Levels In Plasma Samples Research Articles

Inflammatory Proteins in the Amniotic Fluid, Plasma, and Cervicovaginal Fluid for the Prediction of Intra-Amniotic Infection/Inflammation and Imminent Preterm Birth in Preterm Labor.

This study was aimed to develop models using multiple cytokine/chemokine levels in cervicovaginal fluid (CVF) and plasma and widely used noninvasive parameters that have better accuracy for predicting intra-amniotic infection and/or inflammation (IAI) and imminent spontaneous preterm delivery (SPTD, ≤48 hours) in women with preterm labor (PTL). This was a retrospective cohort study of 95 singleton pregnant women with PTL (23-34 weeks) who underwent amniocentesis. Both CVF and plasma samples were obtained at the time of amniocentesis, and serum C-reactive protein (CRP) levels were measured. The amniotic fluid (AF), CVF, and plasma samples were assayed for interleukin (IL)-6, IL-8, IL-10, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1β (MIP-1β) levels using a multiplex immunoassay kit. The levels of most cytokines/chemokines measured in the AF and CVF were significantly higher in the women with than in those without IAI and imminent SPTD, whereas only high-plasma IL-10 level showed a significant association with imminent SPTD. In predicting IAI, proteins in AF had significantly higher areas under the curves (AUCs) than those in CVF and plasma. However, for predicting imminent SPTD, no significant differences in the AUCs of the outcome-associated proteins were observed among the measurements in AF, CVF, and maternal plasma. By using stepwise regression analyses, noninvasive models (using protein levels in CVF and baseline clinical parameters) were developed for the prediction of IAI and imminent SPTD. The AUC of these noninvasive models were similar to those of the invasive models (using AF protein levels and baseline clinical parameters). Noninvasive models based on CVF cytokine/chemokine levels and widely used noninvasive parameters (especially CRP) act as good indicators for predicting the risk of IAI and imminent SPTD in women with PTL. Evaluation of cytokine/chemokine levels in plasma samples did not add valuable information regarding the two outcome measures in the PTL setting. · Markers in either CVF or plasma alone did not have sufficient accuracy for predicting IAI and SPTD.. · Noninvasive models using CVF cytokine and CRP act as effective tools for predicting two outcomes.. · Evaluation of cytokine level in plasma did not add valuable information regarding two outcomes..

Soluble Urokinase Receptor (SuPAR) in COVID-19-Related AKI.

AKI commonly occurs in patients with coronavirus disease 2019 (COVID-19). Its pathogenesis is poorly understood. The urokinase receptor system is a key regulator of the intersection between inflammation, immunity, and coagulation, and soluble urokinase plasminogen activator receptor (suPAR) has been identified as an immunologic risk factor for AKI. Whether suPAR is associated with COVID-19-related AKI is unknown. In a multinational observational study of adult patients hospitalized for COVID-19, we measured suPAR levels in plasma samples from 352 adult patients that had been collected within 48 hours of admission. We examined the association between suPAR levels and incident in-hospital AKI. Of the 352 patients (57.4% were male, 13.9% were black, and mean age was 61 years), 91 (25.9%) developed AKI during their hospitalization, of whom 25 (27.4%) required dialysis. The median suPAR level was 5.61 ng/ml. AKI incidence rose with increasing suPAR tertiles, from a 6.0% incidence in patients with suPAR <4.60 ng/ml (first tertile) to a 45.8% incidence of AKI in patients with suPAR levels >6.86 ng/ml (third tertile). None of the patients with suPAR <4.60 ng/ml required dialysis during their hospitalization. In multivariable analysis, the highest suPAR tertile was associated with a 9.15-fold increase in the odds of AKI (95% confidence interval [95% CI], 3.64 to 22.93) and a 22.86-fold increase in the odds of requiring dialysis (95% CI, 2.77 to 188.75). The association was independent of inflammatory markers and persisted across subgroups. Admission suPAR levels in patients hospitalized for COVID-19 are predictive of in-hospital AKI and the need for dialysis. SuPAR may be a key component of the pathophysiology of AKI in COVID-19.

