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Previous Volume 337:453-458 August 14, 1997 Number 7 Next

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ABSTRACT

Background The atypical cells of nodular lymphocyte-predominant Hodgkin's disease, designated lymphocytic and histiocytic (L&H) cells, have a B-cell phenotype. To clarify the clonality of these cells, we studied rearranged immunoglobulin genes for the variable region of the heavy chain (V_H genes) in individual L&H cells from 11 patients with nodular lymphocyte-predominant Hodgkin's disease. We also studied the expression of immunoglobulin light chains by those cells in six of the same patients.

Methods Single CD20+ L&H cells were isolated from frozen sections by a technique of micromanipulation. The rearranged V_H genes of these cells were amplified by the polymerase chain reaction (PCR), sequenced, and compared with germ-line V_H genes. Immunoglobulin light-chain messenger RNA (mRNA) was detected by in situ hybridization.

Results Of 615 L&H cells isolated from all the frozen sections, 160 yielded PCR products. In each of the 11 patients, the L&H cells that could be evaluated had identically rearranged V_H genes, whether they were isolated from the same nodule, different nodules, or different blocks of tissue. All the V_H sequences derived from the L&H cells were highly mutated (7.5 to 27.2 percent). In two cases the coding capacity of the V_H genes was completely or partially disrupted by mutations. Intraclonal diversity was found in six cases, and monotypic immunoglobulin light-chain mRNA was found in six.

Conclusions The L&H cells of nodular lymphocyte-predominant Hodgkin's disease represent a monoclonal expansion of B cells. The high load of V_H gene mutations and signs of intraclonal diversity suggest a relation between L&H cells and germinal-center B cells at the centroblastic stage of differentiation.

Methods

Tissue Samples

We collected frozen specimens of tissue from patients with typical cases of nodular lymphocyte-predominant Hodgkin's disease that met the criteria of the revised European–American lymphoma²⁰ classification. The samples were obtained from files at the institutes of pathology in London; Toulouse, France; Perugia and Bologna, Italy; and Berlin, Germany. Hyperplastic tonsils were used as controls.

Isolation of Single Cells

Seven-micrometer-thick frozen sections were immunostained for CD20 (L26) or the beta chain of the T-cell receptor (F1). From 25 to 89 CD20+ L&H cells (615 cells in all) and 7 normal T cells (84 in all) were isolated from the frozen tissue from the 11 patients; for 1 patient, two samples obtained nine months apart were studied. In addition, 260 B cells were extracted from two hyperplastic tonsils (158 cells) and seven patients with nodular lymphocyte-predominant Hodgkin's disease (102 cells). In contrast to previous studies of single cells,^12,16,21,22 our method involved suspending the isolated cells in a minimal volume of buffer (less than 250 nl). To check for the presence of contaminating V_H sequences in the buffer, aliquots of at least 1 µl were drawn after the isolation of each cell (959 aliquots in all). Cells were isolated from each section two to six times, depending on the number of L&H cells that could be amplified.

PCR and Analysis of DNA Sequences

To detect single copies of rearranged V_H genes, we used a fully nested PCR with family-specific FW1 primers²³ and a consensus JH primer (LJH)²⁴ in the first amplification and modified family-specific FW2 primers in conjunction with a nested JH primer (VLJH) in the second amplification.^15,24,25 The isolated PCR products were sequenced, and the number of somatic mutations in the amplified V_H region was determined by comparison with corresponding germ-line V_H segments. To establish the coding capacity of the L&H cells, the sequences of the amplification products were translated into the amino acids and studied as described elsewhere.^25,26

In Situ Hybridization

After the linearization of plasmids containing the immunoglobulin kappa and immunoglobulin lambda light-chain constant regions, antisense and sense transcripts labeled with sulfur-35 were generated with T7 or SP6 RNA polymerases (Promega–Biotech, Madison, Wis.). In situ hybridization was performed as described previously.²⁷

Results

Immunoglobulin V_H Rearrangements

Individual CD20+ L&H cells (from 25 to 89 per patient) were isolated from frozen sections of tissue from 11 patients with nodular lymphocyte-predominant Hodgkin's disease and studied by single-copy PCR for rearrangements of the V_H genes (Figure 1A and Table 1). From a total of 615 L&H cells and 260 B cells from hyperplastic tonsils as well as patients with nodular lymphocyte-predominant Hodgkin's disease, 160 (26 percent) and 102 (39 percent) amplification products of appropriate size for rearranged V_H genes were obtained, respectively. Eighty-four T cells isolated from frozen sections of tissue from the 11 patients (with two samples from Patient 1) did not generate V_H amplification products, with one exception (Table 1). Aliquots from the buffer overlying the frozen sections during the process of cell isolation were tested after the selection of each cell, and none of these 959 samples of buffer gave rise to PCR products.

