MPL W515L Research Articles

Acute Panmyelosis with Myelofibrosis: Clinicopathological and Molecular Features of a Rare Malignant Bone Marrow Disease.

Abstract 3101Poster Board III-38 BackgroundAcute panmyelosis with myelofibrosis (APMF) is listed among the category of “AML, not otherwise specified” in the actual WHO classification of hematopoietic tumors. Since specific genetic alterations have not yet been defined, this myeloid neoplasm (MN) remains clinicopathologically assigned. The WHO classification addresses the difficulties concerning the differential diagnosis between APMF, myelodysplastic syndromes (MDS) with excess of blasts and concomitant fibrosis (RAEB-F) and acute megakaryoblastic leukaemia (AMGL). MethodsOur files were searched for patients with rapid-onset MN without a prior history of a myeloproliferative neoplasm or a MDS fulfilling the 2008 WHO criteria of APMF. An age- and gender-matched control group of RAEB-F cases with multilineage dysplasia was retrieved for comparison. Our approach included:i) the careful review of morphological, clinical and laboratory data with special regard to the proportion of immature CD34 + precursors/blasts in bone marrow (BM) biopsies; ii) the grading of BM fibrosis according to the European consensus guidelines; iii) the analysis of the JAK2-V617F, the MPL W515L and the GATA1 mutational status, the cytoplasmic localization of nucleophosphamin1 (NPM1) in BM blasts, and the expression the inhibitor of differentiation protein-2 (Id2) in hematopoietic and stroma cells; iv) the evaluation of the prognostic relevance of these parameters. ResultsFifty-four patients (32 males, 22 females) with a median age of 66, 9 years from our institutions were diagnosed APMF. Sufficient DNA for allele specific PCR for JAK2 genotyping was obtained from paraffin-embedded EDTA-decalcified BMB specimens from 22 APMF and 14 RAEB-F patients. In addition, an analysis of MPL W515L and GATA1 mutations was performed on 15 APMF cases. The relevant clinicopathological and molecular data are summarized in the table.APMFRAEB-FP valueMedianSurvival, median (months)0,0006,5 (*CI: 5,6-7,49)36,3 (CI: 0,9-71,7)MCV, mean (fl)<0,00190,29 (CI: 88,3-92,3)98,3 (CI: 95,2-101,3)LDH, mean ( U/l)<0,001401,8 (CI: 341,6-461,9258,3 (CI: 226,2-290,4)BM fibrosis, (grade)<0,0012,5 (CI: 2,4- 2,7)1,8 (CI: 1,6-2)BM Blasts (%)<0,00131,4 (CI: 27,2-35,5)13,3 (CI: 11,3-14,9)Aberrant NPM1 (%)n.s.12,97%JAK2V617 F + (%)n.s.36%21%MPL W515L +(%)0n.d.GATA1 + (%)0n.d.*CI: 95% confidence intervalSemiquantitative evaluation revealed a decreased expression level of Id2 in erythropoiesis of the APMF samples, while Id2 was increased in the stromal compartment. Within the total APMF cohort, patients aged ≤60 years had a longer OS (p=0,013). A comparison between the Kaplan-Meier curves of the APMF patients who underwent hematopietic cell transplantation (HCT) with those treated by conventional chemotherapy clearly showed that HCT significantly improved OS (median survival 6,1 vs. 11,1 months, p=0,005). Within the non-transplanted group, a bone marrow blast count ≤25% was associated with a longer OS (p=0,006). ConclusionsOur results point to significant differences between APMF and RAEB-F with regard to OS, MCV, LDH, BM fibrosis and BM blast cells. We detected a higher frequency of mutated JAK2 V616F in the MB samples than generally reported in the literature for other categories of AML or MDS. However, the JAK2 V617F mutation status or an aberrant cytoplasmic NPM1 localization were both not predictive of outcome. In contrast to data published for AMGL, no association between a MPL W515L mutation and APMF was observed. Survival was significantly worse in APMF than in RAEB-F. A rapid diagnosis based on a multiparameter approach and a proper management including the option of HCT even in elderly patients seems mandatory for APMF patients. Targeting the fibrotic BM microenvironment which is characterized by an increased Id2 expression may be considered for future therapeutic strategies. DisclosuresSkoda:Genentech Inc.: Consultancy.

