Microvessel Density Measurement Research Articles

Inhibitory effect of endostatin gene therapy combined with phosphorus-32 colloid on tumour growth in Wistar rats.

Eighty healthy male Wistar rats, aged 5 weeks, weighing 100–120 g, were utilized for establishing tumour-bearing models by immediate Walker-256 cancerous ascites injection and randomly divided to four groups (n=20) treated with 0.2 ml solution containing saline, 32P-colloid (0.3 mCi), endostatin gene (20 μg), endostatin gene combined with colloid 32P. The effect of endostatin combined with a small dose of 32P-colloidal on tumour growth in vivo was evaluated and the potential mechanism underlying the combined therapy was explored. We found that 32P-colloid combined with endostatin exhibited higher inhibitory effect upon tumour growth compared with application of 32P-colloid or endostatin alone, although three therapies all significantly inhibited tumour growth compared with saline control group. The higher inhibitory effect of 32P-colloid combined with endostatin upon tumour growth might be attributed to a synergistic effect of inhibiting angiogenesis by endostatin and inducing apoptosis by 32P-colloid, as demonstrated by microvessel density (MVD) and apoptotic index (AI) measurement. Combined therapy of 32P-colloid and endostatin probably serves as a novel and efficacious therapy of tumour growth.

The anti-tumour activity of rLj-RGD4, an RGD toxin protein from Lampetra japonica, on human laryngeal squamous carcinoma Hep-2 cells in nude mice.

The objective of this study is to investigate the antiproliferative activity and mechanism of integrin-binding rLj-RGD4 in a Hep-2 human laryngeal carcinoma-bearing nude mouse model. Human laryngeal squamous carcinoma cells (Hep-2) were inoculated subcutaneously into the axilla of nude mice to generate a Hep-2 human laryngeal carcinoma-bearing nude mouse model. When the Hep-2 xenograft model was successfully established, the animals were randomly separated into five groups. Three groups were treated with different dosages of rLj-RGD4. Cisplatin was administered to the positive control group, and normal saline (NaCl) was administered to the negative control group for 3 weeks. The body weights and the survival of the nude mice were evaluated, and the volumes and weights of the solid tumours were measured. The mechanism underlying rLj-RGD4 inhibition of tumour growth in transplanted Hep-2 human laryngeal carcinoma-bearing nude mice was evaluated by haematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL), measurement of intratumoural microvessel density (MVD), Western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The tumour volumes and weights of the treatment groups were reduced compared with the model group, and survival times were improved by rLj-RGD4 treatment in Hep-2 human laryngeal carcinoma-bearing nude mice. The number of apoptotic Hep-2 human cells and intratumoural MVD significantly decreased after the administration of rLj-RGD4. In the xenograft tissue of animals treated with rLj-RGD4, FAK, PI3K, and Akt expression was unaltered, whereas P-FAK, P-PI3K, Bcl-2, P-Akt, and VEGF levels were down-regulated. In addition, activated caspase-3, activated caspase-9, and Bax levels were up-regulated. rLj-RGD4 exhibits potent invivo activity and inhibits the growth of transplanted Hep-2 human laryngeal carcinoma cells in a nude mouse model. Thus, these results indicate that the recombinant RGD toxin protein rLj-RGD4 may serve as a potent clinical therapy for human laryngeal squamous carcinoma.

Combining dynamic contrast enhanced magnetic resonance imaging and microvessel density to assess the angiogenesis after PEI in a rabbit VX2 liver tumor model

