The aim of this study was to investigate the nucleotide sequence of one distinct fusion transcript of sil-tal1 in childhood T-ALL. The PCR product was cloned into plasmid vector and then sequenced. Genomic DNA was analyzed with PCR using the designed primer pairs representing distinct sequences. The product was sequenced and analyzed with database. The results indicated that 4 different fusion transcripts were detected at cDNA level, in which a part of exons or introns of sil are reserved respectively, and some additions and deletions existed. After analyzing genomic DNA sequence of leukemic cells, the breakpoint in gene sil of this case was proved to be different at DNA level from references. Hence, the sil-tal1 rearrangement was defined to be a new type. It is concluded tal1 rearrangement of leukemic cells in this case is a new type, which expresses classical and at least 3 variant fusion transcripts, presumably caused by extraordinary mechanisms of splicing and transcription in leukemic stem cells.
Base Sequence ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Child ; DNA Breaks ; DNA, Neoplasm ; genetics ; Humans ; Leukemia-Lymphoma, Adult T-Cell ; genetics ; Male ; Oncogene Proteins, Fusion ; genetics ; Proto-Oncogene Proteins ; genetics ; T-Cell Acute Lymphocytic Leukemia Protein 1 ; Transcription Factors

Objective: To assess the clinical characteristics and prognosis of patients with SIL-TAL1-positive T-cell acute lymphoblastic leukemia (T-ALL) . Methods: The clinical data of 19 SIL-TAL1-positive T-ALL patients admitted to the First Affiliated Hospital of Soochow University between January 2014 and February 2022 were retrospectively computed and contrasted with SIL-TAL1-negative T-ALL patients. Results: The median age of the 19 SIL-TAL1-positive T-ALL patients was 15 (7 to 41 years) , including 16 males (84.2%) . SIL-TAL1-positive T-ALL patients had younger age, higher WBC, and hemoglobin compared with SIL-TAL1-negative T-ALL patients. There was no discrepancy in gender distribution, PLT, chromosome abnormality distribution, immunophenotyping, and complete remission (CR) rate. The 3-year overall survival (OS) was 60.9% and 74.4%, respectively (HR=2.070, P=0.071) . The 3-year relapse-free survival (RFS) was 49.2% and 70.6%, respectively (HR=2.275, P=0.040) . The 3-year RFS rate of SIL-TAL1-positive T-ALL patients was considerably lower than SIL-TAL1-negative T-ALL patients. Conclusion: SIL-TAL1-positive T-ALL patients were connected to younger age, higher WBC, higher HGB, and poor outcome.
Adolescent ; Adult ; Humans ; Male ; Young Adult ; Chromosome Aberrations ; Oncogene Proteins, Fusion/genetics* ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; Prognosis ; Recurrence ; Retrospective Studies ; T-Cell Acute Lymphocytic Leukemia Protein 1/genetics* ; T-Lymphocytes ; Female ; Child

Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Chromosome Aberrations ; DNA Nucleotidylexotransferase ; metabolism ; Humans ; Immunoglobulin Light Chains ; metabolism ; Immunoglobulin Light Chains, Surrogate ; Lymphoma, B-Cell ; genetics ; metabolism ; pathology ; Lymphoma, T-Cell ; genetics ; metabolism ; pathology ; Membrane Glycoproteins ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins ; metabolism ; T-Cell Acute Lymphocytic Leukemia Protein 1

OBJECTIVEHematologic relapse remains the greatest obstacle to the cure of acute lymphoblastic leukemia (ALL), especially T-lineage acute lymphoblastic leukemia (T-ALL) in children. Recent studies have shown that patients with increased risk of relapse can be identified by measuring residual leukemic cells, called minimal residual disease (MRD), during clinical remission. Current polymerase chain reaction (PCR) methods, however, for measuring MRD are cumbersome and time-consuming. To improve and simplify MRD assessment, the author developed a real-time quantitative PCR (RQ-PCR) assay for the detection of leukemic cells that harbor the tal-1 deletion. In addition, the author discussed the significance of MRD levels at different stages in treatment and prognosis of children with T-ALL.

