Prognostic Factors in Patients with Advanced Lung Adenocarcinoma Treated with Icotinib
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摘要:目的
探讨晚期肺腺癌表皮生长因子受体(EGFR)突变患者应用表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)盐酸埃克替尼片治疗与预后的关系。
方法入组河北省胸科医院基因检测提示EGFR19、21基因突变且接受盐酸埃克替尼片治疗的晚期肺腺癌患者,分析其临床特征、EGFR基因突变亚型及不同位点与预后的关系。
结果全组共纳入101例晚期肺腺癌患者,EGFR基因19外显子缺失突变(EGFR Del19)58例,21外显子点突变(EGFR L858R)43例。全组患者客观缓解率达63.4%,中位无疾病进展时间(mPFS)和中位生存时间(mOS)分别为13个月和27个月。EGFR Del19对比EGFR L858R及EGFR19突变746~750位点对比其他突变位点的患者mPFS和mOS均增高。多因素分析显示,转移部位数和有无胸膜转移为OS的独立影响因素(P=0.027, P=0.041),转移部位数≤3和无胸膜转移组患者的mOS分别为29个月和27个月。
结论盐酸埃克替尼片治疗晚期肺腺癌患者EGFR不同突变亚型和位点总生存差异不显著,转移部位数≤3和无胸膜转移的患者总生存期更长。
Abstract:ObjectiveTo investigate the relationship between the treatment of EGFR-TKI icotinib and the prognosis of advanced lung adenocarcinoma patients with EGFR mutation.
MethodsPatients with advanced lung adenocarcinoma who had EGFR19 and 21 gene mutations and were treated with EGFR-TKI icotinib were enrolled. The relationships of clinical features, EGFR gene mutation subtypes, and different sites with patients'prognosis were analyzed.
ResultsA total of 101 patients with advanced lung adenocarcinoma were included in this study, including 58 cases (57.4%) of EGFR gene exon 19 deletion mutation (EGFR Del19) and 43 cases (42.6%) of EGFR gene exon 21 point mutation (EGFR L858R). The objective response rate was 63.4%. The mPFS and mOS were 13 months and 27 months, respectively. In addition, the mPFS and mOS of EGFR Del19 and EGFR19 mutation 746-750 were higher than those of EGFR L858R and other EGFR mutations, respectively. Meanwhile, multivariate analysis showed that the number of metastatic sites and pleural metastasis were independent influencing factors of patients'OS (P=0.027; P=0.041). The mOS of patients with the number of metastatic sites ≤3 and without pleural metastasis were 29 and 27 months, respectively.
ConclusionThere is no significant difference found in overall survival of advanced lung adenocarcinoma patients treated with icotinib among different EGFR mutation subtypes and sites. Herein, the overall survival time is longer in patients with less than three metastatic sites and without pleural metastasis.
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Key words:
- Lung cancer /
- EGFR /
- TKI /
- Icotinib /
- Neoplasm metastasis
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0 引言
近年来,我国喉癌发病率及死亡率不断上升,已成为头颈部最常见的恶性肿瘤之一[1],晚期喉癌术后同期放化疗的5年生存率为68.2%,单纯放疗的5年生存率仅为48.9%[2]。放疗在喉鳞癌的治疗中起到至关重要的作用[3]。放射抵抗是导致放疗失败的重要原因[4]。
