The MAGE A1-A10 Expression associated with Histopathological Findings of Malignant or Non-Malignant Cells in Peripheral Lung Tumors.

OBJECTIVE: The objective was to evaluate the expression of melanoma antigen (MAGE) A from A1 to 10 (A1-10) and the individual MAGE A family in the peripheral lung tumors and to analyze its association with histopathological findings. METHODS: A cross-sectional study was conducted on 67 samples of peripheral lung tumor obtained by core biopsies from patients with clinical diagnoses such as lung and mediastinal tumors. The specimens were divided into two, one to perform histopathological diagnosis and the last for mRNA MAGE A examination. A Nested polymerase chain reaction (PCR) was performed using universal primer, MF10/MR10 and MF10/MR12. The collected data were analyzed by appropriate statistical techniques. RESULT: The histopathological finding showed 41 (61.2 %) of specimens as malignant cells and 26 (38.8 %) of specimens as non-malignant cells. MAGE A1-10 was expressed at 47 (70.1 %) and MAGE A1-6 was expressed at 25 (37.3 %) of specimens. In a malignant cell, MAGE A1-10 and MAGE A1-6 were expressed at 33 (80.5 %) and 19 (46.3 %), respectively. In non-malignant cells, MAGE A1-10 and MAGE A1-6 were expressed at 14 (53.9 %) and 6 (23.1 %,) respectively. The MAGE A1-10 and MAGE A8 expressions were significantly associated with histopathological findings of malignant or non-malignant cells. The sensitivity, specificity, and diagnostic accuracy of MAGE A1-10 were 80.5 %, 46.2 %, and 67.2 %, respectively; while for MAGE A8 were 41.5 %, 88.5 %, and 59.7 %, respectively. CONCLUSION: The MAGE A1-10 expression was the most commonly detected and associated with the histopathological finding. Moreover, it was more sensitive and specific and had higher diagnostic accuracy than others. Therefore, the MAGE A1-10 assay may improve the accuracy of the diagnosis of malignancy in peripheral lung tumors.

Reduced cytoplasmic expression of MAGE-A2 predicts tumor aggressiveness and survival: an immunohistochemical analysis.

BACKGROUND: Melanoma antigen gene A2 (MAGE-A2) is one of the most cancer-testis antigens overexpressed in various types of cancers. Silencing the MAGE-A2 expression inhibited the proliferation of prostate cancer (PCa) cells and increased the chemosensitivity. However, the expression pattern of MAGE-A2 in PCa tissue samples and its prognostic and therapeutic values for PCa patients is still unclear. METHODS: In this study, for the first time, the staining pattern and clinical significance of MAGE-A2 were evaluated in 166 paraffin-embedded prostate tissues, including 148 cases of PCa and 18 cases of high-grade prostatic intraepithelial neoplasia (HPIN), by immunohistochemical analysis. RESULTS: The simultaneous expression of both nuclear and cytoplasmic patterns of MAGE-A2 with different staining intensities was observed among studied cases. Increased expression of MAGE-A2 was significantly found in PCa tissues compared to HPIN cases (P < 0.0001). Among PCa samples, the strong staining intensity of nuclear expression was predominantly observed in comparison with cytoplasmic expression in PCa tissues (P < 0.0001). A significant and inverse correlation was found between the cytoplasmic expression of MAGE-A2 and increased Gleason score (P = 0.002). Increased cytoplasmic expression of MAGE-A2 was associated with longer biochemical recurrence-free survival (BCR-FS) and disease-free survival (DFS) of patients (P = 0.002, P = 0.001, respectively). In multivariate analysis, Gleason score and cytoplasmic expression of MAGE-A2 were independent predictors of the BCR-FS (P = 0.014; P = 0.028, respectively). CONCLUSIONS: Taken together, cytoplasmic expression of MAGE-A2 was inversely proportional to the malignant grade and duration of recurrence of the disease in patients with PCa.

Analysis of DNA methylation-driven genes for predicting the prognosis of patients with colorectal cancer.

