Mario Santini

Global Gene Expression Profiling Of Human Pleural Mesotheliomas: Identification of Matrix Metalloproteinase 14 (MMP-14) as Potential Tumour Target

Background The goal of our study was to molecularly dissect mesothelioma tumour pathways by mean of microarray technologies in order to identify new tumour biomarkers that could be used as early diagnostic markers and possibly as specific molecular therapeutic targets. Methodology We performed Affymetrix HGU133A plus 2.0 microarray analysis, containing probes for about 39,000 human transcripts, comparing 9 human pleural mesotheliomas with 4 normal pleural specimens. Stringent statistical feature selection detected a set of differentially expressed genes that have been further evaluated to identify potential biomarkers to be used in early diagnostics. Selected genes were confirmed by RT-PCR. As reported by other mesothelioma profiling studies, most of genes are involved in G2/M transition. Our list contains several genes previously described as prognostic classifier. Furthermore, we found novel genes, never associated before to mesotheliom that could be involved in tumour progression. Notable is the identification of MMP-14, a member of matrix metalloproteinase family. In a cohort of 70 mesothelioma patients, we found by a multivariate Cox regression analysis, that the only parameter influencing overall survival was expression of MMP14. The calculated relative risk of death in MM patients with low MMP14 expression was significantly lower than patients with high MMp14 expression (P = 0.002). Conclusions Based on the results provided, this molecule could be viewed as a new and effective therapeutic target to test for the cure of mesothelioma.

Impact of positive pleural lavage cytology on survival in patients having lung resection for non-small-cell lung cancer: An international individual patient data meta-analysis

OBJECTIVES Pleural lavage cytology is the microscopic study of cells obtained from saline instilled into and retrieved from the chest during surgery for non-small-cell lung cancer. The aims of this study were to collate multi-institutional individual patient data for meta-analysis to determine independence as a prognostic marker and to characterize the impact of positive results on stage-adjusted survival. METHODS We identified 31 publications from 22 centers/research groups that performed pleural lavage cytology during surgery for non-small-cell lung cancer and invited submission of individual patient data. Actuarial survival was calculated using Kaplan-Meier methods, and comparisons were performed using the log-rank test. Cox proportional hazards regression was used to ascertain the covariates associated with survival. RESULTS By January 1, 2008, submissions were received internationally from 11 centers with individual data from 8763 patients. In total, 511 (5.8%) patients had a positive pleural lavage cytology result, and this was shown to be an independent predictor of adverse survival associated with a hazard ratio of 1.465 (1.290-1.665; P < .001) compared with a reference hazard ratio of 1 for a negative result. On statistical modeling, the best adjustment for patients with a positive pleural lavage cytology result was a single increase in the T category assigned to the case, up to a maximum of T4. Correction for differences in survival were obtained in stages IB (P = .315) and IIB (P = .453), with a degree of correction in stage IIIA (P = .07). CONCLUSIONS Pleural lavage cytology should be considered in all patients with non-small-cell lung cancer suitable for resection. A positive result is an independent predictor of adverse survival, and the impact on survival suggests that it may be appropriate to upstage patients by 1 T category.

Analysis of cell cycle regulator proteins in non-small cell lung cancer

Background/Aims: Abnormalities of the proteins involved in cell cycle checkpoints are extremely common among almost all neoplasms. This study aimed to investigate the expression of four components of the cell cycle machinery—p21, p16, p53, and proliferating cell nuclear antigen (PCNA)—in non-small cell lung cancer (NSCLC). Methods: The expression of p21, p16, p53, and PCNA was examined in 68 well characterised NSCLC specimens using immunohistochemistry. The coregulation of these proteins and their influence on survival were analysed using both univariate and multivariate analyses. Results: By univariate analysis, the expression of all the proteins examined, except for PCNA, was significantly correlated with survival. In multivariate analysis, the only immunohistochemical parameter able to influence overall survival was p16, confirming the hypothesis that the RB–p16 tumour suppressor pathway is inactivated in most lung cancer samples. Finally, the group of patients with NSCLC who were negative for both p21 and p16 had a significantly shorter overall survival. Conclusions: These results suggest that loss of control of cell cycle checkpoints is a common occurrence in lung cancers, and support the idea that functional cooperation between different cell cycle inhibitor proteins constitutes another level of regulation in cell growth control and tumour suppression.

Prognostic significance of cyclooxygenase-2 (COX-2) and expression of cell cycle inhibitors p21 and p27 in human pleural malignant mesothelioma

Background: A study was undertaken to analyse the potential prognostic value of the immunohistochemical expression of cyclooxygenase-2 (COX-2) and p27 in 29 malignant mesotheliomas already screened for the expression of p21 and p53. Methods: Immunohistochemistry was used to determine the expression of COX-2 and p27. The correlation with survival of these factors and of p21 and p53 expression was assessed by univariate and multivariate analyses. Results: A positive statistically significant correlation was found between p27 and p21 expression (p<0.0001), but there was a negative correlation between COX-2 expression and both p27 (p = 0.001) and p21 (p<0.0001). No statistically significant correlation was recorded between p53 and all the other immunohistochemical parameters. Univariate analysis showed that overall survival was strongly influenced by p21, p27, and COX-2 expression, but multivariate Cox regression analysis showed that the only immunohistochemical parameter to influence overall survival of patients with mesothelioma was COX-2. Conclusions: These findings suggest that COX-2 expression may be a useful prognostic parameter for mesothelioma.

