Mass Spectrometry-Based Method Targeting Ig Variable Regions for Assessment of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is a systemic malignancy of monoclonal plasma cells that accounts for 10% of hematologic cancers. With development of highly effective therapies for multiple myeloma, minimal residual disease (MRD) assessment has emerged as an important end point for management decisions. Currently, serologic assays lack the sensitivity for MRD assessment, and invasive bone marrow sampling with flow cytometry or molecular methods has emerged as the gold standard. We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. The method detects Ig-derived clonotypic tryptic peptides predicted by sequencing the clonal plasma cell Ig genes. A heavy isotope-labeled Ig internal standard is added to patient serum at a known concentration, the Ig is enriched in a light chain type specific manner, and proteins are digested and analyzed by targeted mass spectrometry. Peptides from the constant regions of the λ or κ light chains, Ig heavy chains, and clonotypic peptides unique to the patient monoclonal Igs are targeted. This technique is highly sensitive and specific for the patient-specific monoclonal Igs, even in samples negative by multiparametric flow cytometry. Our method can accurately and precisely detect monoclonal protein in serum of patients treated for myeloma and has broad implications for management of hematologic patients.

Therapeutic Advances in the Management of Smoldering Myeloma.

BACKGROUND: The International Myeloma Working Group has defined smoldering multiple myeloma (SMM) as the presence of 10%-60% plasma cells in the bone marrow and M-protein (IgG, IgA) ≥3 g/dL without end-organ damage (an increased calcium level, renal failure, anemia, and destructive bone lesions). AREAS OF UNCERTAINTY: Patients considered to have SMM should not have any myeloma-defining events or amyloidosis. Different risks factors classify SMM into low-, intermediate-, or high-risk categories. The rate of progression from SMM to symptomatic myeloma is ∼10% per year during the first 5 years of diagnosis. SMM requires frequent follow-up ∼every 3 months during the first 5 years as compared to monoclonal gammopathy of undermined significance, which usually requires follow-up every 6-12 months after the first year of diagnosis. DATA SOURCES: A literature search was performed from electronic bibliographic databases: MEDLINE (Ovid SP/PubMed), EMBASE, the Cochrane Library (Cochrane Database of Systematic Reviews), and Cochrane Central Register of Controlled Trials and from annual meeting abstracts from inception to May 2017. THERAPEUTIC ADVANCES: This review presents the literature and available data that support or do not support early treatment of high-risk SMM (HR-SMM) and provides evidence-based recommendations for management of SMM patients. Despite emerging data recommending early treatment of HR-SMM, we predict the SMM category may disappear in the near future and patients will be diagnosed with either multiple myeloma or monoclonal gammopathy of undermined significance. CONCLUSIONS: Success with early therapy trials for HR-SMM is largely due to patients meeting current criteria for multiple myeloma that may have been classified as SMM and, therefore, benefitted from therapy. Based on current practices and the literature, SMM should be managed with close follow-up. Based on available data, we suggest SMM to only be treated in clinical trial settings.

Multiple myeloma immunoglobulin lambda translocations portend poor prognosis.

Multiple myeloma is a malignancy of antibody-secreting plasma cells. Most patients benefit from current therapies, however, 20% of patients relapse or die within two years and are deemed high risk. Here we analyze structural variants from 795 newly-diagnosed patients as part of the CoMMpass study. We report translocations involving the immunoglobulin lambda (IgL) locus are present in 10% of patients, and indicative of poor prognosis. This is particularly true for IgL-MYC translocations, which coincide with focal amplifications of enhancers at both loci. Importantly, 78% of IgL-MYC translocations co-occur with hyperdiploid disease, a marker of standard risk, suggesting that IgL-MYC-translocated myeloma is being misclassified. Patients with IgL-translocations fail to benefit from IMiDs, which target IKZF1, a transcription factor that binds the IgL enhancer at some of the highest levels in the myeloma epigenome. These data implicate IgL translocation as a driver of poor prognosis which may be due to IMiD resistance.

Development of a Targeted Mass-Spectrometry Serum Assay To Quantify M-Protein in the Presence of Therapeutic Monoclonal Antibodies.

