scholarly journals PD‐L1 Overexpression Correlates with JAK2 ‐V617F Mutational Burden and Is Associated with 9p Uniparental Disomy in Myeloproliferative Neoplasms

2022 ◽  
Author(s):  
Jelena D. Milosevic Feenstra ◽  
Roland Jäger ◽  
Fiorella Schischlik ◽  
Daniel Ivanov ◽  
Gregor Eisenwort ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Mutational Burden

scholarly journals Overexpression of PD-L1 Correlates with JAK2-V617F Mutational Burden and Is Associated with Chromosome 9p Uniparental Disomy in MPN

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 24-24
Author(s):  
Jelena D. Milosevic Feenstra ◽  
Fiorella Schischlik ◽  
Roland Jäger ◽  
Daniel Ivanov ◽  
Gregor Eisenwort ◽  
...  
Keyword(s):  
Research Funding ◽  
Immune Escape ◽  
Myeloproliferative Neoplasms ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Surface Expression ◽  
Private Company ◽  
Snp Arrays ◽  
Mutational Burden ◽  
Ifn Γ

Myeloproliferative neoplasms (MPN) are characterized by clonal hematopoiesis, hyperproliferation of myeloid cells, hyperinflammation and immune deregulation. The three classical BCR-ABL1-negative MPN are essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The disease is driven by JAK2, CALR or MPL somatic mutations in most patients. Drug resistance is a major problem in MPN. Recent data suggest that MPN cells display certain immune checkpoint molecules that may contribute to resistance, including PD-L1. Antibodies targeting the PD1/PD-L1 axis are highly promising anti-cancer drugs. Their potential use in MPN is being explored but it is unclear which MPN subtypes are most suitable for testing in clinical trials. The aim of our project was to assess PD-L1 expression in disease-initiating neoplastic stem cells (SC) and differentiated cells of MPN patients and to develop therapeutic approaches capable of blocking PD-L1 expression in MPN SC. In a first step, PD-L1 expression was assessed by RNA-sequencing of granulocytes of 106 MPN patients and 15 healthy donors (HD). The cohort included 56 PMF, 33 ET and 17 PV patients. For 102 patients data from Human Genome-wide Affymetrix 6.0 SNP arrays were available. We observed a ~5-fold higher expression of PD-L1 mRNA in patients with PV compared to other MPN (P<.01) or HD (P<.01). JAK2-V617F positive ET patients had higher expression of PD-L1 compared to CALR-mutated ET (p<.005) and the same was observed in PMF (p<.01). Other mutations (TET2, DNMT3A) detected by NGS did not affect PD-L1 expression. Since PD-L1 and JAK2 are located on chromosome 9p24, we looked into our previously published dataset of 400 MPN patients analyzed by SNP arrays and found that in all 195 patients with 9p uniparental disomy (UPD) the aberrations covered both genes. As PD-L1 is more centromeric it could represent the second target of 9pUPD which can precede the acquisition of JAK2-V617F in MPN. Granulocytes in JAK2-V617F positive patients with 9pUPD expressed significantly higher levels of PD-L1 compared to patients without 9pUPD (P<.0001; Figure 1A). Moreover, the JAK2-V617F mutational burden significantly correlated with PD-L1 expression (R=.52, P<.0001; Figure 1B). This correlation was lost when cases with 9pUPD were excluded from the analysis (R=.03, P=.9), indicating that the UPD is relevant for PD-L1 upregulation. To investigate PD-L1 surface expression on MPN SC we analyzed CD34+CD45dimCD38- cells isolated from fresh bone marrow (BM) samples of another 51 MPN patients and 7 HD by flow cytometry (FC). MPN patients showed a significantly higher surface expression of PD-L1 on CD34+CD45dimCD38- cells compared to HD (p<.001; Figure 1C). PD-L1 levels on the SC surface were elevated in both JAK2- and CALR-mutated MPN patients compared to HD (p<.001 and P<.005, respectively). PD-L2 was neither expressed in MPN granulocytes nor on MPN SC. CD4+ and CD8+ T-cells from BM samples of 17 MPN patients expressed the PD-L1 receptor PD-1 as assessed by FC. We cultured ex vivo primary MPN cells from 7 JAK2-V617F positive patients and showed that PD-L1 expression on MPN SC spontaneously decreases in culture, that interferon-gamma (IFN-γ) can promote expression of PD-L1 on these cells, and that ruxolitinib and the BRD4-degrader dBET6 block IFN-γ-induced PD-L1 expression in CD34+CD45dimCD38- MPN SC (P<.05). Together, we show that PD-L1 is overexpressed on the surface of disease-initiating MPN SC, that PD-L1 mRNA is overexpressed in granulocytes of MPN patients and that PD-L1 overexpression in granulocytes correlates with the JAK2-V617F mutational burden. In patients with JAK2-V617F positive MPN, 9pUPD leads to further PD-L1 upregulation either through increasing the mutant JAK2 gene dosage, loss of wt-JAK2,or amplification of PD-L1 allele with higher expression. Our data suggest the possibility that 9pUPD and the subsequent elevation of PD-L1 expression may provide an immune escape mechanism and may contribute to positive selection of JAK2-V617F homozygous SC. Ruxolitinib and dBET6 downregulate PD-L1 expression on MPN SC suggesting a role for the JAK2 and BRD4-MYC pathway. As recent studies revealed an immunogenic potential of JAK2 and CALR mutants, overcoming the disease-mediated immune escape may be of particular importance. Further preclinical and clinical studies are now required to examine the value of PD1/PD-L1 inhibitors in patients with MPN. Disclosures Gisslinger: Celgene: Honoraria; MyeloPro Diagnostics and Research: Honoraria; AOP Orphan Pharmaceuticals AG: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; PharmaEssentia: Honoraria; Janssen-Cilag: Honoraria; Roche: Honoraria. Kralovics:AOP Orphan Pharmaceuticals AG: Honoraria; PharmaEssentia: Honoraria; Qiagen: Honoraria; Novartis: Honoraria; MyeloPro Diagnostics and Research: Current equity holder in private company. Valent:Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

