Low Microvessel Density Research Articles

Relationship of angiogenesis biomarkers and clinical response to bevacizumab (Bev) in persistent or recurrent epithelial ovarian cancer (EOC) and primary peritoneal cancer (PPC) patients treated in a Phase II Gynecologic Oncology Group study

21021 Background: Angiogenesis promotes tumor growth and is associated with poor outcomes in ovarian cancer. Currently, there are no markers to predict or determine response to anti-angiogenesis therapies. Methods: Persistent or recurrent EOC/PPC patients (1–2 prior cytotoxic regimens, measurable disease, and GOG performance status = 2) were treated with Bev (15mg/kg IV q21days) until progression. Tissue biopsies, serum samples and dynamic contrast enhanced MRIs (DC-MRI) were obtained prior to initiation (PT01) and 4th cycle (PT02) of Bev therapy. Tumor vascular endothelial growth factor (VEGF), thrombospondin-1 (TSP-1), and mutant p53 were determined by immunohistochemistry (IHC) and scored using histoscore (HS) analysis ([1 + staining score (0 to 4)] x % positive). Microvessel density (MVD) was determined by counting CD31 IHC hot spots. Serum VEGF levels were obtained by ELISA. Variables were considered as continuous or discontinuous in this training set to establish appropriate cut points to be applied to an ongoing phase III trial (GOG218). Results: From 4/02 to 8/04, 62 eligible patients were enrolled. A 17.7% response rate with 40% of patients with no evidence of disease progression for at least 6 months was previously reported (ASCO 2005). 44 tissue samples from PT01 and 27 from PT02 were evaluable for IHC biomarker levels. Based on current analysis, VEGF HS average was 92 at low MVD counts, while at high MVD counts, VEGF HS average was higher at 159 (p=0.06). In addition, 77% of TSP-1 negative tissues were VEGF positive, while 52% of TSP-1 positive tissues were VEGF positive. Analysis of serum VEGF levels (from 56 PT01 and 39 PT02 specimens) and DCMRIs (49 PT01 and 42 PT02 MRIs) are ongoing. Conclusions: Higher tumorVEGF was associated with increased angiogenesis and lower TSP-1 tissue levels. Results from the biomarkers tested await correlation to the mature survival data which should be available by June 2007. We plan to determine whether an angiogenesis biomarker profile is associated with patient response to Bev therapy. No significant financial relationships to disclose.

Evaluation of VEGF A expression and microvascular density as prognostic factors in extrahepatic cholangiocarcinoma

Objectives Angiogenesis is essential for tumor growth and metastasis. An association between microvessel density, a measure of tumor angiogenesis, and conventional prognostic variables has been shown for many different tumor entities. In extrahepatic cholangiocarcinoma, the VEGF expression and microvessel density have rarely been investigated. Methods Paraffin-embedded specimens from 51 resected adenocarcinomas of the extrahepatic bile duct were immunostained for vascular endothelial growth factor A (VEGF A) and CD 34 to evaluate the microvessel density (MVD). VEGF A staining was evaluated by combining intensity and percentage of positive tumor cells, as low (expression equal or below the median), or high (above the median). Microvessel density was assessed using a method published by Weidner et al. Results Median disease free survival (DFS) of the study group was 12.5 months (range, 1–66.3 months). DFS was calculated in the 39 patients with complete resection. It was significantly better in patients with low microvessel density than DFS in patients with high microvessel density (33 months (range, 3–66.3 months) vs. 21.8 months (range, 1.6–31.6 months); p = 0.022). In contrast, VEGF A expression did not correlate with survival. There was a trend toward a higher VEGF A expression in highly vascularized tumors ( p = 0.08), but failed to reach statistic significance. Conclusions The present study indicates, that vascularisation has an important impact on survival of extrahepatic cholangiocarcinoma patients. Other molecules than VEGF A are probably involved in neovascularization in extrahepatic cholangiocarcinoma.

