Original Article
Hodgkin's Disease with Monoclonal and Polyclonal Populations of Reed–Sternberg Cells
N Engl J Med 1995; 333:901-906October 5, 1995
- Abstract
Background
There is strong evidence that Reed–Sternberg cells have a lymphoid phenotype, but clonally rearranged genes for B-cell and T-cell antigen receptors have not been demonstrable in tumor tissue from most patients with Hodgkin's disease. To elucidate this issue, we assayed single Reed–Sternberg cells from 12 patients with classic Hodgkin's disease of a B-cell immunophenotype to detect rearranged immunoglobulin variable-region heavy-chain (Vh) genes.
Methods
We isolated single Reed–Sternberg cells from frozen sections that had been immunostained for CD30. The rearranged Vh genes of these cells were amplified by the polymerase chain reaction and analyzed by gel electrophoresis and nucleotide sequencing.
Results
In all 12 patients, the Reed–Sternberg cells studied contained rearranged Vh genes. Three patterns were observed: in three patients the rearrangements in each patient were identical, in six patients all the rearrangements were unrelated and unique, and in three patients both identical and unrelated rearrangements were detected. Apparently somatic mutations of Vh genes were present in some Reed–sternberg cells but absent in others.
Conclusions
Reed–Sternberg cells with B-cell phenotypes have rearranged Vh genes; therefore, these cells arise from B cells. The pattern of Vh gene mutations suggests that Reed–Sternberg cells can correspond to either immunologically naive or memory B cells. In half our patients the population of Reed–Sternberg cells was polyclonal; in the other half, monoclonal or mixed cell populations were found. Correlation with the clinical stage suggests that polyclonal Hodgkin's disease can present as a widespread lymphoma.
Media in This Article
Figure 1
Isolation of a Single CD30-Positive Reed–Sternberg Cell from a Section of Tissue from a Patient with Hodgkin's Disease (×600).Figure 2
Amplification Products of the V H Genes Generated by PCR with the use of the FR2A and VLJH Primers.Article Activity
- Article
Hodgkin's disease is generally regarded as a distinct type of malignant lymphoma, because Reed–Sternberg cells are present in an admixture of various nonmalignant cells. Immunologic studies of Reed–Sternberg cells from the nodular-sclerosing, mixed-cellularity, and lymphocyte-depleted types of Hodgkin's disease have revealed the presence of the lymphoid-activation markers CD30 and CD70 in nearly every case,1,2 and that of B-cell or T-cell markers in a substantial proportion of cases.3-7 These findings suggest that Reed–Sternberg cells originate in activated lymphocytes of either the B-cell or T-cell type. Studies of cell lines derived from tissue affected by Hodgkin's disease give further evidence of the lymphoid nature of Reed–Sternberg cells and the existence of B-cell and T-cell types.8-10 However, studies of rearrangements of antigen-receptor genes carried out on whole-tissue DNA from biopsies of patients with Hodgkin's disease were inconclusive, because in most instances the clonal rearrangement expected in a typical lymphoma was not found.11-13 This result may have been due to the scarcity of clonally rearranged Reed–Sternberg cells or to the actual absence of a clonal rearrangement. Studies of karyotypes of cells in metaphase and interphase, DNA content, mutation patterns in the p53 locus, and the terminal repeats of Epstein–Barr virus (EBV) genomes were similarly inconclusive because the results were heterogeneous, applicable in only some cases, or not attributable specifically to Reed–Sternberg cells.10,14-21
A new approach to ascertaining the origin and clonality of Reed–Sternberg cells is the analysis of single Reed–Sternberg cells isolated from tissues affected by Hodgkin's disease. This approach has been used by three groups, but with differing results.22-24 The discrepancies may be due to the small numbers of patients and subtypes of Hodgkin's disease investigated, to differences in isolation methods, or both. In this paper, we report our results with the single-cell assay in 12 patients with Hodgkin's disease whose Reed–Sternberg cells had a B-cell immunophenotype. Our method of isolating immunostained cells directly from frozen sections25 allows a clear morphologic and immunophenotypic identification of Reed–Sternberg cells, permits the reliable collection of single Reed–Sternberg cells, and prevents their contamination by other cells.
Methods
Tissues
Biopsy specimens from 12 patients with Hodgkin's disease (4 with the nodular-sclerosing type and 8 with the mixed-cellularity type) containing CD20-positive Reed–Sternberg cells were obtained from the files in our departments. One hyperplastic tonsil specimen, one specimen of mantle-cell lymphoma, the B-cell line Raji, and the T-cell line HUT102 were used as control tissues and cells.
