The aim of this study was to evaluate the value of CD117/CD11b phenotypic analysis to diagnosis and prognosis of acute promyelocytic leukemia (APL). Three- or four-color flow cytometry with a series of 22 monoclonal antibodies and CD45/Side Scatter (SSC) gating strategy were used to identify immunophenotypic characteristics of APL as compared to CML in chronic phase (CML-CP). PML/RAR alpha fusion gene was detected by using reverse-transcription polymerase chain reaction (RT-PCR) technique. The results showed that MPO, CD13 and CD33 were almost expressed in all patients with APL and CML-CP whereas HLA-DR and CD34, the hematopoietic progenitor cell markers, were rarely expressed. The positive rate of CD15 in APL was significantly lower than those in CML-CP (P < 0.01). CD117 was positive in 78.3% of the APL cases and in none of the cases of CML-CP. On the other hand, CD11b was almost positive in all cases of CML-CP, but only 16.9% of the APL cases were found positive for this antigen. The CD117+ CD11b- phenotype was present in 72.3% of APL cases while none of cases with CML-CP with this phenotype. Almost all of the cases with CML-CP had the phenotype of CD117- CD11b+. CD117- CD11b+ phenotype was detected in all patients recovering from APL with CD117+ CD11b- phenotype at diagnosis and after treatment with all-trans-retinoic acid (ARTA) for 2 months. PML/RAR alpha fusion gene was positive in 80.6% (25/31) of the APL cases, of which, 64% of the cases belonged to the type L while only 36% of the cases were showed type S for this fusion gene. The positive rates of CD117 were 87.5%, 44.4% and 33.3% in type L group, S group and negative group respectively. It is concluded that analysis of both CD117 and CD11b phenotype may be helpful to the diagnosis, therapy and prognosis of APL in children and adults and to differentiation of APL from recovering benign myeloid proliferation.
Adolescent ; Adult ; CD11b Antigen ; analysis ; Child ; Female ; Humans ; Immunophenotyping ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; diagnosis ; genetics ; immunology ; Leukemia, Promyelocytic, Acute ; diagnosis ; genetics ; immunology ; Male ; Oncogene Proteins, Fusion ; genetics ; Prognosis ; Proto-Oncogene Proteins c-kit ; analysis

OBJECTIVETo study the prevalence of ALK, ROS1 and RET fusion genes in non-small cell lung cancer (NSCLC), and its correlation with clinicopathologic features.

METHODSFormalin-fixed and paraffin-embedded tissue sections from samples of 302 patients with NSCLC were screened for ALK, ROS1, RET fusions by real-time polymerase chain reaction (PCR). All of the cases were validated by Sanger DNA sequencing. The relationship between ALK, ROS1, RET fusion genes and clinicopathologic features were analyzed.

RESULTSIn the cohort of 302 NSCLC samples, 3.97% (12/302) were found to contain ALK fusion genes, including 3 cases with E13; A20 gene fusion, 3 cases with E6; A20 gene fusion and 3 cases with E20; A20 gene fusion. There was no statistically significant difference in patient's gender, age, smoking history and histologic type. Moreover, in the 302 NSCLC samples studied, 3.97% (12/302) were found to contain ROS1 fusion genes, with CD74-ROS1 fusion identified in 9 cases. There was no statistically significant difference in patients' gender, age, smoking history and histologic type. One non-smoking elderly female patient with pulmonary adenocarcinoma had RET gene fusion. None of the cases studied had concurrent ALK, ROS1 and RET mutations.

CONCLUSIONSThe ALK, ROS1 and RET fusion gene mutation rates in NSCLC are low, they represent some specific molecular subtypes of NSCLC. Genetic testing has significant meaning to guide clinical targeted therapy.

