Hodgkin lymphoma (HL) is a malignancy of unknown pathogenesis. The malignant Hodgkin and Reed/Sternberg (HRS) cells derive from germinal center B cells (or rarely, T cells) but have a heterogeneous and largely uncharacterized phenotype. Using microarrays, we compared the gene expression profile of four HL cell lines with profiles of the main B cell subsets and B cell non-HLs to find out whether HRS cells, despite their described heterogeneity, show a distinct gene expression, to study their relationship to other normal and malignant B cells, and to identify genes aberrantly or overexpressed by HRS cells. The HL lines indeed clustered as a distinct entity, irrespective of their B or T cell derivation, and their gene expression was most similar to that of EBV-transformed B cells and cell lines derived from diffuse large cell lymphomas showing features of in vitro-activated B cells. Twenty-seven genes, most of which were previously unknown to be expressed by HRS cells, showed aberrant expression specifically in these cells, e.g., the transcription factors GATA-3, ABF1, EAR3, and Nrf3. For five genes, expression in primary HRS cells was confirmed. The newly identified HL-specific genes may play important roles in the pathogenesis of HL, potentially represent novel diagnostic markers, and can be considered for therapeutic targeting.

IntroductionThe hallmark of Hodgkin lymphoma (HL) is the presence of large, mononucleated Hodgkin and multinucleated Reed/Sternberg cells. These cells represent the tumor cells, but usually comprise less than 1% of the cellular infiltrate in the lymphoma tissue (1). Due to the rarity of the Hodgkin and Reed/Sternberg (HRS) cells and their unusual phenotype, the origin of these cells from germinal center (GC) B cells in both the lymphocyte predominant (LP) and the classical subtype of HL could be clarified only recently (reviewed in ref.2). Only in very rare cases, HRS cells of classical HL represent transformed T cells (3, 4). In classical HL, which accounts for 95% of the cases, the pattern of somatic mutations in the rearranged Ig genes suggests that the cells derive from GC B cells that normally would have undergone apoptosis as they acquired unfavorable somatic mutations (5, 6).Despite their GC B cell origin, HRS cells have lost expression of many B cell markers (reviewed in ref. 2). Moreover, expression of markers typical for other hematopoietic lineages is often observed, such as the myelocytic antigen CD15, the cytotoxic T cell/NK cell marker granzyme B, and the dendritic cell-specific chemokine TARC (7-10). Thus, the phenotype of HRS cells in classical HL does not resemble any normal cell type in the body.Besides these and some other reports on selected markers expressed by HRS cells, there is so far no systematic large scale analysis of genes specifically expressed by these cells. A large number of expressed sequence tags was sequenced in a study from HL lines and single HRS cells (11). However, the collection of cells used to generate the expressed sequence tag profile represented a mixture of HRS cells from the classical and LP type of HL, which differ in many aspects (12). Moreover, since only GC B cells were used to analyze differential gene expression, this study was not well suited to identify genes specifically expressed by HRS cells of classical HL in comparison with other normal and malignant B cell populations.

Clonally related composite lymphomas of Hodgkin's lymphoma (HL) and Non-Hodgkin's lymphoma (NHL) represent models to study the multistep transformation process in tumorigenesis and the development of two distinct tumors from a shared precursor. We analyzed six such lymphomas for transforming events. The HLs were combined in two cases with follicular lymphoma (FL), and in one case each with B-cell chronic lymphocytic leukemia, splenic marginal zone lymphoma, mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). In the HL/FL and HL/MCL combinations, BCL2/IGH and CCND1/IGH translocations, respectively, were detected in both the HL and NHL. No mutations were found in the tumor suppressor genes FAS, NFKBIA and ATM. The HL/DLBCL case harbored clonal replacement mutations of the TP53 gene on both alleles exclusively in the DLBCL. In conclusion, we present the first examples of molecularly verified IgH-associated translocations in HL, which also show that BCL2/IGH or CCND1/IGH translocations can represent early steps in the pathogenesis of composite HL/FL or HL/MCL. The restriction of the TP53 mutations to the DLBCL in the HL/DLBCL case exemplifies a late transforming event that presumably happened in the germinal center and affected the fate of a common lymphoma precursor cell towards development of a DLBCL.

Aberrant somatic hypermutation (SHM) has been identified as a mechanism for genomewide instability in diffuse large B-cell lymphoma (DLBCL). To assess whether aberrant SHM plays a role in the molecular pathogenesis of Hodgkin lymphoma (HL), we investigated microdissected neoplastic cells of nodular lymphocyte-predominant HL (NLPHL; n ‫؍‬ 10) and classic HL (cHL; n ‫؍‬ 9) for the presence of mutations in the 5 sequences of 4 previously identified aberrant SHM targets (PIM1, PAX5, RhoH/TTF, c-MYC). Mutations in one or more genes were detected in 80% of NLPHLs and 55% of cHLs, with 50% and 30% of patients carrying mutations in 2 or more genes, respectively. The most frequently involved protooncogene was PAX5, mutated in 7 of 9 patients with NLPHL and 2 of 9 patients with cHL. In total, 34 mutations were detected in NLPHL (frequency, 1.04/1000 bp) and 35 were detected in patients with cHL (frequency, 1.92/1000 bp). Mutations were of somatic origin because they were absent in control T cells and shared most of the features of the immunoglobulin variable (IGV) gene-associated SHM mechanism-ie, single nucleotide substitutions (n ‫؍‬ 63) with rare deletions/insertions (n ‫؍‬ 6) and a predominance of transitions over transversions with preferential targeting motifs. Our finding that NLPHL and cHL are targeted by aberrant SHM, as is DLBCL, suggests that these lymphomas may share common molecular pathogenetic events.