125The most commonly used method is immunohistochemistry,
which is a low-cost technique that can be performed on small
formalin- fixed and paraffin-embedded tissue samples.
Currently, commercially available immunohistochemical anti-
bodies include the HercepTest and A0485 (Dako, Glostrup,
Denmark) rabbit polyclonal antibodies, the SP3 (Labvision;
Thermo Fisher Scientific, Fremont, CA, USA), 4B5 (Ventana
Medical Systems, Tucson, AZ, USA) rabbit monoclonal antibod-
ies, and the CB11 mouse monoclonal antibody (Novocastra,
Newcastle upon Tyne, England). The interpretive problems docu-
mented in the literature emphasize the necessity of expertise in
applying the criteria for assessing HER2 immunohistochemistry in
esophagogastric junction cancer.
●● Deparaffinization—Selected.
●● Cell Conditioning (Antigen Retrieval)—CC1 36 min at
100 °C (HIER).
●● Primary Antibody Incubation —12 min at 36 °C.
●● UltraWash—Selected.
●● Counterstain—Hematoxylin II for 4 min; Bluing Reagent for
4 min.ISH can be performed by two main technologies, bright field
(BRISH) and dark field techniques. Bright field techniques include
chromogenic ISH (CISH) or silver ISH (SISH). Dark field method
includes fluorescence in situ hybridization (FISH). SISH is the
preferred method [ 41 ] and discussed below.There are two methods: one developed by ROCHE/VENTANA
(Tucson, Arizona USA), the SISH technique is a single-probe (a
strand of nucleic acid of specific sequence) assay that uses a dinitro-
phenol (DNP) hapten-labeled probe to identify a specific DNA or
RNA sequence of interest with silver deposition. Secondly, more
recently, a dual DNA probe in situ hybridization assay has been
developed which allows the copy numbers of both HER2 and
chromosome 17 to be enumerated in tumor nuclei on the same
tissue slide section (Fig. 3 ). The INFORM HER2 Dual ISH
(DISH) assay (ROCHE/VENTANA, Tucson, Arizona USA),
uses a cocktail of both DNP-labeled HER2 DNA probes and DIG
(dioxygenin)-labeled chromosome 17 DNA probes.
Staining Protocol for The INFORM HER2 Dual ISH (DISH)
assay performed on Ventana BenchMark ULTRA instrument [ 42 ]:
●● Baking Temperature and Time—63 °C for 20 min.
●● Deparaffinization—Selected.
●● Cell Conditioning (Antigen Retrieval)—Combined HIER and
Enzyme Epitope Retrieval:3.2 Immunohisto‑
chemistry (IHC) (See
Note 1
for Interpretation)
3.2.1 Staining Protocol
for Ventana HER2 (4B5)
Performed on Ventana
BenchMark ULTRA
Instrument with UltraView
Universal DAB Detection
Kit [ 40 ]
3.3 In Situ
Hybridization (ISH)
(See Note 2
for Interpretation)
3.3.1 Silver In Situ
Hybridization (SISH)
HER2