Downregulation of miR-99a/let-7c/miR-125b miRNA cluster predicts clinical outcome in patients with unresected malignant pleural mesothelioma.

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.

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.

Small cell lung cancer transformation and the T790M mutation: A case report of two acquired mechanisms of TKI resistance detected in a tumor rebiopsy and plasma sample of EGFR-mutant lung adenocarcinoma.

The present study describes the case of a 45-year-old man diagnosed with metastatic lung adenocarcinoma, which harbored a deletion within exon 19 of the epidermal growth factor receptor (EGFR) gene. The patient was subsequently treated with gefitinib (250 mg/day orally from May 2013 to March 2014), but developed acquired resistance to the drug following 11 months of treatment. Tumor burden molecular analysis was performed on a tumor rebiopsy and plasma sample, and histological analysis was also performed on the tumor rebiopsy. A small cell transformation retaining the original EGFR mutation was detected in the tumor rebiopsy, while the T790M mutation together with the activating ex19del mutation were identified only in the plasma sample. The patient was treated with cytotoxic chemotherapy (off-label schedule with epirubicin 80 mg/mq and paclitaxel 160 mg/mq every 21 days for 6 cycles) and radiation (50.4 Gy administered in 28 fractions of 1.8 Gy once daily for 5.5 weeks) specific for small cell lung cancer, and may also have benefitted from treatment with a third generation T790M-specific EGFR-TKI. To better describe the mechanisms of resistance to TKI inhibitors and to optimize therapeutic regimens, the simultaneous analysis of tumor biopsies and circulating tumor DNA should be considered.

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.

An MRI Method To Map Tumor Hypoxia Using Red Blood Cells Loaded with a pO2-Responsive Gd-Agent.

Hypoxia is a typical hallmark of many solid tumors and often leads to therapy resistance and the development of a more aggressive cancer phenotype. Oxygen content in tissues has been evaluated using numerous different methods for several imaging modalities, but none has yet reached the required standard of spatial and temporal resolution. Magnetic Resonance Imaging (MRI) appears to be the technique of choice and several pO2-responsive probes have been designed for it over the years. In vivo translation is often hampered in Gd-relaxation agents as it is not possible to separate effects that arise from changes in local concentration from those associated with responsive properties. A novel procedure for the MRI based assessment of hypoxia is reported herein. The method relies on the combined use of Gd-DOTP- and Gd-HPDO3A-labeled red blood cells (RBCs) where the first probe acts as a vascular oxygenation-responsive agent, while the second reports the local labeled RBC concentration in a transplanted breast tumor mouse model. The MRI assessment of oxygenation state has been validated by photoacoustic imaging and ex vivo immunofluorescence. The method refines tumor staging in preclinical models and makes possible an accurate monitoring of the relationship between oxygenation and tumor growth.

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.

Status of Anaplastic Lymphoma Kinase (ALK) in malignant mesothelioma.

BACKGROUND: Malignant mesothelioma (MM) is a particularly aggressive type of primary tumor, associated with exposure to asbestos, and characterized by high mortality. To date, there is no curative therapy for MM. The receptor anaplastic lymphoma kinase (ALK) was found to be mutated in many cases of cancer and used as a target in biological therapies. We investigated whether this pharmacological treatment could also be applicable to MM. MATERIALS AND METHODS: The state of ALK was analyzed by immunohistochemistry and fluorescent in situ hybridization in 63 MM tissue specimens. RESULTS: None of the 63 MM samples showed overexpression or translocation of ALK. CONCLUSION: Our preliminary data exclude the utility of analysis of the ALK gene in MM and suggest that ALK inhibitor therapy is not applicable to MM.

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.

Synthesis and relaxometric characterization of a MRI Gd-based probe responsive to glutamic acid decarboxylase enzymatic activity.

Novel contrast agent based systems, which selectively visualize specific cells, e.g., neurons in the brain, would be of substantial importance for the fast developing field of molecular magnetic resonance imaging (MRI). We report here the synthesis and in vitro validation of a Gd(III)-based contrast agent designed to act as an MRI responsive probe for imaging the activity of the enzyme glutamic acid decarboxylase (GAD) present in neurons. Upon the action of the enzyme, the Gd(III) complex increases its hydration sphere and takes on a residual positive charge that promotes its binding to endogenous macromolecules. Both effects contribute in a synergic way to generate a marked relaxation enhancement, which directly reports enzyme activity and will allow activity detection of GAD positive cells in vitro and in vivo selectively.

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.

Ovarian lymphoma and hydronephrosis.

INTRODUCTION: Ovarian lymphoma is a rare entity, and hydronephrosis from lymphoma is even rarer. Most reports describe a laparoscopic approach to the disease, but we report a case of hydroureteronephrosis associated with ovarian lymphoma managed completely by miniinvasive techniques. CASE REPORT: A 51-year-old woman was referred to us for back pain and renal colic and computed tomography scan findings of right hydroureteronephrosis and a mass in the right mesorectum and uterosacral ligament. After magnetic resonance imaging was performed, the patient underwent laparoscopic adnexectomy and ureterolysis after ureteroscopy and stenting. Histology results showed diffuse B-cell lymphoma of the ovary occluding the ureter without infiltration. The patient has undergone 6 cycles of chemotherapy. DISCUSSION: This is the first report to describe ovarian lymphoma and hydroureteronephrosis managed completely by laparoscopic surgery and endoscopy. Frequency in clinical practice, differential diagnosis, and endoscopic approach are discussed. The advantages of a multidisciplinary endoscopic team are underlined.

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.

In vivo imaging of inhibitory, GABAergic neurons by MRI.

The unambiguous detection of specific neuronal subtypes is up to now only possible with invasive techniques or optical imaging after genetic modification. High field magnetic resonance imaging (MRI) has the ability to visualize the brain structure and anatomy noninvasively, with high resolution--but missing the cell specific and functional information. Here we present a new tool for neuroimaging with MRI, enabling the selective detection of GABAergic neurons under in vivo conditions. The specific imaging contrast is achieved by a novel paramagnetic contrast agent, which responds to the activity of the enzyme glutamic acid decarboxylase--expressed solely by inhibitory neurons. The relaxivity of the complex is increased upon decarboxylation of two glutamic acid moieties, thus allowing increased water access to the inner and outer coordination spheres of the paramagnetic ion. The mechanism and specificity of activation were proven with tissue lysates and further applied to a differentiation protocol for murine embryonic stem cells. The relaxation enhancement was studied quantitatively and revealed decreased longitudinal relaxation times in the inhibitory neuron samples compared to the naïve stem cells in vitro and in vivo. Furthermore, this approach offers not only the discrimination of inhibitory, GABAergic neurons in the brain but also may expand the usefulness of MRI for functional imaging on a cellular level.