Original Article
Origin of Nodular Lymphocyte-Predominant Hodgkin's Disease from a Clonal Expansion of Highly Mutated Germinal-Center B Cells
N Engl J Med 1997; 337:453-458August 14, 1997
- 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.
Media in This Article
Figure 1
Studies of the Clonal Relation among Eight Lymphocytic and Histiocytic (L&H) Cells Isolated from Patient 11.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.Article Activity
- Article
Lymphocyte-predominant Hodgkin's disease,1-3 an indolent disorder associated with a long survival,4 represents approximately 5 percent of all cases of Hodgkin's disease.5 Its histopathological features include an obliterated lymph-node structure that usually contains small numbers of large, dysplastic cells, designated lymphocytic and histiocytic (L&H) cells, and numerous small lymphocytes. There is a nodular subtype of the disease that occurs frequently and a diffuse subtype that occurs rarely.3 L&H cells have consistently been found to express B-cell markers,6-8 but it is unclear whether lymphocyte-predominant Hodgkin's disease is a monoclonal neoplasm or a premalignant or reactive condition with polyclonal L&H cells. Most studies have not found clonal populations of B cells by the analysis of Southern blots or polymerase-chain-reaction (PCR) products of DNA extracts from tissue or single L&H cells that have been isolated,9-16 but some investigators have demonstrated the expression of monotypic immunoglobulin light chains.17-19 These conflicting data probably result from technical difficulties. We used an improved method of isolating cells from frozen sections of tissue from 11 patients with typical cases of nodular lymphocyte-predominant Hodgkin's disease and analyzed their V H gene rearrangements. Six of these patients were also studied for immunoglobulin light-chain transcripts by in situ hybridization. The two approaches yielded congruent results: the pattern of rearranged V H genes in the L&H cells indicated a monoclonal population, and the cells expressed only one type of light chain.
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 (βF1). 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 1AFigure 1
Studies of the Clonal Relation among Eight Lymphocytic and Histiocytic (L&H) Cells Isolated from Patient 11. and Table 1Table 1Clinical Data and Rearrangements of Immunoglobulin Heavy-Chain Genes in L&H Cells from 11 Patients with Nodular Lymphocyte-Predominant Hodgkin's Disease.). 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.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 2Table 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. 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.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.17-19
Previous studies by single-cell PCR of both the nodular lymphocyte-predominant12,16,28 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,32-34 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.
- References
References
1
Lukes RJ ,Craver LF ,Hall TC ,Rappaport H ,Ruben P . Report of the Nomenclature Committee. Cancer Res 1966;26:1311-1311
Web of Science2
Jackson H Jr ,Parker F Jr . Hodgkin's disease. I. General considerations. N Engl J Med 1944;230:1-8
Full Text | Web of Science3
Lukes RJ ,Butler JJ . The pathology and nomenclature of Hodgkin's disease. Cancer Res 1966;26:1063-1083
Web of Science | Medline4
Regula DP Jr ,Hoppe RT ,Weiss LM . Nodular and diffuse types of lymphocyte predominance Hodgkin's disease. N Engl J Med 1988;318:214-219
Full Text | Web of Science | Medline5
Colby TV ,Hoppe RT ,Warnke RA . Hodgkin's disease: a clinicopathologic study of 659 cases. Cancer 1982;49:1848-1858
CrossRef | Web of Science | Medline6
Stein H ,Hansmann ML ,Lennert K ,Brandtzaeg P ,Gatter KC ,Mason DY . Reed-Sternberg and Hodgkin cells in lymphocyte-predominant Hodgkin's disease of nodular subtype contain J chain. Am J Clin Pathol 1986;86:292-297
Web of Science | Medline7
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 L26, a pan B cell marker. Am J Pathol 1988;133:211-217
