The malignant clone in Waldenstrom's macroglobulinemia

The Malignant Clone in Waldenstrom’s Macroglobulinemia Jitra Kriangkum, Brian J. Taylor, Michael J. Mant, Steven P. Treon, Andrew R. Belch, and Linda M. Pilarski The unique IgM VDJ sequence that characterizes the malignant clone in Waldenstrom’s macroglobulinemia (WM), termed clonotypic, was identified for 12 WM patients. The majority of WM patients (92%) had a clonotypic IgM from the VH3 family, with predominantly long CDR3 regions, characteristic of those found in antigen-stimulated populations. Clonotypic IgM transcripts were detected in both blood and bone marrow (BM), clearly identifying a blood-borne compartment of WM. Abnormal numbers of CD20ⴙ B cells were usually detectable and expressed surface IgM. In most cases these cells also expressed surface IgD. Most WM patients lacked detectable CD138ⴙ plasma cells in either blood or BM. Longitudinal analysis suggests that phenotypic identification of B cells in blood of WM patients is insufficient for monitoring disease. Although serum IgM had decreased and clonotypic transcripts were very weak for one patient, the number of CD20ⴙ B cells increased dramatically. The lack of clonotypic transcripts suggests that the majority of these circulating B cells were polyclonal and were not part of the WM clone, indicating that monitoring of clonotypic IgM provides the most accurate identifier of WM cells. Semin Oncol 30:132-135. © 2003 Elsevier Inc. All rights reserved.

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ALDENSTOM’S macroglobulinemia (WM), a slowly progressive clonal lymphoid disorder, involves lymphoplasmacytic bone marrow (BM) proliferation accompanied by a serum monoclonal IgM component, various autoimmune disorders,1 and organomegaly in half of patients.2-4 WM is associated with disseminated mucosa-associated lymphoid type (MALT) lymphoma, primary splenic marginal zone lymphoma, nodal monocytoid lymphoma, and B-cell chronic lymphocytic lymphoma (B-CLL) with lymphoplasmacytoid differentiation.2,4 No molecular studies have confirmed a clonal relationship between these related cell types, but in some cases they shared the same monotypic immunoglobulin.4

From the Departments of Oncology and Medicine, University of Alberta, Cross Cancer Institute, Edmonton, Canada; and the Dana Farber Cancer Institute, Boston, MA. Supported by a Smokler Award from the Research Fund for Waldenstro¨m’s. Address reprint requests to Linda M. Pilarski, PhD, Department of Oncology, University of Alberta and Cross Cancer Institute, 11560 University Ave, Edmonton, Alberta, T6G 1Z2, Canada. © 2003 Elsevier Inc. All rights reserved. 0093-7754/03/3002-0026$30.00/0 doi:10.1053/sonc.2003.50061 132

Phenotypic characterization of WM suggests that the malignant cells express CD19 and CD20, and the plasmacytic components express CD38.5 WM cells in the BM are reported to lack CD5, CD10, and CD23.6 Analysis of immunoglobulin VDJ regions that identify the WM malignant clone indicates that these cells express somatically mutated variable regions.7-9 Evaluation of replacement to silent mutations within the variable regions suggests that WM cells have previously undergone antigen-mediated selection for higher affinity binding,8 but their consistent expression of IgM suggests that they may have failed to undergo immunoglobulin isotype class switching. In a recent molecular study in which a WM patient developed a biclonal IgM␭, the pattern of mutations within the two dominant clones was suggestive of prolonged antigenic selection after the malignant WM transformation event in this patient.9 Our initial work in WM has shown significant abnormalities among circulating B lymphocytes in WM patients,10-12 as well as phenotypically detectable clonal evolution throughout the disease process.11,12 T-lymphocyte profiles in WM are abnormal, with reduced CD4⫹ T cells and abnormal differentiation patterns, but a subset of apparently normal B cells in WM blood have normal repertoires, as do patients with IgM monoclonal gammopathies.11,13 Phenotypically, abnormal circulating B cells in WM are lymphoblastoid cells, as indicated by their light scatter properties, that express the B-cell markers CD19 and CD20, together with CD5, CD9, CD10, CD11b, and CD24, but they lack CD21 and T-cell markers (as expected).6 They also express PCA-1, a plasma cell marker that is detectable on late stage B cells, and LFA-3, an adhesion receptor. These cells are likely to be monoclonal as indicated by their monotypic light chain expression and by the presence of a rearranged IgH gene as detected by Southern blotting.6 The heterogeneity within the population of monotypic lymphoblastoid B cells in WM was dramatically illustrated by staining with monoclonal antibodies to CD45 isoforms. Normal resting B cells express CD45RA, while late-stage B cells and plasma cells express CD45R0. On activation, normal B cells lose CD45RA and acquire expression of CD45R0 through changes in alternative splicSeminars in Oncology, Vol 30, No 2 (April), 2003: pp 132-135

