OBJECTIVETo investigate the application of multiplex ligation-dependent probe amplification (MLPA) in the detection of copy number variations (CNVs) in pediatric ETV6/RUNX1-positive acute lymphoblastic leukemia (ALL), to compare this method with conventional karyotype analysis and fluorescence in situ hybridization (FISH), and to evaluate the value of MLPA.

METHODSThe clinical data of 95 children with ETV6/RUNX1-positive ALL who were treated from January 2006 to November 2012 were analyzed retrospectively, including clinical features, results of karyotype analysis, and results of FISH. CNVs were detected with MLPA.

RESULTSCNVs were detected in 73 (77%), and the median number of CNVs was 1 (range 0-6). The CNVs of EBF1, CDKN2A/2B, PAX5, ETV6, RB1, and BTG1 were detected in more than 10% of all the patients. The changes in the chromosome segments carrying the genes with CNVs detected by MLPA were not detected by conventional karyotype analysis. The coincidence rate between the CNVs in ETV6 gene detected by FISH and those detected by MLPA was 66%.

CONCLUSIONSMLPA is an efficient and convenient method to detect CNVs in children with ETV6/RUNX1-positive ALL.

Adolescent ; Child ; Child, Preschool ; Core Binding Factor Alpha 2 Subunit ; analysis ; DNA Copy Number Variations ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Infant ; Male ; Multiplex Polymerase Chain Reaction ; methods ; Oncogene Proteins, Fusion ; analysis ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics

OBJECTIVETo evaluate the efficiency of one-step multiplex RT-PCR for identifying four common fusion transcripts (TEL/AML1, E2A/PBX1, MLL/AF4 and BCR/ABL) in children with acute lymphoblastic leukemia (ALL).

METHODSTotal RNA was extracted from bone marrow samples of 76 children who were newly diagnosed with ALL between January 2003 and December 2010. These RNAs were analyzed for TEL/AML1, E2A/PBX1, MLL/AF4 and BCR/ABL by one-step multiplex RT-PCR or common nested-multiplex PCR. The PCR products were confirmed by DNA sequencing.

RESULTSTEL/AML1 was found in 12 cases (the length of products was 298 bp in 9 cases and 259 bp in 3 cases), E2A/PBX1 was found in 3 cases (the length of products was 373 bp), BCR/ABL was found in 1 case (the length of products was 2 124 bp), and MLL/AF4 was found in 7 cases (the length of products was 427 bp in 1 case and 673 bp in 6 cases) using one-step multiplex RT-PCR combined with DNA sequencing. The results were consistent with those using common nested-multiplex PCR.

CONCLUSIONSOne-step multiplex RT-PCR may be another alternative for detection of common fusion transcripts in children with ALL.

Child ; Child, Preschool ; Core Binding Factor Alpha 2 Subunit ; genetics ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Infant ; Male ; Multiplex Polymerase Chain Reaction ; methods ; Myeloid-Lymphoid Leukemia Protein ; genetics ; Oncogene Proteins, Fusion ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

Retaining or improving periodontal ligament (PDL) function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J⋅cm(-2). Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Our data showed that LPLI at a dose of 2 J⋅cm(-2) significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J⋅cm(-2) showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration.
Adenine ; analogs & derivatives ; pharmacology ; Adenylyl Cyclase Inhibitors ; Alkaline Phosphatase ; analysis ; genetics ; radiation effects ; Anthraquinones ; Bone Morphogenetic Protein 2 ; genetics ; Calcium ; metabolism ; radiation effects ; Cell Culture Techniques ; Cell Differentiation ; radiation effects ; Cell Line ; Cell Proliferation ; radiation effects ; Coloring Agents ; Core Binding Factor Alpha 1 Subunit ; genetics ; Cyclic AMP ; antagonists & inhibitors ; radiation effects ; Gene Expression ; radiation effects ; Humans ; L-Lactate Dehydrogenase ; analysis ; Lasers, Semiconductor ; Low-Level Light Therapy ; instrumentation ; Osteocalcin ; genetics ; Osteogenesis ; genetics ; radiation effects ; Periodontal Ligament ; cytology ; radiation effects ; Radiation Dosage ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Tetrazolium Salts ; Thiazoles

