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Chapter 5: Reverse-Transcriptase Polymerase Chain Reaction and Multiple Gene Assays
and mutations in tumor tissue specimens, including FFPE samples (Lipson 2012, Takeuchi 2012). In one study, NGS of genomic DNA, involving breakpoints in at least five different genomic loci, detected a complex ALK rearrangement in a sample that was ALK-negative on break-apart FISH (Peled 2012). Experience is limited with determining ALK mutations and gene copy number variations in clinical lung tumor tissue specimens. With use of DNA extracted from FFPE samples of lung tumor tissues, a multiplexed NGS assay detected ALK gene mutations associated with resistance to crizotinib treatment in three of 13 patients with lung cancer (Huang 2013).
Conclusion
RT-PCR to detect EML4-ALK should be designed as comprehensively as possible, with the targets narrowed to the three major variants that account for more than 90% of lung cancers with EML4ALK. RT-PCR may be an adequate tool for confirming the results of IHC and FISH analyses, but is less appropriate for primary screening for ALK rearrangment, especially when the amount of tissue sample is adequate. Novel multiplexed genomic assays, particularly NGS, represent promising clinically applicable methodologies for the detection of ALK fusions, as well as other gene abnormalities, including mutations, copy number gains, and gene expression. However, published data on these assays are limited.