Low Allele Burden Research Articles

Inflammatory crosstalk impairs phagocytic receptors and aggravates atherosclerosis in clonal hematopoiesis in mice.

Clonal hematopoiesis (CH) increases inflammasome-linked atherosclerosis but the mechanisms by which CH mutant cells transmit inflammatory signals to non-mutant cells are largely unknown. To address this question we transplanted 1.5% Jak2VF bone marrow (BM) cells with 98.5% WT BM cells into hyperlipidemic Ldlr-/- mice. Low allele burden (LAB) mice showed accelerated atherosclerosis with increased features of plaque instability, decreased levels of macrophage phagocytic receptors MERTK and TREM2, and increased neutrophil extracellular traps (NETs). These changes were reversed when Jak2VF BM was transplanted with Il1r1-/- BM. LAB mice with non-cleavable MERTK in WT BM showed improvements in necrotic core and fibrous cap formation and reduced NETs. An agonistic TREM2 antibody (4D9) markedly increased fibrous caps in both control and LAB mice eliminating the difference between groups. Mechanistically, 4D9 increased TREM2+PDGFB+ macrophages and PDGF receptor-α positive fibroblast-like cells in the cap region. TREM2 and PDGFB mRNA levels were positively correlated in human carotid plaques and co-expressed in macrophages. In summary, low frequency Jak2VF mutations promote atherosclerosis via IL-1 signaling from Jak2VF to WT macrophages and neutrophils promoting cleavage of phagocytic receptors and features of plaque instability. Therapeutic approaches that stabilize MERTK or TREM2 could promote plaque stabilization especially in CH- and inflammasome-driven atherosclerosis.

Superior detection of low-allele burden Janus kinase 2 V617F mutation and monitoring clonal evolution in myeloproliferative neoplasms using chip-based digital PCR

The JAK2 V617F is a prevalent driver mutation in Philadelphia chromosome-negative myeloproliferative neoplasms (Ph−MPNs), significantly affecting disease progression, immunophenotype, and patient outcomes. The World Health Organization (WHO) guidelines highlight the JAK2 V617F mutation as one of the key diagnostic criterions for Ph−MPNs. In this study, we analyzed 283 MPN samples with the JAK2 V617F mutation to assess the effectiveness of three detection technologies: chip-based digital PCR (cdPCR), real-time quantitative PCR (qPCR), and next-generation sequencing (NGS). Additionally, we investigated the relationship between JAK2 V617F mutant allele burden (% JAK2 V617F) and various laboratory characteristics to elucidate potential implications in MPN diagnosis. Our findings demonstrated high conformance of cdPCR with qPCR/NGS for detecting % JAK2 V617F, but the mutant allele burdens detected by qPCR/NGS were lower than those detected by cdPCR. Moreover, the cdPCR exhibited high sensitivity with a limit of detection (LoD) of 0.08% and a limit of quantification (LoQ) of 0.2% for detecting % JAK2 V617F in MPNs. Clinical implications were explored by correlating % JAK2 V617F with various laboratory characteristics in MPN patients, revealing significant associations with white blood cell counts, lactate dehydrogenase levels, and particularly β2-microglobulin (β2-MG) levels. Finally, a case report illustrated the application of cdPCR in detecting low-allele burdens in a de novo chronic myeloid leukemia (CML) patient with a hidden JAK2 V617F subclone, which expanded during tyrosine kinase inhibitor (TKI) treatment. Our findings underscore the superior sensitivity and accuracy of cdPCR, making it a valuable tool for early diagnosis and monitoring clonal evolution.

Genomic classification and outcomes of young patients with polycythemia vera and essential thrombocythemia according to the presence of splanchnic vein thrombosis and its chronology

To elucidate the role of splanchnic vein thrombosis (SVT) and genomic characteristics in prognosis and survival, we compared patients with polycythemia vera (PV) or essential thrombocythemia (ET) presenting SVT at diagnosis (n = 69, median age 43 years) or during follow-up (n = 21, median age 46 years) to a sex- and age-matched control group of PV/ET without SVT (n = 165, median age 48 years). The majority of patients presenting with SVT at diagnosis were classified as myeloproliferative neoplasm with heterozygous JAK2 mutation (87% of cases vs. 69% in PV/ET control group, p < 0.05), characterized by low JAK2 allele burden and no high-risk mutations. Despite this lower molecular complexity, patients presenting with SVT showed a higher risk of death (HR 3.0, 95% CI 1.5-6.0, p = 0.003) and lower event-free survival (HR 3.0, 95% CI 1.9–4.8, p < 0.001) than age- and sex-matched PV/ET controls. In patients presenting with SVT, molecular high-risk was associated with increased risk of venous re-thrombosis (HR 5.8, 95% CI 1.4–24.0, p = 0.01). Patients developing SVT during follow-up were more frequently allocated in molecular high-risk than those with SVT at diagnosis (52% versus 13%, p < 0.05). In the whole cohort of patients, molecular classification identified PV/ET patients at higher risk of disease progression whereas DNMT3A/TET2/ASXL1 mutations were associated with higher risk of arterial thrombosis. In conclusion, clinical and molecular characteristics are different in PV/ET patients with SVT, depending on whether it occurs at diagnosis or at follow-up. Molecular characterization by NGS is useful for assessing the risk of thrombosis and disease progression in young patients with PV/ET.

