Origins of the malignant clone in typical Waldenstrom's macroglobulinemia

Origins of the Malignant Clone in Typical Waldenstrom’s Macroglobulinemia Surinder S. Sahota, Francesco Forconi, Christian H. Ottensmeier, and Freda K. Stevenson Histopathology and phenotypic considerations in IgMsecreting B-cell tumors allow distinction between different diseases, but insight into pathogenesis is restricted. Here, variable region (V) gene analysis can complement classification but, more importantly, can also reveal disease origins and clonal history. We used VH gene analysis to probe origins in Waldenstrom’s macroglobulinemia (WM), and contrasted these with the less malignant counterpart, IgM-secreting monoclonal gammopathy of undetermined significance (MGUS). Limited data on WM sequences had previously shown evidence for somatic mutation, but with conflicting analysis of intraclonal variation in tumor sequences. To further the investigation, we analyzed seven cases of WM and compared these with three cases of IgM MGUS. In both diseases, VH genes were somatically mutated with no evidence of intraclonal variation, even at the MGUS stage. A sensitive VH gene-probe assay revealed no evidence for isotype switch transcripts in any of the WM and IgM MGUS cases. These findings reveal an origin of WM and IgM MGUS from a IgM cell, which transforms after cessation of somatic mutation but without initiating switch events. In contrast, IgM-secreting multiple myeloma arises at a later stage in differentiation, when isotype switch mechanisms have been engaged. Semin Oncol 30:136-141. © 2003 Elsevier Inc. All rights reserved.

W

ALDENSTROM’S macroglobulinemia (WM) is a specific clinical syndrome commonly associated with a low-grade lymphoplasmacytic lymphoma (LPL), and a characteristic monoclonal serum IgM. WM primarily involves the bone marrow and possibly other lymphoid tissues.1,2 Tumor morphology shows infiltration by small lymphocytes with a variable degree of plasma cell matu-

From the Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division, Southampton University Hospitals, Southampton UK; and Cattedra e U.O. di Ematologia, Ospedale “A. Sclavo”, Universita di Siena, Italy. Supported in part by The Smokler Fellowship Award from Research Fund for Waldenstroms, Ltd, New York, NY, The Leukaemia Research Fund UK, and Tenovus UK. Address reprint requests to Surinder S. Sahota, PhD, Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division, Southampton University Hospitals, Tremona Road, Southampton SO16 6YD, UK. © 2003 Elsevier Inc. All rights reserved. 0093-7754/03/3002-0038$30.00/0 doi:10.1053/sonc.2003.50072 136

ration.2 Immunophenotype and histology considerations assist in delineating WM from other lymphomas with a serum monoclonal IgM.2-4 In contrast, IgM monoclonal gammopathy of undetermined significance (MGUS) is characterized by a monoclonal serum protein, without anemia, lymphadenopathy, or splenomegaly, and no evidence of bone marrow lymphocytosis.5 About 20% of IgM MGUS eventually develop WM, lymphoma, chronic lymphocytic leukemia (CLL), or amyloidosis. Little is known of the mechanisms and pathways of progression and pathogenesis of WM. Immunoglobulin variable region (V) gene analysis provides one means of delineating characteristics most likely to be associated with the cell of origin in B-cell tumors. V genes encode antigen specificity, and affinity for antigen can be increased by an active somatic mutation mechanism.6 This process is generally activated following encounter with antigen and T-cell and cytokine help in follicular germinal centers (GCs). Once a GC reaction is initiated, somatic mutation can begin, introducing nucleotide changes into the functionally rearranged V genes at a high rate.6,7 It is now possible to assess with confidence which B cell has undergone somatic mutation, as the germline genes used to encode V genes have been mapped. V region genes are assembled in early B cells by DNA recombination events to generate a functional V(D)J transcriptional unit. This provides a unique CDR3 sequence that can be used to identify and track B-cell clones. It enables V gene usage to be identified, which can reveal asymmetry of use. Exposure of the tumour cell of origin to somatic mutation in fact allows B-cell tumors to be classified in three categories: those having a pre-GC origin, those residing in the GC, and those having been exposed to the GC and exiting.8 Tumor cells that reside in the GC environment bypass constraints of apoptosis in normal B cells, resulting in tumor progeny being continually targeted by somatic mutation and a consequent intraclonal variation in V gene sequences. Isotype switching from IgM(D) to IgG/A/E also occurs in the GC, but is not confined to that site.7 Seminars in Oncology, Vol 30, No 2 (April), 2003: pp 136-141

