Shared cell of origin in a patient with Erdheim-Chester disease and acute myeloid leukemia

Erdheim-Chester disease (ECD) is a clonal hematopoietic disorder characterized by the accumulation of foamy histiocytes within organs, in particular frequent retroperitoneal involvement, and a high frequency of BRAFV600E mutations. Although ECD is not commonly recognized to have overt peripheral blood (PB) or bone marrow (BM) disease, we recently identified that ECD patients have a high frequency of a concomitant myeloid malignancy. Given this finding and the fact that clonal hematopoiesis frequency precedes development of myeloid malignancies, we conducted a systematic clinical and molecular analysis of the BM from 120 ECD patients. Surprisingly, 42.5% (51/120) of ECD patients had clonal hematopoiesis while 15.8% (19/120) of patients developed an overt hematologic malignancy (nearly all of which were a myeloid neoplasm). The most frequently mutated genes in BM were TET2, ASXL1, DNMT3A, and NRAS. ECD patients with clonal hematopoiesis were more likely to be older (p<0.0001), have retroperitoneal involvement (p=0.02), and harbor a BRAFV600E mutation (p=0.049) than those without clonal hematopoiesis. The presence of the TET2 mutation was associated with a BRAFV600E mutation in tissue ECD lesions (p=0.0006) and TET2 mutant ECD patients were more likely to have vascular involvement than TET2 wild-type ECD patients. Clonal hematopoiesis mutations in ECD were detected in cells derived from CD34+CD38- BM progenitors and PB monocytes but less frequently present in PB B- and T-lymphocytes. These data identify a heretofore unrecognized high frequency of clonal hematopoiesis in ECD patients, reaffirm the development of additional high risk of myeloid neoplasms in ECD, and provide evidence of a BM-based precursor cell-of-origin for many patients with ECD.

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Divergent Epigenomes in Pediatric and Adult Acute Myeloid Leukemia Implicate Cell of Origin and Transcriptional Silencing of Immune Responses As Sources of Clinically Relevant Heterogeneity: A Report from the Children's Oncology Group and NCI/COG Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative

Blood ◽

10.1182/blood.v128.22.1046.1046 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1046-1046

Author(s):

Timothy Junius Triche ◽

Jason E Farrar ◽

Hamid Bolouri ◽

Rhonda E. Ries ◽

Emilia L. Lim ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Mrna Expression ◽

Myeloid Leukemia ◽

Mitotic Checkpoint ◽

Functional Enrichment ◽

Cell Of Origin ◽

Oncology Group ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) carries a poor prognosis across age groups. In children, AML has become the leading cause of leukemia mortality, with only 60% of cases securing long-term remission. In adults, outcomes are far worse, with 5 year survival approaching 24%. The mutational and transcriptional characterization of AML1has not yet translated into improved outcomes for most patients. The TARGET AML project is an effort of Children's Oncology Group (COG) and the National Cancer Institute to characterize molecular abnormalities in pediatric AML. 197 cases were selected for whole genome sequencing (WGS) of diagnostic specimens, 284 cases for mRNA sequencing, 289 cases for DNA methylation arrays, and 721 cases for targeted sequencing (182 assayed by WGS). Most patients (93%) were uniformly treated on COG study AAML0531 or AAML03P1. The Cancer Genome Atlas (TCGA) AML project1characterized 177 comparable adult AMLs with identical assays. DNA methylation changes radically during differentiation of blood cells2, and recurrent pre-leukemic mutations in adult AML3affect DNA methylation and chromatin modifiers. We thus investigated whether differences in cell-of-origin, immune signalling, and regulatory aberrations were captured by focal or regional differences in DNA methylation, within or between adult and pediatric AML patients. In cytogenetically similar TARGET and TCGA AML cases, striking differences in DNA methylation emerge (fig. 1). Pediatric FLT3-mutant AMLs dominate a cluster with normal-progenitor-like DNA methylation. Mutant DNMT3A, RUNX1, and TP53, which selectively favor preleukemic hematopoietic stem cells3,4,5 (HSCs), are common in adult AML, rare in pediatric AML, and tend towards HSC-like hypermethylation. Transcriptional & epigenetic signatures of the cell of origin persist even after leukemic transformation6. Thus we sought to identify the most likely cell of origin for each case. Previous studies of mRNA7 and DNA methylation8 differences in HSCs and progenitor cells (HSPCs), leukemic stem cells (LSCs), and AML blasts allowed us to model these differences in TCGA and TARGET AMLs. RNAseq results revealed many LSC-like cases with aberrant β-catenin signaling and TP53 regulation, distinct from blasts and normal HSPCs (fig. 2a). DNA methylation segregated cases resembling granulocyte/monocyte progenitors (GMPs) from those resembling other HSPC subsets (fig. 2b). DNMT3A mutants strongly associated with HSC/LSC-like mRNA expression, as did most MLL-rearranged AMLs. Nearly all TP53 and RUNX1 mutants presented LSC-like mRNA expression and retained HSC-like methylomes. These results suggest that decades of selective HSC attrition enable cooperating adult-specific mutations to initiate leukemia, while the timescales in pediatric AML favor fusion genes capable of transforming progenitors as well as HSCs. With matched mRNA expression & DNA methylation data from 256 TARGET cases and 156 TCGA cases, we found over 100 genes where DNA methylation accompanied loss of transcription (silencing) in AML but not in normal HSPCs (fig. 3a). Many such genes lie in regions affected by recurrent copy number aberrations, most notably chromosome arms 5q and 19q. Recurrently mutated or deleted genes such as DNMT3A, TET2, SPRY4, and CDKN2A/B are silenced, some mutually exclusively with mutations or CNV. Functional enrichment analyses of silenced genes with DAVID9revealed 4 clusters: NK-cell signaling, innate immune response regulation, transcriptional regulation, and (on chromosome 19q) zinc finger genes involved in Toll-like receptor signaling. Some silencing co-occurs with specific molecular features, but no event was perfectly predicted by any molecular or cytogenetic feature (fig. 3b). Drug-gene interaction mining with DGIDb10 suggests silencing may inform treatment. Silencing of the mitotic checkpoint gene CHFR may confer sensitivity to microtubule inhibitors11, silencing of MGMT suggests greater benefit from alkylating agents12, and demethylating agents may benefit cases with silenced immune response13. Biomarker driven clinical trials will be needed to evaluate these and other markers in pediatric and adult AML, but evidence of independent genetic and epigenetic evolution in AML14supports their continued investigation. This work is dedicated to the late Robert J. Arceci, without whom none of this would have been possible. Disclosures No relevant conflicts of interest to declare.

