above except for the use of goat anti-mouse IgG (Jack- son Immunoresearch ..... Jaskulski D, deRiel JK, Mercer WE, Calabretta B, Baserga. R: Inhibition of ...
American Journal of Pathology, Vol. 138, No. 6, June 1991
Copyright © American Association ofPathologists
Growth Fraction Estimation of Malignant Lymphomas in Formalin-fixed Paraffin-embedded Tissue Using Anti-PONA/Cyclin 19A2 Correlation with Ki-67 Labeling
Onsi W. Kamel, David P. LeBrun, R. Eric Davis, Gerald J. Berry, and Roger A. Warnke From the Department ofPathology, Stanford University Medical Center, Stanford, California
The immunohistochemical detection ofPCNA/Cyclin, a nuclear protein associated with cell proliferation, represents a potentially useful tool for the study of tumor proliferative activity. Previous studies investigating the reactivity of anti-PCNA/Cyclin monoclonal antibody 19A2 have not clearly defined the population ofproliferating cells with which 19A2 reacts in tissue sections. The authors describe a method for detection ofPCNA/Cyclin infor-malin-fixed paraffinembedded tissue using a routine biotin-streptavidin immunohistochemical system that employs an antiIgM, mu-chain-specific second-stage antibody. The authors used this method to study the proliferative activity of 24 malignant lymphomas, consisting of 12 low-grade lymphomas (LGLs) and 12 intermediate-grade lymphomas (IGLs), and five reactive tonsils. 19A2 data was compared with Ki-67 labeling in frozen sections in the same group of case& 19A2 provided easily detectable nuclear staining ofproliferating cells with reactive cells demonstrating varying intensity of staining this latter finding most likely due to the varying nuclear concentration of PCNA/ Cyclin protein during the cell cycle. In tonsils 19A2 reacted with germinal center cells and basal keratinocytes. In the malignant lymphomas, there was good correlation between 19A2 and Ki-67 data (r = 0.90, P < 0.001). The subgroup of LGLs showed a mean PCNA/Cyclin of 26% and a mean Ki-67 of28%. In the subgroup of IGLs, mean PCNA/Cyclin = 54% and mean Ki-67 = 59%. These results indicate that 19A2 detects a fraction of proliferating cells that is
similar to that detected by Ki-67, ie, the growth fraction, and that 19A2 is a reliable marker ofproliferative activity in uniformly handled4 formalin-fixe4 paraffin-embedded tissue. (Am J Pathol 1991,
138:1471-1477)
Proliferating cell nuclear antigen (PCNA/Cyclin) is a 36kd, acidic, non-histone, nuclear polypeptide that is associated with cell proliferation.16 This protein was originally detected during experiments in which serum from patients with systemic lupus erythematosus failed to show immunoreactivity with resting cells but rather reacted with nuclei of proliferating cells in normal human tissue (germinal center cells, cortical thymocytes, spermatogonia of testis) as well as mitogenically stimulated peripheral blood lymphocytes and several cultured cell lines.7 Miyachi et a17 designated this protein proliferating cell nuclear antigen (PCNA). Independently Bravo et al,8 in search for cell cycle phase-specific proteins, identified an acidic nuclear protein of 36 kd that they subsequently called 'cyclin.' Mathews et al,9 in 1984, demonstrated that PCNA and cyclin are identical. Since that time, PCNANCyclin has been demonstrated to be an auxiliary protein to DNA polymerase delta,10 and its presence appears to be necessary for DNA replication.11 The structure of the human PCNA/Cyclin gene has been determined12 and the protein, a 261-amino acid polypeptide with a high content of aspartic and glutamic acids, has been sequenced.13 Recently Ogata et al14 reported the production of two mouse monoclonal antibodies (1 9A2 and 1 9F4) to PCNA/ Cyclin. Although the anti-PCNA/Cyclin monoclonal antiSupported in part by grants 34233 and 33119 from the National Cancer Institute, National Institutes of Health. Accepted for publication February 4, 1991. Address reprint requests to Onsi W. Kamel, MD, Department of Pathology, Stanford University Medical Center, 300 Pasteur Dr., Stanford, CA 94305.
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body 19A2 is now commercially available, there are few studies that have investigated its use in human tissue sections,1"19 and most of these studies have examined the application of 19A2 to frozen tissue or alcohol-fixed tissue sections. Furthermore the population of proliferating cells with which 1 9A2 reacts in tissue sections has not been well defined. This study documents the detection of PCNA/Cyclin in formalin-fixed, paraffin-embedded tissues using a biotinstreptavidin immunohistochemical method that employs an anti-lgM, mu-chain-specific second-stage antibody. Comparison of PCNA/Cyclin data with Ki-67 labeling (an established marker of proliferation in frozen tissue sections) indicates that 1 9A2, like Ki-67, provides an estimate of the growth fraction of a cell population and is a reliable marker for proliferative activity in uniformly handled, formalin-fixed, paraffin-embedded tissue.
Materials and Methods Tissue Specimens Tissue from five tonsils showing reactive hyperplasia and from 24 lymph nodes involved by malignant lymphoma was obtained in saline directly from the operating room or from the Laboratory of Surgical Pathology at Stanford University within 1 hour of surgery. A portion of each tonsil or lymph node was snap frozen and stored at - 70°C until use; another portion of each was fixed in 10% buffered formalin (between 3 and 10 hours) and then routinely processed for paraffin embedding.
