Relationship of miRNA-181b and prognostic factors of myelodysplastic syndrome and the prediction of target genes / 白血病·淋巴瘤

Objective:To investigate the correlation of miRNA-181b (miR-181b) and prognostic factors of myelodysplastic syndrome (MDS), to predict target gene and main biological functions of miR-181b, and to evaluate the risk prediction ability of miR-181b in MDS.Methods:The samples of 131 bone marrow in MDS patients who followed the criteria of World Health Organization (WHO) classification (2016) from the Blood Diseases Hospital, Chinese Academy of Medical Sciences between January 2019 and September 2019 were collected, and the clinical data including routine blood test results, related gene test results of blood diseases were retrospectively analyzed. The expression levels of miR-181b in all bone marrow samples were detected by using quantitative real-time polymerase chain reaction (qRT-PCR). According to the international prognostic scoring system (IPSS), WHO classification-based prognostic scoring system (WPSS) and revised IPSS (IPSS-R), the patients were divided into different groups by the risk grade, and the expression differences of miR-181b in different risk groups were compared, and the correlation between the expressions of miR-181b and partial prognostic factors, including white blood cell (WBC), hemoglobin (Hb), platelet (Plt), absolute neutrophil count(ANC), myeloblast and gene mutations was analyzed. Bioinformatics online tool TargetScan was used to make target gene prediction and the potential function of miR-181b.Results:The expression levels of miR-181b was increased with the increasing risk of IPSS, WPSS and IPSS-R, and there were statistically significant differences in miR-181b expression levels of different risk groups in different scoring systems (all P < 0.01). There was a positive correlation between the expression level of miR-181b and the scores of the three prognostic scoring systems (r was 0.437, 0.368, 0.327; all P = 0.001); miR-181b expression was positively correlated with the proportion of bone marrow myeloblasts ( r = 0.450, P < 0.01) and was negatively correlated with Plt ( r = -0.199, P = 0.024). And miR-18b was not associated with WBC, Hb, ANC, and related gene mutations of blood diseases (all P > 0.05). A total of 1 363 potential target genes of miR-181b were predicted by using bioinformatics, and biological processes of these target genes were mainly enriched in transcription regulation, RNA metabolism regulation. Among them, 22 target genes were related to the hematological malignancies, including RUNX1, ASXL2, NRAS, ATM and KRAS, which have been previously confirmed to be related to MDS. The relative expression level [the median ( P25, P75)] of miR-181b in patients who had those hematological malignancies related to miR-181b target gene mutation (32 cases) was 1.33(0.63, 1.60), which was higher than that in patients without mutation (99 cases) [0.85 (0.49, 1.38)], and the difference was statistically significant ( Z = 2.285, P = 0.022). Conclusions:miR-181b has a correlation with the risk grade of prognostic scoring systems in MDS, and it may be involved in the molecular biology pathogenesis of MDS.

Targeted sequencing analysis of hyper-eosinophilic syndrome and chronic eosinophilic leukemia / 中华血液学杂志

Objective@#Analysis of the molecular characteristics of eosinophilia. @*Methods@#Targeting sequence to 24 patients with chronic eosinophilic leukemia (CEL) with rearrangement of PDGFRA, PDGFRB, or FGFR1 and 62 patients with hyper-eosinophilic syndrome (HES). Mutation annotation and analysis of amino acid mutation using authoritative databases to speculate on possible pathogenic mutation. @*Results@#Thirty-seven kinds of clonal variant were detected from 17 patients with CEL, no recurrent mutation site and hot spot region were found. No pathogenic mutation was detected in 19 patients with PDGFRA rearrangement, but pathogenic mutations of ASXL1, RUNX1 and NRAS were detected from 2 patients with FGFR1 rearrangement who progressed to acute myeloid leukemia and 1 patient with PDGFRB rearrangement who progressed to T lymphoblastic lymphoma, respectively. One hundred and two kinds of clonal abnormalities were detected in 49 patients with HES. The main hot spot mutation regions included: CEBPA Exon1, TET2 Exon3, ASXL1 Exon12, IDH1 Y208C, and FGFR3 L164V. CRRLF2 P224L and PDGFRB R370C point mutations were detected separately in 2 patients with HES who treated with imatinib monotherapy and achieved hematologic remission. @*Conclusion@#The pathogenesis of CEL with PDGFRA, PDGFRB or FGFR1 rearrangement is usually single, and the progression of the disease may involve other driver mutation. A variety of genes with hot mutation regions may be involved in the pathogenesis of HES, and some mutation sites are sensitive to tyrosine kinase inhibitors.

