Characteristics Of IgVH Gene Structure In Small B-cell Lymphomas And Diffuse Large B-cell Lymphomas

Immunoglobulin (Ig) is a very important molecule produced by B cell. The Immunoglobulin heavy chain variable region (IgVH) is formed during B-cell ontogeny by an ordered process of Ig gene rearrangement leading to the assembly of distinct variable(V), diversity(D), and joining(J) gene segments. An additional process of sequence diversification by somatic hyper mutation following antigen encounter occurs in B cells proliferating within the microenvironment of the germinal center (GC) and it increases the immunoglobulin affinity for antigen. Nowadays it is widely accepted that B cell can be divided in to three types according to their SHM states: (1) Pre-germinal center B cells usually exhibit unmutated germinal receptors;(2) Antigen-activated proliferating germinal center B cells show high mutation rates and evidence of ongoing mutations;(3) Post-germinal center memory B cells show mutated but stable receptors. Correspondingly, when B cells neoplastic transformation has occurred, the tumor cells have the same characteristics as their normal counterparts. Because somatic hyper mutation appears to be restricted to Bcells proliferating within the microenvironment of the germinal center, somatically mutated V-region genes are a hallmark of germinal center B cells and their descendants. It has been reported that VH gene familys' distributions are different in some kinds of lymphomas, that means in these kinds of lymphomas, biased VH gene families are used and deeper research of the effect of this moleculargenetic characteristic in the development of lymphomas is worth while. Our study was aimed at acquiring insight into the nature of the immunoglobulin heavy chain variable region sequences in small B-cell lymphomas (SBCLs) and diffuse large B cell lymphomas (DLBCL), to get the relationship between the VH gene structure and development of lymphomas.Material and Methods:1 Material: 110 cases of NHL containing 71 SBCLs and 39 DLBCL were obtained from our affiliated hospitals and other local hospitals from the year of 1997 to 2005. All specimens were fixed in 10% formalin and then embedded in paraffin. Sections were stained by hemotoxylin and eosin and for immunohistochemistrical detection.2 Methods(1) ImmunohistochemistryCD20 (L26) and CD45RA (4KB5) were used to mark the 39 DLBCL and 71 SBCLs for definite diagnosis. Six markers (CD23, CD5, CyclinDl, IgD, BCL-6, FDC) together with the morphological representation were used to distinguish the subgroups of SBCLs.(2) PCR amplification and cloningA seminested strategy was used for the PCR amplification of the VH genes using the FR2A consensus primer, complementary to the conserved framework-2 (FR2) of the variable region, and the LJH and VLJH consensus primers for the J region. Theprimer sequences were as follows: FR2A, 5'- tgg a/gtc cgc/a cag g/cct/c t/ccn gg-3';LJH, 5'-tga gga gac ggt gac c-3' and VLJH, 5'-gtg ace agg gt(a/g/c/t) cct tgg ccc cag-3'. Patient samples were analyzed together with DNA from the Raji cell line as positive control and with a negative control containing all PCR reagents without any template. All those samples amplified successfully were directly sequenced in both directions.Cloning of monoclonal cases was performed with the pGEM T-Easy vector (Promega) using DNA excised and purified from monoclonal bands in the polyacrylamide gels. At least 5 clones were selected at random. Ongoing somatic hypermutation frequency (%) was computed by the following equation: Ongoing somatic hypermutation frequency = somatic mutations / (total nucleotides x total clones).(3) Sequence analysisThe DNA sequences were analyzed for homoiogies in the software, available at the National Center for Biotechnology Information (NCBI). Homology with grem line VH, DH, JH genes were identified using the V-Base database and the IgBlast software of the NCBI.(4) Analysis of intraclonal heterogeneityTo evaluate the presence of ongoing mutations in lymphomas, at least 5 clones from each specimen were selected and sequenced. For evaluation of intraclonal heterogeneity, the following definitions were used: unconfirmed mutation, a substitution mutation observed in only one of the VH clones from the same tumour specimen;comfirmed mutation, a substitution mutation observed in more than one of the VH gene molecular clones from the same tumor specimen. Only the confirmed mutations were considered as evidence of intraclonal heterogeneity;the unconfirmed mutations were instead ascribed to Taq polymerase error.