CTLA-4 in mesothelioma patients: tissue expression, body fluid levels and possible relevance as a prognostic factor.

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.

Evaluation of soluble mesothelin-related peptide as a diagnostic marker of malignant pleural mesothelioma effusions: its contribution to cytology.

Soluble mesothelin-related peptide (SMRP) is regarded as an FDA approved biomarker for the diagnosis and monitoring of pleural malignant mesothelioma (MPM). We detected the SMRP levels in pleural effusions (PE) by means of an ELISA and analyzed their diagnostic relevance to differentiate MPM from benign pathology and from non-MPM pleural metastasis. Comparison with cytology in MPM-PE was also performed. We found that SMRP detection in MPM-PE can help the diagnosis of MPM and provide additional diagnostic value to cytology. We concluded that SMRP test may be incorporated into clinical practice of PE from patients suspicious for MPM.

Potential role of serum mesothelin in predicting survival of patients with malignant pleural mesothelioma.

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.

Determination of Mesothelin Levels in Pleural Effusion Does Not Help Predict Survival of Patients With Malignant Pleural Mesothelioma.

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.

Assessment of RT-PCR detection of human mammaglobin for the diagnosis of breast cancer derived pleural effusions.

The present study investigates the diagnostic significance of human mammaglobin (hMAM) mRNA expression in pleural effusions (PE) from breast cancer (BC) patients. Two hundred and fifty PE samples, including 32 from patients who had diagnosis of BC, 116 from patients with other cancers, and 102 from patients with benign diseases, were subjected to nested reverse-transcription polymerase chain reaction (RT-PCR) for hMAM, and the results were compared with conventional cytology. hMAM was found expressed in 76/250 (30.4%) total PE and in 23/28 (sensitivity of 82.1%) of the PE subgroup owing to metastasis from BC. The specificity for hMAM detection method was 75.7%, whereas accuracy, positive predictive value, and negative predictive value were 76.4%, 30.3%, and 97.1%, respectively. hMAM was also detected in 46/116 (39.6%) PE specimens from other types of cancer and in 7/102 (6.8%) from benign diseases. Comparative analysis of RT-PCR and cytology showed that 14 PE samples from metastatic BC (50%) were positive by both PCR and cytology, 9 (32.1%) were positive only by PCR and 5 (17.9%) were negative by both tests, whereas no cases were found of positive cytology with negative PCR. RT-PCR increased sensitivity of BC effusion detection to 32.1% (McNemar test, P=0.004). We demonstrated that RT-PCR for hMAM test was more sensitive than cytomorphology suggesting that, although hMAM is not BC specific, it may be useful in adjunct to cytology for the routine screening of malignant BC effusions.

Comparison of the Diagnostic Performance of Fibulin-3 and Mesothelin in Patients with Pleural Effusions from Malignant Mesothelioma.

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.

Mesothelin in serum and pleural effusion in the diagnosis of malignant pleural mesothelioma with non-positive cytology.

BACKGROUND/AIM: Mesothelin (SMRP) is regarded as a biomarker of malignant pleural mesothelioma (MPM). Herein, we analyzed the contribution of SMRP detection in pleural effusion and in serum to the diagnosis of MPM with non-positive cytology. MATERIALS AND METHODS: The present study included 52 cases of MPM, 43 of pleural benign lesions and 25 of non-MPM pleural metastases. SMRP was measured by MesoMark ELISA (Cis-Bio International Gif/Yvette; France). RESULTS: In non-positive cytology, effusion-SMRP showed higher diagnostic performance than serum-SMRP. We found 38 out of 52 (73.1%) cases of non-positive cytology MPM, out of which 27 (71.0%) were positive for effusion-SMRP (cut-off=12.70 nM) and 18 (47.4%) for serum-SMRP (cut-off=1.08 nM). When cytology, effusion- and serum-SMRP were used in combination, an overall sensitivity in detection of MPM of 78.9% was achieved. The same sensitivity was obtained by combining cytology with effusion-SMRP alone, whereas the combination of serum-SMRP with cytology led to a sensitivity of 61.5%. CONCLUSION: Detection of both effusion- and serum-SMRP can contribute to improve the diagnosis of MPM with non-positive cytology. However, the analysis of SMRP in effusion makes it unnecessary to test SMRP in the serum.

Clinical value of mesothelin in pleural effusions versus histology by medical thoracoscopy: brief report.

