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Chapter 6: Comparison of Different Assay Platforms for ALK Testing
ALK IHC, if carefully validated, may be considered for screening patients with lung adenocarcinoma (Lindeman 2013). RT-PCR is a highly specific and reliable technique, as it allows for the precise identification of the 5’ partners and breakpoint variants. But atypical ALK variants or fusion partners (such as an irregular variant with insertion or deletion) may be undetected. RT-PCR of EML4-ALK is highly sensitive because the primers will not amplify a product in normal cells, but intact mRNA is poorly preserved in an archived FFPE specimen. The cost of RT-PCR is still being evaluated and depends on the laboratory and the test used. CISH is a new method for detection of ALK rearrangement, with the aim of overcoming some of the disadvantages of FISH (Kim 2011). CISH is a fully automated ISH assay, which provides stable staining and allows detection of ALK gene rearrangement using conventional bright-field light microscopy.
IHC versus FISH
Several studies have compared IHC with FISH. A variety of antibodies have been used for IHC, including two mouse monoclonal antibodies clones, ALK1 (Dako) and 5A4 (Novocastra); a rabbit monoclonal antibody clone D5F3 (Cell Signaling Technology); and a rabbit polyclonal antibody (Invitrogen, Life Technologies). In most studies, FISH has been performed with an ALK break-apart rearrangement probe kit (Abbott Molecular), which has been approved by the US FDA as the companion diagnostic for ALK testing to determine patient eligibility for treatment with crizotinib. ALK1 IHC versus FISH Initially used for diagnosing anaplastic large cell lymphoma, the ALK1 antibody has also been evaluated for the detection of ALK rearrangement in NSCLC. ALK1 IHC and FISH were compared in three large series of adenocarcinoma of the lung or NSCLC (Table 1) (Rodig 2009, Mino-Kenudson 2010, Yi 2011). Compared with FISH, ALK1 IHC seems less sensitive for detecting ALK-rearranged lung cancer than for detecting anaplastic large cell lymphoma, possibly because the ALK fusion protein is expressed at lower levels in NSCLC. ALK1 offers good specificity, but sensitivity has ranged from 67% to 100%. Some specimens have tested positively for ALK on FISH but negatively for ALK on IHC, but no specimens have tested positively on IHC and negatively on FISH (Rodig 2009, Mino-Kenudson
Table 1. Staining Features of IHC with ALK1 Antibody
Study Rodig et al., 2009 MinoKenudson et al., 2010 Yi et al., 2011 No. of ALK1 Specimens Dilution 358 1:2 Antigen Retrieval EDTA (pH 8.0) in pressure cooker EDTA (pH 8.0) in pressure cooker Detection and Amplification System Tyramide amplification and EnVision EnVision Scoring 0 vs. + IHC Positive Threshold IHC Sensitivity (vs. FISH) IHC Specificity (vs. FISH) 100%
>10% 80% with positive amplification tumor cells (40% without) >10% positive tumor cells >0 67%
153
1:2
0, 1+, 2+, or 3+ and % of tumor cells stained 0, 1+, 2+, or 3+
97%
101
1:100
EDTA (pH 8.0) in PT Link
ADVANCE
90%
97.8%
PT Link, EnVision, and ADVANCE are products of Dako.