Plasma Of Healthy Donors Research Articles

Receptor Tyrosine Kinase AXL: A Potential Strategy to Counter Immune Suppression and Dormancy in Multiple Myeloma

Introduction The AXL receptor tyrosine kinase (AXL) has emerged as a promising therapeutic target for cancer therapy. Recent studies revealed a crucial role of AXL signaling in proliferation, survival, dormancy and therapy resistance in different cancers including lung cancer, hepatocellular cancer and AML. In this study, we aimed to investigate the role of AXL in Multiple Myeloma (MM), focusing on myeloma cell dormancy and AXL expression in different cellular components of the bone marrow microenvironment. Material & Methods To investigate dormancy, we used the syngeneic murine 5TGM1 MM model. 5TGM1-GFP+cells were DiD-labeled and injected intravenously in naïve C57BL/KaLwRij mice. At end-stage, GFP+DiD+('dormant', non-proliferating) and GFP+DiD-('proliferating') MM cells were analyzed by flow cytometry for AXL expression. In addition, AXL expression was also analyzed in CD11b+ myeloid cells and in in vitrogenerated macrophages from the 5TMM model. The effects of AXL inhibition by R428 (BGB324|Bemcentinib, Sigma-Aldrich), a highly potent and AXL-specific small molecular inhibitor, on viability and induced apoptosis of MM cells was determined by Cell Titer Glo and AnnexinV/7AAD staining respectively. AXL expression in human myeloma cell lines (HMCL) (JJN3, U266 and LP-1) and murine 5TGM1 cells was analyzed by qRT-PCR and cytospin stainings. Patient cohorts (TT2/TT3) were used to correlate AXL expression and overall survival. Plasma of healthy donors and MM patients was analyzed by ELISA (R&D). Results Using the in vivo5TGM1 dormancy model, we demonstrated an increased expression of AXL (4x higher) in dormant MM cells compared to proliferating MM cells (n=3, p<0,05). Myeloma cell lines (JJN3, U266, 5TGM1) had a very low AXL expression, however, treatment with melphalan induced a more than twofold increase in AXL expression (n=3, p<0.05). The combination of melphalan and R428 significantly increased apoptosis of JJN3 (>10%), U266 (>20%) and LP-1 (>10%) cells compared to single agent therapy (n=6) (p<0.01). Using patient cohorts, we observed that AXL expression correlated with a good overall survival (p=0.006). In addition, plasma samples of patients (n=31) showed a decreased expression of AXL compared to samples of healthy controls (n=9) (p<0.001). This confirms our hypothesis that AXL is associated with dormancy and therefore correlates with a better overall survival. In a second part, we investigated AXL expression in 5TMM-derived myeloid cells and macrophages (n=3). We observed a high expression of AXL in myeloid derived suppressor cells and tumor associated macrophages compared to myeloma cells. In addition, we observed that myeloid cells were much more sensitive to R428 compared to MM cells (n=5, p>0.01). Conclusion We observed that AXL is highly expressed in dormant MM cells and environmental myeloid cells. Despite its association with a good prognosis in MM, AXL serves as an interesting target to eradicate dormant myeloma cells as AXL inhibitors affect viability and induce apoptosis of myeloma cells, especially in combination with melphalan. Therefore, AXL can be considered as a new therapeutic strategy, to target both the immunosuppressive myeloid cells and the residual cancer cells in MM patients. Disclosures No relevant conflicts of interest to declare.

1412P - Analytical performance of the resolution-HRD plasma assay used to identify mCRPC patients with biallelic disruption of DNA repair genes for treatment with niraparib

