Lasho, Terra L.

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

Abstract Background : Recent studies have suggested significant associations between karyotype and certain molecular or phenotypic features in primary myelofibrosis (PMF). In the current study of 835 consecutive patients, we examined the spectrum and prevalence of cytogenetic abnormalities in PMF and their molecular and phenotypic correlates. Methods : PMF diagnosis was according to World Health Organization criteria. Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature. Statistical analyses considered clinical and laboratory parameters obtained at time of cytogenetic studies. Spectrum and frequency of cytogenetic abnormalities : Analyzable metaphases were obtained in 826 (99%) of 835 patients studied; 681(82%) had ≥20 metaphases analyzed. 352 (42.6%) patients had abnormal karyotype, including 240 (68.2%) sole, 64 (18.2%) two and 48 (13.6%) complex; comparison of these groups revealed lower platelet count (p<0.01), higher DIPSS-plus score (p=0.03) and higher percentage of younger patients (p=0.04) with complex abnormalities. Monosomal karyotype was noted in 20 (5.7%) patients. Approximately 150 individual abnormalities were identified; most frequent were 20q- (23.3%), 13q- (18.2%), +8 (11.1%), +9 (9.9%), duplication of chromosome 1q (9.7%) and -7/7q- (7.1%). Other notable abnormalities including i(17q) (1.4%), 12p- (1.1%) and inv(3) (0.6%) were much less frequent. Trisomy 8 was the most frequent in the context of complex abnormality (25%). Among the 500 patients seen within one year of initial diagnosis, 179 (35.8%) had abnormal karyotype, which included 121 (67.6%) sole, 31 (17.3%) two and 27 (15.1%) complex abnormalities; the most common abnormalities were 20q- (24.6%), 13q- (15.1%), +8 (14%) and +9 (10%) whereas 11q- (1.7%), i(17q) (1.1%), inv(3) (0.6%), and 12p- (0.6%) were infrequent. Molecular correlates : 476 patients were annotated for JAK2, CALR and MPL mutations; abnormal karyotype frequencies were 43% in JAK2, 42% CALR, 33% MPL mutated and 34% triple-negative cases (p=0.3). 13q- was associated with mutant CALR (p=0.03) and +9 with mutant JAK2 (p=0.02). Subsets of patients were also screened for ASXL1, EZH2, IDH, SRSF2, U2AF1, and SF3B1 mutations; in all instances, mutational frequencies were higher in patients with normal karyotype, reaching significance with ASXL1 (p=0.02) and U2AF1 (p=0.01). Mutant SRSF2 was associated with 20q- (p=0.02). Phenotypic correlates : Phenotypic correlates included abnormal karyotype with anemia (p=0.02), leukopenia (p<0.01) and thrombocytopenia (p<0.01); complex karyotype with younger age (p=0.04) and thrombocytopenia (p<0.01); leukopenia with 20q-, +8 and -7/7q-; and thrombocytopenia with 20q- and -7/7q-. Cytopenias were less likely to occur with 13q- (p<0.01), which was instead associated with thrombocytosis (p<0.01). 20q- was associated with lower incidence of marked leukocytosis (p=0.02). Trisomy 8 was associated with lower incidences of constitutional symptoms (p<0.01) and marked splenomegaly (p<0.01). Conclusions : The association of 13q- with CALR mutations in PMF might underlie its association with both thrombocytosis and favorable prognosis. The association of +9 with JAK2 mutations might reflect selective clonal advantage through JAK2V617F dosage enhancement or mutation-induced chromosomal instability. The association of 20q- with mutant SRSF2 and thrombocytopenia warrant further clarification of its reported association with favorable prognosis. Disclosures No relevant conflicts of interest to declare.

