Hongwei Wang

MDM4 Overexpressed in Acute Myeloid Leukemia Patients with Complex Karyotype and Wild-Type TP53

Acute myeloid leukemia patients with complex karyotype (CK-AML) account for approximately 10–15% of adult AML cases, and are often associated with a poor prognosis. Except for about 70% of CK-AML patients with biallelic inactivation of TP53, the leukemogenic mechanism in the nearly 30% of CK-AML patients with wild-type TP53 has remained elusive. In this study, 15 cases with complex karyotype and wild-type TP53 were screened out of 140 de novo AML patients and the expression levels of MDM4, a main negative regulator of p53-signaling pathway, were detected. We ruled out mutations in genes associated with a poor prognosis of CK-AML, including RUNX1 or FLT3-ITD. The mRNA expression levels of the full-length of MDM4 (MDM4FL) and short isoform MDM4 (MDM4S) were elevated in CK-AML relative to normal karyotype AML (NK-AML) patients. We also explored the impact of MDM4 overexpression on the cell cycle, cell proliferation and the spindle checkpoint of HepG2 cells, which is a human cancer cell line with normal MDM4 and TP53 expression. The mitotic index and the expression of p21, BubR1 and Securin were all reduced following Nocodazole treatment. Moreover, karyotype analysis showed that MDM4 overexpression might lead to aneuploidy or polyploidy. These results suggest that MDM4 overexpression is related to CK-AML with wild-type TP53 and might play a pathogenic role by inhibiting p53-signal pathway.

The short isoform of the long-type PML-RARA fusion gene in acute promyelocytic leukaemia lacks sensitivity to all-trans-retinoic acid.

Alternative splicing is associated with human disease. In acute promyelocytic leukaemia (APL) patients with the long (L)‐type promyelocytic leukaemia‐retinoic acid receptor α fusion gene (PML‐RARA), three alternative splicing isoforms can be detected: E5(+)E6(+), E5(−)E6(+), and E5(−)E6(−). This study is the first to demonstrate that alternative splicing of L‐type PML‐RARA is associated with time to achieve complete remission (CR) in APL. Higher expression of the E5(−)E6(−) isoform, the short isoform, was related to longer time to achieve CR. Each isoform was constructed into recombinant lentiviral vector and transfected into U937 cells. Compared with the E5(−)E6(+) and E5(+)E6(+) groups, the U937 cells with E5(−)E6(−) showed lower sensitivity to all‐trans‐retinoic acid treatment.

Detection of MPL exon10 mutations in 103 Chinese patients with JAK2V617F-negative myeloproliferative neoplasms

JAK2V617F mutation has been reported in 90% of patients with polycythemia vera (PV) and about 50% of patients with essential thromobocythemia (ET) and primary myelofibrosis (PMF). Recently, acquired mutations in the transmembrane-juxtamembrane region of MPL (MPLW515 mutations) have been reported in approximately 5% of JAK2V617F-negative PMF and about 1% of all cases of ET. MPL is the receptor for thrombopoietin that regulates the production of platelets by bone marrow. It is likely that some mutations more closely related to ET in MPL exon10 may have been missed by current assays. We inferred that there might be other mutations in MPL exon10 for MPN patients in addition to MPLW515 mutations. To investigate its mutation types and prevalence in Chinese patients with myeloproliferative neoplasms (MPN), we performed mutation detection on MPL exon10 in 103 JAK2V617F-negative MPN patients by single strand conformation polymorphism (SSCP) and allele-specific PCR (AS-PCR) combined with sequencing. As a result, one previously unrecognized MPL mutation (12-bp in-frame insertion) was identified in one patient with ET in addition to an MPLW515K mutation identified in one PMF patient. This confirms our hypothesis that BCR/ABL negative and JAK2V617F-negative MPN patients have other mutations besides W515 mutation in MPL exon10 and mutations other than single nucleotide exchange also exist. In addition, MPL mutation was associated with Chinese MPN patients.

Identification of a novel circularized transcript of the AML1 gene.

The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outwardfacing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription. [BMB Reports 2013; 46(3): 163-168]

Droplet digital PCR for BCR/ABL(P210) detection of chronic myeloid leukemia: A high sensitive method of the minimal residual disease and disease progression

This study intended to establish a droplet digital PCR (dd‐PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)‐positive chronic myeloid leukemia (CML), thereby achieving deep‐level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment.

Detection of Promyelocytic Leukemia/Retinoic Acid Receptor α (PML/RARα) Fusion Gene with Functionalized Graphene Oxide

An attempt was made to use functionalized graphene oxide (GO) to detect the Promyelocytic leukemia/Retinoic acid receptor α fusion gene (PML/RARα fusion gene), a marker gene of acute promyelocytic leukemia. The functionalized GO was prepared by chemical exfoliation method, followed by a polyethylene glycol grafting. It is found that the functionalized GO can selectively adsorb the fluorescein isothiocyanate (FITC)-labeled single-stranded DNA probe and quench its fluorescence. The probe can be displaced by the PML/RARα fusion gene to restore the fluorescence, which can be detected by laser confocal microscopy and flow cytometry. These can be used to detect the presence of the PML/RARα fusion gene. This detection method is verified to be fast, simple and reliable.

