Primary Cutaneous Follicle Center Cell Lymphomas and Large B Cell Lymphomas of the Leg Descend from Germinal Center Cells. A Single Cell Polymerase Chain Reaction Analysis
2001; Elsevier BV; Volume: 117; Issue: 6 Linguagem: Inglês
10.1046/j.0022-202x.2001.01543.x
ISSN1523-1747
AutoresSylke Gellrich, Sascha Rutz, Sven Golembowski, C.Y. onder red. van E. Jacobs, Malgorzata von Zimmermann, Pamela Lorenz, Heike Audring, Marcus Muche, Wolfram Sterry, Sigbert Jahn,
Tópico(s)Nail Diseases and Treatments
ResumoPrimary cutaneous B cell lymphomas are defined as non-Hodgkin lymphomas that occur in the skin without extracutaneous involvement for 6 mo after diagnosis. They are characterized by a less aggressive course and better prognosis than their nodal counterparts. According to the European Organization for Research and Treatment of Cancer classification, the major subentities of primary cutaneous B cell lymphoma are follicle center cell lymphomas, immunocytomas, and large B cell lymphomas of the leg, which differ considerably regarding their clinical behavior, the former two being indolent, the latter being of intermediate malignancy. In this study, we applied a single cell polymerase chain reaction approach to analyze immunoglobulin VH/VL genes in 532 individual B lymphocytes from histologic sections of four follicle center cell lymphomas localized on the head and trunk, and four large B cell lymphomas on the leg. We found: (i) in six of eight patients a clonal heavy chain, and in seven of eight patients a clonal light chain rearrangement, all being potentially productive; (ii) no bias in VH gene usage, in four of seven light chain rearrangements the Vκ germline gene IGVK3-20*1 was used; (iii) no biallelic rearrangements; (iv) all VH/VL genes are extensively mutated (mutation rate 5.4–16.3%); (v) intraclonal diversity in six of eight cases (three of each group); and (vi) low replacement vs silent mutation ratios in framework regions indicating preservation of antigen-receptor structure, as in normal B cells selected for antibody expression. Our data indicate a germinal center cell origin of primary cutaneous follicle center cell lymphomas and large B cell lymphomas independent of those belonging to one of these subentities. Primary cutaneous B cell lymphomas are defined as non-Hodgkin lymphomas that occur in the skin without extracutaneous involvement for 6 mo after diagnosis. They are characterized by a less aggressive course and better prognosis than their nodal counterparts. According to the European Organization for Research and Treatment of Cancer classification, the major subentities of primary cutaneous B cell lymphoma are follicle center cell lymphomas, immunocytomas, and large B cell lymphomas of the leg, which differ considerably regarding their clinical behavior, the former two being indolent, the latter being of intermediate malignancy. In this study, we applied a single cell polymerase chain reaction approach to analyze immunoglobulin VH/VL genes in 532 individual B lymphocytes from histologic sections of four follicle center cell lymphomas localized on the head and trunk, and four large B cell lymphomas on the leg. We found: (i) in six of eight patients a clonal heavy chain, and in seven of eight patients a clonal light chain rearrangement, all being potentially productive; (ii) no bias in VH gene usage, in four of seven light chain rearrangements the Vκ germline gene IGVK3-20*1 was used; (iii) no biallelic rearrangements; (iv) all VH/VL genes are extensively mutated (mutation rate 5.4–16.3%); (v) intraclonal diversity in six of eight cases (three of each group); and (vi) low replacement vs silent mutation ratios in framework regions