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Clinical Cancer Research Vol. 8, 502-513, February 2002
© 2002 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Intraclonal Homogeneity of Clonotypic Immunoglobulin M and Diversity of Nonclinical Post-Switch Isotypes in Multiple Myeloma

Insights into the Evolution of the Myeloma Clone1

Brian J. Taylor, Julie A. Pittman, Karen Seeberger, Michael J. Mant, Tony Reiman, Andrew R. Belch and Linda M. Pilarski2

Departments of Oncology and Medicine, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, T6G 1Z2 Canada


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The transformation status and role of clonotypic pre-switch IgM in the evolution of malignant post-switch multiple myeloma (MM) cells is unclear. In this study, we determined the differentiation stage within the B lineage of clonotypic cells from malignant and nonclinical isotype pools by analyzing the frequency and intraclonal diversity of members within each isotype pool.

Results: Immunoglobulin VDJ transcripts were amplified from peripheral blood cells of seven patients with a hemi-nested reverse transcription-PCR with complementarity determining region 1 (CDR1)-specific and constant region primers. Of the 1951 clones screened by patient CDR2/3-specific PCR, 356 of these were sequenced. Intraclonal homogeneity was observed in pre-switch transcripts from four of four informative patients. Transcripts from the IgM pool were relatively frequent in two of four informative patients. Cellular limiting dilution analysis indicated 0.4–25% of peripheral blood mononuclear cells expressed clonotypic IgM for 6 of 15 samples tested. By contrast, significant intraclonal diversity was observed in the nonclinical IgA pool of 1 patient. A genealogical tree of IgA sequences was constructed showing ongoing clonal diversification from sequences with close homology to the germ-line V gene to those resembling the PC sequence. Furthermore, some clones exhibited complete homology with tumor VDJ sequence, plus extra mutations, suggestive of a parallel clonal arm that remains responsive to an antigenic stimulus.

Conclusions: Detection of intraclonal diversity in the post-switch nonclinical isotype pool suggests that remnants of the parent B-cell clone coexist with malignant clonal precursors. The presence of intraclonal homogeneity in the pre-switch IgM pool supports the idea that pre-switch MM cells play a role in malignant events within the MM clone.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The analysis of tumor cell V genes can reveal the stage of B-cell differentiation of many B-cell tumors. The unique VDJ rearrangement of a transformed cell serves as a tumor-specific molecular marker. Subsequent alterations in this marker by somatic hypermutation and isotype class switching activated in the GC3 (1 , 2) can reveal the history of the transformed cell (3) . As such, the origin of a B-cell tumor can be identified in relation to GC events by the degree of mutation and intraclonal mutational differences or intraclonal diversity observed in VDJ sequences. Tumors from early stages of B-cell development that have not entered the GC, such as acute lymphoblastic leukemia, mantle cell lymphoma, and a subset of chronic lymphocytic leukemia, typically have unmutated VDJ showing intraclonal homogeneity (4, 5, 6, 7, 8) . Tumors originating from the GC, such as follicular lymphoma and Burkitt’s lymphoma, show marked mutation of clonotypic VDJ and intraclonal diversity (9, 10, 11, 12, 13) . Tumors originating from post-GC cells, such as lymphoplasmacytoid lymphoma and a subset of chronic lymphocytic leukemia, have mutated VDJ showing intraclonal homogeneity (7 , 8 , 14) . Interestingly, in some cases intraclonal diversity is present in monoclonal gammopathy of undetermined significance patients (15) , suggesting that when MM arises, transformation may occur at the time B cells exit from the GC.

In MM, the IgH VDJ gene rearrangement present in bone marrow plasma cells is a unique tumor-specific marker that unequivocally identifies the malignant clone. Antigen contact areas of the immunoglobulin molecule are termed CDRs and are unique to each individual B-cell clone. The CDR3 is formed only when V, D, and J segments of the IgH gene rearrange to form a functional IgH chain. During development, this IgH rearrangement commits a progenitor cell to B-lineage differentiation. Each pre-B cell expresses a unique immunoglobulin receptor that characterizes its clonal progeny. This receptor undergoes somatic diversification via hypermutation and antigen-driven selection for higher affinity in the GC. During normal B-cell differentiation, early-stage B cells express surface IgM and/or IgD. After antigen-driven selection, these B cells undergo a change in immunoglobulin isotype expression to IgG, IgA, or IgE, termed class switching. For a given clone of B cells, immunoglobulin transcripts of the pre- and post-switch B cells have identical VDJ rearrangements.

