Adolescent ; Fucosyltransferases ; genetics ; Gene Rearrangement ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Male ; Protein-Tyrosine Kinases ; genetics

OBJECTIVETo compare the value of fluorescence in situ hybridization(FISH) with that of immunohistochemistry in detecting ALK gene translocations and ALK fusion protein in anaplastic large cell lymphoma (ALCL) and investigate the possibility of FISH working on paraffin-embedded ALCL tissue.

METHODSDual-color FISH and ALK-1 immunohistochemistry were used to detect ALK gene translocation and ALK fusion protein in 22 paraffin-embedded ALCL cases.

RESULTSThe digestion time of tissue section was a key-point in the operating of FISH in paraffin-embedded tissue. ALK fusion protein expression was detected with ALK-1 antibody in 12 of the 20 systemic ALCL; it was not detected in 2 primary cutaneous ALCL. The Dual-color FISH results were 100% consonant with the immunohistochemical results.

CONCLUSION(1) Generally, immunohistochemical method is the first choice in detecting ALK gene translocation, but when conditions permit, FISH can be the first choice. (2) By the optimization of experimental conditions, FISH can be successfully performed on paraffin-embedded tissue.

Humans ; In Situ Hybridization, Fluorescence ; methods ; Lymphoma, Large-Cell, Anaplastic ; genetics ; pathology ; Paraffin Embedding ; methods ; Protein-Tyrosine Kinases ; genetics ; Receptor Protein-Tyrosine Kinases ; Reproducibility of Results ; Translocation, Genetic

OBJECTIVE: To explore the genetic basis of a child featuring intellectual disability, developmental delay and epilepsy. METHODS: Cytogenetic and molecular analysis including chromosomal karyotyping analysis, single nucleotide polymorphism array (SNP array) and qPCR were performed. RESULTS: The karyotype of the child was determined as 46, XX; SNP array: arr [19]21q22.12q22.13(36 860 195-38 801 482)×1 dn. A heterozygous 1.9 Mb microdeletion was detected at 21q22.12q22.13. qPCR has confirmed deletion of exon 1 of the DYRK1A gene, which has occurred de novo. CONCLUSION A 21q22 deletion was diagnosed with multiple genetic methods. Genotype-phenotype correlation suggested DYRK1A to be a candidate for intellectual disability.
Child ; Developmental Disabilities ; genetics ; Epilepsy ; genetics ; Genetic Association Studies ; Humans ; Intellectual Disability ; genetics ; Karyotyping ; Protein-Serine-Threonine Kinases ; genetics ; Protein-Tyrosine Kinases ; genetics ; Sequence Deletion

The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.
Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Protein-Tyrosine Kinases ; genetics ; Receptor Protein-Tyrosine Kinases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; genetics ; Recombination, Genetic ; Retroviridae ; genetics

BACKGROUND: Thymic carcinomas (TCs) and thymic neuroendocrine neoplasms (TNENs) are two aggressive subtypes of thymic malignancy. Traditional therapy for advanced TCs and TNENs has limited outcome. New genomic profiling of TCs and TNENs might provide insights that contribute to the development of new treatment approaches. METHODS: We used gene panel sequencing technologies to investigate the genetic aberrations of 32 TC patients and 15 TNEN patients who underwent surgery at Shanghai Chest Hospital between 2015 and 2017. Patient samples were sequenced using a 324-gene platform with licensed technologies. In this study, we focused on clinically relevant genomic alterations (CRGAs), which are previously proven to be pathogenic alterations, to identify the pathology-specific mutational patterns, prognostic signatures of TCs and TNENs. RESULTS: The mutational profiles between TCs and TNENs were diverse. The genetic alterations that ranked highest in TCs were in CDKN2A, TP53, ASXL1, CDKN2B, PIK3C2G, PTCH1, and ROS1 , while those in TNENs were in MEN1, MLL2, APC, RB1 , and TSC2 . Prognostic analysis showed that mutations of ROS1, CDKN2A, CDKN2B, BRAF, and BAP1 were significantly associated with worse outcomes in TC patients, and that mutation of ERBB2 indicated shortened disease-free survival (DFS) and overall survival (OS) in TNEN patients. Further investigation found that the prognosis-related genes were focused on signal pathways of cell cycle control, chromatin remodeling/DNA methylation, phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), and receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase (MAPK) signaling. CONCLUSION We profiled the mutational features of 47 Chinese patients with thymic malignancy of diverse pathologic phenotypes to uncover the integrated genomic landscape of these rare tumors, and identified the pathology-specific mutational patterns, prognostic signatures, and potential therapeutic targets for TCs and TNENs.
Humans ; Thymoma ; Protein-Tyrosine Kinases/genetics* ; Proto-Oncogene Proteins/genetics* ; China ; Thymus Neoplasms/pathology* ; Prognosis ; Neuroendocrine Tumors/pathology* ; Mutation/genetics*

