W515L Mutation Research Articles

Development of a Real-Time qPCR Assay for Detection of Common MPL Mutations in Myeloproliferative Neoplasms (MPNS).

Myeloproliferative neoplasms (MPNs) are blood cell disorders, characterized by overproduction of abnormal cells in bone marrow due to stem cell mutation. The proliferations of blood cell are controlled by many genes particularly MPL gene which encodes thrombopoietin receptor, a hematopoietic growth factor involved in the production and regulation of the platelets and multipotent hematopoietic progenitor cells. Acquired mutations including (W515L and W515K) in this gene have been observed in patients with primary myelofibrosis or essential thrombocythemia lacking JAK2 (V617F) mutations. MPL mutation detection is important for MPNs diagnosis, but due to low frequency of mutant allele burden (< 15%) may be missed by already available common assays such as Sanger sequencing. Furthermore, these techniques are costly, time-consuming, and less sensitive. In present study, we aimed to develop sensitive, less time-consuming, and cost-effective real-time PCR assay for the detection of MPL mutations that is based on TaqMan fluorescent probes. DNA was extracted from blood sample of 128 MPNs patients collected and further analysis was performed on TaqMan RT-PCR. Reference curve was obtained for amplified product of MPL gene containing mutated sequence. The predicted sensitivity level was at least 5% mutant allele burden by our developed assay that is much higher than sequencing output. Out of 128, 2 (1.56%) patients harbored W515L mutation and 1 (0.78%) harbored W515K mutation. It was concluded that TaqMan qRT-PCR assay is an efficient, sensitive, cost-effective, and less time-consuming method capable of detecting MPL mutation in MPNs patients. We suggested that this assay might be helpful in investigating mutant allele load in MPNs patients.

JAK2, CALR, MPL, and ASXL1 Mutations in 136 Thai Patients with Philadelphia-Negative Myeloproliferative Neoplasms and Their Correlations with Clinical Outcomes

Background: Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPN) are a group of hematological malignancies, including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). Mutations of JAK2, CALR, MPL, and ASXL1 are associated with carcinogenesis and clinical characteristics of Ph-negative MPN. However, the availability of the data regarding these mutations is relatively limited in Thai population. Objective: To investigate these mutations in Thai Ph-negative MPN patients. Materials and Methods: One hundred thirty-six MPN (48 PV, 72 ET, and 16 PMF) cases were enrolled. Mutations of JAK2 V617F and MPL W515L/K mutations were investigated using allele-specific PCR (AS-PCR) and confirmed by sequencing. CALR and ASXL1 mutations were investigated using Sanger sequencing. Results: The JAK2 V617F mutation was detected in 83.3% of PV, 66.6% of ET, and 50.0% of PMF, and correlated with higher RBC, WBC, and PLT counts in PV. CALR mutations were detected in 16.7% of ET and 12.5% of PMF and associated with a higher PLT count in ET. The MPL W515L mutation was detected in one PMF patient. ASXL1 mutations were detected in 6.3% of PV, 8.3% of ET, and 12.4% of PMF, with c.1954G&gt;A being the preponderant mutational form. ASXL1 mutations increased the risk (RR 27.6) and accelerated the onset of AML transformation. Conclusion: The present study provided the prevalence and clinical correlation of JAK2, CALR, MPL, and ASXL1 mutations among Thai Ph-negative MPN patients. The association of ASXL1 mutations with adverse clinical outcomes suggested the potential usefulness of these mutations as a prognostic marker for Ph-negative MPN patients. Keywords: JAK2, MPL, CALR, ASXL1, Thai, Philadelphia-negative myeloproliferative neoplasm (MPN)

MPL exon 10 mutations other than canonical MPL W515L/K mutations identified by in-house MPL exon 10 direct sequencing in essential thrombocythemia.

MPL exon 10 mutations are one of the driver mutations in essential thrombocythemia (ET) or myelofibrosis (MF). We have established an in-house MPL mutation analysis system, covering the entire region of MPL exon 10 by direct sequencing. Since 2009, MPL exon 10 mutation analysis has been performed for diagnosis of myeloproliferative neoplasms (MPN) without JAK2 V617F or CALR exon 9 mutations. So far, 11 cases of MPL exon 10 mutation have been found in 51 patients with suspected MPN. In patients with ET, we detected five non-canonical MPL mutations including one novel mutation, MPL R514_P518delinsK, and one canonical MPL W515L mutation. Notably, three ET patients without canonical MPL mutations had thrombotic events. Meanwhile, in primary or secondary MF, only canonical MPL W515L/K mutations were found. Further cases need to be examined to elucidate the full MPL mutation profile in MPN. However, our data indicate that analysis of the whole of MPL exon 10 is warranted for the diagnosis of MPL mutations, especially in ET, and that the use of Japanese commercial laboratory tests that only detect canonical MPL W515L/K mutations may miss a significant percentage of MPL exon 10 mutations, which could delay the administration of anti-thrombotic therapy.

