Correlations Between TET2 Mutation and Clinicohematologic Parameters in Myeloproliferative Neoplasms

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1455-1455
Author(s):  
Jung Sook Ha ◽  
Jae Hee Lee ◽  
Sung Gyun Park ◽  
Nam Hee Ryoo ◽  
Dong Suk Jeon ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Direct Sequencing ◽  
Jak2 V617f ◽  
Putative Tumor Suppressor Gene ◽  
Allele Burden ◽  
Frame Shift ◽  
Mutational Status ◽  
Tet2 Mutation ◽  
Disease Specific

Abstract Abstract 1455 Background: Since the acquired somatic mutation, JAK2 V617F, was discovered as a first molecular marker of myeloproliferative neoplasms (MPN), and it has been detected variably in each MPN subtypes. However, JAK2 V617F does not found in all of MPN cases and not necessarily specific to a particular clinicpathologic entity. Recently, mutation of the putative tumor suppressor gene, Ten-Eleven-Translocation-2(TET2), has been identified in MPN patients. However, the frequency of TET2 mutation or its relationship with JAK2 V617F mutation or pathologic function in MPN has not been concluded, yet. The aim of our study was to evaluate the frequency of TET2 in MPN patients, and whether there is any correlation of TET2 mutation with JAK2V617F mutation or the clinicohematologic parameters. Materials and Methods: Total 99 adult MPN patients (18 PV, 62 ET, 11 PMF and 8 MPN unclassified) whose bone marrow cells had been stored from 2007 to 2010 at point of first diagnosis were included in this study. Hematological diagnoses and subtyping were reconfirmed according to the 2008 WHO classification and clinicohematologic datas were collected from patient records. Direct sequencing for TET2(exon3–11) and JAK2 (exons 12 and 14) were performed using an ABI 3730XL DNA analyzer. The JAK2V617F allele burdens were determined by pyrosequencing for samples available and MPL was analyzed by allele-specific PCR. Results: The overall TET2 mutational frequency was 12.1%, and disease-specific mutational frequencies were 22.2% in PV, 9.7% in ET and 18.2% in PMF. The found mutations included 11 mutations, 7 frame-shift (p.Lys95AsnfsX18, p.Gln967AsnfsX40, p.Lys1022GlufsX4, p.Asp1314MetfsX49, p.Gln1534AlafsX43, p.Tyr1618LeufsX4, p.Leu1609GlufsX45), 1 nonsense (p.Gly1735X), 1 missense (Q599R) and 2 splicing mutations (c.3409+1G>T, c.4044+2insT). Those mutations most frequently involved exon 3(four mutations) and exon 11(four mutaions), and rarely intron 3, intron 8 and exon 7. None of the mutations were associated with a karyotypically apparent 4q24 rearrangement. All patients were also screened for JAK2 V617F, and the overall JAK2 V617F positive rate was 68%(94.4% in PV, 69.4% in ET, 45.5% in PMF and 37.5% in MPN, unclassified). All TET2 mutations occurred in JAK2 V617F positive cases. JAK2 exon12 mutation was not found in all patients. MPL W515L was found in one ET patient who also carried JAK2V617F, but not TET2 mutation. Information on JAK2 V617F allele burden was available in 78 patients. Considering all 99 patients, the patient age, hematologic indexes (leukocyte count, neutrophil fraction, lymphocyte fraction, monocyte fraction, Hb, Hct and platelet count), the frequency of organomegaly, marrow fibrosis or thrombotic/hemorrhagic complications were not different according to carrying TET2 mutation. However, TET2 mutation was more frequently found in JAK2 V617F carriers than non-carriers (P=0.008), but JAK2 V617F allele burden did not correlated with the presence of mutant TET2. When analysis was performed for each PV, ET, and PMF (no TET2 mutation in MPN-unclassifiable patients), correlation between TET2 and JAK2 V617F mutational status was not found in each subtypes (P=0.078 in PV, P=0.099 in ET and P=0.182 in PMF). However, the JAK2 V617F allele burden was significantly higher in PMF harboring TET2 mutation than PMF patients did not (88.0 ± 4.3% vs 19.1 ± 28.7%, P=0.034). In statistical analysis for the correlations of clinicohematologic parameters with TET2 mutation in each PV, ET and PMF patients, only a few statistically significant results were identified. The presence of TET2 mutation was correlated with high Hct in PMF (47.4 ± 5.4 vs 25.5 ± 6.2, P=0.037), and TET2 positive ET patients showed relatively higher frequency of organomegaly compared to ET patients without TET2 mutation (50% vs 19.6%, P=0.018). Conclusions: The overall and disease-specific frequencies of TET2 mutation in our study are similar with previous studies, and frame-shift mutation is the most frequent mutation type. There is no specific relationship between JAK2 V617F and TET2 mutation occurrence, but TET2 mutant PMF has higher JAK2 V617F allele burden than non-mutant. TET2 mutation is also associated with a higher Hct in PMF and higher frequency of organomegaly in ET. Larger scale studies involving more MPN patients are needed. Disclosures: No relevant conflicts of interest to declare.