Molecular markers as possible efficacy predictors of targeted therapy for myelofibrosis: single-center study

Background. Targeted therapy is the most promising in the treatment of myelofibrosis, but it is necessary to search for the reasons limiting its effectiveness. There are known factors negatively affecting the development of myelofibrosis, but data on their negative impact in the context of targeted therapy are scarce.Aim. Assessing the impact of cytogenetic and genetic abnormalities on the course and therapy results for primary and secondary myelofibrosis during ruxolitinib therapy.Materials and methods. The prospective study included 106 patients with myelofibrosis in the chronic phase (53 (50 %) men and 53 (50 %) women) who received ruxolitinib at the Moscow City Hematology Center, S.P. Botkin City Clinical Hospital. The median age of patients was 62 (18–84) years. The median disease duration before initiation of ruxolitinib therapy was 79 (1–432) months. Before therapy, genetic studies were performed, including next-generation sequencing. The median duration of ruxolitinib therapy was 33 (1–111) months. The influence of the cytogenetic landscape, driver mutations, allele burden of JAK2 (over time) and CALR, additional mutations on the dynamics of symptoms, spleen size, achievement of hematological response, overall survival, progression-free survival, survival without blast crisis and without progression of myelofibrosis with targeted therapy was assessed.Results. The studied genetic factors did not have a significant correlation with hemogram parameters. The hematological response in patients with JAK2 and CALR mutations compared favorably with the response in the groups with the MPL mutation and triple negative status (TNS). Higher hematological response rate was obtained in the group with initially low allele burden &lt;50 % of JAK2 or CALR. Significant differences in 5-year overall survival were found between groups of patients with TNS and JAK2 and CALR mutations (p &lt;0.05); with CALR allele burden &lt;50 % and ³50 % before initiation of ruxolitinib therapy (p = 0.01); the presence or absence of positive dynamics of the JAK2 allele burden during treatment (p &lt;0.05); additional mutations assigned to different pathogenicity groups (p &lt;0.05); with different number of pathogenic mutations (1 or ³2), the presence or absence of pathogenic mutations in the ASXL1 (p = 0.002) and SETBP1 (p = 0.00001) genes. The 5-year progression-free survival was significantly different in cohorts of patients with or without positive dynamics of the JAK2 allelic load during treatment (p &lt;0.05); additional mutations assigned to different pathogenicity groups (p &lt;0.05); with a different number of pathogenic mutations (1 or ³2), the presence or absence of a pathogenic mutation of the SETBP1 gene (p = 0.003). Progression-free survival did not correlate with the type of driver mutation or its absence; however, all patients with TNS died from myelofibrosis progression. Significant differences in 5-year blast crisis-free survival were observed between groups with JAK2 and MPL mutations (p = 0.001), JAK2 and TNS (p = 0.002); difference in 5-year survival without progression of fibrosis – between groups with pathogenic and benign (p = 0.031); uncertain and benign (p = 0.001) mutations.Conclusion. The study identified genetic markers associated with decreased efficacy of ruxolitinib therapy.

A novel germline hyperactivating JAK2 mutation L604F

Somatic JAK2 mutations are the main molecular cause of the vast majority of polycythemia vera (PV) cases. According to a recent structural model, the prevalent acquired V617F mutation improves the stability of the JAK2 dimer, thereby enhancing the constitutive JAK2 kinase activity. Germline JAK2 mutations usually do not largely alter JAK2 signaling, although they may modulate the impact of V617F. We found an unusual germline JAK2 mutation L604F in homozygous form in a young PV patient, along with a low allele burden JAK2 V617F mutation, and in her apparently healthy sister. Their father with a PV-like disease had L604F in a heterozygous state, without V617F. The functional consequences of JAK2 L604Fmutation were compared with those induced by V617F in two different in vitro model systems: (i) HEK293T cells were transfected with plasmids for exogenous JAK2-GFP expression, and (ii) endogenous JAK2 modifications were introduced into HeLa cells using CRISPR/Cas9. Both mutations significantly increased JAK2 constitutive activity in transfected HEK293T cells. In the second model, JAK2 modification resulted in reduced total JAK2 protein levels. An important difference was also detected: as described previously, the effect of V617F on JAK2 kinase activity was abrogated in the absence of the aromatic residue F595. In contrast, JAK2 hyperactivation by L604F was only partially inhibited by the F595 change to alanine. We propose that the L604F mutation increases the probability of spontaneous JAK2 dimer formation, which is physiologically mediated by F595. In addition, L604F may contribute to dimer stabilization similarly to V617F.

JAK2 V617F Mutation and Associated Chromosomal Alterations Involving JAK2 in Pre-HCT Blood Samples and Transplant Outcomes in Myelofibrosis Subtypes

Introduction: Myelofibrosis (MF) is a BCR-ABL1-negative myeloproliferative neoplasm that develops either de novo (primary myelofibrosis; PMF) or secondary to polycythemia vera (post-PV) or essential thrombocythemia (post-ET). JAK2 V617F is the most common somatic driver mutation in MF known to associate with disease progression. Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment modality for MF. There are limited data regarding the prognostic utility of JAK2 V617F mutation and mosaic chromosomal alterations (mCAs) spanning JAK2 in MF patients undergoing HCT. Methods: The study included 924 patients undergoing HCT for myelofibrosis (634 PMF, 135 post-PV, and 155 post-ET) between 2000-2016. Pre-HCT peripheral blood samples, clinical and post-HCT data were provided by the Center for International Blood and Marrow Transplant Research (CIBMTR) registry and biorepository. We used PacBio Single Molecule Real-Time (SMRT) sequencing to detect the JAK2 V617F mutation, and Illumina Infinium Global Screening Array-24v1-0 to identify mCAs; this analysis focused on mCAs involving JAK2 (9p24.1). We used the Kaplan-Meier estimator to calculate the probability of overall survival (OS), and the cumulative incidence estimator for relapse/disease progression incidence and non-relapse mortality (NRM), where each was treated as a competing event for the other. Cox proportional hazard models were used for multivariable analyses. Analyses were completed separately by MF disease subtype. Follow up started at date of HCT. Results:JAK2 V617F mutation was detected in 562 patients (60.8%) with noted expected differences by disease subtype (57.6% of patients with PMF, 97% of post-PV, and 42.6% of post-ET). Almost all patients with mCAs involving JAK2 region were found to be JAK2 V617F mutation-positive (366 out of 374, 97.9%). In JAK2 V617F positive cases, the frequencies of mCA-positive patients per MF subtypes were as follow: 58.08% of PMF, 89.3% of post-PV, and 56.06% of post-ET. The mean allele burden for JAK2 V617F mutation was 57%, 78%, and 60% in PMF, post-PV, and post-ET, respectively. The probabilities of 3-year post-HCT OS were 55% (95% CI=51-58) for PMF, 59% (95% CI=51-67) for post-PV and 59% (51-66) for post-ET. The corresponding 3-year post-HCT cumulative incidences for relapse were 31% (95% CI=28-35), 31% (95% CI=24-40), and 33% (95% CI=26-41); and for NRM were 29% (95% CI=26-33), 25% (95% CI=18-32), and 23% (95% CI=17-30), for PMF, post-PV, and post-ET, respectively. In PMF, JAK2V617F mutation, JAK2 mCAs, or mutation allele burden were not associated with any of the evaluated HCT outcomes (p>0.05 for all). In post-PV, all four patients without JAK2V617F mutation died in the first 12 months after transplantation. No statistically significant associations between HCT outcomes and JAK2V617F allele burden was noted (p>0.05). For post-ET, patients with high mutation allele burden (³60%) had three-fold excess risk of NRM compared with mutation-negative patients (hazard ratio [HR]=2.9, 95% CI=1.21-6.93, p=0.01). No risk difference was noted between post-ET patients with low allele burden and risk of NRM (HR=0.89, 95% CI=0.34-2.31). No statistically significant association between JAK2V617F mutation status and risk of relapse for high or low mutation allele burden as compared with mutation-negative post ET patients was observed (HR=1.42, p=0.30 in patients with allele burden<60%, and HR= 1.54, p=0.26 for patients with allele burden³60%). JAK2V617F mutation status in post-ET patients was not associated with post HCT OS (HR=1.31, 95% CI=0.78-2.2, p=0.31). Conclusion: The effect of JAK2V617F mutation on post-HCT outcome differs by myelofibrosis subtypes. HCT outcomes in primary and post-PV myelofibrosis were not associated with JAK2 alterations, but high JAK2V617F mutation allele burden in post-ET was associated with elevated risk of non-relapse mortality but did not translate to a statistically significant survival disadvantage. Our findings suggest that allogeneic HCT for myelofibrosis may alleviate the known negative effect of JAK2V617F mutation in those patients, particularly for primary myelofibrosis and post-PV. It is essential to consider evaluating these findings within the context of other MF driver genes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Mydrug Platform Trial: Efficacy and Safety of Daratumumab, Ixazomib, Pomalidomide, and Dexamethasone in Patients with Functionally High-Risk Multiple Myeloma

