Prognostic value of low-level MRD in adult acute lymphoblastic leukemia detected by low- and high-throughput methods

Persistence of minimal residual disease (MRD) after induction/consolidation therapy in acute lymphoblastic leukemia is the leading cause of relapse. The GMALL 07/2003 study used MRD detection by RQ-PCR of clonal immune gene rearrangements with 1x10-4 as discriminating cut-off: levels ≥1x10-4 define molecular failure and MRD-negativity with an assay sensitivity of at least 1x10-4 defines complete molecular response. The clinical relevance of MRD results not fitting in these categories is unclear and termed "molecular not evaluable" (MolNE) towards MRD-based treatment decisions. Within the GMALL 07/03 study, 1019 consecutive bone marrow samples after first consolidation were evaluated for MRD. Patients with complete molecular response had significantly better outcome (five-year overall survival, 5y-OS=85±2%, n=603; five-year disease-free survival, 5y-DFS=73±2%, n=599) compared to patients with molecular failure 5y-OS=40±3%, n=238; 5y-DFS=29±3%, n=208), with MolNE patients in-between (5y-OS=66±4%, 5y-DFS=52±4%, n=178). Of MolNE samples re-analyzed using next-generation sequencing (NGS), patients with undetectable NGS-MRD (n=44; 5y-OS=88±5%, 5y-DFS=70±7%) had significantly better outcome than those with positive NGS-MRD (n=42; 5y-OS=37±8%, 5y-DFS=33±8%). MolNE MRD results are not just borderline values with questionable relevance, but form an intermediate risk group, assignment of which can be further improved by NGS.

Confidence Intervals for Assessing Non-Inferiority with Assay Sensitivity in a Three-Arm Trial with Normally Distributed Endpoints

Various approaches including hypothesis test and confidence interval (CI) construction have been proposed to assess non-inferiority and assay sensitivity via a known fraction or pre-specified margin in three-arm trials with continuous or discrete endpoints. However, there is little work done on the construction of the non-inferiority margin from historical data and simultaneous generalized CIs (SGCIs) in a three-arm trial with the normally distributed endpoints. Based on the generalized fiducial method and the square-and-add method, we propose two simultaneous CIs for assessing non-inferiority and assay sensitivity in a three-arm trial. For comparison, we also consider the Wald-type Bonferroni simultaneous CI and parametric bootstrap simultaneous CI. An algorithm for evaluating the optimal sample size for attaining the pre-specified power is given. Simulation studies are conducted to investigate the performance of the proposed CIs in terms of their empirical coverage probabilities. An example taken from the mildly asthmatic study is illustrated using the proposed simultaneous CIs. Empirical results show that the proposed generalized fiducial method and the square-and-add method behave better than other two compared CIs.

Detection of the omicron variant virus with the Abbott BinaxNow SARS-CoV-2 Rapid Antigen Assay

The US Centers for Disease Control and Prevention recommends rapid testing for SARS-CoV-2 infection as a key element of epidemic control. The Abbott BinaxNow is in widespread use in the United States for self-testing and as part of public health screening campaigns, but has not been evaluated for use with the omicron variant of SARS-CoV-2. We recruited individuals testing positive for COVID-19 PCR at an academic medical center. Anterior nasal swabs were stored in viral transport media and evaluated by viral load quantification and whole genome sequencing. We created serial dilutions from 2.5x103- 2.5x105 viral copies/specimen for two delta and omicron specimens, respectively, and tested each with the BinaxNow assay per manufacturer instructions. Results were interpreted by three readers, blinded to the specimen variant and concentration. All omicron and delta specimens with concentrations of 100,000 copies/swab or greater were positive by the BinaxNow Assay, a concentration similar to previously reported limits of detection for this assay. Assay sensitivity diminished below that. This study demonstrates that Omicron variant SARS-CoV-2 infections are detected by the BinaxNow rapid antigen assay. Additional laboratory and clinical validation assessments are needed to better determine their limits of detection and performance in real-world settings.

