Paired Plasma Samples Research Articles

Using the PCI-IS Method to Simultaneously Estimate Blood Volume and Quantify Nonvitamin K Antagonist Oral Anticoagulant Concentrations in Dried Blood Spots.

Nonvitamin K antagonist oral anticoagulants (NOACs) have emerged as the preferred choice for the treatment of atrial fibrillation (AF). The establishment of a therapeutic range to minimize bleeding and thrombosis is important for personalized treatment of NOACs. The importance of dried blood spots (DBSs) has increased in medical care. An efficient and effective DBS analytical method could facilitate the concentration management of NOACs. The postcolumn infused internal standard (PCI-IS) method was applied to estimate spot volume and quantify dabigatran, rivaroxaban, and apixaban concentrations on DBS cards. The extraction solvent contented 0.1% formic acid and 70% ACN with a successive extraction procedure. Paired DBS and plasma samples from patients undergoing NOAC therapy (n = 269) were used to calculate conversion factors. [13C6]-Rivaroxaban was selected as the PCI-IS. The quantification accuracy for the three NOACs was within 88.9-104.3%. The RSDs of the repeatability and intermediate precision were below 10%. The obtained conversion factors of DBS to plasma concentrations of dabigatran, apixaban, and rivaroxaban were 1.81, 1.59, and 1.31, respectively. Bland-Altman analysis showed that the % differences between predicted and measured plasma concentrations were within a bias of ±20%. The result showed that PCI-IS was an accurate and efficient LC-MS/MS method to simultaneously estimate blood volume and NOAC concentrations on DBS cards. The stability results revealed that the DBS sampling strategy could improve compound stability. The developed method offers a new strategy for the therapeutic drug monitoring of NOACs and may improve the safe use of these drugs.

Circulating miR-21 is Down-Regulated Following Adjuvant Chemotherapy in Breast Cancer Patients

Breast cancer is one of the most prevalent known cancers in women with the incidence rate of annually 7000 cases in Iran. MicroRNAs (miRNAs) are a group of non-coding RNAs with 20–25 nucleotides length, which can effect on cancer development. There is low information about the influence of anti-cancer drugs on the expression levels of miRNAs. The present study is the first investigation to evaluate the down-regulation of circulating microRNA-21 (miR-21) expression in breast cancer patients after treatment with Doxorubicin and 5-fu drugs. The expression levels of miR-21 in paired blood plasma samples of 40 new cases of breast cancer patients, were analyzed by Real-Time Polymerase Chain Reaction (RT-PCR) with 2 repeats for each sample. miR-21 rates were evaluated before and after the treatment with Doxorubicin and 5-FU medicines and data were analyzed using t-test. Comparisons between plasma miRNAs before and after the chemotherapy suggested that the treatment has down regulated the circulating miR-21 expression in both HER2+ (Human Epidermal growth factor Receptor 2 positive) and HER2—breast cancer patients. According to the obtained results, it can be suggested that Doxorubicin and 5-FU drugs significantly decrease the circulating miR-21 expression, which might play a key role in anti-tumor effects (0.15 Fold change, P-value < 0.0001).

Diagnostic test accuracy of droplet digital PCR for the detection of EGFR mutation (T790M) in plasma: Systematic review and meta-analysis

BackgroundT790M mutation was a primary lead cause in the acquired resistance to EGFR-TKIs confirmed in earlier studies. Since the shortcomings of tumor tissue detection are well known, the liquid biopsy is more appropriate to track T790M status. We assessed the accuracy and clinical significance of the droplet digital PCR (ddPCR) detection of T790M mutation in plasma. MethodsWe retrieved PubMed, Embase, Cochrane, and Web of science with no limitation of language and publication year. Summary sensitivity and specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio of detection of EGFR T790M status were calculated from extracted data from included articles. The summary receiver operating curve (SROC), diagnostic odds ratio (DOR), and the area under the summary receiver operating curve (AUC) was used to assess the overall diagnostic accuracy. I2 and meta-regression were used to evaluate heterogeneity and the source of heterogeneity, respectively. ResultWe identified 15 studies in the total search of 1364 reports, including 427 paired tissue and plasma samples. The pooled sensitivity and the pooled specificity were 0.68 (95% CI 0.61–0.75) and 0.85 (95% CI 0.75–0.91) by the bivariate model, respectively. The AUC and the pooled DOR were 0.78 (95% CI 0.74–0.81) and 12 (95% CI 7–22), respectively. None of the cofactors could account for the heterogeneity. ConclusionThe plasma analysis is of a promising performance to screen EGFR-T790M mutation status by ddPCR.

