Background: As many as 25% patients with a histological diagnosis of NSP at a thoracoscopic biopsy are eventually diagnosed with a malignancy, mostly mesothelioma, over the following months. Aim: to evaluate the value of pleural effusion levels of soluble mesothelin-related peptides (PE-SMRP) as a diagnostic tool in patients with histological diagnosis of NSP. Methods: All patients with an exudative pleural effusion and a histological diagnosis of NSP at thoracoscopic biopsies performed between Jenuary 2008 and December 2020 were followed for 18 months. A predefined cut-off level of 20 nM PE-SMRP, based on published data, was used to distinguish high- from low- PE-SMRP patients. A binary logistic regression was used to measure the relative risk of a later diagnosis of cancer. Results: Of the 185 patients with a diagnosis of NSP, 17 (9.2%) were diagnosed with cancer during follow-up (mean 6.38 months). A malignancy was diagnosed in 9 of the 15 high-PE-SMRP patients (60%), and in 8 of the 170 low-PE-SMRP patients (4.7%) (p<0.0001). High-PE-SMRP patients had a higher risk of being diagnosed with mesothelioma at follow-up (OR 31.2, 95% IC 8.5-114.7) (table 1).). Each nM increase in PE-SMRP increased the risk of a final diagnosis of mesothelioma by 1.07-fold (p<0.001). Conclusions: Patients with high levels of PE-SMRP are at increased risk of cancer after an initial histological diagnosis of NSP and might benefit from a closer follow-up.

Literature reports suggest that the host immune system may control Malignant Pleural Mesothelioma (MPM) growth, although its activity is limited by regulatory mechanisms. In this retrospective study, we analyzed the levels of pro-inflammatory (IL-1, IL-6, TNF), immune-regulatory (IL-10) and Th1/CTL-related cytokines (IL-12p70, IFN-γ) in the pleural exudate and their relationship with overall survival (OS) in MPM. Cytokines were quantified by multiplexed immunoassay. Concentrations were dichotomized with respect to the median value. Correlation between cytokine level and OS was assessed using univariate (Kaplan–Meier curves) and multivariate (Cox regression) analyses. Regarding outcome, tumor histology, therapies undergone and IFN-γ were independent prognostic factors of OS in a 72 MPM training cohort. Notably, high concentrations of IFN-γ halved death probability (HR of high vs low IFN-γ concentration = 0.491, 95%CI 0.3–0.8, p = 0.007). Also in patients with epithelioid histology and those receiving at least one line of therapy, high IFN-γ level was an independent factor predictive of OS (HR of high vs low IFN-γ concentration were 0.497, p = 0.007 and 0.324, p = 0.006, respectively). However, these data were not confirmed in a 77 MPM validation cohort, possibly due to the low IFN-γ levels encountered in this population, and the heterogeneous distribution of disease stages between the training and the validation cohorts. None of the other cytokines showed any effect on survival. High level of IFN-γ in pleural effusion may be associated with better survival in MPM patients and potentially serve as a prognostic biomarker. Larger prospective studies are needed to ascertain this hypothesis.

A soluble mesothelin-related peptide (SMRP) is the only FDA-approved biomarker for diagnosis of pleural mesothelioma (PM) and the most used for monitoring treatment. Radiological assessment of PM, based on modified RECIST (mRECIST) criteria, is challenging. This pilot study was designed to evaluate whether SMRP levels correlated over time with mRECIST score. Serial serum samples from PM patients were collected and SMRP levels were measured and compared with the mRECIST score obtained through centralized CT scans by blinded review. The within-patient SMRP-mRECIST relationship over time was estimated through a normal random-effects regression approach applied to the log-transformed mRECIST score. Overall, 58 PM patients were included (46 males and 12 females) with a median age at diagnosis of 67 years (min–max = 48–79), 44 (76%) with epithelioid and 14 (24%) with non-epithelioid histology. The total number of SMRP measurements and CT scans considered for analysis was 183. There was a statistically significant correlation between SMRP and mRECIST score in the 2 cohorts considered both separately and jointly. These results, although exploratory, suggest that SMRP measurement might be considered as an adjunct to monitor PM patients in order to delay CT scans time interval, thus warranting further investigation.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor survival rates. Therefore, it is essential to have effective biological markers predicting the course of the disease and prognosis. The aim of the present study was to highlight the prognostic significance of serum soluble mesothelin-related protein (Se-SMRP) in patients with MPM at diagnosis. Se-SMRP was determined in 60 patients using an ELISA commercial kit. Se-SMRP levels were subdivided into three tertile-based categories and in each category overall survival (OS) indexes were determined using the Kaplan-Meier and Cox regression analyses. The association between Se-SMRP levels and OS was also assessed by restricted cubic spline (RCS) analysis. No notable differences in the Kaplan-Meier probabilities were identified across the Se-SMRP categories (<0.66 nM, 0.66-1.46 nM, >1.46 nM) although an upward trend in death rate ratios (RR) was pointed out by comparing the higher (RR=1.95) and intermediate (RR=1.86) categories with the lower category (RR=1.00). In addition, such an increasing tendency, particularly when the biomarker exceeded 1.0 nM, was confirmed by an RCS function of Se-SMPR levels fitted to survival data using the Cox regression equation. The present study provided evidence in favor of a prognostic value of Se-SMRP in patients with MPM.