Immunophenotyping, Tumor-Cell Fraction, Mitotic Index, and in Situ Hybridization
Sections embedded in paraffin, frozen sections, or both were stained with antibodies against CD30, CD20, CD15, CD10, CD3, CD1a, EBV-encoded latent membrane protein, and terminal deoxynucleotidyl transferase (TdT) from Dako (Glostrup, Denmark) and were stained for T-cell–receptor β-chain (TCRβ) with βF1 (T-cell Sciences, Cambridge, Mass.). In situ hybridization with probes for EBV-encoded nuclear RNA 1 and 2 (EBER 1 and 2) was performed as described elsewhere.26 The number of CD30-positive Reed–Sternberg cells among 2000 cells of other types was determined. The mitotic index of the Reed–Sternberg cells was investigated by counting the number of mitotic figures in 300 CD30-positive Reed–Sternberg cells.
Isolation of Single Cells
Frozen sections 7 μm thick were immunostained for CD30, CD20, or TCRβ. In addition to 5 CD20-positive and 5 TCRβ-positive cells used as controls, at least 20 CD30-positive Reed–Sternberg cells were isolated from each specimen and collected as described by Küppers et al. (Figure 1AFigure 1
Isolation of a Single CD30-Positive Reed–Sternberg Cell from a Section of Tissue from a Patient with Hodgkin's Disease (×600). and Figure 1B).25 All isolations of cells were performed at least twice by different persons.Polymerase Chain Reaction
A nested polymerase chain reaction (PCR) was performed with the consensus primers FR127 and LJH28 in the first round of amplification and the consensus primers FR2A and VLJH for reamplification.28
The first PCR was carried out in the tube to which the single cell had been transferred, with 300 ng of FR1 primers (50 ng each), 75 ng of LJH primer, and 2.5 mmol of magnesium chloride per liter of solution in a total volume of 100 μl. The PCR consisted of 5 cycles at 63°C and 35 cycles at 57°C for the annealing of primers. A 1 percent aliquot of the first amplification product was used as a template for reamplification, with 200 ng of FR2A, 400 ng of VLJH, and 1.5 mmol of magnesium chloride per liter of solution. The second PCR consisted of 40 cycles at 63°C. All other PCR conditions were the same as previously described.29,30
Six microliters of each amplification product was subjected to polyacrylamide-gel electrophoresis and subsequently stained with ethidium bromide.
Analysis of DNA Sequences
The isolation of amplified products and subsequent analysis of DNA sequences were performed as described elsewhere.30 The sequences obtained were compared with each other and with published sequences of immunoglobulin variable-region heavy-chain (Vh) germ lines (GenBank, release 87) and translated into protein. Sequences with substitutions of more than three bases were regarded as somatically mutated, because there is very little polymorphism in the germ-line Vh sequences.31
Results
Control Experiments
The PCR method was capable of detecting identical Vh gene rearrangements in single Raji cells, a culture of monoclonal B cells (Figure 2AFigure 2
Amplification Products of the V H Genes Generated by PCR with the use of the FR2A and VLJH Primers.), whereas DNA from single cells of the T-cell line HUT102 could not be amplified with the Vh gene primers. Approximately 60 percent of single B cells isolated from frozen sections of hyperplastic tonsils and a mantle-cell lymphoma yielded PCR products. The PCR assay revealed unrelated (polyclonal) Vh gene rearrangements in the tonsillar B cells and identical (clonal) rearrangements in the mantle-cell lymphoma cells. Single T cells and buffers that covered the frozen sections during the cell-isolation procedure yielded no amplification products (Figure 2A).PCR and Sequence Analysis of Single Cells Isolated from Hodgkin's Disease Tissues
The material analyzed by PCR from biopsy specimens of tissues affected by Hodgkin's disease included single CD30-positive Reed–Sternberg cells and, as positive and negative controls, single B cells and T cells, respectively. Whereas T cells yielded no amplification products, Vh gene–specific PCR products were obtained from 60 to 70 percent of B cells and about 50 percent of single Reed–Sternberg cells. The failure to obtain such products from the rest of the cells was probably due to the use of tissue sections, which often contain only parts of nuclei, especially in the case of large Reed–Sternberg cells. It proved impossible to increase the yield by increasing the thickness of the sections, which only reduced the quality of immunostaining and made the identification and isolation of single cells less reliable.