Hypoxia contributes to development of recurrent endometrial carcinoma

Tumor hypoxia can trigger the induction of angiogenesis. High microvessel density (MVD) as well as hypoxia-inducible factor-1alpha (HIF-1alpha) have been related to recurrent disease and tumor aggressiveness, respectively. In this study, MVD and hypoxic status were investigated in primary and recurrent endometrial carcinomas. A total of 65 primary tumors of patients with recurrent endometrial carcinoma (n = 40), and without recurrent endometrial carcinoma (n = 25) were studied. Immunohistochemical analysis was performed on paraffin-embedded tumor tissue. MVD was determined by quantitative analysis of CD31/FVIII positive vessels. Tumor hypoxia was estimated by evaluating the expression of the hypoxia-regulated gene HIF-1alphaand its target gene carbonic anhydrase IX (CA-IX). An additional 23 recurrent tumors were available for determination of MVD and HIF-1alpha expression. Effects of hypoxia on tumor protein p53 (TP53) expression were evaluated in the endometrial cancer cell lines (ECC-1), Ishikawa (derived from adenocarcinomas), and AN3CA (derived from a lymph node metastasis). MVD, CA-IX, and HIF-1alpha expression were not significantly different in primary tumors of patients with recurrence compared to the control tumors. The MVD was significantly lower, and HIF-1alpha expression was significantly higher in recurrent tumors when compared with their primary tumors (paired t test, P < 0.05). HIF-1alpha expression correlated well with TP53 expression levels in primary tumors, but not in recurrences. TP53 protein levels were highest in AN3CA cells. Hypoxic conditions induced TP53 protein in ECC-1 and Ishikawa, but not AN3CA cells. We conclude that MVD, CA-IX, and HIF-1alpha expression are not independent prognostic markers for recurrent endometrial carcinoma. The low MVD, increased HIF-1alpha protein levels, dissociation of hypoxia, and TP53 protein induction in the metastatic tumor cells (AN3CA) support a role for hypoxia in the development of recurrent endometrial carcinoma.

A possible role of thymidine phosphorylase expression and5‐fluorouracil increased sensitivity in oropharyngeal cancer patients

Thymidine Pi deoxyribosyltransferase (TP) is an enzyme involved in DNA synthesis up-regulated in tumours and it is also a pro-angiogenic factor. TP cannot activate capecitabine, because capecitabine first needs conversion by carboxylesterase and cytidine deaminase into 5-deoxy-fluorouridine. This compound can be activated by TP to 5-fluorouracil (5-FU). Although TP is not necessary for 5-FU toxicity, experimental data suggest that high levels of TP correlate with an enhanced response to 5-FU therapy. In this study, we have analysed by immunohistochemistry CD34, CD68 and TP positive cells in bioptic samples from 53 patients with T1–3 N0–1 M0 oropharyngeal squamous cell carcinoma (OSC) and from 24 patients with non-dysplastic oropharyngeal leukoplakia (NDOLP). Results showed that the mean of TP-positive cells, CD68 positive macrophages and CD34 positive endothelial cells eval-uated as microvessel density (MVD) was significantly higher in OSC than in NDOLP. Moreover, at a median follow-up of 19 months, patients with TP expression and higher MVD showed a better survival rate as compared to those with low MVD, probably as a consequence of 5-FU-based therapy.We hypothesized a role for TP in oropharyngeal tumourigenesis and 5-FU activation in the adjuvant setting of OSC patients.

