Liquid Biopsy – Possibilities for Monitoring the Therapeutic Response in Non-Small Cell Lung Cancer

Lung cancer is the leading cause of death from malignancy worldwide. Its heterogeneity and tumour biology make treatment considerably more difficult. The introduction of target molecules heralded the beginning of the personalized medicine which tailors medical treatments to the molecular and genetic profile of a patient. Liquid biopsy is an innovative, non-invasive method which is used both for diagnostic purposes and for therapeutic monitoring. Liquid biopsy has the potential to help manage non-small cell lung cancer throughout all stages of this cancer: screening, detection of minimal residual disease to guide adjuvant treatment, early detection of relapse, systemic treatment initiation, monitoring of response to targeted or immune therapy, and the emergence of resistance to applied treatment. At present, the study of circulating tumour DNA is used in clinical practice, but circulating tumour cells, miRNAs, exosomes, and platelets formed in the tumour also show promising results.

COT-3 Exosomal microRNA expression signature in blood and cerebrospinal fluid of glioblastoma patients

Analysis of exosomes derived from plasma or cerebrospinal fluid (CSF) has emerged as a promising biomarker platform for therapeutic monitoring in glioblastoma patients. However, the contents of the various subpopulations of exosomes in these clinical specimens remain poorly defined. Here we characterize the relative abundance of miRNA species in exosomes derived from the plasma and CSF of glioblastoma patients. To this end, we first employed miRNA arrays to measure the expression of exosomal miRNAs in the plasma from glioblastoma patients (n = 24) and healthy volunteers (n = 7) as control. In addition, we performed global miRNA profiling of exosomal miRNAs in the CSF from glioblastoma patients (n = 5) and non-tumoral patients (n = 3; hydrocephalus patients) as control. In plasma derived exosomes, 80 miRNAs were altered by >2-fold in glioblastoma patients compared to controls. In CSF, 92 miRNAs were altered by >2-fold in glioblastoma patients compared to controls. Combined analysis of plasma and CSF revealed a similar fold difference in eight miRNAs. Next, we measured these eight miRNAs expression in in the plasma from pre- and post-operative glioblastoma patients (n = 9). Among these eight miRNAs, we identified only one miRNA (miR-34b-3p) that was upregulated in exosomes from pre-operative glioblastoma patients. Our results suggest that miR-34b-3p might have a potential as a novel diagnostic marker or a therapeutic tool for glioblastoma patients.

Potential Biomarkers for Therapeutic Monitoring and Clinical Outcome in Breast Cancer

Non-coding RNAs are a species of RNA that are not translated to proteins. These include transfer RNAs and ribosomal RNAs, microRNAs, transfer RNA-derived fragments, and long non-coding RNA. It is known that expression levels of some non-coding RNAs included microRNAs are altered in cancer cells or tumor tissues. Moreover, expression profiles of such non-coding RNAs correlate between tissues and body fluids. Therefore, several non-coding RNAs are being used as diagnostic/prognosis biomarkers or therapeutic targets in cancer. In this chapter, we review about representative non-coding RNAs and introduce especially microRNA as diagnosis/prognosis biomarkers and therapeutic targets.

Optimization of Thrombin Generation in Hemophilia a

Background: Hemophilia A (HA) is a bleeding disorder characterized by decreased or absent FVIII. Clinical analysis of coagulation potential in this patient population is classically based on APTT based FVIII assays. Although both the one-stage FVIII assay and the chromogenic FVIII assay can measure FVIII concentrations reliably these types of assays only give insight on the initiation of coagulation. Global coagulation assays, like thrombin generation (TG), can be used to measure the full coagulation spectrum of initiation, amplification and propagation. However the frequently used commercially available TG kits lack sensitivity for measurements of hemophilia plasma within the lower FVIII ranges which are essential in explaining differences in bleeding phenotype. Aim: We aim to optimize the sensitivity of the TG-assay for measurements in hemophilia A patients, especially in the lower FVIII ranges. Methods: In order to minimize patient specific sensitivity a hemophilia A pool plasma (HAPP) was created. Analysis of the influence of pre-analytical variables, like contact activation inhibitors, on the assay was performed. Initiation of coagulation by different reagents was compared for sensitivity towards factor FVIII titrations in patient plasma. Other assay variables like phospholipids and temperature were adjusted to increase sensitivity even further. Results: Commonly used tissue factor (TF) initiated TG at varying concentrations was unable to significantly differentiate in FVIII levels below 20%. In contrast, TG activation with low concentrations of TF in presence of FXIa appeared to be highly sensitive for FVIII changes both in high and low ranges. Additionally, a representative baseline TG-curve in severe HA plasma could only be produced using this dual TF/FXIa-activation. There was a value in the addition of contact activation inhibitors in the assay. Higher phospholipid concentrations seem to benefit this assay setup compared to a TF only setup. Conclusion: TF/FXIa dual activation thrombin generation increased assay sensitivity in severe hemophilia plasma, allows for dose-dependent measurements in low FVIII ranges and provides a solid baseline curve that can be used for further clinical evaluation of coagulation potential and possibly therapeutic monitoring in hemophilia A. Figure 1 Figure 1. Disclosures Hackeng: ACS Biomarker BV: Current Employment, Current equity holder in publicly-traded company; Coagulation Profile BV: Current Employment, Current equity holder in publicly-traded company.

