In Situ Analysis of the Variable Heavy Chain Gene of an IgM/IgG-Expressing Follicular Lymphoma
2002; Elsevier BV; Volume: 160; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64911-5
ISSN1525-2191
AutoresWilhelmina M. Aarts, Richard J. Bende, Jan‐Willem Vaandrager, Philip M. Kluin, Anton W. Langerak, Steven T. Pals, Carel J.M. van Noesel,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoIt is generally assumed that follicular lymphomas (FL) not only morphologically resemble normal germinal centers but have retained some functional characteristics of their non-neoplastic counterparts as well. Recent IgV gene analyses on a panel of FLs however, strongly suggested that FLs do not retain the capacity of somatic hypermutation and are not being selected on basis of the quality of their mIgV regions. To extend these findings, we investigated the follicular organization and class switching in a FL that consisted of both IgM- and IgG-expressing tumor cells with a high somatic mutation load and significant intraclonal VH gene diversity. VH-Cμ and VH-Cγ gene transcripts were amplified and sequenced from samples of approximately 50 tumor cells, isolated from frozen tissue sections by laser microdissection. We identified many different subclones and obtained limited evidence of subclone dominance in individual follicles. Remarkably, several subclones were found scattered over different follicles. All samples contained IgM- and IgG-expressing tumor cells with, in general, non-identical mutation patterns, which is not in support of ongoing class switching. Accordingly, no switch circle recombination products were found. The findings indicate that the neoplastic follicles lack the organization and functions typical of reactive germinal centers. It is generally assumed that follicular lymphomas (FL) not only morphologically resemble normal germinal centers but have retained some functional characteristics of their non-neoplastic counterparts as well. Recent IgV gene analyses on a panel of FLs however, strongly suggested that FLs do not retain the capacity of somatic hypermutation and are not being selected on basis of the quality of their mIgV regions. To extend these findings, we investigated the follicular organization and class switching in a FL that consisted of both IgM- and IgG-expressing tumor cells with a high somatic mutation load and significant intraclonal VH gene diversity. VH-Cμ and VH-Cγ gene transcripts were amplified and sequenced from samples of approximately 50 tumor cells, isolated from frozen tissue sections by laser microdissection. We identified many different subclones and obtained limited evidence of subclone dominance in individual follicles. Remarkably, several subclones were found scattered over different follicles. All samples contained IgM- and IgG-expressing tumor cells with, in general, non-identical mutation patterns, which is not in support of ongoing class switching. Accordingly, no switch circle recombination products were found. The findings indicate that the neoplastic follicles lack the organization and functions typical of reactive germinal centers. During normal B cell maturation, the developing cells are continuously tested for expression and quality of their B cell antigen receptors (BCR).1Rajewsky K Clonal selection and learning in the antibody system.Nature. 1996; 381: 751-758Crossref PubMed Scopus (1374) Google Scholar, 2Lam K-P Kühn R Rajewsky K In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death.Cell. 1997; 90: 1073-1083Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar This principle most obviously rules the germinal center (GC) reaction, where B cells compete to obtain survival signals by binding antigen that is presented at the surface of follicular dendritic cells (FDCs).3Lindhout E Koopman G Pals ST de Groot C Triple check for antigen specificity of B cells during germinal center reactions.Immunol Today. 