Publications by authors named "Anthony D Ho"

356 Publications

Characteristics and outcome of patients with acute myeloid leukaemia and t(8;16)(p11;p13): results from an International Collaborative Study.

Br J Haematol 2021 Mar 2;192(5):832-842. Epub 2021 Feb 2.

Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.

In acute myeloid leukaemia (AML) t(8;16)(p11;p13)/MYST3-CREBBP is a very rare abnormality. Previous small series suggested poor outcome. We report on 59 patients with t(8;16) within an international, collaborative study. Median age was 52 (range: 16-75) years. AML was de novo in 58%, therapy-related (t-AML) in 37% and secondary after myelodysplastic syndrome (s-AML) in 5%. Cytogenetics revealed a complex karyotype in 43%. Besides MYST3-CREBBP, whole-genome sequencing on a subset of 10 patients revealed recurrent mutations in ASXL1, BRD3, FLT3, MLH1, POLG, TP53, SAMD4B (n = 3, each), EYS, KRTAP9-1 SPTBN5 (n = 4, each), RUNX1 and TET2 (n = 2, each). Complete remission after intensive chemotherapy was achieved in 84%. Median follow-up was 5·48 years; five-year survival rate was 17%. Patients with s-/t-AML (P = 0·01) and those with complex karyotype (P = 0·04) had an inferior prognosis. Allogeneic haematopoietic cell transplantation (allo-HCT) was performed in 21 (36%) patients, including 15 in first complete remission (CR1). Allo-HCT in CR1 significantly improved survival (P = 0·04); multivariable analysis revealed that allo-HCT in CR1 was effective in de novo AML but not in patients with s-AML/t-AML and less in patients exhibiting a complex karyotype. In summary, outcomes of patients with t(8;16) are dismal with chemotherapy, and may be substantially improved with allo-HCT performed in CR1.
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http://dx.doi.org/10.1111/bjh.17336DOI Listing
March 2021

Analysis of nonleukemic cellular subcompartments reconstructs clonal evolution of acute myeloid leukemia and identifies therapy-resistant preleukemic clones.

Int J Cancer 2021 Jan 7. Epub 2021 Jan 7.

Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany.

To acquire a better understanding of clonal evolution of acute myeloid leukemia (AML) and to identify the clone(s) responsible for disease recurrence, we have comparatively studied leukemia-specific mutations by whole-exome-sequencing (WES) of both the leukemia and the nonleukemia compartments derived from the bone marrow of AML patients. The T-lymphocytes, B-lymphocytes and the functionally normal hematopoietic stem cells (HSC), that is, CD34 /CD38 /ALDH cells for AML with rare-ALDH blasts (<1.9% ALDH cells) were defined as the nonleukemia compartments. WES identified 62 point-mutations in the leukemia compartment derived from 12 AML-patients at the time of diagnosis and 73 mutations in 3 matched relapse cases. Most patients (8/12) showed 4 to 6 point-mutations per sample at diagnosis. Other than the mutations in the recurrently mutated genes such as DNMT3A, NRAS and KIT, we were able to identify novel point-mutations that have not yet been described in AML. Some leukemia-specific mutations and cytogenetic abnormalities including DNMT3A(R882H), EZH2(I146T) and inversion(16) were also detectable in the respective T-lymphocytes, B-lymphocytes and HSC in 5/12 patients, suggesting that preleukemia HSC might represent the source of leukemogenesis for these cases. The leukemic evolution was reconstructed for five cases with detectable preleukemia clones, which were tracked in follow-up and relapse samples. Four of the five patients with detectable preleukemic mutations developed relapse. The presence of leukemia-specific mutations in these nonleukemia compartments, especially after chemotherapy or after allogeneic stem cell transplantation, is highly relevant, as these could be responsible for relapse. This discovery may facilitate the identification of novel targets for long-term cure.
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http://dx.doi.org/10.1002/ijc.33461DOI Listing
January 2021

Glycogen accumulation, central carbon metabolism, and aging of hematopoietic stem and progenitor cells.

Sci Rep 2020 07 14;10(1):11597. Epub 2020 Jul 14.

Department of Medicine V, Heidelberg University, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany.

Inspired by recent proteomic data demonstrating the upregulation of carbon and glycogen metabolism in aging human hematopoietic stem and progenitor cells (HPCs, CD34+ cells), this report addresses whether this is caused by elevated glycolysis of the HPCs on a per cell basis, or by a subpopulation that has become more glycolytic. The average glycogen content in individual CD34+ cells from older subjects (> 50 years) was 3.5 times higher and more heterogeneous compared to younger subjects (< 35 years). Representative glycolytic enzyme activities in HPCs confirmed a significant increase in glycolysis in older subjects. The HPCs from older subjects can be fractionated into three distinct subsets with high, intermediate, and low glucose uptake (GU) capacity, while the subset with a high GU capacity could scarcely be detected in younger subjects. Thus, we conclude that upregulated glycolysis in aging HPCs is caused by the expansion of a more glycolytic HPC subset. Since single-cell RNA analysis has also demonstrated that this subpopulation is linked to myeloid differentiation and increased proliferation, isolation and mechanistic characterization of this subpopulation can be utilized to elucidate specific targets for therapeutic interventions to restore the lineage balance of aging HPCs.
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http://dx.doi.org/10.1038/s41598-020-68396-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360735PMC
July 2020

Quantitative proteomics reveals specific metabolic features of acute myeloid leukemia stem cells.

Blood 2020 Sep;136(13):1507-1519

Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.