Systemic and central nervous system metabolic alterations in Alzheimer\u2019s disease

BackgroundMetabolic alterations, related to cerebral glucose metabolism, brain insulin resistance, and age-induced mitochondrial dysfunction, play an important role in Alzheimer’s disease (AD) on both the systemic and central nervous system level. To study the extent and significance of these alterations in AD, quantitative metabolomics was applied to plasma and cerebrospinal fluid (CSF) from clinically well-characterized AD patients and cognitively healthy control subjects. The observed metabolic alterations were associated with core pathological processes of AD to investigate their relation with amyloid pathology and tau-related neurodegeneration.MethodsIn a case-control study of clinical and biomarker-confirmed AD patients (n = 40) and cognitively healthy controls without cerebral AD pathology (n = 34) with paired plasma and CSF samples, we performed metabolic profiling, i.e., untargeted metabolomics and targeted quantification. Targeted quantification focused on identified deregulated pathways highlighted in the untargeted assay, i.e. the TCA cycle, and its anaplerotic pathways, as well as the neuroactive tryptophan and kynurenine pathway.ResultsConcentrations of several TCA cycle and beta-oxidation intermediates were higher in plasma of AD patients, whilst amino acid concentrations were significantly lower. Similar alterations in these energy metabolism intermediates were observed in CSF, together with higher concentrations of creatinine, which were strongly correlated with blood-brain barrier permeability. Alterations of several amino acids were associated with CSF Amyloidβ1–42. The tryptophan catabolites, kynurenic acid and quinolinic acid, showed significantly higher concentrations in CSF of AD patients, which, together with other tryptophan pathway intermediates, were correlated with either CSF Amyloidβ1–42, or tau and phosphorylated Tau-181.ConclusionsThis study revealed AD-associated systemic dysregulation of nutrient sensing and oxidation and CNS-specific alterations in the neuroactive tryptophan pathway and (phospho)creatine degradation. The specific association of amino acids and tryptophan catabolites with AD CSF biomarkers suggests a close relationship with core AD pathology.Our findings warrant validation in independent, larger cohort studies as well as further investigation of factors such as gender and APOE genotype, as well as of other groups, such as preclinical AD, to identify metabolic alterations as potential intervention targets.

Asymmetric mutant-enriched polymerase chain reaction and quantitative DNA melting analysis of KRAS mutation in colorectal cancer

Identification of mutant genes in tumor tissues and blood plasma (solid and liquid biopsy samples, respectively) is a necessity for individualized treatment of cancer patients. Here we report the use of a novel mutant-enriched PCR - quantitative DNA melting curve analysis (mePCR-qDMA) with TaqMan probes. The TaqMan probes served as blocking agents during PCR and as hybridization probes during DNA melting curve analyses. The end-point measurement of melt peaks areas by PeakFit software, a nonlinear iterative curve-fitting program, permitted quantification of the mutant/wild-type allele ratios. Approximately 6% and 0.1% of mutant KRAS allele in an excess of wild-type allele is detected with the standard and mePCR-qDMA processes, respectively. The application of the approach was tested for detecting the KRAS codon 12/13 mutation in paired tumor and blood plasma samples from 20 colorectal cancer patients. KRAS mutants were detected in 7 and 18 FFPE tumor samples, and in 3 and 7 plasma samples by the standard and mePCR-qDMA process, respectively. The results were confirmed by Sanger sequencing. This simple, rapid, cost-effective, and quantitative method carried out in a closed-tube format could be applied for the clinical analyses of other cancer genes.

Estimating renal and hepatic clearance rates of organophosphate esters in humans: Impacts of intrinsic metabolism and binding affinity with plasma proteins

The renal and hepatic clearance rates of organophosphate esters (OPEs) in humans were estimated. Six OPEs and their corresponding diester metabolites (mOPEs) were quantified respectively in 30 paired human plasma and urine samples collected in Hengshui, Hebei province, China. The renal clearance rate (CLrenal) of triphenyl phosphate (TPHP), tris(chloroethyl) phosphate (TCEP) and tris(1,3-dichloro-isopropyl) phosphate (TDCIPP) was estimated to be 68.9, 50.9 and 33.3 mL/kg/day, respectively, while it was not calculated for other three OPEs due to the low detection frequencies in human samples. To estimate the clearance rates of the target OPEs, hepatic clearance rates (CLh) of OPEs were extrapolated from their in vitro intrinsic clearance data in human liver microsomes (CLHLM). The calculated CLh values of TCEP and TDCIPP were comparable to their CLrenal, indicating that the in vitro extrapolation method was suitable for estimating the clearance rates of OPEs. The higher binding affinity of TDCIPP with plasma proteins could reduce its renal clearance. The estimated half-lives of Cl-OPEs in human were longer than those of the aryl- and alkyl-OPEs. This study provided a feasible in vitro method to predict the clearance and half-lives of OPEs in human, which is significant for their accurate health risk assessment.

