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.
The advancement of cancer immunotherapy faces barriers which limit its efficacy. These include weak immunogenicity of the tumor, as well as immunosuppressive mechanisms which prevent effective antitumor immune responses. Recent studies suggest that aberrant expression of cancer testis antigens (CTAs) can generate robust antitumor immune responses, which implicates CTAs as potential targets for immunotherapy. However, the heterogeneity of tumor cells in the presence and quantity of CTA expression results in tumor escape from CTA-specific immune responses. Thus, the ability to modulate the tumor cell epigenome to homogenously induce expression of such antigens will likely render the tumor more immunogenic. Additionally, emerging studies suggest that suppression of antitumor immune responses may be overcome by reprogramming innate and adaptive immune cells. Therefore, this paper discusses recent studies which address barriers to successful cancer immunotherapy and proposes a strategy of modulation of tumor-immune cell crosstalk to improve responses in carcinoma patients.
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
Background. Cancer/testis antigens (CTAs) are ideal targets for cancer immunotherapy in virtue of their restricted expression profile in normal tissues. However, CTA-targeted immunotherapy has been rather disappointing clinical setting for CTAs are downregulated by cytosine-phosphate-guanosine (CpG) methylation in their promoter regions, so that tumor cells have low immunogenicity. Methods. We reinduced mouse CTA P1A through demethylation process and generated P1A-specific cytotoxic lymphocytes (CTLs) by immunizing BALB/c (H-2d) mice with dendritic cells pulsed with a P1A-specific peptide and CpG oligodeoxynucleotide (ODN) immune adjuvant. Results. We found that demethylation and CpG ODN immune adjuvant stimulation facilitated DC maturation and enhanced the allogenic capacity of P1A-specific CTLs against target cells both in vitro and in vivo. Conclusions. Our results suggested that CTA induction and immune adjuvant stimulation is a feasible strategy in cancer immunotherapy.
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.
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
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
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.
Recent studies suggest that immunotherapy targeting specific tumor-associated antigens (TAAs) may be beneficial in cancer patients. However, most of these TAAs are tumor type specific and heterogeneous among patients, thus limiting their applications. Here, we describe the de novo induction of a cancer/testis antigen (CTA) for immunotherapy of tumors of various histologies. The murine CTA P1A, normally expressed only in a few tumor lines, could be induced de novo in all P1A-negative cancer lines of eight histologic origins in vitro and in various murine xenografts by systemic administration of 5-aza-2′-deoxycytidine. The induction of P1A expression correlated strongly with demethylation of the CpG island in the promoter region of this gene. The induced antigen was processed and presented properly for recognition by H-2Ld-restricted P1A-specific CTLs. The combination of a demethylating agent and adoptive transfer of P1A-specific CTL effectively treated lung metastases in syngeneic mice challenged with P1A-negative 4T1 mammary carcinoma cells. These data show a novel strategy of combined chemoimmunotherapy of cancer targeting a CTA induced de novo in a broad range of tumor histologies, and support further evaluation of chromatin-remodeling agents for human cancer therapy.
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.
The Cancer/Testis Antigens (CTAs) are a heterogeneous group of proteins whose expression is typically restricted to the testis. However, they are aberrantly expressed in most cancers that have been examined to date. Broadly speaking, the CTAs can be divided into two groups: the CTX antigens that are encoded by the X-linked genes and the non-X CT antigens that are encoded by the autosomes. Unlike the non-X CTAs, the CTX antigens form clusters of closely related gene families and their expression is frequently associated with advanced disease with poorer prognosis. Regardless however, the mechanism(s) underlying their selective derepression and stage-specific expression in cancer remain poorly understood, although promoter DNA demethylation is believed to be the major driver.
Here, we report a systematic analysis of DNA methylation profiling data from various tissue types to elucidate the mechanism underlying the derepression of the CTAs in cancer. We analyzed the methylation profiles of 501 samples including sperm, several cancer types, and their corresponding normal somatic tissue types.
We found strong evidence for specific DNA hypomethylation of CTA promoters in the testis and cancer cells but not in their normal somatic counterparts. We also found that hypomethylation was clustered on the genome into domains that coincided with nuclear lamina-associated domains (LADs) and that these regions appeared to be insulated by CTCF sites. Interestingly, we did not observe any significant differences in the hypomethylation pattern between the CTAs without CpG islands and the CTAs with CpG islands in the proximal promoter.
Our results corroborate that widespread DNA hypomethylation appears to be the driver in the derepression of CTA expression in cancer and furthermore, demonstrate that these hypomethylated domains are associated with the nuclear lamina-associated domains (LADS). Taken together, our results suggest that wide-spread methylation changes in cancer are linked to derepression of germ-line-specific genes that is orchestrated by the three dimensional organization of the cancer genome.
DNA hypomethylation; Cancer/Testis antigens; Lamina attachment domains; Insulator regions
The Cancer-Testis antigens (CTA) are proteins expressed in human germ line and certain cancer cells. CTAs form a large gene family, representing 10% of X-chromosomal genes. They have high potential for cancer-specific immunotherapy. However, their biological functions are currently unknown. Prostate associated genes (PAGE) are characterized as CTAs. PAGE5 is one of six proteins belonging to this protein family, also called CT16.