1997; 18: 573-577Abstract Full Text PDF PubMed Scopus (90) Google Scholar This competition, combined with changes that are introduced in the immunoglobulin variable (IgV) region genes due to somatic hypermutation,4Kocks C Rajewsky K Stable expression and somatic hypermutation of antibody V regions in B cell developmental pathways.Annu Rev Immunol. 1989; 7: 537-559Crossref PubMed Scopus (230) Google Scholar results in clonal evolution of the BCRs and forms the molecular basis of affinity maturation of the humoral immune response. Several in situ studies have provided insight into the selection and differentiation processes that take place within germinal centers.5Küppers R Zhao M Hansmann M-L Rajewsky K Tracing B cell development in human germinal centers by molecular analysis of single cells picked from histological sections.EMBO J. 1993; 12: 4955-4967PubMed Google Scholar, 6Jacob J Przylepa J Miller C Kelsoe G In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells.J Exp Med. 1993; 178: 1293-1307Crossref PubMed Scopus (332) Google Scholar, 7Vora KA Tumas-Brundage K Manser T Contrasting the in situ behavior of a memory B cell clone during primary and secondary immune responses.J Immunol. 1999; 163: 4315-4327PubMed Google Scholar Both anatomically and functionally, germinal centers seem rather closed compartments. It has been shown that, although a single B cell clone can be seeded into multiple GCs, clonal evolution thereafter occurs independently in these GCs.7Vora KA Tumas-Brundage K Manser T Contrasting the in situ behavior of a memory B cell clone during primary and secondary immune responses.J Immunol. 1999; 163: 4315-4327PubMed Google Scholar, 8Jacob J Kelsoe G In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers.J Exp Med. 1992; 176: 679-687Crossref PubMed Scopus (397) Google Scholar The stringency of selection within this environment was demonstrated by the fact that the B cell populations of individual GCs, initially being highly polyclonal, become oligoclonal or even monoclonal within a relatively short time span.5Küppers R Zhao M Hansmann M-L Rajewsky K Tracing B cell development in human germinal centers by molecular analysis of single cells picked from histological sections.EMBO J. 1993; 12: 4955-4967PubMed Google Scholar, 6Jacob J Przylepa J Miller C Kelsoe G In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells.J Exp Med. 1993; 178: 1293-1307Crossref PubMed Scopus (332) Google Scholar, 9Kroese FG Wubbena AS Seijen HG Nieuwenhuis P Germinal centers develop oligoclonally.Eur J Immunol. 1987; 17: 1069-1072Crossref PubMed Scopus (160) Google Scholar Follicular lymphoma (FL) is considered as the prototype of a germinal-center cell-derived B cell non-Hodgkin's lymphoma,10Harris NL Jaffe ES Diebold J Flandrin G Muller-Hermelink HK Vardiman J Lister TA Bloomfield CD World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the clinical advisory committee meeting-Airlie House, Virginia, November, 1997.J Clin Oncol. 1999; 17: 3835-3849Crossref PubMed Scopus (2487) Google Scholar as the centroblast- and centrocyte-like tumor cells proliferate in nodular networks of non-neoplastic FDCs. Many investigators have demonstrated that FLs carry heavily mutated IgV genes.11Stamatopoulos K Kosmas C Papadaki T Pouliou E Belessi C Afendaki S Anagnostou D Loukopoulos D Follicular lymphoma immunoglobulin κ light chains are affected by the antigen selection process, but to a lesser degree than their partner heavy chains.Br J Haematol. 1997; 96: 132-146Crossref PubMed Scopus (53) Google Scholar, 12Noppe SM Heirman C Bakkus MHC Brissinck J Schots R Thielemans K The genetic variability of the VH genes in follicular lymphoma: the impact of the hypermutation mechanism.Br J Haematol. 1999; 107: 625-640Crossref PubMed Scopus (33) Google Scholar, 13Aarts WM Bende RJ Steenbergen EJ Kluin PM Ooms ECM Pals ST van Noesel CJM Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and somatic mutation load.Blood. 