Antitumor activity of ALK1 in pancreatic carcinoma cells

In this study, the authors investigated the expression of activin receptor-like kinase 1 (ALK1) in pancreatic carcinoma and evaluated its potential role as a tumor suppressor in vitro and in vivo. Endogenous ALK1 expression was demonstrated by immunohistochemistry in both pancreatic tumor tissue and peritumoral normal tissue from 6 patients and by RT-PCR in 8/12 established pancreatic cancer cell lines. Ectopic expression of a constitutively active (ca) ALK1 mutant in TGF-beta sensitive PANC-1 and COLO-357 cells augmented transcriptional activation of a Smad2/3 responsive reporter, and slowed down basal growth in vitro. Both effects were further enhanced by TGF-beta/ALK5 stimulation, suggesting largely independent nuclear Smad signaling by both type I receptors. Upon orthotopic transplantation of PANC-1-caALK1 into immunodeficient mice, tumor size was strongly reduced and was associated with a lower microvessel density in the PANC-1-caALK1-derived tumors. In vitro, this mutant efficiently blocked TGF-beta-induced epithelial-to-mesenchymal transdifferentiation and suppressed TGF-beta/ALK5-mediated activation of the p38 MAPK pathway. Mechanistically, caALK1 silenced MyD118, an immediate TGF-beta target gene whose protein product, GADD45beta, couples Smad signaling to p38 activation. These results show that ALK1 activation in pancreatic tumor cells is antioncogenic by inducing ALK5-independent growth inhibition and by blocking TGF-beta/ALK5-mediated epithelial-to-mesenchymal transdifferentiation and, possibly, invasion and metastatic progression.

Inhibition of osteopontin would suppress angiogenesis in gastric cancer

Osteopontin (OPN) plays an important role in tumorigenesis, tumor invasion, and metastasis in many types of cancers, including gastric cancer. Recently, much interest has been focused on the role of OPN in tumor angiogenesis. Our previous studies have shown that OPN is overexpressed, and associated with mean microvessel density in, the tissue samples of patients with gastric cancer. In the present study, we aimed to further determine and provide evidence for the role of OPN in gastric-cancer-associated angiogenesis by diminishing OPN expression in gastric cancer cells using the small interference RNA method, and then evaluate the effects of OPN on gastric cancer-associated angiogenesis by in vivo and in vitro assays. Our results revealed that reduced OPN production by gastric cancer cells would reduce the proliferation, migration, and tube formation of human umbilical vein endothelial cells, and lead to a lower microvessel density, i.e., angiogenesis, in transplanted tumors of mice. These data confirm the positive role of OPN in gastric-cancer-associated angiogenesis.

Prognostic Value of Tumor Architecture, Tumor-Associated Vascular Characteristics, and Expression of Angiogenic Molecules in Pancreatic Endocrine Tumors

It is difficult to predict the biological behavior of pancreatic endocrine tumors (PETs). Our aim was to evaluate the prognostic significance of certain variables in PETs. The following variables were examined in 37 patients with PETs and then compared with other clinicopathologic characteristics: histologic tumor structure; microvessel density (MVD) measured by three different methods, including a unique method involving calculation of solid area MVD; endothelial proliferation; and the immunohistochemical expression of vascular endothelial growth factor-A and CXC chemokine CXCL-12. Intratumoral vascular structures were analyzed by double immunofluorescence using 30-microm-thick sections. The presence of focal and intensive solid growth of tumor cells (large solid nests; P = 0.003), low solid area MVD (P = 0.002), a high endothelial cell proliferation index (EPI; P = 0.005), and high expression of CXCL-12 in PET cells (P = 0.018) were significant unfavorable prognostic indicators. The predominant structure of the overall tumor histology and the expression of vascular endothelial growth factor-A did not separate aggressive PETs. In areas of focal solid growth, tumor-associated blood vessels had obviously low MVD and high EPI, and their structures were poorly formed with highly abnormal features, in comparison with other areas. High expression of CXCL-12 in tumor cells was significantly associated with variables representing tumor growth, hematogenous tumor spread, low MVD, high EPI, and the presence of large solid nests. This study has provided novel findings on the prognostic features of tumor architecture and tumor-associated angiogenesis in PETs. CXCL-12 is the first candidate molecule in association with neoangiogenesis in PETs.

