High Risk Of Leukemic Transformation Research Articles

SF3B1 K700E Alters the Splicing of Metabolism and Stress Response Genes, Attenuating the Effect of DNMT3A Mutations in MDS

Myelodysplastic syndromes (MDS) are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Genomic sequencing studies have demonstrated that MDS develops due to the acquisition of somatic mutations in hematopoietic stem cells (HSCs). Splicing factor 3b subunit 1 ( SF3B1) mutations, one of the most frequent mutations in MDS, are associated with lower-risk disease characterized by ineffective erythropoiesis. Our group and others have shown that mutations in SF3B1 cause aberrant mRNA splicing in human and murine HSCs, an expansion of long-term HSCs (LT-HSCs) in primary mice, but a competitive disadvantage in transplantation assays. DNMT3A, de novo methyltransferase 3A, is an epigenetic regulator mutated in ~10-15% of MDS. DNMT3A mutations are associated with unfavorable outcomes in MDS including a higher risk of leukemia transformation and shorter overall survival. Murine models of Dnmt3a mutations revealed that Dnmt3a-mutant HSCs have increased self-renewal over lineage-specific differentiation. SF3B1 and DNMT3A mutations co-occur more frequently than expected by chance and arise early in MDS pathogenesis, making them attractive therapeutic targets. MDS patients with SF3B1 and DNMT3A mutations have shorter progression-free survival than those with solely SF3B1 mutations. However, the mechanisms by which these mutations cooperate to cause HSC dysfunction and their possible combined role in leukemogenesis are not well understood. To investigate how Dnmt3a and Sf3b1 mutations disrupt HSC function in vivo we crossed two conditional knock-in mouse models of common point mutations in SF3B1 (K700E) and DNMT3A (R787H, murine equivalent to human DNMT3A R882H). We followed monthly blood counts and evaluated stem and progenitor cell composition in the bone marrow at 6 months. We found that Sf3b1/Dnmt3a double mutant mice display a similar phenotype to Sf3b1K700E mice, with a progressive anemia and an expansion in LT-HSCs. However, when assayed for HSC self-renewal potential, whole bone marrow and LT-HSCs isolated from Sf3b1/Dnmt3a mutant mice display increased serial colony formation capacities similar to Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs, 5 to 6 passages), but have a competitive disadvantage in secondary and tertiary transplant assays that is less severe than Sf3b1K700E HSPCs but distinct from the competitive advantage of Dnmt3a mutant HSPCs. Transcriptionally, double mutant HSCs clustered closer to Sf3b1 HSCs with shared expression of 74% of upregulated genes and 64% of downregulated genes and have decreased expression of stress response and metabolism genes. These results indicate Sf3b1 mutations may mitigate the preleukemic phenotype due to Dnmt3a mutations in MDS. To identify factors regulated by SF3B1 mutations that may mitigate the effect of DNMT3A loss on leukemic progression, we generated isogenic DNMT3A KO and SF3B1 K700E single and double mutant K562 cell lines using CRISPR/Cas9. Transcriptome analysis demonstrated that SF3B1K700E/DNMT3AKO double mutant clones clustered with and shared similar upregulated (69%) and downregulated (62%) genes as SF3B1 single mutant clones. Similar to our findings in murine HSPCs, pathway analysis of SF3B1 and SF3B1/DNMT3A mutant clones displayed enrichment of stress response and metabolic pathways. Alternative mRNA splicing analysis identified 13 events (3 alternative 3' splice site, 9 intron retention, and 1 skipped exon) in 8 genes that were upregulated in DNMT3A KO clones and downregulated in SF3B1 and SF3B1/DNMT3A mutant clones. We confirmed decreased protein expression of these mis-spliced genes in SF3B1 single and double mutant clones, but not DNMT3A KO clones. These genes include components of the integrated stress response pathway, which are also mis-spliced in SF3B1 and SF3B1/DNMT3A mutant MDS patient samples. Taken together our data suggest that aberrant splicing by mutant SF3B1 prevents metabolic reprogramming and decreases leukemic progression caused by DNMT3A mutation in MDS.

HMR Mutations Drive Poor Prognosis in Myelofibrosis Patients with Lower JAK2V617F Allele Burden but Not in Those with Higher Allele Burden: Results of a Multicenter Study

