CN114047337A - 一种与肝细胞癌患者预后相关的分子标志物、免疫组化试剂盒及其应用 - Google Patents
一种与肝细胞癌患者预后相关的分子标志物、免疫组化试剂盒及其应用 Download PDFInfo
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Abstract
本发明公开了一种与肝细胞癌患者预后相关的分子标志物、免疫组化试剂盒及其应用,属于生物医学技术领域。本发明通过检测肝细胞癌术后肿瘤组织中MCM10分子的表达水平,能够简便、迅速地反映个体肿瘤中免疫细胞浸润程度和免疫检查点分子表达水平,并初步对患者的预后不良风险进行有效评估。免疫组化染色也证实高MCM10表达具有更多耗竭性CD8+T细胞浸润和更高免疫检查点表达,可有效判断患者免疫治疗反应和预后不良风险,此外通过对肝癌患者OS和RFS进行单因素和多因素COX回归分析与证实MCM10在患者预后的独立预测作用,在临床中能及时对术后的治疗策略提供有价值的参考,有助于肝癌的个体化治疗。
Description
技术领域
本发明属于生物医学技术领域,具体涉及一种与肝细胞癌患者预后相关的分子标志物、免疫组化试剂盒及其应用。
背景技术
肝细胞癌(HCC)是最常见的原发性肝癌,在全球癌症相关死亡原因中排名第三。早期患者的治疗以手术切除为主。然而,在诊断为晚期HCC的患者中,肝切除术后的复发和手术机会的丧失通常导致预后较差。此外,HCC发生在肝硬化/纤维化的背景下,大多数情况下为炎症诱导的癌症提供了范式,大量研究表明,肿瘤浸润淋巴细胞(TILs)显著影响HCC的肿瘤微环境和临床结局。对于诊断为晚期HCC的患者以及其他治疗失败后转入晚期的患者,全身治疗是唯一有效的选择。免疫疗法侧重于免疫检查点抑制剂(ICPs)对免疫系统的“制动”作用,以增强抗肿瘤免疫应答,是全身治疗的重要组成部分。然而,肝癌的预后仍然很差,5年生存率为20%,HCC的分子特征仍不清楚。识别新的免疫相关的生物标志物,肿瘤发生过程中的关键途径,以及选择从肝细胞癌免疫治疗中获益更多的患者,对于临床治疗和生存率的提高至关重要。
MCM10通过将MCM2-7解旋酶和DNA聚合酶α/引物酶复合物结合在一起,并在DNA复制过程中防止DNA损伤中起着关键作用。值得注意的是,MCM10蛋白也被用于某些类型肿瘤的诊断和预后,MCM10在宫颈癌中明显过表达,且随着肿瘤分期的进展,过表达频率增加;髓母细胞瘤中,肿瘤细胞增殖需要MCM10过表达;MCM10也被报道在胶质瘤中过表达,并与肿瘤级别相关;此外,MCM10表达与肺癌患者的OS较差和较差的无复发生存(RFS)有关,MCM10表达增加在细胞周期相关过程中富集;在尿路上皮癌中,MCM10表达异常升高与不良预后相关,无病生存(DFS)较差。在致瘤过程中,细胞过度表达复制因子以驱动肿瘤发生过程中的增殖。因此,MCM10在各种肿瘤样本中普遍上调也就不足为奇,这表明转化细胞依赖于MCM10来防止基因组不稳定性达到致死水平。在之前的一项研究中,MCM10变异被发现与免疫缺陷、自然杀伤细胞(NK)缺乏和限制性心肌病(RCM)相关,这些都与胸腺和脾脏发育不全有关,表明MCM10在免疫调节中具有潜在的作用。然而,MCM10在HCC的致癌转化和免疫调节中的作用尚不清楚。
发明内容
针对现有技术中的不足,本发明的目的在于提供一种与肝细胞癌患者预后相关的分子标志物、免疫组化试剂盒及其应用,基于检测肝细胞癌患者的肿瘤组织中MCM10分子表达可反映肿瘤内免疫细胞浸润和免疫检查点分子表达水平,从而有效预测免疫治疗反应和判断预后不良风险,用于筛选对免疫治疗有效的患者提高治疗效果,促进肝癌患者个体化精准治疗。
为实现上述目的,本发明采用的技术方案是
