結(jié)腸癌轉(zhuǎn)移相關(guān)因子-1和間質(zhì)-上皮細(xì)胞轉(zhuǎn)化因子檢測在肺腺癌患者并發(fā)惡性胸腔積液中的診斷價值

胡　婕　尤青?！O耕耘

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·論著·

結(jié)腸癌轉(zhuǎn)移相關(guān)因子-1和間質(zhì)-上皮細(xì)胞轉(zhuǎn)化因子檢測在肺腺癌患者并發(fā)惡性胸腔積液中的診斷價值

胡婕尤青海孫耕耘

目的探討結(jié)腸癌轉(zhuǎn)移相關(guān)因子-1(MACC1)、間質(zhì)-上皮細(xì)胞轉(zhuǎn)化因子(c-MET)聯(lián)合癌胚抗原(CEA)在肺腺癌患者并發(fā)惡性胸腔積液中的診斷價值。方法將91例胸腔積液患者(良性38例,肺腺癌53例)納入研究,采用 ELISA 法測定血清和胸腔積液中MACC1、c-MET濃度，放射免疫法測定CEA濃度。結(jié)果肺腺癌患者惡性胸腔積液中MACC1、c-MET和CEA濃度明顯高于良性患者胸腔積液中的含量(P<0.05)；兩組患者血清中MACC1和c-MET濃度無統(tǒng)計學(xué)差異(P>0.05)。胸腔積液中MACC1和c-MET含量呈正相關(guān)(r=0.728，P<0.01)。根據(jù)ROC曲線，以MACC1濃度90.98 pg/ml為臨界值, 對肺腺癌患者并發(fā)惡性胸腔積液的診斷靈敏度為62.26%，特異度為84.21%；c-MET濃度757.67 ng/ml為臨界值，肺腺癌患者惡性胸腔積液的診斷靈敏度為52.83%，特異度為84.21%。MACC1聯(lián)合c-MET檢測靈敏度為75.47%，特異度為92.11%；MACC1、c-MET聯(lián)合CEA診斷的靈敏度為98.10%，特異度為100%。 結(jié)論MACC1和c-MET在肺腺癌患者并發(fā)惡性胸腔積液的診斷中具有一定價值，聯(lián)合CEA檢測可明顯提高診斷的靈敏度和特異度。

肺腺癌；結(jié)腸癌轉(zhuǎn)移相關(guān)因子-1；間質(zhì)-上皮細(xì)胞轉(zhuǎn)化因子；癌胚抗原；惡性胸腔積液

惡性胸腔積液(malignant pleural effusions, MPE)因惡性腫瘤侵犯胸膜引起，其中以肺腺癌轉(zhuǎn)移后胸腔積液形成最常見，MPE的產(chǎn)生嚴(yán)重影響患者的生存質(zhì)量，且提示預(yù)后不良[1]。目前臨床對于肺腺癌并發(fā)MPE的診斷僅有癌胚抗原(carcino embryonie antigen, CEA)是其有效的標(biāo)志物，因而有必要探討在肺腺癌并發(fā)MPE診斷中有價值的新型標(biāo)志物。結(jié)腸癌轉(zhuǎn)移相關(guān)因子-1(metastasis-associated in colon cancer-1, MACC1)是近幾年發(fā)現(xiàn)的一個與結(jié)腸癌轉(zhuǎn)移有著密切關(guān)系的因子，通過對間質(zhì)-上皮轉(zhuǎn)化因子(mesenchymal-epithelial transition factor, c-MET)介導(dǎo)的信號通路的異常調(diào)控作用，發(fā)揮促進腫瘤的發(fā)生、侵襲和轉(zhuǎn)移的作用[2]。本研究通過檢測血清和胸腔積液中MACC1、c-MET含量，以探討兩者在肺腺癌并發(fā)MPE中的診斷價值。

對象與方法

一、研究對象

選取安徽醫(yī)科大學(xué)第一附屬醫(yī)院2014年11月至2015年7月呼吸內(nèi)科住院患者，惡性組53例，男31例，女22例，年齡40～90(62.83±13.61)歲，均為肺腺癌并發(fā)MPE(胸水脫落細(xì)胞學(xué)查見腺癌細(xì)胞)；良性組共38例，男30例，女8例，年齡16～84(49.82±22.23)歲，其中肺結(jié)核19例，肺炎13例，心力衰竭2例，肝硬化1例，病因不明漏出液3例。

二、研究方法

1. 標(biāo)本采集： 均在患者治療前采集，抽取晨起空腹靜脈血5 ml，留取首次胸腔穿刺積液10 ml，以離心半徑8 cm在常溫下3 000 r/min 離心10 min，吸取上清液0.5 ml，分裝后放置-20 ℃低溫冷凍保存，期間避免反復(fù)凍融。

