Detection of ALK Gene Rearrangement in Cell-free RNA from Lung Cancer Malignant Pleural Effusion

The aim of this study was to evaluate the feasibility of measuring ALK gene rearrangement in cell-free RNA (cf-RNA) of the supernatant from malignant pleural effusion (MPE). Supernatant, cell blocks, and matched sera samples were collected. Cf-RNA was isolated from the supernatant and sera, and cellular RNA was isolated from cell blocks. The ALK gene rearrangement in the cf-RNA was tested by the real-time polymerase chain reaction. Results showed that the concentration of cf-RNA was higher in the supernatant than in matched sera. ALK status concordance rates were 100% between the supernatant and cell blocks, while they were 0% between sera and cell blocks in ALK gene rearrangement cases. This suggests that using cf-RNA in MPE supernatant, but not in sera, could offer a reliable and robust surrogate strategy for the detection of ALK gene rearrangement.


Introduction
Lung cancer is the leading cause of cancer death among both men and women, of which non-small-cell lung cancer (NSCLC) represents approximately 85% of the total cases diagnosed [1]. Driver gene abnormality tests are the premise of targeted therapy for advanced NSCLC, particularly in the adenocarcinoma subtype [2]. Among these, EGFR gene mutations and ALK gene rearrangement are widely recognized alterations that respond to target agents in NSCLC [3]. Malignant pleural effusion (MPE) is a common manifestation in patient with advanced lung cancer. Most MPE contain tumor cells, which can be used for EGFR and ALK gene status tests [4][5][6][7].
Cell-free nucleic acids are extracellular nucleic acids found in cell-free plasma/serum and other biological fluids [8]. Detection of cell-free nucleic acids (DNA or RNA) using quantitative real-time PCR or quantitative real-time RT-PCR (qRT-PCR) methods have been suggested as a promising diagnostic tool for cancer detection [9]. A number of studies have reported that EGFR mutations can be detected in cell-free DNA (cf-DNA) of malignant pleural effusion rearrangement samples from lung cancer patients [10,11]. ough researchers have showed that there was cellfree RNA (cf-RNA) in MPE [12], it is still not known whether ALK gene rearrangements can be detected by qRT-PCR using cf-RNA. erefore, the subject of the present study was to explore the use of the newly developed method for the detection of ALK gene rearrangement in MPE.

Patients and Samples.
A total of 168 MPE samples were collected and centrifuged. e tumor cell content in cell blocks was sufficient to perform the subsequent immunohistochemistry (IHC) and ALK gene rearrangement test. Twentythree cases of cell-free supernatant and sera (matched with 12 ALK positive rearrangement and 11 ALK negative rearrangement cell blocks) were collected. e diagnosis of NSCLC was based on cytology via a combination of IHC staining results and clinical information. All of the samples were obtained from Guizhou Provincial People's Hospital during November 2015-June 2019. e age at the time of initial diagnosis and smoking status were obtained from the hospital medical records. e study was approval by the Ethics Committees of Guizhou Provincial People's Hospital.

RNA Extraction and ALK Gene Rearrangement Detection.
e cell blocks were embedded by paraffin, and then cellular RNA was extracted by the use of an RNA FFPE Tissue Kit (AmoyDx, Xiamen, China) according to the manufacturer's protocols. e supernatant and sera were firstly centrifuged for 10 min at full speed (8000 g/min), and then cf-RNA was extracted by the use of a Circulate Nucleic Acids Kit (AmoyDx, Xiamen, China) according to the manufacturer's protocols. e quantity of isolated RNA was assessed by using a NanoDrop2000 spectrophotometer ( ermo, L.A., USA). e ALK gene rearrangement was detected by qRT-PCR using an ALK gene rearrangement Detection Kit (AmoyDx, Xiamen, China). e PCR reaction was performed on an Agilent Mx3000P QPCR instrument (Agilent Technologies, Santa Clara, CA). e following PCR procedure was used: an initial reverse transcription at 42°C for 5 min, denaturation at 95°C for 5 min, and then 10 annealing cycles at 95°C for 25 seconds, 64°C for 20 seconds, and 72°C for 20 seconds, followed by 36 extension cycles at 93°C for 25 seconds, 60°C for 35 seconds, and 72°C for 20 seconds to perform the data collection. e quantitative judgment was according to the gene rearrangement fluorescence signal. Assay reactions achieving threshold cycle (Ct) values of ≤35 cycles were considered as positive, otherwise as negative. Amplification of the control gene (beta-actin) in the assay demonstrates the presence of RNA.

