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ORIGINAL ARTICLE  
Year : 2021  |  Volume : 64  |  Issue : 1  |  Page : 107-110
Clinical value of the detection of EGFR gene mutations in pleural effusion cell blocks among patients with non-small-cell lung cancer


1 Department of Clinical Laboratory, Jinhua Municipal Central Hospital, Jinhua, Zhejiang, China
2 Pathology, Jinhua Municipal Central Hospital, Jinhua, Zhejiang, China

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Date of Submission12-Apr-2020
Date of Decision22-May-2020
Date of Acceptance31-May-2020
Date of Web Publication8-Jan-2021
 

   Abstract 


Objective: To evaluate the clinical value of epidermal growth factor receptor (EGFR) detection in pleural effusion cell blocks among patients with non-small-cell lung cancer (NSCLC). Methods: From July 2016 to September 2018, EGFR gene mutations in 40 lung tumor tissue samples and pleural fluid samples from NSCLC patients in Jinhua Municipal Central Hospital were assessed by the amplification refractory mutation system method. The EGFR results of the two types of samples were compared using the paired Chi-square test, and the mutation positive rates in EGFR exons 18, 19, 20 and 21 were compared between the two types of specimens using the four-grid Chi-square test. Results: Among the 40 tissue samples and pleural effusion samples, 21 and 18 cases of EGFR mutations were detected, respectively, and the mutation positive rates were 52.5% and 45%, respectively. The κ value of the consistency test of the two specimens was 0.851. There were no significant differences in the mutation positive rates in EGFR exons 18, 19, 20, and 21 between the two types of specimens. Conclusion: The EGFR results of pleural fluid and tissue samples were in good agreement. Therefore, we can use pleural fluid samples to detect EGFR mutations to guide tyrosine kinase inhibitor treatment for NSCLC patients in whom tumor tissue samples cannot be obtained.

Keywords: Cell block, epidermal growth factor receptor, non-small-cell lung cancer, pleural fluid, gene mutation

How to cite this article:
Zhu Y, Zhu F, Wang L, Zhai X, Shan X. Clinical value of the detection of EGFR gene mutations in pleural effusion cell blocks among patients with non-small-cell lung cancer. Indian J Pathol Microbiol 2021;64:107-10

How to cite this URL:
Zhu Y, Zhu F, Wang L, Zhai X, Shan X. Clinical value of the detection of EGFR gene mutations in pleural effusion cell blocks among patients with non-small-cell lung cancer. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Jan 28];64:107-10. Available from: https://www.ijpmonline.org/text.asp?2021/64/1/107/306511





   Introduction Top


Lung cancer has one of the highest incidence rates and mortality rates globally.[1] Among them, non-small-cell lung cancer (NSCLC) accounts for over 80%.[2] Owing to the lack of typical early symptoms, more than 50% of patients are in the late stage at the time of diagnosis and cannot undergo surgery.[3] Previous studies have confirmed that an epidermal growth factor receptor (EGFR) mutation has a good correlation with the treatment response to an EGFR tyrosine kinase inhibitor (TKI).[4] EGFR expression is strong in NSCLC, and the mutation rate in the Asian population is 30–60%;[5] thus, targeted treatment of an EGFR mutation with TKI can improve the efficacy and survival rate of NSCLC patients.[6] In general, formalin-fixed and paraffin-embedded (FFPE) specimens are used for EGFR detection. However, for patients who have lost the opportunity of surgery or those in whom it is not easy to obtain biopsy specimens, the source of EGFR detection specimens is a problem that needs to be solved. Malignant pleural effusion is a common complication of lung cancer, with an incidence at the time of diagnosis of 8–15%.[7] Pleural effusion occurs in 10–50% of lung cancer patients, especially those with peripheral lung adenocarcinoma.[8] The yield of malignant cells in a malignant effusion sample is approximately 60%;[9] thus, pleural effusion could be a good source of tumor DNA, as even a small amount of soluble DNA in cell-free pleural fluid is sufficient for most molecular analyses.[10] The purpose of this study was to explore the clinical value of EGFR gene mutation detection in pleural effusion cell blocks among NSCLC patients.


