Abstract 2362: Detection of alk rearrangement in advanced non-small cell lung cancer using fluorescent in situ hybridization,immunohistochemistry,reverse transcription-polymerase chain reaction.
Authors: Shi Yuan-kai , Han Xiao-hong , Zhang Ning-ning , Liu Yu-tao , Hao Xue-zhi , Ma Li , Wang Lin , li Dan , Tang Le , Yang Sheng , Lin Hua 
Published: August 2014

Background:ALK (anaplastic lymphoma kinase)gene fusions are detected in 3% to 13% of non-smallcell lung cancer(NSCLC). The ALK FISH assay is the current gold standard assay, but it can be technically challenging and costly.Therefore,other easy-to-use diagnostic modalities are needed to select the suitable patients for the target therapy. We investigated ALK fusions using immunohistochemistry (IHC), reverse transcription-polymerase chain reaction (RT-PCR), and fluorescent in situ hybridization (FISH) in order to compare the three methodologies for detection of ALK fusions.

Methods:139 tumor samples with advanced NSCLC were collected from Cancer Institute/Hospital of Chinese Academy of Medical Sciences. All of these patients who previously received cell cytotoxic regimens therapy had progressive disease. ALK protein was detected by IHC using new clone antibody (D5F3, Cell Signal Techonology, USA). ALK gene rearrangement was evaluated by FISH using Vysis ALK Break Apart FISH Probe kit (Abbott, USA) and by RT-PCR detecting 9 EML4-ALK fusion variants with formalin-fixed paraffin-embedded tissue.

Results:132 samples (95.0%)can be evaluated by FISH,137 samples(98.6%) can be evaluated by IHC and 122samples (87.8%)can be evaluated by RT-PCR in 139 cases.ALK positive samples were34.3% (47/137) by IHC, and ALK rearrangement samples were 32.6% (43/132) by FISH.EML4-ALK variants were detectable by RT-PCR in 32.4% (34 of 122) of specimens,and the variants 1 and variants 3 were the most common variant type.The concordance rate is 93.8% (kappa = 0864,p &lt; 0.001) in the 130 paired samplesof FISH and IHC.75 of 76(98.7%)IHC 0 samples was FISH-,34 of 39 (87.2%) IHC 3+ samples were FISH +, 7 of 8 (87.5%) IHC 2+ samples were FISH +, whereas 1 of 7 (14.3%) IHC 1+ samples was FISH +. Considering FISH as the gold standard, the sensitivity and specificity of IHC were 95.3% and 93.1%, respectively, when 2+ and 3+ were regarded as IHC positive and 0 and 1+ as IHC negative.The concordance rate is 95.7% (kappa = 0.898,p &lt; 0.001) in the 115 paired samples of FISH and RT-PCR . There are 4 negative cases by RT-PCR with ALK positive by FISH,and 1 positive case by RT-PCR with ALK negative by FISH. The sensitivity and specificity of RT-PCR were 89.2% and 98.7% respectively.But in the 120 paired samples of IHC and RT-PCRthe concordance rate is 90.8% (kappa = 0.788,p &lt; 0.001),lower than IHC or RT-PCR with FISH.

Conclusion:IHC can be the next frontier of molecular diagnostics to evaluate ALK fusions in NSCLC screening for ALK targeted therapy.RT-PCR showed relatively lower sensitivity and higher specificity for ALK rearrangement,it can be an alternative diagnostic assay for ALK detection. FISH was the most sensitive method for different ALK fusions.

Citation Format: Yuan-kai Shi, Xiao-hong Han, Ning-ning Zhang, Yu-tao Liu, Xue-zhi Hao, Li Ma, Lin Wang, Dan li, Le Tang, Sheng Yang, Hua Lin. Detection of alk rearrangement in advanced non-small cell lung cancer using fluorescent in situ hybridization,immunohistochemistry,reverse transcription-polymerase chain reaction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2362. doi:10.1158/1538-7445.AM2013-2362