Ml Of Plasma Research Articles

LC-MS/MS determination of glimepiride in human plasma with a high recovery at picogram scale: Pharmacokinetic study after oral administration

Abstract Although glimepiride (GLM) is the first-line treatment of Type II diabetes, low extraction recovery is still a significant limitation in previous plasma analysis methods. An optimized solid-phase extraction method of GLM in human plasma with excellent extraction recovery, 100 ± 0.06%, was achieved using liquid chromatography-electrospray ionization tandem mass spectrometry and Gliclazide (GLZ) as an internal standard. GLM was extracted from 100 µL plasma sample using Sep-Pak® vac 1cc (100 mg) C18 column and methylene chloride: methanol (2: 1, v/v) as eluant. Both GLM and GLZ were monitored by a triple quad mass spectrometer applying positive multiple reaction monitoring mode (+MRM). The protonated precursor ions and product ions of GLM and GLZ were m/z 491(352), and m/z 324 (127), respectively. The detection and measurement of low levels of GLM in human plasma reached to picogram range (limit of detection (LOD) = 60 pg/mL, limit of quantification (LOQ) = 200 pg/mL). The method was validated in terms of selectivity, linearity, recovery, accuracy, and precision. The method was successfully applied to the pharmacokinetic study of GLM following oral administration of 1 mg GLM tablets to 12 healthy volunteers.

High-throughput LC-MS/MS Method for Simultaneous Determination of Pantoprazole and Atorvastatin in Rat Plasma: Application to a Pharmacokinetic Interaction Study.

Pantoprazole and atorvastatin are often used jointly in the clinic. The drug-drug interaction of pantoprazole and atorvastatin is worthy of being investigated. A highly rapid, sensitive, and selective LC-MS/MS method was developed for simultaneous quantification of pantoprazole and atorvastatin in rat plasma. Omeprazole and atorvastatin-d5 were used as the internal standards (ISs) of pantoprazole and atorvastatin, respectively. Simple protein precipitation was used to extract analytes from 50.0 μL plasma samples. The chromatographic separation was achieved on a C18 column and the total chromatographic run time was 3.2 min. Acquisition of mass spectrometric data was performed on a triple-quadrupole mass spectrometer in multiple- reaction-monitoring (MRM) mode with an ESI source using the transition m/z 384→ 200 for pantoprazole and m/z 559.4→ 440.2 for atorvastatin, respectively. The method was validated over the concentration range of 20.0 ∼ 5000 ng/mL for pantoprazole and 1.00 ∼ 250 ng/mL for atorvastatin. All the validation results, including linearity, specificity, precision, accuracy, extraction recovery, matrix effect, and stability, met the acceptance criteria as per FDA guidelines. This method was successfully applied to a pharmacokinetic interaction study in Wistar rats. The results revealed significant evidence for the drug-drug interaction between pantoprazole and atorvastatin.

Ultralow-Fouling Zwitterionic Polyurethane-Modified Membranes for Rapid Separation of Plasma from Whole Blood.

The separation of plasma from blood cells in whole blood is an essential step for many diagnostic and therapeutic applications. However, the current point-of-care plasma separation approaches have not yet satisfied the need for a rapid, high-flux, and low-cost process. Here, we report a portable, low-cost, disposable membrane-based plasma separation device that enables rapid plasma extraction from whole blood. Rapid separation of plasma can be obtained with a simple three-step operation: blood injection, separation, and plasma collection. Our device benefits from the zwitterionic polyurethane-modified cellulose acetate (PCBU-CA) membrane, which can greatly inhibit the surface fouling of blood cells and membrane flux decline. The zwitterionic coating is stable on the membrane surface during blood filtration and leads to a 60% decrease in surface fibrinogen adsorption than a nonmodified membrane surface. The ultralow-blood-fouling properties of the PCBU-CA membrane enable rapid, continuous separation of plasma: within 10 min, the device can yield 0.5-0.7 mL of plasma from 10 mL of whole blood. The extracted plasma is verified as cell-free, exhibits a low hemoglobin level, and has a high protein recovery. Our PCBU-CA membrane provides a pathway for developing a high-efficiency portable plasma separation device that can reduce the time to diagnosis, allow effective patient care, and eventually reduce hospital costs.

A Validated LC-MS/MS Method for the Determination of Mezlocillin in Plasma: An Adapted Method for Therapeutic Drug Monitoring in Children

Background: Mezlocillin is off-label used for the treatment of respiratory infections in children. Therapeutic Drug Monitoring (TDM) data are also limited in children. A sensitive Liquid chromatography- tandem mass spectrometry (LC–MS/MS) method adapted to children was developed and validated for the determination of mezlocillin plasma concentration in the present study. Methods: Mezlocillin, extracted from a volume of 50 μL plasma using acetonitrile, was analyzed on an online LC-MS/MS system with an Agilent 1290 Infinity UHPLC (Agilent Technologies, CA, USA) coupled to an AB SCIEX QTRAP 6500PLUS MS/MS (AB Sciex, Framingham, MA, USA) with ceftiofur as an internal standard. HPLC separation was performed on a C18 column with ultra-pure water and acetonitrile as gradient elution at a flow rate of 0.4 mL/min at 30°C. Analyst TM Version 1.5.2 (Applied Biosystems) was used for data acquisition. The total chromatographic run time was 1.6 min. Results: LC/MS/MS method used for TDM of mezlocillin in children was developed and validated. This assay has a lower limit of quantification of 0.025 μg/mL for mezlocillin with 50 μL plasma. Good linearity was achieved for mezlocillin over the range from 0.025 to 20 μg /mL. The acceptance criteria were met in all cases. Among 36 patients aged between 0.16-1.63 years old, only one patient had detectable trough concentration higher than 1 μg/mL. Conclusion: LC-MS/MS method with 50 μL plasma developed in this study was successfully applied to TDM of mezlocillin in children. The high variability of trough concentration highlighted that TDM is important to optimize mezlocillin therapy in children.