View larger version (148K): [in this window] [in a new window]   Figure 1. Studies of the Clonal Relation among Eight Lymphocytic and Histiocytic (L&H) Cells Isolated from Patient 11. Panel A shows a frozen section of a lymph node from the patient after immunostaining for CD20 (alkaline phosphatase–anti-alkaline phosphatase method, x100), with the reaction products staining red. The white zones surrounded by red rims are the spaces formerly occupied by the L&H cells that were isolated from the section by a micropipette. Among these isolated L&H cells, those that gave rise to VH amplification products are indicated (arrows; the numbering is arbitrary and does not correspond to the order in which the cells were isolated). The boxed portion of the section is shown at higher magnification (inset at upper left, x400). Panel B shows a genealogic tree indicating the somatic mutations in the clonally related VH sequences in the same eight L&H cells, numbered as in Panel A. The germ-line sequence (VH3) and the hypothetical intermediate sequence are also shown. The numbers and positions shown adjacent to the lines indicate the locations of the nucleotide substitutions and their types. The length of the lines corresponds to the number of mutations in the sequence. Uppercase letters denote replacement mutations, and lowercase letters silent mutations. The location numbers follow the system of Cook and Tomlinson.26

 

View this table: [in this window] [in a new window]   Table 1. Clinical Data and Rearrangements of Immunoglobulin Heavy-Chain Genes in L&H Cells from 11 Patients with Nodular Lymphocyte-Predominant Hodgkin's Disease.

 
By gel electrophoresis and sequence analysis, all but 1 of the 160 V_H gene amplification products derived from the L&H cells from the same patient proved to be identical, whether the cells were isolated from the same nodule, different nodules, or different sections of tissue that were analyzed independently. In addition, the two biopsy specimens obtained from Patient 1 nine months apart had identical V_H sequences. In contrast, the V_H gene rearrangements in all the single B cells from hyperplastic tonsils and patients with nodular lymphocyte-predominant Hodgkin's disease were unrelated to each other, to the rearrangements in the L&H cells, and to sequences from a data bank (GenBank release 98).

Somatic Mutations

The rearranged V_H genes of the L&H cells from all 11 patients were found to contain from 11 to 40 nucleotide substitutions (7.5 to 27.2 percent) when they were compared with the corresponding germ-line segments. Most of the mutations were located in complementarity-determining region (CDR) 2 (average mutation rate in CDR2, 21.6 percent), whereas framework region 3 was affected less often (average mutation rate, 8 percent).

Among these clones with hypermutated L&H cells, six had intraclonal diversity (Table 2 and Figure 1B). In the L&H cells from two patients, the coding capacity of the V_H genes was disrupted by the mutations. In Patient 4, the V_H genes of all 13 L&H cells were silenced by two stop codons and a shift in the reading frame. In Patient 7, the coding capacity was only partly lost, because in 11 of the 13 L&H cells there was a stop codon in framework region 3.

View this table: [in this window] [in a new window]   Table 2. Molecular Features of the Immunoglobulin Heavy- and Light-Chain Genes in L&H Cells of 11 Patients with Nodular Lymphocyte-Predominant Hodgkin's Disease.

 
Immunoglobulin Light-Chain Transcripts

In 6 of the 11 patients we used in situ hybridization of frozen tissue to study immunoglobulin light-chain transcripts. In all six patients, the L&H cells had moderately strong labeling for immunoglobulin light-chain kappa messenger RNA, but they were not labeled by the immunoglobulin light-chain lambda antisense probes (Table 2). Both probes for immunoglobulin light chains marked plasma cells strongly and marked a proportion of reactive lymphoid cells weakly. Sense probes produced only background signals.

