GENE PROFILING IDENTIFIES GENES SPECIFIC FOR WELL-DIFFERENTIATED EPITHELIAL THYROID TUMORS*

L.G. PUSKAS1, F. JUHASZ2, A. ZARVA1, L. HACKLER Jr.1 and N.R FARID3✍

1 Laboratory of Functional Genomics, Biology Research Center, Hungarian Academy of Science, Szeged, Hungary2 I Department of Surgery, Medical University of Debrecen, Debrecen, Hungary3✍ Osancor Biotech Inc, 31 Woodland Drive, Watford, Herts WD17 3BY, UK

E-mail: farid@osancor96.fsnet.co.uk* Presented in part at the 86 th Annual Meeting of The Endocrine Society, New Orleans, LO June 16-19, 2004

Received: December 2, 2004; Accepted: December 21, 2004; Published September 5, 2005

Abstract - Thyroid nodules are common. It would very helpful if genetic markers that can diagnose malignancy from fine needleaspiration samples were available. Few such markers has been thus identified and none are specific. Large panels of potential markerscan be screened by microarray technology. Studies done to date have concentrated on single tumor types and thus provide no help inidentifying tumor subtype specific markers. To that end we have studied gene profiles of 5 types of benign and malignant thyroid nodulartissue (multinodular goiter, follicular adenoma, papillary and follicular carcinomas). We have identified 195 genes whose differentialexpression clustered into clinically relevant groups. Twenty-eight genes were selected for further confirmation using real timequantitative polymerase chain reaction. Despite the differences in the microarray panels used, we confirmed the differential regulationof 12 genes previously reported in thyroid cancer, although we found the expression of several genes to be regulated in other histologicaltumor subtypes than originally described. We found, PCSK2, TRIB1, RAP1 GA1 to be specifically overexpressed in follicular cancerand S100A4 and GK2 in papillary carcinoma. SERP1, RNASE 2 and STATA5 were suppressed in papillary thyroid cancer. We havethus identified new potential markers specific to malignant thyroid tumors. It is apparent that a range of nodular thyroid tissue usinglarge tumor sample numbers is necessary to establish robust markers for malignancy and to categorize tumors on the basis of smalltumor samples.

Key words: Microarrays, gene profiling, fine needle biopsy, follicular carcinoma, papillary carcinoma, follicular adenoma, multinodulargoiter

INTRODUCTION

Thyroid epithelial cancer is the most common endocrinemalignancy. Its prevalence has increased ion the last decadeand its rate of increase in women outstripped that of othermalignancies, including breast cancer (http://seer.cancer.gov).The reasons for this increase are not clear. Once thediagnosis is made and the patient is treated along standardguidelines, the outlook is excellent, with the exception ofanaplastic carcinoma, which is a lethal disease (14).

Thyroid cancer usually presents as a nodule.Community surveys show that up to 50% of the populationharbor thyroid nodules by age 60, although a smallproportion (5-7%) are of a size to bring patients to medicalattention (20). Apparently smaller nodules (<1 cm) pickedup incidentally do not necessarily equate to a benign natureand if indeed found to be malignant does not guarantee anindolent course. Approximately 5% of the thyroid nodulesturn out to be cancerous.

The most effective tool in investigating the nature of athyroid nodule is cytology of fine needle aspirates. Thediagnosis of papillary is easily made in 90-95% of adequatespecimens on the basis of cytological and nuclearappearances (3,14). Cytology is of no help in separatingbenign from malignant follicular lesions. There is also aresidue of 10-15% of lesions with indeterminate nature(3,20).

Advances in cancer genomics and more recently in geneexpression profiling (44) hold the promise of providingpotential genetic molecular signatures in terms of geneaberrations and gene transcript abundance respectively tosupplement (if not supercede) cytology in providingdiagnostic and prognostic criteria.

The gene candidate approach (16) uncovered theinvolvement in thyroid cancer of "generic" oncogenes andtumor suppressor genes, not specific for thyroid cancer orapparently thyroid specific gene-rearrangements(RET/PTC and PPARγ/PAX8) that were subsequently

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Cellular and Molecular BiologyTM 51, 177-186 ISSN 1165-158XDOI 10.1170/T617 2005 Cell. Mol. Biol.TM

