Idiopathic Myelofibrosis Patients Research Articles

Chemical Mutagenesis Screen Identifies Novel Drug Resistant JAK2 Variants in V617FJAK2 Mediated MPN and Predict HSP90 Inhibitors As Possible Agents to Overcome Drug Resistance

JAK2 V617F can be identified in the majority of polycythemia vera cases and in 50% of essential thrombocythemia and idiopathic myelofibrosis patients. JAK2 inhibitors including ruxolitinib, fedratinib and lestaurtinib display clinical activity in trials for PV, ET and IMF, and ruxolitinib has recently been approved for the treatment of primary and secondary myelofibrosis. In other malignancies it has been demonstrated that acquired resistance to kinase inhibitors emerge due to secondary resistance mutations in the targeted kinase. To identify JAK2 point mutations mediating ruxolitinib resistance and predict alternatively treatment strategies, we exposed JAK2 V617F expressing Ba/F3 cells to ruxolitinib. Surprisingly, sublines resistant to ruxolitinib at 1000nM, 2000nM and even 4000nM did not harbor point mutations neither in the kinase nor in the pseudokinase domain of JAK2. However, western blot analysis of sublines resistant to ruxolitinib revealed a 45-kDa JAK2 variant together with full length JAK2 V617F protein in 87% of the cases. Sequencing of the short form in drug resistant clones revealed a novel JAK2 variant missing amino acids 76 to 820 resulting in the N-terminal FERM domain directly fused to the kinase domain of JAK2 (FERM-JAK2). FERM-JAK2 was highly resistant to the ATP-competitive JAK2 inhibitors ruxolitinib and fedratinib. Phospho-deficient mutant studies provided evidence that FERM-JAK2 preferentially exist in an inactive state. This would impede drug binding and explain the resistant phenotype of FERM-JAK2.However, in this study we could not identify other genetic TKI resistant JAK2 variants than FERM-JAK2. This is in remarkable contrast to BCR-ABL mediated CML, where more than 90 residues have been described as TKI resistant variants and might indicate that in case of JAK2, the residues which mediate the drug resistance might comprise the JAK2 kinase function. Therefore, we treated JAK2 V617F expressing Ba/F3 cells with a chemical mutagen (ENU) to predict point mutations in JAK2 that might cause resistance towards the ruxolitinib. Surprisingly, after ENU pretreatment the frequency of drug resistant clones increased and most of the drug resistant clones displayed point mutations in the JAK2 kinase domain. In this screen, we identified L902Q, Y931C, L983F as the most frequent mutations and L902Q+R938E, L902Q+N947Q, L902Q+E1028K and L983F+Q959H as minor mutations. All these mutations confer cross-resistance across a panel of different JAK2 kinase inhibitors except L983FJAK2. L983FJAK2 reduces the sensitivity of JAK2-dependent cells to ruxolitinib and JAK inhibitor-1 but does not reduce the sensitivity of cell proliferation against fedratinib, thereby indicated that we identified a compound specific resistant mutations which is not an ATP-competitor specific mutation.Structure based modeling studies suggest that ruxolitinib is held by numerous hydrophobic interactions with residues Leu 855, Val 863, Ala 880, Val 911, Met 929, Leu 932 and Leu 983 that line the binding pocket. The aromatic ring of Tyr 931 is close enough to the pyrimidine ring to have p-p interaction. Surprisingly, this important interaction does not seem to be critical for the orientation of the inhibitor in the binding pocket as the Y931C mutation does not lead to a different binding pose. Leu 902 does not directly interact with ruxolitinib, however, it is close to the binding pocket and its mutation to Gln with a polar side chain significantly disturbs the inhibitor binding, thus explaining the high resistance of this mutation to ruxolitinib. Mutation of Leu 983 to Phe disrupts important hydrophobic interactions (e.g. Ala 880, Val 911, Met 929) with the pyrrolopyrimidine moiety and induces aromatic-aromatic interaction between the new phenyl ring and the pyrrol and pyrazol rings. Finally, our study also provided evidence that HSP90 inhibitors 17-AAG and geldanamycin are highly potent against TKI resistant FERM-JAK2, L902Q, Y931C and L983FJAK2 through JAK2 degradation and inhibition of the JAK2-STAT5 signaling axis. Taken together, our study highlight HSP90 inhibitors as a promising therapeutic agent in drug resistant mutations against tyrosine kinase inhibitors in MPN. DisclosuresNo relevant conflicts of interest to declare.

JAK 2V617F Mutation: Frequency and Relation to Clinical and Laboratory Features of BCR-ABL Negative Myeloproliferative Diseases

In this study, we planned to investigate frequency of the JAK2 V617F mutation and it’s relation with clinical and laboratory findings of BCR-ABL negative myeloproliferative diseases (MPD) which consist of polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Totally 65 patients were included in the study which composed of 28 (43.1 %) PV, 29 (44 %) ET and 8 (12.3 %) IMF patients. Forty one (63%) patients were female and 24 (37%) were male. Mean age of patients was 64.02±12.42. Frequency of the JAK2V617F mutation was found in 25 (89.3%) of PV, 18 (62.1%) of ET and 2 (25%) of IMF patients. We found no difference in gender and frequency of thrombosis, constitutional symptoms, pruritus, hemorrhage, splenomegaly, bone marrow fibrosis, cytoreductive treatment requirement, arterial/venous thrombosis between JAK2 V617F mutated and unmutated PV and ET and IMF patients. When compared homozygous and heterozygous JAK2 V617F mutated PV and ET patients according to these variables, there was no significant difference. There were no statistically significant difference in Hemoglobin (Hb), hematocrit (HCT), leukocyte, lactate dehidrogenase (LDH), EPO and ferritin levels between JAK2 V617F mutated and unmutated PV and ET patients. HCT levels and leukocyte counts were significantly higher in JAK2 V617F mutated than unmutated IMF patients (p=0.046, p=0.046). Other variables were not found different. Comprehensive prospective studies are necessary for determining the relationship of the JAK2 V617F mutation with clinical and laboratory findings .

