Cancer-testis Antigen Genes Research Articles

Cancer/Testis Antigens and Colorectal Cancer, gene expression,

Some antigenic proteins are only normally present in male gametogenic tissues in the testis and notin normal somatic cells. When the genes that encode these proteins are expressed in cancerous cells they are referred to as cancer/testis antigen (CTA) genes. Some CTA genes have been proven to encode immunogenic proteins that have been used as successful immunotherapy targets for various forms of cancer. Thus, there has been a focus over the past two decades to characterise their potential applications for the treatment and/or diagnosis of cancer. Colorectal cancer is among the most common forms of cancer and a leading cause of cancer related death worldwide. It is generally regarded as having low levels of CTA gene expression, compared to some other cancers such as malignant melanoma. There is now scope to revisit this dogma, as an ill-defined cohort of colorectal cancers do exhibit CTA gene expression, potentially exposing these tumours to CTA-based therapeutic and/or diagnostic strategies as they are developed.

BORIS, Brother of the Regulator of Imprinted Sites, Is Aberrantly Expressed in Hepatocellular Carcinoma

The brother of the regulator of imprinted sites (BORIS) is a novel member of the cancer testis antigen gene family, which are normally expressed only in spermatocytes, but abnormally activated in different malignancies. The aim of this study was to explore the expression of BORIS in hepatocellular carcinoma (HCC) and its correlation with the clinicopathologic features and prognosis of HCC. We investigated BORIS expression in HCC cell lines and 105 primary HCC clinical surgical specimens using real-time polymerase chain reaction and Western blot analysis. We further examined the correlation of BORIS with a liver stem cell marker (CD90) in HCC tissues by histochemical double staining. The correlation of BORIS with clinicopathologic features and prognosis of HCC was analyzed using patient data. The expression of BORIS was found in SMMC-7721, BEL-7402, and Huh-7, but not in hep-G2 cells. The expression rate of BORIS was significantly higher in the HCC tissues than in the adjacent noncancerous tissues (p=0.000). BORIS expression was correlated with the tumor size (p=0.000), CD90 expression (p=0.000), and satellite nodule (p=0.000). Kaplan-Meier survival curves showed that patients with positive expression of BORIS had lower overall survival rate (p=0.003). Our data indicate that BORIS may be an auxiliary diagnosis index and a novel favorable prognostic indicator of HCC.

MAGE-C1/CT7 and MAGE-C2/CT10 are frequently expressed in multiple myeloma and can be explored in combined immunotherapy for this malignancy.

The exact function of MAGE-C1/CT7 and MAGE-C2/CT10 is not yet understood in multiple myeloma (MM). However, the homologs MAGE-C1/CT7 and MAGE-C2/CT10 genes encode highly immunogeneic cancer/testis antigens (CTAs) and can be potential targets for T cell-based immunotherapy. MAGE-C1/CT7 and MAGE-C2/CT10 mRNA expression were investigated in MM patients, solitary plasmacytomas, monoclonal gammopathies of undetermined significance (MGUS) and bone marrow (BM) aspirates from healthy donors by RT-PCR. MAGE-C1/CT7 and MAGE-C1/CT10 were expressed in 67 and 59 % of the 46 MM analyzed patients. At least one of the genes was expressed in 76 % of MM cases. Solitary plasmacytoma also showed MAGE-C1/CT7 and MAGE-C2/CT10 expression. MAGE-C1/CT7 and MAGE-C2/CT10 were not expressed in normal BM samples, showing restricted expression of these CTA genes in MM, solitary plasmacytoma and MGUS. In the present study, we found high expression of the homologs MAGE-C1/CT7 and MAGE-C2/CT10 in monoclonal gammopathies and speculate whether these genes might represent a valuable therapeutic option for myeloma, in particular for combined immunotherapy.

High Resolution X Chromosome-Specific Array-CGH Detects New CNVs in Infertile Males

ContextThe role of CNVs in male infertility is poorly defined, and only those linked to the Y chromosome have been the object of extensive research. Although it has been predicted that the X chromosome is also enriched in spermatogenesis genes, no clinically relevant gene mutations have been identified so far.ObjectivesIn order to advance our understanding of the role of X-linked genetic factors in male infertility, we applied high resolution X chromosome specific array-CGH in 199 men with different sperm count followed by the analysis of selected, patient-specific deletions in large groups of cases and normozoospermic controls.ResultsWe identified 73 CNVs, among which 55 are novel, providing the largest collection of X-linked CNVs in relation to spermatogenesis. We found 12 patient-specific deletions with potential clinical implication. Cancer Testis Antigen gene family members were the most frequently affected genes, and represent new genetic targets in relationship with altered spermatogenesis. One of the most relevant findings of our study is the significantly higher global burden of deletions in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p = 8.785×10−6) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.435×10−4).ConclusionsBy the analysis of the X chromosome at the highest resolution available to date, in a large group of subjects with known sperm count we observed a deletion burden in relation to spermatogenic impairment and the lack of highly recurrent deletions on the X chromosome. We identified a number of potentially important patient-specific CNVs and candidate spermatogenesis genes, which represent novel targets for future investigations.

