[Abstract] Objective To investigate the bcl  2, bcl  6, CD10 in diffuse large B-cell lymphoma (diffuse large B  cell lymphoma, DLBCL) Expression and significance. Methods Immunohistochemistry SP method detected 60 cases of diffuse large B-cell lymphoma specimens bcl  expression of bcl  6, CD10 protein expression levels. Results of the expression of bcl  expression of bcl  6, CD10 expression in 60 patients with diffuse large B-cell lymphoma rate were 55.0% (33/60), 48.3% (29/60), 46.7% (28/60) CD10 (+) / expression of bcl-6 (+) in 15 cases, 25%; CD10 (+) / expression of bcl  6 (-) in 13 cases, 21.7%; CD10 (-) / expression of bcl  6 (+) 14 cases, 23.3 %; CD10 (-) / bcl  6 (-) 18 cases, 30%. CD10 (+) / bcl  6 (+) CD10 (-) / bcl  6 (-) bcl  2 expression difference was statistically significant. Conclusion bcl  2 is expected to become DLBCL subclassification indicators.

Key words expression of bcl  2; bcl  6; CD10; diffuse large B-cell lymphoma; immunohistochemistry

Expression of bcl  2, bcl  6, CD10 Protein and Their Significance in Subgroups of Diffuse Large B  cell Lymphoma

    CHEN Yu1, ZHAO Tong2, CAI Qing  fa3, WU Bing  xu1, CHEN Mei  yan1

    1.Department of Pathology, The 95th Military Hospital, Putian 351100, China; 2.Department of Pathology, Nanfang Medical University; 3. Department of Pathology, Affiliated Hospital of Putian UniversityAbstract: Objective To investigate the expression of bcl  2, bcl  6, CD10 protein and their significance in subgroups of diffuse large B  cell lymphoma. Methods Immunohistochemistry technique (SP method) was used to assess the expression levels of bcl  2, bcl  6, CD10 protein in 60 cases of diffuse large B  cell lymphoma. Results Expression of bcl  2, bcl  6 and CD10 protein was found in 33/60 (55.0%), 29/60 (48.3%), 28/60 (46.7%) cases, respectively. The CD10/bcl  6 patterns were as follows: CD10 (+) / bcl  6 (+) 15 cases, 25%; CD10 (+) / bcl  6 (-) 13 cases, 21.7%; CD10 (-) / bcl  6 ( +) 14 cases, 23.3%; CD10 (-) / bcl  6 (-) 18 cases, 30%. Expression levels of the bcl  2 protein in CD10 (+) / bcl  6 (+) pattern was higher than that in CD10 (-) / bcl  6 (-) pattern (P = 0.037). Conclusion The bcl  2 protein may be of value to distinct subgroups of diffuse large B  cell lymphoma.

    Key words: bcl  2; bcl  6; CD10; Diffuse large B  cell lymphoma; Immunohistochemistry

 Diffuse large B-cell lymphoma (diffuse large B  cell lymphoma, DLBCL) is the most common adult high-grade non-Hodgkin’s lymphoma (Non  Hodgkin’s lymphomas, NHL), accounting for about 40% of adult NHL [1  3], although the WHO will be classified as a separate type of disease, but it is actually a class of heterogeneous lymphoma, application of traditional CHOP (cyclophosphamide, doxorubicin, vincristine, splashed Nepal pine) program treatment [2] found that only about half of the patients receive relief, the other half of the patients with either therapy or treatment of relapse after remission, and finally cause death. For this part of the patient, looking for valid indicators to guide the treatment is very important. Some foreign scholars in recent years [4-8] study found differences in the expression of markers of germinal center subclassification of DLBCL, some scholars [9  12] also has an important role in regulation of apoptosis genes in DLBCL classification. By immunohistochemical methods to detect CD10 expression of bcl  6, bcl  2 expression in DLBCL, and explore the expression and clinical significance.

    1 Materials and methods

    1.1 Materials

    Our hospital, Department of Pathology, the First Affiliated Hospital of Putian University, January 2003 to December 2006, the diagnosis of clear cases of complete information DLBCL 60 cases, all cases were neutral formalin-fixed, paraffin section, HE staining. The lymphoma research experts engaged in by two or more re-read, according to the 2001 WHO reclassified lymphoid the hematopoietic tumor classification standard.