175. Assessment of Institutional Uptake of Vancomycin AUC Monitoring One-Year Post Guideline Publication in Hospitals Across the United States

Background A new therapeutic monitoring of vancomycin for serious methicillin resistant Staphylococcus aureus infections guideline was published in March 2020. The guideline recommends a change in monitoring from trough to AUC/MIC based to improve patient outcomes. The purpose of this study was to determine institutional uptake of vancomycin AUC monitoring 1-year post guideline publication in hospitals across the U.S. Methods An electronic survey was created to assess vancomycin AUC monitoring practices and distributed to the American College of Clinical Pharmacy Infections Diseases Practice and Research Network (ACCP IDprn) and American Society of Health System Pharmacists (ASHP). Initial survey distribution (phase 1) occurred May-June 2020 and aimed to serve as baseline data. The survey was re-distributed (phase 2) to the ACCP IDprn and ASHP one year later, May-June 2021. Prior to re-distribution the survey was updated to assess the impact of COVID-19 on uptake. Results were analyzed and reported using descriptive statistics. Chi-Square tests were used to compare categorical data. Results A total of 202 responses to phase 1 and 138 responses to phase 2 were recorded. Significantly more respondents implemented AUC monitoring 1-year post guideline than at baseline (42.8% vs 29.8%, p= 0.013). In both phases, 57% of those who had not implemented AUC monitoring had plans to do so over the next year. Additionally, 46.2% phase 2 respondents reported COVID-19 impacted their ability to transition to AUC monitoring citing issues such as lack of time and inadequate resources. The most common AUC monitoring programs utilized at baseline and 1-year post guideline were purchased Bayesian software (38.3% vs. 35.6%) and homemade software (26.1% vs 23.7%). Perceived challenges to implementing AUC monitoring included cost, difficult use and integration. Conclusion Increased uptake of vancomycin AUC monitoring occurred from baseline to 1-year post guideline publication. However, less than half of hospitals implemented this recommendation. Although COVID-19 impacted a large portion respondents’ ability to implement AUC monitoring, majority plan to transition to vancomycin AUC monitoring over the next year. AUC monitoring should be adapted by all hospitals to optimize vancomycin efficacy and safety. Disclosures All Authors: No reported disclosures

Therapeutic monitoring of mycophenolic acid in renal transplanted patients by a validated HPLC method

Introduction: Mycophenolate mofetil and its active metabolite mycophenolic acid are routinely used as immunosuppressant drugs in solid organ transplantation in a fixed daily dose regimen in association with cyclosporine, tacrolimus and steroids. Therapeutic drug monitoring for mycophenolic acid concentration has been suggested to optimize outcomes by reducing rejection and drug related toxicities in clinical renal transplantation. Aim: To determine the predose concentration of mycophenolic acid in renal transplanted patients by a validated proposed high-performance liquid chromatography (HPLC) method and to estimate the interindividual variability based on the therapeutic target. Materials and methods: An HPLC method combined with protein precipitation has been validated for mycophenolic acid determination in the human plasma obtained from 21 renal transplant recipients. HPLC analysis was carried out using the chromatographic system Agilent Technologies 1200 DAD. Samples were injected manually, and the compounds were separated on a LiChrosphere® select B C18 analytical column. The mobile phase was 45:55 (v/v) acetonitrile-buffer phosphate, pH 2.5, flow rate of 1.0 mL/min and column temperature of 30°C. Detection was performed at 215 nm. Whole blood samples were collected into vacutainers containing EDTA and separated at 6000 g for 10 minutes. A 200-μL aliquot of patient plasma was transferred to a tube, followed by addition of 10 μL of naproxen as internal standard and 400 μL of acetonitrile (v/v) as a protein precipitating agent. Each tube was vortex-mixed for 30 sec and then centrifuged for 10 min at 10000 rpm. 20 μL of the supernatant was injected into the HPLC system for analysis. Results: The method showed appropriate linearity for MPA with correlation coefficient greater than 0.999. High inter-patient variability is observed with 18% of patients within the target trough concentration range, 27% of patients below the target trough concentration range and 54% over the range with risk of toxicity. Conclusions: Therapeutic monitoring of MPA might contribute to a better management of renal transplant recipient with the goal of optimizing therapeutic regimens in order to reduce the risk of rejection and MPA‐related toxicity.