Acute myeloid leukemia is characterized by the accumulation of clonal myeloid blast cells unable to differentiate into mature leukocytes. Chemotherapy induces remission in the majority of patients, but relapse rates are high and lead to poor clinical outcomes. Because this is primarily caused by chemotherapy-resistant leukemic stem cells (LSCs), it is essential to eradicate LSCs to improve patient survival. LSCs have predominantly been studied at the transcript level, thus information about posttranscriptionally regulated genes and associated networks is lacking. Here, we extend our previous report on LSC proteomes to healthy age-matched hematopoietic stem and progenitor cells (HSPCs) and correlate the proteomes to the corresponding transcriptomes. By comparing LSCs to leukemic blasts and healthy HSPCs, we validate candidate LSC markers and highlight novel and potentially targetable proteins that are absent or only lowly expressed in HSPCs. In addition, our data provide strong evidence that LSCs harbor a characteristic energy metabolism, adhesion molecule composition, as well as RNA-processing properties. Furthermore, correlating proteome and transcript data of the same individual samples highlights the strength of proteome analyses, which are particularly potent in detecting alterations in metabolic pathways. In summary, our study provides a comprehensive proteomic and transcriptomic characterization of functionally validated LSCs, blasts, and healthy HSPCs, representing a valuable resource helping to design LSC-directed therapies.
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http://dx.doi.org/10.1182/blood.2019003654DOI Listing
September 2020

Feasibility and Safety of CD19 Chimeric Antigen Receptor T Cell Treatment for B Cell Lymphoma Relapse after Allogeneic Hematopoietic Stem Cell Transplantation.

Biol Blood Marrow Transplant 2020 09 15;26(9):1575-1580. Epub 2020 May 15.

Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany; German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ)/National Centre for Tumor Diseases (NCT), Heidelberg, Germany.

Although CD19-directed chimeric antigen receptor (CAR) T cells have been successfully used after a preceding allogeneic stem cell transplant (alloHCT) in patients with acute lymphoblastic leukemia, little is known about the feasibility and outcome of CAR T cell treatment in patients who have been previously allotransplanted for lymphoma. In a single-center retrospective analysis, course and outcome of all allografted patients treated with CD19 CAR constructs for B cell lymphoma between October 2018 and November 2019 were studied. CAR therapy consisted either of a third-generation CAR (HD-CAR-1) or of commercially manufactured axicabtagene ciloleucel (axi-cel; Gilead, Santa Monica, U.S.). Altogether, 10 CAR T cell dosings using recipient leukapheresis products were performed in 8 patients: 4 patients (2 mantle cell lymphoma, 2 chronic lymphocytic leukemia) received 6 dosings with HD-CAR-1 and 4 patients (all with diffuse large B cell lymphoma) received 4 dosings with axi-cel. Overall, 6 of 8 patients (75%) responded. CAR treatment was well tolerated with grade ≥ 3 cytokine release syndrome and neurotoxicity each being observed after 1 of 10 dosings. A single patient had moderate chronic graft-versus-host disease. Of note, 3 of 4 patients who received axi-cel had ongoing grade ≥ 3 cytopenia 3 months postdosing, whereas prolonged cytopenia was not observed in 9 alloHCT-naive patients who received axi-cel during the same time period. In conclusion, CAR T cell treatment from recipient-derived leukapheresis products after a prior alloHCT appears to be feasible, effective, and safe in patients with B cell lymphoma. Protracted cytopenia after axi-cel treatment is a matter of concern and requires further exploration.
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http://dx.doi.org/10.1016/j.bbmt.2020.04.025DOI Listing
September 2020

Nek2 kinase displaces distal appendages from the mother centriole prior to mitosis.

J Cell Biol 2020 03;219(3)

Centre for Organismal Studies, University of Heidelberg, Heidelberg, Germany.

Distal appendages (DAs) of the mother centriole are essential for the initial steps of ciliogenesis in G1/G0 phase of the cell cycle. DAs are released from centrosomes in mitosis by an undefined mechanism. Here, we show that specific DAs lose their centrosomal localization at the G2/M transition in a manner that relies upon Nek2 kinase activity to ensure low DA levels at mitotic centrosomes. Overexpression of active Nek2A, but not kinase-dead Nek2A, prematurely displaced DAs from the interphase centrosomes of immortalized retina pigment epithelial (RPE1) cells. This dramatic impact was also observed in mammary epithelial cells with constitutively high levels of Nek2. Conversely, Nek2 knockout led to incomplete dissociation of DAs and cilia in mitosis. As a consequence, we observed the presence of a cilia remnant that promoted the asymmetric inheritance of ciliary signaling components and supported cilium reassembly after cell division. Together, our data establish Nek2 as an important kinase that regulates DAs before mitosis.
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http://dx.doi.org/10.1083/jcb.201907136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055001PMC
March 2020

New Class of Crosslinker-Free Nanofiber Biomaterials from Hydra Nematocyst Proteins.

Sci Rep 2019 12 13;9(1):19116. Epub 2019 Dec 13.

Physical Chemistry of Biosystems, Institute of Physical Chemistry, Heidelberg University, 69120, Heidelberg, Germany.

Nematocysts, the stinging organelles of cnidarians, have remarkable mechanical properties. Hydra nematocyst capsules undergo volume changes of 50% during their explosive exocytosis and withstand osmotic pressures of beyond 100 bar. Recently, two novel protein components building up the nematocyst capsule wall in Hydra were identified. The cnidarian proline-rich protein 1 (CPP-1) characterized by a "rigid" polyproline motif and the elastic Cnidoin possessing a silk-like domain were shown to be part of the capsule structure via short cysteine-rich domains that spontaneously crosslink the proteins via disulfide bonds. In this study, recombinant Cnidoin and CPP-1 are expressed in E. coli and the elastic modulus of spontaneously crosslinked bulk proteins is compared with that of isolated nematocysts. For the fabrication of uniform protein nanofibers by electrospinning, the preparative conditions are systematically optimized. Both fibers remain stable even after rigorous washing and immersion into bulk water owing to the simultaneous crosslinking of cysteine-rich domains. This makes our nanofibers clearly different from other protein nanofibers that are not stable without chemical crosslinkers. Following the quantitative assessment of mechanical properties, the potential of Cnidoin and CPP-1 nanofibers is examined towards the maintenance of human mesenchymal stem cells.
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http://dx.doi.org/10.1038/s41598-019-55655-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910907PMC
December 2019

Patients With Malignant Lymphoma and HIV Infection Experiencing Remission After First-Line Treatment Have an Excellent Prognosis.