Aberrant promoter methylation and ensuing abnormal gene expression are important epigenetic mechanisms that contribute to colorectal oncogenesis. Yet, the prognostic significance of such methylation-driven genes in colorectal cancer (CRC) remains obscure. Herein, a total of 181 genes were identified as the methylation-driven molecular features of CRC by integrated analysis of the expression profiles and the matched DNA methylation data from The Cancer Genome Atlas (TCGA) database. Among them, a five-gene signature (POU4F1, NOVA1, MAGEA1, SLCO4C1, and IZUMO2) was developed as a risk assessment model for predicting the clinical outcomes in CRC. The Kaplan-Meier analysis and Harrell's C index demonstrated that the risk assessment model significantly distinguished the patients in high or low-risk groups (p-value < 0.0001 log-rank test, HR: 2.034, 95% CI: 1.419-2.916, C index: 0.655). The sensitivity and specificity were validated by the receiver operating characteristic (ROC) analysis. Furthermore, different pharmaceutical treatment responses were observed between the high-risk and low-risk groups. Indeed, the methylation-driven gene signature could act as an independent prognostic evaluation biomarker for assessing the OS of CRC patients and guiding the pharmaceutical treatment. Compared with known biomarkers, the methylation-driven gene signature could reveal cross-omics molecular features for improving clinical stratification and prognosis.

A new tool for early diagnosis of rheumatoid arthritis using combined biomarkers; synovial MAGE-1 mRNA and serum anti-CCP and RF.

INTRODUCTION: rheumatoid arthritis (RA) is a common autoimmune disease with unknown etiology and pathogenesis. Biomarkers have the potential to aid in the clinical diagnosis of the disease, or to provide means of detecting early signs of the disease. Evaluating Melanoma associated antigen genes (MAGE-1) mRNA expression rate in synovial fluid cells and serum levels of anti-cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) for RA early diagnosis. METHODS: a total of 213 subjects were enrolled in the study, 135 RA patients and 78 normal subjects with traumatic knee joints (control group). Serum RF and anti-CCP were estimated quantitatively using ELISA. MAGE-1 mRNA expression rate was analyzed by RT-PCR. RESULTS: a significant increase in serum levels of RF IgM and anti-CCP in RA patients compared to the controls. A positively significant correlation was found between serum anti-CCP and RF IgM. The expression rate of MAGE-1 mRNA was 100% in RA patients versus the controls (0%). The specificity and the sensitivity of the three biomarkers was 100%. CONCLUSION: the high expression rate of MAGE-1 in synovial fluid cells of RA patients is encouraging its utilization as a diagnostic biomarker for RA. The combined use of MAGE-1 transcript in synovial fluid cells, serum RF and anti-CCP is recommended for improving early diagnostic ability of RA.

CTLA­4 blockade combined with 5­aza­2'­deoxycytidine enhances the killing effect of MAGE­A family common antigen peptide­specific cytotoxic T cells on breast cancer.

Breast cancer is the leading cause of cancer­-associated deaths in women. Combination immunotherapy attracts great interest as a treatment for breast cancer. However, there are no studies on the use of cytotoxic T­lymphocyte antigen 4 (CTLA­4) monoclonal antibody in combination with the melanoma­associated antigen A family (MAGE­As) co­antigen peptide (p248V9) for treating breast cancer, which should be explored. To this aim, in the present study, the samples of 115 patients with breast cancer were collected, and MAGE­As and CTLA­4 levels in breast cancer and adjacent normal tissues were assessed by immunohistochemical staining. The effect of 5­aza­2'­deoxycytidine (5DC) on the expression of MAGE­As in breast cancer cell lines was assessed by reverse transcription­quantitative PCR and western blot assay. Cytotoxic T cells (CTLs) were induced by MAGE­As co­antigen peptide. The specific lytic rate and IFN­Î³ level were examined by CCK­8 assay and ELISA, respectively. It was found that MAGE­As were highly expressed in breast cancer tissues. 5DC treatment promoted the expression of MAGE­As in breast cancer cells. The upregulation of the expression of MAGE­As specifically enhanced the ability of CTLs to kill breast cancer cells. CTLA­4 was highly expressed in breast cancer tissues and cells, and patients with breast cancer exhibiting high expression of CTLA­4 had low overall survival. CTLA­4 promoted the lytic efficiency of CTLs in breast cancer cells, and the combination of an anti­CTLA­4 antibody and 10 µM 5DC exhibited the highest cell lysis ability of CTLs. The present study demonstrated that MAGE­As co­antigen peptide­specific CTLs in combination with an anti­CTLA­4 monoclonal antibody and 5DC, have potent tumor cell­killing effects. It provides a novel theory for the development of breast cancer therapies.