Abstract CT189: Characterization of ESR1 mutations at metastatic relapse and outcome under first line aromatase inhibitor and palbociclib in the PADA-1 trial

Abstract Background: Palbociclib (Pal) combined with an aromatase inhibitor (AI) is a standard of care as first line therapy in ER+ HER2- metastatic breast cancer (MBC). Cell-free DNA (cfDNA) ESR1 mutation (ESR1mut) status prior to treatment initiation may be associated with shorter progression-free survival (PFS) under first line Pal+AI; however, the impact of the exact type of ESR1mut, of their quantitative levels and clonality are unknown. In a subsidiary analysis of the first line PADA-1 phase 3 trial, cfDNA samples previously detected as positive by ddPCR were submitted to NGS; mutations types, level and clonality were correlated with patient outcomes under first line Pal+AI. Methods: PADA-1 (NCT03079011) is a phase III trial testing the clinical utility of real time ESR1mut detection in the blood of patients treated with AI-Pal. Main inclusion criteria are patients with ER+ HER2- MBC, who never received adjuvant AI or completed adjuvant AI for >12 months, with neither prior therapy for MBC nor visceral crisis. ESR1mut are tracked in cfDNA from up to 4ml of plasma by a single ddPCR assay targeting E380, L536, Y537 and D538 hotspots (i.e. >90% of known ESR1 activating mutations) with 0.1% sensitivity (Jeannot et al, Oncogene 2020). We sequenced the ddPCR positive cfDNA samples using a short amplicon-based NGS panel spanning 30 genes, including the full sequence of ESR1. We assessed for the correlation between PFS and ESR1mut type (NGS), absolute level (copy/ml, ddPCR) or variant allelic frequency (VAF, NGS & ddPCR), and clonality (NGS, pending the detection of other driver mutations). Results: Among the 1,017 included patients, N=33 (3.2%) had an ESR1mut detected by ddPCR at inclusion (median VAF= 2.5%, range (0.09-46.6%). 26/33 left-over cfDNA samples were available for NGS. ESR1mut VAFs retrieved by NGS and ddPCR showed an excellent intraclass correlation coefficient (ICC= 0.98; 95% CI [0.89;0.99]). In N=3 samples with low VAF by ddPCR (<1%), NGS was not able to detect ESR1mut. ESR1 codons 380, 536, 537 and 538 were mutated in N=5, 6, 10 and 7 patients, respectively (5 patients (19.2%) having polyclonal mutations). Among evaluable patients, ESR1mut were found clonal and subclonal in 11 (47.8%) and 12 (52.2%) patients, respectively. After a median follow-up of 24.8 months (range 0-41.9 months) (485 PFS events among the 1,017 included patients), ESR1mut detection at inclusion was found to be a prognostic factor for PFS (median PFS = 11.6 months 95% CI [8.3; NR] vs 28.5 months 95% CI [23.3;30.2]; HR= 2.2 95% CI = [1.4;3.4]). ESR1mut type, clonality and baseline levels had no significant additional impact on PFS. Conclusion: Presence of ESR1mut detected in cfDNA at metastatic relapse are associated with a shorter PFS under first line AI and palbociclib. ddPCR and NGS yielded similar quantitative results, while supplementary information obtained by NGS (mutation type and clonality) did not add further prognostic information. Funding: Pfizer, French National Cancer Institute (Grant PRT-K 2020-041) Citation Format: Anne Pradines, Céline Callens, Aurélia Doussine, Ivan Bièche, Jérôme Lemonnier, Marjorie Mauduit, Thomas Bachelot, Florence Dalenc, Alain Lortholary, Barbara Pistilli, Thibault De La Motte Rouge, Renaud Sabatier, Jean-Marc Ferrero, Sylvain Ladoire, Frédérique Berger, François-Clément Bidard. Characterization of ESR1 mutations at metastatic relapse and outcome under first line aromatase inhibitor and palbociclib in the PADA-1 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT189.

Abstract 111: Accurate multi-cancer detection using methylated DNA markers and proteins in plasma

