RUNX1-RUNX1T1 Fusion Gene Research Articles

Analysis of the therapeutic effect of avatinib bridged allogeneic hematopoietic stem cell transplantation on 7 cases of recurrent/refractory RUNX1-RUNX1T1 positive acute myeloid leukemia with KIT mutations

Objective: To evaluate the efficacy of avatinib plus allogeneic hematopoietic stem cell transplantation (allo-HSCT) for the treatment of recurrent/refractory RUNX1-RUNX1T1 positive acute myeloid leukemia (AML) with KIT mutations. Method: A retrospective study was conducted on the clinical data of seven relapsed/refractory AML patients containing the RUNX1-RUNX1T1 fusion gene and KIT mutation who received afatinib plus allo-HSCT treatment at the First Affiliated Hospital of Soochow University from June 2019 to June 2023. Results: The seven AML patients included one male and six females with a median age of 37 (18-56) years. All seven patients had KIT mutations (five positive for D816V and two positive for D816Y) . There were two refractory patients and five relapsed patients (all of whom had bone marrow recurrence) . All patients had to complete at least one course of treatment with afatinib before transplantation. Four patients achieved complete remission (CR) after treatment with afatinib, six patients had negative KIT gene mutations, and one had a decreased KIT gene mutational burden. There were three cases of unrelated identical transplantation and four cases of haploidentical transplantation. All patients received the modified Bu/Cy pretreatment regimen. After transplantation, all patients were successfully implanted and a bone marrow examination showed CR and minimal residual disease turned negative. Five patients exhibited negative fusion genes. Two patients died from infection following transplantation. Conclusion: Afatinib plus allo-HSCT may be an effective and safe new treatment strategy for RUNX1-RUNX1T1 positive AML patients with KIT-D816 mutation.

Two cases of systemic mastocytosis with RUNX1-RUNX1T1 positive acute myeloid leukemia treated with sequential avapritinib after allogeneic hematopoietic stem cell transplantation and literature review

Systemic mastocytosis (SM) with RUNX1-RUNX1T1 positive acute myeloid leukemia (AML) is a rare myeloid tumor with no standard treatment. Two cases of SM patients with RUNX1-RUNX1T1 positive AML treated with sequential avapritinib after allogeneic hematopoietic stem cell transplantation (allo-HSCT) were reported in Henan Cancer Hospital. Mast cell in bone marrow disappeared, C-KIT mutation and RUNX1-RUNX1T1 fusion gene remained negative. Allo-HSCT sequential avapritinib is an effective treatment for SM patients with RUNX1-RUNX1T1 positive AML.

Prognosis of RUNX1-RUNX1T1 Rearrangement in Newly Diagnosed Acute Myeloid Leukemia Patients

Objective: To compare the clinical-hematological (including laboratory and morphological) parameters of newly diagnosed AML patients with RUNX1-RUNX1T1 rearrangement before and after induction therapy. Study Design and Setting: This is a cross-sectional study, Department of Hematology, Armed Forces Institute of Pathology (AFIP), Rawalpindi from December 2021 to December 2022. Methodology: 64 newly diagnosed patients with de novo t (8;21) AML were included. The RUNX1-RUNX1T1 fusion gene was detected using real-time reverse transcriptase polymerase chain reaction (RT-PCR); while t(8;21) was identified through chromosomal/cytogenetic analysis. All the clinical parameters, laboratory variables, blast percentages, and morphological parameters of newly diagnosed AML RUNX1-RUNX1T1 patients were compared before and after therapy. AML induction regimen included the following drugs: cytarabine along daunorubicin. Assessment of these patients was carried out four weeks after induction therapy. Results: The patients' mean age was 60 (ranging from 14 to 85 years), with 46 males and 18 females. Statistical significance was observed in TLC (p-value < 0.001), Hb (p= 0.001), and platelet count(p=0.001) levels. After treatment, the blast size in peripheral blood was reduced to zero and both Auer rods and abnormal granules were absent in bone marrow blasts of patients. The average percentage of eosinophilia decreased from 8.52±1.76 before treatment to 2.42±1.79 after treatment. Conclusions: Our study concluded that the treatment approach(cytarabine along with daunorubicin or idarubicin) for patients with RUNX1-RUNX1T1 AML resulted in improved blood counts, reduced blast cells, Auer rods, and abnormal granules; with a higher rate of complete remission and a lower incidence of relapse.

