Myeloproliferative Neoplasia Research Articles

Clonal Evolution of Myelofibrosis Patients in Relapse after Allogeneic Stem Cell Transplantation Revealed By Whole Exome Sequencing

Introduction: Clonal evolution has been described in various hematologic malignancies. Persistence of one founding clone is the basis for relapse of AML, and diversity of accompanying mutations greatly varies in AML [Ding et al. Nature 2012]. On the contrary, a targeted sequencing approach revealed that in myeloproliferative neoplasia, few additional mutations arise during progression [Lundberg et al. Blood 2014]. The dynamics of clonal evolution has been linked to response to Ruxolitinib therapy [Newberry et al. Blood 2017].Patients and methods: Here, we describe the clonal evolution of 30 pts (median age at alloSCT 60, range 36-75 years) with myelofibrosis (16 primary, 6 post-ET, 7 post-PV MF) who either progressed directly (n=14) or after a period of complete donor hematopoiesis for 417 days (median, range 7-2240 days) after allogeneic stem cell transplantation. Amplicon-based next generation sequencing of a custom panel consisting of 17 different genes (178 amplicons in 2 primer pools, targeting regions in TET2, IKAROS, JAK2, MPL, SH2B3, CBL, IDH1, IDH2, ASXL1, EZH2, SRSF2, SF3B1, DNMT3A, RUNX1, FLT3, NRAS, KRAS) was performed for all pts using a Personalized Genome Machine (PGM, ThermoFisher/ LifeTechnologies). Additional whole exome sequencing was performed for 6 out of these 30 pts, both before alloSCT and at relapse of the disease after alloSCT, using a Proton (ThermoFisher/ LifeTechnologies). Mutations in CALR were determined using digital PCR.Results: Pts carried JAK2 V617F (n=23), CALR L367fs* (n=3), CALR K368fs*, CALR K385fs* (each n=1), or were triple-negative (n=2). The mutations in the driver genes JAK2, CALR, and MPL that were present before alloSCT were also present at relapse. One pt carrying a JAK2 V617F before alloSCT had developed an additional MPL L494V missense mutation at relapse. With targeted amplicon sequencing, a median of 3 nonsynonymous mutations (range 0-13) was found in a median of 2 (0-8) genes before alloSCT. Before alloSCT, 19 pts were mutated in TET2 (I1762V/ L1721W/ P363L/ L34F/ V218M/ H1778R in 13/ 7/ 3/ 2/ 2/ 2 pts, 9 further mutations occurring only once), trailed by ASXL1 (7 pts) and SRSF2 (6 pts). DNMT3A and IDH2 were mutated in 3 pts, each, with the latter exclusively altered at R140. KRAS (G12V, G12H), RUNX1 (D198Y, A424S), SF3B1 (K666N, V695L) were mutated in 2 pts each. Mutations in CBL (C381S), IKZF1 (Q7R), EZH2 (Y733C), SH2B3 (S186I), and IDH1 (R132H) occurred in one pt each. At relapse, using the identical amplicon based sequencing strategy, a median of 3 (0-13) nonsynonymous mutations was found in a median of 2.5 (0-6) genes. Counting gains and losses of nonsynonymous mutations at relapse, a median of 1 (0-9) changes was observed. Identities of gains and losses were more variable when compared to the mutations before alloSCT. Most changes occurred in TET2, followed by ASXL1, SRSF2, RUNX1, and DNMT3A . Taking only pts who had achieved full donor chimerism (n=16) into account, short molecular remission and number of mutational changes correlated (R²=0.013, figure). This correlation was stronger than the correlation between number of mutations before alloSCT and time in molecular remission after alloSCT (R²=0.0001). Notably, of the 6 pts in whom additional whole exome sequencing was performed at first presentation and upon progression, none showed clonal evolution beyond genes depicted in the targeted amplicon sequencing panel.Conclusion: Clonal evolution of myelofibrosis pts who relapse after alloSCT is modest. A net loss of mutations can be found at relapse. In comparison to the situation in MDS and AML, the gain of novel mutations at relapse seems to be moderate in myelofibrosis. Future research has to identify mutational spectra with specific prognostic and predictive value. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development of BCR-ABL1 Transgenic Zebrafish Model Reproducing Chronic Myeloid Leukemia (CML) Like-Disease and Providing a New Insight into CML Mechanisms.

Zebrafish has proven to be a versatile and reliable experimental in vivo tool to study human hematopoiesis and model hematological malignancies. Transgenic technologies enable the generation of specific leukemia types by the expression of human oncogenes under specific promoters. Using this technology, a variety of myeloid and lymphoid malignancies zebrafish models have been described. Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the BCR-ABL1 fusion gene, derived from the t (9;22) translocation causing the Philadelphia Chromosome (Ph). The BCR-ABL1 protein is a constitutively activated tyrosine kinas inducing the leukemogenesis and resulting in an accumulation of immature leukemic cells into bone marrow and peripheral blood. To model Ph+ CML, a transgenic zebrafish line expressing the human BCR-ABL1 was generated by the Gal4/UAS system, and then crossed with the hsp70-Gal4 transgenic line. The new line named (BCR-ABL1pUAS:CFP/hsp70-Gal4), presented altered expression of hematopoietic markers during embryonic development compared to controls and transgenic larvae showed proliferating hematopoietic cells in the caudal hematopoietic tissue (CHT). The present transgenic zebrafish would be a robust CML model and a high-throughput drug screening tool.

Patterns of previous and secondary malignancies in patients with multiple myeloma.

