Michela Paganuzzi

Functional analysis of c-Met/hepatocyte growth factor pathway in malignant pleural mesothelioma

c-Met receptor tyrosine kinase (RTK) has not been extensively studied in malignant pleural mesothelioma (MPM). In this study, c-Met was overexpressed and activated in most of the mesothelioma cell lines tested. Expression in MPM tissues by immunohistochemistry was increased (82%) in MPM in general compared with normal. c-Met was internalized with its ligand hepatocyte growth factor (HGF) in H28 MPM cells, with robust expression of c-Met. Serum circulating HGF was twice as high in mesothelioma patients as in healthy controls. There was a differential growth response and activation of AKT and extracellular signal-regulated kinase 1/2 in response to HGF for the various cell lines. Dose-dependent inhibition (IC50 < 2.5 micromol/L) of cell growth in mesothelioma cell lines, but not in H2052, H2452, and nonmalignant MeT-5A (IC50 > 10 micromol/L), was observed with the small-molecule c-Met inhibitor SU11274. Furthermore, migration of H28 cells was blocked with both SU11274 and c-Met small interfering RNA. Abrogation of HGF-induced c-Met and downstream signaling was seen in mesothelioma cells. Of the 43 MPM tissues and 7 cell lines, we have identified mutations within the semaphorin domain (N375S, M431V, and N454I), the juxtamembrane domain (T1010I and G1085X), and an alternative spliced product with deletion of the exon 10 of c-Met in some of the samples. Interestingly, we observed that the cell lines H513 and H2596 harboring the T1010I mutation exhibited the most dramatic reduction of cell growth with SU11274 when compared with wild-type H28 and nonmalignant MeT-5A cells. Ultimately, c-Met would be an important target for therapy against MPM.

Clinical Significance of Serum Mesothelin in Patients with Mesothelioma and Lung Cancer

Purpose: High levels of serum-soluble mesothelin family proteins (SMRP) have been found to be associated with malignant mesothelioma (MM), but not lung cancer (LC). To verify the clinical role of this marker for both these tumors, we tested serum SMRP in the largest population of thoracic cancers ever assembled. Experimental Design: SMRP blood concentrations were measured in 107 patients with MM, 215 patients with LC, 130 patients with benign respiratory diseases (BRD), and 262 controls. Statistical comparison between mean serum SMRP levels in all groups was done and receiver operating characteristic curves were constructed to evaluate the performance of this marker. Results: SMRP levels were significantly higher in patients with MM and LC than in patients with benign respiratory diseases and controls (P < 0.001). The area under the receiver operating characteristic curve for serum SMRP discriminating MM and controls was 0.77 (95% confidence interval, 0.71-0.83), with a best cutoff of 1.00 nmol/L (sensitivity, 68.2%; specificity, 80.5%). In both MM and LC, serum SMRP levels did not differ significantly between early and late stages. High SMRP levels proved to be an independent negative prognostic factor in patients with MM. Conclusions: Our data confirm that serum SMRP is a promising marker for the diagnosis, prognosis, and clinical monitoring of MM. We found that serum SMRP dosage may prove helpful in LC diagnosis as well. These data may also have positive repercussions on secondary preventive medical strategies for workers previously exposed to asbestos.

CA 19‐9 and CA 50 in Benign and malignant pancreatic and biliary diseases

Serum concentrations of the CA 19‐9 and CA 50 antigens were determined in 129 patients with malignant and benign biliary and pancreatic diseases. Values for the two markers were highly correlated (P < 0.001). The concentrations of CA 19‐9 and CA 50 were positive in 84.6% and 80.7% of patients with pancreatic cancer, respectively. The overall specificity of CA 19‐9 (92.4%) was slightly higher than that of CA 50 (88.5%). The sensitivity of CA 50 (91.3%) was greater than that of CA 19‐9 (73.9%) in patients with diseases of the biliary tract. Elevated concentrations of CA 19‐9 (12.9%) and CA 50 (35.2%) were also found in a number of cases with benign disease, especially in patients with obstructive jaundice. These data suggest that both CA 19‐9 and CA 50 can be useful markers of pancreatic cancer in nonjaundiced patients. The joint use of the two markers does not yield a better diagnostic resolution than the use of either one alone.

