孟 超，高 潔，梁 軍，梁智勇，林巖松

1青島大學(xué)醫(yī)學(xué)院附屬醫(yī)院腫瘤科，青島 266003中國醫(yī)學(xué)科學(xué)院 北京協(xié)和醫(yī)學(xué)院 北京協(xié)和醫(yī)院 2病理科 3核醫(yī)學(xué)科，北京 100730

·論著·

甲狀腺乳頭狀癌并BRAFV600E突變及原癌基因重排蛋白表達(dá)與其侵襲性的關(guān)系

孟 超1，高 潔2，梁 軍1，梁智勇2，林巖松3

1青島大學(xué)醫(yī)學(xué)院附屬醫(yī)院腫瘤科，青島 266003中國醫(yī)學(xué)科學(xué)院 北京協(xié)和醫(yī)學(xué)院 北京協(xié)和醫(yī)院2病理科3核醫(yī)學(xué)科，北京 100730

目的探討B(tài)RAFV600E突變及原癌基因重排 (RET) 蛋白表達(dá)并存與甲狀腺乳頭狀癌(PTC)侵襲性的關(guān)系。方法收集50例PTC患者術(shù)后病理，分別用RT-PCR檢測 BRAFV600E突變，免疫組織化學(xué)SP法檢測RET表達(dá)情況。分析BRAFV600E突變合并RET表達(dá)組(n=24)和BRAFV600E單突變或RET單表達(dá)組 (n=19)的臨床及病理特征。結(jié)果BRAFV600E突變率、 RET表達(dá)率分別為76%(38/50)、56%(28/50)，存在BRAFV600E突變合并RET表達(dá)者占48%(24/50)。與BRAFV600E突變合并RET表達(dá)組比較， BRAFV600E單突變或RET單表達(dá)組腫瘤組織分化程度差，且甲狀腺癌評分系統(tǒng)評分較高(P=0.011，P=0.022)。結(jié)論BRAFV600E突變并存RET表達(dá)時提示PTC腫瘤組織分化差、更易出現(xiàn)實體亞型等侵襲性較高的病理亞型，有可能增加腫瘤相關(guān)死亡風(fēng)險。

BRAFV600E突變；原癌基因重排蛋白表達(dá)；甲狀腺乳頭狀癌；侵襲性

ActaAcadMedSin,2013,35(1):64-68

甲狀腺乳頭狀癌(papillary thyroid cancer, PTC)是甲狀腺惡性腫瘤中最常見的一種類型，發(fā)病率為70%～80%[1]。Xing等[2]對近30篇PTC相關(guān)基因突變文獻(xiàn)分析后總結(jié)其BRAFV600E突變率為29%～83%，即15外顯子第1799位核苷酸T被A取代，導(dǎo)致600密碼子編碼的纈氨酸被谷氨酸替代(V600E)[3-4]，與PTC的局部淋巴結(jié)及遠(yuǎn)處轉(zhuǎn)移相關(guān)[5-6]。原癌基因重排(rearranged during transfection proto-oncogene，RET)蛋白，尤其是RET/PTC活化表達(dá)不僅是PTC的致病因之一，還與其局部侵襲性[7]、預(yù)后及復(fù)發(fā)[8]有關(guān)。目前國內(nèi)外研究二者突變并存的文獻(xiàn)較少，Musholt等[9]對可疑PTC患者的細(xì)針穿刺活檢組織聯(lián)合行BRAFV600E突變及RET/PTC檢測，有助于PTC術(shù)前診斷、惡性程度評估及優(yōu)化手術(shù)方式。Henderson等[10]對PTC復(fù)發(fā)人群的研究結(jié)果提示二者雙突變可能與PTC復(fù)發(fā)有關(guān)。在臨床工作中PTC患者可同時出現(xiàn)BRAFV600E突變及RET表達(dá)，有關(guān)BRAFV600E突變和RET表達(dá)并存與其侵襲性的關(guān)系，目前報道較少。本研究擬對初診PTC患者手術(shù)切除腫瘤組織行BRAF、RET檢測，旨在分析BRAFV600E突變和RET表達(dá)并存與PTC侵襲性之間的關(guān)系。