Table 1 Gene expression in follicular carcinoma*

Gene Description FA FTC PTC MNG

JRKL Jerky homolog-like (mouse) 0.81 0.088 1.48 0.81ADHIB Alcohol dehydrogenase I B(Class!) beta polypeptide NA 0.11 0.55 0.69DSIPI delta sleep inducing peptide, immunoreactor NA 0.15 0.73 0.94NOLC1 nucleolar and coiled-body phosphoprotein 1 NA 0.23 0.50 0.92PROX1 prospero-related homeobox 1 NA 0.25 1.02 0.38SLC25A16 solute carrier family 25 (mitochondrial carrier; Graves disease autoantigen), member 16 1.32 0.27 1.41 NACDO1 cysteine dioxygenase, type I NA 0.27 0.46 1.07SERPINA5 serine (or cysteine) proteinase inhibitor, clade A (α-1 antiproteinase, antitrypsin), member 5 NA 0.28 0.92 NAPIK3CA similar to phosphoinositide 3-hydroxykinase p110-alpha subunit 0.96 0.30 0.84 0.91LILRB4 leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 4 NA 0.30 0.94 NAKCNK1 potassium channel, subfamily K, member 1 NA 0.30 0.72 3.42TRIM31 tripartite motif-containing 31 1.1 0.38 1.49 0.81FOXE1 forkhead box E1 (thyroid transcription factor 2) 0.60 0.39 0.92 1.91MYB v-myb myeloblastosis viral oncogene homolog (avian) 0.96 0.39 0.92 1.92PRLR prolactin receptor 1.73 0.40 1.50 0.26SLC23A2 solute carrier family 23 (nucleobase transporters), member 2 0.42 0.42 0.36 0.47CASP3 caspase 3, apoptosis-related cysteine protease NA 0.44 0.91 0.57ATXN1 ataxin 1 1.09 0.45 1.07 0.52DNAJC3 DnaJ (Hsp40) homolog, subfamily C, member 3 1.10 0.48 0.68 0.52PREP prolyl endopeptidase 0.38 0.49 0.69 1.14ARG2 arginase, type II 1.21 3.48 0.47 3.97IL12B interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40) 1.14 0.49 0.97 5.44RXRG retinoid X receptor, gamma 0.41 0.49 0.66 1.66LMNB1 lamin B1 1.81 2.00 2.05 NDMATR3 matrin 3 1.45 2.04 1.40 0.44ALOX12 arachidonate 12-lipoxygenase 1.44 2.05 1.05 1.50PPIC peptidylprolyl isomerase C (cyclophilin C) 1.00 2.08 1.22 5.78U66048 Hs clone 161455-2-3 B cells from breast carcinoma ND 2.12 0.76 0.58VLDLR very low density lipoprotein receptor 0.98 2.13 0, 91 NDPTK2 PTK2 protein tyrosine kinase 2 1.71 2.13 1.30 6.38RRSA2 0.79 2.17 1.59 0.96SETDB1 SET domain, bifurcated 1 1.17 2.19 0.49 0.70SLC2A5 solute carrier family 2 (facilitated glucose/fructose transporter), member 5 0.64 2.22 1.30 0.53CLCN5 chloride channel 5 (nephrolithiasis 2, X-linked, Dent disease) 1.17 2.22 1.40 NDHMBS hydroxymethylbilane synthase 1.34 2.32 1.86 2.31NOS3 nanos homolog 3 (Drosophila) 0.81 2.34 1.06 1.32BMP7 bone morphogenetic protein 7 (osteogenic protein 1) 0.79 2.40 1.76 3.16PIK3CG phosphoinositide-3-kinase, catalytic, gamma polypeptide ND 2.43 1.63 1.13CAPSNS1 2.34 2.50 0.48 0.87DDX11 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 11 (CHL1-like helicase homolog, S. cerevisiae) 1.08 2.54 0.87 0.77MAPK1 mitogen-activated protein kinase 1 0.47 2.62 1.68 5.15TM7SF2 transmembrane 7 superfamily member 2 1.24 2.64 1.59 1.83MAN2A2 mannosidase, alpha, class 2A, member 2 1 1.61 2.72 1.57 1.38PCSK2 proprotein convertase subtilisin/kexin type 2 0.24 2.88 ND 3.08ACO2 aconitase 2 0.48 3.02 1.71 6.28MYL7 myosin, light polypeptide 7, regulatory 1.10 3.10 1.096 NDFSHB follicle stimulating hormone, beta polypeptide 1.44 3.22 0.67 NDCHD9 chromodomain helicase DNA binding protein 9 1.04 3.30 0.85 1.97BRAD1 0.82 3.32 0.92 NDCA3 carbonic anhydrase III, muscle specific 1.06 3.48 0.98 0.34CYP27B1 cytochrome P450, family 27, subfamily B, polypeptide 1 6.57 3.49 0.87 1.12PTPN9 protein tyrosine phosphatase, non-receptor type 9 1.74 3.49 1.05 0.20YWHAH tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide 1.55 3.56 1.94 1.92INHBB inhibin, beta B (activin AB beta polypeptide) 1.40 3.68 1.01 2.1MYBL2 v-myb myeloblastosis viral oncogene homolog (avian)-like 2 1.30 4.10 0.53 NDGBP2 guanylate binding protein 2, interferon-inducible 1.41 4.21 1.31 NDUPP1 uridine phosphorylase 1 1.40 4.68 1.02 1.62BAMBI BMP and activin membrane-bound inhibitor homolog (Xenopus laevis) ND 5.38 0.71 0.46SLC2A2 solute carrier family 2 (facilitated glucose transporter), member 2 1.50 5.67 2.06 0.65HPD 4-hydroxyphenylpyruvate dioxygenase 1.37 6.88 1.44 NDITIH2 inter-alpha (globulin) inhibitor H2 1.01 19.66 1.11 0.77

*Genes whose expression is significantly reduced (<0.5 fold) or increased in follicular carcinoma (FTC) shown in bold. Expression of thosegenes are also shown for other thyroid specimens studied [follicular adenoma (FA), papillary carcinoma (PTC) and multinodular goiter(MNG)] for comparison. Asimilar schema is adopted for tables 2 and 3. ND: not done; Hs: Homo sapiens. Designation of gene symbols andnames was according to http://www.ncbi.nlm.nih.gov/LocusLink

178 L.G. Laszlo et al.

found to occur in only a percentage of tumors of a givenhistology or not to be entirely specific for thyroid cancer(27). Moreover oncogenes /tumor suppressor genes whosemutations were held to be specific for certain tumorhistiotypes were found to show remarkable convergence indownstream signaling pathways (27), raising questionabout further complexity of those pathways. Given thenature of malignant transformation (19), these new findingsare not surprising, although they introduce confusion in themolecular classification of malignant thyroid lesions.

It is apparent that under the circumstances gene profilingmight have a potentially helpful role in diagnosis (follicularand indeterminate) and prognostication of thyroid tumors.Studies in a number of cancers showed that DNAmicroarrays are valuable in tumor sub-classification,prediction of response to therapy and disease outcome(8,10,18,31,33,40,51). Since we completed this study andwere engaged in its analysis, a number of studies haveappeared on gene expression of thyroid tumors(1,2,5,9,21,34,45). Some have limited themselves to onetumor histiotypes (5,21), raised question about genes whoseexpression distinguishes malignant from benign nodule(34) or those that are diagnostic of follicular carcinomafrom benign adenoma (1,2,5,9). Our study is distinct in thatit examined gene expression in a range of benign andmalignant thyroid nodules.

MATERIALAND METHODS

RNA purificationTotal RNAs pooled from normal human thyroid gland (N = 20), and

from dissected follicular adenomas (N = 8) follicular carcinoma (N = 7),papillary carcinoma (N = 10) and multinodular goitre (N = 9) thyroidglands were purified by SV Total RNA isolation kit (Promega, Madison,WI) according to the manufacturer’s instructions. 200 µg of total RNAfrom placenta and 450 µg of total RNA from thyroid glands wereobtained. The purity of the RNA was confirmed by agarose-gelelectrophoresis.

Construction of the microarraysBriefly, 3200 cDNAs amplified from a Human mixed library were

arrayed in duplicate on amino-silanized slides (Sigma, Budapest,Hungary) with a MicroGrid Total Array System spotter (Biorobotics,Cambridge, UK). The diameter of each spot was ~250 mm. After printing,DNA was UV-crosslinked to the slides with 700 mJ energy (Startalinker,Stratagene, La Jolla, CA). Before hybridization, the slides were blocked in1XSSC, 0.2% SDS, 1% BSA for 30 min at 42°C, rinsed in water, anddried.

Microarray probe preparation and hybridizationThree micrograms of total RNA from each sample was amplified by

a linear antisense amplification method and labeled withCy3 or Cy5fluorescent dyes during reverse transcription. After a 2 hr incubation at37°C, heteroduplexes were purified and denatured, and the mRNA washydrolyzed with NaOH for 15 min at 37°C and neutralized. The labeledcDNA was purified with a PCR purification kit (Macherey & Nagel,Duren, Germany) according to the manufacturer’s instructions. Probesgenerated from control or diseased thyroid glands were dissolved in 15 mlof hybridization buffer (50% formamide/5 x SSC/100 mg/ml salmon

sperm DNA) and applied onto the array after denaturation by heating for1 min. at 90°C. After hybridization at 42°C for 20 hr in a humidhybridization chamber, slides were washed by submersion and agitationfor 10 min in 1x SSC with 0.1% SDS, for 10 min in 0.1 x SSC with 0.1%SDS and for 10 min in 0.05x SSC at room temperature, then rinsed brieflyin deionised water and dried.

