Cancer/testis antigens are considered potential targets for immunotherapy due to their tumor-associated expression pattern. Although recent studies have demonstrated high expression of CT45 in classical Hodgkin's lymphomas (cHL), less is known about the expression pattern of other families of CTAs in cHL. We aim to evaluate the expression of MAGE-A family, MAGE-C1/CT7, MAGE-C2/CT10, NY-ESO1 and GAGE family in cHL and to correlate their expression with clinical and prognostic factors in cHL.
Tissue microarray was generated from 38 cHL archival cases from Pathology Department of Universidade Federal de Sao Paulo. Immunohistochemistry (IHC) was done using the following panel of antibodies: MAGE-A family (MA454, M3H67, 57B and 6C1), GAGE (#26), NY-ESO-1 (E978), MAGE-C1/CT7 (CT7-33) and MAGE-C2/CT10 (CT10#5).
We found CTA expression in 21.1% of our cHL series. Among the tested CTAs, only MAGE-A family 7/38 (18.4%) and MAGE-C1/CT7 5/38 (13.2%) were positive in our cHL samples. We found higher CTA positivity in advanced stage (28.6%) compared to early stage (11.8%) disease, but this difference was not statistically significant. Analysis of other clinicopathological subgroups of cHL including histological subtypes, EBV status and response to treatment also did not demonstrate statistical significant differences in CTA expression.
We found CTA expression in 21.1% of cHL samples using our panel. Our preliminary findings suggest that from all CTAs included in this study, MAGE-A family and MAGE-C1/CT7 are the most interesting ones to be explored in further studies.
Hodgkin's Lymphoma; cancer/testis antigens
Cancer–testis antigens (CTA) comprise a family of proteins, which are physiologically expressed in adult human tissues solely in testicular germ cells and occasionally placenta. However, CTA expression has been reported in various malignancies. CTAs have been identified by their ability to elicit autologous cellular and or serological immune responses, and are considered potential targets for cancer immunotherapy. The breast differentiation antigen NY-BR-1, expressed specifically in normal and malignant breast tissue, has also immunogenic properties. Here we evaluated the expression patterns of CTAs and NY-BR-1 in breast cancer in correlation to clinico-pathological parameters in order to determine their possible impact as prognostic factors.
The reactivity pattern of various mAbs (6C1, MA454, M3H67, 57B, E978, GAGE #26 and NY-BR-1 #5) were assessed by immunohistochemistry in a tissue micro array series of 210 randomly selected primary invasive breast cancers in order to study the diversity of different CTAs (e.g. MAGE-A, NY-ESO-1, GAGE) and NY-BR-1. These expression data were correlated to clinico-pathological parameters and outcome data including disease-free and overall survival.
Expression of at least one CTA was detectable in the cytoplasm of tumor cells in 37.2% of the cases. NY-BR-1 expression was found in 46.6% of tumors, respectively. Overall, CTA expression seemed to be linked to adverse prognosis and M3H67 immunoreactivity specifically was significantly correlated to shorter overall and disease-free survival (p=0.000 and 0.024, respectively).
Our findings suggest that M3H67 immunoreactivity could serve as potential prognostic marker in primary breast cancer patients. The exclusive expression of CTAs in tumor tissues as well as the frequent expression of NY-BR-1 could define new targets for specific breast cancer therapies.
Breast Cancer; Cancer-testis Antigen; NY-BR-1; Immunotherapy; Prognosis
Cancer/Testis Antigens (CTAs) are a promising class of tumor antigens that have a limited expression in somatic tissues (testis, ovary, fetal, and placental cells). Aberrant expression of CTAs in cancer cells may lead to abnormal chromosome segregation and aneuploidy. CTAs are regulated by epigenetic mechanisms (DNA methylation and acetylation of histones) and are attractive targets for immunotherapy in cancer because the gonads are immune privileged organs and anti-CTA immune response can be tumor-specific. Multiple myeloma (MM) is an incurable hematological malignancy, and several CTAs have been detected in many MM cell lines and patients. Among CTAs expressed in MM we must highlight the MAGE-C1/CT7 located on the X chromosome and expressed specificity in the malignant plasma cells. MAGE-C1/CT7 seems to be related to disease progression and functional studies suggests that this CTA might play a role in cell cycle and mainly in survival of malignant plasma cells, protecting myeloma cells against spontaneous as well as drug-induced apoptosis.
Cancer-testis antigens (CTAs) are suitable targets for cancer-specific immunotherapy. The aim of the study is to investigate the expression of CTAs in intrahepatic cholagiocarcinoma (IHCC) and evaluate their potential therapeutic values.