2000; 95: 2922-2929PubMed Google Scholar Moreover, based on the findings of intraclonal IgV gene diversity and the co-presence of different heavy (H) chain switch variants in some FLs, it was concluded that the processes of somatic hypermutation14Levy S Mendel E Kon S Avnur Z Levy R Mutational hot spots in Ig V region genes of human follicular lymphomas.J Exp Med. 1988; 168: 475-489Crossref PubMed Scopus (76) Google Scholar, 15Zelenetz AD Chen TT Levy R Clonal expansion in follicular lymphoma occurs subsequent to antigen selection.J Exp Med. 1992; 176: 1137-1148Crossref PubMed Scopus (152) Google Scholar, 16Zhu D Hawkins RE Hamblin TJ Stevenson FK 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, 17Ottensmeier CH Thompsett AR Zhu D Wilkins BS Sweetenham JW Stevenson FK Analysis of VH genes in follicular and diffuse lymphoma shows ongoing somatic mutation and multiple isotype transcripts in early disease with changes during disease progression.Blood. 1998; 91: 4292-4299PubMed Google Scholar and H chain isotype switching18Zelenetz AD Chen TT Levy R Histologic transformation of follicular lymphoma to diffuse lymphoma represents tumor progression by a single malignant B cell.J Exp Med. 1991; 173: 197-207Crossref PubMed Scopus (96) Google Scholar, 19Raghoebier S Broos L Kramer MHH van Krieken JHJM Kluin-Nelemans JC van Ommen GJB Kluin PM Histological conversion of follicular lymphoma with structural alterations of t(14;18) and immunoglobulin genes.Leukemia. 1995; 9: 1748-1755PubMed Google Scholar remain active in these neoplasms. Finally, as the mutation patterns in the IgV genes of FLs were found to be highly comparable to those in normal antigen-selected B cells, it was proposed that FL cells are, during their malignant growth, still being clonally selected on basis of the antigen-binding capacity of their BCRs.15Zelenetz AD Chen TT Levy R Clonal expansion in follicular lymphoma occurs subsequent to antigen selection.J Exp Med. 1992; 176: 1137-1148Crossref PubMed Scopus (152) Google Scholar, 20Bahler DW Zelenetz AD Chen TT Levy R Antigen selection in human lymphomagenesis.Cancer Res. 1992; 52: 5547s-5551sPubMed Google Scholar, 21Bahler DW Levy R Clonal evolution of a follicular lymphoma: evidence for antigen selection.Proc Natl Acad Sci USA. 1992; 89: 6770-6774Crossref PubMed Scopus (217) Google Scholar, 22Matolcsy A Schattner EJ Knowles DM Casali P Clonal evolution of B cells in transformation from low- to high-grade lymphoma.Eur J Immunol. 1999; 29: 1253-1264Crossref PubMed Google Scholar We have recently studied a large panel of FLs.13Aarts WM Bende RJ Steenbergen EJ Kluin PM Ooms ECM Pals ST van Noesel CJM Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and somatic mutation load.Blood. 2000; 95: 2922-2929PubMed Google Scholar These analyses indicated that FLs may not retain the capacity to somatically mutate. Additionally, we obtained evidence for subclone selection instead of clonal BCR evolution over time.23Aarts WM Bende RJ Bossenbroek JG Pals ST van Noesel CJM Variable heavy chain gene analysis of follicular lymphomas: subclone selection rather than clonal evolution over time.Blood. 2001; 98: 238-240Crossref PubMed Scopus (36) Google Scholar These findings challenged the concept of antigen-driven lymphomagenesis. We here analyze an interesting case of a FL that harbored, in all tumor follicles, IgM+ and IgG+ tumor cells of the same clonal origin. By in situ analyses using laser microdissection we aimed to obtain insight in the composition of the neoplastic follicles, the process of isotype switching and the repertoire of infiltrating T cells within the tumor compartment. The results again indicate that FL cells may have retained less properties of normal GC B cells than is generally assumed. Fresh tissue of FL no. 8 was obtained from surgically removed lymph nodes from the departments of pathology of the Westeinde Hospital, The Hague, the Netherlands and was kindly provided by Dr. E.C.M. Ooms. The patient, a 48-year-old woman, presented with a FL in 1983 (FL 8-′83). Complete remission was achieved after chemotherapy. After irradiation of a local relapse, in 1988, a systemic relapse developed in 1992 (FL 8-′92). As control tissue, fresh human tonsil was used. All tissues had been snap-frozen and stored in liquid nitrogen until usage. Cryostat sections were immunohistochemically stained as described,24Bernsen MR Dijkman HBPM De Vries E Figdor CG Ruiter DJ Adema GJ van Muijen GNP Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.Lab Invest. 