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

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.

Download Full-text

Carlituculin and JAK2 Exon 12 Mutation Screening in Patients with Myeloproliferative Neoplasm’s in Jeddah Region, Saudi Arabia

10.21203/rs.3.rs-849146/v1 ◽

2021 ◽

Author(s):

Heba Alkhatabi ◽

Heyam Abdulqayoom ◽

Raed Alserihi ◽

Raed Felimban ◽

Aisha Elaimi ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Mutation Screening ◽

Gene Mutations ◽

Primary Myelofibrosis ◽

Pcr Analysis ◽

Negative Results ◽

Allele Specific ◽

Pcr Method ◽

The Impact

Abstract PurposeThe JAK2V617F 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 negative MPNs for JAK2V617F. The CALR gene mutations have been reported in patients with negative JAK2V617F, primary myelofibrosis (PMF), and essential thrombocythemia (ET). This study analyzed the impact of JAK2 Exon 12 and CALR common mutations on MPN patients from the Jeddah region.MethodsAn allele-specific polymerase chain reaction (PCR) method was used to screen four common mutations on Exon 12. Consequently, the results showed that none of the patients were positive for the Exon 12 mutation. Direct sequencing and PCR analysis were utilized to screen all patients for CALR. Results The impact of CALR and JAK2 Exon 12 in 65 patients with a variety of MPN symptoms was investigated. This study included patients who had negative results, when previously screened for JAK2V617F. CALR mutations were identified in eight patients out of 65 JAK2V617F and JAK2 Exon 12 negative patients in this study’s cohort. 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. In addition, 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.

Download Full-text

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

Journal of the Medical Association of Thailand ◽

10.35755/jmedassocthai.2021.05.12344 ◽

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)

Download Full-text

STAT5 is Expressed in CD34+/CD38− Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms

Cancers ◽

10.3390/cancers12041021 ◽

2020 ◽

Vol 12 (4) ◽

pp. 1021 ◽

Cited By ~ 3

Author(s):

Emir Hadzijusufovic ◽

Alexandra Keller ◽

Daniela Berger ◽

Georg Greiner ◽

Bettina Wingelhofer ◽

...

Keyword(s):

Stem Cells ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Janus Kinase ◽

Signaling Cascades ◽

Nuclear Compartment ◽

Downstream Signaling ◽

Direct Targeting ◽

Calr Mutations

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38− MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38− MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

Download Full-text

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

Blood ◽

10.1182/blood.v126.23.5215.5215 ◽

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.

Download Full-text

Analysis of JAK2 V617F mutation in Tunisian patients with myeloproliferative neoplasms

European Journal of Inflammation ◽

10.1177/20587392211006538 ◽

2021 ◽

Vol 19 ◽

pp. 205873922110065

Author(s):

Soumaya Chadi ◽

Tarak Dhaouadi ◽

Imen Sfar ◽

Hela Baccouche ◽

Rym Nabli ◽

...

Keyword(s):

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Jak2 V617f Mutation ◽

Pcr Rflp ◽

Tunisian Patients ◽

Healthy Control ◽

V617f Mutation

We aimed to investigate the prevalence of the JAK2 V617F mutation in Tunisian patients with myeloproliferative neoplasms (MPN) and to look for possible associations with diseases’ presentation. In this context, JAK2 V617F polymorphism was detected by PCR-RFLP and direct sequencing in 213 MPN patients (109 with polycythemia vera (PV), 93 with essential thrombocythemia (ET) and 11 with primary myelofibrosis (PMF)), 77 unclassified patients with thrombosis (UPT) and 95 healthy control subjects. The JAK2 V617F mutant allele was present by either PCR-RFLP or direct sequencing in 158 (74.17%) MPN patients while all UPT and controls were negative. Besides, the JAK2 V617F mutation was significantly more frequent in patients with PV 98 (89.9%) than in ET 54 (58.1%) and PMF 6 (54.5%) groups, p < 0.001. Analytic results in MPN patients showed significant associations between the JAK2 SNP and both hemoglobin levels (16.29 ± 3 vs 13.01 ± 3.65) and hematocrit (52.99 ± 8.34 vs 45.37 ± 10.94), p < 0.001 and p < 0.001, respectively. In addition, in the ET subgroup thrombosis was significantly more frequent in patients carrying the V617F mutation (16, (29.6%) vs 3, (7.7%)), p = 0.01. In ET patients, the V617F mutation seems to be predictive of thrombosis occurrence.

Download Full-text

Bone marrow megakaryocytic activation predicts fibrotic evolution of Philadelphia-negative myeloproliferative neoplasms.

Haematologica ◽

10.3324/haematol.2020.264143 ◽

2020 ◽

pp. 0-0

Author(s):

Mattia Schino ◽

Vincenzo Fiorentino ◽

Elena Rossi ◽

Silvia Betti ◽

Monica Di Cecca ◽

...

