MAGE-A3 Research Articles

Validation of autoantibody panel for early detection of lung cancer in Chinese population.

e22143 Background: Autoantibodies is an attractive diagnostic approach for early detection of malignant tumors. Our previous studies found a panel of 7 TAAs(p53, GAGE7, PGP9.5, CAGE, MAGE A1, SOX2,...

Development of a Quantitative Real-Time RT-PCR Assay for the Detection of MAGE-A3–Positive Tumors

Melanoma antigen A3 (MAGE-A3) is a member of the MAGE family of tumor antigens and a relevant candidate for use in cancer immunotherapy. However, not all tumors express MAGE-A3, and closely related members of the MAGE family can be co-expressed with MAGE-A3 in the same tumor. Therefore, in the frame of MAGE-A3 clinical trials, it appeared necessary to evaluate tumors for MAGE-A3 expression with a highly specific quantitative assay to select patients who are eligible for anti-MAGE-A3 immunotherapy treatment. Herein, we describe the development and validation of a quantitative real-time RT-PCR (RT-qPCR) assay for the determination of MAGEA3 gene expression in tumor tissues. In the early phases of development, the designed primers and probe were not able to distinguish between MAGE-A3 and MAGE-A6. To ensure the specificity for MAGE-A3 over MAGE-A6, our strategy was to use a 5'-nuclease probe (or hydrolysis probe). The final assay was shown to be specific and linear within the analytical range, with an acceptable CV for repeatability and intermediate precision. When compared with a reference semiquantitative RT-PCR assay, the two methods were in good agreement, with only 4.23% of the samples giving discordant results. In conclusion, we have developed a MAGE-A3-specific RT-qPCR assay, compatible with a high-throughput setting for the estimation of MAGEA3 gene expression in present and future clinical trials.

Expression of cancer-testis antigens of Mage-a and Mage-b families in mouse embryonic fibroblasts cultured in vitro

Cancer-testis antigens are expressed in the spermatogenic and cancer cells as well as in human and mouse pluripotent stem cells. However, the role of cancer-testis antigens of Mage families in the regulation of cellular processes in embryonic cells is largely unknown. In the present study comparative quantitative analysis of the gene expression of Magea and Mageb families was performed in mouse embryonic somatic cells (mouse embryonic fibroblasts, MEFs), long-term cultured in vitro and exposed to factors that inhibit and stimulate proliferation. The analysis revealed low expression of cancer-testis antigens of Mage families and showed that a lower proliferative activity of MEF at late passages was accompanied by slight up-regulation of the Magea gene expression and down-regutation of Mageb gene expression. However, modulation of the activity of MEK/ERK-signaling pathway and DNA demethylation by 5-azacytidine had no significant effects on the expression of Magea and Mageb genes in M EFs. The most essential changes in the expression levels of Mageb and Magea genes were found only when MEFs were exposed to mitomycin C. In all experimental variants, predominantly cytoplasmic localization of Mage antigens was found in MEFs at the phase of DNA synthesis, as well as at other cell cycle phases. Presumably, in actively proliferating mouse embryonic somatic cells the antigens of Magea and Mageb families can act as co-activators in the regulation of cell proliferation and other cellular functions.

Overexpression of MAGE-A9 predicts unfavorable outcome in breast cancer

Melanoma-associated antigens (MAGEs) are a group of well-characterized members of the cancer/testis antigen (CTA) family, which is one of the largest groups of human tumor-associated antigens. MAGE-A9 is a particular member in the context of the MAGE-A gene family and was defined as presenting prognostic relevance in certain type of human cancer. However, the expression of MAGE-A9 in invasive ductal breast cancer (IDC) and the relationship with the clinical attributes of IDC, especially prognostic characteristic, remain poorly understood. In this present study, one-step quantitative reverse transcription polymerase chain reaction (18 fresh-frozen IDC tissues and corresponding non-cancerous tissues) and immunohistochemistry by tissue microarrays (82 IDC tissue samples and 70 matched tumor-adjacent non-cancerous tissue samples) were performed to characterize expression of the MAGE-A9 gene in IDC. Kaplan–Meier survival and Cox regression analyses were performed to evaluate the prognosis of IDC. The results of qPCR and IHC analysis showed that the expression of MAGE-A9 in IDC was significantly higher than that in corresponding non-cancerous tissue. Moreover, the expression level of MAGE-A9 protein in IDC was significantly related to histological grade (p=0.011) and distant metastasis (p=0.019). Multivariate analysis with the Cox regression model showed that MAGE-A9 protein expression (p=0.009), histological grade (p=0.014), lymph node metastasis (p=0.012) and distant metastasis (p=0.011) were independent prognostic factors for overall survival of IDC patients. The data suggest that MAGE-A9 expression is correlated with malignant attributes of IDC and it may serve as a novel prognostic factor and an ideal candidate for targeted therapy in IDC treatment.