Scanning and data analysisEach array was scanned under a green laser (532 nm for Cy3) or a red

laser (660 nm for Cy 5) by using a ScanArray Lite (GSI Lumonics,Billerica, MA) scanning confocal florescent scanner with 10-mmresolution. Image analysis was performed with SCANLYZE 2 software(www.microarray.org/software.html) of replica spots, local backgroundcorrection was performed as previously described (25).Significant spotshave >0.55 CHGTB2 values in both Cy3 and Cy5 channels. These valuesequal the fraction of pixels in the spot and are >1.5 times the background,which can be used to filter out weak spots and spots where the intensitycomes from a few bright pixels having high mean intensities but lowvalues. Each experiment was performed twice with both florescent dyesfor labeling control and sample to reduce the number of false-positives orfalse-negatives. These false results could derive from the possibleincorporation of the dyes during labeling or from other experimentalvariables introduced by hybridization, washing conditions, or arrayfeatures.The array was repeated once with similar results. Internal controlsto correct for abundance of mRNAand inter-blot normalization were doneusing 4 house-keeping genes.

Real time PCIn the case of 28 genes, gene-expression changes observed by

microarray were confirmed by quantitative real-time PCR using a RotorGene 2000 instrument (Corbett Research, Sydney) with gene-specificprimers. SYBR Green 1 kit was used for quantitative real time PCRaccording to the manufacturer’s protocol (Roche, Mannheim, Germany).Total RNA was reverse transcribed as described above. The cDNA mixwas diluted 10 fold and 2 µl of the dilution mixture was used for real timePCR analysis. The PCR conditions were optimized for each of the primerpairs used to amplify specific genes. The resulting concentration of PCRproducts was normalized by comparison to that of the house-keeping geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Variation in the expression of these selected genes forms the basis ofthis report. A gene is only reported if at least 2/3 of individual samplesexhibited the same trend as the pooled RNA.

RESULTS AND DISCUSSION

A number of studies have reported on gene expressionprofiles in thyroid cancer (1,2,5,9,21,34). Some havefocused on individual tumor histiotypes (1,2,5,9,21), ondifferentiating malignant from benign follicular lesions(5,9) or indeed between benign and malignant tumors (34).The numbers of samples analyzed in the systematic studieswere initially small (1,2,21) but have since increasedconsiderably and have been submitted to sophisticatedanalytical algorithms (9,34). Most of the studies quoted are,however, robust and pass muster for the stringent rulesstipulated for reporting of gene expression studies (4,44).

We have used human cDNA microarray constructed in-house by spotting previously PCR-amplified and purifiedgene-specific samples to study RNAfrom a range of thyroidtissues. We examined samples from multinodular goitre(MNG), autoimmune thyroid disease, papillary carcinoma,

Gene expression in thyroid cancer 179

180 L.G. Laszlo et al.

Table 2 Gene expression of follicular adenoma (FA)*

Gene Description FA FTC PTC MNG

ANK1 ankyrin 1, erythrocytic 0.11 0.96 1.17 NDCD53 CD53 0.15 1.06 3.27 1.61ELF4 E74-like factor 4 (ets domain transcription factor) 0.24 0.69 0.99 0.51ITIH1 inter-alpha (globulin) inhibitor H1 0.23 0.92 0.84 0.67PCSK2 proprotein convertase subtilisin/kexin type 2 0.24 2.87 ND 3.07CFTR cystic fibrosis transmembrane conductance regulator, ATP-binding cassette (sub-family C, member 7) 0.28 0.76 0.80 1.42GLYAT glycine-N-acyltransferase 0.28 0.54 1.83 NDPLXNA3 plexin A3 0.29 0.79 1.02 NDTTC9 tetratricopeptide repeat domain 9 0.29 0.65 1.87 2.33FPR1 formyl peptide receptor 1 0.29 0.82 1.11 NDDPP4 dipeptidylpeptidase 4 (CD26, adenosine deaminase complexing protein 2) 0.29 0.99 1.89 0.70CETP cholesteryl ester transfer protein, plasma 0.31 1.62 1.15 0.64MUTYH mutY homolog (E. coli) 0.31 0.72 0.68 NDNPHS1 nephrosis 1, congenital, Finnish type (nephrin) 0.31 0.86 1.35 2.48ADRA2C adrenergic, alpha-2C-, receptor 0.32 0.72 1.31 0.65TRIB1 tribbles homolog 1 (Drosophila) 0.32 0.96 0.64 NDSCNN1A sodium channel, nonvoltage-gated 1 alpha 0.34 1.35 0.88 1.05C3 complement component 3 0.35 0.53 1.70 3.32C2 complement component 2 0.35 1.89 1.17 10.67TBXAS1 thromboxane A synthase 1 (platelet, cytochrome P450, family 5, subfamily A) 0.35 0.69 1.08 1.68TLE4 transducin-like enhancer of split 4 (E(sp1) homolog, Drosophila) 0.36 0.66 0.81 NDIMPA1 inositol(myo)-1(or 4)-monophosphatase 1 0.36 0.66 0.75 1.01RNASE3 ribonuclease, RNase A family, 3 (eosinophil cationic protein) 0.37 0.72 0.83 0.68GYG2 glycogenin 2 0.37 1.34 1.07 3.70COH1 Cohen syndrome 1 0.39 1.30 1.11 1.68TIMP1 tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor) 0.40 0.70 0.77 2.82CTSL2 cathepsin L2 0.40 1.09 2.84 0.89BTG1 B-cell translocation gene 1, anti-proliferative 0.41 0.54 0.62 2.30THBS1 thrombospondin 1 0.41 0.66 2.38 1.48ZNF267 zinc finger protein 267 0.41 0.55 0.82 0.90RORA RAR-related orphan receptor A 0.41 0.61 0.84 0.75RXRG retinoid X receptor, gamma 0.41 0.50 0.66 1.67CCNT2 cyclin T2 0.42 1.25 1.49 0.47TTC1 tetratricopeptide repeat domain 1 0.42 0.55 0.70 NDDEGS degenerative spermatocyte homolog, lipid desaturase (Drosophila) 0.43 1.01 0.67 1.01DDX10 DEAD (Asp-Glu-Ala-Asp) box polypeptide 10 0.44 1.22 0.76 2.24S100A12 S100 calcium binding protein A12 (calgranulin C) 0.44 0.63 0.67 0.74RFX3 regulatory factor X, 3 (influences HLA class II expression) 0.45 1.56 1.69 0.23KLKB1 kallikrein B, plasma (Fletcher factor) 1 0.45 0.92 0.51 2.30S100A4 S100 calcium binding protein A4 (calcium protein, calvasculin, metastasin, murine placental homolog) 0.45 1.02 9.44 1.44TIMP3 tissue inhibitor of metalloproteinase-3 0.46 0.54 0.74 2.43KIAA0323 KIAA0323 0.47 1.39 0.96 4.04CUBN cubilin (intrinsic factor-cobalamin receptor) 0.49 0.68 0.62 0.53TMSB4X thymosin, beta 4, X-linked 2.01 1.27 1.90 0.92MNDA myeloid cell nuclear differentiation antigen 2.02 0.72 1.30 NDHNMT histamine N-methyltransferase 2.04 1.90 0.69 0.36SLC25A17 solute carrier family 25 (mitochondrial carrier; peroxisomal membrane protein, 34kDa), member 17 2.08 1.41 1.1 1.26DUOX2 dual oxidase 1 2.08 1.71 1.16 1.89SPTAN1 spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) 2.1 1.52 1.34 0.77CANX calnexin 2.15 1.41 1.54 3.61CDK2AP1 CDK2-associated protein 1 2.13 3.81 1.26 1.74CBFA2T1 core-binding factor, runt domain, alpha subunit 2; translocated to, 1; cyclin D-related 2.14 0.84 1.66 1.23PPP2R2B protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform 2.18 1.33 0.77 1.44IL10RA interleukin 10 receptor, alpha 2.18 1.74 1.42 1.81SMYD5 SMYD family member 5 2.26 1.02 1.04 0.57PPP1R12A protein phosphatase 1, regulatory (inhibitor) subunit 12A 2.28 1.93 0.94 0.32DATF1 death associated transcription factor 1 2.33 3.0 0.98 0.51TTN titin 2.34 1.64 2.125 1.41CD164 CD164 antigen, sialomucin 2.34 2.66 0.98 0.57DDB2 damage-specific DNA binding protein 2, 48kDa 2.35 1.23 1.28 NDBANF1 barrier to autointegration factor 1 2.47 1.62 1.82 1.65PDZK10 PDZ domain containing 10 2.49 0.63 0.70 1.94ABCB1 ATP-binding cassette, sub-family B (MDR/TAP), member 1 2.51 1.14 1.19 1.01IMMT inner membrane protein, mitochondrial (mitofilin) 2.53 1.78 0.82 1.58BRCA1 breast cancer 1, early onset 2.60 1.37 0.58 0.69MTRR 5-methyltetrahydrofolate-homocysteine methyltransferase reductase 2.65 2.20 0.77 0.81SFRS5 splicing factor, arginine/serine-rich 5 2.69 0.95 1.06 0.84SUPT6H suppressor of Ty 6 homolog (S. cerevisiae) 2.7 1.90 1.59 1.50BCL2L2 BCL2-like 2 2.76 1.28 1.47 1.95LDHB lactate dehydrogenase B 2.78 2.21 1.87 3.19TK2 thymidine kinase 2, mitochondrial 2.78 1.68 1.55 1.59EP300 E1A binding protein p300 3.08 1.55 0.71 0.89