Web of Science | Medline8
Korkolopoulou P ,Cordell J ,Jones M , et al. The expression of the B-cell marker mb-1 (CD79a) in Hodgkin's disease. Histopathology 1994;24:511-515
CrossRef | Web of Science | Medline9
Angel CA ,Pringle JH ,Naylor J ,West KP ,Lauder I . Analysis of antigen receptor genes in Hodgkin's disease. J Clin Pathol 1993;46:337-340
CrossRef | Web of Science | Medline10
Angel CA ,Pringle JH ,Primrose L ,Lauder I . Detection of immunoglobulin heavy chain gene rearrangements in Hodgkin's disease using PCR. J Clin Pathol 1993;46:940-942
CrossRef | Web of Science | Medline11
Pan LX ,Diss TC ,Peng HZ ,Norton AJ ,Isaacson PG . Nodular lymphocyte predominance Hodgkin's disease: a monoclonal or polyclonal B-cell disorder? Blood 1996;87:2428-2434
Web of Science | Medline12
Delabie J ,Tierens A ,Wu G ,Weisenburger DD ,Chan WC . Lymphocyte predominance Hodgkin's disease: lineage and clonality determination using a single-cell assay. Blood 1994;84:3291-3298
Web of Science | Medline13
Wickert RS ,Weisenburger DD ,Tierens A ,Greiner TC ,Chan WC . Clonal relationship between lymphocytic predominance Hodgkin's disease and concurrent or subsequent large-cell lymphoma of B lineage. Blood 1995;86:2312-2320
Web of Science | Medline14
Greiner TC ,Gascoyne RD ,Anderson ME , et al. Nodular lymphocyte-predominant Hodgkin's disease associated with large-cell lymphoma: analysis of Ig gene rearrangements by V-J polymerase chain reaction. Blood 1996;88:657-666
Web of Science | Medline15
Tamaru J ,Hummel M ,Zemlin M ,Kalvelage B ,Stein H . Hodgkin's disease with a B-cell phenotype often shows a VDJ rearrangement and somatic mutations in the VH genes. Blood 1994;84:708-715
Web of Science | Medline16
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 | Medline17
Stoler MH ,Nichols GE ,Symbula M ,Weiss LM . Lymphocyte predominance Hodgkin's disease: evidence for a kappa light chain-restricted monotypic B-cell neoplasm. Am J Pathol 1995;146:812-818
Web of Science | Medline18
Hell K ,Hansmann ML ,Pringle JH ,Lauder I ,Fischer R . Combination of Hodgkin's disease and diffuse large cell lymphoma: an in situ hybridization study for immunoglobulin light chain messenger RNA. Histopathology 1995;27:491-499
CrossRef | Web of Science | Medline19
Schmid C ,Sargent C ,Isaacson PG . L and H cells of nodular lymphocyte predominant Hodgkin's disease show immunoglobulin light-chain restriction. Am J Pathol 1991;139:1281-1289
Web of Science | Medline20
Harris NL ,Jaffe ES ,Stein H , et al. A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 1994;84:1361-1392
Web of Science | Medline21
Kanzler H, Küppers R, Hansmann ML, Rajewsky K. Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells.J Exp Med 1996;184:1495-505.
22
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 | Medline23
Marks JD ,Tristem M ,Karpas A ,Winter G . Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes. Eur J Immunol 1991;21:985-991
CrossRef | Web of Science | Medline24
Ramasamy I ,Brisco M ,Morley A . Improved PCR method for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell neoplasms. J Clin Pathol 1992;45:770-775
CrossRef | Web of Science | Medline25
Hummel M ,Tamaru J ,Kalvelage B ,Stein H . Mantle cell (previously centrocytic) lymphomas express VH genes with no or very little somatic mutations like the physiologic cells of the follicle mantle. Blood 1994;84:403-407
Web of Science | Medline26
Cook GP ,Tomlinson IM . The human immunoglobulin VH repertoire. Immunol Today 1995;16:237-242
CrossRef | Medline27
Foss HD ,Herbst H ,Oelmann E , et al. Lymphotoxin, tumour necrosis factor and interleukin-6 gene transcripts are present in Hodgkin and Reed-Sternberg cells of most Hodgkin's disease cases. Br J Haematol 1993;84:627-635
CrossRef | Web of Science | Medline28
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 | Medline29
Pascual V, Liu YJ, Magalski A, de Bouteiller O, Banchereau J, Capra JD. Analysis of somatic mutation in five B cell subsets of human tonsil.J Exp Med 1994;180:329-39.
