Publications by authors named "R Maarten Egeler"

200 Publications

Apparent Lack of Derived HLA Class I Presented Neoantigens Hampers Neoplastic Cell Targeting by CD8 T Cells in Langerhans Cell Histiocytosis.

Front Immunol 2019 10;10:3045. Epub 2020 Jan 10.

Immunology Laboratory Willem-Alexander Children's Hospital, Leiden University Medical Center, Leiden, Netherlands.

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50-60% of LCH-patients, the somatic driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like can be a source of neoantigens capable of eliciting effective antitumor CD8 T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8 T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8 T cell density in = 101 LCH-lesions, with mutated lesions displaying significantly lower CD8 T cell:CD1a LCH-cell ratios ( = 0.01) than wildtype lesions. Because LCH-lesional CD8 T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding derived neopeptide (KIGDFGLATK), which indeed displayed strong to intermediate binding capacity to HLA-A03:01 and HLA-A11:01 in an peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several expressing cell lines with various HLA genotypes. While the HLA-A02:01 binding wildtype peptide KIGDFGLATV was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLATK was not detected in the HLA class I peptidomes of two distinct transduced cell lines with confirmed expression of HLA-A03:01 or HLA-A11:01. These data indicate that the predicted HLA class I binding and proteasome-generated neopeptides derived from the protein are not presented by HLA class I molecules. Given that the mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH.
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http://dx.doi.org/10.3389/fimmu.2019.03045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6967030PMC
November 2020

Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell-mediated TNFα signaling.

Sci Transl Med 2017 Dec;9(421)

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy, but the large number of HSCs required limits its widespread use. Host conditioning and donor cell composition are known to affect HSCT outcomes. However, the specific role that the posttransplantation signaling environment plays in donor HSC fate is poorly understood. To mimic clinical HSCT, we injected human umbilical cord blood (UCB) cells at different doses and compositions into immunodeficient NOD/SCID/IL-2Rgc-null (NSG) mice. Surprisingly, higher UCB cell doses inversely correlated with stem and progenitor cell engraftment. This observation was attributable to increased donor cell-derived inflammatory signals. Donor T cell-derived tumor necrosis factor-α (TNFα) was specifically found to directly impair the survival and division of transplanted HSCs and progenitor cells. Neutralizing donor T cell-derived TNFα in vivo increased short-term stem and progenitor cell engraftment, accelerated hematopoietic recovery, and altered donor immune cell compositions. This direct effect of TNFα on transplanted cells could be decoupled from the indirect effect of alleviating graft-versus-host disease (GVHD) by interleukin-6 (IL-6) blockade. Our study demonstrates that donor immune cell-derived inflammatory signals directly influence HSC fate, and provides new clinically relevant strategies to improve engraftment efficiency during HSCT.
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http://dx.doi.org/10.1126/scitranslmed.aag3214DOI Listing
December 2017

Confirmed efficacy of etoposide and dexamethasone in HLH treatment: long-term results of the cooperative HLH-2004 study.

Blood 2017 12 21;130(25):2728-2738. Epub 2017 Sep 21.

Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institute, Stockholm, Sweden.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome comprising familial/genetic HLH (FHL) and secondary HLH. In the HLH-94 study, with an estimated 5-year probability of survival (pSu) of 54% (95% confidence interval, 48%-60%), systemic therapy included etoposide, dexamethasone, and, from week 9, cyclosporine A (CSA). Hematopoietic stem cell transplantation (HSCT) was indicated in patients with familial/genetic, relapsing, or severe/persistent disease. In HLH-2004, CSA was instead administered upfront, aiming to reduce pre-HSCT mortality and morbidity. From 2004 to 2011, 369 children aged <18 years fulfilled HLH-2004 inclusion criteria (5 of 8 diagnostic criteria, affected siblings, and/or molecular diagnosis in FHL-causative genes). At median follow-up of 5.2 years, 230 of 369 patients (62%) were alive (5-year pSu, 61%; 56%-67%). Five-year pSu in children with (n = 168) and without (n = 201) family history/genetically verified FHL was 59% (52%-67%) and 64% (57%-71%), respectively (familial occurrence [n = 47], 58% [45%-75%]). Comparing with historical data (HLH-94), using HLH-94 inclusion criteria, pre-HSCT mortality was nonsignificantly reduced from 27% to 19% ( = .064 adjusted for age and sex). Time from start of therapy to HSCT was shorter compared with HLH-94 (020 adjusted for age and sex) and reported neurological alterations at HSCT were 22% in HLH-94 and 17% in HLH-2004 (using HLH-94 inclusion criteria). Five-year pSu post-HSCT overall was 66% (verified FHL, 70% [63%-78%]). Additional analyses provided specific suggestions on potential pre-HSCT treatment improvements. HLH-2004 confirms that a majority of patients may be rescued by the etoposide/dexamethasone combination but intensification with CSA upfront, adding corticosteroids to intrathecal therapy, and reduced time to HSCT did not improve outcome significantly.
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http://dx.doi.org/10.1182/blood-2017-06-788349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785801PMC
December 2017

Tertiary lymphoid structures are confined to patients presenting with unifocal Langerhans Cell Histiocytosis.

Oncoimmunology 2016 Aug 28;5(8):e1164364. Epub 2016 Mar 28.

Immunology Laboratory, Willem Alexander Children's Hospital, Leiden University Medical Center , Leiden, the Netherlands.

Langerhans cell histiocytosis (LCH) is a neoplastic myeloid disorder with a thus far poorly understood immune component. Tertiary lymphoid structures (TLS) are lymph node-like entities which create an immune-promoting microenvironment at tumor sites. We analyzed the presence and clinical relevance of TLS in n = 104 H&E-stained, therapy-naive LCH lesions of non-lymphoid origin and applied immunohistochemistry to a smaller series. Lymphoid-follicular aggregates were detected in 34/104 (33%) lesions. In line with the lymphocyte recruitment capacity of MECA-79(+) high endothelial venules (HEVs), MECA-79(+)-expressing-LCH lesions (37/77, 48%) contained the most CD3(+) T-lymphocytes (p = 0.003). TLS were identified in 8/15 lesions and contained T-and B-lymphocytes, Follicular Dendritic Cells (FDC), HEVs and the chemokines CXCL13 and CCL21 representing key cellular components and TLS-inducing factors in conventional lymph nodes (LN). Lymphoid-follicular aggregates were most frequently detected in patients presenting with unifocal LCH (24/70, 34%) as compared to patients with poly-ostotic or multi-system LCH (7/30, 23%, p = 0.03). In addition, patients with lymphoid-follicular aggregates-containing lesions had the lowest risk to develop new LCH lesions (p = 0.04). The identification of various stages of TLS formation within LCH lesions may indicate a key role for the immune system in controlling aberrant histiocytes which arise in peripheral tissues.
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http://dx.doi.org/10.1080/2162402X.2016.1164364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007962PMC
August 2016

Bisphosphonates in Langerhans Cell Histiocytosis: An International Retrospective Case Series.

