MAGE-A3 Expression Research Articles

Improving T Cell Therapy for Relapsed EBV-Negative Hodgkin's Lymphoma by Targeting Upregulated MAGE-A4

Abstract 1658Patients with Hodgkin’s Lymphoma (HL) who relapse after hematopoietic stem cell transplant (HSCT) have limited options for long-term cure. We have shown that infusion of cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV+ HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients, an alternative strategy is to target the cancer/testis antigen MAGE-A4 which is present in EBV antigen-negative Hodgkin’s lymphoma tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors. We explored the feasibility of combining adoptive T-cell therapy with epigenetic modification of tumor antigen expression. We first verified whether MAGE-A4-specific CTL could be expanded from autologous and allogeneic donors. Dendritic cells from healthy donors (n=15) or HL patients (n=9) were pulsed with overlapping peptides spanning the MAGE-A4 protein. Healthy donor T-cell lines expanded to sufficient numbers for clinical use and predominantly comprised CD8+ cytotoxic T cells (mean 60%; range 42–81%) and CD4+ T helper cells (mean 20%; range 8–45%). A mean response of 152 interferon gamma (IFNγ) spot forming cells (SFC)/1 × 105 cells (median 78; range 12–635) was observed against MAGE-A4 peptides compared to a mean of 6 IFNγ SFC/1×105 cells (median 4; range 0–30) to irrelevant peptides. Both CD4+ and CD8+ T cell subsets contributed to the interferon response. In T cells expanded from the peripheral blood of patients with HL, a mean of 296 SFC/1×105 cells (range 32–1031) responded to MAGE-A4 peptides compared to a mean of 5 SFC/1×105 cells (range 1–11) responding to irrelevant targets. Five of fifteen CTL lines generated from healthy donors showed specificity for a particular peptide (aa266-285, NPARYEFLWGPRALAETSYV). In contrast, T-cell lines derived from HL patients showed a range of MAGE-A4 epitope specificities. Peripheral blood mononuclear cell and dendritic cell numbers were reduced, and T cells from these patients failed to expand after several stimulations consistent with the profound immune deficiency frequently seen in HL patients. We next evaluated the effect of combining MAGE-CTL with the epigenetic-modifying demethylating agent 2’-deoxy-5’azacytidine. To determine if decitabine would negatively affect the function of the TAA-specific T cells, we examined the effects of the drug on the function of MAGEA4-specific T cells in vitro. Our results showed that treatment of T cells with decitabine – in concentrations that exceed expected levels following drug treatment (1 μM) - did not adversely affect the phenotype and function of MAGE-A4 specific T cells in vitro. To evaluate the in vivo effects of decitabine on tumor antigen specific T cells, patient samples were obtained before and after treatment. In patients who received a decitabine containing regimen (75 mg/m2 daily × 5 days) (n=2), one of whom had clinical responses on PET scan, we observed that MAGE-specific T cells were of greater magnitude and recognized a broader number of MAGE-A4 epitopes after decitabine treatment (see Figure below – a and b are patients with a decitabine containing regimen; c and d are patients receiving no decitabine). In patients (n= 2) who did not receive a decitabine containing regimen, the magnitude of MAGE-specific reactivity was reduced, as measured by IFNγ Elispot. These results suggest that although decitabine may modulate immune function, the effect of other chemotherapies may result in the lack of appreciable endogenous T-cell responses to tumor antigens in these patients. Hence, adoptive transfer of MAGE-A4 specific T cells combined with the administration of epigenetic-modifying drugs to increase expression of the target antigens may improve the efficacy of treatment of relapsed Hodgkin’s Lymphoma. [Display omitted] [Display omitted] Disclosures:Younes:Celgene: Speakers Bureau; Syndax: Research Funding; SBIO: Research Funding; Genentech: Research Funding; Sanofi-Aventis: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau, Travel Expenses; Gilead: Honoraria; Pharmacyclic: Honoraria.

Improving T-cell Therapy for Relapsed EBV-Negative Hodgkin Lymphoma by Targeting Upregulated MAGE-A4

Patients with Hodgkin lymphoma (HL) relapsing after hematopoietic stem cell transplant have limited options for long-term cure. We have shown that infused cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV(+) HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients, an alternative is to target the cancer/testis antigen MAGE-A4 present in EBV antigen-negative HL tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors. We explored the feasibility of combining adoptive T cell therapy with epigenetic modification of tumor antigen expression. We further characterized MAGE-A4-specific T-cell phenotype and function, and examined the effects of the epigenetic modifying drug decitabine on these T cells. Cytotoxic T cells were generated specifically recognizing MAGE-A4 expressed by autologous HL targets and tumor cell lines. Decitabine-previously shown to increase tumor antigen expression in HL-did not compromise MAGE-A4-specific T-cell phenotype and function. In patients treated with decitabine, expanded MAGE-A4-specific T cells had a broader antitumor T cell repertoire, consistent with increased antigen stimulation in vivo. Adoptive transfer of MAGE-A4-specific T cells, combined with epigenetic modifying drugs to increase expression of the protein, may improve treatment of relapsed HL.