Correlation of Cyclooxygenase-2 Expression and Microvessel Density with Prognosis in Transitional Cell Carcinoma of the Upper Urinary Tract

Purpose: The aim of this study was to investigate the relationship of cyclooxygenase (COX)-2 expression and microvessel density (MVD), a reflection of angiogenesis, with prognosis in patients with transitional cell carcinoma of the upper urinary tract (TCC-UUT). Materials and Methods: Formalin-fixed, paraffin-embedded tissue sections of TCC-UUT from 91 patients, who had undergone radical nephroureterectomy, were examined immunohistochemically using antibodies against COX-2 and CD34. MVD was determined with CD34-stained slides. The expression patterns of COX-2 and MVD were compared with the clinicopathological variables. Results: The COX-2 expression was significantly correlated with T stage (p=0.002), N stage (p=0.010), and grade (p=0.027). MVD was also significantly correlated with T stage (p<0.001), N stage (p=0.002), and grade (p=0.001). The COX-2 expression was significantly correlated with MVD (p=0.001). The survival rate of patients with COX-2 positive tumors or high MVD was significantly lower than that of patients with COX-2 negative tumors or low MVD, respectively (p=0.0013, p=0.0312). Univariate analyses identified T stage, N stage, grade, COX-2 expression, and MVD as significant prognostic factors for cancer-specific survival; multivariate analyses indicated that T stage was the only independent prognostic factor. Conclusions: The increased expression of COX-2 and MVD is associated with a worse prognosis in TCC-UUT. The COX-2 expression is correlated with MVD. These results suggest that COX-2 may play an important role in the progression of TCC-UUT and angiogenesis may be affected by COX-2 during the progression of TCC-UUT. (Korean J Urol 2007;48: 376-382)

Prognostic significance of HIF-2α/EPAS1 expression in hepatocellular carcinoma

To evaluate the prognostic significance of HIF-2alpha/EPAS1 expression in hepatocellular carcinoma (HCC). Surgical specimens from 315 patients with HCC as well as 196 adjacent noncancerous lesions and 22 cases of normal liver tissue were investigated by immunohistochemistry (IHC) for HIF-2alpha/EPAS1 using a standard detection system. Correlations with clinicopathological factors, VEGF, microvessel density (MVD), and prognosis were analyzed. Immunoreactivity of HIF-2alpha/EPAS1 was positive in 69.5% of HCC, 55.6% of adjacent noncancerous tissue, and 0% of normal liver tissue. And it was significantly correlated with tumor grade, venous invasion, intrahepatic metastasis, necrosis, and capsule infiltration. Correlation analysis of HIF-2alpha/EPAS1 with angiogenic factor VEGF (P<0.001), and MVD (P=0.016) was also noted. HIF-2alpha/EPAS1 protein was less frequently expressed in low MVD cases, whereas a high rate of expression was noted in cases with both medium and high MVD (P=0.042). By Kaplan-Meier analysis, strong HIF-2alpha/EPAS1 staining (>50% of tumor cells) in HCC correlated with a shortened survival in patients (Cox's regression, P<0.001, r=3.699). We conclude that HIF-2alpha/EPAS1 expression may play an important role in tumor progression and prognosis of HCC. Assessment of HIF-2alpha/EPAS1 expression in HCC may be used as a diagnostic tool and possibly a target in the treatment of HCC.

HYAL1-v1, An Alternatively Spliced Variant of HYAL1 Hyaluronidase: A Negative Regulator of Bladder Cancer

