ER, PR, HER-2/Neu Immunostaining in Cytology: Effect of Varied Fixation on Human Breast Cancer Cell Lines
Olena Dorokhova, Danny van der Helm, Sara Maleki, Kathie Schlesinger, Mark Suhrland, Maja Hrzenjak Oktay. Montefiore Medical Center and Albert Einstein College of Medicine, Bronx, NY; Albert Einstein College of Medicine, Bronx, NY
Background: The ASCO/CAP Expert Panel recommends that all invasive breast carcinomas and breast cancer recurrences be tested for ER, PR and HER-2/neu expression. The guidelines for the testing of surgical specimens by immunohistochemistry (IHC) are well defined, whereas they are lacking for cytological samples. This study evaluates various fixation protocols for optimal receptor testing by IHC of human breast cancer cell lines.
Design: Human breast cancer cell lines MCF-7 (ER/PR positive) and SKBR-3 (overexpressing HER-2/neu) were used. The cells were fixed in 10% neutral buffered formalin and Saccomanno Fixative (SF) for 2, 4, 6, 12, 24, 48, 72 and 96 hours, and embedded in paraffin as cell blocks. IHC was performed on a Dako autostainer using primary monoclonal antibodies for ER (clone 1D5, Dako), PR (clone PGR 636, Dako) and HER-2/neu (Zymed). ER and PR slides were assigned a proportion score (PS; 0-5), an intensity score (IS; 0-3) and a total score (TS=PS+IS). For the evaluation of HER-2/neu staining we used the standard DAKO scoring system ranging from 0 to 3+.
Results: When stained for ER, formalin-fixed cells had TS of 8 for majority time points, while SF-fixed cells had TS ranging from 8 at 6h to 5 at 96h. TS for PR were more variable for both fixatives, varying between 5 and 7 with formalin fixation and between 4 and 7 with SF. HER-2/neu staining for the formalin-fixed cells was uniformly 3+ for all time points. The SF-fixed cells showed patchy 2+ to 3+ staining when fixed up to 24h; however, if cells were fixed more then 24h, the scores were 0-1+.
Conclusions: Human breast cancer cells can be successfully stained for ER, PR and HER-2/neu when fixed in formalin. In contrast to 6-72h fixation recommended for ER/PR staining and 6-48h fixation for HER-2/neu staining for surgical specimens, the fixation time for formalin-fixed cells may be broader - ranging from 2h to 96h. Cells fixed in SF from 2 to 96 hours will also stain well for ER and PR. SF produces variable results for HER-2/neu staining, in particular, SF fixation beyond 24h may cause false negative results.
Tuesday, March 20, 2012 9:30 AM
Poster Session III # 79, Tuesday Morning