Y Guo 1,2, K Karube 1,3, R Kawano 1, T Yamaguchi 1, J Suzumiya 4, G-S Huang 2 and K Ohshima 1

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1 (2005) 19, & 2005 Nature Publishing Group All rights reserved /05 $ otherwise the rest comprises minor groups of heterogeneous disease entities with Bcl2 amplification, Bcl6 translocation or other gene aberrances Y Guo 1,2, K Karube 1,3, R Kawano 1, T Yamaguchi 1, J Suzumiya 4, G-S Huang 2 and K Ohshima 1 1 Department of Pathology, School of Medicine, Fukuoka University, Fukuoka, Japan; 2 Department of Pathology, State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University, Xi an, Shannxi, People s Republic of China; 3 Department of Medicine and Biosystemic Science, Internal Medicine, Medicine and Surgery, Kyushu University Graduate School of Medical Science, Fukuoka, Japan; and 4 Department of Internal Medicine, School of Medicine, Fukuoka University, Fukuoka, Japan Follicular lymphomas (FL) are morphologically classified into grades 1, 2, 3a and 3b by the World Health Organization. Bcl2, Bcl6 and CD10 are phenotypic markers of FL while the Bcl2 t(14;18) and Bcl6 t(3q27) gene translocations are common genetic changes. However, to date, there has been no integrated analysis based on phenotype, grade and genotype from large numbers of FL cases. We graded 261 cases of FL and determined their phenotypes and gene alterations. According to the antigen markers and gene alterations of 147 cases, we classified FL into typical and the others types. The typical group, which includes 69% cases of FL, is characterized by low histological grade (grade 1, 2), coexpression of BCL2 and CD10 and Bcl2 gene translocation. The rest comprises a small part of low-grade FL without Bcl2 gene translocation and high-grade (grade 3a, 3b) FL. These FLs include some heterogeneous disease entities. They are characterized by high histological grade (87%), no definite expression of BCL2 or CD10 and several kinds of gene aberrances including Bcl2 translocation, Bcl6 translocation, Bcl2 amplification or other unknown gene abnormality. Our findings indicate that typical FL presents a homogeneous disease entity whereas the rest comprises heterogeneous diseases entities. (2005) 19, doi: /sj.leu Published online 7 April 2005 Keywords: follicular lymphoma; t(14;18); t(3q27); Bcl2; Bcl6; CD10 Introduction According to the World Health Organization (WHO) system of classification of lymphoma, the disease categories are defined by a combination of morphology, immunophenotype, genetic features and clinical symptoms. Follicular lymphoma (FL) is the most prevalent form of low-grade B-cell lymphoma in adults. 1 In the Revised Europe American Lymphoma (REAL) classification, a grading system (grade 1 3) was introduced in FL, 2 but no specific recommendations for grading criteria were given. The recently introduced WHO classification (Nathwani et al, 2001) recommends a similar three grade system (grade 1, 2, 3a and 3b) based on counting the absolute number of centroblasts. 3 In the Kiel classification, however, lymphoid tumours with a follicular growth pattern or a follicular component are not regarded as one entity. FL grades 1 and 2 are, in most of the cases, equivalent to the low-grade centroblastic centrocytic lymphoma, and WHO grade 3 largely corresponds to follicular (high-grade) centroblastic lymphoma. 4 Correspondence: Dr K Ohshima, Department of Pathology, School of Medicine, Fukuoka University, Nanakuma , Jonan-ku, Fukuoka , Japan; Fax: þ ; ohshima@fukuoka-u.ac.jp Received 15 December 2004; accepted 21 February 2005; Published online 7 April 2005 FL shows marked differences in the incidence rates across geographic regions. Extremely low incidence rates are reported among most Asia countries, and comparatively high rates are reported in North America and Western Europe. As for the hallmark Bcl2 rearrangement of FL, former studies demonstrate a significant gradient in the frequency of bcl-2 gene rearrangements from a relative low rate of 48 to 55% among Asian and European studies to approximately 80% among the series reported from the United States. 