Molecular and Immunological Dissection of Diffuse Large

Leukemia (1999) 13, 1441–1447  1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu ,Molecular and immunological dissection of diffuse large B cell lymphoma: CD5؉ and CD5؊ with CD10؉ groups may constitute clinically relevant subtypes S Harada1,2, R Suzuki1, K Uehira3, Y Yatabe4, Y Kagami3, M Ogura3, H Suzuki5, A Oyama6, Y Kodera7, R Ueda2, Y Morishima3, S Nakamura4 and M Seto1

1Laboratory of Chemotherapy, Aichi Cancer Center Research Institute; 3Department of Hematology and Chemotherapy, 4Department of Pathology and Clinical Laboratories, Aichi Cancer Center Hospital; 2Second Department of Internal Medicine, Nagoya City University School of Medicine; 5Department of Internal Medicine, Okazaki Municipal Hospital; 6Department of Internal Medicine, Aichi Prefectural Hospital; 7Department of Internal Medicine, Japanese Red Cross, Nagoya First Hospital, Japan

Diffuse large B cell lymphoma (DLBL) constitutes the greatest genotypic parameters were found not to be helpful for deline- percentage of adult non-Hodgkin’s lymphomas and represents ating distinctive morphologic subgroups). DLBL accounts for a diverse spectrum of lymphoid neoplasms. Clinicopathologic, the greatest percentage (48%) of adult non-Hodgkin’s lym- phenotypic and genotypic findings were correlated and com- 4 pared for 63 DLBL cases to investigate whether they represent phoma (NHL) among Japanese patients and for about 40% clinically relevant subtypes. They were all cyclin D1 negative of initial NHL diagnoses in the United States.5 Among DLBL, and were phenotypically divided into three groups, ie group I only a few entities, eg primary mediastinal large B cell lym- + − + (CD5 type, n = 11), group II (CD5 CD10 type, n = 19), and phoma characterized by the fact that it has a peculiar disease − − = group III (CD5 CD10 type, n 33). Data were correlated by site and affects young women, are currently regarded as dis- observing the respective gene rearrangement and expression 1 of BCL2 and BCL6. In clinical aspects, the group I cases dem- tinct clinicopathologic diseases although they are not defined onstrated a significantly inferior survival than those of the in terms of detailed phenotypic and genotypic findings. It is other two groups (log-rank test, P = 0.016). Although rearrange- thus essential for a further understanding of DLBL to create ment of BCL2 and BCL6 did not show any inclination to a spe- reproducible criteria in order to delineate their distinct sub- cific subgroup, the immunohistochemical detection of BCL2 types, which should be relevant for future therapy strategies. = was less frequent, at a statistically significant level (P 0.011), Cytogenetic studies and molecular analysis of protoonco- in group II (50%) than in group I (82%) and III (82%) cases. This appears to confirm the unique aspect of the CD5− CD10+ type genes and tumor suppressor genes have provided insights into DLBL, indicating a certain relationship with the normal germi- the pathogenesis of non-Hodgkin’s lymphoma. They are also nal center cells which usually lack BCL2 expression. The BCL6 relevant for diagnosis and establishment of prognosis. There- protein expression was detected in most of the present DLBL fore, much interest has recently been focused on the pathobi- cases (92%) irrespective of this grouping. These data suggest ologic significance of BCL1, BCL2 and BCL6 genes in B cell that the phenotypic delineation by the detection of CD5 and neoplasms. Rearrangement of the BCL2 gene on chromosome CD10 will improve our understanding of DLBL and be helpful band 18q21 has been observed in up to 85% of follicular lym- in a future subgrouping of DLBL. 6–8 Keywords: diffuse large B cell lymphoma; CD5; CD10; BCL2; phomas (FL), while rearrangement of the BCL1 gene on BCL6 chromosome band 11q13 was detected in mantle cell lymphoma (MCL) with anti-cyclin D1 monoclonal antibody.9–12 Although no single genetic