Programmed death-ligand 1 (PD-L1) protein plays a central role in the antitumor immune response, and appears to be a predictor of prognosis and efficacy for PD-L1 and programmed death 1 (PD-1) blockade therapy. The immunoregulatory role and prognostic impact of PD-L1 soluble form (sPD-L1) have been investigated in biological fluids of patients with different tumors. In malignant pleural mesothelioma (MPM), circulating sPD-L1 has been recently reported in patients’ sera, but no data are available in pleural effusions (PE). In our study, we evaluated the baseline expression levels of sPD-L1 in PE from 84 MPM patients and correlated them with PD-L1-status in matched tumors and patients’ overall survival (OS). sPD-L1 in PE was determined by ELISA and tumor PD-L1 by immunohistochemistry. Association of sPD-L1 with OS was estimated using the Cox regression model. We observed that sPD-L1 was variably expressed in all the PE and tended to be higher (by 30%) in patients with PD-L1-positive tumors (cut-off ≥ 1% stained cells) as compared to patients with PD-L1-negative tumors (geometric mean ratio = 1.28, P value = 0.288). sPD-L1 levels were significantly higher than those of sPD-1 (P value = 0.001) regardless of the MPM histotypes and they were positively correlated (r = 0.50, P value < 0.001). Moreover, high PE sPD-L1 concentrations were associated with a trend towards increased OS (hazard ratio 0.79, 95% CL 0.62–1.01, P value = 0.062). Our study documents the presence of sPD-L1 in PE of MPM patients, and suggests its possible biological and prognostic role in MPM.

The evaluation of progression in epithelioid malignant pleural mesothelioma (MPM) is currently performed by monitoring the tumor mass variation by radiological images.1Armato 3rd, S.G. Nowak A.K. Francis R.J. et al.Observer variability in mesothelioma tumor thickness measurements: defining minimally measurable lesions.J Thorac Oncol. 2014; 9: 1187-1194Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar In our previous letter of August 2018 in the Journal of Thoracic Oncology, we reported a case of MPM in which we observed a strong positive correlation between the levels of serum soluble mesothelin-related peptides (Se-SMRPs) and the thickness of the pleura measured by computed tomography (CT) scans.2Vigani A. Pistillo M.P. Giannoni U. et al.Use of serum mesothelin as an indicator of tumor progression in routine clinical practice of malignant pleural mesothelioma.J Thorac Oncol. 2018; 8: e143-e145Google Scholar, 3Robinson B.W. Creaney J. Lake R. et al.Mesothelin-family proteins and diagnosis of mesothelioma.Lancet. 2003; 362: 1612-1616Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 4Hollevoet K. Nackaerts K. Gosselin R. et al.Soluble mesothelin, megakaryocyte potentiating factor, and osteopontin as markers of patient response and outcome in mesothelioma.J Thorac Oncol. 2011; 6: 1930-1937Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Thus, we suggested that Se-SMRP could be a useful marker to evaluate tumor progression in MPM. In the present letter, we have extended our study to other patients and confirmed the Se-SMRP/CT correlation by means of appropriate statistical analyses. We analyzed 10 MPM patients (6 epithelioid, 3 sarcomatoid, and 1 biphasic; 9 male; median age: 67.5 years) recruited at the Oncology Division, ASL5 La Spezia (Italy), between March 2011 and May 2017. Pleural effusion (PE) was present in eight patients at diagnosis whereas in two patients it appeared during the course of the disease. Three MPM patients received first-line therapy (pemetrexed plus cisplatin) and seven also had second-line therapy (pemetrexed plus cisplatin), no patient had extrapleural pneumonectomy, pleurectomy decortication, or treatment with hemithoracic radiotherapy. Se-SMRP levels and pleural thickness showed very similar trends during the follow-up period. However, this overlap was particularly accentuated in seven cases (Fig. 1A, cases 1-7). In contrast, in three cases, the trends diverged at certain time points, in particular those at which patients showed a strong increase in PE (case 8) or appearance of a PE not present at the diagnosis (cases 9 and 10) (data not shown). The association between Se-SMRP and pleural thickness was confirmed by a mixed-effects regression analysis which is able to properly address the within-patient correlation due to the repeated measurements (log-transformed) on the same subject over time.5Fitzmaurice G.M. Laird N.M. Ware J.H. Applied Longitudinal Analysis. Somerset. John Wiley & Sons, New Jersey2004Google Scholar After adjusting for age and disease stage at diagnosis, we observed a statistically significant (geometric) mean increase in pleural thickness of 110% (95% confidence interval [CI]: 16%–280%, p value = 0.014) per unit increase in log-SMRP (Fig. 1B). In conclusion, this study confirms our previous finding of a strong positive association between Se-SMRP and CT. Although CT provides direct information by images on the variation of MPM tumor volume and onset of new metastases, it cannot always be performed.1Armato 3rd, S.G. Nowak A.K. Francis R.J. et al.Observer variability in mesothelioma tumor thickness measurements: defining minimally measurable lesions.J Thorac Oncol. 2014; 9: 1187-1194Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar In contrast, testing Se-SMRP provides faster information and it can be performed together with other routine blood tests at each patient's visit. However, we must keep in mind the limitation of a Se-SMRP test, that is, the possibility that Se-SMRP levels could be influenced by the development of a new PE or by its volume variation. Se-SMRP is detected by the enzyme-linked immunosorbent assay that is a simple and rapid technique which can be performed on a single test, without requiring sophisticated equipment and high costs. Finally, we are strongly led to believe that the determination of Se-SMRP levels can be performed routinely and integrated to CT for monitoring the progression of MPM. This work was supported by grants from AIL (Sezione Francesca Lanzone) and Italian Ministry of Health (5 × 1000 funds 2015 to Dr. Fonana and Dr. Pistillo), Italy. The study was approved by the Liguria Region Ethics Committee (P.R. 207REG2014) and written informed consent was obtained from all patients.