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Aberrant Megakaryocytic/Erythroid Progenitors Contributes To Transformation Of Cbfb-SMMHC Induced Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1652.1652 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1652-1652

Author(s):

Qi Cai ◽

Robin Jeannet ◽

Hongjun Liu ◽

Ya-Huei Kuo

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Cell Of Origin ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Differentiation Potential ◽

Acute Myeloid

Abstract Inv(16)(p13q22) is a recurrent chromosomal rearrangement found in approximately 12% of human acute myeloid leukemia (AML) cases and creates a fusion gene between CBFb and MYH11. The fusion gene encodes a fusion protein CBFß-SMMHC which causes defects in lymphoid and myeloid differentiation. Previous studies also showed that primitive erythropoiesis is impaired by CBFß-SMMHC, however, CBFß-SMMHC knocked-in cells was able to contribute to adult erythropoiesis in chimeric mice. Expressing CBFß-SMMHC in the hematopoietic cells using a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) recapitulates inv(16)-associated AML. Previous studies in this model showed that CBFß-SMMHC expression leads to pre-leukemic hematopoietic alterations, and together with additional cooperative mutations, result in spontaneous myeloid leukemia in mice with a 3-6 month latency. We hypothesized that an expanded cell population at the pre-leukemic stage could be the target of additional mutations, and hence the cell of origin of leukemia initiating cells. To further delineate the pre-leukemic progenitors and leukemia initiating cells, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 for more than 10 generations. Similar to previous studies in the129SvEv strain, expressing CBFß-SMMHC in adult C57BL/6 mice leads to cell number dependent development of AML. Analysis of pre-leukemic bone marrow as early as 2 weeks after induction revealed a 5.7-fold expansion of Pre-Meg/E cells (Pre-Megakaryocyte/Erythrocyte: Lin-cKit+Sca1-CD16-/loCD150+CD105-) compared to similarly treated control mice. While there was a significant increase in Pre-Meg/E population, we did not find significant increase in their proliferation but observed a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset. Methylcellulose-based colony forming assay showed that pre-leukemic Pre-Meg/E had an impaired differentiation potential for erythroid lineage. In vitro erythroid differentiation assay also showed a partial block of differentiation from pre-leukemic Pre-Meg/E progenitors. These Pre-Meg/E like progenitors were able to induce leukemia in the presence of a known cooperative oncoprotein MPL when transplanted in lethally irradiated congenic recipient mice. In summary, our Results suggest that expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells, which can be the target cell of additional mutations contributing to leukemia transformation. Disclosures: No relevant conflicts of interest to declare.

