Publications by authors named "Vincent H J van der Velden"

93 Publications

B-Cell Regeneration Profile and Minimal Residual Disease Status in Bone Marrow of Treated Multiple Myeloma Patients.

Cancers (Basel) 2021 Apr 3;13(7). Epub 2021 Apr 3.

Translational and Clinical Research Program, Cancer Research Center (IBMCC, USAL-CSIC), Cytometry Service (NUCLEUS) and Department of Medicine, University of Salamanca, Institute of Biomedical Research of Salamanca (IBSAL), 37007 Salamanca, Spain.

B-cell regeneration during therapy has been considered as a strong prognostic factor in multiple myeloma (MM). However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated during follow-up. MM (n = 177) and adult (≥50y) healthy donor (HD; n = 14) BM samples were studied by next-generation flow (NGF) to simultaneously assess measurable residual disease (MRD) and residual normal B-cell populations. BM hemodilution was detected in 41 out of 177 (23%) patient samples, leading to lower total B-cell, B-cell precursor (BCP) and normal plasma cell (nPC) counts. Among MM BM, decreased percentages (vs. HD) of BCP, transitional/naïve B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP increased after induction therapy, whereas TBC/NBC counts remained abnormally low. At day+100 postautologous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC cell numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19 nPC counts vs. non-CR specimens. MRD positivity was associated with higher BCP and nPC percentages. Hemodilution showed a negative impact on BM B-cell distribution. Different BM B-cell regeneration profiles are present in MM at diagnosis and after therapy with no significant association with patient outcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers13071704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038337PMC
April 2021

TRB sequences targeting ORF1a/b are associated with disease severity in hospitalized COVID-19 patients.

J Leukoc Biol 2021 Apr 13. Epub 2021 Apr 13.

Department of Immunology, Laboratory Medical Immunology, Erasmus MC University Medical Center, Rotterdam, The Netherlands.

The potential protective or pathogenic role of the adaptive immune response to SARS-CoV-2 infection has been vigorously debated. While COVID-19 patients consistently generate a T lymphocyte response to SARS-CoV-2 antigens, evidence of significant immune dysregulation in these patients continues to accumulate. In this study, next generation sequencing of the T cell receptor beta chain (TRB) repertoire was conducted in hospitalized COVID-19 patients to determine if immunogenetic differences of the TRB repertoire contribute to disease course severity. Clustering of highly similar TRB CDR3 amino acid sequences across COVID-19 patients yielded 781 shared TRB sequences. The TRB sequences were then filtered for known associations with common diseases such as EBV and CMV. The remaining sequences were cross-referenced to a publicly accessible dataset that mapped COVID-19 specific TCRs to the SARS-CoV-2 genome. We identified 158 SARS-CoV-2 specific TRB sequences belonging to 134 clusters in our COVID-19 patients. Next, we investigated 113 SARS-CoV-2 specific clusters binding only one peptide target in relation to disease course. Distinct skewing of SARS-CoV-2 specific TRB sequences toward the nonstructural proteins (NSPs) encoded within ORF1a/b of the SARS-CoV-2 genome was observed in clusters associated with critical disease course when compared to COVID-19 clusters associated with a severe disease course. These data imply that T-lymphocyte reactivity towards peptides from NSPs of SARS-CoV-2 may not constitute an effective adaptive immune response and thus may negatively affect disease severity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/JLB.6COVCRA1120-762RDOI Listing
April 2021

Extensive longitudinal immune profiling reveals sustained innate immune activation in COVID-19 patients with unfavorable outcome

Eur Cytokine Netw 2020 12;31(4):154-167

Department of Clinical Chemistry and Hematology, Amphia Hospital, Breda, the Netherlands.

COVID-19 differs substantially between individuals, ranging from mild to severe or even fatal. Heterogeneity in the immune response against SARS-COV-2 likely contributes to this. Therefore, we explored the temporal dynamics of key cellular and soluble mediators of innate and adaptive immune activation in relation to COVID-19 severity and progression. Forty-four patients with a PCR-proven diagnosis of COVID-19 were included. Extensive cellular (leukocytes and T-lymphocyte subsets) and serological immune profiling (cytokines, soluble cell surface molecules, and SARS-CoV-2 antibodies) was performed at hospital admission and every 3-4 days during hospitalization. Measurements and disease outcome were compared between patients with an unfavorable (IC admission and/or death) and favorable (all others) outcome. Patients with an unfavorable outcome had higher leukocyte numbers at baseline, mostly due to increased neutrophils, whereas lymphocyte and monocyte numbers were reduced. CRP, IL-6, CCL2, CXCL10, and GM-CSF levels were higher at baseline in the unfavorable group, whereas IL-7 levels were lower. SARS-CoV-2 antibodies were more frequently absent in the unfavorable group. Longitudinal analysis revealed delayed kinetics of activated CD4 and CD8 T-lymphocyte subsets in the unfavorable group. Furthermore, whereas CRP, IL-6, CXCL10, and GM-CSF declined in the favorable group, these cytokines declined with delayed kinetics, remained increased, or even increased further in the unfavorable group. Our data indicate a state of increased innate immune activation in COVID19-patients with an unfavorable outcome at hospital admission, which remained over time, as compared with patients with a favorable outcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1684/ecn.2020.0456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937051PMC
December 2020

The miR-200c/141-ZEB2-TGFβ axis is aberrant in human T-cell prolymphocytic leukemia.

