scholarly journals Concomitant Occurrence of Polyclonal Hematopoiesis and Cell-Autonomous Megakaryopoiesis in Triple-Negative Essential Thrombocythemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-28
Author(s):  
Tadaaki Inano ◽  
Marito Araki ◽  
Soji Morishita ◽  
Misa Imai ◽  
Yoshihiko Kihara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Genomic Dna ◽  
Research Funding ◽  
Somatic Mutations ◽  
Triple Negative ◽  
Jak2 V617f ◽  
Sequencing Analysis ◽  
Hematopoietic Stem ◽  
Otsuka Pharmaceutical

Somatic mutations in JAK2, MPL, and CALR are found in approximately 80% of patients with essential thrombocythemia (ET), whereas the remaining patients are negative for disease-defining mutations and are defined as triple-negative (TN). Studies have shown that some patients with TN-ET harbor non-canonical mutations in JAK2 and MPL; however, the failure to identify recurrent mutations in most patients has made the pathogenesis of TN-ET ambiguous (Milosevic Feenstra et al. Blood 2016, Cabagnols et al. Blood 2016). In this study, we screened 483 patients suspected as having ET in a single center, performed mutation analysis for JAK2 V617F, CALR exon 9, and MPL exon 10, and centrally reviewed bone marrow specimens. We identified 23 patients with TN-ET based on the WHO 2016 criteria. Sequencing analysis of these patients revealed non-canonical mutations in JAK2 and MPL in 4 cases. Whole exome-sequencing analysis of genomic DNA from peripheral blood and CD3-positive cells from 9 patients revealed that 2 patients harbored somatic mutations in other genes; 7 patients showed no detectable somatic mutation. A STAT5 reporter assay revealed that unlike JAK2 V617F and MPL W515L, all non-canonical mutants of JAK2 or MPL activated STAT5 similar to wild-type proteins, suggesting that these mutations did not drive the disease. Statistical analysis of clinical records revealed that patients with TN-ET were mostly young (median age of 36.0 years), female (18/23, 78.3%), and had neither a history of thrombosis nor progression to secondary myelofibrosis and leukemia, demonstrating the unique characteristics of TN-ET. The presence of clonal hematopoiesis, analyzed using genomic DNA purified from granulocytes of peripheral blood from female patients in a human androgen receptor assay, revealed that only 1 out of 15 patients was clonal. Hypothesizing that TN-ET was reactive thrombocytosis, the concentrations of cytokines promoting platelet production such as thrombopoietin (TPO) and interleukin-6 (IL-6) in the serum were analyzed. However, no significant differences in concentrations were observed among ET with driver mutations, TN-ET, and healthy individuals. We next examined the capacity of hematopoietic stem cells from patients with TN-ET to form megakaryocytic colonies. CD34-positive cells purified from cryopreserved bone marrow cells were cultured in the absence or presence of TPO. CD34-positive cells derived from patients with TN-ET exhibited an equivalent capacity to form megakaryocytic colonies compared to those from patients with ET harboring a driver mutation, even in the absence of TPO (Figure 1). Thus, in TN-ET, megakaryopoiesis may have been induced in a cell-autonomous manner. In 10 patients with TN-ET with available blood count data, no sign of thrombocytosis was observed before ET development, indicating that thrombocytosis was not hereditary but rather occurred via an alternate mechanism, such as aberrations in epigenomic regulation that induced cellular transformation (Ohnishi et al, Cell 2015). Taken together, TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis. Figure 1 Disclosures Komatsu: Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy.

Ifnα Attenuates the Disease Phenotype and Extends Survival in Mouse Models of MPN

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 53-53
Author(s):  
Harini Nivarthi ◽  
Andrea Majoros ◽  
Eva Hug ◽  
Ruochen Jia ◽  
Sarada Achyutuni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mouse Models ◽  
Jak2 V617f ◽  
Type I ◽  
Type I Ifn ◽  
Hematopoietic Stem ◽  
Exon 9 ◽  
Ifn Treatment

