Triple-negative Myelofibrosis Research Articles

Trisomy 8 Defines a Distinct Subtype of Myeloproliferative Neoplasms Driven by the MYC-Alarmin Axis.

Despite advances in understanding the genetic abnormalities in myeloproliferative neoplasms (MPN) and the development of JAK2 inhibitors, there is an urgent need to devise new treatment strategies, particularly for patients with triple-negative (TN) myelofibrosis (MF) who lack mutations in the JAK2 kinase pathway and have very poor clinical outcomes. Here we report that MYC copy number gain and increased MYC expression frequently occur in TN-MF and that MYC-directed activation of S100A9, an alarmin protein that plays pivotal roles in inflammation and innate immunity, is necessary and sufficient to drive development and progression of MF. Notably, the MYC-S100A9 circuit provokes a complex network of inflammatory signaling that involves numerous hematopoietic cell types in the bone marrow microenvironment. Accordingly, genetic ablation of S100A9 or treatment with small molecules targeting the MYC-S100A9 pathway effectively ameliorates MF phenotypes, highlighting the MYC-alarmin axis as a novel therapeutic vulnerability for this subgroup of MPNs. Significance: This study establishes that MYC expression is increased in TN-MPNs via trisomy 8, that a MYC-S100A9 circuit manifest in these cases is sufficient to provoke myelofibrosis and inflammation in diverse hematopoietic cell types in the BM niche, and that the MYC-S100A9 circuit is targetable in TN-MPNs.

Triple-Negative Myelofibrosis: Disease Features, Response to Treatment and Outcomes

BackgroundMyelofibrosis is the most aggressive subtype among classical BCR::ABL1 negative myeloproliferative neoplasms. About 90% of cases are driven by constitutive activation of 1 of 3 genes impacting the JAK/STAT pathway: JAK2, CALR, and MPL. Triple-negative myelofibrosis (TN-MF) accounts for only 5%-10% of cases and carries the worst outcomes. Little has been described about this subset of disease. Given the marked heterogeneity surrounding disease biology, clonal architecture, clinical presentation, and poor outcomes in TN-MF, identification of features of interest and assessment of treatment response are areas in need of further investigation. Patients and methodsWe collected and evaluated baseline clinical and molecular parameters from 626 patients with a diagnosis of myelofibrosis who presented to the H. Lee Moffitt Cancer Center in Tampa (Florida, US) between 2003 and 2021 and compared them based on presence or absence of the three classical phenotypic driver mutations. ResultsA small proportion of patients (6%) harbored TN-MF which correlated with inferior outcomes, marked by a 4-year reduction in overall survival time compared to the non-TN cohort (mOS 37.4 months vs. 85.7 months; P = .009) and higher rates of leukemic transformation. More pronounced thrombocytopenia and anemia, lower LDH, EPO levels, as well as lower percentage of marrow blasts at baseline were more commonly seen in TN-MF (P < .05). Similarly, patients with TN-MF had higher risk disease per DIPSS+ and GIPSS. Mutations impacting RNA splicing, epigenetic modification and signaling, specifically SRSF2, SETBP1, IDH2, CBL, and GNAS, were more commonly seen among those lacking a classical phenotypic driver. The prevalence of co-mutant ASXL1/SRSF2 clones was significantly higher in TN-MF as was trisomy 8. TN patients had fewer responses (46.2% vs. 63.4%) and shorter duration of response to ruxolitinib. ConclusionTN-MF is invariably associated with significantly decreased survival and more aggressive clinical behavior with higher rates of leukemic transformation and shorter duration of response to ruxolitinib. Mutations impacting RNA splicing, epigenetic modification and signaling (SRSF2, SETBP1, IDH2, CBL, and GNAS) are more common in TN-MF, which likely drive its aggressive course and may account for suboptimal responses to JAK inhibition.

