Search

Contains fulltext : 290820.pdf (Publisher’s version ) (Open Access) Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34(+) CD19(-) ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS. 01 januari 2023

Monitoring of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) is predictive of disease recurrence and may identify patients who benefit from treatment intensification. Current MRD techniques rely on multicolor flow cytometry or molecular methods, but are limited in applicability or sensitivity. We evaluated the feasibility of a novel approach for MRD detection in peripheral blood (PB), which combines immunomagnetic preenrichment and fluorescence-activated cell sorting (FACS) for isolation of CD34+ cells with error-reduced targeted next-generation sequencing (NGS). For clinical validation, we retrospectively analyzed 429 PB and 55 bone marrow (BM) samples of 40 patients with AML or high-risk MDS, with/without molecular relapse based on CD34+ donor chimerism (DC), in complete remission after allogeneic stem cell transplantation. Enrichment of CD34+ cells for NGS increased the detection of mutant alleles in PB ∼1000-fold (median variant allele frequency, 1.27% vs 0.0046% in unsorted PB; P < .0001). Although a strong correlation was observed for the parallel analysis of CD34+ PB cells with NGS and DC (r = 0.8601), the combination of FACS and NGS improved sensitivity for MRD detection in dilution experiments ∼10-fold to levels of 10−6. In both assays, MRD detection was superior using PB vs BM for CD34+ enrichment. Importantly, NGS on CD34+ PB cells enabled prediction of molecular relapse with high sensitivity (100%) and specificity (91%), and significantly earlier (median, 48 days; range, 0-281; P = .0011) than by CD34+ DC or NGS of unsorted PB, providing additional time for therapeutic intervention. Moreover, panel sequencing in CD34+ cells allowed for the early assessment of clonal trajectories in hematological complete remission.

Contains fulltext : 290818.pdf (Publisher’s version ) (Open Access) BACKGROUND: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures. METHODS: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group. RESULTS: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies. CONCLUSIONS: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice. 01 januari 2023

We conducted a prospective clinical trial to investigate the safety and efficacy of plerixafor (P) in allogeneic peripheral blood stem cells (PBSC) donors with poor mobilization response to standard-dose granulocyte colony-stimulating factor (G-CSF), defined by 6 CD34 + cells/kg recipient body-weight (CD34+/kg RBW) after 1st apheresis. A single dose of 240 µg/kg P was injected subcutaneously at 10 p.m. on the day of the 1st apheresis. Thirty-seven allogeneic PBSC donors underwent study treatment. The median CD34+ count in peripheral blood was 15/µl on Day 1 after G-CSF alone, versus 44/µl on Day 2 after G-CSF plus P (p 8 on Day 1 and 2.8 × 108 on Day 2. In contrast to a median yield of only 1.31 × 106 CD CD34+/kg RBW on Day 1, triggering study inclusion, a median of 3.74 × 106 CD CD34+/kg RBW were collected with G-CSF plus P on Day 2. Of 37 donors, 21 reached the target cell count of >4.5 × 106 CD34+/kg RBW (57%, 95%CI 40–73%). No donor experienced a severe adverse event requiring treatment. In conclusion, P might be considered on a case-by-case basis for healthy allogeneic donors with very poor stem cell mobilization success after G-CSF.

5-Azacitidine (AZA) therapy is used in high-risk myelodysplastic syndrome (MDS) patients who often show abnormalities in their immunophenotype. We explored the potential impact of AZA on these immunophenotypic abnormalities in serial bone marrow studies performed in 81 patients from five centers. We compared the immunophenotypic features before and after therapy with AZA, established definitions consistent with flow cytometry immunophenotyping (FCI) improvement, and explored its clinical significance. After a median of 6 cycles of AZA, 41% of patients showed a FCI improvement and this finding associated with best possible clinical response (P < 0.001). FCI improvement also correlated with hematological improvement (HI) (53/78 patients; 68%), independently of their eligibility for stem cell transplantation. Among patients who achieved a HI after 6 cycles of AZA, the probability of maintaining this response at 12 cycles of AZA was twice as large (67%) for those patients who also achieved a FCI improvement after 6 cycles of AZA as compared to patients who did not (33%, P < 0.01). These findings support that monitoring of the immunophenotypic abnormalities during therapy with AZA may assist in redefining the quality of response in patients with MDS.

Background: Somatic mutations in splicing factor 3B subunit 1 (SF3B1) occur in 25% of all myelodysplastic syndromes (MDS) cases and identify a condition characterized by ring sideroblasts, ineffective erythropoiesis, and indolent disease course in lower-risk (LR) MDS. The impact of SF3B1 mutations on erythroid dysregulation has become apparent but little is known about cellular immune phenotypes. Identification of patient characteristics downstream of the pathogenic mutations in SF3B1 may reveal immunological determinants of disease phenotype and response to therapeutic interventions (e.g. luspatercept). In the present study, we applied multiplex immunophenotyping technologies in combination with artificial intelligence (AI)-based analytical approaches to identify genotype-immunophenotype correlations that may affect disease course and clinical outcomes in the SF3B1-MDS subtype. Methods: We performed transcriptomic immune profiling on bone marrow (BM) mononuclear cells (MNCs) from 12 SF3B1-MUT and 10 SF3B1-WT LR-MDS patients using the NanoString nCounter PanCancer Immune Profiling Panel. Besides, viably frozen PBMC samples (8 SF3B1-MUT; 4 SF3B1-WT) were thawed and stained for CyTOF with a 35-marker MaxPar Immune Profiling Panel (Fluidigm) for deep immune profiling. CyTOF data were analyzed with an in-house developed pipeline (ImmunoCluster, Opzoomer et al. 2021) incorporating the FlowSOM algorithm for unsupervised clustering (K=70). We also retrieved flow cytometric data on fresh BM and peripheral blood (PB) samples acquired independently as part of the diagnostic work-up and re-analyzed them using the T-REX pipeline (Barone and Paul et al. 2020). Results: On the transcriptomic level, the SF3B1-MUT subtype showed a distinct inflammatory signature compared to SF3B1-WT (59 DE genes at FDR < 0.05, Fig. 1A & B). GO/KEGG pathway analysis indicated that downregulated genes in SF3B1-MUT patients are involved in antigen processing/presentation (HLA class II transcript, CD8a), regulation of proinflammatory interleukin-1 beta secretion (IL1B, NLRP3), and cellular response to interferon-gamma (HLA class II transcripts, IRF4/8, CCL5, CCL20). Genes that were upregulated in SF3B1-MUT BM MNCs are involved in cell cycle (CDK1, CCND3), proliferation/survival (AXL), iron homeostasis (TFRC), and erythroid differentiation (TAL1). Unbiased interrogation of cellular phenotypes in PB through FlowSOM analysis of CyTOF data revealed an increased abundance of a CD4 + T cell metacluster resembling a central memory (CM) phenotype in SF3B1-MUT compared to SF3B1-WT PBMCs (7.9 vs 4.5% of CD45 +). Independent automated T-REX-based analysis of clinical flow data supported this finding and further indicated expansion of a cluster resembling CM CD8 + T cells in SF3B1-MUT PB. Lower IL1B expression in SF3B1-MUT and the implication of the IL-1/IL-1RAP axis in the inflammatory leukemic niche (De Boer et al. 2020) prompted us to re-analyze clinical flow cytometric data on IL-1RAP expression using the T-REX pipeline. T-REX revealed differences in IL-1RAP-expressing clusters between SF3B1-MUT and SF3B1-WT BM samples (9 SF3B1-MUT; 9 SF3B1-WT). Specifically, we noticed a cluster of IL-1RAP + CD34 - cells (HLA-DR + CD123 +) present in SF3B1-WT but greatly contracted in SF3B1-MUT MDS (Fig. 1C & D). Our data suggest that proinflammatory signaling via the IL-1/IL-1RAP axis is reduced in SF3B1-MUT BM. Aberrant marker expression on SF3B1-MUT monocytes has been reported (Duetz et al. 2020). T-REX analysis of BM CD14 + cells revealed distinct monocyte clusters with differential expression of CD123, CD11b, and HLA-DR. Our initial analysis showed that SF3B1-MUT-specific clusters displayed lower expression of CD123 and CD11b, suggesting that mutations in SF3B1 directly or indirectly affect monocyte phenotypes and most likely function. Conclusions: We provide evidence that the SF3B1-MDS subtype is associated with a distinct inflammatory signature characterized by lower IL1B expression, changed IL-1RAP + clusters, distinct monocyte phenotypes, and preservation of T cell homeostasis. Further investigation of the immune signature in SF3B1-mutated MDS subtype may lead to using personalized immunotherapy strategies in the future. This work also exemplifies the potential of unsupervised strategies for interrogating cellular immune phenotypes in MDS subtypes within clinical flow cytometry datasets. Figure 1 Figure 1. Disclosures Harrison: BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Constellation Pharmaceuticals: Research Funding; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Platzbecker: Celgene/BMS: Honoraria; Janssen: Honoraria; Novartis: Honoraria; AbbVie: Honoraria; Geron: Honoraria; Takeda: Honoraria. Kordasti: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Alexion: Honoraria; Beckman Coulter: Honoraria.