JAK2V617F is the most common driver mutation in myelofibrosis (MF), followed by CALR and MPL alterations. Clinical variables included in the International Prognostic Scoring System (IPSS), Dynamic IPSS (DIPSS), and DIPSS plus, as well as JAK2V617F allele burden are all well-established prognostic factors influencing the survival of MF patients. In addition, ASXL1, EZH2, SRSF2, IDH and U2AF1 mutations are classified as high-molecular risk (HMR) mutations in this context because of their close association with disease progression and leukemic transformation. However, the relationship between JAK2V617F allele burden and HMR mutations remains unclear. To address this, we recruited 122 MF patients (discovery cohort) diagnosed at the National Taiwan University Hospital (Taipei, Taiwan) who had available mutation profile determined by next-generation sequencing. For external validation, 82 MF patients from the Christie Hospital (Manchester, U.K.) and 51 MF patients from the Cleveland Clinic (Ohio, U.S.A.) and Vilnius University Hospital Santaros Klinikos (Vilnius, Lithuania) were enrolled. The median age of the discovery cohort was 61 years. JAK2V617F was the most common driver mutation (64.8%), followed by CALR (18%) and MPL mutations (9%). ASXL1 mutation was the most common (37%) genetic alteration other than driver mutations, followed by TET2 (16%) and EZH2 mutations (12%). Overall, 55 patients (45%) had at least one HMR mutation ( ASXL1, EZH2, SRSF2, IDH and U2AF1). With a median follow-up of 28.2 months, the median overall survival (OS) of the discovery cohort was not reached (NR) and was well risk-stratified by current prognostication systems, including IPSS, DIPSS plus, MIPSS70, and GIPSS. Notably, patients with at least one HMR mutation had significantly shorter leukemia-free survival (LFS) and OS than those without (both p<0.001). Next, we divided the 79 JAK2-mutated patients into lower- and higher-allele burden groups ( JAK2low and JAK2high) using the median (75%), as the cutoff. Concurring with previous reports ( Tefferi et al, Leukemia 2008; and Guglielmelli et al, Blood 2009), JAK2low patients had significantly inferior OS than JAK2high patients (median, 53 months vs NR, p=0.037). Among all JAK2-mutated patients, those with HMR mutations had a significantly shorter OS than those without (50 months vs NR, p<0.001). When considering HMR mutations and JAK2V617F allele burden together, the presence of HMR alterations conferred worse LFS and OS in JAK2low but not in JAK2high patients (HMR vs without HMR among JAK2low patients, p<0.001 and p<0.001; and JAK2high, p=0.822 and p=0.699, respectively). By and large, patients with concurrent HMR mutations and lower JAK2V617F allele burden had a distinctively inferior OS than those with other constellations of genomic lesions ( p<0.001, Figure 1A). This group of patients also had the shortest survival among the total cohort including both JAK2-mutated and wild patients ( p<0.001). The discriminating power of integrating HMR/ JAK2 allele burden for LFS and OS remained valid in the UK cohort (HMR vs without HMR: JAK2low, p=0.013 and p=0.012; and JAK2high, p=0.337 and p=0.600, respectively) and the US/Lithuania cohort (HMR vs without HMR: JAK2low, p=0.016 and p=0.013; and JAK2high, p=0.643 and p=0.446, respectively). Co-occurrence tests in the discovery and validation cohorts revealed that there was no shared mutational co-occurrence pattern among the 3 cohorts, excluding the potential confounding influence of specific concurrent mutations. More strikingly, time-dependent ROC curve analyses suggested that integrating JAK2 allele burden with HMR mutations could be complementary to current risk stratification systems in predicting LFS and OS of MF patients (Figure 1B) across 3 cohorts. Finally, HMR/ JAK2low was identified as an independent adverse prognostic factor by multivariable analysis, irrespective of age, risk stratification systems, and other mutations. In summary, we observe that HMR mutations accompanied by a lower mutant JAK2V617F allele burden identified a distinct MF population with significantly reduced survival and high risk of leukemic transformation, with independent prognostic relevance extrapolated to and validated in two independent cohorts. Experimental studies are ongoing to ascertain the underlying mechanistic basis and resolve the clonal architecture of this distinct MF genomic grouping.

Mutational Profile Enables the Identification of a High-Risk Subgroup in Myelodysplastic Syndromes with Isolated Trisomy 8.

Trisomy 8 (+8) is the most frequent trisomy in myelodysplastic syndromes (MDS) and is associated with clinical heterogeneity and intermediate cytogenetic risk when found in isolation. The presence of gene mutations in this group of patients and the prognostic significance has not been extensively analyzed. Targeted deep sequencing was performed in a cohort of 79 MDS patients showing isolated +8. The most frequently mutated genes were: TET2 (38%), STAG2 (34.2%), SRSF2 (29.1%) and RUNX1 (26.6%). The mutational profile identified a high-risk subgroup with mutations in STAG2, SRSF2 and/or RUNX1, resulting in shorter time to acute myeloid leukemia progression (14 months while not reached in patients without these mutations, p < 0.0001) and shorter overall survival (23.7 vs. 46.3 months, p = 0.001). Multivariate analyses revealed the presence of mutations in these genes as an independent prognostic factor in MDS showing +8 isolated (HR: 3.1; p < 0.01). Moreover, 39.5% and 15.4% of patients classified as low/intermediate risk by the IPSS-R and IPSS-M, respectively, were re-stratified as a high-risk subgroup based on the mutational status of STAG2, SRSF2 and RUNX1. Results were validated in an external cohort (n = 2494). In summary, this study validates the prognosis significance of somatic mutations shown in IPSS-M and adds STAG2 as an important mutated gene to consider in this specific subgroup of patients. The mutational profile in isolated +8 MDS patients could, therefore, offer new insights for the correct management of patients with a higher risk of leukemic transformation.

Sweet Syndrome Associated with Myelodysplastic Syndrome—A Review of a Multidisciplinary Approach

Sweet syndrome (SS) is a rare disease described as a febrile neutrophilic dermatosis with acute onset, the pathogenesis of which has not yet been elucidated. The syndrome is characterized by the sudden onset of erythematous infiltrated papules or plaques located on the upper body and is associated with fever, leukocytosis and neutrophilia. The lesions show a dense dermal infiltration with mature neutrophils. The condition is responsive to systemic steroids. The central nervous system, bones, muscles, eyes, ears, mouth, heart, lung, liver, kidneys, intestines, and spleen may be affected by SS as extracutaneous manifestations. More and more cases have been found to be associated with malignancies, particularly myelodysplastic syndrome, and, less frequently, other hematologic malignancies or solid tumors. Approximately 21% of patients with SS have an associated malignancy and up to 80% of MASS cases are associated with hematological diseases, predominantly myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Myelodysplastic syndrome is a clonal disease of the bone marrow characterized by inefficient hematopoiesis, dysplasia of the bone marrow and peripheral cytopenias. Affected patients have a high risk of leukemic transformation. After analyzing later studies and current practical aspects regarding MDS-related SS, we suggest an algorithm for evaluating these patients.

HOW IMPORTANT IS THROMBOCYTOSIS FOR PROGNOSIS OF MDS WITH 3Q21Q26 SYNDROME?