5 Virtually all cases of FL have cytogenetic abnormalities. The most common, t(14;18) (q32; q21), involving rearrangement of the Bcl2 gene, is present in 70 95% of FL cases. The t(3q27) rearrangement of Bcl6 is present in about 15% of cases. 6 Although t(3q27) is a less common gene alteration in FL, it occurs frequently in FL 3b. 7 The phenotype of FL cells is characterized by the expression of CD10, Bcl2 and Bcl6. CD10 and Bcl6 are regarded as reliable markers to identify germinal centre B-cell lymphoma, in particular FL. 8 Bcl2, an antiapoptosis protein, is expressed in follicles and is a phenotypic marker to differentiate FL from reactive follicular hyperplasia. CD10 and Bcl2 are expressed in most cases of FL but CD10- and Bcl2-negative cases do exist. 9 It has been verified that the pattern of Bcl6 expression is similar in reactive follicles and FL, and in any case, it is expressed in nearly 100% of FLs. 10,11 However, Bcl6 expression has not been well studied in a large cohort of FL cases. There has been some research into the relationship between phenotype and histological grade of FL. FL grade 3 has been reported to express the Bcl2 protein in 50% of patients. 12 CD10 expression is found in % of patients with FL grade 1 and 2, but in only 20% of patients with FL grade Aberrant Bcl2 expression caused by chromosome translocation has been considered the triggering factor in development of FL. Aberrant Bcl2 expression prevents cell death of FL cells originating from follicular centre cells, while at the same time follicular dendritic cells provide them with growth signals. 14 However, in FL without the t(14;18) translocation, Bcl6 rearrangement is considered to play an important role. 9 Some evidence indicates that Bcl6 may function to protect cells against terminal differentiation, and that differentiation block can be oncogenic. 14,15 However, the relationship between Bcl6 rearrangement and its expression is uncertain. How this translocation influences the pathogenesis of FL is still unknown. Although FL is regarded as an independent disease entity by the WHO disease classification, it exhibits various morphologies, phenotypes, genetic aberrations and clinical behaviors. In order to classify FL into more useful pathogenetic subtypes, we characterized in the present study the morphologic spectrum and antigen expression profile based on a large number of FL

2 cases and to correlate these features with their cytogenetic abnormalities. Materials and methods Patient material Tissue specimens were obtained from human lymph nodes filed in the Department of Pathology at Fukuoka University. We selected 261 cases of FL and five cases of reactive follicular hyperplasia (RFH). Each tissue sample was cut into two or more pieces, one of which was frozen in liquid nitrogen and stored at 801C while the other was fixed in formalin and embedded in paraffin for histopathological diagnosis, including immunostaining. Histopathological diagnoses and grading were carried out by two pathologists (GY and OK) based on the World Health Organization Classification of Neoplastic Disease of Lymphoid Tissues. Immunostaining Paraffin sections from each sample were immunostained with monoclonal antibodies against CD10 (Novocastra, Newcastle, UK), Bcl2 (DAKO, Glostrup, Denmark) and Bcl6 (Novocastra). All immunohistochemical reactions were performed using the peroxidase anti-peroxidase method after antigen retrieval as described previously. 16 Immunohistological scoring The percentages of positive cells were averaged to yield an immunohistological score of 0 100%. The following categories were defined: negative (o20% positively stained tumour cells) and positive (Z20% positively stained tumour cells). The staining pattern was classified as either cytoplasmic or nuclear. Fluorescence in situ hybridization In the present study, we used LSI IgH Spectrum Green/LSI Bcl2 Spectrum Orange Dual-Fusion Translocation Probe (Vysis, Downers Grove, IL) and the LSI Bcl6 Dual Color breakapart probe (Vysis) to detect t(14;18) and t(3q27), respectively. Routine fluorescence in situ hybridization (FISH) method on frozen sections and result analysis were performed according to the methods described previously. 