Abstract: Background Acute myeloid leukemia (AML) is a complex, heterogeneous neoplasm characterized by the accumulation of complex genetic alterations that are responsible for the initiation and progression of the disease. Secondary AML (sAML) represents a progression from antecedent hematologic disorders such as myelodysplastic syndromes (MDS) or myeloprolifrative neoplasms (MPN). Certain acquired mutations have been reported to be specific for sAML when compared to primary AML (pAML), but many limitations exist when cytogenetic grouping or other parameters are taken into account. In addition, some mutations have been shown to impact survival in some studies, but not others. Methods We performed targeted deep sequencing on samples from bone marrow and peripheral blood of pts diagnosed with sAML and pAML and treated at our institution between 1/2003-1/2013. Additional data on pAML was added from The Cancer Genome Atlas (TCGA). A panel of 62 gene mutations described as frequently recurrent mutations in myeloid malignancies were assessed. Cytogenetic grouping was defined by CALGB/Alliance criteria. Differences were compared using Fisher's exact test and the Mann-Whitney U test for categorical and continuous variables, respectively. Overall survival (OS) was calculated from the time of diagnosis to last follow up or death. Results: A total of 496 pts included: 273 with pAML and 223 with sAML. Comparing pAML to sAML, pts were younger (median age 59 vs. 68 years, p 〈 .001) and had a higher WBC at diagnosis (13.5 vs. 3.9 X 109/L, p 〈 .001), respectively. Cytogenetic analysis showed significant differences: 58% of pAML pts had normal karyotype (NK) compared to 37% of sAML (p=.002), whereas 24% and 26% of sAML had intermediate risk (other than NK) and complex karyotype ( 〉 3 abnormalities) compared to 11% and 16% for pAML (p 〈 .001, .009), respectively. Mutations in ASXL1 (p 〈 .001), JAK2 (p=.014), CBL (p=.05), BCOR (p=.02), STAG2 (p =.003), SF3B1 (p=.04), SRSF2 (p=.001 ), and U2AF1 (p=.03) were highly specific for the sAML phenotype, whereas mutations in NPM1 (p 〈 .001 ), FLT3 (p 〈 .001), DNMT3A (p 〈 .001), and IDH2 (p=.02) were more specific for pAML. When the analysis was restricted to pts with NK cytogenetics, only ASXL1 (p 〈 .001) remained specific for sAML and DNMT3A (p 〈 .001) for pAML.Further, when the analysis was restricted to pts with unfavorable risk cytogenetics, only ASXL1 (p=.01) remained specific for sAML. No other mutations were specific for pAML. We then evaluated whether the mutations that were specific to each AML phenotype had an impact on OS. We observed different mutations that impacted OS in each phenotype: DNMT3A (HR 1.81, 95% CI 1.28-2.57, p 〈 .001), TP53 (HR 3.1, 95% 1.74-5.53, p 〈 .001), and SUZ12 (HR 3.18, 95% CI 1.01-10, p=.05) led to worse OS in pAML, whereas mutations in EZH2 (HR 2.12, 95% CI 1.07-4.21, p =.03), PRPF8 (HR 2.32, 95% CI 1.20-4.46, p=.01), and TP53 ( HR 2.92, 95% CI 1.69-5.04, p 〈 .001) lead to worse OS in sAML. Different mutations had a different impact on OS when cytogenetic analysis was taken into account. Mutations in FLT3 (HR 2.15, 95% CI 1.37- 3.35, p 〈 .001) and DNMT3A (HR 2.41, 95% CI 1.57-3.70, p 〈 .001) led to worse OS in NK pAML, whereas none of the mutations impacted OS in NK sAML. Further, in pAML with unfavorable cytogenetics, BCOR (HR 2.41, 95% CI 1.57-3.70, p 〈 .001) and TP53 (HR 2.41, 95% CI 1.57-3.70, p 〈 .001) had led to worse OS, whereas BOCR (HR 2.95, 95% CI 1.03-8.50, p 〈 .001), SF3B1 (HR .19, 95% CI .05-.82, p 〈 .001), SUZ12 (HR .12, 95% CI .01-.99, p 〈 .001),and TP53 (HR 1.9, 95% CI 1.09-3.46, p 〈 .001) only impacted OS in sAML. Conclusion Clear genomic variations exist between sAML and pAML. Although some of these genomic changes are more specific to each phenotype in general, this specificity and the impact on OS differed for each cytogenetic subgroup, highlighting the complexity of interpreting genomic information in pts with AML and the need to incorporate both cytogenetic and molecular data in prognosis-driven treatment decisions. Disclosures Sekeres: TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Abstract: MEC (mitoxantrone, etoposide, cytarabine) is a standard regimen for relapsed/ refractory (R/R) acute myeloid leukemia (AML), but outcomes remain poor. The overexpression of proteasomes and constitutive activation of NF-KB in AML cells suggest that proteasome inhibitors (PI) such as bortezomib (Bz) may be effective anti-leukemia therapy. PI or a decoy NF-KB oligonucleotide increase chemosensitivity to both anthracyclines and cytarabine. To test the hypothesis that PI may improve the efficacy of MEC, we conducted a phase 1 trial of Bz in combination with MEC. Here, we present final results of this trial: response rate, toxicity, and correlation of outcomes with mutation analysis. As CD74 expression may identify a subset NF-KB-dependent AML with predicted increased sensitivity to PI (Clin Can Res 2008; 14: 1446-54), we also explored this correlation. Methods: All pts were treated at the Cleveland Clinic from Aug 2010-Mar 2014. This protocol was approved by the