BackgroundMetastatic castration-resistant prostate cancer (mCRPC) patients with DNA repair gene defects (DRD) have a shorter life expectancy than patients without DRD and may benefit from treatment with PARP inhibitors. Niraparib is a highly selective PARP inhibitor, with activity against PARP-1/2 DNA-repair polymerases. Detection of DRD in cell free DNA (cfDNA) isolated from blood is minimally invasive and of special benefit to mCRPC patients, including patients without accessible lesions. The assay would also have the advantage of a shorter turnaround time (TAT) than genotyping of tissue. However, using cfDNA to identify biallelic disruption of DNA-repair genes is technically challenging. MethodsResolution-HRD identifies patients with biallelic pathogenic alterations of the ATM, BRCA1, BRCA2, BRIP1, CHEK2, FANCA, HDAC2, or PALB2 genes, by targeted NGS sequencing of cfDNA. Analytical performance of Resolution-HRD was validated using cfDNA from mCRPC patient plasma, cfDNA from healthy donor plasma, and contrived samples with a wide spectrum of technically challenging genetic aberrations. ResultsThe LOD95 at a cfDNA input level of 40ng ranged from 0.2 to 1.37 for SNVs and indels, and 6-12 for CNL. APA for intra-run and inter-run studies at the 1X LOD was 95% and 95% respectively. No false-positives were detected in any samples from healthy donors (N=60). Resolution-HRD has been validated to give consistent results across the 10-75ng input range. Resolution-HRD is used to identify patients for enrollment in the GALAHAD Phase II Efficacy and Safety Study (64091742PCR2001) of Niraparib in Men with mCRPC and DNA-Repair Anomalies. As of April 2019, over 2000 patients are tested successfully (0.88% failure rate) with a median TAT of 8.6 days (range 5-12 days). ConclusionsThe analytical performance of the Resolution-HRD assay offers highly sensitive, specific and robust test results, and meets analytical requirements for clinical applications. This test is currently being evaluated in several clinical trials for prospective identification of mCRPC patients with DRD for treatment with niraparib. Legal entity responsible for the studyResolution Bioscience. FundingJanssen Research and Development. DisclosureI. Pekker: Shareholder / Stockholder / Stock options, Full / Part-time employment: resolution bioscience. L. Lim: Leadership role, Shareholder / Stockholder / Stock options, Licensing / Royalties, Full / Part-time employment, Officer / Board of Directors: Resolution Bioscience. J.S. Simon: Leadership role, Shareholder / Stockholder / Stock options: Janssen. M. Gormley: Shareholder / Stockholder / Stock options, Full / Part-time employment: Janssen. Z. Li: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. J. Pollak: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. K. Potts: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. S. Watford: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. J. Posey: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. P. Chan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. K. Urtishak: Shareholder / Stockholder / Stock options, Full / Part-time employment: Janssen. K. Garg: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. A. Hosseini: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. M. Li: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: Resolution Bioscience.

1416P - Enhanced access to EGFR molecular testing in NSCLC using a cell-free DNA tube for liquid biopsy

BackgroundThe clinical utility of non-invasive liquid biopsy and ability to accurately detect EGFR mutations in circulating-tumor DNA of patients with NSCLC has been well demonstrated. This expands the pool of patients eligible for molecular testing. The cobas® EGFR Mutation Test v2 (cobas test) is a real-time PCR test for qualitative and semi-quantitative detection of 42 EGFR mutations in DNA from tissue and circulating free DNA (cfDNA) from K2 EDTA plasma. Blood collected in K2 EDTA requires plasma be separated within 8hours, which can be a barrier to molecular testing and thus impact treatment decisions. The Roche Cell-Free DNA Collection Tube (Roche cfDNA) stabilizes blood up to 8 days, allowing greater flexibility in transportation and time to plasma separation. Here the suitability of plasma from Roche cfDNA tubes for use with the cobas test is demonstrated. MethodsTest performance with Roche cfDNA plasma was verified with NSCLC patient specimens or surrogate samples (sheared cell line DNA in healthy donor plasma). Correlation was tested using paired draws in K2 EDTA and Roche cfDNA tubes for 51 NSCLC patients and 20 healthy donor surrogate samples. Limit of detection (LoD) and linearity established with K2 EDTA plasma were verified with Roche cfDNA plasma. Reproducibility was assessed using surrogate samples at two levels with multiple operators, Roche cfDNA tube lots, instruments, days and sites. Blood storage conditions were established at an external laboratory with 6 NSCLC patient specimens. ResultsCompared to K2 EDTA plasma, Roche cfDNA plasma had a PPA, NPA and OPA of 100.0% with the cobas test. LoD for EGFR mutation detection in Roche cfDNA plasma was verified as≤100 cp/mL. Linearity for EGFR mutations in Roche cfDNA plasma was verified. The reproducibility study had a call agreement of≥98.6%. Blood in Roche cfDNA tubes was stable for up to 8 days at 18-25ºC with one excursion of up to 24hours at 15-30ºC prior to separation. ConclusionsUse of the Roche cfDNA tube with the cobas® EGFR Mutation Test v2 provides the flexibility to store blood for up to 8 days prior to separation, with equivalent performance to K2 EDTA plasma, and facilitates use of liquid biopsy for NSCLC patients needing molecular testing. Legal entity responsible for the studyRoche Molecular Systems Inc. FundingAstraZeneca PLC. DisclosureT.E. May: Full / Part-time employment: Roche Molecular Solutions. S.A. Scudder: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche Molecular Solutions. S.J. Joshi: Full / Part-time employment: Roche Molecular Solutions. M. Kohlmann: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca, PLC. N. Shrestha: Full / Part-time employment: Roche Molecular Solutions. N. Lee: Full / Part-time employment: Roche Molecular Solutions. J. Lai: Full / Part-time employment: Roche Molecular Systems Inc. M. Tsourounis: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca PLC. A. Kohlmann: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca PLC. P. O’Donnell: Full / Part-time employment, Spouse / Financial dependant: Roche Molecular Systems. H. Halait: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche Molecular Systems. All other authors have declared no conflicts of interest.