To evaluate the correlation between parameters of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and microvessel density (MVD) measurements in rabbit VX2 liver tumor models after percutaneous ethanol injection (PEI) and to observe influence of PEI on angiogenesis in a rabbit VX2 liver tumor model with dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). Forty five New Zealand white rabbits were used in this study. VX2 tumor tissue blocks were implanted in the left lobe of liver by percutaneous puncture under CT guidance. 2 weeks later, all rabbits underwent conventional MRI (T1WI, T2WI) to determine the successful models. Then those successful implanted VX2 liver tumor models in the study were randomly divided into the control group and the experimental group, the former did not have processing, and the latter underwent PEI under CT guidance. MRI (T1WI, T2WI and DCE-MRI) was performed 1 week later again, the parameters of DCE-MRI (Ktrans, Kep, Ve and iAUC60) of viable tumor portions were observed. Then all the liver samples were processed for hematoxylin and eosin (H&E) staining and immunohistochemical staining for CD31 to determine MVD. At last, data (including DCE-MRI perfusion parameters and MVD) were compared between experimental and control groups, correlation of DCE-MRI perfusion parameters and MVD was evaluated. Twenty six VX2 liver tumor models underwent all examinations (thirteen models for each group) 1 week later after PEI. For the experimental group, the parameters Ktrans (r=0.6382, P=0.0189) and iAUC60 (r=0.6591, P=0.0143) in viable tumor portions were positively moderately correlated with MVD, whereas the parameters Kep (r=0.4656, P=0.1088) and Ve (r=0.2918, P=0.3333) were not correlated with MVD. For the control group, the parameters Ktrans (r=0.6385, P=0.0188) and iAUC60 (r=0.6391, P=0.0187) in viable tumor portions were also positively moderately correlated with MVD, while the parameters Kep (r=0.5518, P=0.0506) and Ve (r=-0.0824, P=0.789) were not correlated with MVD. Ktrans, Kep, Ve, iAUC60 and MVD of residual viable tumors in the experimental group 1 week later after PEI were similar to the viable tumors of the control group (P>0.05). DCE-MRI could be used to evaluate the efficiency of VX2 liver tumor after PEI. The quantitative parameter Ktrans and semi-quantitative parameter iAUC60 of DCE-MRI are correlated with MVD, which can assess tumor angiogenesis noninvasively of VX2 liver tumor model, and ethanol has no significant impact on angiogenesis of viable tumor 1week later after PEI.

Reconsideration of the clinical and histopathological significance of angiogenesis in prostate cancer: Usefulness and limitations of microvessel density measurement.

Angiogenesis plays important roles in tumor growth and cancer cell dissemination in almost all cancers. In prostate cancer, there is general agreement that increased angiogenesis is an important factor in determining tumor development and prognosis in these patients. Microvessel density is recognized as a useful marker for evaluating the angiogenic status of cancer tissues. Many investigators have reported that microvessel density is significantly associated with pathological features and outcomes in prostate cancer patients; however, some researchers have expressed opposing opinions. As the reason for such discrepancy, previous reports have suggested differences in the methodologies of measuring microvessel density in cancer tissues. In the present review, we focus on the variation in such methods, including the selected area and the method used for (semi)quantification. In particular, we discuss the relationship between malignancy potential, tumor progression, and survival and differences in the antibodies used for detection of endothelial cells in detail. We briefly compare the pathological significance and prognostic roles of microvessel density measured using von Willebrand factor, CD31, CD34, and CD105. Based on these analyses, the advantages and limitations of microvessel density measurements in prostate cancer tissues are clarified. Improved "real" and "specific" markers of cancer-related angiogenesis are necessary for better predictions of prognoses and for discussion of treatment strategies for patients with prostate cancer. However, establishment of a satisfactory cancer-related endothelial marker could take a long time. Therefore, knowledge regarding the pathological significance of microvessel density - based on understanding of the advantages and limitations of microvessel density determination methods - is important.

White matter hypoperfusion and damage in dementia: post-mortem assessment.

Neuroimaging has revealed a range of white matter abnormalities that are common in dementia, some that predict cognitive decline. The abnormalities may result from structural diseases of the cerebral vasculature, such as arteriolosclerosis and amyloid angiopathy, but can also be caused by nonstructural vascular abnormalities (eg, of vascular contractility or permeability), neurovascular instability or extracranial cardiac or vascular disease. Conventional histopathological assessment of the white matter has tended to conflate morphological vascular abnormalities with changes that reflect altered interstitial fluid dynamics or white matter ischemic damage, even though the latter may be of extracranial or nonstructural etiology. However, histopathology is being supplemented by biochemical approaches, including the measurement of proteins involved in the molecular responses to brain ischemia, myelin proteins differentially susceptible to ischemic damage, vessel-associated proteins that allow rapid measurement of microvessel density, markers of blood-brain barrier dysfunction and axonal injury, and mediators of white matter damage. By combining neuroimaging with histopathology and biochemical analysis, we can provide reproducible, quantitative data on the severity of white matter damage, and information on its etiology and pathogenesis. Together these have the potential to inform and improve treatment, particularly in forms of dementia to which white matter hypoperfusion makes a significant contribution.

Differences in vascular endothelial growth factor receptor expression and correlation with the degree of enhancement in medulloblastoma.

