Cervical cancer samples in NIRS. Homo sapiens

cervical cancer samples to test radiosensitivity Keywords: comparison between pre-irradiation and mid-irradiation status, correlation to prognosis 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.

用于检测放射敏感性的宫颈癌样本 关键词：放疗前与放疗中期状态对比、与预后的相关性

整体实验设计：

一、患者特征与活检样本
本研究纳入63例经预处理采样后接受治疗的子宫颈肿瘤患者，所有患者均签署知情同意书，符合日本国立放射科学研究所（National Institute of Radiological Sciences）机构伦理审查委员会的相关要求。临床分期与组织学分类依据国际妇产科联盟（International Federation of Gynecology and Obstetrics, FIGO）的标准制定。
25例患者接受常规放射治疗（radiotherapy, RT）：全骨盆照射剂量为30.6 Gy，随后针对宫旁组织加量照射联合中心挡铅，总剂量达50.6 Gy，同时联合192Ir高剂量率腔内近距离放疗。另有28例患者在上述常规放疗基础上联合化疗：每周给予顺铂（CDDP，40mg/m²），共给药5次。剩余10例患者接受碳离子放疗（HIMAC）：全骨盆照射剂量为25 GyE，随后针对宫旁组织及肿瘤加量照射，总剂量达45 GyE。
治疗开始1周后进行第二次活检，具体操作参照文献[14]。此时，常规放疗组与放化疗联合组患者已接受全骨盆照射9 Gy，放化疗联合组已单次给予顺铂，但尚未启动近距离放疗；碳离子放疗组患者已接受12 GyE的照射剂量。
每份活检样本均分为两部分：一半立即置于RNAlater（Ambion, 美国德克萨斯州奥斯汀市）中，加入体积与样本相当的RNAlater；另一半经10%甲醛固定后进行石蜡包埋。
分别于治疗后6个月、1年及2年进行CT扫描，以评估治疗应答情况，分为完全缓解（complete remission, CR）、部分缓解（partial remission, PR）及疾病进展（progressive disease, PD）。
对60例患者完成了超过2年的随访。无任何疾病复发证据的患者定义为应答良好者；带瘤复发、新发转移或因上述事件死亡的患者定义为应答不良者。
本研究从63例病例中选取10例确诊为FIGO ⅢB期鳞状细胞癌（squamous cell carcinoma, SCC）的放化疗联合组患者，以及14例鳞状细胞癌的常规放疗组患者作为训练集，剩余病例作为测试集（详见表1）。

二、总RNA提取、探针制备与微阵列杂交
按照制造商说明书，使用ISOGEN（Nippon gene, 日本东京）及RNeasy总RNA迷你试剂盒（Qiagen, 德国希尔德）纯化总RNA。通过2100生物分析仪（Agilent Technologies, 美国加利福尼亚州帕洛阿尔托）检测28S与18S核糖体RNA的完整性，以验证RNA样本质量。
取2 μg总RNA，按照CodeLink表达分析试剂盒（GE Healthcare, 美国新泽西州皮斯卡塔韦）说明书合成双链cDNA及后续的cRNA。生物微阵列经Cy5-链霉亲和素（Amersham Biosciences, 美国新泽西州皮斯卡塔韦）染色后，使用Agilent双激光微阵列扫描仪（Agilent Technologies, 美国加利福尼亚州帕洛阿尔托）进行扫描。

三、微阵列数据分析
针对每个样本的多组微阵列数据，通过统计相关性检查数据质量。使用CodeLink表达分析软件（Ver4.0；GE Healthcare）对斑点信号进行定量。采用误差加权算法对3~4次重复实验的扫描数据取平均值。进一步使用基因表达分析软件Resolver version 4.0（Rosetta Biosoftware, 美国华盛顿州西雅图）对输出数据进行分析，该软件采用误差建模方法开展微阵列数据分析。分析前先过滤掉所有未检出的信号。本步骤重复采用误差加权算法对3~4次重复实验的扫描数据取平均值。