Inflammatory signals from fatty bone marrow support DNMT3A driven clonal hematopoiesis.

Both fatty bone marrow (FBM) and somatic mutations in hematopoietic stem cells (HSCs), also termed clonal hematopoiesis (CH) accumulate with human aging. However it remains unclear whether FBM can modify the evolution of CH. To address this question, we herein present the interaction between CH and FBM in two preclinical male mouse models: after sub-lethal irradiation or after castration. An adipogenesis inhibitor (PPARγ inhibitor) is used in both models as a control. A significant increase in self-renewal can be detected in both human and rodent DNMT3AMut-HSCs when exposed to FBM. DNMT3AMut-HSCs derived from older mice interacting with FBM have even higher self-renewal in comparison to DNMT3AMut-HSCs derived from younger mice. Single cell RNA-sequencing on rodent HSCs after exposing them to FBM reveal a 6-10 fold increase in DNMT3AMut-HSCs and an activated inflammatory signaling. Cytokine analysis of BM fluid and BM derived adipocytes grown in vitro demonstrates an increased IL-6 levels under FBM conditions. Anti-IL-6 neutralizing antibodies significantly reduce the selective advantage of DNMT3AMut-HSCs exposed to FBM. Overall, paracrine FBM inflammatory signals promote DNMT3A-driven clonal hematopoiesis, which can be inhibited by blocking the IL-6 pathway.

Side-effect management of chimeric antigen receptor (CAR) T-cell therapy.

Chimeric antigen receptor (CAR) T cells directed against the B-cell marker CD19 are currently changing the landscape for treatment of patients with refractory and/or relapsed B-cell malignancies. Due to the nature of CAR T cells as living drugs, they display a unique toxicity profile. As CAR T-cell therapy is extending towards other diseases and being more broadly employed in hematology and oncology, optimal management strategies of side-effects associated with CAR T-cell therapy are of high relevance. Cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and cytopenias constitute challenges in the treatment of patients with CAR T cells. This review summarizes the current understanding of CAR T-cell toxicity and its management.

RET-mediated autophagy suppression as targetable co-dependence in acute myeloid leukemia.

Many cases of AML are associated with mutational activation of receptor tyrosine kinases (RTKs) such as FLT3. However, RTK inhibitors have limited clinical efficacy as single agents, indicating that AML is driven by concomitant activation of different signaling molecules. We used a functional genomic approach to identify RET, encoding an RTK, as an essential gene in multiple subtypes of AML, and observed that AML cells show activation of RET signaling via ARTN/GFRA3 and NRTN/GFRA2 ligand/co-receptor complexes. Interrogation of downstream pathways identified mTORC1-mediated suppression of autophagy and subsequent stabilization of leukemogenic drivers such as mutant FLT3 as important RET effectors. Accordingly, genetic or pharmacologic RET inhibition impaired the growth of FLT3-dependent AML cell lines and was accompanied by upregulation of autophagy and FLT3 depletion. RET dependence was also evident in mouse models of AML and primary AML patient samples, and transcriptome and immunohistochemistry analyses identified elevated RET mRNA levels and co-expression of RET and FLT3 proteins in a substantial proportion of AML patients. Our results indicate that RET-mTORC1 signaling promotes AML through autophagy suppression, suggesting that targeting RET or, more broadly, depletion of leukemogenic drivers via autophagy induction provides a therapeutic opportunity in a relevant subset of AML patients.

An atlas of bloodstream-accessible bone marrow proteins for site-directed therapy of acute myeloid leukemia.

The concept of arming antibodies with bioactive payloads for a site-specific therapy of cancer has gained considerable interest in recent years. However, a successful antibody-based targeting approach critically relies on the availability of a tumor-associated target that is not only preferentially expressed in the tumor tissue but is also easily accessible for antibody therapeutics coming from the bloodstream. Here, we perfused the vasculature of healthy and acute myeloid leukemia (AML)-bearing rats with a reactive ester derivative of biotin and subsequently quantified the biotinylated proteins to identify AML-associated bone marrow (BM) antigens accessible from the bloodstream. In total, >1400 proteins were identified. Overall, 181 proteins were >100-fold overexpressed in AML as compared with normal BM. Eleven of the most differentially expressed proteins were further validated by immunohistochemistry and confocal microscopic analyses, including novel antigens highly expressed in AML cells (for example, adaptor-related protein complex 3 ß2) and in the leukemia-modified extracellular matrix (ECM) (for example, collagen-VI-α-1). The presented atlas of targetable AML-associated BM proteins provides a valuable basis for the development of monoclonal antibodies that could be used as carriers for a site-specific pharmacodelivery of cytotoxic drugs, cytokines or radionuclides to the BM in AML.

