[Show abstract][Hide abstract] ABSTRACT: Immune checkpoint molecules are highly relevant as potential prognostic markers and therapeutic targets in malignant diseases. HVEM belongs to the TNF receptor family and provides stimulatory as well as inhibitory signals depending on the ligand. Abnormal HVEM expression has been described in various malignancies, but the role in AML is unknown. Here we report extensive data on HVEM surface protein expression analyzed by flow cytometry on bone marrow leukemic cells of 169 AML patients at diagnosis. An independent cohort of 512 AML patients was analyzed for HVEM mRNA expression in bone marrow samples by Affymetrix microarrays. Consistently for both cohorts and methods, we show that HVEM was differentially expressed and that expression levels were associated with defined genetic markers. HVEM expression was lower in cases with FLT3-ITD (p = 0.001, p < 0.001), with mutations in NPM1 (p = 0.001, p < 0.001) or with the combination of NPM1 mutation and FLT3 wild type (p = 0.049, p = 0.050), while a biallelic mutation in CEBPA correlated positively with higher HVEM expression (p = 0.015, p < 0.001). In a differential gene expression analysis, we found 13 genes including HOXA9, MEIS1 and MN1 that were closely associated with HVEM expression. Besides, four gene sets closely linked to immunity were enriched in HVEM (high) samples. Finally, high expression of HVEM was associated with a trend toward longer relapse-free survival. The results of this study provide new information on the potential significance of HVEM in AML.
No preview · Article · Sep 2015 · Cancer Immunology and Immunotherapy
[Show abstract][Hide abstract] ABSTRACT: Lately, mTOR inhibitors have gained clinical relevance in malignant lymphoma. Still, rapamycin derivatives may activate a pro-survival feedback loop through PI3K-Akt. In this current study, temsirolimus effectively reduced cell growth in GCB and ABC diffuse large cell B-cell lymphoma (GCB = 30-66%, ABC = 45-57%). Combination treatment with the PI3K-δ inhibitor idelalisib additively effected ABC and GCB lymphoma (GCB = 16-38%, ABC = 25-50%). Since Bruton's Tyrosine Kinase (BTK) plays a significant role for the survival of ABC lymphoma, this study also combined the BTK inhibitor ibrutinib with temsirolimus, which resulted in additive cell growth reduction (ibrutinib 50%, temsirolimus 44%, combination 25%) in ABC lymphoma. In contrast, bortezomib, which has been shown previously to be efficient in ABC lymphoma, revealed an antagonistic effect with temsirolimus in some GCB lymphoma (temsirolimus 53%, temsirolimus + bortezomib 63%). Western blot analysis identified the increase of phosphorylated pro-survival kinases Akt and PDK as a possible underlying mechanism of this interaction.
No preview · Article · Aug 2015 · Leukemia & lymphoma
[Show abstract][Hide abstract] ABSTRACT: T cell function is crucial for the success of several novel immunotherapeutic strategies for the treatment of acute myeloid leukemia (AML). However, changes in phenotype and function of T cells have been described in various hematologic malignancies, mimicking T cell exhaustion known from chronic viral infections. Detailed knowledge about phenotype and function of T cells in AML patients at different stages of the disease is indispensable for optimal development and application of immunotherapeutic strategies for this disease.
We used flow cytometry-based assays to characterize T cell phenotype and function in peripheral blood and bone marrow of AML patients at diagnosis, at relapse after intensive chemotherapy, and at relapse after allogeneic stem cell transplantation (SCT). Surface expression of CD244, PD-1, CD160, and TIM-3 was determined, and proliferation and production of IFN-γ, TNF-α, and IL-2 were measured.
We detected similar expression of inhibitory molecules on T cells from patients at diagnosis and from age-matched healthy controls. At relapse after SCT, however, PD-1 expression was significantly increased compared to diagnosis, both on CD4(+) and CD8(+) T cells. This pattern was not associated with age and cytomegalovirus (CMV) status but with a shift towards effector memory cells in relapsed AML patients. Proliferation and cytokine production assays did not reveal functional defects in T cells of AML patients, neither at diagnosis nor at relapse.
