细胞学标本检测肺癌间变淋巴瘤激酶的探讨及临床研究

摘要

目的

1.探讨细胞学标本在肺癌分子细胞病理学的应用。

2.探讨细针吸取细胞学（Fine Needle Aspiration Cytology，FNAC）标本传统涂片和液基薄层涂片应用免疫细胞化学（Immunocytochemistry，ICC）检测进展期肺腺癌间变淋巴瘤激酶（anaplasitic lymphoma kinase，ALK）蛋白表达的可行性。

方法

1.收集应用细胞学涂片标本进行ALK基因检测的肺癌病例338例，查阅其基本信息、病理形态学结果，基因检测信息（表皮生长因子受体（epidermal growth factor receptor, EGFR）、K-RAS原癌基因（K-RAS oncogene，K-RAS）、棘皮动物微管样蛋白4-间变淋巴瘤激酶（echinoderm microtubule associated protein like 4-anaplasitic lymphoma kinase, EML4-ALK）、Ros原癌基因1（ros oncogene 1, Ros-1）、c-间质表皮转化因子（c-mesenchymal-epidermal transition, c-Met）、美国癌症联合委员会 ( American Joint Committee on Cancer, AJCC)临床分期，治疗情况（手术、放射治疗，化学治疗，靶向治疗）等进行总结。

2.收集肺腺癌淋巴结转移灶FNAC标本147例，包括传统涂片47例及液基薄片100例，进行Ventana ALK（D5F3）ICC检测，并与逆转录聚合酶链反应(reverse transcriptase-polymerase chain reaction, RT-PCR)或荧光原位杂交（fluorescence in situ hybridization, FISH）结果进行对照。

结果

1.338例行ALK融合基因检测病例，阳性率为9.17%（31/338），EGFR、K-RAS突变率分别为47.74%（159/333）、7.50%（25/333），Ros-1基因融合率为4.00%（3/75），c-Met基因扩增率为4.11%（3/73）；276例(81.66%)在首次就诊时已处于ⅢB-Ⅳ期；338例肺癌ALK检测标本中，包括浆膜腔积液中ALK阳性率为5.52%（9/163）；穿刺细胞学标本中ALK阳性率12.57%（22/175）。217例（64.20%）发生基因改变的病例中，共有123例进行了靶向治疗：靶向药物一线、二线治疗的病例分别为76例、47例。另有10例选择试吃或盲吃靶向药物。

2.ALK蛋白在肺腺癌淋巴结转移灶FNAC标本阳性表达率为11.56%（17/147），传统涂片及液基薄片ALK阳性表达率分别为10.64%（5/47），12.00%（12/100）。147例标本中有57例从分子水平进行验证，包括阳性病例17例及阴性病例40例，总符合率为96.49%（55/57）。肺腺癌淋巴结转移灶FNAC标本检测ALK蛋白敏感性为94.12%（16/17），特异性为97.50%（39/40）。传统涂片ALK蛋白检测的敏感性和特异性均为100%（敏感性5/5，特异性10/10），液基薄片ALK蛋白检测敏感性、特异性分别为91.67%（11/12）、 96.67%（29/30）。 

结论

1.对于初次就诊就处于晚期（ⅢB-Ⅳ）肺癌患者或早期肺癌术后进展患者，细胞学标本是进行靶向治疗相关驱动基因检测的可靠标本来源。

2.胸腔积液和淋巴结细针穿刺是常见的细胞学标本，比较于浆膜腔积液，ALK融合基因在淋巴结穿刺细胞学标本中更容易检测到，二者差异有统计学意义。

3.细胞学标本能够提供足够细胞量完成临床靶向治疗相关基因检测。

4.对于无法制成细胞块的细胞学标本，细针穿刺传统涂片及液基薄片均可用来进行ALK（D5F3）ICC检测。

5.对于ALK-ICC检测在液基薄片的应用，由于液基细胞保存液成分可能会导致ALK蛋白表面抗原丢失，导致异质性表达，建议可疑阳性病例进行FISH检测验证。

关键词  肺癌；间变淋巴激酶；细针吸取细胞学；传统涂片；液基薄层涂片

The Clinical Research of Anaplasitic Lymphoma Kinase in Lung Carcinoma in Cytology Specimens

