Response to treatment with chemical and biological inhibitors of c-met mutated form

c-MET is a receptor tyrosine kinase that, after binding with its ligand, hepatocyte growth factor (HGF), activates many signaling pathways, driving proliferation, motility, migration and invasion. Although c-MET is important in the control of tissue homeostasis under normal physiological conditions, it has also been found to be aberrantly activated in human cancers via mutation, amplification or protein overexpression. Activating point mutations were identified in the kinase domain of MET, either in the germline of patients affected by hereditary papillary renal carcinoma (HPRC) or in spontaneously occurring tumors; in particular, nine missense mutations (defined METPRC mutations), leading to constitutive activation of MET protein, have been identified in HPRC families. Given the importance of MET as a target for cancer therapies, clinical trials aimed at inhibiting it through the use of tyrosine kinase inhibitors (TKIs) have recently been started. The aim of project was: (i) to evaluate if METPRC mutants are sensitive to PHA-665752 (a small kinase inhibitor of MET), (ii) if some mutants are insensitive to the inhibitor, to investigate the mechanisms responsible for resistance, (iii) to check if the resistant mutants are still sensitive to other chemicals inhibitors or monoclonal antibodies against MET, (iv) to identify activating point mutations in human surgically resected lung cancers. We have found that some METPRC mutants cannot be inhibited by PHA-665752. Treatment with this TKI does not alter either receptor phosphorylation or MET mutants-induced biological activities (migration, invasion, anchorage-independent growth). We showed that these mutants are insensitive also to JNJ-38877605, a multitargeted tyrosine kinase inhibitor.. When we performed the mutational analysis on lung cancer samples, in one tumor we found the presence of one of the identified“resistant” mutations. To determine whether the mutants resistant to PHA-665752 could be inhibited with other strategies, we treated the mutant-expressing cells with the monoclonal antibody DN30, directed against the extracellular portion of the receptor. Our results showed that DN30 was indeed able to inhibit all the METPRC mutants. In conclusion, we have identified some METPRC mutants which do not respond to the ATP competitive kinase inhibitors. Since the identified METPRC mutations are located in the kinase domain and alter its conformation; it is likely that the competitive inhibitors are unable to interact with the ATP binding site in the context of the mutated receptors; this would render these mutants "resistant" to the action of tyrosine kinase inhibitors. However, these mutated forms still remain responsive to treatment antibodies directed against the MET extracellular portion: This observation is important since the use of monoclonal antibodies represent a therapeutic alternative for patients with tumors carrying MET mutants resistant to TKIs

Response to treatment with chemical and biological inhibitors of c-met mutated form

c-MET is a receptor tyrosine kinase that, after binding with its ligand, hepatocyte growth factor (HGF), activates many signaling pathways, driving proliferation, motility, migration and invasion. Although c-MET is important in the control of tissue homeostasis under normal physiological conditions, it has also been found to be aberrantly activated in human cancers via mutation, amplification or protein overexpression. Activating point mutations were identified in the kinase domain of MET, either in the germline of patients affected by hereditary papillary renal carcinoma (HPRC) or in spontaneously occurring tumors; in particular, nine missense mutations (defined METPRC mutations), leading to constitutive activation of MET protein, have been identified in HPRC families. Given the importance of MET as a target for cancer therapies, clinical trials aimed at inhibiting it through the use of tyrosine kinase inhibitors (TKIs) have recently been started. The aim of project was: (i) to evaluate if METPRC mutants are sensitive to PHA-665752 (a small kinase inhibitor of MET), (ii) if some mutants are insensitive to the inhibitor, to investigate the mechanisms responsible for resistance, (iii) to check if the resistant mutants are still sensitive to other chemicals inhibitors or monoclonal antibodies against MET, (iv) to identify activating point mutations in human surgically resected lung cancers. We have found that some METPRC mutants cannot be inhibited by PHA-665752. Treatment with this TKI does not alter either receptor phosphorylation or MET mutants-induced biological activities (migration, invasion, anchorage-independent growth). We showed that these mutants are insensitive also to JNJ-38877605, a multitargeted tyrosine kinase inhibitor.. When we performed the mutational analysis on lung cancer samples, in one tumor we found the presence of one of the identified“resistant” mutations. To determine whether the mutants resistant to PHA-665752 could be inhibited with other strategies, we treated the mutant-expressing cells with the monoclonal antibody DN30, directed against the extracellular portion of the receptor. Our results showed that DN30 was indeed able to inhibit all the METPRC mutants. In conclusion, we have identified some METPRC mutants which do not respond to the ATP competitive kinase inhibitors. Since the identified METPRC mutations are located in the kinase domain and alter its conformation; it is likely that the competitive inhibitors are unable to interact with the ATP binding site in the context of the mutated receptors; this would render these mutants "resistant" to the action of tyrosine kinase inhibitors. However, these mutated forms still remain responsive to treatment antibodies directed against the MET extracellular portion: This observation is important since the use of monoclonal antibodies represent a therapeutic alternative for patients with tumors carrying MET mutants resistant to TKIs

