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

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.

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Analysis of JAK2 V617F Mutation and Its Clinical Significance in Patients with Thrombocythemia and Other Myeloproliferative Neoplasms in Chinese

Blood ◽

10.1182/blood.v118.21.4687.4687 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4687-4687

Author(s):

Yue Xu ◽

Changxin Yin ◽

Han He ◽

Lingling Shu ◽

Fuqun Wu ◽

...

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Jak2 Mutation ◽

Western Countries ◽

Arms Pcr ◽

V617f Mutation

Abstract Abstract 4687 JAK2 mutation is commonly found in Philadelphia-negative myeloproliferative neoplasms (MPNs). In Western countries, this mutation is found in approximately 96 percent of people with polycythemia vera, half of individuals with essential thrombocythemia or primary myelofibrosis. We used the method of amplification refractory mutation PCR (ARMS-PCR) to investigate MPN patients in China. We focused our study on patients with essential thrombocythemia (ET). ARMS-PCR was used to detect JAK2 V617F mutation in the bone barrow (BM) or peripheral blood of 37 MPN patients, which consisting of 7 ET, 5 polycythemia vera (PV), 5 chronic myeloid leukemia (CML), 5 chronic idiopathic myelofibrosis (CIMF), as well as 15 suspected MPNs. 17 cases of JAK2 V617F mutation (45.9%) were found in 37 patients, including 4 ET (57.1%), 4 PV (80.0%), 3 CIMF (60.0%), 6 suspected MPNs (40.0%). We did not find JAK2 V617F in the patients with CML. Our results indicated that the frequency of JAK2 V617F mutation in bcr/abl-negative MPNs in Chinese is similar to that in MPN patients in Western countries. At the same time, ARMS-PCR can distinguish the mutation is heterozygous or homozygous. Most patients were heterozygous for JAK2 but only a few were homozygous. In conclusion, our study showed that JAK2 V617F mutation frequency in Chinese MPN patients is similar to that in patients with this disorder in the West. It is the major molecular genetic abnormality in bcr-abl negative MPN and it can be used for diagnosis of MPN in China. Disclosures: No relevant conflicts of interest to declare.

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Clinical Implications for Patients with Low JAK2V617F Allele Burden

Blood ◽

10.1182/blood.v120.21.1745.1745 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1745-1745

Author(s):

Marguerite Vignon ◽

Dorota Jeziorowska ◽

Pierre Hirsch ◽

Ollivier Legrand ◽

Nicole Casadevall ◽

...

Keyword(s):

