Intratumoral Microvascular Density Research Articles

Comprehensive Investigation of Angiogenesis, PASS Score and Immunohistochemical Factors in Risk Assessment of Malignancy for Paraganglioma and Pheochromocytoma.

A challenging task in routine practice is finding the distinction between benign and malignant paragangliomas and pheochromocytomas. The aim of this study is to conduct a comparative analysis of angiogenesis by assessing intratumoral microvascular density (MVD) with immunohistochemical (IHC) markers (CD31, CD34, CD105, ERG), and S100 immunoreactivity, Ki67 proliferative index, succinate dehydrogenase B (SDHB) expressiveness, tumor size with one the most utilized score Pheochromocytoma of Adrenal Gland Scales Score (PASS), using tissue microarray (TMA) with 115 tumor samples, 61 benign (PASS < 4) and 54 potentially malignant (PASS ≥ 4). We found no notable difference between intratumoral MVD and potentially malignant behavior. The group of potentially malignant tumors is significantly larger in size, has lower intratumoral MVD, and a decreased number of S100 labeled sustentacular cells. Both groups have low proliferative activity (mean Ki67 is 1.02 and 1.22, respectively). Most tumors maintain SDHB expression, only 6 cases (5.2%) showed a loss of expression (4 of them in PASS < 4 group and 2 in PASS ≥ 4). PASS score is easily available for assessment and complemented with markers of biological behavior to complete the risk stratification algorithm. Size is directly related to PASS score and malignancy. Intratumoral MVD is extensively developed but it is not crucial in evaluating the malignant potential.

Abstract 5260: Combination activity of eribulin liposomal formulation and anti-PD-1 antibody after tumor regrowth during anti-PD-1 antibody treatment in mice

Abstract Eribulin (ERI) has been reported a microtubule dynamics inhibitor with unique tumor microenvironment modulations such as vascular remodeling activity. In the previous meeting (AACR2022, 2023), we reported that ERI and liposomal formulation of ERI (ERI-LF) also have immunomodulatory activity that induces CD8+ T cells via its vascular remodeling activity and ERI-LF showed more potent immune modulation and combination activities with anti-PD-1 antibody (PD-1 Ab) than ERI in syngeneic mice models and humanized mice models. Although immune-checkpoint inhibitors (ICI) are using as a key drug for various types of cancers in clinical setting, several clinical questions regarding suitable therapies after ICI therapy and efficacy of ICI rechallenge are remaining. In this study, we evaluated anti-tumor activities of combination of ERI-LF and PD-1 Ab in two syngeneic transplantation models using mouse non-small lung cancer cells, P-glycoprotein-knockout (Pgp-KO) KLN205 cells and Pgp-KO LL2 cells. As a result, combination of 1mg/kg ERI-LF (Q7D×2) with PD-1 Ab (200 ug/head, twice a week ×2 weeks) showed significant stronger antitumor activity compared with each monotherapy in only Pgp-KO KLN205 model, in which ERI-LF increased intratumoral microvascular density as a vascular remodeling activity, not in Pgp-KO LL2 model. We next conducted continuous treatment of PD-1 Ab in Pgp-KO KLN205 model to investigate tumor regrowth during treatment of PD-1 Ab. In this model, tumor growth was inhibited by PD-1 Ab until about two weeks after initiation of the treatment and then this inhibitory activity disappeared after two weeks even by continuous treatment of PD-1 Ab. We performed flow cytometry analysis (FCM) for tumor-infiltrating immune cells and RNAseq analysis using regrowing tumors compared with non-treatment tumors. Expressions of mRNA of several immune inhibitory receptors were upregulated in tumors with regrowth, although CD4+ cells and CD8+ cells were also increased in FCM using tumor samples. Furthermore, we investigated anti-tumor activity of combination of ERI-LF and PD-1 Ab against tumors which changed from inhibitory phase to regrowth phase during PD-1 Ab treatment in Pgp-KO KLN205 model. The combination of ERI-LF at 1mg/kg (Q7D×3) and PD-1 Ab (200 ug/head, twice a week ×3 weeks) also showed potent anti-tumor activity against tumors with regrowth by prior PD-1 Ab treatment as well as PD-1 Ab-naïve tumor, suggesting that this combination therapy might be effective after prior ICI therapy. Currently, Phase 1b/2 clinical trial of ERI-LF plus nivolumab in patients with selected solid cancers (NCT04078295) is underway. Patients with or without prior ICI therapy were enrolled in a small cell lung cancer cohort of this clinical study. Our preclinical results might provide a scientific rationale for a uniqueness of ERI-LF as a post ICI therapy. Citation Format: Moe Tamura, Yuki Niwa, Taro Semba. Combination activity of eribulin liposomal formulation and anti-PD-1 antibody after tumor regrowth during anti-PD-1 antibody treatment in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5260.

