Contrived Samples Research Articles

Massively multiplex blocker displacement amplification (BDA) for the detection of measurable residual disease in acute myeloid leukemia.

e19045 Background: Next-generation sequencing (NGS) technologies, used to accomplish high throughput characterization of DNA, have an inherent error rate of between 0.1-1%. However, many important DNA sequences possess variant allele frequencies (VAFs) below this rate, potentially precluding their detection and identification by NGS. The previously published blocker displacement amplification (BDA) research technology has enabled selective amplification of sequences with variants, facilitating detection by NGS of these sequences with low VAFs at low read depths. Methods: Here we present the massively multiplex BDA library preparation method for the NGS-based detection of low VAF sequences. By optimizing our previously published Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE) algorithm, we designed a 4 tube assay, comprising up to ~2400 amplicons in each tube, and ~8300 total amplicons. Tube 1 was designed with the highest likelihood of on-target amplification and the lowest likelihood of dimerization or non-specific amplification. While Tube 1 targeted ~1000 regions covering the most-frequently observed mutations in current public databases of patients with acute myeloid leukemia (AML) across more than 200 genes, Tubes 2-4 were designed for enrichment of the full coding sequence of 63 genes. To highlight the performances of SADDLE, coverage uniformity, primer dimerization, and non-specific amplification of tube 1 were compared to those of a naively designed tube. The panel was further tested with contrived and reference samples to demonstrate its analytical performance. Results: The SADDLE algorithm design decreased the first pass amplicon dropout rate more than 10-fold, from 30% to ~2%, and further improved the overall on-target rate compared to the naively designed panel. Through SADDLE, we were able to achieve an 80% median coverage of 63 genes. The resulting panel has a bulk limit of detection (LoD) of below 0.1% VAF. Conclusions: Massively multiplex BDA enabled the NGS characterization of DNA targets with VAFs suitable for detection of AML measurable residual disease (MRD). This technology represents a 100-fold increase in current plexity for the detection of low VAF alleles over our previously published multiplex BDA technology, demonstrating the ability to perform high throughput sequencing of rare mutations.

Biomarker subpanel customization for different use cases in acute myeloid leukemia research.

e19026 Background: Treatment guidance and decision making according to modified RECIST guidelines often depends on characterization of these biomarkers in patients with cancer. Although panels with larger breadths of coverage are useful for initial detection of these biomarkers for evaluation of disease burden and prognosis, their cost-effectiveness decreases as treatment and disease progress; smaller subpanels may yield equally valuable information into disease progress and guide treatment in a more cost-effective manner, potentially leading to more datapoints in decision making in comparison to larger panels. Methods: QBDA is an NGS enrichment research technology that allows the enrichment of rare variants, reducing the sequencing cost while maximizing the sensitivity. Here we introduce a new capability of our platform that allows tailoring sequencing panel breath to the disease stage needs. We demonstrate the ability to generate a 384-plex NGS subpanel from a 2400-plex acute myeloid-leukemia (AML) measurable residual disease (MRD) targeting research panel. Both panels were tested on both wild-type and 0.1% variant allele frequency (VAF) AML MRD contrived samples. Results: Without any optimization of the subpanel itself, the subpanel exhibited similar or better performance to the main panel. The main panel had a ~80% uniformity of coverage whereas the subpanel had a uniformity of coverage exceeding 75%. The subpanel covers fewer genes of interest and requires fewer overall reads. Both panels had a bulk LoD of below 0.1% VAF. Conclusions: This data represents proof-of-concept for the design of small custom panels from large-scale multiplex panels for different use cases, potentially increasing cost-effectiveness.

The Development of a 3D Printer-Inspired, Microgravity-Compatible Sample Preparation Device for Future Use Inside the International Space Station.

Biological testing on the International Space Station (ISS) is necessary in order to monitor the microbial burden and identify risks to crew health. With support from a NASA Phase I Small Business Innovative Research contract, we have developed a compact prototype of a microgravity-compatible, automated versatile sample preparation platform (VSPP). The VSPP was built by modifying entry-level 3D printers that cost USD 200-USD 800. In addition, 3D printing was also used to prototype microgravity-compatible reagent wells and cartridges. The VSPP's primary function would enable NASA to rapidly identify microorganisms that could affect crew safety. It has the potential to process samples from various sample matrices (swab, potable water, blood, urine, etc.), thus yielding high-quality nucleic acids for downstream molecular detection and identification in a closed-cartridge system. When fully developed and validated in microgravity environments, this highly automated system will allow labor-intensive and time-consuming processes to be carried out via a turnkey, closed system using prefilled cartridges and magnetic particle-based chemistries. This manuscript demonstrates that the VSPP can extract high-quality nucleic acids from urine (Zika viral RNA) and whole blood (human RNase P gene) in a ground-level laboratory setting using nucleic acid-binding magnetic particles. The viral RNA detection data showed that the VSPP can process contrived urine samples at clinically relevant levels (as low as 50 PFU/extraction). The extraction of human DNA from eight replicate samples showed that the DNA extraction yield is highly consistent (there was a standard deviation of 0.4 threshold cycle when the extracted and purified DNA was tested via real-time polymerase chain reaction). Additionally, the VSPP underwent 2.1 s drop tower microgravity tests to determine if its components are compatible for use in microgravity. Our findings will aid future research in adapting extraction well geometry for 1 g and low g working environments operated by the VSPP. Future microgravity testing of the VSPP in the parabolic flights and in the ISS is planned.