Pooled Human Immunoglobulin Preparations as Immunomodulating Drugs

It is time to celebrate the 125th anniversary of the first successful attempt to develop and use a specific high-titer antitoxic serum for treating diphtheria, a deadly infectious disease. This was followed by major advances in passive immunotherapy 75 years ago (production of pooled human IgG for subcutaneous injection) and 50 years ago (widespread technology for producing immunoglobulin preparations for intravenous administration). More than 200 tons of pooled human IgG are produced per year worldwide. The preparation is used primarily for IgG substitution in patients with primary and secondary immunodeficiencies, as well as for an immunomodulating treatment of a growing number of autoimmune and inflammatory diseases. These preparations contain the pooled IgG antibody repertoire of a large population of healthy plasma donors. This repertoire includes antibodies that neutralize pathogens and their factors of virulence, anti-idiotypic antibodies, and antibodies to other foreign and own proteins, as well as to carbohydrate antigens. Naturally polyspecific antibodies that are present in all healthy individuals play an important role as a first-line defense against bacteria and viruses. After exposure to protein-modifying agents, some IgG molecules can acquire the ability to bind novel structurally unrelated antigens. This phenomenon is referred to as induced polyspecificity. The list of these protein-modifying molecules was shown to include low-pH buffers, free heme, pro-oxidative ferrous ions, reactive oxygen species, etc. Such modified antibody preparations may have a therapeutic potential, since their administration to animals with experimental sepsis or aseptic systemic response syndromes significantly improved survival rates, while the same dose of the native preparation had no effect. We also hypothesize that the aggressive protein-modifying molecules released in sites of inflammation and tissue damage could also modify the antigen-binding behavior of surface immunoglobulin B cell receptors and the structurally related T cell receptors. This “specificity editing” of both types of receptors may play a major role in the body’s defense mechanisms.

Abstract 2239: Analysis of nucleosomal DNA as an extraction control for plasma-based circulating tumor DNA assays

