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Chronic inflammation is believed to play an important role in the development and disease progression of polycythemia vera (PV). Since an association between gut microbiota, hematopoiesis, and inflammation is well established we hypothesized that patients with PV have a gut microbiota distinct from healthy controls. Recombinant interferon-α2 (IFN) treatment of patients with PV has been shown to be disease-modifying in terms of normalization of elevated blood cell counts in concert with a reduction in the JAK2V617F allelic burden. Therefore, we hypothesized that patients treated with IFN might have a composition of the gut microbiota towards normalization. Herein, via amplicon-based next generation sequencing of the V3-V4 regions of the 16S rRNA gene, we report on an abnormal gut microbiota in 102 patients with PV as compared with 42 healthy controls (HCs). Patients with PV had a lower alpha diversity and a lower relative abundance of several taxa belonging to Firmicutes (45%) compared with HCs (59%, p

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUTMPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUTMPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUTneoepitopes with high affinity are underrepresented in patients with CALRMUTMPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUTMPN would not efficiently respond to a CALRMUTfragment cancer vaccine but would when immunized with a modified CALRMUTheteroclitic peptide vaccine approach. We found that heteroclitic CALRMUTpeptides specifically designed for the MHC-I alleles of patients with CALRMUTMPN efficiently elicited a CALRMUTcross-reactive CD8+T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUTnative peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+T cell response to the CALRMUTfragment upon immunization with a CALRMUTheteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide–based cancer vaccines in patients with CALRMUTMPN.

The effects of smoking on the molecular response (MR) and overall survival (OS) in patients with chronic myeloproliferative neoplasms (MPNs) have not been investigated before. We analysed a historical cohort of 498 consecutive patients diagnosed with MPNs. Moreover, we analysed a subgroup of 270 consecutive patients with MPNs with > 1 measurement of the JAK2V617F variant allele frequency. The data were analysed using Kaplan-Meier plots and Cox regression analysis, along with linear regression models. In all patients, the rate of MR was significantly higher in never-smokers compared with current smokers in the univariate model (HR, 1·9; 95% CI, 1·1-3·3; P = 0·033) and the multivariate model (HR, 1·9; 95% CI, 1·1-3·5; P = 0·029). Similar findings were observed with different cut-off values for a partial MR. A subgroup analysis including only interferon-α2-treated patients showed similar results. In multivariate analyses, the OS was significantly better for never-smokers (HR, 0·46; 95% CI, 0·29-0·75; P = 0·002) than current smokers. The differences were more pronounced in the pegylated interferon-α2-treated patients. However, no significant interaction of interferon-α2 treatment was observed. In conclusion, we found that tobacco smoking reduced the rate of MR and OS in patients with MPNs. Cessation of smoking should be encouraged.

Treatment with PEGylated interferon‐alpha2 (IFN) of patients with essential thrombocythemia and polycythemia vera induces major molecular remissions with a reduction in the JAK2V617F allele burden to undetectable levels in a subset of patients. A favorable response to IFN has been argued to depend upon the tumor burden, implying that institution of treatment with IFN should be as early as possible after the diagnosis. However, evidence for this statement is not available. We present a thorough analysis of unique serial JAK2V617F measurements in 66 IFN‐treated patients and in 6 untreated patients. Without IFN treatment, the JAK2V617F allele burden increased exponentially with a period of doubling of 1.4 year. During monotherapy with IFN, the JAK2V617F allele burden decreased mono‐ or bi‐exponentially for 33 responders of which 28 patients satisfied both descriptions. Bi‐exponential description improved the fits in 19 cases being associated with late JAK2V617F responses. The decay of the JAK2V617F allele burden during IFN treatment was estimated to have half‐lives of 1.6 year for the monoexponential response and 1.0 year in the long term for the bi‐exponential response. In conclusion, through data‐driven analysis of the JAK2V617F allele burden, we provide novel information regarding the JAK2V617F kinetics during IFN‐treatment, arguing for early intervention., Data‐driven mathematics of the kinetics of the JAK2V617F allele burden during treatment with IFN is presented. The half‐life of the JAK2V617F allele burden was predicted as 1.6 year, and 1.0 year in the long term, supporting early treatment with IFN.

