Gangat, Naseema

Introduction: Whereas morphological assessment and cytogenetics have been the cornerstone for the diagnosis of myelodysplastic syndrome (MDS), flow cytometry and mutational analyses are novel, evolving techniques. These diagnostic procedures play a role in identifying pre-MDS cases including idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS). Aim: To assess flow cytometric findings in ICUS and CCUS and their correlation with progression to MDS or myeloid neoplasm (MN), concurrent genetic aberrations, and underlying pathological evolution. Methods: Patients (Pts) who had undergone evaluation to rule out MDS and had retrospectively undergone flow cytometry, cytogenetic, mutational and morphological assessment were included. Flow cytometry results were classified as normal (no abnormality or 1 equivocal result), abnormal (2 abnormalities or at least 1 abnormality and 1 equivocal result), or atypical (1 abnormality or 2-3 equivocal results). Next generation sequencing included mutation analysis of: ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, TERT, TET2, TP53, U2AF1, WT1, ZRSR2. Results: A) Characteristics: 229 pts were assessed (median age 60 and 69% were males). Based on pathologist interpretation, 87 (38%) pts met diagnostic criteria of MDS, 106 (46%) pts had normal/reactive bone marrow (BM), and 36 (16%) pts had BM findings suspicious for MDS. Median time to follow-up was 24 months for MDS pts, and 32 months for pts with cytopenias. B) Morphology: Pts who had a BM biopsy that was suspicious for MDS by pathologist interpretation were more likely to develop MDS compared to pts with normal/reactive pathology report with an incidence rate (IR) of 16% versus 0.9% (p= .0005). Pts who were suspicious for MDS based on the BM biopsy were more likely to have underlying mutations compared to pts with normal/reactive BM (IR 83% versus 69%, p= .2). C) Flow cytometry: In the non-MDS cohort, 96 (67%) pts had normal flow results, 29 (20%) pts had atypical flow, and 17 (12%) pts had abnormal flow results compared to 16 (18%) pts, 18 (21%) pts, and 53 (61%) pts in the MDS cohort, respectively. Of the pts who had normal flow result (n=112), 16 were MDS, 16 were suspicious for MDS/MN, and 80 were normal/reactive. Pts who had biopsy interpretation suspicious for MDS/MN were more likely to have abnormal flow cytometry compared to normal/reactive BM biopsy (IR 55% versus 24%, p=.0003). IR of MDS was 4% in normal flow, 3% in atypical flow, and 12% in abnormal flow (p= .5). Likewise, IR of MN was 4% in normal flow, 20% in atypical flow, and 30% in abnormal flow (p= .1). D) Genetics (cytogenetics and mutation profiling): Cytogenetics was normal in 123 (86%) pts and abnormal in 19 (13%) pts. Pts who had abnormal cytogenetics were more likely to develop MN (IR 57% versus 5%, p= .002). Out of the 50 non-MDS pts tested for NGS, 38 (76%) were positive for mutations. 15 (39%) pts had 1 mutation, 8 (21%) pts had 2, 3 (8%) pts had 3, 6 (16%) pts had 4, 2 (5%) pts had 5, 1 (3%) pt had 7, and 1 (3%) pt had 8 mutations (61% had >1 mutation). Pts with positive mutations were more likely to develop MDS during follow-up compared to pts who did not have mutations (IR 16% versus 0%, p=.06). Additionally, flow cytometric aberrations significantly correlate with the number of mutations (p= .03). MN correlated significantly with BCOR mutation and RUNX1 (IR 60% (p= .008) and 50% (p=.02), respectively. E) Interaction between variables: In pts with positive mutations, flow cytometry did not correlate with progression to MDS with an IR of 17% of MDS in normal flow, 11% in atypical flow, and 17% in abnormal flow, (p= .9). There was no impact on overall survival neither in pts with abnormal flow results vs normal nor in pts with positive mutations vs wild type. Conclusion: Conventional diagnostic techniques remain the cornerstone of MDS diagnosis with flow cytometry and NGS arising as novel techniques that correlate significantly with progression of pre-MDS pts to MDS. Flow had an impact on pathology final interpretation but did not add value in pt with a positive mutation analysis. Larger studies are needed to confirm our findings. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: 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 Background: Current prognostication in primary myelofibrosis (PMF) utilizes MIPSS70 (mutation-enhanced international prognostic scoring system for transplant-age patients), MIPSS70+ version 2.0 (karyotype-enhanced MIPSS70) and GIPSS (genetically-inspired prognostic scoring system, which is based on mutations and karyotype) (JCO 2018;36:310; JCO doi: 10.1200/JCO.2018.78.9867; Leukemia. 2018;doi:10.1038/s41375-018-0107). These contemporary models are inter-complimentary and highly efficient in predicting overall survival. However, specific risk factors for very long survival (e.g. 20+ years) and short-term mortality (i.e. death within 5 years of diagnosis) have not been systematically looked into. Methods: Study patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses were according to the 2016 World Health Organization criteria (Blood. 2016;127:2391). Logistic regression statistics was used to identify predictors of 20-year survival and 5-year mortality. Variables evaluated in the logistic model included those that are currently listed in MIPSS70, MIPSS70+ version 2.0 and GIPSS, as well as age (≤70 vs >70 years) and sex. Receiver operating characteristic (ROC) curves and area under the curve (AUC) were used to estimate predictive accuracy. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations. Results: A total of 1,282 consecutive patients with PMF (median age 65 years, range 19-92; 63% males) were used to identify 26 (2%) patients (median age 51 years, range 28-71; 38% males) who survived their disease for at least 20 years (very long-lived patients) and 626 (49%) patients (median age 68 years, range 27-92; 66% males) who died within 5 years of their diagnosis. Clinically-derived information, including the clinical variables used in MIPSS70 and MIPSS70+ version 2.0, with the exception of fibrosis grade (information available in 60%), was available in 99% of the study population; cytogenetic information was available in 94%, driver mutational status in 71% and the full profile of high molecular risk (HMR) mutations, including ASXL1, SRSF2, U2AF1 Q157, EZH2, IDH1 and IDH2, in 42%. MIPSS70+ version 2.0 risk distribution for the entire study population (n=1,282) was 4% very low risk, 16% low risk, 19% intermediate risk, 40% high risk and 21% very high risk; the corresponding percentages for the 26 very long-lived patients were 14%, 29%, 14%, 43% and 0% and for the 626 short-term survivors were 0%, 4%, 10%, 47% and 39% (p<0.001). Cytogenetic, driver mutation and HMR mutation information was available in 100%, 58% and 27% of the 26 long-term survivors, respectively, and 98%, 63% and 39% of the 626 short-term survivors. Multivariable logistic regression analysis of 20-year survival identified the following seven variables as being favorable: age ≤70 years (p=0.002); female sex (p=0.03); hemoglobin level ≥10 g/dl for women and ≥11 g/dl for men (p=0.03), leukocyte count ≤25 x 109/l (p=0.009), platelet count ≥100 x 109/l (p=0.002), circulating blasts ≤1% (p=0.03) and absence of constitutional symptoms (p=0.04). The particular phenotypic profile exhibited a high degree of predictive accuracy with an estimated AUC of 0.90 (Figure 1a); karyotype and mutations did not retain significance in this particular analysis; in fact, 5 (71%) of the 7 long-term survivors who were informative harbored at least one HMR mutation. A similar analysis for 5-year mortality identified the following risk factors: high molecular risk mutations (p<0.001); unfavorable or very high risk karyotype (p<0.001); absence of type 1/like CALR mutations (p<0.001); age >70 years (p<0.001); constitutional symptoms (p<0.001); hemoglobin level <10 g/dl for women and <11 g/dl for men (p<0.001); leukocyte count >25 x 109/l (p=0.004); and circulating blasts >1% (p=0.001); predictive accuracy was estimated at AUC 0.87 (Figure 1b). Conclusions: The phenotypic profile of long-lived patients in PMF includes young age (≤70 years old), female sex, asymptomatic disease (i.e. no constitutional symptoms) and absence of moderate to severe anemia, thrombocytopenia or leukocytosis and ≤1% circulating blasts; each one of these parameters, with the exception of female sex, were present in ≥80% of long-term survivors. Karyotype and mutations were more relevant in predicting short-term mortality. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Chronic myelogenous leukemia (CML) is a BCR-ABL1 driven myeloid neoplasm, with excellent response rates to tyrosine kinase inhibitors (TKI). TKI such as imatinib (IM), dasatinib (DAS), nilotinib (NIL), bosutinib (BOS), and ponatinib (PON) currently US FDA approved, have dramatically changed survival outcomes. That being said, two main challenges exist; suboptimal responses and TKI intolerance. The reason why some patients tolerate/respond to TKI better than others is unknown, but is potentially explainable based on drug metabolism. The field of pharmacogenomics (PGX) is rapidly evolving, with commercial panel's rapidly assessing metabolic pathways such as cytochrome P450 (CYP), UGTA1A, p-glycoprotein/ABCB1. We carried out this study using a 22 gene-PGX panel to assess potential causes of TKI related intolerance and suboptimal responses in compliant patients. Methods: Twenty-nine Mayo Clinic patients with CML were prospectively recruited after informed consent. Buccal swabs were utilized for PGX testing to assess variations/polymorphisms involving CYP3A4/5, 1A2, 2C9, UGT1A1, amongst others (www.Oneome.com). Three groups of patients were recruited, those with i) TKI intolerance, ii) suboptimal responses by ELN criteria, iii) and newly diagnosed cases, to assess if PGX testing could explain TKI intolerance or resistance, or alter choice of therapy in newly diagnosed cases. A pharmacology review was obtained to interpret reports in all cases. Results: 29 patients with CML were prospectively enrolled, median age 64 years, 15 (52%) male. At last follow up (median 21 months), 3 (10%) deaths and no disease progressions were documented. 82% received first line IM, whereas 18% received first line DAS. Major metabolic pathways assessed for IM included, CYP3A4 and 5, while minor pathways included 2C19, 2C9 and 2D6. Majority (83%) of IM patients had normal major pathways with 17% being intermediate-normal. With regards to the minor pathways, 25% were rapid metabolizers for 2C19, whereas 29% were intermediate and 4% poor metabolizers for 2C9 and 50% and 16% were intermediate and poor metabolizers for 2D6. In all IM treated patients PGX testing did not completely explain intolerance or resistance and did not change clinical decision making. Front line DAS was used in 18% and the major pathway assessed was CYP 3A4, with 80% being normal and 20% being intermediate metabolizers. Once again PGX testing did not completely explain intolerance/resistance and no therapeutic changes were based on PGX analysis. Seventeen (59%) patients received second line TKI therapy (50% each for intolerance and resistance), with DAS (58%) and NIL (29%), being the two most common. Once again for DAS, 90% had normal 3A4 metabolism while 10% were intermediate and for NIL 80% were normal and 20% intermediate. Only one of 2 NIL treated patients with UGTA1A polymorphisms developed indirect hyperbilirubinemia (grade 1). In the second line setting, PGX testing did not completely explain or alter treatment decisions. BOS was used as a third line agent in 3 (>10%) patients, 2 who remained refractory and one with intolerance, and all three were normal CYP3A4 metabolizers. None of these patients developed diarrhea a common side effect of BOS. PON was used in one patient with intermediate CYP3A4 metabolism as a 5th line agent and the patient continues to have refractory disease with no thrombotic events. In all cases, no changes were made to concomitant medications based on PGX testing. Conclusion: In conclusion, while PGX testing has rapidly evolved and become commercially available, in the context of TKI therapy for CML, while it offers useful insights on minor metabolic derangements and side effects like NIL induced hyperbilirubinemia, in our study, currently limited by a small sample size, it did not completely explain TKI intolerance or resistance. Recruitment of additional patients (n=100) and correlations with plasma drug levels are currently ongoing. Table. Table. Disclosures Al-Kali: Novartis: Research Funding. Stewart:Amgen Inc., Celgene, Roche, Seattle Genetics: Research Funding; Amgen Inc., BMS, Celgene, Takeda, Roche, Seattle Genetics, Janssen, Ono: Consultancy.