The Cancer Testis Antigen PRAME As a Biomarker for Solid Tumor Cancer Management

Among the best-characterized tumor antigens are the cancer-testis antigens (CTAs), or germ cell antigens. These are a category of nonmutated genes that are restricted in expression to male germ cells and fetal tissues, but are detected in a variety of cancers and the cells derived from them. CTA gene families, including MAGE, BAGE, GAGE, LAGE/NY-ESO-1 and PRAME (preferentially expressed antigen of melanoma), encode antigenic peptides recognized by T lymphocytes. Numerous members of the CTA gene families are under intense study as targets for immunotherapy and as biomarkers for diagnosis and/or prognosis. Unlike other CTA genes, the PRAME gene (also known as MAPE or OIP4 ) has been shown to be expressed at low levels in ovarian, endometrial and adrenal tissues [1]. Originally characterized as a tumor antigen in a melanoma patient, PRAME is one of the best-characterized CTA family members because of its prevalence in both acute and chronic leukemias, and Hodgkin’s lymphoma [2]. Significant association of PRAME expression has been reported in acute myelogenous leukemia (AML), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloma and chronic myelogenous leukemia (CML). In CML, expression has been shown to correlate with disease progression [2,3]. PRAME has been reported to be an independent prognostic factor in AML, being associated with increased overall survival in patients with a favorable or poor prognoses based on karyotype [2,3]. The other area of promising clinical utility is disease stratification and the detection of residual disease. Although it varies according to disease type, the level of PRAME expression can indicate probable resistance to chemotherapy in lymphomas and AML [4]. PRAME expression has been observed to significantly decrease in

Identification of novel MAGE-A6- and MAGE-A12-derived HLA-A24-restricted cytotoxic T lymphocyte epitopes using an in silico peptide-docking assay.

Many cancer-testis antigen genes have been identified; however, few human leukocyte antigen (HLA)-A24-restricted cytotoxic T cell (CTL) epitope peptides are available for clinical immunotherapy. To solve this problem, novel tools increasing the efficacy and accuracy of CTL epitope detection are needed. In the present study, we utilized a highly active dendritic cell (DC)-culture method and an in silico HLA-A24 peptide-docking simulation assay to identify novel CTL epitopes from MAGE-A6 and MAGE-A12 antigens. The highly active DCs, called α-type-1 DCs, were prepared using a combination of maturation reagents to produce a large amount of interleukin-12. Meanwhile, our HLA-A24 peptide-docking simulation assay was previously demonstrated to have an obvious advantage of accuracy over the conventional prediction tool, bioinformatics and molecular analysis section. For CTL induction assays, peripheral blood mononuclear cells derived from six cases of HLA-A24(+) melanoma were used. Through CTL induction against melanoma cell lines and peptide-docking simulation assays, two peptides (IFGDPKKLL from MAGE-A6 and IFSKASEYL from MAGE-A12) were identified as novel CTL epitope candidates. Finally, we verified that the combination of the highly active DC-culture method and HLA-A24 peptide-docking simulation assay might be tools for predicting CTL epitopes against cancer antigens.

Abstract 1142: The expression profile of cancer/testis antigens (CTAs) in head and neck cancer

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the most frequent types of cancer. Despite significant improvements in the therapeutic strategies, the overall survival rate in 5 years is around 50%. The development of new therapies and their integration into the current forms of treatment (surgery, radiotherapy and chemotherapy) is of great interest to improve the therapeutic efficacy. Immunotherapy may provide an alternative for current treatment approach, since the natural immune response can be stimulated by active immunotherapeutic interventions which may help in the control of the tumor progression and recurrence. A requirement for the development of immunotherapeutic approaches is the identification of immunogenic gene products expressed predominantly in tumor cells. Cancer/testis antigens (CTAs) fulfill this prerequisite as their expression is restricted to tumors with no expression in normal tissues, except germ line cells. In this study we performed a search on Oncomine, SAGE Anatomic Viewer, CTdatabase and PubMed databases to select among the 153 CTAs genes those that might be expressed in HNSCC. This in silico analysis allowed the selection of 60 CTAs genes to have their expression evaluated in 89 tumors and in 20 normal mucosa by RT-PCR. According this analysis, 13 CTAs were specificaly expressed in HNSCC samples (>25% sensitivity) The expression of CTA1 was significantely associated with perineural invasion (p=0,023) and vascular invasion (p=0,003), whereas the presence of CTA2 or CTA3 was correlated with the presence of tumor cells in the lymph nodes (p=0,023 and p=0,037, repectively). Moreover, the CTA4 expression showed to be associated with capsular rupture (p=0,005). We report here the identification of 13 CTAs frequently expressed in HNSCC and the expression of some of them was significantly associated with poor prognostic factors. These results could be important for the development of immunotherapy strategies for treating HNSCC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1142. doi:1538-7445.AM2012-1142

