第二军医大学  2014, Vol. 35 Issue (4): 378-382   PDF    
引用本文
王碧波, 韩一平, 万善志, 黄玲, 马大烈, 郑建明, 李强. p63与表皮生长因子受体突变肺鳞癌患者生存的关系[J]. 第二军医大学学报, 2014, 35(4): 378-382  
WANG Bi-bo, HAN Yi-ping, WAN Shan-zhi, HUANG Ling, MA Da-lie, ZHENG Jian-ming, LI Qiang. Relationship between p63 expression and survival of squamous lung cancer patients with mutatant epidermal growth factor receptor[J]. Academic Journal of Second Military Medical University, 2014, 35(4): 378-382 (in Chinese with English abstract)  
p63与表皮生长因子受体突变肺鳞癌患者生存的关系
王碧波1, 韩一平1, 万善志1, 黄玲2, 马大烈2, 郑建明2, 李强1    
1. 第二军医大学长海医院呼吸内科, 上海 200433;
2. 第二军医大学长海医院病理科, 上海 200433
收稿日期:2013-11-4     接受日期: 2014-1-9.
作者简介：王碧波，硕士生.E-mail：wuxiwangbb@live.cn
通信作者(Corresponding author)：Tel: 021-31161311 E-mail: yphan2006@163.com
摘要：目的 检测表皮生长因子受体（EGFR）在肺鳞癌患者中的突变率，并分析p63与EGFR突变肺鳞癌患者生存的关系。方法 收集2010年2月至2013年3月在我院初治的肺鳞癌患者的组织样本，用直接测序法检测EGFR突变情况，免疫组织化学法检测p63的表达。随访患者，记录患者的总生存时间（OS），评价EGFR突变和p63表达对患者生存的影响。结果 所有病理组织确诊为肺鳞癌的262例患者中，16例（6.1%）有EGFR突变 ，其中外显子19位点缺失7例，外显子21 L858R点突变9例，未见外显子18和20突变。与传统意义上的肺鳞癌患者相比，EGFR突变的肺鳞癌患者女性和非吸烟者所占比例较高。p63部分阳性的EGFR突变患者（n=4）的OS为11.28（95%CI：9.33～15.23）个月，p63阳性的EGFR突变患者（n=11）的OS 为5.93（95%CI：1.75～8.39）个月，两者差异有统计学意义（P=0.039）。结论 肺鳞癌患者EGFR突变率为6.1%，均为外显子19和21敏感突变。p63部分阳性的EGFR突变患者比p63阳性EGFR突变患者生存期更长。
关键词： p63     表皮生长因子受体     肺肿瘤     鳞状细胞癌    
Relationship between p63 expression and survival of squamous lung cancer patients with mutatant epidermal growth factor receptor
WANG Bi-bo1, HAN Yi-ping1, WAN Shan-zhi1, HUANG Ling2, MA Da-lie2, ZHENG Jian-ming2, LI Qiang1    
1. Department of Respiratory Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China;
2. Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Abstract: Objective To detect the mutation rate of epidermal growth factor receptor (EGFR) in squamous carcinoma (SQC) of lung and to exam the relationship between p63 and survival of SQC patients with EGFR mutation. Methods SQC specimens were collected from the patients who received their initial treatment in our hospital between February 2010 and March 2013. The incidence of EGFR mutations was analyzed by automated direct sequencing, and immunohistochemical method was used to observe p63 expression. The patients were followed up and the impact of EGFR mutation and p63 expression upon survival of SQC patients was evaluated. Results Among the 262 histological diagnosed SQC patients, 16 (6.1%) had EGFR mutations, including exon 19 deletion (7 of 16 patients, 43.8%) and L858R point mutation in exon 21 (9 of 16, 56.2%), with no mutation of exon 18 and 20. SQC patients with EGFR mutations, compared with other SQC patients, had more females and non-smokers. The median overall survival (OS) time of 4 EGFR-mutated patients with p63 partial positive expression was 11.28 months (95% CI: 9.33-15.23 months), which was significantly longer than that of 11 EGFR-mutated patients with p63 positive expression (5.93 months [95% CI: 1.75-8.39 months], P=0.039). Conclusion EGFR mutation rate is 6.1% in our study, with all being exon 19 and 21 mutations. Patients presenting partial positive p63 expression have longer survival time than those presenting p63 positive expression.
Key words: p63     epidermal growth factor receptor     lung neoplasms     squamous cell carcinoma    