17,18 Statistical analysis We compared the CD10, Bcl2,Bcl6 expression and Bcl2, Bcl6 translocation between grade 1, 2; grade 3a, 3b; low-grade and high-grade FLs with w 2 test. Results Grade distribution analysis was based on the HE staining of paraffin sections from all of the FL specimens (n ¼ 261). To analyse immunostaining and FISH, we excluded all cases with sceptical results and retained 147 cases with full data and definite results from immunostaining and FISH. Grade distribution In the present study, the grade distribution of FL from 261 cases was as follows: grade 1, 26%; grade 2, 48%; grade 3a, 17% and grade 3b, 9% (total, n ¼ 261). In 147 cases available for advanced analysis for immunostaining and FISH, the grade distribution was as follows: grade 1, 22%; grade 2, 52%; grade 3a, 3b, 26%. Immunostaining Of all 147 cases of FL included in the analysis, 93% were Bcl2 positive. Among the grades of FL, Bcl2-positive cases comprised 97% of grade 1, 96% of grade 2, 80% of grade 3a and 71% of grade 3b cases. CD10 positivity was observed in 81% of the total of 147 cases, comprising 91% of grade 1, 91% of grade 2, 48% of grade 3a and 57% of grade 3b cases. The immunostaining pattern of Bcl6 was different between normal follicles and tumour follicles. In normal follicles, the Bcl6 staining showed uniform intensity covering whole area of follicles whereas in FL the intensity was from weak to strong in the same follicle and some cases were totally negative for the staining. Bcl6 positivity was detected in 71% of the total 147 cases, comprising 69% of grade 1, 68% of grade 2, 80% of grade 3a and 71% of grade 3b cases. Bcl2, Bcl6 and CD10 are therefore useful antigenic markers for FL (Table 1). The rate of CD10 positivity was much higher in low-grade (grade 1 and 2) cases than in high-grade (grade 3a and 3b) cases (Po0.001). The Bcl2 positivity rate was higher in low-grade cases than in high-grade cases (Po0.001). The Bcl6 positivity rate showed no significant difference between low- and highgrade FLs (P ¼ 0.19). We did not find significant difference between grade 1 and 2 or grade 3a and 3b with regard to Bcl2 (grade 1, 2 P ¼ 0.84; grade 3a, 3b P ¼ 0.54), CD10 (grade 1, 2 P ¼ 0.98; grade 3a, 3b P ¼ 0.58) and Bcl6 (grade 1, 2 P ¼ 0.97; grade 3a, 3b P ¼ 0.92) positivity. Fluorescence in situ hybridization In the FISH analysis, t(14;18) positivity was identified in 81% of FL. According to grade, the positivity rate for t(14;18) was grade 1 (100%), grade 2 (92%), grade 3a (44%) and grade 3b (43%). t(3q27) positivity was much lower at 10% for all FL specimens, comprising grade 1 (0%), grade 2 (3%), grade 3a (32%) and grade 3b (36%). Bcl2 duplication was detected in 3% of FLs, comprising grade 1 (0%) (Table 2), grade 2 (1%), grade 3a (8%) and grade 3b (14%). Of all cases studied, 6% was negative for t(14;18), t(3q27) and Bcl2 duplication, and presumably had some other unidentified gene alteration(s). No grade 1 cases fell into this group; however, 5% of grade 2, 20% of grade 3a and 7% of grade 3b cases belonged to this category. More low-grade FL cases carried t(14;18) than did high-grade FL cases (Po0.001). More high-grade FLs carried t(3q27) than did low-grade FLs (Po0.001). We did not find significant difference between grade 1 and 2 or grade 3a and 3b by either t(14;18) (grade 1, 2 P ¼ 0.10; grade 3a, 3b P ¼ 0.94) or t(3q27) (grade 1, 2 P ¼ 0.32; grade 3a, 3b P ¼ 0.81). Phenotype and gene aberrations Table 3 shows the correlation between phenotype and gene aberrance. Bcl2 expression showed close correlation with Bcl2-1059