MM is post-GC B-cell tumor characterized by an infiltration of monoclonal PCs into the bone marrow and production of monoclonal serum immunoglobulin of one predominant isotype, here termed the "clinical isotype," usually IgG or IgA. V gene analysis reveals that malignant PCs share a unique immunoglobulin heavy chain VDJ rearrangement (16, 17, 18) , termed clonotypic, that has undergone isotype switching from Cµ to one of the downstream post-switch constant regions, either C{alpha} or C{gamma}. Earlier stage B cells with clonotypic VDJ rearrangements identical to myeloma PCs have been also been detected, but their role in the disease process is unclear (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) . Sequence analysis of clonotypic VDJ of the clinical isotype reveals strict intraclonal homogeneity for plasma cells and B cells (30, 31, 32, 33) and clonal stability throughout the course of disease (30 , 34) . These data suggest that MM cells are mature, antigen-selected, post-GC B cells no longer exposed to hypermutation mechanisms.

Previous reports have described MM cells that express other isotypes in addition to the clinical one, either pre-switch IgM or D, or post-switch IgG or IgA (21 , 22 , 35, 36, 37) . These are called nonclinical isotypes, and their transformation status and role in the evolution of the MM clone is unclear. Reiman et al. (36) have shown that drug-resistant cells expressing clonotypic IgM correlate strongly with reduced survival as well as with more aggressive disease at the time of diagnosis, indicating their clinical impact. Of particular interest are Cµ isotype variants detectable in IgG and IgA MM, suggestive of a pre-switch origin for MM (21 , 22 , 35 , 36) . Southern blot analysis of VDJ and switch regions of post-switch MM revealed cells of possible pre-switch origin in 35% of patients studied (38) . V gene analysis using amplification strategies involving anchor PCR (35) , CDR3 and Cµ primers (21) , or CDR2 and Cµ primers (22) revealed clonotypic pre-switch cells exhibiting intraclonal homogeneity. This work suggests that both pre-switch and post-switch MM cells are no longer exposed to somatic hypermutation and antigen-driven selection in the GC. Furthermore, clonotypic clinical isotype and nonclinical isotype transcripts were detected in bone marrow B-cells, whereas only clonotypic clinical isotype transcripts were seen in the PC fraction (21) , indicating that in MM, clonotypic cells expressing nonclinical isotypes are expressed by B lymphocytes but are absent from PCs. By contrast, a recent study has challenged the idea of clonotypic pre-switch cells as myeloma precursors with evidence from the murine 5T myeloma model, showing that isotype variants may arise from rare secondary isotype switching or trans-switching events in the terminally differentiated post-switch PCs (39) . Thus, the place of clonotypic B cells expressing nonclinical isotypes, especially IgM, in the MM clone remains to be determined.

In the work reported here, we present an extensive analysis of the frequency of pre-switch clonotypic cells and intraclonal diversity in all isotype compartments of the MM clone using a hemi-nested PCR strategy and CDR1 upstream primers complementary to regions less susceptible to mutation than CDR2 and CDR3 regions. Here we show that pre-switch clonotypic cells are clonally homogeneous in four of four informative patients and frequent in two of four informative patients. Interestingly, we show that the post-switch nonclinical isotype pool of one patient has both homogeneous counterparts to tumor cells expressing the clinical isotype and diverse members that reflect the presumably pretransformed evolution of the MM clone. These results are consistent with the presence of relatively frequent pre-switch MM precursors exhibiting intraclonal homogeneity and co-existing with a considerably less frequent set of pre-switch progenitors detectable only by their progeny, which may maintain the parent B-cell clone from which MM arose.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Samples.
Eighteen MM patients were chosen from our previous study (36) that had clonotypic transcripts of nonclinical isotypes in peripheral blood samples. PBMCs were harvested by Ficoll/Hypaque purification (Amersham Pharmacia Biotech, Piscataway NJ), and total RNA was extracted using Trizol (Invitrogen Life Technologies, Inc., Carlsbad, CA).

