Kappa Chain Gene Rearrangement Research Articles

Correlation between a 10-color flow cytometric measurable residual disease (MRD) analysis and molecular MRD in adult B-acute lymphoblastic leukemia.

Measurable residual disease (MRD) monitoring in acute lymphoblastic leukemia (ALL) is an important predictive factor for patient outcome and treatment intensification. Molecular monitoring, particularly with quantitative polymerase chain reaction (qPCR) to measure immunoglobin heavy or kappa chain (Ig) or T-cell receptor (TCR) gene rearrangements, offers high sensitivity but accessibility is limited by expertise, cost, and turnaround time. Flow cytometric assays are cheaper and more widely available, and sensitivity is improved with multi-parameter flow cytometry at eight or more colors. We developed a 10-color single tube flow cytometry assay. Samples were subject to bulk ammonium chloride lysis to maximize cell yields with a target of 1 × 106 events. Once normal maturation patterns were established, patient samples were analyzed in parallel to standard molecular monitoring. Flow cytometry was performed on 114 samples. An informative immunophenotype was identifiable in all 22 patients who had a diagnostic sample. MRD analysis was performed on 87 samples. The median lower limits of detection and quantification were 0.004% (range 0.0005%-0.028%) and 0.01% (range 0.001%-0.07%) respectively. Sixty-five samples had concurrent molecular MRD testing, with good correlation (r=0.83, p < 0.001). Results were concordant in 52 samples, and discordant in 13 samples, including one case where impending relapse was detected by flow cytometry but not Ig/TCR qPCR. Our 10-color flow cytometric MRD assay provided adequate sensitivity and good correlation with molecular assays. This technique offers rapid and affordable testing in B-ALL patients, including cases where a suitable molecular assay cannot be developed or has reduced sensitivity.

Extraosseous Plasmacytoma of Thyroid Arising in Hashimoto’s Thyroiditis

Plasma cell neoplasms of the thyroid gland are uncommon. They may occur either as a primary extraosseous (extramedullary) plasmacytoma or as secondary involvement by multiple myeloma (MM). Here, we report the case of a 62-year-old female, presenting with goiter and Hashimoto’s thyroiditis, in whom the histologic diagnosis of extraosseous plasmacytoma was unexpected. Histology of the total thyroidectomy specimen showed a diffuse infiltration of well-differentiated plasma cells against a background of Hashimoto’s thyroiditis. By immunohistochemistry, the majority of the plasma cells are positive for IgG heavy chain and kappa light chain (kappa:lambda ratio was about 6-7:1). PCR analysis of the immunoglobulin heavy and kappa chain (IGH, IGK) gene rearrangements showed clonal IGH and IGK gene rearrangements. MM was ruled out by lack of MM-related end organ damage and negative serum protein electrophoresis, immunofixation, and bone marrow biopsy. Although rare, plasmacytoma should be considered in patients presenting with enlarging thyroid gland and autoimmune thyroiditis. Histologic diagnosis and differential diagnoses are comprehensively discussed.

R-m-NHL-BFM-90/R-EPOCH Protocol in the Treatment of Patients with Primary Mediastinal Lymphoma: First Results