Aim: This study evaluated the prognostic value of soluble mesothelin-related protein (SMRP) levels in pleural effusions (PE) from patients with pleural mesothelioma (MPM). Patients and Methods: SMRP level in PE was tested using an enzyme-linked immunosorbent assay (ELISA) in 109 patients with MPM at diagnosis before any treatment. The Kaplan–Meier method and the Cox regression were applied to compare overall survival probabilities across tertile categories of SMRP level. Results: No significant differences in Kaplan–Meier overall survival probabilities among the SMRP categories were found. A statistically non-significant trend for increased death rate ratio (RR) was computed (p=0.327) when the higher (>46.5 nM, RR=1.38) and intermediate (8.5-46.5 nM, RR=1.18) SMRP categories were compared to the lower category (<8.5 nM, RR=1.00). Cox regression modelling including a restricted cubic spline showed a moderately rising non-linear trend in death rate. Conclusion: The SMRP level in PE does not appear to have prognostic significance and its detection is not recommended in routine clinical management of patients with MPM.

Malignant pleural mesothelioma (MPM) is a particularly aggressive tumor; it is asbestos related, arising from mesothelial cells of the pleura. In MPM conventional therapies are often ineffective, and early estimation of tumor progression acquires great clinical importance.1Opitz I. Friess M. Kestenholz P. et al.A new prognostic score supporting treatment allocation for multimodality therapy for malignant pleural mesothelioma: a review of 12 years’ experience.J Thorac Oncol. 2015; 10: 1634-1641Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Generally, progression of the tumor is evaluated by using the radiological criteria of the Response Evaluation Criteria in Solid Tumors, which are based on linear measurements of target and nontarget lesions. However, the application of these criteria in MPM is more difficult than with other cancers because MPM develops as a diffuse or circumferential irregular pleural thickening.2Armato 3rd, S.G. Nowak A.K. Francis R.J. Kocherginsky M. Byrne M.J. Observer variability in mesothelioma tumor thickness measurements: defining minimally measurable lesions.J Thorac Oncol. 2014; 9: 1187-1194Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar The evaluation of tumor progression in MPM is currently performed by taking into account the thickness of the pleura, lymph nodes, and metastatic lesions. Soluble mesothelin-related peptides (SMRPs), which are expressed by normal mesothelial cells and overexpressed in MPM, can be secreted and detected in a patient’s serum (Se-SMRPs).3Robinson B.W. Creaney J. Lake R. et al.Mesothelin-family proteins and diagnosis of mesothelioma.Lancet. 2003; 362: 1612-1616Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar Se-SMRP levels have been found to be significantly increased in about 80% of patients with MPM,3Robinson B.W. Creaney J. Lake R. et al.Mesothelin-family proteins and diagnosis of mesothelioma.Lancet. 2003; 362: 1612-1616Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 4Grigoriu B.D. Chahine B. Vachani A. et al.Kinetics of soluble mesothelin in patients with malignant pleural mesothelioma during treatment.Am J Respir Crit Care Med. 2009; 179: 950-954Crossref PubMed Scopus (60) Google Scholar, 5Creaney J. Francis R.J. Dick I.M. et al.Serum soluble mesothelin concentrations in malignant pleural mesothelioma: relationship to tumor volume, clinical stage and changes in tumor burden.Clin Cancer Res. 2011; 17: 1181-1189Crossref PubMed Scopus (86) Google Scholar, 6Hollevoet K. Nackaerts K. Gosselin R. et al.Soluble mesothelin, megakaryocyte potentiating factor, and osteopontin as markers of patient response and outcome in mesothelioma.J Thorac Oncol. 2011; 6: 1930-1937Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar have been associated with a worse prognosis,4Grigoriu B.D. Chahine B. Vachani A. et al.Kinetics of soluble mesothelin in patients with malignant pleural mesothelioma during treatment.Am J Respir Crit Care Med. 2009; 179: 950-954Crossref PubMed Scopus (60) Google Scholar, 5Creaney J. Francis R.J. Dick I.M. et al.Serum soluble mesothelin concentrations in malignant pleural mesothelioma: relationship to tumor volume, clinical stage and changes in tumor burden.Clin Cancer Res. 2011; 17: 1181-1189Crossref PubMed Scopus (86) Google Scholar, 6Hollevoet K. Nackaerts K. Gosselin R. et al.Soluble mesothelin, megakaryocyte potentiating factor, and osteopontin as markers of patient response and outcome in mesothelioma.J Thorac Oncol. 