Aggerholm, Anni

Ebbesen, Lene H

Hansen, Maria

Haunstrup, Laura M

Severinsen, Marianne T

English

Detection of somatic mutations in cardinal driver genes is a strong argument for diagnosis in classical Philadelphia-negative myeloproliferative neoplasms (MPNs). Driver mutations in Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL), are generally considered mutually exclusive, but several reports have suggested that they coexist in a small subgroup of patients. In this study, we retrospectively searched for CALR mutations in 136 suspected MPN patients with low allelic burden (≤5%) JAK2 V617F. Fifteen patients with concomitant JAK2 V617F and CALR mutations were identified, of whom 10 were diagnosed with essential thrombocytosis (ET). More than 50 different indel mutations in exon 9 of CALR have been reported, with type 1 (52 bp deletion) and type 2 (5 bp insertion) accounting for more than 80% of CALR-mutated MPN cases. Type 1 is generally considered the most common mutation, but, interestingly, our double-mutated ET patients seem to have an inversed ratio between type 1 and type 2 CALR mutations. Our findings support the possibility of coexisting JAK2 V617F and CALR mutations and stress the importance of further molecular screening in MPN patients with low allele frequencies of JAK2 V617F.</p

    Aalborg UniversitetSkewed ratio between type 1 and type 2 calreticulin mutations in essentialthrombocytosis patients with concomitant Janus kinase 2 V617F mutationHaunstrup, Laura M; Ebbesen, Lene H; Hansen, Maria; Severinsen, Marianne T; Aggerholm,AnniPublished in:Experimental HematologyDOI (link to publication from Publisher):10.1016/j.exphem.2018.09.007Creative Commons LicenseCC BY-NC-ND 4.0Publication date:2018Document VersionAccepted author manuscript, peer reviewed versionLink to publication from Aalborg UniversityCitation for published version (APA):Haunstrup, L. M., Ebbesen, L. H., Hansen, M., Severinsen, M. T., & Aggerholm, A. (2018). Skewed ratiobetween type 1 and type 2 calreticulin mutations in essential thrombocytosis patients with concomitant Januskinase 2 V617F mutation. Experimental Hematology, 68, 62-65. https://doi.org/10.1016/j.exphem.2018.09.007General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim. Accepted ManuscriptSkewed ratio between type 1 and type 2 CALR mutations in EssentialThrombocytosis patients with concomitant JAK2 V617F mutationLaura M. Haunstrup , Lene H. Ebbesen , Maria Hansen ,Marianne T. Severinsen , Anni AggerholmPII: S0301-472X(18)30848-8DOI: https://doi.org/10.1016/j.exphem.2018.09.007Reference: EXPHEM 3675To appear in: Experimental HematologyReceived date: 2 July 2018Revised date: 5 September 2018Accepted date: 30 September 2018Please cite this article as: Laura M. Haunstrup , Lene H. Ebbesen , Maria Hansen ,Marianne T. Severinsen , Anni Aggerholm , Skewed ratio between type 1 and type 2 CALRmutations in Essential Thrombocytosis patients with concomitant JAK2 V617F mutation, ExperimentalHematology (2018), doi: https://doi.org/10.1016/j.exphem.2018.09.007This is a PDF file of an unedited manuscript that has been accepted for publication. As a serviceto our customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, andall legal disclaimers that apply to the journal pertain.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTSkewed ratio between type 1 and type 2 CALR mutations in Essential Thrombocytosis patients with concomitant JAK2 V617F mutation Laura M. Haunstrupa, Lene H. Ebbesena, Maria Hansena, Marianne T. Severinsenb, Anni Aggerholma,* aDepartment of Hematology, Aarhus University Hospital, Palle Juul-Jensens Blvd. 99, 8200 Aarhus N, Denmark bDepartment Hematology, Aalborg University Hospital, Mølle Parkvej 4, 9000 Aalborg, Denmark *Corresponding author. Hemodiagnostic Laboratory Dept. Hematology, Aarhus University Hospital, Palle Juul-Jensens Blvd. 99, DK-8200 Aarhus N. Tel: +45 5173 6813. Fax: +45 7846 7598. anniagge@rm.dk Highlights  Dominant CALR clone in Essential Thrombocytosis with low frequency JAK2 V617F.  CALR clone dictates the disease phenotype in ET with concomitant JAK2 V617F.  