Publications by authors named "Joop H Jansen"

121 Publications

The CADM1 tumor suppressor gene is a major candidate gene in MDS with deletion of the long arm of chromosome 11.

Blood Adv 2021 Oct 12. Epub 2021 Oct 12.

Belgian Cancer Registry, Brussels, Belgium.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic stem-cell disorders characterized by ineffective hematopoiesis leading to peripheral cytopenias and in a substantial proportion of cases to acute myeloid leukemia. The deletion of the long arm of chromosome 11, del(11q), is a rare but recurrent clonal event in MDS. Here, we detail the largest series of 113 cases of MDS and myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) harboring a del(11q) analyzed at clinical, cytological, cytogenetic and molecular levels. Female predominance, a survival prognosis similar to other MDS, a low monocyte count and dysmegakaryopoiesis were the specific clinical and cytological features of del(11q) MDS. In most cases, del(11q) was isolated, primary and interstitial encompassing the 11q22-23 region containing ATM, KMT2A and CBL genes. The common deleted region at 11q23.2 is centered on an intergenic region between CADM1 (also known as TSLC1, Tumour Suppressor in Lung Cancer 1) and NXPE2. CADM1 was expressed in all myeloid cells analyzed in contrast to NXPE2. At the functional level, the deletion of Cadm1 in murine Lineage-Sca1+Kit+ cells modifies the lymphoid to myeloid ratio in bone marrow although not altering their multi-lineage hematopoietic reconstitution potential after syngenic transplantation. Together with the frequent simultaneous deletions of KMT2A, ATM and CBL and mutations of ASXL1, SF3B1 and CBL, we show that CADM1 may be important in the physiopathology of the del(11q) MDS, extending its role as tumor-suppressor gene from solid tumors to hematopoietic malignancies.
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http://dx.doi.org/10.1182/bloodadvances.2021005311DOI Listing
October 2021

CD34 progenitor-derived NK cell and gemcitabine combination therapy increases killing of ovarian cancer cells in NOD/SCID/IL2Rg mice.

Oncoimmunology 2021 1;10(1):1981049. Epub 2021 Oct 1.

Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center/Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.

Combining natural killer (NK) cell adoptive transfer with tumor-sensitizing chemotherapy is an attractive approach against recurrent ovarian cancer (OC), as OC is sensitive to NK cell-mediated immunity. Previously, we showed that CD34 hematopoietic progenitor cell (HPC)-derived NK cells can kill OC cells and inhibit OC tumor growth in mice. Here, we investigated the potential of HPC-NK cell therapy combined with chemotherapeutic gemcitabine (used in recurrent OC patients) against OC. We examined the phenotypical, functional, and cytotoxic effects of gemcitabine on HPC-NK cells and/or OC cells and in OC-bearing mice. To this end, we treated OC cells and/or HPC-NK cells with or without gemcitabine and analyzed the phenotype, cytokine production, and anti-tumor reactivity. We found that gemcitabine did not affect the phenotype and functionality of HPC-NK cells, while on OC cells expression of NK cell activating ligands and death receptors was upregulated. Although gemcitabine pre-treatment of OC cells did not improve the functionality of HPC-NK cells, importantly, HPC-NK cells and gemcitabine additively killed OC cells . Similarly, combined HPC-NK cell and gemcitabine treatment additively decreased tumor growth in OC-bearing mice. Collectively, our results indicate that combination therapy of HPC-NK cells and gemcitabine results in augmented OC killing and . This provides a rationale for exploring this therapeutic strategy in patients with recurrent OC.
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http://dx.doi.org/10.1080/2162402X.2021.1981049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489932PMC
October 2021

Peripheral blood cytopenias in the aging general population and risk of incident hematological disease and mortality.

Blood Adv 2021 09;5(17):3266-3278

Department of Hematology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Peripheral blood cytopenias may precede the development of hematological malignancies and frequently pose clinical challenges in the older population. The natural course of (mild) cytopenias during aging and their association with hematological disorders in community-dwelling individuals are not well studied. Within the population-based Lifelines cohort (n = 167729), we studied changes in peripheral blood counts, occurrence of cytopenias, and associated hematological outcomes in the context of aging. Development of hematological malignancies and (cause-specific) mortality were evaluated by linkage to nationwide registries. Anemia and thrombocytopenia emerged with older age, in line with a general age-related decline in these blood counts. For neutropenia, no increase in prevalence with older age was observed. Using standard reference limits to define cytopenias, anemia (hazard ratio [HR], 1.84; 95% confidence interval [CI], 1.59-2.12), thrombocytopenia (HR, 1.58; 95% CI, 1.32-1.89), and, especially the concomitant presence of anemia and thrombocytopenia (HR, 4.75; 95% CI, 2.98-7.55) were associated with inferior overall survival. Only a minor proportion of deaths was explained by diagnosed hematological malignancies, with the majority attributable to other causes. Neutropenia, either isolated (HR, 0.88; 95% CI, 0.73-1.06) or combined with another cytopenia, did not affect overall survival. For individuals aged ≥60 years, 5-year cumulative incidence of hematological malignancies was 0.60% (95% CI, 0.50-0.70), with higher incidences among those with anemia (P < .001) or thrombocytopenia (P < .001) but not neutropenia (P = .201). Highest cumulative incidences of diagnoses and mortality from hematological malignancies were observed in individuals with >1 cytopenia. We conclude that anemia and thrombocytopenia, but not neutropenia, are associated with inferior overall survival of community-dwelling individuals. Hematological malignancies develop in a small fraction of these cases.
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http://dx.doi.org/10.1182/bloodadvances.2021004355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525229PMC
September 2021

Characterization of a genomic region 8 kb downstream of GFI1B associated with myeloproliferative neoplasms.