MYC-Alarmin Axis As a Novel Oncogenic Driver in a Subgroup of Triple Negative Myeloproliferative Neoplasms

Primary Myelofibrosis (PMF) is an aggressive myeloproliferative neoplasm (MPN) characterized by constitutional symptoms, cytopenias, splenomegaly, extramedullary hematopoiesis, bone marrow (BM) fibrosis, and a propensity to transform to acute myeloid leukemia (AML). Despite advances in understanding the underlying genetic abnormalities in MPN and the development of JAK2 inhibitors to treat MF, there is an urgent need to devise new treatment strategies, particularly for triple negative myelofibrosis (TN-MF) cases that lack mutations in the JAK2 kinase pathway and who have inferior outcomes. To identify potential oncogenic drivers in TN-MF, we performed cytogenetic analyses and targeted exome sequencing of 98 genes commonly mutated in myeloid malignancies in 584 MF patients identified from the Total Cancer Care database at Moffitt Cancer Center. Notably, there were no significant differences in the somatic mutation profiles (other than JAK2 activating mutations) between TN vs. JAK2/CALR/MPL mutant MF patients. However, trisomy of chromosome 8 occurs more frequently in TN-MF vs. other subtypes (26.7% vs. 6.2%, p=0.0001). To identify oncogenic drivers in trisomy 8+ TN-MF, we performed scRNA-seq analysis from 18,651 cells ( n=15,850 from 3 normal donors and n=2,801 from a trisomy 8+ TN-MF patient) and identified a total of 1,260 genes that are differentially regulated in hematopoietic stem cells (HSCs) of trisomy 8+ TN-MF compared to normal donors. Among these, 131 genes were located on chromosome 8 and MYC was one of the top 5 genes that are significantly upregulated in trisomy 8+ HSCs. Additional immunohistochemistry staining demonstrated that MYC protein levels in BM cells were significantly higher in trisomy 8+ TN-MF patients vs. trisomy 8- patients. To assess whether MYC might be a driver of MPN, we established an Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC transgenic mouse model that inducibly overexpresses MYC in HSCs following treatment of polyinosinic-polycytidylic acid (pIpC). Forced MYC expression induced profound anemia, monocytosis, megakaryocytic atypia, splenomegaly, increased BM collagen deposition/fibrosis, extramedullary hematopoiesis in spleen and liver, and significantly reduced OS vs. Mx1-Cre +/- wild type controls (median OS 258 days vs. not reached (NR), p&amp;lt;0.0001). MYC also promoted expansion of HSCs, myeloid progenitors, and Gr-1 +/CD11b + mature myeloid cells in BM and spleen; thus, MYC was sufficient to provoke MF-like disease independent of JAK2 pathway mutations. Subsequent scRNA-seq analysis of 25,232 cells ( n=13,552 from control and n=11,680 from a MYC transgenic mouse) revealed that S100a9 mRNA levels were increased by MYC in most major hematopoietic cell types. Supporting oncogenic roles of S100A9 in MYC-driven MF, deletion of S100a9 in Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC;S100a9 -/- mice abolished MYC-induced MF phenotypes and significantly improved OS (median OS NR vs. 225 days, p=0.0345). To test whether inhibition of MYC can suppress MF disease progression in vivo, lethally irradiated CD45.1 +/CD45.2 + WT mice were transplanted with BM cells harvested from CD45.2 + Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC mice that were treated with pIpC 20 weeks prior to transplant. MYCi975, a small molecule that inhibits MYC, reduced MYC and S100a9 protein levels in BM and spleen cells. Further, MYCi975 effectively suppressed MYC-driven MF phenotypes and disease progression, and significantly improved OS (median OS NR vs. 252 days, p=0.0109). In summary, our studies are the first to describe an oncogenic role of MYC in MF pathogenesis, where MYC provokes an alarmin-driven inflammatory circuit, and where the MYC-S100A9 axis represents a therapeutic vulnerability for TN-MF patients. Accordingly, our results provide a strong rationale for testing agents targeting MYC or S100A9 in early phase clinical trials in MPNs having increased MYC levels or activity.