The Predictive Role of the Histopathological Scoring System in Adipose Tumors-Lipoma, Atypical Lipomatous Tumor, and Liposarcoma.

Lipomatous tumors are the most frequent soft tissue neoplasms. Sometimes their differential diagnosis is difficult to perform only by microscopic analysis. This study aims to create a histopathological scoring system and highlight the impact of intratumoral microvascular density. This study was conducted over 10 years. We analyzed the main pathogenic pathways (MDM2 and CDK4), as well as the tumor microvascularization (CD31 and CD34) by immunohistochemical tests. We also analyzed the status of the MDM2 gene by CISH. These data, together with the clinical and histopathological information, were statistically analyzed by appropriate tests. We identified 112 eligible cases, with most of the patients being in their sixth decade of life, with a slight predominance of the female sex. We found important associations like tumor location linked to nuclear pleomorphism severity and microvascularization density correlated with atypia severity. Also, we observed that a maximum diameter of a tumor of at least 69 mm is associated with the presence of tumor necrosis. The score designed in this study shows an increased sensitivity and specificity for the diagnosis of lipomas (100%, respectively, 97%), atypical lipomatous tumors (93.8%, respectively, 82.3%), and liposarcomas (100%, respectively, 90.5%). This present study enhances the present data by bringing to attention the histopathological score with a role in differential diagnosis, as well as in the prediction of immunohistochemical and genetic tests. Also, we highlighted the importance of microvascular density, especially in the diagnosis of liposarcomas.

Expression and clinical significance of CD31, CD34, and CD105 in pulmonary ground glass nodules with different vascular manifestations on CT.

Blood vessel passage on CT exerts a vital part in early diagnosis as well as treatment of carcinoma of the lungs. Intratumoral microvascular density (iMVD) has gradually become the focus of research on biological behavior, appearance, and evolution of malignant tumors nowadays. The aim of this paper was to verify whether there is a correlation between the iMVD and the vascular morphology of ground glass nodules (GGNs). A total of 109 patients with pulmonary GGN were classified into three groups (I,II, and III) according to the vascular morphology on CT, and their expression of CD31-, CD34-, and CD105-labeled iMVD was detected by the streptoavidin–biotin method, statistically analyzing the iMVD values of each group. The expression of CD31, CD34, and CD105 in different lung tissues was significantly different, with remarkably higher iMVD in lung cancer tissues than in adjacent normal lung tissues. In the imaging sort of types I, II, and III according to the means of vascular passage, the iMVD expression of CD31, CD34, and CD105 was significantly different between groups. These data suggest that the presence and the abnormal morphology of vessels seen within GGNs indicate the occurrence and progression of lung cancer in pathology. It offers a strong theoretical foundation for early diagnosis of carcinoma of the lungs, thus providing a more precise clinical diagnosis and prognosis of early-stage lung cancer.

VEGF expression in cetuximab resistance development in patients with squamous cell carcinoma of the tongue and oral mucosa.

e18072 Background: Clinical course and aggressiveness of a tumor, as well as the subtle mechanisms of its pathogenesis are related to vascularization, which in turn is determined by the ability of transformed cells to synthesize a wide range of biologically active substances. Among modern approaches to cancer treatment, special attention is paid to anti-EGFR agents reducing tumor vascularization. The purpose of this study was to analyze VEGF expression in tumor vessels in patients with squamous cell carcinoma of the tongue and oral mucosa developing cetuximab resistance. Methods: The study included 45 patients with squamous cell carcinoma of the tongue and oral mucosa T3-4N0-1M0. The patients were divided into 3 groups: group 1 – all patients before treatment; group 2 – patients after standard chemotherapy with cisplatin and fluorouracil; group 3 – patients after chemotherapy in combination with targeted therapy with cetuximab. Immunohistochemical study was performed on sections from paraffin blocks of tumors using monoclonal murine anti-VEGF antibodies (clone VG1, Thermo Scientific) diluted 1:150. The Reveal Polyvalent HRP-DAB Detection System was used. The significance of differences was assessed using the Mann Whitney and Wilcoxon tests calculated using the STATISTICA 10.0 program (StatSoftInc., USA). Results: Changes in the density of vessels stained with the VEGF marker were studied in tumor cells of patients in all groups. Patients in group 1 had from 3 to 10 vessels in one field of view; in group 2 – from 2 to 6 vessels in 70% of cases (21) and up to 15 vessels in 30% of cases (9); in group 3 – only solitary vessels. The average numbers of microvessels stained with the VEGF marker per field of view were 6.5±1.52; 4.5±1.71; 1.0±0.28, respectively. Quantification of intratumoral microvascular density showed that the minimum number of microvasculature vessels in the field of view was observed in patients who received chemotherapy and targeted therapy with cetuximab. The value was 5 times lower compared to initial values, and 4 times lower than in patients who received only standard chemotherapy (the Mann Whitney test, p &lt; 0.05). The statistical analysis according to the Wilcoxon test on the effect of targeted therapy (comparison between groups 2 and 3) revealed a statistically significant effect of cetuximab for this marker (p = 0.028). Conclusions: Immunohistochemical analysis of the VEGF neoangiogenesis factor in squamous cell carcinoma of the tongue and oral mucosa confirmed the effectiveness of targeted therapy with cetuximab.