Proton scanning and X-ray beam irradiation induce distinct regulation of inflammatory cytokines in a preclinical mouse model

Purpose Conventional X-ray radiotherapy induces a pro-inflammatory response mediated by altered expression of inflammation-regulating cytokines. Proton scanning and X-ray irradiation produce distinct changes to cytokine gene expression in vitro suggesting that proton beam therapy may induce an inflammatory response dissimilar to that of X-ray radiation. The purpose of the present study was to determine whether proton scanning beam radiation and conventional X-ray photon radiation would induce differential regulation of circulating cytokines in vivo. Materials and methods Female CDF1 mice were irradiated locally at the right hind leg using proton pencil beam scanning or X-ray photons. Blood samples were obtained from two separate mice groups. Samples from one group were drawn by retro-orbital puncture 16 months post irradiation, while samples from the other group were drawn 5 and 30 days post irradiation. Concentration of the cytokines IL-6, IL-1β, IL-10, IL-17A, IFN-γ, and TNFα was measured in plasma using bead-based immunoassays. Results The cytokines IL-6, IL-1β, IL-10, IFN-γ, and TNFα were expressed at lower levels in plasma samples from proton-irradiated mice compared with X-ray-irradiated mice 16 months post irradiation. The same cytokines were downregulated in proton-irradiated mice 5 days post irradiation when compared to controls, while at day 30 expression had increased to the same level or higher. X-ray radiation did not markedly change expression levels at days 5 and 30. Conclusions The inflammatory response to proton and X-ray irradiation seem to be distinct as the principal pro-inflammatory cytokines are differentially regulated short- and long-term following irradiation. Both the development of normal tissue damage and efficacy of immunotherapy could be influenced by an altered inflammatory response to irradiation.

Impact of exogenous antithrombin on low molecular weight heparin anti-Xa activity assays in a pediatric and young adult leukemia and lymphoma cohort with variable antithrombin levels.

Low molecular weight heparin (LMWH) remains the most commonly prescribed pediatric anticoagulant. There is debate whether LMWH anti-Xa assays with or without exogenous antithrombin (AT) best reflect anticoagulation effect, and how much discrepancy exists between assay types. We assessed the effect of variable AT activity on LMWH anti-Xa levels in plasma samples from anticoagulated pediatric and young adult acute lymphoblastic leukemia and lymphoma (ALL/L) patients, using two instruments and their commercial kits with and without exogenous AT (ie, four platforms). We analyzed LMWH anti-Xa levels on 60 plasma samples with known AT activity from 12 enoxaparin-treated ALL/L patients, using four commercial kits from Siemens and Stago containing AT or not, on Siemens BCS and Stago STA R Max, respectively. Of 236/240 samples with interpretable results, mean AT activity was 80% (46-138%). Correlation was acceptable for published kit ranges of LMWH anti-Xa levels when comparing kits containing AT (r=0.82, P<.0001), or not (r=0.93, P<.0001), and within a manufacturer (Berichrom to Innovance, r=0.92, P<.0001; Stachrom to STA-Liquid Anti-Xa r=0.98, P<.0001). LMWH anti-Xa levels were lower in platforms without added AT (P<.0001). For Stago kits, this effect increased when AT<70% (P=.001, n=19, mean 56%). Assay variability, measured as mean percent difference, was less pronounced with Stago kits (14.7%, n=49) than Siemens (41.9%, n=50). Although LMWH levels from anti-Xa assays with added AT trend higher than in those without, correlation was fairly good between platforms in pediatric ALL/L plasmas, even when AT activity was <70%.

Identification and Validation of Circulating Micrornas as Prognostic Biomarkers in Pancreatic Ductal Adenocarcinoma Patients Undergoing Surgical Resection.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal and aggressive cancers with a less than 6% five-year survival rate. Circulating microRNAs (miRNAs) are emerging as a useful tool for non-invasive diagnosis and prognosis estimation in the various cancer types, including PDAC. Our study aimed to evaluate whether miRNAs in the pre-operative blood plasma specimen have the potential to predict the prognosis of PDAC patients. In total, 112 PDAC patients planned for surgical resection were enrolled in our prospective study. To identify prognostic miRNAs, we used small RNA sequencing in 24 plasma samples of PDAC patients with poor prognosis (overall survival (OS) < 16 months) and 24 plasma samples of PDAC patients with a good prognosis (OS > 20 months). qPCR validation of selected miRNA candidates was performed in the independent cohort of PDAC patients (n = 64). In the discovery phase of the study, we identified 44 miRNAs with significantly different levels in the plasma samples of the group of good and poor prognosis patients. Among these miRNAs, 23 showed lower levels, and 21 showed higher levels in plasma specimens from PDAC patients with poor prognosis. Eleven miRNAs were selected for the validation, but only miR-99a-5p and miR-365a-3p were confirmed to have significantly lower levels and miR-200c-3p higher levels in plasma samples of poor prognosis cases. Using the combination of these 3-miRNA levels, we were able to identify the patients with poor prognosis with sensitivity 85% and specificity 80% (Area Under the Curve = 0.890). Overall, 3-miRNA prognostic score associated with OS was identified in the pre-operative blood plasma samples of PDAC patients undergoing surgical resection. Following further independent validations, the detection of these miRNA may enable identification of PDAC patients who have no survival benefit from the surgical treatment, which is associated with the high morbidity rates.