Abstract LB293: The evolution of MRD assays: Moving beyond the tumor-Informed bespoke NGS panel

Abstract The non-invasive detection of circulating tumor DNA (ctDNA) from plasma has been shown to have clinical value for detection of minimal residual disease (MRD), identification of emerging resistance to treatment, and predicting treatment response. The ability to detect MRD following curative treatment allows for the stratification of patients with higher risk of disease recurrence. Multiple studies, across hundreds of patients in different indications, including lung and colon cancer, have shown that tumor-informed personalized assays have high sensitivity to detect recurrence. The tumor-informed bespoke panels utilize sequencing of the tumor tissue to identify clonal somatic variants, which are then used to generate a personalized panel to track the variants in patient plasma. Next-generation MRD technologies are aiming to provide greater sensitivity by expanding methods beyond the small, personalized panels. Utilization of increased numbers of somatic variants is predicted to achieve a greater level of sensitivity for detection of MRD. Here we describe, C2I Genomics which provides a novel platform that exploits whole genome sequencing of the tumor tissue and plasma to define a somatic variant signature. An important difference of this method is that it develops a personalized bioinformatic signature rather than a personalized NGS panel. To understand the potential of this novel methodology, we utilized a set of 54 contrived clinical sample dilutions from three indications, with an expected (or engineered) VAF range of 0.2-0.002%, that have commutability to the intended use population. We found that C2I Genomics technology is highly sensitive, where for all returned samples, the assay routinely achieved detection down to the lowest dilution of 0.002% allelic frequency. Overall, the assay resulted in an average detection rate of 93% across all dilutions (n = 54). The aggregate detection rate of several tumor-informed assays, examining the same dilutions, yielded a detection rate of 68%. In summary, the C2I Genomics assay showed robust detection from contrived samples which are representative of clinical trial samples in both cfDNA yields and allele frequency. Citation Format: Paul Labrousse, Daniel Stetson, James Hadfield, Chris Abbosh, Ahmet Zehir, Carl Barrett, Brian Dougherty, Darren Hodgson. The evolution of MRD assays: Moving beyond the tumor-Informed bespoke NGS panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB293.

Abstract 3384: Evaluation of a tumor informed MRD assay with contrived breast cancer samples

Abstract The non-invasive detection of circulating tumor DNA (ctDNA) from plasma has been shown to have clinical value for detection of minimal residual disease (MRD), emergence of resistance, and predicting treatment response. The sensitive detection of MRD following curative treatment allows for the identification of patients destined to recur. Higher assay sensitivities enable the longest lead times to clinical recurrence. Evaluations using several contrived pan-cancer samples have shown that the Inivata RaDaR tumor-informed personalized assay has a high sensitivity to detect ctDNA. The tumor-informed analysis utilizes sequencing of the tumor tissue to identify up to 48 clonal somatic variants, which are selected to generate a personalized panel to track the variants in patient plasma. To evaluate this technology in a disease specific manner, we generated a set of contrived plasma samples from commercial breast cancer patients and healthy individuals. The biological materials allowed us to generate a plasma sample with defined range of tumor content between 0.2 and 0.001% variant allele fraction (VAF). The sample dilutions were intended to recapitulate challenging cell-free DNA inputs that can be encountered in a clinical setting (median input: 5.76 ng [range 0.22 to 103.73]. Through our pan-cancer and breast cancer evaluations, we were able generate both sensitivity metrics along with a small limit of detection (LOD) study. The assay successfully detected tumor fractions in samples tested at 1X and 1.5X of the established LOD95% of 0.0011% VAF. RaDaR was able to detect the majority of MRD+ samples at 0.5X LOD. We will present data showing the level of sensitivity based on using both 2000 and 20000 genomic equivalents. The RaDaR assay showed high sensitivity for pan-cancer as well as ER+ breast cancer, which is known to have a low TMB compared to other subtypes. In this albeit small LoD study the Inivata assay was highly sensitive for the detection of ctDNA which is imperative to confidently detect ctDNA in advance of overt clinical recurrence. Citation Format: Hugh Russell, Paul LaBrousse, Daniel Stetson, Greg Jones, Giovanni Marsico, Tim Forshew, Robert McEwen, Elza De Bruin, Christopher Abbosh, Darren Hodgson, Brian Dougherty, James Hadfield, Carl Barrett. Evaluation of a tumor informed MRD assay with contrived breast cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3384.

