Heterogeneity Of Multiple Myeloma Research Articles

Abstract 2004: Phenotypic and molecular characterization of inter- and intraclonal heterogeneity in multiple myeloma and Waldenstrom macroglobulinemia

Abstract Intra-clonal heterogeneity in malignant plasma (PC) cells and clonal B cells has been reported in both multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM). Further phenotypic and molecular characterization of inter- and intra-clonal genetic complexity together with humoral immunity will enhance our understanding of disease pathogenesis and identify novel therapeutic strategies. By a large cohort of MM (N = 100) and WM (N = 50) patient samples, we studied evolution of bone marrow patient samples from premalignant stages (MGUS and smoldering MM) to malignant stages (MM and WM) versus age-matched healthy donors (HD) by combining immunophenotypic, molecular, and cytogenetic approaches. We examined phenotypic and molecular features of intra-clonal architecture of malignant PC in MM and clonal B cells in WM together with humoral immunity on a single cell level using mass cytometry (CyTOF). CyTOF is based on rare stable earth elemental isotopes-bound to antibodies to target epitopes on and within cells: up to 40 different markers can simultaneously assess immunophenotyping and analysis of signaling in single cell. To investigate phenotypic profile and molecular signature of intra-clonal heterogeneity of PC in MM, high-dimensional analyses revealed that clonal PC clustered separately from B cells with high CD319 and CD47 expression; variable expression of CD52, CD56, CD81, CD44, CD200; and low expression of CD28, CD117, CD338, CD325, and CD243. For example, adhesion CD56 and anti-adhesion CD52 molecules were significantly increased in MM. Clonal PC highly expressed IFR4 and Notch1; variously expressed FGFR3, sXBP-1, KLF4 and c-Myc; and only minimally expressed Bcl-6, WHSC-1 (MMSET) and RARα2. sXBP-1 was significantly upregulated in all MM stages. Furthermore, expression of stem cell markers Sox-2, Oct3/4 and Nestin was detected only at low level in clonal PC, except for higher expression of Nanog. In WM, clonal B cells expressed Bcl-6 (4-36%) and MYD88 (2-27%) by CyTOF analyses. Both MM and WM are characterized by immune dysfunction; therefore we used CyTOF technology to define the complex immune profile in MM and WM patient samples. Therefore, we examined changes in B cell development, T cell subsets, NK and NKT cells as well as monocytic, granulocytic, erythroid and platelet lineages during evolution of both diseases vs. HD on phenotypic and molecular level. For example, plasmacytoid dendritic cells (pDC) were increased in MM with significantly decreased PD-1 expression on pDC in MM compared to MGUS (P = 0.007). Interestingly, PD-1 and its ligand PD-L1 were variably expressed on B cells (2-9% and 3-27%) and PC (0.5-46% and 3-41%) in MM. A better understanding of inter- and intra-clonal heterogeneity together with host immunity on the phenotypic and molecular/signaling level in MM and WM will provide the framework for identifying and validating novel personalized targeted therapies. Citation Format: Jana Jakubikova, Danka Cholujova, Teru Hideshima, Jacob Laubach, Nikhil C. Munshi, Steven P. Treon, Paul G. Richardson, Efstathios Kastritis, David M. Dorfman, Kenneth C. Anderson. Phenotypic and molecular characterization of inter- and intraclonal heterogeneity in multiple myeloma and Waldenstrom macroglobulinemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2004. doi:10.1158/1538-7445.AM2015-2004

Light Chain Escape in 3 Cases: Evidence of Intraclonal Heterogeneity in Multiple Myeloma from a Single Institution in Poland.

We report three cases of light chain escape (LCE) at a single institution in Poland, including an interesting case of biclonal monoclonal gammopathy of undetermined significance (MGUS) that satisfied the criteria for progression to light chain multiple myeloma (LCMM) with a rapid rise in serum free light chain (FLC) levels, following steroidal treatment for simultaneous temporal artery inflammation and polymyalgia rheumatica (PMR). In the three cases discussed, progression of the disease by light chain escape was associated with rapid and severe renal impairment, highlighting the necessity for prompt detection of such free light chain-only producing clones in order to prevent the possible development of irreversible end-organ damage. Interestingly, monitoring of these three patients by serum free light chain assay (sFLC) and retrospective heavy/light chain analysis (HLC) detected this clonal evolution prior to clinical relapse and suggests that these assays represent important additional tools for more accurate monitoring of multiple myeloma patients.