Background: Multiple myeloma (MM) is an incurable malignant neoplasm with a 5-year relative survival rate of 57.9%. Many new agents have improved overall survival; however, some patients rapidly relapse and develop multidrug resistance, which requires the use of new approaches and drug mechanisms to treat. Precision medicine targeting specific genetic abnormalities has the potential to overcome related inherent and/or acquired resistance in patients with MM, but studying this approach has several limitations. Many different mutations occur in MM, and mutation frequency increases with disease progression and relapse, making it difficult to determine dominant genetic abnormalities and assess their relevance to a particular patient. This is a critical factor, as mutations in certain cancer-associated genes (actionable mutations) are targeted by approved drugs for other cancers. Some mutations are found in only a small number of patients. In addition, a substantial subset of patients appear to lack actionable mutations or have low allelic burden, making recruitment to clinical trials studying one specific mutation operationally difficult. To overcome these challenges and understand the role of novel therapies in precision medicine, the MMRF launched MyDRUG, a platform trial of patients with select actionable genetic mutations. Methods: MyDrug (NCT03732703) is an open-label, multicenter, phase 1/2 platform study of adult patients with functionally high-risk MM (defined as early relapse after initial MM treatment), who have received 1-3 prior therapies. Through different subprotocols, patients received targeted agents or immunotherapy/novel agents in combination with a backbone regimen of ixazomib, pomalidomide, and dexamethasone (IPd). Each subprotocol planned to enroll up to 38 patients, and treatment assignment was based on individual genetic sequencing results as determined by Next-Generation Sequencing assay performed in a CLIA-certified Molecular Characterization Laboratory. Approximately 75% of subjects were expected to have actionable mutations (BRAF, NRAS, KRAS, FGFR3, IDH2, CDK activating) or t(11;14) based on historic data, although not all of these would have the necessary allelic burden of ≥30% to qualify for the targeted subprotocols. Patients with no actionable mutation or mutations with allelic burden below 30% were assigned to the Y1 arm and received daratumumab at the standard dose and schedule in combination with IPd. The primary objective of the Y1 sub-protocol was to evaluate the overall response rate (ORR) according to the International Myeloma Working Group consensus criteria. Secondary objectives included assessment of adverse events (AEs). Confidence intervals were computed using the Clopper-Pearson (exact) method. Results: Thirty-eight patients were enrolled to the Y1 daratumumab arm with a median (range) age of 63 (46-79) years, with 16 (42.1%) patients of ages 65-74 and 2 (5.3%) patients ≥75 years. Thirteen (34.2%) patients were female, and 8 (21.1%) patients were Black or African American. The ORR for patients receiving daratumumab plus IPd was 92.1%, and 19 (50%) patients had a very good partial response. Thirty-seven (97.4%) patients experienced treatment-related AEs, 35 (92.1%) of which were related to daratumumab. Among patients receiving treatment, 22 (57.9%) experienced a serious AE; 3 (7.9%) experienced an AE leading to discontinuation; and 2 (5.3%) had fatal AEs. Conclusions: In this platform study of adult patients with treatment refractory, functionally high-risk MM, patients who received daratumumab plus IPd exhibited significant efficacy and a manageable AE profile.