Cytomorphological spectrum, including immunohistochemical results of 16 cases of clear cell sarcoma of soft tissue, along with positive EWSR1 gene rearrangement result in two cases.

OBJECTIVES: To describe the cytopathological features of clear cell sarcomas (CCSs), including immunohistochemical and molecular results, the latter in selected cases. METHODS: Sixteen consecutively diagnosed cases of CCS of soft tissue, over 6-year duration were included. Fine needle aspiration cytology was performed for primary diagnosis in three and for recurrent/metastatic lesions in 12 cases. Cytopathological features in 16 cases (conventional Papanicolaou- and May-Grünwald Giemsa-stained smears) were critically analysed. Corresponding histopathological and immunostained sections were available in 15 cases. Two cases were tested for EWSR1 gene rearrangement by fluorescence in-situ hybridisation. RESULTS: Sixteen tumours occurred in patients with age ranging from 18 to 56 years (median = 33.5); M: F ratio = 1:1; in deep soft tissues, mostly in extremities. Primary cytopathological diagnosis (3 cases) was CCS with a differential diagnosis of melanoma (1 case) and poorly differentiated malignant tumour (2 cases). On review, smears were predominantly hypercellular (n = 14), invariably composed of monomorphic appearing epithelioid/polygonal cells (n = 16), including spindle cells (n = 6); mostly singly scattered (n = 16), in loose clusters (n = 12); with prominent nucleolisation (n = 16); granular to vacuolated, well-defined cytoplasm (n = 12), binucleation/multinucleation (n = 9); mitoses (n = 6); sudden anisonucleosis; racquet-shaped cells (n = 3), against a tigroid background (n = 2), along with focal intracytoplasmic pigment deposition (n = 2). Immunohistochemically, tumour cells were positive for S-100P (15/15), HMB-45 (15/15) and melan-A(6/12). Two cases tested for EWSR1 rearrangement displayed red-green split signals. CONCLUSIONS: This constitutes one of the largest series describing the cytomorphological spectrum of CCS of soft tissue. Certain features, such as singly scattered monomorphic, epithelioid cells with prominent nucleolisation are useful diagnostic clues. Immunohistochemical stains are necessary and molecular testing is further helpful in reinforcing a diagnosis in certain cases. A correct diagnosis has crucial treatment implications.

Inhibition of MAGEA2 regulates pluripotency, proliferation, apoptosis, and differentiation in mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) exhibit self-renewal and pluripotency, can differentiate into all three germ layers, and serve as an essential model in stem cell research and for potential clinical application in regenerative medicine. Melanoma-associated antigen A2 (MAGEA2) is not expressed in normal somatic cells but rather in different types of cancer, especially in undifferentiated cells, such as in the testis, differentiating cells, and ESCs. However, the role of MAGEA2 in mESCs remains to be clarified. Accordingly, in this study, we examined the expression and functions of MAGEA2 in mESCs. MAGEA2 messenger RNA (mRNA) expression was decreased during mESCs differentiation. MAGEA2 function was then evaluated in knockdown mESC. MAGEA2 knockdown resulted in decreased pluripotency marker gene expression in mESCs consequent to increased Erk1/2 phosphorylation. Decreased MAGEA2 expression inhibited mESC proliferation via S phase cell cycle arrest with a subsequent decrease in cell cycle-associated genes Cdk1, Cdk2, Cyclin A1, Cyclin D1, and Cdc25a. Apoptotic mESCs markedly increased along with cleaved forms of caspases 3, 6, and 7 and PARP expression, confirming caspase-dependent apoptosis. MAGEA2 knockdown significantly decreased embryoid body size in vitro when cells were differentiated naturally and teratoma size in vivo, concomitant with decreased ectoderm marker gene expression. These findings suggested that MAGEA2 regulates ESC pluripotency, proliferation, cell cycle, apoptosis, and differentiation. The enhanced understanding of the regulatory mechanisms underlying diverse mESC characteristics will facilitate the clinical application of mESCs.

