ANKL Is Strongly EBV-Related, Frequent In Chromosome Aberrations and Gene Mutations, LMP2A/LMP1-CDK6-MDM2-CD44-Positive and Might Be Sensitive To L-Asparaginase-Containing Regimen

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3012-3012
Author(s):  
Hongsheng Zhou ◽  
Yongjian Deng ◽  
Changxin Yin ◽  
Xiaolei Xue ◽  
Xiangzhao Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Chromosome Aberrations ◽  
Research Funding ◽  
Clinical Course ◽  
Molecular Cytogenetics ◽  
Sh2 Domain ◽  
Clinical Feature ◽  
Hematopoietic Stem ◽  
Aggressive Clinical Course

Abstract Background Aggressive natural killer leukemia (ANKL), previously being classified as large granular lymphocyte (LGL) leukemia or malignant histiocytosis, is a rare subset neoplasm with high aggressive clinical course. Until now, apart from clinical feature, the frequent chromosome aberrations, somatic genes mutations, signal pathway and the optimal treatment regimen are still poor understood. Patient and Methods A retrospective analysis was performed upon review of clinical database, including additional immunohistochemistry (IHC) staining and integrated mutation analysis on reserved samples. Twelve samples of extranodal NK/T-cell lymphoma-nasal type (ENKL-NT) were used in IHC analysis for control. Results A total of 26 cases were collected during 2001 to 2013, including 17 males and 9 females, median age of 28 years old (range 4-76 years old). Most patients presented with acute-onset high swinging fever (n=26), deteriorating jaundice (n=19) and pancytopenia (n=22) at diagnosis. The organ most frequently involved organs were bone marrow (n=25), liver (n=23), spleen (n=22) and gastrointestinal (n=6). Disseminated intravascular coagulopathy (DIC) was present in 15 out of 26 patients, significantly associated with liver involvement and jaundice (p<0.01). Epstein-Barr virus (EBV) viremia was present in 7 of 8 tested cases (5.12x10*,2-5.41x10*,6 copies/mL). The characteristic morphological appearance of ANKL in bone marrow smears were deep purple-red staining granules in cytoplasm and prominent vacuoles located in cytoplasm and/or nucleus. FACS demonstrated ANKL cells were CD2+cCD3+CD7+CD11b+/-CD11c+/-CD29+CD38+/-CD45+CD56+CD86+BCL-2+, CD3-TCR-cCD79a-cMPO-CD5-CD10-CD19-CD25-CD33-CD83-CD123-. Surprisingly, ANKL cells were negative for CD21, an established EBV receptor. Additional IHC on 14 reserved bone marrow samples revealed ANKL cells were strongly positive for LMP2A (n=14), LMP1 (n=11) and EBER (n=10). In ENKL-NT, LMP2A and LMP1 were moderately positive in 5 and 4 out of 12 cases, respectively. EBERs were detected in all ENKT-NT cases. Moreover, IHC analysis showed ANKL cells were strongly stained by CDK6 (n=13), MDM2 (n=14), CD44 (n=13), IFN-γ (n=14), slightly stained by CDK4 (n=2), negative for SH2 domain-containing inositol 5’-phosphatase-1 (SHIP-1), which is responsible for 2B4-mediated inhibition in NK cells. In ENKL-NT samples, CDK6 (n=11), MDM2 (n=9) and CD44 (n=8) were positive; furthermore, SHIP-1 was slightly positive in 4 samples. Chromosome aberrations were present in 7 patients, including dup1(q22q25), inv(3)(p21p25), del(3)(p13), t(3;11)(q21;q23), i(7)(q10), del(7)(q32), del(14)(q24), der(15)t(7;15)(q10;q10), -8, -12,+13,-18,+19,-22. Multiple somatic mutations were detected in 7 cases, including TET2 (D1938E, S69R, V292L, E1207D, G1391C, T1393K, Y1998X), FBXW7 (S427L,Q428K, D431E, R505C, Q508K, G517V, D775Y), CEBPA (E57D, N292K, S85I), FLT3 (M837I, E604X), KRAS (G77R, Q22H, G13D), PAX5 (P93T, A111S), PHF6 (R15S, S155X, V5F), DNMT3A (P602H, H613D), EGFR (E736X, S155X), IDH2 (K166M),SH2B3 (E190X), c-Kit (G812C), JAK1 (D775Y), NOTCH1 (A1701S). Only 1 out of 10 patients obtained partial remission after anthracycline-based or platinum-based regimens (CHOP, CHOP-Bleomycin, CDOP, EPOCH, HyperCVAD and ESHAP). Notably, two cases rapidly achieved durable complete remission after L-asparaginase-containing regimen (CHOP/PEG-L-asp and VDLP). Two patients received allogeneic hematopoietic stem cell transplantation, one maintained durable remission and another died of invasive pulmonary infection before engraftment. The median overall survival of 26 cases was only 7.5 days (range 2-330 days). Conclusions ANKL is Trojan story about EBV and NK cells, being presented with strong evidence of EBV infection/transformation, highly aggressive clinical course and prominent chromosome/genomic instability, which deserves more research efforts to dissect the role of EBV, molecular cytogenetics make-up, signaling pathway of LMP1/LMP2A-PI3K/AKT-CDK6-MDM2 and optimal regimen such as L-asp-contained protocol for this rare leukemia subset. Disclosures: Zhou: Guangzhou Pearl River of Science & Technology New Star (No. 2011J2200069 to HS.Zhou): Research Funding. Liu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding.