Measuring Proviral HIV-1 DNA: Hurdles and Improvements to an Assay Monitoring Integration Events Utilising Human Alu Repeat Sequences

Integrated HIV-1 DNA persists despite antiretroviral therapy and can fuel viral rebound following treatment interruption. Hence, methods to specifically measure the integrated HIV-1 DNA portion only are important to monitor the reservoir in eradication trials. Here, we provide an up-to-date overview of the literature on the different approaches used to measure integrated HIV-1 DNA. Further, we propose an implemented standard-curve free assay to quantify integrated HIV-1 DNA, so-called Alu-5LTR PCR, which utilises novel primer combinations. We tested the Alu-5LTR PCR in 20 individuals on suppressive ART for a median of nine years; the results were compared to those produced with the standard-free Alu-gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1–12) and 14 (5–26) with the Alu-gag and Alu-5LTR, respectively. The ratios between Alu-gag vs Alu-5LTR results were distributed within the cohort as follows: most patients (12/20, 60%) provided ratios between 2–5, with 3/20 (15%) and 5/20 (25%) being below or above this range, respectively. Alu-5LTR assay sensitivity was also determined using an “integrated standard”; the data confirmed the increased sensitivity of the assay, i.e., equal to 0.25 proviruses in 10,000 genomes. This work represents an improvement in the field of measuring proviral HIV-1 DNA that could be employed in future HIV-1 persistence and eradication studies.

Anti-drug Antibody Validation Testing and Reporting Harmonization

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting. Graphical Abstract

Anti-ganglioside Antibodies in Guillain-Barre Syndrome: A Novel Immunoblotting-Panel Assay

Objective: This study aimed to determine the diagnostic efficiency of a novel immunoblotting detection assay for anti-ganglioside antibodies (AGAs) in the Guillain–Barre syndrome (GBS).Method: Serum immunoglobulin (IgG and IgM) of AGAs were measured in 121 participants from a registered cohort study of immune-mediated neuropathies and 29 healthy controls by immunoblotting panel assay. Sensitivity, specificity, and positive predictive value (PPV) of the assay were compared to calculate the diagnostic accuracy.Result: In our cohort, any of the AGAs were positive in 42.4% of the GBS patients. The sensitivity and specificity of AGAs (both IgG and IgM) in the diagnosis of GSB were 42 and 76% while for IgG-AGAs were 35 and 87%. AGAs positivity had a significant association with the AMAN subtype (P = 0.0004), and the sensitivity, specificity of AGAs in AMAN were 86, 69%, respectively with high (AUC = 0.78, p = 0.002) discriminative powers. GM1-IgG AGA was more common and specific to AMAN patients than other GBS forms (p = 0.008).Conclusion: Our novel immunoblotting detection assay could complement GBS diagnosis. IgG-AGAs were more likely to be detected in GBS, and GM1-IgG AGA could assist AMAN diagnosis.

Species Identification and Quantification of Bovine Bone Marrow from Non-Bovine Products using DNA and Protein Fingerprints

Bovine bone marrow is traditionally regarded as a highly nutritious food that has been widely used as a medicinal and healthy food for several decades in China. A large number of adulterated and counterfeit bone marrows from pigs and donkeys have been used in place of bovine bone marrow in commercial products, which are almost identical morphologically to species. Therefore, we explored a feasibility of fingerprinting of multiplex PCR gels combined with imaged SDS-PAGE gels of proteins. Three pairs of specific primers for bovine, pig and donkey were designed according to the conserved sequence in mitochondrial cytochrome b. The optimal reaction conditions for triple PCR were optimized. A modified method was used to extract total proteins and SDS-PAGE gels of proteins were set up. A three-fold PCR detection assay was successfully established to identify three species of bovine, pig and donkey. Three primers have good specificity and high sensitivity. Additionally, the assay sensitivity test confirmed that the extracted DNA concentration was the lowest of bovine bone marrow at 0.1 pg. The assay also showed 100% specificity. The electrophoretogram of SDS-PAGE showed distinct electrophoresis profiles among three species. The established fingerprint of multiplex PCR DNA gels combined with imaged SDS-PAGE gels of proteins has strong specificity and can be quickly and accurately used for the identification of bovine bone marrow. Rapid authentication of bovine bone marrow and differentiation from non-bovine products can be achieved using an improved SDS alkali denaturation method, species-specific PCR assay combined with imaged SDS-PAGE gels of proteins.

