乳腺癌TOP2A基因表达与含蒽环类药物新辅助化疗效果的关系
2017-05-31蒋奕叶新青廖晓明姬逸男唐玮
蒋奕+叶新青++廖晓明++姬逸男+唐玮++叶洪涛
[摘要] 目的 探讨乳腺癌拓扑异构酶ⅡA(TOP2A)基因表达与含蒽环类药物新辅助化疗疗效的关系。 方法 收集广西医科大学附属肿瘤医院乳腺外科2010年2月~2014年3月收治的91例乳腺癌患者新辅助化疗粗针穿刺的病理标本,采用荧光原位杂交(FISH)法检测癌组织中TOP2A与HER2基因表达,采用免疫组织化学法(IHC)检测ER、HER2和TOPOⅡα蛋白的表达,并观察应用含蒽环类药物新辅助化疗后病理情况。 结果 TOP2A基因表达与HER2基因及TOPOⅡα蛋白表达相关(P < 0.05),而与ER、HER2蛋白表达无关(P > 0.05)。經新辅助化疗后TOP2A基因扩增的患者获得的pCR率(50.0%)优于未扩增者(5.5%)(P < 0.05)。 结论 乳腺癌TOP2A基因表达与HER2基因及TOPOⅡα蛋白相关,而与ER、HER2蛋白表达量无关。TOP2A基因扩增程度可能为评估含蒽环类药物新辅助化疗疗效的预测因子。
[关键词] 乳腺癌;新辅助化疗;拓扑异构酶ⅡA;HER2
[中图分类号] R737.9 [文献标识码] A [文章编号] 1673-7210(2016)12(b)-0016-04
Relationship between expression of TOP2A in breast cancer and anthracycline-based neoadjuvant chemotherapy
JIANG Yi1 YE Xinqing2 LIAO Xiaoming1 JI Yi′nan1 TANG Wei1 YE Hongtao3▲
1.Department of Breast Surgery, Tumor Hospital Affiliated to Guangxi Medical University, Guangxi Zhuang Autonomous Region, Nanning 530021, China; 2.Department of Pathology, Tumor Hospital Affiliated to Guangxi Medical University, Guangxi Zhuang Autonomous Region, Nanning 530021, China; 3.Department of Histopathology, Royal National Orthopaedic Hospital, HA7 4LP, UK
[Abstract] Objective To evaluate the relationship between the expression of TOP2A and efficacy of anthracycline-based neoadjuvant chemotherapy in breast cancer. Methods 91 patients with breast cancer who were treated in Department of Breast Surgery, Tumor Hospital Affiliated to Guangxi Medical University from February 2010 to March 2014 were surveied; a core needle biopsy was used to collect samples before and after neoadjuvant chemotherapy, fluorescence in situ hybridization (FISH) was performed to assess expression of TOP2A and HER2 gene, an immunohistochemistry (IHC) was then employed to check the changes of protein expression levels of ER, HER2 and TOPOⅡα. The pathological was observed after neoadjuvant chemotherapy with anthracyclines. Results TOP2A gene expression had relevant with HER2 gene and TOPO Ⅱ alpha protein expression (P < 0.05), and had nothing to do with ER, HER2 protein expression (P > 0.05). Patients with TOP2A gene amplification had a high rate of pCR (50.0%) than in patients with failure TOP2A gene amplification (5.5%). Conclusion TOP2A gene expression in breast cancer has relevant with HER2 gene and TOPO Ⅱ alpha protein expression, and has nothing to do with ER and HER2 protein status. TOP2A gene amplification can be the predictive factor for anthracycline-based chemotherapy.