Chromosomal Instability Causes Genetic and Clonal Heterogeneity in Myeloproliferative Neoplasms and Is Not Restricted to JAK2-V617F Positive Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 178-178
Author(s):  
Roland Jäger ◽  
Damla Olcaydu ◽  
Tiina Berg ◽  
Bettina Gisslinger ◽  
Heinz Gisslinger ◽  
...  
Keyword(s):  
Chromosomal Instability ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Clonal Heterogeneity ◽  
Chromosome 1P ◽  
Clonal Variability ◽  
Genomic Regions ◽  
Chromosome 13Q

Abstract A number of oncogenic mutations have been identified in myeloproliferative neoplasms (MPN) in the past few years. Among these JAK2-V617F is most frequent, followed by mutations of the thrombopoietin receptor MPL and JAK2 exon 12. In addition, cytogenetic lesions occur frequently in MPN detected either at diagnosis or later in the course of the disease. To explore the genetic aberrations present in MPN patients, we performed microarray genotyping using Affymetrix SNP 6.0 arrays in a series of 71 MPN patients with variable presence of JAK2 and MPL mutations. More than half of the analyzed patients exhibited loss of heterozygosity (LOH) in at least one chromosomal region. Complex karyotypes with two and more regions with LOH were detected in 18 patients. Uniparental disomy (UPD) on chromosomes 9p, 1p, 11q, 14q and 17q represented the largest proportion of LOH detected followed by deletions on chromosome 13q, 20q, and 12p. All patients with UPD on chromosome 1p were homozygous for the MPL-W515L mutation. We observed frequent aberrations of chromosome 7 including monosomy, deletions on 7p and 7q, and UPD of 7q. Using microsatellite PCR, we validated the microarray findings and further determined the frequency of these aberrations in a total of 367 MPN patients. Multiple occurrences of individual chromosomal lesions allowed us to define the minimal genomic regions involved in deletions or UPDs. The sizes of the common deleted regions (CDRs) were variable ranging from 9 mega base pairs (Mb) to 0.5 Mb. The CDR on chromosome 7p included only the IKZF1 and FIGNL1 genes previously shown to associate with leukemic transformation. To determine the clonal composition of the hematopoietic progenitor pool of patients with complex karyotypes we genotyped individual BFU-E and CFU-GM colonies in a series of 27 patients. We observed a remarkable clonal heterogeneity at the progenitor cell level. Using four clonal markers we defined 9 different types of clonal structures. In a set of patients, JAK2-V617F or MPL-W515L mutations occurred before the acquisition of chromosomal deletions. Other patients acquired deletions before the acquisition of JAK2-V617F. In summary, our results show that somatic mutations in MPN are not acquired in a predetermined order as seen in other malignancies, but occur randomly. The chromosomal instability in MPN is not caused by JAK2-V617F exclusively, since many patients show aberrations outside of the JAK2-V617F positive clone. Heterogeneity of somatic mutations in MPN leads to high clonal variability within the progenitor pool potentially affecting therapeutic outcome. Thus, targeting JAK2-V617F alone may not lead to restoration of polyclonal hematopoiesis. An individualized therapeutic approach and/or combination therapy might be necessary to achieve clonal remission in MPN.

