Oncogene (2008) 27, 1554–1561 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Molecular profiling related to poor prognosis in thyroid carcinoma. Combining gene expression data and biological information C Montero-Conde1, JM Martı´ n-Campos2,3, E Lerma4, G Gimenez5, JL Martı´ nez-Guitarte6, N Combalı´ a7, D Montaner8, X Matı´ as-Guiu6, J Dopazo8,9,10, A de Leiva11,12, M Robledo1,9 and D Mauricio11,13 1Hereditary Endocrine Cancer Research Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain; 2Research Institute, Santa Creu and Sant Pau Hospital, Barcelona, Spain; 3Biochemistry and Molecular Biology Department, Autonomous University of Barcelona (UAB), Barcelona, Spain; 4Pathology Service, Santa Creu and Sant Pau Hospital, Barcelona, Spain; 5Endocrinology Unit, Hospital of Sabadell, Spain; 6Department of Pathology and Molecular Genetics, Arnau de Vilanova Hospital (IRBLLEIDA), Lleida, Spain; 7Pathology Service, Hospital of Sabadell, Spain; 8Bioinformatics Department, Centro de Investigacio´n Prı´ncipe Felipe (CIPF), Valencia, Spain; 9ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Spain; 10Functional Genomics Node INB, Spain; 11Department of Endocrinology and Nutrition, Santa Creu and Sant Pau Hospital, Barcelona, Spain; 12ISCIII CIBER-BBN, Spain and 13Department of Endocrinology, Arnau de Vilanova Hospital (IRBLLEIDA), Lleida, Spain Undifferentiated and poorly differentiated thyroid tumors provide a potential prognosis predictor as well as targets are responsible for more than half of thyroid cancer for new therapies. patient deaths in spite of their low incidence. Conventional Oncogene (2008) 27, 1554–1561; doi:10.1038/sj.onc.1210792; treatments do not obtain substantial benefits, and the published online 17 September 2007 lack of alternative approaches limits patient survival. Additionally, the absence of prognostic markers for Keywords: anaplastic thyroid cancer; functional profile; well-differentiated tumors complicates patient-specific molecular signature treatments and favors the progression of recurrent forms. In order to recognize the molecular basis involved in tumor dedifferentiation and identify potential markers for thyroid cancer prognosis prediction, we analysed the Introduction expression profile of 44 thyroid primary tumors with different degrees of dedifferentiation and aggressiveness Approximately 122 000 new cases of thyroid carcinoma using cDNA microarrays. Transcriptome comparison of are diagnosed each year worldwide, and more than dedifferentiated and well-differentiated thyroid tumors 90% have a follicular cell origin. Malignant tumors of identified 1031 genes with >2-fold difference in absolute thyroid follicular cells have been classified either as values and false discovery rate of o0.15. According to well-differentiated thyroid carcinoma (WDTC), which known molecular interaction and reaction networks, the is composed of papillary and follicular thyroid carcino- products of these genes were mainly clustered in the ma (PTC and FTC, respectively), or as undifferentiated/ MAPkinase signaling pathway, the TGF-b signaling anaplastic thyroid carcinoma (ATC). The majority of pathway, focal adhesion and cell motility, activation of WDTC are indolent malignancies with a 10-year survival actin polymerization and cell cycle. An exhaustive search of up to 90% (Randolph and Daniels, 2002). In contrast, in several databases allowed us to identify various ATC is one of the most aggressive human malignancies. members of the matrixmetalloproteinase, melanoma Despite its low frequency (o5% of all thyroid carcino- antigen A and collagen gene families within the upregu- mas), ATC is responsible for more than half of thyroid lated gene set. We also identified a prognosis classifier carcinoma deaths, with a mean survivalof 6 months comprising just 30 transcripts with an overall accuracy of after diagnosis (Ain, 1999). However, severalstudies 95%. These findings may clarify the molecular mechan- (Sakamoto et al., 1983; Sobrinho-Simoes et al., 2002) isms involved in thyroid tumor dedifferentiation and confirm the existence of a class of tumors intermediate between WDTC and ATC, namely poorly differentiated thyroid carcinomas (PDTC), which represent entities in Correspondence: Dr M Robledo, Hereditary Endocrine Cancer Group progress from a pre-existing WDTC to ATC, often with Leader, Spanish NationalCancer Centre, MelchorFerna ´ ndez Alma- a similar outcome as ATC. gro 3, E-28029 Madrid, Spain and Dr D Mauricio, Department of Primary treatment of follicular cell-derived tumors, Endocrinology, Arnau de Vilanova Hospital, Av. Alcalde Rovira which has not substantially changed in recent decades, Roure 80, E-25198 Lleida, Spain. 