In contrast to secondary primary malignancies (SPM) following multiple myeloma (MM), less is known about previous malignancies. We therefore conducted a population-based study to assess the patterns of previous malignancies in MM patients as well as the risk for SPM. Using data from the Cancer Registry of Norway, we included 9574 MM patients and 37810 matched control subjects. The association between previous malignancies and a subsequent diagnosis of MM was analysed by a logistic regression model and the risk for SPM by a Cox model. A previous diagnosis of myeloproliferative neoplasia (MPN) (OR 3.57; 95% CI:1.45-8.80) and Hodgkin lymphoma (HL) (OR 3.66; 95% CI: 1.40-9.55) was associated with the subsequent development of MM. For MPN, the association with MM was explained by an excess of primary myelofibrosis (PMF) in the MM group. The overall incidence of a previous malignancy was not different between MM patients and the control subjects (OR 0.93; 95% CI: 0.87-1.00). MM patients had an increased risk for secondary acute myelogenous leukaemia/myelodysplastic syndromes (HR 6.1, 95% CI: 3.9-9.5). A previous diagnosis of HL and PMF was associated with a subsequent diagnosis of MM, whereas the overall incidence of previous cancers was not increased for MM patients.

A Pharmacokinetic/Pharmacodynamic Model of the Action of Hypomethylating Agents in Chronic Myelomonocytic Leukaemia

Chronic myelomonocytic leukaemia (CMML) is a disease that borders between a myelodyplastic syndrome (MDS) and myeloproliferative neoplasia (MPN). It is a progressive condition: about 20% of CMML patients progress to secondary acute myeloid leukaemia (sAML), which has a very poor prognosis. In all cases, normal bone marrow function is compromised with resulting depletion of circulating erythrocytes and platelets. We modelled the dynamics of CMML at two levels: a cytokinetic model was developed that describes bone marrow cell population dynamics for multiple cell lineages, and changes in DNA mutational status that occur in MDS and CMML. This model, in conjunction with pharmacokinetic (PK) models for cytotoxic drugs and hypomethylating agents, was used to study the pharmacodynamics (PD) of these drugs. For the subset of CMML with loss of activity of TET2, our models suggest that TET2 activators may restore normal DNA methylation with a resulting antileukaemic effect. Mouse and human data were used to place bounds on the parameters of our model. Our modelling suggests that both hypomethylation and cytotoxic effects contribute to the antileukaemic activity of decitabine.

Thiol/Disulfide Balance in Older Patients with BCR-ABL Negative Myeloproliferative Neoplasms.

The trio Essential Thrombocytosis (ET), Polycythemia Vera (PV), and Primary Myelofibrosis (PM) are BCR-ABL negative myeloproliferative neoplasms. All three diseases have the risk of transforming into acute leukemia. Oxidative stress and some genetic mutations increase the risk of leukemic transformation. The median age in patients with ET, PV, and MF is around 64 years, and it is expected to exceed 65 in the coming years. Since oxidative stress increases with age, we aimed to evaluate the oxidative stress parameters in older patients with myeloproliferative neoplasms. The study included a total of 160 patients (57 patients with Essential Thrombocytosis, 52 patients with Primary Myelofibrosis, and 51 patients with Polycythemia Vera) and 56 healthy controls, aged 65 and over. Ischemia Modified Albumin (IMA) and thiol parameters (native thiol, total thiol, and disulfide) were studied from serum samples taken at the time of diagnosis. The median age of the patients was 69 (65 - 85) years. Patients had higher levels of IMA and lower levels of thiol compared to the control group (p < 0.001). When evaluated according to disease subgroups, it was observed that the highest IMA levels and the lowest thiol levels were in patients with PM (p < 0.001). Higher IMA levels and lower native thiol levels were found in patients with the ASXL1 mutation (p < 0.001). Serum IMA and thiol levels are also significantly changed in older patients with BCR-ABL negative myeloproliferative neoplasia. Changes in these markers are independent of age. Disease-associated mutations such as ASXL1 can also affect the serum levels of these markers.

High Dose Flamsa, CLAG or FLAG-Based Sequential Conditioning Regimen Followed By Allogeneic Hematopoietic Transplantation Results in Favorable Outcome for High Risk Acute Myeloid Leukemia, Myelodysplastic Syndrome, Myeloproliferative Neoplasia Patients : A Multicenter Study in Singapore