MAGE-A3 expression is an adverse prognostic factor in diffuse large B-cell lymphoma

This study evaluates the prognostic value of MAGE-A3 expression in 28 diffuselarge B-cell lymphoma (DLBCL) patients. A significant associationwas observed between MAGE-A3 expressions, assessed by quantitative real-timeRT-polymerase chain reaction (PCR), with advanced stages of disease (P<0·05).Elevated serum lactate dehydrogenase (LDH) levels and InternationalPrognostic Index (IPI) score were significantly higher in MAGE-A3-positivepatients (P = 0·025 and P = 0·004,respectively). Expression of MAGE-A3 was associated with poor responseto treatment and a significantly shorter overall survival (P<0·001).Our data address new information in the association of MAGE-A3 expressionand poor prognosis in DLBCL patients.

Expression of MAGE-A10 cancer/testis antigens in breast cancer: a retrospective immunohistochemical study

Expression of MAGE-A10 cancer/testis antigens in breast cancer: a retrospective immunohistochemical study

Progestogen can stimulate growth factor production in breast tissue of women with breast cancer

s / The Breast 20 (2011) S12–S55 S24 intensity 2+ and 3+ were considered MAGE-A10 positive. The MAGE-A10 expression was correlated with the other clinico-pathological variables. Results: MAGE-A10 antigens were expressed in 34.1% (56/164), 46.3% (76/ 164) and 17.1% (28/164) of patients respectivly. MAGE-A10 overexpression (intensity 2+ and 3+) was detected in 63.4% (104/164) of patients. No correlation between MAGE-A10 overexpression and tumor size, tumor grade and lymph nodes status was detected. MAGE-A10 overexpression was associated with ER negative (p1⁄40.002), PR negative (p1⁄40.002) and HER-2 negative (p1⁄40.044) tumors. A significant higher expression of MAGE-A10 was found in triple negative breast cancers compared with ER positive/HER-2 negative cancers (p1⁄40.011). MAGE-A10 overexpression was detected in 78.4% (51/65) triple negative cancers, compared with 52/ 94 (55 %) ER-positive/HER-2 negative patients. Conclusion: MAGE-A10 antigens are preferentially expressed in a substantial proportion of TN breast cancer. Considering the limited treatment options for ER/PR/HER2 triple-negative breast cancer, the potential of CT-based immunotherapy should be explored.

UP-01.144 Prognostic Value of MAGE A9 in Renal Cell Carcinoma

Cancer testis (CT) antigens have shown to be associated with tumor progression in various tumors. MAGE A9, a member of this group, has recently been identified as a tumor-associated antigen in renal cell carcinoma (RCC). The purpose of this study was to investigate, whether MAGE A9 expression has a prognostic relevance in RCC.

Cancer-testis gene expression profiling in esophageal squamous cell carcinoma: Identification of specific tumor marker and potential targets for immunotherapy

Cancer-testis antigens (CTAs) are often specifically expressed in cancer cells and under normal conditions are only considered to be expressed in the germ line cells and the placenta. CTAs are potential targets for cancer immunotherapy and therefore necessitates their expression profiling. The expression profile of LAGE1, MAGE-A4 and NY-ESO1, their possible correlations and interaction, and the clinicopathological associations of each marker were studied. RNA was extracted from fresh esophagectomy tissues of 41 esophageal squamous cell carcinoma (ESCC) patients prior to any other therapeutic intervention. The relative mRNA expression of LAGE1, MAGE-A4 and NY-ESO1 was assessed with the real-time reverse transcription-polymerase chain reaction (RT-PCR) 5‘ nuclease assay. The overexpression of LAGE1, MAGE-A4 and NY-ESO1 was found in 39, 90.2 and 41.4% of ESCC samples respectively. Of the patients, 97.5% showed an overexpression of at least one CTA. The relative expression of MAGE-A4 was directly associated with lymph node metastasis and the stage of the tumor (p < 0.05). A significant direct correlation was also detected between the MAGE-A4/LAGE1 and MAGE-A4/NY-ESO1 levels of gene expression. MAGE-A4 is identified as a specific biomarker of ESCC with a possible oncogenic role contributing to tumor progression. Interactions between MAGE-A4, LAGE1 and NY-ESO1 and their significant clinical consequences introduce these CTAs as appropriate targets for a polyvalent cancer vaccine.