Differential regulation of MAGE-A1 promoter activity by BORIS and Sp1, both interacting with the TATA binding protein

BackgroundAs cancer-testis MAGE-A antigens are targets for tumor immunotherapy, it is important to study the regulation of their expression in cancers. This regulation appears to be rather complex and at the moment controversial. Although it is generally accepted that MAGE-A expression is controlled by epigenetics, the exact mechanisms of that control remain poorly understood.MethodsWe analyzed the interplay of another cancer-testis gene, BORIS, and the transcription factors Ets-1 and Sp1 in the regulation of MAGE-A1 gene expression performing luciferase assays, quantitative real-time PCR, sodium bisulfite sequencing, chromatin immunoprecipitation assays and pull down experiments.ResultsWe detected that ectopically expressed BORIS could activate and demethylate both endogenous and methylated reporter MAGE-A1 promoter in MCF-7 and micrometastatic BCM1 cancer cell lines. Overexpression of Ets-1 could not further upregulate the promoter activity mediated by BORIS. Surprisingly, in co-transfection experiments we observed that Sp1 partly repressed the BORIS-mediated stimulation, while addition of Ets-1 expression plasmid abrogated the Sp1 mediated repression of MAGE-A1 promoter. Both BORIS and Sp1 interacted with the TATA binding protein (hTBP) suggesting the possibility of a competitive mechanism of action between BORIS and Sp1.ConclusionsOur findings show that BORIS and Sp1 have opposite effects on the regulation of MAGE-A1 gene expression. This differential regulation may be explained by direct protein-protein interaction of both factors or by interaction of MAGE-A1 promoter with BORIS alternatively spliced isoforms with different sequence specificity. We also show here that ectopic expression of BORIS can activate transcription from its own locus, inducing all its splice variants.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-796) contains supplementary material, which is available to authorized users.

O6.06 * PERIPHERAL T CELL RESPONSES IN GLIOBLASTOMA PATIENTS ARE ASSOCIATED WITH AN IMPROVED SURVIVAL

BACKGROUND: Spontaneous anti-tumor T cell responses have been detected in several tumor types. However, not only in glioblastoma it is unclear if such immune responses influence patient outcome. MATERIAL AND METHODS: To detect such immune responses, occurence and frequencies of memory T-cells were determined in the peripheral blood of GBM patients applying the IFN gamma ELISPOT assay and using corresponding tumor lysates (n = 106) or a set of immunogenic polypeptides (n = 32). Extent of resection was assessed by either intraoperative or early postoperative MRI. In addition, intratumoral infiltration of different T-cell subpopulations at the time of first diagnosis was analyzed by immunohistochemistry (n = 20) and compared to the occurrence of spontaneous immune responses. RESULTS: T-cell responses against autologous tumor lysate were detected in 61/106 patients. Subsequent survival analysis concentrating on IDH1wt glioblastoma revealed a significant difference towards an improved survival for patients with a spontaneous immune response (p = 0.0251). Most interestingly, when stratifying for the extent of surgery those patients, who received a complete tumor resection and showed a spontaneous T cell response, survived significantly longer (p = 0.0002). In contrast, in subtotally resected patients the immune response did not improve survival. In line with these observations, occurrence of memory T cell responses against a set of known GBM-associated antigens (TTR, MAGE A3, S100A9) also trended to a better survival in those GBM patients who responded to all three antigens. Finally, increased frequency of intratumoral T cell infiltrates was also associated with the occurrence of spontaneous immune responses and an improved survival. CONCLUSIONS: The present analysis for the first time depicts on several levels a significant influence of the patient's immune system on the course of the disease in GBM patients. In this regard the most crucial prerequisite seems to be the MRI-based complete tumor resection. Most likely surgical removal of the immunosuppressive tumor environment allows for the beneficial reactivation of pre-existing immune responses.

