Expression Of Melanoma-associated Antigens Research Articles

Correlation between the Expression of Melanoma-Associated Antigen-A3 and Cytology Results on Bronchoalveolar Lavage on Nsclc

Background: Lung cancer is still considered an alarming health problem, causing a major cause of death in malignant disease. The incidence rate is likely to increase and most of it comes with an advanced stage of diagnosis. To improve the healing rate and life expectancy, the detection of lung cancer should be done early, when it is still small and localized. Bronchoalveolar lavage (BAL) cytology is a method of examination that can be used in lung cancer. The expression of Melanoma-Associated Antigen (MAGE) becomes the marker that is sensitive to certain types of cancer, including lung cancer. Objective:To analyze the correlation between expression MAGE-A3 and cytology of BAL specimen on NSCLC. Method: Bronchoscopy was performed on patients with NSCLC lung tumor at pulmonology unit of Dr. Soetomo Teaching Hospital Surabaya Indonesia to collect the BAL. Histologic examination for BAL and MAGE-A3 examination were conducted on patients. Statistical analysis used to determine the correlation between the expression of MAGE A3 and cytology of BAL specimens on NSCLC was fisher’s exact test. Result: We found that there were 7.14% of subjects with BAL cytology had cancer cells (adenocarcinoma). MAGE-A3 expression was positive only in 28.60% of subjects diagnosed with NSCLC. The result of MAGE A3 expression analysis with cytology of BAL specimen showed p = 0.286. Conclusion: There was no significant correlation between MAGE-A3 expression and histopathology type. The results of MAGE A3 RT PCR fluid expression examination showed that most of them were in the negative category.

Differential expression of melanoma-associated antigen A3/6 and associated immune molecules prior to and post treatment with immune checkpoint inhibitors (ICI) in patients with mUC.

433 Background: MAGE is an attractive target for IO given its expression restricted to carcinomas and the testes. Engineered T-cell targeting MAGE A3/6 has shown promise. However, successful treatment is dependent on circumventing the potentially hostile immune microenvironment in metastatic tumors. In this study, we aim to assess the effects of ICI on the immune microenvironment in patients who progressed on ICI. Methods: We obtained FFPE tissue from 16 patients with mUC across 3 institutions. Samples from the primary or metastatic tumor prior to treatment with PD1/PD-L1 inhibitors (ICI) were paired with samples from metastatic sites post-treatment. Differential mRNA expression was assessed using NanoString PanCancerIO360 panel for the main genes of interest ( MAGEA3/6, CD274, HLADP, HLAA, B2M and PDCD1) in addition to immune profiling of the tumor samples. Also, IHC was done on the paired samples to assess DE for the genes of interest using an H-score analyzed via the paired T-test. p-values <0.05 were considered significant and FDR corrected. Results: MAGEA3/A6 mRNA expression was unchanged in the post-treatment samples. In addition, time to progression was not influenced by the mRNA DE. Similarly, IHC expression was not decreased post-treatment. The other genes of interest were also similarly expressed pre and post treatment. Immune cell localization genes CPA3,TPSAB1/B2, HDC, MS4A2 were significantly downregulated post treatment. Genes implicated in cancer cell killing ( MX1,KIT) were similarly downegulated whereas CD276, TLR1 were upregulated. Expression in the post treatment tissue of PTPN1, CES3, ACVR1C, and BAD were associated with increased progression. LY9, PTPRC, CTLA4, HLA-DMA, IL10RA, HLA-DMB, ITGAL, CD48 were associated with decreased progression. Conclusions: Prior ICI treatment does not decrease expression of MAGE A3/6, suggesting that salvage treatments targeting this antigen remain a viable strategy. Suggestion of immune microenvironment alterations were also noted. More work is necessary to understanding the tumor microenvironment in order to design rational treatment combinations and sequences.

