Assessing technical and biological variation in SWATH-MS-based proteomic analysis of chronic lymphocytic leukaemia cells.

Chronic lymphocytic leukaemia (CLL) exhibits variable clinical course and response to therapy, but the molecular basis of this variability remains incompletely understood. Data independent acquisition (DIA)-MS technologies, such as SWATH (Sequential Windowed Acquisition of all THeoretical fragments), provide an opportunity to study the pathophysiology of CLL at the proteome level. Here, a CLL-specific spectral library (7736 proteins) is described alongside an analysis of sample replication and data handling requirements for quantitative SWATH-MS analysis of clinical samples. The analysis was performed on 6 CLL samples, incorporating biological (IGHV mutational status), sample preparation and MS technical replicates. Quantitative information was obtained for 5169 proteins across 54 SWATH-MS acquisitions: the sources of variation and different computational approaches for batch correction were assessed. Functional enrichment analysis of proteins associated with IGHV mutational status showed significant overlap with previous studies based on gene expression profiling. Finally, an approach to perform statistical power analysis in proteomics studies was implemented. This study provides a valuable resource for researchers working on the proteomics of CLL. It also establishes a sound framework for the design of sufficiently powered clinical proteomics studies. Indeed, this study shows that it is possible to derive biologically plausible hypotheses from a relatively small dataset

Exosomal transport of hepatocyte-derived drug-modified proteins to the immune system.

Idiosyncratic drug-induced liver injury (DILI) is a rare, often difficult to predict adverse reaction with complex pathomechanisms. However, it is now evident that certain forms of DILI are immune-mediated and may involve the activation of drug-specific T-cells. Exosomes are cell-derived vesicles that carry RNA, lipids and protein cargo from their cell of origin to distant cells, and may play a role in immune activation. Herein, primary human hepatocytes were treated with drugs associated with a high incidence of DILI (flucloxacillin, amoxicillin, isoniazid and nitroso-sulfamethoxazole) to characterize the proteins packaged within exosomes that are subsequently transported to dendritic cells for processing. Exosomes measured between 50-100 nm and expressed enriched CD63. LC-MS/MS identified 2109 proteins, with 608 proteins being quantified across all exosome samples. Data are available via ProteomeXchange with identifier PXD010760. Analysis of gene ontologies revealed that exosomes mirrored whole human liver tissue in terms of the families of proteins present, regardless of drug treatment. However, exosomes from nitroso-sulfamethoxazole-treated hepatocytes selectively packaged a specific subset of proteins. LC-MS also revealed the presence of hepatocyte-derived exosomal proteins covalently modified with amoxicillin, flucloxacillin and nitroso-sulfamethoxazole. Uptake of exosomes by monocyte-derived dendritic cells occurred silently, mainly via phagocytosis, and was inhibited by latrunculin A. An, amoxicillin-modified 9-mer peptide derived from the exosomal transcription factor protein SOX30 activated naïve T-cells from HLA-A*02:01 positive human donors. Conclusion. This study shows that exosomes have the potential to transmit drug-specific hepatocyte-derived signals to the immune system and provides a pathway for the induction of drug hapten-specific T-cell responses. This article is protected by copyright. All rights reserved

The proteomic analysis of B-cell receptor signaling in chronic lymphocytic leukaemia