Mediterr J Hematol Infect Dis 2016 1;8(1):e2016033. Epub 2016 Jul 1.

Division of Hematology/Oncology and Bone Marrow Transplantation, Department of Pediatrics, The Hospital for Sick Children (SickKids), University of Toronto, Toronto, Ontario, Canada.

Background: Bone is the most common organ of involvement in patients with Langerhans cell histiocytosis (LCH), which is often painful and associated with significant morbidity from pathological fractures. Current first-line treatments include chemotherapy and steroids that are effective but often associated with adverse effects, whereas the disease may reactivate despite an initial response to first-line agents. Bisphosphonates are osteoclast inhibitors that have shown to be helpful in treating bone lesions of LCH. To date, there are no large international studies to describe their role in treating bone lesions of LCH.

Method: We conducted a multicenter retrospective review of 13 patients with histologically proven LCH, who had received bisphosphonates either at diagnosis or at disease reactivation.

Results: Ten patients (77%) had a single system bone disease, and 3 (23%) had bone lesions as part of multisystem disease. Median follow-up time post-bisphosphonate therapy was 4.6 years (range, 0.8 to 8.2 years). Treatment with bisphosphonates was associated with significant pain relief in almost all patients. Twelve (92%) achieved resolution of active bone lesions, and 10 out of them had no active disease for a median of 3.5 years (range, 0.8 to 5 years). One patient did not respond. No major adverse effects were reported in this series.

Conclusion: Bisphosphonates are well-tolerated drugs that can significantly improve bone pain and induce remission in active bone LCH. Future prospective studies evaluating the role of bisphosphonates in LCH are warranted.
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http://dx.doi.org/10.4084/MJHID.2016.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4928520PMC
July 2016

Langerhans cell histiocytosis is a neoplasm and consequently its recurrence is a relapse: In memory of Bob Arceci.

Pediatr Blood Cancer 2016 10 17;63(10):1704-12. Epub 2016 Jun 17.

Division of Medical Oncology, Dana-Farber Cancer Institute, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts.

Langerhans cell histiocytosis (LCH) remains a poorly understood disorder with heterogeneous clinical presentations characterized by focal or disseminated lesions that contain excessive CD1a+ langerin+ cells with dendritic cell features known as "LCH cells." Two of the major questions investigated over the past century have been (i) the origin of LCH cells and (ii) whether LCH is primarily an immune dysregulatory disorder or a neoplasm. Current opinion is that LCH cells are likely to arise from hematopoietic precursor cells, although the stage of derailment and site of transformation remain unclear and may vary in patients with different extent of disease. Over the years, evidence has provided the view that LCH is a neoplasm. The demonstration of clonality of LCH cells, insufficient evidence alone for neoplasia, is now bolstered by finding driver somatic mutations in BRAF in up to 55% of patients with LCH, and activation of the RAS-RAF-MEK-ERK (where MEK and ERK are mitogen-activated protein kinase and extracellular signal-regulated kinase, respectively) pathway in nearly 100% of patients with LCH. Herein, we review the evidence that recurrent genetic abnormalities characterized by activating oncogenic mutations should satisfy prerequisites for LCH to be called a neoplasm. As a consequence, recurrent episodes of LCH should be considered relapsed disease rather than disease reactivation. Mapping the complete genetic landscape of this intriguing disease will provide additional support for the conclusion that LCH is a neoplasm and is likely to provide more potential opportunities for molecularly targeted therapies.
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http://dx.doi.org/10.1002/pbc.26104DOI Listing
October 2016

Overexpression of LMO2 causes aberrant human T-Cell development in vivo by three potentially distinct cellular mechanisms.

Exp Hematol 2016 09 11;44(9):838-849.e9. Epub 2016 Jun 11.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands. Electronic address:

Overexpression of LMO2 is known to be one of the causes of T-cell acute lymphoblastic leukemia (T-ALL) development; however, the mechanisms behind its oncogenic activity are incompletely understood. LMO2-overexpressing transgenic mouse models suggest an accumulation of immature T-cell progenitors in the thymus as the main preleukemic event. The effects of LMO2 overexpression on human T-cell development in vivo are unknown. Here, we report studies of a humanized mouse model transplanted with LMO2-transduced human hematopoietic stem/progenitor cells. The effects of LMO2 overexpression were confined to the T-cell lineage; however, initially, multipotent cells were transduced. Three effects of LMO2 on human T-cell development were observed: (1) a block at the double-negative/immature single-positive stage, (2) an accumulation of CD4(+)CD8(+) double-positive CD3(-) cells, and (3) an altered CD8/CD4 ratio with enhanced peripheral T lymphocytes. Microarray analysis of sorted double-positive cells overexpressing LMO2 led to the identification of an LMO2 gene set that clustered with human T-ALL patient samples of the described "proliferative" cluster. In this article, we demonstrate previously unrecognized mechanisms by which LMO2 alters human T-cell development in vivo; these mechanisms correlate with human T-ALL leukemogenesis.
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http://dx.doi.org/10.1016/j.exphem.2016.06.002DOI Listing
September 2016

Successful Therapy Reduction and Intensification for Childhood Acute Lymphoblastic Leukemia Based on Minimal Residual Disease Monitoring: Study ALL10 From the Dutch Childhood Oncology Group.

J Clin Oncol 2016 08 6;34(22):2591-601. Epub 2016 Jun 6.

Rob Pieters, Peter Hoogerbrugge, and Gertjan Kaspers, Princess Máxima Center for Pediatric Oncology, Utrecht; Rob Pieters, Hester de Groot-Kruseman, Marta Fiocco, and Valerie De Haas, Dutch Childhood Oncology Group, The Hague; Vincent Van der Velden and Jacques Van Dongen, University Medical Center Rotterdam, Rotterdam; Marta Fiocco, Leiden University, Leiden; Henk van den Berg, Academic Medical Center; Gertjan Kaspers, Free University Hospital Amsterdam; Ellen Van der Schoot, Sanquin Research, Amsterdam; Evelien de Bont, University of Groningen, Groningen, the Netherlands; and R. Maarten Egeler, The Hospital for Sick Children, Toronto, Canada.