The serine protease HtrA1 is a novel prognostic factor for human mesothelioma

AIMS The objective of our study was to analyze the potential prognostic value of the expression of the serine protease HtrA1 and of EGFR in 70 malignant mesotheliomas. MATERIALS & METHODS Immunohistochemistry was used to determine the expression of HtrA1 and EGFR. Univariate and multivariate analyses were used to correlate expression of these molecular factors in combination with available clinicopathologic data to patient survival. RESULTS A positive, statistically significant relationship has been recorded between HtrA1 expression level and survival (p < 0.0001). By contrast, a negative relationship has been identified between EGFR expression and survival (p = 0.02). Moreover, extension of the tumor (T) and involvement of lymph nodes (N) advanced status (p = 0.001 and 0.002, respectively), as well as the sarcomatoid histotype (p = 0.005), correlated significantly with poor survival. Finally, by a multivariate Cox regression analysis, the only immunohistochemical parameter that resulted to influence overall survival was HtrA1 (p = 0.0001). Interestingly, the prognostic value of HtrA1 expression was completely independent from EGFR expression (p < 0.0001). CONCLUSION This is the first study of the relationship between HtrA1 expression and survival of mesothelioma patients. The data obtained strongly indicate the utilization of HtrA1 expression as a prognostic parameter for mesothelioma and suggest this serine protease as a possible molecular target for the treatment of malignant mesotheliomas.

Control of post-thoracotomy pain by transcutaneous electrical nerve stimulation: effect on serum cytokine levels, visual analogue scale, pulmonary function and medication

OBJECTIVES Transcutaneous electrical nerve stimulation (TENS) has been used to control post-thoracotomy pain with contrasting results. We aimed to assess the efficacy of TENS on post-thoracotomy pain in relation of four criterion measurements as: (i) cytokines; (ii) pain; (iii) respiratory function and (iv) intake of narcotic medication. METHODS Between January 2008 and October 2010, 58 patients underwent standard posterolateral thoracotomy for resectable lung cancer. Fifty patients were enrolled in the present study and randomized in two groups: TENS group (25 patients) who received postoperatively TENS for 5 days and placebo group (25 patients) without TENS. In both groups (i) serum cytokines (IL-6, IL-10, TNF-α) were measured by ELISA before surgery and at 6, 12, 24, 48, 72, 96 and 120 postoperative hours (POHs); (ii) at the same POHs, the pain score was measured using visual analogue scale (VAS) ranging from 0 to 10 levels; (iii) respiratory function (FEV 1% and FVC % of predicted value) were valuated on 72, 96 and 120 POHs; (iv) the total intake of narcotic medication given during postoperative period of 5 days was recorded. Repeated measures of analysis of variance assess the difference between two study groups. A value of P < 0.05 was considered statistically significant. RESULTS Of the 50 patients enrolled, two patients of TENS group and two patients of the placebo group were lost to follow-up. (i) Serum IL-6 (P = 0.001), IL-10 (P = 0.001) and TNF-α (P = 0.001) levels in TENS group were significantly lower than in the control group; (ii) VAS score in TENS group was significantly lower than in the control group (P < 0.001); (iii) recovery of FEV 1 (P = 0.02) and of FVC (P = 0.02) was statistically better in the TENS group than in control group; (iv) morphine requirement was lower in the TENS group with respect to placebo TENS (P = 0.004). After 48 POHs, no patient required supplementary dose of morphine. TENS group compared with placebo-group presented a significant reduction of non-opioid consumption (P = 0.002). CONCLUSIONS TENS is a valuable strategy to alleviate post-thoracotomy pain with reduction of cytokine production and of analgesic consumption, and with positive effects on pulmonary ventilation function.

Endobronchial treatment of giant emphysematous bullae with one-way valves: a new approach for surgically unfit patients