1998; 78: 1267-1273PubMed Google Scholar using monoclonal antibodies specific for human CD21L (DRC-1, Dako, Glostrup, Denmark), Bcl-2 (clone 124, DAKO), and for Ig heavy and light chain isotypes (all from Dako except for anti-IgM, -κ, and -λ, which were obtained from Becton Dickinson, Erembodegem-Aalst, Belgium). Heavy chain isotype expression of the lymphoma of patient 8 was also assessed by immunofluorescence. Briefly, acetone-fixed tissue sections were incubated at 37°C with the primary antibody (anti-IgM, -IgG, -IgA, -IgD, -κ or -λ; Dako), washed three times in phosphate-buffered saline (PBS), and incubated for 30 minutes with swine-anti-rabbit, labeled with FITC (Dako). Microdissection out of frozen sections was performed with a laser-microbeam system (PALM GmbH, Bernried, Germany). The potential risk of RNA carry-over by cutting was evaluated using slides with membranes on which tissue of two distinct lymphomas was mounted together. These analyses indicated that cell-specific results were obtained and that out of microdissected membrane located adjacently to the tissue no polymerase chain reaction (PCR) products were amplifiable by reverse transcriptase (RT)-PCR (data not shown). Similar results were reported by others even after a brief fixation and staining procedure.24Bernsen MR Dijkman HBPM De Vries E Figdor CG Ruiter DJ Adema GJ van Muijen GNP Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.Lab Invest. 1998; 78: 1267-1273PubMed Google Scholar If, however, microdissection is possible using polarized light only, like in the present study, we use untreated sections to exclude the risk of RNA carry-over. Thus, here 10-μm frozen sections were dried but were left unfixed and unstained before use. For RNA analyses, samples of approximately 50 cells were dissected from lymphoma 8-'83 (Figure 3), catapulted into 3 μl of cDNA reaction mixture (see below), and stored on ice until cDNA synthesis. For DNA samples, approximately 100 cells were dissected from follicles of hematoxylin-stained tissue sections of lymphoma 8-′83 and its relapse, 8-′92 and from normal tonsil. Tissue samples were “catapulted” into 5 μl of water. RNA of bulk material was isolated from frozen tissue sections using the TRIzol reagent (Life Technologies, Breda, the Netherlands) and cDNA was synthesized as described.25Aarts WM Willemze R Bende RJ Meijer CJLM Pals ST van Noesel CJM VH gene analysis of primary cutaneous B cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.Blood. 1998; 92: 3857-3864PubMed Google Scholar From the microdissected samples, cDNA was synthesized without prior RNA isolation. The microdissected samples were incubated in a total volume of 20 μl of cDNA reaction mixture.25Aarts WM Willemze R Bende RJ Meijer CJLM Pals ST van Noesel CJM VH gene analysis of primary cutaneous B cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.Blood. 1998; 92: 3857-3864PubMed Google Scholar The reaction was performed for 15 minutes at 37°C. Subsequently, the enzyme was inactivated during 10 minutes at 95°C. After cDNA synthesis 20 μl of water was added. For DNA samples, 15 μl of 0.25 mol/L proteinase K in 1X Taq buffer was added (20 mmol/L Tris-HCl, 50 mmol/L KCl, pH 8.4). After incubation overnight at 56°C, the enzyme was inactivated for 10 minutes at 95°C. Bulk DNA was isolated from frozen tissue sections using the DNAzol reagent, according to the manufacturer's instructions (Life Technologies). In the first rounds of amplification 1 μl of cDNA reaction mixture was used in a 25 μl PCR reaction volume using a forward primer with specificity for the leader of the VH3 gene family in combination with a reverse primer specific for Cμ (Cμ1-: 5′-CGTATCCGACGGGGAATTCTC-3′), or Cγ.25Aarts WM Willemze R Bende RJ Meijer CJLM Pals ST van Noesel CJM VH gene analysis of primary cutaneous B cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.Blood. 