Enhancing lung cancer diagnosis: electrochemical simultaneous bianalyte immunosensing using carbon nanotubes-chitosan nanocomposite.

A label-free electrochemical bianalyte immunosensor has been designed for simultaneous detection of lung cancer biomarkers (anti-MAGE A2 and anti-MAGE A11) using carbon nanotubes-chitosan (CNT-CHI) composite. To achieve this, acid-functionalized single-walled CNTs were used to prepare CNT-CHI gel and electrodes were fabricated by drop casting method onto graphite surface. Lung cancer biomarkers specific antigens (Ag), i.e., MAGE A2 and MAGE A11, were covalently immobilized onto CNT-CHI/graphite electrode separately for fabrication process. Fabricated immunoelectrodes (MAGE A2/CNT-CHI/graphite and MAGE A11/CNT-CHI/graphite) were characterized at each modification step by cyclic voltammetry (CV), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Both immunoelectrodes showed successful detection of respective analytes (anti-MAGE A2 and anti-MAGE A11) from 5fgmL(-1) to 50ngmL(-1) using differential pulse voltammetry (DPV). Both Ag/CNT-CHI/graphite immunoelectrodes (using MAGE A2 and MAGE A11) were independently capable of distinguishing specific and nonspecific analytes like CD59, D-dimers, etc. Response studies of both immunoelectrodes revealed successful demonstration of simultaneous detection of anti-MAGE A2 and A11 independently in a single experimental run when exposed to a mixture of various analyte concentrations in different combinations irrespective of the presence of other analyte present in the same vessel.

SPONTANEOUS IMMUNE RESPONSES IN GBM PATIENTS AFTER COMPLETE TUMOR RESECTION ARE ASSOCIATED WITH AN IMPROVED SURVIVAL

BACKGROUND: Spontaneous anti-tumor immune responses have been detected in several tumor types. However, not only in glioblastoma it is unclear if such immune responses influence patient outcome. METHODS: To detect such immune responses, occurence and frequencies of memory T-cells were determined in the peripheral blood of GBM patients applying the IFN gamma ELISPOT assay and using corresponding tumor lysates (n = 106) or a set of immunogenic polypeptides (n = 32). Extent of resection was assessed by either intraoperative or early postoperative MRI. In addition, intratumoral infiltration of different T-cell subpopulations at the time of first diagnosis was analyzed by immunohistochemistry (n = 20) and compared to the occurrence of spontaneous immune responses. RESULTS: Altogether, T-cell responses against autologous tumor lysate were detected in 61/106 patients. Subsequent survival analysis concentrating on IDH1wt glioblastoma revealed a significant difference towards an improved survival for patients with a spontaneous immune response (p = 0.0251). Most interestingly, when stratifying for the extent of surgery those patients, who got a complete tumor resection and showed a spontaneous T cell response, survived significantly longer (p = 0.0002). In contrast, in subtotally resected patients the immune response did not improve survival. In line with these observations, occurrence of memory T cell responses against a set of known GBM-associated antigens (TTR, MAGE A3, S100A9) also trended to a better survival in those GBM patients who responded to all three antigens (p = 0.0663). Finally, increased frequency of intratumoral T cell infiltrates was also associated with the occurrence of spontaneous immune responses and an improved survival (p = 0.0113). CONCLUSIONS: The present analysis for the first time depicts on several levels a significant influence of the patient's immune system on the course of the disease in GBM patients. In this regard the most crucial prerequisite seems to be the MRI-based complete tumor resection. Most likely surgical removal of the immunosuppressive tumor environment allows for the beneficial reactivation of pre-existing immune responses. SECONDARY CATEGORY: Tumor Biology.

