scholarly journals Clinical and Laboratory Features of JAK2 V617F, CALR, and MPL Mutations in Malaysian Patients with Classical Myeloproliferative Neoplasm (MPN)

2021 ◽  
Vol 18 (14) ◽  
pp. 7582
Author(s):  
Razan Hayati Zulkeflee ◽  
Zefarina Zulkafli ◽  
Muhammad Farid Johan ◽  
Azlan Husin ◽  
Md Asiful Islam ◽  
...  
Keyword(s):  
Thrombotic Event ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Genetic Profile ◽  
Allele Specific ◽  
Exon 9 ◽  
Calr Mutations

Mutations of JAK2V617F, CALR, and MPL genes confirm the diagnosis of myeloproliferative neoplasm (MPN). This study aims to determine the genetic profile of JAK2V617F, CALR exon 9 Type 1 (52 bp deletion) and Type 2 (5 bp insertion), and MPL W515 L/K genes among Malaysian patients and correlate these mutations with clinical and hematologic parameters in MPN. Mutations of JAK2V617F, CALR, and MPL were analyzed in 159 Malaysian patients using allele-specific polymerase chain reaction, including 76 polycythemia vera (PV), 41 essential thrombocythemia (ET), and 42 primary myelofibrosis (PMF) mutations, and the demographics of the patients were retrieved. The result showed that 73.6% JAK2V617F, 5.66% CALR, and 27.7% were triple-negative mutations. No MPL W515L/K mutation was detected. In ET and PMF, the predominance type was the CALR Type 1 mutation. In JAK2V617F mutant patients, serum LDH was significantly higher in PMF compared to PV and ET. PV has a higher risk of evolving to post PV myelofibrosis compared to ET. A thrombotic event at initial diagnosis of 40.9% was high compared to global incidence. Only one PMF patient had a CALR mutation that transformed to acute myeloid leukemia. JAK2V617F and CALR mutations play an important role in diagnostics. Hence, every patient suspected of having a myeloproliferative neoplasm should be screened for these mutations.

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

2021 ◽  
Vol 104 (5) ◽  
pp. 834-845
Keyword(s):  
Clinical Outcomes ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Thai Population ◽  
Specific Pcr ◽  
Allele Specific ◽  
Calr Mutations

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>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)

Mutation Type As a Major Determinant of Clinical Phenotype in Myeloproliferative Neoplasms Associated with Mutant Calreticulin

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3215-3215 ◽  
Author(s):  
Daniela Pietra ◽  
Elisa Rumi ◽  
Chiara Milanesi ◽  
Christian A Di Buduo ◽  
Marta Bellini ◽  
...  
Keyword(s):  
Amino Acids ◽  
Clinical Phenotype ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Charged Amino Acids ◽  
Calr Mutations ◽  
Mutation Type ◽  
Calr Mutation

