Immunohistochemical expression of metalloproteinases MMP-2 and MMP-9 in abdominal aortic aneurysms: correlation with symptoms and aortic diameter

Our purpose was to investigate the expression of matrix metalloproteinase (MMP)-2 and -9 in all types of abdominal aortic aneurysms (AAA): symptomatic, asymptomatic, and ruptured. MMP-2 and -9 activity was investigated in surgical samples from the arterior wall of 46 AAA, using a standard immunohistochemical technique. The MMP-9 activity was significantly higher in large AAA (>6 cm), but there was no relation between AAA size and MMP-2 activity. Neither MMP-2 nor MMP-9 were related with AAA rupture, other complications or symptoms. The MMP-9 activity in AAA is a determinant of the aneurysm size, but it is not related to clinical manifestations.     

Activated VEGFR2/KDR pathway in tumour cells and tumour associated vessels of colorectal cancer

BACKGROUND: Vascular endothelial cell growth factor (VEGF) acts by phosphorylating specific tyrosine kinase receptors on endothelial cell membrane promoting angiogenesis. The study of the activation status of VEGF receptors in human malignancies has recently become feasible by means of specific monoclonal antibodies recognising the phosphorylated form of these receptors. MATERIALS AND METHODS: In the current study, we investigate the expression of the phosphorylated VEGFR2/KDR receptor in normal colon and colorectal adenocarcinomas in parallel with histopathological parameters, prognosis and the expression of the 'hypoxia inducible factor' HIF1alpha. RESULTS: pVEGFR2/KDR was weakly expressed in the normal colon, but it was expressed strongly in the cytoplasm and nuclei of cancer cells and in the tumour associated vasculature, mainly at the invading tumour edge. pVEGFR2/KDR expression in cancer cells was significantly associated with a tumour diameter > 6 cm (P = 0.04), poor histological differentiation (P = 0.004) and with high CEF1alpha expression (P = 0.05). High pVEGFR2/KDR expressing vascular density was significantly related with a high VEGF and HIF1alpha expression in cancer cells (P = 0.02 and 0.03, respectively). This was also related significantly to high pVEGFR2/KDR expression in cancer cells. In multivariate analysis, the most significant predictors for death were lympho-vascular invasion (P < 0.001) followed by VEGF (P = 0.014), node status (P = 0.015), standard vascular density (P = 0.022) and necrosis (P = 0.032). CONCLUSIONS: pVEGFR2 receptors are largely expressed in colon cancer cells and intratumoural vasculature. As VEGF targeting agents enter the clinical practice, the role of monoclonal antibodies recognising the phosphorylated form of VEGF receptors as predictors of response to targeted therapies should be sought in clinicopathological trials.     

Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenic pathways and independently to poor outcome in non-small cell lung cancer.

Carbonic anhydrase-9 (CA9), a transmembrane enzyme with an extracellular active site, is involved in the reversible metabolism of the carbon dioxide to carbonic acid. Up-regulation of CA by hypoxia and the hypoxia-inducible factor (HIF) pathway has been recently postulated (Wykoff et al. Cancer Res., 60: 7075-7083, 2000). In the present study we examined the expression of this enzyme in non-small cell lung cancer. Of 107 cases analyzed, 39 (36.4%) had strong membrane/cytoplasmic expression of CA9 and were grouped as positive. The staining was confined around areas of necrosis, and a significant association of CA9 expression with the extent of necrosis was noted (P = 0.004). Nevertheless, 38 of 74 cases with focal or extensive necrosis did not express CA9. CA9 expression was more frequent in the squamous cell histology (P = 0.001) and with advanced T stage (P = 0.009). A significant coexpression of CA9 with platelet-derived endothelial cell growth factor and basic fibroblast growth factor receptor expression was noted. Double staining of CA9 with anti-CD31 monoclonal antibody revealed an overall higher microvessel density in the areas expressing CA9 than in negative areas (P = 0.0005). Thirty-one of 38 CA9-positive cases were positive for HIF1a/HIF2a, but HIF positivity was a more common event (68 of 107) and their patterns of expression were diffuse (not confined in the necrotic areas). A direct association of CA9 expression with epidermal growth factor receptor, c-erbB-2, and MUC1 expression was also noted (P < 0.04). Survival analysis showed that CA9 expression is related to poor prognosis. CA9 expression in tumors with low vascularization defined a prognosis similar to the one of patients with highly angiogenic tumors. Multivariate analysis revealed that CA9 expression is a significant prognostic factor independent of angiogenesis. We conclude that CA9 is an important molecule in non-small cell lung cancer, the up-regulation of which occurs in highly hypoxic/necrotic regions of the tumors. The expression of CA9 is linked to the expression of a constellation of proteins involved in angiogenesis, apoptosis inhibition, and cell-cell adhesion disruption, which explains the strong association of CA9 with poor outcome.     

