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568例HER2不确定乳腺癌原位杂交结果分析

蒋依娜, 王博, 周灿, 王鸿雁

蒋依娜, 王博, 周灿, 王鸿雁. 568例HER2不确定乳腺癌原位杂交结果分析[J]. 肿瘤防治研究, 2019, 46(7): 605-609. DOI: 10.3971/j.issn.1000-8578.2019.18.0482
引用本文: 蒋依娜, 王博, 周灿, 王鸿雁. 568例HER2不确定乳腺癌原位杂交结果分析[J]. 肿瘤防治研究, 2019, 46(7): 605-609. DOI: 10.3971/j.issn.1000-8578.2019.18.0482
JIANG Yi'na, WANG Bo, ZHOU Can, WANG Hongyan. In Situ Hybridization Status of 568 Cases of HER2 Equivocal Breast Cancer[J]. Cancer Research on Prevention and Treatment, 2019, 46(7): 605-609. DOI: 10.3971/j.issn.1000-8578.2019.18.0482
Citation: JIANG Yi'na, WANG Bo, ZHOU Can, WANG Hongyan. In Situ Hybridization Status of 568 Cases of HER2 Equivocal Breast Cancer[J]. Cancer Research on Prevention and Treatment, 2019, 46(7): 605-609. DOI: 10.3971/j.issn.1000-8578.2019.18.0482

568例HER2不确定乳腺癌原位杂交结果分析

基金项目: 

国家自然科学基金 81502413

详细信息
    作者简介:

    蒋依娜(1979-),女,硕士,主治医师,主要从事肿瘤分子病理相关研究

    通讯作者:

    王鸿雁,E-mail: whycc0320@126.com

  • 中图分类号: R737.9

In Situ Hybridization Status of 568 Cases of HER2 Equivocal Breast Cancer

More Information
  • 摘要:
    目的 

    比较2013版及2018版美国临床肿瘤学会/美国病理医师学院(ASCO/CAP)乳腺癌HER2检测指南,对免疫组织化学HER2不确定(2+)乳腺癌FISH判读结果的影响及其临床意义。通过与FISH对比,分析DISH的准确性及存在问题。

    方法 

    选取568例免疫组织化学HER2不确定浸润性乳腺癌石蜡包埋标本,使用FISH技术进行HER2基因状态检测,分别采用2013及2018版ASCO/CAP乳腺癌HER2检测指南进行结果判读。随机选取60例样本进行DISH检测,并与FISH对比分析。

    结果 

    依据2013版指南,HER2阳性183例(32.22%)、阴性365例(64.26%)、不确定20例(3.52%)。采用新指南判读标准,HER2阳性率减少了4.58%,阴性率无差别,两种判读标准差异具有统计学意义(P < 0.01)。对60例肿瘤样本的FISH和DISH检测结果比较,两者总符合率为95.0%,阳性符合率为94.7%;阴性符合率为100%,两种方法的检测结果显著相关(r=0.636, P < 0.01)。

    结论 

    2018版ASCO/CAP HER2检测指南与2013版相比,判读标准更明确、分类更精细,对临床用药更具指导作用。DISH技术具有亮视野的优点,与FISH有同样的临床诊断价值。

     

    Abstract:
    Objective 

    To compare the differences between 2013 and 2018 ASCO/CAP HER2 testing guidelines in 568 immunohistochemical HER2 equivocal breast cancer patients and to analyze the accuracy and deficiency of dual-color silver enhanced in-situ hybridization (DISH) by comparing with FISH.

    Methods 

    We selected 568 cases of paraffin-embedded invasive breast cancer specimen which were proved as HER2 equivocal by immunohistochemical (IHC). The HER2 gene amplification status was evaluated by FISH and comparatively analyzed according to both the 2013 and 2018 ASCO/CAP HER2 Testing guidelines respectively. Sixty specimens were randomly selected for DISH detection and compared with FISH.

    Results 

    According to the 2013 guideline, the number of HER2 positive, negative and equivocal cases were 183 (32.22%), 365 (64.26%) and 20 (53.52%), respectively. Compared with the 2013 guideline, HER2 positive rate decreased by 4.58% according to the revised 2018 guideline, and the negative rates were same; the difference of positive rates was statistically significant (P < 0.01). Comparison between FISH and DISH findings in 60 samples, the total, positive and negative coincidence rates were 95.0%, 94.7% and 100%, and the results of the two methods are significantly correlated (r=0.636, P < 0.01).

    Conclusion 

    Compared with 2013 ASCO/CAP guideline, the revised 2018 guidelines is more accurate and instructive for clinical medication. DISH could achieve similar precise results as compared to FISH. Bright field detection is the irreplaceable advantage of DISH.

