Molecular Heterogeneity of Splenic Marginal Zone Lymphomas

Home , Germinal center, Mantle zone, Marginal zone

Leukemia (2001) 15, 628–634  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Molecular heterogeneity of splenic marginal zone lymphomas: analysis of mutations in the 5′ non-coding region of the bcl-6 gene M-S Mateo1, M Mollejo2, R Villuendas3, P Algara1, M Sanchez-Beato3, P Martı´nez1 and MA Piris3

Departments of 1Genetics and 2Pathology, ‘Virgen de la Salud’ Hospital, Toledo; and 3Centro Nacional de Investigaciones Oncologicas, Programa de Patologia Molecular, Instituto de Salud Carlos III, Madrid, Spain

Splenic marginal zone lymphoma (SMZL) has been recognized dromes, the molecular homogeneity of the cases included and as a distinctive type of small B cell lymphoma, and defined on the cellular origin of this tumor. The claimed marginal zone the basis of its morphological, phenotypic, clinical and molecular characteristics. In spite of this, the borders of the entity, the origin of SMZL has therefore now been brought into question, homogeneity of the cases and the presumably cell origin of since it has been recognized that the presence of marginal SMZL remain controversial issues. The frequency of mutation zone differentiation in the spleen is observed in other lympho- in the 5′ non-coding region of the bcl-6 gene has been used as proliferative conditions, and that SMZL, when it infiltrates the a marker of germinal center derivation, which may be used to lymph nodes, lacks this morphological feature.2,5 establish the molecular heterogeneity of different non-Hodgkin The name of this condition, splenic marginal zone lym- lymphoma (NHL) types. This roughly parallels the characteristics and frequency of the somatic hypermutations found in phoma, recognizes the existence of a prominent presence of marginal zone in splenic infiltration, and refers to a hypotheti- the immunoglobulin heavy chain variable region (IgVH) genes. This study analyzed mutations of bcl-6 in the 5′ non-coding cal origin in the marginal zone. The marginal zone of the region in 22 SMZL cases and, for the purpose of comparison human spleen is a B cell compartment concentric to the with different B cell subsets, in microdissected germinal cen- mantle zone, and is composed of medium-sized lymphocytes ters, mantle zones and marginal zone subpopulations from with clear cytoplasm. Their phenotype is IgM+, IgD−, CD23−, reactive splenic lymphoid follicles. A majority of the SMZL − + + 6 cases studied, 19/22 (87%), bear unmutated bcl-6 gene, while CD5 , alkaline phosphatase , in contrast to IgD mantle cells. mutation was only observed in 3/22 (13%) cases. Analysis of Equivalent marginal zone cell populations have also been normal B cell subpopulations showed bcl-6 hypermutation in described in Peyer’s patches, tonsilar intraepithelial crypt 3/10 (30%) germinal center clones, 5/14 (35%) marginal zone cells, palatine tonsils and the lymph nodes.7–10 clones; and unmutated sequences in all clones derived from The investigation of B cell lineage and differentiation has mantle cells. The frequency of these mutations in normal been enormously facilitated by the discovery that B cells in spleen confirms previous findings on the hypermutation IgVH process in normal B cell populations. The data presented here the germinal center suffer somatic hypermutation of bcl-6 and support the existence of molecular heterogeneity in this entity, immunoglobulin heavy chain variable region (IgVH) genes, and give additional results in favor of the hypothesis that, in which in the case of Ig genes increases their affinity with the spite of initial morphological observations, a significant pro- antigens present in the germinal center microenviron- portion of SMZL cases could derive from an unmutated naive ment.11–15 precursor, different from the marginal zone, and possibly Mutation analysis of rearranged IgV genes in microdis- located in the mantle zone of splenic lymphoid follicles. Thus H the marginal zone differentiation of these tumors could be sected marginal zone B cells in the spleen, Peyer’s patches related more with the splenic microenvironment than it is to the and lymph nodes has shown that there are mutated IgVH genes histogenetic characteristics of the tumor. Leukemia (2001) 15, in the majority of cells, as well as evidence of related B cell 628–634. clones.16–19 Keywords: splenic marginal zone lymphoma; bcl-6; somatic Although it was initially assumed that the process of somatic mutation hypermutation in normal B lymphocytes was restricted to Ig loci, including heavy and light chain variable region genes,20 analogous mutations in the bcl-6 gene were further described, Introduction showing promoter-dependent hypermutations in the non- coding first exon, paralleling and almost identical with the