Purpose: Outcome of childhood acute lymphoblastic leukemia (ALL) improved greatly by intensifying chemotherapy for all patients. Minimal residual disease (MRD) levels during the first months predict outcome and may select patients for therapy reduction or intensification.

Methods: Patients 1 to 18 years old with ALL were stratified on the basis of MRD levels after the first and second course of chemotherapy. Thereafter, therapy was substantially reduced in patients with undetectable MRD (standard risk) and intensified in patients with intermediate (medium risk) and high (high risk) levels of MRD. Seven hundred seventy-eight consecutive patients were enrolled. The method of analysis was intention-to-treat. Outcome was compared with historical controls.

Results: In MRD-based standard-risk patients, the 5-year event-free survival (EFS) rate was 93% (SE 2%), the 5-year survival rate was 99% (SE 1%), and the 5-year cumulative incidence of relapse rate was 6% (SE 2%). The safety upper limit of number of observation years was reached and therapy reduction was declared safe.MRD-based medium-risk patients had a significantly higher 5-year EFS rate (88%, SE 2%) with therapy intensification (including 30 weeks of asparaginase exposure and dexamethasone/vincristine pulses) compared with historical controls (76%, SE 6%). Intensive chemotherapy and stem cell transplantation in MRD-based high-risk patients resulted in a significantly better 5-year EFS rate (78%, SE 8% v 16%, SE 8% in controls). Overall outcome improved significantly (5-year EFS rate 87%, 5-year survival rate 92%, and 5-year cumulative incidence of relapse rate 8%) compared with preceding Dutch Childhood Oncology Group protocols.

Conclusion: Chemotherapy was substantially reduced safely in one-quarter of children with ALL who were selected on the basis of undetectable MRD levels, without jeopardizing the survival rate. Outcomes of patients with intermediate and high levels of MRD improved with therapy intensification.
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http://dx.doi.org/10.1200/JCO.2015.64.6364DOI Listing
August 2016

Revised classification of histiocytoses and neoplasms of the macrophage-dendritic cell lineages.

Blood 2016 06 10;127(22):2672-81. Epub 2016 Mar 10.

Clarient Pathology Services, Aliso Viejo, CA.

The histiocytoses are rare disorders characterized by the accumulation of macrophage, dendritic cell, or monocyte-derived cells in various tissues and organs of children and adults. More than 100 different subtypes have been described, with a wide range of clinical manifestations, presentations, and histologies. Since the first classification in 1987, a number of new findings regarding the cellular origins, molecular pathology, and clinical features of histiocytic disorders have been identified. We propose herein a revision of the classification of histiocytoses based on histology, phenotype, molecular alterations, and clinical and imaging characteristics. This revised classification system consists of 5 groups of diseases: (1) Langerhans-related, (2) cutaneous and mucocutaneous, and (3) malignant histiocytoses as well as (4) Rosai-Dorfman disease and (5) hemophagocytic lymphohistiocytosis and macrophage activation syndrome. Herein, we provide guidelines and recommendations for diagnoses of these disorders.
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http://dx.doi.org/10.1182/blood-2016-01-690636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161007PMC
June 2016

Challenges in the harmonization of immune monitoring studies and trial design for cell-based therapies in the context of hematopoietic cell transplantation for pediatric cancer patients.

Cytotherapy 2015 Dec;17(12):1667-74

Blood and Marrow Transplantation Program, Department of Pediatrics, UMC Utrecht, Utrecht, Netherlands; Laboratory of Translational Immunology, UMC Utrecht, Utrecht, Netherlands. Electronic address:

Clinical trials aimed at improving results of hematopoietic cell transplantation (HCT) by adjuvant cell-based interventions in children have been limited by small numbers and pediatric-specific features. The need for a larger number of pediatric HCT centers to participate in trials has resulted in a demand for harmonization of disease-specific clinical trials and immune-monitoring. Thus far, most phase I/II trials select different end points evaluated at disparate time points, making inter-study comparisons difficult and, sometimes, impossible. In this review, we discuss the various aspects that are important to consider for harmonizing clinical trial design as well as the critical elements for standardized (immune)-monitoring protocols in cell-based intervention trials in the context of HCT. Comparison data from trials applying harmonized trial design will lead to optimized immunotherapeutic treatment protocols to maximize clinical efficacy while minimizing toxicity.
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http://dx.doi.org/10.1016/j.jcyt.2015.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472929PMC
December 2015

Activated Conventional T-Cells Are Present in Langerhans Cell Histiocytosis Lesions Despite the Presence of Immune Suppressive Cytokines.

J Interferon Cytokine Res 2015 Oct 18;35(10):831-9. Epub 2015 Sep 18.

1 Immunology Laboratory, Willem Alexander Children's Hospital (WAKZ), Leiden University Medical Center , Leiden, The Netherlands .

Langerhans cell histiocytosis (LCH) lesions are characterized by neoplastic CD1a(+)/Langerin(+) histiocytes (LCH-cells) and display many features of chronic inflammation. Cancer cells can escape immune-surveillance through intra-tumoral secretion of immune-suppressive cytokines. We therefore studied by immunohistochemistry the local cytokine milieu and phenotypic characteristics of T-cells and LCH-cells present in LCH lesions collected from 25 therapy naïve patients. LCH biopsies predominantly expressed interleukin-10 (IL-10) (10/25), transforming growth factor-beta (TGF-β) (9/25), or both cytokines (6/25). The absolute number of CD3(+)T-cells and the CD3(+)FOXP3(-) conventional cell (T-CONV) versus the CD3(+)FOXP3(+) regulatory T-cell (T-REG) was comparable for each suppressive cytokine profile (5:1). IL-10-expressing lesions contained, however, a higher proportion of T-CONV expressing the activation markers CD25 98% (38%-100%) and inducible costimulatory molecule (ICOS) 86% (47%-100%) than lesions wherein solely TGF-β was detected (CD25(+) 20% (6%-54%); ICOS(+) 29% (7%-51%)). Virtually all T-REG expressed CD25 and ICOS in IL-10 lesions, whereas TGF-β(+) lesions contained a lower proportion of ICOS(+) T-REG (P=0.05). IL-10(+) lesions contained more LCH-cells expressing high intensity of ICOS ligand (ICOSL) compared with TGF-β(+) lesions (P=0.03). ICOS expression by lesion-infiltrating T-CONV and T-REG positively correlated to the extent of ICOSL expression by LCH-cells (P=0.004). Our study points out that the combined detection of interlesional IL-10 and ICOSL expression by LCH-cells is associated with the highest prevalence of activated T-CONV. Immune profiling of LCH-affected tissues obtained at the time of diagnosis may set the stage for the development of new types of therapies, which aim at local boosting of immune cells that recognize and eliminate neoplastic LCH-cells.
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http://dx.doi.org/10.1089/jir.2014.0190DOI Listing
October 2015

Transplant Outcomes for Children with T Cell Acute Lymphoblastic Leukemia in Second Remission: A Report from the Center for International Blood and Marrow Transplant Research.

