| 【objective】1. To retrospectively analyzed the clinical, immunophenotypic, cytogenetic, and molecular genetic features of patients with MPAL as defined under WHO-2008 criteria.2. To profile the somatic mutation spectrum and copy number variations(CNVs) responsible for the pathogenesis of MPAL, we performed targeted exome sequencing, Sanger sequencing, and array-CGH on adult patients with MPAL3. To determine the clinical significance and biological effects of MEF2 C gene in acute myelogenous leukemia cells.【Methods】1. This study covered 4780 adult patients(≥14 years of age) presenting with de novo acute leukemia at the First Affiliated Hospital of Soochow University from January 1998 to July 2011. One hundred and seventeen(117/4780; 2.4%) cases fulfilled WHO-2008 criteria for MPAL. The clinical, immunological, cytogenetic characteristics of MPAL were evaluated.2. Bone marrow mononuclear cells for MPAL patients was collected, common 29 kinds of fusion gene were analyzed by multiplex nested RT-PCR for leukemia; Genomic DNA of some patients with MPAL was extracted from frozen bone marrow mononuclear cells using standard procedures. Mutations of C-KIT, NPM1, FLT3-TKD, FLT3-ITD, EZH2, RUNX1, ASXL1, IDH1, IDH2, CBL, WT1, TET2, DNMT3, PHF6, FBXW7, NOTCH1 and ETV6 were analyzed by PCR amplification followed by direct DNA sequencing. Mutation of IKZF1 gene was analyzed through RT-PCR followed by capillary electrophoresis. Other genetic variations for MPAL patients were further tested by high throughput technology including exon trapping and sequencing and array – CGH. Molecular genetic characteristics of the MPAL patients were clarified.3. By real-time fluorescent quantitative PCR technique, MEF2 C gene m RNA expression level of bone marrow mononuclear cells for 83 adult de novo AML patients was detected, including 12 cases of normal control. MEF2 C gene m RNA expression level was compared in AML patients and normal controls, and among AML different subgroups. The correlation between MEF2 C gene m RNA expression level and event-free survival(EFS) and overall survival(OS). MEF2 C overexpression and MEF2 C inhibition sh RNA slow virus carrier were prepared, then these vector transfected myeloid leukemia cell lines Dami, respectively, its effects on cell proliferation was analyzed by the method of absolute count testing.【Results】1. Clinical and laboratory features of MPALAmong 4,780 patients admitted to our institute since 1998, we identified 117 adult patients fulfilling current WHO-2008 criteria for MPAL(60 males and 57 females). The proportion of MPAL patients in this cohort is 2.4%. Median age was 35 years(range 14-81 years). Based on FAB criteria, 40(34%) patients showed AML morphologies, mainly M1 and M5. A total of 51 patients(44%) were classified ALL-L1, the dominant subtype. The remaining 26(22%) cases resisted classification by morphology and were categorized as acute undifferentiated leukemia.Immunophenotyping data showed that 64 of 117 cases(55%) had combined B+My, 38(33%) combined T+My, 14(12%) combined B+T, and one(0.9%) a trilineage(My+B+T), immunophenotype. MPAL cases with B+T immunophenotypes were more commonly seen in males; 11 of 14(79%), when compared to 23 of 38(61%) with T+My(P=0.376) and 26 of 64(41%) with B+My immunophenotypes(P=0.01). CD34 was strongly positive in 82% cases.Of the 92 patients with available karyotypic data, 33(36%) had no detectable chromosomal abnormality, while the remaining 59(64%) showed abnormal karyotypes. Complex karyotypic abnormalities(≥3 aberrations) described the most prolific subclass, found in 22 cases(24%). The t(9;22)(q34;q11)/BCR-ABL1 fusion was present in 14 patients(15%), all with B+My phenotypes. Monosomy 7 was detected in 7 of 92 cases(7.6%). Polysomy 21 was unexpectedly found in 7 of 92(7.6%) cases. Trisomy 8 occurred in 5(5.4%) cases, all with B+My phenotypes. t(v;11q23)/MLL rearrangements were seen in 4(4.3%) patients, of whom 2 had t(11;19)(q23;p13), while one patient each carried t(4;11)(q21;q23) and t(9;11)(p22;q23), respectively. t(10;11)(p15;q21) was present in 3(3.3%) cases, including 2 with T+My