Discussion

It is generally agreed that L&H cells have a B-cell phenotype, but their clonality is controversial. To clarify this issue, we studied the rearranged V_H genes in individual L&H cells isolated from frozen sections of tissue from 11 patients with nodular lymphocyte-predominant Hodgkin's disease. We identified the cells morphologically and with the B-cell marker CD20. The L&H cells in each patient had the same rearrangement of V_H genes. This finding confirmed that L&H cells were B cells (because only B cells contain rearranged V_H genes) and showed that L&H cells arise as a result of clonal expansion. The clonality of these cells was further evidenced by the monotypic expression of immunoglobulin light-chain transcripts, which is in accordance with earlier studies.^17,18,19

Previous studies by single-cell PCR of both the nodular lymphocyte-predominant^12,16,28 and classic types of Hodgkin's disease have had discordant results.^16,22,28 These discrepancies were most likely due to difficulties with the technique of cell isolation, among which problems with the amplification of contaminating V_H sequences are the most serious. We overcame this difficulty by reducing the volume of buffer used in the suspension of single cells. With this technical modification, none of the 959 samples that were drawn from the buffer covering the frozen sections after the isolation of each cell contained V_H-specific amplification products, and only 1 of the 84 T cells isolated from the tissue sections from the patients with nodular lymphocyte-predominant Hodgkin's disease yielded a PCR product. Moreover, 260 B cells isolated from frozen sections of hyperplastic tonsils and samples of tissue affected by the disease gave rise to 102 PCR products with unrelated VH genes.

The CDR3 sequence is the molecular signature of a B cell, because randomly selected nucleotides are inserted in the course of the V_H rearrangement. None of the V_H sequences derived from the clonal L&H cells that we studied were homologous with sequences we studied previously or with CDR3 sequences in the data bank. The single V_H rearrangement in the sample from Patient 3 that was unrelated to the highly mutated clonal population of L&H cells probably originated from a neighboring B cell, as was evidenced by the absence of somatic mutations in the rearranged V_H gene. That two biopsy specimens from Patient 1 obtained nine months apart contained identical V_H rearrangements provides further support for the validity of our method.

Physiologically, somatic mutations are introduced into immunoglobulin V genes at the centroblastic stage of B-cell differentiation in germinal centers during the immune response.^29,30 Through this process, B cells acquire immunoglobulin receptors with higher or lower affinity for the antigen. The high-affinity B cells are selected by antigen for further differentiation into memory B cells and plasma cells.³⁰ These cells have a low-to-moderate number of somatic mutations (up to 6 percent).²⁹ By contrast, receptors on B cells that have highly mutated V genes lose affinity for the antigen and undergo apoptosis.³⁰ In our study, the high load of somatic mutations (7.5 to 27.2 percent) in all 11 cases of nodular lymphocyte-predominant Hodgkin's disease and the signs of ongoing mutation in 6 of these cases suggest that L&H cells are resistant to these physiologic mechanisms, perhaps because of a block in the apoptotic pathway. The presence in two patients of L&H cells with disrupted capacity for coding by immunoglobulin genes supports this theory, because normally such B cells cannot survive.

The evidence that L&H cells are related to B cells in germinal centers or their progeny includes the pattern of V_H gene mutation, the consistent association of L&H cells with progressively transformed germinal centers,³¹ the distribution of L&H cells within these structures, the expression of cell antigens characteristic of the germinal center,^32,33,34 and the resemblance of the L&H cells to large centroblasts. Our findings show that L&H cells arise from a single clone of germinal-center B cells.

Although our data are limited and the light-chain V genes of L&H cells were not sequenced, it appears that the rearranged V genes in L&H cells and those in Reed–Sternberg cells have undergone somatic mutation to a similar extent²¹ (and this study). However, in L&H cells these mutated genes usually retain the potential for translation into protein, whereas in Reed–Sternberg cells they often lose their coding capacity.²¹ Moreover, intraclonal variants seem to be more frequent among L&H cells than among Reed–Sternberg cells. If further investigation substantiates these differences, they would constitute important molecular distinctions between nodular lymphocyte-predominant Hodgkin's disease and classic Hodgkin's disease.

Sponsored by a grant (Ste 318/7-1) from the Deutsche Forschungsgemeinschaft, by a grant (M25/89/St1) from the Deutsche Krebshilfe, and by the Italian Association for Cancer Research (Milan).

We are indebted to H. Lammert, C. Kreschel, H. Protz, E. Berg, and H.-H. Müller for their excellent technical assistance; to P. Korbjuhn, M.D., and L. Pasqualucci, M.D., for their scientific contributions; and to J. Yates for her editorial assistance.