Abstract Background: KIT D816V is considered the driver mutation in systemic mastocytosis (SM) however this mutation alone does not explain the diverse clinical manifestations of SM. While mutations in other myeloid-relevant genes have been reported in SM, their pattern(s) of co-segregation and prognostic value, independent of age and SM subtype, have not been fully defined in a mature dataset. Objectives: In this pilot study of advanced SM patients, we sequenced a panel of myeloid-relevant genes for known pathogenic mutations, to ascertain: (i) the frequency of individual mutations; (ii) the pattern(s) of mutation co-segregation; and (iii) the prognostic value of mutations, both individually and in combination, on overall- (OS) and leukemia-free survival (LFS). Methods: The current study was approved by the Mayo Clinic institutional review board. SM was classified as per World Health Organization criteria. DNA was isolated from archived bone marrow (BM) cell pellets. Next-generation deep sequencing by HiSeq SBS chemistry (Illumina) was performed to investigate the coding regions of 22 genes: KIT, JAK2, CALR, SRSF2, U2AF1, SF3B1, ASXL1, TET2, EZH2, SETBP1, DNMT3A, IDH-1/2, SUZ12, CBL, NRAS, FLT3, PTPN11, RUNX1, CEBPA, TP53 and NPM1. Results: We studied 19 patients with advanced SM; 6 had aggressive SM (ASM) and 13 had SM with an associated hematological disease (SM-AHD). Of the latter, 5 patients (38%) had myeloproliferative neoplasm-unclassified (MPN-u), 3 (23%) chronic myelomonocytic leukemia (CMML), 2 (15%) acute myeloid leukemia (AML), and 1 (8%) each polycythemia vera, myelodysplastic syndrome, and multiple myeloma. The median follow up from diagnosis was 20 months (range 2 to 84). Over this period, 5 patients (26%) developed leukemic transformation and 12 patients (63%) have died. KIT D816V was identified in 9 (47%) patients; 9 of 10 patients who tested negative by deep sequencing were screened by allele-specific PCR (sensitivity ~0.01%), with 8 additional patients testing positive. Thus, overall KIT D816V frequency was 89%. Fourteen (74%) patients harbored at least one additional non-KIT mutation; of these 7 patients had one additional mutation (ASXL1/TET2/SF3B1 = 2 each and JAK2 =1), 3 patients each had two and three additional mutations, and 1 patient had 4 additional mutations. The most frequently mutated genes (other than KIT) were ASXL1/TET2 (5 patients each, 26%), CBL (4 patients, 21%), SETBP1/SF3B1/DNMT3A (2 patients each, 11%) and JAK2/CALR/FLT3/IDH-1/RUNX1/TP53 (1 patient each, 5%). There was no overlap between patients harboring TET2 and ASXL1 mutations however 2 and 1 CBL-mutated patients concurrently harbored TET2 and ASXL1 mutations, respectively. SETBP1 and SF3B1 but not DNMT3A mutations occurred independently of TET2 and ASXL1 mutations. The distribution of mutations was similar between ASM and SM-AHD subgroups. When considered individually, the following non-KIT mutated genes (present in at least 2 patients) were associated with significantly inferior OS: ASXL1, CBL and SETBP1 (p<0.05). When the 3 mutated genes were considered together, only ASXL1 and CBL maintained their significance, independent of age and SM subtype, for inferior OS (Hazard ratio (95% confidence interval)=34.8 (3.1-393.9) and 59.7 (4.1-861.5), respectively). Patients harboring ASXL1 and/or CBL mutations (n=8, 8 deaths, median OS=11 months) had a significantly worse OS as compared to those without either mutation (n=11, 4 deaths, median OS=84 months) (p=0.0002) (Fig 1). When considering the number of non-KIT mutations (zero, one, two, three and four mutations in 5, 7, 3, 3 and 1 patients, respectively), the group with ≥3 mutations (n=4, 4 deaths, median OS=7 months) had a significantly inferior OS as compared to those with <3 mutations (n=15, 8 deaths, median OS=48 months) (p=0.01) (Fig 2). Presence of mutations was not correlated with LFS in this analysis. Conclusions: In this cohort of advanced SM patients, non-KIT mutations were frequent (prevalence 74%). TET2, ASXL1, SETBP1 and SF3B1, but not CBL or DNMT3A, mutations were mutually exclusive. Presence of ASXL1 and CBL mutations was associated with significantly inferior OS (HR=35 and 60, respectively), independent of known prognostic factors. An increasing number of non-KIT mutations (≥3, HR=5.9) was also associated with a significantly worse survival outcome. Sequencing results from a larger cohort will be presented at the meeting. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Pardanani: Stemline: Research Funding.