Eighty-nine IHCC patients were retrospectively assessed for their expression of CTAs and HLA Class I by immunohistochemistry using the following antibodies: MA454 recognizing MAGE-A1, 57B recognizing multiple MAGE-A (MAGE-A3/A4), E978 recognizing NY-ESO-1, and EMR8-5 recognizing HLA class I. The clinicopathological and prognostic significance of individual CTA markers and their combination were further evaluated.
The expression rates of MAGE-A1, MAGE-A3/4 and NY-ESO-1 were 29.2%, 27.0% and 22.5%, respectively. The concomitant expression of CTAs and HLA class I antigen was observed in 33.7% of the IHCC tumors. We found that positive MAGE-3/4 expression correlated with larger tumor size (≥ 5 cm), tumor recurrence and poor prognosis. Moreover, we identified 52 cases (58.4%) of IHCC patients with at least one CTA marker expression, and this subgroup displayed a higher frequency of larger tumor size and a shorter survival than the other cases. Furthermore, expression of at least one CTA marker was also an independent prognostic factor in patients with IHCC.
Our data suggest that specific immunotherapy targeted CTAs might be a novel treatment option for IHCC patients.
The aim of this study was to evaluate the frequency of expression of the cancer-testis antigens (CTAs) NY-ESO-1, MAGE-A4 and SAGE, in renal cell carcinoma (RCC) patients compared to that in head and neck cancer (HNC) patients, which represent a positive control with a high incidence of CTA expression, to identify novel target antigens for immunotherapy. We prospectively examined frozen tissue samples collected from surgery or biopsy from 35 RCC and 40 HNC patients. Total RNA was extracted, and real-time reverse transcription-polymerase chain reaction (RT)-PCR was performed to determine the expression of MAGE-A4, NY-ESO-1 and SAGE. MAGE-A4 was not detected in any of the RCC samples, although a low incidence of NY-ESO-1 (5.7%; 2/35) and SAGE (2.9%; 1/35) expression was observed. No samples demonstrated co-expression of the three CTAs. By contrast, a comparatively high incidence of CTA expression was detected in squamous cell carcinoma (SCC) specimens of HNC patients. The actual incidence was 42.5% (17/40) for MAGE-A4, 20% (8/40) for NY-ESO-1 and 15% (6/40) for SAGE. The incidence of co-expression was 7.5% (3/40) for MAGE-A4 and NY-ESO-1, 7.5% (3/40) for MAGE-A4 and SAGE, 7.5% (3/40) for NY-ESO-1 and SAGE, and 2.5% (1/40) for the CTAs. The number of HNC samples positive for MAGE-A4 was significantly higher compared to that of RCC samples. The remaining two antigens, NY-ESO-1 and SAGE, were expressed at high levels in HNC compared to RCC samples. Limited frequency of CTA (NY-ESO-1, MAGE-A4 and SAGE) expression was demonstrated in RCC compared to HNC samples.
cancer-testis antigen; kidney cancer
Glioblastoma (GBM) confers a dismal prognosis despite advances in current therapy. Cancer-testis antigens (CTA) comprise families of tumor-associated antigens that are immunogenic in different cancers. The aim of this study was to determine the expression profile of a large number of CTA genes in GBM.
We selected, from 153 CTA genes, those genes potentially expressed in GBM. The expression pattern of 30 CTA was then evaluated by RT-PCR in a series of 48 GBM and 5 normal brain samples. The presence of CTCFL protein was also evaluated by immunohistochemical staining.
Among the genes with no expression in normal brain, ACTL8 (57%), OIP5 (54%), XAGE3 (44%) and CTCFL (15%) were frequently expressed in GBM, while over 85% of the tumors expressed at least 1 of these four CTA. Coexpression of two or more CTA occurred in 49% of cases. CTCFL protein expression was detected in 13% of the GBM and was negative in normal brain samples. GBM expressing 3-4 CTA was associated with significantly better overall survival (OS) rates (P = 0.017). By multivariate analysis, mRNA positivity for 3-4 CTA (P = 0.044), radiotherapy (P = 0.010) and chemotherapy (P = 0.001) were independent prognostic factors for OS.
GBM frequently express ACTL8, OIP5, XAGE3 and CTCFL. A relatively high percentage of tumors expressed at least one of these four CTA, opening the perspective for their utility in antigen-specific immunotherapy. Furthermore, mRNA positivity for 3-4 CTA is an independent predictor of better OS for GBM patients.
