Solary, Éric

Abstract Background : The 2016 revision to the World Health Organization classification of myeloid neoplasms has recommended distinction between "proliferative" (WBC ≥ 13 x 10(9)/L) and "dysplastic" (WBC < 13 X 10(9)/L) subtypes of chronic myelomonocytic leukemia (CMML). In addition, CMML is characterized by a spectrum of cytogenetic and molecular abnormalities (Patnaik Leukemia 2014). In the current study, we looked for correlations between gene expression profiles (GEP) and morphologic or molecular categories of previously untreated patients with CMML. Methods : 35 patients with CMML were included in the study. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for 28 myeloid-relevant genes (Patnaik Blood C. J.). RNA sequencing (Stranded Total RNA- Illumina, San Diego, CA), was performed on 9 normal BM controls, and 35 CMML samples (25 peripheral blood (PB) and 10 BM). The RNA-Seq data was analyzed using MAPRSeq v.2.0. Normalization and differential expression analysis was performed using edgeR 2.6.2. We selected genes that were differentially expressed with a False Discovery Rate (FDR) <0.1 and a fold change bigger than 2. Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). Results: Among the 35 study patients, 75% were males and median age was 71 years (range, 55-87). 21 (60%), 9 (25%) and 5 (15%) patients were classified as CMML-0, 1 and 2, respectively. 16 (45%) had a dysplastic while 19 (55%) had a proliferative phenotype. At a median follow-up of 16 months, 5 (14%) deaths and 4 (14%) leukemic transformations were documented. Mutational frequencies included; TET2 45%, ASXL1 45%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, DNMT3A 6%, U2AF1 6%, SF3B1 5%, ZRSR2 4%, Tp53 4%, and IDH2 4%. Abnormal cytogenetics (n=9, 35%) included; -Y 8%, trisomy 8 6%, monosomy 7 3%, del(13q) 3% and monosomal karyotype 3%. i) Differential GEP in PB samples: 25 PB CMML samples were analyzed. Unsupervised GEP demonstrated two unique clusters (figure one); cluster one was enriched with dysplastic CMML (90%) and cluster two with proliferative CMML (64%). Consistent with the difference in the distribution of morphologic categories, in comparison to cluster one, patients in cluster two had a higher white blood count (p=0.003), higher monocyte count (p=0.02) and higher risk prognostication by the Mayo Model (p=0.016); there was no significant difference between the two clusters in terms of age and gender distribution, hemoglobin level, neutrophil and platelet counts, PB or BM blast content, cytogenetic risk stratification, ASXL1, TET2, RAS, CBL and SETBP1 mutational status. Genes significantly upregulated (FDR<1e-5 and fold change ≥ 16x); in cluster two (proliferative CMML) in comparison to cluster one included; PRG2, CTSG, BEX1, CD34, CRISP2, while those significantly down-regulated included (FDR<1e-15 and fold change ≥ 5x); IL2RB, PRF1, GNLY, PDGFRB. IPA analysis identified the following pathways preferentially expressed in cluster two (proliferative CMML) 1) mitotic roles of polo-like kinase (KIF23, WEE-1, PLK1, PLK4), 2) Cell cycle: G2/M checkpoint regulation (CDC25C, WEE-1, AURKA, CDK1), 3) Cell cycle: chromosomal replication (CDC45, RPA3, MCM2, CDC7), and 4) BRCA1 in DNA damage response (BARD1, RBBP8, PLK1, RAD51). Whereas in the cluster one (dysplastic CMML), preferentially expressed pathways included; 1) T-cell receptor signalling (CD247 PRKCQ, CD4, PLCG1, CD28) and 2) STAT3 signalling (SOCS3, MAP3K9). ii) Differential GEP in BM samples: 10 BM CMML samples were analyzed along with 10 normal controls. Samples and controls clustered separately and within the CMML samples two clusters were identified (figure two). These clusters had equal representations of dysplastic (n=5) and proliferative (n=5) CMML sub-types. Additional samples are being processed. Conclusions: Differential gene expression profiling in patients with CMML identifies two unique clusters, clinically demarcated, at least in PB samples, by "proliferative" and "dysplastic" CMML subtypes. The proliferative cluster is enriched in genes involved in cell cycle regulation and DNA damage response; whereas the dysplastic cluster is enriched in T-cell receptor and STAT3 signalling. The prognostic and therapeutic impact of these gene clusters remains to be assessed in a larger group of informative patients. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Al-Kali: Novartis: Research Funding; Celgene: Research Funding.

Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic neoplasm characterized by sustained peripheral blood (PB) monocytosis and an inherent risk for leukemic transformation. Clonal origins of the disease can be detected in hematopoietic progenitor cells (CD34+/CD38-), while the complete spectrum of mutational evolution can be seen in circulating monocytes (CD14+). Cell sorting strategies have been employed to select cells in CMML, and while there are adequate monocyte numbers in the PB, there are very few circulating progenitor cells. In addition, attrition related to the selection process significantly depletes primary cells available for biological experiments and multiomics studies such as RNA-seq, ChIP-seq, ATAC-seq, and DIP-seq. While single-cell methods may be able to overcome this challenge, bulk sequencing methods remain a robust and cost-effective approach. We hypothesized that, secondary to the stem cell origin of this disease and significant myeloproliferation, PB mononuclear cells (MNC) would provide comparable results with regards to transcriptomic analysis, in comparison to cell selection procedures. Methods: Peripheral blood obtained from 15 molecularly annotated patients with WHO-defined CMML was ACK-lysed and subjected to a Ficoll procedure for collection of MNC. MNC were left unsorted (n=5) or further selected for CD34+/CD38- (n=5) and CD14+ (n=5) using a fully automated RoboSep-S (StemCell Technologies) protocol. All samples were then subjected to bulk whole transcriptome shotgun sequencing (using Illumina TruSeq and an Illumina HiSeq 4000). After data quality control, counts of detectable transcripts were log2-normalized and Pearson's product-moment correlation coefficients were calculated to evaluate the correlation between the two cell-sorting strategies and unsorted cells in terms of detectable transcripts. To visualize sample differences log2-normalized transcripts counts were centered and scaled per gene for a select number of genes relevant to myeloid biology as well as a number of housekeeping genes. Results: Fifteen patients with WHO-defined CMML, median age 69 years (55-73 years), 66% male, were included. Next generation sequencing for somatic mutations was performed on PB MNC obtained at CMML diagnosis (Figure 1, top heatmap). Considering the small sample size, mutations were evenly distributed among groups with the exception of ASXL1 (higher frequency in CD14+ and CD34+/CD38- cells), ZRSR2 (higher frequency in unsorted cells), and TET2 (lower frequency in CD14+ cells). The three groups were also well matched with regards to other CMML-related variables such as WHO and FAB morphological subtypes, cytogenetic abnormalities, and risk stratification by the Mayo Molecular Model. Transcriptomic analysis revealed a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD34+/CD38- cells (ρ = 0.96, p < 0.001, top scatterplot). Likewise, there was a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD14+ cells (ρ = 0.91, p < 0.001, bottom scatterplot). The latter correlation was marginally lower, which was explained by increased global gene expression in 3 of the 5 CD14+ samples (bottom heatmap). Increased gene expression in these 3 samples involved key myeloid genes and housekeeping genes known to have stable expression across human tissues alike. In comparison to PB MNC, both cell sorting strategies resulted in significant depletion of primary cells required for other experiments, and for procedures such as ChIP-seq, DIP-seq and ATAC-seq (CD34+/CD38- had greater depletion than CD14+). Additional experiments to assess this strategy for the above mentioned epigenetic studies are currently being planned. Conclusions: Accounting for sample differences, different cell sorting strategies (unsorted, CD34+/CD38- selection, and CD14+ selection) yielded similar results when performing bulk transcriptomic assessments on PB MNC from patients with CMML. For the purpose of gene expression profiling there was no clear advantage with CD34+/CD38- or CD14+ selection. These results support the use of unsorted cells for bulk transcriptomic analysis in CMML. Figure 1 Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Background: Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) in adult populations include several rare hematologic malignancies with similar clinicopathologic features that have a significant impact on patient QOL and lead to increased risk of infection, hemorrhage, anemia and transformation to AML (e.g. CMML, aCML, MDS/MPN-RST, and MDS/MPN-U). The only approved therapies for any of the MDS/MPNs are DNA methyltransferase inhibitors (DNMTis) for patients (pts) with CMML. Due to both the rarity and the heterogeneity of MDS/MPNs, it has been challenging to study in dedicated, prospective studies and thus refining treatment strategies has been difficult and optimal salvage treatments in pts who fail DNMTis have also not been rigorously studied. ABNL-MARRO (A Basket study of Novel therapy for untreated MDS/MPN and Relapsed/Refractory Overlap Syndromes) is an international cooperation providing the framework for clinical trials for MDS/MPN, and explore clinical-pathologic biomarkers. Study Design and Methods: The first study within ABNL-MARRO, ABNL MARRO 001 (AM-001), is an open label, randomized phase 1-2 study that will test 3 novel oral treatment combinations in MDS/MPNs, with each of 3 treatment arms including a novel targeted agent and ASTX727, a fixed dose combination of the DNMTi decitabine and the cytidine deaminase inhibitor cedazuridine to improve decitabine oral bioavailability. The novel agents combined with ASTX727 are: a selective JAK1 inhibitor itacitinib, pan PIM inhibitor INCB053914 and LSD1 inhibitor, INCB059872. Each combination will be tested first in a safety run-in (Phase 1b) to determine the RP2D and schedule of each combination therapy using a 3+3 de-escalation design, and a maximum of four dose decrements will be allowed. After the RP2D and schedule has been determined for a given novel drug combination, efficacy of that combination therapy in MDS/MPN pts will then be evaluated in phase 2 using a Simon's Two-Stage design to allow early discontinuation of any futile treatment regimen and to pursue potentially beneficial combinations in larger cohorts of pts. While each of the targeted agents either has undergone or is currently undergoing safety testing in combination with the DNMTi in myeloid diseases, clear rates of expected responses in MDS/MPN are not known. Retrospective