Triple-negative Myeloproliferative Neoplasms Research Articles

Assessment of Total Antioxidant Capacity, 8-Hydroxy-2'-deoxy-guanosine, the Genetic Landscape, and Their Associations in BCR::ABL-1-Negative Chronic and Blast Phase Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs), namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal stem cell disorders defined by an excessive production of functionally mature and terminally differentiated myeloid cells. MPNs can transform into secondary acute myeloid leukemia (sAML/blast phase MPN) and are linked to alterations in the redox balance, i.e., elevated concentrations of reactive oxygen species and markers of oxidative stress (OS), and changes in antioxidant systems. We evaluated OS in 117 chronic phase MPNs and 21 sAML cases versus controls by measuring total antioxidant capacity (TAC) and 8-hydroxy-2'-deoxy-guanosine (8-OHdG) concentrations. TAC was higher in MPNs than controls (p = 0.03), particularly in ET (p = 0.04) and PMF (p = 0.01). MPL W515L-positive MPNs had higher TAC than controls (p = 0.002) and triple-negative MPNs (p = 0.01). PMF patients who had treatment expressed lower TAC than therapy-free subjects (p = 0.03). 8-OHdG concentrations were similar between controls and MPNs, controls and sAML, and MPNs and sAML. We noted associations between TAC and MPNs (OR = 1.82; p = 0.05), i.e., ET (OR = 2.36; p = 0.03) and PMF (OR = 2.11; p = 0.03), but not sAML. 8-OHdG concentrations were not associated with MPNs (OR = 1.73; p = 0.62) or sAML (OR = 1.89; p = 0.49). In conclusion, we detected redox imbalances in MPNs based on disease subtype, driver mutations, and treatment history.

Molecular Genetic Profile of Myelofibrosis: Implications in the Diagnosis, Prognosis, and Treatment Advancements

Myelofibrosis (MF) is an essential element of primary myelofibrosis, whereas secondary MF may develop in the advanced stages of other myeloid neoplasms, especially polycythemia vera and essential thrombocythemia. Over the last two decades, advances in molecular diagnostic techniques, particularly the integration of next-generation sequencing in clinical laboratories, have revolutionized the diagnosis, classification, and clinical decision making of myelofibrosis. Driver mutations involving JAK2, CALR, and MPL induce hyperactivity in the JAK-STAT signaling pathway, which plays a central role in cell survival and proliferation. Approximately 80% of myelofibrosis cases harbor additional mutations, frequently in the genes responsible for epigenetic regulation and RNA splicing. Detecting these mutations is crucial for diagnosing myeloproliferative neoplasms (MPNs), especially in cases where no mutations are present in the three driver genes (triple-negative MPNs). While fibrosis in the bone marrow results from the disturbance of inflammatory cytokines, it is fundamentally associated with mutation-driven hematopoiesis. The mutation profile and order of acquiring diverse mutations influence the MPN phenotype. Mutation profiling reveals clonal diversity in MF, offering insights into the clonal evolution of neoplastic progression. Prognostic prediction plays a pivotal role in guiding the treatment of myelofibrosis. Mutation profiles and cytogenetic abnormalities have been integrated into advanced prognostic scoring systems and personalized risk stratification for MF. Presently, JAK inhibitors are part of the standard of care for MF, with newer generations developed for enhanced efficacy and reduced adverse effects. However, only a minority of patients have achieved a significant molecular-level response. Clinical trials exploring innovative approaches, such as combining hypomethylation agents that target epigenetic regulators, drugs proven effective in myelodysplastic syndrome, or immune and inflammatory modulators with JAK inhibitors, have demonstrated promising results. These combinations may be more effective in patients with high-risk mutations and complex mutation profiles. Expanding mutation profiling studies with more sensitive and specific molecular methods, as well as sequencing a broader spectrum of genes in clinical patients, may reveal molecular mechanisms in cases currently lacking detectable driver mutations, provide a better understanding of the association between genetic alterations and clinical phenotypes, and offer valuable information to advance personalized treatment protocols to improve long-term survival and eradicate mutant clones with the hope of curing MF.

Impact of mutations on pregnancy outcome in patients with myeloproliferative neoplasms.

The impact of driver and other somatic mutations on pregnancy outcomes is unknown. The purpose of this study was to report the management and outcome of pregnancies in a cohort of myeloproliferative neoplasms (MPN) patients, particularly to evaluate the impact of somatic mutations. The cohort included consecutive patients with MPN who had a least one confirmed pregnancy. The primary outcome was live births. Secondary outcomes were thrombotic and major bleeding events. Between 2010 and 2021, 29 pregnancies occurred in 24 individuals with MPN. Aspirin was used in 24 cases (83%) and interferon alfa in five (17%). There were 24 live births (83%). There were three thrombotic events, two antepartum and one postpartum. Miscarriages and thrombotic events occurred in JAK2-mutated and triple negative, but not CALR-mutated, MPN. Additional somatic mutations were rare, and there were no apparent associations with pregnancy loss or complications. While JAK2 V617F is associated with an increased risk of thrombosis, its impact on pregnancy outcome has been inconsistently reported. The association between triple negative MPN and adverse pregnancy outcome has not previously been reported. While limited by small numbers, this study underscores the importance of describing driver and other mutations to direct optimal antenatal care in individuals with MPN.

