Use Of 46/1 Haplotype Permits To Follow JAK2V617F Clonal Architecture In PV Patients: Clonal Evolution and Impact Of IFNα Treatment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4109-4109
Author(s):  
Salma Hasan ◽  
Bruno Cassinat ◽  
Jean-Pierre Le Couédic ◽  
Fabrizia Favale ◽  
Barbara Monte-Mor ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Myeloproliferative Neoplasms ◽  
Clonal Evolution ◽  
Allele Burden ◽  
Clonal Architecture ◽  
Specific Pcr ◽  
Oncogenic Mutation ◽  
Allele Specific ◽  
Jak2v617f Allele Burden

Abstract Somatic V617F oncogenic mutation of the protein kinase JAK2 is the most prevalent genetic abnormality in the three myeloproliferative neoplasms (MPNs), namely polycythemia vera (PV, 95%), essential thrombocythemia (ET, 55%) and myelofibrosis (MF, 50%). About 30% of PV patients are homozygous for this mutation due to mitotic homologous recombination (HR). JAK2 46/1 haplotype is strongly associated with the cis-aquisition of JAK2V617F mutation. Since, HR involves most of 46/1 haplotype, JAK2V617F and 46/1 tagging SNPs are also reduced to homozygosity. We hypothesized that 46/1 tagging SNPs, which are in complete linkage disequilibrium with JAK2, can serve as a measure of JAK2V617F homozygosity. 46/1 allele burden (X%) can be used to calculate the HR (HR%) that is a measure of JAK2V617F/V617F clones [(X%-50%)x2]. JAK2V617F/V617F frequency and JAK2V617F allele burden (Y%) can be then exploited to calculate the frequency of JAK2V617F/WT [(Y%-RH%)x2] and JAK2WT [100-(JAK2V617F/WT%+ JAK2V617F/V617F)] clones. The purpose of this study was to calculate the clonal frequency of WT, JAK2V617F/WT and JAK2V617F/V617F in progenitors compartments of PV patients. Here, we have dissected the JAK2V617F clonal architecture in 9 PV patients heterozygous for the 46/1 haplotype. First, we measured the global JAK2V617F and 46/1 allele burden in CD34+ cells either by allele-specific PCR or by Ion Torrent sequencing in order to calculate the expected WT, JAK2V617F/WT and JAK2V617F/V617F clones. Next, we compared thees results with the experimental clonal frequency of WT, JAK2V617F/WT and JAK2V617F/V617F clones in individual colonies derived from the CD34+CD38+ compartment. This algorithm was validated in majority of patients. Moreover, we have exploited this formula to the terminally differentiated polynuclear neutrophils (PNN) and found that JAK2V617F/V617Fclones acquire strong amplification during differentiation. Finally, we used this model to assess the therapeutic potential of IFNα in a cohort of 15 PV patients. IFNα exposure resulted in more impressive and rapid decrease of JAK2V617F allele burden of pure JAK2V617F homozygous patients than in pure JAK2V617F heterozygous patients. Calculations revealed this decrease in JAK2V617F is due to preferential targeting of JAK2V617F/V617Fclones in responding patients. These results demonstrate that JAK2 46/1 haplotype can be used to estimate JAK2V617F clonal architecture in PV patients. This simple modeling can be useful to follow the efficacy and specificity of treatment on JAK2V617F clones in MPNs, without needing exploration at the unicellular level. In addition, it suggests that IFNα treatment more specifically targets the JAK2V617F/V617F clone in responding patients. Disclosures: No relevant conflicts of interest to declare.

46/1 Haplotype Permits to Follow JAK2 Homologous Recombination: Modeling JAK2V617F clonal Architecture in PV Patients

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1757-1757
Author(s):  
Salma Hasan ◽  
Jean Pierre Le Couedic ◽  
Fabrizia Favale ◽  
Barbara Monte-Mor ◽  
Catherine Lacout ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Clonal Structure ◽  
Hematopoietic Stem ◽  
Clonal Architecture ◽  
Specific Pcr ◽  
Allele Specific ◽  
Proliferative Advantage

