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Related Collections Brief Reports Neoplasia Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 November 2002, Vol. 100, No. 9, pp. 3419-3422 BRIEF REPORT Chronic lymphocytic leukemia preceded by cold agglutinin disease: intraclonal immunoglobulin light-chain diversity in VH4-34 expressing single leukemic B cells Sarka Ruzickova, Axel Pruss, Marcus Odendahl, Karsten Wolbart, Gerd-Rüdiger Burmester, Jürgen Scholze, Thomas Dörner, and Arne Hansen From Deutsches Rheumaforschungszentrum, Berlin; Department of Transfusion Medicine/Outpatients Department and Department of Rheumatology and Clinical Immunology, Charité University Hospital, Berlin, Germany.     Abstract Top Abstract Introduction Study design Results and discussion References Autoimmune phenomena may precede or accompany lymphoid malignancies, especially B-chronic lymphocytic leukemia (B-CLL). We report a patient with a 7-year history of primary (idiopathic) cold agglutinin (CA) disease in whom B-CLL subsequently developed. Immunophenotyping and single-cell reverse transcription-polymerase chain reaction (RT-PCR) were applied to investigate the origin and diversification of leukemic B cells. The obtained data indicate a memory cell-type origin of the B-CLL cells. Remarkably, the IgV genes of the B-CLL cells showed intraclonal diversity, whereas the mutational pattern of their paired IgVH genes were invariant. Thus, the light-chain-restricted intraclonal diversity in individual leukemic B cells in this patient strongly indicates a differential regulation or selection of the ongoing mutational process. Of note, our findings suggest that this B-CLL had developed from the patient's CA-producing B-cell population. (Blood. 2002;100:3419-3422) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Several humoral autoimmune phenomena have been reported in B-chronic lymphocytic leukemia (B-CLL), including autoimmune hemolytic anemia.1 However, it remains uncertain whether these phenomena arise from the malignant cells or from residual B cells.2,3 In both possibilities, autoimmunity should develop in association with or following malignancy. On the other hand, autoimmune dysregulation is a well-known risk factor of the development of lymphoid malignancies.4 B-CLL developed in a patient with existing chronic B-cell dyscrasia that had become clinically apparent as primary cold agglutinin disease (CAD),5 which is generally regarded as premalignant or low-grade malignant lymphoproliferation.6 Thus, a B-CLL origin from the cold agglutinin (CA)-expressing population was likely. Because the respective autoantigens in CAD, mostly belonging to the I/i system, are widely expressed on healthy red blood cells, high antigenic pressure on the proliferating B cells may be hypothesized. Using single-cell reverse transcription-polymerase chain reaction (RT-PCR) technology, the current study examined whether B-CLL cells express immunoglobulin (Ig) receptors that share features of CAs and whether signs of intraclonal diversification and ongoing antigenic selection can be found. Notably, a light-chain-restricted, antigen-independent intraclonal diversification was found in IgM+/CD5+ B-CLL cells expressing mutated VH4-34/VA27 gene rearrangements.     Study design Top Abstract Introduction Study design Results and discussion References Patient A 56-year-old man sought treatment for a 1-year history of cold-dependent acrocyanosis and acral numbness. Primary CAD5 was diagnosed by the detection of high-titer (1:4000 at 4°C) monoclonal IgM/ CA and the exclusion of CA syndrome secondary to another disease, especially lymphoma. CA specificity was determined as anti-I by reactivity against panels of group 0+ erythrocytes. Results of direct antiglobulin testing using anticomplement sera were positive, and mild hemolytic anemia was observed. The patient responded to the prevention of cold exposition, repeated plasma exchange, and administration of low-dose prednisolone. During a 7-year follow-up, the paraproteinemia level increased slowly, whereas IgG serum levels (10 to 5.5 g/L) decreased. Increasing CA titers (up to 1:512 000) but moderate autoimmune hemolytic anemia (hemoglobin count, 10.4 g/dL) accompanied worsening of the acral symptoms. Platelet count was normal. Increased and sustained absolute levels of small, mature-appearing lymphocytes (6.5 × 109/L-7.2 × 109/L) with coarsely clumped chromatin were observed in the peripheral blood. In the bone marrow, erythroid hyperplasia and nodular infiltrates of CD20+/CD23+/CD5+/IgM+ lymphoid cells (accounting for approximately 40% of all nuclear cells) were detected. The diagnosis of B-CLL7 was confirmed by flow cytometry and by analysis of IgV gene rearrangements.8,9 Approval was obtained from the