Lijun Wen

ETV6 mutation in a cohort of 970 patients with hematologic malignancies

The ETV6 gene (previously known as TEL) belongs to the ETS (E26 transformation specific) family of transcription factors characterized by 2 important domains: the C-terminal Ets domain responsible for specific DNA-binding activities and the N-terminal helix–loop–helix (HLH) oligomerization

T-SPOT.TB for Detection of Tuberculosis Infection among Hematological Malignancy Patients and Hematopoietic Stem Cell Transplant Recipients

The diagnosis of latent Mycobacterium tuberculosis infection (LTBI) is recommended in hematological malignancy patients and before hematopoietic stem cell transplantation (Guidelines for the prevention and management of infectious complications of solid organ transplantation, 2004). Compared to traditional methods such as tuberculin skin test (TST), T-SPOT.TB has been shown to be more specific. In the present study we enrolled 536 patients for whom T-SPOT.TB was performed, among which 295 patients also received the TST test. The agreement (79%) between T-SPOT.TB and TST was poor (?=0.274, P<0.001). The patients with positive T-SPOT.TB results numbered 62 (11.6%), in which only 20 (48.8%) of the 41 receiving the TST test had positive results. A majority of the patients with T-SPOT.TB positive results had some other evidence ofTB, such as TB history, clinical symptoms and an abnormal chest CT scan. Active TB was found in 9 patients, in which 2 had negative TST results. We followed up the patients and no one developed active TB. Our study suggested that the T-SPOT.TB may be more useful for screening LTBI and active TB in hematological malignancy patients and hematopoietic stem cell transplant recipients than the TST test.

High frequency of BTG1 deletions in patients with BCR-ABL1–positive acute leukemia

Deletions affecting the B-cell translocation gene 1 (BTG1) have recently been reported in 9% of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and occur even more frequently in ETV6-RUNX1-positive and BCR-ABL1-positive subgroups. To investigate whether the BTG1 deletions occur in other BCR-ABL1-positive acute leukemias besides BCP-ALL, we analyzed 44 leukemia cases harboring the BCR-ABL1 transcript [32 BCP-ALL, six mixed-phenotype acute leukemia (MPAL), and six chronic myeloid leukemia in B-lineage blast crisis (CML-BC)] by array-based comparative genomic hybridization and reverse transcription-PCR. BTG1 deletions were present in 31.8% of BCR-ABL1-positive acute leukemia patients, including 31.3% of BCP-ALL (10/32), 33.3% of MPAL (2/6), and 33.3% of CML-BC (B-lineage) (2/6) patients. Of note, the intragenic deletion breakpoints, mapping to 5 different positions at the proximal end of the breakpoint, clustered tightly within exon 2 of BTG1, which were located within a stretch of 20 bp from nucleotide 284 to nucleotide 304 and led to truncated BTG1 transcripts. There were no significant differences in the median white blood cell count, hemoglobin concentration, platelet count, bone marrow blast count, sex, age, or overall complete remission rate between patients with and without BTG1 deletions. Taken together, our data suggest that BTG1 deletions might play a role in leukemogenesis of BCP-ALL as well as of BCR-ABL1-positive MPAL and CML-BC (B-lineage).

Transcriptome sequencing reveals CHD1 as a novel fusion partner of RUNX1 in acute myeloid leukemia with t(5;21)(q21;q22)

BackgroundRUNX1/AML1, which is a Runt family transcription factor critical for normal hematopoiesis, is frequently mutated or translocated in a broad spectrum of hematopoietic malignancies.FindingsWe describe here the case of a 54-year-old female developed acute myeloid leukemia with a t(5;21)(q21;q22). Transcriptome sequencing identified the chromodomain-helicase-DNA-binding protein 1 gene, CHD1, as a novel partner gene of RUNX1. Furthermore, the patient was found to harbor FLT3-ITD mutation, which might collaborated with CHD1-RUNX1 in the development of acute myeloid leukemia.ConclusionsWe have identified CHD1 as the RUNX1 fusion partner in acute myeloid leukemia with t(5;21)(q21;q22).