UBE2N是泛素交联酶E2家族的成员之一。泛素交联酶E2家族参与泛素-蛋白酶体复合通路(ubiquitin-proteasome pathway, UPP)对底物蛋白的泛素化降解[5]。泛素-蛋白酶体复合通路在调控细胞周期、增殖、信号转导、DNA损伤修复、免疫应答等方面都发挥着重要作用[6]。UBE2N是DNA损伤修复通路的成员[7],DNA损伤后,UBE2N与泛素连接酶RNF8及RNF168结合后,促进DNA损伤反应中BRCA1及H2AX的泛素化修饰,从而促进下游DNA损伤反应的进行[8]。研究发现泛素交联酶UBE2N可能调控肿瘤细胞的放疗敏感度[9]。本研究沉默喉鳞癌hep-2细胞中UBE2N的表达,通过CCK8法、流式细胞术及克隆形成等实验,验证沉默UBE2N表达后对喉鳞癌细胞放射敏感度的影响,为明确UBE2N在调控喉鳞癌细胞放射敏感度中的作用提供实验依据。
1 材料与方法
1.1 实验材料
DMEM培养基、胎牛血清购自美国Gibco公司;UBE2N siRNA沉默序列和阴性对照序列均由上海吉玛公司合成。RNA反转录试剂盒、PCR扩增试剂盒购自日本TaKaRa公司;蛋白裂解液、蛋白提取试剂盒、BCA蛋白定量试剂盒、细胞周期检测试剂盒、细胞凋亡检测试剂盒均购自南通碧云天生物技术研究所;内参GAPDH购自苏州GenePharma公司。GAPDH抗体、β-actin抗体、UBE2N抗体及各HRP-标记二抗购自英国Abcam公司;ECL化学发光试剂盒购自美国Thermo公司。Lipofectamine 3000购自美国Invitrogen公司;CCK8试剂盒购自日本同仁公司。流式细胞仪购自美国BD公司;荧光定量PCR仪、酶标仪、凝胶成像系统购自美国Bio-Rad公司。相机购自日本Canon公司。
1.2 实验方法
1.2.1 细胞培养
人喉鳞癌hep-2细胞购自中国科学院上海细胞库,用含10%胎牛血清的DMEM培养基,置于37℃含5%CO2的细胞培养箱中培养。
1.2.2 UBE2N RNA沉默质粒转染
实验分为UBE2N干扰组(UBE2N-siRNA)和阴性对照组(NC),UBE2N RNA沉默靶序列为:5′-AUCCAGAUGAUCCAUUAGCAATT-3′,阴性对照序列为:5′-TTCTCCGAACGTGTCACGT-3′。收集细胞,调整细胞密度至2×105/ml,接种于6孔板。待细胞贴壁生长后,按照Lipofectamine 3000说明书操作转染siRNA,转染24、48、72、96 h后,收集细胞进行后续相关实验。
1.2.3 qPCR实验
收集细胞(约1×106个),按照RNA反转录试剂盒说明书操作提取RNA。取总RNA 2μg进行反转录反应,然后进行qPCR反应,PCR反应条件:95℃预变性1 min,95℃变性15 s,60℃退火1 min,72℃延伸1min,40个循环。UBE2N引物序列为:F: 5′-GCGTTTGCTGGCAGAACCAG-3′,R: 5′-CTCAAAGGGGGAATCCTGAGGG-3′;GAPDH引物序列为:F: 5′-CCAACCGCGAGAAGATGA-3′,R: 5′-CCAGAGGCGTACAGGGATAG-3′。以GAPDH为内参,按照2-ΔΔCt法计算UBE2N相对表达量。
1.2.4 Western blot实验
收集细胞(约1×106个),按照试剂盒说明书操作提取蛋白,采用BCA法进行蛋白定量。取10 μg总蛋白用10% SDS-PAGE胶电泳,转膜后置于5%脱脂奶粉中室温封闭2 h。一抗4℃孵育过夜,二抗室温孵育2 h,采用ECL化学发光试剂盒显影。以ImagePro Plus 6.0图像分析软件计算灰度值,将内参蛋白与目的蛋白两者灰度值的比值作为目的蛋白的表达量。
1.2.5 CCK8增殖实验
hep-2细胞经siRNA转染,并用0、2、4、6、8 Gy X线照射或不照射,收集细胞(约1×106个),以每孔3 000个细胞铺到96孔板中,培养24、48、72和96 h后分别向对应的细胞中加入CCK8,将96孔培养板置于37℃恒温箱孵育1 h,用酶标仪测450 nm处吸光度值。
1.2.6 细胞凋亡的测定
hep-2细胞经siRNA转染,并用0、2、4、6、8 Gy X线照射后,收集细胞(约1×106个),预冷PBS洗3次。按照细胞凋亡检测试剂盒说明书操作,采用Annexin V/PI双染,流式细胞仪检测细胞凋亡。
1.2.7 细胞周期的测定
hep-2细胞经siRNA转染,并用0、2、4、6、8 Gy X线照射后,收集细胞(约1×106个),预冷PBS洗3次。70%的冷乙醇4℃固定过夜。按照细胞周期检测试剂盒说明书操作染色,采用流式细胞仪检测细胞周期。
1.2.8 细胞克隆形成实验
hep-2细胞经siRNA转染,收集细胞铺到6孔板中,每孔2 000个细胞。每组3孔平行样本。并用0、2、4、6、8 Gy X线照射后,静置于37℃培养箱中培养14天。14天后培养板中出现肉眼可见克隆时,终止培养,用PBS轻轻冲洗2~3遍,1%结晶紫染色20 min,除去染色液,纯水轻轻洗涤2~3遍,晾干,用相机拍照留底,在显微镜下计数克隆个数,≥50个的细胞团作为一个克隆。根据克隆形成计数结果计算每组细胞克隆形成率。
1.3 统计学方法
使用SPSS 22.0软件分析数据。以上所有实验至少重复三次,数据结果以(x±s)表示,通过配对样本t检验进行计算分析。P < 0.05为差异有统计学意义。
2 结果
2.1 UBE2N siRNA沉默效果检测