Clin Lymphoma Myeloma Leuk 2019 10 30;19(10):e581-e587. Epub 2019 May 30.

Department of Hematology, Oncology and Rheumatology, Heidelberg University, Heidelberg, Germany.

Background: Malignant lymphoma is still the leading cause of death among AIDS-related diseases.

Patients And Methods: We performed a retrospective analysis of 50 HIV-positive lymphoma patients. The median interval between HIV and malignant lymphoma diagnosis was 4 years. Eight patients (16%) had Hodgkin lymphoma and 42 (84%) non-Hodgkin lymphoma. Among non-Hodgkin lymphoma patients, diffuse large B-cell lymphoma (n = 18, 42%), Burkitt lymphoma (n = 11, 26%), and plasmoblastic lymphoma (n = 5, 12%) were the most frequent entities.

Results: Lymphoma was treated according to standard protocols. Forty-four patients (88%) received combination antiretroviral therapy, 2 (4%) were not treated, and in 4 (8%) the HIV treatment status was not clarified. Response to first-line therapy was complete response (CR) in 24 (56%), partial response (PR) in 15 (35%), and stable disease in 1 (2%). Three patients (7%) developed progressive disease, and 9 (18%) experienced relapse after CR or PR. At a median observation period of 31 (range, 0.4-192) months, the 1-, 2-, and 5-year overall survival was 87%, 79%, and 76%, respectively. At univariate analysis, remission status after first-line treatment was predictive of outcome, as the 2-year overall survival was 95%, 66%, and 0 for patients with CR, with PR, and with progressive disease (P < .001). Results of the multivariate analysis revealed lactate dehydrogenase concentration at lymphoma diagnosis (P = .046) and relapse (P = .050) to be independent factors for overall survival.

Conclusion: First-line treatment of lymphoma in HIV positive patients is crucial. Patients who experienced and maintained a first CR had a favorable prognosis.
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http://dx.doi.org/10.1016/j.clml.2019.05.019DOI Listing
October 2019

Evolution of Peripheral Blood Stem Cell Transplantation.

Authors:
Anthony D Ho

Methods Mol Biol 2019 ;2017:1-10

Department of Medicine V (Hematology, Oncology, Rheumatology), Heidelberg University, Heidelberg, Germany.

Blood-derived progenitors have become the predominant source of hematopoietic stem cells for clinical transplantation. The main advantages compared to the bone marrow are as follows: harvesting blood stem cells is less painful for the donor, utilizes much less resources such as operating theater time and general anesthesia, and, above all, is associated with significantly accelerated reconstitution. The latter has ultimately improved patient safety as a consequence of significantly shortened aplastic phase and hence reduced morbidity and mortality after transplantation. Basic and translational research efforts in the 1960s to the mid-1980s have made the first blood stem cell transplantation in Heidelberg in 1985 possible. Diverse groups around the world have contributed to incremental knowledge that culminated in the first successful attempts in blood stem cell transplantation. These efforts have spawned modern research into stem cell biology and the immune modulatory effects of allogeneic transplantations.
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http://dx.doi.org/10.1007/978-1-4939-9574-5_1DOI Listing
March 2020

Treatment of patients with relapsed or refractory CD19+ lymphoid disease with T lymphocytes transduced by RV-SFG.CD19.CD28.4-1BBzeta retroviral vector: a unicentre phase I/II clinical trial protocol.

BMJ Open 2019 05 19;9(5):e026644. Epub 2019 May 19.

Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg, Germany.

Introduction: Chimeric antigen receptor (CAR) T cells spark hope for patients with CD19+ B cell neoplasia, including relapsed or refractory (r/r) acute lymphoblastic leukaemia (ALL) or r/r non-Hodgkin's lymphoma (NHL). Published studies have mostly used second-generation CARs with 4-1BB or CD28 as costimulatory domains. Preclinical results of third-generation CARs incorporating both elements have shown superiority concerning longevity and proliferation. The University Hospital of Heidelberg is the first institution to run an investigator-initiated trial (IIT) CAR T cell trial (Heidelberg Chimeric Antigen Receptor T cell Trial number 1 [HD-CAR-1]) in Germany with third-generation CD19-directed CAR T cells.

Methods And Analysis: Adult patients with r/r ALL (stratum I), r/r NHL including chronic lymphocytic leukaemia, diffuse large B-cell lymphoma, follicular lymphoma or mantle cell lymphoma (stratum II) as well as paediatric patients with r/r ALL (stratum III) will be treated with autologous T-lymphocytes transduced by third-generation RV-SFG.CD19.CD28.4-1BB zeta retroviral vector (CD19.CAR T cells). The main purpose of this study is to evaluate safety and feasibility of escalating CD19.CAR T cell doses (1-20×10 transduced cells/m) after lymphodepletion with fludarabine (flu) and cyclophosphamide (cyc). Patients will be monitored for cytokine release syndrome (CRS), neurotoxicity, i.e. CAR-T-cell-related encephalopathy syndrome (CRES) and/or other toxicities (primary objectives). Secondary objectives include evaluation of function and survival of CD19.CAR T cells and assessment of CD19.CAR T cell antitumour efficacy.HD-CAR-1 as a prospective, monocentric trial aims to make CAR T cell therapy accessible to patients in Europe. Currently, HD-CAR-1 is the first and only CAR T cell IIT in Germany. A third-generation Good Manufacturing Practice (GMP) grade retroviral vector, a broad spectrum of NHL, treatment of paediatric and adult ALL patients and inclusion of patients even after allogeneic stem cell transplantation (alloSCT) make this trial unique.

