Breast Cancer Research and Treatment

, Volume 36, Issue 2, pp 169–180 | Cite as

Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors

  • Noel Weidner


Abundant evidence has shown that tumor growth and metastasis are dependent upon tumor angiogenesis (TA). TA refers to the growth of new vessels toward and within the tumor. Until TA occurs, tumors grow no larger than 2–4 mm in diameter. Also, TA is necessary at the beginning and at the end of the metastatic cascade of events. Thus, it seems reasonable that increasing intratumoral microvascular density (iMVD) might correlate with greater tumor aggressiveness, such as a higher frequency of metastases and/or decreased survival. Indeed, in 1991 my colleagues and I reported a statistically significant association between greater incidence of metastases in patients with breast carcinoma and increasing iMVD. Microvessel density was measured with a light microscope in a single area of invasive tumor (200x field or 0.74 mm2) representative of the highest microvessel density (neovascular “hot spot”). This was done after endothelial cells, lining the microvessels, had been highlighted with anti-factor VIII-related antigen/von Willebrand's factor (F8RA/vWF). Subsequent studies by other investigators, using either anti-F8RA/vWF or other relatively vessel-specific reagents such as anti-CD31, have shown that the association of greater tumor aggressiveness with increasing iMVD exists not only in breast carcinoma, but also in other solid tumors. This article reviews the methods of highlighting intratumoral vessels and describes the techniques for counting these vessels for assessing iMVD.