Mutational Profile of Children with Myeloproliferative Neoplasms

Introduction Myeloproliferative neoplasms (MPNs) are clonal bone marrow disorders of over proliferation driven largely by mutations in the JAK2, CALR, and MPL genes. Mutational profiles including driver and secondary mutations in adult subjects with MPNs have been well studied and recent work has determined that driver mutations occur as early as in utero. There is little described about mutational profiles in pediatric patients with MPNs. Given the importance of clonal evolution and the relevance of MPN-associated mutations for risk assessment, clinical features, and outcomes, it is important to develop an understanding of the mutational spectrum in young subjects. Therefore, we sought to evaluate a pediatric MPN cohort for potential MPN-associated mutations. Methods We isolated mononuclear cells from the blood and/or bone marrow of 29 pediatric patients (median age 10 years, range 2-21) with MPNs. DNA was extracted and sequenced on the HEMEPACT platform, a gene panel including almost 600 genes focused on myeloid malignancies. Variants were called using a combination of variant callers. For single-nucleotide variants (SNV), we used CaVEMan, Mutect, and Strelka, and for small insertions and deletions also used Pindel. VAF was uniformly reported across all called variants using a realignment procedure. Likely artifact variants were filtered out based on: (i) The number of callers calling a given variant and the combination of filters from the triple callers. (ii) Variants with VAF <2% in all samples or less than 3 mutant supporting reads were excluded. Results In this cohort, 18 subjects had essential thrombocytosis (ET), 8 subjects had polycythemia vera (PV), 2 had pre-fibrotic myelofibrosis, and 1 had myelofibrosis. Clinical sequencing identified 9 subjects with ET with a JAK2V617F mutation, 3 ET subjects with CALR mutations, and 6 ET subjects who were "triple negative" (TN) for driver mutations. Six subjects with PV were found to have a JAK2V617F mutation, while 2 had JAK2 exon 12 mutations. Both subjects with pre-fibrotic myelofibrosis had CALR mutations, and the subject with myelofibrosis had a MPL mutation. HEMEPACT identified mutations in 10 additional cancer-associated genes (Figure 1A). After JAK2 and CALR, the most frequently mutated genes were TET2 and SETBP1. Two subjects had TET2 mutations that co-occurred with JAK2V617F (Figure 1B), and both had very low TET2 variant allele frequencies (VAFs). Two subjects had SETBP1 mutations, one co-occurring with JAK2V617F, and 1 with CALR mutation (Figure 1B), both of whom had high SETBP1 VAFs, possibly implying a germline mutation. No mutations in ASXL1, EZH2, or DNMT3A were identified. Of the 6 TN subjects, 3 had mutations detected on HEMEPACT (in CHEK2, CUX1, and RB1). Eight of the 29 subjects (28%) had more than 1 mutation detected, and the average number of mutations was 1.2 per subject. In contrast, in a small cohort of adult (median age 41 years, range 21-82) subjects with ET (n=2) and PV (n=7), 6 of 9 subjects (67%) had more than 1 mutation identified, and the average number of mutations per subject was 2. JAK2V617F variant allele frequency of the adult cohort was 4-times higher than in the pediatric cohort (median=0.16 vs. median=0.04). Conclusion This is one of the largest analyses to date of the mutational spectrum of pediatric MPNs. TET2 and SETBP1 mutations co-occurred with driver mutations, and no DNMT3A or ASXL1 mutations were seen in these young patients. While fewer pediatric than adult subjects had multiple mutations, there were some young patients who had already developed multiple mutations in childhood and it is important to better understand the role clonal hematopoiesis may play in pediatric MPNs. Buccal samples in our pediatric patients showed evidence of contamination and identifying the best way to sample the germline from children with these disorders is crucial for the study of MPNs in young patients. There is much to learn about the mutational spectrum of pediatric MPNs, including the role of germline mutations in disease pathogenesis, drivers of MPN development in triple-negative MPNs, mutation acquisition over time, and role of various mutations in MPN progression and outcomes. With this initial evaluation we have begun the process of answering some of these questions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel modes of MPL activation in triple-negative myeloproliferative neoplasms