Inter and Intra-Clonal Heterogeneity in Multiple Myeloma and Waldenstrom Macroglobulinemia

Introduction: Intra-clonal heterogeneity in malignant plasma (PC) cells and B-cells has recently been reported in both multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM). Further phenotypic and molecular characterization of inter- and intra-clonal genetic complexity will enhance our understanding of disease pathogenesis and identify novel therapeutic strategies.Methods: In this study, we compared normal and malignant PC maturation-associated B-cell subsets using bone marrow samples from individuals with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), newly diagnosed MM, and relapsed/refractory MM versus age-matched healthy donors (HD). We also similarly analyzed WM. In addition to corrupted B-cell lineage, we examined phenotypic and molecular features of intra-clonal architecture (complexity) of malignant PC in MM and clonal B-cells in WM on a single cell level using time-of-flight mass cytometry (CyTOF) technology. CyTOF technology is based on rare stable earth elemental isotopes-bound to antibodies to target epitopes on and within cells: up to 40 different markers on a single cell can simultaneously assess including phenotype, transcription factors, regulatory signaling molecules and enzymes, as well as activation of signaling molecules. The resulting high-dimensional data were analyzed by SPADE, viSNE and Wanderlust software.Results: Our high-dimensional data of clustered analyses showed significantly decreased CD19+CD27- patient cells in MM with cytogenetic abnormalities (cytog+) including del(13q), t(4;14), t(14;16), t(3;14), +1q or t(11;14) versus patient cells in MM without any cytogenetic abnormalities (cytog-; P=0.013). In contrast, there was a significant increase of transitional B cells (CD19+CD27-IgM+CD10-IgDlow) in patients with MM cytog+ vs. MM cytog- (P=0.028). A significant increase of mature (naïve) B cells (CD19+CD27-IgM+CD10-IgD+) was also detected in MM cytog+ versus MM cytog- patients (P=0.013), but not in WM cytog- vs. WM cytog+ (46XY, -Y, +18q, +6p, 14q). Clonal PC (CD19-CD38++CD138+CD45-/dim; either cyk or cyl +) were significantly upregulated in MM cytog+ compared to MM cytog- (P=0.021) by CyTOF analyses. To investigate phenotypic profiles and molecular signature of intra-clonal heterogeneity of PC in MM, high-dimensional analyses by SPADE and viSNE revealed that clonal PC clustered separately from B cells by, virtue of high CD319 and CD47 expression; variable expression of CD52, CD56, CD81, CD44, CD200; and low expression of CD28, CD117, CD338, CD325, and CD243. For example, adhesion CD56 and anti-adhesion CD52 molecules were significantly increased in MM cytog+ compared to MM cytog-. Clonal PC highly expressed IFR4 and Notch1; variously expressed FGFR3, sXBP-1, KLF4 and c-Myc; and only minimally expressed Bcl-6, WHSC-1 (MMSET) and RARa2. sXBP-1 was significantly upregulated in all MM stages compared to HD. Furthermore, expression of stem cell markers including Sox-2, Oct3/4 and Nestin was detected only at low level in clonal PC, except for higher expression of Nanog. In WM, clonal B cells expressed Bcl-6 (4-36%) and MYD88 (2-27%) by CyTOF analyses. Finally, cluster analyses by SPADE and viSNE allows for detection of phenotypic and molecular changes not only in clonal populations but also at distinct B-lineage maturation stages, such as expression of Pax-5 and Bcl-2 on early B cell progenitors. This data represents a cohort of MM (N=35) and WM (N=15) patients; a significantly larger data set of MM (N=100) and WM (N=50) will be presented.Conclusion: This study characterizes the molecular and phenotypic profile associated with inter- and intra-clonal heterogeneity in MM and WM. It not only enhances our understanding of disease pathogenesis, but may allow for individualized targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

