Expression and Prognostic Impact of VEGF, CD31 and αSMA in Resected Primary Lung Cancers.

BACKGROUND/AIM: One of the most important factors concerning cancer growth is angiogenesis. The purpose of this study was to clarify the relationship of maturation of tumor vessels and prognosis of lung cancer. MATERIALS AND METHODS: Immunohistochemical stainings of 125 lung cancers for VEGF, CD31 and α-smooth muscle actin (αSMA) were scored by multiplying the intensity and the frequency from 0 to 12. RESULTS: Adenocarcinomas showed significantly higher staining scores of both VEGF and αSMA than squamous cell carcinomas did. In 42 cases of high CD31 score, five-year survival rate (87%) of patients with lung cancer showing mature tumor vessels was significantly better than that (69%) of patients with immature tumor vessels. CONCLUSION: Not the number of tumor vessels but their maturation may be a prognostic factor of patients with lung cancer. VEGF may not only stimulate proliferation of endothelial cells but also their maturation in differentiated lung cancers.

[Laparoscopic type C1 hysterectomy based on the anatomic landmark of the uterus deep vein and its branches for cervical cancer].

Objective: To introduce the laparoscopic type C1 hysterectomy based on the anatomic landmark of the uterus deep vein and its branched and to evaluate its feasibility and safety for cervical cancer and its effect to bladder function and to provide some reference to simplify the surgical procedures of laparoscopic type C1 hysterectomy. Methods: The clinicopathologic data of the patients with stage ⅠA2~ⅡB cervical cancer and who underwent the laparoscopic C1 hysterectomy based on anatomic landmark of the uterus deep vein and its branches between March 2010 and December 2015 was retrospectively analysed. Results: A total of 99 patients received laparoscopic type C1 hysterectomy based on the anatomic landmark of the uterus deep vein and its branches, in which 93 patients reserved unilateral or bilateral pelvic autonomic nerve successfully, the other 6 patients were transfered to receive type C2 hysterectomy due to adhesions, bleeding or the low possibility of curative resection. The failure rate of the surgery was 6.1% (6/99). The average age of these 93 patients was 44.4±8.2 years (range 25~61 years) and there was one case of stage ⅠA2, 84 stage ⅠB1, 2 stage ⅠB2, 5 stage ⅡA1 and 1 stage ⅡB. The number of patients with squamous cell carcinoma was 67, adenocarcinoma was 19, adenosquamous carcinoma was 3, small cell neuroendocrine carcinoma was 3 and mixed type was 1. The average operation time was 4.1±0.5 h, the average amount of intraoperative blood loss was 103.8±84.0 ml and the mean number of excisional pelvic lymph nodes was 29.7±8.9. There was no patient with positive parametrial margin, positive vaginal margin or intraoperative ureteral injury. The postoperative catheter extraction time was 20.3±8.4 d. The median follow-up time was 20 months (rang 5~44 months), the long-term bladder dysfunction rate was 8.6% (8/93). The numbers of locally uncontrolled and distantly metastasis case were both one and both patients died. The fatality rate were 2.2% (2/93). The two-year disease-free survival and overall survival rate were 97.6% and 96.2%, respectively. Conclusion: Laparoscopic type C1 hysterectomy based on the anatomic landmark of the uterus deep vein and its branches is a safe and feasible treatment method for cervical cancer and it provides a new approach for simplifying the surgical procedures of laparoscopic type C1 hysterectomy.

[Anti-angiogenic factors in thoracic oncology: successes, failures and prospects]. / Traitements antiangiogéniques en oncologie thoracique: succès, échecs et perspectives.

