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Chapter 6: Comparison of Different Assay Platforms for ALK Testing
results with the ALK1 and 5A4 antibodies using an intercalated antibody-enhanced polymer (iAEP) method to amplify signals (Takeuchi 2009). However, discordance has been found in other studies. For example, Murakami et al. reported one discordant case among 12 specimens with ALK rearrangement; the result was negative with 5A4 IHC but positive with D5F3 IHC (Murakami 2012). Conklin et al. compared five combinations of antibody clones and detection systems, and the concordance was highest with 5A4 and D5F3 with the ADVANCE system (Dako), but a heterogeneous positive reaction was detected in a specimen that was ALK negative on FISH (Conklin 2013).
Multiplex or Quantitative RT-PCR versus FISH (with or without IHC)
Although EML4-ALK transcripts were found in the tumor cells of nine NSCLCs but also in the normal lung in only one study (Martelli 2008); the presence of such transcripts in normal lung tissue has not been confirmed by others, which has led to the questioning of those data (Mano 2010, Sasaki 2010). RT-PCR is a highly sensitive method associated with high specificity and no false-positive results; however, there is a risk of false-negative results because of the difficulty in obtaining high-quality RNA from FFPE specimens. Although the false-negative rate of RT-PCR has not been addressed in detail, successful detection of ALK transcripts in a prospective manner has been reported (Soda 2012). In that study, 108 (12%) of 916 specimens were excluded because RNA was of poor quality. EML4-ALK transcripts were detected in 36 specimens, 15 of which were available for IHC; all 15 specimens were ALK positive by IHC. Overall, the sensitivity and specificity of RT-PCR for detection of ALK transcripts, compared with IHC and FISH, are good, ranging from 94% to 100% (Table 6) (Takeuchi 2008, Inamura 2008, Takeuchi 2009, Soda 2013).
Table 6. Comparison of Results of RT-PCR with the Results of Other Methods of ALK Testing
Study Inamura et al., 2008 Takeuchi et al., 2008 Takeuchi et al., 2009 Soda et al., 2012 Type of RT-PCR Multiplex RT- PCR Multiplex RT-PCR Inverse and multiplex RT-PCR Multiplex RT-PCR IHC Antibody ALK1 – 5A4 ALK1 5A4 FISH None FISH-based fusion assay EML4 and KIF5B fusion assay ALK break-apart probe kit RT-PCR Sensitivity (vs. FISH and/or IHC) 100% (vs. IHC) 100% (vs. FISH) 100% (vs. IHC) 100% (vs. IHC) 94% (vs. FISH) RT-PCR Specificity (vs. FISH and/or IHC) 100% (vs. IHC) 100% (vs. FISH) 100% (vs. IHC) 100% (vs. IHC) 100% (vs. FISH)
The ALK1 antibody in the studies by Inamura et al. and Takeuchi et al. (2009) is a product of Dako. The 5A4 antibody in the study by Takeuchi et al. (2009) is a product of Abcam and, in the study by Soda et al., is a product of Nichirei Biosciences, Inc. In the study by Soda et al., the break-apart probe kit is a product of Abbott Molecular.
CISH versus Other Methods
CISH has some advantages over FISH, and studies have shown that the results of CISH are comparable with those of other methods of detecting ALK rearrangement (Table 7) (Kim 2011, Yoshida 2011a, Schildhaus 2013). In one study, the ALK-positive criteria for CISH were the same as those for FISH, but in another study, the researchers found that a different cutoff value for CISH led to better separation of ALK-positive and ALK-negative tumors (Schildhaus 2013, Yoshida 2011a). A new dual technology