資料和方法

標(biāo)本來源選取北京協(xié)和醫(yī)院2011年11月至2012年2月甲狀腺全切除術(shù)后病理確診為PTC并均無遠(yuǎn)處轉(zhuǎn)移的50例患者，男女比為1∶2.1，平均年齡(40.49±11.44)歲。所有患者術(shù)后病理組織學(xué)切片均由2位高年資病理醫(yī)師復(fù)查確認(rèn)為PTC，其中PTC亞型包括濾泡亞型9例、實體亞型10例、淋巴上皮瘤樣亞型及透明細(xì)胞型各1例(因亞型罕見未行分析)。

RT-PCR法檢測PTC組織中BRAFV600E突變

組織DNA提取：嚴(yán)格按QIAamp DNA FFPE Tissue Kit(50)(中美艾德AmoyDX公司)說明書進(jìn)行。紫外光光度計檢測DNA純度及濃度。

PCR擴(kuò)增：取cDNA 5 μl，上下游引物各35 μl，TaqDNA聚合酶0.4 μl等，加入PCR反應(yīng)體系中擴(kuò)增。反應(yīng)條件：95℃ 5 min 1個循環(huán)； 95℃ 25 s，64℃ 20 s，72℃ 20 s 15個循環(huán)； 93℃ 25 s，60℃ 35 s，72℃ 20 s 31個循環(huán)。

PCR產(chǎn)物分析：(1)確定實驗是否成功可信：待測樣品的內(nèi)控內(nèi)對照信號(VIC信號)應(yīng)升起。(2)確認(rèn)未選擇校正熒光參照，按管號順序依次選擇單一突變檢測反應(yīng)管進(jìn)行檢測分析。需要同時選擇陽性質(zhì)控品反應(yīng)孔、無模板對照孔和樣品反應(yīng)孔，然后根據(jù)實際情況調(diào)節(jié)閾值至目的基因FAM擴(kuò)增曲線升起的拐點處，得到突變組的Ct值，即熒光達(dá)到閾值時候的PCR循環(huán)次數(shù)。若樣本的Ct大于或等于28，則樣本為陰性;若樣本的Ct小于28，則樣本為陽性。

免疫組織化學(xué)法檢測RET在PTC組織中的表達(dá)標(biāo)本經(jīng)4%甲醛溶液固定，常規(guī)石蠟包埋，4μm厚連續(xù)切片，分別行HE和免疫組織化學(xué)染色。采用免疫組織化學(xué)SP法(免疫組織化學(xué)試劑購于丹麥DAKO公司，RET鼠抗人單克隆抗體為即用型制劑)，具體操作步驟按試劑說明書進(jìn)行，以1 mmol/L的EDTA pH 9.0 修復(fù)液進(jìn)行高壓鍋抗原修復(fù)，DBS溶液顯色，蘇木精復(fù)染，中性樹膠封閉，鏡下觀察。RET陽性表達(dá)位于細(xì)胞漿，<5%細(xì)胞著色為陰性，≥5%細(xì)胞著色為陽性。

結(jié) 果

PTC腫瘤組織BRAFV600E突變、RET表達(dá)情況76% (38/50)患者檢測到BRAFV600E突變；56% (28/50)患者癌組織中檢測出RET表達(dá)，2例患者癌旁正常組織中檢測出RET蛋白表達(dá)(合并橋本氏甲狀腺炎和結(jié)節(jié)性甲狀腺腫者各1例)；48% (24/50)患者同時存在BRAFV600E突變及RET表達(dá)，19例患者為BRAFV600E單突變或RET單表達(dá)，7例患者既不存在BRAFV600E突變也無RET表達(dá)(統(tǒng)計數(shù)據(jù)時已將其剔除)。

