Clinical importance of different calreticulin gene mutation types in wild-type JAK2 essential thrombocythemia and myelofibrosis patients.

Abstract

The Janus kinase 2 (JAK2) V617F mutation (JAK2 V617F), JAK2 exon 12 mutations and myeloproliferative leukemia virus oncogene W515L/K mutation (MPL W515L/K) have become three major molecular diagnosis criteria for myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) from 2005.1 However, diagnosing MPNs with non-mutated JAK2 and MPL remains a major diagnostic challenge.2–4 Some recent studies have reported calreticulin (CALR) gene mutations in patients with non-mutated JAK2 V617F MPNs.5–7 There are some distinct mutation types in MPN subtypes, but the differences in the clinical significance and prognosis among the different mutation types are obscure.8–10 Here, we report our data on CALR mutation in wild-type (wt) JAK2 MPN on patients. It should also be mentioned that this is undoubtedly the first report regarding CALR mutations in Chinese MPN patients. From January 2008 to December 2013, bone marrow or peripheral blood samples from 301 MPNs patients were collected in the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province, China, including ET (n=222), PV (n=37), PMF (n=33), post-ET MF (PET-MF; n=6), and post-PV MF (PPV-MF; n=3). We also obtained bone marrow samples from 174 patients with other myeloid neoplasms including: myelodysplastic syndrome (MDS; n=8), chronic myelogeneous leukemia (CML; n=55), acute myeloid leukemia (AML; n=104), and chronic myelomonocytic leukemia (CMML; n=7), as well as peripheral blood samples from 121 healthy controls. All participants provided their informed consent. Genomic PCR combined with direct and cloning sequencing was applied to screen CALR mutations. A total of 24.3% (73 of 301) patients with MPNs were found harboring CALR mutations. The CALR mutation was detected in 31.1% (69 of 222) and 12.1% (4 of 33) of patients with ET and PMF, respectively (Figure 1A). Moreover, CALR mutations were found in 57.0% (69 of 121) ET patients with wt JAK2 and 30.8% (4 of 13) PMF patients with wt JAK2. No CALR mutation in patients with PV, PET-MF, PPV-MF (Figure 1A) was found. The CALR mutations have multiple deletions or insertions including: L367fs*46 (33 of 74; 44.6%), K385fs*47 (25 of 74; 33.8%), K368fs*51 (3 of 74; 4.1%), Q365fs*50 (3 of 74; 4.1%), E364fs*49 (2 of 74; 2.7%), K374fs*56 (2 of 74; 2.7%), L367fs*48 (1 of 74; 1.4%), Q365fs*48 (1 of 74; 1.4%), E364fs*55 (1 of 74; 1.4%), K375fs*48 (1 of 74; 1.4%), K375fs*55 (1 of 74; 1.4%), and K377fs*50 (1 of 74; 1.4%). Figure 1. (A) Frequency of CALR mutations in myeloid neoplasms and healthy control. The CALR mutation was detected in 31.1% (69 of 222) and 12.1% (4 of 33) of ET and PMF patients, respectively. Approximately 1% of patients (1 of 104) with AML were found to harbor ... These patients with MPNs were simultaneously examined for the presence of other gene mutations. PV patients were screened for JAK2 V617F and JAK2 exon 12 mutations, while ET and MF patients were screened for JAK2 V617F and MPL W515L/K mutation. Among the total 301 patients with MPNs, 52.2% (157 of 301) were found to harbor JAK2 V617F mutation. Among the 222 patients with ET and 37 patients with PV, 0.9% (2 of 222) were found to harbor MPL W515L/K mutations and 2.7% (1 of 37) to harbor JAK2 exon 12 mutation, respectively (Figure 1B). JAK2 V617F, JAK2 exon 12 mutation, MPL W515L/K mutations and CALR mutations were found exclusively in these MPNs patients. We also screened CALR mutations in 104 AML patients, 55 CML patients, 7 CMML patients, and 8 MDS patients (including 5 refractory cytopenia with multilineage dysplasis, 2 refractory anemia with excess blasts, and one refractory anemia) to investigate whether CALR mutations were present in other myeloid neoplasms. Although most of these patients had negative results, one AML patient (59-years old, male, M2 