Katrin Bartels

Cancer-testis antigens MAGE-C1/CT7 and MAGE-A3 promote the survival of multiple myeloma cells

Background Multiple myeloma is a life-threatening disease and despite the introduction of stem cell transplantation and novel agents such as thalidomide, lenalidomide, and bortezomib most patients will relapse and develop chemoresistant disease. Therefore, alternative therapeutic modes for myeloma are needed and cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 have been suggested to represent a class of tumor-specific proteins particularly suited for targeted immunotherapies. Surprisingly, the biological role of cancer-testis genes in myeloma remains poorly understood. Design and Methods We performed the first investigation of the function of two cancer-testis antigens most commonly expressed in myeloma, MAGE-C1/CT7 and MAGE-A3, using an RNA interference-based gene silencing model in myeloma cell lines. Functional assays were used to determine changes in proliferation, cell adhesion, chemosensitivity, colony formation, and apoptosis resulting from gene-specific silencing. Results We show that the investigated genes are not involved in regulating cell proliferation or adhesion; however, they play an important role in promoting the survival of myeloma cells. Accordingly, knock-down of MAGE-C1/CT7 and MAGE-A3 led to the induction of apoptosis in the malignant plasma cells and, importantly, both genes were also essential for the survival of clonogenic myeloma precursors. Finally, silencing of cancer-testis genes further improved the response of myeloma cells to conventional therapies. Conclusions Cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 play an important role in promoting the survival of myeloma cells and clonogenic precursors by reducing the rate of spontaneous and chemotherapy-induced apoptosis and might, therefore, represent attractive targets for novel myeloma-specific therapies.

Longitudinal Analysis and Prognostic Effect of Cancer-Testis Antigen Expression in Multiple Myeloma

Purpose: Reliable data on the persistence of tumor expression of cancer-testis (CT) antigens over time and consequent analyses of the effect of CT antigen expression on the clinical course of malignancies are crucial for their evaluation as diagnostic markers and immunotherapeutic targets. Experimental Design: Applying conventional reverse transcription-PCR, real-time PCR, and Western blot, we did the first longitudinal study of CT antigen expression in multiple myeloma analyzing 330 bone marrow samples from 129 patients for the expression of four CT antigens (MAGE-C1/CT7, MAGE-C2/CT10, MAGE-A3, and SSX-2). Results: CT antigens were frequently and surprisingly persistently expressed, indicating that down-regulation of these immunogenic targets does not represent a common tumor escape mechanism in myeloma. We observed strong correlations of CT antigen expression levels with the clinical course of myeloma patients as indicated by the number of bone marrow–residing plasma cells and peripheral paraprotein levels, suggesting a role for CT antigens as independent tumor markers. Investigating the prognostic value of CT antigen expression in myeloma patients after allogeneic stem cell transplantation, we found that expression of genes, such as MAGE-C1, represents an important indicator of early relapse and dramatically reduced survival. Conclusions: Our findings suggest that CT antigens might promote the progression of multiple myeloma and especially MAGE-C1/CT7, which seems to play the role of a “gatekeeper” gene for other CT antigens, might characterize a more malignant phenotype. Importantly, our study also strongly supports the usefulness of CT antigens as diagnostic and prognostic markers as well as therapeutic targets in myeloma.

Chemokine CXCL13 is overexpressed in the tumour tissue and in the peripheral blood of breast cancer patients

The abilities of chemokines in orchestrating cellular migration are utilised by different (patho-)biological networks including malignancies. However, except for CXCR4/CXCL12, little is known about the relation between tumour-related chemokine expression and the development and progression of solid tumours like breast cancer. In this study, microarray analyses revealed the overexpression of chemokine CXCL13 in breast cancer specimens. This finding was confirmed by real-time polymerase chain reaction in a larger set of samples (n=34) and cell lines, and was validated on the protein level performing Western blot, ELISA, and immunohistochemistry. Levels of CXCR5, the receptor for CXCL13, were low in malignant and healthy breast tissues, and surface expression was not detected in vitro. However, we observed a strong (P=0.0004) correlation between the expressions of CXCL13 and CXCR5 in breast cancer tissues, indicating a biologically relevant role of CXCR5 in vivo. Finally, we detected significantly elevated serum concentrations of CXCL13 in patients with metastatic disease (n=54) as compared with controls (n=44) and disease-free patients (n=48). In conclusion, CXCL13 is overexpressed within breast cancer tissues, and increased serum levels of this cytokine can be found in breast cancer patients with metastatic disease pointing to a role of CXCL13 in the progression of breast cancer, suggesting that CXCL13 might serve as a useful therapeutic target and/or diagnostic marker in this malignancy.

