Antigen Expression Patterns Correlate With Underlying Genetic Aberrations in Multiple Myeloma

Abstract A poor response to Fas-induced apoptosis is evident in some multiple myeloma (MM) cell lines and primary cells. In this study, we have examined the possibility to increase the sensitivity to Fas-induced apoptosis by pretreatment of MM cells with interferon-γ (IFN-γ) or interferon- (IFN-). Both IFN-γ and IFN- markedly increased the Fas-induced apoptosis in all cell lines tested (U-266-1970, U-266-1984, and U-1958). In the U-266-1970 and U-1958 cell lines, pretreatment with either IFN-γ or IFN- also inhibited proliferation in a dose-dependent manner. In contrast, IFN-γ activation of the Fas death pathway in the U-266-1984 cells was not accompanied by growth inhibition. Incubation with the IFNs increased the Fas antigen expression in one of three cell lines but did not alter the expression of Bcl-2 or Bax. The IFNs are important regulators of growth and survival in MM cells. Our results suggest that activation of Fas-mediated apoptosis is a novel mechanism by which the IFNs exert inhibitory effects on MM cells. © 1998 by The American Society of Hematology.

Download Full-text

Plasmodium falciparum Variant Surface Antigen Expression Patterns during Malaria

PLoS Pathogens ◽

10.1371/journal.ppat.0010026 ◽

2005 ◽

Vol 1 (3) ◽

pp. e26 ◽

Cited By ~ 116

Author(s):

Peter C Bull ◽

Matthew Berriman ◽

Sue Kyes ◽

Michael A Quail ◽

Neil Hall ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Surface Antigen ◽

Expression Patterns ◽

Antigen Expression ◽

Variant Surface Antigen ◽

Surface Antigen Expression

Download Full-text

P855 High-resolution maps of heterogeneous antigen expression in glioblastoma and implications for immunotherapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/lba2019.9 ◽

2020 ◽

Vol 8 (Suppl 1) ◽

pp. A6.1-A6

Author(s):

Michael Barish ◽

Lihong Weng ◽

Dina Awabdeh ◽

Blake Brewster ◽

Massimo D’Apuzzo ◽

...

Keyword(s):

Tumor Heterogeneity ◽

Diversity Index ◽

Expression Patterns ◽

Antigen Expression ◽

Target Antigen ◽

Spatial Distributions ◽

Combination Therapies ◽

Cell Immunotherapy ◽

Color Deconvolution ◽

Car T

BackgroundGlioblastoma (GBM) remains an almost universally fatal brain tumor. While CAR T cell immunotherapy has shown promising clinical efficacy, therapeutic failure may reflect our incomplete understanding of target antigen expression. We previously examined variations in antigen expression at the level of individual patients (inter-patient or inter-tumor heterogeneity), focusing on immunotherapy targets IL13Rα2, HER2 and EGFR. We concluded that antigen expression diverged from expectations from random expression. Because antigen escape may arise from GBM cell heterogeneity, we have mapped target antigen expression within individual tumors (intra-tumor heterogeneity).MethodsSerial sections from a 43 patient cohort were immunostained (DAB with hematoxylin counterstain) for target antigens IL13Rα2, HER2 and EGFR. Each section was annotated directly from the slide by a neuropathologist. Sections were scanned (0.46 µm/pixel; Hamamatsu), and then working within Fiji/ImageJ, images were segmented by color deconvolution into hematoxylin (nuclei) and DAB layers. Images of nuclear layers were aligned, and used to align the DAB layers. Two schemes were used to examine the spatial distributions of the three target antigens. When tumor domains could be identified, we determined expression of each antigen as optical density (OD). In the second scheme, a 10 µm grid was superimposed on each section, and OD was determined for each position and assembled into spreadsheets (Origin v2019b). Maps for expression were generated from the OD in each position.ResultsApproaching these maps from the perspective of antigen escape, we examined the extent to which expression of target antigens was spatially mixed, how rapidly antigen dominance could shift (spatial frequency), and whether spatial distributions were arrayed in a coordinated manner.When tumor domains could be identified, we calculated the Shannon diversity index (H) for each domain within a section. While values of H clustered within some tumors, usually values of H varied widely.The superimposed grid was used to examine heterogeneity within entire tumor sections. Expression was intermixed, and EGFR and IL13Rα2/HER2 displayeds complementary expression patterns. In tumors with large EGFR+ areas, IL13Rα2+/HER2+ areas could overlap, while when EGFR+ areas were smaller, IL13Rα2+ and HER2+ areas were more distinct. Borders could be quite diffuse, or quite sharp (a few cell diameters).ConclusionsOur results indicate that expression of IL13Rα2, HER2 and EGFR is highly heterogeneous and not always spatially distinct. Because GBM tumors adapt to the selection pressures of immunotherapies, we suggest that combination therapies should be designed accordingly, and immunotherapies targeting IL13Rα2/HER2 could benefit from inclusion of EGFR.