Single-Stage and Hemi-Nested PCR.
Reverse transcription was performed using 1 µg of RNA and 100 ng of universal primer oligo-dT15, 5x First Strand buffer (Invitrogen Life Technologies, Inc.), 0.1 M DTT, 0.25 mM dNTPs and 200 units of Superscript reverse transcriptase. Each single-stage or hemi-nested PCR reaction was performed as described by Saiki et al. (40) with equivalent amounts of cDNA in 25 µl of total volume containing Ultrapure water (Sigma Chemical Co.-Aldrich, St. Louis, MO), 10x PCR buffer, 200 µM of each dNTP, 2 mM MgSO4, 10 µM of each primer, and 0.1 unit/reaction tube of high fidelity HiFi Platinum Taq (Invitrogen Life Technologies, Inc.). Standard HiFi Taq cycling conditions were as follows: 94°C 2 min, followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, and 68°C for 30 s, with a final 68°C extension for 10 min. Primers used in these experiments are listed in Table 1Citation . VH leader-specific primers based on work by Campbell et al. (41) were described previously by Reiman et al. (36) . Constant region primers were described in (21) . Patient-specific CDR1, CDR2, and CDR3 primers were derived from the patient clonotypic sequence, identified as described previously by Szczepek et al. (30) .VH leader-specific primers and C region primers were used in single-stage PCR reactions. In hemi-nested PCR experiments, 1 µl of single-stage product was amplified with patient-specific CDR1 sense primers and the respective C region primer in a 25-µl second-stage reaction, under conditions described above.


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  		Table 1 Nucleotide sequences of primers used in this study

 
Cloning and Sequencing.
Amplification products from single-stage or hemi-nested PCR experiments were resolved on 1% low-melting point gels, excised, and then purified on Genelute columns (Sigma Chemical Co.-Aldrich). Purified fragments were cloned using the TOPO TA cloning system (Invitrogen Life Technologies, Inc.) according to the manufacturer’s directions. A two-step approach was used to isolate clonotypic VDJ-C sequences for diversity analysis:

(a) Transformants were screened by patient-specific PCR (using CDR2 and CDR3 primers; see Table 1Citation ) to identify clones containing clonotypic VDJ-C inserts. Instead of optimized PCR reactions with annealing temperatures of 60°C, relaxed PCR reactions with annealing temperatures of 55°C were used. By choosing both weak and strong bands from this PCR, we could identify clonotypic CDR3 and any CDR3 regions that might be altered by somatic hypermutation.

(b) The entire VDJ-C regions of CDR2/3+ clones were then amplified with TOPO-TA vector-specific M13 forward and reverse primers for intraclonal diversity analysis. Both PCR reactions were performed under conditions described for single-stage PCR, with Taq polymerase and appropriate salts substituted for HiFi Taq. Clonotypic M13F/R PCR products were prepared for cycle sequencing by digestion with shrimp alkaline phosphatase and exonuclease III (United States Biochemical Corp., Cleveland, OH) to remove dNTPs and primers and then subjected to sequencing with a fluorescent dideoxy terminator sequencing kit (Applied Biosystems, Foster City, CA) and T7 or M13 reverse primers, according to the manufacturer’s protocol. Sequencing products were resolved on an ABI310 capillary genetic analyzer (Applied Biosystems).

Analysis of Single-Stage and Hemi-Nested Clonotypic Sequences.
All DNA sequences and their corresponding electrophoregrams were manually inspected after fluorescent sequencing to ensure that the automated interpretation of the raw data was correct. Clones were examined for the presence of the clonotypic VDJ sequence by comparison with the diagnosis bone marrow sequence and confirmed for the appropriate constant region sequence. Germ-line VH gene identity, diversity, junctional regions, and mutations were assigned using the V-base sequence directory (42) 4 and DNAPLOT5 web-based alignment programs. Intraclonal diversity was assessed using AutoAssembler (Applied Biosystems) to align cloned patient VDJ sequences with the respective patient bone marrow PC VDJ sequence.

Taq Error Rate and Statistical Analysis.
To ensure that we could distinguish true mutations caused by somatic hypermutation from random incorporation errors attributable to Taq polymerase, we measured the Taq error rate by sequencing the IgH VDJ regions of 26 individual subclones derived from a single original clone. We used the same hemi-nested amplification described for diversity analysis. The error rate from these experiments was 1 of 7722 bases, which agrees well with previous reports (31 , 33) . Fisher’s exact test was used to compare values of mutation frequency in the Taq error control with those calculated for each isotype. Statistical significance was defined as a two-sided P of 0.05 or less.