Background: Myelodysplastic syndromes (MDS) are considered clonal diseases and are diagnosed according to WHO by cytomorphology and cytogenetics. The diagnostic potential of flow cytometric aberrancies has not yet been comprehensively evaluated. Aim: Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies predefined according to European LeukemiaNet (ELN). Methods: 1682 patients undergoing diagnostics for suspected MDS according to WHO 2016 criteria were analyzed in parallel by flow cytometry according to ELN recommendations. Results: Median age was 72 years (18-97). MDS, MPN-RS-T or CMML were confirmed by cytomophology in 1029 (61%) cases, 653 (39%) were non-MDS. IPSS-R data was available in 857 (51%). An overall flow cytometric readout was available in 1679 (99.8%). 1001 (60%) were in agreement with MDS while 678 (40%) were not. Flow cytometric readout significantly correlated with cytomorphologic diagnosis (p Non-MDS cases had a fewer myeloid progenitor cells (MPC) (mean±SD, 0.8±0.9%) compared to low-risk MDS (1.7±2.3%, p3% was strongly associated with MDS/CMML (286/293, 98%, p Neutrophil aberrancies were found more frequently in neoplastic cases than in non-MDS cases (table 1). Again, frequencies of aberrations were higher for high-risk MDS as compared to low-risk MDS while this was not the case for CMML showing frequencies rather similar to low-risk MDS. Frequencies of aberrancies in monocytes revealed a similar figure as in neutrophils with higher rates in neoplastic cases but clearly significant numbers positive in non-MDS cases. Interestingly, frequencies were not higher in high-risk MDS as compared to low-risk MDS. As anticipated, frequencies were highest in CMML (table 1). Regarding erythroid cells only an aberrant percentage of them and aberrant CD71 expression were found in a reasonable number of cases. Importantly, rates of positivity were rather high in non-MDS cases which did not differ from CMML cases (table 1). In order to identify the diagnostic value of each individual aberrancy multivariate analyses were performed in the three subgroups, low-risk MDS, high-risk MDS and CMML, as well as in the total cohort. In low-risk MDS ten aberrancies were independently related to MDS (table 2). Five of these aberrancies were found in MPCs, two each in neutrophils and monocytes and one in erythroid cells. In high-risk MDS 11 aberrancies were independently related to MDS (table 2). Eight were found in MPCs, two in neutrophils, none in monocytes and one in erythroid cells. In CMML 12 aberrancies were independently related to CMML (table 2). Four were found in MPCs, neutrophils and monocytes, respectively, and none in erythroid cells. Considering all these three groups together and all aberrancies identified significantly related to MDS/CMML in at least one group in univariate analysis, multivariate analysis identified 12 aberrancies independently related to MDS/CMML (table 2). Six were found in MPCs, two in neutrophils, three in monocytes and one in erythroid cells. Taking into consideration only aberrancies independently associated with MDS/CMML, three such aberrancies resulted in an 80% agreement with the cytomorphologic diagnosis of MDS/CMML, i.e. 20% concordantly negative and 60% concordantly positive. Importantly, this applies without need of at least two cell compartments being affected as specified in the ELN recommendations. Conclusions: This multicenter prospective evaluation confirms the diagnostic potential of flow cytometric aberrancies. A core set of 17 markers identified as independently related to a diagnosis of MDS/CMML is suggested mandatory for flow cytometric evaluation of suspected MDS. An MPC count >3% should be considered indicative of MDS/CMML. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Van de Loosdrecht: Celgene: Consultancy, Research Funding; Amgen: Consultancy; Roche: Consultancy; Novartis: Consultancy; Alexion: Consultancy.

