| ObjectiveTo investigate the clinical characteristics, cytogenetics, gene mutations and prognosis of chronic neutrophilic leukemia.MethodsA retrospective study of 27 clinical suspected CNL cases were reviewed according to 2008 World Health Organization (WHO) diagnostic criteria. JAK2 V617F mutation was detected by AS-PCR. Identification of the CSF3R exon 14-17, ASXL1 exon 12, SETBP1 exon 4, CALR exon 9 and MPL exon 10 mutants was performed by direct sequencing and next-generation sequencing. The classifications of mutational types were confirmed by plasmid cloning sequencing. Clinical characteristics, cytogenetics, gene mutations and prognosis of the true CNL patients were then analyzed using SPSS 18.0.ResultsIn the 27 cases,16 were finally confirmed as WHO-defined CNL and all of them carried CSF3R T618I. There was also a 17th patient carried CSF3R T618 not meeting WHO 2008 criteria. Two other who met these criteria but also had monoclonal gammopathy with uncertain significance (MGUS) were diagnosed as MGUS related leukocytosis. The rest 8 were excluded because they were diagnosed as infection or tumor related leukocytosis.Of the 16 WHO-defined CNL patients, the median age was 64 (43-80) years with a male predominance of 75%(12/16). The median hemoglobin was 114 (81-154) g/L, with median WBC of 41.20 (26.05-167.70) ×109/L, median PLT of 238 (91-394) × 109/L. The median percentage of immature cells in peripheral blood (PB) was 2 (0-9)%, the median percentage of blasts in PB was 0 (0-0.5)%, the median percentage of blasts in bone marrow (BM) was 0 (0-4.5)%, and the median marrow fibrosis (MF) was 1 (0-3) degrees. There was no cytogenetic abnormalities except t(1,7) (p32, q11),+21 and 14, ps+ for each.All the 16 WHO-defined CNL harbored CSF3R T618I mutation, with a case combined with CSF3R W791X mutation. ASXL1 mutations were identified in 81% (13/16) of the patients with a following mutational frequencies:8 cases of G646WfsX12, 2 cases of Y591X, a S871SfsX4, a R404X and a Q976X; SETBP1 mutations were confirmed in 63%(10/16) of the patients with a following mutational frequencies:4 cases of D868N,3 cases of I871T, a G870S, a G870D and a D874N. Nine of the 16 patients harbored CSF3R T618I, ASXL1 and SETBP1 mutations,4 harbored CSF3R T618I and ASXL1 mutations, and 1 harbored CSF3R and SETBP1 mutations. One of the rest two cases without ASXL1 or SETBP1 mutations carried a CSF3R T618I and a CALR K385fs*47 mutation, which is commonly seen in BCR-ABL and JAK2 V617F negative MPN. The case not meeting the WHO criteria carried CSF3R T618I and SETBP1 mutations with wild type of ASXL1 or CALR mutations. There was no mutation in JAK2 V617F or MPL in the above 17 patients. MGUS, infection and tumor related leukocytosis had no CSF3R, ASXL1, SETBP1, JAK2 V617F, CALR or MPL mutation. All the mutations were heterogeneous except for a SETBP1 G870S mutation.Compared with the wild type, the mutated ASXL1 or SETBP1 patients did not show any statistical difference in sex proportion, age, hemoglobin, WBC,PLT, percentage of immature cells in PB, percentage of blasts in PB, percentage of blasts in BM, and MF degrees. There was also no statistical correlation between ASXL1 and SETBP1 mutations.The median survival of 16 CNL patients was 26 (95%CI 20-32) month. We found the adverse prognostic factor was WBC≥50×109/L at diagnosis versus WBC<50X109/L, with a median survival of 11 and 39 months, respectively. However, there was no statistical difference between sex proportion, age(≥ 60 vs.