Peptide Nucleic Acid (PNA)-Clamping Competitive PCR: A New Tool for the High Sensitive Detection of JAK2V617F Mutation in Myeloproliferative Neoplasms (MPNs)

Abstract 5168 Background:Myeloproliferative neoplasms (MPNs) are a group of diseases characterized by clonal expansion of single or multiple lineages of myeloid subset (i.e. granulocytic, erythroid, megakaryocytic and mast cell). Since 2008, the WHO included the detection of JAK2 mutations (the common V617F and the less common exon 12 mutations), into the diagnostic criteria of MPNs. The specific pathogenic implication of JAK2 mutations in MPNs is still under investigation. Preliminary data emerging from the treatment of Idiopathic Myelofibrosis patients with JAK2 inhibitors have shown only clinically significant benefits (improvement of splenomegaly and constitutional symptoms) but not a clear evidence of disease-modifying activity. Several techniques (e.g. ASO-PCR, ARMS-PCR, Direct sequencing, HRM, DHPLC, etc) have been used to detect the JAK2 V617F mutation, but all of them were low sensitive and time-consuming. We developed a PNA-clamping competitive PCR assay able to detect JAK2 V617F mutation with a very high level of sensitivity and specificity. Methods:In order to promote the selective amplification of the JAK2 V617F mutant allele, a specific PNA oligonucleotide, full matching with the wild-type (wt) sequence of the portion of JAK2 gene containing the V617F mutation, was designed to compete with the reverse primer used in the PCR assay. It was experimentally demonstrated that a PNA concentration of 6 μmol/L occurred for the complete clamping of 100 ng of wt genomic DNA used as template for the PCR reaction. The sensitivity of the assay was determined by a serial dilution (100% through 0.01%) of genomic DNA containing JAK2 V617F mutation, obtained from HEL cell line, with wt DNA obtained from healthy donors. The specificity of PCR products was assessed by sequencing in both forward and reverse directions. The thermal dissociation profile of PNA/DNA and DNA/DNA duplexes was studied by monitoring the Enthalpy as function of the temperature (range 20–100°C), with an heating/cooling rate of 1°C/min. Results:PNA-clamping competitive PCR was able to detect JAK2 V617F mutation with a sensitivity of 0.01%. Thermodynamic studies clearly showed that the melting temperature (Tm) of fully matched PNA/DNA duplex is always higher than Tm of the corresponding DNA/DNA duplex. The enthalpy values for the hybrid PNA/DNA are always greater than the corresponding DNA/DNA duplex and a single mismatch has an energetic cost higher in a PNA/DNA than in DNA/DNA duplex. This energetic cost is more evident in the melting temperature with a ΔTm of 16°C and 7°C for the PNA/DNA and DNA/DNA duplex, respectively. In the PCR assay the PNA complementary to the JAK2 wild type sequence strongly compete with the primer resulting in a complete knock out of the wild type allele. In a blind screening of samples obtained from 308 MPN patients, PNA clamping competitive PCR was able to detect JAK2 V617F mutation in 61.4% cases of Polycythemia Vera, 54.4% of Essential Thrombocythemia, 57.9% of Idiopathic Myelofibrosis and in 21.4% of Ph negative-MPNs, respectively. In 40 unselected patients, PNA clamping PCR results were confirmed by other techniques such as ASO-PCR, ARMS-PCR and direct sequencing. Conclusions:The PNA-clamping competitive PCR assay could be used as convenient, high-sensitive and reliable diagnostic test for detection of JAK2 V617F mutation both on genomic DNA and cDNA samples. As suggested from the thermodynamic studies, a similar technique could be developed for the detection of any known single point mutation, widely contributing to the improvement of molecular diagnostic tests usable in clinical practice. Disclosures:No relevant conflicts of interest to declare.

Mega-dose methylprednisolone (MDMP) for chronic idiopathic myelofibrosis.

I would like to make few comments on Balci and her colleagues’ paper entitled “Complex cytogenetic findings in the bone marrow of a chronic idiopathic myelofibrosis patient” which was published in the recent issue of the Journal. [1]. Although the authors’ cytogenetic findings looks original, it should be considered that chronic idiopathic myelofibrosis (CIF) is a colonal disorder like chronic myeloid leukemia (CML), polycythemia vera, etc. Among them only Philadelphia chromosome (Ph1) is specific for CML, but mutations are present in all of these clonal disorders. I believe since the cytogenetic changes are not pathognomic or specific, these kind of studies should be carried only on research basis. In addition to radiation as mentioned by the authors makes the interpretation of their findings more questionable. On this occasion, I would specifically like to emphasize the MDMP treatment for these patients which seems to be the only approach in the treatment of this disorder. In addition to some stem cell transplantation reports, MDMP is cheap, safe and easily applicable in all conditions. Although we first reported patient with CIF in 1957 from this country [2], we treated almost a dozen of CIF cases without major complications in children and adults since 1983 [3-13]. MDMP treatment was mentioned in several occasions in this journal, we sorry that despite of its international consideration, except ITP treatment,my colleagues did not take it seriously [14].