Introduction Myelodysplastic syndromes (MDS) belong to the most common hematological neoplasms in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. There is increasing evidence that an aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species. Tasquinimod (TASQ, Active Biotech) is a novel, oral small molecular drug with S100A9 inhibitory activity and it is currently investigated in a phase Ib/IIa trial in relapsed/refractory multiple myeloma (NCT04405167). TASQ has demonstrated anti-angiogenic, antitumor and immunomodulatory properties in a broad range of preclinical solid tumor models; however, little is known about its effects in myeloid malignancies. Aim We investigated the role of S100A9 in cellular models of MDS and the potential of TASQ to target S100A9 within the MDS stroma in vitro. Methods Immunohistochemical staining of S100A9, CD271+ mesenchymal stromal cells (MSCs), CD68+ macrophages and CD66b+ neutrophils in BM tissues from MDS patients and healthy donors was performed with multiplex immunohistochemistry and analyzed with the VECTRA imaging system. MSCs from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) in the presence or absence of TASQ (10µM). Subsequently, TLR4 downstreaming molecules such as IRAK1, gasdermin and NF-kB-p65 were analyzed by Western blot. Moreover, the mRNA expression of further proinflammatory molecules (IL-1b, IL-18, caspase1) and PD-L1 was quantified by real-time PCR. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium. Additionally, PD-1 mRNA expression was quantified in cocultured HSPCs. Results Immunohistochemical staining of BM tissue demonstrated S100A9 expression mainly by CD66b+ neutrophils and with less extent by CD68+ macrophages. In line with this, we could not detect relevant S100A9 mRNA expression in cultured MDS or healthy MSCs in vitro. Exposure of MDS and healthy MSCs with S100A9 induced TLR4 downstream signalling as demonstrated by increased expression of IRAK1 and NF-kB-p65. We further detected a higher expression of gasdermin, an inductor of pyroptosis, in S100A9 exposed MSCs. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation. Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1b and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50%. To evaluate the impact on the hematopoietic support of MSCs, we analysed MSC/HSPC cocultures after treatment with S100A9. We observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs. Interestingly, both the number of CAF-C and CFU could be increased by TASQ pre-treatment. Finally, the PD-1 expression in co-cultured HSPCs was regulated in the same way as its ligand in treated MSCs, nominating this interaction as a potential target of S100A9/TASQ in the MDS BM. Conclusion In summary, we provide evidence that the pathological inflammasome activation in the myelodysplastic bone marrow can be rescued by TASQ at least in part by inhibition of the S100A9 mediated TLR4 downstream signalling including NF-kB-p65 transcription and PD-L1 expression. These effects result in an improved hematopoietic support by MSCs, suggesting a potential efficacy to improve cytopenia in low-risk MDS patients. Disclosures Balaian: Novartis: Honoraria. Törngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. Platzbecker: AbbVie: Honoraria; Takeda: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Geron: Honoraria. Röllig: Novartis: Honoraria, Research Funding; Jazz: Honoraria; Janssen: Honoraria; Bristol-Meyer-Squibb: Honoraria, Research Funding; Amgen: Honoraria; AbbVie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

Introduction: Flow cytometry (FCM) as recommended by European LeukemiaNet (ELN), is a co-criterion in MDS diagnostics. The aims of the present study were (1) to test whether the FCM diagnostic parameters have an impact on overall survival (OS); and (2) a computational algorithm could improve the identification of aberrant expression and/or cell population frequencies with prognostic importance. Methods: FCM was performed in bone marrow (BM) of 399 patients cytomorphologically classified as MDS. Cell populations were identified based on ELN recommended 55 parameters (Porwit et al., 2014), including: progenitor cells (PC), granulopoiesis (G), monopoiesis (M), nucleated red cells (NRC) and other cells (e.g. lymphocytes). Reference ranges were based on control BM (C-RR) and a quartile analysis (Q-A), to identify the parameters with an optimum separation of overall survival (OS). OS was assessed following univariate and multivariable Cox proportional hazards regression analysis using log-rank likelihood test: C-RR or Q-A (model A, C) and C-RR or Q-A with adjustment for patients developing acute myeloid leukemia during disease course (model B, D). Multivariate survival analysis, Kaplan Meier curves, and receiver operating characteristic curve (ROC) were used to develop prognostic FCM-scores (FCM-A to D) and tested for their independent prognostic value versus known prognostic parameters (age, sex, IPSS-R) as well as diagnostic FCM-scores (Ogata-, FCSS-, iFS-score). A summary of the analytical pipeline is shown in Figure 1A. Moreover, a sensitivity analysis for TP53 mutation distribution among the scores was performed (n=108) and a reliability analysis exploring the scores distribution among healthy donors (n=77) and MDS (n=399). In addition to expert gating ,T-REX pipeline (Barone, S. et al, 2020) was applied on a subset of patients (n=20) to check the feasibility of using unsupervised machine learning approach in identifying the FCM parameters incorporated in models A-D, and its ability to differentiate between the low (0,1) and high (≥2 ) scored patients. Results: The multivariable regression analysis identified 9 FCM parameters with independent prognostic impact on OS (detailed in Figure 1B). Prognostic FCM-scores based on these independent parameters were calculated for each model and dichotomized into high (≥2) and a low (0-1) scores. Thus, FCM-scores A and B, (reference ranges C-RR), outperformed FCM-scores C and D, (reference ranges Q-A): (FCM-A: HR (95 %CI) 3.20 (2.15 - 4.48); FCM-B: 4.08 (2.54 - 6.55); FCM-C: 2.03 (1.40 - 1.84); FCM-D: 2.30 (1.60 - 3.32)), as well as the diagnostic FCM-scores, as Ogata-score, FCSS and iFS (2.00 (1.29 - 3.11); 1.68 (0.99 - 2.86), 1.56 (0.92 - 2.65)). Interestingly, the new prognostic FCM-scores allowed a better prognostic grading than IPSS-R (HR (95 %CI): 2.37 (1.61-3.49)). Kaplan Meier survival curves stratified by FCM-score A and B showed a highly significant overall survival benefit for patients with a low score (p The TP53 mutation distribution did not differ among low and high score groups (wtTP53 lowScoreA (48.96%)/high ScoreA (51.04%) vs mTP53 lowScoreA (58.33%)/high ScoreA (41.67%) p>0.054). Healthy donors presented a high proportion of low score distribution (>87%) which differed from the MDS group (HD lowScoreA (90.91%)/high ScoreA (9.09) vs MDS lowScoreA (52.13%)/high ScoreA (47.87%) p Conclusion: We have generated two novel prognostic scores based on distinct FCM characteristics which could predict overall survival in MDS patients. FCM-scores A and B discriminate MDS patients with better overall survival outperforming IPSS-R as well as Ogata, FCSS and iFS scores. The T-REX machine learning has independently identified the same differences in models A and B, which highlights the potential of unsupervised approaches for future use in diagnostic FCM labs. Figure 1 Figure 1. Disclosures Farzaneh: Autolus: Consultancy, Current equity holder in publicly-traded company. Harrison: Incyte Corporation: Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Constellation Pharmaceuticals: Research Funding. Platzbecker: Celgene/BMS: Honoraria; Geron: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Takeda: Honoraria; AbbVie: Honoraria. Kordasti: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Alexion: Honoraria; Beckman Coulter: Honoraria.

Despite the rapid development of molecular markers during the last years, the morphology of blood and bone marrow cells still keeps the crucial role for diagnosis and subclassification of the different subtypes of myelodysplastic syndromes (MDS), as recently confirmed by the new 2016 WHO classification [1]. Nevertheless, morphologic diagnosis is not always self-evident. While increased blast cells are often in-line with advanced MDS subtypes, early forms with mild morphological changes can represent a diagnostic challenge. Especially in these cases, morphologic experience is crucial but should be embedded into further information on disease history, exclusion of other underlying diseases, as well as additional cytogenetic, molecular, and immunophenotypic data to confirm diagnosis of MDS.

