Immunoglobulin lambda isotype gene rearrangements in B cell malignancies.

The human immunoglobulin lambda (IGL) locus contains seven J-Clambda gene regions of which only J-Clambda1, J-Clambda2 J-CA3 and J-Clambda7 encode the four Iglambda isotypes, ie Mcg, Ke-Oz-, Ke-Oz+, and Mcp, respectively. We used isotype-specific DNA probes for detection of IGL gene rearrangements in 212 B cell malignancies: 76 precursor B cell acute lymphoblastic leukemias (precursor B-ALL), 74 Iglambda+ chronic B cell leukemias (CBL), 34 Iglambda+ non-Hodgkin lymphomas (B-NHL), and 28 Iglambda+ multiple myelomas (MM). The J-Clambda3 gene region was most frequently involved (50%), followed by J-Clambda2 (38%) and J-Clambda1 (9%). There was no involvement of the J-Clambda4 and J-Clambda5 gene regions. Rearrangements to J-Clambda6 (n= 4) were exclusively found in precursor B-ALL (19% of all IGL rearrangements in precursor B-ALL) and only a single J-Clambda7 recombination was detected in an Iglambda+ B-NHL. In the group of Iglambda+ malignancies, a significant shift was observed from predominant J-Clambda3 usage (54%) in mature surface Iglambda+ malignancies (CBL and B-NHL) to 60% J-Clambda2 usage in Iglambda+ secreting MM. The distribution of IGL isotype rearrangements found in MM resembled the Iglambda isotype protein expression reported in MM patients. Based on these extensive Southern blot data, we suggest that a rapid and efficient detection of clonal IGL gene rearrangements can be obtained when a single Bg/II digest is used in combination with the IGLJ2 probe, which detects clonality in >95% of cases with an Iglambda+ malignancy. Higher percentages (>98%) can be reached by including a second digest (HindIII) that reduces the chance of comigration of rearranged and germline bands. In case of precursor B-ALL we recommend including the IGLJ6 probe for the detection of rearrangements to J-Clambda6.

Ordered recombination of immunoglobulin light chain genes occurs at the IGK locus but seems less strict at the IGL locus.

Regulation of allelic and isotypic exclusion of human immunoglobulin (Ig) light-chain genes was studied in 113 chronic B-cell leukemias as a "single-cell" model that allowed complete analysis of each light chain allele. Our data show that monospecific Ig light chain expression is in about 90% of cases determined by ordered recombination: Igkappa gene (IGK) rearrangements, followed by IGK deletions and Iglambda gene (IGL) rearrangements, resulting in the presence of only one functional Ig light chain rearrangement. In about 10% (10 cases), 2 functional Ig light chain rearrangements (IGK/IGL or IGL/IGL, but not IGK/IGK) were identified. This might be explained by the fact that regulation of the ordered recombination process is not fully strict, particularly when the IGL locus is involved. Unfavorable somatic mutations followed by receptor editing might have contributed to this finding. Eight of these 10 cases indeed contained somatic mutations. In cases with 2 functional Ig light chain rearrangements, both alleles were transcribed, but monospecific Ig expression was still maintained. This suggests that in these cases allelelic exclusion is not regulated at the messenger RNA level but either at the level of translation or protein stability or via preferential pairing of Ig light and Ig heavy chains. Nevertheless, ordered rearrangement processes are the main determinant for monospecific Ig light chain expression.

Identification of immunoglobulin lambda isotype gene rearrangements by Southern blot analysis.

The human immunoglobulin lambda (Ig(lambda)) gene locus contains seven homologous C(lambda) exons which are organized in a tandem array, each of which is preceded by a single J(lambda) gene segment. The J-C(lambda)1, J-C(lambda)2, J-C(lambda)3, and J-C(lambda)7 are functional gene regions and encode for the four Ig(lambda) isotypes, whereas J-C(lambda)4, J-C(lambda)5, and J-C(lambda)6 are non-functional (pseudo) Ig(lambda) gene regions. Recently, we demonstrated that Southern blot analysis with the IGLC3 probe in combined EcoRI/HindIII digests allows detection of approximately 95% of all clonal Ig(lambda) gene rearrangements in B cell malignancies. Although this single probe/enzyme combination is quite effective in detecting Ig(lambda) gene rearrangements, it should be noted that it results in a complex pattern of multiple germline bands of different density, which needs experience for correct interpretation. To improve further the reliable detection and identification of clonal Ig(lambda) gene rearrangements, we developed a new set of seven 'isotype-specific' DNA probes: the IGLC1D probe for the J-C(lambda)1 gene region, the IGLC2D probe for the J-C(lambda)2 gene region, the IGLJ2 probe for the highly homologous J-C(lambda)2 and J-C(lambda)3 gene regions, and the IGLC4D, IGLJ5, IGLJ6, and IGLJ7 probes for the last four J-C(lambda) gene regions, respectively. In combination with optimally chosen digests (ie HindIII, BglII, BamHI, and/or EcoRI) the seven probes indeed allow easy detection and identification of all rearrangements in the seven J-C(lambda) gene regions. The applicability of the probe/enzyme combinations was confirmed upon analysis of clonal 'Ig(lambda)-isotype' gene rearrangements in 40 B lineage malignancies.

Southern blot detection of immunoglobulin lambda light chain gene rearrangements for clonality studies.

Southern blot analysis of immunoglobulin (Ig) genes has proven to be important for detection of clonal rearrangements in patients with lymphoproliferative diseases. To improve the detection of clonal Ig lambda (Ig lambda) gene rearrangements, we carefully determined the precise restriction map of the J-C lambda gene region, developed a suitable C lambda probe (IGLC3), and evaluated relevant restriction enzymes in combination with the IGLC3 probe. For the latter purpose, we selected 75 B cell malignancies with proven expression of Ig lambda protein chains in order to be sure that each malignancy contained at least one clonally rearranged IG lambda allele. Our extensive Southern blot analyses with the IGLC3 probe in EcoRI and/or HindIII digests revealed that combined EcoRI/HindIII digestion detected Ig lambda gene rearrangements in 95% of the 75 patients and 94% of the 98 rearranged alleles. In contrast, HindIII and EcoRI single digests allowed detection of rearrangements in only 78% and 83% of the patients, and 67% and 79% of rearranged alleles, respectively. We conclude that the use of the IGLC3 probe in combined EcoRI/HindIII digests is superior to EcoRI and HindIII single digests. This probe/enzyme combination is informative for clonality studies in approximately 95% of patients.