血浆EGFR突变检测的特点

1、血浆EGFR/KRAS突变检测的方法学和临床应用特点及研究进展肖何肖何放疗中心放疗中心2014-10-161. 血浆游离血浆游离DNA(Circulating Cell-free DNA)来源与高度片段化来源与高度片段化2. 具有中等检测限具有中等检测限(0.1%-3%选择性选择性)方法方法(DHPLC、ARMS)对血浆对血浆EGFR突变的检测突变的检测3. DHPLC或或ARMS法检测临床血浆样本对法检测临床血浆样本对TKI疗效预测的不一致性疗效预测的不一致性4. 具有极低检测限方法具有极低检测限方法(dPCR、ASB-qPCR、PNA-PCR)(0.005%-0.01%)表明血浆游离肿瘤特

2、异性突变表明血浆游离肿瘤特异性突变DNA分子相对丰度的变异性分子相对丰度的变异性目 录Emily Crowley, Federica Di Nicolantonio, Nat. Rev. Clin. Oncol. 2013 Florent Mouliere, Bruno Robert, Plos One 2011, 6(9)通过肿瘤细胞凋亡、坏死等机制释放高度片段化的DNA到血液SW 620 (G12V KRAS突变型突变型)移植瘤小鼠血浆不同移植瘤小鼠血浆不同KRAS扩增片段具有显著扩增片段具有显著不同的浓度，小于不同的浓度，小于100bp片段是肿瘤来源游离片段是肿瘤来源游离DNA的主要成分

3、的主要成分3例转移性结直肠癌病例血浆例转移性结直肠癌病例血浆ctDNA浓度浓度(ng/ml)Luis A. Diaz Jr and Alberto Bardelli, J Clin Oncol 2014, 32:579-586.DHPLC或ARMS法对血浆EGFR热点突变检测以及与组织突变一致性的比较常用突变检测方法灵敏度和对应应用样本类型常用突变检测方法灵敏度和对应应用样本类型NSimultaneousMaterialExtractionMethodSensitivitySpecificityPPVNPVConcordanceCaicun Zhou1121YesPlasmaQIAamp DN

4、A Mini KitDxS EGFR Mutation Test Kit (DxS,Manchester, UK)48.2%95.4%90.0%68.1%73.5%Jie Wang2744YesPlasmaQIAamp DNA Mini KitDHPLC68.4%83.4%70.0%82.3%80.0%Britta Weber3196NoPlasmacobas DNA Sample Preperation KitARMS cobas EGFR Blood Test60.7%96.4%73.9%93.5%91.3%NCT012039174652YesPlasmaQIAamp Circulatin

5、g Nucleic Acid KitARMS(Therascreen EGFR RGQ PCR kit; Qiagen65.7%99.8%98.6%93.8%94.3%1 Xuefei Li, Translational Oncology. 2014,7: 341-348 2 Zheng Huang, doi: 10.1111/j.1759-7714.2012.00133.x 3 Britta Weber, BMC Cancer 2014, 14:294 4 Jean-Yves Douillard, J Thorac Oncol. 2014;9: 13451353DHPLC或或ARMS法检测临

6、床血浆样本对法检测临床血浆样本对TKI疗效预测作用的不一致性疗效预测作用的不一致性一线一线TKI治疗，组织或血浆突变群体中有显著较长的治疗，组织或血浆突变群体中有显著较长的PFS组织突变分类组织突变分类 绿线：绿线：EGFR M+ 红线：红线：EGFR M-血浆突变分类血浆突变分类 绿线：绿线：EGFR M+ 红线：红线：EGFR M-Zheng Huang, doi: 10.1111/j.1759-7714.2012.00133.x Xuefei Li, Translational Oncology. 2014,7: 341-348因血浆检测较高假阴性，血浆因血浆检测较高假阴性，血浆EGFR

7、 M- TKI治疗治疗患者依然患者依然有较好有较好预后或较高缓解率预后或较高缓解率Alain R Thierry, Florent Mouliere, Nature Medicine, 2014 20(4): 430-436Florent Mouliere, Safia El Messaoudi, Translational Oncology (2013) 6, 319328突变等位基因特异性定量PCR分析 (Allele Specific qPCR) 分析结直肠癌KRAS/BRAF突变突变等位基因占总游离突变等位基因占总游离DNA分子百分比从分子百分比从0.5%-64.2%不等，个体间存在较