and one with B+T phenotypes. In 3 cases, translocations not previously reported in leukemia were found: t(7;9)(q32;p24) with B+My phenotype, t(2;9)(q13;q34) with B+T phenotype, and der(9)t(9;11)(p21;q12) with T+My phenotype. A novel fusion between NUP98 and IQCG gene was identified in a MPAL patient with T+My phenotype harboring t(3;11)(q29q13;p15)del(3)(q29), as previously described. Of the patients for whom cytogenetic data were available, 64% showed abnormal karyotypes. Complex karyotypes, t(9;22)(q34;q11) and t(v;11q23), were detected in our study in a substantial proportion of MPAL cases: 24%, 15%, and 4.3% of patients, respectively. This study identified three recurrent cytogenetic changes for the first time in a significant number of MPAL patients: monosomy 7 in 7.6%, polysomy 21 in 7.6%, t(10;11)(p15;q21) in 3.3%.This research compared the relative effectiveness of the combination of AML+ALL therapy and of the CAG regimen in induction therapy on 34 MPAL patients at presentation. MOAP/IOAP/DOAP was administered to 24 MPAL patients, of whom 14(58%) achieved complete remission(CR), while 2 died from severe infection. The remaining 10 patients received induction therapy according to the CAG regimen of whom 7(70%) achieved CR. There was no significant difference in CR rates between the two groups(P=0.802). However, interestingly, 5 of the 6 patients(83%) who failed to respond to MOAP/IOAP/DOAP therapy achieved CR after receiving the CAG regimen. Furthermore, the overall survival rate in 8 patients with MPAL receiving allo-HSCT was longer than that in the patients receiving consolidation chemotherapy(22.0 vs. 9.0 months; P=0.004).2. Molecular genetic patterns of MPALResults of multiplex nested RT-PCR detection: 12 cases(28.6%) patients existed BCR/ABL fusion gene(including chromosome karyotype examination also found the Ph chromosome in 7 patients).3 cases of chromosome analysis in patients with normal karyotype, this study respectively detected SIL/TAL1 fusion gene caused by 1p32 deletion, dup MLL gene rearrangement and MLL/AF9 fusion gene in 1 case. In addition, this study also detected SET/CAN fusion gene in one MPAL patient with t(16, 17) and complicated abnormal karyotype.Frequencies and distribution of gene mutations: In 31 cases with MPAL, a total of 12 mutations(39%) were documented including: IKZF1 deletions in 4 of 31(13%) patients, and mutations affecting EZH2 in 3 of 31(9.7%), ASXL1 in 2 of 31(6.5%), and ETV6, NOTCH1, and TET2 mutations in one of 31(3.2%) patients each. Although mutations of CBL, DNMT3 A, FBXW7, FLT3, IDH1, IDH2, KIT, NPM1, PHF6, RUNX1, and WT1 have been frequently detected in patients with AML or ALL, none were found in this study. With respect to karyotype, cytogenetic abnormalities were detected in 10 of 11 patients with available data. Among these, IKZF1 deletions were found exclusively in 4 of 8(50%) patients with t(9;22)(q34;q11)/BCR-ABL1 fusion and B+My phenotype. To evaluate the clinical impact of gene mutations on patients with MPAL, this study compared the prognosis of 24 MPAL patients undergoing mutation screening. However, no significant difference on overall survival(P=0.158) between patients with and without gene mutations was found.Exome trapping and sequencing analysis of 28 cases with MPAL: this study analyzed DNA from a total of 28 MPAL samples. Results were mapped to Human Reference Genome(Hg19), common SNPs were eliminated. Over 500 previously reported genes with mutations in leukemias and other cancers were assessed. Twelve of the 28 samples(42.9%) had mutations in epigenetic regulatory genes. The most commonly mutated gene was EZH2 with 4 patients(14.3%); DNMT3 A gene mutation was detected in 3 patients(10.7%), all of them with T/myeloid mixed subtype. Other mutations in epigenetic regulatory genes were found, GATA2(2 patients, 7.1%), TET2(2 patients, 7.1%), and IDH1(1 patients, 3.6%). Interestingly, these variations were co-mutated with TP53 and NOTCH1 in some patients. Other genes had not been found to be mutated for KRAS, NRAS, CREBBP, NF1, et al. In this group, four important transcription factor