胸水脱落细胞DNA含量及VEGF, p53和CEA表达在良恶性胸腔积液.doc

Clin ica l Exper ience临床经验胸水脱落细胞DNA含量及VEGF, p53和CEA表达在良恶性胸腔积液鉴别中的意义周箴1 ,陈岗2 ,宣学金2 ,张俭2(1. 上海市肺部肿瘤临床医学中心,上海200030; 2. 上海交通大学附属胸科医院病理科,上海200030)摘要 目的:探讨检测胸水细胞的DNA倍体以及VEGF、p53、CEA表达鉴别良恶性胸腔积液的价值。方法: 73例胸腔积液患者,良性组13例,恶性组60例。图像细胞光度技术( image cytometry, ICM)进行DNA含量检测,免疫组化Envision法检测VEGF、p53、CEA。结果:良性胸水DNA异倍体率仅占23. 1%。恶性胸水的异倍体率占77. 8% ,两者有统计学差异( P = 0.001) 。良性胸腔积液患者VEGF、p53、CEA表达率分别为12. 5%、0、0。恶性胸腔积液VEGF、p53、CEA表达率分别为17. 5% ,17. 5%和68. 4% ,与良恶性胸腔积液中CEA的表达相比有统计学差异( P 0. 001) ,VEGF和p53的表达无显著差异( P = 0.722, P = 0. 198) 。经DNA倍体联合VEGF、p53、CEA检测,敏感率可达90. 4% ,特异度87. 5% ,符合率90%。结论: ICM检测胸水细胞的DNA倍体联合VEGF、p53、CEA表达值得进一步探讨,有可能成为鉴别良恶性胸腔积液的又一方法。关键词 肺肿瘤; 胸腔积液,恶性; DNA; 基因表达; 免疫组织化学; 诊断,鉴别中图分类号 R734. 2; R730. 43文献标识码 A文章编号 100027431 (2007) 0220155203Clin ica l sign if icance of DNA content and VEGF, p53 and CEA expressions in the differentia l diagnosis of ma lig2 nant and ben ign pleura l effusionsZHOU Zhen1 , CHEN Gang2 , XUAN Xue2jin2 , ZHANG J ian2 ( 1. Shanghai Lung Tumor ClinicalMedical Center, Shanghai 200030, China; 2. Department of Pathology, Shanghai Chest Hosp ital of J iaotong University, Shanghai 200030, China) ABSTRACT Objective: To investigate whether nuclear DNA content and vascular endothelial growth factor (VEGF) , p53 andcarcinoembryonic antigen (CEA) exp ressions had a potential value in differential diagnosis of malignant and benign p leural effusions.Methods: Seventy2three patientswith unilateral p leural effusion were divided into 2 group s: benign group ( n = 13) andmalignant group( n = 60). The DNA contentwasmeasured by the image cytometry ( ICM) and the level ofVEGF, p53 and CEA exp ressionswere de2tected by immunohistochemical Envision method. Results: The rates of aneup loid in benign and malignant p leural fluids were 23. 1%and 77. 8% , respectively. The difference between the benign andmalignant p leural effusionswas significant ( P 0. 001). Immunohis2tochemical staining showed that the positive rates of the VEGF, p53, and CEA in benign p leural fluid were 12. 5% , 0, and 0; in ma2 lignant p leura1 fluid were 17. 5% , 17. 5% , and 68. 