Cancer-testis antigen expression and its epigenetic modulation in acute myeloid leukemia†

Cancer‐testis antigens (CTA) represent attractive targets for tumor immunotherapy. However, a broad picture of CTA expression in acute myeloid leukemia (AML) is missing. CTA expression was analyzed in normal bone marrow (BM) as well as in AML cell lines before and after treatment with demethylating agents and/or histone acetylase inhibitors. Presence of selected CTA with a strictly tumor‐restricted expression was then determined in samples of patients with AML before and after demethylating therapy. Screening AML cell lines for the expression of 20 CTA, we identified six genes (MAGE‐A3, PRAME, ROPN1, SCP‐1, SLLP1, and SPO11) with an AML‐restricted expression. Analyzing the expression of these CTA in blast‐containing samples from AML patients (N = 64), we found all samples to be negative for MAGE‐A3 and SPO11 while a minority of patients expressed ROPN1 (1.6%), SCP‐1 (3.1%), or SLLP1 (9.4%). The only CTA expressed in substantial proportion of patients (53.1%) was PRAME. Following demethylating treatment with 5′‐aza‐2′‐deoxycytidine, we observed an increased or de novo expression of CTA, in particular of SSX‐2, in AML cell lines. In AML patients, we detected increased expression of PRAME and induction of SSX‐2 after demethylating therapy with 5‐azacytidine. With the exception of PRAME, CTA are mostly absent from AML blasts. However, demethylating treatment induces strong expression of CTA, particularly of SSX‐2, in vitro and in vivo. Therefore, we propose that CTA‐specific immunotherapy for AML should preferentially target PRAME and/or should be combined with the application of demethylating agents opening the perspective for alternative targets like CTA SSX‐2. Am. J. Hematol., 2011.

The cytokine/chemokine pattern in the bone marrow environment of multiple myeloma patients.

OBJECTIVE The interaction of multiple myeloma (MM) with its bone marrow (BM) microenvironment is important for the homing pattern, survival, and proliferation of malignant plasma cells. We aimed at answering the question which cytokines, chemokines, and growth factors are typically found in the BM of untreated MM patients as well as in MM patients after allogeneic stem cell transplantation (alloSCT). MATERIALS AND METHODS We determined the concentrations of 34 cytokines/chemokines in the supernatants of 10 myeloma cell lines, as well as in the plasma derived from BM and peripheral blood samples of 10 newly diagnosed MM patients, 20 MM patients who had received allogeneic stem cell transplantation (alloSCT), and 20 healthy donors. RESULTS Besides cytokines/chemokines known to be secreted by myeloma cell lines, such as interleukin-1 receptor antagonist (IL-1RA), IL-8, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, and MIP-3α, we also detected significant levels of epidermal growth factor (EGF), hepatocyte growth factor (HGF), IL2R, IL-12p40/p70, IL-22, interferon-γ (IFN-γ)-inducible protein 10 (IP-10), monokine induced by IFN-γ (MIG), and regulated on activation normally T-cell expressed and secreted (RANTES) in culture supernatants. The BM environment in MM patients evidenced elevated concentrations of HGF, IL-2R, IL-16, EGF, IL-1RA, IP-10, MCP-1, and monokine induced by IFN-γ. Additionally, in the BM of MM patients post alloSCT, we found selectively elevated concentration of IL-4, IL-6, IL-8, IL-12p40/p70, and eotaxin. Eotaxin levels were particularly high in patients with chronic graft-vs-host disease. CONCLUSIONS Our study demonstrates characteristic cytokine/chemokine patterns in the BM environment of MM patients before and after alloSCT. Certain factors, such as MIP-1α, MCP-1, HGF, IL-16, IP-10, and eotaxin, might not only be developed into diagnostic instruments and/or predictive biomarkers, but are also potential targets for future myeloma- or graft-vs-host disease-specific therapies.

