Origin and diversification of the clonogenic cell in multiple myeloma: lessons from the immunoglobulin repertoire

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Abstract

The study of immunoglobulin genes in multiple myeloma over the last decade has provided important information regarding biology, ontogenetic assignment, disease evolution, pathogenic consequences and tumor-specific therapeutic intervention. Detailed analysis of VH genes has revealed the clonal relationship between switch variants expressed by the bone marrow plasma cell and myeloma progenitors in the marrow and peripheral blood. Regarding VH usage, a bias was found against the V4-34 gene encoding antibodies with cold agglutinin specificity (anti-l/i), thus explaining in part the absence of autoimmune phenomena in myeloma compared to other B cell lymphoproliferative disorders. However, in some studies a substantial number of cases analyzed were carrying the rearranged Humκv325 Vκ gene, known to be over utilized by B cell chronic lymphocytic leukemia clones and possessing autoantibody binding activity. VH genes accumulate somatic hypermutations following a distribution compatible with antigen selection, but with no intraclonal heterogeneity. The analysis of Vκ genes indicates a bias in usage of VκI family members; somatic hypermutation, in line with antigen selection, of the expressed Vκ genes is higher than any other B cell lymphoid disorder. Similar conclusions were reached for Vλ genes; in this case, the analysis raises the controversial issue of N nucleotide insertion at Vλ–Jλ junctions, apparently as a result of TdT activity. A complementary imprint of antigen selection as evidenced by somatic hypermutation of either the VH or VL clonogenic genes has been observed. The absence of ongoing somatic mutations in either VH or VL genes gives rise to the notion that the cell of origin in myeloma is a post-germinal center memory B cell.

Introduction

The origin of the neoplastic cell in multiple myeloma (MM) has been extensively debated over the last decade. Expression of early hematopoietic differentiation-associated antigens shared by bone marrow (BM) B cell precursors and aberrant phenotypes typical of the myeloid lineage suggested that myeloma progenitors are developmentally very close to the early hematopoietic stem cells.1 However, subsequent studies applying multiparameter flow cytometry and in vitro manipulation have indicated that the candidate MM stem cell is an activated B lymphocyte that most likely has passed through the germinal center (GC) of lymph nodes.

The apparently mature phenotype of the plasma cell population in the BM has been shown to correspond to the terminally differentiated descendant of the circulating pre- plasmacytic B cell compartment. The preferential homing and expansion of myeloma progenitors in the BM is the result of a fine interplay between adhesion molecules expressed by these cells (CD44, CD54, CD56, VLA-4, syndecan-1) and their counter-receptors expressed by BM stromal cells.2 In addition, a whole cytokine cascade (IL-3, IL-6, M-CSF, TNF-β, IL-1β and IL-11) triggered by the interaction between activated myeloma B cell progenitors and BM stromal cells will eventually lead to proliferation, expansion and terminal differentiation of the neoplastic clone in the BM, constituting the natural environment where most of the tumor burden will reside. It is now appreciated that IL-6, derived from either paracrine or autocrine sources, plays an essential role in the progression of MM by promoting growth and survival of the neoplastic cells, as well as preventing programmed cell death pathways including Fas-mediated apoptosis. It was recently demonstrated the one member of the STAT (signal transducer and activator of transcription) family, STAT3, is constitutively activated in BM plasma cells from MM patients. Moreover, IL-6 prevented Fas-mediated apoptosis by upregulating the expression of the anti-apoptotic protein bcl-XL.3 Resistance to Fas-induced apoptotic signals might be generated by mutations of the gene coding for the Fas antigen.4

Recently, Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8; HHV8) was found in BM dendritic cells of patients with MM, but not in malignant plasma cells or dendritic cells of patients with other malignancies or of normal subjects. Viral IL-6, the human homologue of which is a well established growth factor of myeloma cells, was found to be transcribed by the MM BM dendritic cells; this finding provides compelling evidence for the paracrine stimulation and expansion of myeloma cells in the BM microenvironment.5 However, these findings were not replicated by other investigators, thus leaving the whole issue open for discussion.67 The above data support the notion that the BM microenvironment indeed acts as a ‘mouse trap’ by attracting circulating MM progenitors and providing them with the optimal conditions for growth and differentiation. Differentiation to more mature and end-stage cells characterizes the neoplastic B cell population from the peripheral blood to the BM compartment in MM.

