| Background and ObjectivesAcute myeloid leukemia with t(8;21)/AMLI-ETO-positive (AE-AML) is classified as a separate subtype by World Health Organization (WHO), accounting for as high as 5-10% in AML. AE-AML is a heterogeneous myeloid tumor, with high incidence of extramedullary leukemia (EML), C-KIT mutation and additional cytogenetic abnormalities, and apparent difference in disease outcome in different subgroups. Lots of factors are reported to affect on the prognosis of AE-AML, including peripheral white blood cell count, extramedullary infiltration, CD56-positive expression, additional cytogenetic abnormalities, etc. Recent years, more and more scientists pay attention to molecular abnormalities, including gene mutations and overexpression, which play important role in the progression and prognostic effect of the subtype of leukemia.C-KIT mutation, which is one of the adverse prognostic factors in the National Comprehensive Cancer Network (NCCN) guideline, is considered as one of the most important subsequent events, that it highly expresses (up to 12.8%-48%), cooperates with AML1-ETO full length to induce leukemia, and adversely affects on disease outcome. Except that, there are still some other gene mutations including FLT3/ITD and TEL/PDGFRβ and WT1 reported to involve in the the progression of the subtype of leukemia. It was reported that gene mutations were detested with the first generation sequencing in 139 patients with AE-AML, and the results showed that there were 49.6% patients occurred additional gene mutations, of which 16.5% happened≥2 additional mutations, and 66.7% presented clonal evolution of molecular biology at relapse; Moreover, the number of additional gene mutations, C-KIT and ASXL1 mutation were the poor predictors for disease outcome.Therefore, molecular biology abnormalities happen commonly in AE-AML, and play an important role in the progression of the leukemia. Going further to detect the exact incidence of the molecular events in AE-AML and assess the roles of different gene mutations is necessary and good for the understanding of the mechanism of leukemia development and drug resistance, and then provide a basis for the precise treatment of the subtype of leukemia.However, Sanger sequencing, which is the traditional method in previous studies, is an expensive and time-consuming detection method with low sensitivity of about 20%, and low throughput. The limitation of Sanger sequencing has become the bottleneck of the study of gene mutation spectrum in AML. Torrent Ion personal genome sequencing (PGM) is the latest sequencing instrument from Life Thermo Company, with the advantages of high accuracy, fast speed and flexibility. It has been reported to be used in the analyzation of the mutation types of BCR-ABL before treatment decision on the patients with drug resistance.We added 17 AML-specific gene to Ion AmpliSeq Cancer Hotspot Panel V2 and designed a customized Ion AmpliSeqTM Panels for AML. Then we tested 27 patients with CN-AML with the Ion Torrent PGMTM sequencing platform, and evaluated the reliability of the methodology with Sanger sequencing. The results showed 100% consistency of the two methods, proving that a set of reliable and high-throughput method for gene mutation detection in AML with high sensitivity and specificity had been successfully established. In this study, we detected gene mutations at diagnosis and assessed the clonal evolution of molecular biology at relapse in 64 patients with newly diagnosed as AE-AML using the method, in order for better understanding of the gene mutation spectrum in AE-AML, and the further study of the mechanism of leukemia pathogenesis and drug resistance.In addiction, gene overexpression has bad effect on the prognosis of the subtype of leukemia. AML1/ETO9a, the variant of AML1-ETO fusion gene, has been reported to highly express in AE-AML, and be correlated with C-KIT mutations/ overexpression and have poor prognostic effect in AE-AML.Amyloid precursor protein (APP), generated by APP gene which locates to 21q21.3, has been reported to promote cancer cell proliferation and metastasis and its overexpression in oral squamous cell carcinoma, pancreatic cancer and colorectal cancer has an adverse effect on prognosis. APP also highly expresses in AML harboring t(8;21) and plays an important role in AE leukemia, that its overexpression enhances migration of kasumi-1 cells by p-ERK/c-Myc/MMP-2 pathway. Thus, APP must play an important role in AE-AML. In this study, we assessed the clinical significance of APP in the patients with AE-AML; and then went further to its effect on the biological behavior and the related mechanism on kasumi-1 cell model.In view of the