Although somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPNs), little is known about their impact on molecular response to cytoreductive treatment. We performed targeted next-generation sequencing (NGS) on 202 pretreatment samples obtained from patients with MPN enrolled in the DALIAH trial (A Study of Low Dose Interferon Alpha Versus Hydroxyurea in Treatment of Chronic Myeloid Neoplasms; #NCT01387763), a randomized controlled phase 3 clinical trial, and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea. The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (median, 0.29 to 0.07; P < .0001) compared with those not achieving CHR (median, 0.27 to 0.14; P < .0001). In contrast, the CALR variant allele frequency did not significantly decline in those achieving CHR or in those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with hydroxyurea (P = .04). Furthermore, treatment-emergent DNMT3A mutations were significantly enriched in IFNα–treated patients not attaining CHR (P = .02). A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted odds ratio, 5.29; 95% confidence interval, 1.59-17.54; P = .007), as was a mutation in TET2 alone (age-adjusted odds ratio, 3.03; 95% confidence interval, 1.03-9.01; P = .044). At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN exhibited distinct molecular responses; and (2) DNMT3A-mutated clones/subclones emerged on treatment.

Introduction: The Philadelphia-negative myeloproliferative neoplasms (MPN) are associated with driver mutations in JAK2, CALR, and MPL genes. Non-driver mutations affect disease progression and treatment response. Combination therapy with pegylated interferon-alpha2 (IFN) and ruxolitinib has recently been shown to induce hematologic and molecular responses in patients (pts) with MPN. We studied 25 pts by targeted next generation sequencing (NGS) of 42 genes and investigated the impact of somatic mutations (mut) on treatment response. Methods: Twenty-five MPN pts with polycythemia vera (PV) (n=16, all JAK2V617F) and myelofibrosis (MF) (n=9, JAK2V617F (6), CALR (1), MPL (1), triple-negative (1)) participated in the study. NGS was performed on the Illumina platform on DNA from peripheral blood at baseline and after 24 months of combination therapy. Data were analyzed using Biomedical Genomics workbench and VarSeq. Variants with coverage 1% were excluded. A mut with VAF 1% in the paired sample. However, a mut with a number of mutated reads below 7 was considered absent. Only pathogenic mut were reported. Statistical analysis was done using either chi-square test or Wilcoxon signed rank/rank sum test in R 4.0.2. A p-value Results: At baseline and 24 months, PV-pts had a mean number of mut of 2.3 and 2.4, and MF-pts 2.2 and 3.0, respectively. In all pts, 0 and 1 (0/4%) patient had no mut, 10 and 7 (40/28%) had 1 mut, 10 and 9 (40/36%) had 2 mut, and 5 and 8 (20/32%) had ≥ 3 mut at baseline and 24 months, respectively. In PV, 1 (6%) achieved CR and 3 (19%) achieved PR and 12 (75%) achieved NR. In MF, 4 (44%) achieved CR and 1 (11%) achieved PR and 4 either NR, PD, SD or CI. There was no association between pts achieving CR/PR or NR and median number of mut at baseline (CR/PR: 1, range 1-3) or (NR: 2, range 1-8), (p=0.28). Excluding driver mut, there was still no significant association. In all JAK2V617F positive pts, the median JAK2V617F allele burden (%JAK2V617F) decreased from 37% (range: 1.9-95) at baseline to 22.5% (range: 0-85) at 24 months. In pts achieving CR or PR (n=7), the reduction in %JAK2V617F was greater (median: 42% to 18%, p Stratified according to molecular response (MR) (n=4) or non-MR (n=13), median % JAK2V617F at baseline was 40 (21-95) and 43 (42-70) in non-MR and MR, respectively, and 31 (11-85) and 13 (1.8-25) in non-MR and MR, respectively during treatment. At baseline, pts achieving MR had no non-driver mut and pts achieving non-MR had a median number of non-driver mut of 1 (range: 0-7) (p In all pts, there were 30 non-driver mut in 15 genes at baseline and 40 non-driver mut in 19 genes at 24 months of therapy. At baseline and at 24 months, TP53 occurred in 6 and 6 (24%), TET2, ASXL1, RUNX1, or SF3B1 in 2 and 3 (8/12%), and CBL, DNMT3A, or SRSF2 in 2 and 2 (8%) pts, respectively. Eleven new non-driver post-treatment mut occurred in 9 pts and were more prevalent in pts with MF (6/9, 66%) compared to pts with PV (3/16, 19%), p Discussion and Conclusions: Earlier studies have shown an association between poor response to IFN and number of mut or high molecular risk mut. In this study, the presence of non-driver mut was associated with a significantly poorer molecular response. However, there was no association between the presence or type of non-driver mut and clinico-hematologic response. At 24 months, there was an evolution of subclones with a significantly higher number of new subclones appearing in pts with MF compared to pts with PV. However, the VAF was low and none of the acquired mut appeared in the same gene contradictory to another study showing an association between treatment-emergent mut in DNMT3A and treatment with IFN. In conclusion, these data show that a clinico-hematologic response is achievable during combination therapy despite the presence of non-driver mutations at baseline. Disclosures Bjørn: Novartis: Other: Ruxolitnib. Hasselbalch: Novartis, AOP Orphan: Consultancy, Other: Advisory Board. OffLabel Disclosure: Interferon-alpha for treatment

