Wang Jx

CEBPA gene mutation analysis in acute myeloid leukemia

OBJECTIVE To investigate the incidence, molecular features and clinical significance of CCAAT/enhancer binding protein alpha (CEBPA) gene mutation in patients with acute myeloid leukemia (AML). METHODS Mutation analysis of the entire coding region of CEBPA gene in 206 de novo AML patients was performed by using polymerase chain reaction (PCR) followed by sequence analysis and fragment length analysis. RESULTS Of 206 AML patients, 31 (15%) had CEBPA gene mutations, including 23 with double mutations (duCEBPA) and 8 with single mutation (siCEBPA). CEBPA gene mutations presented mainly in M2 subtype or intermediate risk patients. As compared with those with wild type CEBPA gene, patients with mutated CEBPA gene were of higher white blood cell counts [20.92(0.86-351.43)× 10(9)//L vs 8.17(0.47-295.2) × 10(9)/L, P=0.003], higher hemoglobin levels [97.5(51-128) g/L vs 80.5(13-153) g/L, P=0.015] and lower platelet counts [27.5(5-81)× 10(9)//L vs 44(3-548)× 10(9)/L, P=0.004]. Patients with CEBPA gene mutation had higher complete remission (CR) rate than those with wild type (P=0.009). While co-existing of NPM1 and siCEBPA mutations was observed in M5 subtype (2/8, 25%), NPM1 gene mutation was not present in any patients with duCEBPA mutation (0/23, 0%). Dynamic tracking analysis showed that CEBPA mutations disappeared at CR, and the same mutations re-appeared at relapse. When compared to sequence analysis, the coincidence rate of CEBPA mutations detected by fragment length analysis was 100% (54/54). CONCLUSION CEBPA gene mutation is a recurring genetic change in AML patients and has a certain correlation with clinical and laboratory features. It could be reliably used as a potential marker for minimal residual disease follow up. The prognostic significance of co-existing of siCEBPA with NPM1 mutations in patients with AML-M5 subtype needs further investigation.

Analysis of genomic copy number variations in two sisters with primary amenorrhea and hyperandrogenism

OBJECTIVE To analyze genomic copy number variations (CNVs) in two sisters with primary amenorrhea and hyperandrogenism. METHODS G-banding was performed for karyotype analysis. The whole genome of the two sisters were scanned and analyzed by array-based comparative genomic hybridization (array-CGH). The results were confirmed with real-time quantitative PCR (RT-qPCR). RESULTS No abnormality was found by conventional G-banded chromosome analysis. Array-CGH has identified 11 identical CNVs from the sisters which, however, overlapped with CNVs reported by the Database of Genomic Variants (http://projects.tcag.ca/variation/). Therefore, they are likely to be benign. In addition, a -8.44 Mb 9p11.1-p13.1 duplication (38,561,587-47,002,387 bp, hg18) and a -80.9 kb 4q13.2 deletion (70,183,990-70,264,889 bp, hg18) were also detected in the elder and younger sister, respectively. The relationship between such CNVs and primary amenorrhea and hyperandrogenism was however uncertain. RT-qPCR results were in accordance with array-CGH. CONCLUSION Two CNVs were detected in two sisters by array-CGH, for which further studies are needed to clarify their correlation with primary amenorrhea and hyperandrogenism.

[Significance of BIOMED-2 standardized IG/TCR gene rearrangement detection in paraffin-embedded section in lymphoma diagnosis].

OBJECTIVE 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. METHODS 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. RESULTS (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. CONCLUSION 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.

[Dexamethasone and vorinostat cooperatively promote differentiation and apoptosis in Kasumi-1 leukemia cells through ubiquitination and degradation of AML1-ETO].