METHODSA total of 50 consecutively enrolled patients with T-ALL were analysed for detection of leukemic cells harboring the most common tal-1 deletion. Serial dilutions of leukemic DNA were studied to find the sensitivity of detection with RQ-PCR assay. The MRD of 28 samples in clinical remission from 10 patients were quantified by RQ-PCR assay and limiting dilution assay. The results detected by both methods were compared statistically with correlation analysis.

RESULTS(1) A total of 10 patients presented tal-1 deletion involving the sildb1 breakpoint rearranged to tal1db1 in 50 cases with T-ALL. The breakpoints of relapsed samples are the same as those of the corresponding diagnostic samples; (2) The RQ-PCR assay had a sensitivity of detection of one leukemic cell among 100,000 normal cells. In 24 samples, MRD levels > 10(-5) could be detected with both methods. The percentages of leukemic cells measured by the two methods correlated well (r = 0.898, P < 0.001); (3) The MRD levels of 3 patients out of the 8 cases undergoing disciplinary regimen were over 10(-4) at the end of induction chemotherapy. They all relapsed in bone marrow during chemotherapy. The higher the MRD levels, the earlier the relapse. The other 5 patients with MRD levels < 10(-4) had been relapse-free survival (RFS) for 4-59 months, one of whom with increased MRD levels > 10(-4) for twice at the continuation stage had been RFS for 27 months till now.

CONCLUSIONSThe sildb1-taldb1 deletion presents in 20% of T-ALL, and is an ideal PCR marker for its specificity, uniform and stability; The tal-1 RQ-PCR can be used for the rapidly, sensitively and accurately quantitative assessment of MRD in T-ALL with the tal-1 deletion. MRD levels at different stages of chemotherapy have different significance in prognosis and treatment.

Adolescent ; Base Sequence ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Child ; Child, Preschool ; Female ; Gene Deletion ; Humans ; Male ; Molecular Sequence Data ; Neoplasm, Residual ; diagnosis ; Polymerase Chain Reaction ; methods ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; genetics ; mortality ; Prognosis ; Proto-Oncogene Proteins ; genetics ; T-Cell Acute Lymphocytic Leukemia Protein 1

OBJECTIVETo characterize the time course of spontaneous differentiation of in vitro cultured human embryonic stem cells (hESCs) into hematopoietic cells to provide experimental evidence for induction of hematopoietic commitment of hESCs.

METHODSIn human embryoid bodies (hEBs) derived from spontaneous differentiation of chESC3, a hESC cell line we established previously, the expressions of such genes as KDR, Bmi1, Scl and gata2 were detected by RT-PCR every other day during the 12-day differentiation to monitor the process of the hematopoiesis. The hematopoietic stem cell marker CD34 was examined using flow cytometry to evaluate the efficiency of hematopoietic differentiation of the cells on days 6, 8, 10 and 12. The spontaneously differentiated hESCs were seeded in the hematopoietic colony culture system to study the hematopoietic colony forming ability. Immunocytochemical staining for CD45 was performed on the hEBs to examine the emergence of mature hematopoietic cells.

RESULTSThe expressions of the hematopoietic stem cell-related genes KDR and Bmi-1 were detected in the hESCs, and on days 4 to 6, the two genes were upregulated with prolonged cuture of the hEBs. Scl and gata2 gene expressions were detected since 6-8 days of culture and maintained high expressions till day 12. Flow cytometry revealed a gradual increase in CD34-positive cells in the culture, with positivity rates on days 6, 8, 10, and 12 of (1.4-/+0.4)%, (3.4-/+1.3)%, (5.5-/+2.2)%, and (5.1-/+1.7)%, respectively. The numbers of CD43-positive cell colonies on days 6, 8, 10, and 12 were 0, 7-/+2, 37-/+11, and 89-/+29 in each 10(5) cells, respectively. Immunocytochemical staining identified CD45-positive cells on days 10, 12, 15, and 18 in the cell colonies, with the positive cell numbers of 0, 40.5-/+15.09, 178.6-/+55.89, and 253.0-/+52.04, respectively.

CONCLUSIONThe hESCs undergo spontaneous hematopoietic differentiation in 3 stages, including the differentiation into germ layer-specific cells (days 6-8), expansion period of the hematopoitic progenitors (days 8-12), and maturation of the hematopoietic cells (after day 15).