Exploring the Molecular Landscape of Myelofibrosis, with a Focus on Ras and Mitogen-Activated Protein (MAP) Kinase Signaling.

Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) characterized clinically by cytopenias, fatigue, and splenomegaly stemming from extramedullary hematopoiesis. MF commonly arises from mutations in JAK2, MPL, and CALR, which manifests as hyperactive Jak/Stat signaling. Triple-negative MF is diagnosed in the absence of JAK2, MPL, and CALR but when clinical, morphologic criteria are met and other mutation(s) is/are present, including ASXL1, EZH2, and SRSF2. While the clinical and classic molecular features of MF are well-established, emerging evidence indicates that additional mutations, specifically within the Ras/MAP Kinase signaling pathway, are present and may play important role in disease pathogenesis and treatment response. KRAS and NRAS mutations alone are reportedly present in up to 15 and 14% of patients with MF (respectively), and other mutations predicted to activate Ras signaling, such as CBL, NF1, BRAF, and PTPN11, collectively exist in as much as 21% of patients. Investigations into the prevalence of RAS and related pathway mutations in MF and the mechanisms by which they contribute to its pathogenesis are critical in better understanding this condition and ultimately in the identification of novel therapeutic targets.

Training and Validation Cohorts for Predicting the Impact of High Molecular Risk Mutations after Allogeneic Stem Cell Transplantation in Myelofibrosis

Introduction Myelofibrosis (MF) is the most aggressive form of Philadelphia negative myeloproliferative neoplasm and allogeneic hematopoietic stem cell transplantation (HSTC) is the only curative approach, but non relapse mortality limits the indication for HSTC to high-risk patients. The prognostic evaluation of MF patients is evolving during time together with a better understanding of the mutational landscape of this disease. The presence of high molecular risk (HMR) mutations, specifically ASXL1, SRSF2, EZH2, IDH1, IDH2 and U2AF1Q157, harbors a detrimental effect on survival and leukemic transformation and has been incorporated in different prognostic scoring systems. In this multicenter study we aimed to determine the impact of HMR mutations on transplant outcome. Methods We studied two independent cohorts of patients: 44 patients (training, retrospective cohort) were part of a multicenter, randomized GITMO (Gruppo Italiano Trapianti di Midollo Osseo) clinical trial (Patriarca F, BBMT 2019) and 29 patients (validation, prospective cohort) were subsequently recruited at the Bergamo Bone Marrow Transplant Unit. Patients from the training cohort (median age 56, range 36-66) were conditioned with busulfan-fludarabine or thiotepa-fludarabin while patients in the validation cohort (median age 61, range 36-67) were conditioned with a reduced intensity thiotepa-busulfan-fludarabin regimen. The mutational profile obtained on the pre-transplant DNA was performed by sequencing 30 myeloid related genes by applying SOPHia GENETICS Myeloid Solution on Illumina MiniSeq platform. Results In the training retrospective cohort, driver mutations were detected in 40 (29 JAK2/8 CALR/3 MPL) patients, with 4 patients having a triple negative MF. At least one HMR mutation was detected in 43% of patients and an unfavorable karyotype was present in 18% of patients. According to the Mutation-Enhanced International Prognostic Scoring System 70 plus (MIPPS70+) scoring system, 77% of patients were allocated in the high/very high groups. With a median follow up of 4.2 years (range 0.02-9), the 5-years Overall Survival (OS) and Progression Free Survival (PFS) were 61% and 45% respectively. The 5-years Non-Relapse Mortality (NRM) and Cumulative Incidence of Relapse (CIR) were 25% and 30%. In the prospective validation cohort, a driver mutation was detected in all patients (15 JAK2/7 CALR/7MPL). At least one HMR mutation was detected in 48% of patients and an unfavorable karyotype was present in 24% of patients. Patients allocated to the MIPPS70+ high/very group were 52%. After a median follow up of 10.7 months (range 0-44), the 2-years OS and PFS were 61% and 49% respectively. The 2-years NRM and CIR were 33% and 18%. In the training cohort, a higher risk of death and progression was observed in patients with high/very high risk MIPPS70+ prognostic index [HR for OS 4.3 (95% CI 0.6-33.1), HR for PFS 4.1 (95% CI 0.9-16.9)]. Conversely, the presence of HMR mutations did not significantly affect OS, PFS (Figure 1), NRM or CIR. The prognostic significance of MIPSS70+ risk classification was confirmed in the validation cohort, with a higher risk of death and progression for high/very high MIPSS70+ group [OS HR 3.2 (95% CI 0.4-27.1), PFS HR 3.8 (95% CI 0.5-31.4)]. Similarly, in the validation cohort the presence of HMR mutations did not affect transplant outcome, with no impact on OS, PFS (Figure 2), NRM or CIR. In the whole group of 73 patients, the presence of JAK2V617F mutation showed an adverse prognostic impact on CIR [HR 3.4 (95% CI 1-11.8)], while MIPPS70+ confirmed its role on PFS. Conclusions In this study we prospectively confirmed that the presence of HMR mutations did not impact on transplant outcome. Allogeneic stem cell transplantation can lead to a significant cure rate of MF patients, regardless the presence of HMR mutations. Patients in the high/very high MIPSS70+ risk group had a higher risk of death and progression after transplant. Appropriate timing and selection of patients is crucial for transplant outcome in MF. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Triple-Negative Myelofibrosis: Disease Features, Response to Treatment and Outcomes

