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Last Modified, October 02, 2022
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Technical Aspects of Flow Cytometry-based Measurable Residual Disease Quantification in Acute Myeloid Leukemia: Experience of the European LeukemiaNet MRD Working Party

Hemasphere

2022 Roland Walter; Brent Wood

Measurable residual disease (MRD) quantified by multiparameter flow cytometry (MFC) is a strong and independent prognostic factor in acute myeloid leukemia (AML). However, several technical factors may affect the final read-out of the assay. Experts from the MRD Working Party of the European LeukemiaNet evaluated which aspects are crucial for accurate MFC-MRD measurement. Here, we report on the agreement, obtained via a combination of a cross-sectional questionnaire, live discussions, and a Delphi poll. The recommendations consist of several key issues from bone marrow sampling to final laboratory reporting to ensure quality and reproducibility of results. Furthermore, the experiences were tested by comparing two 8-color MRD panels in multiple laboratories. The results presented here underscore the feasibility and the utility of a harmonized theoretical and practical MFC-MRD assessment and are a next step toward further harmonization.

BiTE secretion from in situ-programmed myeloid cells results in tumor-retained pharmacology

J Control Release

2022 Christine Watson; Vaishali Inamdar; Matthias Stephan; Elena Sigmund; Stephanie Weaver; Sirkka Stephan; Fan Zhang; Siyang Hao

Bispecific T-Cell Engagers (BiTEs) are effective at inducing remission in hematologic cancers, but their use in solid tumors has been challenging due to their extreme potency and on-target, off-tumor toxicities in healthy tissue. Their deployment against solid tumors is further complicated by insufficient drug penetration, a hostile tumor microenvironment, and immune escape. To address these challenges, we developed targeted nanocarriers that can deliver in vitro-transcribed mRNA encoding BiTEs to host myeloid cells - a cell type that is actively recruited into the tumor microenvironment. We demonstrate in an immunocompetent mouse model of ovarian cancer, that infusion of these nanoparticles directs BiTE expression to tumor sites, which reshapes the microenvironment from suppressive to permissive and triggers disease regression without systemic toxicity. In contrast, conventional injections of recombinant BiTE protein at doses required to achieve anti-tumor activity, induced systemic inflammatory responses and severe tissue damage in all treated animals. Implemented in the clinic, this in situ gene therapy could enable physicians - with a single therapeutic - to safely target tumor antigen that would otherwise not be druggable due to the risks of on-target toxicity and, at the same time, reset the tumor milieu to boost key mediators of antitumor immune responses.

The Seattle Flu Study: when regulations hinder pandemic surveillance

Nat Med

2021 Debbie Nickerson; Lea Starita; Jay Shendure; Michael Boeckh; Janet Englund; Christina Lockwood

N/A

Cerebrospinal fluid flow cytometry and risk of central nervous system relapse after hyperCVAD in adults with acute lymphoblastic leukemia

Cancer

2021 Elihu Estey; Anna Halpern; Johnnie Orozco; Pamela Becker; Sioban Keel; Philip Stevenson; Roland Walter; Ryan Cassaday; Andrei Shustov; Vivian Oehler; Mary-Elizabeth Percival

BACKGROUND: Potential involvement of the central nervous system (CNS) by acute lymphoblastic leukemia is typically evaluated by a conventional cytospin (CC) of cerebrospinal fluid (CSF). Multiparameter flow cytometry (MFC) is generally more sensitive and specific than morphology, but data to guide its use versus CC are limited. METHODS: This study identified 92 patients who had MFC performed on their initial CSF specimen and received at least 4 cycles of hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with methotrexate and cytarabine (hyperCVAD) as their initial treatment. RESULTS: Eighteen (20%) were CSF+ by MFC at the baseline, and only 6 of these patients were positive by CC. In contrast, 0 of 51 patients who were negative by MFC and had CC available were positive by CC. Despite the receipt of significantly more intra-CSF chemotherapy (P < .001), the cumulative incidence of CNS relapse by MFC was 22% among CSF+ patients versus 5% among those who were CSF- (P = .044). No such association was observed between CNS relapse and CC results (P = .42). None of the 74 CSF- patients became CSF+ during their initial treatment despite being tested a median of 5 times (range, 2-10). CSF positivity by MFC was the factor most strongly associated with CNS relapse in a series of univariate Cox models (hazard ratio, 3.7; P = .067). The initial CSF status by MFC had no significant impact on overall or event-free survival. CONCLUSIONS: MFC of CSF is superior to CC of CSF in identifying adults at high risk for CNS relapse after treatment with hyperCVAD. Surveillance of CSF by MFC has limited utility.