Animals ; Antigens, CD34 ; metabolism ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Cell Culture Techniques ; Cell Differentiation ; Embryonic Stem Cells ; cytology ; metabolism ; GATA2 Transcription Factor ; genetics ; Gene Expression Regulation ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Mice ; Nuclear Proteins ; genetics ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; genetics ; Repressor Proteins ; genetics ; T-Cell Acute Lymphocytic Leukemia Protein 1 ; Time Factors

In order to investigate expression of SCL (stem cell leukemia) gene in bone marrow stromal cells (BMSC) and bone marrow cells from patients with leukemia and normal individuals, bone marrow mononuclear cells from AML (18 cases), CML (17 cases), ALL (7 cases) and normal individuals (33 cases) were cultured long-term in vitro. Nonadherent cells (hematopoietic cells) and amplified adherent cells (BMSC) were collected respectively. RT-PCR-ELISA assay was then performed to detect expression of SCL gene. The expression ratio of SCL gene were analyzed and its expression level was normalized by beta(2)M gene acting as an internal reference for the purpose of semi-quantitative analysis. The results indicated that the expression ratio of SCL gene was lower in BMSC from AML (27.8%) and CML (11.8%) than that in normal controls (69.7%, P < 0.05), while was higher in the nonadherent cells from CML (64.3%) than that in its corresponding BMSC (P < 0.05). Semi-quantitative analysis showed that SCL gene expression level in nonadherent cells from AML was higher than that in its corresponding BMSC (P < 0.05). In conclusion, the low-level expression state of SCL gene in BMSC from patients with AML and CML may be involved in the abnormal regulation of hematopoiesis in myelocytic leukemia.
Adolescent ; Adult ; Basic Helix-Loop-Helix Transcription Factors ; Bone Marrow Cells ; metabolism ; Child ; Child, Preschool ; DNA-Binding Proteins ; genetics ; Female ; Gene Expression ; Humans ; Infant ; Leukemia ; metabolism ; Male ; Proto-Oncogene Proteins ; genetics ; Stromal Cells ; metabolism ; T-Cell Acute Lymphocytic Leukemia Protein 1 ; Transcription Factors ; genetics

OBJECTIVEIn childhood acute lymphoblastic leukemia (ALL), cytogenetics plays an important role in diagnosis, allocation of treatment and prognosis. On the basis of the conventional cytogenetic analysis, molecular methods have improved pediatric hematologists/oncologist's ability to accurately and rapidly perform risk-stratification on patients with childhood ALL during the last few years. The aim of the present study was to assess the demography of cytogenetic abnormalities in childhood ALL.

METHODThe study subjects consisted of 124 newly diagnosed ALL patients younger than 16 years of age, who were diagnosed at the Department of Pediatric Hematology/Oncology, Soochow University Children's Hospital. The diagnosis and FAB subtypes of ALL was determined by Wright-Giemsa-stained bone marrow smears and cytochemical staining. Immunophenotyping of the bone marrow samples was performed by flow cytometry. Multiplex polymerase chain reaction (Multiplex PCR) analysis was performed to detect the 29 most common leukemia translocations for routine molecular diagnostic hematopathology practice, and complement the information gained from conventional cytogenetic analysis.

RESULTSCytogenetic analysis was successful in 112 of 124 children with ALL. Sixty-eight (60%) of them had clonal chromosomal abnormalities. Numerical imbalances consisted of hyperdiploid (> 47 chromosomes, 36 cases), hypodiploid (< 46 chromosomes, 14 cases), pseudodiploidy (18 cases). Chromosomal translocations were observed in 13 patients by conventional cytogenetic analysis. Three cases were found positive for 4; 11 translocation, 3 cases for 9; 22 translocation, 1 case for 1; 19 translocation and 6 cases for other rare translocations. Multiplex-PCR analysis detected 116 of the 124 ALL patients. Thirteen cases of TEL-AML1, 10 cases of rearrangement in the MLL gene, 4 cases of E2A-PBX1, 4 cases of E2A-HLF, 3 cases of BCR-ABL, 2 cases of TLS-ERG, 32 cases of HOX11 were detected by Multiplex PCR in B-lineage leukemias. SIL-TAL1 had been found in 4 of 7 of T-lineage leukemias.