Abstract Purpose: Screening for high prevalence cancers (breast, colorectum and uterine cervix) reduces mortality; however, most cancer deaths arise in unscreened organs. An ideal solution would accurately detect multiple currently unscreened cancers in a single round of testing. We aimed to assess the feasibility of a targeted assay panel of methylated DNA markers (MDMs) and proteins for detection of highly lethal cancers in a multi-cancer case-control study. Experimental Procedures: Prospectively collected plasmas from 180 cases of 6 cancer types (stage A-D liver (n=36), and TNM stage II-IV esophageal (n=18), lung (n=36), ovarian (n=30), pancreatic (n=30), and stomach (n=30)) and 257 smoking status, age-, and gender-matched asymptomatic controls were tested in blinded fashion. To quantify 26 MDMs previously found to be common among many cancers, multiplex PCR followed by LQAS (Long probe Quantitative Amplified Signal) assay was performed on bisulfite converted DNA extracted from 3 mL of plasma. Additionally, 5 proteins were tested from paired serum aliquots and combined with MDMs for a multi-analyte analysis. Two-thirds of the cases and controls were used to develop prediction algorithms, both logistic regression and random forest models, and the remaining 1/3 were used to validate the models. Results: Using stepwise logistic regression, a combination of 3 proteins (CEA, CA-125, CA19-9) and 5 MDMs (ZNF671, GRIN2D, NDGR4, SHOX2, B3GALT6) resulted in an AUC of 0.95 and an overall sensitivity of 87% for all cancers at 95% specificity. The logistic model on the validation set of the samples resulted in an AUC of 0.96 and a sensitivity of 83% with an observed specificity of 94%. The cancer-specific sensitivities ranged from 78% for lung cancer to 90% for ovarian and pancreatic cancer (Table). Random forest and logistic analyses were in agreement. Conclusion: Targeted assay of select MDMs and proteins show high discrimination for multiple cancers in a case control study, which included early stage patients. Performance with 95% confidence intervals of multi-target assay for multi-cancer detectionSensitivitySpecificityModelOverallBy cancer typeLungEsophagealGastricPancreaticLiverOvarianLogisticTraining set87%(79-92%)79%83%90%95%88%85%95%(90-97%)Test set83%(72-91%)75%100%80%80%75%100%94%(87%-98%)Training + Test186%(80-90%)78%89%87%90%83%90%95%(91-97%)Training + Test275%(68-81%)50%89%83%76%81%80%98%(96-99%)Random forest377%(70-83%)58%78%83%77%81%87%95%(92-97%)195% specificity goal, 5 MDMs and 3 proteins.298% specificity goal, 5 MDMs and 3 proteins.3With 500-fold in silico cross-validation on all samples and all markers (26 MDMs + 5 proteins) Citation Format: Slava Katerov, Abram Vaccaro, Jacquelyn Hennek, Jennifer Carlson, William R. Taylor, Douglas Mahoney, John B. Kisiel, Hatim T. Allawi. Accurate multi-cancer detection using methylated DNA markers and proteins in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 111.

Abstract 569: Correlation of variant allele frequency and mean tumor molecules with tumor burden in patients with solid tumors

Abstract Introduction: Two metrics are commonly used to quantify tumor-specific variants in cell-free DNA (cfDNA): the variant allele frequency (VAF), defined as the ratio of the number of variant alleles to wild-type alleles, and mean tumor molecules (MTM)/mL of plasma. The main difference between the two metrics is that molecular disease levels expressed in MTMs account for the total amount of cfDNA. Changes in cfDNA levels, which occur in response to cytotoxic therapies, surgery, and inflammation, have shown to impact the detection and quantification of tumor variants in plasma, leading to misinterpretation of the test results. Here we sought to explore the relationship between VAF and MTM and evaluate the performance of each metric in monitoring molecular residual disease. Methods: We have analyzed 6569 plasma samples from 3389 patients including patients with primary diagnosis of colorectal cancer (n = 2413), breast (n = 170), pancreatic (n = 132), esophageal (n = 86), lung (n = 60), liver (n = 54), other cancers (n = 474) across multiple settings. ctDNA was quantified using a personalized and tumor-informed (bespoke mPCR NGS) assay. We examined the correlation between MTM and VAF for all ctDNA positive plasma samples (n = 1970) from 1283 patients using log-log regression. For patients with longitudinal plasma time points, further analyses on dynamic changes in MTM and VAF was performed to assess the sample-level discrepancy between the two metrics. Furthermore, the absolute values and the dynamic changes in MTM and VAF levels were plotted and compared against clinical truth based on imaging results and patient outcomes. Results: Across all samples median cfDNA concentrations were 7.9 ng/mL (range: 1.3-381.6 ng/mL). Analysis of all ctDNA positive samples revealed a positive correlation between VAF and MTM, with R2 = 0.91 (slope: 0.86). From 3180 longitudinal plasma samples, we found 2.4% (76/3180) of samples, where changes relative to the previous time point were discordant between MTM and VAF. Interestingly, cfDNA concentrations in these samples were significantly altered. In general, cfDNA levels are representative of intrinsic cell turnover. While VAF is directly related to ctDNA, it is inversely related to cfDNA levels. Increased cfDNA levels were seen in the setting of active cytotoxic therapy, perioperatively, and in the setting of advanced disease, suggesting these settings may be prone for VAF to underestimate the true ctDNA burden. Here we provide evidence that failure of VAF to account for the dynamic changes in cfDNA concentration in plasma may result in misinterpretation of the patient's disease burden. This suggests that in certain indications MTM would be more clinically relevant than VAF. Conclusion: Although both VAF and MTM are highly correlated, our analysis shows when cfDNA levels are significantly altered, MTM/mL provides a more accurate measure of molecular disease burden. Citation Format: Antony Tin, Vasily Aushev, Ekaterina Kalashnikova, Raheleh Salari, Svetalana Shchegrova, Gordon Fehringer, Meenakshi Malhotra, Harini Ravi, Himanshu Sethi, Maxim Brevnov, Bernhard Zimmermann, Angel Rodriguez, Paul R. Billings, Alexey Aleshin. Correlation of variant allele frequency and mean tumor molecules with tumor burden in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 569.

