Characterization and use of the novel human multiple myeloma cell line MC-B11/14 to study biological consequences of CRISPR-mediated loss of immunoglobulin A heavy chain.

The genetic abnormalities underlying multiple myeloma (MM) are notoriously complex and intraclonal heterogeneity is a common disease feature. In the current study, we describe the establishment of a monoclonal immunoglobulin A (IgA) kappa (κ) MM cell line designated MC-B11/14. Cytogenetic and fluorescence in situ hybridization analyses of the original and relapse patient samples revealed that the MM clone was nonhyperdiploid and possessed an 11;14 chromosomal translocation. The MC-B11/14 cell line, established from the relapse sample, is tetraploid and houses the t(11;14) abnormality. Given our long-standing interest in Ig function and secretion, we next used CRISPR technology to knock out IgA heavy-chain expression in the MC-B11/14 cells to assess the biological consequences of converting this cell line to one only expressing κ light chains. As expected, secretion of intact IgA was undetectable from MC-B11/14IgA- cells. Sensitivity to pomalidomide treatment was similar between the MC-B11/14WT and MC-B11/14IgA- cells; however, MC-B11/14IgA- cells were found to be significantly more resistant to bortezomib treatment. This study describes the establishment of a new human MM cell line tool with which to study disease biology and the use of CRISPR technology to create a potentially useful model with which to study MM light-chain escape.

Methionine PET Might Be Reliable for the Detection of Low M-Protein-Producing Myelomas.

C-methionine (MET) positron emission tomography (PET) is more sensitive than F-fluorodeoxyglucose (FDG)-PET for detecting myeloma lesion. Many clinical studies support this evidence mainly for multiple myeloma cases of IgG or IgA subtypes. However, this is not confirmed for low monoclonal protein-producing myelomas, such as IgD, IgE, and nonsecretory type. We encountered a 71-year-old man with IgD λ-type myeloma. In this patient, FDG-PET did not reveal any abnormal uptake lesions, whereas MET-PET revealed diffuse bone marrow uptake that relieved after initial chemotherapy. We speculate that the determinants for high serological activity of protein metabolism are transporter system activity or proliferation index.

Epstein-Barr virus infection leads to partial phenotypic reversion of terminally differentiated malignant B cells.

The B cell lymphomas associated with Epstein-Barr virus (EBV) are not limited to any specific stage of B cell differentiation but covers widely different B cell phenotypes. In vitro infection of the virus negative tumors with a recombinant EBV strain has provided important insights into virus-tumor interaction. Here, we investigated the interaction between EBV and terminally differentiated tumor derived B cells, namely multiple myeloma (MM). The in vitro EBV infected MM expressed restricted viral latency. Acquisition of the virus was accompanied by a partial reprogramming to a mature B cell phenotype. Thus, the plasma cell markers syndecan-1 (CD138), Blimp1 and MUM1 were downregulated, while expression of HLADR, CIITA and TCL1, which are normally not expressed in plasmacytoid cells, was upregulated. The silenced transcription factor gene encoding Pax5 and its target BLNK were activated. Significantly, the free lambda light chains secreted in the medium were reduced in EBV infected MM clones. Collectively, these results suggest that the restricted EBV latency can cause at least partial phenotypic reversion of terminally differentiated B tumor cells. We suggest that the restricted EBV latent gene expression may not only be the consequence but the cause of the mature B cell phenotype, actively participating in the virus persistence.

Granulocyte colony stimulating factor-producing multiple myeloma associated with neutrophilia.

We report a case of IgG-kappa multiple myeloma associated with neutrophilia (WBC 31,300/microl, neutrophil 90.5%). Interestingly, the serum level of granulocyte colony stimulating factor (G-CSF) in this patient was elevated to 1,500 pg/ml (normal range: 5.78-27.5). Plasma cells were 35% in the bone marrow and were strongly stained with anti-G-CSF antibody. To directly study the production of G-CSF from plasma cells in this patient, CD138 positive plasma cells were purified from bone marrow of multiple myeloma patients by magnetic sorting. The expression of G-CSF mRNA was observed in CD138 positive plasma cells from this myeloma patient with neutrophilia by RT-PCR. In contrast, the expression of G-CSF mRNA was not detected in CD138 positive plasma cells from the other multiple myeloma patients without neutrophilia and 4 human myeloma cell lines (HS-Sultan, IM9, RPMI8226, U266) by RT-PCR. After the CD138 positive plasma cells were cultured in vitro for 48 h, the production of G-CSF protein was confirmed (71.8 pg/ml) in the supernatant by ELISA. These results indicated plasma cells of this myeloma patient directly produced G-CSF and that this was the primary cause of neutrophilia.