Abstract About 25% of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) carry a somatic mutation of CALR, the calreticulin gene [N Engl J Med. 2013;369:2379-90]. So far, more than 50 different indels in CALR exon 9 have been found, but a 52-bp deletion (type 1 mutation) and a 5-bp insertion (type 2) are the most common lesions. All indels generate a novel C-terminus of the mutant protein, in which the endoplasmic reticulum retention signal KDEL is lost and the negatively charged amino acids are replaced by neutral and positively charged amino acids, disrupting the Ca-binding site. This suggests that both cellular dislocation and impaired Ca-binding activity may be involved in the abnormal proliferation of cells expressing a mutant calreticulin. It is still unclear, however, why the same mutant gene is associated with 2 different disease phenotypes (ET and PMF). In particular, little in known about the effect of the mutant protein on megakaryocyte biology and bone marrow collagen deposition. We studied the relationships between CALR mutation type, megakaryocyte biology, and clinical phenotype in patients with myeloproliferative neoplasms. According to the 2008 WHO criteria, 716 out of 892 patients had ET and 176 had PMF. Overall, 578 (65%) patients carried JAK2 (V617F), 230 (26%) had a CALR indel, and 84 (9%) had nonmutated JAK2 and CALR. Patients with MPL mutations were excluded. Twenty-six different types of CALR lesions were identified: 120 (52%) patients had type 1 mutation, 75 (33%) had type 2, and 35 (15%) carried other indels. The frequency of type 1 mutation was significantly higher in PMF than in ET (71% vs 46%, P=.004). All these variants involved 3 different stretches of negatively charged amino acids, with an increase in the isoelectric points (pI) of the mutant protein. As type 1 and type 2 mutations affected stretch I and III, respectively, the 26 indels were categorized into 3 groups on the basis of the stretch they affected: i) type 1-like (61%), affecting stretch I; ii) type 2-like (36%), stretch III; iii) and other types (3%), stretch II. The pI values were significantly different in the 3 groups (P<.001). The frequency of type-1 like mutations was significantly higher in PMF than in ET (82% vs 55%, P=.001). In vitro differentiated megakaryocytes from CALR-mutant patients displayed a significant increase in the extent of both intracellular Ca2+ release from the endoplasmic reticulum and extracellular Ca2+ entry inside the cytoplasm, as compared with healthy controls. Megakaryocytes carrying type 1-like CALR mutations exhibited the highest amplitude of Ca2+ flows regardless of the type of disease. In ET, impaired Ca2+ homeostasis was accompanied by atypical proplatelet architecture (ie, more branches and bifurcations). With respect to clinical phenotype at diagnosis, ET patients with type 2-like CALR mutation showed a trend towards higher PLT count (P=.063) and lower age (P=.053), and significantly lower LDH values (P=.021) than those with type 1-like mutation. In a hierarchical cluster analysis including demographic, clinical and molecular data, CALR mutation type (1 vs 2) identified the 2 clusters with the highest dissimilarity. Considering all patients, those with type 2-like CALR lesions had a better survival than those with JAK2 (V617F) (96.1% vs 84.4% at 10 years, P=.039), while no difference was found between the 2 CALR mutation types. ET patients with type 2-like CALR mutations showed a lower risk of thrombosis than those with JAK2 (V617F) (P=.010). By contrast, ET patients with type 1-like CALR mutations had a higher risk of myelofibrotic transformation that those with type 2-like CALR mutations (P=.029) and especially those with JAK2 (V617F) (P=.011). Finally, PMF patients with type 1-like CALR variants had a better survival than those with JAK2 (V617F) (80.1% vs 48% at 10 years, P=.008). In summary, abnormalities in megakaryocyte calcium metabolism and proplatelet architecture are found in patients with CALR-mutant myeloproliferative neoplasms, and their extent is related to mutation type. Type 2-like CALR mutations are more likely to be associated with isolated thrombocytosis without bone marrow fibrosis, ie, with an ET phenotype. By contrast, type 1-like CALR mutations are generally associated with bone marrow fibrosis, ie, with a PMF phenotype. Thus, in CALR-mutant myeloproliferative neoplasms, the mutation type is a major determinant of the clinical phenotype. Disclosures No relevant conflicts of interest to declare.

A Case Report on Coexisting JAK2 V617F and Calr exon 9 Mutation in Essential Thrombocythemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5215-5215
Author(s):  
Munazza Rashid ◽  
Rifat Zubair Ahmed ◽  
Shariq Ahmed ◽  
Muhammad Nadeem ◽  
Nuzhat Ahmed ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Diagnostic Algorithm ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Common Mutation ◽  
Hypochondriac Region ◽  
Exon 9 ◽  
Calr Mutations

