CN116660537A - 一种评估三阴乳腺癌复发和预后的生物标志物及其应用 - Google Patents
一种评估三阴乳腺癌复发和预后的生物标志物及其应用 Download PDFInfo
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Abstract
本发明公开了一种评估三阴乳腺癌复发和/或预后的生物标志物，该生物标志物为KLHL22蛋白表达水平。本发明还公开了所述生物标志物在制备评估三阴乳腺癌复发和/或预后的试剂中的用途。本申请首次发现，高风险三阴乳腺癌组织和血液样本中KLHL22蛋白表达水平显著高于低风险三阴乳腺癌样本,检测KLHL22蛋白表达水平可评估三阴乳腺癌复发,并且KLHL22蛋白表达水平可以作为三阴乳腺癌的独立预后生物标志物，提示KLHL22蛋白表达水平可作为评估三阴乳腺癌复发和/或预后的分子标志。本发明为三阴乳腺癌的复发和/或预后评估提供了新的特异性分子标志物，临床应用前景广阔。
Description
技术领域
本发明属于肿瘤分子生物学技术领域，具体涉及一种评估三阴乳腺癌复发和预后的生物标志物及其应用。
背景技术
三阴乳腺癌（TNBC）约占所有乳腺癌的 15-20％,是一个预后非常差的类型。三阴乳腺癌的雌激素受体（ER），孕激素受体（PR）和人表皮生长因子受体 2（HER2）均为阴性，且缺乏早期发现的生物标志物和有效的治疗靶点，因此一经发现，通常被诊断为组织学等级高的晚期肿瘤，难以从内分泌治疗或靶向治疗中获益。此外，三阴乳腺癌患者通常具有高转移和远端复发的风险，是预后最差的一种分型，无病生存期（DFS）和总体生存期（OS）相较于其他分型明显缩短。因此，开发用于评估三阴乳腺癌复发和预后的特异性分子标志物是降低三阴乳腺癌复发转移、提高病人生存率，降低死亡率的关键。
发明内容
基于此，本发明的目的在于克服上述现有技术的不足之处而提供一种评估三阴乳腺癌复发和/或预后的生物标志物，可用于预测三阴乳腺癌复发及预后评估。
为实现上述目的，本发明提供了KLHL22蛋白表达水平作为生物标志物在筛选用于评估三阴乳腺癌复发和/或预后的试剂中的用途。
本发明还提供了KLHL22蛋白表达水平作为生物标志物在制备用于评估三阴乳腺癌复发和/或预后的试剂中的用途。
本发明还提供了检测KLHL22蛋白表达水平的试剂在制备用于评估三阴乳腺癌复发和/或预后的试剂盒中的用途。
优选地，所述检测KLHL22蛋白表达水平的试剂包括能与KLHL22蛋白特异性结合的抗体。
本发明所述KLHL22蛋白表达水平是指：通过免疫组化染色细胞质中KLHL22蛋白显色情况来评估其表达水平。阳性染色细胞呈棕色或黄色。免疫反应性评分采用阳性细胞数和阳性强度评分，评分方法如下:(i)每片取5个视野，以5%的增量从0到100%对阳性肿瘤细胞百分比进行计数，0表示阴性染色。(ii)我们将正强度评分定义为负(0)、弱(1)、中(2)、强(3)四个等级。(iii)从强度和比例(0-300分)中得出总分。然后用ROC曲线确定TNBC中KLHL22表达水平的临界值。另外，通过酶联免疫吸附试验（ELISA）检测血液中KLHL22蛋白表达水平。将保存于-80℃冰箱的全血标本复溶后于1000×g离心20分钟，取上清进行ELISA检测。血清样本中KLHL22蛋白表达水平和样本颜色深浅呈比例关系。绘制标准样品曲线后，将读取的吸光值带入公式得出样本浓度。
本申请发明人首次发现，高风险三阴乳腺癌样本中KLHL22蛋白表达水平显著高于低风险三阴乳腺癌，检测KLHL22蛋白表达水平可评估三阴乳腺癌的复发风险，并且KLHL22蛋白高表达水平与较差的临床分期、较高的复发率显著相关，另外KLHL22蛋白高表达水平与与较短的平均生存时间、总生存期和无进展生存期显著相关，KLHL22蛋白表达水平可以作为三阴乳腺癌的临床分期、复发率以及平均生存时间、总生存期和无进展生存期的独立预后生物标志物，提示KLHL22蛋白表达水平可作为三阴乳腺癌复发和/或预后的分子标志。
本发明还提供了一种评估TNBC复发和/或预后的试剂盒，所述试剂盒包含检测KLHL22蛋白表达水平的试剂。
优选地，所述试剂盒还包括能与所述抗体结合的二抗。
优选地，所述二抗标记有辣根过氧化物酶。
优选地，所述试剂盒还包括3 ,3-二氨基联苯胺四盐酸盐(D)。
相对于现有技术，本发明的有益效果为：本发明为三阴乳腺癌复发和预后评估提供了新的特异性生物标志物，检测所述生物标志物可评估三阴乳腺癌的复发风险和预后情况，具有特异性好、准确性高等优点，临床应用前景广阔。
附图说明
图1为三阴乳腺癌临床病理变量与KLHL22蛋白表达的相关性。
图2为三阴乳腺癌临床组织和血液样本中KLHL22蛋白表达。
图3为KHLH22表达在不同三阴性乳腺癌患者中的Kaplan-Meier生存分析。
图4为单因素生存分析KLHL22表达可作为评估三阴性乳腺癌预后的重要因素。
图5为多因素分析三阴乳腺癌中高KLHL22水平是与总生存期和无进展生存期相关的独立预后因素。
具体实施方式
以下结合说明书附图和具体实施例，对本发明作更详细准确地阐述和解释。以下所述实施例仅作为解释本发明使用，不限制本发明的范围。以下所述的实施例中所使用的实验方法若无特殊说明，均为常规方法；所使用的材料、试剂等，若无特殊说明，均为常规途径或商业途径获得。
实施例1免疫组化法检测复发三阴性乳腺癌样本KLHL22蛋白的表达水平：
1. 材料与方法
1.1收集TNBC患者的临床样本
