Expression and Clinical Significance of Eukaryotic Translation Initiation Factors 5A2 and Ki67 in Intrahepatic Cholangiocarcinoma
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摘要:
目的 分析真核翻译起始因子5A2(eukaryotic translation initiation factor 5A2,EIF5A2)和Ki67在肝内胆管癌组织中表达的临床意义及两者的相关性。 方法 收集成都市第七人民医院肝胆外科、昆明医科大学第二附属医院肝胆胰外科2014年9月至2021年12月118例肝内胆管癌患者术后石蜡切片,采用免疫组化的方法检测EIF5A2和Ki67表达,收集临床病理资料,分析EIF5A2、Ki67高表达与不同病理特征的关系及两者的相关性。 结果 肝内胆管癌组织中EIF5A2高表达60例(50.85%),Ki67高表达81例(68.64%),EIF5A2高表达与患者年龄、性别、肿瘤直径、肿瘤数量无关(P > 0.05),与TNM分期和淋巴转移有关(P < 0.05);肝内胆管癌组织中Ki67高表达与患者年龄、性别、肿瘤数量无关(P > 0.05),与TNM分期、淋巴转移、肿瘤直径有关(P < 0.05),EIF5A2与Ki-67的表达呈正相关(r = 0.285,P = 0.002)。 结论 肝内胆管癌中EIF5A2和Ki-67的高表达与高TNM分期和淋巴转移有关,两者表达呈正相关,有助于预判肝内胆管癌患者的临床分期及预后。 Abstract:Objective To analyze the clinical significance and correlation of eukaryotic translation initiation factor 5A2 (EIF5A2) and Ki67 expression in intrahepatic cholangiocarcinoma tissues. Methods Paraffin sections were collected from 118 patients with intrahepatic cholangiocarcinoma in the Department of Hepatobiliary Surgery of The Seventh People's Hospital of Chengdu and the Department of Hepatobiliary and Pancreatic Surgery of the Second Affiliated Hospital of Kunming Medical University from September 2014 to December 2021. Immunohistochemical methods were used to detect the expressions of EIF5A2 and Ki67, and clinicopathological data were collected. The positive expression of EIF5A2, Ki67 and different pathological features were analyzed. Results EIF5A2 was positively expressed in 60 cases (50.85%) and Ki67 was positively expressed in 81 cases (68.64%). EIF5A2 expression was not related to age, sex, tumor diameter and tumor number (P > 0.05), but related to TNM stage and lymphatic metastasis (P < 0.05). The expression of Ki67 in intrahepatic cholangiocarcinoma tissues was not correlated with age, sex and tumor number (P > 0.05), but with TNM stage, lymphatic metastasis and tumor diameter (P < 0.05). EIF5A2 was positively correlated with the expression of Ki-67 (R = 0.285, P = 0.002). Conclusion The expression of EIF5A2 and KI-67 in intrahepatic cholangiocarcinoma is positively correlated with high TNM stage and lymphatic metastasis, which is helpful to predict the clinical stage and prognosis of patients with intrahepatic cholangiocarcinoma. -