Splenic marginal zone lymphoma (SMZL) has been recog- hypermutation of IgVH genes with respect to mutation fre- nized as a distinctive type of small B cell lymphoma, charac- quency and the involvement of B cell subpopulations.21–23 terized by massive splenic involvement with a peculiar mor- Bcl-6 is a gene located at chromosomal band 3q27, and phology, in which the main findings are a micronodular encodes a POZ/zinc finger transcriptional repressor that is pattern of infiltration, biphasic cytology and the presence of normally expressed in B cells within germinal centers, and marginal zone differentiation.1–3 Although a molecular hall- which is required for germinal center formation and T cell- mark for this entity has yet to be described, a significant per- directed immune response.24,25 Several studies have shown centage of SMZL cases have 7q31-q32 deletions, and all cases that bcl-6 rearrangements occur at an overall frequency of 30– lack the cytogenetic findings that are characteristic of other 40% in diffuse large B cell lymphoma (DLBCL) and 6–14% in small B cell lymphomas.4 In spite of general agreement on the follicular lymphoma (FL).26–28 Somatic mutations in the 5′ main features of this entity, many uncertainties still remain non-coding region of the bcl-6 gene have been detected in concerning its borderline with other lymphoproliferative syn- normal germinal center B cells, as well as in lymphomas with a germinal center phenotype.21–23 These mutations are found independently of bcl-6 rearrangement in DLBCL and FL.29,30 Correspondence: MA Piris, Centro Nacional de Investigaciones Onco- Recent studies indicate that these mutations accumulating in logicas, Programa de Patologia Molecular, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo Km 2, 28220 Majadahonda-Madrid, the regulatory region of the bcl-6 gene may play a role in Spain; Fax: 34 91 5097055 lymphoma progression and in the transformation of FL to more Received 29 June 2000; accepted 18 December 2000 aggressive large cell lymphoma.31,32 Molecular heterogeneity of SMZL M-S Mateo et al 629 In order to clarify the B cell differentiation stage and the monoclonal antibody (PG-B6p) and CD38 protein was cells originating SMZL, we decided to investigate the presence detected with monoclonal antibody (VS38). of hypermutation in the bcl-6 5′ non-coding region gene, After incubation with the primary antibody, immunodetec- while simultaneously analyzing their presumably normal tion was performed with peroxidase-labeled streptavidin cellular counterparts, microdissecting cells from the germinal biotin, using diaminobencidine chromogen as substrate. All center, mantle cells and marginal zone of normal human immunostaining was performed using the TechMate 500 auto- spleen. To avoid uncertainty in the selection of SMZL cases, matic immunostaining device. All the antibodies and the we only analyzed cases that showed a typical histology in labelling system were purchased from Dako, Copenhagen, their splenectomy specimens, thereby excluding cases selec- Denmark. ted exclusively on the basis of peripheral blood cell morphology. Microdissection

Materials and methods Different areas, the germinal center, mantle zone and marginal zone were identified on a 5 µm section stained with a Tissue samples polyclonal antibody directed against IgD (Dako). The sections were digested with 0.5 µg/ml collagenase type IV (Sigma, St Frozen tissue blocks from 22 cases of SMZL were included in Louis, MO, USA) in phosphate-buffered saline (PBS) for 2 h at this study. These cases were selected from the routine and 37°C in a wet chamber. consultation files of the Pathology Laboratory of the Virgen de 300 to 500 cells from each subset were dissected using a la Salud Hospital (Toledo, Spain), based on the availability of hydraulic micromanipulator (Eppendorf) (Figure 2), and frozen material. These cases were diagnosed on the basis of treated with 10 µl of PCR buffer (Boehringer Mannheim, splenic morphology according to the criteria that are Mannheim, Germany) containing 0.25 mg/ml of proteinase K currently used.33,34 (Sigma) and digested at 50°C for 1 h. Proteinase K was inacti- The percentage of malignant cells was estimated by histo- vated at 95°C for 10 min, and 5 µl of the sample was used logical examination and immunohistochemical CD20 for PCR. staining, and at least 70% of cells were tumoral. The frozen tissue block of normal spleen was obtained from a splenectomy specimen obtained incidentally in a routine PCR gastrectomy. Histological examination of this reactive spleen showed preserved morphology (Figure 1); while an IgVH PCR A unique PCR product encompassing fragments E1.10, E1.11 study showed polyclonal amplification. and E1.12 (previously published29) and spanning 790 pb A clinical follow-up was performed in all cases, with a (nucleotides +357 to +1147) (the first nucleotide of the ampli- median range of 35 months. fied bcl-6 gene region, corresponding to the first nucleotide Peripheral blood from eight healthy donors was included in of the sense primer is arbitrarily defined as position +1) was this study as a negative control.

a b Immunostaining techniques

Monoclonal antibody for CD20 (L-26) and polyclonal antibody for IgD were used. Bcl-6 protein was detected with

c d

Figure 2 Frozen sections of a reactive follicle of normal human Figure 1 Reactive follicle from normal spleen. The germinal spleen immunostaining for IgD (a). Microdissection of germinal centre center, mantle and marginal zones can be seen. IgD− (b), mantle zone IgD+ (c) and marginal zone IgD− (d) cells.

