Evaluation of Ki-67 reactivity in neuroblastoma using paraffin embedded tissue

To examine the pattern of reactivity of Ki-67 in neuroblastoma and correlate this with a) clinical prognostic criteria and b) cell cycle statistics (using flow cytometry).

Four micron sections of paraffin embedded (PE) tissue from 55 patients (25 pre chemotherapy and 30 post) were placed on to aminosialinised slides, dewaxed and rehydrated. Slides were pretreated in a microwave oven, endogenous peroxidase activity blocked using 3% hydrogen peroxide and Ki-67 reactivity investigated using a streptavidin/biotin/peroxidase technique. DNA ploidy was also performed from an immediately adjacent section on the same block using a FACScan and Cellfit software.

Ki-67 reactivity was well defined and highly reproducible. Eighteen out of 30 post chemotherapy samples were totally negative, despite evidence of proliferation on flow cytometry and all subsequently died of disease. As interpretation post chemotherapy was therefore deemed unreliable, this group was excluded from analysis. Reactivity in pretreatment samples ranged from 0% to 67%; staining was restricted to the nucleus with a distinct pattern noted in the nucleolus. Ki-67 positivity was lower in aneuploid compared with diploid tumours (mean 26% vs 36%, NS). Among diploid tumours, a lower percentage positivity was noted in those patients with better clinical prognostic parameters. Correlation however between Ki-67 and SG2M phases of cell cycle was poor (RS = 0.39, NS).

Assessment of proliferation in neuroblastoma by Ki-67 reactivity in paraffin embedded tissue is reliable in pretreatment samples and can be incorporated into routine immunohistochemical evaluation. Larger multicentre studies are required to further evaluate Ki-67 reactivity as a prognostic indicator.