Esophageal Adenocarcinoma Methods and Protocols

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a FISH slide. It is also an advantage for the pathologists who prefer

the light microscopes.

Staining protocol for Dako HER2 IQFISH staining procedure

on the Dako Omnis instrument [ 44 ]:

●● Dewax—Clearify™ (clearing agent) 10 min, 38 °C.

●● Target Retrieval (Antigen Retrieval)—ISH Pre-Treatment

Solution (Dako Omnis) 15 min, 97 °C.

●● Wash—ISH Ethanol Solution, 96% (Dako Omnis) 2 × 3 min,

32 °C.

●● Digestion*—ISH Pepsin (Dako Omnis) for 10, 15, or 20 min.

●● Drying—for 15 min, 45 °C.

●● Denaturation—for 10 min, 66 °C.

●● Hybridization—HER2 IQFISH pharmDx™ (Dako Omnis)

75 min, 45 °C.

●● Stringent Wash—ISH Stringent Wash Buffer (Dako Omnis)

10 min, 61 °C.

*Three validated pepsin digestion times can be chosen using

the HER2 IQFISH protocols stated above.

4 Notes

1. Immunohistochemistry interpretation
   Hoffman et al. validated immunohistochemical scoring for
   HER2 protein expression before the landmark ToGA trial [ 45 ].
   The main differences from assessing HER2 in breast cancer are
   that gastric cancers often do not show complete membranous
   staining (i.e., staining is often basolateral) and only five cells in
   an endoscopic biopsy specimen are required to show immuno-
   reactivity to be considered for a positive. Endoscopic biopsies
   are unique samples associated with gastrointestinal tumors in
   contrast to breast carcinomas. Heterogeneity of the overexpres-
   sion/amplification of HER2 is more prevalent in esophagogas-
   tric junction adenocarcinomas and up to 40%. Breast carcinoma
   most often consists of solid nests of tumor cells and HER2 IHC
   positivity requires complete membranous staining creating a
   chicken wire pattern [ 46 ]. In gastric/esophagogastric junction
   cancers, however, lateral membranous staining with linear stain-
   ing at contact sites between two cells and basolateral membra-
   nous staining creating a U-shaped staining pattern is observed
   (Fig. 4 ). This is due to the higher frequency of glandular forma-
   tions in gastric and esophagogastric cancer (intestinal type).
   Luminal sparing with basolateral staining pattern is probably
   secondary to the absence of growth factor receptors at the lumi-
   nal part of the cell. It is important to acknowledge IHC results

HER2