Chronic Lymphocytic Leukaemia (CLL) is the most common adult Leukaemia in the UK, Western Europe and America. It is a malignancy of naive B-cells. The clinical course of patients with CLL is heterogeneous; some patients survive for years without treatment, others die of a chemotherapy resistant disease within two years of presentation. Genomic studies have found little variation in patients showing differing prognosis, suggesting that it is the same disease but with varying outcomes. At present there is no cost effective, reliable and routine clinical test which can distinguish patient prognosis and a "watch and wait" strategy is currently in clinical use. Studies have shown that patients who express mutated IgVH genes on the B-cell receptor (BCR) have a good prognosis, whereas patients who express unmutated IgVH genes have a poor prognosis. However IgVH gene mutational status is an expensive and time consuming test and would not be practical for routine clinical practice. If the B-cell has not been sensitised to a specific antigen (i.e. unmutated IgVH genes on BCR) it is hyper-responsive to stimulation through the BCR by antigen. Stimulation of the BCR may prevent apoptosis of malignant cells; therefore a hyper-responsive BCR is linked to poor prognosis. By artificially stimulating cells and using proteomic techniques we have investigated signaling pathways activated by the BCR to gain a greater understanding of the anti-apoptotic nature of the malignant B-cells and to find potential prognostic biomarkers related to a hyper-active BCR. Protein was extracted from stimulated and unstimulated cells from CLL patients categorised as having a poor prognosis (unmutated IgVH genes and hyper-responsive BCR). The extracts were separated using conventional two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The gels were stained with coomassie blue total protein stain and analysed with statistical software. Proteins with a two-fold (p less than 0.05) change in expression between stimulated and unstimulated samples were excised from the gels and analysed by matrix assisted laser desorption/ionisation with time of flight mass spectrometry (MALDI-TOF-MS). Antibody microarrays were used as a complimentary method to 2D- PAGE and immunoblotting was applied as a verification technique. Changes in protein expression were detected in response to prolonged BCR stimulation. Many of the proteins have had no previous connection with BCR signaling or leukaemia and give a greater insight to the mechanisms of the BCR. Targets found include ones which are associated with the activation of anaplastic lymphoma kinase (ALK), the plasma kallikrein-kinin system (KKS), the AKT-1 pathway, the MAPK pathways, the adenylate kinase system and involvement in the CD40-dependant activation of B-CLL cells. One of the protein targets found (kininogen) was increased by over two-fold in three independent clinical samples after sustained BCR stimulation. If we understand more about the BCR signaling pathways then we may be able to identify potential prognostic biomarkers and novel targets for therapeutic intervention that may inhibit survival of the malignant B-cells

The proteomic analysis of B-cell receptor signaling in chronic lymphocytic leaukaemia

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Proteomic analysis of B-cell receptor signaling in chronic lymphocytic leukaemia reveals a possible role for kininogen

CLL is an incurable disease with variable prognosis. The hyper reactivity of the B-cell receptor (BCR) to unknown antigen ligation plays a pivotal role in CLL-cell survival. We aimed to investigate the BCR signalling pathway using proteomics to identify novel proteins which may have clinical relevance in this disease.Three CLL samples were selected based upon BCR responsiveness, demonstrated by upregulation of phospho-ERK following in vitro stimulation. The differential expression of proteins, upon artificial stimulation of the BCR, was examined in these samples using two-dimensional gel electrophoresis in combination with mass spectrometry. Proteins of interest were subsequently examined using immunoblotting. Proteomic analysis revealed that kininogen, a critical protein of kinin-kallikrein system, was upregulated in all 3 clinical samples upon BCR stimulation. There are 2 forms of kininogen: HMWK and LMWK. The upregulation of LMWK upon BCR stimulation was confirmed by immunoblotting in all 3 of these samples. In a pilot series of 52 unselected CLL samples, 71% demonstrated basal LMWK expression. There was a trend towards shorter median survival in LMWK positive cases (147. months versus 253. months for LMWK negative cases; p=. 0.125). Kininogen may be a novel therapeutic target in CLL and the possible association with prognosis warrants further investigation. Biological significance: We have identified the upregulation of LMWK upon BCR stimulation of CLL samples. There is no previous published research to suggest a link between kininogen and normal B-cells or CLL cells. In 52 unselected CLL samples, 71% demonstrated basal LMWK expression. There was a trend towards shorter median survival in LMWK positive cases. The absence of LMWK protein expression on normal B-cells suggests that this could be a biomarker for CLL and further research should be undertaken. © 2013 Elsevier B.V

COX-2 specific inhibitors enhance the cytotoxic effects of pemetrexed in mesothelioma cell lines

BACKGROUND Although malignant pleural mesothelioma is a rare tumour, its incidence is increasing. The prognosis remains very poor with an average survival of 10 months from diagnosis. The choice of chemotherapy regimens for mesothelioma patients is limited and new approaches are required. COX-2 inhibition induces apoptosis in a variety of tumour cell lines. The cytotoxic effect of conventional drugs may be enhanced by the addition of a COX-2 inhibitor. In order to identify possible new therapeutic approaches we aimed to determine whether the addition of COX-2 inhibitors would enhance the cytotoxic effect of chemotherapeutic agents in mesothelioma cell lines. MATERIALS AND METHODS Three mesothelioma cell lines MSTO-211H, NCI-H2052 and NCI-H2452 were utilised. Using the COX-2 positive A549 lung cancer cell line as control, all cell lines were assayed using an MTT assay with non-specific COX-2 inhibitors (sulindac and flurbiprofen), specific COX-2 inhibitors (DuP-697 and NS-398), and chemotherapeutic agents (cisplatin, vinorelbine and pemetrexed). RESULTS All cell lines exhibited COX-2 expression by western blotting using two antibodies. The addition of either DuP-697 or NS-398 increased the sensitivity to pemetrexed in all cell lines. CONCLUSION These findings suggest that the design of novel pemetrexed-containing combination regimens with increased cytotoxicity may be feasible