Major 5\u2032terminally deleted enterovirus populations modulate type I IFN response in acute myocarditis patients and in human cultured cardiomyocytes

Major 5′terminally deleted (5′TD) group-B enterovirus (EV-B) populations were identified in heart biopsies of patients with fulminant myocarditis or dilated cardiomyopathy suggesting that these 5′TD forms are key drivers of host-cell interaction in EV cardiac infections. To date, early emergence of EV-B 5′TD forms and its impact on type 1 IFN response during acute myocarditis remains unknown. Using quantitative RACE-PCR assay, we identified major EV-B 5′TD RNA populations in plasma or heart samples of acute myocarditis cases. Deletions identified within the 5′ non-coding region of EV-B populations only affected secondary-structural elements of genomic RNA domain I and were distinguished in two major groups based on the extent of RNA structural deletions. Proportions of these two respective EV-B 5′TD population groups were positively or negatively correlated with IFN-β levels in plasma samples of myocarditis patients. Transfection of synthetic CVB3/28 RNAs harboring various 5′terminal full-length or deleted sequences into human cultured cardiomyocytes demonstrated that viral genomic RNA domain I possessed essential immunomodulatory secondary-structural elements responsible for IFN-β pathway induction. Overall, our results highlight the early emergence of major EVB-TD populations which deletions affecting secondary–structures of RNA domain I can modulate innate immune sensing mechanisms in cardiomyocytes of patients with acute myocarditis.

Effect of rare coding variants in the CFI gene on Factor I expression levels.

Factor I (FI) is one of the main inhibitors of complement activity, and numerous rare coding variants have been reported in patients with age-related macular degeneration, atypical hemolytic uremic syndrome and C3 glomerulopathy. Since many of these variants are of unknown clinical significance, this study aimed to determine the effect of rare coding variants in the complement factor I (CFI) gene on FI expression. We measured FI levels in plasma samples of carriers of rare coding variants and in vitro in the supernatants of epithelial cells expressing recombinant FI. FI levels were measured in 177 plasma samples of 155 individuals, carrying 24 different rare coding variants in CFI. In carriers of the variants p.Gly119Arg, p.Leu131Arg, p.Gly188Ala and c.772G>A (r.685_773del), significantly reduced FI plasma levels were detected. Furthermore, recombinant FI expression levels were determined for 126 rare coding variants. Of these variants 68 (54%) resulted in significantly reduced FI expression in supernatant compared to wildtype (WT). The recombinant protein expression levels correlated significantly with the FI level in plasma of carriers of CFI variants. In this study, we performed the most comprehensive FI expression level analysis of rare coding variants in CFI to date. More than half of CFI variants lead to reduced FI expression, which might impair complement regulation in vivo. Our study will aid the interpretation of rare coding CFI variants identified in clinical practice, which is in particular important in light of patient inclusion in ongoing clinical trials for CFI gene supplementation in AMD.

The potential of microRNA-126 in predicting disease risk, mortality of sepsis, and its correlation with inflammation and sepsis severity.