Tumor cells express HYAL1 hyaluronidase, which degrades hyaluronic acid. HYAL1 expression in bladder cancer cells promotes tumor growth, invasion, and angiogenesis. We previously described five alternatively spliced variants of HYAL1 that encode enzymatically inactive proteins. The HYAL1-v1 variant lacks a 30-amino acid sequence that is present in HYAL1. In this study, we examined whether HYAL1-v1 expression affects bladder cancer growth and invasion by stably transfecting HT1376 bladder cancer cells with a HYAL1-v1 cDNA construct. Although HYAL1-v1 transfectants expressed equivalent levels of enzymatically active HYAL1 protein when compared with vector transfectants, their conditioned medium had 4-fold less hyaluronidase activity due to a noncovalent complex formed between HYAL1 and HYAL1-v1 proteins. HYAL1-v1 transfectants grew 3- to 4-fold slower due to cell cycle arrest in the G(2)-M phase and increased apoptosis. In HYAL1-v1 transfectants, cyclin B1, cdc2/p34, and cdc25c levels were > or =2-fold lower than those in vector transfectants. The increased apoptosis in HYAL1-v1 transfectants was due to the extrinsic pathway involving Fas and Fas-associated death domain up-regulation, caspase-8 activation, and BID cleavage, leading to caspase-9 and caspase-3 activation and poly(ADP-ribose) polymerase cleavage. When implanted in athymic mice, HYAL1-v1-expressing tumors grew 3- to 4-fold slower and tumor weights at day 35 were 3- to 6-fold less than the vector tumors (P < 0.001). Whereas vector tumors were infiltrating and had high mitoses and microvessel density, HYAL1-v1 tumors were necrotic, infiltrated with neutrophils, and showed low mitoses and microvessel density. Therefore, HYAL-v1 expression may negatively regulate bladder tumor growth, infiltration, and angiogenesis.

Preclinical evaluation of tumor microvascular response to a novel antiangiogenic/antitumor agent RO0281501 by dynamic contrast-enhanced MRI at 1.5 T

Inhibition of tumor angiogenesis is a promising approach in cancer treatment. The purpose of this study was to evaluate the vascular response of human lung tumor xenografts in vivo to RO0281501, an inhibitor of tyrosine kinase receptors, including vascular endothelial growth factor receptor 2, fibroblast growth factor receptor, and platelet-derived growth factor receptor, using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Human non-small cell lung carcinoma (H460a) xenografts grown s.c. in athymic nu/nu mice were treated p.o. with the antiangiogenic agent RO0281501. Treatment-induced changes in tumor volume, epiphyseal growth plate thickness, and microvessel density assessed by CD31 immunohistochemistry were analyzed. Tumor vascular permeability and perfusion were measured in tumors using DCE-MRI with gadopentetate dimeglumine on a 1.5 T clinical scanner to assess vascular function. Treatment with RO0281501 resulted in significant growth retardation of H460a tumors. RO0281501-treated tumors showed histologic evidence of growth plate thickening and relatively lower microvessel density compared with the controls. Regarding DCE-MRI variables, the initial slope of contrast uptake and Ak(ep) were significantly decreased on day 7 of treatment. RO0281501 is a novel antiangiogenic/antitumor agent, which is active in the H460a xenograft model. Its effects on tumor vasculature can be monitored and assessed by DCE-MRI on a 1.5 T human MR scanner with clinically available gadopentetate dimeglumine contrast, which will facilitate clinical trials with this or similar agents.

Dissociation of Angiogenesis and Tumorigenesis in Follistatin- and Activin-Expressing Tumors

The transforming growth factor-beta superfamily member activin and its antagonist, follistatin, act as a pleiotropic growth factor system that controls cell proliferation, differentiation, and apoptosis. Activin inhibits fibroblast growth factor 2-induced sprouting angiogenesis in vitro (spheroidal angiogenesis assay) and in vivo (Matrigel assay). To further study the role of the activin/follistatin system during angiogenesis and tumor progression, activin- and follistatin-expressing R30C mammary carcinoma cells were studied in mouse tumor experiments. Surprisingly, activin-expressing tumors grew much faster than follistatin-expressing tumors although they failed to induce increased angiogenesis (as evidenced by low microvessel density counts). Conversely, follistatin-expressing tumors were much smaller but had a dense network of small-diameter capillaries. Qualitative angioarchitectural analyses (mural cell recruitment, perfusion) revealed no major functional differences of the tumor neovasculature. Analysis of activin- and follistatin-expressing R30C cells identified a cell autonomous role of this system in controlling tumor cell growth. Whereas proliferation of R30C cells was not altered, follistatin-expressing R30C cells had an enhanced susceptibility to undergo apoptosis. These findings in experimental tumors are complemented by an intriguing case report of a human renal cell carcinoma that similarly shows a dissociation of angiogenesis and tumorigenesis during tumor progression. Collectively, the data shed further light into the dichotomous stimulating and inhibiting roles that the activin/follistatin system can exert during angiogenesis and tumor progression. Furthermore, the experiments provide a critical proof-of-principle example for the dissociation of angiogenesis and tumorigenesis, supporting the concept that tumor growth may not be dependent on increased angiogenesis as long as a minimal intratumoral microvessel density is maintained.