C2028T polymorphism in exon 12 and dinucleotide repeat polymorphism in intron 13 of the HIF-1alpha gene define HIF-1alpha protein expression in non-small cell lung cancer

OBJECTIVES: In this study, we investigated whether polymorphisms of the HIF-1alpha gene may account for the patterns of HIF-1alpha protein expression in non-small cell lung carcinomas (NSCLC) and the expression of HIF-1alpha down-stream proteins. METHODS: Specific HIF-1alpha polymorphisms were assessed in a series of patients with NSCLC: (a) the C to T transition at nucleotide 1744 (position 2028 according to sequence with accession number , which gives rise to Pro/Ser variation at codon 582), (b) the G to A nucleotide substitution at point 1790 (position 2046 according to sequence with accession number , which gives rise to Ala/Thr variation at codon 588), and (c) the dinucleotide GT repeat polymorphism in intron 13. Immunohistochemistry for HIF-1alpha and down-stream proteins (VEGF, LDH-5, GLUT-1) was also performed in tumor material. RESULTS: A strong association of the P582S polymorphism and of GT repeat polymorphism higher than 14/14 with increased HIF-1alpha expression was noted. HIF-1alpha polymorphism did not relate to the expression of the HIF-1alpha downstream proteins analysed, but significant association of HIF-1alpha expression with LDH-5 was confirmed (p=0.008). CONCLUSIONS: HIF-1alpha polymorphisms may have an important impact on HIF-protein stability and, eventually, function.     

Angiogenesis in colorectal cancer: prognostic and therapeutic implications

Angiogenesis is important for tumor growth and metastasis. This account reviews the clinicopathological studies conducted in the field of angiogenesis in colorectal cancer, the methods of assessing vascular-related characteristics in tissue sections and provides a background for the usefulness of antiangiogenic policies along with chemotherapy and radiotherapy. Highly angiogenic colorectal tumors are associated with aggressive histopathological features and poor patients' survival. Similarly, factors stimulating angiogenesis, such as vascular endothelial growth factor (VEGF), thymidine phosphorylase (TP), and others, are commonly related to increased vascular density (VD) and, therefore, to an unfavorable clinical course. Anti-VEGF agents have improved prognosis in patients with metastatic colorectal cancer, when added to standard chemotherapy. It is expected that, in addition to adjuvant chemotherapy and radiotherapy, agents blocking the stimulatory effect of VEGF on endothelial cells would prove beneficial to the patient.     

Node-related factors and survival in node-positive breast carcinomas

The number of positive lymph nodes (+LNs) is the only node-related prognostic factor recognized by the American Joint Committee TNM system for breast cancer. In this study, we evaluated additional node-related prognostic factors in node-positive breast carcinomas. One hundred and thirty-four patients with infiltrating ductal carcinomas and axillary +LNs, who had been treated with modified radical mastectomy, were analyzed. Metastatic lymph nodes were evaluated for traditional prognostic factors, i.e., the number of involved nodes and the extranodal extension, but also for a variety of other node-related characteristics, such as nodal tumor burden and maximum size of tumor deposits, necrosis, stromatogenesis, extranodal vascular involvement, and nodal matting. The results reaffirmed the prognostic significance of metastasis in 1-3 vs. 4-7 axillary lymph nodes and, in addition, revealed the importance of 4 +LNs as a cut-off point for breast tumor aggressiveness, given that survival curves for 4-7 vs. >7 +LNs were indifferent. Interestingly, nodal tumor burden, when extensive, was statistically an adverse prognostic factor. Prognosis was equally poor if metastases to regional nodes showed extensive nodal necrosis or extensive nodal stromatogenesis, if there was extranodal spread, extranodal vascular involvement, or if the axillary +LNs were matted. In multivariate analysis, intranodal necrosis and extranodal vascular involvement were the only node-related features with a significant and independent prognostic function. Node-related pathological features in node-positive breast carcinomas are important in determining prognosis. Intranodal necrosis and extranodal tumor emboli should be considered, in parallel with the number of +LNs, in subsequent treatment design.     

Coexpression of MUC1 glycoprotein with multiple angiogenic factors in non-small cell lung cancer suggests coactivation of angiogenic and migration pathways.

We investigated the expression of MUC1 protein and its relationship to the microvessel density and the expression of thymidine phosphorylase, vascular endothelial growth factor (VEGF), VEGF-receptor KDR, basic fibroblast growth factor (bFGF), and bFGF-receptor (FGFR-2) in non-small cell lung cancer. Although MUC1 expression was found equally in poorly and highly vascularized tumors, a significant coexpression with multiple angiogenic factors and their receptors was noted (P = 0.0002, 0.03, 0.19, 0.10, and 0.01 for thymidine phosphorylase, VEGF, KDR, bFGF, and FGFR-2, respectively). In multiple regression analysis, both angiogenesis and MUC1 expression were independent prognostic variables. The present study suggests the existence of an early genetic event leading to the activation of both migration and angiogenesis pathways in non-small cell lung cancer.     

Platelet-derived endothelial cell growth factor (Thymidine Phosphorylase) expression in lung cancer

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is an enzyme with angiogenic and cell motility properties. Moreover, it is involved in the transformation of fluoropyrimidines into active cytotoxic metabolites. In the present study, the expression of PD-ECGF in normal lung and lung cancer was immunohistochemically evaluated using the P-GF.44C monoclonal antibody. Alveolar and tumoural macrophages were invariably stained and were used as an internal control for assessment of the staining. Alveolar epithelium was always negative, whilst bronchiolar epithelium showed occasional positive reactivity. Normal lung and tumour endothelium was occasionally positive. Positive staining in more than 50 per cent of cells was observed in 23/71 squamous carcinomas (32 per cent), 16/38 (42 per cent) adenocarcinomas, and 2/6 (33 per cent) adenosquamous carcinomas. Differentiated areas and areas of squamous metaplasia were more strongly positive than other tumour areas. All 22 small cell carcinomas and one carcinoid tumour were negative. The present study provides a baseline for future studies in non-small cell lung cancer to correlate PD-ECGF expression with tumour vascularization, prognosis, and response to chemotherapy. © 1997 John Wiley & Sons, Ltd.