Myelodysplastic syndromes (MDSs) represent a heterogeneous hematopoietic stem cell disorder.1 A precise estimation of the prognosis within the various MDS subgroups is essential for tailored therapeutic decisions. Especially, MDS patients with an isolated deletion of the long arm of chromosome 5 (del(5q)) represent a distinct subgroup regarding clinical outcome with a favorable prognosis in the majority of cases.2 Furthermore, they present with characteristic cytomorphological features, such as hypolobated megakaryocytes, macrocytic anemia and a normal or increased peripheral platelet count.3 Recently, flow cytometry (FCM) has been shown to serve as a valuable additional diagnostic and prognostic tool, especially to separate between unilineage and multilineage dysplasia.4, 5 Besides, it is known that abnormal antigen expression on myeloid progenitor cells (myPCs) is associated with a poor outcome.6, 7 In fact, aberrant CD7 expression on myPC of anemic lower-risk MDS patients predicts for a significantly lower response rate to erythropoiesis-stimulating agent (ESA) therapy irrespective of comparable other clinical predictive markers (erythropoietin level, transfusion burden).8 The pathophysiological background for this observation is still unknown. Notably, it has not been shown so far whether these distinct immunophenotypic characteristics correlate with presence and extent of clonal hematopoiesis, which in turn might not be responsive to growth factor stimulation. Therefore, in this study we separated different hematopoietic cell compartments of del(5q) MDS patients by fluorescence-activated cell sorting (FACS) and quantified the respective distribution of clonal burden with interphase fluorescence in situ hybridization (iFISH).

Patients with myelodysplastic syndromes (MDS) harboring a clonal deletion of the long arm of chromosome 5 [del(5q)] display characteristic cytomorphological features.[1][1],[2][2] Flow cytometry (FCM) is currently considered complementary to the microscopic evaluation of bone marrow (BM) smears and

Introduction: Flow cytometry (FCM) is considered as a co-criterion in MDS diagnostics if the main diagnostic methods are not sufficient to clearly diagnose or rule out MDS. The iMDSFlow working group of the European LeukemiaNet worked on recommendations regarding the harmonization of preanalytics, data analysis, as well as of appropriate diagnostic scores. The aims of the present study were (1) to test whether the parameters and reference ranges applied for FCM diagnostics in MDS do have an impact on prognosis; and (2) whether the incorporation of computational algorithms in data mining could further improve the prognostic information of FCM. Methods: FCM diagnostics was performed in bone marrow (BM) of 303 patients cytomorphologically classified as MDS using a Lyse-wash-method and measuring on a FACSCantoII cytometer. For FCM data evaluation, commonly used diagnostic flow-scores - FCSS (Wells et al. 2003), Ogata-score (Ogata et al. 2009), new iFS-score (Cremers et al. 2017), ELN-Red-score (Westers et al. 2017), Red-score (Mathis et al. 2013) - were applied, including the analysis of progenitor cells, granulopoiesis, monopoiesis, and nucleated erythropoiesis. Thus, 55 FCM parameters necessary to assess the mentioned flow-scores and 33 additional FCM parameters have been evaluated. Clinical variables (n=11) including IPSS-R (vLR+LR: 163 pts., Int: 81 pts., HR+vHR: 59 pts.) have also been recorded. Median follow-up time was 28 months (1.5 - 84 mo). Overall survival (OS) was assessed in uni- and multivariate Cox proportional hazards regression analysis using log-rank likelihood test. Reference ranges for FCM parameters have been assessed before using BM of 49 healthy donors. In addition, in order to improve prognostic output, a computational approach was devised that tests every possible combination of binary, ternary, and quaternary marker strata segregation to identify the thresholds that bring about optimum separation of hazard strata in each model based on log-likelihood p-value. Finally, it was assessed whether the FCM variables correlate with IPSS-R and also have independent predictive value in MDS. Results: First, MDS flow-scores and IPSS-R have been tested for OS resulting in a significant association of higher flow-scores with shorter survival, Ogata: median OS: 51 mo vs. not reached (NR), hazard ratio (HR)=2.1, p=0.00084; FCSS: 70 mo vs. NR, HR=2.1, p=0.013; new iFS: 70 mo vs. NR, HR=1.9, p=0.02; IPSS-R: 37 vs. 55 vs. 74 mo; HR=2.4/1.4, p=0.0045. Second, we wanted to explore whether single FCM parameters inherit prognostic information using the diagnostic reference ranges. Of note, 19 of all FCM parameters were significantly associated with OS, e.g. decreased side scatter (granulopoiesis; 37 mo vs. NR, HR=2.4, p=0.000047), decreased % of B-lymphatic progenitors (lyPC; 52 mo vs. NR, HR=2.3, p=0.0013). As a third step, a computational learning algorithm was applied. In 7/19 parameters already showing significant differences in OS while using the diagnostic reference ranges, the algorithm could add information. Moreover, the algorithm optimized thresholding for 17 additional FCM parameters, resulting in clearer survival differences, e.g. decreased side scatter (myPC, 33 vs. 74 mo, HR=2.6 p=0.00002), aberrant CD5 expression (granulopoiesis; 32 vs. 74 mo, HR=2.5 p=0.000045). Next, we performed an independent multivariate Cox model with lasso penalty, considering 14 FCM parameters which in univariate analysis showed a clearer survival difference compared to IPSS-R. Of note, this identified the following as best predictors of OS: IPSS-R, decreased side scatter (granulopoiesis), CD45 MFI (ratio of CD45 MFI lymphocytes : myPC), and decreased % B-lymphatic progenitors. Conclusions: In addition to the known importance of FCM in MDS diagnostics, we provided evidence that FCM has also an important prognostic impact in relation to IPSS-R even in this heterogeneous group of MDS patients. Thus, in univariate analysis a computational learning algorithm was able to refine thresholding resulting in an improved separation of OS curves. Importantly, even in multivariate analysis FCM proved its significance for patient outcome. Further studies should evaluate, whether a flow-score including the most influential variables could add prognostic information to IPSS-R. Disclosures Kordasti: Celgene: Research Funding; Novartis: Research Funding; Boston Biomed: Consultancy; API: Consultancy. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Lenalidomide (LEN) leads to erythroid improvement in the majority of patients with myelodysplastic syndrome and isolated deletion of the long arm of chromosome 5 (MDS-del(5q)). This effect is believed to be exerted via its immunomodulatory properties, although the precise nature is still incompletely understood. We prospectively performed immune profiling in the bone marrow and blood of MDS-del(5q) patients undergoing LEN therapy for a median of 6 cycles. Therapy with LEN led to a significant increase in the median absolute lymphocyte count (1.3-fold, p = 0.013) without changes in the distribution of the T helper cells within the entire compartment. In parallel, the frequency of Treg increased significantly during treatment both in the peripheral blood (5.0 vs. 9.6 %, p = 0.001) and bone marrow (3.4 vs. 8.1 %, p = 0.001). Surprisingly, LEN treatment led to a decrease in TGFbeta levels, both in the peripheral blood (4.9 vs. 2.3 ng/ml, p = 0.039) and bone marrow (4.5 vs. 0.8 ng/ml, p = 0.023). These changes were not associated with an increase in pro-inflammatory Th17 cells. Taken together, our results demonstrate that LEN induces a shift in lymphocytic populations towards immunosuppression in MDS-del(5q) patients.

