Pardanani, Animesh

Objective : Central nervous system (CNS) involvement is a known phenomenon of acute lymphoblastic leukemia. However little is known about the incidence of CNS involvement in acute myeloid leukemia (AML) as routine lumbar punctures are not done if patients are asymptomatic at diagnosis. Our practice has been to obtain lumbar puncture (LP) at or after complete remission (CR) for patients that present with any one of the following high risk features;hyperleukocytosis with WBC >10,000 or blastic crisis, extramedullary disease,monocytic differentiation. Methods: We reviewed our leukemia database at Mayo Clinic to determine the incidence of CNS involvement in patients with high risk features after due IRB approval. We also compared baseline demographic characteristics, blood counts, cytogenetic risk groups, presence of extramedullary disease, relapse rate ,transplant rate and overall survival (OS) among patients with and without CNS involvement. OS estimates were calculated by Kaplan-Meier curves and log-rank testing using JMP v.13. Results: 298 patients with AML were identified with presence of at least 1 risk factor for CNS involvement. The results of LP were only available for 126 (42%) patients. The overall incidence of CNS involvement was 12% (15/126, figure 1). Among the patients with positive disease 3 had CSF examination at diagnosis prior to CR due to the presence of CNS myeloid sarcoma (n=2) and ambiguous lineage myeloid leukemia and were excluded from further evaluation. The incidence of CSF involvement at CR1 was 9.7% (12/123). All patient with CNS positive disease received intrathecal chemotherapy with alternating cytarabine and methotrexate until they cleared their blast and additional 2-4 treatments as per treating physician's discretion .The CNS positive and negative patients were compared and did not show any statistical difference in age, gender, presence of extramedullary disease ,cytogenetic risk group, relapse rate and transplant rate as shown in table1. Molecular data was not available in most of the patients and hence was not included for analysis. The median OS was significantly lower among patients with CNS involvement compared to absence of CNS involvement but was not statistically significant likely due to small sample size (3.9 vs12.8 years (p=0.2), figure 2). The overall CNS relapse rate in patients who did not have a LP done at CR was 5.8%(10/172). Conclusions : CNS evaluation should be done in AML patients with high risk features before declaring CR. CNS disease portends overall a poor prognosis with impaired overall survival and high relapse rate. However the sample size was small and true incidence of CNS disease will need routine diagnostic LP in all high risk patients. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding.

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: 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 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.

Abstract Background: The World Health Organization (WHO) system lists five morphological categories of systemic mastocytosis (SM): indolent (ISM), smouldering (SSM), SM with an associated hematological neoplasm (SM-AHN), aggressive (ASM) and mast cell leukemia (MCL) (Blood. 2016;127:2391). Recent studies have highlighted the prognostic importance of mutations in SM, including ASXL1, RUNX1 and SRSF2 (AJH 2016;91:888;Leukemia 2016;30:2342). In the current study, we reviewed the cytogenetic findings in 348 cases of SM, in order to clarify incidence and prognostic impact of cytogenetic abnormalities, stratified by WHO morphologic subcategories. Methods : Study patients were selected, based on availability of cytogenetic information. Diagnoses of SM and its morphological subcategories were confirmed by both clinical and bone marrow examinations, in line with WHO criteria (Blood. 2016;127:2391). Next-generation sequencing was performed in a subset of the study population. Abnormal karyotype was further distinguished into favorable and unfavorable class based on previously published criteria for myelodysplastic syndromes (MDS) (Blood 2012;120:2454) and myeloproliferative neoplasms (MPN) (Leukemia 208;32:1189). Statistical analyses considered clinical and laboratory data collected at the time of initial diagnosis at the Mayo Clinic, which coincided with collection of bone marrow for cytogenetic studies. Conventional statistics was used for calculation of overall survival and determination of risk factors. JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations. Results: 348 SM patients were included in the current study (median age 59 years; range 18-88 years; 53% males); 142 (41%) constituted ISM and 206 (59%) advanced SM; the latter included 155 (45%) SM-AHN, 49 (14%) ASM and 2 MCL cases. The SM-AHN cases included 39 (11%) SM-MPN, 36 (10%) SM-CMML, 22 (6%) SM-MDS, 22 (6%) SM-myeloid unclassified, 14 (4%) SM-MDS/MPN, 12 (3%) SM-lymphoid malignancy and 10 (3%) SM-acute leukemia. Adverse mutations, including ASXL1 (19% mutated), RUNX1 (3% mutated) and NRAS (3% mutated) were detected in 30 (23%) of 129 cases screened. After a median follow-up of 21 months, 139 (40%) deaths and 6 (5%) leukemic transformations were documented. Karyotype was abnormal in 53 (15%) cases and included unfavorable (n=29; 8%) and favorable (n=24; 7%) abnormalities. Abnormal karyotype incidences were 6% for ISM and 22% for advanced SM (p<0.001). Among advanced SM cases, abnormal karyotype incidences were 26% for SM-AHN and 8% for ASM (p<0.001); one of 2 MCL cases displayed abnormal karyotype. Among SM-AHN cases, abnormal karyotype incidences were 0% for SM-AHN-lymphoid, 28% for SM-AHN-myeloid (p<0.001); the latter included 19% for SM-CMML, 21% for SM-MDS/MPN, 23% for SM-MPN, 36% for SM-MDS and 41% of SM-myeloid-unclassified (p<0.001). Clinical correlative studies disclosed significant associations between abnormal karyotype and male sex (p=0.002), age >60 years (p=0.04), thrombocytopenia (p<0.001; 27% vs 10%) and anemia (p<0.001; 25% vs 6%), but not with the presence of adverse mutations (p=0.19). In univariate analysis, abnormal karyotype was associated with inferior survival (HR 3.0, 95% CI 2.0-4.3) and significance was sustained when analysis was adjusted for two-tiered (advanced vs indolent SM; p<0.01) or multi-tiered (ISM vs ASM vs SM-AHN; p<0.01) WHO subcategories. WHO category-specific analysis clarified prognostic relevance of abnormal karyotype in ASM (HR 4.4, 95% CI 1.0-14.4; p=0.05; figure 1a) and SM-AHN-myeloid (HR 1.9, 95% CI 1.2-2.9; p=0.005; figure 1b); however, the near-significance in ASM was fully accounted for by thrombocytopenia (p value corrected to 0.35) and for SM-AHN-myeloid by thrombocytopenia and anemia (p value corrected to 0.06); further stratification of abnormal karyotype into favorable vs unfavorable categories did not affect the results in ASM but revealed an independent prognostic effect for unfavorable karyotype in SM=AHN-myeloid (p=0.009). Conclusions: Abnormal karyotype in SM clusters with SM-AHN-myeloid. We found no correlation between abnormal karyotype and adverse mutations. Anemia and thrombocytopenia were significantly associated with abnormal karyotype and accounted for the apparent prognostic relevance of the latter in ASM. Unfavorable karyotype carries independent prognostic effect in SM-AHN-myeloid. Disclosures No relevant conflicts of interest to declare.