3 1060 Table 1 The phenotype and grade Grade Phenotype CD 10 Bcl2 Bcl6 + Positivity (%) + Positivity (%) + Positivity (%) a b Total CD10: grade 1, 2 vs grade 3a, 3b, Po0.001; Bcl2: grade 1, 2 vs grade 3a, 3b, Po0.001; Bcl6: grade 1, 2 vs grade 3a, 3b, P ¼ Table 2 Grade Grade and gene aberrance t(14;18) related gene alteration (Po0.001); inversely Bcl6 expression showed no significant correlation with Bcl6 translocation (P ¼ 0.42). Typical subtype and the others Chromosome aberrance t(3q27) + Positivity (%) + Positivity (%) a b Total t(14;18): grade 1, 2 vs grade 3a, 3b, Po0.001; t (3q27): grade 1, 2 vs grade 3a, 3b: Po We separated a specific FL from other cases and we call these cases as typical follicular lymphoma. The criterion for the typical follicular lymphoma is as follows: low-grade (grade 1 and 2) follicular lymphomas with Bcl2/IGH translocation and characterized by Bcl2, CD10 coexpression in the main. In this study, 95% of cases fulfilling this definition of typical follicular lymphoma coexpressed Bcl2 and CD10. The other follicular lymphomas include a small part of low-grade FL without t(14;18) and all of the high-grade (grade 3) FL. They are characterized by high histological grade (in present study the small part of low-grade FLs without t(14;18) were all grade 2), and instead, the genetic alteration is t(14;18), t(3q27), Bcl2 amplification or other unknown genetic changes. The phenotype of this kind of FL includes no definite Bcl2 and CD10 expression (Table 4). In our present analysis, typical FL and the other FL comprised 69 and 31% of all FL cases, respectively. In all, 100% of the grade 1 cases and 92% of the grade 2 cases were typical. Discussion The natural history of FL is that of an indolent growth pattern, but the disease remains essentially incurable when disseminated. As a result of the inconsistent histological criteria for grading FL, many of the reported series may not be directly comparable. The Mann and Berard grading system adopted by the WHO provides a more uniform system of diagnosing different subtypes of FL (grade 1, grade 2, grade 3a and grade 3b), thereby allowing a more consistent approach in defining the patient cohort. 19 In Japan, the distribution of grade 1, grade 2 and grade 3 FL according to Miyazato s report is 38:29:33% (n ¼ 124). 20 However, no subclassification of grade 3 into 3a and 3b has been made in their study. Chau reported the Table 3 Correlation between phenotype and gene aberrance in each histological grade Gene aberrance Phenotype Grade 1 Grade 2 Grade 3a Grade 3b Bcl2 (%) Bcl6 (%) Bcl2 (%) Bcl6 (%) Bcl2 (%) Bcl6 (%) Bcl2 (%) Bcl6 (%) Bcl2 Translocation (n ¼ 119) Bcl2 Amplification (n ¼ 5) Bcl6 Translocation (n ¼ 15) Others (n ¼ 10) Total (n ¼ 147)

4 Table 4 Distribution of the Bcl2 rearrangement and CD10, Bcl2 phenotype in low- and high-grade FL 1061 Histological grade Gene aberrance Bcl2 rearrangement (+) Bcl2 rearrangement ( ) Grade 1,2 n ¼ 102 (94%, total: 108) n ¼ 6 (6%, total: 108) (n ¼ 108) CD10+ Bcl2+ CD10+ Bcl2+ Grade 1, (n ¼ 108) (95%, total: 102) (100%, total: 102) (17%, total: 6) (50%, total: 6) n ¼ 17 (44%, total: 39) n ¼ 22 (56%, total: 39) Grade 3a, 3b CD10+ Bcl2+ CD10+ Bcl2+ (n ¼ 39) (70%, total: 17) (100%, total: 17) (41%, total: 22) (59%, total: 22) distribution of FL grades in Europe as 43:32:25% (n ¼ 215), and the ratio of grade 3a to grade 3b as 4:1. 19 In our present study, the distribution of the three histological subtypes was 26:48:26% (n ¼ 261), and the ratio of grade 3a to grade 3b was 2:1. This is the first Japanese study of histological grade distribution of FL absolutely based on WHO classification criteria. Our results are inconsistent with results of previous studies in Japan and Western countries. Compared with Chau s study, our data has more grade 2 diseases but similar grade 3 cases. The discrepancy between our study and others might lie in the geographic variation, as it is well known that FL is uncommon in Asia countries, with an incidence ranging from 8 to 12% of that for non-hodgkin lymphoma, in contrast to 20 33% in the North America and Western European countries. 