Background There is strong evidence that Reed–Sternbergcells have a lymphoid phenotype, but clonally rearranged genesfor B-cell and T-cell antigen receptors have not been demonstrablein tumor tissue from most patients with Hodgkin's disease. Toelucidate this issue, we assayed single Reed–Sternbergcells from 12 patients with classic Hodgkin's disease of a B-cellimmunophenotype to detect rearranged immunoglobulin variable-regionheavy-chain (Vh) genes.
Methods We isolated single Reed–Sternberg cells from frozensections that had been immunostained for CD30. The rearrangedVh genes of these cells were amplified by the polymerase chainreaction and analyzed by gel electrophoresis and nucleotidesequencing.
Results In all 12 patients, the Reed–Sternberg cells studiedcontained 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 rearrangementswere detected. Apparently somatic mutations of Vh genes werepresent in some Reed–sternberg cells but absent in others.
Conclusions Reed–Sternberg cells with B-cell phenotypeshave rearranged Vh genes; therefore, these cells arise fromB cells. The pattern of Vh gene mutations suggests that Reed–Sternbergcells can correspond to either immunologically naive or memoryB cells. In half our patients the population of Reed–Sternbergcells was polyclonal; in the other half, monoclonal or mixedcell populations were found. Correlation with the clinical stagesuggests that polyclonal Hodgkin's disease can present as awidespread lymphoma.
Hodgkin's disease is generally regarded as a distinct type ofmalignant lymphoma, because Reed–Sternberg cells are presentin an admixture of various nonmalignant cells. Immunologic studiesof Reed–Sternberg cells from the nodular-sclerosing, mixed-cellularity,and lymphocyte-depleted types of Hodgkin's disease have revealedthe presence of the lymphoid-activation markers CD30 and CD70in nearly every case,1,2 and that of B-cell or T-cell markersin a substantial proportion of cases.3,4,5,6,7 These findingssuggest that Reed–Sternberg cells originate in activatedlymphocytes of either the B-cell or T-cell type. Studies ofcell lines derived from tissue affected by Hodgkin's diseasegive further evidence of the lymphoid nature of Reed–Sternbergcells and the existence of B-cell and T-cell types.8,9,10 However,studies of rearrangements of antigen-receptor genes carriedout on whole-tissue DNA from biopsies of patients with Hodgkin'sdisease were inconclusive, because in most instances the clonalrearrangement expected in a typical lymphoma was not found.11,12,13This result may have been due to the scarcity of clonally rearrangedReed–Sternberg cells or to the actual absence of a clonalrearrangement. Studies of karyotypes of cells in metaphase andinterphase, DNA content, mutation patterns in the p53 locus,and the terminal repeats of Epstein–Barr virus (EBV) genomeswere similarly inconclusive because the results were heterogeneous,applicable in only some cases, or not attributable specificallyto Reed–Sternberg cells.10,14,15,16,17,18,19,20,21
A new approach to ascertaining the origin and clonality of Reed–Sternbergcells is the analysis of single Reed–Sternberg cells isolatedfrom tissues affected by Hodgkin's disease. This approach hasbeen used by three groups, but with differing results.22,23,24The discrepancies may be due to the small numbers of patientsand subtypes of Hodgkin's disease investigated, to differencesin isolation methods, or both. In this paper, we report ourresults with the single-cell assay in 12 patients with Hodgkin'sdisease whose Reed–Sternberg cells had a B-cell immunophenotype.Our method of isolating immunostained cells directly from frozensections25 allows a clear morphologic and immunophenotypic identificationof Reed–Sternberg cells, permits the reliable collectionof single Reed–Sternberg cells, and prevents their contaminationby other cells.
Methods
Tissues
Biopsy specimens from 12 patients with Hodgkin's disease (4with the nodular-sclerosing type and 8 with the mixed-cellularitytype) containing CD20-positive Reed–Sternberg cells wereobtained from the files in our departments. One hyperplastictonsil specimen, one specimen of mantle-cell lymphoma, the B-cellline Raji, and the T-cell line HUT102 were used as control tissuesand cells.
