Abstract
Calreticulin (CALR) has been recently detected in BCR-ABL1 and JAK2V617F negative myeloproliferative neoplasms (MPNs). In this study, we used PCR-PAGE and DNA sequencing methods for detection of CALR mutations and analysis of laboratory findings in MPN patients. BCR-ABL1, JAK2V617F, andMPL515 were assessed using RT-PCR and ARMS-PCR methods in 103 suspected MPN patients. CALR was assessed in patients negative for BCR-ABL1 and JAK2V617Fpatients as well as six cell lines (Jurkat, U937, NB4, KCL22, THP1, and K562) using PCR-PAGE and DNA sequencing. Finally, laboratory data of patients were obtained. BCR-ABL1 and JAK2V617F were detected in 39 (37.86 %) and 34 (33 %) MPN patients, respectively. Two patients had combined BCR-ABL1/JAK2V617F mutation. MPL 515 was negative in all the patients. CALR mutation was found in six male JAK2V617F-negative MPN patients (28.6 %, p = 0.07). Platelet count was 1049.5 × 109/L (p = 0.01) in these patients. All patients with type 1 and 2 CALR mutations had platelet counts lower and higher than 1000 × 109/L, respectively. CALR-positive patients had lower WBC count versus JAK2V617F-positive patients. Finally, CALR mutations were negative in all the cell lines. PCR-PAGE detected mutations in a shorter time with lower cost. JAK2V617F may be detected in BCR-ABL1-positive patients and is not specific for these patients. CALR is useful for molecular diagnosis of JAK2V617F- and MPL-negative MPN patients. PCR-PAGE may be a rapid, cost-effective, and simple method, especially in developing countries. CALR mutations are detected in male patients, suggesting different incidences of CALR mutations in male/female populations in different nations and races.
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References
Andrikovics H, Krahling T, Balassa K, Halm G, Bors A, Koszarska M et al (2014) Distinct clinical characteristics of myeloproliferative neoplasms with calreticulin mutations. haematologica 99(7):1184–90
Bibi A, Agarwal NK, Dihazi GH, Eltoweissy M, Van Nguyen P, Mueller GA et al (2011) Calreticulin is crucial for calcium homeostasis mediated adaptation and survival of thick ascending limb of Henle’s loop cells under osmotic stress. Int J Biochem Cell Biol 43(8):1187–97
Cazzola M, Kralovics R (2014) From Janus kinase 2 to calreticulin: the clinically relevant genomic landscape of myeloproliferative neoplasms. Blood 123(24):3714–9
Chen C-C, Gau J-P, Chou H-J, You J-Y, Huang C-E, Chen Y-Y et al (2014) Frequencies, clinical characteristics, and outcome of somatic CALR mutations in JAK2-unmutated essential thrombocythemia. Ann Hematol 93(12):2029–36
Dameshek W (1951) Some speculations on the myeloproliferative syndromes. Blood 6:372–5
Ellison G, Donald E, Mcwalter G, Knight L, Fletcher L, Sherwood J et al (2010) A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples. J Exp Clin Cancer Res 29(1):132
Fateh A, Aghasadeghi M, Siadat SD, Vaziri F, Sadeghi F, Fateh R et al (2016) Comparison of three different methods for detection of il28 rs12979860 polymorphisms as a predictor of treatment outcome in patients with hepatitis C virus. Osong Public Health Res Perspect 7(2):83–9
Ganguly A, Williams C (1997) Detection of mutations in multi-exon genes: comparison of conformation sensitive gel electrophoresis and sequencing strategies with respect to cost and time for finding mutations. Hum Mutat 9(4):339–43
Guglielmelli P, Nangalia J, Green AR, Vannucchi AM (2014) CALR mutations in myeloproliferative neoplasms: hidden behind the reticulum. Am J Hematol 89(5):453–6
Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al (2013) Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 369(25):2379–90
Kuwabara K, Pinsky DJ, Schmidt AM, Benedict B, Brett B, Ogawa S et al (1995) Calreticulin, an antithrombotic agent which binds to vitamin K-dependent coagulation factors, stimulates endothelial nitric oxide production, and limits thrombosis in canine coronary arteries. J Biol Chem 270(14):8179–87
Li B, Xu J, Wang J, Gale RP, Xu Z, Cui Y et al (2014a) Calreticulin mutations in Chinese with primary myelofibrosis. Haematologica 99(11):1697–700
Li C, Wu J, Wang Z, Feng J (2014b) A comparison of direct sequencing and ARMS assay performance in EGFR mutation analysis of non-small cell lung cancer patients. Chinese J Lung Cancer 17(8):606–11
Luo W, Yu Z (2015) Calreticulin (CALR) mutation in myeloproliferative neoplasms (MPNs). Stem Cell Invest 2:16
