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Introduction
A relative increase in classical monocyte fraction (cMo, CD14++CD16-) over 94% of total peripheral blood monocytes, as measured by flow cytometry, distinguishes a CMML from a reactive ...monocytosis with a 94.1% specificity and a 92.8% sensitivity (Selimoglu-Buet, 2015; Talati, 2017; Patnaik, 2017; Selimoglu-Buet, 2017; Hudson, 2018). This phenotype is independent of the absolute monocyte count, WHO subtype of CMML, its dysplastic (MD) vs proliferative (MP) feature and its mutational background. The limitation of this diagnostic tool is the changes in monocyte subset repartition induced by an associated inflammatory disease that increase the fraction of intermediate monocytes (iMo, CD14++CD16+), leading to underestimation of cMo accumulation (Selimoglu-Buet, 2017). The present study explores the prognostic significance of cMo accumulation in CMML.
Patients and methods
Among CMML patients (diagnosis according to 2016 WHO criteria) included in our previous studies, we selected patients diagnosed from June 2012 to March 2017 in four centers in which follow-up was actualized in June 2019. Disease was classified according to WHO 2016 (CMML-0, -1, -2 and MP vs MD subtypes). When possible, the CMML Prognostic Scoring System (CPSS) was calculated. Peripheral blood flow cytometry data were reanalyzed in a blind fashion by a skilled operator. Inflammatory CMML were defined by a cMo percentage <94% associated with a bulbous aspect on flow cytometry profile (Figure 1A). The absolute cMo count in the peripheral blood was determined by combining flow cytometry measurement of cMo fraction and the total monocyte count provided by Complete Blood Count.
Results
One hundred and twenty-nine CMML patients (mean age: 75±10 years, sex ratio: 2.2) were classified into 53 CMML-0 (41%), 58 CMML-1 (45%) and 18 CMML-2 (14%) with 97 dysplastic forms (75%). Eight patients (6%) having received DNA damaging therapies (radiations or chemotherapies) were considered as secondary CMML. Eleven patients (9%) had evolved from a previous myelodysplastic syndrome. The median white blood count, hemoglobin level and platelet count were 9 G/l (IQR: 5.9-12.9), 11.8 g/dl (IQR: 9.7-13.1), and 121 G/l (IQR: 73-212), respectively. The CPSS score, calculated in 119 cases, classified the patients into “Low” (n=55), “Intermediate-1” (n=33), “Intermediate-2 (n=29) and ”High“ (n=2) categories. Twenty of the 114 patients (18%) with available follow-up (median duration: 26.7 months 13.9-36.5) had received hypomethylating agents and 3 (3%) were allografted.
A cMo percentage above the threshold (97.0% IQR: 96.3-98.1) was detected in 112 patients (87%) whereas 17 (13%), referred thereafter as “inflammatory CMML”, displayed cMo<94% (91.6% IQR: 89.9-93.2, p<0.001) with an inflammatory state (C-reactive protein: 8 mg/L IQR: 3.1-20.2 (Figure 1B). Importantly, the absolute count of cMo was similar in inflammatory (1.6 G/l IQR: 1.1- 1.8) and classical (1.7 G/l IQR: 1.2- 2.6, p=0.25) CMML (Figure 1C). Focusing on total monocyte and cMo absolute counts, we did not detect any significant difference between CMML-0, CMML-1 and CMML-2. While cMo percentages were similar in MP and MD subtypes, the total monocyte (6.0 ±6.0 G/l vs 1.7 ±0.7 G/L, p<0.001) and the cMo (5.8 ±5.7 G/L vs 1.6 ±0.7 G/L, p<0.001) counts were increased in MP compared to MD CMML. A poor prognosis CPSS score at diagnosis also correlated with an increase in the absolute cMo count (“Intermediate-2 or High”: 4.5 ±6.0 G/L vs “Low or Intermediate-1”: 2.0 ±1.7 G/L; p <0.001) (Figure 1D).
A Receiver Operator Curve (ROC) defined a cut-off value of 2.0 G/L to identify patients with poorest outcome. Indeed, CMML patients with an absolute cMO count ≥2.0 G/L at diagnosis had a higher risk to progress to acute myeloid leukemia (AML) (HR=0.34 95% CI, 0.12 - 0.91) (Figure 1E). Eventually, patients with an absolute cMO count ≥2.0 G/L at diagnosis had poorer overall survival (OS) compared with patients with a lower absolute cMO count (HR=0.42 95% CI, 0.22 - 0.80; median OS: 31.9 months vs not reached, p=0.009) (Figure 1F).