Complex cytogenetic findings in the bone marrow of a chronic idiopathic myelofibrosis patient.

Chronic idiopathic myelofibrosis is a myeloproliferative disorder characterized by splenomegaly, myeloid metaplasia and reactive bone marrow fibrosis. Karyotype analysis of the bone marrow is an integral part of the diagnosis, especially as a discriminative tool in ruling out reactive conditions. The frequency of clonal cytogenetic anomalies in this disease is the highest among its group, varying between 30 and 75%. Among these, trisomy 1q, 20q-, 13q- and +8 are the most common aberrations. Here we report a 66-year-old male patient whose bone marrow biopsy revealed signs of chronic myeloproliferative changes and dysmegakaryopoiesis. He was administered hydroxyurea treatment, splenic radiotherapy and multiple transfusions. The patient worsened in the following months and the second bone marrow biopsy revealed myelofibrosis. Cytogenetic analysis of this bone marrow sample revealed a complex karyotype reported to be 46,XY,del(9)(q22q34),t(8;17;21)(q22;q21;q22)[23]/46,XY[2], with a previously undefined three-way translocation and deletion in chromosome 9. The patient died shortly thereafter.

Association of V617F Jak2 mutation with the risk of thrombosis among patients with essential thrombocythaemia or idiopathic myelofibrosis: A systematic review

Many studies evaluated the association of V617F Jak-2 with the risk of thrombosis in patients with essential thrombocythaemia, but the results of these studies were inconsistent. Few studies evaluated the association of V617F Jak-2 mutation with the risk of thrombosis in patients with idiopathic myelofibrosis. Therefore, we performed a systematic review of the studies that assessed the risk of thrombosis associated with V617F Jak-2 in patients with ET or IM. We searched MEDLINE and EMBASE databases and reference lists of retrieved articles. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated for each trial and pooled. We included 21 studies involving patients with essential thrombocythaemia and 6 studies patients with idiopathic myelofibrosis. In essential thrombocythaemia patients, V617F Jak-2 was associated with a significant increased risk of thrombosis (OR 1.92, 95% CI 1.45-2.53), both of venous (OR 2.49, 95% CI 1.71-3.61) and arterial (OR 1.77, 95% CI 1.29-2.43) vessels. In idiopathic myelofibrosis patients, the risk of thrombosis associated with V617F Jak-2 tended to be increased (OR 1.76, 95% CI 0.91-3.41). Our systematic review suggests that V617F Jak-2 increases the risk of thrombosis in essential thrombocythaemia patients by about two fold while its role in idiopathic myelofibrosis patients is uncertain.

AML transformation in 56 patients with Ph− MPD in two well defined populations

The Philadelphia chromosome-negative (Ph-) chronic myeloproliferative disorders (MPD) have an inherent tendency for transformation into acute myelogenous leukaemia (AML). The long-term rate of leukaemic transformation in unselected MPD patients was studied in well-defined MPD populations in Gothenburg, Sweden and the Côte d'Or area, Burgundy, France, respectively. Over a median observation time of 15 yr, 56 subjects (7%) out of a total of 795 patients with Ph- MPD transformed to AML. The yearly incidence of AML transformation was 0.38% in polycythaemia vera (PV), 0.37% in essential thrombocythaemia (ET) and 1.09% in idiopathic myelofibrosis (IMF). The incidence of AML development was significantly higher in IMF as compared with both PV and ET (P = 0.002 and P = 0.02, respectively). Six of the patients who developed AML had never been treated with cytoreductive agents and two had only been exposed to interferon. In IMF, the average time from diagnosis to AML transformation was 42 +/- 33 months, which was significantly shorter than for both PV and ET (88 +/- 56 and 76 +/- 57 months; P = 0.0075 and P = 0.027, respectively). The time from diagnosis to AML transformation appears to be a continuous event as regards all three MPD entities. It was shown that 17 out of the 18 patients with PV who developed AML were females; this was true despite the fact that the male/female ratio for the whole PV group was 146/171 (0.85). As regards ET and IMF patients who transformed to AML, the gender ratio showed slight male predominance (1.33 and 1.13, respectively). The average survival time for the 56 MPD patients who developed AML was 4.6 +/- 5.5 (range 0-28) months and did not differ with respect to the three subtypes of pre-AML MPD.

Integrative Analysis of Genomic Aberration and Gene Expression Suggests That MPD with Loss of Heterozygosity or Gain of Chromosome 9 Represents a Distinct Entity

Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) can all be associated with the JAK2V617F gain of function mutation. MPDs are also associated with gross cytogenetic anomalies including genomic gain/loss or loss of heterozygosity (LOH) due to mitotic homologous recombination. To determine whether genomic anomalies may be associated with JAK mutation status/disease phenotype we subjected genomic DNA from the granulocytes of 87 patients with PV, ET or IMF to analysis using high resolution Affymetrix 250K Nsp SNP arrays with an average probe spacing of 12Kb. Firstly this analysis precisely mapped previously identified anomalies in MPD patients such as gain of chromosome 1q chromosome 9 (Chr9), chromosome 8, and deletion of 20q and 13q indicating the robustness of the technique. By removing genomic Copy Number Polymorphisms (CNPs) ascertained from 90 normal individuals from the HapMap dataset, we further detected a number of novel alterations such as frequent gain of Chr9p33–34 harboring among other genes, Notch1. MPD patients heterozygous for the JAK V617F mutation exhibited higher genomic instability than JAK2 V617 homozygous or wild type patients. IMF patients had overall more aberrations than PV or ET patients but unsupervised hierarchical clustering of chromosomal anomalies could not distinguish patients with PV, ET or IMF. Whole chromosomal gain or loss was equally frequent among the three diseases, however IMF patients had more frequent alteration of 10Mb or greater when compared to ET or PV (p<0.01) and ET and IMF patients both more frequently exhibited changes ranging from 1–10Mb in size than PV patients (p<0.01). Correlation of gene expression profiles from the granulocytes of 38 MPD patients with copy number profiles from DNA of the patients indicated that gene expression in regions of copy number gain or loss correlated with gene dosages in general. From 2493 genes residing in regions of copy number change region, 259 genes exhibited a positive correlation between copy number and gene expression. By calculating the t–statistics of expression values between samples with and without chromosomal aberrations we further identified 28 genes that showed a change of at least 1.5 fold (p<0.05), 27 of which were located on chromosome 9. Among these were Smarca2, NOTCH1 JAK2 itself. JAK2V617F expression was highly elevated by gain of Chr9 or Chr9p LOH. LOH on chr9p was detected in 16 patients, the majority of whom were confirmed as homozygous for JAK2V617F. To investigate how the over-expression of JAK2V617F might affect the expression of other genes and pathways, gene expression profiles of patients with Chr9 abnormality including Chr9 gain or Chr9p LOH were compared to samples with a normal copy number for Chr9. We identified 493 genes including 210 up-regulated and 283 down-regulated genes by at least 1.5 fold at a p value of 0.05 after multiple-testing correction. As a control gene expression patterns in JAK2-wildtype MPD granulocytes were compared to expression in normal granulocytes. This analysis identified an almost completely different set of genes; 441/493 (89%) of genes differentially expressed in Chr9 abnormal cases versus Chr9 normal cases were not found in the Chr9 normal versus normal control dataset. Up-regulated genes in Chr9 abnormal cases included CD177, CDK5RAP2, BIRC1, STAT5, TLR4 and down-regulated genes include KLRB1, Myc, RUNX3, P53CSV, Pathway analysis of dysregulated genes in Chr9 gain/LOH cases showed that genes involved in p38/MAP kinase, Toll-like receptor and Fc epsilon RI signaling were up-regulated and the genes involved in pathways of protein synthesis/translation and T cell receptor signaling were down-regulated. Collectively these findings suggest that MPD associated with Chr9 gain or 9pLOH and increased expression of JAK2V617F represents a distinct subset of MPD, characterized by less genomic instability than other cases. Furthermore, increased JAK2 and STAT5 expression and activity in these cases may induce distinct downstream pathways and alterations in gene expression.

Development of High Throughput PCR-Invader Assay to Detect the JAK2 V617F Mutation in Japanese Myeloproliferative Disorders

The classic myeloproliferative disorders (MPD) of polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) are hematopoietic ones characterized by the overproduction of one or more mature myeloid lineages. Although their clonal nature and cytokine independence were recognized a couple of decades ago, their molecular abnormality was not clarified until the identification of the activating JAK2 V617F mutation. Around 95% of patients with PV have the JAK2 V617F mutation. The JAK2 V617F mutation is found in approximately half of ET and IMF patients. ET patients with JAK2 V617F resemble PV patients such as in their increased erythropoiesis and granulopoiesis, and have increased rates of venous thrombosis compared to JAK2 V617F-negative patients. Accurate genotyping of JAK2 is critical for a diagnosis of MPD and can be useful for treatment decisions. However, compared to Western populations, there is limited information available about JAK2 mutation in a Japanese population. This prompted us to analyze JAK2 genotyping utilizing a new method. Although various methods such as direct sequencing, PCR-RFLP, SSCP and DHPLC have been developed for genotyping, none of these provide a rapid, accurate and reproducible system. In this study, a simple assay based on a serial invasive signal amplification reaction assay (PCR-Invader assay) was developed for the detection of JAK2 V617F. The PCR-Invader assay was originally developed for typing SNPs in the human genome. It can detect genotypes in simple 96-well plates without sequence analysis, enzyme digestion or electrophoresis. We analyzed 86 Japanese MPD patients (PV 21, ET 52, IMF 10, others 3). Genomic DNA was extracted from whole blood and the JAK2 V617F mutation then analyzed by the PCR-Invader method. To confirm the accuracy of genotyping by PCR-Invader, the direct sequencing method was also applied to all samples. The percentage of JAK2 V617F mutation-positive patients was PV 81.0%, ET 48.1% and IMF 80.0%. Both white blood cell and platelet counts were higher in V617F-positive PV compared with V617F-negative PV samples (p<0.05). V617F-positive ET samples were accompanied by higher counts of white blood cells and platelets compared with V617F-negative ET ones. However, we did not observe the any relationship between blood cell counts and V617F mutation in IMF; consistent with previous reports. We confirmed that this PCR-Invader method was approximately 20 times more sensitive than direct sequencing. There were no false positive results by PCR-Invader in all 86 samples tested; indicating that PCR-Invader has cost-effectiveness advantages. It has been reported that the activating MPL mutation is observed in some populations of IMF and ET; therefore, we analyzed exon 10 of the MPL genome by direct sequencing. We found that only two V617F-negative ET patients had MPL mutations; W515K and W515-P518delinsKT. There were no MPL mutations in V617F-positive ET patients or all IMF patients. The two ET patients with MPL mutations displayed lower hemoglobin and higher platelets at diagnosis, which is in line with the recently report of the PT-1 study group. In this study we analyzed the JAK2 V617F mutation utilizing the new PCR-Invader assay and undertook MPL mutation by direct sequencing in a Japanese population. Given that some JAK2 inhibitors for MPD patients are now in clinical trials, we expect that this simple PCR-Invader assay and the clinical information about JAK2 and MPL mutations in Japanese MPD patients will be useful to develop clinical trials for JAK2 inhibitors in Japan.