Transcriptome Alterations of Isolated Primary Bone Marrow Endothelial Cells Provide Insigt to MGUS and Multiple Myeloma Pathobiology

Multiple myeloma (MM) is characterized by an abnormal clonal expansion of plasma cells in the bone marrow, production of monoclonal immunoglobulins and finally organ damage (CRAB). The premalignant precursor of MM is Monoclonal gammopathy of undetermined significance (MGUS) and one percent of all MGUS patients progress to MM yearly. The bone marrow microenvironment is thought to play an important role in plasma cell growth, migration, and survival mainly via cytokine secretion and cell-cell interactions. Endothelial cells (ECs) are a major component in the bone marrow microenvironment, they regulate trafficking and homing of hematopoietic progenitor and stem cells. In MM increased bone marrow angiogenesis and recruitment of endothelial progenitors to the bone marrow niche has been reported. However, the specific EC contribution to myelomagenesis is not yet known. This study therefore aimed to investigate transcriptome alterations in prospectively isolated bone marrow ECs from MGUS and MM patients to identify possible disease-stage related changes. We isolated primary ECs from MGUS and MM patients undergoing diagnostic bone marrow aspirations and age-matched healthy donors by FACS. RNA from Lin- CD45- CD71- CD235a- CD271- CD31+ cells of MGUS (n=4) and MM (n=7) patients and healthy donors (n=6) was extracted. Sequencing was done using the Illumina® NextSeq 500/550 High Output Kit v2.5 (300 cycles). Gene expression analysis was performed in R. Differential gene expression analysis (DEseq2) identified 1,507 genes with p adjusted values below 1e-2 that were significantly differentially expressed between the three groups. Hierarchical clustering was done following Ward's method (ward.D2). Unsupervised clustering on the data showed that one MGUS-EC sample clustered with the healthy controls, and that one healthy control sample clustered with the MGUS samples. We therefore decided to restrict the analysis to those samples that clearly clustered separately, to be able to better depict the MGUS-, MM- and healthy EC specific profiles. Further clustering of differential expressed genes into 8 clusters revealed two especially interesting expression patterns. One cluster (#4) contained 102 genes that where higher expressed in the healthy controls with lower expression in MGUS and lowest expression in MM Samples. These genes thus reflect the downregulation during progression from a healthy bone marrow microenvironment to a reduced expression MGUS and further downregulation in MM. Another cluster (#6) showed the opposite pattern, with 105 genes being low or not expressed in healthy controls while the expression was higher in MGUS and highest in MM. Gene sets where further analyzed in the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. Cluster 4 showed a high number of downregulated transmembrane genes. Six genes of the major histocompatibility complex conserved site where identified might indicate a possible immunomodulating effect in disease progression. Furthermore, within cluster 4 we identified a cluster of genes involved in cell adhesion and receptor binding. Cluster 6 most strikingly showed a group of 6 genes of the melanoma-associated antigen (MAGE) gene family that were upregulated with disease progression. MAGE genes which belong to the cancer-testis group of germline genes have previously been reported in MM, as being involved in tumorigenesis, and plasma cell MAGE expression has been associated with chemotherapy resistance. Furthermore, cluster 6 contained a high number of extracellular matrix genes, and genes for proteins having an extracellular region, respectively, hinting towards a differential microenvironment composition upon MM development. Taken together RNA sequencing analysis of prospectively isolated bone marrow endothelial cells identified genes that were specifically upregulated/suppressed in MM-ECs compared to MGUS-ECs and healthy donor-ECs. These genes thus represent potential gene candidates involved in the disruption of normal microenvironment function, thus leading to disease development and progression. Accordingly, studies are underway to investigate selected transcriptional deregulation EC-MM microenvironmental functions in the context of the disease. Disclosures No relevant conflicts of interest to declare.

Expression, Function, and Prognostic Value of MAGE-D4 Protein in Esophageal Squamous Cell Carcinoma.

