基于外周血EGFR突变检测临床意义的深度思考

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1 基于外周血EGFR突变检测临床意义的深度思考
王 洁 北京大学临床肿瘤学院 北京肿瘤医院

2 Time from randomization (months)
IPASS Study:Progression-free survival by EGFR mutation type (ITT population) Exon 19 deletion L858R Gefitinib (n=66) Carboplatin/paclitaxel (n=74) Gefitinib (n=64) Carboplatin/paclitaxel (n=47) 1.0 1.0 HR (95% CI) = (0.255, 0.560) No. events gefitinib, 46 (69.7%) No. events C/P, 65 (87.8%) HR (95% CI) = (0.352, 0.868) No. events gefitinib, 48 (75.0%) No. events C/P, 40 (85.1%) 0.8 0.8 0.6 0.6 Probability of progression-free survival Probability of progression-free survival 0.4 0.4 0.2 0.2 0.0 0.0 4 8 12 16 20 24 4 8 12 16 20 24 Months Months Patients at risk : Gefitinib 66 61 40 18 6 2 64 48 30 13 5 1 C/P 74 56 15 4 2 1 47 39 17 2 Time from randomization (months) Post-hoc Cox analysis with covariates p-values not calculated due to small patient numbers 2

3 SLOG Study:Survival in patients with EGFR mutation+ disease
Median PFS HR n (months) (95% CI) –16.7 Median OS HR n (months) (95% CI) –31.3 1.0 0.8 0.6 0.4 0.2 1.0 0.8 0.6 0.4 0.2 Probability of PFS Probability of OS 14.0 27.0 Time (months) Time (months) Rosell R, et al. N Eng J Med 2009;361:958–67

4 Randomized Study on Japanese Population with EGFR Mutation: NEJGSG002
Gefitinib N=98 P/C N=100 CR 4 PR 69 29 SD 13 50 PD 8 15 NE 6 缓解率 73 (74.5%) 29(29%) P<0.001 HR= % CI: , P<0.001 Kobayashi K, et al ASCO Abstract 8016.

5 生物标记物检测的采样情况 1217 随机患者(100%) 1038 同意检测生物标记物 (85%) 683 提供样本 (56%) 可评估的:
EGFR 突变: 437 (36%) EGFR 基因表达数目: 406 (33%) EGFR 表达: 365 (30%) 1217 随机患者(100%) 不可评估的主要原因包括： 取样困难 样本量不足 只有细胞学样本 样本取材于肿瘤外的其他部位 5

6 WJTOG 3405 Gefitinib 1:1 Overall Survival Docetaxel Cisplatin R
Chemotherapy- naïve stage IIIb/IV NSCLC; EGFR mutation (Exon 19 or 21); PS 0–2; Age ≥18y; R A N D O M I S E Gefitinib Progression Free Survival Docetaxel Cisplatin 1:1 Progression Free Survival Primary endpoint: PFS Secondary endpoint: OS; ORR; QOL; Safety Overall Survival

7 外周血EGFR突变检测 蛋白组学:MALDI-MS 血浆/血清游离DNA CTC: NSCLC循环肿瘤 细胞-中位数74个/微升 CTC

8 血清/血浆游离DNA EGFR突变研究:争议的问题
(敏感性与特异性)? 外周血EGFR突变检测能否预测疗效与生存?

9 Finding EGFR Mutation in Plasma DNA by PCR: Spanish Study
Value Erlotinib therapy, n (%) First-line Second- or third-line 113 (52.1) 104 (47.9) EGFR mutation, n (%) del 19 L858R 135 (62.2) 82 (37.8) EGFR mutation in serum, n (%)* del 19 L858R Not detected 64 (39.0) 33 (20.1) 67 (40.9) Response (all patients)‡ n (%) CR 24 (12.2) PR 115 (58.4) CR / PR 139 (70.6) SD 38 (19.3) PD 20 (10.2) SD / PD 58 (29.4) ‡Evaluated in 197 patients False Negative Rate *Evaluated in the serum of 164 patients CR = complete response; PR = partial response; SD = stable disease; PD = progressive disease Rosell R, et al. N Eng J Med 2009;361:958–67

10 北京肿瘤医院的研究 230 pts with tumor samples for EGFR mutation analysis
DHPLC performed in plasma 102 pts received gefitinib (second line) Bai and Wang JCO 27:2653, 2009 10

11 血浆DNA与原发瘤中EGFR突变的吻合度
False negative Rate=18.8% False Positive Rate=20.2% 吻合度：78% = Bai and Wang JCO 27:2653, 2009 11