Molecular studies of immunoglobulin (Ig) gene rearrangements, initially carried out by Southern blot analysis, demonstrated the clonal relationship between circulating myeloma progenitors and marrow plasma cells, taking advantage of the unique pattern of rearrangement signals identifying each B cell clone. Later, the development of polymerase chain reaction (PCR) technology for the amplification of rearranged Ig genes further facilitated the study and identification of clonal B cell populations in the peripheral blood and BM. This latter technology helped to shed light on the biology of the myeloma cell indicating that it is a post-switch B cell that has undergone antigen selection after traversing the GC and that there is a bias in the usage of certain Ig variable (V) region genes.

Immunoglobulin gene rearrangements

An overview of Ig gene rearrangements

The V region of Ig heavy chain (HC) and light chain (LC) contains three hypervariable segments (CDRs: CDR1, CDR2, CDR3) and four relatively invariant framework regions (FWRs).8 It is encoded by variable (V), diversity (D) (in HC only), and joining (J) gene segments (VJ joining in LCs) which, on the basis of sequence homology, are subgrouped into several, structurally distinct, gene families. The genes encoding the Ig HC, kappa (κ), and lambda (λ) LC locus are located on chromosomes 14q32, 2p11, and 22q11, respectively.91011 In the IgH locus, there are approximately 50 functional VH genes, 30 D genes and six JH genes. In the κ LC locus, there are about 75 functional Vκ and five Jκ genes, whereas 30 functional Vλ and seven Jλ genes are in the λ LC locus. The gene sequences encoding for both HC and LC V region are assembled during B cell ontogeny from individual V, (D) and J segments through a process of DNA rearrangement known as V(D)J recombination. This process is governed by an as yet insufficiently characterized enzymatic complex (the recombinase).1213141516

Antigen contact of the GC newly entering a ‘naive’ B cell through a low-affinity functional surface Ig will lead to a high proliferation rate associated with somatic hypermutation of Ig genes, a process characterized by mutations within V regions at an estimated rate of 10−3 to 10−4/bp per generation. Somatic hypermutation of Ig genes concerns their V region and is the hallmark of antigen selection, a process that will turn a low-affinity sIg expressing naive B cell entering the GC to a high-affinity antibody producer as well as a long-lived recirculating memory B cell (Figure 1). Clustering of mutations occurs predominantly within the complementarity determining regions (CDRs: CDR1, CDR2 and CDR3) of the respective polypeptide's V region, the sites responsible for antigen contact of a particular Ig. Targeting of somatic hypermutation in CDRs is indeed an intrinsic property of this mechanism.17 Interestingly, separation of the intrinsic properties of the somatic hypermutation process from the effects of antigen-driven selection has been achieved in an experimental model where Vκ genes, open to accumulate mutations but not selected for affinity and carried as ‘passenger’ transgenes not contributing to an antigen-specific immune response, exhibited a very strong intrinsic hypermutation hotspot in CDR1.

Figure 1
figure1

 Analysis of Ig V genes hypermutation in lymphoid malignancies offers valuable insight about the ontogenetic stage at which malignant transformation had taken place. A schematic representation of antigen selection and Ig V gene hypermutation operating in various B cell lymphoid malignancies (Hodgkin's disease (HD), follicular lymphoma (FL) and multiple myeloma (MM)) and their corresponding normal cellular counterparts in relation to intra- and post-GC development is demonstrated. Favorable mutations are those that will provide the B cell with slg capable of interacting with antigen, while unfavorable mutations represent those that will lead to a non-slg expressing B cell or a B cell bearing slg with reduced affinity for antigen.

Antigen selection, seen as a dynamic process, is an ongoing phenomenon in the GC, that will eventually lead to the survival of B cells expressing high-affinity Ig V gene mutants, whereas B cell clones with less affinity for antigen are destined to undergo apoptosis18 (Figure 1). When antigen selection is accomplished, the hypermutation process ceases; typically, memory B cells and plasma cell precursors do not accumulate any further hypermutations in their VH and VL genes.