We previously reported that expression of melanoma-associated antigen (MAGE)-D4 mRNA was a prognostic factor for esophageal squamous cell carcinoma (ESCC). The aim of this study was to validate the expression of MAGE-D4 in two additional patient cohorts, and to investigate its biological functions. The role of MAGE-D4 in cell proliferation, adhesion, and migration was determined by gene knockdown experiments in the KYSE590 cell line. MAGE-D4 protein expression was analyzed in ESCC tissues by immunohistochemistry. A second validation cohort consisted of an ESCC mRNA dataset from The Cancer Genome Atlas. Knockdown of MAGE-D4 significantly decreased cell proliferation and migration. Expression of MAGE-D4 protein was significantly associated with disease-free survival. In the second validation cohort, high MAGE-D4 mRNA expression was associated with significantly shorter overall survival and disease-free survival. MAGE-D4 plays an important role in the malignant behavior of ESCC. MAGE-D4 was validated as a prognostic indicator in two independent ESCC patient cohorts.

The expression of melanoma-associated antigen A (MAGE-A) in oral squamous cell carcinoma: an evaluation of the significance for tumor prognosis.

Melanoma-associated antigens A had been detected repeatedly in oral squamous cell carcinoma, but not in healthy mucosa. Additionally, patients with MAGE-A expressing cancers are regarded to have a worse survival prognosis, so that MAGE-A are supposed to be part of carcinogenesis. Which role these antigens fulfill within OSCC is still, up today, largely unknown. This study examines the hypothesis that MAGE-A is being produced in OSCC but not in mucosa tissue and if MAGE-A has any correlation to clinical patient's parameters like tumor size, lymph node metastasis, distant metastasis, overall survival, and recurrence. For this purpose, 50 tumor samples and 39 mucosa samples were analyzed by means of PCR and immunohistochemical staining with the antibody 6C1. Forty of 41 stained tumor samples showed a positive antibody reaction with a maximum staining rate of 53%. Sixteen mucosa samples showed a mild positive reaction. The PCR revealed a linear expression pattern of MAGE-A in which the genes are proportionally expressed in OSCC. We did not find any relationship between MAGE-A and tumor size, overall survival, or recurrence. There was also no connection between MAGE-A and tumor parameters Hif-1 and LDH. Their expression was detected tendentially in tumors with higher staging, advanced lymph node metastasis, and rising age of the patients. The genes MAGE-A3+6 and MAGE-A4 had a statistically significant correlation with lymph node metastasis (p = 0.007 and p = 0.004). Patients got distant metastasis and influence of MAGE-A on metastatic behavior could not be verified. The genes MAGE-A3 and -A4 are consequently qualified as tumor markers in the field of diagnosis and follow-up of OSCC. Two genes have great potential as target proteins in immunotherapy. The genes MAGE-A3+6 and MAGE-A4 had a statistically significant correlation with lymph node metastasis.

MAGE expression in head and neck squamous cell carcinoma primary tumors, lymph node metastases and respective recurrences-implications for immunotherapy

Melanoma associated antigens (MAGE) are potential targets for immunotherapy and have been associated with poor overall survival (OS) in head and neck squamous cell carcinoma (HNSCC). However, little is known about MAGE in lymph node metastases (LNM) and recurrent disease (RD) of HNSCC.To assess whether MAGE expression increases with metastasis or recurrence, a tissue microarray (TMA) of 552 primary tumors (PT), 219 LNM and 75 RD was evaluated by immunohistochemistry for MAGE antigens using three monoclonal antibodies to multiple MAGE family members. Mean expression intensity (MEI) was obtained from triplicates of each tumor specimen.The median MEI compared between PT, LNM and RD was significantly higher in LNM and RD. In paired samples, MEI was comparable in PT to respective LNM, but significantly different from RD. Up to 25% of patients were negative for pan-MAGE or MAGE-A3/A4 in PT, but positive in RD. The prognostic impact of MAGE expression was validated in the TMA cohort and also in TCGA data (mRNA). OS was significantly lower for patients expressing pan-MAGE or MAGE-A3/A4 in both independent cohorts.MAGE expression was confirmed as a prognostic marker in HNSCC and may be important for immunotherapeutic strategies as a shared antigen.

Association of MAGE A1-6 Expression with Lung Cancer Progression.