ALK-Negative Anaplastic Large Cell Lymphomas with 6p25.3 Translocations Show a Histone-Modifying Gene Expression Signature

Abstract 88 Introduction:The biology and genetics of anaplastic large T-cell lymphomas (ALCLs) lacking ALK translocations remain poorly defined. We recently identified recurrent translocations involving the DUSP22-IRF4 locus on 6p25.3 in ALK-negative ALCL (Blood 2011;117:915–9). This translocation is present in about 20% of ALK-negative ALCLs and is absent in other T-cell lymphomas; however, the biologic consequences are unknown. In this study, we utilized gene expression profiling (GEP) to compare T-cell lymphomas with and without this translocation. Methods:cDNA was prepared from total RNA extracted from frozen tissue from 25 T-cell lymphoma patients under a Mayo Clinic IRB-approved protocol as follows: 5 ALK-negative ALCLs with 6p25.3 translocations (2 systemic, 3 cutaneous), 12 ALK-negative ALCLs without 6p25.3 translocations (11 systemic, 3 cutaneous), 5 ALK-positive ALCLs, and 3 CD30-positive peripheral T-cell lymphomas (1 transformed mycosis fungoides and 2 not otherwise specified). Partner loci in cases with 6p25.3 translocations were 7p32.3 in 2, 9p24.3 in 1, and unknown in 2. GEP data (Affymetrix U133 plus 2.0) were analyzed by unsupervised clustering; in addition, a GEP signature was developed using genes with raw values >200 in at least 2 cases with at least 2-fold, statistically significantly different expression between 6p25.3-translocated and non-translocated cases (p<0.05 after Benjamini-Hochberg multiple testing correction). Representative overexpressed genes were confirmed by quantitative real-time (qRT-) PCR. Supervised pathway analysis was performed using signatures derived from other studies or curated from public pathway databases. Results:By unsupervised clustering, the 5 cases with 6p25.3 translocations clustered together (281 probe sets). A GEP signature segregating translocated from non-translocated cases included 283 genes (195 up- and 88 down-regulated). Key features of this signature included altered expression of histone-modifying genes (e.g. EZH2, SUZ12, MLLT3, MLL; HDAC4) and up-regulation of cancer-testis antigen (CTA) genes (e.g. SSX4, TEX14, MAGEA12, SSX1, CTAG2). The signature included down-regulation of DUSP22 but not of other genes on 6p25 in the signature. Additional overexpressed genes were validated by qRT-PCR (relative expression: TEX14, 2.9, p=0.01, t test; MAL, 6.5, p=0.003; CCR8, 7.6, p=0.0004). Supervised pathway analysis demonstrated a high proliferation signature in cases with 6p25.3 translocations (p=0.006 vs. untranslocated cases). Conclusions:ALK-negative ALCLs with translocations involving the DUSP22-IRF4 locus on 6p25.3 have a distinct gene expression signature characterized by alterations of histone-modifying genes and CTA genes (which typically are regulated epigenetically, either through histone modifications or changes in methylation). This signature appears independent of anatomic site (systemic vs. cutaneous) and partner locus (7p32.3 vs. other). DUSP22, encoding a dual-specificity phosphatase involved in modulating mitogen-activated protein kinase (MAPK) signaling, is significantly down-regulated in translocated cases; however, pathway analysis did not show significant alterations in MAPK-associated genes. Other known genes in the region of the breakpoint on 6p25 were not affected substantially. These findings suggest the possibility of an unrecognized function of DUSP22. Contributions of the varying partner loci remain unclear. The distinct GEP signature associated with 6p25.3 translocations provides strong support for their biologic relevance. Finally, the genes involved in the histone-modifying signature show overlap with histone-modifying genes mutated in B-cell lymphomas, suggesting potential commonalities in the biology and possibly targeted treatment of B- and T-cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Abstract 3864: Evaluation of expression of antigens cancer/testis (CTAs) in head and neck squamous cell carcinoma

