External quality assessment (EQA) and alternative assessment procedures (AAPs) in molecular diagnostics: findings of an international survey.

Objectives: Quality management for clinical laboratories requires the establishment of internal procedures including standard operating procedures (SOPs), internal quality control (QC), validation of test results and quality assessment. External quality assessment (EQA) and alternativeassessment procedures (AAPs) are part of the quality hierarchy required for diagnostic testing. The International Organization for Standardization (ISO) document with requirements for conformance ISO 15189 and the Clinical and Laboratory Standards Institute document (CLSI) QMS24 require participation in EQA schemes and AAPs where applicable. The purpose of this study was to perform a global survey of EQA and AAPs for key procedures in molecular diagnostic laboratories. Methods: The Committee for Molecular Diagnostics of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC C-MD) conducted a survey of international molecular laboratories that covered specific topics of molecular diagnostic services as well as methods for EQA and AAPs. The survey addressed the following aspects: (1) usage of laboratory-developed test (LDT), (2) participation in EQA schemes and (3) performance of AAPs. Results: A total of 93 responses from laboratories located in Asia, Europe, the Middle East, North America and South America were received. The majority of the participating laboratories (65.9%) use LDTs and 81.3% stated that it is mandatory for them to participate in EQA programs, while 22% of the laboratories reported not performing AAPs. Thirty-one percent of the laboratories use EQAs for fewer than 50.0% of their reported parameters/analytes. Conclusions: While the majority of laboratories perform EQA and AAPs to improve their quality in molecular diagnostics, the amount of AAPs as quality procedures differs within the laboratories. Further surveys are necessary to clarify the existing needs in additional EQAs and standardized AAPs. The survey will also guide future efforts of the IFCC C-MD for identifying quality practices in need to improve harmonization and standardization within molecular diagnostics.

Polypharmacy: a healthcare conundrum with a pharmacogenetic solution.

The use of multiple medications is growing at an alarming rate with some reports documenting an average of 12-22 prescriptions being used by individuals ≥50 years of age. The indirect consequences of polypharmacy include exacerbation of drug-drug interactions, adverse drug reactions, increased likelihood of prescribing cascades, chronic dependence, and hospitalizations - all of which have significant health and economic burden. While many practical solutions for reducing polypharmacy have been proposed, they have been met with limited efficacy. This highlights the need for a new systematic approach for fine-tuning dispensing of medications. Pharmacogenetic testing provides an empirical and scientifically rigorous approach for guiding appropriate selection of medicines, with the potential to reduce unnecessary polypharmacy while improving clinical outcomes. The goal of this review article is to provide healthcare providers with an understanding of polypharmacy, its adverse effects on the healthcare system and highlight how pharmacogenetic information can be used to avoid polypharmacy in patients.

Corresponding ctDNA and tumor burden dynamics in metastatic melanoma patients on systemic treatment.

Radiographic imaging is the current standard for monitoring progression of tumor-burden and therapeutic resistance in patients with metastatic melanoma. Plasma circulating tumor DNA (ctDNA) has shown promise as a survelience tool, but longitudinal data on the dynamics between plasma ctDNA concentrations and radiographic imaging is lacking. We evaluated the relationship between longitudinal radiographic measures of tumor burden and ctDNA concentrations in plasma on 30 patients with metastatic melanoma on systemic treatment. In 9 patients with no radiographic evidence of disease over a total of 15 time points, ctDNA concentrations were undetectable. In 21 patients with radiographic tumor burden, ctDNA was detected in 81 % of 58 time points. Plasma ctDNA concentrations demonstrated a modest positive correlation with total tumor burden (TTB) measurements (R2= 0.49, p < 0.001), with the greatest degree of correlation observed under conditions of progressive disease (PD) (R2 = 0.91, p = 0.032). Plasma ctDNA concentrations were significantly greater at times of RECIST v1.1 progression (PD; 22.1 % ± 5.7 %) when compared to samples collected during stable disease (SD; 4.99 % ± 3.0 %) (p = 0.012); this difference was independent of total tumor burden (p = 0.997). Changes in plasma ctDNA showed a strong correlation with changes in TTB (R2= 0.88, p<0.001). These data suggest that measurements of plasma ctDNA during therapy are a better surrogate for responding versus non-responding disease compared to absolute tumor burden.