Source Information

From the Institute of Pathology, University Hospital Benjamin Franklin, Free University Berlin, and the Consultation and Reference Center for Lymph Node Pathology and Haematopathology, Berlin, Germany (T.M., M.H., I.A., H.-D.F., H.S.); the Institute of Hematology, University of Perugia, Perugia, Italy (B.F.); the Laboratoire Central d'Anatomie–Pathologie, Hôpitaux de Toulouse, Centre Hospitalier Universitaire Purpan, Toulouse, France (G.D.); the Department of Histopathology, University College London Medical School, London (P.G.I.); and the Secondo Servizio di Anatomia Patologica, Sezione di Emolinfopatologia, Università di Bologna, Bologna, Italy (S.P.).

Address reprint requests to Professor Stein at the Institute of Pathology, Benjamin Franklin University Hospital, Free University Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.

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L&H Cells in Lymphocyte-Predominant Hodgkin's Disease
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• Bauer, K., Zemlin, M., Hummel, M., Pfeiffer, S., Karstaedt, J., Steinhauser, G., Xiao, X., Versmold, H., Berek, C. (2002). Diversification of Ig Heavy Chain Genes in Human Preterm Neonates Prematurely Exposed to Environmental Antigens. J. Immunol. 169: 1349-1356 [Abstract] [Full Text]  
• Rassidakis, G. Z., Medeiros, L. J., Viviani, S., Bonfante, V., Nadali, G.-P., Vassilakopoulos, T. P., Mesina, O., Herling, M., Angelopoulou, M. K., Giardini, R., Chilosi, M., Kittas, C., McLaughlin, P., Rodriguez, M. A., Romaguera, J., Bonadonna, G., Gianni, A. M., Pizzolo, G., Pangalis, G. A., Cabanillas, F., Sarris, A. H. (2002). CD20 Expression in Hodgkin and Reed-Sternberg Cells of Classical Hodgkin's Disease: Associations With Presenting Features and Clinical Outcome. JCO 20: 1278-1287 [Abstract] [Full Text]  
• Pileri, S A, Ascani, S, Leoncini, L, Sabattini, E, Zinzani, P L, Piccaluga, P P, Pileri, A Jr, Giunti, M, Falini, B, Bolis, G B, Stein, H (2002). Hodgkin's lymphoma: the pathologist's viewpoint. J. Clin. Pathol. 55: 162-176 [Abstract] [Full Text]  
• Hopken, U. E., Foss, H.-D., Meyer, D., Hinz, M., Leder, K., Stein, H., Lipp, M. (2002). Up-regulation of the chemokine receptor CCR7 in classical but not in lymphocyte-predominant Hodgkin disease correlates with distinct dissemination of neoplastic cells in lymphoid organs. Blood 99: 1109-1116 [Abstract] [Full Text]  
• Rassidakis, G. Z., Medeiros, L. J., McDonnell, T. J., Viviani, S., Bonfante, V., Nadali, G., Vassilakopoulos, T. P., Giardini, R., Chilosi, M., Kittas, C., Gianni, A. M., Bonadonna, G., Pizzolo, G., Pangalis, G. A., Cabanillas, F., Sarris, A. H. (2002). BAX Expression in Hodgkin and Reed-Sternberg Cells of Hodgkin's Disease: Correlation with Clinical Outcome. Clin. Cancer Res. 8: 488-493 [Abstract] [Full Text]  
• Torlakovic, E., Tierens, A., Dang, H. D., Delabie, J. (2001). The Transcription Factor PU.1, Necessary for B-Cell Development Is Expressed in Lymphocyte Predominance, But Not Classical Hodgkin's Disease. Am. J. Pathol. 159: 1807-1814 [Abstract] [Full Text]  
• Walton, R. M., Hendrick, M. J. (2001). Feline Hodgkin's-like Lymphoma: 20 Cases (1992-1999). Vet Pathol 38: 504-511 [Abstract] [Full Text]  
• Seitz, V., Hummel, M., Anagnostopoulos, I., Stein, H. (2001). Analysis of BCL-6 mutations in classic Hodgkin disease of the B- and T-cell type. Blood 97: 2401-2405 [Abstract] [Full Text]  
