Correspondence
Immunoglobulin V Genes in Reed–Sternberg Cells
N Engl J Med 1996; 334:404-406February 8, 1996
- Article
To the Editor:
Hummel et al. (Oct. 5 issue)1 report the polyclonality of Reed–Sternberg cells in 6 of 12 cases of Hodgkin's disease. In three other cases, there were mixed polyclonal and monoclonal populations of Reed–Sternberg cells, and in three cases the cells were monoclonal. We are concerned about the reported results of this study.
In an analysis of single Reed–Sternberg cells micromanipulated from frozen tissue sections for heavy-chain (VH) and kappa light-chain (Vκ) gene rearrangements, we detected clonal V gene rearrangements in 11 of 12 cases investigated, 3 which have already been reported.2 No rearrangements were found in one case. The clonality of the Reed–Sternberg cells in the lymph nodes analyzed was confirmed by identifying identical rearrangements in cells from different sections in five of the cases. Thus, in 11 of 12 cases, Reed–Sternberg cells represented a clonal population of B-lineage cells. The clonality of the Reed–Sternberg cells was also demonstrated in all 30 cases of Hodgkin's disease by a combination of fluorescence in situ hybridization and immunophenotyping.3
When we reanalyzed in our laboratory one of the specimens classified as polyclonal by Hummel et al., we could not detect any VH rearrangement in 30 Reed–Sternberg cells picked from another section of the same specimen. Surprisingly, an analysis of Vκ gene rearrangements revealed a clonal kappa light-chain rearrangement. One possible interpretation of this finding is that different populations of Reed–Sternberg cells can be found in different areas of a lymph node. On the basis of our analysis of the 12 cases of Hodgkin's disease noted above, however, we consider this explanation very unlikely.
In our experience, about 5 percent of T cells give rise to a polymerase-chain-reaction (PCR) product with immunoglobulin VH gene primers, most likely because of cellular contamination in the micromanipulation procedure. The number of T cells analyzed by Hummel et al. may have been too small to allow an estimation of the frequency of false positive cells. However, such an estimate is critical if polyclonal PCR products are obtained. If a particular rearrangement carried by a clonal population of Reed–Sternberg cells (e.g., because of a somatic mutation) cannot be amplified by the standard set of primers, one will inevitably accumulate false positive polyclonal contaminants. Whether such cellular contamination may account for the results reported by Hummel et al. cannot be determined, because the frequencies of T cells and Reed–Sternberg cells that gave rise to PCR products are not indicated in their report. This makes a critical evaluation of the data impossible.
Cases of Hodgkin's disease with monoclonal populations of Reed–Sternberg cells clearly exist, but the possibility that such cells represent a polyclonal population in a subgroup of cases of Hodgkin's disease or at a particular stage of the disease remains speculative.
3 ReferencesRalf Küppers, Ph.D.
Holger Kanzler
Martin-Leo Hansmann, M.D.
Klaus Rajewsky, M.D.
University of Cologne, 50931 Cologne, Germany1
Hummel M ,Ziemann K ,Lammert H ,Pileri S ,Sabattini E ,Stein H . Hodgkin's disease with monoclonal and polyclonal populations of Reed-Sternberg cells. N Engl J Med 1995;333:901-906
Full Text | Web of Science | Medline2
Kuppers R ,Rajewsky K ,Zhao M , et al. Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. Proc Natl Acad Sci U S A 1994;91:10962-10966
CrossRef | Web of Science | Medline3
Weber-Matthiesen K ,Deerberg J ,Poetsch M ,Grote W ,Schlegelberger B . Numerical chromosome aberrations are present within the CD30+ Hodgkin and Reed-Sternberg cells in 100% of analyzed cases of Hodgkin's disease. Blood 1995;86:1464-1468
Web of Science | Medline
To the Editor:
Hummel et al. examined multinucleated Reed–Sternberg cells isolated from cryostat sections by single-cell PCR for the presence of immunoglobulin heavy-chain gene rearrangements. Monoclonal and polyclonal populations with rearranged heavy-chain genes were demonstrated in all the cases they studied. These findings contrast with ours: we could not show such rearrangements in Reed–Sternberg cells.1 Hummel et al. interpret our results as a failure due to technical factors. Technical factors may indeed explain — at least in part — the different results we obtained.
We have found that isolation of Reed–Sternberg cells from single-cell suspensions2 or cytospin slides3 is the most reliable and reproducible method and carries virtually no risk of DNA contamination. The pattern of expression of Epstein–Barr virus (EBV)–specific genes EBNA-1 and EBNA-2 proved that the isolated cells were indeed Reed–Sternberg cells. Cryostat sections are not well suited to this type of analysis, since there is a high risk of contamination with DNA from reactive lymphocytes by the cryostat knife (which disrupts many cell nucleoli in sections that are 7 μm thick).