Ethics And Dissemination: Ethical approval and approvals from the local and federal competent authorities were granted. Trial results will be reported via peer-reviewed journals and presented at conferences and scientific meetings.

Trial Registration Number: Eudra CT 2016-004808-60; NCT03676504; Pre-results.
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http://dx.doi.org/10.1136/bmjopen-2018-026644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530404PMC
May 2019

Allogeneic hematopoietic cell transplantation improves outcome of adults with t(6;9) acute myeloid leukemia: results from an international collaborative study.

Haematologica 2020 01 19;105(1):161-169. Epub 2019 Apr 19.

Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.

Acute myeloid leukemia (AML) with t(6;9)(p22;q34) is a distinct entity accounting for 1-2% of AML cases. A substantial proportion of these patients have a concomitant -ITD. While outcomes are dismal with intensive chemotherapy, limited evidence suggests allogeneic hematopoietic cell transplantation (allo-HCT) may improve survival if performed early during first complete remission. We report on a cohort of 178 patients with t(6;9)(p22;q34) within an international, multicenter collaboration. Median age was 46 years (range: 16-76), AML was in 88%, -ITD was present in 62%, and additional cytogenetic abnormalities in 21%. Complete remission was achieved in 81% (n=144), including 14 patients who received high-dose cytarabine after initial induction failure. With a median follow up of 5.43 years, estimated overall survival at five years was 38% (95%CI: 31-47%). Allo-HCT was performed in 117 (66%) patients, including 89 in first complete remission. Allo-HCT in first complete remission was associated with higher 5-year relapse-free and overall survival as compared to consolidation chemotherapy: 45% (95%CI: 35-59%) and 53% (95%CI: 42-66%) 7% (95%CI: 3-19%) and 23% (95%CI: 13-38%), respectively. For patients undergoing allo-HCT, there was no difference in overall survival rates at five years according to whether it was performed in first [53% (95%CI: 42-66%)], or second [58% (95%CI: 31-100%); n=10] complete remission or with active disease/relapse [54% (95%CI: 34-84%); n=18] (=0.67). Neither -ITD nor additional chromosomal abnormalities impacted survival. In conclusion, outcomes of t(6;9)(p22;q34) AML are poor with chemotherapy, and can be substantially improved with allo-HCT.
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http://dx.doi.org/10.3324/haematol.2018.208678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939530PMC
January 2020

Allogeneic transplantation in high-risk chronic lymphocytic leukemia: a single-center, intent-to-treat analysis.

Haematologica 2019 07 10;104(7):e304-e306. Epub 2019 Jan 10.

Department of Internal Medicine V, University Hospital of Heidelberg, Heidelberg.

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http://dx.doi.org/10.3324/haematol.2018.209486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601102PMC
July 2019

Modulation of B Cells and Homing Marker on NK Cells Through Extracorporeal Photopheresis in Patients With Steroid-Refractory/Resistant Graft-Vs.-Host Disease Without Hampering Anti-viral/Anti-leukemic Effects.

Front Immunol 2018 8;9:2207. Epub 2018 Oct 8.

Department of Internal Medicine V, University Clinic Heidelberg, Heidelberg, Germany.

Graft-vs.-host disease (GvHD), a severe complication of allogeneic hematopoietic stem cell transplantation, significantly affects the post-transplant morbidity and mortality. Systemic steroids remain the gold standard for the initial management of GvHD. However, up to 60% of patients will not sufficiently respond to steroids. Extracorporeal photopheresis (ECP), a cell-based immunotherapy, has shown good clinical results in such steroid-refractory/resistant GvHD patients. Given its immunomodulatory, but not global immunosuppressive and steroid-sparing capacity, ECP constitutes an attractive option. In the case of GvHD, the balance of immune cells is destroyed: effector cells are not any longer efficiently controlled by regulatory cells. ECP therapy may restore this balance. However, the precise mechanism and the impact of ECP on anti-viral/anti-leukemic function remain unclear. In this study, 839 ECP treatments were performed on patients with acute GvHD (aGvHD) and chronic GvHD (cGvHD). A comprehensive analysis of effector and regulatory cells in patients under ECP therapy included multi-parametric flow cytometry and tetramer staining, Luminex-based cytokine, interferon-γ enzyme-linked immunospot, and chromium-51 release assays. Gene profiling of myeloid-derived suppressor cells (MDSCs) was performed by microarray analysis. Immunologically, modulations of effector and regulatory cells as well as proinflammatory cytokines were observed under ECP treatment: (1) GvHD-relevant cell subsets like CD62L NK cells and newly defined CD19CD20 B cells were modulated, but (2) quantity and quality of anti-viral/anti-leukemic effector cells were preserved. (3) The development of MDSCs was promoted and switched from an inactivated subset (CD33CD11b) to an activated subset (CD33CD11b). (4) The frequency of Foxp3CD4 regulatory T cells (Tregs) and CD24CD38 regulatory B cells was considerably increased in aGvHD patients, and Foxp3CD8 Tregs in cGvHD patients. (5) Proinflammatory cytokines like IL-1β, IL-6, IL-8, and TNF-α were significantly reduced. In summary, ECP constitutes an effective immunomodulatory therapy for patients with steroid-refractory/resistant GvHD without impairment of anti-viral/leukemia effects.
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http://dx.doi.org/10.3389/fimmu.2018.02207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186805PMC
September 2019

Cell-specific proteome analyses of human bone marrow reveal molecular features of age-dependent functional decline.

