Microvessel Density Measurement Research Articles

Correlation between color power Doppler sonographic measurement of breast tumor vasculature and immunohistochemical analysis of microvessel density for the quantitation of angiogenesis.

To record the correlation between color power Doppler sonographic measurement of breast tumor vasculature and immunohistochemical analysis of microvessel density for the quantitation of angiogenesis. Women with palpable breast masses scheduled for excision biopsy were scanned with two- and three-dimensional color power Doppler sonography before and after the administration of a sonographic contrast agent. Vessel counts were performed on two- and three-dimensional sonographic images before and after contrast agent administration. All tumors were surgically removed and underwent immunohistochemical analysis for microvessel density assessment. The sonographic measure of tumor vascularity was correlated with microvessel density. Pathologic examination showed 43 breast cancers and 14 benign breast masses. Higher microvessel density was noted in malignant than benign breast masses (P < .0005). Color power Doppler sonographic measurement of tumor vessel number showed a significant positive correlation with tumor size (P < .05) and progesterone receptor negativity (P < .05). A significant positive correlation was observed between microvessel density and the number of intratumoral blood vessels assessed by both two- and three-dimensional color power Doppler sonography (P < .05). Regression models showed three-dimensional color power Doppler sonography to have a significantly higher correlation with microvessel density when compared with two-dimensional color power Doppler sonography at baseline (P < .005). The administration of a sonographic contrast agent did not improve correlation with microvessel density. A significant correlation was shown between color power Doppler sonographic measurement of tumor vascularity and microvessel density by immunohistochemical analysis. Further improvement in Doppler sonographic techniques to map capillary vessel flow should be explored to improve the current association with pathologic findings.

Flow cytometric quantification of tumour endothelial cells; an objective alternative for microvessel density assessment.

Assessment of microvessel density by immunohistochemical staining is subject to a considerable inter-observer variation, and this has led to variability in correlation between microvessel density and clinical outcome in different studies. In order to improve the method of microvessel density measurement in tumour biopsies, we have developed a rapid, objective and quantitative method using flow cytometry on frozen tissues. Frozen tissue sections of archival tumour material were enzymatically digested. The single-cell suspension was stained for CD31 and CD34 for flow cytometry. The number of endothelial cells was quantified using light scatter- and fluorescence-characteristics. Tumour endothelial cells were detectable in a single cell suspension, and the percentage of endothelial cells detected in 32 colon carcinomas correlated highly (r=0.84, P<0.001) with the immunohistochemical assessment of microvessel density. Flow cytometric endothelial cells quantification was found to be more sensitive especially at lower levels of immunohistochemical microvessel density measurement. The current method was found to be applicable for various tumour types and has the major advantage that it provides a retrospective and quantitative approach to the angiogenic potential of tumours.British Journal of Cancer (2002) 87, 344–347. doi:10.1038/sj.bjc.6600457 www.bjcancer.com© 2002 Cancer Research UK

Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn't tell us.

A substantial number of clinical trials using antiangiogenic therapies are ongoing worldwide. How to achieve the maximum benefit from these therapies and how to monitor patient response are of paramount concern to investigators. There are currently no markers of the net angiogenic activity of a tumor available to aid investigators in the design of antiangiogenic treatment schemes. It stands to reason that quantification of various aspects of tumor vasculature might provide an indication of angiogenic activity. One often-quantified aspect of tumor vasculature is microvessel density. Studies over the last decade have demonstrated the value of using tumor microvessel density as a prognostic indicator for a wide range of cancers. In this context, measurement of microvessel density facilitates assessments of disease stage and the likelihood of recurrence and helps guide treatment decisions. Recently, however, it has been assumed by some investigators that measurements of microvessel density may also reveal the degree of angiogenic activity in a tumor. Based on this assumption, quantification of microvessel density is thought to constitute a surrogate marker for the efficacy of antiangiogenic agents as well as a means by which to assess which patients are good candidates for antiangiogenic therapy prior to treatment. Here we contend that, although microvessel density is a useful prognostic marker, it is not, by itself, an indicator of therapeutic efficacy, nor should it be used to guide the stratification of patients for therapeutic trials. In this review, we discuss the evidence for these assertions and what can and cannot be determined from measurements of microvessel density.

Bone marrow angiogenesis in systemic mast cell disease.