Leukemia Molecular heterogeneity of SMZL M-S Mateo et al 630 amplified by 5′-CCGCTGCTCATGATCATTATTT (sense), and Table 1 Percentage of bcl-6 mutation in SMZL and normal B cell 5′-TAGACACGATACTTCATCTCAT (antisense) primers.22 This subpopulations of the spleen fragment is located downstream of the first bcl-6 non-coding exon, and has been shown to harbor .95% of the mutation Lymphoma Mutated cases (%) in the 5′ non-coding region of the bcl-6 gene detected in B non-Hodgkin’s lymphoma.33 The high-fidelity polymerase Pfu SMZL 3/22 (15) (Promega, Madison, WI, USA) was used to amplify genomic Normal spleen Mutated clones (%) DNA. GC 3/10 (30) The PCR reaction was carried out in a 25 µl volume con- MC 0/15 (0) taining 25 pmol of each primer, 200 µm dNTPs, 10 mm Tris MZ 5/14 (35)

HCl, 50 mm KCl, 1.5 mm MgCl2 and 2 U Taq polymerase (Boehringer Mannheim). Conditions of amplification were as SMZL, splenic marginal zone lymphoma; GC, germinal center; MC, follows: 5 min denaturation at 95°C, followed by 30 denatur- mantle cell; MZ, marginal zone. ation cycles of 30 s each at 95°C, 30 s annealing at 57°C and a 1-min extension at 72°C. There was a final step of extension at 72°C for 5 min performed in a Perkin Elmer 2400 GeneAmp with identical mutational patterns and the other with one PCR system (Norwalk, CT, USA). PCR products were checked additional mutation, interpreted as a possible subclone for yield and size on 2% agarose gels before further analysis. derived from ongoing mutation (Figure 3). The sequence analysis of this case showed two polymorphisms: deletion (∆T) at position +876, and a G-C change at position +753, 29,35 Cloning both of which have already been described. Both changes were present in heterozygosity (data not shown). A cloning strategy was adopted for the analysis of normal B cell subpopulations within the spleen. PCR products were purified using the Wizard PCR Preps kit (Promega) and ligated BCL-6 mutation in SMZL into a PGEM-T easy vector (Promega). Plasmids with the inserts were transformed into JM109 competent cells A total of 22 SMZL cases were investigated for bcl-6 mutation (Promega), checked by PCR, purified using the Wizard Plus by using direct sequencing. Mutation was found in 3/22 (13%) SV Minipreps Kit (Promega), and directly sequenced. cases of SMZL (Table 1). The characteristics of the bcl-6 mutations detected in SMZL are summarized in Table 2, and representative results are shown in Figure 4. Sequencing analysis The majority of mutations (8/10) were localized in a region of 340 bp spanning the E1.10 and E1.11 fragments. The Direct sequencing was performed from both strands using the mutations were represented by single nucleotide substitution same primers as in the amplification, with two additional and one deletion, and no point insertion was observed. The internal oligonucleotides (E1.24 and E1.23).29 The direct substitutions were caused by 44% transition and 55% trans- sequencing procedure was performed using an ABI 310 version mutations. The frequency of mutations in mutated sequencer (Perkin Elmer, Warrington, UK) following the SMZL cases ranged from 1 to 6 over the 790 bp analyzed, manufacturer’s procedure. Mutations were identified by comparison with the new bcl-6 gene germline sequence32,35 which differs in seven positions from the previously published one (Z79581). In all cases of SMZL sequencing, analysis was confirmed by two separate experiments.

Statistical analysis

Survival analyses were performed using the Kaplan–Meier life- table method. Differences between survival curves were determined according to the log-rank test.

