胃癌的个体化治疗鼓楼医院

胃癌的个体化药物治疗 鼓楼医院的经验,刘宝瑞 Liu Baorui Ph.D MD南京大学医学院附属鼓楼医院肿瘤中心Drum Tower Hospital Cancer Center,Nanjing University Medical School南京大学临床肿瘤研究所Clinical Cancer Institute of Nanjing University,南京大学医学院附属鼓楼医院肿瘤中心,住院病区,日间化疗,放射治疗,生物治疗,微创治疗,温热治疗,中药治疗,南京大学临床肿瘤所,细胞室,药分室,病理室,标本室,分子室,动物室,学科简介,学科简介,江苏省医学重点学科江苏省临床重点专科江苏省青年文明号江苏省医学分子技术重点实验室南京大学临床肿瘤研究所,目 录个体化疗简介肿瘤组织为检查对象的个体化治疗外周血浆为检查对象的个体化治疗,目 录个体化疗简介肿瘤组织为检查对象的个体化治疗外周血浆为检查对象的个体化治疗,“标准化疗”的成就与无奈：,当今肿瘤的化疗： 胃癌：FAM FAMtx EAP FP ECF FOLFOX TFP RR: 28%-46% 均50% 肺癌：RR30-40%肿瘤“异质性”：,有效率毒副作用-个体差异很大,个体化疗： 细胞水平药物敏感实验 基因水平DNA水平（突变、甲基化） RNA水平（mRNA、miRNA）,2004 年ASCO预测 ： 未来5-10年将是由当今标准化疗向个体化疗的过渡期2005 年ASCO预测 ： 药物基因指导下“个体化疗”是肿瘤化疗的一场革命2006年ASCO描述： 肿瘤的化疗已经迈入“个体化疗”的新时代2009年ASCO描述： 个体化医疗成为大会主题2010年以后态势： 大样本的论文相继发表,“药物相关基因指导下个体化 疗”,个体化疗,药物遗传学（pharmacogenetics),药物基因组学（pharmacognomics）,药物遗传学：研究与药物反应性/毒性相关的个体间DNA序列/基因多态性/甲基化的差异。,药物基因组学：将全基因组技术（即基因表达数据）用于预测一个患病个体对一个/一组药物的敏感性或抵抗性。,DPD: 二氢嘧啶脱氢酶 TS: 胸腺嘧啶核苷酸合成酶 OPRT:乳清酸磷酸核糖基转移酶 TP:胸(腺嘧啶脱氧核)苷磷酸化酶,氟脲密啶 (5-FU),足叶乙甙（VP16) -MDM2,开普拓（CPT11) -WRN,Colorectal cancers treated with irinotecan,开普拓（CPT11) Topo I,甲氨蝶呤（MTX) - DHFR,中国人群STAT3 rs 4796793位点多态性分布： CC 40.0% （IFNa敏感性较高） CG 46.7% GG 13.3%,干扰素（IFNa）-STAT3,吉西他滨（GEM）-hENT1、hCNT3,紫杉类 (paclitaxel，docetaxel ),1.Cytoskeleton and paclitaxel sensitivity in breast cancer: The role of tubulin Int J Cancer. 2007;120, 20782085 2.Clinical significance of class III beta-tubulin expression and its predictive value for resistance to docetaxel-based chemotherapy in gastric cancer Int J Oncol. 2006 Feb;28(2):375-81,. Microtubule-Associated Protein-tau is a Bifunctional Predictor of Endocrine Sensitivity and Chemotherapy Resistance in Estrogen Receptor Positive Breast Cancer Clin Cancer Res 2007;13(7):2061-7,甲氨蝶呤（MTX) DHFR,ERCC1(excision repair cross - Complementing 1)ERCC1 mRNA水平与铂类的敏感性密切相关，可以当成铂类化疗效果的独立预测指标。BRCA1(breast cancer 1)参与DNA修复，与铂类药物及作用于微管蛋白药物敏感性密切相关XRCC1 ( X-ray repair cross-complementing group 1) XRCC1基因第399位密码子由CGG CAG的变异可以导致编码的氨基酸由Arg Gln。变异型的DNA修复能力提高，对铂类敏感性下降。XPD (xeroderma pigmentosum pomplementary group D)XPD基因第751位密码子由AAG CAG的变异导致氨基酸由Lys Gln。变异型的表型导致DNA修复能力的提高，铂类敏感性下降。GSTP1 (Glutathione-S-transferase P1 ) GSTP1基因密码子105位缬氨酸(Val) 转变为异亮氨基酸(Ile) ，这一氨基酸的替换导致酶活性升高,铂类敏感性下降。,铂类药物疗效相关基因 (总结）,目 录个体化疗简介肿瘤组织为检查对象的个体化治疗外周血浆为检查对象的个体化治疗,1、方法学建立胃癌石蜡包埋组织检查微量的mRNA水平 2、开展胃癌化疗与疗效预后的随访 3、系统检查了如下基因状况 ERCC1 mRNA & protein expression Ts mRNA & protein expression OPRT mRNA & protein expression 筛选提高化疗效果的 XRCC1 Arg399Gln SNP 基因标志 GSTP1 Ile105Val SNP XPD Lls751Gln SNP 4、统计学分析：与疗效及生存时间的关系,胃癌 生物标志筛选 + 临床研究,从1760例胃癌中筛选出完整随访信息的病人,IF：4.0 他引次数：22,胃癌 生物标志筛选 + 临床研究,5、发现： ERCC1 mRNA 与含铂方案疗效与预后有关,发现： XRCC1 Arg399Gln SNP 与含铂方案疗效与预后有关,发现： GSTP1 Ile105Val SNP和XPD Lls751Gln SNP无统计意义 这一发现与西方人群有明显差异,IF：4.51 他引次数：19,6、进一步检查了如下基因mRNA水平 BRCA1 mRNA RAP80 mRNA SUMO调控途径核心基因PIAS1和PIAS4mRNA 发现：BRCA1水平高者含Doc化疗生存时间是低表达者的 2-3倍,IF：14.6,胃癌 生物标志筛选 + 临床研究,国科金,7、癌性腹水开展了系列性基因标志的检查 ERCC1 mRNA BRCA1 mRNA -tubulin mRNA Ts mRNA、 OPRT mRNA cell free miR-152 cell free miR-21 