Infusion reactions after receiving the broadly neutralizing antibody VRC01 or placebo to reduce HIV-1 acquisition: Results from the phase 2b Antibody Mediated Prevention (AMP) randomized trials

J Acquir Immune Defic Syndr

2021 Michelle Karuna; Maija Anderson; John Hural; Carissa Karg; Erika Rudnicki; Larry Corey; April Randhawa; Robert De La Grecca; Simbarashe Takuva; Michal Juraska

BACKGROUND: The AMP studies (HVTN 703/HPTN 081 and HVTN 704/HPTN 085) are harmonized Phase 2b trials to assess HIV prevention efficacy and safety of intravenous infusion of anti-gp120 broadly neutralizing antibody VRC01. Antibodies for other indications can elicit infusion-related reactions (IRRs), often requiring pre-medication and limiting their application. We report on AMP study IRRs. METHODS: From 2016-2018, 2,699 HIV-uninfected, at-risk men and transgender adults in the Americas and Switzerland (704/085) and 1,901 at-risk heterosexual women in sub-Saharan Africa (703/081) were randomized 1:1:1 to VRC01 10 mg/kg, 30 mg/kg, or placebo. Participants received infusions every 8 weeks (n=10/participant) over 72 weeks, with 104 weeks of follow-up. Safety assessments were conducted pre- and post-infusion and at non-infusion visits. A total of 40,674 infusions were administered. RESULTS: Forty-seven participants (1.7%) experienced 49 IRRs in 704/085; 93 (4.8%) experienced 111 IRRs in 703/081 (p<0.001). IRRs occurred more frequently in VRC01 than placebo recipients in 703/081 (p<0.001). IRRs were associated with atopic history (p=0.046) and with younger age (p=0.023) in 703/081. Four clinical phenotypes of IRRs were observed: urticaria, dyspnea, dyspnea with rash, and "other". Urticaria was most prevalent, occurring in 25 (0.9%) participants in 704/085 and 41 (2.1%) participants in 703/081. Most IRRs occurred with the initial infusion and incidence diminished through the last infusion. All reactions were managed successfully without sequelae. CONCLUSIONS: IRRs in the AMP studies were uncommon, typically mild or moderate, successfully managed at the research clinic, and resolved without sequelae. Analysis is ongoing to explore the potential IRR mechanisms.

Estimating cell type composition using isoform expression one gene at a time

Biometrics

2021 Wei Sun

Human tissue samples are often mixtures of heterogeneous cell types, which can confound the analyses of gene expression data derived from such tissues. The cell type composition of a tissue sample may itself be of interest and is needed for proper analysis of differential gene expression. A variety of computational methods have been developed to estimate cell type proportions using gene-level expression data. However, RNA isoforms can also be differentially expressed across cell types, and isoform-level expression could be equally or more informative for determining cell type origin than gene-level expression. We propose a new computational method, IsoDeconvMM, which estimates cell type fractions using isoform-level gene expression data. A novel and useful feature of IsoDeconvMM is that it can estimate cell type proportions using only a single gene, though in practice we recommend aggregating estimates of a few dozen genes to obtain more accurate results. We demonstrate the performance of IsoDeconvMM using a unique dataset with cell type-specific RNA-seq data across more than 135 individuals. This dataset allows us to evaluate different methods given the biological variation of cell type-specific gene expression data across individuals. We further complement this analysis with additional simulations. This article is protected by copyright. All rights reserved.

New interpretable machine-learning method for single-cell data reveals correlates of clinical response to cancer immunotherapy

Patterns (N Y)

2021 Evan Greene; Paul Nghiem; Raphael Gottardo; Leonard D'Amico; Chihiro Moroshima; Nirasha Ramchurren; Martin Cheever; Greg Finak; Steven Fling

We introduce a new method for single-cell cytometry studies, FAUST, which performs unbiased cell population discovery and annotation. FAUST processes experimental data on a per-sample basis and returns biologically interpretable cell phenotypes, making it well suited for the analysis of complex datasets. We provide simulation studies that compare FAUST with existing methodology, exemplifying its strength. We apply FAUST to data from a Merkel cell carcinoma anti-PD-1 trial and discover pre-treatment effector memory T cell correlates of outcome co-expressing PD-1, HLA-DR, and CD28. Using FAUST, we then validate these correlates in cryopreserved peripheral blood mononuclear cell samples from the same study, as well as an independent CyTOF dataset from a published metastatic melanoma trial. Finally, we show how FAUST's phenotypes can be used to perform cross-study data integration in the presence of diverse staining panels. Together, these results establish FAUST as a powerful new approach for unbiased discovery in single-cell cytometry.

