Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer.

An autochthonous model of pancreatic ductal adenocarcinoma (PDA) permitted the analysis of why immunotherapy is ineffective in this human disease. Despite finding that PDA-bearing mice had cancer cell-specific CD8(+) T cells, the mice, like human patients with PDA, did not respond to two immunological checkpoint antagonists that promote the function of T cells: anti-cytotoxic T-lymphocyte-associated protein 4 (α-CTLA-4) and α-programmed cell death 1 ligand 1 (α-PD-L1). Immune control of PDA growth was achieved, however, by depleting carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). The depletion of the FAP(+) stromal cell also uncovered the antitumor effects of α-CTLA-4 and α-PD-L1, indicating that its immune suppressive activity accounts for the failure of these T-cell checkpoint antagonists. Three findings suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) explained the overriding immunosuppression by the FAP(+) cell: T cells were absent from regions of the tumor containing cancer cells, cancer cells were coated with the chemokine, CXCL12, and the FAP(+) CAF was the principal source of CXCL12 in the tumor. Administering AMD3100, a CXCL12 receptor chemokine (C-X-C motif) receptor 4 inhibitor, induced rapid T-cell accumulation among cancer cells and acted synergistically with α-PD-L1 to greatly diminish cancer cells, which were identified by their loss of heterozygosity of Trp53 gene. The residual tumor was composed only of premalignant epithelial cells and inflammatory cells. Thus, a single protein, CXCL12, from a single stromal cell type, the FAP(+) CAF, may direct tumor immune evasion in a model of human PDA.

Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

CD99 is upregulated in placenta and astrocytomas with a differential subcellular distribution according to the malignancy stage.

In the present study, we searched for genes highly expressed in placenta and that could contribute to the establishment and maintenance of a malignant phenotype in different types of tumours, and in astrocytomas in particular. We employed a strategy based on the integration of in silico data from previously generated massively parallel signature sequencing and public serial analysis of gene expression databases. Among 12 selected genes, CD99 exhibited the highest relative mRNA expression in GBM compared to non-neoplastic brain tissues. In a larger cohort of astrocytic tumours, we further demonstrated increased CD99 expression in all malignant grades, with GBMs showing the highest values. These findings were confirmed at the protein level by Western blotting and immunohistochemistry. Additionally, we demonstrated the CD99 localisation profile in astrocytic tumours. Interestingly, CD99 expression was confined to the cytoplasm or membrane in more malignant astrocytomas, in contrast to non-neoplastic brain tissue or non-infiltrative pilocytic astrocytoma, which showed no obvious staining in these structures. Comparison of three GBM cell lines revealed higher CD99 expression at the membrane and higher migratory capacity in the A172 and U87MG lines, but lower CD99 expression and no migratory ability in the T98 line. Knocking down CD99 expression by siRNA decreased significantly the migration of both cell lines. These integrated CD99 gene and protein expression results suggest that CD99 expression in astrocytomas of different malignant grades might contribute to the infiltrative ability and support the importance of CD99 as a potential target to reduce infiltrative astrocytoma capacity in migration and invasion.

Heat shock protein 90-mediated peptide-selective presentation of cytosolic tumor antigen for direct recognition of tumors by CD4(+) T cells.

Tumor Ag-specific CD4(+) T cells play important functions in tumor immunosurveillance, and in certain cases they can directly recognize HLA class II-expressing tumor cells. However, the underlying mechanism of intracellular Ag presentation to CD4(+) T cells by tumor cells has not yet been well characterized. We analyzed two naturally occurring human CD4(+) T cell lines specific for different peptides from cytosolic tumor Ag NY-ESO-1. Whereas both lines had the same HLA restriction and a similar ability to recognize exogenous NY-ESO-1 protein, only one CD4(+) T cell line recognized NY-ESO-1(+) HLA class II-expressing melanoma cells. Modulation of Ag processing in melanoma cells using specific molecular inhibitors and small interfering RNA revealed a previously undescribed peptide-selective Ag-presentation pathway by HLA class II(+) melanoma cells. The presentation required both proteasome and endosomal protease-dependent processing mechanisms, as well as cytosolic heat shock protein 90-mediated chaperoning. Such tumor-specific pathway of endogenous HLA class II Ag presentation is expected to play an important role in immunosurveillance or immunosuppression mediated by various subsets of CD4(+) T cells at the tumor local site. Furthermore, targeted activation of tumor-recognizing CD4(+) T cells by vaccination or adoptive transfer could be a suitable strategy for enhancing the efficacy of tumor immunotherapy.

