Abstract 1904: RNA expression based screening of ALK gene fusion from formalin fixed non-small cell lung cancer samples
Authors: Maddula Krishna , Joshi Mary-Beth , Modur Vijay , Harpole David H. 
Published: May 2015

Abstract
The ALK gene fusion results in the constitutive activation of the oncogenic ALK kinase activity through the expression of 3`ALK gene downstream of fusion partners like EML4 and KIF5B. Due to this rearrangement, fusion mRNA show high expression of 3` regions compared to 5` region of ALK gene and can be used as an indicator for presence of gene fusion. However, due to the low intrinsic expression of normal ALK mRNA, detection of the 5′ region of the ALK mRNA requires a highly sensitive assay system. To achieve this high sensitivity, we adapted the quantitative nuclease protection assay to combine biotinylated tiled probes for 3′ and 5′ ALK mRNA with the use of streptavidin conjugated polymeric horseradish peroxidase for detection. This assay can robustly detect baseline levels of ALK mRNA from normal and ALK-negative NSCLC specimen. We then tested the ability of the assay to measure the over expression of 3′ ALK gene by spiking in 3′ and 5′ in vitro transcribed (IVT) ALK RNAs into normal lung samples. Signal over baseline was detected for ALK5` and ALK3` IVT at attomolar concentrations even in the presence of a ratio of 95:5 in the background of normal lung tissue. Known ALK fusion cell lines also showed exquisite sensitivity for detection of fusion by comparing 5′ and 3′ expression ratios with sample input as few as 65 cells per reaction. We screened 293 (222 Adenocarcinoma, 47 Squamous cell carcinoma and 24 other NSCLC subtypes) surgically resected NSCLC FFPE clinical samples from multiple sources for presence of EML4-ALK fusions. Endogenous ALK mRNA expression was detectable in all samples with 12 (4.09 %) samples showing ALK fusion based on aberrant overexpression of 3′ region compared with 5′ region. All the 12 samples were of the adenocarcinoma or large cell subtype consistent with reported ALK fusion epidemiology (table). Confirmation studies with positives show greater sensitivity of detection of ALK fusion than is possible currently by FISH.
ALK fusion positive samples epidemiologyALK FUSION POSITIVE SAMPLE IDNSCLC SUBTYPEP63 IHCTTF-1 IHCRACESEXAGEHISTOLOGY041535T2(1)ADENO-+CaucasianFemale69NA042210T2ADENO++CaucasianMale61Poorly differentiatedB610155ADENO++AsianFemale35Undifferentiated carcinomaCU2010/22 15825ADENO++CaucasianNA68Adenosquamous carcinomaCU2010/22 16787ADENO++CaucasianMale71Poorly differentiatedCU2010/22 20035ADENO-+CaucasianMale64Adenosquamous carcinoma with clear cell- and signet cell-like structuresCU2010/22 8119ADENO++CaucasianFemale53NACU2010/24 259313ADENO++CaucasianFemale50NARln020161ADENONANAAsianMale27Mucinous branchioalveolar carcinomaRln020207ADENONANAAsianMale59Mucinous branchioalveolar carcinomaRln060392Large CellNANAAsianFemale45Undifferentiated large cellRln060453Large Cell--AsianMale66Poorly differentiated
Citation Format: Krishna Maddula, Mary-Beth Joshi, Vijay Modur, David H. Harpole. RNA expression based screening of ALK gene fusion from formalin fixed non-small cell lung cancer samples. [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 1904. doi:10.1158/1538-7445.AM2014-1904