¹Dept of Immunology, Erasmus University Rotterdam/University Hospital Rotterdam, Rotterdam, The Netherlands

²Dept of Pathology, Academic Medical Center, Amsterdam, The Netherlands

Correspondence to: C J M van Noesel, Dept of Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; Fax: 00-31-20-6960389

Normal IgM^-IgD⁺ CD38⁺ B cells and IgM^-IgD⁺ multiple myelomas (MM) are characterized by C deletion, biased Ig expression and hypermutated IgV regions. The predominant Ig usage has been proposed as resulting from secondary Ig gene rearrangements during extensive clonal expansion in the germinal center environment. Here, four cases of IgD MM were studied to address the question of light chain receptor revision in a 'single cell' model. Detailed analyses of both IGK and IGL alleles of each case were performed by Southern blotting, (RT-) PCR, and sequencing. The expressed IgV genes were extensively mutated and C deletion was confirmed in two cases. In addition, in the four MM a total of six non-functional deletional IGK rearrangements were identified, which proved to be unmutated. We conclude that IgD myelomas indeed originate from (post) germinal center B cells in which, in spite of the fact that they are hypermutated, there is no evidence of receptor revision.

Leukemia (2002) 16, 1358-1361. doi:10.1038/sj.leu.2402513

receptor revision; somatic mutations; IgD; multiple myeloma

Introduction

A minority of normal human B cells and rare B cell malignancies express IgD in the absence of IgM. It has been reported that in the human tonsil, 2-5% of the CD38⁺ germinal center B cells and 6-20% of the plasma cells are IgM^-IgD⁺.^1,2 In the peripheral blood, sIgM^-IgD⁺ CD27⁺ memory B cells have been detected with a frequency of less than 1%.³ Among the B cell malignancies, approximately 1% of multiple myelomas (MM) and 10% of hairy cell leukemias display the IgD-only phenotype.⁴ The lack of IgM in all these cell populations is explained by unusual C-C class 'switch' recombination mediated by sequences in the J_H-C and C-C introns, ie either homologous recombination between two direct 442 bp repeats ( and )^5,6 or non-homologous, so-called S- recombination,⁴ both with deletion of the S-C region and precluding further isotype switching. The IgM^-IgD⁺ B cell populations mentioned share two additional molecular features. First, IgM^-IgD⁺ B cells generally carry a very high load of somatic mutations, most likely as a result of extensive clonal expansion: whereas the frequency of nucleotide substitutions in the V_H genes of normal (post)-germinal center B cells ranges between 2 and 6%,^3,7 the reported frequencies for both the normal and neoplastic single IgD-positive B cells are on average two- to three-fold higher.^1,2,3,5,6,8 Second, the various normal IgM^-IgD⁺ B cells almost exclusively express Ig (>99%).¹ Also in IgM^-IgD⁺ MM, high frequencies of Ig light chain expression have been reported, ranging between 60 and 90%.^{9,10,11,12,13,14}

It has been hypothesized that the biased Ig expression of IgM^-IgD⁺ B cells results from secondary Ig gene rearrangements at the light chain loci in the germinal center environment (ie receptor revision), elicited by unfavorable somatic mutations that cause loss of Ig expression or disturbed pairing of Ig heavy and light chains.² Ig light chain gene rearrangements generally occur in an ordered sequence, starting at the IGK locus followed by IGK deletion and subsequent IGL rearrangements.¹⁵ Therefore, secondary rearrangements are expected to result in skewing towards IGL gene usage.

Evidence for receptor revision as a rescue mechanism in mature human B cells is as yet scarce. Interestingly, occurrence of V_H replacements has recently been claimed in IgM^-IgD⁺ CD38⁺ tonsillar B cells and in synovial-tissue B cells.^16,17 Previous reports, however, could not draw firm conclusions on the issue of receptor revision of Ig light chain genes since this requires analysis of all four Ig light chain alleles, ie of both IGK and both IGL alleles, of individual cells. To address the question of Ig light chain receptor revision in a 'single cell' model, detailed studies on the IGH, IGK and IGL loci of four cases of IgD⁺ MMs were performed using Southern blot (SB), PCR and sequence analyses.

Materials and methods

IGH, IGK and IGL genes were analysed by SB, PCR and sequencing. Fifteen g of genomic DNA was digested with BamHI and BglII, size fractionated in a 0.7% agarose gel and transferred to a nylon membrane. The BamHI filter was successively hybridized with the IGHMU and IGHJ6 probes for detection of IGH rearrangements and C deletion. For detection of the IgM-IgD recombination by PCR primers upstream of and downstream of were used (adapted from Arpin et al²). The BglII filter was hybridized with IGK and IGL probes for determination of the IGK, and IGL gene configurations.^15,18 Family-specific V_H, V, and V primers were used in combination with J_H, J and J primers (genomic PCR) or in combination with C, C or C primers (RT-PCR).^15,19 Clonal (RT-)PCR products were either directly sequenced or cloned in pGEM-T Easy vector followed by sequencing of four to six individual clones.