The curative potential of Type I interferons for patients suffering from Myeloproliferative Neoplasms (MPNs) has been reported and these are the only class of drugs that can lead to reduction of the mutant allelic burden in patients. However, modelling IFN treatment in mice has been challenging. Here, we report the use of murine pegylated IFNα (murine ropeginterferon-a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) to model IFN treatment in transgenic MPN mouse models. We started treating JAK2V617Ff/+;vavCre and control vavCre mice (n=6-8) with PBS or mRopeg (600 ng/mouse/week), by subcutaneous injections from the time they were 4 weeks old. The mice were bled every 2 weeks from the facial vein and the blood parameters were monitored. We observed significant normalization of platelet and WBC counts in Jak2-V617F fl/+ vavCre mice to wild type levels. No effect on hematocrit and hemoglobin level was observed in the Jak2-V617F fl/+ vavCre mice. VavCre control animals showed no sign of negative effect such as cytopenia during the entire treatment course. We observed a highly significant prolongation of the survival of mRopeg treated JAK2V617Ff/+;vavCre mice over a duration of 80 days of treatment. While all the PBS treated JAK2V617Ff/+;vavCre mice died within 60 days, all the mRopeg treated mice were still alive till the end of the treatment duration. We also generated a novel transgenic mouse model that conditionally expresses hybrid mutant CALR protein (murine exons 1-8 and human CALR del52 exon9) from the endogenous murine Calr locus. We bred them into vavCre background (in both heterozyhous and homozygous states) to induce expression of CALR-del52 in hematopoietic cells. Upon Cre recombinase expression, the endogenous murine exon 9 is replaced by the human del52 exon 9 and the expression of the humanized Calr-del52 oncoprotein is detectable by Western blot analysis using mutant CALR specific antibodies. Calr-del52 animals develop an essential thrombocythemia (ET) like phenotype when expressed in a heterozygous state with elevated number of hematopoietic stem cells and megakaryocytes in the bone marrow. In the homozygous state, the thrombocythemia is more severe with splenomegaly and older animals show anemia with increased WBC. Bone marrow histology shows megakaryocytic hyperplasia with no sign of fibrosis up to age of one year. We treated a cohort of animals with 600 ng mRopeg/PBS once a week for 4 weeks. Peripheral blood counts were determined at baseline and at regular intervals during treatment. At the end of treatment, mice were sacrificed, and splenic and bone marrow cells were immunophenotyped and quantified by FACS. We observed correction of thrombocythemia in the homozygous Calr-del52 mice but no unspecific decrease of platelet count in the vavCre mRopeg treated animals. We observed significant specific reduction of the long-term hematopoietic stem cells (LT-HSCs/fraction A) in homozygous CALR-del52 mice. In conclusion, Type I IFN treatment significantly reduces platelet counts to normal levels in both JAK2 and CALR mutant driven MPN mouse models. The prolongation of survival of JAK2V617F transgenic mice upon Type I IFN treatment is particularly remarkable; as no survival data is reported until now in any clinical trials or other animal models. Further experiments are required to understand the mechanism of action of this phenomenon. Disclosures No relevant conflicts of interest to declare.

scholarly journals Absence of Evidence Implicating Hematopoietic Stem Cells As Common Progenitors for DLBCL Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4107-4107
Author(s):  
Max Jan ◽  
Florian Scherer ◽  
David M. Kurtz ◽  
Aaron M Newman ◽  
Henning Stehr ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
B Cells ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Phylogenetic Trees ◽  
Somatic Mutations ◽  
Hematopoietic Stem ◽  
Tumor Biopsies ◽  
Myd88 L265p

Abstract Background: Pre-leukemic hematopoietic stem cells (HSC) have been implicated in AML (Jan et al STM 2012) and also for several lymphoid leukemias including ALL, HCL, and CLL. Separately, relapse of ALL following CD19 CAR-T cell therapy has been associated with lymphomyeloid lineage switch. Finally, healthy persons with clonally expanded HSCs are at increased risk of hematologic malignancies including lymphomas, and in mouse DLBCL models we previously demonstrated the oncogenic sufficiency of BCL6 overexpression in HSC (Green et al 2014 Nat Comm). Nevertheless, the cellular origin of DLBCL in the majority of patients is not definitively known. We sought to investigate the presence of mutations found in DLBCL within matched HSCs. Methods: We deeply genotyped somatic mutations in diagnostic biopsy tissues of 16 patients with DLBCL using CAPP-Seq to a median sequencing depth of 1100x (Newman et al 2014 Nat Med; Scherer et al 2015 ASH). We then profiled each patient for evidence implicating HSCs using somatic mutation lineage tracing, in either direct or indirect fashion. For direct evaluation, we used highly purified, serially FACS-sorted HSCs from grossly uninvolved bone marrow (BM) (n=5; Fig 1a-b). For indirect assessment, we either profiled serial tumor biopsies (n=13), or interrogated sorted cells from terminally differentiated blood lineages (n=7), including peripheral CD3+ T cells, CD14+ Monocytes, and B cells expressing a light-chain discordant to that of tumor isotype. HSCs and differentiated lineages were then interrogated by direct genotyping, using 3 highly sensitive orthogonal quantitative methods, including Myd88 L265P droplet digital PCR (n=6), BCL6 translocation breakpoint qPCR (n=4), and DLBCL CAPP-Seq profiling of 268 genes (n=5). We used the theoretical limit of detection (LOD) genotyping performance for CAPP-Seq (0.001%, Newman et al 2016 Nat Biotech), and established analytical sensitivity of our custom MYD88 ddPCR via limiting dilution (~1%). These LODs met or exceeded the expected limit of sorting impurity by FACS (~1%). For 6 patients experiencing one or more DLBCL relapse, we deeply profiled 13 serial tumor biopsies by CAPP-Seq, and then assessed overlap in somatic mutations and VDJ sequences in biopsy pairs as additional indirect evidence implicating HSCs. Results: We obtained a median of ~2000 sorted HSCs and ~1700 sorted cells from differentiated lineages, and genotyped each population using one or more of the 3 direct genotyping methods described above. Three patients with sufficient cell numbers were profiled both by CAPP-Seq and either ddPCR (n=2) or qPCR (n=1). Surprisingly, we found no evidence implicating HSCs either directly or indirectly in any of the 16 patients, regardless of the assay employed or the cell types/lineages genotyped (e.g., Fig 1b). In 2 patients with MYD88 L265P mutations, we found evidence for MYD88+ B-cells with discordant light chains by ddPCR (~0.1%) potentially implicating common lymphoid precursors (CLPs), but found no evidence for similar involvement of T-cells or monocytes. In 6 DLBCL patients experiencing relapse, tumor pairs profiled by CAPP-Seq (median depth 957) shared 93% of somatic mutations (75-100%, Fig 1c). Such pairs invariably shared clonal IgH VDJ rearrangements (4/4, 100%), thus implicating a common progenitor arising in later stages of B-cell development, not HSCs. Conclusions: We find no evidence to implicate HSCs in the derivation of DLBCL. While formal demonstration of absence of pre-malignant HSCs in DLBCL would require overcoming practical and technical limitations (including number of available HSCs, sorting purity, and genotyping sensitivity), the pattern of shared somatic alterations at relapse makes this highly unlikely. We speculate that unlike lymphoid leukemias, the cell-of-origin for most DLBCLs reside later in B-lymphopoiesis, beyond CLPs. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Disclosures Newman: Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy; Roche: Consultancy; Varian Medical Systems: Research Funding.

