Comprehensive sequential genetic analysis delineating frequency, patterns, and prognostic impact of genomic dynamics in a real-world cohort of patients with lower-risk MDS.

The acquisition of subsequent genetic lesions (clonal evolution, CE) and/or the expansion of existing clones (CEXP) contributes to clonal dynamics (CD) in myelodysplastic syndromes (MDS). Although CD plays an important role in high-risk patients in disease progression and transformation into acute myeloid leukemia (AML), knowledge about CD in lower-risk MDS (LR-MDS) patients is limited due to lack of robust longitudinal data considering the long clinically stable courses of the disease. In this retrospective analysis, we delineate the frequency and the prognostic impact of CD in an unselected real-world cohort of LR-MDS patients. We screened 68 patients with a median follow-up of 40.5 months and a median of 7.5 (range: 2-22) timepoints for CE and CEXP detected by chromosomal banding analysis, fluorescence in situ hybridization, sequencing, and molecular karyotyping. In 30/68 patients, 47 CE events and a CD rate of 1 event per 4 years were documented. Of note, patients with at least 1 CE event had an increased probability for subsequent treatment. Unexpectedly, CE did not correlate with inferior outcomes, which could be reasonably explained by CD detection triggering the subsequent start of a disease-modifying therapy.

Prognostic value of indoleamine 2,3 dioxygenase in patients with higher-risk myelodysplastic syndromes treated with azacytidine.

Hypomethylating agents (HMAs) are widely used in patients with higher-risk myelodysplastic syndromes (MDS) not eligible for stem cell transplantation; however, the response rate is <50%. Reliable predictors of response are still missing, and it is a major challenge to develop new treatment strategies. One current approach is the combination of azacytidine (AZA) with checkpoint inhibitors; however, the potential benefit of targeting the immunomodulator indoleamine-2,3-dioxygenase (IDO-1) has not yet been evaluated. We observed moderate to strong IDO-1 expression in 37% of patients with high-risk MDS. IDO-1 positivity was predictive of treatment failure and shorter overall survival. Moreover, IDO-1 positivity correlated inversely with the number of infiltrating CD8+ T cells, and IDO-1+ patients failed to show an increase in CD8+ T cells under AZA treatment. In vitro experiments confirmed tryptophan catabolism and depletion of CD8+ T cells in IDO-1+ MDS, suggesting that IDO-1 expression induces an immunosuppressive microenvironment in MDS, thereby leading to treatment failure under AZA treatment. In conclusion, IDO-1 is expressed in more than one-third of patients with higher-risk MDS, and is predictive of treatment failure and shorter overall survival. Therefore, IDO-1 is emerging as a promising predictor and therapeutic target, especially for combination therapies with HMAs or checkpoint inhibitors.

Inhibition of PLK1 by capped-dose volasertib exerts substantial efficacy in MDS and sAML while sparing healthy haematopoiesis.

INTRODUCTION: Targeting the cell cycle machinery represents a rational therapeutic approach in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). Despite substantial response rates, clinical use of the PLK inhibitor volasertib has been hampered by elevated side effects such as neutropenia and infections. OBJECTIVES: The primary objective was to analyse whether a reduced dose of volasertib was able to limit toxic effects on the healthy haematopoiesis while retaining its therapeutic effect. METHODS: Bone marrow mononuclear cells (BMMNCs) of patients with MDS/sAML (n = 73) and healthy controls (n = 28) were treated with volasertib (1 µM to 1 nM) or vehicle control. Short-term viability analysis was performed by flow cytometry after 72 hours. For long-term viability analysis, colony-forming capacity was assessed after 14 days. Protein expression of RIPK3 and MCL-1 was quantified via flow cytometry. RESULTS: Reduced dose levels of volasertib retained high cell death-inducing efficacy in primary human stem and progenitor cells of MDS/sAML patients without affecting healthy haematopoiesis in vitro. Interestingly, volasertib reduced colony-forming capacity and cell survival independent of clinical stage or mutational status. CONCLUSIONS: Volasertib offers a promising therapeutic approach in patients with adverse prognostic profile. RIPK3 and MCL-1 might be potential biomarkers for sensitivity to volasertib treatment.