M-protein diagnostics can be compromised for patients receiving therapeutic monoclonal antibodies as treatment in multiple myeloma. Conventional techniques are often not able to distinguish between M-proteins and therapeutic monoclonal antibodies administered to the patient. This may prevent correct response assessment and can lead to overtreatment. We have developed a serum-based targeted mass-spectrometry assay to detect M-proteins, even in the presence of three therapeutic monoclonal antibodies (daratumumab, ipilimumab, and nivolumab). This assay can target proteotypic M-protein peptides as well as unique peptides derived from therapeutic monoclonal antibodies. We address the sensitivity in M-protein diagnostics and show that our mass-spectrometry assay is more than two orders of magnitude more sensitive than conventional M-protein diagnostics. The use of stable isotope-labeled peptides allows absolute quantification of the M-protein and increases the potential of assay standardization across multiple laboratories. Finally, we discuss the position of mass-spectrometry assays in monitoring minimal residual disease in multiple myeloma, which is currently dominated by molecular techniques based on plasma cell assessment that requires invasive bone marrow aspirations or biopsies.

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.

Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

Identification of miRSNPs associated with the risk of multiple myeloma.

Multiple myeloma (MM) is a malignancy of plasma cells usually infiltrating the bone marrow, associated with the production of a monoclonal immunoglobulin (M protein) which can be detected in the blood and/or urine. Multiple lines of evidence suggest that genetic factors are involved in MM pathogenesis, and several studies have identified single nucleotide polymorphisms (SNPs) associated with the susceptibility to the disease. SNPs within miRNA-binding sites in target genes (miRSNPs) may alter the strength of miRNA-mRNA interactions, thus deregulating protein expression. MiRSNPs are known to be associated with risk of various types of cancer, but they have never been investigated in MM. We performed an in silico genome-wide search for miRSNPs predicted to alter binding of miRNAs to their target sequences. We selected 12 miRSNPs and tested their association with MM risk. Our study population consisted of 1,832 controls and 2,894 MM cases recruited from seven European countries and Israel in the context of the IMMEnSE (International Multiple Myeloma rESEarch) consortium. In this population two SNPs showed an association with p < 0.05: rs286595 (located in gene MRLP22) and rs14191881 (located in gene TCF19). Results from IMMEnSE were meta-analyzed with data from a previously published genome-wide association study (GWAS). The SNPs rs13409 (located in the 3'UTR of the POU5F1 gene), rs1419881 (TCF19), rs1049633, rs1049623 (both in DDR1) showed significant associations with MM risk. In conclusion, we sought to identify genetic polymorphisms associated with MM risk starting from genome-wide prediction of miRSNPs. For some mirSNPs, we have shown promising associations with MM risk.

Clinical course and prognosis of non-secretory multiple myeloma.

OBJECTIVE: To determine the prognosis of patients with non-secretory myeloma. METHODS: We studied 124 patients diagnosed with multiple myeloma who had no monoclonal protein detected on serum and urine immunofixation at diagnosis and on all subsequent follow-up testing (non-secretory myeloma). The overall survival (OS) of patients with non-secretory myeloma was compared with 6953 patients with typical myeloma seen during the same time period in whom a monoclonal protein was detected at the time of diagnosis. RESULTS: One hundred and twenty-four patients met criteria for non-secretory multiple myeloma. The median follow-up was 102 months (range, 1-204 months). The median progression-free survival with initial therapy was 28.6 months, and the median OS was 49.3 months. There was a significant improvement in OS since 2001; median survival 43.8 months (prior to 2001) vs. 99.2 months (2001-2012), P < 0.001. OS was superior in patients with a normal baseline FLC ratio (n = 10) compared to patients with an abnormal ratio (n = 19), medians not reached in both groups. Prior to 2001, OS was similar in non-secretory myeloma (n = 86) and secretory myeloma (n = 4011), median 3.6 vs. 3.5 yr, respectively, P = 0.63. However, among patients diagnosed between 2001 and 2012, OS was superior in non-secretory myeloma (n = 36) compared to secretory myeloma (n = 2942), median 8.3 vs. 5.4 yr, respectively, P = 0.03. CONCLUSIONS: Non-secretory myeloma is an uncommon subtype of multiple myeloma. In the last decade, there has been an improvement in the survival of non-secretory myeloma and appears superior to secretory myeloma.

