Aneuploidy ; DNA, Neoplasm ; analysis ; genetics ; Flow Cytometry ; methods ; Humans ; Lymphoma ; diagnosis ; genetics ; immunology ; Lymphoma, B-Cell ; diagnosis ; genetics ; immunology ; Lymphoma, T-Cell ; diagnosis ; genetics ; immunology ; Receptors, Antigen, T-Cell ; analysis ; genetics

Gene rearrangement analysis using Southern-blot hybridization technique is a standard method for evaluating clonal receptor gene rearrangement. Both clonality and lineage can be identified in lymphoid neoplasms by the demonstration of one or more rearranged antigen receptor genes of the immunoglobulin supergene family-immunoglobulin and T-cell receptor genes. To evaluate the diagnostic applicability of antigen receptor gene rearrangements in the diagnosis of malignant lymphomas and leukemias, the authors performed a gene rearrangement analysis of 54 cases by southern blot hybridization technique. One or two clonally rearranged bands were detected in the malignant lymphomas and in the lymphoblastic leukemias with a false-negative rate of 13.8%. No clonal, rearranged band was detected in benign reactive hyperplasias, carcinomas or non-lymphocytic leukemias. Rearrangement analysis could resolve the lineage, clonality and stage of differentiation of malignant lymphoid neoplasms.
Gene Rearrangement ; *Gene Rearrangement, T-Lymphocyte ; *Genes, Immunoglobulin ; Human ; Leukemia/*genetics/immunology ; Lymphoma/*genetics/immunology ; Support, Non-U.S. Gov't

The study was aimed to investigate the fusion gene transcript and immunophenotypic characteristics of the mixed linage leukemia (MLL)-rearranged positive childhood acute lymphoblastic leukemia (ALL). The incidence of MLL rearrangement in 601 cases of ALL patients was detected by the multiple-nested polymerase chain reaction (PCR); the subtypes and features of the fusion gene transcript were analyzed by PCR products sequencing; the immunophenotypic characteristics at diagnosis were compared between the 22 MLL rearrangement positive of ALL patient, 30 negative control which selected randomly from the patients whose fusion gene could not be detected in the same term and 43 pro-B-ALL patients. The results showed that the incidence of MLL positive ALL was 3.66%, constituted 29.9% of the pro-B-ALL. The MLL rearrangement positive 20 B-ALL patients were all CD10 negative; the number of patients who carried CD13, CD33 and CD34 was lower than that of pro-B-ALL who had no fusion gene, whereas the expression of CD20, CD22, CD2, CD5, CD7 showed no difference. 4 kind partner genes of MLL-AF4, AF9, AF10 and ENL were detected. The fusion loci of MLL gene were mainly located at the exon 6, 7, 8 and many kind of fusion loci of MLL may exist in one patient; whereas its partner gene fusion loci were relatively single. A transcript contains a random insert sequence existed in a transcript of one MLL-AF10+ patient. It is concluded that though incidence of MLL rearrangement is low, but it has a variety of fusion transcripts, the ALL patients has unique biological characteristics at immunophenotype and fusion transcript.
Adolescent ; Child ; Child, Preschool ; Gene Rearrangement ; Humans ; Immunophenotyping ; Infant ; Myeloid-Lymphoid Leukemia Protein ; genetics ; immunology ; Oncogene Proteins, Fusion ; genetics ; immunology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology

The idiotypic determinant of surface immunoglobulin of B-cell lymphoma, as a tumor-specific antigen, has proved to be able to induce immune responses. To analyze whether an idiotypic vaccine fused with cytokine can elicit more effectively protective antitumor immunity, an eukaryotic expression plasmid was constructed, which encoded the fusion gene of single-chain variable fragment as a tumor specific antigen against B-cell lymphoma with monocyte chemotactic protein-3 (MCP3) as immunogen to elicit T-cell-dependent protective antitumor immunity, and EGFP (Enhanced Green Fluorescent Protein) gene as a marker to trace the survival, growth, differentiation and expression of the former exogenetic genes. The cDNAs for immunoglobulin (Ig) VH and IgVL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) connected with a (Gly(4)Ser)(3) linker by recombinant PCR method. Then, the fragments of scFv and MCP3 were ligated with a NDAQAPKS spacer by the same method. The results showed that the fusion genes of scFv and MCP3-scFv were inserted into an eukaryotic expression vector pTARGET, and EGFP was cloned into the downstream of scFv and MCP3-scFv respectively. Finally the constructed plasmids were confirmed by sequencing and restriction analysis. In conclusion, a tumor-derived idiotypic DNA vaccine, encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP3) to elicit a T-cell dependent, antitumor immunity, and the EGFP gene was inserted correctly. The DNA vaccine could be used for further study of DNA vaccine against B cell lymphoma in vivo.
Animals ; Cancer Vaccines ; genetics ; immunology ; Cell Line, Tumor ; Chemokine CCL7 ; immunology ; Genetic Vectors ; Immunoglobulin Variable Region ; immunology ; Lymphoma, B-Cell ; genetics ; immunology ; prevention & control ; Mice ; Mice, Inbred BALB C ; Plasmids ; Single-Chain Antibodies ; immunology ; Vaccines, DNA ; genetics ; immunology

OBJECTIVETo evaluate the efficiency of the BIOMED-2 PCR assay and its implication in the diagnosis of mature B-cell non-Hodgkin's lymphomas.

METHODSClinical, morphological and immunohistochemical features of 72 cases of non-Hodgkin's lymphomas were studied, including 25 reactive lymphoid hyperplasia, 37 diffuse large B cell lymphomas (DLBCL) and 35 extranodal marginal zone lymphomas of mucosa associated lymphoid tissues (MALT lymphoma and in addition, 25 cases of reactive lymphoid hyperplasia were used as the controls). DNA was exacted from the paraffin embedded formalin fixed tissue blocks and the quality of DNA was assessed using the BIOMED-2 specimen control reaction. Adequate samples were then analyzed by BIOMED-2 for immunoglobulin heavy and kappa light chain rearrangements.

RESULTSAdequate DNA was obtained in 83 of 97 samples, including 60 mature B cell lymphomas and 23 reactive lymphoid hyperplasia. Clonal B-cell gene rearrangements were detected in 57 of 60 (95%) lymphomas. In contrast, clonal Ig gene rearrangements were not detected in any of the 23 cases of reactive lymphoid hyperplasia.

CONCLUSIONBIOMED-2 assay is highly sensitive and specific for the detection of clonal B cell gene rearrangement using routine paraffin embedded formalin fixed specimens.

Antigens, CD20 ; metabolism ; CD79 Antigens ; metabolism ; DNA, Neoplasm ; genetics ; Gene Rearrangement, B-Lymphocyte ; genetics ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; genetics ; Gene Rearrangement, B-Lymphocyte, Light Chain ; genetics ; Genes, Immunoglobulin ; Humans ; Immunophenotyping ; Lymphoma, B-Cell ; genetics ; immunology ; pathology ; Lymphoma, B-Cell, Marginal Zone ; genetics ; immunology ; pathology ; Lymphoma, Large B-Cell, Diffuse ; genetics ; immunology ; pathology ; Paraffin Embedding ; Pseudolymphoma ; genetics ; immunology ; pathology ; Sensitivity and Specificity

The purpose of this study was to construct the IgHV and IL-2 coexpressed vector. The IgHV gene fragments were obtained from the peripheral blood of patients with lymphoma, and were cloned into eukaryotic expression vector. Meanwhile, the gene fragments of IgHV linked with gene of IL-2 were inserted into pcDNA3.0 to form a fusion gene of IgHV-IL-2. Then fusion genes were transfected into COS cells by Lipofectin and the expression of IL-2 was detected by ELISA. The results showed that the IgHV/pcDNA3.0 expression vector was successfully constructed. The 3' end of IgHV was linked to IL-2 gene, and IL-2 could be correctly expressed. In conclusion, the expression vector of IgHV-IL-2 can express IL-2 correctly in COS cells.
Cancer Vaccines ; immunology ; Eukaryotic Cells ; metabolism ; Genes, Immunoglobulin ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Interleukin-2 ; biosynthesis ; genetics ; Lymphoma ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, DNA ; biosynthesis ; genetics ; immunology