Dual intron-targeted CRISPR-Cas9-mediated disruption of the AML RUNX1-RUNX1T1 fusion gene effectively inhibits proliferation and decreases tumor volume in vitro and in vivo.

Oncogenic fusion drivers are common in hematological cancers and are thus relevant targets of future CRISPR-Cas9-based treatment strategies. However, breakpoint-location variation in patients pose a challenge to traditional breakpoint-targeting CRISPR-Cas9-mediated disruption strategies. Here we present a new dual intron-targeting CRISPR-Cas9 treatment strategy, for targeting t(8;21) found in 5-10% of de novo acute myeloid leukemia (AML), which efficiently disrupts fusion genes without prior identification of breakpoint location. We show in vitro growth rate and proliferation reduction by 69 and 94% in AML t(8;21) Kasumi-1 cells, following dual intron-targeted disruption of RUNX1-RUNX1T1 compared to a non t(8;21) AML control. Furthermore, mice injected with RUNX1-RUNX1T1-disrupted Kasumi-1 cells had in vivo tumor growth reduction by 69 and 91% compared to controls. Demonstrating the feasibility of RUNX1-RUNX1T1 disruption, these findings were substantiated in isolated primary cells from a patient diagnosed with AML t(8;21). In conclusion, we demonstrate proof-of-principle of a dual intron-targeting CRISPR-Cas9 treatment strategy in AML t(8;21) without need for precise knowledge of the breakpoint location.

Detection of RUNX1-RUNX1T1 Fusion Gene in AML Patients by FISH Technique in Iraq

Background: AML with t(8;21)(q22;q22.1) is a balanced translocation that results in the fusion of RUNX1 and RUNX1T1. The translocation product is located on derivative chromosome 8. It has characteristic morphologic and immunophenotypic features and is linked with good prognosis. t(8;21)(q22;q22.1) AML is diagnostic of AML regardless of blast count. Objectives: To detect the RUNX1-RUNX1T1 fusion gene) in patients with AML by FISH technique and to investigate the relation between this chromosomal abnormality and the immunophenotypic markers CD117, cMPO, CD34, CD13, CD64 and CD33, clinical features and haematological parameters (Hb, WBC, blast percentage and platelets). Materials ad Methods: Fifty patients with de novo AML were selected sequentially from Baghdad teaching hospital in medical city from June 2020 till April 2021. History was taken and data were collected for each patient using a questionnaire form that included: name, age, sex, symptoms and physical signs. The data of haematological parameters and CD markers expression were collected from the patients’ diagnostic reports. Results:The RUNX1/RUNX1T1 fusion gene expression was positive in 4 patients representing 8% whereas 46 patients representing 92% of the samples had negative gene expression. Among the 4 positive AML patients for RUNX1/RUNX1T1 fusion gene expression, M2FAB subtype was revealed in all the cases. All the positive cases expressed CD117 and CD34 markers, while it was noted that 3 out of 4 positive cases were having cMPO and CD13 markers and one positive case expressed CD33 and CD64. Conclusion: It’s shown that RUNX1-RUNX1T1 fusion gene frequency in AML Iraqi patients is similar to international reports.

Genomic Analysis Focusing on RUNX1-RUNX1T1 in Japanese Patients with AML: HM-Screen-Japan 01

Genomic Analysis Focusing on RUNX1-RUNX1T1 in Japanese Patients with AML: HM-Screen-Japan 01

An unusual case of chronic lymphocytic leukemia with trisomy 12 presenting with prolymphocytic transformation and t(8;21)(q22;q22).