Cellular Limiting Dilution Assay for Cells Expressing Clonotypic IgM.
The cellular limiting dilution assay was described previously (30 , 43) . Briefly, cells were aliquoted manually into PCR tubes at 1–1000 cells/tube in a 3-fold dilution series, using a direct lysis RT-PCR (30) . After reverse transcription, cDNA was amplified using primers to the Vh family that characterized each patient sequence together with the IgM constant region primer, and the second stage (nested PCR) used primers to patient-specific CDR2/CDR3. To exclude any potential artifacts that could result from "carryover" of cDNA encoding the clinical isotype into the second-stage PCR reaction, only those limiting dilution series that had a higher frequency of clonotypic IgM-expressing cells than of clinical isotype-expressing cells were considered for this analysis. We were able to reproduce the same frequency in a second assay run. The frequency of cells expressing clonotypic IgM was calculated as the number of cells required to detect bands of the correct sized product. Because the transcript level/cell may be low for IgM, it is not known how many IgM-expressing cells must be placed in a tube to score a positive. In relative terms, comparison of numbers between patients is accurate, although the calculated value may underestimate the true frequency.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CDR1-Hemi-Nested PCR Combined with Screening Is a Sensitive Method for Identifying Clonotypic Sequences from Distinct Isotype Compartments.
In this study, our goal was to determine the frequency and degree of intraclonal diversity in the pre- and post-switch clonotypic transcripts of MM patients. We used a strategy that involved: (a) single-stage PCR using VH and C primers; or (b) hemi-nested PCR, using VH and C primers in the first stage, CDR1 and C primers in the second stage, cloning of amplification products, screening of individual colonies by MM clone-specific CDR2-CDR3 PCR, and sequencing of CDR2-CDR3 positives (Fig. 1)Citation . Previous work had shown intraclonal homogeneity of pre-switch clonotypic transcripts using primers specific for CDR2 and CDR3 regions (21 , 22) . These methods may underestimate the extent of intraclonal diversity, because somatic hypermutation concentrated in CDR regions may disrupt primer binding, resulting in disfavored amplification of mutated clonotypic transcripts over other unmutated transcripts. We attempted to minimize this effect by using an upstream CDR1 primer, taking advantage of the tendency of VDJ mutations to be distributed asymmetrically in favor of downstream regions (44 , 45) . It was also important to relax the specificity of the screening process to account for mutations that may occur in CDR2 and CDR3 regions during clonal diversification. This was achieved by reducing the annealing temperature of the MM clone-specific CDR2/3 screening PCR and choosing both faint and strong bands as positives for sequencing. Although this sometimes led to identification of nonclonotypic sequences, it allowed us to isolate diversified clonotypic sequences that might otherwise have gone undetected.



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  		Fig. 1. Experimental design for examining intraclonal diversity in pre- and post-switch clonotypic IgH VDJ.

 
Frequent Pre-Switch Clonotypic Transcripts Are Revealed in Two of Seven Patients by Single-Stage VH Constant Region PCR.
Peripheral blood from 7 MM patients (Table 2)Citation was taken at diagnosis to minimize influences of chemotherapy on clonotypic cell populations. A representative experiment showing patient 1 amplification products resolved by agarose gel electrophoresis is presented in Fig. 2BCitation . The single-stage PCR reactions yielded faint bands compared with the hemi-nested reactions. In general, we expected that screening clones from a single-stage VH-constant region PCR would be difficult because these primers amplify many transcripts from their respective pools in addition to the clonotypic transcripts. For patients 1 and 4–7, strong bands for the clinical isotype and very weak bands for the nonclinical isotypes were observed. Initially, single-stage IgM amplification products from patient 1 were cloned and sequenced, with 0 of 48 clones and 0 of 24 sequences containing clonotypic sequence by patient-specific CDR2/3 PCR and sequence analysis, respectively (Table 3)Citation . Pre-switch clonotypic VDJ transcripts were also undetectable in a single-stage RT-PCR for patients 3 and 5–7. By contrast, 1 of 192 IgM clones screened were clonotypic in patient 4, and all clones screened were clonotypic in patient 2, including IgM. On the basis of their detection in a single-stage RT-PCR for these two patients, IgM pre-switch clonotypic VDJ transcripts were considered to be relatively frequent (~0.5 and 100%, respectively, of total IgM transcripts).


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  		Table 2 Clinical data on MM patients chosen for diversity analysis

 


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  		Fig. 2. Amplification of pre- and post-switch IgH transcripts by single-stage and hemi-nested PCR. A, single-stage PCR includes four separate first-stage PCR reactions with PBMC cDNA as template: VH leader-specific primer and C{alpha}A (from top to bottom, 1), C{delta}A (2), C{gamma}A (3), or CµA (4). Hemi-nested PCR includes four second-stage PCR reactions with first-stage product as template: patient-specific CDR1 and C{alpha}A (from top to bottom, 5), C{delta}A (6), C{gamma}A (7), or CµA (8). B, amplification products of patient 1 single-stage and hemi-nested PCR reactions resolved by agarose gel electrophoresis. Lanes 1–8 correspond to reactions described in A.

