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P5. Immunohistochemical fluoro-chromogenic triple staining method for detection of lymphovascular invasion in primary breast cancer

Teppo Haapaniemi1,2, Taneli Tani2, Jorma Isola1
1) University of Tampere, Tampere, Finland and 2) Päijät-Häme Social and Health Care Group, Lahti, Finland

Background: Lymphovascular invasion (LBVI) predicts poor outcome in primary breast cancer. LBVI is routinely detected using H&E stained sections, although it is not sensitive enough in intratumoral areas of ductal tumors or in lobular carcinomas in general. We have set up a novel triple immunohistochemical staining to improve LBVI detection.

Methods: Samples of lobular (n=14) and ductal (n=61) G2-G3 breast carcinoma were stained with podoplanin (lymphatic endothelium), CD31 (vascular endothelium) and CK-PAN (to demonstrate that intravascular cells are tumor cells). Podoplanin and CD31 were visualized by DAB and Permanent Red, respectively, and cytokeratin by Cy2 fluorochrome. Slides were analyzed as virtual slide scans of stacked images representing bright field and green fluorescence.

Results: The cytokeratin immunofluorescence staining was often found decisive to reveal the nature of cell aggregates localized within the lumina of lymphatic and blood vessels. Using triple immunohistochemistry lymphatic vessel invasion (LVI) was found in 57% of ductal and 50% of lobular carcinomas, whereas blood vessel invasion (BVI) was found in 38% ductal carcinomas and 23% of lobular carcinomas. As expected, H&E was less sensitive. Using H&E, LVI was found in 31% of ductal and 41% of lobular carcinomas, and BVI in 3% of ductal and 7% of lobular carcinomas.

Conclusion: The detection of LBVI was more sensitive with fluoro-chromogenic staining method as compared to conventional H&E staining. The triple IHC was found easy to read using virtual microscopy.

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