Kappa and lambda determination by single colored immunohistochemistry and chromogenic in situ hybridization techniques in B-cell malignancies

Background: kappa and lambda light chains detection in bone marrow trephine sections help in the determination of B-cell clonality through evaluation of light chain restriction

Aim of the Work: was to compare the efficacy of single color detection-based immunohistochemistry [IHC] and chromogenic in situ hybridization [CISH] in evaluating kappa/lambda expression in tissues harboring B-lymphoid lesions

Patients and Methods: Forty patients were enrolled in this study. They were divided into three groups chronic lymphocytic leukemia [CLL/SLL] group I [n=13], non-Hodgkin lymphoma [NHL] group II [n=24] and hairy cell leukemia [HCL] group III [n=3]. The 24 NHL cases comprised of [11 diffuse large B-cell lymphoma, 6 mantle cell lymphomas, 3 marginal zone lymphoma, 2 lymphoplasmacytic lymphoma, 1 follicular lymphomas and 1 Burkitt's lymphoma]. Kappa and lambda light chains were detected in their bone marrow trephine sections using single colored immunohistochemistry, chromogenic in situ hybridization and the results were compared to the flowcytometry as reference method

Results: Light chain restriction [LCR] was detected by FCM in 100% of the cases followed by CISH [52.1%; 12/23] of the cases and finally IHC [43%; 18/40]

Conclusion: Both conventional CISH and IHC are effective in determining monoclonality in cases of mature B- cell neoplasm that has plasmacytic differentiation and with high amount of cytoplasmic Ig light chains such as MZL and LP. However, they are not effective in determining monoclonality in cases with low amount of Ig light chain such as cases of pregerminal and germinal center lymphoma. Yet, CISH is more informative than IHC due to the lack of background staining which allowed for greater discrimination between absence and presence of monoclonality

Application of BIOMED-2 primers in immunoglobulin gene rearrangement analysis of ocular adnexal lymphoma: a pilot study / 中华病理学杂志

<p><b>OBJECTIVE</b>To assess the practical value of BIOMED-2 primers in the diagnosis of ocular adnexal lymphoma by PCR.</p><p><b>METHODS</b>DNA was extracted from 63 formalin-fixed paraffin-embedded (FFPE) ocular adnexal lymphoma specimens. The DNA quality was evaluated by PCR-based amplification of housekeeping gene beta-actin. IgH(B) and IgK(B) primers of BIOMED-2 standardized clonality analysis system were used to evaluate the immunoglobin gene rearrangements. PCR products were analyzed using capillary electrophoresis and GeneScan software.</p><p><b>RESULTS</b>76.2% (48/63) of FFPE samples produced amplifiable DNA for detection of Ig gene rearrangements.Positive detection rates by BIOMED-2 IgH(B) and IgK(B) primers were 79.2% (38/48) and 68.8% (33/48), respectively, with a combined positive detection rate of 91.7% (44/48).</p><p><b>CONCLUSIONS</b>IgH(B) and IgK(B) primers of BIOMED-2 are suitable for the detection of clonal rearrangements of Ig gene using FFPE specimens of ocular adnexal lymphomas.</p>

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.

Diagnostic role of BIOMED-2 multiplex polymerase chain reaction for antigen receptor genes rearrangement in lymphoproliferative disorders / 中华血液学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Fifty-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.</p><p><b>RESULTS</b>Multiplex 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.</p><p><b>CONCLUSION</b>The novel BIOMED-2 multiplex PCR strategy is a rapid, reliable and sensitive approach to detecting clonality in suspected lymphoproliferations, especially in atypical cases.</p>

Assessment of BIOMED-2 assays for detection of clonal Ig gene rearrangements in mature B-cell lymphomas / 中华病理学杂志

<p><b>OBJECTIVE</b>To evaluate the efficiency of the BIOMED-2 PCR assay and its implication in the diagnosis of mature B-cell non-Hodgkin's lymphomas.</p><p><b>METHODS</b>Clinical, 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.</p><p><b>RESULTS</b>Adequate 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.</p><p><b>CONCLUSION</b>BIOMED-2 assay is highly sensitive and specific for the detection of clonal B cell gene rearrangement using routine paraffin embedded formalin fixed specimens.</p>

