|Year : 2012 | Volume
| Issue : 2 | Page : 74-78
Genetic polymorphism study of the BCL-6 gene, in diffuse large B-cell lymphoma
Gamal T Ebid1, Farida H Gad Allah1, Mahmoud M Kamel1, Nagwa H Hassan2, Asmaa A El Leithy2, Magda M Hassan3
1 Department of Clinical Pathology, National Cancer Institute, Cairo University, Egypt
2 Department of Zoology, Faculty of Science, Ain Shams University, Egypt
3 Department of Pathology, National Cancer Institute, Cairo University, Egypt
|Date of Web Publication||13-Sep-2012|
Mahmoud M Kamel
Department of Clinical Pathology, National Cancer Institute, Cairo University, Cairo
Source of Support: None, Conflict of Interest: None
Background: Diffuse large B-cell lymphoma (DLBCL) is characterized by a marked degree of morphologic and clinical heterogeneity. BCL-6 gene rearrangements are the most common frequent chromosomal abnormalities in DLBCL. Genetic modifications of the BCL-6 gene in lymphoma include translocations, deletions, and somatic mutations (SM) of the 5′-non-coding region. Mutations in the 5′-regulatory region of BCL-6 were suggested to play a role in non-Hodgkin's lymphoma (NHL) progression. Materials and Methods: We studied single nucleotide polymorphism (SNP) at position 397(G->C) of the 5'-non-coding regulatory region of BCL-6. We examined 30 patients with DLBCL treated at the National Cancer Institute (NCI), Cairo University, and 21 normal lymph nodes from patients with breast cancer were selected as controls. We used (Polymerase chain reaction- Restriction fragment length polymorphism) PCR-RFLP coupled analysis to detect the polymorphism. Results: From 30 patients with DLBCL, 17 were males (56.7%) and 13 were females (43.3%), the polymorphism G397C in the 5′ region of BCL-6 was detected in seven cases (23.3%). There was no significant association of clinical characteristics of DLBCL patients with this polymorphism, but there a trend for this polymorphism to occur more frequent in patients with aggressive disease. Conclusion: In our study, we found the incidence of the G397C SNP mutations in the 5′-regulatory region of BCL-6 to be 23.3%. No statistically significant association between the G397C SNP in BCL-6 gene with the standard clinico-pathological factors in patients with DLBCL was observed; however, there a trend for this polymorphism to occur more frequent in patients with aggressive disease.
Keywords: Diffuse large B-cell lymphoma, non-Hodgkin′s lymphoma, BCL-6, single nucleotide polymorphism
|How to cite this article:|
Ebid GT, Gad Allah FH, Kamel MM, Hassan NH, El Leithy AA, Hassan MM. Genetic polymorphism study of the BCL-6 gene, in diffuse large B-cell lymphoma. Saudi J Health Sci 2012;1:74-8
|How to cite this URL:|
Ebid GT, Gad Allah FH, Kamel MM, Hassan NH, El Leithy AA, Hassan MM. Genetic polymorphism study of the BCL-6 gene, in diffuse large B-cell lymphoma. Saudi J Health Sci [serial online] 2012 [cited 2021 Apr 11];1:74-8. Available from: https://www.saudijhealthsci.org/text.asp?2012/1/2/74/100950
| Introduction|| |
The etiology of the most cases of non-Hodgkin's lymphoma (NHL) is unknown, although several genetic diseases, environmental agents, and infectious agents have been associated with the development of lymphoma.  The incidence of NHL has doubled over the past two decades in the US and in most other westernized countries.  Diffuse large B-cell lymphoma (DLBCL) represents the most frequent type of NHL, accounting for 30-40% of adult NHL, and often represents the final transformation stage of follicular lymphoma (FL).  In Egypt, DLBCL is the most frequent type of NHL, representing 54.5%. 
DLBCLs represent a diverse group of lymphoid neoplasms and encompass heterogeneous clinical, histologic, immunophenotypic, cytogenetic, and molecular genetics feature.  However, previous attempts to subclassify these neoplasms on morphologic grounds have suffered from irreproducibility, leading to their categorization as one single group in the Revised European American Lymphoma (REAL) classification.  Chemotherapy has greatly improved the outcome for patients with DLBCL. However, approximately 50% of patients are not cured by the standard combination chemotherapy regimens thus highlighting the need to determine parameters identifying patients at high risk for standard therapy failure who may benefit from risk-adjusted therapies. A clinical indicator of prognosis, the International Prognostic Index (IPI), has been constructed and successfully used to define prognostic subgroups in DLBCL. 