30
Rajewsky K . Clonal selection and learning in the antibody system. Nature 1996;381:751-758
CrossRef | Web of Science | Medline31
Hansmann ML ,Fellbaum C ,Hui PK ,Moubayed P . Progressive transformation of germinal centers with and without association to Hodgkin's disease. Am J Clin Pathol 1990;93:219-226
Web of Science | Medline32
Sundeen JT ,Cossman J ,Jaffe ES . Lymphocyte predominant Hodgkin's disease nodular subtype with coexistent “large cell lymphoma“: histological progression or composite malignancy? Am J Surg Pathol 1988;12:599-606
Web of Science | Medline33
Ree HJ ,Neiman RS ,Martin AW ,Dallenbach F ,Stein H . Paraffin section markers for Reed-Sternberg cells: a comparative study of peanut agglutinin, Leu-M1, LN-2, and Ber-H2. Cancer 1989;63:2030-2036
CrossRef | Web of Science | Medline34
Falini B ,Bigerna B ,Pasqualucci L , et al. Distinctive expression pattern of the BCL-6 protein in nodular lymphocyte predominance Hodgkin's disease. Blood 1996;87:465-471
Web of Science | Medline
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1
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CrossRef16
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CrossRef20
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CrossRef21
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CrossRef22
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CrossRef25
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CrossRef26
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CrossRef27
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CrossRef34
Gulfaraz Khan. (2006) Epstein-Barr virus, cytokines, and inflammation: A cocktail for the pathogenesis of Hodgkin's lymphoma?. Experimental Hematology 34:4, 399-406
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Andrew Wirth, Kally Yuen, Michael Barton, Daniel Roos, Kumar Gogna, Gary Pratt, Craig MacLeod, Sean Bydder, Graeme Morgan, David Christie. (2005) Long-term outcome after radiotherapy alone for lymphocyte-predominant Hodgkin lymphoma. Cancer 104:6, 1221-1229
CrossRef36
K. Willenbrock, C. Renné, A. Bräuninger, M.-L. Hansmann. (2005) Pathologie des Hodgkin-Lymphoms. Der Onkologe 11:9, 917-923
CrossRef37
Christoph Renné, José Ignacio Martín-Subero, Martin-Leo Hansmann, Reiner Siebert. (2005) Molecular Cytogenetic Analyses of Immunoglobulin Loci in Nodular Lymphocyte Predominant Hodgkin's Lymphoma Reveal a Recurrent IGH-BCL6 Juxtaposition. The Journal of Molecular Diagnostics 7:3, 352-356
CrossRef38
Timothy J. Sullivan, Kevin Whitehead, Richard Williamson, David Grimes, David Schlect, Ian Brown, Graeme Dickie. (2005) Lymphoproliferative Disease of the Ocular Adnexa: A Clinical and Pathologic Study With Statistical Analysis of 69 Patients. Ophthalmic Plastic & Reconstructive Surgery 21:3, 177-188
CrossRef39
Sarah E Coupland, Frederic Charlotte, George Mansour, Karim Maloum, Michael Hummel, Harald Stein. (2005) HHV-8-Associated T-Cell Lymphoma in a Lymph Node With Concurrent Peritoneal Effusion in an HIV-Positive Man. The American Journal of Surgical Pathology 29:5, 647-652
CrossRef40
