47
Chapter 6: Comparison of Different Assay Platforms for ALK Testing
Table 3. Staining Features of IHC with 5A4 Antibody
Study Jokoji et al., 2010 No. of 5A4 Specimens Dilutiona 254 1:100 Antigen Retrieval Target Retrieval Solution, high pH CC1 solution Detection and Amplification System EnVision FLEX+ and iAEP i-VIEW Scoring 0 vs. + IHC Positive Threshold No score IHC Sensitivity (vs. FISH) 100%
(FISH done on IHC+ specimens only)
IHC Specificity (vs. FISH) 100%
Paik et al. 2011
640
1:30
0, 1+, 2+, or 3+
0, or 1+: negative 2+: equivocal 3+: positive
100%
96%
Kim et al., 2011
465
1:30
CC1 solution
i-VIEW
0, 1+, 2+, or 3+
0, or 1+: 100% negative when score of 2+: equivocal 2+ considered 3+: positive positive >10% positive tumor cells >10% positive tumor cells 1+, 2+ 95%
98%
McLeerFlorin et al., 2012
Lopes et al., 2012 Sholl et al., 2013
a
441
1:50
CC1 solution
ultraView
0, 1+, 2+, or 3+ and % of positive tumor cells No score
100%
62
1:200
Microwave
Novolink
100%
100%
186
1:50
Citrate buffer, (pH 6) in pressure cooker
EnVision FLEX+
0, 1+, or 2+
93%
100%
The 5A4 antibody used in the studies by McLeer-Florin et al., Jokoji et al., and Lopes et al. were from Abcam ; the antibody used in the other studies were from Novocastra. iView (DAB Detection Kit) and OptiView (DAB IHC Detection Kit) are products of Ventana Medical Systems. Novolink (Polymer Detection System) is a product of Leica Biosystems.
Rabbit Polyclonal Antibody IHC versus RT-PCR At least two studies have involved the use of IHC with a rabbit polyclonal antibody (Table 4) (Wong 2009, Chen 2012). In one study, the results were compared with those obtained by EML4-ALK RT-PCR, and concordance was high (Chen 2012). In the other study, 11 adenocarcinomas and one adenosquamous carcinoma that were ALK positive on RT-PCR were also positive on IHC (Wong et al 2009).
Table 4. Staining Features of IHC with ALK Rabbit Polyclonal Antibody
Study Wong et al., 2009 Chen et al., 2012 No. of Specimens
(% of ALK+)
ALK1 Dilution 1:1000
Antigen Retrieval Citrate buffer (pH 6.0) in microwave CC1 solution
Detection/ Amplification System HRP complex ultraView
Scoring None
IHC Positive Threshold Not specified ?2+
IHC Sensitivity (vs. RT-PCR) 100%
IHC Specificity (vs. RT-PCR) Not specified 90%
266 (4.9%)
64 (4.7%)
1:500
0, 1+, 2+, or 3+
100%
CC1 (Tris/borate/ EDTA) is a product of Roche (Basel, Switzerland). ultraView (Universal DAB Detection Kit) is a product of Ventana Medical Systems, Inc. , and HRP (horseradish peroxidase) complex is a product of Dako.
Comparison of Antibody Clones Some authors have directly addressed concordance among antibody clones using the same sample sets, and concordance has been antibody dependent (Table 5) (Rodig 2009, Takeuchi 2009, MinoKenudson 2010, Murakami 2011, Conklin 2013, Selinger 2013). Takeuchi et al. reported comparable