/4.12Up
IGFBPL1IGF-binding protein-like 13472529p13.13.70/3.58Up
CSNK1G3Casein kinase 1, γ314565q232.72/3.20Up
MARK3MAP/microtubule affinity-regulating kinase 3414014q32.32.90/3.13Up
TRAF5TNF receptor-associated factor 571881q323.65/3.01Up
CDC42BPACDC42-binding protein kinase α (DMPK-like)84761q42.113.24/4.69Up

The direction of expression difference ‘Down’ and ‘Up’ indicates underexpression (Down) and overexpression (Up). The first number in the ‘Fold-change’ column represents malignant PC, and the second number represents benign PC. Abbreviations and full names of genes are listed as well as the chromosomal location and gene ID (NCBI online database).

Differentially expressed genes in malignant compared with benign PC

Nineteen spots were detected to be up-regulated, and 113 spots to be down-regulated with at least a twofold difference at the mRNA level in malignant PC compared with benign PC. This represents 15 and 102 unique genes. A selection of these genes is shown in Table 3.

Table 3

Gene sets which distinguish between malignant and benign pheochromocytoma (PC) identified by microarray analysis

Gene symbolsGene title/definitionGene IDChromosomal locationFold changeDirectionComparison (Brouwers et al.)
FOSBFBJ murine osteosarcoma viral oncogene homolog B235419q13.3214.36DownNS
NRGNNeurogranin (protein kinase C substrate, RC3)490011q249.63DownNS
ARCActivity-regulated cytoskeleton-associated protein232378q24.37.21DownND
QPCTGlutaminyl-peptide cyclotransferase (glutaminyl cyclase)257972p22.26.01DownS
CCL4L2Chemokine (C-C motif) ligand 4-like 238837217q126.35DownS
EGR2Early growth response 2 (Krox-20 homolog, Drosophila)195910q21.15.56DownS
RGS1Regulator of G-protein signaling 159961q315.48DownS
PLATPlasminogen activator, tissue53278p124.84DownNS
MAO BMonoamine oxidase B4129Xp11.234.95DownNS
PLAURPlasminogen activator, urokinase receptor532919q134.60DownNS
SPOCK2Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 2980610pter-q25.34.42DownS
GLDNGliomedin34203515q21.24.17DownND
CCL2Chemokine (C-C motif) ligand 2634717q11.2-q124.38DownS
EGR1Early growth response 119585q31.14.05DownS
NNATNeuronatin482620q11.2-q123.56DownNS
CD163L1CD163 molecule-like 128331612p13.33.49DownS
PCDH20Protocadherin 206488113q213.31DownS
H19H19, imprinted maternally expressed untranslated mRNA28312011p15.53.15DownS
FABP5Fatty acid-binding protein 5 (psoriasis-associated), E-FABP3443328q21.133.15DownNS
CITED2Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2103706q23.33.04DownNS
JUNBJun B proto-oncogene372619p13.23.03DownS
DUSP5Dual specificity phosphatase 5184710q252.85DownS
SERPING1Serpin peptidase inhibitor, clade G (C1 inhibitor), member 171011q12-q13.12.82DownNS
MAOAMonoamine oxidase A4128p11.32.67DownNS
BIN1Bridging integrator 12742q12.40DownS
AIFM1Apoptosis-inducing factor, mitochondrion-associated, 19131Xq25-q262.38DownNS
CDH2Cadherin 2, type 1, N-cadherin (neuronal)100018q11.22.22DownS
DKK3Dickkopf homolog 3 (Xenopus laevis)2712211p15.22.12DownNS
CDC26Cell division cycle 26 homolog (Saccharomyces cerevisiae)2461849q322.11DownS
FOSV-fos FBJ murine osteosarcoma viral oncogene homolog235314q24.32.11DownS
IGFBP2IGF-binding protein34852q33-q342.11DownND
CTSOCathepsin O15194q31-q322.09DownST
SEZ6Seizure-related 6 homolog (mouse)12492517q11.25.57UpNS
CASQ2Calsequestrin 2 (cardiac muscle)8451p13.3-p113.63UpNS
STXBP6Syntaxin-binding protein 6 (amisyn)2909114q122.91UpNS
RNF32Ring finger protein 321405457q362.47UpS
MAN1A2Mannosidase, α, class 1A, member 2109051p132.27UpNS
DTWD1DTW domain containing 15698615.q21.22.22UpST
MINAMYC-induced nuclear antigen848643q11.22.10UpS