Long-term glucocorticoid treatment and high relapse rate remain unresolved issues in the real-life management of polymyalgia rheumatica: a systematic literature review and meta-analysis

A systematic review and meta-analysis were conducted, according to the PRISMA methodology, to summarize current evidence on the prevalence and predictors of long-term glucocorticoid (GC) treatment and disease relapses in the real-life management of polymyalgia rheumatica (PMR).Out of 5442 retrieved studies, 21 were eligible for meta-analysis and 24 for qualitative analysis. The pooled proportions of patients still taking GCs at 1, 2, and 5 years were respectively 77% (95%CI 71-83%), 51% (95%CI 41-61%), and 25% (95CI% 15-36%). No significant difference was recorded by distinguishing study cohorts recruited before and after the issue of the international recommendations in 2010. The pooled proportion of patients experiencing at least one relapse at 1 year from treatment initiation was 43% (95%CI 29-56%). Female gender, acute-phase reactants levels, peripheral arthritis, starting GCs dosage, and tapering speed were the most frequently investigated potential predictors of prolonged GC treatment and relapse, but with inconsistent results. Only a few studies and with conflicting results evaluated the potential role of early treatment with methotrexate in reducing the GC exposure and the risk of relapse in PMR.This study showed that a high rate of prolonged GC treatment is still recorded in the management of PMR. The relapse rate, even remarkable, can only partially explain the long-term GC treatment, suggesting that other and not yet identified factors may be involved. Additional research is needed to profile patients with a higher risk of long-term GC treatment and relapse and identify more effective steroid-sparing strategies. Key Points: • High rate of long-term glucocorticoid (GC) treatment is recorded in polymyalgia rheumatica (PMR), being 77%, 51%, and 25% of patients still on GCs after respectively 1, 2, and 5 years. • A pooled relapse rate of 43% at 1 year, even remarkable, can only partially explain the long-term GC treatment in PMR. • Several studies have attempted to identify potential predictors of prolonged treatment with GCs and relapse, but with inconsistent results. • Additional research is needed to profile patients with a higher risk of long-term GC treatment and relapse and identify more effective steroid-sparing strategies

Metabolic reprogramming identifies the most aggressive lesions at early phases of hepatic carcinogenesis

Metabolic changes are associated with cancer, but whether they are just bystander effects of deregulated oncogenic signaling pathways or characterize early phases of tumorigenesis remains unclear. Here we show in a rat model of hepatocarcinogenesis that early preneoplastic foci and nodules that progress towards hepatocellular carcinoma (HCC) are characterized both by inhibition of oxidative phosphorylation (OXPHOS) and by enhanced glucose utilization to fuel the pentose phosphate pathway (PPP). These changes respectively require increased expression of the mitochondrial chaperone TRAP1 and of the transcription factor NRF2 that induces the expression of the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase (G6PD), following miR-1 inhibition. Such metabolic rewiring exclusively identifies a subset of aggressive cytokeratin-19 positive preneoplastic hepatocytes and not slowly growing lesions. No such metabolic changes were observed during non-neoplastic liver regeneration occurring after two/third partial hepatectomy. TRAP1 silencing inhibited the colony forming ability of HCC cells while NRF2 silencing decreased G6PD expression and concomitantly increased miR-1; conversely, transfection with miR-1 mimic abolished G6PD expression. Finally, in human HCC patients increased G6PD expression levels correlates with grading, metastasis and poor prognosis. Our results demonstrate that the metabolic deregulation orchestrated by TRAP1 and NRF2 is an early event restricted to the more aggressive preneoplastic lesions

Assessment Of Circulating Endothelial Cells And Their Progenitors As Potential Biomarkers Of Disease Activity And Damage Accrual In Behçet's Syndrome

