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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

IPASS Study:Progression-free survival by EGFR mutation type (ITT population)

Post-hoc Cox analysis with covariatesp-values not calculated due to small patient numbers

Exon 19 deletion L858R

Time from randomization (months)

HR (95% CI) = 0.377 (0.255, 0.560)

No. events gefitinib, 46 (69.7%)No. events C/P, 65 (87.8%)

Gefitinib (n=66)Carboplatin/paclitaxel (n=74)

HR (95% CI) = 0.553 (0.352, 0.868)No. events gefitinib, 48 (75.0%)

No. events C/P, 40 (85.1%)

66 40 18 6 2 074 15 4 2 1 0

6156

0 4 8 12 16 20 24

GefitinibC/P

Patients at risk :64 30 13 5 1 047 17 2 0 0 0

4839

0 4 8 12 16 20 240.0

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0.6

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1.0

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al Gefitinib (n=64)Carboplatin/paclitaxel (n=47)

Months Months

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0.8

1.0

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MedianOS HR

n (months) (95% CI)

217 27.0 22.7–31.3

SLOG Study:Survival in patients with EGFR mutation+ disease

1.0

0.8

0.6

0.4

0.2

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PF

S

0 12 24 36 48

Time (months)

MedianPFS HR

n (months) (95% CI)

217 14.0 11.3–16.7

1.0

0.8

0.6

0.4

0.2

0

Pro

bab

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y of

OS

0 12 24 36 48

Time (months)

14.0 27.0

Rosell R, et al. N Eng J Med 2009;361:958–67

Randomized Study on Japanese Population with EGFR Mutation: NEJGSG002

Kobayashi K, et al. 2009 ASCO Abstract 8016.

GefitinibN=98

P/CN=100

CR 4 0

PR 69 29

SD 13 50

PD 8 15

NE 4 6

缓解率 73 (74.5%) 29(29%)

P<0.001HR=0.357 95% CI: 0.252-0.507, P<0.001

生物标记物检测的采样情况

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

Docetaxel

Cisplatin

Gefitinib

•Chemotherapy-

naïve stage IIIb/IV

NSCLC;

• EGFR mutation

(Exon 19 or 21);

• PS 0–2;

• Age ≥18y;

Progression

Free

Su

rvival R

A

N

D

O

M

I

S

E 1:1

Primary endpoint: PFS

Secondary endpoint: OS; ORR; QOL; Safety

WJTOG 3405

Progression Free Survival

Overall Survival

外周血 EGFR 突变检测患者血浆中有足够的游离 DNA( 是正常人的 10

倍 ) 。血浆中的游离 DNA 主要由凋亡和坏死的肿瘤细胞产生，其遗传学特性与肿瘤基因组 DNA 相似。

CTC

CTC: NSCLC 循环肿瘤细胞 - 中位数 74 个 / 微升

蛋白组学 :MALDI-MS

血浆 / 血清游离 DNA

外周血 EGFR 突变检测与组织的一致性 ( 敏感性与特异性 )?外周血 EGFR 突变检测能否预测疗效与生

存 ?

血清 / 血浆游离 DNA EGFR 突变研究 : 争议的问题

Finding EGFR Mutation in Plasma DNA by PCR: Spanish Study

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)

CR = complete response; PR = partial response; SD = stable disease; PD = progressive disease

Rosell R, et al. N Eng J Med2009;361:958–67

Value

Erlotinib therapy, n (%) First-line Second- or third-line

113 (52.1)104

(47.9)

EGFR mutation, n (%)del 19L858R

135 (62.2) 82

(37.8)

EGFR mutation in serum, n (%)* del 19L858RNot detected

64 (39.0)

33 (20.1)

67 (40.9)

*Evaluated in the serum of 164 patients

‡Evaluated in 197 patients

False Negative

Rate

40,0030,0020,0010,000,00

Months

1,0

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0,6

0,4

0,2

0,0

Pro

bab

ility

WT

Mutated

EGFR Serum

Time to Progression

Exon 19

50,0040,0030,0020,0010,000,00

Months

1,0

0,8

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0,4

0,2

0,0

Pro

bab

ility

WT

Mutated

EGFR Serum

Time to Progression

Exon 21

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

血浆 DNA 与原发瘤中 EGFR 突变的吻合度

False negativeRate=18.8%


False PositiveRate=20.2%

False PositiveRate=20.2%

==

Bai and Wang JCO 27:2653, 2009

IPASS: Japanese PopulationPatients recruited in Japan (n=233)

cfDNA extracted from pre-dose serum samples

DNA extracted from paraffin-embedded archival tumor tissue

EGFR mutations detected by ARMS

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

and/or

ESMO 2009

cfDNA Tumor tissue

• 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

IPASS:Comparison of EGFR mutation statusin cfDNA and tumor samples

cfDNA, n

EGFR M+

EGFR M-

Total

22

29

51

0

35

35

EGFR M+ EGFR M-

22

64

86

Total

Tumor tissue, n

Patients with known cfDNA and tumorEGFR mutation status (n=86)

• 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

False PositiveRate=0%

False PositiveRate=0%



Plasma DNA as Predictive Biomarker in IPASS (Japanese Subgroup)

Treatment by subgroup interaction test, p=0.0448Japanese ITT population; Cox analysisHR <1 implies a lower risk of progression/death on gefitinib

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HR (95% CI) = 0.29 (0.14, 0.60) p=0.0009

HR (95% CI) = 0.88 (0.61, 1.28)

p=0.5013

EGFR M+ EGFR M-

24 12 4 2 0 022 4 1 0 0 0

2115

GefitinibC/P

70 23 14 7 1 078 24 7 1 1 0

3654

n

Events, n (%)

C/P

22

19 (86.4)

n

Events, n (%)

C/P

78

67 (85.9)

Patients at risk: Months Months

Gefitinib

24

15 (62.5)

Gefitinib

70

51 (72.9)

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2009 年回顾性 230 DHPLC 78 18.8 20.2 69

PFS:P=0.04 OS:P=0.910

IPASS

2009 年回顾性 86 ARMS 66.3 57.7 0 71.2

HR=0.29P=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。

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。

Wang, et al Clin.Can.Res. 2010,

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

2009 WCLC, Okimi et al

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。

2007.8 Iressa 治疗前 2009.5 Iressa 治疗 21 个月后

深度思考（ II ）

治疗对 EGFR 突变状态有无影响 ?

组织（新辅助化疗后）

外周血（一线化疗前后）

0%

10%

20%

30%

40%

50%

治疗前治疗后

疗前44%

化疗前后 EGFR 突变的改变 - 来自北京肿瘤医院的报道

疗后28%

疗前35.7%

疗后28.6%

深度思考（ III ）

什么是最佳的检测方法？

Comparison of Somatic Gene Mutation Analysis Methods

Jimeno et al. JCO 2008

Method 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 quantitativeInsensitiveProlonged 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 Slow turnaround (4 days to 2 weeks from paraffin)

ComplicatedRequires sequencing confirmationConsiderable manual input required

Amplification Refractory Mutation System (ARMS)

Mutation specific polymerase chain reaction/detection

1 Rapid (<2 days from paraffin)

Detects only single specific mutation per reactionRequires specially engineered primer/probe

Abbreviations: MT – Mutant, WT – Wild Type

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