表皮生长因子受体(epidermal growth factor receptor,EGFR)是肿瘤靶向治疗的重要靶标,其酪氨酸激酶抑制剂如吉非替尼、厄洛替尼等治疗非小细胞肺癌(non-small cell lung cancer, NSCLC)尤其是EGFR敏感突变的肺腺癌(adenocarcinoma,ADC)具有显著疗效[1 ,2]。EGFR基因突变率在东亚裔ADC人群中为30%~40%,但在同地区其他组织类型的NSCLC中则较低[3 ,4],因此目前临床上对肺鳞癌(squamous carcinoma,SQC)患者检测EGFR突变的意义尚存争议[5]。

p63是抑癌基因p53家族成员之一,由于启动子不同和3′端剪切方式不同, p63基因可编码多种具有不同活性的异构体。这些异构体主要分为具有反式激活区的TA异构体和N末端截短的ΔNp63α异构体,前者具有p53样活性,可诱导细胞周期停滞和凋亡,后者则抑制p53的功能,促进转化细胞的生长[6,7,8]。我们先前的研究表明,肺SQC中高表达p63,且其表达水平与组织学或细胞学的采样方法以及分化程度无关[9]。本研究检测了EGFR在肺SQC患者中的突变率并分析了p63对EGFR突变肺SQC患者生存的影响。 1 材料和方法 1.1 样本采集

收集2010年2月至2013年3月经病理组织学确诊的初治肺SQC样本。样本来自于经CT引导的细针活检、支气管镜下经支气管的活检或者手术切除样本。 1.2 EGFR基因突变检测

采用直接测序法对EGFR的4个位点(18～21位点)进行检测[10]。应用PCR直接扩增EGFR 18~21位点的基因片段。2%琼脂糖凝胶电泳鉴定扩增片段,通过凝胶成像系统观察PCR产物电泳结果。PCR产物进行正反两个方向测序。分析测序图谱,判断EGFR基因18~21位点外显子区域是否存在突变。 1.3 p63表达检测

主要试剂抗人p63单克隆抗体及EnVision两步法试剂盒均为丹麦DAKO公司产品。组织均经4%中性甲醛溶液固定,常规脱水后石蜡包埋,切成3～4 μm厚的薄片,在乙二胺四乙酸溶液(EDTA,pH 8.0)中进行热诱导抗原修复。然后用3%过氧化氢阻断内源性过氧化氢酶活性,加入抗人p63单克隆抗体(clone 4A4)作为标记,EnVision两步法染色,显微镜下观察。结果判断: 细胞表现出一种弥漫染色或者平均阳性染色面积≥10%定义为免疫反应阳性(+)；细胞染色面积介于1%和9%之间定义为免疫反应部分阳性(部分+)；细胞完全没有被染色定义为免疫反应阴性(-)[11]。 1.4 生存评价

以无疾病进展时间(progression-free survival,PFS)和总生存时间(overall survival,OS)作为生存评价指标。PFS:从确诊疾病到影像学上第1次发生疾病进展或其他任何原因导致死亡的时间;OS:从确诊开始到死亡或末次随访的时间。 1.5 统计学处理

用Kaplan-Meier法估计生存时间,log-rank法分析生存差异。检验水准(α)为0.05。 2 结 果 2.1 肺SQC患者EGFR突变率

共收取267例经病理组织学确诊的肺SQC样本,经2位富有经验的病理学专家复检,排除5例鳞腺混合样本,最终262例样本纳入研究。262例样本中,16例检测到EGFR基因突变,突变率为6.1%。其中外显子19缺失7例(43.8%),外显子21点突变(L858R)9例(56.2%),未检测到外显子18和20突变。 2.2 EGFR突变肺SQC患者的临床病理特征

