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A Short Mutational Hot Spot in the First Intron of BCL-6 Is Associated with Increased BCL-6 Expression and with Longer Overall Survival in Large B-Cell Lymphomas

2002; Elsevier BV; Volume: 160; Issue: 4 Linguagem: Inglês

10.1016/s0002-9440(10)62564-3

1525-2191

María-Jesús Artiga, Ana-Isabel Sáez, Cristina Romero, Margarita Sánchez‐Beato, Marisol Mateo, Concepción Navas, Manuela Mollejo, Miguel Á. Piris,

Acute Lymphoblastic Leukemia research

BCL-6 somatic mutations have been described in normal and tumoral B lymphocytes, associated with germinal center transit. We analyzed mutations in the major mutation cluster of BCL-6 in a series of 45 large B-cell lymphomas (LBCLs) and 15 Burkitt's lymphomas, and their relation to the level of BCL-6 expression and clinical outcome. Mutations in LBCL cases revealed the existence of two distinct, short mutational hot spots, spanning positions 106 to 127 and 423 to 443, in which the mutation frequency was higher than expected (P < 0.001). Mutations in the 423 to 443 subcluster were associated with an increased level of expression, although this was not the case with the 106 to 127 cluster. Additionally, LBCL cases characterized by the presence of mutations in the 423 to 443 cluster showed an increased overall survival (P < 0.05) when compared with the nonmutated LBCL cases in these positions. Burkitt's lymphoma cases showed a slightly lower frequency of mutations with a nonclustered distribution and lacked any relationship with the level of expression or any clinical characteristic. Findings from LBCLs suggest that the 423 to 443 cluster includes a regulatory region that is of importance for BCL-6 expression. Deregulation of BCL-6 expression caused by these mutations could play an important role in lymphoma genesis or progression. BCL-6 somatic mutations have been described in normal and tumoral B lymphocytes, associated with germinal center transit. We analyzed mutations in the major mutation cluster of BCL-6 in a series of 45 large B-cell lymphomas (LBCLs) and 15 Burkitt's lymphomas, and their relation to the level of BCL-6 expression and clinical outcome. Mutations in LBCL cases revealed the existence of two distinct, short mutational hot spots, spanning positions 106 to 127 and 423 to 443, in which the mutation frequency was higher than expected (P < 0.001). Mutations in the 423 to 443 subcluster were associated with an increased level of expression, although this was not the case with the 106 to 127 cluster. Additionally, LBCL cases characterized by the presence of mutations in the 423 to 443 cluster showed an increased overall survival (P < 0.05) when compared with the nonmutated LBCL cases in these positions. Burkitt's lymphoma cases showed a slightly lower frequency of mutations with a nonclustered distribution and lacked any relationship with the level of expression or any clinical characteristic. Findings from LBCLs suggest that the 423 to 443 cluster includes a regulatory region that is of importance for BCL-6 expression. Deregulation of BCL-6 expression caused by these mutations could play an important role in lymphoma genesis or progression. The BCL-6 gene was identified as a result of its involvement in chromosomal translocations in a subset of large B-cell lymphomas (LBCLs) and follicular lymphomas.1Baron BW Nucifora G McCabe N Espinosa RD Le Beau MM McKeithan TW Identification of the gene associated with the recurring chromosomal translocations t(3;14)(q27;q32) and t(3;22)(q27;q11) in B-cell lymphomas.Proc Natl Acad Sci USA. 1993; 90: 5262-5266Crossref PubMed Scopus (275) Google Scholar These translocations deregulate the expression of the BCL-6 gene after substituting the BCL-6 promoter with a variety of other gene promoters.2Yoshida S Kaneita Y Aoki Y Seto M Mori S Moriyama M Identification of heterologous translocation partner genes fused to the BCL6 gene in diffuse large B-cell lymphomas: 5′-RACE and LA-PCR analyses of biopsy samples.Oncogene. 1999; 18: 7994-7999Crossref PubMed Scopus (55) Google Scholar, 3Ye BH Chaganti S Chang CC Niu H Corradini P Chaganti RS Dalla-Favera R Chromosomal translocations cause deregulated BCL6 expression by promoter substitution in B cell lymphoma.EMBO J. 1995; 14: 6209-6217PubMed Google Scholar This gene contains 10 exons and encodes for a 3.8-kb mRNA that is translated into a 706-amino acid protein.4Kawamata N Miki T Fukuda T Hirosawa S Aoki N The organization of the BCL6 gene.Leukemia. 