The melanoma-associated antigen (MAGE) genes are known to be expressed in various kinds of tumors including lung cancer. Although they are studied as targets for immunotherapy and tools for early detection of lung cancer, the correlation between MAGE expression and the prognosis in lung cancer has not been clarified. In this study, we evaluated the relationship between MAGE A1-6 gene expression and the clinical prognosis in lung cancer.Bone marrow aspirations were performed in 60 patients who were diagnosed as lung cancer and underwent lung cancer surgery between 2007 and 2008. Each bone marrow was examined using nested reverse transcription- polymerase chain reaction (RT-PCR) with the MAGE common primer to detect MAGE A1-6. Overall survival rate, disease-free survival rate, recurrence, and distant metastasis were reviewed retrospectively. Survival periods were analyzed using SPSS ver. 20.0.Of the total 60 lung cancer patients, 9 patients (15%) had MAGE A1-6. MAGE A1-6-positive patients showed poor overall survival and overall disease-free survival rates (43.8 ± 26.1, 43.2 ± 26.9 months, respectively) compared with MAGE A1-6-negative patients (54.4 ± 17.2, 44.8 ± 22.1 months, respectively). No significant difference was shown in either survival rates.In conclusion, MAGE A1-6 expression of bone marrow in lung cancer patients correlated with poor survival rates. We suggest that MAGE A1-6 may be considered as a novel prognostic factor for lung cancer which leads to effective follow-up and treatment.

Abstract 2365: Checkpoint inhibitors and a multivalent melanoma vaccine as a novel combinatorial therapy

Abstract Over 1 million cases of skin cancer are diagnosed in the US every year, of which 75% deaths result due to melanoma, one of the deadliest forms of skin cancer, arising from melanocytes. We have isolated five patient derived melanoma cell lines (MEL-V, 3MM, GLM2, Mel2, KFM) and characterized the expression of various melanoma associated antigens (MAAs). The presence of a multitude repertoire of common MAAs (Gp100, MART-1, MAGE-A1, NY-ESO-1, Tyrosinase, TRP-1 (Gp75), TRP-2, Melanotransferrin, CD71, CD146) enabled us to use composite cell membrane preparations of these cells as a multivalent vaccine. To this end, we designed a vaccinia virus vaccine that was composed of membrane lysate preparations of the five primary cells and recombinant IL-2. When tested in a pilot study, delayed type hypersensitivity and a multivalent immune response were noted. In an effort to correlate genetic lesions with MAA expression we observed that in BRAF mutant melanoma cells, treatment with a BRAFv600E inhibitor, PLX4032, enhanced the expression of MAAs making the melanoma cells more visible to the vaccination driven immune response. We also observed that melanoma cells constitutively expressed CTLA-4 as determined by Immunofluorescence and Western Blots. This expression was found on the membranes as well as in the cytoplasm in comparatively high levels on wild type as well as the BRAFV600E mutant melanoma cell lines. Further our results suggest that CTLA-4 expression on melanoma cells promotes the immunosuppressive tumor microenvironment enabling tumor evasion. Given that CTLA-4 monoclonal antibodies such as Ipilimumab are approved for clinical treatment, this study opens up a new avenue into a plausible combinatorial therapy for our vaccinia virus based vaccine with Ipilimumab for BRAF mutant as well as wild type melanoma cells. Citation Format: Rachana R. Maniyar, Neha Tuli, Robert Suriano, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Checkpoint inhibitors and a multivalent melanoma vaccine as a novel combinatorial therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2365.

MAGE-A1-6 expression in patients with head and neck squamous cell carcinoma: impact on clinical patterns and oncologic outcomes.