     

  • 反义寡核苷酸技术(antisense oligonucleotides, ASOs)又称反义技术,是指一种利用一段人工合成或表达的能与RNA或DNA互补结合的寡核苷酸链,抑制目的基因表达的技术[1-2],也可用于干扰肿瘤细胞中特定基因的表达,从而探讨肿瘤发生的机制和开发基因的治疗策略。近来研究显示,微小RNA-21(miR-21)与包括结直肠癌在内的多种肿瘤的发生密切相关[3-8],可能是肿瘤基因治疗的潜在新靶标[9-10]。我们在前期研究中发现,通过ASOs可显著下调人结肠癌SW620细胞中miR-21的表达,并抑制肿瘤的生长[11]。本研究中进一步利用人结肠癌HCT116细胞建立人结肠癌裸鼠实验动物模型,观察ASOs下调miR-21的效果及对人结肠癌细胞体内生长的效应,为开发基于ASOs技术的人结肠癌基因治疗策略提供实验基础。

    12只雌性BALB/c裸鼠,4~5周龄,体质量11~14 g左右,购自北京维通利华实验动物技术有限公司[实验动物许可证编号:SCXK(京)2011-0011],于遵义医学院SPF级免疫学实验室人工饲养。人结肠癌HCT116细胞株购自中国生命科学院,重组质粒p-miR-21-ASOs和对照质粒p-Cont均由本实验室保存。McCoy's 5A培养基、磷酸盐缓冲液(PBS)均购自北京索莱宝科技有限公司,优质胎牛血清购自美国Gibco公司,100×三抗(青霉素-链霉素-两性霉素B)购自上海第四制药股份有限公司;SYBR Premix Ex Taq,Premix Ex Taq Version2.0以及RNAisoTM Plus均购自北京宝日医生物技术有限公司;miR-21探针试剂盒购自美国ABI公司;RevertAidTM First Strand cDNA Synthesis Kit反转录合成试剂盒购自美国Fermentas公司;总RNA提取试剂盒购自北京天根科技有限公司,0.5%Triton-X-100(聚乙二醇辛基苯基醚)购自北京索莱宝科技有限公司;APC标记的兔抗小鼠Ki-67单克隆抗体、DAPI染液均购自美国ThermoFisher公司;兔抗p-Akt、Akt一抗以及兔抗ERK1/2、p-ERK1/2一抗、HRP标记的羊抗兔二抗均购自英国Abcam公司;全蛋白提取试剂盒购自江苏凯基生物公司;ECL显色液、RIPA裂解液、PVDF膜购自中国联科生物技术公司。

    按如下条件培养人结肠癌HCT116细胞株:人结肠癌HCT116细胞株在10%优质胎牛血清、100 u/ml青霉素、100 μg/ml链霉素、0.25 μg/ml两性霉素B及11.9 g/L McCoy's 5A细胞培养液中培养,将人结肠癌HCT116细胞株置于37℃、5%CO2的条件下培养,待HCT116细胞处于对数生长期时,使用0.25%胰蛋白酶37℃消化2 min,离心收集细胞,使用PBS重复清洗3次,计算细胞总数,利用PBS调整HCT116细胞密度为4.5×107个/毫升备用。

    4~5周龄裸鼠购回后先于本实验室SPF级环境下观察并饲养5~7 d。5~7 d天后小鼠饮食精神状况良好,随后接种100 μl密度为4.5×107个/毫升的人结肠癌HCT116细胞于裸鼠右侧腋窝外侧中部皮下,荷瘤后继续饲养并随时观察注射部位有无红肿溃烂、裸鼠的饮食、精神情况以及每只裸鼠荷瘤成功后的成瘤时间。接种后5~7 d出现肉眼可见的肿瘤包块时,即为荷瘤裸鼠模型构建成功。将实验荷瘤小鼠随机分为实验组(p-miR-21-ASOs组)和对照组(p-Cont组),每组6只,随后每隔4 d分别向两组荷瘤小鼠的肿瘤组织中注射100 μg p-Cont质粒和p-miR-21-ASOs质粒,共注射4次,并每隔3 d测量一次裸鼠皮下肿瘤体积。

    观察方法同前期工作[11]