The histone deacetylase inhibitor entinostat (SNDX-275) induces apoptosis in Hodgkin lymphoma cells and synergizes with Bcl-2 family inhibitors

Based on promising invitro and invivo activity of several histone deacetylase inhibitors in Hodgkin lymphoma (HL), we investigated SNDX-275, an oral class 1 isoform-selective histone deacetylase inhibitors in HL-derived cell lines. Proliferation and cell death were examined by MTS assay, Annexin V/propidium iodide, and fluorescence-activated cell sorting analysis. Gene and protein expression were measured by reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemical analysis. A multiplex assay was used to determine cytokines and chemokines. SNDX-275 induced cell death in a dose- and time-dependent manner with an IC(50) at the sub- and lower micromolar range at 72 hours. At the molecular level, SNDX-275 increased histone H3 acetylation, upregulated p21 expression, and activated the intrinsic apoptosis pathway by downregulating the X-linked inhibitor of apoptosis protein. SNDX-275 downregulated expression of antiapoptotic Bcl-2 and Bcl-xL proteins without altering Mcl-1 or Bax levels. Combination studies demonstrated that two Bcl-2 inhibitors (ABT-737 and obatoclax) significantly enhanced the effect of SNDX-275. SNDX-275 modulated the level of several cytokines and chemokines, including interleukin-12 p40-70, interferon-inducible protein-10, RANTES (regulated on activation, normal T expressed and secreted), interleukin-13, interleukin-4, and thymus and activation-regulated chemokine and variably induced the cancer/testis antigen expression of MAGE-A4 and survivin in HL cell lines. SNDX-275 has antiproliferative activity in HL cell lines, involving several mechanisms: induction of apoptosis, regulation of cytokines and chemokines, and alteration of cancer/testis antigens. Clinical investigation of SNDX-275 alone or in combination with Bcl-2 inhibitors is warranted in patients with HL. Phase 2 studies with SNDX-275 in HL are ongoing, and future clinical studies should investigate combinations with SNDX-275.

MAGE-A Inhibits Apoptosis in Proliferating Myeloma Cells through Repression of Bax and Maintenance of Survivin

The type I Melanoma Antigen GEnes (MAGEs) are commonly expressed in cancers, fueling speculation that they may be therapeutic targets with oncogenic potential. They form complexes with RING domain proteins that have E3 ubiquitin ligase activity and promote p53 degradation. MAGE-A3 was detected in tumor specimens from patients with multiple myeloma and its expression correlated with higher frequencies of Ki-67(+) malignant cells. In this report, we examine the mechanistic role of MAGE-A in promoting survival of proliferating multiple myeloma cells. The impact of MAGE-A3 expression on survival and proliferation in vivo was examined by immunohistochemical analysis in an independent set of tumor specimens segregated into two groups: newly diagnosed, untreated patients and patients who had relapsed after chemotherapy. The mechanisms of MAGE-A3 activity were investigated in vitro by silencing its expression by short hairpin RNA interference in myeloma cell lines and primary cells and assessing the resultant effects on proliferation and apoptosis. MAGE-A3 was detected in a significantly higher percentage of relapsed patients compared with newly diagnosed, establishing a novel correlation with progression of disease. Silencing of MAGE-A showed that it was dispensable for cell cycling, but was required for survival of proliferating myeloma cells. Loss of MAGE-A led to apoptosis mediated by p53-dependent activation of proapoptotic Bax expression and by reduction of survivin expression through both p53-dependent and -independent mechanisms. These data support a role for MAGE-A in the pathogenesis and progression of multiple myeloma by inhibiting apoptosis in proliferating myeloma cells through two novel mechanisms.

MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells to facilitate cytotoxic T lymphocyte-mediated tumor cell killing.