*The genes over-expressed or suppressed are shown in bold. TIB1 encocdes the C8FW phosphoprotein, initially described to be induced inthyroid tissue (see text). See also the legend to Table 1.

follicular carcinoma and follicular adenoma and comparedthese to normal perinodular thyroid tissue. Hierarchicalcluster analysis showed clear separation of various clinicalpathological entities according to variation in the expressionof 1322 genes, many of which were unidentified at the time(38). The four groups of tissues considered here wereclearly separated on the clustering dendrogram (38).

We show here that variation in the expression oftranscripts of 195 genes distinguished between follicularbenign and malignant nodules (Table 1, 2) and wascharacteristic of PTC (Table 3).

We selected 28 genes whose expression showedvariation in thyroid tumors for further examination usingquantitative RT-PCR. Given that we used a completelydifferent panel of genes than did Huang et al. (21), we foundremarkable agreement in genes differentially regulated inPTC (Table 4, 5). The expression of some of these geneswas, however, also regulated in other thyroid tissuesexamined in this study, thus (and as expected 16) METexpression was increased in FTC, as was that of LGALS3whereas that of CH13L1 was overexpressed in adenomas,MNG and FTC. That of Cpb/p300-interacting protein wasoverexpressed in FTC but 5 fold less in PTC. DIO 1 whichis down-regulated in PTC was enhanced in FTC and benignnodular tissue, CITED 1 expression was also reduced inadenoma, FTC and MNG but less than in PTC whereasFABP4, suppressed in PTC, appears to up-regulated inMNG.

We report a number of novel genes whose expressionwas differentially regulated in thyroid tumors. Thus, wefound the TIBI 1 (encoding the C8FW phosphoprotein)regulated in the thyroid gland by TSH and EGF (48), to bespecifically overexpressed in FTC and as was PCSK2 thatwas also found to be over-expressed in FTC by Cerruti et al.(9). PCSK2 is a furin involved in the processing ofprohormones (6) but which also involved in the digestion ofpre-metalloproteases and cadherins and might be involvedin tumor invasion and metastatic behavior. S100 calciumbinding protein (S100A4, mts1 or metastasin), the subject ofintense recent interest in carcinogensis (35), wasspecifically increased in PTC. In an independent study, wehave shown that the abundance of S100 binding proteintranscripts to be related to tumor stage and to the proclivityto metastases (52). RAP 1GA1, which is involved in theregulation of cell-adhesion and tumor development (11,12)as well as glycerol kinase 2 (testis specific), a key enzymein adipose tissue metabolism are increased in FTC (Table 4)SEP15, the 15 kDa selenoprotein (28) whose abundance ismodified in transformed cells was increased in PTC but notin FTC and less so in MNG (Table 4).

We found secreted frizzled related protein (SFRP1)(26,47), which is involved in the regulation of wntsignaling, to be specifically reduced in PTC (Table 5). Incontrast, the expression of tight junction protein 1,

important in maintaining cellular polarity (39) wassuppressed specifically in FTC. RNASE2 (eosinophil-derived neurotoxin), which plays a role in allergic reactionsand has an anti retroviral activity is reduced in all tumoroustissues studied (7,36). We speculate that it may be involvedin evasion by tumors of immune cells. KRT 7 important inmaintaining cellular structure is reduced in all exceptfollicular adenomas (22) (Table 5). In an independent study(42), we found that the cannabinoid receptor 2 (CNR2) aswell as MET to relevant to the metastatic behavior ofanaplastic thyroid carcinoma.

Huang et al. (21) who studied PTC identified othergenes, which were either not represented in our panel orwhich we did not find to be differentially expressed. Theyidentified a number of novel genes not previously reportedin any neoplasia including those of the thyroid: ADORA1(adenosine A1 receptor), SCEL (sciellin), ODZ1 (Odz 1,Drosophila), PROS1 (vitamin K-dependent plasma protein S),KIAA0937, CST6 (cystatin E/M), SDC4 (ryudocan coreprotein), P4HA2 (propyl-4-hydroxylase alpha (II) subunit),DUSP6 (dual specificity phosphatase 6), TSSC3 (tumorsuppressing subtransferable candidate 3).

Barden et al. (5) compared RNA from 10 follicularadenomas with those from 9 follicular carcinomas (FTC)and identified 105 genes whose expression significantlydiffered between adenomas and carcinomas (overexpressedin one or the other). Interestingly, very few of the genessuppressed or overexpressed in follicular tumors wereidentified as important in the Huang et al. study (21) or thecurrent study, the only exception being MET. Five genesoverexpressed in FTC were chosen (5) for furtherverification: adrenomedullin, autotoxin, EMMPRIN(extracellular matrix metalloproteinase inducer, now BSGor OK blood group), transforming growth factor α receptorand as noted above MET.