Abstract LB059: Dual methylation and mutation analysis from a single input of plasma cfDNA without sample splitting

Abstract Genomic and epigenetic changes are known to be the underlying mechanism of cancer development. Assessing the genomic (mutation) and epigenetic (methylation) changes has become increasingly important for cancer detection and treatment selection, especially in the field of cfDNA-based liquid biopsy. Targeted Next-Generation Sequencing (NGS) enables in-depth analysis of specific genomic regions that is essential for the clinical application of liquid biopsy. However, current targeted NGS assays are complicated, lengthy, and inefficient. Two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS) from sample to sequencing ready library. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for multi-modal analyses on one sample. Thus, an assay that is capable of both methylation and mutation analysis would be optimal for clinical research and future diagnostic assays.Here, we report the performance of Point-n-Seq, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both methylation and mutation analysis without sample splitting. Custom panels of 10 to 1000s of markers can be designed with >99% first-pass success rate. We conducted both performance validation and multi-center, multi-operator, reproducibility studies. With spike-in titration of cancer cell-line gDNA with known mutation and methylation profile, we achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions.In a pilot study, we designed a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with >350 hotspot mutations in 22 genes. From only 1ml of plasma from late stage CRC patients, we detected cancer-specific methylation signals in all samples tested, and hotspot mutations with clinical implications. Next, a tumor-informed mutation panel covering approximately 100 personalized mutations for each patient was compared to the tumor-independent CRC methylation panel to analyze 23 stage I and II CRC patient plasma samples. The initial results showed that tumor-independent TMS assay achieved a comparable or superior limit of detection compared to the personalized tumor-informed approach. Moreover, to further improve assay sensitivity, metrics for cfDNA size can be integrated into the analysis of the same workflow without sample splitting. The Point-n-Seq kit, with a highly streamlined workflow and dual methylation and mutation analysis, provides a highly sensitive and robust non-invasive method for cancer detection, monitoring and treatment guidance. Citation Format: Grace Q. Zhao, Yun Bao, Heng Wang, Vanita S. Natu, Jayashree Joshi, Yingmin Wang, Elena Arzumanyan, Wanping Wang, Jianmin Wang, John Coller, Shengrong Lin. Dual methylation and mutation analysis from a single input of plasma cfDNA without sample splitting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB059.

Abstract 409: A pre-treatment plasma extracellular vesicle-mRNA signature associates with checkpoint inhibitor pneumonitis in lung cancer patients

Abstract Introduction: Immune checkpoint inhibitors (ICIs) have demonstrated clinical efficacy in non-small cell lung cancer (NSCLC) patients (p). However, ICIs can also trigger a self-reactive response in the adjacent healthy lung tissue that can eventually lead to life-threatening immune-related adverse events (irAEs), like checkpoint inhibitor pneumonitis (CIP). We hypothesized that a pre-treatment state of chronic inflammation or immune system imbalance could predict which p are at higher risk of developing CIP. Methodology: We retrospectively collected pre-ICI-treatment FFPE tumor tissue and matching plasma samples from 17 CIP- and 24 non-CIP lung cancer p. An additional 40 plasma samples, including 3 CIP and 37 non-CIP p were used as a validation cohort. The miRCURY exosome isolation kit (Qiagen) was used for extracellular vesicle (EV) enrichment from 500 μL of plasma and RNA was extracted using TRI-reagent. EV-mRNA was then pre-amplified (10 cycles) using the Low RNA Input Amplification kit (NanoString Technologies). FFPE mRNA was extracted using the High Pure FFPET RNA Isolation Kit (Roche). Gene expression analysis was performed on tissue and EV-derived mRNA using the NanoString nCounter platform with the Human PanCancer IO360 panel, which targets 770 genes related to tumor biology, immune response and microenvironment. Differential expression (DE) analysis was carried out based on the development of CIP. Finally, a classifier was created using a bioinformatic recursive feature elimination and a leave-one-out cross validation algorithm to predict which combination of genes is most effective to predict CIP development. Results: DE analysis revealed 54 differentially expressed genes (DEGs) in pre-treatment tissue of CIP vs. non-CIP p. An 8-gene CIP mRNA signature was able to distinguish between the two cohorts with areas under the ROC curve (AUC) of 0.81-0.95. When analyzing plasma EV samples, we found 57 DEGs. The tissue CIP signature was not translatable to EVs, yielding AUCs of only 0.53-0.54. Therefore, we developed a new 4-gene EV-based mRNA signature that could differentiate CIP vs. non-CIP developing p with AUCs of 0.82-0.90 and an overall accuracy of 89.9%. The negative- and positive predictive values (NPV and PPV) were 92.7% and 78.6%, respectively with a Youden´s index of 0.67. The 4 genes included in the EV signature were upregulated in CIP p and were found to be involved in T-cell activation and immune cell localization to the tumor. Conclusions: We have created a 4-gene EV-mRNA signature that associates with CIP development upon ICI treatment. Our results also indicate that plasma EV-mRNA was non-inferior to invasive tissue biopsy analysis in predicting CIP development. Validation studies in larger patient cohorts are ongoing. Citation Format: Jillian Wilhelmina Bracht, Santiago Viteri-Ramirez, Andrés Aguilar, Silvia Calabuig-Fariñas, Juan José García-Mosquera, Chung-Ying Huang, Elena Duréndez-Sáez, Nicolas Potie, Erika Aldeguer, Ana Gimenez-Capitán, Sonia Rodriguez, Ruth Roman, Cristina Aguado, Sarah Warren, Carlos Camps, Rafael Rosell, Eloisa Jantus-Lewintre, Miguel-Angel Molina-Vila, Maria González-Cao. A pre-treatment plasma extracellular vesicle-mRNA signature associates with checkpoint inhibitor pneumonitis in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 409.