Application of BIOMED- 2 standardized Ig gene rearrangement system in multiple myeloma / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the application of BIOMED- 2 standardized immunoglobulin (Ig) gene rearrangement system in the diagnosis of multiple myeloma (MM), and the significance of clonality analysis by multiplex-PCR amplifications.</p><p><b>METHODS</b>A total of 167 cases of MM bone marrow samples from 2009 to 2013, and 20 cases of reactive plasmacytosis used as the controls were included in this study. Multiplex-PCR amplifications were performed and the Ig gene rearrangements were analyzed using BIOMED-2 standardized clonality analysis system.</p><p><b>RESULTS</b>① Of 167 MM cases, 107 showed IgH VH-JH rearrangement, 33 showed IgH DH-JH rearrangement, and 30% showed IgH DH-JH rearrangement in 60 IgH VH-JH rearrangement negative MM cases. The difference was statistically significant between IgH VH-JH rearrangement positive and negative cases (14.0% vs 30.0%, P=0.032). The total positive rate of IgH VH-JH, IgH DH-JH and IgK was 94.6%. The 20 reactive plasmacytosis (RP) cases showed negative Ig gene rearrangement. 2 of 167 MM cases, 9 (5.4%) showed clonal IgH rearrangement by agarose electrophoresis were confirmed as polyclonality by capillary electrophoresis. ③ Of 53 MM cases who have been detected by Ig gene rearrangement system and fluorescence in situ hybridization (FISH) for IgH simultaneously, 36 showed IgH rearrangement, 26 showed FISH IgH positive, and the difference was statistically significant (67.9% vs 49.1%, P=0.049).</p><p><b>CONCLUSION</b>Combined detection of IgH VH- JH, IgH DH- JH and IgK could improve the positive rate of MM clonality dramatically, and measurement of IgH DH-JH rearrangement was more important in the IgH VH- JH negative cases. Ig gene rearrangement system was a faster and more sensitive method than FISH IgH. Application of BIOMED- 2 standardized immunoglobulin (Ig) gene rearrangement system is of significance for MM diagnosis.</p>

NPM1 and CEBPA mutations in pediatric cytogenetically normal acute myeloid leukemia / 中华儿科杂志

<p><b>OBJECTIVE</b>To evaluate the frequency of the nucleophosmin (NPM1) gene and the CCAAT/enhancer binding protein α gene (CEBPA) through polymerase chain reaction (PCR) array in pediatric patients with cytogenetically normal acute myeloid leukemia (CN-AML) and explore the clinical significances of these mutations.</p><p><b>METHOD</b>Between August 2009 and December 2012, 30 children (<16 years old) with newly diagnosed CN-AML were included. The clinical characteristics were analyzed in these patients. PCR combined with direct sequencing was used to detect NPM1, CEBPA gene mutations. All the data were statistically analyzed using SPSS17.0 software.</p><p><b>RESULT</b>The gene mutations were detected in each of the 30 patients. NPM1 mutation was positive in three patients (10%) with type A mutation, while CEBPA mutation was positive in two patients (6.7%) with double mutations (TAD, bZIP) . Besides, FLT3/ITD mutation was positive in three patients. Patients with NPM1 or FLT3/ITD had a significantly elevated diagnostic WBC count with a median diagnostic WBC count of 102.80×10(9)/L compared with 18.56×10(9)/L for the patients without mutations(t = 2.353, P = 0.043), as well as the marrow blast percentage (94.0% vs. 80.0%, t = 3.804, P = 0.002). The complete remission was achieved in all the 3 patients with NPM1 mutations and 2 patients with CEBPA mutations. All the patients with these mutations also achieved 2-year event-free survival (EFS) and 2-year overall survival (OS), while 2-year EFS and 2-year OS of the other patients were (40.1 ± 11.2)% and (51.8 ± 10.9)% (P = 0.044, 0.091, respectively).</p><p><b>CONCLUSION</b>NPM1 and CEBPA mutations may indicate a favorable prognosis in pediatric CN-AML.</p>

Significance of BIOMED-2 standardized IG/TCR gene rearrangement detection in paraffin-embedded section in lymphoma diagnosis / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the feasibility of detecting lymphoma with the application of BIOMED-2 standardized immunoglobulin/T cell receptor (IG/TCR) gene rearrangement system in formalin fixed paraffin-embedded (FFPE) tissue samples, and to discuss the relationship between the longest amplification fragment of extracted DNA and positive detection rate of different IGH V-J primers.</p><p><b>METHODS</b>DNA was extracted from 50 cases of FFPE tissue samples. Multiplex-PCR amplifications were performed and then the IG/TCR gene rearrangements were analyzed using BIOMED-2 standardized clonality analysis system.</p><p><b>RESULTS</b>(1)When the DNA concentration was diluted to 50-100 ng/μl from 100-500 ng/μl, the proportion of the longest amplification fragment (300-400 bp) of DNA was improved from 10.0% to 90.0% in 30 cases of diffuse large B cell lymphoma (DLBCL) wax roll samples (P<0.01). The positive rate of IGH+IGK was increased from 46.7% to 83.3%, the difference was statistically significant (P=0.006). The lengths of the longest amplification fragments of DNA were all longer than 300 bp in the paraffin section samples of DLBCL. The positive rate of IGH+IGK of these samples was 96.7%. The difference of the positive rate of IGH+IGK between the wax roll samples and the paraffin section samples had no statistical significance (P=0.195). (2)When the concentration of DNA was high, most of the longest amplification fragments of extracted DNA were 100 bp or 200 bp, and the detection rate of short fragment IGH FR3 was more stable than that of long fragment IGH FR1. (3)The clonality analysis of TCRG+TCRB in all 13 cases of peripheral T cell lymphoma samples showed positive results, while no positive IG/TCR clones were found in 7 cases of reactive lymphoid tissue hyperplasia in control group.</p><p><b>CONCLUSION</b>Dilution of DNA is the only method to improve not only the proportion of longest fragment amplification but also the detection rate of clonality. The detection rate of IGH FR3 would not be affected by the concentration of DNA. The application of BIOMED-2 standardized IG/TCR gene rearrangement system in FFPE tissue samples plays an important role in the lymphoma diagnosis.</p>