   Materials and Methods Top


Patient characteristics

The study included 40 consecutive patients with primary NSCLC and malignant pleural effusion at the time of diagnosis in Jinhua Municipal Central Hospital from July 2016 to September 2018. Of the 40 patients, 20 were male patients and 20 were female patients. The age range was 36–83 years, and the mean age was 65.32 ± 11.83 years. The study protocol was approved by the hospital ethics committee, and all patients were informed of the study and agreed to participate.

Main instruments and reagents

The study used the ABI7500 fluorescent quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA), the NanoDrop 2000 microvolume UV spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the FFPE sample DNA extraction kit and EGFR mutation detection kit (amplification refractory mutation system [ARMS]; Xiamen AmoyDx Biomedical Technology Co., Ltd., Xiamen, China).

Cell block of pleural effusion

Fresh pleural effusion was placed in a 4°C refrigerator, and the supernatant was discarded after precipitation. The precipitate was poured into a 50-mL centrifuge tube and centrifuged for 5 minutes at 2000 rpm, and then, the supernatant was discarded. Thereafter, 6 mL of 95% ethanol was added, and the solution was mixed well to fix the cells. The mixture was kept for 2 hours and centrifuged for 5 minutes at 2000 rpm, and then, the supernatant was discarded. The cells at the bottom of the centrifuge tube were sucked onto microscope paper and wrapped, and then, a cell wax block was made synchronously with the tissue sample.

DNA extraction

Initially, the tumor cell quantity in the section was evaluated. It is better to have more than 200 tumor cells or more than 50% tumor cells in the section.[11] If the tumor cell quantity in the pleural effusion is less, each section requires at least 50 tumor cells or more than 30% tumor cells.[5] Ten paraffin sections with 5-μm thickness were placed into 1.5-mL Eppendorf tubes and 1 mL of xylene was added for dewaxing. Thereafter, DNA was extracted with the FFPE sample DNA extraction kit (Xiamen AmoyDx). The purity of the extracted DNA was measured by using a NanoDrop 2000 microvolume UV spectrophotometer to assess the absorbance ratio of 260 nm to 280 nm (A260/A280). A ratio of 1.8–2.0 was considered appropriate, and the final concentration was adjusted to 2 ng/μL.

Detection of EGFR gene mutation

The EGFR mutation detection kit (arms method; Xiamen AmoyDx) was used, and the specific steps were carried out according to the instructions of the kit.

Statistical analysis

The EGFR mutation rates in pleural effusion and tissue samples were compared using the paired Chi-square test, and the κ value was calculated at the same time. The comparison of the EGFR mutation rate between pleural effusion and tissue samples involved a four-grid Chi-square test, and a P value < 0.05 was considered statistically significant. The statistical analyses were performed using spss 22.0 statistical software (IBM Corp., Armonk, NY, USA).


   Results Top


Comparison of the results of EGFR mutation detection between pleural effusion and tumor tissue samples

We detected 21 EGFR mutations in 40 tissue samples (positive rate of 52.5%) and 18 EGFR mutations in 40 pleural effusion samples (positive rate of 45%). The results of EGFR mutation were compared between the two types of specimens (χ2 = 1.33, P < 0.05, κ = 0.851) [Table 1].The lung adenocarcinoma cells in the pleural effusion cell block are shown in [Figure 1], and the EGFR mutation amplification curve is shown in [Figure 2].
Table 1: Comparison of EGFR mutation detection results between pleural effusion and tissue samples (n=40)

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Figure 1: Lung adenocarcinoma cells in pleural effusion cell block (hematoxylin-eosin staining, 200×)

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Figure 2: The amplification plot of EGFR gene mutation with ARMS method

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Comparison of EGFR mutation types between pleural effusion and tumor tissue samples