[Key words] Breast cancer; Neoadjuvant chemotherapy; TOP2A; HER2
蒽环类药物被认为是目前最有效的乳腺癌化疗药物之一,也是新辅助化疗(neoadjuvant chemothera?鄄py,NAC)的常用药物,但并非所有患者均能从中获益[1-3],因而寻找可预测蒽环类NAC疗效的标志物符合个体化治疗的迫切需要。DNA拓扑异构酶ⅡA基因(TOP2A)位于第17号染色体,其编码的拓扑异构酶蛋白(TOPOⅡα)被认为是蒽环类药物作用靶点之一[4],其表达水平与乳腺癌的预后密切相关[5]。ER及HER2是目前临床上评估乳腺癌预后及指导治疗的常用指标[6],研究证实对NAC的疗效有预测作用[7-9],但存在一定的局限性,增加生物診断参数对评估患者预后和指导治疗是必要的。本研究分析91例乳腺癌组织中TOP2A、HER2基因及ER、HER2、TOPOⅡα蛋白的表达,及应用蒽环类药物NAC方案后的疗效,旨在探讨TOP2A与其他预后因子的关系,及其与含蒽环类NAC方案疗效的相关性。
1 资料与方法
1.1 一般资料
收集广西医科大学附属肿瘤医院乳腺外科2010年2月~2014年3月收治的91例乳腺癌患者标本,年龄28~69岁,平均(45.7±2.1)岁,根据TNM分期,Ⅱ期患者51例,Ⅲ期40例,除1例炎性乳癌患者外,所有患者具有可测量病灶,直径范围为1.5~13.3 cm,平均(3.6±1.5)cm。
1.2 治疗及检测方法
所有患者均采用含蒽环类药物的FEC方案NAC 4个周期。具体为:环磷酰胺(500 mg/m2,百特,批号6C103A)、表柔比星(100 mg/m2,辉瑞,批号L43197)、氟尿嘧啶(500 mg/m2,旭东海普,批号FA160308),21 d为1个周期,共4个周期。收集患者NAC前粗针穿刺活检标本及NAC后手术切除标本的石蜡包埋切片。以上试验均经广西医科大学附属肿瘤医院伦理委员会批准,治疗过程及标本去向均告知患者并签署知情同意书。所有标本经4%中性甲醛固定,常规脱水、石蜡包埋,3~4 μm厚的石蜡切片常规脱蜡,其余步骤参见文献[10],免疫组化染色采用Elivision方法染色,ER(克隆号SP1)和HER2(克隆号SP3)抗体购自迈新公司,TOPOⅡα抗体(克隆号3F6)购自中杉金桥公司。荧光原位杂交(FISH)法检测所用HER2探针、TOP2A探针、HER2和TOP2A购自极地基因有限公司。
1.3 免疫组化结果判读
判读方法:每张切片在低倍镜(40×)下观察并确定有代表性的染色阳性视野,选择5个视野,在高倍镜(400×)下观察,计算100个肿瘤细胞中的阳性细胞数,计算百分比率。阳性判定标准:HER2蛋白位于细胞膜,结果采用乳腺癌HER2检测指南(2014版)[11]判读。ER、TOPOⅡα蛋白为定位于细胞核的棕黄色颗粒,<10%的癌细胞核着色为阴性(-),≥10%为阳性。
1.4 FISH结果判定
计数30个癌细胞核中的HER2(绿色)、TOP2A(红色)及17号染色体(蓝色)的荧光信号数,统计比例(30个癌细胞核中绿色或红色信号数/30个癌细胞核中蓝色信号数)。当荧光信号连接成簇时,可不用计算直接视为基因扩增。HER2基因结果判读:HER2/CEP17<2且平均HER2<4信号因子/细胞为无扩增;HER2/CEP17≥2或者平均HER2≥6信号因子/细胞为扩增;若HER2/CEP17<2且4信号因子/细胞≤平均HER2<6信号因子/细胞,则增加计数细胞至100个或重新计数。TOP2A基因结果判读:TOP2A /CEP17<0.8为无扩增,比值>2.0为扩增,介于0.8~2.0时,增加计数细胞至100个或重做FISH实验,重复计数后仍介于0.8~2.0则判为无扩增。以上判读均由两位病理诊断医生在不知病例资料和诊断结果条件下进行。
1.5 疗效评估
NAC后病理完全缓解(pathologic complete remission,pCR)定义为术后乳腺原发灶无浸润性癌且区域淋巴结阴性。
1.6 统计学方法
采用SPSS 15.0统计软件对数据进行分析,基因和蛋白表达及其与NAC疗效的计数资料均采用χ2检验,以P < 0.05为差异有统计学意义。
2 结果
2.1 TOP2A与HER2基因表达相关性
91例标本中,TOP2A扩增比例为19.8%(18/91),HER2扩增比例为28.6%(26/91);HER2扩增患者中TOP2A扩增比例为57.7%(15/26),HER2无扩增患者中TOP2A扩增比例为4.6%(3/65),两者比较差异有统计学意义(P < 0.01),见表1。图1为TOP2A与HER2的FISH检测图像(封三)。
2.2 TOP2A基因扩增与预后相关蛋白表达的相关性
