Sources/Clones
Accurate (MM1), Biogenex (Ki-67, MIB1), Boehringer Mannheim (Ki-67, Ki-S5), Cymbus Bioscience (Ki67), Dako (Ki-67, polyclonal), Diagnostic Biosystems (Ki-67, polyclonal), Immunotech (MIB1), Novocastra (MM1), RDI (Ki67), Serotec (Ki67), and Zymed (7B11).
Fixation/Preparation
Monoclonal Ki-67 is immunoreactive in frozen sections but not in paraffin sections (although there are claims of reactivity following HIER, the results are not consistent). MIB1, Ki-S5 and polyclonal Ki-67 are all immunoreactive in routinely fixed, paraffin-embedded tissues. Immunoreactivity is enhanced following HIER combined with proteolytic digestion. Best results are obtained with retrieval solutions of low pH and at neutral and high pHs.
Background
The Ki-67 antibody was generated against a Hodgkin's disease cell line and was found to identify a nuclear antigen expressed in all non-G phases of the cell cycle, i.e., all proliferating cells. The antigen recognized by Ki-67 is a 345-395 kD non-histone protein complex which is highly susceptible to protease treatment (Gerdes 1991 et al). The gene encoding the Ki-67 protein is localized on chromosome 10 and organized in 15 exons. The center of the gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp, the "Ki-67 repeats", each containing a highly conserved new motif of 66 bp, the "Ki-67 motif". The deduced peptide sequence of this central exon is associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors. Like the latter, the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit3H-thymidine uptake in human tumor cell lines in a dose-dependent manner (Duchrow et al, 1995).
There is a good correlation between the percentage of Ki-67 positive cells in normal tissues and cell kinetic parameters such as3H-thymidine labeling indices although generally, Ki-67 immunostaining gives a higher proliferative index than the Sphase fraction, as defined by flow cytometric analysis or by 3H-thymidine incorporation.
Until recently, the limitation of the Ki-67 antibody was its requirement for frozen tissue. Several antibodies to the Ki-67 antigen are now available which are immunoreactive in routinely fixed sections, namely MIB1, Ki-S5, polyclonal Ki-67 and Ki-S1 (not commercially available to our knowledge). The proliferation indices obtained with all these five antibodies correlated well with that obtained with monoclonal Ki-67 in frozen sections, indicating that they are suitable substitutes with the advantage of being immunoreactive in fixed paraffin-embedded sections (Leong et al, 1995). This was not the case with the antibodies to proliferating cell nuclear antigens PC10 and 19A2, both of which have been demonstrated to be fixation dependent (Leong et al, 1993).
Applications
Numerous studies have compared the Ki-67 proliferation indices in frozen sections with other prognostic parameters such as tumor grade, hormone receptor status and p53 expression. In general, Ki-67 indices have been shown to be of prognostic relevance (Brown and Gatter, 1990; Raymond et al, 1988, Raymond and Leong 1989). Similar studies have now been performed in wax-embedded archival tissues with some of the new antibodies, particularly MIB1, confirming their relevance as prognostic markers (Wintzer et al, 1991; Sahin et al, 1991, Healy et al, 1995; Kindblom et al, 1995; Nawa et al, 1996). Ki-67 counts have also been useful in distinguishing between benign and malignant liver proliferations (Grigioni et al, 1995) and predicting progress of granulosa cell tumors (Costa et al 1996), Barrett's dysplasia (Polkowski et al 1995) and ovarian serous tumors (Garzetti et al, 1995).
Comments
In many cells the Ki-67 antigen appears to be localized to the nucleoli or perinucleolar region, with lighter diffuse nuclear staining in both frozen and fixed sections. When assessing proliferation indices, notable intratumoral heterogeneity will be observed and counts should be taken from the areas of highest proliferation, usually at the periphery of the tumor.
References
•Brown DC, Gatter KC 1990. Monoclonal antibody Ki-67: its use in histopathology. Histopathology 17:489-503.
•Costa MJ, Walls J, Ames P, Roth LM 1996. Transformation in recurrent ovarian granulosa cell tumors: Ki67 (MIB1) and p53 immunohistochemistry demonstrates a possible molecular basis for the poor histopathologic prediction of clinical behavior. Human Pathology 27:274-281.
•Duchrow M, Schluter C, Key G et al 1995. Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins. Archives of Immunology, Therapy and Experimentation 43:117-121.
•Garzetti GG, Ciavattini A, Goteri G et al 1995. Ki67 antigen immunostaining (MIB1 monoclonal antibody) in serous ovarian tumors: index of proliferative activity with prognostic significance. Gynecologic Oncology 56: 169-174.
•Gerdes J, Li L, Schlueter DM 1991. Immunohistochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. American Journal of Pathology 138:867-873.