Approximately half of patients with stage IV neuroblastoma are expected to relapse despite current therapy, and when this occurs, there is little likelihood of achieving a cure. Very few clinical trials have been conducted to determine whether cellular immune responses could be harnessed to fight this tumor, largely because potential tumor antigens for cytotoxic T lymphocytes (CTL) are limited. MAGE-A1, MAGE-A3, and NY-ESO-1 are cancer-testis (CT) antigens expressed on a number of malignant solid tumors, including neuroblastoma, but many tumor cell lines down-regulate the expression of CT antigens as well as major histocompatibility (MHC) antigens, precluding recognition by antigen-specific T cells. If expression of cancer antigens on neuroblastoma could be enhanced pharmacologically, CT antigen-specific immunotherapy could be considered for this tumor. We have demonstrated that the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells following exposure to pharmacologic levels of the demethylating agent 5-aza-2'-deoxycytidine (decitabine, DAC). Expression of NY-ESO-1, MAGE-A1, or MAGE-A3 was induced in 10/10 neuroblastoma cell lines after 5 days of exposure to DAC. Culture of neuroblastoma cell lines with IFN-γ was also associated with an increased expression of either MHC Class I or II by cytofluorometry, as reported by other groups. MAGE-A1, MAGE-A3, and NY-ESO-1-specific CTL were cultured from volunteer donors by stimulating peripheral blood mononuclear cells with dendritic cells pulsed with overlapping peptide mixes derived from full-length proteins, and these CTL preferentially lysed HLA partially matched, DAC-treated neuroblastoma and glioblastoma cell lines. These studies show that demethylating chemotherapy can be combined with IFN-γ to increase the expression of CT antigens and MHC molecules on neuroblastoma cells, and pre-treatment with these agents makes tumor cell lines more susceptible to CTL-mediated killing. These data provide a basis to consider the use of demethylating chemotherapy in neuroblastoma patients, in conjunction with immune therapies that facilitate the expansion of CT antigen-specific CTL.

PREDICT phase II: A trial to prospectively validate a gene signature associated with clinical benefit from MAGE-A3 antigen-specific cancer immunotherapeutic (ASCI) in patients with unresected metastatic cutaneous melanoma.

TPS231 Background: Active immunization towards the tumor-specific antigen MAGE-A3 is a potential new option under investigation to treat cancer. Clinical activity of the MAGE-A3 Antigen-Specific Cancer Immunotherapeutic (ASCI) has been observed in a Phase II trial (NCT00086866) in patients with unresected metastatic cutaneous melanoma. Current data showed that combination of MAGE-A3 recombinant protein with the AS15 immunostimulant induced a robust and long-lasting antibody and CD4 T-cell immune response as well as clinical responses. Retrospective gene expression profiling of the tumor taken prior immunization led to the characterization of a gene signature (GS) associated with clinical benefit (Louahed et al., EORTC-NCI-AACR 2009). Methods: The aim of this open-labeled, single-arm trial (PREDICT, NCT00942162) is to prospectively validate the GS as a predictive biomarker of the clinical outcome of the MAGE-A3 ASCI. Main eligibility criteria are patients with histologically proven measurable MAGE-A3+ metastatic cutaneous melanoma, (unresectable stage III or stage IV M1a melanoma - AJCC 2002), an ECOG performance status of 0-1. Patients will receive a maximum of 24 doses of MAGE-A3 ASCI (recMAGE-A3 + AS15) by i.m. injections over 48 months. The primary endpoint is 1-Year overall survival rate (OSR) in the GS+ population. Main secondary endpoints include 1-Year OSR in global and GS- populations but also the clinical response rate and other clinical activity indicators (PFS, median OS) in these 3 populations. The GS will be determined on the tumor collected for the MAGE-A3 screening. This trial is also an opportunity to validate the technology transfer to support large scale identification of the GS: from micro-array on fresh tissue (NCT00086866) to qRT-PCR on FFPE tumor samples (PREDICT). Data of Jan 10, 2011 showed that 280 patients were screened. Out of the 236 tumor samples valid for MAGE-A3 expression testing, 111 were found MAGE-A3+ with a MAGE-A3 expression rate of 47% and 82 patients were finally enrolled. The identification of the GS status is ongoing for the enrolled population.

DERMA phase III trial of MAGE-A3 antigen-specific cancer immunotherapeutic (ASCI) as adjuvant therapy in patients with MAGE-A3-positive resected stage III melanoma.