本发明提供一种与肝细胞癌患者预后相关的分子标志物,所述分子标志物为MCM10。
本发明还提供上述分子标志物MCM10或其检测试剂在制备辅助诊断肝细胞癌的免疫组化试剂盒中的应用。
优选地,所述免疫组化试剂盒包括上述分子标志物MCM10或其检测试剂,还包括记载有使用方法和判断标准的说明书;其中:
所述使用方法是通过免疫组化染色检测肝细胞癌患者的肿瘤组织中MCM10分子表达水平,反映肿瘤组织内免疫细胞浸润和免疫检查点分子表达水平,辅助判断肝细胞癌患者免疫治疗反应和预后风险;
所述判断标准采用半定量组织学评分:MCM10分子表达水平的免疫组化染色强度评分与染色范围(0-100%)的乘积,其中,MCM10分子表达水平的免疫组化染色强度评分为0:-;0.5:-/+;1:+;1.5:+/++;2:++;2.5:++/+++和3:+++,采用四级量表进行分级:阴性、弱、中度和强度,阴性为0,弱为0-100%,中度为100-150%,强度为150-300%;将阴性和弱MCM10表达定义为低MCM10表达,将中度和强度MCM10表达定义为高MCM10表达;高MCM10表达提示免疫治疗反应和预后风险大,低MCM10表达提示免疫治疗反应和预后风险小。
优选地,高MCM10表达的肿瘤组织中CD8+T细胞浸润和免疫检查点分子表达水平高,低MCM10表达的肿瘤组织中CD8+T细胞浸润和免疫检查点分子表达水平低。
优选地,所述免疫组化试剂盒包括抗人MCM10单克隆抗体、辣根过氧化物酶连接的第二抗体和哈里斯氏苏木精染色剂。
更优选地,所述使用方法包括如下步骤:
(1)收集肝细胞癌患者的手术切除标本,采用低聚甲醛固定,并用石蜡包埋构建组织微阵列芯片;
(2)将组织微阵列芯片切片脱石蜡后,采用抗人MCM10单克隆抗体孵育过夜,再用辣根过氧化物酶连接的第二抗体孵育,之后进行免疫组化染色,即使用哈里斯氏苏木精进行细胞核染色;
(3)、根据免疫组化染色结果采用半定量组织学评分将肝细胞癌患者的组织中MCM10表达水平划分为高MCM10表达和低MCM10表达。
有益效果
通过免疫组化实验证实,高MCM10表达具有更多耗竭性CD8+T细胞浸润和更高免疫检查点表达,可有效判断患者免疫治疗反应和预后不良风险。此外,通过对肝癌患者OS和RFS进行单因素和多因素COX回归分析证实MCM10在患者预后的独立预测作用。总之,采用本发明的上述技术方案,检测肝细胞癌术后肿瘤组织中MCM10分子的表达水平,能够简便、迅速地反映个体肿瘤中免疫细胞浸润程度和免疫检查点分子表达水平,并初步对患者的预后不良风险进行有效评估,从而在临床实践中能及时对术后的治疗策略提供有价值的参考,尤其是在免疫治疗的选择上具有较高的指导意义,有助于肝癌的个体化治疗。
附图说明
图1是实施例中MCM10不同表达组中免疫细胞浸润水平。
图2是实施例中MCM10、CD8+T细胞典型免疫组化表达(左);免疫组化测定不同MCM10表达水平组CD8+T细胞表达水平(中);流式细胞术测定不同MCM10表达水平组CD8+T细胞(右)。
图3是实施例中MCM10表达水平与各种免疫检查点表达水平的相关矩阵(左);不同MCM10表达水平组中TIGIT、CTLA4和CD86的表达水平(右)。
图4是实施例中免疫组化检测182名患者肿瘤组织与正常组织间MCM10表达及CD8+T细胞浸润情况(上);182名患者中MCM10高、低分组间肿瘤内CD8+T细胞浸润情况及MCM10表达与CD8+T细胞浸润的Spearman相关性(下)。
图5是实施例中根据肿瘤内MCM10表达和CD8+T细胞浸润情况分析研究队列的OS(左)和DFS(右)。
具体实施方式
下面通过实施例进一步详细描述本发明,但本发明不局限于这些实施例。
实施例1