gene variants were also found, IKZF1(4 patients, 14.3%), ASXL1(2patients, 7.1%), RUNX1(2patients, 7.1%) and ETV6(1 patient, 3.6%) in another nine cases with MPAL(total incidence: 32.1%). This study found that somatic cell gene variants were found in 75% of adult MPAL patients.Array-based comparative genomic hybridization analysis of 12 MPAL samples: this search performed array-CGH analysis on 12 MPAL samples and found that all had one or more genomic abnormalities. A total of 68 genomic alterations were detected, with a mean of 5.7 genomic alterations per sample. We identified new cryptic copy number changes in MPAL containing interesting potential candidate genes such as deletions of the CDKN2A(4 of 12), IKZF1(3 of 12), MEF2C(2 of 12), together with one each(8.3%) of BCOR, EBF1, KRAS, LEF1, MBNL1, PBX3 and RUNX1.3. Expression, clinical significance and function research of MEF2 C gene in adult patients with AML(1) Detection of MEF2 C gene m RNA expression levels in de novo AML patientsMEF2C gene m RNA expression level in AML patients’ group was higher than that in normal control group(P = 0.0377). According to FAB classification: MEF2 C gene m RNA expression level in FAB-M5 subgroup patients was significantly higher FAB-M1, M2 and M4 subgroup(P value were 0.029, 0.003 and 0.021, respecctively); MEF2 C gene m RNA expression levels in FAB-M3 subtype patients was significantly lower than other groups(P < 0.001);And this gene m RNA expression levels in FAB-M1 / M2 and M1 / M4, M2 / M4 AML patients has no statistical significance(P value were 0.2, 0.055 and 0.279, respecctively).According to chromosome karyotypic classification, MEF2 C gene m RNA expression level in other group of patients with AML was higher than that of CBF- AML and normal karyotypic group(P value were 0.036 and 0.036); MEF2 C gene m RNA expression levels in t(15;17) a group of patients with AML was significantly lower than other karyotypic group(P = 0.003).(2)Through Kaplan-Meier analysis, EFS was significantly superior in low MEF2 C gene m RNA expression in normal karyotypic AML patients group,compared with that in the group of patients with high expression(EFS of 3 years were 56% and 20% respectively)(P = 0.048). OS of 3 years were 40% and 16% respectively(P = 0.07) in two groups with low expression group and high expression. There was no relationship between MEF2 C gene m RNA expression with prognosis in patients with CBF-AML and other chromosome abnormalities(P > 0.05).(3) Using two independent sh RNA sequences effectively inhibited MEF2 C expression in myeloid cell lines Dami cell, silent MEF2 C gene expression made myeloid cell lines Dami cell growth and colony formation ability decline. But in Dami cell lines overexpressing MEF2 C was not found to have obvious influence on the proliferation ability.【Conclusions】1. MPAL is a set of highly heterogeneous disease. The incidence of MPAL is 2.4% in de novo acute leukemia. B-lymphoid and myeloid antigen mixed expression is the most common immunological characteristics. 64.1% of MPAL patients have cytogenetic abnormalities. Complex karyotype and Ph chromosome are relatively common abnormalities. Existing chemotherapy regimens may not be sufficient to treat this kind leukemia and allogeneic hematopoietic stem cell transplantation may improve long-term survival.2. Multiple nested RT-PCR technology can improve the detection rate of gene rearrangement in MPAL patients. We detected high frequency of gene mutations or CNCs in MPAL by targeted exome sequencing, Sanger sequencing, and array-CGH analysis.75% of the patients with MPAL at least have one current common gene mutation, including epigenetics gene mutation rate(42.9%) and important transcription factor gene variants(32.1%).3. We identified recurrent MEF2 C microdeletions in MPAL patients by array-CGH. The MEF2 C expression level was upregulated in AML and high expression of MEF2 C was associated with inferior 3 years’ EFS. Down-regulating of MEF2 C expression inhibited the proliferative and clonogenic abilities of AML cell line Dami. |
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