Predictive value of microvascular density for response to anlotinib in advanced NSCLC.

Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer. This study aimed to categorize the microvessels in advanced NSCLC and determine the relationship between intratumoral microvascular density (MVD) and the efficacy of anlotinib for NSCLC.The clinical data of 68 patients receiving anlotinib as third-line treatment or beyond for advanced NSCLC were retrospectively collected. Microvessels were stained for CD31 and CD34 by using immunohistochemical staining and were classified as undifferentiated (CD31+ CD34−) and differentiated vessels (CD31+ CD34+). The relationship between MVD and anlotinib efficacy and patient prognosis was analyzed.Patients were divided into the high or low MVD groups according to the median MVD of differentiated (9.4 vessels/field) and undifferentiated microvessels (6.5 vessels/field). There were significantly more patients with high undifferentiated-vessel MVD in the disease control group than in the disease progression group (72.7% vs 16.7%, P < .001). Patients with high undifferentiated-vessel MVD had significantly longer median progression-free survival than those with low undifferentiated-vessel MVD (7.1 vs 3.7 months, P < .001).Anlotinib as third- or beyond line therapy is safe and effective for advanced NSCLC. Patients with a higher density of undifferentiated microvessels have better response to anlotinib and longer progression-free survival.

Predictive Biomarkers of Pathological Response to Neoadjuvant Radiochemotherapy for Locally Advanced Soft Tissue Sarcomas

<h3>Purpose/Objective(s)</h3> Soft tissue sarcomas (STS) is a large group of rare cancers which present high intratumoral heterogeneity. Thus, the assessment of response to the neoadjuvant treatment applied for locally advanced STS remains a challenge. We hypothesized that the pathological response (PR) could be predicted using biomarkers of the tumor microenvironment, hypoxia, and DNA damage. <h3>Materials/Methods</h3> We analyzed prospectively collected core biopsy tissue samples from patients who received preoperative 5 × 5 Gy with doxorubicin-ifosfamide for marginally resectable high-grade STS in a phase II clinical trial. We have chosen tumor-infiltrating macrophages (TAMs) and intratumoral microvascular density (IMVD) as biomarkers for the tumor microenvironment. Hypoxia-inducible factor 1-alpha (HIF1A) expression was selected as a biomarker of intratumoral hypoxia. Double-strand DNA breaks were evaluated by expression of the phosphorylated form of H2A histone family member X (gH2AFX). Immunohistochemical analysis (CD163, CD68, CD105, CD34, HIF1A, gH2AFX) was evaluated by experienced sarcoma pathologists blinded to sample identification with a semi-quantitative method. We scored TAMs by counting the number of positive-staining macrophages per mm2: 0-none, 1-low, and 2-high. IMVD were counted in five randomly selected fields at 200x magnification (∼6.9 mm2), and a total number of microvessels included scoring scale: 1 (1-25), 2 (26-50), 3 (51-100), and 5 (> 500). HIF1A and gH2AFX were assessed in H-score scale, including intensity: 0-none, 1-weak, 2-moderate, 3-strong, and percentage of stained cells in each category (range: 0-300). All markers were studied preoperatively, while HIF1A was additionally assessed in postoperative tissues to define hypoxic microenvironment shift after therapy. PR to the treatment was classified using the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group recommendations; grades A, B, C indicated good PR, whereas D and E suggested poor PR. Spearman rank correlation was used to measure the degree of association between biomarkers and PR. <h3>Results</h3> Nineteen patients were eligible for the analysis. We identified a good treatment PR in four cases (grades A = 1, B = 2, C = 1, D = 11, E = 4). High tumor infiltration with TAMs and high IMVD did not correlate with PR. High initial expression of HIF1A correlated with poor PR, whereas high expression of gH2AFX correlated with better PR (Tab. 1). We also confirmed decreased HIF1A expression after therapy between biomarkers and PR. <h3>Conclusion</h3> HIF1A (hypoxia degree) and gH2AFX (DNA damage extent) are candidate biomarkers for PR's prediction after neoadjuvant treatment in STS. Further evaluation in prospective studies is warranted.