Decline in Serum Carcinoembryonic Antigen and Cytokeratin 19 Fragment During Chemotherapy Predicts Objective Response and Survival in Patients With Advanced Nonsmall Cell Lung Cancer

The authors assessed the predictive and prognostic role of decline in the serum levels of carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA 21‐1) during chemotherapy in patients with advanced nonsmall cell lung cancer (NSCLC).

Natural agents targeting the α7-nicotinic-receptor in NSCLC: A promising prospective in anti-cancer drug development†

Nicotinic acetylcholine receptors (nAChR) are expressed on normal bronchial epithelial and nonsmall cell lung cancer (NSCLC) cells and are involved in cell growth regulation. Nicotine induced cell proliferation. The purpose of this study was to determine if interruption of autocrine nicotinic cholinergic signaling might inhibit A549 NSCLC cell growth. For this purpose α‐Cobratoxin (α‐CbT), a high affinity α7‐nAChR antagonist was studied. Cell growth decrease was evaluated by Clonogenic and MTT assays. Evidence of apoptosis was identified staining cell with Annexin‐V/PI. Characterization of the basal NF‐κB activity was done using the Trans‐AM NF‐κB assay colorimetric kit. “In vivo” antitumour activity was evaluated in orthotopically transplanted nude mice monitored by In vivo Imaging System technology. α‐CbT caused concentration‐dependent cell growth decrease, mitochondrial apoptosis caspases‐9 and 3‐dependent, but caspase‐2 and p53‐independent and down‐regulation of basal high levels of activated NF‐κB. α‐CbT treatment determines a significant reduction of tumor growth in nude mice orthotopically engrafted with A549‐luciferase cells (4.6% of living cells vs. 31% in untreated mice). No sign of toxicity was reported related to treatment. These findings suggest that α7‐nAChR antagonists namely α‐CbT may be useful adjuvant for treatment of NSCLC and potentially other cancers.

Serum PDGF-AB in Pleural Mesothelioma

Overexpression of platelet-derived growth factor (PDGF) has been observed in lung and pleural tumors. The aim of this study was to evaluate the diagnostic and prognostic role of serum PDGF in pleural mesothelioma (PM). Four groups of subjects were studied: 93 malignant PM patients, 33 primary non small cell lung cancer patients, 51 subjects exposed to asbestos, defined as high-risk controls, and 24 healthy controls. PDGF-AB mean concentration was higher in PM patients (45.8 ng/ml) than in high-risk controls (33.1 ng/ml) and healthy controls (26.8 ng/ml). Using the cut-off level of 49.8 ng/ml, corresponding to the mean + 2SD of PDGF-AB in healthy controls, 43% of PM patients showed positive PDGF-AB levels. Survival was evaluated in 82 PM patients. At the end of the follow-up (median 9.8 months) 80.5% of patients had died. Median survival was 13.1 and 7.9 months for patients with PDGF-AB lower and higher than the cut-off, respectively. Adjusting for age, sex, histology and platelet count, positive PDGF-AB levels were associated with lower survival (OR = 1.2, 95%CI: 0.9–1.6), even if not significantly so. In conclusion, serum PDGF may represent a useful additional parameter to prognostic factors already available for PM.

Study of pretreatment serum levels of HER‐2/neu oncoprotein as a prognostic and predictive factor in patients with advanced nonsmall cell lung carcinoma

HER‐2/neu tissue overexpression is found in nearly 15% of patients with nonsmall cell lung carcinoma and is reported to affect prognosis adversely in surgical series. However, the prognostic role of serum HER‐2/neu oncoprotein, particularly in patients with advanced lung carcinoma, remains unknown. This study was designed to assess the potential value of measuring serum levels of HER‐2/neu oncoprotein in predicting response to treatment and survival in patients with locally advanced and metastatic nonsmall cell lung carcinoma.