BACKGROUND/AIMS: This study was performed to determine the association between RUNX3 expression and Helicobacter pylori infection in premalignant gastric lesions. METHODS: We examined 107 patients with gastric epithelial dysplasia who had undergone endoscopic mucosal resection or submucosal dissection. All tissue samples were evaluated by RUNX3 staining and subclassified by immunophenotype. H. pylori infection in dysplastic lesions and the normal surrounding tissue was examined by silver staining, and cagA status was assessed by polymerase chain reaction. RESULTS: The loss of RUNX3 expression was observed in 62 cases (57.9%), and an association with H. pylori infection was found in 54 cases (50.5%). The infection rate with the cagA-positive H. pylori strain was 63.0%. In RUNX3-negative lesions, the rate of H. pylori infection (p=0.03) and the frequency of category 4 lesions (according to the revised Vienna classification) were high (p=0.02). In addition, the gastric mucin phenotype was predominant. In RUNX3-negative category 4 lesions, the rate of cagA-positive H. pylori infection rate was high but not significantly increased (p=0.08). CONCLUSIONS: Infection with H. pylori is associated with inactivation of RUNX3 in early gastric carcinogenesis. This mechanism was prominent in gastric cancer with a gastric mucin phenotype.
Adenoma/*chemistry ; Aged ; Antigens, Bacterial/genetics ; Bacterial Proteins/genetics ; Carcinoma/*chemistry ; Cell Transformation, Neoplastic ; Core Binding Factor Alpha 3 Subunit/*analysis ; Female ; Gastric Mucosa/*chemistry/pathology ; Helicobacter Infections/*metabolism ; Helicobacter pylori/*genetics ; Humans ; Male ; Middle Aged ; Mucin 5AC/analysis ; Mucin-2/analysis ; Mucin-6/analysis ; Neprilysin/analysis ; Phenotype ; Precancerous Conditions/*chemistry/pathology ; Stomach Neoplasms/*chemistry

This study was aimed to investigate the c-kit mutation in acute myeloid leukemia (AML) patients with AML1-ETO and analyze its relation with clinical and laboratorial features and prognosis. PCR and sequencing methods were used to detect the c-kit 17 exon mutations in 31 AML patients with AML1-ETO. The relation of the c-kit mutation with clinical features, results of laboratorial examination and prognosis of disease were analyzed. The results showed that the c-kit mutation was found in 14 out of 31 AML patients and the mutation frequency was 45.16%. Male patients had a higher incidence of c-kit mutation than that of female patients (P = 0.020). The proportion of patients with newly diagnosed white blood cell>10×10(9)/L and with extramedullary infiltration in mutated group were higher than those in unmutated group respectively. No significant difference was observed at the age (P = 0.437) and the rate of bone marrow blasts(P = 0.510) between the above mentioned two groups. The difference in complete remission rate (64.29% vs 80%, P = 0.344)and relapse rate (58.33% vs 21.43%, P = 0.054) between c-kit mutated and c-kit unmutated groups were not significant. While the c-kit mutated group had a significant higher death rate as compared with c-kit unmutated group (57.14% vs 20%, P = 0.039). It is concluded that the c-kit mutation is frequent in AML patients with AML1-ETO and the c-kit mutated patients have a poor prognosis. It is important to detect c-kit mutation in routine clinical practice for patient's risk stratification, evaluation of prognosis and selection of effective treatment.
Adolescent ; Adult ; Aged ; Core Binding Factor Alpha 2 Subunit ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; pathology ; Male ; Middle Aged ; Mutation ; Oncogene Proteins, Fusion ; genetics ; Prognosis ; Proto-Oncogene Proteins c-kit ; genetics ; RUNX1 Translocation Partner 1 Protein ; Treatment Outcome ; Young Adult

No abstract available.
Adolescent ; Aged ; Child ; Child, Preschool ; Core Binding Factor Alpha 2 Subunit/analysis/genetics ; Cytogenetic Analysis ; Female ; Fusion Proteins, bcr-abl/analysis/genetics ; Hematologic Neoplasms/*diagnosis/genetics ; Humans ; Infant ; Leukemia, Myeloid, Acute/diagnosis/genetics ; Male ; Middle Aged ; *Multiplex Polymerase Chain Reaction ; Oncogene Proteins, Fusion/analysis/genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis/genetics ; *Reverse Transcriptase Polymerase Chain Reaction

Childhood leukemia bottleneck phenomenon is the most mysterious corollary of the prenatal origin discovery of leukemogenic chromosome translocations. The bottleneck is evidence that leukemia initiation, by in utero acquired chromosome translocations that generate functional fusion genes, is far more common than the incidence rate of corresponding leukemia. For childhood TEL-AML1(+) acute lymphoblastic leukemia (ALL) this equates to approximately 100 times. Practically this means that among a hundred children born with TEL-AML1 fusion gene, only one child will later in its life develop ALL. The key data necessary for unraveling of this mystery were discovered in 2002. It was the level of TEL-AML1(+) cells’ frequency. The bottleneck is caused by the very low body TEL-AML1(+) cell count. Only one out of a thousand B cells carries TEL-AML1 fusion gene. TEL-AML1(+) body cell count is low because TEL-AML1 fusion is generated at cell level of 10(-3) to 10(-4) just during the late fetal lymphopoiesis i.e. after the 36th gestational week.
Child ; Chromosomes, Human, Pair 12 ; Chromosomes, Human, Pair 21 ; Core Binding Factor Alpha 2 Subunit ; analysis ; genetics ; Humans ; Infant, Newborn ; Models, Genetic ; Oncogene Proteins, Fusion ; analysis ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; etiology ; genetics ; Translocation, Genetic