Monitoring of clonal evolution of double C-KIT exon 17 mutations by Droplet Digital PCR in patients with core-binding factor acute myeloid leukemia

C-KIT gene mutations result in the constitutive activation of tyrosine kinase activity, and greatly affect the pathogenesis and prognosis of core-binding factor acute myeloid leukemia (CBF-AML). C-KIT mutations are often found as single point mutations. However, the rate of double mutations has recently increased in AML patients. In this study, we detected six cases (18.8%) harboring double C-KIT exon17 mutations in 75 patients with CBF-AML. The clone composition and dynamic evolution were analyzed by sequencing and droplet digital PCR (ddPCR). Results revealed that these double mutations can be occurred in either the same or different clones. Different clones of double mutations may result in different sensitivity to the treatment of CBF-AML. The clones with N822 mutation responded better to treatment as compared to those with D816 mutation. Moreover, D816 clone was readily transformed into a predominant clone at relapse. Meanwhile, the predominant clones in the same patient may change during the progression of disease. The emerging mutation can originate from a small quantity of clones at diagnosis or newly acquired during the course of disease. Furthermore, patients with double mutations had better overall survival (OS) and event-free survival (EFS) than those with single mutation, but the differences did not reach statistical significance (P > 0.05). The ddPCR is an effective method for monitoring clonal evolution in patients with CBF-AML.

The CD9+CD11b−HLA-DR− immunophenotype can be used to diagnose acute promyelocytic leukemia

To investigate the immunophenotypic characteristics of acute promyelocytic leukemia (APL) and explore the sensitivity and specificity of various antibody combinations for the timely and accurate diagnosis APL.

Clinical significance of droplet digital PCR quantitative monitoring of KIT gene mutation levels in core binding factor leukemia

Sir, The core binding factor acute myeloid leukemia (CBFAML) is the combination of t(8;21)(q22;q22) and inv(16)(p13;q22), which accounts for approximately 5%12% of AML.1 CBFAML patients with KIT gene mutation (CBFAMLCK) (30%50%)have a high likelihood of relapse (RP) after a morphological complete remission (CR), mortality, death rate, and diseasefree survival time, prognosis difference.2-4 However, CBFAMLCK patients have different clinical manifestations after CR, the level of minimal residual disease (MRD) when the patient reaches CR is an important factor that affects the prognosis of the patient independent of age, diagnostic hematology, or molecular genetics.5 At present, monitoring the MRD level of CBFAMLCK patients is mainly conducted using realtime fluorescent quantitative PCR(RTqPCR) to detect the transcription levels of the fusion genes,6,7 but some patients with fusion genes showed no residue but experienced RP. Hence, we explored whether KIT can be used as a valuable MRD detection index. In this study, we used a high sensitive, repeatability, precision, specific, and objective method–Droplet digital PCR(ddPCR) to absolute quantitatively detect and monitor the KIT gene mutation in 20 CBFAMLCK patients to explore the prognostic significance of the KIT gene mutation level as an MRD index. Twenty CBFAMLCK patients with CR were admitted in our hospital from August 2012 to January 2017 and were followed up for 15 months (645 months).There are 12 cases were KIT D816V(c.2447A>T), 3 cases were KIT D816Y (c.2446G>T) and 5 cases were KIT N822K (c.2466 T>G). All patients diagnosed according to the WHO hematopoietic and lymphoid tissue tumor classification criteria and were given standard treatment and achieved CR. All patients provided written informed consent for treatment and research use of their specimens, in accordance with the Declaration of Helsinki.Then, we collected all patients’ bone marrow from every review and extracted the genomic DNA and RNA. DNA template was used for ddPCR and automated analysis the KIT mutation by using the QX200 droplet reader. A droplet number of ≥3 indicated a positive channel test. The KIT mutation level was computed as mt/(mt + wt) × 100%, and results were expressed as copies/μL. Simultaneously, RNA template was used for RTqPCR and analysis the fusion gene by using the ABI 7500. A patient was considered to have achieved molecular remission if the degree of transcript reduction was ≥3 log (a 1000folds transcripts decrease). The fusion gene level was expressed as fusion gene/ABL × 100%. The repeated samples took the average value. Statistical analysis was performed using SPSS19.0 and GraphPad Prism 5.0. Correlation analysis of the two groups was conducted using Spearman correlation analysis or Pearson correlation analysis. The measurement data were inspected using Student’s t test and nonparametric Mann–Whitney U test. χ2 test and Fisher’s exact probability method were used for counting data. The logrank test was used to compare Kaplan–Meier curves of the OS and EFS. Statistical significance was considered at P < 0.05.8 The recurrence rate, survival rate, CRD, EFS, and OS were evaluated. The KIT gene mutation level at CR was 0%–46.54264% (median value: 0.17457%). Six patients sustained hematological remission after chemotherapy; the KIT gene mutation level was 0.07324% ± 0.32116%. The remaining 14 patients relapsed; the KIT gene mutation level was 8.79605% ± 4.44686%. The difference between the two groups was statistically significant (Figure 1). At CR, the correlation coefficient of the KIT gene mutation level and corresponding fusion gene expression level was 0.618. The correlation between the two indexes was statistically significant (correlation coefficient = 0.618, P = 0.004). In CR, the correlation coefficient between the KIT gene mutation level and OS, EFS, and CRD was higher than the corresponding fusion gene expression level, and the accuracy of recurrence was predicted by comparing the two indexes with the work characteristic curve (ROC curve) of the subjects. The AUC values of the KIT gene mutation level and fusion gene expression level were 0.845 and 0.411 respectively (Figure 2).