Results from an IFCC global survey on laboratory practices for the analysis of circulating tumor DNA.

BACKGROUND: The clinical validity of ctDNA analysis as a diagnostic, prognostic and predictive biomarker has been demonstrated in many studies. The rapid spread of tests for the analysis of ctDNA raises questions regarding their standardization and quality assurance. The aim of this study was to provide a global overview of the test methods, laboratory procedures and quality assessment practices using ctDNA diagnostics. METHODS: The Molecular Diagnostics Committee of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC C-MD) conducted a survey among international laboratories performing ctDNA analysis. Questions on analytical techniques, test parameters, quality assurance and the reporting of findings were included. RESULTS: A total of 58 laboratories participated in the survey. The majority of the participating laboratories (87.7 %) performed testing for patient care. Most laboratories conducted their assays for lung cancer (71.9 %), followed by colorectal (52.6 %) and breast (40.4 %) cancer, and 55.4 % of the labs used ctDNA analysis for follow-up/monitoring of treatment-resistant alterations. The most frequent gene analysed was EGFR (75.8 %), followed by KRAS (65.5 %) and BRAF (56.9 %). Participation in external quality assessment programs was reported by only 45.6 % of laboratories. CONCLUSIONS: The survey indicates that molecular diagnostic methods for the analysis of ctDNA are not standardized across countries and laboratories. Furthermore, it reveals a number of differences regarding sample preparation, processing and reporting test results. Our findings indicate that ctDNA testing is being conducted without sufficient attention to analytical performance between laboratories and highlights the need for standarisation of ctDNA analysis and reporting in patient care.

A diagnostic informatics approach for stratifying risk outcome based on combined genotype effects.

BACKGROUND: Diagnostic informatics (DI) in the context of personalized medicine involves the integration of molecular information to provide "actionable" diagnostic and therapeutic strategies. In many cases, retrospective predictions of clinical outcomes affected by multiple genes are complicated by not having the relevant genes measured within the same study. Multiplicative effect modeling is a statistical method for estimating the net effect of ≥ 2 independent variables. The authors demonstrate a DI approach that uses multiplicative-effect modeling to combine genetic information from ≥ 2 independent studies to predict a net clinical outcome. METHODS: As a hypothetical working model, 2 independent studies were selected each reporting on a unique genetic factor proposed to influence the risk of stent thrombosis (ST) among subjects treated with clopidogrel. A multiplicative effect model was used for developing a hypothesis regarding their combined influence on clinical outcome. RESULTS: Application of multiplicative risk modeling yielded a revised estimated risk of outcomes based on combined genotype. In this scenario, combined genotype revised the categorical risk level (high versus low) estimated from single gene effects for 41.5% of the subjects. Further, the maximum relative risk based on single gene effects was increased from 4.54 to 7.84 based on combined genotype. The revised relative risk values in conjunction with combined genotype frequency estimates provides the data necessary to frame a trial hypothesis and conduct appropriate power analysis to estimate the number of subjects needed to test that hypothesis. CONCLUSIONS: This DI approach can be used to generate quantitative hypotheses on multiple gene effects derived from independent genotype studies. This approach is useful for estimating parameters needed in designing future studies to evaluate the net effect of ≥ 2 genetic variants on a common clinical endpoint.

Molecular diagnostics of SARS-CoV-2: Findings of an international survey.