1994; 8: 1327-1330PubMed Google Scholar BCL-6 protein has been identified as being a transcriptional repressor with two important functional domains: six zinc-fingers at the carboxyl terminus that mediate its interaction with DNA, and a BTB/POZ domain, a hydrophobic region, that mediates its interactions with other proteins.5Deweindt C Albagli O Bernardin F Dhordain P Quief S Lantoine D Kerckaert JP Leprince D The LAZ3/BCL6 oncogene encodes a sequence-specific transcriptional inhibitor: a novel function for the BTB/POZ domain as an autonomous repressing domain.Cell Growth Differ. 1995; 6: 1495-1503PubMed Google Scholar The BCL-6 gene has been shown to be a multifunctional gene, regulating important genes involved in B-cell differentiation (blimp-1, IP-10, and others) and cell-cycle control (such as c-myc, p27KIP1, and cyclin D2).6Shaffer AL Yu X He Y Boldrick J Chan EP Staudt LM BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control.Immunity. 2000; 13: 199-212Abstract Full Text Full Text PDF PubMed Scopus (694) Google ScholarIn addition to translocations, the BCL-6 gene experiences somatic mutations in the first intron, 100 bp downstream from the first noncoding exon, analogous to what occurs with IgVH genes, although at a lower frequency.7Migliazza A Martinotti S Chen W Fusco C Ye BH Knowles DM Offit K Chaganti RS Dalla-Favera R Frequent somatic hypermutation of the 5′ noncoding region of the BCL6 gene in B-cell lymphoma.Proc Natl Acad Sci USA. 1995; 92: 12520-12524Crossref PubMed Scopus (321) Google Scholar, 8Pasqualucci L Migliazza A Fracchiolla N William C Neri A Baldini L Chaganti RS Klein U Kuppers R Rajewsky K Dalla-Favera R BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation acting outside Ig loci.Proc Natl Acad Sci USA. 1998; 95: 11816-11821Crossref PubMed Scopus (426) Google Scholar BCL-6 somatic mutations have been described in normal and tumoral B lymphocytes, being more frequently observed in LBCL than in other tumoral types.8Pasqualucci L Migliazza A Fracchiolla N William C Neri A Baldini L Chaganti RS Klein U Kuppers R Rajewsky K Dalla-Favera R BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation acting outside Ig loci.Proc Natl Acad Sci USA. 1998; 95: 11816-11821Crossref PubMed Scopus (426) Google Scholar, 9Capello D Vitolo U Pasqualucci L Quattrone S Migliaretti G Fassone L Ariatti C Vivenza D Gloghini A Pastore C Lanza C Nomdedeu J Botto B Freilone R Buonaiuto D Zagonel V Gallo E Palestro G Saglio G Dalla-Favera R Carbone A Gaidano G Distribution and pattern of BCL-6 mutations throughout the spectrum of B-cell neoplasia.Blood. 2000; 95: 651-659PubMed Google Scholar BCL-6 mutations are considered to be markers of germinal center transit because they are absent in benign and malignant (mantle cell lymphoma) pregerminal center B cells and characteristically present in germinal center lymphocytes and derived tumors.It has been suggested on several occasions that BCL-6 expression is probably deregulated as a consequence of somatic mutation.8Pasqualucci L Migliazza A Fracchiolla N William C Neri A Baldini L Chaganti RS Klein U Kuppers R Rajewsky K Dalla-Favera R BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation acting outside Ig loci.Proc Natl Acad Sci USA. 1998; 95: 11816-11821Crossref PubMed Scopus (426) Google Scholar However, a relationship between the frequency or localization of these mutations and the expression of the protein has not been conclusively demonstrated.Furthermore, consequences of the accumulation of BCL-6 mutations during the genesis and progression of lymphomas have not been clarified sufficiently. The only exception is the higher grade transformation of follicular lymphoma, in which the accumulation of new mutations has been described.10Lossos IS Levy R Higher-grade transformation of follicle center lymphoma is associated with somatic mutation of the 5′ noncoding regulatory region of the BCL-6 gene.Blood. 