Myeloma stem cell concepts, heterogeneity and plasticity of multiple myeloma.

Multiple myeloma (MM) is a haematological malignancy characterized by the accumulation of clonal plasma cells (PCs) in the bone marrow (BM). Although novel therapeutic strategies have prolonged survival of patients, the disease remains difficult to treat with a high risk of relapse. The failure of therapy is thought to be associated with a persistent population of the so-called MM stem cells or myeloma initiating cells (MIC) that exhibit tumour-initiating potential, self-renewal and resistance to chemotherapy. However, the population responsible for the origin and sustainability of tumour mass has not been clearly characterized so far. This review summarizes current myeloma stem cell concepts and suggests that high phenotypic and intra-clonal heterogeneity, together with plasticity potential of MM might be other contributing factors explaining discrepancies among particular concepts and contributing to the treatment failure.

Oligosecretory biclonal multiple myeloma.

Among the plasma cell dyscrasias, non-secretory myeloma is one of the rarest. This diagnosis is based on the absence of monoclonal proteins in the serum and urine. When serum free light chains are trace and the kappa: lambda ratio normal, clonality may however be established by PCR. We present a case of an oligosecretory myeloma confirmed by PCR, which would have hitherto been classified as non-secretory.

Identification of the differentially expressed genes in SP2/0 myeloma cells from Balb/c mice infected with Trichinella spiralis.

To study the molecular mechanism of suppressed growth caused by Trichinella spiralis, an SP2/0 myeloma model was established using parasite-infected Balb/c mice. Suppression subtractive hybridization (SSH) was then utilized to identify differentially expressed genes between tumor cells from the infected and non-infected mice. On the 11th day after infection, 2 × 10(6) SP2/0 myeloma cells were subcutaneously inoculated into 6-8 week old female Balb/c mice in both the experimental and control groups. Twenty-eight days after tumor cell inoculation, the mice were euthanized and the sizes and weights of the tumors were measured. Messenger RNA was isolated and used to perform SSH. Putative differentially expressed genes were identified, sequenced and analyzed by BLASTn. Among the sequences detected which ranged in size between 180 and 850 bp, genes encoding RpL41, NKTR, Rbbp4 and ANXA2 were enriched and considered possible proteins involved in tumor growth inhibition.

The use of CD138 positively selected marrow samples increases the applicability of minimal residual disease assessment by PCR in patients with multiple myeloma.

We have evaluated the use of CD138+ positively selected bone marrow samples to identify a molecular target for minimal residual disease assessment by polymerase chain reaction (PCR) in 25 untreated patients with multiple myeloma. A fraction of each sample was used for CD138+ selection, and the rest served as a reference control. VDJH, DJH, and Kde gene rearrangements were tested for amplification according to the BIOMED-2 Concerted Action. PCR products were directly sequenced in an automated ABI 3130 DNA sequencer using Big-Dye terminators. Within the CD138+ selected group, VDJH rearrangements were detected in all cases (100 %), DJH in 16 (64 %), and Kde in 18 (72 %) cases; whereas in the control samples, VDJH, DJH, and Kde rearrangements were detected in 19 (76 %), 11 (44 %), and 12 (48 %) cases, respectively. After sequencing, 24 (96 %) cases within the CD138+ group had a PCR target for MRD detection compared with 15 (60 %) cases in the control group. We conclude that the use of CD138+ positively selected bone marrow samples increases the applicability of minimal residual disease studies by PCR in patients with multiple myeloma.

Multiple myeloma shows no intra-disease clustering of immunoglobulin heavy chain genes.

BACKGROUND: Characterization of the immunoglobulin gene repertoire has improved our understanding of the immunopathogenesis of lymphoid tumors. Early B-lymphocyte precursors of multiple myeloma are known to exist and might be susceptible to antigenic drive. DESIGN AND METHODS: To verify this hypothesis, we collected a database of 345 fully readable multiple myeloma immunoglobulin sequences. We characterized the immunoglobulin repertoire, analyzed the somatic hypermutation load, and investigated for stereotyped receptor clusters. RESULTS: Compared to the normal immunoglobulin repertoire, multiple myeloma displayed only modest differences involving only a few genes, showing that the myeloma immunoglobulin repertoire is the least skewed among mature B-cell tumors. Median somatic hypermutation load was 7.8%; median length of complementarity determining-region 3 was 15.5 amino acids. Clustering analysis showed the absence of myeloma specific clusters and no similarity with published chronic lymphocytic leukemia or lymphoma subsets. CONCLUSIONS: Analysis of multiple myeloma immunoglobulin repertoire does not support a pathogenetic role for antigen selection in this tumor.