Key words

angiogenesis breast cancer endothelial cell prognosis 


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  1. 1.
    Brem S, Cotran R, Folkman J: Tumor angiogenesis: a quantitative method for histologic grading. J Natl Cancer Inst 48:347–356, 1972.Google Scholar
  2. 2.
    Srivastava A, Laidler P, Hughes LE, Woodcock J, Shedden EJ: Neovascularization in human cutaneous melanoma: a quantitative morphological and Doppler ultrasound study. Eur J Cancer Clin Oncol 22:1205–1209, 1986Google Scholar
  3. 3.
    Srivastava A, Laidler P, Davies R, Horgan K, Hughes LE: The prognostic significance of tumor vascularity in intermediate-thickness (0.76–4.0 mm thick) skin melanoma. Am J Pathol 133:419–423, 1988Google Scholar
  4. 4.
    Weidner N, Semple JP, Welch WR, Folkman J: Tumor angiogenesis and metastasis — correlation in invasive breast carcinoma. N Engl J Med 324:1–8, 1991Google Scholar
  5. 5.
    McComb RD, Jones TR, Pizzo SV, Bigner D: Specificity and sensitivity of immunohistochemical detection of factor VIII/von Willebrand factor antigen in formalin-fixed paraffin-embedded tissue. J Histochem Cytochem 30:371–377, 1982Google Scholar
  6. 6.
    Gown AM, de Wever N, Battifora H: Microwavebased antigenic unmasking. A revolutionary new technique for routine immunohistochemistry. Appl Immunohistochem 1:256–266, 1993Google Scholar
  7. 7.
    Weidner N, Folkman J, Pozza F, Bevilacqua P, Allred EN, Moore DH, Meli S, Gasparini G: Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 84:1875–1887, 1992Google Scholar
  8. 8.
    Bosari S, Lee AKC, DeLellis RA, et al: Microvessel quantitation and prognosis in invasive breast carcinoma. Hum Pathol 23:755–761, 1992Google Scholar
  9. 9.
    Longacre TA, Rouse RV: CD31: A new marker for vascular neoplasia. Adv Anat Pathol 1:16–20, 1994Google Scholar
  10. 10.
    Horak E, Leek R, Klenk N, LeJeune S, Smith K, Stuart N, Greenall M, Stepniewska K, Harris AL: Angiogenesis, assessed by platelet/endothelial cell adhesion molecule antibodies, as indicator of node metastases and survival in breast cancer. Lancet 340:1120–1124, 1992Google Scholar
  11. 11.
    Gasparini G, Weidner N, Bevilacqua P, Maluta S, Dalla Palma P, Caffo O, Barbareschi M, Boracchi P, Marubini E, Pozza F: Tumor microvessel density, p53 expression, tumor size, and peritumoral lymphatic vessel invasion are relevant prognostic markers in node-negative breast carcinoma. J Clin Oncol 12:454–466, 1994Google Scholar
  12. 12.
    Toi M, Kashitani J, Tominaga T: Tumor angiogenesis is an independent prognostic indicator of primary breast carcinoma. Int J Cancer 55:371–374, 1993Google Scholar
  13. 13.
    Visscher DW, Smilanetz S, Drozdowicz S, Wykes SM: Prognostic significance of image morphometric microvessel enumeration in breast carcinoma. Analytical Quant Cytolol Histol 15:88–92, 1993Google Scholar
  14. 14.
    Arihiro K, Inai K, Kurihara K, Takeda S, Kaneko M: Distribution of laminin, type IV collagen, and fibronectin in the invasive component of breast carcinoma. Acta Pathol Jap 43:758–764, 1993Google Scholar
  15. 15.
    Macchiarini P, Fontanini G, Hardin MJ, Hardin MJ, Squartini F, Angeletti CA: Relation of neovasculature to metastasis of non-small-cell lung cancer. Lancet 340:145–146, 1992Google Scholar
  16. 16.
    Macchiarini P, Fontanini G, Dulmet E, de Montpreville V, Chapelier AR, Cerrin J, Le Roy Ladurie F, Dartevelle PG: Angiogenesis: an indicator of metastasis in non-small-cell lung cancer invading the thoracic inlet. Ann Thorac Surg 57:1534–1539, 1994Google Scholar
  17. 17.
    Wakui S, Furusato M, Itoh T, Sasaki H, Akiyama A, Kinoshita I, Asano K, Tokuda T, Aizawa S, Ushigome S: Tumor angiogenesis in prostatic carcinoma with and without bone marrow metastasis: a morphometric study. J Pathol 168:257–262, 1992Google Scholar
  18. 18.
    Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J: Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 143:401–409, 1993Google Scholar
  19. 19.
    Fregene TA, Khanuja PS, Noto AC, Gehani SK, Van Egmont EM, Luz DA, Pienta KJ: Tumor-associated angiogenesis in prostate cancer. Anticancer Res 13: 2377–2381, 1993Google Scholar
  20. 20.
    Brawer MK, Deering RE, Brown M, Preston SD, Bigler SA: Predictors of pathologic stage in prostate carcinoma. Cancer 73:678–687, 1994Google Scholar
  21. 21.
    Sneige N, Singletary E, Sahin A, et al: Multiparameter analysis of potential prognostic factors in node negative breast cancer patients. Modern Pathol 5:18a, 1992Google Scholar
  22. 22.