131 E-mails: [email protected] and [email protected] is mainly based on surgical treatment, ablative I Received 9 May 2007; revised 13 July 2007; accepted 9 August 2007; treatment, and suppressive L-thyroxine administration published online 17 September 2007 (Vini and Harmer, 2002). Benefits obtained from Molecular profiling in thyroid cancer C Montero-Conde et al 1555 chemotherapy and radiation therapy remain only Results marginal(Cooper et al., 2006), and metastases, recurrent disease and poorly differentiated/anaplastic Differential gene expression related to tumor tumors still pose major challenges to clinicians. There dedifferentiation is a lack of alternative treatments for tumors with a Genes found differentially expressed in the two pairwise poor response to conventionaltreatment, or those comparisons ATC/PDTC versus FTC and ATC/PDTC with a poor degree of differentiation. We are thus in versus PTC, were assumed to be functionally related to need of new approaches that may lead to improved dedifferentiation and clinical aggressiveness. Genes disease management. Microarray analysis of gene- differentially expressed in only one of the comparisons expression patterns has provided a way to identify were supposed to be related to the specific etiology of molecular events related to specific tumor types and to each WDTC class. specific cancer processes, such as metastasis, prolifera- Taking into account only transcripts common to both tion and angiogenesis (Glinsky, 2006). Numerous pairwise comparisons, 742 clones corresponding to 594 studies also have demonstrated its potential for the genes and 83 ESTs were overexpressed and 678 clones identification of molecular signatures associated with corresponding to 535 genes and 78 ESTs were under- different clinical features (van de Vijver et al., 2002; expressed in the ATC/PDTC tumor set. Huang et al., 2003), that could help identify novel targets for therapy. In the present study, using one of the largest series Functional profiling: pathways and gene families involved of frozen thyroid carcinoma tumors published to date, in malignancy progression in thyroid cancer we have carried out an extensive microarray analysis The FatiScan test for gene set enrichment analysis was followed by an innovative bioinformatics approach, applied to analyse the transcriptional profiles of ATC/ the GEPAS package (Montaner et al., 2006; http:// PDTC versus WDTC. We observed a significant upregu- www.gepas.org), to process and interpret the results lation of the biological process ‘cell cycle progression’ taking into account the clinical data. We have com- (adjusted P-valueo0.05), and a downregulation of pared the gene-expression profiles, obtained using hormone biosynthesis, a thyroid-specific function the CNIO Oncochip, of ATC and PDTC cases (adjusted P-valueo0.05). ‘Cell cycle progression’ versus WDTC samples. We first selected the genes includes mitosis, DNA replication and repair, micro- differentially expressed in ATC/PDTC versus FTC and tubule and actin-filament-based processes, and chromo- ATC/PDTC versus PTC, as representing dedifferentia- some reorganization and biogenesis. tion and clinical aggressiveness features, and analysed The additional analysis of gene biological function their functional implications by identifying molecular revealed gene enrichment in several pathways and pathways involved. We were able to define cellular KEGG terms. Interestingly, the MAPkinase signaling processes important for malignant progression of thyroid pathway, the TGF-b signaling pathway, ‘focal adhesion cancer. Moreover, we found a prognosis signature that and cell motility’, and ‘activation of actin polymeriza- recognizes not only undifferentiated and poorly differ- tion’ presented an overexpression of core genes for entiated tumors, but also recurrent and metastatic pathway activation and an underexpression for inhibi- WDTC. These results may provide new perspectives for tors of the same processes. Table 1 shows the diagnosis, treatment and design of new therapeutic differentially expressed genes (FDR o0.15, >2-fold) strategies for thyroid carcinoma patients with poor within these functionalcategories. We alsofound that prognosis. some of the regulated genes with similar transcription Table 1 Enriched KEGG pathways with over- and underexpressed genes (FDR-adjustedo0.15 and differences in absolute values>2-fold) in ATC/PDTC versus PTC and versus FTC KEGG pathway term Overexpressed genes Underexpressed genes MAPK signaling AKT2, SOS1, FGFR1, MAPK12, ATF4, STMN1, MYC, ELK1, PLA2G6, NF1, CASP2, DUSP14 and CASP9 pathway TGFB2, RAC1, HRAS, CRKL, ARRB2, MINK1, TGFBR2, NRAS, MAP4K4, MKNK1, MAPK8IP1, CDC25B, MAP2K3, PRKCA and MAP3K8 Cell cycle MCM7, MAD2L1, CDK2, PKMYT1, HDAC2, ORC6L, CDKN2C, GSK3B, E2F2, MCM10,