Various subtypes of melanoma-associated antigens (MAGEs) are expressed in the tumor tissues of patients with head and neck squamous cell carcinoma (HNSCC). However, little data are currently available on how the gene expression of MAGEs impacts clinical patterns and oncologic outcomes. We have therefore evaluated the expression of MAGE-A1-6 (A1-6) subtypes in tumor tissues of patients with HNSCC and the clinical impact of this expression. This was a retrospective review of 53 patients with histologically proven HNSCC of the oral cavity, oropharynx, larynx, or hypopharynx who underwent both treatment and analysis by reverse transcription (RT)-PCR assay with a common primer to identify the expression of MAGE-A1-6 subtypes in the tumor tissue. The clinicopathologic factors and oncologic outcomes of these patients and the correlations of both to MAGE-A1-6 gene expression were analyzed. MAGE-A1-6 subtypes were expressed in the tumor tissues of 37 patients (69.8%). Patient age of≥65years [p=0.031, hazard ratio (HR)4.866] and advanced American Joint Committee on Cancer stage (p=0.035, HR4.291) were independent risk factors for expression of MAGE-A1-6 subtypes. Patients with MAGE-A1-6 expression had lower disease-free survival (p=0.029), disease-specific survival (p=0.070), and overall survival (p=0.017) rates. Overall survival rate was independently associated to chemotherapy (p=0.011, HR2.859), while no surgery (p=0.050, HR2.400) and MAGE-A1-6 expression (p=0.050, HR2.527) showed borderline significance. In our patient group the expression of MAGE-A1-6 subtypes in tumor tissues of patients with HNSCC was correlated with advanced clinical stage of cancer and poor oncologic outcomes. We suggest that gene expression of MAGE-A1-6 subtypes may be considered to be a predictive factor to determine patient treatment or follow-up strategy.

Abstract 3540: Novel targeted combinational therapies for melanoma

Abstract The BRAFV600E mutation is frequently observed in over 60% of melanomas. Development of the small molecule inhibitor PLX4032, which specifically targets the mutated BRAF protein, offers a novel therapeutic approach by effectively blocking the activation of key signaling molecules of the mitogen-activated protein kinase (MAPK) pathway, such as Mek and Erk, effectively leading to arrest in cell proliferation. Provided that MAPK signaling targets the micropthalmia transcription factor (MITF), which is linked to the expression of various melanoma associated antigens (MAAs), we hypothesized that the inhibition of BRAFV600E can modulate the expression of MAAs. Since MAAs are essential in the development of a melanoma vaccine, due to their increased immunogenicity, altering their expression can be a useful immunotherapeutic approach to targeting melanoma. Using patient derived melanoma cell lines, which express various MAAs; our laboratory has developed a vaccinia virus based immunotherapeutic vaccine. These melanoma cell lines have been characterized by their status of BRAFV600E mutation and found to be either homozygous or heterozygous for the mutation. Inhibition of two key MAPK pathway molecules, namely Mek and Erk, denotes that the melanoma cell lines are responsive to PLX4032. Phenotypically, the cells’ invasive and migratory properties were also modulated upon treatment with PLX4032. Moreover, we observed that the expression of various MAAs was enhanced upon treatment with PLX4032. An amplification of gp100, MART-1 and tyrosinase expression on the RNA level as well as on the protein level was observed. Lastly, expression of MMAs was conserved in vivo following the analysis of melanoma cell xenografts in BALB/c nude mice. Based on our observations in that PLX4032 modulates MMA expression, further studies are warranted to determine the clinical effectiveness of combinational immunologic therapy using BRAFV600E inhibitors with our vaccinia virus melanoma cell line derived preparation. Citation Format: Robert Suriano, Neha Y. Tuli, Jan Geliebter, Raj K. Tiwari, Marc Wallack. Novel targeted combinational therapies for melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3540. doi:10.1158/1538-7445.AM2015-3540

Clinical significance of melanoma-associated antigen A1-6 expression in sputum of patients with squamous cell carcinoma of the larynx and hypopharynx.