2011; 6: 1930-1937Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar and have been approved by the U.S. Food and Drug Administration as a marker for the diagnosis and monitoring of epithelioid MPM. The possibility of using Se-SMRP detection to evaluate the tumor progression in MPM has been suggested by Robinson and et al. since 2003.3Robinson B.W. Creaney J. Lake R. et al.Mesothelin-family proteins and diagnosis of mesothelioma.Lancet. 2003; 362: 1612-1616Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar Indeed, they found that Se-SMRP levels correlate with the tumor size, increasing during tumor progression and remaining constant in stable disease. Although all these findings have been confirmed by other reports,4Grigoriu B.D. Chahine B. Vachani A. et al.Kinetics of soluble mesothelin in patients with malignant pleural mesothelioma during treatment.Am J Respir Crit Care Med. 2009; 179: 950-954Crossref PubMed Scopus (60) Google Scholar, 5Creaney J. Francis R.J. Dick I.M. et al.Serum soluble mesothelin concentrations in malignant pleural mesothelioma: relationship to tumor volume, clinical stage and changes in tumor burden.Clin Cancer Res. 2011; 17: 1181-1189Crossref PubMed Scopus (86) Google Scholar, 6Hollevoet K. Nackaerts K. Gosselin R. et al.Soluble mesothelin, megakaryocyte potentiating factor, and osteopontin as markers of patient response and outcome in mesothelioma.J Thorac Oncol. 2011; 6: 1930-1937Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Se-SMRP detection has not yet entered routine clinical practice in many centers. In this letter, we provide an example of the routine application of Se-SMRPs in the evaluation of MPM progression. In particular, we report a correlation between Se-SMRP levels and computed tomography (CT) findings in a patient with epithelioid MPM. In August 2013, a 67-year-old man with history of asbestos exposure presented to our hospital with dyspnea and cough. Chest CT showed a diffuse irregular thickening of the right costal pleura associated with a metastatic subcarinal lymph node. Histopathological examination confirmed that the patient had epithelioid MPM (Union for International Cancer Control stage II and Eastern Cooperative Oncology Group performances status 1), which was treated with six cycles of cisplatin and pemetrexed (for 4 months). He died about 16 months after starting therapy. Before therapy, his Se-SMRP levels, the thickness of his pleura and the length of the short axis of his subcarinal lymph node were 1.23 nM (Fig. 1A), 1.9 mm, and 5.2 mm (Fig. 1B), respectively. In the subsequent 3 months, the patient’s Se-SMRP levels decreased slightly, reaching a minimum of 0.50 nM (see Fig. 1A), after which they continued increasing until his death. At the fifth month, his Se-SMRP level was 0.96 nM, the pleural thickness remained constant, and the subcarinal lymph node appeared further enlarged (the length of the short axis measured 9.3 mm). Fifteen days before the patient’s death, his Se-SMRP levels reached 7.71 nM and the thickness of the pleura and the length of the short axis of the subcarinal lymph node measured 9.4 mm and 20.5 mm, respectively (see Fig. 1A and B). We observed a strong positive correlation between the patient’s Se-SMRP levels and the radiological extent of his disease. In particular, by using the Spearman coefficient, we estimated a correlation of about 0.95 and 0.80 with the pleural thickness and length of the short axis of the subcarinal lymph node, respectively. This case report suggests that Se-SMRP level can be a useful marker to evaluate tumor progression, particularly when CT has not yet been performed or even to indicate that CT should be performed. In conclusion, detection of Se-SMRP provides useful information for management of patients with MPM. Se-SMRP is a practical marker that can be easily and quickly measured even for a single patient during routine checks. Thus, we believe that Se-SMRP level could be integrated with CT images for monitoring the progression of MPM. This study was approved by the Ethics Committee of the Liguria Region (P.R. 207REG2014).