Results and discussion

The configuration of the IGH, IGK and IGL genes in four IgD MMs were studied by SB, PCR and sequencing (Table 1). MM-1 contained a single in-frame IGH gene rearrangement with deletion of the C region caused by a to recombination. In MM-2 and MM-4, no clonal bands could be identified at the DNA level by SB, most likely due to a limited tumor load in the bone marrow samples. However, by RT-PCR and sequencing, single clonal in-frame V_H-C rearrangements were identified. MM-3 contained a biallelic IGH gene rearrangement with a deletion of the C region on one allele, which was in accordance with identification of clonal in-frame V_H-C transcripts, but no V_H-C transcripts by RT-PCR. No second complete V_H-J_H rearrangement was identified by PCR analysis in MM-3 (Table 1). In myelomas 1, 2 and 3 in-frame IGL gene rearrangements were also identified.

All the functional V_H and V genes were indeed exceptionally hypermutated. The highest number of somatic mutations was found in the V_H region of MM-2, ie 89 mutations. MM-1, MM-3 and MM-4 carried 31, 60 and 31 mutations in their V_H genes, respectively (Figure 1). The average mutation frequency in the V_H regions of the four MM was thus 18%, whereas the reported average mutation frequency of the V_H region in non-IgD MM is 8%.^20,21 Also the V regions showed a slightly higher average mutation load (7.5%) than reported for the light chains of non-IgD MM (6%).²¹ In MM-1, 3 and 4, the ratios of replacement (R) vs silent (S) mutations in the framework regions (FR) was approximately 1.5, which is significantly lower than would be expected if the mutations had occurred randomly in the absence of selection.²² However, in MM-2, with the extremely high mutation load of 31.5%, the R/S ratio was 2.4, suggesting that selectional forces to protect the FR regions and thus to maintain the integrity of the overall Ig structure had been less stringent. Still, this MM, like the other three cases, was found to express the IgD protein. Interestingly, a relatively high number of replacement mutations in the FRs has also been noted previously in the hypermutated IgD-only germinal center cells.¹

The IGH sequences of the four cases were analyzed for the presence of hybrid V_H gene segments, indicative of V_H replacement processes.¹⁶ However, we obtained no evidence for this type of receptor revision at the IGH locus.

Analysis of the IGK genes showed that all rearranged IGK alleles involved the kappa deleting element (Kde). This concerned four intronRSS-Kde rearrangements with preserved out-of-frame V-J rearrangements upstream (MM-1, 3 and 4). We detected no somatic mutations in the V- gene segments of any of these four non-functional V-J rearrangements. In MM-2 and the second allele of MM-4, V-Kde rearrangements were found which deleted the complete J-C region. Also IgD MM, although not present in our limited panel, may contain, next to a functional IGK gene rearrangement, a V-J rearrangement at the second allele either or not followed by an intronRSS-Kde rearrangement. Such an Ig gene configuration may in a similar fashion be informative with respect to the topic of receptor revision.

Analysis of the IGL genes showed that the three identified IGL gene rearrangements used three different J gene segments. This implies that the single IgD expression was not associated with a specific Ig isotype.

The detailed information obtained on the IGK and IGL loci indicated that in at least two cases (MM-1 and MM-3) the cells had not expressed another Ig light chain prior to the identified in-frame Ig chains, since all V-J rearrangements found upstream of the intronRSS-Kde recombinations were out-of-frame and not somatically mutated. This is formally not proven for MM-2 and MM-4 as the V-Kde rearrangements precluded analysis of the possibly pre-existing V-J rearrangements.

In conclusion, our data indicate that IgD MM are most likely derived from IgM^-IgD⁺ Ig-positive B cells that were switched to IgD-only by C deletion. In spite of the fact that the IgV regions were clearly hypermutated, proving that the progenitor cells must have undergone extensive clonal expansion in the germinal centers, we find no evidence of receptor revision at the Ig heavy or Ig light chain loci. It thus remains to be established why IgD, in the absence of IgM, is mainly found in association with Ig or alternatively why C deletion occurs preferentially in Ig⁺ B cells and whether the extraordinary level of somatic hypermutation has a causative role in the induction of this unusual C deletion.

Acknowledgements

The authors would like to thank Ms MJH Berends, Westeinde Hospital, The Hague, The Netherlands, Dr V Gupta, Southend Hospital, Westcliff-on-Sea, UK and Dr GJ Ossenkoppele, VU Medical Center, Amsterdam, The Netherlands for providing patient samples, and Barbara H Barendregt for technical assistance.

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Figure 1 Sequences of the V_H regions of the four IgD MM patients (lower line) compared to the most homologous germline V_H gene segments (upper line) according to VBASE (http://www.dnaplot.de). The most homologous sequences were V3-23/DP-47 (V_H3-23, according to IMGT, see Table 1) for MM-1, V3-11/DP-35 (V_H3-11) for MM-2, V4.33/DP-65 (V_H4-31) for MM-3, and V5-51/ DP-73 (V_H5-51) for MM-4. Replacement mutations are indicated by capitals; silent mutations are indicated by lower cases.

Table 1 Summarized data of Ig gene configurations, gene segment usage, and somatic mutations in four IgD MM