張秋怡 吳一龍 周 清

·國家基金研究進(jìn)展綜述·

晚期非小細(xì)胞肺癌T790M突變的檢測(cè)方法及臨床意義*

張秋怡①②吳一龍②周 清②

表皮生長因子受體酪氨酸激酶抑制劑（epidermal growth factor receptor-tyrosine kinase inhibitors，EGFR-TKIs）是EGFR活化突變陽性晚期非小細(xì)胞肺癌（non-small cell lung cancer，NSCLC）患者的有效治療藥物，然而，幾乎所有服藥患者最終將出現(xiàn)耐藥，第20號(hào)外顯子的T790M點(diǎn)突變可能是導(dǎo)致耐藥的機(jī)制之一，如何敏感、準(zhǔn)確地檢測(cè)T790M基因突變是技術(shù)關(guān)鍵，外周血檢測(cè)因創(chuàng)傷小、操作簡便而成為近幾年研究熱點(diǎn)，實(shí)時(shí)定量檢測(cè)血漿T790M突變有望成為監(jiān)測(cè)EGFR-TKI療效的有效途徑。目前，T790M突變的臨床意義尚存爭議，近年來出現(xiàn)的第三代EGFR-TKI對(duì)T790M突變陽性患者的有效率較高，使得克服T790M突變導(dǎo)致的耐藥成為可能。本文就T790M檢測(cè)方法及其臨床意義進(jìn)行綜述。

非小細(xì)胞肺癌 表皮生長因子受體 T790M突變 定量檢測(cè)

隨著轉(zhuǎn)化醫(yī)學(xué)的飛速發(fā)展，晚期非小細(xì)胞肺癌（NSCLC）患者的治療已從化療時(shí)代轉(zhuǎn)向分子靶向治療時(shí)代，其中表皮生長因子受體（epidermal growth factor receptor，EGFR）是最重要的肺癌驅(qū)動(dòng)基因，EGFR-TKIs是針對(duì)此靶點(diǎn)的小分子抑制劑，多項(xiàng)臨床試驗(yàn)已證實(shí)，EGFR基因活化突變的晚期NSCLC患者能從EGFR-TKI顯著獲益［1-2］。然而，幾乎所有服用EGFR-TKI的患者最終將出現(xiàn)耐藥［3］，早期研究發(fā)現(xiàn)，約50%耐藥的患者具有EGFR20號(hào)外顯子第790位密碼子錯(cuò)義突變（即T790M突變）［4］，并且與EGFR-TKI療效密切相關(guān)［5-6］，如何敏感、準(zhǔn)確地檢測(cè)T790M基因突變成為關(guān)注熱點(diǎn)及技術(shù)關(guān)鍵。此外，關(guān)于T790M突變的臨床意義尚存爭議，本文就其突變檢測(cè)方法及其臨床意義進(jìn)行綜述。

1 T790M基因突變的定性檢測(cè)

T790M突變的檢測(cè)方法推陳出新，主要檢測(cè)技術(shù)包括直接測(cè)序法、擴(kuò)增阻滯突變系統(tǒng)法（amplified refractory mutation system，ARMS）、蝎形探針擴(kuò)增阻滯突變系統(tǒng)法（scorpions-ARMS）、突變富集PCR法、克隆雜交法、核酸肽鎖核酸PCR法、變性高效液相色譜法、質(zhì)譜分析法、基質(zhì)輔助激光解析電離飛行時(shí)間質(zhì)譜法（matrix-assisted laser desorption/ionization time of flight mass spectrometry，MALDI-TOF MS）及二代測(cè)序法（next-generation sequencing，NGS）等，其中MALDI-TOF MS、克隆雜交法及NGS除能對(duì)T790M突變定性檢測(cè)外，還能對(duì)其進(jìn)行定量檢測(cè)。