Abstract Background: Among the ten most common cancers are the head and neck tumors with more than 500,000 new cases diagnosed annually worldwide, the majority (90%) represented by squamous cell carcinomas (HNSCC). Despite of the many strategies used in the treatment for HNSCC, the 5 years overall survival rate is close to 50%, and the development of loco-regional recurrence is the main reason for treatment failure. The head and neck cancer surgery is often mutilating and require extensive reconstruction. The development of new therapies and their integration into the current forms of treatment (including surgery, radiotherapy and chemotherapy) is of great interest to improve the therapeutic efficacy. Immunotherapy may provide an alternative for current treatment scheme, since the natural immune response can be stimulated by active immunotherapeutic interventions which may help control tumor progression and recurrence. A requirement for the development of immunotherapeutic approaches is the identification of immunogenic gene products expressed predominantly in tumor cells. Cancer/testis antigens (CTAs) fulfills this prerequisite as their expression is restricted to tumors with no expression in normal tissues, except germ line cells. Methods: In this study we performed a search on Oncomine, SAGE, CTdatabase and PubMed databases to select among 153 CTAs genes those that might be expressed in HNSCC. The expression of selected genes was investigated in 93 HNSCC samples and 10 normal mucosa samples from healthy individuals using reverse-transcription polymerase chain reaction (RT-PCR). The in silico analysis allowed the selection of 30 CTAs genes to be evaluated in tumors and control samples. The RT-PCR assays identified 6 CTAs with high specificity (100%) and frequently expressed in HNSCC samples (39 – 85%). Noteworthy, 99% of the tumor samples expressed at least one of the 6 CTAS, 91% expressed at least two of them, and 71% expressed at least three of these antigens. The six CTAs were expressed simultaneously in 13% of cases. Conclusion: We identified 6 CTAs genes with high expression and specificity in head and neck cancer. This result could be important for the development of immunotherapy strategies for treating HNSCC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3864. doi:10.1158/1538-7445.AM2011-3864

Abstract 2042: Cancer-specific transcription factor BORIS has different effects on expression of its target genes via chromatin structure alterations

Abstract BORIS (Brother of the Regulator of Imprinted Sites), the paralog of insulator CTCF, is male germ line-specific transcription factor. BORIS and CTCF are highly structurally similar and share the same DNA sequence recognition sites. However, unlike widely expressed CTCF, BORIS is repressed in normal tissues and expressed only in testis and in several types of human cancers including head and neck cancers, lung cancers, melanomas and in tumor-derived cell lines. Suprabasin (SBSN) is a protein that is normally expressed only in suprabasal layer of epidermis, and was defined as a potential oncogene in non small cell lung carcinoma. BORIS has been implicated in the activation of cancer-testis antigens (CTA) genes, such as MAGEA1 and NY-ESO-1. Additionally, BORIS is shown to bind hypermethylated DNA sites and alter the DNA methylation state of MAGEA1 promoter. BORIS shows abundant expression in a variety of cancers and its importance in tumorigenesis has been reported. However, there are controversial reports about the role of BORIS on tumorigenesis. Using cell proliferation assays, immunoprecipitation, qPCR, qRT-PCR, QMSP and other techniques we show that SBSN is a newly discovered target of BORIS epigenetic regulation. Induction of low concentrations of BORIS leads to activation of the chromatin around SBSN transcription start site resulting in its 18-35 fold increase in expression in human cancer cell lines, including lung cancer. We have demonstrate that high BORIS concentrations can lead to subsequent repression of SBSN by 2-3 folds via chromatin repression (5-10 fold decrease in presence of active histone modifications, evaluated by ChIP and qPCR) and via DNA hypermethylation (4 folds increase, by QMSP). In a similar manner, overexpression of BORIS leads to eventual repression of other well-known BORIS targets, including NY-ESO-1 and H19. We also show that BORIS induces cell growth at low concentration and inhibits cell growth and colony formation at high concentrations. Our data indicate that BORIS has different effects on target genes depending on its abundance around binding sites. Thus, low BORIS concentration leads to strong target activation and cell proliferation, while higher concentrations leads to repression of the target genes and cell growth inhibition. These data explain the enigmatic controversy of published materials about role of BORIS effect on tumorigenesis. Preliminary data also suggests that there are a group of transcription factors similar to BORIS that can recognize DNA methylation status of the binding sites. Therefore, they might have both stimulating and repressing effects on epigenetic regulation of target genes expression depending on the factor abundance. Molecular analysis of epigenetic mechanisms of action of BORIS and BORIS-like transcription factors might be beneficial for further development of cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2042. doi:10.1158/1538-7445.AM2011-2042

