Analysis of clonotypic switch junctions reveals multiple myeloma originates from a single class switch event with ongoing mutation in the isotype-switched progeny

Multiple myeloma (MM) is a cancer of plasma cells (PCs) expressing immunoglobulin heavy chain (IgH) postswitch isotypes. The discovery of earlier stage cells related to postswitch PCs, called preswitch clonotypic IgM (cIgM) cells led to the hypothesis that cIgM cells may be MM progenitors, replenishing the tumor throughout malignancy. cIgM cells may do this by undergoing class switch recombination (CSR), a process detectable in postswitch PCs as multiple IgH switch junctions associated with a single clonotypic IgH V/D/J. We addressed this with a specific clonotypic-switch polymerase chain reaction (PCR), informative for 32 of 41 cases. Here we made 2 significant discoveries: (1) in all cases, we detected only a single clonotypic switch fragment that persists over time (1-7.6 years), and (2) we detected ongoing mutation upstream of the switch junction in 5 of 6 patients, often targeting the intronic enhancer, a key control region in IgH expression. The presence of a single, unchanging clonotypic switch junction suggests that cIgM cells are not MM-PC progenitors; rather, postswitch PCs arise from a single cIgM cell, and MM-PC progenitors reside in the postswitch population. Furthermore, mutations revealed here provide a new marker to identify MM-PC progenitors and aggressive clones that evolve throughout malignancy.     

A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma

Although the in vitro expansion of the multiple myeloma (MM) clone has been unsuccessful, in a novel three-dimensional (3-D) culture model of reconstructed bone marrow (BM, n = 48) and mobilized blood autografts (n = 14) presented here, the entire MM clone proliferates and undergoes up to 17-fold expansion of malignant cells harboring the clonotypic IgH VDJ and characteristic chromosomal rearrangements. In this system, MM clone expands in a reconstructed microenvironment that is ideally suited for testing specificity of anti-MM therapeutics. In the 3-D model, melphalan and bortezomib had distinct targets, with melphalan targeting the hematopoietic, but not stromal com-partment. Bortezomib targeted only CD138(+)CD56(+) MM plasma cells. The localization of nonproliferating cells to the reconstructed endosteum, in contact with N-cadherin-positive stroma, suggested the presence of MM-cancer stem cells. These drug-resistant CD20(+) cells were enriched more than 10-fold by melphalan treatment, exhibited self-renewal, and generated clonotypic B and plasma cell progeny in colony forming unit assays. This is the first molecularly verified demonstration of proliferation in vitro by ex vivo MM cells. The 3-D culture provides a novel biologically relevant preclinical model for evaluating therapeutic vulnerabilities of all compartments of the MM clone, including presumptive drug-resistant MM stem cells.     

Spontaneous monoclonal immunoglobulin-secreting peripheral blood mononuclear cells as a marker of disease severity in multiple myeloma

Peripheral blood from patients with multiple myeloma (MM) contains a small number of plasma cells related to the bone marrow tumour cells by their cytoplasmic immunoglobulin (Ig), their cell membrane antigen expression and/or their gene rearrangements, but hitherto the monoclonal Ig (M-Ig) production by circulating cells has not been reported. Using a two-colour ELISPOT assay, Ig-secreting cells (Ig-SCs) were detected in the blood of 28 MM and five Waldenstrom's macroglobulinaemia (WM) patients. The number of cells that spontaneously produced an Ig isotype similar to that of the M-Ig in serum was greater than that of the other Ig-SCs. MM patients presented an excess of circulating heavy-chain (alpha or gamma) Ig-SCs (0.38% of the PBMC) with kappa or lambda light chains (0.48%) compared with the number of cells secreting the other heavy- (0.02%) and light-chain isotypes (0.03%). WM patients also presented high numbers of cells secreting the mu-heavy-chain isotype (0.66%). The Ig synthesized in vitro was characterized as monoclonal, and the M-Ig secretory capacity of the peripheral blood cells was similar to that observed for Ig-SCs from polyclonal activated B cells in vivo. The number of these monoclonal cells was significantly increased in patients in an advanced stage of MM (I/II vs. III, P < 0.001) and correlated with the serum beta-2 microglobulin concentration (r = 0. 69; P < 0.0003). The number of M-Ig-SCs in MM patients could be a useful marker for evaluating the progression of multiple myeloma.     