Many growth factors involved in tumor angiogenesis are potential targets in thoracic oncology. This work is a review of the literature on the effectiveness of anti-angiogenic treatments in non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) and malignant pleural mesothelioma (MM). Thirty-four articles and 15 abstracts were identified. Currently, bevacizumab is the only drug that has demonstrated an impact on overall survival in first line treatment for stage IV non-squamous NSCLC, but VEGFR-TKI such as cediranib, aflibercept, vandetanib, pazopanib have shown encouraging results in phase II or III clinical trials. In extensive-disease SCLC and inoperable MM, bevacizumab is the most studied molecule, but again, clinical trials are still ongoing. Current data on potential predictors for efficacy are disappointing, but some biomarkers or radiological techniques might be useful for guiding the use of anti-angiogenic therapies in the future. In conclusion, bevacizumab is the most studied anti-angiogenic agent in thoracic oncology. It is the only approved drug with an indication in first-line and maintenance treatment for stage IV non-squamous NSCLC. The indications for the use of VEGFR-TKI in clinical practice remain to be defined.

Folate hydrolase (prostate-specific membrane [corrected] antigen) 1 expression in bladder cancer subtypes and associated tumor neovasculature.

Folate hydrolase (prostate-specific antigen) 1 (FH(PSA)1), also known as prostate-specific membrane antigen (PSMA), is a transmembrane receptor expressed on prostate cancer cells that correlates with a more aggressive phenotype. Recent studies have demonstrated FH(PSA)1 expression in numerous benign and malignant tissue types, as well as the malignant neovasculature. As FH(PSA)1 represents a diagnostic immunomarker for prostate cancer, we explored its expression pattern in various subtypes of bladder cancer. Immunohistochemical analysis (IHC) of FH(PSA)1 was performed using tissue microarrays constructed from 167 bladder cancers, including 96 urothelial carcinomas (UCCs), 37 squamous cell carcinomas, 17 adenocarcinomas and 17 small cell carcinomas. We used a FH(PSA)1 monoclonal antibody obtained from Dako (clone 3E6, dilution 1:100), which recognizes the epitope present in the 57-134 amino acid region of the extracellular portion of the PSMA molecule. Intensity of IHC staining was scored as 0 (no expression) to 3+ (strong expression), with 2-3+ IHC considered a positive result. FH(PSA)1 demonstrated expression in a subset of bladder cancers and was most common in small cell carcinoma (3/17; 18%), with concurrent expression in non-small cell components in a subset of cases (2/6). FH(PSA)1 expression was less frequent in UCC (3/96; 3%) and adenocarcinoma (2/17; 12%). None of the squamous cell carcinomas demonstrated tumor cell expression of FH(PSA)1. However, all bladder cancers examined expressed FH(PSA)1 in the tumor vasculature, suggesting a potential role for this molecule in mediating new vessel ingrowth. FH(PSA)1 may occasionally be expressed in various subtypes of bladder cancer. These findings suggest cautious use of FH(PSA)1 as a diagnostic marker for prostatic tissue invading the bladder. The finding of FH(PSA)1 in the bladder cancer neovasculature suggests that this molecule may promote tumor growth and may represent a potential new vascular target in this disease.

A hybrid breath hold and continued respiration-triggered technique for time-resolved 3D MRI perfusion studies in lung cancer.

PURPOSE: Assessment of lung cancer perfusion is impaired by respiratory motion. Imaging times for contrast agent wash-out studies often exceed breath hold capabilities, and respiration triggering reduces temporal resolution. Temporally resolved volume acquisition of entire tumors is required to assess heterogeneity. Therefore, we developed and evaluated an MR measurement technique that exceeds a single breath hold, and provides a variable temporal resolution during acquisition while suspending breath-dependent motion. MATERIALS AND METHODS: 20 patients with suspected lung cancer were subjected to perfusion studies using a spoiled 3D gradient echo sequence after bolus injection of 0.07 mmol/kg body weight of Gd-DTPA. 10 acquisitions in expiratory breath hold were followed by 50 navigator-triggered acquisitions under free breathing. Post-processing allowed for co-registration of the 3D data sets. An ROI-based visualization of the signal-time curves was performed. RESULTS: In all cases motion-suspended, time-resolved volume data sets (40 x 33 x 10 cm(3), voxel size: 2.1 x 2.1 x 5.0 mm(3)) were generated with a variable, initially high temporal resolution (2.25 sec) that was synchronized with the breath pattern and covered up to 8 1/2 min. In 7 / 20 cases a remaining offset could be reduced by rigid co-registration. The tumors showed fast wash-in, followed by rapid signal decay (8 / 20) or a plateau. CONCLUSION: The feasibility of a perfusion study with hybrid breath hold and navigator-triggered time-resolved 3D MRI which combines high initial temporal resolution during breath hold with a long wash-out period under free breathing was demonstrated.