CD4+CD25+FOXP3+ T regulatory cells reconstitute and accumulate in the bone marrow of patients with multiple myeloma following allogeneic stem cell transplantation

Peripheral tolerance is largely maintained by immunosuppressive regulatory T cells, which typically co-express CD4, CD25 and FOXP3. This study analyzes lymphocyte subsets in myeloma patients treated with allogeneic stem cell transplantation, and shows that donor-derived regulatory T cells expand faster than conventional CD4+ T cells and have a strong inhibitory function. Background Very little is known about the number and function of immunosuppressive CD4+CD25+FOXP3+ T regulatory cells (Treg) in the human bone marrow and it is unclear whether bone marrow-residing Treg are capable of regenerating following allogeneic stem cell transplantation. This is particularly surprising since the bone marrow represents a major priming site for T-cell responses and Treg play important roles in the prevention of T-cell-mediated graft-versus-host disease and in promoting tumor escape from T-cell-dependent immunosurveillance. Design and Methods Applying flow cytometry, real-time polymerase chain reaction, and functional assays, we performed the first study on bone marrow and peripheral blood Treg in healthy donors as well as multiple myeloma patients before and after allogeneic stem cell transplantation. Results We found that, following the allogeneic transplantation, donor-derived CD4+CD25+FOXP3+ Treg expanded faster than conventional CD4+ T cells, leading to an accumulation of Treg in the bone marrow of transplanted patients who lack relevant thymic function. The reconstituted bone marrow-residing CD4+CD25+FOXP3+ Treg of myeloma patients after allogeneic stem cell transplantation consisted preferably of CD45RA−CCR7− memory T-cells and contained low numbers of T-cell receptor excision cycles, indicating that Treg had indeed expanded outside the thymus. Importantly, bone marrow-residing Treg of newly diagnosed and myeloma patients after allogeneic stem cell transplantation expressed high levels of transforming growth factor β and cytotoxic T-lymphocyte antigen 4, and showed a strong inhibitory function. Conclusions We suggest that allogeneic stem cell transplantation provides a short but significant window of opportunity for CD8+ T cells before an exuberant regeneration of immunosuppressive Treg sets in. Later after transplantation, bone marrow-residing Treg probably contribute to suppressing graft-versus-host disease but may also undermine persistent immune control of multiple myeloma.

Expression of cancer-testis antigens as possible targets for antigen-specific immunotherapy in head and neck squamous cell carcinoma.

Cancer-Testis (CT) antigens are by definition expressed in tumor but not in healthy tissue except testis and might represent ideal targets for antigen-specific immunotherapy. Here, we present the first comprehensive analysis of CT antigen expression in patients with head and neck squamous cell carcinoma (HNSCC). Tumor samples (N=51), and adjacent healthy tissue from patients with HNSCC were analyzed for the expression of 23 genes designated CT antigens using RT-PCR. Patient sera (N=39) were screened for IgG antibody responses against NY-ESO-1, MAGEA3, and SSX2. According to their expression pattern antigens were divided into four groups. ADAM2, LIP1, SLLP1, AKAP3, CTAGE, ZNF165, CAGE, and FTHL17 were expressed in tumor and healthy tissue at comparable frequencies. NY-TLU-57, GAGE1, SAGE1 were expressed more frequently in tumor samples than in healthy tissues. TPTE, LDHC, SPO11 were expressed neither in tumor samples nor in healthy tissue. 9 CT antigens were expressed only in the tumor tissue and may represent ideal candidates for active immunotherapy in HNSCC: MAGEA3 was expressed in 72%, SSX1 in 45%, MAGEC2 in 33%, MAGEC1 in 28%, BAGE in 17%, SSX2 in 16%, SCP1 in 12%, NY-ESO-1 in 6%, and HOM-TES-85 in 4% of cases. 86% of tumor samples expressed at least one, 69% expressed at least two, and 43% expressed at least three of these antigens. Three patients showed an antibody response against NY-ESO-1. In conclusion, we demonstrate here that HNSCC frequently express CT antigens. Furthermore, a relatively high percentage of tumors express more than one CT antigen opening the perspective for polyvalent antigen-specific immunotherapy.

An optimized assay for the enumeration of antigen-specific memory B cells in different compartments of the human body

OBJECT In the framework of our current study we set out to develop an optimized assay for the quantification of antigen-specific B cells in different compartments of the human body. METHODS Mononuclear cells (MNC) derived from the peripheral blood, bone marrow (BM), or human tonsils were incubated with different combinations of stimuli. The stimulated cells and culture supernatants were then applied to IgG-ELISPOT and ELISA read-out assays and tetanus toxoid (TT)-specific B cell responses were quantified. RESULTS We found that a combination of CD40L, CpG, and IL21 was optimal for the induction of TT-specific IgG-producing cells from memory B cell (mBc) precursors. This cocktail of stimuli led to a proliferation-dependent induction of CD19(intermediate)CD38(high)CD138(high)IgD(negative) terminally differentiated plasma cells. Applying our optimized methodology we were also able to quantify mBc specific for cytomegalovirus and influenza virus A. Most importantly, the same method proved useful for the comparison of mBc frequencies between different compartments of the body and, accordingly, we were able to demonstrate that TT-specific mBc preferably reside within tonsillar tissue. CONCLUSION Here, we optimized an assay for the quantification of antigen-specific B cells in different human tissues demonstrating, for example, that TT-specific mBc preferably reside in human tonsils but not in the BM or the peripheral blood. We suggest that our approach can be used for the enumeration of mBc specific for a wide variety of Ag (microbial, tumor-related, auto-antigens), which will lead to significant improvements regarding our knowledge about the biology of humoral immunity.