abnormality has been consistently Introduction associated with DLBL, rearrangement of BCL2 has been reported to occur in 20–30% of patients and to correlate with 13,14 Diffuse large B cell lymphoma (DLBL) is morphologically a reduction in overall or disease-free survival. characterized by a diffuse proliferation of large lymphoid cells The BCL6 gene, located on chromosome 3q27, encodes for with a nuclear size usually larger than that of tissue macro- a zinc-finger protein sharing homology with several transcrip- phages or at least twice the size of small lymphocytes.1 This tion factors that participate in the control of cell proliferation, 15–17 entity includes lymphomas defined as centroblastic or immu- differentiation and organ formation. Clonal BCL6 gene noblastic in the Kiel classification2 and large cell immunoblas- rearrangement appears to be associated with lymphomagen- 15–21 tic or diffuse mixed in the Working Formulation.3 In the REAL esis. DNA analysis has identified the frequency of BCL6 and coming WHO classifications, they are placed in a single rearrangement as 14–20% of NHL cases, and 27–45% of 20–22 category due to the poor reproducibility of attempts to subtype diffuse large cell type lymphoma cases. them. This lymphoma may thus be regarded as a hetero- Immunophenotyping is most relevant for defining the lym- geneous category that includes cases arising de novo or by phocyte subsets, and subsequently the putative normal transformation from low-grade B cell lymphomas, and, counterpart of each type of lymphoid neoplasm. Among the indeed, it exhibits divergent biologic behavior. Approximately B cell lymphomas, CD5 is nearly always expressed in cases 1 of MCL, but rarely in cases of FL, and vice versa for the 40% of this type has extranodal presentation, which suggests + − − + a certain relationship with mucosa-associated lymphoid tissue expression of CD10. CD5 CD10 and CD5 CD10 pheno- (MALT) lymphoma. types are often encountered in DLBL tumors, but their pheno- To our knowledge, no correlation has been reported in the typic link to respectively mantle zone cells and germinal literature between immunophenotypes and histologic sub- center cells remains to be clarified. types of large B cell lymphoma (immunophenotypic and For this study, 63 DLBL cases were immunophenotypically divided into three groups of CD5+, CD5− CD10+, and CD5− CD10− type, all of which have been confirmed to be cyclin D1 negative. We attempted to correlate their clinicopatho- Correspondence: M Seto, Laboratory of Chemotherapy, Aichi Cancer Center Research Institute, Kanokoden 1–1, Chikusa-ku, Nagoya 464– logic features and phenotype with molecular data of BCL2 and 8681, Japan; Fax: +81–52–764–2982/+81–52–763–5233 BCL6 based on the Southern blot and immunohistochemical Received 8 March 1999; accepted 11 May 1999 analyses. Findings from such a study may lead to a better Molecular and immunological dissection of diffuse large B cell lymphoma S Harada et al 1442 understanding of the nature of DLBL lesions, because results (Novocastra Laboratories, Newcastle, UK) was also performed from previously published genotypic studies of these tumors with the ABC method. Lymphoma cell positivity of more than have not been analyzed in relation to the immunophenotypes 10% was adopted as the standard of positivity for these oncog- of lymphoma cells. ene products. Surface immunophenotyping of the lymphoma cells was also performed with the aid of flowcytometry using the follow- Materials and methods ing panel of lymphoid-associated monoclonal antibodies: OKT6 (CD1), OKT11 (CD2), OKT3 (CD3), OKT4 (CD4), OKT8 Patients (CD8), OKIa 1 (Ortho, Raritan, NJ, USA), J5 (CD10) (Coulter, Hialeah, FL, USA), Leu5 (CD2), Leu4 (CD3), Leu3a (CD4), Samples of lymph node or other tumors from 63 patients with Leu1 (CD5), Leu2 (CD8), Leu11 (CD16), Leu7 (CD57), DLBL were collected at the Aichi Cancer Center Hospital and (Becton Dickinson, Mountain View, CA, USA), B1 (CD20), B2 affiliated hospitals. They were selected from 246 consecutive (CD21), B4 (CD19) and NKH1 (CD56) (DAKO). Multi- DLBL cases diagnosed at the Department of Pathology and parameter analysis of gated cell populations during cell sus- Clinical Laboratories, Aichi Cancer Center Hospital, between pension studies was used to provide more definitive immuno- October 1985 and November 1996. The pathologic diagnoses phenotypic information (Consort 30 computer software; were established by three independent hematopathologists Becton Dickinson). (YY, TS and SN) according to the updated Kiel classification and the REAL classification. Patients without an available sam- ple for DNA or phenotypic analyses were excluded from this DNA extraction and Southern blot analysis study. The cases with previous or simultaneous low-grade lymphomas, as well as those diagnosed as specific subtypes, Genomic DNA was extracted from the tumor samples as eg mediastinal B cell lymphoma or pyothorax-associated described previously.25 Briefly, frozen tumor samples were lymphoma were excluded. resuspended in 9.5 ml of lysis buffer (10 mM Tris-HCl pH 7.4, Combined chemotherapeutic regimens containing doxorub- 10 mM EDTA, 150 mM NaCl, 0.4% sodium dodecyl sulfate icin (ie cyclophosphamide, doxorubicin, vincristine and (SDS) and 500 ␮l of 20 mg/ml proteinase K). After overnight prednisolone (CHOP); vincristine, cyclophosphamide, predni- incubation at 37°C, the mixture was extracted twice with solone and doxorubicin (VEPA); or VEPA and methotrexate) phenol/chloroform and the aqueous phase was precipitated were used as the standard treatment for patients with DLBL in with 2.5 volume of ethanol. The precipitate was then washed advanced stages (stages II–IV).23,24 Patients with stage I lym- twice in 70% ethanol. The genomic DNA obtained was dried phoma were treated with either radiotherapy only or with a and dissolved in TE buffer (10 mM Tris-HCl pH 7.4, 1 mM combination of radiation and chemotherapy. EDTA pH 8.0). For Southern blot analysis, 10 ␮g of DNA was We categorized DLBL cases on the basis of the presence or digested with the appropriate restriction enzyme, electrophor- absence of the representative surface markers (CD5 and esed on 0.7% agarose gel, and transferred to nylon mem- CD10) examined by flowcytometry: group I (CD5+, group II branes (Hybond N, Amersham-Japan, Tokyo, Japan). Probes (CD5−, CD10+), and group III (CD5−, CD10−). CD5+, CD10+ were radiolabeled by using a random primer DNA labeling cases were included in group I. kit (Nippon Gene, Toyama, Japan) with [␣-32P] dCTP. After overnight hybridization, filters were washed to a final strin- gency of 0.1 × standard saline citrate (SSC)/0.1% SDS at 60°C Light microscopy and then autoradiographed using intensifying screens.

Tissue samples were fixed in 10% formalin and embedded in paraffin. Sections (5-mm thick) were stained with hematoxylin DNA probes and eosin, Elastica-van Gieson, silver impregnation, periodic acid-Schiff, May–Gru¨nwald-Giemsa, and methyl green-pyro- The BCL2 probes used have been described elsewhere.7 These nine. Imprint smears of the surgically resected specimens were included the 1.2-kilobase (kb) SacI fragment which recognizes stained with May–Gru¨nwald-Giemsa. 5ЈBCL2, the 2.8-kb EcoRI–HindIII fragment of the mbr (major breakpoint cluster region) and the 4.0-kb EcoRI fragment of the mcr (minor breakpoint cluster region). The restriction Immunohistochemistry and flowcytometry enzymes used for the BCL2 gene analysis were EcoRI, HindIII and BamHI. The surface markers of the tumor cells of each case were ana- The BCL6 gene was analyzed by hybrydization of BamHI lyzed as described previously.10,12 In brief, immunoperoxidase and XbaI-digested DNA to the human BCL6 probe Sac 4.0.17 studies were performed on formalin-fixed paraffin sections by In selected cases, a second probe representative of the BCL6 using an avidin–biotin peroxidase complex (ABC) method. A