Immune alterations in lung cancer - the new therapeutic approach

Lung cancer is the main cause of cancer death worldwide. An advanced stage of the disease at the time of diagnosis, observed in the majority of cases, does not allow for introduction of radical treatment or makes the treatment ineffective. Lung cancer as a solid tumour with a very low antigenicity escapes immune surveillance, and cytotoxic cells attack. Cytotoxic lymphocytes play a key role in anticancer defence, but the population of these cells individually differs. Many suppressor and regulatory mechanisms inhibit the recognition of tumour antigens by dendritic and cytotoxic cell activation. The population of regulatory T cells (T regs) plays a crucial role in this inhibition of immune response. Their function depends on the expression of transcription factor Foxp3 and the presence of CTLA-4 molecules. The increased proportion of T regs and high expression of Foxp3 and CTLA-4 on lung cancer cells and tumour infiltrating lymphocytes were observed. Other components of immune response inhibition and tumour promotion are: Th17 cell population, M2 macrophage polarisation, the presence of myeloid derived suppressor cells (MDSCs) and a significantly elevated concentration of cytokines: TGF-b and IL-10 in the tumour microenvironment. The recognition of these mechanisms may have important therapeutic implications. Several types of agents which are capable of modulating the immune response have recently been used in many clinical trials conducted in lung cancer patients, some of them showing efficacy. Lung cancer immunotherapy has two main directions: the first goal is to improve the cytotoxic effect (for example by inhibition of CTLA-4, stimulation of dendritic cell function, inhibition of lymphocyte apoptosis), and the second way is the production of anti-cancer vaccines using known cancer antigens: MAGE A3, MUC1, EGF and TGF-b. Immunotherapy in lung cancer treatment has a character of personalised therapy - there is a need to specify the patient's immune status prior to treatment. The analysis of immune cells and mediators in the peripheral blood allows this, but the more valuable method seems to be bronchoalveolar lavage (BAL) examination with careful assessment of the tumour microenvironment.

Effects of over-expression of melanoma antigen gene A3 on human hepatocellular carcinoma cell lines

Objective To observe effects of over-expression of melanoma antigen gene (MAGE) A3 on the progression,apoptosis,metastasis and invasion in human hepatocellular carcinoma cell lines,and investigate its effect on hepatocellular carcinoma (HCC) progession.Methods The lentivirus vector with recombinant MAGE-A3 gene (LV-MAGE-A3) were constructed and transfected into HepG2 and MHCC-97H cell lines and cell only or transfected with empty vector were as controls.The expression of green fluorescent protein was observed by inverted fluorescent microscope to detect tranduced efficiency.MAGE-A3 gene and protein expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR) and Western-Blot technology.The effects of MAGE-A3 over-expression on proliferation,migration and invasion of HCC were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays,clone formation assays and transwell technique.Cell apoptosis was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and cell cycle was analysed by flow cytometry (FCM).Results After lentivirus vector transfection for 120 hours,the expression of green fluorescent protein was the strongest and most stable in cells.The transfection efficiency was up to 80％.After transfected with LV-MAGE-A3,the expression of MAGE-A3 gene and the protein in HepG2 and MHCC-97 cells was higher than that of controls.The cell numbers of migration and invasion were 187.00 ± 13.52,121.00 ±4.16 in HepG2 and 164.00 ±9.64,59.50 ± 1.00 in MHCC-97,which significantly increased compared to control group (P ＜0.01).The cell numbers of S stage increased by FCM.However,the cell proliferation,colony-forming ability and apoptosis of HCC cell lines had no statistical significance between LV-MAGE-A3 group and control (P ＞ 0.05).Conclusion MAGE-A3 gene promote the abilities of migration and invasion in HCC cell lines.It suggests that MAGE-A3 expression is closely corelated to HCC progression. Key words: Melanoma antigen gene ; Hepatocelluar carcinoma; Migration; Invasion

CD8+ TIL recruitment may revert the association of MAGE A3 with aggressive features in thyroid tumors.

Background. We aimed to investigate a possible role of MAGE A3 and its associations with infiltrated immune cells in thyroid malignancy, analyzing their utility as a diagnostic and prognostic marker. Materials and Methods. We studied 195 malignant tissues: 154 PTCs and 41 FTCs; 102 benign tissues: 51 follicular adenomas and 51 goiter and 17 normal thyroid tissues. MAGE A3 and immune cell markers (CD4 and CD8) were evaluated using immunohistochemistry and compared with clinical pathological features. Results. The semiquantitative analysis and ACIS III analysis showed similar results. MAGE A3 was expressed in more malignant than in benign lesions (P < 0.0001), also helping to discriminate follicular-patterned lesions. It was also higher in tumors in which there was extrathyroidal invasion (P = 0.0206) and in patients with stage II disease (P = 0.0107). MAGE A3+ tumors were more likely to present CD8+ TIL (P = 0.0346), and these tumors were associated with less aggressive features, that is, extrathyroidal invasion and small size. There was a trend of MAGE A3+ CD8+ tumors to evolve free of disease. Conclusion. We demonstrated that MAGE A3 and CD8+ TIL infiltration may play an important role in malignant thyroid nodules, presenting an interesting perspective for new researches on DTC immunotherapy.