Administration of VEGF receptor tyrosine kinase inhibitor increases VEGF production causing angiogenesis in human small-cell lung cancer xenografts.

Angiogenesis is mediated mainly by vascular endothelial growth factor (VEGF), and VEGF causes rapid growth in cancers, including human small-cell lung cancer (SCLC). The anti-angiogenic strategy of treating cancer using VEGF receptor (VEGFR) inhibition is currently of great interest. We tested the effects of the VEGFR2 tyrosine kinase inhibitor (TKI) vandetanib on the proliferation of two kinds of SCLC cell lines: SBC-1 cells, with detectable VEGFR2 expression and MS-1-L cells, without detectable VEGFR2 expression. To evaluate the anti-tumor and anti-angiogenic effects of vandetanib in vivo, we grafted SBC-1 and MS-1-L cells into mice. After a 3-week treatment, we measured the tumor size and histologically evaluated necrosis and apoptosis using H&E and TUNEL staining, respectively. The microvessels in the xenografts were also quantified by immunostaining of CD31. Vandetanib did not affect the proliferation of SBC-1 cells, but stimulated the growth of MS-1-L cells. In the SCLC xenograft model, vandetanib inhibited growth and tumor angiogenesis in a dose-dependent manner in SBC-1 xenografts. Vandetanib inhibited the growth of MS-1-L xenografts at a low dose (<12.5 mg/kg/day), but it did not affect tumor size or change microvessel counts at a higher dose. Interestingly, secretion of VEGF increased significantly in the MS-1-L cell line in the presence of a high dose of vandetanib in vitro. The effects of vandetanib on tumor angiogenesis were different in SBC-1 and MS-1-L cell lines. Production of angiogenic factors such as VEGF by the tumor potentially stimulates tumor angiogenesis and results in the acquisition of resistance to VEGFR TKI.

Prognostic impact of lymphangiogenesis and lymphatic invasion (CD105 expression) in small cell lung carcinoma.

BACKGROUND: Lymphangiogenesis, an essential process in the metastasis of malignant tumors, has not been thoroughly studied. The possibility of using it to define subsets of patients with different prognosis in cancer could be of vital clinical importance. MATERIALS AND METHODS: Fifty patients (5 women, 45 men; mean age, 64.47 years) with SCLC were retrospectively studied. Tumor specimens were stained for CD105, and intratumoral lymphatic microvessel density (ILMVD) and lymphatic invasion were determined. RESULTS: Twenty-five patients were diagnosed with limited and 25 with extensive SCLC. All patients received chemotherapy and 32.7% radiation therapy. A direct association between ILMVD (CD105 expression) and lymphatic invasion was observed (p<0.046). CD105 expression was significantly associated with the stage of the disease (p=0.004) and the presence of metastasis (p=0.05). CONCLUSION: CD105 expression and lymphatic invasion correlated significantly with the clinical parameters and patient outcome, therefore, constituting an important prognostic role in SCLC.

E7080, a novel inhibitor that targets multiple kinases, has potent antitumor activities against stem cell factor producing human small cell lung cancer H146, based on angiogenesis inhibition.