Abstract Abstract 3840 Background: Patients with myeloproliferative neoplasms (MPN) harbor a multitude of somatic mutations involving JAK2, MPL, LNK, CBL, TET2, ASXL1, IDH1, IDH2, EZH2, DNMT3A, IKZF1 and TP53. None of these mutations have been consistently traced back to the ancestral clone and most of them are currently believed to represent secondary genetic events. The higher prevalence of IDH, LNK, IKZF1 and TP53 mutations in blast-phase MPN, as opposed to chronic-phase disease, suggests their pathogenetic contribution to leukemic transformation (Harutyunyan A, et al. N Engl J Med. 2011;364:488). We examined this possibility for TP53 mutations or its SNPs in chronic-phase PMF; TP53 SNPs involving codon 72 have been associated with increased risk in the development of several cancers. Methods: Mayo Clinic MPN database was utilized to identify adult patients (>18 years) with chronic-phase PMF. Study eligibility criteria included availability of bone marrow histology and cytogenetic information at time of referral. In addition, all patients were annotated for IDH, MPL and JAK2 mutational status. Patients with blast-phase disease at the time of their referral were excluded from the study. DNA from bone marrow or peripheral blood was extracted using conventional methods and amplified with one set of primers covering TP53 exons 4 through 9. The amplification products were then sequenced with eight primers using the Big Dye Terminator sequencing kit v1.1 (Applied Biosystems, Foster City, CA) and the ABI 3130xl Genetic Analyzer. Sequences were examined for mutations by the Sequencher software (Gene Codes, Ann Arbor, MI). Any mutation found was confirmed with a second PCR and sequencing reaction. A mutation was considered to be any sequence other than genomic reference sequence NC_000017.10 or a well documented polymorphism such as the rs1042522 in exon 4. Mutations were compared with the IARC p53 Database (http://www-p53.iarc.fr/) and/or the TP53 Mutation Database (http://p53.free.fr/index.html) to determine if the mutation had been reported before. Results I: Baseline characteristics and clinical course A total of 107 patients with chronic-phase PMF were studied. Median age at time of referral was 64 years (range 32–81) and 71% were males. DIPPS-plus risk assignment was low in 13%, intermediate-1 in 15%, intermediate-2 in 38% and high in 34%. Karyotype was normal in 70 (65%) patients, favorable in 26 (24%) and unfavorable in 11 (10%). JAK2, MPL and IDH mutations were detected in 62 (58%), 7 (7%) and 6 (6%) cases, respectively. To date, 74% of patients had died and 15% had documented leukemic transformation. Results II: TP53 mutations/polymorphisms and their clinical relevance TP53 mutations were discovered in only 4 (4%) cases: i) E204E; GAG>GAA (silent exon 6); ii) G245D; GGC>GAC (exon 7); iii) R175H; CGC>CAC (exon 5); and iv) six base insert (GGCGAG) after bp13767 (exon 6). There was no significant difference in presenting features or survival between TP53 mutated and unmutated cases. None of the four patients with TP53 mutation incurred leukemic transformation. TP53 exon 4 SNPs data for codon 72 was available on 104 patients and included 58 (56%) patients with homozygous Arg72Arg, 34 (33%) patients with heterozygous Pro72Arg and 12 (11%) patients with homozygous Pro72Pro. Allele frequencies were 0.7 for G (corresponding to arginine) and 0.3 for C (corresponding to proline), which is similar to the general population (0.65 and 0.35 respectively). There were no significant differences among the three codon 72 genotypes in terms of presenting characteristics or survival. Conclusions: TP53 mutations are infrequent in chronic phase PMF. A higher number of informative cases are needed to accurately define their phenotypic or prognostic effects. We are currently analyzing more cases in this regard. On the other hand, TP53 polymorphisms at codon 72 do not appear to influence phenotype or prognosis in chronic-phase PMF. Disclosures: No relevant conflicts of interest to declare.