由表 1可见,16例EGFR突变肺SQC患者女性比例和非吸烟者比例较非突变患者高(P<0.05)。由表 2可见,所有16例EGFR突变患者中,13例(81.3%)为ⅢB或 Ⅳ期肺SQC,其中3例(23.1%)是女性,7例(53.8%)是非吸烟者。

表 1 EGFR突变与非突变肺SQC患者的临床特征比较Tab 1 Comparison of clinical data between EGFR mutation and non-EGFR mutation in lung SQC patients

表 2 EGFR突变肺SQC患者一般资料、突变类型、 临床病理特征以及生存时间汇总 Tab 2 Clinical data, mutation types, pathologic findings, and survival time of EGFR-mutated lung SQC patients
2.3 肺SQC患者p63的表达

262例样本中,p63阳性(图 1A)247例,p63部分阳性(图 1B)10例,p63阴性5例。p63部分阳性和阴性的15例均由病理学专家再次复核证实为肺SQC。16例EGFR突变样本的p63表达情况见表 2。

图 1 p63在肺SQC中不同免疫活性的表达 Fig 1 Different immunoreactivities of p63 in lung SQC patients SQC: Squamous carcinoma. A: p63 positive sample; B: p63 partially positive sample. Original magnification: ×400
2.4 生存分析

截至2013年5月20日,16例EGFR突变患者无一例失访。随访时间5.23～ 35.93 个月,中位随访时间13.90(95% CI: 8.76~19.04)个月。16例EGFR突变患者中15例(93.8%)疾病进展,进展患者中11例(68.8%)已死亡。16例EGFR突变患者的PFS和OS见表 2。

根据p63免疫反应活性,把16例EGFR突变患者分成3组: p63阴性组1例, p63部分阳性组4例, p63阳性组11例。p63阴性组因只有1例而未进行统计学分析。4例p63部分阳性患者中位PFS为7.76(95% CI:3.12～12.34)个月,中位OS为11.28(95%CI: 9.33～15.23)个月。11例p63阳性患者中位PFS为4.43(95% CI:2.65～6.22)个月,与p63部分阳性患者相比差异无统计学意义(P=0.256)；中位OS为5.93(95%CI: 1.75～8.39)个月,与p63部分阳性患者相比差异有统计学意义(P=0.039,图 2)。

图 2 p63免疫活性与肺SQC EGFR突变患者生存的关系 Fig 2 Relationship between p63 immunoreactivity andsurvival of SQC patients with EGFR mutation SQC: Squamous cell carcinoma; EGFR: Epidermal growth factor receptor; OS: Overall survival
3 讨 论

EGFR在SQC中的突变率文献报道各不相同。Mu等[12]报道肺SQC的EGFR突变率为5.9%(6/102),而Lai等[13]报道282例肺SQC中有41例EGFR突变,突变率为14.5%。然而Hata等[14]研究认为,纯SQC无EFGR突变。他们对原有测得的33例EGFR突变的肺SQC(33/249)进行复检并结合p63、TTF-1等免疫组化指标检测,结果显示这33例EGFR突变肺SQC都混有不同程度ADC成分,因此认为EGFR在肺SQC中的突变是由于肺SQC中混有ADC成分所致。但大多数学者都认为肺SQC确实存在EGFR突变,本研究结果也与此一致。本研究发现,在262例初治肺SQC样本中有16例EGFR基因敏感突变,突变率为6.1%。根据现有结果,我们认为EGFR突变确实发生在肺SQC患者中,EGFR是肺SQC的驱动基因之一。