In this study we show, using bioinformatics, chromatographic and solution state NMR spectroscopic methods, that PAGE5 is an intrinsically disordered protein (IDP).
The study stands out as the first time structural characterization of the PAGE family protein and introduces how solution state NMR spectroscopy can be effectively utilized for identification of molecular recognition regions (MoRF) in IDPs, known often as transiently populated secondary structures.
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
Genes expressed both in normal testis and in malignancies (Cancer/ Testis associated genes – CTA) have become the most extensively studied antigen group in the field of tumour immunology. Despite this, many fundamentally important questions remain unanswered: what is the connection between germ-cell specific genes and tumours? Is the expression of these genes yet another proof for the importance of genome destabilisation in the process of tumorigenesis?, or maybe activation of these genes is not quite random but instead related to some programme giving tumours a survival advantage?
This review collates most of the recent information available about CTAs expression, function, and regulation. The data suggests a programme related to ontogenesis, mostly to gametogenesis. In the "brain-storming" part, facts in conflict with the hypothesis of random CTA gene activation are discussed. We propose a programme borrowed from organisms phylogenetically much older than humans, which existed before the differentiation of sexes. It is a programme that has served as a life cycle with prominent ploidy changes, and from which, as we know, the germ-cell ploidy cycle – meiosis – has evolved. Further work may show whether this hypothesis can lead to a novel anti-tumour strategy.
CTA genes; gametogenesis; polyploidy
Chondrosarcoma has no proven systemic option in the metastatic setting. The development of a non-cross-resistant strategy, such as cellular immunotherapy using antigen-specific T cells would be highly desirable. NY-ESO-1 and PRAME are members of the Cancer Testis Antigen (CTA) family that have been identified as promising targets for T cell therapy. LAGE-1 is a cancer testis antigen 90% homologous to NY-ESO-1, sharing the 157–165 A*0201 NY-ESO-1 epitope with its transcript variant, LAGE-1s. A number of CTA's have been induced using 5-Aza-2-Deoxycitabine (5-Aza-dC) in other cancers. We sought to evaluate the feasibility of targeting chondrosarcoma tumors using NY-ESO-1/LAGE-1s and PRAME specific T cells using 5-Aza-dC to induce antigen expression.
We used 11 flash frozen tumors from the University of Washington tumor bank to test for the expression of NY-ESO-1, PRAME, LAGE-1s and LAGE-1L in chondrosarcoma tumors. Using four chondrosarcoma cell lines we tested the expression of these CTA's with and without 5-Aza-dC treatments. Finally, using NY-ESO-1/LAGE-1s and PRAME specific effectors that we generated from sarcoma patients, we evaluated the ability of these T cells to lyse A*0201 expressing chondrosarcoma cell lines in vitro both with and without 5-Aza-dC treatment.
A minority (36%) of chondrosarcoma tumors expressed either NY-ESO-1 or LAGE-1s at >10% of our reference value and none expressed PRAME at that level. However, in all four of the chondrosarcoma cell lines tested, NY-ESO-1 and PRAME expression could be induced following treatment with 5-Aza-dC including in cell lines where expression was absent or barely detectable. Furthermore, NY-ESO-1/LAGE-1s and PRAME specific CD8+ effector T cells were able to specifically recognize and lyse A*0201 expressing chondrosarcoma cell lines following 5-Aza-dC treatment.
These data suggest that adoptive immunotherapy in combination with 5-Aza-dC may be a potential strategy to treat unresectable or metastatic chondrosarcoma patients where no proven systemic therapies exist.
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
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 Cancer/Testis Antigens (CTAs) are a group of heterogeneous proteins that are typically expressed in the testis but aberrantly expressed in several types of cancer. Although overexpression of CTAs is frequently associated with advanced disease and poorer prognosis, the significance of this correlation is unclear since the functions of the CTAs in the disease process remain poorly understood. Here, employing a bioinformatics approach, we show that a majority of CTAs are intrinsically disordered proteins (IDPs). IDPs are proteins that, under physiological conditions in vitro, lack rigid 3D structures either along their entire length or in localized regions. Despite the lack of structure, most IDPs can transition from disorder to order upon binding to biological targets and often promote highly promiscuous interactions. IDPs play important roles in transcriptional regulation and signaling via regulatory protein networks and are often associated with dosage sensitivity. Consistent with these observations, we find that several CTAs can bind DNA, and their forced expression appears to increase cell growth implying a potential dosage-sensitive function. Furthermore, the CTAs appear to occupy ‘hub’ positions in protein regulatory networks that typically adopt a ‘scale-free’ power law distribution. Taken together, our data provide a novel perspective on the CTAs implicating them in processing and transducing information in altered physiological states in a dosage-sensitive manner. Identifying the CTAs that occupy hub positions in protein regulatory networks would allow a better understanding of their functions as well as the development of novel therapeutics to treat cancer.