We have recently reexamined 10 of our cases with a newly developed seminested PCR employing family-specific oligonucleotides for six of the VH gene families and two sets of eight different oligonucleotides for the six JH segments (Hummel et al. used consensus primers). Again, we were not able to demonstrate VH gene rearrangements. In each of our experiments VH gene–specific PCR products were obtained in 80 percent of the positive controls (single cells from cell lines and B-cell non-Hodgkin's lymphomas). The difference in results may also be due to biologic heterogeneity of the cases examined. The study by Hummel et al. was restricted to cases with the expression of the CD20 antigen on Reed–Sternberg cells, which is present in only a small fraction of cases.4 None of the 10 cases of classic Hodgkin's disease in our latest series (5 with mixed cellularity and 5 with nodular sclerosis) had CD20-positive Reed–Sternberg cells. The question of immunoglobulin heavy-chain gene rearrangements in Reed–Sternberg cells may be resolved by exchanging samples between groups.
4 ReferencesLorenz Trümper, M.D.
Heiner Daus, M.D.
Angela Gause, M.D.
Michael Pfreundschuh, M.D.
University of Saarland, D-66421 Homburg/Saar, Germany1
Roth J ,Daus H ,Trumper L ,Gause A ,Salamon-Looijen M ,Pfreundschuh M . Detection of immunoglobulin heavy-chain gene rearrangement at the single-cell level in malignant lymphomas: no rearrangement is found in Hodgkin and Reed-Sternberg cells. Int J Cancer 1994;57:799-804
CrossRef | Web of Science | Medline2
Trumper LH ,Brady G ,Bagg A , et al. Single-cell analysis of Hodgkin and Reed-Sternberg cells: molecular heterogeneity of gene expression and p53 mutations. Blood 1993;81:3097-3115
Web of Science | Medline3
Roth J ,Daus H ,Gause A ,Trumper L ,Pfreundschuh M . Detection of Epstein-Barr virus DNA in Hodgkin- and Reed-Sternberg-cells by single cell PCR. Leuk Lymphoma 1994;13:137-142
CrossRef | Web of Science | Medline4
Haluska FG ,Brufsky AM ,Canellos GP . The cellular biology of the Reed-Sternberg cell. Blood 1994;84:1005-1019
Web of Science | Medline
To the Editor:
Hummel and his colleagues address the issue of the cell of origin in Hodgkin's disease. All the cases of Hodgkin's disease they selected for study involved a B-cell immunophenotype (on the basis of the presence of CD20-positive Reed–Sternberg cells). In most reports, this subtype accounts for only 5 to 30 percent of all cases of Hodgkin's disease,1-3 and the results are therefore not applicable to most cases of Hodgkin's disease. The Methods section states that the 12 specimens from patients with classic Hodgkin's disease contained CD20-positive Reed–Sternberg cells and that CD30-positive Reed–Sternberg cells were isolated as single cells. Nested PCR was performed to look for rearranged VH gene–specific PCR products. Having demonstrated that about 50 percent of these isolated CD30-positive Reed–Sternberg cells had rearranged VH genes, the authors conclude that Reed–Sternberg cells with B-cell phenotypes have rearranged VH genes. This suggests that the isolated Reed–Sternberg cells coexpress CD20 and CD30. However, the authors do not document such coexpression. In fact, other studies reported in the literature show that the coexpression of CD20 and CD30 by Reed–Sternberg cells is rare.1,2 We think the assumption that Reed–Sternberg cells coexpress CD20 and CD30 is probably erroneous; it must at least be clarified and documented.
3 ReferencesAbdelghani Tbakhi, M.D.
Joseph Sreenan, M.D.
Raymond R. Tubbs, D.O.
Cleveland Clinic Foundation, Cleveland, OH 441951
Zukerberg LR ,Collins AB ,Ferry JA ,Harris NL . Coexpression of CD15 and CD20 by Reed-Sternberg cells in Hodgkin's disease. Am J Pathol 1991;139:475-483
Web of Science | Medline2
Agnarsson BA ,Kadin ME . The immunophenotype of Reed-Sternberg cells: a study of 50 cases of Hodgkin's disease using fixed frozen tissues. Cancer 1989;63:2083-2087
CrossRef | Web of Science | Medline3
Siebert JD ,McClure SP ,Banks PM ,Gulley ML . Hodgkin's disease, mixed cellularity type, with a B-cell immunophenotype: report of a case and literature review. Arch Pathol Lab Med 1995;119:474-479
Web of Science | Medline
Author/Editor Response
The authors reply:
To the Editor: Tbakhi and colleagues state that only 5 to 30 percent of cases of Hodgkin's disease involve CD20-positive Reed–Sternberg cells. This statement is correct. However, it is difficult to understand the authors' statement that the coexpression of CD20 and CD30 by Reed–Sternberg cells is rare, because in the two papers they cite, double-labeling experiments for CD30 and CD20 were not performed. In our single-cell study, we isolated CD30-positive Reed–Sternberg cells from patients with Hodgkin's disease and a B-cell immunophenotype. Since the expression of CD20 but not that of CD301 varies within a given population of Reed–Sternberg cells,2 randomly selected CD30-positive Reed–Sternberg cells may or may not coexpress CD20.