Triple-Negative Myelofibrosis: Disease Features, Response to Treatment and Outcomes

Bone Marrow Multiparametric Magnetic Resonance Imaging with Texture Analysis for Follow-up of Advanced Stage Myelofibrosis after Transplantation

Introduction Hematopoietic cell transplantation (HCT) is a potentially curative treatment in myelofibrosis (MF). Nevertheless, in at least one-third of patients, a reliable parameter for disease response is lacking due to the absence of a molecular marker (in triple-negative MF) or the suboptimal sensitivity for CALR and MPL monitoring with routinely used PCR methods. Trephine biopsy provides a limited histological description, as it does not represent the fibrosis and/or blast eradication status of the entire bone marrow. The aim of this study is to identify magnetic resonance imaging (MRI) biomarkers that reflect response to transplant and may serve as predictors of transplant efficacy. Methods We report the case of a 45-year-old man diagnosed with triple-negative MF who was eligible for HCT following progression after a previous procedure. A pre-transplant trephine biopsy showed grade III fibrosis, according to the WHO 2016 classification. CD34+ cells were found to comprise 10%-15% of cells. He underwent MRI 8 days before bone marrow transplantation from a haploidentical donor. A second, comparative MRI was performed 6 months after HCT; concurrent chimerism was estimated through Short Tandem Repeats (STRs) analysis. A multiparametric protocol was applied, including intravoxel incoherent motion (IVIM) diffusion-weighted imaging, Dixon GRE with fat fraction calculation, and Turbo Spin Echo T1 and STIR sequences. MRI examination was performed on a 3 Tesla MRI machine and included lumbar segments L4 and L5, pelvis, sacral bone, and coccyx. A phased-array surface coil was applied to optimize signal-to-noise ratio. Finally, radiomics and texture analysis were performed on ADC sequences using specific image software post-analysis (Slicer 3D v.4.10.2). Results The selected MRI parameters showed substantial modifications between the first and second exams. T1 and STIR sequences demonstrated a decrease in bone marrow fibrosis (Figure 1). ADC values increased at pre-specified target points (iliac crest, pre-transplant: 0.762, post-transplant: 0.901; femur, pre-transplant: 0.77, post-transplant: 0.808). Moreover, among 300 features analyzed (Figure 2), 20 presented changes potentially related with bone marrow restoration. Concomitant STRs analysis showed a full donor chimerism status. Conclusions To the best of our knowledge, this is the first case of functional, textural analysis-based MRI employed in transplanted MF. Multiparametric MRI data, including anatomical and functional sequences (particularly IVIM with texture analysis of ADC values and Dixon with fat fraction calculation) may represent valuable biomarkers for accurate detection of bone marrow engraftment and chimerism. Disclosures No relevant conflicts of interest to declare.