Clinical Phenotype of Myeloproliferative Neoplasms with Activating CBL-mutations Resembles Chronic Myelomonocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3895-3895
Author(s):  
Juliana Popa ◽  
Susanne Schnittger ◽  
Philipp Erben ◽  
Tamara Weiss ◽  
Ayalew Tefferi ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Clinical Phenotype ◽  
Myeloproliferative Neoplasms ◽  
Direct Sequencing ◽  
Jak2 V617f ◽  
Chronic Myelomonocytic Leukemia ◽  
A Genome ◽  
Length Mutations ◽  
Myelomonocytic Leukemia

Abstract Abstract 3895 Poster Board III-831 A genome-wide single nucleotide polymorphism (SNP) screen led to the identification of 11q aUPD in patients diagnosed with various subtypes of myeloproliferative neoplasms (MPN), e.g. chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML) and myelofibrosis (MF) (Grand et al., Blood 2009;113:6182). Further molecular analyses revealed acquired activating point and length mutations in CBL exons 8 and 9 in 10% of CMML, 8% of aCML and 6% of MF cases. Most variants were missense substitutions in the RING or linker domains that abrogated CBL ubiquitin ligase activity and conferred a proliferative advantage to 32D cells overexpressing FLT3. In this study, 160 patients with BCR-ABL and JAK2 V617F negative MPNs were screened for CBL mutations by PCR and direct sequencing. Eighteen known (Y371H, L380P [2x], C381R, C381Y [2x], C384Y, C396Y, H398P, H398Q, W408C, P417H, F418L, R420Q [5x]) and four new (F378L, G397V, I423N, V430M) missense mutations affecting fourteen residues were identified in 20 patients. Two patients harbored two different mutations. The clinical phenotype could be characterized more precisely in 17 patients. Median age was 68 years (range 59–85) with a slight female predominance (f, n=10; m, n=7). Striking hematological features were leukocytosis (14/17; 82%; median 29,000/μl, range 4,500-141,000) with continuously left-shifted granulopoiesis (blasts, promyelocytes, myelocytes, metamyelocytes) in 85% and elevated monocytes (median 2,500/μl, range 630-10,656) >1,000/μL in 88% (15/17) of patients. Eosinophilia (>1,500/μL) was rare (3/17, 18%). Anemia (normal values: f, Hb <12g/dL; m, Hb <14g/dL) was present in all 17 patients (f, median 10g/dL, range 8.7-11.8; m, median 11.2g/dL, range 8.6-12.9). Platelets did not exceed 300,000/μL in any patient while 11/17 (65%) patients presented with thrombocytopenia (median 125,000/μL, range 18,000-271,000). Splenomegaly was present in 11/17 patients (65%) and LDH was elevated (median 304U/L, range 189-729) in 9/17 patients (52%). Bone marrow histology and immunohistochemistry were available from 12 patients. Relevant features were hypercellularity, marked granulopoiesis and microlobulated megakaryocytes without clusters in 11/12 patients (92%), respectively. Increased fibres were seen in 8/12 (67%) patients of whom one showed severe fibrosis. Clinical follow-up was available from 17 patients. Thirteen patients (76%) have died because of progression to secondary acute myeloid leukemia/blast phase (n=7), cytopenia-related complications (n=2) or for unknown reasons (n=4) after a median of 23 months (range 3-60) following diagnosis. In conclusion, point mutations of CBL exons 8 and 9 are present in approximately 6-12% of BCR-ABL and JAK2 V617F negative MPNs. They are associated with a distinct clinical and hematological phenotype presenting with myeloproliferative features allowing diagnosis of a proliferative subtype of CMML rather than aCML or MF in the majority of cases. Patients with left-shifted leukocytosis, monocytosis, anemia and lack of thrombocytosis who are negative for BCR-ABL and point or length mutations of JAK2 should be routinely screened for CBL mutations. Disclosures: No relevant conflicts of interest to declare.