Generation Of Anti-Tumor CD4+ T Helper Cells Using Genetically-Engineered Dendritic Cells Expressing Leukemia-Associated Antigens

Adoptive transfer of antigen-specific T cells is a potentially curative strategy for patients with solid tumors and leukemia. Most clinical trials of adoptive T cell therapy have used cytotoxic CD8+ T cells recognizing MHC class I-restricted tumor antigens. Despite overwhelming evidence suggesting the fundamental influence of CD4+ T cells on the immune system, clinical experience with tumor-specific CD4+ Th cells is almost non-existent. Unlike most other tissues, bone marrow-derived cells constitutively express MHC class II and CD4+ T cells play crucial role in mediating the curative GVL effect after allogeneic SCT and donor lymphocyte infusion (DLI). Furthermore, experimental evidences suggest that MHC class II-restricted antigenic targets recognized by CD4+ T cells exist in both solid cancers and in hematological malignancies. Therefore adoptive immunotherapy using CD4+ T cells in the setting of leukemia might be especially relevant.The goal of this study is to establish a simplified non-individualized protocol of generating LAA-reactive CD4+ T cells from patients and normal donors for adoptive immunotherapy directed against common leukemia-associated antigens (LAA) expressed in acute myeloid leukemias (AML) and myelodysplastic syndrome (MDS). We isolated naïve and memory CD4+ T cells from 3 normal donors and stimulated with twice at weekly interval with autologous monocytes pulsed with libraries of overlapping 15-amino acid length peptides (pepmixes) derived from WT-1, MAGE A3 and A4, PRAME and SSX2 antigens. At the end of the experiment CD4+ T cells were evaluated for reactivity against each LAA by analyzing their ability to specifically release cytokines (IL-2, TNF-α, and IFNγ) using flow cytometry. LAA-specific cells were found in either naïve or memory-derived CD4+ T cells upon stimulation with relevant pepmixes in all donors tested. However specific cytokine production could not be demonstrated when the same T cells were exposed to LAA-transduced autologous targets (LCL and T cells), raising the possibility that the majority of pepmix-reactive cells recognized epitopes that were not naturally processed. Therefore, as an alternative strategy to induce LAA-specific cells capable of targeting only therapeutically-relevant epitopes, we used autologous dendritic cells (DCs) transduced with a lentiviral vector encoding MAGE A3 antigen. Autologous CD4+ T cells were stimulated with MAGE A3 or mock-transduced DCs at an interval of 7-10 days and tested for their antigen-specific cytokine secretion. At the end of the culture we observed that Th cells expanded in presence of MAGE A3-expressing DCs and contained a significant number of cells possessing specific reactive against MAGE A3 pepmix (Figure), but not to unrelated antigenic targets, suggesting induction of LAA-reactivity against naturally-processed MAGE A3 epitopes. [Display omitted] In summary, we demonstrate the feasibility of generating specific anti-tumor CD4+ T cells using autologous DCs engineered to express a full-length tumor antigen. This approach allows for selective expansion of polyclonal Th cells recognizing only naturally processed MHC class II-restricted epitopes. Therefore, this strategy circumvents the limitation inherent to usage of overlapping peptide libraries that might induce the expansion of high-avidity T cells specific to epitopes that are irrelevant to in vivo recognition of tumor targets. Furthermore, this approach does not rely on a particular pre-defined MHC class II restriction element, thus it is applicable to majority of donors or patients irrespective of their MHC haplotype. Disclosures:No relevant conflicts of interest to declare.