Immunophenotyping, Tumor-Cell Fraction, Mitotic Index, and in Situ Hybridization
Sections embedded in paraffin, frozen sections, or both werestained with antibodies against CD30, CD20, CD15, CD10, CD3,CD1a, EBV-encoded latent membrane protein, and terminal deoxynucleotidyltransferase (TdT) from Dako (Glostrup, Denmark) and were stainedfor T-cell–receptor -chain (TCR) with F1 (T-cell Sciences,Cambridge, Mass.). In situ hybridization with probes for EBV-encodednuclear RNA 1 and 2 (EBER 1 and 2) was performed as describedelsewhere.26 The number of CD30-positive Reed–Sternbergcells among 2000 cells of other types was determined. The mitoticindex of the Reed–Sternberg cells was investigated bycounting the number of mitotic figures in 300 CD30-positiveReed–Sternberg cells.
Isolation of Single Cells
Frozen sections 7 µm thick were immunostained for CD30,CD20, or TCR. In addition to 5 CD20-positive and 5 TCR-positivecells used as controls, at least 20 CD30-positive Reed–Sternbergcells were isolated from each specimen and collected as describedby Küppers et al. (Figure 1A and Figure 1B).25 All isolationsof cells were performed at least twice by different persons.
Figure 1. Isolation of a Single CD30-Positive Reed–Sternberg Cell from a Section of Tissue from a Patient with Hodgkin's Disease (x600).
The tissue section is shown before (Panel A) and after (Panel B) the isolation of the cell. The immunostained Reed–Sternberg cell was cut away from the surrounding cells with a manipulation capillary (MC) and transferred to the reception capillary (RC) without damage to surrounding cells and tissue, as described by Küppers et al.25
Polymerase Chain Reaction
A nested polymerase chain reaction (PCR) was performed withthe consensus primers FR127 and LJH28 in the first round ofamplification and the consensus primers FR2A and VLJH for reamplification.28
The first PCR was carried out in the tube to which the singlecell had been transferred, with 300 ng of FR1 primers (50 ngeach), 75 ng of LJH primer, and 2.5 mmol of magnesium chlorideper liter of solution in a total volume of 100 µl. ThePCR consisted of 5 cycles at 63°C and 35 cycles at 57°Cfor the annealing of primers. A 1 percent aliquot of the firstamplification product was used as a template for reamplification,with 200 ng of FR2A, 400 ng of VLJH, and 1.5 mmol of magnesiumchloride per liter of solution. The second PCR consisted of40 cycles at 63°C. All other PCR conditions were the sameas previously described.29,30
Six microliters of each amplification product was subjectedto polyacrylamide-gel electrophoresis and subsequently stainedwith ethidium bromide.
Analysis of DNA Sequences
The isolation of amplified products and subsequent analysisof DNA sequences were performed as described elsewhere.30 Thesequences obtained were compared with each other and with publishedsequences 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 regardedas somatically mutated, because there is very little polymorphismin the germ-line Vh sequences.31
Results
Control Experiments
The PCR method was capable of detecting identical Vh gene rearrangementsin single Raji cells, a culture of monoclonal B cells (Figure 2A),whereas DNA from single cells of the T-cell line HUT102could not be amplified with the Vh gene primers. Approximately60 percent of single B cells isolated from frozen sections ofhyperplastic tonsils and a mantle-cell lymphoma yielded PCRproducts. The PCR assay revealed unrelated (polyclonal) Vh generearrangements in the tonsillar B cells and identical (clonal)rearrangements in the mantle-cell lymphoma cells. Single T cellsand buffers that covered the frozen sections during the cell-isolationprocedure yielded no amplification products (Figure 2A).
Figure 2. Amplification Products of the VH Genes Generated by PCR with the use of the FR2A and VLJH Primers.
Panel A shows amplification products after 6 percent polyacrylamide-gel electrophoresis and staining with ethidium bromide. The products were derived from single selected normal B cells (lanes 1 through 4) and cytospin preparations of single cells from the Raji cell line (lanes 9 and 10). No amplification products were obtained from single selected T cells (lanes 5 through 8) or overlying buffers (lanes 11 and 12). Panel B shows VH-specific amplification products derived from single Reed–Sternberg cells isolated from tissue from three patients with Hodgkin's disease. In Patient 6 (lanes 1 through 4), all the PCR products differed in length; in Patient 7, products of both identical length (lanes 5 and 6) and different lengths (lanes 7 and 8) were found, whereas in Patient 12 (lanes 9 through 12) all the PCR products were the same length. S denotes a molecular-weight standard, and bp base pairs.