Effect of interferon-α on immunoglobulin production by peripheral blood mononuclear cells in multiple myeloma

Abstract: To test a hypothesis that interferon-α (IFN) treatment might restore normal immunoglobulin (Ig) production in multiple myeloma (MM), the effect of IFN on Ig isotype (IgG and IgA) production by peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) in MM patients was analyzed by ELISA. IFN at a concentration of 1000 U/ml was found to enhance IgA production by PB MNCs in IgA-MM and had a trend to stimulate IgG production in IgG-MM. The effect of IFN on nonparaprotein Ig isotype production was more variable, with mostly neutral or inhibitory effects being seen in both the MM subtypes. In contrast to the influences observed in MM patients, IFN at the same concentration inhibited both IgG and IgA production by PB MNCs in healthy controls. In studying BM cells, IFN was found to reduce IgA production in IgA-MM, but had a neutral effect on IgG production in IgG-MM. In the controls, the production of both the IgG and the IgA isotypes by BM MNCs was decreased by IFN. On the basis of these results it seems that the disease itself somehow affects the Ig-producing cells in MM, when measured as different responses of the cells to exogenous IFN in vitro. The results do not support the hypothesis that IFN treatment could restore normal Ig production in MM patients.     

In multiple myeloma, clonotypic B lymphocytes are detectable among CD19+ peripheral blood cells expressing CD38, CD56, and monotypic Ig light chain [published erratum appears in Blood 1995 Jun 1;85(11):3365]

Multiple myeloma (MM) is characterized by a plasma cell infiltrate of the bone marrow (BM). However, late-stage monotypic B cells have been detected in the blood. This work analyzes the effects of clinical treatment on late stage CD19+ B cells present in 752 blood samples from 152 MM patients. MM patients have 2 to 8 times as many circulating CD19+ cells as do normal donors. Analysis of the Ig heavy chain (IgH) gene rearrangements using polymerase chain reaction indicates that the CD19+ population includes cells sharing the same clonotypic CDR3 region as is detected in the BM plasma cells, for patients analyzed during chemotherapy or in relapse. They are also monotypic as defined by their cytoplasmic or surface expression of Ig kappa or lambda light chain. The light chain restriction is the same as that of the BM plasma cells. Individual patients observed over 1- to 2-year periods exhibit considerable variation in the number of B cells present in blood; this number does not correlate with the concentration of serum monoclonal Ig. The monoclonal blood CD19+ cells are not eliminated by any of the chemotherapy regimens analyzed and remain at high levels during transient remissions. Patients in the progressive phase of disease or in relapse have significantly higher numbers of B cells than do patients in transient remission or untreated patients. During periods when the quantity of blood B cells approaches normal, phenotypically their quality is highly abnormal, with physical and phenotypic heterogeneity. Most B cells express CD45R0, a high density of CD38, and CD56 characteristic of late-stage B or pre-plasma cells. CD38hi blood B cells had a cyclical presence. We conclude that monoclonal B cells in the blood of myeloma patient populations include drug-resistant reservoirs of clonotypic cells that may underlie relapse.     

Molecular monitoring of minimal residual disease in patients with multiple myeloma

Improvement of transplantation strategies and a multitude of emerging novel therapies result in a better treatment outcome in patients with multiple myeloma (MM). This gives rise to the need for sensitive methods to detect minimal residual disease (MRD) in MM. Qualitative molecular monitoring using allele-specific oligonucleotide PCR for the immunoglobulin heavy chain (IgH) is well established to detect clonotypic cells after therapy or in stem cell harvests. Recently, real-time IgH PCR or limiting dilution based PCR assays offer the possibility to quantify the amount of residual tumour cells. In this review, different qualitative and quantitative IgH PCR techniques will be discussed as well as the current clinical role of molecular monitoring of MRD in patients with MM.     