TET2 Mutations Are Frequent in RARS-T.

Abstract 3794Poster Board III-730Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Since JAK2 V617F and MPL W515L mutations are present in a significant proportion of RARS-T patients, many investigators consider this entity to be more closely related to classical MPN. However, a significant minority of patients with RARS-T do not display either JAK2 V617F or MPL W515L mutations. We have studied a cohort of patients with RARS-T (N=20) characterized by the presence of ring sideroblasts, reticulin fibrosis and thrombocytosis (>450×109/L), that lack obvious causes of secondary thrombocytosis. While 8/20 patients harbored the JAK2 V617F, and 3/20 the MPL W515L mutations, the molecular pathogenesis for the remaining 9 patients was unexplained. Activation of JAK2 and MPL is associated with aberrant phospho-STAT5. Cases positive only for phospho-STAT5 may harbor other related, so far unidentified mutations. Many groups have recently observed a frequent area of somatic uniparental disomy (UPD) at 4q24, most commonly encountered in patients with chronic myelomonocytic leukemia (CMML), MDS/MPN, some typical MDS, and secondary acute myeloid leukemia (sAML). Overlapping microdeletions and UPD on 4q24 pointed towards possible mutations in the TET2 gene; such mutations were subsequently found in myeloid malignancies, most significantly MPN and MDS/MPN. Based on these findings, and the established correlation of RARS-T with JAK2 V617F and MPL W515L mutations, we evaluated the mutational status of TET2 in RARS-T patients. SNP-A allowed detection of copy neutral loss of heterozygosity (CN-LOH), such as UPD9p, which is associated with the JAK2 V617F mutation, and UPD1p, associated with MPL W515L. SNP-A facilitated detection of previously cryptic lesions; 11/20 patients showed an abnormal SNP-A-based karyotype (only 3 of these defects were also detected by MC). The new lesions seen by SNP-A included various UPD, such as, 1p, 2p, 3q, 6p, 8p, 9p and 10p. The presence of UPD9p/1p was consistent with homozygous JAK2 V617F/MPL W515L mutations. Likely, duplication of mutated alleles constituted a further permissive event during clinical evolution. However, none of the patients showed a somatic LOH at 4q24, suggesting that biallelic TET2 mutations were not involved in the pathogenesis of RARS-T. Simultaneously, lack of UPD11q suggested that CBL mutations were absent. Indeed, Cbl ring finger domain mutational screening revealed no mutations. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10). However, 4 patients demonstrated abnormal megakaryocytic STAT5 phosphorylation, despite the absence of both JAK2 V617F and MPL W515L mutations. Within this group, a monoallelic TET2 mutation, delC 1480Sfs, was identified. In addition, we found a group of 5 patients without either JAK2 V617F or MPL W515L mutations, and also without association of the aberrant phospho-STAT5 staining. One of these patients had a monoallelic TET2 V1718L mutation; interestingly, another patient's specimen showed two novel non-synonymous SNPs: Y867H and P1723S. In total, 2/19 (11%) patients harbored TET2 mutations. These findings indicate involvement of TET2 mutations in RARS-T pathogenesis. RARS-T cases with MPN-associated mutations may not show obligatory phospho-STAT5 staining. The majority of patients were characterized by lack of splenomegaly, decreased white blood cell counts, increased thrombocytosis, and a normal karyotype. In summary, the majority of RARS-T patients harbor JAK2 V617F and MPL W515L mutations that strongly activate STAT5 phosphorylation. We described herein the third most common mutation in RARS-T, which can occur with or without abnormal STAT5 activation. Disclosures:No relevant conflicts of interest to declare.

SNP-Array Profiling Identifies Complex Aberrations and Candidate Genes in Myeloproliferative Neoplasms with Leukemic Transformation.