TPS232 Background: Clinical activity of the MAGE-A3 Antigen-Specific Cancer Immunotherapeutic (ASCI) was observed in a Phase II study in patients with metastatic melanoma (NCT00086866) and in a Phase II study in patients with completely resected non-small cell lung cancer (NSCLC) (NCT00290355). Based on these positive signals for the activity of MAGE-A3 ASCI, a Phase III trial is currently ongoing in patients with resectable regionally advanced melanoma (DERMA Phase III trial, NCT00796445). Methods: DERMA is a double-blind, placebo-controlled, 2:1 randomized trial of MAGE-A3 ASCI vs. Placebo. Eligible patients must have cutaneous melanoma expressing MAGE-A3 with histologically proven macroscopic nodal disease (AJCC Stage IIIB-C). Central randomization uses an internet-based algorithm accounting for the following factors: Stage of the disease (IIIB, IIIC and IIITx), TNM nodal category (N1, N2 and N3), primary category (Tx-0 or T1-2 or T3 or T4), extracapsular extension (Yes, No), study center, prior treatment with IFN or anti-CTLA-4 (Yes, No). Patients must have had complete surgical resection of all tumor. Disease-Free Survival (DFS) in the overall patient population is the primary endpoint, while overall survival, safety and health-related quality of life (EQ-5D) are secondary endpoints. A previously-defined gene signature associated with MAGE-A3 ASCI benefit is also being validated as a secondary endpoint of this trial (Louahed et al., EORTC-NCI-AACR 2009). The MAGE-A3 ASCI combines the tumor-specific antigen MAGE-A3, delivered as a recombinant protein, with the immunostimulant AS15. A maximum of 13 doses are administered by intramuscular injection over 27 months; the first 5 are given at 3-week intervals followed by 8 at 12-week intervals. This Phase III trial is designed to enroll 1300 patients from 250 centers worldwide. Since Dec 1, 2008, 2426 patients have been screened. From 1917 tumor samples valid for testing the expression of MAGE-A3, 1265 express MAGE-A3 (MAGE-A3 expression rate of 66%). 832 patients are currently enrolled to this study.

MAGRIT phase III trial: MAGE-A3 antigen-specific cancer immunotherapy (ASCI) as adjuvant therapy in patients with completely resected stage IB-IIIA NSCLC.

TPS210 Background: Active immunization with MAGE-A3 Antigen-Specific Cancer Immunotherapeutic (ASCI) showed clinical activity in a randomized Phase II study (NCT00290355) in patients with completely resected non-small cell lung cancer (NSCLC) and in a randomized Phase II study (NCT00086866) in metastatic melanoma. Methods: MAGRIT (NCT00480025) is a double-blind, placebo-controlled, randomized (2:1; MAGE-A3 ASCI vs. Placebo) Phase III trial in patients with pathological stage IB-IIIA NSCLC (AJCC TNM v6). Tumors of eligible patients have been resected by complete anatomic lobectomy/pneumonectomy and must be MAGE-A3+. Patients are allowed to receive up to 4 cycles of adjuvant platinum-based chemotherapy (CT), when clinically indicated, post-operatively and before study treatment. Adjuvant radiotherapy is not permitted. MAGE-A3 ASCI (recombinant MAGE-A3 protein and immunostimulant AS15) or placebo is administered intramuscularly for a total of 13 doses: 5 doses every 3 weeks followed by 8 doses every 12 weeks. Randomization of patients is performed using a central web-based system, stratified for chemotherapy (CT vs. no-CT) and accounting for the following minimization factors: number of CT cycles (1-4), pathological stage (IB vs. II vs. IIIA), type of lymph node sampling (minimal vs. systematic radical resection), ECOG performance status (0, 1 and 2), and smoking status (never-, ex-, or current smoker). Co-primary endpoints are disease-free survival (DFS) in the overall population and DFS in the population that has not received adjuvant CT prior to study treatment. Secondary endpoints include additional clinical efficacy indicators such as overall survival; safety health-related quality of life (EQ-5D), and validation of a predictive gene signature (GS) associated with clinical benefit of the MAGE-A3 ASCI. As of 21 Jan 2011, 9898 patients were screened, 1579 randomized (2270 targeted). 9057 valid tumor samples have been tested for MAGE-A3 expression, 33% were positive. At their last meeting (Sep 2010), The Independent Data Monitoring Committee suggested no safety issue and allowing the trial to continue as planned.