Extended molecular diagnostics is warranted in low frequency JAK2 V617F MPNs. Abstract Detection of somatic mutations in cardinal driver genes is a strong argument for diagnosis in classical Philadelphia-negative myeloproliferative neoplasms (MPN). Driver mutations in JAK2, CALR and MPL, are generally considered mutually exclusive, but several reports have suggested that they co-exist in a small subgroup of patients. In this study we retrospectively analyzed for CALR mutations in 136 suspected MPN patients with low allelic burden (≤5%) JAK2 V617F. Fifteen patients with concomitant JAK2 V617F and CALR mutation were identified of which ten were diagnosed with essential thrombocytosis (ET). More than 50 different indel mutations in exon 9 of CALR have been reported, with type 1 (52 bp deletion) and type 2 (5 bp insertion) accounting for more than 80% of CALR mutated MPN cases. Type 1 is generally considered the most common mutation, but interestingly, our double mutated ET patients seem to have an inversed ratio between type 1 and type 2 CALR mutations. Our findings support the possibility of co-existing JAK2 V617F and CALR mutations and stress the importance of further molecular screening in MPN patients with low allele frequencies of JAK2 V617F.  Keywords Essential thrombocytosis; CALR; JAK2 V617F; myeloproliferative neoplasms; driver mutations Introduction The classical Philadelphia-negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF), are a group of clonal hematopoietic stem cell disorders characterized by the overproduction of terminally differentiated and fully functional hematopoietic cells. Major molecular diagnostic criteria for the MPNs, include presence of somatic mutations in cardinal driver genes; JAK2 V617F or JAK2 exon 12 in PV and JAK2 V617F, CALR exon 9 or MPL exon 10 in ET and PMF[1].  The MPN driver mutations are generally considered mutually exclusive and with detection of the common JAK2 V617F mutation usually no further testing is performed. However, in recent years papers have been published suggesting that driver mutations indeed do co-exist[2-4].  Detection of such 'double mutant' patients could very well have clinical implications. There is a general consensus that the mutation type affects the phenotypic manifestations and prognosis. Patients with JAK2 mutation often exhibit more prominent leukocytosis, lower levels of platelets, higher hemoglobin levels, more thrombotic events and chance of transformation into AML. CALR mutations are characterized by lower risk of thrombosis, lower hemoglobin and leukocyte counts, higher platelets levels and a better overall survival [5, 6].  We wanted to address the issue of double mutants in our patients to ensure adequate molecular characterization for aiding clinicians providing more precise diagnoses and prognostication in patients with PMF and ET. ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTMethods In this study, we retrospectively reanalysed for CALR mutation in all suspected MPN patients initially analysed for JAK2 V617F between January 2012 and April 30th 2015 for whom allele frequencies of the JAK2 mutation was between 0.01% and 5%. 136 patients were positive for JAK2 V617F with allele frequencies in this range. In addition, a smaller test group of 46 patients with JAK2 V617F allele frequencies between 6% and 10% were analysed. The JAK2 V617F analysis was performed with allele specific qPCR with sensitivity at 0.01% as described previously [7]. CALR exon 9 mutations were analysed by fragment length analysis with sensitivity at 2% as described previously [8]. All samples positive for a CALR mutation were confirmed by Sanger sequencing to determine the specific type of CALR exon 9 mutation. Statistical analyses were performed by Fisher's Exact Test and Student's T-test. The study was approved by the local ethical committee and the Danish Data Protection Agency.  Results and discussion We identified 15 patients with concomitant CALR mutation within the group of 136 patients with JAK2 V617F allele frequencies (VAF) ≤5% while none of the 46 patients with JAK2 V617F VAF >5% had a CALR mutation (p=0.013). Clinical data, collected at diagnosis prior to any treatment, and mutational status of the 15 double mutated patients can be seen in Table 1. The identification of concurrent CALR mutation within the group of suspected MPN patients with low JAK2 V617F allele frequencies is in line with previous studies. Mansier et al reported concurrent CALR mutation in only 2 of 412 patients (0.5%) with JAK2 V617F VAF >5% while in 19 of 133 patients (14%) with JAK2 V617F VAF <5% and Usseglio et al observed concurrent JAK2 V617F and CALR/MPL mutation exclusively in patients with JAK2 V617F VAF<5% [2, 3]. Ten of our double mutated patients were diagnosed with ET, three with PMF and two patients were MPN-unclassifiable.  