The Mutation Profile of Calreticulin in Patients with Myeloproliferative Neoplasms and Acute Leukemia

Objective: Calreticulin (CALR) plays important roles in cell proliferation, apoptosis, and immune responses. CALR mutations were described recently in Janus kinase 2 gene (JAK2)-negative or MPL-negative primary myelofibrosis (PMF) and essential thrombocythemia (ET) patients. CALR trails JAK2 as the second most mutated gene in myeloproliferative neoplasms (MPNs). However, little is known about CALR mutation in Chinese patients with leukemia. In the present study, a cohort of 305 Chinese patients with hematopoietic neoplasms was screened for CALR mutations, with the aim of uncovering the frequency of CALR mutations in leukemia and MPNs. Materials and Methods: Polymerase chain reaction and direct sequencing were performed to analyze mutations of CALR in 305 patients with hematopoietic malignancies, including 135 acute myeloid leukemia patients, 57 acute lymphoblastic leukemia patients, and 113 MPN patients. Results: CALR mutations were found in 10.6% (12 of 113) of samples from patients with MPNs. CALR mutations were determined in 11.3% (6 of 53), 21.7% (5 of 23), and 9.1% (1/11) of patients with ET, PMF, and unclassifiable MPN, respectively. Conclusion: We showed that MPN patients carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels compared to those with mutated JAK2. However, all of the leukemia patients had negative results for CALR mutations.

Mutations of CALR were found essential for the diagnosis and prognosis of MPNs in recent years. All caLr mutations seen so far in MPNs mainly involve exon 9 and are somatic insertions or deletions. Two mutation variants (type 1 and type 2) were the most frequent: type 1 (c.1179_1230del) resulted from a 52-bp deletion, more frequent in PMF, and type 2 (c.1234_1235insTTGTC) resulted from a 5-bp TTGTC insertion [1]. Andrikovics et al. demonstrated that caLr mutations are found in about one-fourth of patients with ET or PMF and are associated with distinct clinical characteristics, and another study also found that caLr mutations are associated with younger age, more severe anemia, higher white blood cell (WBC) and platelet counts, lower Dynamic International Prognostic Scoring System Plus scores, and better survival compared to subjects with JaK2 mutations [17,18]. Similar to MPNs, acute leukemia, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), is a group of disorders characterized by abnormal clonal proliferation and immune imbalance. To investigate whether caLr mutations were present in myeloid neoplasms, Andrikovics et al. detected JAK2, CALR, and MPL genes in 289 cases of ET and 99 cases of PMF, and they reported that in ET, 154 (53%) JAK2V617F mutation-positive, 96 (33%) CALR mutationpositive, 9 (3%) MPL mutation-positive, and 30 triple-negative (11%) cases were identified, while in PMF 56 (57%) JAK2V617F mutation-positive, 25 (25%) CALR mutation-positive, 7 (7%) MPL mutation-positive, and 11 (11%) triple-negative cases were identified [18]. Qiao et al. screened caLr mutations in 104 AML patients, 55 chronic myeloid leukemia (CML) patients, 7 chronic myelomonocytic leukemia patients, and 8 myelodysplastic syndrome (MDS) patients. Although most of these patients had negative results, one AML patient was found to harbor a CALR mutation (c.1179_1230del) without JAK2V617F or MPL W515L/K mutations [19].
Unlike AML, ALL is a heterogeneous malignancy caused by the clonal proliferation of lymphocytes. However, no data about the mutation frequency of CALR in ALL patients have been reported to date. Therefore, in the present study, a cohort of 305 Chinese patients with hematopoietic neoplasms was screened for CALR mutations, with the aim of uncovering the frequency of CALR mutations in leukemia and MPNs. The results demonstrate that CALR mutation status is an important diagnostic factor in MPN patients without JAK2 mutation while it is negative in leukemia patients.

Subjects and Ethics Statement
Bone marrow or peripheral blood samples from 113 MPN patients were collected at Qilu Hospital of Shandong University between August 2012 and November 2014, including cases of ET (n=53), polycythemia vera (PV; n=20), PMF (n=23), MDS/MPN (n=6), and unclassifiable MPN (MPN-U; n=11). We also obtained bone marrow samples from 192 patients with other hematopoietic neoplasms including AML (n=135) and ALL (n=57). These patients were all newly diagnosed before treatment. The characteristics of the patients at the time of sampling are presented in Tables 1 and 2. The patients with AML were treated with standard induction chemotherapy (anthracycline and cytarabine). The patients with ALL were treated with standard induction chemotherapy (vincristine, daunorubicin, L asparaginase, and prednisone). Bone marrow mononuclear cells (BMMCs) or peripheral blood mononuclear cells (PBMCs) were obtained from patients using density-gradient centrifugation with the Ficoll-Hypaque technique (Ficoll, Pharmacia LKB Biotechnology Inc., Piscataway, NY, USA). The samples were then stored at -80 °C. The present study was approved by the Ethics Committee of Qilu Hospital, Shandong University (Jinan, China). Written informed consent was obtained from all participants for treatment and the cryopreservation of bone marrow and peripheral blood according to the Declaration of Helsinki.