Several studies have reported the expression of the melanoma-associated antigen (MAGE) gene in head and neck squamous cell carcinoma (HNSCC). In this study, we evaluated the correlations between MAGE expression in sputum and the clinical features and oncologic outcomes of SCC of the larynx and hypopharynx. We performed a retrospective review of 119 patients treated for SCC of the larynx and hypopharynx and analysis of their induced sputum by nested reverse transcription-polymerase chain reaction (RT-PCR) to detect the MAGE-A1-6 gene. The associations between MAGE expression and clinical characteristics were analyzed. Expression of MAGE-A1-6 in sputum was identified in 57 of 119 patients (47.9%), and was independently correlated to double primary cancer (p = .024; odds ratio [OR] = 4.135). Expression of MAGE-A1-6 in sputum was correlated to poor survival. Expression of MAGE-A1-6 in sputum predicts poor oncologic outcome in patients with SCC of the larynx and hypopharynx. © 2015 Wiley Periodicals, Inc. Head Neck 38: E736-E740, 2016.

Abstract A30: Small molecule kinase inhibitor mediated modulation of immunotherapy in melanoma

Abstract As the BRAFV600E mutation is observed in over 60% of melanomas, the development of the small inhibitor, PLX4032, which specifically targets mutated BRAF, represents a novel therapeutic approach. This targeted inhibition of BRAF effectively blocks activation of key signaling molecules of the mitogen activated protein kinase (MAPK) pathway, such as Mek and Erk, thus halting cell proliferation. In addition, since MAPK signaling targets the micropthalmia transcription factor (MITF), which is linked to the expression of various melanoma associated antigens (MAAs), we hypothesize that the inhibition of BRAFV600E can modulate the expression of MAAs thus serving as a viable target for immunotherapy of melanoma. Our lab has developed a vaccinia virus based immunotherapeutic vaccine using patient derived melanoma cell lines which express various MAAs. The melanoma cell lines have been characterized as far as the status of the BRAFV600E mutation and were found to be either homozygous or heterozygous for the mutation and are responsive to PLX4032, as evidenced by inhibition of two key MAPK pathway molecules, namely Mek and Erk. Phenotypically, the cells invasive and migratory properties were also modulated upon treatment with PLX4032. Interestingly was the observation that upon treatment with PLX4032, the expression of various MAAs was enhanced. Expression of gp100, MART-1, and tyrosinase increased both on the RNA level as well as on the protein level. The expression of MMAs were well maintained in vivo following the analysis of melanoma cell xenografts in BALB/c nude mice. We conclude that an effective multivalent immunotherapeutic vaccine requires the use of various MAAs and since PLX4032 upregulates expression of MAAs, further studies are warranted to determine the clinical effectiveness of combining BRAFV600E inhibitors with our melanoma cell line derived preparation. Citation Format: Robert Suriano, Neha Tuli, Jan Geliebter, Raj K. Tiwari, Marc Wallack. Small molecule kinase inhibitor mediated modulation of immunotherapy in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A30.

Melanoma-associated antigen expression and the efficacy of tyrosine kinase inhibitors in head and neck cancer.

Melanoma-associated antigen (MAGE) has been identified in a variety of types of cancer. The expression of several MAGE subgroups is correlated with poor prognosis and chemotherapeutic resistance. One target of chemotherapeutic treatment in head and neck cancer is the epidermal growth factor receptor (EGFR). The efficacy of tyrosine kinase inhibitors (TKI) in the context of melanoma-associated antigens is discussed in the present study. Five human squamous cell carcinoma cell lines were treated with the EGFR TKIs, erlotinib and gefitinib. The efficacy of these agents was measured using a crystal violet assay. Furthermore, the expression levels of MAGE-A1, -A5, -A8, -A9, -A11 and -A12 were determined by reverse transcription-quantitative polymerase chain reaction. The association between TKI efficacy and MAGE-A expression was analyzed by linear regression. The cell lines revealed inhomogeneous expression patterns for the MAGE-A subgroups. Four of the five cell lines demonstrated a good response to erlotinib and gefitinib. However, treatment with erlotinib induced better results than those of gefitinib, and revealed a concentration-dependent effect. The expression of MAGE-A5 and -A11 were significantly correlated with lower efficacy of erlotinib and gefitinib. By contrast, MAGE-A12 was associated with a superior response to these two drugs. One cell line, which expressed all investigated MAGE-A subgroups, was entirely resistant to the two TKIs. These results revealed a notable correlation between MAGE-A5 and -A11 and lower efficacy of EGFR TKIs. Pretreatment analysis of MAGE-A status may therefore aid improvement of chemoprevention using erlotinib and gefitinib in head and neck cancer.