Heavy chain Ig genes

Relationship of the clonogenic MM cell to pluripotent hematopoietic progenitors:

An important achievement of the PCR technology for clonogenic V(D)J genes was the demonstration that positively selected CD34+ hematopoietic progenitors are devoid of clonal MM B cells;1920 this helped to clarify the rather confusing issue concerning the relationship between MM progenitors and early multipotential hematopoietic stem cells (HSC). However, the latter finding has been challenged in more recent investigations, that detected circulating CD34+/CD 19+ B cell progenitors bearing the same Ig gene rearrangements with the myeloma clone.2122 These differences might reflect differences in the sensitivity of the PCR methods applied; alternatively, they may originate from technical errors, related to the cell separation procedure, allowing small amounts of contaminating clonal myeloma cells to be detected by extremely sensitive PCR methods. The quantitative analysis of circulating clonal MM progenitors in the peripheral blood by PCR at diagnosis has demonstrated a strong correlation between the absolute number of these cells and disease activity; furthermore, it has been successful at predicting the progression of disease in patients with smoldering myeloma.23

Origin of the clonogenic MM cell:

Over the last 5 years, three independent groups of investigators have attempted to address the clonal origin of the postulated myeloma stem cell with molecular biology techniques.242526

The only definite proof that B cells at various stages of development are clonally related derived from the analysis of their Ig rearrangements and, more precisely, the unique sequence of their VDJ junctions, which, in case of clonal relationship, should be identical. The application of an allele-specific oligonucleotide (ASO)-based PCR with ASO derived from the sequence of CDR3 analyzed in each case enabled the detection of a B cell population clonally related to the plasma cell tumor.242526

Using this strategy, it became feasible to demonstrate the existence of pre-switch μ/δ isotype bearing B cells clonally identical to γ or α isotype-bearing myeloma cells from the BM of the patients. Sorting of BM B cells into CD45+ and CD38+ populations resulted in lineage-specific expression of the clonally related RNA, with the Cμ and Cδ isotypes expressed by the CD45+ and the Cγ or Cα isotypes by the CD38+ population.24 In addition, sequence analysis of the clonogenic VDJ transcripts indicated that no further somatic mutations had accumulated from the pre- to the post-switch isotype transition. This result indicates that the cell of origin has already undergone antigen selection while traversing the GC; however, it does not justify the conclusion that clonal proliferation and differentiation of the tumor occurs in the absence of antigen selection.24 In two relevant studies,2425 the authors were able to detect pre-switch Cμ-bearing B cells only in the BM and postulated that these represented the precursor population for post-switch Cγ or Cα-bearing myeloma plasma cells. These findings, however, are not definite proof for the existence of clonally related pre- and post-switch clonal populations; in this context, it cannot be ruled out that the same clonogenic myeloma cell contains both Cμ and Cγ/Cα transcripts and, given the relatively low levels of Cμ transcripts within a cell, in certain B cell populations the number of these transcripts is lower than the PCR detection threshold. In another study, Bakkus et al26 analyzed enriched B cell populations from peripheral blood and BM and found in the peripheral blood Cμ and Cα transcripts clonally related to Cγ transcripts in the BM, with the same number of mutations and no intraclonal variation.

Recently, it has been recognized that memory B cells can express IgM+/IgD+, IgM+/IgG+/ ± IgD+ and that IgG+ B cells can revert back to IgM+/IgD+ expressors;27 this supports the intriguing possibility that the cell of origin in myeloma is indeed an antigen-selected memory B cell capable of recirculating from the peripheral blood to the BM and vice versa, as well as terminally differentiating to post-switch IgG+ or IgA+ plasmacytoid and plasma cell. A recently described, almost universal, finding of ‘promiscuous’ translocations into Ig HC switch regions in MM (t(11;14)(q13.3;q32), t(4;14) (p16.3;q32), t(6;14), etc)28 may further support the existence of aberrant switch variants at the mRNA level. The non-Ig translocation partner genes in chromosomes 11q13, 4p16, 6p25, 16q23, 8q24 and 18q21 are cyclin D1, FGFR3 (fibroblast growth factor receptor 3), IRF4 (interferon regulatory factor 4), the basic zipper c-maf transcription factor, c-myc, and bcl-2, respectively. Most of these genes are thought to be transcriptionally activated by coming close the IgH enhancer.29303132 While c-myc is invariantly translocated into IgH switch regions in murine plasmacytomas,33 this was never found to occur in human MM cell lines or tumor material until recently; the selective overexpression of one c-myc allele was ascribed to tumor-specific cis-dysregulation of c-myc in MM by an as yet unknown mechanism.34 However, recent work by Bergsagel's group suggests frequent c-myc translocations as late events associated with the progression of MM.35