We thus conclude that T cell exhaustion does not play a major role in AML. Immunotherapeutic strategies targeting autologous T cells thus have particularly good prospects in the setting of AML.
[Show abstract][Hide abstract] ABSTRACT: The adapter protein metastasis suppressor 1 (MTSS1) is implicated as a tumor suppressor or tumor promoter, depending on the type of solid cancer. Here, we identified Mtss1 expression to be increased in AML subsets with favorable outcome, while suppressed in high risk AML patients. High expression of MTSS1 predicted better clinical outcome of patients with normal-karyotype AML. Mechanistically, MTSS1 expression was negatively regulated by FLT3-ITD signaling but enhanced by the AML1-ETO fusion protein. DNMT3B, a negative regulator of MTSS1, showed strong binding to the MTSS1 promoter in PML-RARA positive but not AML1-ETO positive cells, suggesting that AML1-ETO leads to derepression of MTSS1. Pharmacological treatment of AML cell lines carrying the FLT3-ITD mutation with the specific FLT3 inhibitor PKC-412 caused upregulation of MTSS1. Moreover, treatment of acute promyelocytic cells (APL) with all-trans retinoic acid (ATRA) increased MTSS1 mRNA levels. Taken together, our findings suggest that MTSS1 might have a context-dependent function and could act as a tumor suppressor, which is pharmacologically targetable in AML patients.
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups.
[Show abstract][Hide abstract] ABSTRACT: Wilms Tumor-1 (WT1) expression level has long been found to be implicated in acute myeloid leukemia (AML) prognosis, though this is not reflected in current AML risk stratification. We hypothesized that a gene expression profile (GEP) associated with WT1 expression could be of prognostic value.
We analyzed two publically available AML GEP series in order to identify a gene signature associated with high-WT1 expression (hi-WT1). The first, herein called Netherlands series, comprised of 524 younger adult patients who have been treated according to sequential Dutch-Belgian Hemato-Oncology Cooperative Group and the Swiss Group for Clinical Cancer Research (HOVON/SAKK) AML-04, 04A, 29, 32, 42, and 43 protocols (GSE14468). The second series, herein called Germany series, consisted of 562 younger and older AML patients who were treated in the German AMLCG 1999 trial (GSE37642). We identified the hi-WT1 gene sets by comparing GEP among the highest and lowest quartiles of WT1 expression in both AML studies. About 62% of the probe sets in the Netherlands hi-WT1 set were found to be common with the Germany hi-WT1 set; 97% differed in the same direction. Moreover, a high degree of correlation of the fold differences was found among the two hi-WT1 sets (r2 = 0.81, p < 10-18), collectively suggesting a biological relevance for hi-WT1 gene sets.
In order to assess the prognostic implication of the hi-WT1 set, we used K-Nearest Neighborhood algorithm to generate various lists of hi-WT1 probe sets predicting event-free survival (EFS) as the favorable, and all others (dead, no remission, progressive disease/relapse) as the unfavorable events in the Netherlands series. Stepwise screening of the lists of 10 to 100 probe sets by Cox Regression identified a 16-gene subset of hi-WT1 set with distinct GEP and as the optimal predictor of overall survival (OS) and EFS in the Netherlands series. It comprised of GPR56, FAM30A, NGFRAP1, WBP5, LTK, PTP4A3, CD109, ZC3H12C, PYGB, CHIC1, HAVCR2, TMEM110, HAL, HDAC4, BLVRA, and P2RY2. In this series, the hi-WT1 cluster of patients showed lower 5y-probability of OS (10% vs 44%) and EFS (6% vs 38%) as compared to the remaining clusters. Accordingly, the hi-WT1 cluster showed shorter median OS (8.3 [CI 6.7-9.9] vs 31.3 [CI 17.1-45.5] months, P = 6 x 10-18) and EFS (4.9 [CI 3.2-6.5] vs 14.5 [CI 9.4-19.5] months, P = 3 x 10-16). Although the hi-WT1 cluster was associated with some of the cytogenetic and molecular aberrations including FLT3-ITD, it remained significant for both OS (P = 3 x 10-5) and EFS (P = 3 x 10-6) after adjustment for known AML risk factors.