Potential role of 5-Aza-2′-deoxycytidine induced MAGE-A4 expression in immunotherapy for anaplastic thyroid cancer

Melanoma antigen gene family (MAGE)-A4, a member of the cancer testis antigen family, has been reported in various cancers including melanoma, bladder, head and neck, oral, and lung, and is a potential target for T-cell-receptor-based immunotherapy. Baseline expression levels of the MAGE-A4 gene in thyroid cancer cell lines have not been previously studied thoroughly. Human thyroid cancer cell lines (8505c, HTh7, BCPAP, and TPC-1) were treated with either 10 μmol/L 5'-azacytidine (Aza) or 10 μmol/L 5-AZA-2'deoxycytidine (DAC) and evaluated for various MAGEA gene expression. Later melanoma cell lines A375 and 8505c were treated with PLX4720 in combination with DAC and evaluated for MAGE-A4 expression. Only BCPAP cells expressed moderate levels of MAGE-A3 and MAGE-A6 at baseline. Treatment with DAC/Aza induced the expression of MAGE-A4 and MAGE-A1 in 8505c cells. PLX4720 treatment did not affect MAGE-A4 expression in 8505c cells, but increased its expression in A375 cells. In contrast, addition of PLX4720 to DAC-treated 8505c cells decreased the previously induced MAGE-A4 expression by DAC in these cells. A similar decrease in MAGE-A4 expression by DAC was also seen in 8505cBRAF(-/-) cells. Although DAC treatment resulted in demethylation of the MAGE-A4 promoter in 2 CpG sites, PLX addition to DAC did not affect the demethylation status. Demethylating agents increased the expression of MAGE genes in thyroid cancer cells. The effect of BRAFV600E inhibitors on MAGE-A4 expression suggest the role of downstream MEK/BRAF signaling in its expression apart from promoter demethylation being the sole requirement. Expression of MAGE-A4 may make immunotherapeutic intervention possible in selected patients with thyroid cancer.

Expression of cancer testis antigens and its correlation with clinicopathological parameters in hepatocellular carcinoma

Meeting abstracts Many therapies such as surgery, chemical or physical approaches have been used for treatment of HCC; however, the outcome is still poor. Cancer immunotherapy is considered to be one of the promising strategies in recent years. Expression of CTAs genes including MAGE A3, MAGE A4,

Signal stratification of autoantibody levels in serum samples and its application to the early detection of lung cancer.

Further signal stratification for the EarlyCDT®-Lung test should facilitate interpretation of the test, leading to more precise interventions for particular patients. Samples were measured for the presence of autoantibodies to seven tumor-associated antigens (TAAs) (p53, NY-ESO-1, CAGE, GBU4-5, SOX2, MAGE A4, and HuD). In addition to the current test cut-offs (determined using a previously reported Validation case-control sample set, set A; n=501), new high and low cut-offs were set in order to maximize the test's positive and negative predictive values (PPV and NPV, respectively). All three sets of cut-offs were applied to two confirmatory datasets: (I) the case-control set B (n=751), and (II) Population-derived set C (n=883), and all three datasets combined (n=2,135). For the Validation dataset, cancer/non-cancer positivity for current cut-offs was 41%/9% (PPV =0.109, 1 in 9). The high positive stratum improved this to 25%/2% (PPV =0.274, 1 in 4). The low negative stratum improved this to 8%/23% (NPV =0.990, 1 in 105). This provides a 25-fold difference in lung cancer probability between the highest and lowest groups. The test performs equally well in subjects who fulfilled the entry risk criteria for the National Lung Screening Trial (NLST) and subjects who did not meet the NLST criteria. The EarlyCDT®-Lung test has been converted to a four-stratum test by the addition of high and low sets of cut-offs: patients are thus stratified into four risk categories. This stratification will enable personalization of subsequent screening and treatment programs for high risk individuals or patients with lung nodules.

A combined approach facilitates the reliable detection of human spermatogonia in vitro