949 poster HIGH DOSE RATE BRACHYTHERAPY BOOST FOR RESIDUAL MALIGNANT GLIOMA: CLINICAL RESULTS FROM SINGLE INSTITUTION

sion, by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Findings: The overall rate of MAGE-A3 expression was 41% (37% in Taiwan and 48% in Thailand, from 132 valid samples). MAGEA3 expression was lower in patients with HBV infection (33.3%) than in HCV-infected patients (63.0%). No difference in MAGEA3 expression was noted for the following factors: age, gender, liver cirrhosis, Child-Pugh class, chronic alcohol abuse, number and size of (largest) tumours. No clinical effects on survival were associated with MAGE-A3 expression in this HCC Asian population. The hazard ratio (HR) for the disease-free interval for MAGE-A3-positive versus MAGE-A3-negative patients was 1.06 (p = 0.82; HR adjusted for T stage, tumour number and size, cirrhosis, Child-Pugh score, and ECOG performance status was 1.40 [p = 0.29]). HRs for disease-free and overall survival were 1.03 (p = 0.91) and 0.97 (p = 0.94), respectively; adjusted HRs were 1.32 (p = 0.37) and 1.33 (p = 0.63). However, because of the small number of patients, no subset analysis by stage or other variables that affect disease-free and overall survival could be done. Interpretation: MAGE-A3 can be assessed by RT-PCR on surgically resected HCC. The overall expression rate is sufficient to consider MAGE-A3 a target for active immunotherapy. The relatively higher expression in HCV-infected patients has no explanation, so far. Clinical evaluation of MAGE-A3 antigen-specific cancer immunotherapeutics in early HCC after resection is being discussed. Funding: GlaxoSmithKline Biologicals. D.D., J.L., A.M., and P.T. are employed by GlaxoSmithKline.

Epigenetic modulation of MAGE-A3 antigen expression in multiple myeloma following treatment with the demethylation agent 5-azacitidine and the histone deacetlyase inhibitor MGCD0103

Immunotherapy targeting MAGE-A3 in multiple myeloma (MM) could eradicate highly aggressive and proliferative clonal cell populations responsible for relapse. However, expression of many cancer-testis antigens, including MAGE-A3, can be heterogeneous, leading to the potential for tumor escape despite MAGE-A3-induced immunity. We hypothesized that a combination of the hypomethylating agent 5-azacitidine (5AC) and the histone deacetylase inhibitor (HDACi) MGCD0103 (MGC) could induce MAGE-A3 expression in MAGE-A3-negative MM, resulting in recognition and killing of MM cells by MAGE-A3-specific cytotoxic T lymphocytes (CTL). Gene expression analyses of MAGE-A3 expression in primary MM patient samples at diagnosis and relapse were completed to identify populations that would benefit from MAGE-A3 immunotherapy. MM cell lines were treated with 5AC and MGC. Real-time polymerase chain reaction (PCR) and Western blotting were performed to assess MAGE-A3 RNA and protein levels, respectively. Chromium-release assays and interferon (IFN) secretion assays were employed to ascertain MAGE-A3 CTL specificity against treated targets. Gene expression analysis revealed that MAGE-A3 is expressed in MM patients at diagnosis (25%) and at relapse (49%). We observed de novo expression of MAGE-A3 RNA and protein in MAGE-A3-negative cell lines treated with 5AC. MGC treatment alone did not induce expression but sequential 5AC/MGC treatment led to enhanced expression and augmented recognition by MAGE-A3-specific CTL, as assessed by (51)Cr-release assays (P = 0.047) and enzyme-linked immunosorbent assay (ELISA) for IFN-γ secretion (P = 0.004). MAGE-A3 is an attractive target for immunotherapy of MM and epigenetic modulation by 5AC, and MGC can induce MAGE-A3 expression and facilitate killing by MAGE-A3-specific CTL.

Abstract 5001: BORIS and MageA expression correlate positively in melanoma

Abstract Cancer Testis Antigens (CTAs) are proteins that are normally expressed only in the male germ cells. These proteins are not expressed in other normal somatic tissues but are aberrantly upregulated in a variety of cancers like lung and melanoma. The expression of CTA genes is known to be regulated by promoter methylation. Using an integrative epigenetic screening approach, we have previously shown that CTAs are upregulated in non-small cell lung cancer and head and neck squamous cell carcinoma by promoter hypomethylation. Although it is now well accepted that CTAs are regulated by promoter methylation, the regulatory mechanisms of these genes remains unclear. Transcription factor BORIS (Brother Of the Regulator of Imprinted Sites) or CTCFL has been implicated in the regulation of two CTAs, mageA1 and NY-ESO-1. In the present study, we analyzed the expression of BORIS in a cohort of 16 melanoma tissues using quantitative reverse-transcription PCR. BORIS expression was detected in ∼70% of the tissues analyzed. Taking into account the evidence that BORIS regulates the expression of at least two CTAs, we also analyzed the expression of four CTAs, mageA1, mageA2, mageA3 and mageA4, in these tissues to assess the correlation between BORIS and CTA gene expression. We found that expression of mageA1, A2 and A3 correlated significantly with that of BORIS. We also analyzed two publicly available melanoma gene expression datasets and found that BORIS expression correlated with all four mageA genes analyzed. In our cohort as well as in the published gene expression datasets, the expression of the mageA genes tightly correlate with each other. Analysis of the methylation status of the mageA genes in our cohort showed that the methylation levels of mageA1, A2 and A3 genes also correlated with each other significantly. These observations support the phenomenon of coordinated CTA expression in solid tumors. We also found inverse correlation between the methylation and expression levels of these genes in some of the tissues analyzed. These results indicate that coordinated transcriptional regulation of the mageA genes may occur via promoter methylation dependent as well as independent mechanisms. Given the role of BORIS in the derepression of mageA1 gene via promoter demethylation, we also evaluated the correlation between BORIS expression and CTA promoter demethylation. We found that BORIS expression and mageA gene demethylation correlated in a subset of the tissues analyzed. The association between expression of BORIS and the mageA genes supports a role for BORIS as an effector in the upregulation of these genes. Also, the coordinated expression of the mageA genes suggests a common transcriptional mechanism for their regulation and our study presents evidence that implicates BORIS as a common regulator of the CTAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5001. doi:10.1158/1538-7445.AM2011-5001

