Simultaneous EGFR and VEGF Alterations in Non-Small Cell Lung Carcinoma Based on Tissue Microarrays

Background: Epidermal growth factor receptor (EGFR) overexpression is observed in significant proportions of non-small cell lung carcinomas (NSCLC). Furthermore, overactivation of vascular endothelial growth factor (VEGF) leads to increased angiogenesis implicated as an important factor in vascularization of those tumors.Patients and Methods: Using tissue microarray technology, forty-paraffi n (n = 40) embedded, histologically confirmed primary NSCLCs were cored and re-embedded into a recipient block. Immunohistochemistry was performed for the determination of EGFR and VEGF protein levels which were evaluated by the performance of computerized image analysis. EGFR gene amplification was studied by chromogenic in situ hybridization based on the use of EGFR gene and chromosome 7 centromeric probes.Results: EGFR overexpression was observed in 23/40 (57.5%) cases and was correlated to the stage of the tumors (p = 0.001), whereas VEGF was overexpressed in 35/40 (87.5%) cases and was correlated to the stage of the tumors (p = 0.005) and to the smoking history of the patients (p = 0.016). Statistical significance was assessed comparing the protein levels of EGFR and VEGF (p = 0.043, k = 0.846). EGFR gene amplification was identified in 2/40 (5%) cases demonstrating no association to its overall protein levels (p = 0.241), whereas chromosome 7 aneuploidy was detected in 7/40 (17.5%) cases correlating to smoking history of the patients (p = 0.013).Conclusions: A significant subset of NSCLC is characterized by EGFR and VEGF simultaneous overexpression and maybe this is the eligible target group for the application of combined anti-EGFR/VEGF targeted therapies at the basis of genetic deregulation (especially gene amplification for EGFR)

Morphometric study of NOR's in neoplastic cells of Hodgkin's disease

Eighty-nine lymph nodes from 53 cases of Hodgkin’s disease were studied for the evaluation of (NORs), in formalin fixed, paraffin embedded sections. For determination of morphometric profile of NORs, a semiautomatic image analysis system was used. The parameters that studied were number of NORs in Sternberg-Reed and Hodgkin's cells, area shapes and number of NORs in postcapillary venules and in activated lyphocytes. A high number of NORs were found in neoplastic cells of Hodgkin's disease equal or higher than that observed in non-Hodgkin's lymphomas and various types of carcinomas and sarcomas. The higher number was observed in lymphocyte depleted type and the lower in lymphocyte predominant type. Observations concerning the mean number of NORs in postcapillary venules and activated lymphocytes, gave as a compatibility in the results for the different histological types of the disease, as mentioned above. In conclusion, the findings of this study indicate that neoplastic cells in Hodgkin's disease are highly activated. NORs counting may be a reliable index of studying the activation degree and perhaps the biological behaviour in a great variety of histological material.Μελετήθηκαν σε τομές παραφίνης με την αργυροφιλική τεχνική εντοπισμού των Περιοχών Οργάνωσης του Πυρηνίσκου (ΠΟΠ), 89 λεμφαδένες από 53 περιπτώσει ς νόσου του Hodgkin. Η μελέτη έγινε με Ημιαυτόματο Μορφομετρικό Σύστημα που περιλαμβάνει Η/Υ, μορφομετρική τράπεζα και το κατάλληλο λογισμικό. Οι παράμετροι που μελετήθηκαν ήταν οι περιεκτικότητες των κυττάρων Sternberg Reed και Hodgkin σε ΠΟΠ, οι μεγαλύτεροι διάμετροι, τα εμβαδά και σχήματα των ΠΟΠ καθώς και η περιεκτικότητα σε ΠΟΠ των μετατριχοειδικών φλεβιδίων και των ενεργοποιημένων λεμφοκυττάρων. Τα αποτελέσματα έδειξαν πολύ υψηλή περιεκτικότητα των νεοπλασματικών κυττάρων σε ΠΟΠ αντίστοιχη ή μεγαλύτερη αυτής των υψηλής κακοήθειας μη Hodgkin λεμφωμάτων, καρκινωμάτων, σαρκωμάτων και ενδοκρινικών νεοπλασμάτων. Η μεγαλύτερη περιεκτικότητα με μεγάλη στατιστική διαφορά από τους υπολοίπους τύπους παρατηρείται στο λεμφοπενικό και η μικρότερη στον λεμφοεπικρατητικό. Αντίστοιχα είναι και τα ευρήματα στα μετατριχοειδικά φλεβίδια και στα ενεργοποιημένα λεμφοκύτταρα. Τα αποτελέσματα οδηγούν στο συμπέρασμα ότι τα νεοπλασματικά κύτταρα της νόσου του Hodgkin είναι σε πολύ υψηλά επίπεδα ενεργοποίησης. Αν ο βαθμός ενεργοποίησης αποδειχθεί ότι αποτελεί και δείκτη βιολογικής συμπεριφοράς τότε η καταμέτρηση των ΠΟΠ αποτελεί μέσο βιολογικής πρόβλεψης

Evaluation of topoisomerase IIa expression in pancreatic ductal adenocarcinoma: A pilot study using chromogenic in situ hybridization and immunohistochemistry on tissue microarrays