indicating preservation of antigen-receptor structure, as in normal B cells selected for antibody expression. Our data indicate a germinal center cell origin of primary cutaneous follicle center cell lymphomas and large B cell lymphomas independent of those belonging to one of these subentities. primary cutaneous B cell lymphomas Primary cutaneous B cell lymphomas (PCBCL) occur in the skin without evidence of internal involvement for a period of at least 6 mo after diagnosis (Burg et al., 1977Burg G. Rodt H. Grosse-Wilde H. Netzel B. Fateh-Moghadam A. Braun-Falco O. Enzyme cytochemical and immunocytological studies as a basis for the revaluation of the histology of cutaneous B-cell and T-cell lymphomas.Hamatol Bluttransfus. 1977; 20: 179-187Google Scholar;Kerl and Burg, 1979Kerl H. Burg G. Immunocytomas and immunoblastic lymphomas of the skin.Hautarzt. 1979; 30: 666-672PubMed Google Scholar). Although histologic parameters are comparable, the primary cutaneous lymphomas differ from their nodal counterparts in a more indolent clinical behavior resulting in a more favorable prognosis, and in a better response to less aggressive treatment strategies (Willemze et al., 1997Willemze R. Kerl H. Sterry W. et al.EORTC classification for primary cutaneous lymphomas. a proposal from the Cutaneous Lymphoma Study Group of the European Organization for Research and Treatment of Cancer.Blood. 1997; 90: 354-371PubMed Google Scholar). The group of cutaneous B cell lymphomas is heterogeneous in itself. The lesions of large B cell lymphomas appearing initially on the lower leg in elderly patients contain a higher proportion of large bcl-2 positive centroblasts and less infiltrating reactive T cells. This subentity is characterized by a more aggressive clinical course in comparison with follicle center cell lymphomas localized in head and trunk areas (Vermeer et al., 1996Vermeer M.H. Geelen F.A. van Haselen C.W.V.V. Geerts M.L. van Vloten W.A. Willemze R. Primary cutaneous large B-cell lymphomas of the legs. A distinct type of cutaneous B-cell lymphoma with an intermediate prognosis. Dutch Cutaneous Lymphoma Working Group.Arch Dermatol. 1996; 132: 1304-1308Crossref PubMed Google Scholar;Geelen et al., 1998Geelen F.A. Vermeer M.H. Meijer C.J. Van der Putte S.C. Kerkhof E. Kluin P.M. Willemze R. bcl-2 protein expression in primary cutaneous large B-cell lymphoma is site-related.J Clin Oncol. 1998; 16: 2080-2085PubMed Google Scholar;Bekkenk et al., 1999Bekkenk M.W. Vermeer M.H. Geerts M.L. et al.Treatment of multifocal primary cutaneous B-cell lymphoma: a clinical follow-up study of 29 patients.J Clin Oncol. 1999; 17: 2471PubMed Google Scholar). The latter represent another type of PCBCL histologically characterized by nodular or diffuse infiltrates consisting of variable proportions of centrocytes, a few centroblasts, and many reactive T cells (Willemze et al., 1997Willemze R. Kerl H. Sterry W. et al.EORTC classification for primary cutaneous lymphomas. a proposal from the Cutaneous Lymphoma Study Group of the European Organization for Research and Treatment of Cancer.Blood. 1997; 90: 354-371PubMed Google Scholar). During ontogeny, the B cell's antigen receptor is subject to multiple alterations. Naive B cells carry surface immunoglobulins encoded by unmutated, germline-identical immunoglobulin genes (Hummel et al., 1994Hummel 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: 403-407PubMed Google Scholar). In response to antigen presentation by follicular dendritic cells within the germinal centers, B cells proliferate. As recognition of antigen provides signals for growth and T cell help, those B cells become selected to produce antibodies, which have the highest affinity to bind this antigen (Lindhout et al., 1997Lindhout E. Koopman G. Pals S.T. de Groot C. Triple check for antigen specificity of B cells during germinal centre reactions.Immunol Today. 