Vascular endothelial growth factor (VEGF) is the major proangiogenic factor in many solid tumors. Vascular endothelial growth factor receptor (VEGFR) is expressed in abundance in pediatric patients with medulloblastoma and is associated with tumor metastasis, poor prognosis, and proliferation. Gadolinium enhancement on MRI has been suggested to have prognostic significance for some tumors. The association of VEGF/VEGFR and Gd enhancement in medulloblastoma has never been closely examined. The authors therefore sought to evaluate whether Gd-enhancing medulloblastomas have higher levels of VEGFR and CD31. Outcomes and survival in patients with enhancing and nonenhancing tumors were also compared. A retrospective analysis of patients with enhancing, nonenhancing, and partially enhancing medulloblastomas was performed. Primary end points included risk stratification, extent of resection, and perioperative complications. A cohort of 3 enhancing and 3 nonenhancing tumors was selected for VEGFR and CD31 analysis as well as microvessel density measurements. Fifty-eight patients were analyzed, and 20.7% of the medulloblastomas in these patients were nonenhancing. Enhancing medulloblastomas exhibited strong VEGFR1/2 and CD31 expression relative to nonenhancing tumors. There was no significant difference in perioperative complications or patient survival between the 2 groups. These results suggest that in patients with medulloblastoma the presence of enhancement on MRI may correlate with increased vascularity and angiogenesis, but does not correlate with worse patient prognosis in the short or long term.

CD163 Expression Was Associated with Angiogenesis and Shortened Survival in Patients with Uniformly Treated Classical Hodgkin Lymphoma

BackgroundRecent studies have reported the prognostic value of tissue-associated magrophages (TAMs) in classical Hodgkin lymphoma (cHL). In addition, TAMs are implicated in the tumor angiogenesis. In this study, we examined the prognostic relevance of TAMs in relation to vascular endothelial growth factor (VEGF) expression and angiogenesis in uniformly treated cases of cHL.MethodsDiagnostic tissue from 116 patients with ABVD-treated cHL was evaluated retrospectively by immunohistochemical analysis for CD68, CD163 and VEGF expression and for CD31 expression as a measure of microvessel density (MVD).ResultsHigh CD163 expression (≥35% of cellularity) correlated with VEGF expression (Pearson’s Chi-square test, P = 0.008) and MVD (Spearman correlation coefficient 0.310, P<0.001). High CD163 expression was associated with inferior event-free survival (EFS, P = 0.005) and overall survival (OS, P<0.001) in univariate analysis. In multivariate analysis, high CD163 expression was strongly associated with inferior EFS (P = 0.043) and OS (P = 0.008). Patients with high MVD had a lower OS than those with low MVD, but the difference was not significant (P = 0.071, respectively). While high expression of CD68 was also associated with inferior EFS (P = 0.007), it showed no correlation with VEGF or MVD.ConclusionsOur data confirms that CD163 expression provides independent prognostic information in cHL. The correlation of CD163 with VEGF expression and MVD suggests the role of CD163-positive cells in tumor angiogenesis of cHL.

Dynamic Contrast-Enhanced MR Imaging in a Phase Ⅱ Study on Neoadjuvant Chemotherapy Combining Rh-Endostatin with Docetaxel and Epirubicin for Locally Advanced Breast Cancer

Background: Anti-angiogenesis is a promising therapeutic strategy for locally advanced breast cancer. We performed this phase II trial to evaluate the anti-angiogenesis and anti-tumor effect of rh-endostatin combined with docetaxel and epirubicin in patients with locally advanced breast cancer by dynamic contrast-enhanced magnetic resonance imaging in 70 previously untreated locally advanced breast cancer patients.Methods: The study population was randomly assigned to neoadjuvant chemotherapy with docetaxel and epirubicin (neoadjuvant chemotherapy group) or neoadjuvant chemotherapy combining rh-endostatin with docetaxel and epirubicin (neoadjuvant chemotherapy+rh-endostatin group). The anti-angiogenic and anti-tumor effects of both regimens were evaluated by serial dynamic contrast-enhanced magnetic resonance imaging and microvessel density measurements after final surgery.Results: The results suggested a higher clinical objective response (90.9% vs. 67.7%, P = 0.021) and greater reductions in tumor size (67.2% vs. 55.9%, P = 0.000), Ki-67 proliferation index (32.79% vs. 12.47%, P = 0.000), tumor signal enhanced ratio (64% vs. 48%, P = 0.018), and Ktrans (67% vs. 39%, P = 0.026) in neoadjuvant chemotherapy+rh-endostatin group than those in neoadjuvant chemotherapy group. In addition, the microvessel density value in the neoadjuvant chemotherapy+rh-endostatin group was significantly lower than in the neoadjuvant chemotherapy group (18.67 ± 6.53 vs. 36.05 ± 9.64, P = 0.000). Moreover, the microvessel density value was significantly correlated with Ktrans after neoadjuvant chemotherapy+rh-endostatin treatment (r=0.88, P = 0.00).Conclusions: The neoadjuvant chemotherapy+rh-endostatin treatment significantly repressed angiogenesis in locally advanced breast cancer and synergistically enhanced the anti-tumor effect of neoadjuvant chemotherapy. Serial dynamic contrast-enhanced magnetic resonance imaging data including reductions in tumor size and Ktrans, could provide non-invasive evaluation for chemotherapeutic efficacy and, consequently, optimization of individual chemotherapy for locally advanced breast cancer patients.