Abstract Background: Anagrelide clinical trials in myeloproliferative neoplasms (MPN) were pioneered by the late Murray N. Silverstein M.D.(1928-1998) of the Mayo Clinic and the first publication involving 20 patients, including 17 with essential thrombocythemia (ET), appeared in 1988 (study launched in October 1985) (N Engl J Med 1988;318:1292). A larger study involving 577 patients, including 335 with ET, was subsequently published in 1992 and led to FDA approval in March 1997 (Am J Med 1992;92:69). Anagrelide has also been evaluated, in controlled studies, for its efficacy and safety as first-line therapy for ET; the results of these studies suggested that anagrelide was not inferior to hydroxyurea, in one study (Blood 2013;121:1720), but might have been harmful to patients in, the second study (N Engl J Med 2005;353:33); in the latter study, patients receiving anagrelide experienced higher incidences of arterial thrombosis, bleeding complications and fibrotic progression. These observations raised significant concern regarding an adverse survival impact of anagrelide in ET; unfortunately, the follow-up period of formal controlled studies were too short to accurately address the particular concern. Methods: The current study represents a retrospective examination of the potential impact of anagrelide therapy on survival and disease complication rates in ET. The study population was recruited from a consecutive cohort of adult patients (age ≥18 years) with MPN seen at the Mayo Clinic from 10/27/1967 through 12/29/2017. Diagnosis and determinations of fibrotic and/or leukemic transformations, were in strict accordance with the 2016 World Health Organization criteria (Blood 2016;127:2391). Statistical analyses were based on clinical and laboratory parameters obtained at the time of first referral to the Mayo Clinic which, in the majority of cases, coincided with initial diagnosis. All patients were followed from diagnosis until death or date of last follow-up or contact. Follow-up was until April 2018 and data collected via medical records or in certain cases, by directly contacting patients or their physicians. Survival and time-to-event curves were prepared using the Kaplan-Meier method and compared by the log-rank test. The JMP® Pro 13.0.0 software package was used for all analyses (SAS Institute, Cary, NC, USA). Results: 1,076 patients with ET were considered (median age 58 years; females 63%); international prognostic scoring system for ET (IPSET; Blood 2012;120:1197) risk distribution was 28% high, 42% intermediate and 30% low; driver mutational status was JAK2 61%, CALR 25%, triple-negative 11% and MPL 3%. Comparison of cases diagnosed before (n=433) and after (n=643) the FDA approval date of anagrelide (1997) revealed the former cohort to be younger (median 57 vs 60 years; p=0.003), although the difference in IPSET risk distribution was not significant (p=0.14). Figures 1a, 1b, 1c and 1d illustrate overall (OS), myelofibrosis-free (MFFS), leukemia-free (LFS) and thrombosis-free survival data comparing ET patients diagnosed before and after the 1997 FDA approval date for anagrelide; a significant difference was apparent for OS (p=0.006; HR 1.4, 95% CI 1.1-1.7) and MFFS (p<0.001; HR 4.2, 95% CI 2.7-6.5), in favor of patients diagnosed prior to 1997; during multivariable analysis that included IPSET and sex, the significant difference in both OS and MFFS were sustained; the difference in OS was most apparent after the first decade of diagnosis with 10- and 20-year survival rates of 75% and 50% for diagnosis prior to 1997 vs 72% and 21% for diagnosis between 1997 and 2017 (figure 1a); the 10- and 20-year risk of fibrotic progression was 3% and 15% for diagnosis prior to 1997 and 14% and 46% for diagnosis after 1997 (figure 1b); there was no impact on LFS (p=0.32; Figure 1c) or TFS (p=0.18; Figure 1d). Similarly stratified survival data in polycythemia vera (n=665) and primary myelofibrosis (n=1,282) showed no impact on survival (p=0.3 and 0.17, respectively). Conclusions: In a retrospective, but otherwise unbiased comparative analysis, we show significantly decreased OS and MFFS in ET patients diagnosed after the FDA approval date of anagrelide (1997); the increased risk of fibrotic progression noted is consistent with results of earlier prospective data (N Engl J Med 2005;353:33). Our observations regarding OS requires confirmation in a prospective controlled setting. Disclosures No relevant conflicts of interest to declare.

