Comparison of EML4-ALK fusion gene positive rate in different detection methods and samples of non-small cell lung cancer

Abstract

Objective: To evaluate differences of EML4-ALK positive rates in tissues samples between immunohistochemistry, reverse transcriptase polymerase chain reaction and the next-generation sequencing method. Besides, to compare the differences of EML4-ALK positive rates in blood samples and tissue samples by next-generation sequencing. The results provide a basis for the selection of a suitable EML4-ALK fusion gene detection method. Methods: Immunohistochemistry analysis of EML4-ALK in tumors was performed on samples from 2631 patients with non-small cell lung cancer. The mutation of EML4-ALK in the tissue samples of 399 patients with non-small cell lung cancer was detected by reverse transcription polymerase chain reaction. Next-generation sequencing was used to detect the mutation of EML4-ALK in 1505 non-small cell lung cancer patients, including 1208 tissue samples and 297 blood samples. Results: The positive incidence of EML4-ALK by immunohistochemistry was 7.11% (187/2631). Histologically, 9.51% (170/1787) of the samples were lung adenocarcinomas, and 2.01% (17/844) were squamous cell carcinomas. The positive rate of EML4-ALK was 8.52% (34/399) in 399 patients with non-small cell lung cancer, as detected by reverse transcription polymerase chain reaction; the mutation rate of adenocarcinoma was 11.62% (33/284), and the mutation rate of squamous cell carcinoma was 0.86% (1/115). In 1208 patients with non-small cell lung cancer with tissue samples, the positive rate of EML4-ALK was 4.88% (59/1208), as determined by next-generation sequencing, the mutation rate of adenocarcinoma was 5.84% (58/994), and the mutation rate of squamous cell carcinoma was 0.47% (1/214). The positive rate of EML4-ALK detected by reverse transcription polymerase chain reaction was higher than that detected by immunohistochemistry. Compared with the next-generation sequencing results, the positive rates of EML4-ALK detected by immunohistochemistry and reverse transcription polymerase chain reaction were higher, and the differences were significant (p<0.05). In blood samples from 297 patients with non-small cell lung cancer, the positive rate of EML4-ALK detected by next-generation sequencing was 3.70% (11/297), the mutation rate of adenocarcinoma was 3.82% (10/262), and the mutation rate of squamous cell carcinoma was 2.86% (1/35). The EML4-ALK positive rate of the tissue samples was thus higher than that of the blood biopsy samples. Conclusion: Among the three methods for detecting EML4-ALK, reverse transcription polymerase chain reaction has the highest positive rate, followed by immunohistochemistry, and next-generation sequencing has the lowest positive rate. The positive detection rate of EML4-ALK in tissue samples by next-generation sequencing was higher than that in blood samples.

Keywords: EML4-ALK fusion gene; immunohistochemistry; next-generation sequencing; non-small cell lung cancer; reverse transcription-polymerase chain reaction.

Figure 1

Examples of the immunohistochemical analysis of EML4-ALK in tissue samples. A, B, and C show the negative control, positive control and an EML4-ALK (+) case (strong granular cytoplasmic staining) (×100), respectively; D, E, and F show the negative control, positive control and an EML4-ALK (+) case (strong granular cytoplasmic staining) (×400), respectively.

Figure 2

EML4-ALK in tissue samples were detected by RT-PCR (ARMS method).