studies of DAC or AZA reveal overall response rates (ORR) from 10-56% in CMML (Helbig et al, 2019; Duchman et al, 2018; Coston et al, 2019). The ORR from the ASTX727 single agent phase I study was 30%, but this included only 14% CMML (remainder MDS pts) and 45% of pts with previous treatment with AZA or DAC (Savona et al, 2019). Finally, and importantly, there are insufficient clinical trial data available for pts non-CMML MDS/MPN syndromes. Thus, the criteria for the Simon's Two-Stage design for each arm are based on best estimation from results of above studies. Accordingly, the null hypothesis that the ORR, as per the MDS/MPN Proposed Response Criteria (Savona et al, 2015; combined CR+MR+PR+Clinical benefit - CB), is 35%, will be tested against a one-sided alternative. In the first stage, 14 DNMTI-treatment naïve (TN) pts will be accrued in each AM-001 Arm and will receive treatment at the RP2D and schedule of the novel targeted agent and of ASTX727, as determined in the phase 1. If there are 5 or fewer responses in these 14 pts before the 7th cycle of therapy, the Arm will be stopped for futility. Otherwise, 30 additional pts (including both TN subjects and subjects relapsed after/refractory to other DNMTI-containing therapies) will be accrued for a total of 44. The null hypothesis will be rejected if 21 or more responses are observed in 44 pts before the 7th cycle of therapy. This design yields a type I error rate of 0.05 and power of 80% when the true response rate is 55%. The probability of early termination and expected sample size of any arm is 64% and 25 pts, respectively, assuming low (35% RR) efficacy Beyond the primary objectives of the study to evaluate the safety and efficacy of novel treatment combinations in MDS/MPN, the study will establish the ABNL MARRO infrastructure that leverages the expertise of the MDS/MPN international working group (IWG) for future prospective studies; will forge innovative scientific research that will improve our understanding of pathogenetic etiologies; and will inform the clinical application of diagnosis, risk stratification tools, and response assessments in MDS/MPNs. Disclosures Savona: AbbVie: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Selvita: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Patents & Royalties. Fenaux:Aprea: Research Funding; Jazz: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding. Padron:Incyte: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Sanz:Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Helsinn Healthcare: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffman - La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen - Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onconova: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Santini:Menarini: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Acceleron: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Honoraria. OffLabel Disclosure: ASTX727, LSD1 inh, PIM inh, JAK1 inh in MDS/MPN

Background: CMML is a heterogeneous disease with overlapping features of MDS and MPN. Several risk scores have been proposed in CMML, including CPSS (Such Blood 2013) that identifies 4 risk groups (low, intermediate-1 [int-1], int-2 and high risk) with median overall survival (OS) ranging from 12 months to > 5 years. HSCT is the only curative treatment in CMML, with an expected 20-40% post-transplantation OS. Current guidelines, common to MDS and CMML recommend rapidly transplanting higher-risk CMML and delaying transplant in lower-risk disease (de Witte Blood 2017). Retrospective transplant studies have shown better outcome in lower-risk CMML (Liu et al BBMT 2017). Whether this translates into a survival benefit compared to a delayed transplant strategy requires prospective studies. Multistate models based on retrospective data from transplant and non-transplant patients (pts) have addressed similar issues in MDS (Koreth JCO 2013, Della Porta Leukemia 2017). Through a large collaborative study, we address the question of optimal timing for HSCT in CMML patients through a similar large international collaborative study. Method: We retrospectively selected pts from 2 registries: International MDS/MPN Working Group (IWG cohort, Padron Blood Cancer J 2015) and EBMT with the following criteria: WHO-defined CMML, age ≤ 70y, ECOG 0-2, diagnosis after 2000, available CPSS at the time of diagnosis. Different states were considered in higher- (CPSS int-2 and high) and lower- risk (CPSS low/int-1) pts: diagnosis, AML transformation, transplantation and death. IWG data was used to estimate transition probabilities from diagnosis to AML, transplantation and death and from AML to transplantation and death. EBMT data was used for transplantation to death transitions (Figure 1). Results: 719 and 403 pts were identified in the IWG and EMBT registry, respectively (resp). Median age was 64 (range 16-70) in IWG and 58 (19-70) years in EBMT cohort. Patients were male in 69% and 67% of IWG and EBMT cohorts, respectively (resp). CPSS was low in 22% and 13%, int-1 in 31% and 31%, int-2 in 40% and 45% and high in 8% and 11% of the IWG and EBMT pts, resp. In the IWG cohort, the 1 year cumulative incidence of transformation into AML were 7.3% and 18.2% in lower and higher risk patients, resp. Among the 719 pts from IWG, 102 received HSCT. At the time of diagnosis the expected life time of higher risk (int-2 and high) patients was 25.2 months while it was 44.1 months in lower risk (int-1 and low) patients. Table 1 reports gain or loss in expected life time (until 60 months after diagnosis) for transplantation at different intervals from diagnosis (within 6, 12, 18 and 24 months) for patients who have survived and not transformed to AML until the prediction time point, taking into account CPSS at diagnosis. Expected life time is also given separately for higher risk pts after transformation into AML. There was a modest gain of life expectancy with transplantation in higher risk patients (CPSS int-2 and high) while it was not the case in lower risk patients. The survival gain in higher risk pts with HSCT rose from 1.11 at 6 months to 3.4 months at 24 months. There was also a survival benefit of HSCT in higher risk pts who were transformed to AML during their follow-up which was even higher in terms of gain of months: from 9.89 month if HSCT performed within 6 months to 14.25 months if performed within 18 months. Of note, the loss of survival in lower risk patients who underwent HSCT