Final Results of Phase 2 Clinical Trial of LCL161, a Novel Oral SMAC Mimetic/IAP Antagonist, for Patients with Intermediate to High Risk Myelofibrosis

Final Results of Phase 2 Clinical Trial of LCL161, a Novel Oral SMAC Mimetic/IAP Antagonist, for Patients with Intermediate to High Risk Myelofibrosis

Results from a Phase 2 Study of Navitoclax in Combination with Ruxolitinib in Patients with Primary or Secondary Myelofibrosis

Results from a Phase 2 Study of Navitoclax in Combination with Ruxolitinib in Patients with Primary or Secondary Myelofibrosis

Triple Negative Myelofibrosis and Myelodysplastic Syndrome with Fibrosis: Clinico-Biological Characterization and Correlation with Gene Mutations

Introduction: Triple negative primary myelofibrosis (TN-PMF) and myelodysplastic syndromes with fibrosis (F-MDS) are rare entities, often difficult to distinguish each other. Currently, no specific molecular markers allowing a precise differential diagnosis are available. In this sense, next generation techniques (NGS) might be useful to distinguish between both entities and to refine prognosis.Methods: Thirty-nine patients with TN-PMF (n=16) or F-MDS (n=23) were analyzed, Targeted NGS was performed in 28 cases (10 TN-PMF and 18 F-MDS) using the Sophia Genetics Myeloid Tumor Solution Panel including the following genes: ABL1, ASXL1, BRAF, CALR, CBL,CEBPA, CSF3R,CSNK1A1,DNMT3A, ETV6, EZH2, FLT3,HRAS, IDH1, IDH2, JAK2, KIT, KMT2A, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2, WT1 and ZRSR2. High molecular risk (HMR) was defined by the presence of any mutation in ASXL1, SRSF2, EZH2 or IDH1/2 genes. Clinical and biological data at diagnosis were compared among both groups of patients, including bone marrow cytogenetics, bone marrow histology, and the results from NGS analysis. The probabilities of survival and progression to acute leukemia were estimated by the Kaplan Meier method using the log rank test for comparisons.Results: Median age at diagnosis for the whole series was 63 years (range 42-87). Cytogenetic abnormalities were detected in 16 (57%) out of 28 assessable patients, being classified as low risk (n=17), intermediate risk (n=5), and high risk (n=6) There were no significant differences among TN-PMF and F-MDS regarding age, sex, hematological values, spleen size or cytogenetic risk or the number of blasts in peripheral blood or bone marrow.Mutations were detected by NGS in 26 out of 28 (93%) patients. Median number of mutations was 3 (Range: 1-7). Most frequent mutated genes were: ASXL1 n=10 (36%), TET2 n=7 (25%), SRSF2 n=6 (21%), DNMT3A n=6 (21%), U2AF1 n=5 (18%), ETV6 n=4 (14%), SETBP1 n=3 (11%), RUNX1 n=3 (11%), EZH2 n=2 (7%), TP53 n=2 (7%), ZRSR2 n=2 (7%) and KMT2A n=2 (7%). Mutations in either TET2, U2AF1, SETBP1, TP53 or RUNX1 genes were observed in only 1 patient with TN-PMF in contrast with 12 out of 18 (67%) F-MDS cases (p=0.006). HMR mutations were detected in 12 cases (43%) with 7 of them (21%) carrying two or more HMR mutations. There were no significant differences among TN-PMF and F-MDS regarding the total number of mutations per case or in the presence of HMR mutations.Median survival was 1.6 and 3.7 years for patients with TN-PMF and F-MDS, respectively (p=0.4). Leukocyte count > 25x109/L and presence of blasts ≥ 1% in peripheral blood were associated with a significantly lower survival. High risk cytogenetics and ≥ 2 HMR mutations were associated with a tendency towards lower survival. Median survival was 1.6 years in patients with ≥ 3 mutations in comparison with 8.3 years in those with < 3 mutations, respectively (p=0.03). Progression to acute myeloid leukemia was observed in 11 patients, resulting in a probability of 32% at 3 years. There were no significant differences in the probability of progression to acute leukemia among TN-PMF and F-MFDS. Time to acute leukemia was significantly shorter in patients with ≥ 3 mutations (3-year probability 45% versus 16%, p=0.039).Conclusions: TN-PMF and F-MDS showed a high rate of mutations in myeloid genes with TET2, U2AF1, SETBP1, TP53 or RUNX1 being more frequently mutated in F-MDS. Despite survival being short in both entities, NGS allowed the identification of a subgroup of patients with especially poor prognosis characterized by the presence of ≥ 3 mutations. DisclosuresHernandez Boluda:Novartis: Consultancy; Incyte: Consultancy. Cervantes:Hospital Clinic Barcelona: Employment; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees.

