Multigene Combined Detection by RT-qPCR Using Cytological Specimens

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.

Overview and update on molecular testing in non‐small cell lung carcinoma utilizing endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) samples

“…The same group also reviewed the best cytologic techniques for this kind of analysis and showed that fresh samples obtained by membrane cell collector method had better performance than cell blocks, probably due to the avoidance of contaminants that can hinder DNA or RNA extraction. 87 Sgariglia et al also showed the feasibility of performing multiplex digital color-coded barcode techniques (nCounter) for RNA detection in cytological samples with a 48-gene panel to improve the detection rate. 88…”

“…These authors confirmed similar results with both types of samples and, although the most prevalent molecular abnormalities involved EGFR , ALK and ROS1 , as expected, they retrieved a small number of abnormalities in other genes that are amenable to targeted inhibition. The same group also reviewed the best cytologic techniques for this kind of analysis and showed that fresh samples obtained by membrane cell collector method had better performance than cell blocks, probably due to the avoidance of contaminants that can hinder DNA or RNA extraction 87 . Sgariglia et al also showed the feasibility of performing multiplex digital color‐coded barcode techniques (nCounter) for RNA detection in cytological samples with a 48‐gene panel to improve the detection rate 88 …”