Does a combined approach allow for the unequivocal detection of human germ cells and particularly of spermatogonia in vitro? Based on our findings, we conclude that an approach comprising: (i) the detailed characterization of patients and tissue samples prior to the selection of biopsies, (ii) the use of unambiguous markers for the characterization of cultures and (iii) the use of biopsies lacking the germ cell population as a negative control is the prerequisite for the establishment of human germ cell cultures. The use of non-specific marker genes and the failure to assess the presence of testicular somatic cell types in germ cell cultures may have led to a misinterpretation of results and the erroneous description of germ cells in previous studies. Testicular biopsies were selected from a pool of 264 consecutively obtained biopsies. Based on the histological diagnosis, biopsies with distinct histological phenotypes were selected (n = 35) to analyze the expression of germ cell and somatic cell markers. For germ cell culture experiments, gonadotrophin levels and clinical data were used as selection criteria resulting in the following two groups: (i) biopsies with qualitatively intact spermatogenesis (n = 4) and (ii) biopsies from Klinefelter syndrome Klinefelter patients lacking the germ cell population (n = 3). Quantitative real-time PCR analyses were performed to evaluate the specificity of 18 selected germ cell and 3 somatic marker genes. Cell specificity of individual markers was subsequently validated using immunohistochemistry. Finally, testicular cell cultures were established and were analyzed after 10 days for the expression of germ cell- (UTF1, FGFR3, MAGE A4, DDX4) and somatic cell-specific markers (SMA, VIM, LHCGR) at the RNA and the protein levels. Interestingly, only 9 out of 18 marker genes reflected the presence of germ cells and cell specificity could be validated using immunohistochemistry. Furthermore, VIM, SMA and LHCGR were found to reflect the presence of testicular somatic cells at the RNA and the protein levels. Using this validated marker panel and biopsies lacking the germ cell population (n = 3) as a negative control, we demonstrated that germ cell cultures containing spermatogonia can be established from biopsies with normal spermatogenesis (n = 4) and that these cultures can be maintained for the period of 10 days. However, marker profiling has to be performed at regular time points as the composition of testicular cell types may continuously change under longer term culture conditions. There are significant differences regarding the spermatogonial stem cell (SSC) system and spermatogenesis between rodents and primates. It is therefore possible that marker genes that do not reflect the presence of spermatogonia in the human are specific for spermatogonia in other animal models. While some studies have reported that human SSCs can be maintained in vitro and show characteristics of pluripotency, the germ cell origin and the differentiation potential of these cells were subsequently called into question. This study provides critical insights into possible sources for the misinterpretation of results regarding the presence of germ cells in human testicular cell cultures and our findings can therefore help to avoid conflicting reports in the future. This project was supported by the Stem Cell Network North Rhine-Westphalia and the Innovative Medical Research of the University of Münster Medical School (Grant KO111014). In addition, it was funded by the DFG-Research Unit FOR 1041 Germ Cell Potential (GR 1547/11-1 and SCHL 394/11-2), the BMBF (01GN0809/10) and the IZKF (CRA 03/09). The authors declare that there is no conflict of interest. Not applicable.

Identification of a Titin-Derived HLA-A1–Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells

MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)-A*01-restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.

MAGEA10 Expression in Human Lung Cancer A549 Cells Influenced by Viili Polysaccharides

Objective To discuss the effects and mechanism of MAGEA10 expression in human lung cancer A549 cells influenced by Viili polysaccharides.Methods MTT was used to study the survival impact of non small cell lung cancer(NSCLC) A549 with viili polysaccharides in different concentration at 24 h,48 h and 72 h.qRT-PCR was used to detect the relative expression changes of MAGEA10 mRNA with Viili polysaccharides in different concentration of 10 mg/L,25 mg/L and 50 mg/L compared with those of the normal A549 cells.And Western blot was adopted to detect the antigen expression of MAGEA10 after stimulation of A549 with Viili polysaccharides in different concentration of 10 mg/L,25 mg/L and 50 mg/L.Through the expression of MAGEA10 mRNA and antigen,we discussed the effects and possible mechanism of MAGEA10 expression made by Viili polysaccharides.Results(1) Viili polysaccharides inhibited cell proliferation of A549 and its concentration was negatively related to cell survival within 0 mg/L-50 mg/L and the result was most remarkable at 48 h.(2) Compared with the blank group,the relative expression of MAGEA10 mRNA elevated at the concentration of 50 mg/L.(3) Compared with the blank group,the protein expression of MAGEA10 elevated,at the concentration of 50 mg/L.Conclusion Viili inhibited NSCLC A549 cell proliferation,but the increase of the gene transcription and translation levels of MAGEA10 in A549 cell needs further exploration for those two parts might be interdependent.This study aims to indicate that viili is possible to promote non specific immunity,and increase possibility of cancer cell recognition by CTLs,and possibly initiate apoptotic process of cancer cells if same epitope of MAGEA10 and corresponding TCR encounters.

Molecular biomarkers as predictive factors of pCR for early triple-negative breast cancer.

