Effect of Formalin Fixation Time on Her 2 Expression by Immunohistochemical Technique
SM Dintzis, KH Allison. University of Washington, Seattle, WA
Background: The 2007 American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Her 2 Testing in Breast Cancer include the specification that formalin fixation for fewer than 6 hours or longer than 48 hours is not recommended. Although there is evidence to suggest that pre-fixation tissue handling, type of fixative and fixation time may have an effect on Her2 expression levels as detected by immunohistochemistry, the reason for the choice of 6 and 48 hours for the minimum and maximum formalin fixation times is unclear.
Design: Using the same Her2 over-expressing cell line used to validate the Herceptest, SK-OV-3, we attempt a systematic study of the effect of formalin fixation time on detection of Her2 overexpression by immunohistochemical (IHC) technique. SK-OV-3 cells grown in tissue culture were pelleted and fixed in formalin for times ranging from 10 days to 25 minutes. The formalin fixed cell pellets were then processed according to the our department's standard breast biopsy protocol. Slides were scored for intensity and pattern of Her2 expression.
Results: The overexpression of Her2 in the SK-OV-3 cell line was confirmed using both FISH and immunohistochemical techniques. FISH testing demonstrated the ratio of Her2 to chromosome 17 to be 2.8. SK-OV-3 cell pellets incubated for long (1, 2, 3, 10 day) formalin fixation times showed no significant difference in intensity or proportion of cells exhibiting strong membrane staining by IHC. Cell pellets incubated for 6 hours or less showed a decrease in stain intensity and proportion of cells with membrane staining.
Conclusions: Her2 cell surface receptor as detected by IHC appears to be relatively unaffected by length of formalin fixation time greater than 24 hours. Formalin fixation less than 6 hours may decrease the sensitivity of Her2 testing by IHC. Although we recognize that tissue culture cell pellets do not exactly recapitulate the cellular environment of invasive carcinomas, the use of a cell line standard can help to identify whether weakness and inconsistency in Her2 scoring systems lie in the heterogeneity of formalin fixation times or are instead a reflection of heterogeneity of tumor composition or other aspects of tumor tissue handling.
Tuesday, March 23, 2010 1:00 PM
Poster Session IV # 19, Tuesday Afternoon