BackgroundMicroRNA‐126 (miR‐126) has been investigated in autoimmune diseases and organ failures, whereas its implication in sepsis is rarely reported. Our study initially explored the value of miR‐126 in diagnosing sepsis and predicting disease severity, degree of inflammation, and mortality.MethodsTotally, 208 sepsis patients and 210 healthy controls were enrolled; then, their plasma samples were collected for detecting circulating miR‐126 by quantitative polymerase chain reaction. For sepsis patients, their cytokine levels in plasma samples were detected by enzyme‐linked immunosorbent assay.ResultsmiR‐126 was upregulated in sepsis patients compared with healthy controls, and it was of certain value in distinguishing sepsis patients from healthy controls (AUC: 0.726 (95% CI: 0.678‐0.774)). miR‐126 expression was positively correlated with acute physiology and chronic health evaluation II score, serum creatinine, and C‐reactive protein but not albumin or white blood cell count in sepsis patients. Regarding cytokines, miR‐126 was positively correlated with tumor necrosis factor‐α, interleukin (IL)‐6, and IL‐8, but negatively correlated with IL‐10 in sepsis patients. As for mortality, miR‐126 expression was higher in deaths compared with survivors, and ROC curve displayed that it could predict mortality of sepsis patients to some extent with AUC of 0.619 (95% CI: 0.533‐0.705).ConclusionmiR‐126 potentially serves as an assistant diagnostic and prognostic biomarker for sepsis.

Isoprostanoids Levels in Cerebrospinal Fluid Do Not Reflect Alzheimer's Disease.

Previous studies showed a relationship between lipid oxidation biomarkers from plasma samples and Alzheimer’s Disease (AD), constituting a promising diagnostic tool. In this work we analyzed whether these plasma biomarkers could reflect specific brain oxidation in AD. In this work lipid peroxidation compounds were determined in plasma and cerebrospinal fluid (CSF) samples from AD and non-AD (including other neurological pathologies) participants, by means of an analytical method based on liquid chromatography coupled with mass spectrometry. Statistical analysis evaluated correlations between biological matrices. The results did not show satisfactory correlations between plasma and CSF samples for any of the studied lipid peroxidation biomarkers (isoprostanes, neuroprostanes, prostaglandines, dihomo-isoprostanes). However, some of the analytes showed correlations with specific CSF biomarkers for AD and with neuropsychological tests (Mini-Mental State Examination (MMSE), Repeatable Battery for the Assessment of Neuropsychological Status (RBANS)). In conclusion, lipid peroxidation biomarkers in CSF samples do not reflect their levels in plasma samples, and no significant differences were observed between participant groups. However, some of the analytes could be useful as cognitive decline biomarkers.

Overexpression of tissue factor pathway inhibitor 2 attenuates trophoblast proliferation and invasion in preeclampsia.

Pre-eclampsia (PE) is a disorder of pregnancy characterized by proteinuria and high blood pressure, affecting 2-8% of pregnancies worldwide. Previous studies have shown that PE is closely associated with trophoblast cell dysfunction. Here, we investigated the role of tissue factor pathway inhibitor-2 (TFPI-2) in regulating the biological processes of trophoblast cells. The TFPI-2 levels in plasma samples and placental tissues were tested by ELISA, immunohistochemistry, qRT-PCR, and western blot. HTR8/Svneo cell line was used to simulate the primary trophoblast cells and H/R culture was applied to mimic the oxidative stress state of PE. MTT assay, Annexin V/propidium iodide (PI) apoptosis assay, and transwell assay were used to determine the cell proliferation, apoptosis, and invasion. The expression levels of matrix metalloproteinases (MMPs) were evaluated by western blot. The expression of TFPI-2 was remarkably up-regulated in both the serum and placenta of PE patients. Hypoxia/reoxygenation increased the expression of TFPI-2 in HTR-8/SVneo cell line. TFPI-2 promoted that cell proliferation and inhibited the cell apoptosis of HTR8/SVneo cells in H/R condition. In addition, downregulation of TFPI-2 increased the cell invasion and the expression of MMP2 and MMP9. This study reveals that TFPI-2 plays a crucial role in monitoring the biological function of trophoblast cells, which might provide theoretical basis and therapeutic targets for the treatment of PE.

Structure–bioavailability relationship study of genistein derivatives with antiproliferative activity on human cancer cell