(5) Antigen selectionTo determine the pattern of somatic mutations compatible with antigen selection, 2 different methods were applied. First, the ratio of replacement-silent mutation(R/S) in the CDR2 and FR3 regions was studied. A sequence was considered to be antigen selected when the R/S ratio in the CDR2 was higher than 2.9 and the R/S ratio in the FR3 region was lower than 1.5. Second, only the R/S ratio of the somatic mutations in the FR3 region was considered. A sequence was regarded as antigen selected when the R/S ratio was less than 1.6.ResultsOf 71 case samples of SBCLs, there were 37 marginal zone B-cell lymphomas (MZL) of mucosa-associated lympohoid tissue (MALT) , 9 DLBCL with MALT component, 7 follicular lymphomas (FL), 9 mantle cell lyphomas (MCL), 2 nodal marginal zone B-cell lymphomas (NMZL), 3 splenic marginal zone lymphomas (SMZL) and 4 undetermined cases. The VH gene segments of 37 MALT lymphomas, 9 DLBCL with MALT component and 39 DLBCL were all amplified by PCR, and at last 10 MALT lymphomas and 10 DLBCL got their VH gene successfully amplified and sequenced directedly. We examined MALT and DLBCL component separately from tissue sections of those DLBCL with MALT component, and all the specimens amplified successfully were cloned and sequenced. This process was only successful in 1 case with its MALT cell population and the other with its DLBCL cell population. Other 4 MALT lymphomas and 5 DLBCL were also cloned and sequenced in order to evaluate the presence of ongoing mutations in these lymphomas.(1) In MALT lymphomas, there were 7 cases with VH3 gene faminy, 3 cases with VH4 gene family. The VH3 gene was used in nearly 64% of the potentially functional VH sequences which was somewhat higher than that in normal individuals (-50%). The distribution of VH genes in DLBCL was as follow: VH3 in7 cases, VH4-34 in 4 cases. VH4-34 gene was used in 36% of the small specimen of8 cases. Fact of biased VH family use with overrepresentation of VH4-34 gene was still under further research with extending our specimens. Adopting the rules proposed by Corbett et al, which required at least 10 consecutive nucleotides of identity to assign a D segment;a D gene was identified in 5 MALT lymphomas and 5 DLBCL. In other 3 MALT lymphomas and 2 DLBCL, the assignment was based on less stringent rules: minimal homology of 6 matches in a row or 7 matches interrupted by one mismatch. Priority was given to the homology with a D segment when both D and JH homologous regions overlapped. JH5 was the most frequently used JH gene segment in our cases, followed by JH4 and JH6.(2) The threshold value for distinguishing the unmutated and mutated VH genes was originally reported to be a 98% homology (2% mutation). According to this, all of our MALT patients and 10 DLBCL patients had encountered SHM. 1 of the DLBCL was unmutated. In the 11 cases of MALT lymphomas, The average of somatic mutation frequency was 9.58%, ranging from 2.38%-20.79%. In 11 DLBCL, the average mutation ratio was 7.1%, ranging from 1.18%-14.29%. There was no statistics difference between these two kinds of lymphomas (P=0.227, /=1.246). Most hypermutation events consisted of single base changes, but double and triple substitutions also frequently occurred, mostly in the same codon. In addition to point mutations, 1 case of the MALT lymphomas displayed a 3-base segment gap.(3) All of our patients showed a potentially functional heavy chain rearrangement. According to the two rule mentioned above paragraphs, the pattern of somatic mutations in 9 MALT lymphomas and 6 DLBCL was compatible with antigen selection.(4) Intraclonal microheterogeneity was analyzed in the MALT cell population of 1 DLBCL with MALT component and the DLBCL population of another DLBCL with MALT component. In the former, the ongoing somatic hypermutation frequency was0.35%, but no ongoing SHM was indetified in the later one. Other 4 primary MALT lymphomas and 5 DLBCL were also cloned and sequenced in order to evaluate the presence of ongoing mutations in these lymphomas. There was no intraclonal microheterogeneity in 2 MALT lymphomas (containing 1 tonsil MALT lymphoma) and 2 gastricintestinal DLBCL. In the other 2 MALT lymphomas and 3 DLBCL, ongoing somatic hypermutation was in existence.Conclusions(1) MALT lymphomas came from post-GC B cells, and the average SHM frequency was 9.58%, which was much higher than that reported abroad.(2) DLBCL was a heterogeneity lymphoma. It could come from pre-GC B cells, GC B cells, and post-GC B cells.(3) There was no difference between the mutation frequency of MALT lymphomas and DLBCL.(4) The pattern of ongoing somatic hypermutation in DLBCL with MALT component supported the hypothesis that a single, somatically mutated MALT lymphoma subclone gave rise to the transformed DLBCL, and the accumulation of SHM in the IgVH gene might be the most important factor in this transformation.(5) Most of the MALT lymphomas and DLBCL showed positive antigen selections on analysis of the R/S in IgVH gene.