Abstract 4354: An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples

Abstract Introduction: Hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) breast cancer is the most common type of breast cancer. Patients with HR+/HER2- metastatic breast cancer (mBC) often become resistant to aromatase inhibitors commonly used in endocrine therapy (ET). Estrogen receptor (ESR1) ligand binding domain mutations are frequently detected in HR+ mBC and have been reported to be associated with ET resistance, noting up to 60% patients with mBC will develop resistance to treatment via acquired resistance in ESR1. Recent studies have shown that monitoring of ESR1 mutations in plasma may serve as a predictive biomarker of acquired resistance to ET, showcasing a strong need for sensitive nucleic acid-based assays. We describe a comprehensive methodology for targeted clinical RT-qPCR monitoring of ESR1 mutations in plasma that utilizes both exosomal nucleic acids and cfDNA to report multiple mutations, provide a streamlined workflow, and accommodate a range of inputs from clinically relevant samples. Methods: Exosomal nucleic acids (exosomal DNA and RNA) and circulating cell-free DNA (cfDNA) were co-isolated using the ExoLution Plus Isolation Kit (Exosome Diagnostics). RT-qPCR-based target enrichment was performed using modified QuantideX reagents (Asuragen) and mutations were confirmed on the QuantStudio qPCR Platform (ThermoFisher). Bioinformatic analyses were conducted using custom software. Results: We developed novel technologies and tested greater than samples across key ET resistance mutations, utilizing contrived samples as needed due to availability. Preliminary studies demonstrated that ESR1 exosomal RNA + cfDNA ranged from approximately 9,500 copies to 64,000 copies in a set of 2mL female presumed normal plasma samples; these results suggest that even 2mL plasma could provide sufficient exosomal RNA/DNA + cfDNA for mutation detection at variant frequencies less than or equal to 0.01%. The ESR1 RT-qPCR showed greater than 90% analytical sensitivity (at less than or equal to 1% mutant allele frequency) and greater than90% analytical specificity. Further, the technology detected less than 10 mutant copies for most tested mutants, showing an LOD of at least 0.1% (5 mutant copies in a background of 5,000 WT copies). Conclusion: A fast, efficient, and sensitive ESR1 RT-qPCR panel approach was developed and evaluated, demonstrating the reliable and specific detection of rare variants in liquid biopsy specimens. Importantly, this prototype technology has potential to address several challenges associated with mutation monitoring in liquid biopsies by expanding detection of mutant analytes (exosomal RNA and DNA), improving analytical sensitivity (novel reagents and software), and increasing accessibility (qPCR instrument install base). Citation Format: Sarah Statt, Julie Thibert, Kurt Franzen, Aquiles Sanchez, Liangjing Chen, Megan Yociss, Elliot Hallmark, Melissa Church, Stela Filipovic-Sadic, Gary Latham, Johan Skog. An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4354.

Development and Clinical Evaluation of a Rapid Point of Care Test for Ebola Virus Infection in Humans.

The genus Ebolavirus contains multiple species of viruses that are highly contagious and lethal, often causing severe hemorrhagic fever. To minimize the global threat from Ebola virus disease (EVD), sustainable, field-appropriate tools are needed to quickly screen and triage symptomatic patients and conduct rapid screening of cadavers to ensure proper handling of human remains. The OraQuick® Ebola Rapid Antigen Test is an in vitro diagnostic single-use immunoassay for the qualitative detection of Ebola virus antigens that detects all known species within the genus Ebolavirus. Here, we report the performance of the OraQuick® Ebola Rapid Antigen Test and provide a comparison of its performance with other rapid diagnostic tests (RDTs) for EVD. OraQuick® Ebola demonstrated clinical sensitivity of 84.0% in archived EVD patient venous whole-blood (WB) samples, 90.9% in Ebola virus-infected monkey fingerstick samples, and 97.1% in EVD patient cadaver buccal swabs, as well as clinical specificity of 98.0-100% in venous WB samples and 99.1-100% in contrived saliva samples. It is the only 510(k)-cleared Ebola rapid test, has analytical sensitivity as good as or better than all RDT comparators for EVD, and can detect the Sudan virus. Our data demonstrate that the OraQuick® Ebola Rapid Antigen Test is a sensitive and specific assay that can be used for rapid detection of EBOV in humans and could support efforts for EVD-specific interventions and control over outbreaks.

High levels of factor VIII activity in patients with acquired hemophilia A in remission are associated with unusually low coagulation potentials.