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic tumor originated from hematopoietic stem cells. FLT3 is an important receptor tyrosine kinase in cell signal transduction pathway and one of the common mutated genes in AML. AML patients with FLT3-ITD mutation have a poor prognosis and tendency to relapse. Therefore, early identification of FLT3 gene mutation and selection of appropriate treatment are particularly important. Currently, the small moleculetargeted drugs have been new treatment methods for AML patients with FLT3-ITD mutation, but accompanied drug resistance need to be solved. This paper reviews the mechanism of FLT3 mutation, the clinical significance of FLT3 mutation in AML, FLT3 inhibitors and drug resistance mechanism.
Humans ; Mutation ; Protein Kinase Inhibitors/therapeutic use* ; Signal Transduction ; Receptor Protein-Tyrosine Kinases/therapeutic use* ; Leukemia, Myeloid, Acute/drug therapy* ; fms-Like Tyrosine Kinase 3/genetics*

OBJECTIVEFMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in (70-90)% pediatric patients with acute myeloid leukemia (AML) and appears to confer an adverse prognosis. Although several FLT3-selective small molecule inhibitors and antibodies were developed with varied degrees of success, to address the specificity and resistance, new approaches for specifically targeted FLT3 are needed and RNA interference is a promising choice. The aim of the present study was to investigate the efficacy of suppression of FLT3 induced by small hairpin interfering RNA (shRNA) on myeloproliferation and apoptosis in an acute monocytic leukemia (AMOL) cell line THP-1.

METHODSFLT3-targeted small hairpin interfering RNA (FLT3-shRNA) was designed and synthesized by transcription system in vitro was transfected into THP-1 cells. Firstly FLT3 mRNA level was detected by semi-quantitative RT-PCR and FLT3 protein level was detected by flow cytometry (FCM) to verify the efficacy on FLT3-shRNA interference at 48 h after transfection. Cell growth viability was measured at 24 h, 48 h and 72 h after treatment with CCK-8. The distribution of cell cycle was assayed by FCM, and apoptosis was analyzed by DNA Ladder and Annexin V-FITC Staining at 48 h.

RESULTSFLT3 targeted shRNAs was synthesized successfully and the concentration of 15 nmol/L for 48 h could obtain desirable downregulation of FLT3 expression, the inhibitory percentages of FLT3 mRNA and protein were (72.95 +/- 2.07)% and (65.39 +/- 5.57)%, respectively. The suppression of FLT3 induced by FLT3-shRNA resulted in marked inhibition of cell growth and the inhibitory percentages were (36.66 +/- 3.67)% at 48 h, (35.56 +/- 0.73)% at 72 h. FLT3-shRNA induced the inhibition of cell cycle from G(0)/G(1) phase to S phase, the percentage of sub-G(0)/G(1) phase (65.71 +/- 4.47)% was higher than those in the PBS-control group (52.23 +/- 2.98)%, NC-shRNA control group (51.81 +/- 1.44)%, P < 0.01; the percentage of S phase (25.11 +/- 2.70)% was lower than those in the PBS-control group (34.41 +/- 4.07)% and NC-shRNA control group (32.50 +/- 1.46)%, P < 0.05. Furthermore treatment with FLT3-shRNA for 48 h resulted in clear apoptosis ladder, the percentage of early apoptosis detected by Annexin V-FITC was (18.59 +/- 2.07)% which was significantly higher than that in the PBS-control group (4.00 +/- 0.50)% and the NC-shRNA control group (6.06 +/- 0.70)%, P < 0.001.

CONCLUSIONThe suppression of FLT3 induced by the shRNA can effectively inhibit cell proliferation, and apoptosis induction on THP-1 cells, which indicates that this approach may bear the therapeutic potential on childhood AMOL.