1997; 18: 573-577Abstract Full Text PDF PubMed Scopus (90) Google Scholar). The maturation of affinity is based on amino acid replacements within the antigen binding regions of the immunoglobulin molecule (Berek et al., 1991Berek C. Berger A. Apel M. Maturation of the immune response in germinal centers.Cell. 1991; 67: 1121-1129Abstract Full Text PDF PubMed Scopus (730) Google Scholar;Jacob et al., 1991Jacob J. Kelsoe G. Rajewsky K. Weiss U. Intraclonal generation of antibody mutants in germinal centres.Nature. 1991; 354: 389-392Crossref PubMed Scopus (870) Google Scholar) introduced in the process of somatic hypermutation during proliferation inside the germinal centers (for review seeRajewsky, 1996Rajewsky K. Clonal selection and learning in the antibody system.Nature. 1996; 381: 751-758Crossref PubMed Scopus (1336) Google Scholar). Therefore, conclusions can be drawn from the mutational pattern of immunoglobulin genes in a B cell clone concerning the maturational state of B lymphocytes and the descending lymphomas. When tumors descend from germinal center B cells, the VH/VL genes show a characteristic mutational pattern. As the process of somatic hypermutation is still under way, analysis of the individual VH/VL genes shows a certain degree of intraclonal diversity (Bahler and Levy, 1992Bahler D.W. Levy R. Clonal evolution of a follicular lymphoma: evidence for antigen selection.Proc Natl Acad Sci USA. 1992; 89: 6770-6774Crossref PubMed Scopus (214) Google Scholar;Stamatopoulos et al., 2000Stamatopoulos K. Kosmas C. Belessi C. Stavroyianni N. Kyriazopoulos P. Papadaki T. Molecular insights into the immunopathogenesis of follicular lymphoma.Immunol Today. 2000; 21: 298-305Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Somatically mutated variable region genes in the absence of intraclonal diversity (i.e., the mutational process has stopped) is typical for postgerminal center B cells and the descending tumors. Accordingly, previous studies could ascribe mantle cell lymphomas (Hummel et al., 1994Hummel 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: 403-407PubMed Google Scholar) to a pregerminal center cell origin because of the predominance of unmutated immunoglobulin genes, whereas other NHL entities (for review seeMüller-Hermelink and Greiner, 1998Müller-Hermelink H.K. Greiner A. Molecular analysis of human immunoglobulin heavy chain variable genes (IgVH) in normal and malignant B cells.Am J Pathol. 1998; 153: 1341-1346Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar;Küppers et al., 1999Küppers R. Klein U. Hansmann M.L. Rajewsky K. Cellular origin of human B-cell lymphomas.N Engl J Med. 1999; 341: 1520-1529Crossref PubMed Scopus (555) Google Scholar;Spencer and Dunn-Walters, 1999Spencer J.O. Dunn-Walters D.K. Somatic hypermutation and B-cell malignancies.J Pathol. 1999; 187: 158-163Crossref PubMed Scopus (19) Google Scholar) expressing mutated immunoglobulin genes represent the expansion of germinal center cells, as in follicle center cell lymphomas (Bahler and Levy, 1992Bahler D.W. Levy R. Clonal evolution of a follicular lymphoma: evidence for antigen selection.Proc Natl Acad Sci USA. 1992; 89: 6770-6774Crossref PubMed Scopus (214) Google Scholar;Zhu et al., 1994Zhu D. Hawkins R.E. Hamblin T.J. Stevenson F.K. Clonal history of a human follicular lymphoma as revealed in the immunoglobulin variable region genes.Br J Haematol. 