    从-80℃超低温冰箱取出用75%冰乙醇固定好的冰冻组织切片,室温(15℃~25℃)平衡10 min,PBS缓冲液漂洗3次,每次5 min;山羊血清封闭液封闭1.5 h,PBS漂洗3次,每次10 min;加0.5%Triton-X-100,冰上破膜2 min,PBS漂洗3次,每次10 min;吸弃封闭液,滴加APC标记的兔抗小鼠单克隆Ki-67抗体后置于湿盒内,4℃冰箱避光过夜孵育,次日,取出冰冻切片肿瘤组织用PBS缓冲液漂洗3次,每次10 min;滴加DAPI染液,染色3 min,PBS漂洗3次,每次10 min;滴加含有DAPI染液的封片剂,封片并固定24 h后观察;激光共聚焦显微镜观察细胞染色情况,拍片。

    肿瘤组织中miR-21、细胞周期蛋白依赖性激酶(cyclin- dependent kinase, CDK)表达的检测同前期工作[11]

    蛋白样品的制备及BCA法定量蛋白的检测同前期工作[11]。Western blot检测相关信号通路的变化:(1)电泳:分离胶10%,积层胶5%,80 V跑积层胶,120 V跑分离胶;(2)上样:第一泳道Marker,其余泳道加提取的总蛋白;(3)转膜:PVD膜,36 V,180 min电转;(4)洗膜:用TBS或者PBS洗膜;5 min一次;(5)封闭:用含5%脱脂奶粉的TBST或者PBST封闭1 h;(6)加一抗:加入相关目的蛋白的一抗(稀释比例均为1:2 000),4℃过夜;(7)洗膜:次日用TBST或者PBST洗膜,10 min三次;(8)加二抗:加入HRP标记的相关目的蛋白的二抗(稀释比例均为1:2 000),室温孵育1 h;(9)用PBST洗膜,10 min×3次;(10)用发光试剂(ECl)对膜进行处理,于发光仪上进行蛋白曝光检测。

    实验数据使用GraphPad Prism 5软件进行统计学分析。本研究中各实验数据均独立重复3次,结果以(x±s)表示。其中组间比较采用t检验或单因素方差分析,P < 0.05为差异有统计学意义。

    结果显示,与p-Cont组相比,p-miR-21-ASOs组皮下肿瘤生长显著延缓,且肿瘤组织的体积、大小、重量都明显减小,差异有统计学意义,见图 1

    图  1  重组质粒注射后裸鼠皮下种植瘤的肿瘤体积(A)、大小(B)和重量(C)变化
    Figure  1  Tumor volume(A), size(B) and weight(C) in nude mice after subcutaneous injection of recombinant plasmid
    *: P < 0.05 (P0 day=0.0341, P6 day=0.0202, P9 day=0.0177, P12 day=0.0101); **: P < 0.01(P15 day=0.0024, P18 day=0.0010)

    HE染色显示:p-Cont组肿瘤细胞形态不一,细胞核深染且核呈多形性,核质比加大;而p-miR-21-ASOs组则可见肿瘤组织大面积坏死,见图 2

    图  2  重组质粒局部注射荷瘤裸鼠后肿瘤组织HE染色结果(×400)
    Figure  2  HE staining results of tumor tissues after local injection of recombinant plasmid into xenograft tumor in nude mice (×400)

    结果显示:与p-Cont组相比,p-miR-21-ASOs组肿瘤组织中miR-21的表达水平显著下调,且差异有统计学意义(P < 0.05),见图 3

    图  3  Real-time PCR探针法检测肿瘤组织中miR-21的表达
    Figure  3  Expression of miR-21 in tumor tissues detected by real-time PCR

    免疫荧光共聚焦观察发现:相比于p-Cont组,p-miR-21-ASOs组肿瘤组织中Ki-67的表达水平明显减少(P < 0.01),见图 4

    图  4  p-miR-21-ASOs重组质粒注射荷瘤裸鼠后激光共聚焦检测肿瘤组织中Ki-67的表达
    Figure  4  Expression level of Ki-67 protein after injection of p-miR-21-ASOs recombinant plasmid into xenograft tumor in nude mice detected by immunofluorescence technique

    结果显示:与p-Cont组相比,p-miR-21-ASOs组肿瘤细胞生长周期相关的周期蛋白依赖性激酶CDK2、CDK3、CDK4以及CDK6的表达水平均明显下调,差异有统计学意义(P < 0.05, P < 0.001),见图 5

    图  5  实时荧光定量PCR法检测肿瘤组织中CDKs的表达
    Figure  5  Expression of CDKs in tumor tissues detected by real-time fluorescent quantitative PCR
    **: P < 0.05(PCDK3=0.0308, PCDK6=0.0252); ***: P < 0.001(PCDK2=0.0001, PCDK4=0.0003)

    结果显示:与p-Cont组相比,p-miR-21-ASOs组中p-ERK1/2,p-Akt的蛋白表达水平明显降低(P < 0.05, P < 0.01),见图 6