E7080 is an orally active inhibitor of multiple receptor tyrosine kinases including VEGF, FGF and SCF receptors. In this study, we show the inhibitory activity of E7080 against SCF-induced angiogenesis in vitro and tumor growth of SCF-producing human small cell lung carcinoma H146 cells in vivo. E7080 inhibits SCF-driven tube formation of HUVEC, which express SCF receptor, KIT at the IC(50) value of 5.2 nM and it was almost identical for VEGF-driven one (IC(50) = 5.1 nM). To assess the role of SCF/KIT signaling in tumor angiogenesis, we evaluated the effect of imatinib, a selective KIT kinase inhibitor, on tumor growth of H146 cells in nude mice. Imatinib did not show the potent antitumor activity in vitro (IC(50) = 2,200 nM), because H146 cells did not express KIT. However, oral administration of imatinib at 160 mg/kg clearly slowed tumor growth of H146 cells in nude mice, accompanied by decreased microvessel density. Oral administration of E7080 inhibited tumor growth of H146 cells at doses of 30 and 100 mg/kg in a dose-dependent manner and caused tumor regression at 100 mg/kg. While anti-VEGF antibody also slowed tumor growth, it did not cause tumor regression. These results indicate that KIT signaling has a role in tumor angiogenesis of SCF-producing H146 cells, and E7080 causes regression of H146 tumors as a result of antiangiogenic activity mediated by inhibition of both KIT and VEGF receptor signaling. E7080 may provide therapeutic benefits in the treatment of SCF-producing tumors.

Peripheral lung cancer: relationship between multi-slice spiral CT perfusion imaging and tumor angiogenesis and cyclin D1 expression.

The aim of this study was to investigate the relationship between 16-slice spiral CT perfusion imaging and tumor angiogenesis and cyclin D1 expression in patients with peripheral lung cancer. Fifty-eight patients with peripheral lung cancer underwent 16-slice spiral CT perfusion imaging. The CT perfusion imaging was analyzed for time density curve (TDC), perfusion parametric maps, and the respective perfusion parameters. Correlation between the respective perfusion parameters and immunohistochemical findings of microvessel density measurement and cyclin D1 expression was evaluated.

A selective small molecule inhibitor of c-Met, PHA665752, inhibits tumorigenicity and angiogenesis in mouse lung cancer xenografts.

The c-Met receptor tyrosine kinase is emerging as a novel target in many solid tumors, including lung cancer. PHA-665752 was identified as a small molecule, ATP competitive inhibitor of the catalytic activity of the c-Met kinase. Here, we show that treatment with PHA665752 reduced NCI-H69 (small cell lung cancer) and NCI-H441 (non-small cell lung cancer) tumorigenicity in mouse xenografts by 99% and 75%, respectively. Reduction in tumor size was also observed by magnetic resonance imaging of tumors in mice. PHA665752 inhibited c-Met phosphorylation at the autophosphorylation and c-Cbl binding sites in mouse xenografts derived from non-small cell lung cancer cell lines (NCI-H441 and A549) and small cell lung cancer cell line (NCI-H69). PHA665752 also inhibited angiogenesis by >85% in all the abovementioned cell lines and caused an angiogenic switch which resulted in a decreased production of vascular endothelial growth factor and an increase in the production of the angiogenesis inhibitor thrombospondin-1. These studies show the feasibility of selectively targeting c-Met with ATP competitive small molecule inhibitors and suggest that PHA665752 may provide a novel therapeutic approach to lung cancer.

Bronchial arterial imaging using helical computed tomography.

The bronchial arteries, which provide the systemic arterial supply to the lungs, are involved in a variety of disease processes in humans, including congenital disorders, infection, and pulmonary thromboembolism. In these conditions, the bronchial arteries hypertrophy and bronchial blood flow increases. Consequently, in many disorders, such as bronchiectasis, the bronchial arteries are a frequent source of haemoptysis, which may be massive and life-threatening. Evaluation of the bronchial circulation has typically required invasive imaging with angiography to determine the location of bleeding. Non-invasive assessment of bronchial arterial anatomy and morphology is currently being investigated with the use of helical computed tomography (CT). We evaluated eight patients with various lung diseases with helical CT (GE Medical Systems, LS16, Milwaukee, WI) to determine the imaging features of the bronchial circulation. Non-ionic contrast medium (iopromide) was injected intravenously (80-1000ml/30s) and scanning was triggered once contrast material was present in the pulmonary artery (average delay=15s) or ascending aorta (average delay=20s). Detector collimation (16-row unit) was 10mm. Imaging parameters included a section thickness of 0.6mm, kilovolt peak of 120, 150-440mA, pitch factor of 1.375, matrix of 512x512, and tube rotation time of 0.8s. The images were reconstructed and scanned isotropically (Advantage Workstation 4.1,GE Medical Systems). We conclude that helical computed tomography may provide a non-invasive means of evaluating the bronchial arteries and their role in pulmonary disease processes.

