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

Diagnosis, Differential ; Gene Rearrangement, B-Lymphocyte ; Gene Rearrangement, T-Lymphocyte ; Humans ; Lymphoma ; diagnosis ; genetics ; Lymphoma, Follicular ; diagnosis ; genetics ; Lymphoproliferative Disorders ; diagnosis ; genetics ; Polymerase Chain Reaction ; methods

OBJECTIVETo explore the feasibility of detecting lymphoma with the application of BIOMED-2 standardized immunoglobulin/T cell receptor (IG/TCR) gene rearrangement system in formalin fixed paraffin-embedded (FFPE) tissue samples, and to discuss the relationship between the longest amplification fragment of extracted DNA and positive detection rate of different IGH V-J primers.

METHODSDNA was extracted from 50 cases of FFPE tissue samples. Multiplex-PCR amplifications were performed and then the IG/TCR gene rearrangements were analyzed using BIOMED-2 standardized clonality analysis system.

RESULTS(1)When the DNA concentration was diluted to 50-100 ng/μl from 100-500 ng/μl, the proportion of the longest amplification fragment (300-400 bp) of DNA was improved from 10.0% to 90.0% in 30 cases of diffuse large B cell lymphoma (DLBCL) wax roll samples (P<0.01). The positive rate of IGH+IGK was increased from 46.7% to 83.3%, the difference was statistically significant (P=0.006). The lengths of the longest amplification fragments of DNA were all longer than 300 bp in the paraffin section samples of DLBCL. The positive rate of IGH+IGK of these samples was 96.7%. The difference of the positive rate of IGH+IGK between the wax roll samples and the paraffin section samples had no statistical significance (P=0.195). (2)When the concentration of DNA was high, most of the longest amplification fragments of extracted DNA were 100 bp or 200 bp, and the detection rate of short fragment IGH FR3 was more stable than that of long fragment IGH FR1. (3)The clonality analysis of TCRG+TCRB in all 13 cases of peripheral T cell lymphoma samples showed positive results, while no positive IG/TCR clones were found in 7 cases of reactive lymphoid tissue hyperplasia in control group.

CONCLUSIONDilution of DNA is the only method to improve not only the proportion of longest fragment amplification but also the detection rate of clonality. The detection rate of IGH FR3 would not be affected by the concentration of DNA. The application of BIOMED-2 standardized IG/TCR gene rearrangement system in FFPE tissue samples plays an important role in the lymphoma diagnosis.

Gene Rearrangement, T-Lymphocyte ; Humans ; Lymphoma ; diagnosis ; genetics ; Paraffin Embedding ; V(D)J Recombination

OBJECTIVETo establish a sensitive and effective method for detection of immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangement,and to explore its role in diagnosis and differential diagnosis of lymphoproliferative disorders.

METHODSFifty-eight lymphoid tissue samples from 54 patients with lymphoproliferations were evaluated by the novel BIOMED-2 multiplex polymerase chain reaction (PCR) for antigen receptor genes rearrangement.

RESULTSMultiplex PCR demonstrated monoclonal Ig/TCR gene rearrangements in 22 of 25 (88.0%) B-cell malignancies and 8 of 15 (53.3%) T-cell malignancies. Among 17 benign lymphoproliferations confirmed histopathologically, polyclonal rearrangements were detected in 14 cases (82.4%). In total, the clonality analysis and the final clinico-histopathological diagnosis were concordant in 77.2%. Combination detection of Iglambda and TCR delta gene rearrangements did not increase the detection rate of monoclonal rearrangement of Ig/TCR, but might help to the detection of Iglambda+ or TCR delta+ lymphomas.

CONCLUSIONThe novel BIOMED-2 multiplex PCR strategy is a rapid, reliable and sensitive approach to detecting clonality in suspected lymphoproliferations, especially in atypical cases.

Female ; Gene Rearrangement, B-Lymphocyte, Light Chain ; Gene Rearrangement, T-Lymphocyte ; Humans ; Lymphoproliferative Disorders ; diagnosis ; genetics ; Male ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo evaluate the significance of T-cell antigen receptor (TCR) gene rearrangement among patients with acquired pure red cell aplastic anemia (A-PRCA).