The present study assesses the in vitro and in vivo bioavailability of genistein derivatives, hydroxyalkyl- and glycosyl alkyl ethers (glycoconjugates). Studies were carried out using compounds that exhibit higher in vitro antiproliferative activity in comparison with the parent isoflavone.Based on in vitro experiments using the Parallel Artificial Membrane Permeability Assay (PAMPA) and the Caco-2 cell monolayer permeability model, we found that modification of the isoflavone structure by O-alkylation improved bioavailability in comparison to genistein. Additionally, the structure of the substituent and its position on genistein influenced the type of mechanism involved in the transport of compounds through biological membranes. The PAMPA assay showed that the structure of glycoconjugates had a significant influence on the passive transport of the genistein synthetic derivatives through a biological membrane. Preferentially the glycoconjugates containing O-glycosidic bond were transported and the transport rate decreased as the carbon linker increased. For glycoconjugates, determination of their transport and metabolism through the Caco-2 membrane was not possible due to interaction with the membrane surface, probably by the change of compound structure caused by contact with the cells or degradation in medium.The intestinal absorption and metabolism of genistein and three derivatives, Ram-3, Ram′-3 and Ram-C-4α (Fig. 1), were tested in vivo in rats. We found that in comparison to genistein, glycoconjugates were metabolized more slowly and to a lesser extent. As part of the in vivo research, we performed analysis of compound levels in plasma samples after enzymatic hydrolysis, but in the collected samples, analytes were not observed. We hypothesize that glycoconjugates compounds bind plasma proteins and were removed from the sample.In conclusion, we show that O-functionalization of the natural, biologically active isoflavone genistein can affect biological activity, bioavailability, and the rate of compound metabolism. The position of the substituent, the length of the linker and the structure of sugar moieties provides a tool for the optimization of the derivative's biological properties.

Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study.

Extracellular vesicles (EVs) have attracted increasing attention because of their potential roles in various biological processes and medical applications. However, isolation of EVs is technically challenging mainly due to their small and heterogeneous size and contaminants that are often co-isolated. We have thus designed a two-step magnetic bead-based (2MBB) method for isolation a subset of EVs as well as their microRNAs from samples of a limited amount. The process involves utilizing magnetic beads coated with capture molecules that recognize EV surface markers, such as CD63. Captured EVs could be eluted from beads or lyzed directly for subsequent analysis. In this study, we used a second set of magnetic beads coated with complementary oligonucleotides to isolate EV-associated microRNAs (EV-miRNAs). The efficiencies of 2MBB processes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) with spiked-in exogenous cel-miR-238 molecules. Experimental results demonstrated the high efficiency in EV enrichment (74 ± 7%, n = 4) and miRNA extraction (91 ± 4%, n = 4). Transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) show that captured EVs enriched by 2MBB method could be released and achieved a higher purity than the differential ultracentrifugation (DUC) method (p < 0.001, n = 3). As a pilot study, EV-miR126-3p and total circulating cell-free miR126-3p (cf-miR126-3p) in eight clinical plasma samples were measured and compared with the level of protein markers. Compared to cf-miR126-3p, a significant increase in correlations between EV-miR126-3p and cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) was detected. Furthermore, EV-miR126-3p levels in plasma samples from healthy volunteers (n = 18) and high-risk cardiovascular disease (CVD) patients (n = 10) were significantly different (p = 0.006), suggesting EV-miR126 may be a potential biomarker for cardiovascular diseases. 2MBB technique is easy, versatile, and provides an efficient means for enriching EVs and EV-associated nucleic acid molecules.

Development and Validation of Analytical Method for SH-1242 in the Rat and Mouse Plasma by Liquid Chromatography/Tandem Mass Spectrometry.

SH-1242, a novel inhibitor of heat shock protein 90 (HSP90), is a synthetic analog of deguelin: It was previously reported that the treatment of SH-1242 led to a strong suppression of hypoxia-mediated retinal neovascularization and vascular leakage in diabetic retinas by inhibiting the hypoxia-induced upregulation of expression in hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). In this study, an analytical method for the quantification of SH-1242 in biological samples from rats and mice was developed/validated for application in pharmacokinetic studies. SH-1242 and deguelin, an internal standard of the assay, in plasma samples from the rodents were extracted with methanol containing 0.1% formic acid and analyzed at m/z transition values of 368.9→151.0 and 395.0→213.0, respectively. The method was validated in terms of accuracy, precision, dilution, matrix effects, recovery, and stability and shown to comply with validation guidelines when it was used in the concentration ranges of 1–1000 ng/mL for rat plasma and of 2–1000 ng/mL for mouse plasma. SH-1242 levels in plasma samples were readily determined using the developed method for up to 480 min after the intravenous administration of 0.1 mg/kg SH-1242 to rats and for up to 120 min to mice. These findings suggested that the current method was practical and reliable for pharmacokinetic studies on SH-1242 in preclinical animal species.