肝内胆管癌(intrahepatic cholangiocarcinoma iCCA)是第二高发的原发性肝脏恶性肿瘤,占原发性肝脏恶性肿瘤的10%~15%[1-3]。近年来全世界范围内肝内胆管癌的发病率和死亡率呈不断上升的趋势[4, 5],可能与非病毒性慢性肝病和代谢性疾病的普遍性增高有关[6, 7]。肝内胆管癌病死率高,5 a生存率仅25%~30%[3],其预后远差于肝细胞癌。真核翻译起始因子5A2(eukaryotic translation initiation factor 5A2,EIF5A2)的编码基因位于人3号染色体2区6带[8],该区域的染色体不稳定,极易出现扩增,导致其表达上调。EIF5A2完成翻译后需依次经DHS和DOHH酶催化[9],转化为成熟的、有生物学活性的蛋白分子,可在细胞核和细胞质间穿梭,负责特定mRNA亚群的选择性转运和翻译[10],其在胰腺导管腺癌、结肠癌、肝细胞癌中表达均增高[11],与较低的生存率、高TNM分期、淋巴结转移和肿瘤分化差等恶性表型显著相关[12, 13]。Ki67可反映肿瘤细胞的增殖程度,用于预估患者预后[14]。本研究用免疫组织化学染色观察EIF5A2和Ki67在肝内胆管癌中的表达水平,分析它们在肝内胆管癌中高表达与临床病理特征的关系,现报道如下。
1. 资料与方法
1.1 一般资料
收集成都市第七人民医院肝胆外科、昆明医科大学第二附属医院肝胆外科2014年9月至2021年12月行肝内胆管癌根治手术患者的石蜡切片118例,年龄38~74岁,其中男性60例,女性58例,术后病理切片镜下组织学特点并结合免疫组化(CK7、CK19、Glypican-3)确诊为肝内胆管细胞癌,纳入研究的患者病例资料完整。
1.2 HE和免疫组化染色
对含肿瘤组织的蜡块进行连续切片,取3张连续的组织切片进行HE染色、EIF5A2和Ki67免疫组化染色。HE染色:含肿瘤组织的载玻片在65 ℃烤片机上烘烤25 min,之后浸泡在二甲苯溶液中脱蜡,然后洗涤二甲苯并进行组织水化,苏木精溶液染核5 min,自来水冲洗多余的苏木精溶液,伊红溶液染细胞质2 min,脱水后封片。免疫组化染色:烤片、脱蜡、水化过程同HE染色,肿瘤组织切片均进行乙二胺四已酸抗原热修复,高压锅内煮沸5 min后磷酸缓冲盐溶液(PBS)冲洗组织切片5遍,EIF5A2免疫组化染色:载玻片上滴加EIF5A2多克隆抗体(Abcam,货号:ab227537,稀释比:1∶150),Ki67免疫组化染色使用即用型兔抗人单克隆抗体(福州迈新,产品编号:RMA-0542),组织切片中加入Ki67一抗后放入37 ℃恒温箱孵育1 h,磷酸缓冲盐溶液(PBS)冲洗5遍,切片上滴加山羊抗兔的二抗,在37 ℃恒温箱中孵育0.5 h,磷酸缓冲盐溶液(PBS)冲洗5遍后DAB显色,苏木素溶液染核后封片。
1.3 免疫组化染色结果判读
EIF5A2和Ki67的表达强弱由2名病理科主治医师采用半定量计分法进行分析,(1)评估肿瘤组织着色强度,基本无着色计 0 分(-),染色后呈淡黄色计 1 分(+),呈浅棕色计 2分(++),呈棕褐色计 3 分(+++);(2)评估染色面积,高倍镜视野下以染色阳性区域的面积占整个视野面积的百分比进行计分,面积小于5%计0分,6%~25%计1分,26%~50%计2分,51 %~75%计3分,>76%计4分;将染色强度①与染色面积②相加,所得结果0~3分为低表达,4~7分为高表达。
1.4 统计学处理
数据应用SPSS19.0进行统计分析,计数资料用[n(%)]表示,采用χ2检验;EIF5A2和Ki67表达的相关性分析采用χ2检验,P < 0.05为差异有统计学意义。
2. 结果
2.1 HE染色及EIF5A2和Ki67在肝内胆管癌组织中的表达
118例肝内胆管癌组织标本中HE染色显示,癌细胞核大且深染,异型性明显,细胞质红染,肿瘤细胞排列成管状,肿瘤组织间包含丰富的纤维间质。免疫组化染色显示EIF5A2主要位于肿瘤细胞的细胞质和细胞核中, Ki67在肿瘤细胞的细胞核中表达,见图1。
2.2 EIF5A2和Ki67的表达与肝内胆管癌临床病理特征的关系分析
118例肝内胆管癌组织中EIF5A2高表达60例(50.85%),Ki67高表达81例(68.64%),EIF5A2高表达与患者年龄、性别、肿瘤直径、肿瘤数量无关(P > 0.05),与TNM分期和淋巴转移有关(P < 0.05);肝内胆管癌组织中Ki67高表达与患者年龄、性别、肿瘤数量无关(P > 0.05),与TNM分期、淋巴转移、肿瘤直径有关(P < 0.05),见表1。