Introduction: Allogeneic hematopoietic cell transplant (allo-HCT) is an effective consolidative treatment for patients with certain hematological malignancies and gives the best outcome when done in remission. However, patients with refractory acute myeloid leukemia (AML), certain forms of myeloproliferative neoplasia (MPN), and myelodysplastic syndrome (MDS) deemed unable to achieve remission with standard induction are often excluded from allo-HCT with conventional conditioning regimen as pre-transplant remission could not be achieved. Recently, a sequential transplant approach, as developed by the Munich group, comprising of intensive cytoreductive chemotherapy FLAMSA (fludarabine/amsacrine/cytarabine) to decrease leukemia cell burden shortly prior to conditioning regimen, had been successfully used for high-risk (HR) AML/MDS with promising results. Methods: We studied 56 patients (median age, 52 years; range 17-68) with HR AML (n=45), as defined by refractory, relapsed disease, secondary leukemia, complete remission with adverse-risk cytogenetics according to ELN criteria, or high/very high risk refined Disease Risk Index (DRI), MPN (n=2), and HR MDS (n=9) according to IPSS-R, undergoing allo-HCT using the sequential transplant approach in 2 transplant centers in Singapore between January 2009 and June 2020. The sequential transplant approach combined a cytoreductive chemotherapy, which consisted of either FLAMSA (n=17), FLAG +/- Ida [fludarabine/cytarabine/granulocyte colony stimulating factor (GCSF) +/- idarubicin] (n=23), or CLAG (clofarabine +/- cytarabine +/- GCSF) (n=15), followed by reduced intensity (RIC) (N=48) or myeloablative (MAC) (N=8) conditioning regimen. All patients received peripheral blood stem cell from matched related donors (N=30), mismatched related donors (N=3), matched unrelated donors (N=16), or mismatched unrelated donors (N=7). Post-grafting immunosuppression consisted of calcineurin inhibitor and mycophenolate mofetil in all patients. Thymoglobuline or post-transplant cyclophosphamide were added for GVHD prophylaxis in unrelated donor transplant and mismatched related donors, respectively. Results: The median time to neutrophil &amp;gt; 1000/μL was 11 days (range, 8-19). With a median follow-up of 44 months (range, 1-123), the Kaplan-Meier estimate of overall (OS) and leukemia-free (LFS) survivals at 5 years were 49% (95% CI, 35-62) and 37% (95% CI, 23-52), respectively. The 2-years cumulative incidence of relapse and non-relapse mortality (NRM) were 47% (95% CI, 32-60) and 13% (95% CI, 6-24), respectively. Patients receiving FLAG or CLAG-based sequential regimen showed lower cumulative incidence of NRM (2-year cumulative incidence for NRM: 5% vs 29%; p=0.018), and similar relapse (2-year cumulative incidence for relapse: 49% vs 53%; p=0.64), as compared to patients given FLAMSA-based sequential regimen, resulting in a trend towards more favourable OS (5-year OS: 53% vs 41%; p=0.29) and LFS (5-year LFS: 46% vs 20%; p=0.08). In multivariable analysis, only refined DRI showed significant impact on OS (p=0.04), but has no significant impact on LFS, NRM, and relapse. The 5-year OS for patients with intermediate/high risk and very high risk DRI were 59% and 27%, respectively (p=0.017), and the corresponding 5-year LFS were 46%, and 20% (p=0.06), respectively (Figure 1 &amp; Figure 2). The intensity of conditioning regimen did not significantly impact on OS, LFS, relapse, and NRM. Conclusions: Sequential transplant conditioning with FLAMSA, FLAG or CLAG followed by allo-HCT is an effective strategy in overcoming the dismal prognosis of HR AML, MDS and MPN, and enabling favourable long-term disease-free survival. More studies on effective strategies such as post-transplant maintenance therapy with prophylactic donor lymphocyte infusion, are needed to further eliminate the risk of relapse, without increasing risk of treatment related toxicity. Disclosures No relevant conflicts of interest to declare.

3132 – IMPACT OF THE INFLAMED BONE MARROW NICHE ON THE PROGRESSION OF MYELOPROLIFERATIVE NEOPLASIA

Bcr/Abl-negative myeloproliferative neoplasia (MPN) is characterized by driver mutations in MPL, CALR, or JAK2, as well as an inflammatory microenvironment. The acquisition of clonal abnormalities and secondary mutations in MPN patients can progress the disease to Acute Myeloid Leukemia (AML), resulting in bone marrow (BM) fibrosis and BM failure. For these patients, treatment options fall short and do not include approaches to prevent leukemic transformation. Our group has developed an animal model for inflammation where loss of RBPJ/Notch signaling in the BM niche leads to upregulation of miR155 and an accumulation of proinflammatory cytokines. We propose to use this model (RBPJKO mouse) to study the influence of chronic inflammation on JAK2V617F-driven MPN progression. We hypothesize that chronic inflammation in the BM niche: i) promotes proliferation and genomic instability in hematopoietic cells; and ii) cooperates with preexisting genetic mutations such as JAK2V617F to promote clonal expansion and selection, accelerating disease progression. Our preliminary studies indicate that JAK2V617F hematopoietic cells drive a more aggressive disease when exposed to chronic inflammation following transplant into RBPJKO inflamed BM niches. Indeed, JAK2V617F/RBPJKO recipients exhibited accelerated neutrophilia, higher myeloid skewing and increased bone marrow cellularity and splenomegaly, when compared to JAK2V617F/RBPJWT recipients. By using alkaline comet assay and gH2Ax foci, we also demonstrate that JAK2V617F cooperates with the inflamed BM niche to promote more DNA single- and double-strand breaks in Lin- progenitors in vivo. To further study these events, we optimized an in vitro model in which WT and JAK2V617F BM Lin- progenitors are co-cultured with RBPJKO or RBPJWT BM-derived stroma. JAK2V617F cells showed increased proliferation, mitochondrial ROS accumulation, elevated 53BP1 foci and increased γH2AX MFI when exposed to the inflammatory conditions supported by RBPJKO stroma. Taken together, these data suggest that chronic inflammation can accentuate the MPN phenotype, promote replicative stress and lead to buildup of DNA-damaging ROS in JAK2V617F MPN cells. This work has the potential to provide us with new insights into the mechanisms of MPN progression and the opportunity to identify new therapeutic approaches.

Diagnosis and prognosis are supported by integrated assessment of next-generation sequencing in chronic myeloid malignancies. A real-life study.

Next-generation sequencing (NGS) is used to investigate the presence of somatic mutations. The utility of incorporating routine sequencing to guide diagnosis and therapeutic decisions remains unclear. We report the findings of an observational, multicenter study that aimed to assess the impact of somatic mutation testing by NGS in a reallife setting of chronic myeloid malignancies. A total of 177 patients were enrolled, partitioned into two overlapping groups. In group A (n=94), the indication was to search for clonal hematopoiesis, in a context of suspected myelodysplastic syndrome or myeloproliferative neoplasia. In group B (n=95), the theranostic impact of somatic mutations was studied. A panel of 34 genes was used on DNA extracted from blood or bone marrow samples. Within group A, the detection of clonal hematopoiesis supported the diagnosis of chronic myeloid malignancies for 31 patients while the absence of clonal hematopoiesis ruled out the suspected diagnosis in 47 patients. Within group B, NGS identified prognostically relevant somatic mutations in 32 patients, which had a therapeutic impact in 18 cases. By determining the presence or absence of somatic mutations, the application of NGS in daily practice was found to be useful for an integrated final diagnosis in 83% of the patients. Moreover, the search for somatic mutations had a prognostic impact that led to treatment modification in 19% of the cases. This study outlines the fact that adequate implementation of new investigations may have a significant positive medico-economic impact by enabling appropriate management of patients.