decreased over time: 11.1 months if HSCT is performed within 6 months and 3.7 months if HSCT is performed within 24 months, probably owing to transition to higher-risk CMML at this stage in some pts, a transition that could not be captured in the present dataset. Conclusion: For the first time, using a multistate model, we could provide evidence that the transplant benefit in CMML is restricted to higher-risk pts, in line with findings in MDS and current guidelines. More analyses will be performed to analyze other subgroups of patients, especially regarding their risk (other classification than CPSS, the role of somatic mutation…). More studies are needed to analyze the role of pre HSCT treatment and its potential impact on post-transplant outcome. Disclosures Robin: Novartis Neovii: Research Funding. Beelen:Medac GmbH Wedel Germany: Consultancy, Honoraria. Fenaux:Celgene Corporation: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Aprea: Research Funding. Kroeger:Riemser: Research Funding; Novartis: Honoraria, Research Funding; Medac: Honoraria; DKMS: Research Funding; Neovii: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; JAZZ: Honoraria; Sanofi-Aventis: Honoraria. Nazha:Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Abbvie: Consultancy. Rampal:Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy; Constellation, Incyte, and Stemline Therapeutics: Research Funding. Finke:Riemser: Honoraria, Other: research support, Speakers Bureau; Neovii: Honoraria, Other: research support, Speakers Bureau; Medac: Honoraria, Other: research support, Speakers Bureau. Komrokji:Agios: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau; Incyte: Consultancy. Killick:JAZZ: Honoraria; Celgene: Honoraria; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; ALEXION: Honoraria. Blaise:Molmed: Consultancy, Honoraria; Sanofi: Honoraria; Pierre Fabre medicaments: Honoraria; Jazz Pharmaceuticals: Honoraria. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction: Chronic myelomonocytic leukemia (CMML) is a chronic myeloid malignancy associated with monocytosis, autoimmunity (~30%) & an inherent risk for leukemic transformation. Bone marrow (BM) dendritic cell (DC) populations occur in ~30% of patients, with a poorly defined biological & prognostic role. The malignant immune microenvironment is regulated by indoleamine 2,3-dioxygenase-1 (IDO-1) expressing DCs, which modulate regulatory T (Treg) cells & block their conversion into proinflammatory T helper (Th17)-like cells. IDO-1 is a known immune checkpoint & functions by catabolizing tryptophan, an amino acid essential for T cell function. We hypothesized that distinct IDO-1 expressing DC populations in CMML modulate Tregs & contribute towards immune tolerance & aggressive disease biology. Methods: Primary diagnostic CMML peripheral blood mononuclear cells (PBMC) & BM biopsy specimens were obtained after Mayo Clinic IRB approval. A DC population was defined on H&E stained biopsy sections as focal collections (>10) of cells with characteristic elongated nuclei & cytoplasmic extensions. Transcriptomic & protein expression studies assessing IDO-1 expression were done by previously described methods. In addition, IHC expression of PD-1, PD-L1 & CTLA-4 was also done. IDO-1 promoter methylation studies with DIP-seq were performed. The impact on immune tolerance was assessed using mass cytometry (CyTOF). Results: Cohort: Twenty eight patients with CMML were included in the study, median age 70 (range: 51-80) years; 71% males. Eleven (39%) patients had coexisting autoimmune conditions. Of these, 8 (73%) had detectable DC populations either at diagnosis, or during the course of their disease. At a median follow-up of 46 (95% CI 27, 84) months, there were 14 (50%) deaths & 9 (32%) leukemic transformations.IHC results: Nine (32%) patients were identified to have a DC population at CMML diagnosis. CD123 & TCL1 staining was performed in 5 (56%) patients, with 3 being positive for both, & 2 positive for CD123 only (additional IHC studies ongoing). IDO1 expression by IHC was documented in all 9 (100%) cases (Fig 1A & 1B), while rare populations of PD-1, PD-L1 & CTLA-4 lymphocytes were also seen in all cases. Due to the low DC burdens (median cellularity ≤ 5%) & uniform staining intensity, IHC-based grading was not done. Samples at serial time-points, post-HMA therapy & at the time of blast transformation, were available in 5 & 3 patients respectively. Among the patients who did not have DC populations at diagnosis, 5 (42%) developed them post-HMA therapy, while 3 (50%) developed them at the time of LT. The development of DC populations was associated with loss of response to HMA (50%) & disease progression (50%).Transcriptomic analysis: RNA expression data was available on 7 (25%) patients, of whom only 1 (14%) had DC populations at diagnosis. The IDO-1 RPKM value in the former was higher than the mean pooled value in the latter group (330 versus 74, p=0.05).Methylation studies: DIP-seq was performed on 12 (43%) cases from the primary IHC cohort. Qualitative analysis of IDO-1 promoter hypomethylation was conducted & confirmed in all 9 (100%) cases with 5-mC & 5-hmC marks compared to input as displayed in figure 1C.Immune profiling: CyTOF was performed on 4 CMML samples (3 with IDO-1 expressing DC populations at diagnosis) from the primary IHC cohort & compared to a normal PBMC control. Results confirmed an increase in DC populations (fig 1D& 1E), & reduced % of Th17-like T cells in CMML samples compared to control (1.1 versus 5.07, p=0.05, fig 1F).Clinical correlates & survival analysis: With the exception that CMML patients with DC populations had a higher frequency of NRAS (P=0.007) mutations, the two groups were comparable for cytogenetic & molecular abnormalities. The median OS for the cohort was 45 (95% CI 29, 84) months. CMML patients with IDO-1 expressing DC populations at diagnosis had a shorter median OS, in comparison to those without (median OS 30 vs 45, p=0.03, Kaplan-Meier analysis in fig 1G). Conclusions: In conclusion, we demonstrate that DC populations are seen in ~30% of patients with CMML with a uniform expression of IDO-1 & limited expression of PD-1, PD-L1 & CTLA-4. CMML patients with BM DC populations have a higher frequency of NRAS mutations & DC IDO-1 expression is associated with tumor induced immune tolerance. Additional IHC, genomic & preclinical studies with IDO-1 inhibitors are ongoing. Figure 1. Figure 1. Disclosures Al-Kali: Novartis: Research Funding.