Results

BCL-6 mutation in normal human spleen

A total of 10–15 clones from each cell population were screened for bcl-6 mutation. Mutations were found in 3/10 clones (30%) from the germinal center and 5/14 clones (35%) from the marginal zone. No mutation was found in mantle Figure 3 Representation of the bcl-6 5′ non-coding region zone B cells (0/15 clones) (Table 1). The frequency of mutations in germinal center, mantle zone and marginal zone clones from normal human spleen. The different clones are indicated by mutation in the mutated clones ranged from 0.06 to + circles. Filled circles represent IgD clones from mantle zone and × −2 0.18 10 /bp. empty circles represent IgD− clones from the germinal center and mar- Two B cell clones from the marginal zone could be related ginal zone. Clonal expansion was observed between two marginal to one of the clones present in the germinal center cells (one zone clones.

Leukemia Molecular heterogeneity of SMZL M-S Mateo et al 631 Table 2 Characteristics of mutations of the bcl-6 5′ non-coding ence32,35 was observed from the reported germline (GenBank region in SMZL accession No. Z79581).

Sample Mutation (position)a Mutation frequency (×10−2/bp)b Correlation between bcl-6 mutation and clinical outcome S 202 G!C(440); G!A(440); G!C(478) 0.37 T!C(538); A!G(688); C!G(1047) All three patients with bcl-6 somatic mutation were alive at S 206 del G(438); A!G(516); C!G(779) 0.18 36, 40 and 13 months, respectively, after splenectomy, with- S 222 C!G(917) 0.06 out evidence of tumoral progression. Analysis of the cases without mutations showed an increasing probability of relapse SMZL, splenic marginal zone lymphoma. or progression over time, but comparative analysis of both ser- aThe ﬁrst nucleotide of the bcl-6 cDNA is arbitrarily chosen as ies did not show any signiﬁcant results, due to the small num- + position 1. ber of patients with somatic mutation included in this series. bCalculated as the ratio between mutated nucleotides and bp sequenced. A relationship between CD38 expression and the absence of bcl-6 somatic mutation was not observed in this series, given that CD38 expression was observed in 4/22 cases, one of them with somatic mutation.

Discussion

This study analyzed mutations in the 5′ non-coding region of the bcl-6 gene in SMZL and different cell populations microdissected from human normal spleen. Previous studies have demonstrated that bcl-6 mutations take place in normal germinal center B cells, and they are therefore regarded as a histogenetic marker of B cell transition through the germinal center in the analysis of lymphomas.29,30

Different studies suggest that bcl-6 and IgVH mutations are produced through the same mechanism, in the same cell com- partments,21,22 and that the difference in the mutability of the

IgVH and bcl-6 genes may be attributable to differences in transcription rates or qualitative differences in control elements.22 Thus essentially the lymphoma types in which

IgVH mutations are detected simultaneously display bcl-6 mutations, albeit at a lower frequency.21,36 Controversial ﬁnd- ings have, nevertheless, been recently published for B-CLL,

where the existence of isolated somatic hypermutation of IgVH or bcl-6 genes has been recently demonstrated. This somatic hypermutation was found useful in B-CLL to define a molecular heterogeneity of this entity, which revealed a significant clinical difference.35,36 These findings prompted us to evaluate the frequency and distribution of bcl-6 mutations in SMZL and Figure 4 Direct sequencing analysis of mutations of the 5′ non- microdissected normal B cell subpopulations. coding regions of the bcl-6 gene in SMZL. The sequence of each case In this series of SMZL cases, mutations of the 5′ non-coding of SMZL is matched with the sequence of a normal control (N) dis- region of the bcl-6 gene were detected in only 3/22 (13%) playing germline bcl-6 alleles. The mutations are indicated by arrows, with the position of mutations corresponding to bcl-6 germline of the analyzed cases. The existence of a relative molecular sequence. heterogeneity in this condition could be supported by this observation, in which a minority of cases show bcl-6 somatic mutation, at a frequency (0.05%) which is comparable to that corresponding to a frequency of 0.06 to 0.37 × 10−2/bp of peripheral memory B cells.21–23 This is consistent with the (Table 2). hypothesis that a fraction of this lymphoproliferative syndrome A variety of studies have recognized specific nucleotide derives from post-germinal center cells, while a majority motifs within the Ig genes as intrinsic hotspots for somatic hyp- would derive from naive B cells. A similar variability in the ermutations, and the consensus RGYW (A/G G C/T A/T) frequency of bcl-6 and IgVH somatic mutations has also been appears to be the one that is most frequently mutated. In the observed in a closely related lymphoproliferative condition, B bcl-6 locus, the frequency of G mutations within the RGYW cell chronic lymphocytic leukemia (B-CLL), where cases with motif is inferior to that observed in IgVH. In these cases one IgVH somatic mutation were also characterized by the absence of two G substitutions were flanked by the mutation hot of CD38 expression and a more favorable clinical course.36–39 motif RGYW. A revision of the frequency of CD38 expression and compara- The two polymorphisms detected in the normal spleen were tive analysis of the clinical course in our series failed to ident- present in some SMZL cases, as well as in the DNA from the ify any significant difference, perhaps because of the relatively peripheral blood of healthy donors. small size of the group with bcl-6 mutations. Thus, a more In all cases analyzed, a previously described 7 base differ- comprehensive analysis of a larger group of SMZL cases seems

