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. 1999 Dec;81(8):1363–1370. doi: 10.1038/sj.bjc.6693399

Automatic quantification of microvessel density in urinary bladder carcinoma

K Wester 1, P Ranefall 2, E Bengtsson 2, C Busch 4, P-U Malmström 3
PMCID: PMC2362966  PMID: 10604734

Abstract

Seventy-three TUR-T biopsies from bladder carcinoma were evaluated regarding microvessel density, defined as microvessel number (nMVD) and cross-section endothelial cell area (aMVD). A semi-automatic and a newly developed, automatic image analysis technique were applied in immunostainings, performed according to an optimized staining protocol. In 12 cases a comparison of biopsy material and the corresponding cystectomy specimen were tested, showing a good correlation in 11 of 12 cases (92%). The techniques proved reproducible for both nMVD and aMVD quantifications related to total tumour area. However, the automatic method was dependent on high immunostaining quality. Simultaneous, semi-automatic quantification of microvessels, stroma and epithelial fraction resulted in a decreased reproducibility. Quantification in ten images, selected in a descending order of MVD by subjective visual judgement, showed a poor observer capacity to estimate and rank MVD. Based on our results we propose quantification of MVD related to one tissue compartment. When staining quality is of high standard, automatic quantification is applicable, which facilitates quantification of multiple areas and thus, should minimize selection variability. © 1999 Cancer Research Campaign

Keywords: microvessel density, angiogenesis, urinary bladder, carcinoma, image analysis, automatic quantification