5-Hydroxymethylcytosine of Circulating Cell-Free DNA in Plasma: A Novel Non-Invasive Marker for Progression and Prognosis in Multiple Myeloma

Background: Multiple myeloma (MM) universally features profound disruption of their epigenomes with gene-specific DNA hypermethylation linked with clinically aggressive subtypes and prognosis. To date, DNA methylation (i.e., 5-methycytosine) is the most extensively studied cytosine modification. No study has characterized progression and prognostic significance of 5-hydroxymethylcytosine (5hmC), a mark of active demethylation and gene activation, nor did study evaluate 5hmC in cell-free DNA (cfDNA) as a non-invasive “liquid biopsy” mainly due to technological constraints.Methods: In a proof-of-concept study, we used the nano-hmC-Seal, a highly sensitive chemical labeling technique, and the next-generation sequencing to profile 5hmC in plasma cfDNA samples from 19 newly diagnosed, treatment-naïve European American patients with MM (n=9) and its premalignant precursor conditions - monoclonal gammopathy of undetermined significance (MGUS, n=5) and smoldering multiple myeloma (SMM; n=5) from the University of Chicago between 2011-12. Three of the 9 MM patients developed refractory disease or relapsed within 12 months after diagnosis. We profiled 5hmC with 1-2 ng of DNA extracted from 1ml of plasma for library construction. We obtained ~25 million reads per sample, providing a depth of coverage ~600X in terms of gene bodies. We normalized raw fragment counts using DESeq2.Results: We found that in cfDNA, 5hmC loci were enriched within gene bodies, while depleted in CpG islands. Our analysis, based on cis-regulatory elements from the ENCODE (Encyclopedia of DNA Elements), showed substantial overlap of 5hmC reads with histone modifications marking active transcription.We identified 183 genes containing differential 5hmC in gene bodies at 5% false discovery rate (p<0.001) between MGUS, SMM, and MM patients. Furthermore, 5hmC features in cfDNA showed the potential of separating MM patients at the time of diagnosis according to relapse status (39 genes, p<0.001). Many of these genes were previously implicated in MM, including those found to be differentially methylated (e.g., SYTL1, PSG1, GDF2, DNMT3A, GRP84) in a previous study of CD138+ cells from premalignant MGUS, early, and advanced stages of MM, and healthy control (Heuck CJ, 2013). We are profiling additional ~200 plasma samples from MM patients to 1) validate these preliminary findings, and 2) evaluate independent prognostic value of 5hmC in cfDNA controlling for established prognostic indices. These results will be presented at the ASH meeting.Conclusion: These preliminary findings suggest that 5hmC signatures in cfDNA differ 1) between MM and its two precursor conditions (i.e., MGUS and SMM), and 2) between MM patients by relapse status. [Display omitted] DisclosuresZimmerman:Abbvie: Employment.

CIRCULATING TUMOR DNA LOAD AND TOTAL METABOLIC TUMOR VOLUME IN DIFFUSE LARGE B CELL LYMPHOMA (DLBCL) PATIENTS TREATED BY R‐CHOP – FROM REMARC, A LYSA STUDY

Background: Circulating tumor DNA (ctDNA) quantification has been shown to predict therapeutic benefit in patients (pts) with DLBCL. We recently investigated the prognostic impact of total metabolic tumor volume (TMTV) in DLBCL pts responder to R-CHOP in the REMARC study. In the present ancillary study, we wish to analyze the potential correlation of ctDNA with the outcome and relate these results to functional imaging by PET/CT (TMTV). We also aim to correlate the circulating tumor DNA load with the new score TMTV plus ECOG PS. Patients and Methods: 794 pts were included in the study either at diagnosis (n = 446) or at the end of R-CHOP (n = 347). At the end of R-CHOP, 650 pts achieved a complete response and were randomized to receive 2 years of lenalidomide or placebo. Tumoral cfDNA measurements were performed using a dedicated 36 genes panel with the QiaSeq® technology. CtDNA concentrations were expressed in haploid genome equivalents per mL of plasma (hGE/mL) and calculated by multiplying the mean variant allele frequency (VAF) by the concentration of total cfDNA (Bohers et al. Blood Cancer J 2018).The optimal ctDNA cut-offs for PFS and OS were determined by X-tile analyses and confirmed by a training validation method. Results: A total of 240 pts were analyzed, including 62 non-responder and non-randomized pts (NR-NRP) and 173 responder and randomized pts (REMARC pts). 5 pts had no interpretable ctDNA analysis and were excluded. Among the 235 NR-NRP/REMARC cohort, a ctDNA > 3.74 was present in 14% of pts and was predictive for PFS (HR = 1.68, 95%CI 1.02–2.76, p = 0.0397) and OS (HR = 2.15, 95%CI 1.28-3.61, p = 0.0031). The 3y-PFS for pts with a ctDNA > 3.74 was 47.3% (95%CI: 30%-63%) vs 71.5% (95% CI: 65%-77%) for pts with a ctDNA ≤ 3.74. The 3y-OS for pts with a ctDNA > 3.74 was 56.4% (95%CI: 38%-71%) vs 81.8% (95% CI: 76%-87%) for pts with a ctDNA ≤ 3.74. Among the 173 R-CHOP responder patients (REMARC cohort), the 12 months-relapse free survival since last cycle of R-CHOP for pts with a ctDNA > 3.74 at diagnosis was 61.1% (95%CI: 35%-79%) vs 88.7% (95%CI: 82%-93%) for pts with a ctDNA ≤ 3.74 (p = 0.009). However, a ctDNA > 3.74 was not predictive either for PFS (HR = 1.64, 95%CI: 0.84-3.21, p = 0.14) nor for OS (HR = 1.91 95%CI: 0.93-3.89, p = 0.07). Among the 100 pts with ctDNA and TMTV available, median log ctDNA was found significantly higher in TMTV > 220 (n = 58) vs TMTV < 220 (p = 0.002). However, no significant impact of ctDNA (threshold 3.4) and TMTV(threshold 220) was observed on PFS and OS. Conclusion: Using a routinely targeted sequencing panel for ctDNA measurement, we confirmed the prognostic value of ctDNA concentration to predict outcome in DLBCL patients. For patients responding to R-CHOP, high ctDNA level was significantly associated with high TMTV and early relapse. The research was funded by: BMS Keywords: Diagnostic and Prognostic Biomarkers, Aggressive B-cell non-Hodgkin lymphoma No conflicts of interest pertinent to the abstract.