91例组织中ER蛋白阳性者62例,其TOP2A扩增率为16.1%(10/62);ER蛋白阴性者29例,其TOP2A扩增率为27.6%(8/29),两者比较差异无统计学意义(P=0.263)。HER2蛋白阳性者30例,其TOP2A扩增率为30.0%(9/30);HER2蛋白阴性者61例,其TOP2A扩增率为14.8%(9/61),两者比较差异无统计学意义(P=0.108)。TOPOⅡα蛋白阳性者34例,其TOP2A扩增率为35.3%(12/34);TOPOⅡα蛋白阴性者57例,其TOP2A扩增率为10.5%(6/57),两者比较差异有统计学意义(P=0.006)。见表2。
2.3 TOP2A基因扩增与NAC疗效相关性
91例患者接受NAC后,13例(14.3%)患者达到pCR。TOP2A基因扩增患者达pCR为50.0%(9/18),而无扩增者达pCR为5.5%(4/73),两者比较差异有统计学意义(P=0.008)。
3 讨论
NAC的疗效评估可以获知患者对化疗药物的敏感性,为后续治疗提供重要参考,然而在临床中部分患者的疗效并不理想,治疗期间甚至会出现疾病进展。在NAC前获知患者对化疗药物的敏感性以指导用药至关重要,现尚无确切有效的预测因子,因此探寻NAC的预测因子具有重要意义。
TOP2A基因编码的TOPOⅡα蛋白是蒽环类药物的重要靶点之一。Arriola等[12]通过多因素分析发现,TOP2A可作为一个独立的乳腺癌患者预后预测因子,此外还发现TOP2A扩增与HER2扩增具有明显相关性,共扩增的患者具有更高的总生存率(OS)與无病生存率(DFS)。本研究亦发现,TOP2A扩增乳腺癌组织中HER2扩增的几率高,与Fasching等[13-14]的研究结果一致;而HER2及ER蛋白的表达则与TOP2A扩增水平未见明显相关性,与Mitrovi■的报道相符[15]。本研究还显示,TOP2A扩增患者的TOPOⅡα表达量更高。而Romero等[16]研究表明,TOP2A基因扩增与否与TOPOⅡα的表达无明显相关性(P > 0.05),与本研究结果相反,可能是TOPOⅡα在转录后和翻译过程中受多因素调控,且依赖细胞增殖信号,可能表现为TOP2A不扩增,蛋白质过表达等。以上研究提示,TOP2A的扩增水平与患者预后因子HER2基因的扩增密切相关,可能是潜在的预后评估因子。
近年来,越来越多的研究发现,TOP2A的表达水平与乳腺癌化疗疗效相关。O`Malley等[17]研究表明,接受蒽环类药物化疗的患者中仅有TOP2A扩增的患者可得到OS与DFS的提高。Tanner等[18]研究发现,接受剂量密集的FEC方案的患者中,HER2及TOP2A共扩增组获得较好的DFS。此外,TOP2A还被发现在预测乳腺癌患者NAC疗效中具有重要价值。Wang等[19]研究发现,TOP2A扩增的患者接受NAC后较之未扩增患者能达到更高的pCR率(56.3%比13.8%,P=0.001)。而Moretti等[20]则发现,TOP2A编码的TOPOⅡα亦可作为预测NAC疗效的独立影响因子;Klintman等[21]提出,TOP2A对NAC疗效的影响与ER,PgR及HER2的表达并无相关性。本研究则发现,接受NAC的91例患者中,13例患者达到pCR(14.3%),其中TOP2A基因扩增患者pCR率显著高于无扩增患者,差异有统计学意义(P < 0.05)。结合以上研究说明TOP2A在预测乳腺癌NAC疗效中的重要价值,可能成为指导乳腺癌患者综合治疗的重要因子。
综上所述,乳腺癌TOP2A基因扩增与HER2扩增水平相关,可能是潜在的预后评估因子;且还可能为含蒽环类药物NAC疗效的预测因子,为指导乳腺癌的个体化精准治疗、提高化疗效果提供重要的理论依据,为判断乳腺癌的预后提供重要的参考价值。
[参考文献]
[1] Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths among 75,000 women. Early Breast Cancer Trialists' Collaborative Group[J]. Lancet,1992,339(8785):71-85.
[2] Ross MB,Buzdar AU,Smith TL,et al. Improved survival of patients with metastatic breast cancer receiving combination chemotherapy [J]. Cancer,1985,55(2):341-346.
[3] Tomiak E,Piccart M,Mignolet F,et al. Characterisation of complete responders to combination chemotherapy for advanced breast cancer:aretrospeceive EORTC breast cancer group study [J]. Eur J Cancer,1996,32A(11):1876-1887.