great heterogeneity of the prognosis of AE-AML with a lot of prognostic factors, establishment of a risk stratification system to evaluate the prognostic significance of different indicators and guide the treatment on the patients is much necessary. However, there is some limitation of the related studies, which mainly focus on single or multiple factors on the prognosis. Aim of this study is to establish a new prognostic stratification system with multivariate analysis of different factors, including gene mutations and APP overexpression.Materials and MethodsPart 1Study objectA total of 64 patients with newly diagnosed as AE-AML were involved in the present study from April 2006 to December 2013 in NanFang Hospital. DNA was extracted from bone marrow samples.Methods1. The collection of clinical dataClinical features of the onset, including gender, age, peripheral WBC, HGB, PLT count, bone marrow morphological features, EML, karyotype analysis, immunophenotyping, were collected.2. DNA preparationDNA was isolated from both fresh bone marrow and bone marrow smears using the QIAamp DNA Mini Kit (Qiagen) and manufacturer’s instructions were followed.3. Next-generation sequencing1) Library Preparation For multiplex PCR amplification,10 ng of DNA (quantified by QubitTM Fluorometer) was used and the customized Ion AmpliSeq panel was processed with Ion AmpliSeq Library kit 2.0 according to the manufacturer’s instructions. The resulting amplicons were treated with FuPa Reagent to partially digest the primers and phosphorylate the amplicons. The resulting library was purified and the concentration and size of the library were determined by Ion Library Quantitation Kit.2) Emulsion PCR and template-positive enrichment Sample emulsion PCR, emulsion breaking, and enrichment were performed using the Ion PGMTM Template OT2200 Kit and Dynabeads(?) MyOneTM Streptavidin C1 Beads according to the manufacturer’s instructions. The two steps were carried out on Ion OneTouchTM.3) Sequencing was performed on Ion 316 chips using the Ion PGMTM 200 Sequencing Kit according to the manufacturer’s instructions. The raw signal data were analyzed using Torrent Suite v.4.0.2 (Life Technologies). The pipeline included signaling processing, base calling, quality score assignment, adapter trimming, read alignment to human genome GRCh37 references, mapping quality control, and coverage analysis. Several filtering steps were used to generate final variant calling.Part 2Study objectClinical study:A total of 65 patients with newly diagnosed as AE-AML, of whom 50 cases were studied in Part 1, were involved in this study from April 2006 to December 2013 in NanFang Hospital. Bone marrow samples were collected at diagnosis,1,3,6,12,24,36 and the time of relapse. DNA and RNA were extracted from those samples..In vitro study:Wild-type kasumi-1 cell line, kasumi-1 cell line with transfection of scramble sequence (negative control, NC), kasumi-1 cell line with transfection of siRNA-APP (siAPP).MethodsI. Clinical study1. The collection of clinical dataClinical features of the onset, including gender, age, peripheral WBC, HGB, PLT count, bone marrow morphological features, EML, karyotype analysis, immunophenotyping, were collected.2. Sanger sequencing C-KIT (exons 8,17) and FLT3-ITD specific PCR fragments were analyzed by Sanger sequencing.3. Next-generation sequencing (see Part 1)4. The relative expression of APP and C-KIT gene detection1) Total RNA was extracted from bone marrow mononuclear cells. Global ampliation of cDNA corresponding to all expressed genes was carried out.2) Real-time quantitative (RTQ-PCR was applied to cDNA from each sample. To determine the specificity of PCR product through the melting curve. Take an average of two Ct values 2-ACT value as the specimen relative mRNA expression. Ⅱ. In vitro study1. Detection of the expression of APP and C-KIT mRNA in different groups of kasumi-1 cells with RTQ-PCR method2. Detection of the expression of cell apoptosis and CD117-positive in different groups of kasumi-1 cells with flow cytometry (FCM) method3. Detection of the distribution of cell cycle in different groups of kasumi-1 cells with FCM method4. Detection of the expression of C-KIT, AKT, p-AKT, Bcl-2, Caspase-3, Caspase-9, NF-κB and p53 in different groups of kasumi-1 cells with western blotting analysis5. Detection of C-KIT mutations in different groups of kasumi-1 cells with Sanger sequencingPart 3Study objectA total of 50 patients with newly diagnosed as AE-AML with next-generation sequencing and APP gene quantitative detection, described in Part 1 and Part 2, were involved in this study. Bone marrow samples were collected at diagnosis,1,3,6,12, 24,36 and the time of