Abstract
Although tumor DNA content and proliferation are usually determined by flow cytometry (FCM), quantitative microscopic image analysis is a viable alternative technique that also provides important histologic correlations. To compare these methods, we measured DNA content and proliferation in 54 consecutive breast cancers and 15 benign breast lesions by FCM and IA. DNA content determination was concordant in 49 of 54 cancers measured by FCM and IA. Four of the discordant cases were aneuploid by IA and diploid by FCM. There was good correlation between the DNA index (DI) measured by FCM and IA (r = 0.89, P less than 0.0001). Proliferation was assessed by IA quantitation of Ki-67 and PCNA/Cyclin antibody staining, as well as by flow cytometric S-phase fraction (SPF). Ki-67 positivity was greater in breast cancer than in benign controls (21.6% +/- 13.1% vs. 7.9% +/- 5.6% [P less than 0.0001]), as was PCNA/Cyclin positivity (10.2 +/- 6.7% vs. 2.7 +/- 2.5% [P less than 0.0001]). S-phase fraction measured by FCM was 7.9% +/- 5.7% for carcinomas and 3.17% +/- 2.1% for benign controls (P less than 0.003). Ki-67 and Cyclin staining, as well as SPF, were significantly increased in aneuploid compared to diploid tumors, and increased staining was associated with worsening nuclear grade. There were significant correlations between SPF and Ki-67 staining (r = 0.48, P less than 0.0001) and SPF and Cyclin staining (r = 0.48, P less than 0.0001). We conclude that FCM and IA provide comparable measurements of DNA content, although occasional discrepancies occur. Image analysis provides a valuable alternative method for assessing tumor cell proliferation and may offer certain advantages over FCM.
Full text
PDF









Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Auer G. U., Caspersson T. O., Wallgren A. S. DNA content and survival in mammary carcinoma. Anal Quant Cytol. 1980 Sep;2(3):161–165. [PubMed] [Google Scholar]
- BLACK M. M., SPEER F. D. Nuclear structure in cancer tissues. Surg Gynecol Obstet. 1957 Jul;105(1):97–102. [PubMed] [Google Scholar]
- BLOOM H. J., RICHARDSON W. W. Histological grading and prognosis in breast cancer; a study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer. 1957 Sep;11(3):359–377. doi: 10.1038/bjc.1957.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baisch H., Göhde W., Linden W. A. Analysis of PCP-data to determine the fraction of cells in the various phases of cell cycle. Radiat Environ Biophys. 1975 Jun 13;12(1):31–39. doi: 10.1007/BF02339807. [DOI] [PubMed] [Google Scholar]
- Barnard N. J., Hall P. A., Lemoine N. R., Kadar N. Proliferative index in breast carcinoma determined in situ by Ki67 immunostaining and its relationship to clinical and pathological variables. J Pathol. 1987 Aug;152(4):287–295. doi: 10.1002/path.1711520407. [DOI] [PubMed] [Google Scholar]
- Bravo R., Frank R., Blundell P. A., Macdonald-Bravo H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature. 1987 Apr 2;326(6112):515–517. doi: 10.1038/326515a0. [DOI] [PubMed] [Google Scholar]
- Charpin C., Andrac L., Habib M. C., Vacheret H., Xerri L., Devictor B., Lavaut M. N., Toga M. Immunodetection in fine-needle aspirates and multiparametric (SAMBA) image analysis. Receptors (monoclonal antiestrogen and antiprogesterone) and growth fraction (monoclonal Ki67) evaluation in breast carcinomas. Cancer. 1989 Mar 1;63(5):863–872. doi: 10.1002/1097-0142(19890301)63:5<863::aid-cncr2820630512>3.0.co;2-p. [DOI] [PubMed] [Google Scholar]
- Clark G. M., Dressler L. G., Owens M. A., Pounds G., Oldaker T., McGuire W. L. Prediction of relapse or survival in patients with node-negative breast cancer by DNA flow cytometry. N Engl J Med. 1989 Mar 9;320(10):627–633. doi: 10.1056/NEJM198903093201003. [DOI] [PubMed] [Google Scholar]
- Cornelisse C. J., Van Driel-Kulker A. M. DNA image cytometry on machine-selected breast cancer cells and a comparison between flow cytometry and scanning cytophotometry. Cytometry. 1985 Sep;6(5):471–477. doi: 10.1002/cyto.990060512. [DOI] [PubMed] [Google Scholar]