Elevated factor VIII activity (FVIII:C) is often observed in patients with acquired hemophilia A (PwAHA) in remission. However, comprehensive coagulation potentials in this patient group remain to be investigated. To evaluate comprehensive coagulation potentials in PwAHA. We investigated coagulation function in eleven PwAHA with high FVIII:C (> 150IU/dL) using thrombin generation assay (TGA) and/or rotational thromboelastometry (ROTEM), and compared findings with results obtained from contrived samples generated by spiking recombinant FVIII. The median FVIII:C and FVIII inhibitor titers during remission in enrolled PwAHA were 206IU/dL and 0.44 BU/mL, respectively. In all patients, lag time and time to peak were either prolonged or normal compared to contrived samples corresponding to their FVIII:C. However, higher values of peak thrombin and endogenous thrombin potentials compared to contrived samples were observed in two patients. ROTEM parameters were within normal ranges in all cases. One patient (FVIII:C 171IU/dL) developed venous thrombosis and pulmonary embolism, but TGA parameters showed low or normal coagulation potential compared to contrived samples corresponding to his FVIII:C. PwAHA with high FVIII:C could exhibit lower coagulation potentials than those corresponding to their FVIII:C.

Quantification of capture efficiency, purity, and single-cell isolation in the recovery of circulating melanoma cells from peripheral blood by dielectrophoresis.

This paper describes a dielectrophoretic method for selection of circulating melanoma cells (CMCs), which lack reliable identifying surface antigens and are extremely rare in blood. This platform captures CMCs individually by dielectrophoresis (DEP) at an array of wireless bipolar electrodes (BPEs) aligned to overlying nanoliter-scale chambers, which isolate each cell for subsequent on-chip single-cell analysis. To determine the best conditions to employ for CMC isolation in this DEP-BPE platform, the static and dynamic dielectrophoretic response of established melanoma cell lines, melanoma cells from patient-derived xenografts (PDX) and peripheral blood mononuclear cells (PBMCs) were evaluated as a function of frequency using two established DEP platforms. Further, PBMCs derived from patients with advanced melanoma were compared with those from healthy controls. The results of this evaluation reveal that each DEP method requires a distinct frequency to achieve capture of melanoma cells and that the distribution of dielectric properties of PBMCs is more broadly varied in and among patients versus healthy controls. Based on this evaluation, we conclude that 50 kHz provides the highest capture efficiency on our DEP-BPE platform while maintaining a low rate of capture of unwanted PBMCs. We further quantified the efficiency of single-cell capture on the DEP-BPE platform and found that the efficiency diminished beyond around 25% chamber occupancy, thereby informing the minimum array size that is required. Importantly, the capture efficiency of the DEP-BPE platform for melanoma cells when using optimized conditions matched the performance predicted by our analysis. Finally, isolation of melanoma cells from contrived (spike-in) and clinical samples on our platform using optimized conditions was demonstrated. The capture and individual isolation of CMCs, confirmed by post-capture labeling, from patient-derived samples suggests the potential of this platform for clinical application.

Nanophotonic immunoarray with electrochemically roughened surfaces for handheld detection of secreted PD-L1 to predict immuno-oncology efficacy.

The analysis of secreted protein biomarkers can be a useful non-invasive method of predicting or monitoring cancer therapeutic response. The increased level of soluble programmed cell death protein ligand 1 (sPD-L1) is a promising predictive biomarker for selecting patients who are likely to respond to immune checkpoint immunotherapy. The current established immunoassay for secreted protein analysis is enzyme-linked immunosorbent assay (ELISA). Yet, ELISA is generally still liable to limited detection sensitivity and restricted to bulky chromogenic readout equipment. Herein, we present a designed nanophotonic immunoarray sensor which achieved sPD-L1 analysis at high-throughput, enhanced detection sensitivity and portability. The key benefits of our nanophotonic immunoarray sensor are (i) high-throughput surface-enhanced Raman scattering (SERS) analysis of multiple samples on a singular platform; (ii) improved sPD-L1 detection sensitivity at 1 pg mL-1 (by two orders of magnitude as compared to ELISA) via electrochemically roughened gold sensor surfaces; (iii) fit for handheld SERS detection with miniaturized equipment footprint. We evaluated the analytical performance of the nanophotonic immunoarray sensor and successfully demonstrated quantitative sPD-L1 detection in a cohort of contrived human plasma samples.