Two activating mutations of MPL in triple-negative myeloproliferative neoplasms.

MPLW515K or W515L mutation plays an important role in the pathogenesis of myeloproliferative neoplasms (MPNs) through signaling molecules of the cytokine receptor axis. Besides MPLW515K or W515L, more than 30 atypical MPL mutations have been reported in patients who are negative for JAK2V617F, MPLW515K/L, and CALR mutations. Here, we aimed to identify the disease‐causing mutations in the triple‐negative case of ET. We described two MPL mutations in patients diagnosed with ET by target sequencing the hotspot mutation region of MPL gene. The MPLA497‐L498ins4 is an insertion mutation detected recurrently in ET patients, and the MPLW515RQ516E is a novel double‐point mutation found in an ET patient. Functional studies of MPLA497‐L498ins4 and MPLW515RQ516E revealed that they are gain‐of‐function mutations. Mutants of MPLA497‐L498ins4 and MPLW515RQ516E promoted autonomous proliferation on Ba/F3 cells in the absence of IL‐3. Autonomous activation of TPO‐R without ligand TPO was observed in MPLA497‐L498ins4 and MPLW515RQ516E mutants. Lower percentage of cells in G1 phase and higher percentage of cells in S phase of two atypical MPL mutants were detected after culturing without any cytokines. These two atypical MPL mutations also presented increase in phosphorylation of signaling proteins including JAK2/STAT, PI3K/AKT, and MAPK/RAS. In summary, the MPLA497‐L498ins4 and MPLW515RQ516E are gain‐of‐function mutations which may be novel driving factors participating in the pathogenesis of triple‐negative MPN.

PF657 USE OF A NGS MYELOID PANEL TO FIND CLONALITY IN TRIPLE NEGATIVE ESSENTIAL THROMBOCYTHEMIA. IDENTIFICATION OF NEW MUTATIONS IN DRIVER GENES

Background:Essential Thrombocythemia is a clonal haematopoietic stem cell disorder characterized by hypercellularity of the bone marrow with effective haematopoietic maturation and increased numbers of platelets in the peripheral blood. Although more than 90% of the cases present typical mutations in JAK2, MPL and CALR, the remaining cases can become a diagnostic challenge as it requires the presence of a clonal marker or the absence of evidence for reactive thrombocytosis.Aims:To evaluate the use of a Myeloid Panel for the identification of clonal markers in TN ET cases and try to identify new mutations and prognostic determinants in these patients.Methods:A cohort of 32 ET negative for JAK2 V617F mutation (RT‐PCR), CALR canonic mutations (GENESCAN) and MPL W515K and W515L mutations (RT‐PCR) was selected. Average age at diagnosis was 49,08 (range 14–88). Male:Female ratio was 11:21. Average Haemoglobin, leukocytes and platelets at diagnosis were 14.14 g/dl 8,93 x109/L and 820x109/L, respectively. We performed targeted gene sequencing by NGS (Ion Torrent S5XL System–Thermofisher Scientific) using a panel of 33 (before March 2018) or 43 genes (after March 2018) implicated in leukemia prognosis.Results:With a median follow‐up of 5 years (range 0,02–25,43) 2 patients suffered transformation to acute myeloid leukemia, 1 progression to myelofibrosis and 1 presented with suffered a thrombotic event after diagnosis. We found clonality markers in 19 (59%) out of 32 patients. These presented an average of 1,84 (range 1–3) and 1,68 (range 1–3) mutations and mutated genes, respectively. 6 patients (31,57%) presented mutations in driver genes (JAK2, MPL and CALR). Three of them, family, presented with JAK2 R683G, a mutation previously described in acute lymphoblastic leukemia but not in myeloproliferative neoplasms. Other patient had JAK2 L393 V mutation, of undetermined significance and reported in population databases. Another presented the previously described MPL S505C and W515R mutations. Finally, we identified a new deletion in CALR exon 9 (c.1153_1155delAAG, p.K385del). 17 (89,47%) mutated patients presented mutations in non driver genes. Among those the most frequently mutated ones were TET2 (29%), ETV6 (17,6%), SF3B1 and KMT2A (both 11,7%). We found one mutation in VHL, DNMT3A, WT1, TP53, EPAS, CBL, IDH1, TPHO, KIT, ASXL1, EPOR and KRAS.Summary/Conclusion:Triple Negative TE suppose a diagnostic challenge. Here we show that targeted gene sequencing by a myeloid panel is able to identify clonality in up to 59% of the cases. Also, we have described new mutations in driver genes, gaining knowledge on TE molecular basis. Finally, larger cohorts of TN ETs are needed to clearly define the determinants and prognostic factors of this disease.