Cancer/testis antigens, gametogenesis and cancer.

Cancer/testis (CT) antigens, of which more than 40 have now been identified, are encoded by genes that are normally expressed only in the human germ line, but are also expressed in various tumour types, including melanoma, and carcinomas of the bladder, lung and liver. These immunogenic proteins are being vigorously pursued as targets for therapeutic cancer vaccines. CT antigens are also being evaluated for their role in oncogenesis--recapitulation of portions of the germline gene-expression programme might contribute characteristic features to the neoplastic phenotype, including immortality, invasiveness, immune evasion, hypomethylation and metastatic capacity.

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual.

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

MAGE-A3 is highly expressed in a subset of colorectal cancer patients.

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.

Transcriptome-guided characterization of genomic rearrangements in a breast cancer cell line.

We have identified new genomic alterations in the breast cancer cell line HCC1954, using high-throughput transcriptome sequencing. With 120 Mb of cDNA sequences, we were able to identify genomic rearrangement events leading to fusions or truncations of genes including MRE11 and NSD1, genes already implicated in oncogenesis, and 7 rearrangements involving other additional genes. This approach demonstrates that high-throughput transcriptome sequencing is an effective strategy for the characterization of genomic rearrangements in cancers.

CT-X antigen expression in human breast cancer.

Cancer/testis (CT) genes are predominantly expressed in human germ line cells, but not somatic tissues, and frequently become activated in different cancer types. Several CT antigens have already proved to be useful biomarkers and are promising targets for therapeutic cancer vaccines. The aim of the present study was to investigate the expression of CT antigens in breast cancer. Using previously generated massively parallel signature sequencing (MPSS) data, together with 9 publicly available gene expression datasets, the expression pattern of CT antigens located on the X chromosome (CT-X) was interrogated. Whereas a minority of unselected breast cancers was found to contain CT-X transcripts, a significantly higher expression frequency was detected in estrogen and progesterone receptor (ER) negative breast cancer cell lines and primary breast carcinomas. A coordinated pattern of CT-X antigen expression was observed, with MAGEA and NY-ESO-1/CTAG1B being the most prevalent antigens. Immunohistochemical staining confirmed the correlation of CT-X antigen expression and ER negativity in breast tumors and demonstrated a trend for their coexpression with basal cell markers. Because of the limited therapeutic options for ER-negative breast cancers, vaccines based on CT-X antigens might prove to be useful.

CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens.

The potency of the immune response has still to be harnessed effectively to combat human cancers. However, the discovery of T-cell targets in melanomas and other tumors has raised the possibility that cancer vaccines can be used to induce a therapeutically effective immune response against cancer. The targets, cancer-testis (CT) antigens, are immunogenic proteins preferentially expressed in normal gametogenic tissues and different histological types of tumors. Therapeutic cancer vaccines directed against CT antigens are currently in late-stage clinical trials testing whether they can delay or prevent recurrence of lung cancer and melanoma following surgical removal of primary tumors. CT antigens constitute a large, but ill-defined, family of proteins that exhibit a remarkably restricted expression. Currently, there is a considerable amount of information about these proteins, but the data are scattered through the literature and in several bioinformatic databases. The database presented here, CTdatabase (http://www.cta.lncc.br), unifies this knowledge to facilitate both the mining of the existing deluge of data, and the identification of proteins alleged to be CT antigens, but that do not have their characteristic restricted expression pattern. CTdatabase is more than a repository of CT antigen data, since all the available information was carefully curated and annotated with most data being specifically processed for CT antigens and stored locally. Starting from a compilation of known CT antigens, CTdatabase provides basic information including gene names and aliases, RefSeq accession numbers, genomic location, known splicing variants, gene duplications and additional family members. Gene expression at the mRNA level in normal and tumor tissues has been collated from publicly available data obtained by several different technologies. Manually curated data related to mRNA and protein expression, and antigen-specific immune responses in cancer patients are also available, together with links to PubMed for relevant CT antigen articles.

Genome-wide analysis of cancer/testis gene expression.