Haematologica 2021 Feb 18. Epub 2021 Feb 18.

Department of Immunology, Erasmus University Medical Center, Rotterdam.

T cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small to medium sized pro-lymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide-range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, whereas no clear T cell receptor alpha (TRA) or beta (TRB) CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of miRNAs is aberrant and often heterogeneous in T-PLL. We identified 35 miRNAs that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR-200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFβR3 and aberrant TGF-β1-induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFβ pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNAs in T-PLL and pave the way for new diagnostic targets in this disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2020.263756DOI Listing
February 2021

PML-controlled responses in severe congenital neutropenia with ELANE-misfolding mutations.

Blood Adv 2021 Feb;5(3):775-786

Department of Hematology.

Mutations in ELANE cause severe congenital neutropenia (SCN), but how they affect neutrophil production and contribute to leukemia predisposition is unknown. Neutropenia is alleviated by CSF3 (granulocyte colony-stimulating factor) therapy in most cases, but dose requirements vary between patients. Here, we show that CD34+CD45+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cell lines from patients with SCN that have mutations in ELANE (n = 2) or HAX1 (n = 1) display elevated levels of reactive oxygen species (ROS) relative to normal iPSC-derived HPCs. In patients with ELANE mutations causing misfolding of the neutrophil elastase (NE) protein, HPCs contained elevated numbers of promyelocyte leukemia protein nuclear bodies, a hallmark of acute oxidative stress. This was confirmed in primary bone marrow cells from 3 additional patients with ELANE-mutant SCN. Apart from responding to elevated ROS levels, PML controlled the metabolic state of these ELANE-mutant HPCs as well as the expression of ELANE, suggestive of a feed-forward mechanism of disease development. Both PML deletion and correction of the ELANE mutation restored CSF3 responses of these ELANE-mutant HPCs. These findings suggest that PML plays a crucial role in the disease course of ELANE-SCN characterized by NE misfolding, with potential implications for CSF3 therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2020003214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876869PMC
February 2021

Clinical Implications of Minimal Residual Disease Detection in Infants With -Rearranged Acute Lymphoblastic Leukemia Treated on the Interfant-06 Protocol.

J Clin Oncol 2021 Feb 6;39(6):652-662. Epub 2021 Jan 6.

Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

Purpose: Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of gene rearrangements and poor outcome. We evaluated the value of minimal residual disease (MRD) in infants with -rearranged ALL treated within the Interfant-06 protocol, which compared lymphoid-style consolidation (protocol IB) versus myeloid-style consolidation (araC, daunorubicin, etoposide/mitoxantrone, araC, etoposide).

Materials And Methods: MRD was measured in 249 infants by DNA-based polymerase chain reaction of rearranged , immunoglobulin, and/or T-cell receptor genes, at the end of induction (EOI) and end of consolidation (EOC). MRD results were classified as negative, intermediate (< 5 × 10), and high (≥ 5 × 10).

Results: EOI MRD levels predicted outcome with 6-year disease-free survival (DFS) of 60.2% (95% CI, 43.2 to 73.6), 45.0% (95% CI, 28.3 to 53.1), and 33.8% (95% CI, 23.8 to 44.1) for infants with negative, intermediate, and high EOI MRD levels, respectively ( = .0039). EOC MRD levels were also predictive of outcome, with 6-year DFS of 68.2% (95% CI, 55.2 to 78.1), 40.1% (95% CI, 28.1 to 51.9), and 11.9% (95% CI, 2.6 to 29.1) for infants with negative, intermediate, and high EOC MRD levels, respectively ( < .0001). Analysis of EOI MRD according to the type of consolidation treatment showed that infants treated with lymphoid-style consolidation had 6-year DFS of 78.2% (95% CI, 51.4 to 91.3), 47.2% (95% CI, 33.0 to 60.1), and 23.2% (95% CI, 12.1 to 36.4) for negative, intermediate, and high MRD levels, respectively ( < .0001), while for myeloid-style-treated patients the corresponding figures were 45.0% (95% CI, 23.9 to 64.1), 41.3% (95% CI, 23.2 to 58.5), and 45.9% (95% CI, 29.4 to 60.9).

Conclusion: This study provides support for the idea that induction therapy selects patients for subsequent therapy; infants with high EOI MRD may benefit from AML-like consolidation (DFS 45.9% 23.2%), whereas patients with low EOI MRD may benefit from ALL-like consolidation (DFS 78.2% 45.0%). Patients with positive EOC MRD had dismal outcomes. These findings will be used for treatment interventions in the next Interfant protocol.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1200/JCO.20.02333DOI Listing
February 2021

Standardised immunophenotypic analysis of myeloperoxidase in acute leukaemia.

Br J Haematol 2020 Nov 7. Epub 2020 Nov 7.

Laboratory Medical Immunology (LMI), Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.

Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as ≥20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bjh.17210DOI Listing
November 2020

Increased group 2 innate lymphoid cells in peripheral blood of adults with mastocytosis.

J Allergy Clin Immunol 2021 Apr 20;147(4):1490-1496.e2. Epub 2020 Oct 20.