2000; 96: 635-639PubMed Google Scholar However, this accumulation has not been shown to have any pathogenic association with the mechanism of the progression.The mutation frequency of BCL-6 seems to be high in LBCL. An expression profile that includes the expression of BCL-6 and other germinal center markers has been found to be a reliable predictor of increased overall survival (OS) in LBCL.11Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson Jr, J Lu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Levy R Wilson W Grever MR Byrd JC Botstein D Brown PD Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (7914) Google Scholar Moreover, it has been recently described that the expression of BCL-6 alone can be a reliable predictor of better survival in LBCL.12Lossos IS Jones CD Warnke R Natkunam Y Kaizer H Zehnder JL Tibshirani R Levy R Expression of a single gene, BCL-6, strongly predicts survival in patients with diffuse large B-cell lymphoma.Blood. 2001; 98: 945-951Crossref PubMed Scopus (253) Google Scholar For these reasons, we decided to determine whether BCL-6 mutations are associated with differences in the expression of the BCL-6 protein and the outcome of the patients in a group of LBCL cases. To this end, we analyzed mutations in the major mutation cluster (MMC) of BCL-6 in a series of 45 LBCL cases, relating them to the level of BCL-6 expression and clinical outcome. A group of 15 Burkitt's lymphoma (BL) cases was also included to allow comparison of the effects of mutations in these different lymphoma types.Materials and MethodsTissue Samples, Patients, and Clinical VariablesWe studied 45 LBCL and 15 BL patients who had been diagnosed according to the REAL classification.13Harris NL Jaffe ES Stein H Banks PM Chan JK Cleary ML Delsol G De Wolf-Peeters C Falini B Gatter KC Grogan TM Isaacson PG Knowles DM Mason DY Muller-Hermelink HK Pileri S Piris MA Ralfkiaer E Warnke R A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar For most tests, each histological group was studied separately, making it possible to analyze molecular differences between them. Diagnostic pretreatment samples were analyzed in all cases. Tumoral and reactive specimens were selected from the Pathology Laboratory, Hospital Virgen de la Salud (Toledo, Spain), on the basis of the availability of paraffin-embedded and frozen tissue for molecular studies and the existence of an adequate clinical follow-up (except for four patients, whose clinical history was not available). All patients were treated with chemotherapy regimes including anthracyclines. A clinical follow-up was performed in all cases. Clinical follow-up of patients alive at the time of the study included periods ranging from 11 to 122 months (mean, 75.38 months). Age, clinical stage, performance status, levels of lactate dehydrogenase (LDH) in blood and the number of extranodal sites of the disease were used as outcome criteria, as set out by the International Prognostic Index (IPI).14A predictive model for aggressive non-Hodgkin's lymphoma: the International Non-Hodgkin's Lymphoma Prognostic Factors Project.N Engl J Med. 1993; 329: 987-994Crossref PubMed Scopus (4946) Google Scholar Patients with 0 to 2 unfavorable variables were considered as being low risk, and those with 3 to 5 variables as high risk. Complete remission was defined as the resolution of clinical and radiological evidence of disease for a minimum of 4 weeks.Biopsy specimens of these 60 patients were divided into two parts: one was formalin-fixed and paraffin-embedded for morphological and immunohistochemical studies, while the other was embedded in Optimal Cutting Temperature Tissue-Tek (OCT; Sakura, Zoeterwoude, Netherlands) and preserved at −80°C for molecular studies.DNA extraction from frozen sections was performed following the standard phenol-chloroform protocol. RNA was extracted from frozen sections. Before each DNA or RNA extraction, frozen sections of the specimens were analyzed to ensure that the specimen was representative of the tumor. The percentage of tumoral cells was greater than 80% in all cases.To quantify BCL-6 mRNA expression, we extracted RNA from Raji cells. This cell line was obtained from the American Tissue Type Culture Collection (Rockville, MD), and maintained in RPMI 1640 (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum, 2 mmol/L glutamine, and penicillin-streptomycin (Life Technologies, Inc., Grand Island, NY).Immunostaining TechniquesBCL-6 protein was detected with monoclonal antibody for P6-B6p, a recombinant protein corresponding to amino acids 3 to 484 from DAKO, Glostrup, Denmark (1/10).15Flenghi L Ye BH Fizzotti M Bigerna B Cattoretti G Venturi S Pacini R Pileri S Lo Coco F Pescarmona E A specific monoclonal antibody (PG-B6) detects expression of the BCL-6 protein in germinal center B cells.Am J Pathol. 