1994; 86: 505-512Crossref PubMed Scopus (92) Google Scholar), or of postgerminal center cells as it is the case in multiple myelomas (Bakkus et al., 1992Bakkus M.H. Van Heirman C.R.I. Van Camp B. Thielemans K. Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation.Blood. 1992; 80: 2326-2335PubMed Google Scholar) and monocytoid B cell lymphomas (Küppers et al., 1996Küppers R. Hajadi M. Plank L. Rajewsky K. Hansmann M.L. Molecular Ig gene analysis reveals that monocytoid B cell lymphoma is a malignancy of mature B cells carrying somatically mutated V region genes and suggests that rearrangement of the kappa-deleting element (resulting in deletion of the Ig kappa enhancers) abolishes somatic hypermutation in the human.Eur J Immunol. 1996; 26: 1794-1800Crossref PubMed Scopus (54) Google Scholar). Recent data byAarts et al., 1998Aarts W.M. Willemze R. Bende R.J. Meijer C.J. Pals S.T. van Noesel C.J. VH gene analysis of primary cutaneous B-cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.Blood. 1998; 92: 3857-3864PubMed Google Scholar, for PCBCL indicate a germinal center cell origin of these skin-located neoplasias. Until recently, different molecular biologic techniques were used to study the immunoglobulin gene expression in malignant B cells, such as mRNA libraries (Aarts et al., 1998Aarts W.M. Willemze R. Bende R.J. Meijer C.J. Pals S.T. van Noesel C.J. VH gene analysis of primary cutaneous B-cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.Blood. 1998; 92: 3857-3864PubMed Google Scholar), genomic gene libraries (Hammer et al., 1993Hammer E. Sangueza O. Suwanjindar P. White C.R.J. Braziel R.M. Immunophenotypic and genotypic analysis in cutaneous lymphoid hyperplasias.J Am Acad Dermatol. 1993; 28: 426-433Abstract Full Text PDF PubMed Scopus (49) Google Scholar), and microdissection techniques followed by polymerase chain reaction (PCR) of genomic bulk DNA and hybridization techniques (Cerroni et al., 2000Cerroni L. Arzberger E. Pütz B. et al.Primary cutaneous follicle center cell lymphoma with follicular growth pattern.Blood. 2000; 95: 3922-3928PubMed Google Scholar). All these techniques have disadvantages due to artificial immunoglobulin gene combination, PCR errors, or low hybridization probe specificity. Such problems can be overcome by applying a single cell approach (Ehlich and Küppers, 1995Ehlich A. Küppers R. Analysis of immunoglobulin gene rearrangements in single B cells.Curr Opin Immunol. 1995; 7: 281-284Crossref PubMed Scopus (14) Google Scholar;Küppers et al., 1997Küppers R. Hansmann M.L. Rajewsky K. Micromanipulation and PCR analysis of single cells from tissue sections,.in: Weir D.M. Blackwell C. Herzenberg L.A. Handbook of Experimental Immunology. Vol. 4. Blackwell Science, Oxford1997: 206.1-206.4Google Scholar), consisting of micromanipulation and single cell PCR, which allows the amplification and analysis of different genes within individual B cells that were mobilized from a definite localization in a certain tissue environment, such as the skin (Golembowski et al., 2000Golembowski S. Gellrich S. von Zimmermann M. et al.Clonal evolution in a primary cutaneous follicle center B cell lymphoma revealed by single cell analysis in sequential biopsies.Immunobiology. 