Biol Blood Marrow Transplant 2015 Dec 29;21(12):2154-2159. Epub 2015 Aug 29.

Stem Cell Transplant Program, Department of Pediatrics, Vanderbilt University, Nashville, TN.

Survival for children with relapsed T cell acute lymphoblastic leukemia (T-ALL) is poor when treated with chemotherapy alone, and outcomes after allogeneic hematopoietic cell transplantation (HCT) is not well described. Two hundred twenty-nine children with T-ALL in second complete remission (CR2) received an HCT after myeloablative conditioning between 2000 and 2011 and were reported to the Center for International Blood and Marrow Transplant Research. Median age was 10 years (range, 2 to 18). Donor source was umbilical cord blood (26%), matched sibling bone marrow (38%), or unrelated bone marrow/peripheral blood (36%). Acute (grades II to IV) and chronic graft-versus-host disease occurred in, respectively, 35% (95% confidence interval [CI], 27% to 45%) and 26% (95% CI, 20% to 33%) of patients. Transplant-related mortality at day 100 and 3-year relapse rates were 13% (95% CI, 9% to 18%) and 30% (95% CI, 24% to 37%), respectively. Three-year overall survival and disease-free survival rates were 48% (95% CI, 41% to 55%) and 46% (95% CI, 39% to 52%), respectively. In multivariate analysis, patients with bone marrow relapse, with or without concurrent extramedullary relapse before HCT, were most likely to relapse (hazard ratio, 3.94; P = .005) as compared with isolated extramedullary disease. In conclusion, HCT for pediatric T-ALL in CR2 demonstrates reasonable and durable outcomes, and consideration for HCT is warranted.
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http://dx.doi.org/10.1016/j.bbmt.2015.08.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654112PMC
December 2015

The presence of CXCR4 CD1a cells at onset of Langerhans cell histiocytosis is associated with a less favorable outcome.

Oncoimmunology 2016 Mar 31;5(3):e1084463. Epub 2015 Aug 31.

Immunology Laboratory, Willem Alexander Children's Hospital/Leiden University Medical Center, Leiden, the Netherlands; Division of Hematology/Oncology, Hospital for Sick Children/University of Toronto, Toronto, Canada.

Purpose: Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder characterized by tissue accumulating CD1a histiocytes which frequently carry somatic mutations. Irrespective of mutation status, these LCH-cells display constitutively active kinases belonging to the MAPK pathway. We evaluated, in retrospect, the contribution of individual components of the MAPK-activating and chemotaxis-promoting TNF-CXCR4-CXCL12 axis to LCH manifestation and outcome.

Experimental Design: CXCR4, CXCL12 and TNF protein expression was immunohistochemically analyzed in 70 LCH-affected biopsies. The presence of CXCR4CD1a cells in peripheral blood (PB) and/or bone marrow (BM) samples was evaluated by flowcytometry in 13 therapy-naive LCH-patients.

Results: CXCL12 was detected in 68/70 (97%) biopsies. CXCR4LCH-cells were present in 50/70 (71%) biopsies; their presence was associated with higher levels of intralesional TNF. Circulating CD1aCXCR4 cells were detected in 4/13 (31%) therapy-naïve LCH-patients which displayed BRAF (2/4), MAP2K1 (1/4) or no (1/4) mutations in their tissues. These CD11c co-expressing CD1aCXCR4cells migrated to CXCL12 in chemotaxis assays. Lesional CXCR4LCH-cells were detected in 18/20 cases who presented with LCH manifestation at multiple sites and in 5/23 (22%) patients who developed additional lesions after initially presenting with a single lesion. The CXCR4 status at onset proved to be an independent risk factor for LCH reactivation in multivariate analysis (odds ratio 10.4, = 0.034).

Conclusions: This study provides the first evidence that CXCR4 is involved in the homing and retention of LCH-cells in CXCL12-expressing tissues and qualifies CXCR4 as a candidate prognostic marker for less favorable disease outcome.
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http://dx.doi.org/10.1080/2162402X.2015.1084463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323006PMC
March 2016

Gene-expression and in vitro function of mesenchymal stromal cells are affected in juvenile myelomonocytic leukemia.

Haematologica 2015 Nov 20;100(11):1434-41. Epub 2015 Aug 20.

Department of Pediatrics, Immunology, Hematology/Oncology and Hematopoietic Stem Cell Transplantation, Leiden University Medical Center, the Netherlands.

An aberrant interaction between hematopoietic stem cells and mesenchymal stromal cells has been linked to disease and shown to contribute to the pathophysiology of hematologic malignancies in murine models. Juvenile myelomonocytic leukemia is an aggressive malignant disease affecting young infants. Here we investigated the impact of juvenile myelomonocytic leukemia on mesenchymal stromal cells. Mesenchymal stromal cells were expanded from bone marrow samples of patients at diagnosis (n=9) and after hematopoietic stem cell transplantation (n=7; from 5 patients) and from healthy children (n=10). Cells were characterized by phenotyping, differentiation, gene expression analysis (of controls and samples obtained at diagnosis) and in vitro functional studies assessing immunomodulation and hematopoietic support. Mesenchymal stromal cells from patients did not differ from controls in differentiation capacity nor did they differ in their capacity to support in vitro hematopoiesis. Deep-SAGE sequencing revealed differential mRNA expression in patient-derived samples, including genes encoding proteins involved in immunomodulation and cell-cell interaction. Selected gene expression normalized during remission after successful hematopoietic stem cell transplantation. Whereas natural killer cell activation and peripheral blood mononuclear cell proliferation were not differentially affected, the suppressive effect on monocyte to dendritic cell differentiation was increased by mesenchymal stromal cells obtained at diagnosis, but not at time of remission. This study shows that active juvenile myelomonocytic leukemia affects the immune response-related gene expression and function of mesenchymal stromal cells. In contrast, the differential gene expression of hematopoiesis-related genes could not be supported by functional data. Decreased immune surveillance might contribute to the therapy resistance and progression in juvenile myelomonocytic leukemia.
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http://dx.doi.org/10.3324/haematol.2015.126938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825298PMC
November 2015

Humanized Chronic Graft-versus-Host Disease in NOD-SCID il2rγ-/- (NSG) Mice with G-CSF-Mobilized Peripheral Blood Mononuclear Cells following Cyclophosphamide and Total Body Irradiation.