[MALT lymphoma producing IgG-kappa type M-protein].

A 72-year-old woman, who has been administered prednisolone and azathioprine with diagnoses of idiopathic thrombocytopenic purpura (ITP) and autoimmune hepatitis (AIH), underwent a complete medical examination because of monoclonal gammopathy (IgG-kappa). Tumors were found in the ileum and descending colon. Pathological examination of biopsy specimens suggested a diagnosis of marginal zone B-cell lymphoma of the MALT type with a high-grade component. Flow cytometric analysis by two-color staining revealed that the neoplastic B cells expressed CD38, CD19, IgG and kappa, but not CD5 or CD10. There were no abnormal plasma cells in bone marrow smears. The patient achieved complete remission after receiving three cycles of THP-COP chemotherapy, which resulted in a decrease of the IgG level to within the normal range. These findings indicated that monoclonal IgG-kappa might be produced by lymphoma cells. However, the relationship of the immunosuppressive agents to the pathogenesis of the MALT lymphoma remains to be clarified.

Ectopic expression of fibroblast growth factor receptor 3 promotes myeloma cell proliferation and prevents apoptosis.

The t(4;14) translocation occurs in 25% of multiple myeloma (MM) and results in both the ectopic expression of fibroblast growth factor receptor 3 (FGFR3) from der4 and immunoglobulin heavy chain-MMSET hybrid messenger RNA transcripts from der14. The subsequent selection of activating mutations of the translocated FGFR3 by MM cells indicates an important role for this signaling pathway in tumor development and progression. To investigate the mechanism by which FGFR3 overexpression promotes MM development, interleukin-6 (IL-6)-dependent murine B9 cells were transduced with retroviruses expressing functional wild-type or constitutively activated mutant FGFR3. Overexpression of mutant FGFR3 resulted in IL-6 independence, decreased apoptosis, and an enhanced proliferative response to IL-6. In the presence of ligand, wild-type FGFR3-expressing cells also exhibited enhanced proliferation and survival in comparison to controls. B9 clones expressing either wild-type FGFR3 at high levels or mutant FGFR3 displayed increased phosphorylation of STAT3 and higher levels of bcl-x(L) expression than did parental B9 cells after cytokine withdrawal. The mechanism of the enhanced cell responsiveness to IL-6 is unknown at this time, but does not appear to be mediated by the mitogen-activated protein kinases SAPK, p38, or ERK. These findings provide a rational explanation for the mechanism by which FGFR3 contributes to both the viability and propagation of the myeloma clone and provide a basis for the development of therapies targeting this pathway.

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

To test a hypothesis that interferon-alpha (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.

Interleukin-10 is a proliferation factor but not a differentiation factor for human myeloma cells.

Because interleukin-10 (IL-10) is a potent differentiation factor of human B cells into mature plasma cells, we investigated its effect on human malignant plasma cells. IL-10 did not induce any differentiation and increase in Ig synthesis in four human IL-6-dependent malignant plasma cell lines. However, it stimulated the proliferation of two of four cytokine-dependent cell lines in the absence of IL-6 and IL-10-dependent myeloma cell lines have been obtained. The myeloma cell growth activity of IL-10 was unaffected by anti-IL-6 and anti-IL-6R antibodies. Similarly, IL-10 stimulated (P = .001) the proliferation of freshly-explanted myeloma cells in IL-6-deprived cultures of tumor samples from patients with active multiple myeloma (MM) and produced twice as many myeloma cells in these cultures. Again, this cytokine was unable to induce further differentiation (assessed by rate of Ig production) of fresh myeloma cells. A very sensitive enzyme-linked immunosorbent assay (ELISA; 1 pg/mL) only rarely detected IL-10 in the sera of MM patients (3 of 89). On the contrary, serum IL-10 was detected in 60% of patients with plasma cell leukemia (12 of 20). These data show that IL-10 is an IL-6-unrelated growth factor for malignant plasmablastic cells. This cytokine could be involved in the late phase of MM in vivo.

Rearrangement of a VH-associated LINE-1 element with the expressed IgH cluster in a murine myeloma cell line.