5 There are three common gene abnormalities involved in FL, that is, the Bcl2/IGH translocation, Bcl6 translocation and Bcl2 duplication, respectively. Bcl2/IGH is the most frequent gene alteration and is regarded as the hallmark genetic change in FL. As Albinger-Heygi et al 21 pointed out, detection rates were significantly lower in Europe (41 61%) and in the Far East (32 39%), using both cytogenetic and molecular Southern blot techniques, in comparison with the USA. More recent analyses, however, suggest that the variation in the incidence of t(14;18) in FL across studies may be technical rather than real. These new data were obtained using methods that eliminate most false-negative results by detecting rearrangements involving Bcl2 sequences outside of the MBR and mcr. 22 FISH analysis for t(14;18) is a new method with very high sensitivity and specificity. In the present study, we found t(14;18) positivity in 81% of total FL cases, which is similar with that in the Western world. So we consider that the discrepancy between our results and those of other groups in Japan is mainly because of technique differences. Bcl6 translocation is another important gene alteration involved in FL. Although it is a relatively rare cytogenetic aberration in FL, it has been noted as a frequent gene alteration in grade 3 lymphomas in recent studies. 7,23 Bcl6 translocation occurs at a frequency of 6 14% in FL in the West and 12.5% in Japan Our result of 10% is similar with studies from the West and Japan. Bcl2, Bcl6 and CD10 are frequently expressed antigen markers in FL. Bcl2 protein expression has been described in FL in association with the t(14;18) chromosomal translocation. The majority of FL express Bcl2 protein, ranging from nearly 100% in grade 1 to 75% in grade In the present study, Bcl2 expression was observed in 97% of grade 1, 96% of grade 2 and 77% of grade 3 FL. It is well known that CD10 and Bcl6 are recognized as characteristic markers for normal follicles and FL. 13,29 The Bcl6 expression pattern in FL is similar to that in normal follicles. 8,10 Its expression frequency has been reported high (94 to 100%) in FL. 8 In the present study, Bcl6 was positive in 71% of FL. The expression pattern of Bcl6 in FL was different to that in normal follicles. In FL cases, the intensity of Bcl6 staining was from weak to strong and some cases were totally negative for Bcl6 (data not shown). We do not know why the Bcl6 expression was not as same as it is in normal follicles. Bcl6 expression might be altered during the lymphomagenesis. CD10 is another marker molecule for lymphocytes derived from follicles. Its expression was lower in grade 3 than in grade 1 and grade 2 FLs. 13 In our study, CD10 was positive in 90% of grade 1 and grade 2 cases, which is consistent with previous assay results (70 100%). However, CD10 positivity was observed in 53% of grade 3 cases, which was higher than previously reported (20%). 13 Cases with t(14;18) almost invariably showed expression of Bcl2, whereas Bcl2 was expressed in cases lacking the translocation. In a recent study, 89% of FL associated with t(14;18) stained for Bcl2 protein, whereas 25% of FL lacking t(14;18) showed Bcl2 immunoreactivity. 30 In our present study, 99% of Bcl2 translocation cases expressed Bcl2, and 60% of cases lacking the Bcl2 translocation also expressed Bcl2. In the latter group, Bcl2 amplification comprised 17% of the cases. Only one case with Bcl2 translocation was negative for Bcl2 expression. The existence of tumours that are Bcl2-positive, t(14;18)- and Bcl2 amplification-negative is not surprising, as it has long been recognized that Bcl2 expression is upregulated independently of Bcl2 gene rearrangement in many other forms of lymphoma. 22 With respect to the association between Bcl6 translocation and expression, it has been verified that Bcl6 expression has no relationship with Bcl6 translocation. Moreover, Jardin et al 9 found that the 3q27- translocated group had a significantly lower level of mrna expression as compared to FL without 3q27 rearrangement. In our present study, Bcl6 protein expression showed no correlation with Bcl6 translocation, which is in agreement with the former stuidies. 31 Bcl2 and CD10 expression and Bcl2 translocation are regarded as the hallmarks of FL, and are therefore used extensively in clinical diagnosis. Indeed, we observed these characteristics in the majority of FL cases in our analysis. FL lacking these markers are easily mis-diagnosed on this basis. From our data, we found that low-grade FLs were significantly different from high-grade FLs in such gene aberrance and phenotype. However, between grade 1 and grade 2 or grade 3a and 3b cases there was no significant difference in CD10, Bcl2 expression and Bcl2, Bcl6 translocation. One of the recent study indicated lower CD10 expression,

