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IASLC ATLAS OF ALK TESTING IN LUNG CANCER
2010, Conklin 2013). Others have applied a simple evaluation of the positivity, defining ALK-positive expression as more than 10% of tumor cells being positive, regardless of the intensity (Rodig 2009, Mino-Kenudson 2010, McLeer-Florin 2012, Martinez 2013, Sholl 2013). More recently, however, Takeuchi found that almost all cancer cells were stained in more than 300 ALK-rearranged lung cancers that were tested by IHC with the 5A4 antibody and the intercalated antibody-enhanced polymer (iAEP) method (Takeuchi 2013). This staining homogeneity suggests that, in ALK-rearranged lung cancers, all tumor cells harbor the ALK gene rearrangement. Until further data are available regarding false-positive IHC results, for high-throughput screening with ALK IHC, which is currently proposed in most publications, pathologists should confirm any positive signal by the reference technique, fluorescence FISH. However, an increasing number of patients who have tumors that test positively on IHC and negatively on FISH (according to strictly defined criteria) have had a good response to crizotinib therapy (Peled 2012). Another consideration is the reproducibility of ALK IHC results among different laboratories and pathologists. At the time of writing, two IHC protocols seem to be validated. In one study, using the Ventana ALK IHC kit (Ventana Medical Systems, Inc.), the reproducibility among seven international pathologists was evaluated; in a binary classification using the defined Ventana standard operating procedure, the reproducibility among the observers was 95% and 97% for both positive and negative results (Hirsch 2013). The other is the European Thoracic Oncology Platform (ETOP) protocol using the 5A4 antibody (Novocastra) (Thunnissen 2012c), in which 12 laboratories stained, either manually or with use of an automated procedure, the same tumors in a consistent manner. (See Chapter 9 for more detailed information on standardization.)
Practical Implementation of ALK IHC
Pathologists should be familiar with various artifacts that may lead to false-positive staining: light cytoplasmic stippling in alveolar macrophages
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