Optimization of the scanning technique and diagnosis of pulmonary nodules with first-pass 64-detector-row perfusion VCT

The aims of this study were to optimize the scanning technique of first-pass 64-detector-row perfusion volume computed tomography imaging, to evaluate the effectiveness and stability of this scan protocol, and lastly to evaluate the differential diagnosis ability of perfusion imaging in solitary pulmonary nodules (SPNs). A total of 144 patients with SPNs underwent perfusion scan with 64-slice spiral CT scanner. The CT perfusion imaging was analyzed for time-density curve, perfusion parametric maps, and the respective perfusion parameters. We then analyzed the main factors concerning the imaging quality and evaluated the effectiveness of scan protocol by determining the receiver operating characteristic (ROC) curve, diagnostic efficacy, and odds ratio as well as the stability of scan protocol by consistency analysis. Immunohistochemical findings of microvessel density measurement and vascular endothelial growth factor expression were evaluated. The total sensitivity, specificity, accuracy, positive predictive value, negative predictive value, likelihood ratio, and the area under ROC curve during 5-45-s scan period were 78.95%, 82.4%, 80.6%, 83.3%, 77.8%, 4.620, 0.280, and 0.840, respectively, and Kappa value was 0.894. The diagnostic efficacy of CT pulmonary perfusion was significantly higher than during 0-40-s scan period. The parameter values in different nodules were different. The optimized 5-45-s scan period of CT pulmonary perfusion imaging is effective in pathologic diagnosis and has good stability, worthy of being popularized. Lung perfusion CT could be a promising and feasible method for differentiation of SPNs.

Spectral CT imaging of myocardial infarction: preliminary animal experience

To evaluate the capability of spectral CT imaging to detect the different stages and angiogenesis of myocardial infarction (MI). MI was surgically induced in 40 rabbits that were evenly divided into four stages of MI: 6 h (6H), 3 days (3D), 7 days (7D) and 14 days (14D). Spectral CT was performed at 10 s, 1 min and 3 min after intravenous contrast medium administration. CD31 immunohistochemistry was used for the microvessel density (MVD) measurement. Iodine concentrations in the myocardium were measured and normalised to the aorta as nIC. The relationships between infarcted myocardial nIC and MVD were analysed. The nIC of infarct myocardium decreased at 10 s and increased in late-phase CT images. There were significant differences between the 6H and other groups (P ( 6H-3D ) = 0.01, P ( 6H-7D ) = 0.01, P ( 6H-14D ) = 0.00). There was a significant difference in the MVD of infarct myocardium between the two groups except in the 7D and 14D groups (P = 0.08). In the 10-s phase, the nIC of infarct myocardium was negatively correlated with MVD (r = -0.54, P = 0.00), whereas in the late phases, there was a positive correlation between them (r = 0.57, P = 0.00 in the 1-min phase, r = 0.48, P = 0.00 in the 3-min phase). Spectral CT imaging of the myocardium can be used to evaluate the different stages and angiogenesis of MI.

WE-C-217BCD-04: Multimodality Imaging of Breast Cancer Experimental Lung Metastasis.