2000; 201: 631-644Crossref PubMed Scopus (6) Google Scholar). For this study, this technique was chosen to characterize the heavy (VH) and light (VL) chain variable region genes in eight patients with PCBCL, four of them suffering from follicle center cell lymphomas localized on the head and trunk areas, the others with large B cell lymphomas of the leg. We compare V-gene usage and mutational pattern between the two subentities of PCBCL on the level of single tumor B cells. Our data provide evidence for a germinal center cell origin of PCBCL. This study included two groups of patients suffering from PCBCL. Group I consisted of four patients with follicle center cell lymphomas with head and trunk localization Figure 1a, whereas group II included four patients with large B cell lymphomas of the leg Figure 1b. The course of disease differed markedly between the two groups. Whereas two patients in group II died within 1–1.5 y after diagnosis, all four patients in group I have a time course of more than 5 y with repeated treatment, such as surgery, radiation, or immune therapy (not shown in detail), achieving partial or complete remission. Preliminary data from patients IL and UK Table I were presented elsewhere (Gellrich et al., 1997Gellrich S. Golembowski S. Audring H. Jahn S. Sterry W. Molecular analysis of the immunoglobulin VH gene rearrangement in a primary cutaneous immunoblastic B-cell lymphoma by micromanipulation and single-cell PCR.J Invest Dermatol. 1997; 109: 541-545Crossref PubMed Scopus (20) Google Scholar;Golembowski et al., 2000Golembowski S. Gellrich S. von Zimmermann M. et al.Clonal evolution in a primary cutaneous follicle center B cell lymphoma revealed by single cell analysis in sequential biopsies.Immunobiology. 2000; 201: 631-644Crossref PubMed Scopus (6) Google Scholar). Tissue material from patients DG and AZL (large B cell lymphoma of the leg) was kindly provided by Dr M.H. Vermeer and Prof R. Willemze (Amsterdam, the Netherlands). None of the patients showed internal lymphoma involvement at the time when biopsies were taken, investigated by lymph node sonography, computed tomography, and blood and bone marrow analysis. In all patients the typical histologic picture was found Figure 2. In follicle center cell lymphomas of the head and trunk area, nodular or diffuse infiltrates with sparing of the epidermis were observed. The lesions contained a variable mixture of centrocytes and centroblasts with many infiltrating reactive T cells. Patients with large B cell lymphomas of the leg showed a rather monotonous infiltrate with a higher proportion of centroblasts, large centrocytes, lymphoblasts, multilobated cells, and only a few reactive T cells. Immunohistochemical analysis showed the expression of the B cell marker CD20 on most infiltrating cells, and in most cases the expression of the proliferation marker Mib-1. The overexpression of one particular light chain type suggests a clonal B cell expansion. Large B cell lymphomas of the leg differed from follicle center cell lymphomas by positive staining for bcl-2 of the tumor B cells Table I.Table IPatient data and immunohistochemical analysis of eight cases of PCBCLaMarker expression was indicated with respect to the proposed tumor cell population. M, male; F, female; CBCL-HT, follicle center cell lymphoma localized in the head/trunk area; CBCL-LL, large B cell lymphoma of the leg; ND, not determined.PatientAge, sexLocalizationCD20bcl-2CD30Mib-1CD5CD10κλUK58, MCBCL-HAT+––100%––+–WS84, MCBCL-HAT+––100%––+–LB55, MCBCL-HAT+––90%–––+GG77, FCBCL-HAT+––100%––+–IL49, FCBCL-LL++–100%–––+VM75, FCBCL-LL++–100%––+–DG58, FCBCL-LL++–ND–––+AZL69, FCBCL-LL++–ND––––a Marker expression was indicated with respect to the proposed tumor cell population. M, male; F, female; CBCL-HT, follicle center cell lymphoma localized in the head/trunk area; CBCL-LL, large B cell lymphoma of the leg; ND, not determined. Open table in a new tab Figure 2Histology of cutaneous B cell lymphomas. (1) follicle center cell lymphoma localized at the head; (2) large B cell lymphoma. (A) HE stain; (B) CD20; (C) GIEMSA; (D) bcl-2. Scale bar: (A, B) 50 μm; (C, D) 200 μm.View Large Image Figure ViewerDownload (PPT) Frozen skin sections (10 µm thick) were