Abstract Background : Cytogenetic abnormalities occur in approximately a third of patients with primary myelofibrosis (PMF) and 5-20% of those with essential thrombocythemia (ET) or polycythemia vera (PV). Abnormal karyotype in PV and specific cytogenetic abnormalities in PMF have been shown to adversely affect survival (Leukemia. 2018;32:1189; Br J Haematol. 2017 Jun 9. doi: 10.1111/bjh.14798). In the current retrospective study, we considered 650 Mayo Clinic patients with myeloproliferative neoplasms (MPN), including ET, PV and PMF, with at least two documented bone marrow (BM) biopsies, in order to examine the incidence and pattern of changes in karyotype and their clinical correlates. Methods : Study patients were recruited from the Mayo Clinic, Rochester, MN, USA, based on documentation of at least two serial BM biopsies. Diagnoses were according to the 2016 World Health Organization criteria (Blood. 2016;127:2391). BM biopsy #1 referred to the baseline karyotype at time of initial diagnosis/referral and BM biopsy #2 to the first repeat BM biopsy after diagnosis. In addition to documenting the presence or absence of changes in karyotype, a notation was made regarding disease phase, at the time of the repeat biopsy, in order to allow accurate interpretation of the data and evaluation of survival impact. Results: 650 patients with MPN, including 153 ET, 105 PV and 392 PMF, were included in the current study and had undergone at least two BM biopsies; 227 patients had three, 108 four, 48 five, 19 six, and 4 seven repeat BM biopsies during their clinical course. Cytogenetic clonal evolution in patients with normal karyotype at baseline : Baseline karyotype was normal in 466 (72%) patients, including 139 (91%) ET, 91 (87%) PV and 236 (60%) PMF. Clonal evolution, which constituted a change from "normal" to "abnormal" karyotype, was documented in 16 (12%) patients with ET, 22 (24%) with PV and 75 (33%) with PMF; the latter included favorable karyotype in 11% of the cases, unfavorable in 12% and very high risk (VHR) in 10%. 11 (69%) of the 16 documented cases of clonal evolution in ET, 10 (45%) of 22 in PV and 11 (15%) of 75 in PMF were accompanied by clinically evident progression to blast or fibrotic phase disease, at the time of the repeat BM biopsy. Cytogenetic clonal evolution in patients with abnormal karyotype at baseline : Baseline karyotype was abnormal in 187 (28%) patients, including 17 (9%) ET, 14 (13%) PV and 156 (40%) PMF. Additional changes in karyotype, during serial BM biopsies, were documented in 6 (35%) ET, 5 (36%) PV and 71 (46%) PMF patients. In PMF, the pattern of changes in karyotype included "favorable" to "favorable" in 7%; "favorable" to "unfavorable" in 13%; "favorable" to "VHR" in 5%; "unfavorable" to "unfavorable" in 13%; "unfavorable" to "VHR" in 3%; and "VHR" to "VHR" in 6%. All 11 (100%) patients with ET or PV who underwent clonal evolution displayed clinically overt disease transformation into leukemic or fibrotic phase disease, at the time of the repeat BM biopsy. Leukemic transformation at the time of the repeat BM biopsy was evident in 13 (18%) of the 71 PMF cases with clonal evolution. Survival impact of cytogenetic clonal evolution without change in disease phase : The survival impact of cytogenetic clonal evolution was examined under the following provisions: i) analysis was limited to events occurring at BM biopsy #2 with survival calculated from the date of BM biopsy #2; ii) patients with leukemic or fibrotic transformation at time