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ing patterns.9 Multicolor analysis of WM circulating B cells showed that the majority of monoclonal B cells expressed CD45RA and a smaller subset expressed CD45R0.9,12 For two other WM patients, the majority of monoclonal B cells were CD45R0, indicating heterogeneity in differentiation stage within the WM clone within and between patients. Furthermore, analysis of CD45 isoform expression revealed that the WM clone is dynamic, with the WM clone as a whole undergoing apparently continuous differentiation over time.12 This suggests that throughout treatment, evolution of the malignant WM clone favors the emergence of predominantly later stage B cells. Although a clonal relationship between the B-cell subpopulations in WM is likely, there is no direct evidence that addresses this issue. Nor is there evidence to indicate whether or not the clonal evolution observed is prognostically good or bad. This dynamic differentiation over time and in the face of treatment clearly has potentially profound implications for clinical management of the disease. In effect, the clinical therapeutic arsenal must hit a “moving target.” PHENOTYPIC ANALYSIS OF WM

Analysis of 12 patients indicated that abnormally high numbers of CD20⫹ B-lineage cells were detectable in blood and/or bone marrow (BM) of all patients (Figs 1 and 2). Although morpholog-

Fig 1. Percentage of CD20ⴙ cells in mononuclear cell population of BM and peripheral blood of WM patients. Means ⴞ SE are 23% ⴞ 5.2% in BM and 20% ⴞ 4.9% in blood as compared to 2.4% ⴞ 0.6% and 4.5% ⴞ 0.8% in normal controls. Bar represents the normal average.

Fig 2. CD20 and C138 on bone marrow cells from WM. BMC were stained with CD20-fluorescien isothiocyanate (FITC), CD138-phycoerythrin (PE), and CD45perCP in threecolor immunofluorescence, or with sIgM-FITC and sIgD-biotin followed by streptavidin-PE, or with appropriate isotypematched control antibodies.

ically WM is often characterized by plasmacytoid cells, for most patients, very few CD138⫹ cells (1% to 5%) were detectable in either blood or in BM (Fig 2): nearly all were also CD20⫹. If plasma cells are present, they lack syndecan-1 expression (CD138) and express CD20, distinguishing them from plasma cells in multiple myeloma. Nearly all CD20⫹ cells in blood and BM coexpressed surface (s)IgM and sIgD (Fig 2). For one patient, BM CD20⫹ B cells were sIgM⫹D⫹, but a large subset of CD20⫹ cells in the blood were sIgM⫹D⫺. Analysis of CD45 and CD20 expression revealed considerable heterogeneity among B cells. For the BM sample shown in Fig 2, there are three distinct CD20⫹ populations: CD20⫹45hi, CD20lo45hi, and CD20lo45lo. Molecular analysis is required to determine whether or not all of the subpopulations

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Table 1. Summary of VH Gene Usage and Hypermutation in WM Clones Clonal Transcript Detected (no. pts/total) VH Gene Usage (no. pts/total)

Bone Marrow

Blood

JH Gene Usage (no. pts/total)

␮

␦

␮

␦

% Hypermutation, Mean ⫾ SD (range)

JH4 (8/12)

11/11

4/4

7/7

3/3

6.37% ⫾ 4.7% (0%-18.5%)

VH3 (11/12) Abbreviation: pts, patients.

detectable in blood and BM of WM patients are clonally related. These abnormally high numbers of B cells may reflect the presence of malignant clonal populations, or alternatively may reflect dysregulation of normal B lymphopoiesis and accumulation of abnormal but polyclonal B cells in blood and/or BM. THE CLONOTYPIC IgM VDJ SIGNATURE IN WM