PLoS One 2015 15;10(7):e0133216. Epub 2015 Jul 15.

Hematology/Oncology/BMT, The Hospital for Sick Children, Toronto, ON, Canada; Developmental Stem Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada.

Chronic graft-versus-host disease (cGvHD) is the major source of late phase morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Humanized acute GvHD (aGvHD) in vivo models using NOD-SCID il2rγ-/- (NSG) mice are well described and are important tools for investigating pathogenicity of human cells in vivo. However, there have been only few reported humanized cGvHD mouse models. We evaluated if prolonged inflammation driven by low dose G-CSF-mobilized human PBMCs (G-hPBMCs) would lead to cGvHD following cyclophosphamide (CTX) administration and total body irradiation (TBI) in NSG mice. Engraftment was assessed in peripheral blood (PB) and in specific target organs by either flow cytometry or immunohistochemistry (IHC). Tissue samples were harvested 56 days post transplantation and were evaluated by a pathologist. Some mice were kept for up to 84 days to evaluate the degree of fibrosis. Mice that received CTX at 20mg/kg did not show aGvHD with stable expansion of human CD45+ CD3+ T-cells in PB (mean; 5.8 to 23.2%). The pathology and fibrosis scores in the lung and the liver were significantly increased with aggregation of T-cells and hCD68+ macrophages. There was a correlation between liver pathology score and the percentage of hCD68+ cells, suggesting the role of macrophage in fibrogenesis in NSG mice. In order to study long-term survival, 6/9 mice who survived more than 56 days showed increased fibrosis in the lung and liver at the endpoint, which suggests the infiltrating hCD68+ macrophages may be pathogenic. It was shown that the combination of CTX and TBI with a low number of G-hPBMCs (1x106) leads to chronic lung and liver inflammation driven by a high infiltration of human macrophage and mature human T cells from the graft, resulting in fibrosis of lung and liver in NSG mice. In conclusion this model may serve as an important pre-clinical model to further current understanding of the roles of human macrophages in cGvHD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133216PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503770PMC
May 2016

Reactivation of Human Herpes Virus-6 After Pediatric Stem Cell Transplantation: Risk Factors, Onset, Clinical Symptoms and Association With Severity of Acute Graft-Versus-Host Disease.

Pediatr Infect Dis J 2015 Oct;34(10):1118-27

From the *Department of Pediatrics, †Department of Medical Microbiology, ‡Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands; and §Division of Hematology/Oncology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada.

Background And Methods: To study clinical symptoms, timing and consequences of human herpesvirus-6 (HHV-6) reactivation after pediatric allogeneic stem cell transplantation (SCT), HHV-6 was investigated by plasma polymerase chain reaction in a cohort of 106 pediatric SCT recipients.

Results: HHV-6 viremia was detected post-SCT in 48% of the patients with a median time of onset at 20 days after SCT. In week 3 and 4 post-SCT, HHV-6 is the most common infectious agent detected. In up to 30% of the patients with fever of unknown origin, HHV-6 was the only detected infectious agent to explain fever. Patients transplanted with an unrelated donor or receiving serotherapy were at increased risk of HHV-6 reactivation. The onset of HHV-6 reactivation coincided with the appearance of lymphocytes and monocytes in peripheral blood. Treatment with alemtuzumab (MabCampath) delayed both lymphocyte and monocyte engraftment and, concomitantly, onset of HHV-6 reactivation was delayed in those cases. HHV-6 reactivation was not associated with an increased incidence of acute graft-versus-host disease (GvHD). However, progression to grade II-IV GvHD was in 9 of 10 patients associated with HHV-6 reactivation before GvHD (P = 0.006) and HHV-6 was the only infection with such an association.

Conclusions: HHV-6 frequently reactivates after pediatric SCT around the time of mononuclear cell engraftment and is associated with an increased severity of GvHD. HHV-6 may explain fever of unknown origin in 30% of the patients early after SCT. Assessment of HHV-6 reactivation in patients early after SCT can be instrumental for clinical decision making.
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http://dx.doi.org/10.1097/INF.0000000000000824DOI Listing
October 2015

Significant Transplantation-Related Mortality from Respiratory Virus Infections within the First One Hundred Days in Children after Hematopoietic Stem Cell Transplantation.

Biol Blood Marrow Transplant 2015 Oct 25;21(10):1802-7. Epub 2015 Jun 25.

Division of Haematology/Oncology/BMT, the Hospital for Sick Children, University of Toronto, Ontario, Canada.

Respiratory viral infections (RVI) are important in hematopoietic stem cell transplantations (HSCT) and knowledge regarding incidence, morbidity, mortality, and long-term pulmonary complications is limited. We report a study to evaluate incidence and outcomes, both short and long-term, of RVI in children receiving HSCT. Between January 2000 and December 2012, 844 patients underwent hematopoietic stem cell transplantation (HSCT) at the Hospital for Sick Children: 491 were allogeneic and 353 were autologous. When screening for causes of death in the first year after HSCT in the 844 patients, we found that RVI as a cause of death was only evident in the first 100 days after HSCT. Fifty-four (6.5%) patients were found to have an RVI within the first 100 days after HSCT (allogeneic = 32, autologous = 22). Upper and lower respiratory tract infections were documented in 31 (57%) and 23 (43%) patients, respectively. Viruses were parainfluenza (35%), respiratory syncytial virus (28%), influenza (22%), adenovirus (7%), human metapneumovirus (4%), coronavirus (2%), and rhinovirus (2%). Three patients relapsed with their primary disease before day 100 and were excluded. The overall mortality for the remaining 51 patients was 10% (allogeneic = 4, autologous = 1). All 5 deaths were directly attributable to RVI and all 5 deaths occurred in patients with a lower respiratory tract infection. The remaining patients were followed for a median of 4.3 years (range, 1.4 to 11.8) and no chronic pulmonary complications were observed. A clear seasonal pattern for contracting RVI was evident with 65% of total RVI occurring between October and March (35 of 427 versus 19 of 417, P = .03). Given the significant mortality from RVI and the challenges in preventing them, choosing the time to start HSCT, whenever possible, may help prevent RVI and improve outcomes.
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http://dx.doi.org/10.1016/j.bbmt.2015.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7110880PMC
October 2015

Performance of Busulfan Dosing Guidelines for Pediatric Hematopoietic Stem Cell Transplant Conditioning.