Inhibition of c-Met and prevention of spontaneous metastatic spreading by the 2-indolinone RPI-1.

Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. We investigated the biochemical and biological effects of the tyrosine kinase inhibitor RPI-1 on the human lung cancer cell lines H460 and N592, which express constitutively active Met. RPI-1-treated cells showed down-regulation of Met activation and expression, inhibition of HGF/Met-dependent downstream signaling involving AKT, signal transducers and activators of transcription 3 and paxillin, as well as a reduced expression of the proangiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. Cell growth in soft agar of H460 cells was strongly reduced in the presence of the drug. Furthermore, RPI-1 inhibited both spontaneous and HGF-induced motility/invasiveness of both H460 and human endothelial cells. Targeting of Met signaling by alternative methods (Met small interfering RNA and anti-phosphorylated Met antibody intracellular transfer) produced comparable biochemical and biological effects. Using the spontaneously metastasizing lung carcinoma xenograft H460, daily oral treatment with well-tolerated doses of RPI-1 produced a significant reduction of spontaneous lung metastases (-75%; P < 0.001, compared with control mice). In addition, a significant inhibition of angiogenesis in primary s.c. tumors of treated mice was observed, possibly contributing to limit the development of metastases. The results provide preclinical evidence in support of Met targeting pharmacologic approach as a new option for the control of tumor metastatic dissemination.

Small adenocarcinoma of the lung: prognostic significance of central fibrosis chiefly because of its association with angiogenesis and lymphangiogenesis.

To clarify the reason why central fibrosis (CF) is an important histological prognostic factor in small adenocarcinoma (SA) of the lung, tumor tissues from 50 patients with SA < or = 2 cm in diameter were investigated using immunohistochemical and in situ hybridization analysis for factors relating to extracellular matrix and vessels. CF was observed in 33/50 cases (66%). In adenocarcinoma areas, positive activity was observed with both primary antibodies and probes for matrix metalloproteinase-2 (MMP-2) in 11/50 patients (22%), membrane-type 1 matrix metalloproteinase (MT1-MMP) in 39/50 patients (78%) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in 49/50 patients (98%). In CF areas, the positive activity of fibroblastic cells was seen for only TIMP-2 in 32/33 patients (97%). In CF areas, both CD34-positive (blood and lymphatic) vessels and D2-40-positive lymphatic vessels were semiquantitatively increased in 16/33 patients (48.5%) by immunohistochemistry. Tumors with increased vessel density were associated with statistically lower disease-free survival curves compared with tumors without increased vessels. Lymphatic vessels in some CF showed intravasation by carcinoma cells. In conclusion, CF could be an important histological prognostic factor in SA chiefly because of its association with angiogenesis and lymphangiogenesis.

[Prognostic role of microvascularization in surgically treated lung cancer]. / A mikrovaszkularizáltság prognosztikai jelentôsége operált tüdôrák esetén.

OBJECTIVE: The aim of our study was the determination of microvascularization and its prognostic significance in lung cancer patients. METHODS: Histological sections were prepared from paraffin-embedded tissues removed from the peripheral part of the tumor of 450 radically operated non-small cell and small cell lung cancer patients. Immunohistochemical staining was performed with antibody against factor VIII-associated antigen. During computer imaging, the absolute and relative parameters of vascularization were determined, as was the density of tumor cells situated to the nearest neighboring vessels. The results were compared with TNM status, the cell type and survival. RESULTS: T2 and T4 tumors demonstrated an enhanced vascularization, however, except for the surface fraction, statistically significant difference was not found. The microvascularization parameters did not differ significantly between tumors with different N status. In small cell lung cancer cases, the vascularization was stronger than in non-small cell lung cancer cases, while cell density was lower, however, these differences did not prove statistically significant. The survival rate decreased significantly with the increasing tumor cell density in the interval of 0-20 microm. CONCLUSIONS: A clear connection could not be demonstrated between vascularization and the appearance of lymph node metastases. The density of tumor cells measured in the direct vicinity of vessels proved an important prognostic factor.