A Novel Somatic Mutation in IKZF1 Is Common in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant for Myelofibrosis

Background:Myeloproliferative neoplasms (MPN) including polycythemia vera, essential thrombocytosis, and primary and secondary myelofibrosis (MF) are characterized by driver mutations in JAK2, CALR, and MPL. Additional somatic variants can be seen. Currently allogeneic hematopoietic stem cell transplant (HCT) is the only treatment with curative potential, but it is associated with a high risk of mortality, exceeding 50% in some patient subsets. The risk of disease progression usually outweighs the risk of transplant in patients with advanced MF. Here, we have evaluated the mutational profile using a targeted next-generation sequencing panel of 54 genes involved in myeloid neoplasms in patients with MF treated with HCT.Patients and Methods:48 patients with a diagnosis of primary (n=18) or secondary (n=30) MF who received a HCT at our center and from whom genomic DNA was available from pre-HCT marrow aspirates were evaluated. DNA was isolated from whole marrow aspirates obtained prior to HCT. Paired-end indexed libraries were sequenced at an average read depth of 1000x on Illumina MiSeq platform. Sequences were aligned to hg19 using the Burrows-Wheeler Aligner, and nucleotide variants were identified using the Genome Analysis Toolkit and Picard tools. Variants were annotated with ANNOVAR. Variants present at >5% variant allele frequency (VAF) with at a minimum of 25 variant reads were selected for further analysis. Variants were called "mutations" if they resulted in (1) non-synonymous changes in amino acid sequence, including nonsense and missense mutations, (2) the variant was known to be associated with hematologic malignancies in COSMIC database, or (3) the variant was present at < 0.5% in 1000 genomes and ExAC databases. Comparisons between clinical variables and mutations were analyzed with chi-square tests, adjusted for multiple testing.Results:The average age of the 48 patients was 55 years (range 23-72) and 52% of patients were female. Disease risk was estimated with DIPSS-Plus (High 33%, INT-2 53%, INT-1 7%, Low 7%). Unfavorable cytogenetics were present in 27% of patients. Donors included a matched sibling donor in 30% (n=14), a matched unrelated donor in 60% (n=29), and an alternative donor in 10% (n=5). At 3 years, overall survival was 52%, 17% of patients had relapsed disease, and 31% had non-relapse mortality.In our cohort, 38% had a mutation in CALR (n=15 type-I and n=3 type-II), 29% in JAK2 (n=14), 0% in MPL, and 33% were triple-negative (n=16; no evidence of CALR, JAK2, or MPL mutation). The average VAF of CALR mutations was 50% (range 20-82%) and JAK2 mutations was 58% (range 25-81%). In addition to driver mutations, 94% of patients had at least one other mutation in 26 of the 54 genes sequenced. On average, patients had 2.1 other mutations (range 0-5 mutations in addition to driver mutation). The most common other mutations were in ASXL1 (31%), IKZF1 (23%), ETV6 (17%), and U2AF1 (17%). The VAF of these other mutations spanned 9-76%.Patients with triple-negative MF were more likely to have primary MF (p=0.019) and platelets <100 (p<0.001), than patients with JAK2 and CALR mutations. Patients with ≥3 other mutations were more likely to have hemoglobin <10 (p=0.02) and a higher rate of relapse (p<0.01) than patients with <3 other mutations. Patients with ASXL1 mutations were more likely to have peripheral blood blasts (p<0.001).Mutations in ASXL1, ETV6, and U2AF1 have been previously reported in myeloid malignancies. Like other groups, we found ASXL1 mutations associated with worse prognosis. Here we report a novel, recurrent mutation in the gene IKZF1. All patients with IKZF1 mutation carried the same insertion of 11 nucleotides leading to an early frameshift and ultimate nonsense mutation (L92fs), which would lead to deletion of the zinc-finger domain. Such a mutation likely leads to loss of IKZF1 tumor suppressor function.Conclusions:Somatic mutations, in addition to driver mutations, are common in patients with MF treated with HCT. Mutation profiling in this patient subset may improve relapse risk prediction. Loss of heterozygosity on chromosome 7 mapping to the IKZF1 locus has been found by others to be associated with leukemic transformation in patients with MPN (Jaeger et al. 2010. Leukemia. 24: 1290). Given their frequency in this high risk MF population, further study of the role of IKZF1 mutations in MPN is warranted. DisclosuresRadich:Novartis: Consultancy, Other: laboratory contract; Bristol-MyersSquibb: Consultancy; TwinStrand: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy.

CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons

Calreticulin (CALR) mutations were recently described in JAK2 and MPL unmutated primary myelofibrosis (PMF) and essential thrombocythemia. In the current study, we compared the clinical, cytogenetic and molecular features of patients with PMF with or without CALR, JAK2 or MPL mutations. Among 254 study patients, 147 (58%) harbored JAK2, 63 (25%) CALR and 21 (8.3%) MPL mutations; 22 (8.7%) patients were negative for all three mutations, whereas one patient expressed both JAK2 and CALR mutations. Study patients were also screened for ASXL1 (31%), EZH2 (6%), IDH (4%), SRSF2 (12%), SF3B1 (7%) and U2AF1 (16%) mutations. In univariate analysis, CALR mutations were associated with younger age (P<0.0001), higher platelet count (P<0.0001) and lower DIPSS-plus score (P=0.02). CALR-mutated patients were also less likely to be anemic, require transfusions or display leukocytosis. Spliceosome mutations were infrequent (P=0.0001) in CALR-mutated patients, but no other molecular or cytogenetic associations were evident. In multivariable analysis, CALR mutations had a favorable impact on survival that was independent of both DIPSS-plus risk and ASXL1 mutation status (P=0.001; HR 3.4 for triple-negative and 2.2 for JAK2-mutated). Triple-negative patients also displayed inferior LFS (P=0.003). The current study identifies 'CALR(-)ASXL1(+)' and 'triple-negative' as high-risk molecular signatures in PMF.