Biochim Biophys Acta Mol Basis Dis 2021 Dec 31;1867(12):166259. Epub 2021 Aug 31.

Department of Laboratory Medicine, Laboratory of Hematology, Radboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen 6525GA, the Netherlands. Electronic address:

A genomic locus 8 kb downstream of the transcription factor GFI1B (Growth Factor Independence 1B) predisposes to clonal hematopoiesis and myeloproliferative neoplasms. One of the most significantly associated polymorphisms in this region is rs621940-G. GFI1B auto-represses GFI1B, and altered GFI1B expression contributes to myeloid neoplasms. We studied whether rs621940-G affects GFI1B expression and growth of immature cells. GFI1B ChIP-seq showed clear binding to the rs621940 locus. Preferential binding of various hematopoietic transcription factors to either the rs621940-C or -G allele was observed, but GFI1B showed no preference. In gene reporter assays the rs621940 region inhibited GFI1B promoter activity with the G-allele having less suppressive effects compared to the C-allele. However, CRISPR-Cas9 mediated deletion of the locus in K562 cells did not alter GFI1B expression nor auto-repression. In healthy peripheral blood mononuclear cells GFI1B expression did not differ consistently between the rs621940 alleles. Long range and targeted deep sequencing did not detect consistent effects of rs621940-G on allelic GFI1B expression either. Finally, we observed that myeloid colony formation was not significantly affected by either rs621940 allele in 193 healthy donors. Together, these findings show no evidence that rs621940 or its locus affect GFI1B expression, auto-repression or growth of immature myeloid cells.
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http://dx.doi.org/10.1016/j.bbadis.2021.166259DOI Listing
December 2021

Molecular landscape and prognostic impact of FLT3-ITD insertion site in acute myeloid leukemia: RATIFY study results.

Leukemia 2021 Jul 28. Epub 2021 Jul 28.

Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany.

In acute myeloid leukemia (AML) internal tandem duplications of the FLT3 gene (FLT3-ITD) are associated with poor prognosis. Retrospectively, we investigated the prognostic and predictive impact of FLT3-ITD insertion site (IS) in 452 patients randomized within the RATIFY trial, which evaluated midostaurin additionally to intensive chemotherapy. Next-generation sequencing identified 908 ITDs, with 643 IS in the juxtamembrane domain (JMD) and 265 IS in the tyrosine kinase domain-1 (TKD1). According to IS, patients were categorized as JMDsole (n = 251, 55%), JMD and TKD1 (JMD/TKD1; n = 117, 26%), and TKD1sole (n = 84, 19%). While clinical variables did not differ among the 3 groups, NPM1 mutation was correlated with JMDsole (P = 0.028). Overall survival (OS) differed significantly, with estimated 4-year OS probabilities of 0.44, 0.50, and 0.30 for JMDsole, JMD/TKD1, and TKD1sole, respectively (P = 0.032). Multivariate (cause-specific) Cox models for OS and cumulative incidence of relapse using allogeneic hematopoietic cell transplantation (HCT) in first complete remission as a time-dependent variable identified TKD1sole as unfavorable and HCT as favorable factors. In addition, Midostaurin exerted a significant benefit only for JMDsole. Our results confirm the distinct molecular heterogeneity of FLT3-ITD and the negative prognostic impact of TKD1 IS in AML that was not overcome by midostaurin.
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http://dx.doi.org/10.1038/s41375-021-01323-0DOI Listing
July 2021

Prevalence, predictors, and outcomes of clonal hematopoiesis in individuals aged ≥80 years.

Blood Adv 2021 04;5(8):2115-2122

Department of Hematology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Clonal hematopoiesis (CH), characterized by a fraction of peripheral blood cells carrying an acquired genetic variant, emerges with age. Although in general CH is associated with increased mortality and morbidity, no higher risk of death was observed for individuals ≥80 years. Here, we investigated CH in 621 individuals aged ≥80 years from the population-based LifeLines cohort. Sensitive error-corrected sequencing of 27 driver genes at a variant allele frequency ≥1% revealed CH in the majority (62%) of individuals, independent of gender. The observed mutational spectrum was dominated by DNMT3A and TET2 variants, which frequently (29%) displayed multiple mutations per gene. In line with previous results in individuals ≥80 years, the overall presence of CH did not associate with a higher risk of death (hazard ratio, 0.91; 95% confidence interval, 0.70-1.18; P = .48). Being able to assess the causes of death, we observed no difference between individuals with or without CH, except for deaths related to hematological malignancies. Interestingly, comparison of mutational spectra confined to DNMT3A and TET2 vs spectra containing other mutated genes, showed a higher risk of death when mutations other than DNMT3A or TET2 were present (hazard ratio, 1.48; 95% confidence interval, 1.06-2.08; P = .025). Surprisingly, no association of CH with cardiovascular morbidity was found, irrespective of clone size. Further, CH associated with chronic obstructive pulmonary disease. Data on estimated exposure to DNA damaging toxicities (ie, smoking, a history of cancer [as a proxy for previous genotoxic therapy], and job-related pesticide exposure) showed an association with spliceosome and ASXL1 variants, but not with DNMT3A and TET2 variants.
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http://dx.doi.org/10.1182/bloodadvances.2020004062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095141PMC
April 2021

Low relapse risk in poor risk AML after conditioning with 10-day decitabine, fludarabine and 2 Gray TBI prior to allogeneic hematopoietic cell transplantation.