Background. Response to the induction chemotherapy is an important predictor of favorable outcome in aggressive lymphomas. As it was shown recently (Kurtz et al.2018) combination of interim PET/CT (iPET/CT) negativity and early molecular response detected by cell-free DNA (cfDNA) analysis, robustly stratify EFS and OS in patients (pts) with diffuse large B-cell lymphoma. There is no consensus on the most optimal program for primary mediastinal lymphoma (PML) treatment. Both R-CHOP+\-RT and R-DA-EPOCH are highly effective frontline options for PML, but about 10-15% of pts develop relapse/progression mainly within 2 years from diagnosis. We hypothesize that intensification of induction with 2 courses of modified R-NHL-BFM-90 program (R-m-NHL-BFM-90), followed by 4 courses of R-EPOCH without dose escalation might improve treatment outcome in PML. Aim. To assess the efficacy of R-m-NHL-BFM-90/R-EPOCH regimen for untreated PML pts by means of PET/CT and cfDNA evaluation. Methods. Overall, 18 pts were enrolled in prospective study between 2015 and 2019 years; median age 34 (19-50); M/F=4/14; Ann Arbor stage &gt;I in 18 (100%); adverse factors in 12 pts (bulky disease&gt;10 cm in 11 pts, involvement of the soft tissues in 9 pts and breast in 4 pts, pleural effusion in 6 pts, elevated lactate dehydrogenase level in 10 pts). The treatment plan consisted of: (i) induction of remission (blocks A and B of R-mNHL-BFM-90 program); (ii) consolidation with 4 courses of R-EPOCH. PET/CT was performed in all pts before therapy, after 2 courses of induction and after the end of therapy. B-cell clonality was assessed detecting immunoglobulin heavy chain gene rearrangements IGH (VH-JH FR2) and immunoglobulin light kappa chain gene rearrangement (IGK), by the use of multiplex Biomed-2 primer system for PCR and Gene-Scan analysis. cfDNA was obtained before the start of the treatment, after the induction and after the end of therapy in 4 pts. DNA concentration was measured by real time PCR with beta-globin primers. Results. All 18 pts completed induction phase. After 2 courses of R-mNHL-BFM-90 PET/CT Deauville score (DS) were DS1-3 in 9 pts (50%) and DS4 in 9 pts (50%). 16 pts completed treatment; from these 12 pts received all 4 courses of R-EPOCH. 2 pts received 2 courses and 2 pts only 1 course due to hematologic toxicity grade 3-4 and infectious complications. After the end of therapy PET/CT were DS1-3 in 15 out of 16 (94%) pts. Only 1 patient response was assessed as DS4; after she received autologous HSCT PET/CT negativity was achieved. cfDNA assessment at all stages of treatment was available in 4 patients (Table 1). In all cases before treatment there were no clonal rearrangements in blood samples. Clonal rearrangements in cfDNA before treatment and in tumor samples were the same in all cases. After induction treatment in one case B-cell clonality persisted in cfDNA, but after the end of the treatment there was no amplification of cfDNA from all pts. Overall, with a median follow-up of 14 months (range 1-40) 16/16 pts (100%) are alive in complete remission. Of note, all 4 pts who received only 1-2 courses of R-EPOCH are in complete remission for 4, 13, 16 and 17 months. Conclusions. Our results suggest that R-m-NHL-BFM-90/R-EPOCH protocol is highly effective for treatment of patients with PML. Given our results, we consider the possibility of reducing the number of cycles of chemotherapy. We suggest that detection of the tumor markers in cfDNA could be used as a biomarker to adapt treatment strategies in the future. Disclosures No relevant conflicts of interest to declare.

Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions.

Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains and probed the Protein Data Bank to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda, and heavy chain gene rearrangements are correlated within donors but can differ between donors. This indicates that terminal deoxynucleotidyl transferase may work with differing efficiencies between different people but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response.

Pax-5 is essential for kappa sterile transcription during Ig kappa chain gene rearrangement.

Pax-5 is the key regulator in B cell development. Pax-5-deficient mice show defects in B cell commitment and recombination of IgH chain gene rearrangement from DJ to VDJ. Previously, we found that Pax-5 bound to KI and KII sites, which play a crucial role in kappa-chain gene rearrangement. However, the function of Pax-5 in Ig kappa chain gene rearrangement has not been investigated. To address this issue, we newly established pre-BI cell lines expressing the pre-B cell receptor from Pax-5-deficient mice and used them in an in vitro culture system, in which kappa-chain gene rearrangement is induced by removing IL-7. By examining the Pax-5-deficient pre-BI (knockout (KO)) cells, we show in this study that, despite recombination-activating gene 1 and 2 expression, these KO cells did not rearrange the kappa-chain gene following the absence of kappa sterile transcription. Consistent with these data, fluorescent in situ hybridization analyses revealed that the J(kappa) locus in KO cells was located at the nuclear periphery as a repressive compartment. Transfection of KO cells with Pax-5 constructs indicated that the transactivation domain of Pax-5 was required for kappa sterile transcription and kappa-chain gene rearrangement. Moreover, the hormone-inducible system in KO cells demonstrated that Pax-5 directly functioned in kappa sterile transcription. These results indicate that Pax-5 is necessary for kappa sterile transcription during Ig kappa chain gene rearrangement.