Genomic DNA Isolation, Polymerase Chain Reaction Amplification, and Sequencing
Genomic DNA samples from BMMCs or PBMCs of patients were extracted using the TIANGEN DNA Extraction Kit (TIANamp Genomic DNA Kit, Beijing, China). Oligonucleotide primers targeting exon 9 of CALR were used to amplify a 377-bp product:

The Profile of CALR Mutations in MPN Patients
Mutant CALR in MPNs is a result of frameshift mutations, caused by exon 9 deletions or insertions; the type 1 variant, a 52-bp deletion (c.1179_1230del), and type 2 variant, a 5-bp TTGTC insertion (c.1234_1235insTTGTC), constitute more than 80% of these mutations. In our study, a total of 10.6% of patients (12 of 113) with MPNs were demonstrated to harbor CALR mutations. The CALR mutation was found in 11.3% (6 of 53) of ET, 21.7% (5 of 23) of PMF, and 9.1% (1/11) of MPN-U patients, respectively (Table 3). Moreover, CALR mutations were found in 24.0% of JAK2V617F-negative ET patients (6 of 25) and 35.7% of JAK2V617F-negative PMF patients (5 of 14). No CALR mutation was found in patients with PV. For mutation types, a total of 5 distinct variants of CALR mutation, including 4 deletions and 1 insertion, were identified (Figure 1). c.1179_1230del, which resulted from a 52-bp deletion, and c.1234_1235insTTGTC, which resulted from a 5-bp insertion, were the most frequent CALR mutations. The two mutations accounted for 50% (6 of 12) and 25% (3 of 12) in all cases with mutant CALR, respectively. For ET patients, the two mutations were 50% (3 of 6) and 50% (3 of 6), respectively. For PMF patients, the two mutations were 60% (3 of 5) and 0% (0 of 5), respectively. Moreover, we also identified other kinds of deletions of CALR genetic variation: c.1239_1257del (1/12) and c.1183_1228del (1/12) were found  in ET patients, and c.1183_1216del (1/12) was found in a MPN-U patient.

The Profile of CALR Mutations in Leukemia Patients
To investigate whether CALR mutations were present in other hematopoietic neoplasms, we screened 135 patients with AML and 57 patients with ALL. However, no CALR exon 9 mutations were found in any of these patients. One single nucleotide polymorphism (SNP) of CALR, rs143880510 (Figure 2), was found in one ALL patient.
ET patients with mutant CALR were significantly younger (44.0±15.1 years; p<0.001) than those with mutant JAK2 (56.2±12.9). No significant difference was identified between ET patients with mutant CALR and mutant JAK2 in terms of sex (Table 4). There was no significant difference in sex, age, WBC count, or platelet count between PMF patients with mutant CALR and mutant JAK2 (Table 4).

Discussion
Since the first description of myeloproliferative diseases by Dameshek in 1951 [20], there has been a consecutive progression in the understanding of these disease conditions characterized by abnormal bone marrow hyperplasia. Apart from the characterization of the Philadelphia chromosome in CML, the discovery of JAK2V617F mutation in 2005 [21,22] is the most thrilling development in the molecular diagnosis of Phnegative MPNs. The subsequently reported somatic mutation in    JAK2 exon 12 [23], though much less prevalent in the patients, is considered as another robust molecular marker for Ph-negative MPNs, and especially for PV patients.
The mutations in JAK2, MPL, and CALR are driver mutations, and they all activate the JAK2 pathway, but additional recurrent somatic mutations in several genes (TET2, ASXL1, DNMT3A, CBL,  LNK, IDH1/2, IKF1, EZH2, TP53, SRSF2), encoding transcriptional and epigenetic regulators and signaling proteins, occur in MPNs. These additional mutations modulate disease progression and can also occur as primary mutations, but it is now convincingly demonstrated that MPNs can be initiated from a single JAK2V617F hematopoietic stem cell.
JaK mutations have also emerged in other hematologic diseases, and the majority of the pathogenic mutations in JAK2 (also in JaK1 and JAK3) localize in or near the pseudokinase domain.  [29].
No CALR mutations were found in 62 patients with ALL [2]. However, little attention has been paid to AML and no data about the mutation frequency of CALR in Chinese ALL patients have been reported until now. Therefore, we screened 135 AML patients and 57 ALL patients. However, no CALR exon 9 mutations were found in any of these patients. Only one of the leukemia patients was found to have a CALR SNP, rs143880510.
To date, detection of CALR mutations in peripheral blood has been used as a diagnostic tool in the same way that tests for JAK2 mutations have simplified and improved the accuracy of diagnosis of patients with MPNs. However, in order to develop novel therapeutic drugs, further research is needed to explore  the relationship between the pathogenesis of MPNs and the function of CALR.

Conclusion
In summary, our data from this cohort of Chinese patients with MPNs confirmed that CALR mutations were novel molecular markers in JAK2V617F-negative MPNs. Patients with the c.1179_1230del and c.1234_1235insTTGTC mutations have shown distinct clinical characteristics, but further research is required to confirm this result.