Nat Commun 2018 10 1;9(1):4004. Epub 2018 Oct 1.

Molecular Medicine Partnership Unit (MMPU), Meyerhofstrasse 1, Heidelberg, D69117, Germany.

Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms remain elusive. Here, we present proteome-wide atlases of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) and five other cell populations that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified is assessed in 59 human subjects from different ages. As the HPCs become older, pathways in central carbon metabolism exhibit features reminiscent of the Warburg effect, where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation. Ageing causes alterations in the bone marrow niche too, and diminishes the functionality of the pathways involved in HPC homing. The data represent a valuable resource for further analyses, and for validation of knowledge gained from animal models.
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http://dx.doi.org/10.1038/s41467-018-06353-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167374PMC
October 2018

CD38 as Immunotherapeutic Target in Light Chain Amyloidosis and Multiple Myeloma-Association With Molecular Entities, Risk, Survival, and Mechanisms of Upfront Resistance.

Front Immunol 2018 20;9:1676. Epub 2018 Jul 20.

Medizinische Klinik V, Universitätsklinikum Heidelberg, Heidelberg, Germany.

Monoclonal antibodies against the cell surface antigen CD38, e.g., isatuximab, daratumumab, or Mor202, have entered the therapeutic armamentarium in multiple myeloma due to single agent overall response rates of 29 vs. 36 vs. 31%, effectivity in combination regimen, e.g., with lenalidomide or bortezomib plus dexamethasone, and tolerable side effects. Despite clinical use, many questions remain. In this manuscript, we address three of these: first, upfront CD38 target-expression in AL-amyloidosis, monoclonal gammopathy of unknown significance (MGUS), asymptomatic, symptomatic, and relapsed multiple myeloma. Second, relation of CD38-expression to survival, disease stages, molecular entities, and high-risk definitions. Third, alternative splicing or lack of CD38-expression as potential mechanisms of upfront resistance. We assessed CD138-purified plasma cell samples from 196 AL-amyloidosis, 62 MGUS, 259 asymptomatic, 764 symptomatic, and 90 relapsed myeloma patients, including longitudinal pairs of asymptomatic/symptomatic ( = 34) and symptomatic/relapsed myeloma ( = 57) regarding interphase fluorescence hybridization ( = 1,380), CD38-expression by gene expression profiling ( = 1,371), RNA-sequencing ( = 593), and flow cytometry ( = 800). Samples of normal bone marrow plasma cells ( = 10), memory B-cells ( = 9), polyclonal plasmablastic cells ( = 9), and human myeloma cell lines ( = 54) were used as comparators. CD38 was expressed in all malignant plasma cell samples, but significantly lower compared to normal plasma cells with small but significant downregulation in longitudinal sample pairs. Higher CD38 expression was associated with the presence of t(4;14) and high-risk according to the UAMS70-gene score, lower expression was associated with del17p13 and hyperdiploidy in symptomatic myeloma as well as t(11;14) in asymptomatic myeloma. Higher CD38-expression was associated with slower progression to symptomatic and relapsed myeloma and better overall survival in the latter two entities. CD38 expression, t(4;14), del17p13, and gain of 1q21 are independently prognostic in multivariate analysis. By contrast, high CD38-expression is associated with adverse survival in AL-amyloidosis. Regarding mechanisms of upfront anti-CD38-treatment resistance, lack of CD38-expression and alternative splicing of receptor binding-sites could be excluded. Here, of the two protein coding CD38-transcripts CD38-001 (eight-exon, full length) and CD38-005 (truncated), CD38-001 conveyed >97% of reads spanning the respective CD38 splice junction.
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http://dx.doi.org/10.3389/fimmu.2018.01676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062598PMC
July 2018

Author Correction: BCAT1 restricts αKG levels in AML stem cells leading to IDH-like DNA hypermethylation.

Nature 2018 08;560(7718):E28

Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), 69120, Heidelberg, Germany.

In Extended Data Fig. 1a of this Letter, the flow cytometry plot depicting the surface phenotype of AML sample DD08 was a duplicate of the plot for AML sample DD06. Supplementary Data 4 has been added to the Supplementary Information of the original Letter to clarify the proteome data acquisition and presentation. The original Letter has been corrected online.
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http://dx.doi.org/10.1038/s41586-018-0403-9DOI Listing
August 2018

Selective contrast-enhanced computed tomography is appropriate in diffuse large B-cell lymphoma therapy response assessment.

Eur J Haematol 2018 Jul 24. Epub 2018 Jul 24.

Department of Hematology, Oncology and Rheumatology, Heidelberg University, Heidelberg, Germany.

Objective: Although not the gold standard, contrast-enhanced CT of neck, thorax, and abdomen/pelvis is routinely performed in diagnosis and response assessment of DLBCL. PD during first-line treatment is a relatively rare event. The question arises if the imaging of initially involved regions only might be sufficient for response evaluation.

Method: We retrospectively analyzed the data of 167 DLBCL patients who had an extensive contrast-enhanced CT scan at first diagnosis. The majority of patients (n = 128, 77%) was treated with R-CHOP. Therapy response was assessed as interim and end of treatment staging by contrast-enhanced CT.

Results: The overall response rate at the end of treatment was 94%. None of the patients showed involvement of new sites at interim staging. As a major finding, none of the patients showed an involvement of sites, which were not initially involved. Four patients developed PD during first-line chemotherapy/after mid-treatment staging and 31 relapsed. A conclusive comparison between initial and PD/relapse DLBCL involvement was possible in 27 patients: 8 patients did and 19 patients did not show additional/new sites of involvement compared to first diagnosis.