The direction of expression difference ‘Down’ and ‘Up’ indicates underexpression (Down) and overexpression (Up). Abbreviations and full names of genes are listed as well as chromosomal location and gene ID (NCBI online database). S, significant; NS, not significant; ST, statistical trend (P<0.1).

Unsupervised hierarchical cluster

Transcriptional profiles distinguished between malignant and benign PCs and identified several differentially expressed transcripts as demonstrated by cluster analysis (as shown in Fig. 1). The majority of the differentially expressed genes were down-regulated.

Figure 1
Figure 1

Supervised hierarchical cluster in malignant and benign pheochromocytomas. On the left, malignant PCs (P1, P2, P7, P9, and P10) and on the right, benign PCs (P3, P4, P5, P6, and P8) are shown. Expression differences are indicated by the color (red, overexpressed (threefold difference); green, underexpressed (threefold difference) compared with the average expression of all samples). Genes that revealed a differential expression in malignant and benign PC by the microarray analysis are listed in the right margin. Full colour version of this figure available via http://dx.doi.org/10.1677/ERC-09-0118.

Citation: Endocrine-Related Cancer 17, 3; 10.1677/ERC-09-0118

Analysis of datatset with gene set enrichment analysis (GSEA), DAVID, and EASE

We identified 50 gene sets, which discriminate between malignant and benign PC with a P value <0.01. If we reset the significance level to a P value of 0.05, the number of gene sets increased to 135. A core enrichment was shown for eight gene sets: ZNF198, UBE2N, CHR17P11, BRACX UP, CSNK1A1, WNT pathway, and SUFU. Related to the Wnt pathway, TSGs (CREBBP), kinases (GSK3β, NLK, and MAP3K7), oncogenes (CTNNB1, MYC, and CCND1), and transcription factors (TCF1, HDAC1, and PPARD) were shown to be significantly overexpressed in malignant PC.

Validation of microarray analysis by RT-PCR

Some of the genes which were differentially expressed in malignant and benign PC were subjected to further analysis to ensure the validity of the microarray. Expression levels of calsequestrin, neurogranin, NNAT, SPOCK2, EGR2, and MAOB were confirmed by RT-PCR in 6 malignant and 19 benign PCs (P1–P25) as shown in Fig. 2. ERCC1, EGR1, and MAOA did not reveal statistically different levels of expression in malignant and benign PC. P values were calculated by the Mann–Whitney test, and are shown in Fig. 2. The discriminant analysis (leave-one-out) showed that 78% of cross-validated grouped cases were classified correctly.

Figure 2
Figure 2

Comparative quantification of calsequestrin (CASQ2), neurogranin (NGRN), Sparc, EGR2, MAOB, and Sez6 in 25 PCs (6 malignant/19 benign) by RT. The relative expression levels of the above mentioned genes are shown as whisker plots. The left bar of each diagram stands for benign PC, and the right bar stands for malignant PC. The statistical significance (t-test) is included in each diagram.

Citation: Endocrine-Related Cancer 17, 3; 10.1677/ERC-09-0118

Comparison with the study of Brouwers et al.