ASXL1 mutations are frequent in de novo AML with trisomy 8 and confer an unfavorable prognosis

Acute myeloid leukemia (AML) is a heterogeneous group of genetically diverse hematopoietic malignancies with variable response to treatment.[1] Among the clinical and laboratory characteristics which influence the response to treatment and the survival of patients, cytogenetics at diagnosis represents the most important prognostic variable. Trisomy 8 is one of the most frequent recurring numerical chromosome aberrations in AML, occurring at a frequency of 10%.[2] It presents either as the sole cytogenetic abnormality or in combination with a large variety of different cytogenetic abnormalities. Patients with sole trisomy 8 are mostly classified as having an intermediate prognosis.[3,4] Additional sex combs like-1 (ASXL1) gene, encodes a highly conserved protein that belongs to the enhancer of trithorax and polycombgenes, a gene family with dual functions in both epigenetic activation and repression of gene transcription.[5] Frameshift and stop mutations were found that were predicted to lead to loss of the carboxyterminal plant homeodomain finger on the protein level.[6] ASXL1 mutations (ASXL1-mut) have been detected in about 6–30% of AML.[7–10] Recent reports have demonstrated that ASXL1-mut are more frequent in patients with aberrant karyotype especially in cases with trisomy 8.[11,12] These suggested that ASXL1-mut might play an important role in the patients with trisomy 8. However, the roles of ASXL1-mut in these patients are still unclear. Here, we investigated the occurrence and prognostic impact of ASXL1-mut in AML patients with trisomy 8 aberration. A total of 78 patients with trisomy 8 as the sole aberration or accompanied with other anomalies enrolled from 2007 to 2013 in Jiangsu Institute of Hematology were retrospectively analyzed in this study which was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University following Declaration of Helsinki. The median age was 43 years (12–84 years), and the cohort was all Chinese with the majority of male (51.3%). Historically, analyses have divided patients into favorable, intermediate, and unfavorable cytogenetic groups.[13,14] The same classification scheme was used for this analyses. Here, favorable subgroup consisted of 9 cases with trisomy 8 in combination with t(15;17) or t(8;21), the intermediate group comprised of 41 cases with trisomy 8 as the sole aberration or accompanied with one anomaly which did not include the favorable cytogenetic abnormalities, and the unfavorable category included 28 patients with complex (two or more) aberrations. Only patients of de novo AML with trisomy 8 who received intensive treatment were included into the prognostic analyses. Chromosome analyses were performed according to standard protocols using R-banding. The chromosomes were classified according to the International System for Human Cytogenetic Nomenclature (ISCN, 2005). Genomic DNA was prepared from purified fraction of mononuclear cells after Ficoll density centrifugation. Then PCR amplification of the entire coding region of ASXL1 exon 12 was performed followed by direct bidirectional DNA sequencing. The positive mutation samples were analyzed twice to confirm the results. Only frameshift and nonsense mutations of ASXL1 were analyzed in this study. All statistical analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL). Comparisons of continuous and dichotomous variables were based on the Kruskal–Wallis test and chi-square test respectively. Survival curves were calculated for overall survival (OS) and event-free survival (EFS) according to Kaplan–Meier. Comparisons between groups were based on the twosided log-rank test. Statistically significant tests were identified with p value less than 0.05. The frequency of ASXL1-mut was identified in 19.23% (15/78) of AML patients with trisomy 8 as the sole aberration or accompanied with other anomalies by sequencing

MLL-SEPT5 Fusion Transcript in Two de novo Acute Myeloid Leukemia Patients With t(11;22)(q23;q11)