Conclusion: Our retrospective analysis provides a rationale for selective imaging of initially involved DLBCL sites for therapy response assessment.
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http://dx.doi.org/10.1111/ejh.13150DOI Listing
July 2018

Simple Physical Model Unravels Influences of Chemokine on Shape Deformation and Migration of Human Hematopoietic Stem Cells.

Sci Rep 2018 Jul 13;8(1):10630. Epub 2018 Jul 13.

Center for Integrative Medicine and Physics, Institute for Advanced Studies, Kyoto University, 606-8501, Kyoto, Japan.

We studied the dynamic behavior of human hematopoietic stem cells (HSC) on the in vitro model of bone marrow surfaces in the absence and presence of chemokine (SDF1α). The deformation and migration of cells were investigated by varying the chemokine concentration and surface density of ligand molecules. Since HSC used in this study were primary cells extracted from the human umbilical cord blood, it is not possible to introduce molecular reporter systems before or during the live cell imaging. To account for the experimental observations, we propose a simple and general theoretical model for cell crawling. In contrast to other theoretical models reported previously, our model focuses on the nonlinear coupling between shape deformation and translational motion and is free from any molecular-level process. Therefore, it is ideally suited for the comparison with our experimental results. We have demonstrated that the results in the absence of SDF1α were well recapitulated by the linear model, while the nonlinear model is necessary to reproduce the elongated migration observed in the presence of SDF1α. The combination of the simple theoretical model and the label-free, live cell observations of human primary cells opens a large potential to numerically identify the differential effects of extrinsic factors such as chemokines, growth factors, and clinical drugs on dynamic phenotypes of primary cells.
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http://dx.doi.org/10.1038/s41598-018-28750-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045678PMC
July 2018

A prospective study on serum Cytokeratin (CK)-18 and CK18 fragments as biomarkers of acute hepato-intestinal GVHD.

Leukemia 2018 12 27;32(12):2685-2692. Epub 2018 Jun 27.

Department of Hematology and Oncology, University of Heidelberg, Heidelberg, Germany.

Apoptotic intestinal crypt cells are pathognomonic of acute intestinal graft versus host disease (GVHD). Serum levels of the apoptotic degradation product cytokeratin-18 fragments (CK18F) were associated with acute hepato-intestinal GVHD. Here we present a prospective clinical observational trial (NCT00935324) investigating serum levels of total CK18 (tCK18) and apoptotic CK18F to predict imminent acute hepato-intestinal GVHD and response to treatment. Total (t)CK18 and CK18F kinetics were measured before transplantation and in weekly intervals thereafter. In total 109 patients were enrolled. Acute hepato-intestinal GVHD grade I-IV was suspected in 36 patients (33%) at a median of 56 days post-transplant, 12 of these patients developed steroid-refractory GVHD. Both tCK18 and apoptotic CK18F increased at GVHD onset, and distinguished patients with suspected acute hepato-intestinal GVHD who were negative in intestinal histology. In patients with clinical acute hepato-intestinal GVHD, tCK18 significantly raised already 7-14 days before symptom onset. In receiver operator characteristics, areas under the curve at GVHD onset were 0.927 (p < 0.001) for tCK18 and 0.875 (p < 0.001) for apoptotic CK18F for patients with proven hepato-intestinal acute GVHD. This prospective study validates CK18F and highlights tCK18 as specific biomarkers suitable for improving prediction and diagnosis of suspected imminent and clinically manifest acute hepato-intestinal GVHD.
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http://dx.doi.org/10.1038/s41375-018-0183-0DOI Listing
December 2018

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

Blood 2018 09 21;132(12):1225-1240. Epub 2018 Jun 21.

Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.

, , and are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34 cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators and aberrantly spliced target genes of and mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.
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http://dx.doi.org/10.1182/blood-2018-04-843771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172604PMC
September 2018

Distinct Activities of Glycolytic Enzymes Identify Chronic Lymphocytic Leukemia Patients with a more Aggressive Course and Resistance to Chemo-Immunotherapy.

EBioMedicine 2018 Jun 5;32:125-133. Epub 2018 Jun 5.

Department of Medicine V, University Hospital Heidelberg, Heidelberg, Germany; National Center for Tumor Diseases (NCT), German Cancer Consortium (DKTK), Heidelberg, Germany. Electronic address:

A higher capacity to grow under hypoxic conditions can lead to a more aggressive behavior of tumor cells. Determining tumor activity under hypoxia may identify chronic lymphocytic leukemia (CLL) with aggressive clinical course and predict response to chemo-immunotherapy (CIT). A metabolic score was generated by determining pyruvate kinase and lactate dehydrogenase, key enzymes of glycolysis, ex vivo in primary CLL samples under normoxic and hypoxic conditions. This score was further correlated with clinical endpoints and response to CIT in 96 CLL patients. 45 patients were classified as metabolic high risk (HR), 51 as low risk (LR). Treatment-free survival (TFS) was significantly shorter in HR patients (median 394 vs 723 days, p = .021). 15 HR patients and 14 LR patients received CIT after sample acquisition. HR patients had a significantly shorter progression-free survival after treatment compared to LR patients (median 216 days vs not reached, p = .008). Multivariate analysis evaluating age, IGHV, TP53 deletion or mutation and 11q22-23 deletion besides the capacity of tumor cells to grow under severe hypoxic conditions identified the metabolic profile as the strongest independent risk factor for shorter TFS (hazard ratio 2.37, p = .011). The metabolic risk can provide prognostic and predictive information complementary to genetic biomarkers and identify patients who might benefit from alternative treatment approaches.
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http://dx.doi.org/10.1016/j.ebiom.2018.05.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021262PMC
June 2018

CD7 is expressed on a subset of normal CD34-positive myeloid precursors.