Osteoblastic niche supports the growth of quiescent multiple myeloma cells

AbstractThe heterogeneity of multiple myeloma (MM) contributes to variable responses to therapy. In this study, we aim to correlate the heterogeneity of MM to the presence of quiescent cells using the PKH26 dye. We tracked the rare quiescent cells in different niches of the bone marrow by allowing the cells to cycle in vivo. Surprisingly, quiescent PKH+ MM cells prefer to reside within the osteoblastic niches of the bone marrow (PKH+/OS) rather than the vascular (VS) niches or the spleen. These cells (PKH+/OS) displayed enhanced stemlike properties by forming colonies in semisolid medium. PKH+ cells were highly tumorigenic compared with PKH– cells and were resistant to a variety of drugs. However, the levels of drug resistance were somewhat similar regardless of where the PKH+ cells were isolated. Our data indicate that osteoblastic niches support the growth of quiescent PKH+ cells and allow them to have stemlike functions.

Widespread Genetic Heterogeneity in Multiple Myeloma: Implications for Targeted Therapy

We performed massively parallel sequencing of paired tumor/normal samples from 203 multiple myeloma (MM) patients and identified significantly mutated genes and copy number alterations and discovered putative tumor suppressor genes by determining homozygous deletions and loss of heterozygosity. We observed frequent mutations in KRAS (particularly in previously treated patients), NRAS, BRAF, FAM46C, TP53, and DIS3 (particularly in nonhyperdiploid MM). Mutations were often present in subclonal populations, and multiple mutations within the same pathway (e.g., KRAS, NRAS, and BRAF) were observed in the same patient. In vitro modeling predicts only partial treatment efficacy of targeting subclonal mutations, and even growth promotion of nonmutated subclones in some cases. These results emphasize the importance of heterogeneity analysis for treatment decisions.

Implications of heterogeneity in multiple myeloma.

Multiple myeloma is the second most common hematologic malignancy in the world. Despite improvement in outcome, the disease is still incurable for most patients. However, not all myeloma are the same. With the same treatment, some patients can have very long survival whereas others can have very short survival. This suggests that there is underlying heterogeneity in myeloma. Studies over the years have revealed multiple layers of heterogeneity. First, clinical parameters such as age and tumor burden could significantly affect outcome. At the genetic level, there are also significant heterogeneity ranging for chromosome numbers, genetic translocations, and genetic mutations. At the clonal level, there appears to be significant clonal heterogeneity with multiple clones coexisting in the same patient. At the cell differentiation level, there appears to be a hierarchy of clonally related cells that have different clonogenic potential and sensitivity to therapies. These levels of complexities present challenges in terms of treatment and prognostication as well as monitoring of treatment. However, if we can clearly delineate and dissect this heterogeneity, we may also be presented with unique opportunities for precision and personalized treatment of myeloma. Some proof of concepts of such approaches has been demonstrated.

Heterogeneity of outcome with single-agent carfilzomib: all relapsed/refractory myelomas are not created equal

Since the year 2000, there has been a continued improvement in survival outcomes for multiple myeloma (MM) patients,1 attributable to the clinical development and inclusion of novel agents in upfront therapy, and improvement in high-dose therapy administration.2 There has been a growing body of literature furthering the biological understanding of the disease. There is a clear recognition of both inter-patient and intra-patient genomic heterogeneity in multiple myeloma,3 although the academic community is still trying to comprehend specific phenotypic presentations such as primary plasma cell leukemia4 and extramedullary disease.5 Even with effective upfront therapeutic strategies, which seemingly help a greater proportion of MM patients achieve a ‘complete’ remission by biochemical and pathology criteria, relapses do occur sooner or later that require subsequent lines of therapy. There is a growing list of classes of novel agents that are disease-relevant and showing promise in early clinical investigations in the relapsed/refractory MM (RRMM) setting. These classes include histone deacytelase inhibitors, heat-shock protein inhibitors, kinesin spindle protein inhibitors, immune-therapeutics and ubiquitin-proteasome pathway targeting agents.6 We have witnessed the Food and Drug Administration approval of two novel agents in the past 12 months, namely carfilzomib and pomalidomide, in the RRMM setting. It is highly likely that several novel agents will acquire Food and Drug Administration approval for RRMM over the next 5 years. The challenges in this setting would then be to determine which class of drugs may be more suitable for an individual patient, whether it would be better to sequence or combine different drug classes, and whether or not it would be possible to achieve ‘functional cure’ by making RRMM a chronic disease for most patients.