The Investigation of JAK2 and C-MPL Mutations of Myeloproliferative Disorders Patiens

Most studies have shown that JAK2V617F was found in nearly all the patients with polycythemia vera (PV) and half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). However, JAK2 exon 12 mutations in JAK2V61F-negetive PV patiens and MPLW515L/K mutations in IMF patiens implied other pathogenesis in these myeloproliferative disorders (MPD). Two parts were included in our research to investigate the JAK2 and c-MPL mutations of MPD patients. Firstly, to evaluate the JAK2 exon 12 mutations and its clinical significance in patients with PV, polymerase chain reaction was used to amply the region of JAK2 exon 12 and direct sequencing was performed to detect mutations of JAK2 exon 12. Clinical features of the PV patients with JAK2 exon 12 mutations and JAK2V617F mutation were retrospectively reviewed. The results showed that the JAK2 exon 12 mutations were detected in 3 of 12 JAK2V617F-negtive PV patients (25.0%). Three JAK2 exon 12 mutations were H538QK539L, K539L and N542-E543del respectively. The patients carrying the mutations of JAK2 exon 12 displayed higher hemoglobin(223.6±15.0 vs190.3±20.2g/l, P<0.05), lower white blood cell counts (7.8±2.3 vs16.2±6.7 *109/l, P<0.05)compared with JAK2V617F-positive PV patients (n=31). Secondly, allele specific polymerase chain reaction (AS-PCR) and gene sequencing were used to detected JAK2V617F, MPLW515L and four types of JAK2 exon 12 mutations (F537-K539delinsL, H538QK539L, K539L, N542-E543del) in 30 IMF patients. Results showed that 15 of 30 (50.0%) patients had JAK2V617F mutation and 2 of 30 (6.7%) patients had MPLW515L mutation. JAK2 exon 12 mutations were not detected in IMF patients. Literature reviews show that this is the first report of JAK2 and c-MPL mutations in a number of Chinese patients with BCR/ABL-negative MPD. It is concluded that the JAK2 exon 12 mutations exist in PV patient and the patients with JAK2 exon 12 mutations are characterized by isolated erythrocytosis. The frequency of MPLW515L mutations is high in IMF, which supports recent studies.

Analysis of Idiopathic Myelofibrosis Initiating Cell in NOD/SCID/IL2rgKO Mice

Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These cells are thought to secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and the fibrotic changes in the BM lead to extramedullary hematopoiesis and increased frequency of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate from an abnormality at the level of hematopoietic stem cell (HSC), this has not been experimentally addressed using primary human IMF samples. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we employed the newborn NOD/SCID/IL2rg-null xenotransplantation model that efficiently supports engraftment of normal and malignant human stem cells. We purified PB CD34+ cells and PB CD34+CD38− cells from four IMF patients, and intravenously transplanted the purified cells into newborn NOD/SCID/IL2rg-null recipients. In long-term observation of the recipient mice, we analyzed human CD45+ hematopoietic cell chimerism both in the PB and in the BM, suppression of murine normal hematopoiesis, and the fibrotic changes in the BM. Twelve out of nineteen recipients transplanted with patient CD34+ cells or CD34+CD38− cells exhibited human hematopoietic engraftment, and the frequency of CD33+ myeloid cells (82.5+/−12.2% among the engrafted CD45+ cells) was higher than that in the recipients transplanted with normal HSCs. These CD33+ cells expressed other myelo-monocytic markers such as CD14, CD11b, CD15, and HLA-DR. BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of fibroblasts and the presence of human megakaryocytes, recapitulating the clinical features of IMF. In the 7 remaining recipients, PB hCD45 chimerism was < 1.5% at thirty-two weeks and decreased over time and fibroblast proliferation could not be demonstrated in the BM at forty weeks. With FISH analysis using human X probe and immuno-staining using anti-TGF-beta1 antibody, we could confirm the TGF-beta1 production of human megakaryocytes in the recipient BM. To investigate the origin of BM fibroblasts, we performed FISH analysis using human X chromosome probe and mouse centromeric probe, and immuno-staining using anti-CD45 and anti-vimentin antibodies. Of one hundred fifty fibroblasts examined, one hundred thirty six cells (90.7%) were of human origin. These findings demonstrate that the IMF-initiating cells are contained within the CD34+CD38− HSC fraction and these cells possess differentiation capacity to fibroblasts. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and the development of novel therapeutic agents for IMF.

Prevalence of the JAK2‐V617F mutation in Taiwanese patients with chronic myeloproliferative disorders

The Janus kinase-2 (JAK-2) V617F mutation has been recently reported in patients with myeloproliferative disorders (MPD), which is believed to underlie growth factor hypersensitivity displayed by haematopoietic progenitors in these disorders. However, its frequency has been rarely determined in Taiwanese patients. The frequency of JAK2-V617F mutation in patients with polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis (IMF) was determined in the DNA from the peripheral blood leucocytes of 108 patients by genomic polymerase chain reaction and restriction enzyme-based assay. The JAK2-V617F mutation could be detected in 28 of 33 polycythaemia vera patients (85%), 29 of 49 essential thrombocythaemia patients (59%) and 2 of 6 IMF patients (33%), but was not detected in 11 patients with myelodysplastic syndrome or another 10 with other haematological diseases. The presence of the JAK2 mutation was associated with specific MPD disease subtypes (P = 0.007), longer disease duration (P = 0.005), splenomegaly (P = 0.019), a higher white blood cell count (P = 0.002) and a higher haemoglobin level (P = 0.036). However, the overall risk of thrombosis or bleeding was not affected by the presence of the JAK2 mutation (32 vs 17%; P = 0.22). The JAK2-V617F mutation can be frequently detected in the Taiwanese patients with MPD disorders and therefore should be incorporated into the initial evaluation of patients suspected of MPD.