Metastatic breast cancer (MBC) is incurable. The clinical gold standard for assessing tumor microvessel density (MVD), an independent prognostic marker in MBC, is CD 105 staining. The goal of this study is to develop a positron emission tomography (PET)/near-infrared fluorescent (NIRF) probe for imaging of CD105 expression in MBC (i.e. non-invasive measurement of MVD), as well as other applications such as early detection of metastasis, intraoperative guidance, etc. TRC105, a chimeric anti-CD105 mAb, was dual-labeled with a NIRF dye and 89 Zr to yield 8 9 Zr-Df-TRC105-800CW. Luciferase-transfected 4T1 murine breast cancer cells were injected intravenously into female BALB/c mice to establish a lung MBC model. Bio luminescence imaging (BLI) was carried out to non- invasively monitor the lung tumor burden. Comprehensive in vivo/ex vivo studies were performed to investigate 8 9 Zr-Df-TRC105-800CW in this MBC model. Cetuximab was used as an isotype-matched control. Radiolabeled TRC105 has high tumor uptake in many tumor types in addition to MBC (e.g. pancreatic/prostate cancer and brain tumor), revealing broad clinical potential for TRC105-based agents. FACS analysis of HUVECs showed no difference in CD 105 binding between TRC105 and Df- TRC105-800CW. PET imaging revealed that 4T1 lung tumor uptake of 89 Zr-Df-TRC105-800CW was 8.7±1.4,10.9±0.5, and 9.7±1.1 %ID/g at 4, 24, and 48 h post-injection (n = 4), with excellent tumor contrast. Bio distribution studies, blocking, control studies with 8 9 Zr-Df-cetuximab- 800CW, ex vivo BLI/PET/NIRF imaging, and histology all confirmed CD 105 specificity of the tracer. NIRF imaging-guided removal of 4T1 tumors with Df-TRC105-800CW in a subcutaneous model was also straightforward. We report the first PET/NIRF imaging of CD105 expression in a MBC model. Broad clinical potential of TRC105- based agents was shown in many tumor types, which also enabled early detection of small metastases and provided intraoperative guidance for tumor removal.

Value of DCE-MRI and FDG-PET/CT in the prediction of response to preoperative chemotherapy with bevacizumab for colorectal liver metastases

Background:The purpose of this study was to assess the role of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and 18F-fluorodeoxyglucose positron emission tomography computed tomography (FDG-PET/CT) for evaluation of response to chemotherapy and bevacizumab and for prediction of progression-free survival (PFS) in patients with metastatic colorectal cancer (mCRC) with potentially resectable liver lesions.Methods:A total of 19 mCRC patients were treated with FOLFOX/FOLFIRI and bevacizumab followed by surgery. Dynamic contrast-enhanced magnetic resonance imaging and FDG-PET/CT were performed before treatment and after cycle 5. PET results were quantified by calculating maximum standardised uptake value (SUVmax) whereas area under the enhancement curve (AUC), initial AUC (iAUC) and the endothelial transfer constant (Ktrans) were used to quantify DCE-MRI. Pathological analysis of the resection specimen was performed, including measurement of microvessel density (MVD) and proliferation index.Results:Both AUC and iAUC were significantly decreased following bevacizumab therapy (median change of 22% (P=0.002) and 40% (P=0.001) for AUC and iAUC, respectively). Progression-free survival benefit was shown for patients with >40% reduction in Ktrans (P=0.019). In the group of radiological responders, the median baseline SUVmax was 3.77 (IQR: 2.88–5.60) compared with 7.20 (IQR: 4.67–8.73) in nonresponders (P=0.021). A higher follow-up SUVmax was correlated with worse PFS (P=0.012). Median MVD was 10.9. Progression-free survival was significantly shorter in patients with an MVD greater than 10, compared with patients with lower MVD (10 months compared with 16 months, P=0.016).Conclusion:High relative decrease in Ktrans, low follow-up SUVmax and low MVD are favourable prognostic factors for mCRC patients treated with bevacizumab before surgery.

Vascular markers CD31, CD34, actin, VEGFB, and VEGFR2, are prognostic markers for malignant development in benign endometrial polyps