stained with an anti-CD20 antibody (Dako Diagnostika, Hamburg, Germany) using biotinylated Fab-anti-mouse and streptavidin–alkaline phosphatase complexes (Dako) as developing reagents. Control sections were stained with anti-CD3 antibodies. Only in patient GG, were the infiltrate obviously contained only a few tumor B cells, a κ chain staining was carried out taking into consideration the statistical overexpression of this light chain. After washing, bound alkaline phosphatase was visualized by staining with New Fuchsin. The slides were counterstained with hematoxylin. Single cells were mobilized under the microscope (Nikon, Düsseldorf, Germany) with the help of a hydraulic micromanipulator (Narishige, Tokyo, Japan) using 600 × magnification. Thereafter, single cells were aspirated into a micropipette (Narishige), put into 20 µl PCR buffer supplemented by 1 ng per µl rRNA (both from Boehringer, Mannheim, Germany) and stored at -20°C. Photographs were taken before and after the micromanipulation of each cell. A seminested PCR approach was applied to amplify simultaneously the immunoglobulin heavy and light chain gene rearrangements of isolated single B lymphocytes. To this end, sets of family-specific primers for the framework region (FR) I of the variable VH, Vκ, and Vλ genes were used together with two sets of nested primers binding to the joining (J) region of each locus. All primers were synthesized according toKüppers et al., 1993Küppers R. Zhao M. Hansmann M.L. Rajewsky K. Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections.EMBO J. 1993; 12: 4955-4967PubMed Google Scholar,Küppers et al., 1995Küppers R. Willenbrock K. Rajewsky K. Hansmann M.L. Detection of clonal lambda light chain gene rearrangements in frozen and paraffin-embedded tissues by polymerase chain reaction.Am J Pathol. 1995; 147: 806-814PubMed Google Scholar) and Bräuninger et al., 1999Bräuninger A. Küppers R. Spieker T. et al.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. 1999; 93: 2679-2687PubMed Google Scholar. Before PCR, the single cells were incubated with 0.25 mg per ml proteinase K (Boehringer Mannheim) for 55 min at 50°C, and additionally at 95°C for 10 min (enzyme inactivation). The reaction mix (50 µl) for the first round of amplification contained: PCR buffer, 2.5 µM MgCl2, 200 µM of each deoxyribonucleoside triphosphate, 7 nM of each primer, and 3.5 U Expand High Fidelity Taq-polymerase (all chemicals from Boehringer Mannheim). PCR amplification was carried out on a Personal Cycler (Biometra, Göttingen, Germany): one cycle at 95°C for 2 min, 65°C for 1 min (when Taq polymerase was added), 72°C for 1 min, followed by 35 cycles at 95°C for 30 s, 59°C for 30 s, 72°C for 1 min, and final extension at 72°C for 5 min. Further amplification of IgH and Igκ genes was performed as previously described (Gellrich et al., 1997Gellrich S. Golembowski S. Audring H. Jahn S. Sterry W. Molecular analysis of the immunoglobulin VH gene rearrangement in a primary cutaneous immunoblastic B-cell lymphoma by micromanipulation and single-cell PCR.J Invest Dermatol. 1997; 109: 541-545Crossref PubMed Scopus (20) Google Scholar;Golembowski et al., 2000Golembowski S. Gellrich S. von Zimmermann M. et al.Clonal evolution in a primary cutaneous follicle center B cell lymphoma revealed by single cell analysis in sequential biopsies.Immunobiology. 