2. 標(biāo)本檢測 ：血清及胸水CEA含量由安徽醫(yī)科大學(xué)第一附屬醫(yī)院檢驗科采用放射免疫法測定。MACC1和c-MET含量應(yīng)用ELISA試劑盒(批號：MACC1試劑盒為E-14431，c-MET試劑盒為E-12829，由合肥欣樂生物有限公司提供)，操作嚴(yán)格按試劑盒說明書進行。

三、統(tǒng)計學(xué)方法

用SPSS20.0統(tǒng)計軟件進行分析，采用Kolmogorov-Smirnov test確定所得數(shù)據(jù)是否符合正態(tài)分布，所有數(shù)據(jù)用表示。兩組變量的比較采用t檢驗，相關(guān)性分析用Pearson相關(guān)分析，判斷敏感度及特異度采用受試者工作曲線(ROC曲線)，以P<0.05為差異有統(tǒng)計學(xué)意義。

結(jié)　　果

一、胸腔積液和血清中MACC1、c-MET和CEA含量測定結(jié)果比較

肺腺癌組MACC1、c-MET、CEA濃度均明顯高于良性組(P<0.05)。兩組血清CEA濃度差異有統(tǒng)計學(xué)意義(P<0.01)，惡性組明顯高于良性組。血清中MACC1和c-MET濃度肺腺癌組高于良性組，但差異無統(tǒng)計學(xué)意義(P>0.05)。根據(jù)Pearson相關(guān)分析得知胸腔積液中MACC1和c-MET含量呈正相關(guān)(r=0.728，P<0.01)，見表1。在惡性組胸腔積液CEA低于診斷臨界值的4例樣本中，有3例的MACC1高于診斷臨界值。

項　目良性(n=38)惡性(n=53)P值t值胸水　MACC172．53±20．93105．49±47．64<0．053．993　c?MET603．44±156．93797．03±270．84<0．054．294　CEA1．46±1．08539．68±275．00<0．0512．05血清　MACC1111．86±33．86127．99±83．700．337-0．97　c?MET795．48±184．65842．78±388．860．295-1．06　CEA2．14±1．26115．33±79．190．002-3．39

注：MACC1：結(jié)腸癌轉(zhuǎn)移相關(guān)因子-1；c-Met：間質(zhì)-上皮細(xì)胞轉(zhuǎn)化因子; CEA:癌胚抗原

二、不同因素與肺腺癌并發(fā)惡性胸腔積液中MACC1、c-MET和CEA濃度變化的相關(guān)性

不同性別、年齡(以60歲分界)、是否吸煙、有無淋巴結(jié)和遠(yuǎn)處轉(zhuǎn)移的肺腺癌并發(fā)惡性胸腔積液中MACC1、c-MET及CEA含量差異無統(tǒng)計學(xué)意義(P>0.05)，見表2。

表2　53例惡性胸腔積液MACC1、c-MET的濃度與其臨床特征的關(guān)系±s)

注：MACC1：結(jié)腸癌轉(zhuǎn)移相關(guān)因子-1；c-Met：間質(zhì)-上皮細(xì)胞轉(zhuǎn)化因子

三、MACC1、c-MET和CEA的診斷價值

應(yīng)用ROC曲線分析實驗所得數(shù)據(jù),約登指數(shù)最大值對應(yīng)的切點為診斷的最佳臨界點。胸腔積液MACC1濃度90.98 pg/ml為最佳臨界點(靈敏度62.26%，特異度84.21%，曲線下面積AUC為0.787)；胸腔積液c-MET濃度757.67 ng/ml為最佳臨界值(靈敏度為52.83%，特異度為84.21%，AUC為0.728)；CEA濃度1.95 ng/ml為最佳臨界值(靈敏度為92.45%，特異度為92.11%，AUC為0.953)。見圖1，表3。聯(lián)合檢測：MACC1和c-MET聯(lián)合靈敏度為75.47%，特異度為92.11%；MACC1和c-MET聯(lián)合CEA靈敏度為98.10%，并聯(lián)特異度為97.37%，見表4。

表3　MACC1、c-MET及CEA單項對肺腺癌并發(fā)惡性胸腔積液的診斷效能(%)

注：MACC1：結(jié)腸癌轉(zhuǎn)移相關(guān)因子-1；c-MET：間質(zhì)-上皮細(xì)胞轉(zhuǎn)化因子；CEA：癌胚抗原

表4　MACC1、c-MET及CEA聯(lián)合對肺腺癌并發(fā)惡性胸腔積液的診斷效能(%)