- Dressler L. G., Seamer L. C., Owens M. A., Clark G. M., McGuire W. L. DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens. Cancer. 1988 Feb 1;61(3):420–427. doi: 10.1002/1097-0142(19880201)61:3<420::aid-cncr2820610303>3.0.co;2-0. [DOI] [PubMed] [Google Scholar]
- Fallenius A. G., Auer G. U., Carstensen J. M. Prognostic significance of DNA measurements in 409 consecutive breast cancer patients. Cancer. 1988 Jul 15;62(2):331–341. doi: 10.1002/1097-0142(19880715)62:2<331::aid-cncr2820620218>3.0.co;2-8. [DOI] [PubMed] [Google Scholar]
- Fallenius A. G., Franzén S. A., Auer G. U. Predictive value of nuclear DNA content in breast cancer in relation to clinical and morphologic factors. A retrospective study of 227 consecutive cases. Cancer. 1988 Aug 1;62(3):521–530. doi: 10.1002/1097-0142(19880801)62:3<521::aid-cncr2820620314>3.0.co;2-f. [DOI] [PubMed] [Google Scholar]
- Franklin W. A., Bibbo M., Doria M. I., Dytch H. E., Toth J., DeSombre E., Wied G. L. Quantitation of estrogen receptor content and Ki-67 staining in breast carcinoma by the microTICAS image analysis system. Anal Quant Cytol Histol. 1987 Aug;9(4):279–286. [PubMed] [Google Scholar]
- Garcia R. L., Coltrera M. D., Gown A. M. Analysis of proliferative grade using anti-PCNA/cyclin monoclonal antibodies in fixed, embedded tissues. Comparison with flow cytometric analysis. Am J Pathol. 1989 Apr;134(4):733–739. [PMC free article] [PubMed] [Google Scholar]
- Gerdes J., Lelle R. J., Pickartz H., Heidenreich W., Schwarting R., Kurtsiefer L., Stauch G., Stein H. Growth fractions in breast cancers determined in situ with monoclonal antibody Ki-67. J Clin Pathol. 1986 Sep;39(9):977–980. doi: 10.1136/jcp.39.9.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gerdes J., Lemke H., Baisch H., Wacker H. H., Schwab U., Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol. 1984 Oct;133(4):1710–1715. [PubMed] [Google Scholar]
- Grogan T. M., Lippman S. M., Spier C. M., Slymen D. J., Rybski J. A., Rangel C. S., Richter L. C., Miller T. P. Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki-67. Blood. 1988 Apr;71(4):1157–1160. [PubMed] [Google Scholar]
- Haag D., Feichter G., Goerttler K., Kaufmann M. Influence of systematic errors on the evaluation of the S phase portions from DNA distributions of solid tumors as shown for 328 breast carcinomas. Cytometry. 1987 Jul;8(4):377–385. doi: 10.1002/cyto.990080406. [DOI] [PubMed] [Google Scholar]
- Kallioniemi O. P., Blanco G., Alavaikko M., Hietanen T., Mattila J., Lauslahti K., Lehtinen M., Koivula T. Improving the prognostic value of DNA flow cytometry in breast cancer by combining DNA index and S-phase fraction. A proposed classification of DNA histograms in breast cancer. Cancer. 1988 Nov 15;62(10):2183–2190. doi: 10.1002/1097-0142(19881115)62:10<2183::aid-cncr2820621019>3.0.co;2-b. [DOI] [PubMed] [Google Scholar]
- Kamel O. W., Franklin W. A., Ringus J. C., Meyer J. S. Thymidine labeling index and Ki-67 growth fraction in lesions of the breast. Am J Pathol. 1989 Jan;134(1):107–113. [PMC free article] [PubMed] [Google Scholar]
- Kaudewitz P., Braun-Falco O., Ernst M., Landthaler M., Stolz W., Gerdes J. Tumor cell growth fractions in human malignant melanomas and the correlation to histopathologic tumor grading. Am J Pathol. 1989 May;134(5):1063–1068. [PMC free article] [PubMed] [Google Scholar]
- Kurki P., Lotz M., Ogata K., Tan E. M. Proliferating cell nuclear antigen (PCNA)/cyclin in activated human T lymphocytes. J Immunol. 1987 Jun 15;138(12):4114–4120. [PubMed] [Google Scholar]
- Kurki P., Ogata K., Tan E. M. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry. J Immunol Methods. 1988 Apr 22;109(1):49–59. doi: 10.1016/0022-1759(88)90441-3. [DOI] [PubMed] [Google Scholar]
- Kurki P., Vanderlaan M., Dolbeare F., Gray J., Tan E. M. Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle. Exp Cell Res. 1986 Sep;166(1):209–219. doi: 10.1016/0014-4827(86)90520-3. [DOI] [PubMed] [Google Scholar]