Abstract 2608▪▪This icon denotes an abstract that is clinically relevant.Poster Board II-584 Background:Myeloproliferative neoplasms (MPN) represent a heterogeneous group of acquired hematopoietic stem cell disorders. Clonality leads to exceeding production of myeloid cells resulting in an inherent tendency for thrombotic and hemorrhagic complications as well as transformation into acute myeloid leukemia (sAML). While vascular complications predominantly account for the morbidity in essential thrombocythemia (ET) and polycythemia vera (PV), the mortality of MPN is significantly related to leukemic transformation. Secondary AML occurs more frequently in primary and secondary myelofibrosis (PMF and SMF) than in ET and PV, and the risk for leukemic transformation increases with the duration of the disease. The molecular basis underlying the progression of MPN is poorly understood. Clonal evolution due to genomic instability is considered to play an important role. Aim:To identify genomic lesions associated with leukemic transformation, we applied 250K single-nucleotide polymorphism (SNP) arrays that allow for genome-wide screening of both copy-number alterations (CNAs) and copy-neutral runs of homozygosity (ROH) at high resolution. Method:An unpaired SNP-array analysis of 23 sAML samples was performed [former diagnosis: ET, n=5; PV, n=7; PMF, n=9; SMF, n=2;]. An own set of 30 reference samples was used for normalization. CNAs and ROH were analyzed by CNAG 3.0 software. Aberrations were compared with the 250K SNP-array dataset of 151 MPN patients [ET, n=45; PV, n=45; PMF, n=47, SMF, n=14]. In one sAML patient corresponding SNP-array data from the time of ET diagnosis were available. Results:CNAs were present in 15 of 23 (65%) sAML patients. Thirty-five percent of cases (n=8) exhibited complex genomic aberrations with up to 20 CNAs in one patient (range 5–20). The most frequent larger (>5 Mb) CNAs were trisomy 8 (n=7), gain of 1q, loss of 5q, and deletion in 6p25-pter (16.2–26.7 Mb) and 20q11-q13 (13.6–16.9 Mb) in three cases each, followed by gain in 3q24-qter (51.7 and 54.1 Mb) in two patients. Of note, one case with deletion in 17p12-pter (64.4 Mb) encompassing TP53 and a second with gain in 21q22.12-qter (11.8 Mb) were identified; in the latter one the proximal breakpoint of the gain was located at RUNX1. Smaller CNAs (<5 Mb) were restricted to single cases with four cases exhibiting micro-deletions ranging from 0.7 to 2.7 Mb in size. Interestingly, three chromosomal regions harbour single genes: 11p11.2 (FOLH1), 18q21.2 (TCF4), and 21q22.12 (RUNX1). ROH comprising the terminal end of the chromosome were detectable in 13 of 23 (57%) sAML cases. The most frequent ROH included the JAK2 locus in 9p24 (n=6; 15.6–38.7 Mb), followed by ROH in 17p13-pter (16.3 and 17.7 Mb) covering TP53 and an overlapping segment in 1p32-pter (53 Mb) affecting MPL in two cases each. All cases with 9p ROH were JAK2 V617F mutated, whereas the MPL W515L mutation was found in one of the two 1p ROH cases. Moreover, sequencing analyses in both patients with ROH in 17p revealed TP53 missense mutations in exon 7 and exon 8, respectively. In addition, non-recurrent ROH covering the long arm of chromosome 7, 11, and 21 as well as ROH in the chromosomal segments 14q32-qter (12.6 Mb) and 17q31-qter (31 Mb) were identified. In one sAML patient SNP-array data performed at the time of ET were available for comparative analysis. While 20q deletion was present as sole aberration in ET, complex genomic aberrations (7 CNAs) were identified after development of sAML. Merging the results from our recent 250K SNP-array analysis (Stegelmann et al., Blood 2008; 112: Abstract #2794) on 61 PMF and SMF cases with data from this study, we were able to identify a second case with micro-deletion in 12q24. The commonly deleted region of both cases is 1.3 Mb in size and encompasses TCF1 as a novel recurrent aberration in MPN. Conclusion:In summary, our data on a large series of well-defined sAML cases that evolved from MPN demonstrate that 250K SNP-array profiling is an excellent tool to identify genomic aberrations. In contrast to MPN, genomic alterations in sAML are characterized by a marked complexity reflecting both genomic instability and genetic heterogeneity of sAML. However, in our study several regions of interest including recurrently affected candidate genes such as TCF1, RUNX1, and TP53 were identified that need to be further investigated on a single gene level. Disclosures:No relevant conflicts of interest to declare.