Abstract Abstract 1757 Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) disorders characterized by excess proliferation of one or several myeloid lineages. More than 95% polycythemia vera (PV) and 50–60% essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients harbor a somatic 1849 G>T mutation in JAK2 gene. Moreover about 30% of PV patients are homozygous for this mutation due to a loss of heterozygosity after a mitotic homologous recombination (HR). Among 92 haplotypes of chromosome 9p, 46/1 haplotype is strongly associated with the cis-aquisition of JAK2V617F mutation. The purpose of this study was to estimate the clonal frequency of WT, JAK2V617F/+ and JAK2V617F/V617F in progenitors compartments. Here, we have modeled the JAK2V617F clonal architecture in 9 PV patients heterozygous for the 46/1 haplotype by using the level of JAK2 and the 46/1 haplotype as a marker to follow HR. First we measured the global JAK2V617F and 46/1 allele burden in CD34+ cells either by allele-specific PCR or by Ion Torrent sequencing in order to calculate the expected WT, JAK2V617F/+ and JAK2V617F/V617F clones. Next, we compared the results with the experimental clonal frequency of WT, JAK2V617F/+ and JAK2V617F/V617F cells in individual colonies derived from the CD34+CD38+ compartment. In majority of patients, the observed values corresponded to the expected values suggesting that JAK2 46/1 haplotype can be used to estimate JAK2V617F clonal structure in PV patients. In three JAK2 46/1 heterozygous hemochromatosis patients used as controls, no JAK2 46/1 homozygous clone was observed showing that 46/1 haplotype itself was not responsible for HR. Furthermore, we have studied the proliferative advantage of the mutated clones in patients. No proliferative advantage of JAK2V617F clone has been observed in between CD34+CD38− and CD34+CD38+ progenitors stages whereas strong amplification of JAK2V617F clone was found in terminally differentiated polynuclear neutrophils (PNN). Moreover, during evolution of MPN in one patient, we observed an amplification of the JAK2V617F/V617F clone in both the CD34+CD38− and CD34+CD38+cell compartments suggesting acquisition of a proliferative advantage of the homozygous clone over time. This simple modeling could help to understand the effect of treatments on the JAK2V617F clonal structure without working at the unicellular level. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

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.

Milestones of Post Transplant Monitoring of JAK2 p.V617F Positive MPN Patients.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3462-3462
Author(s):  
Thoralf Lange ◽  
Anja Edelmann ◽  
Uwe Siebolts ◽  
Claudia Nehring ◽  
Nadja Jäckel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Molecular Monitoring ◽  
Assay Sensitivity ◽  
Bone Marrow Biopsies ◽  
Allele Burden ◽  
Allele Specific

Abstract Abstract 3462 Detection of JAK2 p.V617F point mutation plays an important role in the initial diagnosis of myeloproliferative neoplasms (MPN) and also in the monitoring of JAK2 p.V617F positive MPN following allogeneic stem cell transplantation (SCT). Therefore it is of great interest to optimize molecular monitoring techniques and to define milestones for clinical decisions after SCT. We analyzed 146 probes of 14 patients with a median age of 61 (range 52–70) and with JAK2 p.V617F positive MPN prior and post SCT after reduced (n=10) or myeloablative conditioning (n=4) using related (n=6) or unrelated (n=8) donors. DNA from 70 peripheral blood (PB) samples and 76 bone marrow biopsies (BMB) were analyzed both by a quantitative allele-specific oligonucleotides (ASO) PCR assay (sensitivity 0.01% JAK2 p.V617F mutated alleles per HCK) and a allele specific quantitative real-time (Q) PCR (sensitivity 1%) followed by a JAK2 p. V617F wild type blocking (WTB) PCR in case of a negative result (sensitivity 0.001%). 144 samples has been analyzed with both assays and showed 100% and 89% concordance in samples containing ≥1% and <1% JAK2 p. V617F, respectively. In order to select the optimal compartment, 26 BMB and simultaneously drawn PB samples showed 77%, 92% and 100% concordance for negative or positive results by ASO, (WTB) Q-PCR or both methods. A total of 146 samples from 14 patients, among them 40 samples prior allo-SCT, were analyzed for molecular follow-up. A cut-off of >1% (ASO) and/ or a JAK2 p.V617F positive result in the QPCR was seen in 7 patients. Six (86%) died due to relapse and disease persistence (n=5) or TRM with significant persistence of JAK2 p.V617F. In contrast, all patients with engraftment post allo-SCT exhibited episodes of molecular negativity if the JAK2 p.V617F allele burden was < 1% one month after SCT. We conclude that (1) adequate monitoring post allo-SCT requires a sensitivity of at least 0.01%, (2) monitoring can be performed on full blood samples as peripheral blood and bone marrow biopsies showed identical results, (3) a cut-off of > 1% JAK2 p.V617F allele burden one month post allo-SCT indicates an adverse course of the disease Disclosures: No relevant conflicts of interest to declare.