Pro-inflammatory cytokines associate with NETosis during sickle cell vaso-occlusive crises

Recurring episodes of acute pain, also referred to as vaso-occlusive crises (VOC), are characteristic of sickle cell disease (SCD), during which pro-inflammatory cytokines, chemokines, adhesion markers and white cell count, some already elevated at steady state, increase further. Hydroxyurea (HU) is licensed by the FDA for reducing frequency of VOCs in SCD; increased fetal hemoglobin (HbF) together with reduction of the neutrophil count and circulating inflammatory markers, contribute to its clinical efficacy.Here, using paired plasma samples from HbSS patients (in steady-state and VOC) we determined that despite HU treatment, the SCD environment remained highly inflammatory and particularly at VOC, triggered neutrophil activity. While neutrophil extracellular traps (NETs) induction by the steady state plasmas were comparable to that of plasma from healthy donors, the NETs response triggered by crisis plasmas was significantly increased over that of the steady state (P = 0.0124*). Levels of IL-6 and IL-1α, IL-1ra/IL1F3 and adhesion molecule P-selectin were significantly increased in the VOC plasma when compared with steady state plasma. Higher levels of IL-6 and IL-1ra were also found in the crises samples that yielded an increased NETs response suggesting that increased NETs production associated with increased levels of the inflammatory products of the IL-6 family and regulators of IL-1 family of cytokines during sickle VOCs.

The Blood Microbiome Predicts Acute Graft-Versus-Host Disease after Stem Cell Transplantation

Introduction Translocation of intestinal bacteria across impaired mucosal barriers has long been believed to occur following exposure to chemotherapy. Consistent with this, we and others have previously reported that expansions of potentially pathogenic bacteria within the gastrointestinal microbiome precedes bloodstream infection. While the blood microbiome represents a rich area for investigation, detailed unbiased characterization of the blood microbiome has not previously been possible. Here, we sought to investigate the relationship between the blood and stool microbiome in patients undergoing allogeneic hematopoietic stem cell transplantation (HCT) who are at high risk for gut barrier dysfunction, severe infection, and the immunological complications of transplantation including graft-versus-host disease (GVHD). We show preliminary data suggesting that the blood microbiome may hold biomarkers for gut integrity. Subsequently, it may offer microbiological data supporting causative organisms in "culture-negative" fevers, or in itself, perform an immunomodulatory role acting as a damage/pathogen associated molecular pattern (DAMP/PAMP). Methods We sequenced serially-collected plasma and stool samples (n = 61 unique samples of each type) from a cohort of 19 patients who underwent allogeneic HCT at our center. Microbial cell-free DNA (mcfDNA) was extracted from plasma and sequenced using a next generation sequencing assay (Karius, Inc, Redwood City, CA). After sequences are processed, mcfDNA abundances are reported in molecules per microliter. Stool samples underwent were profiled using 16S-targeted sequencing (V4-V5 region) on the Illumina MISEQ platform and analyzed using the DADA2 pipeline. Association of blood microbial burden with clinical factors was assessed using a Wilcoxon rank sum test. Results We confirmed a high sequence homology between the DNA found in plasma and stool samples, thus demonstrating that circulating mcfDNA is gut-derived in these patients. By comparing microbial sequences from paired plasma and stool samples collected equivalent time points during the neutropenic nadir after HCT, we observed a correlation between the abundance of bacterial mcfDNA in plasma and DNA from the same microbes in stool. Of note, this occurred independently of conditioning regimen intensity, and was observed in patients who had received myeloablative, non-myeloablative and reduced intensity therapy. We assessed the association of bacterial mcfDNA burden (only considering sequences common to stool sequences) with various clinical factors. Translocation was significantly higher in patients who experienced pre-engraftment mucositis (any observed grade) compared with those who did not (p = 0.02, n = 5 in the mucositis group, any grade, and 12 in the mucositis-free group), but there was no relationship between the degree of translocation and HCT-CI, conditioning intensity, donor type, age or pre-engraftment fevers. We next asked whether translocation events were associated with acute GVHD (aGVHD) by assessing a subgroup of patients who received unmodified grafts (peripheral blood stem cell or bone marrow; n = 10) and tracked the degree of translocation from the gut to the blood stream pre-transplant, during the neutropenic nadir, and following engraftment. As shown in Figure 1, we observed low pre-transplant translocation (as quantified by the total bacterial DNA abundance in plasma where sequences were also shared in stool samples), and a marked increase during neutropenic nadir. Remarkably, even in this small cohort, we observe a higher burden of translocation in the post-engraftment period in patients who subsequently develop aGVHD, while it decreased to baseline levels in those who do not (n = 7 in the aGVHD group; n = 3 aGVHD-free; p = 0.05). Conclusions Here we demonstrate the first use of a culture-free molecular assay to track the blood microbiome and identify features of the circulating mcfDNA that correlate with the microbial DNA in stool samples. Despite the small sample size, these data suggest that the maintenance of a high mcfDNA-burden beyond the neutropenic nadir is associated with subsequent GVHD development, and provides some evidence for early gut barrier dysfunction that permits translocation in these patients. Disclosures Blair: Karius, Inc: Employment. Peled:Seres Therapeutics: Other: IP licensing fees, Research Funding. Giardina:Seres Therapeutics: Other: Salary funding. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Hollemon:Karius, Inc: Employment. Ho:Karius, Inc: Employment. Bercovici:Karius, Inc: Employment. Ahmed:Karius, Inc: Employment. Hong:Karius, Inc: Employment. Giralt:Celgene: Consultancy, Research Funding; Takeda: Consultancy; Sanofi: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. van den Brink:Therakos: Consultancy, Honoraria; Merck &amp; Co, Inc.: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Acute Leukemia Forum (ALF): Consultancy, Honoraria; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Flagship Ventures: Consultancy, Honoraria; Juno Therapeutics: Other: Licensing; Evelo: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria.

