for Cancer Research-Cancer Institute,

Cell-free microRNAs: potential biomarkers in need o

Deletions of CDKN2A and MTAP Detected by Copy-Number Variation Array Are Associated with Loss of p16 and MTAP Protein in Pleural Mesothelioma

CDKN2A deletion is a common alteration in pleural mesothelioma (PM) and frequently associated with co-deletion of MTAP. Since the standard detection method for CDKN2A deletion and FISH analysis is relatively expensive, we here investigated the suitability of inexpensive p16 and MTAP IHC by comparing concordance between IHC and OncoScan CNV arrays on samples from 52 PM patients. Concordance was determined using Cohen's kappa statistics. Loss of CDKN2A was associated with co-deletion of MTAP in 71% of cases. CDKN2A-MTAP copy-number normal cases were also IHC positive in 93% of cases for p16 and 100% for MTAP, while homozygous deletion of CDKN2A-MTAP was always associated with negative IHC for both proteins. In cases with heterozygous CDKN2A-MTAP loss, IHC expression of p16 and MTAP was negative in 100% and 71%, respectively. MTAP and p16 IHC showed high sensitivity (MTAP 86.5%, p16 100%) and specificity (MTAP 100%, p16 93.3%) for the detection of any gene loss. Loss of MTAP expression occurred exclusively in conjunction with loss of p16 labeling. Both p16 and MTAP IHC showed high concordance with Oncoscan CNV arrays (kappa = 0.952, p < 0.0001, and kappa = 0.787, p < 0.0001 respectively). We recommend combined MTAP and p16 immunohistochemistry to confirm the diagnosis of PM

Tumor Immune Microenvironment and Genetic Alterations in Mesothelioma

Malignant pleural mesothelioma (MPM) is a rare and fatal disease of the pleural lining. Up to 80% of the MPM cases are linked to asbestos exposure. Even though its use has been banned in the industrialized countries, the cases continue to increase. MPM is a lethal cancer, with very little survival improvements in the last years, mirroring very limited therapeutic advances. Platinum-based chemotherapy in combination with pemetrexed and surgery are the standard of care, but prognosis is still unacceptably poor with median overall survival of approximately 12 months. The genomic landscape of MPM has been widely characterized showing a low mutational burden and the impairment of tumor suppressor genes. Among them, BAP1 and BLM are present as a germline inactivation in a small subset of patients and increases predisposition to tumorigenesis. Other studies have demonstrated a high frequency of mutations in DNA repair genes. Many therapy approaches targeting these alterations have emerged and are under evaluation in the clinic. High-throughput technologies have allowed the detection of more complex molecular events, like chromotripsis and revealed different transcriptional programs for each histological subtype. Transcriptional analysis has also paved the way to the study of tumor-infiltrating cells, thus shedding lights on the crosstalk between tumor cells and the microenvironment. The tumor microenvironment of MPM is indeed crucial for the pathogenesis and outcome of this disease; it is characterized by an inflammatory response to asbestos exposure, involving a variety of chemokines and suppressive immune cells such as M2-like macrophages and regulatory T cells. Another important feature of MPM is the dysregulation of microRNA expression, being frequently linked to cancer development and drug resistance. This review will give a detailed overview of all the above mentioned features of MPM in order to improve the understanding of this disease and the development of new therapeutic strategies

Posttranscriptional regulation controls calretinin expression in malignant pleural mesothelioma