374. Evaluation of the ChromaCode HDPCR™ RV6 RUO Assay

Abstract Background In December of 2019, an outbreak of pneumonia was reported to the World Health Organization. The etiology of this outbreak was identified as novel coronavirus (SARS-CoV-2), which has resulted in a global pandemic. The persistence of circulating SARS-CoV-2 leads to the need for a test that can detect and differentiate SARS-CoV-2 from common causes of respiratory illnesses such as influenza and respiratory syncytial virus (RSV). ChromaCode’s HDPCR™ RV6 RUO Assay is a real time PCR test that provides qualitative detection of SARS-CoV-2, Influenza A, Influenza B, and RSV A/B from upper respiratory specimens. An evaluation in an infectious disease molecular laboratory was performed and presented herein. Methods A combination of residual and contrived samples were used to evaluate the ChromaCode HDPCR™ RV6 RUO assay. Residual upper respiratory samples tested with the standard of care were enrolled in this study to evaluate RV6 RUO. A total of 201 samples originally tested using Diasorin Simplexa® tests were enrolled: 17 assisted mid-turbinate, 15 contrived Flu B samples, and 179 nasopharyngeal swabs. The contrived samples were made utilizing dilutions of Zeptometrix Influenza B material (PN 0810253CF LN 325137) in negative NPS matrix to achieve final concentrations of 0.02-0.04 TCID50/mL. Results The ChromaCode RV6 RUO Assay had comparable results to the standard of care test. The assays had a 99.0% concordance for the Influenza A target, 100% concordance for the Influenza B target, 97.5% concordance for the RSV A/B target, and 99.5% concordance for the SARS-CoV-2 target. Conclusion There are several tools available for distinguishing between common respiratory illnesses, which now include SARS-CoV-2. This study demonstrated the viability of the HDPCR RV6 RUO Assay to be a solution for distinguishing between common respiratory illnesses. RV6 RUO showed a high degree of concordance with the standard of care test and highlighted the flexibility of the RV6 RUO assay to be used with multiple upper respiratory sample types. The HDPCR RV6 RUO Assay and the accompanying ChromaCode Cloud software is a user-friendly solution with minimal setup time and rapid data analysis. Disclosures Kristin Fathe, PhD, ChromaCode, Inc: Work there with stock options.

Ligation-based assay for variant typing without sequencing: Application to SARS-CoV-2 variants of concern.

COVID-19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. A ligation-based typing assay was developed to detect known variants of severe acute respiratory syndrome virus 2 (SARS-CoV-2) by identifying the presence of characteristic single-nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real-time polymerase chain reaction (PCR) instrumentation for assay performance. The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for the classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold standard of viral genome sequencing. It achieved an average limit of detection of 7.4× 104 genome copies/mL in contrived nasopharyngeal samples. The ligation-based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by the sequence alignment of clinical samples. The assay demonstrates the potential for robust variant typing with performance comparable with next-generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource dependency and generalizability could expand access to variant classification information for pandemic surveillance.

Development and validation of blood tumor mutational burden reference standards.

Tumor mutational burden (TMB), measured by exome or panel sequencing of tumor tissue or blood (bTMB), is a potential predictive biomarker for treatment benefit in patients with various cancer types receiving immunotherapy targeting checkpoint pathways. However, significant variability in TMB measurement has been observed. We developed contrived bTMB reference materials using DNA from tumor cell lines and donor-matched lymphoblastoid cell lines to support calibration and alignment across laboratories and platforms. Contrived bTMB reference materials were developed using genomic DNA from lung tumor cell lines blended into donor-matched lymphoblastoid cell lines at 0.5% and 2% tumor content, fragmented and size-selected to mirror the size profile of circulating cell-free tumor DNA with TMB scores of 7, 9, 20, and 26 mut/Mb. Variant allele frequency (VAF) and bTMB scores were assessed using PredicineATLAS and GuardantOMNI next-generation sequencing assays. DNA fragment sizes in the contrived reference samples were similar to those found within patient plasma-derived cell-free DNA, and mutational patterns aligned with those in the parental tumor lines. For the 7, 20, and 26 mut/Mb contrived reference samples with 2% tumor content, bTMB scores estimated using either assay aligned with expected scores from the parental tumor cell lines and showed good reproducibility. A bioinformatic filtration step was required to account for low-VAF artifact variants. We demonstrate the feasibility and challenges of producing and using bTMB reference standards across a range of bTMB levels, and how such standards could support the calibration and validation of bTMB platforms and help harmonization between panels and laboratories.