    图  6  Western blot检测肿瘤组织中总Akt、ERK1/2和Akt、ERK1/2磷酸化的水平
    Figure  6  Expression levels of total Akt, ERK1/2, p-Akt and p-ERK1/2 in tumor tissues analyzed by Western blot
    *: Pp-Akt=0.0440, **: Pp-ERK1/2=0.0095, compared with p-Cont group

    近来的研究发现,ASOs技术可广泛用于多种临床疾病发生机制探讨和治疗策略开发[12-18]。对于肿瘤基因治疗来说,利用ASOs技术干预特定基因表达来影响肿瘤生长方面的研究也取得了重要进展[19-20]。我们在新近工作中也发现利用真核载体过表达ASOs干预miR-21表达可显著抑制人结肠癌SW620细胞的体内生长[11]。本研究中,我们进一步发现,真核载体过表达ASOs也可有效抑制人结肠癌HCT116细胞中miR-21的表达,同时肿瘤细胞体内生长受到抑制。范钰等[21]用ASOs下调人喉癌Hep-2细胞中miR-21的表达,发现肿瘤细胞的生长、侵袭能力明显减弱。吴子晏等[22]研究发现ASOs干预miR-21联合顺铂在体内、外能显著抑制人骨肉瘤MG-63细胞生长,并促进细胞凋亡,显示了一定的治疗效应。这些研究结果提示,利用ASOs技术干预miR-21表达可能是一种能有效影响特定肿瘤生长的手段,为后续开发基于ASOs的人结肠癌基因治疗策略提供了重要工作基础。

    研究显示,miR-21可通过对多种靶分子和信号途径传递的调节来调控人结肠癌的生长[23-28]。在前期工作中,我们也发现ASOs下调miR-21表达后,肿瘤细胞生长相关信号途径Akt和ERK1/2明显改变,与其靶分子PTEN的表达变化有关[4, 10]。本研究进一步发现ASOs下调miR-21表达后,肿瘤组织存在坏死,肿瘤细胞的增殖能力明显减弱,同时,肿瘤组织中p-Akt和p-ERK1/2等蛋白的表达水平均明显下调,进一步提示了miR-21对Akt和ERK1/2等信号途径的调控作用。现已知,细胞内CDKs家族分子的表达改变与肿瘤细胞生长相关。本研究也发现肿瘤细胞内CDKs家族成员表达水平显著减低。鉴于已有的研究表明,肿瘤细胞生长相关信号途径Akt和ERK1/2与细胞内CDKs家族成员表达间存在密切联系[29-31]。因此,推测ASOs干预miR-21表达后对人结肠癌细胞体内生长的抑制效应机制,包括肿瘤组织坏死的变化,与Akt和ERK1/2等信号途径传递改变,进而影响CDKs家族分子的表达有关。然而,鉴于不同实验条件的差异性,ASOs下调miR-21后抑制人结肠癌细胞体内生长的确切分子机制仍有待后续研究阐明。

    总之,本研究发现ASOs可显著下调人结肠癌动物模型中肿瘤组织miR-21的表达,同时肿瘤体内生长受到明显抑制,该效应与Akt和ERK1/2等信号途径传递变化有关,为后续深入探讨miR-21调控肿瘤生长的分子机制,以及开发基于ASOs技术的人结肠癌基因治疗策略提供前期实验基础。

    作者贡献
    蒋依娜: 实验设计、部分FISH及DISH实验操作、数据整理及分析、论文撰写及修改
    王博: 统计学分析
    周灿: 临床资料整理及分析
    王鸿雁: 实验及论文指导
  • 图  1   HER2基因扩增阳性的浸润性乳腺癌

    Figure  1   HER2 positive invasive breast cancer specimen tested by FISH and DISH

    图  2   HER2基因扩增不确定的浸润性乳腺癌

    Figure  2   HER2 equivocal invasive breast cancer specimen tested by FISH and DISH

    图  3   FISH与DISH检测结果不一致病例

    Figure  3   Inconsistent case tested by FISH and DISH

    表  1   568例免疫组织化学不确定的浸润性乳腺癌HER2 FISH检测结果

    Table  1   FISH results of HER2 in 568 cases of immunohistochemical HER2 equivocal invasive breast cancer

    下载: 导出CSV

    表  2   FISH和DISH方法检测HER2基因状态的比较

    Table  2   Comparison of HER2 gene status between FISH and DISH test

    下载: 导出CSV
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出版历程
  • 收稿日期:  2019-01-08
  • 修回日期:  2019-04-03
  • 网络出版日期:  2024-01-12
  • 刊出日期:  2019-07-24

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