Comprehensive analysis of isolated der(1;7)(q10;p10) in a large international homogenous cohort of patients with myelodysplastic syndromes.

The karyotype is a strong independent prognostic factor in myelodysplastic syndromes (MDS). Since the implementation of the new comprehensive cytogenetic scoring system for MDS, chromosome 7 anomalies are no longer generally assigned to poor risk features but are thoroughly separated. However, der(1;7)(q10;p10), hereinafter der(1;7), is merged into the group labeled "any other single" and belongs to the intermediate risk group, just by definition due to lack of adequate clinical data. The aim of our international collaborative was to clarify the "real" prognostic impact of der(1;7) on a homogenous and well-documented data base. We performed detailed analysis of 63 MDS patients with isolated der(1;7) constituting the largest cohort hitherto reported. Furthermore, clinical data are compared with those of patients with isolated del(7q) and isolated monosomy 7. Median overall survival (OS) of patients with der(1;7) is 26 months (hazard ratio (HR) 0.91 for del(7q) vs der(1;7) and 2.53 for monosomy 7 vs der(1;7)). The der(1;7) is associated with profound thrombocytopenia most probably causing the reduced OS which is in striking contrast to the low risk for AML transformation (HR 3.89 for del(7q) vs der(1;7) and 5.88 for monosomy 7 vs der(1;7)). Molecular karyotyping indicates that der(1;7) is generated in a single step during mitosis and that a chromosomal imbalance rather than a single disrupted gene accounts for malignancy. Thus, the current cytogenetic scoring system assigning isolated der(1;7) to the intermediate risk group is now confirmed by a sufficient data set.

Serum Vitamin D Levels in Patients with Myelodysplastic Syndromes: A Retrospective Single-Center Analysis.

BACKGROUND/AIMS: There is growing evidence supporting the role of innate immune deregulation and inflammation in the pathogenesis of myelodysplastic syndromes (MDS). Vitamin D (VD) is known to be involved in various immune and epigenetic processes. This analysis aimed to evaluate serum VD levels in patients with MDS and to analyze associations between serum VD levels and disease characteristics. METHODS: Serum levels of 25-hydroxyvitamin D3 (25(OH)-D3), the major form of VD in human serum, were measured by chemiluminescence immunoassay in 62 unselected patients with MDS. Associations between serum 25(OH)-D3 levels and disease characteristics were analyzed using Kendall's tau and two-sided p values. RESULTS: The median serum 25(OH)-D3 level was markedly reduced (17.5 ng/mL). Patients with lower-risk disease features had lower serum 25(OH)-D3 levels than patients with higher-risk disease features with regard to medullary blast counts (16 vs. 31 ng/mL, p < 0.001), the revised international prognostic scoring system (13 vs. 30.5 ng/mL, p = 0.001), and blood counts. CONCLUSIONS: We show that patients with lower-risk disease characteristics exhibit lower serum VD levels than patients with higher-risk disease characteristics. Whether these findings might reflect innate immune deregulation has to be investigated in further studies.

Venetoclax with azacitidine targets refractory MDS but spares healthy hematopoiesis at tailored dose.

Patients with Myelodysplastic Syndromes (MDS) and secondary Acute Myeloid Leukemia (sAML) have a very poor prognosis after failure of hypomethylating agents (HMA). Stem cell transplantation is the only effective salvage therapy, for which only a limited number of patients are eligible due to age and comorbidity. Combination therapy of venetoclax and azacitidine (5-AZA) seems to be a promising approach in myeloid malignancies, but data from patients with HMA failure are lacking. Furthermore, a considerable concern of combination regimens in elderly AML and MDS patients is the toxicity on the remaining healthy hematopoiesis. Here, we report in vitro data showing the impact of venetoclax and 5-AZA, alone or in combination, in a larger cohort of MDS/sAML patients (n = 21), even after HMA failure (n = 13). We especially focused on the effects on healthy hematopoiesis and the impact on colony forming capacity as a parameter for long-term effects. To the best of our knowledge, we show for the first time that venetoclax in combination with capped dose of 5-AZA targets cell malignancies, while sparing healthy hematopoiesis.