Dear Editor, Rearrangements involving mixed lineage leukemia (MLL) are common chromosome aberrations in infant, pediatric and adult acute leukemia, which are generally associated with poor prognosis. To date, more than 79 partner genes have been identified [1]. MLL fusion partners can be classified into four groups: nuclear proteins (MLLT3, MLLT10, and MLLT1), cytoplasmatic proteins (GAS7, SH3GL1, EPS15, and MLLT4), histone acetyltransferases (EP300, CREBBP), and the septin family (SEPT2, SEPT5, and SEPT6) [2]. In pediatric and adult AML, the most frequent fusion partners are represented by MLLT3-AF9 (9p22), MLLT10-AF10 (10p12), ELL (19p13.1), MLLT4-AF6 (6q27), and MLLT1-ENL (19p13.3) [1, 3]. However, as a partner gene, the SEPT5 gene has been reported in only five AML cases (Table 1). Here we present two cases of de novo AML with MLL-SEPT5 transcript. Chromosomal analysis revealed a karyotype of 46, XX/XY, t(11;22)(q23;q11.2). Reverse transcription polymerase chain reaction (RT-PCR) analysis indicated an MLL-SEPT5 fusion transcript. Sanger sequencing of the PCR product confirmed the fusion between MLL and SEPT5. Notably, a new fusion transcript between MLL exon 8 and SEPT5 intron 2 was detected in one of the patients. Patient 1 was a 21-yr-old man who presented with fever, oral ulcer, and tonsillitis. His hemoglobin level was 8.5 g/dL, white blood cell count was 35.45 ×10/L, and platelet count was 29×10/L. Bone marrow aspiration was hypercellular, containing 81% blasts. Immunophenotypic analysis showed that the blasts were positive for CD45, CD33, CD117, CD15, and HLADR, but negative for other markers. Cytogenetic studies revealed the karyotype of 46, XY, t(11;22)(q23;q11.2) [8]/46,XY [2]. Fluorescence in situ hybridization analysis using a MLL-specific probe showed a split in the MLL gene (Fig. 1). He received three courses of induction chemotherapy in our hospital followed by a mother-to-son haploidentical bone marrow transplantation. To date, he is in complete remission. To validate MLL-SEPT5 existence, we performed RT-PCR and direct DNA sequencing. Total RNA was extracted from the bone marrow cells using TRIzol reagent (Invitrogen, Paisley, UK) and chloroform. cDNA was obtained by RT-PCR using the M-MLV reverse transcriptase (Promega, Madison, WI, USA). The resulting cDNA was amplified by PCR using 2×Hieff PCR Master Mix (Yaeson, Shanghai, China) and the following primers: 5 -GCTCCACCCATCAAACCAAT-3 from exon 5 of MLL and 5 -TTCTTCTCAATGTCCACCGT-3 from exon 4 of SEPT5 (Fig. 2). PCR products were sequenced on both strands by using an Applied Biosystems ABI 3730 XL DNA analyzer (Thermo Fisher Scientific,

Inhibitory effect of magnetic Fe 3 O 4 nanoparticles coloaded with homoharringtonine on human leukemia cells in vivo and in vitro

Homoharringtonine (HHT), a natural cephalotaxine alkaloid, has been used in the People’s Republic of China for treatment of leukemia for >3 decades. Here, we employed magnetic Fe3O4 nanoparticles (MNP-Fe3O4) to improve the therapeutic effect of HHT and investigated its biological effects. Within a certain range of concentrations, the HHT-MNP-Fe3O4 showed a more enhanced inhibitory effect on the selected myeloid leukemia cell lines than HHT alone. Compared with HHT, HHT-MNP-Fe3O4 could induce more extensive apoptosis in leukemia cells, which also showed more pronounced cell arrests at G0/G1 phase. HHT-MNP-Fe3O4 enhanced antitumor activity by downregulating myeloid cell leukemia-1, which could inhibit the activation of caspase-3 and poly-ADP-ribose polymerase. In vivo experiments using tumor-bearing animal models showed that the mean tumor volume with HHT-MNP-Fe3O4 was significantly smaller than that with HHT alone (193±26 mm3 versus 457±100 mm3, P<0.05), while the mean weight was 0.67±0.03 g versus 1.42±0.56 g (P<0.05). Immunohistochemical study showed fewer myeloid cell leukemia-1-stained cells in mice treated with HHT-MNP-Fe3O4 than with the controls. These findings provide a more efficient delivery system for HHT in the treatment of hematological malignancy.