Department of Pulmonary Medicine, Erasmus MC, Rotterdam, The Netherlands; Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands. Electronic address:

Background: Systemic mastocytosis is a hematological disease in which aberrant mast cells accumulate because of gain-of-function mutations in the KIT receptor. Group 2 innate lymphoid cells (ILC2s) are effector cells of type 2 immune responses that also express KIT and colocalize with mast cells at barrier tissue sites. In mouse models, mast cell-ILC2 crosstalk can drive local inflammation. However, a possible role for ILC2s in the pathophysiology of mastocytosis remains unexplored.

Objective: We sought to characterize circulating ILC2s in a clinically diverse cohort of patients with mastocytosis.

Methods: We included 21 adults with systemic mastocytosis and 18 healthy controls. Peripheral blood ILC2 abundance and phenotype were analyzed by flow cytometry and correlated to clinical characteristics, including the presence of the D816V KIT mutation.

Results: ILC2 levels were significantly higher in D816V patients with mastocytosis compared with D816V- patients or healthy controls. We observed increased proportions of KIT ILC2s among patients with mastocytosis, regardless of D816V status. Patients with skin involvement and itch showed the highest levels of ILC2s, which was independent from atopy or serum tryptase levels. Allele-specific quantitative PCR showed that the vast majority of ILC2s did not carry the D816V mutation.

Conclusions: Our findings suggest a role for ILC2s and pathogenic ILC2-mast cell crosstalk in mastocytosis. We hypothesize that a high cutaneous D816V mast cell burden alters the skin microenvironment to induce chronic local ILC2 activation and their dissemination into the circulation. Activated ILC2s could contribute to skin symptoms by producing inflammatory mediators and by further augmenting mast cell mediator release.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2020.09.037DOI Listing
April 2021

A phase 1 study of inotuzumab ozogamicin in pediatric relapsed/refractory acute lymphoblastic leukemia (ITCC-059 study).

Blood 2021 Mar;137(12):1582-1590

Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.

This phase 1 study investigated the recommended phase 2 dose (RP2D) of inotuzumab ozogamicin (InO), a CD22-directed antibody-drug conjugate, in pediatric patients with multiple relapsed/refractory (R/R) CD22+ acute lymphoblastic leukemia (ALL). Patients (age ≥1 year or <18 years) received 3 doses of InO (days 1, 8, and 15) per course. Dose escalation was based on dose-limiting toxicities (DLTs) during course 1. Dose level 1 (DL1) was 1.4 mg/m2 (0.6, 0.4, 0.4 mg/m2) and DL2 was 1.8 mg/m2 (0.8, 0.5, 0.5 mg/m2). Secondary end points included safety, antileukemic activity, and pharmacokinetics. Twenty-five patients (23 evaluable for DLTs) were enrolled. In course 1, the first cohort had 1 of 6 (DL1) and 2 of 5 (DL2) patients who experienced DLTs; subsequent review considered DL2 DLTs to be non-dose-limiting. Dose was de-escalated to DL1 while awaiting protocol amendment to re-evaluate DL2 in a second cohort, in which 0 of 6 (DL1) and 1 of 6 (DL2) patients had a DLT. Twenty-three patients experienced grade 3 to 4 adverse events; hepatic sinusoidal obstruction syndrome was reported in 2 patients after subsequent chemotherapy. Overall response rate after course 1 was 80% (95% confidence interval [CI], 59% to 93%) (20 of 25 patients; DL1: 75% [95% CI, 43% to 95%], DL2: 85% [95% CI, 55% to 98%]). Of the responders, 84% (95% CI, 60% to 97%) achieved minimal residual disease (MRD)-negative complete response, and 12-month overall survival was 40% (95% CI, 25% to 66%). Nine patients received hematopoietic stem cell transplantation or chimeric antigen receptor T cells after InO. InO median maximum concentrations were comparable to simulated adult concentrations. InO was well tolerated, demonstrating antileukemic activity in heavily pretreated children with CD22+ R/R ALL. RP2D was established as 1.8 mg/m2 per course, as in adults. This trial was registered at https://www.clinicaltrialsregister.eu as EUDRA-CT 2016-000227-71.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020007848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995290PMC
March 2021

Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study.

Mod Pathol 2021 01 30;34(1):59-69. Epub 2020 Sep 30.

Department of Immunology, Laboratory for Medical Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r > 0.95 for all cell types in PB and r > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41379-020-00677-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806506PMC
January 2021

Flowcytometric evaluation of cerebrospinal fluid in childhood ALL identifies CNS involvement better then conventional cytomorphology.

Leukemia 2020 Aug 27. Epub 2020 Aug 27.

Department of Immunology, Laboratory Medical Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41375-020-01029-9DOI Listing
August 2020

Mediating effect of soluble B-cell activation immune markers on the association between anthropometric and lifestyle factors and lymphoma development.

Sci Rep 2020 08 14;10(1):13814. Epub 2020 Aug 14.

Division of Environmental Epidemiology, Institute for Risk Assessment Sciences, Utrecht University, P.O. Box 80178, 3508 TD, Utrecht, The Netherlands.