Somatic hypermutation of MM VH genes:

Analysis of clonogenic Ig-VH genes in MM has shown high R:S mutation ratios in CDRs and low R:S mutation ratios in FWRs of the VH sequence.2636 CDRs are the structures responsible for the formation of the antigen binding loops; thus, it is conceivable that a high R:S mutation ratio in these regions would result in amino acid replacements that might ultimately lead to improved affinity for antigen of the particular B cell clone and its rescue from apoptosis. On the other hand, a low R:S ratio in FWRs is again compatible with antigen selection, since the FWR regions, while not contacting the antigen, are responsible for the three dimensional conformation of the CDRs and therefore any excessive load of R mutations would negate the ability of the VH to bind antigen.

In order to evaluate whether the number of R in the CDRs is significantly higher than the number expected to occur randomly, the binomial probability model adopted by Shlomchik et al37 was applied; this model takes into account the relative sizes of the CDRs and FWRs in VH, Vκ and Vλ. Chang and Casali38 modified the above model, assessing each VH and VL gene sequence codon by codon and that the R mutation frequencies (Rf) for certain V genes are inherently higher than expected in CDRs as computed according to the inherent ability of their codons to mutate.38

The binomial model postulates that the distribution of mutations in the CDRs and FWRs, provided mutations occur by chance, depends on: (1) the relative sizes of these regions (0.25 for the CDRs and 0.75 for the FWRs); (2) the assumption that the ratio of R to S in a protein region not under functional constraint should be close to 2.9 (however, this ratio should change if analysis is carried out according to the proposals of Chang and Casali, which takes into account the inherent tendency of certain codons, particularly those of CDR1, to mutate with a higher frequency irrespectively of antigen selection); and (3) the assumption that twice as many R mutations occur in the FWRs, since some of these having deleterious effects will be lost from a clone of antibody-producing cells and will not be included in the total number of R mutations observed in FWRs. Therefore, the total number of V region mutations would be distributed as follows: n = RCDR + S + 2RFWF. Therefore, the probability (P) of k·R mutations to occur in the CDRs for a total number of mutations equal to n is calculated by the equation P = [N!/k!(N − k)!]qk(1 − q)N−k, where q is the chance for an R mutation to occur in CDRs.

The largest study concerning somatic hypermutation in MM demonstrated a significant distribution of R mutations in CDRs, compatible with antigen selection, in about 22% of cases.36 However, this low incidence of mutations, simply based on statistical arguments, might indeed be an underestimation of a key biological phenomen.39 It is possible that a minimum number of amino acid substitutions in CDRs might ultimately confer a great increase in affinity for antigen and favor selection; this has been convincingly shown in hybridomas producing monoclonal antibodies, where a single amino acid replacement in CDRs by site-directed mutagenesis may cause a 10-fold rise in affinity.40 Therefore, it is not impossible to have a relatively modest R:S mutation ratio in CDRs and yet attain optimal conditions for high-affinity antigen binding. Moreover, a statistically significant low R:S ratio in FWRs in such situations might in itself constitute a surrogate measure for non-random distribution of mutations suggestive of selection by antigen. If these considerations are taken into account (clones with a few R mutations in CDRs but a large number of mutations in FWRs) then in the above cited study, evidence for the operation of antigen selection in MM could be obtained in 58% of cases, a fact also pointed out by the authors of that report.36

Monoclonal gammopathy of undetermined significance (MGUS), a much more common condition in the general population than MM, is considered pre-malignant with up to one third of MM cases suggested as emerging from MGUS. MGUS has been shown to harbor clonogenic B cells with ongoing mutations of the VH region genes resulting in intraclonal heterogeneity;41 this finding indicates that the cell of origin is indeed a GC B cell. In a recent study, KSHV has been detected in 2/8 patients with MGUS; the authors hypothesized that KSHV might be required for the transformation from MGUS to MM by sustaining the growth of malignant plasma cells.5 Moreover, translocations involving the 14q32 region leading to illegitimate IgH rearrangements were observed at a similar incidence to MM by FISH analysis.42 Therefore, MGUS and MM should be regarded as a continuous spectrum in a multistep transformation process.41 Delineation of the above fundamental issues regarding the cell of origin in MM would more likely require analysis of V(D)J Ig gene rearrangements by PCR in isolated single cell populations, with an approach similar to the one applied in Hodgkin's disease.43