MAGE-A1 in lung adenocarcinoma as a promising target of chimeric antigen receptor T cells.

BACKGROUND: Cancer/testis antigens (CTAs) are a special type of tumor antigen and are believed to act as potential targets for cancer immunotherapy. METHODS: In this study, we first screened a rational CTA MAGE-A1 for lung adenocarcinoma (LUAD) and explored the detailed characteristics of MAGE-A1 in LUAD development through a series of phenotypic experiments. Then, we developed a novel MAGE-A1-CAR-T cell (mCART) using lentiviral vector based on our previous MAGE-A1-scFv. The anti-tumor effects of this mCART were finally investigated in vitro and in vivo. RESULTS: The results showed striking malignant behaviors of MAGE-A1 in LUAD development, which further validated the rationality of MAGE-A1 as an appropriate target for LUAD treatment. Then, the innovative mCART was successfully constructed, and mCART displayed encouraging tumor-inhibitory efficacy in LUAD cells and xenografts. CONCLUSIONS: Taken together, our data suggest that MAGE-A1 is a promising candidate marker for LUAD therapy and the MAGE-A1-specific CAR-T cell immunotherapy may be an effective strategy for the treatment of MAGE-A1-positive LUAD.

MAGE cancer-testis antigens protect the mammalian germline under environmental stress.

Ensuring robust gamete production even in the face of environmental stress is of utmost importance for species survival, especially in mammals that have low reproductive rates. Here, we describe a family of genes called melanoma antigens (MAGEs) that evolved in eutherian mammals and are normally restricted to expression in the testis (http://MAGE.stjude.org) but are often aberrantly activated in cancer. Depletion of Mage-a genes disrupts spermatogonial stem cell maintenance and impairs repopulation efficiency in vivo. Exposure of Mage-a knockout mice to genotoxic stress or long-term starvation that mimics famine in nature causes defects in spermatogenesis, decreased testis weights, diminished sperm production, and reduced fertility. Last, human MAGE-As are activated in many cancers where they promote fuel switching and growth of cells. These results suggest that mammalian-specific MAGE genes have evolved to protect the male germline against environmental stress, ensure reproductive success under non-optimal conditions, and are hijacked by cancer cells.

Signature microRNAs and long noncoding RNAs in laryngeal cancer recurrence identified using a competing endogenous RNA network.

The aim of the present study was to identify novel microRNA (miRNA) or long noncoding RNA (lncRNA) signatures of laryngeal cancer recurrence and to investigate the regulatory mechanisms associated with this malignancy. Datasets of recurrent and nonrecurrent laryngeal cancer samples were downloaded from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus database (GSE27020 and GSE25727) to examine differentially expressed miRNAs (DE­miRs), lncRNAs (DE­lncRs) and mRNAs (DEGs). miRNA­mRNA and lncRNA­miRNA networks were constructed by investigating the associations among these RNAs in various databases. Subsequently, the interactions identified were combined into a competing endogenous RNA (ceRNA) regulatory network. Feature genes in the miRNA­mRNA network were identified via topological analysis and a recursive feature elimination algorithm. A support vector machine (SVM) classifier was established using the betweenness centrality values in the miRNA­mRNA network, consisting of 32 optimal feature­coding genes. The classification effect was tested using two validation datasets. Furthermore, coding genes in the ceRNA network were examined via pathway enrichment analyses. In total, 21 DE­lncRs, 507 DEGs and 55 DE­miRs were selected. The SVM classifier exhibited an accuracy of 94.05% (79/84) for sample classification prediction in the TCGA dataset, and 92.66 and 91.07% in the two validation datasets. The ceRNA regulatory network comprised 203 nodes, corresponding to mRNAs, miRNAs and lncRNAs, and 346 lines, corresponding to the interactions among RNAs. In particular, the interactions with the highest scores were HLA complex group 4 (HCG4)­miR­33b, HOX transcript antisense RNA (HOTAIR)­miR­1­MAGE family member A2 (MAGEA2), EMX2 opposite strand/antisense RNA (EMX2OS)­miR­124­calcitonin related polypeptide α (CALCA) and EMX2OS­miR­124­Î³­aminobutyric acid type A receptor Î³2 subunit (GABRG2). Gene enrichment analysis of the genes in the ceRNA network identified that 11 pathway terms and 16 molecular function terms were significantly enriched. The SVM classifier based on 32 feature coding genes exhibited high accuracy in the classification of laryngeal cancer samples. miR­1, miR­33b, miR­124, HOTAIR, HCG4 and EMX2OS may be novel biomarkers of recurrent laryngeal cancer, and HCG4­miR­33b, HOTAIR­miR­1­MAGEA2 and EMX2OS­miR­124­CALCA/GABRG2 may be associated with the molecular mechanisms regulating recurrent laryngeal cancer.