Abstract Myeloproliferative Neoplasms (MPNs) are a heterogeneous group of clonal disorders derived from multipotent hematopoietic myeloid progenitors. Classic "BCR-ABL1-negative" MPNs is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation. The most common mutation in the MPNs PV, ET and PMF is JAK2 V617F. JAK2 V617F can be detected in about 95% of patients with PV while remaining 5% of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one third of patients with ET or PMF do not carryany mutation in JAK2 or MPL. In December 2013 mutations were described in calreticulin (CALR) gene in 67-71% and 56-88% of JAK2 V617F and MPL negative patients with ET and PMF, respectively. Since this discovery, CALR mutations have not only been recommended to be included in the diagnostic algorithm for MPNs, but also CALR exon 9 mutations have been recognised to have clinical utility as mutated patients have a better outcome than JAK2 V617F positive patients.CALR mutations have also been reported to be mutually exclusive with JAK2 V617F or MPL mutations. According to our knowledge so farthere have been only six reports published,which described patients harbouring concurrent JAK2 V617F and CALR exon 9 mutations; seven ET, three PMF, one PV and one MPN-U. In the present study we are reporting ET patient with coexisting JAK2 V617F and CALR exon 9 mutations from our center. In July 2011, 55-years-old female patient was referred to our hospital with a history of gradual elevation of platelet counts accompanied with pain in right hypochondriac region and feet. Bone Marrow aspirate consisted of 'Stag-horn' appearance Megakarocytes. Multiple platelets aggregates and islands were seen throughout the aspirate smear. ARMS-PCR for JAK2 V617F mutation was positive whereas bidirectional Sanger sequencing for CALR exon 9 exhibited c.1214_1225del12 (p.E405_D408del) mutation pattern. Disclosures No relevant conflicts of interest to declare.

Clinical Relevance of Molecular Markers in Taiwan Chinese Patients with Primary Myelofibrosis: Endogenous Erythroid Colony Growth Is the Most Important Predictor of Outcomes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4083-4083
Author(s):  
Ming-Chung Kuo ◽  
Tzung-Chih Tang ◽  
Tung-Liang Lin ◽  
Po-Nan Wang ◽  
Po Dunn ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Clinical Relevance ◽  
Triple Negative ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Important Predictor ◽  
Over Expression ◽  
Allele Burden ◽  
Calr Mutations

Abstract Background and purpose: The clinical features and molecular markers of primary myelofibrosis (PMF) in Asian population have rarely been reported. We examined the clinical relevance of molecular markers in a large cohort of PMF patients in Taiwan. Methods: Bone marrow or blood samples at initial diagnosis from 145 patients consecutively diagnosed with PMF based on WHO criteria in Chang Gung Memorial Hospital-Linkou, Taiwan, were examined. EEC assay was performed in a serum free culture system. PRV-1 mRNA expression in granulocytes was measured by real-time RQ-PCR TaqMan assay. Pyrosequencing was used to detect JAK2 V617Fand its allele burden as well as 46/1 rs12343867 genotype in granulocytes. Mutational analysis of MPL (exon 10) was performed by PCR assay followed by direct sequencing. CALR (exon 9) mutations were screened by GeneScan analysis followed by sequencing for those with length changes. Ten of 20 patients progressed to secondary AML (sAML) had matched paired diagnosis and sAML samples available for comparative analysis. Results: Of the 145 patients with PMF, the median age was 64 years, 76 were male, IPSS low risk 25, Int I 23, Int II 41, and high risk 56 patients. In a median follow-up of 35.8 months (range 1.1 to 275.5 months), 20 patients progressed to sAML, 88 patients died with a median overall survival (OS) of 67.4 months. JAK2 V617F was detected in 52% (74/143) patients, CALR mutations in 30% (41/135) (type1 n=29; type 2 n=5; and others n=7), MPL mutations in 4% (5/141) (n=2/2/1 for W515L/K/A), and 11.0% of PMF patients were triple-negative. The incidence of 46/1 haplotype in 112 patients analyzed was TT 32 %, CT 36 %, and CC 32 %; C-allele frequency was significantly higher in PMF compared with 50 normal subjects (50% vs. 24%; P< 0.0001).EEC growth was detected in 48.9% (45/92) of patients examined. PRV-1 over-expression was present in 40% (28/70) of patients. Of the 10 matched paired PMF/sAML samples, 6 patients had CALR mutations with similar allele burden at both phases of disease whereas sAML evolved from a non-JAK2 V617F clone in one of the 3 patients carrying JAK2 V617F at diagnosis. Patients with EEC growth or PRV-1 over-expression were significantly associated with younger age, higher WBC and platelet counts. EEC-positive patients had higher Hb level and lower circulating blasts. JAK2 V617F was closely associated with higher WBC and platelet counts whereas patients with CALR mutations had lower WBC counts. None of these molecular markers had a correlation with constitutional symptom, IPSS, occurrence of thrombosis or risk of sAML transformation. EEC growth conferred a favorable leukemia-free survival (LFS) (P =0.019) and OS (P =0.013) compared with those without EEC. PRV-1 over-expression was associated with better OS (P =0.036). JAK2 V617F and MPL mutations did not influence LFS and OS. Allele burden of JAK2 V617F had no impact on outcomes. CALR mutations were associated with a favorable OS compared with mutation-negative patients (P =0.034). There were no difference in outcomes between type 1 and type 2 mutations of CALR. Patients with triple-negative mutations had a significantly inferior OS (P =0.020). CT genotype (46/1) was associated with shorter LFS (P =0.026). EEC growth was strongly associated with PRV-1 over-expression and JAK2 V617F mutation, whereas EEC formation and CALR mutations were mutually exclusive. In multivariate analysis, EEC growth was the most important predictor for LFS (HR 0.058; 95% CI: 0.005-0.676, P =0.023) and OS (HR 0.21; 95% CI 0.076-0.581, P =0.003) among the molecular markers; CALR mutations also held favorable OS (HR 0.245; 95% CI 0.085-0.709, P =0.009). Conclusions: Approximately 90% of PMF patients in Taiwan had JAK2 V617F, CALR, or MPL mutations, half were associated with C-allele genotype, 78% had EEC growth and /or PRV-1 over-expression. EEC growth was the most important independent factor for predicting better outcomes and CALR mutations also conferred a favorable OS. (Grant support: NSC96-2314-B-182-003, CMRPG330303, OMRPG3C0021, and MOHW103-TD-B-111-09) Disclosures No relevant conflicts of interest to declare.