scholarly journals CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adult mice

Blood ◽  
2011 ◽  
Vol 117 (2) ◽  
pp. 451-458 ◽  
Author(s):  
Mamiko Noda ◽  
Yoshiki Omatsu ◽  
Tatsuki Sugiyama ◽  
Shinya Oishi ◽  
Nobutaka Fujii ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Development ◽  
Hematopoietic Stem ◽  
Adult Mice ◽  
Multipotent Progenitors ◽  
Chemokine Signaling ◽  
Chemokine Ligand

Abstract Natural killer (NK) cells are granular lymphocytes that are generated from hematopoietic stem cells and play vital roles in the innate immune response against tumors and viral infection. Generation of NK cells is known to require several cytokines, including interleukin-15 (IL-15) and Fms-like tyrosine kinase 3 ligand, but not IL-2 or IL-7. Here we investigated the in vivo role of CXC chemokine ligand-12 (CXCL12) and its primary receptor CXCR4 in NK-cell development. The numbers of NK cells appeared normal in embryos lacking CXCL12 or CXCR4; however, the numbers of functional NK cells were severely reduced in the bone marrow, spleen, and peripheral blood from adult CXCR4 conditionally deficient mice compared with control animals, probably resulting from cell-intrinsic CXCR4 deficiency. In culture, CXCL12 enhanced the generation of NK cells from lymphoid-primed multipotent progenitors and immature NK cells. In the bone marrow, expression of IL-15 mRNA was considerably higher in CXCL12-abundant reticular (CAR) cells than in other marrow cells, and most NK cells were in contact with the processes of CAR cells. Thus, CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adults, and CAR cells might function as a niche for NK cells in bone marrow.

scholarly journals Dnmt3a Is Essential for Hematopoietic Stem Cell Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 386-386 ◽  
Author(s):  
Grant A. Challen ◽  
Deqiang Sun ◽  
Mira Jeong ◽  
Min Luo ◽  
Jaroslav Jelinek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Dna Methyltransferase ◽  
Donor Cell ◽  
The Self ◽  
Blood Cell Count ◽  
Serial Transfer ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Differentiation Capacity