Intention of the study: The prevalence of endometrial polyps has been demonstrated in between 10% and 35% of all women, but knowledge regarding malignant potential within polyps is limited. Even though premalignant and malignant changes have been reported in up to 24% of all cases, no objective tissue markers have ever been developed for routine diagnostics to select high risk cases. As vascular changes and activation of endometrial angiogenesis has been demonstrated in former studies, our main objective was to evaluate different members of the angiogenic pathway as potential risk factors for cancer development. Patients and methods: Formalin-fixed, paraffin-embedded tissue from 15 women with benign endometrial polyps, and 16 women diagnosed with endometrial cancer were included. Immunohistochemical investigation with antibodies against VEGF, VEGF-B, VEGFR2, VEGFR3, CD31, CD34, actin, and factorVIII was performed, followed by evaluation of staining intensity of microvessels, evaluation of H-score in glands (cell membrane, cytoplasm) and stroma, and measurement of micro vessel density. Results: Expression of CD31 in microvessels was significantly stronger in cancers compared to endometrial polyps (P = 0.006 for arterioles, P = 0.038, for venyles, and P = 0.002 for capillaries, respectively), whereas, a reverse change was shown for CD34. Expression of actin in capillary walls was also significantly increased in cancers compared to polyps (P = 0.002). No significant difference was found for staining intensity in microvessels (arterioles, venyles or capillaries) in endometrial benign polyps compared with endometrial cancers for VEGF, VEGFB, VEGFR2, VEGFR3, or Factor VIII. Also no difference in H-score values between benign polyps and endometrial cancers could be detected in glandular epithelium, in epithelial cell membrane or in stroma for VEGFR3, CD31 or Factor VIII. Conclusions: The present study strongly indicates that activation of angiogenesis differs in benign endometrial polyps and endometrial cancers. Thus, immunohistochemical expression of specific angiogenic markers may be of great importance as prognostic factors in the routine diagnostics of this lesion. The ratio between stromal expression of CD34 and actin might be of particular interest to select polyps with increased malignant potential.

Characterization of Spatial Distribution of Myeloma Cells and Their Microenvironment in Bone Marrow; A Computer Assisted Study Using Novel Whole Slide Image Analysis Technology

Abstract Abstract 2889 The ability to characterize distribution of neoplastic hematopoietic cells and their progenitors in their native microenvironment is emerging as an important challenge and potential therapeutic target in many disease areas, including multiple myeloma. The goal of the study is to develop a method of analyzing neoplastic cells in their hematopoietic niche context using readily available tissue sections and standard histology approaches. We applied this method to study myeloma cells spatial distribution within a given distance of bone, blood supply, or other cell type, and can be generalized to any class of hematopoietic progenitor stem cell and tissue type. We utilized consecutive cut sections combined with whole slide scanning and novel image analysis to provide quantitative data on bone marrow microenvironment. Consecutive bone marrow core biopsy sections from multiple myeloma patients were stained for CD34 (endothelial cells) and CD138 (myeloma plasma cells). Bone to plasma cell analysis: We applied pattern recognition image analysis to find all bone areas, followed by a cell based analysis to identify all plasma cells. We then computationally grew a distance out from bone, at increments of 5 microns, and classified myeloma cells as either inside or outside of this distance, and computed the percentages of myeloma cells near bone. Vessel to plasma cell analysis: We identified endothelial cells using an image analysis thresholding technique, followed by an algorithm that groups neighboring endothelial cells into vessels. The number of vessels was reported as a microvessel density, and the percentage of hematopoietic cells within a given distance of vessels was also calculated for each sample. We then looked at three distances (15, 20, and 25 microns) in twelve representative samples as a threshold to stratify the neoplastic cells based on their distance from vessels. The image analysis markup for the pattern recognition approach in bone is shown in Figure 1, and the vessel image analysis approach is described in Figure 2. Using 12 bone marrow sections, we determined the best distance for discriminating between low and highly vascularized samples was 20 microns (Table 1). The correlation between computer based microvessel density (MVD) analysis and percentage of hematopoietic cells in proximity to vessels (MVP) is shown in Figure 3. We demonstrate the feasibility of analyzing the spatial distribution of plasma cells, and possibly other hematopoietic / stem cells in their microenvironment, as characterized by the distance to both bone and vessels in bone marrow. The distance of 20 microns was determined empirically as providing the most separation of data between high and low vascularized samples, suggesting a potential differential oxygenation point. While the MVD measurement had a high degree of correlation with MVP, MPV measures a different biological phenomena, namely the percentage of tissue that is well oxygenated, and would only be perfectly correlated if the vessels were exactly evenly distributed in a sample. Preliminary observations appear that there is substantially greater information in the MVP measurement particularly between the range of 400 to 600 vessels / mm2, on samples that would be considered highly vascularized. Further work is ongoing to characterize a large patient cohort and correlate various microenironment variables with clinical outcome and other standard laboratory measurements. Disclosures: Landis: Flagship Biosciences: Employment. Lange:Flagship Biosciences: Employment. Potts:Flagship Biosciences: Employment.