EXTH-65. PLASMA AND CEREBROSPINAL FLUID PHARMACOKINETICS COMPARISON OF BRAF AND MEK INHIBITORS FOLLOWING SINGLE AND EXTENDED ADMINISTRATION IN A PRE-CLINICAL NON-HUMAN PRIMATE MODEL

Abstract BACKGROUND Aberrations in the MAPK signaling pathway are present in adult and pediatric CNS tumors. The BRAF and MEK inhibitors Dabrafenib, Vemurafenib, Selumetinib and Trametinib, alone or in combination, have demonstrated clinical efficacy. However, minimal CSF penetration has been demonstrated in pre-clinical models with single dose administration. CSF penetration of these agents was compared following single and extended oral administration in a nonhuman CSF Lateral Reservoir model to determine if CSF penetration increases at steady state. METHODS Agents were administered orally, to each subject (n=9), in single or multiple doses, as serial single agent studies, with washout periods between studies. The following dosages (Human equivalent dose HED) were utilized in the studies: Single Dosage-Selumetinib 2.5 mg/kg (50.0 mg/m2), Dabrafenib 8.0 mg/kg (160 mg/m2), Vemurafenib 26 mg/kg (520 mg/m2), Trametinib 0.06 and 0.12 mg/kg (1.2 and 2.4 mg/m2). Extended dosages-Selumetinib 2.5 mg/kg BID x 3 days (50.0 mg/m2), Dabrafenib 4.0 mg/kg BID x 2 days (160 mg/m2/day), Vemurafenib 26 mg/kg BID x 3 days (520 mg/m2), Trametinib 0.05 mg/kg x 10 days BID (1.0mg/m2). Paired serial plasma and CSF samples were collected. Agents were quantified by uHPLC-MS/MS. Pharmacokinetic parameters were calculated via noncompartmental methods. Plasma exposure reported as Area Under the Curve (AUC) and CSF penetration as ratio of CSF:plasma AUC. Statistical significance determined by t-test. RESULTS Single administration AUCinf/D (hr*ng/ml/mg) vs. extended administration AUCtau/D (hr*ng/ml/mg) for each agent: Vemurafenib-Plasma 30.9–351.4 vs.29.2–379.7, CSF-non-quantifiable; Dabrafenib-Plasma 29.3–102.6 vs.3.4–58.2, CSF-0.12–0.54 vs 0.04–0.19, % CSF:Plasma-0.3–0.84 vs.0.32–1.3; Trametinib-Plasma AUC indeterminable, CSF-non-quantifiable; Selumetinib-Plasma 69.8–133.0 vs.26.5–81.2; CSF-AUC indeterminable vs. 0.37–0.86, % CSF:Plasma-Indeterminable vs. 0.78–1.4. T-test: A downward trend in Dabrafenib and Selumetinib plasma AUC and a marked upward trend in Selumetinib CSF AUC. CONCLUSIONS With the exception of Selumetinib, the CSF exposure of BRAF and MEK inhibitors studied did not markedly increase following extended dosing.