Eur J Haematol 2018 Sep 4;101(3):318-325. Epub 2018 Jul 4.

Department of Hematology, Oncology and Rheumatology, University of Heidelberg, Heidelberg, Germany.

Objective: To improve monitoring of myeloid neoplasms by flow cytometry-based minimal residual disease (MRD) analysis, we analyzed the significance of leukemia-associated immunophenotype (LAIP) markers in 44 patients.

Methods: In a pilot study cohort, peripheral blood or bone marrow samples from 13 patients with myeloid neoplasms and one case of B lymphoblastic leukemia in complete hematologic remission after allogeneic bone marrow or stem cell transplantation were subjected to selection for leukemia-specific phenotypes by fluorescence-activated cell sorting using individual marker combinations, followed by PCR-based chimerism analysis.

Results: The feasibility of this method could be demonstrated, with selection being successful in 12 cases, including two cases where mixed chimerism was found exclusively in sorted cells. Interestingly, four specimens displayed full donor chimerism in cells expressing the presumably aberrant combination CD34 /CD7 . Further analyses, including assessment of an independent cohort of 25 patients not affected by neoplastic bone marrow infiltration, revealed that normal myeloid precursors usually include a population coexpressing CD34, CD13, CD33, and CD7.

Conclusion: We conclude that the combination CD34 /CD7 might not be suitable as an LAIP for MRD diagnostics and that a subset of normal myeloid precursors in the bone marrow expresses CD7.
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http://dx.doi.org/10.1111/ejh.13100DOI Listing
September 2018

Author Correction: Dynamic cellular phenotyping defines specific mobilization mechanisms of human hematopoietic stem and progenitor cells induced by SDF1α versus synthetic agents.

Sci Rep 2018 Apr 30;8(1):6996. Epub 2018 Apr 30.

Physical Chemistry of Biosystems, Institute of Physical Chemistry, Heidelberg University, 69120, Heidelberg, Germany.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-25253-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928094PMC
April 2018

Platelet Count before Peripheral Blood Stem Cell Mobilization Is Associated with the Need for Plerixafor But Not with the Collection Result.

Transfus Med Hemother 2018 Jan 4;45(1):24-31. Epub 2017 Oct 4.

Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden-Württemberg - Hesse, Mannheim, Germany.

Background: A low platelet count before mobilization has recurrently been identified as risk factor for poor mobilization.

Methods: To determine the relevance of this finding for peripheral blood stem cell (PBSC) mobilization, including pre-emptive or rescue plerixafor in the case of poor mobilization, we retrospectively analyzed all patients undergoing PBSC collection at our institution between January 2014 and December 2015 (n = 380).

Results: In total, 99% of the patients (377/380) successfully collected a minimum of 2 × 10 CD34+ cells/kg body weight sufficient for a single transplant. Rescue or pre-emptive plerixafor was administered to 11% of the patients (42/380). No correlations between the platelet count before mobilization and the number of peripheral blood CD34+ cells or the CD34+ cell collection result were detected in the entire population or the subgroups according to diagnosis (newly diagnosed multiple myeloma, relapsed multiple myeloma, lymphoma, amyloid light-chain amyloidosis, sarcoma, or germ cell tumor). However, patients requiring pre-emptive or rescue plerixafor had a significantly lower platelet count before mobilization (217/nl vs. 245/nl; p = 0.004).

Conclusion: With the current state of the art PBSC mobilization strategies, the platelet count before mobilization was not associated with the CD34+ cell collection result but was associated with the need for pre-emptive or rescue application of plerixafor.
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http://dx.doi.org/10.1159/000478911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836245PMC
January 2018

Quantification of number of CD38 sites on bone marrow plasma cells in patients with light chain amyloidosis and smoldering multiple myeloma.

Cytometry B Clin Cytom 2018 09 20;94(5):611-620. Epub 2018 Apr 20.

Department of Hematology, Oncology and Rheumatology, Heidelberg University, Heidelberg, Germany.

Background: Recent approaches in multiple myeloma (MM) treatment have targeted CD38. As antigen expression levels on plasma cells (PCs) were demonstrated to affect response to monoclonal antibody (mAb) treatment, a precise characterization of PC phenotype is warranted.

Methods: Anti-CD38 mAb (isatuximab) was tested for antibody-dependent cellular cytotoxicity (ADCC) in MM cell lines. Quantification of the number of sites (NOS) of CD38 on bone marrow PCs and other immune cells obtained from light chain (AL) amyloidosis (n = 46) and smoldering multiple myeloma (SMM) patients (n = 19) was performed with two different quantitative flow cytometry (QFCM) applications.

Results: ADCC activity of isatuximab was observed in cell lines with >100 × 10 CD38-NOS only. The average PC CD38-NOS was 153 ± 53 × 10 in AL amyloidosis and 138.7 ± 53 × 10 in SMM patients. Eight (17%) AL amyloidosis and 4 (21%) SMM patients showed a PC CD38-NOS level <100 × 10 . In four AL amyloidosis and two SMM patients <10% of PCs had a CD38-NOS ≥100 × 10 . The CD38-NOS identified on bone marrow lymphocytes, monocytes, and granulocytes was two log units below the CD38-NOS on PCs (P < 0.001). No significant differences in CD38-NOS expression levels on any of the analyzed PC subpopulations in AL amyloidosis and SMM patients were identified.

Conclusion: Levels of CD38 expression affect the isatuximab-mediated ADCC in vitro. As PCs of patients with AL amyloidosis and SMM do not homogenously express high CD38 our data provide a rationale for assessment of CD38-NOS in patients with PC disorders prior to anti-CD38 treatment. © 2018 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21636DOI Listing
September 2018

The impact of stem cell transplantation on the natural course of peripheral T-cell lymphoma: a real-world experience.