locus, Sac 0.8, was also used (kindly provided by Dr R panel of monoclonal antibodies consisting of CD3, UCHL- Dalla-Favera, College of Physicians and Surgeons, Columbia 1/CD45RO, L26/CD20, Ber-H2/CD30, CD79a (Dako, Copen- University, New York, NY, USA). hagen, Denmark) and MT1/CD43 (Bioscience Products, Emmenbru¨ke, Switzerland) was used. Tumors were con- sidered to be of B cell origin when the neoplastic cells Statistical analysis expressed CD20 or CD79a and lacked T cell-associated markers (CD3, CD43, and/or CD45RO). A T cell lineage was Overall survival was calculated from the date of diagnosis assigned if the opposite pattern of reactivity was observed. until the patients’ death or last follow-up. Survival curves were Immunohistochemistry on paraffin sections of plotted with the method of Kaplan and Meier26 and were com- PRAD1/cyclin D1 (5D411), BCL2 (Dako), and BCL6 pared with the log-rank test.27 Associations between our Molecular and immunological dissection of diffuse large B cell lymphoma S Harada et al 1443 groupings and initial clinical features and gene alterations Gene rearrangement and immunohistochemical were analyzed with the two-sided chi-squared test. analysis of BCL6

Rearranged BCL6 was found in 16 out of 63 (25%) cases: in group I (2/11, 18%), group II (3/19, 16%) and group III (11/33, Results 33%), but this relatively high incidence in group III was not statistically significant (Table 1). Furthermore, in the group III Patients’ characteristics cases, eight of the 18 (44%) DLBLs occurring in the extranodal sites showed alteration of the BCL6 gene. The frequency was relatively higher than that (3/15, 20%) of those in nodal sites, The clinical features of the 63 cases are summarized in but the difference was not statistically significant. The anatom- Table 1. There were 31 men and 32 women with a median ical sites of the extranodal lesions with BCL6 rearrangement age of 57 years at diagnosis (range, 21–89 years). The disease were the stomach (three of four, 75%), the tonsil (two of three, was in advanced stages (III or IV) at diagnosis in 39 (62%) of 67%), the colon (one of three, 33%), the breast (one of five, the patients and in an early clinical stage (stage I or II) in 24 20%), and other (one of three, 33%). (38%). In most cases, the lymphoadenopathy was superficial, Immunohistochemical examination of the BCL6 protein but 30 patients (48%) presented with extranodal lesions as the showed a positive reaction in 57 cases (92%): 10 out of 11 major sites of disease at diagnosis (Table 1). These extranodal (91%) in group I, 17 out of 18 (94%) in group II and 30 out lesions included one in the arm, six in the breast, five in the of 33 (91%) in group III (Figure 1c). Only one case (No. 8) colon, two in the nasal cavity, one on the skin, one in the soft stained negatively for BCL6 in spite of the rearrangement of tissue, one in the subcutis, two in the spleen, five in the stom- BCL6 (Table 1). ach, one in the testis, one in the thyroid and four in the tonsil. An enzyme-linked immunosorbent assay of the serum obtained from all patients did not reveal any antibodies to Correlation among the clinical, phenotypic, genetic human T cell leukemia/lymphoma virus type 1, nor did an and immunohistochemical findings analysis of their life styles suggest any risk factors for acquired immunodeficiency disease. None of the cases showed occur- Clinical, phenotypic, genetic and immunohistochemical fea- rence of a second neoplasm during their entire clinical course. tures of these 63 patients are summarized in Table 2. The Twenty-two of the 63 patients were alive at the last follow- Kaplan–Meier survival curves for the three groups of DLBL up, with a median follow-up time of 47 months from the diag- divided by phenotype are shown in Figure 2. The overall sur- nosis of DLBL. Fifty-two (83%) of the 63 patients achieved vival of group I was significantly inferior to those of groups II complete response with