In an attempt to identify genes whose expressiondistinguished FTC from FA, Cerruti et al. (9) used serialanalysis of gene expression (SAGE) to focus with real timePCR on 4 genes that correctly categorized benign asopposed to malignant follicular thyroid lesions. Two ofthese genes were previously described by Barden et al. (5)to be upregulated in FTC (DDTI3) and FA(putatative Emu 1),although only the former was found to discriminatebetween FTC and FA. We found ARG 2, whose expressionwas reported to be increased in FTC (9) but not in FA, alsoto be upregulated in PTC (Table 2). So while AGR2 lacksbroad histocytic specificity, it is diagnostically helpful in thecontext of follicular tumors. Our studies also support aspecific increase in the expression of ACO I (aconitase 1)(Table 3) as well as PCSK2 in FTC (Table 4), although wehave at the time pursued only the latter with confirmatoryreal time PCR.

Aldred et al. (1,2) selected genes found to bedifferentially regulated in FTCs; they chose specifically

Gene expression in thyroid cancer 181

Table 3 Gene expression in papillary thyroid carcinoma*

Gene Description FA FTC PTC MNG

IFIT3 interferon-induced protein with tetratricopeptide repeats 3 0.54 0.83 0.31 0.69TNFSF10 Fas (TNFRSF6)-associated via death domain 0.52 0.61 0.32 0.52FEZ1 fasciculation and elongation protein zeta 1 (zygin I) 0.78 0.80 0.32 0.06HSHUR7SEQ UV-B repressed sequence, HUR 7 0.53 0.65 0.33 0.50REL v-rel reticuloendotheliosis viral oncogene homolog (avian) 0.52 0.60 0.35MED6 mediator of RNA polymerase II transcription, subunit 6 homolog (yeast) 0.95 0.65 0.35 0.63SIAT4A sialyltransferase 4A (beta-galactoside alpha-2, 3-sialyltransferase) 1.14 0.96 0.37 0.69HYAL3 hyaluronoglucosaminidase 3 1.05 0.49 0.37TAF1A TATA box binding protein (TBP)-associated factor, RNA polymerase I, A, 48kDa 0.57 0.59 0.39 0.65MLLT4 myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 4 0.64 1.03 0.39 0.89CLDN10 claudin 10 0.54 0.44 0.40 0.60ZNF297B zinc finger protein 297B 0.71 0.73 0.41 0.48DPF1 D4, zinc and double PHD fingers family 1 0.51 0.71 0.41 0.59KIF2C kinesin family member 2C 0.82 0.54 0.43PCDH7 BH-protocadherin (brain-heart) 0.66 0.67 0.45 5.61SCAMP1 secretory carrier membrane protein 1 0.72 0.70 0.45 0.36HMGB2 high-mobility group box 2 0.72 0.50 0.46 0.85FABP5 fatty acid binding protein 5 (psoriasis-associated) 0.89 0.86 0.47 1.85TAC1 tachykinin, precursor 1 (substance K, substance P, neurokinin 1, neurokinin 2, neuromedin L, 0.92 1.42 0.47 3.56

neurokinin α neuropeptide K, neuropeptide γ)ARG2 arginase, type II 0.94 2.48 0.47 3.97CAPNS1 calpain, small subunit 1 2.35 2.48 0.48 0.87LSAMP limbic system-associated membrane protein 0.30 0.52 0.49CASP10 caspase 10, apoptosis-related cysteine protease 0.90 0.57 0.49 0.70CTSG cathepsin G 0.67 0.74 0.49 0.097SETDB1 SET domain, bifurcated 1 1.17 2.19 0.49 0.70HGD homogentisate 1, 2-dioxygenase (homogentisate oxidase) 0.74 0.54 0.50 0.79GTF3C1 general transcription factor IIIC, polypeptide 1, alpha 220kDa 1.39 1.41 2.02 1.77FRG1 FSHD region gene 1 0.77 1.44 2.02HLA-A major histocompatibility complex, class I, A 1.24 1.88 2.02 7.73ADAM17 a disintegrin and metalloproteinase domain 17 (tumor necrosis factor, α, converting enzyme) 1.15 2.02 0.98GTF3C2 general transcription factor IIIC, polypeptide 2, beta 110kDa 2.64 1.3 2.03 0.47EIF2B2 eukaryotic translation initiation factor 2B, subunit 2 beta, 39kDa 0.86 0.70 2.04 1.06SLC6A6 Solute carrier family 6 (neurotransmitter transporter, taurine), member 6 1.26 1.85 2.05 1.30LMNB1 lamin B1 2.34 1.78 2.05 1.64ACVR1 activin A receptor, type I 1.19 1.61 2.10 6.82ITGB3BP integrin beta 3 binding protein (beta3-endonexin) 1.17 1.44 2.10 0.78RPL21 ribosomal protein L21 1.76 1.37 2.13 0.36MEF2 MADS box transcription enhancer factor 2, polypeptide A (myocyte enhancer factor 2A) 1.35 1.14 2.13 1.17SCN7A sodium channel, voltage-gated, type I, alpha 1.35 1.15 2.14 0.82MTHFD2 methylene tetrahydrofolate dehydrogenase (NAD+ dependent), methenyltetrahydrofolate cyclohydrolase 0.87 0.93 2.15 1.27M6PR mannose-6-phosphate receptor (cation dependent) 1.81 1.74 2.17 2.84EGR3 early growth response 3 1, 61 1.97 2.26FNTB farnesyltransferase, CAAX box, beta 0.93 1.90 2.27 2.02EED embryonic ectoderm development 1.20 1.75 2.33 1.66KIAA0555 Jak and microtubule interacting protein 2 1.36 0.69 2.34 0.99ITGAE integrin, alpha E (antigen CD103, human mucosal lymphocyte antigen 1; alpha polypeptide) 1.78 0.99 2.34 NDTHBS1 thrombospondin 1 0.41 0.67 2.34 1.98HLA-DRB1 major histocompatibility complex, class II, DR beta 1 1.07 1.87 2.35 1.88DLD dihydrolipoamide dehydrogenase (E3 component of pyruvate dehydrogenase complex, 1.76 1.90 2.41 1.67

2-oxo-glutarate complex, branched chain keto acid dehydrogenase complex)TNFAIP2 tumor necrosis factor, alpha-induced protein 2 1.07 1.00 2.44 1.42BCKDHA branched chain keto acid dehydrogenase E1, alpha polypeptide (maple syrup urine disease) 0.85 1.46 2.45 84NAPA N-ethylmaleimide-sensitive factor attachment protein, alpha 1.43 1.39 2.49 NDMRCL3 myosin regulatory light chain MRCL3 1.39 1.27 2.55 1.94CASP1 caspase 1, apoptosis-related cysteine protease (interleukin 1, beta, convertase) 1.44 1.95 2.67 1.98SNX19 sorting nexin 19 1.34 1.26 2.73 2.22IL1RL1LG interleukin 1 receptor-like 1 ligand 4.44 1.92 2.82 1.52RTCD1 RNA terminal phosphate cyclase domain 1 1.90 1.96 2.93 1.85ATF7 activating transcription factor 7 1.49 1.41 3.40 0.85CYP1A1 cytochrome P450, family 1, subfamily A, polypeptide 1 1.45 1.35 3.50 0.86MECP2 methyl CpG binding protein 2 (Rett syndrome) 1.44 2.03 3.86 0.63RNASEH1 ribonuclease H1 2.45 1.60 4.21 0.87RIN2 Ras and Rab interactor 2 1.68 1.14 4.64 2.45

* For explanation see the legend to Tables 1 and 2.

