An Expression Signature Classifies Chemotherapy-Resistant Pediatric Osteosarcoma Michelle B. Mintz, 1,3 Rebecca Sowers, 6 Kevin M. Brown, 2 Sara C. Hilmer, 4 BethAnne Mazza, 7 Andrew G. Huvos, 8 Paul A. Meyers, 7 Bonnie LaFleur, 10 Wendy S. McDonough, 1 Michael M. Henry, 4 Keri E. Ramsey, 1 Cristina R. Antonescu, 8 Wen Chen, 7 John H. Healey, 9 Aaron Daluski, 11 Michael E. Berens, 1 Tobey J. MacDonald, 5 Richard Gorlick, 6 and Dietrich A. Stephan 1 1 Neurogenomics Division and 2 Genetic Basis of Human Disease Division, Translational Genomics Research Institute, Phoenix, Arizona; 3 Department of Genetics, George Washington University; 4 Research Center for Genetic Medicine and 5 Center for Cancer Research, Children’s National Medical Center, Washington, District of Columbia; 6 Department of Pediatrics, Children’s Hospital at Montefiore, Bronx, New York; Departments of 7 Pediatrics, 8 Pathology, and 9 Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York; 10 Department of Preventative Medicine, Vanderbilt University Medical Center, Nashville, Tennessee; and 11 Department of Orthopedics, Cornell University Medical Center, Ithaca, New York Abstract Osteosarcoma is the most common malignant bone tumor in children. Osteosarcoma patients who respond poorly to chemotherapy are at a higher risk of relapse and adverse outcome. Therefore, it was the aim of this study to identify prognostic factors at the time of diagnosis to characterize the genes predictive of poor survival outcome and to identify potential novel therapeutic targets. Expression profiling of 30 osteosarcoma diagnostic biopsy samples, 15 with inferior necrosis following induction chemotherapy (Huvos I/II) and 15 with superior necrosis following induction chemotherapy (Huvos III/IV), was conducted using Affymetrix U95Av2 oligonucleotide microarrays. One hundred and four genes were found to be statistically significant and highly differen- tially expressed between Huvos I/II and III/IV patients. Statistically significant genes were validated on a small independent cohort comprised of osteosarcoma xenograft tumor samples. Markers of Huvos I/II response predominantly were gene products involved in extracellular matrix (ECM) microenvironment remodeling and osteoclast differentiation. A striking finding was the significant decrease in osteoprote- gerin, an osteoclastogenesis inhibitory factor. Additional genes involved in osteoclastogenesis and bone resorption, which were statistically different, include annexin 2 , SMAD, PLA2G2A , and TGFB1 . ECM remodeling genes include desmo- plakin , SPARCL1 , biglycan , and PECAM. Gene expression of select genes involved in tumor progression, ECM remodeling, and osteoclastogenesis were validated via quantitative reverse transcription-PCR in an independent cohort. We propose that osteosarcoma tumor–driven changes in the bone microenvi- ronment contribute to the chemotherapy-resistant phenotype and offer testable hypotheses to potentially enhance thera- peutic response. (Cancer Res 2005; 65(5): 1748-54) Introduction Osteosarcoma is a primary malignant tumor of the bone. Osteosarcoma accounts for f5% of childhood tumors in the United States and is the most common malignant bone tumor in children (1). Over the past three decades, advances in treatment have been responsible for improved limb salvage, reduced metastases, and overall higher survival rates (1). Multiagent dose- intensive chemotherapy regimens have resulted in long-term disease-free survival rates of f60% to f76% in patients with localized disease. Osteosarcoma patients whose tumors respond poorly to chemotherapy are at a higher risk of relapse and adverse outcome. Therefore, it is imperative to identify prognostic factors at the time of diagnosis to detect chemotherapy-resistant tumors and to generate a modified treatment regimen. The standard therapy regimen of high-grade osteosarcoma includes induction multiagent chemotherapy followed by surgical resection and postoperative chemotherapy (2). Induction therapy allows for treatment of micrometastatic disease, tumor shrinkage, and decreased tumor vascularity, thus facilitating the surgical removal of the tumor (3). The percentage of necrotic tissue following induction chemotherapy is classified using the Huvos grading system, where the various levels of necrosis reflect the effectiveness of the given therapy (Table 1; ref. 4). Patients with <90% tumor necrosis following induction therapy are classified as inferior responders, or Huvos grade I/II (1). To date, aside from metastatic lesions at presentation, histologic response to chemo- therapy is the most dependable and reproducible prognostic indicator of the probability of disease-free survival (1). The degree of necrosis following definitive surgery remains the only consistent prognostic factor in the majority of patients presenting with apparently localized disease. As treatment regimens have evolved over time, numerous clinical trials have attempted to increase the disease-free survival rate for poorly responding patients with intensified postoperative therapy. However, no survival benefit has been convincingly shown through the administration of more intensified therapy to poor responders. This conclusion has been reached by several independent clinical trials (5–7). This suggests that there may be an innate biological difference between responsive and nonresponsive tumors. Although Huvos grading is a powerful predictor of survival, the prognostic marker is ineffective in altering the outcome of chemoresistant tumors, as this indicator can only be determined after therapy has already been given. For patients who display inferior histologic response to chemotherapy, it is crucial to determine the biological factors that drive lack of response at the time of diagnosis. The importance of this is twofold: First, to identify a genetic fingerprint that will distinguish patients as good or poor responders before therapy; second, to identify potential Requests for reprints: Dietrich A. Stephan, Neurogenomics Division, Translational Genomics Research Institute, 445 North Fifth Street, Phoenix, AZ 85004. Phone: 602- 343-8727; Fax: 602-334-8844; E-mail: dstephan@tgen.org. I2005 American Association for Cancer Research. Cancer Res 2005; 65: (5). March 1, 2005 1748 www.aacrjournals.org Research Article Research. on May 5, 2021. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from