A Novel NGS Assay to Detect Any KMT2A fusion Transcript at Low Levels

Introduction: Measurable residual disease (MRD) detection is associated with relapse. Leukemias with KMT2A rearrangement (KMT2Ar) have an adverse prognosis with more than 80 possible fusion partner genes (FPG) identified. With the advent of effective targeted therapies for KMT2Ar leukemia such as menin inhibitors, there is an unmet need for sensitive assays to detect these various fusions, not only to guide and monitor therapy, but also to predict and intercept relapse. Multicolor flow cytometry (MFC) is the only readily available test for MRD in KMT2Ar acute myeloid leukemia (AML), not requiring prior knowledge of exact fusions and breakpoints for design of patient-specific assays. Standard next-generation sequencing (NGS) methods cannot accurately detect genetic alterations at low frequencies because of inherent technical errors and the need for high sequencing depth. Blocker Displacement Amplification (BDA) technology enables selective detection of such rare alterations with NGS (Wu LR, Nat Biomed Eng 2017). Here, we demonstrate the effective detection of KMT2Ar and its various FPG with a novel NGS assay leveraging BDA. Methods: The NuProbe RNA fusion proprietary technology uses BDA (FusionBDA) where primers flank any known exon junction of cDNA corresponding to transcripts of the gene of interest, while non-extensible oligonucleotides (blockers) span the wildtype (WT) exon junction, therefore suppressing amplification of WT while differentially amplifying fusion junctions. As a result, final libraries are enriched with reads mapping to fusion genes. This allows de novo calls and minimizes read requirements. We designed a custom panel targeting 35 exons of KMT2A (forward and reverse) and developed a pipeline using 3 previously established fusion detection methods (Arriba, STAR-Fusion, FusionCatcher). We validated this assay in a KMT2Ar cell line. Limit-of-detection (LoD) was assessed by blending RS4;11 (leukemia with KMT2A::AFF1) with HT-1197 cells (bladder cancer, no KMT2Ar), to attain contrived samples with 0.001% to 100% tumor fractions (400ng input). Limit-of-input (LoI) was assessed using cDNA with 1- 400ng RNA from 100% and 0.1% tumor fraction samples. In a blinded analysis, we tested 50 bone marrow samples from 35 patients with KMT2Ar AML, chosen deliberately with various 11q23 translocations including rare ones. Results: Following LoD experiment, KMT2Ar was detected accurately down to 0.005% tumor fraction (Figure 1). Subsequently, LoI assay revealed the ability to detect KMT2Ar in 100% VAF tumor fraction samples using as low as 1ng of input RNA and using 400ng in 1% VAF samples. Among samples with morphologic evidence of leukemia, FusionBDA detected various KMT2Ar in 44/45 samples (97.8%) except 1 sample with a t(11;19)(q23;p13.1) (Table 1). This sample had a suboptimal amount and quality of cDNA loaded. We evaluated concordance of predicted FPG based on bands by cytogenetics (CG) and the published literature on KMT2A recombinome. We found that FusionBDA identified the same predicted FPG (same band) in 41/45 samples (91%). In samples with various t(11;19) translocations, different FPG were identified all located on 11p13 within a few bands from ones by CG, likely representing the accurate FPG given the low resolution of CG and the associated error in determining arm band accurately. Finally, we explored the potential of this assay for MRD detection by examining 5 samples at morphologic remission following treatment. All samples where fluorescence in situ hybridization (FISH) detected a KMT2A fusion had FusionBDA+ (31/31 samples including 1 in morphologic remission). 1 sample with no detectable KMT2A fusion by FISH and MRDneg by MFC, had detectable fusion by FusionBDA, indicating possibly an improved sensitivity. 3 samples with MRD+ by MFC also had detectable KMT2Ar by FusionBDA (lowest MRD+ level by flow was 0.5%). 1 sample was MRD+ by MFC and had a detectable KMT2Ar by FusionBDA in 2/3 callers (detectable by FusionCatcher and StarFusion not Arriba), therefore not meeting our stringent criteria though likely concordant with MRD by MFC. Further analysis in the MRD setting is planned. Conclusion: FusionBDA is a novel, sensitive, and specific assay for KMT2Ar acute leukemia. This assay is able to agnostically detect KMT2Ar with various fusions at a predicted sensitivity of at least 0.005%, therefore allowing both MRD detection and target identification. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Detecting Exogenous Contamination of Clinical Blood Samples with Crystalloid Solutions: A Data Driven Approach to Derive and Validate Delta Checks

Abstract Pre-analytical error may occur when collecting blood samples from intravenous (IV) lines. Blood samples can be inadvertently diluted by exogenous solutions if an insufficient amount of blood is discarded prior to collection. Rules built in the laboratory information system or middleware can be used to alert laboratory staff to specimens that require co-investigation with the clinical team to determine if sample dilution is possible or likely. Currently, however, there is a lack of literature to guide laboratorians on how to define quality rules of this nature, particularly for detecting subtle, rather than gross contamination by commonly administered IV-fluids. Accordingly, the primary objective of this study is to derive sensitive and specific, multivariate delta checks to detect more subtle blood sample contamination by commonly used crystalloid solutions. To derive the rules, we began with in vitro experiments by spiking increasing volumes of major IV-fluids (normal saline (NS), lactated ringers (LR), and 5% dextrose (D5W)) into clinical blood samples that were collected from healthy donors (n=3). Crystalloid solutions were serially spiked into blood samples at 10% (solution:sample) increments for NS and LR from 10% up to 90%, and in 5% increments for D5W, up to 50%. Basic metabolic panel (BMP) analytes were measured and compared between neat and contrived samples. All testing was performed on Roche Cobas 8000 chemistry auto-analyzers. Based on in vitro data, we derived multivariate delta checks using analytes in a BMP that would reflect 10-40% contamination by a given fluid. We then performed a retrospective data analysis on more than 28 thousand, serially collected BMP results to identify samples that were flagged by these rules. Chart review was then performed to identify EHR-based evidence of temporally associated administration of crystalloid solutions through a peripheral IV access line. Increasing sample dilution from the in vitro study showed significant changes in several BMP analytes across all fluid types. With respect to NS, the most significant changes relative to baseline were observed with potassium, calcium, and chloride, while for LR, the most significant changes relative to baseline were observed with glucose, bicarbonate, and calcium. For D5W, significant changes were observed with glucose, calcium, chloride and sodium. Accordingly, delta check rules were implemented using relative changes of the aforementioned analytes. On chart review, we found 80-100% of flagged samples came from patients with administration of that given fluid in the last 12-24 hours. The results from this study support delta checks that consider multiple analytes, and fluid-specific bias profiles, as this may provide a more sensitive and specific approach to detecting contamination of clinical blood samples by crystalloid solutions. Further evaluation using retrospective data analysis will be used to validate these rules sensitivity and specificity followed by prospective testing before implementation.