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Selected References

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  1. Arakawa A., Soh S., Chakraborty S., Scardino P. T., Wheeler T. M. Prognostic significance of angiogenesis in clinically localized prostate cancer (staining for Factor VIII-related antigen and CD34 Antigen. Prostate Cancer Prostatic Dis. 1997 Sep;1(1):32–38. doi: 10.1038/sj.pcan.4500204. [DOI] [PubMed] [Google Scholar]
  2. Barbareschi M., Gasparini G., Morelli L., Forti S., Dalla Palma P. Novel methods for the determination of the angiogenic activity of human tumors. Breast Cancer Res Treat. 1995;36(2):181–192. doi: 10.1007/BF00666039. [DOI] [PubMed] [Google Scholar]
  3. Bochner B. H., Cote R. J., Weidner N., Groshen S., Chen S. C., Skinner D. G., Nichols P. W. Angiogenesis in bladder cancer: relationship between microvessel density and tumor prognosis. J Natl Cancer Inst. 1995 Nov 1;87(21):1603–1612. doi: 10.1093/jnci/87.21.1603. [DOI] [PubMed] [Google Scholar]
  4. Butler H. B., Sheaf E. W. ANOCI-ASSOCIATION IN THE PREVENTION OF SHOCK AND POST-OPERATIVE DISCOMFORTS: AN EXPERIENCE OF 143 CASES. Br Med J. 1914 Jul 18;2(2794):113–115. doi: 10.1136/bmj.2.2794.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chodak G. W., Haudenschild C., Gittes R. F., Folkman J. Angiogenic activity as a marker of neoplastic and preneoplastic lesions of the human bladder. Ann Surg. 1980 Dec;192(6):762–771. doi: 10.1097/00000658-198012000-00012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Choi H. K., Jarkrans T., Bengtsson E., Vasko J., Wester K., Malmström P. U., Busch C. Image analysis based grading of bladder carcinoma. Comparison of object, texture and graph based methods and their reproducibility. Anal Cell Pathol. 1997;15(1):1–18. doi: 10.1155/1997/147187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dickinson A. J., Fox S. B., Persad R. A., Hollyer J., Sibley G. N., Harris A. L. Quantification of angiogenesis as an independent predictor of prognosis in invasive bladder carcinomas. Br J Urol. 1994 Dec;74(6):762–766. doi: 10.1111/j.1464-410x.1994.tb07122.x. [DOI] [PubMed] [Google Scholar]
  8. Dinney C. P., Babkowski R. C., Antelo M., Perrotte P., Liebert M., Zhang H. Z., Palmer J., Veltri R. W., Katz R. L., Grossman H. B. Relationship among cystectomy, microvessel density and prognosis in stage T1 transitional cell carcinoma of the bladder. J Urol. 1998 Oct;160(4):1285–1290. [PubMed] [Google Scholar]
  9. Duarte I. G., Bufkin B. L., Pennington M. F., Gal A. A., Cohen C., Kosinski A. S., Mansour K. A., Miller J. I. Angiogenesis as a predictor of survival after surgical resection for stage I non-small-cell lung cancer. J Thorac Cardiovasc Surg. 1998 Mar;115(3):652–659. doi: 10.1016/S0022-5223(98)70331-9. [DOI] [PubMed] [Google Scholar]
  10. Fisher C. J., Gillett C. E., Vojtesek B., Barnes D. M., Millis R. R. Problems with p53 immunohistochemical staining: the effect of fixation and variation in the methods of evaluation. Br J Cancer. 1994 Jan;69(1):26–31. doi: 10.1038/bjc.1994.4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995 Jan;1(1):27–31. doi: 10.1038/nm0195-27. [DOI] [PubMed] [Google Scholar]
  12. Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst. 1990 Jan 3;82(1):4–6. doi: 10.1093/jnci/82.1.4. [DOI] [PubMed] [Google Scholar]
  13. Fox S. B., Leek R. D., Weekes M. P., Whitehouse R. M., Gatter K. C., Harris A. L. Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis. J Pathol. 1995 Nov;177(3):275–283. doi: 10.1002/path.1711770310. [DOI] [PubMed] [Google Scholar]
  14. Fox S. B. Tumour angiogenesis and prognosis. Histopathology. 1997 Mar;30(3):294–301. doi: 10.1046/j.1365-2559.1997.d01-606.x. [DOI] [PubMed] [Google Scholar]
  15. Green M. A., Sviland L., Malcolm A. J., Pearson A. D. Improved method for immunoperoxidase detection of membrane antigens in frozen sections. J Clin Pathol. 1989 Aug;42(8):875–880. doi: 10.1136/jcp.42.8.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grossfeld G. D., Ginsberg D. A., Stein J. P., Bochner B. H., Esrig D., Groshen S., Dunn M., Nichols P. W., Taylor C. R., Skinner D. G. Thrombospondin-1 expression in bladder cancer: association with p53 alterations, tumor angiogenesis, and tumor progression. J Natl Cancer Inst. 1997 Feb 5;89(3):219–227. doi: 10.1093/jnci/89.3.219. [DOI] [PubMed] [Google Scholar]
  17. Hollingsworth H. C., Kohn E. C., Steinberg S. M., Rothenberg M. L., Merino M. J. Tumor angiogenesis in advanced stage ovarian carcinoma. Am J Pathol. 1995 Jul;147(1):33–41. [PMC free article] [PubMed] [Google Scholar]
  18. Jaeger T. M., Weidner N., Chew K., Moore D. H., Kerschmann R. L., Waldman F. M., Carroll P. R. Tumor angiogenesis correlates with lymph node metastases in invasive bladder cancer. J Urol. 1995 Jul;154(1):69–71. [PubMed] [Google Scholar]
  19. Jarkrans T., Vasko J., Bengtsson E., Choi H. K., Malmström P. U., Wester K., Busch C. Grading of transitional cell bladder carcinoma by image analysis of histological sections. Anal Cell Pathol. 1995 Mar;8(2):135–158. [PubMed] [Google Scholar]