Cadmium-binding proteins in human blood plasma

Blood is the transmission medium for metal contaminants to and from bodily organs; as such, it can provide useful and reliable information about their bio-kinetics as they're distributed throughout the body. Metals can interact with endogenous proteins present in the blood, and these metal-protein complexes often dictate the fates of the introduced metals. The aim of this study was to investigate cadmium-binding protein characteristics in normal human plasma. Cadmium-binding plasma proteins in two different groups: normal human plasma (n = 29), and normal paired maternal and fetal umbilical cord plasmas (n = 3), were analyzed. In order to detect cadmium-binding plasma proteins present in low concentrations, blood plasma samples were first depleted of their two most abundant proteins - albumin and immunoglobulin G. Both the crude and depleted plasma samples were analyzed using column gel electrophoresis in conjunction with Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). One cadmium-binding protein was detected in 11 of 29 normal plasma samples and all three paired maternal and cord plasma samples. This protein was further identified as apolipoprotein A-I by high-resolution mass spectrometry. To the best of our knowledge, this is the first study to reveal cadmium-binding proteins in real human blood plasma, which is extremely critical to our understanding of cadmium transportation and accumulation in human blood.

Assessment of candidate biomarkers in paired saliva and plasma samples from oral cancer patients by targeted mass spectrometry

For oral cancer, numerous saliva- and plasma-derived protein biomarker candidates have been discovered and/or verified; however, it is unclear about the behavior of these candidates as saliva or plasma biomarkers. In this study, we developed two targeted assays, MRM and SISCAPA-MRM, to quantify 30 potential biomarkers in both plasma and saliva samples collected from 30 healthy controls and 30 oral cancer patients. Single point measurements were used for target quantification while response curves for assay metric determination. In comparison with MRM assay, SISCAPA-MRM effectively improved (>1.5 fold) the detection sensitivity of 11 and 21 targets in measurement of saliva and plasma samples, respectively. The integrated results revealed that the salivary levels of these 30 selected biomarkers weakly correlated (r < 0.2) to their plasma levels. Five candidate biomarkers (MMP1, PADI1, TNC, CSTA and MMP3) exhibited significant alterations and disease-discriminating powers (AUC = 0.914, 0.827, 0.813, 0.77, and 0.753) in saliva sample; nevertheless, no such targets could be found in plasma samples. Our data support the notion that saliva may be more suitable for the protein biomarker-based detection of oral cancer, and the newly developed SISCAPA-MRM assay could be applied to verify multiple oral cancer biomarker candidates in saliva samples. SignificanceIn this work we systematically determined the abundance of 30 selected targets in the paired saliva and plasma samples to evaluate the utility of saliva and plasma samples for protein biomarker-based detection of oral cancer. Our study provides significant evidence to support the use of saliva, but not blood samples, offer more opportunity to achieve the success of protein biomarker discovery for oral cancer detection.

Clinical, laboratory and immune aspects of Zika virus-associated encephalitis in children

ObjectiveTo evaluate the clinical, laboratory, and immune characteristics of Zika virus (ZIKV)-associated encephalitis in pediatric patients after the epidemic in Huila, southern Colombia. MethodsA pediatric neuro-surveillance hospital study was conducted in a referral health center in southern Colombia, from October 2016 to October 2017. Cases of encephalitis were confirmed by nucleic acid amplification tests and serological methods in cerebrospinal fluid (CSF), plasma, and/or urine. Levels of six cytokines were evaluated by flow cytometry. Patients underwent daily clinical and laboratory follow-up. ResultsTwenty children with probable encephalitis were included for further studies and 16 of them were confirmed. Four cases of bacterial meningoencephalitis (Streptococcus pneumoniae, group B Streptococcus, Staphylococcus epidermidis, and Escherichia coli) and 12 cases of viral encephalitis were identified, six of them associated with ZIKV infection. Other viral encephalitis cases were caused by herpes viruses (n=3), enterovirus (n=2), and dengue virus type 2 (DENV-2; n=1) infections. ZIKV-associated encephalitis symptoms subsided faster than those of patients with encephalitis caused by other agents. CSF analysis revealed lymphocytic pleocytosis. Compared to healthy controls, children with ZIKV-associated encephalitis presented modest plasma interleukin (IL)-10 but not IL-2, IL-4, IL-6, interferon gamma (IFN-γ), or tumor necrosis factor alpha (TNF-α). Cytokine expression was differentially regulated, as dramatically elevated IL-6, IL-10, and IFN-γ levels were observed in CSF but not in paired plasma samples in one of the patients with ZIKV detectable in CSF. ConclusionsThis study provides evidence that ZIKV is responsible for pediatric encephalitis in endemic areas, and the local presence of the virus may induce cephalic but not systemic expression of cytokines.