Angiogenesis in a human neuroblastoma xenograft model: mechanisms and inhibition by tumour-derived interferon-γ

Tumour progression in neuroblastoma (NB) patients correlates with high vascular index. We have previously shown that the ACN NB cell line is tumorigenic and angiogenic in immunodeficient mice, and that interferon-γ (IFN-γ) gene transfer dampens ACN tumorigenicity. As IFN-γ represses lymphocyte-induced tumour angiogenesis in various murine models and inhibits proliferation and migration of human endothelial cells, we have investigated the antiangiogenic activity of tumour-derived IFN-γ and the underlying mechanism(s). In addition, we characterised the tumour vasculature of the ACN xenografts, using the chick embryo chorioallantoic membrane assay. We show that the ACN/IFN-γ xenografts had a lower microvessel density and less in vivo angiogenic potential than the vector-transfected ACN/neo. The vascular channels of both xenografts were formed by a mixed endothelial cell population of murine and human origin, as assessed by the FICTION (fluorescence immunophenotyping and interphase cytogenetics) technique. With respect to ACN/neo, the ACN/IFN-γ xenografts showed more terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive human and murine endothelial cells, suggesting that inhibition of angiogenesis by IFN-γ was dependent on the induction of apoptosis, likely mediated by nitric oxide. Once the dual origin of tumour vasculature is confirmed in NB patients, the xenograft model described here will prove useful in testing the efficacy of different antiangiogenic compounds.

Vascularity and angiogenesis as predictors of growth in optic pathway/hypothalamic gliomas

The authors' aim in conducting this study was to investigate retrospectively the prognostic significance of angiogenic features in optic pathway/hypothalamic gliomas (OPHGs) in children. Patients were identified in whom a diagnosis of OPHG was made using pathological analysis at the Toronto Hospital for Sick Children between 1985 and 2002. Tumor specimens were reviewed for diagnostic accuracy and adequacy of the specimen. Sections were immunostained with factor VIII to assess microvessel density (MVD). A ratio of alpha-smooth muscle actin to factor VIII immunostaining was calculated to arrive at a vascular maturity index (VMI). Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) immunostaining were performed to evaluate angiogenic factors. In addition, the MIB-1 labeling index (LI) was used to assess proliferation. These factors were evaluated with respect to progression-free survival (PFS). Forty-one of 60 patients originally identified had adequate samples and follow up for inclusion in the study. Of these, eight patients had coexisting neurofibromatosis Type 1. Twenty-eight patients experienced tumor progression after the initial treatment (surgery with or without adjuvant treatment). Thirty-eight patients are still alive. A high MVD (> 21 vessels/1.2 mm2) was associated with a significantly higher rate of progression compared with a low MVD (< 21 vessels/1.2 mm2; p = 0.017). Microvessel density was also predictive of reduced PFS on multivariate analysis stratified for extent of resection (p = 0.04), and VMI as well as intensity and distribution of VEGF and VEGFR staining and the MIB-1 LI were not significantly associated with PFS. These findings suggest that MVD is the best current predictor of PFS in incompletely resected OPHGs. This information highlights the importance of angiogenesis in regard to low-grade gliomas.

