Evidence that Hodgkin and Reed-Sternberg cells in Hodgkin disease do not represent cell fusions

Abstract

IntroductionIn most cases of classical Hodgkin disease (HD), Hodgkin and Reed-Sternberg (HRS) cells carry clonal immunoglobulin (Ig) gene rearrangements and thus derive from B cells. 1 Specifically, the pattern of somatic mutations in the rearranged Ig genes indicates that these cells represent transformed preapoptotic germinal center B cells. 1,2 In rare cases, HRS cells derive from T cells. 3,4 However, the morphology and immunophenotype of HRS cells is untypical for B and also T cells. HRS cells lack expression of most B-cell markers, 5,6 and molecules typical for other lineages are regularly expressed by these cells. 5,7 Another typical feature of HRS cells is their abnormal karyotype. Numerical chromosomal abnormalities with additional copies of several chromosomes are not only observed in the multinucleated Reed-Sternberg cells but also in the mononuclear Hodgkin cells. 8,9 Based on these observations, it has been speculated that HRS cells might represent cell fusions. [10][11][12][13] For example, the coexpression of B and dendritic cell markers was taken as an indication that these cells derive from a fusion of a B lymphocyte with a dendritic cell. To clarify this matter, we analyzed single HRS cells from 5 cases of HD with 2 rearranged IgH alleles for the presence of additional IgH alleles in germline configuration as a footprint for a fusion of a B cell with a non-B cell. Likewise, one case of T-cell-derived HD with 2 rearranged T-cell receptor (TCR)␤ loci was analyzed for additional TCR␤ germline alleles. Study design Patients and tissuesThe 6 cases investigated here were previously analyzed for Ig and/or TCR␤ V gene rearrangements (Table 1). They were selected from a collection of 26 cases of HD analyzed by us. Two V H region genes had been amplified from 5 of the cases, and 1 case analyzed for D H J H joints harbored such a joint besides a V H D H J H joint. Micromanipulation and single-cell PCRSingle HRS cells were micromanipulated from immunostained frozen tissue sections. 2,14 Before gene-specific polymerase chain reaction (PCR) was carried out, genomic DNA of single cells was preamplified with a random 15-mer primer. 15 IgH and TCR␤ VDJ and DJ gene rearrangements and fragments specific for germline configuration of IgH and TCR␤ loci were amplified by seminested PCR and sequenced ( Figure 1). 2,3,16 To rule out that germline polymorphisms at the binding sites for the IgH germlinespecific PCR primers (D H 7-27 and J H 1) hamper amplification of germline fragments, fragments covering these sites were amplified from whole-tissue DNA of cases 2 to 5 (not shown). No polymorphisms were detected at the primer binding sites. In cases 2, 3, and 5, successful amplification of both IgH alleles was confirmed by a polymorphism near the D H 7-27 gene. For patient 1, single micromanipulated T cells of the tumor tissue and, for patient 6, micromanipulated B cells were used for control amplification of germline-specific IgH or TCR␤ fragments. For case 1, IgH germlinespecific amplificates were obtained from 6 of 1...

Incomplete cytokinesis and re-fusion of small mononucleated Hodgkin cells lead to giant multinucleated Reed–Sternberg cells

“…2G). Importantly, we found that cell fusion occurs preferentially between cells sharing the same ancestor, explaining why it has been impossible to detect this phenomenon with genetic approaches (17).…”

“…2). Although cell fusion was ruled out as the origin of RS cells and endomitosis is the currently accepted mechanism for RS cell development (17,33), we have clearly demonstrated that re-fusion of daughter cells is the initial step in RS cell formation (Fig. 2B), which occurs concurrently with the average division time point of the bulk population (Fig.…”

“…Moreover, a mixing experiment of dual fluorescent-labeled cells of the HL cell line L1236 provided evidence against cell fusion as the mechanism giving rise to RS cells (18). Thus, endomitosis instead of cell fusion has been proposed as the mechanism for RS cell formation in HL (17,18). But endomitosis by definition means mitosis leading to polyploidy within a cell without nuclear division, and thus the proposed mechanism should have been…”

“…Cell fusion of mononuclear Hodgkin cells has been explored as a mechanism for RS cell generation (15); however, a molecular analysis of primary HRS cells excluded the possibility that the HRS cell clone as such or the RS cells are derived from the fusion of different cells (e.g., a B cell and a non-B cell) (17). Moreover, a mixing experiment of dual fluorescent-labeled cells of the HL cell line L1236 provided evidence against cell fusion as the mechanism giving rise to RS cells (18).…”

Aberrant expression of cell cycle regulators in Hodgkin and Reed–Sternberg cells of classical Hodgkin's lymphoma

“…In contrast to micronuclei, which are not observed in Hodgkin and ReedSternberg cells, and which are caused by chromosome malsegregation during mitosis, multinucleation is caused by cytokinesis defects or cell fusion. Kuppers et al 28 have investigated the mechanism leading to multinucleation in Hodgkin and ReedSternberg cells and reported that it is not caused by cellular fusion. The large nuclei present in Hodgkin and Reed-Sternberg cells may, therefore, be caused by cytokinesis defects or by DNA over-replication.…”