conventional chemotherapy, and III (P = 0.016 by log-rank test), while the latter two groups although 31 of them relapsed 1–74 months after the time of showed identical survival. When the overall survival of group diagnosis. I was compared with the mixed group of groups II and III, ie CD5+ vs CD5− groups, the survival of group I becomes more significant (P = 0.004 by log-rank test, data not shown). The Immunohistochemical analysis of cyclin D1 group I cases were also characterized by a high incidence of bone marrow involvement, which was significant in terms of the 2 × 3 chi-square test for independence (4/11, P = 0.045) In several previous reports, including one of ours,11 nuclear and of the chi-square test (P = 0.041) in comparison with the positive immunostaining of cyclin D1 has been shown to other two groups. reflect cyclin D1 mRNA overexpression and to be relevant for The prognoses of the present cases did not correlate with the diagnosis of MCL. Based on this observation, all of the the genetic or immunohistochemical data obtained in this present cases were examined by cyclin D1 immunohisto- study. However, a lower incidence of BCL2 expression in chemistry and were found to be completely negative (Table 1). group II was demonstrated to be significant according to the 2 × 3 chi-square test for independence (P = 0.039) and the chi- square test in comparison with the other two groups (P = 0.011). Gene rearrangement and immunohistochemical In order to compare our present data with those previously analysis of BCL2 published, all cases were statistically analyzed as a whole regardless of phenotype. None of the various clinical features BCL2 gene rearrangement was detected in six out of 63 (10%) including age, sex, clinical stage, histologic subtype cases: in group I (1/11, 9%), group II (1/19, 5%) and group III (immunoblastic or not), site of origin (nodal or extranodal), (4/33, 12%). The mbr probe demonstrated rearrangement in bone marrow involvement, and complete response (data not five of them, the 5Ј BCL2 probe in one and the mcr probe in shown) correlated significantly with either BCL2 or BCL6 none (Table 1). The frequency of BCL2 gene rearrangement in rearrangements. The complete response rate in BCL6 DLBL was similar to that reported in a previous study of ours.8 immunostaining-negative cases was found to be significantly The BCL2 protein expression was also immunohistochem- less than that in BCL6-positive cases (P = 0.019, chi-square ically identified in 45 out of 62 (73%) cases: in group I (9/11, test), although the number of BCL6-negative cases was small. 82%), group II (9/18, 50%) and group III (27/33, 82%). All of the six cases with BCL2 rearrangements also demonstrated strong expression of the BCL2 protein (Figure 1a). However, Discussion among the BCL2-positive cases, there was no significant difference in the strength of BCL2 expression between the cases Lymphoma classification based on morphology and cytologic with and without BCL2 rearrangements. composition alone is not necessarily suitable because com- Molecular and immunological dissection of diffuse large B cell lymphoma S Harada et al 1444 Table 1 Patients’ presentation and results of immunostain/Southern analysis on 63 DLBL cases

Case No. Sex/Age Major sites of disease Ann Arbor stage BCL1 BCL2 Southern BCL6 Southern Outcome at presentation (I–IV) (Immunostain) (Immunostain) (Immunostain)

Group I (CD5+) 1 F/38 Lymph node IV (−)G(−, Ͻ10%) G (+, Ͼ90%) DOD 6 yr 2 M/51 Lymph node III (−)G(+, Ͼ90%) G (+, 60%) AW 5.5 yr 3 F/72 Lymph node IV (−)G(+, Ͼ90%) G (+, Ͼ90%) DOD 6 mo 4 F/61 Lymph node III (−)G(+, 80%) G (+, 20%) DOD 9 mo 5 F/71 Lymph node III (−)G(+, 70%) G (+, 50%) DOD 1.5 yr 6 F/82 Lymph node III (−)G(+, Ͼ90%) G (+, 70%) DOD 1.5 yr 7 M/58 Lymph node IV (−)G(+, Ͼ90%) G (+, 70%) DOD 2 yr 8 F/36 Lymph node IV (−)G(+, Ͼ90%) R (−, Ͻ10%) DOD 1 yr 9 F/46 Colon III (−)R(+, Ͼ90%) R (+, 60%) DOD 2 yr 10 M/61 Spleen III (−)G(+, 80%) G (+, 50%) AW 1.5 yr 11 M/27 Stomach II (−)G(−, Ͻ10%) G (+, Ͼ90%) DOD 3.5 yr

