Prevalence of JAK2V617F, CALR in Philadelphia Positive and Negative Myeloproliferative Neoplasm

Background: Myeloproliferative neoplasms (MPNs) are heterogeneous disorders with a variety of genetic abnormalities. We aim to assess the prevalence of Calreticulin (CALR) and JAK2 mutations in Iranian MPNs. Materials and Methods: In a cross-sectional study, CALR and JAK2 mutations among 130 MPNs patients, including 78 Philadelphia chromosome-negative (MPN-) and 52 Philadelphia chromosome-positive (MPN+) as well as 51 healthy control subjects, were investigated by GAP-PCR. Results: In MPN- group JAK2 and CALR gene mutations were found in 64.1% and 7.7%, respectively, that 5.1% were positive for both mutations, and 2.6% had only CALR mutation. In polycythemia vera (PV) patients 90% had JAK2 mutation, which was significantly higher than other MPN- or MPN+ patients. Most of the MPN+ patients had neither mutation in CALR nor JAK2 (70% CALR-/JAK2-). Among all patients’ groups, the prevalence of CALR+ mutation in either rs1450785140 (4 cases) or rs765476509 (5 cases) position was not statistically different. Conclusion: These results showed a low prevalence of CALR mutations in all types of MPNs in the Iranian population that its frequency may influence by ethnicity and genetic diversity. CALR mutation may be seen in JAK2 negative cases, also. The PV had the highest JAK2 mutation with a 90 percent positivity rate among MPNs cases. [GMJ.2021;10:e2127]

VEGFA rs3025020 Polymorphism Contributes to CALR-Mutation Susceptibility and Is Associated with Low Risk of Deep Vein Thrombosis in Primary Myelofibrosis

Background Single nucleotide polymorphisms (SNPs) in vascular endothelial growth factor A (VEGFA) are associated with susceptibility to several diseases including cancer. Correlations between VEGFA rs3025020 genotypes with clinical and laboratory features of primary myelofibrosis (PMF) are unstudied. Methods DNA was analyzed by real-time polymerase chain reaction for VEGFA rs3025020 genotypes in a cohort of 844 subjects with PMF and in two cohorts of normal subjects (N = 247 and N = 107). Results Frequency of rs3025020 minor allele (T) was not significantly different in subjects with PMF compared with normals; however, the T-allele was more frequent in PMF subjects with a calreticulin (CALR)-mutated genotype compared with normals (35 vs. 27%; OR = 1.47 [95% CI, 1.09, 1.98] p = 0.011), especially in subjects with a CALR-type 2/type 2-like mutation (43 vs. 27%; OR = 2.01 [1.25, 3.24] p = 0.004). CALR mutants with the rs3025020 TT genotype had higher CXCR4 expression on CD34-positive blood cells, and those who carried CT/TT genotypes had lower platelet concentrations compared with other genotypes at diagnosis. Overall, subjects with the rs3025020 CT/TT genotype had a lower cumulative incidence of deep vein thrombosis in typical sites (1.6 vs. 4.2%; OR = 0.37 [0.15, 0.90] p = 0.029) and longer interval from diagnosis to first thrombosis (HR = 0.37 [0.14, 0.95] p = 0.039). Conclusion Persons with PMF and the VEGFA rs3025020 minor T-allele are more likely to have a CALR mutation compared with other somatic driver mutations and lower cumulative incidence and hazard for deep vein thrombosis in typical sites.

PEAR1 Genetic Variants in Essential Thrombocythemia: A New Study of The Prevalence and Association of PEAR1 Variants with Hematological Parameters and ET Mutations

Objective: Essential thrombocythemia (ET) is a type of myeloproliferative neoplasm characterized by the expansion of the megakaryocytic/platelet line. Given the undeniable role of genetic variations in the pathogenesis of ET, as well as the proven effects of PEAR1 SNPs on platelet function, the innovative purpose of this study is to investigate the prevalence of PEAR1 variants (rs12041331 and rs12566888) and their relationship to hematological parameters and ET-related mutations.Materials and Methods: We studied 105 ET patients and analyzed ET patients' mutational profiles, including JAK2 V617F mutation (detected by Allele-specific PCR), CALR, and MPL mutations (both through PCR amplification). Two SNPs of the PEAR1 gene were assessed through ARMS-PCR, and the Sanger method was used for the validation of ARMS-PCR amplification.Results: The prevalence of rs12041331 and rs12566888 in ET patients were 43.9% and 38.5%, respectively, and rs12041331 was significantly associated with increased platelet counts (P-Value: 0.02). A significant relationship was also found between the rs12041331 and CALR mutation (P-Value: 0.03). Platelet count was higher in CALR+ patients (934.45 ×10 9/L ± 265.35 SD) than in JAK2 + patients (790.11 ×10 9/L ± 265.35 SD). Conversely, other hematological parameters and thrombosis were higher in JAK2 + patients than the CALR + patients.Conclusions: Our findings reinforce the idea that rs12041331 and rs12566888 may be associated with ET, and rs12041331 is significantly associated with increased platelet count. Besides, the prevalence of ET-related mutations in patients with rs12041331 and rs12566888 was almost similar; however, only CALR mutation had a significant relationship with rs12041331.