549 Unified carcinogenesis theory

Azanucleoside DNA-hypomethylating agents have remarkable clinical activity in myelodysplastic syndromes and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs) encoding immunogenic proteins are not expressed in almost all normal tissues and many tumor types, but are consistently derepressed by epigenetically active agents in various cancer cell lines. Since the expression of CTA genes is usually very low or absent in myeloid leukemias, we treated various AML cell lines with 5-aza-2′-deoxycytidine (DAC) and quantified mRNA expression of the CTAs NY-ESO-1, MAGEA1, MAGEA3 and MAGEB2. Consistent time- and dose-dependent reactivation of all 4 CTA genes was observed, with maximum mRNA levels 72–144 h after treatment start. As determined by RNA microarray analyses, numerous other CTA genes (all located on the X-chromosome) were also derepressed in a time-dependent fashion by DAC. NY-ESO-1 derepression was confirmed at the protein level. By Elispot and chromium release assays we showed that the de novo expressed NY-ESO-1 protein was naturally processed and presented in a time- and dose-dependent fashion up to 8 days after the start of DAC treatment, and converted the cell lines susceptible to antigen-specific recognition by CD8+ T-cell clones. In conclusion, NY-ESO-1 and numerous other CTAs localized on the X-chromosome are readily and transiently derepressed in AML cell lines treated with DAC. The susceptibility of DAC-treated AML cell lines to antigen-specific T-cell recognition has clear implications for future clinical trials combining DAC and specific immunotherapy in AML.

Abstract 587: Development of brain tumor-specific targeted system by using retroviral-mediated gene therapy with SSX4 promoter

Abstract Despite many efforts to develop effective therapy, the outcome of malignant glioma remains poor. Previously, we reported the eradication of mouse glioma by retroviral-mediated gene therapy. In this study, a tumor-specific targeting system was studied to develop the effective and safe gene therapy. We first searched for genes expressing at high frequency in brain tumors but not in normal human astrocyte (NHA) among cancer testis antigen (CTA) genes by in silico and RT-PCR screening. MAGEA3 and SSX4 were identified as tumor-specific genes. The promoter activity was measured in glioma, telomerase-immortalized fibroblast and NHA cells. The SSX4 promoter but not MAGEA3 showed the tumor-specific activity. In order to define a useful tumor-specific promoter for targeting in context of retroviral-mediated gene therapy, the SSX4 promoter were used to restrict the expression of suicide gene HSVtk in retroviral vector. The glioma cell lines transduced with the retroviral vector were killed efficiently by addition of ganciclovir (GCV), but telomerase-immortalized fibroblast BJ-5ta cells were not. Mouse glioma RSV-M cells transduced with the retroviral vector were transplanted into S.C. of syngeneic mouse. The administration of GCV suppressed the tumor growth completely. These results suggest that trans-acting factors may play a role for tumor-specific activity of SSX4 promoter while the expression of most CTA genes is regulated by epigenetic factors and the retrovirus vector expressing HSVtk under control of SSX4 promoter is a promising therapeutic reagent for malignant brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 587.

The DNA demethylating agent 5-aza-2′-deoxycytidine induces expression of NY-ESO-1 and other cancer/testis antigens in myeloid leukemia cells

Azanucleoside DNA-hypomethylating agents have remarkable clinical activity in myelodysplastic syndromes and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs) encoding immunogenic proteins are not expressed in almost all normal tissues and many tumor types, but are consistently derepressed by epigenetically active agents in various cancer cell lines. Since the expression of CTA genes is usually very low or absent in myeloid leukemias, we treated various AML cell lines with 5-aza-2′-deoxycytidine (DAC) and quantified mRNA expression of the CTAs NY-ESO-1, MAGEA1, MAGEA3 and MAGEB2. Consistent time- and dose-dependent reactivation of all 4 CTA genes was observed, with maximum mRNA levels 72–144 h after treatment start. As determined by RNA microarray analyses, numerous other CTA genes (all located on the X-chromosome) were also derepressed in a time-dependent fashion by DAC. NY-ESO-1 derepression was confirmed at the protein level. By Elispot and chromium release assays we showed that the de novo expressed NY-ESO-1 protein was naturally processed and presented in a time- and dose-dependent fashion up to 8 days after the start of DAC treatment, and converted the cell lines susceptible to antigen-specific recognition by CD8+ T-cell clones. In conclusion, NY-ESO-1 and numerous other CTAs localized on the X-chromosome are readily and transiently derepressed in AML cell lines treated with DAC. The susceptibility of DAC-treated AML cell lines to antigen-specific T-cell recognition has clear implications for future clinical trials combining DAC and specific immunotherapy in AML.