A Phenotypic Screen for Small-Molecule Inhibitors of Constitutively Active Mutant Thrombopoietin Receptor Implicated in Myeloproliferative Neoplasms.

Rather than a Janus Kinase 2 inhibitor (ruxolitinib), a specific thrombopoietin receptor (TpoR) inhibitor would be more specific for the treatment of myeloproliferative neoplasms due to TpoR mutations. A cell-based phenotypic approach to identify specific TpoR inhibitors was implemented and a library of 505,483 small molecules was screened for inhibitory effects on cells transformed by TpoR mutants. Among the identified hits are two analogs of 3-(4-piperidinyl) indole. The analogs showed about five-fold preferential inhibition of cell viability towards Ba/F3 cells expressing the TpoR W515L mutation compared to the parental cells. There was no significant difference in inhibition of cell viability between the TpoR wild type and the TpoR W515L mutant cells. Preferential inhibition of viability was observed in Ba/F3 cells expressing erythropoietin receptor (EpoR) when stimulated with Epo compared to stimulation with interleukin-3 (IL3). The indole analog inhibited ex vivo colony formations of primary bone marrow cells from heterozygous JAK2 V617F knock-in mice. Drug combination treatment study was performed using ruxolitinib and the indole analog. Drug synergistic effects were observed when cells were stimulated to proliferate through both the IL3 and TpoR pathways. Our compound specifically targets monoamine receptors in the rhodopsin-like receptor family of G protein-coupled receptor. This screen has identified a monoamine receptor inhibitor that can inhibit viability of cells with active TpoR or EpoR signalings. Drug synergism with ruxolitib is demonstrated.

Concurrent MPL W515L and Y591D mutations in a patient with myelofibrosis

Concurrent MPL W515L and Y591D mutations in a patient with myelofibrosis

Concomitant BCR-ABL1 positive chronic myelogenous leukemia emerging in a patient with MPL W515L associated primary myelofibrosis

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by proliferation of one or more cell lineages in the bone marrow. At present, the main criterion in the 2008 World Health Organization classification of MPNs is the presence of an underlying genetic abnormality. These mutations are generally mutually exclusive except for rare reports in the literature. We report for the first time a detailed analysis of the clinical, histologic and cytogenetic/molecular features of a patient who initially presented with MPL W515L positive primary myelofibrosis and over the course of five years developed an MPN associated with both BCR-ABL1 and MPL W515L mutation. We discuss the diagnostic challenges and therapeutic implications of concomitant BCR-ABL1 translocation with MPL W515L mutation. Multiple genetic alterations may simultaneously coexist in patients exhibiting features of myeloproliferative disorders.

Mpl Gain-of-Function Mutations Can be Classified By Differential Subcellular Processing, Molecular Mechanisms, Mode of Inheritance and Clinical Impact