P606: META-ANALYSIS OF LOW ALLELIC BURDEN C481 BTK-MUTATIONS CALLS FOR CAUTION IN IBRUTINIB RESISTANCE EVALUATION

Background: Introduction of the orally administered tyrosine kinase inhibitor ibrutinib, a drug approved for relapsed or refractory mantle cell lymphoma in late 2013 and chronic lymphocytic leukemia in 2014, has changed treatment algoritms for B-cell malignancies and improved progression-free survival. Ibrutinib irreversibly targets Bruton’s tyrosine kinase (BTK), thus interrupting B-cell receptor signaling. However, it is known that mutations in the BTK gene, located on the X chromosome, can mediate acquired resistance, specifically modifying cysteine residue 481 to serine (C481S, X:101356176, GRCh38). Aims: Despite this knowledge, it is still unknown how low mutational burden can contribute to general resistance towards ibrutinib treatment. Thus, we performed a meta-analysis based on previous cardinal studies to investigate the collective and gender-specific distribution of variant allele frequencies. Methods: We implemented meta-analysis of single-nucleotide variant calls from seven studies involving whole-exome sequencing (Woyach et al. 2014 and 2017, Maddocks et al. 2015, Ahn et al. 2017, Kanagal-Shamanna et al. 2019, Gángó et al. 2020, Bödör et al. 2021) on mutational profiling concerning ibrutinib resistance and CLL. Four of these studies contained gender specific data. Computational analyses, statistics, and visualization were performed in Wolfram Language, R, and GraphPad Prism. Results: From the available data included in these studies, we were able to base our analyses on 226 non-BTK mutations and 170 BTK mutations within a variant allele frequency (VAF) range of 0.0001–0.9, of which 93 mutation candidates from males (VAF < 0.9) and 77 from females (VAF < 0.44) were observed. Extrapolative analysis of allele frequency distribution from all other variants (excl. BTK) in the studies revealed an exponential increase in the number of low burden variants below 10% VAF (R2>0.99). These variants were not significantly different between genders (PMann-Whitney=0.24, nmale=130, nfemale=73). In contrast, a significant difference was observed between male variant allele frequencies (PMann-Whitney=0.005) and females owing to BTK hemizygosity in males. However, this significance persisted when female BTK mutations were transformed to reflect a single copy (VAF/2, PMann-Whitney=0.015, ΔVAF=0.05) for gender comparison and even when observations for males were alternatively transformed to reflect two copies (2*VAF, PMann-Whitney<0.001, ΔVAF=0.19). The previously discovered threshold of 10% was set as the detection limit. Summary/Conclusion: Defining specific molecular contributions in drug resistance is imperative for the correct use of targeted therapy. It has been questioned how the low allelic burden of BTK mutations plays a role. Therefore, it is crucial to investigate the lower limit of detection. The meta-analysis suggests that variants below a certain threshold, here empirically 10%, may in many cases be false-positive variants, in agreement with other reports involving WES in general, introduced by erroneous base calling, PCR errors, etc. Although intriguing, it remains to elaborate whether the haploinsufficiency of BTK in males skew in the degree of ibrutinib resistance.

Understanding the Secondary Transformation of JAK2 Myeloproliferative Neoplasms: Genomic Analysis

Myeloproliferative neoplasms (MPNs) comprise of a group of clonal stem cell disorders characterized by proliferation of red blood cells (RBC; polycythemia vera or PV), platelets (essential thrombocytosis or ET) or white blood cells (WBC; primary myelofibrosis or PMF). Biggest challenge with MPNs is the leukemic transformation that occurs in 1-2%, 4-5%, and 20-30% of patients over a 10-year period in ET, PV, and PMF, respectively. These secondary leukemia patients are associated with dismal prognosis, with median survival of 6 months. Mutations in JAK2 have been observed in the majority of MPN patients, and though JAK2 mutation seems to be the phenotypic driver of MPNs, recent evidence suggest clonality and mutational events antecedent to JAK2 are responsible for leukemic transformation. To better understand the impact of somatic mutations that contribute to transformation of MPN to leukemia, and the clonal architecture of these mutations, we conducted this study. The collection of bone marrow samples, saliva samples and clinical data of 48 clinically JAK2 positive MPN patients (i.e. JAK2 V617F PCR positive) was carried out at Seoul National University Hospital, and this study was approved by the institutional review board. Written informed consents were obtained from all patients in accordance with the Declaration of Helsinki. The bone marrow samples obtained at chronic MPN state were subjected to target sequencing. Target genes providing diagnostic or prognostic information in myeloid malignancies were selected, and the final gene panel consisted of 2228 amplicons covering 47 genes. Amplified targets were sequenced on the Illumina TruSeq Amplicon. The mean coverage depth was 1056X. For 3 patients with sequential paired samples at MPN diagnosis and at secondary transformation, whole genome sequencing (WGS) was performed using Illumina HiSeq2000. Fisher's exact test was used to compare the mutation frequency of single genes in different MPN subgroups, while Levene's test was used to for comparison of mutation frequency of gene groups. Two-sided t-test was used to evaluate the statistical significance. There were 18 ET patients (37.5%), 21 PV patients (43.8%) and 9 PMF patients (18.8%), and the median age at MPN diagnosis was 63 years for the total cohort. Not surprisingly, PMF was associated with most secondary transformations (P=0.030). About 89.6% of the patients carried somatic mutations other than JAK2. The most frequently observed mutations included TP53, PTCH1, ASXL1, TET2, CALR, EZH2, KMT2A, MPL and SETBP1. Interestingly, majority of the patients carrying ASXL1 were PV. PMF patients more frequently carried mutated splicing genes per patient (p=0.005), while PV patients seem to carry more mutated epigenetic regulator genes per patient, especially the chromatin modifiers (P=0.077). In addition, somatic mutations in TP53 and NF1 are associated with increased risk of secondary transformation and these mutations seem to be present already in the chronic MPN state at a low allelic burden. The WGS results showed the number of mutations at chronic MPN state versus at secondary transformation did not significantly differ, suggesting novel mutation rate is low in MPN and the secondary transformation is indeed driven by expansion of already existent small clones. Efforts to effectively incorporate next generation sequencing into MPN diagnosis and follow-up will be helpful for recognition of patients at high risk of secondary transformation, and can ultimately lead to better clinical outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Predominant Prognostic Significance of NOTCH1 Mutation Defined by Emulsion PCR in Chronic Lymphocytic Leukemia.

