Application of contrast-enhanced ultrasonography for large cell neuroendocrine carcinoma in the urinary bladder: a case report.

BACKGROUND: Large cell neuroendocrine carcinoma (LCNEC) of the urinary bladder is an uncommon malignant bladder tumor, and the overall prognosis is poor. Contrast-enhanced ultrasound (CEUS) provides a new effective modality for tumor detection and diagnosis. CASE PRESENTATION: A 30-year-old man complained of repeated painless gross haematuria for half a month. Conventional ultrasound demonstrated a hypoechoic solitary lesion with hyperechoic margins measuring 3.4 × 3.1 cm in the anterior wall of the bladder. Superb microvascular imaging (SMI) showed a strong flow signal in the mass. CEUS revealed that the lesion was characterized by hyper-enhancement in the early phase and hypo-enhancement in the late phase. The entire bladder wall was disrupted by homogeneous hyper-enhanced tumor tissue on CEUS. Time-intensity curves (TICs) showed a rapid wash-in with a high maximum signal intensity (SI) and quick wash-out. Finally, partial cystectomy was performed and the pathological examination confirmed the diagnosis of LCNEC with invasion into the whole layer of the bladder wall. CONCLUSION: This case suggested that CEUS was a valuable imaging method to detect and diagnose LCNEC in the bladder, and that CEUS can provide information related to the depth of wall invasion and the microvasculature.

Relation between vascular patterns visualized by Narrow Band Imaging (NBI) videobronchoscopy and histological type of lung cancer.

Narrow Band Imaging (NBI) videobronchoscopy is a new technique for visualization of microvascular changes in bronchial mucosa. The primary aim of this study was to evaluate relation between vascular patterns visualized by NBI and histology of lung cancer. We prospectively evaluated 65 patients with suspected lung cancer scheduled for bronchoscopy. NBI followed conventional WL videobronchoscopy. After identification of endoscopically visible tumor, NBI was used to determine predominant type of pathological vascular pattern (dotted, tortuous, abrupt-ending blood vessels-Shibuya descriptors). All the lesions were biopsied and histologically confirmed. There were 81.5 % male and 18.5 % female patients evaluated in the study. Lung cancer was confirmed in all patients; 63.1 % were diagnosed with squamous cell lung cancer (SCC), 24.6 % had adenocarcinoma, 9.2 % had small-cell (SCLC) and 3.1 % large-cell lung cancer (LC). Dotted blood vessels were significantly (p < 0.000) associated with adenocarcinoma, identified in 68.4 % adenocarcinoma and 31.6 % SCC. Tortuous blood vessels were identified in 72 % SCC, 8 % adenocarcinoma, 12 % SCLC and 8 % of LC. Tortuous blood vessels were significantly (p < 0.000) associated with SCC. Abrupt-ending vessels were identified in 81 % SCC, 14.3 % SCLC and 4.8 % adenocarcinoma and were significantly associated (p < 0.000) with SCC. Dotted visual pattern of blood vessels identified during NBI videobronchoscopy is highly suggesting adenocarcinoma histology of lung cancer. Tortuous and abrupt-ending blood vessels visualized under NBI videobronchoscopy significantly suggest squamous cell histology of lung cancer. Large-scale studies should be designed in order to determine true relation between visual appearance and histology in lung cancer.

Increased NDRG1 expression is associated with advanced T stages and poor vascularization in non-small cell lung cancer.