Fast cancer uptake of 99mTc-labelled bombesin (99mTc BN1).

In human blood, breakdown of gastrin-releasing peptide and other bombesin-related peptides occurs in less than 15 min. This quick enzymatic cleavage might impair the diagnostic use of labelled bombesin (BN). 99mTc-labelled bombesin (99mTc BN1) was injected intravenously and dynamic uptake data were acquired for diagnosing 26 cancers of different origin: 15 breast, 3 prostate, 5 colo-rectal, 1 pancreas, 2 small cell lung cancers and 1 gastrinoma. Background subtracted tumour uptake data were plotted against time and fitted with known mathematical functions. Twenty-three out of 26 cancers showed rapid increase of radioactivity followed by a radioactivity plateau, with some oscillations around the average plateau value. The time to 80% of max activity (T80) was the reference parameter to measure and to compare the uptake speeds. The slowest T80 was 7 min in one T1b breast cancer, gastrinoma reached T80 in 5 min and node-positive prostate cancers in 2 min. N+ breast cancers showed T80 at 3.62 +/- 0.75 min, N- breast cancers at 5.5 +/- 0.88 min (p < 0.02). When all the tumours were considered, N+ tumours showed T80 at 2.68 +/- 1.03 min and N- cancers at 5.5 +/- 0.82 min. In all the cancer types, the uptake of 99mTc BN was faster than 10 min. This result shows the ability of 99mTc BN to image tumours. The faster uptake by N+ versus N- cancers probably depends on the higher blood flow in N+ cancers.

Specific occlusion of murine and human tumor vasculature by VCAM-1-targeted recombinant fusion proteins.

BACKGROUND: The tumor vasculature is increasingly recognized as a target for cancer therapy. We developed and evaluated recombinant fusion proteins targeting the coagulation-inducing protein soluble tissue factor (sTF) to the luminal tumor endothelial antigen vascular cell adhesion molecule 1 (VCAM-1, CD106). METHODS: We generated fusion proteins consisting of sTF fused to antibody fragments directed against mouse or human VCAM-1 and characterized them in vitro by flow cytometry, surface plasmon resonance, and two-stage coagulation assays. Their therapeutic effects were tested in three human xenograft tumor models: L540rec Hodgkin lymphoma, Colo677 small-cell lung carcinoma, and Colo677/HDMEC small-cell lung carcinoma with human vasculature. Toxicity was analyzed by histologic examination of organs and determination of laboratory blood parameters. RESULTS: The fusion proteins bound VCAM-1 with nanomolar affinities and had the same coagulation activity as an sTF standard. Xenograft tumor-bearing mice treated with fusion protein (FP) alone or in combination with lipopolysaccharide (FP/L) or doxorubicin (FP/D) exhibited tumor-selective necrosis (L540rec tumors: 74% tumor necrosis [95% confidence interval {CI} = 55% to 93%] with FP/L versus 13% tumor necrosis [95% CI = 4% to 22%] with vehicle; Colo677 tumors: 26% [95% CI = 16% to 36%] with FP versus 8% [95% CI = 2% to 14%] with vehicle); tumor growth delay (Colo677/HDMEC: mean tumor weights after 3 days = 42 mg in FP-treated mice versus 71 mg in vehicle-treated mice, difference = 29 mg, 95% CI = 8 to 100, Mann-Whitney P = .008); and some tumor regressions (one of seven FP-treated Colo677 tumor-bearing mice and two of seven FP/D-treated mice). The fusion protein was well tolerated. CONCLUSIONS: Recombinant tissue factor-based fusion proteins directed against an intraluminal tumor endothelial cell marker induce tumor-selective intravascular coagulation, tumor tissue necrosis, and tumor growth delay.