与传统意义上的肺SQC患者相比,EGFR突变患者的临床特征显示有更多的女性(37.5% vs 3.7%)和非吸烟者(62.5% vs 26.0%),提示EGFR突变的肺SQC患者可能与传统的肺SQC患者有着不同的遗传背景。本研究中EGFR突变类型以及各类型百分比(外显子19缺失,43.8%；L858R,56.2%),与其他作者[3,12-13]研究结果大致相同。有文献报道EGFR基因在SQC中存在非敏感位点突变,包括外显子21 N826S、A859T、L861Q、V843I、K860E等 [15,16,17,1819],但报道呈现单个、非重复性。因此,对于检测敏感位点L858R和外显子19缺失以外的非敏感位点,是不必要和不经济的。

p63是免疫组化诊断肺SQC的常用指标,本研究发现p63与EGFR之间似乎对肺SQC患者生存有影响:p63部分阳性EGFR突变患者的OS为11.28(95%CI: 9.33～15.23)个月,比p63阳性EGFR突变患者的OS (5.93个月,95%CI: 1.75～8.39个月)更长(P=0.039)。p63和EGFR两者的关系可能与胰岛素样生长因子结合蛋白3(IGFBP-3)相关。IGFBP-3是EGFR和p63下游信号转导的共同产物。EGFR能够活化下游信号转导通路,其中包括磷脂酰肌醇3激酶/蛋白激酶B (PI3K/Akt)通路,它能够刺激细胞增殖以及阻止肿瘤细胞凋亡[20]。IGFBP-3作为PI3K/Akt的转录产物,主要起到阻止肿瘤细胞凋亡的作用,从而促进肿瘤的生长繁殖[21]。而p63亚基之一的ΔNp63α同样能激活下游信号转导通路PI3K/Akt,ΔNp63α介导的转录产物IGFBP-3可使过度表达p63的肿瘤细胞免于凋亡[6]。由此推测,检测SQC细胞中IGFBP-3的含量可以评估EGFR和p63的表达活性,进而更好地评价患者的生存状况。

p63免疫活性与肺SQC患者生存关系的报道较少,结论不一。Pelosi等[22]报道p63的免疫活性与患者OS和PFS无关,而Massion等[23]指出肺SQC患者中p63的免疫强度与患者生存时间呈正相关,免疫染色强度越高越有利于患者的生存。我们对EGFR突变肺SQC患者进行生存分析,结果发现p63部分阳性的 EGFR突变患者比p63阳性EGFR突变患者生存期更长,提示p63免疫强度与EGFR突变肺SQC患者生存时间呈负相关。由于EGFR突变样本例数较少,且生存分析结果受其他多重因素综合影响,本研究结果还需要更大样本来进行验证。 4 利益冲突