Abstract 4150: CK-19, MAGE-A3, HER-2 and TWIST-1 expression by RT-qPCR in CTCs of primary breast cancer patients after positive immune-magnetic selection using anti-EpCAM and anti-MUC1 magnetic beads

Abstract Background: Circulating tumor cells (CTCs) are suggested as potential surrogate markers for minimal residual disease, the precursor of metastatic disease. Furthermore, to be able to disseminate and metastasize, CTCs must be able to perform epithelial-mesenchymal transition (EMT). The aim of our study was to evaluate the sensitivity of RT-qPCR for the detection and characterization of CTCs analyzing the expression of CK-19, MAGE-A3, HER-2 and TWIST-1 as a transcription factor for EMT in blood of early breast cancer patients after positive immunomagnetic selection using a combination of anti-EpCAM and anti-MUC1 magnetic beads. Patients and Methods: Two × 5 ml blood samples were collected before surgery and CTCs were isolated by positive immunomagnetic selection using anti-EpCAM and anti-MUC1 coated magnetic beads (AdnaTest Breast Cancer Select, AdnaGen AG, Langenhagen, Germany). CK-19, MAGE-A3, HER-2 and PBGD transcripts were simultaneously quantified by multiplex RT-qPCR while TWIST-1 transcript by single RT-qPCR in the LightCycler 2.0 (Roche Diagnostics). All primers and probes were designed to match the assay conditions, such as amplicon sizes, and melting temperatures. Before proceeding to patients’ samples, the RT-qPCR assay was evaluated in respect to sensitivity, specificity, and reproducibilty. To ensure that amplifiable material was present in all specimens and to avoid false-negative results, real-time amplification of the reference gene porphobilinogen deaminase (PBGD) was performed for all samples. A total number of 436 samples were first evaluated for RNA quality by PBGD expression and finally 293 samples that were of good RNA quality were further processed. Results: CK-19 was detected in 117/293 (39.9%), HER-2 in 35/293 (12.0%), MAGE-A3 in 25/293 (8.5%), and TWIST-1 in 16/293 (5.5 %) of all samples, respectively. One gene was detected in 135/293 (46.1%) of the patients, two genes in 23/293 (7.8%) of the patients, three genes in 3/293 (1.0%) patients and four genes in 0/293 (0 %) of the patients, respectively. In total, 161/293 (54.9%) patients were found positive for at least one gene expression, while in 132/293 (45.05%) patients we didn't detect CTCs at all. Discussion: Our study has revealed a high sensitivity when using a combination of immunomagnetic separation and RT-qPCR, as well as a remarkable heterogeneity of gene expression for these genes between early breast cancer patients. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4150. doi:10.1158/1538-7445.AM2011-4150

Expression of MAGE-A and NY-ESO-1 cancer/testis antigens in medullary breast cancer: a retrospective immunohistochemical study

AimTo immunohistochemically evaluate the expression of MAGE-A1, MAGE-A, and NY-ESO-1 cancer/testis (C/T) tumor antigens in medullary breast cancer (MBC) tumor samples and to analyze it in relation to the clinicopathological features.MethodsThis retrospective study included samples from 49 patients: 40 with typical MBC and 9 with atypical MBC. Tumor specimens were obtained from patients operated on in the University Hospital for Tumors and the Sisters of Mercy University Hospital, Zagreb, Croatia, from 1999 to 2005. Standard immunohistochemistry was used on archival paraffin-embedded MBC tissues.ResultsMAGE-A1, MAGE-A, and NY-ESO-1 antigens were expressed in 33% (16/49), 33% (16/49), and 22% (11/49) of patients, respectively. No difference between the groups with and without C/T tumor antigen expression in age at diagnosis, tumor size, axillary lymph node metastasis, adjuvant therapy, and HER-2 expression was identified. Significantly more patients died in the MAGE-A-positive group than in the MAGE-A-negative group (P = 0.010), whereas a borderline significance was found between MAGE-A1-positive and the MAGE-A1-negative group (P = 0.079) and between NY-ESO-1-positive and NY-ESO-1-negative group (P = 0.117). Overall survival, as evaluated by the Kaplan-Meier curves, was lower in MAGE-A1- (P = 0.031), MAGE-A- (P = 0.004), NY-ESO-1-positive groups (P = 0.077).ConclusionExpression of C/T antigens may represent a marker of potential prognostic relevance in MBC.

