Molecular characterization of the response to chemotherapy in conventional osteosarcomas: Predictive value of HSD17B10 and IFITM2 S ebastien Salas 1,2 * , Pascal J ez equel 3 , Loic Campion 4 , Jean-Laurent Deville 1,2 , Fr ed eric Chibon 5 , Catherine Bartoli 2 , Jean-Claude Gentet 6 , Catherine Charbonnel 3,4 , Wilfried Gouraud 3,4 , Brigitte Voutsinos-Porche 2 , Anne Brouchet 7 , Florence Duffaud 1 , Dominique Figarella-Branger 2,8 and Corinne Bouvier 2,8 1 Service Oncologie M edicale, Hoˆpital de la Timone, Assistance Publique-Hopitaux de Marseille (AP-HM), Marseille, France 2 UMR 911, Facult  e de M edecine de Marseille, Marseille, France 3 Unit  e Mixte de G enomique du Cancer/D epartement de Biologie Oncologique - CLCC Ren e Gauducheau, Nantes, France 4 DIEM, CLCC Ren e Gauducheau, Nantes, France 5 D epartement de pathologie, CLCC Bergoni  e, Bordeaux, France 6 Service d’Oncologie p ediatrique, Hoˆpital de la Timone, APHM, Marseille, France 7 Service d’Anatomopathologie, Hoˆpital Rangueil, CHU Toulouse, France 8 Service d’Anatomopathologie, Hoˆpital de la Timone, AP-HM, Marseille, France The therapy regimen of high-grade osteosarcoma includes chemo- therapy followed by surgical resection and postoperative chemo- therapy. The degree of necrosis following definitive surgery remains the only reliable prognostic factor and is used to guide the choice of postoperative chemotherapy. The aim of this study was to find molecular markers able to classify patients with an os- teosarcoma as good or poor responders to chemotherapy before beginning treatment. Gene expression screening of 20 nonmeta- static high-grade osteosarcoma patients was performed using cDNA microarray. Expression of selected relevant genes was vali- dated using QRT-PCR. Immunohistochemistry on tissue microar- rays sections of 73 biopsies was performed to investigate protein expression. Fluorescent in situ hybridization was performed for RPL8 gene. We have found that HSD17B10 gene expression was up-regulated in poor responders and that immunohistochemistry expression of HSD17B10 on biopsy before treatment was correlated to response to chemotherapy. Other results include correlation of IFITM2, IFITM3, and RPL8 gene expression to chemotherapy response. A statistical correlation was found between polysomy 8 or gain of RPL8 and good response to chemo- therapy. These data suggest that HSD17B10, RPL8, IFITM2, and IFITM3 genes are involved in the response to the chemotherapy and that HSD17B10 may be a therapeutic target. RPL8 and IFITM2 may be useful in the assessment at diagnosis and for strat- ifying patients taking part in randomized trials. ' 2009 UICC Key words: conventional osteosarcoma; predictive markers; response to chemotherapy; cDNA microarray; QRT-PCR, FISH; immunohistochemistry Osteosarcoma, the most common type of primary bone cancer is a rare disease. Approximately 900 new cases of osteosarcoma are diagnosed each year in the United States (http://www.cancer. org/docroot/home/index.asp) and 200 in France, including 150 in children (http://www.fnclcc.fr/sor/SSP/CancersEnfant/PeauTissus Soutien/Osteosarcome). Adjuvant and neoadjuvant chemotherapy have significantly improved the long-term survival rate for patients with osteosarcoma. 1–3 Nevertheless, recurrent disease still occurs in about 30–40% of patients and more than 70% of them die of their tumor, despite second-line treatment. The standard therapy regimen of high-grade osteosarcoma includes induction by multiagent chemotherapy followed by surgi- cal resection and postoperative chemotherapy. 4 The percentage of necrotic tissue following induction chemotherapy is classified using the Huvos grading system. 5 Patients with <90% tumor necrosis following induction therapy are classified as poor res- ponders, or Huvos Grade I/II 6 whereas more than 90% necrosis corresponds to Huvos Grade III and complete necrosis to Huvos Grade IV. Good responders correspond to Huvos Grade III/IV. The degree of necrosis remains the only reliable prognostic factor for the patients presenting with localized disease and is used to guide the choice of postoperative chemotherapy. Numerous clinical trials have attempted to increase the disease- free survival rate for poorly responding patients with intensified postoperative therapy. 1,7–10 Intensified first-line chemotherapy regimens could improve prognosis, but the risk is over-treatment of patients who could benefit from less aggressive regimens. 1,7–10 No survival benefit has been convincingly shown through the administration of more intensified therapy to poor responders. This suggests that there may be an innate biological difference between good responsive and poor responsive tumors. Therefore, there is a need to identify at the time of diagnosis predictive fac- tors for the response to chemotherapy. The aim of this study was to identify by cDNA microarray technology a genetic fingerprint that predicts response to therapy. Only few studies comparing gene expression profiles of pretherapeutic biopsies of responsive or nonresponsive patients with an osteosarcoma are available in the literature. 11–13 This may be because of the rarity of the tumor and to the difficulty to get frozen specimens from preoperative material. Previous studies have shown that in chemoresistant tumors, genes involved in osteoclastogenesis, extracellular matrix remodeling, bone development, tumor progression, drug resistance and angiogenesis are up-regulated. 11–16 It is intriguing that there is apparently no overlap between the gene sets identified by the dif- ferent groups of investigators. We have found in this study that HSD17B10 gene expression was significantly up-regulated in the group of poor responders and that immunohistochemical expression of HSD17B10 on biopsy prior treatment was correlated to the response to chemotherapy. Other relevant results include correlation of IFITM2, IFITM3 and RPL8 gene expression to the response to chemotherapy. Material and methods Patients and tumor specimens Eighty-one patients treated for osteosarcoma at the La Timone hospital and at the Rangueil Hospital, between 1993 and 2007 in the pediatric and adult oncological departments, were included in this study. All tumors arose de novo. All patients received preop- erative polychemotherapy with high-dose Methotrexate. Eighty patients received preoperative and postoperative chemotherapy derived from SFOP OS94 regimen 17 and 1 patient had preopera- tive chemotherapy only. Histological diagnosis was made on Grant sponsors: Philippe Daher’s Foundation (‘‘Appel d’offre APHM 2005’’) and the Institut National du Cancer (INCA). *Correspondence to: Service d’Oncologie M edicale Adulte CHU Timone, APHM, Marseille, France Timone, 264, Rue Saint Pierre 13385 Marseille, France. Fax: 133-4-91-38-76-58. E-mail: sebastien.salas@ap-hm.fr Received 12 July 2008; Accepted after revision 31 March 2009 DOI 10.1002/ijc.24457 Published online 25 March 2009 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 125, 851–860 (2009) ' 2009 UICC Publication of the International Union Against Cancer