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

Open AccessPublished:October 04, 2018DOI:https://doi.org/10.1016/j.exphem.2018.09.007

      Highlights

      • There is a dominant calreticulin (CALR) clone in essential thrombocytosis (ET) with low-frequency Janus kinase 2 (JAK2) V617F mutation.
      • CALR clone dictates the disease phenotype in ET with concomitant JAK2 V617F mutation.
      • Extended molecular diagnostic tests are warranted in low-frequency JAK2 V617F myeloproliferative neoplasm (MPN) patients.
      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.
      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 the presence of somatic mutations in cardinal driver genes: Janus kinase 2 (JAK2) V617F or JAK2 exon 12 in PV and JAK2 V617F, calreticulin (CALR) exon 9, or thrombopoietin receptor (MPL) exon 10 in ET and PMF [
      • Rumi E
      • Cazzola M
      Diagnosis, risk stratification, and response evaluation in classical myeloproliferative neoplasms.
      ].
      The MPN driver mutations are generally considered mutually exclusive and, after detection of the common JAK2 V617F mutation, usually no further testing is performed. However, in recent years, studies have suggested that driver mutations indeed do coexist [
      • 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.
      ,
      • Usseglio F
      • Beaufils N
      • Calleja A
      • Raynaud S
      • Gabert J
      Detection of CALR and MPL mutations in low allelic burden JAK2 V617F essential thrombocythemia.
      ,
      • Nussenzveig RH
      • Pham HT
      • Perkins SL
      • Prchal JT
      • Agarwal AM
      • Salama ME
      Increased frequency of coexisting JAK2 exon-12 or MPL exon-10 mutations in patients with low JAK2(V617F) allelic burden.
      ].
      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 a higher chance of transformation into acute myeloid leukemia. CALR mutations are characterized by s lower risk of thrombosis, lower hemoglobin and leukocyte counts, higher platelet levels, and better overall survival [
      • 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.
      ,
      • Rotunno G
      • Mannarelli C
      • Guglielmelli P
      • et al.
      Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia.
      ]. We wanted to address the issue of double mutants in our patients to ensure adequate molecular characterization for aiding clinicians in providing more precise diagnoses and prognostication in patients with PMF and ET.

