Objectives: The advanced non-small cell lung cancer (NSCLC) patients with pleural effusion have no opportunity for surgery treatment. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the first-line drugs for these patients with EGFR-sensitive mutation. However, the disease progression and drug update during or after treatment of EGFR-TKIs bring more challenges and puzzles to clinical diagnosis and treatment, which inevitably requires archived pleural cell samples for EGFR re-examination or comparative study. Understanding the DNA quality of archived pleural fluid samples and effectively using archival data of pleural fluid cells are of great significance for tracing the origin of cases and basic medical research. This study aims to evaluate the consistency of EGFR mutant gene expression between the 2 methods, and to explore a reliable way for preserving cytological data and making full use of cytological archival data via cell HE staining smear and cell paraffin section.
Methods: A total of 57 pleural fluid cytology cases in the Department of Pathology of China Aerospace Center Hospital from October 2014 to April 2021 were selected. Tumor cells were detected by cell HE staining smears and immunohistochemical staining for TTF-1 and Napsin A in the paired cell paraffin sections. There were more than 200 tumor cells in cell HE staining smear and the proportion of tumor cells were ≥70% in matched cell paraffin sections. Patients with 2 cell smears (one for cell data retention and the other for DNA extraction) were selected as the research subjects, and 57 pleural fluid samples were enrolled. EGFR gene mutation was detected by amplification refractory mutation system-polymerase chain reaction in 57 paired cell HE staining smears and cell paraffin sections. DNA concentration was 2 ng/μL. Cell HE smear was amplified side-by-side with DNA samples from paired cell paraffin sections. Result determination was according to the requirements of the reagent instructions. The external control cycle threshold (Ct) value of the No. 8 well of the samples to be tested was between 13 and 21, which was considered as successful and reliable samples. When the Ct value of EGFR gene mutation was <26, it was considered as positive; when the Ct value was between 26 and 29, it was critical positive; when the Ct value was equal or more than 29, it was negative. ΔCt value was the difference between mutant Ct value and externally controlled Ct value. The smaller the ΔCt value was, the better the quality of DNA of the detected sample was.
Results: Among the 57 pleural effusion samples, 42 patients were hospitalized with pleural effusion as the first symptom, accounting for 73.7% (42/57). EGFR mutation was detected in 37 samples [64.9% (37/57)]. The mutation rate for 19del was 37.8% (14/37) while for L858R was 48.6% (18/37). Females were 56.7% (21/37) of mutation cases. The mutation consistency rate of cell HE staining smear and matched cell paraffin sections was 100%. The ΔCt values of cell HE staining smears were less than those of matched cell paraffin sections. The mutation Ct values of 37 cytological samples were statistically analyzed according to the preservation periods of the years of 2014-2015, 2016-2017, 2018-2019, and 2020-2021. There were significant differences in cell paraffin section in the years of 2014-2015 and 2016-2017 compared with the years of 2018-2019 and 2020-2021, while no significant differences were found in cell HE staining smear. Statistical analysis of externally controlled Ct values of 57 cytological samples showed that there were significant differences between cell HE staining smears and cell paraffin section in the years of 2014-2015 and 2016-2017, compared with the years of 2018-2019 and 2020-2021. The mutational Ct values of 37 paired cell blocks and smears were all <26, and the externally controlled Ct values of 57 paired cell paraffin sections and HE staining smears were all between 13 and 21.
Conclusions: The DNA quality of cell HE smears and matched cell paraffin section met the qualified requirements. Two methods possess show an excellent consistency in detecting EGFR mutation in NSCLC pleural fluid samples. The DNA quality of cell HE staining smear is better than that of cell paraffin sections, so cell HE staining smear can be used as important supplement of the gene test source. It should be noted that the limitation of cell HE staining smears is non-reproducibility, so multiple smears of pleural fluid are recommended to be prepared for multiple tests.
目的: 伴发胸腔积液的晚期非小细胞肺癌(non-small cell lung cancer，NSCLC)患者已失去手术机会，表皮生长因子受体(epithelial growth factor receptor，EGFR)酪氨酸激酶抑制剂(tyrosine kinase inhibitors，TKIs)是EGFR敏感突变的晚期NSCLC患者的一线用药。但EGFR-TKIs治疗时或治疗后出现的疾病进展以及药物的更新迭代给临床诊治带来了挑战，需要对存档的胸腔积液细胞样本进行EGFR复检或比对研究。本研究采用细胞HE染色涂片与细胞蜡块两种制作方法对NSCLC胸腔积液样本的EGFR突变基因进行检测，分析两种保存方式、保存时间对胸腔积液细胞DNA质量的影响，以期探索出一条保存细胞学资料及充分利用细胞学档案资料的可靠途径。方法: 选取2014年10月—2021年4月航天中心医院病理科接收的胸腔积液样本57例，所有样本同时制作细胞HE染色涂片与细胞蜡块。将57例配对的细胞HE染色涂片与细胞蜡块采用扩增阻碍突变系统-聚合酶链反应(amplification refractory mutation system-polymerase chain reaction，ARMS-PCR)技术进行EGFR基因突变检测。DNA检测浓度为2 ng/μL，细胞HE涂片与配对细胞蜡块DNA样本并排进行扩增。结果判读按试剂说明书要求，待测样本的8号孔外控循环阈值(cycle threshold，Ct值)在13~21之间为样本合格，EGFR基因突变Ct值<26时，判断为阳性；26≤Ct值<29为临界阳性，Ct值≥29为阴性。ΔCt值为突变Ct值与外控信号Ct值的差值。比较细胞HE染色涂片与细胞蜡块检测为阳性的ΔCt值。同时将57例患者按时间段分为4组：2014—2015年(n=10)，2016—2017年(n=20)，2018—2019年(n=17)，2020—2021年(n=10)。比较不同时间段的检测结果。结果: 57例晚期NSCLC患者中以胸腔积液为首发症状入院者42例，占73.7%；57例中37例发生EGFR突变，突变率64.9%；19del突变和L858R突变为主要的突变类型，分别占37.8%(14/37)和48.6%(18/37)；37例突变患者中女性占56.7%(21/37)；细胞涂片与配对的细胞蜡块突变一致率为100%；细胞HE染色涂片的ΔCt值小于配对细胞蜡块的ΔCt值(t=4.526，P<0.001)。37例配对细胞蜡块与细胞HE染色涂片的突变Ct值均<26。4个时间段中，2014—2015年、2016—2017年、2018—2019年的细胞蜡块突变基因Ct均值均高于2020—2021年(P<0.05)；4个时间段的细胞HE染色涂片突变Ct均值差异无统计学意义(P>0.05)。57例配对细胞蜡块与细胞HE染色涂片的外控Ct值均在13~21之间。2014—2015年、2016—2017年的细胞蜡块与细胞HE染色涂片的外控Ct值均高于2018—2019年、2020—2021年(均P<0.05)。结论: 细胞HE染色涂片与细胞蜡块检测NSCLC胸腔积液样本EGFR突变具有极好的一致性，且细胞HE染色涂片的DNA质量优于细胞蜡块，但细胞HE染色涂片的局限在于不可复制性，建议在胸腔积液标本制作中进行多张涂片，以满足多次检测的需求。.
Keywords: cell HE staining smear; cell paraffin section; epithelial growth factor receptor; non-small cell lung cancer; pleural fluid.