Bone Marrow Transplant 2021 08 6;56(8):1964-1970. Epub 2021 Apr 6.

Department of Hematology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.

Patients with poor risk acute myeloid leukemia (AML) have a dismal outcome. We hypothesized that combining decitabine with a standard non-myeloablative (NMA) conditioning regimen prior to allogeneic hematopoietic cell transplantation (allo HCT), might decrease the relapse incidence. We conducted a multicenter prospective phase II study (NCT02252107) with 10-day decitabine (20 mg/m/day) integrated in a standard non-myeloablative conditioning regimen (3 days fludarabine 30 mg/m with 2 Gray total body irradiation (TBI)). Patients with AML ≥ 18 years in 1st (in)complete remission (CR/CRi) with a poor or very poor risk profile, as defined by the HOVON-132 protocol, were eligible. Results: Forty-six patients (median age 60; range 23-74) were included. Median follow up time was 44 months (range 31-65 months). The cumulative 1-year incidence of relapse and NRM were respectively 23% and 11%. Incidence of grade III-IV acute graft-vs-host-disease (GVHD) and severe chronic GVHD were 13% and 20%, respectively. One-year OS was 70%. Application of ELN 2017 risk classification to the study cohort revealed a cumulative one-year relapse rate of respectively 31% and 13% for the adverse and intermediate risk patients. To conclude, the 10-day DEC/FLU/TBI conditioning regimen prior to allo HCT in poor risk AML patients is effective and feasible.
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http://dx.doi.org/10.1038/s41409-021-01272-3DOI Listing
August 2021

Clinically applicable CD34-derived blood dendritic cell subsets exhibit key subset-specific features and potently boost anti-tumor T and NK cell responses.

Cancer Immunol Immunother 2021 Nov 1;70(11):3167-3181. Epub 2021 Apr 1.

Department of Laboratory Medicine, Laboratory of Hematology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 8, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands.

Allogeneic stem cell transplantation (alloSCT), following induction chemotherapy, can be curative for hemato-oncology patients due to powerful graft-versus-tumor immunity. However, disease recurrence remains the major cause of treatment failure, emphasizing the need for potent adjuvant immunotherapy. In this regard, dendritic cell (DC) vaccination is highly attractive, as DCs are the key orchestrators of innate and adaptive immunity. Natural DC subsets are postulated to be more powerful compared with monocyte-derived DCs, due to their unique functional properties and cross-talk capacity. Yet, obtaining sufficient numbers of natural DCs, particularly type 1 conventional DCs (cDC1s), is challenging due to low frequencies in human blood. We developed a clinically applicable culture protocol using donor-derived G-CSF mobilized CD34 hematopoietic progenitor cells (HPCs) for simultaneous generation of high numbers of cDC1s, cDC2s and plasmacytoid DCs (pDCs). Transcriptomic analyses demonstrated that these ex vivo-generated DCs highly resemble their in vivo blood counterparts. In more detail, we demonstrated that the CD141CLEG9A cDC1 subset exhibited key features of in vivo cDC1s, reflected by high expression of co-stimulatory molecules and release of IL-12p70 and TNF-α. Furthermore, cDC1s efficiently primed alloreactive T cells, potently cross-presented long-peptides and boosted expansion of minor histocompatibility antigen-experienced T cells. Moreover, they strongly enhanced NK cell activation, degranulation and anti-leukemic reactivity. Together, we developed a robust culture protocol to generate highly functional blood DC subsets for in vivo application as tailored adjuvant immunotherapy to boost innate and adaptive anti-tumor immunity in alloSCT patients.
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http://dx.doi.org/10.1007/s00262-021-02899-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8505305PMC
November 2021

Distinct bone marrow immunophenotypic features define the splicing factor 3B subunit 1 (SF3B1)-mutant myelodysplastic syndromes subtype.

Br J Haematol 2021 05 25;193(4):798-803. Epub 2021 Mar 25.

Department of Hematology, Cancer Center Amsterdam, Amsterdam UMC, Location VUmc, Amsterdam, the Netherlands.

Splicing factor 3B subunit 1 (SF3B1) mutations define a distinct myelodysplastic syndromes (MDS) patient group with a relatively favourable disease course and high response rates to luspatercept. Few data are available on bone marrow phenotype beyond ring sideroblasts in this subgroup of patients with MDS. In the present study, we identified immunophenotypic erythroid, myelomonocyte and progenitor features associated with SF3B1 mutations. In addition, we illustrate that SF3B1-mutation type is associated with distinct immunophenotypic features, and show the impact of co-occurrence of a SF3B1 mutation and a deletion of chromosome 5q on bone marrow immunophenotype. These genotype-phenotype associations and phenotypic subtypes within SF3B1-MDS provide leads that may further refine prognostication and therapeutic strategies for this particular MDS subgroup.
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http://dx.doi.org/10.1111/bjh.17414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8252736PMC
May 2021

The Effect of Mutation on the DNA Damage Response and Nonsense-Mediated mRNA Decay in Cancer.

Front Oncol 2020 29;10:609409. Epub 2021 Jan 29.

Department of Experimental Immunology, Amsterdam University Medical Centers, Location AMC, University of Amsterdam, Amsterdam, Netherlands.