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

Abstract Abstract 3850 Background: We have recently shown that primary myelofibrosis (PMF) was associated with increased plasma levels of IL-1b/IL-1RA/IL-2R/IL-6/IL-8/IL-10/IL-12/IL-13/IL-15/TNF-α/G-CSF/INF-α/MIP-1α/MIP-1b/HGF/IP-10/MIG/MCP-1/VEGF in PMF (J Clin Oncol 2011; 29: 1356). In addition, increased levels of IL-8, IL-2R, IL-12, IL-15 and IP-10 were independently associated with inferior survival. In the current study, we focused on the plasma cytokine profile in polycythemia vera (PV) and examined how it compares with PMF or in a cohort of PV patients whose samples were collected during follow-up. We also looked for phenotypic and prognostic correlates. Methods: Study inclusion for patients with PV required availability of archived plasma within one year of diagnosis. Samples from PV patients seen at different points post-diagnosis were also collected, in order to compare their cytokine profile with that of patients whose samples were collected within one year of diagnosis. Standard procedures were followed to centrifuge peripheral blood samples at 4°C and store aliquots at −80°C. Concentrations of 30 plasma cytokines/chemokines were analyzed in duplicates using Multiplex Bead-based Luminex Technology (Invitrogen, Carlsbad, CA, USA): IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, EGF, eotaxin, FGF-b, GM-CSF, G-CSF, HGF, IFN-α, IFN-γ, IP-10, MCP-1, MIG, MIP-1α, MIP-1β, RANTES, TNF-α, and VEGF. Measurements were performed on a Luminex 200 analyzer (Luminex Corporation, Austin, TX, USA) and resulting data were evaluated using STarStation Software Version 2.3 (Luminex Corporation, Austin, TX, USA). Results I: Plasma cytokines in PV (n=36 for samples collected within one year of diagnosis and n=40 for samples collected beyond that time point) vs. PMF (n=92) vs. normal controls (n=35) There was no significant difference between the three groups in the plasma levels of the following cytokines: IL-1β, IL-2, IL-4, EGF, and IL-17. PV and normal controls had similar levels of IL-2R, IL-5, FGF-b, and TNF-α; all four cytokines were elevated in PMF. The levels of the following cytokines were equally increased in PV and PMF: IL-1RA, IL-7, HGF, MIG, and VEGF. The following cytokines were also elevated in both PMF and PV but predominantly so in PV (MIP-1α) or PMF (IL-6, IL-8, IL-12, IL-13, IL-15, MIP-1β, IP-10, and MCP-1). Eotaxin and GM-CSF were elevated in PV but not PMF. Compared to normal controls, significantly lower levels were noted for IL-10 in PV and IFN-α and IFN-γ for PMF. IFN-α and IFN-γ levels were both elevated in PV. Comparison of PV samples collected within one year of diagnosis (n =36) vs. those collected beyond that time point (n =40) showed significantly increased levels of IL-8 (p=0.0005) and borderline significant increases in IP-10 (p=0.09) and IL-2R (p=0.09). Results II: Clinical correlates of plasma cytokines in PV Among the above-listed cytokines that were abnormally increased or decreased in PV, significant correlations were seen with sex (IP-10 and GM-CSF), age (IP-10, eotaxin), platelet count (MIG), hemoglobin level (IL-6), arterial thrombosis (IFN-γ, IL-1RA, VEGF, MIP-1α), and abnormal karyotype (IL-13, MIP-1α). None of the cytokines correlated with leukocyte count, venous thrombosis, pruritus or microvascular symptoms. The following, treated as continuous variables, were associated with inferior survival: IL-7, HGF, IFN-α, MCP-1, GM-CSF and IL-10 (p<0.05 for all). The number of events and sample size were too small to perform multivariable analysis. Conclusions: Plasma cytokine levels in PV are markedly abnormal and show both similarities and differences with PMF; the similarities became more pronounced in