Philip Brown, Teresa Marafioti, Rajko Kusec, Alison H. Banham. (2005) The FOXP1 Transcription Factor is Expressed in the Majority of Follicular Lymphomas but is Rarely Expressed in Classical and Lymphocyte Predominant Hodgkin’s Lymphoma. Journal of Molecular Histology 36:4, 249-256
CrossRef41
David M. Dorfman, Eun Sook Hwang, Aliakbar Shahsafaei, Laurie H. Glimcher. (2005) T-bet, a T cell–associated transcription factor, is expressed in Hodgkin's lymphoma. Human Pathology 36:1, 10-15
CrossRef42
Eva-Maria Schneider, Emina Torlakovic, Albert Stühler, Volker Diehl, Hans Tesch, Bernd Giebel. (2004) The early transcription factor GATA-2 is expressed in classical Hodgkin's lymphoma. The Journal of Pathology 204:5, 538-545
CrossRef43
P Lin, L J Medeiros, R B Wilder, L V Abruzzo, J T Manning, D Jones. (2004) The activation profile of tumour-associated reactive T-cells differs in the nodular and diffuse patterns of lymphocyte predominant Hodgkin's disease. Histopathology 44:6, 561-569
CrossRef44
Joseph D Khoury, Dan Jones, Marwan A Yared, John T Manning, Lynne V Abruzzo, Fredrick B Hagemeister, L Jeffrey Medeiros. (2004) Bone Marrow Involvement in Patients With Nodular Lymphocyte Predominant Hodgkin Lymphoma. The American Journal of Surgical Pathology 28:4, 489-495
CrossRef45
Hitoshi Hanamoto, Takashi Nakayama, Hajime Miyazato, Sumio Takegawa, Kunio Hieshima, Yoichi Tatsumi, Akihisa Kanamaru, Osamu Yoshie. (2004) Expression of CCL28 by Reed-Sternberg Cells Defines a Major Subtype of Classical Hodgkin's Disease with Frequent Infiltration of Eosinophils and/or Plasma Cells. The American Journal of Pathology 164:3, 997-1006
CrossRef46
Roman K Thomas, Daniel Re, Jürgen Wolf, Volker Diehl. (2004) Part I: Hodgkin's lymphoma—molecular biology of Hodgkin and Reed-Sternberg cells. The Lancet Oncology 5:1, 11-18
CrossRef47
Jinfen Wang, Clive R. Taylor. (2003) Apoptosis and Cell Cycle-related Genes and Proteins in Classical Hodgkin Lymphoma. Applied Immunohistochemistry & Molecular Morphology206-213
CrossRef48
Theodora Papadaki, Kostas Stamatopoulos. (2003) Hodgkin disease immunopathogenesis: long-standing questions, recent answers, further directions. Trends in Immunology 24:9, 508-511
CrossRef49
G Pillai, F Pezzella, K Gatter. (2003) 'Follicular' pattern of bone marrow involvement in lymphocyte-predominant Hodgkin's disease. Histopathology 43:2, 203-205
CrossRef50
Cabot, Richard C.Harris, Nancy Lee, McNeely, William F., Shepard, Jo-Anne O., Ebeling, Sally H.Ellender, Stacey M.Peters, Christine C., Lehmann, Leslie E., Anupindi, Sudha, Harris, Nancy Lee, . (2003) Case 18-2003. New England Journal of Medicine 348:24, 2443-2451
Full Text51
Patricia Uherova, MD, Riccardo Valdez, MD, Charles W. Ross, MD, Bertram Schnitzer, MD, William G. Finn, MD. (2003) Nodular Lymphocyte Predominant Hodgkin Lymphoma: An Immunophenotypic Reappraisal Based on a Single-Institution Experience. American Journal of Clinical Pathology 119:2, 192-198
CrossRef52
Riccardo Valdez, MD, William G. Finn, MD, Patricia Uherova, MD, Bertram Schnitzer, MD, Charles W. Ross, MD. (2003) Nodular Lymphocyte Predominant Hodgkin Lymphoma: An Immunophenotypic Reappraisal Based on a Single-Institution Experience. American Journal of Clinical Pathology 119:2, 192
CrossRef53