The interaction between the c-Mpl receptor and its ligand thrombopoietin (TPO) on the cell surface is crucial for the regulation of thrombopoiesis. Several mutations in the c-Mpl receptor gene have been linked to a gain-of-function resulting in thrombocytosis. We have analyzed the known gain-of-function mutations in the extracellular part of the Mpl receptor, K39N and P106L, as well as the S505N, W515K and W515L mutations in the transmembrane and juxtamembrane region, respectively. Interestingly, the latter mutations can occur as autosomal dominant and/or as somatic mutations and are known to be associated with myeloproliferative malignancies and AML, whereas the abundant K39N and the P106L mutations are the cause of autosomal recessive hereditary thrombocytosis without a known predisposition to hematologic malignancies. To date, these differences in clinical impact and mode of inheritance are poorly understood.Starting from these clinical observations, we have performed functional analyses of the described gain-of-function mutations to address the key functional properties that might explain the observed clinical differences. Three crucial stages of the c-Mpl receptor life cycle were addressed: (1) post-translational processing of the immature receptor protein and its subcellular distribution, (2) membranous expression of the mature receptor and (3) receptor internalization upon stimulation with its ligand TPO.We first analyzed the post-translational processing of the normal, the K39N and the P106L mutated receptor in comparison with receptors carrying the S505N, the W515K and W515L mutations in a HeLa cell culture model. The normal, the K39N, S505N, W515K and W515L mutated c-Mpl receptors were properly glycosylated during their transport through the Golgi apparatus, whereas the P106L mutated receptor did not enter the Golgi and was not fully glycosylated. The K39N mutant was fully glycosylated but did show different running behavior on the SDS Gel, most likely caused by post-translational modifications different from glycosylation. The S505N, the W515K and the W515L mutated receptors displayed stable surface expression in confocal microscopy and FACS analysis, whereas the P106L mutated receptor was not detectable on the cell surface. After stimulation with TPO, a decrease in mean receptor surface protein could be observed for the wild type and all mutants that were expressed on the surface, namely S505N, W515K and W515L, however not significant (p>0.05). Interestingly, our functional analyses of the TPO/c-Mpl signaling pathways in TPO stimulated c-Mpl transfected BA/F3 cells showed activation of the ERK1/2 pathway in all mutants but only weaker activation of the PI3K/m-TOR and Stat3/5 signaling pathways for the P106L mutant. By contrast, cells transfected with the wild type, the S505N, W515K and W515L c-Mpl mutants showed predominant up-regulation of the PI3K/m-TOR and Stat3/5 pathways.These results show that first, both impaired and regular receptor glycosylation and correlating subcellular distribution may occur in c-Mpl gain-of function mutants. Second, the c-Mpl gain-of-function mutants differ substantially in surface expression levels. Third, our results suggest differences in the maintenance of the TPO negative feedback loop across c-Mpl gain-of-function mutants. Indeed, in contrast to P106L, it seems likely that the TPO negative feedback-loop is preserved in the S505N, the W515K and the W515L mutants. In line with this, highly elevated TPO serum levels have only been described for P106L, but not for the other gain-of-function mutations. We hypothesize that maintenance of the TPO negative feedback-loop is sufficient to prevent dysregulation of TPO levels but not transmission of a harmful c-Mpl gain-of-function effect. Instead, the predominant activation of the PI3K/m-TOR and Stat3/5 pathways might explain the different propensity to induce hematopoietic malignancy.In summary, our findings suggest the existence of different disease causing molecular mechanisms behind the mutations' respective clinical correlates and provide the basis for an important extension to the current classification of c-Mpl mutations that is primordially based on clinical observations. DisclosuresNo relevant conflicts of interest to declare.

The mutation profile of JAK2, MPL and CALR in Mexican patients with Philadelphia chromosome-negative myeloproliferative neoplasms

By using molecular markers, it is possible to gain information on both the classification and etiopathogenesis of chronic myeloproliferative neoplasias (MPN). In a group of 27 Mexican mestizo patients with MPNs, we studied seven molecular markers: the BCR/ABL1 fusion gene, the JAK2 V617F mutation, the JAK2 exon 12 mutations, the MPL W515L mutation, the MPL W515K mutation, and the calreticulin (CALR) exon 9 deletion or insertion. Patients with the BCR/ABL1 fusion gene were excluded. We studied 14 patients with essential thrombocythemia (ET), eight with polycythemia vera (PV), four with primary myelofibrosis (MF), and one with undifferentiated MPN. We found twelve individuals with the JAK2 V617F mutation; five of them had been clinically classified as PV, five as ET, and one as MF. One patient with the MPL W515L was identified with a clinical picture of ET. Five patients with the CALR mutation were identified, four ET and one MF. No individuals with either the MPL W515K mutation or the JAK2 exon 12 mutations were identified. The most consistent relationship was that between PV and the JAK2 V617F mutation (p=.01). Despite its small size, the study shows much less prevalence of JAK2 mutation in PV, ET and MF, which does not match international data.

The Predominant Activation of Alternative Signaling Pathways Explains the Differential Propensity of Different c-Mpl Gain-of-Function Mutations to Promote Myeloproliferative Malignancies