2000; 201: 631-644Crossref PubMed Scopus (6) Google Scholar). For amplification of the λ-chain rearrangement, aliquots of the first round were reamplified using only one of the Vλ-specific primers plus a set of more internally binding Jλ primers in separate reactions for each Vλ gene family. The reaction mix (50 µl) for the second round of amplification contained 1 µl of the amplification product from the first round, PCR buffer, 1.5 (IgH) or 2.5 µM (IgκH/Igλ) MgCl2, 200 µM of each deoxyribonucleoside triphosphate, 7 nM (IgH) or 50 nM (Igκ/Igλ) of each primer, and 5 U (IgH) or 1.25 U (Igκ/Igλ) Taq-polymerase (all Perkin Elmer). The second round cycle program was carried out on a TRIO-Thermoblock (Biometra) and consisted of one cycle at 95°C for 2 min, 68°C for 5 min (addition of Taq polymerase), 72°C for 1 min, 45 cycles at 95°C for 1 min, 61°C (VH1, VH2, VH5, VH6, Vκ, Vλ) or 65°C (VH3, VH4) for 30 s, 72°C for 1 min, and a final extension at 72°C for 5 min. A 5 µl aliquot of the reaction mixture was analyzed on a 2% agarose gel. Strong attention was paid to avoid any contamination with DNA. The micromanipulation as well as the first and second round of PCR were carried out separately in different rooms. As negative controls, T cells picked from CD3-stained adjacent sections were used during the same experiment. PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), and used for direct sequencing (see below). In order to exclude an allelic polymorphism for subsequent mutational analysis, the patient's specific germline VH gene rearranged in the lymphoma B cells was cloned and sequenced in three cases (patients UK, LB, and IL). To this end, genomic DNA was required. We used paraffin-embedded skin tissue, which was first deparafinized and then treated with proteinase K. Primers were designed to amplify the proposed germline genes, one annealing to the leader sequence of the respective VH gene family, VH5 for patient UK (5′-CCC CTG ATT CAA ATT TTG TGT CTC C-3′); VH4 for patient LB (5′-TGG TGG CRG CTC CCA GAT GTG A-3′), and VH3 for patient IL (5′-CTA GGA AGA TTG AGT GTG TGT GGA T-3′), and the second primer specific for the respective recombination signal sequence (5′-CTC GGG GCT GGT TTC TCT CAC TGT G-3′ (patient UK), 5′-GGG CTC ACA CTC ACC TCC CCT CAC T-5′ (patient LB), and 5′-CTC ACA CTG ACT TCC CCT CAC TGT G-3′ (patient IL). The PCR products were purified with the QIAquick Gel Extraction Kit (Qiagen) and then cloned using the TA-Cloning Kit (Invitrogen, Leek, Netherlands) according to the manufacturer's protocols, and finally sequenced (see below). To analyze the lymphoma cell-specific sequence on the transcriptional level, mRNA was isolated from cryopreserved tissue of patients UK and LB using the Oligotex Direct mRNA-Kit (Qiagen). Then, cDNA was prepared with the Superscript Preamplification System (Gibco BRL, Eggenstein, Germany). For PCR amplification the following primer pairs were used: 5′ the respective family specific VH primer (see section single cell PCR), and 3′ a primer specific for the CH1 domain of one of the human immunoglobulin isotypes (cµ: 5′-CCA AGC TTA GAC GAG GGG GAA AAG GGT T-3′; Cγ: 5′-CAC AAG CTT GAA GTA GTC CTT GAC CAG GC-3′; Cδ: 5′- TGG CCA GCG GAA GAT CTC CTT CTT-3′; Cα: 5′-CCC GGA GGC ATC CTG GCT GGG-3′; Cε: 5′-GGG TCG ACA GTC ACG GAG GTG GCA TT-3′). The PCR products were purified with the QIAquick Gel Extraction Kit (Qiagen) and then cloned using the TA-Cloning Kit (Invitrogen), again according to the manufacturer's protocols. Five (UK) and eight (LB) clones were sequenced. DNA sequencing was performed with the Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) on a Sequencing System 373 (Applied Biosystems, Weiterstadt, Germany). Amplificates from single cell PCR were used for direct sequencing. To this end, the respective V gene family specific primer, used in the second round PCR, was applied to the sequencing reaction. Once the rearranged J segment became obvious, the respective internal J-specific primer was used to verify sequence information. For plasmid sequencing, the primers T7 (5′-AAT ACG ACT CAC TAT AGG GCG-3′) and M13r (5′-TTC ACA CAG GAA ACA GCT ATG ACC-3′) were used. All sequences were analyzed online using the Software “DNAPLOT” (created by H.