OBJECTIVE To probe the effects of dexamethasone (DEX) combined with histone deacetylase (HDAC) inhibitor vorinostat on inhibiting proliferation and inducing differentiation and apoptosis in Kasumi-1 leukemia cells, and its possible mechanisms in order to provide a theoretical basis for the treatment of AML1-ETO positive AML. METHODS The cell survival, differentiation and apoptosis rates were tested by MTT or flow cytometry analysis after Kasumi-1 cells were treated by DMSO, DEX (20 nmol/L), vorinostat (1 μmol/L) or DEX (20 nmol/L) in combination with vorinostat (1 μmol/L). WB and IP-WB were performed to detect AML1-ETO and its ubiquitination. RESULTS Treatment with the combination of DEX and vorinostat for 48 h led to statistically significant differences of inhibited proliferation [(42.06±8.20)%], increased differentiation [(52.83±8.97)%] and apoptosis [(52.92±2.53)%] of Kasumi-1 cells when compared with vorinostat [(33.82±9.41)%, (43.93±9.04)% and (42.98±3.01)%, respectively], DEX [(17.30±3.49)%, (22.53±4.51)% and (19.57±2.17)%, respectively] or control [(6.96±0.39)%, (21.73±2.03)% and (6.96±0.39)%, respectively]. Also significant ubiquitination and decreased AML1-ETO protein in Kasumi-1 cells after the combination treatment over single agent or control were observed. CONCLUSION The results indicated that DEX and vorinostat could synergistically inhibit the Kasumi-1 cells proliferation, induce Kasumi-1 cells differentiation and apoptosis through ubiquitination and degradation of AML1-ETO.

[Acute myeloid leukemia with t(11;12)(p15;q13) translocation: two cases report and literature review].

OBJECTIVE To investigate the clinical and laboratory features of acute myeloid leukemia (AML) with t(11;12)(p15;q13) translocation. METHODS Two cases of AML with t(11;12)(p15;q13) translocation were reported and the related literatures were reviewed. RESULTS The diagnosis of AML-M3 was supported by morphological, cytochemical staining and electron microscope tests. A rare t(11;12)(p15;q13) translocation, but not classical t(15;17)(q22;q12) translocation and PML- RARα fusion gene, was detected in both cases. Both of the patients were refractory to differentiation induction therapy such as retinoic acid and arsenic trioxide. CONCLUSION AML is a group of heterogeneous disease derived from hematopoietic stem cell. Cytogenetic characteristic is important for diagnosis, prognosis stratification and therapy selection. Because of the heterogeneity of clinical and molecular features, it is unsuitable to classify AML with t(11;12)(p15;q13) as AML with recurrent cytogenetic aberration. This group of disease may benefit from allogeneic hematopoietic stem cell transplantation.

[Outcomes of younger than 60 years old adults with Ph/BCR-ABL positive acute lymphoblastic leukemia: a single center clinical trial of BDH ALL 2000/02].

OBJECTIVE To explore the treatment options for younger than 60 years old adults with Ph /BCR-ABL positive acute lymphoblastic leukemia (Ph⁺ ALL). METHODS From January 2001 to June 2012, 42 adult patients were enrolled in the study. All patients received standard VDCP±L ±imatinb (IM) as induction therapy followed by intensive consolidation of modified Hyper-CVAD/MA±IM. At complete remission 1 (CR1), patients with appropriate donor received allogeneic hematopoietic stem cell transplantation (allo-HSCT), the others sequentially received intensive consolidation ±IM and autologous HSCT (ASCT) at molecular CR (MCR), then MM±VP±IM as maintenance therapy. Overall survival (OS), disease free survival (DFS) and relapse rate (RR) were analyzed. RESULTS CR rate after 1 cycle of induction chemotherapy was 83.3%. 39(92.9%) patients achieved CR. The median DFS and OS were (22.0±3.5) and (37.0±5.3) months respectively, with cumulative RR of (43.7±9.7)% during a median follow-up of 26.5(8-75) months. All 7 patients in CT group relapsed. Two patients received IM pre- and post-ASCT maintained MCR for 35 and 12 months after ASCT. But the other 3 ASCT recipients without IM died of relapse within 1 year. The transplant-related mortality rate in allo-HSCT group was 12.5%. The estimated 3-year OS in allo-HSCT (n=16), ASCT (n=5) and CT (n=7) groups were (66.7±12.2)%, (25.0±21.7)% and (16.7±15.2)%, respectively (P=0.014); meanwhile, the estimated 3-year DFS in those groups were of (56.3±12.4)%, (26.7±22.6)% and 0, respectively (P=0.002). CONCLUSION IM combined with intensive chemotherapy significantly increased the CR rate with the improved quality of CR, which highlighted the feasibility of SCT. Allo-HSCT could decrease relapse to produce favorable OS and DFS in CR1 of young adults with Ph⁺ ALL. ASCT combined IM might be the treatment of choice for those achieved MCR but without donors.