The identification of a somatic mutation associated with myeloid malignancy is of diagnostic importance in myeloproliferative neoplasms (MPNs). Individuals with no mutation detected in common screening tests for variants in JAK2, CALR, and MPL are described as 'triple-negative' and pose a diagnostic challenge if there is no other evidence of a clonal disorder. To identify potential drivers that might explain the clinical phenotype, we used an extended sequencing panel to characterise a cohort of 44 previously diagnosed triple-negative MPN patients for canonical mutations in JAK2, MPL and CALR at low variant allele frequency (found in 4/44 patients), less common variants in the JAK-STAT signalling pathway (12 patients), or other variants in recurrently mutated genes from myeloid malignancies (18 patients), including hotspot variants of potential clinical relevance in eight patients. In one patient with thrombocytosis we identified biallelic germline MPL variants. Neither MPL variant was activating in cell proliferation assays, and one of the variants was not expressed on the cell surface, yet co-expression of both variants led to thrombopoietin hypersensitivity. Our results highlight the clinical value of extended sequencing including germline variant analysis and illustrate the need for detailed functional assays to determine whether rare variants in JAK2 or MPL are pathogenic.

Gene expression profiling of CD34(+) cells from patients with myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the increased proliferation of hematopoietic stem cell precursors and mature blood cells. Mutations of Janus kinase 2 (JAK2), Calreticulin (CALR) and MPL (myeloproliferative leukemia virus) are key driver mutations in MPN. However, the molecular profile of triple negative MPN has been a subject of ambiguity over the past few years. Mutations of, methylcytosine dioxygenase TET2, polycomb group protein ASXL1 and histone-lysine N-methyltransferase EZH2 genes have accounted for certain subsets of triple negative MPNs but the driving cause for majority of cases is still unexplored. The present study performed a microarray-based transcriptomic profile analysis of bone marrow-derived CD34(+) cells from seven MPN samples. A total of 21,448 gene signatures were obtained, which were further filtered into 472 upregulated and 202 downregulated genes. Gene ontology and protein-protein interaction (PPI) network analysis highlighted an upregulation of genes involved in cell cycle and chromatin modification in JAK2V617F negative vs. positive MPN samples. Out of the upregulated genes, seven were associated with the hematopoietic stem cell signature, while forty-seven were associated with the embryonic stem cell signature. The majority of the genes identified were under the control of NANOG and E2F4 transcription factors. The PPI network indicated a strong interaction between chromatin modifiers and cell cycle genes, such as histone-lysine N-methyltransferase SUV39H1, SWI/SNF complex subunit SMARCC2, SMARCE2, chromatin remodeling complex subunit SS18, tubulin β (TUBB) and cyclin dependent kinase CDK1. Among the upregulated epigenetic markers, there was a ~10-fold increase in MYB expression in JAK2V617F negative samples. A significant increase in total CD34 counts in JAK2V617F negative vs. positive samples (P<0.05) was also observed. Overall, the present data showed a distinct pattern of expression in JAK2V617F negative vs. positive samples with upregulated genes involved in epigenetic modification.

Two activating mutations of MPL in triple-negative myeloproliferative neoplasms.

MPLW515K or W515L mutation plays an important role in the pathogenesis of myeloproliferative neoplasms (MPNs) through signaling molecules of the cytokine receptor axis. Besides MPLW515K or W515L, more than 30 atypical MPL mutations have been reported in patients who are negative for JAK2V617F, MPLW515K/L, and CALR mutations. Here, we aimed to identify the disease‐causing mutations in the triple‐negative case of ET. We described two MPL mutations in patients diagnosed with ET by target sequencing the hotspot mutation region of MPL gene. The MPLA497‐L498ins4 is an insertion mutation detected recurrently in ET patients, and the MPLW515RQ516E is a novel double‐point mutation found in an ET patient. Functional studies of MPLA497‐L498ins4 and MPLW515RQ516E revealed that they are gain‐of‐function mutations. Mutants of MPLA497‐L498ins4 and MPLW515RQ516E promoted autonomous proliferation on Ba/F3 cells in the absence of IL‐3. Autonomous activation of TPO‐R without ligand TPO was observed in MPLA497‐L498ins4 and MPLW515RQ516E mutants. Lower percentage of cells in G1 phase and higher percentage of cells in S phase of two atypical MPL mutants were detected after culturing without any cytokines. These two atypical MPL mutations also presented increase in phosphorylation of signaling proteins including JAK2/STAT, PI3K/AKT, and MAPK/RAS. In summary, the MPLA497‐L498ins4 and MPLW515RQ516E are gain‐of‐function mutations which may be novel driving factors participating in the pathogenesis of triple‐negative MPN.

