Summary
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.
Similar content being viewed by others
Article PDF
Change history
16 November 2011
This paper was modified 12 months after initial publication to switch to Creative Commons licence terms, as noted at publication
References
Arakawa, A, Soh, S, Chakraborty, S, Scardino, PT & Wheeler, TM (1997). Prognostic significance of angiogenesis in clinically localized prostate cancer (staining for Factor VIII-related antigen and CD34 antigen). Prost Cancer Prost Dis 1: 32–38.
Barbareschi, M, Gasparini, G, Morelli, L, Forti, S & Dalla Palma, P (1995a). Novel methods for the determination of the angiogenic activity of human tumors. Breast Cancer Res Treat 36: 181–192.
Barbareschi, M, Weidner, N, Gasparini, G, Morelli, L, Forti, S, Eccher, C, Fina, P, Caffo, O, Leonardi, E, Mauri, F, Bevilacqua, P & Dalla Palma, P (1995b). Microvessel density quantification in breast carcinomas. Assessment by light microscopy vs. a computer-aided image analysis system. Appl Immunohistochem 3: 75–84.
Bochner, BH, Cote, RJ, Weidner, N, Groshen, S, Chen, SC, Skinner, DG & Nichols, PW (1995). Angiogenesis in bladder cancer: relationship between microvessel density and tumor prognosis. J Natl Cancer Inst 87: 1603–1612.
Chodak, GW, Haudenschild, C, Gittes, RF & Folkman, J (1980). Angiogenic activity as a marker of neoplastic and preneoplastic lesions of the human bladder. Ann Surg 192: 762–771.
Choi, HK, Jarkrans, T, Bengtsson, E, Vasko, J, Wester, K, Malmstrom, PU & Busch, C (1997). Image analysis based grading of bladder carcinoma. Comparison of object, texture and graph based methods and their reproducibility. Anal Cell Pathol 15: 1–18.
de Jong, JS, van Diest, PJ & Baak, JP (1995). Heterogeneity and reproducibility of microvessel counts in breast cancer. Lab Invest 73: 922–926.
Dickinson, AJ, Fox, SB, Persad, RA, Hollyer, J, Sibley, GN & Harris, AL (1994). Quantification of angiogenesis as an independent predictor of prognosis in invasive bladder carcinomas. Br J Urol 74: 762–726.
Dinney, CP, Babkowski, RC, Antelo, M, Perrotte, P, Liebert, M, Zhang, HZ, Palmer, J, Veltri, RW, Katz, RL & Grossman, HB (1998). Relationship among cystectomy, microvessel density and prognosis in stage T1 transitional cell carcinoma of the bladder. J Urol 160: 1285–1290.
Duarte, IG, Bufkin, BL, Pennington, MF, Gal, AA, Cohen, C, Kosinski, AS, Mansour, KA & Miller, JI (1998). Angiogenesis as a predictor of survival after surgical resection for stage I non-small-cell lung cancer. J Thorac Cardiovasc Surg 115: 652–658.
Fisher, CJ, Gillett, CE, Vojtesek, B, Barnes, DM & Millis, RR (1994). Problems with p53 immunohistochemical staining: the effect of fixation and variation in the methods of evaluation. Br J Cancer 69: 26–31.
Folkman, J (1990). What is the evidence that tumors are angiogenesis dependent? [editorial]. J Natl Cancer Inst 82: 4–6.
Folkman, J (1995). Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1: 27–31.
Fox, SB (1997). Tumour angiogenesis and prognosis. Histopathology 30: 294–301.
Fox, SB, Leek, RD, Weekes, MP, Whitehouse, RM, Gatter, KC & Harris, AL (1995). Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis. J Pathol 177: 275–283.
Green, MA, Sviland, L, Malcolm, AJ & Pearson, AD (1989). Improved method for immunoperoxidase detection of membrane antigens in frozen sections. J Clin Pathol 42: 875–880.
Grossfeld, GD, Ginsberg, DA, Stein, JP, Bochner, BH, Esrig, D, Groshen, S, Dunn, M, Nichols, PW, Taylor, CR, Skinner, DG & Cote, RJ (1997). Thrombospondin-1 expression in bladder cancer: association with p53 alterations, tumor angiogenesis, and tumor progression. J Natl Cancer Inst 89: 219–227.
Hawke, CK, Delahunt, B & Davidson, PJT (1996). The prognostic value of microvessel density in bladder cancer. J Urol 155: 690A (abstract 1517)
Hollingsworth, HC, Kohn, EC, Steinberg, SM, Rothenberg, ML & Merino, MJ (1995). Tumor angiogenesis in advanced stage ovarian carcinoma. Am J Pathol 147: 33–41.
Jaeger, TM, Weidner, N, Chew, K, Moore, DH, Kerschmann, RL, Waldman, FM & Carroll, PR (1995). Tumor angiogenesis correlates with lymph node metastases in invasive bladder cancer. J Urol 154: 69–71.
Jarkrans, T, Vasko, J, Bengtsson, E, Choi, HK, Malmstrom, PU, Wester, K & Busch, C (1995). Grading of transitional cell bladder carcinoma by image analysis of histological sections. Anal Cell Pathol 8: 135–158.