BACKGROUND: In the current COVID-19 pandemic, early and rapid diagnosis of potentially infected and contagious individuals enables containment of the disease through quarantine and contact tracing. The rapid global expansion of these diagnostic testing services raises questions concerning the current state of the art with regard to standardization of testing and quality assessment practices. The aim of this study was to provide a global overview of the test methods, laboratory procedures and quality assessment practices used for SARS-CoV-2 diagnostics. METHODS: The Molecular Diagnostics Committee of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC C-MD) initiated a survey among international laboratories performing molecular genetic detection of SARS-CoV-2. Questions on quality assurance, variant testing, sequencing and the transmission of findings were included in the survey. RESULTS: A total of 273 laboratories from 49 countries participated in the survey. The majority of the participating laboratories (92.2%) use reverse transcriptase polymerase chain reaction (RT-PCR). The majority of participating laboratories do not conduct testing to identify SARS CoV-2 variants. Participation in external quality assessment programs was reported by the majority of laboratories, however, 33.2% of the laboratories reported not participating in external quality assurance programmes. CONCLUSIONS: Based on the survey, molecular diagnostic methods for SARS-CoV-2 detection are clearly not standardized across different countries and laboratories. The survey found an array of responses in regard to sample preparation, collection, processing and reporting of results. This work suggests quality assurance is insufficiently performed by diagnostic laboratories conducting SARS-CoV-2 testing.

Enhancing the compatibility of BioCaRGOS silica sol-gel technology with ctDNA extraction and droplet digital PCR (ddPCR) analysis.

Previously, our group had demonstrated long term stabilization of protein biomarkers using BioCaRGOS, a silica sol-gel technology. Herein, we describe workflow modifications to allow for extraction of cell free DNA (cfDNA) from primary samples containing working concentrations of BioCaRGOS, as well as the compatibility of BioCaRGOS with droplet digital PCR (ddPCR) analysis for pancreatic cancer biomarkers i.e., KRAS circulating tumor DNA (ctDNA). Preliminary attempts to extract ctDNA from BioCaRGOS containing samples demonstrated interference in the extraction of primary samples and the interference with ddPCR analysis when BioCaRGOS was directly introduced to stabilize sample extracts. In our modified technique, we have minimized the interference caused by methanol with ddPCR by complete removal of methanol from the activated BioCaRGOS formulation prior to addition to the biospecimen or ctDNA extract. Interference of the silica matrix present in BioCaRGOS with ctDNA extraction was eliminated through the introduction of invert filtration of the sample prior to extraction. These modifications to the workflow of BioCaRGOS containing samples allow for use of BioCaRGOS for stabilization of trace quantities of nucleic acid biomarkers such as plasma ctDNA, while retaining the capability to extract the biomarker and quantify based on ddPCR.

A Shared Diagnostic Stewardship Approach toward Improving Autoimmune Encephalopathy Send-out Testing Utilization.

BACKGROUND: For many laboratories, autoimmune encephalopathy (AE) panels are send-out tests. These tests are expensive, and ordering patterns vary greatly. There is also a lack of consensus on which panel to order and poor understanding of the clinical utility of these panels. These challenges were presented to our newly formed, multidisciplinary, diagnostic stewardship committee (DSC). Through this collaboration, we developed an algorithm for ordering AE panels; combining diagnostic criteria with practice guidelines. METHODS: We analyzed test-ordering patterns in 2018 and calculated a true-positive rate based on clinical presentation and panel interpretation. An evidence-based approach was combined with input from the Department of Neurology to synthesize our algorithm. Efficacy of the algorithm (number of panels ordered, cost, and true positives) was assessed before and after implementation. RESULTS: In 2018, 77 AE-related panels were ordered, costing $137 510. The true-positive rate was 10%, although ordering multiple, similar panels for the same patient was common. Before implementing the algorithm (January 1-July 31, 2019), 55 panels were ordered, costing $105 120. The total true-positive rate was 3.6%. After implementation, 23 tests were ordered in a 5-month period, totaling $50 220. The true-positive rate was 13%. CONCLUSION: With the DSC-directed mandate, we developed an algorithm for ordering AE panels. Comparison of pre- and postimplementation data showed a higher true-positive rate, indicating that our algorithm was able to successfully identify the at-risk population for AE disorders. This was met with a 43% decrease in the number of tests ordered, with total cost savings of $25 000 over 5 months.

Long term storage of miRNA at room and elevated temperatures in a silica sol-gel matrix.