Distinction between lymphoma-like lesions and lymphoma of uterine cervix: a clinicopathologic study of 26 cases / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic features and differential diagnosis of lymphoma-like lesions and lymphoma of uterine cervix.</p><p><b>METHODS</b>Clinical data and hematoxylin and eosin-stained slides of 10 cases of lymphoma-like lesion and 16 cases of lymphoma of uterine cervix were reviewed. Immunohistochemical study for B- and T-cell markers and light chains (kappa, lambda) were performed on paraffin sections. The rearrangement status of immunoglobulin heavy chain (IgH) gene was analyzed with semi-nested polymerase chain reaction in 4 cases lymphoma-like lesion and 4 cases of lymphoma of uterine cervix.</p><p><b>RESULTS</b>The age of patients with lymphoma-like lesion ranged from 24 to 54 years (medium = 43 years). The lesion generally presented with cervical erosion or polyp. Microscopically, it is characterized by focal or diffuse superficial infiltration of immunoblast-like large B cells intermingled with a polymorphic population of inflammatory cells, including plasma cells, eosinophils and neutrophils. Maturation of the transformed large B cells was also noticed. On the other hand, the age of the patients with lymphoma of uterine cervix varied from 28 to 78 years (medium = 58 years). Cervical mass or diffuse enlargement of cervix were the commonest clinical findings. The cases included 12 examples of diffuse large B-cell lymphoma and 4 examples of follicular lymphoma. The former was characterized by a diffuse monomorphic population of large atypical lymphoid cells, while neoplastic follicles were identified in the latter. Neither polymorphic inflammatory infiltrates nor maturation phenomenon was found. The immunostaining for kappa and lambda light chains was inconclusive. Molecular study showed clonal rearrangement of IgH gene in all cases of cervical lymphoma, as well as 2 cases of lymphoma-like lesion.</p><p><b>CONCLUSIONS</b>The distinction between lymphoma-like lesion and lymphoma of uterine cervix depends primarily on the clinical and histopathologic features. Assay for rearrangement of IgH gene may be helpful in differential diagnosis, though monoclonality can be detected in some benign lesions as well.</p>

Use of immunoglobulin heavy chain and kappa light chain gene rearrangements by PCR for molecular diagnosis of minimal residual disease in Iranian children suffering from beta-precursor acute lymphoblastic leukemia

Diversity of IgH and IgK molecules is generated during B and T Lymphocyte differentiation through the rearrangement of variable, diversity, junction and constant gene segments. Additionally, random insertion and deletions of nucleotides between gene segments make unique sequences which are cell or clone specific. Similar IgH and IgK genes rearranged in normal cells of lymphoid leukemia cases can be used as a marker of clonality and for evaluation of minimal residual disease [MRD]. The purpose of this study is to evaluate the pattern of IgH chain and IgK gene rearrangements using polymerase chain reaction [PCR] in beta-precursor acute lymphoblastic leukemias [ALL] to follow the MRD at day 14, day 28 [end of remission induction], week 10, 3-6 months and 6-12. month after the initiation of treatment. In our prospective study bone marrow aspirates of 183 children at the mean age of 63.6 months with diagnosis of acute leukemia were collected at admission before any chemotherapy. After reviewing cytomorphology and immunophenotyping, only 140 cases with diagnosis of beta-precursor ALLs were selected for study. Mononuclear cells including leukemic blasts were isolated by density gradient. After DNA extraction, IgH and IgK [V[K] I-IV / Kde] were amplified by consensus primers using PCR. PCR products were analyzed after heteroduplex analysis and polyacrylamide gel electrophoresis [silver stain]. The DNA sequences were compared and aligned with the sequences homologous for IgH and IgK published by Gene Bank. The follow up specimens were collected at day 14, day 28 [end of remission induction], day 45-month 3, and 3-6 months and 6-12 months after initiation of treatment. After routine cytomorphologic analysis, similar PCR was done on follow up extracted DNAs in parallel with diagnosis DNA. MRD was considered to be approved positive if bands similar to those at the time of diagnosis were present. Statistical analysis using SPSS software [version 11.5] was performed. 90.5% of patients had clonal IgH gene rearrangements. Monoclonal, biclonal and oligoclonal patterns were observed in 57.8%, 34.9% and 5.5% of patients with IgH [CDR III] rearrangement, respectively. Clonal patterns of IgK-Kde were detected in 59 [67%; n: 88] of BP-ALLs. According to cytomorphology about 92% of patients were in complete remission. MRD positivity decreased from more than 90% to 20% using different gene rearrangements in defined time points. Four patients who relapsed during follow up were MRD positive using 1-3 rearrangements and all except one were in clinical remission. Clonal rearrangement of IgH had a pattern similar to other populations. IgK was slightly more frequent than previously reported and the VKI [25%] was the most common type. These differences can be explained by different techniques, DNAs and clonality markers. According to the results, these clonal markers can be used in diagnosis and follow up of MRD

Co-expression of immunoglobulin light chain kappa and lambda in gastric carcinoma cell / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the expression of immunoglobulin light chain kappa and lambda (Igkappa and Iglambda) in gastric carcinoma cell and their co-expression.</p><p><b>METHODS</b>Igkappa and Iglambda of 22 human gastric carcinoma specimens embedded in paraffin were monitored through immunohistochemical method-LSAB method.</p><p><b>RESULTS</b>Among 22 gastric carcinoma specimens, both Igkappa and Iglambda were positive in 17 (77.3%), only Igkappa was positive in 2 (9.1%), only Iglambda was positive in 1 (4.5%), both Igkappa and Iglambda negative in 2 (9.1%). The expression of Igkappa and Iglambda in human gastric carcinoma cell showed significant close correlation (chi(2) = 5.49, P < 0.05).</p><p><b>CONCLUSION</b>Co-expression of immunoglobulin light chain kappa and lambda in gastric carcinoma cell is common, which suggests that the activation mechanism of immunoglobulin gene in gastric carcinoma cell may be different from that in B-lymphocytes. Study on co-expression of immunoglobulin light chain kappa and lambda in gastric carcinoma is promising.</p>