Recent studies have shown the Philadelphia-negative myeloproliferative neoplasms (MPN) to be massively underdiagnosed and often preceded by a long pre-diagnostic phase of several years, in which many patients suffer serious vascular events. In this review, we focus on the urgent need for earlier diagnosis and treatment of MPN. Such efforts are foreseen to decrease morbidity and mortality for the individual patients and potentially reduce costs for health and social care systems.

BACKGROUND Hydroxyurea (HU) treatment of patients with essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) (MPNs) normalizes elevated blood cell counts within weeks in the large majority of patients. Studies on the impact of HU upon the kinetics of the JAK2V617F allele burden, leukocyte, and platelet counts over time are scarce. PURPOSE Using data-driven analysis as a novel tool to model the kinetics of the JAK2V617F allele burden and blood cell counts over time during treatment with HU. MATERIAL AND METHODS Using serial measurements of JAK2V617F and correlation analysis of routine hematological values (the Hb-concentration, leukocyte count, platelet count, and lactic dehydrogenase), we present a detailed description and analysis of the kinetics of the JAK2V617F, leukocyte, and platelet counts and lactic dehydrogenase in 27 patients (PV = 18; ET = 7; PMF = 2), who were followed in the Danish randomized trial (DALIAH). To further analyze the JAK2V617F kinetics, we use a machine learning clustering algorithm to group the response patterns. RESULTS Response patterns were highly heterogeneous, with clustering resulting in 3 groups and 3 outliers. In the large majority of patients, HU treatment was initially associated with a modest decline in the JAK2V617F allele burden in concert with a decline in leukocyte and platelet counts. However, HU did not induce a sustained and continuous decrease in the JAK2V617F allele burden. CONCLUSION Using data-driven analysis of the JAK2V617F allele burden, leukocyte, and platelet kinetics during treatment with HU, we have shown that HU does not induce a sustained decrease in the JAK2V617F allele burden and neither induces sustained normalization of elevated cell counts in MPN patients. Our results may explain why MPN patients during treatment with HU still have a substantially increased risk of thrombosis.

(1) Background: myeloproliferative neoplasms (MPNs) are slowly developing hematological cancers characterized by few driver mutations, with JAK2V617F being the most prevalent. (2) Methods: using mechanism-based mathematical modeling (MM) of hematopoietic stem cells, mutated hematopoietic stem cells, differentiated blood cells, and immune response along with longitudinal data from the randomized Danish DALIAH trial, we investigate the effect of the treatment of MPNs with interferon-&alpha, 2 on disease progression. (3) Results: At the population level, the JAK2V617F allele burden is halved every 25 months. At the individual level, MM describes and predicts the JAK2V617F kinetics and leukocyte- and thrombocyte counts over time. The model estimates the patient-specific treatment duration, relapse time, and threshold dose for achieving a good response to treatment. (4) Conclusions: MM in concert with clinical data is an important supplement to understand and predict the disease progression and impact of interventions at the individual level.