institutional review board of the Charité University Hospital for these studies. Informed consent was provided according to the Declaration of Helsinki. Cytometric analysis Peripheral blood mononuclear cells (PBMCs) were prepared as described10 from heparinized venous blood samples kept at 37°C. CD19+ B cells were enriched by negative immunomagnetic separation (Miltenyi Biotec, Bergish-Gladbach, Germany). Incubation with antibodies, propidium iodide staining, and flow cytometric analysis were performed as reported previously.10 Single-cell cDNA synthesis and IgV gene analysis For analysis of IgV gene mRNA transcripts, individual CD19+ B cells from the expanded CD5+/sIgM+ population were sorted (FACSVantage, Becton Dickinson, CA) into single wells containing modified 1× RT-PCR buffer (5 mM dithiothreitol, 400 ng oligo-dT18, 0.2 mM dNTP, 1% Triton, 10 U RNAsin, 40 U avian myeloblastosis virus reverse transcriptase). First-strand cDNA was generated at 42°C for 60 minutes. VH and V mRNA transcripts were amplified by family-specific nested PCR protocols4,11,12 using 5 µL cDNA in the first round. VJ gene rearrangements were amplified from wells with clonally related heavy-chain transcripts. Moreover, the protooncogene bcl-2 mRNA was specifically amplified13 from these wells to further characterize the clonally related cells. To delineate disease stage, before and after diagnosis of B-CLL, cDNAs from metachronously collected enriched CD19+ B cells and single sorted B cells were subjected to specific nested PCR protocols to identify clonal IgVH rearrangements4,11 and bcl-2 and bcl-2/IgH fusion transcripts.13,14 Following column purification, all PCR products were directly sequenced using the BigDye Termination Sequencing Kit (Perkin-Elmer, Foster City, CA) and were analyzed with an automated sequencer (ABI 377; Perkin-Elmer). Sequence alignments were performed by DNAPLOT version 2.0.1 (University of Cologne, Germany) using the VBASE Sequence Directory15 or GenBank. Analysis of mutations was performed as described.16     Results and discussion Top Abstract Introduction Study design Results and discussion References The demonstration of an expanded monotypic B-cell population is central to the diagnosis of B-CLL. In this study, the diagnosis of B-CLL was confirmed by the detection of a markedly expanded CD19+/CD20+/CD5+/CD27+/sIgM+/CD10 peripheral blood population (comprising approximately 98% of all CD19+ cells), including a striking  light-chain restriction (Figure 1).7-9 No sIgG, sIgD, or sIgA was detected. View larger version (25K): [in this window] [in a new window]   Figure 1. Flow cytometric analysis of the expanded peripheral blood B-cell population. Patient PBMCs were stained for CD19 and CD27 and gated according to light-scatter properties. Propidium iodide and CD19 expression were used to identify viable CD19+ cells. Cells were counterstained for CD20, CD5, CD10, sIgM, sIg light chain, and sIg light chain, respectively, and were plotted against CD27. By single-cell RT-PCR, these leukemic B cells were found to express mutated IgVH/V gene pairings, indicating their memory cell-type origin.17,18 Twenty-eight clonally related Cµ-transcripts were analyzed, each encompassing a mutated VH4-34 gene segment (mutational frequency, 7.7%) rearranged to D3-22 and JH3 segments, with a unique 42-base pair (bp) complementarity determining region (CDR)3. Invariantly, all of these VH rearrangements shared the same mutations. The ratios of replacement (R) and silent (S) mutations determined for the FRs and CDRs were 4:7 (0.6) and 4:1 (4.0), respectively. A significant scarcity of FR R mutations, as observed in the patient's leukemic heavy-chain transcripts, is regarded to reflect negative selection of R mutations in these regions to maintain the functional stability of the expressed immunoglobulin molecule.16 Coexpressed light-chain transcripts were identified in 21 of the 28 analyzed leukemic B cells. All expressed mutated VA27-J2 rearrangements, with the same 27-bp CDR3 lacking any indication of TdT activity. Twelve light-chain rearrangements displayed an identical mutational pattern (mutational frequency, 4.8%). The R/S ratios determined for FRs and CDRs were 5:1 (5.0) and 3:2 (1.5), respectively. Most notably, 9 rearrangements displayed additional bp exchanges in their FRs or CDRs (Figure 2a) that were confirmed by several PCR reamplifications from the original wells. These nucleotide exchanges appeared to reflect ongoing mutations because their frequency (13/5271 bp; 2.5 × 103/bp) was significantly higher than the estimated PCR error rate12 (1 × 104/bp; P < .001; 2 analysis), and no variations were found in 7420 bp on the heavy-chain level. The intraclonal relationship of cells expressing diverse VA27 transcripts is given in a genealogical