Abstract Background: The prognostic significance of cytogenetic abnormalities in chronic myelomonocytic leukemia (CMML) was recently revisited (AJH, 89; 813-818, 2014 and Blood April, 2013). Using a large Mayo Clinic-French Consortium database, we analyzed the molecular and prognostic correlates of cytogenetic abnormalities in CMML. Methods: CMML diagnosis was according to World Health Organization criteria. Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature. Statistical analyses considered clinical and laboratory parameters obtained at time of cytogenetic studies. Results: Spectrum and frequency of cytogenetic abnormalities: A total of 409 patients participated in this study including, 268 (66%) from the Mayo Clinic and 141 (34%) from the French CMML consortium. Of these, 396 (97%) had ≥20 metaphases and 13 (3%) had ten to 19, analyzed. One hundred and fifteen (30%) patients displayed an abnormal karyotype, including 82 (71%) sole, 20 (17%) two and 13 (11%) complex abnormalities. The most common abnormalities were; +8 (23%), -Y (20%), -7/7q- (14%), 20q- (8%), +21 (8%) and der (3q) (8%). Other cytogenetic abnormalities included 5q-, 12p-, 13q- and i(17q), present at a much lower frequency (0.9-4%). Phenotypic correlates: Abnormal vs normal karyotype was associated with older age (p=0.03), hemoglobin<10 g/dL (p=0.0009), white blood cell count (WBC) >15 x 109/L (p=0.02), absolute neutrophil count (ANC) >10 x 109/L (p=0.03), absolute lymphocyte count (ALC) >2.5 x109/L ( p=0.04), peripheral blood (PB) blast ≥1% (p<0.0001), bone marrow (BM) blast ≥10% (p<0.0001) and circulating immature myeloid cells (IMC) (p=0.0003). +8 (p=0.01), +21 (p=0.03) and der (3q) (p=0.03) were associated with hemoglobin <10 g/dL. -Y was associated with older age (p=0.04), lower PB (p=0.04) and BM (p=0.02) blasts. -7/7q was associated with leukocytosis (p=0.005), neutrophilia (p=0.04), and higher PB blasts (p=0.004). 20q- was associated with thrombocytopenia (p=0.04). Molecular correlates: ASXL1 mutations were associated with abnormal karyotype (p=0.04) and SRSF2 with normal karyotype (p=0.02). In comparison to other abnormal karyotypes, the incidence of ASXL1 mutations was lower in –Y (P=0.04) and der(3q) (p=0.03). U2AF1 mutations were associated with monosomal karyotype (p=0.03) and SF3B1 with der (3q) (p<0.0001). Prognostic relevance : Median follow-up was 1.8 years with 244 (60%) deaths and 79 leukemic transformations (19%). A step-wise survival analysis resulted in three distinct cytogenetic risk categories (Figure 1): high (complex and monosomal karyotype), intermediate (all abnormalities not in high or low risk) and low (normal, sole -Y and sole der (3q)); the corresponding median survivals were 0.2 (HR 8.1, 95% CI 4.6-14.2), 1.7 (HR 1.7, 95% CI 1.2-2.3). In multivariable analysis, the particular cytogenetic risk stratification remained significant in the context of Mayo molecular model (p<0.0001), MDAPS (p<0.0001), and the GFM risk model (P<0.0001). The Mayo-French cytogenetic risk model was also effective in predicting leukemic transformation with HR of 10.9 (95% CI 4.2-27.8) for high and 2.2 (95% CI 1.3-3.7) for intermediate risk groups. Conclusion: Cytogenetic abnormalities are seen in approximately 30% of patients with CMML and display significant associations with certain molecular and phenotypic characteristics. We describe a novel cytogenetic prognostic model for both over-all and leukemia free survival in CMML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract Background : TET2, located on chromosome 4q24, is frequently mutated in the majority of patients with chronic myelomonocytic leukemia (CMML). TET2 has 11 exons, and variations, especially in exon 3 have been described as a part of age related clonal hematopoiesis (Jaiswal NEJM 2015). In CMML, somatic TET2 mutations in the absence of ASXL1 mutations (ASXL1wt/TET2mt) were previously shown to impart a favorable outcome on survival (Patnaik BCJ 2015). In the current larger CMML patient cohort, we describe the number and type of TET2 mutations and examine their phenotypic and prognostic effects. Methods : 261 patients with CMML were included in the study. All patients had bone marrow (BM) biopsies and cytogenetics performed at diagnosis. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for the following genes; TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, KRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1. TET2 (NM_001127208.2) coverage extended from exons 3-11, with frame shift, non-sense, and missense variations considered pathogenic. Previously annotated single nucleotide polymorphisms (http//www.hapmap.org) were considered non-pathogenic. Results: Among the 261 study patients, 65% were males and median age was 70 years (range, 28-91). 