臨床病理特征BRAFV600E突變和RET表達(dá)并存組(G1組)患者腫瘤組織呈中分化者明顯高于二者單表達(dá)組(G2組)(P=0.011)；G1組患者實體亞型者明顯高于G2組(P=0.020)；甲狀腺乳頭狀癌評分系統(tǒng)(MACIS評分)高于G2組(P=0.022)。G1組中患者腫瘤侵出甲狀腺包膜與周圍組織黏連例數(shù)明顯高于G2組(P=0.036)(表1、圖1)。

表 1 兩組患者臨床病理資料比較

G1組：BRAFV600E突變合并RET表達(dá)組；G2組：BRAFV600E單突變或RET單表達(dá)組；RET:原癌基因重排；Nx：未被評估的局部結(jié)節(jié)

GroupG1: concurrent BRAFV600Eand RET protein expression; GroupG2: only one of BRAFV600Emutation and RET protein expression; RET: rearranged during transfection proto-oncogene; Nx：regional nodes cannot be assessed

G1組：BRAFV600E突變合并RET表達(dá)組；G2組：BRAFV600E單突變或RET單表達(dá)組GroupG1: concurrent BRAFV600E and RET protein expression; GroupG2: only one of BRAFV600E mutation and RET protein expression圖 1 兩組患者腫瘤組織實體亞型、中分化程度及包膜侵犯發(fā)生率Fig 1 Percentages of solid variant, moderately differentiation and extrathyroid invasion in two groups

討 論

PTC是甲狀腺惡性腫瘤中最常見的類型[1]，經(jīng)甲狀腺全切或次全切除術(shù)輔以選擇性131I治療及甲狀腺激素抑制治療后，大多數(shù)患者預(yù)后較好，但仍約30%復(fù)發(fā)率[11]。包膜侵犯、頸部淋巴結(jié)轉(zhuǎn)移可使PTC腫瘤組織侵襲性增強(qiáng)，導(dǎo)致患者10年生存率下降[12]。研究顯示PTC患者BRAFV600E突變率為29%～83%[2]，RET/PTC重排發(fā)生率為17%～44%[13]。二者均與PTC患者局部侵襲性、預(yù)后及復(fù)發(fā)相關(guān)[5-8,14]。有關(guān)BRAFV600E突變合并RET表達(dá)對PTC侵襲性影響的報道較少，Musholt等[9]證實聯(lián)合檢測BRAFV600E突變及RET/PTC異常表達(dá)有助于PTC術(shù)前細(xì)針穿刺活檢組織診斷、惡性程度評估及優(yōu)化手術(shù)方式；針對復(fù)發(fā)性PTC的研究顯示BRAFV600E突變和RET/PTC重排并存可能與PTC復(fù)發(fā)及惡性程度有關(guān)[10]。本研究首次觀察了PTC患者初次手術(shù)病理BRAFV600E突變并存RET表達(dá)與PTC侵襲性的關(guān)系，結(jié)果顯示BRAFV600E突變和RET表達(dá)并存組(G1組)患者包膜外侵犯率明顯高于單一突變或表達(dá)組(G2組)，提示兩者并存時腫瘤組織的侵襲性更高。

PTC患者腫瘤組織的侵襲性與其分化程度及病理亞型類型有關(guān)。RET原癌基因高表達(dá)，RAS/BRAF/MAPK信號通路持續(xù)激活被認(rèn)為是PTC發(fā)病的主要分子機(jī)制之一，影響PTC腫瘤組織的分化程度及亞型[15]。Kakudo等[16]研究提示中分化甲狀腺癌患者10年生存率較經(jīng)典型甲狀腺癌者下降約20%，提示其預(yù)后較差。實體亞型是PTC病理特殊組織學(xué)亞型，屬于侵襲性強(qiáng)、預(yù)后差的類型，其患者頸部淋巴結(jié)及遠(yuǎn)處轉(zhuǎn)移率、復(fù)發(fā)率高[16-18]。本研究G1組PTC患者腫瘤組織呈中分化者明顯高于G2組，同時實體亞型者明顯高于G2組，提示BRAFV600E突變和RET表達(dá)并存時使PTC腫瘤組織分化程度降低、更易出現(xiàn)實體亞型等侵襲性較高的病理亞型。