The IPI takes into account factors that are mostly linked to patient characteristics (age, performance status) and to disease extension and growth (disease stage, lactate dehydrogenase [LDH] levels, and extent of extranodal involvement). However, it is clear that differences in clinical features and in treatment responses of DLBCL are owing to the marked genetic and molecular heterogeneity that underlies disease aggressiveness and tumor progression. Several molecular abnormalities such as BCL-2, , survivin expression,  and p53 mutations , were identified as prognostic indicators of DLBCL.
The BCL-6 proto-oncogene has been originally identified because of its involvement in chromosomal translocations affecting band 3q27 in B-lineage DLBCL. , The BCL-6 protein is a POZ/zinc finger transcriptional repressor, which, in the B-cell lineage, is expressed selectively by germinal center (GC) B cells but not by immature B-cell precursors or differentiated plasma cells.  The BCL-6 gene encodes a transcriptional repressor protein acting on a broad spectrum of target genes implicated in T and B cell maturation, cell-cycle control, apoptosis, or inflammation. Its expression is crucial for the GC formation, where its downregulation is necessary for the post-GC B cell maturation. 
BCL-6 expression is common in low-grade FL, but is rare in other indolent B-cell lymphoid disorders as, e.g., small lymphocytic, mantle cell and marginal zone lymphomas, and thus may be a useful adjunct in classification of indolent lymphomas.  Deregulated BCL-6 expression, by translocations, deletions, and somatic mutations (SM) of the 5′-non-coding region, contributes to lymphomagenesis in part by functional inactivation of p53. 
These mutations accumulating in the regulatory region of the BCL-6 gene could play a role in lymphoma progression and in the transformation of FL to more aggressive DLBCL.  One of these hot spots is single nucleotide polymorphism (SNP) at position 397(G->C) of the 5′-non-coding regulatory region of BCL-6. 
The present study was conducted to assess the expression of SNP at the position 397 of the 5′-non-coding regulatory region of BCL-6 and to verify if there is any association between this mutation and some standard clinical and prognostic factors in Egyptian patients with DLBCL.
| Materials and Methods|| |
This study included 30 patients with DLBCL treated at the National Cancer Institute, Cairo University. In all cases, the diagnosis was confirmed by histopathology and by positivity of tumor cells for B-cell marker CD20. Classification was done according to the World Health Organization classification. The control group was composed of 21 normal lymph nodes from patients with breast cancer.
A small section (not more than 25 mg) of paraffin-embedded LN was deparraffinized in xylene and the fragments were recovered by centrifugation. They were washed in absolute ethanol. The pellet was incubated at 37°C for 10-15 min until the ethanol had evaporated and then the tissue pellet was re-suspended in 180 mL buffer tissue lysis buffer from Qiagen (ATL). DNA was then extracted using the QIAamp DNA Mini Kit. DNA concentration and purity were estimated by spectrophotometry.
Polymerase chain reaction
Polymerase chain reaction (PCR) was performed in 25 mL of final volume using 2 mg of genomic DNA, 1 mM of each oligonucleotide primer, and 12.5 mL PCR master mix (2x concentrated), containing Taq DNA polymerase, QIAGEN PCR buffer (with 3 mM MgCl 2 ), and 400 mM of each dNTP. The primers used were as follows:
Forward primers (5′-TTTGGAAAGGAAGGTGGAGGA-3′)
Reverse primers (5′-AGAGATCACAAGCCGTACGCA-3′)
Conditions of PCR cycling were: Initial denaturation at 94°C for 3 min followed by 10 cycles at 94°C for 30 s and then annealing with touch down from 64°C to 55°C (1 degree per cycle) and elongation at 72°C for 60 s then 25 cycles of the same cycling parameters with annealing temperature of 60°C for 30 s. PCR products (183 bp) were subjected to digestion by BtgI, at 37°C for overnight incubation, where the mutations abolish the restriction site. Horizontal agarose gel electrophoresis and staining with ethidium bromide was used for visualization and assessment of the results (wild G/G 121+62 bp, homozygous C/C183 bp and heterozygous G/C 183, 121, 62 bp).