Biomarkers of chemotherapy resistance in breast cancer identified by proteomics: current status

This review describes and discusses the advantages and limitations of proteomic approaches in the identification of biomarkers associated with chemotherapy resistance. Both gel-based (two-dimensional polyacrylamide gel electrophoresis) and gel-free (shotgun and quantitative) mass spectrometry approaches are discussed. Non-mass spectrometry approaches including antibody microarray platforms are described as complementary proteomic strategies. Methods for technical confirmation and clinical validation of putative biomarkers are presented. Use of this proteomic toolbox in the quest for biomarkers of chemotherapy resistance in breast cancer is reviewed. Technical aspects of sample selection, acquisition, storage and analysis are discussed and putative biomarkers identified through proteomic approaches are presented. (C) 2010 Elsevier Ireland Ltd. All rights reserved

Proteomic (antibody microarray) exploration of the molecular mechanism of action of the specific COX-2 inhibitor DuP 697

We have previously shown that specific COX-2 inhibitors, including DuP 697, have anti-proliferative effects on mesothelioma cells and potentiate the cytotoxicity of pemetrexed. Here, we used a novel proteomic approach to explore the mechanism of action of this agent. COX-2-positive cell lines MSTO-211H (mesothelioma) and A549 (lung cancer) were exposed to DuP 697 for 72 h. Drug carrier only was added to control cells. Extracted proteins from treated and control cells were analysed using a comparative proteomic platform. Differentially expressed proteins, identified by the Panorama Xpress Profiler725 antibody microarray were submitted to Ingenuity Pathway Analysis. A total of 32 unique differentially expressed proteins were identified with a significant (>1.8-fold) difference in expression between treated and untreated cells in at least one cell line. Five molecules, BCL2L1 (Bcl-xL), BID, CHUK (IKK), FASLG and RAF1, were mapped to the Apoptosis Signaling pathway following Ingenuity Pathway Analysis. BCL2L1 (Bcl-xL) and BID were analysed using immunoblotting and differential expression was confirmed. Proteomic (antibody microarray) analysis suggests that the mechanism of action of DuP 697 may be exerted via the induction of apoptosis. The antibody microarray platform can be utilised to explore the molecular mechanism of action of novel anticancer agents

780. Incidence and Prevalence of Nontuberculous Mycobacterial Lung Disease in US Medicare, 2008–2015

Abstract Background Previous research has reported nontuberculous mycobacterial lung disease (NTMLD) prevalence of 47 per 100,000 among Medicare beneficiaries ≥65 years in 2007, with an average increase of 8.2% annually between 1997 and 2007. In this study, we have evaluated NTMLD incidence and prevalence in Medicare between 2008 and 2015. Methods Patients diagnosed for NTMLD with an ICD9 031.0 were identified from the Medicare database (N≈30 million yearly), not including the Part C portion. Individuals who incurred at least 2 medical claims ≥30 days apart between 2007 and 2015 were considered as a positive NTMLD case, yielding 58,294 patients. All individuals fulfilling the case definition each calendar year were considered as prevalent cases. Incident cases included those meeting case criteria and who did not have a Medicare claim for NTMLD in the prior year. Poisson regression was used to estimate yearly confidence intervals. ARIMA models were used to forecast incidence and prevalence over 2016–2025. Results Patients with NTMLD in the Medicare database had a mean age of 74 (standard deviation: ±10) years. Sixty-nine percent were women and 89% white. Yearly NTMLD incidence increased from 20.7 (95% CI: 20.2–21.3) in 2008 to 28.1 (27.5–28.7) in 2013 per 100,000 Medicare beneficiaries and leveled to 27.6 (26.9–28.2) in 2014 and 25.9 (25.3–26.5) in 2015 per 100,000. Yearly NTMLD prevalence increased throughout the observation period from 41.6 (40.9–42.3) in 2008 to 63.1 (62.2–64.0) in 2015 per 100,000 Medicare beneficiaries. Incidence was 28.1 vs. 14.7 per 100,000 in 2015 in Medicare beneficiaries ≥65 years vs. those <65 years, respectively. Prevalence was 70.2 vs. 27.9 per 100,000 in 2015 in Medicare beneficiaries ≥65 years vs. those <65 years, respectively. In 2015, incidence and prevalence were higher in women than men (33.9 vs. 16.0/100,000 and 86.2 vs. 34.6/100,000, respectively) and among individuals of Asian origin compared with White (41.1 vs. 27.6/100,000 and 89.4 vs. 68.7/100,000, respectively). The 10-year incidence and prevalence forecasts were presented in figures. Conclusion In US Medicare beneficiaries, NTMLD incidence increased from 2008 through 2013 and leveled off in more recent years, while NTMLD prevalence continued to rise through 2015. Disclosures K. L. Winthrop, Insmed Incorporated: Scientific Advisor, Consulting fee and Research grant. T. Marras, Insmed Incorporated: Investigator, Consulting fee and Research grant, Horizon Pharmaceuticals: Consultant, Consulting fee, Red Hill: Consultant, Consulting fee, AstraZeneca: CME, Speaker honorarium. G. Eagle, Insmed Incorporated: Employee, Salary. R. Zhang, Insmed Incorporated: Consultant, Consulting fee. P. Wang, Insmed Incorporated: Employee, Salary. E. Chou, Insmed Incorporated: Employee, Salary. Q. Zhang, Insmed Incorporated: Employee, Salary