Thrombopoiesis is tightly regulated by the binding of thrombopoietin (THPO) to its receptor c-Mpl that leads to the clearance of THPO from the plasma thus establishing a negative feedback loop. Several mutations in the c-Mpl receptor gene have been linked to a gain-of-function resulting in thrombocytosis. We focused on a comparison of gain-of-function mutation in the extracellular part of the c-Mpl receptor, where ligand binding and receptor dimerization occur, with the S505N, W515K and W515L mutations in the transmembrane and juxtamembrane region, respectively. Interestingly, the latter mutations are known to also promote myeloproliferative malignancies and AML, whereas the P106L mutation causes hereditary thrombocytosis without a known predisposition to hematologic malignancies.We have now performed functional analyses of these gain-of-function mutations to address the question of how the different propensity to induce malignancy can be explained. We first analyzed the post-translational processing of the normal and the P106L mutated receptor in comparison to the receptors carrying the S505N, W515K and W515L mutations in transfected HeLa and BA/F3 cells. The normal and the S505N, W515K and W515L mutated c-Mpl receptors were properly glycosylated during their transport through the Golgi apparatus, whereas the P106L mutated receptor did not enter the Golgi and was not fully glycosylated. The plasma membrane expression, assayed by confocal microscopy and FACS, of the S515N, W515K and W515L mutated receptors was comparable to the normal receptor, whereas the P106L mutated receptor was not detectable on the cell surface. Functional analyses of the THPO/c-Mpl signaling pathways in THPO stimulated c-Mpl transfected BA/F3 cells showed activation of the ERK1/2 pathway in all mutants but only weaker activation of the PI3K/m-TOR and Stat3/5 signaling pathways for the P106L mutant. By contrast, cells transfected with the normal receptor gene and the S505N, W515K and W515L c-Mpl mutants showed predominant up-regulation of the PI3K/m-TOR and Stat3/5 pathways.These results show that (1) the activation of c-Mpl by THPO does not absolutely require surface expression of the receptor (2) different c-Mpl gain-of-function mutations activate separable downstream pathways and (3) the predominant activation of the PI3K/m-TOR and Stat3/5 pathways correlates with the propensity to induce hematopoietic malignancy.Further, it is interesting to note that the c-Mpl P106L gain-of-function mutant is known to cause 10 to 20-fold elevated thrombopoietin (THPO) plasma levels in patients and is shown here not to be properly glycosylated and transported to the cell surface. By contrast, patients with the other gain-of-function mutations analyzed here show normal or even reduced THPO plasma concentrations and normal post-translational processing and cell surface expression.In conclusion, we propose that c-Mpl gain-of-function mutations exert their effect by predominantly activating either the PI3K/m-TOR and Stat3/5 or the ERK signaling pathways and that the predominant activation of PI3K/m-TOR and Stat3/5 correlates with the propensity to induce malignancy. Furthermore, THPO clearance and maintenance of the negative feedback loop regulating THPO plasma levels, but not signaling activity of the c-Mpl receptor necessarily require proper glycosylation, intracellular trafficking and cell surface expression. DisclosuresNo relevant conflicts of interest to declare.

An MPL W515L mutation in refractory anemia with ringed sideroblasts associated with marked thrombocytosis: A case report.

The current study presents the case of a 63-year-old patient exhibiting refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), who was positive for the MPL W515L mutation, but negative for the JAK2 V617F mutation. Following diagnosis, the patient remained asymptomatic for over three years, however, in August 2012, the patient relapsed and was administered with supportive treatment in the form of subcutaneous darbepoetin α at a dose of 300 μg/week, which resulted in an increased hemoglobin concentration, allowing the patient to remain transfusion-independent. The MPL W515L mutation has been reported in two previous cases of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) with ringed sideroblasts, however, to the best of our knowledge, the current report is the first to present a case of RARS-T with an MPL W515L mutation. A clinical trial designed to evaluate the efficacy of a targeted agent against the JAK2 V617F mutation is currently ongoing, with the aim of providing a novel therapeutic strategy for treating MDS/MPN patients. As MPL is located upstream of the JAK-STAT signaling pathway, it is a possible therapeutic target in MDS/MPN patients positive for an MPL W515L mutation, but negative for a JAK2 V617F mutation.

Monitoring residual disease in the ph-negative myeloproliferative neoplasms post-allogeneic stem cell transplantation: more mutations and more methodologies.

Monitoring residual disease in the ph-negative myeloproliferative neoplasms post-allogeneic stem cell transplantation: more mutations and more methodologies.

Familial Essential Thrombocythemia Associated with MPL W515L Mutation in Father and JAK2 V617F Mutation in Daughter.

Familial essential thrombocythemia features the acquisition of somatic mutations and an evolution similar to the sporadic form of the disease. Here we report two patients—father and daughter—with essential thrombocythemia who displayed a heterogeneous pattern of somatic mutations. The JAK2 V617F mutation was found in the daughter, while the father harbored the MPL W515L mutation. This case report may constitute further proof that in familial essential thrombocythemia there are other, still undefined, constitutional, inherited genetic factors predisposing to the acquisition of various somatic mutations (e.g., JAK2 V617F and MPL).

MPL W515L mutation in pediatric essential thrombocythemia

Essential thrombocythemia (ET) is extremely rare in the pediatric population. In most patients no molecular abnormality can be found, with about 40% of pediatric patients harboring a JAK2 V617F mutation. Another recurrent mutation, involving a W to L or K transversion at MPL codon 515, has been reported in about 3-8% of adult ET patients. Herein we describe this mutation in a pediatric patient.