The MPC11 mouse myeloma cell line (IgG2b kappa) has yielded numerous variants in Ig heavy-chain production. One such variant, E5.7A14, fails to produce gamma 2b heavy chain but still produces kappa light chain. Comparison of the restriction maps of E5.7A14 and MPC11 has shown that in E5.7A14, the expressed MPC11VH gene has been deleted and replaced by a different DNA segment. Cloning and sequencing of the rearranged heavy-chain gene has identified the new DNA segment as a virtually full-length LINE-1 element that, in germ line, apparently lies in a inverted transcriptional orientation downstream of a previously unknown member (pseudogene) of the VH3609 gene family. The LINE-1 rearrangement was associated with an inversion of a 2-kb segment of the J-C gamma 2b intron and a deletion of switch sequences. The nature of the rearrangements and the sequences at recombination and inversion breakpoints suggest that the rearrangement event was mediated via class switch mechanisms. This is one of a limited number of reports that both characterizes a LINE rearrangement and localizes the germ-line origin of the particular LINE element involved.

Characterization and expression of alternatively spliced IgE heavy chain transcripts produced by peripheral blood lymphocytes.

We have investigated the IgE heavy chain isoforms produced in vivo by analyzing the epsilon mRNA species present in unstimulated PBL and expressing them individually in a myeloma cell line. Seven epsilon mRNA species were identified by using reverse transcription-PCR, cloning, and sequencing analysis. These species included the classical secreted (epsilon CH4-S) and membrane-bound (epsilon CH4-M1'-M2) IgE and five alternatively spliced epsilon transcripts. At the protein level, the five alternatively spliced epsilon transcripts (epsilon CH4*, epsilon CH4-M2', epsilon CH4'-1, epsilon CH4'-1-M2, and epsilon CH3-13-CH4) corresponded to four epsilon heavy chain isoforms, in which various parts of the CH4 domain were replaced by new stretches of amino acids at the carboxyl termini. The same epsilon mRNA species also were present in the IgE producing myeloma cell line U266. However, except for the classical membrane and secreted IgE, the corresponding proteins could not be identified. To further characterize the epsilon CH4-S, epsilon CH4*, epsilon CH4-M2', epsilon CH4'-1, and epsilon CH4-M1'-M2 species, we expressed them as chimeric mouse/human anti-4-hydroxy-5-iodo-3-nitrophenacetyl Abs in a mouse myeloma cell line. Only the classical secreted and membrane isoforms were found to be secreted or expressed on the cell surface, respectively, and the other forms were retained within the cells and degraded. These data suggest that some of the epsilon mRNA isoforms produced by PBL are aberrantly spliced mRNAs, the protein products of which are eliminated by post-translational events.

Hematopoietic growth factors stimulate paraprotein isotype production by bone marrow mononuclear cells in an aggressive case of multiple myeloma.

We describe a patient with multiple myeloma (MM), whose bone marrow (BM) cells were capable of spontaneous paraprotein isotype secretion, which could be strongly stimulated by hematopoietic growth factors (GFs), such as interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and IL-3. Ig production by BM cells from another five MM patients and four control patients with non-malignant hematological diseases could not be stimulated by these GFs. The results indicate that GFs, at least in some instances, can activate tumoral plasma cells in patients with MM. This possibility should be taken into account when the utility and effectiveness of GFs in the treatment of MM is evaluated.

Mechanisms of the escape phase of myeloma.

In multiple myeloma the duration of plateau is an important clinical and biological determinant of quality of life and survival. During plateau phase, the tumour is in an indolent state, as manifested by a low labelling index of plasma cells and other proliferative markers, e.g. the thymidine kinase level. The mechanism by which plasma cells escape from this indolent phase to a more aggressive phase of this disease is unknown, but a number of possible mechanisms have been postulated. These include loss of immunoregulation, clonal evolution, cytokine dysfunction and oncogene activation or tumour suppressor gene dysfunction. As current chemotherapy protocols do not appear to be able to eradicate the malignant clone, understanding the nature of the indolent phase of the malignant clone and the reasons for its escape from this phase are very important and may provide new options for disease control.

Nonsecretion of myeloma protein in spite of an increase in tumor burden by chemotherapy.

A unique case of IgA kappa myeloma is presented. While the myeloma cells had secreted a large quantity of IgA kappa monoclonal protein, they were induced to stop secreting the monoclonal protein by cyclophosphamide and vincristine, in spite of a remarkable increase in tumor burden. The absence of intracytoplasmic IgA kappa was clearly evidenced by the immunofluorescence technique using anti-IgA and anti-kappa monoclonal antibodies.