patients whose samples were examined at later stages of their disease. The current study suggests prognostic implications that warrant validation in a larger number of patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1751 Because IDH mutations are frequent in blast-phase myeloproliferative neoplasms (MPN), they might contribute to leukemic transformation. We examined this possibility in 301 consecutive patients with chronic-phase primary myelofibrosis (PMF). All study patients were fully characterized for karyotype, JAK2 and MPL mutational status, and Dynamic International Prognostic Scoring System-plus (DIPSS-plus) risk status. DNA from bone marrow or peripheral blood was used to screen for IDH1 and IDH2 mutations, by direct sequencing and/or high resolution melting (HRM) assay. Mutant IDH was detected in 12 patients (4%): seven IDH2 (five R140Q, one R140W and one R172G) and five IDH1 (three R132S and two R132C). MPL exon 10 was mutated in 18 patients (6.3%) and constituted W515L in 14 patients, W515K in 3 and a frameshift mutation in 1 patient. JAK2V617F was detected in 169 (56%) patients. Six patients displayed both JAK2V617F and IDH mutations (IDH2R140Q in 2 patients, IDH2R140W in 1 and IDH1R132S in 3); JAK2V617F allele burden was 1%, 7%, 22%, 27%, 30% and 96%, respectively. One patient displayed both IDHR140Q and MPLW515R. One-hundred and seven (36%) patients were negative for all three mutations. The 12 IDH-mutated patients were clinically compared to patients belonging to the three other molecular subgroups: mutated for JAK2 only (n=164), mutated for MPL only (n=18) and unmutated for all three (n=107). The four molecular subgroups were remarkably similar in their phenotype with few exceptions; IDH-mutated patients were significantly older than those with no mutations (p=0.04) whereas age distribution was similar between patients with mutant IDH, MPL or JAK2. In univariate analysis, overall survival (OS) for IDH-mutated patients was significantly shorter than those for JAK2-mutated (p=0.03), MPL-mutated (p=0.047) or unmutated (p=0.0009) patients. IDH-mutated patients also showed significantly shorter leukemia-free survival (LFS), compared to those with mutant JAK2 (p=0.0008), mutant MPL (p=0.02) or no mutations (p=0.001). After accounting for age, the presence of mutant IDH remained a significant disadvantage for both OS (p=0.04) and LFS (p=0.005). Multivariable analysis of OS that included risk categorization per DIPSS-plus confirmed the independent prognostic relevance of mutant IDH (p=0.03): HR for patients with no mutations =0.39, 95% CI 0.2–0.75; HR for JAK2-mutated patients =0.50, 95% CI 0.27–0.95; HR for MPL-mutated patients =0.53, 95% CI 0.23–1.2. A similar analysis for LFS that included risk factors for LT (i.e. unfavorable karyotype and platelet count <100 × 109/L) as covariates also confirmed the prognostic relevance of mutant IDH (p=0.003): HR for patients with no mutations =0.16; 95% CI 0.06–0.46; HR for JAK2-mutated patients =0.18; 95% CI 0.06–0.48; HR for MPL-mutated patients =0.09; 95% CI 0.01–0.76). Further analysis disclosed that the negative OS and LFS effect of mutant IDH was most pronounced in the presence (p=0.0002 and <0.0001, respectively) as opposed to the absence (p=0.34 and 0.64, respectively) of concomitant JAK2V617F expression. Analysis of paired samples obtained during the chronic and blast phases of the disease was possible in 2 IDH-mutated patients and showed the presence of both IDH and JAK2 mutations at both time points. Our observations suggest that IDH mutations in PMF are independent predictors of leukemic transformation and raise the possibility of leukemogenic collaboration with JAK2V617F. Disclosures: No relevant conflicts of interest to declare.