Recurrent mutations in splicing factor 3B subunit 1 () have been identified in several malignancies and are associated with an increased expression of 3' cryptic transcripts as a result of alternative branchpoint recognition. A large fraction of cryptic transcripts associated with mutations is expected to be sensitive for RNA degradation nonsense-mediated mRNA decay (NMD). Several studies indicated alterations in various signaling pathways in SF3B1-mutated cells, including an impaired DNA damage response (DDR) in chronic lymphocytic leukemia (CLL). In this study, we investigated isogenic cell lines and treatment naïve primary CLL samples without any and/or defect, and found no significant effects of mutations on the ATM/p53 response, phosphorylation of H2AX and sensitivity to fludarabine. Cryptic transcripts associated with mutation status were observed at relatively low levels compared to the canonical transcripts and were validated as target for mRNA degradation NMD. Expression of cryptic transcripts increased after NMD inhibition. In conclusion, our results confirm involvement of NMD in the biological effects of mutations. Further studies may elucidate whether -mutant patients could benefit from NMD modulatory agents.
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http://dx.doi.org/10.3389/fonc.2020.609409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880055PMC
January 2021

Multicenter Next-Generation Sequencing Studies between Theory and Practice: Harmonization of Data Analysis Using Real-World Myelodysplastic Syndrome Data.

J Mol Diagn 2021 03 23;23(3):347-357. Epub 2020 Dec 23.

Institute of Medical Informatics, University of Münster, Münster, Germany.

In the age of personalized medicine, genetic testing by means of targeted sequencing has taken a key role. However, when comparing different sets of targeted sequencing data, these are often characterized by a considerable lack of harmonization. Laboratories follow their own best practices, analyzing their own target regions. The question on how to best integrate data from different sites remains unanswered. Studying the example of myelodysplastic syndrome (MDS), we analyzed 11 targeted sequencing sets, collected from six different centers (n = 831). An intersecting target region of 43,076 bp (30 genes) was identified; whereas, the original target regions covered up to 499,097 bp (117 genes). Considering a region of interest in the context of MDS, a target region of 55,969 bp (31 genes) was identified. For each gene, coverage and sequencing data quality was evaluated, calculating a sequencing score. Analyses revealed huge differences between different data sets as well as between different genes. Analysis of the relation between sequencing score and mutation frequency in MDS revealed that most genes with high frequency in MDS could be sequenced without expecting low coverage or quality. Still, no gene appeared consistently unproblematic for all data sets. To allow for comparable results in a multicenter setting analyzing MDS, we propose to use a predefined target region of interest and to perform centralized data analysis using harmonized criteria.
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http://dx.doi.org/10.1016/j.jmoldx.2020.12.001DOI Listing
March 2021

TIGIT blockade enhances functionality of peritoneal NK cells with altered expression of DNAM-1/TIGIT/CD96 checkpoint molecules in ovarian cancer.

Oncoimmunology 2020 11 8;9(1):1843247. Epub 2020 Nov 8.

Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center, Nijmegen, The Netherlands.

Advanced ovarian cancer (OC) patients have a poor 5-year survival of only 28%, emphasizing the medical need for improved therapies. Adjuvant immunotherapy could be an attractive approach since OC is an immunogenic disease and the presence of tumor-infiltrating lymphocytes has shown to positively correlate with patient survival. Among these infiltrating lymphocytes are natural killer (NK) cells, key players involved in tumor targeting, initiated by signaling via activating and inhibitory receptors. Here, we investigated the role of the DNAM-1/TIGIT/CD96 axis in the anti-tumor response of NK cells toward OC. Ascites-derived NK cells from advanced OC patients showed lower expression of activating receptor DNAM-1 compared to healthy donor peripheral blood NK cells, while inhibitory receptor TIGIT and CD96 expression was equal or higher, respectively. This shift to a more inhibitory phenotype could also be induced by co-culturing healthy donor NK cells with OC tumor spheroids, and on intraperitoneally infused NK cells in SKOV-3 OC bearing NOD/SCID-IL2Rγnull (NSG) mice. Interestingly, TIGIT blockade enhanced degranulation and interferon gamma (IFNγ) production of healthy donor CD56 NK cells in response to OC tumor cells, especially when DNAM-1/CD155 interactions were in place. Importantly, TIGIT blockade boosted functional responsiveness of CD56 NK cells of OC patients with a baseline reactivity against SKOV-3 cells. Overall, our data show for the first time that checkpoint molecules TIGIT/DNAM-1/CD96 play an important role in NK cell responsiveness against OC, and provides rationale for incorporating TIGIT interference in NK cell-based immunotherapy in OC patients.
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http://dx.doi.org/10.1080/2162402X.2020.1843247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657585PMC
November 2020

Paroxysmal nocturnal hemoglobinuria caused by CN-LOH of constitutional PIGB mutation and 70-kbp microdeletion on 15q.