Apoptosis ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Child ; Humans ; Leukemia, Monocytic, Acute ; enzymology ; pathology ; Protein-Tyrosine Kinases ; metabolism ; RNA Interference ; physiology ; RNA, Small Interfering ; pharmacology ; Receptor Protein-Tyrosine Kinases ; metabolism ; fms-Like Tyrosine Kinase 3 ; metabolism

OBJECTIVETo detect the expressions of receptor tyrosine kinases (RTKs) mRNA and protein and to explore potentially promising tumor markers and conceivable drug target in bladder cancer.

METHODSThe expressions of RTKs mRNA and protein in tissue from invasive urothelial carcinoma of the bladder were examined by real-time quantitative PCR array and cytokine antibody array, with normal bladder tissue as control. The Results were analyzed using bioinformatic approaches.

RESULTSThe expressions of TGFA, STAB1, SERPINE1, ANGPT2, SPINK5, ANGPTL1, PROK1, MDK, CXCL9, GRN, RUNX1, VEGFA, and TGFB1 were obviously upregulated in bladder cancer tissue, while those of EDIL3, PTN, CCL2, PDGFD, FGF13, KITLG, FGF2, SERPINF1, and TNF were downregulated. ALK, Btk, EphB2, ErbB4, PDGFR-α, ROS, Tie-2, Tyk2, and VEGFR3 were over-expressed in bladder cancer, while FRK, Fyn, IGF-IR, Insulin R, Itk, JAK1, JAK3, and LCK were low-expressed.

CONCLUSIONVascular endothelial growth factor/platelet-derived growth factor-targeted therapies may play an active role in treating carcinoma of bladder.

Carcinoma, Transitional Cell ; metabolism ; Humans ; RNA, Messenger ; genetics ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism ; Urinary Bladder Neoplasms ; metabolism

OBJECTIVETo investigate mutations of anaplastic lymphoma kinase (ALK) in Chinese children with neuroblastoma (NB).

METHODSGenomic DNA was extracted from 22 cases of paraffin-embedding NB tumor tissues. Gene mutations in the exons 20-26 which were mutational hotspots of ALK were analyzed by PCR-DNA direct sequencing.

RESULTSA novel synonymous mutation C3586T (Leu1196Leu) and a known synonymous mutation C3375A (Gly1125Gly) were found and located at exon 23 and exon 21 of ALK respectively. There were 10 cases (46%) of known synonymous mutation C3375A in 22 cases of NB. The C3375A allelic frequency was 27%. No statistically significant correlation was found between mutation C3375A and clinical parameters of NB such as age, sex, metastasis and tumor differentiation. Mutation was not found in the other 5 exons.

CONCLUSIONSA novel ALK gene synonymous mutation C3586T was identified using PCR-DNA sequencing. A known mutation C3375A in ALK was successfully identified in children, and its incidence is not influenced by the clinical features of childhood NB.

Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; Neuroblastoma ; genetics ; Polymerase Chain Reaction ; Receptor Protein-Tyrosine Kinases ; genetics

AIMTo observe the effect of FAK-related non-Kinase (FRNK) plasmid on hepatic stellate cell (HSC) proliferation stimulated by fibronectin (FN).

METHODSFRNK plasmid was transfected into HSC with transient liposomal transfection. The proteins of FRNK, FAK and p-FAK(Tyr397) were assayed by Western blotting analysis. The proliferation of HSC was evaluated by improved MTT assay, and cell cycle pattern was determined by flow cytometry (FCM).

RESULTS(1) The expression of FRNK protein increased after FRNK transfected HSC, and it was at 48 h that the expression of FRNK protein was the highest (P < 0.01). The protein level of FAK was no significant difference between before FRNK plasmid transfection and after transfection (P > 0.05). The expression of p-FAK(Tyr397) protein was down-regulated after FRNK had been transfected in HSC, (P < 0.01). (2) The HSC proliferation inhibition rates at 12 h, 24 h and 48 h after FRNK transfection were 20.07%, 26.16%, 29.77%, respectively (P < 0.01). (3) Compared with the non-FRNK plasmid group, the FRNK-transfected HSCs almost arrested in G0/G1 phase (71.4 +/- 2.81 vs 48.9 +/- 1.66, P < 0.01).

CONCLUSIONAfter FRNK were transfected successfully in HSCs using lipofectamine, the phosphorylation of FAK was inhibited. The HSC proliferation was restrained in a time-dependent manner and the HSC was arrested in G0/G1 phase.

Cell Line ; Cell Proliferation ; Fibronectins ; Hepatic Stellate Cells ; cytology ; Humans ; Phosphorylation ; Plasmids ; genetics ; Protein-Tyrosine Kinases ; genetics ; Transfection