Leukemia (2002) 16, 1857–1862  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu BCL-6 mRNA expression in higher grade transformation of follicle center lymphoma: correlation with somatic mutations in the 5 regulatory region of the BCL-6 gene IS Lossos 1 , R Warnke 2 and R Levy 1 1 Division of Oncology, Department of Medicine, Stanford University Medical Center, Stanford, CA, USA; and 2 Department of Pathology, Stanford University Medical Center, Stanford, CA, USA Follicle center lymphoma (FCL) is an indolent low-grade B cell non-Hodgkin’s lymphoma (NHL) that frequently transforms to aggressive diffuse large B cell lymphoma (DLBCL). Histological transformation of FCL is commonly associated with accumu- lation of secondary genetic alterations. The BCL-6 gene is com- monly implicated in the pathogenesis of DLBCL and its expression may be altered by clonal rearrangements and somatic point mutations in its 5 non-translated regulatory region. Recently, somatic mutations of the BCL-6 gene were associated with the transformation process. Here, we examined BCL-6 mRNA expression and BCL-6 mutations in paired biopsies from the same patients obtained at the time of FCL diagnosis and after transformation. BCL-6 mRNA expression markedly increased upon transformation (1.9- to 4.8-fold) in three cases, remained unchanged in one case and decreased compared to the diagnosis FCL specimens in four cases. The three specimens that demonstrated an increase in the BCL-6 mRNA expression upon transformation harbored BCL-6 gene mutations in the 5 region of the first intron that overlapped with the previously reported negative regulatory region of the gene. Accumulation of new mutations in this region was not observed in DLBCL biopsies in which the BCL-6 mRNA expression did not increase. The present study demonstrates that although BCL-6 gene mutations do accumulate during the transformation process and, depending on their location within the first intron, may deregulate BCL-6 mRNA expression, increase in BCL-6 mRNA expression is not uniformly required for transformation from FCL to DLBCL. Leukemia (2002) 16, 1857–1862. doi:10.1038/sj.leu.2402578 Keywords: follicle center lymphoma; transformation; mutations Introduction Follicle center lymphoma (FCL) is a low-grade non-Hodgkin’s lymphomas (NHL) characterized by long survival and a rela- tively indolent clinical course. Nonetheless, transformation to a higher grade aggressive lymphoma occurs in 25–60% of patients. 1,2 The transformation is usually associated with a rap- idly progressive clinical course and short survival. The mol- ecular mechanisms underlying the process of morphologic transformation and clinical progression of FCL are presently unknown, however their identification is critical for the improved understanding of the pathogenesis of these lym- phomas. Secondary genetic abnormalities associated with his- tological transformation of FCL include genome wide genetic instability, 3 nonrandom chromosomal changes, 4–6 c-myc gene rearrangement, 7,8 p53 tumor suppressor gene mutations, 9,10 p16 and p15 inactivation by deletions, mutations and hyper- methylation 11,12 and somatic mutations of the translocated BCL-2 gene. 13 However, these secondary aberrations are markedly heterogeneous and are each observed in only a sub- set of transformed lymphomas, suggesting that other molecu- Correspondence: R Levy, Stanford University School of Medicine, Division of Oncology, CCSR Building, Room 1105, Stanford, CA 94305–5306, USA; Fax: 650 725–1420 Received 1 September 2001; accepted 18 February 2002 lar mechanisms must be implicated in FCL transformation. Recently, we and others described the accumulation of somatic mutations in the 5 non-translated regulatory region of the BCL-6 gene during transformation. 14–16 The BCL-6 gene is located at chromosome 3q27 and enco- des a 96KD nuclear protein with six C-terminal zinc finger motifs and an N-terminal POZ/ZIN domain homologous to a family of zinc finger proteins. 17 This gene is commonly impli- cated in the pathogenesis of diffuse large B cell lymphomas (DLBCL) and its expression may be altered by clonal rearrangements and somatic point mutations of its 5 non- translated regulatory region. 18–24 However, only 33% of the mutant alleles found in primary DLBCL caused BCL-6 over- expression. 25 The nature of the somatic mutations leading to the changes in the BCL-6 expression has not been reported in this abstract. 25 Consequently, the observed accumulation of the somatic mutations in the 5 non-translated regulatory region of the BCL-6 gene during higher grade transformation of FCL to DLBCL may play a functional role in the process of transformation. We have now examined and correlated BCL- 6 mRNA expression and somatic mutations in the 5 non- translated regulatory region of the BCL-6 gene in sequential biopsy specimens from FCL patients that underwent morpho- logic transformation to DLBCL. Materials and methods Tumor specimens Sequential biopsy specimens from eight patients with FCL that underwent morphologic transformation to DLBCL were selec- ted for this study. All lymphoma specimens were classified according to the Revised European–American Lymphoma Classification 26 (Table 1) and were routinely immunopheno- typed by flow cytometry for expression of immunoglobulin (Ig) heavy and light chains and B and T cell markers. Percentage of the tumor cells in each specimen was estimated from the light chain restriction in each specimen as determined by flow cytometry (goat F(ab) 2 FITC anti-human  and  antibodies; Biosource International, Camarillo, CA, USA). Lymph node histology at the time of transformation was classified as DLBCL in all the specimens. In three cases (IL105B, IL114C and IL119B) no remaining follicles were observed while in the other transformed cases some remaining scattered follicles were seen in addition to the classical DLBCL pathological findings. RNA isolation and real-time PCR measurement of the BCL-6 mRNA expression Total cellular RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instruc-