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.

Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 350-350
Author(s):  
Kyung-Hee Chang ◽  
Amitava Sengupta ◽  
Ramesh C Nayak ◽  
Angeles Duran ◽  
Sang Jun Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Negative Regulator ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

Bone Marrow (BM) Microenviroment Factors As Early Markers of Response in Patients with Newly Diagnosed Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Treated with Nilotinib

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1570-1570
Author(s):  
Santa Errichiello ◽  
Simona Caruso ◽  
Concetta Quintarelli ◽  
Biagio De Angelis ◽  
Novella Pugliese ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Myelogenous Leukemia ◽  
Hematopoietic Progenitor Cell ◽  
Newly Diagnosed ◽  
Hematopoietic Stem ◽  
Optimal Response

Abstract Introduction Tyrosine Kinase Inhibitors (TKI) have completely changed the scenario of CML and dramatically improved the outcomes. Thus, early identification of patients expecting poor outcome is crucial to offer alternative TKI regimens or in some selected cases stem cell transplantation before disease progression may occur. The Evaluating Nilotinib Efficacy and Safety in Trial as First-Line Treatment (ENEST1st) is a phase 3b is an open-label study of nilotinib 300 mg twice daily (BID) in adults with newly diagnosed BCR-ABL positive CP-CML. Aim of the ENEST1st sub-study N10 was to investigate BM microenvironment markers that regulate leukemic stem cells in the bone marrow (BM) niche of Nilotinib-treated patients. Methods The study enrolled patients in 21 Italian ENEST1st participating centers. Response was based on ELN recommendations (Baccarani M, et al. Blood 2013 122:872-884). In an interim analysis, molecular and cytogenetic response by 24 months was assessed. Mononuclear cells were collected from BM and PB samples at the screening visit (V0) and after 3 months of treatment (V4). RT-qPCR for the expression of 10 genes (ARF, KIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the stemness and hematopoietic stem cells survival signaling regulation was conducted. RT-qPCR data were normalized by the expression of GUS mRNA (normalized copy number, NCN). Plasma samples were collected at different time points from both BM or PB samples. Concentrations of 20 different analytes, including IL-1a, IL-3, M-CSF, SCF, SDF1-a, TRAIL, HGF, PDGF-bb, IL1b, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, G-CSF, GM-CSF, MIP-1a, TNF-a, and VEGF, were simultaneously evaluated using commercially available multiplex bead-based sandwich immunoassay kits. Results 33 out of 37 patients enrolled were available for an interim molecular analysis at 24 months: an optimal response was achieved in 25 patients, a warning response in 5 patients and a failure response in 3 patients. We observed a significant correlation between the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) or cytokine signaling (SET) and patient outcome. Indeed, NANOg and SET mRNA were significantly down-regulated in PB samples at diagnosis of patients with optimal response compared to patients with warning/failure response (NANOg mRNA: 0.3±0.25 NCN vs 0.6±0.7 NCN, respectively; p=0.05; SET mRNA: 0.2±0.3 NCN vs 2.3±4.2 NCN, respectively; p=0.03). We also investigated the plasma level of several factors involved in the hematopoietic stem cells (HSCs). Some of these markers showed a significant correlation with patient's outcome when evaluated at diagnosis in either PB or BM samples. Indeed, high level of IL12 (in the BM samples), or HGF, mCSF and SCF (in the PB samples) were associated to a worst prognosis markers, since significantly correlating with no MMR@12months (IL12, p=0.03), intermediate/high Socal score (mCSF, p=0.03; SCF, p=0.03), no reduction of MMR below to 1 at 3 month (SCF, p=0.04) or warning/failure response to Nilotinib treatment (HGF, p=0.03; SCF, p=0.04). Indeed, we find a lower levels of PDGFb, SDF1, TNFa, TRAIL (in the BM samples), and HGF, SDF1, TRAIL (in the PB samples) in those patients with intermediate/high Hasford or Sokal score (PDGFb, p=0.0007; SDF1, p=0.02), warning/failure response to Nilotinib treatment (HGF, p=0.03) or lacking of MMR4.0 (SDF1, p=0.01; TNFa, p=0.02; TRAIL, p=0.05). Conclusion/Summary Taken together, our results suggest that the expression analysis of genes involved in cell pluripotency (NANOg) and/or cell signaling (SET) at baseline, may indicate early achievement of deep molecular response in shown CML-CP patients treated with nilotinib. In addition, in patients with optimal response to Nilotinib, high concentration of SDF-1, TRAIL (inversely correlated with BCR-ABL, and associated to an higher susceptibility to apoptosis in the leukemic blasts) were observed as well as BM TNF (cell-extrinsic and potent endogenous suppressor of HSC activity). A lower concentration of several factors associated to hematopoietic progenitor cell growth and survival (including HGF, SCF and IL12) were observed compared to patients failing to achieve response to Nilotinib. These data strongly suggest that stromal microenvironment supports the viability of BCR-ABL cells in BM niches through direct feeding, or environment releasing of survival factors. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Galimberti:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Profiling the Genomic Landscape at the Early Stages of CLL: Low Genomic Complexity and Paucity of Driver Mutations in MBL and Indolent CLL

