Abstract 4656: The novel mRNA in situ hybridization method for the detection of ALK, RET, ROS1 and NTRK1 mRNA in non-small cell lung cancer
Authors: Hirai Noriko , Sasaki Takaaki , Minami Yoshinori , Ohsaki Yoshinobu 
Published: May 2015

Abstract
[Background]In recent years, tumor genotyping is critical for making decisions of non-small cell lung cancer (NSCLC) treatment. However genetic rearrangement of ALK, RET, ROS1 and NTRK1 were identified in the subset of NSCLC patients, these clinical characteristics were still remained to be clear. Immunohistochemistry (IHC) is widely used as a simple and quick method for screening for protein expression of these oncogenes. Fluorescence in situ hybridization (FISH) or RT-PCR are performed consecutively to confirm the genotyping. However it is often taking time and expensive and it is needed to consolidate novel substitute methods.
[Purpose]We performed the in situ hybridization (ISH) analysis to detect cellular mRNA in formalin-fixed paraffin-embedded (FFPE) tissue samples of lung cancer. We also conducted IHC with the use of ALK, RET, ROS1 and NTRK1 specific antibodies and investigate a comparative study to assess the usefulness of mRNA-ISH to detect expression of these mRNA.
[Materials and Methods]We made tissue micro array (TMA) slides using 40 FFPE samples of resected lung adenocarcinoma with known epidermal growth factor receptor (EGFR) mutation status by PCR (PNA-LNA PCR clamp method). The mRNA in situ hybridization was employed using RNA scope R 2.0 Regant Kit with targeting probes. IHC staining was conducted by antibodies using 5A4 and D5F3 for ALK, RET01 for RET, D4D6 for ROS1, and 14G6 for NTRK1.
[Results]All of the IHC stained cases were clearly detected by mRNA-ISH respectively. There were some cases which were only visualized by ISH, each case was partially positive and not totally of the tumor.
ALK: 5% of cases (2 out of 40) were detected in mRNA-ISH and were strongly stained with both 5A4 and D5F3. RET: 20% of cases (8/40) were detected in mRNA-ISH. Two cases (5% of total) had positive RET01 staining, while 6 cases had negative staining. (3 had no other genetic alterations, 2 had EGFR-L858R confirmed by PCR, and 1 had ALK by IHC and FISH.) ROS1: 20% (8/40) were detected in mRNA-ISH. Three cases (7.5% of total) presented positive D4D6 staining, while 5 had negative staining. (3 had no other alterations, 1 had EGFR-E746-A750 and 1 had EGFR-L858R by PCR.) NTRK1: 17.5% of cases (7/40) were detected in mRNA-ISH. 1 case (2.5% of total) presented positive 14G6 staining, while 6 cases had negative staining. (2 had no other alterations, 2 had EGFR-L858R, 1 had EGFR-E746-A750, and 1 had ALK.)
[Conclusion] The novel mRNA in situ hybridization method combined with traditional IHC could improve detection rate of ALK, RET, ROS1 and NTRK1 gene alterations in NSCLC FFPE samples.
Citation Format: Noriko Hirai, Takaaki Sasaki, Yoshinori Minami, Yoshinobu Ohsaki. The novel mRNA in situ hybridization method for the detection of ALK, RET, ROS1 and NTRK1 mRNA in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2014-4656