Calretinin (CALB2) is a diagnostic and prognostic marker in malignant pleural mesothelioma (MPM). We previously reported that calretinin expression is regulated at the mRNA level. The presence of a medium-sized (573 nucleotide) 3′ untranslated region (3′UTR) predicted to contain binding sites for miR-30a/b/c/d/e and miR-9 as well as an adenine/uridine-rich element (ARE) in all three transcripts arising from the CALB2 gene, suggests that calretinin expression is regulated via posttranscriptional mechanisms. Our aim was to investigate the role of the CALB2-3′UTR in the posttranscriptional regulation of calretinin expression in MPM. CALB2-3′UTR was inserted downstream of the luciferase reporter gene using pmiRGLO vector and reporter expression was determined after transfection into MPM cells. Targeted mutagenesis was used to generate variants harboring mutated miR-30 family and ARE binding sites. Electrophoretic mobility shift assay was used to test for the presence of ARE binding proteins. CALB2-3′UTR significantly decreased luciferase activity in MPM cells. Analysis of mutation in the ARE site revealed a further destabilization of the reporter and human antigen R (HuR) binding to the ARE sequence was detected. The mutation of two miR-30 binding sites abolished CALB2-3′UTR destabilization effect; a transient delivery of miR-30e-5p mimics or anti-miR into MPM cells resulted in a significant decrease/increase of the luciferase reporter expression and calretinin protein, respectively. Moreover, overexpression of CALB2-3′UTR quenched the effect of miR-30e-5p mimics on calretinin protein levels, possibly by sequestering the mimics, thereby suggesting a competitive endogenous RNA network. Finally, by data mining we observed that expression of miR-30e-5p was negatively correlated with the calretinin expression in a cohort of MPM patient samples. Our data show the role of (1) adenine-uridine (AU)-binding proteins in calretinin stabilization and (2) miR-30e-5p in the posttranscriptional negative regulation of calretinin expression via interaction with its 3′UTR. Furthermore, our study demonstrates a possible physiological role of calretinin’s alternatively spliced transcripts

Expression of phosphorylated ribosomal protein S6 in mesothelioma patients - correlation with clinico-pathological characteristics and outcome: results from the European Thoracic Oncology Platform (ETOP) Mesoscape project

Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Although histology and pathologic stage are important prognostic factors, better prognostic biomarkers are needed. The ribosomal protein S6 is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway involved in protein synthesis and cell proliferation. In previous studies, low phosphorylated S6 (pS6) immunoreactivity was significantly correlated with longer progression-free survival (PFS) and overall survival (OS) in PM patients. We aimed to correlate pS6 expression to clinical data in a large multi-centre PM cohort as part of the European Thoracic Oncology Platform (ETOP) Mesoscape project. Tissue Micro Arrays (TMAs) of PM were constructed and expression of pS6 was evaluated by a semi-quantitatively aggregate H-score. Expression results were correlated to patient characteristics as well as OS/PFS. pS6 IHC results of 364 patients from 9 centres, diagnosed between 1999 and 2017 were available. The primary histology of included tumours was epithelioid (70.3%), followed by biphasic (24.2%) and sarcomatoid (5.5%). TMAs included both treatment-naïve and tumour tissue taken after induction chemotherapy. High pS6 expression (181 patients with H-score>1.41) was significantly associated with less complete resection. In the overall cohort, OS/PFS were not significantly different between pS6-low and pS6-high patients. In a subgroup analysis non-epithelioid (biphasic and sarcomatoid) patients with high pS6 expression showed a significantly shorter OS (p < 0.001, 10.7 versus 16.9 months) and PFS (p < 0.001, 6.2 versus 10.8 months). In subgroup analysis, in non-epithelioid PM patients high pS6 expression was associated with significantly shorter OS and PFS. These exploratory findings suggest a clinically relevant PI3K pathway activation in non-epithelioid PM which might lay the foundation for future targeted treatment strategies

Tumor Immune Microenvironment and Genetic Alterations in Mesothelioma

Malignant pleural mesothelioma (MPM) is a rare and fatal disease of the pleural lining. Up to 80% of the MPM cases are linked to asbestos exposure. Even though its use has been banned in the industrialized countries, the cases continue to increase. MPM is a lethal cancer, with very little survival improvements in the last years, mirroring very limited therapeutic advances. Platinum-based chemotherapy in combination with pemetrexed and surgery are the standard of care, but prognosis is still unacceptably poor with median overall survival of approximately 12 months. The genomic landscape of MPM has been widely characterized showing a low mutational burden and the impairment of tumor suppressor genes. Among them, BAP1 and BLM are present as a germline inactivation in a small subset of patients and increases predisposition to tumorigenesis. Other studies have demonstrated a high frequency of mutations in DNA repair genes. Many therapy approaches targeting these alterations have emerged and are under evaluation in the clinic. High-throughput technologies have allowed the detection of more complex molecular events, like chromotripsis and revealed different transcriptional programs for each histological subtype. Transcriptional analysis has also paved the way to the study of tumor-infiltrating cells, thus shedding lights on the crosstalk between tumor cells and the microenvironment. The tumor microenvironment of MPM is indeed crucial for the pathogenesis and outcome of this disease; it is characterized by an inflammatory response to asbestos exposure, involving a variety of chemokines and suppressive immune cells such as M2-like macrophages and regulatory T cells. Another important feature of MPM is the dysregulation of microRNA expression, being frequently linked to cancer development and drug resistance. This review will give a detailed overview of all the above mentioned features of MPM in order to improve the understanding of this disease and the development of new therapeutic strategies

When RON MET TAM in Mesothelioma: All Druggable for One, and One Drug for All?

Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer with a poor survival rate. Treatment options are limited at best and drug resistance is common. Thus, there is an urgent need to identify novel therapeutic targets in this disease in order to improve patient outcomes and survival times. MST1R (RON) is a trans-membrane receptor tyrosine kinase (RTK), which is part of the c-MET proto-oncogene family. The only ligand recognized to bind MST1R (RON) is Macrophage Stimulating 1 (MST1), also known as Macrophage Stimulating Protein (MSP) or Hepatocyte Growth Factor-Like Protein (HGFL). In this study, we demonstrate that the MST1-MST1R (RON) signaling axis is active in MPM. Targeting this pathway with a small molecule inhibitor, LCRF-0004, resulted in decreased proliferation with a concomitant increase in apoptosis. Cell cycle progression was also affected. Recombinant MST1 treatment was unable to overcome the effect of LCRF-0004 in terms of either proliferation or apoptosis. Subsequently, the effect of an additional small molecular inhibitor, BMS-777607 (which targets MST1R (RON), MET, Tyro3, and Axl) also resulted in a decreased proliferative capacity of MPM cells. In a cohort of MPM patient samples, high positivity for total MST1R by IHC was an independent predictor of favorable prognosis. Additionally, elevated expression levels of MST1 also correlated with better survival. This study also determined the efficacy of LCRF-0004 and BMS-777607 in xenograft MPM models. Both LCRF-0004 and BMS-777607 demonstrated significant anti-tumor efficacy in vitro, however BMS-777607 was far superior to LCRF-0004. The in vivo and in vitro data generated by this study indicates that a multi-TKI, targeting the MST1R/MET/TAM signaling pathways, may provide a more effective therapeutic strategy for the treatment of MPM as opposed to targeting MST1R alone

Haemolysis during Sample Preparation Alters microRNA Content of Plasma

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers

Expression of phosphorylated ribosomal protein S6 in mesothelioma patients - correlation with clinico-pathological characteristics and outcome: results from the European Thoracic Oncology Platform (ETOP) Mesoscape project

Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Although histology and pathologic stage are important prognostic factors, better prognostic biomarkers are needed. The ribosomal protein S6 is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway involved in protein synthesis and cell proliferation. In previous studies, low phosphorylated S6 (pS6) immunoreactivity was significantly correlated with longer progression-free survival (PFS) and overall survival (OS) in PM patients. We aimed to correlate pS6 expression to clinical data in a large multi-centre PM cohort as part of the European Thoracic Oncology Platform (ETOP) Mesoscape project. Tissue Micro Arrays (TMAs) of PM were constructed and expression of pS6 was evaluated by a semiquantitatively aggregate H-score. Expression results were correlated to patient characteristics as well as OS/PFS. pS6 IHC results of 364 patients from 9 centres, diagnosed between 1999 and 2017 were available. The primary histology of included tumours was epithelioid (70.3%), followed by biphasic (24.2%) and sarcomatoid (5.5%). TMAs included both treatment-naive and tumour tissue taken after induction chemotherapy. High pS6 expression (181 patients with H-score>1.41) was significantly associated with less complete resection. In the overall cohort, OS/PFS were not significantly different between pS6-low and pS6-high patients. In a subgroup analysis nonepithelioid (biphasic and sarcomatoid) patients with high pS6 expression showed a significantly shorter OS (p< 0.001, 10.7 versus 16.9 months) and PFS (p < 0.001, 6.2 versus 10.8 months). In subgroup analysis, in non-epithelioid PM patients high pS6 expression was associated with significantly shorter OS and PFS. These exploratory findings suggest a clinically relevant PI3K pathway activation in non-epithelioid PM which might lay the foundation for future targeted treatment strategies