Blood Adv 2020 11;4(22):5755-5761

Research Institute for Microbial Diseases and WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell (HSC) disorder characterized by defective synthesis of the glycosylphosphatidylinositol (GPI) anchors as a result of somatic mutations in the X-linked PIGA gene. The disease is acquired. No constitutional PNH has been described. Here, we report familial PNH associated with unusual inflammatory symptoms. Genetic analysis revealed a germline heterozygous PIGB mutation on chromosome 15 without mutations in PIGA or any of the other genes involved in GPI biosynthesis. In vitro data confirmed that transfection of the mutant PIGB could not restore the surface expression of GPI-anchored proteins (APs) in PIGB-deficient Chinese hamster ovary cells. Homozygosity was caused by copy number-neutral loss of heterozygosity (CN-LOH) of the germline PIGB mutation, leading to deficient expression of GPI-APs in the affected blood cells of the index patient and her mother. The somatic event leading to homozygosity of the germline mutant PIGB gene involved a 70-kbp microdeletion of chromosome 15q containing the TM2D3 and TARSL2 genes, which was implicated in chromosome 15q mosaicism. Interestingly, we detected the deletion in both the patient and her mother. A sister of the mother, who carried the same germline PIGB mutation but without this microdeletion involving TM2D3 and TARSL2, did not have a PNH clone or CN-LOH. In conclusion, we describe PNH caused by CN-LOH of a germline heterozygous PIGB mutation in a patient and her mother and hypothesize that the 70-kbp microdeletion may have contributed to the PNH clone in both.
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http://dx.doi.org/10.1182/bloodadvances.2020002210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686886PMC
November 2020

Malignant Transformation Involving Mutations Identified in a Leukemic Progression Model of Severe Congenital Neutropenia.

Cell Rep Med 2020 Aug 25;1(5):100074. Epub 2020 Aug 25.

Department of Hematology, Erasmus University Medical Center, Rotterdam 3015 CN, the Netherlands.

Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor () mutations in the neutropenic phase, followed by mutations in before AML becomes overt. To investigate how the combination of CSF3 therapy and and mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in / mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN.
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http://dx.doi.org/10.1016/j.xcrm.2020.100074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659587PMC
August 2020

CopyDetective: Detection threshold-aware copy number variant calling in whole-exome sequencing data.

Gigascience 2020 11;9(11)

Institute of Medical Informatics, University of Münster, Albert-Schweitzer-Campus 1, Building A11, Münster 48149, Germany.

Background: Copy number variants (CNVs) are known to play an important role in the development and progression of several diseases. However, detection of CNVs with whole-exome sequencing (WES) experiments is challenging. Usually, additional experiments have to be performed.

Findings: We developed a novel algorithm for somatic CNV calling in matched WES data called "CopyDetective". Different from other approaches, CNV calling with CopyDetective consists of a 2-step procedure: first, quality analysis is performed, determining individual detection thresholds for every sample. Second, actual CNV calling on the basis of the previously determined thresholds is performed. Our algorithm evaluates the change in variant allele frequency of polymorphisms and reports the fraction of affected cells for every CNV. Analyzing 4 WES data sets (n = 100) we observed superior performance of CopyDetective compared with ExomeCNV, VarScan2, ControlFREEC, ExomeDepth, and CNV-seq.

Conclusions: Individual detection thresholds reveal that not every WES data set is equally apt for CNV calling. Initial quality analyses, determining individual detection thresholds-as realized by CopyDetective-can and should be performed prior to actual variant calling.
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http://dx.doi.org/10.1093/gigascience/giaa118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604644PMC
November 2020

Transcription factor 4 (TCF4) expression predicts clinical outcome in RUNX1 mutated and translocated acute myeloid leukemia.

Haematologica 2020 09 1;105(9):e454-457. Epub 2020 Sep 1.

Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Centrer, Nijmegen, the Netherlands.

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http://dx.doi.org/10.3324/haematol.2019.232827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556620PMC
September 2020

Midostaurin in patients with acute myeloid leukemia and FLT3-TKD mutations: a subanalysis from the RATIFY trial.

Blood Adv 2020 10;4(19):4945-4954

Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus der TU Dresden, Dresden, Germany; and.

The results from the RATIFY trial (ClinicalTrials.gov: NCT00651261; CALGB 10603) showed that midostaurin combined with standard chemotherapy significantly improved outcomes in patients with FMS-like tyrosine kinase 3 (FLT3)-mutated acute myeloid leukemia (AML), compared with placebo. In this post hoc subgroup analysis from the trial, we evaluated the impact of midostaurin in 163 patients with FLT3-tyrosine kinase domain (TKD) mutations. At a median follow-up of 60.7 months (95% CI, 55.0-70.8), the 5-year event-free survival (EFS) rate was significantly higher in patients treated with midostaurin than in those treated with placebo (45.2% vs 30.1%; P = .044). A trend toward improved disease-free survival was also observed with midostaurin (67.3% vs 53.4%; P = .089), whereas overall survival (OS) was similar in the 2 groups. Patients with AML and NPM1mut/FLT3-TKDmut or core binding factor (CBF)-rearranged/FLT3-TKDmut genotypes had significantly prolonged OS with or without censoring at hematopoietic cell transplantation (HCT), compared with NPM1WT/CBF-negative AMLs. The multivariable model for OS and EFS adjusted for allogeneic HCT in first complete remission as a time-dependent covariable, revealed NPM1 mutations and CBF rearrangements as significant favorable factors. These data show that NPM1 mutations or CBF rearrangements identify favorable prognostic groups in patients with FLT3-TKD AMLs, independent of other factors, also in the context of midostaurin treatment.
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http://dx.doi.org/10.1182/bloodadvances.2020002904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556122PMC
October 2020

Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes.

Nat Med 2020 10 3;26(10):1549-1556. Epub 2020 Aug 3.

Department of Hematology, Democritus University of Thrace Medical School, Alexandroupolis, Greece.