表 1 EIF5A2和Ki67表达水平与肝内胆管癌临床病理特征的关系[n(%)]Table 1. Relationship between EIF5A2 and Ki67 expression levels and clinicopathological features of intrahepatic cholangiocarcinoma [n (%)]临床特征 n EIF5A2 Ki67 高表达(n = 60) 低表达(n = 58) χ2 P 高表达(n = 81) 低表达(n = 37) χ2 P 年龄(岁) ≥53 60 34(56.67) 26(43.33) 1.654 0.198 38(63.33) 22(36.67) 1.600 0.206 < 53 58 26(44.83) 32(55.17) 43(74.14) 15(25.86) 性别 男 49 29(59.18) 20(40.82) 2.330 0.127 31(63.27) 18(36.73) 1.126 0.298 女 69 31(44.93) 38(55.07) 50(72.46) 19(27.54) TNM分期 III/IV 38 27(71.05) 11(28.95) 9.156 0.002* 32(84.21) 6(15.79) 6.310 0.012* I/II 80 33(41.25) 47(58.75) 49(61.25) 31(38.75) 淋巴转移 是 35 23(65.71) 12(34.29) 4.400 0.036* 29(82.86) 6(17.14) 4.670 0.031* 否 83 37(45.58) 46(55.42) 52(62.65) 31(37.35) 肿瘤直径(cm) ≥5 87 48(55.17) 39(44.83) 2.478 0.115 65(74.71) 22(25.29) 5.666 0.017* < 5 31 12(38.71) 19(61.29) 16(51.61) 15(48.39) 肿瘤数量 多发 15 9(60.00) 6(40.00) 0.576 0.448 11(73.33) 4(26.67) 0.176 0.675 单发 103 51(49.51) 52(50.49) 70(67.96) 33(32.04) *P < 0.05。 2.3 EIF5A2和Ki67在肝内胆管癌中表达的相关性
118例肝内胆管癌免疫组化染色结果中EIF5A2与Ki-67两种蛋白均为高表达者49例,占 EIF5A2高表达患者的81.67%(49/60),EIF5A2高表达而Ki-67低表达的肝内胆管癌组织切片11例;EIF5A2低表达但Ki-67呈高表达的患者有32例,占EIF5A2低表达患者的55.17%(32/58),两者均阴性者26例;由χ2检验得知,EIF5A2的表达与Ki-67的表达呈正相关,差异具有统计学意义(r = 0.285,P = 0.002),见表2。
表 2 EIF5A2和Ki67在肝内胆管癌中表达的相关性Table 2. Correlation of EIF5A2 and Ki67 expression in intrahepatic cholangiocarcinomaKi67 EIF5A2 r P 高表达
(n = 60)低表达
(n = 58)高表达(n = 81) 49 32 0.285 0.002 低表达(n = 37) 11 26 *P < 0.05。 3. 讨论
肝内胆管癌(intrahepatic cholangiocarcinoma iCCA)主要起源于胆管上皮细胞[15-16],也可能起源于肝祖细胞[17],近期的研究表明肝内胆管癌也可能起源于HA-tag阳性的肝细胞[18]。形态学可分为肿块型、管周浸润型和管内型,依据分子学特征分为增殖型和炎症型[19]。我国学者以肝内胆管癌蛋白质组学的表达差异,将肝内胆管癌分为炎症型、间质型、代谢型和分化型[20]。肝内胆管癌对放化疗敏感性差[21],缺乏有效的靶向治疗和免疫治疗,手术切除是唯一可达到根治的治疗方式[22],但手术后仍极易出现转移和复发,造成了患者5 a生存率低,绝大多数肝内胆管癌患者死亡是由于肿瘤复发转移导致的,一项国际多中心的研究结果显示肝内胆管癌行根治性肝切除后复发率高达71%[23]。因此更好地了解肝内胆管癌复发转移的分子机制对于改善患者预后是极为重要的。
EIF5A2是一个新的候选癌基因,可促进肿瘤细胞增殖及侵袭转移[9, 24],在人类恶性消化道肿瘤中有所涉及,我们前期的研究其在肝内胆管癌组织中表达增高[25],但它与肝内胆管患者的癌临床病理特征的相关性尚未见报道。既往研究发现,EIF5A2的高表达与这些胃癌患者的高TNM分期、淋巴和远处转移和较差的总生存期相关[12];结肠癌中EIF5A2的过表达促进了肿瘤的转移[13];此外Tang等[26]报道,与未发生转移或静脉浸润的HCC组织相比,发生转移或静脉浸润的HCC组织中EIF5A2的表达显著增加。笔者的研究结果发现EIF5A2的表达与年龄、性别、肿瘤直径、肿瘤数量无关(P > 0.05),与TNM分期和淋巴转移有关(P < 0.05),说明EIF5A2增强了肝内胆管癌细胞的侵袭转移能力,与既往研究基本一致。一些肿瘤研究中报道了EIF5A2增强癌细胞侵袭转移能力的机制,结肠癌研究中发现高水平的EIF5A2有助于c-myc结合到MTA1的启动子区,进而上调MTA1蛋白的表达,从而增强了结肠癌细胞的运动性和侵袭性[27]。基质金属蛋白酶在肿瘤细胞侵袭转移中起关键性的作用[28, 29],皮肤黑色素瘤的研究发现肿瘤细胞核中EIF5A2增多与强MMP2 表达直接相关[30],促进肿瘤侵袭转移。肝细胞癌中EIF5A2 可能通过激活 p38 MAPK 和 JNK/c-Jun 通路增强 MMP-2 活性促进血管形成[31],促进肿瘤经血道转移。EIF5A2在肝内胆管癌中高表达,临床病理特征分析发现其促进了肿瘤细胞的侵袭转移能力,笔者将进一步研究EIF5A2促肝内胆管癌细胞的侵袭转移的分子机制。