本实施例的研究对象为182例肝细胞癌患者术后肿瘤组织,纳入及排除标准为:(1)无其他恶性肿瘤病史;(2)术前未曾接受过癌症治疗,术前未发现肝外转移;(3)除病毒性肝炎外没有感染或其他炎症的迹象;(4)根治性切除;(5)术后病理明确诊断为肝细胞癌。
采用的免疫组化染色方法步骤如下:
(1)收集肝细胞癌患者的手术切除肿瘤组织标本,进行低聚甲醛固定,石蜡包埋以构建组织微阵列芯片。
(2)收集肝细胞癌患者的临床病理信息和术后随访数据
常规的临床病理信息包括年龄、性别、有无腹水和肝硬化,术前实验室指标和术后病理报告。其中,术前实验室指标包括丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、乙型肝炎病毒表面抗原(HBsAg)、甲胎蛋白(AFP)、癌胚抗原(CEA)和糖类抗原19-9(CA19-9)水平。术后病理报告包括肿瘤数量、大小、包膜、分级和微血管浸润等内容。
随访第1年每3个月进行1次,随后第2年每3-6个月进行1次,然后每年1次。随访内容包括血常规、肝功能检查、血清肿瘤标志物检测、腹部超声和胸片,对怀疑复发的患者实施了增强CT或磁共振成像,OS和RFS分别定义为从手术日期到死亡或复发日期(或最后一次随访)的时间间隔。
(3)肿瘤组织MCM10分子免疫组化染色
将脱石蜡的肝细胞癌组织切片浸入3%的H2O2中以淬灭内源性过氧化物酶活性,然后与抗人MCM10单克隆抗体(抗体稀释比例为1:100)在4℃下孵育过夜,第二天将切片与辣根过氧化物酶第二抗体和二氨基联苯胺溶液一起孵育,用哈里斯氏苏木精染剂进行细胞核染色。随后,由两名病理学医师独立进行半定量组织学评分,最终的MCM10表达水平为染色强度评分(0:-;0.5:-/+;1:+;1.5:+/++;2:++;2.5:++/+++;3:+++)与染色范围(0-100%)的乘积。最终,MCM10表达水平使用四级量表进行分级:阴性(0),弱(0-100%),中度(100-150%)和强度(150-300%),将阴性和弱MCM10表达定义为低MCM10表达,将中度和强度MCM10表达定义为高MCM10表达。为验证MCM10在促进耗竭性CD8+T细胞浸润的作用,同时测定肿瘤组织中的CD8+T细胞的浸润水平。
(4)肿瘤MCM10分子表达水平与肝细胞癌患者预后的关系
根据182例肝细胞癌患者的临床随访信息计算相应的OS值和RFS值,然后对高MCM10表达和低MCM10表达的患者的OS和RFS进行Kaplan–Meier生存分析绘制生存曲线,并采用Log-rank检验差异有无统计学意义。
实验结果
图1是在MCM10不同表达水平中免疫细胞的浸润水平,发现MCM10的表达与CD8+T细胞浸润显著相关。利用手术切除的八对新鲜肿瘤样本测定肿瘤和癌旁组织中MCM10免疫组化表达水平和CD8的表达水平(图2),结果提示肿瘤中MCM10的表达水平明显高于癌旁组织,并且高MCM10表达组的患者肿瘤组织内耗竭性CD8+T细胞浸润明显升高,同时MCM10高表达与免疫检查点表达正相关(图3)。
为验证MCM10在HCC中的重要作用,检测包含182名患者的研究队列中MCM10的瘤内表达和CD8+T细胞的浸润程度,发现MCM10的相对表达在肿瘤明显高于正常组织(P=0.002),而CD8+T细胞在正常组织明显高于肿瘤组织(P<0.001)。根据MCM10的瘤内表达将患者分为MCM10高表达组和低表达组,MCM10高表达组肿瘤内CD8+T细胞表达明显高于MCM10低表达组(P=0.029),并且CD8+T细胞与MCM10瘤内表达呈显著正相关(图4)。MCM10高表达组中较高的AFP水平(P=0.021)和较晚的TNM分期(P=0.030)更常见(表1)。根据单变量分析,CA19-9、肿瘤大小、肿瘤分化、微血管侵犯、AST、TNM分期、瘤内CD8+ T细胞和MCM10表达与患者OS相关,而AFP、腹水、肿瘤大小、AST、TNM分期、MCM10表达与患者RFS相关。MCM10、AFP和肿瘤大小是预测OS的独立因素(表2)。