Outcome of allogeneic stem cell transplantation for AML and myelodysplastic syndrome in elderly patients (⩾60 years).

Allogeneic stem cell transplantation (SCT) remains the best curative option for patients with refractory AML or with high-risk myelodysplastic syndrome (MDS). For decades, age alone had been widely used as the primary criterion to assess eligibility for allogeneic SCT; however, prospective studies to evaluate allogeneic SCT in elderly patients are still limited. A total of 187 patients (median age of 64 years, range 60-77 years) with AML (87%) or MDS (13%) transplanted between 1999 and 2014 were included in this retrospective analysis. Relapse-free survival (RFS) and overall survival (OS) at 3 years were 32% (95% confidence interval (CI): 25-39%) and 35% (95%CI: 27-42%), respectively. Overall survival was 49% (95%CI: 35-64%) in AML patients who were transplanted in first complete remission (CR1), but even patients with active disease did benefit from transplantation, showing an OS at 3 years of 30% (95%CI: 20-40%). Multivariate analysis revealed disease- and patient-specific risk indices as independent prognostic factors for OS and non-relapse mortality (NRM). In conclusion, our monocenter results indicate that patients should not be generally withheld from allogeneic SCT because of age or disease status only. Specific risk models incorporating disease status and disease-specific risk factors at the time of transplantation as well as existing comorbidities are helpful tools to assess transplantation-associated risk factors of elderly patients.

Enantiomer-specific and paracrine leukemogenicity of mutant IDH metabolite 2-hydroxyglutarate.

Canonical mutations in IDH1 and IDH2 produce high levels of the R-enantiomer of 2-hydroxyglutarate (R-2HG), which is a competitive inhibitor of α-ketoglutarate (αKG)-dependent enzymes and a putative oncometabolite. Mutant IDH1 collaborates with HoxA9 to induce monocytic leukemia in vivo. We used two mouse models and a patient-derived acute myeloid leukemia xenotransplantation (PDX) model to evaluate the in vivo transforming potential of R-2HG, S-2HG and αKG independent of the mutant IDH1 protein. We show that R-2HG, but not S-2HG or αKG, is an oncometabolite in vivo that does not require the mutant IDH1 protein to induce hyperleukocytosis and to accelerate the onset of murine and human leukemia. Thus, circulating R-2HG acts in a paracrine manner and can drive the expansion of many different leukemic and preleukemic clones that may express wild-type IDH1, and therefore can be a driver of clonal evolution and diversity. In addition, we show that the mutant IDH1 protein is a stronger oncogene than R-2HG alone when comparable intracellular R-2HG levels are achieved. We therefore propose R-2HG-independent oncogenic functions of mutant IDH1 that may need to be targeted in addition to R-2HG production to exploit the full therapeutic potential of IDH1 inhibition.

Increasing intensity of therapies assigned at diagnosis does not improve survival of adults with acute myeloid leukemia.

We randomized 3375 adults with newly diagnosed acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome to test whether increasingly intensive chemotherapies assigned at study-entry and analyzed on an intent-to-treat basis improved outcomes. In total, 1529 subjects <60 years were randomized to receive: (1) a first course of induction therapy with high-dose cytarabine and mitoxantrone (HAM) or with standard-dose cytarabine, daunorubicin and 6-thioguanine (TAD) followed by a second course of HAM; (2) granulocyte-colony stimulating factor (G-CSF) or no G-CSF before induction and consolidation courses; and (3) high-dose therapy and an autotransplant or maintenance chemotherapy. In total, 1846 subjects ⩾60 years were randomized to receive: (1) a first induction course of HAM or TAD and second induction course of HAM (if they had bone marrow blasts ⩾5% after the first course); and (2) G-CSF or no G-CSF as above. Median follow-up was 7.4 years (range, 1 day to 14.7 years). Five-year event-free survivals (EFSs) for subjects receiving a first induction course of HAM vs TAD were 17% (95% confidence interval, 15, 18%) vs 16% (95% confidence interval 14, 18%; P=0.719). Five-year EFSs for subjects randomized to receive or not receive G-CSF were 19% (95% confidence interval 16, 21%) vs 16% (95% confidence interval 14, 19%; P=0.266). Five-year relapse-free survivals (RFSs) for subjects <60 years receiving an autotransplant vs maintenance therapy were 43% (95% confidence interval 40, 47%) vs 40 (95% confidence interval 35, 44%; P=0.535). Many subjects never achieved pre-specified landmarks and consequently did not receive their assigned therapies. These data indicate the limited impact of more intensive therapies on outcomes of adults with AML. Moreover, none of the more intensive therapies we tested improved 5-year EFS, RFS or any other outcomes.