Abstract Purpose: Challenges to accurate quantitative measurements of circulating tumor DNA (ctDNA) include variable contamination with large DNA fragments released during mononuclear cell lysis, and potential variability in extraction efficiency. We investigated optimal procedures for plasma processing and the potential utility of a DNA spike-in as a process control for extraction efficiency. Methods: Initial experiments analyzed the potential benefits of different centrifugation protocols (1600 x g vs. 1600 x g followed by 16000 x g) for plasma separation by analyzing the size distribution of extracted DNA using a BioAnalyzer. In subsequent experiments we used droplet digital PCR (ddPCR) to compare differences in extraction efficiency between nucleosomal DNA (nDNA) and genomic DNA (gDNA) spiked into healthy donor plasma and extracted using the QIAamp DSP Circulating NA Kit (Qiagen). To evaluate the properties of the purification columns with respect to the yield of different DNA sizes, we compared the plasma results to experiments extracting purified nDNA and gDNA spiked into PBS instead of plasma. We investigated the potential competitive effect of large DNA on the extraction efficiency of small DNA by mixing nDNA with commercially available human gDNA in different concentrations. To assess if nDNA, when spiked into patient plasma, would track with endogenous ctDNA, we spiked a pool of plasma from patients with BRAFV600E mutations with known quantities of nDNA purified from NRASQ61K mutant cells (CHP212). The spiked plasma pool was divided and extracted using multiple separate columns. Copies/ml and mutant fraction were determined by ddPCR assays for BRAFV600E and NRASQ61K. Coefficients of variation were also calculated. Results: Comparison of different centrifugation speeds for plasma processing before and after freeze-thaw indicated that a 2nd high-speed centrifugation (16000 x g), either before or after a freeze-thaw cycle, minimizes large DNA in plasma samples prior to ctDNA extraction. In initial spike-in experiments extraction efficiency for nDNA was 87.7% compared to 42% for gDNA. We obtained similar results when purifying the nDNA and gDNA from PBS instead of plasma. We observed greater variability in nDNA extraction efficiency when increasing amounts of gDNA were added as a contaminant. Finally, we found that the slight, paralell variations in extraction yields of externally spiked NRASQ61K mutant nDNA and endogenous BRAFV600E ctDNA in 7/9 replicate extractions. The overall coefficients of variation (total copies/mL) for 9 extractions for BRAFV600E and NRASQ61K were 3.9 and 4.2 respectively. Conclusion: A second centrifugation step helps reduce potential contamination of ctDNA with large DNA, and may improve the recovery of ctDNA from plasma samples. Nucleosomal DNA can potentially be used as a process control for extracting ctDNA from plasma samples. Citation Format: Mahrukh M. Syeda, George Karlin-Neumann, Iman Osman, David Polsky. Analysis of nucleosomal DNA as an extraction control for plasma-based circulating tumor DNA assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2239.

Abstract 2282: Progress toward non-invasive oncology diagnostics: Evaluation of cfDNA next-generation sequencing assays

Abstract Introduction Surgical biopsy or excisional resection is a standard practice in oncology diagnosis that requires a detectable tumor mass and can be highly invasive, limiting its role in the very early detection, as well as ongoing monitoring efforts, of genetic aberrations relevant to tumor biology. In contrast to surgical biopsies, liquid biopsies provide a readily accessible source of circulating tumor DNA, albeit in a highly diluted state that necessitates a very sensitive and robust method of detection. With recent advents in molecular barcoding and bioinformatics, next-generation sequencing (NGS) has emerged as a platform for probing this liquid oncology space. Herein, we evaluate the ability of the Oncomine™ cell-free DNA (cfDNA) Breast, Colon, and Lung NGS panels to reliably detect clinically relevant genomic alterations in a large background of non-mutated genetic material. Methods A total of 197 individual libraries were prepared across all three cfDNA panels according to the manufacturer protocols. Libraries were manually normalized, pooled, and loaded onto the ThermoFisher IonChef/S5xL system for bead-templating and sequencing. Samples included a mix of commercially available cfDNA controls, plasma samples, and cell lines spiked into healthy donor plasma, and were selected to cover a wide range of clinically relevant mutations and mimic the conditions inherent to real patient specimens. Isolation of cfDNA was performed on both Qiagen and Kingfisher platforms. Raw variant call files were generated using IonReporter and processed through a custom filtering pipeline to generate the final results. Results Nucleic acid isolations yields were comparable for cfDNA samples isolated using the Qiagen or Kingfisher extraction methods, with both yielding a 40-60% DNA recovery rate when known DNA quantities were spiked into plasma background. Library yield mapped to NGS panel size, and the greatest variability was observed for the Colon cfDNA panel. The sensitivity for all Oncomine™ cfDNA assays exceeded 92%, and was highly dependent on sample input, with robust detection at the 0.1% allele frequency requiring up to 50 ng of input cfDNA. A single false positive was present in one HapMap sample analyzed with the breast cfDNA panel. Conclusion The Oncomine™ cfDNA panels provide accurate detection of variants in cfDNA down to 0.1% variant allele frequency. However, increased DNA inputs are required to achieve this highly-sensitive level of detection. Although sensitivity is reduced at lower inputs, the ability to generate high-quality libraries from as little as 10 ng DNA ensures sufficient material can be obtained for sequencing from even low-yielding samples. Overall, these results indicate that the Oncomine™ cfDNA assays can aid in the very early diagnosis and monitoring of tumor populations via minimally-invasive blood-based diagnostics. Citation Format: Nathan Riccitelli, Nancy Valencia, Ashley Beams, Sarah Johnson, Reinhold Pollner. Progress toward non-invasive oncology diagnostics: Evaluation of cfDNA next-generation sequencing assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2282.