Leukemia Molecular heterogeneity of SMZL M-S Mateo et al 632 to be warranted by this study, in order to search for other the transcriptional activity of bcl-6.29,30,44 The level of clinical, morphological and phenotypical traits linked to this expression of bcl-6 in these cases did not differ substantially molecular heterogeneity. from expression in non-mutated cases, where bcl-6 is mainly These results contrast with previous studies in which cases expressed in the germinal center, and at lower intensity in of SMZL and splenic lymphoma with villous lymphocytes marginal zone cells. (SLVL), an overlapping condition, showed somatic mutations In this study, mutations in the bcl-6 gene have been found 40–43 of IgVH genes. This difference could be derived from the in benign germinal centers as well as marginal zone cells of different criteria used to select cases, the difficulty of accu- normal spleen. These findings confirm previous data on the

rately recognizing cases of SMZL, or it may also be dependent frequency of IgVH mutations in the marginal zone cells of the on the choice of the bcl-6 gene in this series, instead of IgVH. spleen, thereby confirming that at least a significant pro- To avoid bias deriving from a low representation of tumoral portion of splenic marginal zone B cells are composed of cells in the analyzed samples, we only studied DNA extracted memory cells, which is consistent with their IGM+ IgD− immu- from splenic samples in which the percentage of B cells was nophenotype, suggesting that these cells are post-germinal shown to represent over 70% of the cells included in the sam- center B cells (Table 3).16,18 This study has also shown evi- ple. At the same time the diagnosis of all cases was confirmed dence of clonal expansion within the marginal zone. How- in spleen sections, using restrictive histological and immuno- ever, the mantle cells of the spleen that were analyzed, selec- phenotypical criteria. ted on the basis of their IgD expression, show a germinal The low frequency of bcl-6 mutations in this series differs configuration of the bcl-6 gene. This datum coincides with