收集2005年至2013年之间本院TNBC患者146例，包括临床病理信息和TNBC组织和血液样本。入选依据为及肿瘤标本病理诊断符合TNBC的患者。通过电话询问或查阅住院病例等方式收集临床资料，随访时间为5年。
1.2 免疫组化和ELLSA检测KLHL22表达情况
对石蜡切片进行免疫组化染色，使用了如下抗体：抗KLHL22单克隆抗体(1:500稀释; abs132394)。首先，我们将包埋的组织块依次切割成3μm厚的切片，然后用组织薄片在二甲苯中干燥和去蜡。接下来，通过分级酒精将载玻片再水化，并浸泡在3%过氧化氢中10分钟，以阻断内源性过氧化物酶活性。然后，在pH为9.0的乙二胺四乙酸（EDTA）抗原回收液中高压蒸煮3分钟。为减少非特异性反应，载玻片与5%牛血清白蛋白孵育15分钟后，与小鼠抗KLHL22单克隆抗体在37°C下孵育50分钟。然后将载玻片与二抗(Envision, k5007)在37°C下浸泡30分钟，然后用3,3-二氨基联苯胺染色。最后用苏木精复染。对于血液组织标本，我们使用了如下试剂盒：人Kelch-like蛋白22(KLHL22试剂盒)ELISA试剂盒（E7176h），将保存于-80℃冰箱的全血标本复溶后于1000×g离心20分钟，取上清进行ELISA检测。分别设空白孔、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品最终稀释度为5倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。用封板膜封板后置37℃温育30分钟。将30倍浓缩洗涤液用蒸馏水30倍稀释后备用。小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。每孔加入酶标试剂50μl，空白孔除外。用封板膜封板后置37℃温育30分钟。小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显色10-20分钟。每孔加终止液50μl，终止反应（此时蓝色立转黄色）。以空白孔调零，450nm波长依序测量各孔的吸光度（OD值）。测定应在加终止液后15分钟以内进行。血清样本中KLHL22蛋白表达水平和样本颜色深浅呈比例关系。
2. 免疫组化评分和ELISA表达水平比较
KLHL22染色主要发生在细胞质中，阳性染色细胞呈棕色或黄色。免疫反应性评分采用阳性细胞数和阳性强度评分，评分方法如下:(i)每片取5个视野，以5%的增量从0到100%对阳性肿瘤细胞百分比进行计数，0表示阴性染色。(ii)我们将正强度评分定义为负(0)、弱(1)、中(2)、强(3)四个等级。(iii)从强度和比例(0-300分)中得出总分。然后用ROC曲线确定TNBC中KLHL22表达水平的临界值。对患者的临床病理资料不知情的三名独立的病理学家对KLHL22表达进行评分。所有分析均由至少两名病理学家在同一实验室进行。当至少有两个与诊断结果一致时选择该值。如果他们有不同的意见，将重复诊断并讨论以达成共识。诊断符合率约为75%，证明该方法具有较高的重复性。对于ELISA试验，根据说明书绘制标准样品曲线后，将样本读取的吸光值带入公式得出样本浓度。并进行T检验统计分析。
3. 结果
我们在130个初治三阴性乳腺癌(pTNBC)和16个复发三阴性乳腺癌(rTNBC)组织上进行免疫组化染色，以探索pTNBC和rTNBC之间KLHL22蛋白表达的差异。ROC曲线确定KLHL22表达的临界值为190。结果如图1和2所示，免疫组化分析结果显示，KLHL22蛋白在TNBC中的高表达率为56.85%(83/146),在复发三阴性乳腺癌样本组织和血液标本中呈现高KLHL22表达水平（P＜0.05）。进一步分析发现，KLHL22表达与原发肿瘤及局部淋巴结状况、临床分期、复发呈显著正相关(P＜0.05)（图3）。Kaplan-Meier生存分析显示，低KLHL22表达的患者比高KLHL22表达的患者平均生存时间更长(147.93个月vs 90.1个月; P＜0.05)（图4）。多因素分析发现，KLHL22水平、P53表达、临床分期是影响总生存（OS）的独立预后因素(P＜0.05)，临床分期和KLHL22水平是影响无进展生存（PFS）的独立预后因素(P＜0.05)（图5）。这些结果表明KLHL22蛋白高表达水平与TNBC的局部复发显著相关，并且KLHL22表达可以作TNBC中OS和PFS的独立预后生物标志物。综上所述，通过免疫组化检测KLHL22蛋白的表达是鉴别TNBC患者预后的有效工具。我们的研究也提供了证据，证明KLHL22可能是TNBC的潜在治疗靶点。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (5)
1.KLHL22蛋白表达水平作为生物标志物在制备用于评估三阴性乳腺癌预后的试剂中的用途。
2.如权利要求1所述的用途，其特征在于，所述评估三阴性乳腺癌预后为评估三阴性乳腺癌复发。
3.检测KLHL22蛋白表达水平的试剂在制备用于评估三阴性乳腺癌预后的试剂盒中的用途。
4.如权利要求3所述的用途，其特征在于，所述评估三阴性乳腺癌预后为评估三阴性乳腺癌复发。
5.根据权利要求3或4所述的用途，其特征在于，所述检测KLHL22蛋白表达水平的试剂包括能与KLHL22蛋白特异性结合的抗体。
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