Statistical Analysis.
Data were analyzed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). e chi-square test, Friedman test, and paired Student's t-test were performed to analyze variables, where appropriate. In addition, Kappa statistic and McNemar's test were performed to determine consistency between the supernatant, sera, and cell blocks. p < 0.05 was considered statistically significant.

Patient Features.
In total, 168 cases with MPE were eligible for analysis. e ALK gene rearrangement status was assessed by analysis of RNA extracted from cell blocks using the qRT-PCR method. e clinicopathological features of the patients are shown in Table 1. e current ALK positive rate was 7.1% (12/168) in patients with MPE. Further analysis found that the positive rate was significantly higher in patients with age under 60 years (14.1%) than in patients with age over 60 years (2.4%) (p � 0.012; Table 1).

Cf-RNA in Supernatant and Sera.
To determine whether delayed MPE supernatant processing would lead to the degradation of cf-RNA, 23 patients with MPE supernatant were collected for cf-RNA assay. e samples of MPE supernatant were placed in 4°C icebox at different time points (0, 24, and 48 hours) and then processed. Results showed that no significant difference was found for the concentrations of cf-RNA at three time points (p � 0.632; Figure 1). We further detected the ALK gene rearrangement by qRT-PCR, nevertheless, there was no difference between the three time points too (Figure 2). We also tested the RNA   concentration in the matched sera of patients with MPE (n � 23); however, the level of cf-RNA was very low in sera than in the supernatant (p < 0.0001; Figure 3).  Figure 4). However, the detection of ALK gene rearrangement was wholly negative in all matched sera (McNemar's p < 0.001; kappa � 0.000; Table 2; Figure 4).

Discussion
MPE is a common manifestation of advanced NSCLC. For many patients, MPE can be used for driver gene status tests, including EGFR gene mutation and ALK gene rearrangement [4,6,7,13]. Recently studies have showed that the cf-DNA of MPE could be used for the EGFR mutation test [10,11]. However, whether the cf-RNA of MPE is appropriate for ALK gene rearrangement testing is not yet known and was therefore the subject of the current study.  We firstly detected the concentration of cf-RNA and ALK gene rearrangement in different processing time points (0, 24, and 48 hours), and the results showed that there was no difference between them (Table 1; Figure 2). at indicates that there will be plenty of time to process cf-RNA in clinical working practice for MPE samples. In addition, we found that the concentration of cf-RNA was very low in matched sera than in the supernatant (Figure 3). ese findings suggest low level of cf-RNA burden in sera. Previous studies have showed that MPE cell blocks can be used for ALK gene rearrangement detection [2,4]. Hence, in this study, we compared the mutation status in cf-RNA of sera and the supernatant to the mutation status in the cell blocks from the same patients. Our findings showed that the ALK status concordance rates were 100% between the supernatant and cell blocks, but 0% between sera and cell blocks in ALK gene rearrangement cases (Table 2; Figure 4). We speculated that very low level cf-RNA in sera maybe the cause of negative results, while high concentration of cf-RNA provide enough material to ensure the sensitivity testing ALK gene rearrangement status in the supernatant. So, we think that cf-RNA in MPE supernatant samples is one of the proper substrates for ALK gene rearrangement testing. In the future, cf-RNA and cf-DNA in the MPE supernatant  would be an attractive alternative source supplying useful information about the mutation status of ALK, EGFR, and other genes.
In conclusion, this study explored a newly developed method for the detection of ALK gene rearrangement in MPE. Cf-RNA in the supernatant would be one potential substitute for molecular diagnostics.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
Mingliang Chu and Yanqiu Zhu contributed equally to this work.