根据队列中肿瘤内MCM10表达和CD8+T细胞浸润情况进行生存分析:依据肿瘤内MCM10表达及CD8+T细胞浸润情况将患者进一步分为三组:组Ⅰ(MCM10高);组Ⅱ(MCM10低,CD8+T细胞高);组Ⅲ(MCM10低&CD8+T细胞低),采用Kaplan-Meier分析评估临床结果,发现与来自组Ⅱ的患者相比,来自组Ⅰ的患者的OS和DFS明显更差,与来自组Ⅲ的患者相比,来自组Ⅱ的患者的OS明显更好。然而,组Ⅰ和组Ⅲ之间的OS和DFS没有显著差异(图5)。
表1:临床特征与肿瘤内MCM10表达的关系
表2:OS和DFS相关的单因素和多因素COX分析
通过上述实施例和分析结果进一步证实MCM10在肝细胞癌患者的肿瘤组织中表达明显高于癌旁组织,并与耗竭性CD8+T细胞高浸润和免疫检查点高表达呈正相关,MCM10作为有效的预后预测指标,有助于评估肝细胞癌的免疫特征和预后风险,在临床实践中可用于指导肝癌患者免疫治疗管理,判断肝细胞癌患者术后生存时间,推动肝癌个体化治疗方案制定。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.一种与肝细胞癌患者预后相关的分子标志物,所述分子标志物为MCM10。
2.权利要求1所述分子标志物MCM10或其检测试剂在制备辅助诊断肝细胞癌的免疫组化试剂盒中的应用。
3.根据权利要求2所述的应用,其特征在于,所述免疫组化试剂盒包括上述分子标志物MCM10或其检测试剂,还包括记载有使用方法和判断标准的说明书;其中:
所述使用方法是通过免疫组化染色检测肝细胞癌患者的肿瘤组织中MCM10分子表达水平,反映肿瘤组织内免疫细胞浸润和免疫检查点分子表达水平,辅助判断肝细胞癌患者免疫治疗反应和预后风险;
和/或所述判断标准采用半定量组织学评分:MCM10分子表达水平的免疫组化染色强度评分与染色范围(0-100%)的乘积,MCM10分子表达水平的免疫组化染色强度评分为0:-;0.5:-/+;1:+;1.5:+/++;2:++;2.5:++/+++和3:+++,采用四级量表进行分级:阴性、弱、中度和强度,阴性为0,弱为0-100%,中度为100-150%,强度为150-300%;将阴性和弱MCM10表达定义为低MCM10表达,将中度和强度MCM10表达定义为高MCM10表达;高MCM10表达提示免疫治疗反应和预后风险大,低MCM10表达提示免疫治疗反应和预后风险小。
4.根据权利要求3所述的应用,其特征在于,高MCM10表达反映肿瘤组织中CD8+T细胞浸润和免疫检查点分子表达水平高,低MCM10表达反映肿瘤组织中CD8+T细胞浸润和免疫检查点分子表达水平低。
5.根据权利要求3所述的应用,其特征在于,所述免疫组化试剂盒包括抗人MCM10单克隆抗体、辣根过氧化物酶连接的第二抗体和哈里斯氏苏木精染色剂。
6.根据权利要求3至5任一项所述的应用,其特征在于,所述使用方法包括如下步骤:
(1)收集肝细胞癌患者的手术切除标本,采用低聚甲醛固定,并用石蜡包埋构建组织微阵列芯片;
(2)将组织微阵列芯片切片脱石蜡后,采用抗人MCM10单克隆抗体孵育过夜,再用辣根过氧化物酶连接的第二抗体孵育,之后进行免疫组化染色,即使用哈里斯氏苏木精进行细胞核染色;
(3)、根据免疫组化染色结果采用半定量组织学评分将肝细胞癌患者的组织中MCM10表达水平划分为高MCM10表达和低MCM10表达。
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