Biol Blood Marrow Transplant 2015 Aug 11;21(8):1471-8. Epub 2015 May 11.

Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada; Department of Pharmacy, The Hospital for Sick Children, Toronto, Ontario, Canada; Child Health Evaluative Sciences, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada. Electronic address:

Achievement of a busulfan area-under-the-concentration versus time curve (AUC) of 900 to 1500 μM·min is associated with improved hematopoietic stem cell transplant (HSCT) outcomes. Multiple pediatric busulfan dosing guidelines aim to achieve this target. The authors' objective was to describe the AUCs achieved after simulated dosing using available pediatric i.v. busulfan dosing guidelines. The health records of children who received i.v. busulfan for HSCT conditioning at The Hospital for Sick Children were reviewed. Busulfan AUCs were calculated for each patient based on plasma busulfan concentrations using either a 1-compartment model or a validated limited-sampling strategy. Published pediatric busulfan dosing guidelines were identified. Initial busulfan doses were determined for all patients using each dosing guideline and total body weight (TBW). For overweight patients (TBW-to-ideal body weight [IBW] ≥ 1.25), initial busulfan doses were also determined using IBW and adjusted IBW (IBWadj). The resulting AUCs were simulated. The proportion of subjects (TBW/IBW < 1.25, TBW/IBW ≥ 1.25, and infants) with an AUC within target (900 to 1500 μM·min) after dosing simulation with each guideline was compared. One hundred eleven children (mean age, 6.2 years [SD, ±5.2]) who received i.v. busulfan were included. When dosing with each of the 12 i.v. busulfan dosing guidelines identified was simulated using TBW in 97 non-overweight patients, the proportion of patients with an AUC within the target range varied from 51% to 74% and from 45% to 64% in infants. Use of IBW or IBWadj to calculate initial busulfan doses in overweight children improved the performance of most guidelines. Current busulfan dosing guidelines vary in their ability to achieve AUCs within the target range. For children who are not overweight, we recommend 1 of 3 high-performing guidelines that allow individualization of the target busulfan AUC. Use of either IBW or IBWadj in overweight children improves the performance of most guidelines. Regardless of the guideline used, therapeutic drug monitoring is essential to verify achievement of the target AUC.
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http://dx.doi.org/10.1016/j.bbmt.2015.05.006DOI Listing
August 2015

Serum Krebs Von Den Lungen-6 as a Biomarker for Early Detection of Bronchiolitis Obliterans Syndrome in Children Undergoing Allogeneic Stem Cell Transplantation.

Biol Blood Marrow Transplant 2015 Aug 8;21(8):1524-8. Epub 2015 May 8.

Programs of Physiology & Experimental Medicine, Research Institute, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology and Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada.

Bronchiolitis obliterans syndrome (BOS) is a devastating complication after allogeneic stem cell transplantation (allo-SCT). Early identification of high-risk patients is pivotal for success. Lung proteins, KL-6, CCSP, SP-A, and SP-D, measured in the serum may identify high-risk patients for BOS earlier than pulmonary function tests (PFTs) can identify changes or clinical symptoms. Lung proteins were measured in patients' serum at baseline and at 1, 3, 6, 9, 12, 18, and 24 months after transplantation along with history, clinical examination, and PFTs. Serum levels of lung proteins were also measured in healthy control subjects. The primary endpoint was the development of BOS confirmed by pathological biopsy or National Institutes of Health criteria. Between September 2009 and September 2011, 39 patients were enrolled. Six children developed BOS at a median time of 200 days (range, 94 to 282). KL-6 levels were low in control subjects, at a median of .1 U/mL (range, .1 to 1.5). Pre-SCT and 1-month KL-6 levels were significantly higher in surviving patients who developed BOS (n = 6) versus those who did not (n = 18) (pre-SCT: mean, 32.6 U/mL [IQR, 9.7 to 89.3] versus 5.8 U/mL [IQR, 2.1 to 12.6], P = .03; at 1 month: mean, 52.5 U/mL [IQR, 20.2 to 121.3] versus 11.4 U/mL [IQR, 5.7 to 36.0], P = .04). Three- and 6-month KL-6 levels continued to be higher in BOS group but were not statistically significant. CCSP, SP-A, and SP-D were not predictive. KL-6 measured in the serum of children receiving allo-SCT may identify patients at high risk for the development of BOS. These patients will benefit from intensive surveillance protocol and early therapy before irreversible lung damage.
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http://dx.doi.org/10.1016/j.bbmt.2015.04.021DOI Listing
August 2015

MAP2K1 and MAP3K1 mutations in Langerhans cell histiocytosis.

Genes Chromosomes Cancer 2015 Jun 31;54(6):361-8. Epub 2015 Mar 31.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA.

Langerhans cell histiocytosis (LCH) is now understood to be a neoplastic disease in which over 50% of cases have somatic activating mutations of BRAF. However, the extracellular signal-related (ERK) pathway is activated in all cases including those with wild type BRAF alleles. Here, we applied a targeted massively parallel sequencing panel to 30 LCH samples to test for the presence of additional genetic alterations that might cause ERK pathway activation. In 20 BRAF wild type samples, we found 3 somatic mutations in MAP2K1 (MEK1) including C121S and C121S/G128D in the kinase domain, and 56_61QKQKVG>R, an in-frame deletion in the N-terminal regulatory domain. All three variant proteins constitutively phosphorylated ERK in in vitro kinase assays. The C121S/G128D and 56_61QKQKVG>R variants were resistant to the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib in vitro. Within the entire sample set, we found 3 specimens with mutations in MAP3K1 (MEKK1), including two truncation mutants, T779fs and T1481fs; T1481fs encoded an unstable and nonfunctional protein when expressed in vitro. T779fs was present in a specimen carrying BRAF V600E. The third variant was a single nucleotide substitution, E1286V, which was fully functional and is likely a germline polymorphism. These results indicate that LCH cells can harbor additional genetic alterations in the RAS-RAF-MEK pathway which, in the case of MAP2K1, may be responsible for ERK activation in a wild type BRAF setting. The resistance of some of these variants to trametinib may also have clinical implications for the combined use of RAF and MEK inhibitors in LCH.
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http://dx.doi.org/10.1002/gcc.22247DOI Listing
June 2015

Role of NKG2D, DNAM-1 and natural cytotoxicity receptors in cytotoxicity toward rhabdomyosarcoma cell lines mediated by resting and IL-15-activated human natural killer cells.