Differences in proportion and dynamics of recipient hematopoiesis following hematopoietic cell transplantation in CML and IMF

Since decades myeloablation followed by allogeneic stem cell transplantation offered the only opportunity to cure leukemia patients and only recently the development of STI571 created a further alternative in chronic myeloid leukemia (CML). While among all leukemias this transplantation regimen had the best outcome in CML, trials with reduced intensity conditioning regimens (RIC) were rather humbling and recurrence of the neoplastic clone occurred frequently. However, the same therapy in patients with idiopathic myelofibrosis (IMF) resulted in a more favorable outcome. Therefore, long-term mixed chimerism (mCh) was determined on bone marrow (BM) biopsies derived from five IMF patients and from eight CML patients of the pre STI era following sex-mismatched transplantation. All patients presented lasting hematologic remission and were matched concerning age, sex and appearance of GvHD. Analysis of late transplant period (day +100) revealed a concentration of host cells within the CD34+ precursor cell compartment in both diseases. However, in IMF BM biopsies only up to 8% recipient CD34+ precursors but in CML biopsies up to 26% recipient CD34+ precursors were detected. Taken into account that in CML up to 10% of the host BM CD34+ precursors bear the BCR-ABL translocation our data suggest that the neoplastic CD34+ progenitor cell population might dispose of better strategies to escape immune surveillance in CML than in IMF.

Clonal Dominance at the Progenitor Cell Level Defines Clinical Phenotype More Accurately Than the Neutrophil Allele Burden in JAK2V617F-Positive MPD Patients.

The chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET) and idiopathic myelofibrosis (IMF) differ phenotypically but share the same mutation, JAK2V617F. To determine how the same mutation yields three different phenotypes, we examined the relationship between disease phenotype and the quantitative JAK2V617F allele burden in both CD34+ cells and neutrophils. The study population consisted of 126 PV, 41 ET and 18 IMF JAK2V617F-positive patients with a median disease duration of 8 years. The JAK2V617F allele burden was determined by allele-specific quantitative PCR using genomic DNA from neutrophils and circulating purified CD34+ cells; in 6 ET, 6 IMF and 13 PV patients, erythroid colonies cloned from CD34+ cells were genotyped for JAK2V617F. As we previously reported, the mean neutrophil JAK2V617F allele burdens were lower in ET compared to PV and IMF (38% vs 66%; p<.001). In PV but not in ET, the neutrophil JAK2V617F allele burden correlated directly with the reticulocyte count (R=.452, p<.001), the white cell count (R=.519; p<.001) and splenomegaly (R=0.378; p<.001) and inversely with the platelet count (R=0.323; p=0.004). In both ET and PV, the median CD34+ cell JAK2V617F allele burdens were lower than the corresponding neutrophil allele burdens (25% vs 39% in ET, p=.04, and 56% vs 64% in PV, p=.01), but these were equivalent in IMF (77% and 77%). The median CD34+ cell allele burden was lower in ET patients compared to PV or IMF patients (p<.001). In contrast to neutrophils in which there was no correlation between allele burden and disease duration (R = 0.067; p = ns), in PV the CD34+ cell allele burden correlated with disease duration (R=0.358; p = 0.035), and more strongly correlated with the white cell count (R=.725; p<0.001) and spleen size (R=0.635; p<0.001) than the neutrophil allele burden. BFU-e clonogenic assays identified JAK2 wild-type progenitors in ET and PV patients in whom the CD34+ JAK2V617F allele burden was lower than the neutrophil allele burden, and did not identify wild-type progenitors in PV and IMF subjects in whom the CD34+ cell and neutrophil allele burdens were similar. Based on this observation, we defined clonal dominance at the progenitor level as a difference of 10 or less between the neutrophil and CD34+ allele burdens. Clonal dominance was present in 22% of ET, 53% of PV and 90% of IMF patients, and was independent of the clonal genotype as clonal dominance was present with heterozgous only (ET, PV and IMF), homozygous only (PV or IMF) or mixtures of heterozygous and homozygous clones (PV, IMF). Within PV, clonally dominant subjects had significantly longer disease durations (11 vs 5.5 years, p=.01), higher white cell counts (18.2 vs 11.1, p=.02), larger spleens (4 vs 0 cm below the costal margin, p=.003), and were more commonly receiving chemotherapy than the nonclonally dominant PV patients (46% vs 22%, p=.02). Importantly, the neutrophil allele burden did not differ between clonally dominant and nondominant PV patients (mean 72.8 vs 68.7%, p=0.506). We conclude that the extent of JAK2V617F CD34+ cell clonal dominance is a major determinant of disease class within the MPD, and within PV, is a major determinant of extramedullary disease and leukocytosis. The extent of clonal dominance at the progenitor level cannot be determined from the neutrophil allele burden alone, and thus limits the utility of the neutrophil allele burden as a disease marker.