<60 years), anemia at diagnosis (male less than 120g/L and female less than 110g/L was considered as anemia, P=0.063), ASXL1 mutated or not, SETBP1 mutated or not, existence of immature cells in PB or not,≥2% of BM blasts or not, MF≥1 degree or not and existence of cytogenetic abnormalities or not in the prognostic analysis. ConclusionsCSF3R T618I should be included in the major diagnostic criteria of CNL to improve diagnostic accuracy and may have therapeutic implications. CSF3R T618I was commonly combined with ASXL1 and SETBP1 mutations in CNL patients. Cytogenetic abnormalities were not common at the diagnosis of CNL. The overall survival of CNL patients was 26 months, and WBC≥50×109/L at diagnosis indicated poorer prognosis.ObjectiveTo investigate the mutational status of ASXL1, SETBP1, TET2 and SRSF2, and analyze the prognostic factors of chronic myelomonocytic leukemia (CMML).MethodsA retrospective study of 141 CMML patients were reviewed according to 2008 World Health Organization (WHO) diagnostic criteria. Identification of the ASXL1 exon 12, SETBP1 exon 4, TET2 exon 3-11 and SRSF2 exon 1 mutants was performed by direct sequencing. The classifications of mutational types were confirmed by plasmid cloning followed. Statistical analysis of clinical and laboratory characteristics and prognosis were then performed using SPSS 18.0.ResultsOf the 141 WHO-def ined CMML patients, the median age was 63 (18-85) years with a male predominance of 67%. The median hemoglobin was 88 (43-166) g/L, with median WBC of 21.88 (3.01-117.57) × 109/L, median ANC of 7.07 (0.30-66.91) ×109/L, median AMC of 3.72(1.02-57.72) ×109/L, median PLT of 78 (4-1001)×X109/L.ASXL1 (frameshift and nonsense mutations only) mutations were identified in 46% (65/141) of the patients with 59 frameshift and 7 nonsense mutations. SETBP1 mutations were detected in 18% (25/141) with all the cases of missense mutations. TET2 mutations were detected in 33%(46/141), with 22 of frameshift,19 missense and 7 nonsense mutations, while there were a case of missense and nonsense mutations and another of all the 3 mutational types. SRSF2 mutations were detected in 29% (41/141) with 38 cases of missense and 3 of frameshift mutations. There was also mutational concomitance between ASXL1 and SETBP1, and similar between TET2 and SRSF2.Compared with the wild type, the mutated TET2 patients showed elder in age and less in bone marrow blasts, and the mutated SRSF2 patients showed elder in age, higher in hemoglobin, WBC, ANC and AMC. To the contrast, the mutated ASXL1 or SETBP1 patients did not show any statistical difference in age, bone marrow blasts, hemoglobin, WBC, ANC, AMC and PLT over their wild type, respectively.In multivariable analysis of 141 CMML patients, hemoglobin<100 g/L, existence of circulating immature myeloid cells and ASXL1 mutations separated predicted poorer survival. The median overall survival (MS) stratified by Mayo Prognostic Model were unreached,28 and 18 months of low, intermediate and high risk, respectively. MS stratified by Molecular Mayo Model were unreached,55,25 and 15 months of low, intermediate-1, intermediate-2 and high risk, respectively. The Molecular Mayo Model was better in predicting MS than the Mayo Prognostic Model (the-2 log likelihood were 627 and 654, respectively, P=0.001).In a separate multivariable analysis that included the Mayo Prognostic Model as a single variable along with ASXL1wt/TET2wt, the respective hazard ratios of ASXLlmut/TET2mut, ASXLlmut/TET2wt and ASXLlwt/TET2mut were 4.7 (95% CI 2.2-10.3; P=0.000),2.2 (95% CI 1.1-4.2; P=0.025) and 1.3 (95% CI 0.6-2.5; P=0.521). ConclusionsThe mutational frequencies of ASXL1, SETBP1, TET2 and SRSF2 were 46%, 18%,33%and 29%, respectively. About 3 of 4 patients harbored at least one of the above gene mutations. SRSF2 mutations were correlated with the myeloid cells of CMML to acquire growth advantage. SRSF2 mutations was not associated with overall survival and disease progression. Mutated ASXL1 was a separated factor predicting poorer survival in multivariable analysis, and the addition of ASXL1 mutations improved the effectiveness of Mayo prognostic model in predicting survival. Additional TET2 mutations predicted poorer survival in ASXL1 mutated CMML patients. |
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