The use of quantitative real-time reverse transcriptase PCR for 5' and 3' portions of ALK transcripts to detect ALK rearrangements in lung cancers.
OBJECTIVE
Approximately 3% to 7% of non-small cell lung cancers (NSCLC) harbor an ALK fusion gene, thus defining a tumor group that may be responsive to targeted therapy. The breakpoint in ALK consistently occurs at exon 20 and EML4 or other fusion partners, thus driving a strong expression of ALK kinase domain and resulting in an unbalanced expression in 5' and 3' portions of ALK transcripts. We have developed a rapid and accurate method by simultaneously detecting the expression in 5' and 3' portions of ALK mRNA.


METHODS
Quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to examine expression levels of the 5' and 3' portions of ALK transcripts in177 NSCLCs, in which EGFR, KRAS, HER2, and BRAF mutations were absent. If unbalanced ALK mRNA expression was seen, ALK rearrangement was assumed to exist. ALK FISH was used to confirm the accuracy of qRT-PCR. RT-PCR and 5' RACE coupling sequencing identified the fusion variants.


RESULTS
Real-time RT-PCR showed excellent sensitivity and specificity (100% and 100%, respectively) for detection of ALK rearrangements in resected specimens. In addition, six novel ALK fusion variants were identified, including one KIF5B-ALK (E17;A20) and five EML4-ALK variants (E6a;A19, E6a/b ins 18;A20, E17b ins 39;A20, E10a/b, E13;A20, and E17 ins 65;A20).


CONCLUSIONS
Real-time RT-PCR is a rapid and accurate method for diagnosing ALK-rearranged lung cancers. Coupling of 5' RACE to this method should further facilitate rapid identification of novel ALK fusion genes.
Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, 270 Dong-An Road, Shanghai 200032, China.
Rui
R
Wang
Yunjian
Y
Pan
Chenguang
C
Li
Haichuan
H
Hu
Yang
Y
Zhang
Hang
H
Li
Xiaoyang
X
Luo
Jie
J
Zhang
Zhaoyuan
Z
Fang
Yuan
Y
Li
Lei
L
Shen
Hongbin
H
Ji
David
D
Garfield
Yihua
Y
Sun
Haiquan
H
Chen
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000077548:Anaplastic Lymphoma Kinase; D005091:Exons; D006801:Humans; D016547:Kinesins / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; C522504:EML4-ALK fusion protein, human; C488101:KIF5B protein, human; D015514:Oncogene Proteins, Fusion; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases; D016547:Kinesins