Brain cancer; Glioblastoma; GBM; Cancer/Testis antigens; CTA expression
Primary testicular lymphoma (PTL) is a rare and lethal disease. The most common histological subtype is diffuse large B-cell lymphoma (DLBCL). Standard treatments are frequently ineffective. Thus, the development of novel forms of therapy is urgently required. Specific immunotherapy generating immune responses directed against antigen predominantly expressed by cancer cells such as cancer-testis antigens (CTA) may provide a valid alternative treatment for patients bearing PTL, alone or in combination with current therapies.
Three monoclonal antibodies (mAbs), 77B recognizing MAGE-A1, 57B recognizing an epitope shared by multiple MAGE-A CTA (multi-MAGE-A specific) and D8.38 recognizing NY-ESO-1/LAGE-1 were used for immunohistochemical staining of 27 PTL, including 24 DLBCL.
Expression of MAGE-A1 was infrequently detectable in DLBCL specimens (12.50%), whereas multi-MAGE-A and NY-ESO-1/LAGE-1 specific reagents stained the cytoplasms of tumor cells in DLBCL specimens with higher frequencies (54.17% and 37.50%, respectively) with different expression levels.
These results suggest that MAGE-A and NY-ESO-1/LAGE-1, possibly in combination with other CTA, might be used as targets for specific immunotherapy in DLBCL.
Primary testicular lymphoma; DLBCL; Cancer/testis antigens; MAGE-A; NY-ESO-1; Immunotherapy
The MAGE-C1/CT7 encodes a cancer/testis antigen (CTA), is located on the chromosomal region Xq26–27 and is highly polymorphic in humans. MAGE-C1/CT7 is frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. MAGEC1/CT7 expression is restricted to malignant plasma cells and it has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function this protein in the pathophysiology of MM is not yet understood. Our objectives were (1) to clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle in myeloma and (2) to evaluate the impact of silencing MAGE-C1/CT7 on myeloma cells treated with bortezomib. Myeloma cell line SKO-007 was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time quantitative PCR and western blot. Functional assays included cell proliferation, cell invasion, cell cycle analysis and apoptosis. Western blot showed a 70–80% decrease in MAGE-C1/CT7 protein expression in inhibited cells (shRNA-MAGE-C1/CT7) when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. However, we found a decreased percentage of cells in the G2/M phase of the cell cycle among inhibited cells, but not in the controls (p<0.05). When myeloma cells were treated with bortezomib, we observed a 48% reduction of cells in the G2/M phase among inhibited cells while controls showed 13% (empty vector) and 9% (ineffective shRNA) reduction, respectively (p<0.01). Furthermore, inhibited cells treated with bortezomib showed an increased percentage of apoptotic cells (Annexin V+/PI-) in comparison with bortezomib-treated controls (p<0.001). We found that MAGE-C1/CT7 protects SKO-007 cells against bortezomib-induced apoptosis. Therefore, we could speculate that MAGE-C1/CT7 gene therapy could be a strategy for future therapies in MM, in particular in combination with proteasome inhibitors.
The type I melanoma antigen gene (MAGE) proteins CT7 (MAGE-C1) and MAGE-A3 are commonly expressed in multiple myeloma (MM), and their expression correlates with increased plasma cell proliferation and poor clinical outcome. They belong to the cancer-testis antigen (CTAg) group of tumor-associated proteins, some of which elicit spontaneous immune responses in cancer patients. CT7 and MAGE-A3 are promising antigenic targets for therapeutic tumor vaccines in myeloma; therefore, it is critical to determine if they are immunogenic in MM patients. We analyzed cellular and humoral immune responses against CTAgs in patients with plasma cell dyscrasias: MM, monoclonal gammopathy of undetermined significance (MGUS), and Waldenström’s macroglobulinemia (WM). Bone marrow lymphocytes from two of four untreated MM patients exhibited CT7-specific cellular immune responses as measured by an autologous cellular immunity assay, the first such immune response to CT7 to be reported in cancer patients. Sera from 24 patients were screened by ELISA for humoral immune responses to CTAgs. Two patients with MM demonstrated positive titers, one for MAGE-A1 and the other for SSX1. These data demonstrate that CTAgs, particularly CT7, are immunogenic in MM patients and merit further exploration as targets of immunological therapy in MM.