Elevated c-myc messenger RNA in multiple myeloma cell lines.

Oncogene analyses of four human myeloma cell lines provided no indication of gene amplification or rearrangement using DNA probes for the met, raf, abl, mos, erb B, Her-2-neu, fos, myb-7, fms, L-myc, sis, and myb-1 genes. However, a consistent elevation of up to 23-fold in the level of c-myc mRNA was observed in all of the cell lines studied. No restriction fragment length polymorphism (in exons one, two, or three) or c-myc gene amplification has as yet been demonstrated to account for the c-myc mRNA elevation. The c-myc mRNA has a half-life of 25 min which is comparable to that observed in other systems. The elevation in c-myc mRNA is further evidence for the role of the c-myc proto-oncogene in the pathogenesis of myeloma.

Structural correlates of a regulatory idiotope.

The A48RI expressed on the ABPC48 and UPC10 beta 2----6 fructosan-binding myeloma proteins is a conformational antigenic determinant encoded by V genes deriving from the VHX24 and VK10 families. In the preimmune repertoire the clones using VHX24 genes rarely express A48 idiotopes, clearly demonstrating that this regulatory idiotope is a minor or silent idiotope. Furthermore, these same VHX24-utilizing preimmune clones are frequently associated with the VK1 gene family which is highly represented in the neonatal and adult repertoires. The clonal expansion occurring subsequent to neonatal injection of minute amounts of anti-Id antibodies leads to selective expansion of A48Id+ clones associated with class switching. Few somatic mutations are observed in preimmune clones, or in those expanded by anti-Id antibodies. The fact that few mutations were observed in the IgG1 clones obtained from animals injected with anti-A48Id antibodies after birth indicates that, in contrast to antigen-induced class-switching, the anti-Id-induced switching is not associated with a highly active mutational process. In contrast to the preimmune clones, or those expanded by anti-Id (in the absence of antigenic stimulation) in which VHX24 is associated with VK regions deriving from various gene families, the clones expanded by anti-Id and fructan resemble A48 by using VHX24 and VK10 genes. Few apparent mutations were also observed in these IgM or IgG3 clones expressing A48 idiotopes. The A48 RI can be expressed on clones producing antibodies specific for various self and foreign antigens, and encoded by V genes deriving from various VH and VK families. These results indicate that key contacting residues bearing A48 conformational idiotypic determinants can be made up by various VH-VK combinations. A comparison of the VH and VL sequences of A48 RI+ mAbs showed that many of the observed somatic mutations could be correlated to decreased IDA10 binding. This comparison allowed identification of specific idiotope-determining regions of VH and VK which could represent contacting residues with anti-idiotypic antibodies. The contributions of these regions to the expression of the A48Id was tested by generating a transfectoma antibody expressing the rearranged VHJ558 gene of the ricin 45 hybridoma and the VK10-Ars-a gene of the 36-65 hybridoma. This transfectoma antibody expresses the idiotope recognized by IDA10 and confirms the conformational nature of this idiotope. There are three amino acid residues shared by VHX24 and VHJ558 antibodies expressing the A48 RI which are important for its expression.(ABSTRACT TRUNCATED AT 400 WORDS)

BSF-2/IL-6 does not augment Ig secretion but stimulates proliferation in myeloma cells.

Human myeloma cells were highly purified from bone marrow aspirates of 21 patients with advanced immunoglobulin G (IgG)-type multiple myeloma. B-cell stimulatory factor 2 (BSF-2)/interleukin-6 (IL-6) was originally characterized as a cytokine that can enhance immunoglobulin secretion from activated normal B cells and increase the expression of secretory-type Ig mRNA in these B cells, but that does not augment proliferation of activated B cells. However, recombinant IL-6 (rIL-6) could not enhance M-protein (IgG) secretion in freshly isolated myeloma cells in vitro but could augment proliferation of myeloma cells, although myeloma cells constitutively expressed IL-6 receptors. Furthermore, expression of secretory-type IgG (gamma-chain) mRNA in myeloma cells was not changed in the presence of IL-6. These results show that IL-6 is not an enhancing factor in Ig secretion from myeloma cells, and thus signal transduction through IL-6 in myeloma cells may be altered as opposed to activated B cells.