cell free miR-146a .,胃癌 生物标志筛选 + 临床研究,23种miRNAs,胃癌 生物标志筛选 + 临床研究,再次证实： BRCA1与紫衫疗效显著相关 ERCC1与铂类疗效显著相关,发现： miR-152 与紫衫疗效具有相关性,胃癌个体化治疗 BREC-CHINA 前瞻性临床研究,BREC-AGC,胃癌“生物标志指导下个体化疗”的随机对照前瞻性研究,伦理批文,CRF报告表,T2-T3 RAP80 (T1-T2 BRCA1),T2-T3 RAP80 (T3 BRCA1),Gem/Cis,Docetaxel,Docetaxel/Cis,T1 RAP80(T1-T3 BRCA1),CONTROL,Docetaxel/Cis,Advanced NSCLC,1:1,EXPERIMENTAL,肺癌治疗,启动肺癌个体化化疗BREC-CHINA前瞻性多中心临床研究,目 录个体化疗简介肿瘤组织为检查对象的个体化治疗外周血浆为检查对象的个体化治疗,新一代血清生物标志 -在肿瘤个体化药物治疗中的应用前景,刘宝瑞 Ph.D MD南京大学医学院附属鼓楼医院肿瘤中心南京大学临床肿瘤研究所,目 录,一、血清中含有大量肿瘤生物信息 二、血清中分子靶向药物生物标志 三、血清中化疗药物疗效生物标志 四、南京大学附属鼓楼医院的探索,一、血清中含有大量肿瘤生物信息,传统意义上的生物标志 目的：肿瘤诊断和随访 回顾性预测治疗效果 内容：血清蛋白标志 CEA AFP CA125 CA199 CY211 CA242 CA724 PSA fPSA,一、血清中含有大量肿瘤生物信息,2. 新一类血清生物标志药物疗效标志及预后标志 目的：前瞻性预测药物治疗效果及肿瘤患者的预后 predictive marker prognostic marker 内容： 外周血循环肿瘤细胞 外周血循环特定的DNA 外周血循环特定的RNA 外周血循环特定的miRNA,一、血清中含有大量肿瘤生物信息,文献综述循环肿瘤细胞及游离DNA,Circulating nucleic acids as a potential source for cancer biomarkers. Russia. Curr Mol Med. 2010 Mar;10(2):142-65.Since the association of circulating DNA level changes with tumor growth was discovered many attempts have been made to develop the sensitive and robust blood-based tests for early tumor diagnostics. Both genomic as well as mitochondrial DNA quantification in the circulation have been extensively evaluated as a diagnostic and prognostic tool to monitor cancer therapy. Cell-free DNA bearing the same genetic and epigenetic changes as the tumor tissues were shown to be detectable in plasma / serum of cancer patients indicating the principal possibility to create the minimally invasive diagnostic tests based on tumor-specific DNA markers. Apart from circulating DNA, tumor-derived RNA in plasma / serum was found to be a promising approach for the development of cancer markers. Results of the last two years establish the quantification of the tumor-derived microRNAs in plasma / serum as an extremely promising approach for cancer diagnostics.,一、血清中含有大量肿瘤生物信息,文献综述循环DNA RNAmiRNA,一、血清中含有大量肿瘤生物信息,文献血浆RNA与mRNA,一、血清中含有大量肿瘤生物信息,Chemosensitivity profile assay of circulating cancer cells: prognostic and predictive value in epithelial tumors. Roma, Italy. Int J Cancer 2010;126(10):2437-47. The prognostic value associated with the detection of circulating tumor cells (CTCs) in metastatic breast cancer by the CellSearch technology raise additional issues regarding the biological value of this information. We postulated that a drug-resistance profile of CTCs may predict response to chemotherapy in cancer patients and therefore could be used for patient selection. One hundred 5 patients with diagnosis of carcinoma were enrolled in a prospective trial. CTCs were isolated from peripheral blood, and positive samples were evaluated for the expression of a panel of genes involved in anticancer drugs resistance. Sensitivity of the test: able to predict treatment response in 98% of patients. Specificity of the test: 100%; no sample from healthy subject was positive for the presence of CTCs. We identified a drug-resistance profile of CTCs, which is predictive of response to chemotherapy, independent of tumor type and stage of disease. This approach may represent a first step toward the individualization of chemotherapy in cancer patients.,文献循环肿瘤细胞的个体化疗标志,一、血清中含有大量肿瘤生物信息,二、血清中分子靶向药物疗效标志,1. 