A comparison of the gene lists from both microarrays revealed a total of 4709/15233 (30.9%) genes that matched. Brouwers et al. found a total of 1126 genes that were up-regulated, and 1633 that were down-regulated in all assessed malignant PC compared with benign PC. In primary metastatic/malignant PC, Brouwers et al. found a total of 1263 genes that were up-regulated, and 1808 genes that were down-regulated versus benign PC. The overall conformance of Brouwers et al. and the present microarray analysis is illustrated by a Venn diagram in Fig. 3. The group of all malignant PCs is referred as Brouwers_1 in Fig. 3 (yellow), while the primary malignant PCs are referred as Brouwers_4 in Fig. 3 (purple). This takes into account that a subgroup of PC patients present with DM at diagnosis (Brouwers_4), while others develop metastases in further follow-up (these are included in Brouwers_1). The meaning of the color code is explained in detail in the legend of Fig. 3. Details on the comparison with Brouwers data for distinct genes are given in Table 3, which either shows confirmation (S, significant) or difference (NS, not significant) of the two datasets. Eighteen of the 39 genes (present on both chips) were significantly differentially expressed in both analyses (see also Table 3). Interestingly, 5/11 down-regulated genes that overlapped are cation binders (secreted protein acidic and rich in cysteine (SPARC), SP100 nuclear antigen, vav2 oncogene, EMR3, RCHY1), and 5/11 genes are known to play role in the cell cycle (SPARC, SP100 nuclear antigen, vav2 oncogene, NFARC1, p78). It has to be kept in mind that only 704/1962 (35.9%) of up-regulated genes and 886/2691 (32%) of down-regulated genes of the total NIH dataset (number in parentheses) were found on the chip used for the present study.

Figure 3
Figure 3

Venn diagram with the overlap between the microarray of the present study and the NIH dataset (Brouwers et al. 2006). On the left diagram underexpressed genes in malignant PC, and on the right overexpressed genes in malignant PC are shown. Brouwers 1 and 4 represent all malignant tumors (1) and all primary malignant tumors (4). Slater represents data of the present study. Colors (yellow, purple, and turquoise) are assigned to genes, which are underexpressed or overexpressed without an intersection between the three groups (Slater, Brouwers_1 and 4). Overlapping genes are indicated by the colors orange, green, red, and blue. The number of genes which are assessed by both chips (Slater/Brouwers) are shown without parentheses. If genes were only on the array of Brouwers et al., number is given in parentheses. Full colour version of this figure available via http://dx.doi.org/10.1677/ERC-09-0118.

Citation: Endocrine-Related Cancer 17, 3; 10.1677/ERC-09-0118

IHC for calsequestrin

IHC revealed an increased expression of calsequestrin at the protein level in ten malignant PCs (P1, P3–5, P19, P31, and P33–36) compared with ten benign PCs (P9, P12, P15, P24, P26–30, and P32). Whereas only one of the ten benign PCs showed strong calsequestrin expression, seven of ten malignant PCs did (P=0.01). Clinical data are summarized in Tables 1 and 3. Positive staining was localized to the cytoplasm as in the positive control (murine heart muscle). Figure 4 shows the typical expression difference in a benign and a malignant PC. Sustenacular cells stained positive in malignant and benign PC, and were used as internal control.

Figure 4
Figure 4

Immunostaining for calsequestrin in two malignant and two benign PCs. Immunostaining for calsequestrin in two different beingn and malignant PCs. Note the intense staining (brown) in malignant PC compared with benign PC. Nonneoplastic sustentacular cells were stained in both malignant and benign PCs. Full colour version of this figure available via http://dx.doi.org/10.1677/ERC-09-0118.

Citation: Endocrine-Related Cancer 17, 3; 10.1677/ERC-09-0118

Discussion

Malignancy in PC can only be established accurately if DM are present. Neither histology nor clinical patterns can distinguish between patients with truly benign PC and patients who will develop DM in further follow-up. DM in patients with apparently benign PC occur in up to 7% of cases (Khorram-Manesh et al. 2005). The treatment of metastatic PC is an unresolved problem as response to chemotherapy, radiotherapy, and I131-MIBG is very limited and results in 5-year survival rate of ∼40% (Remine et al. 1974, Safford et al. 2003, Khorram-Manesh et al. 2005). However, long-term survival of more than 20 years has been reported, reflecting the different biological subtypes of malignant PCs (Jarvelainen & Viikari 2001, Yoshida et al. 2001).

Gene expression profiling is an attractive approach to obtain detailed insights into the biology of PC. It offers a possibility to identify both molecular markers for malignancy and potential targets for novel therapies. Therefore, several studies have assessed malignant and benign PC by gene expression profiling (Anouar et al. 2006, Brouwers et al. 2006, Thouennon et al. 2007).