Abstract

Objective

1. To explore the application of cytology specimens in molecular pathology of lung cancer.

2.To explore the feasibility of anaplasitic lymphoma kinase(ALK) immunocytochemistry (ICC) analysis on conventional smears and liquid-based slides of fine needle aspiration cytology（FNAC）in advanced lung adenocarcinoma.

Methods

1.338 cases of lung cancer which ALK fusion gene were detected using cytology specimens were collected, and the basic information, pathological results, gene detection information ,which included ALK, Epidermal Growth Factor Receptor(EGFR),K-RAS, Ros-1, and c-Mesenchymal-Epidermal Transition(c-Met), American Joint Committee on Cancer clinical stages and treatment(Surgery, radiotherapy, chemotherapy, targeted therapy) were summarized.

2.147 cases of lymph node metastasis lesions of advanced lung adenocarcinoma, including 100 of liquid -based slides and 47 of conventional smears, are collected in this study. The expression of ALK fusion protein was detected by Roche Ventana ICC technology, compared with the ALK gene fusion assessed by Fluorescence in Situ Hybridization(FISH), reverse transcriptase-polymerase chain reaction(RT-PCR).

Results

1. In 338 cases, the positive rate of ALK fusion gene was 9.17% (31/338), the mutation rate of EGFR and K-RAS was 47.74% (159/333), 7.50% (25/333), the fusion rate of Ros-1 was 4% (3/75), the amplification rate of c-Met was 4.11% (3/73).And 276 of 338 cases had been in stage III B- IV at first visit. The positive rate of ALK in the serous cavity effusion was 5.52% (9/163), while in the FNAC specimens was 12.57% (22/175). In 217 cases (64.20%) with genetic alterations, 123 cases were targeted therapy. Drug targeting first-line and second-line treatment of patients were 76 47 cases,respectively.10 cases of blind tasting or otherwise choose to eat targeted drugs.

2.The positive rate of ALK(D5F3) fusion protein expression in advanced lung adenocarcinoma FNAC was 11.56%（17/147）while in conventional smears and liquid-based cytology reached 10.64%（5/47）and12.00%（12/100）respectively. Among all 147 cases, there are 57 cases referred to corresponding molecular methods verified, including 17 positive cases and 40 negative cases. The coincidence rate reached 96.49%（55/57）overall, while sensitivity and specificity in ALK(D5F3) fusion protein expression in advanced lung adenocarcinoma FNAC are 94.12%（16/17）,97.5%（39/40）respectively. The sensitivity and specificity in conventional smears both reached 100%(5/5 in sensitivity and 10/10 in specificity) while 91.67%(11/12) and 96.67%(29/30) respectively in liquid-based cytology slides.

Conclusion

1.The patients with advanced (IIIB-IV) lung cancer at the first time or postoperative progressing, the cytology specimen is a reliable source of gene detection.

2. The pleura effusion and lymph node FNAC were common specimens in the advanced lung cancer, and ALK fusion genotype were more likely to appear in the cytological specimens compared with the serous cavity effusion, the difference between them were statistically significant.

3. Cytological specimens can provide enough cell volume to complete clinical targeted therapy related gene detection.

4.Conventional smears and liquid-based cytology in FNAC samples can be used in the detection of ALK(D5F3) ICC analysis of advanced lung adenocarcinoma, especially for those who have no chance in making cell block to perform gene analysis.

5. For the application of ALK ICC in liquid-based specimens, the composition of the liquid based cell preservative may lead to the loss of cell surface antigen and often demonstrated heterogeneous expression. It is suggested that FISH test should be carried out when necessary.

[key words] lung neoplasm; ALK; fine needle aspiration cytology; Conventional smears; Liquid-Based cytology slides