Mangaonkar, Abhishek A.

Introduction: Isocitrate dehydrogenase 1 and 2 (IDH1/2) are metabolic enzymes in the citric acid cycle, producing alpha-ketoglutarate (αKG). Mutations in specific regions of these genes have been characterized in gliomas, AML and chronic myeloid neoplasms such as MDS and MPN. These mutations produce an oncometabolite 2-hydroxyglutarate (2HG) that disrupts the epigenetic phenotype of myeloid cells promoting oncogenesis. While this process has been characterized for arginine hot spot (R132/R140) mutations, there exist variants of unclear significance (IDHVUS), with potential pathogenic predictions using in silico assessment models. With the availability of mutant IDH inhibitors in practice, it has become important to characterize these VUS. We carried out this study to functionally interrogate potentially pathogenic IDHVUS in MDS and MPN. Methods: After institutional approval, bone marrow (BM) DNA of patients with MDS and MPN, including overlap syndromes were subjected to next generation sequencing (29-gene, including the entire coding region of IDH1/2) by previously described methods (Patnaik et al., BCJ 2016). A sub-set of patients with available plasma [IDH wild type (IDHWT), bona fide IDH mutations (IDHMT) and IDHVUS with predicted pathogenicity (≤0.01% minor allele frequency, and predicted damaging in silico scores)] were submitted for mass spectrometry (LC-MS) based detection of IDH-related metabolites; D and L isomers of 2HG, αKG, and total 2HG. The impact of variations on protein secondary structure was assessed by the GorIV prediction algorithm. Potential alterations within the tertiary structure of the proteins was evaluated by in silico mutagenesis. Results: One hundred and seventeen patients were included in the study; median age 68 years (range, 39-94), 68% male. Of these, 40 (34%) had MDS, 42 (36%) MPN, and 35 (30%) MDS/MPN overlap syndromes. Eighty six (74%) had IDHMT, including n=28 (24%) with IDH1MT (R132C/G/H/S) (11 MDS, 8 MPN, 9 MDS/MPN) and n=59 (50%) with IDH2MT (R140G/Q/W) (22 MDS, 20 MPN, 17 MDS/MPN). IDHVUS were detected in 32 (27%) patients, including 1 IDH1MT patient, and involved IDH1 in 14 (12%) cases and IDH2 in 18 (15%) cases. All IDHVUS were missense, except for a 2 amino acid insertion in IDH2, with the most frequent VUS being IDH1Y183C (7%) and IDH2T352P (7%). While there were no phenotypic differences between IDH1/2MUT and IDH1/2VUS, in comparison to patients with IDH1/2MT, those with IDH1VUS were more likely to have higher WBC counts (median 8.1 vs 3.6 x109/L, p=0.0322), higher neutrophil counts (median 3.3 vs 1.3 x109/L, p=0.0203), and lower BM blast % (median 1 vs 3%, p=0.02061). Compared to IDH1/2MT patients, those with sole IDH1/2VUS more frequently had mutations in SF3B1 (p=0.0167), while they were associated with a lower frequency of SRSF2 mutations (p=0.0002). Interestingly, TET2 mutations were detected in only 5% of patients with IDHMT and 16% of IDHVUS patients, although without reaching statistical significance. LC-MS analysis on 75 plasma samples (n=43 IDHMT, n=9 IDHVUS, n=20 IDHWT, 3 normal) revealed a significant increase in total 2HG levels in IDHMT vs IDHVUS (p=0.0089), and IDHWT (p=0.0020) cases (Figure 2A-D). While there were no significant differences in αKG levels between the groups, the D-2HG enantiomer levels were higher in IDHMT samples vs IDHVUS (p=0.0199) and IDHWT (p=0.0055). On examination of the crystal structures of the IDH genes using PDB, while we observed a direct interaction between R132/R140/R172 and the catalytic core, the IDHVUS were located away from this active center and were hence thought to be allosteric in nature (Figure 2F). These VUS however, did demonstrate predictive steric clashes in the tertiary structure context of IDH1 using the PyMol artificial mutagenesis tool. The absence of wild-type crystal structure for IDH2precluded a similar analysis for IDH2. Median overall survival (OS) of the entire cohort (Figure 1) was 36 (range, 0-190) months, with OS being inferior in IDH1/2MT patients (34 months) vs those with IDH1/2VUS (50 months, p=0.0255). Conclusion: Our study demonstrates that in spite of predictive pathogenicity of IDH1/2VUS using in silico predictive tools and crystal studies, the lack of involvement of the arginine hot spot precludes IDH neomorphic activity, as shown by normal 2HG levels. In addition, in comparison to IDHMT patients, those with IDHVUS had fewer BM blasts and a better survival. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Introduction: Short telomere syndromes (STS) are accelerated aging syndromes affecting hematopoietic, pulmonary, hepatobiliary and/or immunological systems. Clinical assessment of age-appropriate telomere length (TL) is performed using flow cytometry & fluorescence in-situ hybridization (flowFISH). Screening for germline variants in STS-related genes is guided by flowFISH-determined centile categories of TL, with screening recommended for TL <1st centile or 1-10th centile in lymphocytes (L) or granulocytes (G). However, the utility of genetic testing for patients with TL >10th centile and integration of clinical phenotype with flowFISH data in predictive algorithms is currently unclear. Methods: FlowFISH testing was done at reference laboratories in Vancouver (Repeat Diagnostics; Canada) & Johns Hopkins University (JHU, USA). Salient clinical features were pre-determined as, personal history of premature hair greying (onset at age < 30 years), idiopathic pulmonary fibrosis (IPF) or IPF/emphysema overlap (in smokers), cryptogenic cirrhosis or NRH, unexplained cytopenias &/or immunodeficiency, & family history of the above (in >1 1st or 2nd degree relatives). Clinical likelihood score (CLS) was assigned as low (1), intermediate (int, 2) or high (>2), based on the number of aforementioned clinical features present prior to flowFISH testing. Genetic testing was performed using either an in-house or commercial bone marrow failure-specific next generation sequencing (NGS) panel or whole exome sequencing (WES), and data for known variants affecting telomerase or telomeric function (TERT, TERC, DKC1, TINF2, NHP2, NOP10, TCAB1, NAF1, & RTEL1) was recorded. Results: One hundred forty-nine patients at our institution underwent TL assessment at Repeat diagnostics (n=38) and JHU (n=111) laboratories, respectively. Median age was 56 (range: 7-79) years; 88 (59%) being males. Significant family history was present in 40 (27%) patients, while premature greying of hair was present in 13 (9%) patients. Organ-specific clinical features included unexplained cytopenias (n=89, 60%) IPF (n=71, 48%), cryptogenic cirrhosis or NRH (n=21, 14%), & unexplained immunodeficiency (n=14, 9%). CLS stratification included low (n=74, 50%), int (n=54, 36%), & high (n=21, 14%), with higher CLS significantly correlating with lower delta TL for L (p=0.0005) but not G (p=0.3). Genetic testing was performed in 51 (35%) patients (NGS-51, WES-1) among which 13 (26%) patients had a telomere-associated variant; 5 (10%) pathogenic (pv, all TERT). CLS alone was unable to predict likelihood of finding a telomere-associated variant (p=0.4). Based on age-appropriate centile categorization of L & G TL (information for both available in 134 patients), patients were stratified into six groups (table 1). TL <1st centile in L: This group was further divided into two groups; TL<1st centile in both L & G [A1, n=7, CLS low-3 (43%), int-2 (29%), & high-2 (29%)] and TL <1st centile in L and 1-50th centile in G (A2, n=2, CLS low & high) patients. Among the 4 (57%) patients who underwent genetic (NGS-3, WES-1) testing, 3 (75%) had TERTpv in A1 subgroup and 1 A2 subgroup patient had a VUS in TERT.TL <1st centile in G, 1-10th centile in L (n=18): This group included 9 (50%) low, 8 (44%) int and 1 (6) high CLS patients, of which only 1 of 8 NGS-tested patients had TERTpv.TL 1-10th centile in L or G: This group was divided into; 1-10th centile in both L & G [C1, n=28, CLS low-3 (11%), int-14 (50%), high-11(39%)] of whom 8 (22%) underwent NGS with no pathogenic variants but 3 VUS in RTEL1, NAF1 & PARN genes, and 1-10th centile in L, >1-90th centile in G [C2, n=36, CLS low-19 (53%), 16 (44%), 1 (3%)] of whom 8 (22%) underwent NGS with 1 TERTpv and 2 VUS in TINF2TL >10th centile in L & 1-90th centile in G (n=43, 32%): CLS stratification in this group included 27 (63%) low, 12 (28%) int, 4 (9%) high. NGS testing was done in 13 (30%) patients [CLS low-9(69%), int 2(15%), high 2 (15%)], of whom only 2 (15%) had VUS in TINF2 and TERT gene, but no pathogenic variants (figure 1). Conclusion: Our study demonstrates the importance of using a flowFISH assay based predictive algorithm to screen patients with suspected STS for telomere-related genetic alternations, in comparison to a clinical likelihood score. We also demonstrate a limited role for genetic testing in patients with lymphocyte TL >10th centile, regardless of the clinical likelihood score. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

BACKGROUND: CMML is a myelodysplastic/myeloproliferative neoplasm with a median survival of 32 months and no therapies that improve its natural history. We have previously demonstrated that CMML bone marrow mononuclear cells (BMNCs) are hypersensitive to GM-CSF and that the GM-CSF axis is a viable therapeutic target (Padron et al., Blood 2013). Lenzilumab is a novel, humaneered IgG1κ monoclonal antibody, with high affinity for human GM-CSF that has activity in preclinical models of CMML. We report a Phase 1 clinical trial testing the safety and preliminary efficacy of this agent in CMML. METHODS: The study was approved by scientific and ethical review boards. This was a multicenter Phase 1 study designed to evaluate the safety and determine the recommended phase 2 dose of lenzilumab in subjects with CMML. Dose escalation proceeded using a standard 3+3 study design to determine the maximum tolerated dose (MTD). Three dose cohorts included 200 mg, 400 mg, and 600 mg, were given IV on day 1 and 15 of cycle 1 and then only on day 1 of subsequent 28-day cycles. Key inclusion criteria included a WHO-defined diagnosis of CMML and a platelet count greater than 20 x103 cells/dL. Response was evaluated utilizing the MDS/MPN International Working Group Criteria (Savona Blood 2015). Pharmacokinetic analysis and pharmacodynamics were evaluated by pSTAT5 by flow cytometry. RESULTS: Between July 2016 and June 2018, a total of 15 patients were enrolled. The median age at study entry was 74 years (range 52-85) and 80% were male. Nine patients were classified as CMML-0, 3 as CMML-1, and 3 as CMML-2. Seventy three percent of patients had normal cytogenetics or -Y. The most commonly mutated genes at screening included TET2 60%, ASXL1 53%, SRSF2 47%, and RAS pathway (i.e. NRAS or CBL) mutations 40%. Nine patients were previously treated with hypomethylating agents and/or experimental therapies, 3 were treated with hydroxyurea only, and 3 were untreated. The mean Hgb was 9.7g/dL (7.6-14g/dL), the mean platelet count was 147 x103 cells/dL (16-942 x103 cells/dL), and 66% of cases were MPN-CMML by the French-American-British classification at study entry. Three patients were enrolled at each dose level and an additional 6 patients were enrolled at 600mg as planned. Consistent with prior studies of lenzilumab, no dose limiting toxicities were identified and no treatment emergent grade 3 or 4 toxicities were reported. The mean duration on therapy was 221.8 days (14-787 days) and the majority of patients discontinued study drug because of disease progression or lack of response (69%). Five of 15 (33%) patients enrolled achieved clinical