Conclusion
These results demonstrate that in CMML patients, regardless of the relative cMo percentage, a high absolute count of circulating cMo at diagnosis ≥2.0 G/L correlates with a poor outcome, including a higher risk of AML progression and shorter survival.
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Fenaux:Jazz: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Aprea: Research Funding. Wagner Ballon:Alexion: Consultancy, Honoraria.
Acute myeloid leukemias (AML) comprise a genetically diverse group of human hematologic malignancies with a generally poor prognosis and modes of pathogenesis that have been difficult to investigate. ...Historic evidence and more recent findings have identified hematopoietic stem cell programs as major targets of mutations that predispose to and/or initiate a multistep process of transformation. However, details of the changes involved and how they interact in emerging human leukemic cells have been particularly challenging to characterize. This is due to an emerging appreciation of the heterogeneity in events that impact the differentiation processes of normal human hematopoietic cells, as well as difficulties in recreating the full leukemogenic alterations in the actual human cells affected, to avoid discrepancies inherent in studying model organisms. Here we describe several models of de novo human leukemogenesis that illustrate their power and the novel results they can generate. These include our recent discovery of a novel “latent” leukemic state that can be obtained in MYC-transduced human cord blood cells regenerating a normal spectrum of lymphoid and myeloid progeny in transplanted immunodeficient mice, but that can then be rapidly activated to an aggressive form of AML by in vivo exposure to a single human growth factor. These findings portend the future utility of de novo human leukemogenesis models as new platforms for elucidating shared molecular mechanisms responsible for different stages of human leukemogenesis that may be initiated by different mutations or exposure to different microenvironmental conditions. The flexibility and consistency of these models also make them attractive for identifying and testing new treatment strategies targeting mechanisms required for disease manifestation.
Acute myeloid leukemias (AMLs) are heterogenous diseases often resulting from the acquisition of multiple genetic alterations that deregulate hematopoietic precursor proliferation and block normal ...differentiation. Chronic myeloid leukemia offers a unique opportunity to identify molecular mechanisms that interfere with normal differentiation in the context of a highly proliferative hematopoietic stem cell clone that produces massive number of functional differentiated myeloid cells due to the presence of a BCR-ABL1 fusion gene. Since the NUP98-HOXA9 (NA9) fusion gene has been identified in some CML blast crisis patients or de novo AML, we asked whether a humanized model of CML progression to AML would result from its lentiviral-mediated introduction into primary CML CD34+ cells from 3 chronic phase CML patients in which >97%, >96%, and 44% of the CD34+ longterm culture-initiating cells were Ph+/BCR-ABL1+.
In vitro experiments showed the NA9-transduced cells produced a hugely increased number of granulo-monocytic progenitors in long-term cultures (up to 1000-fold, p=0.03; t-ratio test) and enhanced the serial replating activity of directly clonogenic cells as compared to matched samples of cells transduced with a control vector. In vivo experiments showed that 90% of sublethally irradiated NOD-SCID IL2Rgc-null mice expressing human IL3, GM-CSF and SCF constitutively that were transplanted with these same NA9-transduced cells developed evidence of a progressed myeloproliferative neoplasia. This included tissue infiltrates of eosinophils, basophils and mastocytes, exclusive myeloid differentiation and signs of an imminently fatal leukemia between 8 and 27 weeks post-transplant in 50% of cases, although an excess blast population was not seen in these.
RNA-sequencing of cells analyzed just 48 hours after transduction revealed a signature of 53 genes that were more highly expressed in CD34+ CML cells carrying the NA9 fusion gene in comparison to control CML cells. This signature included many genes expressed by hematopoietic stem cells (HSCs), such as 5‘ HOXA genes, 3‘ HOXB genes, PBX3, MEIS1, ARID5B, AHR, REL, BMP6, GDF10, SFRP5, PPBP, PLA2G4A suggesting that NA9 induces the expression of a partial HSC program in later CML progenitor types that make up the bulk of the CD34+ CML population. Consistent with this hypothesis, the NA9 signature separated the HSCs, CMPs and GMPs of chronic CML patients as well as those of normal subjects (GSE47927; p=1x10-6, p=2x10-4 respectively). Most genes in the NA9 signature were also found to be over-expressed in CD34+ cells of patients in the accelerated phase and in the blast phase of CML, including ARID5B, AHR, STARD9, TOX, or FOXP1 (p=9.10-8; ANOVA) (GSE4170). The NA9 signature was also significantly enriched in transcripts of genes that are over-expressed in the blasts of AML patients carrying NPM1 mutations or MLL fusions and was predictive of overall survival in the AML cohort of the Cancer Genome Atlas (p=0.02; log-rank test, n=200).
