Analysis of TCR gene rearrangements for diagnosis of T-cell lymphomas / 临床与实验病理学杂志

Purpose To discuss the TCR gene rearrangements in the diagnosis of T-cell lymphomas. Methods Formalin-fixed and paraffin-embedded samples including 30 cases of T-cell lymphomas and 30 cases of reactive lymphoid hyperplasia were chosen for ex-tracting genomic DNA and PCR amplification using 56 BIOMED-2 primers. PCR products were analyzed by heteroduplex and polyacryl-amide gel electrophoresis. Results In all 30 cases of T-cell lymphomas, 25 cases (83. 3%) showed TCRβ gene monoclonal rear-rangements, 28 cases (93. 3%) of TCRγ gene monoclonal rearrangements, 4 cases (13. 3%) of TCRδ gene monoclonal rearrange-ments. 29 cases (96. 7%) with TCRβ+TCRγ+TCRδ gene monoclonal rearrangements were detected. but no clonal TCR gene rear-rangements were found in 30 cases of reactive lymphoid hyperplasia. Conclusions The detection of TCR gene rearrangements using BIOMED-2 primers is a useful assistant method for the diagnosis of T-cell lymphomas.

Detection of immunoglobulin and TCR gene rearrangements by PCR using BIOMED-2 multi-plex protocols in T lymphoblastie lymphoma and acute lymphoblastic leukemia / 临床与实验病理学杂志

Purpose To investigate the sensitivity of BIOMED-2 primer system in T lymphoblastic lymphoma ( T-LBL) and acute lym-phoblastic leukemia ( ALL) patients immunoglobulin ( Ig) and T-cell receptor ( TCR) gene rearrangement, and to analyze the co-rear-rangement pattern. Methods Amplification of rearranged Ig and TCR gene was performed in standard PCR in 35 T-LBL/ALL pa-tients. PCR products were analyzed by heteroduplex and polyacrylamide gel electrophoresis. Results 16 cases (45. 7%) of 35 sam-ples were detected to have TCR gene rearrangements, including 6 cases (37. 5%) of TCRβgene monoclonal rearrangements, 4 cases (25. 0%) of TCRγ gene monoclonal rearrangements, 3 cases (18. 8%) of TCRβ and TCRγ gene double rearrangements, 2 cases (12. 5%) of TCRδ gene monoclonal rearrangements and 1 case (6. 3%) of TCRγand TCRδgene double rearrangements were detec-ted. 4 cases (11. 4%) of 35 samples detected to have clonal immunoglobulin and TCR gene rearrangements. 11 cases (39. 3%) of 28 T-LBL patients were detected to have TCR gene rearrangements, 6 cases (85. 7%) of 7 T-ALL have TCR gene rearrangements. Con-clusions BIOMED-2 multiplex PCR analysis strategy is a useful technique in the T-LBL patients.

Diagnostic Utility of a Clonality Test for Lymphoproliferative Diseases in Koreans Using the BIOMED-2 PCR Assay

BACKGROUND: A clonality test for immunoglobulin (IG) and T cell receptor (TCR) is a useful adjunctive method for the diagnosis of lymphoproliferative diseases (LPDs). Recently, the BIOMED-2 multiplex polymerase chain reaction (PCR) assay has been established as a standard method for assessing the clonality of LPDs. We tested clonality in LPDs in Koreans using the BIOMED-2 multiplex PCR and compared the results with those obtained in European, Taiwanese, and Thai participants. We also evaluated the usefulness of the test as an ancillary method for diagnosing LPDs. METHODS: Two hundred and nineteen specimens embedded in paraffin, including 78 B cell lymphomas, 80 T cell lymphomas and 61 cases of reactive lymphadenitis, were used for the clonality test. RESULTS: Mature B cell malignancies showed 95.7% clonality for IG, 2.9% co-existing clonality, and 4.3% polyclonality. Mature T cell malignancies exhibited 83.8% clonality for TCR, 8.1% co-existing clonality, and 16.2% polyclonality. Reactive lymphadenitis showed 93.4% polyclonality for IG and TCR. The majority of our results were similar to those obtained in Europeans. However, the clonality for IGK of B cell malignancies and TCRG of T cell malignancies was lower in Koreans than Europeans. CONCLUSIONS: The BIOMED-2 multiplex PCR assay was a useful adjunctive method for diagnosing LPDs.

Application of BIOMED-2 system for the detection of IGH gene rearrangement in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue lymphoma diagnosis / 白血病·淋巴瘤

Objective To evaluate the efficiency of BIOMED-2 system in detecting IGH gene clonal rearrangement and application in diagnosis of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) lymphoma. Methods Forty-five cases were collected, including 36 MALT lymphomas from different organs, 3 extranodal lymphoid tissue proliferative lesions and 6 severe gastritis associated with H pylori. DNA was extracted from the formalin fixed paraffin embedded blocks of these cases and the quality of DNA was assessed using the BIOMED-2 specimen mixed control primers. IGH gene clonal rearrangement was detected using IGH VH-JH primers. The sensitivity and specificity of BIOMED-2 PCR were analyzed.Results Adequate DNA (≥ 300 bp) was obtained in 31 of 45 samples (including 22 MALT lymphomas, 3 lymphoid tissue proliferative lesions and 6 severe gastritis), and the DNA from the other 14 samples was degraded seriously. 16 of 22 MALT lymphomas were positive of IGH clonal rearrangement with the sensitivity of 72.7 ％.In contrast,none of 6 severe gastritis was positive with the specificity of 100 ％ IGH and clonal rearrangement were detected in one of 3 lymphoid tissue proliferative lesions. Conclusion BIOMED-2 assay is an effient and reliable method for diagnosis and differential diagnosis of MALT lymphoma which is important for clinical practical value.