Transplantation of Primary Human CD34+CD38 - Hematopoietic Stem Cells Recapitulates Idiopathic Myelofibrosis in the NOD/scid/IL2rgKO Mice.

Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These cells are thought to secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and the fibrotic changes in the BM leads to extramedullary hematopoiesis and increased frequency of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate from an abnormality at the level of hematopoietic stem cell (HSC), this has not been experimentally addressed using primary human IMF samples. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we employed the newborn NOD/SCID/IL2rg-null xenotransplantation model that efficiently supports engraftment of normal and malignant human stem cells. We purified PB CD34+ cells and PB CD34+CD38- cells from six IMF patients, and intravenously transplanted the purified cells into newborn NOD/SCID/IL2rg-null recipients. In long-term observation of the recipient mice, we analyzed human CD45+ hematopoietic cell chimerism both in the PB and in the BM, suppression of murine normal hematopoiesis, and the fibrotic changes in the BM. Six out of thirteen recipients transplanted with patient HSCs exhibited human hematopoietic engraftment, and CD33+ myeloid cells accounted for 80.5+/−9.41% of all the engrafted CD45 + population (as compared with the recipients transplanted with normal HSCs). BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of fibroblasts and the presence of human megakaryocytes, recapitulating the clinical features of IMF. In the 7 remaining recipients, PB hCD45 chimerism was < 1.5% at thirty-two weeks and decreased over time and fibroblast proliferation could not be demonstrated in the BM at forty weeks. To investigate the origin of BM fibroblast, we performed FISH analysis using human and mouse centromeric probes and immuno-staining using anti-CD45 and anti-vimentin antibodies. Of sixty fibroblasts examined, fifty-four cells were of human origin. These findings demonstrate that the IMF-initiating cells are contained within the CD34+CD38- HSC fraction and these cells possess differentiation capacity to fibroblasts. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and the development of novel therapeutic agents for IMF.

Clinical Significance of MPL Mutations in Essential Thrombocythemia: Analysis of the PT-1 Cohort.

Activating mutations in MPL, the thrombopoietin receptor, were originally described in idiopathic myelofibrosis (IMF) and subsequently in a small minority of patients with essential thrombocythemia (ET). The prevalence and clinical significance of MPL mutations in ET has not been fully characterised. Using prospectively acquired diagnostic and follow-up data from the large MRC PT-1 study, we describe the clinical characteristics, complications and laboratory features of these mutations. Sequencing the entire MPL cDNA from 19 JAK2 wild-type ET patients did not reveal any mutations outside exon 10. Sequencing MPL exon 10 DNA in 88 ET and 112 IMF patients revealed MPLW515L (n=11), MPLW515K (n=3) and MPLS505N (n=2) mutations. The MPLS505N mutation, previously associated with inherited thrombocythemia, was clearly an acquired mutation in both patients (1 ET and 1 IMF). Sensitive PCR-based methods were then developed for all three mutant MPL alleles and used to genotype 776 patients with ET from the PT-1 trial. MPL mutations were detected in 4% of all patients, comprising 9% of JAK2 wild-type patients. MPL mutant patients were older at diagnosis than both JAK2 wild-type (p=0.0001) and JAK2V617F patients (p=0.09). Compared to JAK2V617F patients, MPL mutant patients had lower hemoglobin and higher platelet levels at diagnosis (p=0.0001 and p=0.006 respectively). There were no differences in diagnostic blood counts between MPL mutant and JAK2 wild-type patients. Diagnostic trephine histology for JAK2V617F (n=168), JAK2 wild-type (n=130) and MPL mutant patients (n=13) was reviewed by three independent histopathologists blinded to mutation status. MPL mutant samples were less cellular than JAK2V617F and JAK2 wild-type samples (p=0.0001 and p=0.003 respectively). Compared to both JAK2V617F and JAK2 wild-type samples, MPL mutant samples had lower erythroid cellularity (p=0.0006 and p=0.005 respectively) and granulocyte cellularity (p=0.006 and p=0.05 respectively). There were no differences in megakaryocyte cellularity or reticulin grade between the three groups. MPL mutant patients had an increased risk of venous thrombosis after trial entry when compared to JAK2 wild-type patients (p=0.02); the rate of venous thrombosis in the MPL mutant group was comparable to the JAK2V617F group. The mutant allele burden (assessed quantitatively by pyrosequencing) was significantly higher in patients with MPLW515K compared to patients with MPLW515L mutations (p=0.0001), suggesting biological differences in the transforming abilities of the two mutations. In colony assays using patient mononuclear cells the MPLW515L mutation was associated with cytokine-independent growth of megakaryocyte colonies but not with erythropoietin-independent erythroid colony growth (4/4 patients). Of 48 MPL mutant patients, only one also carried the JAK2V617F mutation by allele-specific PCR. Taken together, our results describe the prevalence, clinical features and laboratory manifestations of MPL mutations in the context of a large prospective study of ET.