Development and validation of a UHPLC-MS/MS method to measure cefotaxime and metabolite desacetylcefotaxime in blood plasma: a pilot study suitable for capillary microsampling in critically ill children.

Critical illness has been shown to affect the pharmacokinetics of antibiotics, which can lead to ineffective antibiotic exposure and the potential emergence of resistant bacteria. The lack of studies describing antibiotic pharmacokinetics in critically ill children has led to significant off-label dosing. This is, in part, due to the ethical and physiological challenges of removing frequent, large-volume samples from children. Capillary microsampling facilitates the collection of small volumes of blood samples to conduct clinical pharmacokinetic studies. A sensitive, rapid, and accurate ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) bioanalytical method to measure cefotaxime and desacetylcefotaxime in 2.8μL of plasma was developed and validated. Plasma samples were treated with acetonitrile and analytes were separated using a Kinetex C8 (100 × 2.1mm) column. The chromatographic separation was established using a gradient method, with the mobile phases consisting of acetonitrile and ammonium acetate. An electrospray ionization source interface operated in a positive mode for the multiple reaction monitoring MS/MS analysis of cefotaxime, desacetylcefotaxime, and deuterated cefotaxime (internal standard). The bioanalytical method using microsample volumes met requirements for method validation for both analytes. Cefotaxime had precision within ± 7.3% and accuracy within ± 5% (concentration range of 0.5 to 500mg/L). Desacetylcefotaxime had precision within ± 9.5% and accuracy within ± 3.5% (concentration range of 0.2 to 10mg/L). The bioanalytical method was applied for the quantification of cefotaxime and its metabolite to 20 capillary microsamples collected at five time points in one dosing interval from five critically ill children.

Minimal residual disease by circulating tumor DNA analysis for colorectal cancer patients receiving radical surgery: An initial report from CIRCULATE-Japan.

3608 Background: Circulating tumor DNA (ctDNA) analysis can be used to predict the risk of recurrence by detecting molecular residual disease (MRD) in patients with colorectal cancer (CRC). We are conducting a prospective observational study to monitor MRD status in patients with clinical stage II–IV or relapsed CRC amenable to radical surgical resection (GALAXY study), as part of the CIRCULATE-Japan, a nationwide ctDNA-guided precision adjuvant therapy project. Methods: Analysis of ctDNA is being performed at pre- and post-surgery timepoints and will continue periodically for up to 2 years using Signatera, a personalized, tumor-informed ctDNA assay that is designed to track 16 patient-specific somatic variants based on whole-exome sequencing of tumor tissue. The association of peri-operative ctDNA status with clinicopathological characteristics was investigated. Results: As of January 13, 2021, 941 patients have been enrolled in the GALAXY study, of which 400 patients had their pre-operative ctDNA status evaluated. Of the 400 patients, baseline ctDNA was detected in 92% (367/400) of the patients: consisting of 35 patients with pathological stage (pStage) I, 135 with pStage II, 152 with pStage III, and 78 with pStage IV or relapsed disease (pStage IV/R). Patient-specific Signatera assays targeting 16 variants were designed for 100% of the patients. Out of the 6400 designed variants 99.3% passed quality control in the plasma analysis and produced the final results. Among 4425 genes selected for 400 patients, 3330 genes were selected for only one patient, while TP53 was the most commonly selected in 113 patients (28%). Median ctDNA levels, measured in mean tumor molecules per mL of plasma and ctDNA detection rate, stratified by stage are presented in table. Positive ctDNA status post-surgery was significantly associated with advanced pStage, pT and pN, and lymphovascular invasion. Of the 13 patients with recurrence, 10 were detected with a positive ctDNA at 4-weeks post-surgery, before confirmation of recurrence by the radiological imaging. Conclusions: Preoperative ctDNA detection rates were observed to be in &gt;90% in patients with pStage II–III by personalized ctDNA assay based on unique somatic variants, specific to each patient. ctDNA- based MRD detected post-surgery (4W) was significantly associated with certain known clinicopathological factors for recurrence with ctDNA positivity associated with a very short-term of recurrence. Clinical trial information: 000039205. [Table: see text]

A dual target sequencing solution to assess genomic and epigenomic alterations in cell-free DNA with no sample splitting.