PurposeNOTCH1mut represents a new prognostic marker in chronic lymphocytic leukaemia (CLL). The low sensitivity of the current methods may increase the risk of false-negative results, particularly in patients with low NOTCH1mut allelic burden. This study compared two methods of the NOTCH1mut assessment including droplet digital PCR (ddPCR) and amplification-refractory mutation system PCR (ARMS-PCR) untreated CLL patients.Patients and MethodsThis study included 319 untreated CLL patients. Two PCR-based methods; ddPCR and ARMS-PCR were performed to assess the mutational status of NOTCH1. The Mann–Whitney, Fisher’s exact test, Kruskal–Wallis, Kaplan–Meier, Log rank tests and multivariate Cox proportional hazard regression model were used to analyze collected data.ResultsWe proved that ddPCR increased the detectability of the NOTCH1mut compared to ARMS-PCR in CLL (18.55% vs 6%). We showed a shorter time to first treatment (TTFT) in the NOTCH1mut group of patients compared to the NOTCH1wt defined by ddPCR (1.5 vs 33 months, p=0.01). The TTFT survival curves analysis in subgroups divided according to the mutational status of IGHV and NOTCH1 assessed by ddPCR discriminated group with the best prognosis: IGHVmutNOTCH1wt. Multivariate analysis revealed that the mutational status of IGHV represented an independent prognostic factor for TTFT, while NOTCH1mut determined by ddPCR constituted as a dependent prognostic factor for TTFT.ConclusionThe selection of the precise method of NOTCH1mut detection as ddPCR might significantly improve prognostic stratification of CLL patient. Assessment of IGHV might be relevant to more accurate discrimination of prognostic groups of CLL patients, especially in harboring NOTCH1mut irrespective of the quantity of allelic burden.

Low JAK2 V617F Allele Burden in Ph-Negative Chronic Myeloproliferative Neoplasms Is Associated with Additional CALR or MPL Gene Mutations

JAK2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9, and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Ph-negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of proliferative signal transduction and therefore, after the emergence of one type of mutation, other types should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Our study includes DNA samples from 1958 patients with clinical evidence of MPN, admitted to the National Research Center for Hematology for genetic analysis between 2016 and 2019. In 315 of 1402 cases (22.6%), CALR mutations were detected. In 23 of these 315 cases (7.3%), the JAK2 V617F mutation was found in addition to the CALR mutation. In 16 from 24 (69.6%) cases, with combined CALR and JAK2 mutations, V617F allele burden was lower than 1%. A combination of JAK2 V617F with MPL W515L/K was also observed in 1 out of 1348 cases, only. JAK2 allele burden in this case was also lower than 1%. Additional mutations may coexist over the low background of JAK2 V617F allele. Therefore, in cases of detecting MPNs with a low allelic load JAK2 V617F, it may be advisable to search for other molecular markers, primarily mutations in exon 9 of CALR. The load of the combined mutations measured at different time points may indicate that, at least in some cases, these mutations could be represented by different clones of malignant cells.

Pacritinib demonstrates spleen volume reduction in patients with myelofibrosis independent of JAK2V617F allele burden

AbstractMyelofibrosis (MF) has heterogeneous clinical manifestations, with some patients exhibiting a myelodepletive phenotype characterized by cytopenias and an absent or low JAK2V617F allele burden. Ruxolitinib may be less effective in these patients. We assessed the efficacy of pacritinib, a JAK2/IRAK1 inhibitor, in MF patients with low JAK2V617F allele burden. In this post hoc analysis of the PERSIST-1 and -2 trials, patients with MF randomized to pacritinib or best available therapy (BAT) were stratified by JAK2V617F allele burden quartile for spleen response of ≥35% and improvement in total symptom score of ≥50%. Five hundred thirty-six patients were included. Patients with lower JAK2V617F allele burden had smaller baseline spleens and lower hemoglobin and platelet counts as compared with higher allele burden patients. Among pacritinib-treated patients, spleen responses were observed across all JAK2V617F allele burden quartiles and in JAK2V617F− disease. No spleen responses were observed among BAT-treated patients with allele burden ≤50% or JAK2V617F− disease. The intention-to-treat response rate was significantly higher on the pacritinib arm for JAK2V617F− disease (23.0% vs 0%; P = .033), and for the lowest allele burden quartiles (0%-25%: 20.9% vs 0%, P < .001; 25%-50%: 15.4% vs 0%, P = .020). There were significantly more symptom responders with pacritinib vs BAT in the 0% to 25% and 25% to 50% cohorts. Pacritinib treatment led to superior spleen and symptom burden reduction compared with BAT in patients with absent or low JAK2V617F allele burden, suggesting that pacritinib may be uniquely suited for patients with myelodepletive MF.

RNA sequencing as an alternative tool for detecting measurable residual disease in core-binding factor acute myeloid leukemia

DNA sequencing-based measurable residual disease (MRD) detection has shown to be clinically relevant in AML. However, the same methodology cannot be applied to fusion gene-driven subtypes of AML such as core-binding factor AML (CBF-AML). Here in this study, we evaluated the effectiveness of using DNA and RNA sequencing in MRD detection and in tracking clonal dynamics in CBF-AML. Using RNA-seq, we were able to quantify expression levels of RUNX1-RUNX1T1 and CBFB-MYH11 at diagnosis and their levels of reduction during remission (P < 6.3e−05 and P < 2.2e−13). The level of reduction of RUNX1-RUNX1T1 as measured by RNA-seq and qPCR were highly correlated (R2 = 0.74, P < 5.4e−05). A decision tree analysis, based on 3-log reduction of RUNX1-RUNX1T1 and cKIT-D816mut at diagnosis, stratified RUNX1-RUNX1T1 AML patients into three subgroups. These three subgroups had 2-year overall survival rates at 87%, 74%, and 33% (P < 0.08) and 2-year relapse incidence rates at 13%, 42%, and 67% (P < 0.05). On the other hand, although low residual allelic burden was common, it was not associated with long-term outcome, indicating that mutation clearance alone cannot be interpreted as MRD-negative. Overall, our study demonstrates that the clinical utility of RNA sequencing as a potential tool for MRD monitoring in fusion gene-driven AML such as RUNX1-RUNX1T1 AML.

