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Chapter 5: Reverse-Transcriptase Polymerase Chain Reaction and Multiple Gene Assays
ALK EML4-ALK (E13;A20), v1 EML4-ALK (E20;A20), v2 EML4-ALK (E6;A20), v3 EML4-ALK (E14;del49A20), v4 EML4-ALK (E2;A20), v5 EML4-ALK (E13;ins69A20), v6 EML4-ALK (E14;del14A20), v7 EML4-ALK (E15del19;del20A20), v4' EML4-ALK (E18;A20), v5' KIF5B-ALK (K24;A20) KIF5B-ALK (K15;A20) KIF5B-ALK (K17;A20) KLC1-ALK (K9;A20)
e13 e13 e20 e6
e20 e20 e20
e20 e14 e2 del49e20
e20
ins69e20 e14 e15del19 del14e20
e18 del20e20 e24 e15 e17 e20 e20 e20
e20 e9 e20
Figure 1. Wild-type ALK and various types of ALK fusions in lung cancer: five regular variants (v1, v2, v3, v5, and v5') and four irregular variants (v4, v6, v7, and v4') of EML4-ALK, three variants of KIF5B-ALK, and KLC1-ALK. Darker regions in the fusion partner genes (EML4, KIF5B, and KLC1) represent coiled-coil domains, and those in ALK represent the transmembrane domain (orange) and the kinase domain (red).
an ALK inhibitor. From a different perspective, therapeutic efficacy with ALK inhibitors may vary according to fusion partners and/or fusion variants, as has been suggested by studies showing that L858R point mutation and EGFR exon 19 deletion affect transformation activity in vitro and clinical efficacy of treatment with EGFR TKIs. If such differences are found to be clinically relevant in ALKpositive lung cancer, RT-PCR may be beneficial. RT-PCR enables and is best suited for the examination of specimens that are not amenable to tissue blocks, such as bronchial washing fluid, sputum, blood, body cavity effusion, and other body fluids (Soda 2012). For patients with confirmed ALK rearrangement, RT-PCR using cell-free RNA or circulating tumor cells in fluid samples is a powerful and minimally invasive tool to monitor disease progression. However, the presence of tumor cells (or tumor-derived RNA) in fluid samples is often difficult to confirm, which increases the risk that specimens with no tumor cells will be misdiagnosed as negative for ALK rearrangement. In principle, if fluid samples are used for primary screening, only samples confirmed to be positive for cancer cells should be examined, but the high sensitivity of RT-PCR is then no longer advantageous.
Comparative Quantitative RT-PCR
IHC can be used to detect ALK rearrangements because ALK is not expressed in normal lung tissues;