Storage of biospecimens in their near native environment at room temperature can have a transformative global impact, however, this remains an arduous challenge to date due to the rapid degradation of biospecimens over time. Currently, most isolated biospecimens are refrigerated for short-term storage and frozen (-20 °C, -80 °C, liquid nitrogen) for long-term storage. Recent advances in room temperature storage of purified biomolecules utilize anhydrobiosis. However, a near aqueous storage solution that can preserve the biospecimen nearly "as is" has not yet been achieved by any current technology. Here, we demonstrate an aqueous silica sol-gel matrix for optimized storage of biospecimens. Our technique is facile, reproducible, and has previously demonstrated stabilization of DNA and proteins, within a few minutes using a standard benchtop microwave. Herein, we demonstrate complete integrity of miRNA 21, a highly sensitive molecule at 4, 25, and 40 °C over a period of ∼3 months. In contrast, the control samples completely degrade in less than 1 week. We attribute excellent stability to entrapment of miRNA within silica-gel matrices.

Current and future challenges in quality assurance in molecular diagnostics.

The development and performance of molecular genetic assays has required increasingly complex quality assurance in recent years and continues to pose new challenges. Quality management officers, as well as academic and technical personnel are confronted with new molecular genetic parameters, methods, changing regulatory environments, questions regarding appropriate validation, and quality control for these innovative assays that are increasingly applying quantification and/or multiplex formats. Yet, quality assurance and quality control guidelines are still not widely available or in some circumstances have become outdated. For these reasons, the need for solutions to provide test confidence continues to grow. In order to integrate new test procedures into existing quality assurance measures, the ISO 15189 guideline can serve as an orientation. The ISO 15189 guideline describes requirements for medical laboratories and thus includes those performing molecular diagnostics. This article gives an overview of the possibilities and challenges in quality assurance of molecular parameters and shows possible solutions.

Comparison of Tissue Molecular Biomarker Testing Turnaround Times and Concordance Between Standard of Care and the Biocartis Idylla Platform in Patients With Colorectal Cancer.

OBJECTIVES: Management of colorectal cancer warrants mutational analysis of KRAS/NRAS when considering anti-epidermal growth factor receptor therapy and BRAF testing for prognostic stratification. In this multicenter study, we compared a fully integrated, cartridge-based system to standard-of-care assays used by participating laboratories. METHODS: Twenty laboratories enrolled 874 colorectal cancer cases between November 2017 and December 2018. Testing was performed on the Idylla automated system (Biocartis) using the KRAS and NRAS-BRAF cartridges (research use only) and results compared with in-house standard-of-care testing methods. RESULTS: There were sufficient data on 780 cases to measure turnaround time compared with standard assays. In-house polymerase chain reaction (PCR) had an average testing turnaround time of 5.6 days, send-out PCR of 22.5 days, in-house Sanger sequencing of 14.7 days, send-out Sanger of 17.8 days, in-house next-generation sequencing (NGS) of 12.5 days, and send-out NGS of 20.0 days. Standard testing had an average turnaround time of 11 days. Idylla average time to results was 4.9 days with a range of 0.4 to 13.5 days. CONCLUSIONS: The described cartridge-based system offers rapid and reliable testing of clinically actionable mutation in colorectal cancer specimens directly from formalin-fixed, paraffin-embedded tissue sections. Its simplicity and ease of use compared with other molecular techniques make it suitable for routine clinical laboratory testing.

Interactive modeling for ongoing utility of pharmacogenetic diagnostic testing: application for warfarin therapy.

BACKGROUND: The application of pharmacogenetic results requires demonstrable correlations between a test result and an indicated specific course of action. We developed a computational decision-support tool that combines patient-specific genotype and phenotype information to provide strategic dosage guidance. This tool, through estimating quantitative and temporal parameters associated with the metabolism- and concentration-dependent response to warfarin, provides the necessary patient-specific context for interpreting international normalized ratio (INR) measurements. METHODS: We analyzed clinical information, plasma S-warfarin concentration, and CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) and VKORC1 (vitamin K epoxide reductase complex, subunit 1) genotypes for 137 patients with stable INRs. Plasma S-warfarin concentrations were evaluated by VKORC1 genotype (-1639G>A). The steady-state plasma S-warfarin concentration was calculated with CYP2C9 genotype-based clearance rates and compared with actual measurements. RESULTS: The plasma S-warfarin concentration required to yield the target INR response is significantly (P < 0.05) associated with VKORC1 -1639G>A genotype (GG, 0.68 mg/L; AG, 0.48 mg/L; AA, 0.27 mg/L). Modeling of the plasma S-warfarin concentration according to CYP2C9 genotype predicted 58% of the variation in measured S-warfarin concentration: Measured [S-warfarin] = 0.67(Estimated [S-warfarin]) + 0.16 mg/L. CONCLUSIONS: The target interval of plasma S-warfarin concentration required to yield a therapeutic INR can be predicted from the VKORC1 genotype (pharmacodynamics), and the progressive changes in S-warfarin concentration after repeated daily dosing can be predicted from the CYP2C9 genotype (pharmacokinetics). Combining the application of multivariate equations for estimating the maintenance dose with genotype-guided pharmacokinetics/pharmacodynamics modeling provides a powerful tool for maximizing the value of CYP2C9 and VKORC1 test results for ongoing application to patient care.