Influence of JAK2 46/1 haplotype in the natural evolution of JAK2V617F allele burden in patients with myeloproliferative neoplasms

2012 ◽  
Vol 36 (3) ◽  
pp. 324-326 ◽  
Author(s):  
Alberto Alvarez-Larrán ◽  
Anna Angona ◽  
Luz Martínez-Avilés ◽  
Beatriz Bellosillo ◽  
Carlos Besses
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Natural Evolution ◽  
Allele Burden ◽  
Jak2v617f Allele Burden

Detection of Imatinib-Resistant BCR-ABL Mutants in Drug-Naïve Patients: Correlation with Disease Phase and Clonal Evolution but Not with Response to Treatment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 273-273
Author(s):  
Michael W. Deininger ◽  
Stephanie Willis ◽  
Thoralf Lange ◽  
Shannon McWeeney ◽  
Sandra Otto ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Clonal Evolution ◽  
Drug Binding ◽  
Disease Stage ◽  
Wild Type ◽  
Imatinib Resistance ◽  
Specific Pcr ◽  
Blastic Phase ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Background: Point mutations in the kinase domain (KD) of BCR-ABL that impair drug binding are a common cause of imatinib resistance. In some cases the mutations present at relapse were detected in pretherapeutic samples, suggesting selection in the presence of imatinib and a possible association with disease progression. To test this hypothesis, we studied the presence of KD mutations in imatinib-naïve patients in various disease phases. Methods and patients: we developed fluorescent allele-specific RT-PCR assays for 8 common KD mutations (Q252H, Y253F, Y253H, E255K, E255V, T315I, M351T, F359V), comprising approximately 75% of mutations detected in patients, using universal BCR and mutation-specific ABL primers. Mutation-complementary oligonucleotides were further modified to favor amplification of mutant over wild type. Sensitivity and specificity were optimized in serial dilutions of mutant in wild type, using plasmid DNA or cDNA extracted from BaF3 cells expressing BCR-ABL constructs. The assays reproducibly detected mutations with a sensitivity of at least 10−4 – 10−5. To avoid false-positive results cDNA was prepared in a physically separate lab that never handled recombinant mutant BCR-ABL. Negative controls included “blanks”, K562 cells (positive for wild type BCR-ABL) and HL60 cells (BCR-ABL-negative). Pretherapeutic samples from 20 patients in first chronic, 24 in accelerated and 21 in blastic phase were studied. Results: Mutations were detected in 13 samples from blastic phase (5) or from accelerated phase (8) but in none from chronic phase. Mutations were: T315I (7), Y253F (2), 253H (2), Q252H (1) and F359V (1). Sequencing of the same material detected F359V in one sample (consistent with the allele specific PCR), and K247R in another (positive for T315I by allele-specific PCR) but was wild type in the remaining 10 samples with good quality readings. Multivariate analysis of baseline factors revealed significant and independent correlations between mutation detection and disease phase (P = 0.0008) and clonal cytogenetic evolution (P = 0.0004). In contrast, mutation detection was not an independent predictor of failure to achieve complete hematologic response (CHR) or major cytogenetic response (MCR) and was not correlated with overall survival. Four patients with mutations achieved CHR and 3 MCR; 4 patients failed to achieve CHR, 1 died from neutropenic sepsis and 1 discontinued because of skin toxicity. Thus far, follow-up specimens during imatinib therapy have been sequenced in 6 patients. In 2 cases (T315I and Q252H) the identical mutation was detected, one case (T315I) showed K247R (detected prior to therapy by sequencing but not included in the allele specific assays), and 3 cases were wild type BCR-ABL. Conclusion: We provide direct evidence that the presence of BCR-ABL KD mutantions is correlated with disease stage. Moreover, the strong association with clonal evolution suggests that genomic instability may be responsible for both phenomena. However, not all mutants that were detected prior to therapy were subsequently selected in the presence of imatinib, suggesting that some mutations may occur in cells without self-renewal capacity or that additional factors may be required to confer a fully resistant phenotype.

Klotho Polymorphisms and Priapism In Sickle Cell Disease.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1653-1653 ◽  
Author(s):  
Claudia R Lustosa Souza ◽  
Marily M Azevedo Shimmoto ◽  
Perla Vicari ◽  
Martha Mariana A S Arruda ◽  
Marina Roizenblatt ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Vascular Function ◽  
Conflicts Of Interest ◽  
Significant Proportion ◽  
Cell Disease ◽  
Specific Pcr ◽  
Allele Specific ◽  
Male Patients ◽  
Allele Specific Pcr

