Bioluminometric Assay for Relative Quantification of Mutant Allele Burden: Application to the Oncogenic Somatic Point Mutation JAK2 V617F

2009 ◽  
Vol 81 (20) ◽  
pp. 8596-8602 ◽  
Author(s):  
Vaya Tsiakalou ◽  
Margarita Petropoulou ◽  
Penelope C. Ioannou ◽  
Theodore K. Christopoulos ◽  
Emmanuel Kanavakis ◽  
...  
Keyword(s):  
Point Mutation ◽  
Mutant Allele ◽  
Jak2 V617f ◽  
Relative Quantification ◽  
Allele Burden ◽  
Somatic Point Mutation

scholarly journals Distinct Clinical, Laboratory, and Molecular Features of Myeloproliferative Neoplasm Patients Presenting with Splanchnic Vein Thrombosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3121-3121
Author(s):  
Joan How ◽  
Stephen T. Oh ◽  
Kathryn M. Trinkaus
Keyword(s):  
Risk Factors ◽  
Board Of Directors ◽  
Mutant Allele ◽  
Jak2 V617f ◽  
Continuous Variables ◽  
Disease Phenotype ◽  
Advisory Committees ◽  
Allele Burden ◽  
Vein Thrombosis ◽  
Cell Counts

Abstract BACKGROUND: Myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and primary meylofibrosis (PMF), are frequently associated with splanchnic vein thromboses (SVT). Risk factors for SVT in MPN patients differ from risk factors for all-cause thrombosis. This is likely due to differing disease mechanisms at play, and suggest a separate disease phenotype for MPN/SVT patients. While several studies have characterized the features of MPN patients with SVT, a direct comparison of MPN/SVT versus all MPN patients has been lacking. METHODS: We performed a retrospective, cross-sectional analysis of patients at Barnes-Jewish Hospital from 2000-2014 with MPN and SVT. Patients were identified using ICD-9 codes in the electronic medical record. 52 available patients with both MPN and SVT were included. Randomly selected 134 patients with MPNs only were used as controls. Clinical and laboratory variables were compared between the two groups. Quantitative JAK2 V617F allele burdens were available in 20 patients. As continuous variables were not normal in distribution, a Mann-Whitney U test was performed. Non-continuous variables were compared with an N-1 Chi-squared test to accommodate rare events. All p-values were corrected for multiple testing. RESULTS: MPN/SVT patients were significantly younger at time of MPN diagnosis (median age 47 vs 57 years, p=0.003). MPN/SVT patients were more likely to have splenomegaly (83% vs 30%, p=0.003), deep vein thrombosis (37% vs 15%, p=0.003), and concomitant thrombophilia (17% vs 2%, p=0.003). MPN/SVT patients had a higher proportion of females (63% vs 54%), but this finding did not reach significance. However, PV/SVT patients had a significantly higher proportion of females compared to PV alone (67% vs 37%, p=0.02). There were no significant differences in JAK2 mutation status, race, smoking status, presence of stroke or coronary artery disease risk factors. MPN/SVT patients had significantly lower hemoglobin (13.1 vs 14.6, p=0.024), hematocrit (39.3 vs 43.5, p=0.027), and platelet count (513 vs 698, p=0.003) at time of MPN diagnosis. When analysis was restricted to PV, only hemoglobin (14.6 vs 17.24, p=0.007) and hematocrit (44.3 vs 50.68, p=0.012) were significantly lower in SVT patients. No significant differences in cell counts were detected in ET and PMF patients. MPN/SVT patients had significantly lower JAK2 mutant allele burdens, with no MPN/SVT patient having an allele burden greater than 10% (p=0.019) (Figure 1). In contrast, mutant allele burdens for MPN patients ranged from 0.1 to 99.7%, with median allele burden being 36.3%. DISCUSSION: This is the first study to directly compare clinical and laboratory features of MPN patients with and without SVT. Our results confirm that MPN/SVT patients are younger, and within PV are more likely to be female. We also demonstrate that MPN/SVT patients have lower cell counts and lower JAK2 mutant allele burdens, findings not previously shown in the literature. MPN/SVT patients are more likely to have splenomegaly, concurrent thrombophilia, and additional DVT. These results indicate that MPN/SVT patients exhibit a disease phenotype distinct from MPN patients without SVTs. While the nature of this study is retrospective and causality cannot be definitively established, the findings of younger age, lower laboratory values, and lower JAK2 allele burden are consistent with the hypothesis that MPN/SVT patients present early in disease. It is possible that in MPN/SVT patients, other environmental and host factors (such as concurrent thrombophilia), in combination with early MPN disease, result in the first manifestation of SVT. These findings have important implications, as investigating the natural course of MPN/SVT patients would allow insight into MPN disease pathogenesis. These findings also suggest that SVTs in MPN patients are not solely mediated by elevated cell counts. In addition, while the presence of the JAK2 V617F mutation likely does affect thrombotic risk, the finding of lower allele burdens in MPN/SVT patients suggests that additional interactions mediate SVT development. These interactions are likely multifactorial and include both environmental and genetic factors. Of particular interest would be the presence of yet unidentified driver mutations present in MPN/SVT patients. Disclosures Oh: Incyte: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Clinical relevance of JAK2 (V617F) mutant allele burden