VH GENES IN WALDENSTROM’S MACROGLOBULINEMIA

137

Switching to a selected isotype depends on availability of appropriate cytokine and other factors.9 It may involve genetic rearrangement with deletion of CH genes, or alternatively can be mediated by RNA processing.10,11 There is in vitro evidence for somatic mutation to precede isotype switch, and to continue post-switch.12 Analysis of isotype switch events is readily feasible via V gene probes, and provides another level of assessing maturational status in tumor cells.8 Specificity is built-in using CDR3-based oligonucleotides. The presence of tumor-derived isotype-switched transcripts can reveal whether tumor cells have activated switch mechanisms, at least at the RNA level. It is an assay with greater sensitivity than Southern blotting, and can detect switched transcripts arising either from CH genes rearranged by DNA recombination or by RNA processing events, which will not be identifiable by analysis at the chromosome level.

formed lines from WM patients secreting IgM with cold agglutinin (CA) activity had also indicated somatic mutation.15 Consistent with this reactivity, WM VH genes were encoded by the V4-34 gene segment, a mandatory requirement for CA activity.15 V␬ gene use in WM had also been analyzed in some cases.16 Intraclonal variation in WM remained unresolved, as analysis of only the CDR3FR4 regions was restrictive. Furthermore, a single WM case was reported where VH gene sequences revealed intraclonal heterogeneity.17 To resolve the stage of arrest in WM, we analyzed the VH gene status of seven cases of typical WM and compared them with three cases of IgM MGUS. Unselected patients from the Hematology clinic were studied. Diagnosis of WM (cases WM1 through WM7) and MGUS (cases MG1 through MG3) disease was made using established clinical and laboratory criteria.1-3,5 All patients had a raised monoclonal serum IgM. Tumor VH genes were identified as previously described from bone marrow– derived mononuclear cells.18 This method of identifying tumor V genes has been validated by evidence from use of anti-idiotypic antibodies and more recently by xenogeneic antibody responses to recombinant tumor-derived V genes.19,20 In each of our cases, potentially functional inframe tumor-derived VH sequences were identified (Table 1 and Fig 1). Although cohort size is limited, there was no observed bias in VH gene usage. Each WM and MGUS tumor VH gene revealed

V GENE ANALYSIS IN WM

Maturation of normal B cells can give rise to IgM-secreting B cells derived either from a primary immune responses associated with unmutated V genes, or from a IgM⫹ memory B cell that has undergone somatic mutation.7,13 Early findings from V gene analysis in WM had suggested origins from a somatically mutated B cell, but data were restricted to evaluation of a short fragment of tumor VH genes, from CDR3 to FR4.14 A few VH sequences from Epstein-barr virus (EBV)-trans-

Table 1. Analysis of WM and IgM MGUS Tumor-Derived VH Genes

% Homology

FR

CDR

JH

TumorDerived Clones/Clones Screened

92.8 91.0 95.5 93.9 89.4 88.4 93.2 92.0 91.2 86.9

6/9 (0.7) 9/3 (3.0) 5/3 (1.7) 5/1 (5.0) 7/7 (1.0) 10/8 (1.3) 12/4 (3.0) 7/8 (0.9) 12/5 (2.4) 14/9 (1.6)

6/0 (⬁) 9/5 (1.8) 4/1 (4.0) 8/4 (2.0) 12/3 (4.0) 9/4 (2.3) 4/0 (⬁) 7/2 (3.5) 5/4 (1.3) 12/3 (4.0)

