Multigene Combined Detection by RT-qPCR Using Cytological Specimens

Author:

Ma Yang,Zhao Jingxia,Du Yun,Wang Rui,Ji Xiaokun,Wu Juan,Guo Xiao,Liu Ying,Zhang Yan

Abstract

<b><i>Objective:</i></b> The aim of the study was to investigate the mutation status of multiple driver genes by RT-qPCR and their significance in advanced lung adenocarcinoma using cytological specimens. <b><i>Materials and Methods:</i></b> 155 cytological specimens that had been diagnosed with lung adenocarcinoma in the Fourth Hospital of Hebei Medical University were selected from April to November 2019. The cytological specimens included serous cavity effusion and fine-needle aspiration biopsies. Among cytological specimens, 108 cases were processed by using the cell block method (CBM), and 47 cases were processed by the disposable membrane cell collector method (MCM) before DNA/RNA extraction. Ten drive genes of EGFR, ALK, ROS1, BRAF, KRAS, NRAS, HER2, RET, PIK3CA, and MET were combined detected at one step by the amplification refractory mutation system and ABI 7500 RT-qPCR. <b><i>Results:</i></b> The purity of RNA (<i>p</i> = 0.005) and DNA (<i>p</i> = 0.001) extracted by using the MCM was both significantly higher than that extracted by using the CBM. Forty-seven cases of fresh cell specimens processed by the MCM all succeeded in multigene detections, while of 108 specimens processed by the CBM, 6 cases failed in multigene detections. Among 149 specimens, single-gene mutation rates of EGFR, ALK, ROS1, RET, HER2, MET, KRAS, NRAS, BRAF, and PIK3CA mutations were 57.71%, 6.04%, 3.36%, 2.68%, 2.01%, 2.01%, 1.34%, 0.67%, 0% and 0% respectively, and 6 cases including 2 coexistence mutations. We found that mutation status was correlated with gender (<i>p</i> = 0.047), but not correlated with age (<i>p</i> = 0.141) and smoking status (<i>p</i> = 0.083). We found that the EGFR mutation status was correlated with gender (<i>p</i> = 0.003), age (<i>p</i> = 0.015) and smoking habits (<i>p</i> = 0.007), and ALK mutation status was correlated with age (<i>p</i> = 0.002). <b><i>Conclusion:</i></b> Compared with the CBM, the MCM can improve the efficiency of DNA/RNA extraction and PCR amplification by removing impurities and enriching tumor cells. And we speculate that the successful detection rate of fresh cytological specimens was higher than that of paraffin-embedded specimens. EGFR, ALK, and ROS1 mutations were the main driver mutations in patients with advanced lung adenocarcinoma. We speculate that EGFR and ALK are more prone to concomitant mutations, respectively. Targeted therapies for patients with coexisting mutations need further study.

Publisher

S. Karger AG

Subject

General Medicine,Histology,Pathology and Forensic Medicine

Reference24 articles.

1. Wang K, Chen R, Feng Z, Zhu YM, Sun XX, Huang W, et al. Identification of differentially expressed genes in non-small cell lung cancer. Aging. 2019;11(23):11170–85.

2. Ellison G, Donald E, McWalter G, Knight L, Fletcher L, Sherwood J, et al. A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples. J Exp Clin Cancer Res. 2010;29(1):132.

3. Shi X, Wu H, Lu J, Duan H, Liu X, Liang Z. Screening for major driver oncogene alterations in adenosquamous lung carcinoma using PCR coupled with next-generation and Sanger sequencing methods. Sci Rep. 2016;6(1):22297–306.

4. Gao J, Wu H, Shi X, Huo Z, Zhang J, Liang Z. Comparison of next-generation sequencing, quantitative PCR, and sanger sequencing for mutation profiling of EGFR, KRAS, PIK3CA and BRAF in clinical lung tumors. Clin Lab. 2016;62(4):689–96.

5. Shaozhang Z, Ming Z, Haiyan P, Aiping Z, Qitao Y, Xiangqun S. Comparison of ARMS and direct sequencing for detection of EGFR mutation and prediction of EGFR-TKI efficacy between surgery and biopsy tumor tissues in NSCLC patients. Med Oncol. 2014;31(5):926.

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