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.

Laboratory characterizations on 2007 cases of monoclonal gammopathies in East China.

Monoclonal gammopathies are characterized by the presence of monoclonal immunoglobulin in patients with or without evidence of multiple myeloma (MM), macroglobulinemia, amyloidosis (AL), or a related plasma cell proliferative disorder. This study aims to evaluate laboratory diagnostic characters of monoclonal gammopathies and investigates the correlation between monoclonal gammopathies and transforming growth factor beta1 (TGFbeta1). Immunofixation electrophoresis (IFE), serum protein electrophoresis (SPE), nephelometry and urine light chain ELISA were used for laboratory identification of monoclonal immunoglobulins. Plasma TGFbeta1 was detected with double-antibodies ELISA. Lightcycler was used for single nucleotide polymorphism (SNP) analysis. Totally 2,007 cases of monoclonal immunoglobulin (M protein) were identified in 10,682 samples. The isotypes of M protein were IgG type 47.1%, IgA 23.0%, IgM 8.7%, IgD 5.3%, free light chain kappa 6.1%, lambda 9.8%. In reference to IFE, the coherency of diagnosis was serum light chain ratio (kappa/lambda ) 94.4%, quantitation of Igs 83%, light chain quantitation 80.9%, and urine light chain ratio (kappa/lambda) 58.0%. Plasma TGFbeta1 was elevated significantly compared to normal control. The allelic frequency of codon 10 (C>T) was neither associated with the existence of the M protein nor with the M protein isotype. Monoclonal gammopathies can be identified with the combination of IFE, SPE, Igs quantitation and urine light chain determination. Although TGFbeta1, an important cytokine in immune regulation, was elevated in monoclonal gammopathies, the SNPs in coding region of TGFbeta1 gene did not confer susceptibility to the development of monoclonal gammopathies in this study.

Specific targeting to murine myeloma cells of Cyt1Aa toxin from Bacillus thuringiensis subspecies israelensis.

Multiple myeloma is currently an incurable cancer of plasma B cells often characterized by overproduction of abnormally high quantities of a patient-specific, clonotypic immunoglobulin "M-protein." The M-protein is expressed on the cell membrane and secreted into the blood. We previously showed that ligand-toxin conjugates (LTC) incorporating the ribosome-inactivating Ricin-A toxin were very effective in specific cytolysis of the anti-ligand antibody-bearing target cells used as models for multiple myeloma. Here, we report on the incorporation of the membrane-disruptive Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis into LTCs targeted to murine myeloma cells. Proteolytically activated Cyt1Aa was conjugated chemically or genetically through either its amino or carboxyl termini to the major peptidic epitope VHFFKNIVTPRTP (p87-99) of the myelin basic protein. The recombinant fusion-encoding genes were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis through the shuttle vector pHT315. Both chemically conjugated and genetically fused LTCs were toxic to anti-myelin basic protein-expressing murine hybridoma cells, but the recombinant conjugates were more active. LTCs comprising the Cyt1Aa toxin might be useful anticancer agents. As a membrane-acting toxin, Cyt1Aa is not likely to induce development of resistant cell lines.

A VH4-34+ myeloma protein with weak autoreactivity.

It has been suggested that VH4-34 gene segment expression is counter-selected in multiple myeloma (MM) due to a self-tolerance mechanism. We cloned and sequenced a VH4-34 gene segment from bone marrow mononuclear cells of a stage III MM patient. We show that VH4-34 was expressed by the serum IgA myeloma (M)-protein, as demonstrated by reactivity with the VH4-34 specific 9G4 mAb and mass spectrometry (MS). The M-protein had weak reactivity with nuclei. These results demonstrate that VH4-34 may be expressed in secreted IgA M-protein with weak autoreactivity. Thus, counter-selection of VH4-34 is pronounced but not absolute in MM. Mechanisms of how VH4-34 can occasionally be expressed in MM and clinical implications are discussed.