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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.

Introduction Increased susceptibility to infections in MDS is due to neutropenia as well as functional impairment of neutrophils. We investigated whether neutrophil dysfunction includes diminished formation of neutrophil extracellular traps (NETs). The latter are networks of extracellular fibers, primarily composed of DNA, which bind pathogens. This component of the immune system's first line of defense was first described in 2004 by Zychlinsky and coworkers. Formation of NETS is triggered by reactive oxygen species (ROS), which in neutrophils are mainly produced by NADPH-oxidase and myeloperoxidase (MPO). Methods Fluorescence microscopy was used to show NET formation in patients with MDS (n=12) (1 MDS del(5q), 1 MDS-RS-SLD, 1 MDS-RS-MLD, 5 MDS-MLD, 2 MDS-EB-1, 2 MDS-EB-2) in comparison to an age-adjusted normal control group (n=15). Neutrophils were isolated from peripheral blood by density gradient centifugation, sedimented on cover slips in 24-well plates, and incubated with phorbol-12-myristate-13-acetate (PMA) for 30 and 180 minutes to stimulate NET formation. Neutrophil elastase was detected by primary anti-NE (Abcam) plus secondary goat anti-rabbit Alexa 488 (FITC), histones by primary anti-histone (Millipore) plus secondary goat anti-mouse Alexa 568 (PE), and cell nuclei by DNA staining with DAPI. Fluorescent images were loaded into Image J/FIJI Software (https://imagej.nih.gov/ij/) for automatic cell detection and assessment of the proportion of cells undergoing NET formation, detectable by positive histone staining. Flow cytometry was used to further characterize NET formation and demonstrate the production of ROS by NADPH-oxidase and MPO. Intracellular MPO staining was positive for 96.7% and 99.1% of neutrophils in controls and MDS patients, respectively. Reactive oxygen species (ROS) were detected using dihydro-rhodamin 123 (DHR) for hydrogen peroxide, hydroethidine (HE) for superoxide anion, and 3`-(p-aminophenyl) fluorescein (APF) and 3`-(hydroxyphenyl) fluorescein (HPF) for hypochloride (HOCl). In order to block certain components of cellular ROS production, the following inhibitors were used: 4-aminobenzoic hydrazide (ABAH) to block MPO; 4-dimethylamino-antipyrine (AP) to block MPO and quench ROS; and diphenylen-iodonium chloride (DPI) to inhibit NADPH oxidase and mitochondrial respiratory complex I. Flow cytometry data were analysed using CellQuest Software (Becton Dickinson) and FCS Express Reader (De Novo Software). Results By fluorescence microscopy, marked morphological abnormalities of NET formation were observed. PMA-stimulated neutrophils of patients with MDS generated significantly fewer NETs than neutrophils from controls (increase from 17% to 67% vs. increase from 17% to 85% of cells, respectively) (p=0.02), see Fig. 1. MDS neutrophils showed significantly less cellular swelling after stimulation with PMA (p=0.04). Histone staining showed a more condensed chromatin in MDS neutrophils prior to stimulation (smaller mean fluorescence intensity (MFI) for PE; p=0.05). Upon stimulation with PMA, the decrease in MFI of DAPI, indicating chromatin decondensation, was significantly less in MDS neutrophils than controls (delta MFI 3467 vs. 4687, p=0.03). In addition, assessment of neutrophil elastase showed that its release from cytoplasmic granules (decreasing MFI for FITC) was diminished in patients with MDS (p=0.00002). On flow cytometry, forward scatter confirmed a significant difference in cell swelling after PMA stimulation (p=0.02). Sideward scatter showed a smaller decrease in granularity after stimulation with H2O2 (p=0.002). The morphological abnormalities did not seem to be attributable to diminished ROS production since measurement of PMA-stimulated ROS production using HE, and assessing oxidative burst activity using DHR did not reveal significant differences. However, H202-stimulated production of HOCl by MPO was lower in MDS patients, albeit only when MPO activity was simultaneously impeded by inhibitors (ABAH and AP). Inhibition of MPO activity was more easily achieved in patients with MDS (p=0.01), corroborating the notion of a partial MPO defect in MDS. Conclusions Our results indicate that NET formation is significantly impaired in MDS neutrophils. Although we found abnormalities of MPO-dependent generation of HOCl, impaired ROS production may not be the only cause of deficient NETosis in MDS. Figure Disclosures Gattermann: Novartis: Honoraria, Research Funding.