benefit by MDS/MPN IWG criteria with 3 platelet responses, 1 neutrophil response, and 1 spleen response. An additional patient had bone marrow myeloblast reduction from 6% to 1% which allowed that patient to undergo allogeneic stem cell transplantation. Clinical response was not statistically associated with somatic mutations or changes in pSTAT5 between screening and cycle 3. However, 3 of 4 patients with NRAS mutation achieved clinical benefit or had clinical meaningful bone marrow myeloblast reductions. CONCLUSION: Lenzilumab is well tolerated in patients with CMML, with no grade 3 or 4 treatment emergent adverse events or DLTs reported. Durable clinical benefit was achieved in 33% of patients and one patient was bridged to allogenic transplant, providing proof of concept that GM-CSF inhibition has activity in CMML. The favorable safety and activity profile of lenzilumab warrants future evaluation as part of a combination regimen targeted to specific subtypes more likely to respond, including patients with NRAS mutations. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Sallman:Celgene: Research Funding, Speakers Bureau; Celyad: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Jazz: Research Funding. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding. Komrokji:celgene: Consultancy; Agios: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau; pfizer: Consultancy; DSI: Consultancy; Incyte: Consultancy. Lo:Humanigen: Employment. Durrant:Humanigen: Employment. Chappell:Humanigen: Employment. Ahmed:Humanigen: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: Germline RUNX1 mutations/deletions result in a Familial Platelet Disorder with propensity to Myeloid Malignancy (FPDMM); an autosomal dominant condition characterized by thrombocytopenia, qualitative platelet defects, and myeloid clonal evolution (MDS and AML). The advent of next generation sequencing (NGS) has allowed detection of clonal cytopenias of unclear significance (CCUS) prior to bone marrow (BM) morphological changes. Somatic RUNX1 mutations (RUNX1MT) can be seen in myeloid malignances, including MDS/MPN overlap syndromes, with an indeterminate prognostic impact. We carried out this study to define the landscape of clonal evolution in FPDMM patients and to compare germline RUNX1MT with somatic mutations. Methods After IRB approval, families with RUNX1MT FPDMM (detected by germline sequencing and deletion/duplication assays) and patients with 2016, WHO-defined MDS/MPN overlap syndromes (except JMML) were included in the study. Index patients with FPDMM underwent BM biopsies and in select cases NGS at diagnosis and then as indicated for follow up, or at disease progression. All MDS/MPN overlap syndrome patients underwent a 29-gene panel NGS assay on BM samples obtained at diagnosis by previously described methods (Patnaik et al., BCJ 2016). Results Eight index patients with FPDMM were included; median age at diagnosis 58 (range, 0-71) years, 3 (38%) males (Figure A). Four (50%) had intragenic deletions involving multiple exons in 3 cases and 2 nucleotides in 1 case, 1 (13%) each had missense, frameshift and splice site mutations, while 1 (13%) patient had a whole gene deletion (Figure C). Notably, in 4 (100%) of 4 patients with large deletions, NGS testing was negative for RUNX1 abnormalities. Four (50%) index patients presented with a myeloid neoplasm; JAK2, SRSF2, ASXL1 mutant CMML, MDS/MPN-U with trisomy 8, AML with del7q and normal karyotype AML (negative NGS). Three (38%) index patients had CCUS at diagnosis with TET2 (67%) being the most common acquired mutation, and others including SRSF2, EZH2, and SF3B1. Cumulatively, myeloid clonal evolution was seen in 7 (88%) patients. Three (38%) index patients (AML-2, CMML-1) underwent allogeneic HCT, and at last follow up were in remission. Median age at myeloid clonal evolution was 61 (range, 4-71) years; median