Jaap M. Middeldorp, Antoinette A.T.P Brink, Adriaan J.C van den Brule, Chris J.L.M Meijer. (2003) Pathogenic roles for Epstein–Barr virus (EBV) gene products in EBV-associated proliferative disorders. Critical Reviews in Oncology/Hematology 45:1, 1-36
CrossRef54
Bradley C. Ekstrand, Sandra J. Horning. (2002) Lymphocyte predominant Hodgkin’s disease. Current Oncology Reports 4:5, 424-433
CrossRef55
Takehiko Mori, Junichi Tamaru, Michihide Tokuhira, Shigehisa Mori, Hiroshi Suzuki, Toru Abe, Tsutomu Takeuchi. (2002) Rapid Proliferation of a Different Clone Early after Autologous Peripheral Blood Stem Cell Transplantation for Non-Hodgkin’s Lymphoma. International Journal of Hematology 76:3, 287-289
CrossRef56
M Fraga, A García-Rivero, L Sánchez-Verde, J Forteza, M A Piris. (2002) T-cell/histiocyte-rich large B-cell lymphoma is a disseminated aggressive neoplasm: differential diagnosis from Hodgkin's lymphoma. Histopathology 41:3, 216-229
CrossRef57
Naoya Nakamura, Naoyoshi Mori, Koichi Ohshima, Tetsuo Kuze, Yuko Hashimoto, Yoshikazu Sasaki, Asumi Shirakawa, Michiko Sato, Masafumi Abe. (2002) Epstein-Barr Virus–Positive Hodgkin/Reed-Sternberg–Like B Cell in Non-Hodgkin Lymphoma: Nucleotide Sequence of the Amplified Immunoglobulin Heavy-Chain Variable Region Gene by the Single-Cell Polymerase Chain Reaction Technique. Diagnostic Molecular Pathology 11:2, 83-89
CrossRef58
Paul J. Kurtin. (2002) Lymphocyte Predominant Hodgkin Lymphoma. Pathology Case Reviews 7:3, 97-104
CrossRef59
Antonino Carbone, Annunziata Gloghini, Donatella Aldinucci, Valter Gattei, Riccardo Dalla-Favera, Gianluca Gaidano. (2002) Expression pattern of MUM1/IRF4 in the spectrum of pathology of Hodgkin's disease. British Journal of Haematology 117:2, 366-372
CrossRef60
Dan Jones. (2002) Dismantling the Germinal Center: Comparing the Processes of Transformation, Regression, and Fragmentation of the Lymphoid Follicle. Advances in Anatomic Pathology 9:2, 129-138
CrossRef61
Steven H. Kroft, MD. (2001) Lymphoma Transformation. American Journal of Clinical Pathology 116:6, 811-814
CrossRef62
Emina Torlakovic, Anne Tierens, Hien D. Dang, Jan Delabie. (2001) The Transcription Factor PU.1, Necessary for B-Cell Development Is Expressed in Lymphocyte Predominance, But Not Classical Hodgkin’s Disease. The American Journal of Pathology 159:5, 1807-1814
CrossRef63
Dennis D. Weisenburger, Grant Wu, MD, James Z. Huang, MD, Wing C. Chan, MD, Kwang Hwa Park, MD, Toshiyuki Ohno, MD. (2001) The Tumor Cells in Nodular Lymphocyte-Predominant Hodgkin Disease Are Clonally Related to the Large Cell Lymphoma Occurring in the Same Individual: Direct Demonstration by Single Cell Analysis. American Journal of Clinical Pathology 116:4, 506-511
CrossRef64
Lorenzo Cerroni, Helmut Kerl. (2001) Cutaneous Follicle Center Cell Lymphoma, Follicular Type. The American Journal of Dermatopathology 23:4, 370-373
CrossRef65
Eric D. Hsi, Subramanian Yegappan. (2001) Lymphoma Immunophenotyping: A New Era in Paraffin-Section Immunohistochemistry. Advances in Anatomic Pathology 8:4, 218-239
CrossRef66