Sustained B-cell activation is an important mechanism contributing to B-cell lymphoma (BCL). We aimed to validate four previously reported B-cell activation markers predictive of BCL risk (sCD23, sCD27, sCD30, and CXCL13) and to examine their possible mediating effects on the association between anthropometric and lifestyle factors and major BCL subtypes. Pre-diagnostic serum levels were measured for 517 BCL cases and 525 controls in a nested case-control study. The odds ratios of BCL were 6.2 in the highest versus lowest quartile for sCD23, 2.6 for sCD30, 4.2 for sCD27, and 2.6 for CXCL13. Higher levels of all markers were associated with increased risk of chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). Following mutual adjustment for the other immune markers, sCD23 remained associated with all subtypes and CXCL13 with FL and DLBCL. The associations of sCD23 with CLL and DLBCL and CXCL13 with DLBCL persisted among cases sampled > 9 years before diagnosis. sCD23 showed a good predictive ability (area under the curve = 0.80) for CLL, in particular among older, male participants. sCD23 and CXCL13 showed a mediating effect between body mass index (positive) and DLBCL risk, while CXCL13 contributed to the association between physical activity (inverse) and DLBCL. Our data suggest a role of B-cell activation in BCL development and a mediating role of the immune system for lifestyle factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-70790-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429856PMC
August 2020

Robust FCS Parsing: Exploring 211,359 Public Files.

Cytometry A 2020 11 15;97(11):1180-1186. Epub 2020 Jul 15.

Laboratory Medical immunology, Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.

When it comes to data storage, the field of flow cytometry is fairly standardized, thanks to the flow cytometry standard (FCS) file format. The structure of FCS files is described in the FCS specification. Software that strictly complies with the FCS specification is guaranteed to be interoperable (in terms of exchange via FCS files). Nowadays, software interoperability is crucial for eco system, as FCS files are frequently shared, and workflows rely on more than one piece of software (e.g., acquisition and analysis software). Ideally, software developers strictly follow the FCS specification. Unfortunately, this is not always the case, which resulted in various nonconformant FCS files being generated over time. Therefore, robust FCS parsers must be developed, which can handle a wide variety of nonconformant FCS files, from different resources. Development of robust FCS parsers would greatly benefit from a fully fledged set of testing files. In this study, readability of 211,359 public FCS files was evaluated. Each FCS file was checked for conformance with the FCS specification. For each data set, within each FCS file, validated parse results were obtained for the TEXT segment. Highly space efficient testing files were generated. FlowCore was benchmarked in depth, by using the validated parse results, the generated testing files, and the original FCS files. Robustness of FlowCore (as measured by testing against 211,359 files) was improved by re-implementing the TEXT segment parser. Altogether, this study provides a comprehensive resource for FCS parser development, an in-depth benchmark of FlowCore, and a concrete proposal for improving FlowCore. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.24187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754493PMC
November 2020

41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo.

J Immunother Cancer 2020 06;8(1)

Biomedicine, Research Institute Against Leukemia Josep Carreras, Barcelona, Catalunya, Spain

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative.

Methods: We set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs.

Results: Here, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs.

Conclusions: This study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/jitc-2020-000845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292050PMC
June 2020

Autologous haematopoietic stem-cell transplantation versus bortezomib-melphalan-prednisone, with or without bortezomib-lenalidomide-dexamethasone consolidation therapy, and lenalidomide maintenance for newly diagnosed multiple myeloma (EMN02/HO95): a multicentre, randomised, open-label, phase 3 study.

Lancet Haematol 2020 Jun 30;7(6):e456-e468. Epub 2020 Apr 30.

Department of Haematology, Aarhus University Hospital, Aarhus, Denmark.

Background: The emergence of highly active novel agents has led some to question the role of autologous haematopoietic stem-cell transplantation (HSCT) and subsequent consolidation therapy in newly diagnosed multiple myeloma. We therefore compared autologous HSCT with bortezomib-melphalan-prednisone (VMP) as intensification therapy, and bortezomib-lenalidomide-dexamethasone (VRD) consolidation therapy with no consolidation.

Methods: In this randomised, open-label, phase 3 study we recruited previously untreated patients with multiple myeloma at 172 academic and community practice centres of the European Myeloma Network. Eligible patients were aged 18-65 years, had symptomatic multiple myeloma stage 1-3 according to the International Staging System (ISS), measurable disease (serum M protein >10 g/L or urine M protein >200 mg in 24 h or abnormal free light chain [FLC] ratio with involved FLC >100 mg/L, or proven plasmacytoma by biopsy), and WHO performance status grade 0-2 (grade 3 was allowed if secondary to myeloma). Patients were first randomly assigned (1:1) to receive either four 42-day cycles of bortezomib (1·3 mg/m administered intravenously or subcutaneously on days 1, 4, 8, 11, 22, 25, 29, and 32) combined with melphalan (9 mg/m administered orally on days 1-4) and prednisone (60 mg/m administered orally on days 1-4) or autologous HSCT after high-dose melphalan (200 mg/m), stratified by site and ISS disease stage. In centres with a double HSCT policy, the first randomisation (1:1:1) was to VMP or single or double HSCT. Afterwards, a second randomisation assigned patients to receive two 28-day cycles of consolidation therapy with bortezomib (1·3 mg/m either intravenously or subcutaneously on days 1, 4, 8, and 11), lenalidomide (25 mg orally on days 1-21), and dexamethasone (20 mg orally on days 1, 2, 4, 5, 8, 9, 11, and 12) or no consolidation; both groups received lenalidomide maintenance therapy (10 mg orally on days 1-21 of a 28-day cycle). The primary outcomes were progression-free survival from the first and second randomisations, analysed in the intention-to-treat population, which included all patients who underwent each randomisation. All patients who received at least one dose of study drugs were included in the safety analyses. This study is registered with the EU Clinical Trials Register (EudraCT 2009-017903-28) and ClinicalTrials.gov (NCT01208766), and has completed recruitment.