Melanoma-associated antigen-A and programmed death-ligand 1 expression are associated with advanced urothelial carcinoma.

BACKGROUND: Melanoma-associated antigen-A (MAGE-A) and programmed-death ligand 1 (PD-L1) are present in urothelial carcinoma (UC). We assessed survival outcomes in patients with MAGE-A and PD-L1 expression. METHODS: MAGE-A and PD-L1 expression on neoplastic cells was analyzed using tissue microarrays from patients with UC. We compared differential expression between disease stage and grade. MAGE-A and PD-L1 co-expression was subcategorized. Fisher's exact test was done for categorical variables followed by univariable and multivariable analysis of recurrence-free survival (RFS) and progression-free survival (PFS). RESULTS: Co-expression of MAGE+/PD-L1+ was higher in advanced disease; however, only MAGE+/PD-L1- was associated with shorter RFS [hazard ratio (HR) 1.89; 95% confidence interval (CI) 1.19-2.99; p = .006]. MAGE+/PD-L1+ was associated with the worst PFS (HR 17.1; 95% CI 5.96-49.4; p ≤ .001). MAGE-A expression was more prevalent with high-grade (p = .015), and higher-stage ≥ pT2 (p = .001) disease. The 5-year RFS was 44% for MAGE+ versus 58% for MAGE- patients. On multivariable analysis, MAGE+ was also associated with shorter RFS (HR 1.55; 95% CI 1.05-2.30; p = .03). Similarly, MAGE+ was associated with shorter PFS (HR 3.12; 95% CI 1.12-8.68; p = .03). CONCLUSION: MAGE-A and PD-L1 expression is increased in advanced disease and associated with shorter PFS. Furthermore, MAGE-A expression was significantly associated with higher-grade and -stage disease and associated with shorter RFS and PFS. The worse prognosis associated with MAGE-A+/PD-L1+ provides evidence that a combinatorial treatment strategy co-targeting MAGE/PD-L1 might be feasible. Further studies are needed to validate these findings.

miR­34a regulates the chemosensitivity of retinoblastoma cells via modulation of MAGE­A/p53 signaling.

The present study aimed to explore the combined role of microRNA (miR)-34a, melanoma antigen-A (MAGE­A) and p53 in altering the chemosensitivity of retinoblastoma (RB) cells. Human RB and adjacent tumor tissues, as well as human RB cell lines (HXO­Rb44, SO­Rb50, Y79 and WERI­Rb-1) were used. In addition, four chemotherapeutic drugs, including carboplatin, etoposide, Adriamycin and vincristine, were used to treat the cell lines, in order to evaluate the sensitivity of RB cells. Furthermore, miR­34a expression was detected by reverse transcription-quantitative polymerase chain reaction, and western blotting was implemented to quantify expression levels of MAGE­A and p53. A luciferase reporter gene assay was used to validate the targeted association between miR­34a and MAGE­A. The results indicated that SO­Rb50 cells exhibited the highest resistance to carboplatin, Adriamycin and vincristine (P<0.05), whereas HXO­Rb44 cells revealed the highest inhibition rate in response to etoposide (P<0.05) out of the four cell lines. Furthermore, reduced miR­34a expression and increased MAGE­A expression significantly elevated the survival rate and viability of SO­Rb50 cells following drug treatment (all P<0.05). miR­34a was also demonstrated to directly target MAGE­A, thereby significantly promoting the viability of RB cells and depressing apoptosis (P<0.05). p53, which was subjected to modulation by miR­34a and MAGE­A, also significantly reduced the proliferation rate of RB cells (P<0.05). In conclusion, the miR­34a/MAGE­A/p53 axis may be conducive to enhancing the efficacies of chemotherapeutic treatments for RB.