[4] 杨燕华,王树森.乳腺癌中TOP2A与蒽环类药物关系的研究进展[J].中国普外基础与临床杂志,2014,21(8):1029-1033.
[5] Biesaga B,Niemiec J,Ziobro M. BCL-2,topoisomerase IIα,microvessel density and prognosis of early advanced breast cancer patients after adjuvant anthracycline-based chemotherapy [J]. J Cancer Res ClinOncol,2014,140(12):2009-2019.
[6] Brunelli M,Manfrin E,Bria E,et al. HER2/neu gene determination in women screened for breast carcinoma:how screening program s reduce the skyrocketing cost of targeted therapy [J]. Anticancer Res,2013,33(9):3705-3710.
[7] 陈丽荣.乳腺癌新辅助化疗的临床意义和病理学评价[J].中华病理学杂志,2010,39(4):218-221.
[8] 雷蕾,王晓稼,杨红健,等.乳腺癌分子分型在新辅助化疗疗效和预后中的预测作用[J].中国肿瘤,2012,21(11):868-873.
[9] Pedrini JL,Franealacei SR,Casales SM,et al. The effect of neoadjuvant chemotherapy on hormone receptor status,HER2/neu and prolactin in breast cancer [J]. Tumori,2011, 97(6):704-710.
[10] 王桂秋,周英琼,宫丽平,等.用荧光原位杂交在石蜡切片上检测t(11;18)和涉及bcl-10基因染色体易位的方法[J].中华病理学杂志,2007,36(7):494-495.
[11] 《乳腺癌HER2检测指南(2014版)》编写组.乳腺癌HER2检测指南(2014版)[J].中华病理学杂志,2014, 43(4):262-267.
[12] Arriola E,Marchio C,Tan DS,et al. Genomic analysis of the HER2/TOP2A amplicon in breast cancer and breast cancer cell lines [J]. Lab Invest,2008,88(5):491-503.
[13] Fasching PA,Weihbrecht S,Haeberle L,et al. HER2 and TOP2A amplification in a hospital-based cohort of breast cancer patients:associations with patient and tumor char?鄄acteristics [J]. Breast Cancer Res Treat,2014,145(1):193-203.
[14] Knoop AS,Knudsen H,Balslev E,et al. Retrospective analysis of topoisomeraseⅡa amplifications and deletions as predictive markers in primary breast cancer patients randomly assigned to cyclophosphamide,methotrexate,and fluorouracil or cyclophosphamide,epirubicin,and fluorouracil:Danish Breast Cancer Cooperative Group [J]. J Clin Oncol,2005,23(30):7483-7490.
[15] Mitrovi■ O,■oki■ V,■iki■ D,et al, Correlation between ER,PR,HER-2,Bcl-2, p53,proliferative and apoptotic indexes with HER-2 gene amplification and TOP2A gene amplification and deletion in four molecular subtypes of breast cancer [J]. Target Oncol,2014,9(4):367-379.
[16] Romero A,Martín M,Cheang MC,et al. Assessment of Topoisomerase Ⅱ α status in breast cancer by quantitative PCR,gene expression microarrays,immunohistochemistry,and fluorescence in situ hybridization [J]. Am J Pathol,2011,178(4):1453-1460.
[17] O′Malley FP,Chia S,Tu D,et al. Topoisomerase Ⅱ alpha and responsiveness of breast cancer to adjuvant chemotherapy [J]. J Natl Cancer Inst,2009,101(9):644-650.
[18] Tanner M,J?覿rvinen P,Isola J. Amplification of HER-2/neu and topoisomerase Ⅱalpha in primary and metastatic breast cancer [J]. Cancer Res,2001,61(14):5345-5348.
[19] Wang J,Xu B,Yuan P,et al. TOP2A amplification in breast cancer is a predictive marker of anthracycline-based neoadjuvant chemotherapy efficacy [J]. Breast Cancer Res Treat,2012,135(2):531-537.
[20] Moretti E,Desmedt C,Biagioni C,et al. TOP2A protein by quantitative immunofluorescence as a predictor of response to epirubicin in the neoadjuvant treatment of breast cancer [J]. Future Oncol,2013,9(10):1477-1487.
[21] Klintman M,Buus R,Cheang MC,et al. Changes in Expression of Genes Representing Key Biologic Processes after Neoadjuvant Chemotherapy in Breast Cancer,and Prognostic Implications in Residual Disease [J]. Clin Cancer Res,2016,22(10):2405-2416.
(收稿日期:2016-09-04 本文編辑:王红双)