Findings: Between Feb 25, 2011, and April 3, 2014, 1503 patients were enrolled. 1197 patients were eligible for the first randomisation, of whom 702 were assigned to autologous HSCT and 495 to VMP; 877 patients who were eligible for the first randomisation underwent the second randomisation to VRD consolidation (n=449) or no consolidation (n=428). The data cutoff date for the current analysis was Nov 26, 2018. At a median follow-up of 60·3 months (IQR 52·2-67·6), median progression-free survival was significantly improved with autologous HSCT compared with VMP (56·7 months [95% CI 49·3-64·5] vs 41·9 months [37·5-46·9]; hazard ratio [HR] 0·73, 0·62-0·85; p=0·0001). For the second randomisation, the number of events of progression or death at data cutoff was lower than that preplanned for the final analysis; therefore, the results from the second protocol-specified interim analysis, when 66% of events were reached, are reported (data cutoff Jan 18, 2018). At a median follow-up of 42·1 months (IQR 32·3-49·2), consolidation therapy with VRD significantly improved median progression-free survival compared with no consolidation (58·9 months [54·0-not estimable] vs 45·5 months [39·5-58·4]; HR 0·77, 0·63-0·95; p=0·014). The most common grade ≥3 adverse events in the autologous HSCT group compared to the VMP group included neutropenia (513 [79%] of 652 patients vs 137 [29%] of 472 patients), thrombocytopenia (541 [83%] vs 74 [16%]), gastrointestinal disorders (80 [12%] vs 25 [5%]), and infections (192 [30%] vs 18 [4%]). 239 (34%) of 702 patients in the autologous HSCT group and 135 (27%) of 495 in the VMP group had at least one serious adverse event. Infection was the most common serious adverse event in each of the treatment groups (206 [56%] of 368 and 70 [37%] of 189). 38 (12%) of 311 deaths from first randomisation were likely to be treatment related: 26 (68%) in the autologous HSCT group and 12 (32%) in the VMP group, most frequently due to infections (eight [21%]), cardiac events (six [16%]), and second primary malignancies (20 [53%]).

Interpretation: This study supports the use of autologous HSCT as intensification therapy and the use of consolidation therapy in patients with newly diagnosed multiple myeloma, even in the era of novel agents. The role of high-dose chemotherapy needs to be reassessed in future studies, in particular in patients with undetectable minimal residual disease after four-drug induction regimens including a monoclonal antiboby combined with an immunomodulatory agent and a proteasome inhibitor plus dexamethasone.

Funding: Janssen and Celgene.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S2352-3026(20)30099-5DOI Listing
June 2020

Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting.

Br J Haematol 2020 09 2;190(6):891-900. Epub 2020 Apr 2.

Department of Hematology, Amsterdam UMC, Vrije Universiteit Amsterdam, Cancer Center Amsterdam, Amsterdam, the Netherlands.

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection. Here we present the validation experiments of the assay in several large AML research centres, both in Europe and the United States. Variability within instruments and sample processing showed high correlations between different instruments (R  > 0·91, P < 0·001). Multi-centre testing introduced variation in reported LSC percentages but was found to be below the clinical relevant threshold. Clear gating protocols resulted in all laboratories being able to perform LSC assessment of the validation set. Participating centres were nearly unanimously able to distinguish LSC (>0·03% LSC) from LSC (<0·03% LSC) despite inter-laboratory variation in reported LSC percentages. This study proves that the LSC assay is highly reproducible. These results together with the high prognostic impact of LSC load at diagnosis in AML patients render the one-tube LSC assessment a good marker for future risk classification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bjh.16594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540683PMC
September 2020

More precisely defining risk peri-HCT in pediatric ALL: pre- vs post-MRD measures, serial positivity, and risk modeling.

Blood Adv 2019 11;3(21):3393-3405

Children's Center for Cancer and Blood Diseases, Children's Hospital Los Angeles, Los Angeles, CA.

Detection of minimal residual disease (MRD) pre- and post-hematopoietic cell transplantation (HCT) for pediatric acute lymphoblastic leukemia (ALL) has been associated with relapse and poor survival. Published studies have had insufficient numbers to: (1) compare the prognostic value of pre-HCT and post-HCT MRD; (2) determine clinical factors post-HCT associated with better outcomes in MRD+ patients; and (3) use MRD and other clinical factors to develop and validate a prognostic model for relapse in pediatric patients with ALL who undergo allogeneic HCT. To address these issues, we assembled an international database including sibling (n = 191), unrelated (n = 259), mismatched (n = 56), and cord blood (n = 110) grafts given after myeloablative conditioning. Although high and very high MRD pre-HCT were significant predictors in univariate analysis, with bivariate analysis using MRD pre-HCT and post-HCT, MRD pre-HCT at any level was less predictive than even low-level MRD post-HCT. Patients with MRD pre-HCT must become MRD low/negative at 1 to 2 months and negative within 3 to 6 months after HCT for successful therapy. Factors associated with improved outcome of patients with detectable MRD post-HCT included acute graft-versus-host disease. We derived a risk score with an MRD cohort from Europe, North America, and Australia using negative predictive characteristics (late disease status, non-total body irradiation regimen, and MRD [high, very high]) defining good, intermediate, and poor risk groups with 2-year cumulative incidences of relapse of 21%, 38%, and 47%, respectively. We validated the score in a second, more contemporaneous cohort and noted 2-year cumulative incidences of relapse of 13%, 26%, and 47% (P < .001) for the defined risk groups.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2019000449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855112PMC
November 2019

Comments on EuroFlow standard operating procedures for instrument setup and compensation for BD FACS Canto II, Navios and BD FACS Lyric instruments.