The types of EGFR mutations in pleural effusion samples were 19DEL in 8 cases (20%), 20INS in 1 case (2.5%), G719X in 1 case (2.5%), and L858R in 8 cases (20%), with 19DEL and L858R accounting for 88.9% (16/18) of the total mutations. The types of EGFR mutations in tumor tissue samples were 19DEL in 9 cases (22.5%), 20INS in 1 case (2.5%), G719X in 1 case (2.5%), and L858R in 10 cases (25%), with 19DEL and L858R accounting for 90.5% (19/21) of the total mutations. There were no significant differences in the mutation positive rates of EGFR exons 18, 19, 20, and 21 between the two types of samples [Table 2].
Table 2: Comparison of EGFR mutation types between pleural effusion and tumor tissue samples

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   Discussion Top


The use of molecular agents targeting EGFR is important in the treatment of advanced NSCLC. The validity of EGFR-TKI-targeted therapy is related to the presence of functional EGFR mutations in NSCLC. Therefore, detection of EGFR mutations is essential to predict whether patients can benefit from EGFR-TKI-targeted therapy.[12] Most EGFR mutations in NSCLC patients are detected with FFPE. However, for some advanced patients in whom FFPE samples cannot be obtained by operation or biopsy, it is urgently needed to find alternative samples for EGFR detection.

At present, pleural fluid and blood are important specimens for EGFR detection, instead of tissues.[13],[14],[15] Compared with blood specimens, pleural effusion specimens have more tumor cells and a higher positive rate. Our study showed that there was no significant difference in the EGFR positive rate between pleural effusion specimens and FFPE specimens among 40 NSCLC patients. The κ value was 0.851, indicating good consistency between the two types of specimens.

Pleural effusion is a kind of body fluid sample that is easy to obtain. It is associated with low cost, easy operation, less damage, and less complications. Traditional pleural effusion cytology cannot preserve samples for a long time or provide more effective information. The diagnosis level stays at “tumor cells found” or “tumor cells not found.” Compared with traditional smear, the most important advantage of a cell block is that it can provide multiple sections, which are suitable for further immunohistochemistry and molecular research.[14] The production process of a cell block can not only enrich tumor cells,[16] but also help in the evaluation of the tumor cell quantity in the pleural effusion specimen, which is conducive to the quality control of EGFR detection. In addition, pleural effusion can collect samples for dynamic monitoring of EGFR mutations in multiple periods, which is of great value for the detection of T790M resistant mutations caused by targeted drug therapy.

The number of cancer cells in pleural effusion is sometimes small, so gene detection in pleural effusion has a high requirement regarding sensitivity of the method. We used the ARMS method to detect EGFR mutations. This method has the advantages of high sensitivity, speed, and accuracy. The detection process is carried out in a closed reaction tube, which effectively prevents contamination, and the reaction process can be monitored in real time. This method can detect 29 mutation types in EGFR exons 18, 19, 20, and 21. The results showed that the most common EGFR mutations in NSCLC were L858R mutations in exon 21 and 19DEL mutations in exon 19. The proportions of these two mutations in pleural effusion and tumor tissue samples were 88.9% and 90.5%, respectively, which were similar to the results reported in the literature.[17] In this study, EGFR mutations were positive in three tissue samples, without corresponding mutations in pleural effusion samples, and this may be related to tumor heterogeneity. Malignant pleural effusion can involve pleural metastasis of a malignant tumor or pleural effusion caused by a malignant tumor of the pleura itself. Thus, there may be tumor heterogeneity between the metastasis and primary focus.[18]

In summary, a pleural effusion cell block has the advantages of reliable results, easy accessibility, and dynamic monitoring in multiple periods. It has important clinical value for the detection of EGFR among NSCLC patients in whom tumor tissue specimens cannot be obtained.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69:7-34.  Back to cited text no. 1
    
2.
Novello S, Barlesi F, Califano R, Cufer T, Ekman S, Levra MG, et al. Metastatic non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2016;27:v1-27.  Back to cited text no. 2
    
3.
Magnini D, Fuso L, Varone F, D'Argento E, Martini M, Pecoriello A, et al. Molecular testing in EBUS-TBNA specimens of lung adenocarcinoma: A study of concordance between cell block method and liquid-based cytology in appraising sample cellularity and EGFR mutations. Mol Diagn Ther 2018;22:723-8.  Back to cited text no. 3
    