4% , respectively. The difference in CEA between the benign and malignant p leu2 ral effusion was significant ( P 15% 以上的超二倍体细胞群(即S期细胞)和一个明显的四倍体细胞峰位诊为异位体阳性(图1)。图1DNA含量检测图Fig. 1DNA con ten t by ICMA: DNA dip loid; B: DNA aneup loid1. 2. 2免疫组化VEGF、p53、CEA的检测免疫组化Envision法, VEGF、p53、CEA单克隆抗体及DA2KO Envision试剂盒均购自长岛公司。阳性判断标准:试剂盒中阳性片为阳性对照, PBS取代一抗为阴性对照。VEGF、CEA以细胞质(膜)染色呈棕色视为阳性。p53以细胞核染色呈棕色视为阳性。其表达的判断以染色强度和阳性细胞百分率之和进行评估 2 。染色强度: 0 分,为无色; 1 分,淡黄为弱阳性; 2分,深黄为中等阳性; 3分,棕色为强阳性。阳性细胞百分率: 0分,为无阳性细胞; 1分,为阳性细胞 75%。两项之和: 0分为( - ) , 2分为( + ) , 34分为( + + ) , 57分为( + + + ) (图2) 。图2VEGF、p53和CEA在胸水细胞中的表达( Env ision, 400)F ig. 2Expression of VEGF, p53 and CEA in pleuraeffusion( Env ision, 400)A: Expression of VEGF; B: Exp ression of p53; C: Expression of CEA1. 3统计学方法采用SPSS 11. 0软件分别作2检验及Fishers精确检验,以P 0. 05为差异有统计学意义。2结果2. 1ICM对良恶性细胞中DNA含量的定性分析73例患者共进行了ICM检测68例, 13例良性疾病患者的胸腔积液,异倍体率仅占23. 1%。而临床诊断肺癌的60例肺癌患者胸水中检测了55例患者的胸水异倍体率占77. 8%, 两者有统计学差异( P = 0. 001) ,其中,以非小细胞肺癌(NSCLC)的胸水异倍体率较高为80. 8% ( P = 0. 002) ,鳞、腺癌间无显著差异(表1) 。DNA 定量分析能客观地判断良恶性胸水, 尤其是非小细胞肺癌的恶性胸水。DNA倍体灵敏度77. 8% ( 42 /55 ) ,特异度76. 9%(10 /13) ,阳性预测值( PPV)为93. 3% ( 42 /45) ,阴性预测值(NPV)为43.5% (10 /23) ,符合率77. 6%(52 /73) 。表1DNA含量在良恶性胸腔积液中表达Table 1Expression of DNA con ten t in pleura flu ids ofd ifferen t or ig in s n (%) DNA dip loidy DNA aneup loidy P value Benign 10 (76. 9) 3 (23. 1)Lung cancer 13 (22. 2) 42 (77. 8) 0. 001 SCLC 4 (100) 0NSCLC 9 (17. 6) 42 (80. 8) 0. 002Squamous 1 (50. 0) 1 (50. 0) Non2squamous 8 (16. 3) 41 (83. 7) 0. 325 SCLC: Small cell lung cancer; NSCLC: Non2small cell lurg cancer2. 2胸腔积液的免疫组化检测检测VEGF、p53、CEA。13例良性疾病患者胸腔积液中检测8例,表达率分别为12. 5% , 0, 0。60例肺癌患者胸腔积液中检测57 例, 表达率分别为17. 5% , 17. 5%和68. 4% (表2) ,良恶性胸腔积液中CEA的表达有显著差异( P 0. 001) ,VEGF和p53的表达无统计学意义( P = 0. 671, P = 0. 211) 。表2VEGF、p53、CEA在良恶性胸腔积液中表达Table 2Expression of VEGF, p53 and CEA in pleura flu ids of d ifferen t or ig in s n (%) Exp ression Benign Malignant P valueVEGF - 7 (87. 5) 47 (82. 5)65+ 1 (12. 5) 10 (17. 5) 0. 722p53 - 8 (100) 47 (82. 5)65+ 010 (17. 5) 0. 198CEA - 8 (100) 18 (31. 6)65+ 039 (68. 4) 0. 001DNA combinedbiomarkers60 - 7 (87. 5) 5 (9. 5)+ 1 (12. 