UNLABELLEDOBJECTIVE To study the clinical and biological characteristics and prognosis of t(8;21)/AML1-ETO-positive childhood acute myeloid leukemia (AML).

METHODSThe clinical data of 55 children who were diagnosed as t (8; 21)/AML1-ETO-positive AML were retrospectively studied. Event-free survival (EFS), disease-free survival (DFS), and overall survival (OS) rates were estimated by the Kaplan-Meier method. Prognostic factors were evaluated by COX regression analysis software.

RESULTSOf the 55 patients, 4 patients gave up treatment after the diagnosis was confirmed and 4 patients were lost to follow-up after the first chemotherapy course. The remaining 47 patients received a double-induction therapy. The total complete remission (CR) rate was 71% and 94% after the first and second chemotherapy course, respectively. The disease was relapsed in 10 patients (21%). The 5-year EFS, DFS and OS rates were (56.1 ± 7.9)%, (59.8 ± 8.1)%, and (72.0 ± 8.1)%, respectively. Multivariate analysis showed that age was an independent risk factor for the long-term prognosis. The older children had a greater risk of experiencing an accident or death (P<0.05). The 5-year OS rate in 27 patients with regular consolidation chemotherapy was significantly higher than 13 patients with irregular chemotherapy after CRz [(47.5 ± 17.1)% vs (38.9 ± 17.3)%; P<0.01].

CONCLUSIONSChildhood t(8;21)/AML1-ETO-positive AML is a highly heterogeneous disease, with a high CR rate and a good long-term prognosis. Age is one of the important factors affecting the long-term therapeutic effect. Regular consolidation chemotherapy applied after CR usually is helpful.

Adolescent ; Bone Marrow Examination ; Child ; Child, Preschool ; Chromosomes, Human, Pair 21 ; Chromosomes, Human, Pair 8 ; Core Binding Factor Alpha 2 Subunit ; analysis ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; mortality ; Male ; Oncogene Proteins, Fusion ; analysis ; Prognosis ; RUNX1 Translocation Partner 1 Protein ; Translocation, Genetic

This study was aimed to investigate various factors influencing long-term survival in adult AML patients with fusion gene aml1/eto positive. A single institutional retrospective study with long-term follow-up was performed to better define the prognostic factors for AML patients with aml1/eto positive. Newly diagnosed 89 adult AML patients with aml1/eto positive were followed up for 1 to 42 months (median 24 months) from January 2004 to July 2008. Univariate and multivariate analysis of potential factors influencing survival and prognosis were carried out by using Log-Rank and Cox regression method, including sex, age, initial WBC counts, extramedullary leukemic disease, central nervous system leukemia (CNSL), chromosome aberrations, immunophenotype, first induction regimen, chemotherapy course to complete remission (CR), time from induction therapy to CR, negative or positive rate of aml1/eto and allogeneic hematopoietic stem cell transplantation and so on. The results showed that the estimated 5-year overall survival (OS) and relapse-free survival (RFS) were (50.0 +/- 2.3)% and (47.0 +/- 1.9)% respectively in follow-up of 89 patients for 1 - 42 months (mean 24 months). Univariate analysis revealed that initial WBC counts, CNSL, chemotherapy course to CR, time from induction therapy to CR, persistent negative in remission and allogeneic hematopoietic stem cell transplantation were important prognostic factors for long-term surviva1. Multivariate study demonstrated that initial WBC counts, CNSL, CD56 positive, negative or positive rate of aml1/eto, time from induction therapy to CR, persistent negative result of RT-PCR assay in remission and allogeneic hematopoietic stem cell transplantation were all critical factors in relation to OS and RFS. It is concluded that Chinese adult AML patients with fusion gene aml1/eto positive have some different characteristics as compared with patients from other countries, a relatively poor outcome is observed in patients, HSCT should be recommended to adult AML patients.
Adolescent ; Adult ; Core Binding Factor Alpha 2 Subunit ; genetics ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; mortality ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; Prognosis ; RUNX1 Translocation Partner 1 Protein ; Retrospective Studies ; Survival Analysis ; Young Adult

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