    Sahin AA, Sneige N, Ordonez GN, et al: Tumor angiogenesis detected byUlex europaeus agglutinin 1 lectin (UEA1) and factor VIII immunostaining in node-negative breast carcinoma (NNBC) treated by mastectomy: prediction of tumor recurrence. Modern Pathol 6:19a, 1993Google Scholar
  23. 23.
    Obermair A, Czerwenka K, Kurz C, Buxbaum P, Schemper M, Sevela P: Influence of tumoral microvessel density on the recurrence-free survival in human breast cancer: preliminary results. Onkologie 17:44–49, 1994Google Scholar
  24. 24.
    Gasparini G, Weidner N, Bevilacqua P, Maluta S, Boracchi P, Testolin A, Pozza F, Folkman J: Intratumoral microvessel density and p53 protein: correlation with metastasis in head-and-neck squamous-cell carcinoma. Int J Cancer 55:739–744, 1993Google Scholar
  25. 25.
    Mikami Y, Tsukuda M, Mochimatsu I, Kokatsu T, Yago T, Sawaki S: Angiogenesis in head and neck tumors. Nip Jib Gak Kai 96:645–50, 1991Google Scholar
  26. 26.
    Albo D, Granick MS, Jhala N, Atkinson B, Solomon MP: The relationship of angiogenesis to biological activity in human squamous cell carcinomas of the head and neck. Ann Plast Surg 32:588–594, 1994Google Scholar
  27. 27.
    Petruzzelli GJ, Snyderman CH, Johnson JT, Myers EN: Angiogenesis induced by head and neck squamous cell carcinoma xenografts in the chick embryo chorioallantoic membrane model. Ann Otol Rhinol Laryngol 102:215–221, 1993Google Scholar
  28. 28.
    Vesalainen S, Lipponen P, Talja M, Alhava E, Syrjanen K: Tumor vascularity and basement membrane structure as prognostic factors in T1–2M0 prostatic adenocarcinoma. Anticancer Res 14:709–714, 1994Google Scholar
  29. 29.
    Saclarides TJ, Speziale NJ, Drab E, Szeluga DJ, Rubin DB: Tumor angiogenesis and rectal carcinoma. Dis Colon Rectum 37:921–926, 1994Google Scholar
  30. 30.
    Olivarez D, Ulbright T, DeRiese W, Foster R, Reister T, Einhorn L, Sledge G: Neovascularization in clinical stage A testicular germ cell tumor: prediction of metastatic disease. Cancer Res 54:2800–2802, 1994Google Scholar
  31. 31.
    Hollingsworth HC, Steinberg SM, Kohn E, Bryant B, Merino MJ: Tumor angiogenesis in advanced stage ovarian cancer. Mod Pathol 7:89a (Abst#514), 1994Google Scholar
  32. 32.
    Jaeger TM, Weidner N, Chew K, Moore DH, Kerschmann RL, Waldman FM, Carroll PR: Tumor angiogenesis and lymph node metastases in invasive bladder carcinoma. J Urol 151:348A (#482), 1994Google Scholar
  33. 33.
    Barnhill RL, Fandrey K, Levy MA, Mihm MC, Hyman B: Angiogenesis and tumor progression of melanoma. Quantitation of vascularity in melanocytic nevi and cutaneous melanoma. Lab Invest 67:331–337, 1992Google Scholar
  34. 34.
    Barnhill RL, Levy MA: Regressing thin cutaneous malignant melanomas (≤1.0 mm) are associated with angiogenesis. Am J Pathol 143:99–104, 1993Google Scholar
  35. 35.
    Nasser I, Tahan S: Angiogenesis in melanocytic lesions and its relationship to tumor progression. Mod Pathol 6:35A (#190), 1993Google Scholar
  36. 36.
    Fallowfield ME, Cook MG: The vascularity of primary cutaneous melanoma. J Pathol 164:241–244, 1991Google Scholar
  37. 37.
    Cockerell CJ, Sonnier G, Kelly L, Patel S: Comparative analysis of neovascularization in primary cutaneous melanoma and Spitz nevus. Am J Dermatopathol 16:9–13, 1994Google Scholar
  38. 38.
    Graham CH, Rivers J, Kerbel RS, Stankiewicz KS, White WL: Extent of vascularization as a prognostic indicator in thin (<0.76 mm) malignant melanomas. Am J Pathol 145:510–514, 1994Google Scholar
  39. 39.
    Ewaskow SP, Collins CA, Conrad EU, Gown AM, Schmidt RA: Quantitative assessment of blood vessel density and size in soft-tissue tumors. Mod Pathol 6:6A (#12), 1993Google Scholar
  40. 40.
    Li VW, Folkerth RD, Watanabe H, Yu C, Rupnick M, Barnes P, Scott RM, Black PM, Sallan SE, Folkman J: Microvessel count and cerebrospinal fluid basic fibroblast growth factor in children with brain tumors. Lancet 334:82–86, 1994Google Scholar
  41. 41.
    Van Hoef MEHM, Knox WF, Dhesi SS, Howell A, Schor AM: Assessment of tumor vascularity as a prognostic factor in lymph node negative invasive breast cancer. Eur J Cancer 29A:1141–1145, 1993Google Scholar
  42. 42.
    Hall NR, Fish DE, Hunt N, Goldin RD, Guillou PJ, Monson JRT: Is the relationship between angiogenesis and metastasis in breast cancer real? Surg Oncol 1:223–229, 1992Google Scholar
  43. 43.
    Weidner N: The relationship of tumor angiogenesis and metastasis with emphasis on invasive breast carcinoma.In: Weinstein RS (ed) Advances in Pathology and Laboratory Medicine. Mosby Year Book, Chicago, 1992, vol 5, pp 101–121Google Scholar
  44. 44.