Perivascular cell coverage of intratumoral vasculature is a predictor for bevacizumab efficacy in metastatic colorectal cancer.

PurposeTumor vessels supported by perivascular cells have been implicated in the failure of some anti-angiogenic agents. The relationship between perivascular cell coverage (PC) and bevacizumab efficacy in metastatic colorectal cancer (mCRC) was analyzed.Patients and methodsA total of 284 consecutive mCRC patients who received first-line chemotherapy with or without bevacizumab from 2007–2014 in Sun Yat-Sen University Cancer Center were analyzed. Immunohistochemical double-stain for the perivascular cell marker alpha-smooth muscle actin and endothelial cell (cluster of differentiation 31) was performed to characterize the intratumoral microvascular density. Multispectral image capturing and computerized image analyses were used to quantify the microvessels supported by the perivascular cells. The patients were divided into high and low PC group according to a median cutoff value of 0.55.ResultsNo significant differences in overall survival (OS) and progression-free survival (PFS) were noted between the high and low PC group. In the low PC group, the patients with bevacizumab treatment had favorable OS (P=0.03), but without PFS benefit. In the high PC group, neither OS nor PFS was significantly different between the B+C and C subgroup. Tumors with perineural invasion had high PC (P=0.03).ConclusionThe data showed that a low PC value could be a predictor for bevacizumab benefit.

Contrast harmonic EUS for the prediction of pancreatic neuroendocrine tumor aggressiveness (with videos)

Contrast harmonic EUS (CH-EUS) has the ability to depict tumor microvasculature. Decreased microvascular density has been identified as a factor associated with tumor aggressiveness. Weaimed to study the accuracy of CH-EUS for the prediction of pancreatic neuroendocrine tumor (PNET)aggressiveness. Between June 2009 and March 2015, all consecutive patients with histology-proven PNETs and CH-EUS examination were included. Nine endosonographers blindly analyzed all videos. CH-EUS tumor aggressiveness was defined as a heterogeneous enhancement at the early arterial phase. The final diagnosis of tumor aggressiveness was defined as follows: G3 tumors, morphologic and/or histologic findings of metastatic disease in G1/G2 tumors. Diagnostic values were calculated. Intratumoral microvascular density and fibrosis were assessed on pathologic specimens. Eighty-one tumors were included, of which 26 were aggressive (32.1%). In CH-EUS 35 tumors (43.2%) had a heterogeneous enhancement. The overall accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of CH-EUS for the diagnosis of tumor aggressiveness were 86%, 96%, 82%, 71%, and 98%, respectively. The interobserver agreement among the 9 endosonographers was good (k= .66). The intraobserver agreement was excellent for the junior (κ= .83) and senior (κ= .82) endosonographers. Heterogeneous tumors at CH-EUS corresponded to fewer vascular and more fibrotic tumors (P< .01). CH-EUS is accurate in the prediction of PNET aggressiveness and could be a decision-making element in their management.

Assessment of Microvascular Density in the Neuroendocrine Tumors of the Pancreas: a Correlation with Multidetector Computed Tomography (MDCT) Features and Tumor Grade