AIMS/HYPOTHESIS: Plasma ATP is a potent vasodilator and is thought to play a role in the local regulation of blood flow. Type 2 diabetes is associated with reduced tissue perfusion. We aimed to examine whether individuals with type 2 diabetes have reduced plasma ATP concentrations compared with healthy control participants (case-control design).METHODS: We measured femoral arterial and venous plasma ATP levels with the intravascular microdialysis technique during normoxia, hypoxia and one-legged knee-extensor exercise (10 W and 30 W) in nine participants with type 2 diabetes and eight control participants. In addition, we infused acetylcholine (ACh), sodium nitroprusside (SNP) and ATP into the femoral artery to assess vascular function and ATP signalling.RESULTS: Individuals with type 2 diabetes had a lower leg blood flow (LBF; 2.9 ± 0.1 l/min) compared with the control participants (3.2 ± 0.1 l/min) during exercise (p CONCLUSIONS/INTERPRETATION: These findings demonstrate that individuals with type 2 diabetes have lower plasma ATP concentrations during exercise and hypoxia compared with control individuals, and this occurs in parallel with lower blood flow. Moreover, individuals with type 2 diabetes have a reduced vasodilatory response to infused ATP. These impairments in the ATP system are both likely to contribute to the reduced tissue perfusion associated with type 2 diabetes.TRIAL REGISTRATION: ClinicalTrials.gov NCT02001766.

Pegylated interferon-α (peg-IFNa) treatment induces molecular responses (MR) in patients with myeloproliferative neoplasms (MPNs), including partial MR (PMR) in 30–40% of patients. Here, we compared the efficacy of IFNa treatment in JAK2V617F- vs. calreticulin (CALR)-mutated cells and investigated the mechanisms of differential response. Retrospective analysis of MPN patients treated with peg-IFNa demonstrated that patients harboring the JAK2V617F mutation were more likely to achieve PMR than those with mutated CALR (p = 0.004), while there was no significant difference in hematological response. In vitro experiments confirmed an upregulation of IFN-stimulated genes in JAK2V617F-positive 32D cells as well as patient samples (peripheral blood mononuclear cells and CD34+ hematopoietic stem cells) compared to their CALR-mutated counterparts, and higher IFNa doses were needed to achieve the same IFNa response in CALR− as in JAK2V617F-mutant 32D cells. Additionally, Janus-activated kinase-1 (JAK1) and signal transducers and activators of transcription 1 (STAT1) showed constitutive phosphorylation in JAK2V617F-mutated but not CALR-mutated cells, indicating priming towards an IFNa response. Moreover, IFN-induced growth arrest was counteracted by selective JAK1 inhibition but enhanced by JAK2 inhibition. In conclusion, our data suggest that, clinically, higher doses of IFNa are needed in CALR-mutated vs. JAK2V617F-positive patients and we suggest a model of JAK2V617F-JAK1/STAT1 crosstalk leading to a priming of JAK2V617F-positive cells to IFNa resulting in differential sensitivity.

