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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.

Abstract Background : Myeloproliferative leukemia virus oncogene (MPL)-mutated essential thrombocythemia (ET) is uncommon, with a cited incidence of less than 5%, while that of MPL-mutated primary myelofibrosis (PMF) is at least twice as frequent (Blood. 2006;108:3472;Blood.2008;112:141). MPL-mutated ET cohorts also have higher reported rates of fibrotic progression than their MPL wild type counterparts (Blood. 2014;124:2507). These observations suggest the possibility that some instances of MPL-mutated ET might actually represent prefibrotic PMF. Methods : Patients were recruited from institutional databases of the Mayo Clinic, Rochester, MN, USA. Diagnoses were according to the 2016 World Health Organization (WHO) criteria (Blood. 2016;127:2391). Study inclusion criteria required the availability of bone marrow biopsy slides for central review by one of the authors (C.A.H.). Central pathology review included assessment of bone marrow cellularity and extent of tri-lineage proliferation, megakaryocyte morphology and grading of reticulin fibrosis. Data was additionally collected from MPL-mutated patients with PMF, for comparison. Standard statistical methods were used for statistical analysis, including calculation of survival data (JMP® Pro 13.0.0, SAS Institute, Cary, NC, USA). Results: A total of 665 patients with ET were annotated for their driver mutational status; 18 (2.7%) were reported out as being MPL-mutated; by comparison, among 867 patients with PMF, 47 (5.4%) were signed out as MPL-mutated. Among the 18 cases with MPL-mutated ET, bone marrow slides were available for central pathology review in 14 patients (Table 1). The latter were subsequently reassigned the diagnosis of either prefibrotic PMF (n=8; 57%) or were felt to be morphologically consistent with true WHO-defined ET (n=6; 43%). Comparison of these two distinct histopathological patterns, i.e. true ET vs reassigned prefibrotic PMF, was respectively characterized by lower (median 35%, range 30-50) vs higher (median 65%, range 40-80) bone marrow cellularity (P<0.001), ET (n=6) vs PMF (n=8) consistent megakaryocyte morphology (P<0.001) and presence of tri-lineage proliferation (0% vs 100%; P<0.001); in contrast the degree of reticulin fibrosis was similar between the two (P=0.1) (Table 2). The reassigned prefibrotic PMF (n=8), vs confirmed ET (n=6), cases also displayed a higher frequency of increased serum levels of lactate dehydrogenase (60% vs 0%; P=0.02), higher likelihood of displaying hemoglobin levels below the sex-adjusted reference range values (29% vs 0%; P=0.1), leukoerythroblastosis (14% vs 0%; P=0.2), constitutional symptoms (13% vs 0%; P=0.2) and a higher incidence of thrombosis history at presentation (38% vs 0%; P=0.04) (Table 2). Interestingly, reassigned prefibrotic PMF also displayed a narrower MPL mutational spectrum compared to those confirmed as ET (MPLW515L/K incidence 100% vs 60%; P=0.04). The incidences of abnormal karyotype and high risk molecular mutations (ASXL1, SRSF2 and U2AF1) were similar between the two. We documented a higher incidence of post-diagnosis thrombosis in prefibrotic PMF (25% vs 0%; P=0.1) but similar rates of leukemic transformation (0% for both) and fibrotic progression (38% vs 33%); when all 665 ET patients were assessed for myelofibrosis-free survival, MPL-mutated cases (prior to central review) displayed significantly worse outcome, compared to patients with other driver mutations (Figure 1a). Median overall survival in confirmed MPL-mutated ET, ET re-classified as PMF, and MPL-mutated PMF was not reached, 11.6 and 5.3 years, respectively (confirmed ET vs PMF, P=0.01; ET re-classified vs PMF, P=0.04; confirmed ET vs ET re-classified as PMF, P=0.54) (Figure 1b). Conclusions: The current study suggests that the majority of routinely-assigned MPL-mutated ET probably represents prefibrotic PMF, when morphologically scrutinized. Prior to central pathology review, we show a higher rate of fibrotic progression in MPL-mutated ET, compared to patients with other driver mutations; accordingly, after central pathology review, the similar rate of fibrotic progression between morphologically confirmed MPL-mutated ET and those reassigned as prefibrotic PMF further suggests the latter to be biologically more akin to PMF, despite its ET-consistent morphologic features. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Vitamin B1 (Thiamine) deficiency might result in Wernicke's encephalopathy (WE); the latter was reported in clinical studies with the JAK2 inhibitor fedratinib resulting in disruption of clinical development, despite efficacy in patients with myelofibrosis (MF) (JAMA Oncol, 2015, 5). Our objectives were i) to determine the incidence of thiamine deficiency in patients with myeloproliferative neoplasms (MPN), ii) assess clinical correlations of thiamine level and iii) determine the impact of JAK2 inhibitor therapy on thiamine level in MPN. Methods: After Institutional review board approval, patients referred with a suspected diagnosis of MPN were enrolled. All clinical and laboratory variables including treatment details were collected at the time of referral. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of thiamine diphosphate in whole blood was performed at the time of referral. Reference range for thiamine level in whole blood was 70-180 nmol/L. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all statistical analysis. Results: A total of 115 patients were prospectively enrolled and included 28 patients without MPN and 87 with MPN. 87 MPN patients (median age, 65 years, 52% males) included 32 with primary myelofibrosis (PMF), 17 with polycythemia vera (PV), 17 with essential thrombocythemia (ET), 11 with post PV MF, 8 with post ET MF, and 2 with MPN-U. The MPN and non-MPN patients were similar in their gender distribution, pattern of alcohol use, malnutrition and multivitamin use (p=0.24, 0.12, 0.54 and 0.78 respectively), although the former were older (p=0.09). Median thiamine level of the study patients was 167 nmol/L (range; 60-442 nmol/L) with only two (1.7%) female patients displaying level below the normal reference range. One belonged to the non-MPN group (thiamine level 68 nmol/l); a 26 year old female with erythroid leukemia in the absence of alcohol use or malnutrition; the second patient (thiamine level 60 nmol/l) was a 38 year old female with ET, CALR type 2 mutated on hydroxyurea, without a history of alcohol use or malnutrition. Overall, both MPN and non-MPN patients displayed similar thiamine level (p=0.89). Clinical correlations of thiamine level in MPN Median thiamine level was similar amongst post ET MF, ET, PMF, PV, and post PV PMF patients (127, 162, 166, 190 and 209 nmol/L respectively, p=0.13). No significant effect on thiamine level was apparent for age, gender, hemoglobin, platelet count, dynamic international prognostic scoring system (DIPSS) score, unfavorable cytogenetics, palpable splenomegaly, or constitutional symptoms. Interestingly, thiamine level was significantly higher in JAK2 mutated cases (median 177 nmol/L vs 145 nmol/L in unmutated cases, p=0.03). Furthermore, when analysis was restricted to PMF patients, thiamine level positively correlated with JAK2V617F mutant allele burden (p=0.01) and leukocyte count > 10 x 109/L (p=0.03). JAK2 mutated PMF patients with leukocyte count > 10 x 109/L depicted significantly higher thiamine levels (median 313 nmol/L) compared to JAK2 mutated patients without leukocytosis (median 164 nmol/L) and JAK2 unmutated patients with or without leukocytosis (median 192 nmol/L and 143 nmol/L respectively)(p=0.004, Figure 1a). Impact of treatment on thiamine level Treatment with hydroxyurea was documented in 35 patients with no significant impact on thiamine level (p=0.28). 27 patients were on JAK inhibitors (median duration at referral; 41 months); 12 patients on ruxolitinib and 15 MF patients on momelotinib as part of a clinical trial. Thiamine level was similar amongst JAK inhibitor treated vs untreated patients (median 166 vs 169.5 nmol/L respectively, p=0.72). Annual thiamine measurements were performed on 15 patients treated with momelotinib (median duration 52 months, range 18-60 months). Despite fluctuations in thiamine levels, only two patients were noted to have thiamine level below normal (65 and 69 nmol/L at year 3), which subsequently improved to 101 and 121 nmol/L by year 4, while still on treatment (Figure 1b). Conclusions: The current study confirms the rarity of subnormal thiamine levels in MPN and the limited impact of treatment on thiamine levels, including with JAK2 inhibitors. Our novel observation regarding the correlation of thiamine level with JAK2V617F mutant allele burden and leukocyte count requires further exploration. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Young patients with myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF), constitute a distinct, steadily growing subpopulation (Eur J Haematol. 2017;98:85). Most available data, including current prognostic models, are excerpted from older populations and may not accurately mirror the unique phenotypic and prognostic patterns seen in younger patients. The objective of the current study was to comprehensively assess the phenotypic features and long-term outcomes in MPN patients age ≤ 40 years. Methods: The study population was recruited from a master database of MPN patients seen at our institution between 1967 and 2017. Diagnoses were in accordance with 2016 World Health Organization criteria (Blood. 2016;127:2391). Data was abstracted from the time of referral, which coincided in the majority with the time/within one year of diagnosis. Additional molecular data was obtained based on availability of next-generation sequencing (NGS) information. Conventional statistics was utilized for group comparisons and calculation of survival data. Results: The study population was recruited from a total of 3,023 MPN patients: 665 PV, 1076 ET, and 1282 PMF. Of these, 361 (12%) were ≤ 40 years of age at the time of initial diagnosis; the corresponding incidences were 12% in PV (n=79), 20% in ET (n=219) and 5% in PMF (n= 63). Young PV patients (n=79): Median age (range) was 32 years (18-40) and males constituted 54% (Table 1). Over a median follow-up of 11.3 years, fibrotic transformation rates were 22% vs 25% and 10% in those 41-60 and >60 years of age, respectively (P<0.001). Leukemic conversion rates were balanced across age categories (4% vs 5% vs 3% for successively older cohorts) (P=0.4). Interestingly, young patients had lower rates of arterial (P<0.001) and higher of venous thrombosis (P=0.01) prior to/at diagnosis vs older cohorts while events post-diagnosis were comparable across age groups (P=0.6). Young ET patients (n=219): Median age (range) was 32 years (18-40) with a minority (31%) constituted of males. During a median follow-up of 13 years, fibrotic progressions rates were 16% vs 16% and 9% in those aged 41-60 and >60 years, respectively (P<0.001). The leukemic conversion rate (2%) was analogous to that of older cohorts (5% and 3%, respectively) (P=0.1). Young patients also experienced significantly fewer arterial thrombotic events prior to/at diagnosis (6% vs 14% and 18%) and after diagnosis (8% vs 16% and 22%) compared to older cohorts (P<0.0001). Young PMF patients (n=63): Median age (range) was 37 years (19-40) with a preponderance of males (59%). High risk karyotype was not seen in young patients vs in 5% and 8% of those 41-60 and > 60 years of age, respectively (P=0.004). During median follow-up of 7.1 years, leukemic conversions were recorded in 10% of young patients vs 10% and 9% of respectively older cohorts (P=0.9). Young subjects also experienced fewer arterial thrombotic events prior to/at diagnosis (2% vs 6% and 13%) and lower rates of venous thrombosis after diagnosis (2% vs 10% and 4%) compared to cohorts 41-60 and > 60 years of age, respectively (P<0.0001). NGS data disclosed significant clustering of high risk molecular mutations with successively older subgroups (≤ 40 vs 41-60 vs > 60 years); ASXL1: 19 vs 40 vs 45% (P=0.005), SRSF2: 6 vs 8 vs 18% (P=0.0004), and U2AF1: 3 vs 11 vs 19% (P=0.004) (Table 1). Survival data Kaplan-Meier survival curves stratified by age bracket (≤ 40 vs 41-60 vs > 60 years) disclosed median OS in successively older age groups to be 37, 22, and 10 years for PV (Figure 1A), 35, 22, and 11 years for ET (1B), and 20, 8, and 3 years for PMF (1C); exposing significantly longer median survival estimates in younger patients, systematically across all disease