Application Of Ig Light Chains Clonal Rearrangement Analysis In B Cell Lymphomas

Diagnosis of lymphomas is mainly based on morphological and immunohistochemical techniques due to it's many subtypes and classification, Southern blot analysis was considerd as gold standard for diagnosis of lymphoma, however there are some pitfalls of these methods. Morphology and immunohistochemitry is helpful in diagnosis of lymphomas, but there are still difficulties for diagnosis of some cases. It is good specificity and sensitivity of Southern blot analysis for diagnosis of lymphoma by molecular pathological techiques; however Southern blot analysis (SBA) of the IgH gene was one of the first molecular genetic tools used in the diagnostic scenario. However, given the numerous limitations associated with SBA, over the last few years it has been supplanted by polymerase chain reaction (PCR) analysis of this locus.PCR is given all of the features that identify PCR as a more practical test for most diagnostic laboratories (including faster turn-around time, lower cost, and absence of reliance on radioactive materials).The diagnosis of lymphoid malignancies can be supported by clonality assessment based on the fact that, in principle, all cells of a malignancy have a common clonal origin. Normal B and T cells undergo immunoglobulin gene rearrangement and T cell receptor gene rearrangemtn. Lymphoinoid reactive proliferation diseases demonstrate polyclonal Ig gene rearrangement and (or) TCR gene rearrangement. PCR analysis is todetect Ig gene rearrangement and TCR gene rearrangement.Some problems fall in molecular diagnosis of lymphoma in clinical practice: Firstly, the Ig heavy (IgH) chain gene has been the preferred target because most B cell tumours have rearranged IgH genes and becomes a diagnostic tool widely used in the investigation of B-cell lymphomas. The majority of lymphoid malignancies encountered are of B cell lineage. Consequently, analysis of immunoglobulin (Ig) gene rearrangements is one of the most frequently ordered molecular hematopathology assays and the major reason: the IgH gene rearranges before the light chain genes and some B cell malignancies, typically precursor lymphoblastic leukemias and lymphomas, have not yet rearranged their light chain genes. Some B cell malignant lymphomas such as precursor lymphocytoblast leukemias and lymphomas.Secondly, some difficulties exist in application of IgH rearrangement analysis of lymphomas, such as false positivity, false negativity, biclonal rearrangement and oligoclonal rearrangement. Countires are seeking some measures to solve these problems. BIOMED-2 Concerted Action BMH4-CT98-3936 project is one measure, 7 European countries including 47 institutes are involved. The aim of BIOMED-2 is to standardize and normalize, but it' s too complicated, the PCR systems are involved too much primers such as 21 primers for IgH PCR. The researchers was performing IgH rearrangement analysis of lymphomas, So a few researches were focused on IgL rearrangement analysis.In theory, SHM of CDRs in IgL was less than that in IgH. Our laboratory tries to seek significance of IgL rearrangement analysis of lymphomas.Thirdly, combination FR2A and FR3A PCR is a simple and effective assay of IgH gene rearrangement, but overall sensitivity of these approaches does not exceed 80% even when using multiple primer sets, particularly in germinal center (GC) and post-GC B-cell origin lymphomas, ranging from 50% to 76%.The failure of IgH gene rearrangements detection in this significant proportion of B-cell lymphomas is mostly due to somatic hypermutaion, chromosomal translocation, incomplete translocation and other technical factors, etc.SHM of Ig gene allows antibody and target antigen affinity mature and lead to Bcell selection. The SHM maybe occurred FR, and FR is the target of the majority family and universal primers. Mutation causes loss of some target sequence and change of location of target and finally impacts optimal primers match. Universal and family primers in generally are optimal for considerable genes but show relatively low homology for some genes.Concurrent concept of lymphoma is malignant, so precise