Cell-free DNA diagnostics: current and emerging applications in oncology.

Liquid biopsy is a noninvasive dynamic approach for monitoring disease over time. It offers advantages including limited risks of blood sampling, opportunity for more frequent sampling, lower costs and theoretically non-biased sampling compared with tissue biopsy. There is a high degree of concordance between circulating tumor DNA mutations versus primary tumor mutations. Remote sampling of circulating tumor DNA can serve as viable option in clinical diagnostics. Here, we discuss the progress toward broad adoption of liquid biopsy as a diagnostic tool and discuss knowledge gaps that remain to be addressed.

Avoidable drug-gene conflicts and polypharmacy interactions in patients participating in a personalized medicine program.

AIM: Determine the ability of a pharmacogenetic service, PRIMER, to identify drug-gene (DGI) and drug-drug interactions (DDI) in patients across multiple conditions. PRIMER consists of patient selection criteria, a gene panel and actionable guidance for DGIs and DDIs. RESULTS: The average patient was prescribed 12 medications. PRIMER identified significant DGIs in 73% of patients tested, with 43% having more than one DGI. DDIs were found in 87% of patients. The most common actionable DGIs were for opioid, psychotropic and cardiovascular medications. CONCLUSION: The pairing of patient selection criteria, a multigene panel with evidence-based interpretation and review of DDIs maximizes the patients tested who have actionable benefit and alerts physicians to potentially critical adjustments needed for the patient's medication regimen.

Analysis of ligand binding by bioaffinity mass spectrometry.

BACKGROUND: Ligand binding is commonly analyzed using various immunoassays that are generally time-consuming and some may require secondary antibodies or gel electrophoresis which are also time-consuming and sometimes subjective. We introduced various examples for a more rapid approach using pre-activated surface chips which are analyzed by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Specific applications presented in this study include immobilization of antigen, antibody or oligo DNA on pre-activated chips with subsequent identification of the binding antibodies, antigens or DNA binding proteins to demonstrate the universal utility of this novel approach. METHODS: BSA-digoxin conjugate (BSA-Dig), anti-digoxin antibody, anti-urinary trypsin inhibitor (uTi) antibody, or a double stranded oligo nucleotide based on the nucleotide sequence between -91 and -10 of the human CYP 450 2E1 promoter were immobilized on the Ciphergen pre-activated surface chips. Anti-digoxin antibody, BSA-digoxin conjugate, uTi, and CYP450 2E1 promoter binding protein were captured on the chip and identified by SELDI-TOF MS. RESULTS: A protein with 141kDa was identified from anti-digoxin serum using BSA-Dig chips. This binding was competitively inhibited by addition of digoxin. Using anti-digoxin antibody, a peak at approximately 66kDa was detected in the preparation of BSA-Dig. This peak was also inhibited by free digoxin, suggesting BSA-Dig is detected. uTi fragments with approximately 3kDa to approximately 30kDa in the standard and urine samples were captured on the chip by anti-uTi antibody. Finally, we identified a 95-kDa CYP 450 2E1 promoter binding protein in HeLa cells nuclear extracts. CONCLUSIONS: Bioaffinity SELDI-TOF MS is a powerful and versatile approach for analysis of ligands. It eliminates tracer-labeled secondary antibodies and allows for determination of molecular weights of binding proteins and their ligands directly. This approach may also be considered for the detection of enzymes, receptors, or any other specific ligands.