Azacitidine in combination with intensive induction chemotherapy in older patients with acute myeloid leukemia: The AML-AZA trial of the Study Alliance Leukemia.

DNA methylation changes are a constant feature of acute myeloid leukemia. Hypomethylating drugs such as azacitidine are active in acute myeloid leukemia (AML) as monotherapy. Azacitidine monotherapy is not curative. The AML-AZA trial tested the hypothesis that DNA methyltransferase inhibitors such as azacitidine can improve chemotherapy outcome in AML. This randomized, controlled trial compared the efficacy of azacitidine applied before each cycle of intensive chemotherapy with chemotherapy alone in older patients with untreated AML. Event-free survival (EFS) was the primary end point. In total, 214 patients with a median age of 70 years were randomized to azacitidine/chemotherapy (arm-A) or chemotherapy (arm-B). More arm-A patients (39/105; 37%) than arm-B (25/109; 23%) showed adverse cytogenetics (P=0.057). Adverse events were more frequent in arm-A (15.44) versus 13.52 in arm-B, (P=0.26), but early death rates did not differ significantly (30-day mortality: 6% versus 5%, P=0.76). Median EFS was 6 months in both arms (P=0.96). Median overall survival was 15 months for patients in arm-A compared with 21 months in arm-B (P=0.35). Azacitidine added to standard chemotherapy increases toxicity in older patients with AML, but provides no additional benefit for unselected patients.

Mtss1 is a critical epigenetically regulated tumor suppressor in CML.

Chronic myeloid leukemia (CML) is driven by malignant stem cells that can persist despite therapy. We have identified Metastasis suppressor 1 (Mtss1/MIM) to be downregulated in hematopoietic stem and progenitor cells from leukemic transgenic SCLtTA/Bcr-Abl mice and in patients with CML at diagnosis, and Mtss1 was restored when patients achieved complete remission. Forced expression of Mtss1 decreased clonogenic capacity and motility of murine myeloid progenitor cells and reduced tumor growth. Viral transduction of Mtss1 into lineage-depleted SCLtTA/Bcr-Abl bone marrow cells decreased leukemic cell burden in recipients, and leukemogenesis was reduced upon injection of Mtss1-overexpressing murine myeloid 32D cells. Tyrosine kinase inhibitor (TKI) therapy and reversion of Bcr-Abl expression increased Mtss1 expression but failed to restore it to control levels. CML patient samples revealed higher DNA methylation of specific Mtss1 promoter CpG sites that contain binding sites for Kaiso and Rest transcription factors. In summary, we identified a novel tumor suppressor in CML stem cells that is downregulated by both Bcr-Abl kinase-dependent and -independent mechanisms. Restored Mtss1 expression markedly inhibits primitive leukemic cell biology in vivo, providing a therapeutic rationale for the Bcr-Abl-Mtss1 axis to target TKI-resistant CML stem cells in patients.

Treatment strategies in patients with AML or high-risk myelodysplastic syndrome relapsed after Allo-SCT.

