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 epidemiology 
ALK FUSION POSITIVE SAMPLE ID NSCLC SUBTYPE P63 IHC TTF-1 IHC RACE SEX AGE HISTOLOGY 
041535T2(1) ADENO Caucasian Female 69 NA 
042210T2 ADENO Caucasian Male 61 Poorly differentiated 
B610155 ADENO Asian Female 35 Undifferentiated carcinoma 
CU2010/22 15825 ADENO Caucasian NA 68 Adenosquamous carcinoma 
CU2010/22 16787 ADENO Caucasian Male 71 Poorly differentiated 
CU2010/22 20035 ADENO Caucasian Male 64 Adenosquamous carcinoma with clear cell- and signet cell-like structures 
CU2010/22 8119 ADENO Caucasian Female 53 NA 
CU2010/24 259313 ADENO Caucasian Female 50 NA 
Rln020161 ADENO NA NA Asian Male 27 Mucinous branchioalveolar carcinoma 
Rln020207 ADENO NA NA Asian Male 59 Mucinous branchioalveolar carcinoma 
Rln060392 Large Cell NA NA Asian Female 45 Undifferentiated large cell 
Rln060453 Large Cell Asian Male 66 Poorly differentiated 
ALK fusion positive samples epidemiology 
ALK FUSION POSITIVE SAMPLE ID NSCLC SUBTYPE P63 IHC TTF-1 IHC RACE SEX AGE HISTOLOGY 
041535T2(1) ADENO Caucasian Female 69 NA 
042210T2 ADENO Caucasian Male 61 Poorly differentiated 
B610155 ADENO Asian Female 35 Undifferentiated carcinoma 
CU2010/22 15825 ADENO Caucasian NA 68 Adenosquamous carcinoma 
CU2010/22 16787 ADENO Caucasian Male 71 Poorly differentiated 
CU2010/22 20035 ADENO Caucasian Male 64 Adenosquamous carcinoma with clear cell- and signet cell-like structures 
CU2010/22 8119 ADENO Caucasian Female 53 NA 
CU2010/24 259313 ADENO Caucasian Female 50 NA 
Rln020161 ADENO NA NA Asian Male 27 Mucinous branchioalveolar carcinoma 
Rln020207 ADENO NA NA Asian Male 59 Mucinous branchioalveolar carcinoma 
Rln060392 Large Cell NA NA Asian Female 45 Undifferentiated large cell 
Rln060453 Large Cell Asian Male 66 Poorly 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