Survival Pathways Stabilized By Proteasome Inhibition Reveal PIM2 As a Novel Target For Potentiating The Anti-Myeloma Activity Of Carfilzomib

In recent years, new agents for multiple myeloma treatment (e.g., proteasome inhibitors) have become more efficacious, yet nearly all patients eventually relapse and develop refractory disease. Growing evidence suggests that clonal heterogeneity in multiple myeloma may constitute the basis for treatment resistance. Therefore, a multi-pronged approach with novel agents is needed to increase the efficacy of standard therapy and prevent or overcome resistance to standard treatments. We have undertaken a research effort to discover novel targets that potentiate the anti-tumor effects of proteasome inhibition in myeloma cells. We hypothesized that proteins that are stabilized in tumor cells following proteasome inhibition likely constitute components of both pro-apoptotic and pro-survival pathways. A mass spectrometry approach, referred to as UbiScan®, was employed to determine the identity and levels of cellular proteins modified with ubiquitin. MM cell lines (U266 and NCI-H929) were treated with either carfilzomib (CFZ) or bortezomib (BTZ) for 1 hour and the ubiquitome was profiled at 1 and 3 hours after culture in drug-free media. A concentration of 125 nM was chosen in order to reflect physiologically relevant drug and target inhibition levels and to induce cell death in ∼80% of cells after 48 hours. Approximately 400 proteins showed similar increases in ubiquitination with CFZ or BTZ. One of these proteins was PIM2, a serine/threonine proto-oncogene required for plasma cell proliferation that is highly expressed in multiple myeloma cell lines. We determined that ubiquitination on PIM2 was occurring at lysine 61, which is known to be associated with proteasomal degradation. Four hours after exposure to CFZ, PIM2 ubiquitination increased 34.6 and 24.9 fold in U266 and H929 cells, respectively, and similar changes were measured following BTZ treatment. Western blot analysis of CFZ-treated cells showed a dose-dependent accumulation of total PIM2 protein, confirming that the increase in ubiquitination correlated with protein accumulation. Next, we employed a siRNA-mediated knockdown approach to study the role of PIM2 in proteasome inhibitor mediated-cell death. Knockdown of PIM2 caused a 20% - 50% decrease in viability in both myeloma cell lines. When CFZ was added 48 hours after siRNA transfection, a significant and dose-dependent decrease in viability was observed, suggesting a synergistic interaction. Based on these results, we tested the combination of CFZ and (Z)-5-(4-propoxybenzylidene)thiazolidine-2,4-dione (PIM1/2 inhibitor), which is known to inhibit both PIM1 and PIM2. The PIM1/2 inhibitor decreased levels of phosphorylation on 4E-BP1, a downstream target, confirming its activity in cells. Chemical inhibition of PIM2 potentiated the effect of CFZ in both MM cell lines. These data suggest that the combination of targeting PIM2 and the proteasome will be efficacious in the treatment of multiple myeloma. Disclosures: Lin: Onyx Pharmaceuticals, Inc.: Employment. Lowe:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Lerner:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Arastu-Kapur:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership.