human; multiple myeloma; CT antigens; cellular immunity; humoral immunity
The type I melanoma antigen gene (MAGE) proteins CT7
(MAGE-C1) and MAGE-A3 are commonly expressed in multiple myeloma (MM),
and their expression correlates with increased plasma cell proliferation
and poor clinical outcome. They belong to the cancer-testis antigen
(CTAg) group of tumor-associated proteins, some of which elicit
spontaneous immune responses in cancer patients. CT7 and MAGE-A3
are promising antigenic targets for therapeutic tumor vaccines in
myeloma; therefore, it is critical to determine if they are immunogenic
in MM patients. We analyzed cellular and humoral immune responses
against CTAgs in patients with plasma cell dyscrasias: MM, monoclonal
gammopathy of undetermined significance (MGUS), and Waldenström's
macroglobulinemia (WM). Bone marrow lymphocytes from two of four
untreated MM patients exhibited CT7-specific cellular immune responses
as measured by an autologous cellular immunity assay, the first
such immune response to CT7 to be reported in cancer patients. Sera
from 24 patients were screened by ELISA for humoral immune responses to
CTAgs. Two patients with MM demonstrated positive titers, one for
MAGE-A1 and the other for SSX1. These data demonstrate that CTAgs,
particularly CT7, are immunogenic in MM patients and merit further
exploration as targets of immunological therapy in MM.
myeloma; CT antigens; cellular immunity; humoral
AIM: To evaluate the diagnostic value of cancer-testis antigen (CTA) mRNA in peripheral blood samples from hepatocellular carcinoma (HCC) patients.
METHODS: 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 α-fetoprotein (AFP) in these patients was also determined.
RESULTS: 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 μg/L) for HCC, and was negative (AFP ≤ 20 μg/L) or positive with a low concentration (20 μg/L < AFP ≤ 400 μg/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.
CONCLUSION: Detecting MAGE-1, SSX-1 and CTp11 mRNA in PBMC improves the total diagnostic rate of HCC.
Hepatocellular carcinoma; α-fetoprotein; Cancer-testis antigen; Diagnosis; Nested reverse transcripts-polymerase chain reaction
Cancer-testis antigens (CTAs) such as MAGE are selectively expressed in various types of human neoplasms but not in normal tissues other than testis. This characteristic feature of CTAs makes them promising antigens for cancer-specific immunotherapy. A critical requirement for this therapy is identification of promising antigens. In this study, we investigated the expression of 6 genes recently identified by serological analysis of antigens by recombinant expression (SEREX) libraries: NY-ESO-1, LAGE-1, SCP-1, SSX-1, SSX-2, and SSX-4, in many surgical samples of gastrointestinal and breast carcinomas using reverse transcription-polymerase chain reaction. We found relatively high expression of SCP-1 (23.5%) and SSX-4 (20.6%) in gastric carcinoma, LAGE-1 (39.1%) and NY-ESO-1 (23.9%) in oesophageal carcinoma, and SCP-1 (34.1%) in breast carcinoma. We also found frequent synchronous expression with MAGE, including LAGE-1 (46.2%) in oesophageal carcinoma, SSX-4 (46.7%) in gastric carcinoma, and SCP-1 (38.3%) in breast carcinoma. Immunohistochemical analysis of the tumour samples expressing both MAGE-4 and NY-ESO-1 genes demonstrated differences in distribution between MAGE-4 and NY-ESO-1 in serial sections. We concluded that NY-ESO-1, LAGE-1, SCP-1 and SSX-4 genes may be promising candidates for cancer-specific immunotherapy in addition to MAGE, and that polyvalent cancer vaccines may be useful in cases of heterogeneous expressions of CTA genes in gastrointestinal and breast carcinomas. © 2001 Cancer Research Campaign http://www.bjcancer.com
MAGE; tumour-rejection antigens; cancer-testis antigen; immunotherapy; cancer vaccine
Cancer-Testis Antigens (CTAs) are immunogenic proteins that are poor prognostic markers in non-small cell lung cancer (NSCLC). We investigated expression of CTAs in NSCLC and their association with response to chemotherapy, genetic mutations and survival.
We studied 199 patients with pathological N2 NSCLC treated with neoadjuvant chemotherapy (NAC; n = 94), post-operative observation (n = 49), adjuvant chemotherapy (n = 47) or unknown (n = 9). Immunohistochemistry for NY-ESO-1, MAGE-A and MAGE-C1 was performed. Clinicopathological features, response to neoadjuvant treatment and overall survival were correlated. DNA mutations were characterized using the Sequenom Oncocarta panel v1.0. Affymetrix data from the JBR.10 adjuvant chemotherapy study were obtained from a public repository, normalised and mapped for CTAs.