Surface molecule CD229 as a novel target for the diagnosis and treatment of multiple myeloma

Background To date, multiple myeloma remains an incurable malignancy due to the persistence of minimal residual disease in the bone marrow. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cell populations. We, therefore, investigated functionally relevant immunoreceptors specifically associated with myeloma cells as well as their clonogenic precursors. Design and Methods Potential target proteins were identified using antibody arrays against phosphorylated immunoreceptors with lysates from myeloma cell lines. CD229 expression was confirmed in primary myeloma cells by reverse transcriptase polymerase chain reaction, western blot, fluorescence-activated cell sorting, and immunohistochemistry. Apoptosis, clonogenic growth, and sensitivity to chemotherapy were determined following short-interfering RNA-mediated downregulation of CD229. Antibody-dependent cellular and complement-dependent cytotoxicity were analyzed using a monoclonal antibody against CD229 to demonstrate the antigen’s immunotherapeutic potential. Results Our screening assay identified CD229 as the most strongly over-expressed/phosphorylated immunoreceptor in myeloma cell lines. Over-expression was further demonstrated in the CD138-negative population, which has been suggested to represent myeloma precursors, as well as on primary tumor cells from myeloma patients. Accordingly, CD229 staining of patients’ bone marrow samples enabled the identification of myeloma cells by flow cytometry and immunohistochemistry. Down-regulation of CD229 led to a decreased number of viable myeloma cells and clonal myeloma colonies, and enhanced the anti-tumor activity of conventional chemotherapeutics. Targeting CD229 with a monoclonal antibody resulted in complement- and cell-mediated lysis of myeloma cells. Conclusions Our results demonstrate that the immunoreceptor CD229 is specifically over-expressed on myeloma cells including their clonogenic precursors and contributes to their malignant phenotype. Monoclonal antibodies against this protein may represent a promising diagnostic and immunotherapeutic instrument in this disease.

Role of Interleukin 16 in Multiple Myeloma

BACKGROUND Multiple myeloma is a malignancy characterized by the expansion of a plasma cell clone that localizes to the human bone marrow. Myeloma cells and bone marrow stromal cells produce soluble factors that promote the survival and progression of multiple myeloma. Interleukin 16 (IL-16) is involved in regulating the migration and proliferation of normal leukocytes. However, the role of IL-16 in human cancers, including multiple myeloma, is unclear. METHODS We investigated IL-16 expression in cell lines (n = 10) and in the bone marrow of myeloma patients (n = 62) and healthy bone marrow donors (n = 12) by quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry. Transfection of two human multiple myeloma cell lines with small interfering RNAs was used to examine the effect of IL-16 gene silencing on apoptosis by flow cytometry, on proliferation by bromodeoxyuridine incorporation, and on colony formation. Protein neutralization assays were performed by treating multiple myeloma cells with a monoclonal antibody against the carboxyl-terminal fragment of IL-16. All statistical tests were two-sided. RESULTS IL-16 was strongly overexpressed in the bone marrow of myeloma patients compared with healthy donors. Myeloma cell lines as well as primary tumor cells from myeloma patients constitutively expressed IL-16 and its receptors CD4 and/or CD9 and spontaneously secreted soluble IL-16. Silencing of IL-16 reduced the proliferative activity of myeloma cells by approximately 80% compared with untreated cells (mean relative proliferative activity IL-16 siRNA vs untransfected cells, EJM cells: 20.1%, 95% confidence interval [CI] = 14.3% to 26.0%, P = .03; KMS-12-BM cells: 22.8%, 95% CI = 5.5% to 40.0%, P = .04), and addition of a recombinant carboxyl-terminal IL-16 peptide reversed that effect. A monoclonal antibody directed against IL-16 or its receptors had a comparably strong growth-inhibiting effect on the tumor cells. CONCLUSIONS IL-16 is an important growth-promoting factor in multiple myeloma and a candidate for novel diagnostic, prognostic, and therapeutic applications for this incurable human malignancy.