Evaluation of Intact Parathyroid Hormone Levels in Plasma Samples: A Comparative Study Using Serum Samples

Evaluation of Intact Parathyroid Hormone Levels in Plasma Samples: A Comparative Study Using Serum Samples

Plasma metabolomics in tuberculosis patients with and without concurrent type 2 diabetes at diagnosis and during antibiotic treatment

Tuberculosis (TB) and type 2 diabetes mellitus (DM), a major TB risk factor, are both accompanied by marked alterations in metabolic processes. Dissecting the specific metabolic changes induced by disease through metabolomics has shown potential to improve our understanding of relevant pathophysiological mechanisms of disease, which could lead to improved treatment. Targeted tandem liquid chromatography–mass spectrometry (LC-MS/MS) was used to compare amine and acylcarnitine levels in plasma samples of patients with TB or TB-DM from Indonesia at time of diagnosis and during antibiotic treatment. Partial least squares discrimination analysis (PLS-DA) showed good separation of patient groups. Amine levels were strongly altered in both disease groups compared to healthy controls, including low concentrations of citrulline and ornithine. Several amino acid ratios discriminated TB from controls (phenylalanine/histidine; citrulline/arginine; kynurenine/tryptophan), possibly reflecting changes in indoleamine-pyrrole 2,3-dioxygenase (IDO) and nitric oxide synthase (NOS) activity. Choline, glycine, serine, threonine and homoserine levels were lower in TB-DM compared to TB, and, in contrast to other analytes, did not normalize to healthy control levels during antibiotic treatment. Our results not only provide important validation of previous studies but also identify novel biomarkers, and significantly enhance our understanding of metabolic changes in human TB and TB-DM.

In-tube solid-phase microextraction with a dummy molecularly imprinted monolithic capillary coupled to ultra-performance liquid chromatography-tandem mass spectrometry to determine cannabinoids in plasma samples

A selective and sensitive method that uses automated in-tube solid-phase microextraction coupled to ultra-performance liquid chromatography–tandem mass spectrometry (in-tube SPME/UHPLC–MS/MS) was developed to determine cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) in plasma samples. A new dummy molecularly imprinted monolithic capillary (MIP monolith) for in-tube SPME was prepared by in situ polymerization in a fused silica capillary; hydrogenated cannabidiol was employed as dummy template. Fourier Transform Infrared Spectroscopy (FTIR) confirmed that the synthesis reagents were incorporated into the polymer chain. On the basis of the microscopy images (scanning electron microscopy – SEM and transmission electron microscopy – TEM), the MIP monolithic phase presented larger pores than the non-imprinted monolithic phase (NIP monolith), as well as a skeleton comprising clusters consisting of microspheres. By optimizing the polymerization conditions, the MIP monolith specifically recognized CBD and Δ9-THC. The MIP monolith had CBD and Δ9-THC sorption capacity of 148.05 and 44.49 ng cm−3, respectively. The capillary was reused over fifty times without significant changes in its extraction efficiency. For both CBD and Δ9-THC, in-tube SPME/UHPLC–MS/MS presented linear range from 10 to 300 ng mL−1, precision with coefficient of variation (CV) values ranging from 0.2% to 19.1% (LLOQ), and accuracy with relative standard deviation (RSD) values spanning from −9.3% to 19.6% (LLOQ). The developed method was successfully applied to determine cannabinoid levels in plasma samples from volunteer patients in treatment with CBD.

Short Diagnosis-to-Treatment Interval Is Associated with Higher Levels of Circulating Tumor DNA in Aggressive B-Cell Non-Hodgkin Lymphoma