Identification of a novel fusion gene, RUNX1-PRPF38A , in acute myeloid leukemia

Sir, acute myeloid leukemia (AML) is a group of hematologic malignancies originated from the malignant clone of the hematopoietic stem cell (HSC). Some malignant transformation of HSC derived from the fusion genes was initiated by chromosomal translocations. The RUNX1 gene (also known as AML1 or CBFa2) is one of the most frequent targets of chromosomal translocations in human acute leukemia. Genetic translocation alterations involving the RUNX1 gene represent critical events in the development of acute leukemia. Located at chromosome band 21q22, RUNX1 gene encodes a transcription factor that heterodimerizes with the human core binding factor (CBF) b protein to form a CBF complex which regulates a number of genes important for hematopoiesis, such as interleukin-3, granulocyte–macrophage colony-stimulating factor, macrophage colony-stimulating factor, myeloperoxidase, neutrophil elastase, and subunits of Tcell antigen receptors. At least three different isoforms of RUNX1 gene have been described, including AML1a, AML1b, and AML1c [1]. Proteins AML1b and AML1c have an evolutionarily conserved runt homology domain (RHD) at the N-terminus and a transactivation domain (TA) at the C-terminus. RHD interacts with several hematopoietic factors. TA directly interacts with co-activators (CBP/p300, MOZ) and co-repressors (Sin3A) modulating the transcription activity of RUNX1 gene [2]. By contrast, the protein AML1a exhibits RHD but lacks the TA domain. The function of RUNX1 is hypothesized to be mediated by AML1b and AML1c, which have the same function. To date, more than 20 distinct RUNX1 gene fusions have been reported in hematologic malignancies. The presence of the RUNX1-RUNX1T1 (also known as AML1ETO) chimeric product resulting from t(8;21) defines patients as AML with t(8;21)(q22;q22); RUNX1RUNX1T1 is observed in approximately 12–15% of cases of AML[3]. In addition, a number of fusion partners of RUNX1 have been cloned, including the following: YTHDF2 at 1p35, ZNF687 at 1q21.2, MDS1/EVI1 at 3q26, FGA7 at 4q28, SH3D19 at 4q31.3, USP42 at 7p22, FOG2 at 8q23, TRPS1 at 8q24, TEL (ETV6) at 12p13, MTG16 at 16q24 and PRDX4 at Xp22, and HMGB2 at 4q31 [4, 5]. It is noted that 50RUNX1-30USP42 can occur through an insertion mechanism rather than translocation [6]. In the present study, a novel chromosomal translocation, t (1;21)(p32;q22), was identified in a 57-year-old woman with de novo AML using banding cytogenetic analysis and 30 rapid amplification of cDNA ends’ analysis in this study. Existence of fusion transcripts was further confirmed by reverse transcription–polymerase chain reaction and sequencing analysis. The novel partner gene of RUNX1 located at 1p33-p32.1 showed significant homology with the known gene PRPF38A. The predicted RUNX1-PRPF38A chimeric protein contained an N-terminal half of RUNX1, including a RHD fused to the 50 noncoding region of PRPF38A gene. Based on existing literature, chromosome rearrangements involving the RUNX1 locus and a locus on the short arm of chromosome 1 at 1p32 have been described in three other patients with AML [7–9]. The 57-year-old woman with acute leukemia was admitted to the Second Hospital of Shanxi Medical University. Complete blood count showed the following results: hemoglobin, 74 g/L; platelets, 66 9 10; and total leukocyte count, 30 9 10 with 95% blast cells. BM was hypercellular with 90% blast cells. Immunophenotype analysis revealed positivity for CD33, CD13, HLA-DR, CD7, and cMPO, as well as negativity for cCD3, CD19, CD117, CD34, CD14, CD15, and CD11b and CBFb/MYH11 ( ), RUNX1-RUNX1T1 ( ). A trypsin–Giemsa banding technique was performed on unstimulated BM cells cultured for 24 h according to the standard procedures, which showed a reciprocal translocation between chromosomes 1 and 21. The patient was diagnosed with AML, not otherwise specified, acute myelomonocytic leukemia, and given MA (MIT, Ara C) protocol. Hematological remission was not achieved after one course of induction chemotherapy. The patient was released from the hospital and died 8 months after diagnosis. Written informed consent was obtained from the patient, and the study obtained approval at the hospital’s ethics committee.