Immunohistochemistry Five-micron-thick paraffin sections were deparaffinized and rehydrated and endogenous peroxidase was blocked by 3% H202 for 5 minutes followed by a brief wash in phosphate-buffered saline (PBS). Tissue sections were incubated with the anti-PCNA/Cyclin monoclonal antibody 19A2 (American Biotech, Plantation, FL) for 30 minutes at room temperature (optimal dilution 1:5000). The second-stage antibody was an anti-lgM, muchain-specific, biotin-conjugated antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA), used at a dilution of 1:400 (in PBS in Coplin jars), in which sections were incubated for 45 minutes at 4°C. This was followed by incubation with peroxidase-conjugated streptavidin (Jackson lmmunoresearch Laboratories, Inc.), used at a dilution of 1:400 (in PBS in Coplin jars), for 45 minutes at 4°C. Diaminobenzidine was used as a chromogen, followed by copper sulfate to darken the re-
action product and absolute methanol to fix the precipitate. Each step was followed by a brief wash in PBS. The sections were lightly counterstained with methylene blue. For Ki-67 staining, cryostat sections were fixed in acetone at 4°C for 10 minutes. Ki-67 antibody (Harald Stein, Berlin), used at a dilution of 1:5000, was applied to tissue sections for 30 minutes at room temperature. The rest of
the staining procedure was similar to that described above except for the use of goat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc.) as the secondstage antibody.
Scoring of 19A2 and Ki-67 Scoring of 19A2 and Ki-67 was performed using a standard light microscope equipped with an ocular reticle (magnification, x15) and a x40 objective. The sections were scanned at low power to determine the areas that were most evenly and heavily labeled. Only germinal centers were scored in the reactive tonsils and only the neoplastic follicles were scored in follicular lymphomas. All reactive cells were counted as positive regardless of the intensity of staining. In each case, 1000 cells were counted and the fraction of positive cells was determined. The cases were scored without knowledge of the diagnosis or other labeling parameters.
Results 19A2 and Ki-67 in Reactive Human Tonsils Our initial studies with 19A2 in formalin-fixed, paraffinembedded tissue sections showed that use of the antilgM second-stage antibody was critical to the reliable detection of PCNA/Cyclin in this setting. These studies, carried out on reactive human tonsils, showed strong nuclear reactivity of germinal center cells and basal cells of the squamous epithelium when the anti-lgM secondstage antibody was used. In contrast, no reactivity or only weak and often focal reactivity was detected with the use of the anti-lgG second-stage antibody. Studies using serial dilutions of anti-lgG and anti-lgM secondary antibodies confirmed these results. Although higher concentrations of the anti-lgG slightly increased 19A2 detection, background staining was also increased and nuclear reactivity remained less than that seen with the anti-lgM. The results that follow were obtained using the anti-lgM, mu-chain-specific detection, as has been described in Materials and Methods. Reactivity of 19A2 was distinct and it was generally not a problem to determine whether a particular nucleus was reactive, although there was a clear gradation in the
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cleaved and large cell lymphoma (5), follicular large cell lymphoma (4), diffuse mixed small and large cell lymphoma (1), and diffuse large cell lymphoma (7). Twelve cases were classified as low-grade lymphomas (LGLs) and 12 as intermediate-grade lymphomas (IGLs) according to the Working Formulation.2' The 1 9A2 and Ki-67 data for malignant lymphomas is summarized in Table 1. In the subgroup of LGLs, mean PCNA/Cyclin = 26% and mean Ki-67 = 28%. In the subgroup of IGLs, mean PCNA/Cyclin = 54% and mean Ki-67 = 59%. 1 9A2 and Ki-67 reactivity was localized to and often highlighted the neoplastic follicles in cases of follicular lymphoma (Figure 2). As in the reactive tonsils, variability in intensity of nuclear staining was also clearly demonstrated in the lymphomas (Figure 3). There was good correlation between 1 9A2 and Ki-67 data (r = 0.90, P < 0.001; Figure 4).
intensity of staining. The labeling was confined to the nucleus of cells with the exception of cells in mitosis, which showed faint cytoplasmic staining. Serial dilution studies of 1 9A2 showed 1:5000 to be the optimum dilution, providing nuclear staining in the greatest number of cells and little or no background or cytoplasmic staining. 19A2 and Ki-67 showed similar labeling of reactive tonsils. Both antibodies reacted with germinal center cells, labeling approximately 50% (35% to 75%) of germinal center cell nuclei. The fraction of labeled germinal center cells differed in different germinal centers within the same tonsil and in different reactive tonsils. In addition, both 1 9A2 and Ki-67 labeled many of the basal keratinocytes of the tonsilar squamous epithelium. Scattered interfollicular lymphoid cells also showed nuclear reactivity with both antibodies (Figure 1).
19A2 and Ki-67 in Malignant Lymphomas
Discussion The group of 24 lymphomas included the following histologic subtypes: small lymphocytic lymphoma (1), follicular small cleaved lymphoma (6), follicular mixed small
In the context of cell proliferation, tumors are considered to consist of three populations of cells: proliferating