Adenocarcinoma ; Aged ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; Female ; Gene Fusion ; Genetic Testing ; Humans ; Lung Neoplasms ; Mutation ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-ret ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism ; Sequence Analysis, DNA ; Smoking

Objective: To investigate the clinicopathological features, immunophenotype, molecular features and differential diagnosis of primary synovial sarcoma of the lung (PSSL). Methods: Twelve cases of PSSL were collected at Henan Provincial People's Hospital, during May 2010 and April 2021, and their clinicopathological parameters were summarized. SS18-SSX, H3K27Me3, and SOX2 were added to the original immunomarkers to evaluate their diagnostic value for PSSL. Results: The age of 12 patients when diagnosed ranged from 32 to 75 years (mean of 50 years). There were 7 males and 5 females, 2 left lung cases and 10 right lung cases. Of the 6 patients who underwent surgical resection, five cases were confined to lung tissue (T1), one case had mediastinal invasion (T3), two cases had regional lymph node metastasis (N1), and none had distal metastasis. Microscopically, 11 cases showed monophasic spindle cell type and one case showed biphasic type composed of mainly epithelial cells consisting of cuboidal to columnar cells with glandular and cribriform structures. It was difficult to make the diagnosis by using the biopsy specimens. Immunohistochemistry (IHC) showed CKpan expression in 8 of 12 cases; EMA expression in 11 of 12 case; TLE1 expression in 8 of 12 cases; S-100 protein expression in two of 12 cases; various expression of bcl-2 and vimentin in 12 cases, but no expression of SOX10 and CD34 in all the cases. The Ki-67 index was 15%-30%. The expression of SS18-SSX fusion antibody was diffusely and strongly positive in all 12 cases. SOX2 was partially or diffusely expressed in 8 of 12 cases, with strong expression in the epithelial component. H3K27Me3 was absent in 3 of 12 cases. SS18 gene translocation was confirmed by fluorescence in situ hybridization (FISH) test in all 12 samples. Six cases underwent surgery and postoperative chemotherapy, while the other six cases had chemotherapy alone. Ten patients were followed up after 9-114 months, with an average of 41 months and a median of 26 months. Five patients survived and five died of the disease within two years. Conclusions: PSSL is rare and has a broad morphological spectrum. IHC and molecular tests are needed for definitive diagnosis. Compared with current commonly used IHC markers, SS18-SSX fusion antibody has better sensitivity to PSSL, which could be used as an alternative for FISH, reverse transcription-polymerase chain reaction or next generation sequencing in the diagnosis of PSSL.
Male ; Female ; Humans ; Adult ; Middle Aged ; Aged ; Biomarkers, Tumor/analysis* ; Sarcoma, Synovial/diagnosis* ; In Situ Hybridization, Fluorescence ; Histones/genetics* ; Proto-Oncogene Proteins/metabolism* ; Oncogene Proteins, Fusion/genetics* ; Repressor Proteins/metabolism* ; Lung/pathology* ; Lung Neoplasms

OBJECTIVETo evaluate the possibility of applying oligonucleotide microarrays for detection of the fusion genes in childhood acute lymphoblastic leukemia (ALL).

METHODSTo detect five types of fusion genes emerging frequently in childhood ALL including TEL/AML1, E2A/PBX1, BCR/ABLp190, BCR/ABLp210, MLL/AF4, probes were designed, synthesized and spotted on the chemical-material-coated-glass plates in array. The total RNAs were extracted from patients' bone marrow or peripheral blood cells at the beginning of diagnosis, analyzed by multiplex nested reverse-transcription-polymerase chain reaction (RT-PCR), and labeled by fluorescein. The products of RT-PCR were hybridized with microarray in order to detect specific types of fusion genes in leukemia cells.

RESULTSDistinctive hybridization signals were obtained for internal positive control and specific types of fusion genes. TEL/AML1 gene was found positive in 2 of the 36 cases, E2A/PBX1 gene in 3, BCR/ABLp190 gene in 2, BCR/ ABLp210 gene in one, and MLL/ AF4 gene in one. The results of the microarray and RT-PCR were consistent.

CONCLUSIONThe microarray-based assay could screen 5 types of chromosome structural aberrations and the splice variants at the same time. It can provide reliable and helpful information for patient stratification, evaluation of therapeutic effects and prediction of prognosis in childhood ALL, although there are both advantages and disadvantages in applying this new method.

Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Oligonucleotide Array Sequence Analysis ; Oncogene Proteins, Fusion ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

In order to explore the application of real-time quantitative reverse-transcription polymerase chain reaction (Q-PCR) for detecting PML/RARalpha gene transcripts in patients with acute promyelocytic leukemia (APL), the bone marrow samples from 46 newly diagnosed APL patients were collected for analysis. Three plasmids containing cDNA fragments of the bcr1-, bcr3-form PML/RARalpha and ABL control gene were constructed respectively. The ABI Prism 7500 Sequence Detection System using Taqman fluorogenic probes was used to quantify target gene. PML/RARalpha mRNA was detected by Q-PCR in 46 APL patients and 40 non-APL patients. The normalized quotient (NQ) of PML/RARalpha mRNA was calculated as followings: NQ = PML/RARalpha mRNA copy numbers/ABL mRNA copy numbers. Immunophenotype of acute promyelocytic leukemia was determined by four-color flow cytometry. The results showed that the coefficients of variation (CV) of inter-day assay and intra-day assay by using Q-PCR were 1.58% and 0.88% respectively. Q-PCR could detect reproducibly 5 copies of target gene per 100 ng RNA and no pseudopositive results were found. The median NQ of PML/RARalpha mRNA was 0.450 (0.084 - 1.082) in 46 APL patients. There was no indication of any correlation of PML/RARalpha mRNA type with age, sex, hemoglobin, platelet count, percentage of promyelocytes in bone marrow detected by morphology or flow cytometry, PML/RARalpha NQ, or signs of clinically diagnosed coagulation/bleeding disorders. Compared with bcr1-form cases, bcr3-form cases had more M(3v) phenotype (42.9% vs 9.4%, P = 0.015) and higher WBC count (9.35 x 10(9)/L vs 2.15 x 10(9)/L, P = 0.038). APL cells could be classified into large side scatter population (L-SSC) and non-large side scatter population (NL-SSC) in CD45/SSC histogram of flow cytometry. 87.50% patients with bcr1-form showed L-SSC phenotype and 64.29% patients with bcr3-form showed NL-SSC phenotype. It is concluded that a sensitive Q-PCR method is established. The median NQ of PML/RARalpha mRNA was 0.450 in newly diagnosed APL patients. There was no significant difference about PML/RARalpha mRNA expression of both bcr3-form and bcr1-form APL patients. Type of PML/RARalpha transcripts is related with the morphology and immunophenotype.
Adolescent ; Adult ; Aged ; Child ; Female ; Genes, abl ; genetics ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; genetics ; metabolism ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; analysis ; genetics ; Phenotype ; RNA, Messenger ; analysis ; genetics ; Receptors, Retinoic Acid ; analysis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods

This study was purposed to establish new method for detecting CBFB-MYH11 fusion gene in acute myeloid leukemia (AML) and to evaluate its value in clinical use. All fusion types of reported CBFB-MYH11 fusion gene were defined by search of references and databank, then the primers and probes were designed on this basis, and 3 positive plasmids and negative cell line as control were established. GUSB gene was also amplified as an internal reference. The primer/probe sets were tested with 3 positive plasmids and HL-60 cDNA using quantitative real-time PCR (qPCR) assays, which were then combined as a multiplex qPCR for simultaneous detection of CBFB-MYH11 and GUSB. After optimization, the multiplex qPCR assay demonstrated both high sensitivity (10 copies for all the 3 plasmids) and high specificity. Finally, the multiplex qPCR assay was clinically evaluated with 58 AML patients, and 4 CBFB-MYH11-positive cases (6.9%) were detected, involving A type (3 cases) and J type (1 case). By comparison, the multiplex qPCR assay showed results concordant with sequencing results, and detected one case that was missed by cytogenetic analysis. It is concluded that a novel qPCR method for screening of CBFB-MYH11 fusion gene in AML is established. This method is fast, comprehensive, sensitive, specific, reliable, and should consider to be a robust tool for identification and management of AML patients with CBFB-MYH11 fusion gene.
Case-Control Studies ; Core Binding Factor beta Subunit ; analysis ; genetics ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Myosin Heavy Chains ; analysis ; genetics ; Oncogene Proteins, Fusion ; analysis ; genetics ; Real-Time Polymerase Chain Reaction