CA 19-9 assay in patients with extrahepatic cholestatic jaundice.

Serum concentrations of the CA 19-9 tumour marker were determined in 35 patients with histologically proven bilio-pancreatic malignancies associated with obstructive jaundice and in 35 patients with benign extrahepatic jaundice due to choledocholithiasis. At a cut-off level of 37 U/ml the sensitivity of this assay was 82.8%, but the specificity was very low (45.7%). Thus CA 19-9 can not be employed to differentiate between malignant and benign extrahepatic jaundice. Serial samples of CA 19-9 were achieved in 7 patients with benign and in 6 patients with malignant biliary obstruction, before and after the disappearance of jaundice. Serum concentrations of this tumour-antigen returned to normal concurrently with the bilirubin values only in patients with benign obstruction, remaining unchanged in all cases of malignancies. The data suggest that patients with extrahepatic jaundice should be evaluated by other examinations or by collecting serial samples for this assay.

Serum anti-p53 autoantibodies in pleural malignant mesothelioma, lung cancer and non-neoplastic lung diseases.

Alterations of the p53 gene may lead to the production of detectable autoantibodies (p53-Abs) in cancer patients. In order to evaluate the association of p53-Abs with pleuropulmonary diseases, four groups of subjects were analyzed by ELISA for serum p53-Abs, in the framework of a molecular epidemiologic study. Two of 30 pleural malignant mesothelioma patients (MM; 6.7%) and 8/48 lung cancer patients (LC; 16.7%) were seropositive, while all 51 healthy controls (HC) were negative. Two of 55 (3.6%) at-risk controls (RC) with non-malignant respiratory diseases were positive and were not subsequently diagnosed any cancer. The difference was statistically significant between LC and RC or HC (P = 0.01), but not between MM and any other group. No correlation was found with age, sex, cancer stage or histology, cigarette smoking or occupational exposure. A longer survival (not significant) was shown in seropositive LC but not in MM. p53 expression in tumor tissue was also evaluated in a subgroup of MM. In conclusion, the presence of detectable p53-Abs in serum was associated in a statistically significant proportion of cases with LC but only occasionally with MM. The longer survival among positive LC patients and the presence of two seropositive among patients with non-neoplastic respiratory diseases should be further investigated.

Osteopontin is not a specific marker in malignant pleural mesothelioma

BACKGROUND AND AIMS Osteopontin (OPN) is an integrin-binding protein recently shown to be related to tumorigenesis, progression and metastasis in different experimental models of malignancy. Malignant pleural mesothelioma (MPM) is a fatal disease in which the prognosis remains very poor and the knowledge of predictive factors for outcome is insufficient. The identification of new molecules involved in cancer initiation and development is a fundamental step for improving the curability of this kind of tumor. The purpose of this study is to define the role of OPN in the diagnosis of MPM by determining its prognostic and diagnostic value. METHODS A group of 24 surgically staged MPM subjects was compared with a group of 31 subjects with nonmalignant pulmonary diseases, and with 37 healthy controls. Tumor tissue was analyzed for OPN by immunohistochemical tests, and plasma OPN levels were measured by an enzyme-linked immunosorbent assay. RESULTS Plasma OPN levels were not significantly higher in either of the patient groups compared with the control group. Immunohistochemical analysis revealed OPN staining of tumor cells in 21 of 24 MPMs. Receiver operating characteristic curve/area under the curve (ROC/AUC) analysis comparing the plasma OPN levels in the healthy group with those of MPM patients showed 40% sensitivity and 100% specificity at a cutoff value of 60.8 ng of OPN per milliliter (AUC 0.6). CONCLUSION Plasma OPN levels do not discriminate between chronic inflammatory and malignant lung diseases and staining intensity in MPM specimens does not correlate with OPN plasma levels.