Disruption of the ASXL1 Gene Is Prevalent In PMF: Analysis of Molecular Genetics and Clinical Phenotypes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3074-3074
Author(s):  
Brady L Stein ◽  
Donna M Williams ◽  
Michael A McDevitt ◽  
Christine L. O'Keefe ◽  
Ophelia Rogers ◽  
...  
Keyword(s):  
Molecular Genetics ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Single Nucleotide Polymorphism Array ◽  
Myeloproliferative Neoplasms ◽  
Direct Sequencing ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Wild Type

Abstract Abstract 3074 Background: The myeloproliferative neoplasms, PV, ET and PMF, share phenotypic features and molecular lesions, yet PMF distinguishes itself by its unfavorable natural history and rate of leukemic evolution. These distinctions may occur as a result of cooperating genomic lesions specific to PMF compared to PV or ET. We performed single nucleotide polymorphism array (SNP-A)-based karyotyping in 210 MPN patients and identified 20q11 deletions in 10% of PMF cases and in none of the PV or ET cases. The 20q11 deletion region spanned 1,662 KB and encompassed 37 genes, of which ASXL1 was included. To test whether ASXL1 contained lesions in the MPN cohort at large, we directly sequenced key regions of the ASXL1 gene in 65 PMF, 11 PV and 14 ET cases, as well as 7 controls from the SNP-array cohort. Genomic DNA from neutrophils and in select cases, purified CD34+ cells was used for both SNP-A and direct sequencing. Clinical parameters were correlated with genomic findings and the quantitative JAK2 V617F neutrophil allele burden Molecular genetics: 26/65 (40%) of PMF cases had abnormalities in ASXL1 (4 deletions, 22 mutations) whereas none of the 32 PV, ET or control cases had such lesions. The majority of ASXL1 sequence variations were nonsense lesions including the previously reported 1934dupG which comprised 30% of all of the mutations. The residual ASXL1 allele in all 20q11 deletion cases containing the ASXL1 gene was intact. In three PMF cases, more than one distinct ASXL1 mutation was identified, and cloning experiments on two of those cases indicated that the lesions were biallelic. Using banked samples, we observed the acquisition of an ASXL1 lesion over time, and established that ASXL1 lesions detected in 2 post ET-MF cases were also detected at low levels in the ET phase of the MPN. Genotype/Phenotype Correlations: ASXL1 deletions and mutations were prevalent in de novo PMF (37%), post PV-PMF (20%) post ET-PMF (62%) and in PMF/AML (33%). ASXL1 mutations did not associate with chemotherapy exposure as the prevalence of hydroxyurea use was similar in patients with and without mutations, and ASXL1 –mutation positive cases were present in patients who had never received any form of chemotherapy. There was no dependence upon JAK2 status as the presence of ASXL1 mutations were identified in JAK2 V617F-negative cases (9/26); JAK2 V617F-heterozygous cases (10/26); and JAK2 V617F-homozygous cases (7/26). Based on results of SNP-A, patients with ASXL1 mutations were equally as likely to have uniparental disomy (involving 9p or other regions) and loss/gain abnormalities (>1MB) compared to those without ASXL1 mutations. There were no differences in sex, age, or disease duration between PMF patients with and without ASXL1 mutations. In the ASXL1-mutant group, there was a trend toward a lower median white blood cell count (8 vs. 12.5 k/cu mm; p=0.3) and hemoglobin (9.7 vs. 11 g/dl; p=0.3) compared to ASXL1-wild-type patients. Furthermore, those PMF patients with ASXL1 mutations were significantly more likely to have received anemia-directed therapy (transfusion, erythropoietin, immunomodulating agents, steroids) compared to those without mutations (15/26 (58%) vs. 11/39 (23%); p=0.02). Post ET-MF patients comprised 31% (8/26) of ASXL1-mutant cases, compared to only 10% (4/39) ASXL1- wild-type cases (p=0.03). However, the presence of an ASXL1 mutation did not associate with an accelerated transition rate from ET to MF; among the 12 post ET-MF cases in the cohort, the median time of transition from ET to MF was 15.5 years in those with ASXL1 mutations compared to 7 years in those with ASXL1 wild-type status (p=0.02). Conclusion: Disruption of the ASXL1 gene occurs in 40% of PMF cases. The association of ASXL1 lesions, due to either mutation or deletion, suggests that ASXL1 haplo-insufficiency is associated with a PMF phenotype in the context of other known and unknown lesions, and that disruption of ASXL1 function may directly contribute to the pathophysiology and clinical complications of primary and secondary myelofibrosis. These data support the concepts that cooperative lesions in addition to JAK2 V617F are critical in generating PMF, that PMF is molecularly more complex than either PV or ET, and that the transition of PV or ET to PMF is associated with the acquisition of genomic lesions, such as ASXL1, that are present in PMF at large. Disclosures: No relevant conflicts of interest to declare.