Contamination of clinical blood samples with crystalloid solutions: An experimental approach to derive multianalyte delta checks

BackgroundLaboratorians are left unguided by a paucity of literature on how to configure rules for the detection of intravenous (IV) fluid contamination in blood samples. We designed a study to determine the in vitro effect of increasing blood sample contamination from commonly used crystalloid solutions and how these observations can guide the derivation of multianalyte delta checks to detect such pre-analytical error. MethodsIn this study, we spiked increasing volumes of commonly used IV fluids (normal saline (NS), lactated ringers (LR), and 5% dextrose) into blood samples that were collected from healthy donors. Routine chemistry analytes were measured and compared between neat and contrived samples. From these observations, we derived several permutations of multianalyte delta checks using the basic metabolic panel framework and evaluated rule performance using retrospective data. ResultsThe wet chemistry experiments showed that increasing the volume of crystalloid solution contamination significantly changed several analytes. Subsequently derived multianalyte delta check procedures were applied to retrospective data. For all IV fluids tested, smaller magnitudes of analyte change resulted in more samples flagged. ConclusionMultianalyte delta checks may be an effective method for the detection of IV fluid contamination.

Singleplex, multiplex and pooled sample real-time RT-PCR assays for detection of SARS-CoV-2 in an occupational medicine setting

For workplaces which cannot operate as telework or remotely, there is a critical need for routine occupational SARS-CoV-2 diagnostic testing. Although diagnostic tests including the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (CDC Diagnostic Panel) (EUA200001) were made available early in the pandemic, resource scarcity and high demand for reagents and equipment necessitated priority of symptomatic patients. There is a clearly defined need for flexible testing methodologies and strategies with rapid turnaround of results for (1) symptomatic, (2) asymptomatic with high-risk exposures and (3) asymptomatic populations without preexisting conditions for routine screening to address the needs of an on-site work force. We developed a distinct SARS-CoV-2 diagnostic assay based on the original CDC Diagnostic Panel (EUA200001), yet, with minimum overlap for currently employed reagents to eliminate direct competition for limited resources. As the pandemic progressed with testing loads increasing, we modified the assay to include 5-sample pooling and amplicon target multiplexing. Analytical sensitivity of the pooled and multiplexed assays was rigorously tested with contrived positive samples in realistic patient backgrounds. Assay performance was determined with clinical samples previously assessed with an FDA authorized assay. Throughout the pandemic we successfully tested symptomatic, known contact and travelers within our occupational population with a ~ 24–48-h turnaround time to limit the spread of COVID-19 in the workplace. Our singleplex assay had a detection limit of 31.25 copies per reaction. The three-color multiplexed assay maintained similar sensitivity to the singleplex assay, while tripling the throughput. The pooling assay further increased the throughput to five-fold the singleplex assay, albeit with a subtle loss of sensitivity. We subsequently developed a hybrid ‘multiplex-pooled’ strategy to testing to address the need for both rapid analysis of samples from personnel at high risk of COVID infection and routine screening. Herein, our SARS-CoV-2 assays specifically address the needs of occupational healthcare for both rapid analysis of personnel at high-risk of infection and routine screening that is essential for controlling COVID-19 disease transmission. In addition to SARS-CoV-2 and COVID-19, this work demonstrates successful flexible assays developments and deployments with implications for emerging highly transmissible diseases and future pandemics.

An improved, simple and field-deployable CRISPR-Cas12a assay for the detection of SARS-CoV-2.

The RT-PCR is the most popular confirmatory test for SARS-CoV-2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR-based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high-quality lateral flow strips. The main goal of this study was to improve the Cas12a-based SARS-CoV-2 DETECTR method and develop a portable and field-deployable system to reduce the recurring consumable cost. Specific regions of N and E genes from SARS-CoV-2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop-mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a-based trans-cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT-LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans-cleavage reaction into a single ready-to-use mix. A portable, cost-effective battery-operated instrument, CRISPR-CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples. The simplified CRISPR-based SARS-CoV-2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR-CUBE. Significant improvement in Cas12 method, coupled with simple visualization of end point makes the method and instrument deployable at the point-of-care (POC) for SARS-CoV-2 detection, without any recurrent cost for the lateral flow strips which is used in other POC methods.