1998; 92: 3857-3864PubMed Google Scholar Next, a nested PCR was performed using 2.5 μl of the first PCR product in a 25-μl reaction. PCR reactions were performed with a VH3 primer that anneals in the FRI region (VH3FR: 5′-TCCCTGAGACTCTCCTGTG-3′) combined with the appropriate reverse primer either Cμ or Cγ2.25Aarts WM Willemze R Bende RJ Meijer CJLM Pals ST van Noesel CJM VH gene analysis of primary cutaneous B cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.Blood. 1998; 92: 3857-3864PubMed Google Scholar The PCR consisted of 50 cycles under conditions that were the same as described previously for the CDR3-specific PCR.25Aarts WM Willemze R Bende RJ Meijer CJLM Pals ST van Noesel CJM VH gene analysis of primary cutaneous B cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.Blood. 1998; 92: 3857-3864PubMed Google Scholar PCR products were analyzed on a 1% standard agarose gel (Sigma, St. Louis, MO). All PCR reactions and the subsequent sequencing reactions were performed in duplicate to obtain a reliable consensus sequence of a sample. Specificity of the PCR results was further controlled using many buffer-only samples to which no cDNA was added. These controls were in all instances negative. Quantification and subsequent stratification of the amount of amplifiable DNA was performed using a PCR on the β2 microglobulin gene (β2m). The primers used were 5′-AGCATTCAGACTTGTCTTTCAG-3′ and 5′-GATGCTGCTTACATGTCTCG-3′, which yield a product of 776 base pairs from DNA. The PCR protocol for this β2m-PCR was the same as for the amplification of the VH gene. Sγ-Sμ switch circle fragments were amplified in 25-μl reaction mixtures from 250 ng or less DNA, using the upstream Iγ1/2 primer, in combination with the downstream Sμ primer.26Cerutti A Zan H Schaffer A Bergsagel L Harindranath N Max EE Casali P CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plamacytoid phenotypic differentiation in a human monoclonal IgM+IgD+ B cell line.J Immunol. 1998; 160: 2145-2157PubMed Google Scholar As positive controls, 10 copies of artificial construct plasmids27Malisan F Brière F Bridon J-M Harindranath N Mills FC Max EE Banchereau J Martinez-Valdez H Interleukin-10 induces immunoglobulin G isotype switch recombination in human CD40-activated naive B lymphocytes.J Exp Med. 1996; 183: 937-947Crossref PubMed Scopus (144) Google Scholar were amplified. All plasmids (Sγ1-Sμ, Sγ2-Sμ, Sγ3-Sμ, and Sγ4-Sμ) could be amplified using this PCR approach (data not shown). The PCR samples were first incubated at 95°C for 4 minutes, then 40 cycles of 1 minute at 95°C, 1 minute of 68°C and 1 minute of 72°C were performed, followed by 10 minutes at 72°C. Next, a nested PCR was performed with 2 μl of PCR product of the first PCR in a 25-μl PCR, using the Sγ and Sμ primers.26Cerutti A Zan H Schaffer A Bergsagel L Harindranath N Max EE Casali P CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plamacytoid phenotypic differentiation in a human monoclonal IgM+IgD+ B cell line.J Immunol. 1998; 160: 2145-2157PubMed Google Scholar The same PCR mixtures were used as for the amplification of the VH gene, except that 100 nmol/L of each primer was used and Taq platinum polymerase (Life Technologies). With this PCR, one switch circle copy in at least 1000 cells is detectable. The PCR products were analyzed on a 1% agarose gel (Sigma) and subsequently sequenced to confirm their origin. The configuration of the constant heavy chain gene locus of both time points of FL 8 was analyzed by DNA fiber fluorescence in situ hybridization (FISH) as described.28Vaandrager J-W Schuuring E Kluin-Nelemans JC Dyer MJS Raap AK Kluin PM DNA fiber fluorescence in situ hybridization analysis of immunoglobulin class switching in B cell neoplasia: aberrant CH gene rearrangements in follicle center-cell lymphoma.Blood. 1998; 92: 2871-2878PubMed Google Scholar Probes for the J region, the different constant region genes and the BCL-2 locus were used. At least 20 fibers representative for each configuration were analyzed out of tissue of each time point. The T cell receptor-γ (TCRG) gene rearrangements were amplified from DNA of microdissected samples. The first PCR was performed with the PCR primers as described,29Pongers-Willemse MJ Seriu T Stolz F d'Aniello E Gameiro P Pisa P Gonzalez M Bartram CR Panzer-Grumayer ER Biondi A San Miguel JF van Dongen JM Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets. Report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia.Leukemia. 