[Detection of heterogeneity and evolution of subclones in t(8;21) AML by QM-FISH].

OBJECTIVE To explore the heterogeneous subclones in acute myeloid leukemia (AML) with t(8;21) by quantitative multicolor- fluorescence in situ hybridization (QM-FISH), and to figure out whether there is putative ancestral relationship among different subclones. METHODS Bacterial artificial chromosomes (BAC) clones that contain the targeted genes including AML1, ETO, WT1, p27 and c-kit were searched in the data base UCSC Genome Bioinformatics. Multicolor FISH probes were prepared by linking fluorescein labeled dUTP or dCTP to targeted genes by nick translation. Bone marrow mononuclear cells from t (8;21) AML patients are dropped on to the wet surface of glass slides after hypotonic treatment and fixation. After hybridization, the fluorescence signals were captured by Zeiss fluorescence microscope. The copy number of AML1, ETO, WT1, p27, c- kit and the AML1-ETO fusion gene in AML1-ETO positive cells was counted. The cells with same signals were defined as a subclone. Various subclones were recorded and their proportions were calculated, and their evolutionary relationship was deduced. The subclones in matched primary and relapsed samples were compared, the evolution of dominant clones were figured out and the genomic abnormality that is associated with relapse and drug resistance were speculated. RESULTS In this study, 36 primary AML with t(8;21) cases and 1 relapsed case paired with the primary case were detected. In these 36 primary cases, 4 cases (11.1%) acquired additional AML1-ETO fusion signal, 3(8.3%) had additional AML1 signal, 4(11.1%) had additional ETO signal, 20(55.6%) had additional WT1 signal, 15(41.7%) had additional p27 signal and 14(38.9%) had additional c-kit signal. In addition, 10(27.8%) displayed AML1 signal deletion, and such an aberration represents statistic significance in male patients. It seems that male patients usually accompany AML1 signal deletion. Of 36 cases, 28(77.8 %) harbored at least 2 subclones (ranged from 2 to 10). According to the genetic signature of subclones, we can assemble a putative ancestral tree, and the genetic architecture is linear or branching. In particular, the clonal architecture of the relapsed sample exhibited significant clonal evolution compared to its paired sample at diagnosis, including proportion changes in dominant clone, subclone disappearance and appearance of new dominant clones. CONCLUSION Genomic abnormality is very diverse in t(8;21) AML. Subclones have linear or complex branching evolutionary histories, and clonal architecture is dynamic.

Characteristics of karyotypes and gene mutations for elder acute myeloid leukemia