VH gene usage in MM:

The analysis of VH gene usage in MM has revealed a relative over-representation of VH1–69, VH3–9, VH3–23 and VH3–30 genes;44 in contrast, certain genes (VH3–49, VH3–53 and VH4.21 (VH4–34)), which are rearranged with increased frequency in normal circulating B cells, autoimmune diseases and other B cell neoplasms were under-represented. In particular, the VH4.21 (VH4–34) gene, was not found rearranged in any of the 72 MM patients analyzed in one study.44 However, VH4–34 is markedly over-represented in the normal B cell repertoire: ≈8%, in CLL 4–22%; in ALL, 9–33%; and in diffuse large cell lymphoma (DLCL) ≈65%.45464748 VH4–34 encodes autoantibodies with cold agglutinin specificity (anti-I/i);49 collectively, the above data provide a plausible explanation, at the molecular level, for the paucity of autoimmune phenomena in MM.44

Light chain Ig genes

VL gene usage in MM:

Vκ or Vλ LC contribute to the 3-D conformational orientation of the antigen-binding site. It is, therefore, important to examine whether somatic hypermutation of the Vκ or Vλ LC genes is of equal and/or complementary significance to that of the VH genes with regard to antigen selection of the particular B cell clone in MM. In addition, VL gene mutation and family usage studies might provide important biologic information for the possibility of a biased repertoire, similarly to that which has been demonstrated regarding VH genes.

The rearranged Vκ genes identified in five published reports5051525354 were compared for differences and similarities regarding individual gene usage by MM clonogenic B cells (Table 1). A preference for over-representation of the O8–18 and O2–12 Vκ genes, which belong to the VκI family was observed. The VκI family members appear to be utilized more frequently than others in MM, but with similar frequencies to normal peripheral blood, fetal bone marrow and malignant B cells.55 The solitary VκIV family gene was found to be used in about 20% of cases when data from three of these studies are combined (see also Table 1). Sahota et al53 observed a strikingly high usage of the A27 (Humκv325) Vκ gene in 33% of their cases. In sharp contrast, this gene has not been found rearranged in any of the cases analyzed by us,50 Wagner et al51 and Cannell et al.52 Humκv325 has been described to rearrange with a very high frequency in B-CLL cells irrespectively of whether it is expressed or not (ie abortively rearranged in λ-bearing CLLs);56 it is believed to represent a Vκ gene derived from the autoimmune repertoire of CD5+ B cells expressing Igs with rheumatoid factor activity. In total, 27 Vλ genes have been analyzed in three of the above studies.505354 However, no definite conclusion can yet been reached regarding their preferential distribution in the existing Vλ gene families or biased usage of certain Vλ genes in MM.

Table 1  Collective data on Vκ gene usage in MM from various reports

CDR3 in rearranged MM VL genes:

CDR3 formation in rearranged VL genes differs substantially from that of VH genes. While CDR3 in VH genes is partially encoded by D gene segments flanked by random non-templated (N) nucleotides as a result of TdT activity, which creates imprecision of joining at the sites of VH-to-D and D-to-JH junctions, CDR3 in Vκ genes is encoded almost entirely by the Vκ region. However, additional nucleotides in the Vκ-Jκ junctions may be germline derived or inserted by the activity of TdT during active gene rearrangement in pre-B cells. Usually, no TdT activity is detected in B cells at the ontogenetic timing of κ gene rearrangement. Nevertheless, Ehlich et al15 demonstrated TdT activity during active Vκ–Jκ rearrangements at the pre-B cell stage in mice unable to rearrange Ig HC genes. Additional nucleotide sequences with properties of N segments have occasionally been observed in Vκ–Jκ junctions fitting neither the germline derivation nor the N-region insertion modes. In this context, other mechanisms may apply, such as slippage replication and somatic hypermutation targeting the additional nucleotides.11