Enhanced expression of cancer testis antigen genes in glioma stem cells

Cancer stem cells are an important target for effective therapy, since they show tumorigenicity, chemoresistance, and radioresistance. We isolated cancer stem cells from glioma cell lines and tissues and examined the expression of cancer testis antigen (CTA) genes as potential target molecules for cancer vaccine therapy. CTA genes were highly and frequently expressed in cancer stem cells compared with differentiated cells. In addition, histone acetylation levels in the promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, while DNA methylation analysis of the promoter regions revealed hypomethylation in cancer stem cells. This epigenetic difference between cells leads to heterogeneous expression of CTA genes in the tumor mass, which consists of cells at various levels of differentiation. Moreover, the expression level of HLA class I antigens was not affected by the differentiation status, suggesting that CTA genes may present as surface antigens in cancer stem cells. Taken together, these findings suggest that CTA genes may be attractive candidates for targeted vaccine therapy against cancer stem cells in glioma patients.

Diagnostic value of cancer-testis antigen mRNA in peripheral blood from hepatocellular carcinoma patients

To evaluate the diagnostic value of cancer-testis antigen (CTA) mRNA in peripheral blood samples from hepatocellular carcinoma (HCC) patients. Peripheral blood samples were taken from 90 patients with HCC before operation. Expression of melanoma antigen-1 (MAGE-1), synovial sarcoma X breakpoint-1 (SSX-1), and cancer-testis-associated protein of 11 kDa (CTp11) mRNA in peripheral blood mononuclear cells (PBMC) was tested by nested reverse transcripts-polymerase chain reaction (RT-PCR). Serum alpha-fetoprotein (AFP) in these patients was also determined. The positive rate of MAGE-1, SSX-1 and CTp11 transcripts was 37.7%, 34.4%, 31.1% in PBMC samples, and 74.4%, 73.3%, 62.2% in their resected tumor samples, respectively. The positive rate for at least one of the transcripts of three CTA genes was 66.7% in PBMC samples and 91.1% in their resected tumor samples. MAGE-1, SSX-1 and/or CTp11 mRNA were not detected in the PBMC of those patients from whom the resected tumor samples were MAGE-1, SSX-1 and/or CTp11 mRNA negative, nor in the PBMC samples from 20 healthy donors and 10 cirrhotic patients. Among the 90 patients, the serum AFP in 44 patients met the general diagnostic standard (AFP > 400 microg/L) for HCC, and was negative (AFP < or = 20 microg/L) or positive with a low concentration (20 microg/L < AFP < or = 400 microg/L) in the other patients. The positive rate for at least one of the transcripts of three CTA genes in PBMC samples from the AFP negative or positive patients with a low concentration was 69.2% and 45.0%, respectively. Of the 90 patients, 71 (78.9%) were diagnosed as HCC by nested RT-PCR and serum AFP. Although the positive rate for at least one of the transcripts of three CTA genes in PBMC samples from 53 patients at TNM stage III or IV was obviously higher than that in PBMC samples from 37 patients at stage I or II (77.9% vs 51.4%, P = 0.010), the CTA mRNA was detected in 41.7% and 56.0% of PBMC samples from HCC patients at stages I and II, respectively. Detecting MAGE-1, SSX-1 and CTp11 mRNA in PBMC improves the total diagnostic rate of HCC.

Cancer/testis (CT) antigens: Potential targets for immunotherapy

Cancer/testis (CT) antigens are protein antigens with normal expression restricted to adult testicular germ cells, and yet are aberrantly activated and expressed in a proportion of various types of human cancer. At least a subset of this group of antigens has been found to elicit spontaneous humoral and cell-mediated immune responses in cancer patients, raising the possibility that these antigens could be cancer vaccine targets. More than 100 CT antigen genes have been reported in the literature, with approximately 30 being members of multigene families on the X chromosome, so-called CT-X genes. Most CT-X genes are expressed at the spermatogonia stage of spermatogenesis, and their functions are mostly unknown. In cancer, the frequency of CT antigen expression is highly variable among different tumor types, but is more often expressed in high-grade late-stage cases in general. Cancer vaccine trials based on CT antigens MAGE-A3 and NY-ESO-1 are currently ongoing, and these antigens may also play a role in antigen-specific adoptive T-cell transfer and in the immunomodulation approach of cancer therapy.