Bone Marrow ◽

Clinical Phenotype ◽

Myeloproliferative Neoplasms ◽

Kinase Domain ◽

Jak2 Mutation ◽

Allele Burden ◽

Vein Thrombosis ◽

Myeloid Neoplasms ◽

Exon 10 ◽

Jak2v617f Allele Burden

Abstract Abstract 1745 In classical Philadelphia-negative myeloproliferative neoplasms (MPN), JAK2V617F is considered as a driver mutation when the threshold of 1% JAK2V617F/JAK2total allele burden is reached. However a lower ratio is sometimes detected with highly sensitive assays. We investigated the clinical significance of such minor clones by describing the characteristics of 27 patients with a low JAK2V617F allele burden ranging from 0.1% to 0.99%. Material and Methods A commercially available quantitative ASO-PCR assay of 0.1% sensitivity (MutaQuant® kit, Ipsogen) was used. Two thousand five hundred consecutive blood samples were sent to our lab for JAK2V617F mutation between 2009 and 2012. Total blood DNA was extracted by an automated standardized procedure (Qiasymphony®, Qiagen). All samples were tested in duplicate. The 27 samples of our cohort were controlled using a second assay of 0.01% sensitivity (Larsen et al, BJH 2007). Thirty samples from healthy donors were also tested. High resolution melting curve (HRM) analysis of JAK2 exon 14 ruled out the possibility of an additional mutation hampering the annealing of a primer. Patients with a known classical MPN clinical phenotype were also tested for JAK2 exons 12–17 (entire pseudo-kinase domain) or for MPL exon 10 depending on the context. Results Laboratory Findings Among the 2500 samples, 735 (29.4%) were positive above 1%, 27 (1.1%) had low JAK2V617F allele burden ranging from 0.12 to 0.99%. The patient with the lowest ratio (0.12%) was not confirmed by the second assay and therefore was excluded from the study. This allowed the median to settle at 0.40%. No associated mutations were found in the JAK2 pseudo-kinase domain in patients with polycythemia vera (PV) and in MPL exon 10 in patients with essential thrombocytosis (ET) and primary myelofibrosis (PMF). Healthy patients were all tested JAK2V617F negative. Clinical Aspects The cohort included 19 men and 7 women ranging from 28 to 95 years of age (median 63 years old). Two patients had secondary acute myeloid leukaemia following JAK2V617F positive MPN indicating the presence of residual JAK2V617F cells and the negativity of the myeloblastic population. Thirteen patients (50%) had a classical MPN with a median ratio of 0.36%: 7 ET, 5 PV and 1 PMF according to WHO 2008 criteria. However a bone marrow biopsy was available for only two patients (1 ET, 1 PMF). None of them had received pegylated interferon alpha-2a. Four patients had a prior history of thrombosis: two strokes, one pulmonary embolism, two portal vein thrombosis (PVT). For one PV patient, a 6 months follow-up blood and bone marrow sample confirmed a low allele burden in the same range (0.4%) and in vitro Epo-independant erythroid colonies were observed. Five patients had other chronic myeloid neoplasms (two myelodysplastic/myeloproliferative neoplasms, one chronic eosinophilic leukaemia, one chronic myeloid leukaemia, one refractory anaemia with ring sideroblasts). Among these five, four had an abnormal karyotype. We did not observe any thrombotic event in these patients. We cannot conclude on hematological diagnosis for the last six patients: four patients were screened for JAK2 mutation because of PVT. One patient had chronic polycythemia in a context of alcohol and tobacco abuse. One patient had homozygous hemochromatosis with a normal haemoglobin level in spite of repeated phlebotomies. Discussion In this single centre study low JAK2V617F allele burden represented 1% of all samples sent for JAK2V617F study and 3.5% of JAK2V617F positive patients. Seventeen patients (65%) had classical MPN or splanchnic vein thrombosis. To our knowledge PV patients with such low JAK2V617F allele burden have not been reported in the absence of associated JAK2 pseudo-kinase domain mutation. A larger screen for cooperating mutations responsible for the PV phenotype is under process. In the context of other chronic myeloid neoplasms, the JAK2V617F mutation is thought to belong to a more complex clonal architecture mostly implicating chromatin remodeling genes. Here, the presence of a JAK2 mutation could argue in favour of clonal haematopoiesis. In conclusion the clinical phenotype of low JAK2V617F patients overlaps with classical JAK2V617F MPN. The technical implications might be challenging for molecular diagnostic platforms. More data are needed to further characterize these patients. Disclosures: No relevant conflicts of interest to declare.

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Correlations Between TET2 Mutation and Clinicohematologic Parameters in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v120.21.1455.1455 ◽

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.

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The Role Of Deregulated HMGA2 Expression With Promoter Methylation Of p16 In Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v122.21.1606.1606 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1606-1606

Author(s):

Kayo Shirado Harada ◽

Kazuhiko Ikeda ◽

Kazuei Ogawa ◽

Hideyoshi Noji ◽

Hideo Kimura ◽

...

Keyword(s):