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3214-3214 ◽  
Author(s):  
Andreas Agathangelidis ◽  
Viktor Ljungström ◽  
Lydia Scarfò ◽  
Claudia Fazi ◽  
Maria Gounari ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Research Funding ◽  
Somatic Mutations ◽  
Driver Mutations ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Driver Genes ◽  
Hematopoietic Stem ◽  
Genomic Complexity ◽  
Targeted Deep Sequencing

Abstract Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B cell lymphocytosis (MBL), characterized by the presence of monoclonal CLL-like B cells in the peripheral blood, yet at lower numbers than those required for the diagnosis of CLL. MBL is distinguished into low-count (LC-MBL) and high-count (HC-MBL), based on the number of circulating CLL-like cells. While the former does not virtually progress into a clinically relevant disease, the latter may evolve into CLL at a rate of 1% per year. In CLL, genomic studies have led to the discovery of recurrent gene mutations that drive disease progression. These driver mutations may be detected in HC-MBL and even in multipotent hematopoietic progenitor cells from CLL patients, suggesting that they may be essential for CLL onset. Using whole-genome sequencing (WGS) we profiled LC-MBL and HC-MBL cases but also CLL patients with stable lymphocytosis (range: 39.8-81.8*109 CLL cells/l) for >10 years (hereafter termed indolent CLL). This would refine our understanding of the type of genetic aberrations that may be involved in the initial transformation rather than linked to clinical progression as is the case for most, if not all, CLL driver mutations. To this end, we whole-genome sequenced CD19+CD5+CD20dim cells from 6 LC-MBL, 5 HC-MBL and 5 indolent CLL cases; buccal control DNA and polymorphonuclear (PMN) cells were analysed in all cases. We also performed targeted deep-sequencing on 11 known driver genes (ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, FBXW7) in 8 LC-MBL, 13 HC-MBL and 7 indolent CLL cases and paired PMN samples. Overall similar mutation signatures/frequencies were observed for LC/HC-MBL and CLL concerning i) the entire genome; with an average of 2040 somatic mutations observed for LC-MBL, 2558 for HC-MBL and 2400 for CLL (186 for PMN samples), as well as ii) in the exome; with an average of non-synonymous mutations of 8.9 for LC-MBL, 14.6 for HC-MBL, 11.6 for indolent CLL (0.9 for PMN samples). Regarding putative CLL driver genes, WGS analysis revealed only 2 somatic mutations within NOTCH1, and FBXW7 in one HC-MBL case each. After stringent filtering, 106 non-coding variants (NCVs) of potential relevance to CLL were identified in all MBL/CLL samples and 4 NCVs in 2/24 PMN samples. Seventy-two of 110 NCVs (65.5%) caused a potential breaking event in transcription factor binding motifs (TFBM). Of these, 29 concerned cancer-associated genes, including BTG2, BCL6 and BIRC3 (4, 2 and 2 samples, respectively), while 16 concerned genes implicated in pathways critical for CLL e.g. the NF-κB and spliceosome pathways. Shared mutations between MBL/CLL and their paired PMN samples were identified in all cases: 2 mutations were located within exons, whereas an average of 15.8 mutations/case for LC-MBL, 8.2 for HC-MBL and 9 for CLL, respectively, concerned the non-coding part. Finally, 16 sCNAs were identified in 9 MBL/CLL samples; of the Döhner model aberrations, only del(13q) was detected in 7/9 cases bearing sCNAs (2 LC-MBL, 3 HC-MBL, 2 indolent CLL). Targeted deep-sequencing analysis (coverage 3000x) confirmed the 2 variants detected by WGS, i.e. in NOTCH1 (n=1) and FBXW7 (n=1), while 4 subclonal likely damaging variants were detected with a VAF <10% in POT1 (n=2), TP53 (n=1), and SF3B1 (n=1) in 4 HC-MBL samples. In conclusion, LC-MBL and CLL with stable lymphocytosis for >10 years display similar low genomic complexity and absence of exonic driver mutations, assessed both with WGS and deep-sequencing, underscoring their common low propensity to progress. On the other hand, HC-MBL comprising cases that may ultimately evolve into clinically relevant CLL can acquire exonic driver mutations associated with more dismal prognosis, as exemplified by subclonal driver mutations detected by deep-sequenicng. The existence of NCVs in TFBMs targeting pathways critical for CLL prompts further investigation into their actual relevance to the clinical behavior. Shared mutations between CLL and PMN cells indicate that some somatic mutations may occur before CLL onset, likely at the hematopoietic stem-cell level. Their potential oncogenic role likely depends on the cellular context and/or microenvironmental stimuli to which the affected cells are exposed. Disclosures Stamatopoulos: Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses. Ghia:Adaptive: Consultancy; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding.