scholarly journals JAK2 V617F Mutational Burden and Clinical Findings Relationship in Myeloproliferative Neoplasms

2021 ◽  
Author(s):  
Cigdem Yuce Kahraman ◽  
Gulden Sincan ◽  
Abdulgani Tatar
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Spleen Size ◽  
Jak2 Mutation ◽  
Laboratory Findings ◽  
Treatment Protocols ◽  
Mutational Burden ◽  
Mutation Burden ◽  
Significant Difference

Abstract Background: Philadelphia-negative chronic myeloproliferative neoplasms(MPN) are associated with various genetic abnormalities. JAK2 V617F mutation is the most common one and important for diagnosis. We aimed to evaluate JAK2 mutation status and clinical parameters relationship of the MPN patients referred to our clinic.Methods and Results: We evaluate 143 JAK-2 positive patients diagnosed with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2 mutational burden was higher in PV and PMF than ET. Laboratory findings were different in MPN groups and higher –lower JAK2 mutational burden groups. JAK2 mutational burden was correlated with spleen size and LDH level, particularly in PMF. There was no significant difference in age, gender, jak2 mutation burden and laboratory findings in patients with and without thrombosis and bleeding. Common treatment protocols were acetylsalicylic acid (ASA) + hydroxyurea, ASA and ASA + phlebotomy and others respectively. JAK2 mutational burden, mean age and LDH level were higher significantly in the patients treated with ASA+ hydroxyurea than the patients treated with ASA.Conclusion: We speculate that if the spleen size in MPN is as large as the massive splenomegaly and the LDH level is high, the JAK2 mutation burden may tend to be higher. This relationship is more pronounced for PMF.

Association Between EZH2 and Other Acquired Mutations In Myelofibrosis and Myelodysplastic/Myeloproliferative Neoplasms

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 625-625
Author(s):  
Thomas Ernst ◽  
Joannah Score ◽  
Claire E Hidalgo-Curtis ◽  
Amy V Jones ◽  
Andreas Hochhaus ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Uniparental Disomy ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Mutational Status ◽  
Significant Difference ◽  
Chromosome 7Q ◽  
Secondary Myelofibrosis