Promoter Methylation ◽

Blood Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Expression Levels ◽

Allele Burden ◽

Polycomb Group Genes ◽

Micro Rnas ◽

Jak2v617f Allele Burden

Abstract Myeloproliferative Neoplasms (MPNs) are characterized by clonal proliferative hematopoiesis with increased mature blood cells. The signal-activating mutations such as JAK2V617F increase blood cells, but it remains uncertain how an abnormal hematopoietic cell clone expands in MPNs. We have recently showed that overexpression of the high mobility group AT-hook 2 (HMGA2) causes proliferative hematopoiesis with providing a clonal growth advantage to hematopoietic cells in mice (Ikeda et al, Blood, 2011), suggesting the possibility that HMGA2 contributes to the pathogenesis of MPNs. However, since only a few studies have evaluated expression of HMGA2 mRNA in patients with MPNs, the role of HMGA2 in the pathogenesis of MPNs is yet unclear. MPNs also show mutations in epigenetic modifiers involving DNA methylation such as polycomb group genes (PcG) and aberrant expressions of micro RNAs (miRNA) that negatively regulate expressions of targeted genes. Interestingly, deficiency in either PcG-related BMI1 (Oguro et al, J Exp Med, 2012) or let-7-family miRNA (Mayr et al, Science, 2007) causes deregulation of HMGA2 expression, leading to its oncogenic activity in part by negatively regulating tumor suppressor p16. Thus, in this study, to clarify the role of HMGA2 in MPNs, we investigated expression of HMGA2 mRNA in peripheral granulocytes of 56 patients with MPNs including 23 polycythemia vera (PV), 26 essential thrombocythemia (ET) and 7 primary myelofibrosis (PMF) along with clinical findings, JAK2V617F allele burden, expressions of BMI1 mRNA and let-7-family miRNAs, and promoter methylation of p16. Quantitative RT-PCR (qPCR) showed significantly higher expression of HMGA2 mRNA relative to internal control HPRT1 mRNA in PMF (mean ± SD; 31.7 ± 42.8, p<0.01), but not PV (15.7 ± 53.2) or ET (2.14 ± 7.70), compared with 12 healthy volunteers (HV; 0.431 ± 0.366). In addition, deregulated HMGA2 expression (>1.2), which was determined as relative expression level above mean + 2SD of HMGA2 mRNA in 12 HV, was most frequently detected in patients with PMF [7/7 (100%)] (p<0.01), compared with PV [5/23 (21.7%)] and ET [6/26 (23.1%)]. We also found a significant positive correlation in expression levels of HMGA2 mRNA with serum LDH values (r=0.531, p<0.01) rather than JAK2V617F allele burden (r=0.25, p=0.08). These data suggested that expression of HMGA2 mRNA independently correlated with disease phenotype and status in MPNs. We next explored the cause of deregulated expression of HMGA2 mRNA and found lower expression of let-7a (0.19 ± 0.13 vs. 0.42 ± 0.39, p=0.04) and -7c (0.57 ± 0.60 vs. 1.14 ± 0.94, p=0.06) rather than -7b (p=0.2) by qPCR, in patients with deregulated expression of HMGA2 mRNA compared with other patients. However, HMGA2-involved chromosomal abnormality in 12q13-15 was not detected in any patient, and there was no difference in expression of BMI1 mRNA between patients with deregulated expression of HMGA2 mRNA and other patients. Thus, decreased expression of let-7 miRNAs might contribute to deregulated expression of HMGA2 mRNA in MPNs. Finally, we investigated correlation of deregulated expression of HMGA2 mRNA with promoter methylation of p16. Methylation-specific PCR assay detected promoter methylation of p16 in 17/56 (30.4%) patients with MPNs. Strikingly, patients with deregulated expression of HMGA2 mRNA significantly more often showed promoter methylation of p16 compared with other patients [10/18 (55.6%) vs. 7/38 (18.4%), p<0.01]. Furthermore, patients with promoter methylation of p16 showed higher expression levels of HMGA2 mRNA than patients without the methylation, especially in patients with PMF (2.33 ± 0.90 vs. 70.9 ± 38.3, p=0.01). In conclusion, deregulated expression of HMGA2 in association with decreased expression of let-7 miRNAs may play a crucial role in the pathogenesis of MPNs possibly through p16. Disclosures: No relevant conflicts of interest to declare.

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Study of the Relation Between the Time Course of the JAK2V617F Allele Burden and the Clinical Evolution in Patients with Myeloproliferative Neoplasms (MPN); A Single Center Study

Blood ◽

10.1182/blood.v112.11.5250.5250 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5250-5250

Author(s):

Marta Anna Sobas ◽

Manuel Pérez-Encinas ◽

Celsa Quinteiro ◽

Teresa González ◽

Sandra Suaréz ◽

...

Keyword(s):