Pax-5 is identical to EBB-1/KLP and binds to the VpreB and lambda5 promoters as well as the KI and KII sites upstream of the Jkappa genes.

During B cell differentiation, the pre-B cell stage plays a significant role in immunoglobulin gene rearrangement in the context of the allelic exclusion and kappa chain gene rearrangement. We previously reported that the early B cell-specific binding protein (EBB)-1 transcription factor binds to the promoters of two pre-B cell-specific genes, VpreB and lambda5, and regulates their pre-B cell-specific expression. Here, we demonstrate that EBB-1 binds to the KI and KII sites in the upstream of Jkappa region, which are crucial for kappa chain gene rearrangement. Gene transfer and gel-shift assays demonstrate that EBB-1 is identical to Pax-5 and binds to promoters of VpreB and lambda5 as well as the KI and KII sites. Our results suggest that Pax-5 plays an important role in the coordinate regulation of several immunoglobulin gene family members that are crucial in B cell development.

Primary Centroblastic/Centrocytic Lymphoma of the Skin Detection of B-Cell Monoclonality by Polymerase Chain Reaction

A 60-year-old woman was examined for erythematous plaques and nodules on the back that had appeared 1 year earlier. Histologic examination of a skin biopsy specimen showed a dense, diffuse infiltrate throughout the dermis and subcutis, composed mainly of centroblasts and centrocytes. Immunohistochemistry confirmed positivity of the neoplastic cells for L26 (CD20) and LN1 (CDw75) antibodies and negativity for UCHL1 (CD45RO), polyclonal anti-CD3, anti-kappa, and anti-lambda antibodies. Polymerase chain reaction (PCR) analysis on paraffin-embedded tissue sections of the cutaneous lesion showed immunoglobulin heavy-chain (VDJ) and kappa-chain gene rearrangement. Routine laboratory studies were within normal limits. The staging workup of the patient (bone marrow biopsy, computed tomographic scans of the chest and abdomen) showed no abnormalities. Based on clinicopathologic findings and molecular analysis, a diagnosis of primary cutaneous centroblastic/centrocytic lymphoma was made. The patient was treated with intra- and perilesionally administered recombinant interferon alpha-2a (3 x 10(6) IU) three times a week for 2 months. Complete response was achieved, and no evidence of recurrence has been observed after 18 months of follow-up.

Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.

Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replaced this enhancer by a neomycin resistance (neoR) gene in embryonic stem (ES) cells. B cells heterozygous for the mutation undergo V kappa-J kappa recombination exclusively in the intact Ig kappa locus but not in the mutated Ig kappa locus. Homozygous mutant mice exhibited no rearrangements in their Ig kappa loci. However, splenic B cell numbers were only slightly reduced as compared with the wild-type, and all B cells expressed lambda chain bearing surface Ig. These findings demonstrate that rearrangement in the Ig kappa locus is not essential for lambda gene rearrangement. We also generated homozygous mutant mice in which the neoR gene was inserted at the 3' end of the Ig kappa intron enhancer. Unexpectedly, mere insertion of the neoR gene showed some suppressive effect on V kappa-J kappa recombination. However, the much more pronounced inhibition of V kappa-J kappa recombination by the replacement of the Ig kappa intron enhancer suggests that this enhancer is essential for V kappa-J kappa recombination.

Vχ gene segments rearranged in chronic lymphocytic leukemia are distributed over a large portion of the Vχ locus and do not show somatic mutation

The structure of the human V kappa locus has been thoroughly investigated, but how the germ-line V kappa gene segment repertoire is actually sampled in kappa chain gene rearrangements is not known. In order to begin to answer this question we have polymerase chain reaction (PCR) amplified the rearranged V kappa genes from 26 kappa-expressing cases of chronic lymphocytic leukemia (CLL), followed by cloning and sequencing of the PCR product. All four V kappa gene families were represented amongst rearranged genes. In 25 out of 32 cases, the sequence of the rearranged gene matches perfectly that of 1 of 11 different known germ-line V kappa genes, indicating that no somatic mutation has occurred. Of the remaining 7 rearranged V kappa genes, 4 differ from known germ-line genes by only one or two amino acid residues; and 3 differ from each other and from all known sequences by 5 or more residues, suggesting that somatic mutation has occurred in these 3 cases. We conclude that: (a) in at least three-quarters of cases the rearranged genes are unmutated; (b) there is preferential usage of individual V kappa genes but not of V kappa gene families; and (c) the V kappa genes used are widely dispersed in the V kappa locus.

Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion.

Burnet's clonal selection theory suggests that each B lymphocyte is committed to a single antibody specificity. This is achieved by a programme of somatic rearrangements of the gene segments encoding antibody variable (V) regions, in the course of B-cell development. Evidence from immunoglobulin-transgenic mice and immunoglobulin-gene-transfected transformed pre-B cells suggest that the membrane form of the immunoglobulin heavy (H) chain of class mu (microns), expressed from a rearranged H-chain (IgH) locus, may signal allelic exclusion of the homologous IgH locus in the cell and initiation of light (L)-chain gene rearrangement in the Ig kappa loci. We report here that targeted disruption of the membrane exon of the mu chain indeed results in the loss of H-chain allelic exclusion. But, some kappa chain gene rearrangement is still observed in the absence of micron expression.

Lack of feedback inhibition of V kappa gene rearrangement by productively rearranged alleles.

Circular DNAs excised by immunoglobulin kappa chain gene rearrangements were cloned and characterized. 16 of 17 clones examined were double recombination products containing a V kappa-J kappa rearrangement (coding joint) as well as the reciprocal element (signal joint) of another V kappa-J kappa rearrangement. These products suggested multiple recombination, primary inversion, and secondary excision. In primary events, 5 of 16 translational reading frames were in-phase. Thus, V kappa gene rearrangement may not be inhibited by the presence of a productively rearranged allele. An unusually large trinucleotide (P) insertion forming a palindrome of 12 nucleotides was also observed in one of the coding joints.

A shared kappa reciprocal fragment and a high frequency of secondary Jk5 rearrangements among influenza hemagglutinin specific B cell hybridomas.

Ig kappa-chain gene rearrangement results in the displacement or loss of the DNA immediately 5' of Jk. This retained DNA is found on a different size fragment than in the germline (a reciprocal fragment), and contains the reciprocal joint of rearranged Vk and Jk genes, the back-to-back fusion of the heptamer/nonamer recombination signals. B cells of independent origin rarely have reciprocal fragments of the same size. However, we report that 9 of 15 B cell hybridomas of independent origin have reciprocal fragments of the same size (8-kb BamHI fragments) unrelated to their productive rearrangements. An 8-kb reciprocal fragment has also occurred on about 25% of the kappa alleles of normal splenic B cells. We find that the reciprocal fragments in two of these hybridomas contain the reciprocal joints of Jk1 genes and different Vk8 genes. In addition, we find that at least 8 of the 12 Jk4 or Jk5 expressing hybridomas have undergone double recombinations on their productive kappa alleles. The implications of these findings on the high frequency of 8-kb reciprocal rearrangements and on Vk rearrangement are discussed.

B-cell lymphoma marking only with anti-epithelial. Membrane antigen

A large cell malignancy identified as a B-lymphoma only by virtue of mu and kappa chain gene rearrangement additionally displayed profuse plasmalemma marking with anti-epithelial membrane antigen (EMA). Affinity absorption of the antibody with N-acetyl-galactosamine but not with N-acetylglucosamine or d-galactose effectively removed the lymphoma reactive antibodies. Soy bean agglutinin but not wheat germ agglutinin demonstrated lectin activity towards an epitope on the lymphoma membrane. Finally, digestion of cryostat sections of the lymphoma with N-acetylgalactosaminidase effectively removed the sugar epitope involved in EMA marking. Such data taken together would tend to indicate a membrane positioned complex carbohydrate with galactosamine dominancy. By analogy to the purported "late differentiation antigen secretory function" of EMA this lymphoma may be close to plasma cell stage.

Light-chain gene expression before heavy-chain gene rearrangement in pre-B cells transformed by Epstein-Barr virus.