This stem cell signature was also associated with an increase in the number of H3K27ac marks (on average 7805 ± 960 peaks for NA9 vs 5888 ± 2739 peaks for control) at 48 hours post-transduction. De novo H3K27ac peaks in NA9+ CD34+ cells were located in proximal and distal enhancers of 993 genes (GREAT parameters TSS ± 2kb from TSS and 100kb max extension). These were significantly enriched genes in the GM-CSF signaling pathway (MySigDB, binomial p-value=2.19x10-8) and that are upregulated in granulocytes and monocytes following LPS exposure. H3K27ac peaks also matched with GATA1, RELA, MEF2A and IKZF1 transcription factor binding sites previously mapped by ChIPSeq experiments (adjusted p-value <0.05). Finally, we identified super-enhancers in 12 genes among which were PBX3, ANGPT1, MBNL1 and PRKACB.
Overexpression of the NA9 fusion gene in chronic phase CD34+ CML cells thus appears to reprogram the expression of HSC genes as well as those associated with GM-CSF pathway activation and inflammatory responses via H3K27 acetylation of associated loci leading to a picture of advanced accelerated phase/disease progression but not the complete differentiation arrest seen in terminal blast crisis or frank AML. These findings highlight the multiplicity of biologically important molecular alterations that can result from a single epigenetic perturbation but, nevertheless, are insufficient to create an overt AML phenotype.
Turhan:novartis: Honoraria, Research Funding; Incyte: Consultancy, Honoraria.
Several fusion genes such as BCR::ABL1, FIP1L1::PDGFRA, and PML::RARA are now efficiently targeted by specific therapies in patients with leukemia. Although these therapies have significantly ...improved patient outcomes, leukemia relapse and progression remain clinical concerns. Most myeloid next-generation sequencing (NGS) panels do not detect or quantify these fusions. It therefore remains difficult to decipher the clonal architecture and dynamics of myeloid malignancy patients, although these factors can affect clinical decisions and provide pathophysiologic insights. An asymmetric capture sequencing strategy (aCAP-Seq) and a bioinformatics algorithm (HmnFusion) were developed to detect and quantify MBCR::ABL1, μBCR::ABL1, PML::RARA, and FIP1L1::PDGFRA fusion genes in an NGS panel targeting 41 genes. One-hundred nineteen DNA samples derived from 106 patients were analyzed by conventional methods at diagnosis or on follow-up and were sequenced with this NGS myeloid panel. The specificity and sensitivity of fusion detection by aCAP-Seq were 100% and 98.1%, respectively, with a limit of detection estimated at 0.1%. Fusion quantifications were linear from 0.1% to 50%. Breakpoint locations and sequences identified by NGS were concordant with results obtained by Sanger sequencing. Finally, this new sensitive and cost-efficient NGS method allowed integrated analysis of resistant chronic myeloid leukemia patients and thus will be of interest to elucidate the mutational landscape and clonal architecture of myeloid malignancies driven by these fusion genes at diagnosis, relapse, or progression.
