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IntroductionThe majority of hematopoietic progenitor cells (HPCs) reside in the bone marrow surrounded by a complex, highly organized microenvironment. Under normal conditions, a small number of HPCs are released into the peripheral blood. Agents with distinct cellular targets and biologic activities can induce the mobilization of HPCs into blood, including hematopoietic growth factors, chemotherapeutic agents, and chemokines. 1,2 Recently, mobilized peripheral blood HPCs have become the principal cellular source for reconstitution of the hematopoietic system following myeloablative therapy. Currently, granulocyte colony-stimulating factor (G-CSF) is the most widely used agent to induce HPC mobilization due to its potency, predictability, and safety. 3 However, the mechanisms responsible for G-CSF-induced HPC mobilization have not been defined.We previously showed that G-CSF receptor (G-CSFR) expression on HPCs is not required for their mobilization by G-CSF, suggesting that G-CSF induces HPC mobilization indirectly through the generation of trans-acting signals. 4 The nature of the transacting signals that mediate G-CSF-induced HPC mobilization is unknown; however, accumulating evidence suggests that interaction of CXCL12 (stromal-derived factor 1 [SDF-1]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), may play an important role in regulating G-CSF-induced HPC mobilization. CXCL12 is a CXC chemokine constitutively produced in the bone marrow by stromal cells. 5 Studies of CXCL12-or CXCR4-deficient mice have established that these genes are necessary for the normal migration of HPCs from the fetal liver to the bone marrow and in the efficient retention of myeloid precursors in the adult bone marrow. 6,7 Moreover, treatment with AMD-3100, a specific antagonist of CXCR4, induces rapid and robust HPC mobilization in both humans and mice. 8,9 Finally, we and others showed that CXCL12 protein expression in the bone marrow is significantly decreased following G-CSF treatment. [10][11][12] Collectively, these data suggest a model in which disruption of CXCL12/CXCR4 signaling is a key step in G-CSF-induced HPC mobilization.The mechanisms mediating the G-CSF-induced decrease in CXCL12 protein expression in the bone marrow have not been For personal use only. on May 12, 2018. by guest www.bloodjournal.org From defined. Previous reports suggested that neutrophil elastase (NE) and cathepsin G (CG) might regulate CXCL12 protein expression in the bone marrow through proteolytic cleavage of CXCL12. 10,11 However, mice genetically lacking NE and CG display normal G-CSF-induced HPC mobilization, and the expected decrease in bone marrow CXCL12 protein was observed. 13 Thus, the G-CSFinduced decrease in CXCL12 protein expression in the bone marrow does not require these proteases. It is possible that other proteases can compensate for the loss of NE and CG. Alternatively, nonproteolytic mechanisms may regulate CXCL12 expression in the bone marrow during G-CSF-induced HPC mobilization.In this study, we characterize G-CSF...

BACKGROUND As consolidation therapy for acute myeloid leukemia (AML), allogeneic hematopoietic stem-cell transplantation provides a benefit in part by means of an immune-mediated graft-versus-leukemia effect. We hypothesized that the immune-mediated selective pressure imposed by allogeneic transplantation may cause distinct patterns of tumor evolution in relapsed disease. METHODS We performed enhanced exome sequencing on paired samples obtained at initial presentation with AML and at relapse from 15 patients who had a relapse after hematopoietic stem-cell transplantation (with transplants from an HLA-matched sibling, HLA-matched unrelated donor, or HLA-mismatched unrelated donor) and from 20 patients who had a relapse after chemotherapy. We performed RNA sequencing and flow cytometry on a subgroup of these samples and on additional samples for validation. RESULTS On exome sequencing, the spectrum of gained and lost mutations observed with relapse after transplantation was similar to the spectrum observed with relapse after chemotherapy. Specifically, relapse after transplantation was not associated with the acquisition of previously unknown AML-specific mutations or structural variations in immune-related genes. In contrast, RNA sequencing of samples obtained at relapse after transplantation revealed dysregulation of pathways involved in adaptive and innate immunity, including down-regulation of major histocompatibility complex (MHC) class II genes (HLA-DPA1, HLA-DPB1, HLA-DQB1, and HLA-DRB1) to levels that were 3 to 12 times lower than the levels seen in paired samples obtained at presentation. Flow cytometry and immunohistochemical analysis confirmed decreased expression of MHC class II at relapse in 17 of 34 patients who had a relapse after transplantation. Evidence suggested that interferon-γ treatment could rapidly reverse this phenotype in AML blasts in vitro. CONCLUSIONS AML relapse after transplantation was not associated with the acquisition of relapse-specific mutations in immune-related genes. However, it was associated with dysregulation of pathways that may influence immune function, including down-regulation of MHC class II genes, which are involved in antigen presentation. These epigenetic changes may be reversible with appropriate therapy. (Funded by the National Cancer Institute and others.)

IMPORTANCETests that predict outcomes for patients with acute myeloid leukemia (AML) are imprecise, especially for those with intermediate risk AML.OBJECTIVES To determine whether genomic approaches can provide novel prognostic information for adult patients with de novo AML. DESIGN, SETTING, AND PARTICIPANTS Whole-genome or exome sequencing was performed on samples obtained at disease presentation from 71 patients with AML (mean age, 50.8 years) treated with standard induction chemotherapy at a single site starting in March 2002, with follow-up through January 2015. In addition, deep digital sequencing was performed on paired diagnosis and remission samples from 50 patients (including 32 with intermediate-risk AML), approximately 30 days after successful induction therapy. Twenty-five of the 50 were from the cohort of 71 patients, and 25 were new, additional cases. EXPOSURES Whole-genome or exome sequencing and targeted deep sequencing. Risk of identification based on genetic data. MAIN OUTCOMES AND MEASURES Mutation patterns (including clearance of leukemia-associated variants after chemotherapy) and their association with event-free survival and overall survival. 10.5 (7.5-22.2) 42.2 (20.6-not estimable) 2.86 (1.39-5.88) 19.3 (7.5-42.3) 46.8 (22.6-not estimable) 2.88 (1.11-7.45) CONCLUSIONS AND RELEVANCEThe detection of persistent leukemia-associated mutations in at least 5% of bone marrow cells in day 30 remission samples was associated with a significantly increased risk of relapse, and reduced overall survival. These data suggest that this genomic approach may improve risk stratification for patients with AML.

Expression of the G-CSF receptor on bone marrow monocytes is sufficient to trigger HSC mobilization in response to G-CSF, in part via effects on osteoblast lineage cells.