N-myc downstream regulated gene 1 (NDRG1) is a member of the N-myc downstream regulated gene family which belongs to the alpha/beta hydrolase superfamily. Earlier studies have shown its association with inhibition of tumor metastasis. However, its function in malignant tumors is not fully enunciated. Recently there was increasing evidence that NDRG1 is involved in stress responses. In the current study, we examined the expression of NDRG1 and its correlation with clinicopathological factors and microvessel density (MVD) in non-small cell lung cancer (NSCLC) using immunohistochemistry (IHC). NDRG1 expression in NSCLC (71/115, 61.7%) was higher than that in normal lung tissues (32/115, 27.8%) (p < 0.05). NDRG1 expression in NSCLC cells was found in cytoplasm (63/115, 54.8%), nuclear (24/115, 20.9%) and cell membrane (13/115, 11.3%). NDRG1 expression in NSCLC with advanced T stages (T2-4) (63/84, 75.0%) was significantly higher than that with T1 stage (8/31, 25.8%) (P < 0.05). No other clinicopathological factors including lymph node metastasis were found to be associated with NDRG1 expression (p > 0.05). Moreover increased NDRG1 expression was associated with lower MVD in NSCLC (P < 0.05). MVD in adenocarcinoma (33.4 ± 8.4/HP) was significantly higher than that in squamous cell carcinoma (SCC) (19.3 ± 8.1/HP) (P < 0.05). No other clinicopathological factors were associated with MVD in NSCLC (p > 0.05). The present findings indicate an increase of NDRG1 expression with the progress of tumour extent which may be due to unbalanced tumor oxygenation on account of poor vascularization in NSCLC.

Significance of the presence of microscopic vascular invasion after complete resection of Stage I-II pT1-T2N0 non-small cell lung cancer and its relation with T-Size categories: did the 2009 7th edition of the TNM staging system miss something?

INTRODUCTION: The aim of this study was to assess the significance of microscopic vascular invasion (MVI) in a population of resected patients with early-stage non-small cell lung cancer (NSCLC), along with an analysis of the effect of the combination of MVI and tumor size for the T-size categories T1a-T2b according to the 2009 7th edition of the tumor, node, metastasis (TNM) classification. METHODS: From January 1993 to August 2008, 746 patients with pT1-T2N0 NSCLC received resection at our institution. MVI was ascertained using histopathological and immunohistochemical techniques. RESULTS: MVI was observed in 257 patients (34%). Prevalence was higher in adenocarcinoma (ADK) than in squamous cell carcinoma (p = 0.002). A significant correlation was found between MVI and ADK (p = 0.03), increased tumor dimension (p = 0.05), and the presence of tumor-infiltrating lymphocytes (p = 0.02). The presence of MVI was associated with a reduced 5-year survival overall (p = 0.003) and in ADK (p = 0.0002). In a multivariate survival analysis, MVI was an indicator of poor survival overall (p = 0.003) and in ADK (p = 0.0005). In each T category (T1a-T2b) of the 2009 TNM staging system, survival of MVI+ patients was significantly lower than the corresponding MVI- patients; T1a and T1b MVI+ patients had a survival similar to MVI- T2 patients. CONCLUSIONS: The finding of MVI in pT1-T2N0 NSCLC is frequent. MVI correlates with adenocarcinoma histotype, increased tumor dimensions, and tumor-infiltrating lymphocytes. The presence of MVI is an independent negative prognostic factor. In our experience, MVI was a stronger prognostic indicator than T size in T1a-T2b categories according to the 2009 TNM staging system.

Apelin expression in human non-small cell lung cancer: role in angiogenesis and prognosis.

INTRODUCTION: The recently discovered bioactive peptide, apelin, has been demonstrated to stimulate angiogenesis in various experimental systems. However, its clinical significance and role in tumor vascularization have not yet been investigated in a human malignancy. Therefore, our aim was to study whether apelin expression is associated with angiogenesis and/or tumor growth/behavior in human non-small cell lung cancer (NSCLC). METHODS: A total of 94 patients with stage I-IIIA NSCLC and complete follow-up information were included. Apelin expression in human NSCLC samples and cell lines was measured by quantitative reverse-transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Effects of exogenous apelin and apelin transfection were studied on NSCLC cell lines in vitro. In vivo growth of tumors expressing apelin or control vectors were also assessed. Morphometric variables of human and mouse tumor capillaries were determined by anti-CD31 labeling. RESULTS: Apelin was expressed in all of the six investigated NSCLC cell lines both at the mRNA and protein levels. Although apelin overexpression or apelin treatments did not increase NSCLC cell proliferation in vitro, increasing apelin levels by gene transfer to NSCLC cells significantly stimulated tumor growth and microvessel densities and perimeters in vivo. Apelin mRNA levels were significantly increased in human NSCLC samples compared with normal lung tissue, and high apelin protein levels were associated with elevated microvessel densities and poor overall survival. CONCLUSIONS: This study reveals apelin as a novel angiogenic factor in human NSCLC. Moreover, it also provides the first evidence for a direct association of apelin expression with clinical outcome in a human cancer.