Myeloma progenitors in the blood of patients with aggressive or minimal disease: engraftment and self-renewal of primary human myeloma in the bone marrow of NOD SCID mice

The myelomagenic capacity of clonotypic myeloma cells in G-CSF mobilized blood was tested by xenotransplant. Intracardiac (IC) injection of NOD SCID mice with peripheral cells from 5 patients who had aggressive myeloma led to lytic bone lesions, human Ig in the serum, human plasma cells, and a high frequency of clonotypic cells in the murine bone marrow (BM). Human B and plasma cells were detected in BM, spleen, and blood. Injection of ex vivo multiple myeloma cells directly into the murine sternal BM (intraosseus injection [IO]) leads to lytic bone lesions, BM plasma cells, and a high frequency of clonotypic cells in the femoral BM. This shows that myeloma has spread from the primary injection site to distant BM locations. By using a cellular limiting dilution PCR assay to quantify clonotypic B lineage cells, we confirmed that peripheral myeloma cells homed to the murine BM after IC and IO injection. The myeloma progenitor undergoes self-renewal in murine BM, as demonstrated by the transfer of human myeloma to a secondary recipient mouse. For 6 of 7 patients, G-CSF mobilized cells from patients who have minimal disease, taken at the time of mobilization or after cryopreservation, included myeloma progenitors as identified by engraftment of clonotypic cells and/or lytic bone disease in mice. This indicates that myeloma progenitors are mobilized into the blood by cyclophosphamide/G-CSF. Their ability to generate myeloma in a xenotransplant model implies that such progenitors are also myelomagenic when reinfused into patients, and suggests the need for an effective strategy to purge them before transplant.     

Quantitation of minimal residual disease in multiple myeloma using an allele-specific real-time PCR assay

OBJECTIVE: To develop a real-time PCR method, based on the 5'nuclease TaqMan technology, for quantitation of clonal cells in multiple myeloma (MM). MATERIALS AND METHODS: The real-time quantitative PCR method incorporates both an allele-specific oligonucleotides (ASO) primer and an ASO dual-labeled fluorogenic probe (ASO TaqMan probe). The ASO primer and probe corresponded to the complementary determining region 3 (CDR3) of the rearranged immunoglobulin heavy chain gene (IgH). With the use of a sequence detector, PCR product accumulation was measured through the ASO TaqMan probe. The real-time PCR method was compared with flow cytometric quantitation of myeloma plasma cells. RESULTS: The application of the real-time quantitative ASO IgH PCR method is illustrated by a sequential analysis of minimal residual disease (MRD) in bone marrow (BM) samples from myeloma patients undergoing peripheral blood stem cell (PBSC) transplantation. The real-time PCR method was able to quantitate residual malignant cells in BM samples from patients who were considered to be in complete remission. Further, it was illustrated that a potential problem in determining tumor cell content in myeloma BM samples is the heterogeneous infiltration of the marrow. CONCLUSION: The application of the real-time PCR method provides a sensitive, highly specific, and reproducible quantitation of myeloma cells.     

多发性骨髓瘤血清单克隆免疫球蛋白及IgG亚类检测

目的：探讨多发性骨髓瘤（MM）患者血清单克隆免疫球蛋白（M－蛋白）及IgG亚类的分布。方法：用免疫固定电泳对236例MM患者血清中M－蛋白进行免疫球蛋白重链及轻链定性分析,并用单扩散法对其中76例IgG型MM进行IgG亚类分析。结果：236例中,IgG型104例（44．07％）,IgA型60例（25．42％）,IdD型22例（9．32％）,轻链型47例（19．92％）,未分泌型3例（1．27％）。对其中76例IgG型MM研究发现：IgG161例（80．26％）,IgG26例（7．89％）,IgG1-IgG2双克隆1例（1．32％）,IgG3 8例（10．53％）,未见IgG4。结论：血清M－蛋白重链及轻链分类及IgG亚类的检测对MM的免疫分型、指导临床治疗及判断疾病预后具有一定的意义。     

Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish

During the analysis of Ig superfamily members within the available rainbow trout (Oncorhynchus mykiss) EST gene index, we identified a unique Ig heavy-chain (IgH) isotype. cDNAs encoding this isotype are composed of a typical IgH leader sequence and a VDJ rearranged segment followed by four Ig superfamily C-1 domains represented as either membrane-bound or secretory versions. Because teleost fish were previously thought to encode and express only two IgH isotypes (IgM and IgD) for their humoral immune repertoire, we isolated all three cDNA isotypes from a single homozygous trout (OSU-142) to confirm that all three are indeed independent isotypes. Bioinformatic and phylogenetic analysis indicates that this previously undescribed divergent isotype is restricted to bony fish, thus we have named this isotype "IgT" (tau) for teleost fish. Genomic sequence analysis of an OSU-142 bacterial artificial chromosome (BAC) clone positive for all three IgH isotypes revealed that IgT utilizes the standard rainbow trout V(H) families, but surprisingly, the IgT isotype possesses its own exclusive set of D(H) and J(H) elements for the generation of diversity. The IgT D and J segments and tau constant (C) region genes are located upstream of the D and J elements for IgM, representing a genomic IgH architecture that has not been observed in any other vertebrate class. All three isotypes are primarily expressed in the spleen and pronephros (bone marrow equivalent), and ontogenically, expression of IgT is present 4 d before hatching in developing embryos.     

Real-time polymerase chain reaction in multiple myeloma: quantitative analysis of tumor contamination of stem cell harvests

OBJECTIVE: Autologous transplantation of bone marrow (BM) and peripheral blood progenitor cells (PBPC) is commonly used for treatment of multiple myeloma (MM). Although both stem cell sources harbor residual clonal cells, a quantitative evaluation of their level of tumor contamination (LTC) still needs to be performed through highly accurate and reproducible approaches. In this study, we used a validated real-time polymerase chain reaction (PCR) strategy to evaluate LTC of BM and PBPC samples obtained from MM patients. MATERIALS AND METHODS: The patients underwent two different mobilization courses (defined as early or late course) following two cycles of cyclophosphamide 5 g/m(2). LTC was evaluated by measuring the number of clonal immunoglobulin heavy-chain rearrangements followed by normalization of samples using the GAPDH gene. RESULTS: Overall, 26 PBPC and 12 BM samples were analyzed. Main results are as follows. 1) PBPC harvests are less contaminated than BM samples taken immediately after each mobilization course (median difference 2.68 logs; range 1.7 to 4.6) (p < 0.0001). 2) LTC of PBPC harvests has only minimal variation among different leukaphereses performed during the same mobilization course (median difference 0.45 logs; range 0.22 to 1.2). 3) No difference was observed among PBPC and BM samples obtained after the late mobilization course as compared to the early mobilization course (median reduction 0.21 logs; range -0.39 to 1.3) (p = 0.84). 4) In PBPC but not in BM samples, there is a clear overestimation of the percentage of plasma cells when flow cytometric evaluation of CD38(bright) cells is compared to real-time PCR results. This suggests that in PBPC, most CD38(bright) cells do not belong to the neoplastic clone. CONCLUSIONS: Real-time PCR using the IgH rearrangement proved an effective tool for monitoring LTC in stem cell harvests from MM patients. The smaller LTC of PBPC harvests supports the role of PBPC as stem cell rescue for MM patients compared to BM cells.     