Purpose To assess the optimal cumulative dose of cytarabine for treatment of young adults with acute myeloid leukemia (AML) within a prospective multicenter treatment trial. Patients and Methods Between 1996 and 2003, 933 patients (median age, 47 years; range 15 to 60 years) with untreated AML were randomly assigned at diagnosis to receive cytarabine within the first consolidation therapy at either a intermediate-dose of 12 g/m2 (I-MAC) or a high-dose of 36 g/m2 (H-MAC) combined with mitoxantrone. Autologous hematopoietic stem-cell transplantation or intermediate-dose cytarabine (10 g/m2) were offered as second consolidation. Patients with a matched donor could receive an allogeneic transplantation in a risk-adapted manner. Results After double induction therapy including intermediate-dose cytarabine (10 g/m2), mitoxantrone, etoposide, and amsacrine, complete remission was achieved in 66% of patients. In the primary efficacy analysis population, a consolidation with either I-MAC or H-MAC did not result in significant differences in the 5-year overall (30% v 33%; P = .77) or disease-free survival (37% v 38%; P = .86) according to the intention-to-treat analysis. Besides a prolongation of neutropenia and higher transfusion demands in the H-MAC arm, rates of serious adverse events were comparable in the two groups. Conclusion In young adults with AML receiving intermediate-dose cytarabine induction, intensification of the cytarabine dose beyond 12 g/m2 within first consolidation did not improve treatment outcome.

Introduction Recently, progress has been made in the treatment of patients with higher risk myelodysplastic syndromes (HR MDS) and acute myeloid leukemia (AML). Nevertheless, patients failing hypomethylating agents (HMA) have a dismal prognosis and very limited treatment options. Targeting CD123 on leukemic stem cells (LSC) is one promising approach in MDS and AML. Talacotuzumab (TAL, JNJ-56022473) is an IgG1 monoclonal antibody targeting CD123 preferentially via antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer cells (NKs). Aim The SAMBA trial, a phase II study of the German and French MDS study groups within the EMSCO network assessed the overall hematological response rate after 3 months of single agent TAL treatment in AML or HR MDS patients failing hypomethylating agents (HMAs). Methods TAL was given IV at a dose of 9 mg/kg once every two weeks for a total of 6 infusions, responders received up to 20 additional infusions. After the first 3 months, overall hematological response rate (either CR, PR, marrow-CR, HI, SD) was evaluated by bone marrow biopsy. The study was accompanied by an immune monitoring via flow cytometric analysis to investigate the distribution of T- and NK cells in peripheral blood (PB) and bone marrow (BM) at the time of screening and during therapy in comparison with healthy, age-matched controls. Results 24 patients (19 AML and 5 HR MDS) with a median age of 77 (range 71-90) years, who either failed to achieve complete- (CR), partial response (PR), hematological improvement (HI) or relapsed after HMA therapy were included in the study. After TAL administration, 14 patients could be assessed for response after 4 infusions and 10 patients after 6 infusions. The overall response rate (ORR) was 20.8% including 1 complete remission (CRi), 1 patient with hematologic improvement (HI-E) and additionally 3 patients with disease stabilization. The median duration of response in these patients was 3 months (range 3-14 months). Two patients are still on treatment, one patient despite losing objective response (14 months) and one patient with disease stabilization (13 months). The median overall survival for the entire cohort of patients was 3.2 months (range 0.4-11.2 months). In 10 patients (41.6%), therapy with TAL resulted in grade 3/4 infusion related side effects (pneumonia, n=1; infusion-related reaction, n=8; septic shock, n=1). Before treatment initiation, patients had lower levels of CD56dim NK-cells in PB (82% vs. 89% of NK-cells; p=0.069) expressing significantly more inhibiting NK-cell receptors like KIR2DL2 (8.8% vs. 3.2% of NK-cells; p We could not detect any difference in NK-cell levels in responding patients compared to non-responders. Interestingly, pre-treatment expression (MFI and percentage) of CD123 on immature myeloid derived suppressor cells (iMDSC) was higher in responders than in non-responders (p Conclusion Single agent TAL has limited efficacy in patients with advanced myeloid malignancies failing HMA. Expression of CD123 on immature MDSCs might serve as a biomarker of response for future anti-CD123 targeted approaches. Disclosures Götze: Celgene: Honoraria, Research Funding; JAZZ Pharmaceuticals: Honoraria; Novartis: Honoraria; Takeda: Honoraria, Other: Travel aid ASH 2017. Krönke:Celgene: Honoraria. Middeke:Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Fenaux:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding. Schlenk:Pfizer: Research Funding, Speakers Bureau. Ades:JAZZ: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; silent pharma: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Platzbecker:Celgene: Research Funding.

Introduction: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis due to genetic and functional abnormalities of hematopoietic stem and progenitor cells (HSPCs). Accumulating evidence now also points to the bone marrow microenvironment (BMME) as a key mediator of MDS pathophysiology. Increased levels of transforming growth factor beta (TGF-β) superfamily ligands, including growth differentiation factor 11 (GDF-11), in the bone marrow have been linked to ineffective erythropoiesis and activation of SMAD2/3 signalling in MDS. Luspatercept (ACE-536) is a novel recombinant fusion protein containing modified activin receptor type IIB linked to the fragment crystallisable (Fc) domain of human immunoglobulin G1 and is a first-in-class erythroid maturation agent. Luspatercept binds to selected TGF-β superfamily ligands, including GDF-11 and activin B, restoring late-stage erythropoiesis in MDS mice and patients. Whether luspatercept modulates the BMME is unknown. Aim: We investigated the potential impact of luspatercept on the biology of mesenchymal stromal cells (MSCs). Methods: MSCs from patients with either high-or low-risk MDS and age-matched healthy donors (HD) were treated with GDF-11 in the presence or absence of RAP-536, a homologue of luspatercept harboring the same activin receptor IIB domain. Subsequently, Smad signaling pathway activation was analyzed by Western blot. Moreover, MSC phenotype, stromal derived factor-1 (SDF-1/CXCL12) expression and secretion as well as the osteogenic differentiation potential were recorded. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with GDF-11 ± RAP-536 before freshly isolated CD34+ HSPCs were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly. After four weeks, the HSPCs were seeded in methylcellulose medium to perform a CFU-GEMM assay. Further, engraftment and migration potential of co-cultured HSPCs was analyzed in zebrafish embryos in vivo. Results: GDF-11/RAP-536 treatment did not impact viability, proliferation, and growth pattern of MSCs. The osteogenic differentiation was significantly improved by RAP-536 treatment which was shown by a 2.3-fold increase of ALP activity. GDF-11 induced Smad2/3 phosphorylation in MSCs which was inhibited by RAP-536 to about 50% of activity. The MSC phenotype characterized by the expression of CD73, CD90, CD105, CD146, CD29 and CD44 was not significantly influenced by RAP-536 treatment. Interestingly, the chemokine SDF-1 which plays an important role for the interaction and support of HSPCs was significantly up-regulated in MDS and HD MSCs by RAP-536 at both the mRNA (1.9-fold) and the protein level (2.1-fold; 313.0±35.0 vs. 649.7±76.8 pg/ml, *p< 0.05, n=3). This restored SDF-1 secretion of MSCs resulted in functional effects of HSPCs when co-cultured with MSCs. The number of CAF-C was significantly higher after two, three, and four weeks of HSPCs cultured on pre-treated MSC layers (12.0-fold / 3.4-fold / 1.75-fold). The phenotypical analysis of HSPCs demonstrated increased expression levels of CXCR4 in the adherent fraction. Addition of the CXCR4 antagonist AMD3100 blocked enhanced colony formation by RAP-536 confirming the previous observation on SDF-1 modulation. Clonal growth of CAF-C-derived HSPCs after four weeks of co-culture on un- or pre-treated MSC monolayers was analyzed in a secondary colony-forming cell assay (CFU) for an additional 14 days. The frequency of CFUs was increased in all lineages with significant differences in total colony numbers (15±5.8 vs. 35.8±14.7, *p< 0.05, n=6). These results were validated in vivo in a zebrafish model. HSPCs were co-cultured with GDF-11 and RAP-536 pre-treated MDS-MSCs for one week before injection into the zebrafish circulation. One and two days post-injection, a significantly higher cell proportion could be detected in the caudal hematopoietic (CHT) region as a result of RAP-536 pre-treatment (Fig.1). Conclusion: These data provide first evidence that RAP-536 has also the capacity to modulate MSCs which might contribute to the restoration of hematopoiesis in MDS. Disclosures Platzbecker: Celgene: Research Funding.