182 L.G. Laszlo et al.

Gene expression in thyroid cancer 183

genes mapping to regions of loss of heterozygozity (LOH) (1).Five genes (caveolin-1, caveolin-2, GDF10/BMP3b,glypican-3 a novel chordin-like) were downregulated, thelatter 3 being involved in bone morphogenesis signaling.On the other hand, the expression of the those 3 genes werealso downregulated in benign adenomas and multinodulargoitre and glypican-3 in PTC suggesting that they might beearly events in pathologic thyroid cell growth. The authorsalso found PPARγgene expression reduced in FTCs (2) andattributed this change in expression to changes in one ormore regulatory proteins acting upstream form the PPARγgene. We found a key bone morphogenic factor BMP7 to bedownregulated in FA but overexpressed in FTC and MNG.By the same token, BAMBI (Table 1) an inhibitor of tumorresponse to tumor necrosis factor beta signaling is clearlyincreased in FTC (41). Both BMP7 and BAMBI are part ofthe transforming growth factor-beta super-family.

There is differential expression in a number ofmolecules involved in different biological functions. Theseinclude cancer-related genes (REL and MTTL4 decreased inPTC, BBRCA and BCL2L2 increased in FA; whereasRRASS2 and MYBL2 were increased in FTC), genes

involved in the regulation of apoptosis (CASP decreasedand DDX11 increased in FTC, DDX10 is decreased andDATF1 increased in FA whereas in PTC CASP10 wasdecreased and CASP1 increased)(29), genes promoting intissue modulation within and around tumors and tumorinvasion (TIMP 1, TIMP 3 and TIBS1 decreased in FA, thesecond was increased in MNG and TIBS 1 in PTC, whichalso exhibit an increase in ADAM 17) (43,46) . Theexpression of some thyroid-specific genes appears to bespecifically modulated in follicular tumors (DUOX2increased in FA, SLC25A16 and FOXE1 decreased in FTC;also see Table 5) (13,30,50) (Tables 1-3). Our studies showthe modulation products and receptor of immune cells e.g.LILRB4, ITIH 1 and 2, IL10RA, IFIT 3 (38), undoubtedly asa result of tumor editing (15,17,37), although the interfacebetween tumors and the immune system may be morecomplex in that products of antigen presenting cells arebeing increasingly incriminated in promoting tumor growth(33). Within that context, we were surprised by theincreased expression of MHC Class I and more so Class IImolecules (Table 3), specifically in PTC, until it has latelyfound that the re-arranged RET/PTC (Rearranged in

Table 4 Genes over-expressed in thyroid tumors by real-time PCR

Gene and Description Chromosomal FA FTC PTC MNGLocation

DDP4* 2q24.3 -1.43 -1.87 6.76 1.29Dipeptidyl peptidase IVFNI* 2q34 1.86 1.44 8.76 1.74Fibronectin 1LGALS3* 14q21-q22 1.00 4.75 34.22 4.61Galectin 3MET* 7q31 1.00 1.42 5.98 1.00met proto-oncogeneCH13L1* 1q32.1 4.58 3.43 9.84 6.84Cartilage glycoprotein 39CITED1* Xq31.1 1.41 4.59 25.99 1.95Cpb/p300-interacting transactivatorTRIB1 8q24.13 1.01 27.8 1..32 1.19tribbles homolog 1 (Drosophila)GSTT2 22q11.23 2.56 1.61 3.38 -1.10Glutathione S-transferase theta 2S100A4 1q21 -3.48 -1.85 3.52 1.56S100 calcium binding protein 1DATF1 20q13.33 2.98 2.39 2.86 -1.87Death associated transcription factor 1RAP1 GA1 1p36.1-p35 -1.07 3.32 1.00 1.00GTPase activating protein 1SEP15 1q31 9.18 2.63 8.86 1.8915kD selenoproteinILF2 1q22 4.28 3.54 4.89 1.98Interleukin enhancer binding factor 2PCSK2+ 20p11.2 1.00 59.71 1.08 1.00Proprotein convertase subtilsin/kexin type 2GK2 4q13 2.56 1.20 2.57 -4.80Glycerol kinase (testis specific)STIM1 11p15.5 2.09 4.30 2.78 9.89Stromal interaction protein 1CD63 12q12-q13 3.50 3.31 3.46 2.93Melanoma 1 antigen

* These genes products were previously reported to be over-expressed in PTC (21). +Also found to be expressed in FTC in Cerruti et al. (9)

184 L.G. Laszlo et al.

Transformation/papillary thyroid carcinoma) gene actuallyincreases the transcription of these molecules (22).

The study of gene expression by of thyroid tumors isstarting to yield genes whose regulation is specific tothyroid tumors or subtypes thereof, allowing diagnosis andprognostication form fine needle biopsy samples. While theprotocols for performing gene expression are relativelyeasy, the analysis and interpretation of the results are timeconsuming and may be difficult to interpret. Headway incorroborating and prospectively testing the specificity ofgenes for thyroid tumor histiotypes is being made.Independent corroboration of differential expression isessential and, in our opinion, by replication in differentsample sets in the same or independent laboratories. Giventhe diverse pathways to malignancy (19) unexpected genesand pathways (34) to justify a "candidate gene panel"approach in trying to develop robust tests to diagnosemalignancy in a thyroid nodule, predict histology andsubsequent tumor behavior and response to therapy.

Ideally, a multicenter study in which large numbers [30-50]of well characterized thyroid tumors is necessary to provideus with the data-base. The application of statistical methodssuch as that described by Wright et al. (49), will allowcomparison of the results from the bank of tumors proposedabove to previously studied tumors whose array results arein the public domain, and from tumors that may beindependently studied in the future to hone in on a limitednumber of genes important in tumor classification,prognostication and adjuvant therapy targeting.

REFERENCES

1. Aldred, M.A., Ginn-Pease, M.E., Morrison, C.D., Popkie, A.P.,Gimm, O., Hoang-Vu, C., Krause, U., Dralle, H., Jhiang, S.M., Plass,C. and Eng, C., Caveolin-1 and Caveolin-2, together with three bonemorphogenetic protein-related genes, amy encode novel tumorsuppressors down-regulated in sporadic follicular thyroidcarcinogenesis. Cancer Res. 2003, 63: 2864-2871.

2. Aldred, M.A., Morrison, C., Grimm, O., Hoang-Vu, C., Krause, U.,Dralle, H., Jhiang, S. and Eng, C., Peroxisome proliferators-activatedreceptor gamma is frequently downregulated in a diversity ofsporadic nonmedullary thyroid carcinoma. Oncogene 2003, 22:3412-3416.

3. Asa S.L., The pathology of thyroid cancer in molecular events infollicular thyroid tumors. In: Molecular Basis of Thyroid Cancer,Farid, N.R. (ed.), Kluwer Acad. Press, Boston, 2004, pp. 23-67.