Introduction/ Background The biologic course of pancreatic neuroendocrine tumors (NET) can be accurately predicted on the basis of pathology parameters which, unfortunately, cannot be fully assessed until the entire tumor has been resected. When obvious landmarks of malignancy (metastases, vascular invasion) are lacking, tumour size is the only reliable preoperative parameter used to differentiate between presumably benign and malignant pancreatic NETs. It is generally accepted that pancreatic NETs exceeding 2 cm in their diameter may have borderline pathology or even malignancy. Anyway, up to 10% of tumors with diameter less than 2 cm are although malignant. That is why there is need in studies focusing on additional imaging characteristics, such as contrast-enhanced MDCT, useful for the aggressiveness assessment. Aims To determine whether it is possible to predict pancreatic NET Grade and microvascular density (MVD) according to contrast-enhanced MDCT findings. Methods A retrospective study was conducted and 70 patients with pancreatic NETs were retrieved from the institutional archives. Twenty eight of them had met the including criteria (a - patients underwent surgery in our Institution and b - patients underwent preoperative dynamic abdominal CT within 30 days prior surgery) and comprised the study population. Thus, research included 15 patients with functioning and non-functioning pancreatic NETs Grade 1 and 13 of patients with Grade 2 (according to the WHO classification of 2010). Radiology All preoperative CT examinations were performed by a 256-slice CT scanner. Dynamic CT images, including non-enhanced, arterial, portal venous and delay phase images, were obtained for all patients. For contrast-enhanced CT, 90–120 mL of high concentration contrast mediaiomeprol 400 (Iomeron 400; Bracco Imaging SpA, Milan, Italy). CT images were reconstructed with a section thickness of 1-2 mm. Pathology For identification of vessels, regardless lymphatic or venous, immunohistochemistry with CD34 (clone QBEnd/10, CellMarque) was performed on formalin-fixed paraffin-embedded (FFPE) tumor blocks. Image analysis Density of vessels was defined on 3 fields at x 200 (~ 1,8 mm2). The choice of fields of vision was carried out among sites with visually greatest density of vessels (“hot spots”). Determined total number of the pixels belonging to vessels by the Color Threshold tool (ImageJ) and divided this number into total of pixels in the image. Results The median of density of vessels at patients with NET Grade 1 made 8% ± 2% whereas in group of patients with NET Grade 2 this indicator made 5 ± 4%. The difference was statistically significant (p <0,05). Mean arterial enhancement ratio was 1.66(0.42) in Grade 1 and 1.04(0.39) in Grade 2 pancreatic NET (p<0.01); and correlated with intratumoral MVD (r=0.61, p<0.005) and tumor grade (p<0.01). Vascular invasion, metastases and pancreatic duct dilatation were observed in two cases, both in G2 tumors. Arterial enhancement ratio <1.1, size≥20 mm, ill-defined borders and tumor non-homogeneity showed 83%, 74%, 70% and 56% accuracy in diagnosing G2 tumor respectively, while the accuracy of two of these criteria used in combination was 91%. Contrast-enhanced MDCT-features of pancreatic NET correlate with MVD and can predict tumor grade during preoperative staging.

Prognostic angiogenic markers (endoglin, VEGF, CD31) and tumor cell proliferation (Ki67) for gastrointestinal stromal tumors.

To evaluate the correlation between the immunoexpression of angiogenic markers [CD31, CD105 and vascular endothelial growth factor (VEGF)], proliferative index (Ki67), and prognosis of patients with gastrointestinal stromal tumors (GIST). This is a retrospective study of 54 GIST cases. Medical records were searched to obtain the GIST patients' demographic and clinical data, and paraffin-embedded blocks of tumor samples were retrieved from the hospital archives to conduct a new immunohistochemical evaluation. The tumor samples of GIST patients were subject to immunohistochemical evaluation for endoglin (CD105), CD31, VEGF, and Ki67 expression. The CD105 and CD31 intratumoral microvascular density (IMVD) was measured using automated analysis. We determined the correlation between the immunoexpression of CD105, CD31, VEGF, Ki67 and prognosis. In addition, we conducted a cutoff analysis using the receiver-operating characteristic curve. VEGF positivity was classified as either null/weak or strong. Ki67 was evaluated using a cutoff of 5% positive cells. The prognosis was classified as good (patient alive without recurrence) or poor (patient with recurrence/death). The distribution of tumor sites among the 54 analyzed samples was as follows: 27 (50%) in the stomach, 20 (37.1%) in the small intestine, 6 (11.1%) in the colon, and 1 (1.8%) in the esophagus. The size of the tumors ranged from 2 to 33 cm (median: 8 cm); in 12 cases (22.2%), the tumor was below 5 cm at the largest diameter, but in 42 cases (77.7%), the tumor was larger than 5 cm. The means of CD105 and CD31 were significantly higher in the group with poor prognosis (P < 0.001). The cut-off values of CD105 (> 1.2%) and CD31 (> 2.5%) in the receiver-operating characteristic curve were related to a poorer prognosis. Cases with a better prognosis showed significantly null/weak staining for VEGF (P < 0.001). Ki-67 expression of ≥ 5% was strongly correlated with a worse prognosis (P < 0.001). In the multivariate analysis, CD105 was the variable that most strongly correlated with prognosis. The IMVD cutoff values for the angiogenic markers CD105 and CD31, may be prognostic factors for GIST, in addition to VEGF and Ki67.