Molecular Monitoring of Minimal Residual Disease in Patients with Multiple Myeloma

Improvement of transplantation strategies and a multitude of emerging novel therapies result in a better treatment outcome in patients with multiple myeloma (MM). This gives rise to the need for sensitive methods to detect minimal residual disease (MRD) in MM. Qualitative molecular monitoring using allele-specific oligonucleotide PCR for the immunoglobulin heavy chain (IgH) is well established to detect clonotypic cells after therapy or in stem cell harvests. Recently, real-time IgH PCR or limiting dilution based PCR assays offer the possibility to quantify the amount of residual tumour cells. In this review, different qualitative and quantitative IgH PCR techniques will be discussed as well as the current clinical role of molecular monitoring of MRD in patients with MM.     

Successive switching of antibody isotypes expressed within the lines of a B-cell clone.

An antigen-stimulated B cell can differentiate to form a clone of cells that secrete antibodies with the IgM, IgG1, and IgA isotypes. We have examined the sequence of isotype expression by proliferating cell lines within a clone by directly staining for immunoglobulin in the cytoplasm of clonal daughter cells. All of the cones selected for analysis originally secreted IgM as well as other isotypes, as determined by radioimmunoassay of culture fluids. Cell staining showed that (i) at least 25% of the cells contained more than one isotype, indicating that cells can switch expression of isotypes during clonal expansion; (ii) some cells contained both IgM and IgA without detectable IgG1, and some cells contained both IgM and IgG1 without detectable IgA, suggesting that cells can switch from IgM directly to IgG1 or IgA; and (iii) some cells contained both IgG1 and IgA, indicating that cell lines can undergo two successive switches from IgM to the IgG1 and IgA isotypes. Using serological markers for allotypic determinants on the constant region of heavy chains, we have also shown that heterozygous B cells specific for phosphorylcholine generated clones of cells that secreted IgG1 and IgA antibodies that were derived from the expression of genes on only one parental chromosome. Assuming that the gene coding for the IgA isotype is the last gene in the gene cluster coding for heavy chain isotypes, we have proposed a model of successive, but not necessarily stepwise, switching of isotypes within B-cell lines, leading to the eventual accumulation of cells expressing IgA.     

Evaluation of the kinetics of the bone marrow tumor load in the course of sequential high-dose therapy assessed by quantitative PCR as a predictive parameter in patients with multiple myeloma

The aim of this investigation was to examine the possible clinical significance of the kinetics of bone marrow (BM) tumor load during the course of sequential high-dose therapy (HDT) as assessed by quantitative PCR in patients with multiple myeloma. In 20 patients with multiple myeloma (MM) treated with two consecutive cycles of HDT followed by autologous peripheral blood stem cell transplantation (PBSCT), clonotypic cells in the peripheral blood (PB) and BM were quantitated by PCR using allele-specific oligonucleotides (ASO) prior to the first, immediately prior to the second, and after the second HDT. The median proportion of clonotypic cells in the BM was 1.27% before the first HDT (range, 0.03-70%), 0.17% after the first (range, 0.001-22%), and 0.05% after the second HDT (range, 0.00009-1.44%). The median number of circulating clonotypic cells was 65/ml (range, 0.9-10842) prior to HDT, 2.7/ml (range, 0-315) after the first, and 3.5/ml PB (range, 0.7-97) after the second HDT. While the median BM tumor load decreased during the first (P = 0.03) and second (P = 0.044) HDT cycles, only the first cycle resulted in a reduction of clonotypic cells in the PB (P = 0.00078 and P= 1.0, respectively). In seven patients, the BM tumor load did not decrease below the initial level after one or two cycles of HDT. All of these patients developed progressive disease (median, 19 months post first cycle; range, 10-21). Of the remaining 13 patients, only four relapsed (18, 19, 21 and 22 months after the first cycle of HDT), while nine remain in response (median followup, 29 months; range, 18-41) (log-rank test P = 0.0009). Our results indicate that the kinetics of the BM tumor load is a predictive parameter in patients with MM and identifies those patients who could benefit from further therapy including new treatment modalities.     