Direct modulation of the bone marrow mesenchymal stromal cell compartment by azacitidine enhances healthy hematopoiesis.

Mesenchymal stromal cells (MSCs) are crucial components of the bone marrow (BM) microenvironment essential for regulating self-renewal, survival, and differentiation of hematopoietic stem/progenitor cells (HSPCs) in the stem cell niche. MSCs are functionally altered in myelodysplastic syndromes (MDS) and exhibit an altered methylome compared with MSCs from healthy controls, thus contributing to disease progression. To determine whether MSCs are amenable to epigenetic therapy and if this affects their function, we examined growth, differentiation, and HSPC-supporting capacity of ex vivo-expanded MSCs from MDS patients in comparison with age-matched healthy controls after direct treatment in vitro with the hypomethylating agent azacitidine (AZA). Strikingly, we find that AZA exerts a direct effect on healthy as well as MDS-derived MSCs such that they favor support of healthy over malignant clonal HSPC expansion in coculture experiments. RNA-sequencing analyses of MSCs identified stromal networks regulated by AZA. Notably, these comprise distinct molecular pathways crucial for HSPC support, foremost extracellular matrix molecules (including collagens) and interferon pathway components. Our study demonstrates that the hypomethylating agent AZA exerts its antileukemic activity in part through a direct effect on the HSPC-supporting BM niche and provides proof of concept for the therapeutic potential of epigenetic treatment of diseased MSCs. In addition, our comprehensive data set of AZA-sensitive gene networks represents a valuable framework to guide future development of targeted epigenetic niche therapy in myeloid malignancies such as MDS and acute myeloid leukemia.

Efficacy of azacitidine is independent of molecular and clinical characteristics - an analysis of 128 patients with myelodysplastic syndromes or acute myeloid leukemia and a review of the literature.

Azacitidine is the first drug to demonstrate a survival benefit for patients with MDS. However, only half of patients respond and almost all patients eventually relapse. Limited and conflicting data are available on predictive factors influencing response. We analyzed 128 patients from two institutions with MDS or AML treated with azacitidine to identify prognostic indicators. Genetic mutations in ASXL1, RUNX1, DNMT3A, IDH1, IDH2, TET2, TP53, NRAS, KRAS, FLT3, KMT2A-PTD, EZH2, SF3B1, and SRSF2 were assessed by next-generation sequencing. With a median follow up of 5.6 years median survival was 1.3 years with a response rate of 49%. The only variable with significant influence on response was del(20q). All 6 patients responded (p = 0.012) but survival was not improved. No other clinical, cytogenetic or molecular marker for response or survival was identified. Interestingly, patients from poor-risk groups as high-risk cytogenetics (55%), t-MDS/AML (54%), TP53 mutated (48%) or relapsed after chemotherapy (60%) showed a high response rate. Factors associated with shorter survival were low platelets, AML vs. MDS, therapy-related disease, TP53 and KMT2A-PTD. In multivariate analysis anemia, platelets, FLT3-ITD, and therapy-related disease remained in the model. Poor-risk factors such as del(7q)/-7, complex karyotype, ASXL1, RUNX1, EZH2, and TP53 did not show an independent impact. Thus, no clear biomarker for response and survival can be identified. Although a number of publications on predictive markers for response to AZA exist, results are inconsistent and improved response rates did not translate to improved survival. Here, we provide a comprehensive overview comparing the studies published to date.

Selective inhibition of BCL-2 is a promising target in patients with high-risk myelodysplastic syndromes and adverse mutational profile.