J Immunol Methods 2019 12 23;475:112680. Epub 2019 Oct 23.

CLIP, Department of Paediatric Hematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic. Electronic address:

This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jim.2019.112680DOI Listing
December 2019

Lossless Compression of Cytometric Data.

Cytometry A 2019 10 20;95(10):1108-1112. Epub 2019 Aug 20.

Laboratory Medical Immunology, Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.

Nowadays, most cytometrists apply lossless compression by storing their FCS files in ZIP archives. Unfortunately, ZIP only achieves modest space savings in cytometric data, due to DEFLATE being used as the underlying lossless compression algorithm (LCA). Presumably, other modern LCA can outperform DEFLATE, especially in terms of space savings. Twenty-one codecs (programs implementing LCA) were evaluated in 167,131 publicly available FCS files. Within floating-point data, as produced by modern instruments, most favorable compression ratios (CRs) were achieved by ZPAQ (median 0.469), BCM (median 0.523), and LZMA (median 0.545). In comparison, the DEFLATE-based codecs only achieved median CR of 0.728 under the most optimal conditions. By default, ZIP offers nine compression level (CL) settings, where lower ZIP-CL optimizes for time efficiency, while higher ZIP-CL optimizes for space efficiency. Interestingly, the third ZIP-CL already resulted in near optimal CR in 90% of the files with floating-point data, as produced by digital cytometers. LZMA is well established, widely supported, and actively maintained (in sharp contrast to ZPAQ and BCM) and therefore arguably the most attractive alternative for ZIP. Within floating-point data, by shifting from ZIP (under optimal conditions) to LZMA (at default settings), the median CR can be improved by 25%. Based on our results, cytometrists can benefit from state-of-the-art compression by choosing the appropriate codec for their situation. Our results are likely to speed-up the adaptation of modern codecs, as CR around 0.5 were beyond all expectations, and such space savings will benefit the field of cytometry. © 2019 International Society for Advancement of Cytometry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.23879DOI Listing
October 2019

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR.

Methods Mol Biol 2019 ;1956:199-228

Second Medical Department, University Hospital Schleswig-Holstein, Kiel, Germany.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL) and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. MRD diagnostics performed by real-time quantitative PCR (RQ-PCR) is still the gold standard and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy chain gene (IGHV). Chromosomal translocations like the t(14;18) translocation in FL and t(11;14) translocation in MCL can be used as MRD target in selected lymphoma subtypes. In patients with B-cell chronic lymphocytic leukemia, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10 shall be achieved.MRD diagnostics targeting the IGHV gene is complex and requires extensive knowledge and experience because the junctional regions of each lymphoma have to be identified before the patient-specific RQ-PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation (SHM) within the rearranged IG heavy variable (IGHV) gene occurring as during B-cell development of germinal center and post-germinal center lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. RQ-PCR-based methods can reach a good sensitivity (≤10). This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IGHV gene rearrangement and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-9151-8_9DOI Listing
July 2019

Combined cellular and soluble mediator analysis for improved diagnosis of vitreoretinal lymphoma.

Acta Ophthalmol 2019 Sep 27;97(6):626-632. Epub 2019 Jan 27.

Department of Immunology, Laboratory Medical Immunology, Erasmus MC, Rotterdam, the Netherlands.

Purpose: Primary vitreoretinal lymphoma [(P)VRL]) is a rare malignancy of the eye localized in the retina, vitreous or choroid. Here, we aim to determine the value of the combination of innovative diagnostic methods for accurate differentiation between (P)VRL and non-(P)VRL in patients with suspect uveitis or vitritis.

Methods: Multicolour flow cytometric immunophenotyping of cells in the vitreous samples was performed using the EuroFlow small sample tube. Additionally, cytokines/chemokines and growth factors were measured in the vitreous specimens using a multiplex immunoassay. Data were evaluated in predefined clinical subgroups using omniviz unsupervised Pearson's correlation visualization and unsupervised heatmap analysis.

Results: A total of 53 patients were prospectively included in the period 2012-2015. In the (P)VRL subgroup (n = 10), nine cases showed aberrant surface membrane immunoglobulin (SmIg) light chain expression. In the non-(P)VRL group (n = 43) clearly skewed SmIg light chain expression was observed in two multiple sclerosis-related uveitis cases, but not in other uveitis types. Soluble mediator measurement revealed high interleukin (IL)-10/IL-6 ratios, and high IL-1RA levels in 9/10 (P)VRL cases, but not in any non-(P)VRL case. Further correlation and heatmap analysis revealed a minimal signature of cellular parameters (CD19+ B cells, aberrant SmIg light chain expression) and cytokine parameters (IL-10/IL-6 ratio >1, high IL-10, high IL-1 RA, high monocyte chemotactic protein-1, high macrophage inflammatory protein-1β) to reliably distinguish (P)VRL from non-(P)VRL.