Leukemic evolution of polycythemia vera and essential thrombocythemia: genomic profiles predict time to transformation

AbstractAmong myeloproliferative neoplasms, polycythemia vera (PV) and essential thrombocythemia (ET) are the 2 entities associated with the most chronic disease course. Leukemic evolution occurs rarely but has a grim prognosis. The interval between diagnosis and leukemic evolution is highly variable, from a few years to >20 years. We performed a molecular evaluation of 49 leukemic transformations of PV and ET by targeted next-generation sequencing. Using a hierarchical classification, we identified 3 molecular groups associated with a distinct time to leukemic transformation. Short-term transformations were mostly characterized by a complex molecular landscape and mutations in IDH1/2, RUNX1, and U2AF1 genes, whereas long-term transformations were associated with mutations in TP53, NRAS, and BCORL1 genes. Studying paired samples from chronic phase and transformation, we detected some mutations already present during the chronic phase, either with a significant allele burden (short-term transformation) or with a very low allele burden (especially TP53 mutations). However, other mutations were not detected even 1 year before leukemic transformation. Our results suggest that the leukemic transformation of PV and ET may be driven by distinct time-dependent molecular mechanisms.

MPN-376: The Clinical Impact of the Burden of JAK2V617F Mutated Allele in Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Context: Several studies reported the impact of JAK2V617F allele burden on phenotypic properties and clinical course in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs). We aimed to analyze the association of mutated JAK2V617F allele burden with clinical phenotype and laboratory characteristics in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. Objective: A total of 202 consecutive Philadelphia-negative MPN patients, 118 with ET and 84 with PMF, were included in this study. Methods: We used a real-time, semi-quantitative polymerase chain reaction (PCR) with JAK2 MutaScreen assay (Ipsogen, Luminy Biotech, Marseille, France) to screen JAK2V617 mutations and mutant allele burden. Results: According to the JAK2V617F status, ET patients were stratified into three groups: JAK2V617F mutation-negative (n=54) and JAK2V617F-positive with mutant allele burden in the lower quartile (n=59) and upper quartile (n=5) ranges. ET patients with upper quartile JAK2V617F allele burden had significantly lower Hgb levels (p=0.001), lower Htc levels (p=0.001), and higher LDH levels (p=0.008) as compared to the other two groups. Also, ET patients with upper quartile JAK2V617F allele burden had significantly increased spleen size and higher bleeding complications (p=0.034; 0.002; respectively). We observed significant association between ET patients with high JAK2V617F allele burden and higher death rates (p=0.007). There was a trend towards higher incidence of venous thrombosis in ET patients with upper quartile JAK2V617F allele burden (p=0.057). According to JAK2V617F status, PMF patients were stratified into three groups: JAK2V617F mutation-negative (n=25) and JAK2V617F-positive with mutant allele burden in the lower quartile (n=41) and upper quartile (n=18) ranges. Comparison across all three groups revealed significant associations between upper quartile allele burden and higher leukocyte counts (p=0.001). Also, PMF patients with upper quartile JAK2V617F allele burden had significantly increased spleen size compared to the other two groups (p=0.018). We observed significant association between PMF patients with low JAK2V617F allele burden and higher incidence of venous thrombosis (p=0.035). Conclusion: Our study reported that presence of higher V617F allele burden in ET is associated with defined haematological and clinical markers indicative of a more aggressive behavior while, in PMF, low allele burden was found to be associated with higher incidence of venous thrombosis.

Clinical Characteristics and Cardioavscular Events in Patients with Esential Thrombocythemia with

Introduction The clinical characteristics, treatment, cardiovascular events (CVE) and evolution of patients diagnosed with JAK2 V617F positive essential thrombocythemia (ET) with low allele burden (LAB) are scarcely studied. Its presence in people without a confirmed diagnosis of malignant hemopathy is called clonal hematopoiesis of uncertain significance (CHIP) and confers higher risk of developing CVE. The objective of this study was to compare the clinical characteristics and CVE of a series of JAK2 V617F-positive ET patients with &lt;10% (LAB) vs. ≥10% allele burden (HAB), from the GEMFIN (Grupo Español de Enfermedades Mieloproliferativas Crónicas Filadelfia Negativas) Group. Methods From the database of the GEMFIN group, 410 ET patients were JAK2 V617F positive, 89 (21.7 %) with LAB and 321 (78.3%) with HAB. The clinical characteristics, treatment (cytoreduction, antiagregation, anticoagulation, JAK inhibitor), CVE (before, at and after diagnosis) and evolution to myelofibrosis (MF) or acute myeloid leukemia (AML) of these two groups of patients were compared. Results LAB and HAB groups did not significantly differ regarding the main clinical characteristics (i.e cardiovascular risk factors [CVRF] and International Prognostic Score for Thrombosis in Essential Thrombocythemia [IPSET] score) except for the median platelet count: LAB 636 x109/L [436- 2500] vs HAB 687 x109/L[440-1980L], p=0.035). CVE after diagnosis of ET were more frequent in patients with HAB (41/137, 30%) than in patients with LAB (5/48, 10%), p=0.007. Only one LAB patient with CVE had JAK2 allele burden &gt;5%. Treatments received by both groups were not significantly different. None of the patients from both groups progressed to AML, whereas 1/48 vs. 6/137 of patients evolved to MF. Median follow-up of patients with LAB and HAB was 3.4 years [0.1-17.7] and 4.3 years [0.1-27.8], respectively (Table 1). Conclusions In these series of ET patients from the GEMFIN group, patients with LAB had significantly lower median platelet count at diagnosis and less CVE after diagnosis than patients with HAB, although CVRF and IPSET scores and treatment approach were similar. The clinical behavior of LAB patients may resemble that of individuals with CHIP. The therapeutic algorithm of ET patients with LAB may be somehow different than that of patients with HAB and therefore, might be revised. Disclosures Bellosillo: Astra-Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biocartis: Honoraria; Merck-Serono: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Hoffman â€"La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; ThermoFisher: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; BMS: Honoraria. Hernandez Boluda:Incyte: Other: Travel expenses paid. Pérez:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

The Oral JAK2/IRAK1 Inhibitor Pacritinib Demonstrates Spleen Volume Reduction in Myelofibrosis Patients Independent of JAK2V617F Allele Burden