Mast cells may participate in tumor angiogenesis through the release of angiogenic cytokines from their secretory granules. To gain additional insight into the role of mast cells in bone marrow angiogenesis, we performed a semiquantitative measurement of bone marrow microvessel density in 52 consecutive adult patients with systemic mast cell disease (SMCD). The results were examined for potential correlations with mast cell expression of angiogenic cytokines and with other histologic features of the bone marrow. Standard immunohistochemical methods were used to visualize bone marrow microvessels (CD34 staining) and mast cell expression of transforming growth factor-beta, basic fibroblast growth factor, and their respective receptors. An increase in microvessel density was demonstrated in 32 of the 52 patients (62%) with SMCD, and the degree of bone marrow angiogenesis did not correlate with either the mast cell expression pattern of the study cytokines or the presence (23 patients) or absence (29 patients) of an associated hematologic disorder. In the 29 patients without an associated hematologic disorder, microvessel density was correlated significantly with the presence of an abnormal pattern of hematopoiesis but not with the degree of bone marrow involvement by mast cells. Furthermore, areas occupied by mast cell lesions were often devoid of neovascularization. We conclude that bone marrow angiogenesis characterizes a percentage of patients with SMCD and that the pathogenesis may not necessarily be linked to the mast cells themselves.

A computer image analysis system for microvessel density measurement in solid tumours.

Microvessel density (MVD) is a widely used surrogate measure of angiogenesis in pathological specimens and tumour models. Measurement of MVD can be achieved by several methods. Automation of counting methods aims to increase the speed, reliability and reproducibility of these techniques. The image analysis system described here enables MVD measurement to be carried out with minimal expense in any reasonably equipped pathology department or laboratory. It is demonstrated that the system translates easily between tumour types which are suitably stained with minimal calibration. The aim of this paper is to offer this technique to a wider field of researchers in angiogenesis.

Angiogenesis of Endometrial Carcinomas Assessed by Measurement of Intratumoral Blood Flow, Microvessel Density, and Vascular Endothelial Growth Factor Levels

Angiogenesis of Endometrial Carcinomas Assessed by Measurement of Intratumoral Blood Flow, Microvessel Density, and Vascular Endothelial Growth Factor Levels

Angiogenesis of endometrial carcinomas assessed by measurement of intratumoral blood flow, microvessel density, and vascular endothelial growth factor levels

Objective: To evaluate the relationship between blood flow in the tumor assessed by color Doppler ultrasound, microvessel density, and vascular endothelial growth factor levels in endometrial carcinoma. Methods: Forty-nine patients undergoing surgery for endometrial carcinoma were enrolled. Transvaginal color Doppler ultrasound was performed preoperatively and the lowest resistance index (RI) in the tumor was recorded for analysis. Vascular endothelial growth factor in the tumor was quantified by enzyme immunoassay. The microvessel density of the excised tumor was assessed immunohistochemically. The relationships between the corresponding RI, microvessel density, and vascular endothelial growth factor level of the tumor tissues and clinical and pathologic parameters were analyzed. Results: Significantly lower RIs were noted in tumors of stage II or greater (0.37 compared with 0.50, P < .001), of high histologic grade (grade 3) (0.34 compared with 0.49, P = .004), with deep myometrial invasion (one-half depth or greater) (0.39 compared with 0.49, P = .002), with lymphovascular emboli (0.38 compared with 0.49, P < .001), or with lymph node metatasis (0.30 compared with 0.49, P < .001) compared with stage I tumors and tumors of histologic grade 1 or 2, with superficial myometrial invasion, without lymphovascular emboli, or with no lymph node metastasis. Increased vascular endothelial growth factor levels and microvessel density (×200 field) also were detected in tumors of stage II or greater (975 compared with 129 pg/mg, P = .014; and 88 compared with 61, P = .018, respectively), with lymphovascular emboli (1138 compared with 120 pg/mg, P = .002; and 86 compared with 63, P = .023), or with lymph node metastasis (1011 compared with 95 pg/mg, P < .001; and 98 compared with 61, P = .019). Resistance index, microvessel density, and vascular endothelial growth factor levels in the tumor showed linear correlations (RI compared with microvessel density: r = −.32, P = .03; RI compared with vascular endothelial growth factor levels: r = −.40, P = .004; microvessel density compared with vascular endothelial growth factor levels: r = .36, P = .011). Conclusion: Blood flow assessed by color Doppler ultrasound has histologic and biologic correlations with angiogenesis and vascular endothelial growth factor levels and might play an important role in predicting tumor progression and metastasis in endometrial carcinoma.