METHODSFor 16 patients with A-PRCA, an immunosuppressive regimen based on cyclosporin A (CsA) was applied. Rearrangement of the TCR gene was detected by PCR, and T lymphocyte subsets in peripheral blood specimens was detected with flow cytometry.

RESULTSFive patients had presented with TCR clonal rearrangement and were positive for both TCR γ and TCR δ. The blood of 13 patients have returned to normal with the treatment, which included 3 cases with bone marrow returning to normal. In 7 cases, the red cell hyperplasia of bone marrow is still down, but has increased with the treatment. Three patients were close to cure, 7 showed remission, 3 were improved, but 3 were ineffective. The rate of effective treatment in those with TCR rearrangement (2/5) was significantly lower than that those without (11/11, chi-square=8.123, P < 0.05). Compared with those without the TCR gene rearrangement, the Th cells and proportion of Th/Ts were significantly lower, while the Ts cell (CD3+CD8+) were significantly higher in those with the rearrangement (P < 0.05).

CONCLUSIONTCR gene rearrangement may play a role in the pathogenesis of A-PRCA. CsA is effective for the treatment of A-PRCA, but patients presenting clonal TCR gene rearrangement may response poorly to the treatment.

Adult ; Aged ; Female ; Gene Rearrangement, T-Lymphocyte ; Humans ; Male ; Middle Aged ; Red-Cell Aplasia, Pure ; etiology ; genetics ; immunology

Gene Rearrangement, T-Lymphocyte ; Genes, T-Cell Receptor ; genetics ; Genotype ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Lymphoma, Non-Hodgkin ; genetics ; Polymerase Chain Reaction ; methods

OBJECTIVETo investigate immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements in bone marrow or peripheral blood of patients with non-Hodgkin's lymphoma (NHL), and to explore the potential clinical significance.

METHODSThe Ig/TCR gene rearrangements in bone marrow or peripheral blood of 139 NHL patients were analyzed by using BIOMED-2 multiple primers system and Multiplex PCR assay.

RESULTSIg clonality was detected in 85.4% (70/82) of chronic lymphocytic leukemia (CLL), including 46.3% (38/82) IgH rearrangement, 62.2% (51/82) IgK rearrangement and 1.2% (1/82) IgL rearrangement, and in 39.4% (13/33) of other categories of B-lineage NHL (B-NHL), including 33.3% (11/33) IgH and 39.4% (13/33) IgK rearrangements. TCR clonality was detected in 50.0% (12/24) of all definite T-lineage NHL (T-NHL), including 8.3% (2/24) TCRB and 45.8% (11/24) TCRG, no TCRD was detected. The detection rate of gene rearrangements of NHL diversed in different clinical stages \[36.4% in early stage (Ann Arbor stage I and II) and 45.6% in late stage (III and IV)\] and in different degrees of malignancy (40.0% indolent NHL and 45.6% aggressive NHL), but no obvious statistical significance was obtained (P > 0.05). The detection rate of bone marrow invasions of NHL (except CLL) with examinations of bone marrow smear under the microscope was 12.3% (7/57), much lower than the clonality testing (43.9%, 25/57) (P < 0.05). Sensitivity test showed that the sensitivity of malignant clonality testing by multiplex PCR was 3.12% - 6.25%.

CONCLUSIONSThe detection rate of gene rearrangements diverses in different clinical stages and degrees of malignancy of NHL, but the correlation has not been proved in this research. The sensitivity of multiplex PCR-based clonality testing is enhanced with the combination of BIOMED-2 primers system. It is more sensitive than the morphological examinations of bone marrow smear in detecting bone marrow invasions, and may provide a powerful strategy in the routine diagnosis and assessment after treatment.