Abstract Background U2AF1 mutations occur in approximately 16% of patients with primary myelofibrosis (PMF) and were significantly associated with anemia, thrombocytopenia, older age, JAK2V617F, ASXL1 mutations and normal karyotype (Leukemia 2014;28:431); furthermore, U2AF1 mutations were associated with inferior survival in univariate but not multivariable analysis. In the current study, we looked for the possibility of a differential effect from U2AF1 mutation variants on these observations in PMF. Methods Study patients fulfilled the 2016 WHO criteria for the diagnosis of PMF (Blood. 2016;127:2391). Additional selection criteria included the availability of U2AF1 mutational status. Previously published methods (Leukemia 2014;28:431), including targeted next generation sequencing (Blood 2015 126:354), were used to screen for U2AF1 and other prognostically-relevant mutations. Statistical analyses considered clinical and laboratory parameters obtained at time of initial referral to the Mayo Clinic. Results Patient characteristics: U2AF1 mutational status was available for 457 patients and 72 (16%) harbored one of several mutation variants: these mutations affected residue Q157 in 44 (10%) patients, S34 in 24 (5%) and the remaining 1% displayed other variants. The 44 patients with U2AF1Q157 mutations included 23 with Q157P, 18 with Q157R and 3 with Q157-Y158insYE. The 24 patients with U2AF1S34 included 16 with S34F and 8 with S34Y. Only one patient harbored both Q157 and S34 mutations (Q157R, S34Y). Among all 457 study patients, median age was 63 years, 64% were males, dynamic international prognostic scoring system (DIPSS)-plus (JCO 2011;29:392) risk distributions were 13% low, 18% intermediate-1, 38% intermediate-2 and 32% high and driver mutation distributions were 54% JAK2, 22% CALR type 1/ type1-like, 4% CALR type 2/type 2-like, 7% MPL and 13% triple-negative. Cytogenetic studies were available in 449 patients: 39% abnormal and 10% unfavorable. All 457 patients were screened for ASXL1 mutations with 37% mutated, 450 for SRSF2 mutations with 15% mutated, 403 for SF3B1 with 8% mutated, 366 for IDH1/2 with 5% mutated and 369 for EZH2 with 4% mutated. Median follow-up was 4.4 years and during this period, 318 (70%) deaths and 53 (12%) leukemic transformations were documented. Phenotypic correlates of U2AF1 mutation variants: Because of the relatively small number of informative cases with specific mutations, we classified all patients into Q157 (n=44) and S34 (n=24) mutation variants and compared them with the 385 U2AF1 un-mutated cases. First, we confirmed our previous observations regarding the association between U2AF1 mutations in general and older age (p=0.0003), JAK2V617F (p<0.0001), ASXL1 mutations (p=0.0002), normal karyotype (p=0.03), hemoglobin <10 g/dL (p<0.0001) and platelet count <100 x 10(9)/L (p<0.0001); when the two U2AF1 mutation categories were analyzed separately, the corresponding p values for Q157 were 0.0005, 0.001, <0.0001, 0.04, <0.0001 and <0.0001 and for S34 were 0.12, 0.001, 0.41, 0.41, <0.0001 and 0.67. Phenotypic correlates of U2AF1 mutation variants: In univariate analysis, survival was adversely affected by U2AF1Q157 (p<0.0001; HR 2.2, 95% CI 1.6-3.1) but not by U2AF1S34 (p=0.8; HR 1.1, 95% CI 0.6-1.8) mutations (Figure 1a). Furthermore, the negative survival effect of U2AF1Q157 mutations was independent of anemia, thrombocytopenia, ASXL1, SRSF2, IDH1/2 and EZH2 mutations, as well as driver mutational status; multivariable analysis that included all molecular alterations identified U2AF1Q157 (HR 1.6, 95% CI 1.1-2.3), ASXL1 (HR 2.3, 95% CI 1.8-3.5), SRSF2 (HR 1.6, 95% CI 1.2-2.2) and absence of CALR type-1/like (HR 2.6, 95% CI 1.8-3.5) mutations as independent risk factors for survival. Finally, the survival effect of U2AF1Q157 mutations was independent of DIPSS-plus in patients with hemoglobin ≥10 g/dL (HR 2.6, 95% CI 1.3-5.3; p=0.007) (Figure 1c) but not in those with hemoglobin <10 g/dL (p=0.98) (Figure 1b). Conclusion Both U2AF1Q157 and U2AF1S34 mutation variants in PMF are associated with JAK2V617F and severe anemia whereas only the former is associated with ASXL1 mutations and thrombocytopenia. More importantly, U2AF1Q157, but not U2AF1S34, mutation variants in PMF are predictive of inferior survival, independent of other adverse mutations, and, in the absence of severe anemia, independent of DIPSS-plus. Disclosures No relevant conflicts of interest to declare.