The structural changes involving the long arm of chromosome 3 at bands 3q21 and 3q26.2 in the form of inversion are named paracentric inversion inv(3)(q21q26.2) and in myeloid neoplasms have long been recognized, but are rare. The 3q21q26 syndrome usually occurs in a high-risk myelodysplastic syndrome (MDS) or the setting of acute myeloid leukemia (AML) and is most commonly reported as inv(3)(q21q26.2). Myeloid neoplasms with inv(3) are rare disorders with an incidence of 1% in MDS and AML. Thus, this report aims to show a patient with MDS and high platelets count who presented inv(3)(q21q26.2). A 72-year-old woman looked for medical attention due to fatigue and weakness. The patient reported a history of smoking for 41 years and denied any exposure to toxic agents. At physical examination, only pale was detected. A complete blood count revealed hemoglobin 7g/dL, MCV = 94 fL, leukocytes 5,600/mm3, neutrophils 2,242/mm3 and thrombocytosis with a platelet count of 514,000/mm3. Bone marrow aspirate showed dyserythropoiesis in 30% of cells and 6,5% blasts. The bone marrow cytogenetic analysis showed 46,XX,inv(3)(q21q26.2)[16]/46,XX[4]. The diagnosis of MDS with excess blasts - 1 was established according to the 2016 World Health Organization classification and International Prognostic Scoring System was very high. While waiting for beginning treatment, the patient died of respiratory failure due to COVID-19. Myelodysplastic syndrome with inv(3)(q21q26.2) is a rare aggressive disorder that occurs in less than 1% of all MDS cases and has been associated with a poor outcome: chemoresistance, high risk of leukemic transformation and short survival. Our case showed thrombocytosis with a platelet count of 514,000/mm3. The incidence of thrombocytosis in MDS has been reported in 8% of cases with platelets > 400 ×109/L. The major report which evaluated thrombocytosis in MDS studied 2,042 cases, detecting high platelets count in 5% of cases (102/2,042). It appears that thrombocytosis does not adequately capture the aggressive nature of inv(3)(q21q26.2) in MDS but still plays an important role in the pathogenesis of this heterogeneous and dynamic disease. Our patient reported herein showed dyserythropoiesis in 30% of cells and 6,5% blasts, but nothing was detected regarding megakaryocytic lineage. Our patient died very soon after diagnosis due to viral infection. Thrombocytosis is an unusual clinical feature in MDS associated with inv(3)(q21q26.2) and the unfavorable prognosis of inv(3) is independent of thrombocytosis.

Estrogens revert neutrophil hyperplasia by inhibiting Hif1α-cMyb pathway in zebrafish myelodysplastic syndromes models

Myelodysplastic syndromes (MDS) are characterized by daunting genetic heterogeneity and a high risk of leukemic transformation, which presents great challenges for clinical treatment. To identify new chemicals for MDS, we screened a panel of FDA-approved drugs and verified the neutrophil hyperplasia inhibiting role of 17β-estradiol (E2, a natural estrogen) in several zebrafish MDS models (pu.1G242D/G242D, irf8Δ57Δ/57 and c-mybhyper). However, the protective mechanism of estrogen in the development of hematological malignancies remains to be explored. Here, analyzing the role of E2 in the development of each hematopoietic lineage, we found that E2 exhibited a specific neutrophil inhibiting function. This neutrophil inhibitory function of E2 is attributed to its down-regulation of c-myb, which leads to accelerated apoptosis and decreased proliferation of neutrophils. We further showed that knockdown of hif1α could mimic the neutrophil inhibiting role of E2, and hif1α overexpression could reverse the protective function of E2. Collectively, our findings highlight the protective role of E2 on MDS by inhibiting hif1α-c-myb pathway, suggesting that E2 is a promising and effective drug for hematopoietic tumors associated with abnormal neutrophil hyperplasia.

P998: REGULATION OF BCL-X SPLICING BY SRC KINASES INHIBITORS IN MYELOPROLIFERATIVE NEOPLASMS

Background: Myeloproliferative neoplasms (MPN) are a group of incurable diseases that include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Both ET and PV are associated with prolonged clinical courses, with symptoms that affect their quality of life, and risk of progression to MF and acute leukemia. MF is the MPN subtype with the most adverse prognosis and is associated with a high risk of leukemic transformation. The FDA approved the use of ruxolitinib, a JAK1/2 kinase inhibitor, that proved to be effective in reducing symptoms and spleen volume, but not in inducing complete molecular remission. Although hematopoietic stem cell transplantation involves a high risk of mortality, it is nowadays the only curative treatment for MF. Therefore, it is relevant to identify new therapeutic strategies to improve the clinical outcome of MPN. Recently, there has been a growing interest in Bcl‐xL, the long isoform encoded by alternative splicing of the Bcl‐x gene, that acts as an anti‐apoptotic regulator. Bcl-xL appears to be up-regulated in MPN patients and we previously showed that its modulation correlates with the clinical severity of MPN subgroups. We also demonstrated that ABT-737, a specific Bcl-xL inhibitor, has a synergistic effect in combination with Ruxolitinib in HEL cell lines and MPN patients’ cells, suggesting that Bcl-xL inhibition could increase the power of treatment (Petiti et al. J Cell Mol Med. 2020). Unfortunately, ABT-737 is characterized by poor bioavailability, so a new approach to target Bcl-xL must be investigated. Paronetto et al. found that tyrosine phosphorylation of Sam68 in cancer cells may protect them from apoptosis by altering the Bcl-xS/L ratio in favor of Bcl-xL. Intriguingly, c-Src is one of the tyrosine kinases that phosphorylate Sam68 and several papers indicated its involvement in the MPN pathogenesis, without explaining the molecular mechanism. Aims: We aimed to molecular investigate the mechanism linking c-Src kinase activity with the deregulation of Bcl-x splicing in MPN, intending to consider c-Src kinase inhibitors, widely bioavailable, as indirect modulators of Bcl-xL to improve MPNs outcome. Methods: HEL cell lines, JAK2 V617F-mutated in homozygosis, has been incubated with different concentrations of ruxolitinib (JAK1/2 inhibitor) and dasatinib (SRC kinases inhibitor) at different concentrations and time points. Then, proliferation, and apoptosis were evaluated. Subsequently, the c-Src pathway, Sam68, and Bcl-xL/S ratio were investigated at both RNA and protein levels. Results: Performing in vitro study on HEL cell lines, we identified that dasatinib induced apoptosis and inhibition of proliferation in HEL cell lines, with a synergistic effect in combination with ruxolitinib. We also showed that dasatinib alone inhibited c-Src and Sam68 phosphorylation and down-modulated Bclx-L protein expression. Combination treatment with ruxolitinib and dasatinib increased both pro-apoptotic Cleaved Caspase 3 and BAX protein expression. Furthermore, we noted that treatment with ruxolitinib alone decreases the mRNA levels of Bclx-L, but also of the Bcl-xS isoform. On the contrary, combined drug therapy modulated the Bcl-xL/S mRNA ratio in favor of the pro-apoptotic Bcl-xS isoform. Summary/Conclusion: Although further studies will be necessary to better understand the c-Src role in Bcl-x splicing regulation in MPN, our preliminary data suggest that indirect downmodulation of Bcl-xL through SRC kinases inhibitors in combination with standard therapy could be useful in the future for the treatment of MPN patients.