Group II (CD5−, CD10+) 12 F/31 Lymph node II (−)G(−, Ͻ10%) G (+, 60%) AW 3 yr 13 M/56 Lymph node II (−)G(−, Ͻ10%) G (+, Ͼ90%) AW 2 yr 14 M/83 Lymph node I (−)G(−, Ͻ10%) G (+, 60%) AW 3.5 yr 15 F/61 Lymph node IV (−)G(−, Ͻ10%) G (+, Ͼ90%) DOD 3 mo 16 M/67 Lymph node III (−)G(+, 20%) G (+, 80%) DOD 4 yr 17 M/56 Lymph node II (−)G(−, Ͻ10%) G (+, 70%) AW 9.5 yr 18 F/68 Lymph node III (−)R(+, Ͼ90%) G (+, 80%) AW 6 yr 19 M/21 Lymph node IV (−)G(+, Ͼ90%) G (+, Ͼ90%) DOD 6 mo 20 F/89 Lymph node IV (−)G(+, 50%) R (+, 70%) AW 6 mo 21 M/57 Lymph node II (−)G(+, 80%) G (+, 60%) AW 4 yr 22 F/71 Arm IV (−)G(+, Ͼ90%) R (+, 70%) AW 3 yr 23 F/55 Breast IV (−)G(+, Ͼ90%) G (+, 70%) DOD 2 yr 24 M/53 Colon IV (−) G (ND) G (ND) DOD 10 mo 25 M/51 Nasal cavity II (−)G(−, Ͻ10%) G (−, Ͼ10%) DOD 3 mo 26 M/56 Skin IV (−)G(+, Ͼ90%) G (+, 50%) DOD 6 yr 27 M/54 Spleen IV (−)G(−, Ͻ10%) G (+, Ͼ90%) AW 7 yr 28 M/59 Testis III (−)G(−, Ͻ10%) G (+, 60%) DOD 6.5 yr 29 F/56 Thyroid I (−)G(−, Ͻ10%) R (+, Ͼ90%) AW 4.5 yr 30 M/52 Tonsil II (−)G(+, Ͼ90%) G (+, Ͼ90%) AW 8.5 yr

Group III (CD5−, CD10−) 31 F/76 Lymph node IV (−)G(−, Ͻ10%) G (+, 50%) DOD 7 mo 32 F/61 Lymph node IV (−)R(+, 50%) G (+, 70%) AW 7.5 yr 33 M/52 Lymph node II (−)R(+, Ͼ90%) G (+, 60%) AW 6.5 yr 34 F/58 Lymph node III (−)G(+, Ͼ90%) G (−, Ͻ10%) DOD 4.5 yr 35 M/54 Lymph node IV (−)G(+, 70%) G (−, Ͻ10%) DOD 9 mo 36 M/58 Lymph node II (−)G(+, Ͼ90%) G (+, 80%) AW 4.5 yr 37 F/76 Lymph node III (−)G(+, 50%) R (+, 50%) DOD 2.5 yr 38 M/47 Lymph node I (−)G(+, 80%) G (+, Ͼ90%) AW 3 yr 39 M/72 Lymph node IV (−)G(+, Ͼ80%) G (+, 90%) DOD 4 mo 40 M/39 Lymph node II (−)Ra (+, Ͼ90%) R (+, Ͼ90%) DOD 4.5 yr 41 F/65 Lymph node III (−)G(+, Ͼ90%) G (+, Ͼ90%) AW 7 yr 42 F/48 Lymph node IV (−)G(+, Ͼ90%) G (+, 30%) AW 4.5 yr 43 F/46 Lymph node II (−)G(+, 50%) G (+, 50%) DOD 3.5 yr 44 M/53 Lymph node II (−)G(+, 80%) G (+, 70%) AW 7 yr 45 F/59 Lymph node IV (−)G(+, Ͼ90%) R (+, 80%) AW 2 yr 46 F/38 Breast II (−)G(+, Ͼ90%) G (+, Ͼ90%) AW 1 yr 47 F/64 Breast III (−)G(+, 80%) G (+, 70%) AW 9.5 yr 48 F/60 Breast I (−)G(+, 80%) G (+, 50%) AW 5 yr 49 F/46 Breast IV (−)G(+, Ͼ90%) R (+, 70%) AW 2.5 yr 50 F/46 Breast II (−)G(+, Ͼ90%) G (+, Ͼ90%) DOD 2.5 yr 51 M/60 Colon II (−)G(+, 80%) G (+, 60%) AW 6 yr 52 F/34 Colon IV (−)G(+, Ͼ90%) G (−, Ͻ10%) DOD 6.5 yr 53 M/68 Colon IV (−)G(+, Ͼ90%) R (+, 70%) DOD 1 mo 54 M/61 Nasal cavity II (−)G(+, 70%) R (+, 50%) DOD 1 yr 55 M/59 Soft tissue III (−)G(+, Ͼ90%) G (+, 50%) DOD 1.5 yr 56 M/56 Stomach II (−)G(−, Ͻ10%) R (+, 80%) AW 2 yr 57 F/53 Stomach IV (−)G(−, Ͻ10%) R (+, Ͼ90%) AW 3 yr 58 F/65 Stomach II (−)G(+, 60%) G (+, 80%) AW 5.5 yr 59 M/46 Stomach II (−)G(+, Ͼ90%) R (+, 70%) DOD 1.5 yr 60 M/62 Subskin III (−)G(−, Ͻ10%) G (+, 70%) DOD 1 yr 61 F/66 Tonsil II (−)G(−, Ͻ10%) R (+, 50%) DOD 4.5 yr 62 M/34 Tonsil III (−)G(−, Ͻ10%) G (+, Ͼ90%) DOD 10 yr 63 F/53 Tonsil III (−)R(+, Ͼ90%) R (+, 70%) AW 9 yr

DOD, died of disease; AW, alive and well; ND, not done; G, germ line; R, rearranged. a5Јbcl-2 is rearranged. Molecular and immunological dissection of diffuse large B cell lymphoma S Harada et al 1445 Table 2 Clinicopathologic characteristics, BCL2 and BCL6 analyses by immunophenotypic subgroup

Group I Group II Group III P value (n = 11) (n = 19) (n = 33)

Age (Ͼ60 yr) 5 (45)a 6 (32) 11 (33) 0.716 Sex (male) 4 (36) 12 (63) 15 (45) 0.302 Stage (III, IV) 10 (91) 11 (58) 18 (55) 0.090 Histologic type 2 (18) 4 (21) 11 (33) 0.485 (immunoblastic) Site of origin (extranodal) 3 (27) 9 (47) 18 (55) 0.292 BM involvement (yes) 4 (36) 4 (21) 2 (6) 0.045 Complete response (yes) 8 (73) 16 (84) 28 (80) 0.640 a BCL2 rearrangement 1 (9) 1 (5) 4 (12) 0.719 BCL2 immunostaining 9 (82) 9 (50)b 27 (82) 0.039 (positive) BCL6 rearrangement 2 (18) 3 (16) 11 (33) 0.313 BCL6 immunostaining 10 (91) 17 (94b) 30 (91) 0.898 (positive)

aNumber in parenthesis indicates percentage. bNumbers examined were 18 cases.