P1.14-11 A Prospective Multicenter Study of Target-Capture Deep Sequencing in Paired Tissue and ctDNA to Guide EGFR-Mutated Lung Cancer Treatment

TKIs have significantly improved the survival of NSCLC pts carrying sensitive mutations. However, heterozygous responses were observed. We conducted a prospective multicenter clinical trial to explore factors associated with the efficacy of EGFR-TKI, and assess the mutation and TMB concordance between plasma and tissue NGS. Paired tumor and plasma samples were obtained from treatment naïve advanced NSCLC pts whenever applicable. DNA was sequenced by target-capture deep sequencing of 1021 tumor-related genes (pan-cancer panel). PFS was estimated using Kaplan-Meier method and compared using log-rank test. Tissue TMB (tTMB) and plasma TMB (bTMB) analysis interrogated SNVs/Indels with VAF ≥3% and ≥0.5%, respectively. TMB-H pts were identified with ≥9 muts/Mb. From Feb. 2017 to Jan. 2019, 262 advanced NSCLC pts were enrolled from 12 centers. In 224 pts with paired tumor and plasma samples, 144 had EGFR sensitive mutations in tumor samples (L858R, 46%; Ex19Indel, 42%), of whom, 106 (74%) had the identical mutations detected in plasma. The detection rate of tissue EGFR mutations in paired plasma was significantly higher in pts with extrathoracic metastasis (81% vs. 61%, p = 0.03). In 38 pts lacking paired samples, 20 pts had EGFR sensitive mutations detected. Thus, 164 pts were identified as EGFR positive by either plasma or tissue NGS. One hundred of them were treated EGFR TKIs (ORR: 70%, mPFS: 20 mo). The ORR was affected by EGFR subtypes (Ex19Indel vs. L858R: 72% vs. 45%, p = 0.02), concomitant CNV/fusion (with vs. without: 11% vs. 68%, p = 0.002) and CDKN2A mutations (with vs. without: 0% vs. 66%, p = 0.007). Mutations in p53 pathway (p = 0.02), CDK12/13 (p = 0.0002), concomitant CNV/fusion (p = 0.003), and high number of alterations (≥ 5) (p = 0.003) significantly shortened mPFS. tTMB was correlated with bTMB (rPearson = 0.9, p <0.0001), with a concordance rate of 90% for TMB-H and TMB-L classification. Interestingly, 9.8% of the EGFR positive pts were bTMB-H, and mPFS was shorter in bTMB-H pts (6 mo, 95% CI: 5 - NR) than in bTMB-L pts (NR, 95%: 13 - NR) (p = 0.2). Deep sequencing with the pan-cancer panel effectively detected mutations and evaluated TMB in both tissue and plasma with a high consistence. Moreover, the presence of structure variation, high tumor heterogeneity and concomitant mutations in genes such as CDKN2A were associated with worse prognosis. Further studies of predictive factors are ongoing (NCT03059641).

1417P - Analysis of circulating tumour DNA in paired plasma and sputum samples of EGFR-mutated NSCLC patients

BackgroundLiquid biopsy is a minimally invasive strategy to detect genetic alterations in non-small cell lung cancer (NSCLC) patients. In particular, it is clinically useful for the detection of the EGFR T790M resistance mutation in advanced EGFR-mutated NSCLC patients who had progressed under treatment with an EGFR-TKI. While plasma analysis is commonly performed, sputum could be another potential source for circulating tumor DNA (ctDNA) in NSCLC, which has not yet been entirely evaluated. MethodsPaired plasma and sputum samples from 23 patients with EGFR-mutated lung adenocarcinoma were analyzed for EGFR exon 19 deletions (del19), L858R, and T790M mutations by droplet digital PCR (ddPCR). All patients had histologically confirmed lung adenocarcinoma with EGFR mutations in their initial tissue biopsy at diagnosis. ResultsTissue genotyping at diagnosis revealed 18 patients with EGFR del19 and 5 patients with L858R mutations. Among the 18 EGFR del19-positive patients, 8 showed concordant test results in plasma and sputum (2 positive, 6 negative), 6 had del19-positive plasma samples but del19-negative sputum samples, 4 were del19 negative in plasma and del19 positive in sputum. Among 5 L8585R-positive patients, 4 had concordant test results in plasma and sputum (3 positive, 1 negative), one patient was L858R positive in the plasma sample but L858R negative in sputum. In 10 out of 23 patients the T790M mutation was assessed in paired samples, 9 showed concordant test results in plasma and sputum (3 positive, 6 negative), one patient was T790M positive only in the sputum sample. ConclusionsOur results show a high concordance between plasma and sputum for EGFR L858R and T790M mutation testing. A lower concordance was observed for EGFR del19. Thus liquid biopsy from sputum is a promising strategy that should be further evaluated. Clinical trial identificationGS1-EK4/479-2017, 20.07.2017 EK-18-172-0918, 08.11.2018. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Predicting and Defining Steroid Resistance in Pediatric Nephrotic Syndrome Using Plasma Proteomics