Trümper and colleagues claim that the IgH rearrangements that we demonstrated in single Reed–Sternberg cells stem from contaminating B-cell–derived DNA. This possibility can at least be ruled out in those cases in which Reed–Sternberg cells with identically rearranged IgH genes were found, because the rearrangements of the corresponding single reactive B cells proved to be unrelated. This finding is in agreement with the results reported by Küppers et al.3 and makes the existence of Reed–Sternberg cells with rearranged immunoglobulin genes highly likely.
Both the single-cell studies performed by Küppers et al.3 and those performed by our group show that monoclonal B-cell–derived cases of Hodgkin's disease exist. However, two sets of studies have discrepant results with regard to the occurrence of polyclonal populations of Reed–Sternberg cells. Küppers and colleagues speculate that our demonstration of Reed–Sternberg cells with polyclonal rearrangements may be due to the primers we used, which are ineffective because of somatic V gene mutations and which consequently bind to B-cell–derived DNA contaminants. This concern is not justified, since the reanalysis of our polyclonal cases with additional sets of primers (i.e., family-specific, VH, Vκ, and framework-III consensus primers) led in two of six cases to the detection of an additional population of Reed–Sternberg cells with an identical Vκ rearrangement in one case (this case is also mentioned by Küppers et al.) and a VH5 rearrangement in another. However, in two of the four remaining cases, the additional primer sets revealed — as in the previous investigations — only Reed–Sternberg cells with unrelated (polyclonal) rearrangements. In the two other cases no amplifiable material was left. Numerous T-cell and buffer controls (20 to 30 samples per assay) did not contain notable contamination.
Thus, our additional experiments support the view that cases of Hodgkin's disease with polyclonally rearranged Reed–Sternberg cells occur — a finding in complete agreement with the results recently reported by Delabie and colleagues4 — although at a lower percentage than previously assumed.
4 ReferencesMichael Hummel, Ph.D.
Theresa Marafioti, M.D.
Harald Stein, M.D.
Freie Universität Berlin, 12200 Berlin, Germany1
Schwab U ,Stein H ,Gerdes J , et al. Production of a monoclonal antibody specific for Hodgkin and Sternberg-Reed cells of Hodgkin's disease and a subset of normal lymphoid cells. Nature 1982;299:65-67
CrossRef | Web of Science | Medline2
Pinkus GS ,Said JW . Hodgkin's disease, lymphocyte predominance type, nodular -- further evidence for a B cell derivation: L & H variants of Reed-Sternberg cells express L2b, a pan B cell marker. Am J Pathol 1988;133:211-217
Web of Science | Medline3
Kuppers R ,Rajewsky K ,Zhao M , et al. Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. Proc Natl Acad Sci U S A 1994;91:10962-10966
CrossRef | Web of Science | Medline4
Delabie J, Tierens A, Gavriil T, et al. Single-cell study of the lineage and clonality of Reed-Sternberg cells in nodular sclerosis Hodgkin's disease. Presented at the Third International Symposium on Hodgkin's Lymphoma, Cologne, Germany, 1995.
- Citing Articles (5)
Citing Articles
1
Lian-hua Kim, Suat-cheng Peh, Sibrand Poppema. (2003) Dual variant of Epstein-Barr virus in Hodgkin/Reed-Sternberg cells: Single-cell PCR study onlatent membrane protein-1 gene. International Journal of Cancer 107:2, 250-255
CrossRef2
Wing C. Chan. (2001) The Reed-Sternberg cell in classical Hodgkin's disease. Hematological Oncology 19:1, 1-17
CrossRef3
U. Hasse, M. Tinguely, E. O. Leibundgut, J.-F. Cajot, A. M. Garvin, A. Tobler, B. Borisch, M. F. Fey. (1999) Clonal Loss of Heterozygosity in Microdissected Hodgkin and Reed-Sternberg Cells. JNCI Journal of the National Cancer Institute 91:18, 1581-1583
CrossRef4
Antonio Pinto, Valter Gattei, Vittorina Zagonel, Donatella Aldinucci, Massimo Degan, Angela Iuliis, Francesca Maria Rossi, Francesca Tassan Mazzocco, Cristiana Godeas, Maurizio Rupolo, Dalisa Poletto, Annunziata Gloghini, Antonino Carbone, Hans-Jürgen Gruss. (1998) Hodgkin’s disease: A disorder of dysregulated cellular cross-talk. Biotherapy 10:4, 309-320
CrossRef5
Ralf Küppers, Klaus Rajewsky. (1998) THE ORIGIN OF HODGKIN AND REED/STERNBERG CELLS IN HODGKIN'S DISEASE. Annual Review of Immunology 16:1, 471-493
CrossRef