Triple-Negative Myeloproliferative Neoplasms Vs. Calr, JAK2 or MPL-Mutated Myeloproliferative Neoplasms: Distinct Molecular Characteristics

Background: We compared the clinical, cytogenetic, molecular features, and telomere lengths of patients with triple negative MPN and MPN with any of CALR, JAK2 or MPL mutations.Methods: Target capture sequencing of 87 genes and molecular cytogenetic studies were performed in 61 Korean patients with MPN. Also, we searched the newly reported mutations and novel mutations in triple negative MPN [JAK2-G335D (germline), JAK2-F556V, JAK2-G571S (germline), JAK2-V625F (germline), MPL-T119I, MPL-S204F/P, MPL-E230G, MPL-V285E (germline), MPL-R321W (germline), MPL-Y591D, MPL-S505N and MPL-W515R]. We compared clinical and molecular characteristics between two groups. Additionally, we performed telomere quantitative FISH for 48 patients' samples and measured telomere/centromere ratios of them.Results: Among 61 patients, 13 patients showed mutations in CALR, 34 in JAK2, and 3 in MPL. All of JAK2 mutation and CALR mutation site were reported sites, but 2 among 5 mutation site of MPL were novel mutation [D128N, D261Y]. Twelve patients showed triple negative (7 of PMF 7, 2 of ET, and 3 of MPN-U) - they showed 8 different mutation sites among 6 different genes (ASXL1, DNMT3A, NPM1, POLG, SRSF2, and ZMYM3). NPM1, POLG, and ZMYM3 mutations were seen only in triple negative patients. NPM1 mutation was significantly higher in triple negative MPN (P=0.0301). In telomere length study, there was no statistical difference between triple negative group (T/C ratio mean 12.5) and CALR, JAK2 or MPL mutated group (T/C ratio mean 10.0). Although, MPN patients with telomere length shorter than normal control's lower 10% (7.04) showed poor prognosis (P=0.0045).Conclusions: Patients with triple negative MPN are characterized by high frequency of NPM1 mutation and less number of somatic mutations. Since the mutational analysis for diagnostic purposes is limited to exons 14 of JAK2, exon 10 of MPL and exon 9 of CALR at present, search for JAK2 and MPL mutations in other sites are essential in triple negative MPNs.Keywords: Myeloproliferative neoplasms, next-generation sequencing, triple negative MPN, chromosome, FISH, telomere [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident.

Myeloproliferative neoplasms (MPNs) driver mutations are usually found in JAK2, MPL, and CALR genes; however, 10%-15% of cases are triple negative (TN). A previous study showed lower rate of JAK2 V617F in primary myelofibrosis patients exposed to low doses of ionizing radiation (IR) from Chernobyl accident. To examine distinct driver mutations, we enrolled 281 Ukrainian IR-exposed and unexposed MPN patients. Genomic DNA was obtained from peripheral blood leukocytes. JAK2 V617F, MPL W515, types 1- and 2-like CALR mutations were identified by Sanger Sequencing and real time polymerase chain reaction. Chromosomal alterations were assessed by oligo-SNP microarray platform. Additional genetic variants were identified by whole exome and targeted sequencing. Statistical significance was evaluated by Fisher's exact test and Wilcoxon's rank sum test (R, version 3.4.2). IR-exposed MPN patients exhibited a different genetic profile vs unexposed: lower rate of JAK2 V617F (58.4% vs 75.4%, P = .0077), higher rate of type 1-like CALR mutation (12.2% vs 3.1%, P = .0056), higher rate of TN cases (27.8% vs 16.2%, P = .0366), higher rate of potentially pathogenic sequence variants (mean numbers: 4.8 vs 3.1, P = .0242). Furthermore, we identified several potential drivers specific to IR-exposed TN MPN patients: ATM p.S1691R with copy-neutral loss of heterozygosity at 11q; EZH2 p.D659G at 7q and SUZ12 p.V71 M at 17q with copy number loss. Thus, IR-exposed MPN patients represent a group with distinct genomic characteristics worthy of further study.

Roles of germline JAK2 activation mutation JAK2 V625F in the pathology of myeloproliferative neoplasms

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors.

Clinical Utility of Next-generation Sequencing in the Management of Myeloproliferative Neoplasms: A Single-Center Experience.

Although next-generation sequencing (NGS) has helped characterize the complex genomic landscape of myeloid malignancies, its clinical utility remains undefined. This has resulted in variable funding for NGS testing, limiting its accessibility. At our center, targeted sequencing (TAR-SEQ) using a 54-gene NGS myeloid panel is offered to all new patients referred for myeloid malignancies, as part of a prospective observational study. Here, we evaluated the diagnostic, prognostic, and potential therapeutic utility of clinical grade TAR-SEQ in the routine workflow of 179 patients with myeloproliferative neoplasms (MPN). Of 13 patients with triple negative (TN) MPN, who lacked driver mutations in JAK2, CALR, and MPL, TAR-SEQ confirmed clonal hematopoiesis in 8 patients. In patients with intermediate-risk myelofibrosis (MF), TAR-SEQ helped optimize clinical decisions in hematopoietic cell transplant (HCT)-eligible patients through identifying a high molecular risk (HMR) mutation profile. The presence of an HMR profile favored HCT in 9 patients with intermediate-1 risk MF. Absence of an HMR profile resulted in a delayed HCT strategy in 10 patients with intermediate-2 risk MF, 7 of which were stable at the last follow-up. Finally, TAR-SEQ identified patients with various targetable mutations in IDH1/2 (4%), spliceosome genes (28%), and EZH2 (7%). Some of these patients can be potential candidates for future targeted therapy trials. In conclusion, we have demonstrated that TAR-SEQ improves the characterization of TN MPN, can be integrated in clinical practice as an additional tool to refine decision making in HCT, and has the potential to identify candidates for future targeted therapy trials.