Microvessel Density But Not Neoangiogenesis Is Associated with 18F-FDG Uptake in Human Atherosclerotic Carotid Plaques

The vulnerable atherosclerotic lesion exhibits the proliferation of neovessels and inflammation. The imaging modality 2-deoxy-2-[(18)F]fluoro-D: -glucose positron emission tomography ((18)FDG-PET) is considered for the identification of vulnerable plaques. The purpose of this study was to compare the gene expression of neoangiogenesis and vulnerability-associated genes with (18)FDG uptake in patients undergoing carotid endarterectomy. Human atherosclerotic carotid artery plaques from symptomatic patients were used for gene expression analysis by quantitative PCR of vascular endothelial growth factor (VEGF) and integrin α(V) and integrin β(3) subunits, genes essential during neoangiogenesis. We also evaluated the gene expression of CD34, a measure of microvessel density (MVD), as well as CD68, MMP-9, and cathepsin K, genes of major importance in plaque vulnerability. Gene expression analysis was compared with (18)FDG-PET. VEGF and integrin α(V)β(3) gene expression did not correlate with (18)FDG uptake, whereas CD34 gene expression exhibited an inverse correlation with (18)FDG uptake. Additionally, we established that markers of vulnerability were correlated with (18)FDG uptake. Neoangiogenesis is not associated with (18)FDG uptake, whereas MVD and markers of vulnerability correlate with (18)FDG uptake. The absence of correlation between markers of neoangiogenesis and (18)FDG uptake suggests a temporal separation between the process of neoangiogenesis and inflammatory activity.

A Phase II Randomized Trial of Gefitinib Alone or with Tegafur/Uracil Treatment in Patients with Pulmonary Adenocarcinoma Who had Failed Previous Chemotherapy

Tegafur/uracil (UFT) is suitable for metronomic chemotherapy because of its underlying antiangiogenesis mechanism. This study aimed to assess the efficacy of adding daily oral UFT to gefitinib treatment in patients with pulmonary adenocarcinoma who had failed previous chemotherapy. Taiwanese patients who had adenocarcinoma of the lung and failed previous chemotherapy were randomized into gefitinib 250 mg daily alone (G) or plus daily oral UFT (GU). From November 2005 to August 2009, 115 patients were enrolled. There were 58 patients in the G arm and 57 in the GU arm. One-year progression-free survival (PFS) was 18% in the G arm and 36.7% in the GU arm (p = 0.03). Fifty-four patients had tissue samples available for tumor epidermal growth factor receptor (EGFR) sequence analysis: 16 classical mutations and 8 wild types in the G arm, and 20 classical mutations and 10 wild-types in the GU arm. The addition of UFT significantly improved PFS in patients with EGFR mutations (14.4 versus 7.6 months, p = 0.0061). Forty-three patients underwent tumor tissue microvessel density measurement, and a trend favoring the addition of UFT to gefitinib treatment was found in those with low microvessel density (median PFS: 11.8 versus 2.8 months, p = 0.0536). The median survival time was 18.3 months in the G arm and 23.6 months in the GU arm (p = 0.381). Gefitinib plus UFT treatment had better PFS than gefitinib alone treatment. Gefitinib is effective in patients with EGFR mutations, and the addition of UFT treatment produced better PFS in these patients with mutations.

Minimally invasive assessment of tumor angiogenesis by fine needle aspiration and flow cytometry

The development of a new, less invasive, and more rapidly implemented method of quantifying endothelial cell density in tumors could facilitate experimental and clinical studies of angiogenesis. Therefore, we evaluated the utility of tumor fine needle aspiration (FNA) coupled with flow cytometry for assessment of tumor angiogenesis. Samples were obtained from cutaneous tumors of mice using FNA, then immunostained and assessed by flow cytometry to determine the number of CD31(+) endothelial cells. Results of the FNA/flow cytometry technique were compared with quantification of tumor microvessel density using immunohistochemistry. The ability of the FNA/cytometry technique to quantify the effects of anti-angiogenic therapy and to monitor changes in tumor angiogenesis over time in individual tumors was also determined. We found that endothelial cell percentages determined in tumor tissue aspirates by flow cytometry correlated well with the percentages of endothelial cells determined in whole tumor digests by flow cytometry and with tumor microvessel density measurements by immunohistochemistry. Moreover, we found that repeated FNA sampling of tumors did not induce endothelial cell changes. Interestingly, by employing repeated FNA sampling of the same tumors we were able to observe a sudden and marked decline in tumor angiogenesis triggered when tumors reached a certain size. Thus, we conclude that the FNA/flow cytometry technique is an efficient, reproducible, and relatively non-invasive method of rapidly assessing tumor angiogenesis, which could be readily applied to evaluation of tumor angiogenesis in clinical settings in humans.