5 1062 less Bcl2 translocation and more Bcl6 translocation in grade 3b than in grade 3a. Furthermore, Bcl6 translocation seemed dominant gene aberrance in grade 3b 7 (the ratio of frequency between Bcl2 and Bcl6 translocation is 1:3.5). We did not find the same results in our data. Anyway our ratio between Bcl2 translocation and Bcl6 translocation in grade3b is similar with another recent study 23 (1:0.8). Since the low- and high-grade FL showed different phenotype and gene aberrance characters, we sought to classify FLs into typical and the other FL pathogenetic subtypes based on histological grade, phenotype and Bcl2 translocation. The criteria for typical follicular lymphoma was as follows: low histological grade FLs with t(14;18). The others includes lowgrade FLs lacking t(14;18) and all of the high-grade FLs. Bcl2 translocation, Bcl6 translocation, Bcl2 amplification and other unknown gene alterations comprise the genetic alterations of this type of FL. Typical FL is characterized by Bcl2 and CD10 coexpression, while the others do not always exhibit such coexpression. Typical FL covered most cases of low-grade FL and presented a homogeneous disease entity with definite morphologic character, phenotype and gene aberrance. The other FLs were almost composed of high-grade FL (87%) and presented a heterogeneous disease entity. Typical and the other FLs exhibit differential gene alterations, phenotypes and histological grades, which might reflect their different pathogenesis. Bcl2 translocation might play a key role in the pathogenesis of typical FL. Bcl2 expression correlates closely with Bcl2 gene alteration. The aberrant Bcl2 expression resulting from chromosome translocation provides an antiapoptotic signal, resulting in a growth advantage for these cells at the follicular centre cell stage. However, Bcl2 translocation alone is not sufficient for cells to become malignant, the multigenetic (also epigenetic) alterations are likely to be required for FL development. 14 Our results indicate that the other FL cases express four types of genetic alterations involved in the pathogenesis, that is, Bcl2 translocation, Bcl6 translocation, Bcl2 amplification and other unknown gene alterations. Bcl2 translocation can only explain part of the pathogenesis of this type of FL. Moreover, the pathogenesis maybe different from that of typical FL. Among the FLs with t(14;18), the low-grade FL tend to have a simple karyotype. However, the lymphoma that exhibited a high-grade histology had a larger number of secondary changes and a high degree of polyploidy. 32 Bcl6 translocation is the other important gene alteration in the other FL. Bcl6 is an important gene involved in lymphocyte growth, differentiation and apoptosis. However, it remains unclear how Bcl6 gene rearrangement influences the protein expression and thus the pathogenesis of FL. In conclusion, in present study we found that low-grade FL with t(14;18) presented a homogeneous disease entity with definite histological grade, phenotype and gene aberrance. The other FLs presented a heterogeneous disease entity with no definite CD10 and Bcl2 expression. They included several kinds of gene aberrances and were almost composed of high-grade FLs. The pathogenesis and pathological findings of typical FL have been understood well but that of the others are still needed to explore. Acknowledgements This study was supported in part by the Japan China Sasakawa Medical Fellowship and Grant-in-Aid for Cancer Research 16-6 from the Ministry of Health, Labor and Welfare of Japan. References 1 The Non-Hodgkin s lymphoma Classification Project. A clinical evaluation of the International Lymphoma Study Group classification of non-hodgkin s lymphoma. 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