Co-existence of myeloproliferative neoplasias and β-thalassemia with IVS-2-654 mutation—a case report

Thalassemia and myeloproliferative neoplasias (MPNs) are recognized as two separate diseases. Thalassemia is a hemolytic disease caused by abnormal goblin genes, which results in the deficiency of globin chains. MPNs are clonal hematopoietic stem cell diseases characterized by the proliferation of multiple myeloid lineages. The coexistence of thalassemia and myeloproliferative neoplasia are rarely reported. We herein describe a case of a 24-year-old woman, previously diagnosed as β-thalassemia, with a lifelong history of thrombocytosis. She was subsequently diagnosed as low-risk essential thrombocythemia (ET), one of the MPNs. The patient was treated with folic acid supplementation and Iron-chelating therapy, without aspirin or cytoreductive therapy. Up to date, no thrombotic events or disease-related bleeding are happening to the patient, who remains in good health. Furthermore, we found three novel genes mutations EP300, CUX1, and FGFR3 after next-generation sequencing. We presume that the mutations of these genes in β-thalassemia might have an impact on the happening of ET and therefore need further investigations.

Current treatment of myeloproliferative neoplasias: three scenarios

Current treatment of myeloproliferative neoplasias: three scenarios

MOLECULAR GENETIC ABNORMALITIES IN THE GENOME OF PATIENTS WITH Ph-NEGATIVE MYELOPROLIFERATIVE NEOPLASIA AFFECTED BY IONIZING RADIATION AS A RESULT OF THE CHORNOBYL NUCLEAR ACCIDENT.

to determine the frequency of major somatic mutations in the JAK2, MPL and CALR genes in the genomeof patients with Ph-negative myeloproliferative neoplasms that occur in individuals who have been exposed to ionizing radiation as a result of the Chornobyl accident. Molecular genetic analysis of genomic DNA samples isolated from blood was performed in90 patients with Ph-negative myeloproliferative neoplasia (MPN) with a history of radiation exposure and 191patients with spontaneous MPN utilizing allele-specific polymerase chain reaction (PCR). The presence of major mutations in the genes JAK2, CALR and MPL was revealed in patients with MPN witha history of radiation exposure with a frequency 58.9 % (53 of 90), 12.2 % (11 of 90), and 0 % respectively, and without exposure with frequency 75.4 % (144 of 191), 3.1 % (6 out of 191) and 1.6 % (3 out of 191) respectively.Mutations JAK2 V617F in patients with spontaneous MPN were observed in each clinical form: polycythemia vera (PV),essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations were detected exclusively inpatients with PMF and ET, significantly more often in groups with a radiation exposure history (18.9 % and 33.3 %,vs. 4.2 % and 6.5 %) than without one. At the same time, the occurence of MPL mutations was determined only inpatients with spontaneous MPN in 1.6 % of casees. Triple negative mutation status of genes JAK2, MPL and CALR prevailed in the group of patients with MPN with a history of radiation exposure and was 27.8 %, against 16.2 % inpatients without radiation exposure (p = 0.05). Genomic research of patients with Ph-negative MPN revealed features of molecular genetic damage inthose patients who were exposed to IR as a result of the Chornobyl accident and those with spontaneous MPN. Thedata obtained by determining of JAK2, MPL and CALR genes mutational status in the genome of patients with MPN isnecessary to expand the understanding of the mechanism of leukogenesis, especially caused by radiation.

Hematobiological Profile of Patients with Chronic Myeloid Leukemia at the Diagnosis in Yaound&amp;#233;: A Cross-Sectional Study

Background: Chronic Myeloid Leukemia (CML) is a myeloproliferative blood neoplasia, characterized by the presence of a translocation between chromosomes 9 and 22 leading to the formation of the Philadelphia chromosome. Data on the biological profile of patients with CML at diagnosis are still lacking in sub-Saharan Africa, particularly in Cameroon. Methods: A cross-sectional study was carried out from January 2001 to July 2016 among patients recently diagnosed with CML at the Yaounde University Teaching Hospital, the Yaounde Central Hospital and the Yaounde General Hospital. Analyzed variables included socio-demographic, clinical presentation, the diagnosis means, biological parameters (hematological and biochemical). Sampling was consecutive. Results: We included 132 (76 males) patients with CML with a median age of 39.2 years at diagnosis. The 31 - 45 years age group was the most represented, with 40.9% of the study population. A risk factor was found in only 5 (3.8%) of patients. Clinical manifestations were recorded in only 27 (20.45%) patients, with fatigue being the commonest (10.6%). Almost all patients (128, 96.9%) have performed the karyotype while 22 (16.7%) have performed fluorescence in situ hybridization (FISH) and 4 (3.0%) the PCR. At diagnosis, 66% of the patients were in the chronic phase (CP), 11.3% in accelerated phase (AP), and 22.7% in blast crisis (BC). All patients presented hyperleukocytosis, with a white blood cell mean of 128,362/mm3. Anemia was common (77.3%), usually moderate (61.4%). Thrombocytopenia was rare (8.3%), as far as basophilia (1.2%). Among those patients, mean values of creatinine, Glutamic pyruvate transaminase (GPT) and glycemia were normal while activated partial thromboplastin time (APTT), prothrombin time (PT), plasma uric acid level, gamma glutamic transferase (GGT), lactate dehydrogenase (LDH), and inflammatory parameters (ESR and CRP) were increased. Conclusion: Patients with CML presented at their diagnosis hyperleukocytosis and anemia as hematological clues. Other biological anomalies include increased signs of cellular destruction (plasma uric acid level, LDH), coagulation perturbation and inflammatory syndrome. The chronic phase of the disease was common.

