Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML.

Targeted immunotherapy in acute myeloid leukemia (AML) is challenged by the lack of AML-specific target antigens and clonal heterogeneity, leading to unwanted on-target off-leukemia toxicity and risk of relapse from minor clones. We hypothesize that combinatorial targeting of AML cells can enhance therapeutic efficacy without increasing toxicity. To identify target antigen combinations specific for AML and leukemic stem cells, we generated a detailed protein expression profile based on flow cytometry of primary AML (n = 356) and normal bone marrow samples (n = 34), and a recently reported integrated normal tissue proteomic data set. We analyzed antigen expression levels of CD33, CD123, CLL1, TIM3, CD244 and CD7 on AML bulk and leukemic stem cells at initial diagnosis (n = 302) and relapse (n = 54). CD33, CD123, CLL1, TIM3 and CD244 were ubiquitously expressed on AML bulk cells at initial diagnosis and relapse, irrespective of genetic characteristics. For each analyzed target, we found additional expression in different populations of normal hematopoiesis. Analyzing the coexpression of our six targets in all dual combinations (n = 15), we found CD33/TIM3 and CLL1/TIM3 to be highly positive in AML compared with normal hematopoiesis and non-hematopoietic tissues. Our findings indicate that combinatorial targeting of CD33/TIM3 or CLL1/TIM3 may enhance therapeutic efficacy without aggravating toxicity in immunotherapy of AML.

Tyrosine kinase inhibition increases the cell surface localization of FLT3-ITD and enhances FLT3-directed immunotherapy of acute myeloid leukemia.

The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regard to oncogenic alterations that do not only serve as prognostic markers but also as therapeutic targets in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Because of sequence-dependent protein conformational changes FLT3-ITD tends to autophosphorylate and displays a constitutive intracellular localization. Here, we analyzed the effect of tyrosine kinase inhibitors (TKIs) on the localization of the FLT3 receptor and its mutants. TKI treatment increased the surface expression through upregulation of FLT3 and glycosylation of FLT3-ITD and FLT3-D835Y mutants. In T cell-mediated cytotoxicity (TCMC) assays, using a bispecific FLT3 × CD3 antibody construct, the combination with TKI treatment increased TCMC in the FLT3-ITD-positive AML cell lines MOLM-13 and MV4-11, patient-derived xenograft cells and primary patient samples. Our findings provide the basis for rational combination of TKI and FLT3-directed immunotherapy with potential benefit for FLT3-ITD-positive AML patients.

A four-gene LincRNA expression signature predicts risk in multiple cohorts of acute myeloid leukemia patients.

Prognostic gene expression signatures have been proposed as clinical tools to clarify therapeutic options in acute myeloid leukemia (AML). However, these signatures rely on measuring large numbers of genes and often perform poorly when applied to independent cohorts or those with older patients. Long intergenic non-coding RNAs (lincRNAs) are emerging as important regulators of cell identity and oncogenesis, but knowledge of their utility as prognostic markers in AML is limited. Here we analyze transcriptomic data from multiple cohorts of clinically annotated AML patients and report that (i) microarrays designed for coding gene expression can be repurposed to yield robust lincRNA expression data, (ii) some lincRNA genes are located in close proximity to hematopoietic coding genes and show strong expression correlations in AML, (iii) lincRNA gene expression patterns distinguish cytogenetic and molecular subtypes of AML, (iv) lincRNA signatures composed of three or four genes are independent predictors of clinical outcome and further dichotomize survival in European Leukemia Net (ELN) risk groups and (v) an analytical tool based on logistic regression analysis of quantitative PCR measurement of four lincRNA genes (LINC4) can be used to determine risk in AML.

Persistence of pre-leukemic clones during first remission and risk of relapse in acute myeloid leukemia.

Some patients with acute myeloid leukemia (AML) who are in complete remission after induction chemotherapy harbor persisting pre-leukemic clones, carrying a subset of leukemia-associated somatic mutations. There is conflicting evidence on the prognostic relevance of these clones for AML relapse. Here, we characterized paired pre-treatment and remission samples from 126 AML patients for mutations in 68 leukemia-associated genes. Fifty patients (40%) retained ⩾1 mutation during remission at a variant allele frequency of ⩾2%. Mutation persistence was most frequent in DNMT3A (65% of patients with mutations at diagnosis), SRSF2 (64%), TET2 (55%), and ASXL1 (46%), and significantly associated with older age (P<0.0001) and, in multivariate analyses adjusting for age, genetic risk, and allogeneic transplantation, with inferior relapse-free survival (hazard ratio, 2.34; P=0039) and overall survival (hazard ratio, 2.14; P=036). Patients with persisting mutations had a higher cumulative incidence of relapse before, but not after allogeneic stem cell transplantation. Our work underlines the relevance of mutation persistence during first remission as a novel risk factor in AML. Persistence of pre-leukemic clones may contribute to the inferior outcome of elderly AML patients. Allogeneic transplantation abrogated the increased relapse risk associated with persisting pre-leukemic clones, suggesting that mutation persistence may guide postremission treatment.Leukemia accepted article preview online, 18 December 2017. doi:10.1038/leu.2017.350.