BACKGROUND:In the literature, there exist conflicting data on the value of fibulin-3 (FBLN3) for the diagnosis of pleural effusion (PE) in malignant pleural mesothelioma (MPM). Therefore we compared the diagnostic performance of FBLN3 against that of soluble mesothelin-related peptide (SMRP) in a cohort of Italian patients.MATERIALS AND METHODS:FBLN3 and SMRP were detected in PE from 33 patients with MPM, 64 with pleural benign lesions and 23 with non-MPM pleural metastases using a commercial enzyme-linked-immunosorbent(ELISA)-assay kit according to manufacturers' instructions.RESULTS:Levels of FBLN3 were similar in PE from MPM and PE from other pathologies (geometric mean=68.1 vs. 66.2 ng/ml; p=0.872) in contrast to SMRP levels, which were significantly higher in PE from MPM (geometric mean=14.6 vs. 3.2 nM; p<0.001). Receiver operating characteristic analysis confirmed that SMRP showed a good performance (area under the curve=0.79, p<0.001), whereas FBLN3 was not able to discriminate MPM from other pathologies (area under the curve=0.44, p=0.838).CONCLUSION:FBLN3 detection in PE, in contrast to SMRP detection, is not useful as a biomarker for the diagnosis of PE from MPM.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal overall survival (OS) and to date no molecular markers are available to guide patient management. This study aimed to identify a prognostic miRNA signature in MPM patients who did not undergo tumor resection. Whole miRNA profiling using a microarray platform was performed using biopsies on 27 unresected MPM patients with distinct clinical outcome: 15 patients had short survival (OS<12 months) and 12 patients had long survival (OS>36 months). Three prognostic miRNAs (mir-99a, let-7c, and miR-125b) encoded at the same cluster (21q21) were selected for further validation and tested on publicly available miRNA sequencing data from 72 MPM patients with survival data. A risk model was built based on these 3 miRNAs that was validated by quantitative PCR in an independent set of 30 MPM patients. High-risk patients had shorter median OS (7.6 months) as compared with low-risk patients (median not reached). In the multivariate Cox model, a high-risk score was independently associated with shorter OS (HR=3.14; 95% CI, 1.18-8.34; P=0.022). Our study identified that the downregulation of the miR-99a/let-7/miR-125b miRNA cluster predicts poor outcome in unresected MPM.

Malignant mesothelioma (MM) is an aggressive tumor, with poor prognosis and limited possibility of treatment. MMNG HOS Transforming gene (MET) is a proto-oncogene located in the 7q31 that encodes the high-affinity receptor for hepatocyte growth factor (HGF). MET tyrosine-kinase was recently proposed for a targeted therapy and clinical trials are in progress in many tumors. MET amplification identifies a subgroup of patients potentially able to respond to HGF/MET inhibitors and may represent an element of resistance for anti-EGFR inhibitor therapy. The aim of this study was to evaluate MET amplification and expression in MM. The protocol of this study was approved by the Liguria Region Ethics Committee (P.R. 207REG2014) and the written informed consent was obtained from all the patients. We analyzed 109 MM (67 male; 65 epithelioid, 26 sarcomatoid, 14 biphasic, 2 desmoplastic, 2 papillary). Seventy-nine MM were from a tissue microarray (MS801 and MS 1001, US Biomax Inc, Rockville, MD, USA), 12 cases of formalin-fixed paraffin-embedded tissues were from, IRCCS AOU San Martino-IST (Genova) and 18 tissues from ASL N°5 (La Spezia). MET gene amplification was investigated by FISH using MET/CEP7 probe cocktail (Vysis MET Spectrum Red FISH Probe Kit reagent/Vysis CEP 7 (D7Z1) SpectrumGreen Probe, both reagents from Abbott Molecular, Des Plaines, IL USA). Immunohistochemistry was performed by Anti-c-Met Antibody IHC-plus™ LS-B2812 (LSBio, Seattle, WA). By using the UCCC-scored system we found one epithelioid MET amplification (MET to CEP7 ratio ≥2 or at least 15 copies of MET signals in ≥10% of the tumor cells). In contrast, 8/109 (7.3%) MM (6 epithelioid, 1 sarcomatoid, 1 biphasic) showed high MET polysomy (according to mean ≥4 copies/cells in ≥40% of tumor cells) in a range of 4-10 spots of MET gene in about 60-80% of tumor cells (Table 1). Immunohistochemistry showed that amplification was associated with moderate expression of MET protein in cytoplasm and membrane of MM cells. In contrast, high gene polysomy resulted always associated with low staining of MET protein. Amplification and high polysomy of MET, associated with c-MET receptor expression may be present in MM. These preliminary observations might represent the basis for designing new clinical trials assessing MET targeting agents in MM. Moreover, the possibility of MET amplification should be considered before starting MM patient treatment with the anti-EGFR targeted inhibitors.

Background:MPM is an aggressive cancer showing a high mortality due to ineffective therapies. Prognostic biomarkers such as soluble mesothelin (SM) may play a role in treatment choice and clinical management of patients. Aim: we evaluated the prognostic role of SM levels in serum (S-SM) and pleural effusion (P-SM) at diagnosis on overall survival (OS) of MPM patients. Methods: we studied 109 MPM at diagnosis. SM was measured by the “MesoMark” kit. The level of P-SM was determined in all patients while S-SM only in 43 (39.5%) cases. OS probabilities were estimated using the Kaplan-Meier method within three categories of S-/P-SM obtained according to tertiles of their distributions. The role of each biomarker on OS was assessed through the Cox regression and expressed as death rate ratio (HR), adjusted for gender, age, stage and histotype. Likelihood ratio test was used to evaluate statistical significance. Results: the median follow-up for all patients was 13.7 months with median OS time=14.4 months (95% CL=11.8-17.5). According to tertiles, the nM categories <8.5, 8.5-46.7, >46.7 of P-SM did not show differences in OS. In contrast, considering the nM categories <0.66, 0.66-1.46, >1.46 of S-SM pointed out a continuous increase of HR value (HR=1 vs HR=1.85 vs HR=2.95). Moreover, although no statistical significance was found, by the Cox modelling, we established a remarkable association between log-scaled S-SM and death rate, with a steep increase for S-SM levels >0.51 (HR=1.50, 95%CL=0.54-4.16). (Fig.1,2) Conclusions: S-SM levels>0.51 at diagnosis appear to have a prognostic significance whereas P-SM levels did not seem to influence OS.