PURPOSE: To explore the potential role of circulating endothelial cells (CECs) and their progenitors (EPCs) as biomarkers of disease activity and damage accrual in patients with Behçet's syndrome (BS), by using a standardised and reliable flow cytometry protocol. PATIENTS AND METHODS: CECs and EPCs were assessed in 32 BS patients and 11 gender/age/smoking habits matched healthy controls (HC). They were identified by flow cytometry as alive/nucleated/CD45-negative/CD34-bright/CD146-positive and alive/nucleated/CD45-negative/CD34-bright/CD309-positive events, respectively. In BS patients, demographic and clinical features, including disease activity (assessed by Behçet's disease current disease activity form, BDCAF) and irreversible damage accrual (by the vasculitis damage index, VDI) were recorded. Uni- and multivariate analysis were performed to compare the CECs and EPCs concentrations in BS vs HC and to identify potential associations with demographic or clinical features. RESULTS: The CECs concentration was significantly higher in the BS patients than HCs [median (IQR) 15.0 (7.5-23.0) vs 6.0 (2.0-13.0) CECs/mL, p=0.024]. In BS patients, no significant associations were found between CECs and demographic features, present and past clinical manifestations, BDCAF score and ongoing treatment. A significant association was observed between CECs and organ damage, as assessed by the VDI (rho 0.356, p=0.045). Higher levels of CECs were especially associated with vascular damage [median (IQR) 23.0 (14.0-47.0) vs 13.0 (6.0-19.0) CECs/mL, p=0.011], including arterial aneurysm and stenosis, complicated venous thrombosis, cerebrovascular accident. The concentration of EPCs did not significantly differ between the BS and HC [median 26.5 (13.0-46.0) vs 19.0 (4.0-42.0) EPCs/mL, p=0.316] and no significant associations were observed between their levels and any clinical characteristic. CONCLUSION: Our study suggests that the CECs concentration is significantly higher in BS than healthy subjects, and it mainly correlates with vascular damage. A longitudinal extension of the present study on a wider cohort would be useful to validate the potential role of CECs as a marker or, hopefully, predictor of vascular damage in BS

YAP activation is an early event and a potential therapeutic target in liver cancer development

Background and Aims: Although the growth suppressor Hippo pathway has been implicated in hepatocellular carcinoma (HCC) pathogenesis, it is unknown at which stage of hepatocarcinogenesis its dysregulation occurs. We investigated in early rat and human preneoplastic lesions whether overexpression of the transcriptional co-activator Yes-associated protein (YAP) is an early event. Methods: The experimental model used is the Resistant-Hepatocyte (R-H) rat model. Gene expression was determined by qRT-PCR or immunohistochemistry. Forward genetic experiments were performed in human HCC cells and in murine oval cells. Results All foci of preneoplastic hepatocytes generated in rats 4 weeks after diethylnitrosamine (DENA) treatment, displayed YAP accumulation. This was associated with down-regulation of the β-TRCP ligase, known to mediate YAP degradation, and of microRNA-375, targeting YAP. YAP accumulation was paralleled by up-regulation of its target genes. Increased YAP expression was also observed in early dysplastic nodules and adenomas in humans. Animal treatment with verteporfin (VP), which disrupts the formation of the YAP–TEAD complex, significantly reduced preneoplastic foci and oval cell proliferation. In vitro experiments confirmed that VP-mediated YAP inhibition impaired cell growth in HCC and oval cells; notably, oval cell transduction with wild type or active YAP conferred tumorigenic properties in vitro and in vivo. Conclusions: These results suggest that i) YAP overexpression is an early event in rat and human liver tumorigenesis; ii) it is critical for the clonal expansion of carcinogen-initiated hepatocytes and oval cells, and, iii) VP-induced disruption of YAP-TEAD interaction may provide an important approach for the treatment of YAP-overexpressing cancers