Phenotypic variations and new mutations in JAK2 V617F–negative polycythemia vera, erythrocytosis, and idiopathic myelofibrosis

The chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis, and idiopathic myelofibrosis (IMF), are characterized by a spectrum of clinical features and linked by common genetic lesions in JAK2 and MPL. However, the clinical phenotypes in genetically undefined MPD patients are similar to those patients with JAK2 and MPL lesions. We, therefore, sought to determine whether there were JAK2 or MPL lesions in a well-defined, JAK2 V617F-negative MPD cohort, and to determine if clinical associations could be identified based on variations identified in these genes. We examined the JAK2 and MPL genes in JAK2 V617F-negative PV, IMF, and idiopathic erythrocytosis patients for sequence variations. We identified two previously unrecognized JAK2 mutations and three previously unrecognized MPL mutations in JAK2 V617F-negative PV, erythrocytosis, and IMF patients. We identified JAK2 exon 12 lesions in 30% of JAK2 V617F-negative PV patients, and either JAK2 V617F or JAK2 exon 12 lesions in 9% of erythrocytosis patients. In IMF, in addition to the MPL gene mutation, W515K, we identified three additional mutations: 204P and two intervening sequence transitions, IVS 11/12 and 10/11. While the clinical phenotype of JAK2 exon 12 lesions in the MPD was predominantly erythroid, there was significant disease spectrum overlap between JAK2 V617F and JAK2 exon 12 mutations. By contrast, MPL gene mutations were not associated with erythrocytosis, but segregated primarily with the phenotypes of thrombocytosis, extramedullary disease, myelofibrosis, and osteosclerosis.

Detection of JAK2V617F mutation in patients with myeloproliferative disorders with TaqMan-MGB probe

To develop a novel platform for detection of the JAK2V617F mutation in patients with myeloproliferative disorders (MPD) by real-time quantitative PCR. TaqMan-MGB probe was constructed. Peripheral blood samples were collected from 374 MPD patients, 76 with polycythemia vera (PV), 38 with chronic myelogenous leukemia (CML), and 115 with essential thrombocythemia (ET), and 19 with idiopathic myelofibrosis (IMF). Peripheral blood samples from 65 patients with acute myelogenous leukemia (AML), 30 patients with acute lymphoblastic leukemia, 8 patients with chronic lymphoblastic leukemia, and 7 patients with non-Hodgkin's lymphoma and 16 cases of normal donor bone marrow were used as controls. Genomic DNA or RNA was extracted and reversely transcrtibed into cDNA. TaqMan-MGB probe was used to detect the JAK2V617F mutant in MPD. Furthermore, 168 specimens underwent allele specific PCR and 8 specimens underwent sequencing. This method was used on both DNA and cDNA specimens from 38 MPD patients simultaneously so as to test the consistency. The JAK2V617F mutation rates of the PV, ET, and IMF patients were 53 (70%), 59 (51%), (58%) respectively. JAK2V617F mutation was found in only one of the 65 AML patients and was not identified in other control specimens. Both the results of allele specific PCR and of sequencing were consistent with the result of TaqMan-MGB probe method. JAK2V617F mutation is widespread in Chinese MPD patients. Real-time quantitative PCR with TaqMan MGB probe can be used for rapid and accurate detection of the JAK2V617F mutation.

Behavior of CD34 + cells isolated from patients with polycythemia vera in NOD/SCID mice

We investigated if polycythemia vera (PV) peripheral blood (PB) CD34+ cells contain cells capable of engrafting nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and if the JAK2V617F mutational burden of these cells alters their behavior in NOD/SCID mice. CD34+ cells isolated from patients with PV, idiopathic myelofibrosis (IM), or granulocyte colony-stimulating factor-mobilized normal donors were transplanted into sublethally irradiated NOD/SCID mice. Cells engrafted into the NOD/SCID mice were analyzed flow cytometrically using lineage-specific antibodies. Genomic DNA was extracted from granulocytes, CD34+ cells, and sorted human CD45(+) cells purified from the bone marrow cells of these mice to examine their JAK2V617F mutational burdens. Multilineage human cell engraftment was observed in mice transplanted with CD34+ cells from mobilized normal volunteers, IM patients and PV patients with high JAK2V617F burden, but not in mice receiving grafts from PV patients with low JAK2V617F burden. The differentiation program of engrafting PV CD34+ cells with high JAK2V617F burden was remarkably different than that of IM CD34+ cells. The JAK2V617F allele frequency in the human CD45+ cells isolated from the mice receiving CD34+ cells was lower than that observed in the CD34+ cell grafts, indicating the persistence of a JAK2V617F negative compartment of stem cells. We conclude that PB CD34+ cells from PV patients with high JAK2V617F burden and patients with IM contain NOD/SCID repopulating cells, and that differentiation program of IM and PV CD34+ cells are dramatically different.

The clinical implications of JAK2V617F mutation in idiopathic myelofibrosis

17542 Background: JAK2V617F is a novel molecular mechanisms of BCR/ABL-negative MPD especially polycythemia vera. However, its role may not be well clarified in idiopathic myelofibrosis (IMF). Methods: To investigate its prevalence and clinical significance in Chinese IMF patients, Allele-Specific PCR (AS-PCR) in combination with sequence analysis were introduced to screen JAK2V617F mutation in our research. Genomic DNA of 12 IMF patients was extracted from peripheral blood mononuclear cells, and AS-PCR was firstly adopted to amplify the exon 12 of JAK2 gene which harbored V617F mutation. The positive samples with JAK2V617F mutation were further confirmed by sequence analysis. In addition, the clinical features of JAK2V617F positive and negative subgroup were compared. Results: The JAK2V617F mutation was identified in 6 IMF patients (50%, 6/12) by AS-PCR and sequence analysis. Its presence is significantly associated with a higher platelet counts, megakaryocyte counts of bone marrow and incidence rate of thrombosis in IMF patients. Conclusions: AS-PCR is a sensitive and accurate technique in identification of JAK2V617F mutation. JAK2V617F mutation of our research is similar to other reports. Moreover, the clinical process of JAK2V617F positive subgroup may be more invasive in comparison with JAK2V617F negative subgroup. No significant financial relationships to disclose.