Epigenetic Manipulation of Cancer Testis Antigen (CTA) Expression: A Strategy for Manipulating the Graft-Versus Leukaemia Response in Patients Allografted for Haematological Malignancies

An immunologically mediated graft-versus-tumor (GVT) underlies the curative effect of reduced intensity allografts in acute myeloid leukaemia (AML) and other haematologic malignancies. Cancer testis antigens (CTA) represent a family of immunodominant proteins that are variably expressed in haematological malignancies and represent a potential target of a GVT response. Importantly a number of CTA genes demonstrate promoter hypermethylation in solid tumours which can be reversed using demethylating agents such as azacitidine. We have therefore examined whether donor T cell responses to CTAs are observed in patients allografted for AML and multiple myeloma (MM) and whether epigenetic therapies can be used to manipulate T cell mediating killing of haemopoietic targets.We screened 37 patients with AML and 8 patients with MM, who had not received demethylating agent treatment, for T cell responses to 25 peptides derived from 10 CTA genes, including BAGE, LAGE, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-C2, RAGE-1, by interferon-γ cytokine secretion assay (IFN-γ CSA). We found CTA specific T cells in 11.1% of patients (5 of 45) with frequencies ranging from 0.0005% to 0.2% (median 0.024%). Subsequently, we studied expression of 15 cancer-testis antigens (CTA) at the RNA and protein levels in AML, MM and Hodgkins' lymphoma cell lines before and after exposure to demethylating agent 5-aza-2′-deoxycytidine (Aza) and histone deacetylase inhibitor sodium valproate (VPA), as single agents and in combination. We found that expression of CTAs HAGE, SACA3, SPANXB, LAGE, XAGE, MAGEA3 could be induced in a dose dependent manner by Aza alone but not with VPA alone. We also observed that expression induced by Aza treatment was increased further by combination treatment with VPA. Induction of CTAs was confirmed in vitro in primary AML cells and in vivo in 2/6 patients on VPA/Aza trial. Furthermore, using interferon-γ ELISA assay we observed that Aza-induced expression of the CTA MAGEA3 in MM cell lines was accompanied by increased recognition by MAGEA3-specific T cells.These studies confirm the importance of members of the CTA family as targets of a T cell mediated immune response. Our data demonstrate that the expression of these putative immunodominant antigens in haematological malignancies in disease such as AML and myeloma, can be significantly up-regulated by epigenetic therapies with functional increases in target cell killing and support the use of adjunctive epigenetic therapies after allogeneic transplantation with the aim of augmenting a GVL response. A Phase II clinical trial combining post transplant azacitidine with a reduced intensity allograft is currently ongoing in patients with AML.

Cancer Testis Antigens Residing on the X-Chromosome Are Preferentially Derepressed in Myeloid Leukemic Cells by the DNA Demethylating Agent 5-Aza-2'-Deoxycytidine (DAC).

Introduction: Azanucleoside DNA demethylating agents upregulate large sets of genes in vitro. Cancer testis antigens (CTAs) are a growing group of immunogenic proteins that provide attractive targets for cancer-specific immunotherapy in solid tumors. In contrast, only a very limited number of studies has been performed on epigenetic regulation of CTAs in myeloid leukemia. A single report suggested specific in vivo induction of CTA mRNAs in blasts from AML and MDS pts treated with intermediate-dose Decitabine (DAC; Sigalotti et al., Blood 101:4644–6, 2003). We wished to extend this finding by examining kinetics and spectrum of CTA and leukemia-associated antigen (LAA) regulation by DAC in myeloid cell lines and primary AML blasts.Materials and Methods: Myeloid cell lines were treated over 72 hrs with 50–200 nM DAC. RNA was isolated from peripheral blood blasts of AML pts treated with DAC (135mg/m2 over 72 hours within the 00331 phase II trial) before and during early phase of the treatment (= days 2, 5 from start of DAC) when circulating blasts were still >40% (mean 75%). Expression analyses were performed by Western Blot (NY-ESO-1), qPCR (NY-ESO-1, MAGEA1, MAGEA3, Myeloblastin), and by HG-U133plus 2.0 mRNA microarray.Results: The five myeloid cell lines Kasumi-1, U937, HL60, K562 and NB4 did not express NY-ESO-1 protein. Upon treatment with DAC, NY-ESO-1 was markedly de-repressed in 3/5 cell lines (Kasumi-1, U937, HL60). Induction of expression was dose- and time-dependent, with maximum induction at day 6, and reversible with prolonged culturing up to day 21. No derepression was seen when U-937 and HL-60 were treated with equitoxic low concentrations of cytarabine (not a Dnmt inhibitor). MAGEA1, MAGEA3 and the LAA Myeloblastin (MBN) were also induced by DAC in Kasumi-1, with the strongest effect observed for NY-ESO-1 and the least for MBN (high baseline expression). Expression analyses were extended to all 67 CTAs present on the U133plus array (45 located on the X-chromosome, 22 autosomally), of which 11/67 (17%) were induced >2 fold in Kasumi-1. Notably, all 11 CTA genes were X-chromosomal, whereas 0/22 autosomal CTAs showed reinduction (p= 0.008 by Fisher-Exact test). 2/9 LAA genes (22%) were also reinduced. In primary AML blasts from 9 pts, DAC treatment resulted in >2fold induction of 3 CTAs and 5 LAAs. qPCR demonstrated NY-ESO-1 upregulation in 2/5 patients, with a maximum at day 5 from start of treatment.Conclusions: Marked derepression of NY-ESO-1 and other CTAs by treatment of myeloid cell lines with an azanucleoside DNA demethylating agent occurred preferentially with genes residing on the X chromosome, in line with a major role of DNA methylation for their regulation. Derepression by DAC was also observed in vivo, albeit less marked and occurring early during treatment. One of the therapeutic effects of low-dose DNA demethylating agents on leukemic myeloid blasts may be mediated via upregulation of antigens rendering the malignant cells more immunogenic.