Ourania Nicolatou-Galitis, Theodora Papadaki, Maria Moshovi, Joanna J. Kamma, Cathrine Van Vliet-Constantinidou, Constantine Tsoumakas, Antonis Kattamis, Fotini Tzortzatou-Stathopoulou. (2001) Gingival Overgrowth as the Initial Paraneoplastic Manifestation of Hodgkin's Lymphoma in a Child. A Case Report. Journal of Periodontology 72:1, 107-112
CrossRef67
Hitoshi Kiyoi, Tomoki Naoe. (2001) Immunoglobulin variable region structure and B-Cell malignancies. International Journal of Hematology 73:1, 47-53
CrossRef68
Tetuo Kuze, Naoya Nakamura, Yuko Hashimoto, Yoshikazu Sasaki, Masafumi Abe. (2000) The Characteristics of Epstein-Barr Virus (EBV)-positive Diffuse Large B-Cell Lymphoma: Comparison between EBV+ and EBV- Cases in Japanese Population. Cancer Science 91:12, 1233-1240
CrossRef69
G.A. Hale, G.L. Phillips. (2000) Allogeneic stem cell transplantation for the non-Hodgkin’s lymphomas and Hodgkin’s disease. Cancer Treatment Reviews 26:6, 411-427
CrossRef70
Michael Hummel, Harald Stein. (2000) Clinical relevance of immunoglobulin mutation analysis. Current Opinion in Oncology 12:5, 395-402
CrossRef71
A CHOTT, M RADERER. (2000) New developments in extracutaneous lymphomas. Seminars in Cutaneous Medicine and Surgery 19:2, 149-156
CrossRef72
P. G. Isaacson. (2000) The Current Status of Lymphoma Classification. British Journal of Haematology 109:2, 258-266
CrossRef73
Watanabe, Yamashita, Nakayama, Hasegawa, Kojima, Nagasawa, Mori. (2000) Varied B-cell immunophenotypes of Hodgkin/Reed-Sternberg cells in classic Hodgkin's disease. Histopathology 36:4, 353-361
CrossRef74
Nora C. Pirisi-Hauck, Hans-Dieter Foss, Juergen Baier, Sandor Kurunczi. (2000) Simultaneous Occurrence of Autoimmune Enteropathy and Recurrent Deep Venous Thrombosis. Journal of Pediatric Gastroenterology and Nutrition 30:3, 324-329
CrossRef75
L. M. Staudt. (2000) The Molecular and Cellular Origins of Hodgkin's Disease. Journal of Experimental Medicine 191:2, 207-212
CrossRef76
Maurice P. H. M. Jansen, Anton H. N. Hopman, Fredrik J. Bot, Annick Haesevoets, Marian J. P. L. Stevens-Kroef, Jan Willem Arends, Andrea Jox, J
rgen Wolf, Frans C. S. Ramaekers, Harry C. Schouten. (1999) Morphologically normal, CD30-negative B-lymphocytes with chromosome aberrations in classical Hodgkin's disease: the progenitor cell of the malignant clone?. The Journal of Pathology 189:4, 527-532
CrossRef77
Epstein, Franklin H., , Küppers, Ralf, Klein, Ulf, Hansmann, Martin-Leo, Rajewsky, Klaus, . (1999) Cellular Origin of Human B-Cell Lymphomas. New England Journal of Medicine 341:20, 1520-1529
Full Text78
Michele Spina, Simonetta Sandri, Umberto Tirelli. (1999) Hodgkin’s disease in HIV-infected individuals. Current Opinion in Oncology 11:6, 522
CrossRef79
Kevin G Kempers, Joseph W Foote, Robert L Zimmerman, John K Choi. (1999) Hodgkin's disease presenting as a submental mass. Journal of Oral and Maxillofacial Surgery 57:11, 1363-1366
CrossRef80
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
CrossRef81
Andreas Sarris, Richard Ford. (1999) Recent advances in the molecular pathogenesis of lymphomas. Current Opinion in Oncology 11:5, 351
CrossRef82
A. van den Berg, L. Visser, S. Poppema. (1999) High Expression of the CC Chemokine TARC in Reed-Sternberg Cells. The American Journal of Pathology 154:6, 1685-1691
CrossRef83