BACKGROUND Selection biases can impair the generalizability of clinical trials. Studies investigating aggressive diseases such as Diffuse Large B-cell Lymphoma (DLBCL) can be particularly affected by such biases since clinical urgency and need for therapy may not allow the requisite extensive screening and consent processes for trials. Diagnosis-to-Treatment Interval (DTI) has recently been proposed as a novel metric to capture this phenomenon (Maurer et al, JCO, 2018), and short DTI is associated with both adverse clinical factors and adverse clinical outcomes. Intriguingly, DTI was independent of clinical risk factors like the International Prognostic Index (IPI) suggesting that widely applied prognostic scores do not adequately reflect risk factors considered for clinical decision making. In this study, we aim to assess whether pretreatment levels of circulating tumor DNA (ctDNA) are associated with shorter DTI and may constitute an objective measure of clinical urgency. METHODS We quantified pretreatment ctDNA levels in plasma samples from 178 patients treated in 5 US and European centers for large cell lymphoma (DLBCL, Follicular lymphoma grade 3b, or High-grade-B-cell-lymphoma) using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) as previously described (Kurtz, JCO 2018; Scherer, STM 2016). Pretreatment ctDNA levels were correlated with DTI, clinical factors and treatment outcome. RESULTS Pretreatment ctDNA was detectable in 175/178 cases. Median number of single nucleotide variants (SNV) detected per patient was 129 (range 0-628). Pretreatment ctDNA levels ranged from 0 - 1.4 x105 haploid genome equivalents per milliliter of plasma (hGE/ml, median 239). Median DTI was 19 days (range 0-141, Figure 1A) and was similar in distribution to 2 previously described cohorts from the US and Europe (Maurer et al, JCO 2018). Shorter DTI was associated with higher ctDNA levels (RS=-0.39, P= 1.4 x10-7, Figure 1B). Patients with longer DTI had improved Event-Free Survival (EFS, Hazard Ratio (HR) for DTI: 0.9/week, P= 0.03). However, this association was lost when adjusting for pretreatment ctDNA levels (HR for DTI: 0.95/week, P= 0.39; HR for log10(ctDNA): 1.7, P= 5.8 x10-5). In a multivariate analysis including DTI, ctDNA and IPI, only ctDNA levels were significantly associated with EFS (HR for log10(ctDNA): 1.6, P= 0.002, n=178, Figure 1C). Pretreatment ctDNA levels remained the only prognostic factor for EFS in a second multivariate analysis also considering pretreatment metabolic tumor volume (MTV, HR for log10(ctDNA): 1.8, P= 0.01, n=93, Figure 1D). DISCUSSION Shorter DTI is associated with higher pretreatment ctDNA levels in patients with aggressive B-cell lymphomas. When comparing to established factors (DTI, IPI, MTV), pretreatment ctDNA levels appear to best predict clinical outcomes. This suggests that quantification of ctDNA better reflects disease burden and treatment urgency than existing clinical biomarkers. Pretreatment ctDNA level may therefore be a valuable metric for disease aggressiveness of patients included in clinical trials, and may help identify studies suffering from selection bias. This may be particularly useful for noncontrolled Phase I/II single arm trials, but also for stratification in randomized trials. Disclosures Kurtz: Roche: Consultancy. Dührsen:Alexion: Honoraria; Novartis: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Honoraria; Takeda: Consultancy, Honoraria; Celgene: Research Funding; CPT: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Teva: Honoraria; Roche: Honoraria, Research Funding. Hüttmann:Takeda: Honoraria; Gilead: Honoraria; University Hospital Essen: Employment. Westin:Juno: Other: Advisory Board; Novartis: Other: Advisory Board, Research Funding; Janssen: Other: Advisory Board, Research Funding; Kite: Other: Advisory Board, Research Funding; Curis: Other: Advisory Board, Research Funding; Celgene: Other: Advisory Board, Research Funding; 47 Inc: Research Funding; Unum: Research Funding; MorphoSys: Other: Advisory Board; Genentech: Other: Advisory Board, Research Funding. Gaidano:AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sunesys: Consultancy, Honoraria. Rossi:Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Diehn:Novartis: Consultancy; BioNTech: Consultancy; AstraZeneca: Consultancy; Quanticell: Consultancy; Roche: Consultancy. Alizadeh:Pfizer: Research Funding; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Roche: Consultancy.