In order to validate our findings, we performed a supervised clustering of the Germany AML series using the 16-gene signature. The hi-WT1 cluster predicted both adverse OS and relapse-free survival (RFS) (Fig. 1), which remained statistically significant after adjustment for known AML risk factors (Table 1). The median OS was 7.1 (CI 5.7-8.5) months for the hi-WT1 cluster as compared to 20.1 (CI 15.2- 25.0) months for other cases (P = 2 x 10-13), and the median RFS was 5.8 (CI 4.8-6.8) vs 20.3 (CI 10.6-30.0) months, respectively (P = 2 x 10-11). Moreover, the rate of complete remission was significantly lower in hi-WT1 cluster as compared to other clusters (42% vs 61%, P = 2 x 10-5). The positive (PPV) and negative predictive value (NPV) of the marker for prediction of adverse OS were 90% and 34%, respectively. These values were found to be 88% and 38%, respectively, for prediction of adverse RFS. MetaCore analysis identified the Antigen Presentation by MHC-II as the most implicated biological pathway in hi-WT1 sets, with many genes downregulated in the pathway.
In brief, we identified a 16-gene signature associated with WT1 expression and demonstrated its adverse and independent prognostic impact in adult AML patients. These promising results should be validated in further trials and provide new clues to the molecular mechanisms underlying WT1 regulation.
[Show abstract][Hide abstract] ABSTRACT: This largest prospective multicenter trial for adult patients with Burkitt lymphoma/leukemia aimed to prove efficacy and feasibility of a short-intensive chemotherapy combined with the anti-CD20 antibody Rituximab. 363 patients 16 to 85 years old (median 42 yrs) were recruited in 98 centers from 8/2002 until 06/2011. Treatment consisted of 6 five-day chemotherapy cycles with high-dose methotrexate, high-dose cytosine arabinoside, cyclophosphamide, etoposide, ifosphamide, corticosteroids, and triple intrathecal therapy. Patients >55 years received a reduced regimen. Rituximab was given d -1 before each cycle and twice as maintenance, for a total of 8 doses. The complete remission rate was 88% (319/363), the overall survival (OS) at 5 years 80%, and the progression-free survival (PFS) 71%, with significant difference between adolescents (≥15-25 yrs), adults (26-55 yrs) and elderly (>55 yrs) patients with an OS of 90%, 84%, and 62%. Full treatment could be applied in 86% of the patients. Most important prognostic factors were IPI (0-2 vs. 3-5, p=0.0005), aaIPI (0-1 vs. 2-3, p=0.0001), and gender (male vs. female, p=0.004). The high cure rate in this prospective trial with a substantial number of participating hospitals demonstrates the efficacy and feasibility of chemoimmunotherapy, also in elderly patients. The study is registered to www.clinicaltrials.gov as NCT00199082.
[Show abstract][Hide abstract] ABSTRACT: Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients.
[Show abstract][Hide abstract] ABSTRACT: NPM1 mutations represent frequent genetic alterations in patients with acute myeloid leukemia (AML) associated with a favorable prognosis. Different types of NPM1 mutations have been described. The purpose of our study was to evaluate the relevance of different NPM1 mutation types with regard to clinical outcome. Our analyses were based on 349 NPM1-mutated AML patients treated in the AMLCG99 trial. Complete remission rates, overall survival and relapse-free survival were not significantly different between patients with NPM1 type A or rare type mutations. The NPM1 mutation type does not seem to play a role in risk stratification of cytogenetically normal AML.