A KLF4-DYRK2–mediated pathway regulating self-renewal in CML stem cells

AbstractLeukemia stem cells are a rare population with a primitive progenitor phenotype that can initiate, sustain, and recapitulate leukemia through a poorly understood mechanism of self-renewal. Here, we report that Krüppel-like factor 4 (KLF4) promotes disease progression in a murine model of chronic myeloid leukemia (CML)-like myeloproliferative neoplasia by repressing an inhibitory mechanism of preservation in leukemia stem/progenitor cells with leukemia-initiating capacity. Deletion of the Klf4 gene severely abrogated the maintenance of BCR-ABL1(p210)–induced CML by impairing survival and self-renewal in BCR-ABL1+ CD150+ lineage-negative Sca-1+ c-Kit+ leukemic cells. Mechanistically, KLF4 repressed the Dyrk2 gene in leukemic stem/progenitor cells; thus, loss of KLF4 resulted in elevated levels of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2), which were associated with inhibition of survival and self-renewal via depletion of c-Myc protein and p53 activation. In addition to transcriptional regulation, stabilization of DYRK2 protein by inhibiting ubiquitin E3 ligase SIAH2 with vitamin K3 promoted apoptosis and abrogated self-renewal in murine and human CML stem/progenitor cells. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling p53- and c-Myc–mediated regulation of survival and self-renewal in CML cells with leukemic-initiating capacity that can be targeted with small molecules.

Shared Genomic Alterations in Patients with Co-Existing Myeloproliferative Neoplasms and Angioimmunoblastic T-Cell Lymphoma

Background: Myeloproliferative neoplasia (MPN) and angioimmunoblastic T-cell lymphoma (AITL) are two distinct hematologic cancers. However, we have shown that patients with both malignancies probably occur with frequencies higher than expected in the background population (1,2). AITL tumors show a high frequency (30-70%) of mutations in RHOA and epigenetic modifier genes such as DNMT3A, TET2 and IDH2. The latter genomic alterations are similar to those found in myeloid diseases such as MPN. This observation has raised questions on whether the two hematologic malignancies may share genomic aberrations (e.g. at progenitor level) suggesting a possible pathogenetic relationship between these diseases. Aim: To further investigate these questions, we explored the mutational landscape in MPN and AITL tumors occurring in the same host. Methods: From a Danish cohort of 97 patients with co-existing MPN and lymphoma identified through the Danish Lymphoma and Pathology Registries and immunohistochemically validated by tertiary center hematopathologists, five patients with MPN and concurrent and/or subsequent AITL were included. Paired DNA samples obtained from lymph node biopsies (AITL component), bone marrow aspirates (MPN component) and saliva specimens (normal reference DNA) were analyzed by whole exome sequencing. A minimum variant allele frequency threshold of 10% was applied. However, mutations in epigenetic modifier genes, RHOA and JAK2, were also included, if found at slightly lower frequencies. Results: Figure 1 shows an overview of the identified mutations. At a VAF% cut-off value of 10 and a median coverage of 30, mutations shared between the MPN and AITL specimens were found within the DNMT3A, TET2, and IDH2 genes. Mutations in the JAK2 gene were identified in all MPN and in some of the AITL samples. NOTCH2 mutations were frequent and some were shared between the MPN and AITL samples. Two patients with essential thrombocytosis and AITL had the same NOTCH2 (C19W) and NCOR1 (K178N) mutation. Most of the identified genomic alterations were inactivating missense mutations. Conclusion: Co-existence of MPN and AITL occurs and may not be a random event. We studied tumor tissues from patients diagnosed with both diseases, and identified recurrent shared and non-shared mutations in samples from both types of cancers. These mutations may contribute to the development of the two malignancies in the same host, partly through common steps at precursor level (e.g. DNMT3A) and partly more downstream at a more lineage-specific level (e.g. IDH2). The functional role of the observed NOTCH2 and NCOR1 mutations is currently under investigation. Holst J et al. Blood 2017, 130 (S1): 1525 Frederiksen H et al. Blood 2011;118(25):6515-6520 Disclosures Tam: Takeda: Consultancy; Paragon Genomics: Consultancy.

JAK2V617F Clonal Hematopoiesis Stratifies By Peripheral Blood Counts.