First report of t(8;21)(q22;q22) in a patient with CLL. RUNX1‐RUNX1T1 fusion gene resulting from the translocation may have played a role in the prolymphocytic transformation.

High EVI1 Expression Predict Adverse Outcome in Children with Acute Myeloid Leukemia: A Multicenter, Nonrandomized Clinical Study in China

Backgroud and purpose In clinical studies in adult acute myeloid leukemia (AML), high ecotropic virus integration-1 (EVI1) gene expression has shown to be an independent prognostic factor. However, the predictive value of high EVI1 expression (EVI1+) in children with AML is little known. The purpose of this study was to investigate frequency and prognostic significance of high EVI1 expression in children with AML. Patients and methods Total of 403 newly diagnosed childhood AML patients from 7 centers of south of China from November 2014 to May 2020 were included in the study. A diagnostic assay detecting multiple EVI1 splice variants was developed to determine the relative EVI1 expression by single real-time quantitative polymerase chain reaction in the 403 newly diagnosed pediatirc patients with AML younger than 14 years. Patients were treated with C-HUANAN-AML15 protocol. In the C-HUANAN-AML15 protocol, tandem 2 courses of FLAG-IDA regimen, or sequential DAE(3+5+10) and DAE(3+5+8) were applied as induction chemotherapy. One course of Homoharringtonine (substitution of Amsacrine in MRC-AML 15 protocol)/ Cytarabine/ Etoposide and one course of Mitoxantrone/ Cytarabine in consolidation chemotherapy were uniform in both groups. 75 patients selected hematopoietic stem cell transplantation (HSCT) in first CR. The median follow-up period was 20 (0.8 to 67.7) months up to July 2020. The similarity of clinical data was analyzed by the chi square test and COX proportional hazard function model. Complete remission (CR) rates, event-free survival (EFS) and overall survival (OS) were compared by Log-Rank chi square test. Results EVI1+ was found in 36/403 (8.9%) of children with de novo AML. The clinical features of the patients with EVI1+(n=36) compared with EVI1-(n=367) AML were summarized in Table 1. No significant differences in sex, white blood cell count, and age were noted between EVI1+and EVI1- AML. When studying the relationship between EVI1+ and conventional classification criteria such as morphology (FAB classification) and cytogenetic data, EVI1+ was predominantly found in FAB M7, t(9;11) and other t(v;11q23), as well as complex karyotype, but mutually exclusive with t(8;21), inv(16)/t(16;16), CEBPA, NPM1 or C-KIT mutations. Furthermore, EVI1+ was significantly associated with unfavoralbe cytogenetic risk. Twenty-five patients (22 cases in the EVI1-group and 3 cases in the EVI1+ group) who gave up treatment or transferred to other hospital when treatment was not completed were excluded, and the remaining 378 AML children with standard treatment were included in the survival analysis. Univariate analysis showed that initial white blood cell count (≥50×109/L), RUNX1-RUNX1T1 fusion gene, FLT3-ITD mutation and EVI1+ affected 5-year EFS (P=0.000, 0.001, 0.016, 0.011). Multivariate prognostic analysis with COX proportional hazard function model for EFS identified EVI1+ as an independent prognosis factor (HR0.531, 95%CI 0.298-0.945, P=0.032). Patients with EVI1+ AML (n=12) who received HSCT in first CR had better 5-year EFS, but the difference was not statistically significant (46.103% vs. 56.250%, P=0.33). Conclusion It could be concluded that EVI1+ can be found in ~10% of pediatric AML in China. It is predominantly found in intermediate to unfavorable cytogenetic subtypes and predicts adverse outcome. EVI1 screening at diagnosis should be included in risk stratification of pediatric AML. Whether pediatric patients with EVI1+ AML can benefit from HSCT in first CR needs further reasearch. Disclosures No relevant conflicts of interest to declare.