Germline variants in DNA repair genes, including BRCA1/2, may cause familial myeloproliferative neoplasms

AbstractThe molecular causes of myeloproliferative neoplasms (MPNs) have not yet been fully elucidated. Approximately 7% to 8% of the patients carry predisposing genetic germline variants that lead to driver mutations, which enhance JAK-STAT signaling. To identify additional predisposing genetic germline variants, we performed whole-exome sequencing in 5 families, each with parent-child or sibling pairs affected by MPNs and carrying the somatic JAK2 V617F mutation. In 4 families, we detected rare germline variants in known tumor predisposition genes of the DNA repair pathway, including the highly penetrant BRCA1 and BRCA2 genes. The identification of an underlying hereditary tumor predisposition is of major relevance for the individual patients as well as for their families in the context of therapeutic options and preventive care. Two patients with essential thrombocythemia or polycythemia vera experienced progression to acute myeloid leukemia, which may suggest a high risk of leukemic transformation in these familial MPNs. Our study demonstrates the relevance of genetic germline diagnostics in elucidating the causes of MPNs and suggests novel therapeutic options (eg, PARP inhibitors) in MPNs. Furthermore, we uncover a broader tumor spectrum upon the detection of a germline mutation in genes of the DNA repair pathway.

Abstract 2093: Alternative splicing in hematopoietic stem cells is affected by the loss of DNMT3A

Abstract Myelodysplastic syndromes are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Survival ranges from months to years and bone marrow transplantation remains the only curative option. Large sequencing studies have revealed that somatic mutations in epigenetic regulators are one of the most commonly affected pathways in MDS. DNMT3A, a de novo methyltransferase is mutated in ~10-15% of MDS patients. Murine models of DNMT3A loss have shown that hematopoietic stem cells favor self-renewal over differentiation. Loss of function mutations in DNMT3A can lead to reduction in methylation resulting in differentially methylated regions (DMRs). The effects of altered DNA methylation at promoters and enhancers on gene expression in DNMT3A mutant cells are well characterized but do not completely explain the gene expression patterns in these cells. Previous studies have shown that DNA methylation within intragenic regions affects mRNA splicing through interactions with RNA polymerase II and DNA binding proteins. Therefore, our study aimed to determine whether altered methylation caused by DNMT3A loss affects mRNA splicing and gene expression. To address this, we used CRISPR/Cas9 to genetically knockout DNMT3A in K562 cells. Findings were further evaluated using lineage- c-kit+ hematopoietic stem and progenitor cells (HSPC) from a DMNT3A conditional knock in mouse model of the most common MDS-associated DNMT3A point mutation, R878H. Our central hypothesis is that aberrant methylation caused by loss of function mutations in DNMT3A leads to changes in RNA splicing resulting in differential isoform expression of genes involved in stem cell self-renewal and hematopoiesis. Using a combination of RNA sequencing and whole genome bisulfite sequencing we found increased alternative splicing in DNMT3A mutant cells compared to wildtype cells. Integration of methylation and splicing revealed that 15-20 percent of the alternative splicing events were associated with changes in methylation. We found that many of the genes aberrantly spliced were involved in cell cycle regulation, transcriptional regulation, and hematopoietic stem cell differentiation. In addition, isoform usage analysis revealed that genes involved in RNA processing were perturbed. We identified similar splicing patterns and affected pathways in murine HSPCs and K562 human cells. Notably, the type of splicing events changed with age. Our results show that DNMT3A mutant cells have distinct alternative splicing events and suggest an aberrant epigenome can cause dysregulated gene expression through splicing. These findings shed light onto additional mechanisms by which epigenetic regulator mutations lead to MDS development. Citation Format: LaShanale Wallace, Ana Leal-Cervantes, Jacquelyn Myers, Yiping Fan, Koral Campbell, Juan Martin Barajas, Aparna Calindi, Chandra Rolle, Baranda Hansen, Shondra Miller, Esther Obeng. Alternative splicing in hematopoietic stem cells is affected by the loss of DNMT3A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2093.

Characteristics and Survival of Patients with Chronic Phase Myelofibrosis and Elevated Blasts (5-9%), and the Effect of Therapy with JAK2 Inhibitor Ruxolitinib