JAK2 V617F and CALR double mutation have been previously observed in both ET and PMF but with at preponderance for ET as also observed in the current study [3, 9, 10]. More than 50 different indel mutations in exon 9 of CALR have been found with type 1 (52 bp deletion) and type 2 (5 bp insertion) as the most common variants. In our double mutated patients we identified five type 1 (33%) and ten type 2 (66%) CALR mutations and in the ET group 3 patients were found positive for CALR mutation type 1 (30%) and seven were positive for type 2 (70%). More type 1 (53%) than type 2 (32%) CALR mutations are found in MPN patients in general [8] while the rest are classified as either type 1-like, type2-like or 'other types', based on the remaining stretches of negatively charged amino acids in the mutant proteins [11]. The distribution of CALR mutations is hence 57% type 1-like and 39% type 2-like in ET, and 83% type 1-like and 15% type 2-like in PMF [11]. The clinical significance of type 1 versus type 2 CALR mutations is, however, debated. A recent report indicate that type 1 mutations are associated with PMF or ET with an increased risk of myelofibrotic transformation, while type 2 mutations are associated with ET, low risk of thrombosis and indolent clinical course[11]. Another report on ET patients showed that patients with type 2 variants displayed significantly higher platelet count but that the two CALR mutation variants were similar in their hemoglobin level, leukocyte count, IPSET (International Prognostic Scoring System for Essential Thrombocythemia) scores and in overall and thrombosis-free survival [12]. A third report suggests that the prognostic advantage of CALR mutations in PMF might be confined to type 1 or type 1-like CALR variants [13].  The skewed frequencies of type 1 versus type 2 mutations observed in the present study could, however, indicate that double mutated patients constitute a distinct MPN entity. This hypothesis is supported by a recent report that found an association between concurrent JAK2 V617F and CALR/MPL mutation and higher age compared to single mutated ET patients [9]. However, this could not be confirmed in our cohort where the  mean age of our double mutated ET patients was 64.1 years while 59.3 years in 66 ET patients diagnosed at our institution with CALR mutation alone (p=0.31). Our double mutated ET patients had median thrombocyte count at 1195 x106/l. Erythrocyte volume fraction was within normal range for all ET patients (not shown), one patient had slightly lowered hemoglobin level, and all except one ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTpatient had moderate increased lactate dehydrogenase (Table 1). Two of 3 ET patients with type 1 mutation experienced deep venous thrombosis (DVT) or Non ST-Elevation Myocardial Infarction (non-STEMI) two years prior to diagnosis while only one patient out of seven with type 2 mutation had DVT two years post diagnosis. Even though this is a small study, these results could imply that type 1 CALR mutations are more frequently associated with thrombosis than type 2 CALR mutations as also suggested previously [11]. None of the ET patients progressed to myelofibrosis during a median follow-up time at 59 months and all ET patients were alive at end of follow-up. We searched for CALR mutations in 136 patients with low allele burden JAK2 V617F (≤5%) and identified CALR mutations with allele frequencies in the range 9 - 44% in 15 (11%) of these patients, stressing that the CALR clone is the dominant one in our double mutated cases in accordance with previous studies [2, 3]. This finding, in combination with the high thrombocyte count and few thrombotic events in ET patients with type 2 mutation, indicate that the CALR clone and mutation subtype dictate the disease phenotype in double mutant patients. Future studies are warranted for clarifying the etiology of double mutated MPN patients and whether mutations arise in the same or two different tumor clones. In conclusion, our data stress the importance of extending the molecular screening in suspected MPN patients with low allele frequencies of JAK2 V617F (e.g ≤3%) to also include CALR in order to confirm the MPN diagnosis and refine prognostication.  