significantly from the percentage of bcl-6 mutation that other others previously obtained in the analysis of the IgVH and bcl- studies have observed in marginal zone lymphoma–mucosa- 6 genes, which corroborate that mantle cells in the spleen are associated lymphoid tissue (MZL-MALT), DLBCL and FL, naive B cells.16 where the frequency of mutations ranges from 33% to 60%; The data presented here seem to support the hypothesis this is slightly more frequent than in mantle cell lymphoma that, in spite of initial morphological observations, a signifi- (MCL) (5%, 10%) and slightly lower than the percentages of cant proportion of SMZL cases could derive from an unmu- bcl-6 mutations observed in chronic lymphocytic leukemia tated naive precursor other than the marginal zone, and poss- (CLL) (15–42%) (Table 3).21,31,35,36 ibly situated in the mantle zone of the splenic lymphoid Previous studies have suggested that bcl-6 mutations may follicles. This is consistent with the high frequency of IgD have functional significance, based on their frequency and expression by SMZL cells. This has already been proposed as clustering in the proximity of the bcl-6 promoter. This is sup- an explanation for the lack of marginal zone differentiation ported by in vitro studies showing that mutations may alter observed in the lymph nodes involved by SMZL, and the frequent presence of marginal zone differentiation in spleens involved by a variety of types of non-Hodgkin lymphoma. This Table 3 Incidence of mutations of the bcl-6 5′ non-coding region in normal B cells and B cell lymphomas suggests that such marginal zone differentiation could be dependent on the microenvironmental features of the lym- Type of cell References phoid follicles in the spleen, rather than the histogenetic characteristics of the tumor. Normal cell Mutated cells (%) + + PBL cells IgM /IgD 022 Acknowledgements Tonsil MC IgM+/IgD+ 5.7; 4 21, 23 Spleen MC IgD+ 0 This study PBL cells IgA, IgG 30–42 22 This study was supported by grants (FIS 99/0705) from the Tonsil GC B cells 32; 54 21, 23 Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y − Spleen GC B cells IgD 30 This study Consumo, and 1FD97–0431, from the Comision Interminis- Spleen MZ cells 35 This study terial de Ciencia y Tecnologia, Spain. Dr D Capello IgM+/IgD− (Department of Medical Sciences, University of Torino at B cell lymphomas Mutated Novara, Italy) for her assistance in the interpretation of results; cases (%) Drs T Flores, L Bernardo, M Morente and M Medina from the MCL 5;4.8;10 21, 23, 31 Pathology Departments at hospitals in Salamanca, Gerona, CLL 15; 30; 36; 21, 31, 35, 36 Guadalajara and Seville (SPAIN), for kindly providing the 42 cases included in this series. SMZL 13 This study SLL 22 31 HCL 25 31 BL 37 31 References LPL 40 31 MZL-MALT 42; 33 23, 31 1 Harris NL, Jaffe NS, Stein H, Banks PM, Chan JKC, Cleary M, FL 37; 42; 60 21, 23, 31 Delsol G, Wolf-Peters CD, Falini B, Gatter KC, Grogan TM, Isaac- DLBCL 59 21 son PG, Knowles DM, Masson SY, Muller-Hermelink HK, Pileri Primary splenic DLBCL 60 31 SA, Piris MA, Ralfkiaer E, Warnke RA. A revised European–Amer- ican classification of lymphoid neoplasms: a proposal from the PBL, peripheral blood lymphocytes; MC, mantle cells; GC, germinal International Lymphoma Study Group. Blood 1994; 84: 1361– center; MZ, marginal zone; MCL, mantle cell lymphoma; CLL, 1392. chronic lymphocytic leukemia; SMZL, splenic marginal zone lym- 2 Mollejo M, Menarguez J, Lloret E, Sanchez A, Campo E, Algara phoma; SLL, small lymphocytic lymphoma; HCL, hairy cell leuke- P, Cristobal E, Sanchez E, Piris MA. Splenic marginal zone lym- mia: BL, Burkitt’s lymphoma; LPL, lymphoplasmocytoid lymphoma; phoma, a distinctive type of low-grade B-cell lymphoma: a clinic- MZL-MALT, marginal zone lymphoma–mucosa-associated lymph- opathological study of 13 cases. Am J Surg Pathol 1995; 19: oid tissue; DLBCL, diffuse large B cell lymphoma. 1146–1157.