Abstract Introduction: Patients with accelerated phase (AP) myelofibrosis (MF) (10-19% blasts in bone marrow [BM] or peripheral blood [PB]) have higher risk of leukemic transformation and shorter overall survival (OS) than those in the chronic phase ( Objective: We aimed to identify clinical characteristics, and survival, as well as evaluate effect of JAK2 inhibitor ruxolitinib (RUX) on outcome of patients with 5-9% PB / BM blasts seen at MD Anderson Cancer Center (MDACC). Methods: Medical charts of 1199 patients who presented to MDACC between years 1984 - 2015 with MF and available blast percentage in PB and BM, were retrospectively evaluated. All relevant clinical characteristics were collected at the time of referral. Chronic phase (CP) was defined as Results: Eighty five percent of patients (n=1020) were in CP; 10% (n=123) in CP-e, and 5% (n=56) in AP. Clinical characteristics and demographics are shown in Table 1. Patients with CP-e had similar clinical characteristics as patients in AP, and furthermore, both had higher white blood cell counts, lower hemoglobin and platelets, more frequent splenomegaly, systemic symptoms, and presence of abnormal and unfavorable karyotype (Caramazza, Leukemia 2011) than patients in CP. After median follow-up of 27 months (range, 1-251), 50% (n=594) of patients have died. Fourteen percent of patients (n=169) were untreated while being followed at MDACC. Among the remaining 1030 patients, 52% (n=533), 16% (n=162), and 12% (n=126) have received one, two, and ≥3 therapies. RUX was used in 30% of patients (n=328): in 289 (28%), 33 (27%) and 6 (11%) patients in CP, CP-e and AP, respectively. The median overall survival (OS) of the entire group was 48 months (range, 40-65). The median OS for patients in CP, CP-e and AP was 56, 34, and 23 months, respectively (p As the next step we analyzed the impact of RUX on OS in each of the groups. Patients who were exposed to RUX have superior overall survival within CP as well as CP-e groups, with respective OS of 61 vs 52 months in CP (with vs without RUX; p=0.002, HR 0.85, 95% CI 0.69-0.97), and 54 vs 27 months in CP-e (with vs without RUX; p=0.001, HR 0.50, 95% CI 0.29-0.85, Graph 1B, C). Furthermore, OS of patients in CP-e who were exposed to RUX has reached OS of patients in CP group, both with and without RUX, with respective OS of 54, 61, and 52 months (p>0.05, Graph 2B, C). RUX had no impact on OS among AP patients (with and without RUX, 23 vs 23.4 months, p>0.05, graph not shown). Progression to AML occurred in total of 139 (9%) patients, with overall incidence of 3.96 cases per 100 persons-years. Nine percent (n=93), 20% (n=25), and 39% (n=22) of patients have progressed to AML among CP, CP-e, and AP groups, respectively, (p Conclusions: RUX, the only FDA approved JAK2 inhibitor for patients with MF, has a potential to significantly improve an outcome of patients with 5-9% blast percentage whose clinical characteristics and outcome appear to be worse than patients with CP MF and Download : Download high-res image (181KB) Download : Download full-size image Table 1 . Disclosures Pemmaraju: abbvie: Research Funding; cellectis: Research Funding; novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria; stemline: Consultancy, Honoraria, Research Funding; roche diagnostics: Consultancy, Honoraria; affymetrix: Research Funding; Incyte Corporation: Consultancy, Honoraria. Daver: Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Immunogen: Research Funding; Pfizer Inc.: Consultancy, Research Funding; Incyte Corporation: Honoraria, Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Otsuka America Pharmaceutical, Inc.: Consultancy; Kiromic: Research Funding; Jazz: Consultancy. Cortes: Sun Pharma: Research Funding; ImmunoGen: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; BMS: Consultancy, Research Funding. Kantarjian: Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Delta-Fly Pharma: Research Funding; Pfizer: Research Funding. Verstovsek: Gilead: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Lilly Oncology: Research Funding; Bristol Myers Squibb: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding; Pfizer: Research Funding; Astrazeneca: Research Funding; Celgene: Research Funding; Blueprint Medicines Corp: Research Funding; Bristol Myers Squibb: Research Funding; Genentech: Research Funding; Promedior: Research Funding; Astrazeneca: Research Funding; Pfizer: Research Funding; Galena BioPharma: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Galena BioPharma: Research Funding; CTI BioPharma Corp: Research Funding; NS Pharma: Research Funding; Roche: Research Funding; CTI BioPharma Corp: Research Funding; Seattle Genetics: Research Funding; Roche: Research Funding; Lilly Oncology: Research Funding; Blueprint Medicines Corp: Research Funding.

Loss of Toll-like Receptor 2 Results in Accelerated Leukemogenesis in the NUP98-HOXD13 Mouse Model of MDS

The myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis and a high risk of leukemic transformation. Stem cell transplantation is the only curative treatment available to a subset of patients and is associated with significant morbidity and mortality; thus, new therapies are needed. Multiple studies have shown a correlation between MDS and enhanced toll-like receptor 2 (TLR2) signaling, suggesting this might contribute to disease pathogenesis. In fact, an inhibitory TLR2 antibody is currently in clinical trial for use in MDS (OPN-305, Opsona Therapeutics). Despite enthusiasm for TLR2 as a potential drug target, the role of TLR2 in MDS remains unclear. Increased TLR2 signaling has been associated with low-risk disease, higher rates of apoptosis associated with improved prognosis, and longer survival (Zeng et al ., Exp Cell Res 2016; Wei et al., Leukemia 2013), suggesting TLR2 may, in fact, have a protective role in MDS. Thus, we are exploring how TLR2 signaling regulates cell fate in MDS to shape efforts to target this pathway in patients.To elucidate the contribution of TLR2 signaling to MDS pathogenesis, we determined the effects of TLR2 loss in a mouse model of MDS (expressing the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter). These “ NHD13” mice recapitulate many features of human MDS and die of leukemia or cytopenias (Lin et al ., Blood 2005). Similar to CD34+ cells in humans, we found a nearly two-fold increase of TLR2 on NHD13 HSCs as compared to controls. NHD13 mice were crossed to Tlr2-/- mice to generate NHD13;Tlr2-/-, NHD13;Tlr2+/+, Tlr2-/-, and wild-type (WT) groups. Interestingly, loss of TLR2 in NHD13 mice did not confer an improvement in peripheral cytopenias, but was associated with accelerated leukemogenesis and significantly worse survival (p=0.016, with a median survival of 335 days in NHD13;Tlr2-/- mice vs. 370 days for NHD13;Tlr2+/+ mice; in both cohorts, leukemia was the primary cause of death- 12/20 for NHD13;Tlr2-/- and 10/13 for NHD13;Tlr2+/+). An even more significant acceleration of leukemogenesis and death was observed in NHD13 mice crossed to mice lacking myeloid differentiation primary response gene 88 (MyD88), an adaptor required for most TLR signaling, including TLR2 (p<0.0001, with a median survival of 218 days in NHD13;MyD88-/- mice vs. 370 days in NHD13 mice).To investigate the potential mechanism by which TLR2 signaling loss accelerates leukemogenesis in NHD13 mice, hematopoietic stem and progenitor cells (c-Kit+, Sca-1+, Lin-; HSPCs) of premalignant NHD13;Tlr2+/+ and NHD13;Tlr2-/- mice were evaluated for cell cycling, cell death, and DNA damage. While loss of TLR2 in the NHD13 mice did not confer a significant difference in HSPC cycling, it did reduce cell death (HSPCs of NHD13+;Tlr2+/+ mice display 18.4% Annexin V+ cells, while those of NHD13+;Tlr2-/- mice display only 6.2%, p=0.0008). An assessment of caspase-1 and caspase-3/7 activities showed a significant reduction in activated caspase-1 in the HSPCs of NHD13;Tlr2-/- mice compared to NHD13; Tlr2+/+ mice (average luminescence units: 7310 vs. 10164), suggesting that TLR2 specifically promotes inflammasome-dependent cell death in the NHD13 mice. RNA sequencing of sorted HSPCs also demonstrated an enrichment of apoptosis-related gene pathways in the NHD13 compared to the NHD13;Tlr2-/- cells. Moreover, the accumulated HSPCs in the NHD13;Tlr2-/- mice showed elevated levels of γH2AX staining as compared to NHD13; Tlr2+/+ mice and WT controls, indicating TLR2 loss promotes the survival of damaged HSPCs.As apoptosis of HSPCs is characteristic of low-risk MDS and is lost as the disease progresses to leukemia, these data suggest that some TLR2 signaling may be protective against leukemic transformation via promotion of apoptosis of damaged premalignant HSCs. Further supporting this idea, we found that in 38% of cases evaluated, TLR2 surface expression was lost from the malignant cells upon transformation to acute leukemia in the NHD13 mice. Thus, down-regulation of TLR2 may be a later event in the progression of MDS to acute leukemia, and complete inhibition of this pathway may, in fact, facilitate the survival of pre-leukemic HSPCs. Ongoing experiments are aimed at elucidating the mechanism by which TLR2 signaling promotes premalignant cell death in MDS and understanding how TLR2 is down-regulated upon transformation to leukemia. DisclosuresNo relevant conflicts of interest to declare.