154 (59%), 64 (25%) and 43 (16%) patients were classified as CMML-0, 1 and 2, respectively. At a median follow-up of 23 months, 174 (67%) deaths and 37 (14%) leukemic transformations were documented. Mutational frequencies ≥4% were encountered in; ASXL1 45%, TET2 42%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, DNMT3A 6%, U2AF1 6%, SF3B1 5%, ZRSR2 4%, Tp53 4% and IDH2 4%. i) TET2 mutations type, number and phenotypic correlates: TET2 mutations were seen in 109 (42%) patients; these included frameshift 33 (30%), nonsense 29 (27%) and missense 11 (10%) variants whereas 36 (33%) had more than one type of mutation. Overall, 57 (52%) patients had more than 1 TET2 mutations: 52 (48%) patients had 1, 46 (42%) 2 and 11 (10%) ≥3 TET2 mutations. Among all 109 TET2 mutated patients, 65% were male, and median age was 71 years with no significant difference in age and gender distribution between mutated and un-mutated cases, or type of TET2 mutations; however, older patients were more likely to carry multiple TET2 mutations (p=0.01). Compared to their un-mutated counterparts, TET2 mutated cases were less likely to have a low hemoglobin (p<0.001), circulating immature myeloid cells (IMC) (p=0.001), peripheral blood (PB) (p=0.009) and BM blasts (p=0.009), and have high-risk stratification per Mayo Molecular Model (p<0.001); these differences were not affected by the type or number of TET2 mutations. TET2 mutated cases were more likely to have a higher frequency of SRSF2 (p=0.004) and lower frequency of ASXL1 (p=0.03), Tp53 (p=0.04) and IDH1/2 mutations (p<0.001); these associations were also not affected by the type or number of TET2 mutations. ii) Impact on survival: Median survival for the entire cohort (n=261) was 24 months. In univariate analysis, survival was superior in TET2 mutated (median 33 months) versus wild-type (median 21 months) patients [p=0.03; HR 1.3 95% CI 1.12-1.86] (figure 1A). This survival difference remained significant after adjustment for age (p=0.04), leukocyte count (p=0.017), absolute monocyte count (p=0.02), absolute lymphocyte count (p=0.02), platelet count (p=0.015), circulating IMC (p=0.03), DNMT3A (p=0.02) and ASXL1 (p=0.045) mutations; however, significance was lost after adjustment for abnormal karyotype (p=0.32) and the Mayo Molecular Model (p=0.0003). These observations were not affected by the type or number of TET2 mutations. Finally, our previous observation regarding the survival advantage of ASXL1wt/TET2mt versus other genotypes was most apparent for patients with multiple TET2 mutations (p=0.02) (figure 1B). Conclusions: TET2 mutations are common in CMML and constitute approximately equal proportions of frameshift and non-sense mutations, while missense mutations are less frequent. Majority of TET2 mutated CMML cases harbor more than one mutant variant. Regardless, the relevance of type and number of TET2 mutations in CMML was limited to an association between older age and number of mutations and the latter with possibly improved survival in the absence of clonal ASXL1 mutations. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction: CMML is a clinically heterogeneous myeloid neoplasm hallmarked by the coexistence of dysplastic and proliferative patho-clinical features, which can include cytopenias, constitutional symptoms, splenomegaly, and leukocytosis. However, according to the FAB classification schema, CMML may be differentiated in dysplastic and proliferative subgroups only by the presence of leukocytosis in the latter (WBC ≥13 x 109/L). We hypothesize that incorporation of other clinically discriminating features may yield a more informative CMML stratification system. To address this, we propose three distinct CMML categories and explore their clinical relevance leveraging our existing international CMML database (Padron E et al. Blood Cancer J. 20151). Method: 1622 WHO-defined CMML cases diagnosed between 1973 and 2014 were collected from eight large cancer centres that include up to 80 discrete data elements as previously described1. Cases were placed into three clinically distinct groups and the Pearson Chi-Square test and the Kruskal-Wallis test were applied respectively to compare categorical and continuous characteristics. The Kaplan-Meier (KM) method was used to estimate median OS and the log rank test was used to compare survival curves.Cox models whereapplied to obtain univariate and adjusted hazard ratios. Identification of optimal cut-off values for continuous variable was supported by graphical inspection of martingale residuals from the null Cox model. Statistical analyses were done in SPSS v23 and R v.3.3.0. Results: We propose three categories to delineate clinically distinct CMML subtypes: (1) Myelodysplastic (MD)-CMML: WBC≤10 x 109/L, PB-immature myeloid cells (IMC) = 0%, no splenomegaly (2) MD/MP-CMML: WBC 10-20 x 109/L or WBC ≤10 x 109/L but PB-IMC>0% and/or splenomegaly (3) Myeloproliferative (MP)-CMML: WBC >20 x 109/L. A recursive partitioning approach was used to identify the WBC cut points, with splenomegaly and IMC added to more accurately depict the MPN aspect of CMML. Numbers of patients included in the MD-, MD/MP-, and MP-CMML subcategories were 319 (19.7%), 789 (48.6%) and 514 (31.7%), respectively (Table 1). According to the FAB criteria, the MD/MP group included 521 (66%) MD- (WBC ≤13 x 109/L) and 268 (34%) MP-CMML (WBC>13 x 109/L) patients suggesting that the proposed classification clinically reclassifies FAB-defined CMML. Within the MD/MP group, 344 patients (21.2%) had a WBC <10 x 109/L but with IMC>0% and/or splenomegaly. Comparison of overall survival (OS) among proposed groups demonstrated that this classification schema was capable of discriminating