• Franke, S., Wlodarska, I., Maes, B., Vandenberghe, P., Delabie, J., Hagemeijer, A., De Wolf-Peeters, C. (2001). Lymphocyte predominance Hodgkin disease is characterized by recurrent genomic imbalances. Blood 97: 1845-1853 [Abstract] [Full Text]  
• Zemlin, M., Bauer, K., Hummel, M., Pfeiffer, S., Devers, S., Zemlin, C., Stein, H., Versmold, H. T. (2001). The diversity of rearranged immunoglobulin heavy chain variable region genes in peripheral blood B cells of preterm infants is restricted by short third complementarity-determining regions but not by limited gene segment usage. Blood 97: 1511-1513 [Abstract] [Full Text]  
• Brauninger, A., Yang, W., Wacker, H.-H., Rajewsky, K., Kuppers, R., Hansmann, M.-L. (2001). B-cell development in progressively transformed germinal centers: similarities and differences compared with classical germinal centers and lymphocyte-predominant Hodgkin disease. Blood 97: 714-719 [Abstract] [Full Text]  
• Stein, H., Marafioti, T., Foss, H.-D., Laumen, H., Hummel, M., Anagnostopoulos, I., Wirth, T., Demel, G., Falini, B. (2001). Down-regulation of BOB.1/OBF.1 and Oct2 in classical Hodgkin disease but not in lymphocyte predominant Hodgkin disease correlates with immunoglobulin transcription. Blood 97: 496-501 [Abstract] [Full Text]  
• Flavell, K J, Murray, P G (2000). Hodgkin's disease and the Epstein-Barr virus. Mol. Pathol. 53: 262-269 [Abstract] [Full Text]  
• Sotlar, K, Marafioti, T, Griesser, H, Theil, J, Aepinus, C, Jaussi, R, Stein, H, Valent, P, Horny, H-P (2000). Detection of c-kit mutation Asp 816 to Val in microdissected bone marrow infiltrates in a case of systemic mastocytosis associated with chronic myelomonocytic leukaemia. Mol. Pathol. 53: 188-193 [Abstract] [Full Text]  
• Seitz, V., Hummel, M., Marafioti, T., Anagnostopoulos, I., Assaf, C., Stein, H. (2000). Detection of clonal T-cell receptor gamma-chain gene rearrangements in Reed-Sternberg cells of classic Hodgkin disease. Blood 95: 3020-3024 [Abstract] [Full Text]  
• Falini, B., Fizzotti, M., Pucciarini, A., Bigerna, B., Marafioti, T., Gambacorta, M., Pacini, R., Alunni, C., Natali-Tanci, L., Ugolini, B., Sebastiani, C., Cattoretti, G., Pileri, S., Dalla-Favera, R., Stein, H. (2000). A monoclonal antibody (MUM1p) detects expression of the MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells. Blood 95: 2084-2092 [Abstract] [Full Text]  
• Marafioti, T., Hummel, M., Foss, H.-D., Laumen, H., Korbjuhn, P., Anagnostopoulos, I., Lammert, H., Demel, G., Theil, J., Wirth, T., Stein, H. (2000). Hodgkin and Reed-Sternberg cells represent an expansion of a single clone originating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription. Blood 95: 1443-1450 [Abstract] [Full Text]  
• Staudt, L. M. (2000). The Molecular and Cellular Origins of Hodgkin's Disease. JEM 191: 207-212 [Full Text]  
• Marafioti, T., Hummel, M., Anagnostopoulos, I., Foss, H.-D., Huhn, D., Stein, H. (1999). Classical Hodgkin's Disease and Follicular Lymphoma Originating From the Same Germinal Center B Cell. JCO 17: 3804-3809 [Abstract] [Full Text]  
• Kuppers, R., Klein, U., Hansmann, M.-L., Rajewsky, K. (1999). Cellular Origin of Human B-Cell Lymphomas. NEJM 341: 1520-1529 [Full Text]  
• Emmerich, F., Meiser, M., Hummel, M., Demel, G., Foss, H.-D., Jundt, F., Mathas, S., Krappmann, D., Scheidereit, C., Stein, H., Dorken, B. (1999). Overexpression of I Kappa B Alpha Without Inhibition of NF-kappa B Activity and Mutations in the I Kappa B Alpha Gene in Reed-Sternberg Cells. Blood 94: 3129-3134 [Abstract] [Full Text]  