一直以來，直接測(cè)序法是EGFR突變的標(biāo)準(zhǔn)檢測(cè)方法［1，7］，也有學(xué)者用于血液檢測(cè)，但其過程繁瑣、耗時(shí)長，且敏感性很低［8-9］。ARMS法靈敏度可達(dá)0.1%～1%［10-11］，SARMS法是將ARMS法與Scorpion實(shí)時(shí)PCR相結(jié)合的一種新技術(shù)，其經(jīng)全基因組擴(kuò)增后能提高T790M突變陽性檢出率［12］。Arcila等［13］學(xué)者用核酸肽鎖核酸PCR法檢測(cè)EGFR-TKI耐藥肺癌患者的T790M突變，檢出率高達(dá)68%，用MALDI-TOF MS檢測(cè)T790M突變的陽性率為25%～32%，其中Su等［6，14］的結(jié)果經(jīng)過二代測(cè)序驗(yàn)證；Fujita等［15］用CH方法檢測(cè)pre-TKI活化突變患者的T790M陽性率達(dá)79%。提示SARMS法敏感度和特異性較高，且已有商品試劑盒，操作方便、重復(fù)性好，適合應(yīng)用于臨床。NGS是近年發(fā)展起來的高通量深度測(cè)序技術(shù)群，可一次性對(duì)幾百萬條至十億條DNA分子進(jìn)行并行測(cè)序，但其對(duì)稀有突變及結(jié)構(gòu)變異不敏感，且價(jià)格昂貴，不適于廣泛應(yīng)用。

2 T790M基因突變的定量檢測(cè)

Zhou等［16］率先提出EGFR-TKI療效不僅與EGFR突變狀態(tài)有關(guān)，而且與突變豐度有關(guān)。目前，定量檢測(cè)T790M突變技術(shù)有限，文獻(xiàn)報(bào)道主要有數(shù)字PCR法（digital PCR，dPCR）、實(shí)時(shí)熒光定量PCR法（real time quantitative PCR，qPCR）和NGS法。

Nakamura等［17］采用突變偏差PCR聯(lián)合猝滅探針法［mutation-biased PCR quenching probe（MBP-QP）method，MBP-QP］方法實(shí)現(xiàn)了血漿T790M的動(dòng)態(tài)定量檢測(cè)。Taniguchi等［18］用BEAMing（beads，emulsion，amplification，magnetics）技術(shù)定量檢測(cè)44例患者血漿的EGFR耐藥突變，檢出率為43.5%，敏感性可達(dá)0.01%［18］。BEAMing是由Diehl等［19-20］提出的一種基于磁珠和微乳液的固相數(shù)字PCR技術(shù)，其敏感性僅受限于PCR過程中引入的突變（此種情況在PCR中較常見），故其敏感性比其他方法高。Oxnard等［21］用微滴式數(shù)字PCR（droplet digital PCR，ddPCR）檢測(cè)NSCLC患者血漿T790M突變，敏感度為0.5%～0.05%，特異性達(dá)100%，可在影像學(xué)進(jìn)展前16周檢測(cè)出血漿T790M突變，并且與19號(hào)外顯子缺失突變量呈平行的變化關(guān)系。

qPCR在常規(guī)PCR基礎(chǔ)上增加熒光染料和探針而實(shí)現(xiàn)相對(duì)定量，根據(jù)Ct值及標(biāo)準(zhǔn)曲線計(jì)算出初始DNA模版相對(duì)量，受擴(kuò)增效率影響。而dPCR是一種核酸分子絕對(duì)定量技術(shù)，其不依賴于Ct值及標(biāo)準(zhǔn)曲線、不受擴(kuò)增效率影響，擴(kuò)增結(jié)束后通過直接計(jì)數(shù)或者泊松分布公式計(jì)算每個(gè)反應(yīng)單元格的平均濃度（含量），能將誤差控制在5%以內(nèi)，dPCR更精確［22］、更易檢測(cè)稀有基因突變以及不易被抑制［23-24］，對(duì)于檢測(cè)存在大量背景野生型的罕見突變具有重大意義，但其設(shè)備昂貴，對(duì)操作人員要求高，難以在臨床推廣。

3 T790M檢測(cè)的臨床意義

目在EGFR-TKI治療前、疾病進(jìn)展后以及治療過程中檢測(cè)T790M突變各有臨床意義。治療前對(duì)患者腫瘤組織進(jìn)行T790M檢測(cè)可能預(yù)測(cè)EGFR-TKI的療效。Su等［6］研究發(fā)現(xiàn)，治療前同時(shí)發(fā)生EGFR L858R突變或19外顯子缺失及T790M突變患者的無進(jìn)展生存劣于僅發(fā)生L858R突變或19號(hào)外顯子缺失患者，但優(yōu)于不存在上述突變患者，故該研究認(rèn)為原發(fā)T790M突變是EGFR-TKI療效的不良預(yù)測(cè)因子［6］，此結(jié)果與Rosell等［25］研究一致。相比定性檢測(cè)，定量檢測(cè)T790M突變能更精確指導(dǎo)臨床，Lee等［14］研究發(fā)現(xiàn)，在服用EGFR-TKI前伴T790M突變患者中，高豐度組比低豐度組有著更短的無進(jìn)展生存期及總生存期，提示治療前T790M的突變豐度與EGFR-TKI療效及預(yù)后相關(guān)。