Endoglin (CD105) as a prognostic factor in head and neck squamous cell carcinoma

Endoglin (CD105) is a proliferation-associated protein abundantly expressed in angiogenic endothelial cells. Recent studies revealed that CD105 is intensively expressed in tumor vasculature, whereas intratumoral microvessel density (MVD) determined with the use of antibodies to CD105 has been found to be an important prognostic indicator for the outcome in a number of malignancies. In the current study, we investigated endoglin expression and evaluated MVD in 108 patients with head and neck squamous cell carcinoma. Endoglin was intensively expressed in intratumoral blood vessels, whilst lymphatics were rarely positive for CD105. High microvessel density was associated with a more aggressive tumor phenotype, including advanced clinical stage (p = 0.008) and the presence of lymph node metastasis at the time of diagnosis (p = 0.02). When microvessel counts were assessed for their prognostic values (high vs low MVD), there was a statistically significant difference in the overall survival among patients with tumors of the oral cavity and larynx (p < 0.001) and in the disease-free survival among patients with tumors of the lower lip (p = 0.01). The prognostic impact of microvessel density was not dependent on clinical stage or lymph node status. The results of the current study suggest that CD105 is a promising target for tumor imaging and prognosis.

Dynamic MR Imaging of Breast Lesions: Correlation with Microvessel Distribution Pattern and Histologic Characteristics of Prognosis

To evaluate the association of dynamic enhancement parameters of benign and malignant breast lesions at magnetic resonance (MR) imaging with microvessel distribution and histologic prognostic tumor characteristics. Regional review board approval and informed consent were obtained. Surgical resection specimens of breast lesions (32 benign, 86 malignant) in 118 patients (age range, 28-86 years; mean, 58 years) who had undergone dynamic T1-weighted MR imaging of both breasts were included in the study. Different MR enhancement parameters and microvessel density (MVD) distribution were determined. In malignant lesions, TNM stage, tumor grade, proliferative activity, and hormone receptor expression were determined. Spearman correlation coefficients; Wilcoxon, Fisher exact, Kruskal Wallis, and chi(2) tests; and logistic regression analysis were used for evaluation. Malignant lesions exhibited a higher ratio of microvessels in tumor periphery versus tumor center than did benign lesions (P < .0005). High vessel ratios (P = .001) and low central vessel numbers (P = .007) were associated with high tumor grade. In malignant lesions, initial enhancement ratios of periphery to center of lesion correlated with the corresponding microvessel ratios (r = 0.61). Yet, a high peripheral MVD was not associated with strong peripheral enhancement (r = -0.09). High enhancement ratios, washout rates, and early enhancement peaks were associated with unfavorable, albeit not significant, prognostic indicators. Visible rim enhancement was the most accurate prognostic enhancement criterion for estrogen receptor status (P = .007), tumor grade (P = .06), and lymph node status (P = .046). Washout was the best discriminating criterion for proliferative activity. The different enhancement behaviors of malignant and benign breast lesions cannot be explained by MVD alone; however, a low MVD in the center of carcinoma is reflected quantitatively by a high enhancement ratio and qualitatively by rim enhancement, with an implication of adverse prognosis.

The ratio of membrane-bound form Flt-1 mRNA to VEGF mRNA correlates with tumor angiogenesis and prognosis in non-small cell lung cancer

Fms-like tyrosine kinase 1 (Flt-1), a receptor for vascular endothelial growth factor (VEGF), have two isoforms: membrane-bound form (mFlt-1) and soluble form. In the present study, we quantitatively evaluated expression level of mFlt-1 mRNA and VEGF mRNA in non-small cell lung cancer, and demonstrated the clinical significance of the ratio of mFlt-1 mRNA to VEGF mRNA ( mFlt-1/VEGF). High mFlt-1/VEGF tumor showed a significantly lower microvessel density ( P=0.004), and patients with high mFlt- 1/ VEGF tumor had a significantly favorable survival ( P=0.037). Thus, the ratio of mFlt- 1 mRNA to VEGF mRNA was inversely correlated with tumor angiogenesis, and was a significant prognostic factor.