Cancer Immunol Immunother 2015 May 18;64(5):573-83. Epub 2015 Feb 18.

Section Immunology, Hematology, Bone Marrow Transplantation and Autoimmune Diseases, Department of Pediatrics, Leiden University Medical Center (LUMC), PO Box 9600, 2300 RC, Leiden, The Netherlands,

Children with advanced stages (relapsed/refractory and stage IV) of rhabdomyosarcoma (RMS) have a poor prognosis despite intensive chemotherapy and autologous stem cell rescue, with 5-year survival rates ranging from 5 to 35 %. Development of new, additional treatment modalities is necessary to improve the survival rate. In this preclinical study, we investigated the potential of resting and cytokine-activated natural killer (NK) cells to lyse RMS cell lines, as well as the pathways involved, to explore the eventual clinical application of (activated) NK cell immunotherapy. RMS cell lines (n = 3 derived from embryonal RMS and n = 2 derived from alveolar RMS) were susceptible to cytolysis mediated by resting NK cells, and this susceptibility was significantly increased using IL-15-activated NK cells. Flow cytometry and cytolytic assays were used to define the activating and inhibitory pathways of NK cells involved in recognizing and lysing RMS cells. NKG2D and DNAM-1 receptor-ligand interactions were essential in cytolysis by resting NK cells, as simultaneous blocking of both pathways resulted in almost complete abrogation of the cytotoxicity. In contrast, combined blocking of DNAM-1 and NKG2D only led to partial reduction of the lytic activity of IL-15-activated NK cells. In this respect, residual lysis was, at least partly, mediated by pathways involving the natural cytotoxicity receptors NKp30 and NKp46. These findings support further exploration of NK cell-based immunotherapy as adjuvant modality in current treatment strategies of RMS.
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http://dx.doi.org/10.1007/s00262-015-1657-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4412555PMC
May 2015

Haematopoietic stem cell transplantation for refractory Langerhans cell histiocytosis: outcome by intensity of conditioning.

Br J Haematol 2015 Jun 27;169(5):711-8. Epub 2015 Mar 27.

CIBMTR® (Center for International Blood and Marrow Transplant Research), Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA.

Patients with Langerhans cell histiocytosis (LCH) refractory to conventional chemotherapy have a poor outcome. There are currently two promising treatment strategies for high-risk patients: the first involves the combination of 2-chlorodeoxyadenosine and cytarabine; the other approach is allogeneic haematopoietic stem cell transplantation (HSCT). Here we evaluated 87 patients with high-risk LCH who were transplanted between 1990 and 2013. Prior to the year 2000, most patients underwent HSCT following myeloablative conditioning (MAC): only 5 of 20 patients (25%) survived with a high rate (55%) of transplant-related mortality (TRM). After the year 2000 an increasing number of patients underwent HSCT with reduced intensity conditioning (RIC): 49/67 (73%) patients survived, however, the improved survival was not overtly achieved by the introduction of RIC regimens with similar 3-year probability of survival after MAC (77%) and RIC transplantation (71%). There was no significant difference in TRM by conditioning regimen intensity but relapse rates were higher after RIC compared to MAC regimens (28% vs. 8%, P = 0·02), although most patients relapsing after RIC transplantation could be salvaged with further chemotherapy. HSCT may be a curative approach in 3 out of 4 patients with high risk LCH refractory to chemotherapy: the optimal choice of HSCT conditioning remains uncertain.
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http://dx.doi.org/10.1111/bjh.13347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433436PMC
June 2015

Expression of the immune regulation antigen CD70 in osteosarcoma.

Cancer Cell Int 2015 18;15:31. Epub 2015 Mar 18.

Department of Pediatrics, Leiden University Medical Center, 2333ZA Leiden, The Netherlands.

Osteosarcoma is the most frequent bone cancer in children and young adults. The outcome of patients with advanced disease is dismal. Exploitation of tumor-immune cell interactions may provide novel therapeutic approaches. CD70-CD27 interactions are important for the regulation of adaptive immunity. CD70 expression has been reported in some solid cancers and implicated in tumor escape from immunosurveillance. In this study, expression of CD70 and CD27 was analyzed in osteosarcoma cell lines and tumor specimens. CD70 protein was expressed on most osteosarcoma cell lines (5/7) and patient-derived primary osteosarcoma cultures (4/6) as measured by flow cytometry. In contrast, CD70 was detected on few Ewing sarcoma cell lines (5/15) and was virtually absent from neuroblastoma (1/7) and rhabdomyosarcoma cell lines (0/5). CD70(+) primary cultures were derived from CD70(+) osteosarcoma lesions. CD70 expression in osteosarcoma cryosections was heterogeneous, restricted to tumor cells and not attributed to infiltrating CD3(+) T cells as assessed by immunohistochemistry/immunofluorescence. CD70 was detected in primary (1/5) but also recurrent (2/4) and metastatic (1/3) tumors. CD27, the receptor for CD70, was neither detected on tumor cells nor on T cells in CD70(+) or CD70(-) tumors, suggesting that CD70 on tumor cells is not involved in CD27-dependent tumor-immune cell interactions in osteosarcoma. CD70 gene expression in diagnostic biopsies of osteosarcoma patients did not correlate with the occurrence of metastasis and survival (n = 70). Our data illustrate that CD70 is expressed in a subset of osteosarcoma patients. In patients with CD70(+) tumors, CD70 may represent a novel candidate for antibody-based targeted immunotherapy.
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http://dx.doi.org/10.1186/s12935-015-0181-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365554PMC
March 2015

Despite differential gene expression profiles pediatric MDS derived mesenchymal stromal cells display functionality in vitro.

Stem Cell Res 2015 Mar 28;14(2):198-210. Epub 2015 Jan 28.