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.

The JAK2 46/1 Haplotype Analysis In Essential Thrombocythaemia and Polycythaemia Rubra Vera Reveals That CC Genotype Is Associated with a Higher JAK2V617F and c-MPL W515 Allele Burden

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1977-1977
Author(s):  
Shameem Mahmood ◽  
Louise Mellish ◽  
Nicholas Lea ◽  
Austin G Kulasekararaj ◽  
Atiyeh Abdallah ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Association Studies ◽  
Statistical Significance ◽  
Jak2 V617f ◽  
Tagging Snp ◽  
Jak2 Mutation ◽  
Allele Burden ◽  
Significant Difference ◽  
Jak2v617f Allele Burden

Abstract Abstract 1977 First 2 authors contributed equally. Background: Genomic-wide association studies have identified the germline 46/1 haplotype as a predisposing allele associated with JAK2V617F positive myeloproliferative neoplasms (MPN). The present study analysed data on 856 JAK2V617F positive patients, 326 of which had complete clinical data. Aims: To evaluate the JAK2 46/1 haplotype frequencies, JAK2V617F allele burden, c-MPL 515 mutation and risk of transformation. Methods: Genomic DNA from whole peripheral blood or bone marrow patient samples was analysed as follows: JAK2V617F allele burden by Q-PCR, JAK2 exon 12 mutations by Q-PCR and PCR fragment analysis, MPL W515 L and K mutations by allele specific PCR. The 46/1 JAK2 mutation susceptibility haplotype (46/1) tagging SNP rs12343867 (susceptibility allele C) were analysed by pyrosequencing. Results: The allele frequency for the 46/1 tag SNP rs1234867 in the 856 patients was calculated for the total JAK2V617F cohort (0.48) and the clinical entities ET (0.34) and PRV (0.44) confirming that the 46/1 haplotype is greatly over represented in JAK2V617F MPD patients as compared to published the control population (Wellcome Trust Case Control Consortium (WTCCC) (0.24). The Analysis of the 856 patients demonstrated that JAK2V617F and c-MPL W515L/K mutations co-existed in 16 patients(1.9%), the incidence of c-MPL W515L being twice as common as the c-MPL W515K mutations. There was no correlation between these mutations and age or 46/1 haplotype status. The JAK2V617F allele burden (AB) was lower in the c-MPL mutant patients, the average JAK2AB 31%. 3 out 4 c-MPL patients for which clinical information was available had a diagnosis of ET. No JAK2 exon 12 mutations were found in any of the 859 JAK2V617F positive samples suggesting that co-existing JAK2 exon 14 and exon 12 mutations are extremely rare. The genotypic data in ET patients showed: C/C 12%, C/T 44%, T/T 44% and their respective JAK2V617 allele burden (AB) were 46%, 32%, 29%. The genotype data in PRV patients: C/C 18%, C/T 53%, T/T 28.6% and their respective AB were 47%, 31% and 39%. The median AB was 32% (n=121) for ET and 37% (n=103) for PRV. Within a cohort of 255 patients (ET=138, PRV=117) 4% of ET and 6% of PRV patients transformed to acute myeloid leukaemia or myelofibrosis with no predominant haplotype association. In the ET patients, the median AB was 35%, there was no significant difference in the JAK2 V617F AB between those who transformed or not (p=0.45). Interestingly, on the whole ET group C/C genotype patients were more likely to have an allele burden >50% (p=0.058). In the PRV patients, the median AB was 48%. Again, the C/C genotype, PRV patients were more likely to have an AB>50% (p=0.06), although not reaching statistical significance. Conclusions: The 46/1 haplotype in both clinical entities ET and PRV demonstrated a higher allele burden in the C/C genotype in comparison to the other genotypes. No predominant haplotype predicted the risk of transformation to a more aggressive disease such as MF or AML. The analysis also showed that c-MPL W515K/L mutations can co-exist with JAK2V617F. The c-MPL W515K/L mutations did not exhibit a positive correlation with a preferential 46/1, but was associated with a lower allele burden. No co-existing exon 12 and exon 14 mutations were found, suggesting the rarity of this occurrence. Disclosures: No relevant conflicts of interest to declare.

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.

Clonal Evolution of Gene Mutations Involving DNA Methylation in the Progression of CMML to Secondary AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4120-4120
Author(s):  
Hsiao-Wen Kao ◽  
Ming-Chung Kuo ◽  
Po-Nan Wang ◽  
Jin-Hou Wu ◽  
Tung-Huei Lin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Detection Sensitivity ◽  
Additional Gene ◽  
Allele Burden ◽  
Tet2 Mutation ◽  
Idh1 And Idh2 Mutations

Abstract Background and Aim: The molecular pathogenesis of progression of chronic myelomonocytic leukemia (CMML) to secondary acute myeloid leukemia (sAML) remains incompletely understood. Genemutations involving DNA methylation in the transformation of CMML to sAML were investigated in matched paired CMML/sAML bone marrow samples to determine the roles of TET2, DNMT3A, IDH1 and IDH2 mutations in the evolution of CMML to sAML. Material and Methods: 106 CMML (63 CMML-1 and 43 CMML-2) patients were analyzed for TET2, DNMT3A, IDH1, and IDH2 mutations at the initial diagnosis. 33 patients had paired CMML/sAML bone marrow samples for comparative analyses. Mutational analysis of TET2 was performed by PCR followed by direct sequencing for PCR products amplified with primer pairs covering the whole coding sequences (exons 3-11). DNMT3A mutations (exons 2-23) were screened by denaturing high-performance liquid chromatography (DHPLC) with adding GC-clamps to the primers to facilitate mutation detection.Samples with abnormal DHPLC profile were then directly sequenced. The hot spots of IDH1 and IDH2 genes on exon 4 were PCR-amplified from gDNA and subjected for direct sequencing. Additional gene mutations were analyzed by PCR-based assays with direct sequencing. The allele burden of gene mutations was measured at both CMML and sAML phases by pyrosequencing with a detection sensitivity of 5%. Results: The frequencies of TET2, DNMT3A, IDH1 and IDH2 in 106 CMML patients were 39.8% (41/103), 8.5% (9/106), 0% (0/106), and 7.5% (8/106), respectively. These epigenetic gene mutations were mostly mutually exclusive. Of the 31 paired CMML/sAML samples examined for DNMT3A, 4 had DNMT3A mutations at diagnosis; the mutation status, patterns and allele burden remained unchanged at sAML phases. None of 33 patients acquired IDH1 mutation and one acquired IDH2 mutation during sAML progression. Of the 30 patients with paired samples analyzed for TET2 mutation, 12 patients had TET2 mutations at both CMML and sAML phases; 10 patients retained the same TET2 mutations with stable allele burden, one patient had clonal expansion of TET2 mutation, and the other patient acquired 3rdTET2 mutation at sAML progression along with expansion of a preexisted TET2 mutant clone and one stable TET2 mutant subclone. Another patient harboring TET2 mutation at CMML diagnosis lost themutation at sAML progression. Acquisition of additional gene mutations during sAML evolution was detected in 5 TET2-mutated patients, including RUNX1, CEBPA, FLT3- ITD, JAK2 V617F, NPM1, SRSF2, and CSF3R, either alone or in combination. Conclusions: Our results showed that TET2 and DNMT3A mutational status and allele burden remained unchanged during the progression of CMML to sAML except that rare patients might have expansion or emergence of TET2 subclone at sAML phase. Acquisition of additional gene mutations occurred in half of TET2-mutated patients during the progression of CMML to sAML. Grant support: NHRI- EX103-10003NI and MOHW103-TD-B-111-09 Disclosures No relevant conflicts of interest to declare.