Mahdieh N, Rabbani B (2013) An overview of mutation detection methods in genetic disorders. Iran J Pediatr 23(4):375–88
Michalak M, Groenendyk J, Szabo E, Gold L, Opas M (2009) Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem J 417:651–66
Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al (2013) Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 369(25):2391–405
Ogden CA, Hoffmann PR, Bratton D, Ghebrehiwet B, Fadok VA, Henson PM (2001) C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med 194(6):781–96
Raghavan M, Wijeyesakere SJ, Peters LR, Del Cid N (2013) Calreticulin in the immune system: ins and outs. Trends Immunol 34(1):13–21
Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L et al (2014) Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood 123(10):1552–5
Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD et al (2014) JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood 123(10):1544–51
Shaozhang Z, Ming Z, Haiyan P, Aiping Z, Qitao Y, Xiangqun S (2014) Comparison of ARMS and direct sequencing for detection of EGFR mutation and prediction of EGFR-TKI efficacy between surgery and biopsy tumor tissues in NSCLC patients. Med Oncol 31(5):1–9
Shirane S, Araki M, Morishita S, Edahiro Y, Takei H, Yoo Y et al (2014) JAK2, CALR, and MPL mutation spectrum in Japanese myeloproliferative neoplasms patients. Haematologica
Stift G, Pachner M, Lelley T (2003) Comparison of RAPD fragment separation in agarose and polyacrylamide gel by studying Cucurbita species. Cucurbit Genet Coop Rep 26:62–5
Szabo E, Qiu Y, Baksh S, Michalak M, Opas M (2008) Calreticulin inhibits commitment to adipocyte differentiation. J Cell Biol 182(1):103–16
Tefferi A (2010) Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia 24(6):1128–38
Tefferi A, Wassie EA, Lasho TL, Finke C, Belachew AA, Ketterling RP et al (2014a) Calreticulin mutations and long-term survival in essential thrombocythemia. Leukemia 28(12):2300–3
Tefferi A, Lasho T, Finke C, Belachew A, Wassie E, Ketterling R et al (2014b) Type 1 vs type 2 calreticulin mutations in primary myelofibrosis: differences in phenotype and prognostic impact. Leukemia 28(7):1568–70
Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A et al (2009) The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 114(5):937–51
Vecoli C, Prevost F, Ververis J, Medeiros A, O’Leary G (1983) Comparison of polyacrylamide and agarose gel thin-layer isoelectric focusing for the characterization of beta-lactamases. Antimicrob Agents Chemother 24(2):186–9
Acknowledgments
This work was financially supported by grant no. Th94/3 from vice chancellor for Research Affairs of Ahvaz Jundishapur University of Medical Sciences. This paper is issued from thesis of Reza Shirzad, MSc student of hematology and blood banking. We wish to thank all our colleagues in Golestan Hospital and Allied Health Sciences School, Ahvaz Jundishapur University of Medical Sciences.
Authors’ contributions
N.S. and R.Sh. have conceived the manuscript and revised it; R.Sh, J.M.A., and N.S. wrote the manuscript; M.S., A.S.M., and A.A. provided clinical data and provided pathological diagnoses and information; R.Sh and Z.T. performed the technical tests.
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The authors declare that they have no conflict of interest.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the local ethics committee of the Ahvaz Jundishapur University of Medical Sciences and with the 1964 Helsinki declaration. Written informed consent was obtained from all patients and normal individuals.
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Highlights
• Assessment of CALR can be used for diagnosis of BCR-ABL1, JAK2V617F, and MPL 515 negative MPN patients with platelet counts higher than 600 × 109/L.
• PCR-PAGE can be used for detection of CALR mutations.
• Frequency of CALR mutations in male/female may be different in different nations and races.
• JAK2V617F mutation can be detected in BCR-ABL1-positive MPN patients.
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Shirzad, R., Tahan-nejad, Z., Mohamadi-asl, J. et al. High platelet count and high probability of CALR detection in myeloproliferative neoplasms. Comp Clin Pathol 26, 25–33 (2017). https://doi.org/10.1007/s00580-016-2343-9
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DOI: https://doi.org/10.1007/s00580-016-2343-9