The Analysis of JAK2 and MPL Mutation as Well as JAK2 SNPs in MPN Patients by MassARRAY Assay.

Abstract 4989 IntroductionRecent studies have shown that JAK2 V617F, MPL W515L/K and JAK2 exon 12 mutations underlie the major molecular pathogenesis of myeloproliferative disorders (MPN). MethodsTo ascertain the real prevalence of these mutations and the influence of genetic susceptibility in Chinese MPN patients, we applied Allele-Specific Polymerase Chain Reaction (AS-PCR), directly sequencing and MassARRAY assay into our study. ResultsThe positive rate of JAK2 V617F in polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) was 82.0%, 36.6% and 51.1% individually. We also found one ET patient, two PMF patients harboring MPL W515L mutation, and three PV patients harboring JAK2 exon 12 mutations. All of these patients were confirmed as JAK2 V617F negative. Moreover, clinical data demonstrated that PV patients with JAK2 exon 12 mutations had higher hemoglobin and lower age as well as WBC than PV patients with JAK2 V617F. In addition, through analysis of 4 polymorphic loci of JAK2 gene, no significant difference of distribution frequency was found among PV, ET and PMF patients. Distribution frequency of haplotype was not found to have significant difference among PV, ET and PMF patients either. ConclusionWe conclude that JAK2 V617F is major molecular pathogenesis in Chinese MPN patients. MPL W515L mutation and JAK2 exon 12 mutations can also be found in JAK2 V617F negative MPN patients. DisclosuresNo relevant conflicts of interest to declare.

Molecular characterization of chronic myeloproliferative neoplasias in México

By using novel molecular markers, it is possible to gain information on both the classification and the pathophysiology of the chronic myeloproliferative neoplasia (MPN). In a group of 36 Mexican mestizo patients with MPN, we studied five molecular markers: The BCR/ABL1 fusion gene, the JAK2 V617F mutation, the JAK2 exon 12 mutations, the MPL W515L mutation and the MPL W515K mutation; 17 patients with essential thrombocythemia (ET), eight with polycythemia vera (PV), four with primary mielofibrosis (MF), five with undifferentiated MPN, one with primary erythrocytosis and one with familial thrombocytosis. Patients with the BCR/ABL1 fusion gene were excluded. Twelve individuals with the JAK2 V617F mutation were found; 11 of them had been clinically classified as PV and one had been classified as MF. One patient with the MPL W515L was identified with a clinical picture of ET. No individuals with either the MPL W515K mutation or the JAK2 exon 12 mutations were identified. Of the 17 individuals with ET, six (35%) had the JAK2 V617F mutation and one (6%) was found to have the MPL W515L mutation. Of the eight individuals with PV, five displayed the JAK2 V617F mutation, whereas of the four patients with MF, one had the JAK2 V617F mutation. The most consistent relationship was that between PV and the JAK2 V617F mutation (p=0·08). In the diagnosis and classification of the MPN, in addition to the newly identified molecular markers, clinical and laboratory data are still very important.

MPL W515L/K mutations in 343 Chinese adults with JAK2V617F mutation‐negative chronic myeloproliferative disorders detected by a newly developed RQ‐PCR based on TaqMan MGB probes