Converting Prescriptions to 90-day Supply Increases Specialty Pharmacy Technician Efficiency

The mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder “phenotype” (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels (r = 0.898; P < .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.

Neurofilament Light Chain (NfL) Levels in CSF, Serum, and Plasma of Healthy Donors Using the Quanterix NfL Advantage Kit™ (P1.9-032)

Neurofilament Light Chain (NfL) Levels in CSF, Serum, and Plasma of Healthy Donors Using the Quanterix NfL Advantage Kit™ (P1.9-032)

Sugars make the difference – Glycosylation of cardiodepressant antibodies regulates their activity in dilated cardiomyopathy

BackgroundCardiodepressant antibodies contribute to cardiac dysfunction in dilated cardiomyopathy (DCM). Changes in immunoglobulin G (IgG) glycosylation modulate the activity of various autoimmune diseases and influence disease activity as well as severity of various autoimmune diseases. We hypothesized that alterations in IgG glycosylation are involved in the disease course of DCM. Methods and resultsIgG glycosylation was analyzed in plasma samples of 50 DCM patients using a lectin-based ELISA. Negative inotropic (cardiodepressant) activity (NIA) of antibodies was assessed by measuring the effect of purified DCM-IgG on the shortening of isolated rat cardiomyocytes by means of a video-edge detection system. IgG obtained from plasma of healthy blood donors served as control.DCM-IgG contained significantly less sialic acid (−25%) and galactose (−16%; both P < 0.001), but showed no significant differences in core-fucosylation compared to controls. Interestingly, IgG with NIA displayed a lower percentage of sialylation (−16%, P < 0.001) core-fucosylation (−15%, P = 0.015) and galactosylation (−10%, P = 0.129) than IgG without NIA. The extent of NIA was directly associated with IgG sialylation (r = 0.68; P < 0.001) and galactosylation (r = 0.37; P = 0.001). ConclusionReduced sialylation and galactosylation of IgGs enhances their cardiodepressant activity in DCM indicating that changes in IgG glycosylation may be involved in the pathogenesis of DCM.

The Unique Single-Stranded cfDNA Species in Retinoblastoma Patients Blood Plasma: Beyond New HPLC Technology

A significant population of ultrashort (50n-150n) single-stranded DNA fragments were found in exosome-free blood plasma of retinoblastoma patients (6.84 ng ml-1), but not in plasma of healthy donors. An original high resolution HPLC technique has been proposed to reveal and characterize this peculiarity. To solve this task, a novel molecular size exclusion-anion exchange analytical technique was developed. Its applicability to diagnostics and oncogenesis research is quizzed here.

Ultrashort ssDNA in Retinoblastoma Patients Blood Plasma Detected by a Novel High Resolution HPLC Technique: a Preliminary Report.

A significant population of ultrashort (50-150n) single-stranded DNA fragments were found in exosome-free blood plasma of retinoblastoma patients (6.84 ng mL-1), but not in plasma of healthy donors. An original high resolution HPLC technique has been proposed to reveal and characterize this peculiarity. To solve this task, a novel molecular size exclusion - anion exchange analytical technique was developed. Its applicability to diagnostics and oncogenesis research is quizzed here.

Pooled Human Immunoglobulin Preparations as Immunomodulating Drugs

It is time to celebrate the 125th anniversary of the first successful attempt to develop and use a specific high-titer antitoxic serum for treating diphtheria, a deadly infectious disease. This was followed by major advances in passive immunotherapy 75 years ago (production of pooled human IgG for subcutaneous injection) and 50 years ago (widespread technology for producing immunoglobulin preparations for intravenous administration). More than 200 tons of pooled human IgG are produced per year worldwide. The preparation is used primarily for IgG substitution in patients with primary and secondary immunodeficiencies, as well as for an immunomodulating treatment of a growing number of autoimmune and inflammatory diseases. These preparations contain the pooled IgG antibody repertoire of a large population of healthy plasma donors. This repertoire includes antibodies that neutralize pathogens and their factors of virulence, anti-idiotypic antibodies, and antibodies to other foreign and own proteins, as well as to carbohydrate antigens. Naturally polyspecific antibodies that are present in all healthy individuals play an important role as a first-line defense against bacteria and viruses. After exposure to protein-modifying agents, some IgG molecules can acquire the ability to bind novel structurally unrelated antigens. This phenomenon is referred to as induced polyspecificity. The list of these protein-modifying molecules was shown to include low-pH buffers, free heme, pro-oxidative ferrous ions, reactive oxygen species, etc. Such modified antibody preparations may have a therapeutic potential, since their administration to animals with experimental sepsis or aseptic systemic response syndromes significantly improved survival rates, while the same dose of the native preparation had no effect. We also hypothesize that the aggressive protein-modifying molecules released in sites of inflammation and tissue damage could also modify the antigen-binding behavior of surface immunoglobulin B cell receptors and the structurally related T cell receptors. This "specificity editing" of both types of receptors may play a major role in the body's defense mechanisms.