IntroductionNephrotic syndrome (NS) is a characterized by massive proteinuria, edema, hypoalbuminemia, and dyslipidemia. Glucocorticoids (GCs), the primary therapy for >60 years, are ineffective in approximately 50% of adults and approximately 20% of children. Unfortunately, there are no validated biomarkers able to predict steroid-resistant NS (SRNS) or to define the pathways regulating SRNS.MethodsWe performed proteomic analyses on paired pediatric NS patient plasma samples obtained both at disease presentation before glucocorticoid initiation and after approximately 7 weeks of GC therapy to identify candidate biomarkers able to either predict steroid resistance before treatment or define critical molecular pathways/targets regulating steroid resistance.ResultsProteomic analyses of 15 paired NS patient samples identified 215 prevalent proteins, including 13 candidate biomarkers that predicted SRNS before GC treatment, and 66 candidate biomarkers that mechanistically differentiated steroid-sensitive NS (SSNS) from SRNS. Ingenuity Pathway Analyses and protein networking pathways approaches further identified proteins and pathways associated with SRNS. Validation using 37 NS patient samples (24 SSNS/13 SRNS) confirmed vitamin D binding protein (VDB) and APOL1 as strong predictive candidate biomarkers for SRNS, and VDB, hemopexin (HPX), adiponectin (ADIPOQ), sex hormone–binding globulin (SHBG), and APOL1 as strong candidate biomarkers to mechanistically distinguish SRNS from SSNS. Logistic regression analysis identified a candidate biomarker panel (VDB, ADIPOQ, and matrix metalloproteinase 2 [MMP-2]) with significant ability to predict SRNS at disease presentation (P = 0.003; area under the receiver operating characteristic curve = 0.78).ConclusionPlasma proteomic analyses and immunoblotting of serial samples in childhood NS identified a candidate biomarker panel able to predict SRNS at disease presentation, as well as candidate molecular targets/pathways associated with clinical steroid resistance.

Plasma extracellular vesicle microRNAs for pulmonary ground-glass nodules

ABSTRACTIn this study, we evaluated the diagnostic value and molecular characteristics of plasma extracellular vesicles (EVs)-derived miRNAs for patients with solitary pulmonary nodules (SPNs), particularly ground-glass nodules (GGNs). This study was registered at www.clinicaltrials.gov under registration number NCT03230019. Small RNA sequencing was performed to assess plasma EVs miRNAs in 59 patients, including 12 patients with benign nodules (2017, training set). MiRNA profiles of 40 an additional individuals were sequenced (2018, validation set). Overall, 16 pure GGNs, 21 mixed GGNs, and 42 solid nodules were included, with paired post-operative plasma samples available for 20 patients. The target miRNA/reference miRNA ratio was used to construct a support vector machine (SVM) model. The SVM model with the best specificity showed 100% specificity in both the training and validation sets independently. The model with the best sensitivity showed 100% and 96.9% sensitivity in the training and validation sets, respectively. Principal component analysis revealed that pure GGN distributions were distinct from those of solid nodules, and mixed GGNs had a diffuse distribution. Among differentially expressed miRNAs, miR-500a-3p, miR-501-3p, and miR-502-3p were upregulated in tumor tissues and enhanced overall survival. The SVM classifier accurately distinguished malignant GGNs and benign nodules. The distinct profile characteristics of miRNAs provided insights into the feasibility of EVs miRNAs as prognostic factors in lung cancer.

A clinical and epidemiological survey of the largest dengue outbreak in Southern Taiwan in 2015

ObjectivesThis study examined the epidemiological, clinical, and immunological characteristics of the 2015 dengue outbreak in Taiwan. MethodsClinical data were collected from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients. A phylogenetic tree was used to analyze the source of the outbreak strain. Paired plasma samples from DF/DHF patients were used for antibody-dependent enhancement (ADE) assay and cytokine multiplex biometric immunoassay to validate the immunological mechanism. ResultsThis outbreak mainly occurred in two of the southern cities of Taiwan: Tainan (n=22 777; 52%) and Kaohsiung (n=19 784; 45%). A high DHF death rate was noted (34.6%). The case (DHF) and control (DF) study indicated that older age (>60 years), type II diabetes, and hypertension were risk factors correlated with the development of DHF (p< 0.0001). The phylogenetic tree results suggested that the outbreak-associated strain was dengue virus serotype 2 and cosmopolitan genotype, forming a stable cluster with the isolates from Thailand and Indonesia (bootstrap value of 99%). Cytokine analyses demonstrated that levels of interleukin (IL)-6, IL-4, IL-13, IL-1β, interferon gamma (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly higher in DHF patients compared to DF patients (p< 0.001). The ADE assay showed that diluted plasma containing preexisting dengue antibodies from DHF patients significantly enhanced dengue infection (p< 0.05). ConclusionThe results suggest that older age, type II diabetes, hypertension, immunological cytokine dysregulation, and preexisting dengue antibodies inducing ADE infection are correlated with dengue severity. This study also indicates that the largest dengue outbreak in Taiwan might have been a result of imported DF from dengue epidemic regions.