Acquired mutations in the juxtamembrane region of MPL (W515L or W515K), the receptor for thrombopoietin, have been reported in patients with primary essential thrombocythemia (ET) or primary myelofibrosis (PMF). The mutations were detected by the newly developed real-time quantitative PCR (RQ-PCR) with TaqMan MGB probes and followed by the sequencing analysis. DNA samples were from 343 Chinese adults with JAK2V617F mutation-negative chronic myeloproliferative disorders (cMPDs). Reference curves were obtained using cloned fragments of MPL containing either the wild-type or MPL W515L or MPL W515K mutated sequence; the predicted sensitivity level was at least 0.5%(0.1-0.5%) for MPL W515L and 0.5%(0.2-0.5%) for MPL W515K mutant allele in a wild-type background. The detection rates of MPL W515 mutations were 3.5% in 199 ET patients (7/199), 12.5% in 24 PMF patients (3/24) and 5.6% in 36 cMPD-unclassed patients (2/36), respectively. No MPL W515 mutations were detected in 32 polycythemia vera (PV) patients, 40 chronic myeloid leukaemia (CML) patients, 12 hypereosinophilic syndrome (HES) patients and 29 normal volunteers. The mean calculated burden of MPL mutant alleles using RQ-PCR for MPL W515L/K was 24.88 +/- 14.80% (range, 1.10-56.32%). MPL W515L/K patients presented lower haemoglobin levels, compared with the patients with JAK2V617F mutation-positive cMPDs (p < 0.01). The results demonstrated that RQ-PCR was a reliable and sensitive method for large-scale screening of the MPL W515L/K mutation in patients suspected to have a cMPD. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy.

Acquired 1p Uniparental Disomy Is Associated with Biallelic MPL W515L Mutation in RARS-T.

Refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative diseases (MDS/MPD). Since JAK2 V617F mutation is present in a significant proportion of RARS-T patients (Szpurka et al. Blood, 2006), many investigators consider this entity to be more closely related to classical MPD. However, a significant minority of patients with RARS-T do not display a JAK2 V617F mutation. We have studied a cohort of patients with RARS-T (N=18) characterized by the presence of ringed sideroblasts, reticulin fibrosis and thrombocytosis (>600×109/L), that lack obvious causes of secondary thrombocytosis. While 8/18 patients harbored a JAK2 V617F mutation, a molecular pathogenesis for the remaining patients was unexplained. The successful application of SNP-A to characterize the genomic lesions in MDS prompted us to use this technology to study RARS-T. SNP-A allows detection of copy neutral loss of heterozygosity such as UPD9p which is associated with JAK2 V617F mutation. SNP-A facilitated detection of previously cryptic lesions; 9/18 patients showed an abnormal SNP-A-based karyotype often involving multiple lesions (only 5 of these defects were detected by metaphase cytogenetics). The new lesions seen by SNP-A included gains of chromosome 11p, 20q and 21q; deletion of 2p and various areas of UPD including 1p, 9p, 6p, 2p and 8p. SNP-A allowed identification of seemingly invariant UPD1p in 4/18 patients. As this region includes the Mpl gene, we analyzed patients for the presence of MPL W515L/K mutations which have been described in MPD. We did not find any patients with MPL W515K, however MPL W515L mutation was present in 2/4 RARS-T patients with UPD1p; another patient showed monoallelic MPL W515L variant. In addition, 1 patient with UPD1p harbored both JAK2 V617F and MPL W515L mutations. To further delineate the molecular lesion we analyzed all patients for the presence of abnormal STAT5 activation. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10); unexplained STAT5 activation was found in only 4 cases, pointing towards a molecular defect involving this pathway. In these 4 patients, and in 1 additional with UPD1p who did not harbor MPL W515L mutation, we searched for other genes which might explain the pathogenesis of this disease by potentially causing aberrant activation of STAT5. We sequenced Jak1T478S, Jak1V623A and Ntrk1S677N as well as the transmembrane, juxtamembrane and kinase domain of Tie1, Epha2 and Ephb2 genes, but no mutation was found. In addition, we found a group of phospho-STAT5-negative patients (N=4) that showed typical genetic features of myelodysplasia e.g. del(5), +8 and partial loss of chromosome X; these cases are probably best considered to be of MDS origin rather than MPD. To our knowledge, our work is the first description of biallelic MPL W515L mutation and UPD1p found in RARS-T patients. This data is important for understanding the clonal selection process and pathophysiology of activating mutations in MDS/MPD. Overall, our studies demonstrate that somatic UPD1p is associated with homozygous MPL W515L mutation in MDS/MPD cases. Localizing areas of somatic UPD by SNP-A may help identify candidate genes within the shared regions that are likely targets for mutations.