Effectiveness and Survival of 4 Courses of Chemotheray in 321 Children with Acute Myeloid Leukemia in China: A Multicenter, Nonrandomized Clinical Study

Objectives Increasing the intensity of chemotherapy can improve long-term survival in young patients with acute myeloid leukemia, but the number of appropriate courses is still worth exploring. We prospectively used MRC-AML 15 protocol as backbone to designed the C-HUANAN-AML15 protocol, which including 2 courses of inductions and 2 courses of consolidation to treat new diagnosed acute myeloid leukemia children in order to adapt to China's actual situation. The midterm outcome of effectiveness and survival was analyzed in the multicenter of south of China. Patients and methods Total of 321 newly diagnosed childhood AML patients from 9 centers of south of China from November 2014 to December 2018 were included in the study. 194 boys and 127 girls, with median age 6 (0.5-14) years old were enrolled. The median follow-up period was 16 (4 to 73) months up to July 2019. Risk stratifications were based on genetic abnormalities and response to induction chemotherapy . One hundred and seven patients (33.3%),146 patients (45.5%),68 patients (21.2%) were classified as low-risk group (LR), intermediate-risk group (IR) and high-risk group (HR) respectively. The protocol C-HUANAN-AML15 protocol including tandem 2 courses of FLAG-IDA regimen (marked as group A, n=225) , or sequential DAE(3+5+10) and DAE(3+5+8) regimen (marked as group B, n=96) were applied to induction chemotherapy. One course of Homoharringtonine (substitution of Amsacrine in MRC-AML 15 protocol)/ Cytarabine/ Etoposide and one course of Mitoxantrone/ Cytarabine in consolidation chemotherapy were uniform in both groups. 57 patients (39 patients in group A and 18 patients in group B, P=0.752) selected hematopoietic stem cell transplantation (HSCT) after 2-3 courses of chemotherapy. In the 264 patients who only underwent chemotherapy, the LR/IR/HR ratio in the A and B group was 65(34.9%),82(44.1%),39(21.0%) and 34(43.6%),37(47.4%),7(9.0%) respectively (P=0.056). The similarity of clinical data was analyzed by the chi square test and COX proportional hazard function model. Complete remission (CR) rates, treatment related mortalities (TRM), event-free survival (EFS) and overall survival (OS) were compared by Log-Rank chi square test. Results The CR rates in A and B group were 87.6% and 83.3% after 1st course induction regimen, and 92.4% and 89.2% after 2nd course induction regimen, which were not significantly different (P=0.375, 0.337). The 3-year EFS and OS of all patients were 65.8±3.2% and 70.3±3.4% respectively. The 3-year EFS and OS of 264 patients who only received chemotherapy was 65.4±7.4% and 71.7±3.4%. The 3-year EFS of HR, IR and LR in patients who received only chemotherapy were 40.3±8.8%, 62.0±5.2% and 83.5±4.3% respectively (P=0.000). The 3-year OS of HR, IR and LR in patients who received only chemotherapy were 46.9±9.0%, 69.1±5.1% and 86.1±4.0% respectively (P=0.000). The 3-year EFS of A and B group in patients who received only chemotherapy was 70.8±4.0% and 56.7±6.3% (P=0.059); The 3-year OS of A and B group in patients who received only chemotherapy was 78.7±3.6% and 58.6±6.4% (P=0.005). The 3-year EFS and OS of 57 patients who received HSCT was 60.2±8.9% and 65.4±7.4%(P=0.477, 0.821). The EFS of IR and HR patients who received HSCT were superior than that of who received only chemotherapy, but they were not significantly different (78.6±8.6%vs62.4±5.1%, P=0.121; 44.4±12.9%vs38.9±8.8%, P=0.208). Univariate analysis showed that initial white blood cell count (≥50×109/L), RUNX1-RUNX1T1 fusion gene, EVI1 gene positive, FLT3-ITD mutation, no CR after 1st course and 2nd course induction affected 3-year EFS (all P<0.05). Multivariate prognostic analysis with COX proportional hazard function model for EFS showed that EVI1 gene (HR0.397, 95%CI 0.201-0.785, P=0.008) and no CR after 2nd course induction (HR0.319, 95%CI 0.134-0.763, P=0.010) were independent risk factors. Conclusion This prospectively clinical study indicated that intensive induction with FLAG-IDA or DAE followed only 2 courses of consolidation chemotherapy was effective in the LR and IR group, and could be utilized as first line treatment in childhood AML. EVI1 gene positive and no CR after 2nd induction course were independent risk factors for EFS. More effective strategies are needed to be explored for HR group patients, EVI1 gene positive patients and those who cannot achieve CR after 2nd course induction. Figure Disclosures No relevant conflicts of interest to declare.