Non-relapse mortality after Allo-SCT has significantly decreased over the last years. Nevertheless, relapse remains a major cause for post SCT mortality in patients with AML and high-risk myelodysplastic syndrome (MDS). In this retrospective single-center analysis, we have analyzed the treatment outcomes of 108 patients with AML or MDS, who relapsed after Allo-SCT. Seventy of these patients (65%) were treated with salvage therapies containing chemotherapy alone, allogeneic cell-based treatment or the combination of both. Thirty-eight patients (35%) received palliative treatment. Median OS after diagnosis of relapse was 130 days. Compared with patients who received chemotherapy alone, response to salvage therapy was significantly improved in patients treated with a combination of chemo- and allogeneic cell-based therapy (CR rate 57% vs 13%, P=0.002). Among risk factors concerning pretreatment characteristics, disease status before first Allo-SCT, and details of transplantation, only the time interval from Allo-SCT to relapse was an independent predictor of response to salvage therapy and OS. These data confirmed that time to relapse after transplantation is an important prognostic factor. Up to now, only patients eligible for treatment regimens containing allogeneic cell-based interventions achieved relevant response rates.

NF-κB/STAT5/miR-155 network targets PU.1 in FLT3-ITD-driven acute myeloid leukemia.

Almost 30% of all acute myeloid leukemias (AML) are associated with an internal tandem duplication (ITD) in the juxtamembrane domain of FMS-like tyrosine kinase 3 receptor (FLT3). Patients with FLT3-ITD mutations tend to have a poor prognosis. MicroRNAs (miRNAs) have a pivotal role in myeloid differentiation and leukemia. MiRNA-155 (MiR-155) was found to be upregulated in FLT3-ITD-associated AMLs. In this study, we discovered that FLT3-ITD signaling induces the oncogenic miR-155. We show in vitro and in vivo that miR-155 expression is regulated by FLT3-ITD downstream targets nuclear factor-κB (p65) and signal transducer and activator of transcription 5 (STAT5). Further, we demonstrate that miR-155 targets the myeloid transcription factor PU.1. Knockdown of miR-155 or overexpression of PU.1 blocks proliferation and induces apoptosis of FLT3-ITD-associated leukemic cells. Our data demonstrate a novel network in which FLT3-ITD signaling induces oncogenic miR-155 by p65 and STAT5 in AML, thereby targeting transcription factor PU.1.

Treatment of rare co-occurrence of Epstein-Barr virus-driven post-transplant lymphoproliferative disorder and hemophagocytic lymphohistiocytosis after allogeneic stem cell transplantation.

In both conditions, post-transplant lymphoproliferative disorder (PTLD) and hemophagocytic lymphohistiocytosis (HLH), infection with Epstein-Barr virus (EBV) is a key mechanism: almost all PTLD in allogeneic stem cell transplantation (alloSCT) is caused by EBV-related neoplastic lymphoproliferation, and secondary HLH is most frequently triggered by EBV infection. Therefore, concomitant EBV-driven PTLD and HLH early after alloSCT require an approach to eliminate EBV and balance immune activation simultaneously. We report on a patient who developed simultaneous PTLD and signs of HLH on day 64 after alloSCT. Treatment was comprised of stopping cyclosporine, short-course dexamethasone, and 3 courses of rituximab. The patient showed full recovery and complete remission of lymphadenopathy. This result indicates that immediate reduction in EBV-carrying B cells by rituximab, suppression of general inflammation, and parallel support of reconstitution of long-term T-cell function, might be an appropriate therapeutic approach in this rare situation.

Origins of aberrant DNA methylation in acute myeloid leukemia.

Aberrant DNA methylation patterns are a characteristic feature of cancer including myeloid malignancies such as acute myeloid leukemia (AML). The mechanisms behind aberrant DNA methylation have long remained obscure. New genome-wide studies have elucidated the genome and epigenome of solid tumors and AML. Molecular subtypes of AML were found to exhibit highly distinct DNA methylation profiles. Clonal evolution patterns of AML were recently dissected and might shape epigenetic dysregulation. Also, recurrent mutations in epigenetic modifying enzymes were identified in AML and linked to distinct DNA methylation signatures. The genetic background, thus, takes center stage as a driver of epigenetic dysregulation in AML. First mechanistic insights into the dysregulation of DNA methylation by recurrent mutations have already been gained. Other studies suggest that epigenomic plasticity and aging-associated changes in DNA methylation also contribute extensively to aberrant DNA methylation in cancer. Epigenetic dysregulation, therefore, seems to also occur independently of the genetic background. Furthermore, global changes in chromatin conformation and nuclear organization have also been proposed as potential contributors to aberrant DNA methylation. This review will summarize and discuss current concepts regarding the mechanisms behind aberrant DNA methylation in cancer and specifically AML.