scholarly journals Calr Mutational Status in Egyptian Patients with Myeloproliferative Neoplams

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5388-5388
Author(s):  
Eman A. Soliman ◽  
Samah I. El-Ghlban ◽  
Abdelaleem H. Abdelaleem ◽  
Sherin Abdel-Aziz ◽  
Sameh Shamaa ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Response To Therapy ◽  
Hcv Infection ◽  
Number Of Patients ◽  
Significant Difference ◽  
Egyptian Patients ◽  
Calr Mutations

It has been known that the insertion/deletion mutation of CALR gene is the second deriver mutation in myeloproliferative neoplasm (MPN) of essential thrompocythemia (ET) and primary myelofibrosis (PMF). As the molecular workup has been incorporated for the prospective screening and diagnosis of MPN in our Oncology Center. An Egyptian 87 cohort of patients with non-mutated JAK2 (58 ET and 29 MF) were investigated using polymerase chain reaction (PCR) as a pilot study. We found that 37 out of 87 patients (42%) were carrying CALR mutations (30 out of ET (52%) and 7 out of MF (24%)). Sanger sequencing was used to determine the type of CALR mutations in all positive patients and we found that 13 out of 37 (35%) had type 1/type 1-like and 36 out of 37 (97%) with type 2/type 2-like. This CALR mutation profile in Egyptian patients appear different from the western status as type2/type 2-like is the highest in our patients (97%) versus 35-45% and type1/type 1-like was 35% versus 55-65% compared to western results. Meanwhile, the clinical course and phenotype of our cohort of patients is not similar to that in western as there is no significant difference of overall survival between type1/type1-like and type2/type2-like. This finding might be due to the different environmental and genetic backgrounds of Egyptian population. A part of it might be related to the HCV infection as 12 out of 37 (32%) had HCV infection. Further study is in progress on a large number of patients to correlate that with the clinicopathological status, response to therapy and the mechanistic pathway of oncogenic transformation. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Pathogenic CALR Exon 9 Mutation in a Patient with Essential Thrombocythemia