外周血EGFR突变 2. 胸水EGFR突变 3. 外周血Kras突变 4. 外周血Her2mRNA水平,二、血清中分子靶向药物疗效标志,1. 外周血EGFR突变（广州）,Detection of epidermal growth factor receptor mutations in plasma by mutant-enriched PCR assay for prediction of the response to gefitinib in patients with non-small-cell lung cancer. He C, Liu M, Zhou C, Guangzhou China. Int J Cancer. 2009；125(10):2393-9.Specimen source and methods for EGFR mutation analysis are limited by tissue availability and technical feasibility in clinical application. Therefore, the current study is designed to establish a blood-based approach for the assessment of EGFR mutations in NSCLC patients, in particular the advanced stage, and to test its clinical application. Plasma samples were obtained from the enrolled 134 NSCLC patients. The detection rate of the EGFR exon19 deletions and exon21 L858R was 49.3% (66/134) by the blood-based, mutant-enriched polymerase chain reaction. In the paired tumor and plasma samples, the detected mutant types of each pair respectively by direct sequencing and mutant-enriched polymerase chain reaction were concordant in 17 of 18 (94.4%). In the patients treated with gefitinib as a second-line therapy, those with plasma EGFR mutation have a prolonged median progression-free survival compared with those with EGFR wild type (7.609 vs. 2.877 months, p = 0.002). On comparing the efficacy of gefitinib with that of docetaxel, it was found that the median progression-free survival was significantly longer for patients treated with gefitinib than those with docetaxel in those harboring plasma EGFR mutation (7.609 vs. 3.192 months, p = 0.006). These results suggest that the blood-based EGFR mutations test has the ability to provide a reliable guidance for clinical decision making for the treatment of the advanced NSCLC patients.,二、血清中分子靶向药物疗效标志,1. 外周血EGFR突变（北京）,Epidermal growth factor receptor mutations in plasma DNA samples predict tumor response in Chinese patients with stages IIIB to IV non-small-cell lung cancer. Bai H, Mao L, Wang HS, Beijing Cancer Hospital J Clin Oncol. 2009 Jun 1;27(16):2653-9Plasma DNA samples and matched tumors from 230 patients with stages IIIB to IV NSCLC were analyzed for EGFR mutations in exons 19 and 21 by using denaturing high-performance liquid chromatography. We compared the mutations in the plasma samples and the matched tumors and determined an association between EGFR mutation status and the patients clinical outcomes prospectively.In 230 patients, we detected 81 EGFR mutations in 79 (34.3%) of the patients plasma samples. We detected the same mutations in 63 (79.7%) of the matched tumors. Sixteen plasma (7.0%) and fourteen tumor (6.1%) samples