Aberrant Methylation Status of Tumour Suppressor Genes in Ovarian Cancer Tissue and Paired Plasma Samples.

Ovarian cancer is a highly heterogeneous disease and its formation is affected by many epidemiological factors. It has typical lack of early signs and symptoms, and almost 70% of ovarian cancers are diagnosed in advanced stages. Robust, early and non-invasive ovarian cancer diagnosis will certainly be beneficial. Herein we analysed the regulatory sequence methylation profiles of the RASSF1, PTEN, CDH1 and PAX1 tumour suppressor genes by pyrosequencing in healthy, benign and malignant ovarian tissues, and corresponding plasma samples. We recorded statistically significant higher methylation levels (p < 0.05) in the CDH1 and PAX1 genes in malignant tissues than in controls (39.06 ± 18.78 versus 24.22 ± 6.93; 13.55 ± 10.65 versus 5.73 ± 2.19). Higher values in the CDH1 gene were also found in plasma samples (22.25 ± 14.13 versus 46.42 ± 20.91). A similar methylation pattern with positive correlation between plasma and benign lesions was noted in the CDH1 gene (r = 0.886, p = 0.019) and malignant lesions in the PAX1 gene (r = 0.771, p < 0.001). The random forest algorithm combining methylation indices of all four genes and age determined 0.932 AUC (area under the receiver operating characteristic (ROC) curve) prediction power in the model classifying malignant lesions and controls. Our study results indicate the effects of methylation changes in ovarian cancer development and suggest that the CDH1 gene is a potential candidate for non-invasive diagnosis of ovarian cancer.

Bronchoalveolar Lavage Fluid from COPD Patients Reveals More Compounds Associated with Disease than Matched Plasma.

Smoking causes chronic obstructive pulmonary disease (COPD). Though recent studies identified a COPD metabolomic signature in blood, no large studies examine the metabolome in bronchoalveolar lavage (BAL) fluid, a more direct representation of lung cell metabolism. We performed untargeted liquid chromatography–mass spectrometry (LC–MS) on BAL and matched plasma from 115 subjects from the SPIROMICS cohort. Regression was performed with COPD phenotypes as the outcome and metabolites as the predictor, adjusted for clinical covariates and false discovery rate. Weighted gene co-expression network analysis (WGCNA) grouped metabolites into modules which were then associated with phenotypes. K-means clustering grouped similar subjects. We detected 7939 and 10,561 compounds in BAL and paired plasma samples, respectively. FEV1/FVC (Forced Expiratory Volume in One Second/Forced Vital Capacity) ratio, emphysema, FEV1 % predicted, and COPD exacerbations associated with 1230, 792, eight, and one BAL compounds, respectively. Only two plasma compounds associated with a COPD phenotype (emphysema). Three BAL co-expression modules associated with FEV1/FVC and emphysema. K-means BAL metabolomic signature clustering identified two groups, one with more airway obstruction (34% of subjects, median FEV1/FVC 0.67), one with less (66% of subjects, median FEV1/FVC 0.77; p < 2 × 10−4). Associations between metabolites and COPD phenotypes are more robustly represented in BAL compared to plasma.

Determination of Essential and Toxic Elements in Cattle Blood: Serum vs Plasma.

Simple SummaryKnowledge of type of blood sample (serum or plasma) for determination of essential and toxic elements is essential for assessing the health and nutritional status of the animals and the herd. We demonstrate that both plasma and serum samples are suitable and interchangeable for the determination of most of the essential and toxic elements in blood in cattle. Nevertheless, we must take into account that the concentrations of Cu and Se are lower in the serum than in plasma.This study was designed to evaluate the influence of type of blood sample (serum or plasma) on essential and toxic element analysis in cattle. Paired plasma and serum samples (n = 20) were acid digested, and the concentrations of As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn. Mo, Ni, P, Pb, Sb, Se, Sr and Zn were determined by inductively coupled plasma mass spectrometry (ICP-MS). The study findings indicate that plasma and serum samples appear suitable and interchangeable for the determination of most of the essential and toxic elements in blood in cattle. The only exceptions are Cu and Se, the concentrations of which were significantly lower (40.9 and 29.9% respectively) in serum than in plasma. Some of the Cu in blood samples from bovine ruminants is known to be sequestered during clotting. However, further research on Se in ruminants and other animal species is warranted. Finally, the significantly higher Mn (9.9%) concentrations in serum than in plasma may have been caused by haemolysis of some samples. Special attention should be paid to preventing haemolysis of samples during collection and processing, in order to prevent overestimation of elements present at high concentrations inside erythrocytes (i.e., Fe, Mn and Zn).