The JAK2 GGCC (46/1) Haplotype in Myeloproliferative Neoplasms: Causal or Random?

The germline JAK2 haplotype known as “GGCC or 46/1 haplotype” (haplotypeGGCC_46/1) consists of a combination of single nucleotide polymorphisms (SNPs) mapping in a region of about 250 kb, extending from the JAK2 intron 10 to the Insulin-like 4 (INLS4) gene. Four main SNPs (rs3780367, rs10974944, rs12343867, and rs1159782) generating a “GGCC” combination are more frequently indicated to represent the JAK2 haplotype. These SNPs are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPN) positive for both JAK2 V617 and exon 12 mutations. The association between the JAK2 haplotypeGGCC_46/1 and mutations in other genes, such as thrombopoietin receptor (MPL) and calreticulin (CALR), or the association with triple negative MPN, is still controversial. This review provides an overview of the frequency and the role of the JAK2 haplotypeGGCC_46/1 in the pathogenesis of different myeloid neoplasms and describes the hypothetical mechanisms at the basis of the association with JAK2 gene mutations. Moreover, possible clinical implications are discussed, as different papers reported contrasting data about the correlation between the JAK2 haplotypeGGCC_46/1 and blood cell count, survival, or disease progression.

PSTAT3/pSTAT5 Signaling Patterns in Molecularly Defined Subsets of Myeloproliferative Neoplasms.

BCR/ABL1-negative myeloproliferative neoplasms (MPNs) are characterized by recurrent mutations in JAK2, CALR, and MPL, each of which has been reported to alter JAK/STAT signaling pathways. This report characterizes JAK/STAT signaling patterns in molecularly defined subsets of MPN utilizing immunohistochemistry for pSTAT3 and pSTAT5. Analysis of 30 BCR/ABL1-negative, nonpolycythemia vera MPN identified 15 (50%) with JAK2 V617F, 2 with MPL mutations (7%), and 8 with CALR mutations (27%). All mutations were mutually exclusive, except for 1 case with concurrent JAK2 V617F and CALR mutations. pSTAT3 staining in megakaryocyte nuclei was found in 4 cases (13%) and was not significantly associated with mutation status. pSTAT5 staining in megakaryocyte nuclei was found in 16 cases (53%), as was significantly associated with JAK2 V617F versus CALR mutation (P=0.009). Erythroid staining for pSTAT5 was seen exclusively in "triple-negative (TN)" cases lacking JAK2 V617F, MPL, and CALR mutations (P=0.006, TN vs. other genotypes), and pSTAT5 staining in megakaryocyte nuclei was seen in 2 TN cases. pSTAT5 staining in TN MPN suggests that other unknown abnormalities in this pathway may contribute to the pathogenesis of these cases. Furthermore, the demonstration of distinct STAT staining patterns in molecularly defined MPN suggests that these mutations result in divergent signaling events that may contribute to the biological and prognostic differences in these molecular subsets of MPN.

Targeted next-generation sequencing identified novel mutations in triple-negative myeloproliferative neoplasms.

Mutations in JAK2, MPL and CALR genes have been identified in the majority of myeloproliferative neoplasm (MPN) patients, and patients negative for these three mutations are the so-called triple-negative (TN) MPN. In this study, we examined the mutational profiles of 16 triple-negative MPN patients including 7 essential thrombocythemia (ET), 1 primary myelofibrosis and 8 polycythemia vera (PV). Targeted next-generation sequencing was performed using the ACTOnco Comprehensive Cancer Panel (Ion AmpliSeq Comprehensive Cancer Panel, Life Technologies) to target all coding exons of 409 cancer-related genes. Overall, 30 nonsynonymous somatic mutations were detected in 12 (75%) patients with a range of 1-5 mutations per sample. Notably, one ET patient was found to have JAK2V617F and KITP551L mutations at very low allele frequency. One MPLP70L and 1 MPLM602T mutations were identified each in 1 ET and 1 PV, respectively. Other recurrent mutations were also identified including KMT2C, KMT2D, IRS2, SYNE1, PDE4DIP, SETD2, ATM, TNFAIP3 and CCND2. In addition, germline mutations were also found in some cancer-related genes. Copy number changes were rare in this cohort of TN MPNs. In conclusion, both somatic and germline mutations can be detected in TN MPN patients.