Haematologica ◽  
2009 ◽  
Vol 94 (1) ◽  
pp. 7-10 ◽  
Author(s):  
F. Passamonti ◽  
E. Rumi
Keyword(s):  
Clinical Relevance ◽  
Mutant Allele ◽  
Jak2 V617f ◽  
Allele Burden

scholarly journals Prevalence of the JAK2 V617F Mutation in Patients with Peripheral Arterial Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3165-3165
Author(s):  
Elena Kinz ◽  
Klaus Gasser ◽  
Axel Muendlein ◽  
Andreas Leiherer ◽  
Michael Steurer ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Arterial Disease ◽  
Jak2 V617f Mutation ◽  
Allele Burden ◽  
Cell Counts ◽  
V617f Mutation ◽  
Artery Disease ◽  
Peripheral Arterial

Abstract Introduction: The acquired JAK2 V617F mutation is common in patients with myeloproliferative neoplasms and increases thrombotic risk. We previously showed that JAK2 V617F is also found in healthy subjects as well as in patients with coronary artery disease (0.6% and 1.3%, respectively). Peripheral arterial disease (PAD) is an important manifestation of diffuse atherosclerosis and PAD patients are at exceptionally high risk for cardiovascular events, showing a worse prognosis than that of patients with coronary artery disease Due to the close relation of the JAK2 V617F mutation to thrombotic events we hypothesized that this mutation may play an important role in the risk management of PAD patients. However, prevalence of JAK2 V617F or of occult myeloproliferative neoplasms is unknown in PAD patients. Methods: In the present study we determined the prevalence of JAK2 V617F in a cohort of 287 patients with sonographically proven PAD. JAK2 mutational status from 997 age-matched healthy people was available from a previous study. JAK2 V617F screening and quantification of allele burden in both cohorts was performed with allele-specific quantitative real-time PCR. Results: From a total of 287 PAD patients samples, 9 (3.1%) were tested positive for JAK2 V617F mutation corresponding to a 5-fold, highly significant increase compared with healthy people (p<0.001). Mutant allele burden of JAK2 V617F positive samples was ranging between 0.2% and 96.2% (median=0.75%). Generally, our study showed no significant association of the JAK2 V617F mutation with abnormal blood cell counts. However, the patient with the highest mutant allele burden showed elevated hemoglobin values (> 18.5 g/dL) indicating polycythemia vera (PV). Conclusion: We conclude that the prevalence of JAK2 V617F mutation is significantly increased in PAD patients compared to the general population. For this reason mutation analysis should be considered in PAD patients with abnormal blood cell counts to identify occult myeloproliferative neoplasms and to adjust therapeutic treatment, possibly reducing the risk of future vascular complications. Disclosures No relevant conflicts of interest to declare.

Relationship Between Granulocyte JAK2 (V617F) Mutant Allele Burden and Risk of Progression to Myelofibrosis in Polycythemia Vera: a Prospective Study of 338 Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 751-751
Author(s):  
Francesco Passamonti ◽  
Elisa Rumi ◽  
Daniela Pietra ◽  
Chiara Elena ◽  
Emanuela Boveri ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Mutant Allele ◽  
Multivariable Analysis ◽  
Jak2 V617f ◽  
Continuous Variable ◽  
Free Survival ◽  
Who Criteria ◽  
Allele Burden ◽  
Risk Of Progression

Abstract Abstract 751 An identical gain-of-function mutation of JAK2 is found in about 95% of patients with polycythemia vera (PV). According to a two-step model [N Engl J Med. 2005 Apr 28;352(17):1779-90], the occurrence of JAK2 (V617F) gives rise to a clone that is heterozygous and expands to replace hematopoietic cells without the JAK2 mutation. A mitotic recombination in a hematopoietic cell that is heterozygous for JAK2 (V617F) later generates uniparental disomy and 9pLOH. The daughter cell that is homozygous for JAK2 (V617F) gives rise to a new clone that expands and replaces the previous heterozygous clone. Therefore, variable proportions of JAK2 (V617F) mutant alleles are found in myeloid cell populations from PV patients. A mutant allele dosage effect on phenotype has been described, and PV patients with high mutant allele burden have been found to have a more severe disease. Patients with post-PV myelofibrosis have the highest mutant allele burdens [median value of about 90% - Blood. 2008 Apr 1;111(7):3383-7]. Interestingly, JAK2 (V617F) activates circulating granulocytes, and by this means likely plays a role in the constitutive mobilization of CD34-positive cells into peripheral blood that characterizes the transformation of PV into post-PV myelofibrosis [Blood. 2006 May 1;107(9):3676-82]. Since all these observations may suggest that the mutant allele burden contributes to determining the myelofibrotic transformation of PV, we examined PV patients enrolled in a prospective observational cohort study. As of August 10, 2009, 338 patients diagnosed with PV according to the 2008 WHO criteria have been enrolled in this study. Of these patients, 320 (94.7%) carried JAK2 (V617F), 14 (4.1%) had JAK2 exon 12 mutations, and 4 (1.2%) did not carry JAK2 (V617F) nor exon 12 mutations despite a typical PV phenotype. Of the 320 patients carrying JAK2 (V617F), 146 were enrolled at diagnosis and 174 at follow-up. Patients were routinely treated with phlebotomy and low dose aspirin, while those at high risk for thrombosis (history of previous thrombosis and/or age greater than 60 years) were given also cytoreductive therapy. Diagnosis of post-PV myelofibrosis was based on the IWG-MRT criteria, while diagnosis of myelodysplastic syndrome or acute myeloid leukemia (AML) was done according the 2008 WHO criteria. In order to accurately assess the granulocyte mutant allele burden, we refined a previously described quantitative real-time polymerase chain reaction (qRT-PCR)-based allelic discrimination assay. This assay is now routinely calibrated using defined standards [Haematologica. 2009 Jan;94(1):38-45] and has a sensitivity equal to 0.2% mutant alleles. Within 320 JAK2 (V617F)-positive patients, the median mutant allele burden was 47% (range 1.1-100%); 167 (52%) patients had less than 50%, while 153 (48%) had more than 50% mutant alleles. PV patients at diagnosis had significantly lower mutant allele burdens than those enrolled in the study at follow-up (P = .002). During the study period, disease transformation occurred in 18 patients. Eight patients, all with more than 50% JAK2 (V617F) mutant alleles at study entry, progressed to post-PV myelofibrosis, while 10 patients developed AML. Since about half of the patients were enrolled at follow-up, survival analyses were carried out accounting for left censoring of the observation. Cox proportional hazard regression showed that the JAK2 mutant allele burden, analyzed as a continuous variable, was related to hematologic transformation-free survival (HR: 1.025, 95% CI 1.005-1.046; P = .015). By categorizing the mutant allele burden, patients with more than 50% mutant alleles had a significantly worse hematologic transformation-free survival (HR: 6.54, 95% CI 1.47-29.1; P = .013) compared with those with lower mutant allele burden. After adjusting for age in a multivariable analysis, the 50% cutoff retained statistical significance (P = .048). With respect to the risk of progression to post-PV myelofibrosis, the JAK2 mutant allele burden, considered as a continuous variable, was significantly related to myelofibrosis-free survival (HR: 1.04, 95% CI: 1.004-1.08; P = .029). In a multivariable analysis with allele burden and age as covariates, the mutant allele burden showed an independent effect on myelofibrosis-free survival (P= .038). By contrast, the risk of developing AML was not significantly related to the mutant allele burden. In conclusion, the findings of this study suggest that a high mutant allele burden represents a risk factor for progression to myelofibrosis in patients with PV. Disclosures: No relevant conflicts of interest to declare.

Clonal Analysis of SF3B1, JAK2 and MPL in Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 172-172
Author(s):  
Ilaria Ambaglio ◽  
Anna Gallì ◽  
Daniela Pietra ◽  
Matteo G Della Porta ◽  
Marta Ubezio ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Refractory Anemia ◽  
Wild Type ◽  
Mutation Status ◽  
Allele Burden ◽  
Ring Sideroblasts ◽  
Exon 10

Abstract Abstract 172 Somatic mutations of the RNA splicing machinery have been recently identified in patients with myelodysplastic syndrome (MDS). In particular, a strong association has been found between SF3B1 mutation and the MDS subtype defined as refractory anemia with ring sideroblasts (RARS). Similarly, within myelodysplastic/myeloproliferative neoplasms (MDS/MPN) a high prevalence of SF3B1 mutations has been reported in the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). These findings strongly support a causal relationship between SF3B1 mutations and ring sideroblasts. Interestingly, a high proportion of RARS-T patients also harbor JAK2 and/or MPL mutations. The available evidence suggests that somatic mutations of SF3B1 might be an early pathogenetic event determining myelodysplastic features, and that subsequent occurrence of JAK2 and/or MPL mutations may cause the myeloproliferative phenotype. In this work, we studied the mutation status of SF3B1, JAK2 and MPL in circulating granulocytes and bone marrow cells from RARS-T patients. We also studied the in vitro growth of hematopoietic progenitors (BFU-E, CFU-GM), and genotyped individual colonies to examine the mutation status of the above genes. The coding exons of SF3B1 were screened using massively parallel pyrosequencing. A real time PCR-based allelic discrimination assay was used for the detection of JAK2 (V617F), while Sanger sequencing was employed for JAK2 exon 12 and MPL exon 10 mutation analysis. Twenty-eight patients affected with RARS-T were assessed for SF3B1, JAK2 and MPL exon 10 mutation status. Eighteen patients (64%) showed somatically acquired mutation of SF3B1. The median mutant allele burden was 43%, consistent with the presence in the majority of patients of clonal hematopoiesis characterized by a dominant clone carrying a heterozygous SF3B1 mutation. Fourteen patients carried the JAK2 (V617F) mutation (median allele burden 6.5%, range 0.4–29.5%), while one had a JAK2 exon 12 mutation. In 13 cases, the JAK2 mutation was detected at the time of diagnosis, whereas in 2 patients, who had a typical RARS phenotype and were negative for JAK2 mutations at clinical onset, JAK2 (V617F) was detected 18 and 32 months after diagnosis, respectively, and concomitantly with a progressive increase in platelet count. Four patients, two of whom were JAK2 (V617F)-positive, carried the MPL (W515L) mutation (median allele burden 27.5%, range 25–50%). Concomitant mutations of SF3B1 and JAK2 or MPL were observed in 8 cases. Seven patients carried an SF3B1 mutation and JAK2 (V617F), while one carried SF3B1 (K700E), JAK2 (V617F), and MPL (W515L). In all these cases, the SF3B1 mutant allele burden was higher than that of JAK2 or MPL, indicating the existence of an SF3B1-mutated dominant clone with minority JAK2- or MPL-mutated clones. We genotyped individual colonies from peripheral blood in 2 patients with concomitant mutations. In a patient with granulocyte SF3B1 and JAK2 mutant allele burdens equal to 45% and 8%, respectively, SF3B1 (H662Q) was detected in 9 of 11 colonies, three of which also carried JAK2 (V617F); the remaining two colonies had wild type SF3B1 and JAK2. These data are consistent with the existence of a dominant hematopoietic clone carrying the SF3B1 mutation and the subsequent emergence of a JAK2-mutated subclone. The other patient, who was initially SF3B1- mutated and JAK2 wild type, at the time of colony assay had a mutant allele burden equal to 50% and 1% for SF3B1 (K700E) and JAK2 (V617F), respectively. Forty-three of 45 colonies were heterozygous for SF3B1 (K700E) and wild type for JAK2. The opposite pattern was observed in the remaining 2 colonies, which carried just JAK2 (V617F). These data indicate the coexistence of two distinct clones, a dominant one carrying the SF3B1 mutation and a minority one carrying JAK2 (V617F). In summary, these observations suggest that the occurrence of an SF3B1 mutation represents an early event in patients with RARS-T, likely causing mitochondrial iron overload, ring sideroblasts, ineffective erythropoiesis and anemia, typical myelodysplastic features. The subsequent occurrence of a somatic mutation of JAK2 or MPL involves the emergence of minority clones and the acquisition of myeloproliferative features. JAK2- mutated clones may emerge as subclones of the dominant SF3B1-mutated clone or as independent clones. Disclosures: No relevant conflicts of interest to declare.

JAK2 (V617F)-Positive Essential Thrombocythemia and Polycythemia Vera Are Different Expressions Of a Genotypic/Phenotypic Continuum

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1592-1592
Author(s):  
Elisa Rumi ◽  
Daniela Pietra ◽  
Chiara Elena ◽  
Ilaria Casetti ◽  
Emanuela Sant 'Antonio ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Median Time ◽  
Essential Thrombocythemia ◽  
Cumulative Incidence ◽  
Mutant Allele ◽  
Clinical Phenotype ◽  
Jak2 V617f ◽  
Wild Type ◽  
Allele Burden ◽  
Wbc Count

Abstract Background About 95% of patients with polycythemia vera (PV) and 60-70% of those with essential thrombocythemia (ET) carry the unique JAK2 (V617F) mutation. Previous observations suggest that JAK2 (V617F)-positive ET and PV form a biological continuum, in which the degree of erythrocytosis is determined by physiological and genetic factors. Aims In this work, we studied the natural history of JAK2 (V617F)-positive ET and PV with the aim of establishing whether the two disorders indeed represent different phenotypic expression of a genotypic/phenotypic continuum. Methods We identified 1269 patients diagnosed with ET or PV at our Division between 1980 and 2012, for whom at least one DNA sample was available. The JAK2 (V617F) mutation was assessed using allele-specific quantitative PCR. As patients carrying JAK2 exon 12 or MPL mutations were excluded, the final study population included 1214 patients, 719 of whom with ET (463 JAK2 mutated, 256 JAK2 wild-type) and 495 with PV. Results I: presenting features The clinical phenotype of ET patients at diagnosis differed according to JAK2 mutational status. JAK2 mutated ET presented with older age at diagnosis, higher hemoglobin (Hb) level and white blood cell (WBC) count, lower platelet (PLT) count and erythropoietin level compared to JAK2 wild-type ET (Wilcoxon rank-sum test: P<.001 in all comparisons). The median V617F allele burden was significantly lower in JAK2 mutated ET than in PV (18.4% vs 43.4%, P<.001). A mutant allele burden greater than 50% was observed in 2% of patients with JAK2 mutated ET and 41.5% of those with PV (Fisher exact test: P<.001). In both JAK2 mutated ET and PV, the mutant allele burden was directly related to WBC count and Hb level. Results II: PV evolution Evolution to PV was observed in 53 JAK2 mutated ET patients (incidence 95% CI: 1.4-2.4 per 100 p-years) vs none of the 256 JAK2 wild-type ET (incidence 95% CI: 0-0.2 per 100 p-years), resulting in a significantly different occurrence. The median time to PV evolution was 54 months (range 3.5-220). The cumulative incidence of PV evolution in JAK2 mutated ET patients at 15 years was 28.8% (95% CI: 20.7-37.3; Figure 1). PV evolution was significantly associated with higher JAK2 allele burden at diagnosis (Cox regression HR=1.04, P<.001). Based on the hypothesis that PV patients might have had a silent “pre-PV phase”, we did an ad hoc search for any complete blood count (CBC) collected before diagnosis. Among PV patients, 177 (36%) had a previous CBC, collected at a median time of 22 months (range 1-305) before PV diagnosis. A normal CBC was observed in 15% of patients; the remaining subjects showed thrombocytosis (≥450 x 109/L) and/or leukocytosis (≥10 x 109/L) and/or erythrocytosis. The median time to PV onset was significantly shorter in patients showing at least one CBC abnormality than in those with normal CBC (24 vs 48 months, P=.011). Results III: clinical course The median follow-up was 5.1 years (range, 0-32 years). JAK2-mutated ET and PV did not differ in terms of cumulative incidence of thrombosis (25.3% vs 33.7% at 15 years, P=.35; Figure 2A) and had similar overall survival (OS) (90.3% vs 82.6% at 15 years, P=.29; Figure 2B). Conversely, JAK2 wild-type ET showed a better OS in comparison with both JAK2 mutated ET (P=.028) and PV (P=.004) and a lower incidence of thrombosis (12.7% at 15 years) than JAK2 mutated ET (P=.002) and PV (P<.001). A similar cumulative incidence of disease progression (leukemia and myelofibrosis) was observed in JAK2 mutated (11.7% at 15 years) and JAK2 wild-type ET (12.1% at 15 years), whereas a higher cumulative incidence was observed in PV (26% at 15 years; P=.011 and P=.007 when compared with JAK2 mutated ET and JAK2 wild-type ET respectively). Conclusions This study supports the hypothesis that JAK2 mutated ET and PV are different expressions of a genotypic/phenotypic continuum, in which the mutant allele burden contributes to determine the clinical phenotype. The risk of progression from JAK2 mutated ET to PV is about 2% per year. This work was supported by grant #1005 from Associazione Italiana per la Ricerca sul Cancro (AIRC) “Special Program Molecular Clinical Oncology 5x1000” to AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative - http:www-progettoagimm.it). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effects of the Sympathicomimetic Agonist Mirabegron on Disease Course, Mutant Allele Burden, Marrow Fibrosis, and Nestin Positive Stem Cell Niche in Patients with JAK2-Mutated Myeloproliferative Neoplasms. a Prospective Multicenter Phase II Trial SAKK 33/14

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3108-3108 ◽  
Author(s):  
Beatrice Drexler ◽  
Jakob Passweg ◽  
Martin Bigler ◽  
Alexandre PA Theocharides ◽  
Nathan Cantoni ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Phase Ii ◽  
Mutant Allele ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Nerve Fibers ◽  
Allele Burden ◽  
Sympathetic Nerve Fibers ◽  
Bone Marrow Histology ◽  
Cell Niche

Abstract Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by aberrant proliferation of erythroid, megakaryocytic and myeloid lineages. They are associated with decreased survival, thromboembolic complications, hemorrhage and leukemic transformation. MPN can be subdivided into polycythemiavera(PV), essentialthrombocythemia(ET) and primary myelofibrosis (PMF). The JAK2-V617F mutation is present in 70-80% of all MPN patients. MPN is initiated and maintained by mutated hematopoietic stem and progenitor cells (HSPC). Bone marrow mesenchymal stem cells expressing the intermediate filament proteinnestin(nestin+ MSCs) that are innervated by sympathetic nerve fibers constitute an important component of the stem cell niche and regulate normal HSCs. Thesenestin+ MSCs are strongly reduced in bone marrow of JAK2-V617F positive MPN patients and in mice expressing JAK2-V617F due to damage of the sympathetic nerve fibers triggered by cytokines from the mutant cells. In a JAK2-V617F mouse model of MPN, treatment with a beta-3sympathicomimeticagonist corrected the damage inflicted by the MPN clones on their niches and ameliorated the MPN phenotype. To test the potentially beneficial effect on disease-control by modulating bone marrow niche cells in patients with MPN, we performed a phase II trial with the beta-3sympathicomimeticagonistmirabegron. Patients and Methods: The trial consisted ofmirabegrontreatment with 25 mg daily during the first week, followed by 50 mg daily for at least 24 weeks. Patients with acytohistologicallyconfirmed diagnosis of MPN and a JAK2-V617F allele burden &gt;20% in granulocytes at study entry were eligible, if not treated with JAK2 inhibitors or interferon. Reduction of the JAK2-V617F mutant allele burden ³50% in granulocytes was defined as the primary end point. Secondary end points included changes in blood counts or MPN related symptoms. As a side study, bone marrow biopsies were quantified fornestin+ MSCs, fibrosis and CD34+ HSPCs. N=39 patients have been accrued in 10 institutions in Switzerland. Eight (21%) had ET, 22 (56%) PV, and 9 (23%) PMF. N=27 (69%) were male, the median age was 62 (Q1-Q3 53-72) years. Median mutated allele burden at study onset was 52% (Q1-Q3 33-73%). All patients had prior treatment, N=28 (72%) patients hadcytoreductivetreatment, the remaining patients hadantiaggregation, anticoagulation or phlebotomy. Results: No patient reached the primary endpoint of 50% reduction in allele burden, one patient achieved a 25% reduction by 24 weeks of treatment. Adverse events were mostly grade I or II on the CTCAE scale. Three patients had grade III events: two were considered to be at least possibly related to study medication. In the side study, 24 patients agreed to bone marrow biopsy prior to and at the end ofmirabegrontreatment and for 20 patients both measurements are available. In these patients an increase in thenestin+ MSCs cells from a median of 1.09 (Q1-Q3 0.38-3.27)/mm2 to 3.95 (Q1-Q3 1.98-8.79)/mm2 (p&lt;0.0001, Wilcoxon signed-rank test) and a slight decrease of myelofibrosis from a median grade of 1.00 (Q1-Q3 0.50-3.00) to 0.75 (Q1-Q3 0.50-2.00) (p=0.02), were observed. The mean change in thenestin+ cells from baseline to week 24 was 3.52 (95% confidence interval 1.65-5.39)/mm2. Morphometric changes in thenestin+ MSCs were significant for PV (n=13, p=0.007) and PMF (n=5, p=0.04). Bone marrow CD34+ cells slightly increased from a median 2.50 (Q1-Q3 2.00-3.25) to 3.00 (Q1-Q3 2.00-3.75) (p=0.06). Conclusion: In this prospective phase II clinical trial treatment with the beta-3-sympathicomimetic agonistmirabegronfor 24 weeks failed to achieve the primary endpoint to reduce the JAK2-V617F mutant allele burden &gt;50% in patients with MPN. However, an increase in thenestin+ MSCs in bone marrow and a slight decrease of myelofibrosis were found, which will be further investigated. Figure 1 Bone marrow histology of a patient before (week 0) and at the end ofmirabegron treatment (week 24). Upper panel,reticulin fibers are stained black by silver impregnation (Gomori). Lower panel, immunohistochemistry staining with antibodies against humannestin protein (brown staining). Note decrease inreticulin fibrosis and increase innestin+ cells after 24 weeks of treatment. Magnification: 200x. Figure 1. Bone marrow histology of a patient before (week 0) and at the end ofmirabegron treatment (week 24). Upper panel,reticulin fibers are stained black by silver impregnation (Gomori). Lower panel, immunohistochemistry staining with antibodies against humannestin protein (brown staining). Note decrease inreticulin fibrosis and increase innestin+ cells after 24 weeks of treatment. Magnification: 200x. Disclosures Theocharides: Novartis: Consultancy, Honoraria. Rüfer:Novartis: Consultancy, Speakers Bureau. Benz:Celgene: Consultancy. Tzankov:Novartis: Speakers Bureau; Abbott: Speakers Bureau. Skoda:Novartis: Consultancy, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Consultancy, Speakers Bureau.