Download Full-text

MicroRNA Changes Occur in Multiple Myeloma Cells in the Context of Bone Marrow Milieu.

Blood ◽

10.1182/blood.v114.22.1785.1785 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1785-1785 ◽

Cited By ~ 1

Author(s):

Roccaro M. Roccaro ◽

Antonio Sacco ◽

Abdel Kareem Azab ◽

Feda Azab ◽

Hai Ngo ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Expression Patterns ◽

Mirna Gene ◽

Marrow Stromal Cells ◽

Stem Loop ◽

Mirna Profiling ◽

Qrt Pcr

Abstract Abstract 1785 Poster Board I-811 Background We and Others have previously demonstrated that primary multiple myeloma (MM) cells are characterized by a specific microRNA (miRNA) signature compared to the related normal plasmacell counterpart; and that miRNAs play a crucial role in regulating MM pathogenesis. Nevertheless, miRNA changes that occur in MM cells in the context of the bone marrow microenvironment have not been previously examined. Therefore, characterization of miRNA profiling of MM cells in conjunction with bone marrow stromal cells (BMSCs) is important to better understand the underlying molecular changes that lead to initiation and progression of this disease. Methods We performed miRNA-expression-profiling of MM cell lines (MM.1S; RPMI8226) that were co-cultured with primary BMSCs obtained from 5 MM patients, using liquid phase Luminex microbead miRNA profiling (Luminex, Austin, TX). The expression patterns of unfiltered data were performed using unsupervised hierarchical clustering of samples, based on centroid linkage and 1-correlation distance metric, using dChip (www.dchip.org). To further define those miRNAs differentially expressed between groups (patients vs normal), the data were filtered on significance of differences using ANOVA test, (P < 0.05). Microbead-miRNA profiling data were validated data by stem-loop qRT-PCR. To identify specific predicted miRNA-targeted mRNAs, TargetScan, PicTar, and miRanda algorithms were used. Results miRNA profiling of MM cells cultured with primary BMSCs (MM+BMSC system) differs from MM cells which were not grown in contact with primary BMSCs (MM cells alone). Specifically, we observed increased expression of miRNA-450, -432*, -299-5p, -409-3p, -29b, -542-5p, -184, -517*, -218, 128b, -142-5p and -211 (P<0.05) in MM cells obtained from the MM+BMSC system, compared to MM cells alone. Stem-loop qRT-PCR was performed on matched samples and showed expression patterns similar to those observed in miRNA analysis. Using algorithms commonly used to predict human miRNA gene targets (miRanda; TargetScan; PicTar), predicted targets of the increased miRNAs included negative regulators of NFkB, PI3K/Akt/mTOR, and MAPK/ERK signaling pathways, such as PTEN, KSR2, TWEAK, and DUSP; as well as tumor suppressors (MCC, TSSC1, TUSC1, FBW7, RHOBTB), pro-apoptotic factors and cyclin-dependent kinases inhibitors. These data demonstrate that bone marrow stromal cells exert a modulatory effect on miRNA profiling in MM cells, which results in promoting MM cell growth and reducing MM cell survival. Disclosures Ghobrial: Millennium : Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Download Full-text

Identification Of Novel Alternative Splice Variants Of Sirtuins In Multiple Myeloma: Therapeutic Implications

Blood ◽

10.1182/blood.v122.21.3121.3121 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3121-3121

Author(s):

Antonia Cagnetta ◽

Michele Cea ◽

Sophia Adamia ◽

Yu-Tzu Tai ◽

Teru Hideshima ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Splice Variants ◽

