Chromosomal Translocation & Rearrangement In The Pathogenesis Of B-cell Lymphoma

Analysis of Immunoglobulin heavy chain variable region (IgVH) genes in lymphomas has been ongoing since the 1980s, and it became apparent that this analysis could help calssify B-cell malignancies as pre-germinal center(GC), GC or post-GC derived. In 1999 it was discovered that chronic lymphocytic leukemia could be divided into two prognostic subgroups based on the presence or absence of somatic mutation. These studies renewed interest in the field and characterization of the Ig genes in different entities has been accomplished since then in larger cohorts of patients.The examination of the variable heavy (VH) chain of Ig gene, which is usually rearranged and expressed as a unique clonal surface Ig receptor in majority of B cell lymphomas is a useful approach to trace the stage of neoplastic transformation.Previous studies of diffuse large B-cell lymphoma (DLBCL) demonstrated that the majority of these tumors contain mutated VH genes in a pattern that suggested antigen selection pressure. However, it is not known whether the mutation process is still ongoing. What's more, it is unclear whether the presence of ongoing somatic mutation distinguishes between DLBCL derived from different stages of ontogenesisor whether it is a property of malignant cells irrespective of their developmental origin. To answer these questions, we divided our DLBCL cases into two subgroups and analyzed the relationship between DLBCL groups and the presence of ongoing mutation.Since the description of the translocation t(8;14) in Burkitt's lymphoma in 1972, evidence has accumulated that chromosomal translocations are prime importance in the genesis of B-cell lymphomas. Chromosomal translocations are closed associated with the morphologic features, immunophenotyping and clinical data of NHL cases. Chromosomal translocations usually result in activation of proto-oncogenes or creation of new fusion proteins that alter the expression of specific oncogenes.The most frequent translocation in human lymphoma is t(14;18), which is detected in 85%-90% of cases of follicular lymphoma(FL). The t(14;18) is believed to play a crucial role in the pathogenesis of FL because it deregulates the expression of the antiapoptotic gene BCL-2 by bringing it into proximity of the immunoglobulin heavy chain gene enhancer. It seems that deregulation of BCL-2 is more easily to establish a malignant phenotype, which is supported by data obtained from transgenic mice.The t(14;18) is considered to be the major pathogenetic mechanism in FL. The translocation is also demonstrated in up to 30% of DLBCL cases. Excluding known transformed FL, the translocation has been demonstrated in 18%-20% of primary DLBCL cases. The significance of the t(14;18) in DLBCL is unclear. Therefore we examined our patients with DLBCL to determine the role of the t(14:18) in the pathogenesis of DLBCL.The t(14;18) translocation, with downstream overexpression of the antiapoptotic BCL-2 protein, is the hallmark of FL. However, this potentially oncogenic mutation has also been identified in 50%-70% of healthy persons in North America and Western Eurepe. In the mutihit theory of tumorigenesis, translocation of the BCL-2 gene isconsidered to be the first somatic mutation, and additional mutations are needed for development of FL. From this viewpoint, the low incidence of FL in Chinese individuals may be due to the low frequency of BCL-2/IgH translocation in the general population. Accordingly, we investigated the relationship between the frequency of BCL-2/IgH translocation in peripheral blood cells of healthy Chinese individuals in Zhejiang area and the low incidence of FL.MATERIALS1. Blood samples: Blood samples were collected from 196 healthy Han nationality individuals in Zhejiang area.2. Lymphoma samples: 34 cases of primary DLBCL were obtained from our affiliated hospitals and other local hospitals in Zhejiang Province froml997-2004. All of the specimens were fixed in 10% formalin, embedded paraffin, and serial sections were made for hemotoxylin and eosin stain. All cases were grouped again according to the WHO classification of tumors of haematopoietic and lymphoid tissues (2001).METHODS1. DNA isolation: Genome DNA of mononuclear cells were salted out from blood samples and traditional hydroxybenzene-chloroform methods were performed to extract the genome DNA from lymphoma samples. ?2. Immunohistochemistry: We performed immunohischemical stains on paraffin-embedded tissues with a panel of antibodies (CD10, BCL-6 and MUM1). Immunohistochemistry results were used to subclassify the cases into germinal center (GC) subtypes and non-GC subtypes. Monoclonal antibody BCL-2 was also used on paraffin-embedded tumor tissues.3. Analysis of IgVH gene: After the detection of IgVH gene with primer of FR2A, direct DNA sequencing was performed. To evaluate for the presence of ongoingmutations in primary DLBCL, 5 GC subtypes of DLBCL specimens and 5 non-GC subtypes of DLBCL specimens selected randomly were examined. The VH chain amplicons obtained from a independent PCR reaction were cloned into a TA cloning vector. At least S molecular clones were sequenced per tumor sample.4. BCL-2/IgH gene translocation: Single-pair primer PCR was used to detect the BCL-2/IgH gene translocation in DLBCL tumor samples. Nested-PCR and direct DNA sequencing were used to detect the BCL-2/IgH gene translocation in peripheral blood cells.5. Statistical analysis: SPSS software was applied for statistical analysis. The results were analyzed by x2 test, Fisher's exact test and Spearman's rank correlation.RESULTS1. In all 34 DLBCL cases, 8 cases are considered to be GC subtypes and 26 cases are non-GC subtypes.2. In all 34 DLBCL cases, IgH gene clonality is detected in 22 of 34 cases by using primer FR2A. IgH gene clonality is detected in 5 of 8 GC subtypes of DLBLC cases.3. 23 VH genes are detected in 22 DLBLC cases;the VH genes are from 4/7 VH gene families with VH3, the largest family used most often. Analysis of VH mutations in comparison to the most closely related germline gene discloses that the average mutation frequency is 7.72%(range 0-18.6%).4. Four of the five cases classified as GC subtypes have ongoing somatic mutations. Whereas non of the 5 randomly selected non-GC subtypes of DLBCL cases shows ongoing somatic mutation. The difference between them is significant.5. Bcl-2/IgH translocation is present in 4 of 34 DLBCL cases. Among them, 3 cases belong to GC subtypes and 1 case belongs to non-GC subtypes.Two cases with a translocation express BCL-2 protein, however, the other two cases with atranslocation don't express BCL-2 protein.6. In our sample the frequency of Bcl-2/IgH translocation in Chinese individuals is 9.66%, much lower than that in North America and Europe countries.7. The breakpoints tend to fall into 3 clusters region: bp3055, 3116 and 3165. Usage of J6 segment is most common.8. There are biclonal lymphocytes of Bcl-2/IgH rearrangements in the same individual.CONCLUSIONS1. The presence of ongoing somatic mutation is a property of germinal cencer subtypes of DLBCL.2. Bcl-2/IgH translocation is a property of germinal center subtypes of DLBCL. The BCL-2 protein expression shows no dependence on the presence of BCL-2/IgH translocation.3. The low frequency of Bcl-2/IgH translocation in healthy Chinese individuals may be one of the reasons for the difference in the incidence of FL between China and Western countries.