institution’s review board. Eligibility included: age 18-70 yrs, R/R AML, cardiac ejection fraction ≥ 45%. CD74 was assessed by flow cytometry using CD45 PE (BD Biosciences San Jose, CA) and CD74-Alexa 488 (AbD Serotec Raleigh, NC). A myeloid panel mutational analysis was performed on extracted DNA in pts with banked samples (n=26). All pts received 1 cycle of MEC: mitoxantrone (6 mg/m2/d), etoposide (80 mg/ m2), and cytarabine (1000 mg/ m2) Days 1-6. Bz was administered IV on Days 1, 4, 8, and 11. Dose was escalated using a standard 3 x 3 design. Dose levels (DL) were: -1 (0.40 mg/ m2), 1 (0.70 mg/ m2), 2 (1.0 mg/ m2), and 3 (1.3 mg/m2). Response was defined by IWG criteria (Cheson, 2006). The maximum tolerated dose (MTD) of Bz with MEC was 1.0 mg/m2 (Advani et al, ASH 2012, Abstract 3595). Results: Of 35 pts enrolled, the median age was 55 yrs (range 33-69), 13 (38%) were male, and median baseline WBC was 4.0 K/ µL (range 0.82-84.7). The median time from initial diagnosis of AML to enrollment was 8.4 months (range 1.1-88.2) and 6 pts (17%) had an antecedent hematologic disorder. Salvage status (S) at enrollment: S1 (24 pts, 69%), S2 (7 pts, 20%), S4 (4 pts, 11%). Nine pts (26%) were refractory to all prior therapies, and 3 pts (9%) had received prior allogeneic hematopoietic cell transplant (AHCT). Adverse cytogenetics per CALGB/ Alliance 8461 criteria occurred in 19% of pts at study entry and 15 of 26 pts (58%) had poor-risk molecular mutations (RUNX1, ASXL1, TET2, p53, IDH1, MECOM, FLT3 ITD). Ten pts were enrolled on DL1, 13 pts on DL2, 11 pts on DL3, and 1 pt died prior to treatment. Overall, 3 pts (9%) died during induction. In addition to febrile neutropenia and Gr 4 hematologic toxicity, the most commonly reported adverse events (AEs) were metabolic, constitutional, gastrointestinal (GI), and dermatologic, with the majority of these being Gr 1 or 2. GI toxicity was the only reported AE attributable to Bz: 12 pts had constipation or ileus (10: Gr 1 or 2; 2: Gr 3 or 4). Seventeen of the 33 evaluable pts (52%) have achieved a complete remission (CR) or complete remission with incomplete count recovery (CRi); with 1 pt inevaluable due to donor lymphocyte infusion. The estimated median overall survival was 7.2 months; median duration of response was 10.3 months. DL did not correlate with response. Eleven pts (32%) went on to receive AHCT. Among pts with poor-risk molecular mutations, 64% achieved CR/ CRi. Inhibition of NF-KB signaling in leukemia cells with mutated RUNX1 efficiently blocks growth and development of leukemia (Blood 2011; 118: 6626-37). Of the 5 pts with RUNX1 mutations, 3 (60%) achieved CR/ CRi, suggesting that Bz may have promising clinical benefit in this difficult subset of pts. Among the 17 pts with CD74 expression testing who were evaluable for response, the mean CD74 expression trended higher in non-responding pts (32.6%) than in responders (11.1%) (p=0.14). Conclusions: The combination of MEC/Bz was well-tolerated and resulted in high response rates, even within a molecularly-defined poor risk population of pts with R/R AML. Our data do not confirm the expectation that higher CD74 expression would correlate with response in this R/ R AML cohort, but larger pt numbers are needed. These results, especially in pts with poor-risk mutations, support development of a randomized study to address the benefit of adding Bz to MEC in the treatment of R/R AML. Disclosures Advani: Takeda: Research Funding. Carew:Takeda: Research Funding. Sekeres:Celgene Corp.: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

Abstract: Patients with therapy-related acute lymphoblastic leukemia (t-ALL) represent a small subset of acute lymphoblastic leukemia (ALL) patients who received genotoxic therapy (ie, chemotherapy or radiation) for a prior malignancy. These patients should be distinguished from patients with de novo ALL (dn-ALL) and ALL patients who have a history of prior malignancy but have not received cytotoxic therapies in the past (acute lymphoblastic leukemia with prior malignancy [pm-ALL]). We report a retrospective multi-institutional study of patients with t-ALL (n = 116), dn-ALL (n = 100), and pm-ALL (n = 20) to investigate the impact of prior cytotoxic therapies on clinical outcomes. Compared with patients with pm-ALL, t-ALL patients had a significantly shorter interval between the first malignancy and ALL diagnosis and a higher frequency of poor-risk cytogenetic features, including KMT2A rearrangements and myelodysplastic syndrome-like abnormalities (eg, monosomal karyotype). We observed a variety of mutations among t-ALL patients, with the majority of patients exhibiting mutations that were more common with myeloid malignancies (eg, DNMT3A, RUNX1, ASXL1), whereas others had ALL-type mutations (eg, CDKN2A, IKZF1). Median overall survival was significantly shorter in the t-ALL cohort compared with patients with dn-ALL or pm-ALL. Patients who were eligible for hematopoietic cell transplantation had improved long-term survival. Collectively, our results support t-ALL as a distinct entity based on its biologic and clinical features.