Abstract Abstract 386 Aberrant genomic DNA methylation patterns are widely reported in human cancers but the prognostic value and pathological consequences of these marks remain uncertain. CpG methylation is catalyzed by a family of DNA methyltransferase enzymes comprised of three members – Dnmt1, Dnmt3a and Dnmt3b. Mutations in the de novo DNA methyltransferase enzyme DNMT3A have now been reported in over 20% of adult acute myeloid leukemia (AML) and 10–15% of myelodysplastic syndrome (MDS) patients. However, analysis of promoter methylation and gene expression in these patients has thus far failed to yield any mechanistic insight into the pathology of DNMT3A mutation-driven leukemia. In this study, we have used a conditional knockout mouse model to study the role of Dnmt3a in normal hematopoiesis. Hematopoietic stem cells (HSCs) from Mx1-Cre:Dnmt3afl/fl mice were serially transplanted into lethally irradiated recipient mice to study the effect of loss of Dnmt3a on HSC self-renewal and differentiation. We show that loss of Dnmt3a progressively impedes HSC differentiation over four-rounds of serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Examination of the bone marrow post-transplant revealed that control HSCs showed a gradual decline in their ability to regenerate the HSC pool at each successive round of transplantation, while in contrast Dnmt3a-KO HSCs show a remarkably robust capacity for amplification, generating 40,000 – 100,000 HSCs per mouse. Quantification of peripheral blood differentiation on a per HSC basis demonstrated in the absence of Dnmt3a, a cell division is more likely to result in a self-renewal rather than differentiation fate (Figure 1). Using semi-global reduced representation bisulfite sequencing (RRBS), we show that Dnmt3a-KO HSCs manifest both increased and decreased methylation at distinct loci, including dramatic CpG island hypermethylation. Global transcriptional analysis by microarray revealed that Dnmt3a-KO HSCs show upregulation of HSC multipotency genes coupled with simultaneous downregulation of early differentiation factors (e.g. Flt3, PU.1, Mef2c), likely inhibiting the initial stages of HSC differentiation. Upregulation of key HSC regulators including Runx1, Gata3 and Nr4a2 was associated with gene-body hypomethylation and activated chromatin marks (H3K4me3) in Dnmt3a-KO HSCs. Finally, we show that Dnmt3a-KO HSCs are unable to methylate and transcriptionally repress these key HSC multipotency genes in response to chemotherapeutic ablation of the hematopoietic system, leading to inefficient differentiation and manifesting hypomethylation and incomplete repression of HSC-specific genes in their limited differentiated progeny. In conclusion, we show that Dnmt3a plays a specific role in permitting HSC differentiation, as in its absence, phenotypically normal but impotent stem cells accumulate and differentiation capacity is progressively lost. This differentiation-deficit phenotype is reminiscent of Dnmt3a/Dnmt3b-null embryonic stem (ES) cells while markedly distinct from that of Dnmt1-KO HSCs which show premature HSC exhaustion and lymphoid-deficient differentiation, demonstrating distinct roles for the different DNA methyltransferase enzymes in HSCs. In light of the recently-identified DNMT3A mutations in AML and MDS patients, these studies are the first biological models linking mutation of Dnmt3a with inhibition of HSC differentiation which may be one of the first pathogenic steps occuring in such patients.Figure 1Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal.Figure 1. Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal. Disclosures: Issa: Novartis: Honoraria; GSK: Consultancy; SYNDAX: Consultancy; Merck: Research Funding; Eisai: Research Funding; Celgene: Research Funding; Celgene: Honoraria; J&J: Honoraria.

scholarly journals The Necessary for Long-Term Follow-up of Mutated Genes and Clonal Evolutions in Multiple Myeloma Combined with cfDNA Assay

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5515-5515
Author(s):  
Yuko Mishima ◽  
Yuji Mishima ◽  
Masahiro Yokoyama ◽  
Noriko Nishimura ◽  
Yoshiharu Kusano ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Genomic Dna ◽  
Research Funding ◽  
Somatic Mutations ◽  
Clinical Course ◽  
Bone Marrow Aspiration ◽  
Long Term Follow Up

