Clinical importance of different calreticulin gene mutation types in wild-type JAK2 essential thrombocythemia and myelofibrosis patients
Clinical importance of different calreticulin gene mutation types in wild-type JAK2 essential thrombocythemia and myelofibrosis patients
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DOI：
10.3324/haematol.2014.109199
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发表时间：
2014-07
期刊：
影响因子：
10.1
通讯作者：
C. Qiao;Chao Sun;Ouyang Yuan;Jujuan Wang;S. Qian;Jian-yong Li;Su-jiang Zhang
中科院分区：
文献类型：
--
作者：
C. Qiao;Chao Sun;Ouyang Yuan;Jujuan Wang;S. Qian;Jian-yong Li;Su-jiang Zhang
Janus激酶2（JAK2）V617F突变（JAK2 V617F）、JAK2外显子12突变和骨髓增殖性白血病病毒癌基因W515L/K突变（MPL W515L/K）已成为骨髓增殖性肿瘤（MPNs）的三大分子诊断标准，包括真性红细胞增多症（PV）、原发性血小板增多症（ET）和原发性血小板增多症（ET）。自 2005 年起骨髓纤维化 (PMF).1 然而，诊断具有非突变 JAK2 和 MPL 的 MPN 仍然是一个主要的诊断挑战。2–4 最近的一些研究报道了非突变 JAK2 V617F MPN 患者的钙网蛋白 (CALR) 基因突变。5–7 MPN 亚型中有一些不同的突变类型，但不同突变类型之间的临床意义和预后差异并不明显。 8–10在这里，我们报告了患者野生型 (wt) JAK2 MPN 中 CALR 突变的数据。还值得一提的是，这无疑是中国MPN患者CALR突变的首例报道。 2008年1月至2013年12月，收集中国江苏省南京医科大学第一附属医院301例MPNs患者的骨髓或外周血样本，其中ET（n=222）、PV（n=37）、PMF（n=33）、ET后MF（PET-MF；n=6）和PV后MF（PPV-MF；n=3）。我们还采集了 174 名其他骨髓肿瘤患者的骨髓样本，包括：骨髓增生异常综合征 (MDS；n=8)、慢性粒细胞白血病 (CML；n=55)、急性粒细胞白血病 (AML；n=104) 和慢性粒单核细胞白血病 (CMML；n=7)，以及 121 名健康对照者的外周血样本。所有参与者均提供了知情同意书。应用基因组PCR结合直接测序和克隆测序来筛选CALR突变。总共 24.3%（301 名 MPN 患者中的 73 名）被发现携带 CALR 突变。在 ET 和 PMF 患者中，分别有 31.1%（222 名中的 69 名）和 12.1%（33 名中的 4 名）检测到 CALR 突变（图 1A）。此外，在 57.0%（121 例中的 69 例）携带 wt JAK2 的 ET 患者和 30.8%（13 例中的 4 例）携带 wt JAK2 的 PMF 患者中发现了 CALR 突变。 PV、PET-MF、PPV-MF 患者中未发现 CALR 突变（图 1A）。 CALR 突变具有多个缺失或插入，包括：L367fs*46（74 个中的 33 个；44.6%）、K385fs*47（74 个中的 25 个；33.8%）、K368fs*51（74 个中的 3 个；4.1%）、Q365fs*50（74 个中的 3 个；4.1%）、 E364fs*49（74 个中的 2 个；2.7%）、K374fs*56（74 个中的 2 个；2.7%）、L367fs*48（74 个中的 1 个；1.4%）、Q365fs*48（74 个中的 1 个；1.4%）、E364fs*55（74 个中的 1 个；1.4%）、 K375fs*48（74 个中的 1 个；1.4%）、K375fs*55（74 个中的 1 个；1.4%）和 K377fs*50（74 个中的 1 个；1.4%）。图 1.(A) 骨髓肿瘤和健康对照中 CALR 突变的频率。 ET 和 PMF 患者中分别有 31.1%（222 名中的 69 名）和 12.1%（33 名中的 4 名）检测到 CALR 突变。大约 1% 的 AML 患者（104 名中的 1 名）被发现携带...这些患有 MPN 的患者同时接受了其他基因突变的检查。对PV患者进行JAK2 V617F和JAK2外显子12突变筛查，对ET和MF患者进行JAK2 V617F和MPL W515L/K突变筛查。在总共 301 名 MPN 患者中，52.2%（301 名中的 157 名）被发现携带 JAK2 V617F 突变。