PCR products of Vh genes were obtained from individual Reed–Sternberg cells isolated from all 12 patients with Hodgkin's disease. Gel electrophoresis and analysis of Vh gene sequences revealed three patterns (Table 1Table 1
Analysis of Single Reed–Sternberg Cells from 12 Patients with Classic Hodgkin's Disease with a B-Cell Immunophenotype, Performed to Detect VH Gene Rearrangements. and Figure 2B). In three patients (Patients 10, 11, and 12), all the amplification products of Reed–Sternberg cells from a given biopsy specimen had the same length and sequence. In six patients (Patients 1 through 6), the lengths and Vh gene sequences of the amplification products from each biopsy specimen differed. In the remaining three patients (Patients 7, 8, and 9), some PCR products had the same lengths and sequences, whereas other products had different ones. Each experiment was performed at least twice by two persons, with identical results in each case.Comparison of Sequences and Translation into Protein
We compared the Vh gene sequences we studied with germ-line sequences in the GenBank data bank to identify individual Vh genes and infer the presence of Vh mutations in Reed–Sternberg cells (Table 1, and material on deposit with the National Auxiliary Publications Service [NAPS]). The population of Vh genes that the Reed–Sternberg cells had rearranged resembled the population used by normal B cells.32 Mutations of Vh genes varied greatly, from none to sequences that appeared to be highly mutated within the same patient and between different patients. One patient (Patient 6), with a polyclonal population of Reed–Sternberg cells, had only wild-type Vh sequences.
The DNA sequences of the Vh genes from the Reed–Sternberg cells were potentially translatable into protein, and were thus functional, with the exceptions of a deletion of 23 base pairs (in Patient 12) that resulted in a frame shift, and of three other sequences, each in a different patient, in which the translation broke off in the N region of the rearranged gene.
Correlation of Rearrangement Patterns with Other Features
The histologic, immunophenotypic, and other features shown in Table 1 and Table 2Table 2
Clinical, Histologic, and Immunohistologic Features of 12 Patients with Classic Hodgkin's Disease Whose Reed–Sternberg Cells Were Positive for CD20 and CD30 and Negative for CD3 and T-Cell Receptor b. indicate that the Reed–Sternberg cells from all the patients expressed CD30 and, in variable quantity and density, the B-cell marker CD20. Early lymphoid-cell markers, such as CD1a, CD10, and TdT, were not detected (data not shown). The Reed–Sternberg cells of all four patients with nodular sclerosing Hodgkin's disease contained unrelated (polyclonal) Vh gene rearrangements and lacked detectable transcripts of EBER 1 and 2. The average mitotic index of the Reed–Sternberg cells was significantly lower (2.3 percent) in the polyclonal group, with late mitotic figures almost totally absent in three instances, as compared with the monoclonal group (mitotic index, 10.2 percent).Discussion
We conducted a PCR analysis of the rearranged Vh genes in single Reed–Sternberg cells with B-cell immunophenotypes that were isolated from 12 patients with classic Hodgkin's disease. We focused on this B-cell type of Reed–Sternberg cells, because with whole-tissue DNA we found a striking positive correlation between the expression of the B-cell marker CD20 on Reed–Sternberg cells and the presence of clonal Vh gene rearrangements.29 However, analysis of whole-tissue DNA cannot determine whether the clonal rearrangements are derived from Reed–Sternberg cells or from other cells in the biopsy specimens. Therefore, we turned to the analysis of individual Reed–Sternberg cells. With this approach, we found rearranged Vh genes in Reed–Sternberg cells in all 12 patients studied. Our results, and the demonstration of immunoglobulin-gene rearrangements in single Reed–Sternberg cells in two other studies,22,24 suggest that technical factors may have contributed to the failure of Roth et al.23 to obtain Vh PCR products from single Reed–Sternberg cells in any of their patients. By molecular means, our experiments show the B-cell origin of Reed–Sternberg cells that express the B-cell antigen CD20.
The pattern of Vh gene rearrangements in the 12 patients we studied was heterogeneous. In three patients, all the Reed–Sternberg cells isolated from the same biopsy specimen had identical Vh rearrangements. This result is consistent with the molecular findings in a monoclonal B-cell lymphoma. We were surprised, however, by the unrelated Vh rearrangements in other patients. The rearranged Vh genes of some Reed–Sternberg cells were identical, whereas those of the others in the same tissue sample were unrelated. These findings indicate a polyclonal proliferation of Reed–Sternberg cells in six patients and mixed populations of both monoclonal and polyclonal cells in the other three patients. Typically, the B cells in a reactive lymph node consist of a polyclonal population, whereas in a lymphoma the B cells are monoclonal. The heterogeneous pattern of Vh gene rearrangement can explain most of the differing results of previous studies using DNA that was extracted either from tissue samples or from enriched populations of Reed–Sternberg cells,33 as well as the heterogeneous findings of analyses of the DNA content of Reed–Sternberg cells.20,21 The reproducibility of our results diminishes the possibility that they represent methodologic artifacts.
