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Prepublished online as a Blood First Edition Paper on January 16, 2003; DOI 10.1182/blood-2002-09-2825. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-09-2825v1 101/10/4137    most recent 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 Zojer, N. Articles by Sahota, S. S. Search for Related Content PubMed PubMed Citation Articles by Zojer, N. Articles by Sahota, S. S. Related Collections Immunobiology Neoplasia Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 May 2003, Vol. 101, No. 10, pp. 4137-4139 NEOPLASIA Brief report Patterns of somatic mutations in VH genes reveal pathways of clonal transformation from MGUS to multiple myeloma Niklas Zojer, Heinz Ludwig, Michael Fiegl, Freda K. Stevenson, and Surinder S. Sahota From the Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division, Southampton University Hospitals, Southampton, United Kingdom; the First Department of Internal Medicine and Medical Oncology, Wilhelminenspital, Vienna, Austria; and the Division of Hematology and Oncology, Department of Internal Medicine, University Hospital Innsbruck, Innsbruck, Austria.     Abstract Top Abstract Introduction Study design Results and discussion References Monoclonal gammopathy of undetermined significance (MGUS) can transform to multiple myeloma (MM). In myeloma, mutated VH genes with sequence homogeneity reveal a postfollicular origin. Previously, some MGUS cases showed mutated VH genes with intraclonal variation, indicating an earlier stage of arrest. We investigated progression from 2 of 2 MGUS to MM, in which VH genes confirmed clonal evolution. In one MGUS case, intraclonal heterogeneity was evident, and transformation to myeloma occurred rapidly with apparent homogeneity in the emergent clone. However, residual MGUS-derived sequences were detectable at this time. Heterogeneity in MGUS does not associate with benign disease, but it indicates an origin from a tumorigenic cell, most likely surface immunoglobulin+, undergoing somatic mutation. The remaining case displayed intraclonal homogeneity at the MGUS stage, conceivably resulting from a self-cloning outgrowth from MGUS with heterogeneity. Transformation can occur at either MGUS stage, but it involves a single cell in which somatic mutation is then silent. (Blood. 2003;101:4137-4139) © 2003 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Monoclonal gammopathy of undetermined significance (MGUS) is defined by key criteria: a plasma cell infiltration of less than 10% of bone marrow mononuclear cells, serum monoclonal M-protein of equal to or less than 30 g/L, absence of lytic lesions, related anemia, hypercalcemia, and renal insufficiency.1 MGUS varies in disease outcome, in some cases transforming to multiple myeloma (MM). Little is known concerning the underlying pathways of this progression, or indeed if the same clone is always involved. In part, these questions are hampered by the paucity of suitable tumor material for longitudinal analysis. More recently, evolution from MGUS to MM has been shown to exhibit a chromosome 13q abnormality.2,3 Analysis of immunoglobulin variable (V) region genes now provides a means of identifying salient features of B-cell tumor biology. Tumor V genes retain an imprint of the clonal history of the cell of origin.4 They can reveal the impact of somatic mutation, most likely to have occurred in the germinal center (GC).5 This is initiated in vitro by ligation of surface immunoglobulin (sIg) in normal and neoplastic B cells.6-8 Tumors residing in the GC display intraclonal variation in V gene sequence patterns, a product of ongoing mutational activity at this site.4,9 Extensive data have characterized a postfollicular origin for MM, with more than 100 VH gene sequences displaying homogeneity of clonal tumor-derived sequences.4 In contrast, 3 of 7 MGUS revealed significant intraclonal variation in VH sequence in tumor clones, suggesting at least in a subset of MGUS a maintenance of the mutational mechanism.10 We now report 2 cases, evaluated during progression from MGUS to myeloma by VH gene analysis. Our findings reveal that the same tumor clone evolves to malignant status, and, importantly, analysis of somatic mutations suggests different pathways of disease progression.     