Abstract Abstract 625 We recently identified EZH2 as the major target of chromosome 7q acquired uniparental disomy (aUPD) in myeloproliferative neoplasm (MPN) and myelodysplastic syndromes (MDS). To determine the prevalence of EZH2 mutations we screened all coding exons for mutations in total of 624 cases with myeloid disorders (MPN, n=157; MDS, n=154; MDS/MPN, n=219; AML, n=54, CML in transformation, n=40) and found 49 monoallelic or biallelic EZH2 mutations in 42 individuals, most commonly MDS/MPN (27/219; 12%), primary or secondary myelofibrosis (4/30; 13%) and MDS (9/154; 6%). To determine if EZH2 mutations might co-operate with other known abnormalities or whether they might be mutually exclusive, we tested the mutational status of TET2, ASXL1, CBL, RUNX1, CEBPA, FLT3, NPM1, and WT1 in 187 of the 219 MDS/MPN cases that were screened for EZH2. We also tested an additional cohort of 52 primary myelofibrosis cases for both EZH2 and JAK2 V617F mutations. Of the 187 MDS/MPN cases (CMML, n=97; atypical CML, n=68; MDS/MPN-U, n=22), mutations were seen most frequently in TET2 (67/187; 36%), followed by ASXL1 (38/187, 20%; not including cases with the controversial c.1934dupG variant), RUNX1 (27/187; 14%), EZH2 (25/187; 13%), CBL (22/175; 13%), FLT3 (8/187; 4%), CEBPA (7/187; 4%), NPM1 (6/187; 3%) and WT1 (2/187; 1%). Sixty six (35%) cases tested negative for mutations in all 9 genes. Of the 25 cases with EZH2 mutations, 22 (88%) had mutations in at least one other gene, most frequently TET2 (n=11) and ASXL1 (n=10). EZH2 mutations were also seen in combination with mutations in CBL (n=5), CEBPA (n=4), RUNX1 (n=3) and FLT3 (n=2), however there was no significant difference in the frequency of other mutations on comparison of EZH2 mutated and EZH2 unmutated cases. When the analysis was restricted to the 10 cases with homozygous EZH2 mutations, a similar heterogeneity was observed with mutations in CBL, RUNX1, CEPBA and TET2 only (n=1 for each gene), ASXL1 only (n=2), TET2+ASXL1 (n=1), TET2+ASXL1+RUNX1 (n=1) or no other mutation (n=2). Analysis of CFU-GM from one case that tested positive for both EZH2 and TET2 mutations revealed a complex pattern with an EZH2 mutation clearly preceding the sequential acquisition of two TET2 mutations. Of the 82 primary and secondary myelofibrosis cases, 9 (11%) tested positive for an EZH2 mutation. Of these, 5 were positive for JAK2 V617F and 4 were negative. In 2 cases both EZH2 and JAK2 V617F were homozygous indicating that the predominant clone must harbor both mutations. Overall, these data indicate a complex interaction between different abnormalities with little indication of co-operativity or functional redundancy. Whilst these observations will need to be refined by detailed analysis of single clones, they do suggest that the development of both myelofibrosis and MDS/MPN requires functional alterations in multiple pathways. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Worse Outcomes in Males with MPN Are Associated with Qualitative and Quantitative Differences in Non-Driver Mutational Burden

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 837-837
Author(s):  
Theodoros Karantanos ◽  
Evan M Braunstein ◽  
Shruti Chaturvedi ◽  
Jerry L. Spivak ◽  
Linda Resar ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Cox Regression ◽  
Myeloproliferative Neoplasms ◽  
Multivariable Analysis ◽  
Jak2 V617f ◽  
Driver Mutation ◽  
Molecular Characteristics ◽  
Free Survival ◽  
Mutational Burden ◽  
Male Sex