Multiplex CRISPR/Cas9 genome editing in hematopoietic stem cells for fetal hemoglobin reinduction generates chromosomal translocations

Mol Ther Methods Clin Dev

2021 Stefan Radtke; Clare Samuelson; Savannah Cook; Keith Jerome; Hans-Peter Kiem; Olivier Humbert

Sickle cell disease and β-thalassemia are common monogenic disorders that cause significant morbidity and mortality globally. The only curative treatment currently is allogeneic hematopoietic stem cell transplantation, which is unavailable to many patients due to a lack of matched donors and carries risks including graft-versus-host disease. Genome editing therapies targeting either the BCL11A erythroid enhancer or the HBG promoter are already demonstrating success in reinducing fetal hemoglobin. However, where a single locus is targeted, reliably achieving levels high enough to deliver an effective cure remains a challenge. We investigated the application of a CRISPR/Cas9 multiplex genome editing approach, in which both the BCL11A erythroid enhancer and HBG promoter are disrupted within human hematopoietic stem cells. We demonstrate superior fetal hemoglobin reinduction with this dual-editing approach without compromising engraftment or lineage differentiation potential of edited cells post-xenotransplantation. However, multiplex editing consistently resulted in the generation of chromosomal rearrangement events that persisted in vivo following transplantation into immunodeficient mice. The risk of oncogenic events resulting from such translocations therefore currently prohibits its clinical translation, but it is anticipated that, in the future, alternative editing platforms will help alleviate this risk.

Machine learning modeling of genome-wide copy number alteration signatures reliably predicts IDH mutational status in adult diffuse glioma

Acta Neuropathol Commun

2021 Patrick Cimino; Linda Shapiro; Eric Holland

Knowledge of 1p/19q-codeletion and IDH1/2 mutational status is necessary to interpret any investigational study of diffuse gliomas in the modern era. While DNA sequencing is the gold standard for determining IDH mutational status, genome-wide methylation arrays and gene expression profiling have been used for surrogate mutational determination. Previous studies by our group suggest that 1p/19q-codeletion and IDH mutational status can be predicted by genome-wide somatic copy number alteration (SCNA) data alone, however a rigorous model to accomplish this task has yet to be established. In this study, we used SCNA data from 786 adult diffuse gliomas in The Cancer Genome Atlas (TCGA) to develop a two-stage classification system that identifies 1p/19q-codeleted oligodendrogliomas and predicts the IDH mutational status of astrocytic tumors using a machine-learning model. Cross-validated results on TCGA SCNA data showed near perfect classification results. Furthermore, our astrocytic IDH mutation model validated well on four additional datasets (AUC = 0.97, AUC = 0.99, AUC = 0.95, AUC = 0.96) as did our 1p/19q-codeleted oligodendroglioma screen on the two datasets that contained oligodendrogliomas (MCC = 0.97, MCC = 0.97). We then retrained our system using data from these validation sets and applied our system to a cohort of REMBRANDT study subjects for whom SCNA data, but not IDH mutational status, is available. Overall, using genome-wide SCNAs, we successfully developed a system to robustly predict 1p/19q-codeletion and IDH mutational status in diffuse gliomas. This system can assign molecular subtype labels to tumor samples of retrospective diffuse glioma cohorts that lack 1p/19q-codeletion and IDH mutational status, such as the REMBRANDT study, recasting these datasets as validation cohorts for diffuse glioma research.

Calibration of two validated SARS-CoV-2 pseudovirus neutralization assays for COVID-19 vaccine evaluation

Sci Rep

2021 Michelle Karuna; Christine Posavad; Julie McElrath; Lindsay Carpp; Yunda Huang; Larry Corey; Jia Jin Kee; Peter Gilbert; John Hural

Vaccine-induced neutralizing antibodies (nAbs) are key biomarkers considered to be associated with vaccine efficacy. In United States government-sponsored phase 3 efficacy trials of COVID-19 vaccines, nAbs are measured by two different validated pseudovirus-based SARS-CoV-2 neutralization assays, with each trial using one of the two assays. Here we describe and compare the nAb titers obtained in the two assays. We observe that one assay consistently yielded higher nAb titers than the other when both assays were performed on the World Health Organization's anti-SARS-CoV-2 immunoglobulin International Standard, COVID-19 convalescent sera, and mRNA-1273 vaccinee sera. To overcome the challenge this difference in readout poses in comparing/combining data from the two assays, we evaluate three calibration approaches and show that readouts from the two assays can be calibrated to a common scale. These results may aid decision-making based on data from these assays for the evaluation and licensure of new or adapted COVID-19 vaccines.