MAGEA1 inhibits the expression of BORIS via increased promoter methylation.

Melanoma-associated antigen A1 (MAGEA1) and BORIS (also known as CTCFL) are members of the cancer testis antigen (CTA) family. Their functions and expression-regulation mechanisms are not fully understood. In this study, we reveal new functions and regulatory mechanisms of MAGEA1 and BORIS in breast cancer cells, which we investigated in parental and genetically manipulated breast cancer cells via gene overexpression or siRNA-mediated downregulation. We identified the interaction between MAGEA1 and CTCF, which is required for the binding of MAGEA1 to the BORIS promoter and is critical for the recruitment of DNMT3a. A protein complex containing MAGEA1, CTCF and DNMT3a was formed before or after conjunction with the BORIS promoter. The binding of this complex to the BORIS promoter accounts for the hypermethylation and repression of BORIS expression, which results in cell death in the breast cancer cell lines tested. Multiple approaches were employed, including co-immunoprecipitation, glutathione S-transferase pull-down assay, co-localization and cell death analyses using annexin V-FITC/propidium iodide double-staining and caspase 3 activation assays, chromatin immunoprecipitation and bisulfite sequencing PCR assays for methylation. Our results have implications for the development of strategies in CTA-based immune therapeutics.

Tumor suppressor let-7a inhibits breast cancer cell proliferation, migration and invasion by targeting MAGE-A1.

Let-7 was one of the earliest discovered miRNAs and while it reportedly acts as a tumor suppressor in various solid tumors, its function in breast cancer has not been fully studied. Therefore, we examined let-7a and MAGE-A1 expression in breast tissues by qRT-PCR and found that let-7a expression significantly correlates with larger tumor size, higher histological grade (p<0.05) and is significantly lower in patients with Her-2-positive cancers and Ki-67 >14% (p=0.028 and p=0.023). MAGE-A1 expression incidence is 50.8% (33/65) and it inversely correlates with let-7a expression (p=0.008). let-7a inhibition of breast cancer cell proliferation, migration and invasion was also observed in in vitro cell culture experiments, and dual-luciferase reporter assays showed that melanoma-associated antigen A1 (MAGE-A1) was its target gene; the target comprised bases 451-457 of the 3'UTR region of the MAGE-A1 mRNA. RT-qPCR and Western blot analyses showed that let-7a inhibited MAGE-A1 expression at both the nucleic acid and protein levels. In our final co-transfection experiment, we targeted MAGE-A1 in a breast cancer cell line and observed that let-7a inhibited cell proliferation, migration and invasion. These combined results confirm that let-7a functions as a tumor suppressor by targeting MAGE-A1 in breast cancer and it therefore provides a novel target in breast cancer clinical treatment.

Cancer Neoepitopes for Immunotherapy: Discordance Between Tumor-Infiltrating T Cell Reactivity and Tumor MHC Peptidome Display.

Tumor-infiltrating lymphocytes (TIL) are considered enriched for T cells recognizing shared tumor antigens or mutation-derived neoepitopes. We performed exome sequencing and HLA-A*02:01 epitope prediction from tumor cell lines from two HLA-A2-positive melanoma patients whose TIL displayed strong tumor reactivity. The potential neoepitopes were screened for recognition using autologous TIL by immunological assays and presentation on tumor major histocompatibility complex class I (MHC-I) molecules by Poisson detection mass spectrometry (MS). TIL from the patients recognized 5/181 and 3/49 of the predicted neoepitopes, respectively. MS screening detected 3/181 neoepitopes on tumor MHC-I from the first patient but only one was also among those recognized by TIL. Consequently, TIL enriched for neoepitope specificity failed to recognize tumor cells, despite being activated by peptides. For the second patient, only after IFN-γ treatment of the tumor cells was one of 49 predicted neoepitopes detected by MS, and this coincided with recognition by TIL sorted for the same specificity. Importantly, specific T cells could be expanded from patient and donor peripheral blood mononuclear cells (PBMC) for all neoepitopes recognized by TIL and/or detected on tumor MHC-I. In summary, stimulating the appropriate inflammatory environment within tumors may promote neoepitope MHC presentation while expanding T cells in blood may circumvent lack of specific TIL. The discordance in detection between physical and functional methods revealed here can be rationalized and used to improve neoantigen-targeted T cell immunotherapy.