The aim of this study based on the third phase of the architecture of diagnostic research was to assess the sensitivity and specificity of soluble mesothelin-related peptide (SMRP) in pleural exudative effusions (PE) compared to the histology obtained by medical thoracoscopy as the diagnostic gold standard examination. We assessed 104 consecutive thoracoscopies. SMRP concentrations were obtained using an ELISA test. We had 34 mesotheliomas (25 epithelioid and 9 sarcomatoid), 35 pleural metastases, and 35 benign diseases. PE-SMRP were significantly higher in patients with epitheliomorphic mesothelioma (mean ± SD 46.55 ± 44.29 nM) than in patients with sarcomatoid mesothelioma (16.11 ± 25.02 nM; p = 0.061), pleural metastasis (7.52 ± 10.77 nM; p < 0.0001), or benign diseases (5.82 ± 8.86 nM; p < 0.0001). Using ROC curve analysis, PE-SMRP offered an AUC of 0.767 in its ability to differentiate between patients with mesothelioma and all other diagnoses. The diagnostic sensitivity and specificity of PE-SMRP for distinguishing mesothelioma from all other causes of pleural effusion, at a cut-off value of 19.6 nM, were 58.8 and 97.1 %, respectively. PE-SMRP higher than the assumed cut-off of 19.6 nM were observed in 18/25 (72.0%) epitheliomorphic mesotheliomas, 2/9 (22.2%) sarcomatoid mesotheliomas, 5/35 (14.3%) pleural metastases, and 1/35 (2.9%) benign diseases. We conclude that PE-SMRP adds some clinical information in the work-up of patients with a PE of unknown origin: (1) thoracoscopy should always be done in patients with a positive mesothelin; (2) a negative mesothelin does not exclude a malignant disease.

Mesothelin is more useful in pleural effusion than in serum in the diagnosis of pleural mesothelioma.

BACKGROUND/AIM: Soluble mesothelin-related peptide (SMRP) is regarded as a biomarker of malignant pleural mesothelioma (MPM). Herein, we compared the diagnostic performances of SMRP in matched pleural effusion (PE-SMRP) and serum (S-SMRP). MATERIALS AND METHODS: Diagnosis on pleural biopsies was performed for all patients including 43 with MPM, 23 with non-MPM pleural metastases (MTS) and 36 with benign (BNG) pleural diseases. SMRP was measured by a MesoMark ELISA (Cis-Bio International Gif/Yvette; France). RESULTS: Using the receiver operating characteristic (ROC) analysis, 12.70 and 1.08 nM were detected as cut-off values to optimal discrimination for PE-SMRP and S-SMRP, respectively. PE-SMRP showed a better diagnostic accuracy than S-SMRP in MPM vs. MTS+BNG (area under the ROC curve=81.6 vs. 70.5; sensitivity=69.8% vs. 46.5%; specificity=88.1% vs. 84.7%; diagnostic odds ratio (DOR)=17.1 vs. 4.8). In S-SMRP-negative patients, PE-SMRP maintained an acceptable performance (Sensitivity=47.8%; DOR=8.3; p=0.001), whereas in PE-SMRP-negative patients, S-SMRP performed very poorly (Sensitivity=15.4%; DOR=1.2; p=0.858). CONCLUSION: PE-SMRP detection has a superior diagnostic accuracy than S-SMRP detection in the diagnosis of MPM.

Clinical relevance of human mammaglobin mRNA in pleural effusion from patients undergoing thoracoscopy: a pilot study.

BACKGROUND: human mammaglobin (hMAM) expression has been reported in pleural effusions (PE). The aim of this study was to assess the clinical relevance of hMAM mRNA in PE from patients who underwent thoracoscopy.
 MATERIAL AND METHODS: A total of 288 patients with PE were studied, 155 of which were diagnosed with malignant and 133 with non-malignant diseases by thoracoscopy. Cells from PE were analyzed by nested hMAM RT-PCR. Statistical analyses were performed to evaluate the diagnostic performance parameters (DPP), the association between hMAM expression and benign or malignant status and the relative risk of cancer for patients with negative thoracoscopy showing hMAM positivity.
 RESULTS: hMAM mRNA was found in 68/288 (23.6%) PE samples of which 51 were from the 155 patients diagnosed with malignant diseases and 17 were from the 133 patients diagnosed with non-malignant diseases. A significant correlation between hMAM expression and malignancy was found (OR=3.04) and the DPP were as follows: sensitivity=32.9%, specificity=87.2%, accuracy=58.0%, positive predictive value=75.0% and negative predictive value=52.7%. Among the patients with negative thoracoscopy (n=133), 5/17 (29.4%) hMAM-positive patients had or developed a tumor during the 18-month follow up period, as compared to 10/116 (8.6%) hMAM-negative patients (relative risk of 4.6 for developing a malignancy).
 CONCLUSION: These findings suggest a possible application of hMAM RT-PCR detection in PE as to identify a false-negative thoracoscopy in non-specific pleuritis.