Background: The human gut microbiota (the population of microorganisms present) is important for digestion of food but also for development of the host immune system and protection against pathogens. Changes in the gut microbiota are linked to several inflammatory diseases such as diabetes, atopic diseases and Alzheimer's disease. Polycythemia vera (PV) is one of the Philadelphia chromosome negative classical myeloproliferative neoplasms (MPNs), which also include essential thrombocythemia (ET) and primary myelofibrosis (PMF). MPNs are increasingly recognized as inflammatory driven diseases. The role of the gut microbiota in patients with MPNs is largely unknown. In a small study (n=25) the microbiota of MPN patients had higher levels of Prevotellaceae compared to healthy controls, and differed significantly in composition between patients treated with hydroxyurea and ruxolitinib. Since MPNs are likely to be driven by chronic inflammation and the gut microbiota influences the immune system, investigations of the PV-microbiota are highly relevant. We compared the microbiota in a cross-sectional study of patients with PV stratified into five different treatment groups. Method and Material: Patients above 18 years diagnosed with PV, according to the 2016 World Health Organization (WHO) classification, were invited to participate in the study. The exclusion criteria were: pregnancy, use of antibiotics within the last 2 months, change in treatment within the last 3 months or inability to understand the oral and written information. Clinical and biochemical data for each patient were collected retrospectively and included co-morbidities, smoking status, anti-inflammatory treatment, hypertension, haematological parameters, haematological treatment, body mass index (BMI), among others. Stool samples, no more than 6 hours old were stored at -80°C. DNA was extracted by using the EMAG® Nucleic acid extraction system, [bioMérieux] according to the manufacturer's instructions. The bacterial microbiota was characterized by amplicon-based next generation sequencing of the V3-V4 region of the 16S ribosomal unit using a MiSeq instrument, Illumina. BION was used to assign taxonomic classification. The patients were divided into 5 groups according to treatment: no treatment (n=18), hydroxyurea (n=33), PEG-interferon-α2 (IFN) (n=23), IFN combined with ruxolitinib (COMBI) (n= 21) and patients treated with other combinations e.g. ruxolitinib, anagrelide, hydroxyurea combined with IFN hydroxyurea combined anagrelide, or hydroxyurea combined with ruxolitinib, (n=11). The alpha diversity was measured using the Shannon diversity index, and compared with a pairwise Wilcoxon test adjusted for multiple testing. Beta diversity (difference between the samples) was visualised by a PCoA plot, and compared using an ANOSIM test. Differential abundance analysis was performed by Linear discriminant analysis Effect Size (LEfSe). Results: In total, 116 patients with PV were included. Of these, 106 fulfilled the inclusion criteria (49 women and 57 men) and had a median age of 68 years (range: 31 - 85). The five treatment groups did not differ in alpha diversity. The bacterial composition differed slightly between IFN group and no treatment group (p=0.032, R=0.075), and between IFN and COMBI group (p= 0.031, R=0.054). Patients from the no treatment group had a higher relative abundance of the Bacteroides genus (39%) compared to the IFN group (14.7%) (p=0.003) and hydroxyurea group (23.1%) (p=0.047), but not the COMBI group (30.1%). A lower abundance of the Bacteroides genus was found in the IFN group compared to the COMBI group (p=0.004) and compared to the hydroxyurea group (p=0.04). Due to the small number of patients treated with other combinations, these were not included in the differential abundance analysis. Conclusions: Among the five treatment groups in patients with PV, the alpha diversity of the microbiota were similar, but the relative abundance of the Bacteroides genus in patients not receiving any treatment compared to patients treated with IFN and hydroxyurea was higher. A lower abundance of Bacteroides genus was seen in the IFN group compared to the COMBI group and compared to the hydroxyurea group. Whether these differences are explained as a treatment response to IFN or clinical parameters, such as comorbidities, age JAK2 allele burden or BMI need further investigations. Disclosures Hasselbalch: Novartis, AOP Orphan: Consultancy, Other: Advisory Board.