Dear Editor, Chronic myelomonocytic leukemia (CMML) has been classified into a new category of myelodysplastic/myeloproliferative diseases (MDS/MPN) according to the last WHO classification of myeloid malignancies [1]. However, some patients with CMML show proliferative features (MP-CMML) such as leukocytosis and organomegaly, making them clinically and biologically similar to myeloproliferative neoplasms (MPN). At biological level, JAK2V617F mutations in MP-CMML have been described at a lower frequency than in MPN [2]. Recently, CALR gene mutations have been described in 67–88 % JAK2V617F and MPL-negative MPN; these cases also have a good prognosis [3–7]. The role of CALR mutations in CMML is not well known, and the few studies carried out so far have not focused on MP subtype [3, 4, 8]. The objective of this study was to characterize the frequency, type, and prognostic implications of CALR mutations in MP-CMML. CALR exon 9 mutations were studied in 30 patients (22 males, median age 66 years) diagnosed of CMML according toWHO classification [1], and all of them belonged to MP-CMML according to the French-American-British Co-operative Leukaemia Group (Table 1). All patients provided informed consent for sample collection. Biological analyses were carried out in accordance with the Declaration of Helsinki. Mutation analysis was performed as previously described [3]. An informative result was obtained in 29/30 patients, and no CALR exon 9 mutation was found. Although a small percentage of mutated MP-CMML patients was expected [4], no mutations were detected in the samples studied. These results are in concordance with those of Klampfl T et al. [3] and Hou et al. [8], who found no CALR mutations in 64 and 48 CMML patients, respectively (subtype not specified), but they do differ from the results of Nangalia et al., who found 1/33 CMML mutated patient (subtype not specified) [4]. There is also another study [9] of other MDS/ MPN groups (atypical CML and unclassifiable MDS/MPN) where CALR mutations were not seen in either entity (n=30). Therefore, outside essential thrombocythemia and primary myelofibrosis, CALR mutations are only found in (1) a few patients with MDS/MPN, most of them defined as having refractory anemia with ring sideroblasts associated with marked thrombocytosis, even though the percentage described is very variable depending on the series (0 to 12.5 %) [3, 4, 10] and (2) a few MDS patients, mainly with refractory anemia with ring sideroblasts, refractory anemia, and refractory anemia with excess blasts [3, 4, 8, 10]. This observation could indicate a relationship between CALR mutations and the thrombocytosis phenotype within myeloid neoplasms, suggesting that calreticulin mutations primarily affect the biology of megakaryocytes [11]. L. Zamora (*) :M. Cabezon : S. Marce : L. Palomo : F. Milla : E. Feliu : B. Xicoy ICO Badalona, Hospital Germans Trias i Pujol, Institut de Recerca contra la Leucemia Josep Carreras, Badalona, Spain e-mail: lzamora@iconcologia.net

Introduction: Lenalidomide (LEN) has shown impressive single agent activity in MDS with del(5q). In patients with non-del(5q) MDS the response rate is much lower (around 26%) and response duration is shorter. Valproic acid (VPA), an anticonvulsive drug and histone deacetylase inhibitor, has shown promising activity, improving cell counts in lower risk MDS. In our previous study, the overall response rate in that subset of patients was 29%. Here we report final results of a phase II trial of VPA and LEN combination therapy in patients with IPSS low or intermediate-I MDS without del(5q).The primary objective of the trial was to evaluate efficacy and safety of the combination of LEN and VPA, which are both effective in lower-risk MDS. Patients/Methods: A total of 26 patients were evaluated for eligibility. 23 patients were enrolled in the trial. VPA was given continuously according to serum level (target range: 50-110 µg/l), LEN was given at 10 mg/d for 21 days per 28 day cycle. Only 12 patients finished the originally planned 16 week treatment period, mainly due to hematological toxicity. Median age was 66 (48-81) years. 16 patients had RCMD, 3 RCMD-RS, 1 RA, and 3 RAEB I. The IPSS score was low in 11 and int-I in 12 patients. IPSS-R score was low in 8, intermediate in 15, and high in 1 patient. Most patients (n=19) had a normal karyotype. Only 4 patients showed an aberrant karyotype, including isolated +8 in two, and t(1;2)(p36;p21) and del(11q)(q21q24) in one patient each. Analysis of molecular markers (DNMT3A, EZH2, CBL, RUNX1, TET2, JAK2, FLT3, MLL-PTD, KRAS, NRAS, ASXL1, IDH1, IDH2, TP53, SF3B1, SRSF2) has been performed in 10 out of 23 patients as yet. Results: Response according to International Working Group (IWG 2006) criteria was observed in 9 of 23 patients, including 3 complete remission (CR) dysplastic, 1 marrow CR with hematologic improvement- erythroid (HI-E), and 5 stable disease (SD) with HI-E. 13 patients had stable disease and 1 showed progressive disease. Patients with hematologic improvement achieved transfusion independence. Median response duration was 8 (6-20) months. The most relevant side effect was hematological toxicity. 12 patients exhibited grade 3/4 neutropenia (8 grade 1/2) and 12 patients grade 3/4 thrombocytopenia (4 grade 1/2). After 6 patients had been included, the LEN starting dose was reduced from 10 to 5 mg. Of 12 patients who completed the full treatment period of at least 16 weeks, 8 (67%) responded. In the first 2 patients we observed an interesting response pattern. Both developed grade 3/4 neutropenia and thrombocytopenia together with unspecific symptoms of infection. Both had to stop study treatment. One developed a tri-lineage response for 13 months without restarting treatment. The other continued on VPA alone and responded for 6 months. Such a pattern of acute toxicity and response was no longer observed after the LEN starting dose had been reduced. Out of 8 patients with a platelet count Conclusion: We found a response rate of 39% (9/23) according to IWG criteria. There were no unusual or unexpected non-hematologic toxicities. However, bone-marrow toxicity was more severe than expected and required a reduction of the LEN starting dose. In our view, hematological toxicity limits the tolerability of LEN/VPA combination therapy in patients with lower-risk MDS. Disclosures Kuendgen: Celgene: Research Funding, Travel expenses Other. Off Label Use: Lenalidomide and valproic acid are not approved for lower risk MDS treatment without del5q. Lübbert:Ratiopharm: study drug supply VPA, study drug supply VPA Other; Celgene: Travel support, Travel support Other. Schlenk:Celgene: Research Funding, Speakers Bureau. Platzbecker:Celgene: Research Funding. Haferlach:MLL: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment. Germing:Celgene: Honoraria, Research Funding. Gattermann:Celgene: Research Funding; Novartis: Honoraria.