subtypes (P<0.001). When appraised as a function of MPN subtype, survival data confirmed the significantly reduced OS in PMF relative to PV and ET (P<0.001), though PMF patients aged ≤ 40 years were still projected to have a reasonably favorable median life expectancy of 20 years (Figure 2). Conclusions: Young MPN patients comprise a unique disease subset defined by an attenuated-risk cytogenetic and mutational backdrop compared to their older counterparts. Caution must be exercised in counseling and managing this population as historical 'one-size-fits all' data fails to reflect the true natural history and disease biology in the young. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Subnormal serum erythropoietin (EPO) level is widely accepted as a minor diagnostic criterion for polycythemia vera (PV) and has retained its value in the revised 2016 World Health Organization (WHO) classification scheme of myeloid neoplasms (Blood. 2016;127:2391). While over 80% of PV patients exhibit subnormal EPO values (Haematologica. 2004;89:1194), very little is known about the incidence, phenotypic connotation, or prognostic significance of serum EPO levels in essential thrombocythemia (ET). Methods: Study patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnosis of ET was in strict accordance with the 2016 WHO criteria (Blood. 2016;127:2391) and only patients with EPO measurements at diagnosis or during their chronic phase were considered under the following provisions: i) no evidence of myelofibrotic or leukemic transformation; ii) no treatment with cytoreductive agents at the time of sample collection (and for those with EPO sampling at diagnosis, no prior exposure to cytoreductive therapy); iii) no hemoglobin levels below sex-adjusted values for anemia; and iv) systematic exclusion of all cases of masked PV. Serum erythropoietin levels were measured using Mayo-verified standard immune-enzymatic assay consistent with CLIA requirements with reference range between 4-21 mIU/mL. Standard statistical methods and time-to-event curves prepared using the Kaplan-Meier method were used to compare EPO-stratified groups. The JMP® Pro 13.0.0 software package was used for all analyses (SAS Institute, Cary, NC, USA). Results: Among 177 informative ET patients (median age 51 years, range 18-88; 71% females), median serum EPO concentrations were 5.2 mIU/mL (range <1-25) overall and 2.6 mIU/mL (range <1-3.9) and 7.2 mIU/mL (range 4-25) in those with subnormal versus normal/above normal EPO levels, respectively (p<0.0001). The majority of EPO measurements were at the time or within one year of diagnosis (67%), with the remainder obtained at referral or during the chronic clinical course. Risk stratification disclosed low, intermediate, and high risk status in 45%, 33% and 23% of patients, respectively, with no appreciable differences between EPO-stratified subsets. Subnormal serum EPO levels clustered clinically with JAK2 mutations (95% vs 60%; p<0.0001), higher hemoglobin (median 14.7 vs 13.8 g/dl, range: 12.1-17.8 vs 12-17.1; p<0.0001) and leukocyte counts (median 9.7 vs 8.2 x 109/l, range: 4-28.1 vs 3.9-22; p=0.0001), and arterial thrombosis after diagnosis (13% vs 5%; p=0.04). In addition, all three documented conversions to PV (p=0.01) and none of the six fibrotic progressions (p=0.02) occurred in patients with subnormal serum EPO levels (Table 1). While serum EPO level had no bearing on overall survival data (p=0.41) (Figure 1A), subnormal circulating EPO concentrations were significantly associated with superior myelofibrosis-free survival (p=0.02) (Figure 1B) but conversely, inferior polycythemia-free survival (p=0.03) (Figure 1C). Conclusions: We conclude that subnormal serum EPO levels are relatively frequent in ET, cluster with disease features that are reminiscent of polycythemia vera, and might be associated with a lower risk of fibrotic progression. Disclosures No relevant conflicts of interest to declare.

