Expression Profiles of Osteosarcoma That Can Predict Response to Chemotherapy
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Research Article Expression Profiles of Osteosarcoma That Can Predict Response to Chemotherapy Tsz-Kwong Man,1 Murali Chintagumpala,1 Jaya Visvanathan,1 Jianhe Shen,1 Laszlo Perlaky,1 John Hicks,2 Mark Johnson,3 Nelson Davino,3 Jeffrey Murray,4 Lee Helman,5 William Meyer,6 Timothy Triche,7 Kwong-Kwok Wong,1 and Ching C. Lau1 1Departments of Pediatrics, Texas Children’s Cancer Center; Departments of 2Pathology and 3Orthopedic Surgery, Texas Children’s Hospital/Baylor College of Medicine, Houston, Texas; 4Cook Children’s Medical Center, Fort Worth, Texas; 5Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland; 6University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; and 7Children’s Hospital Los Angeles, Los Angeles, California Abstract the resected tumor specimen is assessed for the degree of necrosis, Osteosarcoma is the most common malignant bone tumor in which is a reliable and the only significant prognostic factor in children. After initial diagnosis is made with a biopsy, patients with nonmetastatic disease and is used to guide the choice treatment consists of preoperative chemotherapy followed by of postoperative chemotherapy. Patients whose tumors display z definitive surgery and postoperative chemotherapy. The 90% necrosis (good or favorable response) have an excellent degree of tumor necrosis in response to preoperative prognosis and continue to receive chemotherapy similar to the chemotherapy is a reliable prognostic factor and is used to preoperative regimen. Patients whose tumors display <90% guide the choice of postoperative chemotherapy. Patients necrosis (poor or unfavorable response) have a much higher risk with tumors, which reveal z90% necrosis (good responders), of relapse and poor outcome even after complete resection of the have a much better prognosis than those with <90% necrosis primary tumor (2). To improve the outcome of the poor (poor responders). Despite previous attempts to improve the responders, attempts are usually made to use postoperative outcome of poor responders by modifying the postoperative chemotherapy regimens that are different from the preoperative chemotherapy, their prognosis remains poor. Therefore, there regimen by the addition or replacement of a chemotherapeutic is a need to predict at the time of diagnosis patients’ response agent. Such attempts in the past have been unsuccessful (1, 3) to preoperative chemotherapy. This will provide the basis for partly because the degree of necrosis is known only after 8 to 10 developing potentially effective therapy that can be given at weeks of preoperative therapy. It is possible that resistant tumor the outset for those who are likely to have a poor response. cells have additional time to either metastasize to the lungs or Here, we report the analysis of 34 pediatric osteosarcoma evolve further during the period when ineffective preoperative samples by expression profiling. Using parametric two-sample chemotherapy is given. Therefore, there is a need to identify at the t test, we identified 45 genes that discriminate between good time of initial diagnosis the patients who are likely to have a poor and poor responders (P < 0.005) in 20 definitive surgery response to standard preoperative therapy and therefore a poor out- samples. A support vector machine classifier was built using come eventually. Therapies tailored to improve the outcome for these predictor genes and was tested for its ability to classify those patients identified at the time of diagnosis to have a poor out- initial biopsy samples. Five of six initial biopsy samples that come can then be instituted at the outset when the chance for had corresponding definitive surgery samples in the training success is potentially higher. Although several other prognostic set were classified correctly (83%; confidence interval, 36%, factors have been proposed for predicting the long-term outcome 100%). When this classifier was used to predict eight of osteosarcoma patients, most are still controversial or have not independent initial biopsy samples, there was 100% accuracy been tested in large prospective studies (4–11). (confidence interval, 63%, 100%). Many of the predictor genes Recently, application of microarray technology to classify and are implicated in bone development, drug resistance, and diagnose various types of tumors has yielded promising results tumorigenesis. (Cancer Res 2005; 65(18): 8142-50) (12, 13). However, the use of this technology to predict response to chemotherapy in pediatric solid tumors is still in its infancy. In this Introduction study, we developed a multigene predictive model to classify good and poor responders