Manuela Födinger, Karin Winkler, Christine Mannhalter, Andreas Chott. (1999) Combined Polymerase Chain Reaction Approach for Clonality Detection in Lymphoid Neoplasms. Diagnostic Molecular Pathology 8:2, 80-91
CrossRef84
Bräuninger, Andreas, Hansmann, Martin-Leo, Strickler, John G., Dummer, Reinhard, Burg, Günther, Rajewsky, Klaus, Küppers, Ralf, . (1999) Identification of Common Germinal-Center B-Cell Precursors in Two Patients with Both Hodgkin's Disease and Non-Hodgkin's Lymphoma. New England Journal of Medicine 340:16, 1239-1247
Full Text85
Anne F. Lauritzen, Peter Horn Møller, Trine Nedergaard, Per Guldberg, Klaus Hou-Jensen, Elisabeth Ralfkiaer. (1999) Apoptosis-related genes and proteins in Hodgkin's disease. APMIS 107:7-12, 636-644
CrossRef86
Roberto Chiarle, Antonello Podda, Gabriel Prolla, Jerry Gong, G.Jeanette Thorbecke, Giorgio Inghirami. (1999) CD30 in Normal and Neoplastic Cells. Clinical Immunology 90:2, 157-164
CrossRef87
Jens Krugmann, Hans Feichtinger, Richard Greil, Falko Fend. (1999) Thymic Hodgkin's Disease — A Histological and Immunohistochemical Study of Three Cases. Pathology - Research and Practice 195:10, 681-687
CrossRef88
Jo Spencer, Deborah K. Dunn-Walters. (1999) Somatic hypermutation and B-cell malignancies. The Journal of Pathology 187:2, 158-163
CrossRef89
Thomas Rüdiger, German Ott, Maria Michaela Ott, Sigrid Maria Müller-Deubert, Hans Konrad Müller-Hermelink. (1998) Differential Diagnosis Between Classic Hodgkin's Lymphoma, T-Cell-Rich B-Cell Lymphoma, and Paragranuloma by Paraffin Immunohistochemistry. The American Journal of Surgical Pathology 22:10, 1184-1191
CrossRef90
Kevin Hollowood, John R. Goodlad. (1998) Germinal centre cell kinetics. The Journal of Pathology 185:3, 229-233
CrossRef91
Christos Kosmas, Kostas Stamatopoulos, Theodora Papndoki, Chrysoula Belessi, Xenophon Yataganas, Dimitra Anagnostou, Dimitris Loukopoulos. (1998) Somatic hypermutation of immunoglobulin variable region genes: focus on follicular lymphoma and multiple myeloma. Immunological Reviews 162:1, 281-292
CrossRef92
Ulf Klien, Tina Goasens, Motthias Fischer, Holger Kanzler, Andreas Braeuninger, Klaus Rajewsky, Ralf Kuppers. (1998) Somatic hypermutation in normal and transformed human B cells. Immunological Reviews 162:1, 261-280
CrossRef93
Audrey E. Evans, Margaret H. Collins, Eli Glatstein, G. Peter Feola. (1998) Hodgkin disease?. Medical and Pediatric Oncology 30:4, 252-258
CrossRef94
Freda Stevenson, Surinder Sahota, Delin Zhu, Christion Ottensmeier, Caroline Chapman, David Oscier, Terry Hamblin. (1998) Insight into the origin and clonal history of B-cell tumors as revealed by analysis of immunoglobulin variable region genes. Immunological Reviews 162:1, 247-259
CrossRef95
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
CrossRef96
(1998) L&H Cells in Lymphocyte-Predominant Hodgkin's Disease. New England Journal of Medicine 338:11, 763-765
Full Text97
Andrea Jox, Jürgen Wolf, Volker Diehl. (1997) Hodgkin's disease biology: recent advances. Hematological Oncology 15:4, 165-171
CrossRef98
Schwartz, Robert S., . (1997) Hodgkin's Disease — Time for a Change. New England Journal of Medicine 337:7, 495-496
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