Study design Top Abstract Introduction Study design Results and discussion References Patients Case 1 presented with advanced lung cancer with pulmonary involvement and a lesion in TH10. Immunofixation identified a trace IgA band. Bone-marrow (BM) biopsy (sample 1/1) revealed normal composition, with plasma cells (PCs) lower than 5%. Magnetic resonance imaging revealed infiltration of most vertebra 4 months later. Serum M-component was demonstrated (total IgA, 64 g/L; M level, not determined [ND]), with 80% PCs in BM (sample 1/2). A retrospective classification of TH10 could not rule out an isolated plasmacytoma at first biopsy; however, this had excluded any clinical marrow involvement. Further, postmortem examination revealed disseminated myeloma and confirmed concomitant squamous-cell cancer with multiple pulmonary lesions, suggesting a possible metastatic TH10 spread. Case 2 initially had an M-gradient in her serum electrophoresis (total IgG, 15.7 g/L; M level, ND; PCs, 5% of nucleated BM cells; sample 2/1) that progressed over 67 months (IgG, 26.6 g/L; BM PCs, 30%; sample 2/2). Cell preparation and VH gene analysis BM aspirates were taken for magnetic activated cell sorting of CD138+ cells.11 Total RNA isolation from CD138+ cells (1 × 104-2.6 × 106 cells) and cDNA preparation using oligo-dT or constant region (CH) primer were as described.12 cDNA was amplified using VH1-7 leader primers with 3' IgG or IgA primer. Cloning, sequence analysis, and VH gene alignments were as reported.12 For VH genes, between 16 to 30 clones/sample were sequenced from 2 separate polymerase chain reactions (PCRs). For each MGUS and MM sample, 246 to 258 bp of the tumor CH were amplified and cloned to establish the Taq error rate using single 5' complementary determining region 3 (CDR3) and C or C primers (916-3690 bp analyzed/sample). Taq error depends primarily upon the initial amount of template available. We used the same cDNA preparation and identical amounts of template input for VH and CH amplification. This corrected for variations in amount of RNA isolated and efficiency of cDNA synthesis, reflected in the range of Taq error observed in CH (3.4 × 104 to 1.6 × 103 bp1). Replicate analysis confirmed Taq error.     Results and discussion Top Abstract Introduction Study design Results and discussion References Case 1 utilized V3-11 and case 2 V4-30.2 in sequential samples. These showed evidence for extensive somatic mutations (nucleotide sequences have been deposited in the European Molecular Biology Laboratory database; accession numbers AJ536045-AJ536051). We observed intraclonal variation in sequence at the MGUS stage in case 1. Mutations in codons 107T>C and 120A>G were identified in 2 respective clones obtained from separate PCRs (Figure 1). Additional single nucleotide changes were also observed (not shown), with a mutational frequency (1.6 × 103 bp1) exceeding Taq errors in CH (3.4 × 104 bp1; 11 clones from 2 PCRs). This confirms heterogeneity in MGUS.10 It implicates a cell that is undergoing continual somatic mutation following tumorigenic arrest. This may be occurring in the GC environment, suggesting a less differentiated but sIg+ cell.6-8 This cell has acquired a proliferative potential, perhaps due to a chromosomal event 1 (Figure 2). It allows progeny with heterogeneous sequences to survive and migrate to the marrow to differentiate further. This heterogeneity also exists in clonally derived Cµ transcripts, demonstrated in only one MGUS case to date,10 and implicating the stage of isotype switch in event 1.  View larger version (12K): [in this window] [in a new window]   Figure 1. Intraclonal heterogeneity observed in tumor VH gene sequences at the MGUS stage in case 1 is identified as residual, persisting clones at the myeloma stage of disease. The mutational frequency of variations in clonal sequences exceeded Taq error in sample 1/1 from the MGUS stage, with repeated nucleotide changes seen in 2 clones from separate PCRs (codons 107 and 120) confirming intraclonal heterogeneity. In sample 1/2 from the MM stage, some of these mutations were again identified (codons 68 and 107), clearly excluding Taq error. The mutational frequency of intraclonal variation, however, was comparable with the background Taq misincorporation rate in this sample, suggesting a cessation of somatic mutation at the MM stage. Most likely, variant clones here represent residual MGUS cells, as disease evolution was remarkably rapid. Only informative codon sequences at the nucleotide level are shown, with homology to the donor germ line gene V3-11 depicted as dashes. Mutations repeatedly observed in MGUS and MM samples are marked with an asterisk. Numbers of clones with each sequence varied (n = 1-26). FR3 indicates framework region 3; and