Leukemia Molecular heterogeneity of SMZL M-S Mateo et al 633 3 Isaacson PG, Piris MA. Splenic marginal zone lymphoma. Adv Louie DC, Offit K, Chaganti RSK, Dalla-Favera R. BCL-6 protein Anat Pathol 1997; 4: 191–201. is expressed in germinal centre B cells. Blood 1995; 86: 45–53. 4 Mateo M, Mollejo M, Villuendas R, Algara P, Sanchez-Beato M, 25 Onizuka T, Moriyama M, Yamochi T, Kuroda T, Kazama A, Kana- Martıńez P, Piris MA. 7q31–32 allelic loss is a frequent finding in zawa N, Sato K, Kato T, Ota H, Mori S. BCL-6 gene product, a 92- splenic marginal zone lymphoma. Am J Pathol 1999; 154: to 98-kD nuclear phosphoprotein, is highly expressed in germinal 1583–1589. centre B cells and their neoplasic counterparts. Blood 1995; 86: 5 Piris MA, Mollejo M, Campo E, Menarguez J, Flores T, Isaacson 28–37. PG. A marginal zone pattern may be found in different varieties 26 Lo Coco F, Ye B, Lista F, Corradini P, Offit K, Knowles DM, of non-Hodgkin’s lymphoma: the morphology and immunohisto- Chaganti RSK, Dalla-Favera R. Rearrangements of the BCL6 gene logy of splenic involvement by B cell lymphomas simulating in diffuse large cell non-Hodgkin’s lymphoma. Blood 1994; 83: splenic marginal zone lymphoma. Histopathology 1998; 33: 1757–1759. 230–239. 27 Bastard C, Deweindt C, Kerchaert J-P, Lenormand B, Rossi A, Pez- 6 Van Krieken JHJM, Te Velde. Normal histology of the human ella F, Fruchart C, Duval C, Monconduit M, Tilly H. LAZ3 spleen. Am J Surg Pathol 1998; 12: 777–785. rearrangements in non-Hodgkin’s lymphoma: correlation with his- 7 Spencer J, Kinn T, Isaacson PG. Human Peyer’s patches. An immu- tology, immunophenotype, karyotype, and clinical outcome in nohistochemical study. Gut 1986; 27: 405–410. 217 patients. Blood 1994; 83: 2423–2427. 8 Morente M, Piris MA, Orradre JL, Rivas C, Villuendas R. Human 28 Otsuki T, Yano T, Clark HM, Bastard C, Kerckaert J-P, Jaffe ES, tonsil intraepithelial B cells: a marginal zone related subpopul- Raffels M. Analysis of LAZ3 (BCL-6) status in B cell non-Hodgkin’s ation. J Clin Pathol 1992; 83: 323–341. lymphomas: results of rearrangement and gene expression studies 9 Spencer J, Kinn T, Pulford KAF, Mason DY, Isaacson PG. The and a mutational analysis of coding region sequences. Blood human gut contains a novel population of B lymphocytes which 1995; 85: 2877–2884. resemble marginal zone cells. Clin Exp Immunol 1985; 62: 29 Migliazza A, Martinotti S, Chen W, Fusco C, Ye BH, Knowles DM, 607–612. Offit K, Chaganti RSK, Dalla-Favera R. Frequent somatic hyper- 10 Van Krieken JHJM, Schilling CV, Kluin M, Lennert K. Splenic mar- mutation of the 5’ noncoding region of the BCL6 gene in B-cell ginal zone lymphocytes and related cells in the lymph node: a lymphoma. Proc Natl Acad Sci USA 1995; 92: 12520–12524. morphologic and immunohistochemical study. Hum Pathol 1989; 30 Gaidano G, Carbone A, Patore C, Capello D, Migliazza A, Glogh- 20: 320–325. ini A, Roncella S, Ferrarini M, Saglio G, Dalla-Favera R. Frequent ′ 11 Berek C, Berger A, Apel M. Maturation of the immune response mutation of the 5 noncoding region of the BCL-6 gene in acquired in germinal centres. Cell 1991; 67: 1121–1129. immunodeficiency syndrome-related non-Hodgkin’s lymphomas. 12 Klein U, Ku¨ppers R , Rajewsky K. Human IgM+IgD+ B cells, the Blood 1997; 89: 3755–3762. 31 Capello D, Vitolo U, Pasqualucci L, Quattrone S, Migliaretti G, major B cell subset in the peripheral blood, express Vk genes with no or little somatic mutation throughout life. Eur J Immunol 1993; Fassone L, Ariatti C, Vivenza D, Gloghini A, Pastore C, Lanza C, 23: 3272–3277. Nomdedeu J, Botto B, Freilone R, Buonaluto D, Zagonel V, Gallo 13 Ku¨ppers R, Zhao M, Hansmann ML, Rayewsky K. Tracing B cell E, Palestro G, Saglio G, Dalla-Favera R, Carbone A, Gaidano G. development in human germinal centres by molecular analysis of Distribution and pattern of BCL-6 mutations throughout the spec- trum of B cell neoplasia. Blood 2000; 95: 651–659. single cells picked from histological sections. EMBO J 1993; 12: ′ 4955–4967. 32 Lossoa IS, Levy R. Mutations analysis of the 5 noncoding regulat- 14 Pascual V, Liu Y-J, Magalski A, Bouteiller O, Banchereau J, Capra ory region of the BCL-6 gene in non-Hodgkin lymphoma: evi- JD. Analysis of somatic mutation in five B cell subsets of human dence for recurrent mutations and intraclonal heterogeneity. tonsil. J Exp Med 1994; 180: 329–339. Blood 2000; 95: 1400–1405. 