Brain-reactive autoantibodies in neuropsychiatric systemic lupus erythematosus

IntroductionThe pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is widely unknown, and the role of autoantibodies is still undetermined.MethodsTo identify brain-reactive autoantibodies possibly related to NPSLE, immunofluorescence (IF) and transmission electron microscopy (TEM) on rat and human brains were performed. ELISA was used to reveal the presence of known circulating autoantibodies, while western blot (WB) was applied to characterize potential unknown autoantigen(s).ResultsWe enrolled 209 subjects, including patients affected by SLE (n=69), NPSLE (n=36), Multiple Sclerosis (MS, n=22), and 82 age- and gender-matched healthy donors (HD). Autoantibody reactivity by IF was observed in almost the entire rat brain (cortex, hippocampus, and cerebellum) using sera from NPSLE and SLE patients and was virtually negative in MS and HD. NPSLE showed higher prevalence (OR 2.4; p = 0.047), intensity, and titer of brain-reactive autoantibodies than SLE patients. Most of the patient sera with brain-reactive autoantibodies (75%) also stained human brains. Double staining experiments on rat brains mixing patients’ sera with antibodies directed against neuronal (NeuN) or glial markers showed autoantibody reactivity restricted to NeuN-containing neurons. Using TEM, the targets of brain-reactive autoantibodies were located in the nuclei and, to a lesser extent, in the cytoplasm and mitochondria. Given the high degree of colocalization between NeuN and brain-reactive autoantibodies, we assumed NeuN was a possible autoantigen. However, WB analysis with HEK293T cell lysates expressing or not expressing the gene encoding for NeuN protein (RIBFOX3) showed that patients’ sera carrying brain-reactive autoantibodies did not recognize the NeuN corresponding band size. Among the panel of NPSLE-associated autoantibodies (e.g., anti-NR2, anti-P-ribosomal protein, antiphospholipid) investigated by ELISA assay, only the anti-β2-glycoprotein-I (aβ2GPI) IgG was exclusively found in those sera containing brain-reactive autoantibodies.ConclusionIn conclusion, SLE and NPSLE patients possess brain-reactive autoantibodies but with higher frequency and titers found in NPSLE patients. Although many target antigens of brain-reactive autoantibodies are still undetermined, they likely include β2GPI

Brain-reactive autoantibodies in neuropsychiatric systemic lupus erythematosus

IntroductionThe pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is widely unknown, and the role of autoantibodies is still undetermined. MethodsTo identify brain-reactive autoantibodies possibly related to NPSLE, immunofluorescence (IF) and transmission electron microscopy (TEM) on rat and human brains were performed. ELISA was used to reveal the presence of known circulating autoantibodies, while western blot (WB) was applied to characterize potential unknown autoantigen(s). ResultsWe enrolled 209 subjects, including patients affected by SLE (n=69), NPSLE (n=36), Multiple Sclerosis (MS, n=22), and 82 age- and gender-matched healthy donors (HD). Autoantibody reactivity by IF was observed in almost the entire rat brain (cortex, hippocampus, and cerebellum) using sera from NPSLE and SLE patients and was virtually negative in MS and HD. NPSLE showed higher prevalence (OR 2.4; p = 0.047), intensity, and titer of brain-reactive autoantibodies than SLE patients. Most of the patient sera with brain-reactive autoantibodies (75%) also stained human brains. Double staining experiments on rat brains mixing patients' sera with antibodies directed against neuronal (NeuN) or glial markers showed autoantibody reactivity restricted to NeuN-containing neurons. Using TEM, the targets of brain-reactive autoantibodies were located in the nuclei and, to a lesser extent, in the cytoplasm and mitochondria. Given the high degree of colocalization between NeuN and brain-reactive autoantibodies, we assumed NeuN was a possible autoantigen. However, WB analysis with HEK293T cell lysates expressing or not expressing the gene encoding for NeuN protein (RIBFOX3) showed that patients' sera carrying brain-reactive autoantibodies did not recognize the NeuN corresponding band size. Among the panel of NPSLE-associated autoantibodies (e.g., anti-NR2, anti-P-ribosomal protein, antiphospholipid) investigated by ELISA assay, only the anti-& beta;2-glycoprotein-I (a & beta;2GPI) IgG was exclusively found in those sera containing brain-reactive autoantibodies. ConclusionIn conclusion, SLE and NPSLE patients possess brain-reactive autoantibodies but with higher frequency and titers found in NPSLE patients. Although many target antigens of brain-reactive autoantibodies are still undetermined, they likely include & beta;2GPI

Psychometric Properties of Patient Global Assessment in Psoriatic Arthritis: A Systematic Literature Review