relapse. DNA and RNA were extracted from those samples.Methods1. The follow-up and collection of clinical data Clinical features of the onset, including gender, age, peripheral WBC, HGB, PLT count, bone marrow morphological features, EML, karyotype analysis, immunophenotyping, were collected. Follow up of the early response, including hematologic and molecular remission, OS and RFS.2. Next-generation sequencing (see Part 1)3. The relative expression of APP gene detection (see Part 2)4. Induction regimens:anthracycline combined with cytarabine "3+7" for 1-2 courses; Consolidation therapy:All patients received standard-dose Ara-C-based (SDAC-based) or median-dose Ara-C-based (MDAC-based) regimens for consolidation after complete remission.ResultPart11. The incidence of gene mutations in AE-AML 47 out of 64 patients occurred median 2 (1-14) types of additional gene mutations, with the incidence rate of 73.4%, of which 14 cases happened 1 type of gene mutation and 33 cases happened≥2 types of mutations. A total of 29 kinds of gene mutations with mainly heterogeneous mutated types were detected.31 patients occurred KIT gene mutations, the highest gene mutations detected in the subtype of leukemia, including 13 cases of D816,5 cases of N822,7 cases of M541,4 cases of Pro518Leu and 1 cases of R815_D816ins R815_D816ins. Followed by 19 cases of ASXL1 mutations,11 cases of DNMT3L and MET mutations,9 cases of KMT2A and MLH1 mutations,8 cases of DNMT3A mutations,6 cases of DNMT3B, PAX5 and FBXW7 mutations, and 5 cases of NRAS mutations, etc.2. Different effect of different gene mutations on the clinical featuresObviously increased incidence of EML (9/19 vs.11/44, P=0.071) and CD56-positive expression (18/19 vs.23/44, P=0.001) in the ASXL1 mutated group than in the wild-type group. Significantly higher bone marrow blast [median 52.3 (13.0-94.0)% vs.35.0(13.0-93.)%, P=0.039] in the DNMT3L mutated group than in the wild-type group. Significantly increased CD19-positive expression (7/9 vs. 21/54, P=0.030) and apparently decreased incidence of additional chromosome abnormalities (1/7 vs.23/55, P=0.069) in the KMT2A mutated group than in the wild-type group.3. The clonal evolution of molecular biology at relapse in AE-AML Six of 11 patients occurred clonal evolution of molecular biology at relapse, with the occurrence rate of 54.5%; of whom five cases obtained new clone, including the KMT2A, KIT and TET2 mutations, and three cases experienced clonal deletion, including KIT and NRAS mutations. KIT gene mutations appeared the most frequently in the process of clonal evolution that two cases obtained the mutation and two lost.Part 21. The clinical significance of APP gene in AE-AML1) APP gene was correlated with C-KIT (exon 8,17) mutations/overexpression 17 out of 65 patients happened C-KIT mutations, of which 13 in the APP-H group, and 4 in the APP-L group. There was statistically different between the two groups (P=0.009).9 out of 50 patients experienced C-KIT (exon 10) mutations, of which 4 in the APP-L group and 5 in the APP-H group (P=0.976). No significant difference was found in the other gene mutations between the two groups. Furthermore, Pearson rank correlation analysis showed that the expression levels of APP gene were positively correlated with C-KIT mRNA expression (r=0.621, P<0.001).2) APP gene affected on the other clinical characteristics A significantly higher white blood cell (WBC) count (29.3±4.0×109/L vs. 18.1±2.8×109/L, P=0.011), bone marrow cellularity rate (85.8%±2.2% vs. 80.2%±2.3%, P=0.039) and incidence of EML (11/32 vs.3/33, P=0.013) were found in the APP-H patients than in the APP-L patients. No significant difference in the other clinical characteristics was observed between the two groups.2. APP involved in the regulation of cell apoptosis but not proliferation in kasumi-1 cellscell apoptosis, both early and late, as estimated by analyzing Annexin-V, increased significantly when APP was knocked down in kasumi-1 cells, in that the early and late apoptosis rate in the siAPP cells were 29.00%±0.98% and 19.80%±1.51%, respectively; when compared with 21.43%±0.86% and 12.33%±0.75% in the wild type cells and 21.67%±0.78% and 12.90%±1.25% in the NC cells, respectively, there was statistically different (Early apoptosis:F=71.927, P<0.001; Late apoptosis rate:F=35.239, P<0.001, respectively). In parallel, western blotting analysis revealed apparently reduced levels of the anti-apoptotic protein Bcl-2 with increased levels of activated caspase-3 and caspase-9 in the siAPP cells, suggesting that APP involved in the regulation of cell apoptosis in kasumi-1 cell.However, no obvious difference in the cell cycle distribution was observed among the