Abstract: Background Achieving a complete remission (CR) in patients with newly diagnosed acute myeloid leukemia (AML) after induction chemotherapy with cytarabine and an anthracycline (7+3) remains an important treatment goal associated with better overall survival (OS). Approximately 25-30% of younger, and up to 50% of older patients (pts) fail to achieve CR. AML pts with residual leukemia at day 14 receive a second cycle of the same regimen; whether these pts have worse survival than pts not requiring re-induction is unclear. Information on pts with primary refractory AML and the best treatment strategy in this setting are limited. Methods Pts with newly diagnosed AML treated at our institution between 1/2000 and 1/2015 were included. Pts received standard induction chemotherapy with cytarabine for 7 days and an anthracycline for 3 days (7+3). Bone marrow biopsies were obtained at day 14 and a second cycle of the same regimen (7+3 for younger adults, 5+2 for older adults) was given to pts with residual leukemia (blasts 〉 5%). All responses were assessed at day 30 +/- 5 days post induction. Response was defined as CR and CR with incomplete hematologic recovery (CRi) or platelet recovery (CRp) per International Working Group (IWG) 2003 response criteria. Cytogenetic risk stratifications were based on CALGB/Alliance criteria. OS was calculated from the time of diagnosis to time of death or last follow up. A panel of 62 gene mutations that have been described as recurrent mutations in myeloid malignancies was used to evaluate whether genomic data can be used to predict response. Results: Among 227 pts with AML, 123 received 7+3 and had clinical and mutational data available. Median age was 60 years (range, 23-82). Median baseline WBC was 8.2 X 109/L (range, 0.3-227), hemoglobin 8.9 g/L (range, 4.7-13.8), platelets 47 X 109/L (range, 9-326), and BM blasts 46% (range, 20-95). Cytogenetic risk groups were: favorable in 12 (10%), intermediate in 68 (56%) [normal karyotype in 44 (36%)], and unfavorable in 42 (34%). A total of 93 pts (76%) responded, 69 (74%) received 1 cycle of induction and 24 (26%) required re-induction at day 14 due to residual leukemia. A total of 39 pts (32%) received allogeneic stem cell transplant (ASCT): 18 (46%) from a matched sibling donor, 16 (41%) from a matched unrelated donor and 5 (13%) had an umbilical cord transplant. With a median follow up of 13.5 months, the median OS for the entire group was 13 months (m, range, 0.1-120). The median OS for pts who failed 1-2 cycles of 7+3 was significantly worse than pts who responded (median 2.6 vs 16.9 m, p = 0.002). When pts undergoing ASCT were censored, the median OS was 2.3 vs 9.9 m, p= 0.003, respectively. Overall, 33 pts (27%) had residual leukemia at day 14 and received re-induction, 24 (72%) achieved a response at day 30+/- 5 days. The median OS for pts who received re-induction was inferior compared to pts who did not (10.1 vs. 16.1 months, p= 0.02). When pts who received ASCT were censored, the OS was similar (8.5 vs. 7.4 months, p = 0.49, respectively). Among the 30 pts with persistent disease following induction therapy at day 30, 11 (37%) died from induction complications, 6 (20%) received salvage therapy with mitoxantrone/etoposide/cytarabine, 3 (10%) received high dose cytarabine, 2 (7%) received azacitidine, and 8 (27%) received best supportive care. Among pts who received salvage chemotherapy 56% achieved CR and proceeded with ASCT. Two pts had ASCT with residual leukemia and relapsed within 3 m of ASCT. Pts who received ASCT after induction failure had a significantly better OS compared to non-transplant pts (median OS 22.0 vs. 1.4 months, p 〈 0.001, respectively); however, this benefit was only seen in pts who had ASCT in CR. We then investigated if genomic mutations can predict response or resistance to chemotherapy. Out of the 62 genes tested, only a TP53 mutation was associated with resistance, p = 0.02. Further, pts with TP53 mutations had significantly inferior OS compared to TP53 wild type regardless of ASCT status (1.4 vs 14.8 m, p 〈 0.001) Conclusion: Pts with newly diagnosed AML who fail induction chemotherapy with a 7+3 regimen have a poor outcome. Re-induction with the same regimen at day 14 for residual leukemia converted most non-responders to responders, but was associated with worse OS. ASCT improves outcome only in pts who achieve CR with salvage therapy. TP53 mutations predicted resistance to chemotherapy with 7+3. Disclosures Carew: Boehringer Ingelheim: Research Funding. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Abstract: Recently, rare somatic nonsense PHF6 mutations and deletions have been reported in patients with T-ALL, AML and blast crisis CML. Germ line PHF6 mutations have been described in Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation and somatic deformities. Patients with BFLS have been also reported to develop leukemia, suggesting PHF6 mutations may predispose to cancer. PHF6 is a highly conserved 41kDa protein showing ubiquitous expression in a variety of tissues, including bone marrow, CD34+ cells and leukocytes. The function and molecular pathogenesis in hematological disorders is unknown. PHF6 has been suggested to be a tumor suppressor gene (TSG) involved in the control of rRNA synthesis. Recent CHIPseq experiments showed that PHF6 binds upstream of the regulatory sequence of RUNX1. In an index case of a young adult female patient with proliferative CMML with dysmorphic features, we have identified remarkable GL mosaicism for PHF6 mutation (p.K44fs), confirmed by deep sequencing of bone marrow, CD3+ cells, spleen and skin tissue. Subsequently, we screened patients with myeloid neoplasms by targeted multi-amplicon sequencing to determine the prevalence and distribution of PHF6 gene alterations. Sequencing results from 1122 cases were analyzed (778 by targeted deep sequencing and 344 by whole exome sequencing). In total, we identified 45 cases with PHF6 mutations, 32 of which were frameshift or nonsense mutations. Previously, PHF6 have been included in screening panels by Haferlach et al., (Leukemia 2014) and Papaemmanuil et al., (Blood 2013) and somatic mutations were found in 24/944 and 21/738 cases of MDS, respectively. The somatic nature of these defects was confirmed by analysis of non-clonal CD3+ lymphocytes, Thus the incidence of PHF6 mutations ranges from 4.3% in current study to 2.8% and 2.5% reported by others and are most frequently observed among patients with secondary AML (33%), suggesting that PHF6 mutations are not uncommon driver events in myeloid neoplasia. Gender distribution showed a strong male predominance (76%), indicating that retention of a single copy of PHF6 may be protective. There was no significant sex difference in the transcriptional expression of PHF6 itself. The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (p=.08). The most commonly associated somatic mutations were in RUNX1 (p=.001) and IDH2 (p=.008). Concomitant PHF6 and RUNX1 mutations are associated with a poor prognosis in AML, and occur predominantly in males. There was no association observed between low expressors of PHF6 and RUNX1 mutations or RUNX1 expression levels. Conversely, RUNX1 mutant cases without somatic PHF6 mutations were not observed to have low transcriptional PHF6 levels. Subsequent analysis of clonal architecture using variant allelic frequency calculations and serial sampling suggested that mutated PHF6 may function as a founder driver gene in proportion of cases, while RUNX1 mutations are acquired as secondary events. Recent studies proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at the G2/M phase and an increase in DNA damage. To delineate a possible pathophysiological pathway involving PHF6 we compared transcriptional expression profiles of low expressors to those with normal levels of PHF6. The most notably deregulated group of genes were clustered to a functionally related group of ribosomal RNA proteins (p 〈 .00001). To better understand functional properties of PHF6 we conducted PHF6 specific immuno-precipitation followed by mass spectrometric fingerprinting on K562 cells to identify protein partners. We have identified a novel association of PHF6 in RNA degradation/stability and ribosomal proteins, including MOV and PABPC families. In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms and arefrequently associated with RUNX1/IDH2 mutations. Our functional in vitro studies, along with recently published reports, suggest an association of PHF6 deficiency with transcriptional regulation and thereby provide a basis for a transcriptional repressor phenotype conveyed by ancestral lesions, consistent with a role for PHF6 as a TSG. Disclosures Levine: Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.

Abstract: Chromosomal abnormalities can be founder lesions (e.g., t (8; 21), inv (16), inv (3)), initiate or advance disease progression (both founder and secondary hits e.g., ASXL1, TP53, RUNX1) or can be obligatory secondary hits (FLT3, NPM1). Hence, the rank of these mutations may determine the biological properties and clinical outcomes. However, while many mechanistic studies have been undertaken without identifying the key pathogenetic factors resulting from SF3B1 mutations, important biological clues can be derived from the consequences of SF3B1 alterations in the context of the clonal architecture of myeloid neoplasia (MN). SF3B1 mutant patients often have a homogeneous phenotype with isolated erythroid dysplasia, ring sideroblasts (RS) and favorable prognoses. Studies in primary MDS cells have suggested that SF3B1 mutations are initiating lesions and provide a marked clonal advantage to MDS-RS cells by propagating from rare lympho-myeloid hematopoietic stem cells. However, there is significant diversity of clinical phenotypes and outcomes including the observation that the disappearance of RS can be observed during the disease course of clonal MN and might suggest cellular shifts due to acquisition of additional hits. In such scenarios, the cell's fate in the context of SF3B1 mutations is pre-defined by the predominance of expanded hits. We took advantage of our detailed database of molecularly and clinical annotated cases with MN to study the SF3B1 mutatome and describe whether the clonal nature (ancestral vs. secondary) might change the clinical and phenotypic trajectories of MDS cells and whether the concatenation of mutations decreases the competitiveness of SF3B1 clones, leading to the dominance of other driver genes and subsequently to clonal evolution. The clonal hierarchy was resolved using our in-house designed VAF-based bioanalytic method and confirmed by the PyClone pipeline, which showed a high level of concordance. We first assigned clonal hierarchy to SF3B1 mutations by using VAFs (adjusted for copy number and zygosity) and classifying the mutations into dominant (if a cutoff of at least 5% difference between VAFs existed), secondary (any subsequent sub-clonal hit) and co-dominant hits (if the difference of VAFs between two mutations was 〈 5%). In total, we identified 140 dominant (SF3B1DOM), 121 secondary (SF3B1SEC) and 74 co-dominant SF3B1 mutations. For the purpose of this study, we set