Microvessel density and the association with single nucleotide polymorphisms of the vascular endothelial growth factor receptor 2 in patients with colorectal cancer

The measurement of microvessel density (MVD) is a widely accepted method for assessing the neoangiogenetic activity in neoplasia. The aim of the present study was to compare MVD with single nucleotide polymorphisms (SNPs) in the vascular endothelial growth factor receptor (VEGFR)-1 and VEGFR-2 genes and, furthermore, with quantitative measurements of the receptors in colorectal cancer (CRC) tissue. Prognosis was also assessed. Blood and tissue were collected from 110 patients surgically resected for CRC. SNPs were analysed from genomic DNA by polymerase chain reaction. MVD was assessed by immunohistochemistry using CD34 and CD105 combined with caldesmon in order to identify also immature vessels. Microvessels were counted in three fields of vision, and the mean MVD was used for statistical analysis. The VEGFR-2 1192 C/T and -604 T/C SNPs were associated with the MVD assessed by CD105. The median MVD score for the 1192 CC genotype was significantly lower compared to the CT + TT genotypes (p = 0.002). The median MVD score for the -604 CC genotype was significantly higher compared to the TT + TC genotypes (p = 0.009). A possible association, although non-significant, was demonstrated for the CD34-positive microvessels. The 1192 CC genotype and the -604 TT + TC genotypes correlated with improved survival. This is the first report on correlations between SNPs in the VEGF receptor genes and MVD in patients with CRC. Associations were shown between two SNPs in the VEGFR-2 gene and the CD105-positive microvessels indicating an impact on neoangiogenesis. Moreover, an association between the SNPs and survival was demonstrated. The clinical implications of these findings need further investigations.

Differential Impact of Familial Hypercholesterolemia and Combined Hyperlipidemia on Vascular Wall and Network Remodeling in Mice

Genetic familial hypercholesterolemia (FH) and combined hyperlipidemia (FCH) are characterized by elevated plasma low-density lipoprotein (LDL) (FH) and LDL/triglycerides (FCH), with mouse models represented by LDL receptor (LDLR) and apolipoprotein E (ApoE) gene deletion mice, respectively. Given the impact of FH and FCH on health outcomes, we determined the impact of FH/FCH on vascular structure in LDLR and ApoE mice. LDLR, ApoE and control mice were utilized at 12-13 and 22-23 weeks when gracilis arteries were studied for wall mechanics and gastrocnemius muscles were harvested for microvessel density measurements. Conduit arteries and plasma samples were harvested for biochemical analyses. Arteries from ApoE and LDLR exhibited blunted expansion versus control, reduced distensibility and left-shifted stress versus strain relation (LDLR > ApoE). Microvessel density was reduced in ApoE and LDLR (ApoE > LDLR). Secondary analyses suggested that wall remodeling in LDLR was associated with cholesterol and MCP-1, while rarefaction in ApoE was associated with tumor necrosis factors-alpha, triglycerides and vascular production of TxA(2). Remodeling in ApoE and LDLR appears distinct; as that in LDLR is preferential for vascular walls, while that for ApoE is stronger for rarefaction. Remodeling in LDLR may be associated with cellular adhesion, while that in ApoE may be associated with pro-apoptotsis and constrictor prostanoid generation.

P26: Performance of a novel automated microvessel analysis algorithm across whole slide digital images

The ability to evaluate microvessels in tissues is important across many therapeutic areas in both preclinical studies as well as clinical trials which seek potential biomarkers of vascular injury. There is an urgent need for more accurate, reproducible microvessel measurements that do not require tedious, manual counting under a microscope. The time spent by a pathologist in training others to manually count vessels and to review the work is a bottleneck in this area. The Aperio Microvessel Analysis tool, an image analysis tool that identifies and counts vessels stained with an endothelial cell marker, was compared to manual counting for its accuracy and precision in mouse matrigel whole slide images. Manual counting of blood vessels by two PhD scientists had a low precision rate with a concordance of only 60% compared to precision by the automated approach of 87.4%. Expected error rates averaged 13.6% for statistics that were based on individual vessels (e.g. vessel perimeter or individual vessel area) and 12.2% for microvessel density measurements. Other advantages to automated microvessel analysis on whole slides include speed; both the scanning and analysis time can be accomplished in minutes. Additional advantages include quantitative vessel measurements and statistics which are calculated by the software algorithm. Further work will expand the validation to other tissue types and will compare concordance between trained pathologists and between trained pathologists and automated analysis.