MACIS評分主要依據(jù)患者年齡、原發(fā)腫瘤直徑、局部侵犯情況、是否完全切除及遠(yuǎn)處轉(zhuǎn)移進(jìn)行計算。有報道MACIS評分>6時可作為PTC患者死亡率的預(yù)測指標(biāo)之一，其分值高，提示PTC患者腫瘤組織的侵襲性增強(qiáng)，生存率降低[19]。本研究G1組PTC患者M(jìn)ACIS評分較G2組高，分析其原因主要與本組患者包膜侵犯率較高有關(guān)，提示BRAFV600E突變和RET表達(dá)并存時，患者腫瘤組織侵襲性增高，增加了腫瘤相關(guān)死亡風(fēng)險。

將G2組進(jìn)一步分為兩個亞組，即BRAFV600E突變或RET單表達(dá)組，兩組患者病理資料差異無統(tǒng)計學(xué)意義，可能與患者病例數(shù)較少有關(guān)，在今后研究中將增加病例數(shù)繼續(xù)對其進(jìn)一步觀察。

綜上，PTC患者初次術(shù)后病理BRAFV600E突變和RET表達(dá)并存時與腫瘤組織的侵襲性明顯相關(guān)，使其具有更高包膜侵犯率，導(dǎo)致分化程度變差且更易出現(xiàn)實體亞型等侵襲性較高的病理亞型，有可能增加腫瘤相關(guān)死亡風(fēng)險。

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Invasive Properties of Papillary Thyroid Cancer with Concurrent BRAFV600EMutationand Rearranged during Transfection Proto-oncogene Protein Expression

MENG Chao1, GAO Jie2, LIANG Jun1, LIANG Zhi-yong2, LIN Yan-song3

1Department of Oncology, the Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, China2Department of Pathology,3Department of Nuclear Medicine, PUMC Hospital，CAMS and PUMC, Beijing 100730, China

LIN Yan-song，Tel：010-69155610,E-mail:linyansong68@yahoo.com.cn

Objective To investigate the aggressive properties of papillary thyroid cancer (PTC) with concurrent BRAFV600Emutation and rearranged during transfection (RET) proto-oncogene protein expression.MethodsFifty pathologically confirmed PTC patients who had

thyroidectomy were enrolled in this study. BRAFV600Emutation was detected by real time polymerase chain reaction (RT-PCR), while RET protein expression was measured by immunohistochemical SP method. Clinical and pathological features were compared between the concurrent BRAFV600Emutation and RET protein expression group (n=24) and BRAFV600Emutation or RET protein expression alone group (n=19). Seven patients were ruled out from the final analysis due to the absence of either BRAFV600Emutation or RET protein expression.ResultsOf these 50 patients, BRAFV600Emutation and RET protein expression were detected in 38 patients (76%) and 28 patients (56%), respectively. Concurrent BRAFV600Emutation and RET expression was detected in 24 patients (48%). Compared with the concurrent BRAFV600Emutation and RET protein expression group, the BRAFV600Emutation or RET protein expression alone group had relatively poorer tissue differentiation and higher prognostic score (P=0.011，P=0.022).ConclusionPTC patients with concurrent BRAFV600Emutation and RET expression present poorer differentiation, more highly aggressive variant in carcinoma tissues, and higher cancer-related mortality risk.

BRAFV600Emutation; tyrosine kinase receptor protein expression; papillary thyroid cancer; aggressiveness

國家自然科學(xué)基金(30970850)和衛(wèi)生行業(yè)科研專項項目(201202012)Supported by the National Natural Sciences Foundation of China (30970850)and the Research Special Fund of the Health Industry(201202012)；第一、二位作者對本文貢獻(xiàn)一致The first two authors contributed equally to this article

林巖松 電話：010-69155610，電子郵件：linyansong68@yahoo.com.cn

R736.1

A

1000-503X(2013)01-0064-05

10.3881/j.issn.1000-503X.2013.01.012

2012-04-25)