Decitabine and Interferon-γ enhance neuroblastoma cells susceptibility to CTL-mediated killing (156.26)

Abstract MAGE-A1, MAGE-A3, and NY-ESO-1 are cancer-testis (CT) antigens expressed on a number of malignant solid tumors, including neuroblastoma, but many tumor cell lines down-regulate the expression of CT antigens as well as major histocompatibility (MHC) antigens, precluding recognition by antigen specific T cells. If expression of cancer antigens as well as MHC molecules could be enhanced pharmacologically, the efficacy of CT antigen specific immunotherapy could be improved. We have demonstrated that the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells following exposure to the demethylating agent 5-aza-2’-deoxycytidine (decitabine, DAC) in 10/10 neuroblastoma cell lines. Culture of neuroblastoma cell lines with IFN-γ was associated with an increased expression of either MHC Class I or II by cytofluorometry, with MHC Class I upregulated in 10/10 of these cell lines, although to varying degrees. MAGE-A1, MAGE-A3, and NY-ESO-1 specific CTL were cultured from volunteer donors by stimulating peripheral blood mononuclear cells with dendritic cells pulsed with overlapping peptide mixes derived from full length proteins, and these CTL lysed HLA partially matched, DAC treated neuroblastoma and glioblastoma cell lines. These studies confirm that DAC and IFN-γ can be used to increase the expression of CT antigens and MHC molecules on neuroblastoma cells, and pre-treatment with these agents makes tumor cell lines more susceptible to CTL mediated killing.

MAGE-A3/4 and NY-ESO-1 antigens expression in metastatic esophageal squamous cell carcinoma

In the present study we analyzed immunohistochemical expression of MAGE-A 3/4 and NY-ESO-1 in 55 samples of esophageal squamous cell carcinomas (ESCC) and their respective lymph node metastases. To our knowledge this is the first study to assess and compare the expression of these antigens in ESCC lymph node metastases.Fifty (90.9%) primary ESCC were positive for MAGE-A 3/4 and 53 (96.6%) were positive for NY-ESO-1. MAGE-A 3/4 was expressed in all lymph node metastases and the intensity of expression was high in a majority of cases. NY-ESO-1 was negative in 2 (7.1%) lymph nodes metastases, while the reaction was predominantly moderate in the positive group. In primary tumors MAGE-A 3/4 showed a significantly higher intensity of expression compared to NY-ESO-1 (P=0.047), while in lymph node metastases the intensity of expression was not significantly different (P=0.387). Primary tumors with and without lymph node metastases showed no significant differences in MAGE-A 3/4 (P=0.672) and NY-ESO-1 (P=0.444) expression. Intensity of MAGE-A 3/4 (P=0.461) and NY-ESO-1 (P=0.414) expression in primary tumors was not significantly different compared to the expression in their respective lymph nodes metastases. Expression of MAGE-A 3/4 in primary tumors showed significant positive correlation with primary tumor expression of NY-ESO-1 (P=0.021) but no significant correlation with the expression of MAGE-A 3/4 in lymph node metastases (P=0.056). Expression of NY-ESO-1 in primary tumors showed significant positive correlation with the expression of NY-ESO-1 in lymph node metastases (P=0.001) and significant negative correlation with patients' age (P<0.001). Expression of MAGE-A 3/4 and NY-ESO-1 in primary tumors and lymph node metastases showed no significant correlation with prognostic parameters such as tumor grade and TNM stage (P>0.05). We have shown different levels of MAGE-A 3/4 and NY-ESO-1 expression in almost all specimens of primary tumor and lymph node metastases, suggesting that ESCC may be possible target of immunotherapy and anti-tumor vaccination. High levels of expression in lymph node metastases indicate possible clinical benefit of postoperative vaccine with MAGE-A3 and NY-ESO-1 in advanced stage of disease.