Figure 2 Overall survival of patients (Kaplan–Meier method) in groups I, II and III. c lymphoma and intravascular lymphomatosis, have been Figure 1 Immunohistochemical staining of BCL2 (a and b) and BCL6 (c) protein in DLBL. (a) A case with high BCL2 expression show- defined in terms of their peculiar clinicopathologic features. ing strong labelling of virtually all tumor cells. (b) The tumor cells Previous studies have reported the detection of BCL1 and show no positivity for BCL2. Note the BCL2-positive small reactive BCL2 rearrangements in a small percentage of DLBL cases. lymphocytes which may be regarded as internal positive controls. (c) BCL6 rearrangements have also been reported as one of the All tumor cells show nuclear positivity for BCL6. Original magnifi- most frequent gene alterations in DLBLs.28 Patients with BCL6 × cation 350. genetic alterations were subsequently described to have a better clinical outcome,19 but this has been contradicted by other pletely different biologic entities can have relatively similar studies.20,29 All clinical characteristics were also evenly dis- appearances. Therefore, definition of disease entity has to be tributed between patients with rearranged and those with based on a combination of clinical, histologic, phenotypic germ-line BCL6 in such studies.20,29 Thus, it is still contro- and genotypic features. In practice, the representative types of versial whether the BCL6 gene can be a useful marker for low-grade B cell tumors have been phenotypically and geno- defining a subset of DLBLs. In an attempt to establish definite typically defined in addition to an analysis of their morpho- dividing lines among DLBL cases, three groups have been logic features:1 MCL has a CD5+ phenotype and has cyclin D1 identified in the present study on the basis of their surface overexpression activated by t(11;14); FL has a CD5− CD10+ markers: group I (CD5+ CD10− CD20+ type), group II (CD5− phenotype with abnormal expression of BCL2 by t(14;18); and CD10+ CD20+ type), and group III (CD5− CD10− CD20+ type). marginal zone B cell lymphoma of MALT type has a CD5− This grouping is based on the hypothesis that the expression CD10− phenotype often with t(11;18) or trisomy 3 which of CD5 and CD10 may be related to developmental stages of remains to be genetically clarified. Until now, however, the mantle zone cell and follicular center cell, or certain subsets attempts to delineate a specific group among DLBL cases on of B cell neoplasms, ie MCL and FL. To our knowledge, this is genetic and phenotypic grounds have resulted in failure1 the first report of a correlative analysis of clinical, phenotypic, although a few distinct subtypes, eg mediastinal large cell genotypic and immunohistochemical findings for DLBL cases. Molecular and immunological dissection of diffuse large B cell lymphoma S Harada et al 1446 The present study highlights the clinical significance of nostic significance. The BCL6 gene rearrangement also identifying the CD5 phenotype of DLBLs. The group I cases showed no significant relationship with survival, which was of this phenotype were characterized by a more aggressive in keeping with the other investigations, while a immunohisto- clinical course and more frequent bone marrow involvement chemical negativity of BCL6 was associated with a poor prog- than those in groups II and III. All cases were cyclin D1 nega- nosis (P = 0.004). This should be confirmed in a larger-scale tive, and most expressed the BCL2 and BCL6 proteins, study since the number of BCL6 unstained cases was limited although only a few cases showed their genetic alterations. in the present study. They were also distinct from other CD5+ low-grade tumors, It is noteworthy that case No. 8 showed BCL6 gene that is, MCL and CLL, in that they showed a high frequency rearrangement in spite of less than 10% BCL6-positive tumor of somatic mutations in the heavy chain variable region of the cells. This case simultaneously