Objective: Patient global assessment (PtGA) is a patient-reported outcome (PRO) that reflects a patient's judgment of their health/disease activity (DA). The objective of this systematic literature review was to assess the psychometric properties of PtGA in psoriatic arthritis (PsA). Methods: Research articles reporting the assessment of psychometric properties of PtGA in PsA, listed in PubMed and extracted according to the Outcome Measures in Rheumatology (OMERACT) Filter 2.1 and the Consensus-based Standards for the Selection of Health Measurement Instruments (COSMIN) terminology, were selected. Validity was assessed for comprehensiveness (content), correlation with other DA instruments (construct), and with quality of life measurements (criterion). A metaanalysis regarding construct validity was performed. Correlations between PtGA variations and other indices' variations (external responsiveness) and PtGA variations after treatment (internal responsiveness) were collected. Data on the formulation of PtGA and its discordance with physician global assessment (PGA) were also collected. Methods: Of 60 articles analyzed (comprising 17,453 patients), 44 were observational studies and 16 were trials. PtGA was assessed through 27 different formulations. In all the retrieved studies, PtGA assessed DA, and in 3 studies, PtGA was assessed as a variable of global health status. The correlation between PtGA and PROs was strong (ρ > 0.50), whereas with other DA indices and PGA, it ranged from weak to moderate (ρ 0.20-0.50). Three studies described a positive discordance (PtGA > PGA). Responsiveness, assessed in 24 studies, showed a strong correlation with joint count index variations (ρ 0.51-0.52). Conclusion: PtGA is a valid and responsive tool in PsA. Correlations were higher with PROs and weaker with DA composite indices and PGA. PGA was usually scored lower than PtGA. A standardized formulation of PtGA would be useful

Molecular modifications induced by mud-bath therapy in patients with osteoarthritis

Background Mud-bath therapy (MBT) is a non-pharmacological approach commonly used to treat osteoarthritis (OA). Several data indicate that MBT improve patient's symptoms (1), exerting a beneficial effect on pain and joint function, although the biological mechanisms involved in the therapeutic response are poorly defined. Objectives This study aimed to find molecular changes (proteins and mRNA variations) in patients with OA after MBT treatment. Methods The study included 39 patients whit primary diffuse osteoarthritis, assigned to receive a cycle of mud-bath therapy over a period of 2 weeks added to usual pharmacologic treatment. Whole blood and serum were collected before and after standard MBT treatment: for each time points two pools of patients sera were analyzed by the direct antigen-labeling technology (RayBio® Biotin Label-based Antibody Array, RayBiotech) obtaining a broad, panoramic view of protein expression. Using this semi-quantitative technique up to 1000 target proteins was simultaneously detected, making this approach ideally suited for proteomic studies. Again, pooled samples of mRNA were used to investigate genes expression and to perform the transcriptomic analysis using an high-resolution array design that contains >6.0 million distinct probes, covering coding and non-coding transcripts (GeneChip® HTA 2.0, Affymetrix). Results At the end of mud-bath therapy, using first a semi-quantitative approach, we observed in both biological replicates increased levels (>1.5 fold change) of: inhibin beta A subunit (INHBA), activin A receptor type 2B (ACVR2B), angiopoietin-1 (ANGPT1), beta-2-microglobulin (B2M), growth differentiation factor 10 (BMP-3b/GDF10), C-X-C motif chemokine ligand 5 (CXCL5), fibroblast growth factor 2 (FGF2), fibroblast growth factor 12 (FGF12), oxidized low density lipoprotein receptor 1 (OLR1), matrix metallopeptidase 13 (MMP13). We observed that some increased proteins belongs to the same family or pathway: INHBA, ACVR2B and BMP-3b are members of TGF-Beta superfamily; CXCL5 and MMP13 participate into IL-17 signaling pathway; FGF2 and FGF12 are proteins of FGF family. We are currently performing the transcriptome analysis, and the next step will be to correlate detected proteins with mRNA levels considering also post-transcriptional or epigenetic modifications. Finally we will validate these findings with other quantitative techniques (like ELISA and RT-PCR). Conclusions Our first proteomic and broad spectrum analysis suggest the implication of molecular pathways involved in a wide variety of cellular functions such as gene expression modulation, differentiation, angiogenesis, tissue repair, acute and chronic inflammatory response which may explain the beneficial effects of MBT observed in osteoarthritis. This “omic” approach (proteomic and transcriptomic), generated a huge amount of data that is currently under statistical and bioinformatic analysis. References Forestier R, Desfour H, Tessier JM, et al. Spa therapy in the treatment of knee osteoarthritis: a large randomised multicentre trial. Ann Rheum Dis. 2010;69:660–5