在 222 名 ET 患者和 37 名 PV 患者中，分别发现 0.9%（222 名患者中的 2 名）携带 MPL W515L/K 突变，2.7%（37 名患者中的 1 名）携带 JAK2 外显子 12 突变（图 1B）。 JAK2 V617F、JAK2 外显子 12 突变、MPL W515L/K 突变和 CALR 突变仅在这些 MPN 患者中发现。我们还筛查了 104 名 AML 患者、55 名 CML 患者、7 名 CMML 患者和 8 名 MDS 患者（包括 5 例难治性血细胞减少伴多系发育不良、2 例难治性贫血伴原始细胞过多和 1 例难治性贫血）的 CALR 突变，以研究其他髓系肿瘤中是否存在 CALR 突变。尽管大多数患者的结果均为阴性，但一名 AML 患者（59 岁，男性，M2 亚型）被发现携带 CALR 突变 (L367fs*46)，但不存在 JAK2 V617F 和 MPL W515L/K 突变（图 1A）。该患者既往无 MPN 或 MDS 病史、Fms 相关酪氨酸激酶 3 内部串联重复、v-kit Hardy-Zuckerman 4 猫肉瘤病毒癌基因同源突变、核磷蛋白突变。 CCAAT/增强子结合蛋白α突变均为阴性，细胞遗传学分析显示核型正常。此外，在 121 名健康对照中未检测到 CALR 突变（图 1A）。对于突变类型，在我们的患者中总共鉴定出 12 种不同的 CALR 突变变异，包括 11 种缺失和 1 种插入。由 52 bp 删除产生的 L367fs*46 和由 5 bp 插入产生的 K385fs*47 是最常见的 CALR 突变。在所有 CALR 突变病例中，这两种突变分别占 44.6%（74 例中的 33 例）和 33.8%（74 例中的 25 例）。对于 ET 患者，这两种突变分别为 14.0%（222 人中的 31 人）和 10.4%（222 人中的 23 人）。对于 PMF 患者，这两种突变分别为 3.0%（33 例中的 1 例）和 6.1%（33 例中的 2 例）。 ET和PMF患者两种突变类型差异无统计学意义（分别为P=0.137和P=0.645）。此外，在CALR突变阳性ET患者中，这两种突变分别为44.9%（69人中的31人）和33.3%（69人中的23人）（图1C），在CALR突变阳性PMF患者中，这两种突变分别为25%（4人中的1人）和50%（4人中的2人）（分别为P=0.793和P=0.888）。 CALR 突变样本中几乎没有其他突变类型（图 1C）。与 JAK2 V617F 患者相比，具有 CALR 突变的 ET 患者明显更年轻 (P<0.001)，白细胞 (WBC) 计数较低 (P<0.001)、血红蛋白 (Hb) 水平较低 (P=0.002) 和血小板 (PLT) 计数较高 (P<0.001)。具有突变 CALR 和 JAK2 V617F 的 ET 患者在性别和血栓事件方面没有显着差异（表 1）。同样，CALR 突变的 PMF 患者的 Hb 水平低于 JAK2 V617F（P=0.001）。具有突变CALR和JAK2 V617F的PMF患者之间的性别、年龄、WBC计数、PLT计数或血栓事件没有显着差异（表1）。对于不同CALR突变的ET患者，CALR L367fs*46较JAK2 V617F年龄较小（P=0.020）、WBC计数较低（P<0.001）和Hb水平较低（P=0.002）。此外，与 JAK2 V617F 相比，CALR K385fs*47 的 ET 患者年龄更低（P<0.001），WBC 计数更低（P<0.001），Hb 水平更低（P=0.025），PLT 计数更高（P=0.005）（表 2）。表 1. CALR 和 JAK2 突变的原发性血小板增多症和原发性骨髓纤维化患者的临床特征。表2.不同类型CALR和JAK2突变的原发性血小板增多症患者的临床特征。使用 Kaplan-Meier 曲线分析 ET 和 PMF 患者的总生存 (OS) 率。在具有突变 CALR 的 ET 和 PMF 患者中观察到更长的 OS，但在 wt CALR 中则没有（分别为 P=0.511 和 P=0.729）（表 1 和图 1D）。根据 ET 的危险分层系统，11 CALR 突变组的 ET 患者与 JAK2 V617F 突变组或 wt CALR 组的 ET 患者之间存在显着性差异（均 P<0.001）（表 1）。总之，我们从这一大群中国 MPN 患者中获得的数据证实了 CALR 突变是 wt JAK2 MPN 中的新型分子标志物。值得注意的是，CALR、JAK2 和 MPL W515L/K 突变分析的结合有助于 MPN 的诊断。5-7 MPN 患者的不同 CALR 突变具有不同的临床特征。携带L367fs*46和K385fs*47突变的患者表现出良好的预后，但还需要进一步的研究来证实这一结果。鉴于我们的患者组中没有 JAK2/MPL/CALR 突变的 MPN 患者的相对比例，应进行进一步研究以发现新的分子畸变。
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.