Endoglin Expression as a Measure of Microvessel Density in Cervical Cancer

Endoglin Expression as a Measure of Microvessel Density in Cervical Cancer

Endoglin expression as a measure of microvessel density in cervical cancer

Objective: To evaluate endoglin, a membrane protein and member of the transforming growth factor β-1 receptor complex, as an endothelial marker of angiogenesis in cervical cancer tissues. Methods: Tumor tissue was collected from 31 surgically treated stage IB nonbulky (under 5 cm) cervical cancer subjects, and samples were fixed in formalin and embedded in paraffin. Endoglin was stained on 5-μm slide sections by the DAKO Catalyzed Signal Amplification method (DAKO Corporation, Carpinteria, CA). Factor VIII was stained by standard immunohistochemistry. Positively stained microvessels were counted in “hot spots” at 200× magnification. Clinical data were correlated with vessel counts by Spearman correlation. Mean differences in counts were tested using paired t tests. Results: This staining method for endoglin identified significantly more vessels than the factor VIII method (mean 92 ± 45 versus 33 ± 16, P < .001). Endoglin and factor VIII counts correlated significantly with deep stromal invasion (Spearman rho 0.466 and 0.522, respectively, P < .05); however, only endoglin counts correlated significantly with lymph node metastases (rho = .495, P < .01). Conclusion: Endoglin is stimulated in tumor angiogenesis and might be relatively more specific than commonly used endothelial markers. The endoglin system was more sensitive for staining capillaries in neoplastic cervical tissue, better predicted lymph node metastases, and should be widely applicable for the study of other tumors.

Quantitative vascularity of breast masses by Doppler imaging: regional variations and diagnostic implications.

Seventy-four biopsy proven breast masses were imaged by color and power Doppler imaging to evaluate vascular pattern of malignant and benign breast masses. The images were analyzed for vascularity. The measurements were made over the entire mass as well as regionally at its core, at its periphery, and in the tissue surrounding it. The surgical specimens were analyzed for microvessel density. The diagnostic performance of Doppler sonographic vascularity indices was evaluated by receiver operating characteristic analysis. The malignant masses were 14 to 54% more vascular than the benign masses. Both types of masses were more vascular by ultrasonography than the tissue surrounding them. Whereas benign masses were 2.2 times more vascular than the surrounding tissue, the malignant masses were 5.0 times more vascular. In a subset of patients the regional vascularity at the core, periphery, and surrounding tissue by Doppler imaging exhibited a strong correlation (R2 > 0.9) with the corresponding histologic microvessel density measurements. Although the malignant masses exhibited a strong gradient in vascularity, core > periphery > surrounding tissue, the benign masses had relatively uniform distribution of vascularity. The area under the receiver operating characteristic curve (A(Z)) for the Doppler indices ranged from 0.56 +/- 0.07 to 0.65 +/- 0.07. A nonlinear analysis including age-specific values of Doppler indices improved the diagnostic performance to A(Z) = 0.85 +/- 0.06. In conclusion, quantitative Doppler imaging when used in combination with a nonlinear rule-based approach has the potential for differentiating between malignant and benign masses.

Immunohistochemical measurement of tumor vascular endothelial growth factor in breast cancer. A more reliable predictor of tumor stage than microvessel density or serum vascular endothelial growth factor.

Microvessel density counting is commonly proposed as a method of assessing angiogenesis. However, results have been difficult to reproduce because of many methodological inconsistencies. Vascular endothelial growth factor (VEGF), an angiogenic regulator, is also a poor prognostic indicator in breast cancer, correlating in many studies with microvessel density. In this study, VEGF and microvessel density counting were examined as methods of assessing angiogenesis in breast cancer and correlated with tumor stage. A representative tumor section was stained with anti-CD34 and anti-VEGF. Microvessel density was evaluated using the manual "hot-spot" procedure and a semiquantitative image analysis system. Serum VEGF levels were available from an additional nine patients. Results were analyzed using Kendall's tau correlation. Tumor stage correlated with tumor VEGF, but not with microvessel "hot-spot" or vessel counts. There was no correlation between scores obtained from the manual or semiquantitative methods. Serum VEGF did not correlate with either tumor VEGF or tumor stage. The prognostic importance of VEGF in invasive breast cancer is associated with tumor stage. Measurement of tumor VEGF, as an indicator of angiogenesis, is more reliable prognostically than measurement of microvessel density or serum VEGF. Routine measurement of microvessel density in breast cancer is less reliable.

NO and de novo mammalian angiogenesis: further evidence that NO inhibits bFGF-induced angiogenesis while not influencing VEGF165-induced angiogenesis.