scholarly journals From Bone Marrow to Mobilized Peripheral Blood Stem Cells: The Circuitous Path to Clinical Gene Therapy for Fanconi Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2208-2208
Author(s):  
Pamela S Becker ◽  
Jennifer Adair ◽  
Grace Choi ◽  
Anne Lee ◽  
Ann Woolfrey ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Research Funding ◽  
Hematopoietic Stem ◽  
Post Infusion ◽  
Clinical Gene Therapy

Abstract For decades, it has remained challenging to achieve long-term engraftment and correction of blood counts using gene-modified hematopoietic stem cells for Fanconi anemia. Toward this goal, our group conducted preclinical studies using a safety modified lentiviral vector encoding full-length cDNA for FANCA in normal and affected patient hematopoietic progenitor cells, and in a mutant mouse model that supported the IND for a gene therapy clinical trial for Fanconi anemia, complementation group A (NCT01331018). These studies led us to incorporate methods such as addition of N-acetylcysteine and hypoxic incubation during transduction. Because of the low stem cell numbers of Fanconi patients and initial difficulty with using plerixafor off-label for mobilization, we began our study with bone marrow as the source of stem cells. Due to concerns regarding secondary cancers, no conditioning was administered prior to infusion of gene-modified cells. The US Food and Drug Administration approved adult patients initially, but later permitted pediatric patient enrollment with a minimum age of 4 years. The primary objective of our phase I trial was safety. Secondary objectives included in vitro correction of mitomycin C (MMC) sensitivity, procurement of sufficient cell numbers, and ultimately, long-term correction of blood counts in recipients. Eligibility included absolute neutrophil count ≥0.5, hemoglobin ≥8, platelet count ≥20,000, lack of matched family donor, adequate organ function, and not meeting criteria for diagnosis of MDS. Our three enrolled patients were ages 22, 10, and 5 years. All demonstrated defects in the FANCA gene, with two patients sequenced and one patient diagnosed by complementation. Due to in-process learning and the later addition of plerixafor mobilization to the protocol, three different laboratory procedures were used to prepare the gene-modified product for each patient. Cell products were CD34+ selected bone marrow, bone marrow mononuclear cells depleted of red cells by hetastarch, and G-CSF and plerixafor mobilized cells depleted of red blood cells and cells bearing lineage markers, respectively. Transduction efficiencies were 17.7, 42.7 and 26.3% of colony forming cells (CFC) in 0 nM MMC, and 80, 100, and 100% of CFC in 10 nM MMC. Growth of hematopoietic colonies in MMC indicated functional correction of the FANCA defect. The 1st patient received 6.1×10e4, the 2nd 2.9×10e5, and the 3rd 4.3×10e6 CD34+ cells/kg. Serious adverse events included cytopenias in all patients, and hospital admission for fever due to viral upper respiratory infection in one patient. The patients remain alive at 46, 38, and 12 months after receipt of gene-modified cells. Due to worsening cytopenias, the third patient underwent hematopoietic cell transplant from an unrelated donor 10 months after infusion of gene-modified cells. To date, he has done well with transplant, and no indication that prior gene therapy impacted the outcome. The blood counts for the first 2 patients who have not undergone allogeneic transplant remain stable at 1,111 and 1,077 days post infusion compared to the first blood counts when they arrived at our center. For the 1st patient, vector was detectable in white blood cells (WBC) up to 21 days, in the 2nd up to 582 days, and the 3rd up to 81 days post infusion. Thus, in these patients, despite dramatic improvement in cell dose during the study, there was lack of persistence in detection of gene-modified WBCs beyond 1.5 years. A number of factors may have contributed, including lack of conditioning, in vitro cell manipulation including cytokine exposure, inability to transduce primitive hematopoietic stem cells, and paucity of long-term repopulating cells at the ages of the patients, suggesting earlier collection may be beneficial. This study is now closed to enrollment. Valuable information gained as a result of this study will contribute to future clinical gene therapy trials. Current work focuses on how to evaluate stem cell fitness prior to attempting gene therapy, minimizing manipulation required for gene correction and/or in vivo genetic correction and non-chemotherapy-based conditioning to facilitate engraftment. We would like to personally thank each patient and their families for participating in this study, as we could not have learned these lessons without their support. Disclosures Becker: GlycoMimetics: Research Funding; Abbvie: Research Funding; Amgen: Research Funding; BMS: Research Funding; CVS Caremark: Consultancy; Trovagene: Research Funding; Rocket Pharmaceuticals: Research Funding; Novartis: Research Funding; Pfizer: Consultancy; JW Pharmaceuticals: Research Funding. Adair:Miltenyi Biotec: Honoraria; RX Partners: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Kiem:Rocket Pharmaceuticals: Consultancy; Homology Medicine: Consultancy; Magenta: Consultancy.