      Methods

      In this study, we retrospectively reanalyzed for CALR mutation in all suspected MPN patients initially analyzed for JAK2 V617F between January 2012 and April 2015 and for whom allele frequencies of the JAK2 mutation were between 0.01% and 5%. A total of 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 analyzed. The JAK2 V617F analysis was performed with allele specific qPCR with sensitivity at 0.01% as described previously [
      • 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.
      ]. CALR exon 9 mutations were analyzed by fragment length analysis with sensitivity at 2% as described previously [
      • Klampfl T
      • Gisslinger H
      • Harutyunyan AS
      • et al.
      Somatic mutations of calreticulin in myeloproliferative neoplasms.
      ]. 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 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%, whereas 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 consistent with previous studies. Mansier et al. reported concurrent CALR mutation in only two of 412 patients (0.5%) with JAK2 V617F VAF >5%, whereas in 19 of 133 patients (14%) with JAK2 V617F VAF <5%. Usseglio et al. observed concurrent JAK2 V617F and CALR/MPL mutation exclusively in patients with JAK2 V617F VAF<5% [
      • 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.
      ,
      • Usseglio F
      • Beaufils N
      • Calleja A
      • Raynaud S
      • Gabert J
      Detection of CALR and MPL mutations in low allelic burden JAK2 V617F essential thrombocythemia.
      ].
      Table 1Clinical and mutational characteristics of the 15 patients with concomitant JAK2 V617F and CALR mutation
      Patient IDSexDiagnosisAge atdiagnosisJAK2V617F(VAF%)CALRmutationCALR(VAF%)Hemoglobin(mmol/L)Platelets( × 109/L)Leukocytes( × 109/L)LDH (U/L)ThrombosisFollow-up(months)
      1MET750.02Type 1448.8107611321non-STEMI69
      2FET580.02Type 1289.212736289DVT62
      3FUnclassifiable850.01Type 2278.58599.133533
      4FPMF490.01Type 1159.32296.9289DVT43
      5FET690.06Type 2359.410108.622852
      6FET540.09Type 2337.713547.321956
      7MPMF670.1Type 1407.467411.149148
      8FET720.5Type 2418.2158412.729255
      9MET470.01Type 2439.2188110378204
      10MET610.02Type 2449.59066.5NA154
      11MET770.03Type 2368.011319.7412DVT40
      12MET520.1Type 2419.712599190145
      13FPMF (5q-)741Type 2255.8738.557046
      14MUnclassifiable573Type 299.86177.818243
      15MET763Type 1328.671510.227449
      VAF=variant allele frequency, LDH=lactate dehydrogenase, non-STEMI=non ST-elevation myocardial infarction, NA=not available. Numbers in bold font indicate levels outside of the normal range.
      Ten of our double-mutated patients were diagnosed with ET, three with PMF and two who were MPN unclassifiable. JAK2 V617F and CALR double mutation have been previously observed in both ET and PMF, but with a preponderance for ET, as was also observed in the current study [
      • Usseglio F
      • Beaufils N
      • Calleja A
      • Raynaud S
      • Gabert J
      Detection of CALR and MPL mutations in low allelic burden JAK2 V617F essential thrombocythemia.
      ,
      • 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.
      ,
      • Kang MG
      • Choi HW
      • Lee JH
      • et al.
      Coexistence of JAK2 and CALR mutations and their clinical implications in patients with essential thrombocythemia.
      ].
      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 10 type 2 (66%) CALR mutations and, in the ET group, three 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 [
      • Klampfl T
      • Gisslinger H
      • Harutyunyan AS
      • et al.
      Somatic mutations of calreticulin in myeloproliferative neoplasms.
      ], whereas the rest are classified as either type 1-like, type 2-like, or “other type,” based on the remaining stretches of negatively charged amino acids in the mutant proteins [
      • Pietra D
      • Rumi E
      • Ferretti VV
      • et al.
      Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms.
      ]. The distribution of CALR mutations is thus 57% type 1-like and 39% type 2-like in ET and 83% type 1-like and 15% type 2-like in PMF [
      • Pietra D
      • Rumi E
      • Ferretti VV
      • et al.
      Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms.
      ]. 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, whereas type 2 mutations are associated with ET, low risk of thrombosis, and an indolent clinical course [
      • Pietra D
      • Rumi E
      • Ferretti VV
      • et al.
      Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms.
      ]. 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, International Prognostic Scoring System for Essential Thrombocythemia scores, and overall and thrombosis-free survival [
      • Tefferi A
      • Wassie EA
      • Guglielmelli P
      • et al.
      Type 1 versus Type 2 calreticulin mutations in essential thrombocythemia: a collaborative study of 1027 patients.
      ]. 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 [
      • Tefferi A
      • Lasho TL
      • Finke C
      • et al.
      Type 1 vs type 2 calreticulin mutations in primary myelofibrosis: differences in phenotype and prognostic impact.
      ].
      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 with single-mutated ET patients [
      • 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.
      ]. However, this could not be confirmed in our cohort, in which the mean age of our double-mutated ET patients was 64.1 years, whereas in 66 ET patients diagnosed at our institution with CALR mutation alone, it was 59.3 years (p = 0.31). Our double-mutated ET patients had a median thrombocyte count 1195 × 106/L. Erythrocyte volume fraction was within the normal range for all ET patients (not shown). One patient had a slightly lowered hemoglobin level and all except one patient had moderate increased lactate dehydrogenase (Table 1). Two of three ET patients with type 1 mutation experienced deep venous thrombosis (DVT) or non ST-elevation myocardial infarction 2 years prior to diagnosis, whereas only one patient out of seven with type 2 mutation had DVT 2 years after 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 was suggested previously [
      • Pietra D
      • Rumi E
      • Ferretti VV
      • et al.
      Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms.
      ]. None of the ET patients progressed to myelofibrosis during a median follow-up time at 59 months and all ET patients were alive at the 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 of 9% to 44% in 15 (11%) of these patients, stressing that the CALR clone is the dominant one in our double-mutated cases, which is consistent with previous studies [
      • 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.
      ,
      • Usseglio F
      • Beaufils N
      • Calleja A
      • Raynaud S
      • Gabert J
      Detection of CALR and MPL mutations in low allelic burden JAK2 V617F essential thrombocythemia.
      ]. 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-mutated patients. Future studies are warranted to clarify the etiology of double-mutated MPN patients and to determine 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.

      Acknowledgments

      The authors thank Dorte Melsvik, Marianne Bomberg, Karin Brændstrup, and Bodil Andersen for expert technical assistance and Johannes Frasez Sørensen for help with statistical analyses.

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