2019 ◽  
Vol 51 (3) ◽  
pp. 306-309
Author(s):  
Jee-Soo Lee ◽  
Ho Young Kim ◽  
Miyoung Kim ◽  
Young Kyung Lee
Keyword(s):  
Essential Thrombocythemia ◽  
Frameshift Mutation ◽  
Myeloproliferative Neoplasms ◽  
Case Reports ◽  
Individual Case ◽  
First Case ◽  
Exon 9 ◽  
Calr Mutations

Abstract The clinical phenotypes and prognoses of CALR-mutant myeloproliferative neoplasms depend on the mutation type. The 2 most common mutations, type 1 (52-bp deletion) and type 2 (5-bp insertion), account for 85% of CALR-mutated neoplasms. The former confers a myelofibrotic phenotype, and the latter is associated with a low risk of thrombosis and an indolent clinical course. Individual case reports for patients with novel pathogenic CALR mutations are rare. Herein, we present the first case in the literature, to our knowledge, of a 63-year old ethnic Korean man with essential thrombocythemia who was diagnosed with a novel +1-bp frameshift mutation in CALR, which was predicted to exhibit a type 2–like phenotype.

scholarly journals Calreticulin and JAK2 Exon 12 Mutation Screening in Patients with Myeloproliferative Neoplasms’ in Jeddah Region, Saudi Arabia

2021 ◽  
pp. 420-428
Author(s):  
Heba Alkhatabi ◽  
Heyam Abdulqayoom ◽  
Raed Alserihi ◽  
Raed Felimban ◽  
Aisha Elaimi ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Pcr Analysis ◽  
Allele Specific ◽  
Pcr Method ◽  
Calr Mutations ◽  
The Impact

Background: The JAK2 V617F mutation’s discovery has largely facilitated the comprehension of the myeloproliferative neoplasms’(MPNs) pathogenesis. In recent times, calreticulin (CALR) mutations have been detected in patients with JAK2V617F negative primary myelofibrosis (PMF), and essential thrombocythemia (ET). Methods: This study analyzed the impact of JAK 2 Exon 12 and CALR common mutations in 65 patients with JAK2V617F negative MPN from the Jeddah region. An allele-specific polymerase chain reaction (PCR) method was used to screen four common mutations on Exon 12 and direct sequencing and PCR analysis were utilized to screen all patients for CALR. Results: None of the patients were positive for the Exon 12 mutation and eight patients were positive for CALR mutations. Conclusions: This is the first Saudi Arabian research that focused on screening CALR hotspot mutations and this mutation exists. This fact highlights the importance of implementing diagnostic screening of CALR on MPN patients, in general, and patients with high platelet count, in particular. Further screening of other predisposing genetic markers might facilitate the identification of an important genetic variant, which could aid in the understanding of disease pathogenesis.

scholarly journals Modeling Calreticulin-Mutant Myeloproliferative Neoplasms with Isogenic Induced Pluripotent Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4319-4319 ◽  
Author(s):  
Wei Wang ◽  
Tiansu Wang ◽  
Andriana G. Kotini ◽  
Camelia Iancu-Rubin ◽  
Ronald Hoffman ◽  
...  
Keyword(s):  
Research Funding ◽  
Ex Vivo ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Mutant Type ◽  
C Terminus ◽  
Calr Mutations

Abstract Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.

Frequency and Prognosis of JAK2 V617F, Calr, MPL and ASXL1 Mutations in Primary Myelofibrosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5562-5562
Author(s):  
Marc Sorigue ◽  
Marta Cabezón ◽  
Olga Garcia ◽  
Patricia Velez ◽  
Silvia Marce ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Jak2 V617f Mutation ◽  
Prognostic Impact ◽  
Wild Type ◽  
Exon 9 ◽  
V617f Mutation ◽  
Calr Mutations ◽  
Exon 10