In our study, we identified a set of 130 genes, which could distinguish between malignant and benign PC. To validate our results, we confirmed the differential expression by RT-PCR, and we increased the sample size to 29 PCs, including the ten PCs, which were subjected to the microarray analysis.

In addition, an unsupervised clustering clearly could discriminate malignant from benign PC while blinded to clinical characteristics. This indicates that gene expression profiling may serve as an approach to classify PC and to evaluate prognosis. Based on expression data, patients can be subjected to closer follow-up or adjuvant therapy in the future.

Our study shows that the majority of genes were under-expressed in malignant PC (119/132), which is in the line with previous studies (Anouar et al. 2006, Brouwers et al. 2006, Thouennon et al. 2007). Interestingly, several of these genes encode neuroendocrine factors (neurogranin and NNAT), enzymes involved in catecholamine metabolism (MAOA) and peptide processing (QPCT, SERPING1). The decreased expression may reflect a loss of differentiation in malignant PC, which possibly arises from immature neuroendocrine precursor cells. According to Nakamura & Kaelin (2006), neural crest precursor cells fail to undergo apoptosis during embryonic development. This is supported by the findings of Powers et al. (2007) who reported a neural progenitor profile in NF1 knock out mice PCs.

With SPARC, protocadherin 20 (PCDH20), and N-cadherin, we identified matricellular proteins that are known regulators of metastasis in the context of epithelial-to-mesenchymal transition (EMT). SPARC was down-regulated in malignant PC, which is consistent with its role in the inhibition of angiogenesis and tumor growth in neuroblastomas (Chlenski et al. 2002, 2006). Powers found SPARC to be up-regulated in PC compared with normal adrenal medulla (11.1-fold) as did we in our study (3.9-fold, data not shown; Powers et al. 2007). N-cadherin was also found to be down-regulated in a neuroblastoma cell line, and is a target gene of RASSF1A (Agathanggelou et al. 2003). PCDH20 belongs to a subfamily of the cadherin superfamily, displays features of a TSG, and is found to be down-regulated in small cell lung cancer (Imoto et al. 2006). Also, an activation of the WNT pathway might indicate a role of EMT in the malignant transformation of PC, as activation of the WNT/β-catenin pathway is known to provoke EMT in colorectal cancer (Brabletz et al. 2005).

Seizure-related 6 homolog (SEZ6) was one of the few up-regulated genes in malignant PC, and is reported to serve as a prognostic marker for lung cancer (Ishikawa et al. 2006). Calsequestrin was also up-regulated in malignant PC, as confirmed by RT and IHC (Fig. 4). It is normally expressed in heart muscle, and plays role in subcellular calcium storage. The function in the context of PC is unknown, but one could speculate that an impaired calcium homeostasis might influence intracellular signaling.

Two candidates for TSGs are BIN1 and NNAT, which are both under-expressed in malignant PC. NNAT is overexpressed in neuroblastoma with a favorable diagnosis, while BIN1 expression is reported to be reduced in neuroblastomas with unfavorable biological features (Tajiri et al. 2003, Higashi et al. 2007). Interestingly, we found SUMO, a small-ubiquitin-like modifier, to be up-regulated in benign and malignant PC. SUMO controls diverse cellular functions of protein targets including transcription factors and other regulators that maintain cellular homeostasis. Additionally, SUMO plays important roles in genomic stability and DNA repair (Kim & Baek 2006).

Chromosomal losses of 6q and 17p are frequently observed in malignant PC (Dannenberg et al. 2000). Focusing on chromosomal locations of genes that showed decreased expression in malignant PC, we identified CITED 2 and phosphatase and actin regulator 2 (PHACTR2) on chromosome 6q, but none on chromosome 17p.