Introduction)Somatic mutations in multiple myeloma (MM) are strongly related to the clinical outcome and clonal evolution over the clinical course, and are a major problem. From a clinical viewpoint, although numerous novel drugs have been utilized, achieving long-lasting and complete remission remains difficult. Recent studies have elucidated the mutated genes using next-generation sequencing, and have examined how clonal change can be acquired in myeloma. In this study, we traced the transition of the somatic mutations of bone marrow tumor cells in patients with MM over a long-term follow-up. Furthermore, we compared the somatic mutations found in serum cell-free DNA (cfDNA) and mutated genes obtained from bone marrow myeloma cells. Material and Methods)Patients diagnosed with multiple myeloma who provided written informed consent to participate in the study were enrolled. Patients were treated by immuno-chemotherapy with or without radiation between 2000 and 2017 at our institute. Bone marrow aspiration and biopsy were performed at the time of diagnosis and upon disease progression. Around the time of bone marrow aspiration, serum was obtained from a peripheral blood sample for cfDNA analysis. Myeloma cells were separated from bone marrow samples with MicroBeads of CD138 antibody and genomic DNA was extracted. The peripheral blood samples derived from myeloma patients. The cfDNA was extracted from the serum using a Maxwell RSC cfDNA Plasma kit. Using genomic DNA derived from cfDNA and bone marrow, multiplex polymerase chain reaction (PCR) was performed, and a sequence library was then constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using an Ion AmpliSeq DesignerTM. 126 targeted genes were selected. The genomes were sequenced using the Ion ProtonTM System. This protocol was approved by the institutional review board and the Genomic Review Board of the Japanese Foundation for Cancer Research. Result)We followed 7 patients' long term-clinical course and the transition of mutations (8.5 year average). The expression of myeloma driver genes, such as RAS, BRAF, and MYC, were not critical. We did, however, detect a relationship between an increase in the dominant mutated gene, such as TP53, DIS3, FAM46C, KDM6B, and EGR1 and poor prognosis in patients with myeloma. Next, we calculated the cfDNA concentrations from 34 cases. The cfDNA concentrations were significantly higher than 10 control cases (average 62.0 ng/mL (0-200 ng/mL) and 8.18 ng/mL (4.3-14.1 ng/mL), P=0.0046). The 2.5 year-progression free survival (PFS) during the first treatment of MM were tend to be poorer in the group with cfDNA>50 ng/mL (72.9%) than the group with cfDNA<50 ng/mL(25.9%), however there are no statistical significance (P = 0.15).We caluculated concordance rate of derived mutations from bone marrow MM cells and cfDNA in 7 cases. The somatic mutations found in serum cell-free DNA (cfDNA) and bone marrow MM cells were determined the correlation coefficients. However, there are few difference expression pattern in each source. In cfDNA assay, CREEP, EGR1, HDAC4, HDAC6, and JMJD1C were highly expressed as 57.1% (4/7) - 85.7% (6/7), and these results were almost the same as those for bone marrow MM cells. On the other hand, KDM1A (85.7%), PI3KCD (71.4%), and KDM3B (57.1%) were highly detected in cfDNA, although those were not frequently expressed in bone marrow. Discussion)Our data demonstrate the importance of the long-term follow-up of somatic mutations during the clinical course of myeloma. Serum cfDNA is a useful alternative source for detecting somatic mutations in MM patients during long-term follow-up. Disclosures Mishima: Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Yokoyama:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Nishimura:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy; Celgene K.K.: Honoraria. Hatake:Celgene K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Honoraria. Terui:Bristol-Myers Squibb K.K.: Research Funding; Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria.

scholarly journals CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1054-1054 ◽  
Author(s):  
Hongxing Liu
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Lymph Nodes ◽  
Conflicts Of Interest ◽  
Sh2 Domain ◽  
Janus Kinase ◽  
Gingival Hyperplasia ◽  
Hematopoietic Stem ◽  
T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals Idiopathic myelofibrosis mimicking hemolytic anemia

2012 ◽  
Vol 2 (4) ◽  
pp. 324-327
Author(s):  
R Baral ◽  
G Aryal ◽  
KC Shiva Raj
Keyword(s):  
Bone Marrow ◽  
Clinical Course ◽  
Myeloproliferative Disorder ◽  
Severe Anemia ◽  
Idiopathic Myelofibrosis ◽  
Blood Picture ◽  
Bone Marrow Fibrosis ◽  
Hematopoietic Stem ◽  
Chronic Myeloproliferative Disorder ◽  
Marrow Fibrosis

Idiopathic Myelofibrosis is an infrequent chronic myeloproliferative disorder characterized by varying degrees of bone marrow fibrosis and extra medullary hematopoiesis, with the fibrosis being a reactive phenomenon to a neoplastic proliferation of a pluripotent hematopoietic stem cell. Idiopathic Myelofibrosis is heterogeneous in presentation and clinical course, with anemia being one of the most important problems. We present a case of a 59 year old male who presented with severe anemia, the peripheral blood picture mimicking hemolysis with numerous schistocytes and teardrop cells.Journal of Pathology of Nepal (2012) Vol. 2, 323-327DOI: http://dx.doi.org/10.3126/jpn.v2i4.6888

Frequent CD7 Antigen Loss in Aggressive Natural Killer-Cell Leukemia Can Be a Useful Diagnostic Marker.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2400-2400
Author(s):  
Sun-Hee Kim ◽  
Eun-Hyung Yoo ◽  
Hee-Jin Kim ◽  
Won Seog Kim
Keyword(s):  
Bone Marrow ◽  
Natural Killer Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Diagnostic Marker ◽  
Clinical Course ◽  
Killer Cell ◽  
Cell Leukemia ◽  
Laboratory Findings ◽  
Antigen Loss