1999; 13: 110-118Crossref PubMed Scopus (321) Google Scholar except that Taq platinum polymerase (Life Technologies) was used instead of AmpliTaq Gold polymerase (PE Biosystems, Foster City, CT). For the second, nested PCR, the sequencing primers29Pongers-Willemse MJ Seriu T Stolz F d'Aniello E Gameiro P Pisa P Gonzalez M Bartram CR Panzer-Grumayer ER Biondi A San Miguel JF van Dongen JM Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets. Report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia.Leukemia. 1999; 13: 110-118Crossref PubMed Scopus (321) Google Scholar were used with the same PCR protocol, with exception that 2.0 mmol/L MgCl2 was used and 0.25 μmol/L of each primer. The products were first analyzed on a 1% agarose gel and subsequently by heteroduplex analysis.30Langerak AW Szczepanski T van der Burg M Wolvers-Tettero ILM van Dongen JJM Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations.Leukemia. 1997; 11: 2192-2199Crossref PubMed Scopus (186) Google Scholar Briefly, the PCR products were denatured for 5 minutes at 95°C, then they were allowed to reanneal at 4°C for at least 1 hour. The resulting homo- and/or heteroduplexes were analyzed on a 6% non-denaturing polyacrylamide gel and visualized by staining with ethidium-bromide. Both strands of the PCR products were either directly sequenced with an ABI sequencer (Perkin Elmer Corporation, Norwalk, CT) using the dye-terminator cycle-sequencing kit (Perkin Elmer), or first cloned into pCR2.1-TOPO vectors (Invitrogen, Groningen, the Netherlands) and transformed into TOP10 bacteria (Invitrogen). Subsequently, both strands of the inserts of a clone were sequenced. We previously analyzed the VH and variable light (VL) chain genes of a large panel of FLs of different Ig isotypes. Among these, two biopsies of FL 8 were included.13Aarts WM Bende RJ Steenbergen EJ Kluin PM Ooms ECM Pals ST van Noesel CJM Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and somatic mutation load.Blood. 2000; 95: 2922-2929PubMed Google Scholar Interestingly, in all follicles of the presentation biopsy of 1983 (FL 8-′83) both IgM/κ and IgG/κ tumor cells were found by immunofluorescence as well as immunohistochemically (Figure 1). Molecular analyses revealed that these assumed isotype switch variants indeed carried the same VDJ rearrangement which contained 30 and 35 somatic mutations compared to the germ-line gene V3–23, respectively. This FL expressed the L16 Vκ chain gene, which harbored 20 somatic mutations (data not shown). At the second time point (FL 8-′92), 9 years later, only IgG-expressing tumor cells were found of the same clonal origin (data not shown). In the V3–23 gene, 35 somatic mutations were found compared to the germ-line gene, 30 of which were shared with the IgM and IgG sequences of the first time point. In the κ light chain one extra replacement mutation was present (data not shown). At both time points, significant sequence variation was found between cloned VH products: the means of this intraclonal variation were 5.0, 4.0, and 1.3 mutations/clone compared respectively to the consensus IgM and IgG sequences of FL 8-′83 and the consensus IgG sequence of FL 8-'92, as determined on crude tissue.13Aarts WM Bende RJ Steenbergen EJ Kluin PM Ooms ECM Pals ST van Noesel CJM Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and somatic mutation load.Blood. 