Triple Negative Myeloproliferative Neoplasms (MPNs) Patients Show Low MPN10 Score and Lower Grades of Bone Marrow Fibrosis

Introduction: The vast majority ofmyeloproliferative neoplasms (MPNs) patients are characterized by a molecular genetic background and by variable symptoms reflecting disease burden that may correlate with prognosis.Aim: To study the impact of triple negative status of driver gene mutations: Janus kinase 2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia virus oncogene (MPL) on disease burden and its correlation with symptom severity (MPN10 score) and degree of bone marrow (BM) fibrosis in MPNs patients.Patients and Methods: MPN Symptom Assessment Form Total Symptom Score (MPN-SAF TSS) was assessed as median of 10 items: fatigue, concentration, early satiety, inactivity, night sweats, itching, bone pains, abdominal discomfort, weight loss and fever. JAK2V617F and MPL W515exon 10 mutations were performed by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) while CALR exon 9 insertion/deletion was detected by high-resolution melting (HRM) curve analysis.Results: 100 MPNs patients (54 males and 46 females): 18 polycythemia vera (PV), 41 essential thrombocythemia (ET), 24 primary myelofibrosis (PMF), 10 Post-ET/PV-myelofibrosis (post-ET/PV-MF) and 7 myelodysplastic/myeloproliferative neoplasms (MDS/MPN) were included. Median age at diagnosis was 55 years (17-75) and was lower in ET than PV and PMF patients; 44 (19-75) years vs. 56 (34-70) years and 56 (20-75) years, respectively (p=0.06). JAK2 mutation was positive in 15 (85%) PV patients, 14 (34%) ET patients, 15 (62%) PMF patients, 8(80%) post-ET/PV-MF patients and zero (0%) MDS/MPN patients (p<0.001). CALR mutation was positive in zero (0%) PV patients, 10 (24%) ET patients, 4 (17%) PMF patients, zero (0%) Post-ET/PV-MF patients and zero (0%) MDS/MPN patients (p<0.05). MPL mutation was positive in zero (0%) PV patients, 2 (5%) ET patients, 1(4%) PMF patients, zero (0%) Post ET/PV-MF patients and zero (0%) MDS/MPN patients. Twenty four/93 (26%) patients were triple negative; 15 ET (16%), 3 PV (3%), 6 PMF (6%). Median MPN10 score was 21 (4-45) in ET versus 37.5 (25-56) in PV, 54 (15-80) in PMF and 59 (45-75) in Post-ET/PV-MF (p<0.001). Median MPN10 score was 25 (10-50) in triple negative patients vs. 40 (4-80) in MPNs patients showing at least one driver mutation positivity (p<0.001). BM fibrosis was present in 6 (15%) patients with triple negative vs. 33 (85%) patients showing at least one molecular marker positivity (p=0.007). Out of 52 patients having splenomegaly; seven (13.5%) patients were triple negative vs. 45 (87%) patients with at least one gene mutation (p<0.001). Out of the 24 triple negative patients, 19 (80%), 4 (16%), 1 (3%) and 0(0%) had BM fibrosis grades 0, 1, 2 and 3 vs. 36 (52%), 7 (10%), 12 (17%), 14 (20%) out of 69 patients with at least one gene mutation, respectively (p=0.002). After a median follow-up period of 16 months (3-151), overall survival (OS) was 95%. OS in PV and ET patients was 100 % versus 83 % in PMF patients (p=0.08). OS in triple negative group was 100% versus 94% in the gene mutations group (p=0.387).Conclusion: Driver gene mutations show an impact on disease symptoms and burden. Triple negative MPNs patients in our cohort have significantly low MPN10 score and less BM fibrosis which may indicate a more indolent disease course. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chasing down the triple-negative myeloproliferative neoplasms: Implications for molecular diagnostics.

The majority of patients with classical myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia, and primary myelofibrosis harbor distinct disease-driving mutations within the JAK2, CALR, or MPL genes. The term triple-negative has been recently applied to those MPN without evidence of these consistent mutations, prompting whole or targeted exome sequencing approaches to determine the driver mutational status of this subgroup. These strategies have identified numerous novel mutations that occur in alternative exons of both JAK2 and MPL, the majority of which result in functional activation. Current molecular diagnostic approaches may possess insufficient coverage to detect these alternative mutations, prompting further consideration of targeted exon sequencing into routine diagnostic practice. How to incorporate these illuminating findings into the expanding molecular diagnostic algorithm for MPN requires continual attention.