Abstract Aggressive natural killer-cell leukemia (ANKL) is a rare neoplasm characterized by systemic proliferation of NK cells with rapidly progressive clinical course and fatal outcome. Because of the aggressive clinical course, rapid and accurate diagnosis of ANKL is critical. However, the differential diagnosis of NK cell lymphoproliferative disorders including hemophagocytic lymphohistiocytosis is still challenging in the absence of a distinct diagnostic hallmark. Furthermore, cases with a low burden of malignant cell polpuation makes it more difficult. To find any diagnostic markers in ANKL, we analyzed clinical data and laboratory findings from bone marrow studies in Korean patients with bone marrow involvement of ANKL. From January 2000 to July 2007, a total of 20 cases were diagnosed with ANKL based on morphologic and immunophenotypic findings from bone marrow studies. The leukemic cells were surface CD3–CD16/56+ large granular lymphocytes with pale or lightly basophilic cytoplasm containing azurophilic granules. We retrospectively analyzed clinical features and laboratory findings including complete blood count (CBC), Epstein-Barr virus (EBV) status, serum lactate dehydrogenase (LDH) level, immunophenotype, and cytogenetic results from medical records. There were 6 (30%) women and 14 (70%) men with a median age of 44 years (range, 2–70 years). Hepatomegaly (70%), splenomegaly (60%), and lymphadenopathy (30%) were frequently observed. Peripheral blood counts were variable; anemia (hemoglobin &lt;10g/dL) was predominant in 14 patients and thrombocytopenia (platelet &lt;100×109/L) in 16. The proportion of leukemic NK cells ranged 3∼70%. EBV was detected in 15 of 18 cases (83%) by EBV in situ hybridization or EBV quantitative PCR. Cytogenetic studies were performed in 18 cases, and karyotypic abnormalities were observed in 50% (9/18). There were no recurrent cytogenetic abnormalities, except 6q abnormalities observed in 4 cases (4/18, 22%). The immunophenotype of the leukemic NK cells by flow cytometry was cytoplasmic CD3+, surface CD3−, CD16/56+, CD2+, and CD5−. Most cases were CD4− (13/16, 81%) and CD8− (11/14, 79%). Of note, loss of CD7 antigen was observed in 10 patients (10/20; 50%) (normal NK cells: CD2+, CD7+, and CD5−). There were no significant differences in clinical or laboratory parameters between the CD7+ and CD7− groups. All three cases with deletion of 6q revealed absent expression of CD7. When the CD7 loss was combined with cytogenetic abnormalities, clonal markers could be identified in 75% of ANKL cases. We observed frequent CD7 antigen loss in our series of Korean patients with ANKL. This characteristic immunophenotypic finding can provide a reliable and timely information as a diagnostic marker in ANKL along with cytogenetic findings. Therefore, immunophenotypic analysis of the expression of CD7 should be included in the diagnostic workup of NK cell neoplasms.

The Effects of Rapamune on NK Cell Post Bone Marrow Transplantation in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4858-4858
Author(s):  
Guanghua Chen ◽  
De Pei Wu ◽  
Ming Zhen Yang ◽  
Xiao Wen Tang ◽  
Ai-ning Sun
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Nk Cells ◽  
Marrow Transplantation ◽  
Nk Cell ◽  
Immunosuppressive Agents ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Hematopoietic Stem ◽  
Effector Cells

Abstract Natural killer(NK) cells are innate effector cells of the immune system, believed to limit viremia and tumor burden before the onset of adaptive T and B cell immunity. NK cells are potent effector cells in allogeneic bone marrow transplantation. NK cell activity is partially controlled through interactions between killer Ig-like receptors on NK cells and their respective HLA class I ligands. Immunosuppressive agents including cyclosporin, FK506 and Rapamune are utilized in clinical hematopoietic stem cell transplantation. Little is known about the effects of immunosuppressive agents on NK cells post bone marrow transplantation. The in vivo effects of Rapamune on NK cells were determined in an allogeneic bone marrow transplantation model. Splenic NK cell levels in recipients treated with Rapamune decrease markedly. NK cell proliferation and function are significantly decreased in the presence of Rapamune. Studying the differential effects of immunosuppressive drugs on NK cell function is critical in clinical hematopoietic stem cell transplantation.