33 Isaacson PG, Matutes E, Burke M, Catovsky D. The histopathology 15 Klein U, Kuppers R, Rajewsky K. Evidence for a large compartment of splenic marginal lymphoma with villous lymphocytes. Blood of IgM-expressing memory B cells in humans. Blood 1997; 89: 1994; 84: 3828–3834. 1288–1298. 34 Mollejo M, Lloret E, Menarquez J, Piris MA. Lymph node involve- 16 Dunn-Walters DK, Isaacson PG, Spencer J. Analysis of mutations ment by splenic marginal zone lymphoma morphological an in immunoglobulin heavy chain variable region genes of microdis- immunohistochemical features. Am J Pathol 1997; 21: 772–780. sected marginal zone (MGZ) B cells suggests that the MGZ of 35 Sahota S, Davis Z, Hamblin TJ, Stevenson FK. Somatic mutation of human spleen is a reservoir of memory B cells. J Exp Med 1995; bcl-6 genes can occur in the absence of Vh mutations in chronic 182: 559–566. lymphocytic leukemia. Blood 2000; 95: 3534–3540. 17 Dunn-Walters DK, Isaacson PG, Spencer J. Sequence analysis of 36 Capello D, Fais F, Vivenza D, Migliaretti G, Chiorazzi N, Gaidano rearranged IgV genes from microdissected human Peyer’s patch H G, Ferrarini M. Identification of three subgroups of B cell chronic marginal zone B cells. Immunology. 1996; 88: 618–624. lymphocytic leukemia based upon mutations of BCL-6 and IgV 18 Tierens A, Delabie J, Michiels L, Vandenberghe P, Wolf-Peeters genes. Leukemia 2000; 14: 811–815. CD. Marginal-zone B cells in the human lymph node and spleen 37 Naylor M, Capra JD. Mutational status of Ig VH genes provides show somatic hypermutations and display clonal expansion. clinically valuable information in B-cell chronic lymphocytic leu- Blood 1999; 93: 226–234. kaemia. Blood 1999; 94: 1837–1839. 19 Stein K, Hummel M, Korbjuhn P, Foss HD, Anagnostopoulos, Mar- 38 Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buch- afioti T, Stein H. Monocytoid B cells are distinct from splenic mar- binder A, Budman D, Dittmar K, Kolitz J, Lichtman SM, Schulman ginal zone cells and commonly derive from unmutated naive B P, Vinciguerra VP, Rai KR, Ferrarini M, Chiorazzi N. IgV gene cells and less frequently from postgerminal centre B cells by poly- mutation status and CD38 expression as novel prognostic indi- clonal transformation. Blood 1999; 94: 2800–2808. cators in chronic lymphocytic leukaemia. Blood 1999; 94: 20 Rajewsky K. Clonal selection and learning in the antibody system. 1840–1847. Nature 1996; 381: 751–758. 39 Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. 21 Pasqualucci L, Migliazza A, Fracchiola N, William C, Neri A, Bal- Unmutated Ig VH genes are associated with a more aggressive dini L, Chaganti RSK, Klein U, Ku¨ppers R, Rajewsky K, Dalla-Fav- form of chronic lymphocytic leukaemia. Blood 1999; 94: 1848– era R. BCL-6 mutations in normal germinal centre B cells: evi- 1854. dence of somatic hypermutation acting outside Ig loci. Proc Natl 40 Dunn-Walters DK, Boursier L, Spencer J, Isaacson PG. Analysis of Acad Sci USA 1998; 95: 11816–11821. immunoglobulin genes in splenic marginal zone lymphoma sug- 22 Shen HM, Peters A, Baron B, Zhu X, Storb U. Mutations of BCL- gests ongoing mutation. Hum Pathol 1998; 29: 585–593. 6 gene in normal B cells by the process of somatic hypermutation 41 Miranda RN, Cousr JB, Hammer RD, Collins RD, Vnencak-Jones of Ig genes. Science 1998; 280: 1751–1752. CL. Somatic mutation analysis of IgH variable regions reveals that 23 Peng HZ, Du MQ, Koulis A, Aiello A, Dogan A, Pan LX, Isaacson tumor cells of most parafollicular (monocytoid) B-cell lymphoma, PG. Nonimmunoglobulin gene hypermutation in germinal centre splenic marginal zone B-cell lymphoma, and some hairy cell leu- B cells. Blood 1999; 93: 2167–2172. kaemia are composed of memory B lymphocytes. Hum Pathol 24 Cattoretti G, Chang C-C, Cechova K, Zhang J, Ye BH, Falini B, 1999; 30: 306–312.

Leukemia Molecular heterogeneity of SMZL M-S Mateo et al 634 42 Tierens A, Delabie J, Pittaluga S, Driessen A, Wolf-Peeters CD. phocytes involves B cells with extensively mutated Ig heavy chain Mutation analysis of the rearranged immunoglobulin heavy chain variable region genes. Blood 1995; 85: 1603–1607. genes of marginal zone cell lymphomas indicates an origin from 44 Migliazza A, Ye H, Frachiolla N, Neri A, Baldini L, Chaganti RSK, different marginal zone B lymphocyte subsets. Blood 1998; 91: Dalla-Favera R. Mutations of the 5′ non-coding region of the BCL- 2381–2386. 6 are common in B cell malignancies and can deregulate BCL-6 43 Zhu D, Oscier DG, Stevenson FK. Splenic lymphoma with lym- expression. Blood 1997; 90: 780 (Abstr.).

Leukemia