Cytosolic phospholipase A2: targeting cancer through the tumor vasculature.

PURPOSE: In vascular endothelial cells, low doses of ionizing radiation trigger the immediate activation of cytosolic phospholipase A2 (cPLA2). This event initiates prosurvival signaling that could be responsible for radioresistance of tumor vasculature. Thus, the development of radiosensitizers targeting these survival pathways may enhance tumor response to radiation therapy. Arachidonyltrifluoromethyl Ketone (AACOCF3), a specific cPLA2 inhibitor, was studied as a potential radiosensitizer. EXPERIMENTAL DESIGN: Vascular endothelial cells (3B11 and MPMEC) and lung tumor cells (LLC and H460) were treated with 1 micromol/L AACOCF3 for 30 minutes prior to irradiation. Treatment response was evaluated by clonogenic survival, activation of extracellular signal-regulated kinase 1/2 (ERK1/2), tubule formation, and migration assays. For in vivo experiments, mice with LLC or H460 tumors in the hind limbs were treated for 5 consecutive days with 10 mg/kg AACOCF3 administered daily 30 minutes prior to irradiation. Treatment response was assessed by tumor growth delay, Power Doppler Sonography, and immunohistochemistry. RESULTS: In cell culture experiments, inhibition of cPLA2 with AACOCF3 prevented radiation-induced activation of ERK1/2 and decreased clonogenic survival of irradiated vascular endothelial cells but not the lung tumor cells. Treatment with AACOCF3 also attenuated tubule formation and migration in irradiated vascular endothelial cells. In both tumor mouse models, treatment with AACOCF3 prior to irradiation significantly suppressed tumor growth and decreased overall tumor blood flow and vascularity. Increased apoptosis in both tumor cells and tumor vascular endothelium was determined as a possible mechanism of the observed effect. CONCLUSION: These findings identify cPLA2 as a novel molecular target for tumor sensitization to radiation therapy through the tumor vasculature.

Tumor cavitation during therapy with antiangiogenesis agents in patients with lung cancer.

PURPOSE: Treatment of lung cancer patients with antiangiogenesis agents is a new promising paradigm. Tumor cavitation is frequently noted in these patients, but the clinical significance of this finding has not been fully determined. Our purposes were to evaluate the frequency, imaging characteristics, and clinical outcome of patients receiving antiangiogenesis agents who develop tumor cavitation, and correlate these findings with therapy related adverse events, especially hemoptysis. METHODS: Retrospective analysis of lung cancer patients treated with antiangiogenesis agents in MD Anderson Cancer Center between June 1998 and June 2005. Clinical data were retrieved from medical records, and chest imaging findings were documented. RESULTS: One hundred and twenty-four patients were treated in 10 different trials. All patients had advanced lung cancer and failed previous chemotherapy. Seventeen patients developed tumor cavitation during the trial (14%; median time to event, 1.8 months; range, 0.7-6.2 months), 16 patients (13%) had preexisting cavitary tumors, and 91 (73%) did not develop cavitation. Cavity formation was more common with squamous cell histology (p = 0.04) but was not associated with hemoptysis (p = 0.12), tumor location (central versus peripheral), imaging characteristics, progression-free survival (p = 0.56), or overall survival (p = 0.33). Hemoptysis was noted in five patients (median time to event, 1.3 months; range, 0.8-2.9 months). One of five patients with hemoptysis was fatal in a cavitary squamous cell tumor. Additional adverse events were hypertension, rash, and proteinuria, none associated with cavitation. CONCLUSION: Development of tumor cavitation is not rare in lung cancer patients treated with antiangiogenesis agents, but the clinical implications are minimal in most cases.

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.

Interleukin-8 in non-small cell lung carcinoma: relation with angiogenic pattern and p53 alterations.