The WM clonotypic IgM VDJ for each selected WM patient was derived from BM mRNA using primers to VH leader sequences and C␮. Patientspecific primers were designed and tested. For two patients, single-cell reverse-transcriptase polymerase chain reaction (RT-PCR) was used to successfully confirm that the IgH VDJ identified detects the clonotypic rearrangement in BM cells. The characteristics of the clonotypic IgM WM sequences identified suggest an unusual progenitor population for WM. Unexpectedly, 11 of 12 WM clones were of the VH3 family, and most utilized JH4 (Table 1). Sahota found that five of seven WM clonotypic sequences were VH3,14 consistent with our observations. Eight of 12 WM patients (67%) had long CDR3 regions (defined as 40 or more nucleotides), characteristic of those found in antigen-stimulated populations. Others have shown a VH3 predominance in some IgM B-cell malignancies (IgM myeloma and IgM CLL) but unlike WM, these malignancies have relatively short CDR3 regions.15,16 Clonotypic VDJ regions in WM had extensive somatic mutation (Table 1) with mainly replacement mutations, although for one WM patient a germline VH3 sequence was identified as clonotypic. For seven of seven patients, clonotypic IgM transcripts were readily detected in both blood and BM, clearly identifying a blood-borne compartment of WM. For four of four patients, using an assay that had been rigorously optimized to detect clonotypic IgM, clonotypic

IgD was detected in blood and/or BM, but postswitch clonotypic isotypes were not detected. ABNORMAL NUMBERS OF CD20ⴙ B CELLS ARE SOMETIMES POLYCLONAL

One patient has been followed longitudinally by analysis of cell phenotype and of the molecular signature in both blood and BM (Fig 3). At diagnosis, this WM patient has a serum IgM level of 13.5 g/L, and 24% CD20⫹ B-lineage cells in BM. In the blood, the numbers of CD20⫹ B cells were within the normal range. Analysis of the molecular signature showed that the BM had abundant clonotypic IgM transcripts. Despite the relatively low number of B cells in blood, clonotypic IgM was still readily detectable although at a likely lower copy number than in BM. Thus, WM colonization of the blood, that is readily detected by molecular analysis of the IgM VDJ signature sequence is not necessarily accompanied by abnor-

Fig 3. In some cases elevated numbers of B cells in WM represent apparently polyclonal B cells. Longitudinal analysis of WM B cells. (A) Detection of clonal transcripts as determined by RT-PCR using patient-specific CDR2 and CDR3 primers for patient no. 1. (B) Percentage of CD20ⴙ BMC or peripheral blood mononuclear cells (BL) and the amount of serum monoclonal protein of the indicated samples. Samples 1.1 and 1.2 were taken approximately 6 months apart.

THE MALIGNANT CLONE IN WALDENSTROM’S

mal numbers of B cells. Six months later, the disease had responded to treatment as evidenced by a serum IgM of 5.8 g/L (Fig 3). Furthermore, clonotypic IgM transcripts has become undetectable in blood; this was specific to the IgM VDJ as transcripts for a housekeeping gene (␤2-microglobulin) were readily detected, confirming that mRNA from these cells was intact. Despite the reduction in monoclonal IgM and lack of clonotypic transcripts, circulating B cells had increased by more than threefold (Fig 3). These circulating B cells were likely to be polyclonal cells, unrelated to the WM. Normal B lymphopoiesis may have been dysregulated, resulting in abnormally high numbers of normal B cells in the blood. This confirms that analysis of clonotypic signatures provides the most accurate indicator of WM involvement in blood and BM. CONCLUSIONS

WM is a complex disease characterized by apparent morphological heterogeneity within the malignant clone. The extent to which the diverse subtypes of lymphocytes, lymphoblastoid cells, and plasmacytoid cells participate in, and contribute to, the disease progression is unknown. A potential confounding factor in diagnosis, treatment, and research is that the characteristics of the disease at one point in time may be radically different from those at another phase of development. The identification of the IgH VDJ molecular signature for the WM clone in each patient enables unequivocal identification of otherwise heterogeneous populations of cells as being clonotypic. This is particularly important for the characterization and monitoring of circulating WM cells. If, as appears likely to date, populations of clonotypic WM cells colonize the blood, this opens the way for development of noninvasive blood based testing to monitor the WM clone as it develops during therapy. Only through analysis of molecular signatures can the extent of minimal disease be monitored and early relapse be detected. In addition, with the impending availability of sensitive tests to detect the WM signature, we have the potential to identify novel therapies that may be better able to eradicate clonotypic populations. REFERENCES 1. Dimopoulos M, Galani E, Matsouka C: Waldenstro¨ m’s macroglobulenimia. Hematol Oncol Clin North Am 13:13511366, 1999

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