CTLA-4 function as a negative regulator of T cell-mediated immune response is well established, whereas much less is known about the immunoregulatory role of its soluble isoform (sCTLA-4). No data are available on CTLA-4 expression and prognostic impact in malignant pleural mesothelioma (MPM). We investigated, by immunohistochemistry, CTLA-4 expression in tumor tissues and, by ELISA, sCTLA-4 levels in sera and matched pleural effusions from 45 MPM patients. Prognostic effect of CTLA-4 expression on overall survival (OS) was assessed through Cox regression and prognostic significance expressed as death rate ratio (HR). We found that 56.0 % of MPM tissues expressed CTLA-4 with variable intensity and percentage of positive cells estimated by the immunoreactive score. sCTLA-4 levels were significantly higher in sera (S-sCTLA-4) than in pleural effusions (PE-sCTLA-4) (geometric mean ratio = 2.70, P value = 0.020). CTLA-4 expression at the tissue level was higher in the epithelioid histological subtype than in the sarcomatoid, whereas at the serum level, it was higher in the sarcomatoid subtype. A homogeneous favorable prognostic effect was found for CTLA-4 overexpression in tissue, serum and pleural effusion. Interestingly, only the PE-sCTLA-4 was found to be a statistically significant positive prognostic factor (HR = 0.37, 95 % CI = 0.18–0.77, P value = 0.007). Indeed, PE-sCTLA-4 correlated with CTLA-4 expression in tissues, whereas this latter expression showed a weak association with OS. To confirm our findings, further experimental evidences obtained from a larger cohort of MPM patients are required. However, our results would indicate a positive correlation of PE-sCTLA-4 levels and OS in MPM patients.

In our October 2015 letter in the Journal1Varesano S. Salvi S. Boccardo S. et al.Amplification of MET in a patient with malignant pleural mesothelioma.J Thorac Oncol. 2015; 10: e103-e104Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar we reported a patient with malignant mesothelioma (MM) with amplification of the MET gene associated with MET receptor expression. This finding suggests that the inhibition of MET might be used as a targeted therapy also in selected patients with MM.2Kawakami H. Okamoto I. Okamoto W. et al.Targeting MET amplification as a new oncogenic driver.Cancers (Basel). 2014; 22: 1540-1552Crossref Google Scholar We now report a MET copy number analysis in patients with MM scored by the Union for International Cancer Control criteria proposed for stratification of non–small cell lung cancer according to the EGFR fluorescence in situ hybridization (FISH) assay and also used by Go et al.3Go H. Jeon Y.K. Park H.J. et al.High MET gene copy number leads to shorter survival in patients with non-small cell lung cancer.J Thorac Oncol. 2010; 5: 305-313Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar for the scoring of MET. MET status was classified as FISH-positive and -negative according to the frequency of MM cells with specific copy numbers of the MET gene and chromosome 7 centromere (CEP7).3Go H. Jeon Y.K. Park H.J. et al.High MET gene copy number leads to shorter survival in patients with non-small cell lung cancer.J Thorac Oncol. 2010; 5: 305-313Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar This study was approved by the Liguria Region Ethics Committee, and written informed consent was obtained from all patients. We analyzed 60 patients with MM (male, 66.7%; median age, 60.0 years [range, 5–85 years]), including patients with epithelioid (n = 36), sarcomatoid (n = 12), biphasic (n = 8), desmoplastic (n = 2), and papillary (n = 2) subtypes. Thirty cases of MM were from a tissue microarray (MS801; US Biomax Inc, Rockville, MD), 12 cases of formalin-fixed paraffin-embedded tissues were from the Unit of Pathology, IRCCS A.O.U. San Martino–IST (Genova, Italy), and 18 cases were from the Division of Histopathology (ASL5, La Spezia, Italy). We found 5 FISH-positive cases (8.3%), of which one epithelioid MM had MET amplification (about 8 MET signals on >70% of cells; MET/CEP7 ratio = 4.0; Fig. 1A) and four epithelioid MMs showed high polysomy of MET (range of 4–10 spots of MET in about 60%–80% of MM cells; a representative case is shown in Fig. 1D). All the other 55 FISH-negative cases (91.7%) were disomic for MET (a representative case is shown in Fig. 1G). As in the previously reported case, IHC analysis showed that amplification was associated with moderate expression of MET protein in cytoplasm and membrane of MM cells (Fig. 1B). In contrast, high gene polysomy resulted in low staining of MET protein (Fig. 1E). In our study, we found that MET amplification is a rare event in patients with MM (1.7% of total cases) in contrast to MET polysomy, which occurs more frequently (6.7% of total cases). The biological impact of MET polysomy on cancer cells has not been well established. However, highly polysomic status in MM might be predictive for targeted therapy. Indeed, Catenacci et al.4Catenacci D.V. Henderson L. Xiao S.Y. et al.Durable complete response of metastatic gastric cancer with anti-Met therapy followed by resistance at recurrence.Cancer Discov. 