Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.
BACKGROUND
Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain.


METHODS
Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed.


RESULTS
Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian.


CONCLUSIONS
Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.
Department of CardioVascular and Thoracic Surgery, Chang Gung Memorial Hospital at Linko, and Chang Gung University College of Medicine, Taoyuan, Taiwan.
Yi-Cheng
YC
Wu
Il-Chi
IC
Chang
Chi-Liang
CL
Wang
Tai-Di
TD
Chen
Ya-Ting
YT
Chen
Hui-Ping
HP
Liu
Yen
Y
Chu
Yu-Ting
YT
Chiu
Tzu-Hua
TH
Wu
Li-Hui
LH
Chou
Yi-Rong
YR
Chen
Shiu-Feng
SF
Huang
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000077548:Anaplastic Lymphoma Kinase; D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D066246:ErbB Receptors / Q000235:genetics; D005260:Female; D015321:Gene Rearrangement; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016547:Kinesins / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D008297:Male; D015201:Meta-Analysis as Topic; D008875:Middle Aged; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013624:Taiwan; D055815:Young Adult; D018631:ras Proteins / Q000235:genetics
C522504:EML4-ALK fusion protein, human; C488101:KIF5B protein, human; C117307:KRAS protein, human; D015514:Oncogene Proteins, Fusion; D011518:Proto-Oncogene Proteins; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; C512478:EGFR protein, human; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D016547:Kinesins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins