The sympathomimetic agonist mirabegron did not lower JAK2-V617F allele burden, but restored nestin-positive cells and reduced reticulin fibrosis in patients with myeloproliferative neoplasms: results of phase II study SAKK 33/14.
ABSTRACT: The β-3 sympathomimetic agonist BRL37344 restored nestin-positive cells within the stem cell niche, and thereby normalized blood counts and improved myelofibrosis in a mouse model of JAK2-V617F-positive myeloproliferative neoplasms. We therefore tested the effectiveness of mirabegron, a β-3 sympathomimetic agonist, in a phase II trial including 39 JAK2-V617F-positive patients with myeloproliferative neoplasms and a mutant allele burden more than 20%. Treatment consisted of mirabegron 50 mg daily for 24 weeks. The primary end point was reduction of JAK2-V617F allele burden of 50% or over, but this was not reached in any of the patients. One patient achieved a 25% reduction in JAK2-V617F allele burden by 24 weeks. A small subgroup of patients showed hematologic improvement. As a side study, bone marrow biopsies were evaluated in 20 patients. We found an increase in the nestin+ cells from a median of 1.09 (interquartile range 0.38-3.27)/mm2 to 3.95 (interquartile range 1.98-8.79)/mm2 (P<0.0001) and a slight decrease of reticulin fibrosis from a median grade of 1.0 (interquartile range 0-3) to 0.5 (interquartile range 0-2) (P=0.01) between start and end of mirabegron treatment. Despite the fact that the primary end point of reducing JAK2-V617F allele burden was not reached, the observed effects on nestin+ mesenchymal stem cells and reticulin fibrosis is encouraging, and shows that mirabegron can modify the microenvironment where the JAK2-mutant stem cells are maintained. (Registered at clinicaltrials.gov identifier: 02311569).
Project description:The myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), differ phenotypically, but share the same JAK2(V617F) mutation. We examined the relationship of the quantitative JAK2(V617F) allele burden to MPD disease phenotype among the three MPD classes and within PV.We measured the JAK2(V617F) allele percentage in genomic DNA from neutrophils, CD34(+) cells, and cloned progenitors in 212 JAK2(V617F)-positive MPD patients and correlated the allele burdens to both disease class and disease features.In ET and PV, mean CD34(+) cell JAK2(V617F) allele burdens were lower than the corresponding neutrophil allele burdens, but these were equivalent in PMF. JAK2(WT) progenitors were present in ET and PV when the CD34(+) JAK2(V617F) allele burden was lower than the neutrophil allele burden, but not in PV and PMF subjects in whom the CD34(+) cell and neutrophil allele burdens were similar. CD34(+) cell JAK2(V617F) clonal dominance, defined as coherence between the CD34(+) cell and neutrophil JAK2(V617F) allele burdens, was present in 24% of ET, 56% of PV, and 93% of PMF patients, and was independent of the CD34(+) cell JAK2(V617F) genotype. Clonally dominant PV patients had significantly longer disease durations, higher white cell counts, and larger spleens than nondominant PV patients.We conclude that the extent of JAK2(V617F) CD34(+) cell clonal dominance is associated with disease phenotype within the MPD and, in PV, is associated with extramedullary disease, leukocytosis, and disease duration.
Project description:Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2 (V617F) mutations. The outcomes of these cases are critically influenced by the transition from JAK2 (V617F) heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2 (V617F), is highly desirable. In this study, we present an approach to assess the JAK2 (V617F) burden from genomic DNA (gDNA) and complementary DNA (cDNA) using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR). Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2 (V617F) spaced from one template for JAK2(Wild Type) were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2(V617F) burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.53 ± 4.2% and 51.46 ± 4.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation) using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2 (V617F) expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2 (V617F) from heterozygosity to homozygosity.
Project description:Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-??Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-??Cq method-the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.
Project description:Proinflammatory cytokines such as TNF? are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNF? in promoting clonal dominance of JAK2(V617F) expressing cells in MPN. We show that JAK2(V617F) kinase regulates TNF? expression in cell lines and primary MPN cells and TNF? expression is correlated with JAK2(V617F) allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNF? while colony formation by JAK2(V617F)-positive progenitor cells is resistant or stimulated by exposure to TNF?. Ectopic JAK2(V617F) expression confers TNF? resistance to normal murine progenitor cells and overcomes inherent TNF? hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNF? limits clonal expansion and attenuates disease in a murine model of JAK2(V617F)-positive MPN. Altogether our data are consistent with a model where JAK2(V617F) promotes clonal selection by conferring TNF? resistance to a preneoplastic TNF? sensitive cell, while simultaneously generating a TNF?-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.
Project description:Activating alleles of Janus kinase 2 (JAK2) such as JAK2(V617F) are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5muM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2(V617F) allele burden, JAK2(V617F) cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2(V617F) cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.
Project description:Patients with essential thrombocythemia may carry JAK2 (V617F), an MPL substitution, or a calreticulin gene (CALR) mutation. We studied biologic and clinical features of essential thrombocythemia according to JAK2 or CALR mutation status and in relation to those of polycythemia vera. The mutant allele burden was lower in JAK2-mutated than in CALR-mutated essential thrombocythemia. Patients with JAK2 (V617F) were older, had a higher hemoglobin level and white blood cell count, and lower platelet count and serum erythropoietin than those with CALR mutation. Hematologic parameters of patients with JAK2-mutated essential thrombocythemia or polycythemia vera were related to the mutant allele burden. While no polycythemic transformation was observed in CALR-mutated patients, the cumulative risk was 29% at 15 years in those with JAK2-mutated essential thrombocythemia. There was no significant difference in myelofibrotic transformation between the 2 subtypes of essential thrombocythemia. Patients with JAK2-mutated essential thrombocythemia and those with polycythemia vera had a similar risk of thrombosis, which was twice that of patients with the CALR mutation. These observations are consistent with the notion that JAK2-mutated essential thrombocythemia and polycythemia vera represent different phenotypes of a single myeloproliferative neoplasm, whereas CALR-mutated essential thrombocythemia is a distinct disease entity.
Project description:BACKGROUND: Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are myeloproliferative neoplasms (MPNs) characterized in most cases by a unique somatic mutation, JAK2 V617F. Recent studies revealed that JAK2 V617F occurs more frequently in a specific JAK2 haplotype, named JAK2 46/1 or GGCC haplotype, which is tagged by rs10974944 (C/G) and/or rs12343867 (T/C). This study examined the impact of single nucleotide polymorphisms (SNPs) of the JAK2 locus on MPNs in a Japanese population. METHODS: We sequenced 24 JAK2 SNPs in Japanese patients with PV. We then genotyped 138 MPN patients (33 PV, 96 ET, and 9 PMF) with known JAK2 mutational status and 107 controls for a novel SNP, in addition to two SNPs known to be part of the 46/1 haplotype (rs10974944 and rs12343867). Associations with risk of MPN were estimated by odds ratios and their 95% confidence intervals using logistic regression. RESULTS: A novel locus, rs4495487 (T/C), with a mutated T allele was significantly associated with PV. Similar to rs10974944 and rs12343867, rs4495487 in the JAK2 locus is significantly associated with JAK2-positive MPN. Based on the results of SNP analysis of the three JAK2 locus, we defined the "GCC genotype" as having at least one minor allele in each SNP (G allele in rs10974944, C allele in rs4495487, and C allele in rs12343867). The GCC genotype was associated with increased risk of both JAK2 V617F-positive and JAK2 V617F-negative MPN. In ET patients, leukocyte count and hemoglobin were significantly associated with JAK2 V617F, rather than the GCC genotype. In contrast, none of the JAK2 V617F-negative ET patients without the GCC genotype had thrombosis, and splenomegaly was frequently seen in this subset of ET patients. PV patients without the GCC genotype were significantly associated with high platelet count. CONCLUSIONS: Our results indicate that the C allele of JAK2 rs4495487, in addition to the 46/1 haplotype, contributes significantly to the occurrence of JAK2 V617F-positive and JAK2 V617F-negative MPNs in the Japanese population. Because lack of the GCC genotype represents a distinct clinical-hematological subset of MPN, analyzing JAK2 SNPs and quantifying JAK2 V617F mutations will provide further insights into the molecular pathogenesis of MPN.
Project description:JAK2 V617F is the most common mutation in myeloproliferative neoplasms (MPNs) and is a major diagnostic criterion. Mutation quantification is useful for classifying patients with MPN into subgroups and for prognostic prediction. Droplet digital PCR (ddPCR) can provide accurate and reproducible quantitative analysis of DNA. This study was designed to verify the correlation of ddPCR with pyrosequencing results in the diagnosis of MPN and to investigate clinical implications of the mutational burden.Peripheral blood or bone marrow samples were obtained from 56 patients newly diagnosed with MPN or previously diagnosed with MPN but not yet indicated for JAK2 inhibitor treatment between 2012 and 2016. The JAK2 V617F mutation was detected by pyrosequencing as a diagnostic work-up. The same samples were used for ddPCR to determine the correlation between assays and establish a detection sensitivity cut-off. Clinical and hematologic aspects were reviewed.Forty-two (75%) and 46 (82.1%) patients were positive for JAK2 V617F by pyrosequencing and ddPCR, respectively. The mean mutated allele frequency at diagnosis was 37.5±30.1% and was 40.7±31.2% with ddPCR, representing a strong correlation (r=0.9712, P<0.001). Follow-up samples were available for 12 patients, including eight that were JAK2 V617F-positive. Of these, mutational burden reduction after treatment was observed in six patients (75%), consistent with trends of hematologic improvement.Quantitative analysis of the JAK2 V617F mutation using ddPCR was highly correlated with pyrosequencing data and may reflect the clinical response to treatment.
Project description:<h4>Background</h4>Primary myelofibrosis (PMF) is an acquired clonal disease of the hematopoietic stem cell compartment, characterized by bone marrow fibrosis, anemia, splenomegaly and extramedullary hematopoiesis. About 60% of patients with PMF harbor a somatic mutation of the JAK2 gene (JAK2-V617F) in their hematopoietic lineage. Recently, a splicing isoform of JAK2, lacking exon 14 (JAK2?14) was described in patients affected by myeloproliferative diseases.<h4>Materials and methods</h4>By using a specific RT-qPCR method, we measured the ratio between the splicing isoform and the JAK2 full-length transcript (JAK2+14) in granulocytes, isolated from peripheral blood, of forty-four patients with PMF and nine healthy donors.<h4>Results</h4>We found that JAK2?14 was only slightly increased in patients and, at variance with published data, the splicing isoform was also detectable in healthy controls. We also found that, in patients bearing the JAK2-V617F mutation, the percentage of mutated alleles correlated with the observed increase in JAK2?14. Homozygosity for the mutation was also associated with a higher level of JAK2+14. Bioinformatic analysis indicates the possibility that the G>T transversion may interfere with the correct splicing of exon 14 by modifying a splicing regulatory sequence.<h4>Conclusions</h4>Increased levels of JAK2 full-length transcript and a small but significant increase in JAK2 exon 14 skipping, are associated with the JAK2-V617F allele burden in PMF granulocytes. Our data do not confirm a previous claim that the production of the JAK2?14 isoform is related to the pathogenesis of PMF.
Project description:Polycythemia vera is the ultimate phenotypic consequence of the V617F mutation in Janus kinase 2 (encoded by JAK2), but the extent to which this mutation influences the behavior of the involved CD34+ hematopoietic stem cells is unknown.We analyzed gene expression in CD34+ peripheral-blood cells from 19 patients with polycythemia vera, using oligonucleotide microarray technology after correcting for potential confounding by sex, since the phenotypic features of the disease differ between men and women.Men with polycythemia vera had twice as many up-regulated or down-regulated genes as women with polycythemia vera, in a comparison of gene expression in the patients and in healthy persons of the same sex, but there were 102 genes with differential regulation that was concordant in men and women. When these genes were used for class discovery by means of unsupervised hierarchical clustering, the 19 patients could be divided into two groups that did not differ significantly with respect to age, neutrophil JAK2 V617F allele burden, white-cell count, platelet count, or clonal dominance. However, they did differ significantly with respect to disease duration; hemoglobin level; frequency of thromboembolic events, palpable splenomegaly, and splenectomy; chemotherapy exposure; leukemic transformation; and survival. The unsupervised clustering was confirmed by a supervised approach with the use of a top-scoring-pair classifier that segregated the 19 patients into the same two phenotypic groups with 100% accuracy.Removing sex as a potential confounder, we identified an accurate molecular method for classifying patients with polycythemia vera according to disease behavior, independently of their JAK2 V617F allele burden, and identified previously unrecognized molecular pathways in polycythemia vera outside the canonical JAK2 pathway that may be amenable to targeted therapy. (Funded by the Department of Defense and the National Institutes of Health.).