Synergistic Action of the Human Inhibitory KIR Antibody IPH2102, and the Human CD38 Antibody Daratumumab to Enhance the Lysis of Primary Multiple Myeloma (MM) Cells in the Bone Marrow Mononuclear Cells (MNCs) From Myeloma Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1865-1865
Author(s):  
Inger S. Nijhof ◽  
Michel de Weers ◽  
Pascale Andre ◽  
Berris van Kessel ◽  
Henk M. Lokhorst ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Human Monoclonal Antibody ◽  
Cell Activity

Abstract Abstract 1865 Despite significant improvements in the treatment of multiple myeloma (MM), this progressive malignancy of antibody-producing clonal plasma cells is still considered incurable. New innovative treatments need to be developed to improve long term outcomes. Recent successes of CD20 antibodies in the clinical lymphoma management indicate that targeted immunotherapy can represent a powerful therapeutical strategy for hematological malignancies. Towards developing a similar strategy for MM, we have recently generated a novel human monoclonal antibody, daratumumab (DARA), which targets the CD38 molecule expressed at high levels on MM cells. We have demonstrated that DARA mediates the lysis of CD38+ MM cells via direct apoptosis, complement mediated lysis and antibody-dependent cell mediated cytotoxicity (ADCC). Natural killer (NK) cells appeared important effector cells mediating the ADCC effect. Since NK cell activity against tumor cells is regulated by the balance of signals generated by inhibitory or activating receptors of NK cells (KIRs), we now explored whether blocking the inhibitory KIRs would improve the NK cell mediated DARA dependent lysis of MM cells. Thus, we evaluated the potential benefits of combining DARA with a novel human anti KIR monoclonal antibody, IPH2102, which blocks the inhibitory KIR2DL1/2/3 receptors (HLA-C specific KIRs), and has been shown to augment NK cell function against MM cells. We recently developed FACS-based ex vivo MM cell lysis assays, in which DARA-dependent NK cell-mediated lysis of MM cells can be directly measured in bone marrow MNCs, thus without separating the malignant cells from autologous NK cells and other accessory cells. Using these, we investigated whether the addition of IPH2102 would augment the DARA dependent lysis of MM cells. As expected, DARA induced lysis of MM cells in bone marrow MNCs isolated from MM patients (n=10). Mean lysis at 10 μg/ml DARA was 27.6% (range 11.3–48.1%). IPH2102 showed little or no lysis of MM cells (at 0.3, 1, 3 and 10 μg/ml) in this setting. The combination of 10 μg/ml IPH2102 with 3 and 10 μg/ml DARA significantly enhanced cytotoxicity against primary MM tumor cells compared to DARA alone (p=0.013 and p=0.028 respectively). Mean lysis of MM tumor cells at 10 μg/ml DARA and 10 μg/ml IPH2102 was 38%. These data confirm our previous findings that NK-cell mediated killing is an important mechanism of action of DARA. We demonstrate a clear synergy between DARA and IPH2102 to achieve effective lysis of MM cells directly in the bone marrow MNC of MM patients, indicating that complementary effects may be achieved by combining IPH2102 and DARA in clinical MM management. Disclosures: Weers: Genmab: Employment. Andre:Innate Pharma: Employment. Lokhorst:Genmab: Research Funding. Parren:Genmab: Employment. Morel:Innate Pharma: Employment. Mutis:Genmab: Research Funding.

Leukemia-Induced Alterations in Bone Marrow Cytokine and Chemokine Levels Contribute to Altered Stem Cell Lodgment and Impairment of Normal Stem Cell Growth in CML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 962-962
Author(s):  
Bin Zhang ◽  
Yin Wei Ho ◽  
Tessa L. Holyoake ◽  
Claudia S Huettner ◽  
Ravi Bhatia
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mouse Model ◽  
Hematopoietic Stem Cell ◽  
Stromal Cells ◽  
Research Funding ◽  
Mrna Levels ◽  
Hematopoietic Stem ◽  
Tnf Α ◽  
Selective Impairment