Data were analyzed using SPSSwin statistical package version 15. Qualitative data were expressed as frequency and percentage. Chi-square test or Fisher's exact test was used to examine the relation between qualitative variables. Risk was estimated using odds ratio with its 95% confidence interval. P-value less than 0.05 was considered significant.
| Results|| |
In the present study, there was 17 male (56.7%) and 13 female (43.3%), with a male to female ratio of 1.3:1, and the age of our patients ranged from 28 to 76 years, with a median of 54.5 years. B-symptom were present in 14 patients (46.7%), bone marrow involvement in three (10%) and high LDH in 15 patients (50%). Performance status (PS) was I in 14 (46.7%) patients, II in 12 (40%) patients and III in four (13.3%) patients.
Microscopic examination of hematoxylin/eosin-stained slides revealed that four patients (13.3%) were in stage I, 10 patients in stage II (33.3%), 12 patients (40%) in stage III and four patients (13.3%) in stage IV. Twenty-one patients (70%) presented with nodal involvement only, nine patients (30%) had extranodal involvement at presentation, and nine patients (30%) presented with bulky disease.
Allelic C frequency was found to be higher in the patients group compared with the control group, as the distribution of the G397C SNP in BCL-6 in DLBCL in the 5′ region of BCL-6 was detected in seven cases (23.3%) who were considered as the mutated group while 23 cases (76.7%) showed no allelic polymorphism, and those were considered as the unmutated patients; only one case of the 21 controls (4.8%) was found to have allelic polymorphism (P = 0.119) [Figure 1].
|Figure 1: BCL-6 G397C single nucleotide polymorphism. Lane 1, 5-8: Wild type, Lane 2-4: Heterozygous|
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In our study, we used multiple statistical tests to observe the relation between the occurrences of G397C SNP in the 5′-non-coding regulatory region of BCL-6 with the slandered prognostic factors (age, sex, LDH level, B-symptoms, stage, bone marrow involvement, presence of bulky disease, nodal presentations) in our patients with DLBCL. There was a trend for the mutation to occur more commonly in patients with some aggressive features such as proportion of the mutated gene was higher in patients with extranodal involvement (44.4%) than in those with nodal involvement (14.3%); also, higher in patients with high LDH (26.7%) than in those with normal LDH and higher in patients with positive B-symptoms (28.6%) than in those without B-symptoms (18.8%), but this trend did not reach the statistically significant level [Table 1].
|Table 1: Relation between BCL-6 SNP G397C mutation and clinical features within the DLBCL patients' group|
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As regarding the genotype, in our study, we found the proportion of mutated GC genotype to be higher in our patients as it was present in seven patients (23.3%) compared with only one control (4.8%) (P = 0.119), and the unmutated genotype GG was expressed in 23 patients (76.7%) and in 29 of the control group (95.2%). These data are expressed in [Table 2].
|Table 2: The genotype GG/GC and GC/GG in our studied patients and control groups|
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| Discussion|| |
SNP changes occurring in the coding regions and/or in the regulatory regions of the genes may affect function or expression of their proteins products. In NHL, SNPs of several genes have been previously related to lymphoma susceptibility, such as genes coding inflammation regulatory cytokines (lL1, lL10, TNFαα), , mismatch repairing elements (hMSH2),  oncoproteins (H-ras1)  and/or the BCL-6 proto-oncogene, which is involved in the genesis of NHL. 
Mutations in the 5′-regulatory region of BCL-6 were suggested to play a role in NHL progression. Rearrangements that may occur due to chromosomal translocations, deletions, and/or SM of the 5′-non-coding regulatory region of the BCL-6 gene are potential mechanisms for altering its expression in NHL. 
BCL-6 plays a crucial role during both normal B-cell maturation and lymphomagenesis, as downregulation of BCL-6 is necessary for lymphocytes within the GC to differentiate into memory B cells or plasma cells or to undergo selective apoptosis upon antigen stimulation. Accordingly, it has been hypothesized that constitutional or acquired mutations of putative functional elements may impact on lymphoma predisposition or lymphoma outcome. 
BCL-6 translocations typically juxtapose the 5′ regulatory region of BCL6 to the promoter region of a constitutively expressed partner gene such as the immunoglobulin (IG) genes, while leaving the coding domain of BCL6 intact, resulting in constitutive BCL-6 expression. This, in turn, blocks the normal differentiation of GC B cells and thus contributes to lymphomagenesis.  Also, BCL-6 represses p53 by directly binding to its promoter and allows GC B cells to tolerate the physiologic DNA breaks required for Ig class-switch recombination and somatic hypermutations. 