diagnosis and classification of lymphoma can firmly support for effective treatment. When researchers were encountered problems of low detection rate of clonal IgVH gene rearrangement, people focused on IgL gene rearrangement analysis recently.In theory,V region of IgH is mainly antigen binding site, so it's more variable or SHM than that of IgL. So amplification of IgL gene rearrangement is an ideal aid to IgH gene rearrangement analysis. Some reports provide information IgL can improve detection rate of B cell lymphoma.It had been reported of dual rearrangements in lymphoma long before. It means both IgH and TCR gene clonal rearrangements simultaneously occur in a simple. Furthermore, it is named biclonal rearrangement which refers to 2 alleles of IgH simultaneously rearrange in B-NHL, so is T-NHL. The notion of dual rearrangements in our study is the former. More attention is recently paid to dual rearrangements. Both at home and abroad there are little papers about whether lymphomas of dual rearrangements by PCR method take place dual rearrangements truly and what pathogenesis is.PCR analysis of IgH and TCRγ gene rearrangement had been carried out in 77 cases of B-NHL to evaluate detection rate of clonality gene rearrangement in our lab. Addition of IgL gene rearrangement assay to IgH gene rearrangement assay and to evaluate the significance of IgL gene rearrangement analysis in B-NHL.Materials and Methods Materials1. NHL specimens: 77 cases of B-NHL were obtained from our affiliated hospitals and other local hospitals from 1997 to 2007. All specimens were fixed in 10% formalin and then embedded in paraffin. Sections were stained by hemotoxylin and eosin and by IHC. All cases were confirmed as B- NHL on the basis of their immunophenotypes and morphologies according to the WHO classification.2. Control specimens: 2 cases of reactive lymph nodes from our affiliated hospitals. Methods1. DNAs from paraffin embedded tissues were prepared by phenol-chloroform extraction method. DNA was extracted from peripheral blood specimen by high salt method.2. Our study analyzed the rearrangement pattern of IgH and TCR gene with primers of FR3A, FR2A and TCRγ in a group of 77 B-NHLs. PCR products were evaluated using firstly 2% agarose gel and then 8% denaturing polyacrylamide gel for electrophoresis.3. IgK PCR analysis was performed in a group of 77 B-NHLs. PCR products were evaluated using 8% denaturing polyacrylamide gel for electrophoresis and sequencing was followed.4. IgL PCR analysis for cases of dual rearrangement PCR products were evaluated using 8% denaturing polyacrylamide gel for electrophoresis and sequencing was followed.Results1. The detection rate of IgH gene clonality was 62.3% (48/77) by primer FR3A B-NHL; using the combination of primer FR3A and FR2A, the detection rate of IgH clonality increased to 75.3% (58/77).2. The combination of the two IgH gene primers with the multiplex TCRγ gene PCR allowed preliminary detection of dual rearrangements in 6.5% (5/77) of NHL, and verified by sequencing. The cases of dual rearrangement were B cell immunophenotype by IHC. Of 5 cases, there were 2 DLBCLs, 2 MALT-MZLs and 1 FL.3. The detection rate of Igκ gene clonality was 11.7% (9/77), detection rate of Igλ gene clonality was 6.5% (5/77) and detection rate of IgL gene clonality was 18.2%(14/77),Combination of IgH and IgL gene clonality analysis increased the detection rate from 75.3% to 84.5%. Conclusions1. The detection rate of IgH gene clonality is 62.3 % by primer FR3A in B-NHL; with the combination of primer FR3A and FR2A, the detection rate of IgH clonality increases to 75.3%.2. The detection rate of Igκ gene clonality was 11.7% (9/77) , detection rate of Igλgene clonality was 6.5%, detection rate of IgL gene clonality was18.2%. Combination of IgH and IgL gene clonality analysis increased the detection rate from 75.3% to 84.5%.3. The combination of the two IgH gene primers with the multiplex TCRγ gene PCR allowed detection of dual rearrangements in 6.5% of NHL. Pathological types are DLBCL, MALT-MZL and FL.4. IgH gene and TCR gene rearrangements must be detected simultaneously in a suspect sample in order to fidelity and integrity result.