N nucleotide insertions in the Vκ–Jκ junctions were detected in 5/11 (45%) of MM clones by our group39 and ranged from 14 to 33% in the other published studies.51525354 In contrast, there was a discordance between the studies regarding N region diversity at Vλ–Jλ junctions.535457 Functional Vλ–Jλ rearrangements have not been found to carry N nucleotide insertions at the junction or deletions in the Jλ gene segment in normal human B cells958 and very rarely in the mouse B cell repertoire.59 A recent report, dealing with the molecular mechanisms that affect the generation of the human Vλ and Jλ gene repertoire in normal peripheral B cells,60 demonstrated that the majority of B cells clones harbored N nucleotides at Vλ–Jλ junctions. The apparent controversy regarding the aforementioned data may possibly be resolved if one takes into account the reactivation of the recombinase machinery (RAG1 and RAG2 genes) together with TdT in GC B cells undergoing receptor editing.61

Somatic hypermutation in MM Vκ and Vλ genes:

In our experience, many cases (6/11; 55%) demonstrated extensively mutated Vκ gene sequences.50 The pattern of mutations in the Vκ rearranged genes revealed an asymmetric distribution in CDRs compared to FWRs. Most of the mutations occurring in FWRs were S, whereas mutations in the CDRs were more frequently of the R type. It is noteworthy that, in all studies, serine (Ser) residues represent mutational ‘hotspots’ in many cases analyzed. For instance, Ser31 has been identified as a mutational hotspot in CDR1.1751 Similarly, in a limited number of functional Vκ genes, 2/4 demonstrated a distribution of mutations suggestive of antigen selection,51 whereas this proved to be the case in 3/9 (33%) and 3/17 (18%) clonogenic Vκ genes in two other studies.5354

In five cases from our study, mutations were scattered throughout the Vκ region with no asymmetry in the distribution of R mutations in favor of the CDRs. This finding is not consistent with the operation of an antigen-driven process; rather, it raises the possibility that in certain Ig expressing mature B cells–the normal counterparts of the clonogenic cells of certain late B cell lymphoproliferative disorders—it is sufficient for the antigen selection mechanism to operate at the level of the VH region. Alternatively, it may imply that the neoplastic event occurred before selection by the antigen had taken place and was potent enough to prevent apoptosis in the absence of antigen selection. The above findings support the notion that the nature of the myeloma B cell progenitors is heterogeneous and this heterogeneity may be implicated in the clinical spectrum of the disease.

Analysis of Vλ genes revealed significant clustering of mutations within CDRs in 1/6 (17%),53 2/6 (33%)57 and 3/16 (19%)54 cases in the three studies reported.

In total, when both Vκ and Vλ genes used by MM cases are concerned, 8/17 (47%) in our study and 4/15 (27%) in the study of Sahota et al53 demonstrated an R/S mutation distribution compatible with the notion that the clonogenic B cell precursors of MM have been selected by antigen. The cohort of patients analyzed by Kiyoi et al54 does not appear to be representative of what happens in most series since the authors included four cases (out of 31; 12%) of IgD myeloma, a proportion much higher than the usual 1–2% of this Ig subclass in most clinical series. IgD myelomas represent a subgroup with a very high load of mutations within VH genes (up to 80) with almost all expressing λ-LCs and corresponding to IgM/IgD+ GC B cells that have undergone a peculiar Cμ-to-Cδ switch recombination.6263 In the study of Kiyoi et al,54 3/4 IgD MM were of the λ-subtype and one of these carried an unusually high load of mutations within the Vλ region.