 

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  		Table 3 Intraclonal diversity analysis of clonotypic VDJ transcripts from distinct isotype compartments in MM patients

The results of intraclonal diversity analysis in seven MM patients are tabulated below. Ig, patient clinical isotype; germ-line VH genes were identified using the Vbase WWW program described in "Materials and Methods." Single-stage and hemi-nested PCR strategies are explained in Fig. 2ACitation . The frequency of clonotypic VDJ sequences was determined by PCR using patient-specific CDR2 and CDR3 primers and confirmed by sequence analysis of PCR-positive clones. The values for mutations in the VDJ region was obtained by enumerating nucleotide differences between the PC sequence and cloned sequences. The mutation frequency was obtained by dividing the number of mutations by the total number of nucleotides examined. Dashes indicate experiments or calculations that were not done, either when no clonotypic sequences were isolated or in the case of patient 2, when no single-stage C{gamma} amplification product was amplified.

 
Amplification and Intraclonal Diversity Analysis of Infrequent Clonotypic Isotype Variant VDJ Transcripts by CDR1 Hemi-Nested PCR.
Our subsequent effort to isolate clonotypic VDJ transcripts from the nonclinical isotype pools focused on the CDR1 hemi-nested PCR reactions. For each patient, we targeted the pre-switch IgM and post-switch clinical isotype compartments first, followed by the other isotype compartments (Table 3)Citation . We had the most success with patient 1, identifying transcripts from all four isotype compartments tested, and the least success with patients 5–7, in which no pre-switch clonotypic transcripts were identified. The lack of identifiable transcripts in patients 5–7 is most likely attributable to low frequency as opposed to high mutation in the CDR regions. Although faint positive bands were picked from the CDR2-CDR3 screen for these patients, none of these were identified as clonotypic on sequencing analysis. This marks the limit of sensitivity for the CDR1 hemi-nested PCR approach.

Intraclonal Homogeneity Is Observed in Pre-Switch Clonotypic IgM Transcripts from Four of Four Informative MM Patients.
The results of intraclonal diversity analysis in the MM patients of this study are summarized in Table 3Citation . We were able to amplify and sequence pre-switch IgM clonotypic transcripts in four of seven MM patient PBMC samples. Clonotypic VDJ regions from single-stage or hemi-nested clones were sequenced and compared with the closest germ-line VH gene and the bone marrow PC sequence (Fig. 3)Citation . The degree of intraclonal diversity (designated mutation frequency in Table 3Citation ) was calculated as the number of nucleotide differences between the PC VDJ sequence and the cloned sequences divided by the total number of nucleotides assessed. The highest intraclonal diversity in the pre-switch IgM compartment was seen with patient 1 clonotypic IgM sequences, with four nucleotide differences between the PC and cloned sequences in the VDJ regions of 17 clones. This was not significantly different from our measured Taq error rate (P = 0.07). Intraclonal diversity in the pre-switch IgM compartment was comparable with that seen in the clonotypic VDJ of the clinical isotype compartment (see Fig. 4Citation for alignment). One replacement mutation is shared between the IgM and IgG clonotypic sequences at position 61. Patients 2–4 had fewer differences between analyzed clonotypic VDJ and PC sequences, again, not significantly different from the Taq error rate. This indicates stringent and consistently detectable intraclonal homogeneity in the pre-switch clonotypic compartment of MM.



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  		Fig. 3. Alignment of patient 1 IgM clonotypic VDJ-C sequences. Heavy chain VDJ segments are aligned from CDR1 to the beginning of the constant chain sequence. The germ-line sequence with the closest homology to the clonotypic sequence is the topmost sequence of the alignment, DP-78. PC, clonotypic PC IgH sequence derived from the diagnosis bone marrow. Numbered sequences, clonotypic sequences containing mutations. Sequence identity is indicated with -; mutations are indicated with either A, C, G, or T; differences between the PC sequence and individual clonotypic sequences are designated with a black rectangle. Amino acids corresponding to silent mutations are shown in lowercase; replacement mutations in are shown in uppercase, below the sequence alignment.

 


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  		Fig. 4. Alignment of patient 1 IgG clonotypic VDJ-C sequences. See Fig. 3Citation for details.