Progression of solid tumors, including NSCLC, is associated with increase in MVC (microvessel count), as a measure of tumor angiogenesis resulting from an imbalance between angiogenic factors and inhibitors. However, since tumor angiogenesis is a multi-step process under the control of various molecules, the mechanism of angiogenesis has not been fully clarified. Interleukin (IL)-8 has been shown to have a potential angiogenic effect in vitro and in vivo, and is overexpressed in several human solid cancers. Among the various angiogenic factors, vascular endothelial growth factor (VEGF) has been shown to correlate with a high MVC and with adverse prognosis in several human cancers, including NSCLC. Alterations of p53 suppressor gene are the most common genetic changes found in malignant tumors; several studies examined the link between aberrant p53 and angiogenesis in lung cancer, but only a few studies report data regarding a relation between p53 mutations and IL-8 expression. In this study we observed a correlation between IL-8 mRNA expression, intratumoral MVC and VEGF mRNA expression levels; furthermore, an aberrant p53 status was related to IL-8 expression. However, in our samples IL-8 levels did not significantly affect prognosis of NSCLC; more studies are required to elucidate the precise role of IL-8 in a large series of patients with non-small cell lung carcinoma.

Carbonic anhydrase IX in early-stage non-small cell lung cancer.

PURPOSE: Tumor hypoxia is associated with poor prognosis and increased tumor aggressiveness. Carbonic anhydrase (CA) IX, an endogenous marker for tumor hypoxia, catalyzes the hydration of carbon dioxide into carbonic acid and contributes to the pH regulation of tumor cells. Therefore, CA IX might allow tumors to acclimate to a hypoxic microenvironment, promoting tumor cell proliferation. We hypothesized that CA IX expression is related to tumor cell proliferation and poor disease-free survival in patients with early-stage non-small-cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: CA IX expression was measured in 75 resected NSCLC tumors to assess prognostic implications for disease-free survival. The relationship of CA IX expression with microvessel density (MVD) and proliferation (Ki-67) index was assessed via colocalization analysis. RESULTS: All patients had operable NSCLC (stage I, 58; stage II, 17). CA IX expression was present in 54 (72%) of 75 patients and was associated with tumor necrosis (P < 0.05). CA IX-positive tumor areas showed greater cell proliferation as measured by Ki-67 index (P < 0.05) and less MVD (P < 0.05) than did CA IX-negative areas in colocalization analysis. The percentage of CA IX-positive tumor cells was significantly related to postoperative recurrence and poor disease-free survival (P < 0.05). Ki-67 index and pathologic stage were also independent prognostic factors for worse disease-free survival (P < 0.05). CONCLUSIONS: CA IX expression of tumor cells may be an indicator for poor disease-free survival in early-stage NSCLC.

Overexpression of Ang-2 mRNA in non-small cell lung cancer: association with angiogenesis and poor prognosis.

Overexpressed Angiopoietin-2 (Ang-2) derived mainly from cancer cells was reported to promote tumor angiogenesis. The aim of this study was to evaluate the expression of Ang-2 in non-small cell lung cancers (NSCLCs). We investigated Ang-2 expression in 77 patients with NSCLC who underwent curative tumor resection by means of reverse transcription-polymerase chain reaction (RT-PCR). We also determined whether or not expression of Ang-2 mRNA correlates with immunohistochemical assays of Ang-2 protein and microvessel density (MVD) level. The level of Ang-2 mRNA expression was presented by the relative yield of each gene to the S14 mRNA, respectively. Ang-2 mRNA expression in NSCLC was significantly greater than that in non-cancerous normal lung (p=0.0178). The Ang-2 mRNA expression was significantly associated with lymph node metastasis, stage, Ang-2 protein, and microvessel density (MVD) level (p=0.0009 for lymph node metastasis; p=0.0002 for stage; p<0.0001 for Ang-2 protein; p<0.0001 for MVD). With regard to prognosis, the overall and stage I survival rates for patients in the high Ang-2 mRNA expression group were significantly poorer when compared with the low Ang-2 mRNA expression group (p<0.0001 for overall; p=0.0201 for stage I). Furthermore, expression of Ang-2 mRNA was an independent predictor of prognosis by multivariate analysis (p=0.0028). These data indicate that Ang-2 may contribute to tumor angiogenesis and progression and that Ang-2 gene expression can serve as a useful prognostic marker in NSCLC.