122. All-Cause Mortality Increased With Nontuberculous Mycobacterial Lung Disease in US Medicare

Abstract Background Nontuberculous Mycobacterial Lung Disease (NTMLD) is a chronic, debilitating, and progressive disease. This study evaluates all-cause mortality in patients with NTMLD in the US Medicare. Methods Patients (n = 43,394) were identified from the Medicare database (excluding Part C) based on physician claims for NTMLD on ≥2 separate occasions ≥30 days apart between 2007 and 2015. About 12% patients were <65 years and qualified for Medicare due to disability. A control cohort (n = 84,814) was randomly selected and matched to the NTMLD sample by age and sex. The NTMLD diagnosis date was assigned to the matched controls as an index date. Poisson and Cox regression were used to derive descriptive rates and adjusted risk of mortality accounting for baseline comorbidities of pulmonary, immune, cardiovascular, cancer, and other disorders. Results Mean age was 74 (±10) years and 68% were female in both NTMLD and control cohorts. Mean Charlson comorbidity index (CCI) was 2.9 (standard deviation ±2.6) in NTMLD vs. 1.3 (±1.9) in control cohort. In Medicare members ≥65 years, mean age was 76 (±7) years and 70% were female. Mean CCI was 2.8 (±2.5) in NTMLD cohort vs. 1.4 (±2.0) in control cohort. In Medicare members <65, mean age was 53 (±10) and 49% were female. Mean CCI was 3.8 (±3.3) in NTMLD vs. 1.1 (±1.9) in the control. Observed yearly mortality rates were 9.8% in NTMLD vs. 4.7% in control cohort (rate ratio [RR] = 2.1; 95% CI: 2.03–2.13). In ≥65 Medicare members, the observed rates were 9.7% in NTMLD vs. 5.0% in control cohort (RR = 2.0; 1.9–2.0). In Medicare members <65, the observed rates were 10.4% in NTMLD vs. 2.5% in control cohort (RR = 4.1; 3.8–4.5). Compared with the Asian race, observed mortality was higher in NTMLD patients of Native American (hazard ratio [HR] = 1.69, 1.30–2.19), Black (HR = 1.23; 1.08–1.39), Hispanic (HR = 1.27, 1.07–1.51), or White (HR = 1.18, 1.06–1.31) race (Figure 1). Mortality rates were elevated with NTMLD relative to controls in all age categories from ≥65 years (Figure 2). Adjusted mortality increased with NTMLD by 35% overall (HR = 1.35; 1.3–1.4), by 23% in age group ≥65 (HR = 1.23, 1.19–1.27), and almost doubled in age group <65 (HR = 1.97, 1.80–2.15). Conclusion Among US Medicare enrollees, NTMLD was associated with a 35% increased risk of mortality overall. Disclosures T. Marras, Insmed Incorporated: Investigator, Consulting fee and Research grant. Horizon Pharmaceuticals: Consultant, Consulting fee. Red Hill: Consultant, Consulting fee. AstraZeneca: CME, Speaker honorarium. Q. Zhang, Insmed Incorporated: Employee, Salary. G. Eagle, Insmed Incorporated: Employee, Salary. P. Wang, Insmed Incorporated: Employee, Salary. R. Zhang, Insmed Incorporated: Consultant, Consulting fee. E. Chou, Insmed Incorporated: Employee, Salary. K. L. Winthrop, Insmed Incorporated: Consultant and Scientific Advisor, Consulting fee and Research grant