CD34(+) lineage specific donor cell chimerism for the diagnosis and treatment of impending relapse of AML or myelodysplastic syndrome after allo-SCT.

After allo-SCT, analysis of CD34(+) lineage-specific donor cell chimerism (DCC) is a sensitive method for monitoring minimal residual disease in patients with AML or myelodysplastic syndrome (MDS) with CD34 expression. To substantiate evidence of whether immune interventions in patients with impending relapse, defined by incomplete lineage-specific DCC, may prevent hematological relapse, we performed a retrospective nested case control study. Unsorted and lineage-specific DCC were measured in 134 patients. Forty-three patients had an incomplete CD34(+)-DCC with no other evidence of relapse. After immediate tapering of immunosuppressive treatment (30 patients) and/or infusion of donor lymphocytes (10 patients), 21 patients remained in remission (conversion to complete lineage-specific DCC) and 22 relapsed. Relapse-free survival at 3 years of the 91 patients with stable DCC and of the 43 patients with incomplete DCC was 74% (95% confidence interval (CI), 64-83%) and 40% (95% CI, 24-58%), respectively. OS rates were 79% (95% CI, 70-88%) and 52% (95% CI, 35-69%), respectively. These results, with 49% of patients with impending relapse successfully treated with immune intervention, highly suggest that analysis of CD34(+)-DCC is an important tool for monitoring and the management of AML and MDS patients after allo-SCT.

High-dose chemotherapy with autologous PBSC transplantation for poor prognosis germ cell tumors: a retrospective monocenter analysis of 44 cases.

Germ cell cancer (GCC) is curable in metastatic stages. The International Germ Cell Cancer Collaborative Group (IGCCCG) reports a poor prognosis subgroup with a 5-year survival of 48%. High-dose chemotherapy with PBSC transplantation (HD-PBSCT) in these patients showed promising results in phase II, but failed to show significant advantage in randomized trials. We report our monocenter series of all poor and selected intermediate prognosis germ cell tumor patients treated with multiple-course HD-PBSCT and secondary surgery of remaining tissue. We performed a retrospective analysis of our complete series of 44 patients (40 poor prognosis and 4 intermediate prognosis) treated by HD-PBSCT as part of first-line therapy from 1999 to 2010. The CR rate after up to four cycles of HD-PBSCT and radical resection of residual manifestations was 73%. The 3-year survival rate was 79.5% (median follow-up of 51.5 months; range: 7-143 months). Disease-related death rate was 16%. HD-PBSCT-related death did not occur. One patient died postsurgery. Multiple courses of HD-PBSCT with radical secondary surgery is safe and effective in poor prognosis metastatic GCC. Despite disappointing phase III studies it is of high interest to further study this field.

Induction therapy of AML with ara-C plus daunorubicin versus ara-C plus gemtuzumab ozogamicin: a randomized phase II trial in elderly patients.

BACKGROUND: Chemotherapy for elderly patients with acute myeloid leukemia (AML) results in a median overall survival (OS) of ≤ 1 year. Elderly patients often present with cardiac comorbidity. Gemtuzumab ozogamicin (GO) is active in elderly (≥ 60 years) patients with relapsed AML with low cardiac toxicity. PATIENTS AND METHODS: This randomized phase II study compared a standard combination of ara-C and daunorubicin (DNR; 7+3) versus ara-C plus gemtuzumab ozogamicin (7+GO) as the first course of induction therapy. Primary objectives were comparison of blast clearance on day 16, event-free survival (EFS), and remission duration. OS, complete remission (CR), and tolerability were secondary objectives. RESULTS: One hundred and nineteen patients with de novo AML, treatment-related AML, AML with a history of myelodysplastic syndrome (MDS), or high-risk MDS entered the study. Median age of 115 patients (intent-to-treat population) was 69 years. Protocol outlined a second course 7+3 for patients without blast clearance and two courses of high-dose ara-C consolidation upon CR. Both treatments were equally effective in blast clearance, CR, EFS, remission duration, or OS (median: 7+3, 9 months; 7+GO, 10 months). Induction death rate was higher in the GO group due to veno-occlusive disease. CONCLUSION: The study did not show significant superiority of 7+GO over standard 7+3.