Differential effects of hydroxyurea and INC424 on mutant allele burden and myeloproliferative phenotype in a JAK2-V617F polycythemia vera mouse model

Blood ◽  
2013 ◽  
Vol 121 (7) ◽  
pp. 1188-1199 ◽  
Author(s):  
Lucia Kubovcakova ◽  
Pontus Lundberg ◽  
Jean Grisouard ◽  
Hui Hao-Shen ◽  
Vincent Romanet ◽  
...  
Keyword(s):  
Mouse Model ◽  
Competitive Advantage ◽  
Polycythemia Vera ◽  
Mutant Allele ◽  
Jak2 V617f ◽  
Blood Parameters ◽  
Therapeutic Agents ◽  
Wild Type ◽  
Allele Burden ◽  
Key Points

Key Points JAK2-V617F cells show a competitive advantage over wild-type cells in BM transplantation assays. A preclinical mouse model allows the examination of the effects of therapeutic agents on blood parameters and JAK2-V617F mutant allele burden.

scholarly journals Using the Minor Variant Finder software to identify and quantify the allelic burden level of somatic mutations in oncohematologic diseases

2020 ◽  
Vol 15 (2) ◽  
pp. 85-91
Author(s):  
T. N. Subbotina ◽  
I. E. Maslyukova ◽  
A. A. Faleeva ◽  
P. A. Nikolaeva ◽  
A. S. Khazieva ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Sanger Sequencing ◽  
Quantitative Assessment ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Mutant Gene ◽  
Positive Sample ◽  
Allele Burden ◽  
Minor Variant ◽  
Allelic Burden

Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes.

scholarly journals A comparison of qPCR and ddPCR used for quantification of the JAK2 V617F allele burden in Ph negative MPNs

2018 ◽  
Vol 97 (12) ◽  
pp. 2299-2308 ◽  
Author(s):  
Dorota Link-Lenczowska ◽  
Niels Pallisgaard ◽  
Sabrina Cordua ◽  
Magdalena Zawada ◽  
Sylwia Czekalska ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Allele Burden
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