Somatic mutations in genes such as ASXL1, RUNX1, TP53 or EZH2 adversely affect the outcome of patients with myelodysplastic syndromes (MDS). Since selective BCL-2 inhibition is a promising treatment strategy in hematologic malignancies, we tested the therapeutic impact of ABT-199 on MDS patient samples bearing an adverse mutational profile. By gene expression, we found that the level of pro-apoptotic BIM significantly decreased during MDS disease progression in line with an acquired resistance to cell death. Supporting the potential for ABT-199 treatment in MDS, high-risk MDS patient samples specifically underwent cell death in response to ABT-199 even when harbouring mutations in ASXL1, RUNX1, TP53 or EZH2. ABT-199 effectively targeted the stem- and progenitor compartment in advanced MDS harbouring mutations in ASXL1, RUNX1, TP53 or EZH2 and even proved effective in patients harbouring more than one of the defined high-risk mutations. Moreover, we utilized the protein abundance of BCL-2 family members in primary patient samples using flow cytometry as a biomarker to predict ABT-199 treatment response. Our data demonstrate that ABT-199 effectively induces apoptosis in progenitors of high-risk MDS/sAML despite the presence of adverse genetic mutations supporting the notion that pro-apoptotic intervention will hold broad therapeutic potential in high-risk MDS patients with poor prognosis.

Outcome of Lower-Risk Patients With Myelodysplastic Syndromes Without 5q Deletion After Failure of Erythropoiesis-Stimulating Agents.

Purpose Most anemic patients with non-deleted 5q lower-risk myelodysplastic syndromes (MDS) are treated with erythropoiesis-stimulating agents (ESAs), with a response rate of approximately 50%. Second-line treatments, including hypomethylating agents (HMAs), lenalidomide (LEN), and investigational drugs, may be used after ESA failure in some countries, but their effect on disease progression and overall survival (OS) is unknown. Here, we analyzed outcome after ESA failure and the effect of second-line treatments. Patients and Methods We examined an international retrospective cohort of 1,698 patients with non-del(5q) lower-risk MDS treated with ESAs. Results Erythroid response to ESAs was 61.5%, and median response duration was 17 months. Of 1,147 patients experiencing ESA failure, 653 experienced primary failure and 494 experienced relapse after a response. Primary failure of ESAs was associated with a higher risk of acute myeloid leukemia (AML) progression, which did not translate into an OS difference. Of 450 patients (39%) who received second-line treatment, 194 received HMAs, 148 received LEN, and 108 received other treatments (MISC), whereas 697 received RBC transfusions only. Five-year AML cumulative incidence was 20.3%, 20.3%, and 11.3% for those receiving HMAs, LEN, and MISC, respectively ( P = .05). Five-year OS for patients receiving HMA, LEN, and MISC was 36.5%, 41.7%, and 51%, respectively ( P = .21). In a multivariable analysis adjusted for age, sex, revised International Prognostic Scoring System score, and progression at ESA failure, there was no significant OS difference among the three groups. Conclusion In this large, multicenter, retrospective cohort of patients with non-del(5q) lower-risk MDS treated with ESAs, none of the most commonly used second-line treatments (HMA and LEN) significantly improved OS. Early failure of ESAs was associated with a higher risk of AML progression.

Targeted positron emission tomography imaging of CXCR4 expression in patients with acute myeloid leukemia.