1041 Background: Early triple-negative breast cancer (TNBC) patients (p) without pathologic complete response (pCR) after neoadjuvant chemotherapy (NCT) have unsuccessful prognosis. Predictive factors for pCR are necessary in order to improve the treatment choice. The aims of the study are to determine the expression of different biomarkers (BM) in the initial biopsy (IB) of TNBC, to analyze the relationship between the BM expression and pCR, and to determine the expression changes of BM after NCT. Methods: We reviewed retrospectively the medical records of 49 TNBC p treated with NCT between 2001 and 2011 at two institutions. Expression of 14 BM in the IB and after NCT was independently analyzed by inmunohistochemistry by two pathology specialists. Staining intensity 0-1 + was considered as negative expression, and 2-3-4 + as positive. Ki 67&gt;13% was interpreted as positive. Results: Forty-nine p with a median age of 47 years (27-79) were evaluated. Twenty-seven p (55%) had grade 3. Tumor stages were T1(2%), T2(26%), T3(39%), and T4(33%). 38p (77%) were node positive. Five p (10%) received anthracyclines and 42p (86%) anthracyclines plus taxanes. Fourteen p (29%) presented pCR, 27p (55%) partial response, 4p (8%) stable disease, 2p (4%) progressive disease, and 2p (4%) were not evaluable. The BM expression in the IB was: CD44 (88%), CK 5/6 (27%), EGFR ( 0%), Ki 67 (73%), Wt-1 (10%), p-Akt (24%), HER2 (19%), NY-ESO-1 (11%), MAGE A1 (0%), HER3 (14%), BRCA1 (84%), PTEN (12%), IGFR1 (12%) and AR (14%). Diferentially expressed BM in IB for p with and without pCR, respectively, were p-Akt 0/8(0%) vs 5/13(38%) p=0.11, CK 5/6: 4/9 (44%) vs 2/15 (13%) p=0.15 and Ki 67: 7/7(100%) vs 10/17(59%) p=0.06. The Table shows the BM expression before and after NCT for p without pCR. Conclusions: Tumor samples of TNBC show high expression of CD44, ki67, and BRCA1. Most of BM has a decrease in expression after NCT. CK 5/6, Ki 67, and p-Akt could be predictive factor for pCR, although larger prospective studies are needed. [Table: see text]

Phase I/II clinical trials of dendritic cell-based immunotherapy in patients with the biochemical relapse of the prostate cancer.

e16002 Background: In order to have the highest impact on the control of tumor growth, cancer immunotherapy should be initiated as early in the disease course as possible. In prostate cancer, the effect of cancer immunotherapy at the minimal residual disease stage can be evaluated in patients with the biochemical relapse detected by ultrasensitive PSA measurements. We performed a Phase I/II clinical trial of dendritic cell based immunotherapy in patients with the biochemical relaps of the prostate cancer. Methods: Patients with the prostate cancer in the biochemical failure after radical prostatectomy or primary radiotherapy were enrolled in the study. Study medication consisted of 12 doses of 1x107 dendritic cells pulsed with killed prostate cancer cell line LNCap (DCVAC/PCa), injected s.c. in 4 weeks intervals. Primary goal of the study was to assess the safety. Secondary goals were PSA kinetics and presence of tumor antigen specific immune response in the peripheral blood. Study is registered as EudraCT 2009-017259-91. Results: Twenty patients were enrolled in the study and evaluated after receiving complete treatment cycle of DCVAC/PCa. Administration of DCVAC/PCa did not lead to any significant side effects. Continuous cancer immunotherapy by DCVAC/PCa significantly prolonged the PSA doubling time (PSA-DT) in all treated patients, on average there was a 3,43-fold increase of PSA-DT after the completion of the treatment. Seven of 20 patients had stable PSA levels during the treatment duration. Mean PSA-DT increased from 8,15 months before the initiation of cancer immunotherapy to 52,64 months at the completion of 12 doses, p&lt;0.0015. Sustained T cells responses against PSA, NY-ESO1, MAGE A1 and MAGE A3 were detected in the peripheral blood of enrolled patients. Conclusions: This study indicates that the continuous cancer immunotherapy with dendritic cells loaded with killed LNCAP cell line represents efficient treatment modality for prostate cancer patients with biochemical relapse. This study supports the use of immunotherapy early in the course of the disease, provided that relevant surrogate endpoints predictive of improved prognosis of early stage patients will be identified. Clinical trial information: 2009-017259-91.