Another characteristic of the hypermutation machinery with respect to rearranged VJλ genes is the targeting of Jλ regions; a finding that has been observed in clonal VJλ genes of MM.54 It is well recognized that the intronic enhancer of Ig (Ein) located between J and C regions in VH and Vκ genes (missing in Vλ), acts as a sort of boundary for somatic hypermutation and relatively, but not absolutely, prevents the occurrence of mutations downstream. However, in Vλ genes, where the Ein is lacking, the mutation tract is extended even further into the Cλ exon, although at a 100-fold less frequency of that in the Vλ region.64

Amyloidogenic Vκ and Vλ genes in MM and their hypermutation:

Ig LC-related amyloidosis (AL amyloidosis) refers to B cell lymphoproliferative diseases that share the common feature of extracellular deposition of pathogenic insoluble fibrillar deposits, consisting of free monoclonal κ or λ LC, in organs and tissues. The main constituent of AL deposits is the LC-Ig V region (Vκ or Vλ) and less frequently parts of the C region or whole Ig. Members of the Vκ1 (O8–18 and L18) and Vλ VI gene families are found rearranged with increased frequency in cases of AL amyloid.6566 Since Vλ VI-bearing LCs account for less than 5% of normal serum Igs, but are encountered in the vast majority of λ LC-related cases of amyloidosis, it has been suggested that Vλ proteins in this subgroup (Vλ VI) are associated with an invariant amyloid formation tendency.67 In our series of molecularly analyzed MM cases, two patients had clinical evidence of AL amyloidosis; one with a L11 gene and the other with a IGL V6S1 gene belonging to VκI and VλVI families, respectively.57 A recent comprehensive study analyzing the hypermutation of 14 amyloidogenic LCs has shown that 50% demonstrated evidence of distribution of mutations compatible with antigen selection; more specifically, 40% of Vκ genes and 56% of Vλ genes.68 More importantly, the authors described that, in their series, 44% of λ LC cases carried the VλIII.1 Vλ gene and 60% κ LC cases carried Vκ genes, members of the VκI family.68 Therefore, the VλIII.1 gene represents a new candidate to be added to the list of existing LC genes known to possess amyloidogenic potential.

Antigen selection imprint on MM Ig genes

According both to our data and to that of others, the rearranged VH and VL genes of the clonogenic B cells in MM almost always show extensive somatic mutations, with high R:S ratios in CDRs, implying that in this malignancy the neoplastic B cell precursor has already undergone antigen selection and both its HC and LC have participated in the process. In contrast, in FL, a disorder typically arising from GC B cells with strong indication for antigen-selected mutations in clonogenic VH genes, we have demonstrated that the respective Vκ genes are relatively unmutated or in some cases bear less antigen-selected mutations and are more likely at a stage of differentiation where they have not yet been the target of the somatic hypermutation machinery.69 (Figure 1). Moreover, since CDR3 formation might play a pivotal role in antigen binding and selection, our analysis of D gene reading frames (RF) in B cell lymphoid malignancies revealed a consistent pattern of usage in RF2 and RF3 both for MM and FL;70 it is noteworthy that similar RF usage was observed in D gene segments paricipating in the t(14;18) and t(11;14) chromosomal translocations.7172

Generally, the nature of somatic mutations in hypermutating B cells indicates a preference for transitions over transversions with purines being preferentially targeted over pyrimidines, suggesting strand bias; mutations are concentrated mainly in CDRs and most often are single nucleotide substitutions.7273747576 However, investigators have reported deletions or insertions of base stretches of varying length in hypermutating B cells and have concluded that this is a byproduct of the somatic hypermutation machinery within the GC rather than a result of V(D)J recombination in earlier stages of B cell ontogeny.7778 Furthermore, it appears that several types of oncogene translocations and Ig HC/LC trancations in heavy chain disease and AL amyloidosis are the result of this phenomenon.77 In our series, we have decribed a single case of six base deletion in FWR2 in an otherwise potentially functional Vκ gene in FL yielding a 4–5% incidence for the analyzed Vκ genes in both FL and MM, which is similar to that observed by others in normal GC and post-GC B cells in the process of somatic hypermutation.6979

A recent important observation53 pointed out that comparison of VH and VL sequences derived from the same MM B cell clone yielded significant clustering of mutations for antigen selection in CDRs of either the VH or VL gene, but never in both. However, no physiological analogue to this phenomenon has been observed, at least when normal antigen-selected B cells are concerned. Similarly, with regard to FLs in our previous studies,80 the majority of cases exhibited significant clustering of mutations either in VH or Vκ genes, but not in both, thus supporting the suggestion, that a complementary imprint of antigen selection witnessed by VH and VL tumor-derived sequences might constitute an important event during B cell ontogeny.