Department of Pediatrics, Section Immunology, Hematology/Oncology and Hematopoietic Stem Cell Transplantation, Leiden University Medical Center, Leiden, The Netherlands.

Pediatric myelodysplastic syndrome (MDS) is a heterogeneous disease covering a spectrum ranging from aplasia (RCC) to myeloproliferation (RAEB(t)). In adult-type MDS there is increasing evidence for abnormal function of the bone-marrow microenvironment. Here, we extensively studied the mesenchymal stromal cells (MSCs) derived from children with MDS. MSCs were expanded from the bone-marrow of 17 MDS patients (RCC: n=10 and advanced MDS: n=7) and pediatric controls (n=10). No differences were observed with respect to phenotype, differentiation capacity, immunomodulatory capacity or hematopoietic support. mRNA expression analysis by Deep-SAGE revealed increased IL-6 expression in RCC- and RAEB(t)-MDS. RCC-MDS MSC expressed increased levels of DKK3, a protein associated with decreased apoptosis. RAEB(t)-MDS revealed increased CRLF1 and decreased DAPK1 expressions. This pattern has been associated with transformation in hematopoietic malignancies. Genes reported to be differentially expressed in adult MDS-MSC did not differ between MSC of pediatric MDS and controls. An altered mRNA expression profile, associated with cell survival and malignant transformation, of MSC derived from children with MDS strengthens the hypothesis that the micro-environment is of importance in this disease. Our data support the understanding that pediatric and adult MDS are two different diseases. Further evaluation of the pathways involved might reveal additional therapy targets.
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http://dx.doi.org/10.1016/j.scr.2015.01.006DOI Listing
March 2015

Histologic patterns of thymic involvement in Langerhans cell proliferations: a clinicopathologic study and review of the literature.

Pediatr Dev Pathol 2015 Mar-Apr;18(2):127-38. Epub 2015 Jan 28.

1 Department of Pathology, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Thymic involvement by Langerhans cell histiocytosis (LCH) has been described mainly in isolated case reports. A description of the histopathologic patterns of LCH proliferations in the thymus, together with therapeutic implications, has not, to our knowledge, been previously addressed. The pathology consultation files at Children's Hospital of Pittsburgh of the University of Pennsylvania Medical Center were reviewed for cases of thymic involvement by LCH. Relevant cases in the literature were also reviewed, and the histopathology and clinical course of those cases were collected. Nine consultation cases of thymic involvement were reviewed, together with 23 cases in the literature, which provided adequate pathologic description and ancillary confirmation (n  =  32), revealing 4 distinct pathologic groups. Group 1 showed microscopic collection of hyperplastic LCH-like cells in incidental thymectomies of patients without LCH disease, requiring no further treatment (n  =  7; 22%). Group 2 showed solitary and/or cystic LCH of the thymus with gland disruption, and at least 3 cases resolved without systemic therapy (n  =  10; 31%). Group 3 showed more variable thymic involvement in multisystemic LCH disease, with either a medullary restricted pattern or more diffuse gland involvement, requiring adjuvant therapy and having a higher mortality rate (n  =  13; 41%). Group 4 showed a mixed histiocytic lesion with a concurrent LCH and juvenile xanthogranuloma-like proliferation (n  =  2; 6%). Thymic involvement in LCH is quite rare. Based on our cases and those in the literature, we propose 4 distinct pathologic groups of thymic involvement in Langerhans cell proliferations with relevance for diagnosis and treatment.
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http://dx.doi.org/10.2350/15-01-1593-OA.1DOI Listing
May 2015

Major ABO incompatible BMT in children: determining what residual volume of donor red cells can safely be infused following red cell depletion.

Bone Marrow Transplant 2015 Apr 26;50(4):536-9. Epub 2015 Jan 26.

Division of Haematology/Oncology, The Hospital for Sick Children, Toronto, Ontario, Canada.

Major ABO incompatible BM transplantation carries a risk of acute haemolysis. Red cell depletion reduces this risk but not all incompatible RBC (iRBCs) are removed and in children the residual volume can be significant relative to body weight. We sought to determine the volume of iRBCs that can be safely given to children. All patients receiving fresh BM from a donor with a major ABO blood group mismatch between January 2000 and July 2013 at the Hospital for Sick Children, Toronto, were included. Seventy-eight patients were identified. The median volume of iRBCs transfused was 1.6 mL/kg (range 0.1-10.6 mL/kg). Thirty-five patients had minor haemolytic events and five patients had clinically significant adverse events. Two patients, who received 3.66 and 3.9 mL iRBCs/kg, developed renal impairment and in one case hypoxia and hyperbilirubinaemia. One patient had mild hypotension that resolved with i.v. fluid. Two patients developed hypotension secondary to sepsis and unrelated to BM infusion. Although signs of haemolysis occur, with appropriate hydration and monitoring of renal function, clinically significant adverse events related to the infusion of ABO incompatible BM are rare, and, in this study, were only seen in patients receiving >3 mL/kg of iRBCs per kg.
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http://dx.doi.org/10.1038/bmt.2014.309DOI Listing
April 2015

Sustained Engraftment of Cryopreserved Human Bone Marrow CD34(+) Cells in Young Adult NSG Mice.

Biores Open Access 2014 Jun;3(3):110-6

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center , Leiden, The Netherlands .

Hematopoietic stem cells (HSCs) are defined by their ability to repopulate the bone marrow of myeloablative conditioned and/or (lethally) irradiated recipients. To study the repopulating potential of human HSCs, murine models have been developed that rely on the use of immunodeficient mice that allow engraftment of human cells. The NSG xenograft model has emerged as the current standard for this purpose allowing for engraftment and study of human T cells. Here, we describe adaptations to the original NSG xenograft model that can be readily implemented. These adaptations encompass use of adult mice instead of newborns and a short ex vivo culture. This protocol results in robust and reproducible high levels of lympho-myeloid engraftment. Immunization of recipient mice with relevant antigen resulted in specific antibody formation, showing that both T cells and B cells were functional. In addition, bone marrow cells from primary recipients exhibited repopulating ability following transplantation into secondary recipients. Similar results were obtained with cryopreserved human bone marrow samples, thus circumventing the need for fresh cells and allowing the use of patient derived bio-bank samples. Our findings have implications for use of this model in fundamental stem cell research, immunological studies in vivo and preclinical evaluations for HSC transplantation, expansion, and genetic modification.
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http://dx.doi.org/10.1089/biores.2014.0008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048975PMC
June 2014