This study was purposed to investigate the immunological effects of modified dendritic cells (DCs) in inducing cytotoxic T cells (CTLs) effect against lymphoma cells. The DCs were derived from human peripheral blood and transfected with recombined adenovirus vector carrying survivin gene, Western blot was used to detect the expression of survivin, the lactate dehydrogenase (LDH) release test was used to determine the cytotoxicity of CTLs, the mixed lymphocyte reaction (MLR) was used to measure the ability to proleferate allo-lymphocyte by DCs, ELISA was used to assay IL-12 level in supernatant. The results showed that the expression of survivin in transfected dentritic cells was confirmed by Western blot analysis. In MLR assay, DCs transfected with Ad-survivin could induce higher allogeneic lymphocytes reaction at the ratios of 1:5, 1:10, 1:50 and 1:100. DCs transfected with Ad-survivin had much higher activity of CTL to CA46 cells than control DCs. The levels of IL-12 of supernatants containing DCs transfected with Ad-survivin were significantly higher than that in the control group. It is concluded that DCs transfected with Ad-survivin can induce CTL response in vitro against lymphoma cells.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; immunology ; Dendritic Cells ; immunology ; Genetic Vectors ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Interleukin-12 ; metabolism ; Lymphoma ; immunology ; Microtubule-Associated Proteins ; genetics ; immunology ; Recombination, Genetic ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Tumor Cells, Cultured

To evaluate the expression of cytoplasmic CD79a (CyCD79a) and other commonly used B-lymphoid immunomarkers including cytoplasmic CD22 (CyCD22), CD19, CD20 and CD10 in various acute leukemia cells and to define the most sensitive and specific markers in the diagnosis of precursor B-cell acute lymphoblastic leukemia (pB-ALL), the immunophenotypic data from 221 de novo adult and pediatric acute leukemia patients as studied using multi-parameter flow cytometry in addition to routine morphologic and enzyme cytochemical assay, were retrospectively analyzed. Cytogenetic and/or molecular biological data in all 45 cases of acute promyelocytic leukemia (APL) and 13 cases of acute leukemia suspected as AML with the fusion genes such as AML1/ETO and CBFbeta/MYH11 were investigated. The results showed that CyCD79a and CyCD22 were the most sensitive and specific markers respectively for pB-ALL. Expression of CyCD79a was seen in 100% of 58 cases of pB-ALL. At the same time, none (0%) of all 147 cases of acute myeloid leukemia (AML) and 15 cases of precursor T-cell acute leukemia (pT-ALL) was positive for CyCD22. The conclusion is made that united detection of CyCD79a and CyCD22 is the optimal immune combination for the diagnosis pB-ALL and the distinguishing pB-ALL with AML and pT-ALL.
Acute Disease ; B-Lymphocytes ; immunology ; Biomarkers, Tumor ; immunology ; CD79 Antigens ; immunology ; Cytoplasm ; immunology ; Flow Cytometry ; Humans ; Immunophenotyping ; Karyotyping ; Leukemia, Myeloid ; genetics ; immunology ; pathology ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; metabolism ; pathology ; Sialic Acid Binding Ig-like Lectin 2 ; immunology

OBJECTIVETo explore the clinicopathologic features and diagnosis of splenic T-cell and NK-cell neoplasms.

METHODSNine cases of splenic T-cell and NK-cell neoplasms were collected and studied by morphology, immunophenotyping, EBER in situ hybridization and TCR-gamma gene rearrangement. Antibodies used were as follows: CD45RO, CD3epsilon, CD3, CD4, CD8, CD56, TIA-1, GranzymeB, CD30, Ki-67 and CD20.

RESULTSAmong the 9 cases, hepatosplenic T-cell lymphoma (HSTCL) and extranodal nasal type NK/T-cell lymphoma (NK/TCL) were both of 4 cases, and the remaining one was peripheral T-cell lymphoma, unspecified (PTL, unspecified). Follow up data were available for 7 cases. Five patients including 2 with HSTCL, 2 with extranodal nasal type NK/TCL and one with PTL, unspecified died, with survival times ranged from 1 to 10 months. The other two patients are still alive, one with NK/TCL (two months+) and one with HSTCL (14+ months).