scholarly journals Impact of Genomic Alterations on Outcomes in Myelofibrosis Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2301-2301 ◽  
Author(s):  
Raajit K. Rampal ◽  
Roni Tamari ◽  
Nan Zhang ◽  
Caroline Jane McNamara ◽  
Franck Rapaport ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Triple Negative ◽  
Unrelated Donor ◽  
Reduced Intensity Conditioning ◽  
Hematopoietic Stem ◽  
Disease Characteristics ◽  
Donor Type ◽  
The Impact ◽  
Reduced Intensity

Abstract Introduction: The impact of genomic alterations, such as mutations in ASXL1, on the risk of disease progression and leukemic transformation in patients with myelofibrosis (MF) is well established. Further, emerging data suggests that the number and type of mutations may impact response to therapies such as ruxolitinib or imetelstat. Allogeneic hematopoietic stem cell transplant (allo-HSCT) remains the only potentially curative treatment for MF patients. However, the impact of somatic mutations on overall survival (OS) and relapse-free survival (RFS) is poorly understood. Using next-generation sequencing of pre-transplant blood and bone marrow samples from a well clinically-annotated cohort of MF patients who underwent allo-HSCT, we sought to determine the impact of mutational burden on outcomes. Methods: A multicenter retrospective analysis of a cohort of 84 patients was carried out. This included 52 patients treated on the MPD-RC 101 prospective study (NCT00572897), 18 patients treated at Prince Margaret Hospital, and 14 patients treated at Memorial Sloan Kettering Cancer Center. Patient and transplant characteristics are displayed in Table 1. DNA was extracted from pre-transplant bone marrow aspirate samples or peripheral blood samples. High-throughput sequencing of a panel of genes was performed. Average coverage of 829x (standard deviation of ±130) was obtained. Mutect was utilized to call single point variants (comparing our samples to a pool of normal samples) and PINDEL was used to call short insertions and deletions. We excluded all mutations present in at least one database of known non-somatic variants (DBSNP and 1000 genomes) and absent from COSMIC. Univariate Cox regression and Kaplan-Meier graphics were used to investigate the association of patient, transplant, and disease characteristics with OS and RFS. Results: JAK2V617F was the most frequent mutation detected in 41(48.8%) patients (Table 2). Eighteen patients (21.4%) had triple negative disease (negative for JAK2, MPL, and CALR mutations). Univariate analysis included the following: patient characteristics (age, gender), transplant characteristics (related vs. unrelated donor, matched vs. mismatched donor and myeloablative vs. reduced intensity conditioning) and disease characteristics (DIPSS and presence of mutations). Decreased OS was associated with unrelated donor status (HR 2.09, 95% CI: 1.03-4.23, p=0.04), reduced intensity conditioning (HR 4.21, 95% CI: 1.01-17.59, p=0.049), triple negative disease (HR 2.09, 95% CI: 1.02-4.30, p=0.04), and presence of U2AF1 (HR 2.53, 95% CI: 1.10-5.81, p=0.03) or SUZ12 mutations (HR 3.92, 95% CI: 1.19-12.21, p=0.02). Decreased RFS was associated with unrelated donor status (HR 2.27, 95% CI: 1.16-4.45, p=0.02), and the presence of SUZ12 mutation (HR 6.97, 95% CI: 2.37-20.49, p<0.001). A descriptive decrease in RFS in patients with U2AF1 (HR 2.15, 95% CI: 0.94-4.88, p=0.07) was observed but did not reach statistical significance. Importantly, mutations previously reported to be associated with reduced OS and RFS in the non-transplant setting, such as ASXL1, EZH2, IDH1/2, and SRSF2, were not associated with poorer outcomes in this analysis in transplanted patients. In an exploratory multivariate analysis including donor type (related vs. unrelated) and presence of U2AF1 and SUZ12 mutations, there was a significantly reduced OS and RFS in patients who harbor these mutations regardless of donor type (OS: HR 5.30, 95% CI: 2.08-13.47, p<0.001; RFS: HR 5.49, 95% CI: 2.27-13.30, p<0.001). In patients without the above mutations, having an unrelated donor was associated with worse OS (HR 2.55, 95% CI: 1.09-5.96, p=0.03) and RFS (HR 2.61, 95% CI: 1.17-5.83, p=0.02, Figure 1). Conclusions: Our analysis demonstrates that mutations previously associated with poor prognosis in MF, such as ASXL1, do not appear to confer a worsened prognosis in patients undergoing allo-HSCT, suggesting transplant may be able to overcome the impact of these mutations. However, mutations in SUZ12 and U2AF1 are associated with reduced OS in univariate and multivariate analysis (together with donor type). Further studies with larger cohorts of patients are indicated to validate these findings, and to elucidate the impact of these mutations on disease biology. Disclosures Rampal: Incye and CTI: Consultancy. Mascarenhas:Janssen: Research Funding; CTi Biopharma: Research Funding; Promedior: Research Funding; Merk: Research Funding; Incyte: Research Funding. Mesa:Galena: Consultancy; Gilead: Research Funding; Promedior: Research Funding; Incyte: Research Funding; CTI Biopharma: Research Funding; Celgene: Research Funding; Ariad: Consultancy; Novartis: Consultancy. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding.

