Expression of C-terminal ALK, RET, or ROS1 in lung cancer cells with or without fusion

Abstract

Background: Genetic alterations, including mutation of epidermal growth factor receptor or v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog and fusion of anaplastic lymphoma kinase (ALK), RET proto-oncogene (RET), or v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1), occur in non-small cell lung cancers, and these oncogenic drivers are important biomarkers for targeted therapies. A useful technique to screen for these fusions is the detection of native carboxy-terminal (C-terminal) protein by immunohistochemistry; however, the effects of other genetic alterations on C-terminal expression is not fully understood. In this study, we evaluated whether C-terminal expression is specifically elevated by fusion with or without typical genetic alterations of lung cancer.

Methods: In 37 human lung cancer cell lines and four tissue specimens, protein and mRNA levels were measured by capillary western blotting and reverse transcription-PCR, respectively.

Results: Compared with the median of all 37 cell lines, mRNA levels at the C-terminus of all five of the fusion-positive cell lines tested (three ALK, one RET, and one ROS1) were elevated at least 2000-, 300-, or 2000-fold, respectively, and high C-terminal protein expression was detected. In an ALK fusion-positive tissue specimen, the mRNA and protein levels of C-terminal ALK were also markedly elevated. Meanwhile, in one of 36 RET fusion-negative cell lines, RET mRNA levels at the C-terminus were elevated at least 500-fold compared with the median of all 37 cell lines, and high C-terminal protein expression was detected despite the absence of RET fusion.

Conclusions: This study of 37 cell lines and four tissue specimens shows the detection of C-terminal ALK or ROS1 proteins could be a comprehensive method to determine ALK or ROS1 fusion, whereas not only the detection of C-terminal RET protein but also other methods would be needed to determine RET fusion.

Keywords: ALK; Alectinib; EML4; Fusion; Immunohistochemistry; Lung cancer; RET; ROS1; Rearrangement.

Fig. 1

RT-PCR analysis of ALK, RET, or ROS1 mRNA in 37 cancer cell lines. The mRNA expression of ALK (a), RET (b), or ROS1 (c) was determined by RT-PCR using the aqman probes (Additional file 2: Tables S2). Exons of each probe are shown on the X-axis. The relative mRNA expression was calculated as the ratio of the normalized value with GAPDH mRNA to the median of that in 37 cell lines

Fig. 2

Western blotting analysis in 37 lung cancer cell lines. The protein expression of ALK, RET, ROS1, KIF5B, or CCDC6 and phosphorylation of ALK, RET, or ROS1 in 37 cell lines was determined by western blotting using the antibodies (Additional file 3: Tables S3). Both actin and COX4 were used as loading controls

Fig. 3

RT-PCR and western blotting analysis in four tissue specimens. The mRNA expression of ALK in the four tumor tissue specimens shown in Table 2 was determined by RT-PCR using the Taqman probes (Additional file 2: Tables S2). Exons of each probe are shown on the X-axis. The relative mRNA expression was calculated as the ratio of the normalized value with GAPDH mRNA to the median of that in the four tissue specimens (a). The protein expression of ALK, RET, ROS1, KIF5B, or CCDC6 and phosphorylation of ALK, RET, or ROS1 in the tumor tissue specimens were determined by western blotting using the antibodies (Additional file 3: Tables S3) (b). The relative EML4 mRNA expression at exon 1/2 in NAT specimens was calculated as the ratio of the normalized values with GAPDH mRNA to that in tumor tissue specimens (c). Each bar represents the mean + SD (n = 3)