Prediction of Sensitivity of Rectal Cancer Cells in Response to Preoperative Radiotherapy by DNA Microarray Analysis of Gene Expression Profiles Toshiaki Watanabe, 1 Yasuhiro Komuro, 1 Tomomichi Kiyomatsu, 1 Takamitsu Kanazawa, 1 Yoshihiro Kazama, 1 Junichiro Tanaka, 1 Toshiaki Tanaka, 1 Yoko Yamamoto, 1 Masatoshi Shirane, 2 Tetsuichiro Muto, 3 and Hirokazu Nagawa 1 1 Department of Surgical Oncology, University of Tokyo Hospital, Hongo, Bunkyo-ku, Tokyo, Japan; 2 Product Research Department, Chugai Pharmaceutical Co. Ltd., Kajiwara, Kamakura, Kanagawa, Japan; and 3 The Cancer Institute Hospital of JFCR, Ariake, Kotoh-ku, Tokyo, Japan Abstract Preoperative radiotherapy has been widely used to improve local control of disease and to improve survival in the treat- ment of rectal cancer. However, the response to radiotherapy differs among individual tumors. Our objective here was to identify a set of discriminating genes that can be used for characterization andprediction of response to radiotherapy in rectal cancer. Fifty-two rectal cancer patients who underwent preoperative radiotherapy were studied. Biopsy specimens were obtained from rectal cancer before preoperative radio- therapy. Response to radiotherapy was determined by histo- pathologic examination of surgically resected specimens and classified as responders or nonresponders. By determining gene expression profiles using human U95Av2 Gene Chip, we identified 33 novel discriminating genes of which the expres- sion differed significantly between responders and nonres- ponders. Using this gene set, we were able to establish a new model to predict response to radiotherapy in rectal cancer with an accuracy of 82.4%. The list of discriminating genes included growth factor, apoptosis, cell proliferation, signal transduction, or cell adhesion–related genes. Among 33 discriminating genes, apoptosis inducers (lumican, thrombo- spondin 2, and galectin-1) showed higher expression in responders whereas apoptosis inhibitors (cyclophilin 40 and glutathione peroxidase) showed higher expression in non- responders. The present study suggested the possibility that gene expression profiling may be useful in predicting response to radiotherapy to establish an individualized tailored therapy for rectal cancer. Global expression profiles of responders and nonresponders may provide insights into the development of novel therapeutic targets. (Cancer Res 2006; 66(7): 3370-4) Introduction Rectal cancer remains one of leading causes of cancer mortality. Preoperative radiotherapy has been widely used as a major treatment modality to improve local control of the disease as well as to improve survival (1–4). However, response to radiotherapy differs among individual tumors. If we can predict response to radiotherapy before treatment, other treatment modalities such as chemoradiotherapy can be considered for tumors that may not respond to radiotherapy alone. We have previously shown that a few molecular markers could be used to select good candidates among colon cancer patients for adjuvant chemotherapy (5). However, recent advances in expression genomics by DNA microarray have made it possible to analyze tens of thousands of genes at a time and have shown that expression profiles of cancer cells may be used for distinguishing responders and nonresponders to a given drug, as well as predicting toxicity and adverse effects of drugs (6). Until now, there have only been a limited number of studies of expression genomics aimed at predicting response to radiotherapy or chemo- radiotherapy in cancer treatment (7, 8). In rectal cancer, this is the first report using DNA microarray for predicting response to radiotherapy. In this report, to predict response radiotherapy, we examined the gene expression profiles of rectal cancer cells by DNA microarray before preoperative radiotherapy was done. Our objective here was to identify a set of discriminating genes that could be used for characterization of responders and nonresponders to preoperative radiotherapy in rectal cancer. We were able to identify a novel set of genes of which the expression differed significantly between responders and nonresponders. Using this set of genes, we established a new model to predict response to radiotherapy with a high rate of accuracy. Furthermore, we did an ontology analysis of discriminating genes and showed a character- istic signature between responders and nonresponders. This may provide insights into the development of novel therapeutic targets in preoperative radiotherapy for rectal cancer. Materials and Methods Patient Samples Informed consent was obtained from rectal cancer patients for the collection of specimens, and the study protocol was approved by the Ethics Committee of The University of Tokyo. Fifty-two rectal cancer patients who approved to receive preoperative radiotherapy were studied. We prospec- tively collected biopsy specimens during colonoscopic examination from rectal cancer before starting preoperative radiotherapy (Fig. 1). Paralleled tumor specimens were formalin fixed and paraffin embedded for histologic examination and other specimens were used for RNA extraction. Samples were used for RNA extraction when paralleled specimens contained at least 70% tumor cells as previously described (ref. 9; see Supplementary Fig. S1). Samples were snap-frozen in liquid nitrogen and stored at 80jC until use. All patients received a total dose of 50.4 Gy of radiation and underwent standardized curative resection, following an interval of 4 weeks after radiotherapy. Response to Radiotherapy Response to radiotherapy was determined by histopathologic exami- nation of surgically resected specimens based on a semiquantitative Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Toshiaki Watanabe, Department of Surgical Oncology, University of Tokyo Hospital, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Phone: 81-3-5800-8653; Fax: 81-3-3811-6822; E-mail: toshwatanabe@yahoo.co.jp. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-3834 Cancer Res 2006; 66: (7). April 1, 2006 3370 www.aacrjournals.org Priority Report