Isocitrate dehydrogenase 2 mutations correlate with leukemic transformation and are predicted by 2-hydroxyglutarate in myelodysplastic syndromes.

The myelodysplastic syndromes (MDS) are a group of hematologic disorders characterized by the presence of somatically mutated hematopoietic stem cells (HSCs) that increase the risk of progression to secondary acute myeloid leukemia (sAML). Mutations in isocitrate dehydrogenase (IDHmut) are thought to correlate with the increased production of the oncogenic protein 2-hydroxyglutarate (2-HG) in AML. The aim of this study was to examine whether serum 2-HG has utility as a prognostic biomarker, and whether elevated 2-HG levels are predictive of IDH mutations in patients with MDS. Genetic profiling was utilized to determine the genetic composition of a large cohort of MDS patients, including the presence or absence of IDH1 or IDH2 mutations (n = 281). Serum 2-HG levels were detected by liquid chromatography-tandem mass spectrometry. In the current study of MDS patients, elevated serum 2-HG levels were predictive of inferior overall- and leukemia-free survival irrespective of IPSS risk grouping. Higher serum 2-HG levels predicted the presence of IDH mutations. IDH2mut patients had a higher risk of leukemic transformation. The co-occurrence of DNMT3A or SRSF2 mutations was found to be increased in IDH2mut patients. IDH2 mutations were associated with significantly worse OS and LFS amongst patients with low-risk MDS by IPSS grouping. The noted predictive value of serum 2-HG levels and IDH2 mutations on OS and LFS support the use of biomarkers and/or underlying cytogenetics in novel prognostic scoring systems for MDS.

Reduced Cell Division Control Protein 42 Activity Compromises Hematopoiesis-Supportive Function of Fanconi Anemia Mesenchymal Stromal Cells.

Hematopoietic stem cells preserve their ability to self-renew and differentiate to different lineages in the bone marrow (BM) niche, which is composed in large part by BM stromal cells. Studies have shown that altered signaling in the BM niche results in leukemia initiation or progression. Fanconi anemia (FA) is an inherited BM failure syndrome associated with extremely high risk of leukemic transformation. By using two FA mouse models, here we have investigated the hematopoiesis-supportive function of FA BM mesenchymal stroma cells (MSCs). We found that MSCs deficient for Fanca or Fancc gene are defective in proliferation and prone to undergo senescence in vitro. Mechanistically, we show that the activity of cell division control protein 42 (Cdc42), a Rho GTPase known to be a critical regulator for cytoskeleton organization, is significantly reduced in FA MSCs. Furthermore, we demonstrate that this reduction in Cdc42 activity plays a causal role in defective hematopoiesis-supportive function of the FA MSCs. The progenies of wild-type hematopoietic stem and progenitor cells cocultured on FA MSCs exhibit compromised self-renewal capacity both in vitro and in vivo. Genetic correction of FA deficiency restores Cdc42 activity and improves the hematopoiesis-supportive capacity of FA MSC. Finally, ectopic expression of a constitutively active Cdc42 mutant, Cdc42F28L, or pretreatment with Wnt5a, increases the active Cdc42 level and rescues the hematopoietic supportive defects of FA MSCs. Taken together, our results identify a novel link between Cdc42 activity and the hematopoiesis-supportive function of MSCs and suggest that a niche-specific increase of Cdc42 activity may be beneficial for FA therapy. Stem Cells 2018;36:785-795.

Leukemic transformation in patients with myeloproliferative neoplasms: a population-based retrospective study.

This study determined the epidemiology of developing leukemic transformation in patients with myeloproliferative neoplasms (MPN). We utilized the Surveillance, Epidemiology and End Results 13 database to identify 83 cases of leukemic transformation in MPN (n = 9335). The 5-year cumulative incidence of leukemic transformation was higher in male versus female (2.17 vs 1.09%; p < 0.001), and in myelofibrosis (2.19%; 95% CI: 1.36-3.34%), compared with essential thrombocythemia (0.37%; 95% CI: 0.19-0.65%) and polycythemia vera (0.72%; 95% CI: 0.46-1.07%; p < 0.001). Patients had a median survival of 2 months after leukemic transformation, worse in older patients and without any impact of prior MPN subtypes. Myelofibrosis has a higher risk of leukemic transformation. Overall survival is dismal regardless of MPN subtypes.

How We Treat Patients With Polycythemia Vera

How We Treat Patients With Polycythemia Vera

The immune receptor Trem1 cooperates with diminished DNA damage response to induce preleukemic stem cell expansion.

Fanconi anemia (FA) is an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Here we investigate the relationship between DNA damage response (DDR) and leukemogenesis using the Fanca knockout mouse model. We found that chronic exposure of the Fanca-/- hematopoietic stem cells to DNA crosslinking agent mitomycin C in vivo leads to diminished DDR, and the emergence/expansion of pre-leukemia stem cells (pre-LSCs). Surprisingly, although genetic correction of Fanca deficiency in the pre-LSCs restores DDR and reduces genomic instability, but fails to prevent pre-LSC expansion or delay leukemia development in irradiated recipients. Furthermore, we identified transcription program underlying dysregulated DDR and cell migration, myeloid proliferation, and immune response in the Fanca-/- pre-LSCs. Forced expression of the downregulated DNA repair genes, Rad51c or Trp53i13, in the Fanca-/- pre-LSCs partially rescues DDR but has no effect on leukemia, whereas shRNA knockdown of the upregulated immune receptor genes Trem1 or Pilrb improves leukemia-related survival, but not DDR or genomic instability. Furthermore, Trem1 cooperates with diminished DDR in vivo to promote Fanca-/- pre-LSC expansion and leukemia development. Our study implicates diminishing DDR as a root cause of FA leukemogenesis, which subsequently collaborates with other signaling pathways for leukemogenic transformation.