1995; 147: 405-411PubMed Google Scholar The proliferation index was evaluated using nuclear antigen Ki67 expression, detected with the MIB1 antibody from Immunotech (Marseille, France).Immunostaining techniques were performed in paraffin-embedded tissue sections. For antigen retrieval, before incubation with the antibodies (Ab), the slides were heated in a pressure cooker for 3 minutes in 0.01 mol/L of sodium citrate solution. Additionally, the slides were digested with proteinase K for 10 minutes at room temperature.After incubation with the primary Ab, immunodetection was performed with biotinylated anti-mouse immunoglobulins, followed by peroxidase-labeled streptavidin (LSAB-DAKO, Denmark) and diaminobenzidine chromogen as substrate. All immunostaining was performed using the Techmate 500 (DAKO) automatic immunostaining device.Incubations omitting the specific Ab, or with unrelated Abs, were performed to provide controls of the technique. The quality of the staining was checked on every slide, using BCL-6-positive reactive lymphocytes as an internal control.Quantitative StudiesAll scoring and interpretations of immunohistochemical results were performed independently by two of the authors (AIS and MJA) without knowledge of the clinical variables or the results of the molecular analysis.High-magnification fields were chosen for the evaluation of BCL-6 and MIB1 expression, focusing on tumoral areas and counting up to 300 cells. All immunoreactive cells were considered to be positive. A manual cell-counting procedure was used so that all nontumoral subpopulations could be excluded on the basis of their cell morphology.The intensity of BCL-6 immunostaining was classified into low, intermediate, or high groups when the signal was lower, similar, or higher, respectively, than reactive T lymphocytes in the tumor.For survival analysis of BCL-6 expression, a cutoff of 50% of positive cells was used because this threshold divided the series into groups of similar size (median of percentage of BCL-6-positive cells: 50) and had the advantage of being easily reproducible.Real-Time Polymerase Chain Reaction (PCR)Total RNA extraction was performed using a protocol based on Trizol (Life Technologies, Inc.). cDNA was synthesized with avian myeloblastosis virus (AMV) retrotranscriptase (Promega, Madison, WI), according to manufacturer's instructions.Real-time PCR was developed using the TaqMan technology, in an ABI Prism 7700 Sequence Detector System (PE Applied Biosystems, Norwalk, CT). As a control of the quality and quantity of the RNA, GAPDH gene was amplified in parallel with that of BCL-6. The primers and probes used in this study have been described previously.12Lossos IS Jones CD Warnke R Natkunam Y Kaizer H Zehnder JL Tibshirani R Levy R Expression of a single gene, BCL-6, strongly predicts survival in patients with diffuse large B-cell lymphoma.Blood. 2001; 98: 945-951Crossref PubMed Scopus (253) Google Scholar Both probes were labeled at the 5′ end with 6-carboxy-fluorescein phosphoramidite (FAM), and at the 3′ end with 6-carboxy-tetramethyl-rhodamine (TAMRA) as quencher. The reactions were not multiplexed.Each sample was measured from two different RNA extractions, each at two different dilutions, and each dilution in triplicate. To compare different experiments, in every PCR a standard curve (composed of different dilutions of a cDNA from Raji cells) and a calibrator (cDNA from a reactive tonsil) were derived, as suggested in the ABI 7700 User Bulletin 2 (PE Applied Biosystems). The conditions of the reactions were those recommended in the ABI 7700 User Bulletin 2 (PE Applied Biosystems).Sequencing of BCL-6A unique PCR product, 791 bp long, was amplified using 5′-CCGCTGCTCATGATCATTATTT-3′ and 5′-TAGACACGATACTTCATCTCAT-3′ primers. This fragment is located downstream of the first noncoding exon of BCL-6 and includes the entire MMC region.The PCR reaction was performed in a 50-μl total volume containing 50 pmol of each primer, 0.1 mmol/L dNTP, 1.5 mmol/L MgCl2, and 2 U Taq Platinum (Life Technologies, Inc.). Conditions for amplification were as follows: 94°C, 5 minutes denaturation; 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C, and 1 minute at 72°C; and a final extension step at 72°C for 10 minutes. PCR was performed in a Perkin Elmer 9700 GeneAmp PCR System (Norwalk, CT).PCR products were purified by using the Microcon PCR kit (Millipore, Bedford, MA). Both strands were then directly sequenced, using the same primers as for the amplification and two additional internal oligonucleotides, in an ABI 370 (Perkin Elmer Applied Biosystems, Warrington, UK), following the manufacturer's procedure. Mutations were identified by comparison with the BCL-6 germline sequence (GenBank accession number AF191831). To control for potential Taq errors, all PCR and sequencing procedures were performed twice.Statistical AnalysisStatistical study of the correlation between distributions was performed using either Fisher's exact test for categorical variables, the Kruskal-Wallis test for single-ranked data, and the Pearson correlation for double-ranked data.The clinical variables analyzed in the survival studies were those included in the IPI14A predictive model for aggressive non-Hodgkin's lymphoma: the International Non-Hodgkin's Lymphoma Prognostic Factors Project.N Engl J Med. 1993; 329: 987-994Crossref PubMed Scopus (4946) Google Scholar (measured as 0 to 2, 3 to 5), these being: age (≤60 versus >60 years), gender (female versus male), clinical stage (I + II versus III + IV), and LDH (normal versus >normal). Survival curves were calculated by the Kaplan-Meier method and compared by the log-rank test.16Kaplan EL Meier P Nonparametric estimation for incomplete observations.J Am Stat Assoc. 1958; 53: 457-481Crossref Scopus (47677) Google Scholar, 17Mantel N Evaluation of survival and two new rank order statistics arising in its consideration.Cancer Chemother Rep. 1966; 50: 163-170PubMed Google Scholar Actuarial survival curves [OS and disease-free survival (DFS)] were calculated using the Kaplan-Meier method. Statistical significance was calculated using the log-rank test. Cox's proportional hazard univariate analysis18Abraira V Pérez de Vargas A Métodos Multivariantes en Bioestadística.in: de EC Ramón Areces, Madrid1996Google Scholar was also performed, providing estimates of the confidence interval and the relative risk (RR) in terms of survival.To identify the factors that might be of independent significance in influencing survival (OS and DFS), a Cox backward proportional hazard model was fitted.19Cox D Regression models and life tables.J R Stat Soc (B). 1972; 34: 187-220Google Scholar Variables included in the maximal models were IPI (0 to 2, 3 to 5) and presence of mutations inside the 423 to 443 cluster. The low-risk IPI and presence of mutations in the 423 to 443 region, found to be associated with higher survival probability, were taken as reference levels. All P values were two-sided, and values of 0.05 or less were considered to indicate statistical significance. SPSS 10.0 for Windows was used for all statistical analyses (SPSS Inc., Chicago, IL).ResultsBCL-6 ExpressionAn initial screening of BCL-6 protein expression was performed in reactive lymphoid tissue. The pattern of expression was similar to that described before: germinal center cells were variable although they generally stained strongly for BCL-6. Mantle and interfollicular cells were mostly negative, although some scattered cells were present in these areas (Figure 1A).BCL-6 protein expression was then analyzed by immunohistochemistry in 52 cases of aggressive NHL, including 40 LBCLs and 12 BLs (Figure 1A). In the other eight cases, the results could not be evaluated because of the lack of adequate internal controls, which are usually provided by BCL-6-positive reactive lymphocytes. Variation in signal intensity and number of BCL-6-positive cells was observed and there was a strong positive correlation between the two variables (r = 0.437, P = 0.001, Spearman correlation). Therefore, we only considered the number of positive cells in the quantification of BCL-6 expression.BCL-6 reactivity represented by fewer than 20% of positive cells was found in 5 cases (all LBCL samples), whereas 13 cases (8 LBCL and 5 BL) had more than 80% positive cells. Most cases (35 of 52) showed an intermediate level of BCL-6 expression. Table 1 and Figure 1 summarize the immunostaining results. There were only slight, nonsignificant differences in the distribution of expression levels between LBCL and BL cases.Table 1Summary of BCL-6 IHC ResultsLBCLBLTotalCases401252Mean53.6 (±27.69)64.0 (±28.51)55.9 (±27.95)Range2–10023–1002–100Values of mean and range are expressed as percentages of the number of BCL-6-positive cells in the total number of cells scored. Standard deviation is indicated in brackets.LBCL, large B-cell lymphoma; BL, Burkitt's lymphoma. Open table in a new tab BCL-6 protein expression was then contrasted with BCL-6 mRNA expression as measured by real-time PCR. We analyzed a group of 17 tumor samples from our series with variable protein expression detected by immunohistochemistry, and 4 reactive tonsil samples and 2 mantle cell lymphoma samples as controls (Figure 1B). LBCL samples showed a good correlation among protein and mRNA expression levels with the exception of a discrepancy in five cases. The only striking finding was the detection in MCL cases of mild, but distinct BCL-6 mRNA expression levels, thus confirming the previous observations by Lossos and co-workers.12Lossos IS Jones CD Warnke R Natkunam Y Kaizer H Zehnder JL Tibshirani R Levy R Expression of a single gene, BCL-6, strongly predicts survival in patients with diffuse large B-cell lymphoma.Blood. 2001; 98: 945-951Crossref PubMed Scopus (253) Google ScholarBCL-6 MutationsSequencing analysis was performed in 45 LBCL and 14 BL cases identifying a wide variety of mutations that were present in the majority of cases (80% LBCL and 71% BL were mutated). Table 2 shows a summary of the mutational analysis. The frequency of mutations in our series was 3.7 × 10−3 mutations/bp (3.8 × 10−3 mutations/bp in LBCL cases and 3.7 × 10−3 mutations/bp in BL cases).Table 2Description of the BCL-6 MMC Mutations Found in this SeriesDiagn.MutationsDiagn.MutationsLBCLG-C (397)LBCLWTLBCLG-A (243), G-A (244), G-C (397), G-A (448), G-C (518)LBCLA-G (96), G-C (125), G-A (448), A-G (455), C-T (519), C-A (586), T-C (753)LBCLC-G (88), T-C (89), A-C (108), G-C (122), C-G (169), C-T (172), C-A (173), G-A (211), T-C (218), A-G (226), G-A (240), G-A (241), T-C (272), G-C (397), C-G (423), C-T (425), G-A (434), G-A (441), C-G (446), G-A (448), G-A (588), G-C (618)LBCLG-C (397)LBCLT-C (120), G-T (122), A-C (195), T-G (349), T-G (350), C-A (487), C-G (495), C-T (561), A-G (616), C-G (728), T-A (744)LBCLG-C (397), C-G (438), A-G (696)LBCLINSERC C (36)LBCLWTLBCLG-C (397), DEL T (520)LBCLG-C (397), DEL T (520)LBCLA-G (106), A-T (116), G-C (122), A-G (208), C-T (348), A-G (445), G-C (448)LBCLG-C (122), DEL T (520)LBCLT-C (750)LBCLC-G (423)LBCLT-A (79)LBCLT-A (110), A-G (112), DEL C (164), C-G (166), C-T (216), DUPL (383–413), G-C (414), G-A (431), C-G (435), C-T (548)LBCLC-G (88), T-C (111), G-C (335), G-C (369), G-A (373), G-A (385), T-G (737)LBCLG-C (122)LBCLC-T (602)LBCLA-T (176), G-T (177), C-G (423), DEL T (520)LBCLWTLBCLWTLBCLWTLBCLDEL T (520)LBCLT-A (360), C-G (429), C-G (514), C-T (515), DEL T (520), T-G (573), T-C (632)LBCLG-C (397), DEL T (520)LBCLG-A (122), C-G (423), T-G (439), G-A (518), G-A (588)LBCLDEL T (520)LBCLWTLBCLWTBLT-C (43), T-G (110), G-C (122), C-T (123), T-G (133), T-G (182), T-C (194), T-C (266), G-C (398), C-A (432), G-A (464), G-A (502), DEL T (520)LBCLWTBLDEL T (520)LBCLDEL T (520)BLT-C (87), G-A (398), A-C (401), C-T (495), T-C (517), G-A (736), INSERC T (755)LBCLG-A (443)BLDEL T (485), C-A (514), DEL T (520)LBCLT-G (450), T-C (451)BLG-C (397)LBCLG-A (122), G-A (125), C-T (127), G-A (298), C-T (507)BLWTLBCLA-G (112), G-C (122), G-A (400)BLDEL T (520)LBCLA-G (615)BLWTLBCLDEL T (520), T-C (582), G-A (643), A-G (727)BLA-G (90), A-G (112), A-T (191), A-G (196), G-C (397), C-T (574)LBCLWTBLWTLBCLG-C (397), C-G (493), C-A (432), G-A (598)BLDEL T (520)LBCLDEL T (520)BLWTLBCLC-G (54), T-C (201), C-A (251), G-C (397), A-C (504), T-C (512), G-A (626), A-T (657)BLDEL T (520)LBCLDEL T (520)BLT-G (349), T-G (350), C-G (366), T-A (367), C-G (459)Nucleotide I was arbitrarily assigned at the start of the MMC (position +356 of the gene), according to the previously published sequence (GenBank accession number AF191831). Polymorphisms are indicated in grey. Open table in a new tab Our series included 9 LBCL and 4 BL nonmutated cases (20% and 28%, respectively), 15 LBCL and 5 BL cases (33% and 36%, respectively) with one mutation, and 21 LBCL and 5 BL cases (47% and 36%, respectively) with more than two mutations (up to 22 sequence changes) (Figure 2). The mutations present in the series were mostly punctual substitutions. Two cases showed punctual deletions, two cases had a punctual insertion, and one case featured a long tandem duplication (31 bp) (See Table 2 for details). In agreement with previous studies,7Migliazza A Martinotti S Chen W Fusco C Ye BH Knowles DM Offit K Chaganti RS Dalla-Favera R Frequent somatic hypermutation of the 5′ noncoding region of the BCL6 gene in B-cell lymphoma.Proc Natl Acad Sci USA. 1995; 92: 12520-12524Crossref PubMed Scopus (321) Google Scholar, 20Mateo MS Mollejo M Villuendas R Algara P Sanchez-Beato M Martinez P Piris MA Molecular heterogeneity of splenic marginal zone lymphomas: analysis of mutations in the 5′ non-coding region of the bcl-6 gene.Leukemia. 2001; 15: 628-634Crossref PubMed Scopus (24) Google Scholar there were two polymorphisms in the MMC at positions 397 (C to G) and 520 (del T). Changes in these nucleotides were observed in 42% of LBCL cases and 57% of BL cases. Additionally, seven recurrent mutations were identified in more than one patient (at positions 88, 112, 349, 350, 423, 432, 588). Moreover, mutations to different nucleotides appeared at eight positions (110, 122, 125, 398, 448, 495, 514, 518) (recurrently mutated positions). These positions can thus be considered to be mutational hot spots. Hypothetical polymorphisms in these positions were excluded through the study of nontumoral DNA for the changes in positions 88, 112, 423, 432, 448, 518, 588. Other possible polymorphisms had already been excluded by previous studies.7Migliazza A Martinotti S Chen W Fusco C Ye BH Knowles DM Offit K Chaganti RS Dalla-Favera R Frequent somatic hypermutation of the 5′ noncoding region of the BCL6 gene in B-cell lymphoma.Proc Natl Acad Sci USA. 1995; 92: 12520-12524Crossref PubMed Scopus (321) Google Scholar, 8Pasqualucci L Migliazza A Fracchiolla N William C Neri A Baldini L Chaganti RS Klein U Kuppers R Rajewsky K Dalla-Favera R BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation acting outside Ig loci.Proc Natl Acad Sci USA. 1998; 95: 11816-11821Crossref PubMed Scopus (426) Google Scholar, 10Lossos IS Levy R Higher-grade transformation of follicle center lymphoma is associated with somatic mutation of the 5′ noncoding regulatory region of the BCL-6 gene.Blood. 2000; 96: 635-639PubMed Google Scholar, 20Mateo MS Mollejo M Villuendas R Algara P Sanchez-Beato M Martinez P Piris MA Molecular heterogeneity of splenic marginal zone lymphomas: analysis of mutations in the 5′ non-coding region of the bcl-6 gene.Leukemia. 2001; 15: 628-634Crossref PubMed Scopus (24) Google Scholar, 21Gaidano G Carbone A Pastore C Capello D Migliazza A Gloghini A Roncella S Ferrarini M Saglio G Dalla-Favera R Frequent mutation of the 5′ noncoding region of the BCL-6 gene in acquired immunodeficiency syndrome-related non-Hodgkin's lymphomas.Blood. 1997; 89: 3755-3762PubMed Google Scholar, 22Gaidano G Capello D Cilia AM Gloghini A Perin T Quattrone S Migliazza A Lo Coco F Saglio G Ascoli V Carbone A Genetic characterization of HHV-8/KSHV-positive primary effusion lymphoma reveals frequent mutations of BCL6: implications for disease pathogenesis and histogenesis.Genes Chromosom Cancer. 1999; 24: 16-23Crossref PubMed Scopus (88) Google Scholar, 23Lossos IS Levy R Mutation analysis of the 5′ noncoding regulatory region of the BCL-6 gene in non-Hodgkin lymphoma: evidence for recurrent mutations and intraclonal heterogeneity.Blood. 2000; 95: 1400-1405PubMed Google ScholarFigure 2Distribution of MMC BCL-6 mutations per case. The number of mutations per case varied from 0