Background:Patients with clonal hematopoiesis (CH) in the absence of WHO-classified myeloid disease are of special interest given their increased prevalence with age, predisposition to morbid cardiovascular complications, and amplified risk of overt hematologic malignancy. Pts are often stratified by normal peripheral blood counts into clonal hematopoiesis of indeterminate potential (CHIP), or those with unexplained cytopenias as clonal cytopenias of undetermined significance (CCUS). However, less is known about pts with elevated counts and clonal hematopoiesis who do not fulfill WHO criteria for any myeloproliferative neoplasia (MPN). We leveraged Vanderbilt University Medical Center's unique biobank, BioVU, to identify the prevalence of JAK2V617Facross 48,000 pts to evaluate the clinical changes in progression from CH to overt myeloid disease. Methods:To develop a reference JAKV617Ftraining set, next generation sequencing via Illumina Trusight Myeloid Panel (NGS) was performed on BioVU samples (N=133) from pts with confirmed myeloproliferative malignancy. Of those pts, 78 harbored JAK2V617Fwith a range of variant allele frequencies (VAF). Matched samples in this training set (N=133) were also analyzed via Infinium® Expanded Multi-Ethnic Genotyping Array (MEGAEX). SNP array JAK2V617Fvariant intensity was extracted (rs77375493; NM_004972.3(JAK2): c.1849G&gt;T (p.Val617Phe). A regression model was built using NGS VAF as a dependent variable and MEGAEX intensity data as independent variable (r2=0.9931).Based on this model, we imputed JAK2V617FVAF for all 48,000 pts in our cohort. Pts with JAK2V617Fwere subdivided into: clinically confirmed myeloid disease, or JAK2V617Fwithout a diagnosis of MPN. Upon review of the EMR, the latter group was further dived into: 1) probable undiagnosedMPN, 2) CHIP, 3) CCUS, or 4) CH with associated elevated peripheral blood counts (CHAPbc). Only lab values after the date of JAK2V617Fdetection were included. Confirmed malignancy was defined by WHO classification of disease. Pts with evidence of possible WHO classified PV or ET with Hgb &gt;18.5g/dl in men, &gt;16.5g/dl in women, or PLT count &gt;450k/mcl regardless of gender were classified as probable undiagnosedMPN. CHIP was defined as JAK2V617Fwithout abnormal counts across a patient's EMR lifetime, except when confounding events, e.g. trauma surgery or overt iron deficiency anemia, incorrectly skewed values. CCUS was defined as JAK2V617Fin the presence of unexplained cytopenias; hemoglobin (Hgb) &lt;13.5g/dl men or &lt;12g/dl women, leukocyte count (WBC) &lt;3.9x10^3/uL or platelet (PLT) &lt;135 x10^3/mcL. We classified pts with elevated blood counts who did not meet the WHO classification of MPN [e.g. WBC &gt;10.7 regardless of gender, Hgb 18-18.5 g/dL in men or 16.0-16.5 g/dL in women with maximum Hgb no greater than 18.5g/dl in men and 16.5g/dl in women, or PLT count between 371-450k/mcl regardless of gender (and no values &gt;450k/mcl)] as CHAPbc. Results:We identified 410 of 48,000 pts who harbored JAK2V617F(0.85% prevalence). Of those, 270/410 had clinically diagnosed hematologic malignancy including primary myelofibrosis (PMF) (79), ET (48), PV (43) and Ph-MPN NOS (29). MDS (29), AML (15), NHL (16), plasma cell dyscrasias (5), CML (3), other (3). There were 19/410 with insufficient clinical data to determine diagnosis. The remaining 121/410 JAK2V617Fpts did not have a related diagnosis. Figure 1a demonstrates imputed VAF differences in JAK2V617Fbetween known MPN vs. CH (including undiagnosed MPN) by age (mean VAF 0.44 vs. 0.17 respectively P&lt;0.001). We identified several undiagnosed MPNs (22), which subdivided into polycythemia predominant (1), thrombocythemia predominant (16) or a combination of polycythemia and thrombocythemia (5). The remaining 99/410 cases were CH,with CHIP (55), CCUS (29), and CHAPbc (15). Ranges of blood counts appear to be a continuous variable among JAK2V617Fpts with CH or undiagnosed MPN (Fig 1b-1d). Summary: We used an unbiased approach to identify the prevalence of JAK2V617Facross all pts at a single institution. In this cohort, hematologic malignancy and CH did stratify by imputed VAF. Further, within JAK2V617FCH, CHAPbc may be differentiated from CHIP by clinical phenotype and further investigation will be required to determine its impact on patient outcomes. Disclosures Savona: Sunesis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Patents &amp; Royalties; Selvita: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding.

High Dose Flamsa, CLAG or FLAG-Based Sequential Conditioning Regimen Followed By Allogeneic Hematopoietic Transplantation Results in Favorable Outcome for High Risk Acute Myeloid Leukemia, Myelodysplastic Syndrome, Myeloproliferative Neoplasia Patients: A Multicenter Study in Singapore

Introduction: Allogeneic haematopoietic cell transplant ( allo-HCT) is an effective consolidative treatment for patients with certain haematological malignancies and gives the best outcome when done in remission. However patients with refractory acute myeloid leukemia (AML) or some myeloproliferative neoplasia (MPN) and myelodysplastic syndrome (MDS) deemed unable to achieve remission with standard induction are often excluded from allo HCT with conventional conditioning regimen as pre-transplant remission could not be achieved. Recently, a sequential transplant approach, as developed by the Munich group, comprising of intensive cytoreductive chemotherapy FLAMSA (Fludarabine/Amsacrine/Cytarabine) to decrease leukemia cell burden shortly prior to conditioning regimen, has been successfully used for high-risk (HR) AML/MDS with promising results. Methods: We studied 48 patients (median age 53 years, range 26 - 68) with high risk AML (n=38), as defined by refractory, relapsed disease, secondary leukemia, complete remission with adverse-risk cytogenetics according to ELN criteria, or high/very risk refined Disease Risk Index (DRI), MPN(n=2) and HR MDS (n=8) according to IPSS-R, undergoing allo-HCT using the sequential transplant approach in 2 transplant centers in Singapore between January 2009 and October 2018. The sequential transplant approach combined a cytoreductive chemotherapy, which consisted of either FLAMSA (n=17), FLAG +/- Ida (fludarabine/Cytarabine/Granulocyte Colony Stimulating factor (GCSF) +/- Idarubicin] (n=23), or CLAG (Clofarabine / Cytarabine / GCSF) (n=8), followed by reduced intensity (RIC) (N=43) or myeloablative (MAC) (N=5) conditioning regimen. All patients received peripheral blood stem cell from matched related donors (N=27), matched unrelated donors (N=14), or mismatched unrelated donors (N=7). Post-grafting immunosuppression consisted of calcineurin inhibitor and mycophenolate mofetil in all patients. Thymoglobuline was added for GVHD prophylaxis for unrelated donor transplant. Results: The median time to neutrophil &gt; 1000/μL was 10 days (range, 9-25). With a median follow-up of 48 months (range, 9 to 111 months), the Kaplan-Meier estimate of overall survival (OS) and leukemia-free (LFS) at 5 years were 48 % (95% CI, 33-62), 45% (95% CI, 30-58), respectively. At 2 years cumulative incidence of relapse and non-relapse mortality (NRM) were 51% (95% CI, 33-67) and 15% (95% CI, 7-27), respectively. Patients receiving FLAG or CLAG based sequential regimen showed, lower cumulative incidence of NRM (2-year CI NRM: 7% vs 40%; p=0.037), and similar relapse (2 year CI relapse: 49% vs 53%; p=0.63) as compared to patients given FLAMSA-based sequential regimen, resulting in a trend towards more favourable OS (5 year OS: 53% vs 40%; p=0.33) and LFS (5 year LFS: 54% vs 35%; p=0.23). In multivariable analysis, only refined DRI showed significant impact on OS (p=0.04), but has no significant impact on LFS, NRM and relapse. The 5-year OS for patients with intermediate/high risk and very high risk DRI were 60% and 24%, respectively (p=0.024), and the corresponding 5-year LFS were 57%, and 27% (p=0.065), respectively (Figure 1 &amp; Figure 2). The intensity of conditioning regimen did not significantly impact on OS, LFS, relapse and NRM. Conclusions: Sequential transplant conditioning with FLAMSA, FLAG or CLAG followed by allo-HCT is an effective strategy in overcoming the dismal prognosis of high risk AML, MDS and MPN, and enabling favourable long-term disease free survival. More studies on effective strategies such as post-transplant maintenance therapy with prophylactic donor lymphocyte infusion, are needed to further eliminate the risk of relapse, without increasing risk of treatment related toxicity. Disclosures No relevant conflicts of interest to declare.