Abstract INTRODUCTION: Several mutations have been described in patients with BCR-ABL1 –negative chronic myeloproliferative neoplasms, including primary myelofibrosis (PMF). The most frequent mutation is JAK2 V617F, followed by calreticulin exon 9 (CALR), MPL exon 10 and ASXL1 exon 12. Currently, less than 10% of patients lack molecular marker. CALR and ASXL1 mutations have been consistently found to have favorable and unfavorable prognostic implications, respectively. The aim of this study was to describe the frequency and prognostic impact of JAK2 V617F, CALR, MPL and ASXL1 mutations in patients diagnosed with PMF in 6 Spanish hospitals. METHODS: To detect the presence of JAK2 V617F mutation, an allele-specific PCR using TaqMan probes was used. Screening for insertions and deletions in CALR gene was performed with 6-FAM labeled primers spanning exon 9 and CALR mutations were described by Sanger sequencing. Sanger sequencing was also used to detect MPL exon 10 and ASXL1 exon 12 mutations. RESULTS: Sixty-eight patients were included in the study. All of them were screened for JAK2 and CALR mutations. Forty-five of them (66%) were positive for the JAK2 V617F mutation, while 11/68 (16%) were positive for CALR mutations. Of the 11 CALR mutations, 10 were JAK2 wild-type. MPL exon 10 mutation analysis was only performed in JAK2 wild-type patients and was positive in 4/23 patients (17%), and all of them were CALR wild-type. At the time of submission, ASXL1 exon 12 has been assessed in 18 patients (analysis in the rest of them is currently ongoing). ASXL1 mutations have been found in 3/18 (17%) patients, two of them also with a CALR mutation and the other one with the JAK2 V617F mutation. All three cases were indel mutations. Overall, no mutation was detected in 9/68 (13%) patients. JAK2 V617F, CALR and MPL mutations had no prognostic impact on overall survival. The effect of ASXL1 mutation on prognosis (with and without CALR mutation) will be assessed once all samples have been sequenced. CONCLUSION: JAK2 V617F, CALR and MPL mutations were found in our series of PMF patients in the same proportion found in larger series. ASXL1 has so far been found in a smaller percentage but the entire series of patients will need to be sequenced before reaching definitive conclusions. Studying these genes, only 13% of patients with PMF did not have a clonal marker. None of the studied mutations had prognostic significance. ACKNOWLEDGMENTS: The authors would like to thank Diana Dominguez for her excellent technical assistance and to the grant 2014 SGR225 (GRE), Generalitat de Catalunya. Disclosures No relevant conflicts of interest to declare.

A Greater Mutational Complexity May Contribute to the Differential Prognostic Impact of Type 1/Type 1-like Versus Type 2/Type2-like Calreticulin Mutations in Primary Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1627-1627
Author(s):  
Paola Guglielmelli ◽  
Giada Rotunno ◽  
Tiziana Fanelli ◽  
Annalisa Pacilli ◽  
Laura Calabresi ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Chronic Phase ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Driver Mutations ◽  
Prognostic Impact ◽  
Advisory Committees ◽  
Significant Difference