JH, joining (H) gene. View larger version (21K): [in this window] [in a new window]   Figure 2. Pathways of clonal progression from MGUS to multiple myeloma. Mutational patterns in VH genes define diversity in MGUS, with some cases showing intraclonal heterogeneity. Plasma cells in these MGUS cases most likely derive by maturation of a less differentiated sIg+ cell that is undergoing somatic mutation and has acquired a chromosomal event 1 to gain a proliferative potential. Transformation to myeloma can clearly occur in MGUS displaying heterogeneity, but does so in a single cell in which somatic mutation is silent and that acquires neoplastic event 2. This transformation may be rapid, and residual MGUS clones can still be identified at this time but will eventually be lost by clonal outgrowth as myeloma is homogeneous. A self-cloning process may also lead to the MGUS cases identified by intraclonal stability of sequence, which are more common. These cases can then transform to myeloma as a result of event 2. VDJ indicates functionally rearranged variable-diversity-joining gene. Following transformation to myeloma in this patient (sample 1/2; Figure 1), the observed ongoing mutational activity appeared to be silenced, as the frequency of single nonclonal mutations (1.5 × 103 bp1) was similar to Taq error (1.6 × 103 bp1). However, we observed 2 mutational changes at codons 68T>C and 107T>C in single clones at the MM stage that were identical to mutations at the early stage of disease (Figure 1). These 2 mutations were detected in samples taken at 2 different disease stages, negating random PCR error. Most likely, they represent residual MGUS clones as transformation was rapid. In the absence of paired samples, a very low level of nonshared mutations in some MM clones would have been assigned as Taq error in V gene analysis. Reports of stability of myeloma V gene sequence indicate the rarity of identifying such persisting residual clones by current technology.4 However, there have been sporadic reports that hint at this possibilty. In 1 of 4 MM cases with more than 40% tumor cells, 2 clonally-related subclones with heterogeneity were observed,13 possibly reflecting a narrowing of a wider variation that existed previously. In a further MM case, significant intraclonal diversity was noted in the IgA variant transcripts but not the tumor isotype, suggesting emergence of a dominant clone but with residual variants.14 Given that MM is clonally homogeneous even in Cµ tumor-derived sequences,15-17 this suggests that the final transformation event (event 2, Figure 2) occurs in one cell of the population, at a site where somatic mutation is silent, possibly the bone marrow. In case 2, we observed intraclonal homogeneity at both the MGUS and MM stage, confirming previous observations in 4 of 7 MGUS cases.10 A self-cloning phenomenon may underlie this observed sequence homogeneity in MGUS, being analyzed at a stage where hypermutation has ceased and heterogeneity has been lost because of clonal outgrowth. Transformation to myeloma then occurs at the invariant stage, involving event 2 (Figure 2). VH gene analysis has revealed diversity in MGUS and has suggested that transformation may involve different pathways. MGUS may derive from an tumorigenic sIg+ cell undergoing somatic mutation, which differentiates into Ig-secreting plasma cells. This population may persist and possibly self clone to homogeneity. However, it is susceptible to further transforming events that can occur in one cell at the heterogeneous or homogeneous stage, giving rise to homogeneous MM that is independent of the sIg+ population. Progression to myeloma then involves factors intrinsic to this cell.     Acknowledgments We wish to thank Dr Judith Schuster and Irene Assmann for assistance in cell sorting.     Footnotes Submitted September 19, 2002; accepted December 23, 2002. Prepublished online as Blood First Edition Paper, January 16, 2003; DOI 10.1182/ blood-2002-09-2825. Supported by a Novartis-UICC Translational Cancer Research Fellowship (funded by Novartis AG, Switzerland), the European Society for Medical Oncology (ESMO), the International Myeloma Foundation (United Kingdom) and The Leukaemia Research Fund (United Kingdom). 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: Surinder S. Sahota, Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division, Southampton University Hospital Trust, Southampton, SO16 6YD, United Kingdom; e-mail: s.s.sahota{at}soton.ac.uk.     