Aryl Hydrocarbon Receptor (AHR) is an ubiquitous basic helix-loop-helix transcription factor, which is ligand-activated and involved in numerous biological processes including cell division, cell ...quiescence and inflammation. It has been shown that AHR is involved in normal hematopoietic progenitor proliferation in human cells. In addition, loss of AHR in knockout mice is accompanied by a myeloproliferative syndrome-like disease, suggesting a role of AHR in hematopoietic stem cell (HSC) maintenance. To study the potential role of AHR pathway in CML progenitors and stem cells, we have first evaluated the expression of AHR in UT-7 cell line expressing BCR-ABL. AHR expression was highly reduced in UT-7 cell expressing BCR-ABL as compared to controls. AHR transcript levels, quantified in primary peripheral blood CML cells at diagnosis (n = 31 patients) were found to be significantly reduced compared to healthy controls (n = 15). The use of StemRegenin (SR1), an AHR antagonist, induced a marked expansion of total leukemic cells and leukemic CD34+ cells by about 4- and 10-fold respectively. SR1-treated CML CD34+ cells generated more colony-forming cells and long-term culture initiating cell (LTC-IC)-derived progenitors as compared to non-SR1-treated counterparts. Conversely, treatment of CML CD34+ cells with FICZ, a natural agonist of AHR, induced a 3-fold decrease in the number of CD34+ cells in culture after 7 days. Moreover, a 4-day FICZ treatment was sufficient to significantly reduce the clonogenic potential of CML CD34+ cells and this effect was synergized by Imatinib and Dasatinib treatments. Similarly, a 3-day FICZ treatment contributed to hinder significantly the number of LTC-IC-derived progenitors without synergistic effect with Imatinib. The analysis of molecular circuitry of AHR signaling in CML showed a transcriptional signature in CML derived CD34+ CD38- primitive cells with either low or high levels of AHR, with an upregulation of myeloid genes involved in differentiation in the "AHR low" fraction and an upregulation of genes involved in stem cell maintenance in the "AHR high" fraction. In conclusion, these findings demonstrate for the first time that down-regulation of AHR expression, a major cell cycle regulator, is involved in the myeloproliferative phenotype associated with CML. AHR agonists inhibit clonogenic and LTC-IC-derived progenitor growth and they could be used in leukemic stem cell targeting in CML.
Abstract We report here the first use of whole genome sequencing (WGS) to examine the initial clonal dynamics in an unusual patient with chronic myeloid leukemia (CML) who presented in chronic phase ...(CP) with doubly marked BCR-ABL1+ / JAK2 V617F -mutant cells and over a 9 year period progressed into an accelerated phase (AP) and then terminal blast phase (BP). WGS showed the diagnostic cells also contained mutations in ASXL1, SEC23B , MAD1L1 and RREB1, as well as 12,000 additional uncommon DNA variants. WGS of endothelial cells generated from circulating precursors revealed many of these were shared with the CML clone. Surprisingly, WGS of induced pluripotent stem cells (iPSCs) derived from the AP cells revealed only 6 additional coding somatic mutations despite retention by their hematopoietic progeny of the parental AP cell levels of BCR-ABL1 expression and sensitivity to imatinib and pimozide. Limited analysis of BP cells showed independent subclonal progression to homozygosity of the MAD1L1 and RREB1 variants. MAD1L1 and SEC23B mutations were also identified in 2/101 cases of myeloproliferative neoplasms but not in 42 healthy subjects. These findings challenge historic concepts of clonal evolution in CML.
Chronic myeloid leukemia is a clonal myeloproliferative neoplasm defined by the presence of BCR-ABL fusion gene. This oncogenic event occurs in a hematopoietic stem cell (HSC) involved in CML ...initiation, maintenance, relapse and progression. Several evidences suggest that inflammatory pathways may participate to the pathophysiology of CML as well as disease progression to blast crisis. It has been shown that NFKB/REL pathway is constitutively activated both in BCR-ABL positive leukemic cell lines as well as in primary blast cells from CML-BC patients. More recent works identified IL6 as key cytokine acting on CML multipotent progenitors and their normal bystander counterpart to favor their differentiation toward the myeloid lineage. In addition, high levels of autocrine TNFα secretion by quiescent CML stem/progenitor cells activate NFKB pathway and promote their survival. Although all of these observations are linked to inflammatory processes, a focused analysis of inflammatory pathways in primary CML stem cells has not been performed so far. In the present study we undertook a text-mining strategy using pubmed e-querying to generate an exhaustive set of genes linked to inflammation. Then we integrated transcriptome analysis of highly purified CML stem cells to evaluate the contribution of these genes in CML development and progression.
Methods : We queried 6 key words (Inflammation, macrophages, inflammatory response, chemokines, leukocytes and interleukins) that returned a total of 332000 hits in Pubmed. A raw gene set of 918 genes was found significantly associated (p<0.05) with these hits. Using R-package, we applied a false discovery rate correction that decreased the set to 588 relevant genes. The expression level of this gene set was then analyzed in previously reported microarray data (GEO accession: GSE47927) of highly purified normal cord blood CD34+CD38-CD90+ HSCs (CB; n=3), chronic phase (CP; n= 6), accelerated phase (AP; n =4) and Blast crisis (BC; n=2) CML cells.