Background Recombinant Interferon Alpha-2a (r-IFNα) is a potent immunomodulating agent, which has been used off-label for the treatment of polycythemia vera (PV) for more than three decades and has been demonstrated to induce high rates of clinical, hematological and molecular responses. Only few studies have compared efficacy and safety of r-IFNα vs. hydroxyurea (HU), which is considered first line therapy for PV patients > 60 years in most parts of the world. However, recent studies have provided encouraging results for the treatment of PV with r-IFNα compared to HU irrespective of age (R. Hoffmann 2016; H. Gisslinger 2018). Aims To examine the difference in efficacy and safety of low-dose r-IFNα in PV patients ≤ 60 or > 60 years of age compared to HU > 60 years of age. Methods Ninety newly diagnosed or previously phlebotomized PV patients only (WHO 2008) were enrolled in the DALIAH trial (NCT01387763). All patients provided written informed consent. Patients ≤ 60 years were randomized (I:I) to r-IFNα-2a (Pegasys®) or to r-IFNα-2b (PegIntron®) whereas patients > 60 years were randomized (I:I:I) to either r-IFNα-2a, r-IFNα-2b or to HU. The starting dose of r-IFNα-2a and r-IFNα-2b was 45 or 35 µg/week, respectively. The HU dose was 500 to 2000 mg/day. Patients randomized to r-IFNα who presented with major thrombosis or platelets > 1500 109/L received HU from inclusion and until normalization of the platelet count. Efficacy assessment consisted of the clinicohematological and the molecular response rates by intention to treat analysis (ITT) using the European Leukemia Net (ELN) 2009 criteria. JAK2V617F analysis was performed by qPCR. Groups were compared by Fisher's Exact Test. Results Three-year analysis was available in all but five patients (n=85) at time of abstract submission (Table 1). The analysis was performed after a median of 36 months (range: 33-39 months). The median treatment dose was 684 mg/day (IQR: 131 - 942) for HU, 51 μg/week (IQR: 30-90 μg/week) and 54 μg/week (IQR: 30-66 μg/week) for r-IFNα-2a age ≤ 60 and > 60, respectively, and 41 μg/week (IQR: 29-45 μg/week) and 36 μg/week (IQR: 31-37 μg/week) for r-IFNα-2b age ≤ 60 and > 60. The overall clinicohematological response rate (ORR) was 68% (13/19) for HU, 42% (14/33) for r-IFNα ≤ 60 years and 39% (13/33) for r-IFNα > 60 years. The partial clinicohematological response rate (PHR) and the complete clinicohematological response rate (CHR) was 53% (10/19) and 16% (3/19) for HU, 9% (3/33) and 33% (11/33) for r-IFNα ≤ 60 years and 9% (3/33) and 30% (10/33) for r-IFNα > 60 years. Neither the ORR, CHR nor the PHR was significantly different between the three groups. Maintenance of CHR from first occurrence to data analysis after 36 months was 11% (2/19) for HU, 21% (7/33) for r-IFNα ≤ 60 years and 18% (6/33) for r-IFNα > 60 years. Forty-seven JAK2V617F positive patients were available for molecular response analysis after 36 months of therapy. A partial molecular response (PMR) was detected in 21% (4/19) of HU treated patients and in 24% (7/29) of r-IFNα treated patients ≤ 60 years and 18% (6/33) of r-IFNα > 60 years. Notably, 7% (2/29) of the r-IFNα treated patients ≤ 60 years obtained a complete molecular response (CMR). The median JAK2V617F reduction from baseline was 38% (IQR: 31-63%) for HU, 79% (IQR: 59-92%) for r-IFNα ≤ 60 years and 73% (IQR: 49-97%) for r-IFNα > 60 years. There was no statistically significant difference in the PMR between groups. Discontinuation of treatment for any reason after 36 months of therapy was 21% (4/19) for HU, 52% (17/33) for r-IFNα ≤ 60 years and 45% (15/33) for r-IFNα > 60 years. Toxicity related discontinuation was 5% (1/19) for HU and 30% (10/33) for both r-IFNα ≤ 60 and > 60 years. Grade 3-4 AEs occurred in 32% (6/19) of HU treated patients, 27% (9/33) in r-IFNα treated patients ≤ 60 years and in 42% (14/33) r-IFNα treated patients > 60 years. SAEs were reported in 21% (4/19) for HU, 9% (3/33) for r-IFNα ≤ 60 years and 24% (8/33) for r-IFNα > 60 years. The numbers of grade 3-4 AEs as well as SAEs were comparable between groups. Conclusion After 36 months of therapy CHR was non-significantly higher in PV patients treated with r-IFNα compared to HU by ITT, irrespective of age. Also, maintenance of CHR was longer for r-IFNα. However, ORR was non-significantly higher for HU. PMR was almost similar between the three groups but the median JAK2V617F reduction was greater for r-IFNα. Toxicity related discontinuation from study therapy was higher for r-IFNα compared to HU. Disclosures Stentoft: Bristol-Myers Squibb: Research Funding; Merck Sharp&Dohme: Research Funding. Hasselbalch:Novartis: Research Funding.

A novel mechanism-based model - the Cancitis model - describing the interaction of blood cancer and the inflammatory system is proposed, analyzed and validated. The immune response is divided into two components, one where the elimination rate of malignant stem cells is independent of the level of the blood cancer and one where the elimination rate depends on the level of the blood cancer. A dimensional analysis shows that the full 6-dimensional system of nonlinear ordinary differential equations may be reduced to a 2-dimensional system - the reduced Cancitis model - using Fenichel theory. The original 18 parameters appear in the reduced model in 8 groups of parameters. The reduced model is analyzed. Especially the steady states and their dependence on the exogenous inflammatory stimuli are analyzed. A semi-analytic investigation reveals the stability properties of the steady states. Finally, positivity of the system and the existence of an attracting trapping region in the positive octahedron guaranteeing global existence and uniqueness of solutions are proved. The possible topologies of the dynamical system are completely determined as having a Janus structure, where two qualitatively different topologies appear for different sets of parameters. To classify this Janus structure we propose a novel concept in blood cancer - a reproduction ratio R. It determines the topological structure depending on whether it is larger or smaller than a threshold value. Furthermore, it follows that inflammation, affected by the exogenous inflammatory stimulation, may determine the onset and development of blood cancers. The body may manage initial blood cancer as long as the self-renewal rate is not too high, but fails to manage it if an inflammation appears. Thus, inflammation may trigger and drive blood cancers. Finally, the mathematical analysis suggests novel treatment strategies and it is used to discuss the in silico effect of existing treatment, e.g. interferon-α or T-cell therapy, and the impact of malignant cells becoming resistant.