Spliceosome Gene Mutations Are Frequently Found In JAK2 Negative Myelofibrosis and Associated With Worse Clinical Outcomes

Myelofibrosis (MF), a Philadelphia chromosome negative myeloproliferative neoplasm (MPN) requires clonal markers for accurate diagnosis according to the 2008 World Health Organization classification scheme. However, molecular mutations in Janus Kinase 2 (JAK2) are found in only 50% of MF patients (pts). Various somatic mutations identified in other myeloid cancers have also been found in MF with pre-existing JAK2 V617F mutations. We previously reported in 57 pts diagnosed with Triple Negative MF (TN-MF: JAK2 exon12/14 and wild type (WT) MPL) the absence of mutations in genes commonly identified in other myeloid malignancies, specifically TET2, DNMT3A, CBL, IDH1/2, SH3B2 (LNK), N/KRAS and EZH2. Only somatic mutations in ASXL1 were identified in a small cohort of TN-MF (16%). Mutations in components of the RNA splicing machinery have been recently identified in varying frequencies in different myeloid malignancies. We performed RNA-sequencing on 2 samples (JAK2 mutant (MUT) and 1 JAK2 WT) and found that 2 spliceosome genes (U2AF1 and SF3B1) had higher mRNA expression levels in the JAK2 WT (MFC=0.76 and 0.82, respectively) compared to JAK2 MUT patient. We hypothesized that somatic mutations in RNA splicing factor genes occur in TN-MF pts and may be helpful in the biological characterization of this group of pts. Therefore, we isolated DNA from bone marrow (BM) or peripheral blood mononuclear cells from a cohort of MF pts (N=132; JAK2 MUT:75 and JAK2 WT:57) and performed Sanger sequencing for SF3B1 (exons 13-16), U2AF1 (exons 2, 6 and 7), and SRSF2 (exons 1 and 2). Baseline characteristics of pts and clinical data including hematologic parameters, BM results, and presence of splenomegaly by palpation were collected. Pts were stratified based on the Dynamic International Prognostic Scoring System-Plus risk as high=40, Int-2=51, Int-1=11 and low-risk=5 pts. The mean duration of follow-up was 16.5 months in JAK2 MUT and 12.8 months in JAK2 WT. Spliceosome mutations were found in 35/132 (27%) (SRSF2=17%, U2AF1=8% and SF3B1=2%) of pts. Interestingly, the frequency of spliceosome mutations in JAK2 WT was higher (31%; SRSF2=26%, U2AF1= 5%, SF3B1=0) compared to JAK2 MUT (18%; SRSF2=10%, U2AF1=6%, SF3B1=2%) pts. Of note, the 2 pts with SF3B1 mutations (K700E) had ring sideroblast in the BM (occasional and 50%). A large number of WT JAK2 patients (50%) had concomitant SRSF2 and ASXL1 mutations. Both SRSF2 and ASXL1 have been associated with poor prognosis in MF. Indeed, in our JAK2 WT cohort, pts who harbored SRSF2 mutations had a higher mean percentage of BM blasts compared to the ones that were SRSF2 WT (5.6% vs 1.8%; P=.006). Moreover, JAK2 WT pts carrying spliceosome mutations had more severe anemia as shown by lower hemoglobin (8.98 g/dL vs 10.5) and higher leukocyte counts (27.5 x109/L vs 20.5) compared to WT cases. Furthermore, we noticed that they also had higher frequency of RBC (Red Blood Cells) transfusions compared to WT (60% vs 40%) cases. Spleen examination by palpation below the left sub-costal margin showed that JAK2 WT cases carrying spliceosome mutations had larger splenomegaly compared to WT cases [10.1 cm (range 4-26), vs 8.7cm (range 2-20); P=0.05]. In conclusion, molecular alterations in the spliceosome machinery are frequently found in MF pts who are WT for JAK2/MPL and are associated with higher BM blast percentage, more severe anemia, higher leukocyte counts, transfusion dependence and more prominent splenomegaly. The observation of appreciable frequency of spliceosome mutations in JAK2 WT MF pts opens the possibility of using spliceosome inhibitors in the management of this disease group. Disclosures:No relevant conflicts of interest to declare.