Multiple MAGE-A genes as surveillance marker for the detection of circulating tumor cells in patients with ovarian cancer

Ovarian cancer is a leading cause of death among gynecologic malignancies. In this study, we reported the expression of melanoma-associated antigens A (MAGE-A) genes in peripheral blood from 80 patients with ovarian cancer and 30 healthy donors. MAGE-As expression was associated with the factors indicating poor prognosis. The expressions of MAGE-As and each individual MAGE-A genes were also associated with low overall survival of patients with ovarian cancer. Our results suggested MAGE-A genes may have the potential to be surveillance markers for the detection of circulating tumor cells and represent a poor prognosis for patients with ovarian cancer.

Ipilimumab induces simultaneous regression of melanocytic naevi and melanoma metastases

Ipilimumab blocks cytotoxic T-lymphocyte-associated antigen (CTLA)-4, potentiating the antimelanoma T-cell host response. Ipilimumab has been shown to improve overall survival in patients with previously treated metastatic melanoma. CTLA-4 antibodies generate immune responses to the melanoma-associated antigens Melan-A, NY-ESO-1 and glycoprotein (gp)100 in metastatic melanoma. Digital epiluminescence microscopy (DELM) is a noninvasive method permitting the monitoring of the morphology of melanocytic lesions over time. A 50-year-old man with metastatic melanoma received four ipilimumab injections after failure of dacarbazine chemotherapy. Positron emission tomography revealed regression of pulmonary metastases, and simultaneously, DELM showed regression of several melanocytic naevi. On histological examination of the regressing naevi, prominent CD8+, CD4+ and CD45R0 lichenoid lymphohistiocytic infiltrates were seen, whereas nonregressing naevi were almost free of inflammatory infiltrate. Expression of melanoma-associated antigens in benign melanocytic naevi may explain the induction of naevus regression by ipilimumab. DELM could represent a valuable noninvasive method to monitor ipilimumab efficacy.

Dendritic Cells Loaded With mRNA Encoding Full-length Tumor Antigens Prime CD4+ and CD8+ T Cells in Melanoma Patients

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient's human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8(+) and CD4(+) T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8(+) response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4(+) response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8(+) and CD4(+) T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.

The Immune Response to Melanoma Is Limited by Thymic Selection of Self-Antigens

The expression of melanoma-associated antigens (MAA) being limited to normal melanocytes and melanomas, MAAs are ideal targets for immunotherapy and melanoma vaccines. As MAAs are derived from self, immune responses to these may be limited by thymic tolerance. The extent to which self-tolerance prevents efficient immune responses to MAAs remains unknown. The autoimmune regulator (AIRE) controls the expression of tissue-specific self-antigens in thymic epithelial cells (TECs). The level of antigens expressed in the TECs determines the fate of auto-reactive thymocytes. Deficiency in AIRE leads in both humans (APECED patients) and mice to enlarged autoreactive immune repertoires. Here we show increased IgG levels to melanoma cells in APECED patients correlating with autoimmune skin features. Similarly, the enlarged T cell repertoire in AIRE−/− mice enables them to mount anti-MAA and anti-melanoma responses as shown by increased anti-melanoma antibodies, and enhanced CD4+ and MAA-specific CD8+ T cell responses after melanoma challenge. We show that thymic expression of gp100 is under the control of AIRE, leading to increased gp100-specific CD8+ T cell frequencies in AIRE−/− mice. TRP-2 (tyrosinase-related protein), on the other hand, is absent from TECs and consequently TRP-2 specific CD8+ T cells were found in both AIRE−/− and AIRE+/+ mice. This study emphasizes the importance of investigating thymic expression of self-antigens prior to their inclusion in vaccination and immunotherapy strategies.

Melanoma-Associated Antigen (MAGE) Expression in the Normal Mucosa around Colorectal Cancer after Curative Resection: Presence of Undetectable Free Cancer Cells?