siAPP, NC and wild type cells with FCM analysis, indicating that proliferation was not correlated with APP expression in AE leukemia.3. The knockdown of APP downregulated C-KIT expression We analyzed the difference of CD117-positive expression and C-KIT mRNA expression levels among the siAPP, NC and wild type cells and found that both CD117 (by flow cytometry analysis, siAPP:81.05%±1.38%, wild type: 91.28%±1.98%, NC:92.15%±1.56%, respectively, F=45.27, P=0.000) and C-KIT mRNA expression levels (by QRT-PCR analysis, siAPP:0.05273±0.00873, wild type:0.09008±0.00712, NC:0.08707±0.00676, respectively, F=22.46, P=0.002) in the siAPP kasumi-1 cells decreased significantly, as compared with those in the wild type and NC kasumi-1 cells. The result of western blotting analysis that protein C-KIT decreased when APP was knocked down further confirmed the involvement of APP in the regulation of C-KIT expression.4. The involvement of APP in the regulation of PI3K/AKT signaling pathway Western blotting analysis revealed obviously decreased levels of AKT phosphorylation concomitant with reduced transcription factors p53 and NF-κB and antiapoptosis factor Bcl-2 in the si APP kasumi-1 cells, as compared with the levels in the NC and wild type kasumi-1 cells, suggesting that APP mediates kasumi-1 cell apoptosis via PI3K/AKT pathway.Part 31. Disease outcomeThe two-cycle cumulative remission rate was 46/48. The total complete remission (CR) rate was 48/48. The cumulative major molecular remission (MMR) rate after two courses of consolidation was 21/45. With a median follow-up of 23(4-85) months, the cumulative recurrence rate was 24/48, and the cumulative mortality rate was 16/48.2. The prognostic factors in AE-AML1) The impact factors for the early treatment response Additional gene mutations, especially mutations in DNMT3A, MLH1 and C-KIT (exon 8,17) had significant effect on MMR after two courses of consolidation, while other clinical features did not have.2) The effect factors for relapse free survival and overall survival in AE-AML Univariate factors analysis results showed that the clinical features of onset, including extramedullary leukemia, bone marrow cellularity rate ≥90%, APP overexpression, additional gene mutations, especially mutations in MET, ASXL1, DNMT3A, MLH1 and C-KIT (exon 8,17), significantly affected on relapse free survival (RFS) and overall survival (OS). In addition, KMT2A mutation was an adverse factor for RFS, while FBXW7 had poor effect on OS. Furthermore, MDAC-based chemotherapy as consolidation regimen after CR and MMR after two courses of consolidation therapy were both protective factors for RFS and OS. Multivariate analysis showed that ASXL1 mutations and APP overexpression were the adverse factors for RFS, and MET mutation was the only poor factor for both RFS and OS. MMR after two courses of consolidation therapy was the independent protective factor for RFS and OS.3. The establishment of risk stratification system in AE-AMLAccording to the results of multivariate analysis, we defined the integral value of MET mutations, APP overexpression, ASXL1 mutations and not reveiving MMR after two courses of consolidation as one point of eacht. According to the total integral value, the patients were divided into score 0 group (n= 9), score 1 group (n = 16), score 2 group (n=13) and score 3～4 group (n= 10). The RFS rate of the three groups were 61.5%8.2%,61.9%±6.2%,34.2%±6.0% and 14.8%±2.2%(P<0.001), respectively; and the OS rate were 64.5%±6.1%,84.2%±4.6%,32.6%±6.9% and 14.8%±2.2%(P<0.001), respectively.Conclusion1.73.4% patients with AE-AML occurred additional gene mutations. Ion Torrent PGMTM detection platform is a specific and high-throughput method with more sensitivity for gene mutation spectrum assessment in AML. C-KIT mutations were the most frequent types of gene mutations detested in this series, followed by ASXL1 mutations. Different mutated genes had different clinical significance. Most patients experienced molecular clonal evolution at relapse.2. APP gene was correlated with C-KIT (exon 8,17) mutations. APP regulated cell apoptosis but not cell proliferation in AE leukemia. The regulation mechanism could be that APP cooperates with C-KIT mutation to reduce cell apoptosis via PI3K/AKT pathway.3. ASXL1 mutations and APP overexpression were the adverse factors for RFS, and MET mutation was the only poor factor for both RFS and OS. MMR after two courses of consolidation therapy was the independent protective factor for RFS and OS. The risk stratification system directed by the factors of gene mutations, APP overexpression and MRD monitoring could be more prognosis predictive. |
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