Abstract Background The Dynamic International Prognostic Scoring System (DIPSS)-plus currently provides the most comprehensive prognostic model for primary myelofibrosis (PMF) (JCO 2011;29:392). DIPSS-pluse uses eight independent predictors of inferior survival: age >65 years, hemoglobin <10 g/dL, leukocyte count >25 x 109/L, circulating blasts ³1%, constitutional symptoms, unfavorable karyotype, red cell transfusion need and platelet count <100 x 109/L. More recently, type 1/type 1-like CALR driver mutational status and ASXL1 andSRSF2 mutations were identified as additional DIPSS-plus-independent risk factors for survival in PMF (JAMAOncol. 2015;1:97). Based on our earlier preliminary observation (Leuk Res. 2007;31:1503), we conducted the current study to fully examine the additional prognostic relevance of monocytosis (absolute monocyte count ≥1 x 10(9)/L) in PMF. Methods Study patients fulfilled the 2016 WHO criteria for the diagnosis of PMF, including both pre-PMF and overt PMF (Blood. 2016;127:2391). Additional selection criteria included the availability of absolute monocyte count (AMC) and percentage at time of referral. Targeted next generation sequencing was used to screen for prognostically-relevant mutations (Blood 2015 126:354). Statistical analyses considered clinical and laboratory parameters obtained at time of initial referral to the Mayo Clinic. Results Patient characteristics: The study population included 454 patients (median age 64 years; 62% males); 293 (64%) patients harbored JAK2 mutations, 74 (16%) type 1/like CALR, 16 (3%) type 2/like CALR, 24 (5%) MPL and 47 (10%) were "triple-negative". DIPSS-plus risk distribution was 29% high, 38% intermediate-2, 17% intermediate-1 and 15% low; 27% displayed red cell transfusion-dependency, 29% constitutional symptoms, 70% palpable splenomegaly and 36% abnormal karyotype, including 13% with unfavorable karyotype. 301 patients were screened for ASXL1 mutations with 39% mutated, 297 for SRSF2 mutations with 15% mutated, 286 for U2AF1 with 18% mutated, 253 for SF3B1 with 6% mutated, 229 for EZH2 with 5% mutated and 110 for IDH1/2 with 7% mutated. Clinical, cytogenetic and molecular comparisons between PMF patients with and without monocytosis: Among all study patients, 376 (83%) displayed AMC <1 x 10(9)/L; the remaining had either moderate (AMC between 1 and 3 x 10(9)/L; n=65) or marked (AMC >3 x 10(9)/L; n=13) monocytosis. Comparison of these three groups revealed that monocytosis was significantly associated with older age (p=0.0001), higher leukocyte count (p<0.0001), lower platelet count (p=0.01), higher circulating blast percentage (p=0.0002) and higher DIPSS-plus risk distribution (p=0.01). There were no significant differences in the distribution of driver mutations, ASXL1, SRSF2, U2AF, SF3B1, EZH2 or IDH1/2 mutations or incidence of unfavorable karyotype. Survival analysis: After a median follow up of 3.2 years, 265 (58%) deaths and 38 (8%) leukemic transformations were documented. In univariate analysis, all 8 variables included in DIPSS-plus were significantly associated with shortened survival (p<0.001 in all instances). Significant risk factors in univariate analysis also included AMC >3 x 10(9)/L (p<0.0001; HR 5.6, 95% CI 3.1-10.0), AMC 1 to 3 x 10(9)/L (p<0.0001; HR 2.1, 95% CI 1.5-2.9), absence of CALR type 1/type 1-like (p<0.0001) and presence of ASXL1 (p<0.0001) or SRSF2 (p<0.0001) mutations. The significant difference in survival between the three AMC categories (i.e. <1, 1-3 and >3 x 10(9)L) was independent of each one of the eight DIPSS-plus variables, DIPSS-plus, driver mutational status, ASXL1 and SRSF2 mutations (p<0.0001 in all instances). The survival difference between patients with and without monocytosis was evident in both high/intermediate-2 and low/intermediate-1 DIPSS-plus risk groups (Figure). Patients with monocytosis were also more likely to undergo leukemic transformation (HR 3.0, 95% CI 1.4-6.5) and this effect was also independent of karyotype, driver mutational status, ASXL1 or SRSF2 mutations. Conclusion The current study identifies monocytosis as a powerful risk factor for both overall and leukemia-free survival in PMF and the effect was independent of currently established prognostic models and genetic risk factors. It was particularly noteworthy to document similar distribution of driver mutations among the three monocyte categories. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Abstract Background Polycythemia Vera (PV) constitutes one of the three BCR-ABL1-negative myeloproliferative neoplasms and is characterized by clonal erythrocytosis and the almost invariable presence of JAK2 mutation. An absolute monocyte count (AMC) of ≥1 x 10(9)/L defines chronic myelomonocytic leukemia (CMML) but can also be seen in other myeloid disorders including myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). The presence or development of monocytosis has previously been shown to confer poor prognosis in both primary myelofibrosis (PMF), which is one of the three BCR-ABL1-negative MPN (Leukemia Research 2007;31:1503, Mod Pathol. 2013 Feb;26(2):204) and MDS (Haematologica. 1997;82:25). In the current study, we examined the clinical, prognostic and molecular correlates of monocytosis in PV. Methods Study patients were selected from our institutional database of MPN and fulfilled the 2008 World Health Organization (WHO) criteria for the diagnosis of PV (Blood. 2009;114:937). Cytogenetic and mutational analyses were performed according to conventional methods (Leukemia. 2014;28:2206) any assignment as unfavorable karyotype was per PMF criteria (Leukemia. 2011;25:82). Mutation screening included TET2, ASXL1 and SRSF2 because of their known association with CMML (Leukemia. 2014;28:2206) Statistical analyses considered clinical and laboratory parameters obtained at time of diagnosis. Results Patient characteristics: Analysis was conducted on 587 patients (median age 60 years; 48% males) who met WHO criteria for diagnosis of PV. Amongst them, accurate documentation of AMC was available in 237 patients, cytogenetic information in 239, and ASXL1, TET2 and SRSF2 mutational status in 133 patients. Median (range) values were for AMC 0.6 x 10(9)/L (0-4.7) and leukocytes 11.6 x 10(9)/L (3.8-171.6). 31% of 506 informative patients had palpable splenomegaly, 34% of 551 had microcirculatory symptoms, 30% of 566 had pruritus, 8% of 504 had erythromelalgia, 42% of 581 had hypertension, 9% of 584 had diabetes and 11% of 575 were active tobacco users. 25% of the patients presented with history of thrombosis and 22% developed thrombosis after diagnosis. Cytogenetic findings were abnormal in 19%, of whom 20% were unfavorable. TET2, ASXL1 and SRSF2 mutations were documented in 18%, 11% and 3%, respectively. During follow-up, 224 (38%) patients died and median follow-up for living patients was 109 months. Median survival was 16 years and leukemic or fibrotic transformations were documented in 4% and 14%, respectively. Comparison of patients with and without monocytosis: Among 237 informative patients, 32 (14%) displayed monocytosis (AMC ≥1 x 10(9)/L) at time of diagnosis. PV patients with monocytosis were older (p=0.006) and displayed higher leukocyte count (p<0.0001) and higher incidences of leukocytosis (p=0.024) and unfavorable cytogenetic abnormalities (p=0.02). There was no association between monocytosis and mutations for TET2 (p=0.1), ASXL1 (p=0.7) and SRSF2 (p=0.3) or thrombosis before (p=0.9) or after (p=0.5) diagnosis (p=0.5), palpable splenomegaly (p=0.6), pruritus (p=0.7) or microcirculatory symptoms (p=0.1). Among the 237 PV patients in whom information regarding AMC was available, 70 (30%) died during follow-up and 49 (21%), 23 (10%), 9 (4%) developed thrombosis, leukemic transformation or fibrotic progression, respectively. In univariate analysis, overall (p=0.009; HR 2.0, 95% CI 1.2-3.4) but not leukemia-free (p=0.79), myelofibrosis-free (p=0.13) or thrombosis-free (p=0.48) survivals were different between patients with or without monocytosis. Furthermore, the significant difference in survival was no longer apparent when analysis was adjusted for age (p=0.13), unfavorable karyotype (0.17) or leukocytosis (p=0.06). Conclusions Monocytosis (AMC ≥1 x 10(9)/L) is not infrequent in PV (14%). However, the presence of monocytosis does not appear to represent a significantly different phenotype in terms of molecular characteristics although it is associated with older age, leukocytosis and unfavorable karyotype. The latter associations account for the inferior survival seen in patients with monocytosis. Disclosures No relevant conflicts of interest to declare.