Overall design |
Patients Characteristics and Biopsy Samples
The subjects were 63 patients with uterine cervix tumors who had been treated after pretreatment sampling. All provided informed consent in accordance with the Institutional Ethical Review Committee at National Institute of Radiological Sciences. Clinical stages and histological classification were based on criteria of the International Federation of Gynecology and Obstetrics. Twenty-five patients received conventional radiotherapy (RT) consisting of 30.6 Gy to the whole pelvis, plus an additional dose to parametria with central shielding to complete 50.6 Gy, along with 192Ir high dose-rate intracavitary brachytherapy. The other 28 patients received radiotherapy as same as RT and chemotherapy consisting of five weekly administrations of CDDP (40mg/m2). The other 10 patients received carbon-ion radiotherapy (HIMAC) for local tumor consisting of 25 GyE to the whole pelvis plus an additional dose to parametria and tumors to complete 45 GyE. A second biopsy was taken one week after the start of therapy as described elsewhere (14). At that point, patients in CRT and RT groups had received 9 Gy of whole pelvic irradiation and CRT group had received a single dose of CDDP, but brachytherapy had not started yet. HIMAC group had received 12 GyE. One half of each sample was placed immediately in RNAlater (Ambion, Austin, TX, USA) at half volume of specimen, and the other half was fixed in 10% formalin and embedded in paraffin. A CT scan at 6 months, 1, and 2 years was used to diagnose a response of therapy as complete remission (CR), partial remission (PR), or progressive disease (PD). The prognosis was followed up for more than 2 years in 60 patients. Patients, who did not develop any evidence of disease, were defined as good responders, and patients, who were alive with recurred tumor or newly developed metastasis, or died of them, were defined as poor responders. From 63 cases, 10 CRT who were diagnosed as FIGO stage IIIB with squamous cell carcinoma (SCC) and 14 RT cases with SCC were selected as training set, and the rest of cases were used for test samples (Table 1).
Extraction of Total RNA, Probe Preparation and Microarray Hybridization
We purified total RNA using ISOGEN (Nippon gene, Tokyo, Japan) followed by the RNeasy Total RNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturers' recommendations. The quality of RNA samples was verified by examining the integrity of 28S and 18S rRNA using 2100 Bioanalyzer (Agilent Technologies, Palp Alto, CA, USA). Double-stranded cDNA and subsequent cRNA was synthesized from 2 micrograms of total RNA using the CodeLink Expression Assay Kit (GE Healthcare, Piscataway, NJ, USA) according to manufacturer's instructions. Bioarrays were stained with Cy5-streptavidin (Amersham Biosciences, Piscataway, NJ, USA) and scanned using the Agilent dual-laser Microarray Scanner (Agilent Technologies, Palo Alto, CA, USA).
Analysis of Microarray Data
We examined all data from multiple arrays for each sample, using statistical correlation as a means of checking quality. Spot signals were quantified using CodeLink Expression Analysis Software (Ver4.0; GE Healthcare). We averaged scan data from 3-4 replicate experiments using an error-weighted algorithm. An output data was further analyzed by the Resolver version 4.0, a gene expression analysis software (Rosetta Biosoftware, Seattle, WA, USA). This software employs an error-modeling approach for the analysis of microarray data. All non-detection calls were filtered prior to performing analysis. We averaged scan data from 3-4 replicate experiments using an error-weighted algorithm.
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