Secondary Acute Myeloid Leukemia: Demographic, Physiopathogenic, Clinical and Therapeutic Comparative Study

INTRODUCTION Secondary Acute Myeloid Leukemia (s-AML) evolves from a previous hematopoietic clonal disease such as Myelodysplastic Syndromes (MDS/AML), myeloproliferative neoplasia (NPM/AML), medullary insufficiencies - aplastic anemia - (AA/AML) or exposure to chemo or radiotherapy (t-AML). The objective of this work is to describe and highlight the demographic, pathophysiologic and clinical-therapeutic characteristics of s-AML patients compared with p-AML. METHODS This is a retrospective cohort study based on the casuistry from 34 reference centers in Latin America during a period of 10 years (JAN10'-MAY19'). Patients ≥18 years old with primary AML, excluding the promyelocytic subtype (p-AML), and s-AML were admitted. Age, gender, performance status, comorbidity, cytogenetics, mutations, AML subtypes, extramedullary compromise, treatments and overall survival (OS) were analyzed. Statistically, Graph Pad Prism version 5.00 and, SPSS version 17 were used. RESULTS One thousand eleven patients with newly diagnosed AML were recruited, 693 (68.5%) corresponded to p-AML and 318 (31.5%) to s-AML. The demographic differences between p-AML and s-AML are shown on Table 1. Subtypes of s-AML: t-AML (18.5%), MDS/AML (58.2%), NMP/AML (13.5%), AA/AML (5.7%) and others s-AML (4.1%). Global median age was 58 years (R 18-93) and male 52%. Extramedullary compromise in CNS (3.2%) and other organs (5.5%). Seven hundred ninety-three cytogenetic studies were evaluable (based on MRC classification): High (22.3%), Intermediate (68.3%) and Low Risk (9.3%). FLT3 mutation was more frequently found in p-AML. In s-AML, the multivariate study showed short overall survival associated with ECOG ≥2 (HR:2.0), white blood count ≥ 50x109/L at diagnosis (HR:1.9), poor risk karyotype (HR:1.6) and age over 60 years (HR:1.5). At least, 883 patients received treatment (Table 2). During this study period, 211 patients (21%) were transplanted; 49 (23%) were s-AML; histoidentical related donor (46%), haploidentical (39%), non-related (8%) and autologous (7%). The median survival for all AML was 11.0 months with a statistically significant difference in favor of the p-AML (Figure 1). CONCLUSION Performance status (by ECOG ≥2), age ≥60, level of leukocytes a ≥50x109/L, poor risk karyotype and s-AML subtype at diagnosis had a significant worse impact on overall survival. Most patients with s-AML came from MDS, they were older and their incidence increased as the population aged. They presented more comorbidities and worse performance status. Undoubtedly, our findings showed that s-AML is a distinct high risk subset of myeloid disorder with adverse prognosis and represents a therapeutic challenge. Disclosures No relevant conflicts of interest to declare.

NUP98-HOXA9-Initiates Molecular and Biologic Features of Disease Progression in a Humanized Model of CML