JAK2V617F allele burden in patients with myeloproliferative neoplasms carrying Trisomy 9, and its relationship with clinical phenotypes

ET to PV to MF [1]. Previous reports have suggested that in ET and PV, a higher allele burden is associated with adverse clinical phenotype, disease complications and progression to MF [2]. Trisomy 9 and gains on 9p are among the most frequent cytogenetic abnormalities in both PV and ET, together with trisomy 8, del(20q) and del(13q). Rice et al. demonstrated that patients (3/32 patients with PV, 2/24 with PMF and 1/31 with ET) with trisomy 9 showed a tendency to elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage [3]. Here, we investigated a total of 1,361 patients diagnosed with BCR-ABL1 negative MPNs in our division from June 2009 to April 2015; diagnosis was established following the 2008 WHO criteria. Nine patients (3/501 patients with PV, 5/1035 with ET and 1/100 with PMF) were detected an abnormal karyotype of trisomy 9, showing all JAK2V617F mutation positive. To study the variation tendency of JAK2V617F allele burden in patients with MPNs carrying trisomy 9, another 65 patients with normal karyotype and JAK2V617F mutation (21 patients with PV and 44 patients with ET) were retrospectively analyzed in this study. The diagnosis, clinical data and cytogenetic analyses of the patients with trisomy 9 are listed in Supplementary Table 1. Patients with trisomy 9 had significantly higher WBC count (19.05 × 10/L vs 11.53 × 10/L, p < 0.001). There was no significant differences in terms of thrombotic complications and splenomegaly among the two groups (Supplementary Table 3a). The present study was approved by the ethics committee of the First Affiliated Hospital of Soochow University, following the Declaration of Helsinki. To study changes of the allele burden of JAK2V617F in MPN patients with trisomy 9, a real-time quantitative polymerase chain reaction testing for JAK2V617F allele burden was performed on genomic DNA of these Polycythemia Vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are BCR-ABL1 negative myeloproliferative neoplasms (MPNs) characterized by mutually exclusive Janus kinase 2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia virus oncogene (MPL) mutations. JAK2V617F is the most frequent mutation, with frequencies of approximately 95–97 % in PV, 50–60 % in ET, and 55–65 % in PMF. The somatic mutation of JAK2V617F leading to a valine → phenylalanine change at position 617 in the JH2 pseudokinase domain, results in constitutive activation of the JAK-STAT pathway. This in turn leads to proliferation of hematopoietic cells. JAK2 is located in chromosome 9p24.1. JAK2V617F can be present in a heterozygous or a progressively homozygous state, the latter is most frequently driven by mitotic homologous recombination between the JAK2-WT and JAK2V617F alleles, resulting in 9p uniparental disomy (9pUPD). Previous studies have demonstrated that 9pUPD is an important route to duplication of the JAK2V617F mutant allele [1]. Homozygosity for this mutation was reported with increasing frequency in the continuum from

Clinical and molecular features of acute promyelocytic leukemia with variant retinoid acid receptor fusions.

Acute promyelocytic leukemia (APL) is a unique disease entity in acute myeloid leukemia (AML), characterized by the expansion of leukemic cell block at the promyelocytic stage. The vast majority of APL patients bear t(15;17)(q24;q21) involving the promyelocytic leukemia ( PML ) gene at chromosome

Identification of novel recurrent CPSF6-RARG fusions in acute myeloid leukemia resembling acute promyelocytic leukemia

TO THE EDITOR: Retinoic acid receptor γ (RARG) is a member of the nuclear receptor superfamily and shares high homology (90%) with retinoic acid receptor α (RARA) and retinoic acid receptor β (RARB).[1][1] So far, little is known about RARB or RARG fusion. Such et al reported the first case of