The Overall Analysis of JAK2 and MPL Mutation Status in Chinese MPD Patients by MassARRAY Assay

In recent three years the new notable progress associated with myeloproliferative disorder (MPD) was the identification of JAK2 and MPL mutations, which have been confirmed to be the major molecular mechanism and marker of BCR/ABL negative MPD. Multiple techniques including allele-specific PCR, conventional DNA sequencing, pyrosequencing as well as BsaXI restriction analysis have been used to detect these mutations. The previous data of Shanghai Group had demonstrated the sensitivity and preliminary result of JAK2 V617F in 162 Chinese patients with MPD using MassARRAY assay. To generally identify the frequency of these mutations in Chinese MPD patients, we continued to employ this technique-MassARRAY assay to detect JAK2 V617F, JAK2 K539L as well as MPL W515L mutation in a larger scale of Chinese MPD patients. A total of 204 Chinese MPD patients were enrolled in our study. These patients were referred to polycythemia vera (PV) (n=55), essential thrombocythemia (ET) (n=110), primary myelofibrosis (PMF) (n=29) and hypereosinophilic syndrome (HES) (n=10). 2 ml peripheral blood was obtained with informed consent and genomic DNA was isolated. The diagnosis of MPD was established according to the 2001 WHO diagnostic criteria. Finally, 187 patient samples with good signal can be analyzed for JAK2 V617F mutation. Among the available 101 signals of ET patients, 30 were heterozygous mutation and 9 were homozygous mutation. Among 52 PV patients, 25 were heterozygous mutation and 14 were homozygous mutation. Among 25 PMF patients, 9 were heterozygous mutation and 5 were homozygous mutation. None of HES patients was found harboring JAK2 V617F. In addition, we identified a PV patient harboring JAK2 K539L but not V617F mutation (1/52, 1.9%), and an ET patient harboring MPL W515L mutation (1/101, 1%). Both results were further confirmed by sequence analysis. Hence, we concluded that JAK2 V617F underlie the major molecular pathogenesis of Chinese MPD patients especially PV, however, JAK2 K539L and MPL W515L are rare events in these patients.

Frequency and clinical features of the JAK2 V617F mutation in pediatric patients with sporadic essential thrombocythemia

Pediatric essential thrombocythemia (ET) is a rare and heterogenous disease entity. While several recent studies have focused on the role of the JAK2 V617F mutation in pediatric ET, the frequency of pediatric ET cases with this mutation and the associated clinical features remain unclear. We examined six childhood cases who had been diagnosed with ET according to WHO criteria (onset age: 0.2-14 years) for the presence of the JAK2 V617F mutation, MPLW515L mutation and JAK2 exon 12 mutations. Two sensitive PCR-based methods were used for the JAK2 V617F genotyping. We also examined the expression of polycythemia rubra vera-1 (PRV-1), which is a diagnostic marker for clonal ET. We found that three of the six cases had the JAK2 V617F mutation and that all six cases expressed PRV-1 in their peripheral granulocytes. Neither MPL W515L mutation nor JAK2 exon 12 mutations was detected in the patients without JAK2 V617F mutation. The two patients who developed thrombocythemia during infancy were JAK2 V617F-negative. These findings suggest that the JAK2 V617F mutation is not rare in childhood sporadic ET cases, and that these cases might be older and myeloproliferative features.

UPD1p indicates the presence of MPL W515L mutation in RARS-T, a mechanism analogous to UPD9p and JAK2 V617F mutation

UPD1p indicates the presence of MPL W515L mutation in RARS-T, a mechanism analogous to UPD9p and JAK2 V617F mutation