Acute myeloid leukemias (AMLs) are heterogenous diseases often resulting from the acquisition of multiple genetic alterations that deregulate hematopoietic precursor proliferation and block normal differentiation. Chronic myeloid leukemia offers a unique opportunity to identify molecular mechanisms that interfere with normal differentiation in the context of a highly proliferative hematopoietic stem cell clone that produces massive number of functional differentiated myeloid cells due to the presence of a BCR-ABL1 fusion gene. Since the NUP98-HOXA9 (NA9) fusion gene has been identified in some CML blast crisis patients or de novo AML, we asked whether a humanized model of CML progression to AML would result from its lentiviral-mediated introduction into primary CML CD34+ cells from 3 chronic phase CML patients in which &gt;97%, &gt;96%, and 44% of the CD34+ longterm culture-initiating cells were Ph+/BCR-ABL1+. In vitro experiments showed the NA9-transduced cells produced a hugely increased number of granulo-monocytic progenitors in long-term cultures (up to 1000-fold, p=0.03; t-ratio test) and enhanced the serial replating activity of directly clonogenic cells as compared to matched samples of cells transduced with a control vector. In vivo experiments showed that 90% of sublethally irradiated NOD-SCID IL2Rgc-null mice expressing human IL3, GM-CSF and SCF constitutively that were transplanted with these same NA9-transduced cells developed evidence of a progressed myeloproliferative neoplasia. This included tissue infiltrates of eosinophils, basophils and mastocytes, exclusive myeloid differentiation and signs of an imminently fatal leukemia between 8 and 27 weeks post-transplant in 50% of cases, although an excess blast population was not seen in these. RNA-sequencing of cells analyzed just 48 hours after transduction revealed a signature of 53 genes that were more highly expressed in CD34+ CML cells carrying the NA9 fusion gene in comparison to control CML cells. This signature included many genes expressed by hematopoietic stem cells (HSCs), such as 5' HOXA genes, 3' HOXB genes, PBX3, MEIS1,ARID5B, AHR, REL, BMP6, GDF10, SFRP5, PPBP,PLA2G4A suggesting that NA9 induces the expression of a partial HSC program in later CML progenitor types that make up the bulk of the CD34+ CML population. Consistent with this hypothesis, the NA9 signature separated the HSCs, CMPs and GMPs of chronic CML patients as well as those of normal subjects (GSE47927; p=1x10-6, p=2x10-4 respectively). Most genes in the NA9 signature were also found to be over-expressed in CD34+ cells of patients in the accelerated phase and in the blast phase of CML, including ARID5B, AHR, STARD9, TOX, or FOXP1 (p=9.10-8; ANOVA) (GSE4170). The NA9 signature was also significantly enriched in transcripts of genes that are over-expressed in the blasts of AML patients carrying NPM1 mutations or MLL fusions and was predictive of overall survival in the AML cohort of the Cancer Genome Atlas (p=0.02; log-rank test, n=200). This stem cell signature was also associated with an increase in the number of H3K27ac marks (on average 7805 ± 960 peaks for NA9 vs 5888 ± 2739 peaks for control) at 48 hours post-transduction. De novo H3K27ac peaks in NA9+ CD34+ cells were located in proximal and distal enhancers of 993 genes (GREAT parameters TSS ± 2kb from TSS and 100kb max extension). These were significantly enriched genes in the GM-CSF signaling pathway (MySigDB, binomial p-value=2.19x10-8) and that are upregulated in granulocytes and monocytes following LPS exposure. H3K27ac peaks also matched with GATA1, RELA, MEF2A and IKZF1 transcription factor binding sites previously mapped by ChIPSeq experiments (adjusted p-value &lt;0.05). Finally, we identified super-enhancers in 12 genes among which were PBX3, ANGPT1, MBNL1 and PRKACB. Overexpression of the NA9 fusion gene in chronic phase CD34+ CML cells thus appears to reprogram the expression of HSC genes as well as those associated with GM-CSF pathway activation and inflammatory responses via H3K27 acetylation of associated loci leading to a picture of advanced accelerated phase/disease progression but not the complete differentiation arrest seen in terminal blast crisis or frank AML. These findings highlight the multiplicity of biologically important molecular alterations that can result from a single epigenetic perturbation but, nevertheless, are insufficient to create an overt AML phenotype. Disclosures Turhan: novartis: Honoraria, Research Funding; Incyte: Consultancy, Honoraria.

Arterial and venous thrombosis by high platelet count and high hematocrit: 108 521 individuals from the Copenhagen General Population Study

BackgroundIt is unclear whether high platelet count or high hematocrit predict risk of thrombosis in individuals from the general population. ObjectivesWe tested the hypothesis that individuals from the general population with high platelet count or high hematocrit have high risk of arterial and venous thrombosis. MethodsWe prospectively followed 108 521 individuals from The Copenhagen General Population Study for a median of 8 years. Platelet count and blood hematocrit were measured at study entry. Results and ConclusionMultivariable adjusted hazard ratios for individuals with platelet counts in the top 5 percentiles (>398 × 109/L) vs in the 25th‐75th percentiles (231‐316 × 109/L) were 1.77 (95% confidence interval [CI], 1.38‐2.24) for arterial thrombosis in the brain (38 and 26 events/10 000 person‐years) and 0.82 (95%, 0.61‐1.11) for arterial thrombosis in the heart (23 and 28 events/10 000 person‐years). For individuals with hematocrit values in the top 5 percentiles (women/men: >45/>48%) vs the 25th‐75th percentiles (women/men: 38.1‐42/41.1‐45%), hazard ratios were 1.27 (95% CI, 0.91‐1.75) for arterial thrombosis in the brain (40 and 26 events/10 000 person‐years) and 1.46 (95% CI, 1.06‐2.00) for arterial thrombosis in the heart (43 and 25 events/10 000 person‐years). Neither high platelet count nor high hematocrit was associated with risk of venous thromboembolism. When excluding individuals with myeloproliferative neoplasia from the main analyses, results on risk of thrombosis were similar. In this prospective study, high platelet counts were associated with 1.8‐fold risk of arterial thrombosis in the brain, whereas high hematocrit was associated with 1.5‐fold risk of arterial thrombosis in the heart.

Feasibility of tumor‑derived exosome enrichment in the onco‑hematology leukemic model of chronic myeloid leukemia.

Due to the discovery of their role in intra-cellular communications, exosomes, which carry information specific to the cell of origin, have garnered considerable attention in cancer research. Moreover, there is evidence to suggest the possibility of isolating different exosome sub-populations based on target antigens at the cell surface. Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the break-point cluster region-proto-oncogene 1 tyrosine-protein kinase (BCR-ABL1) fusion-gene, derived from the t (9;22) translocation. Tyrosine kinase inhibitors (TKIs) target BCR-ABL1 protein and induce major or deep molecular responses in the majority of patients. Despite the fact that several studies have demonstrated the persistence of leukemic cells in the bone marrow niche, even following treatment, TKIs prolong patient survival time and facilitate treatment-free remission. These characteristics render CML a plausible model for investigating the feasibility of tumor-derived exosome fraction enrichment. In the present study, patients in the chronic phase (CP) of CML were treated with TKIs, and the quantification of the BCR-ABL1 exosomal transcript was performed using digital PCR (dPCR). The possibility of tumor-derived exosomes enrichment was confirmed, and for the first time, to the best of our knowledge, the detection of the BCR-ABL1 transcript highlighted the presence of active leukemic cells in patients with CP-CML. According to these findings, tumor-derived exosomes may be considered a novel tool for the identification of active leukemic cells, and for the assessment of innovative monitoring focused on the biological functions of exosomes in CML.