NY-ESO-1 was expressed in 50/199 (25%) samples. Expression of NY-ESO-1 in the NAC cohort was associated with significantly increased response rates (P = 0.03), but not overall survival. In the post-operative cohort, multivariate analyses identified NY-ESO-1 as an independent poor prognostic marker for those not treated with chemotherapy (HR 2.61, 95% CI 1.28–5.33; P = 0.008), whereas treatment with chemotherapy and expression of NY-ESO-1 was an independent predictor of improved survival (HR 0.267, 95% CI 0.07–0.980; P = 0.046). Similar findings for MAGE-A were seen, but did not meet statistical significance. Independent gene expression data from the JBR.10 dataset support these findings but were underpowered to demonstrate significant differences. There was no association between oncogenic mutations and CTA expression.
NY-ESO-1 was predictive of increased response to neoadjuvant chemotherapy and benefit from adjuvant chemotherapy. Further studies investigating the relationship between these findings and immune mechanisms are warranted.
Neoplastic populations with stem cell potential have been most recently identified in human cutaneous melanoma, and initially characterized for their phenotypic profile. Being melanoma stem cells (MSC) the most desirable target of therapeutic intervention, we asked whether they express the epigenetically-regulated Cancer Testis Antigens (CTA) on which melanoma immunotherapy is increasingly focusing. Reverse transcription-PCR analyses identified the presence of the large majority of investigated CTA (i.e., MAGE, GAGE, NY-ESO and SSX families) in different MSC populations. MSC expressed MAGE-A proteins as detected by western blot; noteworthy, the distribution of MAGE-A proteins was highly homogeneous within given MSC populations as shown by confocal immunofluorescence. Promoter methylation studies unveiled a homogeneously-demethylated MAGE-A3 promoter that paired MAGE-A3 expression in MSC. Altogether these findings demonstrate that MSC can be efficiently targeted by CTA-directed immunotherapeutic approaches, and suggest that epigenetic patterns most likely drive the expression of CTA in MSC as previously shown for melanoma cells.
cancer stem cells; melanoma; immunotherapy; DNA methylation; cancer testis antigens
Pluripotent stem cells can differentiate into various lineages but undergo genetic and epigenetic changes during long-term cultivation and, therefore, require regular monitoring. The expression patterns of cancer-testis antigens (CTAs) MAGE-A2, -A3, -A4, -A6, -A8, -B2, and GAGE were examined in undifferentiated human embryonic stem (hES) cells, their differentiated derivatives, teratocarcinoma (hEC) cells, and cancer cell lines of neuroectodermal and mesodermal origin. Undifferentiated hES cells and embryoid body cells expressed MAGE-A3, -A6, -A4, -A8, and GAGEs while later differentiated derivatives expressed only MAGE-A8 or MAGE-A4. Likewise, mouse pluripotent stem cells also express CTAs of Magea but not Mageb family. Despite similarity of the hES and hEC cell expression patterns, MAGE-A2 and MAGE-B2 were detected only in hEC cells but not in hES cells. Moreover, our analysis has shown that CTAs are aberrantly expressed in cancer cell lines and display low tissue specificity. The identification of CTA expression patterns in pluripotent stem cells and their derivatives may be useful for isolation of abnormally CTA-expressing cells to improve the safety of stem-cell based therapy.
The expression of Cancer/Testis (CT) antigens in some tumors and restricted expression in normal tissue make CT antigens attractive vaccine targets. We evaluated the expression of MAGE-A3, PLAC1, GAGE, and CTAG2 in a series of colorectal cancers (CRC). CT mRNA expression was determined via quantitative PCR on paired tumors and normal tissue samples from 82 CRC patients. In addition, plasma antibody titers specific to MAGE-A3, PLAC1, GAGE, and CTAG2 were determined via ELISA. Tissue expression of MAGE-A3 was assessed via a standard IHC protocol. The Student’s t-test was used for statistical analysis (significance p < 0.05). Tumor expression of MAGE-A3, CTAG2, and GAGE was compared to the levels of expression in testis. The percentage of samples that had a tumor vs. testis expression ratio above 0.1% was: MAGE-A3 (28%) and CTAG2 (17%) but no tumor presented GAGE expression levels above 0.1%. The expression levels of PLAC1 in tumors were compared to the levels in placenta, and in 12.8% of the samples analyzed, these levels were above 0.1%. Sero-reactivity specific for MAGE-A genes and PLAC1 was noted in 2.4% and 2.6% of patients, respectively. MAGE-A3 and PLAC1 may hold promise as vaccine targets for CRC. Further study is warranted.