 
A High Frequency of Clonotypic IgM-expressing Cells in PBMCs for Some MM Patients.
A high frequency of clonotypic IgM- expressing cells in PBMCs correlates with active disease status. The sequencing analysis above indicates the frequency of clonotypic IgM transcripts in the pool of aggregate IgM transcripts. To quantify the number of cells expressing clonotypic IgM transcripts, we performed a direct lysis cellular limiting dilution assay on 15 PBMC samples, randomly selected from those having detectable clonotypic IgM in purified RNA from 106 PBMCs. The frequency of PBMCs expressing clonotypic IgM ranged from <0.03 to 25% (Table 4)Citation . Although Table 4Citation represents a set of patients additional to those analyzed above, the frequency estimates are consistent, with some patients having undetectable cells with clonotypic IgM (<0.03%; 9 of 15) and those with relatively high frequency (0.4–25%; 6 of 15).


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  		Table 4 A high frequency of clonotypic IgM+ B cells in some MM patients

A cellular limiting dilution assay for clonotypic IgM was performed for PBMCs from 15 randomly selected samples from 11 patients to analyze the frequency of cells expressing clonotypic IgM transcripts as indicated in "Materials and Methods." All samples had detectable clonotypic IgM when purified RNA from 106 cells was analyzed.

 
Intraclonal Diversity in Nonclinical Clonotypic IgA Transcripts from Patient 1.
Significant intraclonal diversity was found in the IgA nonclinical isotype pool for patient 1. Of the 276 clones obtained, 35 were clonotypic (13%), and 9 of these contained significant nucleotide differences from the PC VDJ sequence (26% of clonotypic IgA sequences; Table 3Citation ; Fig. 5Citation ). Insertions of five triplet codons in clones H10 and HP4 and one triplet codon in H05 and HT4 were observed in the CDR2 region. These insertions maintain the VDJ reading frame and share close homology with upstream codons 53–57 in the case of the H10 and HP4 sequences shown in Fig. 5Citation . Interestingly, clone HR2{gamma}, isolated from the clinical isotype IgG pool, shares complete homology with the H05 VDJ region, including the insertion at CDR2. A notable hotspot for mutation was observed at codon position 40, with clones H10, H05, HP4, HV2, X2, and HR2{gamma} all sharing one or two nucleotide differences from the PC sequence at this position. Six of nine clonotypic IgA sequences contained one or two nucleotide differences from the PC sequence, resulting in amino acid replacements in the translated protein. Finally, clones HT2 and G3 share the closest homology with the PC sequence, with one nucleotide difference in each. Thus, the members of the nonclinical isotype compartment in this patient show significant clonal diversification, not just in random single mutations but also in hotspot mutations and nucleotide insertions. This suggests that antigen-driven selection and perhaps ongoing somatic mutation may be operative in this nonclinical isotype compartment.



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  		Fig. 5. Alignment for patient 1 IgA diverse heavy chain VDJ segments. See Fig. 3Citation for details. Clone HR2{gamma}, designated in bold, is an IgG clone that showed exact homology to IgA clone H05. Nucleotide insertions are indicated in bold.

 
Genealogy of the IgA Compartment of Patient 1.
Although a definitive history of clonotypic cells from the IgA compartment is difficult to determine, distinct relationships are evident from comparison with the PC sequence (Fig. 6)Citation . Clones H10 and HP4 seem to arise from an earlier and perhaps separate progenitor from the remainder of the clones. Inspection of the VDJ sequence with emphasis on the CDR2 regions of these clones reveals that they have not incurred any of the mutations that mark the PC sequence, and they have additional nucleotide insertions. This implies divergence from the main group early in the evolution of the MM clone, possibly before malignant transformation events. Members of the second group have VDJ sequences with closer homology to the PC sequence, and some divergence is evident early in this lineage with clone H05 and its homologous IgG counterpart HR2{gamma} (designated in bold in Fig. 5Citation ). On the basis of its mutational pattern, HR2{gamma} is not considered an example of intraclonal heterogeneity of post-switch tumor cells; rather, it seems to have arisen as part of the normal evolution of earlier stages of the clone. The MM tumor clone encompasses 35 IgA clones with complete homology to the PC IgG VDJ sequence (possibly posttransformation events) and five additional clones with minor sequence differences, HT4, HT2, HV2, G3, and X2. Most striking of these is clone HT4, which has five single-nucleotide substitutions and an insertion of a triplet codon close to the CDR2 region. Clones HT4, and to a lesser extent HT2, and G3 comprise a population of post-switch nonclinical clonotypic cells that seem to have been exposed to additional somatic hypermutation, suggesting they may represent a later stage of the original parent B-cell clone that gave rise to MM. No IgM counterparts for these mutated isotype switch variants of the MM tumor clone were detected by single-stage or hemi-nested PCR, although these might be rare. A genealogical tree of diversified clonotypic IgA sequences for patient 1 is presented in Fig. 6Citation .