Rapid flow cytometric detection of aberrant cytoplasmic localization of nucleophosmin (NPMc) indicating mutant NPM1 gene in acute myeloid leukemia

Purpose: Gemtuzumab ozogamicin (GO) has been associated with an increased risk of liver sinusoidal obstruction syndrome (SOS) when applied within 3 months of allogeneic hematopoietic cell transplantation (HCT). We hypothesized that GO might be safe and effective as part of a reduced-intensity conditioning regimen as salvage therapy of CD33+ acute myeloid leukemia. Experimental Design: Thirty-one patients with acute myeloid leukemia which relapsed following conventional therapy (n = 15), autologous (n = 3), or allogeneic (n = 13) HCT were included in a prospective phase I/II trial. The preparative regimen contained 6 and 3 mg/m2 of GO on days −21 and −14 before transplantation, leading to a reduction of marrow blasts in 18 patients (58%). Eight patients received further cytoreductive chemotherapy before conditioning therapy was initiated. Fludarabine-based reduced-intensity (n = 11) or nonmyelablative (n = 16) conditioning and peripheral blood stem cell infusion from related (n = 6) or unrelated (n = 21) donors could be done in 27 patients during cytopenia. Results: Primary engraftment occurred in all evaluable patients. Only one case of reversible hepatic sinusoidal obstruction syndrome was documented. Non–relapse mortality until day 100 was 22% (n = 6). The probabilities of overall and disease-free survival at 24 months were 39% and 35%, respectively. Relapse of leukemia occurring between 2 and 24 months after transplantation (median, 8 months) was the major reason for treatment failure and death. Conclusion: These data suggest that GO can be combined with reduced-intensity conditioning even after previous autologous or allogeneic HCT.

Endothelial progenitor cells (EPC) are involved in endothelial repair and maintenance. Dysfunction of EPC may contribute to accelerated arteriosclerosis in chronic kidney disease. Kidney transplantation (KTx) improves both survival and endothelial function of dialysis patients. In a prospective study, we tested to which extent KTx changes EPC biology. We studied number and function (migratory activity, adhesion to extracellular matrix proteins and to mature endothelial cells [EC]) of EPC in 20 patients during dialysis and 3, 6, 9 and 12 months after KTx. Twenty-two healthy volunteers served as matched controls. Circulating precursor populations were measured by flow cytometric analysis. Cytokines relevant for EPC mobilization were monitored. Compared to the dialysis state, KTx increased the migration of EPC to approximately 2-fold. Adhesion to fibronectin and to collagen type IV was significantly increased after KTx. An improved adhesion rate of EPC to mature EC was observed. The number of EPC decreased. The amount of precursor populations showed no difference compared to the pretransplant state. Our study shows an improved function of EPC after KTx. This finding indicates an improved potential for endothelial repair which in turn may contribute to enhanced endothelial function and reduced cardiovascular morbidity after KTx.

BACKGROUND A single injection of pegfilgrastim has been shown to be equivalent to daily filgrastim in enhancing neutrophil recovery after chemotherapy, whereas the experiences with pegfilgrastim in mobilization of peripheral blood progenitor cells (PBPCs) are limited. STUDY DESIGN AND METHODS Forty unselected patients with lymphoma or multiple myeloma were treated with different chemotherapy regimens followed by 6 mg of pegfilgrastim for mobilization of autologous PBPCs. Patients with an inadequate mobilization (blood CD34+ cells

Background: Rheumatoid arthritis (RA) is associated with increased morbidity and mortality attributable to accelerated atherosclerosis and cardiovascular events. Objective: To determine the role played by endothelial progenitor cells (EPC) in the defence system against arteriosclerosis. Methods: The number and function of EPC in 13 young patients with RA with low disease activity (DAS28 3.5 (0.3)) and 13 healthy control subjects was studied. Endothelial function was investigated by agonist-induced, endothelium dependent vasodilatation measured by the forearm blood flow technique. Migratory activity and adhesion of EPC to tumour necrosis factor α (TNFα) activated mature endothelial cells and components of the extracellular matrix were tested in vitro. Putative precursor populations (CD34 + , CD34 + /CD133 + , and CD34 + /KDR + haematopoietic stem cells) were measured by flow cytometric analysis. Results: Acetylcholine-induced, endothelium dependent vasodilatation was reduced by about 50% in patients with RA, indicating endothelial dysfunction, whereas endothelium-independent vasodilatation in response to glyceryl trinitrate was at control level. Significantly reduced numbers of EPC were found in the patients compared with controls. Migratory activity of EPC was decreased in patients with RA. Adhesion to mature endothelial cells after activation with TNFα was enhanced only in controls. The adhesion to matrix proteins and the number of putative precursor cell lineages was comparable in both groups. Conclusion: Endothelial dysfunction in patients with RA with low grade inflammation is associated with a reduced number and partial dysfunction of EPC. Further studies are needed to explore whether interventions that potentially ameliorate the number and function of EPC also improve endothelial function in these patients.

Chronic myelomonocytic leukemia (CMML) is classified by the WHO as an overlap syndrome of myelodysplastic syndrome and myeloproliferative neoplasia (MDS/MPN) because it shares features of both enti...

Reply to: ‘Flow cytometric identification of acute myeloid leukemia with limited differentiation and NPM1 type A mutation: a new biologically defined entity’ by Kern et al.

