Flow cytometric DNA analysis with cytokeratin gating of formalin-fixed deparaffinized breast cancer nuclei.
The "gold standard" inflow cytometric DNA analysis of breast cancer uses fresh tumor cells simultaneously labeled for cytokeratin (CK) and DNA. We developed a 2-parameter CK-DNA flow assay suitable for archival, paraffin-embedded tissue (PT). Six anti-CK monoclonal antibodies were tested by immunocytochemistry and our assay for staining of nuclei extracted from PT breast cancers by combination pepsin-trypsin digestion. Clone CAM 5.2 was inadequate for PT nuclear suspensions, but a cocktail of 2 anti-CK clones (AE1/AE3 and KL-1) distinguished epithelial from nonepithelial nuclei in 2-parameter flow dot plots. We studied 82 routine PT breast tumors by our assay and used a univariate flow DNA histogram based on fresh biopsy tissue for comparison. Three histogram data quality indicators were improved. A trend toward higher S-phase fractions was found for DNA diploid PT tumors, although when inflammation was evident histologically, the increment in S-phase fraction with gating was often marked. CK gating identified PT tumors containing concurrent CK-positive DNA diploid and nondiploid populations (27 of 56 DNA nondiploid histograms). By excluding nonepithelial nuclei, 2-color CK-DNA flow methods may increase the accuracy of ploidy and S-phase fraction measurements. Our method appears superior to previous techniques using clone CAM 5.2 for labeling of archival breast cancers.
Department of Pathology, United Hospital, St Paul, Minnesota 55102, USA.
S R
SR
McCormick
A A
AA
Peters
J B
JB
Schrauth
D000911:Antibodies, Monoclonal / Q000276:immunology; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005404:Fixatives / Q000494:pharmacology; D005434:Flow Cytometry; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007633:Keratins / Q000276:immunology / Q000378:metabolism*; D016612:Paraffin Embedding; D011003:Ploidies; D012189:Retrospective Studies
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde; D007633:Keratins