转染48 h后qPCR检测结果显示:UBE2N-siRNA组UBE2N mRNA表达(0.459±0.044)明显受到抑制,与NC组(1.000)比较,差异有统计学意义(F=0.10, P=0.01),见图 1A。转染72 h后Western blot结果显示:UBE2N siRNA显著抑制了UBE2N蛋白水平,见图 1B。上述结果表明UBE2N siRNA能够有效抑制hep-2细胞UBE2N表达。
2.2 沉默UBE2N促进hep-2细胞增殖
CCK8检测结果显示:经siRNA沉默24 h后, UBE2N-siRNA组和NC组细胞活力指数分别为(0.31±0.02)和(0.26±0.02)(P=0.042);48 h后, 细胞活力指数分别为(0.49±0.10)和(0.29±0.02)(P=0.037);72 h后, 细胞活力指数分别为(0.35±0.00)和(0.23±0.02)(P=0.003);96 h后, 细胞活力指数分别为(0.27±0.03)和(0.20±0.00)(P=0.009)。说明沉默UBE2N显著提高hep-2细胞增殖能力,见图 2。
2.3 沉默UBE2N促进辐射后hep-2细胞增殖
经0、2、4、6、8 Gy射线照射48 h后,CCK8法结果显示:4、6 Gy射线照射后,UBE2N-siRNA组hep-2细胞增殖能力显著高于NC组(F4Gy=0.27, P4Gy=0.04; F6Gy=0.63, P6Gy=0.03),见图 3。提示沉默UBE2N会增强喉鳞癌细胞放射抵抗。
2.4 细胞周期实验结果
经0、2、4、6、8 Gy射线照射后,NC组和UBE2N-siRNA组G1期细胞比例差异无统计学意义(均P > 0.05);未经射线照射(0 Gy),UBE2N-siRNA组G2期细胞比例明显高于NC组(P=0.043);但经2、6 Gy射线照射后,UBE2N-siRNA组G2期细胞比例明显低于NC组(均P < 0.05);经0、4、6 Gy射线照射后,UBE2N-siRNA组S期细胞比例明显高于NC组(均P < 0.05),见图 4、表 1。结果表明经辐射处理后,沉默UBE2N会促进DNA合成,并部分解除G2阻滞,提示沉默UBE2N会增强喉鳞癌细胞放射抵抗。
表 1 沉默UBE2N对经X射线处理后的hep-2细胞周期的影响Table 1 Effects of UBE2N silence on cell cycle of hep-2 cells after X-ray treatment2.5 细胞凋亡实验结果
沉默UBE2N并经0、2、4、6、8 Gy射线照射后,Annexin V/PI双染法结果显示:UBE2N干扰组细胞总凋亡率(早期凋亡+晚期凋亡)显著低于NC组(均P < 0.05),见图 5、表 2。结果证明沉默UBE2N降低辐射诱导的喉鳞癌细胞凋亡。
表 2 沉默UBE2N对X射线诱导的细胞凋亡的影响Table 2 Effects of UBE2N silence on X-ray-induced cell apoptosis2.6 克隆形成实验结果
沉默UBE2N并经0、2、4、6 Gy射线照射后,UBE2N-siRNA组细胞克隆形成能力显著强于NC组(均P < 0.05),见图 6、表 3。提示沉默UBE2N会增强喉鳞癌细胞放射抵抗。
表 3 沉默UBE2N对X射线处理后的细胞克隆形成能力的影响Table 3 Effects of UBE2N silence on cell clone formation ability after X-ray treatment3 讨论
近年研究显示,泛素交联酶与肿瘤放疗抵抗密切相关。研究发现使用一种特异性抑制小分子抑制剂NSC697923抑制UBE2N后,P53及JNK通路激活,神经母细胞瘤细胞、弥漫性大B淋巴瘤细胞的增殖减慢并促进其凋亡,故而推测UBE2N可能是这两种肿瘤的潜在治疗靶点之一[10-11]。张喜梅等在人喉鳞癌细胞株中发现UBE2N在放疗抵抗株hep-2R中表达明显高于亲本细胞hep-2,提示泛素交联酶UBE2N可能调控肿瘤细胞的放射敏感度[9],但未做相关功能实验进行验证。
细胞周期调控是决定细胞放射敏感度的一个关键因素。辐射诱导DNA损伤,使细胞周期产生阻滞,如G2/M期阻滞[12],为DNA修复提供充足的时间[13]。本研究发现在细胞周期实验中,hep-2细胞转染UBE2N siRNA后,其S期显著延长,而G2期显著缩短,增殖实验也表明沉默UBE2N增强hep-2细胞增殖,因此UBE2N调控细胞增殖和细胞周期可能是调节放射敏感度的重要机制。
细胞凋亡是影响细胞放射敏感度的重要原因,是一种潜在的放疗增敏机制[14-15],辐射可通过多种途径诱导细胞凋亡。本研究通过AnnexinV/PI双染法证实,经X线照射后,转染UBE2N siRNA较转染阴性对照NC的hep-2细胞凋亡比例明显减少。克隆形成率是反映细胞群体依赖性和增殖能力的重要指标。本研究发现经X线照射后,UBE2N沉默组细胞克隆形成率显著升高,提示沉默UBE2N能显著降低喉鳞癌细胞群体依赖性,并增强喉鳞癌细胞增殖能力。
综上所述,UBE2N通过调控细胞增殖、细胞周期进展、细胞克隆形成能力和细胞凋亡来影响喉鳞癌细胞放射抵抗。本研究在细胞水平探讨了UBE2N对人喉鳞癌细胞放射抵抗的影响,但相关动物实验和机制研究并未完成,因此,在体内外探讨UBE2N在人喉鳞癌细胞放射敏感度的机制是我们下一步需要完成的内容,以期为喉鳞癌放疗增敏提供新的可靠靶点。
Competing interests: The authors declare that they have no competing interests.作者贡献:张红斌:研究设计、统计分析、论文撰写朱玲玲:数据汇总、统计分析、文献查阅谢炯、才虹美、姬巧霞、梁香存、李华、王愿:资料收集、文献查阅赵敏:研究设计、审阅、论文修改 -
表 1 Ⅳ期肺腺癌患者EGFR19、21基因突变的临床特征(n=101)