Tumor protein p53 (TP53) is the most frequently mutated gene in cancer. In patients with myelodysplastic syndromes (MDS), TP53 mutations are associated with high-risk disease, rapid transformation to acute myeloid leukemia (AML), resistance to conventional therapies and dismal outcomes. Consistent with the tumor-suppressive role of TP53, patients harbor both mono- and biallelic mutations. However, the biological and clinical implications of TP53 allelic state have not been fully investigated in MDS or any other cancer type. We analyzed 3,324 patients with MDS for TP53 mutations and allelic imbalances and delineated two subsets of patients with distinct phenotypes and outcomes. One-third of TP53-mutated patients had monoallelic mutations whereas two-thirds had multiple hits (multi-hit) consistent with biallelic targeting. Established associations with complex karyotype, few co-occurring mutations, high-risk presentation and poor outcomes were specific to multi-hit patients only. TP53 multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System (IPSS-R). Surprisingly, monoallelic patients did not differ from TP53 wild-type patients in outcomes and response to therapy. This study shows that consideration of TP53 allelic state is critical for diagnostic and prognostic precision in MDS as well as in future correlative studies of treatment response.
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http://dx.doi.org/10.1038/s41591-020-1008-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8381722PMC
October 2020

PD-L1 siRNA-mediated silencing in acute myeloid leukemia enhances anti-leukemic T cell reactivity.

Bone Marrow Transplant 2020 12 11;55(12):2308-2318. Epub 2020 Jun 11.

Department of Laboratory Medicine - Laboratory of Hematology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands.

Acute myeloid leukemia (AML) is an immune-susceptible malignancy, as demonstrated by its responsiveness to allogeneic stem cell transplantation (alloSCT). However, by employing inhibitory signaling pathways, including PD-1/PD-L1, leukemia cells suppress T cell-mediated immune attack. Notably, impressive clinical efficacy has been obtained with PD-1/PD-L1 blocking antibodies in cancer patients. Yet, these systemic treatments are often accompanied by severe toxicity, especially after alloSCT. Here, we investigated RNA interference technology as an alternative strategy to locally interfere with PD-1/PD-L1 signaling in AML. We demonstrated efficient siRNA-mediated PD-L1 silencing in HL-60 and patients' AML cells. Importantly, WT1-antigen T cell receptor PD-1 2D3 cells showed increased activation toward PD-L1 silenced WT1 AML. Moreover, PD-L1 silenced AML cells significantly enhanced the activation, degranulation, and IFN-γ production of minor histocompatibility antigen-specific CD8 T cells. Notably, PD-L1 silencing was equally effective as PD-1 antibody blockade. Together, our study demonstrates that PD-L1 silencing may be an effective strategy to augment AML immune-susceptibility. This provides rationale for further development of targeted approaches to locally interfere with immune escape mechanisms in AML, thereby minimizing severe toxicity. In combination with alloSCT and/or adoptive T cell transfer, this strategy could be very appealing to boost graft-versus-leukemia immunity and improve outcome in AML patients.
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http://dx.doi.org/10.1038/s41409-020-0966-6DOI Listing
December 2020

Cell composition and expansion strategy can reduce the beneficial effect of AKT-inhibition on functionality of CD8 T cells.

Cancer Immunol Immunother 2020 Nov 5;69(11):2259-2273. Epub 2020 Jun 5.

Department of Laboratory Medicine, Laboratory of Hematology, Radboud Institute of Molecular Life Sciences, Radboud university medical center, Geert Grooteplein Zuid 8, 6525 GA, Nijmegen, The Netherlands.

AKT-inhibition is a promising approach to improve T cell therapies; however, its effect on CD4 T cells is insufficiently explored. Previously, we and others showed that AKT-inhibition during ex vivo CD8 T cell expansion facilitates the generation of polyfunctional T cells with stem cell memory-like traits. However, most therapeutic T cell products are generated from lymphocytes, containing CD4 T cells that can affect CD8 T cells dependent on the Th-subset. Here, we investigated the effect of AKT-inhibition on CD4 T cells, during separate as well as total T cell expansions. Interestingly, ex vivo AKT-inhibition preserved the early memory phenotype of CD4 T cells based on higher CD62L, CXCR4 and CCR7 expression. However, in the presence of AKT-inhibition, Th-differentiation was skewed toward more Th2-associated at the expense of Th1-associated cells. Importantly, the favorable effect of AKT-inhibition on the functionality of CD8 T cells drastically diminished in the presence of CD4 T cells. Moreover, also the expansion method influenced the effect of AKT-inhibition on CD8 T cells. These findings indicate that the effect of AKT-inhibition on CD8 T cells is dependent on cell composition and expansion strategy, where presence of CD4 T cells as well as polyclonal stimulation impede the favorable effect of AKT-inhibition.
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http://dx.doi.org/10.1007/s00262-020-02612-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568704PMC
November 2020

In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states.

Nat Cell Biol 2020 05 4;22(5):534-545. Epub 2020 May 4.

Department of Cell Biology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.
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http://dx.doi.org/10.1038/s41556-020-0508-xDOI Listing
May 2020

SF3B1-mutant MDS as a distinct disease subtype: a proposal from the International Working Group for the Prognosis of MDS.

Blood 2020 07;136(2):157-170

Amsterdam University Medical Center, Amsterdam, The Netherlands.