Predictive Value of Intratumoral Microvascular Density in Patients with Advanced Non-small Cell Lung Cancer Receiving Chemotherapy Plus Bevacizumab

The use of bevacizumab combined with chemotherapy represents a recent advance in clinical oncology for significantly improving the survival of patients who have non-small cell lung cancer (NSCLC). There is an unmet need for biomarkers that can predict response to such treatment and identify patients sensitive to it. Our study was designed to investigate the predictive value of intratumoral microvascular density (MVD) in patients with NSCLC treated with bevacizumab. Sixteen patients with NSCLC who underwent chemotherapy combined with bevacizumab were included into this study. Paraffin-embedded tumor samples were sectioned and stained immunohistochemically for the blood vessel markers CD34 and CD31 to characterize the intratumoral vasculature. A computerized image analysis program was used to quantitatively calculate the intratumoral MVD. Treatment response was evaluated by computed tomography scanning. Two types of blood vessels, undifferentiated (CD31*/CD34*) and differentiated (CD34*), were identified. A positive correlation was found between the largest percentage of tumor shrinkage and the MVD of undifferentiated (CD31*/CD34*) vessels, with Spearman correlation coefficient being 0.576 (p = 0.019). No correlation between tumor shrinkage and differentiated vessel MVD (CD34*) was found. Moreover, seven of the eight patients with more undifferentiated vessels showed a partial response, versus only one of the seven patients with fewer undifferentiated vessels (p = 0.009). There are two major types of microvessel in lung cancer vasculature. The MVD of undifferentiated vessels is a favorable predictor for patients with NSCLC treated with a chemotherapy regimen plus bevacizumab, with a higher MVD value correlating with better treatment response. Further studies are needed to verify the predictive role of MVD in treatment of NSCLC with bevacizumab.

Recombinant adenovirus vector-mediated human MDA-7 gene transfection suppresses hepatocellular carcinoma growth in a mouse xenograft model

Hepatocellular carcinoma is one of the most common tumors in the world. The purpose of the present study was to investigate the inhibitory effects of adenoviral transduction of human melanoma differentiation-associated gene −7 (MDA −7) gene on hepatocellular carcinoma, so as to provide a theoretical basis for gene therapy of the disease. The human MDA −7 gene was cloned into replication-defective adenovirus specific to HepG2 cells using recombinant virus technology. RT-PCR and Western blotting assays were used to determine the expression of human MDA −7 mRNA and MDA −7 protein in HepG2 cells in vitro. Induction of apoptosis by overexpression of the human MDA −7 gene was determined by flow cytometry. In-vivo efficacy of adenoviral delivery of the human MDA −7 gene was assessed in nude mice bearing HepG2 cell lines in vivo by determining inhibition of tumor growth, VEGF and CD34 expression, and microvascular density (MVD). The results showed that AdGFP/MDA −7 induced apoptosis of HepG2 cells in vitro and significantly inhibited tumor growth in vivo (P < 0.05). The intratumoral MVD decreased significantly in the treated tumors (P < 0.05). We conclude the recombination adenovirus AdGFP/MDA −7 can effectively express biologically active human MDA −7, which leads to inhibition of hepatocellular carcinoma growth.

Assessment of the prognostic significance of endoglin (CD105) in clear cell renal cell carcinoma using automated image analysis

The behavior of clear cell renal cell carcinoma can be difficult to predict. Angiogenesis has proven to be a useful prognostic indicator in different malignancies. Endoglin (CD105) is a new marker of angiogenesis found to have prognostic utility in various tumors. Here, we provide the first automated digital assessment of intratumoral microvascular density in clear cell renal cell carcinoma using endoglin and CD31 and assess their utility as predictors of clinical outcome. Both endoglin and CD31 expression showed association with advanced tumor stage (P = .025 and P = .011, respectively). There was a significant correlation between CD31 and tumor grade (P = .034). Kaplan-Meier survival curves showed that patients with higher endoglin expression had significantly shorter progression-free survival (P = .010). Patients with higher CD31 expression tended to have a worse prognosis, although this was not statistically significant (P = .082). In univariate analysis using endoglin as a continuous variable, increased endoglin was strongly associated with reduced survival (hazard ratio, 1.74; 95% CI, 1.39-2.18; P = <.001). CD31 also correlated with poor outcomes (hazard ratio, 1.52; 95% CI, 1.24-1.86; P = .001). There was no correlation between CD31 and endoglin expression (r = -0.090, P = .541). Receiver operating characteristic analysis showed the area under the curve to be 0.749 for endoglin and 0.550 for CD31. In conclusion, increased endoglin and CD31 expression are associated with a higher tumor stage and decreased progression-free survival. Our automated approach overcomes many limitations of manual quantification. Advances in digital assessment of immunohistochemical markers can be helpful in standardizing the evaluation of tumor biomarkers.