A novel ELISA for the detection of active form of plasminogen activator inhibitor-1 based on a highly specific trapping agent

Plasminogen activator inhibitor-1 (PAI-1) is a labile molecule that exists in four different forms: active, latent, cleaved and target bound form. Although there have been many methods to measure the total PAI-1, the measurement of active form of PAI-1 antigen is still challenging. Here we developed a novel ELISA to detect the active form of PAI-1 based on a highly specific PAI-1 capturing agent which binds to active PAI-1 with high affinity. We also used a highly stable PAI-1 mutant as an assay calibrator to enhance the method's reproducibility. This ELISA has the advantage of measuring both the antigen level and activity of PAI-1 at the same time. The assay had a sensitivity of 0.167 ng/ml and a working range of 0.195–25 ng/ml. The intra- and inter-assay variations were 6.7% and 11.3% respectively. The mean recovery of spiked standard was 102%. We used this strategy to measure the active PAI-1 level in plasma of healthy donors, and had an interesting observation: the PAI-1 level reduced by half after plasma storage for 6 h at room temperature. This finding represents the first observation of activity loss in plasma PAI-1 samples, and may explain large variations in PAI-1 levels (0–100 ng/ml) observed in human samples using commercial assays.

Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway

AbstractThe mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder “phenotype” (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels (r = 0.898; P < .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.

Conjugation of urate-derived electrophiles to proteins during normal metabolism and inflammation

Urate is often viewed as an antioxidant. Here, we present an alternative perspective by showing that, when oxidized, urate propagates oxidative stress. Oxidation converts urate to the urate radical and the electrophilic products dehydrourate, 5-hydroxyisourate, and urate hydroperoxide, which eventually break down to allantoin. We investigated whether urate-derived electrophiles are intercepted by nucleophilic amino acid residues to form stable adducts on proteins. When urate was oxidized in the presence of various peptides and proteins, two adducts derived from urate (Mr 167 Da) were detected and had mass additions of 140 and 166 Da, occurring mainly on lysine residues and N-terminal amines. The adduct with a 140-Da mass addition was detected more frequently and was stable. Dehydrourate (Mr 166 Da) also formed transient adducts with cysteine residues. Urate-derived adducts were detected on human serum albumin in plasma of healthy donors. Basal adduct levels increased when neutrophils were added to plasma and stimulated, and relied on the NADPH oxidase, myeloperoxidase, hydrogen peroxide, and superoxide. Adducts of oxidized urate on serum albumin were elevated in plasma and synovial fluid from individuals with gout and rheumatoid arthritis. We propose that rather than acting as an antioxidant, urate's conversion to electrophiles contributes to oxidative stress. The addition of urate-derived electrophiles to nucleophilic amino acid residues, a process we call oxidative uratylation, will leave a footprint on proteins that could alter their function when critical sites are modified.

Open ADAMTS13 Conformation in Immune-Mediated Thrombotic Thrombocytopenic Purpura Is Induced By Anti-ADAMTS13 Autoantibodies and Corresponds with an Ongoing ADAMTS13 Pathology