5) 47 (90. 4) 0. 0012. 3胸腔积液中DNA倍体与肿瘤标记物联合检测60 例患者全部检测DNA 倍体、VEGF、p53 和CEA,良恶性胸腔积液经DNA倍体联合VEGF、p53及CEA检测有统计学意义( P 0. 001) 。平行试验(4项指标中任何一项阳性即为阳性)的敏感率可达90. 4% (47 /52) ,特异度87. 5% (7 /8) ,符合率90%(54 /60) 。3讨论目前肺部肿瘤的发生率在不断提高,鉴别良恶性胸腔积液是临床工作中常见问题。诊断肺癌的金指标是组织、细胞学的病理诊断。但这是人为地把连续演变的肿瘤细胞分成良性,交界性和恶性,往往带有一定的主观倾向性。而且常因细胞数太少或细胞形态问题出现“核异型”等,不能作出肯定的诊断,给临床诊疗带来困难。1994年就有利用流式细胞分析( FCM)进行胸腔积液脱落细胞DNA定量分析,鉴别良恶性胸腔积液 3 。在正常细胞中DNA的含量是恒定的,人正常细胞是二倍体,恶性细胞在多数情况下染色体数增加,其DNA含量也增加,称为异倍体。根据这个特点,测定DNA含量可作为诊断癌细胞的客观依据。当前国际上已利用图像处理系统( IAS)和图像细胞系统客观地从微观水平来诊断。其中, ICM可用来测定细胞经染色后的光密度进行DNA 分析 4 。FCM作DNA 检测主要把肿瘤组织制成单个细胞悬液后才能加以测量,这种悬液中常包含许多非肿瘤成分,如间质细胞、血管内皮及白细胞等;还由于分离不彻底以致可能含多个细胞的细胞团或分离过度而产生核的碎片等。在分离过程中不能对成分进行选择,因此会影响测量结果的准确性。ICM的特点是在光镜观察下可以选择细胞,这样就可以摒除那些非肿瘤细胞或其他会影响结果的成分,而且即使细胞的量很少也能测量 1 。据报道 5 :痰DNA异倍体作为标志物诊断肺癌,异倍体阳性率达40% ,与结核对照组、健康人对照组有显著差异,而且高于常规痰脱落细胞学检查。本研究证实利用ICM检测DNA含量异倍体阳性率在良恶性胸腔积液中有显著差( P = 0. 001) 。DNA倍体灵敏度77. 8%,特异度76. 9%,阳性预测值( PPV)为93. 3%,阴性预测值(NPV)为43. 5%,符合率77.6%。尤其诊断非小细胞肺癌的癌性胸水更准确,因小细胞肺癌的胸水部分原因是因肿大淋巴结压迫,以致淋巴回流障碍所致,而非肿瘤侵犯胸膜所致。VEGF是一种具有内皮细胞特异性的有丝分裂原,对血管生成有强烈诱导作用。VEGF在肺癌胸膜转移导致恶性胸腔积液生成具有重要的作用。但是免疫组化VEGF半定量检测在胸水细胞中的表达较低,在良恶性胸水中均有表达,分别为12. 5%, 17.5%,对鉴别良恶性胸腔积液作用有限。Thiekett等 6 采用EL ISA定量研究不同病因胸水中的 VEGF含量发现癌性胸水中VEGF含量明显高于非癌性胸水,与本研究不一致。可能因本研究样本量较少并检测方法不同,未能体现VEGF表达在良恶性胸水鉴别的价值,与Kaya等 7 的检查结果相似。p53 基因突变存在于各种实体瘤细胞中, 约50%的肺癌组织中可检测到p53基因突变。但胸水细胞中的p53基因突变其阳性率仅17. 5% ,与张贺龙等 8 的研究结果相似。虽然鉴别良恶性胸水在统计学上没有意义,但可以看到良性胸水无表达,可以作为恶性胸水的筛选。CEA作为癌胚抗原在良性胸水中无表达,而恶性胸水表达率达68. 4%,在鉴别良恶性胸水有统计学意义( P 0. 001) 。通过ICM对胸水细胞的DNA检测联合VEGF、P53和CEA的检测,敏感率可达90. 4%,特异度87.5%,与常规胸水脱落细胞学检查相比,符合率达90%。值得进一步探讨,有可能成为鉴别良恶性胸腔积液的又一方法。第2期. 周箴,等. 胸水脱落细胞DNA含量及VEGF, P53和CEA表达在良恶性胸腔积液鉴别中的意义157 J . Proc N atl Acad Sci USA, 2003, 100 (17) : 979129796. 17 GROSSM, YANG R, TOP I, et al. PIASy2mediated rep ression ofthe androgen recep tor is independent of sumoylation J . Onco2gene, 2004, 23 (17) : 305923066. 18 JOHNSON ES. Protein modification by SUMO J . Annu Rev B io2chem , 2004, 73: 3552382. 19 SCHMIDTD, MULLER S. Members of the PIAS family act as SU2MO ligases for c2Jun and p53 and rep ress p53 activity J . ProcN atl Acad Sci USA , 2002, 99 (5) : 287222877. 