Neuroendocrine lung tumors: grade correlates with proliferation but not angiogenesis.

Angiogenesis has been implicated in the progression of human neoplasia from benign precursor to invasive and metastatic phenotypes. The acquisition of dominant oncogenes in preneoplastic cells in vitro and in vivo has been associated with the increased ability of tumor cells to secrete angiogenic mediators and recruit blood vessels. However, in a subset of benign lesions, high levels of angiogenesis have been found before the conversion to invasive and metastatic phenotypes. In many of these benign lesions, dominant oncogenic pathways are activated first; then as malignant potential is acquired, there is a loss of nuclear tumor suppressor genes, such as p53 and p16. We studied neuroendocrine lung tumors (NLT) ranging from typical and atypical carcinoid tumors to large cell neuroendocrine and small cell carcinomas in order to determine whether angiogenesis (as assessed by mean vessel density) and proliferation rates (as assessed by MIB-1 nuclear immunohistochemical staining) correlate with tumor type. We found that increased rates of proliferation, but not angiogenesis, correlate with tumor type. The association of increased proliferation and tumor type may prove to be clinically useful and shed light on the role of sequential oncogenic alterations in NLT.

Endothelial cells and angiogenesis intensity in lung cancer.

We focused our studies on single endothelial cells (ECs) scattered in extracellular matrix in lung cancer tumors. Neovascularization was evaluated in 100 tumors obtained from patients operated for lung cancer, in relation to histological type, tumor differentiation and clinical stage of the disease. Angiogenic objects (single endothelial cells and microvessels) were identified by immunohistochemistry using monoclonal antibodies against von Willebrand factor. The count of angiogenic objects per 1 mm2 in each section was determined in a "hot spot" located at the margin of the tumor. We used an arbitrary scale of angiogenesis intensity: 1 - 0-200, 2 - 201-400, 3 - >400 angiogenic objects/mm2. A majority (57%) of the examined cases belonged to the group 2. The angiogenesis intensity measured by the single EC numbers/mm2 correlates with the histological type and the differentiation of the tumors. There was no such a correlation when the angiogenesis intensity was measured by counting total angiogenic objects (microvessels + EC) number/mm2. Single EC number/mm2 in different histological types of cancer were as follows: 162+/-121 in squamous cell (SqCC), 194+/-71 in adenocarcinoma (AdC), 225+/-145 in large cell (LCC), 264+/-52 in small cell (SCC), 279+/-173 in combined cancer. The differences between the EC counts in the different histological types of lung cancers were statistically significant in the following pairs: SqCC vs SCC (p=0.0233) and AdC vs SCC (p=0.0409). The correlation between EC count in the "hot spot" and the grade of tumor differentiation was statistically significant for G1 vs G4 (p=0.0007) and G1 vs G2 (p=0.0411). Our results suggest that higher numbers of EC/mm2 may confirm rapid development of angioneogenesis. These relations should be examined in larger series of cases.

Immunoreactivity for thyroid transcription factor-1 in stage I non-small cell carcinomas of the lung.