MAGE-A3; tumor expression; colorectal cancer; Cancer/Testis antigens
Immunotherapy is theoretically an attractive therapeutic option for patients with hematological malignancies. Various laboratory studies suggested the importance of the choice of tumor antigen for successful immunotherapy. Cancer-testis antigens (CTAs) are potentially suitable molecules for tumor vaccines of hematological malignancies because of their high immunogenicity in vivo, even in cancer-bearing patients, and their relatively restricted normal tissue distribution. Tumor cell kill using a CTA-based immunotherapy will, therefore, be more specific and associated with less toxicities when compared to chemotherapy. Many CTAs have been identified in various hematologic malignancies. In this review, we will take the readers through the journey of hopes and the disappointments arisen from the discovery of CTAs. We will describe the features of CTAs and their expression in hematologic malignancies. We will also discuss the mechanisms regulating the expression of these CTAs, from a primary regulatory mechanism involving DNA methylation to secondary controls by cytokines. Finally, we will address the potential obstacles that will prevent the successful use of CTAs as targets for tumor immunotherapy.
Cancer-testis antigens; hematological malignancies; DNA methylation; heterogeneity of antigen expression
Cancer/testis antigens (CTAs) are a group of tumour-associated antigens (TAAs) that display normal expression in the adult testis—an immune-privileged organ—but aberrant expression in several types of cancers, particularly in advanced cancers with stem cell-like characteristics. There has been an explosion in CTA-based research since CTAs were first identified in 1991 and MAGE-1 was shown to elicit an autologous cytotoxic T-lymphocyte (CTL) response in a patient with melanoma. The resulting data have not only highlighted a role for CTAs in tumorigenesis, but have also underscored the translational potential of these antigens for detecting and treating many types of cancers. Studies that have investigated the use of CTAs for the clinical management of urological malignancies indicate that these TAAs have potential roles as novel biomarkers, with increased specificity and sensitivity compared to those currently used in the clinic, and therapeutic targets for cancer immunotherapy. Increasing evidence supports the utilization of these promising tools for urological indications.
Cancer-testis (CT) antigen genes might promote the progression of multiple myeloma (MM). CT antigens may act as diagnostic and prognostic markers in MM, but their expression levels and clinical implications in this disease are not fully understood. This study measured the expression levels of four CT antigen genes in Chinese patients with MM and explored their clinical implications.
Real-time quantitative polymerase chain reaction (qPCR) was used to quantify the expression of MAGE-C1/CT7, MAGE-A3, MAGE-C2/CT10 and SSX-2 mRNA in 256 bone marrow samples from 144 MM patients.
In the newly diagnosed patients, the positive expression rates were 88.5% for MAGE-C1/CT7, 82.1% for MAGE-C2/CT10, 76.9% for MAGE-A3 and 25.6% for SSX-2. The expression levels and the number of co-expressed CT antigens correlated significantly with several clinical indicators, including the percentage of plasma cells infiltrating the bone marrow, abnormal chromosome karyotypes and the clinical course.
MAGE-C1/CT7, MAGE-A3, MAGE-C2/CT10 and SSX-2 expression levels provide potentially effective clinical indicators for the auxiliary diagnosis and monitoring of treatment efficacy in MM.
Cancer-testis antigen gene; Multiple myeloma; Real-time quantitative polymerase chain reaction
Immunotherapy targeting MAGE-A3 in multiple myeloma (MM) could eradicate highly aggressive and proliferative clonal cell populations responsible for relapse. However, expression of many cancer-testis antigens, including MAGE-A3, can be heterogeneous, leading to the potential for tumor escape despite MAGE-A3-induced immunity. We hypothesized that a combination of the hypomethylating agent 5-azacitidine (5AC) and the histone deacetylase inhibitor (HDACi) MGCD0103 (MGC) could induce MAGE-A3 expression in MAGE-A3-negative MM, resulting in recognition and killing of MM cells by MAGE-A3-specific cytotoxic T lymphocytes (CTL).
Gene expression analyses of MAGE-A3 expression in primary MM patient samples at diagnosis and relapse were completed to identify populations that would benefit from MAGE-A3 immunotherapy. MM cell lines were treated with 5AC and MGC. Real-time polymerase chain reaction (PCR) and Western blotting were performed to assess MAGE-A3 RNA and protein levels, respectively. Chromium-release assays and interferon (IFN) secretion assays were employed to ascertain MAGE-A3 CTL specificity against treated targets.
Gene expression analysis revealed that MAGE-A3 is expressed in MM patients at diagnosis (25%) and at relapse (49%). We observed de novo expression of MAGE-A3 RNA and protein in MAGE-A3-negative cell lines treated with 5AC. MGC treatment alone did not induce expression but sequential 5AC/MGC treatment led to enhanced expression and augmented recognition by MAGE-A3-specific CTL, as assessed by 51Cr-release assays (P = 0.047) and enzyme-linked immunosorbent assay (ELISA) for IFN-γ secretion (P = 0.004).