MPL W515L mutation in patients with myeloproliferative diseases

18019 Background: The BCR-ABL negative chronic myeloproliferative disorders (MPD) include polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with idopathic myelofibrosis (IMF). Although clonal hematopoiesis was observed in these disorders more than three decades ago, the molecular etiology of these disorders was not known until recently when several groups reported a somatic mutation in the JAK2 tyrosine kinase (JAK2 V617F) in most patients with PV and in a subset of patients with ET and IMF. As JAK2 V617F, MPL W515L is a novel acquired mutation that induces constitutive cytokine-independent activation of the JAK-STAT pathway in MPD. The discovery of this mutation provides a novel mechanism for activation of signal transduction in hematopoietic malignancies. Methods: To investigate its prevalence in Chinese patients with MPD, we introduced allele-specific PCR (AS- PCR) combined with sequence analysis to simultaneously screen MPL W515L and JAK2 V617F mutations in 190 MPD patients. Results: The results indicated that MPL W515L mutation was found to be harbored in only one of 102 ET patients (1.0%) and was not detected in PV, IMF, chronic myelogenous leukemia (CML) patients. Sixty eight BCR/ABL-negative MPD patients (46.3%) were found harbouring JAK2 V617F mutation (PV, 62.5% ET, 42.1% IMF 38.1%). Furthermore, MPL W515L and JAK2 V617F mutations were not detected in patients of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplastic syndromes (MDS) and CML. Conclusions: MPL W515L mutations may contribute to the primary molecular pathogenesis mechanism of Chinese patients with ET. No significant financial relationships to disclose.

Phospho-STAT5 expression pattern with the MPL W515L mutation is similar to that seen in chronic myeloproliferative disorders with JAK2 V617F

Abnormal nuclear megakaryocytic staining for phospho-STAT5 (pSTAT5) correlates with JAK2 V617F mutational status in non-chronic myelogenous leukemia chronic myeloproliferative disorders. However, a proportion of wild-type JAK2 non-chronic myelogenous leukemia chronic myeloproliferative disorders cases also demonstrate this abnormal pSTAT5 expression pattern. We report a patient with a JAK2 V617F-negative myeloproliferative/myelodysplastic syndrome who had abnormal megakaryocytic pSTAT5 expression and a MPL W515L mutation. The patient was a 71-year-old man with anemia and thrombocythemia on laboratory examination. His peripheral blood smear demonstrated occasional dysplastic neutrophils. Bone marrow biopsy revealed hypercellular marrow with features consistent with myeloproliferative/myelodysplastic syndrome. Immunohistochemistry for pSTAT5 showed abnormal nuclear megakaryocyte positivity. Cytogenetic analysis revealed a normal karyotype, fluorescence in situ hybridization for BCR-ABL was negative, and JAK2 genotyping demonstrated wild-type JAK2. However, MPL genotyping showed a MPL W515L mutation. Abnormal nuclear megakaryocytic staining for pSTAT5 expression, previously associated with the JAK2 V617F mutation, is also associated with MPL W515L, likely reflecting activation of the JAK-STAT signaling pathway.

MPL W515L mutation in Chinese patients with myeloproliferative diseases

As JAK2 V617F, MPL W515L is a novel acquired mutation that induces constitutive cytokine-independent activation of the JAK-STAT pathway in myeloproliferative disorders (MPD). The discovery of this mutation provides a novel mechanism for activation of signal transduction in hematopoietic malignancies. To investigate its prevalence in Chinese patients with MPD, we introduced allele-specific PCR (AS-PCR) combined with sequence analysis to simultaneously screen MPL W515L and JAK2 V617F mutations in 190 MPD patients. MPL W515L mutation was found to be harbored in only one of 102 patients, who had essential thrombocythemia (ET, 1.0%) and was not detected in patients with polycythemia vera (PV), idiopathic myelofibrosis (IMF), and chronic myelogenous leukemia (CML). Sixty-eight BCR/ABL-negative MPD patients (46.3%) were found harboring JAK2 V617F mutation (PV, 62.5%; ET, 42.1%; IMF 38.1%). Furthermore, MPL W515L and JAK2 V617F mutations were not detected in patients of acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndromes, and CML. It has been shown that MPL W515L mutations may contribute to the primary molecular pathogenesis of Chinese patients with ET.

Relation between JAK2 V617F Mutation and Chronic Myeloproliferative Disorders.