Design of Artificial Immunogens Containing Melanoma-associated T-cell Epitopes.

OBJECTIVE: Immunotherapy based on induction of T-cell responses is a promising approach to cancer treatment. The study aims to design artificial epitope-based immunogens, DNA vaccine candidates against melanoma and evaluate their ability to stimulate tumor cytotoxicity of ex vivo generated T-cells. METHODS: The original computational methods were used for predicting T-cell epitopes and designing polyepitope melanoma antigens. Artificial genes encoding the target antigens were cloned into DNA vaccine plasmid vector. Target gene expression was confirmed both at transcriptional and translational level in HEK-293T cells transfected with DNA-vaccine constructs. Dendritic cells were generated from adherent peripheral blood mononuclear cells of HLA-A*02:01+ donors. Cytotoxic activity of effector lymphocytes stimulated in co-culture with autologous antigen-presenting dendritic cells towards melanoma Mel Is cells was assessed with lactate dehydrogenase release assay. The proportion of granzyme B producing CD8+ T-cells was estimated using intracellular cytokine staining and flow cytometry. RESULTS: Two DNA vaccine constructions were created - pMEL-TCI and pMEL-A0201 - encoding polypeptides containing T-cell epitopes of six immunodominant melanoma antigens (NY-ESO-1, MART1, MAGE-A1, MAGE-A11, MAGE-A3, and MAGE-C1). Dendritic cells transfected with DNA vaccine constructs were found to stimulate both tumor cytotoxicity mediated by autologous lymphocytes and granzyme B production by CD8+ T-cells, and pMEL-A0201 was found to be the most efficient. CONCLUSION: The described approach may become a common platform for designing immunotherapeutic vaccines against oncological diseases.

Tumor suppressive miR-6775-3p inhibits ESCC progression through forming a positive feedback loop with p53 via MAGE-A family proteins.

Accumulating evidences indicate that microRNAs (miRNAs) play vital roles in multiple diseases, including cancer. In the present study, we showed that miR-6775-3p plays a tumor suppressive role in esophageal squamous cell carcinoma (ESCC). High expression miR-6775-3p is associated with good clinical outcomes of ESCC patients. Over-expression of miR-6775-3p inhibited tumor growth and liver metastasis of ESCC xenograft tumors. Enforced expression of miR-6775-3p inhibited ESCC cell proliferation, migration, and invasion. KEGG pathway analysis revealed that miR-6775-3p was associated with the genes on "pathway in cancer". Mechanically, miR-6775-3p inhibited the expression of tumor antigens MAGE-A family through direct binding the 3'UTR region of MAGE-A mRNAs, and attenuated MAGE-A-inhibited transcriptional activity of tumor suppressor p53. In addition, miR-6775-3p also directly inhibits its host gene SLC7A5 which has been reported to play oncogenic roles in cancer progression. Interestingly, miR-6775-3p and its host gene SLC7A5 were directly transcriptionally induced by p53. Thus, for the first time, our study proposed a novel positive feedback regulation between miR-6775-3p and p53 via MAGE-A family, which plays crucial role in ESCC progression.

Specific Features of Transcription Activity of Cancer-Testis Antigens in Patients with Metastatic and Non-Metastatic Breast Cancer.