Impaired class switch recombination (CSR) in Waldenstrom macroglobulinemia (WM) despite apparently normal CSR machinery

Analysis of clonotypic isotype class switching (CSR) in Waldenstr?m macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (MGUS) reveals a normal initial phase of B-cell activation as determined by constitutive and inducible expression of activation-induced cytidine deaminase (AID). Switch mu (Smu) analysis shows that large deletions are not common in WM or IgM MGUS. In CD40L/IL-4-stimulated WM cultures from 2 patients, we observed clonotypic IgG exhibiting intraclonal homogeneity associated with multiple hybrid Smu/Sgamma junctions. This suggests CSR had occurred within WM cells. Nevertheless, the estimated IgG/IgM-cell frequency was relatively low (1/1600 cells). Thus, for the majority of WM B cells, CSR does not occur even when stimulated in vitro, suggesting that the WM cell is constitutively unable to or being prevented from carrying out CSR. In contrast to WM, the majority of IgM MGUS clones exhibit intraclonal heterogeneity of IgH VDJ. Furthermore, most IgM MGUS accumulate more mutations in the upstream Smu region than do WM, making them unlikely WM progenitors. These observations suggest that switch sequence analysis may identify the subset of patients with IgM MGUS who are at risk of progression to WM.     

Advances in biology of multiple myeloma: clinical applications

There appear to be 2 pathways involved in the early pathogenesis of premalignant monoclonal gammopathy of undetermined significance (MGUS) and malignant multiple myeloma (MM) tumors. Nearly half of these tumors are nonhyperdiploid and mostly have immunoglobulin H (IgH) translocations that involve 5 recurrent chromosomal loci, including 11q13 (cyclin D1), 6p21 (cyclin D3), 4p16 (fibroblast growth factor receptor 3 [FGFR3] and multiple myeloma SET domain [MMSET]), 16q23 (c-maf), and 20q11 (mafB). The remaining tumors are hyperdiploid and contain multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21, but infrequently have IgH translocations involving the 5 recurrent loci. Dysregulated expression of cyclin D1, D2, or D3 appears to occur as an early event in virtually all of these tumors. This may render the cells more susceptible to proliferative stimuli, resulting in selective expansion as a result of interaction with bone marrow stromal cells that produce interleukin-6 (IL-6) and other cytokines. There are 5 proposed tumor groups, defined by IgH translocations and/or cyclin D expression, that appear to have differences in biologic properties, including interaction with stromal cells, prognosis, and response to specific therapies. Delineation of the mechanisms mediating MM cell proliferation, survival, and migration in the bone marrow (BM) microenvironment may both enhance understanding of pathogenesis and provide the framework for identification and validation of novel molecular targets.     

High-dose sequential chemoradiotherapy in multiple myeloma: residual tumor cells are detectable in bone marrow and peripheral blood cell harvests and after autografting

Based on preliminary encouraging results in terms of response rate and survival, high-dose chemoradiotherapy has gained considerable interest in the treatment of patients with multiple myeloma (MM). We have evaluated the presence of residual myeloma cells in 15 of 18 patients enrolled in a high-dose sequential (HDS) chemoradiotherapy program followed by autografting. Our analysis has been performed both on bone marrow (BM) and peripheral blood (PB) cell harvests and after autografting. As it has been recently shown that B cells clonally related to malignant plasma cells are detectable in MM patients, we have developed a polymerase chain reaction (PCR)-based strategy to detect both residual B cells and plasma cells using clone-specific sequences derived from the rearrangement of Ig heavy chain (IgH) genes. The complementarity-determining regions (CDR) of IgH genes have been used to generate tumor-specific primers and probes. The constant (C) region usage defined the differentiation stage of residual myeloma cells. We report that plasma cells were detectable in PB and BM cell harvests and after transplantation in all assessable patients, irrespective of disease status. B cells were detectable in a consistent proportion of BM and PB samples at diagnosis, but only in one case at the time of PB and BM cell harvests. These cells became sometimes detectable after transplantation. Whether residual myeloma cells are clonogenic and contribute to relapse is currently unknown, and further investigations are required.