Cancer/Testis Antigen Expression Panel Incorporating MAGEC1 and BAGE2 Predicts Multiple Myeloma Disease Stage and Severity

To provide a single molecular assay that could be used to easily stage Multiple Myeloma patients at diagnosis, we investigated the association between the simultaneous expression of 7 Multiple Myeloma-associated Cancer/Testis Antigens and biochemical parameters that are currently used for disease staging. We analysed the mRNA expression of MAGEC1, MAGEA3/A6, BAGE2, PRAME, NYESO1, SSX2 and PAGE by qualitative reverse transcription PCR using RNA extracted from diagnostic bone marrow samples from 39 patients covering the Multiple Myeloma disease continuum and compared this to levels of key biochemical parameters at diagnosis. We found that the Cancer/Testis Antigen panel was expressed in a specific order that was specifically associated with the severity of disease. This allowed the Cancer/Testis Antigens expression profile to successfully place the patient clearly into either stage I or stage III of the disease, with further sub-stratification in the stage III grouping. In addition, we putatively identified MAGEC1 expression as a confirmatory diagnostic marker for symptomatic Multiple Myeloma and clearly associated BAGE2 expression exclusively with stage III disease. We also demonstrated the novel finding of PAGE expression in Multiple Myeloma, with an association with more advanced disease. We suggest that this particular molecular Cancer/Testis Antigen panel can be used at diagnosis as a single test to clearly stage patient

Molecular and functional profiling identifies therapeutically targetable vulnerabilities in plasmablastic lymphoma

Plasmablastic lymphoma (PBL) represents a rare and aggressive lymphoma subtype frequently associated with immunosuppression. Clinically, patients with PBL are characterized by poor outcome. The current understanding of the molecular pathogenesis is limited. A hallmark of PBL represents its plasmacytic differentiation with loss of B-cell markers and, in 60% of cases, its association with Epstein-Barr virus (EBV). Roughly 50% of PBLs harbor a MYC translocation. Here, we provide a comprehensive integrated genomic analysis using whole exome sequencing (WES) and genome-wide copy number determination in a large cohort of 96 primary PBL samples. We identify alterations activating the RAS-RAF, JAK-STAT, and NOTCH pathways as well as frequent high-level amplifications in MCL1 and IRF4. The functional impact of these alterations is assessed using an unbiased shRNA screen in a PBL model. These analyses identify the IRF4 and JAK-STAT pathways as promising molecular targets to improve outcome of PBL patients

The Use of MAGE C1 and Flow Cytometry to Determine the Malignant Cell Type in Multiple Myelom

The malignant cell phenotype of Multiple Myeloma (MM) remains unclear with studies proposing it to be either clonotypic B or proliferating plasma cells. Cancer/testis antigen MAGE C1 is being extensively studied in MM and it has been suggested that it is involved in the pathogenesis of the cancer. Therefore, we report on the use of MAGE C1 to determine the malignant cell phenotype in MM using flow cytometry. Bone marrow aspirate (BM) and peripheral blood (PB) was collected from twelve MM patients at diagnosis, as well as three MM disease-free controls. Mononuclear cells were isolated using density-gradient centrifugation, and stabilized in 80% ethanol, before analysis via flow cytometry using relevant antibodies against B cell development cell-surface markers and nuclear MAGE C1. MAGE C1 expression was observed consistently in the early stem cells (CD34+) and early pro-B to pre-B cells (CD34+/−/CD19+), as well as the proliferating plasma cells in both the MM PB and BM, while no expression was observed in the corresponding control samples. Monoclonality indicated a common origin of these cell types suggesting that the CD34+/MAGE C1+ are the primary malignant cell phenotype that sustains the downstream B cell maturation processes. Furthermore, this malignant cell phenotype was not restricted to the BM but also found in the circulating PB cells. Introductio

Size clonality analysis of the VDJ region from matched CD19⁺/CD34⁺/MAGE C1⁺ (pro-B to pre-B cells) and CD138⁺/MAGE C1⁺ (proliferating plasma cells) cell populations.

<p>Using capillary electrophoresis and specific primers for the CDR3 region cell clonality in MM subpopulations was investigated. As controls, the PB of a donor was analysed (A), as well as the CD19⁺/CD34⁺ isolated population from a donor BM sample (B), both showing polyclonal populations as expected. Comparison of PCR amplicons from the pro-B to pre-B cells and proliferating plasma cells isolated from BM of a MM patient with a large proliferating plasma cell population (MM9, 89%) indicated clonality of these two cell types (C and D).</p

Statistical analysis of MAGE C1 expression in various B cell sub populations.