overall survival (OS) from date of myeloid clonal evolution was 12 (range, 7-53) months. For the comparative analysis, 499 patients with MDS/MPN overlap syndromes were included; 382 (77%) with CMML, 48 (10%) with MDS/MPN-RS-T (MDS/MPN with ring sideroblasts and thrombocytosis), 17 (3%) with aCML (atypical CML), and 52 (10%) with MDS-MPN-U (unclassifiable). Median age at diagnosis was 71 (range, 18-99) years, and 329 (66%) were male. RUNX1MT were detected in 57 (11%) patients; 43 (11%) with CMML, 5 (30%) with aCML, and 9 (17%) with MDS-MPN-U. No RUNX1MT were found in MDS-MPN-RS-T. Overall, 62 RUNX1MT were identified in 57 patients, with 52 (39 CMML, 4 aCML, 9 MDS-MPN-U) and 5 (4 CMML, 1 aCML) patients harboring 1 and 2 RUNX1MT, respectively. RUNX1MT spanned the entire coding sequence without specific hotspots (Figure B): 27 (44%) were missense, 2 (3%) involved the splicing sites, 5 (8%) were nonsense and 28 (45%) were frameshift. There was only one RUNX1MT (R273K), seen in both germline and somatic patients (Figures B and C). Compared to wild type patients, RUNX1MT patients with MDS/MPN overlap syndromes had significantly lower platelet counts (median 72 vs 117 x109/L, p<0.0001), without any other phenotypic differences. In the somatic RUNX1MT cohort, co-occurring mutations included ASXL1 (54%), SRSF2 (54%), TET2 (49%), and NRAS (28%), with NRAS (28% vs 13%, p=0.0028), and SRSF2 (54% vs 39%, p=0.0218) mutations clustering with RUNX1MT. Median OS of the entire cohort was 29 (range, 0-170) months. On univariate analysis, while there was a trend for an inferior OS in RUNX1MTvs wild type patients (20 vs 29 months, p=0.0523), the leukemia-free survival was clearly inferior (p=0.0175, Figure D). Conclusion RUNX1 MT FPDMM is a germline predisposition syndrome with a high rate of myeloid clonal evolution (88%), including clonal cytopenias (38%). Germline RUNX1MT have minimal overlap with somatic mutations seen in myeloid malignancies, with NGS testing often missing RUNX1 deletions. Somatic RUNX1MT in myeloid malignancies are significantly associated with thrombocytopenia and an inferior leukemia-free survival. Figure Disclosures 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 : Chronic myelomonocytic leukemia (CMML), a myeloid neoplasm with overlapping features of myelodysplastic syndromes and myeloproliferative neoplasms (MPN), is characterized by peripheral blood (PB) monocytosis and a risk for leukemic transformation (LT). Gene mutations commonly seen in CMML include, TET2 (~60%), SRSF2 (~50%) and ASXL1 (~40%). MPN associated-driver mutations such as JAK2V617F (~10%), MPL (<1%) and CALR (<1%) are uncommon, and in fact, the 2016 World Health Organization (WHO) guidelines state that the presence of these mutations tends to support a diagnosis of MPN with monocytosis. We carried out this study to assess the i) clinical correlates and ii) survival outcomes of MPN associated-driver mutations in CMML. Methods : 323 molecularly annotated patients with WHO-defined 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 30 myeloid relevant genes, by previously described methods. Results: Among the 323 study patients, 67% were males and median age was 71 years (range, 18-95). Twenty five (8%) patients had CMML with MPN associated-driver mutations; 24 (96%) with JAK2V617F and 1(4%) with a MPLW515L mutation. There were no patients with CALR or JAK2 exon 12 mutations. Additional signal-pathway mutations included NRAS (15%), KRAS (4%), CBL (14%), PTPN11 (3%), CSF3R (1%), and SH2B3 (<1%). i) Phenotypic correlates: The median age of CMML patients with MPN-associated driver mutations was 71 years and 66% were male. The WHO morphological subtypes included CMML-0 60%, CMML-1 24% and CMML-2 16%; while 91% of patients had a normal karyotype. The distribution of mutations included TET2 75%, SRSF2 54%, ASXL1 48%, RUNX1 20%, NRAS and EZH2 12% each, SETBP1 