Table 1 Clinical characteristics of stage Ⅳ lung adenocarcinoma patients with EGFR 19 and 21 gene mutations (n=101)
表 2 基于Cox模型对所有单因素变量进行多因素分析的结果
Table 2 Results of multivariate analysis of all univariate variables based on Cox model
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[1] Gao S, Li N, Wang S, et al. Lung Cancer in People's Republic of China[J]. J Thorac Oncol, 2020, 15(10): 1567-1576. doi: 10.1016/j.jtho.2020.04.028
[2] Zhang YL, Yuan JQ, Wang KF, et al. The prevalence of EGFR mutation in patients with non-small cell lung cancer: a systematic review and meta-analysis[J]. Oncotarget, 2016, 7(48): 78985-78993. doi: 10.18632/oncotarget.12587
[3] Shi Y, Au JS, Thongprasert S, et al. A Prospective, Molecular Epidemiology Study of EGFR Mutations in Asian Patients with Advanced Non-Small-Cell Lung Cancer of Adenocarcinoma Histology (PIONEER)[J]. J Thorac Oncol, 2014, 9(2): 154-162. doi: 10.1097/JTO.0000000000000033
[4] Tu HY, Ke EE, Yang JJ, et al. A comprehensive review of uncommon EGFR mutations in patients with non-small cell lung cancer[J]. Lung Cancer, 2017, 114: 96-102. doi: 10.1016/j.lungcan.2017.11.005
[5] Patel N, Wu P, Zhang H. Comparison of gefitinib as first- and second-line therapy for advanced lung adenocarcinoma patients with positive exon 21 or 19 del epidermal growth factor receptor mutation[J]. Cancer Manag Res, 2017, 9: 243-248. doi: 10.2147/CMAR.S138643
[6] Akamatsu H, Koh Y, Okamoto I, et al. Clinical significance of monitoring EGFR mutation in plasma using multiplexed digital PCR in EGFR mutated patients treated with afatinib (West Japan Oncology Group 8114LTR study)[J]. Lung Cancer, 2019, 131: 128-133. doi: 10.1016/j.lungcan.2019.03.021
[7] Lynch TJ, Bell DW, Sordella R, 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(21): 2129-2139. doi: 10.1056/NEJMoa040938
[8] Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib[J]. Proc Natl Acad Sci U S A, 2004, 101(36): 13306-13311. doi: 10.1073/pnas.0405220101
[9] Paez JG, Jänne PA, Lee JC, et al. EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy[J]. Science, 2004, 304(5676): 1497-1500. doi: 10.1126/science.1099314
[10] Bergqvist M, Christensen HN, Wiklund F, et al. Real world utilization of EGFR TKIs and prognostic factors for survival in NSCLC during 2010-2016 in Sweden: A nationwide observational study[J]. Int J Cancer, 2020, 146(9): 2510-2517. doi: 10.1002/ijc.32596
[11] Lee CK, Davies L, Wu Y, et al. Gefitinib or Erlotinib vs Chemotherapy for EGFR Mutation-Positive Lung Cancer: Individual Patient Data Meta-Analysis of Overall Survival[J]. J Natl Cancer Inst, 2017, 109(6): 1-9.
