Lasho, Terra L.

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.

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.

Abstract Background: Type 1 calreticulin (CALR) variants comprise ~70% of all CALR mutations in primary myelofibrosis (PMF) and form a distinct phenotypic and prognostic disease subset (Leukemia. 2014;28:1568). Determinants of long-term outcome have not, however, been systematically appraised in this population. The current study documents the natural history, molecular correlates, and independent predictors of overall (OS), leukemia-free (LFS), and thrombosis-free (TFS) survival in CALR type 1/like-mutated myelofibrosis. Methods: Patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses were consistent with World Health Organization (PMF, fibrotic/leukemic transformations) (Blood. 2016;127:2391) and International Working Group for Myeloproliferative Neoplasms Research and Treatment criteria (post-essential thrombocythemia (ET) MF) (Leukemia. 2008;22:437). Laboratory and clinical data were retrospectively abstracted corresponding to time of referral (PMF) or myelofibrotic transformation (post-ET MF). Conventional prognostic scoring was as previously outlined (Blood. 2010;115:1703; J Clin Oncol. 2018;36:1769). Recipients of allogeneic stem cell transplant were censored at the time of transplant. Standard statistical methods were used for all analyses using the JMP® Pro 13.0.0 software package (SAS Institute, Cary, NC, USA). Results: A total of 162 consecutive patients with CALR type 1/like-mutated myelofibrosis were identified: 139 (86%) with PMF and 23 (14%) with post-ET MF with median age 55 years (range 23-85), 62% male. The PMF and post-ET MF cohorts displayed similar phenotypic features with the exception of higher platelet counts (median 443 vs 340 x 109/l; P=0.02) in post-ET MF (Table 1). The most frequent co-existing mutations were ASXL1 (n=47; 37%) and SRSF2 (n=4; 3%), with ASXL1 seen more frequently in PMF (39% vs 11% post-ET MF; P=0.07). Over a median follow-up of 6 years (range 0-25 years), a total of 20 (12%) leukemic transformations and 66 (41%) deaths were recorded, with no significant differences between the PMF and post-ET MF cohorts (P=0.16 and 0.11, respectively). Kaplan-Meier survival estimates revealed comparably favorable median OS in both CALR type 1/like-mutated variants: not yet reached and 13 years in post-ET MF vs PMF, respectively (P=0.7) (Figure 1A). Multivariable analysis disclosed moderate to severe sex-adjusted anemia (P<0.001), ≥2% circulating blasts (P<0.001), very high risk (VHR) karyotype (P<0.001), age >70 years (P=0.006), and constitutional symptoms (P=0.008) to be independent predictors of inferior OS in CALR type 1/like-mutated PMF (Table 2). LFS was significantly shortened in the presence of IDH1 mutations (P=0.01) and platelets <100 x 109/l (P=0.02) while IDH2 mutations (P=0.02), leukocytosis ≥11 x 109/l (P=0.03) and history of arterial thrombosis (P=0.04) were independent predictors of shortened TFS. Importantly, myelofibrosis variant (primary vs post-ET) did not influence survival (P=0.7) or complication rates (P=0.8 for LFS, P=0.09 for TFS) (Table 2). The karyotype- and mutation-enhanced international prognostic scoring system (MIPSS70+ version 2.0), was effective in risk stratifying type 1/like CALR-mutated PMF (P-values 0.01 to <0.0001), with the exception of low vs intermediate (P=0.22) and intermediate vs high risk (P=0.49) (Figure 1C). The detrimental influences of unfavorable/VHR karyotype and ASXL1 mutations were confirmed (Figure 2A-B) while borderline adverse and prognostically neutral effects were seen on OS for U2AF1 (n=116; P=0.05) and SRSF2 (n=120; P=0.98) respectively (Figure 2C-D). Conclusions: The current study documents analogous disease patterns in primary and post-ET CALR type 1/like-mutated MF and provides information on determinants of long-term survival. Disclosures No relevant conflicts of interest to declare.