Detection Limit of Monoclonal B-Cells Using Multiplex PCR and Laser-Induced Fluorescence Capillary Electrophoresis

BACKGROUND: The identification of monoclonality has been widely used for making diagnoses of lymphoproliferative lesions. Awareness of the sensitivity and detection limit of the technique used would be important for the data to be convincing. METHODS: We investigated the minimum requirement of cells and sensitivity of gel electrophoresis (GE) and laser-induced fluorescence capillary electrophoresis (LFCE) for identifying IgH gene rearrangement using BIOMED-2 protocols. DNA extracted from Raji cells were diluted serially with peripheral blood mononuclear cells (PBMNCs) DNA. DNA from mixtures of diffuse large B-cell lymphoma (DLBCL) and reactive lymph nodes were also serially diluted. RESULTS: For Raji cells, the detection limit was 62 and 16 cell-equivalents for GE and LFCE, respectively. In the condition with PBMNCs mixture, 2.5% and 1.25% of clonal cells was the minimum requirement for GE and LFCE, respectively. In 23% of DLBCL cells in tissue section, the detection limit was 120 and 12 cell-equivalents for GE and LFCE, respectively. In 3.2% of DLBCL cells, that was 1,200 and 120 cell-equivalents for GE and LFCE, respectively. CONCLUSIONS: These results show that LFCE method is more sensitive than GE and the sensitivity of clonality detection can be influenced by the amount of admixed normal lymphoid cells.

Analysis of Immunoglobulin Gene Rearrangement: Comparison between BIOMED-2 Multiplex PCR and Conventional Nested PCR

BACKGROUND: Immunoglobulin (Ig) gene rearrangement analysis is a useful additional tool to detect clonality of B-lymphoproliferative disease and the method to detect immunoglobulin gene rearrangement is required the high sensitivity and specificity. BIOMED-2 multiplex PCR was designed for the evaluation of molecular clonality of lymphoid lesions. We evaluated the usefulness of the BIOMED-2 multiplex PCR by comparing it with conventional nested PCR. METHODS: Sixteen patients with malignant lymphoma and 5 with reactive lymph nodes were examined to assess the sensitivity, specificity, and accuracy between conventional nested PCR and BIOMED-2. All 3 tests performed using the BIOMED-2 kit for immunoglobulin (Ig) heavy chain (IGH) gene, Igkappa light chain (IGK) gene, and Iglambda light chain (IGL) gene, were used to analyze clonality. RESULTS: Both the methods showed 100% specificity (95% confidence interval, 56.6-100.0). The combination of IGH and IGK BIOMED-2 tests with or without IGL revealed the highest sensitivity (87.5%; range, 64.0-96.5%) and accuracy (90%; range, 0.70-0.97). Compared to the conventional method, the BIOMED-2 test for IGH showed a higher sensitivity (62.5%; range, 38.6-81.5%) and accuracy (71%, 0.50-0.86). CONCLUSIONS: These results suggest that, compared to the conventional method, BIOMED-2 has higher sensitivity and allows for easier interpretation while evaluating the clonality of B-lymphoproliferative disease.

Detection of immunoglobulin gene rearrangements by PCR using BIOMED-2 multiplex protocols in acute lymphoblastic leukemia patients / 中华微生物学和免疫学杂志

Objective To investigate the sensitivity of BIOMED-2 primer system in adult acute lymphoblastic leukemia (AIJ,) patients Ig gene rearrangement, and to analyze their frequency, corearrangement pattern, utilization of V, D and J genes and composition of junctional regions. Methods Amplification of rearranged IgH (complete and incomplete), IgK, IgK-Kde and IgL was performed in standard PCR in 29 adult ALL patients. Monoclonal PCR products were subjected directly to DNA sequencing. Sequences were identified by comparison with all known human Ig germline sequences to analyze the recombination patterns, somatic mutations and germline gene segments usage. Results IgH, incomplete IgH, IgK, igK-Kde and Igl, rearrangements were found with positive rate of 70.8%, 12.5% , 29.2% , 25.0% and 0 of B-ALL patients, respectively. All B-ALL patients displayed at least one pattern of Ig gene rearrangements. In TALL, one of five patients was found with incomplete IgH rearrangement, two patients were found with IgK rearrangements and two patients were PCR-negative. The sequence analysis showed that the most frequently used V, D, J segments in adult B-ALL patients were from VH3/VH4 families, DH3 family and JH6 family, respectively. Four of five IgK rearrangement used VκI family. 23.5% B-ALL IgH contained scattered replacement mutations with replacement to silent substitution ratio < 1 in complementarity determining regions. Conclusion BIOMED-2 multiplex PCR analysis strategy is a reliable and useful technique in the adult BALL patients.

A Case of Lambda-expressing Pulmonary MALT Lymphoma with Dual Clonal Rearrangements of Kappa and Lambda Immunoglobulin Light Chain Gene / 대한진단검사의학회지

A 70-yr-old woman was hospitalized with a history of dry cough. Bronchial endoscopy and transbronchial lung biopsy were performed. However, the findings of histopathology and immunohistochemistry were not sufficient to decide whether the lesion was benign or malignant, because of the presence of crush artifacts in the biopsy specimens. We performed B-cell clonality studies using BIOMED-2 multiplex PCR (InVivoScribe Technologies, USA) to detect clonal rearrangements in the immunoglobulin gene. The results of multiplex PCR showed clonal rearrangements of both kappa and lambda immunoglobulin light chain genes. The findings of immunochemistry revealed that the lesion expressed lambda light chain, but not kappa light chain. Based on the clinical, pathologic, and molecular findings, this case was diagnosed as pulmonary MALT lymphoma. We report the first case in Korea of lambda-expressing MALT lymphoma that is shown to have dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene by multiplex PCR.