The chronic myeloproliferative disorders (cMPDs) are a group of clonal malignant tumors of the hematological system, and derived from the pluripotential hemopoietic stem cells, manifested as the proliferation of one or more series of cells in the bone marrow as well as the occurrence of excessive mature or naive cells in the peripheral blood. It was reported by several research groups that there was the acquired JAK2 V617F mutation in the majority of the PV patients and in a part of the ET or PMF patients, which provided the new ideas for the investigation of the pathogenesis of BCR/ABL- cMPDs. In the present study, the JAK2 V617F mutation was detected in a larger collection of Chinese cMPD patients, to give a picture of the incidence of JAK2 V617F mutation in the Asian pollutions. A total of 523 patients with the cMPDs, including 278 males and 245 females, were analyzed. Their median age was 50 years old (7 to 83 years old). According to the WHO diagnostic criteria, among the 523 patients were 88 cases of CML at the chronic phase (including 59 males and 29 females with a median age of 40 years old), 25 of CML complicated with myelofibrosis, 116 of PV (64 males and 52 females; a median age of 53; among them were six cases of PV with the secondary myelofibrosis), 153 of ET (63 males and 91 females; a median age of 50; 15 cases of ET with the secondary myelofibrosis), 142 of PMF (71 males and 71 females; a median age of 53.5), four of unclassified CMPD (CMPD-U) (2 males and 2 females; a median age of 60), seven of high eosinophil syndrome (HES) (6 males and 1 female; a median age of 32), and 13 of chronic eosinophilic leukemia (13 males; a median age of 34). In addition, 140 of healthy adults were included in the control group. Allele-specific PCR (ASP) was applied to identify JAK2 V617F mutation, the mutation status was analyzed by PCR-RFLP, and the results were confirmed by sequence analysis. The mutation load was calculated by the ratio of T/G. Then explore the correlation between the allele load and the clinical, hematologic features. To those without JAK2 V617F, MPL W515L mutation was analyzed. JAK2 V617F was detected in 66%(346/523) of all patients (94%(109/116) in PV, 79%(122/153) in ET, 78%(111/142) in PMF, 75%(3/4) in CMPD-U and 14%(1/7) in HES).Majority of patients carried JAK2 V617F mutation were heterozygous, homozygote was found in only 5 cases (4 in PV and 1 in ET). The mutation load in majority patients (71.5%) was low, PV>ET>MF when compared with mutation load (p=0.003). Hemoglobin level was significantly related to high mutation load in PV (p=0.033, r=0.203). Bone marrow megakaryocyte counts were found to be marked increased in ET with high JAK2 V617F loads (P=0.024, r=0.205), and hepatomegaly in PMF was also significiently associated with high JAK2 V617F mution load (p=0.003, 0.001)(p=0.001, r=0.315). Oue data showed that1.Majority of cMPD patients, especialy with PV, carried JAK2 V617F mutation, but JAK2 V617F was absent in CML;2.98% of JAK2 V617F mutation occurs in a heterozygous status, only 4 patients with PV and 1 with ET were homozygouse.;3.PV> ET> MF when compared with mutation load. High JAK2 V617F loads were found to be significantly associated with higher hemoglobin level in PV and higher bone marrow megakaryocyte counts in ET;4.The correlation between hepatomegaly and JAK2 V617F mutation load were also found in PMF.

Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis

AbstractThe MPL (W515L and W515K) mutations have been detected in granulocytes of patients suffering from certain types of primitive myelofibrosis (PMF). It is still unknown whether this molecular event is also present in lymphoid cells and therefore potentially at the hematopoietic stem cell (HSC) level. Toward this goal, we conducted MPL genotyping of mature myeloid and lymphoid cells and of lymphoid/myeloid progenitors isolated from PMF patients carrying the W515 mutations. We detected both MPL mutations in granulocytes, monocytes, and platelets as well as natural killer (NK) cells but not in T cells. B/NK/myeloid and/or NK/myeloid CD34+CD38−-derived clones were found to carry the mutations. Long-term reconstitution of MPL W515 CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice was successful for as long as 12 weeks after transplantation, indicating that MPL W515 mutations were present in HSCs. Moreover, the 2 MPL mutations induced a spontaneous megakaryocytic growth in culture with an overall normal response to thrombopoietin (TPO). In contrast, erythroid progenitors remained EPO dependent. These results demonstrate that in PMF, the MPL W515L or K mutation induces a spontaneous megakaryocyte (MK) differentiation and occurs in a multipotent HSCs.