4. Baldi, P. and Hatfield, G.W., DNA Microarrays and GeneExpression from Experiments to Data Analysis, CambridgeUniversity Press, 2002.

5. Barden, C.B., Shister, K.W., Zhu, B., Guiter, G., Greenblatt, D.Y.,Zeiger, M.A. and Fahey III, T.J., Classification of follicular thyroidtumors by molecular signature: results of gene profiling. Clin.Cancer Res. 2003, 9: 1792-1800.

6. Bassi, D.E., Mahloogi, H. and Klein-Szanto, A.J., The proproteinfurin and PACE-4 play a significant part in tumor progression. Mol.Carcino. 2000, 28: 63-69.

7. Brenner, S.A., The past as the key to the present: Resurrection ofancient proteins from eosinophils. Proc. Natl. Acad. Sci. USA 2002,99: 4760-4761.

8. Carr, K.M., Bittner, M. and Trent, J.M., Gene-expression profiling inhuman cutaneous melanoma. Oncogene 2003, 22: 3076-3080.

9. Cerutti, J.M., Delcelo, R., Amadei, M.J., Nakabashi, C., Maciel,R.M.B., Peterson, B., Shoemaker, J. and Riggins, G.J., Apreoperative diagnostic test that distinguishes benign from malignantthyroid carcinoma based on gene expression. J. Clin. Invest. 2004,

Table 5 Genes whose expression is repressed in thyroid tumors by real time PCR

Gene and Description Chromosomal FA FTC PTC MNGLocation

TPO* 2p25 -1.23 -1.19 -7.67 -1.56Thyroid peroxidaseDIO I* 1p32-p33 4.63 2.27 -3.46 4.67Type I iodothyronine deiodinaseCRABP1* 15q24 -3.45 -5.58 -22.76 -4.32CRAB1 cellular retinoic acid binding proteinFABP4* 8q21 1.89 -2.56 -1.96 4.22Fatty acid binding protein 4TJP1 15q13 -1.62 -7.47 1.32 1.42Tight junction protein 1RNASE 2 14q24-q31 -3.03 -4.63 -2.39 -6.33Eosinphil-derived neurotoxinSFRP1 8p12-p11.1 -1.23 1.45 -4.28 -1.45Secreted frizzeled related proteinCDC42 1p36.1 2.07 -1.75 1.37 -2.46Cell division cycle 42KRT7 17q11.2 -1.56 -2.64 -3.24 -4.59Keratin, Type II cytoskeletal 7CCL19 9q13 1.13 1.12 1.76 -3.89EBI 1-ligand chemokineSTATA5 17q11.2 -1.23 1.45 -4.38 -1.45Signal transducer and activator of transcription 5A

*These genes products were previously reported to be suppressed in PTC (21) Negative values denote the degree of repression of geneproduct compared to a pool of normal thyroid tissue.

Gene expression in thyroid cancer 185

113: 1234-1242.10. Cooper, C.S., Application of microarray technology in breast cancer

research. Breast Cancer Res. 2001, 3: 158-175.11. Daumke, O., Weyand, M., Chakarebarti, P.P., Vetter, I.R. and

Wittinghof, A., Protein Rap1 GAP uses a catalytic aspargine. Nature2004, 429: 197-201.

12. De Buryn, K.M., Rangarajan, S., Reedquist, K.A., Figdor, C.G. andBos, J.L., The small GTPase Rap1 is required for Mn (2+)- andantibody-induced LFA-1 and VLA-4-mediated cell adhesion. J. Biol.Chem. 2003, 277: 29468-29476.

13. De Deken, X., Wang, D., Dumont, J.E. and Miot, F., Characterizationof ThOX proteins as components of the thyroid H(2)O(2)-generatingsystem. Exp. Cell Res. 2002, 273: 187-196.

14. DeLellis, R.A., Lloyd, R.V., Heitz, P.U. and Eng, C. (eds.), Pathologyand Genetics: Tumors of Endocrine Organs, World HeathOrganization Classification of Tumours, IARC Press, Lyon 2004, pp.49-123.

15. Dunn, G.P., Old, L.J. and Schreiber, R.D., The three Es of cancerimmunoediting. Annu. Rev. Immunol. 2004, 22: 329-360.

16. Farid, N.R., Shi, Y. and Zou, M., Molecular basis of thyroid cancer.Endocrin. Rev. 1994, 15: 202-232.

17. Gupta, S., Patel, A., Folstad, A., Fenton, C., Dinauer, C.A., Tuttle,R.M., Conran, R. and Francis, G.L., Infiltration of differentiatedthyroid carcinoma by proliferating lymphocytes is associated withimproved disease-free survival for children and young adults. J. Clin.Endocrinol. Metabol. 2001, 80: 1346-1345.

18. Guo, Q.M., DNA microarray and cancer. Curr. Opin. Oncol. 2003,15: 36-43.

19. Hahn, W.C. and Weinberg, R.A., Rules for making human tumorcells. N. Engl. J. Med. 2002, 347: 1593-1603.

20. Hegedus, L., The thyroid nodule. N. Engl. J. Med. 2004, 351: 1764-1771.

21. Huang, Y., Prasad, M., Lemon, W.J., Hempel, H., Wright, F.A.,Kornacker, K., LiVolsi, V., Frankel, W., Kloos, R.T., Eng. C.,Pellegata, N.S. and de la Chapelle, A., Gene expression in papillarythyroid carcinoma reveals highly consistent profiles. Proc. Natl.Acad. Sci. USA 2001, 98: 15044-15049.

22. Hwang, E.S., Kim, D.W., Song, J.H., Park, K.C., Park, S.H., Yun, H.-J., Kimm, J.M. and Shong, M., Regulation of signal transducer andactivator of transcription 1 (STAT1) and STAT1-dependent genes byRET/PTC (rearranged in transformation/papillary carcinoma)oncogenic tyrosine kinase. Mol. Endocrinol. 2004, 18: 2672-2684.

23. Kato, Y., Ying, H., Willingham, M.C. and Cheng, S.-Y., A tumorsuppressor role for thyroid hormone ? receptor in a mouse model ofthyroid carcinogenesis. Endocrinology 2004, 145: 4430-4438.

24. Kirfel, J., Magin, T.M. and Reichelt, J., Keratins: a structural scaffoldwith emerging functions. Cell. Mol. Life Sci. 2003, 60: 56-71.

25. Kitajka, J., Puskas, G.L., Zvaram A., Hackler Jr., L., Barcelo-Cobling, T., Yeo, Y.K. and Farkas, T., The role of n-polyunsaturatedfatty acids in the brain: modulation of rat brain gene expression bydietary n-fatty acids. Proc. Natl. Acad. Sci. USA 2002, 99: 2619-2624.

26. Ko, J., Ryu, K.S., Lee, Y.H., Na, D.S., Kim, Y.S., Oh, Y., M., Kim,I.S. and Kim, J.W., Human frizzled-related protein is down-regulatedand induces apoptosis in human cervical cancer. Exp. Cell Res. 2002,280: 280-287.