Using the non-surgical rat mesenteric window angiogenesis assay, we studied the systemic effect of (i) the nitric oxide (NO)-releasing vasodilator isosorbide-5-mononitrate (ISMN) and (ii) the NO-synthase inhibitor L-NAME on angiogenesis induced by the intraperitoneal injection of bFGF and VEGF165. The response was assessed objectively and quantitatively by microscopic morphometry and image analysis in terms of the vascularized area (VA; a measurement of microvessel spatial extension), the microvascular length (MVL; a composite measurement of microvessel density), the total microvascular length (TMVL=VA x MVL), the number of microvessel segments per unit tissue volume (No. MS), the length of the microvessel segments (Le. MS) and the degree of microvessel tortuosity (MVT). Additional architectural features of the network were assessed in terms of variables introduced here: the number of microvessel branching points per unit tissue volume (No. BP), the index of interconnecting microvessel loop formation (In. LF), the index of microvessel intersection (In. IS), the number of microvessel sprouts per unit tissue volume (No. SP) and their length (Le. SP). In bFGF-mediated angiogenesis, L-NAME significantly, augmented angiogenesis, whereas ISMN significantly inhibited angiogenesis. By contrast, neither L-NAME nor ISMN affected the angiogenic response to VEGF165.

Dynamic contrast-enhanced MRI studies in human tumours.

There has been much recent public and scienti¢c interest in the possibility that drugs could attack the blood supply to tumours. Press statements by the US National Cancer Institute and the Cancer Research Campaign in the UK have focused attention on tumour neovascularity. The importance of tumour angiogenesis is well known to clinical oncologists but is less familiar to radiologists. This article serves to highlight the potential value of contrast-enhanced MRI in probing the functional aspects of angiogenesis in human cancers [1]. Angiogenesis is a complex process, critical to the growth and metastasis of malignant tumours. Angiogenesis is mediated by factors released by tumours and/or tumour-associated in£ammatory cells [2]. Microvessel density measurements using immuno-histochemical staining techniques are used to measure angiogenic activity. In many types of tumour, including breast, lung, prostate, and head and neck cancers, microvessel density correlates with clinical stage and acts as an independent prognostic factor [3]. Many radiological studies are able to depict the increased vascularity of tumours. Recently, quanti¢cation of tumour vascularity has been undertaken using imaging methods, including colour Doppler ultrasound and dynamic contrast-enhanced MRI (dMRI). MR imaging after the administration of a contrast agent that is retained in the blood can be used to estimate the plasma volume of a tissue. Most recent attempts to assess tissue vascularity in humans have used extracellular contrast agents. These agents, based on gadolinium chelates, readily pass from the intravascular space into the interstitium, thereby giving rise to parenchymal enhancement. Numerous studies have shown that malignant tumours frequently demonstrate faster and higher levels of enhancement than do normal surrounding tissues. This observation has been the basis for diagnostic MR studies for many years, but the underlying pathophysiological processes and quanti¢cation of these studies have not been clinically emphasized. The introduction of fast imaging routines and developments in tracer kinetic modelling for dMRI studies now permit a fuller understanding of the physiological basis of contrast enhancement in human tumours. With optimal data collection, sequences can be designed to be sensitive to tissue perfusion and blood volume (so-called T2* methods) and/or permeability and extracellular leakage space (so-called T1 methods). Such techniques permit the functional aspects of tumour neovascularity to be assessed in vivo in a non-invasive and repeatable way. Other advantages of MR methods include good spatial resolution and the ability to incorporate physiological data acquisition into routine patient studies.

Clinical significance of the determination of angiogenic factors

INTRODUCTION ANGIOGENIC ACTIVITY is known to depend upon a balance between positive and negative angiogenesis regulators [ 11. The alteration of this balance by mechanisms, such as the induction of vascular endothelial growth factor (VEGF), expression of TP53 or RAS mutations, SRC overexpression, and downregulation of thrombospondin-1 expression by TP53 mutation, can switch the angiogenic phenotype and facilitate the growth of solid tumours [2-51. Currently, measurement of intratumoral microvessel density (IMD) by immunocytochemistry appears to be the most reliable method of measuring angiogenic activity [6]. However, the determination of levels of each endothelial growth regulator in tumour tissues and in serum or urine can provide more information on the angiogenic characteristics of each tumour and might be an alternative method for evaluating angiogenic activity. For example, the level of intratumoral VEGF varies greatly between tumours and can be a valuable prognostic factor [7]. The endogenous angiogenesis inhibitor, angiostatin, which can cause tumour regression in human tumour xenografts models, has been detected in serum [8]. Since the biological properties of each endothelial growth factors is different, it will be of particular importance to know the role of each factor in human tumours. Therefore, we have focused on the determination of angiogenic factors, particularly positive endothelial growth regulators including VEGF, platelet-derived endothelial cell growth factorithymidine phosphorylase (I’D-ECGFITP), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and metalloproteinases (MM&), in breast cancer patients. In this article, we review our experience and the most relevant data in the literature on the clinical significance of the determination of these endothelial growth regulators in human tumours.