Abstract Abstract 962 Specialized bone marrow (BM) microenvironmental niches are essential for hematopoietic stem cell (HSC) lodgment and maintenance. However microenvironmental interactions of leukemia stem cells (LSC) are poorly understood. Although chronic myelogenous leukemia (CML) results from HSC transformation by the BCR-ABL gene, the role of the microenvironment in modulating leukemia development is not known. We employed the SCL-tTA-BCR/ABL mouse model of CML to investigate the LSC interactions with the BM microenvironment. In this model, targeted expression of the BCR-ABL gene in murine HSC via a tet-regulated SCL promoter results in development of a chronic phase CML-like disorder. We have reported that LSC capacity is restricted to BCR-ABL+ cells with long-term hematopoietic stem cell (LTHSC) phenotype(LSK Flt3-CD150+CD48-) (Blood 2010 116:1212A). LSC numbers are reduced in the BM but increased in the spleen of CML mice compared with LTHSC from control mice, suggesting that LSC have altered niche interactions. LSC also demonstrate altered trafficking with significant reduction in homing of IV injected LSC to BM, and markedly increased egress of intrafemorally injected LSC to the spleen, potentially related to reduced CXCL12 levels in the BM of CML mice. In addition, levels of several chemokines and cytokines, including MIP1α, MIP1β, MIP2, IL-1α, IL-1β, TNF-α, G-CSF and IL-6, were increased in CML BM, related to increased production by malignant hematopoietic cells. We investigated whether altered chemokine and cytokine expression was associated with altered capacity of the CML BM microenvironment to support LTHSC engraftment. LTHSC from control mice or LSC from CML mice were transplanted into irradiated CML or control recipients. There was reduced engraftment of both control LTHSC and CML LSC in the BM of CML compared to control recipients at 2 weeks after transplantation, associated with reduced homing to CML BM, potentially related to low BM CXCL12 levels. The numbers of control LTHSC in the BM of CML recipient mice remained low at 4 weeks post-transplantation, whereas the numbers of CML LSC increased to numbers similar to those seen in the BM of control recipients. Culture with CML BM supernatants (SN) resulted in impaired growth of control LTHSC compared to control BM SN. In contrast the growth of CML LSC was similar following culture with CML and control BM SN. Culture with individual factors at concentrations similar to those observed in CML BM (16ng/ml MIP1α, 8ng/ml MIP1β, 2.5ng/ml IL-1α, 3.5ng/ml IL-1β, 0.05ng/ml TNF-α) resulted in significantly reduced growth of normal LTHSC compared with CML LSC. These results indicate that diffusible factors produced by leukemic cells in the CML BM environment selectively inhibit normal LTHSC compared to CML LSC growth. Exposure of a murine stromal cell line to CML BM SN resulted in reduced CXCL12 mRNA levels compared to BM SN from control mice. The cytokine G-CSF, which was increased in CML BM SN, has been reported to reduce CXCL12 transcription. We observed significant reduction of CXCL12 mRNA levels in stromal cells cultured with G-CSF (0.2ng/ml), supporting a potential role for increased G-CSF production by leukemia cells in reduced CXCL12 production by CML BM stromal cells and reduced LSC retention in the BM. We evaluated whether defects in microenvironmental function in CML were affected by imatinib treatment. Treatment of CML mice with imatinib (200mg/kg/day, 2 weeks) led to reduction in MIP1α, MIP1β, IL-1β, and IL-6 levels in BM cells. Engraftment of normal LTHSC was significantly enhanced in BM of CML recipients pre-treated with imatinib. Results obtained with the mouse model were validated using specimens obtained from CML patients. CXCL12 mRNA levels were significantly reduced in human CML compared to normal MNCs, whereas expression of MIP1α, MIP-2, IL-1α and IL-1β were increased in CML MNCs, consistent with results obtained with the mouse model. Coculture with CML MNC conditioned medium (CM) resulted in selective impairment of growth of normal CD34+CD38- primitive progenitors compared to CM from normal MNC, but did not inhibit growth of CML progenitors. We conclude that leukemia-induced alterations in BM cytokine and chemokine levels contribute to altered LSC lodgment and to selective impairment of growth of normal LTHSC in the CML BM microenvironment, leading to a relative growth advantage for CML LSC over normal LTHSC and expansion of the leukemic clone. Disclosures: Holyoake: Novartis: Research Funding; Bristol Myers Squibb: Research Funding.