Clinical, pathological and genetic features of follicular lymphoma grade 3A: a joint analysis of the German low-grade and high-grade lymphoma study groups GLSG and DSHNHL.

BACKGROUND: Histologically, follicular lymphoma (FL) grades 1, 2 and 3A are composed of two distinct cell types, centroblasts and centrocytes. FL grade 3B is composed only of centroblasts and has been shown to differ in immunophenotype and genetics from FL that contain centrocytes. We aimed to understand the pathogenetic and clinical relation between FL grade 3A to FL grade 1/2 on the one hand and FL grade 3B on the other hand. PATIENTS AND METHODS: Trial patients with long-term follow-up and diagnosis of FL grade 3 were selected and samples underwent a second central pathological review using a multiple-observer approach to assess grading. RESULTS: Interobserver variability for diagnosing FL grade 3 was high. FL grade 3A frequently harbored areas of FL grade 1/2 within the same tissue specimen. FL grade 3B rarely coexisted with grade 1/2 or 3A, suggesting divergent pathogenesis. There was no statistically significant difference in outcome between 47 cases of FL grade 3A and 14 cases of grade 3B. Compared with grade 1/2 FL, both groups showed longer progression-free survival without late events, especially after immunochemotherapy; this outcome difference was retained after adjustment for clinical prognostic factors. The subgroup of FL grade 3A with an additional FL grade 1/2 component or a translocation t(14;18) showed a poorer outcome. In contrast, the FL grade 3A lacking t(14;18) and of localized stage resembled the pediatric type of FL and showed a very good outcome. FL3 with MYC breaks showed a poor outcome. CONCLUSIONS: The results suggest that first-line immunochemotherapy might allow long-lasting remissions in a subgroup of FL grade 3A similar to diffuse large B-cell lymphoma. Within FL3A, prognostic subgroups can be identified by analyzing for coexisting FL1/2 and MYC breaks.

[Current treatment strategies for follicular lymphoma]. / Aktuelle Therapiestrategien beim follikulären Lymphom.

In the last few years major changes have taken place in the management of follicular lymphoma (FL) leading to a substantial improvement in the prognosis of patients suffering from this disease. For patients with limited disease stages I and II radiotherapy can lead to long-term remission, while it still remains unclear whether this actually represents a cure; therefore, new approaches are investigating the combination of irradiation with an anti-CD20 antibody. For patients with advanced stages III and IV and a low tumor burden, watch and wait is still the first choice of management. For advanced stages III and IV and a high tumor burden, immunochemotherapy followed by 2 years of rituximab maintenance therapy is the worldwide accepted standard. New antibodies and new agents targeting oncogenic pathways or having immunomodulatory activity provide attractive new therapeutic options, which are currently being explored in clinical phase II/III studies. These approaches provide the perspective to establish chemotherapy-free therapy concepts in the near future.

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.

Similar clinical features in follicular lymphomas with and without breaks in the BCL2 locus.

Approximately 15% of follicular lymphomas (FLs) lack breaks in the BCL2 locus. The aim of this study was to better define molecular and clinical features of BCL2-breakpoint/t(14;18)-negative FLs. We studied the presence of BCL2, BCL6 and MYC breaks by fluorescence in situ hybridization and the expression of BCL2, MUM1, CD10, P53 and Ki67 in large clinical trial cohorts of 540 advanced-stage FL cases and 116 early-stage disease FL patients treated with chemotherapy regimens and radiation, respectively. A total of 86% and 53% of advanced- and early-stage FLs were BCL2-breakpoint-positive, respectively. BCL2 was expressed in almost all FLs with BCL2 break and also in 86% and 69% of BCL2-breakpoint-negative advanced- and early-stage FLs, respectively. CD10 expression was significantly reduced in BCL2-breakpoint-negative FLs of all stages and MUM1 and Ki67 expression were significantly increased in BCL2-break-negative early-stage FLs. Patient characteristics did not differ between FLs with and without BCL2 breaks and neither did survival times in advanced-stage FLs. These results suggest that the molecular profile differs to some extent between FLs with and without BCL2 breaks and support the notion that FLs with and without BCL2 breaks belong to the same lymphoma entity.

Blockade of the PD-1/PD-L1 axis augments lysis of AML cells by the CD33/CD3 BiTE antibody construct AMG 330: reversing a T-cell-induced immune escape mechanism.

Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, P<0.0001). This phenomenon was cytokine-driven as the sole addition of interferon (IFN)-γ and tumor necrosis factor-α also induced expression. Through blockade of the PD-1/PD-L1 interaction, AMG 330-mediated lysis (n=9, P=0.03), T-cell proliferation (n=9, P=0.01) and IFN-γ secretion (n=8, P=0.008) were significantly enhanced. The combinatorial approach was most beneficial in settings of protracted AML cell lysis. Taken together, we have characterized a critical resistance mechanism employed by primary AML cells under AMG 330-mediated proinflammatory conditions. Our results support the evaluation of checkpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity.