A Multicenter Nonrandom Clinical Study Using Induction Regimen of FLAG-IDA or Dae in Treatment of 101 Children with Newly Diagnosed CBF-AML in South of China: Clinical Responses and the Midterm Report of Survival

OBJECTIVE: Patients with core binding factor (CBF) AML generally have a favorable prognosis, but the appropriate intensity of chemotherapy and number of courses are still key points for further exploration by the different leukemic group. Our prospective multicenter clinical study was designed to use high intensity, 4-courses of chemotherapy (backbone of MRC AML 15 protocol) to treat childhood CBF-AML and to observe the feasibility and effectiveness in China. METHODS: Total of 101 children with CBF-AML from 9 centers of south of China were enrolled on the C-HUANAN Children AML 2015 protocol from 2015.1 to 2018.11, accounting for 30.6% of total AML (non-APL) cases. There were 59 boys and 42 girls, aged between 2- 14 years old. The diagnosis of CBF-AML was established by PCR-based detection of RUNX1-RUNX1T1 and CBFβ-MYH11 fusion gene transcripts or by FISH based detection of t(8;21) and inv(16) aberration, respectively. Nine cases were combined with FTL3-ITD. Eleven cases were combined with ASXL1 mutation, and 23 cases were combined with c-KIT mutation. The induction regimen were non-randomized to DAE group and FLAG-IDA group. The induction regimen of DAE group including coures 1 (DAE 3+5+10), course 2 (DAE 3+5+8) . The induction regimen of FLAG-IDA including two courses of FLAG-IDA. Consolidation treatment was same and consisted of 2 courses: 1.HAE or MACE; 2.MidAC. Follow-up observation was then started after 4 courses of treatment. All of patients were followed-up to 06/30/2019, median follow-up time was 25 months. RESULTS: The overall complete remission rate was 93.1% after two courses of induction, 93.8% in the DAE group and 92.8% in the FLAG-IDA group. There was no statistical difference between the two groups. The MRD negative rate (<0.1%) detected by the flow cytometry after first course was 57/87 (65.5%) and 67/77 (87.0%) after second course respectively. The RUNX1-RUNX1T1 or CBFβ-MYH11 gene negative rate was 28/60 (46.7%) and 25/43 (58.1%) after first course and second course of treatment respectively .The overall survival rate (OS) was 83.2%, and the leukemia-free survival rate was 81.1%.Among them, the OS and LFS of the DAE group were 76.4% and 73.2%, respectively, and the OS and LFS of the FLAG-IDA group were 86.3% and 84.6%, respectively( P= 0.331 and 0.241).The OS and LFS of the RUNX1-RUNX1T1 group were 81.8% and 79.2%, respectively.The OS and LFS of the CBFβ/MYH11 group were both 88.9%( P= 0.542 and 0.403). The OS and LFS in CBF-AML with FLT3-ITD positive were both 62.2%. The OS and LFS in CBF-AML without FLT3-ITD were 85.3% and 82.9%, respectively (P=0.045 and 0.081),there was significantly inferior OS in CBF-AML with FLT3-ITD subgroup. Among them, the OS was 60.0% and 85.9% in RUNX1-RUNX1T1 with or without FLT3-ITD group respectively( P=0.041), there was significantly inferior OS in RUNX1-RUNX1T1 with FLT3-ITD subgroup. There were no significant differences in OS and LFS between the CBF with or without C-kit and ASXL1 genes. The overall relapse was 10.9%, and treatment related mortality was 6.9%. Conclusion: The childhood CBF-AML accounted for 30.6% of children's newly diagnosed AML in the South China Children's AML Group. The complete remission rate reached to 93.1% by using 2 courses of intensive induction regimen of DAE or FLAG-IDA. Only 2 courses of consolidation chemotherapy were administered. Two years OS and LFS were higher than 80%.There were significant difference in OS of CBF-AML with FLT3-ITD, and RUNX1-RUNX1T1 AML with FLT3-ITD. There were no significant differences in that of LFS. The overall LFS was close to the outcome of the MRC AML 15 protocol, but there was a gap in OS. The main reason was that the most of relapsed patients abandoned treatment. On other hand the treatment related mortality need to be further reduced. Figure Disclosures No relevant conflicts of interest to declare.