JH 4b JH 4b JH 6c JH 6b JH 6b JH 3b JH 4b JH 4b JH 4b JH 1

7/10 6/8 8/10 9/9 17/18 11/11 8/24 9/10 10/12 10/12

R/S Mutation Patient

VH Family

Germline Gene

WM1 WM2 WM3 WM4 WM5 WM6 WM7 MG1 MG2 MG3

V H1 VH 3 VH 3 VH 3 VH 3 VH 3 VH 1 VH 3 VH 3 VH 3

V1-02 V3-30 V3-53 V3-9 V3-23 V3-72 V1-02 V3-72 V3-74 V3-23

Data on cases WM1-7 are from Sahota et al.21

Tumor Clones Sequenced 7 6 6 9 16 11 8 8 8 8

138

SAHOTA ET AL

Fig 1. Deduced amino acid sequences of tumor-derived VH genes from WM and IgM MGUS cases, indicating extent of somatic mutation and primer design for alternative transcripts. Comparisons for WM (WM1-7) and IgM MGUS (MG1-3) are made with the closest germline VH gene: uppercase, replacement (R) mutation (underlined in JH); lower case, silent (S) mutation. Each mutation was defined by nucleotide exchanges in a single codon, with successive mutations leading in some cases to two or three distinct R or S events. These are shown as vertically aligned amino acid changes at specific sites. Nucleotide mutations in the last codons of tumor VH genes were only assessed if followed by at least a 2-bp homology to the germline gene. Tumor VH sequence-specific 5ⴕ-primers to assess switched transcripts are double-underlined in individual sequences. Data on cases WM1-7 are from Sahota et al.21

the presence of extensive somatic mutations, indicating exposure of the cell of origin to somatic hypermutation. This confirms previous and more recent observations of V gene status in WM.14-16,22,23 In three of seven WM and two of three MGUS cases (Table 1), an overall R:S ratio of mutations ⬍1.7 in FRs was indicative of selective pressure to maintain structural integrity.24 Although the nature of the antigen is unclear, BCR structure is preserved in WM for potential interactions in situ. We observed no evidence for any intraclonal variation in VH sequence in any of the WM cases, placing the cell of origin as a mature IgM⫹ B cell that arises at a stage where mutational activity has ceased. A similar finding of a lack of intraclonal heterogeneity has recently been confirmed in three cases of WM,25 and now in two further reports.22,23 This also appears to be the case in the less malignant IgM MGUS, although the numbers of cases analyzed need to be expanded. Our findings therefore indicate that typical WM and IgM MGUS derive from a post-follicular cell, with the

static patterns of somatic mutation also found in IgM-secreting myeloma and in typical isotypeswitched myeloma.8 Curiously, arrest in a proportion of cases of isotype-switched MGUS can apparently occur at an earlier stage of differentiation, as indicated by mutational intraclonal heterogeneity in VH genes,26 a feature of follicular lymphoma cells exposed to a continual influence of the mutator mechanism in the GC environment.8 We assessed whether tumor-associated isotype switch events had occurred in WM and IgM MGUS, to map the stage of arrest further. By employing a strategy shown to identify such transcripts in other B-cell tumors in small cohort studies,27,28 no tumor-derived isotype-switch variants could be identified in six WM and three MGUS cases. This indicates that transformation occurred prior to isotype switch in an IgM⫹ B-cell and that tumor cells do not activate switch mechanisms during persistence of the tumor clone. Absence of switch events in four of four WM cases has more recently been confirmed by V gene analysis in a separate study.22

VH GENES IN WALDENSTROM’S MACROGLOBULINEMIA

139

Fig 2. Schematic representation of proposed pathways in the clonal origins of WM. Both IgM MGUS and WM are derived from a cell of origin that has undergone somatic mutation, with arrest following cessation of mutational activity. In IgM MGUS, the cell of origin has a proliferative advantage (Event 1), with progression to malignant WM disease requiring a neoplastic event (Event 2). Steps in this pathway are as yet unknown (“?”). Transformation does not involve isotype switch events. In contrast, origins in IgM myeloma are comparable, but here switch mechanisms have been engaged. Heterogeneity in isotype-switched MGUS, which has also undergone Event 1, can evolve to MM following Event 2, with a clonal outgrowth phenomenon likely. Clonal homogeneity may already be apparent in switched MGUS, with progression to myeloma contingent on at least Event 2. BN, B cell (normal); BT, B cell (tumor).

CLONAL ORIGINS IN WM

Cessation of somatic mutation prior to tumor arrest in WM and IgM MGUS suggests origins from a cell that has exited the GC. Since these tumors localize to the bone marrow, this may be a putative site of arrest. Somatic mutation appears not to occur in the bone marrow, as observed in myeloma cells from presentation to plateau phase of disease.8 Normal IgM⫹ memory B cells can clearly home to the marrow, and retain the capacity to differentiate further to secrete IgM,29 raising the possibility that transformation may occur in such a cell (Fig 2). Since in WM this can be a cell expressing V4-34– encoded immunoglobulin with CA activity,15 immediate differences in origins with myeloma emerge, as these malignant plasma

cells do not utilize V4-34 to produce IgM or IgG/ A.8 Environmental signals will continue to impact on tumor cells, and those cells that commit to isotype switch expose the genome to potential aberrant events during recombination. Certain tumors, such as typical myeloma and IgM-secreting myeloma, show evidence for isotype variants within the clone (Fig 2),8 and for these there is evidence for illegitimate switching as a causal mechanism in transformation.30 In WM and IgMsecreting MGUS, this is not the case, and the question arises whether these cells have a defective switch mechanism, or have not received the appropriate stimuli. Analysis of 14q32 by fluorescence in situ hybridization (FISH) and Southern blots in WM indicate an absence of IgH rearrange-