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  		Fig. 6. A genealogy of clonotypic VDJ sequences of the IgA compartment of patient 1. Circles represent individual clones designated by the code used in Fig. 3Citation 4Citation 5Citation , or hypothetical intermediates, designated A–F. Clones are arranged based on the accumulation of mutations described in Fig. 5Citation . The number of mutations from a hypothetical precursor A are indicated with a + beside each clone.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The emphasis of this work was on using V gene sequence analysis to identify components of the MM clone relative to the GC, where somatic hypermutation is activated, and MM differentiation stages immediately thereafter, such as post-GC memory B cells and their isotype-switched progeny. Our main strategy involved a sensitive CDR1-C region hemi-nested PCR, which allowed us to assess intraclonal diversity in key regions, such as CDR2, where many single mutations and insertions were observed. We examined intraclonal diversity in MM cells of seven patients, screening in total 1951 clones and analyzing 356 VDJ-C sequences. We focused on peripheral blood cells, which may be more likely to contain clonotypic B cells (21 , 22) . We found that clonotypic IgM transcripts were present in four of seven patients, occurring at a high frequency in two of seven patients. Cellular limiting dilution analysis indicated a frequency of 0.4–25% PBMCs expressing clonotypic IgM for 6 of 15 samples tested. The VDJ regions expressed by these cells exhibited somatic mutation and rigid intraclonal homogeneity. However, intraclonal diversity was observed in the IgA compartment of one MM patient, suggesting that malignant and nonmalignant clonotypic lineages may coexist.

Intraclonal homogeneity suggests that clonal expansion is independent of normal antigen-driven selection and diversification, as predicted for malignant cells. The clonotypic VDJ transcripts of pre-switch cells showed intraclonal homogeneity, suggesting that the most frequent component of the clonotypic IgM compartment is that within the malignant arm of the MM clone. This underscores previous studies that have characterized post-switch MM cells as post-GC cells based on strict intraclonal homogeneity (31, 32, 33) and expands on studies showing intraclonal homogeneity of VDJ transcripts in pre-switch isotype variants using CDR2 and CDR3 primers (21 , 22) . We have shown that pre-switch components of the MM clone are detectable and have a VDJ sequence identical to that of autologous plasma cells. This is as expected if pre-switch cells are part of the malignant clone but unexpected if they represent remnants of the presumptively normal parent B-cell clone that gave rise to MM.

A novel finding from this study, which has not been reported previously in MM, was the presence of significant diversity in the clonotypic VDJ transcripts of the post-switch nonclinical isotype pool. In the IgA pool of patient 1, silent and replacement mutations and three different kinds of insertional events were detected in the CDR2 region, suggesting ongoing somatic hypermutation, a characteristic predicted of any remnants of the original B-cell clone that gave rise to MM. Insertions and deletions of nucleotides in both CDR1 and CDR2 regions have been described extensively (46) and may involve upstream sequence motifs that form loop intermediates during "replication slippage" (reviewed in Ref. 47 ). These events have been reported in VDJ regions in follicular lymphoma (48) , and kappa light chain regions in MM (49) . They have been localized to germinal center cells, and post-germinal center memory cells (46) . Perhaps the most interesting observation comes from clones expressing VDJ transcripts with the closest homology to the post-switch tumor VDJ transcripts. Strikingly, one clone of this group (HT4) had accumulated all of the mutations of the PC sequence, plus five substitutions and an insertion of a triplet codon just upstream of the CDR2 region. The presence of an insertion event in addition to mutations decreases the chance that the diversity in this clone arises from Taq error, and places it in the category of germinal center or post-germinal center memory cells. This clone seems to represent a late stage within the presumptively normal arm of the MM clone. Such late stage clones capable of re-entering the germinal center are probably not transformed, suggesting that at least two types of clonotypic IgM precursors exist. One type exhibits intraclonal homogeneity and likely gives rise to malignant post-switch tumor cells also having strict intraclonal homogeneity. The second, infrequent type of pre-switch clonotypic cells are likely to have intraclonal heterogeneity and are detected by their presumptive progeny, identified as post-switch variants exhibiting intraclonal diversity.