Background: To investigate the role of genomics in determining response and resistance to front line treatment in MPN, we performed somatic mutational profiling of the DALIAH trial, a randomized controlled phase III trial of interferon versus hydroxyurea in newly diagnosed MPN patients. Methods: We performed genomic analyses on 202 pre-treatment primary MPN samples obtained from patients enrolled on the DALIAH trial (NCT01387763) and 135 samples obtained after 24 months of treatment. Genomic profiling comprised targeted next generation sequencing (NGS) of 100 genes, selected on the basis of their known or suspected involvement in the pathogenesis of myeloid malignancies, and 1609 informative single nucleotide polymorphisms (SNPs) on chromosome 9p. Clinicohematological response was determined by central review using ELN 2009 (ET, PV and pre-MF) and EUMNET 2005 (PMF) criteria. We evaluated the association of somatic mutations with clinical parameters and with attainment of clinicohematological complete response (CR) at 24 months. Results: Prior to treatment, 191 of 202 (95%) patients had somatic mutations, including 93% with canonical MPN phenotypic drivers: JAK2 (74%), CALR (14%), and MPL (5%). Among those with JAK2 mutations 37% had JAK2 copy-neutral loss of heterozygosity (JAK2 CN-LOH). Patients with PV were more likely to have JAK2 CN-LOH (p = 0.0001) as compared with patients with other MPN subtypes. At baseline, patients with JAK2 CN-LOH had significantly higher hemoglobin (p = 0.0001), higher white blood cell count (WBC, p = 0.002) and lower platelet count (p=0.0001) than patients without JAK2 CN-LOH. Mutations in TET2 (24%), DNMT3A (16%), and ASXL1 (10%) were the most frequent co-occurring non-MPN phenotypic driver mutations and they occurred across all MPN subtypes. In addition, 5% of patients had spliceosome gene mutations, and 6% had mutations in genes involved in RAS/MAPK signaling. Patients with TET2, DNMT3A or ASXL1 mutations were significantly older than patients without these mutations (p= 0.0001) and there was a significant association between the presence of a TET2, DNMT3A or ASXL1 mutation and prior stroke (p = 0.004). There were no other significant associations between somatic mutation status and baseline clinical characteristics. The probability of attaining clinicohematological CR at 24 months was independent of baseline somatic mutations. Among patients with JAK2 mutations who remained on interferon treatment at 24 months, those with CR had a greater reduction in mean variant allele fraction (VAF) (28% to 8%, p Among patients who remained on treatment for 2 years, 44 mutations in 35 patients were newly detected or expanded on serial sampling. DNMT3A mutations were the most frequently acquired, accounting for 41% of new mutations. ASXL1, TET2, PPM1D, TP53, IDH2, and CBL mutations were also recurrently acquired. 97% of patients who acquired new mutations were JAK2-mutant. Among those with acquired DNMT3A mutations, 85% were treated with interferon, and 23% had CR at 24 months. Among those that acquired non-DNMT3A mutations, 38% were treated with interferon and 47% had CR at 24 months. The VAF of newly detected mutations was low (median 1.5%), and half of the patients with newly acquired mutations had at least 50% reduction in JAK2V617F VAF, suggesting that new mutations could either have arisen independently or be subclonal to the dominant JAK2-mutant clone. Conclusions: Using sequential genomic analyses of a phase III clinical trial of interferon versus hydroxyurea in MPN patients, we found mutation-specific and treatment-specific patterns of response. We uncovered distinct patterns of response to interferon in JAK2-mutant MPN as compared with CALR-mutant MPN. We found that DNMT3A mutations were the most frequent acquired mutations at 24 months and that these were enriched in patients treated with interferon. In aggregate, these results provide insights into molecular response and resistance to interferon and inform the clinical use of interferon in MPN patients. Disclosures Hansen: Alexion: Research Funding. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Hasselbalch:Novartis: Research Funding; AOP Orphan Pharmaceuticals: Other: Data monitoring board. Lindsley:Jazz Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding. Mullally:Janssen: Research Funding.