CONCLUSIONSplenic T-cell and NK-cell neoplasms are a group of uncommon lymphomas with heterogeneous clinicopathologic features and poor prognosis. A definite diagnosis must depend on clinical manifestations, histopathology, immunophenotype and TCR gene rearrangement analysis.

Adolescent ; Adult ; Child ; Female ; Follow-Up Studies ; Gene Rearrangement ; Humans ; Immunophenotyping ; Lymphoma, Extranodal NK-T-Cell ; genetics ; immunology ; pathology ; Lymphoma, T-Cell, Peripheral ; genetics ; immunology ; pathology ; Male ; Middle Aged ; Splenic Neoplasms ; genetics ; immunology ; pathology

OBJECTIVETo evaluate the diagnostic role of nuclear expression of bcl-10 protein in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) type.

METHODSOne hundred and forty cases of MALT lymphoma were collected from Cancer Hospital of Fudan University (including 38 cases from stomach, 35 cases from ocular adnexa, 16 cases from intestine, 15 cases from skin, 15 cases from salivary gland, 14 cases from lung, 3 cases from thyroid and 4 cases from other sites). Ten cases of reactive follicular hyperplasia of tonsil, 5 cases of reactive lymphoid hyperplasia of orbit and 143 cases of non-Hodgkin's lymphoma other than MALT lymphoma (including 20 cases of NK/T cell lymphoma, 20 cases of follicular lymphomas, 20 cases of anaplastic large cell lymphomas, 20 cases of nodal diffuse large cell B-cell lymphoma (DLBCL), 10 cases of gastric diffuse large B-cell lymphoma, 13 cases of nodal marginal zone B-cell lymphoma, 12 cases of mantle cell lymphoma, 11 cases of splenic marginal zone B-cell lymphoma, 6 cases of angioimmunoblastic T-cell lymphoma, 6 cases of peripheral T-cell lymphoma, not otherwise specified, 3 cases of small lymphocytic lymphoma, 1 case of lymphoplasmacytic lymphoma and 1 case of plasmacytoma were used as controls. Immunohistochemical study for bcl-10, as well as dual staining with CD20, was performed by EnVision method in paraffin sections.

RESULTSIn reactive follicular hyperplasia of tonsil, bcl-10 was moderately or strongly expressed in the cytoplasm of germinal center B cells, while the mantle cells were negative and the marginal zone cells and paracortical T cells showed weak staining. In the 5 cases of reactive lymphoid hyperplasia of orbit, 2 were bcl-10-negative and the remaining 3 expressed bcl-10 in the cytoplasm of germinal center B cells. As for non-MALT lymphomas, 3 gastric DLBCL showed nuclear expression. The remaining cases showed variable cytoplasmic staining. In some cases of lymphoma, bcl-10 was expressed in tumor cells but not in reactive lymphoid cells. On the other hand, 92.1% (129/140) of MALT lymphoma were bcl-10 positive. Among those cases, 54.3% (76/140) showed cytoplasmic positivity and 37.9% (53/140) showed nuclear positivity. The nuclear positivity rate of bcl-10 in different anatomic sites was different. The staining was most intense in MALT lymphoma of ocular adnexa. Dual staining with CD20 showed that the bcl-10-positive cells were also CD20-positive, though the number of bcl-10-positive cells were less than that of CD20-positive cells.

CONCLUSIONSBcl-10 expression in lymphoid hyperplasia is a universal phenomenon. Cytoplasmic expression of bcl-10 is seen in many different kinds of non-Hodgkin's lymphoma and reactive lymphoid conditions. In some cases of lymphoma, bcl-10 is expressed in tumor cells but not in reactive lymphoid cells, suggesting a possible role of abnormal bcl-10 expression in tumorgenesis. Nuclear expression of bcl-10 is seen mainly in MALT lymphoma, especially when occurring in ocular adnexa and lung. This is in contrast to loss of bcl-10 expression in residual germinal center cells.

Adaptor Proteins, Signal Transducing ; genetics ; Antigens, CD20 ; immunology ; B-Cell CLL-Lymphoma 10 Protein ; Cell Nucleus ; genetics ; Cytoplasm ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphocytes ; pathology ; Lymphoma, B-Cell, Marginal Zone ; genetics ; immunology ; pathology ; Palatine Tonsil ; pathology ; Pseudolymphoma ; genetics