Natural Killer (NK) Cell Reconstitution After Haploidentical Unmanipulated Bone Marrow Transplantation with Reduced Intensity Conditioning

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4557-4557 ◽  
Author(s):  
Isabel Gonzalez-Gascon y Marin ◽  
Ana Maria Perez-Corral ◽  
Jorge Gayoso ◽  
Javier Anguita ◽  
Cristina Pascual ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Median Number ◽  
Reduced Intensity Conditioning ◽  
Hematopoietic Stem ◽  
Reduced Intensity

Abstract Abstract 4557 BACKGROUND: Natural killer (NK) cells are innate immune effectors that directly lyse virally infected or malignant cells. There are 2 different subsets of NK cells with distinct phenotypic and functional characteristics: the CD56dim subset, which composes 90% of peripheral blood NK cells and has a cytotoxic function, and the CD56bright subset, which cooperates with dendritic cells and T cells in lymph nodes to secrete interferon and promote adaptive immune responses. NK cells are the first donor-derived lymphocyte subset to reconstitute after hematopoietic stem cell transplantation, reaching normal levels after 1 month. Nearly all phenotyping studies of NK subsets after haploidentical hematopoietic stem cell transplantation (HHSCT) reveal a rapid reconstitution of NK cells towards the CD56bright subset. In addition, Y.-J. Chang et al found the highest 2-year survival in patients with a high number of CD56bright NK cells after unmanipulated HHSCT. We analyzed reconstitution of the NK compartment between days 90 and 180 after unmanipulated bone marrow HHSCT with reduced intensity conditioning (RIC). METHODS: Six adults received unmanipulated bone marrow HHSCT after RIC (fludarabine 30 mg/m2 [day –6 to –2], cyclophosphamide 14.5 mg/kg [day –6 and –5], and busulfan i.v. 3.2mg/kg [day –3]) at our institution between July 2007 and July 2010. Prophylaxis for acute graft-versus-host disease (GvHD) consisted of cyclophosphamide 50mg/kg (days +3 and +4) and cyclosporine A and mycophenolate mofetil from day +5 onwards. We monitored the reconstitution kinetics of circulating NK cells (CD56+, CD3–), and the CD56bright and CD56dim subsets by multiparametric flow cytometry (FC 500 Beckman® Coulter) at day +90 and day +180 after transplantation. Patient characteristics and clinical outcomes are shown in Table 1. 6 patients who underwent allogeneic HLA-identical sibling HSCT with RIC during the same period were used as controls. RESULTS: After HHSCT, NK cells reached normal levels in all patients but one at day +90, with a median number of NK cells of 111/mm3 (range, 25–195/mm3). At day +180 the median number of NK cells was 92/mm3 (range, 4–272/mm3). When we analyzed the absolute number of CD56bright and CD56dim subsets at day +90, we observed 2 patterns: Two patients showed skewed NK cell reconstitution towards CD56bright (Patient no. 3: 54 CD56bright/mm3; 11 CD56dim/mm3. Patient no. 4: 70 CD56bright/mm3; 17 CD56dim/mm3). Three patients reconstituted with a CD56dim/CD56bright ratio towards the CD56dim cell subset, similar to that of healthy adults (Patient no. 1: 17 CD56bright/mm3; 178 CD56dim/mm3. Patient no. 5: 9 CD56brigh/mm3; 135 CD56dim/mm3. Patient no. 6: 20 CD56bright/mm3; 116 CD56dim/mm3). One patient did not achieve adequate NK cell reconstitution (Patient no. 2: 15 CD56bright/mm3; 10 CD56dim/mm3). In contrast, in the control group, an increase in the CD56bright NK cell subset was not observed in any of the patients at any point. It is worth noting that 2 of the 3 patients with better clinical outcome (no GvHD, no relapse), namely patients no. 3 and no. 4 were the ones with skewed NK cell reconstitution towards the CD56bright NK cell subset. The other patient with a better clinical outcome (patient no. 6) had a normal CD56dim/CD56bright ratio at day +90. However, he showed an early CD56bright reconstitution (363 CD56bright/mm3; 34 CD56dim/mm3) in an additional determination on day +30. NK cell subsets reconstitution kinetics is shown in Figure 1. CONCLUSIONS: In our experience, NK cell reconstitution is adequate after RIC unmanipulated bone marrow HHSCT. Some patients recovered with a high proportion of CD56bright NK cells, as previously reported in other studies on HHSCT. Although limited by the sample size, our results are consistent with the previously observed survival advantage of patients with high early levels of CD56bright NK cells after unmanipulated haploidentical transplantation. Disclosures: No relevant conflicts of interest to declare.

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