2011; 1: 573-579Crossref PubMed Scopus (103) Google Scholar reported a case of durable complete response in metastatic gastric cancer treated with anti–MET-TKI receptor monoclonal antibody (Onartuzumab MetMAb) in which the primary tumor had high MET polysomy.4Catenacci D.V. Henderson L. Xiao S.Y. et al.Durable complete response of metastatic gastric cancer with anti-Met therapy followed by resistance at recurrence.Cancer Discov. 2011; 1: 573-579Crossref PubMed Scopus (103) Google Scholar Until today, the use of biological therapies in patients with MM, such as anti-MET drugs, was performed only in vitro—for instance, by inhibiting the MET receptor with PHA-665752 or with Perifosine (Keryx Biopharmaceuticals, New York, NY), which directly inhibits the EGFR/MET–AKT axis.5Smolen G.A. Sordella R. Muir B. et al.Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.Proc Natl Acad Sci U S A. 2006; 103: 2316-2321Crossref PubMed Scopus (442) Google Scholar, 6Pinton G. Manente A.G. Angeli G. et al.Perifosine as a potential novel anti-cancer agent inhibits EGFR/MET-AKT axis in malignant pleural mesothelioma.PLoS One. 2012; 7: e36856Crossref PubMed Scopus (35) Google Scholar Therefore, the presence of MET gene amplification and polysomy associated with MET receptor overexpression reinforces new possibilities of treatments also in vivo. However, whether patients with MM could benefit from MET-targeted therapies remains to be established in clinical trials. EGFR Status in Mesothelioma: Possible Implications for the Efficacy of Anti-EGFR and Anti-MET TherapiesJournal of Thoracic OncologyVol. 11Issue 6PreviewDespite the current conventional multimodal treatments, to date there are no effective therapies for malignant mesothelioma (MM), and to improve survival, novel therapeutic strategies are required. New hope for a cure might arise from the biological anticancer targeted therapy against the receptor tyrosine kinases that may be activated after chromosome aberration. In our previous letter of February 2016 in the Journal of Thoracic Oncology, we reported the analysis, by fluorescence in situ hybridization, of MNNG HOS Transforming gene (MET) copy number showing high polysomy in 6.7% of patients with MM accompanied by staining of the protein to mesenchymal-epithelial transition factor (MET) in the cytoplasm and membrane of tumor cells. Full-Text PDF Open Archive

Despite the current conventional multimodal treatments, to date there are no effective therapies for malignant mesothelioma (MM), and to improve survival, novel therapeutic strategies are required. New hope for a cure might arise from the biological anticancer targeted therapy against the receptor tyrosine kinases that may be activated after chromosome aberration. In our previous letter of February 2016 in the Journal of Thoracic Oncology, we reported the analysis, by fluorescence in situ hybridization, of MNNG HOS Transforming gene (MET) copy number showing high polysomy in 6.7% of patients with MM accompanied by staining of the protein to mesenchymal-epithelial transition factor (MET) in the cytoplasm and membrane of tumor cells.1Varesano S. Salvi S. Boccardo S. et al.MET gene status in malignant mesothelioma by fluorescent in situ hybridization.J Thorac Oncol. 2016; 11: e28-e30Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar We concluded that those patients might be candidates for treatment with MET tyrosine kinase inhibitors (TKIs).1Varesano S. Salvi S. Boccardo S. et al.MET gene status in malignant mesothelioma by fluorescent in situ hybridization.J Thorac Oncol. 2016; 11: e28-e30Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar In this study, we have evaluated, by fluorescence in situ hybridization, the epidermal growth factor receptor (EGFR) gene copy number status on the same set of previously obtained paraffin-embedded MM tissue samples.1Varesano S. Salvi S. Boccardo S. et al.MET gene status in malignant mesothelioma by fluorescent in situ hybridization.J Thorac Oncol. 2016; 11: e28-e30Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar EGFR is implicated in the development of many cancers, including MM. Indeed, EGFR dysregulation plays a role in tumor differentiation, tumor proliferation, cell migration, and survival. Currently, EGFR is a key gene for targeted therapies with TKIs such as gefitinib and erlotinib in many tumors comprising NSCLC. As previously reported, this study was also approved by the Liguria Region Ethics Committee, and written informed consent was obtained from all patients.1Varesano S. Salvi S. Boccardo S. et al.MET gene status in malignant mesothelioma by fluorescent in situ hybridization.J Thorac Oncol. 2016; 11: e28-e30Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Using the University Colorado Cancer Center–scored system,2Varella-Garcia M. Diebold J. Eberhard D.A. et al.EGFR fluorescence in situ hybridization assay: guidelines for application to non-small-cell lung cancer.J Clin Pathol. 