Cancer/Testis Antigens; Intrinsically Disordered Proteins; Dosage Sensitivity; 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.
Light-chain amyloidosis (AL) is a plasma cell dyscrasia closely related to multiple myeloma. In multiple myeloma, the cancer-testis antigens (CTAs) CT7 (MAGE-C1), CT10 (MAGE-C2) and MAGE-A CTAs are expressed in up to 80% of cases. In this study, we investigated the expression and immunogenicity of several CTAs in patients with AL amyloidosis in a total of 38 bone marrow specimens by employing standard immunohistochemistry techniques on paraffin-embedded archival tissues. Plasma samples from 35 patients (27 with matched bone marrow samples) were also analyzed by ELISA for sero reactivity to a group of full-length CTA proteins. CT7 was present in 25/38 (66%) while CT10 was demonstrated in 3/38 and GAGE in 1/38 AL amyloid cases. The expression pattern was mostly focal. There were no significant differences with regard to organ involvement, response to treatment, or prognosis in CTA positive compared to negative cases. None of the specimens showed spontaneous humoral immunity to CT7, but sero reactivity was observed in individual patients to other CTAs. This study identifies CT7 as the prevalent CTA in plasma cells of patients with AL amyloidosis. Further analyses determining the biology of CTAs in AL amyloidosis and their value as potential targets for immunotherapy are warranted.
AL amyloidosis; cancer-testis antigens; stem cell transplantation
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 introduction of serum prostate-specific antigen (PSA) in the 1980s has dramatically altered and benefited the initial diagnosis of prostate cancer. However, the widespread use of PSA testing has resulted in overdetection and overtreatment of potentially indolent disease. Thus, a clinical dilemma today in the management of prostate cancer is to discern men with aggressive disease who need definitive treatment from men whose disease are not lethal. Although several serum and tissue biomarkers have been evaluated during the past decade, improved markers are still needed to enhance the accuracy, with which patients at risk can be discerned and treated more aggressively. The cancer/testis antigens (CTAs) are a group of proteins that are restricted to the testis in the normal adult, but are aberrantly expressed in several types of cancers. Because of their restricted expression pattern, the CTAs represent attractive biomarker candidates for cancer diagnosis/prognosis. Furthermore, several studies to date have reported the differential expression of CTAs in prostate cancer. Here, we review recent developments that demonstrate the potential of the CTAs as biomarkers to discern the aggressive phenotype of prostate cancer.
cancer/testis antigens; DNA microarrays; prostate cancer; prostate carcinoma tumor antigen
Testis-specific transcription factor BORIS (Brother of the Regulator of Imprinted Sites), a paralog and proposed functional antagonist of the widely expressed CTCF, is abnormally expressed in multiple tumor types and has been implicated in the epigenetic activation of cancer-testis antigens (CTAs). We have reported previously that suprabasin (SBSN), whose expression is restricted to the epidermis, is epigenetically derepressed in lung cancer. In this work, we establish that SBSN is a novel non-CTA target of BORIS epigenetic regulation. With the use of a doxycycline-inducible BORIS expressing vector, we demonstrate that relative BORIS dosage is critical for SBSN activation. At lower concentrations, BORIS induces demethylation of the SBSN CpG island and disruption and activation of chromatin around the SBSN transcription start site (TSS), resulting in a 35-fold increase in SBSN expression in the H358 human lung cancer cell line. Interestingly, increasing BORIS concentrations leads to a subsequent reduction in SBSN expression via chromatin repression. In a similar manner, increase in BORIS concentrations leads to eventual decrease of cell growth and colony formation. This is the first report demonstrating that different amount of BORIS defines its varied effects on the expression of a target gene via chromatin structure reorganization.
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 (CTAs) were first discovered as immunogenic targets normally expressed in germline cells, but differentially expressed in a variety of human cancers. In this study, we used an integrative epigenetic screening approach to identify coordinately expressed genes in human non-small cell lung cancer (NSCLC) whose transcription is driven by promoter demethylation.
Our screening approach found 290 significant genes from the over 47,000 transcripts incorporated in the Affymetrix Human Genome U133 Plus 2.0 expression array. Of the top 55 candidates, 10 showed both differential overexpression and promoter region hypomethylation in NSCLC. Surprisingly, 6 of the 10 genes discovered by this approach were CTAs. Using a separate cohort of primary tumor and normal tissue, we validated NSCLC promoter hypomethylation and increased expression by quantitative RT-PCR for all 10 genes. We noted significant, coordinated coexpression of multiple target genes, as well as coordinated promoter demethylation, in a large set of individual tumors that was associated with the SCC subtype of NSCLC. In addition, we identified 2 novel target genes that exhibited growth-promoting effects in multiple cell lines.
Coordinated promoter demethylation in NSCLC is associated with aberrant expression of CTAs and potential, novel candidate protooncogenes that can be identified using integrative discovery techniques. These findings have significant implications for discovery of novel CTAs and CT antigen directed immunotherapy.