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CHAPTER 4: Immunohistochemistry
ALK protein expression may be increased in some situations without ALK rearrangement, and the IHC results may be positive with a negative (or atypical) pattern on ALK FISH (Figure 8) (also see Chapter 3). In addition, lack of ALK protein expression despite gene amplification has been reported (Pelosi 2012, Salido 2011). For example, Pelosi et al. reported that a subset of sarcomatoid carcinoma had amplification of the ALK gene, but ALK protein expression was not detected using two different antibodies. The clinical implications of ALK gene copy number gain associated with protein expression needs to be elucidated further (Salido 2011, Kim 2013). Histologically, mucin-containing cells such as signet ring cells require careful interpretation of ALK immunoreactivity. A thin membranous positive pattern on ALK IHC may be masked by intracellular mucin vacuole (Figure 9), and the positive pattern may then be difficult to detect in the signet ring cells (Rodig 2009, Yoshida 2011b, Popat 2012). Some researchers have noted membranous staining, particularly in the apical portion, in FISH-negative cancer (Murakami 2012, Mino-Kenudson [personal communication]). This finding was not specific to cancer cells and was also seen in some nontumor cells, such as reactive type II pneumocytes. In addition, some neuroendocrine carcinomas have also been associated with positive reactions (Murakami 2012, Nakamura 2013).
Table 3. Potential Pitfalls in Interpreting the Results of IHC
Mucinproducing cells Cytoplasm is masked by intracellular mucin, with absence of ALK protein, leading to negative staining or marginally membranous-like staining, and a falsenegative interpretation. Nonspecific membranous staining, particularly prominent in the apical portion, is seen occasionally. This finding is not specific to tumor cells and is also seen in normal pneumocytes. Some squamous cell carcinomas, large cell neuroendocrine carcinomas, and normal ganglion cells show positive reactions. Depending on the amplification system used, some background can be found on extracellular mucin and within the cytoplasm of alveolar macrophages and bronchial cells.
Membranous staining
Neuroendocrine cells Nonspecific mucin staining
A
B
Figure 8. An example of ALK protein expression (left) with atypical pattern on FISH (diffuse single green signals; right).
Figure 9. A tumor with variation in morphology and staining. H & E staining shows (A) an area with many signet ring cells, and (B) area with solid pattern with few signet ring cells. ALK 5A4 immunohistochemistry shows (C) small cytoplasmic rim low (+1) and (D) high (+2/+3) staining intensities (magnification, x40).