Expression Patterns ◽

Critical Role ◽

Sequencing Analysis ◽

Biological Processes ◽

Newly Diagnosed ◽

Cloning And Sequencing ◽

Aberrant Expression

Abstract Background Alternative splicing (AS) is a normal epigenetic event with a critical role in the regulation of gene expression. Previous studies showed increased AS in Multiple Myeloma (MM) cells, suggesting the need to assess both expression level of genes and post translational modifications, mediating overall gene function. The NAD-dependent deacetylases Sirtuins (SIRTs), mammalian homologues of the yeast Sir2, modulate various biological processes including metabolism, cell survival, development, chromatin dynamics, or DNA repair. Recent microarray profiling data using newly diagnosed patients with MM, suggests clinical relevance of such deacetylases since their level predicts for both progression free and overall survival. Among SIRTs family membersSIRT-5, SIRT-6 and SIRT-7 transcript levels positively correlated with disease progression (from MGUS to active MM). These studies provide the rationale for further examining the biological processes including epigenetic changes, mutations, or AS events that contribute to aberrant expression of SIRTs in MM. Methods Purified RNA from MM cell lines, newly diagnosed MM patient cells, as well as peripheral blood mononuclear cells (PBMCs) from normal healthy donors was subjected to SIRT expression analysis. Specifically, SIRTs-specific primers were developed and optimized using standard RT-PCR conditions. RNA integrity was confirmed using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. Aberrant splicing of SIRT-5, SIRT-6, and SIRT-7 was confirmed by cloning and sequencing, followed by analysis of their expression patterns in MM patients versus normal PBMCs. Results We found that SIRTs genes are frequently miss-spliced in MM patients. To our knowledge, this is the first report showing SIRTs miss-splicing event in MM. Through cloning and sequencing analysis, we identified novel spliced isoforms of SIRT-5, SIRT-6 and SIRT-7; these occurred as a result of aberrant AS within exon-12, exon-4 and and exon-5, respectively. Specifically, exon skipping was noted in SIRT-6 and SIRT-7 variants, via cryptic 5 prime or 3 prime splice sites on exon 12 and/or through partial retention of an intron created SIRT-5 variants. The novel spliced forms were widely expressed in MM cell lines and primary cells, without significance occurrence in normal PBMCs. Our preliminary data show that even though these novel isoforms exhibit reduced deacetylase activity versus full-length variants, this characteristic may impart distinct functional outcome. Finally, in support of above studies, our analysis of MM patient samples suggest that AS among SIRTs is associated with poor clinical outcome in MM patients. Conclusion In the current study, we have identified novel transcript variants of SIRT-5, SIRT-6 and SIRT-7 in MM cells. These aberrant isoforms allow for generating transcripts that encode for dysfunctional proteins, which in turn, may contribute to the genetic heterogeneity in MM. Ongoing studies are delineating the function of these newly identified splice variants of SIRTs and their association with MM progression. Overall, our studies will provide basis for utilizing SIRTs variants as prognostic markers and/or as novel therapeutic targets in MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Chauhan:Vivolux: Consultancy.

Download Full-text

Role of immune-related conditions in smoldering myeloma and MGUS.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.8104 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 8104-8104 ◽

Cited By ~ 2

Author(s):

Mary Kwok ◽

Neha Korde ◽

Elisabet E. Manasanch ◽

Manisha Bhutani ◽

Irina Maric ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

High Risk ◽

Mayo Clinic ◽

Plasma Cells ◽

Risk Model ◽

Expression Patterns ◽

Autoimmune Disorder ◽

Epidemiological Studies ◽

Smoldering Myeloma

8104 Background: Recent guidelines emphasize tailored follow-up and the need for clinical trials for high-risk smoldering myeloma (SMM). Emerging evidence from epidemiological studies suggests that immune-related conditions play a role in the causation of myeloma precursor disease (SMM and monoclonal gammopathy of undetermined significance; MGUS) and are of clinical importance for the risk of developing multiple myeloma. The aim of our study is to assess whether there is an altered biology in SMM/MGUS patients with preceding immune-related conditions. Methods: From our ongoing prospective SMM/MGUS natural history study, we evaluated 56 SMM and 60 MGUS patients. Information on autoimmunity was identified at baseline. All patients underwent extensive clinical and molecular characterization. At baseline, all patients underwent bone marrow biopsy evaluation using immunohistochemistry and multi-color flow cytometry of plasma cells. We assessed expression patterns of adverse plasma cell markers (CD56 and CD117), and applied risk models based on serum immune markers and bone marrow findings. Results: Among enrolled SMM and MGUS patients, 7 (12%) and 9 (15%) had a preceding autoimmune disorder. We found SMM patients with (vs. without) a preceding autoimmune disorder to have a substantially lower rate of CD56 (28% vs. 61%) and CD117 (28% vs. 61%) expressing plasma cells. When we compared the same markers in MGUS patients, CD56 and CD117 expression patterns were similar among patients with vs. without preceding autoimmunity (10% vs. 17%, and 50% vs. 48%). Using the Mayo Clinic risk model, none of the SMM patients with a preceding autoimmune disorder had high-risk features; in contrast, 3/41 (7%) of those without a preceding autoimmune disorder were high-risk SMM. Using the Mayo Clinic risk model, none of the MGUS patients were high-risk independent of autoimmune status. Conclusions: Our prospective clinical study found SMM patients with preceding immune-related conditions to have less adverse biology, supportive of epidemiological studies suggesting the risk of developing multiple myeloma is substantially lower in these patients.

Download Full-text