Acute myeloid leukemia originates from leukemia-initiating cells that reside in the protective bone marrow niche. CXCR4/CXCL12 interaction is crucially involved in recruitment and retention of leukemia-initiating cells within this niche. Various drugs targeting this pathway have entered clinical trials. To evaluate CXCR4 imaging in acute myeloid leukemia, we first tested CXCR4 expression in patient-derived primary blasts. Flow cytometry revealed that high blast counts in patients with acute myeloid leukemia correlate with high CXCR4 expression. The wide range of CXCR4 surface expression in patients was reflected in cell lines of acute myeloid leukemia. Next, we evaluated the CXCR4-specific peptide Pentixafor by positron emission tomography imaging in mice harboring CXCR4 positive and CXCR4 negative leukemia xenografts, and in 10 patients with active disease. [(68)Ga]Pentixafor-positron emission tomography showed specific measurable disease in murine CXCR4 positive xenografts, but not when CXCR4 was knocked out with CRISPR/Cas9 gene editing. Five of 10 patients showed tracer uptake correlating well with leukemia infiltration assessed by magnetic resonance imaging. The mean maximal standard uptake value was significantly higher in visually CXCR4 positive patients compared to CXCR4 negative patients. In summary, in vivo molecular CXCR4 imaging by means of positron emission tomography is feasible in acute myeloid leukemia. These data provide a framework for future diagnostic and theranostic approaches targeting the CXCR4/CXCL12-defined leukemia-initiating cell niche.

Frequency of del(12p) is commonly underestimated in myelodysplastic syndromes: Results from a German diagnostic study in comparison with an international control group.

In myelodysplastic syndromes (MDS), deletion of the short arm of chromosome 12 (del(12p)) is usually a small abnormality, rarely detected as a single aberration by chromosome banding analysis (CBA) of bone marrow metaphases. Del(12p) has been described in 0.6 to 5% of MDS patients at initial diagnosis and is associated with a good to intermediate prognosis as a sole anomaly according to current scoring systems. Here, we present the results of a systematic del(12p) testing in a German prospective diagnostic study (clinicaltrials.gov: NCT01355913) on 367 MDS patients in whom CD34+ peripheral blood cells were analysed for the presence of del(12p) by sequential fluorescence in situ hybridization (FISH) analyses. A cohort of 2,902 previously published MDS patients diagnosed by CBA served as control. We demonstrate that, using a sensitive FISH technique, 12p deletion occurs significantly more frequently in MDS than previously described (7.6% by CD34+ PB-FISH vs. 1.6% by CBA, P < 0.001) and is often associated with other aberrations (93% by CD34+ PB-FISH vs. 60% by CBA). Additionally, the detection rate can be increased by repeated analyses in a patient over time which is important for the patient´s prognosis to distinguish a sole anomaly from double or complex aberrations. To our knowledge, this is the first study to screen for 12p deletions with a suitable probe for ETV6/TEL in 12p13. Our data suggest that the supplement of a probe for the detection of a 12p deletion to common FISH probe panels helps to avoid missing a del(12p), especially as part of more complex aberrations.

Validation of cytogenetic risk groups according to International Prognostic Scoring Systems by peripheral blood CD34+FISH: results from a German diagnostic study in comparison with an international control group.

International Prognostic Scoring Systems are used to determine the individual risk profile of myelodysplastic syndrome patients. For the assessment of International Prognostic Scoring Systems, an adequate chromosome banding analysis of the bone marrow is essential. Cytogenetic information is not available for a substantial number of patients (5%-20%) with dry marrow or an insufficient number of metaphase cells. For these patients, a valid risk classification is impossible. In the study presented here, the International Prognostic Scoring Systems were validated based on fluorescence in situ hybridization analyses using extended probe panels applied to cluster of differentiation 34 positive (CD34(+)) peripheral blood cells of 328 MDS patients of our prospective multicenter German diagnostic study and compared to chromosome banding results of 2902 previously published patients with myelodysplastic syndromes. For cytogenetic risk classification by fluorescence in situ hybridization analyses of CD34(+) peripheral blood cells, the groups differed significantly for overall and leukemia-free survival by uni- and multivariate analyses without discrepancies between treated and untreated patients. Including cytogenetic data of fluorescence in situ hybridization analyses of peripheral CD34(+) blood cells (instead of bone marrow banding analysis) into the complete International Prognostic Scoring System assessment, the prognostic risk groups separated significantly for overall and leukemia-free survival. Our data show that a reliable stratification to the risk groups of the International Prognostic Scoring Systems is possible from peripheral blood in patients with missing chromosome banding analysis by using a comprehensive probe panel (clinicaltrials.gov identifier:01355913).

