Table 3 Human epidermal growth factor receptor 2 (HER2) testing recommendations in gastric cancer, (a) immunohistochemistry and (b) in situ hybridization

Testing recommendations

 • Representative surgical samples or an adequate number of viable biopsy specimens (ideally six to eight) are required

 ○ If few biopsies are available, all viable specimens should be tested

 • Immunohistochemistry should be the initial HER2 testing methodology for gastric cancer and bright-field methodologies are preferred wherever possible

 ○ HER2-positive per European Medicines Agency license: immunohistochemistry 3+ or immunohistochemistry 2+/fluorescence in situ hybridization-positive or immunohistochemistry 2+/silver in situ hybridization-positive

 ○ Borderline immunohistochemistry 1+/immunohistochemistry 2+ cases and samples with focal and intense membranous reactivity in <10% cells may also be retested with fluorescence in situ hybridization or silver in situ hybridization (scores for both assays should be indicated separately on the report)

 • Validated immunohistochemistry HER2 assays should be used

Scoring recommendations

 • Due to the tumor heterogeneity (focal areas of positivity) and incomplete membrane staining commonly seen in gastric cancer, the gastric cancer-specific scoring criteria should be adhered to:

 ○ Surgical specimen cutoff: complete, basolateral, or lateral membranous reactivity in ≥10% of cells

 ○ Biopsy specimen cutoff: complete, basolateral, or lateral membranous reactivity in ≥5 clustered cells

 • The ‘magnification rule’ should be used in conjunction with the scoring criteria

 • Borderline cases (immunohistochemistry 1+/immunohistochemistry 2+ or focal staining in <10% cells) that score fluorescence in situ hybridization-positive or silver in situ hybridization-positive may be considered HER2-positive (scores for both assays should be indicated separately on the report)

(b) In situ hybridization

Testing recommendations

 • Tumor samples classified as immunohistochemistry 2+ should be retested by fluorescence in situ hybridization or silver in situ hybridization to assess HER2 status

 • Silver in situ hybridization is a more suitable methodology than fluorescence in situ hybridization for assessing HER2 status in gastric tumor samples as it is a bright-field methodology and thus allows for rapid identification of HER2-positive tumor foci within a heterogeneous sample

 • Validated in situ hybridization HER2 assays should be used

Scoring recommendations

 • The definition of fluorescence in situ hybridization or silver in situ hybridization positivity in gastric or gastro–esophageal junction cancer is a HER2:chromosome 17 ratio of ≥2.0

 • The entire case should be screened for amplified regions (particularly important for fluorescence in situ hybridization samples where a bright-field image is not available)

 • At least 20 evaluable, non-overlapping cells in the invasive component should be counted initially

 • In borderline amplification cases, ∼20 additional cells should be recounted or scoring should be performed in an alternative area of tissue

 • The overall HER2 gene count is important:

 ○ >6 HER2 gene copies using single probe: considered positive

 ○ Four to six HER2 gene copies: dual probe test advised and the ratio should be recalculated by counting an additional 20 cells

Ensuring quality and timely HER2 testing results

 • The use of validated immunohistochemistry and in situ hybridization tests is strongly recommended and appropriate controls should be included in each run

 • Turnaround time from initial diagnosis to reporting of results should ideally not exceed 5 working days and a multidisciplinary approach is required

 • Centralized testing is recommended wherever possible and all laboratories should participate in validated quality assurance programs