注：MACC1：結(jié)腸癌轉(zhuǎn)移相關(guān)因子-1；c-MET：間質(zhì)-上皮細(xì)胞轉(zhuǎn)化因子；CEA：癌胚抗原

討　　論

MACC1是位于人染色體7p21.1上的MACC1基因編碼的蛋白質(zhì)，由Stein等[3]在2009年首次報道，并發(fā)現(xiàn)其是HGF(肝細(xì)胞生長因子)/c-MET信號通路的重要調(diào)節(jié)因子，對結(jié)腸癌的轉(zhuǎn)移和生長起到重要調(diào)控作用。MACC1在多種實體腫瘤組織中有高表達，如肝癌，乳腺癌，直腸癌，肺癌等[4-7]。Chundong等[8]通過檢測197例肺癌手術(shù)患者的癌組織中的MACCl表達水平，隨訪發(fā)現(xiàn)復(fù)發(fā)組MACC1表達水平明顯高于未復(fù)發(fā)組(82.50%vs. 65.50%)，提示MACC1對肺癌轉(zhuǎn)移起到調(diào)控作用。臨床研究發(fā)現(xiàn)MACC1mRNA和蛋白在非小細(xì)胞肺癌組織中高表達并提示預(yù)后不良，MACC1mRNA在非小細(xì)胞肺癌外周血中亦有高表達，在發(fā)生淋巴結(jié)轉(zhuǎn)移患者中水平更高[9-10]。故MACC1水平的檢測在肺癌的診斷中可能有一定幫助。

圖1胸腔積液MACC1、c-MET和CEA的ROC曲線

c-MET是由原癌基因c-met編碼的HGF的細(xì)胞膜特異性受體，主要在各種上皮細(xì)胞中表達[11]。HGF/c-MET信號通路主要調(diào)節(jié)細(xì)胞的增殖、分化、收縮、運動及分裂等多種生物學(xué)行為。該通路在癌細(xì)胞中的調(diào)控機制不同于正常細(xì)胞。研究發(fā)現(xiàn)c-MET介導(dǎo)的異常信號轉(zhuǎn)導(dǎo)在多種腫瘤包括肺癌中起著重要作用，HGF依賴的c-MET信號通路活化胞質(zhì)內(nèi)多種信號通路，如PI3K /AKT、Ras-Rac/Rho、MAPK及Stat3信號通路等，從而介導(dǎo)腫瘤發(fā)生、侵襲和轉(zhuǎn)移、血管新生、上皮-間質(zhì)轉(zhuǎn)化等過程[12-13]。有研究分析發(fā)現(xiàn)，c-MET在非小細(xì)胞肺癌組織中約61%呈高表達，其中約67%為肺腺癌[14]。也有多項研究發(fā)現(xiàn)，抑制c-MET表達后肺癌細(xì)胞的增殖和侵襲能力下降，從而證實c-MET與肺癌細(xì)胞侵襲生長緊密相關(guān)[15-16]。目前c-MET已被認(rèn)為是非小細(xì)胞肺癌治療的有效靶點，并有其針對性的靶向藥物出現(xiàn)[17-18]。

MACC1在轉(zhuǎn)錄水平上增強c-MET的表達，兩者結(jié)合后通過擴增或變異的方式激活下游傳導(dǎo)通路，而不依賴與HGF的結(jié)合，因此MACC1作為誘導(dǎo)轉(zhuǎn)移的HGF/c-MET通路的重要調(diào)節(jié)因子也發(fā)揮促進腫瘤細(xì)胞增殖、侵襲和轉(zhuǎn)移的作用[19]。本研究結(jié)果顯示胸腔積液MACC1和c-MET含量呈正相關(guān)，亦提示MACC1和c-MET間的緊密聯(lián)系。最近由Kokostynska等[20]通過對345例患者胸腔積液中c-MET的含量分析發(fā)現(xiàn)，非小細(xì)胞肺癌組MPE的c-MET較良性組明顯高表達，其靈敏度為75.2%，特異度為89.9%，與本研究結(jié)果較為相似。目前尚未見有文獻報道MACC1在肺癌患者外周血及惡性胸腔積液中的表達水平。本研究結(jié)果提示肺腺癌并發(fā)惡性胸腔積液中MACC1的表達水平明顯高于良性胸腔積液，其靈敏度為62.26%，特異度為84.21%，在肺腺癌并發(fā)惡性胸腔積液的診斷中有一定價值。

CEA是目前廣泛應(yīng)用于臨床的腫瘤標(biāo)志物，它是一種外分泌型的糖蛋白，最早發(fā)現(xiàn)于人的結(jié)腸癌組織中[21-23]。研究證實，CEA主要來源于腫瘤組織，在正常組織和良性病變組織中僅有少量合成和分泌[24-26]。在各型肺癌中，其對肺腺癌的診斷價值最高。本研究結(jié)果與既往研究結(jié)果一致，CEA在肺腺癌并發(fā)惡性胸腔積液中具有較高診斷價值。