The 2016 revision of the World Health Organization classification of tumors of hematopoietic and lymphoid tissues is characterized by a closer integration of morphology and molecular genetics. Notwithstanding, the myelodysplastic syndrome (MDS) with isolated del(5q) remains so far the only MDS subtype defined by a genetic abnormality. Approximately half of MDS patients carry somatic mutations in spliceosome genes, with SF3B1 being the most commonly mutated one. SF3B1 mutation identifies a condition characterized by ring sideroblasts (RS), ineffective erythropoiesis, and indolent clinical course. A large body of evidence supports recognition of SF3B1-mutant MDS as a distinct nosologic entity. To further validate this notion, we interrogated the data set of the International Working Group for the Prognosis of MDS (IWG-PM). Based on the findings of our analyses, we propose the following diagnostic criteria for SF3B1-mutant MDS: (1) cytopenia as defined by standard hematologic values, (2) somatic SF3B1 mutation, (3) morphologic dysplasia (with or without RS), and (4) bone marrow blasts <5% and peripheral blood blasts <1%. Selected concomitant genetic lesions represent exclusion criteria for the proposed entity. In patients with clonal cytopenia of undetermined significance, SF3B1 mutation is almost invariably associated with subsequent development of overt MDS with RS, suggesting that this genetic lesion might provide presumptive evidence of MDS in the setting of persistent unexplained cytopenia. Diagnosis of SF3B1-mutant MDS has considerable clinical implications in terms of risk stratification and therapeutic decision making. In fact, this condition has a relatively good prognosis and may respond to luspatercept with abolishment of the transfusion requirement.
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http://dx.doi.org/10.1182/blood.2020004850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362582PMC
July 2020

Harnessing natural killer cells for the treatment of ovarian cancer.

Gynecol Oncol 2020 06 5;157(3):810-816. Epub 2020 Apr 5.

Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Medical Center, Nijmegen, the Netherlands.

Introduction: Adoptive cellular immunotherapy could be an interesting new treatment option for ovarian carcinoma (OC), as research has demonstrated that OC is an immunogenic disease. In particular, natural killer (NK) cells have attracted attention due to their ability to kill tumor cells without prior sensitization. The therapeutic value of allogeneic NK cells has been first observed in hematological cancers and is increasingly being explored in solid tumors.

Methods: To substantiate the rationale for NK cell therapy in OC we performed a literature search in the Pubmed database and in the international trial register clinicaltrials.gov with attention for the effect of OC on NK cell function, the effect of current treatment on NK cell biology and the evidence on the therapeutic value of NK cell therapy against OC.

Results: In six clinical trials only 31 OC patients have been reported that received NK cell adoptive transfer. The majority of patients reached stable disease after NK cell therapy, with a mild pattern of side effects. In patients who received repeated infusions, more complete responses are described. All reported studies investigated the intravenous infusion of NK cells. Whereas the studies that are currently recruiting, investigate intraperitoneal infusion of allogeneic NK cells.

Conclusion: In this review the pre-clinical evidence and current trials on NK cell immunotherapy in OC patients are summarized. Furthermore, challenges that have to be overcome for NK cell adoptive therapy to have a significant impact on disease outcome are discussed.
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http://dx.doi.org/10.1016/j.ygyno.2020.03.020DOI Listing
June 2020

Mutational spectrum and dynamics of clonal hematopoiesis in anemia of older individuals.

Blood 2020 04;135(14):1161-1170

Department of Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Anemia is a major and currently poorly understood clinical manifestation of hematopoietic aging. Upon aging, hematopoietic clones harboring acquired leukemia-associated mutations expand and become detectable, now referred to as clonal hematopoiesis (CH). To investigate the relationship between CH and anemia of the elderly, we explored the landscape and dynamics of CH in older individuals with anemia. From the prospective, population-based Lifelines cohort (n = 167 729), we selected all individuals at least 60 years old who have anemia according to World Health Organization criteria (n = 676) and 1:1 matched control participants. Peripheral blood of 1298 individuals was analyzed for acquired mutations at a variant allele frequency (VAF) of 1% or higher in 27 driver genes. To track clonal evolution over time, we included all available follow-up samples (n = 943). CH was more frequently detected in individuals with anemia (46.6%) compared with control individuals (39.1%; P = .007). Although no differences were observed regarding commonly detected DTA mutations (DNMT3A, TET2, ASXL1) in individuals with anemia compared with control individuals, other mutations were enriched in the anemia cohort, including TP53 and SF3B1. Unlike individuals with nutrient deficiency (P = .84), individuals with anemia of chronic inflammation and unexplained anemia revealed a higher prevalence of CH (P = .035 and P = .017, respectively) compared with their matched control individuals. Follow-up analyses revealed that clones may expand and decline, generally showing only a subtle increase in VAF (mean, 0.56%) over the course of 44 months, irrespective of the presence of anemia. Specific mutations were associated with different growth rates and propensities to acquire an additional hit. In contrast to smaller clones (<5% VAF), which did not affect overall survival, larger clones were associated with increased risk for death.
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http://dx.doi.org/10.1182/blood.2019004362DOI Listing
April 2020

Impact of NPM1/FLT3-ITD genotypes defined by the 2017 European LeukemiaNet in patients with acute myeloid leukemia.

Blood 2020 01;135(5):371-380

Department of Internal Medicine I, University Hospital Carl Gustav Carus, Dresden, Germany.