Conclusion: Here, we show the power of a combined cellular and proteomics strategy for detecting (P)VRL in vitreous specimens, especially in cases with minor cellular (P)VRL infiltrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/aos.14036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796208PMC
September 2019

Immunophenotypic measurable residual disease (MRD) in acute myeloid leukemia: Is multicentric MRD assessment feasible?

Leuk Res 2019 01 27;76:39-47. Epub 2018 Nov 27.

Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.

Flow-cytometric detection of now termed measurable residual disease (MRD) in acute myeloid leukemia (AML) has proven to have an independent prognostic impact. In a previous multicenter study we developed protocols to accurately define leukemia-associated immunophenotypes (LAIPs) at diagnosis. It has, however, not been demonstrated whether the use of the defined LAIPs in the same multicenter setting results in a high concordance between centers in MRD assessment. In the present paper we evaluated whether interpretation of list-mode data (LMD) files, obtained from MRD assessment of previously determined LAIPs during and after treatment, could reliably be performed in a multicenter setting. The percentage of MRD positive cells was simultaneously determined in totally 173 LMD files from 77 AML patients by six participating centers. The quantitative concordance between the six participating centers was meanly 84%, with slight variation of 75%-89%. In addition our data showed that the type and number of LAIPs were of influence on the performance outcome. The highest concordance was observed for LAIPs with cross-lineage expression, followed by LAIPs with an asynchronous antigen expression. Our results imply that immunophenotypic MRD assessment in AML will only be feasible when fully standardized methods are used for reliable multicenter assessment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.leukres.2018.11.014DOI Listing
January 2019

CD34CD38 leukemic stem cell frequency to predict outcome in acute myeloid leukemia.

Leukemia 2019 05 12;33(5):1102-1112. Epub 2018 Dec 12.

Department of Hematology, VU University Medical Center, Cancer Center Amsterdam, Amsterdam, The Netherlands.

Current risk algorithms are primarily based on pre-treatment factors and imperfectly predict outcome in acute myeloid leukemia (AML). We introduce and validate a post-treatment approach of leukemic stem cell (LSC) assessment for prediction of outcome. LSC containing CD34+CD38- fractions were measured using flow cytometry in an add-on study of the HOVON102/SAKK trial. Predefined cut-off levels were prospectively evaluated to assess CD34+CD38-LSC levels at diagnosis (n = 594), and, to identify LSC/LSC (n = 302) and MRD/MRD patients (n = 305) in bone marrow in morphological complete remission (CR). In 242 CR patients combined MRD and LSC results were available. At diagnosis the CD34CD38 LSC frequency independently predicts overall survival (OS). After achieving CR, combining LSC and MRD showed reduced survival in MRD/LSC patients (hazard ratio [HR] 3.62 for OS and 5.89 for cumulative incidence of relapse [CIR]) compared to MRD/LSC, MRD/LSC, and especially MRD/LSC patients. Moreover, in the NPM1mutant positive sub-group, prognostic value of golden standard NPM1-MRD by qPCR can be improved by addition of flow cytometric approaches. This is the first prospective study demonstrating that LSC strongly improves prognostic impact of MRD detection, identifying a patient subgroup with an almost 100% treatment failure probability, warranting consideration of LSC measurement incorporation in future AML risk schemes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41375-018-0326-3DOI Listing
May 2019

CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping.

Cytometry B Clin Cytom 2019 03 18;96(2):134-142. Epub 2018 Nov 18.

Laboratory Medical immunology (LMI), Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.

Background: While it is known that CD123 is normally strongly expressed on plasmacytoid dendritic cells and completely absent on nucleated red blood cells, detailed information regarding CD123 expression in acute leukemia is scarce and, if available, hard to compare due to different methodologies.

Methods: CD123 expression was evaluated using standardized EuroFlow immunophenotyping in 139 pediatric AML, 316 adult AML, 193 pediatric BCP-ALL, 69 adult BCP-ALL, 101 pediatric T-ALL, and 28 adult T-ALL patients. Paired diagnosis-relapse samples were available for 57 AML and 19 BCP-ALL patients. Leukemic stem cell (LSC) data was available for 32 pediatric AML patients. CD123 expression was evaluated based on mean fluorescence intensity, median fluorescence intensity, and percentage CD123 positive cells.

Results: EuroFlow panels were stable over time and between laboratories. CD123 was expressed in the majority of AML and BCP-ALL patients, but absent in most T-ALL patients. Within AML, CD123 expression was lower in erythroid/megakaryocytic leukemia, higher in NPM1 mutated and FLT3-ITD mutated leukemia, and comparable between LSC and leukemic blasts. Within BCP-ALL, CD123 expression was higher in patients with (high) hyperdiploid karyotypes and the BCR-ABL fusion gene. Interestingly, CD123 expression was increased in BCP-ALL relapses while highly variable in AML relapses (compared to CD123 expression at diagnosis).