Background. Deficient ADAMTS13 activity (TS13:act <10%) induced by anti-ADAMTS13 autoantibodies (autoAbs) causes immune-mediated thrombotic thrombocytopenic purpura (iTTP). Recently we showed that an open ADAMTS13 conformation is characteristic for acute iTTP patients, while folded ADAMTS13 was found in 78% of iTTP patients in remission with an TS13:act >50%. However, also iTTP patients in remission with a persistent (<10%) or moderately restored (10-50%) TS13:act have been described, but their ADAMTS13 conformation is unknown. Intriguingly, the factor responsible for inducing open ADAMTS13 in iTTP patients remains elusive. Identifying the cause of open ADAMTS13 in iTTP will help better understand the pathophysiology of iTTP and could help appreciate the prognosis and better manage the prevention of subsequent relapses.Aim. Determine ADAMTS13 conformation in plasma of iTTP patients during acute TTP and remission when TS13:act is <10%, moderately restored (10-50%) or >50% and investigate if anti-ADAMTS13 autoAbs induce conformational changes in ADAMTS13.Methods. TS13:act was determined in 120 iTTP plasma samples from 4 different centers (Marseille, Milan, Budapest, Utrecht). Samples were categorized according to the presence of clinical symptoms (acute versusremission) and their TS13:act in remission (>50%, 10-50%,<10%). Next, ADAMTS13 conformation was determined in all samples using our ADAMTS13 conformation ELISA. Additionally, presence of anti-ADAMTS13 autoAbs was also determined via ELISA. Finally, IgG's from 18 acute iTTP plasma samples were purified and added to folded ADAMTS13 from healthy donor (HD) plasma to test whether iTTP IgG's are able to induce the open HD ADAMTS13 conformation.Results. Of the 120 iTTP plasma samples, 46 were obtained during the acute (clinical signs present) and 74 during the remission phase (clinical signs absent). Further subdividing remission samples showed that TS13:act was >50% in 41, 10-50% in 14 and <10% in 19 samples. ADAMTS13 was open in 98% (45/46) of the acute samples and folded in 71% (29/41) of the remission samples with TS13:act >50%, confirming our previous results. Interestingly, ADAMTS13 was open in 93% and 89% of remission samples with TS13:act 10-50% and <10%, respectively (chi square, P<0.0001). Since anti-ADAMTS13 autoAbs influence TS13:act in iTTP patients, we next could demonstrate that open ADAMTS13 conformation was linked with presence of anti-ADAMTS13 autoAbs (chi square, P<0.0001) suggesting that anti-ADAMTS13 autoAbs could be a factor able to induce an open ADAMTS13 conformation in iTTP patients. To further test this hypothesis, we purified IgG's from 18 acute iTTP plasma's with open ADAMTS13 and added them to HD plasma containing closed ADAMTS13, where 14 of the 18 patient IgG pools (78%) did induce the open conformation in HD ADAMTS13, indicating that patient anti-ADAMTS13 autoAbs indeed can induce conformational changes in ADAMTS13.Conclusion. We show that ADAMTS13 is not only in the open conformation in iTTP patient plasma during the acute phase but also in remission when TS13:act is <10% or 10-50%. Hence, the presence of open ADAMTS13 in those remission patients indicates that the underlying pathophysiology is still ongoing, emphasizing the need for a close monitoring of those patients. In addition, anti-ADAMTS13 autoAbs were identified as a factor responsible for the change in conformation in ADAMTS13 in iTTP. DisclosuresPeyvandi:Octapharma US: Honoraria; Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Novo Nordisk: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Roche: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Roche: Speakers Bureau; Roche: Speakers Bureau; Grifols: Speakers Bureau; Sobi: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy; Novo Nordisk: Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Kedrion: Consultancy; Kedrion: Consultancy; Grifols: Speakers Bureau; Octapharma US: Honoraria. Coppo:Ablynx: Consultancy. Veyradier:LFB: Other: Investigator. Vanhoorelbeke:Shire: Consultancy; Ablynx: Consultancy.

Plasma exosomes stimulate breast cancer metastasis through surface interactions and activation of FAK signaling

The interaction between malignant cells and surrounding healthy tissues is a critical factor in the metastatic progression of breast cancer (BC). Extracellular vesicles, especially exosomes, are known to be involved in inter-cellular communication during cancer progression. In the study presented herein, we aimed to evaluate the role of circulating plasma exosomes in the metastatic dissemination of BC and to investigate the underlying molecular mechanisms of this phenomenon. Exosomes isolated from plasma of healthy female donors were applied in various concentrations into the medium of MDA-MB-231 and MCF-7 cell lines. Motility and invasive properties of BC cells were examined by random migration and Transwell invasion assays, and the effect of plasma exosomes on the metastatic dissemination of BC cells was demonstrated in an in vivo zebrafish model. To reveal the molecular mechanism of interaction between plasma exosomes and BC cells, a comparison between un-treated and enzymatically modified exosomes was performed, followed by mass spectrometry, gene ontology, and pathway analysis. Plasma exosomes stimulated the adhesive properties, two-dimensional random migration, and transwell invasion of BC cells in vitro as well as their in vivo metastatic dissemination in a dose-dependent manner. This stimulatory effect was mediated by interactions of surface exosome proteins with BC cells and consequent activation of focal adhesion kinase (FAK) signaling in the tumor cells. Plasma exosomes have a potency to stimulate the metastasis-promoting properties of BC cells. This pro-metastatic property of normal plasma exosomes may have impact on the course of the disease and on its prognosis.