aside co-dominant SF3B1 mutations. Focusing on SF3B1DOM and SF3B1SEC, SF3B1DOM were often associated with a normocellular bone marrow compared to SF3B1SEC (n=42 vs. 26; P=0.02) and were less likely enriched in multi-dysplastic myeloid cells (29% vs. 53%; P=0.01). As such, SF3B1DOM tended to be more frequently detected in lower-risk MDS (P=0.05) in the subtypes of MDS-RS and MLD-RS (RS≥15%: 67% vs. 41%; P=0.01) compared to other disease subtypes. Twenty-three percent of patients with SF3B1SEC had secondary acute myeloid leukemia (sAML) (P=0.03). SF3B1SEC patients tended to have a lower median platelet count than patients with SF3B1DOM (97 vs. 130 x 109/L; P=0.05). SF3B1SEC was also more associated with bi-cytopenia compared to SF3B1DOM (52% vs. 36%; P=0.01). No specific association was found between SF3B1 clonal nature and cytogenetic abnormalities, suggesting that additional mutations might be the main contributors in the evolution of MDS to AML. Of note, patients with SF3B1SEC had half OS compared to patients with SF3B1DOM (SF3B1SECvs. SF3B1DOM: 15.9 mo. vs. 39.7 mo., P= 0.0001), suggesting that in cases evolving to AML, expanding hits might have dramatically skewed the favorable nature of SF3B1 mutations. Indeed, mutations preceding SF3B1 mainly affected lineage-restricted genes associated with repression of erythroid programs (RUNX1, 23%), terminal monocytic differentiation (TET2, 9%), transcriptional corepressors (BCOR/L1, 8%) and development of leukemia (DNMT3A, 8%). In conclusion, our study of the clonal architecture of SF3B1 mutations highlights that clonal progression of cases with MN harboring SF3B1 mutations might be inferred by the rank of additional genetic lesions cooperating with SF3B1. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Advani:Abbvie: Research Funding; Macrogenics: Research Funding; Pfizer: Honoraria, Research Funding; Amgen: Research Funding; Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy. Nazha:Tolero, Karyopharma: Honoraria; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Abbvie: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Abstract: Different CSF3R mutations (CSF3RMT) result in aberrant G-CSF signaling pathways and are linked to a wide range of myeloid disorders. Loss-of-function mutations in its extracellular domain cause severe congenital neutropenia (SCN). Activating mutations in the juxtamembrane region have been associated with a variety of myeloid malignancies. Truncating mutations in the cytoplasmic domain are associated with SCN cases that progress to MDS or AML. In this study, we evaluate the extent to which different CSF3RMT associate with disease onset, progression to leukemia and neutrophil counts in patients (pts) diagnosed with myeloid malignancies. We identified CSF3RMT cases in a cohort of 1400 pts [median age 71 years (yrs)]. We analyzed somatic and germline mutational patterns, and cross-sectional correlation with other gene mutations in CSF3RMT. A stringent algorithm based on conserved amino acid residues and alterations of protein features was used to predict the pathogenic significance of CSF3RMT. We identified 44 CSF3RMT: 33 germline (CSF3RGL) and 11 somatic (CSF3RS) variants. Most CSF3RGL were found in pts (median age 63 yrs) with MDS or related conditions (87% of all mutant cases), conversely these mutations were present in 5% (n= 22/424) of MDS, 3% (n= 7/244) MDS/MPN and 〈 1% (n= 3/392) of AML and in 1 out of 3 pts with aCML tested. Mutations were mostly missense and located between the cytoplasmic (58%: M696T, R698C (isoform III), D732N, P733T, S744F, Y752*, E808K), and extracellular (42%: C131Y, E149Q, A208V, Q216H, D320N, E405K, S413L, Y562H) domains. No mutations were detected in the juxtamembrane domain. Variants were grouped in Tier-1 (61%: C131Y, E149Q, A208V, Q216H, D320N, E405K, S413L, Y562H Y752*, E808K) and Tier-2 (variants with uncertain significance, 39%: S413L, M696T, R689C, D732N, P733T, S744F). E808K and R698C were the most common amino acid changes in Tier-1 (53%) and Tier-2 (44%), respectively. A total of 4/7 pts with E808K progressed to AML (but none with R698C), supporting previous observations that E808K (or E785K) represents a pathogenic variant predisposing to leukemia. A total of 46% (n=14) of pts with CSF3RGL had neutropenia [median 0.9x109/L (0.02-1.22x109/L)] at the time of sampling. Two pts diagnosed with a prior cancer manifested sustained neutropenia before the diagnosis of MDS and MDS/MPN. G-CSF was administered in 21% of pts. Alterations in -7/7q- were common (21%). Some pts also harbored other somatic mutations in NF1 (15%), DNMT3A (12%), SETBP1 (12%), or U2AF1 (12%). Of note, 1 patient carried mutations in WAS and GATA2 and another carried a mutation in VPS45, which have been previously associated with SCN/MDS. The patient with aCML harbored also a CSF3RS (T615A). Overall combined allelic burden in pts cohort was 2% vs. 1.6% expected allelic burden in control populations for the same variants (P=.02). CSF3R S were found in 11 pts (median age 71 yrs) with AML or MDS related conditions (73% of all mutant cases), conversely these mutations were present in 1.4% (n= 6/424) of AML, 〈 1% in MDS (n= 2/244) and MDS/MPN (n= 1/392) and in 2/3 pts with aCML tested. Mutations were missense in 63% of pts, T618I being most recurrent (n=5/11; 45%). Frameshifts accounted for 36% of the mutations and were localized in the cytoplasmic domain (Q741*, Q749*, Y752*, Q768*). All mutations were heterozygous. At the time of sampling 3/11 pts had leukocytosis and 3/11 had neutropenia. Mutations were distributed between the juxtamembrane domain (55%) and the