    1.2 Methods

    Anti-biotin  peroxidase connection method (SP) immunohistochemical staining of paraffin sections streptomycin germinal center markers (CD10, bcl  6), the inhibitor of apoptosis gene bcl  2 detection, using antibody Source clone, working concentration shown in Table 1. Immunohistochemistry kit was purchased from the Fuzhou Maixin Biotechnology Co., Ltd.. Antigen-fix solution pH9.0 ethylenediamine tetra acetic acid (EDTA), CD20 0.01mol / L citrate buffer (pH 6.0). As a negative control antibody was replaced with PBS, tonsil tissue as a positive control. DAB color, lining of hematoxylin stained. CD10/bcl  6 classification as a germinal center-like basis.

    1.3 The results of determination

    Positive result criteria: CD20, CD10 positive positioning in the cell membrane, bcl  6 positive positioning in the nucleus, bcl  2 positive mainly located in the cytoplasm, and> 20% of tumor cells staining positive ruled.

    1.4 statistical methods

    SPSS10.0 statistical analysis software χ2 test, P <0.05 difference was statistically significant.

    Table 1 antibodies name, sources and concentration

    Tab 1 Immunohistochemical study:

    antibodies and conditions of use

    AntibodyCloneSourseDilutionCD20L26Maxim1: 20bcl  2100/D5Maxim1: 1bcl  6P1F6Neo  marker1: 1CD1056C6Zymed1: 1

    2 Results

    2.1 clinical and pathological findings

    60 cases of DLBCL patients, aged 16 to 91 years old, with an average age of 52.9 years old. 29 males and 31 females, the male to female ratio of 0.94:1. Involvement of lymph nodes in 27 patients (45.0%), pure extranodal involvement in 33 cases (55.0%), mainly found in the gastrointestinal tract, central nervous system, submandibular gland, testicles. The reference to the 2001 WHO lymphoid hematopoietic tumor classification criteria, histologic variants mainly centroblastic, accounting for 86.7%, as shown in Table 2. Table 260 cases of DLBCL histological variant

    2.2 The expression of the protein

    60 cases of DLBCL, CD20 positive, CD10 positive rate of 46.7% (28/60), the expression of bcl  6 positive rate of 48.3% (29/60), bcl  2 positive rate of 55.0% (33/60). bcl  6/CD10 mode of CD10 (+) / expression of bcl  6 (-) in 13 cases, 21.7%; CD10 (-) / bcl  6: CD10 (+) / bcl  6 (+) 15 cases, 25%; (+) 14 cases, 23.3%; CD10 (-) / expression of bcl  6 (-) of 18 cases, 30%.

    2.3 CD10/bcl  6 and bcl  2 expression

    CD10 (+) / bcl  6 (+) 15 cases, bcl  2 (+) 3 cases, the positive rate of 20.0%; CD10 (-) / expression of bcl  6 (-) 18 cases, bcl  2 (+) 10 cases, the positive rate of 55.6%. The expression of both a statistically significant difference (χ 2 = 4.332, P <0.05). CD10 (+) 28 case, 10 cases were positive for expression of bcl  positive rate of 57.1%; CD10 (-) 32 cases, 17 cases of expression of bcl  2, the positive rate of 53.1%; Both bcl  2 expression was no statistical difference 0.05)。">significance (χ2 = 0.097, P> 0.05). expression of bcl  6 (+) 27 cases, 9 cases were positive for expression of bcl  2, the positive rate of 33.3%; expression of bcl  6 (-) 33 cases, 25 cases were bcl  2, the positive rate of 75.8%; Both bcl  2 expression difference was statistically significant (χ2 = 10.884, P <0.05).