12 IPASS: Japanese Population
Patients recruited in Japan (n=233) DNA extracted from paraffin-embedded archival tumor tissue cfDNA extracted from pre-dose serum samples and/or EGFR mutations detected by ARMS cfDNA Tumor tissue EGFR M+: 1/21 mutationsa (n=46) EGFR M-: 0/21 mutations (n=148) EGFR M unknownc: (n=39) EGFR M+: 1/29 mutationsb (n=56) EGFR M-: 0/29 mutations (n=35) EGFR M unknownc: (n=142) Comparison of cfDNA vs tumor tissue EGFR mutations based on 22 mutations analyzed for cfDNA 5 patients had a known mutation result by tumor tissue but not cfDNA 108 patients had a known mutation result by cfDNA but not by tumor tissue 86 patients had a known mutation status by both tumor tissue and cfDNA ESMO 2009 12

13 IPASS:Comparison of EGFR mutation status in cfDNA and tumor samples
Patients with known cfDNA and tumor EGFR mutation status (n=86) Tumor tissue, n EGFR M+ EGFR M- Total cfDNA, n EGFR M+ EGFR M- Total 22 29 51 35 22 64 86 False Positive Rate=0% False negative Rate=57.7% No false positive results Specificity and positive predictive value 100% 29/51 (56.9%) of tumor EGFR M+ were cfDNA EGFR M- (false negatives) Sensitivity 43.1% (22/51), negative predictive value 54.7% (35/64) 57/86 (66.3%) concordance Japanese ITT population 13

14 Plasma DNA as Predictive Biomarker in IPASS (Japanese Subgroup)
EGFR M+ EGFR M- 1.0 1.0 HR (95% CI) = 0.29 (0.14, 0.60) p=0.0009 HR (95% CI) = 0.88 (0.61, 1.28) p=0.5013 0.8 0.8 0.6 0.6 Probability of progression-free survival 0.4 0.4 0.2 0.2 0.0 0.0 4 8 12 16 20 24 4 8 12 16 20 24 Months Months Patients at risk: Gefitinib 24 21 12 4 2 70 36 23 14 7 1 C/P 22 15 4 1 78 54 24 7 1 1 n Events, n (%) Gefitinib 24 15 (62.5) C/P 22 19 (86.4) n Events, n (%) Gefitinib 70 51 (72.9) C/P 78 67 (85.9) Treatment by subgroup interaction test, p=0.0448 Japanese ITT population; Cox analysis HR <1 implies a lower risk of progression/death on gefitinib 14

15 血清/血浆游离DNA EGFR突变临床预测意义研究
研究名称 生物标志分析/证据级别 病例数 检测方法 组织/外周血一致性% 假阴性% 假阳性% 预测RR 预测PFS/OS Rosell 2009年 回顾性 164 ARMS 59.1 40.9 --- 70 PFS:HR=1.48, P=0.044, OS:HR=1.50, P=0.910 北京肿瘤医院 230 DHPLC 78 18.8 20.2 69 PFS:P=0.04 OS:P=0.910 IPASS 86 66.3 57.7 71.2 HR=0.29 P=0.0009 以上三组研究对外周血分析而言均为回顾性研究 且检测方法、病人基线条件不一。但结果显示若利用更加敏感的 检测方法，假阳性率较低。需前瞻性研究验证。

16 Wang, et al Clin.Can.Res ,

17 深度思考（I） 外周血与组织EGFR突变检测结果不一致的原因? 肿瘤组织内的异质性 原发灶与转移灶的异质性

18 2009 WCLC, Okimi et al

19 患者,女,65岁,右下肺周围型低分化腺癌术后(IIb)3年肺内、脑转移。
Iressa 治疗前 Iressa治疗21个月后

20 深度思考（II） 治疗对EGFR突变状态有无影响?

21 化疗前后EGFR突变的改变-来自北京肿瘤医院的报道
35.7% 疗后 28.6% 疗前 44% 疗后 28%

22 深度思考（III） 什么是最佳的检测方法？

23 Comparison of Somatic Gene Mutation Analysis Methods
Principle Sensitivity (MT/WT; %) Turnaround Disadvantages Direct Sequencing Non-mutation-specific determination of test case nucleotide sequence and comparison with normal sequence 20-50 Slow turnaround (4 days to 2 weeks from paraffin) Poorly quantitative Insensitive Prolonged turnaround Allele specific probe Polymerase chain reaction/selective detection 10 Rapid (<2 days from paraffin) Relatively low sensitivity High resolution melting analysis, confirmed by direct sequencing Sequences with mutations hybridize at different, fixed temperatures 3-5 Complicated Requires sequencing confirmation Considerable manual input required Amplification Refractory Mutation System (ARMS) Mutation specific polymerase chain reaction/detection 1 Detects only single specific mutation per reaction Requires specially engineered primer/probe Abbreviations: MT – Mutant, WT – Wild Type Jimeno et al. JCO 2008

24 未来方向 积极开展以外周血分子标志严格分层的前瞻多中心研究 建立规范化\标准化系列分子检测平台 探索新的治疗靶基因及相关药物 THANKS!