Tryptophan at the transmembrane–cytosolic junction modulates thrombopoietin receptor dimerization and activation

Dimerization of single-pass membrane receptors is essential for activation. In the human thrombopoietin receptor (TpoR), a unique amphipathic RWQFP motif separates the transmembrane (TM) and intracellular domains. Using a combination of mutagenesis, spectroscopy, and biochemical assays, we show that W515 of this motif impairs dimerization of the upstream TpoR TM helix. TpoR is unusual in that a specific residue is required for this inhibitory function, which prevents receptor self-activation. Mutations as diverse as W515K and W515L cause oncogenic activation of TpoR and lead to human myeloproliferative neoplasms. Two lines of evidence support a general mechanism in which W515 at the intracellular juxtamembrane boundary inhibits dimerization of the TpoR TM helix by increasing the helix tilt angle relative to the membrane bilayer normal, which prevents the formation of stabilizing TM dimer contacts. First, measurements using polarized infrared spectroscopy show that the isolated TM domain of the active W515K mutant has a helix tilt angle closer to the bilayer normal than that of the wild-type receptor. Second, we identify second-site R514W and Q516W mutations that reverse dimerization and tilt angle changes induced by the W515K and W515L mutations. The second-site mutations prevent constitutive activation of TpoR W515K/L, while preserving ligand-induced signaling. The ability of tryptophan to influence the angle and dimerization of the TM helix in wild-type TpoR and in the second-site revertants is likely associated with its strong preference to be buried in the headgroup region of membrane bilayers.

Pomalidomide in Myelofibrosis with Cytopenia: First Results of the Mpnsg 01–09 Study (ClinicalTrials.gov Identifier: NCT00949364).

AbstractAbstract 2840 Background:Pomalidomide in a single arm phase-I/II study and one randomized four arm phase-II study in primary myelofibrosis (MF) and post-polycythemia vera/essential thrombocythemia (post-PV/ET) MF showed efficacy in particular with respect to improvement in anemia. To date, pomalidomide has been evaluated in MF at two dose levels, 0.5mg and 2.0 mg/day. Aims:To evaluate clinical efficacy of pomalidomide alone and in combination with prednisolone (PRED) in patients with primary or post-PV/ET MF and cytopenia. Methods:The main inclusion criteria for primary or post-PV/ET MF patients were red blood cell (RBC)-transfusion-dependence or hemoglobin <10 g/dl, and/or thrombocytopenia <50/nl and/or neutropenia <1.0/nl; patients >=50 years were eligible. Treatment consisted of pomalidomide (POM) 2mg/day; prednisone (PRED) 30mg/day was added in patients who did not respond (≤ stable disease) within three months of therapy. The primary endpoint was response assessed by IWG criteria and extended by the criterion red blood cell transfusion-independence (Gale RP et al., Leuk Res. 2011). Concurrent hydroxyurea in patients with proliferative disease and aspirin 100 mg/d in patients with platelets between 50/nl and 1000/nl were administered. The statistical design of the study was based on the Simon optimal two-stage design. Here we report on the first stage of the study. Median follow-up according to the method of Korn was 18 months. Results:Thirty-eight patients were treated with POM 2 mg/d, the median age was 71 years (range 51–83), 34% were female. Twenty-seven had primary and 11 post-PV/ET MF. Disease stage at study-entry according to the DIPSS was high-risk in 13 (34%), intermediate-2 risk in 22 (58%) and intermediate-1 risk in 3 (8%). Incidence of high-risk cytogenetics, JAK2 V617F mutation and MPL W515L mutation were 29% (10/34), 55% (21/38) and 18% (7/38), respectively. Twenty-seven patients (71%) were RBC-transfusion- and 7 (18%) platelet-transfusion dependent. Median duration of treatment with POM was 11.4 months with 5 patients continue on treatment 24+ months. PRED was added after 3 months in 19 of 28 eligible patients. POM dose-reduction (n=8, 1mg/d; n=2, 0.5mg/d) was performed after a median time of 34 days (range 3–308 days) due to fatigue (n=2), thrombo- and/or neutropenia (n=7), rash (n=1). Seven patients with high risk characteristics (n=6 RBC-transfusion dependent, n=4 high risk cytogenetics) experienced transformation into blast phase (BP), the actuarial probability of transformation to BP measured from diagnosis was 6.0% (SE 4.1%) at 2 years and 22.4% (SE 8.4%) at 5 years. Response was observed in 14 patients (37%) after a median time of 4.8 months (n=1 complete remission, n=5 clinical improvement-platelets, n=3 clinical improvement-erythrocytes, n=5 red cell transfusion-independence); 8 responders received concomitant PRED and responded after a median of three months (range 0.8–11.7 months) of the addition of PRED. In 5 of 14 responders POM dose was reduced due to toxicity, notably before response occurred. Responses were observed within the first 3 months (n=4), between month 4 to 6 (n=4) and beyond month 6 (n=6) with the latest response seen at 12.7 months. There was no correlation between response and JAK2/MPL mutation status or cytogenetics. Basophilia defined as greater than 50% increase in absolute basophil count at month 3 was in trend associated with response (p=0.06). Conclusions:POM with or without PRED in patients with different risk groups of patients with primary and post-PV/ ET MF was effective with a response rate of 37%. Based on results of this first cohort the protocol was amended; i) POM dose has been adjusted to 0.5 mg/d, ii) up-front randomization of PRED at month 4 or month 7 in patients without response but stable disease to single agent POM was introduced. Disclosures:Schlenk:Celgene: Research Funding. Off Label Use: Pomalidomide is so far not approved for the treatment of primary and secondary myelofibrosis.