References Top Abstract Introduction Study design Results and discussion References 1. Kyle RA, Therneau TM, Rajkumar SV, et al. A long-term study of prognosis in monoclonal gammopathy of undetermined significance. N Engl J Med. 2002;346:564-569[Abstract/Free Full Text]. 2. Bernasconi P, Cavigliano PM, Boni M, et al. Long-term follow up with conventional cytogenetics and band 13q14 interphase/metaphase in situ hybridization monitoring in monoclonal gammopathies of undetermined significance. Br J Haematol. 2002;118:545-549[CrossRef][Medline] [Order article via Infotrieve]. 3. Kaufmann H, Ackerman J, Noesslinger T, et al. Deletion of chromosome 13q is a frequent abnormality in multiple myeloma evolving from a preexisting monoclonal gammopathy of undetermined significance [abstract]. Blood. 2002;100:103a. 4. Stevenson FK, Sahota SS, Ottensmeier CH, Zhu D, Forconi F, Hamblin TJ. The occurrence and significance of V gene mutations in B cell-derived human malignancy. Adv Cancer Res. 2001;83:81-116[Medline] [Order article via Infotrieve]. 5. Berek C. The development of B cells and the B-cell repertoire in the microenvironment of the germinal center. Immunol Rev. 1992;126:5-19[Medline] [Order article via Infotrieve]. 6. Denepoux S, Razanajaona D, Blanchard D, et al. Induction of somatic mutation in a human B cell line in vitro. Immunity. 1997;6:35-46[Medline] [Order article via Infotrieve]. 7. Razanajaona D, Denepoux S, Blanchard D, et al. In vitro triggering of somatic mutation in human naive B cells. J Immunol. 1997;159:3347-3353[Abstract]. 8. Zan H, Cerutti A, Dramitinos P, Schaffer A, Li Z, Casali P. Induction of Ig somatic hypermutation and class switching in a human monoclonal IgM+ IgD+ B cell line in vitro: definition of the requirements and modalities of hypermutation. J Immunol. 1999;162:3437-3447[Abstract/Free Full Text]. 9. Zhu 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;86:505-512[Medline] [Order article via Infotrieve]. 10. Sahota SS, Leo R, Hamblin TJ, Stevenson FK. Ig VH gene mutational patterns indicate different tumor cell status in human myeloma and monoclonal gammopathy of undetermined significance. Blood. 1996;87:746-755[Abstract/Free Full Text]. 11. Schuster-Kolbe J, Ludwig H, Adolf GR, Heider KH. Expression of CD44 isoforms on isolated bone marrow plasma cells and peripheral CD19+ B cells of patients with multiple myeloma and healthy individuals. Leuk Lymphoma. 1999;34:95-103[Medline] [Order article via Infotrieve]. 12. Forconi F, Sahota SS, Raspadori D, Mockridge CI, Lauria F, Stevenson FK. Tumor cells of hairy cell leukemia express multiple clonally related immunoglobulin isotypes via RNA splicing. Blood. 2001;98:1174-1181[Abstract/Free Full Text]. 13. Biggs DD, Kraj P, Goldman J, et al. Immunoglobulin gene sequence analysis to further assess B-cell origin of multiple myeloma. Clin Diagn Lab Immunol. 1995;2:44-52[Abstract]. 14. Taylor BJ, Pittman JA, Seeberger K, et al. Intraclonal homogeneity of clonotypic immunoglobulin M and diversity of nonclinical post-switch isotypes in multiple myeloma: insights into the evolution of the myeloma clone. Clin Cancer Res. 2002;8:502-513[Abstract/Free Full Text]. 15. Billadeau D, Ahmann G, Greipp P, Van Ness B. The bone marrow of multiple myeloma patients contains B cell populations at different stages of differentiation that are clonally related to the malignant plasma cell. J Exp Med. 1993;178:1023-1031[Abstract]. 16. Corradini P, Boccadoro M, Voena C, Pileri A. Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to C mu sequence in immunoglobulin (IgG)- and IgA-secreting multiple myelomas. J Exp Med. 1993;178:1091-1096[Abstract]. 17. Bakkus MH, Van Riet I, Van Camp B, Thielemans K. Evidence that the clonogenic cell in multiple myeloma originates from a pre-switched but somatically mutated B cell. Br J Haematol. 1994;87:68-74[Medline] [Order article via Infotrieve]. © 2003 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: T. M. Tancred, A. R. Belch, T. Reiman, L. M. Pilarski, and J. Kirshner Altered Expression of Fibronectin and Collagens I and IV in Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance J. Histochem. Cytochem., March 1, 2009; 57(3): 239 - 247. [Abstract] [Full Text] [PDF] D. Gonzalez, M. van der Burg, R. Garcia-Sanz, J. A. Fenton, A. W. Langerak, M. Gonzalez, J. J. M. van Dongen, J. F. San Miguel, and G. J. Morgan Immunoglobulin gene rearrangements and the pathogenesis of multiple myeloma Blood, November 1, 2007; 110(9): 3112 - 3121. [Abstract] [Full Text] [PDF] W. M. Kuehl and P. L. 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