Curative surgical resection is of great importance and some trials have been performed to identify free undetectable cancer cells using molecular markers. The aim of this study is to investigate melanoma-associated antigen (MAGE) expression in normal mucosa around colorectal cancer and its clinical significance.? From October 2003 to June 2004, we collected 46 colorectal cancer and matched normal mucosal tissues within 20 mm, 20 to 50 mm and more than 50 mm from tumors after the curative operation. Twenty-two mucosal tissues were harvested from patients with benign colorectal diseases as controls. MAGE expression was assayed using nested RT-PCR of MAGE A1-6 mRNA.? The MAGE expression rates in cancer tissue and adjacent normal mucosa were 65.2%, 6.5% (<20 mm), 2.2% (20-50 mm) and 0.0% (>50 mm), respectively, while MAGE was not expressed in the mucosa of benign diseases. The MAGE-positive cases in the normal mucosa around tumors were located in the left colon or rectum, and one patient showed anastomotic mucosal site recurrence.? MAGE expression in normal-appearing mucosa around colorectal cancer showed some clinical findings suggesting the presence of undetectable free cancer cells after curative resection.

Induction of a CD8+ T-cell response to the MAGE cancer testis antigen by combined treatment with azacitidine and sodium valproate in patients with acute myeloid leukemia and myelodysplasia

AbstractEpigenetic therapies, including DNA methyltransferase and histone deacetylase inhibitors, represent important new treatment modalities in hematologic malignancies, but their mechanism of action remains unknown. We reasoned that up-regulation of epigenetically silenced tumor antigens may induce an immunologically mediated antitumor response and contribute to their clinical activity. In this study, we demonstrate that azacitidine (AZA) and sodium valproate (VPA) up-regulate expression of melanoma-associated antigens (MAGE antigens) on acute myeloid leukemia (AML) and myeloma cell lines. In separate studies, we observed that prior exposure to AZA/VPA increased recognition of myeloma cell lines by a MAGE-specific CD8+ cytotoxic T-lymphocyte (CTL) clone. We therefore measured CTL responses to MAGE antigens in 21 patients with AML or myelodysplasia treated with AZA/VPA. CTL responses to MAGE antigens were documented in only 1 patient before therapy; however, treatment with AZA/VPA induced a CTL response in 10 patients. Eight of the 11 patients with circulating MAGE CTLs achieved a major clinical response after AZA/VPA therapy. This is the first demonstration of a MAGE-specific CTL response in AML. Furthermore, it appears that epigenetic therapies have the capacity to induce a CTL response to MAGE antigens in vivo that may contribute to their clinical activity in AML. This study was registered at http://isrctn.org as #ISCTN68418952.

Comparison of three culture media for the establishment of melanoma cell lines

Melanoma cell lines are useful tools for the analysis of tumor-specific lymphocytes which are injected to patients treated by adoptive immunotherapy. So they have been established previously (with an efficacy of 47%) in Roswell Park Memorial Institute (RPMI) medium enriched with fetal calf serum (FCS). In order to improve the probability of establishing melanoma cell lines, we compared two FCS-free media with the original FCS medium. Ten melanoma-invaded lymph nodes were tested for their ability to grow in three different culture media: RPMI with FCS; RPMI with human serum (HS); serum-free X-vivo 15 (X15). For each medium, we compared the following criteria: percentage of lines obtained; period of establishment; cell morphology; expression of melanoma-associated antigens and surface molecules. More cell lines were obtained with HS and X15 media compared to FCS medium (7/10, 5/10 and 4/10, respectively). The time period to establish a stable line was similar for the three media. No morphological differences were observed in cells derived from the same tumor sample in the different media. With the X15 medium, cells generally expressed lower levels of melanocytic differentiation antigens and surface molecules. The growth of melanoma cell lines in FCS-free culture media appears possible and advantageous, with an increased probability of obtaining autologous tumor cell lines. Furthermore the cells obtained could be used as multiple antigenic sources in active or adoptive immunotherapy protocols.