Human mammaglobin transcript amplification for differential diagnosis in a breast cancer metastatic to dura mater.

BACKGROUND: In breast cancer (BC), metastases to the central nervous system usually arise in women with advanced disease. Diagnosis of leptomeningeal (LM) metastasis is based on neurological symptoms, imaging studies and cytological detection of malignant cells in the cerebrospinal fluid (CSF). However, often these approaches are not sensitive enough to recognize leptomeninges involvement and subsequently to make a diagnosis of LM carcinomatosis. This study investigated the employment of reverse transcriptase-polymerase chain reaction (RT-PCR) for the human mammaglobin (hMAM) gene in a case of BC with cerebral metastases in which the involvement of the leptomeninges was in doubt. MATERIALS AND METHODS: Amplification of hMAM mRNA was performed from CSF cells by RT-PCR. RESULTS: No amplification of hMAM was obtained from the CSF cells. CONCLUSION: RT-PCR for human mammaglobin mRNA of the CSF in BC patients with brain metastases may aid clinical determination of LM involvement and consequently the choice of the most effective therapy regimens for affected patients.

Detection of circulating tumour cells in breast cancer patients using human mammaglobin RT-PCR: association with clinical prognostic factors.

BACKGROUND: So far discordant results regarding the significance of tumour cells circulating in peripheral blood (CTCs) of breast cancer (BC) patients have been reported. Our aim was to evaluate the association of indirect CTC detection by amplification of human mammaglobin (hMAM) gene expression with traditional prognostic markers of clinical outcome in BC at the time of diagnosis. PATIENTS AND METHODS: Peripheral blood samples from 190 patients with invasive and 12 patients with in situ BC, before therapy and/or surgery, from 184 patients with benign breast disease and from 146 healthy volunteers were tested for hMAM expression by a nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Correlations between CTCs and age at diagnosis, tumour type and size, grading, lymph node involvement, oestrogen and progesterone receptor status, HER-2/neu expression and Ki-67/MIB-1 labelling index were assessed through the odds ratio (OR) point estimates, considering OR >2.0 or <0.5 as being clinically relevant. ORs and their corresponding 95% confidence limits (95% CL) were obtained by logistic regression analysis. RESULTS: Expression of hMAM was found only in peripheral blood of patients with invasive BC (9.5%) and multivariate logistic regression analysis indicated its association with lymph node involvement (pN1-pN3 vs. pN0, OR=5.6, 95% CL=1.4-22.6; p=0.009), tumour size (pT2-pT4 vs. pT1, OR=2.3, 95% CL=0.6-9.0; p=0.207) and negative ER status (OR=2.5, 95% CL=0.6-10.0; p=0.227). CONCLUSION: Our data show that CTC detection in invasive BC at the time of diagnosis is associated with poor prognosis and may also be used as an additional prognostic indicator.

Diagnosis and origin determination of malignant pleural effusions through the use of the breast cancer marker human mammaglobin.

As was reported that human mammaglobin (hMAM) may be expressed in malignant pleural effusions (PEs), we investigated the relevance of hMAM reverse-transcriptase polymerase chain reaction (RT-PCR) for their diagnosis and determination of primary origin. Two hundred and twenty-eight malignant (132 male, 96 female) and 185 benign (132 male, 53 female) PEs were investigated. Statistical analyses evaluated the diagnostic performance parameters in all PEs and in cytologically negative malignant PEs, the association between hMAM and benign or malignant status by the direct index of correlation [diagnostic odds ratio (DOR)], chi test, and P value (P). In addition, the discriminative diagnostic power of hMAM expression, independently in breast cancer, lung cancer (LC), malignant mesothelioma (MM), and other cancers was evaluated. In the entire patient population, hMAM was detected in 45.6% and 5.4% of malignant and benign PEs, respectively, in the male group in 41.7% and 4.5% and in the female group in 51.0% and 7.5% of malignant and benign PEs, respectively. A statistically significant correlation between hMAM and malignancy was found in the entire population (DOR=14.68, P<0.001) and in the male (DOR=15.00, P<0.001) or female (DOR=12.77, P<0.001) groups. hMAM RT-PCR increased the diagnostic rate of malignant PEs as it allowed us to detect as malignant 32.1% of cytologically negative PEs. In female patients the positivity of hMAM indicated with higher probability (50.8%) the origin of PEs from breast cancer but lower probability from LC (17%), MM (9.4%), or other cancers (15.1%), whereas in male patients it indicated with similar probability (about 40%) the origin from LC or MM. Our results suggest that hMAM RT-PCR may provide information both in the diagnosis of PE and in the search for the primary site of neoplasia, either in male or female patients.