Intra-Tumoral Heterogeneity In Multiple Myeloma Gleaned From Gene Expression Profiling Analysis Of Plasma Cells and Bone Marrow Biopsies: Comparison Of Random Bone Marrow Samples and CT-Guided Fine Needle Aspirations Of MRI-Defined Focal Lesions

Intra-tumoral heterogeneity (ITH) is increasingly viewed as the Achilles heel of treatment failure in malignant disease including multiple myeloma (MM). Most MM patients harbor focal lesions (FL) that are recognized on MRI long before bone destruction is detectable by conventional X-ray examination. Serial MRI examinations show that eventually 60% of patients will achieve resolution of FL (MRI-CR). However, this will lag behind the onset of a clinical CR by 18 to 24 months, thus attesting to the biological differences between FL and diffuse MM growth patterns. Consequently, we performed concurrent gene expression profiling (GEP) analyses of plasma cells (PC) from both random bone marrow (RBM) via iliac crest and FL. Our primary aims were to first compare the molecular profiles of FL vs. RBM, second to determine if ITH existed (as defined molecular subgroup and risk), and finally to investigate if the bone marrow micro-environment (ME) contained a biologically interesting signature.A total of 176 patients were available for this study with a breakdown of: TT3 (n=23), TT4 for low-risk (n=131) and TT5 for high-risk MM (n=22). Regarding the molecular analyses of PCs, GEP-based risk (GEP-70, GEP-5) and molecular subgroup correspondence were examined for commonalties and differences between RBM and FL. A “filtering” approach for ME genes was also developed and bone marrow biopsy (BMBx) GEP data derived from this method is under analysis.PC risk correspondence between FL and RBM was 86% for GEP70 and 88% for the GEP5 model. Additionally, 82% had a molecular subgroup concordance, however, they did differ among subgroups (p=0.020) by Fisher's Exact Test. A lower concordance was noted in the CD2, LB, and PR subgroups (67%, 69%, 73%, respectively). GEP70 and GEP5 risk concordance between RBM and FL samples by molecular subgroup was also examined. The overall correlation coefficients were 0.619 (GEP70) and 0.597 (GEP5). The best correspondence was noted for CD1, MF and PR subgroups especially for the GEP5 model. HY, LB and MS showed intermediate correlations, while CD2 fared worst with values of only 0.322 for GEP70 and 0.267 for GEP5 model. Figure 1 portrays these data in more detail for the GEP70 and GEP5 models. Good correlations were noted between RBM and FL based risk scores in case of molecular subgroup concordance (left panels) in both GEP5 and GEP70 risk models, whereas considerable scatter existed in case of subgroup discordance (right panels). The clinical implications in TT4 regarding RBM and FL derived risk and molecular subgroup information, viewed in the context of standard prognostic baseline variables are portrayed in Table 1. High B2M levels at both cut-points imparted inferior OS and PFS as did low hemoglobin. Although present in 42% of patients, cytogenetic abnormalities (CA) did not affect outcomes. FL-based GEP5-defined high-risk designation conferred poor OS and PFS. B2M>5.5mg/L and FL-derived GEP5 high-risk MM, pertaining to 29% and 11% of patients, survived the multivariate model for both OS and PFS. Next, in examining PC-GEP differences among RBM and FL sites, 199 gene probes were identified with a false discovery rate (FDR) of 1x10-6. Additionally, 55 of the 199 belong to four molecular networks of inter related genes associated with: lipid metabolism, cellular movement, growth and proliferation, and cell-to-cell interactions. Multivariate analysis identified the GEP5 high risk designation of focal lesion PCs to be significantly prognostic with a HR=3.73 (p=0.023). [Display omitted] Disclosures:No relevant conflicts of interest to declare.

CD32B Expression Reflects Phenotypic and Functional Heterogeneity in Multiple Myeloma (MM)