Conclusions: Authors evaluated CD123 expression in a large cohort of acute leukemia patients, based on standardized and reproducible methodology. Our results may facilitate stratification of patients most likely to respond to CD123 targeted therapies and serve as reference for CD123 expression (in health and disease). © 2018 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.b.21745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587863PMC
March 2019

Next-generation antigen receptor sequencing of paired diagnosis and relapse samples of B-cell acute lymphoblastic leukemia: Clonal evolution and implications for minimal residual disease target selection.

Leuk Res 2019 01 22;76:98-104. Epub 2018 Oct 22.

Department of Immunology, Erasmus MC, University Medical Center Rotterdam, The Netherlands. Electronic address:

Antigen receptor gene rearrangements are frequently applied as molecular targets for detection of minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukemia patients. Since such targets may be lost at relapse, appropriate selection of antigen receptor genes as MRD-PCR target is critical. Recently, next-generation sequencing (NGS) - much more sensitive and quantitative than classical PCR-heteroduplex approaches - has been introduced for identification of MRD-PCR targets. We evaluated 42 paired diagnosis-relapse samples by NGS (IGH, IGK, TRG, TRD, and TRB) to evaluate clonal evolution patterns and to design an algorithm for selection of antigen receptor gene rearrangements most likely to remain stable at relapse. Overall, only 393 out of 1446 (27%) clonal rearrangements were stable between diagnosis and relapse. If only index clones with a frequency >5% at diagnosis were taken into account, this number increased to 65%; including only index clones with an absolute read count >10,000, indicating truly major clones, further increased the stability to 84%. Over 90% of index clones at relapse were also present as index clone at diagnosis. Our data provide detailed information about the stability of antigen receptor gene rearrangements, based on which we propose an algorithm for selecting stable MRD-PCR targets, successful in >97% of patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.leukres.2018.10.009DOI Listing
January 2019

Usability of femoral head bone marrow to verify reference ranges for the assessment of myelodysplasia by flow cytometry.

Int J Lab Hematol 2018 Dec 2;40(6):726-733. Epub 2018 Sep 2.

Department of Immunology, Laboratory Medical Immunology, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, the Netherlands.

Background: A flow cytometry score (FCS) based on four parameters has been proposed for analysis of myelodysplastic syndromes patients with <5% blasts. We evaluated whether bone marrow aspirate samples isolated from femoral heads could be used for verification of cutoff values of the individual parameters score (IPS), contributing to the FCS and compared the applicability of FCS parameters in two centers.

Study Design And Methods: Bone marrow cells were obtained from femoral heads of patients who underwent hip replacement surgery. The score of the 4 individual parameters of the cell samples were obtained independently in two centers, using their own facilities and methods. Flow cytometry data files were subsequently exchanged and reanalyzed in the other center. The resulting four data sets were compared to assess reproducibility and outcomes in both centers.

Results: Twenty-nine of 40 bone marrow samples contained sufficient cells for analysis. Proposed cutoff values for 3 of 4 individual parameters were appropriate. All 29 samples showed a positive individual parameters score (IPS:1) in 1 of the 4 obtained data sets. Most differences in IPS scores were a result of reanalyzing the data file in the other center, rather than data acquisition. FCS: ≥2 were observed in 11 samples.

Conclusion: Bone marrow samples from femoral heads are appropriate for verification of the proposed reference cutoff values of the individual parameters. Proposed reference cutoff values needed to be adjusted for reliable interpretation. Standardization of both data acquisition and data analysis is necessary for obtaining uniform results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ijlh.12911DOI Listing
December 2018

Low-dose cytarabine to prevent myeloid leukemia in children with Down syndrome: TMD Prevention 2007 study.

Blood Adv 2018 07;2(13):1532-1540

Department of Pediatric Hematology and Oncology, Martin Luther University Halle-Wittenberg, Halle, Germany.

Approximately 5% to 10% of children with Down syndrome (DS) are diagnosed with transient myeloproliferative disorder (TMD). Approximately 20% of these patients die within 6 months (early death), and another 20% to 30% progress to myeloid leukemia (ML-DS) within their first 4 years of life. The aim of the multicenter, nonrandomized, historically controlled TMD Prevention 2007 trial was to evaluate the impact of low-dose cytarabine treatment on survival and prevention of ML-DS in patients with TMD. Patients received cytarabine (1.5 mg/kg for 7 days) in case of TMD-related symptoms at diagnosis (high white blood cell count, ascites, liver dysfunction, hydrops fetalis) or detection of minimal residual disease (MRD) 8 weeks after diagnosis. The 5-year probability of event-free and overall survival of 102 enrolled TMD patients was 72 ± 5% and 91 ± 3%, respectively. In patients eligible for treatment because of symptoms (n = 43), we observed a significantly lower cumulative incidence (CI) of early death as compared with symptomatic patients in the historical control (n = 45) (12 ± 5% vs 33 ± 7%, = .02). None of the asymptomatic patients in the current study suffered early death. However, the treatment of symptomatic or MRD-positive patients did not result in a significantly lower CI of ML-DS (25 ± 7% [treated] vs 14 ± 7% [untreated], = .34 [per protocol analysis]; historical control: 22 ± 4%, = .55). Thus, low-dose cytarabine treatment helped to reduce TMD-related mortality when compared with the historical control but was insufficient to prevent progression to ML-DS. This trial was registered at EudraCT as #2006-002962-20.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2018018945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039662PMC
July 2018