Cancer-testis antigens, effective markers of tissue malignant transformation, are characterized by heterogonous transcription depending on the pathological features of breast cancer. We performed screening of transcription profile of cancer-testis antigens specific for breast tumor tissues in female patients with and without regional metastasis. The relative expression of 16 genes (MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEB1, MAGEB2, GAGE1, GAGE3, GAGE4, MAGEC1, BAGE, XAGE3, NY-ESO1, SSX2, SYCP1, and PRAME1) was analyzed by RT-qPCR method in biopsy specimens of the mammary gland tissues obtained during surgery from 25 patients. Differential transcription activity of cancer-testis antigens genes was observed in patients with metastatic (enhanced expression of MAGEA2, MAGEB1, and XAGE3 genes) and non-metastatic (enhanced expression of GAGE3 and PRAME1 genes) breast cancer.

Immunization with tumor neoantigens displayed on T7 phage nanoparticles elicits plasma antibody and vaccine-draining lymph node B cell responses.

The aim of this preclinical study was to evaluate T7 bacteriophage as a nanoparticle platform for expression of neoantigens that could allow rapid generation of vaccines for potential studies in human cancer patients. We have generated recombinant T7 phage vaccines carrying neoepitopes derived from mutated proteins of B16-F10 melanoma tumor cells. With the single mutated amino acid (AA) centered, peptides were expressed on the outer coat of T7 phage. All peptides with 11 and 34 AAs were successfully expressed. Trimers of the 11-AA peptides were successfully expressed in only 3 of 8 peptides. The 11-AA peptide was better in stimulating antibodies selective for the mutated region than the longer 34-AA peptide. We observed a dose response for vaccines which provides an initial framework of the minimum phage required for vaccination. A single injection with phage-peptide vaccines in both monomer and trimer formats produced significant immune responses in mice on day 21, as assessed by lymph node cell counts, next generation sequencing (NGS), and plasma titers against T7 phage and vaccine peptides. A trimer provided no additional serum response to the monomer format. Immunization of mice with a mixture of 8 different peptide vaccines resulted in antibodies to most of the peptides. It was encouraging that induced antibodies had higher binding to the mutated peptides compared to the corresponding normal peptides. The NGS of lymph node cells demonstrated a low B cell receptor diversity and clonal hyperpolarization in vaccine-draining lymph nodes in comparison to those in unvaccinated mice nodes. The NGS data also revealed phenomenal increase in IgG and other class-switched antibodies following vaccination. These results agree with the higher plasma titers of IgG antibodies against T7 phage and vaccine peptides. Antibodies bound whole B16-F10 cells, lysates and multiple bands on Western blot. This indicates that these vaccine peptides successfully induced antibodies that bind full proteins from which the vaccine peptides were derived. We demonstrate a preclinical platform for rapid production of vaccines that can deliver mutated peptides and stimulate an appropriate B cell response. We anticipate further research in utilizing the cells from a tumor or vaccine draining lymph node as a resource for therapeutic anticancer reagents.

Gas­filled ultrasound microbubbles enhance the immunoactivity of the HSP70­MAGEA1 fusion protein against MAGEA1­expressing tumours.

Advanced malignant melanoma is characterized by rapid development, poor prognosis and insensitivity to chemoradiotherapy. Immunotherapy has become one of the primary clinical treatments for malignant melanomas. In recent decades, identifying specific tumour antigens and the enhanced immunoactivity of tumour vaccines has become critical for engineering successful tumour vaccines. As a widely used vaccine carrier, heat shock protein 70 (HSP70) clearly increases the immunogenicity of tumour antigens, such as melanoma­associated antigen A1 (MAGEA1). Based on previous studies, gas­filled ultrasound microbubbles (MBs) were engineered to carry an HSP70­MAGEA1 fusion protein (FP). Following subcutaneous injection around the lymphatic nodes the FP was directly released into the lymph nodes under ultrasonic imaging. The results indicated that the microbubbles enhanced the immunoactivity of FPs more effectively than HSP70­MAGEA1 fusion alone. Additionally, HSP70­MAGEA1 delivered via microbubbles clearly inhibited and delayed the growth of MAGEA1­expressing B16 melanomas in mice and improved the survival times of these animals compared with the fusion protein alone. The results of the present study demonstrated that controlled MBs enhance the immunoactivity of FPs and also highlights novel, potential vaccine carriers and a new strategy for engineering controllable tumour vaccine designs.