RUNX1/ETO blocks selectin-mediated adhesion via epigenetic silencing of PSGL-1.

RUNX1/ETO (RE), the t(8;21)-derived leukemic transcription factor associated with acute myeloid leukemia (AML) development, deregulates genes involved in differentiation, self-renewal and proliferation. In addition, these cells show differences in cellular adhesion behavior whose molecular basis is not well understood. Here, we demonstrate that RE epigenetically silences the gene encoding P-Selectin Glycoprotein Ligand-1 (PSGL-1) and downregulates PSGL-1 expression in human CD34+ and murine lin- hematopoietic progenitor cells. Levels of PSGL-1 inversely and dose-dependently correlate with RE oncogene levels. However, a DNA-binding defective mutant fails to downregulate PSGL-1. We show by ChIP experiments that the PSGL-1 promoter is a direct target of RE and binding is accompanied by high levels of the repressive chromatin mark histone H3K27me3. In t(8;21)+ Kasumi-1 cells, PSGL-1 expression is completely restored at both the mRNA and cell surface protein levels following RE downregulation with short hairpin RNA (shRNA) or RE inhibition with tetramerization-blocking peptides, and at the promoter H3K27me3 is replaced by the activating chromatin mark H3K9ac as well as by RNA polymerase II. Upregulation of PSGL-1 restores the binding of cells to P- and E-selectin and re-establishes myeloid-specific cellular adhesion while it fails to bind to lymphocyte-specific L-selectin. Overall, our data suggest that the RE oncoprotein epigenetically represses PSGL-1 via binding to its promoter region and thus affects the adhesive behavior of t(8;21)+ AML cells.

Sequential therapy combining clofarabine and T-cell-replete HLA-haploidentical haematopoietic SCT is feasible and shows efficacy in the treatment of refractory or relapsed aggressive lymphoma.

Prognosis is poor for patients with biologically aggressive Non-Hodgkin lymphoma (NHL), refractory to chemotherapy or relapsed after autologous transplantation, especially when no disease control before allogeneic transplantation is achieved. In 16 patients (median age 53, median prior regimes 5) with relapsed or refractory non-remission NHL, we analysed retrospectively the efficacy of a sequential therapy comprising clofarabine re-induction followed by a reduced-intensity conditioning with fludarabine, CY and melphalan, and T-cell-replete HLA-haploidentical transplantation. High-dose CY was utilized post-transplantation. All patients engrafted. Early response (day +30) was achieved in 94%. Treatment-related grade III-IV toxicity occurred in 56%, most commonly transient elevation of transaminases (36%), while there was a low incidence of infections (19% CMV reactivation, 19% invasive fungal infection) and GVHD (GVHD: acute III-IV: 6%; mild chronic: 25%). One-year non-relapse mortality was 19%. After a median follow-up of 21 months, estimated 1- and 2-year PFS was 56 and 50%, respectively, with 11 patients (69%) still alive after 2 years. In summary, sequential therapy is feasible and effective and provides an acceptable toxicity profile in high-risk non-remission NHL. Presumably, cytotoxic reinduction with clofarabine provides enough remission time for the graft-versus lymphoma effect of HLA-haploidentical transplantation to kick in, even in lymphomas that are otherwise chemo-refractory.

Framework for timing of the discussion about forgoing cancer-specific treatment based on a qualitative study with oncologists.

BACKGROUND: Many patients with advanced cancer receive aggressive chemotherapy close to death and are referred too late to palliative or hospice care. AIM: The aim of this study was to investigate oncologists' and oncology nurses' perceptions of the optimal timing for discussions about forgoing cancer-specific therapy at the End-of-Life (EOL) and the reasons that might hinder them. DESIGN: Qualitative in-depth interviews with oncologists and oncology nurses were carried out. The empirical data were evaluated from a normative perspective. SETTING/PARTICIPANTS: Twenty-nine physicians and nurses working at the Department of Hematology and Oncology of a German university hospital were interviewed. RESULTS: Health-care professionals differed considerably in their understanding of when to initiate discussions about forgoing cancer-specific therapy at the EOL. However, their views could be consolidated into three approaches: (1) preparing patients gradually throughout the course of disease (anticipatory approach) which is best suited to empower patient self-determination in decision-making, (2) waiting until the patient him/herself starts the discussion about forgoing cancer-specific treatment, and (3) waiting until all tumor-specific therapeutic options are exhausted. CONCLUSION: The empirically informed ethical analysis clearly favors an approach that prepares patients for forgoing cancer-specific therapy throughout the course of disease. Since the last two approaches often preclude advance care planning, these approaches may be less ethically acceptable. The proposed framework could serve as a starting point for the development of concrete recommendations on the optimal timing for EOL discussions.