Abstract BACKGROUND. Calreticulin (CALR) mutations (mut) are associated with a IPSS/DIPSSplus and mutated ASXL1-independent favorable outcome in patients (pts) with primary myelofibrosis (PMF) compared to those harboring JAK2 V617F and MPL W515 mut and those lacking driver mutations ("triple negative", TN). Spliceosome mut were reported to be less represented in CALR - mut pts compared with other genotypes (Leukemia 2014;28:1472), but no other molecular or cytogenetic correlate is known. It is also debated whether there are prognostic differences between type 1/type 1 like (Ty1; a 52 bp deletion; p.L367fs*46) and type 2/type 2 like (Ty2; a 5 bp TTGTC insertion; p.K385fs*47) CALR mut. Tefferi et al (Blood 2014;124:2465) reported that only Ty1 was prognostically favorable, while either the opposite or no effect was found in other studies (Leukemia 2015; 29:249). AIMS and METHODS. To evaluate the prognostic relevance of different types of CALR mut in a series of 396 PMF pts (2008 WHO criteria) seen in our center. CALR mut were analyzed by high-resolution capillary electrophoresis and confirmed by Sanger sequencing. Deep sequencing based on Ion Torrent PGM platform was used to analyze mutations in 17 genes previously shown to recur at discrete frequencies in chronic phase and leukemic transformation (LT) of PMF. Comparisons of quantitative variables between groups were carried out by the nonparametric Wilcoxon rank-sum test. RESULTS. 251 pts (63.4%) harbored JAK2 V617F mut, 21 (5.3%) MPL W515 mut, 74 (18.7%) CALR mut, of which 53 (71.6%) Ty1 and 21 (28.4%) Ty2; 50 pts (12.6%) were TN. There was no statistically significant difference between Ty1 and Ty2 for common hematologic and clinical variables. Conversely, both Ty1 and Ty2 differed from JAK2 V617F mut counterpart for younger age, lower leukocyte and higher platelet counts; males were more represented among Ty1 pts than other genotypes. Among CALR Ty1, more pts were in lower (low+Intermediate-1) IPSS risk categories (80%) compared to Ty2 (62%; P=0.04). With a median follow-up of 6.4 and 5.4 yrs for CALR mut Ty1 and Ty2 pts, respectively, there were less pts who died among Ty1 (17%) compared to Ty2 (47.6%) (P=0.007). Median survival of CALR Ty1 was 26.4 yr (range, 15.5-37.3) vs 7.4 yr (4.6-10.2) for CALR Ty2, 7.2 yr (5.7-8.6) for JAK2 V617F and 2.0 (1.6-2.4) for TN (P<.0001). The corresponding hazard ratio, taking CALR Ty1 pts as the reference, was 4.9 (95% CI, 1.8-12.9), 6.0 (95% CI, 2.7-13.4) and 20.6 (95% CI, 8.9-48.4) for CALR Ty2, JAK2 V617F and TN pts, respectively. In a multivariable Cox proportional hazard regression model, CALR Ty2 (HR 4.4, 95% CI 1.6-11.7), JAK2 V617F (HR 3.8, 95% CI 1.7-8.7) and TN (HR 9.2, 95%CI 3.8-22.2) all retained IPSS-independent prognostic impact on survival. Since some subclonal mutations are prognostically relevant in PMF (Leukemia 2013; 27:1861), we compared the mutational profile of 17 such genes, functionally clustered as epigenetic (DNMT3A, EZH2 TET2, IDH2, IDH1, ASXL1), of thespliceosome (SF3B1, SH2B3 U2AF1, SRSF2) orassociated with LT (cKIT, RUNX1, NRAS, KRAS, IKZF1, TP53 CBL) in 44 and 20 CALR mut Ty1 and Ty2 pts, respectively. There was a greater proportion of pts harboring at least one mutated gene among CALR Ty2 than Ty1 (70% vs 56.8%; P=0.02). Also, the proportion of pts with >2 mut was significantly greater in CALR mut Ty2 compared to Ty1 (40% vs 18%; P=0.01); in particular, 15% of CALR Ty2 mut pts had >3 mut compared to 2.2% in Ty1 (P=0.04). Frequency of individual mutations in Ty2 vs Ty1 was: epigenetic, 50% vs 50%; spliceasome 30% vs 4.5% (P=0.009); LT 30% vs 13.6% (P=0.06). Of note, SF3B1 mutations were 10-fold more represented in CALR mut Ty2 (25%) compared to Ty1 (2.2%; P<0.01). TP53 mutations were found in 10% of Ty2 and 0 in Ty 1. CONCLUSIONS. These findings support previous report that the prognostic advantage of CALR mutation in PMF regards only pts harboring type 1/type 1-like abnormalities. A greater mutational complexity involving subclonal mutations, particularly in genes of the spliceosome with an unusually high incidence of SF3B1 mutations, as well as increased number of mutated genes, may contribute to the dismal outcome of CALR mut Ty2 pts. Disclosures Vannucchi: Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Shire: Speakers Bureau.

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