Abstract 1723: Elucidation of novel therapeutic targets for acute myeloid leukemias with RUNX1-RUNX1T1 fusion

Abstract The RUNX1-RUNX1T1 fusion is a frequent chromosomal alteration in acute myeloid leukemias (AMLs). Although RUNX1-RUNX1T1 fusion protein has pivotal roles in the development of AMLs with the fusion, RUNX1-RUNX1T1 fusion protein is difficult to target since it lacks kinase activities. Here, we used a sophisticated bioinformatic tool to elucidate targetable signaling pathways in AMLs with RUNX1-RUNX1T1 fusion. After analysis of 93 AMLs from the TCGA database, the expressions of 293 genes were significantly correlated with the expression of the RUNX1-RUNX1T1 fusion gene. Based on the 293 genes, the COX, VEGFR, PDGFR, and FGFR pathways are predicted to be specifically activated in AMLs with RUNX1-RUNX1T1 fusion. Compared with the AML cells without the fusion, the specific activations of the AML cell lines with RUNX1-RUNX1T1 fusion were confirmed by the increased phosphorylation of VEGFRs, PDGFRs, and FGFRs. Moreover, the in vitro proliferation of AML cells with RUNX1-RUNX1T1 fusion decreased significantly more than that of AML cells without the fusion when the pathways were inhibited pharmacologically. The results indicate that novel targetable signaling pathways could be identified by the analysis of the gene expression features of AMLs with non-targetable genetic alterations. The elucidation of specific molecular targets for AMLs that have a specific genetic alteration would promote personalized treatment of AMLs and improve treatment outcomes for AML patients in clinic. Note: This abstract was not presented at the meeting. Citation Format: Jae Won Yun, Yoon Kyung Bae, So Yeong Cho, Hee-Jin Kim, Do-Hyun Nam, Sun-Hee Kim, Sejong Chun, Kyeung Min Joo, Hye Won Lee, Woong-Yang Park. Elucidation of novel therapeutic targets for acute myeloid leukemias with RUNX1-RUNX1T1 fusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1723.

Elucidation of Novel Therapeutic Targets for Acute Myeloid Leukemias with RUNX1-RUNX1T1 Fusion.

The RUNX1-RUNX1T1 fusion is a frequent chromosomal alteration in acute myeloid leukemias (AMLs). Although RUNX1-RUNX1T1 fusion protein has pivotal roles in the development of AMLs with the fusion, RUNX1-RUNX1T1, fusion protein is difficult to target, as it lacks kinase activities. Here, we used bioinformatic tools to elucidate targetable signaling pathways in AMLs with RUNX1-RUNX1T1 fusion. After analysis of 93 AML cases from The Cancer Genome Atlas (TCGA) database, we found expression of 293 genes that correlated to the expression of the RUNX1-RUNX1T1 fusion gene. Based on these 293 genes, the cyclooxygenase (COX), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR) pathways were predicted to be specifically activated in AMLs with RUNX1-RUNX1T1 fusion. Moreover, the in vitro proliferation of AML cells with RUNX1-RUNX1T1 fusion decreased significantly more than that of AML cells without the fusion, when the pathways were inhibited pharmacologically. The results indicate that novel targetable signaling pathways could be identified by the analysis of the gene expression features of AMLs with non-targetable genetic alterations. The elucidation of specific molecular targets for AMLs that have a specific genetic alteration would promote personalized treatment of AMLs and improve clinical outcomes.