Sensitivity and Robustness of Spatially Dependent Thrombin Generation and Fibrin Clot Propagation

Blood coagulation is a delicately regulated space- and time-dependent process that leads to the formation of fibrin clots preventing blood loss upon vascular injury. The sensitivity of the coagulation network was previously investigated without accounting for transport processes. To investigate its sensitivity to coagulation factor deficiencies in a spatial reaction-diffusion system, we combined an in vitro experimental design with a computational systems biology model. Clot formation in platelet-free plasma supplemented with phospholipids was activated with identical amounts of tissue factor (TF) either homogeneously distributed (concentration 5 pM, homogeneous model) or immobilized on the surface (surface density 100 pmole/m2, spatially heterogeneous model). Fibrin clot growth and thrombin concentration dynamic in space were observed using video microscopy in plasma of healthy donors or patients with deficiencies in factors (F) II, FV, FVII, FVIII, FIX, FX, or FXI. In the spatially heterogeneous model, near-activator thrombin generation was decreased in FV-, FVII-, and FX-deficient plasma. In the homogeneous model, clotting was not registered in these samples. The simulation and experiment data showed that the coagulation threshold depended on the TF concentration. Our data indicate that the velocity of spatial clot propagation correlates linearly with the concentration of thrombin at the clot wave front but not with the overall thrombin wave amplitude. Spatial clot growth in normal plasma at early stages was neither reaction nor diffusion limited but became diffusion limited later. In contrast, clot growth was always diffusion limited in FV-, FVII-, and FX-deficient plasma and reaction limited in FVIII-, FIX-, and FXI-deficient plasma. We conclude that robustness of the spatially heterogeneous coagulation system was achieved because of the combination of 1) a local high TF surface density that overcomes activation thresholds, 2) diffusion control being shared between different active factors, and 3) an early saturated stimulus-response dependence of fibrin clot formation by thrombin.

Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERTmutant DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERTmutant tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERTmutant DNA in tumors and plasma of cancer patients.

Evaluation of Preanalytical Conditions and Implementation of Quality Control Steps for Reliable Gene Expression and DNA Methylation Analyses in Liquid Biopsies.

Liquid biopsy provides important information for the prognosis and treatment of cancer patients. In this study, we evaluated the effects of preanalytical conditions on gene expression and DNA methylation analyses in liquid biopsies. We tested the stability of circulating tumor cell (CTC) messenger RNA by spiking MCF-7 cells in healthy donor peripheral blood (PB) drawn into 6 collection-tube types with various storage conditions. CTCs were enriched based on epithelial cell adhesion molecule positivity, and RNA was isolated followed by cDNA synthesis. Gene expression was quantified using RT-quantitative PCR for CK19 and B2M. We evaluated the stability of DNA methylation in plasma under different storage conditions by spiking DNA isolated from MCF-7 cells in healthy donor plasma. Two commercially available sodium bisulfite (SB)-conversion kits were compared, in combination with whole genome amplification (WGA), to evaluate the stability of SB-converted DNA. SB-converted DNA samples were analyzed by real-time methylation-specific PCR (MSP) for ACTB, SOX17, and BRMS1. Quality control was assessed using Levey-Jennings graphs. RNA-based analysis in CTCs is severely impeded by the preservatives used in many PB collection tubes (except for EDTA), as well as by time to analysis. Plasma and SB-converted DNA samples are stable and can be used safely for MSP when kept at -80 °C. Downstream WGA of SB-converted DNA compensated for the limited amount of available sample in liquid biopsies. Standardization of preanalytical conditions and implementation of quality control steps is extremely important for reliable liquid biopsy analysis, and a prerequisite for routine applications in the clinic.