2000; 95: 2922-2929PubMed Google Scholar To assay the distribution of the subclones within the tissue of FL 8, we microdissected a total of 47 samples from 19 neoplastic follicles from frozen tissue sections (Figure 2). Cells were microdissected from unstained tissue sections. Of each sample, containing approximately 50 cells, the VH genes were amplified and sequenced. All PCR and sequence reactions were carried out in duplicate and consensus sequences thus obtained of each sample were compared. The overall PCR efficiency was 86%. All VH PCR products contained the known VDJ rearrangement of this FL. From 5 of 47 samples the duplicates were not reproducible (the IgM-derived sequences of sample 11a and 17b, and the IgG-derived sequences of sample 9c, 15b, and 20b), ie, point mutation differences were observed, most likely due to the presence of too many different subclones without clear dominance of one of them. The sequences that could be reproduced are depicted in Figure 3. The variation found within the IgM and IgG sequences of the samples was 2.7 and 3.1 somatic mutations per IgVH sequence, compared to the IgM- or IgG-derived consensus sequences established in crude tissue analyses, respectively.13Aarts WM Bende RJ Steenbergen EJ Kluin PM Ooms ECM Pals ST van Noesel CJM Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and somatic mutation load.Blood. 2000; 95: 2922-2929PubMed Google Scholar In 9 instances (concerning 5 IgM and 4 IgG sequences), a second sample derived from the same follicle was assayed. In two of the five paired IgM samples, the obtained VH sequences were identical (3a and b, 13a and b), suggesting that these follicles were dominated by a specific subclone. However, in the three other sample pairs non-corresponding subclones were identified with respectively 1 (samples 4a and b), 3 (8a and e) and 6 (14a and b) nucleotide differences. All four paired IgG-derived sequences yielded non-identical sequences with either five (samples 3a and b and samples 4a and b) or one (samples 11a and b and samples 13 a and b) nucleotide differences (Figure 3). To get more insight into (sub)clonal diversity, three to eight bacterial clones were made from the PCR products of samples 2μ, 3aμ, 3bμ, and 9aγ, and the respective inserts were sequenced (Figure 4). Significant intraclonal variation was found, which ranged from 1 mutation per clone (sample 3aμ) to 4.6 mutations per clone (sample 9aγ). It must be emphasized that in these calculations Taq errors may be present. We determined the frequency of Taq polymerase errors to be 0.14% in our experimental setup, which would amount to an average of 0.4 mutations per sequence for these clones.25Aarts WM Willemze R Bende RJ Meijer CJLM Pals ST van Noesel CJM VH gene analysis of primary cutaneous B cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.Blood. 1998; 92: 3857-3864PubMed Google Scholar In sample 3aμ and 3bμ, these clonal analyses were concordant with the consensus sequences obtained by directly sequencing the PCR products derived from these samples (Figure 4). However, in sample 2μ and 9aγ, the consensus sequence derived from the clones differed from the directly sequenced product. This may be erroneous, due to the low number of analyzed clones, or be a reflection of polyclonality. It was striking that several subclones were found in more than one follicle (Figure 3, Figure 5). The IgM+ subclone of sample 3a and 3b was also found in samples 2 and 4a and closely resembled the subclones of sample 1, 4b and 20a (one nucleotide difference). These follicles are located adjacently (Figure 3, Figure 5). However, the same samples yielded quite different IgG sequences, not or less clearly related to each other. Furthermore, the IgM sequences of sample 8a, 11b, and 12 were the same, and closely resembled the IgM sequences of 7 and 9a (one nucleotide difference). The IgM-sequences of 13a and 13b were the same as the IgM sequence from 6, and the 17a IgM-sequence was the same as the IgM sequence from sample 19. Similarly, an identical IgG+ subclone was found in samples 11b, 13a, and 14b and in samples 2 and 9a. In all these cases no clear zonation was apparent (Figure 3, Figure 5). Immunohistochemically, no clear compartmentalization was found of the IgM- and IgG-expressing cells. Accordingly, PCR analyses revealed that all dissected samples contained both IgM and IgG transcripts (data not shown). In all samples, exc
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