of osteosarcoma in response to preoperative Osteosarcoma is the most common malignant bone tumor in chemotherapy using gene expression profiling. We used a slightly f children and accounts for 60% of malignant bone tumors different approach from previously published works (14). We first diagnosed in the first two decades of life (1). After the diagnosis is identified a molecular signature of chemoresistance by comparing made by an initial biopsy, standard treatment involves the use of the expression profiles of the definitive surgery samples of the good multiagent chemotherapy, definitive surgery of the primary tumor, responders with those of the poor responders, which, in principle, and postoperative chemotherapy. At the time of definitive surgery, have been enriched for resistant cells. We then tested the hypothesis that this predictor signature of chemoresistance could recognize the resistant cells present in the initial biopsy of some of the same Note: Supplementary data for this article are available at Cancer Research Online cases used in the first analysis (definitive surgery samples), although (http://cancerres.aacrjournals.org/). Requests for reprints: Ching C. Lau, Texas Children’s Hospital, 6621 Fannin Street, these resistant cells might have constituted only a small fraction of MC 3-3320, Houston, TX 77030-2399. Phone: 832-824-4543; Fax: 832-825-4038; E-mail: the primary tumor. Finally, we tested the ability of this signature to [email protected]. I2005 American Association for Cancer Research. predict chemoresistance in an independent set of initial biopsies doi:10.1158/0008-5472.CAN-05-0985 and found that there was 100% accuracy in its prediction. Cancer Res 2005; 65: (18). September 15, 2005 8142 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Chemoresistance Signature in Osteosarcoma Materials and Methods initial biopsy or a definitive surgery specimen. The initial biopsy samples were obtained at the time of diagnosis before the initiation of preoperative Patients and tumor samples. The clinical information of the chemotherapy. The definitive surgery samples were collected after the osteosarcoma samples used in this study is summarized in Table 1. All completion of preoperative chemotherapy. The good responders were samples were collected through institutional review board–approved defined as patients whose tumors had z90% necrosis in response to protocols at four centers (Texas Children’s Hospital/Baylor College of preoperative chemotherapy as determined by histologic examination at the Medicine, Cook Children’s Medical Center, Pediatric Branch of the National time of definitive surgery and poor responders had <90% necrosis. In this Cancer Institute, and University of Oklahoma Health Sciences Center) after study, the percentage necrosis in the poor responders ranged from 5% to informed consents were signed. All but three patients (10, 20, and 26) were 86%. Five of the 28 patients were diagnosed with metastatic disease at enrolled in the same treatment protocol, the schema of which is shown in presentation. Immediately after collection, tumor specimens were snap Fig. 1. All patients, including the three not enrolled in the protocol, received frozen in liquid nitrogen and stored at À80jC until RNA extraction. All the same preoperative chemotherapy consisting of cisplatin, doxorubicin, samples used for RNA extraction were immediately adjacent to the frozen and high-dose methotrexate, except patients 3, 4, 8, and 24, who received sections used for diagnostic purpose and were representative of the only cisplatin and doxorubicin. All pathologic diagnoses were centrally corresponding tumors. All initial biopsy specimens were confirmed to reviewed by a single pathologist (J.H.). A total of 34 samples (14 initial contain >80% tumor cells. biopsies and 20 definitive surgery specimens) were included in this study, RNA extraction. Total RNA was extracted from tissues and cultured cells which were obtained from 28 individual patients, 18 males and 10 females. using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufac- The age of the patients ranged from 9 to 22 years. Six patients contributed turer’s protocol. Osteosarcoma samples were homogenized in TRIzol two samples each, both initial biopsies and definitive surgery specimens, reagent using 1 mL per 50 to 100 mg tissue. Normal human osteoblast whereas the remaining 22 patients contributed one sample each, either an primary culture cells obtained from Clonetics (San Diego, CA) were used as Table 1. Clinical information of the osteosarcoma samples used in this study Patient Tumor ID IB/DS Age (y) Gender Metastatic Histologic Recurrence Follow-up (mo) Status status response 1 197 IB 12 F N PR Yes 60 NED* 221 DS 2 207 IB 13 M N PR No 59 NED 236 DS 3 278 IB 13