scholarly journals Age Distribution and Pattern of Myeloid Marrow Mutations in Patients (pts) with Higher-Risk Myelodysplastic Syndromes (HR-MDS) after Failure of Hypomethylating Agents (HMAs)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5257-5257
Author(s):  
Ghulam J Mufti ◽  
Lewis R. Silverman ◽  
Steven Best ◽  
Steve Fructman ◽  
Nozar Azarnia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Overall Survival ◽  
Stem Cell ◽  
Research Funding ◽  
Somatic Mutations ◽  
Phase Iii ◽  
Elderly Subjects ◽  
Normal Peripheral Blood ◽  
Marrow Stem Cell

Abstract Background: The aging marrow stem cell demonstrates more somatic mutations when compared to younger marrow stem cells. These abnormalities have been noted in pts with MDS, as well as in patients with normal peripheral blood counts (Steensma et al, Blood 2015; Jaiswal et al, N Engl J Med 2014). Mutations are rarely detected in people <40 years of age, but increase each decade thereafter (Jaiswal et al, N Engl J Med 2014). Certain mutations (eg, DNMT3A, TET2, ASXL1, and SF3B1) are most prevalent in the oldest pts, and approximately 2% of pts with mutations associated with leukemia or lymphoma have age-related hematopoietic clonal expansion, which increases to 5-6% among patients ≥70 years of age (Xie et al, Nat Med 2014). In another study, 10% of elderly subjects had clonal hematopoiesis with somatic mutations and this number increased with increasing age (Genovese et al, N Engl J Med 2014). In a randomized, Phase III study with intravenous rigosertib (ONTIME) in patients with MDS failing HMA therapy, a much higher proportion of pts with bone marrow mutations was observed. The most frequent mutations were as follows: SRSF2 (28% of pts), TP53 (22%), ASXL1 (19%), SF3B1 (14%), and TET2 (14%) (Mufti et al, Blood 2014). Given that MDS is a disease of the elderly, and the importance of somatic mutations for diagnosis, prognosis, and (potentially) targeted therapy, we explored the correlation between age and type of somatic bone marrow mutation found in pts entered into ONTIME. Methods: We evaluated the bone marrow mutations in patients with MDS who were enrolled in ONTIME after failing to respond to a previous HMA. Bone marrow genomic DNA was isolated from single microscopic slides from 153 pts from ONTIME and subjected to sequence analysis of a "myeloid panel" comprising of 24 selected loci known to be frequently mutated in MDS and AML (Mufti et al, Blood 2014). We investigated these 24 myeloid abnormalities for their frequency in the identified age cohorts prior to study randomization to explore the correlation between age and the somatic mutation identified, specifically looking at pts older or younger than the mean age of pts with MDS in ONTIME (75 years). Results: Approximately 45% of patients had 1 mutation and an equal number had >1 (Figure 1). Table 1 shows the most frequent clonal myeloid mutations in ONTIME, based on age above and below 75 years (the median age in ONTIME).Table 3.Incidence (%) of Patients with Specific Mutations, Age Above and Below 75 YearsMutation< 75 years (N=60)≥ 75 years (N=51)Total (N=111)Fisher's Exact Test P-valueSRSF22729280.83TP532518220.37ASXL12018190.81SF3B11316140.79U2AF11212121.00TET21216140.59RUNX1814110.38DNMT3A812100.75In a separate analysis, the number of months from diagnosis of MDS and duration of prior HMA treatment did not appear to influence the pattern of mutations (Table 2). Table 2.Mutations by Months Since MDS Diagnosis and Duration of Prior HMAMutationNMonths from MDS Diagnosis median (range)Duration of HMA (mo) median (range)All analyzed pts11118.5 (0.1-116)8.9 (1.2-65)TP532414.9 (0.7-116)13.0 (1.2-36)SF3B11629.4 (7.5-63)13.0 (1.2-36)TET21522.6 (0.1-63)11.4 (2.0-36)SRSF23117.2 (6.6-116)6.4 (3.0-35)ASXL12115.7 (4.9-66)8.2 (2.8-44)DNMT3A1115.1 (7.4-36)6.5 (4.2-30)Conclusions: Somatic mutations are common in marrow stem cells from patients with HR-MDS. Over 45% of patients had 1 mutational abnormality, and 44% had >1. Of note, patients under and over the median age of pts with MDS had a similar mutational pattern, which was not influenced by either length of time since diagnosis of MDS or prior treatment with an HMA. In this analysis, the mutational genomic abnormalities in the MDS marrow stem cell were similar among younger and older patients with MDS, suggesting the underlying pathogenic mechanisms causing these abnormalities are also similar irrespective of patient age. Figure 1. Number of Mutations Per Patient Figure 1. Number of Mutations Per Patient Figure 2. Overall Survival in ONTIME by Number of Marrow Stem Cell Mutations Figure 2. Overall Survival in ONTIME by Number of Marrow Stem Cell Mutations Disclosures Mufti: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Honoraria, Patents & Royalties: co-patent holder on combination of rigosertib and azacitdine, Research Funding. Best:Onconova Therapeutics Inc: Research Funding. Fructman:Onconova Therapeutics Inc: Employment. Azarnia:Onconova Therapeutics Inc: Employment. Petrone:Onconova Therapeutics Inc: Employment.