140

SAHOTA ET AL

ments, confirming absence of isotype switch events by deletional recombination.31 By contrast, IgM myeloma typically exhibit 14q32 rearrangements, with t(11;14) predominating.32 Clearly, tumors can arrest at different points of maturation. Occasional tumors such as lymphoplasmacytoid lymphoma expressing dual IgM⫹IgG⫹ isotypes, with identical or distinct mutational patterns,33 suggest a transitional stage of arrest. The numbers of IgM MGUS cases assessed here are low, but point to a separate and distinct pathway from isotype-switched MGUS, some of which display intraclonal heterogeneity (Fig 2). These post-switch MGUS can, however, transform to multiple myeloma, as shown by tracking progression in individual patients by VH genes.34 In this progression, clonal outgrowth may occur to yield tumor homogeneity at the myeloma stage, in a cell that has acquired the full complement of neoplastic events. It is conceivable that such a cloning phenomenon has already occurred in other isotype-switched MGUS prior to transformation to myeloma.26 In comparison, clonal homogeneity in VH genes may be a feature of IgM MGUS disease and is mirrored at the malignant WM stage. A spectrum of post-follicular tumors can clearly be delineated by V gene analysis, with apparent differences in disease origins. REFERENCES 1. Jaffe ES, Harris NL, Stein H, et al (eds): WHO Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC Press, 2001 2. Harris NL, Jaffe ES, Stein H, et al: A revised EuropeanAmerican classification of lymphoid neoplasms: A proposal from the International Lymphoma Study Group. Blood 84: 1361-1392, 1994 3. Morel P, Monconduit M, Jacomy P, et al: Prognostic factors in Waldenstrom macroglobulinelia: A report on 232 patients with the description of a new scoring system and its validation on 253 other patients. Blood 96:852-858, 2000 4. Owen RG, Johnson SA, Morgan GJ: Waldenstrom’s macroglobulinemia: Laboratory diagnosis and treatment. Hematol Oncol 18:41-49, 2000 5. Kyle RA, Rajkumar SV: Monoclonal gammopathies of undetermined significance. Hematol Oncol Clin North Am 13:1181-1202, 1999 6. Berek C: The development of B cells and the B-cell repertoire in the microenvironment of the germinal center. Immunol Rev 126:5-19, 1992 7. MacLennan IC: Germinal centers. Ann Rev Immunol 12:117-139, 1994 8. Stevenson FK, Sahota SS, Ottensmeier CH, et al: The