Ki67是评价细胞增殖的重要指标,也是实体瘤诊断和判断预后的生物标志物,既往研究发现肝内胆管癌组织中Ki67的表达远高于正常肝内胆管组织[32],本研究中Ki67在肝内胆管癌阳性表达率为68.64%(81/118),其表达与年龄、性别、肿瘤数量无关(P > 0.05),与TNM分期、淋巴转移、肿瘤直径有关(P < 0.05),与既往研究基本一致[33],此外Qiang等[14]在肝内胆管癌的研究还发现高表达的Ki67与血管浸润相关。在卵巢癌中发现,EIF5A2的过表达与肿瘤分化差、高pT/pN和FIGO分期以及Ki-67的阳性率升高均有显著的正相关[34],同样,笔者通过Spearman相关性分析得知,肝内胆管癌中EIF5A2与Ki-67的表达呈正相关,差异具有统计学意义(r = 0.285,P = 0.002),肝内胆管癌中EIF5A2是否直接调控Ki-67及具体的调控机制还有待进一步研究。
综上所述,肝内胆管癌组织中EIF5A2、Ki67的阳性率较高,两者的表达呈正相关,其表达与高TNM分期和淋巴转移有关,说明EIF5A2和Ki67促进了肝内胆管癌的发生及侵袭转移,有助于预判肝内胆管癌患者的临床分期及预后。
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表 1 EIF5A2和Ki67表达水平与肝内胆管癌临床病理特征的关系[n(%)]
Table 1. Relationship between EIF5A2 and Ki67 expression levels and clinicopathological features of intrahepatic cholangiocarcinoma [n (%)]
临床特征 n EIF5A2 Ki67 高表达(n = 60) 低表达(n = 58) χ2 P 高表达(n = 81) 低表达(n = 37) χ2 P 年龄(岁) ≥53 60 34(56.67) 26(43.33) 1.654 0.198 38(63.33) 22(36.67) 1.600 0.206 < 53 58 26(44.83) 32(55.17) 43(74.14) 15(25.86) 性别 男 49 29(59.18) 20(40.82) 2.330 0.127 31(63.27) 18(36.73) 1.126 0.298 女 69 31(44.93) 38(55.07) 50(72.46) 19(27.54) TNM分期 III/IV 38 27(71.05) 11(28.95) 9.156 0.002* 32(84.21) 6(15.79) 6.310 0.012* I/II 80 33(41.25) 47(58.75) 49(61.25) 31(38.75) 淋巴转移 是 35 23(65.71) 12(34.29) 4.400 0.036* 29(82.86) 6(17.14) 4.670 0.031* 否 83 37(45.58) 46(55.42) 52(62.65) 31(37.35) 肿瘤直径(cm) ≥5 87 48(55.17) 39(44.83) 2.478 0.115 65(74.71) 22(25.29) 5.666 0.017* < 5 31 12(38.71) 19(61.29) 16(51.61) 15(48.39) 肿瘤数量 多发 15 9(60.00) 6(40.00) 0.576 0.448 11(73.33) 4(26.67) 0.176 0.675 单发 103 51(49.51) 52(50.49) 70(67.96) 33(32.04) *P < 0.05。 表 2 EIF5A2和Ki67在肝内胆管癌中表达的相关性
Table 2. Correlation of EIF5A2 and Ki67 expression in intrahepatic cholangiocarcinoma
Ki67 EIF5A2 r P 高表达
(n = 60)低表达
(n = 58)高表达(n = 81) 49 32 0.285 0.002 低表达(n = 37) 11 26 *P < 0.05。 -
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