Expression of Cancer Testis Antigens as Potential Targets for Immunotherapy Is Retained Following Treatment in Multiple Myeloma.

Background: Multiple myeloma (MM) remains an incurable malignancy, but new treatment modalities can achieve durable remissions in most cases. Remission is generally regarded as a window of opportunity for active immunotherapy, aimed at eradicating residual disease. For immunotherapy, and vaccination in particular, the expression of cancer testis antigens (CTAs) in MM has emerged as a key target that is essentially tumor-specific. These CTAs are expressed at disease presentation, many confirmed also at the protein level. However, the question has remained whether treatment might critically alter the expression profile of CTAs. To address this, we have examined the expression levels of a large panel of known CTAs using gene expression profiles (GEPs) from patient cohorts in clinical trials at defined stages: at disease presentation in HOVON-65 and post-treatment in the APEX, SUMMIT and CREST trials.Methods and results: The APEX, SUMMIT and CREST trials have been described by Mulligan et al. (Blood , 109, 3177; 2007) and data consist of GEPs obtained from 264 patients with relapse following prior therapies (median number of therapies, 3; min-max, 1–14). In the HOVON-65 study, GEPs of 204 previously untreated myeloma cases were obtained. For comparison of both studies, original raw data files (CEL-files) were obtained and analyzed using Partek software. After normalization and merging, the expression of 62 CTA genes and gene clusters was evaluated. 63 probe sets were used, of which 51 correspond to 50 unique genes and 12 correspond to clusters of cancer antigen genes. Normalized expression levels for 24/62 genes/gene clusters were well above cut-off, and for remainder (38/62) were generally low with medians below 30. High median expression values, in the top 10% of CTA expression values, were found for SSX2, MAGE-A3 and MAGE-C1. Genes in the bottom 10% include MAGE-B1 and SPACA3. Notably, of 62, the fold change in expression level before and after treatment was at most 1.5 in any of the genes studied, indicating that the expression of the majority of CTAs is unchanged by treatment. For 8 genes/gene clusters, significantly decreased expression was found in relapsed cases compared to untreated MM, including GAGE3 and a gene cluster containing the GAGE1 gene.Conclusion: These clinical trial data indicate that targeting CTAs with immunotherapy during first remission or after relapse of MM remains a viable option.

DNA Hypomethylation Arises Later in Prostate Cancer Progression than CpG Island Hypermethylation and Contributes to Metastatic Tumor Heterogeneity

Hypomethylation of CpG dinucleotides in genomic DNA was one of the first somatic epigenetic alterations discovered in human cancers. DNA hypomethylation is postulated to occur very early in almost all human cancers, perhaps facilitating genetic instability and cancer initiation and progression. We therefore examined the nature, extent, and timing of DNA hypomethylation changes in human prostate cancer. Contrary to the prevailing view that global DNA hypomethylation changes occur extremely early in all human cancers, we show that reductions in (5me)C content in the genome occur very late in prostate cancer progression, appearing at a significant extent only at the stage of metastatic disease. Furthermore, we found that, whereas some LINE1 promoter hypomethylation does occur in primary prostate cancers compared with normal tissues, this LINE1 hypomethylation is significantly more pronounced in metastatic prostate cancer. Next, we carried out a tiered gene expression microarray and bisulfite genomic sequencing-based approach to identify genes that are silenced by CpG island methylation in normal prostate cells but become overexpressed in prostate cancer cells as a result of CpG island hypomethylation. Through this analysis, we show that a class of cancer testis antigen genes undergoes CpG island hypomethylation and overexpression in primary prostate cancers, but more so in metastatic prostate cancers. Finally, we show that DNA hypomethylation patterns are quite heterogeneous across different metastatic sites within the same patients. These findings provide evidence that DNA hypomethylation changes occur later in prostate carcinogenesis than the CpG island hypermethylation changes and occur heterogeneously during prostate cancer progression and metastatic dissemination.