Abstract Abstract 1653 Background: Sickle cell disease (SCD) is a monogenic disorder with phenotypic heterogeneity, possibly determined by polymorphisms (SNPs) in genes whose products modify the pathophysiology of the disease. Priapism is one of the most common vaso-occlusive complications of SCD, and it occurred in more than 30% of males. The Klotho (KL) gene appears to be associated with vascular function and nitric oxide biology and the presence of SNPs could affect its function. Association between KL and priapism in SCD patients was suggested by Nolan et al. in 2004. However, other authors could not confirm this finding (Elliot et al., 2007). Objective: We decided to evaluate the relevance of SNPs rs2249358, rs211234 and rs9536314 to the occurrence of priapism in patients with SCD followed at Outpatient clinic at Escola Paulista de Medicina/UNIFESP. Methods: Forty male patients with SCD were enrolled, 39 (97.5%) with sickle cell anemia (SS) and one (2.5%) SC hemoglobinopathy. The manifestation of priapism was identified through analyses of medical records. The SNP rs2249358 was identified by PCR followed by restriction with XbaI. The other SNPs, rs211234 and rs9536314, were analyzed by allele specific PCR. Statistical analysis: t test, Chi2 or Fisher. This study was approved by Ethical Committee, and all patients agreed in participate. Results: The median age of the patient was 28.5 years-old (20-68 y.o.). Fourteen out of 40 patients had priapism (35%), each one with SS disease. The group of patients with priapism were older (32.5 y.o., 25–68 y.o.) than the group without this manifestation (27.5 y.o., 20–56 y.o.) (p=0.03). There was no statistical difference in the distribution of the SNPs rs211234 and rs9536314 between the two groups of patients (p=0.51 and p= 0.09, respectively). Regarding the distribution of SNP rs2249358, the group with priapism presented 8 individuals (57.1%) with GG genotype, 5 (35.7%) with AA and 1 (7.17%) with AG, whereas in the group without priapism, the distribution was different: 5 (19.2%) with GG, 7 (26.9%) with AA and 14 (53.8%) with AG genotype (p=0.0212). When we compare the presence of at least one A allele (AA or A-) with the G allele in homozygosis (GG), we observed that the A allele has a protector effect (OR: 0.1786; CI: 0.04232–0.7535) (p=0.031). Conclusions: In a relatively small group of patients with SCD, it was observed a significant proportion of individuals with priapism, which reinforces the importance of this manifestation. We also observed correlation between SNP rs2249358 of KL gene and priapism, as suggested previously. Disclosures: No relevant conflicts of interest to declare.

Incidence of the JAK2V617F Mutation In Mexican Patients with Myeloproliferative Neoplasms (MPNs)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5180-5180
Author(s):  
Gregorio Ignacio ◽  
Rosa María Arana-Trejo ◽  
Verónica Gónzalez ◽  
Maria Paula Hérnandez ◽  
Yolanda Lugo ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Direct Sequencing ◽  
Jak2v617f Mutation ◽  
Normal Blood ◽  
Base Substitution ◽  
Blood Samples ◽  
Specific Pcr ◽  
Dna And Rna ◽  
Mexican Patients

Abstract Abstract 5180 Introduction: The V617F mutation in JAK2 gene has been described in approximately 50–90% of patients with ET, MF AND PV [essential trombocythaemia, idiopathic myelofibrosis and policithemia vera]; but has also been reported, albeit at a lower frequency, in patients with other myeloid malignancies such as atypical CML, CMML, AML, MDS, JMML and CNL. A single G>T base substitution in exon 12 results in the conversion of valine to a phenylalanine aminoacid at position 617 of the JAK2 gene. The identification in this study were using techniques such as allele-specific PCR, RFLP-PCR and direct sequencing; for to determine the incidence in Mexican patients with MPNs. Patients and Methods: The JAK2V617F mutation was determined in 88 patients and 5 normal blood samples for healthy individuals as controls. About the patients, 60 were cataloged like MPNs and 28 patients with features suggestive of MPNs vs CML. Samples for bone marrow or peripherical blood were taken either at time of diagnosis of MPNs or during treatment with cytoreductive or anti-thrombotic agents. DNA and RNA were extracted using the QIAamp DNA and RNeasy mini kit (Qiagen) and amplified by the three techniques mentioned for JAK2V617F and by nested RT-PCR for BCR/ABL. Results: The five normal blood samples for controls were negative for JAK2V617F mutation and to BCR/ABL. Patients had median age 65 years (47–85 years old), 46% male and 54% female. In de overall patients: 60 patients with MPNs all were BCR/ABL negative and 20 (33%) had JAK2V617F. In the 28 patients with likely MPNs vs CML, 23 were BCR/ABL positive/JAK2 negative, two had the coexistence of both genetics defects [BCR/ABL+ and JAK2V617F+] and 3 BCR/ABL and JAK2 negative. Finally the patiens with JAK2V617F+, were 12 ET, PV 1, MF 2, CML 2, and 5 continued like MPNs. Discussion: The incidence of the JAK2V617F in this study for MPNs patiens were 33% and the incidence varied between MPNs subtype. Less than ten cases of BCR/ABL+ CML with JAK2V617F have been published; we report two patients with the coexistence and we agree with previous reports that screening for JAK2V617F mutation should be considered in any BCR/ABL+ CML patients and the clinical outcome will be define in long period. Disclosures: No relevant conflicts of interest to declare.

Clinical Implications for Patients with Low JAK2V617F Allele Burden

Blood ◽  
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.

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.

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