Nevertheless, our data seem to be at variance with the results obtained by several other groups. In the study of single cells by Delabie et al.,24 only polyclonal populations of Reed–Sternberg cells were detected, whereas Küppers et al.22 found only monoclonal tumor cells. This discrepancy is probably due to the small numbers of patients — three and four, respectively — included in these two studies. A study of the DNA of Reed–Sternberg cells20 has been interpreted to indicate that the cells have a monoclonal origin. However, it is possible that some of the patients in that study, especially those without aneuploidy, chromosomal aberrations, or p53 mutations, had Reed–Sternberg cells of polyclonal origin. Most studies of EBV genomes in Hodgkin's disease33 have found evidence of monoclonal EBV episomes with a molecular probe of the terminal-repeat region of the virus.34 However, this probe has also found monoclonal EBV episomes in some samples of hyperplastic (polyclonal) lymphoid tissue35 and in some cases of HIV-related immunoblastic lymphomas with polyclonal populations of rearranged immunoglobulin genes.36
The rearranged Vh genes in the three patients with monoclonal gene rearrangements that we studied had somatic mutations. In each patient, all the Reed–Sternberg cells isolated had the same distinctive mutations. The heterogeneous pattern of the Vh gene mutations in five of the six patients with polyclonal Hodgkin's disease indicates that in a given tissue sample the Reed–Sternberg cells were not only unrelated, but also probably derived from B cells in different stages of maturation; those without somatic mutations could correspond to B cells that had not yet entered the germinal center, whereas those with Vh gene mutations may have originated from memory B cells that had left the germinal center.37
If the Reed–Sternberg cell is indeed the neoplastic component of Hodgkin's disease, then our finding of polyclonal Reed–Sternberg cells conflicts with current concepts of tumorigenesis. Monoclonal neoplasms grow through continuous mitotic divisions and give rise to identical progeny. In the case of polyclonal Reed–Sternberg cells, the mechanism of cell growth must differ. Quantitative and qualitative analyses of mitosis indeed revealed that the mitotic index was far lower in the patients with polyclonal cells than in those with monoclonal cells. Moreover, three of the six patients with polyclonal Reed–Sternberg cells, but none of the three with monoclonal Reed–Sternberg cells, lacked late mitotic figures, suggesting a disturbance of the mitotic process, of cytokinesis, or both. These observations suggest that polyclonal populations of Reed–Sternberg cells arise from the continuous recruitment of unrelated B lymphocytes. Such a mechanism would be predicated on the susceptibility of certain B cells to be transformed into Reed–Sternberg cells (a process perhaps mediated by genetic instability); a transforming agent or agents, such as EBV; and an immune defect that impairs the elimination of aberrant cells. There is evidence of each of these elements in Hodgkin's disease.38-42
The classification of Hodgkin's disease as polyclonal or monoclonal may have clinical implications. For example, the patients with polyclonal Reed–Sternberg cells may respond better to chemotherapy43 than those with monoclonal Reed–Sternberg cells. Our study shows that the presence of B-cell markers on Reed–Sternberg cells does not constitute an example of aberrant gene expression, but indicates a real relation between those cells and B cells. We therefore conclude that there are B-cell types of Hodgkin's disease and that some of them contain polyclonal populations of Reed–Sternberg cells. Further studies of single cells may clarify the origin of Reed–Sternberg cells that express T-cell antigens or of those that lack both B-cell and T-cell antigens.
Supported by the Deutsche Krebshilfe, Mildred–Scheel–Stiftung; the Berliner Krebsgesellschaft; and a grant from the Italian Association for Cancer Research (Milan).
NAPS See NAPS document no. 05246 for three pages of supplementary material. Order from NAPS c/o Microfiche Publications, P.O. Box 3513, Grand Central Station, New York, NY 10163-3513.
We are indebted to B. Kalvelage, C. Kreschel, and H.-H. Müller for excellent technical assistance; to Ioannis Anagnostopoulos, M.D., for valuable assistance with the Discussion section, and to Joannah Caborn for her editorial assistance. This paper comprises part of the doctoral thesis of Ms. Ziemann.
Source Information
From the Institute of Pathology, Klinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany (M.H., K.Z., H.L., H.S.); and the Hematopathology Unit, Second Service of Pathological Anatomy, University of Bologna, Bologna, Italy (S.P., E.S.).
Address reprint requests to Professor Stein at the Institute of Pathology, Klinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.
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