Kohlberger, PD, Obermair, A, Sliutz, G, Heinzl, H, Koelbl, H, Breitenecker, G, Gitsch, G & Kainz, C (1996). 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 105: 705–710.
Lambkin, HA, Mothersill, CM & Kelehan, P (1994). Variations in immunohistochemical detection of p53 protein overexpression in cervical carcinomas with different antibodies and methods of detection. J Pathol 172: 13–18.
Lee, AHS, Happerfield, LC & Bobrov, LG (1997). Comparison of four endothelial markers for assessing angiogenesis in carcinoma of the breast. J Cell Pathol 2: 67–73.
Leong, AS-Y & Gilham, PN (1989). The effects of progressive formaldehyde fixation on preservation of tissue antigens. Pathology 21: 266–268.
McDermott, N, Farah, N, Milburn, C, Butler, D, Kay, E, Barry Walsh, C & Leader, M (1997). MIB1 staining in archival material: problems with immunostaining in older paraffin embedded tissue may limit its predictive value. J Cell Pathol 2: 113–115.
Malmstrom, PU, Wester, K, Vasko, J & Busch, C (1992). Expression of proliferative cell nuclear antigen (PCNA) in urinary bladder carcinoma. Evaluation of antigen retrieval methods. Apmis 100: 988–992.
Malmstrom, PU, Rintala, E, Wahlqvist, R, Hellstrom, P, Hellsten, S & Hannisdal, E (1996). Five-year followup of a prospective trial of radical cystectomy and neoadjuvant chemotherapy: Nordic Cystectomy Trial I. The Nordic Cooperative Bladder Cancer Study Group [see comments]. J Urol 155: 1903–1906.
Martin, L, Green, B, Renshaw, C, Lowe, D, Rudland, P, Leinster, SJ & Winstanley, J (1997). Examining the technique of angiogenesis assessment in invasive breast cancer. Br J Cancer 76: 1046–1054.
Morgan, JM, Navabi, H, Schmid, KW & Jasani, B (1994). Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol 174: 301–307.
Nakanishi, K, Hiroi, S, Kawai, T & Torikata, C (1997). Expression of platelet-derived growth-factor B-chain mRNA and tumor angiogenesis in invasive transitional cell carcinoma of the upper urinary tract. Mod Pathol 10: 341–347.
Penfold, CN, Partridge, M, Rojas, R & Langdon, JD (1996). The role of angiogenesis in the spread of oral squamous cell carcinoma. Br J Oral Maxillofac Surg 34: 37–41.
Philp, EA, Stephenson, TJ & Reed, MW (1996). Prognostic significance of angiogenesis in transitional cell carcinoma of the human urinary bladder. Br J Urol 77: 352–357.
Ranefall, P, Egevad, L, Nordin, B & Bengtsson, E (1997). A new method for segmentation of colour images applied to immunohistochemically stained cell nuclei. Anal Cell Pathol 15: 145–156.
Ranefall, P, Wester, K, Busch, C, Malmström, P-U & Bengtsson, E (1998). Automatic quantification of microvessels using unsupervised image analysis. Anal Cell Pathol 17: 83–92.
Vermeulen, PB, Gasparini, G, Fox, SB, Toi, M, Martin, L, McCulloch, P, Pezzella, F, Viale, G, Weidner, N, Harris, AL & Dirix, LY (1996). Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation. Eur J Cancer 32a: 2474–2484.
Wagner, BM (1993). To immunostain or not to immunostain, that is the question [editorial]. Mod Pathol 6: 113
Weidner, N (1995a). Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Res Treat 36: 169–180.
Weidner, N (1995b). Intratumor microvessel density as a prognostic factor in cancer [comment]. Am J Pathol 147: 9–19.
Weidner, N, Semple, JP, Welch, WR & Folkman, J (1991). Tumor angiogenesis and metastasis – correlation in invasive breast carcinoma. N Engl J Med 324: 1–8.
Weidner, N, Folkman, J, Pozza, F, Bevilacqua, P, Allred, EN, Moore, DH, Meli, S & Gasparini, G (1992). Tumor angiogenesis: a new significant and independent indicator in early-stage breast carcinoma. J Natl Cancer Inst 84: 1875–1887.
Wold, LE, Corwin, DJ, Rickert, RR, Pettigrew, N & Tubbs, RR (1989). Interlaboratory variability of immunohistochemical stains. Results of the cell markers survey of the College of American Pathologists. Arch Pathol Lab Med 113: 680–683.
Author information
Authors and Affiliations
Rights and permissions
From twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
About this article
Cite this article
Wester, K., Ranefall, P., Bengtsson, E. et al. Automatic quantification of microvessel density in urinary bladder carcinoma. Br J Cancer 81, 1363–1370 (1999). https://doi.org/10.1038/sj.bjc.6693399
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.bjc.6693399
Keywords
This article is cited by
-
Automatic identification of angiogenesis in double stained images of liver tissue
BMC Bioinformatics (2009)