<p>An unpaired 2-tailed Student t-test was used to determine statistical significance between the different cell populations expressing MAGE C1 as indicated, with <i>p</i>-values <0.05 considered to be statistically significant. <b>A</b>: Comparison of the CD34⁺/MAGE C1⁺ cell population size found in the BM between the MM and donor groups. <b>B</b>: Comparison of the CD34⁺/MAGE C1⁺ cell population size found in the PB between the MM and donor groups. <b>C</b>: Comparison of the CD19⁺/MAGE C1⁺ and CD27⁺/MAGE C1⁺ cell population sizes in the BM of the MM group. <b>D</b>: Comparison of the CD19⁺/MAGE C1⁺ and CD20⁺/MAGE C1⁺ cell population sizes in the PB of the MM group. *** = 0.0001 <<i>p</i>< 0.001. **** = <i>p</i>-value <0.0001.</p

Examples of flow cytometric analysis of the BM of a donor (A) and two MM patients (B and C) to determine the different B cell lineage cell populations expressing MAGE C1.

<p>Mononuclear cells from donor and MM patient BM were stained with different antibodies to determine the cell phenotype and stage of B cell maturation where MAGE C1 was expressed. <b>A and B</b>: Density plots (SS and CD45) were used to selectively gate around specific cell populations including plasma cells (R1), stem/immature B lymphocytes (R2) and mature B lymphocytes (R3). Using various antibodies specific to the cells of interest gated in R1, R2 and R3, cells with positive antibody expression were further selectively gated (R4) and MAGE C1 expression was determined with histograms. <b>A</b>: Donor BM indicated that all selected cell populations were negative for MAGE C1 expression. <b>B</b>: MAGE C1 expression was observed in the stem/immature B lymphocytes and not in the non-proliferating plasma or mature B lymphocytes of the MM patient. <b>C</b>: Density plots (SS and CD45) were used to selectively gate the plasma cells (R1) and these cells were further confirmed with CD138 (R2). The proliferation index of the plasma cells was determined with Ki-67 expression and this was correlated to MAGE C1 expression via histograms, respectively. MAGE C1 expression was only observed in the proliferating plasma cells (Ki-67>20%). 1: Plasma cells, 2: Stem/immature B lymphocytes, 3: Lymphocytes, 4: Residual monocytes, 5: Residual Granulocytes.</p

Summary of MM patient characteristics at time of diagnosis.

<p>MM: MM patient BM sample; D: Donor BM sample</p><p>¹Sex was indicated as M (male) or F (female)</p><p>²Durie-Salmon staging system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120734#pone.0120734.ref001" target="_blank">1</a>]</p><p>³Calcium >2.56 mmol/l; Renal: creatinine >90 μmol/l (F), >110 μmol/l (M); anemia: Hb <12 g/dl (F), <13 g/dl (M); bone lesions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120734#pone.0120734.ref045" target="_blank">45</a>]</p><p>⁴Immunoglobulin G or A, Lambda (Igλ) or Kappa (Igκ) light chain staining of the plasma cells</p><p>Summary of MM patient characteristics at time of diagnosis.</p

Schematic representation of the co-expression of B cell maturation markers and MAGE C1 in the relevant PB B-lymphocyte sub-populations of MM patients and donors.

<p>MAGE C1 expression was observed in the very early stages of B cell maturation involving the CD34⁺ and CD19⁺ as well as the proliferating plasma cells in the PB. Additionally no MAGE C1 expression was observed in the late B cells (CD20⁺) in the PB. The percentage indicated is the number of cells in a specific cell population expressing MAGE C1 (as defined by a specific cell-surface antigen). Kappa/Lambda light chain expression as well as CD117, CD10 and CD27 antibodies were not investigated in the PB. MM 1 to 12 as well as D1 to 3 indicates the different MM patient and donor profiles, respectively.</p

Schematic representation of the co-expression of B cell maturation markers and MAGE C1 in the relevant BM B-lymphocyte sub-populations of MM patients and donors.

<p>MAGE C1 expression was observed in the very early stages of B cell maturation involving the CD34⁺, CD117⁺, CD10⁺ and CD19⁺ cells (stem cells to early-mid immature B cells) in the BM, but was absent when light chain expression was expressed (naive and germinal centre B cells). Positive MAGE C1 expression was also observed in the proliferating plasma cell population, but not in the preceding CD27⁺ memory cells present in the BM. The percentage indicated is the number of cells in a specific cell population expressing MAGE C1 (as defined by a specific cell-surface antigen). MM BM samples are ordered according to increasing MAGE C1⁺ expression on the plasma cells. MM 1 to 10 as well as D1 to 3 indicates the different MM patient and donor profiles, respectively.</p