• Stein, K., Hummel, M., Korbjuhn, P., Foss, H.-D., Anagnostopoulos, I., Marafioti, T., Stein, H. (1999). Monocytoid B Cells Are Distinct From Splenic Marginal Zone Cells and Commonly Derive From Unmutated Naive B Cells and Less Frequently From Postgerminal Center B Cells by Polyclonal Transformation. Blood 94: 2800-2808 [Abstract] [Full Text]  
• Hasse, U., Tinguely, M., Leibundgut, E. O., Cajot, J.-F., Garvin, A. M., Tobler, A., Borisch, B., Fey, M. F. (1999). Clonal Loss of Heterozygosity in Microdissected Hodgkin and Reed-Sternberg Cells. JNCI J Natl Cancer Inst 91: 1581-1583 [Full Text]  
• van den Berg, A., Visser, L., Poppema, S. (1999). High Expression of the CC Chemokine TARC in Reed-Sternberg Cells : A Possible Explanation for the Characteristic T-Cell Infiltratein Hodgkin's Lymphoma. Am. J. Pathol. 154: 1685-1691 [Abstract] [Full Text]  
• Brauninger, A., Hansmann, M.-L., Strickler, J. G., Dummer, R., Burg, G., Rajewsky, K., Kuppers, R. (1999). Identification of Common Germinal-Center B-Cell Precursors in Two Patients with Both Hodgkin's Disease and Non-Hodgkin's Lymphoma. NEJM 340: 1239-1247 [Abstract] [Full Text]  
• Brauninger, A., Kuppers, R., Spieker, T., Siebert, R., Strickler, J. G., Schlegelberger, B., Rajewsky, K., Hansmann, M.-L. (1999). Molecular Analysis of Single B Cells From T-Cell-Rich B-Cell Lymphoma Shows the Derivation of the Tumor Cells From Mutating Germinal Center B Cells and Exemplifies Means by Which Immunoglobulin Genes Are Modified in Germinal Center B Cells. Blood 93: 2679-2687 [Abstract] [Full Text]  
• Carbone, A., Gloghini, A., Larocca, L. M., Antinori, A., Falini, B., Tirelli, U., Dalla-Favera, R., Gaidano, G. (1999). Human Immunodeficiency Virus-Associated Hodgkin's Disease Derives From Post-Germinal Center B Cells. Blood 93: 2319-2326 [Abstract] [Full Text]  
• Aster, J. C. (1999). Lymphocyte-Predominant Hodgkin's Disease: How Little Therapy Is Enough?. JCO 17: 744-744 [Full Text]  
• Diehl, V., Sextro, M., Franklin, J., Hansmann, M.-L., Harris, N., Jaffe, E., Poppema, S., Harris, M., Franssila, K., van Krieken, J., Marafioti, T., Anagnostopoulos, I., Stein, H. (1999). Clinical Presentation, Course, and Prognostic Factors in Lymphocyte-Predominant Hodgkin's Disease and Lymphocyte-Rich Classical Hodgkin's Disease: Report From the European Task Force on Lymphoma Project on Lymphocyte-Predominant Hodgkin's Disease. JCO 17: 776-776 [Abstract] [Full Text]  
• Vockerodt, M., Soares, M., Kanzler, H., Kuppers, R., Kube, D., Hansmann, M.-L., Diehl, V., Tesch, H. (1998). Detection of Clonal Hodgkin and Reed-Sternberg Cells With Identical Somatically Mutated and Rearranged VH Genes in Different Biopsies in Relapsed Hodgkin's Disease. Blood 92: 2899-2907 [Abstract] [Full Text]  
• Carbone, A., Gloghini, A., Gaidano, G., Franceschi, S., Capello, D., Drexler, H. G., Falini, B., Dalla-Favera, R. (1998). Expression Status of BCL-6 and Syndecan-1 Identifies Distinct Histogenetic Subtypes of Hodgkin's Disease. Blood 92: 2220-2228 [Abstract] [Full Text]  
• Irsch, J., Nitsch, S., Hansmann, M.-L., Rajewsky, K., Tesch, H., Diehl, V., Jox, A., Kuppers, R., Radbruch, A. (1998). Isolation of viable Hodgkin and Reed-Sternberg cells from Hodgkin disease tissues. Proc. Natl. Acad. Sci. USA 95: 10117-10122 [Abstract] [Full Text]  
• Kuppers, R., Rajewsky, K., Braeuninger, A., Hansmann, M.-L., Hummel, M., Stein, H., Marafioti, T., Anagnostopoulos, I. (1998). L&H Cells in Lymphocyte-Predominant Hodgkin's Disease. NEJM 338: 763-765 [Full Text]  
• Schwartz, R. S. (1997). Hodgkin's Disease -- Time for a Change. NEJM 337: 494-496 [Full Text]