of BM biopsy #2 were excluded from analysis; and iii) clonal evolution for the purposes of survival analysis constituted changes from "normal" to "abnormal", for ET and PV, while the type of changes were taken under consideration for PMF. Under these stipulations, clonal evolution appeared to affect survival in general, although statistical significance was apparent for only ET and PMF (Figures 1a, 1b and 1c). Conclusions: Cytogenetic clonal evolution in ET and PV is infrequent, in the absence of clinically overt disease transformation, and is more likely to occur in PMF. Such events, when documented in chronic phase disease, appeared to predict shortened survival in all three MPNs, which was most apparent in ET and with emergence of VHR or unfavorable abnormalities in PMF. These observations warrant prospective studies that account for indication bias. Disclosures No relevant conflicts of interest to declare.

Abstract Background: The selective AURKA inhibitor alisertib (MLN8237) exhibits disease modifying activity in murine models of myelofibrosis by eradicating atypical megakaryocytes resulting in reduction of marrow fibrosis (Nat Med 2015). Here, we present long term follow-up results from the investigator initiated pilot study of alisertib in patients with myelofibrosis (clinical trials.gov Identifier NCT 02530619). Methods: 24 patients with DIPSS intermediate 1, intermediate-2, or high risk myelofibrosis who were in need of therapy, refractory/intolerant or unlikely to respond to JAK inhibitors with neutrophil count ≥ 1 x109/L, and platelet count ≥ 50 x109/L, received alisertib (provided by Millennium Pharmaceuticals Inc) at a dose of 50 mg twice daily for one week every 21 days. Toxicity assessment was performed by the standard common terminology criteria (Version 4.0). Response was assessed by the international working group for myelofibrosis research and treatment (IWGMRT) criteria. Correlative studies included assessments of JAK2V617F, CALR, and MPL mutant allele burden, degree of fibrosis and GATA1 expression in bone marrow samples obtained pre and post therapy. Results: We enrolled 17 patients with primary myelofibrosis, 4 with post essential thrombocythemia myelofibrosis and 3 with post polycythemia vera myelofibrosis. Median age was 72 years with 66% males. 79% of patients were DIPSS intermediate risk, and the remainder were high risk with 15 patients (62.5%) having received prior JAK inhibitor therapy. Driver mutational status was as follows; 58% JAK2V617F, 29% CALR, and 13% MPL mutated. At study entry, 54% of patients demonstrated palpable splenomegaly ≥ 5 cm below the left costal margin, 54% were transfusion dependent with all patients experiencing constitutional symptoms. At the time of data cut-off, patients received a median of 7.5 cycles (range; 1-29 cycles) of therapy. The 7 patients presently on study have received a median of 23 cycles (range; 8-29 cycles). Reasons for treatment discontinuation included progressive disease/lack of response in 11 (65%) patients, toxicity in 4 (24%) patients and refusal of further therapy in 2 (11%) patients.Safety and Efficacy assessments The most common treatment-emergent grade 3/4 adverse events included neutropenia (42%), thrombocytopenia (29%) and anemia (21%), with 4% each experiencing neutropenic fever, diarrhea, vertigo, elevated creatinine and elevated alanine aminotransferase. 