FDA Cleared Companion Diagnostics (CDx) Tests (IDH1, IDH2 and FLT3) for Acute Myeloid Leukemia (AML) Patient Care

Acute myeloid leukemia (AML) is one of the most lethal blood cancers from which nearly 10,000 people die in the United States each year. While therapies for other blood cancers have made some progress, the standard of care for AML, a combination of toxic chemotherapies, has changed very little over the past four decades. In recent years the US Food and Drug Administration (FDA) has been very active in approving targeted therapeutic drugs for AML patients including Midostaurin (Rydapt; 2017) and Gilteritinib (Xospata;2018) for FLT3 mutations; Enasidenib (Idhifa; 2017) for IDH2 mutations and Ivosidenib (Tibsovo; 2018) for IDH1 mutations. Additionally, the Leukemia and Lymphoma Society's Beat AML Master Clinical Trial has shown that waiting for molecular results prior to treatment decision leads to better outcomes. Versiti Blood Center of Wisconsin Diagnostics laboratory which is certified under the Clinical Laboratory Improvement Amendments (CLIA) and qualified to perform high complexity clinical laboratory testing has performed the verification studies and offers two companion diagnostics tests for IDH1 and IDH2 mutations for AML patients. Also, in collaboration with Invivoscribe Inc., the AML patient can be tested for Leukostrat CDx FLT3 mutations assay so that the same AML patient can get three CDx test results leading to available drug therapy treatment decision making by physicians. IDH1 CDX Test: IDH1 CDx is indicated as an aid in identifying AML patients with an IDH1 mutation for treatment with ivosidenib (TIBSOVO®). Mutations in codon R132 of IDH1 can be found in 6% to 10% of AML patients. The IDH1 CDx test detects five IDH1 mutations R132H (CAT), R132C (TGT), R132G (GGT), R132S (AGT), and R132L (CTT) using PCR technology with homogeneous real-time fluorescence detection. The assay sensitivity for these five IDH1 mutations is 100% at variant allele frequencies of 2% and higher and 98% or greater at variant allele frequencies of 1% and higher. This test has been approved by the FDA as companion diagnostic device (PMA number P170041). IDH2 CDx: IDH2 CDx is indicated as an aid in identifying AML patients with an IDH2 mutation for treatment with IDHIFA® (enasidenib). Mutations in the R140 and R172 codons of IDH2 8% to 19% of AML patients.The IDH2 CDX test detects nine IDH2 mutations (R140Q, R140L, R140G, R140W, R172K, R172M, R172G, R172S, and R172W) using PCR technology with real-time fluorescent detection. The assay sensitivity for these nine IDH2 mutations is 99.8% or greater at variant allele frequencies of 2% and higher or 93.5% or greater at variant allele frequencies of 1% and higher. This test has been approved by the FDA as companion diagnostic device (PMA number P170005). FLT3 CDx: The FLT3 Leukostrat® CDx Assay is the FDA approved (PMA number P160040) predictive test for the efficacy of midostaurin (RYDAPT®) therapy in all AML patients, regardless of cytogenetics and efficacy of gilteritinib (XOSPATA ® ) therapy in relapsed or refractory AML patients. FLT3 is one of the most commonly mutated genes in AML with 30% of patients at the time of diagnosis 1. The most prevalent type of FLT3 mutation is an internal tandem duplication (ITD) in the juxtamembrane domain. The second most common mutation type in the FLT3 gene is a tyrosine kinase domain (TKD) point mutation in the codon for an aspartate (D835) or an isoleucine (I836) residue. The LeukoStrat® CDx FLT3 Mutation Assay is a PCR-based, in vitro diagnostic test designed to detect internal tandem duplication (ITD) mutations and the tyrosine kinase domain (TKD) mutations D835 and I836 in genomic DNA extracted from mononuclear cells obtained from peripheral blood or bone marrow aspirates of patients diagnosed with AML. Versiti Blood Center sends the patient specimens to Invivoscribe Inc. where the LeukoStrat® CDx FLT3 Mutation Assay is performed and the interpretive comments are included in the patient report by Versiti. From our experience pathologists and treating physicians want molecular test results as fast as possible, especially for the actionable gene mutations in IDH1, IDH2 and FLT3. The IDH1 CDx, IDH2 CDx and FLT3 CDx tests are highly sensitive and Versiti provides average turn around time of 3 business days which enable rapid decision making on the recently available drug therapies for AML patients. We strongly recommend that the IDH1 CDx, IDH2 CDx and FLT3 CDx tests should be performed on all AML patients for better care. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: In recent years the US Food and Drug Administration (FDA) approved targeted therapeutic drugs for AML patients including Midostaurin (Rydapt; 2017) and Gilteritinib (Xospata;2018) for FLT3 mutations; Enasidenib (Idhifa; 2017) for IDH2 mutations and Ivosidenib (Tibsovo; 2018) for IDH1 mutations.

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.