8%, SF3B1, U2AF1, CBL, PTPN11, FLT3-TKD, & Tp53 4% each, respectively. Risk stratification by the Mayo Molecular Model included high 16%, intermediate-2 32%, intermediate-1 36% and low risk 16%, respectively. In comparison to CMML patients without MPN associated-driver mutations, those with, had a higher hemoglobin (HB, p=0.001) and hematocrit (p=0.001), were more likely to have leukocytosis (p=0.02) and elevated LDH levels (p=0.0002), less likely to have thrombocytopenia (p=0.003), more likely to have a "proliferative" CMML phenotype (p=0.01) with palpable splenomegaly (p=0.0007), and more likely to have mutations involving TET2 (p=0.02). Six (2%) thrombotic events were documented in the cohort; 2 (8%) in patients with MPN-driver mutations and 4 (1%) in those without (p=0.07). There were no differences between the two groups with regards to the degree of monocytosis, PB and BM blasts, BM cellularity, BM megakaryocytic atypia, and BM fibrosis. ii) Survival outcomes: At last follow up 219 (68%) deaths and 55 (17%) LT were documented, of which 14 (56%) deaths and 2 (8%) LT occurred in the MPN-driver mutation group. The median OS for the entire cohort was 28 months (22-32); 31 months for CMML patients with MPN associated-driver mutations and 24 months for those without (p=0.4). On a univariate analysis, survival was adversely impacted by low HB (p<0.0001), high white blood count (p=0.0009) and absolute monocyte count (p=0.0004), circulating immature myeloid cells (p=0.02) and blasts (p=0.005), BM blasts (p=0.02), palpable splenomegaly (p=0.03), abnormal karyotype (p=0.001), presence of DNMT3A (p=0.006), ASXL1 (p=0.009), EZH2 (p=0.03) and Tp53 (p=0.02) mutations and the absence of TET2 (p=0.0009) mutations. Survival was not affected by the presence of JAK2V617F (p=0.4) or MPL (p=0.46) mutations. On a multivariable analysis that included the aforementioned significant variables, only HB <10 gm/dl (p=0.0016), presence of PB blasts (p=0.01), presence of DNMT3A (p=0.003) and the absence of TET2 mutations (p=0.02) retained significance. The presence of MPN associated-driver mutations did not impact LFS (p=0.36). Conclusions: The occurrence of MPN associated-driver mutations in CMML is infrequent (~8%) and is largely restricted to JAK2V617F. CMML patients with MPN-associated driver mutations have a higher HB/hematocrit, are more likely to have proliferative features and palpable splenomegaly, and are less likely to be thrombocytopenic. There was a trend towards an increased thrombotic risk, with no impact of these mutations on overall and leukemia free survival. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Recent reports from the thalassemia literature suggest that serum ferritin may be a poor and possibly misleading measure of total iron store in heavily iron overloaded patients. Moreover, the relationship between plasma ferritin and body iron stores is distorted by ascorbate deficiency, fever, infection, inflammation, and hepatic dysfunction, all of which occur in patients with sickle cell disease (SCD). On the other hand, following the results of multiple studies that demonstrated a high correlation of hepatic iron overload determination by magnetic resonance imaging to the values found in specimens from liver biopsy, R2* liver MRI has emerged as the best noninvasive yet highly sensitive and specific method for measuring the level of iron in the liver. The goal of this study was to determine the correlation of liver iron by liver R2* MRI with serum ferritin and by extrapolation assess whether serum ferritin remains to be a useful clinical marker of iron overload in patients with SCD. We also sought to determine the correlation of liver iron concentration with abnormalities in the liver function tests. Methods: We conducted a retrospective analysis of 31 patients with sickle cell disease and transfusional iron overload who