Cooperation of germ line JAK2 mutations E846D and R1063H in hereditary erythrocytosis with megakaryocytic atypia

AbstractThe role of somatic JAK2 mutations in clonal myeloproliferative neoplasms (MPNs) is well established. Recently, germ line JAK2 mutations were associated with polyclonal hereditary thrombocytosis and triple-negative MPNs. We studied a patient who inherited 2 heterozygous JAK2 mutations, E846D from the mother and R1063H from the father, and exhibited erythrocytosis and megakaryocytic atypia but normal platelet number. Culture of erythroid progenitors from the patient and his parents revealed hypersensitivity to erythropoietin (EPO). Using cellular models, we show that both E846D and R1063H variants lead to constitutive signaling (albeit much weaker than JAK2 V617F), and both weakly hyperactivate JAK2/STAT5 signaling only in the specific context of the EPO receptor (EPOR). JAK2 E846D exhibited slightly stronger effects than JAK2 R1063H and caused prolonged EPO-induced phosphorylation of JAK2/STAT5 via EPOR. We propose that JAK2 E846D predominantly contributes to erythrocytosis, but is not sufficient for the full pathological phenotype to develop. JAK2 R1063H, with very weak effect on JAK2/STAT5 signaling, is necessary to augment JAK2 activity caused by E846D above a threshold level leading to erythrocytosis with megakaryocyte abnormalities. Both mutations were detected in the germ line of rare polycythemia vera, as well as certain leukemia patients, suggesting that they might predispose to hematological malignancy.

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms

AbstractEssential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed “triple negative.” We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders.

Whole Exome Sequencing Identifies Novel MPL and JAK2 M utations in Triple Negative Myeloproliferative Neoplasms

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic myeloproliferative neoplasms (MPN) characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. Most of the cases are sporadic and driven by somatic mutations, although familial clustering is observed. The most common mutation affecting 50-60% of the cases is JAK2-V617F, while 25-30% of the patients carry somatic mutations in exon 9 of CALR. MPL exon 10 mutations affect ~5% of the cases. JAK2, CALR and MPL mutations are mutually exclusive and account for >90% of ET and PMF cases. In 12% of ET and 5% of PMF cases the disease drivers remain unknown. These patients are termed as triple negative. The mutational analysis for diagnostic purposes is limited to exons 14 of JAK2, exon 10 of MPL and exon 9 of CALR. The aim of this study was to identify disease causing mutation in triple negative cases of ET and PMF.To identify the somatic mutations that are potential disease drivers in triple negative MPN we performed whole exome sequencing (WES) on paired samples from the tumor and control tissue of 4 patients with ET and 4 patients with PMF. We identified somatic mutations in 3/8 analyzed cases. In two PMF cases we identified somatic mutations in genes relevant for MPN- TET2, ASXL1, CBL, SRSF2 and a mutation in MPL-S204P. We did not identify a novel recurrent mutation. In the 5 cases without somatic mutations, we looked for germline mutations in genes relevant for MPN. We identified germline mutations MPL-V285E and JAK2-G571S in one PMF case and one case of ET, respectively. SNP microarray analysis for presence of chromosomal aberrations revealed a uniparental disomy of chromosome 6p in the case with MPL -V285E mutation, suggesting clonal hematopoiesis. To determine the frequency of MPL and JAK2 mutations outside exons 10 and 14 in triple negative MPN, we performed Sanger sequencing of all coding exons of MPL in 62 patients and of JAK2 in 49 patients. We detected variants outside exon 10 of MPL in 6/62 cases (9.7%). MPL-T119I, MPL-S204F, MPL-E230G and MPL-Y591D were somatic mutations, while MPL-R321W was germline. We identified an additional patient with MPL-S204P mutation, however the control tissue was not available. JAK2 variants were found in 4/49 cases (8.1%). JAK2-G335D and JAK2-V625F were germline mutations, while for the patients with JAK2-F556V and JAK2-G571S the control tissue was unavailable. In total, we identified non-canonical MPL mutations in 8/70 (11.4%) and JAK2 mutations in 5/57 (8.8%) triple negative cases of ET and PMF. All mutations were heterozygous. The mutations in MPL and JAK2 were mutually exclusive in our patient cohort.The expression of identified MPL mutants did not induce cytokine independent growth of Ba/F3 cells, but the MPL-Y591D expressing cells showed marked hypersensitivity to TPO compared to the wild type. Using a luciferase reporter assay in JAK2-deficient gamma 2A cells, where we transiently expressed the wild type or mutant MPL cDNAs, JAK2, STAT5, STAT5 reporter Spi-Luc, and pRL-TK for transfection control, we could demonstrate that all identified MPL mutations lead to constitutive activation of JAK/STAT signaling. As the detection of activity required longer times (48h) than for the MPL-W515K (24h), we concluded that the identified mutations have a milder effect of the function of MPL. By Western immunodetection we could demonstrate that expression of JAK2-F556V and JAK2-V625F in Ba/F3-MPL cells, lead to the increased phosphorylation of STAT5 in the absence of cytokines. We also observed increased sensitivity to TPO in the Ba/F3 MPL cell lines expressing JAK2-F556V and JAK2-V625F. JAK2-V625F and JAK2-F556V are mild gain-of-function mutations, while JAK2-G335D and JAK2-G571S do not seem to alter the function on the JAK2 protein.The results of our study suggest that sequencing of all coding exons of MPL and JAK2 is recommended for the diagnostic work-up of the ET and PMF patients who do not carry other more common mutations. The lack of evidence for clonal disease in 50% of the triple negative cases and presence of germline mutations suggests that a proportion of cases are likely to be hereditary MPN-like disorders. Application of whole genome sequencing or RNA sequencing for fusion oncogene detection will likely fill in the gap of the remaining triple negative MPN cases with clonal hematopoiesis in which we did not identify a recurrent driving mutation using WES. DisclosuresGisslinger:AOP ORPHAN: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Consultancy; Geron: Consultancy; Janssen Cilag: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Genomic Profile of Patients with Triple Negative (JAK2, CALR and MPL) Essential Thrombocythemia and Primary Myelofibrosis