Abstract Background: We have long introduced the concept of host genetic variations in the phenotypic diversity of myeloproliferative neoplasms (MPN) (Blood 2008;111:2785). Previous studies have established an association between JAK2 mutations in myeloproliferative neoplasms (MPN) and the germline GGCC (46/1) haplotype, which constitutes a string of single nucleotide polymorphisms (SNPs) near the JAK2 gene that are inherited together on chromosome 9p (reviewed recently;Int J Mol Sci. 2018; 19: 1152). In 2010, we reported an association between shortened survival in primary myelofibrosis (PMF) and nullizygosity for the JAK2 46/1 haplotype (Leukemia 2010; 24:105), although our findings were not confirmed in another study (Leukemia 2010; 24:1533). Others have reported an association with splanchnic vein thrombosis, that was not accounted for by JAK2 mutations (Ann Hematol 2014;93:1845). In the current study, we have increased the number of informative cases to 414 (from 130 reported in 2010), in order to revisit with the phenotypic and prognostic relevance of the JAK2 46/1 haplotype in PMF. Methods : Study patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses PMF and its leukemic transformation were confirmed by both clinical and bone marrow examinations, in line with the 2016 World Health Organization criteria (Blood. 2016;127:2391). Screening for the JAK2 46/1 haplotype included rs12343867 SNP genotyping, as previously detailed (Leukemia 2010; 24:105), and using a commercially available TaqMan SNP genotyping assay (Applied Biosystems Inc., Foster City, CA, USA). Statistical analyses considered clinical and laboratory data collected at the time of initial PMF diagnosis or Mayo Clinic referral point. Conventional statistics was used for confirming phenotypic associations and calculation of overall (OS) and leukemia-free (LFS) survival. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations. Results: 414 patients with PMF (median age 63 years; 63% males) were included in the current study; among 324 evaluable cases, MIPSS70+ version 2.0 risk distribution was 18% very high risk, 41% high risk, 19% intermediate risk, 18% low risk and 4% very low risk. Driver mutation distribution was 63% JAK2, 17% type 1-like CALR, 3% type 2-like CALR, 7% MPL and 10% triple-negative. JAK2 46/1 haplotype was documented in 69% of the study patients, including 25% in homozygous and 44% in heterozygous state. Driver mutation frequency in patients homozygous/heterozygous/nullizygous for the 46/1 haplotype was 78%/60%/56% JAK2, 10%/20%/18% type 1-like CALR, 3%/2%/5% type 2-like CALR, 4%/8%/7% MPL and 6%/10%/14% triple-negative (p=0.02). The three 46/1 haplotype groups were phenotypically mostly similar, with the exception of platelet count (p=0.02) and leukocyte count (p=0.003), which were both higher with homozygous 46/1 haplotype. In univariate analysis, nullizygosity for the JAK2 46/1 haplotype was associated with inferior overall survival (HR 1.5, 95% CI 1.1-1.9; figure 1a); this survival effect was most pronounced in JAK2 mutated cases (figure 1b; p<0.001), as opposed to CALR/MPL mutated cases (figure 1c; p=0.48) or triple-negative cases (figure 1d; p=0.27). Multivariable analysis that included age and other genetic risk factors, including karyotype, driver mutational status and presence of high molecular risk mutations, such as ASXL1 and SRSF2, confirmed the independent prognostic contribution of nullizygosity for the 46/1 haplotype (p=0.02; HR 1.4, 95% CI 1.1-1.8). Nullizygosity for 46/1 also remained significant in the context of the recently unveiled genetics-based prognostic model, GIPSS (genetically-inspired prognostic scoring system) (p=0.04) (Leukemia.2018 doi: 10.1038/s41375-018-0107-z), but not in the context of MIPSS70+ version 2.0 (karyotype and mutation-enhanced international prognostic scoring system for transplant-age patients) (p=0.4). (JClinOncol.2018 doi: 10.1200/JCO.2018.78.9867). Leukemia-free survival was not affected by the 46/1 haplotype (p=0.6). Conclusions: The current study confirms the association of nullizygosity for the JAK2 GGCC (46/1) haplotype with inferior survival in PMF, primarily in JAK2-mutated cases; the observed survival effect was independent of currently acknowledged genetic risk factors, including karyotype and high molecular risk mutations. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Current drug therapy in primary myelofibrosis (PMF) offers palliative value only, including alleviation of splenomegaly, constitutional symptoms and anemia; hydroxyurea (HU), JAK2 inhibitors and interferon (IFN)-α are used for splenomegaly; JAK2 inhibitors for constitutional symptoms; and immunomodulatory drugs (IMiDs), erythropoiesis-stimulating agents (ESAs) and androgens for anemia. The current study constitutes a retrospective evaluation of specific drug response in anemia or splenomegaly in PMF. 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). For the purposes of the current study, conventional response criteria were modified to reflect clinical benefit assessment without strict adherence to criteria designed for clinical trials; accordingly, spleen response was evaluated only in patients with palpable splenomegaly and was defined as a minimum 50% reduction in palpable spleen size, regardless of response duration; anemia response was evaluated only in patients with hemoglobin level <10 g/dl and was defined as achieving transfusion-indpendence lasting for at least one month or an increase in hemoglobin of 2 g/dl, regardless of response duration. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all statistical calculations. Results: 432 cytogenetically- and molecularly-annotated patients with PMF were accessed in order to identify 333 patients who received first-line treatment with HU (n=97), JAK2 inhibitors (n=41), IFN-α (n=22), IMiDs (n=58), androgens (n=19), ESAs (n=54) and various other drugs (n=42), and were evaluable for response; driver mutational status was JAK2 59%, CALR type 1/like 20%, CALR type 2/like 4%, MPL 7% and triple-negative 10%; karyotype included very high risk (VHR) 6%, unfavorable 18% and favorable 76%; 60% of the patients harbored high molecular risk (HMR) mutations including ASXL1 (45%), SRSF2 (18%), U2AF1Q157 (10%), EZH2 (4%), IDH2 (4%) and IDH1 (2%). MIPSS70+ version 2.0 risk distribution was very high 17%, high 46%, intermediate 21%, low 14% and very low 2%. Overall, 249 patients were evaluable for spleen response, including 218 that were treated with the specific drugs analyzed in the current study. Anemia response was evaluated in 222 patients, including 194 (105 transfusion-dependent) that were treated with the specific drugs analyzed in the current study. Predictors of response to HU: Spleen response to HU was more likely in the presence of HMR mutations (37% vs 11%; p=0.02); however, responses were mixed and limited to presence of ASXL1 (40% vs 17%; p=0.06) or SRSF2 (47% vs 22%; p=0.1) mutations while none of 8 patients with either U2AF1Q157 or IDH2 mutations responded. None of 5 patients with VHR karyotype and only 1 (9%) of 11 patients with platelet count <100 x 109/l responded. Anemia responses to HU were infrequent. Overall response rate to HU was predicted by the absence of U2AF1Q157 mutations (64% vs 0%; p=0.007). Predictors of response to JAK2 inhibitors: Spleen response to JAK2 inhibitors was more likely in female patients (87% vs 50%; p=0.01) and in the presence of CALR type 1/like mutations (90% vs 57%; p=0.06). Anemia responses to JAK2 inhibitors were largely unpredictable. In order to further verify the aforementioned observed associations, we accessed data from a previous formal clinical trial of momelotinib (JAK2 inhibitor); among 91 evaluable patients, spleen response was higher in the presence of CALR mutations (73% vs 37%; p=0.009) and female sex (49% vs 39%, p=NS). Predictors of response to other agents: Spleen response to IFN-α was unlikely in the presence of ≥2% circulating blasts (0/6 responded) or presence of ASXL1 mutations (0/7 responded). IFN-α was ineffective for the treatment of anemia. Spleen responses to treatment with IMiDs, androgens or ESAs were unusual while anemia response to all three agents was not predicted by either genetic or clinical markers. Conclusions: Our observations, which require additional examination in a prospective setting, suggest limited value of genetic and clinical markers in predicting response to currently available drugs for PMF; this is consistent with the non-specific mechanism of action for these drugs. CALR (JAK inhibitors) and U2AF1Q157 (HU) mutations respectively predict favorable or unfavorable spleen response. Disclosures No relevant conflicts of interest to declare.