are being followed at the Augusta University Comprehensive Sickle Cell Center. Serum ferritin, hepatic R2* MRI liver iron concentration, hepcidin level, and liver function tests (AST, ALT and total bilirubin) were assessed for correlation. We used the Pearson correlation coefficient to determine the relationship between the various variables with hepatic R2* MRI. Results: Serum ferritin levels showed a statistically significant positive correlation with R2* hepatic MRI (r = 0.479 with p = 0.0085) in the patients with SCD and transfusional iron overload . We also saw a positive correlation, although not statistically significant, between hepcidin level and liver iron concentration by liver MRI(r=0.493 with P= 0.399). This may be due to the small sample size of the patients who had hepcidin levels available. On the other hand, no correlation was detected between abnormalities in liver function tests and liver iron concentration. The correlation between liver iron concentration(LIC) and AST was 0.045 with p = 0.816 and the correlation between LIC and ALT was 0.233 with p = 0.224. Conclusion: While R2* MRI is the the most accurate method to diagnose and monitor response to therapy in SCD patients with transfusional iron overload, we have found a statistically significant positive correlation with serum ferritin values. Thus, where R2* liver MRI is unavailable, serum ferritin remains a clinically useful tool that can be used in the diagnosis and monitoring of iron overload in sickle cell patients, despite its limitations. As previously reported, we did not find any correlation between LIC and liver function abnormalities in this population (Harmatz et al, 2000) These findings suggest that patients with SCD may have a different response to iron overload in comparison to patients with thalassemia or hereditary hemochromatosis. Disclosures Kutlar: Bluebird Bio: Other: DSMB Member; Sancilio: Other: DSMB Chair; Novartis: Consultancy, Honoraria, Other: Personal fees, Research Funding.

Key Points This article characterizes a cryptic IGH/CCND1 rearrangement in MCL by NGS. Mate-pair sequencing can help in accurately diagnosing MCL in cases of cyclin-D1–positive B-cell lymphoma with negative CCND1 FISH studies.

AbstractHypomethylating agents (HMA) are currently the only FDA approved therapy for patients with chronic myelomonocytic leukemia (CMML). In the current retrospective study, we assessed response rates as adjudicated by the IWG (International Working Group) MDS (myelodysplastic syndrome) and MDS/MPN myeloproliferative neoplasm overlap syndrome response criteria, in 121 CMML patients treated with Azacitidine (AZA, n = 56) and Decitabine (DAC, n = 65). The overall response rates were 41% by the IWG MDS (AZA‐ 45%, DAC‐39%), and 56% by the IWG MDS/MPN (AZA‐56%, DAC‐58%) response criteria, with CR (complete remission) rates of <20% for both agents, by both criteria. There were no significant differences in response rates between proliferative and dysplastic CMML. Moreover, 29% of CMML patients in a CR with HMA progressed to AML (blast transformation), underscoring the limited impact of these agents on disease biology. Progression after HMA response was associated with a median overall‐survival (OS) of 8 months, while median OS in patients with primary HMA failure was 4 months. Lower serum LDH levels (<250 Units/L) were associated with HMA responses by both criteria; while ASXL1 and TET2 mutational status had no impact. HMA treated patients had a longer median OS (31 vs 18 months; P = .01), in comparison to those treated with conventional care regimens (excluding observation only patients), without any differences between AZA vs DAC (P = .37). In conclusion, this study highlights the inadequacies of HMA therapy in CMML, retrospectively validates the IWG MDS/MPN response criteria and underscores the need for newer, rationally derived therapies.