    Ordonez NG, Batsakis JG: Comparison ofUlex europaeus I lectin and factor VIII-related antigen in vascular lesions. Arch Pathol Lab Med 108:129–132, 1984Google Scholar
  45. 45.
    Lee AKC, DeLellis RA, Wolfe HJ: Intramammary lymphatic invasion in breast carcinomas. Evaluation using ABH isoantigens as endothelial markers. Am J Surg Pathol 10:589–594, 1986Google Scholar
  46. 46.
    Bettelheim R, Mitchell D, Gusterson BA: Immunocytochemistry in the identification of vascular invasion in breast cancer. J Clin Pathol 37:364–366, 1984Google Scholar
  47. 47.
    Kiyoshi M, Rosai J, Burgdorf WHC: Localization of factor VIII-related antigens in vascular endothelial cells using an immunoperoxidase method. Am J Surg Pathol 4:273–276, 1980Google Scholar
  48. 48.
    Mukai K, Rosai J: “Factor VIII-related antigen: an endothelial marker.In: DeLellis RA (ed) Advances in Immunohistochemistry. Masson Publishing USA, New York, 1984, pp 253–261Google Scholar
  49. 49.
    Nagle RB, Witte MH, Martinez AP, Witte CL, Hendrix MJC, Way D, Reed K: Factor VIII-associated antigen in human lymphatic endothelium. Lymphology 20:20–24, 1987Google Scholar
  50. 50.
    Lee AKC, DeLellis RA, Silverman ML, Wolfe HJ: Perspectives in pathology. Lymphatic and blood vessel invasion in breast carcinoma: a useful prognostic indicator. Hum Pathol 17:984–987, 1986Google Scholar
  51. 51.
    Svanholm H, Nielsen K, Hauge P: Factor VIII-related antigen and lymphatic collecting vessels. Virchows Arch [Pathol Anat] 404:223–228, 1984Google Scholar
  52. 52.
    Ordonez NG, Brooks T, Thompson S, Batsakis JG: Use ofUlex europaeus agglutinin I in the identification of lymphatic and blood vessel invasion in previously stained microscopic slides. Am J Surg Pathol 11:543–550, 1987Google Scholar
  53. 53.
    Jain RK: Delivery of novel therapeutic agents in tumors: physiologic barriers and strategies. J Natl Cancer Inst 81:570–576, 1989Google Scholar
  54. 54.
    Tanigawa N, Kanazawa T, Satomura K, et al: Experimental study on lymphatic vascular changes in the development of cancer. Lymphology 14:149–154, 1981Google Scholar
  55. 55.
    Zeidman I, Copeland B, Warren S: Experimental studies on the spread of cancer in the lymphatic system. II. Absence of lymphatic supply in carcinoma. Cancer 8:123–127, 1955Google Scholar
  56. 56.
    Gilchrist RK: Surgical management of advanced cancer of the breast. Arch Surg 61:913–929, 1950Google Scholar
  57. 57.
    Papadimitriou JM, Woods AE: Structural and functional characteristics of the microcirculation in neoplasms. J Pathol 116:65–72, 1975Google Scholar
  58. 58.
    Barsky SH, Baker A, Siegal GP, Togo S, Liotta LA: Use of anti-basement membrane antibodies to distinguish blood vessel capillaries from lymphatic capillaries. Am J Surg Pathol 7:667–677, 1983Google Scholar
  59. 59.
    Schlingemann RO, Rietveld FJR, Kwaspen F, van de Kerkhof PCM, de Waal RMW, Ruiter DJ: Differential expression of markers for endothelial cells, pericytes, and basal lamina in the microvasculature of tumors and granulation tissue. Am J Pathol 138:1335–1347, 1991Google Scholar
  60. 60.
    Traweek ST, Kandalaft PL, Mehta P, Battifora H: The human hematopoietic progenitor cell antigen (CD34) in vascular neoplasia. Am J Clin Pathol 96:25–31, 1991Google Scholar
  61. 61.
    Herlyn M, Clark WH, Rodeck U, Mancianti ML, Jambrosic J, Koprowski H: Biology of tumor progression in human melanocytes. Lab Invest 56:461–74, 1987Google Scholar
  62. 62.
    DeYoung BR, Wick MR, Fitzgibbon JF, Sirgi KE, Swanson PE: CD31: An immunospecific marker for endothelial differentiation in human neoplasms. Appl Immunohistochem 1:97–100, 1993Google Scholar
  63. 63.
    van de Rijn M, Rouse RV: CD34: A review. Appl Immunohistochem 2:71–80, 1994Google Scholar
  64. 64.
    Wang JM, Kumar S, Pye D, Haboubi N, Al-Nakib L: Breast carcinoma: comparative study of tumor vasculature using two endothelial-cell markers. J Natl Cancer Inst 86:386–388, 1994Google Scholar
  65. 65.
    Wang JM, Kumar S, Pye D, van Agthoven AJ, Krupinski J, Hunter RD: A monoclonal antibody detects heterogeneity in vascular endothelium of tumors and normal tissues. Int J Cancer 54:363–370, 1993Google Scholar
  66. 66.
    Barbareschi M, Gasparini G, Weidner N, Morelli L, Forti S, Eccher C, Fina P, Leonardi E, Mauri F, Bevilacqua P, Dalla Palma P: Microvessel density quantification in breast carcinomas: assessment by manual vs. a computer-assisted image analysis system. Appl Immunohistochem, in pressGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Noel Weidner
    • 1
  1. 1.Department of PathologyUniversity of CaliforniaSan FranciscoUSA

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