Introduction: The presence of a molecular driver mutation such as JAK2 V617F is a major criterion for diagnosing myeloproliferative neoplasms. JAK2 V617F allelic burden (percentage of cells harboring the mutation) has been associated with varying disease phenotypes and outcomes. Myelofibrosis (MF) with low allelic burden is associated with a myelodepletive phenotype, which includes shorter overall survival, anemia, leukopenia, and lesser degree of splenomegaly. Some JAK inhibitors utilized in patients with low allelic burden (&lt;50%) may achieve smaller spleen volume reduction (SVR), therefore, treatment of the lower allelic burden population remains clinically challenging. Pacritinib is an oral JAK2/IRAK1 inhibitor with demonstrated efficacy based on SVR from two phase 3 studies in MF patients including those with severe thrombocytopenia (PERSIST-1 [P1] and PERSIST-2 [P2]). These studies were unique in that the combined P1/P2 median allelic burden is 47% as compared to other phase 3 studies of JAK2 inhibitors with a median allelic burden &gt;80%. To better evaluate the impact of allelic burden on pacritinib-induced SVR in MF patients, a retrospective analysis was performed on the combined data from the P1 and P2 studies. Methods: Patients ≥18 years of age with primary or secondary MF, palpable splenomegaly ≥5 cm below the left costal margin, and DIPSS risk category of intermediate-1, intermediate-2, or high-risk were eligible for the studies. Patients with any baseline platelet count were eligible for P1, while P2 included only those with baseline counts ≤100,000/μL. Prior JAK inhibitor therapy was allowed in P2, but not P1. Patients in P1 received pacritinib 400 mg once daily (QD) or best available therapy (BAT); those in P2 received pacritinib 400 mg QD, pacritinib 200 mg twice daily (BID), or BAT. In both studies, BAT consisted of physician-selected treatments for MF (including symptom-directed treatment or watch and wait); treatment with ruxolitinib was allowed as BAT only in P2. The percentage of patients achieving ≥35% SVR at week 24 was a primary endpoint for both studies. This retrospective analysis assessed those patients with JAK2 V617F for possible relationship between allelic burden and SVR at 24 weeks. Results: JAK2 V617F allelic burden data were available for 363 patients that received pacritinib and 173 patients that received BAT. 19.6% of BAT subjects received ruxolitinib during study. Pacritinib demonstrated similar SVR across all allelic burden quartiles, including the lowest quartile. In contrast, SVR responses with BAT were more frequently observed in those patients with higher allelic burden (&gt;75-100% quartile; Figure 1). The percentage of patients with ≥35% SVR was significantly higher with pacritinib compared with BAT for those with an allele burden ≤50% (allele burden &gt;0-25%: pacritinib=18/86 [21%], BAT=0/46, P&lt;0.001; allele burden &gt;25-50%: pacritinib=14/91 [15%], BAT=0/33, P=0.020); there was no significant difference in the &gt;50-75% (pacritinib=9/53 [17%], BAT=1/26 [4%], P=0.153) and &gt;75-100% quartiles (pacritinib=14/72 [20%], BAT=5/49 [10%], P=0.209). Conclusions: Myelofibrosis (MF) with JAK2 V617F allelic burden &lt;50% is associated with a myelodepletive phenotype and a worse prognosis. MF patients with low allelic burden may have a non-JAK mediated disease which could explain why other JAK inhibitors have been shown to be less effective in patients with low allelic burden. Pacritinib provides similar SVR across all levels of allelic burden, including levels &lt;50% unlike BAT (which included ruxolitinib). This data suggests that pacritinib may provide benefit over a wider range of patients with MF compared to other JAK inhibitors. Disclosures Verstovsek: Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy. Scott:Celgene: Consultancy; Novartis: Consultancy; Agios: Consultancy; Incyte: Consultancy. Taylor:Baxalta: Research Funding; CTI BioPharma: Employment, Equity Ownership. Mascarenhas:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Promedior: Research Funding; Merus: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Preliminary Results from CPX-351 Italian Compassionate Use Program Show High Response Rate and Good Tolerability in Poor Prognosis AML Patients

Background: Therapy-related acute myeloid leukemia (t-AML) or AML evolving from a myelodisplastic syndrome are characterized by a low response rate to conventional chemotherapy and high relapse rate with poor overall survival (OS) despite intensive treatment and allogeneic stem cell transplantation consolidation (HSCT). CPX-351 is a dual-drug liposomal encapsulation of cytarabine and daunorubicin, in a fixed synergistic 1:5 molar ratio. CPX-351 has been approved by FDA for the treatment of patients affected by t-AML or AML with myelodisplasia-related changes (MRC-AML) based on the results of a randomized phase III trial where the drug was compared with conventional 3+7 induction (Lancet et al, JCO 2018). Notably, CPX-351 proved to increase survival probabilities in comparison with standard chemotherapy even among patient achieving complete remission (CR) and proceeding to HSCT, suggesting that CPX-351 may allow deeper responses. However, few information is available on minimal residual disease (MRD) assessment after CPX-351 treatment, or on the impact of molecular alterations on response probability. Aims: The aim of this study was to evaluate the clinical activity of CPX-351 in a real life setting, with particular focus on molecular characterization at diagnosis and MRD evaluation in responding patients. Methods: Seventy five patients were enrolled in a compassionate use program (CUP) in 37 Italian Hematology Centers. CUP started on December 2018 and closed on June 2019. Data collection began on July 2019 and was completed, at the time of writing, for 25/75 patients, enrolled in 9 Centers. Median age was 69 years (56-73), 10 patients were female. Molecular and MRD analysis were performed in each Center as per internal standard. MRD was assessed in most Centers with multicolor flow cytometry. NPM1 mutation was found in 2/22 assessed patients, FLT3-ITD in 3/22, with low allelic burden in 2/3 patients. TP53 mutations have been found in 4/12 patients.Four patients had complex Karyotype, one had isolated del(7q) whereas the remaining 19 had normal karyotype. Six patients had t-AML; 15 patients were previously diagnosed with MDS and 5 of them had already received hypomethilating agents for a median of 5 cycles (2-49). European Leukemia net risk score was low in 2 (8%), intermediate in 12 (48%) and high in 11 (44%) patients. Most patients (20/25) had relevant comorbidities upon enrollment, mostly COPD, diabetes and/or hypertension. As per CUP inclusion criteria, all patients had normal left ventricular function at the time of enrollment (defined by a normal ejection fraction). Results: Induction-related mortality was 2/25 (8%). Fourteen patients experienced grade &amp;gt;1 extra hematological adverse event during induction (mainly infections). Alopecia was observed in 4/25 patients (16%).Response was assessed in 20 patients:2 patients died during induction and 3 patients were not yet evaluated for response at the time of analysis. CR or CRi was observed in 19/22 (86.3%). MRD was performed in 11 patients, with 4 of them achieving flow MRD negativity after first cycle (defined as &amp;lt;0.1%, Minetto et al. BJH 2019). Median time to neutrophil and platelets recovery in responding patients were 29 (18-60) and 24.5 (19-60) days, respectively. Among responding patients, 10 received further CPX-351 consolidation, which was very well tolerated. One patient proceeded directly to HSCT, one is currently waiting for HSCT to be performed and one patient received a conventional chemotherapy consolidation. The remaining patients are currently waiting for administration of consolidation therapy with CPX-351. Four patient had completed so far all planned consolidation therapy with CPX-351 and 2/4 of them were flow MRD negative at the end of therapy. Of note, 3/4 patients with mutated TP53 achieved MRD negative CR. After a median follow-up of 5 months, relapse was observed in 2/20 responding patients and 21 patients are alive at the time of analysis. Conclusions: Our preliminary data confirm the high clinical activity and good tolerability of CPX-351 in a challenging AML cohort. The high median age and the high incidence of severe comorbidities did not result in unacceptable risk of death during induction. With the limitation of very small numbers, CPX-351 showed good antileukemic activity among TP53 mutated patients. Disclosures No relevant conflicts of interest to declare.