scholarly journals Whole Transcriptome Sequencing Identifies Novel Pathways Associated with Paroxysmal Nocturnal Hemoglobinuria- Increased CXCR2 Expression in PNH Granulocytes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3608-3608
Author(s):  
Kohei Hosokawa ◽  
Sachiko Kajigaya ◽  
Keyvan Keyvanfar ◽  
Danielle M. Townsley ◽  
Bogdan Dumitriu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Lymphoid Cells ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cxcr2 Expression ◽  
Healthy Donors

Abstract Background. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder that arises from hematopoietic stem cells (HSCs). PNH is caused by a somatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A), responsible for a deficiency in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). PNH is a clonal disease that originates from HSCs, as the originating PIGA mutation is present in cells of multiple lineages, including myeloid, erythroid, and lymphoid cells. However, a critical question regarding PNH that has yet to be fully explained despite several decades of research is the mechanism responsible for clonal expansion of PIGA -mutant cells in bone marrow failure. Using RNA-seq, we identify pathways, coding and non-coding RNA transcripts, splice variants, or single nucleotide variants and other alterations that may relate to the selective advantage of PNH clone. Method. Blood samples were obtained after informed consent from patients with 14 PNH and 18 age-matched healthy donors. From PNH patients and healthy donors, 4 samples were used for RNA sequencing 6 samples were used for validation by flow cytometry. The liquid FLAER method was used for the detection of PNH-type granulocytes. For RNA extraction, granulocytes were sorted for CD11b+ FLAER+ granulocytes, CD11b+ FLAER- granulocytes. For bone marrow staining, cells not expressing lineage markers were separated into five subpopulations: Long-term hematopoietic stem cells (LT-HSC; Lin- CD34+ CD38- CD90+), short-term hematopoietic stem cells (ST-HSC; Lin- CD34+ CD38- CD90-), common myeloid progenitors (CMP; Lin- CD34+ CD38+ CD123+ CD45RA-), granulocyte-monocyte progenitors (GMP; Lin- CD34+ CD38+ CD123+ CD45RA+) and megakaryocyte-erythrocyte progenitors (MEP; Lin- CD34+ CD38+ CD123- CD45RA-). Results and Discussion. First, RNA expression levels in CD11b+ FLAER+ and CD11b+ FLAER- populations of PNH patients were analyzed using RNA sequencing. Expression levels of 7 mRNAs (CSF2RB, ACSL1, FCGR3B, IL1RN, CXCR2, TREM1, and TNFRSR10C) were significantly upregulated (> 3 FC, P < 0.01) in CD11b+ FLAER- cells of PNH patients compared with CD11b+ FLAER+ cells. To validate the differential expression observed in GPI-AP- granulocytes from PNH patients, protein expression levels of CSF2RB, FCGR3B, CXCR2, TREM1, and TNFRSF10C were assessed by flow cytometry. In CD11b+ FLAER- granulocytes of 6 PNH patients, increased expression of CXCR2 was validated, whereas decreased expression of FCGRB and TNFRSF10C were validated compared with CD11b+ FLAER+ granulocytes and that of healthy controls. Low expression FCGRB and TNFRSR10C in CD11b+ FLAER- granulocytes were considered to be reasonable, as these were GPI-APs. Next, we examined whether increased CXCR2 expression in PNH-type cells was validated in different peripheral blood cell populations. Increased CXCR2 expression in PNH-type cells was confirmed in granulocyte and monocyte populations, not in T cell or B cell population. We checked the expression levels of CXCR1 and CXCR2, which are closely related receptors that recognize CXC chemokines. CXCR2 expression was significantly different between normal and PNH-type cells in granulocytes and monocytes, and CXCR1 expression was significant only for granulocytes. To address the difference of CXCR2 expression levels between normal and PNH-type cells in more undifferentiated cells, we next examined the CXCR2 expression levels in bone marrow hematopoietic stem cells. Expression of CXCR2 was weakly expressed in hematopoietic stem cells and progenitors, both in normal and PNH-type cells, suggesting that difference of CXCR2 expression between normal and PNH-type cells is evident only in differentiated myeloid cells, not in hematopoietic stem cells or lymphoid cells. Conclusion. We provide evidence for increased expression of CXCR2 in PNH-type granulcoytes and monocytes by RNA-seq and flow cytometry. The differential expression of CXCR2 might partly explain the dominance of PNH clones in myeloid cells in patients. CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell-enriched cell fractions in MDS and AML (Schinke C, et al, Blood, 2015). Understanding the mechanism of increased CXCR2 expression in PNH-type cells may offer new therapeutic strategies and novel mechanistic insight into the pathophysiology of PNH. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.

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