  20. Kohlberger P. D., Obermair A., Sliutz G., Heinzl H., Koelbl H., Breitenecker G., Gitsch G., Kainz C. Quantitative immunohistochemistry of factor VIII-related antigen in breast carcinoma: a comparison of computer-assisted image analysis with established counting methods. Am J Clin Pathol. 1996 Jun;105(6):705–710. doi: 10.1093/ajcp/105.6.705. [DOI] [PubMed] [Google Scholar]
  21. Lambkin H. A., Mothersill C. M., Kelehan P. Variations in immunohistochemical detection of p53 protein overexpression in cervical carcinomas with different antibodies and methods of detection. J Pathol. 1994 Jan;172(1):13–18. doi: 10.1002/path.1711720105. [DOI] [PubMed] [Google Scholar]
  22. Leong A. S., Gilham P. N. The effects of progressive formaldehyde fixation on the preservation of tissue antigens. Pathology. 1989 Oct;21(4):266–268. doi: 10.3109/00313028909061071. [DOI] [PubMed] [Google Scholar]
  23. Malmström P. U., Rintala E., Wahlqvist R., Hellström P., Hellsten S., Hannisdal E. Five-year followup of a prospective trial of radical cystectomy and neoadjuvant chemotherapy: Nordic Cystectomy Trial I. The Nordic Cooperative Bladder Cancer Study Group. J Urol. 1996 Jun;155(6):1903–1906. [PubMed] [Google Scholar]
  24. Malmström P. U., Wester K., Vasko J., Busch C. Expression of proliferative cell nuclear antigen (PCNA) in urinary bladder carcinoma. Evaluation of antigen retrieval methods. APMIS. 1992 Nov;100(11):988–992. doi: 10.1111/j.1699-0463.1992.tb04030.x. [DOI] [PubMed] [Google Scholar]
  25. Martin L., Green B., Renshaw C., Lowe D., Rudland P., Leinster S. J., Winstanley J. Examining the technique of angiogenesis assessment in invasive breast cancer. Br J Cancer. 1997;76(8):1046–1054. doi: 10.1038/bjc.1997.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Morgan J. M., Navabi H., Schmid K. W., Jasani B. Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol. 1994 Dec;174(4):301–307. doi: 10.1002/path.1711740410. [DOI] [PubMed] [Google Scholar]
  27. Nakanishi K., Hiroi S., Kawai T., Torikata C. Expression of platelet-derived growth-factor B-chain mRNA and tumor angiogenesis in invasive transitional cell carcinoma of the upper urinary tract. Mod Pathol. 1997 Apr;10(4):341–347. [PubMed] [Google Scholar]
  28. Penfold C. N., Partridge M., Rojas R., Langdon J. D. The role of angiogenesis in the spread of oral squamous cell carcinoma. Br J Oral Maxillofac Surg. 1996 Feb;34(1):37–41. doi: 10.1016/s0266-4356(96)90133-3. [DOI] [PubMed] [Google Scholar]
  29. Philp E. A., Stephenson T. J., Reed M. W. Prognostic significance of angiogenesis in transitional cell carcinoma of the human urinary bladder. Br J Urol. 1996 Mar;77(3):352–357. doi: 10.1046/j.1464-410x.1996.08475.x. [DOI] [PubMed] [Google Scholar]
  30. Ranefall P., Egevad L., Nordin B., Bengtsson E. A new method for segmentation of colour images applied to immunohistochemically stained cell nuclei. Anal Cell Pathol. 1997;15(3):145–156. doi: 10.1155/1997/304073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ranefall P., Wester K., Busch C., Malmström P. U., Bengtsson E. Automatic quantification of microvessels using unsupervised image analysis. Anal Cell Pathol. 1998;17(2):83–92. doi: 10.1155/1998/490585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vermeulen P. B., Gasparini G., Fox S. B., Toi M., Martin L., McCulloch P., Pezzella F., Viale G., Weidner N., Harris A. L. Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation. Eur J Cancer. 1996 Dec;32A(14):2474–2484. doi: 10.1016/s0959-8049(96)00379-6. [DOI] [PubMed] [Google Scholar]
  33. Wagner B. M. To immunostain or not to immunostain, that is the question. Mod Pathol. 1993 Mar;6(2):113–113. [PubMed] [Google Scholar]
  34. Weidner N. Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Res Treat. 1995;36(2):169–180. doi: 10.1007/BF00666038. [DOI] [PubMed] [Google Scholar]
  35. Weidner N., Folkman J., Pozza F., Bevilacqua P., Allred E. N., Moore D. H., Meli S., Gasparini G. Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst. 1992 Dec 16;84(24):1875–1887. doi: 10.1093/jnci/84.24.1875. [DOI] [PubMed] [Google Scholar]
  36. Weidner N. Intratumor microvessel density as a prognostic factor in cancer. Am J Pathol. 1995 Jul;147(1):9–19. [PMC free article] [PubMed] [Google Scholar]
  37. Weidner N., Semple J. P., Welch W. R., Folkman J. Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma. N Engl J Med. 1991 Jan 3;324(1):1–8. doi: 10.1056/NEJM199101033240101. [DOI] [PubMed] [Google Scholar]
  38. Wold L. E., Corwin D. J., Rickert R. R., Pettigrew N., Tubbs R. R. Interlaboratory variability of immunohistochemical stains. Results of the cell markers survey of the College of American Pathologists. Arch Pathol Lab Med. 1989 Jun;113(6):680–683. [PubMed] [Google Scholar]
  39. de Jong J. S., van Diest P. J., Baak J. P. Heterogeneity and reproducibility of microvessel counts in breast cancer. Lab Invest. 1995 Dec;73(6):922–926. [PubMed] [Google Scholar]

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