Application value of quantitative monitoring of RUNX1-RUNX1T1 fusion gene in pediatric acute myeloid leukemia

Objective To explore the prognostic value of quantitative monitoring of RUNX1-RUNX1T1 fusion gene in pediatric t(8; 21)/RUNX1-RUNX1T1 positive acute myeloid leukemia (AML). Methods A total of 81 newly diagnosed AML children with t (8; 21)/RUNX1-RUNX1T1 positive were enrolled in the People′s Hospital, Peking University, between August 2005 and January 2016.RUNX1- RUNX1T1 gene copy number of all the patients was analyzed by real-time quantitative PCR (qPCR) technology at diagnosis and after therapy in all patients.Cumulative incidence of relapse rate (CIR), event-free survival (EFS) rate and overall survival (OS) rate were estimated by Kaplan-Meier method and prognostic factors were evaluated by COX regression. Results The level of RUNX1-RUNX1T1 gene on diagnosis was used as the baseline to determine whether the level of gene after treatment had a more than 3 logarithmic (3 log) reduction.After 2 courses of induction therapy, the patients with a more than 3 log reduction of RUNX1-RUNX1T1 transcript levels(≥3 log)had better EFS rate (82.4% vs.57.6%, χ2=7.454, P<0.01), and better OS (93.6% vs.59.3%, χ2=9.703, P<0.01), compared to the patients with a less than 3 log reduction(<3 log). Multivariate analysis showed that 3 log reduction in RUNX1- RUNX1T1 transcript levels after 2 courses of induction therapy was an independent prognostic factor for EFS rate [hazard ratio(HR)=4.223, 95% confidence interval(CI): 1.507-11.836, P<0.01]and OS rate (HR=5.002, 95%CI: 1.282-19.516, P<0.05). When periodically monitoring the RUNX1-RUNX1T1 gene, 63 out of the 81 children patients were monitored for more than 6 times.The patients who had a more than 3 log reduction of gene before, but then those whose gene transcript level rose more than 1 log level were divided into group A, and the remaining patients were divided group B, and the difference of CIR was statistically significant between group A and group B (46.7% vs.4.7%, P<0.01). Conclusions RUNX1-RUNX1T1 gene copy number was detected with qPCR method in pediatric t(8; 21)/RUNX1-RUNX1T1 positive AML, which can determine the treatment effect, predict the recurrence of patients and assess long-term prognosis.Thus it has great clinical application value. Key words: Leukemia, myeloid, acute; Gene fusion, RUNX1-RUNX1T1 fusion gene; Neoplasm, residual; Survival analysis; Child

Decoding of exon splicing patterns in the human RUNX1–RUNX1T1 fusion gene

The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1–RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1–RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1–RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by “exons-hubs” of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of “exons-hubs”. Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain “attractiveness” of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1–RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process.

RUNX1T1/MTG8/ETO gene expression status in human t(8;21)(q22;q22)-positive acute myeloid leukemia cells

The RUNX1-RUNX1T1 fusion gene, a product of the nonhomologous balanced translocation t(8;21)(q22;q22), is a complex genetic locus. We performed extensive bioinformatic analysis of transcription initiation as well as transcription termination sites in this locus and predicted a number of different RUNX1T1 transcripts. To confirm and quantify the RUNX1T1 gene expression, we analyzed samples from seven acute myeloid leukemia (AML) patients and from the Kasumi-1 cell line. We found variable activity of the four predicted RUNX1T1 promoters located downstream of the chromosome breakpoint. Nineteen alternative RUNX1T1 transcripts were identified by sequencing at least seventeen of which predictably can be translated into functional proteins. While the RUNX1T1 gene is not expressed in normal hematopoietic cells, it may participate in t(8;21)(q22;q22)-dependent leukemic transformation due to its multiple interactions in cell regulatory network particularly through synergistic or antagonistic effects in relation to activity of RUNX1-RUNX1T1 fusion gene.