Background: Treatment of polycythemia vera (PV) patients with hydroxyurea (HU) normalizes elevated blood cell counts within weeks in the large majority of patients. Studies on the impact of HU upon the kinetics of the JAK2V617F allele burden, leukocyte and platelets over time are scarce, and results have been reported highly heterogeneous. Purpose: Using data driven analysis as a novel tool to model the kinetics of the JAK2V617F allele burden and blood cell counts over time during treatment with HU. Material and Methods: Using serial measurements of JAK2V617F and correlation analyzes of routine hematological values (Hb-concentration, leukocyte count, platelet count, lactic dehydrogenase), we present a detailed description and analysis of the kinetics of the JAK2V617F, leukocyte and platelet counts and lactic dehydrogenase in 29 PV patients who were followed in the Danish randomized trial (DALIAH) comparing the efficacy of pegylated interferon-alpha2 (IFN) versus HU in patients older than 60 years. Results: Response patterns were highly heterogeneous. In the large majority of patients, HU treatment was initially associated with a modest decline in the JAK2V617F allele burden in concert with a decline in leukocyte and platelet counts. However, HU did not induce a sustained and continuous decrease in the JAK2V617F allele burden in any patient as previously reported in patients being treated with IFN. Importantly, HU treatment was neither able to induce a sustained normalization of elevated leukocyte and platelet counts, although counts were temporarily normalized in most patients during the first months of therapy. The fluctuating leukocyte and platelet counts contrast the sustained normalization of cell counts in the large majority of patients during long term treatment with IFN. Discussion and Conclusions: Using data-driven analysis of the JAK2V617F allele burden, leukocyte and platelet kinetics during treatment with HU, we have shown that HU does not induce a sustained decrease in the JAK2V617F allele burden and neither induces sustained normalization of elevated cell counts in PV patients. Our results may explain why PV patients during treatment with HU still have a substantially increased risk of thrombosis. Based upon our previous and present findings, obtained by mathematical modelling and data driven analysis studies, a rational and cost-effective treatment might be a combination therapy of HU and IFN, both being used for decades in the treatment of PV but according to the findings in our studies now being proposed to be combined in the initial treatment period, since their combined effects might be highly efficacious and are foreseen to have the potential to minimize the risk of thrombosis and bleeding. By these studies, we have also proven mathematical modelling and data driven analysis to be highly important tools to decipher novel treatment modalities for patients suffering from MPN cancers but likely other cancers as well. Disclosures Hasselbalch: Novartis: Research Funding; AOP Orphan Pharmaceuticals: Other: Data monitoring board. OffLabel Disclosure: Interferon-alpha2 for the treatment of myeloproliferative neoplasms.

High levels of cardiovascular fitness (CRF) and physical activity (PA) are associated with decreased mortality and risk to develop metabolic diseases. The independent contributions of CRF and PA to metabolic disease risk factors are unknown. We tested the hypothesis that runners who run consistently >50 km/wk and/or >2 marathons/yr for the last 5 years have superior metabolic fitness compared to matched sedentary subjects (CRF, age, gender, and BMI). Case-control recruitment of 31 pairs of runner-sedentary subjects identified 10 matched pairs with similar VO2max(mL/min/kg) (similar-VO2max). The similar-VO2maxgroup was compared with a group of age, gender, and BMI matched pairs who had the largest difference in VO2max(different-VO2max). Primary outcomes that defined metabolic fitness including insulin response to an oral glucose tolerance test, fasting lipids, and fasting insulin were superior in runners versus sedentary controls despite similar VO2max. Furthermore, performance (velocity at VO2max, running economy), improved exercise metabolism (lactate threshold), and skeletal muscle levels of mitochondrial proteins were superior in runners versus sedentary controls with similar VO2max. In conclusion subjects with a high amount of PA have more positive metabolic health parameters independent of CRF. PA is thus a good marker against metabolic diseases.

Recent studies have shown the Philadelphia-negative myeloproliferative neoplasms (MPN) to be massively underdiagnosed and often preceded by a long pre-diagnostic phase of several years, in which many patients suffer serious vascular events. In this review, we focus on the urgent need for earlier diagnosis and treatment of MPN. Such efforts are foreseen to decrease morbidity and mortality for the individual patients and potentially reduce costs for health and social care systems.