27. Kroll, T.G., Molecular events in follicular thyroid tumors. In:Molecular Basis of Thyroid Cancer, Farid, N.R. (ed.), Kluwer Acad.Press, Boston, 2004, pp. 85-106.

28. Kumaraswamy, E., Malykh, A., Korotkovm K.V., Kozyavkinm, S.,Hu, Y., Kwon, S.Y., Moustafa, M.E., Carlson, B.A., Berry, M.J., Lee,B.J., Hatfied, D.L., Diamond, A.M. and Gladyshev, V.N., Structure-relationship of the 15-kDa seleoprotein gene. Possible role of theprotein in cancer. J. Biol. Chem. 2000, 275: 5540-5547.

29. Los, M. and Walczak, H. (ed.), Caspsaes: Their Role in Cell Death

and Cell Survival, Kluwer Acad., Dordrecht, 2003.30. Macchia, P.E., Mattei, M.G., Lapi, P., Fenzi, G. and Di Lauro, R.,

Cloning, chromosomal localization and identification ofpolymorphisms in the human thyroid transcription factor 2 gene(TITF2). Biochimie 1999, 81: 433-440.

31. Macgregor, P.F., Gene expression in cancer: The application ofmicroarrays. Exp. Rev. Mol. Diagn 2003, 3: 185-200.

32. Macgregor, P.F. and Squire, J.A., Application of microarrays toanalysis of gene expression in cancer. Clin. Chem. 2002, 48: 1170-1177.

33. Marx, J., Inflammation and cancer: The link grows stronger. Science2004, 306: 966-968.

34. Mazzanti, C., Zeiger, M.A., Costourous, N., Umbrick, C., Westra,W.H., Smith, D., Somervell, H., Bevliacqua, G., Alexander, H.R. andLibutt, S.K., Using gene expression profiling to differentiate benignfrom malignant thyroid tumors. Cancer Res. 2004, 64: 2989-2903.

35. Mazzucchelli, L., Protein S100A4: Too long overlooked bypathologists. Am. J. Pathol. 2001, 160: 7-13.

36. McDevitt, A.L., Deming, M.S., Rosenberg, H.F. and Dyer, K.D.,Gene structure and enzymatic activity of mouse eosinophil-associated ribonuclease 2. Gene 2001 267: 23-30.

37. Powell Jr., D.J., Eisenlohr, L.C. and Rosthein, J.L., A thyroid tumor-specific antigen formed by the fusion of two self antigen. J. Immunol.2003, 170: 861-869.

38. Puskas, L.G., Juhasz, F., Zavra, A., Hackler Jr., L., Kacszur, V. andFarid, N.R., The distinction between Graves’ disease andHashimoto’s thyroiditis by gene profiling. J. Endocr. Genet. in press,Accepted 2005.

39. Rao, R.K., Baurov, S., Rao, V.U., Karnaky Jr., K.J. and Gupta, A.,Tyrosine phosphorylation and dissociation of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the cytoskeleton by oxidativestress. Biochem. J. 2002, 368: 471-481.

40. Schwaenen, C., Wessendorf, S., Kestler, H.A., Dohner, H., Lichter,P. and Bentz, M., DNA microarray analysis in malignantlymphomas. Ann. Hematol. 2003, 82: 323-332.

41. Sekiya, T., Adachi, S., Kohu, K., Yamada, T., Higuchi, O., Furukawa,Y., Nakamura, Y., Nakamura, T., Tashiro, K., Kuhara, S., Ohwada, S.and Akiyama, T., Identification of BMP and activin membrane-bound inhibitor (BAMBI), an inhibitor of transforming growthfactor-beta signaling, as a target of the beta-catenin pathway incolorectal tumor cells. J. Biol. Chem. 2004, 279: 6840-6846.

42. Shi, Y., Parhar, R.S., Zou, M., Baieti, E., Kessie, G., Farid, N.R.,Alzahrani, A. and Al-Mohanna, F.A., Gene therapy of anaplasticcarcinoma with a single chain interleukin 12 fusion protein. Hum.Gene Ther. 2004, 14: 1741-1751.

43. Shi, Y. and Zou, M., Martix metalloproteases in thyroid cancer In:Molecular Basis of Thyroid Cancer, Farid, N.R. (ed.), Kluwer Acad.,Boston, 2004, pp. 179-190.

44. Staudt, L.M. and Brown, P.O., Genomic view of the immune system.Annu. Rev. Immunol. 2000, 18: 829-859.

45. Takano, T., Hasegawa, Y., Matsuzuka, F., Miyauchi, A., Yoshida, H.,Higaashiyama, T., Kuma, K. and Amino, N., Gene expressionprofiles in thyroid carcinomas. Br. J. Cancer 2000, 83: 1495-1502.

46. Tanaka, K., Sonoo, H., Kurebayashi, J., Nomura, T., Ohkubo, S.,Yamamoto, Y. and Yamamoto, S., Inhibition of infiltration andangiogenesis by thrombospondin-1 in papillary thyroid carcinoma.Clin. Cancer Res. 2002, 8: 1125-1131.

47. Uren, A., Reichsman, F., Anest, V., Taylor, W.G., Muraiso, K.,Bottaro, D.P., Cumberledge, S. and Rubin, J.S., Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasicmodulator of wnt signaling. J. Biol. Chem. 2000, 275: 4374-4382,

48. Wilkin, F., Suarez-Huerta, N., Robaye, B., Peetermans, J., Libert, F.,Dumont, J.E. and Maenhaut, C., Characterization of aphosphoprotein whose mRNA is regulated by the mitogenicpathways in dog thyroid cells. Eur. J. Biochem, 1997, 248: 660-668.

186 L.G. Laszlo et al.

49. Wright, G., Tan, B., Rosenwald, A., Hurt, E.H., Wiester, A. andStaudt, L.M., Agene expression-based method to diagnose clinicallydistinct subgroups of diffuse large B cell lymphoma. Proc. Natl.Acad. Sci. USA 2003, 100: 9991-9996.

50. Zarrilli, R., Oates, E.L., McBride, O.W., Lerman, M.I., Chan, J.Y.,Santisteban, P., Ursini, M.V., Notkins, A.L. and Kohn, L.D.,Sequence and chromosomal assignment of a novel cDNA identifiedby immunoscreening of a thyroid expression library: similarity to afamily of mitochondrial solute carrier proteins. Mol. Endocrinol.1989, 3: 1498-1505.

51. Zvara, A., Heckler Jr., L., Nagy, Z.B., Micsik, T. and Puskas, L.G.,New molecular methods for classification, diagnosis and therapyprediction of hemtological malignancies. Pathol. Oncol. Res. 2002,8: 231-240.

52. Zou, M., Famluski, K.S., Parhar, R.S., Baitei, E., Al-Mohanna, F.A.,Farid, N.R. and Shi, Y., Microarray analysis of metastatis-associatedgene expression profiling in a murine model of thyroid carcinomapulmonary metastasis: Identification of S100A4 (Mts1) geneoverexpressionas a poor prognostic marker for thyroid carcinoma. J.Clin. Endocrinol. Metabol. 89: 6146-6154.