Zempolich K, Fuhrman C, Milash B, Flinner R, Greven K, Ryu J, Forbes A, Kerlin K, Nichols R C, Gaffney D K
University of Utah and Huntsman Cancer Institute, USA.
Gynecol Oncol. 2008 May;109(2):275-9. doi: 10.1016/j.ygyno.2008.01.027. Epub 2008 Mar 4.
To evaluate gene expression patterns in patients with advanced cervix cancer before and during chemoradiation in a multi-institutional cooperative group setting.
RTOG C0128 was designed as a Phase II trial of radiation therapy with concomitant chemotherapy and Celecoxib at 400 mg twice daily for one year. Tumor samples were obtained for microarray gene expression analysis before treatment and at the time of the first implant (paired sample). RNA was extracted, linearly amplified, and purity was assessed by gel electrophoresis. Each sample was hybridized against a universal RNA mixture on a customized spotted array consisting of >10,000 genes. Gene expression pre-treatment was compared with clinical characteristics. Changes in gene expression following radiation were assessed within the paired samples (same patient) and then compared across all paired samples. Data were normalized using the AROMA software, and clustering analysis was performed using Ward's method in Spotfire. Differences in paired samples were calculated with Significance Analysis of Microarrays (SAM).
From August 2001 to March 2004, 84 patients were accrued to the trial. Tissue was obtained prior to initiation of therapy from 34 patients (40%). FIGO stages of the patients providing tissue were IB (23%), II (57%), and IIIA-IVA (20%). RNA quality was sufficient in 22 pre-treatment and 14 post-treatment samples. Among pre-treatment samples, no significant differences in gene expression were observed by FIGO stage, age, or race. However, between comparison of histologic subtypes (adenocarcinoma, n=5; squamous cell carcinoma, n=17) demonstrated 45 genes differentially expressed with a false discovery rate of 0.018. Cluster analysis segregated unpaired samples into 2 groups: 18/22 comprising pre-treatment samples and 10/14 in group 2 representing post-treatment samples. In all 13 paired samples, gene expression after chemoradiation was significantly upregulated in 91 genes and downregulated in 251 genes (false discovery rate of 0.0018). Genes significantly upregulated included bax, cdk inhibitor 1, MMP2, and adhesion molecules PECAM1, VCAM1, and ICAM2. Genes significantly downregulated included topoisomerase II alpha, myc, H2AX, MSH2, RAD51, RAD53, PCNA, and cell cycle-regulating molecules chk1, CDK2, cyclinB1, cyclin D3, cdc2, and cdc25.
Microarray analysis was successfully performed in a multi-institutional cooperative group trial. Gene expression significantly correlated with histology, but not stage, age or race. Cluster analysis identified two groups of gene expression profiles correlating with pre or post-treatment acquisition of tissue. Notably, paired samples showed significant changes in gene expression following chemoradiation, including several downregulated radiation response genes. Further analysis comparing gene expression to clinical outcomes, acute and late toxicities awaits maturation of clinical data. Hopefully, this data will lead to the development of molecularly based therapies.
在多机构合作组环境中评估晚期宫颈癌患者在放化疗前及放化疗期间的基因表达模式。
RTOG C0128被设计为一项II期试验，采用放射治疗联合化疗以及每日两次服用400毫克塞来昔布，持续一年。在治疗前和首次植入时（配对样本）获取肿瘤样本用于微阵列基因表达分析。提取RNA，进行线性扩增，并通过凝胶电泳评估纯度。每个样本与由超过10,000个基因组成的定制点样阵列上的通用RNA混合物进行杂交。将治疗前的基因表达与临床特征进行比较。在配对样本（同一患者）内评估放疗后基因表达的变化，然后在所有配对样本之间进行比较。使用AROMA软件对数据进行归一化处理，并在Spotfire中使用Ward方法进行聚类分析。使用微阵列显著性分析（SAM）计算配对样本中的差异。
从2001年8月至2004年3月，84名患者入组该试验。34名患者（40%）在开始治疗前获取了组织。提供组织的患者的国际妇产科联盟（FIGO）分期为IB期（23%）、II期（57%）和IIIA-IVA期（20%）。22个治疗前样本和14个治疗后样本的RNA质量足够。在治疗前样本中，按FIGO分期、年龄或种族未观察到基因表达的显著差异。然而，在组织学亚型（腺癌，n = 5；鳞状细胞癌，n = 17）的比较中，显示有45个基因差异表达，错误发现率为0.018。聚类分析将未配对样本分为两组：第1组18/22个为治疗前样本，第2组10/14个为治疗后样本。在所有13个配对样本中，放化疗后基因表达在91个基因中显著上调，在251个基因中显著下调（错误发现率为0.0018）。显著上调的基因包括bax、细胞周期蛋白依赖性激酶抑制剂1、基质金属蛋白酶2以及黏附分子PECAM1、VCAM1和ICAM2。显著下调的基因包括拓扑异构酶IIα、myc、H2AX、错配修复蛋白2、RAD51、RAD53、增殖细胞核抗原以及细胞周期调节分子chk1、细胞周期蛋白依赖性激酶2、细胞周期蛋白B1、细胞周期蛋白D3、细胞周期蛋白依赖性激酶2和细胞周期蛋白磷酸酶25。
在多机构合作组试验中成功进行了微阵列分析。基因表达与组织学显著相关，但与分期、年龄或种族无关。聚类分析确定了两组与治疗前或治疗后获取的组织相关的基因表达谱。值得注意的是，配对样本显示放化疗后基因表达有显著变化，包括几个下调的放射反应基因。将基因表达与临床结果、急性和晚期毒性进行进一步比较分析有待临床数据成熟。希望这些数据将推动基于分子的疗法的发展。