3043 Background: Assessing the genomic and epigenomic changes on plasma cell-free DNA (cfDNA) using next-generation sequencing (NGS) has become increasingly important for cancer detection and treatment selection guidance. However, two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS), let alone one that analyzes both mutation and methylation information in one assay. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for conducting multi-omic analysis. Thus, an assay that is capable of both genomic and epigenomic analysis would be advantageous for clinical research and future diagnostic assays. Methods: Here, we report the performance of Point-n-SeqTM dual analysis, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both genomic and epigenomic analysis without sample splitting. With custom panels of tens to thousands of markers designed with &gt; 99% first-pass success rate, we conducted both performance validation and multi-center, multi-operator, reproducibility studies. Using spike-in titration of cancer cell-line gDNA with known mutation and methylation profiles, Point-n-Seq assay achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions. Benchmarked with conventional targeted sequencing and methylation sequencing, Point-n-Seq solution also demonstrated improved performance, speed and shortened hands-on time. Results: In a pilot clinical study, a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with &gt; 350 hotspot mutations in 22 genes were used in the dual assay. Using 1ml of plasma from late-stage CRC patients, cancer-specific methylation signals were detected in all samples tested, and oncogenic mutations. In an early-stage cohort (33 stage I/II CRC patient ), comparison of the analysis between tumor-informed, personalized-mutation panels (̃100 private SNVs) for each patient and the tumor-independent CRC methylation panels were conducted. The initial results showed that tumor-independent TMS assay achieved a comparable detection compared to the personalized tumor-informed approach. Moreover, cfDNA size information (fragmentome) is also integrated into the analysis of the same Point-n-Seq workflow to improve the assay sensitivity. Conclusions: Point-n-Seq dual analysis is poised to advance both research and clinical applications of early cancer detection, minimal residual disease (MRD), and monitoring.

A highly sensitive and selective UPLC-MS/MS assay for the determination of enarodustat (JTZ-951) in human plasma

Enarodustat, a potent, orally bioavailable, selective inhibitor of hypoxia inducible factor-Prolyl hydroxylase (HIF-PH), has been approved recently in Japan for the treatment of anemia in patients with chronic kidney disease (CKD). To evaluate the pharmacokinetics of enarodustat, a bioanalytical assay in human plasma was needed for the quantitation of enarodustat for both healthy subjects and patients with CKD. The UPLC-MS/MS method for the quantitation of enarodustat was initially validated in a bioanalytical laboratory in Japan to support clinical studies conducted in Japan, and then was transferred and validated in a bioanalytical laboratory in United States to support clinical studies conducted here. A cross-validation was successfully performed between the two bioanalytical laboratories using both quality control (QC) samples and incurred study samples. Enarodustat was fortified with its isotopically labeled internal standard in a 25 µL plasma aliquot and extracted with protein precipitation. The chromatographic separation was achieved on an Acquity UPLC BEH C18 (1.7 µm, 2.1 × 50 mm) column with gradient elution. The calibration curve range for the assay was 1.00–500 ng/mL. Assay precision, accuracy, linearity, selectivity, sensitivity and analyte stability covering sample storage and analysis were established. No interferences were observed from medications that may be co-administered along with enarodustat. The validated UPLC-MS-MS method at the US bioanalytical laboratory has been successfully applied to eight clinical studies for the determination of enarodustat concentrations in human plasma for both healthy subjects and patients with CKD.

Biophysical Phenomenon-Based Separation of Platelet-Poor Plasma from Blood

The extraction or isolation of biological entities on a microfluidic platform has gained considerable attention in disease diagnostics, biomedical research, and point-of-care applications. The isolation of platelet-poor plasma from blood is the first and an essential step for applications in coagulation studies. The widely known clinical methods of platelet-poor plasma separation are based on centrifugation, which require a long processing time, high power, skilled personnel, and are incompatible with point-of-care devices. The microdevice utilized in this study facilitates the employment of various biophysical phenomena, hydrodynamic forces acting on cells, and geometrical features of the microdevice for its operation. The developed microdevice is compact in size, easy to fabricate, reliable, and exhibits clog-free operation. The microdevice isolates platelet-poor plasma with a purity of 94.7 ± 1.90%, having a platelet count of 968 ± 15 per μL plasma, while operating at an inlet flow rate of 0.4 mL/min. Furthermore, biological assessment of the platelet-poor plasma extracted from the microdevice confirms that the sample is completely free from white blood cells and have no impact on the shape and size of the platelets after microfluidic processing.

Ultrasensitive Detection of BRAF Mutations in Circulating Tumor DNA of Patients With Metastatic Thyroid Cancer

Background: Liquid biopsy is a promising technology that can offer various advantages for molecular testing over tissue-based approaches. Most studies trying to address the utility of liquid biopsy in thyroid cancer have failed so far to achieve satisfactory rates of detection of relevant mutations. In this study, we examined a newly developed approach for ultrasensitive detection of oncogenic mutations in thyroid cancer using BRAF mutation as a proof-of-concept. In an exploratory analysis, we also correlated our findings with clinical outcomes and with levels of standard of care biomarkers. Methods: We included a group of patients with metastatic thyroid carcinoma. Cell free DNA (cfDNA) was isolated from an average of 2 ml of plasma and from matched formaldehyde fixed paraffin tissue blocks (FFPB) that were obtained from prior surgery. Extracted DNA was subject to preamplification of mutant copies using Q5 High-Fidelity PCR kit. Digital droplet PCR was performed on pre-amplified purified DNA where BRAF mutated allele frequencies (AF) were measured using BioRad ddPCR Qx200. Results: Thirty-three patients were included in our study with a median age at diagnosis of 62. Our method achieved a sensitivity of detection of 47.6% and a specificity of 80%. Mutant BRAF V600E was detected in cfDNA of 54.5% of patients (n=18) compared to 80.8% in isolated DNA from matched FFPB. Median overall survival (OS) was shorter in patients with wild type (WT) BRAF in both ctDNA and tissue (127m vs 218m, p=0.015; 116m vs 223m, p=0.004). Thyroglobulin (Tg) levels did not correlate with BRAF mutations either quantitatively or qualitatively. In the papillary thyroid carcinoma-classic variant cohort (n=20), however, patients with cfDNA mutant BRAF were more likely to have elevated Tg (90.9% versus 44.4% respectively, p=0.05). Conclusions: Our study provided a proof of concept for a newly developed technique to provide high sensitivity of mutation detection in thyroid cancer. The achieved sensitivity of detection is the highest to date using liquid biopsy in this tumor type. While we addressed only BRAF mutations in our study, the same approach can potentially be used for other mutations as well, likely changing the paradigm for use of liquid biopsy in thyroid cancer.