-H. Althaus, University of Cologne, Germany) and compared with the gene bank “IMGT, the international ImMunoGeneTics database” (Lefranc, 1997Lefranc M.P. Unique database numbering system for immunogenetic analysis.Immunol Today. 1997; 18: 509Abstract Full Text PDF PubMed Scopus (91) Google Scholar) (http://www.imgt.cines.fr:8104; initiator and coordinator: Marie-Paule Lefranc, Montpellier, France) for determining the most similar germline VH/VL genes. A total of 532 single B cells was isolated by micromanipulation from skin biopsies of eight PCBCL patients. Applying a single cell PCR approach, clonally related immunoglobulin heavy chain gene rearrangements (VHDJH) were obtained in three of four follicle center cell lymphomas, and three of four large B cell lymphomas of the leg. (All sequences were submitted to EMBL/GenBank database. Accession numbers: IGHV5-51UK–Y12817; UKVH5A–Y11556; UKVH5B–Y11557; LBVH4A–AJ292947; LBVH4B–AJ292948; LBVH4C–AJ292949; GGVH1–AJ292938; ILVH3–Y10020; VMVH3A–AJ292939; VMVH3B–AJ292940; DGVH6A–AJ292941; DGVH6B–AJ292942; DGVH6C–AJ292943; DGVH6D–AJ292944; DGVH6E–AJ292945; DGVH6F–AJ292946; UKVK3A–AJ223760; UKVK3B–AJ223761; WSVK3A–AJ292955; WSVK3B–AJ292956; WSVK3C–AJ292957; WSVK3D–AJ292958; LBVL3A–AJ292950; LBVL3B–AJ292951; LBVL3C–AJ292952; LBVL3D–AJ292953; LBVL3E–AJ292954; GGVK3–AJ292937; VMVK3–AJ292936; DGVL2A–AJ292934; DGVL2B–AJ292935; AZLVK1A–AJ292931; AZLVK1B–AJ292932; AZLVK1C–AJ292933.) Amplification failed in patients WS and AZL, whereas light chain gene rearrangements could be obtained in these cases. From patient IL (large B cell lymphoma of the leg), we were not able to amplify a clonal light chain gene rearrangement Table II. Possible reasons for failure of amplification include somatic point mutations at sites of primer annealing or some kind of selective DNA degradation. The latter is rather unlikely, regarding the successful amplification of light chain rearrangements in case of failure of heavy chain amplification and vice versa but cannot be ruled out because of the localization of the immunoglobulin loci on different chromosomes.Table IIClonal immunoglobulin heavy and light chain gene rearrangements detected by single cell PCR in skin sections of PCBCL patientsPatientNo. of cells analyzedClonal IgH rearrangementNo. of clonal IgH sequencesaNumbers of clonal IgH and IgL gene sequences are indicated separately for each of the identified subclones. IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain. Immunoglobulin gene nomenclature according toLefranc (1997);for VH genes seePallares et al (1999);for VK genes seeBarbie and Lefranc (1998);for VL genes seePallares et al (1998).Clonal IgL rearrangement (VLJL)No. of clonal IgL sequencesaNumbers of clonal IgH and IgL gene sequences are indicated separately for each of the identified subclones. IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain. Immunoglobulin gene nomenclature according toLefranc (1997);for VH genes seePallares et al (1999);for VK genes seeBarbie and Lefranc (1998);for VL genes seePallares et al (1998).No. of cells with both clonal IgH and IgL sequences(VHDJH)UK129IGHV5–51UK–D6-13–JH5b19 (UKVH5A)IGKV3-20*1–JK26 (UKVK3A)413 (UKVH5B)5 (UKVK3B)WS100––IGKV3-20*1–JK23 (WSVK3A)–5 (WSVK3B)12 (WSVK3C)4 (WSVK3D)LB126IGHV4-59*3–D7-27–JH444 (LBVH4A)IGLV3-21*2–JL2/JL328 (LBVL3A)241 (LBVH4B)1 (LBVL3B)1 (LBVH4C)1 (LBVL3C)3 (LBVL3D)1 (LBVL3E)GG18IGHV1-69*1–D3-22–JH48IGKV3
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