- Kurtin P. J., Pinkus G. S. Leukocyte common antigen--a diagnostic discriminant between hematopoietic and nonhematopoietic neoplasms in paraffin sections using monoclonal antibodies: correlation with immunologic studies and ultrastructural localization. Hum Pathol. 1985 Apr;16(4):353–365. doi: 10.1016/s0046-8177(85)80229-x. [DOI] [PubMed] [Google Scholar]
- Lage J. M., Driscoll S. G., Yavner D. L., Olivier A. P., Mark S. D., Weinberg D. S. Hydatidiform moles. Application of flow cytometry in diagnosis. Am J Clin Pathol. 1988 May;89(5):596–600. doi: 10.1093/ajcp/89.5.596. [DOI] [PubMed] [Google Scholar]
- Lellé R. J., Heidenreich W., Stauch G., Gerdes J. The correlation of growth fractions with histologic grading and lymph node status in human mammary carcinoma. Cancer. 1987 Jan 1;59(1):83–88. doi: 10.1002/1097-0142(19870101)59:1<83::aid-cncr2820590119>3.0.co;2-i. [DOI] [PubMed] [Google Scholar]
- McDivitt R. W., Stone K. R., Craig R. B., Palmer J. O., Meyer J. S., Bauer W. C. A proposed classification of breast cancer based on kinetic information: derived from a comparison of risk factors in 168 primary operable breast cancers. Cancer. 1986 Jan 15;57(2):269–276. doi: 10.1002/1097-0142(19860115)57:2<269::aid-cncr2820570214>3.0.co;2-d. [DOI] [PubMed] [Google Scholar]
- Merkel D. E., Dressler L. G., McGuire W. L. Flow cytometry, cellular DNA content, and prognosis in human malignancy. J Clin Oncol. 1987 Oct;5(10):1690–1703. doi: 10.1200/JCO.1987.5.10.1690. [DOI] [PubMed] [Google Scholar]
- Olszewski W., Darzynkiewicz Z., Rosen P. P., Schwartz M. K., Melamed M. R. Flow cytometry of breast carcinoma: I. Relation of DNA ploidy level to histology and estrogen receptor. Cancer. 1981 Aug 15;48(4):980–984. doi: 10.1002/1097-0142(19810815)48:4<980::aid-cncr2820480421>3.0.co;2-7. [DOI] [PubMed] [Google Scholar]
- Opfermann M., Brugal G., Vassilakos P. Cytometry of breast carcinoma: significance of ploïdy balance and proliferation index. Cytometry. 1987 Mar;8(2):217–224. doi: 10.1002/cyto.990080216. [DOI] [PubMed] [Google Scholar]
- Roggendorf W., Schuster T., Peiffer J. Proliferative potential of meningiomas determined with the monoclonal antibody Ki-67. Acta Neuropathol. 1987;73(4):361–364. doi: 10.1007/BF00688260. [DOI] [PubMed] [Google Scholar]
- Sasaki K., Matsumura K., Tsuji T., Shinozaki F., Takahashi M. Relationship between labeling indices of Ki-67 and BrdUrd in human malignant tumors. Cancer. 1988 Sep 1;62(5):989–993. doi: 10.1002/1097-0142(19880901)62:5<989::aid-cncr2820620525>3.0.co;2-u. [DOI] [PubMed] [Google Scholar]
- Schnitt S. J., Connolly J. L., Harris J. R., Hellman S., Cohen R. B. Pathologic predictors of early local recurrence in Stage I and II breast cancer treated by primary radiation therapy. Cancer. 1984 Mar 1;53(5):1049–1057. doi: 10.1002/1097-0142(19840301)53:5<1049::aid-cncr2820530506>3.0.co;2-o. [DOI] [PubMed] [Google Scholar]
- Schwartz B. R., Pinkus G., Bacus S., Toder M., Weinberg D. S. Cell proliferation in non-Hodgkin's lymphomas. Digital image analysis of Ki-67 antibody staining. Am J Pathol. 1989 Feb;134(2):327–336. [PMC free article] [PubMed] [Google Scholar]
- Thorud E., Fosså S. D., Vaage S., Kaalhus O., Knudsen O. S., Børmer O., Shoaib M. C. Primary breast cancer. Flow cytometric DNA pattern in relation to clinical and histopathologic characteristics. Cancer. 1986 Feb 15;57(4):808–811. doi: 10.1002/1097-0142(19860215)57:4<808::aid-cncr2820570421>3.0.co;2-#. [DOI] [PubMed] [Google Scholar]
- Ueda T., Aozasa K., Tsujimoto M., Ohsawa M., Uchida A., Aoki Y., Ono K., Matsumoto K. Prognostic significance of Ki-67 reactivity in soft tissue sarcomas. Cancer. 1989 Apr 15;63(8):1607–1611. doi: 10.1002/1097-0142(19890415)63:8<1607::aid-cncr2820630827>3.0.co;2-1. [DOI] [PubMed] [Google Scholar]
- Walker R. A., Camplejohn R. S. Comparison of monoclonal antibody Ki-67 reactivity with grade and DNA flow cytometry of breast carcinomas. Br J Cancer. 1988 Mar;57(3):281–283. doi: 10.1038/bjc.1988.60. [DOI] [PMC free article] [PubMed] [Google Scholar]