PCR and Sequence Analysis of Single Cells Isolated from Hodgkin's Disease Tissues
The material analyzed by PCR from biopsy specimens of tissuesaffected by Hodgkin's disease included single CD30-positiveReed–Sternberg cells and, as positive and negative controls,single B cells and T cells, respectively. Whereas T cells yieldedno amplification products, Vh gene–specific PCR productswere obtained from 60 to 70 percent of B cells and about 50percent of single Reed–Sternberg cells. The failure toobtain such products from the rest of the cells was probablydue to the use of tissue sections, which often contain onlyparts of nuclei, especially in the case of large Reed–Sternbergcells. It proved impossible to increase the yield by increasingthe thickness of the sections, which only reduced the qualityof immunostaining and made the identification and isolationof single cells less reliable.
PCR products of Vh genes were obtained from individual Reed–Sternbergcells isolated from all 12 patients with Hodgkin's disease.Gel electrophoresis and analysis of Vh gene sequences revealedthree patterns (Table 1 and Figure 2B). In three patients (Patients10, 11, and 12), all the amplification products of Reed–Sternbergcells from a given biopsy specimen had the same length and sequence.In six patients (Patients 1 through 6), the lengths and Vh genesequences of the amplification products from each biopsy specimendiffered. In the remaining three patients (Patients 7, 8, and9), some PCR products had the same lengths and sequences, whereasother products had different ones. Each experiment was performedat least twice by two persons, with identical results in eachcase.
Table 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.
Comparison of Sequences and Translation into Protein
We compared the Vh gene sequences we studied with germ-linesequences in the GenBank data bank to identify individual Vhgenes and infer the presence of Vh mutations in Reed–Sternbergcells (Table 1, and material on deposit with the National AuxiliaryPublications Service [*]). The population of Vh genes that theReed–Sternberg cells had rearranged resembled the populationused by normal B cells.32 Mutations of Vh genes varied greatly,from none to sequences that appeared to be highly mutated withinthe same patient and between different patients. One patient(Patient 6), with a polyclonal population of Reed–Sternbergcells, had only wild-type Vh sequences.
The DNA sequences of the Vh genes from the Reed–Sternbergcells were potentially translatable into protein, and were thusfunctional, with the exceptions of a deletion of 23 base pairs(in Patient 12) that resulted in a frame shift, and of threeother sequences, each in a different patient, in which the translationbroke off in the N region of the rearranged gene.
Correlation of Rearrangement Patterns with Other Features
The histologic, immunophenotypic, and other features shown inTable 1 and Table 2 indicate that the Reed–Sternberg cellsfrom all the patients expressed CD30 and, in variable quantityand 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 nodularsclerosing Hodgkin's disease contained unrelated (polyclonal)Vh gene rearrangements and lacked detectable transcripts ofEBER 1 and 2. The average mitotic index of the Reed–Sternbergcells was significantly lower (2.3 percent) in the polyclonalgroup, with late mitotic figures almost totally absent in threeinstances, as compared with the monoclonal group (mitotic index,10.2 percent).
Table 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.
Discussion
We conducted a PCR analysis of the rearranged Vh genes in singleReed–Sternberg cells with B-cell immunophenotypes thatwere 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 correlationbetween the expression of the B-cell marker CD20 on Reed–Sternbergcells and the presence of clonal Vh gene rearrangements.29 However,analysis of whole-tissue DNA cannot determine whether the clonalrearrangements are derived from Reed–Sternberg cells orfrom other cells in the biopsy specimens. Therefore, we turnedto the analysis of individual Reed–Sternberg cells. Withthis approach, we found rearranged Vh genes in Reed–Sternbergcells in all 12 patients studied. Our results, and the demonstrationof immunoglobulin-gene rearrangements in single Reed–Sternbergcells in two other studies,22,24 suggest that technical factorsmay have contributed to the failure of Roth et al.23 to obtainVh PCR products from single Reed–Sternberg cells in anyof their patients. By molecular means, our experiments showthe B-cell origin of Reed–Sternberg cells that expressthe B-cell antigen CD20.
The pattern of Vh gene rearrangements in the 12 patients westudied was heterogeneous. In three patients, all the Reed–Sternbergcells isolated from the same biopsy specimen had identical Vhrearrangements. This result is consistent with the molecularfindings 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 wereidentical, whereas those of the others in the same tissue samplewere unrelated. These findings indicate a polyclonal proliferationof Reed–Sternberg cells in six patients and mixed populationsof both monoclonal and polyclonal cells in the other three patients.Typically, the B cells in a reactive lymph node consist of apolyclonal population, whereas in a lymphoma the B cells aremonoclonal. The heterogeneous pattern of Vh gene rearrangementcan explain most of the differing results of previous studiesusing DNA that was extracted either from tissue samples or fromenriched populations of Reed–Sternberg cells,33 as wellas the heterogeneous findings of analyses of the DNA contentof Reed–Sternberg cells.20,21 The reproducibility of ourresults diminishes the possibility that they represent methodologicartifacts.
