摘要
目的：研究细胞学标本间变性淋巴瘤激酶（ ALK）蛋白表达及基因融合检测的规范化流程。方法收集北京市7所三级甲等医院晚期肺腺癌细胞学标本，按照设计的规范化流程检测ALK蛋白表达及基因融合状态。制作福尔马林固定石蜡包埋（ FFPE）细胞学蜡块，应用免疫组化（ IHC）方法鉴别肿瘤细胞的来源与分型；应用Ventana IHC ALK（ D5F3）检测ALK蛋白的表达；应用实时荧光定量PCR（ qRT-PCR）方法检测表皮生长因子受体（ EGFR）基因野生型标本中ALK基因融合，并对无法制作细胞学蜡块的细胞学标本进行ALK基因融合检测；Ventana IHC ALK（ D5F3）检测阳性标本应用荧光原位杂交（FISH）方法验证ALK基因的融合状态。分析Ventana IHC ALK（D5F3）检测阳性并采用克唑替尼治疗患者的疗效。结果229例细胞学标本符合肺腺癌诊断，其中207例标本采用至少1种方法检测并获得ALK蛋白表达和基因融合检测结果，检测率为90．4％（207／229）。203例标本成功制作FFPE细胞学蜡块，应用Ventana IHC ALK（D5F3）检测ALK蛋白表达的成功率为100．0％，阳性率为10．3％（21／203）。98例EGFR基因野生型肺腺癌FFPE细胞学标本采用qRT-PCR方法检测ALK基因融合，96例检测成功（97．96％）。以qRT-PCR方法为标准，Ventana IHC ALK（D5F3）检测ALK蛋白表达的灵敏度为100．0％，特异性为98．7％。2种检测方法一致率为94．7％（ Kappa＝0．967， P＜0．001）。 FISH方法对Ventana IHC ALK（ D5F3）表达阳性的结果进行验证，2例寡肿瘤细胞学标本无法获得明确判读结果。6例Ventana IHC ALK（ D5F3）检测ALK蛋白表达阳性的患者选用克唑替尼治疗，有效5例。2例细胞学蜡块应用4％甲醛固定72 h后，Ventana IHC ALK（ D5F3）染色强度明显减弱；10例患者分别用FFPE细胞学蜡块标本切片和刮取细胞学甩片中细胞提取总RNA，qRT-PCR检测ALK基因融合结�
Objective The aim of this study was to establish a standardized protocol for detection of ALK protein expression and gene fusion in cytologic specimens. Methods Lung adenocarcinoma cytologic specimens were collected from seven hospitals in Beijing city. A detection protocol for ALK protein expression and gene fusion was designed according to the results of comparative experiment. Ventana immunohistochemical （ IHC） ALK（ D5F3） detecting ALK protein expression was performed in 203 prepared 〈br〉 formalin-fixed paraffin-embedded （ FFPE） cell blocks. ALK gene fusion in 98 EGFR gene wild type cytologic specimens and in 4 bronchoalveolar lavage fluid （ BL ） samples was detected by quantitative reverse transcription polymerase chain reaction （qRT-PCR）. ALK gene fusion in the Ventana IHC ALK （D5F3） positive samples was further tested by fluorescence in situ hybridization （ FISH） . Six patients with ALK IHC-positive result were followed up to analyze the responses of crizotinib therapy. Comparative experiments：（ 1） Comparison of the results of 4% neutral buffered formalin fixed for different time （ 24 h, 48 h, 72 h） on the Ventana IHC ALK （D5F3） staining was conducted in two cases of IHC ALK positive FFPE cell blocks;（2） Comparing qRT-PCR results for ALK fusion in samples from FFPE cell blocks and cytospin prepared slides in 10 cases of lung adenocarcinoma cytologic specimens. Results Among the specimens examined using the standardized protocol recommended by this study, 229 cases of cytologic specimens met the diagnostic criteria of lung adenocarcinoma. Among them, 207 cases obtained ALK gene test results （ by at least one method）, with an ALK test ratio of 90.4% （207/229）.FFPE cell blocks were successfully prepared in 203 cases, Ventana IHC ALK （ D5F3） were successfully performed in all the 203 FFPE cell blocks （ 100%） , and the ALK protein positive detection rate was 10.3% （21/203）. ALK fusion was tested in 98 FFPE cytologic samples of EGFR wild types
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2015年第10期742-748,共7页
Chinese Journal of Oncology
基金
北京市科学技术委员会研究基金（D141100000214003）
关键词
肺肿瘤
腺癌
间变性淋巴瘤激酶
细胞学技术
免疫组织化学
克唑替尼
Lung neoplasms
Adenocarcinoma
Anaplastic lymphoma kinase
Cytological techniques
Immunohistochemistry
Crizotinib
