Diffuse Large B-Cell Lymphoma with Extra BCL-2 Gene Signals Detected by FISH Analysis Is Associated with “Non-Germinal Center Phenotype” Defined by Immnohistochemistry.
Authors: Kusumoto Shigeru , Kobayashi Yukio , Sekiguchi Naohiro , Tanimoto Kazuki , Onishi Yasushi , Yokota Yukiko , Watanabe Takashi , Maeshima Akiko , Ishida Takashi , Inagaki Hiroshi , Matsuno Yoshihiro , Ueda Ryuzo , Tobinai Kensei 
Published: October 2019

Abstract
Introduction: Amplification and translocation of bcl-2 gene have been detected in a certain subset of diffuse large B-cell lymphoma (DLBCL). Recently, it has been reported that tissue microarray might be able to divide DLBCL into significant subgroups similar to those determined by cDNA array. The correlations among bcl-2 protein expression, gene translocation or amplification, and molecular signature determined by cDNA array are poorly understood.
Materials and methods: This study included 25 cases with de novo nodal DLBCL which were diagnosed between June 1997 and December 2001, and had been followed up at a single institution (NCCH). There were 12 males and 13 females with a median age at diagnosis of 60 years (range, 24 to 83 years). All the tissue samples were formalin-fixed paraffin sections obtained by biopsy before chemotherapy. Interphase fluorescence in situ hybridization (FISH) analysis were performed to evaluate the bcl-2 gene status using IGH/BCL2 and CEP 18 centromere probe (Vysis). We named a “additional chromosome 18” when extra BCL2 gene signals were observed in each tumor cells in proportion to extra signals of CEP18 probe, and “gene amplification” when extra BCL2 signals were observed without additional CEP18 signals. To define each case as “Germinal Center B-cell (GCB) phenotype” or “non-GCB phenotype” a panel of 3 antigens, CD10, bcl-6, and MUM-1 was evaluated following the algorithm reported by Hans CP et al (Blood.2004;103:275). Immunoreactivity was judged to be positive if 20% or more of the tumor cells were stained, and strongly positive if 50% or more.
Results: CD10, bcl-6, and MUM1 were positive in 5/25 (20%), 25/25 (100%), and 17/25 (68%) cases, respectively. Nineteen of 25 (76%) cases were positive, 13 of 25 (52%) strongly positive for the bcl-2 protein staining. Of 25 cases in total, 8 cases (32%) were classified into “GCB phenotype” and 17 cases (68%) were classified into “non-GCB phenotype”. FISH analysis revealed that t(14;18) was detected in 2 of 8 cases (25%) of “GCB phenotype”, but in none of 17 cases of “non-GCB phenotype”. Extra BCL2 gene signals were detected in 7 of 25 (28%); “additional chromosome 18” (n=5), “gene amplification” (n=1), and “additional chromosome 18 + gene amplification” (n=1). “Additional chromosome 18” and “gene amplification” were exclusively detected in “non-GCB phenotype” DLBCL, and these cases showed strongly positive bcl-2 staining except for one. Thirteen cases with strongly positive bcl-2 staining included t(14;18) (n=2) and extra signals of BCL2 (n=6). No cases showed both t(14;18) and extra BCL2 gene signals, simultaneously.
Conclusion: Our results showed t(14;18) is found among cases with “GCB phenotype”, whereas, additional chromosome 18 and/or bcl-2 gene amplification, among “non-GCB phenotype”. The mechanism for overexpression of bcl-2 protein in de novo DLBCL varies between “GCB phenotype” and “non-GCB phenotype” defined by immunohistochemistry.