showed more than 90% posi- immunoglobulin gene as described by us and others.30,31 The tivity in BCL2 staining, suggesting that the reduced expression number of the group I cases appears to be relatively small, of BCL6 may not be affected by prolonged formal in fixation. which may represent the incidence of this phenotype in DLBL. Indeed, the relation between BCL6 immunostaining and gene The clinical significance of CD5+ phenotype in DLBL should rearrangement is currently not well clarified. It has been indi- be confirmed in a larger number of the cases examined. As cated that activation of the BCL6 gene is essential for B cells well, further investigation is needed for clarifying the putative to give rise to germinal center formation37 and the BCL6 pro- normal counterpart of this group. tein has been immunohistochemically detected in germinal There were no significant differences in clinical features and center B cells.38 Thus, it is possible that the positive staining overall survival between groups II and III. However, the inci- of BCL6 without BCL6 gene rearrangement may indicate the dence of BCL2 expression in group II (50%, Figure 1b) was origin of DLBL to be follicular center cells. Further studies are significantly lower than that in groups I and III (82%) as well required to clarify the relationship of BCL6 protein expression as that in FLs (110/127, 87%) (unpublished data). Normal fol- with normally regulated and deregulated expression resulting licular center cells have a CD5− and CD10+ immunopheno- from BCL6 gene rearrangement. type and rarely express BCL2.32,33 The tumor cells of CD5− Our present study shows the importance of surface pheno- CD10+ CD20+ BCL2− type in group II might thus be assumed typing of CD5 and CD10 with reference to the expression of to have the biologic property of follicular center cells, oncogenes and clinical outcomes. Further investigations with although half of the cases in group II showed positive BCL2 other phenotypic or molecular markers are warranted for new immunostaining. Only one of the eight BCL2+ cases in this insights into the development of malignant lymphomas and to group (case No. 18, Table 1) featured BCL2 gene rearrange- help establish more effective therapeutic strategies. ment, which resulted in BCL2 expressed as expected. How- ever, the mechanism of BCL2 expression without BCL2 gene rearrangement in the other seven cases is unknown. One Acknowledgements possibility is that they may have been derived from BCL2+ 32 cells occasionally found in germinal centers, while an alter- We would like to thank Drs Y Hosokawa, T Joh, M Kume, T native explanation is that they might have originated from the + 34 Akagi and N Hamajima for their valuable discussions, and we other normal counterparts which were BCL2 . Monni et al greatly appreciate the technical assistance of Mss H Suzuki, also suggested the possibility that BCL2 expression is caused M Sugiyama and Y Maeda. This work was supported in part by gene amplification at follicular center cell stage B cells, by a Grant-in-aid for the Second-term Comprehensive 10-year which can hardly be detected by Southern blot analysis. A Strategy for Cancer Control from the Ministry of Health and method such as fluorescence in situ hybridization, which can Welfare, a Grant-in-aid for Science on Primary Areas (Cancer cover much larger regions, may also be useful for examining Research), a Grant-in-aid for the Encouragement of Young whether these cases feature BCL2 translocation. Although the Scientists from the Ministry of Education, Science and Culture, present study failed to show clinical relevance for the group Japan, a Grant-in-aid from Uehara Memorial Foundation, and in question, a less positive BCL2 expression might be an by the Bristol-Myers Squibb Unrestricted Biomedical Research important feature for future therapy strategies aiming at Grants Program. apoptosis. 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