the CMML natural history (Figure 1). In comparison to MD/MP-CMML, the unadjusted OS Hazard Ratio (HR) was 0.60 (95% CI 0.49-0.73) for MD-CMML and 1.57 (95% CI 1.36-1.81) for MP-CMML (p<0.001 for both). This difference was retained after adjusting for BM-Blasts (< vs ≥5%), IPSS or CPSS cytogenetics (High-risk vsInterm/low-risk), and elevated levels of LDH (HR was 0.73 with 95% CI 0.57-0.93, p=0.011 for MD-CMML, and 1.33 with 95% CI 1.12-1.58, p=0.001 for MP-CMML, respectively). We next explored whether each proposed group may have distinct variables that uniquely govern its prognosis. Peripheral blood blasts >5%, RBC- and PLT-transfusion-dependence predicted poor OS only in the MD and MD/MP-subcategories (p<0.005), but had no impact in the MP-CMML. Male gender and elevated LDH were only significantly associated with prognosis in the MD/MP group (p<0.05 and p<0.005, respectively) while absolute lymphocyte count >2.5 x 109/L and absolute monocyte count >10 x109/L were uniquely predictive for shorter survival in the MP-CMML subcategory (p<0.005). Notably, we additionally identified gene mutations that uniquely predicted prognosis in each proposed group (Table 2). Conclusions: We demonstrate that our proposed 3-group clinical classification schema is capable of independently stratifying prognosis. Further, our analysis identified clinical and genetic variables that uniquely govern each group's prognosis, suggesting independent clinical behaviour. Further investigations are warranted to validate these groups. Disclosures Jabbour: ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Fenaux:Celgene, Janssen,Novartis, Astex, Teva: Honoraria, Research Funding. Kantarjian:Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau.

Abstract Background Chronic myelomonocytic leukemia (CMML) is a clonal stem cell disorder with overlapping features between myelodysplastic syndromes and myeloproliferative neoplasms. Numerous models exist for CMML prognostication, with more recent studies suggesting (JCO 201; 31:2428) or refuting (Leukemia 2013; 27:1504) the prognostic contribution of ASXL1 mutations. Furthermore, SETBP1 mutations were recently shown to be associated with shortened overall survival (OS) in CMML (Leukemia 2013; 10:1038). In the current international study, we examine these issues in a larger cohort of 431 patients. Methods 431 patients with WHO-defined CMML were included in the study. 235 (55%) were seen at the Mayo Clinic from 1997 through 2012. The remainders were from the French CMML registry (JCO 201; 31:2428). All patients underwent bone marrow (BM) examination and cytogenetic evaluation at diagnosis. DNA analysis for spliceosome component mutations (SRSF2, SF3B1 and U2AF1), ASXL1 and SETBP1 mutations were carried out on BM specimens obtained at diagnosis. In order to address the aforementioned discrepancy regarding the prognostic impact of ASXL1 mutations, relevant analyses in the Mayo cohort were first performed with and without inclusion of missense ASXL1 mutations. ASXL1 mutations from the French cohort did not include missense mutations. We evaluated the prognostic relevance of ASXL1 and SETBPI mutations, as well as several other clinical and laboratory parameters including those previously identified by the MDAPS (Blood 2002;99:840) the Spanish cytogenetic risk stratification (Haematologica 2011;96:375), and the Mayo prognostic model (Leukemia 2013;27;1504). Results Among the 431 study patients, 286 (66%) were males and median age was 73 years (range, 17-93 years). There were 368 (85%) patients with CMML-1 and the remainder had CMML-2. At a median follow-up of 23 months, 260 (60%) deaths and 70 (16%) leukemic transformations were documented. Median survivals were 38 months for CMML-1 and 24 months for CMML-2 (p=0.11). Mutational frequencies were 44% (173/390) for SRSF2, 6% (23/379) for SF3B1, 7% (27/387) for U2AF1, 38% (164/411) for ASXL1 (excluding missense mutations), and 5% (21/431) for SETBP1. Risk stratification was, based on i) Mayo prognostic model: 172 (40%) high, 151 (35%) intermediate and 94 (25 %) low risk, ii) MDAPS: 15 (3%) high, 73 (17%) intermediate-2, 125 (29%) intermediate-1 and 218 (50%) low risk and iii) Spanish cytogenetic stratification system: 316 (73%) low, 43 (10%) intermediate and 50 (12%) high risk. In the Mayo cohort, univariate analysis revealed that the exclusion of missense mutations changed the prognostic impact of ASXL1 mutations from non-significant (p=0.08) to significant (p=0.001). Accordingly, all subsequent analyses excluded missense ASXL1 mutations. In univariate analysis, lower hemoglobin (p<0.0001), lower platelet count (p=0.0027), higher absolute monocyte count (AMC) (p<0.0001), higher absolute lymphocyte count (ALC) (p=0.0002), circulating immature myeloid cells (IMC) (P<0.0001), cytogenetic risk stratification (p<0.0001) and ASXL1 mutations (p<0.0001) were significant for OS. In multivariable analysis, lower hemoglobin (p=0.0001; RR 2, 99% CI 1.6-2.6), lower platelet count (p=0.002; RR 1.5, 99% CI 1.2-1.9), higher AMC (p=0.0002; RR 2.2, 99% CI 1.6-3.1) and ASXL1mutations (p=0.0009; RR 1.9, 99% CI 1.5-2.4) retained their independent negative prognostic impact. Similarly, in univariate analysis, leukemia-free survival (LFS) was