Myelodysplastic Syndromes and Other Precursor Myeloid Neoplasms in the Era of Genomic Medicine (Mini Review)

Myeloid neoplasm are derived from precursor cells of myeloid lineage and are composed of a broad spectrum of hematopoietic malignancies. The nature of the myeloid precursors is largely under-investigated until the recent application of next generation sequencing (NGS) technology for genome-wide analysis of myeloid neoplasms. It is important to define precursor myeloid neoplasms mediated by molecular signatures including driver gene mutations essential in disease initiation as well as acquired genetic alterations that play a role in disease progression. In addition to myelodysplastic syndrome with a high risk of leukemic transformation, there are newly proposed early precursor disorders with the potential to evolve into myeloid neoplasms [e.g., clonal hematopoiesis of indeterminate potential (CHIP), and clonal cytopenias of undetermined significance (CCUS)]. Furthermore, certain predisposing germline mutations (e.g. CEBPA, DDX41, RUNX1, ETV6 and GATA) have been recognized with predisposition to develop into myeloid neoplasms. This review paper aims to provide a brief summary of novel concepts of early precursor lesions that could lead to myeloid neoplasms, potential molecular prognostic indicators for MDS, and updated sub-classification of myelodysplastic syndromes according to the 2016 revision of World Health Organization (WHO).

Mutational Landscape of Patients with Myelofibrosis That Do Not Harbor Mutations in JAK2, MPL and Calreticulin Driver Genes

Background. In primary myelofibrosis (PMF), mutations of JAK2, CALR and MPL driver genes can be detected in about 60%, 20% and 5% of the patients (pts), respectively. Therefore, about 10% of the pts lack any of the 3 driver mutations and are operationally called "triple negative" (TN). TN pts present a higher risk of developing anemia and thrombocytopenia, suffer from reduced overall survival compared to other genotypes, particularly to CALR type1/type1-like mutations, and may be at higher risk of leukemic transformation (Blood 2014; 124:1062; Leukemia 2014; 28:1472; Blood 2014;124:2465).Aims The aim of the study was to analyse the molecular landscape of TN PMF pts by genotyping a set of myeloproliferative-neoplasms associated genes whose mutated status was shown to be prognostically relevant in previous studies (Leukemia 2013;27:1861; Blood 2014;123:2220; Leukemia 2014;28:1804;Leukemia 2014;28:1494).Methods. Diagnosis ofPMF was made according to WHO2008 criteria. All pts provided informed written consent. Previously published methods were used to screen mutations involving JAK2, MPL and CALR. A deep sequencing custom panel was designed to genotype the following genes: EZH2, ASXL1, IDH1/2, SRSF2, TP53, TET2, RUNX1, CBL, NRAS, KRAS, DNMT3A, SF3B1, IKZF1, NFE2, SH2B3. Analysis was performed using the Ion torrent PGM platform. Comparisons of quantitative variables between groups were carried out by the nonparametric Wilcoxon rank-sum test. The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression.Results. We analysed 28 TN PMF collected at the time of diagnosis. Median age was 66.7y, 57% were male. Median follow up was 2.1y (0.3-14.5). Overall, 8 patients (28.6%) progressed to AML. Death occurred in 20 pts (71.4%) after a median follow up of 2.3y (1.4-3.3y); progression to leukemia was the cause of death in 5 pts (25%).Overall, 22 pts (78.6%) presented at least one mutation in any of the 15 genes of the panel; 14 pts (50%) presented at least 2 mutations in different genes. The frequency of mutated genes was: SRSF2 39.3%, ASXL1 28.6%, EZH2 21.4%, NRAS 21.4%, TET2 10.7%, CBL 10.7%, IDH 3.6%, DNMT3A 3.6%, SH2B3 3.6%, U2AF1 3.6%. Twenty pts (71.4%) were classified as high molecular risk (HMR: ie, any mutated gene of ASXL1, EZH2, SRSF2, IDH1/2), a proportion significantly higher (P<0.01) than among JAK2 V617F (31.5%), CALR Type1/1-like (22.0%) and CALR Type2-type2-like (5.0%) (Leukemia 2013; 27:1861). Mutated genes were grouped into 3 different pathways: epigenetic regulation (ASXL1, EZH2, TET2, IDH), splicing machinery (SRSF2, U2AF1) and leukemic transformation (NRAS, DNMT3A, SH2B3, CBL). The most frequently mutated pathway was the epigenetic one with mutations in 14 pts (63.6%) of which 3 pts (21.4%) had 2 or more mutated genes of the pathway; 12 pts (54.5%) presented mutations in the splicing machinery and of these 8 pts (66.7%) had 2 or more mutated genes of the pathway; genes involved in leukemic transformation were mutated in 11 pts (50%) and 10 of 11 (90.9%) had 2 or more mutated genes. In three cases (13.6%) all 3 pathways were concurrently involved.Among the mutated genes, SRSF2 was associated with shorter survival [1.9y (1.6-2.2)] compared to pts with un-mutated SRSF2 [3.2y (0.9-5.4y)] (HR 2.3, 95%CI 0.9-6.4). SRSF2 mutations were also associated with shorter leukemia free survival (LFS): LFS not reached in un-mutated pts compared to 2.2y (1.8-2.7y) for mutated pts, with a HR=4.5 (95%CI 10.3-19.9). We also found that in pts with grade 1 bone marrow fibrosis the splicing and the leukemic pathway were more frequently mutated compared to grade 2-3 fibrosis (57% vs 28.5% and 50% vs 28.5% respectively).Conclusions. "Triple-negative" pts with PMF present high rate of mutations of MPN-associated genes, most of them are classified as "high molecular risk" and harbor >2 mutations. Mutated SRSF2 was particularly associated with shorter LFS. Such complex molecular landscape might help to explain the negative outcome of TN PMF pts. DisclosuresVannucchi:Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Speakers Bureau.