    3 Discussion

    DLBCL is a highly aggressive the heterogeneity lymphoma, there are obvious differences in the clinical manifestations, morphological characteristics, phenotype, and genetic features, but if the early diagnosis of powerful chemotherapy, nearly half of the patients receive remission . People in the past commonly the size of the international prognostic index (IPI) values ​​prognosis, but clinical the application proved most of DLBCL IPI value for intermediate values ​​[13, 14], it is difficult to judge the prognosis. Abroad since 2000 using cDNA microarray technology [4  7] found that the gene level DLBCL can be divided into three types: (1) germinal center-like (germinal center B  cell  like, GCB): Gene Expression similar to normal germinal center B cells; (2) the activated B cell-like (activated B  cell  like, ABC): the gene expression characteristics similar to activated peripheral blood B cells; (3) type 3 (type 3). And found that the the GCB prognosis significantly better than ABC and type 3, so DLBCL simply divided into two types: GCB and non  GCB, former represents a better prognosis type, the latter on behalf of the poor prognosis of type. CDNA microarray technology requires fresh or frozen specimens were used to extract enough RNA chip prices are relatively expensive and difficult to promote clinical applications. In recent years, the direction of the research to the protein level, and found that the expression of CD10, the expression of bcl  6 patients with a better prognosis.

    CD10, acute lymphoblastic leukemia common antigen, is a membrane-associated neutral endopeptidase enzyme; lymphocyte differentiation process, it is expressed in pre-B cells and germinal center B cells, about 75% of pre-B lymphoid The mother cell leukemia, and the majority of follicular lymphoma express CD10, CD10 expression and t (14:18) chromosome found in the genetic analysis of ectopic, so can be used as a marker of germinal center cell [15]. This group CD10-positive rate of 46.7% (28/60), higher than the the foreign reports [11, 16], and may be associated with this group of patients centroblastic.

    bcl  6, zinc finger protein, is a nuclear transcription factor belonging to regulate cell proliferation, differentiation and organ development, the formation of the zinc finger protein family. bcl  6 gene in the chromosome 2q27 translocation associated with DLBCL first found expression in mature B cells in germinal centers, but no good in immature precursor cells or differentiated plasma cells in B-cell differentiation and germinal center formation plays an important role. bcl  6 gene control inflammatory cell infiltration, the physiological role of lymphokines expression and germinal center formation, can lead to the occurrence of the NHL as a transcription factor to inhibit further differentiation and maturation of germinal center B lymphocytes [17], can also be used as germinal center cell marker. Studies have shown that the combination of CD10 and bcl  6 to determine the GCB type of DLBCL is an effective indicator of good prognosis [4  6,15]. This group bcl  6 positive rate was 48.3% (29/60), and close to the reported in the literature. Reported 60 cases of DLBCL, CD10 (+) / bcl  6 (+), 15 patients (25%), lower than the Bai M (32%) [18]. CD10 (-) / bcl  6 (-) 18 patients (30%), Bai M (31%) reported similar [18]. CD10 (+) / bcl  6 (-) or CD10 (-) / bcl  6 (+) cases accounted for 38%, the prognosis for patients with this part, the indicators need to be further explored.

    A large number of studies have shown that by the number of gene regulation of apoptosis bcl  2 gene is an important regulator of apoptosis genes, it is first found in the B lymphocytes of the human body, is a proto-oncogene, can inhibit apoptosis. They play a regulatory role in the mitochondria involved in apoptosis pathway, can control mitochondrial apoptotic factors such as cytochrome C release. [8,13,19] reported that expression of bcl  2 high-expression is more common in DLBCL (30% to 60%), of which 20% to 30% of cases bcl  2 gene translocations, namely t (14; 18) lead bcl  2 high expression of the remaining cases and the expression of bcl-2 over-expression of bcl  2 gene amplification. , Bcl  2 high expression of the prognosis was significantly worse in DLBCL [4,14,20], and resistance to chemotherapy [19]. The group of bcl  2 positive rate was 55.7% (33/60), similar to [8,13] and abroad, most of the research results. CD10 (+) / bcl  6 (+) 15 cases, bcl-2 positive rate was 20% in the expression of bcl  6 (-) / CD10 (-) 18 cases, bcl-2 positive rate was 55.6%, the two expressed by the difference was statistically significant, indicating that the expression of germinal center marker cases bcl  2 low expression, and germinal center markers cases expressed bcl  2 high expression, suggesting that of bcl  6/CD10 with bcl  2 in combination may DLBCL subclassification indicators.

    In this group of cases of CD10 (+) and CD10 (-), bcl-2 expression was no significant difference, bcl  6 (+) and expression of bcl-6 (-) cases bcl  2 expression differences were statistically sense, whether prompted bcl  6 (-) cases prognosis based on the expression of bcl  2 expression of bcl  6  2 expression and expression of bcl whether certain pending further study.