1541. A Novel and Fast Liquid Chromatography Method for Determination of Fluoroquinolones in Human Plasma

BackgroundFluoroquinolones (FQs) are frequently used antimicrobial agents. Considering that the FQs exhibit concentration-dependent bactericidal activity, concentrations of FQs in the biological fluids must be monitored to ensure treatment success. The literature search revealed that there is no method for the determination of levofloxacin (LEV), ciprofloxacin (CIP), moxifloxacin (MOX), and gemifloxacin (GEM) in plasma up to date. Consequently, the aim of this study was to develop and validate a new high-performance liquid chromatography (HPLC) method for determination of these FQs in plasma and evaluate effects of concomitant drugs on plasma FQ concentrations of patients.MethodsBlank plasma samples spiked with FQs were employed for method validation studies. Validation studies were conducted in accordance with the recommendations of the US FDA. In order to demonstrate feasibility of method, 5 patients with polypharmacy, receiving orally CIP, LEV, or MOX as part of their treatment were included in the study. Blood samples were collected at two different times, just before and 2 hours after the second drug administration.ResultsThe separation of FQs was accomplished within 7.5 minutes. The method was linear in the range of 0.1–10 µg/mL with the correlation coefficient >0.99. The RSD at four concentration levels (0.1, 0.3, 4, and 8 µg/mL) was less than 7% with accuracy in the range of 91.8–111.9%.The method was applied to the determination of CIP, LEV, and MOX levels in plasma samples of 5 patients with polypharmacy. Determined CIP and LEV levels were in accordance with literature. On the other hand, MOX concentration 2 hours after administration in plasma of one patient was found to be 6.1 ± 0.1 µg/mL which was higher than previously reported maximum plasma concentration of MOX (4.5 µg/mL). The patient had hypoalbuminemia and MOX is approximately 50% bound to serum proteins. Due to low level of albumin, the level of free MOX in plasma may be increased.ConclusionA simple, fast, and reliable HPLC method was developed and validated for the determination of LEV, CIP, MOX, and GEM in plasma. It is suitable therapeutic drug monitoring of these FQs and can be applied to other pharmacokinetic and toxicological studies.Disclosures All authors: No reported disclosures.

Biochemical parameters and oxidative stress markers in Tunisian patients with periodontal disease

BackgroundOxidative stress is involved in many diseases including diabetes and cancer. Numbers of studies have suggested its involvement in the pathogenesis of periodontal diseases. The aim of this study was to evaluate the levels of biochemical parameters and oxidative stress markers in plasma of healthy and chronic periodontitis patients.MethodsOne hundred thirty subjects were divided into two groups; patients (mean age = 42 ± 13.6 y.o) and control (mean age = 44.8 ± 12.6 y.o). Patients and healthy subjects were free from any infection, coronary or heart disease, diabetes or liver failure. Total cholesterol, LDLc, HDLc, Triglycerides (TG), creatinine, uric acid (UA), glucose and urea levels as well as the activities of enzymatic antioxidants such as catalase, glutathione reductase (GR) and total antioxidant capacity (TAOC), were measured in plasma samples using colorimetric assays. Statistical differences between groups were determined by Student’s t-test and p ≤ 0.05 was considered as significant.ResultsPeriodontitis patients exhibited significant decrease in the activities of catalase, TAOC, GR and TG, cholesterol, LDLc, glucose, HDLc, uric acid levels in plasma samples in comparison with healthy subjects. However, no statistically significant differences in the levels of creatinine and urea were observed between the two groups.ConclusionThe reduction of plasma antioxidant activities (Catalase, TAOC, GR) may have a role in the pathogenesis of periodontal diseases. Our findings suggest a decrease in the host capacity to control the damage caused by oxidative stress. Therefore, therapeutic strategies, aiming at modulating the oxidative stress could be considered as potential tools for the prevention or treatment of periodontal diseases and their potential systemic effects on the general health.

Validation of Circulating Tumor DNA Assays for Detection of Metastatic Melanoma.

The detection of cell-free, circulating tumor DNA (ctDNA) in the blood of patients with solid tumors is often referred to as "liquid biopsy." ctDNA is particularly attractive as a candidate biomarker in the blood. It is relatively stable after blood collection, can be easily purified, and can be quantitatively measured with high sensitivity and specificity using advanced technologies. Current liquid biopsy research has focused on detecting and quantifying ctDNA to (1) diagnose and characterize mutations in a patient's cancer to help select the appropriate treatment; (2) predict clinical outcomes associated with different treatments; and (3) monitor the response and/or progression of a patient's disease. The diagnostic use of liquid biopsies is probably greatest in tumors where the difficulty and/or risk of obtaining a tissue specimen for molecular diagnostics is high (e.g., lung, colon). In metastatic melanoma, however, obtaining a tissue sample for molecular diagnostics is not typically a major obstacle to patient care plans; rather predicting treatment outcomes and monitoring a patient's disease course during therapy are considered the current priorities for this cancer type. In this chapter we describe an approach to the validation of ctDNA detection assays for melanoma, focusing primarily on analytical validation, and provide methods to guide the use of droplet digital PCR assays for measuring ctDNA levels in plasma samples.