showed unique mutations. In the 102 patients who failed platinum-based treatment and who were treated with gefitinib, 22 (59.5%) of the 37 with EGFR mutations in the plasma samples, whereas only 15 (23.1%) of the 65 without EGFR mutations, achieved an objective response (P = .002). Patients with EGFR mutations had a significantly longer progression-free survival time than those without mutations (P = .044) in plasma.EGFR mutations can be reliably detected in plasma DNA of patients with stages IIIB to IV NSCLC and can be used as a biomarker to predict tumor response to TKIs.,二、血清中分子靶向药物疗效标志,1. 胸水EGFR突变（日本）,EGFR mutation status in tumour-derived DNA from pleural effusion fluid is a practical basis for predicting the response to gefitinib. Kimura H, Japan. Br J Cancer. 2006；95(10):1390-5.We obtained 43 samples, which was the cell-free supernatant of pleural fluid, from Japanese NSCLC patients, and examined them for EGFR mutations. The epidermal growth factor receptor mutation status was determined by a direct sequencing method (exons 18-21 in EGFR). EGFR mutations were detected in 11 cases (E746_A750del in seven cases, E746_T751del insA in one case, L747_T751del in one case, and L858R in two cases). A comparison between the EGFR mutant status and the response to gefitinib in the 27 patients who received gefitinib revealed that all seven patients with partial response and one of the seven patients with stable disease had an EGFR mutation. No EGFR mutations were detected in the patients with progressive disease. The results suggest that DNA in pleural effusion fluid can be used to detect EGFR mutations and that the EGFR mutation status may be useful as a predictor of the response to gefitinib.,二、血清中分子靶向药物疗效标志,1. 外周血及胸水EGFR突变（上海）,Prediction of epidermal growth factor receptor mutations in the plasma/pleural effusion to efficacy of gefitinib treatment in advanced non-small cell lung cancer.Jian G, Songwen Z, Caicun Z. J Cancer Res Clin Oncol. 2010； 136(9):1341-7. The free DNA was isolated from the plasma of 56 cases and pleural effusion of another 32 cases of advanced NSCLC. Five common types of EGFR mutations were analyzed by LightCycle PCR with Taqman-MGB probes.EGFR gene mutations were found in 22 of all the 88 (25%) NSCLC patients (23.2% of 56 plasma samples, 28.1% of another 32 pleural effusion samples). The EGFR mutations in the serum and the pleural effusion from advanced NSCLC patients can be detected with LightCycle PCR using Taqman-MGB probes. The mutations highly predict the efficacy of gefitinib in advanced NSCLC.,二、血清中分子靶向药物疗效标志,2. 外周血HER2mRNA（意大利）,二、血清中分子靶向药物疗效标志,Detection of occult HER2 mRNA-positive tumor cells in the peripheral blood of patients with operable breast cancer: evaluation of their prognostic relevance.Apostolaki S Greece. Breast Cancer Res Treat. 