negatively affected by age (p=0.0015), lower hemoglobin (p=0.0002), lower platelet count (p=0.0002), higher AMC (p<0.0001), higher ALC (p=0.0001), circulating IMC (p<0.0001), BM blasts (p<0.0001), and cytogenetic risk stratification (p=.0002). ASXL1 (p=0.17) and SETBP1(p=0.87) mutations were not found to be significant. In multivariable analysis, lower platelet count (p=0.0005), higher AMC (P=0.0042), circulating IMC (P=0.008) and cytogenetic risk stratification (p=0.009) retained their independent negative prognostic impact. Conclusions In the current international study of a large cohort of patients with CMML, we confirm and clarify the independent prognostic relevance of ASXL1 mutations. The relatively high frequency of ASXL1 mutations in CMML warrants its inclusion in contemporary prognostic models. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Most patients with chronic myelomonocytic leukemia (CMML) are elderly and age >65 years is an adverse prognostic factor (JCO 2013;31:2428). Our objective in the current study was to examine risk factors for survival and treatment outcome in younger patients (age ≤65 years), with emphasis on molecular and cytogenetic markers. Methods: DNA analysis for SRSF2, SF3B1, U2AF1, ASXL1 and SETBP1 mutations was carried out on specimens obtained at time of diagnosis. We used both conventional and revised (see accompanying ASH 2014 abstract) cytogenetic risk models to query their application in the current study population. Treatment data included outcome of allogeneic stem cell transplant (HCT) and treatment with hypomethylating agents (HMA), including azacitidine (AZA) and decitabine (DAC). Results: Survival: 259 patients (median age 59 years, range 18-65; 75% males) with WHO-defined CMML and age≤ 65 years were included in the study: 108 from the French consortium, 81 Mayo Clinic, 47 Moffitt Cancer Center and 23 University of Milan; 217 (84%) patients had CMML-1 and the remainder CMML-2. At a median follow-up of 26 months, 124 (48%) deaths and 79 (31 %) leukemic transformations were documented. Mutational frequencies were 34% (61/178) for ASXL1, 45% (74/165) for SRSF2, and 4% (7/168) for SETBP1. Cytogenetic data was available in 248 of 259 study patients; 56 (23%) patients displayed abnormal karyotype and the revised cytogenetic risk designations included 196 (80%) low, 29 (12%) intermediate, and 23 (9%) high. In univariate analysis, lower hemoglobin (p<0.0001), higher WBC (p=0.005), higher monocyte count (AMC) (P=0.004), lower platelet count (p=0.004), presence of circulating immature myeloid cells (IMC) (p=0.004), presence of circulating blasts (p<0.0001), bone marrow (BM) blasts ≥10% (p=0.005), WHO morphological subtype (p=0.0003), Spanish cytogenetic risk stratification (p=0.0002), revised Mayo cytogenetic risk stratification (p<0.0001), SRSF2 (p=0.03) and ASXL1 (p=0.03) mutational status predicted shortened survival. In multivariable analysis, lower hemoglobin (p<0.0002), presence of circulating blasts (p<0.0001), SRSF2 (p=0.03) and ASXL1mutational status (p=0.04) retained their negative prognostic impact. A similar analysis identified circulating IMC (p<0.0001), presence of circulating blasts (p<0.0001), and BM blast % (p=0.0002) as predictors of blast transformation (BT). Response to hypomethylating agents: Seventy-two (28%) patients received HMA (AZA-46, DAC-26, both-6). In the AZA group, median number of cycles was 5 (range; 1-44), median duration of therapy 5 months (range;1-59) with 4 (6%) receiving DAC on progression and 26 (42%) proceeding with HCT. Over-all response rate (ORR) was 25% (6/24) and included complete response (CR) in 2 (8%) patients, partial response (PR) in 2 (8%) and hematological improvement in 2 (8%). In the DAC group, median number of cycles was 6 (2-25), median duration of therapy 5 months (range;1-49), with 2 (8%) receiving AZA on progression; ORR was 50% (7/14) and included CR in 4 (29%) patients, PR in 1 (7%) and HI in 2 (14%). ASXL1 (p=0.4), SRSF2 (p=0.7) mutational status or karyotype (p=0.5 for Spanish and 0.3 for revised Mayo) did not correlate with response to HMA. Allogeneic stem cell transplant: 50 patients underwent HCT. Conditioning regimen was reported in 41 patients and included myeloablative (MA) in 14 (34%) (median age 51 years, 27-62) and reduced intensity (RIC) in 27 (66%) (median age 48 years, 18-65). Donor source was documented in 40 patients and included matched related in 19 (48%), matched unrelated in 12 (30%), mismatched unrelated in 4, haploidentical in 2 and umbilical cord transplant in one. 27 (63%) patients had chronic GVHD (50% in MA group and 80% in the RIC group). Among 40 evaluable patients, 23 (57%) achieved CR, 8 (21%) relapsed and 8 (21%) died from transplant-related complications. ASXL1 (p=0.9), SRSF2 (p=0.5) mutational status or karyotype (p=0.2 for Spanish and 0.3 for revised Mayo) did not affect transplant outcome. Conclusions: Survival in young CMML patients is poor and even worse in the presence of SRSF2 or ASXL1 mutations, anemia or circulating blasts. Treatment outcome was more impressive with transplant than it was with HMA and neither was influenced by karyotype or ASXL1/SRSF2 mutational status. Disclosures No relevant conflicts of interest to declare.