Patients with acute myeloid leukemia (AML) harboring FLT3 internal tandem duplications (ITDs) have poor outcomes, in particular AML with a high (≥0.5) mutant/wild-type allelic ratio (AR). The 2017 European LeukemiaNet (ELN) recommendations defined 4 distinct FLT3-ITD genotypes based on the ITD AR and the NPM1 mutational status. In this retrospective exploratory study, we investigated the prognostic and predictive impact of the NPM1/FLT3-ITD genotypes categorized according to the 2017 ELN risk groups in patients randomized within the RATIFY trial, which evaluated the addition of midostaurin to standard chemotherapy. The 4 NPM1/FLT3-ITD genotypes differed significantly with regard to clinical and concurrent genetic features. Complete ELN risk categorization could be done in 318 of 549 trial patients with FLT3-ITD AML. Significant factors for response after 1 or 2 induction cycles were ELN risk group and white blood cell (WBC) counts; treatment with midostaurin had no influence. Overall survival (OS) differed significantly among ELN risk groups, with estimated 5-year OS probabilities of 0.63, 0.43, and 0.33 for favorable-, intermediate-, and adverse-risk groups, respectively (P < .001). A multivariate Cox model for OS using allogeneic hematopoietic cell transplantation (HCT) in first complete remission as a time-dependent variable revealed treatment with midostaurin, allogeneic HCT, ELN favorable-risk group, and lower WBC counts as significant favorable factors. In this model, there was a consistent beneficial effect of midostaurin across ELN risk groups.
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http://dx.doi.org/10.1182/blood.2019002697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993016PMC
January 2020

Ring sideroblasts in AML are associated with adverse risk characteristics and have a distinct gene expression pattern.

Blood Adv 2019 10;3(20):3111-3122

Department of Hematology, Cancer Research Center Groningen, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Ring sideroblasts (RS) emerge as result of aberrant erythroid differentiation leading to excessive mitochondrial iron accumulation, a characteristic feature for myelodysplastic syndromes (MDS) with mutations in the spliceosome gene SF3B1. However, RS can also be observed in patients diagnosed with acute myeloid leukemia (AML). The objective of this study was to characterize RS in patients with AML. Clinically, RS-AML is enriched for ELN adverse risk (55%). In line with this finding, 35% of all cases had complex cytogenetic aberrancies, and TP53 was most recurrently mutated in this cohort (37%), followed by DNMT3A (26%), RUNX1 (25%), TET2 (20%), and ASXL1 (19%). In contrast to RS-MDS, the incidence of SF3B1 mutations was low (8%). Whole-exome sequencing and SNP array analysis on a subset of patients did not uncover a single genetic defect underlying the RS phenotype. Shared genetic defects between erythroblasts and total mononuclear cell fraction indicate common ancestry for the erythroid lineage and the myeloid blast cells in patients with RS-AML. RNA sequencing analysis on CD34+ AML cells revealed differential gene expression between RS-AML and non RS-AML cases, including genes involved in megakaryocyte and erythroid differentiation. Furthermore, several heme metabolism-related genes were found to be upregulated in RS- CD34+ AML cells, as was observed in SF3B1mut MDS. These results demonstrate that although the genetic background of RS-AML differs from that of RS-MDS, they have certain downstream effector pathways in common.
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http://dx.doi.org/10.1182/bloodadvances.2019000518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849935PMC
October 2019

Intraperitoneal infusion of ex vivo-cultured allogeneic NK cells in recurrent ovarian carcinoma patients (a phase I study).

Medicine (Baltimore) 2019 Feb;98(5):e14290

Department of Laboratory Medicine, Laboratory of Hematology, Radboud Institute of Molecular Life Sciences, Nijmegen, The Netherlands.

Introduction: Recurrent ovarian carcinoma has dismal prognosis, but control of disease and prolonged survival are possible in some patients. The estimated 5-year survival is 46% for all stages of ovarian cancer, and only 28% for metastasized disease. Notably, the majority of women with ovarian cancer are diagnosed with stage III or IV disease with a high recurrence rate. As most women with relapsed or metastatic cancer will die of progressive disease, there is an urgent need for novel therapeutic strategies. The primary aim of our study is to evaluate safety and toxicity of intraperitoneal infusion of ex vivo-expanded natural killer cells (NK), generated from CD34+ umbilical cord blood (UCB) progenitor cells, with and without a preceding non-myeloablative immunosuppressive conditioning regimen in patients suffering from recurrent ovarian cancer. The secondary objectives are to compare the in vivo lifespan, expansion, and biological activity of intraperitoneally infused NK cell products with or without preparative chemotherapy, as well as evaluate effects on disease load.

Methods: In this phase I safety trial, 12 patients who are suffering from recurrent ovarian cancer, detected by a significant rise in serum level of CA-125 on two successive time points, will be included. Prior to UCB-NK cell infusion, a laparoscopy is performed to place a catheter in the peritoneal cavity. The first cohort of three patients will receive a single intraperitoneal infusion of 1.5-3×10 UCB-NK cells, generated ex vivo from CD34+ hematopoietic progenitor cells obtained from an allogeneic UCB unit, without a preparative chemotherapy regimen. The second group of three patients will be treated with a similar dose of UCB-NK cells following a preparative four days non-myeloablative immunosuppressive conditioning regimen with cyclophosphamide and fludarabine (Cy/Flu). If no severe toxicity is seen in these 6 patients, an extension cohort of 6 patients will be included to answer the secondary objectives.

Discussion: This study investigates the safety of a promising new cellular therapy in a group of patients with a poor prognosis. Demonstration of safety and in vivo expansion capacity of allogeneic UCB-NK cells in the absence of Cy/Flu pretreatment will provide rationale for UCB-NK cell infusion after regular second-line chemotherapy.
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http://dx.doi.org/10.1097/MD.0000000000014290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380776PMC
February 2019
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