8、大差异不等，个体间存在较大差异(100倍倍)，中位值为，中位值为10.47%Kazuya Taniguchi, Junji Uchida, Clin Cancer Res; 2011, 17(24); 780815.BEAMing (微乳滴磁珠PCR法) Activating Mutation Resistant MutationGroup1 PD after TKI treatmentGroup2 Never treated 初诊初诊NSCLC患者患者EGFR激活突变等位激活突变等位基因基因比例主要比例主要集中在集中在1%以内以内Stage IVGeneMutation descriptio

9、nTum Freq (%)Cf DNA Freq (%)#23YEGFRchr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del410.2#56YERBB2chr17:37880981ins12; ENST00000269571:c.2310_2311ins12; p.E770_A771ins12430.3#31YEGFRchr7:55259524TA; ENST00000344576:c.2582TA; p.L861Q590.4#52EGFRchr7:55242469del18; ENST00000344576:c.

10、2239_2256del; p.L747_S752delND0.4#62YEGFRchr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del550.4#19YERBB2chr17:37880981ins12; ENST00000269571:c.2310_2311ins12; p.E770_A771ins12400.5#45YEGFRchr7:55241707; ENST00000344576:c.2155GA; p.G719S120.5#9YKRASchr12:25398284CA; ENST00000256078:c

11、.35GT; p.G12V110.6#58EGFRchr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del560.6#67YEGFRchr7:55242465del15;ENST00000344576:c.2235_2249del;p.K746_A750del950.9#21YEGFRchr7:55241708GC; ENST00000344576:c.2156GC; p.G719A191#20YEGFRchr7:55259515TG; ENST00000344576:c.2573TG; p.L858R831.3#10

12、YEGFRchr7:55259515TG; ENST00000344576:c.2573TG; p.L858R121.5#34KRASchr12:25398284CT; ENST00000256078:c.35GA; p.G12D271.5#50YEGFRchr7:55259515TG; ENST00000344576:c.2573TG; p.L858R82#17YEGFRchr7:55259515TG; ENST00000344576:c.2573TG; p.L858R93#47YEGFRchr7:55259515TG; ENST00000344576:c.2573TG; p.L858R15

13、4#5YEGFRchr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del217#4YEGFRchr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del1910#49YERBB2chr17:37880981ins12; ENST00000269571:c.2310_2311ins12; p.E770_A771ins122511#51YEGFRchr7:55242464del15; ENST00000344576:c.2234_2248del; p.

14、K745_A750del5911#11YEGFRchr7:55242465del15;ENST00000344576:c.2235_2249del;p.K746_A750delND12#37PIK3CAchr3:178936092AC; NM_006218.1:c.1634AC; p.E545AND13#8YEGFRchr7:55242465del15;ENST00000344576:c.2235_2249del;p.K746_A750del2017#53YKRASchr12:25398284CT; ENST00000256078:c.35GA; p.G12D1118#55YEGFRchr7:

15、55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del8619.5#3YEGFRchr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del2131#22YEGFRchr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del1437#2YEGFRchr7:55241708GC; ENST00000344576:c.2156GC; p.G719A6244Sbastien Couraud,

16、 Felipe Vaca-Paniagua, Clin Cancer Res 2014;20:4613-4624.深度测序法 (Deep Sequencing)EGFR激活突变等位基因频率从激活突变等位基因频率从0.2%-44%，中位值中位值2.0%血浆激活突变频率和配对肿瘤组织突变频血浆激活突变频率和配对肿瘤组织突变频率率没有没有相关性相关性(rho=-0.227, P=0.797)总 结1. 血浆游离血浆游离DNA高度片段化，所有基于游离高度片段化，所有基于游离DNA的的PCR方法必须建立在扩方法必须建立在扩增小片段基础上增小片段基础上2. ARMS和和DHPLC在检测灵敏度上更适合穿刺样本或细胞学样本，或者胸在检测灵敏度上更适合穿刺样本或细胞学样本，或者胸水沉渣包埋物水沉渣包埋物3. 由于由于DNA的片段化以