Monitoring residual disease during therapy in multiple myeloma

PCR-based strategies for rearranged VDJ Ig genes were applied for the detection of residual clonogenic myeloma cells in the BM and peripheral blood as well as the assessment of contamination of peripheral stem cell autografts by neoplastic MM B cells. PCR for Ig genes is now a powerful tool in assessing the frequency of clonogenic malignant B cells at different stages of experimental treatment programs8182 employing sequential intensive chemotherapy regimens, HSC mobilization and finally high-dose chemotherapy regimens rescued by the cryopreserved HSC.8384 Moreover, recently, selection of CD34+ circulating hematopoietic progenitors after mobilization with chemotherapy and colony-stimulating factors, in order to negatively select myeloma cells, enabled the reduction of 2–3 logs of clonogenic B cells in the autograft; in certain cases, this has resulted in elimination of the contaminating malignant cells. However, persistence of clonal myeloma cells within the CD34+ mobilized fraction makes relevant the requirement of purging the graft.82 In addition, after a single course of high-dose chemotherapy rescued by autologous HSC almost all patients remain positive by PCR.8185 Recent developmental work employing double high-dose chemoradiotherapy86 with autologous HSC transplantation has demonstrated that this strategy can induce molecular remissions defined by sensitive PCR methods for rearranged Ig V genes.87 Therefore, sensitive/quantitative PCR methods can provide a means of assessing the degree of cytoreduction after allo- or auto-grafting in MM.

Idiotypic vaccines in MM

An important area of clinical investigation related to Ig V region genes represents various immunotherapeutic approaches directed against the slg of malignant B cells. The idiotype (id) of the slg expressed by the malignant B cell clone constitutes an almost unique tumor-specific antigen. Rescue and isolation of the monoclonal Ig from each patient's tumor cells has offered the opportunity to apply it as a tumor-specific id vaccine in patients with B cell NHL.88 Updated results of this early pilot study in 41 patients indicated that freedom from disease progression and survival were significantly longer in those patients who mounted an anti-tumor id immune response compared to those who did not or to those not vaccinated. Transfer of anti-id T cell immunity from the donor to a patient with advanced MM undergoing allogeneic bone marrow transplantation (BMT) was achieved after active immunization of donor with purified patient's monoclonal Ig.89 It was therefore concluded that immunity against tumor-derived id determinants can be transferrable from the donor to the recipient after allogeneic BMT.

Isolation of patient-specific id Ig has been simplified by PCR amplification, cloning and coexpression of VH and VL genes from the malignant B cell clone assembled together with a flexible linker as a single-chain Fv (scFv).90 Alternatively, live DNA vaccines based on vectors carrying VH and VL genes in appropriate delivery vehicles can be used in order to generate potent anti-id T cell immunity.91 Potentiation of cellular immunity against tumor-derived id has been achieved by id/granulocyte–macrophage colony-stimulating factor (GM-CSF) fusion protein in B cell lymphoma.92 Dendritic cells as carriers of whole id-Ig or VH/VL peptide fragments may also represent a viable option for the development of potent vaccination strategies.93 Id vaccination strategies in MM using antigen (id)-pulsed dendritic cells appear particularly relevant in the post-autologous HSC transplant setting, where optimal conditions for successful immunotherapy are offered by the minimal amount of residual disease.

Conclusions

The study of Ig genes in MM has helped to elucidate important biological questions, concerning the nature of the clonogenic cell, its molecular archeology related to antigen selection, ontogenetic location as a memory B cell and pathophysiology of major disease manifestations. In addition, these research efforts provided tools for the detection of minimal residual disease after high-dose chemoradiotherapy/stem cell transplantation strategies and for devising tumor-specific idiotypic vaccines for active immunization, which aim at retargeting a potent immune response against myeloma cells.

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Acknowledgements

CK and KS were supported by grants from the Secretariat for Research and Technology EPET/603 and AXIA 13. NS was supported from the Secretariat for Research and Technology program AXIA 13.

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Kosmas, C., Stamatopoulos, K., Stavroyianni, N. et al. Origin and diversification of the clonogenic cell in multiple myeloma: lessons from the immunoglobulin repertoire. Leukemia 14, 1718–1726 (2000) doi:10.1038/sj.leu.2401908

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Keywords

  • immunoglobulin genes
  • somatic hypermutation
  • multiple myeloma

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