2009；117(3):525-34. To evaluate whether HER2 mRNA could be used as a marker of circulating tumor cells (CTCs) .A nested RT-PCR assay was developed and used for the detection of HER2 mRNA-positive CTCs. Blood from 216 women with early breast cancer obtained before adjuvant treatment was tested for HER2 mRNA-positive cells to assess their prognostic value. Nested RT-PCR for HER2 mRNA showed high sensitivity whereas no HER2 mRNA-positive cells could be identified in the blood of healthy donors. HER2 mRNA-positive CTCs were detected in 53 (24.5%) of 216 patients and HER2 mRNA detection was associated with reduced disease-free survival (DFS; P 0.0001) and overall survival (OS; P = 0.004). In multivariate analysis, detection of HER2 mRNA-positive CTCs emerged as independent prognostic factor for DFS (P = 0.0001) and OS (P = 0.003). HER2 mRNA could be a valuable prognostic marker for the detection of CTCs in early breast cancer patients.,2. 外周血HER2mRNA（希腊）,二、血清中分子靶向药物疗效标志,2. 外周血HER2mRNA (美国）,Circulating tumor cells in HER-2 positive metastatic breast cancer patients treated with trastuzumab and chemotherapy.Nunes RA, Li X, Kang SP, Dana-Farber Cancer Institute, USA. Int J Biol Markers. 2009;24(1):1-10.The goal of this paper is to present a sensitive and specific methodology of detecting CTCs in women with HER-2 positive metastatic breast cancer, and to examine its role as a marker that tracks disease response during treatment with trastuzumab-containing regimens. The study included patients with HER-2-positive metastatic breast cancer enrolled on two different clinical protocols using a trastuzumab-containing regimen. Our study supports the prognostic and predictive role of the detection of CTCs in treatment of HER-2-positive metastatic breast cancer patients.,二、血清中分子靶向药物疗效标志,Origin and prognostic value of circulating KRAS mutations in lung cancer patients. Gautschi O, Huegli B, Ziegler A, USA. Cancer Lett. 2007 ;254(2):265-73. Because of the current controversy on the origin and clinical value of circulating KRAS codon 12 mutations in lung cancer, we screened 180 patients using a combined restriction fragment-length polymorphism and polymerase chain reaction (RFLP-PCR) assay. We detected KRAS mutations in 9% plasma samples and 0% matched lymphocytes. Plasma KRAS mutations correlated significantly with poor prognosis. We validated the positive results in a second laboratory by DNA sequencing and found matching codon 12 sequences in blood and tumor in 78% evaluable cases. These results support the notion that circulating KRAS mutations originate from tumors and are prognostically relevant in lung cancer.,3. 外周血Kras突变（美国）,Potential clinical significance of a plasma-based KRAS mutation analysis in patients with advanced non-small cell lung cancer.Wang S, An T, Wang J, Beijing Cancer Hospital P 0.001). Among 120 patients who received EGFR-TKI treatment, the response rate was only 5.3% (1 of 19) for patients with plasma KRAS mutation compared with 29.7% for patients with no KRAS mutation in plasma DNA (P = 0.024). The median progression-free survival time of patients w