Detecting measurable residual disease beyond 10−4 by an IGHV leader-based NGS approach improves prognostic stratification in CLL

AbstractThe sensitivity of conventional techniques for reliable quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10−4. Measuring MRD <10−4 could help to further distinguish between patients with CLL with durable remission and those at risk of early relapse. We herein present an academically developed immunoglobulin heavy-chain variable (IGHV) leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL. We demonstrate, based on measurements in contrived MRD samples, that the linear range of detection and quantification of our assay reaches beyond MRD 10−5. If provided with sufficient DNA input, MRD can be detected down to MRD 10−6. There was high interassay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative polymerase chain reaction (PCR) (r = 0.92 [95% confidence interval {CI}, 0.86-0.96]) and droplet digital PCR (r = 0.93 [95% CI, 0.88-0.96]) on contrived MRD samples. In a cohort of 67 patients from the CLL11 trial, using MRD 10−5 as a cutoff, undetectable MRD was associated with superior progression-free survival (PFS) and time to next treatment. More important, deeper MRD measurement allowed for additional stratification of patients with MRD <10−4 but ≥10−5. PFS of patients in this MRD range was significantly shorter, compared with patients with MRD <10−5 (hazard ratio [HR], 4.0 [95% CI, 1.6-10.3]; P = .004), but significantly longer, compared with patients with MRD ≥10−4 (HR, 0.44 [95% CI, 0.23-0.87]; P = .018). These results support the clinical utility of the IGHV leader-based NGS assay.

P652: MEASURING MINIMAL RESIDUAL DISEASE BEYOND 10-4 THROUGH IGHV LEADER-BASED NEXT GENERATION SEQUENCING IMPROVES PROGNOSTIC STRATIFICATION IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background: The sensitivity of conventional techniques for reliable quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10-4. Measuring MRD <10-4 could help to further distinguish between CLL patients with durable remission and those at risk of early relapse. Aims: To develop an academically sourced IGHV leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL. Methods: The IGHV leader primer set developed by the EuroClonality-NGS working group was used to amplify all IGH rearrangements present in an end of treatment (EOT) DNA pool. The IGH clonal target was amplified in a single-step PCR and subsequent sequencing was performed on the Illumina MiSeq platform. To calculate MRD depth, NGS output was annotated through in the ARResT/Interrogate immunoprofiler. Technical validation of the IGHV leader-based assay was performed on contrived MRD samples, created by serial dilution of pretreatment CLL DNA pooled PBMC DNA from healthy donors. Clinical validation of the IGHV leader-based assay was performed on EOT samples from the CLL11 trial (NCT01010061). Results: In 176/183 (96%) measurements on contrived samples, the CLL-specific rearrangement was detected. The limit of detection and limit of quantitation were estimated at 3.4 [95% CI 1.9-16.0] and 3.8 [95% CI 1.7-8.2] malignant cells per assay, respectivelly. The limit of blank was found to be 0. Linearity was established in the MRD 10-2-10-5 range (r=0.94 [95%CI 0.91-0.96]). Of note, when provided with sufficient DNA input, MRD could even be detected down to MRD 10-6. There was high inter-assay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative PCR (r=0.92, [95%CI 0.86-0.96]) and droplet digital PCR (r=0.93, [95%CI 0.88-0.96]). In a cohort of 67 patients from the CLL11 trial, using 5μg DNA input and MRD 10-5 as a cut-off, undetectable MRD (uMRD) was associated with superior progression-free survival (PFS) and time to next treatment. Importantly, deeper MRD measurement allowed for additional stratification of patients with MRD <10-4 but ≥10-5. Patients with MRD in this range had a significantly shorter PFS, compared to patients with uMRD <10-5, but significantly longer, compared to patients with MRD ≥10-4 (median PFS: ≥10-4, 10.4 months; <10-4 but ≥10-5, 27.5 months; uMRD <10-5, NR, 4-year PFS rate: ≥10-4, 66.7%; <10-4 but ≥10-5, 25.0%; uMRD <10-5, 7.2%, P<0.001) (Figure 1). Image:Summary/Conclusion: We here present an academically-developed, IGHV leader-based NGS assay for the detection and quantification of MRD in CLL beyond MRD 10-4. The assay has high sensitivity and is quantitative and linear up to MRD 10-5 when using 5μg DNA input. Measurement to MRD 10-6 is feasible, conditional on sufficient DNA input. The deeper MRD measurements enabled by the IGHV leader-based NGS assay resulted in improved stratification of CLL patients following treatment.