Microvessel count and vascular endothelial growth factor in renal cell carcinoma

Angiogenesis plays an important role in the growth of solid tumors. The objective of this study is to investigate the relationship between microvessel density and mRNA measurement for vascular endothelial growth factor (VEGF) as well as the kinase domain receptor (KDR) in renal cell carcinoma (RCC). From 1992 to 1994, expression of mRNAs of VEFG, KDR and microvessel count by Factor VIII immunohistochemical staining were examined in 22 cases of RCC. Northern blot hybridization demonstrated that 18 tumors (82%) expressed a higher level of VEGF gene than corresponding normal renal tissues (T/N > 1) and that the expression of VEGF gene in the tumors correlated well with that of KDR gene (r = 0.780, p = 0.001). Most of the tumor cells positively reacted to immunohistochemical staining for VEGF, but all of the normal renal tissue elements did not. RT-PCR analysis revealed that mRNAs of VEGF expressed in the tumors were of soluble subclasses. The mean number of microvessels (< 50 micrometers in diameter) in the tumors was 62.7 +/- 39.1/mm2 (range 13.7-133.1) and significantly correlated with the expression of VEGF gene (r = 0.636, p = 0.001). The results indicate that RCC cells actively produce soluble VEGF and strongly suggest that VEGF and its receptor KDR cooperate to enhance the angiogenesis of this tumor.

Angiogenesis in Bladder Cancer: Relationship Between Microvessel Density and Tumor Prognosis

Tumor stage, histologic grade, and regional lymph node status are currently used to obtain prognostic information about bladder cancers. However, additional prognostic indicators are needed to aid clinicians in selecting patients who would benefit most from specific therapies. A majority of studies assessing the prognostic value of measuring tumor angiogenesis (i.e., measurement of tumor microvessel densities) have found a positive association between increasing microvessel densities and worsening prognosis. We explored the relationship between established prognostic indicators and the extent of tumor-associated angiogenesis in patients with invasive transitional cell carcinoma (TCC) of the bladder, and we determined whether tumor microvessel density measurement could be used independently to predict bladder tumor behavior. Tumor tissue was obtained from 164 patients with invasive primary TCC of the bladder. The extent of tumor-associated angiogenesis in this tissue was evaluated by immunohistochemical methods using HPCA-1, a mouse monoclonal antibody directed against the endothelial cell antigen, CD34. The number of microvessels in a 200x microscopic high-power field (hpf) containing the area of greatest neovascularization within or immediately adjacent to each tumor was determined. The patient population was then divided into three equivalently sized groups, with tumors containing low (< or = 64), intermediate (65-99), or high (> or = 100) numbers of microvessels per hpf. Kaplan-Meier product limit estimates of overall survival and the complement of cumulative incidence curves for recurrence-free survival were plotted. When analyzing survival or recurrence, the logrank test was used to compare groups of patients with and without stratification according to tumor stage. Analysis of variance was used to test for an association between microvessel density and established prognostic variables. Reported P values are from two-sided tests. Microvessel density was significantly associated with disease-free (P < .0001) and overall (P = .0007) survival. The estimated probabilities of recurrence at 5 years were 19% (95% confidence interval [CI] = 8-29), 56% (95% CI = 43-69), and 68% (95% CI = 55-81) for patients with lowest, intermediate, and highest microvessel counts, respectively. Overall survival at 5 years was estimated to be 68% (95% CI = 56-81), 44% (95% CI = 30-57), and 34% (95% CI = 21-47) for the same three patient groups. Microvessel density was associated with disease progression in patients with organ-confined tumors, tumors extending through the bladder wall, and tumors that had spread to regional lymph nodes. Tumor angiogenesis was found to be an independent prognostic indicator when evaluated in the presence of histologic grade, pathologic stage, and regional lymph node status. Tumor angiogenesis, as determined by microvessel density measurement, is an independent prognostic indicator for patients with invasive TCC of the bladder.