2009; 62: 970-977Crossref PubMed Scopus (107) Google Scholar we did not find any EGFR amplification (EGFR-to-CEP7 ratio ≥ 2 or ≥15 copies of EGFR signals in at least 10% of the tumor cells2Varella-Garcia M. Diebold J. Eberhard D.A. et al.EGFR fluorescence in situ hybridization assay: guidelines for application to non-small-cell lung cancer.J Clin Pathol. 2009; 62: 970-977Crossref PubMed Scopus (107) Google Scholar). In contrast, four of 60 MMs (6.7%), all epithelioid, showed high EGFR polysomy (mean of ≥4 copies/cell in ≥40% of tumor cells2Varella-Garcia M. Diebold J. Eberhard D.A. et al.EGFR fluorescence in situ hybridization assay: guidelines for application to non-small-cell lung cancer.J Clin Pathol. 2009; 62: 970-977Crossref PubMed Scopus (107) Google Scholar) in a range of four to 10 spots of EGFR gene in approximately 60% to 80% of tumor cells (Fig. 1A). All EGFR-positive MM cells showed also overexpression of membrane and cytoplasmic EGFR protein (Fig. 1B). Therefore, these patients might be treated with EGFR TKIs.3Toffalorio F. de Marinis F. Conforti F. et al.Erlotinib efficacy in NSCLC patients with high polysomy of chromosome 7 and EGFR/KRas wild-type tumors.J Thorac Oncol. 2015; 10: 392-396Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar EGFR gene maps on the 7p11-12 locus of chromosome 7. On 7q21-31 locus of chromosome 7 there is located also the MET gene and, as expected, all the four EGFR high polysomy MM cases observed were the same that we had previously reported to show MET gene high polysomy.1Varesano S. Salvi S. Boccardo S. et al.MET gene status in malignant mesothelioma by fluorescent in situ hybridization.J Thorac Oncol. 2016; 11: e28-e30Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar We believe that the simultaneous high EGFR and MET polysomy, which is associated with the coexpression of related protein receptors, might have implications for the efficacy of anti-EGFR and anti-MET TKI monotherapies. Our hypothesis is supported by reported evidence that, in cell lines, simultaneous activation of both EGFR and MET may allow MM cells to acquire resistance against single-agent TKIs, including PHA-665752 and crizotinib.4Brevet M. Shimizu S. Bott M.J. et al.Coactivation of receptor tyrosine kinases in malignant mesothelioma as a rationale for combination targeted therapy.J Thorac Oncol. 2011; 6: 864-874Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Moreover, in other tumors such as lung adenocarcinoma with EGFR mutation, MET FISH-positive status predicts poor clinical outcome after TKI gefitinib treatment,5Noro R. Seike M. Zou F. et al.MET FISH-positive status predicts short progression-free survival and overall survival after gefitinib treatment in lung adenocarcinoma with EGFR mutation.BMC Cancer. 2015; 15: 31Crossref PubMed Scopus (24) Google Scholar and in gastric carcinoma cell lines, the EGFR activation pathway is one of the mechanisms of the development of resistance to the MET TKI PHA-665752 or crizotinib.6Qi J. McTigue M.A. Rogers A. et al.Multiple mutations and bypass mechanisms can contribute to development of acquired resistance to MET inhibitors.Cancer Res. 2011; 71: 1081-1091Crossref PubMed Scopus (175) Google Scholar Thus, our data suggest that the simultaneous inhibition of EGFR and MET by TKIs might be a better choice than monotherapies for treatment of MM showing high chromosome 7 polysomy. To date, the combined inhibition of EGFR and MET are under study in vivo and in vitro in many tumors. In vitro studies on MM cell lines show that treatment with a combination of EGFR and MET TKIs can suppress proliferation and/or survival of MM cells.7Kawaguchi K. Murakami H. Taniguchi T. et al.Combined inhibition of MET and EGFR suppresses proliferation of malignant mesothelioma cells.Carcinogenesis. 2009; 30: 1097-1105Crossref PubMed Scopus (69) Google Scholar In conclusion, we think that chromosome 7 copy number gain might be one of the predictive markers for combined targeted therapy against both EGFR and MET pathways. However, whether chromosome 7 polysomy might have a clinical application needs to be evaluated in controlled trials. This work was supported by grants from Ricerca Sanitaria Regione Liguria 2009, AIL (Sezione Francesca Lanzone) La Spezia, and Comitato Assistenza Malati e Lotta Contro i Tumori, Sarzana (to Dr. Roncella) and the Italian Ministry of Health (5×1000 fund, 2011) (to Dr. Pistillo). MET Gene Status in Malignant Mesothelioma Using Fluorescent In Situ HybridizationJournal of Thoracic OncologyVol. 11Issue 2PreviewIn our October 2015 letter in the Journal1 we reported a patient with malignant mesothelioma (MM) with amplification of the MET gene associated with MET receptor expression. This finding suggests that the inhibition of MET might be used as a targeted therapy also in selected patients with MM.2 Full-Text PDF Open Archive