Introduction: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are myeloproliferative neoplasms (MPN) with similar driver mutations. The three hallmark molecular alterations in these diseases are JAK2, MPL and CALR mutations. Patients with myelodysplastic/myeloproliferative (MPN/ MDS) neoplasms such as refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) also present with the same hallmark genetic changes. Nevertheless, roughly 10% of these patients do not present mutations in neither of these genes. Recent data suggest that triple negative patients have a more aggressive clinical course. While the molecular alterations present in patients with MPN have been extensively studies, the genomic profile of triple negative TE/PMF/RARS-T has not been described. To better characterize these patients we performed whole exome / genome sequencing of paired granulocytes and skin from 15 triple negative MPN patientsMethods: A total of 15 patients with triple negative MPN or MPN/ MDS [PMF (N=6)/TE (N=8)/RARS-T (N=1)] were analyzed. DNA was extracted from CD66b+ magnetic bead selected granulocytes (EasySep, Stem Cell Technologies) and matched skin biopsies with QiaAmp DNA Mini kit (Qiagen).Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University). Tumor coverage was 150x and germline coverage was 60x. The combined output of these 3 softwares was further filtered by in-house criteria in order to reduce false-positive calls (minimum coverage at both tumor/germline ≥8 reads; fraction of reads supporting alternate allele ≥5% in tumor and ≤10% in germline; ratio of allele fraction tumor:germline >2). All JAK2 and CALR mutations were validated through Sanger sequencing. Validations of other somatic mutations are under way at this point.Results: First we asked whether other hematopoietic related genes could be responsible for the pathogenesis of the triple negative cases. With that goal we searched for high confidence mutations in genes that are mutated in at least 1% of patients with hematopoietic tumors on COSMIC (catalog of somatic mutations in cancer) database and also genes known to be recurrently mutated in myeloid malignancies. Only 6 out of 15 patients presented mutations in other myeloid related genes. The diagnosis of these patients were PMF=4, TE=2. The hematopoietic related genes mutated in these patients were: ASXL1 (n=4), CUX1 (n=3), NRAS (n=2) and ATM, CBL, CSFR3, CREBBP, DNMT3A, ETV6, EZH2, JARID2, MLL2, PHF6, SRSF2, STAG2, TET2, GNAS, U2AF1 (n=1).Noteworthy, we have found one known oncogenic mutation in CSFR3, an alteration supposed to be specific for chronic neutrophilic leukemia and atypical CML, in a patient with ET, and an oncogenic mutation in GNAS in a patient with PMF. In addition, the patient with RSRA-T had a putative oncogenic mutation in PTPN11Remarkably, the average number of hematopoietic related mutations in these patients was 5, significantly higher than the total number of mutations found in another cohort of patients with either JAK2 (average = 1.7) or CALR mutations (average = 1.9). Although our numbers are small, we may speculate that the high incidence of ASXL1 mutations (28%) associated with a high number of prognostically detrimental mutations can partially explain the worse outcomes associated with triple negative MPN. Noteworthy is also the high prevalence of CUX1 mutations in this subset of patients (21%) when compared to other myeloid malignancies in general.Regarding the other 9 patients for whom no hematopoietic mutations could be identified, 6 patients had ET, 2 patients PMF and one patient had RARS-T.Conclusion: We have shown that: i-patients with triple negative MPN are molecularly heterogeneous, with one group presenting a high number of hematopoietic related mutations, ii-the most common mutations present in these patients are ASXL1, CUX1 and NRAS, iii-The majority of these patients do not present mutations in hematopoietic related genes, what suggests that non-described molecular mechanisms are operating in these patients. DisclosuresNo relevant conflicts of interest to declare.