所有作者声明本文不涉及任何利益冲突。

参考文献
[1] Lee V W, Schwander B, Lee V H.Effectiveness and cost-effectiveness of erlotinib versus gefitinib in first-line treatment of epidermal growth factor receptor-activating mutation-positive non-small-cell lung cancer patients in Hong Kong[J].Hong Kong Med J, 2013, 10:2129-2139.
[2] Keam B, Kim D W, Park J H, Lee J O, Kim T M, Lee S H, et al.How molecular understanding affects to prescribing patterns and clinical outcome of gefitinib in non-small cell lung cancer? 10 year experience of single institution[J].Cancer Res Treat, 2013, 45:178-185.
[3] Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Seto T, et al.Updated overall survival results of WJTOG 3405, a randomized phase Ⅲ trial comparing gefitinib (G) with cisplatin plus docetaxel (CD) as the first-line treatment for patients with non-small cell lung cancer harboring mutations of the epidermal growth factor receptor (EGFR)[J].J Clin Oncol, 2012, 30 (Suppl):Abstr 7521.
[4] Mok T S, Wu Y L, Yu C J, Zhou C, Chen Y M, Zhang L, et al.A randomized placebo-controlled phase Ⅲ study of intercalated erlotinib with gemcitabine/platinum in first-line advanced non-small cell lung cancer (NSCLC):FASTACT-Ⅱ[J].J Clin Oncol, 2012, 30 (Suppl):Abstr.7520.
[5] Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba I I, et al.Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers[J].J Natl Cancer Inst, 2005, 97:339-346.
[6] Chen X.The p53 family:same response, different signals?[J].Mol Med Today, 1999, 5:387-392.
[7] Flores E R, Tsai K Y, Crowley D, Sengupta S, Yang A, McKeon F, et al.p63 and p73 are required for p53-dependent apoptosis in response to DNA damage[J].Nature, 2002, 416:560-564.
[8] Di Como C J, Urist M J, Babayan I, Drobnjak M, Hedvat C V, Teruya-Feldstein J, et al.p63 expression profiles in human normal and tumor tissues[J].Clin Cancer Res, 2002, 8:494-501.
[9] Wang B, Han Y, Zang J.Expression of p63 in squamous cell carcinoma of the lung and its diagnostic significance:a meta-analysis[J].J Cancer Res Updates, 2012, 1:228-238.
[10] Lynch T J, Bell D W, Sordella R, Gurubhagavatula S, Okimoto R A, Brannigan B W, et al.Activating mutations in the epidermal growth factor receptor underlying responsiveness of non small-cell lung cancer to gefitinib[J].N Engl J Med, 2004, 350:2129-2139.
[11] Yanagita E, Imagawa N, Ohbayashi C, Itoh T.Rapid multiplex immunohistochemistry using the 4-antibody cocktail YANA-4 in differentiating primary adenocarcinoma from squamous cell carcinoma of the lung[J].Appl Immunohistochem Mol Morphol, 2011, 19:509-513.
[12] Mu X L, Li L Y, Zhang X T, Wang M Z, Feng R E, Cui Q C, et al.Gefitinib-sensitive mutations of the epidermal growth factor receptor tyrosine kinase domain in Chinese patients with non-small cell lung cancer[J].Clin Cancer Res, 2005, 11:4289-4294.
[13] Lai Y, Zhang Z, Li J, Sun D, Zhou Y, Jiang T, et al.EGFR mutations in surgically resected fresh specimens from 697 consecutive Chinese patients with non-small cell lung cancer and their relationships with clinical features[J].Int J Mol Sci, 2013, 14:24549-24559.
[14] Hata A, Katakami N, Yoshioka H, Kunimasa K, Fujita S, Kaji R, et al.How sensitive are epidermal growth factor receptor-tyrosine kinase inhibitors for squamous cell carcinoma of the lung harboring EGFR gene-sensitive mutations?[J].J Thorac Oncol, 2013, 8:89-95.
[15] Sugio K, Uramoto H, Ono K, Oyama T, Hanagiri T, Sugaya M, et al.Mutations within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking[J].Br J Cancer, 2006, 94:896-903.
[16] Mitsudomi T, Yatabe Y.Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer[J].Cancer Sci, 2007, 98:1817-1824.
[17] Chou T Y, Chiu C H, Li L H, Hsiao C Y, Tzen C Y, Chang K T, et al.Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer[J].Clin Cancer Res, 2005, 11:3750-3757.
[18] Han S W, Kim T Y, Hwang P G, Jeong S, Kim J, Choi I S, et al.Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib[J].J Clin Oncol, 2005, 23:2493-2501.
[19] Pallis A G, Voutsina A, Kalikaki A, Souglakos J, Briasoulis E, Murray S, et al.'Classical' but not 'other' mutations of EGFR kinase domain are associated with clinical outcome in gefitinib-treated patients with non-small cell lung cancer[J].Br J Cancer, 2007, 97:1560-1566.
[20] Lopez-Chavez A, Carter C A, Giaccone G.The role of KRAS mutations in resistance to EGFR inhibition in the treatment of cancer[J].Curt Opin Investig Drugs, 2009, 10:1305-1314.
[21] Barbieri C E, Perez C A, Johnson K N, Ely K A, Billheimer D, Pietenpol J A.IGFBP-3 is a direct target of transcriptional regulation by DeltaNp63alpha in squamous epithelium[J].Cancer Res, 2005, 65:2314-2320.
[22] Pelosi G, Pasini F, Olsen Stenholm C, Pastorino U, Maisonneuve P, Sonzogni A, et al.p63 immunoreactivity in lung cancer:yet another player in the development of squamous cell carcinomas?[J].J Pathol, 2002, 198:100-109.
[23] Massion P P, Taflan P M, Jamshedur Rahman S M, Yildiz P, Shyr Y, Edgerton M E, et al.Significance of p63 amplification and overexpression in lung cancer development and prognosis[J].Cancer Res, 2003, 63:7113-7121.