scholarly journals The Frequency of Calr and MPL Gene Mutations in Jak2 V617F - Positive Chronic Myeloproliferative Neoplasms in Russia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5400-5400
Author(s):  
Tatiana V Makarik ◽  
Adhamjon O Abdullaev ◽  
Sergei M. Kulikov ◽  
Elena E Nikulina ◽  
Svetlana A Treglazova ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Cell Clone ◽  
Myeloproliferative Neoplasms ◽  
Gene Mutations ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Allele Burden ◽  
Chronic Myeloproliferative Neoplasms ◽  
V617f Mutation ◽  
Calr Mutations

Background. Ph-negative chronic myeloproliferative neoplasms (MPNs) are characterized by proliferation of one or more myeloid cell lineages and include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Somatic Jak2, MPL and CALR gene mutations are responsible for more than 90% of NPM cases. These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Aim. To evaluate frequencies of MPL and CALR mutations in Jak2 positive MPN cases for Russian cohort of patients. Methods. Archival DNA samples from MPN patients followed up at the National Research Center for Hematology between 2014 and 2019 included into retrospective study. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantified by real-time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity >= 3%). MPL W515L/K mutations were assessed by in-house allele specific PCR. All cases were tested for phi-negativity using BCR-ABl p210 PCR kit from Interlabservice (Russia). Results. At least one of the mutations was found in 3863 cases. Jak2 V617F mutation - 3385 cases (87.6%); CALR insertion or deletion - 471 case (12.2%); MPLW515L/K mutation - 31 case (0.8%). We have found 28 cases (0.7%) with Jak2 and CALR mutations combined and 3 cases (0.1%) with Jak2 and MPL mutations in the cohort studied. Matched measures were obtained at least twice at different time points during the course of disease for these cases. No cases with simultaneous CALR and MPL mutations were detected. In 23 from 31 (74%) cases with combined mutations Jak2 V617F allele burden was lower than 3%. Among cases with combined mutations 5 were diagnosed with PV, 8 - with ET, 8 - with PMF and 10 with unclassified MPN. No correlations between diagnosis, mutation combination or allele burden were found. Conclusions. Based on the data, obtained on retrospective DNA samples we cannot state whether combined mutations are present in different clones of myeloid cells or in one. Indirectly, the fact that more often mutations in CALR and MPL genes were found in the cases with a low Jak2 V617F allele burden may indicate that additional mutations occur in the "competing" cell clone. Further prospective studies with mutation monitoring over the therapy are required to assess the value of combined mutations for MPN pathogenesis. Disclosures No relevant conflicts of interest to declare.

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.

The JAK2 V617F mutational status and allele burden – authors’ reply

2015 ◽  
Vol 136 (3) ◽  
pp. 691-692 ◽  
Author(s):  
Martyna Borowczyk ◽  
Marzena Wojtaszewska ◽  
Krzysztof Lewandowski ◽  
Lidia Gil ◽  
Maria Lewandowska ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Allele Burden ◽  
Mutational Status
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