The prognostic impact of c-KIT mutation in systemic mastocytosis associated with acute myeloid leukaemia patients

We evaluate the haematopathologic features of systemic mastocytosis associated with acute myeloid leukaemia (SM-AML) and the prognostic role of c-KIT mutation. Total 11 patients were enrolled. Cytochemistry using toluidine blue and tryptase was positive, as was immunohistochemistry for CD117 and CD25 on clustered mast cells; however, CD2 was expressed in only nine cases. In 10 cases, RUNX1-RUNX1T1 fusion gene was detected, and one patient presented with a t(5;6)(q22;q23) translocation at diagnosis. The c-KIT mutation D816V was detected in six patients. Patients with c-KIT mutations had higher relapse and death rates than those without; 4/5 (80.0%) and 5/6 (83.3%) vs. 1/5 (20%) and 2/5 (40%), respectively. Overall survival was also significantly shorter in cases with, than those without, c-KIT mutations. To identify rare cases of SM-AML, which have a dismal prognosis, c-KIT mutation study and careful examination for the presence of clustered mast cell infiltration by immunochemistry should be performed.

Identification of a new translocation that disrupts the RUNX1 gene in a patient with de novo acute myeloid leukemia

Translocation (8;21)(q22;q22)/RUNX1-RUNX1T1 is a molecular marker that is usually associated with a favorable outcome in both pediatric and adult patients with acute myeloid leukemia (AML). The present report describes the results of hematologic, cytogenetic, and fluorescence in situ hybridization analysis of a case of AML with maturation in a 23-year-old woman. Cytogenetic analysis revealed a balanced translocation involving chromosomal band 21q22, which disrupts the RUNX1 gene, and 10q22, with the following karyotype: 45,X,-X,t(10;21)(q24;q22)[cp16]/46,XX [4]. Interphase FISH showed, in 67% of the 300 interphase nuclei analyzed, three signals for RUNX1 and two RUNX1T1, but no signals corresponding to RUNX1-RUNX1T1 fusion gene. These results were corroborated by RT-PCR, which revealed negative results for the amplification of RUNX1-RUNX1T1 fusion gene. The patient was refractory to conventional and salvage chemotherapy regimens and early relapsed after unrelated donor bone marrow transplantation (BMT), dying of pneumonia, acute respiratory failure, and sepsis on day +80 after BMT, 1 year after diagnosis.

Quantitative monitoring of single nucleotide mutations by allele-specific quantitative PCR can be used for the assessment of minimal residual disease in patients with hematological malignancies throughout their clinical course

Background Monitoring of minimal residual disease (MRD) in patients with hematological malignancies is important for evaluating the patients' therapeutic response and risk of relapse. Single nucleotide mutations associated with leukemogenesis can be considered as applicable MRD markers. Methods We developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) for FLT3 2503G > T, KIT 2446G > T, and KIT 2447A > T and compared the change in the expression levels of the FLT3 or KIT mutations assessed by AS-qPCR to those of the RUNX1–RUNX1T1 fusion gene and WT1 by conventional quantitative PCR. Results The AS-qPCR using primers including template-mismatched nucleotide or template-mismatched nucleotide plus locked nucleic acid substituted nucleotide provided higher selectivity for mutant nucleotides. The change in the expression levels of the FLT3 or KIT mutations at the time of relapse and just after hematopoietic stem cell transplantation correlated well with that of the RUNX1–RUNX1T1 fusion gene and WT1. Moreover, during complete remission, only AS-qPCR could detect low-level expression of residual mutations. Conclusions The AS-qPCR for analyzing single nucleotide mutations contributes to the monitoring of MRD in patients without recurrent fusion gene throughout the clinical course and thus broadens the spectrum of patients in whom MRD can be monitored.