JAK2 V617F, MPL W515L and JAK2 exon 12 mutations in Chinese patients with primary myelofibrosis

JAK2 V617F, MPL W515L and JAK2 exon 12 mutations are novel acquired mutations that induce constitutive cytokine-independent activation of the JAK-STAT pathway in myeloproliferative disorders (MPD). The discovery of these mutations provides novel mechanism for activation of signal transduction in hematopoietic malignancies. This research was to investigate their prevalence in Chinese patients with primary myelofibrosis (PMF). We introduced allele-specific PCR (AS-PCR) combined with sequence analysis to simultaneously screen JAK2 V617F, MPL W515L and JAK2 exon 12 mutations in 30 patients with PMF. Fifteen PMF patients (50.0%) carried JAK2 V617F mutation, and only two JAK2 V617F-negative patients (6.7%) harbored MPL W515L mutation. None had JAK2 exon 12 mutations. Furthermore, these three mutations were not detected in 50 healthy controls. MPL W515L and JAK2 V617F mutations existed in PMF patients but JAK2 exon 12 mutations not. JAK2 V617F and MPL W515L and mutations might contribute to the primary molecular pathogenesis in patients with PMF.

Single Center Experience with Screening for JAK2 V617F, cMPL W515L, cMPL W515K, and JAK2 L611V Mutations in MPN. Rarity of JAK2 L611V Does Not Justify Its Routine Screening

Abstract 5167The myeloproliferative neoplasms (MPNs) polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF) are caused by an unregulated clonal proliferation of cells derived from a pluripotent stem cell. The exon 14 JAK2 V617F somatic mutation and cMPL mutations in codon 515 of exon 10 have provided excellent diagnostic tools to distinguish MPNs from other diseases with similar clinical features. JAK2 V617F mutations are present in ∼95% of PV patients and ∼50% of ET or PMF patients. Other somatic mutations have also been found in JAK2 exon 12 in patients with PV. Recently, a new somatic mutation of JAK2 in exon 14, L611V, was reported in 3 out of 168 PV patients in cis with JAK2 V617F (Cleyrat Leukemia 2010).Two cMPL mutations, cMPL W515L and cMPL W515K, have been identified in MPNs. The cMPL W515L mutation is reported to be present in ET and PMF patients, while cMPL W515K in some PMF patients. We designed and validated a new rapid and sensitive allele-specific quantitative PCR assay (AS-qPCR) for determination of the JAK2 L611V mutation. In our design, we included a locked nucleic acid and a mismatch in the allelic specific primers in order to enhance allelic discrimination in the PCR reaction. The assay was sensitive to mutant frequencies of >2% in the background of wild-type alleles. Allelic discrimination of the assay is 14 cycles for wild type and 11 cycles for mutant alleles.We studied 584 patients referred with clinical diagnoses of MPN belonging to two different groups: 1) 390 patients, who, based on clinical and laboratory evaluations, had diagnoses of PV (n=74), ET (n=60), MF (n=30), other unspecified MPNs (n=189), and other non-PV patients with erythrocytosis (n=37); 2) DNA from patients referred to ARUP Laboratories with otherwise unspecified clinical diagnoses of MPN who were found to be 196 JAK2 V617F-positive but appropriate clinical and other testing was not available for classification. The first group was tested for the JAK2 V617F, JAK2 L611V, cMPL W515L and W515K mutations. Out of 390 patients, 37% (148/390) were JAK2 V617F positive (over >90% of PV patients), 9 (ET or PMF) patients carried the cMPL W515L mutation, and 1 PMF patient had 93% allelic burden for the cMPL W515K mutation. While cMPL W515L mutation was not previously reported in PV, one PV patient was positive for both the cMPL W515L and JAK2 V617F mutations. Of the 10 cMPL W515L mutations, two MF patients carried JAK2 V617F mutations concurrently. No JAK2 L611V mutation was detected in this cohort. The additional 196 JAK2 V617F-positive samples from the patients with unspecified MPNs were also screened for the JAK2 L611V mutation and none were positive.We conclude that the rarity of the JAK2 L611V mutation does not justify its routine screening. We suggest that those patients who fulfill the WHO criteria for PV should be further evaluated by more productive laboratory studies such as screening for JAK2 exon 12 mutations, clonality studies in females and if negative assays for germline mutations such as EPOR and VHL gene mutations. Disclosures:No relevant conflicts of interest to declare.