Intraclonal heterogeneity (IH) in MM may provide a basis for the selection of resistance to therapy that occurs over time. Model systems to study IH in MM have not been defined so we focused on the expression of CD32B, the low-affinity inhibitory Fcγ-receptor that is a key participant in normal plasma cell apoptosis and a potential target for monoclonal antibody therapy. At diagnosis, CD138+ MM marrow cells are largely CD32B+ (n=72; median 94%, range 1–100) while at relapse the CD32B+ MM subpopulation is significantly smaller in both bone marrow (n=25, median 83%, (10–100), P = 0.02) and extramedullary sites (n=8, median 17% (0–78); P << 0.01). Among human MM cell lines only two of ten tested expressed cell-surface CD32B. We hypothesize that the CD32B phenotype of MM subpopulations contributes to the functional heterogeneity that is critical to the selection of resistance, and sought a model system to study CD32B-related IH. The KMS-12BM cell line (BM), a human MM line from marrow, has CD32B+ and CD32B− subpopulations as MM marrows do, while the KMS-12PE cell line (PE), from the pleural fluid of the same patient, is completely CD32B negative, as is often the case in extramedullary MM. Both BM and PE cells share t(11;14), del 17p and other cytogenetic abnormalities, and both contain intact CD32B genes (FCGR2B). We used 3 populations, BM CD32B+, BM CD32B− and PE (CD32B−), as well as sorted patient MM cells, to study the IH associated with CD32B. RNAi studies demonstrated that the expression of FCGR2B in BM cells is dependent on the Ets family transcription factor PU.1, while PE cells do not express PU.1. RT-PCR of patient MM cells sorted into paired CD32B+ and CD32B− fractions showed PU.1 and FCGR2B (CD32B) expression only in CD138+/CD32B+ cells. Cell-tracking studies with CFSE showed that BM CD32B+ cells gave rise to BM CD32B− cells but that the reverse did not occur. Cell-culture studies showed that BM CD32B+ cells have significantly lower apoptotic rates than BM CD32B− cells, while the latter display greater dependence on insulin for survival and on IGF-1 and MIP-1α for proliferation. In order to better understand the differences between CD32B+ and CD32B− populations, we performed transcript profiling. BM CD32B+ cells have significantly higher expression of the activin inhibin, while BM CD32B− cells overexpress insulin-response genes such as the kinase Akt2. Compared to BM cells, PE cells overexpress germ-cell transcription factors and inflammasome-related genes, while BM cells overexpress tetraspanins (CD9, CD37, CD53) and insulin-response genes. Of interest, there are also significant differences in the expression of BMP and Wnt-related genes (but not Hh or Notch genes). BM cells overexpress BMPR1A, FZD2 and Wnt5A while PE cells overexpress CRIM1, FZD1 and LEF1. These data indicate that functional IH exists in MM, that the KMS-12 cell lines provide a useful model for its study, and that MM may originate in a CD32B+ cell that begets CD32B− heirs. We continue to study PU.1 and the generation of CD32B− cells by CD32B+ cells; to validate transcript data using sorted patient specimens; and to assess with RNAi the contributions of BMP, Wnt and insulin-response genes to the survival and proliferation of CD32B+ and CD32B− cells, seeking novel therapeutic targets on which selection of resistance may depend.

FDG-PET predicts disease burden in multiple myeloma

7619 Background: Because of the clinical and biologic heterogeneity of multiple myeloma, staging systems have been developed based on recognition of various prognostic factors. The recent integration of FDG-PET into the diagnostic algorithm may also have prognostic implications. Our aim was to evaluate the value of FDG-PET in determining the extent of disease as a staging tool in MM and to compare PET results with the established staging systems and laboratory data. Methods: Thirty seven patients with MM (mean age: 62±12) underwent a whole body FDG-PET scan at initial staging prior to therapy. FDG uptake was qualitatively scored in the following patterns: minimally diffuse (score 1), focal (score 2), multifocal (score 3), diffuse (score 4) and extramedullary (score 5). Standardized uptake values (SUVmax) were obtained in 780 osseous and extramedullary sites for quantitative evaluation. Additionally, mean SUVmax was calculated in 14 representative anatomic sites in the axial and appendicular skeleton in 20 age-matched control patients with no osseous malignancy. Ratios of the mean lesion SUVmax to the mean SUVmax of corresponding osseous sections from the control group were calculated. A score was obtained for each patient to reflect disease severity by adding the ratio for each lesion. Qualitative and quantitative results were correlated with the tumor burden (paraproteins, free light chains in the serum) and staging scores of Durie-Salmon (D-S) and the international staging systems (ISS). Results: There was good correlation between the quantitative score and both D-S staging and the ISS. However, correlation was stronger with the D-S system compared to that with ISS (r=0.44, p=0.007 vs. r=0.33, p=0.06). There was also good correlation between the quantitative score and the tumor burden (r=0.37, p&lt;0.05). The qualitative scoring system produced a good correlation with the tumor burden (r=0.35, p&lt;0.05). There was no correlation between the qualitative scoring system and D-S staging or ISS (P&gt;0.05). Conclusions: FDG-PET is useful in assessing the extent of disease as a staging tool at initial diagnosis in MM. The scoring system we have developed appears to correlate with the established staging systems and the level of free light chains which may contribute to the prognostic evaluation of patients with MM. No significant financial relationships to disclose.

