Analysis of proliferative grade in glial neoplasms using antibodies to the Ki-67 defined antigen and PCNA in formalin fixed, deparaffinized tissues

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Abstract
In an effort to optimize immunocytochemical methods to evaluate cell kinetics in brain tumors, we studied two newly-developed antibodies which react with formalin resistant epitopes of Proliferating Cellular Nuclear Antigen (PCNA) and Ki-67. These results were compared with standard flow cytometric cell cycle data from the same tumor specimens to determine if these methods correlate with each other, and whether retrospective analysis using these antibodies is feasible for cell kinetic analysis of brain tumors. Thirty-one specimens of glial tumors submitted for flow cytometry during 1992 were also reacted with antibodies to PCNA (PC-10) and Ki-67 (MIB-1). Flow cytometry scores for S-phase Fraction were compared with immunocytochemical scores for both antibodies, using an arbitrary rating of 1 (low, < 4%), 2 (intermediate, 4–6%), 3 (high, > 6%), and 1 (< 25% positive), 2 (26–75% positive), 3 (> 75% positive), respectively. MIB-1 results were found to correlate significantly with the S-phase fraction as determined by flow cytometry. The MIB-1 data showed a trend toward underestimating, i.e., lower scores, the proliferative index compared with flow cytometry. There was less of a correlation between PC-10 antibody scores and flow cytometry S-phase fraction, as PC-10 immunostaining typically overestimated the proliferative rate of brain tumors when compared with flow cytometry. There was an exact correlation between PC-10 and MIB-1 in only 4 cases, whereas in the remaining specimens, PC-10 results were always higher than MIB-1. These new immunostaining methods, which react with formalin fixed deparaffinized tissue and require microwave pre-treatment to optimize the results, have demonstrated their usefulness in retrospectively analyzing tumor cell kinetics. For one antibody, namely MIB-1, excellent correlation with standard flow cytometric analysis was achieved. Further studies comparing patient outcome with immunoreactivity to both Ki-67 and PCNA proteins is now possible and may be done on large numbers of achived specimens.

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