Gao et al. BMC Cancer 2011, 11:177 http://www.biomedcentral.com/1471-2407/11/177 Page 3 of 8 and hydrated in graded alcohols. After antigen retrieval in 0.01M citrate buffer (pH 6.0), sections were treated with endogenous peroxidase confining liquid (Beijing CoWin Biotech Co., Ltd., Lot. CW0117) for 10 min. Sections were rinsed and incubated with TUBB3 and TP (Beijing CoWin Biotech Co., Ltd.) monoclonal anti- bodies for 60 min, respectively. After rinsing in phos- phate buffered saline (PBS), the sections were incubated with general type IgG-HRP Polymer (Beijing CoWin Biotech Co., Ltd., Lot. CW0117) for 10 min, followed by chromogenic 3,3’-Diaminobenzidine tetra- hydrochloride dihydrate (DAB) for about 2-7min. Finally, sections were conterstained with hematoxylin for 1 min followed by dehydrated in graded alcohols, cleared in xylene, and covered with coverslips. Each experiment included negative control. Sections were examined and scored by two independent professional pathologists of pathology department without any knowledge of this study. TP protein was distributed in cytoplasm and nuclear, TUBB3 in cytoplasm. Staining was graded for intensity of staining according to pre- vious description [31]. Briefly, intensity of staining (1, weak; 2, moderate; 3, strong) and percentage of cells stained (1, 0%~10%; 2, 11%~50%; 3, 51%~100%) were calculated. At last, the expression levels were considered to be positive or negative based on the median staining score (intensity score plus percentage score) as following: negative for score ≤ 3, positive for score > 3. Table 1 Patient Demographics and Clinical Characteristics Characteristic Sex Male female Age, years Median Range KPS 90-100 70-80 Sites of metastatic disease Liver Lung Lymph nodes Peritoneum Others* Histological differentiation# Poor Good 20 13 23 10 12 1 33 6 8 25 8 57 27-74 Statistical Analysis According to TP and TUBB3 protein expression levels, patients were divided into two groups (positive and negative). The relationships between TP, TUBB3 expres- sions and response to capecitabine plus paclitaxel or cis- platin were analyzed using Fisher’s exact test. Kaplan- Meier curves and log-rank test were used to analyze the association between expression levels of biomarkers and survival. Statistical analysis was done using SPSS 13.0 (SPSS Inc, Chicago, Illinois, USA). Results Patient Demographics Fifty-one patients were included in this study between July 2003 to December 2008 in our hospital with male 34, female 17, median age 57 years (range 27-75 years). Thirty-three patients (male 20, female 13, median age 57 years [range 27-74 years]) received capecitabine plus paclitaxel with a median 6 cycles of chemotherapy (cohort 1) and eighteen patients (male 14, female 4, median age 57 years [range 42-75 years]) received cape- citabine plus cisplatin with a median 6 cycles of che- motherapy (cohort 2). The characteristics of 51 patients are presented in Table 1. Response Evaluation and Survival Up to February 2010, all patients had been evaluable for response and 43 patients died. The overall response rate (CR+PR) in 51 patients was 43.1%, with 22 partial Cohort 1 (n = 33) No. of Patients Cohort 2 (n = 18) No. of Patients Total (n = 51) No. of Patients % 60.6 39.4 69.7 30.3 36.4 3.0 100 18.2 24.2 75.8 24.2 NOTE: *Including ovary, adrenal glands, subcutaneous of abdominal wall, pelvic cavity, bone and thoracic cavity. #Poor, including poorly differentiated adenocarcinoma, signet ring cell carcinoma and mucinous adenocarcinoma; Good, including moderate-well differentiated adenocarcinoma. KPS, Kamofsky performance status. 14 4 14 4 6 4 17 3 4 11 7 57 42-75 % 77.8 22.2 77.8 22.2 33.3 22.2 94.4 16.7 22.2 61.1 38.9 34 17 37 14 18 5 50 9 12 36 15 57 27-75 % 66.7 33.3 72.5 27.5 35.3 9.8 98.0 17.6 23.5 70.6 29.4