Microsphere-Based Microfluidic Device for Plasma Separation and Potential Biochemistry Analysis Applications.

The development of a simple, portable, and cost-effective plasma separation platform for blood biochemical analysis is of great interest in clinical diagnostics. We represent a plasma separation microfluidic device using microspheres with different sizes as the separation barrier. This plasma separation device, with 18 capillary microchannels, can extract about 3 μL of plasma from a 50 μL blood sample in about 55 min. The effects of evaporation and the microsphere barrier on the plasma biochemical analysis results were studied. Correction factors were applied to compensate for these two effects. The feasibility of the device in plasma biochemical analysis was validated with clinical blood samples.

Effect of Berberine Isolated from Barberry Species by Centrifugal Partition Chromatography on Memory and the Expression of Parvalbumin in the Mouse Hippocampus Proper.

Neurodegenerative diseases associated with memory disturbances are important health issues occurring due to a prolonged life span. This article presents the results of a study targeting the emergence of a drug candidate with antiamnesic properties. The effect of berberine (BBR), an isoquinoline alkaloid isolated from the overground parts of Berberis sibirica Pall., on memory and expression of parvalbumin in the mouse hippocampus proper were determined. High-purity BBR was isolated by centrifugal partition chromatography from a methanolic extract from B. sibirica by using a methyl-tert-butyl ether and water (1:1 v/v) solvent system with 10 mmol/L of triethylamine and hydrochloric acid. In an in vivo study, we assessed the influence of the chronic administration of BBR on different stages of memory-related responses in mice. Our results indicated that the chronic administration of BBR in a higher dose (5 mg/kg) improves long-term memory acquisition in mice, as determined in the passive avoidance test. The hippocampal CA1–CA3 fields showed an increased number of parvalbumin-immunoreactive neurons (PV-IR) and nerve fibers as compared to the control. No significant changes in the dentate gyrus were observed between the groups. The HPLC-ESI-QTOF-MS/MS analysis of the biological material revealed the content of BBR as 363.4 ± 15.0 ng (4.11% of RSD) per brain, 15.06 ± 0.89 ng (5.91% of RSD) per hippocampus, and 54.45 ± 1.40 (4.05% of RSD) ng in 100 µL plasma. The study showed that BBR could be a factor influencing the expression of PV in hippocampal neurons. We speculate that BBR may modulate the level of Ca2+ in neurons and thus potentially act as a neuroprotective factor against neuronal damages.

Plasma miRNA Biomarkers in Limited Volume Samples for Detection of Early-stage Pancreatic Cancer.

Early detection of pancreatic ductal adenocarcinoma (PDAC) is key to improving patient outcomes; however, PDAC is usually diagnosed late. Therefore, blood-based minimally invasive biomarker assays for limited volume clinical samples are urgently needed. A novel miRNA profiling platform (Abcam Fireplex-Oncology Panel) was used to investigate the feasibility of developing early detection miRNA biomarkers with 20 μL plasma from a training set (58 stage II PDAC cases and 30 controls) and two validation sets (34 stage II PDAC cases and 25 controls; 44 stage II PDAC cases and 18 controls). miR-34a-5p [AUC = 0.77; 95% confidence interval (CI), 0.66-0.87], miR-130a-3p (AUC = 0.74; 95% CI, 0.63-0.84), and miR-222-3p (AUC = 0.70; 95% CI, 0.58-0.81) were identified as significantly differentially abundant in plasma from stage II PDAC versus controls. Although none of the miRNAs individually outperformed the currently used serologic biomarker for PDAC, carbohydrate antigen 19-9 (CA19-9), combining the miRNAs with CA 19-9 improved AUCs from 0.89 (95% CI, 0.81-0.95) for CA 19-9 alone to 0.92 (95% CI, 0.86-0.97), 0.94 (95% CI, 0.89-0.98), and 0.92 (95% CI, 0.87-0.97), respectively. Gene set enrichment analyses of transcripts correlated with high and low expression of the three miRNAs in The Cancer Genome Atlas PDAC sample set. These miRNA biomarkers, assayed in limited volume plasma together with CA19-9, discriminate stage II PDAC from controls with good sensitivity and specificity. Unbiased profiling of larger cohorts should help develop an informative early detection biomarker assay for diagnostic settings. PREVENTION RELEVANCE: Development of minimally invasive biomarker assays for detection of premalignant disease and early-stage pancreatic cancer is key to improving patient survival. This study describes a limited volume plasma miRNA biomarker assay that can detect early-stage resectable pancreatic cancer in clinical samples necessary for effective prevention and clinical intervention.