Extracellular Vesicles Move Toward Use in Clinical Laboratories.

Extracellular vesicles (EVs) are membranous particles found in a variety of biofluids that encapsulate molecular information from the cell, which they originate from. This rich source of information that is easily obtained can then be mined to find diagnostic biomarkers. This article explores the current biological understanding of EVs and specific methods to isolate and analyze them. A case study of a company leading the charge in using EVs in diagnostic assays is provided.

Clinical Utility and Economic Impact of CYP2D6 Genotyping.

Pharmacogenetics examines an individual's genetic makeup to help predict the safety and efficacy of medications. Practical application optimizes treatment selection to decrease the failure rate of medications and improve clinical outcomes. Lack of efficacy is costly due to adverse drug reactions and increased hospital stays. Cytochrome P450 2D6 (CYP2D6) metabolizes roughly 25% of all drugs. Detecting variants that cause altered CYP2D6 enzymatic activity identifies patients at risk of adverse drug reactions or therapeutic failure with standard dosages of medications metabolized by CYP2D6. This article discusses the clinical application of pharmacogenetics to improve care and decrease costs.

Liquid Biopsies in Oncology and the Current Regulatory Landscape.

There is a profound need in oncology to detect cancer earlier, guide individualized therapies, and better monitor progress during treatment. Currently, some of this information can be achieved through solid tissue biopsy and imaging. However, these techniques are limited because of the invasiveness of the procedure and the size of the tumor. A liquid biopsy can overcome these barriers as its non-invasive nature allows samples to be collected over time. Liquid biopsies may also allow earlier detection than traditional imaging. Liquid biopsies include the analysis of circulating tumor cells (CTCs), cell-free nucleic acid (cfNA), or extracellular vesicles obtained from a variety of biofluids, such as peripheral blood. In this review, we discuss different liquid biopsy types and how they fit into the current regulatory landscape.

Prospective dosing of warfarin based on cytochrome P-450 2C9 genotype.

Cytochrome P-450 2C9 (CYP2C9) polymorphisms (CYP2C9*2 and CYP2C9*3) reduce the clearance of warfarin, increase the risk of bleeding, and prolong the time to stable dosing. Whether prospective use of a retrospectively developed algorithm that incorporates CYP2C9 genotype and nongenetic factors can ameliorate the propensity to bleeding and delay in achieving a stable warfarin dose is unknown. We initiated warfarin therapy in 48 orthopedic patients tailored to the following variables: CYP2C9 genotype, age, weight, height, gender, race, and use of simvastatin or amiodarone. By using pharmacogenetics-based dosing, patients with a CYP2C9 variant achieved a stable, therapeutic warfarin dose without excessive delay. However compared to those without a CYP2C9 variant, patients with a variant continued to be at increased risk (hazard ratio 3.6, 95% confidence interval 1.4-9.5, p = 0.01) for an adverse outcome (principally INR > 4), despite pharmacogenetics-based dosing. There was a linear relationship (R(2) = 0.42, p < 0.001) between the pharmacogenetics-predicted warfarin doses and the warfarin maintenance doses, prospectively validating the dosing algorithm. Prospective, perioperative pharmacogenetics-based dosing of warfarin is feasible; however, further evaluation in a randomized, controlled study is recommended.

What is next in pharmacogenomics? Translating it to clinical practice.

Pharmacogenomics (PG) holds promise for transforming medical therapeutics but the details of how the promise will become reality are still vague. In this article, we focus on the role that laboratory medicine, as a discipline, might play in transitioning the application of pharmacogenomics into the healthcare system and begin to frame a perspective on how PG may be viewed in this context. Development of clinical diagnostic tests usually evolves as a continuum of information starting with the discovery of a potential biological marker through to its routine use in clinical practice. This process has traditionally been rooted in the practice of laboratory medicine and, importantly, includes the development of testing strategies to optimize the predictive value of single or a combination of biological markers. In this context, we also discuss a perspective on some future strategies that may prove useful in advancing the application of PG, including the need for an evidenced-based approach and the potential role of proteomics as a means to drive more comprehensive strategies.