CONCLUSIONSSixty-eight cases of ALL showed chromosomal aberrations. Multiplex PCR positivity was detected in 59 (50%) of the 116 ALL patients studied. Multiplex PCR combined with chromosomal analysis uncovered chromosomal abnormalities in 95 of 124 (77%) of ALL patients and supplemented each other in detecting chromosomal abnormalities.

Adolescent ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Child ; Child, Preschool ; Chromosome Aberrations ; Core Binding Factor Alpha 2 Subunit ; genetics ; Cytogenetic Analysis ; DNA-Binding Proteins ; genetics ; Female ; Fusion Proteins, bcr-abl ; genetics ; Gene Fusion ; genetics ; Homeodomain Proteins ; Humans ; Immunophenotyping ; methods ; Infant ; Karyotyping ; Male ; Myeloid-Lymphoid Leukemia Protein ; genetics ; Oncogene Proteins, Fusion ; genetics ; Polymerase Chain Reaction ; Pre-B-Cell Leukemia Transcription Factor 1 ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Proto-Oncogene Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; T-Cell Acute Lymphocytic Leukemia Protein 1 ; Translocation, Genetic

The hematopoietic system of the mouse arises from extraembryonic mesoderm that migrate through primitive streak to the presumptive yolk sac at day 7.0 of gestation. However, the mechanisms regulating mesoderm commitment to hematopoietic lineages remain poorly understood. Previous studies demonstrated that the development kinetics and growth factor responsiveness of hematopoietic precursors derived from embryonic stem cells (ES cells) is similar to that found in the yolk sac, indicating that the onset of hematopoiesis within the embryoid bodies (EBs) parallels that found in the embryo. Furthermore, in vitro differentiation of ES cells to hematopoietic cells is valuable for establishment of therapeutic clone against a variety of hematological disorders. Despite the identification of multipotential hematopoietic progenitors in EBs, a subset of more primitive progenitors, identical to the high proliferative potential colony-forming cells (HPP-CFC) derived from human and murine hematopoietic tissues, have not been clearly identified regarding particular their replating potential in vitro. HPP-CFC is among the most primitive hematopoietic multipotent precursors cultured in vitro. In this study, our aim was to investigate the in vitro and in vivo hematopoietic capacity of HPP-CFC within the day 12 EBs, rather than the expansion of more committed progenitors. In this study the HPP-CFC could be detected within EBs differentiated for 5 to 14 days of murine ES cells, but the development dynamics of the HPP-CFC differed greatly among distinct serum lots. Qualitatively HPP-CFC is capable of forming secondary colonies. As to our expectation the ES cells-derived HPP-CFC demonstrated similar regeneration capacity to those from yolk sac, giving rise to secondary granulocyte, erythrocyte, macrophage and mast cells, however largely differed from the counterparts of adult bone marrow. In addition, by RT-PCR ES cells-derived HPP-CFC were found to express transcription factors associated closely with stem cell proliferation including SCL, GATA-2 and AML1 as well as various receptors of hematopoietic growth factors such as c-kit, GM-CSF receptor and interleukin 3 receptor et al. Finally, in order to understand the in vivo hematopoietic capacity of the ES cells-derived HPP-CFC, spleen colony-forming unit (CFU-S) assay was performed. Nevertheless, typical CFU-S was not observed after transplantation of the day 12 EB cells or HPP-CFC colonies into lethally irradiated adult murine. In conclusion the HPP-CFC differentiated from murine ES cells displayed robust hematopoietic activity in vitro, however their in vivo reconstitution ability was not detected. The difference between in vitro and in vivo hematopoietic activities of ES cells-derived primitive hematopoietic precursors deserves further investigation.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Cell Differentiation ; genetics ; physiology ; Colony-Forming Units Assay ; Core Binding Factor Alpha 2 Subunit ; genetics ; Embryonic Stem Cells ; cytology ; GATA2 Transcription Factor ; genetics ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Mice ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Receptors, Interleukin-3 ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; T-Cell Acute Lymphocytic Leukemia Protein 1