Jak2 V617f ◽

Initial Increase ◽

Jak2 Mutation ◽

Allele Burden ◽

Clinical Evolution ◽

Clinical Control ◽

Cytoreductive Treatment ◽

The Mean ◽

Jak2v617f Allele Burden

Abstract Introduction: The JAK2V617F mutation is frequent in MPN, however its clinical implication is still in debate. There are few publications that analyze changes in JAK2V617F allele burden. Methods: We performed a single centre study on 65 patients (19 polycythemia vera (PV), 42 essential thrombocythemia (ET) and 4 primary myelofibrosis (PMF)) all of them with at least two JAK2V617F determination separated by minimum 12 months. The JAK2 mutation was determinated in DNA from peripheral blood granulocytes (in 3 cases from bone marrow smear) by MutaScreenTMKit (IPSOGEN). Results: The mean follow-up period was 26 months (range 12–176). In 42/65 (64.6%) patients JAK2 mutation was positive and in 24/42 (57%) changes in JAK2V617F allele burden (>10%) were observed. Interestingly, in 7/18 (39%) patients who showed an increase of JAK2V617F allele burden progression was observed. In this group, one case of PMF with initial increase of JAK2V617F allele burden during transformation to acute myeloid leukemia (AML) and posterior decrease after transformation* and one case of ET with an increase during transformation to myelofibrosis (MF) and posterior conversion to JAK2V617F negativity after allo-HSCT** were observed (table1). 36/42 (85.7%) of JAK2V617F positive patients were under cytoreductive treatment: 30 with hydroxyurea (HDU), 1 anagrelide (ANA) and 5 mixed. There was no decrease of JAK2-V617F allele burden in patients without cytoreductive treatment. Data of patients with HDU treatment are presented in table 2. 3/5 of patients with mixed cytoreduction showed an increase of JAK2V617F allele burden: all of them presented bad clinical control but no data of progression were observed. None of JAK2V617F negative patients (n=23: 1 PV, 2 PMF, 20 ET) became positive for the mutation with the mean follow-up period of 19.37 months. There were no cases of transformation in this group of patients. Conclusions: According to our study, JAK2V617F allele burden can change during the follow-up. Increase (or stability) of JAK2V617F allele burden are more frequent in transformation from ET to PV or MF; in cases of progression to AML we can observe decrease, stability or initial increase with posterior decrease of JAK2V617F allele burden. Decrease of JAK2 allele burden can be related to cytoreductive treatment. Increase of JAK2V61F in patients with cytoreductive treatment can be related to transformation or bad clinical control. More prospective studies are needed to conclude whether changes in JAK2-V617F allele burden could be useful to predict transformation of MPN patients. Table1. Changes of JAK2V617F allele burden and clinical evolution in JAK2V617F positive patients. JAK2 Clinical evolution (N=42) STABILITY PROGRESSION DECREASE 6/42 (14%) 5 1 [1 PV-AML] STABILITY 18/42 (33%) 15 3 [1 PV-MF; 1 ET-MF; 1 ET-AML] INCREASE 18/42 (43%) 11 7 [3 ET-PV; 3 ET-MF (one case with posterior allo-HSCT**); 1 PMF-AML*] Table2. Changes of JAK2V617F allele burden in JAK2V617F positive patients with HDU and no cytoreductive treatment. Treatment JAK2V617F: changes of allele burden DECREASE STABILITY INCREASE Untreated - 4 2 [ET-PV] HDU newly started 2 5 [PV-MF and ET-AML after 14 and 7 years of treatment respectively] 7 [2 ET-PV; PMF-AML*] HDU already treated 3 [PV-AML] 7 6 [3 ET-MF (one case with posterior allo-HSCT**)]

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JAK2 V617F Mutational Burden and Clinical Findings Relationship in Myeloproliferative Neoplasms

10.21203/rs.3.rs-305849/v1 ◽

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.

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

Blood ◽

10.1182/blood.v116.21.625.625 ◽

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.

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Clinical and Biological Characteristics According to the Burden of JAK2V617F Mutated Allele in BCR-ABL Negative MPNs

Blood ◽

10.1182/blood.v120.21.1437.1437 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1437-1437

Author(s):

Lurdes Zamora ◽

Marta Cabezon ◽

Olga Garcia ◽

Esther Alonso ◽

Silvia Marce ◽

...

Keyword(s):