Xenon-Inhalation Computed Tomography for Noninvasive Quantitative Measurement of Tissue Blood Flow in Pancreatic Tumor

The purpose of this prospective study was to demonstrate the ability to measure pancreatic tumor tissue blood flow (TBF) with a noninvasive method using xenon inhalation computed tomography (xenon-CT) and to correlate TBF with histological features, particularly microvascular density (MVD). TBFs of pancreatic tumors in 14 consecutive patients were measured by means of xenon-CT at diagnosis and following therapy. Serial abdominal CT scans were obtained before and after inhalation of nonradioactive xenon gas. TBF was calculated using the Fick principle. Furthermore, intratumoral microvessels were stained with anti-CD34 monoclonal antibodies before being quantified by light microscopy (×200). We evaluated MVD based on CD34 expression and correlated it with TBF. The quantitative TBF of pancreatic tumors measured by xenon CT ranged from 22.3 to 111.4 ml/min/100 g (mean ± SD, 59.6 ± 43.9 ml/min/100 g). High correlation (r = 0.885, P < 0.001) was observed between TBF and intratumoral MVD. Xenon-CT is feasible in patients with pancreatic tumors and is able to accurately estimate MVD noninvasively.

Endogenous Angiogenesis Inhibitor Blocks Tumor Growth via Direct and Indirect Effects on Tumor Microenvironment

Tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) belongs to a small family of endogenous proteins that inhibits a group of enzymes, the matrix metalloproteinases (MMPs). TIMP-2 inhibits endothelial cell proliferation and migration in vitro and angiogenesis in vivo, through MMP-dependent and -independent mechanisms. However, little is known regarding the contribution of these mechanisms to the antitumor effects of TIMP-2. Using a retroviral delivery system, we stably overexpressed TIMP-2 and its mutant Ala+TIMP-2 (devoid of MMP inhibitory activity) in human adenocarcinoma A549 cells. Using real time PCR, and enzyme-linked immunosorbent assay (ELISA), we confirmed enhanced TIMP-2 expression and its MMP inhibitory activity by reverse zymography. In vitro, growth assays suggested that TIMP-2 and Ala+TIMP-2 did not alter basal cell proliferation rates, however, tumor cell migration and invasion were inhibited. In vivo, both TIMP-2 and Ala+TIMP-2 A549 xenografts exhibited reduced growth rate, CD31 immunostaining indicated decreased intratumoral microvascular density, and TUNEL demonstrated enhanced tumor cell apoptosis. Immunoblotting and immunohistochemical analyses of A549 xenograft tissues with either phospho-FAK (Tyr397) or phospho-AKT (Ser473) showed decreased activation in both TIMP-2 and Ala+TIMP-2 tumor cells. We conclude that TIMP-2-mediated inhibition of tumor growth occurs, at least in part, independently of MMP inhibition, and is a consequence of both direct effects of TIMP-2 on tumor cells and modulation of the tumor microenvironment.

Expression of VEGF, EGFR, and IL-6 in Gastric Adenomas and Adenocarcinomas by Endoscopic Submucosal Dissection

The degree of intratumoral microvascular density is thought to affect tumor metastasis and prognosis in various human cancers, including gastric cancer. Despite recent medical advancements, gastric adenoma or adenocarcinoma remains a considerable therapeutic challenge. Endoscopic submucosal dissection (ESD) is a more recent approach that is now commonly used for radical resection of gastric adenoma and adenocarcinoma. The expression of vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and interleukin-6 (IL-6) are related to the prognosis of gastric adenocarcinoma. However, the expression of these factors in gastric adenoma/adenocarcinoma following ESD has not been clearly evaluated. Here, we report on our study of the expression of VEGF, EGFR, and IL-6 by immunohistochemical staining in extracted tissue from adenoma or adenocarcinoma of the stomach by ESD and subsequent evaluation of the correlation of VEGF, EGFR, and IL-6 with other clinicopathological parameters. The patient cohort consisted of 102 patients with adenoma or adenocarcinoma of the stomach. Immunohistochemical staining for VEGF and IL-6 was significantly higher in both high grade dysplasia and adenocarcinoma than in low grade dysplasia (P < 0.05). There was significant correlation between histological grade and intensity of immunohistochemical staining of VEGF (P = 0.039). Histological differentiation of adenocarcinoma was related to IL-6 expression (P = 0.028). The immunoreactivity of VEGF and IL-6 increased significantly in lesions >2 cm compared to lesions <2 cm (P < 0.05). The immunohistochemical expression of IL-6 and VEGF can be considered to be useful for clinical diagnosis and follow-up of adenoma or adenocarcinoma of the stomach.