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

To investigate the immunological and other clinical characteristics in TEL/AML1+ childhood B-acute lymphoblastic leukemia (B-ALL), immunophenotyping was performed with three-color flow cytometry, and the expression of TEL-AML1 fusion gene was detected with nested RT-PCR. Diagnosis was made according to FAB and MIC criteria. The results showed that (1) among 119 children with B-ALL, 22 (18.5%) were TEL-AML1 positive and classified as L2 morphological subtype. In TEL-AML1+ group, positive rate and score of PAS, which were 65% and 121 respectively, were all higher than that of TEL-AML1- group (P < 0.05); (2) compared with TEL-AML1- group, no significant difference was found in age, gender, white cell count and blasts count in peripheral blood of TEL-AML1+; (3) in TEL-AML1+ group, 21 out of 22 (95.5%) were common ALL, as compared with TEL-AML1- group, the positive rate of CD13 was higher (59.1%, 13/22) and the positive rate of CD20 was lower (22.7%, 5/22) than that in TEL-AML1- group, respectively (P < 0.05), and the mean fluorescence index of CD10 and HLA-DR significantly increased to 92.80 and 53.61, respectively (P < 0.05). It is concluded that TEL-AML1 rearrangement is a frequent molecular abnormality in childhood ALL. Leukemic blasts with this anomaly have special immunophenotypic characteristics. These characteristics may be useful in detection of minimal residual leukemia.
Antigens, CD20 ; analysis ; Burkitt Lymphoma ; genetics ; immunology ; CD13 Antigens ; analysis ; Child ; Child, Preschool ; Core Binding Factor Alpha 2 Subunit ; genetics ; Female ; HLA-DR Antigens ; analysis ; Humans ; Immunophenotyping ; Infant ; Male ; Oncogene Proteins, Fusion ; genetics

To investigate the relationship between the single nucleotide polymorphism (SNPs) of the bcr and abl gene and chronic myelogeous leukemia (CML), the 9 sequence-tagged sites (STS) in bcr and abl gene were screened by DNA pooling and denaturing high performance liquid chromatography (dHPLC), and the results were varified by sequencing. The results showed that the polymorphism sites were detected in 4 out of the 9 STS fragments and there were 3 bases different from the reference sequence found in 3 fragments. In conclusion, the novel SNP in U07000 fragment shows significantly different frequencies between CML and controled people.
Chromatography, High Pressure Liquid ; methods ; Fusion Proteins, bcr-abl ; genetics ; Genes, abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins c-bcr ; genetics ; Sequence Analysis, DNA ; Sequence Tagged Sites

A fusion gene between echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) has been identified in non-small cell lung cancers (NSCLCs). Although a few studies have evaluated EML4-ALK fusion genes in Korean NSCLCs, the prevalence of different EML4-ALK fusion variants has yet to be clearly assessed. Herein, we have examined the profiles of EML4-ALK fusion gene variants in Korean patients of NSCLCs. EML4-ALK fusion genes have been detected in 10 (6.0%) of 167 patients of NSCLCs and in 9 (7.4%) of 121 patients of adenocarcinoma. Of the 10 patients with fusion genes identified, 8 (80%) were E13;A20 (variant 1) and 2 (20%) were E6;A20, with an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). These results indicate that the profiles of EML4-ALK fusion gene variants in Korean patients of NSCLC may differ from those in other ethnic populations. Herein, we describe for the first time the profiles of EML4-ALK fusion variants of Korean patients with NSCLCs.
Adenocarcinoma/diagnosis/genetics ; Aged ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Carcinoma, Non-Small-Cell Lung/diagnosis/*genetics ; Exons ; Female ; Humans ; Introns ; Lung Neoplasms/diagnosis/*genetics ; Male ; Middle Aged ; Oncogene Proteins, Fusion/chemistry/*genetics ; Republic of Korea ; Sequence Analysis, RNA ; Smoking