OBJECTIVE To investigate the incidence of karyotypes and gene mutations for elder acute myeloid leukemia and to explore the relationship between each other. METHODS Clinical data and bone marrow samples of elder AML patients were collected. Karyotype and gene mutation (FLT3, NPM1, C-Kit, CEBPα, DNMT3A) test were performed, characteristics of karyotypes and gene mutations were analysed. RESULTS The incidence of better risk karyotype was 16.6%, in which the incidences of t(15;17), t(8;21) and inv (16)/t(16;16) were 3.90%, 10.73%, and 1.95% respectively; the incidence of intermediate risk karyotype was 72.2%, in which the incidence of normal karyotype was 57.86%; the incidence of poor risk karyotype was 11.20%, in which the incidence of of MLL/11q23, complex karyotype and monosomal karyotype were 1.95%, 6.34%, 5.85% respectively; the incidences of FLT3, NPM1, C-Kit, CEBPα, DNMT3A mutation were 12.57%, 22.06%, 2.16%, 14.71%, 15.71% respectively. Compared with patients older than 60 years, patients with age of 55-60 years were with less complex karyotype (1.09% vs 10.62%)(P=0.003) and monosomal karyotype (2.17% vs 8.85%)(P=0.032), and more t(8;21)(17.39% vs 5.31%)(P=0.008) and inv (16)/t(16;16)(4.35% vs 0.00%)(P=0.045). CONCLUSION For older AML patients, great difference in the distribution of karyotyes was found between the patients older than 60 years and patients with age of 55-60 years, while no such characteristics was found for gene mutations. Good elucidation of karyotypes and gene mutations are key for the treatment of older acute myeloid leukemia patients.

Expression of cMPO in 502 cases of acute myeloid leukemia (AML) and its diagnosis significance in AML subtypes

目的 探讨髓过氧化物酶（cMPO）在急性髓系白血病（AML）患者中的表达及其在诊断分型中的意义。 方法 采用CD45/SSC双参数散点图设门方法对502例AML患者进行八色流式细胞术免疫表型分析，观察患者白血病细胞cMPO表达的阳性率和阳性强度。 结果 502例AML患者cMPO总体阳性率为58.0%，其中阳性占21.5%，弱阳性占34.1%，部分阳性占2.4%；阴性占42.0%。各亚型中，AML伴t(15;17）（q22;q12）/PML-RARα的cMPO阳性率最高，为100%，阳性强度多数接近正常粒细胞水平；其次为AML伴t(8;21）（q22;q22）/RUNX1-RUNX1T1，阳性率为91.4%，阳性强度多为弱阳性；AML微分化型和急性巨核细胞白血病患者cMPO表达皆为阴性；余各亚型阳性率在22.7%~76.2%。 结论 各亚型AML cMPO的阳性率及阳性强度存在显著差异。OBJECTIVE To investigate the myeloperoxidase (cMPO) expression pattern by flow cytometry (FCM) in patients with acute myeloid leukemia (AML) and its role in classifying AML. METHODS Eight- color multiparametric FCM with CD45/SSC gating was used to determine the cMPO expression in 502 AML patients. RESULTS The positive rate of cMPO in all patients was 58.0%, in which the proportion of normal positivity, dim positivity and partial positivity was 21.5%, 34.1% and 2.4%, respectively. The remaining case (42.0%) were all negative. In AML with t (15;17）(q22;q12)/PMLRARα, the positive rate was the highest (100%) and the intensity was similar to that of the normal granular leukocytes, followed by AML with t (8;21（q22;q22/RUNX1-RUNX1T1, the positive rate was 91.4% and the intensity was mostly dim. AML with minimal differentiation and acute megakaryoblastic leukemia were all cMPO negative. The positive rates of cMPO in the remaining subtypes were between 22.7% and 76.2%. CONCLUSION The positive rate and intensity of cMPO were significantly different among different subtypes of AML.

[Autologous hematopoietic stem cell transplantation in adults with Philadelphia chromosome-negative acute lymphoblastic leukemia: a single center experience from the BDHALL2000/02 protocol].

