Delay to Formalin Fixation Effect on HER2 Test in Breast Cancer by Dual-color Silver-enhanced in Situ Hybridization (Dual-ISH)

Background: HER2 status is an integral part of breast cancer management. One of the variables that negatively affects HER2 test is delay to formalin fixation (DFF). The purpose of this study is to determine the effect of progressive DFF on HER2-dual color in situ hybridization (Dual-ISH).

Materials And Methods: Ten palpable invasive breast cancers were included in the study. For each case, the procured tumor was divided into 8 parts and consecutively fixed after 0, 10, 30 minutes, 1, 2, 4, and 8 hours; 1 section was kept in saline and stored overnight (ON) at 4°C. Two tissue microarray blocks were constructed. Then, HER2 was tested with Dual-ISH. HER2 and CEP17 signal and HER2/CEP17 ratio was calculated in 10 cells for each core. The percentage of cells that had complete loss of signal was calculated. The percentage of cells that had nuclear bubbling and/or background staining was also calculated.

Results: DFF had no statistically significant effect on HER2/CEP17 ratio, absolute number of HER2, or CEP17 signals. However, 1 case became falsely amplified in ON sample. The trend of the percentage of cells that lost signal with time of DFF was statistically significant (P<0.001), more notably after 1 hour of DFF. Nuclear bubbling and/or haze or dust was seen in 4 cases (P=0.009). These changes started to appear at 30 minutes to 1 hour DFF (P=0.025 and 0.003, respectively).

Conclusions: DFF has no statistically significant effect on the HER2/CEP17 ratio tested by Dual-ISH. However, significant artifacts could occur in samples fixed after 1 hour of DFF time.