MAGE-A3 is an attractive target for immunotherapy of MM and epigenetic modulation by 5AC, and MGC can induce MAGE-A3 expression and facilitate killing by MAGE-A3-specific CTL.
5-azacitidine; cancer-testis antigen; demethylation; epigenetics; histone deactylase inhibitor; hypomethylation; MAGE-A3; MGCD0103; multiple myeloma
Cancer-testis antigens (CTAs) represent an expanding class of tumor-associated proteins defined on the basis of their tissue-restricted expression to testis or ovary germline cells and frequent ectopic expression in tumor tissue. The expression of CTA in MHC class I-deficient germline cells makes these proteins particularly attractive as immunotherapeutic targets because they serve as essentially tumor-specific antigens for MHC class I-restricted CD8+ T cells. Moreover, because CTAs are expressed in many types of cancer, any therapeutic developed to target these antigens might have efficacy for multiple cancer types. Of particular interest among CTAs is the synovial sarcoma X chromosome breakpoint (SSX) family of proteins, which includes ten highly homologous family members. Expression of SSX proteins in tumor tissues has been associated with advanced stages of disease and worse patient prognosis. Additionally, both humoral and cell-mediated immune responses to SSX proteins have been demonstrated in patients with tumors of varying histological origin, which indicates that natural immune responses can be spontaneously generated to these antigens in cancer patients. The current review will describe the history and identification of this family of proteins, as well as what is known of their function, expression in normal and malignant tissues, and immunogenicity.
To observe mRNA expression of tumor-specific antigen MAGE, BAGE and GAGE in epithelial ovarian cancer tissues and cell lines, to explore the relationship between gene expression and diagnosis, treatment and prognosis of ovarian cancer, and to evaluate the feasibility of their gene products as markers, and an immunotherapy target for ovarian cancer.
mRNA expression of MAGE-1, MAGE-3, GAGE-1/2 and BAGE were determined by reverse transcription polymerase chain reaction (RT-PCR) in 14 cases of normal ovarian tissue, 20 cases of ovarian benign tumor specimens, 41 cases of ovarian cancer specimens, and ovarian cancer cell lines SKOV3, A2780, and COC1.
MAGE, GAGE and BAGE genes were not expressed in normal ovarian tissue. In benign tumors, only the MAGE gene was expressed; the expression rate of this gene in benign tumors was 15% (3/20). In ovarian cancer tissues, MAGE-1 and MAGE-3 was highly expressed, with expression rates of 53.7% (22/41) and 36.6% (15/41), while GAGE-1/2 and BAGE had relatively low expression, with rates of 26.8% (11/41) and 14.6% (6/41). In metastatic lesions of ovarian cancer, only MAGE-1 and BAGE were expressed, with expression rates of 28.6% (2/7) and 14.3% (1/7). The positive expression rates of MAGE-1 and MAGE-3 in serous cystadenocarcinoma were significantly higher than that in other types of ovarian cancer (P < 0.05). Gene expression rate was not correlated with menopause or lymph node metastasis. Positive expression of MAGE-1 and MAGE-3 was positively correlated with tumor differentiation and the clinical stage of the ovarian cancer. In addition, the positive expression rate of BAGE was significantly higher in ovarian cancer patients with ascites (P < 0.05). The mRNA expression profiles of MAGE, GAGE and BAGE in ovarian carcinoma cell lines SKOV3, A2780 and COC1 varied, but there was at least one gene expressed in each cell line.
Tumor-specific antigen MAGE, BAGE and GAGE may play a role in the occurrence and development of ovarian cancer. These genes can be used as one of the important indicators for early diagnosis, efficacy evaluation and prognostic determination of ovarian cancer.
The Cancer/Testis Antigens (CTAs) are an important group of proteins that are typically restricted to the testis in the normal adult but are aberrantly expressed in several types of cancers. As a result of their restricted expression patterns, the CTAs could serve as unique biomarkers for cancer diagnosis/prognosis. The aim of this study was to identify promising CTAs that are associated with prostate cancer (PCa) recurrence following radical prostatectomy (RP).
The expression of 5 CTAs was measured by quantitative multiplex real-time PCR using prostate tissue samples obtained from 72 patients with apparently clinically localized PCa with a median of two years follow-up (range, 1 to 14 years).