A limitation of this study is the fact that some of the apparently benign PCs that were used for the microarray may turn out to be malignant in further follow-up. As reported in the literature, DM can develop 20 years after surgery (Tanaka et al. 1993), and only lifelong follow-up can assure that the PC is truly benign. Another limitation is the small sample number, which underlines the ultimate need for multicenter studies. To reevaluate our data, we compared it with the data of Brouwers et al. (2006). Thirty-nine of 191 (20%) down-regulated genes were also decreased expressed in Brouwers analysis (see Fig. 3), while only 4/55 (7%) of up-regulated genes were confirmed by Brouwers. The limited comparibility (different array platforms) must be carefully considered when interpretate these results. Only ∼30% of genes assessed by our array platform were also found on the array chip used by Brouwers et al. Different array platforms (affymetrix versus agilant) were applied, and therefore, their numeric results could not be directly combined to select significant markers. The resulting gene sets were therefore generated by simple intersection on their Unigene symbol annotation. At least, the normal adrenal medulla was obtained from patients who underwent adrenalctomy due to adrenocortical disease (primary hypercortisolism/primary hyperaldosteronism). Thus, effects of cortisol on gene expression in the adrenal medulla have only been reported so far for tyrosinhydroxylase and catecholamin synthesizing enzymes, this may have influenced our data (Kvetnansky et al. 1995). Under different circumstances, where adrenal medulla could be obtained (Organ donor, cancer-associated nephrectomy), changes in gene expression appear to be likely, as even histological have been described (Gelfman 1964, Cuschieri 1969, Masterson & Mostafa 1998).

Future studies should validate marker genes and elaborate pathways by functional studies. The development of tissue microarrays with paraffin-embedded PC samples would provide an instrument for testing different prognostic markers when clinical data on follow-up, especially the development of DM, is available.

In conclusion, we performed a microarray analysis to discriminate between malignant and benign PC and identified a set of genes that clearly classify PC. This study offers a useful database for further elaboration of prognostic markers in PCs, and provides insights into the biology of this disease.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.

Acknowledgements

We thank Brunhilde Chaloupka for excellent technical assistance and Michael Krause for assistance with the F-chip. In addition, we thank Prof Pacak and Dr Brouwers from the NIH who made their microarray data available to us. Florian Finkernagel helped us with the comparative statistical analysis of the NIH dataset.

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  • Supervised hierarchical cluster in malignant and benign pheochromocytomas. On the left, malignant PCs (P1, P2, P7, P9, and P10) and on the right, benign PCs (P3, P4, P5, P6, and P8) are shown. Expression differences are indicated by the color (red, overexpressed (threefold difference); green, underexpressed (threefold difference) compared with the average expression of all samples). Genes that revealed a differential expression in malignant and benign PC by the microarray analysis are listed in the right margin. Full colour version of this figure available via http://dx.doi.org/10.1677/ERC-09-0118.

  • Comparative quantification of calsequestrin (CASQ2), neurogranin (NGRN), Sparc, EGR2, MAOB, and Sez6 in 25 PCs (6 malignant/19 benign) by RT. The relative expression levels of the above mentioned genes are shown as whisker plots. The left bar of each diagram stands for benign PC, and the right bar stands for malignant PC. The statistical significance (t-test) is included in each diagram.

  • Venn diagram with the overlap between the microarray of the present study and the NIH dataset (Brouwers et al. 2006). On the left diagram underexpressed genes in malignant PC, and on the right overexpressed genes in malignant PC are shown. Brouwers 1 and 4 represent all malignant tumors (1) and all primary malignant tumors (4). Slater represents data of the present study. Colors (yellow, purple, and turquoise) are assigned to genes, which are underexpressed or overexpressed without an intersection between the three groups (Slater, Brouwers_1 and 4). Overlapping genes are indicated by the colors orange, green, red, and blue. The number of genes which are assessed by both chips (Slater/Brouwers) are shown without parentheses. If genes were only on the array of Brouwers et al., number is given in parentheses. Full colour version of this figure available via http://dx.doi.org/10.1677/ERC-09-0118.

  • Immunostaining for calsequestrin in two malignant and two benign PCs. Immunostaining for calsequestrin in two different beingn and malignant PCs. Note the intense staining (brown) in malignant PC compared with benign PC. Nonneoplastic sustentacular cells were stained in both malignant and benign PCs. Full colour version of this figure available via http://dx.doi.org/10.1677/ERC-09-0118.

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