Interlaboratory Quality Control Round of MPL Mutation Detection in Fourteen European Laboratories: A MPN&MPNr-EuroNet Study,

Abstract 3859▪▪This icon denotes a clinically relevant abstractThe MPL gene is located on chromosome 1p34 and encodes the thrombopoietin receptor. It includes 12 exons and is a key factor for growth and survival of megacaryocytes. Acquired mutations in this gene activate the thrombopoietin receptor constitutively. MPL515 somatic mutations are stem cell-derived events that involve both myeloid and lymphoid progenitors. Two distinct exon 10 mutations are found in 15% of JAK2-V617F negative myeloproliferative neoplasms (MPN),), i.e. 3% of essential thrombocythemia (ET) and 10% of primary myelofibrosis (PMF).: W515L, W515K and the rare W515A variant. A hereditary mutation, S505N, is associated with familial thrombocytosis.MPL mutation detection is a helpful new tool to detect clonality in JAK2 V617F negative MPN and to establish the diagnosis of MPN. As many laboratories use very different methods and interpretations, standardization is highly warranted. Particularly the methodology is diverse and the results need to be comparable, requiring comprehensive testing. This quality control project was established within workgroup 2 of the MPN&MPNr-EuroNet network, (www.mpneuronet.eu, supported by the European program COST (CoOperation in Science and Technology)). The lab from the ‘Hopital H. Mondor AP-HP Paris’ provided 29 samples containing randomized concentrations (between 100% and 1%) of the four mutations MPL W515L, W515K, W515A and S505N. The plasmids used for this quality control experiment spanned exons 9, intron 9 and exon10 of the MPL with S505N, W515L, W515K and W515A mutants diluted first with wild-type plasmid gene and then diluted in human genomic DNA. Thirteen European laboratories tested these 29 samples, each using their own chosen methods (14 altogether). The following methods were used: Mutascreen W515L/K Kit (Ipsogen, France):(n=4), allelic discrimination real-time PCR (n=2), high resolution melting (HRM) (n=7) and sequencing (n=2, 1 Sanger, 1 pyrosequencing)).There were no false positive results in any of the labs. All labs using the Mutascreen W515L/K Kit detected all W515L and W515K mutations, from 100% mutated down to 1% mutated plasmids. The allelic discrimination assays which were also designed for W515L and W515K only, detected the mutations down to 2%. The HRM methods were all designed differently. All except one (which did not recognize S505N) detected all 4 mutations with a sensitivity, down to 5% mutated plasmids, with few exceptions detecting either lower or higher amounts. The Sanger sequencing and pyrosequencing assays had a detection limit of 5–10%, with the pyrosequencing assay not being designed for the S505N mutation.All participating labs detected the most frequent MPL mutations in MPN W515L and W515K, with many designs not including W515A and S505N. Achieved sensitivities differed between methods with cutoffs of 1% to 10% (1.5% for the Ipsogen kit). Most laboratories reported the results as either positive or negative. However, the percentages of mutated alleles reported by a few labs differed greatly from each other and from the original dilutions (range 2–50 times) In conclusion, these results show that the diverse methods for MPL mutation detection used by different European labs yielded comparable specificity with varying sensitivity. Smaller clones might be missed by the less sensitive methods, and quantification of mutated alleles should be interpreted very carefully until standardised reference material for MPL mutation testing will be available. More extensive interlaboratory testing including patient samples is needed to identify the most robust assays suitable for diagnostic mutation detection and particularly for quantification of mutated alleles. Disclosures:No relevant conflicts of interest to declare.