V gene analysis of homogeneous and diverse MM transcripts has provided insight into the time of malignant transformation by giving us a rare glimpse at the co-evolution of normal and malignant lineages of the MM clone. The B lineage from which MM originated appears to co-exist with its malignant daughter cells. Thus pre-switch progenitors may be divided into normal and malignant arms by a transformation event (Fig. 7)Citation , leading respectively to rarely detected normal post-switch clonal progeny, or to frequent malignant progeny of the post-switch clinical isotype. The malignant population would include (a) homogeneous and frequent pre-switch cells that may already be predisposed to switching to the clinical isotype, (b) homogeneous and frequent post-switch cells of the clinical isotype, and perhaps (c) homogeneous and infrequent post-switch cells of the nonclinical isotype. The normal population would be expected to include diverse and presumably infrequent clonotypic pre- and post-switch cells. If a transformation event occurs late in the evolution of the MM clone, the normal arm may be more developed, and consequently diverse members would be detectable (e.g., patient 1). For some patients, transformation events occurring early in the evolution of the MM clone may preclude the development and detection of a normal arm, unless this population undergoes a parallel evolution concurrent with the malignant arm.



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  		Fig. 7. Model of transformation in MM. This model diagrams the ancestry of normal and malignant arms of the MM clone from their original progenitor compartment. Normal IgM clones (open circles) generate infrequent diverse IgA and IgG clones; transformed IgM clones (shaded circles) generate frequent post-switch clinical isotype cells that show intraclonal homogeneity and infrequent homogeneous nonclinical isotypes. Both are presumed to have originally derived from a normal, antigen-dependent progenitor population, with a transformation event giving rise to the malignant arm of the MM clone.

 
MM is a post-switch disease characterized by malignant plasma cells expressing clinical isotypes. The role of the clonotypic cells that express nonclinical isotypes in malignant disease remains uncharacterized. However, we have recently shown that nonclinical isotypes, especially pre-switch IgM, are directly related to outcomes in MM (36) . Persistent clonotypic IgM expression in MM patients was associated with more advanced disease at diagnosis and poorer survival than infrequent or absent clonotypic IgM expression. The work by Reiman et al. (36) could be indicative of a malignant pre-switch progenitor for MM or alternatively could represent a nonmalignant population that in some undefined way parallels disease severity. To begin to distinguish between these alternatives, we have shown here that the predominant compartment of clonotypic IgM in MM patients exhibits tight intraclonal homogeneity. This is predicted if pre-switch cells represent a malignant compartment of MM but is unexpected if they represent remnants of the parent B cell clone that gave rise to MM. Our detection of an infrequent compartment of post-switch cells likely to have arisen from presumptively normal pre-switch progenitors in one MM patient suggests normal and malignant arms of the MM clone may co-exist, although in both pre-switch and post-switch compartments the malignant arm of the MM clone is dominant. Consistent with the strong correlation between drug-resistant B cells expressing clonotypic IgM and advanced disease or reduced survival (36) the work reported here provides strong support for the idea that the malignant arm of the MM clone includes pre-switch, memory B cells expressing clonotypic IgM transcripts.


   ACKNOWLEDGMENTS
 
We thank the MM patients who contributed to this study, and Angie Battochio, Jennifer Carpenter, John Hanson, Eva Pruski, Tim Sousa, Agnes Szczepek, and Jennifer Szdylowski for their expert assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Funded by R01 CA80963 from the National Cancer Institute. Back

2 To whom requests for reprints should be addressed, at Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, T6G 1Z2 Canada. Phone: (780) 432-8925; Fax: (780) 432-8928; E-mail: lpilarski{at}gpu.srv.ualberta.ca Back

3 The abbreviations used are: GC, germinal center; MM, multiple myeloma; CDR, complementarity determining region; VDJ-C, variable, diversity, joining constant region; PC, plasma cell; VH, heavy chain variable region; dNTP, deoxynucleotide triphosphate; RT-PCR, reverse transcription-PCR; C{alpha}, {delta}, {gamma}, and µ, IgA, IgD, IgG, and IgM constant regions, respectively; DH-JH, D-J junction of immunoglobulin heavy chain; PBMC, peripheral blood mononuclear cell. Back

4 Internet address: http://www.mrc-cpe.cam.ac.uk/imt-doc/vbase-home-page.html. Back

5 Internet address: http://www.mrc-cpe.cam.ac.uk/imt-doc/DNasearch.html. Back

Received 6/16/00; revised 10/29/01; accepted 11/ 6/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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