Clinical Data ◽

Conflicts Of Interest ◽

Leukocyte Count ◽

Myeloproliferative Neoplasms ◽

Hemoglobin Level ◽

Jak2v617f Mutation ◽

P Value ◽

Continuous Variables ◽

Jak2 Mutation ◽

Allele Burden

Abstract Abstract 1437 JAK2 mutation testing and karyotye are routinely used for diagnosis of myeloproliferative neoplasms (MPNs) but they have not been incorporated into risk stratification. Although JAK2V617F mutation in MPNs has been one of the most seminal medical discoveries in recent years, it is not clear the importance of the amount of JAK2V617F allele. Some studies correlate the JAK2 allele burden with a higher hemoglobin level, leukocyte count, splenomegaly and thrombosis and more probability of transformation to MF or AML. The aim of this study was to determine whether cytogenetic data, JAK2 mutation status and allele burden correlates with cytological subtypes, clinical complications or provide prognostic information. Methods A retrospective study was conducted with samples centralized in a unique laboratory since 2005. A total of 526 patients were included (median age 63; 243 males) with classic MPNs (348 ET, 135 PV, 43 PMF) fulfilling 2008 WHO criteria and in accordance with the Declaration of Helsinki. Conventional cytogenetic was performed in bone marrow samples obtained at diagnosis (n=205) and at progression to MF or AML (n=46). DNA was extracted from peripheral blood using QIAamp DNA mini kit (Qiagen). All samples were coded and assayed blindly in duplicate to detect JAK2V617F mutation with an allele-specific PCR using TaqMan allelic discrimination, with 2 specific probes to measure the respective fluorescence of each allele. Then, JAK2 MutaQuant assay (Ipsogen, Luminy Biotech) was used to detect the JAK2V617F quantity by real-time PCR, detecting fluorescent signals using double-dye hyrolysis oligonucleotide probes with calibration standards at 4 different concentrations. Homozygous (HOZ) ratio was considered when percentage was higher than 50. Laboratory (hemoglobin, WBC and platelet counts) and clinical data (constitutional symptoms, splenomegaly, complications, OS and DFS) were collected. For continuous variables parametric and non parametric statistics were used. Survival analysis was performed using Kaplan-Meier estimate and log-rank tests were used for comparisons. The χ2 and Fisher's exact tests were used to analyze differences in the distribution of variables among patient subsets. p-value less than 0.05 were considered statistically significant. Results Aberrant karyotypes were seen in 15/205 (7%) cases at diagnosis (4% in ET and PV and 40% in PMF). At progression to MF or AML we have cytogenetic studies in 22 patients, and 10 (45%) harbor alterations. A total of 283 patients (64%) were JAK2V617F, 61% ET (4% HOZ), 75% PV (28% HOZ) and 55% PMF (16% HOZ). The median value of JAK2V617F was 26% (range, 1–99.9%). No correlation was seen between JAK2 and karyotype at diagnosis, but 7/9 patients with aberrant karyotype at progression had JAK2V617F mutation. JAK2 correlations with laboratory and clinical data are summarized in Table 1 and 2. Conclusions JAK2V617F is associated with a more pronounced myeloproliferative phenotype (higher hemoglobin level, platelet or leukocyte count). Patients with JAK2V617F HOZ have a higher probability of splenomegaly. Hematological complications do not depend on JAK2 ratio but mutated patients have more probability of thrombosis or hemorrhage. OS and PFS do not depend on JAK2 status, but patients with JAK2V617F heterozygous seem to have a slightly better outcome. Disclosures: No relevant conflicts of interest to declare.

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Evaluation of the JAK2V617F mutational burden in patients with philadelphia chromosome negative myeloproliferative neoplasms: A single-center experience

Balkan Journal of Medical Genetics ◽

10.2478/bjmg-2019-0021 ◽

2019 ◽

Vol 22 (2) ◽

pp. 31-36

Author(s):

M Popova-Labachevska ◽

I Panovska-Stavridis ◽

A Eftimov ◽

Nestorovska A Kapedanovska ◽

L Cevreska ◽

...

Keyword(s):

Real Time ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Treatment Decision ◽

Published Data ◽

Single Mutation ◽

Mutational Load ◽

Jak2 Mutation ◽

Allele Burden ◽

Jak2v617f Allele Burden

AbstractThe identification of the JAK2V617F mutation in several distinct myeloproliferative neoplasms (MPNs) raised the question how one single mutation incites expression of at least three different clinical phenotypes, i.e., polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In order to further evaluate already published data on the correlation between mutant JAK2V617F allele burden and specific hematological and clinical parameters, we tested the level of the JAK2 mutation in 134 JAK2+ patients with different MPNs. The patients were diagnosed according to the 2008 WHO criteria and followed for a median of 48 months. The JAK2 V617F quantification was done with a real time polymerase chain reaction (real time-PCR) method. The median allele burden was lowest in ET (25.8%), followed by 34.6% in PV and 51.8% in PMF patients (p<0.01). There was statistically significant association between the mutational load of 10.0-50.0% and blood count parameters in the PV patients (p<0.05). In PMF patients the mutational load was in correlation with older age and leukocyte count that were higher in patients with the mutational load of 10.0-50.0% and >50.0% compared to those with a mutational load of <10.0%. There were no statistically significant associations between the allele burden and blood counts in the ET cohort. Our study confirmed an association between the JAK2V617F allele burden and the distinct MPN phenotypes, indicating unfavorable prognosis in patients with a higher JAK2 allele burden. Our results suggest that JAK2 quantification should be incorporated in the diagnostic work-up of MPN patients as a useful tool for optimal treatment decision.

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

Blood ◽

10.1182/blood-2019-124767 ◽

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.

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