scheme19 (Figure 2B). To further delineate the origin of these clonally related cells, the presence of the protooncogene bcl-2 mRNA20 was analyzed by specific nested PCR and DNA sequencing. Notably, bcl-2 transcripts were detected in invariant and variant clonally related single cells in the stage of B-CLL, further indicating their relationship to the same population. By contrast, clonally related single-sorted B cells (4 of 96; 4.2% of sorted cells) obtained before the diagnosis of B-CLL did not express bcl-2 transcripts. Moreover, enriched CD19+ B cells obtained at the time of B-CLL were positive for bcl-2 and bcl-2/IgH fusion transcripts, whereas those collected before the time of B-CLL were negative for both products, suggesting that this transformation step occurred between these time points.20 View larger version (34K): [in this window] [in a new window]   Figure 2. Sequence analysis of the V transcripts from individual VH4-34 expressing/IgM+/CD5+ B-CLL cells. (A) Intraclonal light-chain diversity in 21 clonally related VA27-J2 transcripts. VA27 was identified as the closest germline sequence.15 Dashes indicate sequence identity. Somatic mutations are shown either as small letters for silent mutations or as capital letters for replacement mutations. Positions of CDRs and FRs are marked according to Kabat et al.28 The numbers of V rearrangements carrying the same sequences are indicated. All V and corresponding VH sequences have been reported to GenBank (accession numbers AY057999-AY058047). (B) Genealogical tree of cells expressing additional mutations in clonally related VA27-J2 transcripts, as demonstrated in panel A. Numbers within each circle represent the codon position of the respective V rearrangements. Mutated nucleotides are underlined, and silent or replacement substitutions are shown as small or capital letter, respectively. Resultant R/S ratios of CDRs and FRs, respectively, for each of the clonally related VA27-J2 transcripts are also given. To our knowledge, this is the first report of intraclonal variable light-chain diversification in IgM+/CD5+ B-CLL.21,22 However, given that the common and diverse mutations were randomly scattered throughout the V segments lacking a preferential accumulation of R mutations in the CDRs, the ongoing mutational process seemed to be antigen independent. In this regard, our findings are in line with those of a recent study suggesting that antigen may no longer be able to play a significant role in the clonal expansion of CD5+/IgM+ B-CLL cells.22 One explanation for the variable light-chain-restricted intraclonal heterogeneity in our study might be an ongoing antigen-independent but differential mutational process affecting only the light-chain locusfor example, because of differences in accessibility or genetic abnormalities.23 In this context, preliminary data show that approximately 10% of patients with B-CLL have mutations in their rearranged variable light-chain genes only.18 Whether these findings belong to a distinct subset of patients, however, remains uncertain. Remarkably, clinical findings, shared immunoglobulin isotypes with the paraprotein, and VH4-34/VIII expression24-26 suggest that the patient's leukemic cells had developed from his CA-expressing population. This assumption is supported by the detection of the same monoclonal and unique VH4-34/CDR3/Cµ sequence in the patient's B cells obtained before diagnosis of B-CLL lacking bcl-2/IgH fusion transcripts and in B cells obtained at the stage of B-CLL with bcl-2/IgH translocation. Notably, VH4-34 expression is crucial in the specific recognition of the anti-I/i system,24-26 whereas the HCDR3 and light chain are thought to modify the fine specificity and affinity of binding.26 Moreover, expansions of CD5+/CD20+ B cells in CAD6,27 and, conversely, B-CLL cells producing CA2 have been reported. Although antibodies using VH4-34 do not necessarily have anti-I/i specificity25 and although VH4-34 usage in IgM+ B-CLL cells is frequent,17,18 selection by the I-antigen might have contributed to the distinct mutational pattern of the patient's expanded CD5+/sIgM+ B-cell population. In conclusion, single-cell RT-PCR provides a powerful tool for analyzing IgV gene expression in cellular (sub)populations of heterogenous lymphoproliferative diseases, such as B-CLL, to enable further insight into the association with known autoimmune phenomena.     Footnotes Submitted September 21, 2001; accepted June 13, 2002. S.R. was a fellow of the Competence Network Rheumatology at the Department of Medicine/Rheumatology and Clinical Immunology, Berlin, Germany. T.D. and A.H. contributed equally to this work. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Arne Hansen, Outpatients Department of Medicine, Charité University Hospital, Schumanstrasse 20/21, 10117 Berlin, Germany; e-mail: arne.hansen{at}charite.de.     