Expansion of Prior Existing TP53 Mutated Clones in Polycythemia Vera Patients Treated with Idasanutlin

Expansion of Prior Existing TP53 Mutated Clones in Polycythemia Vera Patients Treated with Idasanutlin

Significant Impact of the Molecular Profile on the Prognosis of Patients with Myeloproliferative Neoplasms and Splanchnic Vein Thromboses

Introduction: Myeloproliferative neoplasms (MPN) are the most frequent underlying causes of splanchnic vein thromboses (SVT), including Budd-Chiari syndrome (BCS), portal, splenic and mesenteric vein thromboses. This subgroup of MPN patients with SVT (MPN/SVT) has been shown to have distinct clinical and molecular features like younger age and lower JAK2V617Fmutant allele burden (the most predominant driver mutation in MPN/SVT patients) and are often considered as early stages of MPN. However, there is no study that has investigated the contribution of NGS data to the risk stratification of these MPN/SVT patients. Main objective of this study was to evaluate the impact of the presence of additional mutations to the driver mutation in the long term outcome of MPN patients with SVT. Patients and methods: Over the past 10 years, a total of 286 patients with SVT have been referred to our center for diagnostic assessment of an underlying MPN. Among them, a diagnosis of MPN (WHO criteria) was finally made in 197. Full molecular analyses by NGS was available in 61 of these MPN/SVT patients. The molecular profiling of patients was performed using a Capture-based custom NGS panel provided by Sophia Genetics. This panel comprised the coding sequences of 36 genes involved in MPN. Bioinformatics analysis was performed by Sophia Genetics, with a detection limit of variants set at 1%. We compared (i) MPN/SVT patients to our local cohort of 1371 MPN patients without SVT with full NGS data available; (ii) MPN/SVT patients with and without additional mutation to the driver mutation. Results: Median follow-up of MPN/SVT patients was 11 years, 62% were females, 33% had BCS and 67% portal vein thrombosis (Table 1). All patients received anticoagulants and 58 (95%) a cytoreductive therapy after the diagnosis of MPN. The type of MPN was polycythemia vera in 69%, essential thrombocythemia in 25% and myelofibrosis in 6%. The driver mutation was JAK2V617F in 57 (93%), and CALR mutation in 4 patients, respectively, and no patient had MPL mutation. By NGS, an additional mutation was identified in 27 (44%) patients, while 34 patients had only the driver mutation. The most frequent additional mutations were in TET2 (23%), ASXL1 (8%), DNMT3A (7%), IDH1/2 (5%), and LNK (5%) genes. EZH2, SRSF2 and TP53 mutations were found in only 1 patient, respectively. When compared to NGS data from 1371 MPN patients, there was no significant difference in the frequency of each additional mutation, except a slightly lower incidence of ASXL1 mutations in MPN/SVT patients (8% vs 23% in MPN patients, p=0.07). Of note, MPN/SVT patients were younger at MPN diagnosis than the control MPN cohort (44 vs 61 years, p&amp;lt; 10-5). Clinical characteristics were not significantly different between MPN/SVT patients with and without additional mutations (Table 1), including age at SVT or at MPN diagnosis, type of MPN or type of SVT, use of cytoreductive therapy, and median follow-up (11 years in both groups). In contrast, MPN/SVT patients with additional mutations had a significantly higher median JAK2V617F mutant allele burden than those with only JAK2 mutation (34% vs 11%, p=0.003, Figure 1); and higher risk of hematological transformation to myelofibrosis or acute leukemia or death (30% vs 6%, p=0.017). Only few patients experienced recurrence of thrombosis during follow up, with no difference according to the mutational profile (3 (11%) and 6 (17%) in patients with and without additional mutations, respectively). Conclusion: This is to our knowledge the first study assessing the impact of molecular abnormalities in the long term outcome of MPN/SVT patients showing a possible prognostic role for NGS data in these patients. Our results suggest that MPN/SVT patients with only JAK2V617F mutation and a low mutant allele burden (below 50%) are at low risk of adverse hematological outcome. In contrast, NGS may identify a group of patients with additional mutations at very high long term risk of unfavorable hematological evolution (30% transformation or death after 11 years of median follow-up, that is unexpected in such a young MPN population). In this group, a disease modifying therapy like interferon or other targeted therapies should be proposed when possible to reduce this risk of transformation. Disclosures Kiladjian: Novartis: Honoraria, Research Funding; Celgene: Consultancy; AOP Orphan: Honoraria, Research Funding.