Molecular cytogenetic monitoring from CD34+ peripheral blood cells in myelodysplastic syndromes: first results from a prospective multicenter German diagnostic study.

The gold standard of cytogenetic analysis in myelodysplastic syndromes (MDS) is conventional chromosome banding (CCB) analysis of bone marrow (BM) metaphases. Most aberrations can also be detected by fluorescence-in situ-hybridization (FISH). For this prospective multicenter German diagnostic study (www.clinicaltrials.gov: #NCT01355913) 360 patients, as yet, were followed up to 3 years by sequential FISH analyses of immunomagnetically enriched CD34+ peripheral blood (PB) cells using comprehensive FISH probe panels, resulting in a total number of 19,516 FISH analyses. We demonstrate that CD34+ PB FISH correlates significantly with CCB analysis and represents a feasible method for a reliable non-invasive cytogenetic monitoring from PB.

A limited number of 5-azacitidine cycles can be effective treatment in MDS.

Hypomethylating agents, such as 5-azacitidine (5-AZA) and 5-aza-2'-deoxycytidine (decitabine), have recently been approved for the treatment of myelodysplastic syndromes (MDS). Several randomized trials have shown favorable results concerning response rate, survival, transformation to acute leukemia, and quality of life. In these trials, treatment was administered continuously until progression. In the retrospective study presented here, we evaluated the outcome of patients with higher risk MDS or secondary acute myeloid leukemia (sAML) treated with a limited number of 5-AZA cycles. A total of 32 patients received 5-AZA alone (n=30) or in combination with valproic acid and all-trans retinoic acid (n=2). 5-AZA was administered subcutaneously at a fixed dose of 75 mg/m(2) daily for 7 days and repeated every 28 days. 5-AZA was given for a median of four courses. Treatment was continued for two more cycles as consolidation in patients who had achieved complete remission (CR), marrow CR, or stable disease with hematologic improvement. The overall response rate was 50% according to the modified International Working Group criteria. Complete remissions were achieved in 15.6% and stable disease in 34.4% of patients. Peripheral blood counts normalized in 6.3% of patients while hematologic improvement was achieved in 25%. The median time to AML in responding patients was 45 weeks, while AML occurred after a median of 14 weeks in non-responding patients (P= .038). The median survival of all patients was 60 weeks; the median survival of responders was 74 weeks compared with 26 weeks in non-responders (P= .047). In this retrospective analysis, 5-AZA was associated with a survival advantage in responding patients with higher risk MDS or sAML. These favorable results suggest that patients may benefit even from a limited number of courses of 5-AZA. A randomized controlled clinical trial is required to prospectively validate these findings.

Induction of apoptosis by Legionella pneumophila in mammalian cells requires the mitochondrial pathway for caspase activation.

Legionella pneumophila, the agent of human Legionnaire's disease is a Gram-negative, rod-shaped bacterium. During infection, the bacteria invade human cells and replicate intracellularly. L. pneumophila can induce apoptosis in human myeloid and epitheloid cells and this may contribute to the development of pathology and disease. However, the molecular mechanism of apoptosis induction is still uncertain. Here we investigate this process. Legionella efficiently induced apoptosis in myeloid cells, T cells and fibroblasts. Induction of apoptosis involved activation of the initiator caspase-9 and effector caspases. Caspase activity was required for cell death. Analysis of mutant cells showed that the death receptor pathway was not involved in Legionella-induced apoptosis. Surprisingly, caspase activity was found almost exclusively in cells that did not harbor bacteria. Infection with Legionella caused the activation of the pro-apoptotic protein Bax and the release of cytochrome c. Mouse embryonic fibroblasts deficient for Bax and/or Bak were protected from Legionella-induced caspase activation. These results show a clear contribution of the mitochondrial pathway to Legionella-induced apoptosis.