In our study, we assessed the expression of SNP at the position 397 of the 5′-non-coding regulatory region of BCL-6 and found that 23.3% of our 30 patients with DLBCL had a genetic polymorphism G397C in the 5′-non-coding regulatory region of the BCL-6 gene as compared with 4.8% in the control group (P = 0.119). There was no significant association between this polymorphism and other standard clinicopathologic factors, although the occurrence of this mutation was higher in some high-risk group of patients (as patients with high LDH, patients with positive B-symptoms and patient with extronodal disease).
Consistent with our result, Berglund  found that the frequency of the BCL6397C allele did not differ significantly in 120 de novo DLBCLs patients compared with the control group and, furthermore, he did not find any difference between patients with the BCL6397GG genotype and BCL6397GC/CC genotype in age, sex, clinical stage, and overall survival. This is also in accordance with Susova,  who stated that SNP in the 5′-flanking region of BCL6 is not associated with increased risk of non-Hodgkin's lymphoma.
Our results are in agreement with that of Pescarmona,  who detected BCL-6 rearrangements in 23/80 cases (28.8%) and Bastard et al.  who reported that 31 of the 114 patients (27%) showed BCL-6 rearrangements; both authors did not find any significant difference in the outcome.
In our work, we found that the proportion of the mutated GC gene is higher in patients with extranodal presentation (44.4%) than in those with nodal involvement only (14.4%). The same was stated by Kramer et al.,  who found the BCL-6 gene rearrangements in 36/116 (31%) patients with DLBCL and reported that these rearrangements were more often noted in patients with extronodal (36%) presentation compared with those with primary nodal disease (28%). They also reported no significant correlation with disease stage, lymphadenopathy, or bone marrow involvement, and both Disease-free survival (DFS) and overall survival (OS) were not influenced by BCL-6 rearrangements.
Although lower frequency was detected by Vitolo,  who reported BCL-6 rearrangement in 11/71 patients (15%), he stated that patients with rearranged bcl-6 tended to have a more aggressive disease than patients with germ-line bcl-6. However, there were no differences in the 3-year survival rates between the two groups. Also, Kawasaki  found structural alterations of bcl-6 in 22/137 (16.1%) patients with de novo DLBCL. Such differences could be explained by the differences in patient cohorts as well as technical factors related to staining, interpretation, and scoring of positive results. 
On the other hand, a higher frequency was found in only one study in which Offit  reported rearranged bcl-6 in 23/40 (57.5%) cases of DLBCL, and the status of the bcl-6 gene was an independent prognostic marker of survival and freedom from disease progression in a multivariate model and added predictive value to established prognostic signs.
As regards BCL-6 protein expression, the frequency of BCL-6 + cases varied in many reported studies. Winter et al.  reported BCL-6 protein expression in 77% of the cases, which is comparable to the 72% frequency reported by Colomo et al.  and the 63% frequency reported by Lossos et al.,  but higher than the 56% frequency reported by Hans et al.  Many authors reported that BCL-6 protein expression alone or in combination with other germinal center markers predicted a favorable outcome in DLBCL. ,,
Remarkable progress has been made in understanding the biological heterogeneity of DLBCL and in improving survival for DLBCL patients with the use of novel combinations of chemotherapy and immunotherapy. Therefore, future approaches to DLBCL management will use molecular signatures identified through gene expression profiling to provide prognostic information and to isolate therapeutic targets like addition of monoclonal antibody, rituximab, to CHOP to markedly improve outcomes to DLBCL patients who relapse or those with high-risk disease.  One of these studies use combined computer-aided drug design that could induce expression of BCL-6 target genes and kill BCL-6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was non-toxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients. 
In conclusion, we have reported a 23.3% incidence of the G397C SNP mutations in the 5'-regulatory region of BCL-6 in DLBCL in a cohort of Egyptian patients. No statistically significant association was encountered between the G397C SNP in BCL-6 gene and the standard clinicopathological factors in patients with DLBCL; however, a trend for this polymorphism to occur more frequently in patients with aggressive disease was observed. Future studies integrating both rearrangement of BCL-6 and BCL-6 protein expression over a large number of patients, studying their incidence, association, prognostic significance, and their effect to response to stranded therapy and new therapeutic targets, are required.
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[Table 1], [Table 2]