[12] Ke EE, Zhou Q, Zhang QY, et al. A Higher Proportion of the EGFR T790M Mutation May Contribute to the Better Survival of Patients with Exon 19 Deletions Compared with Those with L858R[J]. J Thorac Oncol, 2017, 12(9): 1368-1375. doi: 10.1016/j.jtho.2017.05.018
[13] Mok TS, Cheng Y, Zhou X, et al. Improvement in Overall Survival in a Randomized Study That Compared Dacomitinib With Gefitinib in Patients With Advanced Non-Small-Cell Lung Cancer and EGFR-Activating Mutations[J]. J Clin Oncol, 2018, 36(22): 2244-2250. doi: 10.1200/JCO.2018.78.7994
[14] Soria JC, Ohe Y, Vansteenkiste J, et al. Osimertinib in Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer[J]. N Engl J Med, 2018, 378(2): 113-125. doi: 10.1056/NEJMoa1713137
[15] Rossi S, D'Argento E, Basso M, et al. Different EGFR Gene Mutations in Exon 18, 19 and 21 as Prognostic and Predictive Markers in NSCLC: A Single Institution Analysis[J]. Mol Diagn Ther, 2016, 20(1): 55-63. doi: 10.1007/s40291-015-0176-x
[16] Liu HL, Han G, Peng M, et al. Efficacy of EGFR tyrosine kinase inhibitors in non-small cell lung cancer patients harboring different types of EGFR mutations: A retrospective analysis[J]. J Huazhong Univ Sci Technolog Med Sci, 2017, 37(6): 864-872.
[17] Ng SK, Sun CK, Ting KB, et al. Prognostic factors of EGFR-mutated metastatic adenocarcinoma of lung[J]. Eur J Radiol, 2020, 123: 108780. doi: 10.1016/j.ejrad.2019.108780
[18] Taniguchi Y, Tamiya A, Nakahama K, et al. Impact of metastatic status on the prognosis of EGFR mutation-positive non-small cell lung cancer patients treated with first-generation EGFR-tyrosine kinase inhibitors[J]. Oncol Lett, 2017, 14(6): 7589-7596.
[19] Yoshimura A, Yamada T, Tsuji T, et al. Prognostic impact of pleural effusion in EGFR-mutant non-small cell lung cancer patients without brain metastasis[J]. Thorac Cancer, 2019, 10(3): 557-563. doi: 10.1111/1759-7714.12979
[20] Wang TF, Chu SC, Lee JJ, et al. Presence of pleural effusion is associated with a poor prognosis in patients with epidermal growth factor receptor-mutated lung cancer receiving tyrosine kinase inhibitors as first-line treatment[J]. Asia Pac J Clin Oncol, 2017, 13(4): 304-313. doi: 10.1111/ajco.12658
[21] Okauchi S, Watanabe H, Yamada H, et al. The Prognosis of Lung Cancer With Different Metastatic Patterns[J]. Anticancer Res, 2020, 40(1): 421-426. doi: 10.21873/anticanres.13969