cytoplasmic domain (45%). Mutations in the extracellular domain were not detected. Pts with sAML mostly carried mutations in the juxtamembrane domain (67%), those with MDS carried only in cytoplasmic domain, and those with MDS/MPN or aCML carried mutations in both the juxtamembrane and extracellular domains. There was one somatic and one RUNX1GL mutation. The cytogenetic abnormalities -7/7q- were detected in 18% (2/11) of cases. Interestingly, T618I was found solely in pts with sAML. Focusing on associations between CSF3RMT and mutations in the class III receptor tyrosine kinases CSF1R, FLT3, and KIT we identified only FLT3 to be co-mutated with CSF3RMT. All 3 pts (2 CSF3RGL and 1 CSF3RS) with such co-mutations evolved to AML. In sum, we found that CSF3RGL do not commonly co-occur with CSF3RS, suggesting that the neutropenia observed at the sampling time most likely is causative of undetected GL variants and/or is representative of a long unrecognized disease. Disclosures Nazha: MEI: Consultancy. Carraway:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau; FibroGen: Consultancy. Santini:Otsuka: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract: CMML is heterogeneous clinically (a varying degree of dysplastic or proliferative clinical features) and in terms of its molecular pathogenesis. Analysis of the spectrum of genomic lesions in CMML may contribute to understanding of the pathogenesis and help identify certain mutations as diagnostic biomarkers. Apart of the mere presence of a somatic lesion, phenotypic features may be shaped by initial hits. Conversely sub-clonal events may determine the phenotype or progression. Finally, initial hits may predetermine e.g., mutator phenotype, or differentiation block and therefore selecting for specific sub-clonal hits. We have selected a large CMML cohort to establish generalizable pathogenetic and clinical associations to account for molecular heterogeneity and serial samples have to be analyzed to assess clonal dynamics and hierarchy. The study group consisted of 242 patients, including 150 CMML cases (96 CMML-1, 27 CMML-2, 27 post CMML sAML) and JMML (N =92); 15 patients were studied serially. We also used comparison cohorts of M4/M5 AML (N =64) and advanced (N= 231) and low risk MDS (N= 199) serving as risk adjusted match for CMML subtypes. The CMML entity was further sub-classified based on clinical parameters and pathomorphologic features, 57% dysplastic (MD-CMML) and 43% proliferative form (MP-CMML). Analysis was performed using WES (paired germ line/tumor samples) and multiamplicon NGS targeting top 60 most commonly affected genes. For clonal architecture analysis, cross-sectional variant allelic frequency (VAF) concept based analysis was performed including assessment of affected genes by ranking of the corresponding clonal burden rather than the absolute cellular frequency. The results of this analysis were confirmed in serial samples to identify expanding, declining and stable subclones. Comparison of mutational spectra between the disease entity show profound differences in morphologically similar entities as particularly evident in comparison of CMML to JMML (TET2, ASXL1) or to lesser degree low risk MDS and CMML1 while progression in advanced cases was often associated with similar spectrum of additional subclonal events. However, the differences were more striking when clonal hierarchy was assessed to identify dominant/codominant and subclonal mutational events. We found that top 4 dominant/codominant clonal events, included TET2 (56%), SRSF2 (42%), ASXL1 (46%), DNMT3A (45% of patients), while in MDS corresponding frequency of these dominant events was TET2 (15%) SRSF2 (8%), ASXL1 (11%) and DNMT3A (8%), with most common ancestral events ranked SF3B1, TET2, ASXL1 etc. The clinical importance of these dominant events in CMML is highlighted by their impact on survival in KM analysis (p=.018). Our analysis also demonstrated that for certain founding events not pathognomonic for CMML either codominant or subsequent subclonal events determine the phenotypic features (1st generation) or progression (2nd generation). For instance initial TET2 in CMML was followed often by SRSF2 or in conjunction, RAS pathway mutations while MDS was driven by TET2, SF3B1, TP53, and many other events. Progression in our cohort was driven in both CMML and MDS by ASXL1, RUNX1, NPM1. When other common mutations were categorized by their role in individual patients 27% EZH2, 20% of CBL and 22% of SETBP1 were dominant. Serial analysis further qualified the cross-sectional analysis and allowed for categorization of subclonal events. For instance, CMML-1 cases initially presented with dominant TET2 followed by subtype specific subclonal SRSF2 and progression event IDH2 progressed to sAML with new NPM1 acquisition and expansion of IDH2 c lone. In our serial sample analysis we observed that increasing ASXL1 and RUNX1 clones correspond to clinical progression, ancestral events may remain stable (TET2, SRSF2, SETBP1), while non-permissive subclones can smolder or even decline. In sum, deep NGS allows for identification of specific ancestral events, which may determine the subsequent secondary mutational events in CMML. Classification of CMML based on ancestral events and subclonal events rather than on the global mutational spectrum correlates with clinical features and prognosis and may contribute to further clinical resolution of CMML based on the presence of specific founder mutations ultimately help establish therapeutic interventions. Disclosures Sekeres: Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.

