121The samples that may be tested are endoscopic esophageal biop-
sies, esophagostomy specimens, and specimens from metastatic
lesions that include both biopsies and cytology samples (ascitic and
pleural fluids and fine-needle aspiration cytology samples of meta-
static lymph nodes) [ 3 , 20 ].To date, validation of the HER2 testing methods has largely
focused on histological samples obtained from endoscopic biopsies
of the primary tumor [ 20 , 21 ]. There are several issues related to
testing in endoscopic biopsies. As most patients with esophageal
carcinoma present with advanced disease and are not surgical can-
didates, endoscopic biopsy may be the only diagnostic tissue mate-
rial available for testing. Endoscopic biopsy should be used in an
optimal fashion to determine HER2 status of the carcinoma. The
important pre-analytical issues to consider about the suitability of
endoscopic biopsies for testing are the adequacy of the materials,
fixation- related issues, and cost concerns. Endoscopic biopsy
should be representative of the tumor with adequate number of
tumor fragments. Recent studies have shown that five or more
tumor fragments are required in gastric and esophagogastric junc-
tion adenocarcinomas as a prerequisite for accurate results [ 22 –
24 ]. This is due to known heterogeneity in gastric/esophagogastric
junction carcinomas. Heterogeneity rate has been reported up to
30–38% of HER2- positive carcinomas [ 25 ]. Although this has not
been clearly defined for pure esophageal adenocarcinoma, more
tumor biopsy fragments are likely providing more accurate results.
One advantage of endoscopic biopsies is that formalin penetration
is faster and the specimen is likely to be better fixed compared to
the gastric resections. In addition, the cost of testing small endo-
scopic biopsies will be less when compared to large resection
blocks.There are inherent pre-analytical issues that need consideration in
resection specimens. Main issue is adequate tumor fixation, which
has major implications on the process of HER2 antibody retrieval.
Delay in formalin fixation after specimen collection as well as pro-
longed fixation times may affect HER2 testing results. HER2 test-
ing in resection is costly when compared to biopsies, as tumor
tissue penetration needs higher quantities of the antibody. For
research purposes, tissue microarray is an alternative to overcome
high costs although not recommended for clinical testing. In brief,
the tissue microarray is a novel technology for analysis of molecular
markers in a large number of tumors at the same time [ 26 ] (see
Chapter 10 , Fig. 1 ). Therefore, antibody cost could be reduced
using this technique. The main drawback in this method is unavoid-
able heterogeneity in gastric or esophagogastric junction cancers
resulting in potential false-negative results related to selective and
limited sampling.2.2 Selection
of Samples
and Fixation of Tissue
2.2.1 Endoscopic Biopsy
2.2.2 Resections
HER2