DETECTION OF CALR MUTATIONS USING HIGH RESOLUTION MELTING CURVE ANALYSIS (HRM-A); APPLICATION ON A LARGE COHORT OF GREEK ET AND MF PATIENTS
Main Article Content
Keywords
Leukemia
Abstract
Background and Objectives
Somatic mutations in the calreticulin gene (CALR) are detected in approximately 70% of patients with essential thrombocythemia (ET) and primary or secondary myelofibosis (MF), lacking the JAK2and MPLmutations. To determine the prevalence of CALRframeshift mutations in a population of MPN patients of Greek origin, we developed a rapid low-budget PCR-based assay and screened samples from 5 tertiary Haematology units. This is a first of its kind report of the Greek patient population that also disclosed novel CALRmutants.
Methods
MPN patient samples were collected from different clinical units and screened for JAK2and MPLmutations after informed consent was obtained. Negative samples were analyzed for the presence of CALRmutations. To this end, we developed a modified post Real Time PCR High Resolution Melting Curve analysis (HRM-A) protocol. Samples were subsequently confirmed by Sanger sequencing.
Results
Using this protocol we screened 173 MPN, JAK2and MPLmutation negative, patients of Greek origin, of whom 117 (67.63%) displayed a CALRexon 9 mutation. More specifically, mutations were detected in 90 out of 130 (69.23%) essential thrombocythaemia cases (ET), in 18 out of 33 (54.55%) primary myelofibrosis patients (pMF) and in 9 out of 10 (90%) cases of myelofibrosis secondary to ET (post-ET sMF). False positive results were not detected. The limit of detection (LoD) of our protocol was 2%. Furthermore, our study reavealed 6 rare novel mutations which are to be added in the COSMIC database.
Conclusions
Overall, our method could rapidly and cost-effectively detect the mutation status in a representative cohort of Greek patients; the mutation make-up in our group was not different from what has been published for other national groups.
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References
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2. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, Vassiliou GS, Bench AJ, Boyd EM, Curtin N, Scott MA, Erber WN, Green AR; Cancer Genome Project. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365(9464): 1054-1061. available at: https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(05)71142-9/abstract (Aug. 2018).
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4. Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L, Score J, Seear R, Chase AJ, Grand FH, White H, Zoi C, Loukopoulos D, Terpos E, Vervessou EC, Schultheis B, Emig M, Ernst T, Lengfelder E, Hehlmann R, Hochhaus A, Oscier D, Silver RT, Reiter A, Cross NC. Widespread occurrence of the JAK2V617F mutation in chronic myeloproliferative disorders. Blood 2005; 106(6): 2162-2168. available at: http://www.bloodjournal.org/content/106/6/2162.long (Aug. 2018).
5. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli A, Cazzola M, Skoda RC. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352(17): 1779-1790. available at: https://www.nejm.org/doi/full/10.1056/NEJMoa051113 (Aug. 2018).
6. Levine RL, Loriaux M, Huntly BJ, Loh ML, Beran M, Stoffregen E, Berger R, Clark JJ, Willis SG, Nguyen KT, Flores NJ, Estey E, Gattermann N, Armstrong S, Look AT, Griffin JD, Bernard OA, Heinrich MC, Gilliland DG, Druker B, Deininger MW. The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia. Blood 2005; 106(10): 3377-3379. available at: http://www.bloodjournal.org/content/106/10/3377/tab-figures-only (Aug. 2018).
7. Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR, Futreal PA, Erber WN, McMullin MF, Harrison CN, Warren AJ, Gilliland DG, Lodish HF, Green AR. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med 2007; 356(5): 459-468. available at: https://www.nejm.org/doi/full/10.1056/NEJMoa065202 (Aug. 2018).
8. Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M, Cuker A, Wernig G, Moore S, Galinsky I, DeAngelo DJ, Clark JJ, Lee SJ, Golub TR, Wadleigh M, Gilliland DG, Levine RL. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med 2006; 3(7): e270. available at: https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.0030270 (Aug. 2018).
9. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, Harris NL, Le Beau MM, Hellström-Lindberg E, Tefferi A, Bloomfield CD. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009; 114(5): 937-951. available at: http://www.bloodjournal.org/content/early/2009/04/08/blood-2009-03-209262 (Aug. 2018).
10. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, Them NC, Berg T, Gisslinger B, Pietra D, Chen D, Vladimer GI, Bagienski K, Milanesi C, Casetti IC, Sant'Antonio E, Ferretti V, Elena C, Schischlik F, Cleary C, Six M, Schalling M, Schönegger A, Bock C, Malcovati L, Pascutto C, Superti-Furga G, Cazzola M, Kralovics R. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 2013; 369(25): 2379-2390. available at: https://www.nejm.org/doi/full/10.1056/NEJMoa1311347 (Aug. 2018).
11. Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, Avezov E, Li J, Kollmann K, Kent DG, Aziz A, Godfrey AL, Hinton J, Martincorena I, Van Loo P, Jones AV, Guglielmelli P, Tarpey P, Harding HP, Fitzpatrick JD, Goudie CT, Ortmann CA, Loughran SJ, Raine K, Jones DR, Butler AP, Teague JW, O'Meara S, McLaren S, Bianchi M, Silber Y, Dimitropoulou D, Bloxham D, Mudie L, Maddison M, Robinson B, Keohane C, Maclean C, Hill K, Orchard K, Tauro S, Du MQ, Greaves M, Bowen D, Huntly BJP, Harrison CN, Cross NCP, Ron D, Vannucchi AM, Papaemmanuil E, Campbell PJ, Green AR. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 2013; 369(25): 2391-2405. available at: https://www.nejm.org/doi/full/10.1056/NEJMoa1312542 (Aug. 2018).
12. Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM, Bloomfield CD, Cazzola M, Vardiman JW. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016; 127(20): 2391-2405. available at: http://www.bloodjournal.org/content/early/2016/04/11/blood-2016-03-643544 (Aug. 2018).
13. Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L, Fanelli T, Bosi A, Vannucchi AM; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative Investigators. Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood 2014; 123(10): 1552-1555. available at: http://www.bloodjournal.org/content/123/10/1552 (Aug. 2018).
14. Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD, Them NC, Berg T, Elena C, Casetti IC, Milanesi C, Sant'antonio E, Bellini M, Fugazza E, Renna MC, Boveri E, Astori C, Pascutto C, Kralovics R, Cazzola M; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative Investigators. JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood 2014; 123(10): 1544-1551. available at: http://www.bloodjournal.org/content/123/10/1544?sso-checked=true (Aug. 2018).
15. Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, Hanson CH, Maffioli M, Caramazza D, Passamonti F, Pardanani A. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia 2014; 28(7): 1472-1477. available at: https://www.nature.com/articles/leu20143 (Aug. 2018).
16. Tefferi A, Guglielmelli P, Lasho TL, Rotunno G, Finke C, Mannarelli C, Belachew AA, Pancrazzi A, Wassie EA, Ketterling RP, Hanson CA, Pardanani A, Vannucchi AM. CALR and ASXL1 mutations-based molecular prognostication in primary myelofibrosis: an international study of 570 patients. Leukemia 2014; 28(7): 1494-1500. available at: https://www.nature.com/articles/leu201457 (Aug. 2018).
17. Nangalia J, Green TR. The evolving genomic landscape of myeloproliferative neoplasms. Hematology Am Soc Hematol Educ Program 2014; 2014(1): 287-296. available at: http://asheducationbook.hematologylibrary.org/content/2014/1/287.long (Aug. 2018).
18. Bench AJ, White HE, Foroni L, Godfrey AL, Gerrard G, Akiki S, Awan A, Carter I, Goday-Fernandez A, Langabeer SE, Clench T, Clark J, Evans PA, Grimwade D, Schuh A, McMullin MF, Green AR, Harrison CN, Cross NC; British Committee for Standards in Haematology. Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2V617F and other relevant mutations. Br J Haematol 2013; 160(1): 25-34. available at: https://onlinelibrary.wiley.com/doi/abs/10.1111/bjh.12075 (Aug. 2018).
19. Bilbao-Sieyro C, Santana G, Moreno M, Torres L, Santana-Lopez G, Rodriguez-Medina C, Perera M, Bellosillo B, de la Iglesia S, Molero T, Gomez-Casares MT. High resolution melting analysis: a rapid and accurate method to detect CALR mutations. PLoS One 2014; 9(7): e103511. available at: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103511 (Aug. 2018).
20. Chi J, Nicolaou KA, Nicolaidou V, Koumas L, Mitsidou A, Pierides C, Manoloukos M, Barbouti K, Melanthiou F, Prokopiou C, Vassiliou GS and Costeas P. Calreticulin gene exon 9 frameshift mutations in patients with thrombocytosis. Leukemia 2014; 28(5): 1152-1154. available at: https://www.nature.com/articles/leu2013382 (Aug. 2018).
21. Jones AV, Ward D, Lyon M, Leung W, Callaway A, Chase A, Dent CL, White HE, Drexler HG, Nangalia J, Mattocks C, Cross NC. Evaluation of methods to detect CALR mutations in myeloproliferative neoplasms. Leuk Res 2015; 39(1): 82-87. available at: https://www.lrjournal.com/article/S0145-2126(14)00371-3/abstract (Aug. 2018).
22. Vannucchi AM, Rotunno G, Bartalucci N, Raugei G, Carrai V, Balliu M, Mannarelli C, Pacilli A, Calabresi L, Fjerza R, Pieri L, Bosi A, Manfredini R, Guglielmelli P. Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value. Leukemia 2014; 28(9): 1811-1818. available at: https://www.nature.com/articles/leu2014100 (Aug. 2018).
23. Vossen RH, Aten E, Roos A, den Dunnen JT. High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum Mutat 2009; 30(6): 860-866. available at: http://www.bloodjournal.org/content/111/8/4418 (Aug. 2018).
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