Predictive values of intratumoral microvascular density (MVD) in the patients (pts) with advanced NSCLC and metastatic colorectal cancer (mCRC) receiving chemotherapy plus bevacizumab

e14618 Background: The using of bevacizumab (VEGFR inhibitor) combined with chemotherapy represents a most noticeable recent advance in clinical oncology for significantly improving pts’ survival in several tumor types. There is an unmet need for seeking biomarker(s) to predict the treatment response of bevacizumab and identify the pts sensitive to the treatment. Our study was designed to investigate the predictive value of intratumoral MVD in NSCLC and mCRC pts treated with bevacizumab. Method: Sixteen NSCLC (stage IIIB/IV) and 15 mCRC pts who underwent chemotherapy (paclitaxel+carbo for NSCLC and IFL for CRC, respectively) combined with Bevacizumab were included into this study. The paraffin-embedded tumor samples (13/16 NSCLC and all CRC samples were collected from primary tumor) were sectioned and stained immunohistochemically for blood vessel markers (CD34 and CD31) to identify the characteristics of intratumoral vasculature. A computerized image analysis program was used to quantitatively calculate the intratumoral MVD (Yao et al., Clin Cancer Res 2007). Treatment response was evaluated by computed tomography scanning. Results: Two types of blood vessel, undifferentiated (CD31+/CD34-) and differentiated (CD34+), were identified. For the 16 NSCLC pts, a positive correlation was found between the largest tumor shrinkage percentage (evaluate in the primary tumors and metastases) and the total (CD31+) MVD as well as the undifferentiated (CD31+/CD34-) MVD, with Spearman's correlation coefficients equaled to 0.567 (p=0.022) and 0.576 (p=0.019), respectively. However, no significant correlation between tumor shrinkage and MVD was found in CRC pts, in which the treatment response was evaluated in metastatic lesions while the MVD was calculated in primary tumor. Conclusion: This exploratory analysis suggests higher total vessel and undifferentiated vessel MVD appeared to be predictive of bevacizumab treatment advantage for advance NSCLC pts.Our study also indicated that the MVD of primary lesions did not correlate with the treatment response of metastatic lesions in CRC pts. Further studies are needed to verify the predictive role of MVD in the treatment of bevacizumab in NSCLC. [Table: see text]

Pancreatic Endocrine Tumors: Tumor Blood Flow Assessed with Perfusion CT Reflects Angiogenesis and Correlates with Prognostic Factors1

To prospectively correlate multidetector computed tomographic (CT) perfusion measurement of pancreatic endocrine tumors with tumor microvascular density (MVD) assessed by using histologic techniques and to determine whether perfusion CT parameters differ between tumor grades. Institutional review board approval and informed consent were obtained. Thirty-six patients (15 men, 21 women; mean age, 53 years; range, 18-78 years) with resectable pancreatic endocrine tumors underwent presurgical dynamic perfusion CT. Twenty-eight (78%) of 36 patients were included in the study group; eight were excluded because of artifacts that were not compatible with perfusion postprocessing. Multidetector CT perfusion data were analyzed to calculate tumor and normal pancreatic blood flow, blood volume, mean transit time, and permeability-surface area product. Multidetector CT perfusion parameters were compared with intratumoral MVD by using the Spearman correlation coefficient and with World Health Organization (WHO) classification, tumor size, tumor proliferation index, hormonal profile, and presence of metastases by using Mann-Whitney tests. High correlation (r = 0.620, P < .001) was observed between tumor blood flow and intratumoral MVD. Blood flow was significantly higher (P = .02) in the group of benign tumors (WHO 1) than in the groups of tumors of indeterminate prognosis (WHO 2) or well-differentiated carcinomas (WHO 3). Blood flow was significantly higher in tumors with a proliferation index of 2% or less (P = .005) and in those without histologic signs of microscopic vascular involvement (P = .008). Mean transit time was longer in tumors with lymph node (P = .02) or liver (P = .05) metastasis. Perfusion CT is feasible in patients with pancreatic endocrine tumors and reflects MVD. Perfusion CT measurements are correlated with histoprognostic factors, such as proliferation index and WHO classification.

Tumor Recovery by Angiogenic Switch from Sprouting to Intussusceptive Angiogenesis after Treatment with PTK787/ZK222584 or Ionizing Radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved alpha-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated alpha-smooth muscle actin-expression during recovery reflected the recruitment of alpha-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1alpha expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.