Results: Among the 588 genes related to inflammation we found 70 genes differentially expressed between the four groups (normal, CP, AP and BC, p<0.01; ANOVA test). Enrichment analysis confirmed 29 up regulated genes (NES = 2.12; p<0.0001) among which IL-6, PARP1, IL1R2, IRF5, IRF8, IL20. 39 genes such as STAT3, STAT4, CD47, CXCR4 IL-11, IL15, TLR-1, were down-regulated in CML CD34+CD38-CD90+ (all phases) as compared with normal HSCs (NES = -2,58; p<0.0001). Using principal component analysis on the 70 inflammatory deregulated genes we identified 10 genes such as IRAK1, IL1R2, VEGF and ESAM that discriminate "all phase" CML samples from normal HSCs (Dim 2 = 22.7%). Another inflammatory gene subset (n=26 genes) comprising IL6, REL, CXCR4, CXCL2, IL11, TLR1, IL1R2, PPARA highly separated CML stem cells according to the disease phase. The later gene set highly separates CP and AP-CML stem cells from BC-CML stem cell (Dim 1 = 50.3%). We next performed a random forest analysis with machine learning (1000 trees) and found that the inflammatory transcript level that best predicted CML phase was REL transcription factor. The expression of 413 genes were found positively correlated with REL expression in CP, AP and BC-CML CD34+CD38-CD90+ cells (r>0.75 and p-value <0.001). A search using JASPAR and TRANSFAC database identified a significant enrichment of NFKB1 and RELA binding motif in the promoter regions of these 413 genes (p<0.00001) among which several regulatory factors of hematopoietic stem cell biology.
Conclusion : Using a bio-integrative approach we identified a specific inflammatory signature in CD34+CD38-CD90+ CML stem cells. This inflammatory network is highly altered in blast crisis suggesting its contribution to disease evolution. We identified REL overexpression as a good predictor for disease progression to blast crisis and found NFKB1and RELA (p=3.2x10-13) as the best REL target candidates. RELA/NFKB1 was previously shown to be constitutively activated in CML and Ph+ ALL and this analysis suggests that this may also take place in the most primitive subset of CML cells although REL may be the main partner of NFKB in CML stem cells. These results which are currently validated using functional assays, could lead to identification of novel therapeutic strategies.
Turhan:Bristol Myers Squibb: Consultancy; Novartis: Research Funding.
We aimed to study the prognostic impact of the mutational landscape in primary and secondary myelofibrosis. The study included 479 patients with myelofibrosis recruited from 24 French Intergroup of ...Myeloproliferative Neoplasms (FIM) centers. The molecular landscape was studied by high-throughput sequencing of 77 genes. A Bayesian network allowed the identification of genomic groups whose prognostic impact was studied in a multistate model considering transitions from the 3 conditions: myelofibrosis, acute leukemia, and death. Results were validated using an independent, previously published cohort (n = 276). Four genomic groups were identified: patients with TP53 mutation; patients with ≥1 mutation in EZH2, CBL, U2AF1, SRSF2, IDH1, IDH2, NRAS, or KRAS (high-risk group); patients with ASXL1-only mutation (ie, no associated mutation in TP53 or high-risk genes); and other patients. A multistate model found that both TP53 and high-risk groups were associated with leukemic transformation (hazard ratios HRs 95% confidence interval, 8.68 3.32-22.73 and 3.24 1.58-6.64, respectively) and death from myelofibrosis (HRs, 3.03 1.66-5.56 and 1.77 1.18-2.67, respectively). ASXL1-only mutations had no prognostic value that was confirmed in the validation cohort. However, ASXL1 mutations conferred a worse prognosis when associated with a mutation in TP53 or high-risk genes. This study provides a new definition of adverse mutations in myelofibrosis with the addition of TP53, CBL, NRAS, KRAS, and U2AF1 to previously described genes. Furthermore, our results argue that ASXL1 mutations alone cannot be considered detrimental.
•Mutations of TP53 and high-risk genes (EZH2, CBL, U2AF1, SRSF2, IDH1, IDH2, NRAS or KRAS) are adverse prognostic factors in myelofibrosis.•ASXL1 isolated mutations (ie, without TP53 or high-risk mutations) have no prognostic impact in myelofibrosis.
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