A NEW TYPE-2-LIKE CALR MUTATION IN ESSENTIAL THROMBOCYTHEMIA

CALR mutations, together with JAK-2 and MPL ones, are recognized as “driver” mutations in Philadelphia-negative chronic myeloproliferative neoplasms (MPNs). Most frequent CALR mutations are Type-1 deletions (45-55% of cases) and type-2 insertion (32-42% of cases). These mutations are usually associated with younger age, higher platelet counts, lower leukocyte counts, lower hemoglobin levels and a higher incidence of transformation from ET to MF. Recognizing and describing cases with different mutations can be useful to create a database that might help clinicians to include these patients in risk categories and to guide the appropriate therapeutic choices. We report a case of a 77-years old woman who presented a new type-2 like CALR mutation.

JAK2 V617F Mutation Negative Polycythemia Diagnosed By IGF-1R, EEC Formation , and IRS2 over Expressions

We previously reported that by flow cytometry study of mononuclear cells (peripheral blood) of IGF-1 R (insulin growth factor -1 receptor) can help in differentiating polycythemia vera (PV) from secondary erythrocytosis (PLoS ONE 11(11): e016529), in patients negative for JAK2V617F mutation who requires frequent phlebotomy. EEC (Endogenous erythroid colony formation) formation which was shown to be related to IGF-1 signaling was employed as a minor criteria of PV prior to 2016 WHO criteria for the diagnosis of PV. IRS-2, an IGF-1 activation downstream signaling protein was found to be a docking protein, specific for erythropoietin-induced erythropoiesis. Current studies were designed to correlation between IGF-1R over-expression to EEC (Endogenous erythroid colony formation) formation and IRS-2 over expression in those patients who required frequent phlebotomy with elevated IGF-1R and JAK2 mutation negative patients. Materials and Methods: Patients: 8 patients withsecondary polycythemia (JAK2V617F mutation negative), and 12 MPN patients (6 ET, 3 PV and 3 MF) were studied. Of the 12 MPN patients, 11 were JAK2 mutation positive and 1 was CALR mutation positive. All 20 patients had elevated IGF-1 R. Methods: 1) IGF-1R expression: 106 cells blood mononuclear cells were incubated with 10 μL of PE-conjugated control or anti-IGF1R IgG (R&D Systems) for 30 minutes. Fluorescent intensity was detected by a flow cytometry. 2) EEC formation: Erythroid colony cultures using Methyl cellulose based medium without (MethoCult H4230) or with pre-inclusion of cytokines (MethoCult H4230, including erythropoitin) from StemCell Technologies Inc. were used. On day 10-12, colonies were scored for Burst forming unit-erythroid (BFU-e), Colony forming unit-erythroid (CFU-e), and Colony forming unit cells (CFU-c) according to the manufacturer's graphic guide. 3) IRS-2 Expression: RT-PCR Predesigned primers for IRS-2 and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR blood mononuclear cells was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of IRS-2 was compared with each internal control and the final patient to normal control ratio was the average of the three. Results: 7 out of 8 patients with secondary PV, were found to have EEC formation. 12 MPN patients had increased expression of IRS-2 (Fig 1). 2 patients with secondary PV, so far studied also were found to have increased expression of IRS-2. Conclusion: 1) In patients with secondary PV (JAK2 mutation negative and increased IGF-1 and requiring frequent phlebotomy0, 7 out of 8 (87%) were found to have EEC formation. This suggests that IGF-1 by Flow cytometry can replace EEC as a supplement to the diagnosis of PV. 2) In JAK2 or CALR mutation positive patients , IGF-1R over-expression were found to have to have IRS-2 over-expression , 2 patients with secondary PV so far studied were also found to have elevated IRS-2 signaling. This suggests that the etiology of erythrocytosis in these secondary PV is likely through IRS-2 pathway. Further studies on this is still in progress. Disclosures No relevant conflicts of interest to declare.

Philadelphia+ Chronic Myeloid Leukemia with CALR Mutation: A Case Report and Literature Review

Myeloproliferative neoplasms (MPNs) are classified as chronic myeloid leukemia (CML) and Philadelphia chromosome-negative MPN. In MPN cases, the presence of a <i>BCR-ABL1</i> translocation with a coexisting mutation is exceptionally rare. Herein, we report the first documented patient with CML harboring <i>CALR</i> mutation in Korea. A 33-year-old woman was referred to our hospital in February 2015 with splenomegaly, leukocytosis, and thrombocytosis. She was diagnosed with CML and started receiving nilotinib. In October 2015, a major molecular response was observed, but thrombocytosis persisted. A repeat bone marrow (BM) examination revealed no specific findings. However, as thrombocytosis worsened, we changed nilotinib to dasatinib. In May 2019, owing to persistent thrombocytosis, we repeated the BM examination and found <i>CALR</i> mutation (15.97%) on the MPN–next generation sequencing (NGS) test. We then retrospectively performed repeat MPN-NGS testing using the BM aspirate sample obtained in 2015 and found <i>CALR</i> mutation (10.64%).