Loss of Y chromosome (-Y) is described as an age related cytogenetic polymorphism as well as an abnormality associated with clonal diseases mainly Myelodysplastic Syndromes (MDS) reported in up to 15% of these patients. However, it often remains an uncertainty dissecting these two conditions especially in patients with mild dysplasia and a low percentage of abnormal metaphases. The aim of our study was to investigate whether flow cytometry (FCM) allows for a valid discrimination between MDS and age related presence of a loss of Y chromosome. Bone marrow aspirates of 46 patients, suspicious for MDS, with a median age of 76 years and a cytogenetically detected -Y as single abnormality (>75% threshold for assignment to MDS; Wiktor et al. 2011) were investigated. According to cytomorphology MDS could be diagnosed in 26 patients (IPSS-R verylow/low=22, intermediate=3, high/very high=1) and CMML in 6 patients. MDS was excluded in 14 patients (11 IDUS, 1 aplastic anemia, and 2 pts. with reactive changes only). Further routine diagnostic procedures included FCM according to WHO and ELNet iMDS-Flow criteria. Thus, FCM was performed using an 8-color antigen panel measured on a FACS-Canto II cytometer. MDS were defined according to the current FCM-scores developed by Wells et al. (normal/mild: 0-1, moderate: 2-3, or severe: > 4; Blood 2003) and Ogata et al. (low: 0-1, high: >1; Haematologica 2009). In case of an intermediate Wells-score only, we demanded cross lineage antigen expression in myeloid progenitors as criteria for MDS/CMML. The median percentage of abnormal metaphases presenting with -Y was 52% (range: 12%-100%). Applying the above mentioned threshold of 75% abnormal metaphases as criteria for the presence of MDS, only 14 patients (30%) could clearly be allocated to MDS. Because of the present diagnostic incertainty we performed a comprehensive FCM investigation, applying Wells- and Ogata-Score. Thus, in 26 patients (63%) MDS was likely applying FCM. Furthermore, 4 patients with a normal/mild Wells-score plus a low Ogata-score were considered as "non-MDS". Applying cytomorphology 70% (32/46) of all -Y patients have been diagnosed as MDS. Of note, MDS diagnosis could be made by FCM or cytomorphology in an equal extent in the groups of more vs. less of 75% cytogenetically abnormal metaphases: 64% vs. 63% and 86% vs. 63%, respectively. Finally, we searched for patients meeting FCM plus cytomorphologic MDS criteria. Of importance, even in the group with less abnormal metaphases 50% of the patients showed a concordant FCM and cytomorphologic assignment to MDS or non-MDS vs. 64% concordance in the high cytogenetically aberrant group. Patients with a loss of Y chromosome as single cytogenetic abnormality often meet FCM and cytomorphologic criteria for MDS diagnosis independent from the load of abnormal metaphases. This study demonstrates that FCM can significantly contribute to MDS diagnosis in this specific subgroup of patients with -Y and often only mild dysplasia. Disclosures No relevant conflicts of interest to declare.

Recently, we have established a 5-parameter flow cytometry (FCM)-based score allowing for a precise prediction a deletion (5q) in therapy naïve MDS patients. The aim of this prospective study was to test, whether this FCM-based profiling is at least equal to the cytogenetics/FISH-based del(5q) detection for monitoring response to treatment. Overall, 229 FCM investigations were performed in 57 patients with MDS and del(5q) (IPSS-R very low/low: n=24, int: n119, high/very high n=22) including 39 patients with isolated del(5q) or one additional cytogenetic abnormality. The majority of analyses were performed in patients receiving lenalidomide or azacitidine (n=28 and n=21 patients), or in patients receiving chemotherapy and/or allogeneic transplantation (n=3), or growth factors (n=5). The del(5q)-FCM-score includes the following 5 parameters: myeloid progenitors (myPC > 2%) = 3 points, CD45 MFI ratio (lymphocytes: myPC ≤ 7.0) = 10, SSC ratio (granulocytes : lymphocytes < 6.0) = 2, CD71 (≤ 20%) on granulocytes = 1.5, and female gender = 1.5; a score ≥ 15.0 indicates the presence of del(5q). A standardized FCM (lyse-stain-wash) and cytogenetics/FISH were performed according to ELN guidelines at the TU of Dresden, VUMC of Amsterdam, UH of Bristol, and UH of Guadalajara. Before therapy, in 61 cytogenetic/FISH analyses in 40 MDS patients a del(5q) was detectable. In 53/561 (87%) FCM measurements, performed in parallel, a del(5q) could be predicted using the del(5q)-FCM-score. Monitoring response to treatment, a cytogenetic complete response (CCR) with the disappearance of del(5q) was accompanied by the disappearance of the typical del(5q)-FCM-profile in all of the 50 (19 patients) FCM measurements (sensitivity=100%). Otherwise, in patients without CCR the presence of del(5q) could be predicted by FCM in 96/118 measurements (specificity=81%). Aiming at a still higher specificity, in the following analyses we included only patients who presented with a typical del(5q)-FCM-score before therapy (33 patients; 54 measurements; median score=16.5) resulting in the above described sensitivity of 100% (32 measurements; median score=13.0) as well as a remarkably high specificity of 97% (59/61 measurements; median score=16.5) for response prediction. Next, we compared cytogenetics/FISH and del(5q)-FCM-score with further methods for response monitoring as cytomorphology, two well established diagnostic FCM scores: FCSS (flow cytometry scoring system; Wells et al. 2003) and the diagnostic score (Ogata et al. 2009), as well as the assessment of hematological improvement (HI). Thus, cytomorphology and FCSS, analyzing dyspoiesis of multiple cell lineages, showed a CR or disappearance of a MDS typical FCSS in less than the half of all investigations being in cytogenetic CR (sensitivity: 41% and 38%), but those two methods revealed a high specificity (98% and 97%). On the other side, the analysis of HI was high sensitive (91%) but not specific (49%). The use of the diagnostic Ogata score, considering almost only abnormalities of the myeloid progenitors, ended up with a slightly lower sensitivity (91%) and specificity (89%) as cytogenetics/FISH and the del(5q)-FCM-score. Finally, response monitoring using the del(5q) FCM-score allowed for a better separation of overall survival compared to cytogenetics/FISH (p=0.039 vs. p=0.1725). Flow cytometry-based detection of a del(5q)-specific immunophenotype is feasible and can serve as a rapid tool for response monitoring during treatment with disease-modifying drugs. At the moment, we test whether the better separation of the patients' survival curves applying the FCM-score holds true in a larger patient cohort to be analyzed within the ELNet iMDS-Flow working group. Disclosures Platzbecker: TEVA: Honoraria, Research Funding.

Concurrent JAK2(V617F) mutation and BCR-ABL translocation within committed myeloid progenitors in myelofibrosis

Item does not contain fulltext An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

This study evaluated azacitidine as treatment of minimal residual disease (MRD) determined by a sensitive donor chimerism analysis of CD34(+) blood cells to pre-empt relapse in patients with CD34(+) myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT). At a median of 169 days after HSCT, 20/59 prospectively screened patients experienced a decrease of CD34(+) donor chimerism to80% and received four azacitidine cycles (75 mg/m(2)/day for 7 days) while in complete hematologic remission. A total of 16 patients (80%) responded with either increasing CD34(+) donor chimerism to ≥80% (n=10; 50%) or stabilization (n=6; 30%) in the absence of relapse. Stabilized patients and those with a later drop of CD34(+) donor chimerism to80% after initial response were eligible for subsequent azacitidine cycles. A total of 11 patients (55%) received a median of 4 (range, 1-11) additional cycles. Eventually, hematologic relapse occurred in 13 patients (65%), but was delayed until a median of 231 days (range, 56-558) after initial decrease of CD34(+) donor chimerism to80%. In conclusion, pre-emptive azacitidine treatment has an acceptable safety profile and can substantially prevent or delay hematologic relapse in patients with MDS or AML and MRD after allogeneic HSCT.