OBJECTIVE We aimed to evaluate the feasibility and short-term efficacy of endobronchial treatment with one-way valves for giant emphysematous bulla in surgically unfit patients. METHODS Nine consecutive patients with giant emphysematous bulla were enrolled in the last 3 years. Endobronchial valves were placed in the segmental bronchi to functionally isolate the airway that supplied the bulla, favouring the deflation of the bulla and its atelectasis. Mean value ± standard deviation of forced expiratory volume in 1s (FEV1), preoperative forced vital capacity (FVC) and residual volume (RV) were: 1.0 ± 0.2l (35 ± 9.9%), 1.5 ± 0.5l (42 ± 12%) and 5.5 ± 0.7 l (23 1 ± 32%), respectively; and the values for diffusion capacity for carbon monoxide was 31 ± 4.6% and for the 6-min walk test (6 MWT) was 156 ± 92 m); all patients required supplemental oxygen at rest. The St. Georges Respiratory Questionnaire (SGRQ) score was 85 ± 4.6. RESULTS At 24-48 h after the procedure, the mean value of FEV1 (from 35% to 47%, p < 0.01), FVC (from 42% to 52%, p < 0.01), diffusion lung capacity for carbon monoxide (DLCO) (from 31% to 33%, p < 0.05) and 6 MWT (from 156 m to 281 m, p < 0.01) significantly improved with respect to baseline value. Conversely, mean value of total lung capacity (TLC) (from 157% to 123%, p < 0.01) RV (from 231% to 158%, p < 0.01) and SGRQ score (from 85 to 37, p < 0.01) was significantly lower than baseline data; these changes were preserved during the entire follow-up. CONCLUSION Our preliminary data confirm the feasibility and the potential efficacy of this strategy with significantly immediate improvement of respiration and quality of life, which remains stable during 6 months of follow-up.

Lung mast cells are a source of secreted phospholipases A2

BACKGROUND Secreted phospholipases A(2) (sPLA(2)s) are released in plasma and other biologic fluids of patients with inflammatory, autoimmune, and allergic diseases. OBJECTIVE We sought to evaluate sPLA(2) activity in the bronchoalveolar lavage fluid (BALF) of asthmatic patients and to examine the expression and release of sPLA(2)s from primary human lung mast cells (HLMCs). METHODS sPLA(2) activity was measured in BALF and supernatants of either unstimulated or anti-IgE-activated HLMCs as hydrolysis of oleic acid from radiolabeled Escherichia coli membranes. Expression of sPLA(2)s was examined by using RT-PCR. The release of cysteinyl leukotriene (LT) C(4) was measured by means of enzyme immunoassay. RESULTS Phospholipase A(2) (PLA(2)) activity was higher in the BALF of asthmatic patients than in the control group. BALF PLA(2) activity was blocked by the sPLA(2) inhibitors dithiothreitol and Me-Indoxam but not by the cytosolic PLA(2) inhibitor AZ-1. HLMCs spontaneously released a PLA(2) activity that was increased on stimulation with anti-IgE. This PLA(2) activity was blocked by dithiothreitol and Me-Indoxam but not by AZ-1. HLMCs constitutively express mRNA for group IB, IIA, IID, IIE, IIF, III, V, X, XIIA, and XIIB sPLA(2)s. Anti-IgE did not modify the expression of sPLA(2)s. The cell-impermeable inhibitor Me-Indoxam significantly reduced (up to 40%) the production of LTC(4) from anti-IgE-stimulated HLMCs. CONCLUSIONS sPLA(2) activity is increased in the airways of asthmatic patients. HLMCs express multiple sPLA(2)s and release 1 or more of them when activated by anti-IgE. The sPLA(2)s released by mast cells contribute to LTC(4) production by acting in an autocrine fashion. Mast cells can be a source of sPLA(2)s in the airways of asthmatic patients.

SMO Gene Amplification and Activation of the Hedgehog Pathway as Novel Mechanisms of Resistance to Anti-Epidermal Growth Factor Receptor Drugs in Human Lung Cancer

Purpose: Resistance to tyrosine kinase inhibitors (TKI) of EGF receptor (EGFR) is often related to activation of other signaling pathways and evolution through a mesenchymal phenotype. Experimental Design: Because the Hedgehog (Hh) pathway has emerged as an important mediator of epithelial-to-mesenchymal transition (EMT), we studied the activation of Hh signaling in models of EGFR-TKIs intrinsic or acquired resistance from both EGFR-mutated and wild-type (WT) non–small cell lung cancer (NSCLC) cell lines. Results: Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-mutated HCC827-GR cells, we found SMO (the Hh receptor) gene amplification, MET activation, and the functional interaction of these two signaling pathways. In HCC827-GR cells, inhibition of SMO or downregulation of GLI1 (the most important Hh-induced transcription factor) expression in combination with MET inhibition exerted significant antitumor activity. In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation. In addition, the inhibition of SMO by the use of LDE225 sensitizes EGFR-WT NSCLC cells to standard chemotherapy. Conclusions:This result supports the role of the Hh pathway in mediating resistance to anti-EGFR-TKIs through the induction of EMT and suggests new opportunities to design new treatment strategies in lung cancer. Clin Cancer Res; 21(20); 4686–97. ©2015 AACR.

Primary cardiac T-cell lymphoma

Primary cardiac lymphoma (PCL), defined as a lymphoma clinically mimicking cardiac disease, with the bulk of the tumor located intrapericardially, is extremely rare in immunocompetent patients. Clinical manifestations vary depending on sites of involvement in the heart and include chest pain, arrhythmias, pericardial effusion, and heart failure. Diagnosis is often difficult and may require invasive procedures; in some cases, diagnosis is not made until autopsy. Histologically, nearly all cases of PCL reported thus far have been of B-cell origin. In this report, we describe a case of PCL of T-cell origin in an adult immunocompetent patient, the second reported in the literature to the best of our knowledge, and provide a brief overview of the features of previously published PCL cases.