•Grigioni WF, Fiorentino M, D'Errico A et al 1995. Overexpression of c-met proto-oncogene product and raised Ki67 index in hepatocellular carcinomas with respect to benign liver conditions. Hepatology 21: 1543-1546.
•Healy E, Angus B, Lawrence CM, Rees JL 1995. Prognostic value of Ki67 antigen expression in basal cell carcinomas. British Journal of Dermatology 133:737-741.
•Kindblom LG, Ahlden M, Meis-Kindblom JM, Stenman G 1995. Immunohistochemical and molecular analysis of p53, MDM2, proliferating cell nuclear antigen and Ki67 in benign and malignant peripheral nerve sheath tumours. Virchows Archives 427:19-26.
•Leong AS-Y, Milios J, Tang SK 1993. Is immunolocalization of proliferating cell nuclear antigen (PCNA) in paraffin sections a valid index of cell proliferation? Applied Immunohistochemistry 1: 127-135.
•Leong AS-Y, Vinyuvat S, Suthipintawong C, Milios J 1995. A comparative study of cell proliferation markers in breast carcinomas. Journal of Clinical Pathology: Molecular Pathology 48:M83-M87.
•Nawa G, Ueda T, Mori S et al 1996. Prognostic significance of Ki67 (MIB1) proliferation index and p53 over-expression in chondrosarcomas. International Journal of Cancer 69:86-91.
•Polkowski W, Van Lanschot JJ, Ten Kate FJ et al 1995. The value of p53 and Ki67 as markers for tumor progression in the Barrett's dysplasia-carcinoma sequence. Surgical Oncology 4:163-171. tumor progression in the Barrett's dysplasia-carcinoma sequence. Surgical Oncology 4:163-171.
•Raymond WA, Leong AS-Y 1989. The relationship between growth fractions and oestrogen receptors in human breast carcinoma, as determined by immunohistochemical staining. Journal of Pathology 158: 203-211.
•Raymond WA, Leong AS-Y, Bolt JW et al 1988. Growth fractions in human prostatic carcinoma determined by Ki-67 immunostaining. Journal of Pathology 156:161-167.
•Sahin AA, Ro J, Ro JY, et al 1991. Ki-67 immunostaining in node-negative stage I/II breast carcinoma.
•Significant correlation with prognosis. Cancer 68: 549-557.
•Wintzer HO, Zipfel I, Schulte-Monting J et al 1991. Ki-67 immunostaining in human breast tumours and its relationship to prognosis. Cancer 67:21-428.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Accurate (MM1), Biogenex (Ki-67, MIB1), Boehringer Mannheim (Ki-67, Ki-S5), Cymbus Bioscience (Ki67), Dako (Ki-67, polyclonal), Diagnostic Biosystems (Ki-67, polyclonal), Immunotech (MIB1), Novocastra (MM1), RDI (Ki67), Serotec (Ki67), and Zymed (7B11).
Fixation/Preparation
Monoclonal Ki-67 is immunoreactive in frozen sections but not in paraffin sections (although there are claims of reactivity following HIER, the results are not consistent). MIB1, Ki-S5 and polyclonal Ki-67 are all immunoreactive in routinely fixed, paraffin-embedded tissues. Immunoreactivity is enhanced following HIER combined with proteolytic digestion. Best results are obtained with retrieval solutions of low pH and at neutral and high pHs.
Background
The Ki-67 antibody was generated against a Hodgkin's disease cell line and was found to identify a nuclear antigen expressed in all non-G phases of the cell cycle, i.e., all proliferating cells. The antigen recognized by Ki-67 is a 345-395 kD non-histone protein complex which is highly susceptible to protease treatment (Gerdes 1991 et al). The gene encoding the Ki-67 protein is localized on chromosome 10 and organized in 15 exons. The center of the gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp, the "Ki-67 repeats", each containing a highly conserved new motif of 66 bp, the "Ki-67 motif". The deduced peptide sequence of this central exon is associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors. Like the latter, the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit3H-thymidine uptake in human tumor cell lines in a dose-dependent manner (Duchrow et al, 1995).
There is a good correlation between the percentage of Ki-67 positive cells in normal tissues and cell kinetic parameters such as3H-thymidine labeling indices although generally, Ki-67 immunostaining gives a higher proliferative index than the Sphase fraction, as defined by flow cytometric analysis or by 3H-thymidine incorporation.
Until recently, the limitation of the Ki-67 antibody was its requirement for frozen tissue. Several antibodies to the Ki-67 antigen are now available which are immunoreactive in routinely fixed sections, namely MIB1, Ki-S5, polyclonal Ki-67 and Ki-S1 (not commercially available to our knowledge). The proliferation indices obtained with all these five antibodies correlated well with that obtained with monoclonal Ki-67 in frozen sections, indicating that they are suitable substitutes with the advantage of being immunoreactive in fixed paraffin-embedded sections (Leong et al, 1995). This was not the case with the antibodies to proliferating cell nuclear antigens PC10 and 19A2, both of which have been demonstrated to be fixation dependent (Leong et al, 1993).