To assess the optimal cumulative dose of cytarabine for treatment of young adults with acute myeloid leukemia (AML) within a prospective multicenter treatment trial.Between 1996 and 2003, 933 patients (median age, 47 years; range 15 to 60 years) with untreated AML were randomly assigned at diagnosis to receive cytarabine within the first consolidation therapy at either a intermediate-dose of 12 g/m² (I-MAC) or a high-dose of 36 g/m² (H-MAC) combined with mitoxantrone. Autologous hematopoietic stem-cell transplantation or intermediate-dose cytarabine (10 g/m²) were offered as second consolidation. Patients with a matched donor could receive an allogeneic transplantation in a risk-adapted manner.After double induction therapy including intermediate-dose cytarabine (10 g/m²), mitoxantrone, etoposide, and amsacrine, complete remission was achieved in 66% of patients. In the primary efficacy analysis population, a consolidation with either I-MAC or H-MAC did not result in significant differences in the 5-year overall (30% v 33%; P = .77) or disease-free survival (37% v 38%; P = .86) according to the intention-to-treat analysis. Besides a prolongation of neutropenia and higher transfusion demands in the H-MAC arm, rates of serious adverse events were comparable in the two groups.In young adults with AML receiving intermediate-dose cytarabine induction, intensification of the cytarabine dose beyond 12 g/m² within first consolidation did not improve treatment outcome.

Donor lymphocyte infusions have been effective in patients with chronic myeloid leukemia (CML) relapsing after allogeneic stem cell transplantation, but their use is associated with the risk of graft-versus-host disease. We investigated the effects of prophylactic infusion of in vitro-generated donor T cells reactive against peptides derived from CML-associated antigens. Fourteen CML patients received conditioning therapy followed by CD34+-selected peripheral blood stem cells from matched siblings (n = 7) or unrelated (n = 7) donors. Donor-derived mature dendritic cells generated in vitro from CD14+ monocytes were loaded with human leukocyte Ag-restricted peptides derived from PR1, WT1, and/or B-cell receptor–ABL and used to repetitively stimulate donor CD8+ T cells in the presence of IL-2 and IL-7. Stimulated T cells were infused 28, 56, and 112 days after transplantation. Thirteen patients are alive and 7 remain in molecular remission (median follow-up, 45 months). Interestingly, all 4 patients receiving CD8+ T cells displaying marked cytotoxic activity in vitro and detectable peptide-reactive CD8+ T cells during follow-up have not experienced graft-versus-host disease or relapse. Our study reveals that prophylactic infusion of allogeneic CD8+ T cells reactive against peptides derived from CML-associated antigens is a safe and promising therapeutic strategy. This trial was registered at www.clinicaltrials.gov as #NCT00460629.

Therapeutic application of natalizumab, an anti-CD49d Ab, in patients with multiple sclerosis (MS) has been associated with increased levels of circulating CD34+ progenitors. We analyzed the frequency, phenotype and functional activity of CD34+ HSC in blood and BM of patients with MS who were treated with natalizumab. Compared with healthy controls and untreated MS patients, natalizumab treatment increased CD34+ cells in the peripheral blood 7-fold and in BM 10-fold. CD34+ cells derived from blood and marrow of natalizumab-treated patients expressed less of the stem cell marker CD133, were enriched for erythroid progenitors (CFU-E) and expressed lower levels of adhesion molecules than G-CSF-mobilized CD34+ cells. The level of surface CXCR-4 expression on CD34+ cells from patients treated with natalizumab was higher compared with that of CD34+ cells mobilized by G-CSF (median 43.9 vs 15.1%). This was associated with a more than doubled migration capacity toward a chemokine stimulus. Furthermore, CD34+ cells mobilized by natalizumab contained more mRNA for p21 and less for matrix metallopeptidase 9 compared with G-CSF-mobilized hematopoietic stem cell (HSC). Our data indicate that G-CSF and CD49d blockade mobilize different HSC subsets and suggest that both strategies may be differentially applied in specific cell therapy approaches.

Analysis of donor chimerism is an important diagnostic tool to assess the risk of relapse after allogeneic stem cell transplantation, especially in patients lacking a specific marker suitable for monitoring of minimal residual disease. We prospectively investigated the predictive value of donor chimerism analyses in sorted CD34(+) peripheral blood cells in 90 patients with acute leukemia and myelodysplastic syndrome. The cumulative incidence of relapse after four years was significantly increased in cases with decreasing or incomplete CD34(+) donor chimerism (57% vs. 18%, p=0.0001). Multivariate analysis confirmed decreasing CD34(+) donor chimerism as an independent predictor of relapse and inferior survival. The interval between a decrease of CD34(+) chimerism of less than 80% and hematologic relapse was 61 days (range 0-567). Monitoring of CD34(+) donor chimerism in the peripheral blood allows prediction of imminent relapse after allogeneic stem cell transplantation even when a disease-specific marker is lacking.

Background: Loss of HLA-DR and CD34 is a well-known characteristic of malignant promyelocytes in acute promyelocytic leukemia (APL). However, this immunophenotype is not specific for APL. The purpose of this study was to investigate whether further biological characterization of the HLA-DRneg acute myeloid leukemia patients would allow more clearly define criteria to separate APL from non-APL patients. Methods: Immunophenotyping, cytogenetics, molecular analyses, and cytomorphology were prospectively performed within routine leukemia diagnostics of 800 patients included in different prospective acute myeloid leukemia multicenter trials. Results: Beside 60 APL, an additional 62 HLA-DRneg non-APL patients were identified. The main differential characteristics of HLA-DRneg non-APL included high CXCR-4 expression in most patients and almost all leukemia cells, a significantly higher proportion of patients presenting with NPM1 mutations as well as the significant association with cup-like nuclear morphology. The biological distinctness of both leukemia subtypes was further emphasized by the complete absence of aberrant CD2 expression and increased leukocyte and platelet counts in HLA-DRneg non-APL patients. Even in the CD34pos subgroup of HLA-DRneg non-APL all those features contributed in at least the same way to the separation from APL. Conclusions: The results of the present study show that an immunophenotypic, molecular, and cytomorphologic separation of both HLA-DRneg leukemia subgroups is possible indicating that both groups are biologically distinct. © 2009 Clinical Cytometry Society

We investigated cup-like nuclear morphology of acute myeloid leukemia blasts in 266 randomly selected patients and its association with hematologic findings, disease markers and outcome data. Cup-like acute myeloid leukemia was diagnosed in 55 patients (21%). It was associated with female sex, high white blood cell and blast cell counts, normal karyotype, and low CD34 and HLA-DR expression. Mutations of FLT3, NPM1 or both were detected in 84.9% compared with 58.1% in cases without this morphology (p=0.001). There was no influence on response to treatment or survival. Therefore, cup-like nuclear morphology is an indicator of normal karyotype and should guide more specific molecular analyses.