目的 探讨自体造血干细胞移植（AHSCT）治疗年轻成人Ph−急性淋巴细胞白血病（ALL）的疗效。 方法 纳入56例于2000年1月至2007年12月接受BDHALL2000/02方案治疗并于CR1期行AHSCT的成人（15~60岁）Ph−ALL患者，对其进行生存和预后影响因素分析。 结果 56例患者中标危、中危和高危者分别为23例（41.1%）、19例（33.9%）和14例（25.0%）。中位随访75(7~177）个月。5年总生存（OS）、无事件生存（EFS）、无复发生存（RFS）、复发率分别为（51.8±6.7）%、（51.8± 6.7）%、（60.5±6.9）%、（39.1±6.9）%。标、中、高危组患者的5年OS率分别为（60.9±10.2）%、（52.6± 11.5）％和（35.7±12.8）%，EFS率分别为（60.9±10.2）%、（52.6±11.5）％和（35.7±12.8）%，RFS率分别为（68.3±9.9）%、（62.5±12.1）％和（44.9±14.1）%，复发率分别为（31.7±9.9）%、（37.5±12.1）％和（55.1± 14.1）%。标危和中危组、中危和高危组患者的上述指标比较差异均无统计学意义（P值均>0.05）；标危组患者的OS、EFS率高于高危组（P值分别为0.040和0.029），而RFS和复发率差异则无统计学意义（P值均>0.05）。对年龄≥35岁、完全缓解时间超过5周、初诊白细胞水平、免疫表型（B/T）、伴髓系表达、超二倍体染色体核型、复杂核型、完全缓解至AHSCT间隔时间、预处理方案是否包含TBI等进行单因素分析，均未显示对预后存在影响（P值均>0.05）。 结论 年轻成人Ph−ALL患者经BDHALL2000/02方案治疗可以获得较高的缓解率，缓解后给予早期序贯强化/巩固治疗后进行AHSCT疗效显著，是标危、中危组及无合适供者的高危组患者的合适选择。OBJECTIVE To evaluate the results of autologous hematopoietic stem cell transplantation (auto-HSCT) in adults with Philadelphia chromosome-negative acute lymphoblastic leukemia (Ph-ALL). METHODS From January 2000 to December 2007, the clinical data of auto-HSCT in adults Ph-ALL with complete remession (CR) 1 according to BDHALL2000/02 protocol were analyzed. RESULTS A total of 56 patients were enrolled and the probabilities of standard risk, intermediated risk and high-risk group were 41.1%, 33.9%, and 25.0%, respectively. After a median follow-up of 75 months (range 7-177 months), the 5-year overall survival (OS), events free survival (EFS) and relapse free survival (RFS) were (51.8 ± 6.7)%, (51.8 ± 6.7)%, and (60.5 ± 6.9)%, respectively. And the 5-year accumulative relapse rate was (39.1 ± 6.9)%. The 5-year OS of standard risk, intermediate risk, high-risk group were (60.9 ± 10.2)%, (52.6 ± 11.5)%, and (35.7 ± 2.8)%, respectively. The 5-year RFS among three groups were (68.3 ± 9.9)%, (62.5 ± 12.1)%, and (44.9 ± 14.1)%, respectively. The 5-year EFS among three groups were (60.9 ± 10.2)%, (52.6 ± 11.5)%, and (35.7 ± 12.8)%, respectively. The 5-year accumulative relapse rate among three groups were (31.7 ± 9.9)%, (37.5 ± 12.1)%, and (55.1 ± 14.1)%, respectively. There was no statistical significance of any survival rates between standard and intermediate risk groups, just as intermediate and high-risk groups. The OS and EFS in standard risk group were superior to those in high-risk group (P=0.040 and P=0.029, respectively), while there was no statistical significance of RFS and accumulative relapse rate between the two groups. The clinical factors listed below did not influenced the prognosis in the univariate analysis (P>0.05), including more than 5 weeks reaching to CR, WBC count at diagnosis, different immunophenotype (T or B cells), myeloid antigen expression, hyperdiploid chromosome karyotype, complex chromosome abnormality, conditioning regimen with or without TBI, duration between transplantation and diagnosis. CONCLUSION Ph-ALL adults could achieve a satisfactory CR and better survial according to BDHALL2000/02 protocol followed by auto-HSCT, especially for the standard or intermediate risk group, and no-donors high-risk patients.