Importance of formalin fixing conditions for HER2 testing in gastric cancer: immunohistochemical staining and fluorescence in situ hybridization

BACKGROUND: Accurate and reliable assessment of human epidermal growth factor receptor type 2 (HER2) status is important for selecting patients with gastric cancer who may benefit from trastuzumab treatment. Here we examined the impact of formalin fixing conditions on HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in xenografted tumor tissues. METHODS: Xenografted tumor tissues of the human gastric cancer cell lines NCI-N87, SCH, and SNU-16 were collected and kept at room temperature for 0, 6, or 24 h before being fixed with 10 % neutral buffered formalin (NBF) for 24 h or 5, 7, or 10 days and embedded in paraffin. Use of 10 % NBF, 20 % NBF, or nonbuffered formalin as fixative was investigated. RESULTS: The HER2 IHC scores for NCI-N87, SCH, and SNU-16 tumors were 3+, 2+, and 1+, respectively, when specimens were fixed with 10 % NBF for 24 h immediately after resection of the tumors. Specimens left for longer than 6 h before fixation had shrinkage of the tumor periphery and decreased immunostaining intensity in this region in all specimens. In SCH and SNU-16 specimens, starting fixation 24 h after tumor tissue collection induced autolysis and reduction of the number of stained cells, and 10-day-fixation lowered the HER2 score. Prolongation of fixation time did not affect FISH results, but if samples were left for more than 6 h before fixation, the FISH score was strongly reduced in SCH specimens (2.3 to 1.3). Reduced IHC staining intensity was observed with 20 % NBF and nonbuffered formalin compared to 10 % NBF. CONCLUSIONS: The time to and length of fixation of tumor specimens can affect HER2 IHC and FISH scores. The fixative used can affect IHC results.