Gene Expression Profiling of Plasma Cell Dyscrasias: The Role of IGH Translocations in the Heterogeneity of Multiple Myeloma.

Multiple Myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy ofundetermined significance, MGUS) or progress from intra-medullary to extra-medullaryforms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analyzed the gene expression profiles of plasma cells isolated from 7 MGUS, 39 MM and 6 PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct signatures have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis related functions. In addition, we identified a set of cancer germ-line antigens specifically expressed in a sub-group of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.

Genetic heterogeneity in multiple myeloma

In the past decade, many progresses have been made in our knowledge of the genetics of multiple myeloma. The use of molecular cytogenetic techniques has led to the identification of several recurrent (cyto)genetic abnormalities, representing either prognostic markers, or novel therapeutic targets. More global analyses of this genetic heterogeneity using expression array technologies should further extend our understanding of the disease, hopefully enabling some improvements in the treatment of the patients. The goal of this minireview is to summarize these recent advances.

Tumor Cell Heterogeneity in Multiple Myeloma: Antigenic, Morphologic, and Functional Studies of Cells From Blood and Bone Marrow

Tumor cells from six patients with immunoglobulin G (IgG) multiple myeloma were analyzed for surface antigens, cytoplasmic paraprotein, morphology, and response to various culture conditions. The tumor marker was the paraprotein idiotype. Low numbers of tumor cells were found in the blood of most of the patients. In some patients, the circulating tumor cells were solely B lymphocytes, whereas in other patients, they were lymphoid, lymphoplasmacytoid, and plasmacytoid. Dual surface antigen analysis of blood and bone marrow cells confirmed that the tumor may be composed of a spectrum of cell types. Thus, cells may range from surface-idiotype+,CD19+,CD20+, PCA-1-,cytoplasmic-idiotype- lymphocytes, to CD19-,PCA-1+,cytoplasmic-idiotype+ plasma cells that are surface-idiotype- or weakly surface-idiotype+. In one patient, some of the tumor cells co-expressed surface idiotype and CD10. The tumor B lymphocytes were activated in vitro to synthesize paraprotein by pokeweed mitogen (PWM), and by low molecular weight B cell growth factor (BCGF). In contrast, spontaneous synthesis of paraprotein by more mature tumor cells was inhibited by agents that also inhibit nonmyeloma plasma cells. These agents included PWM, gamma interferon, and phorbol ester. The results demonstrate that in multiple myeloma there exist different tumor cell types that are similar, by a variety of criteria, to normal B lineage cells at different stages of differentiation. Thus, further evidence is provided for the hypothesis of myeloma cell differentiation.