subtype) was found to harbor CALR mutation (L367fs*46) without JAK2 V617F and MPL W515L/K mutations (Figure 1A). This patient had no previous history of MPN or MDS, Fms-related tyrosine kinase 3 internal tandem duplication, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog mutation, nucleophosmin mutation. CCAAT/enhancer binding protein alpha mutation was all negative and cytogenetics analysis showed normal karyotype. In addition, no CALR mutation was detected in the 121 healthy controls (Figure 1A). For mutation types, a total of 12 distinct variants of CALR mutation, including 11 deletions and one insertion, were identified in our patients. L367fs*46, which resulted from a 52-bp deletion, and K385fs*47, which resulted from a 5-bp insertion, were the most frequent CALR mutations. The two mutations accounted for 44.6% (33 of 74) and 33.8% (25 of 74) in all cases with mutant CALR, respectively. For ET patients, the two mutations were 14.0% (31 of 222) and 10.4% (23 of 222), respectively. For PMF patients, the two mutations were 3.0% (1 of 33) and 6.1% (2 of 33), respectively. There was no significant difference in the two mutation types between patients with ET and PMF (P=0.137 and P=0.645, respectively). Moreover, the two mutations were 44.9% (31 of 69) and 33.3% (23 of 69) in CALR mutation positive ET patients (Figure 1C), as well as 25% (1 of 4) and 50% (2 of 4) in CALR mutation positive PMF patients (P=0.793 and P=0.888, respectively). There were few other mutation types in the CALR-mutated samples (Figure 1C). ET patients with mutant CALR were significantly younger (P<0.001) and had lower white blood cell (WBC) counts (P<0.001), lower hemoglobin (Hb) levels (P=0.002), and higher platelet (PLT) counts (P<0.001) than patients with JAK2 V617F. No significant difference can be identified between ET patients with mutant CALR and JAK2 V617F in terms of sex and thrombotic events (Table 1). Similarly, PMF patients with mutant CALR showed lower Hb level (P=0.001) than JAK2 V617F. There was no significant difference in sex, age, WBC count, PLT counts or thrombotic events between PMF patients with mutant CALR and JAK2 V617F (Table 1). For different CALR mutations in ET patients, younger age (P=0.020), lower WBC count (P<0.001), and lower Hb level (P=0.002) were observed in CALR L367fs*46 than JAK2 V617F. In addition, ET patients with CALR K385fs*47 showed lower age (P<0.001), lower WBC counts (P<0.001), lower Hb levels (P=0.025) and higher PLT counts (P=0.005) than JAK2 V617F (Table 2). Table 1. Clinical features of essential thrombocythemia and primary myelofibrosis patients with CALR and JAK2 mutation. Table 2. Clinical characteristics of essential thrombocythemia patients with different types of CALR and JAK2 mutation. The overall survival (OS) rates of patients with ET and PMF were analyzed using the Kaplan-Meier curve. Longer OS was observed in ET and PMF patients with mutant CALR, but not wt CALR (P=0.511 and P=0.729, respectively) (Table 1 and Figure 1D). According to the risk stratification system in ET,11 there was a significant difference between patients with ET in the CALR-mutated group and JAK2 V617F mutant group or wt CALR group (both P<0.001) (Table 1). In summary, our data from this large cohort of Chinese patients with MPNs confirmed CALR mutations were novel molecular markers in wt JAK2 MPNs. It should always be noted that the combination of CALR, JAK2, and MPL W515L/K mutation analysis could contribute to the diagnosis of MPNs.5–7 Different CALR mutations of patients with MPNs had distinct clinical characteristics. Patients with the L367fs*46 and K385fs*47 mutations have shown a favorable prognosis, but further research is required to confirm this result. Given the relative proportion of MPN patients without JAK2/MPL/CALR mutations in our patient group, further investigation should be carried out to find novel molecular aberrations.