The expression of CTAs namely, CEP55, NUF2, PBK and TTK were significantly higher while PAGE4 was significantly lower in patients with recurrent disease. All CTAs with the exception of TTK were significantly correlated with the prostatectomy Gleason score, but none were correlated with age, stage, or preoperative PSA levels. In univariate proportional hazards models, CEP55 (HR = 3.59, 95% CI: 1.50-8.60), p = 0.004; NUF2 (HR = 2.28, 95% CI: 1.11-4.67), p = 0.024; and PAGE4 (HR = 0.44, 95% CI: 0.21-0.93), p = 0.031 were significantly associated with the risk of PCa recurrence. However, the results were no longer significant after adjustment for prostatectomy Gleason score.
To our knowledge, this is the first study to identify CTAs as biomarkers that can differentiate patients with recurrent and non-recurrent disease following RP and underscores its potential impact on PCa prognosis and treatment.
Ovarian cancer is one of the neoplasms affecting the reproductive tract associated with high mortality rate because of limited therapeutic options and an elevated incidence of chemoresistance and recurrence. In this context, immunotherapy may constitute a promising approach to improve survival rates and clinical outcome, raising the need for specific target antigens. Cancer-testis antigens (CTAs) are considered promising candidates in this sense because they are aberrant expressed by various malignancies but not by non-transformed tissue, with the exception of testes. Here, we examined the expression and potential to promote humoral immune responses of a novel CTA, A-kinase anchor protein 4 (AKAP4), among 38 ovarian carcinoma patients. Our results reveal that AKAP4 was expressed at both the mRNA and protein levels in 89% (34/38) of ovarian carcinoma tissue specimens but not in 21 matched adjacent non-cancerous tissues. In addition, a humoral response against AKAP4 was detected in 58% (22/38) of ovarian carcinoma patients by ELISA. In particular, 65% (22/34) patients bearing an AKAP4-expressing tumor exhibited circulating anti-AKAP4 antibodies. Interestingly, the majority of specimens were categorized as ovarian serous adenocarcinoma and serous papillary carcinoma, of which 93% (28/30) and 100% (6/6), respectively, expressed AKAP4. A humoral response against AKAP4 was detected in 79% (19/24) and 67% (4/6) of ovarian serous adenocarcinoma and serous papillary carcinoma patients, respectively. The presence of circulating anti-AKAP4 antibodies suggests the AKAP4 is highly immunogenic in ovarian serous carcinoma patients. Our study lays the foundations for exploring AKAP4 as a potential target for the immunotherapy of ovarian cancer.
AKAP4; cancer-testis antigen; immunotherapy; ovarian serous carcinoma; vaccine
Background: The application of lymphoscintigraphy followed by sentinel lymph node (SN) biopsy to patients with primary melanoma has revolutionised the ability to identify accurately, yet conservatively, those patients who harbour occult nodal metastases. The molecular detection of SN micrometastases facilitates the cost effective analysis of the entire SN using multiple markers. Currently, a lack of marker specificity is the main barrier preventing the molecular evaluation of SN tissue from becoming clinically applicable.
Aims: To develop a reproducible multimarker reverse transcription-polymerase chain reaction (RT-PCR) assay, with the emphasis on achieving high specificity for the accurate detection of melanoma metastases in nodal tissue.
Methods: Three pigment cell specific (PCS) markers—tyrosinase, Pmel-17, and MART-1—and one cancer testis antigen (CTA)—MAGE-3—were selected for use in a multimarker RT-PCR assay. The conditions for this assay were optimised.
Results: High specificity was achievable for each marker by optimising the PCR cycle number such that unwanted transcripts (that is, illegitimate transcripts and/or specific transcripts from other low abundance nodal cell types) remained undetectable in appropriate controls (normal visceral nodes). Tyrosinase was 100% specific at 40 PCR cycles, MAGE-3 and MART-1 at 35 PCR cycles, and Pmel-17 at 30 PCR cycles. Tyrosinase proved to be the most sensitive marker, detecting 10 melanoma cells in 0.1 g of nodal tissue.
Conclusions: Excellent reproducibility of the entire nodal processing and RT-PCR protocol for the detection of very low numbers of melanoma cells in nodal tissue was shown, although there is a risk of false positives using the PCS markers alone, because of an approximate 4–8.5% incidence rate of nodal nevi in melanoma draining SNs (these nevi being absent in all other normal nodes). MAGE-3 was shown to be the only marker that is not expressed by melanocytes. However, because not all melanomas express MAGE-3, it is recommended that more emphasis should be placed on the development of a panel of CTA markers to ensure a zero false positive rate and to provide optimum detection.
melanoma; prognosis; reverse transcription-polymerase chain reaction; sentinel lymph node; specificity; metastasis