Ann Hematol 2018 Jul 16;97(7):1241-1250. Epub 2018 Mar 16.

Department of Medicine V, University of Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany.

The role of autologous stem cell transplantation (autoSCT) as consolidating treatment for peripheral T-cell lymphoma (PTCL) is unsettled. The aim of this analysis was to investigate the impact of autoSCT in the upfront setting by intent-to-treat and to study salvage strategies after relapse. Retrospective follow-up of all patients aged 18-70 years and treated at our institution for ALK-PTCL diagnosed between 2001 and 2014. Of 117 eligible patients, diagnosis was PTCL-NOS in 34, ALCL ALK- in 31, AITL in 28, and other PTCL in 24 patients. Disregarding 20 patients who received first-line treatment externally, upfront autoSCT was not intended in 34 due to comorbidity, higher age, low IPI, physician's decision or unknown reasons (nITT), while intent-to-transplant (ITT) was documented in 63 patients. ITT was not associated with significant benefits for 5-year progression-free survival (PFS) and overall survival (OS) with 46 and 23% in the ITT group vs. 42 and 25% in the nITT group, even after multivariate adjustment for confounders. Altogether, 91 of all 117 patients relapsed or progressed. Thirty-one patients managed to proceed to salvage allografting and achieved a 5-year OS of 52%. In contrast, all 7 patients receiving salvage autoSCT relapsed and died, and only 3 of the 51 patients not eligible for SCT salvage survived. In this study, a significant benefit of intending first-line autoSCT over non-transplant induction in patients with ALK-PTCL did not emerge. Most patients fail first-line treatment and have a poor outlook if salvage alloSCT cannot be performed.
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http://dx.doi.org/10.1007/s00277-018-3288-7DOI Listing
July 2018

Mathematical modeling of the impact of cytokine response of acute myeloid leukemia cells on patient prognosis.

Sci Rep 2018 02 12;8(1):2809. Epub 2018 Feb 12.

Institute of Applied Mathematics, Interdisciplinary Center of Scientific Computing and BIOQUANT Center, Heidelberg University, Im Neuenheimer Feld 205, 69120, Heidelberg, Germany.

Acute myeloid leukemia (AML) is a heterogeneous disease. One reason for the heterogeneity may originate from inter-individual differences in the responses of leukemic cells to endogenous cytokines. On the basis of mathematical modeling, computer simulations and patient data, we have provided evidence that cytokine-independent leukemic cell proliferation may be linked to early relapses and poor overall survival. Depending whether the model of cytokine-dependent or cytokine-independent leukemic cell proliferation fits to the clinical data, patients can be assigned to two groups that differ significantly with respect to overall survival. The modeling approach further enables us to identify parameter constellations that can explain unexpected responses of some patients to external cytokines such as blast crisis or remission without chemotherapy.
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http://dx.doi.org/10.1038/s41598-018-21115-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809606PMC
February 2018

Dynamic cellular phynotyping defines specific mobilization mechanisms of human hematopoietic stem and progenitor cells induced by SDF1α versus synthetic agents.

Sci Rep 2018 01 30;8(1):1841. Epub 2018 Jan 30.

Physical Chemistry of Biosystems, Institute of Physical Chemistry, Heidelberg University, 69120, Heidelberg, Germany.

Efficient mobilization of hematopoietic stem and progenitor cells (HSPC) is one of the most crucial issues for harvesting an adequate amount of peripheral HSPC for successful clinical transplantation. Applying well-defined surrogate models for the bone marrow niche, live cell imaging techniques, and novel tools in statistical physics, we have quantified the functionality of two mobilization agents that have been applied in the clinic, NOX-A12 and AMD3100 (plerixafor), as compared to a naturally occurring chemokine in the bone marrow, SDF1α. We found that NOX-A12, an L-enantiomeric RNA oligonucleotide to SDF1, significantly reduced the adhesion of HSPC to the niche surface mediated via the CXCR4-SDF1α axis, and stretched the migration trajectories of the HSPC. We found that the stretching of trajectories by NOX-A12 was more prominent than that by SDF1α. In contrast, plerixafor exhibited no detectable interference with adhesion and migration. We also found that the deformation of HSPC induced by SDF1α or plerixafor was also drastically suppressed in the presence of NOX-A12. This novel technology of quantitative assessment of "dynamic phenotypes" by physical tools has therefore enabled us to define different mechanisms of function for various extrinsic factors compared to naturally occurring chemokines.
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http://dx.doi.org/10.1038/s41598-018-19557-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789976PMC
January 2018

Differences in Expansion Potential of Naive Chimeric Antigen Receptor T Cells from Healthy Donors and Untreated Chronic Lymphocytic Leukemia Patients.

Front Immunol 2017 10;8:1956. Epub 2018 Jan 10.

Cellular Immunotherapy, GMP Core Facility, Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg, Germany.

Introduction: Therapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of naive (T) vs. effector (T) T cells, T cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the T/T ratio of CART cells.

Materials And Methods: CART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays.

Results: IL-7/IL-15 preferentially induced differentiation into T, stem cell memory (T: naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (T), CD56+ and CD4+ T regulatory (T) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CART cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CART cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CART cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CART cells in untreated CLL patients. Final T/T ratio stayed <0.3 despite stimulation condition for patients, whereas this ratio was >2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CART expansion.

Conclusion: Untreated CLL patients might constitute a challenge for long-lasting CART effects since only a low number of T among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the T/T ratio within the CART product.
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http://dx.doi.org/10.3389/fimmu.2017.01956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767585PMC
January 2018