4.
Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 2004;304:1497-500.  Back to cited text no. 4
    
5.
Brachtel EF, Iafrate AJ, Mark EJ, Deshpande V. Cytomorphological correlates of epidermal growth factor receptor mutations in lung carcinoma. Diagn Cytopathol 2007;35:257-62.  Back to cited text no. 5
    
6.
Kogure Y, Shigematsu F, Oki M, Saka H. T790M Correlates with longer progression-free survival in non-small cell lung carcinomas harboring EGFR mutations. In Vivo 2018;32:1199-204.  Back to cited text no. 6
    
7.
Antony VB, Loddenkemper R, Astoul P, Boutin C, Goldstraw P, Hott J, et al. Management of malignant pleural effusions. Eur Respir J 2001;18:402-19.  Back to cited text no. 7
    
8.
Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947-57.  Back to cited text no. 8
    
9.
Maskell NA, Butland RJ; Pleural Diseases Group, Standards of Care Committee, British Thoracic Society. BTS guidelines for the investigation of a unilateral pleural effusion in adults. Thorax 2003;58:ii8-17.  Back to cited text no. 9
    
10.
Kawahara A, Azuma K, Sumi A, Taira T, Nakashima K, Aikawa E, et al. Identification of non-small-cell lung cancer with activating EGFR mutations in malignant effusion and cerebrospinal fluid: Rapid and sensitive detection of exon 19 deletion E746-A750 and exon 21 L858R mutation by immunocytochemistry. Lung Cancer 2011;74:35-40.  Back to cited text no. 10
    
11.
Chinese Expert Group for Epidermal Growth Factor Receptor Gene Mutation Detection in Non-Small Cell Lung Carcinoma. Consensus on epidermal growth factor receptor gene mutation detection in non-small cell lung carcinoma. Zhonghua Bing Li Xue Za Zhi 2016;45:217-20.  Back to cited text no. 11
    
12.
Choi YW, Choi JH. Does the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor differ according to the type of EGFR mutation in non-small cell lung cancer? Korean J Intern Med 2017;32:422-8.  Back to cited text no. 12
    
13.
Jeon SH, Kim HW, Kim BN, Kang N, Yeo CD, Park CK, et al. Comparison of PNA clamping-assisted fluorescence melting curve analysis and PNA clamping in detecting EGFR mutations in matched tumor tissue, cell block, pleural effusion and blood of lung cancer patients with malignant pleural effusion. In Vivo 2019;33:595-603.  Back to cited text no. 13
    
14.
Gupta V, Shukla S, Husain N, Kant S, Garg R. A comparative study of cell block versus biopsy for detection of epidermal growth factor receptor mutations and anaplastic lymphoma kinase rearrangement in adenocarcinoma lung. J Cytol 2019;36:13-7.  Back to cited text no. 14
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15.
Asaka S, Yoshizawa A, Nakata R, Negishi T, Yamamoto H, Shiina T, et al. Utility of bronchial lavage fluids for epithelial growth factor receptor mutation assay in lung cancer patients: Comparison between cell pellets, cell blocks and matching tissue specimens. Oncol Lett 2018;15:1469-74.  Back to cited text no. 15
    
16.
Wang Y, Liu Z, Yin H, Hu J, Zhong S, Chen W, et al. Improved detection of EGFR mutations in the tumor cells enriched from the malignant pleural effusion of non-small cell lung cancer patient. Gene 2018;644:87-92.  Back to cited text no. 16
    
17.
Xu H, Sun W, Zhang G, Cheng Y. Detection of epidermal growth factor receptor mutation in non-small-cell lung carcinoma using cytological and histological specimens. J BUON 2015;20:142-5.  Back to cited text no. 17
    
18.
Morgillo F, Della Corte CM, Fasano M, Ciardiello F. Mechanisms of resistance to EGFR-targeted drugs: Lung cancer. ESMO Open 2016;1:e000060.  Back to cited text no. 18
    

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Correspondence Address:
Fang Zhu
Department of Pathology, Jinhua Municipal Central Hospital, NO.365 Renmin East Road, Jinhua, Zhejiang - 321 000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_360_20

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