Nevertheless, our data seem to be at variance with the resultsobtained by several other groups. In the study of single cellsby Delabie et al.,24 only polyclonal populations of Reed–Sternbergcells were detected, whereas Küppers et al.22 found onlymonoclonal tumor cells. This discrepancy is probably due tothe small numbers of patients — three and four, respectively— included in these two studies. A study of the DNA ofReed–Sternberg cells20 has been interpreted to indicatethat the cells have a monoclonal origin. However, it is possiblethat some of the patients in that study, especially those withoutaneuploidy, chromosomal aberrations, or p53 mutations, had Reed–Sternbergcells of polyclonal origin. Most studies of EBV genomes in Hodgkin'sdisease33 have found evidence of monoclonal EBV episomes witha molecular probe of the terminal-repeat region of the virus.34However, this probe has also found monoclonal EBV episomes insome samples of hyperplastic (polyclonal) lymphoid tissue35and in some cases of HIV-related immunoblastic lymphomas withpolyclonal populations of rearranged immunoglobulin genes.36
The rearranged Vh genes in the three patients with monoclonalgene rearrangements that we studied had somatic mutations. Ineach patient, all the Reed–Sternberg cells isolated hadthe same distinctive mutations. The heterogeneous pattern ofthe Vh gene mutations in five of the six patients with polyclonalHodgkin's disease indicates that in a given tissue sample theReed–Sternberg cells were not only unrelated, but alsoprobably derived from B cells in different stages of maturation;those without somatic mutations could correspond to B cellsthat had not yet entered the germinal center, whereas thosewith Vh gene mutations may have originated from memory B cellsthat had left the germinal center.37
If the Reed–Sternberg cell is indeed the neoplastic componentof Hodgkin's disease, then our finding of polyclonal Reed–Sternbergcells conflicts with current concepts of tumorigenesis. Monoclonalneoplasms grow through continuous mitotic divisions and giverise to identical progeny. In the case of polyclonal Reed–Sternbergcells, the mechanism of cell growth must differ. Quantitativeand qualitative analyses of mitosis indeed revealed that themitotic index was far lower in the patients with polyclonalcells than in those with monoclonal cells. Moreover, three ofthe 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 themitotic process, of cytokinesis, or both. These observationssuggest that polyclonal populations of Reed–Sternbergcells arise from the continuous recruitment of unrelated B lymphocytes.Such a mechanism would be predicated on the susceptibility ofcertain B cells to be transformed into Reed–Sternbergcells (a process perhaps mediated by genetic instability); atransforming agent or agents, such as EBV; and an immune defectthat impairs the elimination of aberrant cells. There is evidenceof each of these elements in Hodgkin's disease.38,39,40,41,42
The classification of Hodgkin's disease as polyclonal or monoclonalmay have clinical implications. For example, the patients withpolyclonal Reed–Sternberg cells may respond better tochemotherapy43 than those with monoclonal Reed–Sternbergcells. Our study shows that the presence of B-cell markers onReed–Sternberg cells does not constitute an example ofaberrant gene expression, but indicates a real relation betweenthose cells and B cells. We therefore conclude that there areB-cell types of Hodgkin's disease and that some of them containpolyclonal populations of Reed–Sternberg cells. Furtherstudies of single cells may clarify the origin of Reed–Sternbergcells that express T-cell antigens or of those that lack bothB-cell and T-cell antigens.
Supported by the Deutsche Krebshilfe, Mildred–Scheel–Stiftung;the Berliner Krebsgesellschaft; and a grant from the ItalianAssociation for Cancer Research (Milan).
We are indebted to B. Kalvelage, C. Kreschel, and H.-H. Müllerfor excellent technical assistance; to Ioannis Anagnostopoulos,M.D., for valuable assistance with the Discussion section, andto Joannah Caborn for her editorial assistance. This paper comprisespart of the doctoral thesis of Ms. Ziemann.
* See NAPS document no. 05246 for three pages of supplementarymaterial. Order from NAPS c/o Microfiche Publications, P.O.Box 3513, Grand Central Station, New York, NY 10163-3513.
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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A correction has been published: N Engl J Med 1999;340(5):394.
-chain (TCR
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