The Effect of Incubation of Rectal Biopsies on Measures of Proliferation Using Proliferating Cell Nuclear Antigen in Comparison with 5ri’

Rectal epithelial cell kinetics are used as intermediate markers for coborectal cancer and relate to risk. In this study, measures of proliferation using direct immunohistochemistry for proliferating cell nuclear antigen (PCNA) were compared to in vitro labeling by bromodeoxyuridine (BrdUrd) and incubated biopsies that were later stained for PCNA (PCNA-I) in human rectal biopsies. The study group consisted of 20 sets of biopsies from 12 subjects participating in an intervention trial. Fresh nomncubated biopsies were fixed in methacarn and stained immunohistochemically for PCNA (clone 19A2). In parallel biopsies, BrdUrd was incorporated into the DNA of S-phase cells during a 2-h incubation at 37#{176}C under hyperbaric conditions and localized by immunohistochemistry. Additionally, biopsies were incubated under hyperbaric conditions for 2 h at 37#{176}C, fixed in methacarn, and stained for PCNA (PCNA-I). There was a highly significant difference in the labeling index between the three methods (P < 0.01), but there was no significant difference between subjects (P 0.439). The mean labeling index was 2.3 ± 0.1 % for PCNA, 2.9 ± 0.1% for PCNA-I, and 4.1 ± 0.1% for BrdUrd. The proportion of labeled cells in the top twofifths was significantly higher (P = 0.01) for BrdUrd (5.5 ± 0.8%) and PCNA-I (6.4 ± 1.1%) compared to PCNA (3.1 ± 0.6%), and a significant difference was seen between subjects (P = 0.038). PCNA-I and BrdUrd methods had similar crypt heights with 73.5 ± 1.8 and 71.2 ± 1.3 cells/crypt column, respectively, but were significantly shorter (P < 0.001) than PCNA with 83.4 ± 1.5 cells/crypt column, indicating a loss of cells during organ culture. The simplicity of the PCNA technique, which avoids potential perturbations occurring during Received 1 2/2/96; revised 6/ I 2/97; accepted 6/26/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ads’ertisernent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. t Supported in part by Kellogg (Australia) Proprietary Ltd. 2 To whom requests for reprints should be addressed, at Department of Medicine. University of Melboume, The Royal Melboume Hospital, Parkville, Victoria 3050, Australia. organ culture, has considerable appeal as a marker for colorectal cancer risk, but additional studies are needed to correlate PCNA with neoplastic risk.