Conflict of interest disclosure: The authors declare no competing financial interests. Acknowledgements Thanks to Dorte Melsvik, Marianne Bomberg, Karin Brændstrup and Bodil Andersen for expert technical assistance and Johannes Frasez Sørensen for support with statistical analyses. Table 1. Clinical and mutational characteristics of the fifteen patients with concomitant JAK2 V617F and CALR mutation. VAF: variant allele frequency, LDH: lactate dehydrogenase, DVT: deep venous thrombosis, non-STEMI: non ST-Elevation Myocardial Infarction, NA: not available. Numbers in bold font indicate levels outside normal range. Patient ID Sex Diagnosis Age at diagnosis JAK2V617F (VAF%) CALR mutation CALR (VAF%) Hemoglobin (mmol/L) Platelets (x109/L) Leukocytes (x109/L) LDH (U/L) Thrombosis Follow-up (months) 1 M ET 75 0.02 Type 1 44 8.8 1076 11 321 non-STEMI 69 2 F ET 58 0.02 Type 1 28 9.2 1273 6 289 DVT 62 3 F Unclassifiable 85 0.01 Type 2 27 8.5 859 9.1 335  33 4 F PMF 49 0.01 Type 1 15 9.3 229 6.9 289 DVT 43 5 F ET 69 0.06 Type 2 35 9.4 1010 8.6 228  52 6 F ET 54 0.09 Type 2 33 7.7 1354 7.3 219  56 7 M PMF 67 0.1 Type 1 40 7.4 674 11.1 491  48 8 F ET 72 0.5 Type 2 41 8.2 1584 12.7 292  55 9 M ET 47 0.01 Type 2 43 9.2 1881 10 378  204 10 M ET 61 0.02 Type 2 44 9.5 906 6.5 NA  154 11 M ET 77 0.03 Type 2 36 8.0 1131 9.7 412 DVT 40 ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT12 M ET 52 0.1 Type 2 41 9.7 1259 9 190  145 13 F PMF (5q-) 74 1 Type 2 25 5.8 73 8.5 570  46 14 M Unclassifiable 57 3 Type 2 9 9.8 617 7.8 182  43 15 M ET 76 3 Type 1 32 8.6 715 10.2 274  49 References 1. Rumi E, Cazzola M. Diagnosis, risk stratification, and response evaluation in classical myeloproliferative neoplasms. Blood. 2017;129:680-692.  2. Mansier O, Migeon M, Etienne G, Bidet A, Lippert E. JAK2V617F and CALR double mutations are more frequently encountered in patients with low JAK2V617F allelic burdens. Leuk Lymphoma. 2016;57:1949-1951.  3. Usseglio F, Beaufils N, Calleja A, Raynaud S, Gabert J. Detection of CALR and MPL Mutations in Low Allelic Burden JAK2 V617F Essential Thrombocythemia. J Mol Diagn. 2017;19:92-98.  4. Nussenzveig RH, Pham HT, Perkins SL, Prchal JT, Agarwal AM, Salama ME. Increased frequency of co-existing JAK2 exon-12 or MPL exon-10 mutations in patients with low JAK2(V617F) allelic burden. Leuk Lymphoma. 2016;57:1429-1435.  5. Rumi E, Pietra D, Ferretti V, et al. JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood. 2014;123:1544-1551.  6. Rotunno G, Mannarelli C, Guglielmelli P, et al. Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood. 2014;123:1552-1555.  7. Larsen TS, Christensen JH, Hasselbalch HC, Pallisgaard N. The JAK2 V617F mutation involves B- and T-lymphocyte lineages in a subgroup of patients with Philadelphia-chromosome negative chronic myeloproliferative disorders. Br J Haematol. 2007;136:745-751.  8. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369:2379-2390.  9. Mansier O, Luque Paz D, Ianotto JC, et al. Clinical and biological characterization of MPN patients harboring two driver mutations, a French intergroup of myeloproliferative neoplasms (FIM) study. Am J Hematol. 2018;93:E84-E86.  10. Kang MG, Choi HW, Lee JH, et al. Coexistence of JAK2 and CALR mutations and their clinical implications in patients with essential thrombocythemia. Oncotarget. 2016;7:57036-57049.  11. Pietra D, Rumi E, Ferretti VV, et al. Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms. Leukemia. 2016;30:431-438.  12. Tefferi A, Wassie EA, Guglielmelli P, et al. Type 1 versus Type 2 calreticulin mutations in essential thrombocythemia: a collaborative study of 1027 patients. Am J Hematol. 2014;89:E121-4.  13. Tefferi A, Lasho TL, Finke C, et al. Type 1 vs type 2 calreticulin mutations in primary myelofibrosis: differences in phenotype and prognostic impact. Leukemia. 2014;28:1568-1570.  

Skewed ratio between type 1 and type 2 calreticulin mutations in essential thrombocytosis patients with concomitant Janus kinase 2 V617F mutation

https://vbn.aau.dk/ws/files/303002905/Skewed_ratio_between_type_1_and_type_2_CALR_mutations_in_Essential_Thrombocytosis_patients_with_concomitant_JAK2_V617F_mutation.pdf

Skewed ratio between type 1 and type 2 calreticulin mutations in essential thrombocytosis patients with concomitant Janus kinase 2 V617F mutation

Abstract

Similar works

Full text

Available Versions

VBN