本研究結(jié)果提示胸腔積液中的MACC1及c-MET在肺腺癌并發(fā)惡性胸腔積液中均有一定診斷價值，兩者在肺腺癌并發(fā)惡性胸腔積液患者血清中濃度較良性組高，但其差異無統(tǒng)計學(xué)意義，可能與MACC1和c-MET的組織起源和表達強度有關(guān),也可能與樣本量、誤差及偏倚等因素有關(guān),需要擴大樣本量等進一步研究。根據(jù)統(tǒng)計分析MACC1聯(lián)合c-MET，MACC1、c-MET聯(lián)合CEA可提高診斷的靈敏度和特異度。本研究同時發(fā)現(xiàn)在惡性組胸腔積液CEA低于診斷臨界值的4例樣本中，有3例的MACC1高于診斷臨界值，故對于MACC1能否在CEA發(fā)生漏診時作為補充診斷的假設(shè)尚有待擴大樣本量進一步探討。

綜上所述，MACC1和c-MET對肺腺癌并發(fā)惡性胸腔積液的診斷具有一定價值，兩者聯(lián)合CEA可明顯提高診斷的靈敏度和特異度。本實驗因時間和地點限制具有其局限性：①MPE病理類型只收集了肺腺癌；②樣本量較小；③無隨訪，未能分析肺腺癌患者治療前后MACC1及c-MET水平的變化及與患者總生存期有無相關(guān)性。故對于MACC1及c-MET能否作為肺腺癌患者并發(fā)惡性胸腔積液的診斷指標(biāo)應(yīng)用于臨床，還有待進一步研究。

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(本文編輯：張大春)

胡婕，尤青海，孫耕耘. 結(jié)腸癌轉(zhuǎn)移相關(guān)因子-1和間質(zhì)-上皮細(xì)胞轉(zhuǎn)化因子檢測在肺腺癌患者并發(fā)惡性胸腔積液中的診斷價值[J/CD]. 中華肺部疾病雜志： 電子版， 2016， 9(2)： 120-124.

Diagnostic value of combined measurement of metastasis-associated in colon cancer 1 and mesenchymal-epithelial transition factor in lung adenocarcinoma patients with malignant pleural effusion

HuJie,YouQinghai,SunGengyun.

DepartmentofRespiratoryMedicine,theFirstAffiliatedHospitalofAnhuiMedicalUniversity,Hefei230022,ChinaCorrespondingauthor：SunGengyun,Email:sungengyun@tom.com

ObjectiveTo investigate the diagnostic value of combined measurement of metastasis-associated in colon cancer 1(MACC1), carcino embryonie antigen(CEA) and mesenchymal-epithelial transition factor(c-MET) in lung adenocarcinoma patients with malignant pleural effusion. MethodsNinety-one patients with pleural effusions were observed, including 53 cases of malignant pleural effusions from lung adenocarcimoma and 38 cases of benign effusions. MACC1 and c-MET levels in plasma and pleural effusion were detected by ELISA. CEA was detected by radioimmunoassay. ResultsThe expressions of MACC1, c-MET and CEA in the group of patients with malignant pleural effusion(MPE) from lung adenocarcinoma were significantly higher than those in the benign group(P<0.05). There was no significant difference of the expression of MACC1 and c-MET in plasma for the two groups(P>0.05). The expression of MACC1 was correlated significantly positive with the expression c-MET in pleural effusion(r:0.728,P<0.01).A receiver operating characteristic (ROC) curve analysis was carried out to assess the value of MACC1, c-MET and CEA in MPE from lung adenocarcinoma patients. The cut-of values for MACC1, c-MET and CEA were defined at 90.98 pg/ml, 757.67 ng/ml and 1.95 ng/ml, respectively. And the sensitivities of MACC1, c-MET and CEA were 62.26%, 52.83% and 92.45%, the specificities were 84.21%, 84.21% and 92.11%, respectively. The sensitivity and specificity of combined detection of MACC1 and c-MET increased to 75.47% and 92.11%, respectively. Moreover, for MACC1, c-MET and CEA, the sensitivity and specificity combined detection increased to 98.10% and 100%, respectively. ConclusionsMACC1 and c-MET can be used as the reference indexes for the differentiation of benign pleural effusions and malignant pleural effusions from lung adenocarcinoma patients. Combined examination with CEA can significantly improve the diagnostic value.

Lung adenocarcinoma;Metastasis-associated in colon cancer 1;Mesenchymal-epithelial transition factor;Carcino embryonie antigen;Malignant pleural effusion

10.3877/cma.j.issn.1674-6902.2016.02.002

國家臨床重點?？平ㄔO(shè)項目基金(2012·649)

230022 合肥，安徽醫(yī)科大學(xué)第一附屬醫(yī)院呼吸內(nèi)科

孫耕耘，Email: sungengyun@tom.com

R563，R743

A

2015-10-08)