20 UNGUREANU D, VANHATUPA S, GRONHOLM J, et al. SU2MO21 conjugation selectively modulates STAT12mediated gene re2sponses J . B lood, 2005, 106 (1) : 2242226. 21 L IANG M, MELCH IOR F, FENG X H, et al. Regulation ofSmad4 sumoylation and transforming growth factor2 signaling byp rotein inhibitor of activated STAT1 J . J B iol Chem , 2004, 279(22) , 22857222865. 22 LONG J, WANG G, MATSUURAL I, et al. Activation of Smadtranscrip tional activity by p rotein inhibitor of activated STAT3( P IAS3) J . Proc N atl Acad SciUSA, 2004, 101 (1) : 992104. 23 BETZ A, LAMPEN N, MARTINEK S, et al. A Drosophila PIAShomologue negatively regulates stat92E J . Proc N atl Acad SciUSA , 2001, 98 (17) : 956329568. 24 HAR I KL, COOK KR, KARPEN GH. The Drosophila Su ( var)2210 locus regulates chromosome structure and function and en2codes a member of the PIAS p rotein family J . GenesDev, 2001,15 (11) : 133421348. 25 ROTH W, SUSTMANN C, KIESL INGER M, et al. P IASy2defi2cientmice disp laymodest defects in IFN andWnt signaling J . JImm unol, 2004, 173 (10) : 618926199. 26 MEL INO G, LU X, GASCO M, et al. Functional regulation ofp73 and p63: development and cancer J . Trends B iochem Sci,2003, 28 (12) : 6632670. 27 MEGID ISH T, XU JH, XU CW. Activation of p53 by p rotein in2hibitor of activated Stat1 ( P IAS1) J . J B iol Chem , 2002, 277(10) : 825528259. 28 MUNARR IZ E, BARCAROL ID, STEPHANOU A, et al. PIAS21is a checkpoint regulatorwhich affects exit from G1 and G2 by su2moylation of p73 J . M ol Cell B iol, 2004, 24 ( 24 ) : 10593210610. 29 YANG Y, L I C C, WEISSMAN AM. Regulating the p53 systemthrough ubiquitination J . Oncogene, 2004, 23 ( 11 ) : 209622106. 30 M IYAUCH I Y, YOGOSAWA S, HONDA R, et al. SumoylationofMdm2 by p rotein inhibitor of activated STAT ( P IAS) and Ran2BP2 enzymes J . J B iol Chem , 2002, 277 (51) : 50131250136. 31 W IBLE BA, WANGLM, KURYSHEV YA, et al. Increased K +efflux and apop tosis induced by the potassium channel modulatoryp rotein KchAP /PIAS3B in p rostate cancer cells J . J B iol Chem ,2002, 277 (20) : 32 WANG LM, BANERJEE S. Differential PIAS3 exp ression in hu2man malignancy J . Oncol