[Show abstract][Hide abstract] ABSTRACT: Background
The aim of this study was to analyze the long-term survival of AML patients with CEBPA mutations.Patients and methodsWe investigated 88 AML patients with a median age of 61 years and (1) cytogenetically normal AML (CN-AML), (2) monoallelic (moCEBPA) or biallelic (biCEBPA) CEBPA mutation, and (3) intensive induction treatment. 60/88 patients have been described previously with a shorter follow-up.ResultsMedian follow-up time was 9.8 years (95% CI: 9.4-10.1 years) compared to 3.2 and 5.2 years in our former analyses. Patients with biCEBPA survived significantly longer compared to those with moCEBPA (median overall survival (OS) 9.6 years vs. 1.7 years, p¿=¿0.008). Patients¿¿¿60 years and biCEBPA mutations showed a favorable prognosis with a 10-year OS rate of 81%.Both, bi- and moCEBPA-mutated groups had a low early death (d60) rate of 7% and 9%, respectively. Complete remission (CR) rates for biCEBPA- and moCEBPA-mutated patients were 82% vs. 70% (p¿=¿0.17). biCEBPA-mutated patients showed a longer relapse free survival (RFS) (median RFS 9.4 years vs. 1.5 years, p¿=¿0.021) and a lower cumulative incidence of relapse (CIR) compared to moCEBPA-mutated patients. These differences in OS and RFS were confirmed after adjustment for known clinical and molecular prognostic factors.Conclusions
In this long-term observation we confirmed the favorable prognostic outcome of patients with biCEBPA mutations compared to moCEBPA-mutated CN-AML. The high probability of OS (81%) in younger patients is helpful to guide intensity of postremission therapy.
[Show abstract][Hide abstract] ABSTRACT: Isolated trisomy 13 (AML+13) is a rare chromosomal abnormality in acute myeloid leukemia (AML), and its prognostic relevance is poorly characterized. We analyzed the clinical course of 34 AML+13 patients enrolled in the German AMLCG-1999 and SAL trials and studied their biological characteristics by exome sequencing, targeted sequencing of candidate genes and gene expression profiling. Relapse-free (RFS) and overall survival (OS) of AML+13 patients were inferior compared to other ELN Intermediate-II patients (n=855) (median RFS, 7.8 vs 14.1 months, p=0.006; median OS 9.3 vs. 14.8 months, p=0.004). Besides the known high frequency of RUNX1 mutations (75%), we identified mutations in spliceosome components in 88%, including SRSF2 codon 95 mutations in 81%, of AML+13 patients. Moreover, recurring mutations were detected in ASXL1 (44%) and BCOR (25%). Two patients carried mutations in CEBPZ, suggesting that CEBPZ is a novel recurrently mutated gene in AML. Gene expression analysis revealed a homogenous expression profile including upregulation of FOXO1 and FLT3 and downregulation of SPRY2. This is the most comprehensive clinical and biological characterization of AML+13 to date, and reveals a striking clustering of lesions in a few genes, defining AML+13 as a genetically homogenous leukemia subgroup with alterations in a few critical cellular pathways. These studies were registered at clinicaltrials.gov, identifiers: AMLCG-1999: NCT00266136; AML96: NCT00180115; AML2003: NCT00180102; and AML60+: NCT00893373.
[Show abstract][Hide abstract] ABSTRACT: Cyclic cytotoxic maintenance therapy can be applied to patients with AML in post-remission. We studied the immune status of AML patients in complete remission and the effect of maintenance therapy on different immune cell populations. Patients in complete remission had reduced NK, TH and Treg counts and a reduced NK activation capacity. In the course of cytotoxic maintenance therapy, NK counts further declined, while TH and Treg cells increased, with lower proliferative potential of TH cells. We conclude that immunotherapeutic approaches in post-remission have to consider reduced NK cell function and further impairment of cellular immune responses during cytotoxic therapy.
No preview · Article · Jun 2014 · Leukemia Research