Keyword(s):

Bone Marrow ◽

Myeloproliferative Neoplasms ◽

Rapid Evolution ◽

Progression Free Survival ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Serum Levels ◽

Chronic Myeloproliferative Neoplasms ◽

Calr Mutations ◽

Wbc Count

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have been traditionally considered as indistinctly slowly progressing conditions; recent evidence proves that a subset of cases have a rapid evolution, so that MPNs’ prognosis needs to be personalized. We identified a new morphological parameter, defined as Megakaryocytic Activation (M-ACT) based on the coexistence of megakaryocytic emperipolesis, megakaryocytes (MK) clusters formation and evidence of arrangement of collagen fibers around the perimeter of MK. We retrospectively analyzed the bone marrow biopsy of two MPNs cohorts of patients with polycythemia (PV) (n=64) and non-PV patients [including essential thrombocythemia (ET), and early/prefibrotic primary myelofibrosis (PMF)] (n=222). M-ACT showed a significant correlation with splenomegaly, white blood cell (WBC) count, and LDH serum levels in both groups, with JAK2 V617F allele burden in PV patients, and with CALR mutations, and platelet count in non-PV patients. Progression-free survival, defined as PV-to-secondary MF progression and non-PV-to-overt PMF, was worse in both PV and early/prefibrotic PMF patients with M-ACT in comparison to those without M-ACT (P<.0001). Interestingly, M-ACT was not found in the subgroup of ET patients. In conclusion, M-ACT can be helpful in the differential diagnosis of MPNs and can represent a new morphologic parameter with a predictive value for progression of MPNs.

Download Full-text

Frequency and Molecular Characteristics of Calreticulin Gene (CALR) Mutations in Patients with JAK2-Negative Myeloproliferative Neoplasms

Acta Haematologica ◽

10.1159/000366263 ◽

2014 ◽

Vol 133 (2) ◽

pp. 193-198 ◽

Cited By ~ 10

Author(s):

Marzena Wojtaszewska ◽

Małgorzata Iwoła ◽

Krzysztof Lewandowski

Keyword(s):

Data Analysis ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Molecular Characteristics ◽

New Mutation ◽

Clinical Data Analysis ◽

Leukocyte Counts ◽

Exon 9 ◽

Calr Mutations

In 2013, Nangalia et al. and Klampfl et al. found a recurrent and abundant mutation in the calreticulin gene (CALR), mutually exclusive with JAK2 and MPL alterations. At present, the data concerning the new mutation, i.e. its prevalence, allele burden and clinical significance, are scarce. We report the incidence and molecular characteristics of CALR mutations in a group of 184 Polish patients with myeloproliferative neoplasms (MPNs). Clinical data analysis revealed significant differences between JAK2 V617F-mutated and CALR-mutated groups. In essential thrombocythemia patients, hemoglobin levels and leukocyte counts were significantly higher in JAK2-positive than in CALR-positive patients (p = 0.023 and p = 0.017, respectively), but the CALR-positive patients had significantly higher platelet counts (p = 0.022). Patients harboring CALR mutations were also younger at the time of diagnosis (p = 0.039). In primary myelofibrosis patients, the degree of anemia was less severe in those who were CALR exon 9 mutation-positive than in those who were JAK2 V617F-positive (p = 0.048). © 2014 S. Karger AG, Basel

Download Full-text

Calreticulin Gene Mutations in Suspicion of Myeloproliferative Neoplasms: A Single Institution Experience

Blood ◽

10.1182/blood.v124.21.5569.5569 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5569-5569

Author(s):

Amin Ben Lassoued ◽

Nathalie Beaufils ◽

Caroline Bonnefoy ◽

Jean Gabert

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Molecular Diagnostic ◽

Platelet Counts ◽

Study Population ◽

Calr Mutations

Abstract Recently, Klampfl et al. and Nangalia et al. reported recurrent somatic mutations in the calreticulin (CALR) gene in patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. All CALR mutations described to date are either deletions or insertions, and occur in exon 9 and result in a frameshift leading to a loss of KDEL sequence (endoplasmic reticulum retention motif) and multiple calcium biding sites with a new basic (instead of acidic) C terminal region. The aim of this work is to know how far the CALR mutations can fill the molecular diagnostic gap for myeloproliferative neoplasms (MPNs) left by JAK2 and MPL mutations. The study was approved by the local ethics committee. Our study population consists of 155 patients with platelet counts > 500 G/L sampled between 2012 and 2013 for suspicion of MPNs. Only JAK2 V617F negative patients were included. Analyses were performed using genomic DNA isolated from peripheral blood. Patients were screened for CALR mutations using a High Resolution Melting (HRM) and then the precise mutations were characterized by Sanger sequencing which is a new approach for this test. A total of 21 (13.5%) patients with CALR mutations were detected with 8 distinct variants. Among CALR mutations, type 1 (52 basepair deletion, c.1092_1143del) and type 2 (5 basepair insertion, c.1154_1155insTTGTC) were the most common (respectively 7 (33%) and 7 (33%) cases). Three patients had 2 concurrent mutations (insertion+substitution / Deletion+substitution / deletion +insertion). We found a deletion that has not been reported so far to our knowledge (c.1125_1147del). Sequencing is ongoing for 4 patients. Compared with negative patients, CALR-mutated patients demonstrated markedly higher platelet counts (median count: 820 vs 543 G/L). Disclosures No relevant conflicts of interest to declare.

Download Full-text

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

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18147582 ◽

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.

Download Full-text