INTRODUCTION: The chronic myeloproliferative neoplasms (MPN) have variable courses and outcomes despite similar driver mutations. We have found that males present more commonly with primary myelofibrosis (PMF) as opposed to essential thrombocytosis (ET) independently of driver mutation and age, and have demonstrated the independent negative impact of male sex in transformation to secondary MF (sMF), AML, and overall survival. The aim of this study was to examine both driver and non-driver mutational burden as a factor in presentation and outcome in the MPN. PATIENTS AND METHODS: 652 individuals with ET, PV or PMF (394 females and 258 males; 311 with ET, 252 with PV and 89 with PMF) were enrolled in our prospective observational cohort between 2005-2019, with a median follow up of 9 years (Q25-75% 4, 15 years). All JAK2 V617F -positive patients were genotyped for quantitative JAK2 V617F variant allele fractions (VAF) at enrollment and over time, and 76 patients (41 females and 35 males, 43 with ET, 23 with PV and 10 with PMF) had additional sequencing, examining 63 genes implicated in myeloid neoplasms. Multivariable cox regression was used to examine the associations of sex, age and molecular characteristics with venous thrombosis, cerebrovascular events, and MF/AML transformation. Univariate cox regression and Kaplan-Meier (KM) were used to assess the effect of JAK2 V617F VAF change/year on survival. Multivariable logistic regression was used to evaluate the associations of sex, age, phenotype and number of additional somatic mutations. RESULTS: Venous thromboembolism was less common in males (OR 0.44, 95% CI 0.26 - 0.76, P=0.004) independent of age, driver mutation, and MPN diagnosis. Arterial ischemic events were more common in males (OR 1.86, 95% CI 1.14 - 3.02, P=0.013) independent of driver mutation, and initial MPN diagnosis. Male sex was a predictor of sMF transformation (HR 1.5, 95% CI 1.02 - 2.2, P=0.04) independent of age and phenotype at diagnosis, and driver mutation, and transformation to AML (HR 2.63, 95% CI 1.21 - 5.67, P=0.014) independent of age at diagnosis and driver mutation. These results were confirmed with KM analysis (P=0.013 and P=0.018 respectively) (Figures 1A - B). The neutrophil JAK2 V617F VAF or its change/year were not associated with survival in the entire cohort (HR 1, 95% CI 0.99 - 1.01, P=0.082 and HR 1.05, 95% CI 0.97 - 1.12, P=0.22 respectively). However, JAK2 V617F VAF or its change/year was associated with survival in females (HR 1.01, 95% CI 1 - 1.02, P=0.044 and HR 1.12, 95% CI for HR 1.01 - 1.25, P=0.04 respectively). KM analysis confirmed that yearly increases of JAK2 V617F VAF higher than 0.5/year were associated with worse survival only in females (P=0.013). Multivariable analysis showed that males had a higher number of additional somatic mutations (Coef 1.14, 95% CI 0.15 - 2.13, P=0.024) independent of age and diagnosis at the time of sequencing (Figures 1C). Analysis of individual mutations (Figure 1C) showed that males have higher prevalence of ASXL1 (22.9% vs 2.4%, P=0.01) and DNMT3A mutations (11.4% versus 7.3%), mutations associated with MDS/MPN phenotype (CBL, KRAS, EZH2, U2AF1), (22.9% vs 7.3%) and concurrent JAK2 and CALR mutations (11.4% vs 0%, P=0.04). CONCLUSIONS: Males with MPN have higher risk of sMF and AML transformation accounting for their worse survival. Males with MPN are less dependent on the JAK2 V617F VAF as an outcome determinant compared to females, but instead carry a higher risk non-driver mutational burden compared to females. Both quantity and quality of non-driver mutational burden differ between males and females with MPN, with males harboring higher risk burden that underlies higher risk presentation and outcomes. Figure 1. Males have higher frequency of somatic mutations additional to their MPN driver mutation independent of age and MPN diagnosis at the time of the NGS. A. KM analysis showing that males have shorter sMF-free survival (P=0.013). B. KM analysis showing that males have shorter AML-free survival (P=0.018). C. The percentage of males with 1 or more additional somatic mutations is higher compared to females across all MPN at the time of the NGS (ET, PV, PMF, sMF). D. The analysis of the specific additional somatic mutations in our cohort showed that males have higher frequency of high-risk CHIP-related mutations (ASXL1, DNMT3A), mutations in genes related to MDS/MPN phenotype and concurrent mutations in JAK2 and CALR. Disclosures Chaturvedi: Shire/Takeda: Research Funding; Alexion: Consultancy; Sanofi: Consultancy.