Tumor cell heterogeneity in multiple myeloma: antigenic, morphologic, and functional studies of cells from blood and bone marrow

Tumor cells from six patients with immunoglobulin G (IgG) multiple myeloma were analyzed for surface antigens, cytoplasmic paraprotein, morphology, and response to various culture conditions. The tumor marker was the paraprotein idiotype. Low numbers of tumor cells were found in the blood of most of the patients. In some patients, the circulating tumor cells were solely B lymphocytes, whereas in other patients, they were lymphoid, lymphoplasmacytoid, and plasmacytoid. Dual surface antigen analysis of blood and bone marrow cells confirmed that the tumor may be composed of a spectrum of cell types. Thus, cells may range from surface-idiotype+,CD19+,CD20+, PCA-1-,cytoplasmic- idiotype- lymphocytes, to CD19-,PCA-1+,cytoplasmic-idiotype+ plasma cells that are surface-idiotype- or weakly surface-idiotype+. In one patient, some of the tumor cells co-expressed surface idiotype and CD10. The tumor B lymphocytes were activated in vitro to synthesize paraprotein by pokeweed mitogen (PWM), and by low molecular weight B cell growth factor (BCGF). In contrast, spontaneous synthesis of paraprotein by more mature tumor cells was inhibited by agents that also inhibit nonmyeloma plasma cells. These agents included PWM, gamma interferon, and phorbol ester. The results demonstrate that in multiple myeloma there exist different tumor cell types that are similar, by a variety of criteria, to normal B lineage cells at different stages of differentiation. Thus, further evidence is provided for the hypothesis of myeloma cell differentiation.

Prognostic Relevance of Cellular Morphology in Multiple Myeloma

Morphological characteristics of tumor cells have been employed in the prognosis of lymphomas and solid tumors. This report documents an attempt to predict survival from the known cytologic heterogeneity in multiple myeloma. Myeloma cells in bone marrow smears from patients at diagnosis were evaluated by assigning them to morphologically defined categories. Cox's multivariate regression model for censored survival data was used to generate optimal weights, which served as coefficients in two regression equations to estimate death risk from cellular morphology. Step-wise procedures excluded redundant parameters. "Myeloma morphology score" (MMS) discriminates significantly (p less than 0.0001) among 3 stages, with median survival times of 42.5, 30.7, and 9.1 mo. For clinical routine application, "myeloma progression score" (MPS), the weight sum of the proportion of plasmablasts and the extent of bone marrow plasma cell infiltration, is suggested as a simple prognostic tool. Its discriminative power is very high [p less than 10(-9)]. Median survival times of greater than 71.5, 23.4, and 6.1 mo were found for good, moderate, and poor risk groups, respectively. However, staging is not confined to three subgroups, grouping is flexible, and pairs of data can be matched. This fact may prove to be valuable in designing prognosis-controlled clinical trials or theoretical studies on cellular differentiation. Preliminary results suggest changes in morphology due to disease progression and/or the effect of therapy on tumor kinetics. Most importantly, staging according to MPS or MMS may facilitate the adaption of therapy to the current state of the disease in patients with multiple myeloma.

Prognostic relevance of cellular morphology in multiple myeloma

Morphological characteristics of tumor cells have been employed in the prognosis of lymphomas and solid tumors. This report documents an attempt to predict survival from the known cytologic heterogeneity in multiple myeloma. Myeloma cells in bone marrow smears from patients at diagnosis were evaluated by assigning them to morphologically defined categories. Cox's multivariate regression model for censored survival data was used to generate optimal weights, which served as coefficients in two regression equations to estimate death risk from cellular morphology. Step-wise procedures excluded redundant parameters. “Myeloma morphology score” (MMS) discriminates significantly (p less than 0.0001) among 3 stages, with median survival times of 42.5, 30.7, and 9.1 mo. For clinical routine application, “myeloma progression scorex201D; (MPS), the weight sum of the proportion of plasmablasts and the extent of bone marrow plasma cell infiltration, is suggested as a simple prognostic tool. Its discriminative power is very high [p less than 10(-9)]. Median survival times of greater than 71.5, 23.4, and 6.1 mo were found for good, moderate, and poor risk groups, respectively. However, staging is not confined to three subgroups, grouping is flexible, and pairs of data can be matched. This fact may prove to be valuable in designing prognosis-controlled clinical trials or theoretical studies on cellular differentiation. Preliminary results suggest changes in morphology due to disease progression and/or the effect of therapy on tumor kinetics. Most importantly, staging according to MPS or MMS may facilitate the adaption of therapy to the current state of the disease in patients with multiple myeloma.