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The Cell of Origin in NPM1 Positive Acute Myeloid Leukemia (AML) Is Prognostic

Blood ◽

10.1182/blood.v128.22.1690.1690 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1690-1690

Author(s):

Mark Iscoe ◽

Kelly J Norsworthy ◽

Gabriel Ghiaur ◽

B. Douglas Smith ◽

Christopher Gocke ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Retrospective Cohort ◽

Cohort Analysis ◽

Newly Diagnosed ◽

Cell Of Origin ◽

Retrospective Cohort Analysis ◽

Cd34 Expression ◽

Acute Myeloid

Abstract Mutations in the nucleophosmingene (NPM1) are common in patients with cytogenetically-normal acute myeloid leukemia (AML) and have been associated with positive outcomes. Although most leukemic stem cells (LSCs) arise from CD34+ primitive progenitors, our recent data suggest that a fraction arise from less primitive CD34- progenitors and seem to have a better prognosis (Gerber et al. Haematologica 2016). Conversely, poor-risk AMLs such as those harboring internal tandem duplications of the FMS-like tyrosine kinase 3 (FLT3-ITD) or with poor-risk cytogenetics appear to arise from LSCs with a primitive hematopoietic stem cell (HSC) phenotype. NPM1-mutated AMLs can be either CD34- or CD34+, and have been associated with variable prognoses. We hypothesize that the cell of origin, CD34- or CD34+, of NPM1-mutated AML has prognostic implications. A retrospective cohort analysis was conducted using electronic medical record data from patients aged 18 or older with newly-diagnosed NPM1-mutated AML treated with intensive induction chemotherapy at Johns Hopkins Hospital from 2008-2015. NPM1 and FLT3-ITD mutations were determined from PCR assays on diagnostic samples. We (Gerber et al. Haematologica2016) and others have shown that CD34+ cells represent normal, non-leukemia progenitors when they comprise <1% of mononuclear cells (MNCs); thus, >/= 1% MNCs was the cut-off used to distinguish CD34+ from CD34- AMLs. Clinical outcomes including complete remission (CR) rate were compared using Chi-squared tests and event-free survival (EFS) and overall survival (OS) were estimated per the Kaplan-Meier method, using Wilcoxon tests to determine the impact of CD34 status. Of 46 consecutive, newly diagnosed NPM1-positive patients treated between 2008-15, 36 (78%) had normal cytogenetics and 37 (80%) had de novo AML. 26/46 (57%) had CD34+ disease; of these, 18/26 (69%) had FLT3-ITD mutations, compared to 9/20 (45%) with CD34- AML (p=0.10). In the CD34- patients with FLT3-ITD, the mean allelic ratio (mutant to wild type allele) ±SEM was 0.26±.08 compared to 2.3±1.0 in the CD34+ patients (p=0.07). Comparing CD34+ and CD34- patients, there were no significant differences in mean age at diagnosis (52 vs 59), sex, initial hemoglobin, platelet count, or lactate dehydrogenase, but CD34+ patients had significantly higher baseline white blood cell count (mean 78 vs 38 x 103 cells/mm3; p=0.04). There was a trend toward lower CR rates following induction for CD34+ patients (69 vs 89%; p=0.11), and similar proportions of patients underwent bone marrow transplantation (BMT) (50% vs 37%, p=0.38). However, CD34+ patients had significantly shorter EFS (figure 1; median 8.0 vs 34.3 months; p=0.04), and a trend toward inferior OS (15.4 vs 35.4 months; p=0.36). The lack of statistical significance in OS may reflect effective salvage by allogeneic transplantation. In conclusion, in this small retrospective cohort analysis of NPM1-mutated AML patients, CD34 expression was associated with significantly reduced EFS and, among FLT3-ITD mutated patients, a trend toward higher allelic ratios. Hence, CD34 positivity may be an early indicator of poor prognosis in NPM1+ AML and requires further characterization to determine its full prognostic and predictive value in guiding AML therapy. These data support evidence that the biology of AML is not only a function of genetic mutations, but also in part a functionof the stage of hematopoietic differentiation at which the leukemogenic mutations develop. Figure 1 EFS of NPM1-mutated AML patients by CD34 expression. Median follow-up of 23 months. EFS was significantly shorter for CD34+ patients (median 8.0 vs 34.3 months; p=0.01). Figure 1. EFS of NPM1-mutated AML patients by CD34 expression. Median follow-up of 23 months. EFS was significantly shorter for CD34+ patients (median 8.0 vs 34.3 months; p=0.01). Disclosures No relevant conflicts of interest to declare.

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