occurrence and significance of V gene mutations in B cellderived human malignancy. Adv Cancer Res 83:81-116, 2001 9. Stavnezer J, Sirlin S, Abbott J: Induction of immunoglobulin isotype switching in cultured I.29 B lymphoma cells. Characterization of the accompanying rearrangements of heavy chain genes. J Exp Med 161:577-601, 1985 10. Matsuoka M, Yoshida K, Maeda T, et al: Switch circular DNA formed in cytokine-treated mouse splenocytes: Evidence for intramolecular DNA deletion in immunoglobulin class switching. Cell 62:135-42, 1990 11. Fujieda S, Lin YQ, Saxon A, et al: Multiple types of chimeric germ-line Ig heavy chain transcripts in human B cells: Evidence for trans-splicing of human Ig RNA. J Immunol 157:3450-3459, 1996 12. Zan H, Cerutti A, Dramitinos P, et al: 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 162: 3437-3447, 1999 13. Klein U, Kuppers R, Rajewsky K: Evidence for a large compartment of IgM-expressing memory B cells in humans. Blood 89:1288-1298, 1997 14. Aoki H, Takishita M, Kosaka M, et al: Frequent somatic mutations in D and/or JH segments of Ig gene in Waldenstrom’s macroglobulinemia and chronic lymphocytic leukemia (CLL) with Richter’s syndrome but not in common CLL. Blood 85: 1913-1919, 1995 15. Pascual V, Victor K, Lelsz D, et al: Nucleotide sequence analysis of two IgM cold agglutinins. Evidence that the VH4-21 gene segment is responsible for the major cross-reactive idiotype. J Immunol 146:4385-4391, 1991 16. Wagner SD, Martinelli V, Luzzato L: Similar patterns of V␬ gene usage but different degrees of somatic mutation in hairy cell leukemia, prolymphocytic leukemia, Waldenstrom’s macroglobulinemia, and myeloma. Blood 83:3647-3653, 1994 17. Ciric B, VanKeulen V, Rodriguez M, et al: Clonal evolution in Waldenstrom’s macroglobulinemia highlights functional role of B-cell receptor. Blood 97:321-323, 2001 18. Hawkins RE, Zhu D, Ovecka M, et al: Idiotypic vaccination against human B-cell lymphoma. Rescue of variable region gene sequences from biopsy material for assembly as single-chain Fv personal vaccines. Blood 83:3279-3288, 1994 19. Stevenson FK, Spellerberg MB, Treasure J, et al: Differential usage of an Ig heavy chain variable region gene by human B-cell tumors. Blood 82:224-230, 1993 20. Forconi F, King CA, Sahota SS, et al: Insight into the potential for DNA idiotypic fusion vaccines designed for patients by analyzing xenogeneic anti-idiotypic antibody responses. Immunology 106:1-12, 2002 21. Sahota SS, Forconi F, Ottensmeier CH, et al: Typical Waldenstrom macroglobulinemia is derived from a B-cell arrested after cessation of somatic mutation but prior to isotype switch events. Blood 100:1505-1507, 2002 22. Kriangkum J, Taylor BJ, Mant MJ, et al: The malignant clone in Waldenstrom’s macroglobulinemia. Semin Oncol 30: 132-135, 2003 23. Schop RFJ, Fonseca R: Genetics and cytogenetics of Waldenstrom’s macroglobulinemia. Semin Oncol 30:142-145, 2003 24. Dorner T, Foster SJ, Brezinschek HP, et al: Analysis of the targetting of the hypermutational machinery and the im-

VH GENES IN WALDENSTROM’S MACROGLOBULINEMIA

pact of subsequent selection on the distribution of nucleotide changes in human VHDJH rearrangements. Immunol Rev 162: 161-171, 1998 25. Shiokawa S, Suehiro Y, Uike N: Sequence and expression of ␮ and ␦ transcripts in patients with Waldenstrom’s macroglobulinemia. Am J Hematol 68:139-143, 2001 26. Sahota SS, Leo R, Hamblin TJ, et al: Ig VH mutational patterns indicate different tumor cells status in human myeloma and monoclonal gammopathy of undetermined significance. Blood 87:746-755, 1996 27. Sahota SS, Garand R, Mahroof R, et al: VH gene analysis of IgM-secreting myeloma indicates an origin from a memory cell undergoing isotype switch events. Blood 94:1070-1076, 1999 28. Forconi F, Sahota SS, Raspadori D, et al: Tumor cells of hairy cell leukemia express multiple clonally related immunoglobulin isotypes via RNA splicing. Blood 98:1174-1181, 2001 29. Paramithiotis E, Cooper MD: Memory B lymphocytes migrate to bone marrow in humans. Proc Natl Acad Sci USA 94:208-212, 1997

141

30. Bergsagel PL, Chesi M, Nardini E, et al: Promiscuous translocations into immunoglobulin heavy chain switch regions in multiple myeloma. Proc Natl Acad Sci 13931-13936, 1996 31. Schop RF, Kuehl WM, Van Wier SA, et al: Waldenstrom macroglobulinemia neoplastic cells lack immunoglobulin heavy chain locus translocations but have frequent 6q deletions. Blood 100:2996-3001, 2002 32. Avet-Loiseau H, Garnad R, Lode L, et al: 14q32 translocations discriminate IgM multiple myeloma from Waldenstrom’s macroglobulinemia. Semin Oncol 30:153-155, 2003 33. Sahota SS, Garand R, Bataille R, et al: VH gene analysis of clonally related IgM and IgG from human lymphoplasmacytoid B-cell tumors with chronic lymphocytic leukemia features and high serum monoclonal IgG. Blood 91:238-243, 1998 34. Zojer N, Ludwig H, Fiegl M, et al: Patterns of somatic mutations in VH genes reveal pathways of clonal transformation from MGUS to multiple myeloma. Blood (in press)