Epstein-Barr virus transformation of B-cell-depleted bone marrow cells from human fetuses allowed us to identify novel cell types characterized by the expression of immunoglobulin kappa or lambda light chain without heavy chains. Four kappa-only clones with normal karyotype were obtained and examined for their immunoglobulin gene configurations and expression. All four clones had kappa-chain gene rearrangements at either one or both alleles, but the heavy-chain gene loci in these clones either were in germ-line context or had undergone only D-JH rearrangements (D and JH represent diversity and joining gene segments). All clones contained kappa mRNA of normal size at levels consistent with the protein level, except for one clone that no longer produced kappa protein. No mu mRNA or immunoglobulin heavy-chain molecules were detected in any of the kappa+ clones. The results suggest that the mu heavy-chain protein is not an obligatory prerequisite for light-chain gene rearrangements.

Rearrangement and expression of rabbit immunoglobulin kappa light chain gene in transgenic mice.

To determine whether a foreign unrearranged immunoglobulin gene can be functionally rearranged and expressed in vivo, a rabbit b9 kappa light chain gene construct containing a single germ-line kappa chain variable (V) region gene (V kappa), the five kappa chain joining (J) segments (J kappa), and the kappa chain constant (C) region germ-line gene (C kappa) was introduced into fertilized mouse eggs. Eleven transgenic mice carrying 1-30 copies of the rabbit kappa gene construct were obtained. Rearrangement of the transgene due to V kappa-J kappa recombination was observed in the spleen of all the mice lines analyzed. Only the J kappa 1 and J kappa 2 segments, which have canonical sequences for rearrangement and high-level expression, are utilized in assembly of the rabbit gene. V kappa-J kappa 1 and V kappa-J kappa 2 joining was also observed in the thymus but not in nonlymphoid tissue. Polyadenylylated rabbit kappa chain transcripts of 1.2 kilobases were found in the spleen of the transgenic mice. The level of transcription was low despite a high level of rearrangement. Three transgenic mice lines secreted kappa light chains encoded by the foreign rabbit gene. Serum rabbit kappa chains were associated with mouse mu and gamma 1 heavy chains. However, hybrid antibody molecules containing both rabbit and mouse kappa light chains were also found in the serum of these animals. These results suggest that, although the rabbit kappa chain gene construct contains the necessary sequences for gene assembly, sequences implicated in stage- and tissue-specific regulation of kappa chain gene rearrangement are either missing or not recognized by mouse lymphoid cells.

Heterogeneity of acute undifferentiated leukemia at the immunoglobulin and T-cell receptor genes level.

Phenotypic markers of leukemic cells from 76 children with acute leukemia were examined. Of these cases, 7 were diagnosed as acute undifferentiated leukemia (AUL) whose leukemic cells were negative for myeloperoxidase and did not react with lineage-specific or lineage-associated monoclonal antibodies. Then, we analyzed the configuration of both immunoglobulin (Ig) and T-cell receptor beta-chain (T beta) gene in these 7 AUL cases. Three cases had no rearrangement of Ig or T beta genes which was suggestive of non-lymphoid origin of these cases. In contrast, 4 cases showed rearrangements of Ig and/or T beta genes. One of these 4 cases demonstrated T beta gene rearrangement with retention of the germline configuration of Ig genes. Two cases with both Ig and T beta gene rearrangements also showed kappa-chain gene rearrangements. These findings indicate the heterogeneity of AUL at the DNA level, and may cast new light on the early differentiation of hematopoietic progenitor cells.

Kappa-chain gene rearrangement in an apparent T-lineage lymphoma.

We describe a 10-yr-old boy with T-lineage non-Hodgkin's lymphoma. He had a mediastinal mass, swollen supraclavicular lymph nodes, and pleural effusion. A supraclavicular lymph node biopsy under light microscopy showed a malignant lymphoma of diffuse lymphoblastic type. Most of the cells taken from the malignant pleural effusion expressed T cell-associated antigens such as Leu-1 and OKT 8. To confirm these antigens as T-lineage lymphoma, we examined genomic DNA from malignant cells obtained from the pleural effusion. As was expected, T cell receptor beta-chain gene rearrangements were demonstrated. However, when the immunoglobulin gene organization was analyzed, we detected rearrangements in both the heavy- and kappa-chain genes. To our knowledge, this is the first case in which kappa-chain gene rearrangement was detected in apparent T-lineage cells. These findings provide important information relating to determination of the cellular lineage of lymphoid malignancy.