22 patients were considered for response evaluation with 4 of 14 patients (29%) with palpable splenomegaly ≥ 5 cm achieving a spleen response, 1 of 13 patients (8%) becoming transfusion independent, and 5 of 22 patients (23%) experiencing symptom response with ≥ 50% reduction in the MPN-SAF total symptom score. However, when response assessment was restricted to 13 patients who had received a minimum of 5 cycles of therapy, spleen responses were observed in 4 of 7 (57%) patients, 1 of 5 (20%) achieved transfusion independence and 5 of 13 (38%) achieved symptom response. All patients presenting with leukocytosis (n=4) and thrombocytosis (n=2) had resolution with therapy. Of the 7 patients presently on study, four patients continue to demonstrate symptom response, two patients with both spleen and symptom response, and another patient with sustained anemia response. Correlative assessments We compared the intensity of staining of GATA1, a factor that is required for maturation, in sequential bone marrow biopsies from six patients at baseline and after a minimum of five cycles and observed a striking increase in the numbers of GATA1-positive megakaryocytes in five of six cases (Figure 1a). In addition, we observed a one grade reduction in marrow fibrosis in 4 of 6 paired samples (Figure 1b). This reduction in fibrosis was accompanied by sustained responses to the drug. Finally, we compared JAK2, MPL or CALR mutant allele burden in eight paired baseline and cycle 5 or 6 samples and observed decreases in 4 of 8 patients (Figure 1c). Conclusions: Alisertib is safe and well tolerated in patients with myelofibrosis with prolonged administration up to 1.7 years. In addition to providing clinical benefit, alisertib restored normal morphology and GATA1 expression in atypical megakaryocytes and reduced marrow fibrosis and mutant allele burdens. These findings demonstrate that AURKA inhibition should be further explored as a therapeutic option in myelofibrosis. Figure 1. Figure 1. Disclosures Swords: AbbVie: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding. Frankfurt:Celgene, Jazz, Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees. Altman:Cyclacel: Other: payment to the institution to conduct clinical trial work; Epizyme: Other: payment to the institution to conduct clinical trial work; Ariad: Other: payment to the institution to conduct clinical trial work; Bayer: Other: payment to the institution to conduct clinical trial work; Celator: Other: payment to the institution to conduct clinical trial work; FujiFilm: Other: payment to the institution to conduct clinical trial work; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: payment to the institution to conduct clinical trial work; Agios: Other: Payment to the institution to conduct the trial ; Astellas Pharma: Other; Genetech: Other: Payment to the institution to conduct clinical trial work; Syros: Membership on an entity's Board of Directors or advisory committees; Incyte: Other: payment to the institution to conduct clinical trial work; GSK: Other: payment to the institution to conduct clinical trial work; Immune Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Boeringer Ingelheim: Other: payment to the institution to conduct clinical trial work; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: payment to the institution to conduct clinical trial work. Rampal:Celgene: Honoraria; Stemline: Research Funding; Incyte: Honoraria, Research Funding; Constellation: Research Funding; Jazz: Consultancy, Honoraria. Giles:Actuate Therapeutics Inc: Employment, Equity Ownership. Crispino:Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.