疾病進(jìn)展后檢測(cè)T790M突變有助于了解EGFR-TKI的耐藥機(jī)制，為制訂克服耐藥策略提供依據(jù)。目前針對(duì)T790M靶點(diǎn)的第三代EGFR-TKI，如AZD9291［26］、CO-1686［27］、HM61713等藥物快速涌現(xiàn)，疾病進(jìn)展后T790M突變狀態(tài)與生存的關(guān)系正在探索中，Oxnard等［28］的研究顯示，T790M突變陽性患者的進(jìn)展后生存期顯著長于無T790M突變患者。因此，治療后T790M突變盡管是EGFR-TKI的耐藥原因，但也可能是進(jìn)展后生存的良好預(yù)后因子。

在EGFR-TKI治療期間連續(xù)動(dòng)態(tài)檢測(cè)血漿T790M突變狀態(tài)，可以為預(yù)測(cè)療效、監(jiān)測(cè)耐藥提供更多信息。Oxnard等［28］報(bào)道了在EGFR敏感突變患者中血漿連續(xù)檢測(cè)T790M突變與影像學(xué)腫瘤變化的關(guān)系，發(fā)現(xiàn)血漿T790M在影像學(xué)疾病進(jìn)展的16周前已經(jīng)出現(xiàn)，提示在EGFR-TKI治療過程中動(dòng)態(tài)監(jiān)測(cè)T790M基因狀態(tài)有可能預(yù)測(cè)耐藥，不僅可以指導(dǎo)晚期NSCLC患者的治療，而且有助于探究T790M突變誘導(dǎo)的耐藥機(jī)制及其異質(zhì)性［21］。

4 結(jié)語

T790M突變是EGFR-TKI耐藥的研究熱點(diǎn)，其耐藥機(jī)制、異質(zhì)性及臨床意義仍需進(jìn)一步探討，未來使用更敏感、準(zhǔn)確、簡便的方法檢測(cè)T790M突變，如用敏感特異的方法實(shí)時(shí)定量檢測(cè)血漿T790M突變，可能監(jiān)測(cè)EGFR-TKI療效及進(jìn)一步揭示T790M突變?cè)谀退幹械淖饔?，隨著第三代EGFR-TKI藥物的研究進(jìn)展，克服T790M導(dǎo)致的耐藥成為可能。

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（2014-06-24收稿）

（2014-08-01修回）

（本文編輯：楊紅欣）

Methodology and clinical significance of detecting EGFR-T790M mutation in advanced non-small cell lung cancer

Qiuyi ZHANG1,2,Yilong WU2,Qing ZHOU2

Qing ZHOU;E-mail:gzzhouqing@126.com
1Southern Medical University,Guangzhou 510080;2Guangdong People's Hospital and Guangdong Academy of Medical Sciences, Guangzhou 510080,China.
This work was supported by grants from the General Project of the National Natural Science Foundation of China(No.81172090) and the Guangzhou Pearl River New Scientific and Technological Star Special Project(2011J2200043).

Epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)comprise an effective therapy for advanced non-small cell lung cancer patients with EGFR-activating mutations.Unfortunately,most patients eventually develop resistance to EGFR-TKIs,probably due to a secondary point mutation of EGFR T790M.Thus,a sensitive method for accurate detection of T790M mutation is essential.Peripheral blood detection has gained our attention because it is convenient,making dynamic noninvasive quantitative detection of T790M mutation an optimal means of monitoring the efficacy of EGFR-TKIs.To date,the clinical significance of T790M mutation and EGFR-TKI resistance remains controversial.Several EGFR-TKIs targeting EGFR mutation,which have been introduced in recent years,showed better response in patients with T790M mutation,indicating that T790M may be a biomarker for conquering resistance.This review introduces the methodology of T790M detection and its role in clinical practice.

non-small cell lung cancer,epidermal growth factor receptor,T790M mutation,quantitative detection

10.3969/j.issn.1000-8179.20141068

①南方醫(yī)科大學(xué)（廣州市510080）；②廣東省醫(yī)學(xué)科學(xué)院，廣東省人民醫(yī)院

*本文課題受國家自然科學(xué)基金面上項(xiàng)目（編號(hào)：81172090）和廣州市珠江科技新星專項(xiàng)（編號(hào)：2011J2200043）資助

周清 gzzhouqing@126.com

張秋怡 在讀碩士研究生。專業(yè)方向?yàn)榉伟┓肿影邢蛑委煛?/p>

E-mail：116984600@qq.com