Thyroid transcription factor-1 (TTF-1) is a nuclear protein regulating the transcriptional activity of lung-specific genes in the normal and neoplastic bronchioloalveolar cells. It has been implicated in the normal growth and development of the lung, and the disruption of the TTF-1 locus leads to neonatal death with pulmonary hypoplasia. We evaluated retrospectively the prevalence and clinical significance of TTF-1 immunoreactivity in 222 patients with stage I non-small cell lung carcinoma (NSCLC) with a follow-up time of at least 5 years, and we investigated its relationship with other markers of tumor growth, namely cell proliferation and angiogenesis. TTF-1 immunoreactivity was documented by using the commercially available monoclonal antibody 8G7G3/1 in 72% of 97 adenocarcinomas, 5% of 119 squamous cell carcinomas, and in the glandular component of two adenosquamous carcinomas. Four large cell carcinomas were completely unreactive. In adenocarcinomas, but not squamous cell carcinomas, TTF-1 immunoreactivity correlated significantly with microvessel density (p = 0.04) and inversely with the tumor proliferation fraction assessed by Ki-67 immunostaining (p = 0.03). Also, TTF-1-immunoreactive adenocarcinomas showed a trend for a size less than 3 cm (p = 0.08). TTF-1 expression was not related to specific growth patterns, tumor grade, or tumor cell typing. TTF-1 immunoreactivity did not significantly affect patient survival, although patients with more than 75% immunoreactive neoplastic cells showed a trend for longer overall and disease-free survival. Our findings suggest that TTF-1 could be involved in the development of small pulmonary adenocarcinomas, but it has not prognostic implications in patients with stage I NSCLC.

Immunohistochemical study on tumor angiogenic factors in non-small cell lung cancer.

BACKGROUND: In order to elucidate the roles of tumor angiogenesis in lung carcinogenesis, the expressions of several angiogenic factors in lung carcinoma tissues were examined. MATERIALS AND METHODS: Tissue specimens from 112 cases of resected non-small cell lung cancer (NSCLC) were studied. The expressions of platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) were examined immunohistochemically. Microvessel density (MVD) was also evaluated. RESULTS: VEGF-positive cases were observed more frequently in advanced stage lung cancers than in early cancers, and VEGF-positive tumors had higher MVD than VEGF-negative tumors, while such differences were not observed for PD-ECGF. In squamous cell carcinoma, the patients with high-MVD tumor had significantly worse survival than those with low-MVD tumor. CONCLUSIONS: Our results suggest that VEGF plays an important role in angiogenesis of lung cancers, while the contribution of PD-ECGF may be limited.

Studies on angiogenesis intensity in lung cancer in aspect of its correlation with histological type of tumor and clinical stage.

Angiogenesis intensity in lung cancer, in compliance with histological types, tumor differentiation and different clinical stage of disease, was evaluated. The group of 65 patients, 34-73 years old (average 58), who have been operated, were examined. Microvessels were highlighted by immunohistochemical method staining of endothelial cells for factor VIII-von Willebrand. Microvessel and single endothelial cell count per 1 mm2 in each section was determined using light microscope, synchronized with camera and IBM-AT computer (LUCIA-NIKON program for morphometric studies). All cases were divided into three groups depending on angiogenesis intensity: Io-0-200, IIo-201-400, IIIo-400 angiogenic objects/mm2 (microvessels-MV plus endothelial cells-EC). Majority (57%) of examined cases were found in IIo group. The results of studies on angiogenic objects number (MV+EC) per 1 mm2 in different histological type of cancer were following: 248.97 +/- 114.72 in squamous cell, 253.18 +/- 81.32 in adenocarcinoma, 284.04 +/- 114.27 in large cell, 388.02 +/- 117.73 in small cell, 385.27 +/- 210.92 in combined cancer. In each group of lung cancer with different TNM and clinical stages was found that the angiogenic objects number depends on T tumor feature, mainly in EC count analysis (T1-148.61 +/- 113.21, T2-179.38 +/- 100.57, T3-199.52 +/- 137.70, T4-253.18 +/- 108.60). Obtained data were analyzed with of t Student's test. The differences between angiogenic objects number in the groups with different histological type of lung cancer were no statistically significant, although were near threshold value in pairs squamous cell versus small cell (p = 0.0545) and adenocarcinoma versus small cell (p = 0.0611). The differences of EC counts in the same pairs were statistically significant: p = 0.0247 (squamous cell versus small cell) and p = 0.0380 (adenocarcinoma versus small cell). The correlation between angiogenic objects number and grade of tumor differentiation was statistically significant for G1 group versus G2 (p = 0.0380) and G1 versus G4 (p = 0.0008), in comparison to G2 versus G4-p = 0.0688. The remaining results were not statistically significant. Obtained data are no final because the examined groups of cases were not numerous enough. The dependences should be examined in large series of cases.