TET2 Mutations Are Frequent in RARS-T.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3794-3794
Author(s):  
Hadrian Szpurka ◽  
Anna M. Jankowska ◽  
Hideki Makishima ◽  
Nelli Bejanyan ◽  
Eric D. Hsi ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Significant Proportion ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Refractory Anemia ◽  
Common Mutation ◽  
Cell Counts ◽  
Mutational Status ◽  
Ring Sideroblasts

Abstract Abstract 3794 Poster Board III-730 Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Since JAK2 V617F and MPL W515L mutations are present in a significant proportion of RARS-T patients, many investigators consider this entity to be more closely related to classical MPN. However, a significant minority of patients with RARS-T do not display either JAK2 V617F or MPL W515L mutations. We have studied a cohort of patients with RARS-T (N=20) characterized by the presence of ring sideroblasts, reticulin fibrosis and thrombocytosis (>450×109/L), that lack obvious causes of secondary thrombocytosis. While 8/20 patients harbored the JAK2 V617F, and 3/20 the MPL W515L mutations, the molecular pathogenesis for the remaining 9 patients was unexplained. Activation of JAK2 and MPL is associated with aberrant phospho-STAT5. Cases positive only for phospho-STAT5 may harbor other related, so far unidentified mutations. Many groups have recently observed a frequent area of somatic uniparental disomy (UPD) at 4q24, most commonly encountered in patients with chronic myelomonocytic leukemia (CMML), MDS/MPN, some typical MDS, and secondary acute myeloid leukemia (sAML). Overlapping microdeletions and UPD on 4q24 pointed towards possible mutations in the TET2 gene; such mutations were subsequently found in myeloid malignancies, most significantly MPN and MDS/MPN. Based on these findings, and the established correlation of RARS-T with JAK2 V617F and MPL W515L mutations, we evaluated the mutational status of TET2 in RARS-T patients. SNP-A allowed detection of copy neutral loss of heterozygosity (CN-LOH), such as UPD9p, which is associated with the JAK2 V617F mutation, and UPD1p, associated with MPL W515L. SNP-A facilitated detection of previously cryptic lesions; 11/20 patients showed an abnormal SNP-A-based karyotype (only 3 of these defects were also detected by MC). The new lesions seen by SNP-A included various UPD, such as, 1p, 2p, 3q, 6p, 8p, 9p and 10p. The presence of UPD9p/1p was consistent with homozygous JAK2 V617F/MPL W515L mutations. Likely, duplication of mutated alleles constituted a further permissive event during clinical evolution. However, none of the patients showed a somatic LOH at 4q24, suggesting that biallelic TET2 mutations were not involved in the pathogenesis of RARS-T. Simultaneously, lack of UPD11q suggested that CBL mutations were absent. Indeed, Cbl ring finger domain mutational screening revealed no mutations. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10). However, 4 patients demonstrated abnormal megakaryocytic STAT5 phosphorylation, despite the absence of both JAK2 V617F and MPL W515L mutations. Within this group, a monoallelic TET2 mutation, delC 1480Sfs, was identified. In addition, we found a group of 5 patients without either JAK2 V617F or MPL W515L mutations, and also without association of the aberrant phospho-STAT5 staining. One of these patients had a monoallelic TET2 V1718L mutation; interestingly, another patient's specimen showed two novel non-synonymous SNPs: Y867H and P1723S. In total, 2/19 (11%) patients harbored TET2 mutations. These findings indicate involvement of TET2 mutations in RARS-T pathogenesis. RARS-T cases with MPN-associated mutations may not show obligatory phospho-STAT5 staining. The majority of patients were characterized by lack of splenomegaly, decreased white blood cell counts, increased thrombocytosis, and a normal karyotype. In summary, the majority of RARS-T patients harbor JAK2 V617F and MPL W515L mutations that strongly activate STAT5 phosphorylation. We described herein the third most common mutation in RARS-T, which can occur with or without abnormal STAT5 activation. Disclosures: No relevant conflicts of interest to declare.

Expression level and differential JAK2-V617F–binding of the adaptor protein Lnk regulates JAK2-mediated signals in myeloproliferative neoplasms

Blood ◽  
2010 ◽  
Vol 116 (26) ◽  
pp. 5961-5971 ◽  
Author(s):  
Fanny Baran-Marszak ◽  
Hajer Magdoud ◽  
Christophe Desterke ◽  
Anabell Alvarado ◽  
Claudine Roger ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Adaptor Protein ◽  
Jak2 V617f ◽  
Negative Control ◽  
Sh2 Domain ◽  
Expression Level ◽  
Activating Mutations ◽  
Thrombopoietin Receptor ◽  
Dependent Cell

Abstract Activating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F–dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F–binding regulate JAK2-mediated signals in MPNs.

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