Background/Aims: To co-evaluate topoisomerase IIa ( Topo IIa) protein expression and gene status in pancreatic ductal adenocarcinoma, determining the potential prognostic impact of its alterations. Methods: Using tissue microarrays, 50 sporadic, primary pancreatic ductal adenocarcinomas were cored twice and re-embedded into one paraffin block with a core diameter of 1 mm. Immunohistochemistry and chromogenic in situ hybridization were performed in serial tissue sections for the detection of protein expression levels, chromosome 17 and Topo IIa gene status, respectively. Finally using a semi-automated image analysis system we evaluated the levels of protein expression. Results: A significant proportion of the tumors showed Topo IIa overexpression (32/50 or 64%). Gene amplification and deletion were detected in 9 and 4 cases, respectively, associated with protein overexpression. Aneuploidy regarding chromosome 17 was observed in 19/50 tumors and correlated with poor survival rate ( Cox regression test: p = 0.001). Topo IIa protein expression was strongly correlated with stage ( p = 0.021) and grade ( p = 0.034). Conclusions: Topo IIa gene amplification correlates with protein overexpression, but not vice versa. This is a crucial observation for the application of targeted chemotherapies, such as anthracyclines, only in subgroups of patients, according to molecular deregulation criteria and not only to immunohistochemical results. Also, chromosome 17 and not Topo IIa gene instability can be used as a potential independent prognostic factor. Copyright (c) 2007 S. Karger AG, Basel and IAP

Expression of CTGF and TNFa in alveolar macrophages of patients with idiopathic pulmonary fibrosis before and after treatment with azathioprine or interferon-γ-1b

SUMMARY.Backgrou nd: Idiopathic pulmonary fibrosis (IPF) is a fatal lungdisorder the aetiology of which is unknown and for which there isno effective therapy. Connective tissue growth factor (CTGF) andtumour necrosis factor alpha (TNFα) have been reported to participatesignificantly in the pathogenesis of the disease. The role of alveolarmacrophages in the expression of these cytokines remains unclear.Materials and Methods: Samples of bronchoalveolar lavagefluid (BALF) derived from 20 newly diagnosed patients with IPF beforeand after 6 months of treatment with either interferon (IFN-γ-1b) andprednisolone (10 patients) or azathioprine (AZA) and prednisolone(10 patients) and from 10 normal subjects (control group) were usedfor the analysis of CTGF and TNFα protein expression in the alveolarmacrophages. The effectiveness of the two drug regimes on thepulmonary function tests (FEV1, FVC, DLCO) and PaO2 and PaCO2 ofthe patients with IPF was investigated. Results: Decreased CTGFprotein expression was detected in the patients with IPF comparedwith the control group (p=0.001). TNFα expression in IPF patientsdid not differ from that of the normal control subjects. Neither ofthe drug regimes affected the protein expression of these factorsorthe pulmonary function parameters. Co nclusion: These findingssuggest that the alveolar macrophages are not the main source ofCTGF and TNFα in IPF. Treatment with either AZA or IFN-γ-1b did notresult in any significant change in the protein expression of thesefactors. Pneumon 2011, 24(2):149-156

Decreased Apoptotic Rate of Alveolar Macrophages of Patients with Idiopathic Pulmonary Fibrosis

Introduction. Increased apoptosis of epithelial cells and decreased apoptosis of myofibroblasts are involved in the pathogenesis of IPF. The apoptotic profile of alveolar macrophages (AMs) in IPF is unclear. Aim. To investigate whether AMs of patients with IPF exhibit a different apoptotic profile compared to normal subjects. Methods. We analyzed, by immunohistochemistry, the expression of the apoptotic markers fas, fas ligand , bcl-2, and bax in AM obtained from bronchoalveolar lavage fluid (BALF) of 20 newly diagnosed, treatment-naive IPF patients and of 16 controls. Apoptosis of AM was evaluated by Apoptag immunohistochemistry. IPF patients received either interferon-g and corticosteroids or azathioprine and corticosteroids for six months. Results. BALF AMs undergoing apoptosis were significantly less in IPF patients. No difference was found in the expression of fas or fas ligand, bcl-2 and bax between IPF and control group. No difference was found between the respiratory function parameters of the two treatment groups after six months. A positive correlation was found between the number of bcl-2 positive stained macrophages and DLCO after treatment. Conclusions. The decreased apoptotic rate of AM of patients with IPF is not associated with decreased expression of apoptosis mediators involved in the external or internal apoptotic pathway

Expression of Hypoxia-Inducible Factor (HIF)-1a-Vascular Endothelial Growth Factor (VEGF)-Inhibitory Growth Factor (ING)-4- axis in sarcoidosis patients

Abstract Background Sarcoidosis is a granulomatous disorder of unknown etiology. The term of immunoangiostasis has been addressed by various studies as potentially involved in the disease pathogenesis. The aim of the study was to investigate the expression of the master regulator of angiogenesis hypoxia inducible factor (HIF)-1a – vascular endothelial growth factor (VEGF)- inhibitor of growth factor 4-(ING4) - axis within sarcoid granuloma. Methods A total of 37 patients with sarcoidosis stages II-III were recruited in our study. Tissue microarray technology coupled with immunohistochemistry analysis were applied to video-assisted thoracoscopic surgery (VATS) lung biopsy samples collected from 37 sarcoidosis patients and 24 controls underwent surgery for benign lesions of the lung. Computerized image analysis was used to quantify immunohistochemistry results. qRT-PCR was used to assess HIF-1a and ING4 expression in 10 sarcoidosis mediastinal lymph node and 10 control lung samples. Results HIF-1a and VEGF-ING4 expression, both in protein and mRNA level, was found to be downregulated and upregulated, respectively, in sarcoidosis samples compared to controls. Immunohistochemistry coupled with computerized image analysis revealed minimal expression of HIF-1a within sarcoid granulomas whereas an abundant staining of ING4 and VEGF in epithelioid cells was also visualized. Conclusions Our data suggest an impairment of the HIF-1a – VEGF axis, potentialy arising by ING4 overexpression and ultimately resulting in angiostasis and monocyte recruitment within granulomas. The concept of immunoangiostasis as a possible protection mechanism against antigens of infectious origin needs further research to be verified.</p