Applications
Numerous studies have compared the Ki-67 proliferation indices in frozen sections with other prognostic parameters such as tumor grade, hormone receptor status and p53 expression. In general, Ki-67 indices have been shown to be of prognostic relevance (Brown and Gatter, 1990; Raymond et al, 1988, Raymond and Leong 1989). Similar studies have now been performed in wax-embedded archival tissues with some of the new antibodies, particularly MIB1, confirming their relevance as prognostic markers (Wintzer et al, 1991; Sahin et al, 1991, Healy et al, 1995; Kindblom et al, 1995; Nawa et al, 1996). Ki-67 counts have also been useful in distinguishing between benign and malignant liver proliferations (Grigioni et al, 1995) and predicting progress of granulosa cell tumors (Costa et al 1996), Barrett's dysplasia (Polkowski et al 1995) and ovarian serous tumors (Garzetti et al, 1995).
Comments
In many cells the Ki-67 antigen appears to be localized to the nucleoli or perinucleolar region, with lighter diffuse nuclear staining in both frozen and fixed sections. When assessing proliferation indices, notable intratumoral heterogeneity will be observed and counts should be taken from the areas of highest proliferation, usually at the periphery of the tumor.
References
•Brown DC, Gatter KC 1990. Monoclonal antibody Ki-67: its use in histopathology. Histopathology 17:489-503.
•Costa MJ, Walls J, Ames P, Roth LM 1996. Transformation in recurrent ovarian granulosa cell tumors: Ki67 (MIB1) and p53 immunohistochemistry demonstrates a possible molecular basis for the poor histopathologic prediction of clinical behavior. Human Pathology 27:274-281.
•Duchrow M, Schluter C, Key G et al 1995. Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins. Archives of Immunology, Therapy and Experimentation 43:117-121.
•Garzetti GG, Ciavattini A, Goteri G et al 1995. Ki67 antigen immunostaining (MIB1 monoclonal antibody) in serous ovarian tumors: index of proliferative activity with prognostic significance. Gynecologic Oncology 56: 169-174.
•Gerdes J, Li L, Schlueter DM 1991. Immunohistochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. American Journal of Pathology 138:867-873.
•Grigioni WF, Fiorentino M, D'Errico A et al 1995. Overexpression of c-met proto-oncogene product and raised Ki67 index in hepatocellular carcinomas with respect to benign liver conditions. Hepatology 21: 1543-1546.
•Healy E, Angus B, Lawrence CM, Rees JL 1995. Prognostic value of Ki67 antigen expression in basal cell carcinomas. British Journal of Dermatology 133:737-741.
•Kindblom LG, Ahlden M, Meis-Kindblom JM, Stenman G 1995. Immunohistochemical and molecular analysis of p53, MDM2, proliferating cell nuclear antigen and Ki67 in benign and malignant peripheral nerve sheath tumours. Virchows Archives 427:19-26.
•Leong AS-Y, Milios J, Tang SK 1993. Is immunolocalization of proliferating cell nuclear antigen (PCNA) in paraffin sections a valid index of cell proliferation? Applied Immunohistochemistry 1: 127-135.
•Leong AS-Y, Vinyuvat S, Suthipintawong C, Milios J 1995. A comparative study of cell proliferation markers in breast carcinomas. Journal of Clinical Pathology: Molecular Pathology 48:M83-M87.
•Nawa G, Ueda T, Mori S et al 1996. Prognostic significance of Ki67 (MIB1) proliferation index and p53 over-expression in chondrosarcomas. International Journal of Cancer 69:86-91.
•Polkowski W, Van Lanschot JJ, Ten Kate FJ et al 1995. The value of p53 and Ki67 as markers for tumor progression in the Barrett's dysplasia-carcinoma sequence. Surgical Oncology 4:163-171. tumor progression in the Barrett's dysplasia-carcinoma sequence. Surgical Oncology 4:163-171.
•Raymond WA, Leong AS-Y 1989. The relationship between growth fractions and oestrogen receptors in human breast carcinoma, as determined by immunohistochemical staining. Journal of Pathology 158: 203-211.
•Raymond WA, Leong AS-Y, Bolt JW et al 1988. Growth fractions in human prostatic carcinoma determined by Ki-67 immunostaining. Journal of Pathology 156:161-167.
•Sahin AA, Ro J, Ro JY, et al 1991. Ki-67 immunostaining in node-negative stage I/II breast carcinoma.
•Significant correlation with prognosis. Cancer 68: 549-557.
•Wintzer HO, Zipfel I, Schulte-Monting J et al 1991. Ki-67 immunostaining in human breast tumours and its relationship to prognosis. Cancer 67:21-428.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