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 : Survival prediction in essential thrombocythemia (ET) and polycythemia vera (PV) is currently based on clinically-derived risk variables, including age, thrombosis history and leukocyte count (Blood. 2012;120:1197; Leukemia. 2013;27:1874). We have previously reported on the prognostic contribution of mutations, in both ET and PV (Blood Adv. 2016;1:21). The current study examines the possibility of integrating genetic and clinical information for predicting overall (OS), leukemia-free (LFS) and myelofibrosis-free (MFFS) survival in ET and PV. Methods : Study patients were recruited from the Mayo Clinic, Rochester, MN, USA and the University of Florence, Florence, Italy, based on availability of next-generation sequencing (NGS)-derived mutation information. Diagnoses were according to the 2016 World Health Organization criteria (Blood. 2016;127:2391). NGS-detected coding region variants were filtered through the Exome Aggregation Consortium (ExAC) database and annotated by the Catalogue of Somatic Mutations in Cancer (COSMIC) database as mutants or variants of uncertain significance (VUS). Conventional statistics was employed for outcome analysis and assessment of model performance. Results: 906 molecularly-annotated patients (416 from Mayo and 490 from Florence), including 502 ET and 404 PV cases, were included in the current study. The Mayo/Florence cohorts included 270/232 ET (median age 57/54 years, 60%/59% females) and 146/258 PV (median age 63/58 years, 52%/44% females) patients; median follow-up was 9.9/12.9 years for ET and 10.7/12.4 years for PV; during this time 39%/38% deaths and 4.4%/6.5% leukemic and 16%/33% fibrotic progressions were documented for ET, with the corresponding figures for PV being 50%/26%, 4.8%/3% and 13%/36%. Cytogenetic information was available in 84%/63% ET and 80%/49% PV cases in the Mayo/Florence cohorts. In both Mayo and Florence cohorts, multivariable analysis of mutations identified primarily spliceosome mutations to adversely affect OS (SF3B1 and SRSF2 in ET and SRSF2 in PV) and MFFS (U2AF1 and SF3B1 in ET); in addition, TP53 mutations was associated with leukemic transformation in ET, in both patient cohorts. Other mutations with independently significant contribution, validated in one but not both cohorts, included EZH2 (OS and LFS in ET), IDH2 (OS and LFS in PV) and RUNX1 (LFS in PV and ET). For the purposes of the current study, mutations considered "adverse" for subsequent analysis and development of prognostic models required validation in both Mayo and Florence cohorts. Age-adjusted, all-inclusive multivariable analysis of genetic and clinical variables identified the following as independent risk factors for OS, in both Mayo and Florence cohorts: ET ─ SRSF2/SF3B1 mutations, age >60 years and male sex; PV ─ SRSF2 mutations, age >67 years and leukocyte count ≥11 x 109/l. In addition, multivariable analysis flagged leukocytosis in ET (Florence cohort only) and abnormal karyotype in PV (Mayo cohort only) as additional factors. Development of mutation-enhanced international prognostic systems for ET and PV In order to optimize the number of informative cases, the two Mayo and Florence databases were subsequently combined and subjected to multivariable analysis that confirmed the independent survival effect, in ET, of SRSF2/SF3B1 mutations (HR 2.8, 95% CI 1.8-4.3), age >60 years (HR 6.7, 95% CI 4.8-9.4) and male sex (HR 1.8, 95% CI 1.4-2.4) and, in PV, of SRSF2 mutations (HR 7.0, 95% CI 2.3-17.4), age >60 years (HR 5.7, 95% CI 3.3-10.1), and leukocyte count ≥11 x 109/l (HR 2.4, 95% CI 1.5-3.9); the combined analysis also flagged independent prognostic contribution from abnormal karyotype in PV (HR 2.1, 95% CI 1.1-3.6). HR-weighted risk point allocation resulted in new, mutation-enhanced 4-tiered risk models for ET (MIPSS-ET based on mutations, age and sex; figure 1a) and PV (MIPSS-PV based on mutations, karyotype, leukocyte count and age; figure 1b). Both models displayed high predictive accuracy and were internally validated by bootstrapping. The combined analysis also confirmed the impact of TP53 mutations on LFS (figure 1c) and U2AF1/SF3B1 mutations on MFFS (figure 1d), in ET. Conclusions: Spliceosome mutation information enhances survival prediction in ET and PV and identifies those at risk for fibrotic progression. TP53 mutations predict leukemic transformation in ET. Disclosures No relevant conflicts of interest to declare.