Cytogenetics of Patients with Myelodysplasic Syndrome in Venezuela

The myelodysplastic syndromes are clonal hematological disorders of the stem Cell, and clinically characterized by ineffective hematopoiesis and morphological dispoyesis. The affected patients have a high risk of leukemic transformation. In a total of (n=416) patients studied, (n=247) patients (59.3%) corresponded with a female gender, with an average age of = 59 years (limits 15-99 yrs), and (n=169) male patients (40.6%), with average age of =62 years (limits 09-89 yrs). In (n = 295) studied patients, 71% had normal karyotype 46, XX or 46, XY, where in 16% (n = 66) of the samples cell growth was not attained. In (n = 54) of patient samples, 13% showed karyotype changes, including those frequently reported for MDS; and 11.1% (n = 6) with -Y, 11q 1.8% (n = 1), del20q 3.7% (n = 2), +8 3.7% (n = 2), - 21 1.8% (n = 1), del 7q 1.8% (n = 1), inv3 1.8% (n = 1), complex karyotypes 12.9 % (n = 7) cases. Other alterations include, simple anomalies: +2 1.8% (n = 1), +13 5.5% (n = 3), del17q 1.8% (n = 1), del4p 1.8 %(n=1), del9q 3.7% (n = 2), der21q 3.7 %(n = 2), del17p3 3.7% (n = 2), t (1; 7) 1.8% (n = 1), t (4; 12) 1.8% (n = 1), rob13 1.8% (n = 1); 14, r2 1.8%(n = 1); double anomalies, delXq and del10q (n = 1), del18p and del13q (n = 1). Among other findings, polymorphisms in (n=15) patients (27.7%), such as 1qh + (n = 2), 9qh + (n = 4), 16qh + (n = 3), 14PS + (n = 1), 15ps + (n = 2) , 22ps + (n = 1), inv9 (n = 2). According with IPSS-R karyotypes, samples were classified as very favorable 12.9% (n = 7), pro 3.7% (n = 4), Intermediate 40.7% (n = 22), 1.8% unfavorable ( n = 1) and very favorable 18.5% (n = 12.9). DisclosuresNo relevant conflicts of interest to declare.

Driver Mutations and Prognosis in 1118 Patients with Primary Myelofibrosis

▪Background: In primary myelofibrosis (PMF), ̴ 88% of patients harbor JAK2, CALR or MPL, with respective mutational frequencies of approximately 60%, 22% and 6%; ̴ 12% are wild type for all three driver mutations and are labelled as being "triple negative" (Blood. 2014;124:2507). It is now well-established that CALR-mutated patients with PMF display significantly better survival, compared to all the other mutational categories; in addition, preliminary data have suggested inferior survival for "triple-negative" cases (Leukemia. 2014;28:1472; Blood. 2014;124:1062) and the possibility that the favorable impact of CALR mutations might be restricted to CALR type 1 or type 1-like variants (Blood. 2014;124:2465). In the current study, we sought to clarify the prognostic impact of driver mutations, and CALR variants, on overall (OS) and leukemia-free (LFS) survival, in a two-center study involving 1118 patients.Methods: A total of 1118 patients with PMF from the Mayo Clinic (n =722) and University of Florence, Italy (n =396) were included in the current study. Study patients were selected based on availability of mutation information. PMF diagnosis was according to World Health Organization criteria (Blood. 2009;114:937). Previously published methods were used for CALR, JAK2 and MPL mutation analyses and determination of CALR variants (Blood. 2014;124:2465). Kaplan-Meier survival analysis was considered from the date of first referral for the Mayo cohort and date of diagnosis for the Florence cohort. Leukemic transformation events replaced death as the uncensored variable during LFS analysis and Cox proportional hazard regression model was used for multivariable analysis.Results:Analysis was first conducted on the Mayo cohort of 722 patients (median age 64 years; 64% males). DIPSS-plus risk distributions were 14% low, 17% intermediate-1, 37% intermediate-2 and 33% high. All patients were annotated for JAK2/CALR/MPL mutations as well as CALR variants; 477 harbored JAK2, 139 CALR and 41 MPL mutations; 65 patients were triple-negative. The 139 CALR -mutated patients included type 1/type 1-like (n =115) and type 2/type 2-like (n =24).At a median follow-up time of 3.1 years, 439 (61%) deaths and 63 (8.7%) blast transformations were documented. In univariate analysis, survival in patients with CALR type 1/type 1-like mutations was significantly longer than every other mutational category: HR (95% CI) were for triple-negative 2.7 (1.8-4.0), JAK2 2.7 (2.0-3.7), CALR type 2/type 2-like 2.3 (1.3-4.2) and MPL 1.9 (1.1-3.1) (Figure 1); these differences were sustained during multivariable analysis that included ASXL1 mutations (n =480) and DIPSS-plus, and were also apparent in the Florence cohort of 396 cases that included 251 JAK2, 53 CALR type 1/type 1-like, 21 CALR type 2/type 2-like, 50 triple-negative and 21 MPL mutated cases (Figure 2). There was no difference in survival among the Mayo cohort across mutational categories not including CALR type 1/type 1-like variants (p=0.33). Because specific driver mutations in PMF differentially cluster with age and sex, multivariable analysis including these two parameters was also performed and confirmed the significant survival advantage of CALR type 1/type 1-like over triple-negative, JAK2 and CALR type 2/type 2-like mutated cases.Based on the above information, we divided driver mutational categories into "type 1/type 1-like" (median survival 10.3 years) and "all other mutational categories" (median survival 3.9 years; p<0.01). The difference in survival between these two groups remained significant (HR 2.1, 95% CI 1.5-2.9) during multivariable analysis that included both DIPSS-plus (P<0.01) and ASXL1 mutation (HR 1.8, 95% CI 1.4-2.2). LFS was similar between these two groups (p=0.4) but it was inferior in triple-negative cases compared to CALR type 1/type 1-like variants (HR 2.8, 95% CI 1.3-6.2), MPL (HR 4.6; 95% CI 1.04-20.4) and JAK2 (HR 2.5, 95% CI 1.3-4.8) but not CALR type 2/type 2-like variants (p=0.56). There was no difference in LFS among mutational categories not including triple-negative cases (p=0.33).Conclusions: In PMF, driver mutations might be classified into two prognostically distinct categories: "favorable" (type 1/type 1-like) and "unfavorable" (all other mutational categories). In addition, triple-negative cases might be at a higher risk of leukemic transformation. [Display omitted] [Display omitted] DisclosuresPardanani:Stemline: Research Funding. Vannucchi:Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.