Adult ; Aged ; Aged, 80 and over ; Female ; Gene Rearrangement, T-Lymphocyte ; Genes, T-Cell Receptor ; genetics ; Humans ; Immunoglobulins ; genetics ; Lymphoma, Non-Hodgkin ; genetics ; Male ; Middle Aged ; Polymerase Chain Reaction

Child ; Gene Rearrangement, T-Lymphocyte ; Humans ; Neoplasm, Residual ; diagnosis ; Polymerase Chain Reaction ; methods ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; Receptors, Antigen, T-Cell ; genetics

OBJECTIVETo provide the evidence of RQ-PCR-based assessment of minimal residual disease (MRD), the clonal immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements were identified in newly diagnosed adult patients with acute lymphoblastic leukemia (ALL) by multiplex PCR protocols.

METHODSForty newly diagnosed adult patients with B-lineage (B-) and T cell (T-) ALL were involved in this study. All DNA samples were obtained from the bone marrow (BM) mononuclear cells (MNC). IgH, IgK, TCRB, TCRG and TCRD gene rearrangements were detected by BIOMED-2 multiplex PCR protocols, which included 96 different primers and 14 multiplex PCR tubes.

RESULTSThe clonal immunoglobulin (Ig) rearrangements were found in 96% of B-ALL, 86% being IgH and 14% IgK. While in T-ALL, clonal TCR rearrangements were found in all of the patients, 83% being TCRB, 78% TCRG and 39% TCRD. More than two clonal markers were found in 91% of B-ALL and 89% of T-ALL patients.

CONCLUSIONSThe detection rate of clonal rearrangements using the BIOMED-2 14 multiplex PCR tubes is high, which can detect virtually all clonal B and T-cell proliferations. It can be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Adult ; Gene Rearrangement, T-Lymphocyte ; Humans ; Immunoglobulins ; genetics ; Neoplasm, Residual ; diagnosis ; genetics ; Polymerase Chain Reaction ; methods ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics

The purpose of this study was to analyze the gene rearrangement pattern of immunoglobulin and T-cell receptor (Ig/TR) and its clinical characteristics in three children with SET-NUP214 fusion gene positive leukemia/lymphoma. The transcript of SET-NUP214 fusion gene was detected by RT-nested PCR. The pattern of Ig/TR gene rearrangement was analyzed by using the BIOMED-2 multiplex PCR assays. Allelic-specific primers were designed for further monitoring the minimal residual disease (MRD). The results indicated that the fusion site located between exon 7 of SET and exon 18 of NUP214 at mRNA level in the three patients. The diagnoses were made as the mixed phenotype of acute leukemia (MPAL) for patients 1, acute T-lymphoblastic leukemia (T-ALL) for patients 2, and stage IV T-lymphoblastic lymphoma (T-LBL) for patients 3, respectively. Patient 1 responded to chemotherapy very poorly and relapsed at month 6 after hematopoietic stem cell transplantation. Patient 2 had high MRD (> 10(-2)) at the end of inducing remission therapy (day 33) which implied poor outcome, and died of toxic epidermal necrolysis and sequent serious infection. Patient 3 achieved hematological complete remission (CR) and MRD negative at day 15 and day 33 respectively. The duration of CR lasted for 30 months. Clonal TR gene rearrangements were detected in all the three patients. The rearrangements of TRD, TRG and TRB were found in patient 1 and 3. The rearrangements of TRD, TRB, IgH and IgK Kde were detected in patient 2. All the 6 TRB rearrangements detected were incomplete rearrangements, whereas 85.7% and 14.3% of the TRD, and TRG rearrangements were complete and incomplete, respectively. It is concluded that the transformation of SET-NUP214(+) leukemia/lymphoma cells may occur after the rearrangements of TRD and TRG and shortly after TRB rearrangement. The leukemia/lymphoma cells of patient 1 and 2 are more immature which may be related with poor outcome or response to chemotherapy.
Child ; Female ; Gene Fusion ; Gene Rearrangement, T-Lymphocyte ; Histone Chaperones ; genetics ; Humans ; Immunoglobulins ; genetics ; Male ; Neoplasm, Residual ; diagnosis ; genetics ; Nuclear Pore Complex Proteins ; genetics ; Oncogene Proteins, Fusion ; genetics ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Transcription Factors ; genetics