Radiation-induced alterations in microvessel density as predictor of treatment efficiency in oropharyngeal cancer

In this prospective study we have evaluated the predictive role of intratumoral microvessel density (MVD) for the therapeutic response and progression of inoperable oropharyngeal cancer (OPCC) treated with radiotherapy. Thirty-five OPCC patients were enrolled in this study. Biopsies from the primary tumor were taken before and after 20 Gy irradiation. Pathological factors such as histological grade, mitotic activity index and MVD were determined in both samples. Correlations of these factors with response to therapy, progression-free and overall survival were analyzed after a follow-up period of a minimum of 50 months. Objective response and survival was independent of the pretreatment MVD of OPCC. On the other hand, objective response was significantly affected by stage and low posttreatment MVD. Response to irradiation, and therapy-induced low postirradiation MVD were significant indicators of better overall and progression-free survival. We have shown in this small exploratory study that the anti-angiogenic effect of irradiation has a predictive value of the success of radiotherapy in locally advanced OPCC and can be used to select a radioresistant patient population which might require a more aggressive protocol.

Characterization of a von Hippel Lindau Pathway Involved in Extracellular Matrix Remodeling, Cell Invasion, and Angiogenesis

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene results in highly vascularized tumors, making the VHL tumor syndrome an ideal system to study the mechanisms of angiogenesis. VHL operates along two pathways with the first involving hypoxia-inducible factor-alpha degradation and down-regulation of its proangiogenic target genes vascular endothelial growth factor and platelet-derived growth factor-beta, and the second pathway promoting extracellular matrix (ECM) assembly. Secretion of proangiogenic factors was shown to be a primary inducer of angiogenesis. Here, we show that loss of ECM assembly correlates with tumor angiogenesis in VHL disease. Upon inactivation of the VHL-ECM assembly pathway, we observe tumors that are highly vascularized, have a disrupted ECM, and show increased matrix metalloproteinase-2 activity. Loss of the VHL pathway leading to hypoxia-inducible factor-alpha degradation results in tumors with increased vascular endothelial growth factor levels but with surprisingly low microvessel density, a tightly assembled ECM and low invasive ability. We conclude that loss of ECM integrity could promote and maintain tumor angiogenesis by providing a route for blood vessels to infiltrate tumors.

Prognostic Value of Tumor Angiogenesis and Microvascular Invasion in Renal Cell Carcinoma

Purpose: This study was performed to evaluate the impact of microvessel density (MVD), a reflection of tumor angiogenesis, and microvascular invasion (MVI) on the prognosis of patients with renal cell carcinoma (RCC). Materials and Methods: Formalin-fixed, paraffin-embedded tissue sections of RCC from 81 patients who had undergone radical nephrectomy were stained immunohistochemically for CD34, which decorate endothelial cells, in order to assess MVD and MVI. The immunostaining results of MVD and MVI were compared with the clinicopathological variables. Results: Twenty-two patients had either synchronous or metachronous metastases and fourteen patients died during the follow-up. MVD was significantly correlated with only metastasis (synchronous or metachronous; p=0.020). MVI was significantly correlated with tumor size (p=0.005), TNM stage (p<0.001), T stage (p<0.001), M stage (p=0.001), and metastasis (synchronous or metachronous; p=0.007). MVD was not significantly associated with MVI (p=0.232). The survival rate of patients with higher MVD or MVI-positive tumors was significantly lower than that of patients with lower MVD or MVI-negative tumors, respectively (p<0.0001, p=0.0002). Multivariate analyses indicated that tumor size, M stage and MVI were independent prognostic factors for cancer-specific survival. MVD was not an independent factor. Conclusions: MVD and MVI were associated with metastasis and a worse prognosis in RCC, which suggests that tumor angiogenesis and MVI may play an important role in the progression of RCC. MVI was an independent prognostic factor for cancer-specific survival. (Korean J Urol 2006;47: 111-117) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