References Top Abstract Introduction Study design Results and discussion References 1. Mauro FR, Foa R, Cerretti R, et al. Autoimmune hemolytic anemia in chronic lymphocytic leukemia: clinical, therapeutic, and prognostic features. Blood. 2000;95:2786-2792[Abstract/Free Full Text]. 2. Sthoeger ZM, Sthoeger D, Shtalrid M, Sigler E, Geltner D, Berrebi A. Mechanism of autoimmune hemolytic anemia in chronic lymphocytic leukemia. Am J Hematol. 1993;43:259-264[Medline] [Order article via Infotrieve]. 3. Ishida F, Saito H, Kitano K, Kiyosawa K. Cold agglutinin disease by autoanti-i blood type antibody associated with B cell chronic lymphocytic leukemia. Int J Hematol. 1998;67:69-73[Medline] [Order article via Infotrieve]. 4. Bahler DW, Swerdlow SH. Clonal salivary gland infiltrates associated with myoepithelial sialadenitis (Sjögren's syndrome) begin as nonmalignant antigen-selected expansion. Blood. 1998;91:1864-1872[Abstract/Free Full Text]. 5. Schubothe H. The cold hemagglutinin disease. Semin Hematol. 1966;3:27-47[Medline] [Order article via Infotrieve]. 6. 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Cambridge United Kingdom: MRC Centre for Protein Engineering; 1999. 16. Chang B, Casali P. The CDR1 sequences of a major proportion of human germline IgVH genes are inherently susceptible to amino acid replacement. Immunol Today. 1994;15:367-373[CrossRef][Medline] [Order article via Infotrieve]. 17. Fais F, Ghiotto F, Hashimoto S, et al. Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J Clin Invest. 1998;102:1515-1525[Medline] [Order article via Infotrieve]. 18. Damle RN, Wasil T, Fais F, et al. IgV gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood. 1999;94:1840-1847[Abstract/Free Full Text]. 19. Shlomchik MJ, Marshak-Rothstein A, Wolfowicz CB, Rothstein TL, Weigert MG. The role of clonal selection and somatic mutation in autoimmunity. Nature. 1987;328:805-811[CrossRef][Medline] [Order article via Infotrieve]. 20. Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol. 124:1-6. 21. Korganow AS, Martin T, Weber JC, et al. Molecular analysis of rearranged VH genes during B cell chronic lymphocytic leukemia: intraclonal stability is frequent but not constant. Leuk Lymphoma. 1994;14:55-69[Medline] [Order article via Infotrieve]. 22. Schettino EW, Cerutti A, Chiorazzi N, Casali P. Lack of intraclonal diversification in Ig heavy and light chain V region genes expressed by CD5+IgM+ chronic lymphocytic leukemia B cells: a multiple time point analysis. J Immunol. 1998;160:820-830[Abstract/Free Full Text]. 23. Oscier DG, Thompsett A, Zhu D, Stevenson FK. Differential rates of somatic hypermutation in VH genes among subsets of chronic lymphocytic leukemia defined by chromosomal abnormalities. Blood. 1997;89:4153-4160[Abstract/Free Full Text]. 24. Silberstein LE, Jefferies LC, Goldman J, et al. Variable region gene analysis of pathologic human autoantibodies to the related i and I red blood cell antigens. Blood. 1991;78:2372-2386[Abstract/Free Full Text]. 25. Pascual V, Victor K, Spellerberg M, Hamblin TJ, Stevenson FK, Capra JD. VH restriction among human cold agglutinins: the VH4-21 gene segment is required to encode anti-I and anti-i specificities. J Immunol. 1992;149:2337-2344[Abstract]. 26. Li Y, Spellerberg MB, Stevenson FK, Capra JD, Potter KN. The I binding specificity of human VH4-34 (VH4-21) encoded antibodies is determined by both VH framework region 1 and complementarity determining region 3. J Mol Biol. 1996;256:577-589[CrossRef][Medline] [Order article via Infotrieve]. 27. Natoya A, Hirayama S, Takano H, et al. Chronic cold agglutinin disease accompanied with an increase of CD20+/CD5+ cells: a case report. Rinsho Ketsueki. 1994;35:881-885[Medline] [Order article via Infotrieve]. 28. Kabat EA, Wu TT, Perry HM, Gottesmann KS, Foeller C. Sequences of proteins of immunological interest. Vol 1. 5th ed. Bethesda, MD: National Institutes of Health; 1991. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: A. D. Volkheimer, J. B. Weinberg, B. E. Beasley, J. F. Whitesides, J. P. Gockerman, J. O. Moore, G. Kelsoe, B. K. Goodman, and M. C. Levesque Progressive immunoglobulin gene mutations in chronic lymphocytic leukemia: evidence for antigen-driven intraclonal diversification Blood, February 15, 2007; 109(4): 1559 - 1567. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Erratum (v101,p1676) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ruzickova, S. Articles by Hansen, A. Search for Related Content PubMed PubMed Citation Articles by Ruzickova, S. Articles by Hansen, A. Related Collections Brief Reports Neoplasia Social Bookmarking                   What's this?

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