Abstract: Background The Revised International Prognostic Scoring System (IPSS-R) was developed to risk stratify untreated patients (pts) with myelodysplastic syndromes (MDS). It has since been validated in pts treated with a single line of therapy; however, these approaches do not reflect typical MDS pts who receive different types of treatment in different sequences. Recently, genome sequencing technologies have identified recurrent somatic mutations that impact survival in MDS. We propose and validate a modification to the IPSS-R to include mutational data that can improve its predictive power at diagnosis regardless of the initial or subsequent therapies and at any time during the disease course. Methods Clinical and mutational data from MDS pts diagnosed between 1/2000-1/2012 were analyzed. A panel of 62 gene mutations obtained by next generation targeted deep sequencing which has been described as recurrent mutations in myeloid malignancies was included. Pts who underwent hematopoietic cell transplant (HCT) were censored at the time of transplant. A Cox proportional multivariate analysis including age, IPSS-R score and mutations that are present in 〉 10 pts was used to select independent prognostic factors. The fit of the proposed model to the data was assessed by using the concordance (c-) index. Results A total of 508 pts were included and divided into two cohorts, training (333 pts, used to build the new model), and validation (175 pts, used to validate it). Median age of the training cohort was 68 years (range, 20-87); 214 pts (64%) had de novo MDS, 39 (12%) had prior antecedental hematologic disorders, 37 (11%) secondary MDS, and 43 (13%) had chronic myelomonocytic leukemia. Pts received 0-7 lines of therapy: 15% did not receive any treatment, 85% received at least one treatment, 40% received 〉 2 treatments, 20% received 〉 3 treatments and 14% underwent HCT. First line therapies included: growth factors (30%), azacitidine+/- combinations (32%), decitabine+/- combinations (7%), lenalidomide (5%), investigational agents (5%), chemotherapy (2%), and immunosuppressive therapy (4%). Per IPSS-R, median OS for very low, low, intermediate, high, and very high was 35.5, 31.8, 19.1, 17.9, and 6.9 months (m), respectively, Figure1A. To minimize bias in pt selection, the validation cohort samples were randomly selected and sequenced after the development of the new model. Among the 62 gene mutations, 24 were present in 〉 10 pts in the training cohort: TET2 (17%), ASXL1 (15%), SF3B1 (14%), STAG2 (11%), DNMT3A (11%), RUNX1 (10%), U2AF1 (9%), GPR98 (8%), ZRSR2 (7%), BCOR (6%), TP53 (5%), NF1 (5%), EZH2 (5%), APC (5%), SUZ12 (5%), CBL (4%), PRPF8 (4%), NRAS (3%), CUX1 (3%), DDX54 (3%), IDH1 (3%), KDM6A (3%), PHF6 (3%), and SETBP1 (3%). A cox proportional hazard analysis including age, IPSS-R score, and the 24 genes mutations listed above identified the following as independent prognostic factors: age, IPSS-R, EZH2, SF3B1, and TP53. The linear predictive Cox model score obtained using the fitted coefficients of each prognostic factor wasage x.04 + IPSS-R score x.3 + EZH2 x.7 + SF3B1 x.5 + TP53 x 1 which translated to 4 prognostic groups: low, intermediate-1, intermediate-2, and high with median OS of 37.4, 23.2, 19.9, and 12.2 m, respectively, p 〈 .001, Figure1B with significant improvement in the C-index of the new model (.74) observed compared to the IPSS-R (.57). The model was then applied to the validation cohort with significant ability to distinguish prognostic groups for OS (p 〈 .0001) (Figure1C) despite differences between training and validation cohorts in IPSS-R risk categories (p =.04) and treatment history. To validate whether the new model can be applied at any time during disease course, we sequenced paired samples from 53 MDS pts at different time points (diagnosis, after treatment failure, and at the time of AML progression). The median time from diagnosis to sample 1 was 5.6 m (range, 0-56) and to sample 2 was 18.2 m (range, 5-94.6). The new model was able to predict the OS at each time point (Figure 1D shows IPSS-Rm at sample1 and 1E at sample2). Conclusion We propose a modification of the IPSS-R scoring system that incorporates mutational data and enhances its predictive ability in pts with MDS regardless of initial or subsequent treatments. This model is dynamic and valid at varying time points of a pt's disease course. Figure 1. OS by IPSS-R and IPSS-Rm in training, validation, and paired samples cohorts Figure 1. OS by IPSS-R and IPSS-Rm in training, validation, and paired samples cohorts Disclosures Sekeres: TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Abstract: Background: Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2MT) occur in up to 20% of MDS, MDS/MPN overlap and (s)AML. These mutations induce a metabolic rewiring of cancer cells that may impose metabolic vulnerabilities which can be pharmacologically targeted. In carcinoma cells, IDH1MT sensitize cells to oxidative stress induced by irradiation and treatment with electron transport chain complex I inhibitors, such as phenformin and metformin. Conversely, IDH1MT inhibitors protect IDH1MT cells against these treatment modalities. As a consequence, IDH1MT inhibitors and cytotoxic drugs should not be used together in patients with IDH1MT carcinoma and this has clinical relevance since IDH1/2MT inhibitors are already in phase I clinical trials. The underlying mechanism is that IDH1MT dramatically reduce the cellular production capacity of NADPH, an important antioxidant, and IDH1MT inhibitors restore the NADPH production capacity. Of note, IDH1 and IDH2 are the most important providers of NADPH in most cells. Methods: Primary IDH1/2MT, IDH1/2WT and TET2MT AML cells (n=15) were cultured and treated with the IDH1MT inhibitor AGI-5198, the IDH2MT inhibitor AGI-6780, irradiation, phenformin, metformin, daunorubicin or cytarabine. Cell numbers were counted 5 days after therapy exposure. In addition, cells were adhered to microscopy slides and maximal IDH1/2 and G6PD activity was determined using quantitative enzyme cytochemistry. ROS levels were fluorometrically measured using flow cytometry. Results: We observed that IDH1/2MT do not sensitize primary AML cells to irradiation, phenformin, metformin, daunorubicin or cytarabine and IDH1/2MT inhibitors had no effect on therapy sensitivity in primary AML cells. Because it is argued that inactivating TET2MT function downstream of IDH1/2MT, TET2MT AML cells served as a negative control and showed no sensitization neither.Although IDH1/2MT greatly reduce the IDH1/2-mediated NADPH production capacity in AML cells (P & lt; 0.0001), IDH1/2 are not important NADPH providers in myeloid cells and the contribution of glucose-6-phosphate dehydrogenase (G6PD) to the total NADPH production pool is much larger (maximal G6PD activity is ~4 times higher than maximal IDH1/2 activity, Fig. 1). This contrasts the situation in glioblastoma, where IDH1/2 provide 65% of all NADPH. Large-scale mRNA-sequencing in AML and glio(blasto)ma datasets revealed no differences in IDH1/2 and G6PD expression between types of cancer, suggesting that post-translational modifications perform key roles in regulating the maximal activity of these enzymes (Fig. 2). To investigate the implications of these results on cellular redox potentials, we performed ROS measurements after treatment with irradiation and observed that IDH1/2MT do not increase ROS in AML cells, whereas IDH1/2MT do increase ROS in glioma cells. Discussion: Increased sensitivity to oxidative stress is not a metabolic vulnerability in IDH1/2MT AML and this is likely due to the fact that maximal NADPH production capacity is affected to only a small extent in IDH1/2MT AML. Therapies that increase oxidative stress, such as irradiation and metformin, counteract with IDH1/2MT inhibitors in glio(blasto)ma cells because there, IDH1/2MT have profound implications on cellular NADPH production capacities and ROS levels. Our results suggest that IDH1/2MT do not induce sensitivity to cytotoxic drugs in AML cells and thatIDH1/2MT inhibitors can be safely used adjuvantly to the AML standard of care daunorubicin and cytarabine, or whole-body irradiation in the context of bone marrow transplantations. These data are crucial for phase II and III clinical trial designs ofIDH1/2MT inhibitors. G6PD (a, c, e, g) and IDH1 activity (b, d, f, h) staining of IDH1R132 non-mutated (a, b, e, f) and mutated (c, d, g, h) primary AML cells (a-d) and glioblastoma (e-h) cryostat sections. The amount of blue color (nitro BT-formazan) directly reflects IDH1/2 and G6PD activity (production of NADPH). Figure 1. IDH1MT reduce maximal IDH1/2 activity, but IDH1/2 is a more important NADPH producer in glioblastoma than in AML compared with G6PD. Figure 1. IDH1MT reduce maximal IDH1/2 activity, but IDH1/2 is a more important NADPH producer in glioblastoma than in AML compared with G6PD. Figure 2. mRNA sequencing reveals no differences in IDH1, IDH2 and G6PD expression between AML, low-grade glioma (LGG) and glioblastoma (GBM). Figure 2. mRNA sequencing reveals no differences in IDH1, IDH2 and G6PD expression between AML, low-grade glioma (LGG) and glioblastoma (GBM). Disclosures No relevant conflicts of interest to declare.