Cancer/Testis (CT) genes, normally expressed in germ line cells but also activated in a wide range of cancer types, often encode antigens that are immunogenic in cancer patients, and present potential for use as biomarkers and targets for immunotherapy. Using multiple in silico gene expression analysis technologies, including twice the number of expressed sequence tags used in previous studies, we have performed a comprehensive genome-wide survey of expression for a set of 153 previously described CT genes in normal and cancer expression libraries. We find that although they are generally highly expressed in testis, these genes exhibit heterogeneous gene expression profiles, allowing their classification into testis-restricted (39), testis/brain-restricted (14), and a testis-selective (85) group of genes that show additional expression in somatic tissues. The chromosomal distribution of these genes confirmed the previously observed dominance of X chromosome location, with CT-X genes being significantly more testis-restricted than non-X CT. Applying this core classification in a genome-wide survey we identified >30 CT candidate genes; 3 of them, PEPP-2, OTOA, and AKAP4, were confirmed as testis-restricted or testis-selective using RT-PCR, with variable expression frequencies observed in a panel of cancer cell lines. Our classification provides an objective ranking for potential CT genes, which is useful in guiding further identification and characterization of these potentially important diagnostic and therapeutic targets.

The cancer testis antigens CABYR-a/b and CABYR-c are expressed in a subset of colorectal cancers and hold promise as targets for specific immunotherapy.

INTRODUCTION: Calcium-binding tyrosine phosphorylation-regulated protein (CABYR) is expressed in the human germ line but not in adult human tissues, thus, it is considered a cancer testis protein. The aim of this study is to evaluate the CABYR isoforms: a/b and c mRNA expression in colorectal cancer (CRC) and to determine if these proteins hold promise as vaccine targets. MATERIALS AND METHODS: CABYR mRNA expression in a set of normal human tissues, including the testis, were determined and compared using semi-quantitative PCR. As regards the tumor and normal mucosal samples from study patients, RNA was extracted and cDNA generated after which quantitative PCR was carried out. Analysis of CABYR protein expressions by immunohistochemistry in tumor and normal colon tissues was also performed. RESULTS: A total of 47 paired CRC and normal tissue specimens were studied. The percent of patients with a relative expression ratio of malignant to normal (M/N) tissues over 1 was 70% for CABYR a/b and 72% for CABYR c. The percent with both a M/N ratio over 1 and expression levels over 0.1% of testis was 23.4% for CABYR-a/b and 25.5% for CABYR c. CABYR expression in tumors was further confirmed by immunohistochemistry. CONCLUSIONS: CABYR a/b and c hold promise as specific immunotherapy targets, however, a larger and more diverse group of tumors (Stage 1-4) needs to be assessed and evaluation of blood for anti-CABYR antibodies is needed to pursue this concept.

Dissecting the transcriptional networks underlying breast cancer: NR4A1 reduces the migration of normal and breast cancer cell lines.

INTRODUCTION: Breast cancer currently accounts for more than one-quarter of all female cancers and, despite the great progress in treatment observed in the past few years, the need for identification of new gene targets that can be used for diagnosis, prognosis and therapy is evident. A previous study identified the transcription factor NR4A1 as a gene upregulated in primary breast cancer compared with normal tissue by microarray analysis and sequencing technologies. The purpose of the study was to identify the role of NR4A1 in normal mammary epithelial and breast cancer cell biology. METHODS: NR4A1 expression in breast tumours was assessed by semiquantitative and real-time PCR using RNA from normal and tumour samples or breast cancer cell lines. Immunohistochemistry on tissue microarrays was performed to check NR4A1 protein expression in breast tumours. MCF-10A and 226L normal mammary epithelial cells as well as the tumour lines PMC42, ZR-75-1 and MDA-MB-231 were transduced with full-length NR4A1, and the ability of NR4A1-overexpressing cells to migrate was tested using scratch wound or transwell migration assays. Proliferation was measured using the MTT and BrdU assays, while apoptosis was determined by the Annexin V assay. The ability of the cells to adhere to extracellular matrix was tested by adhesion assays and integrin cell surface expression was measured by flow cytometry. Activation of the FAK as well as ERK1/2 and PI3K pathways was checked by western blotting. RESULTS: Breast tissue microarray analysis showed NR4A1 expression in primary tumours, which was reduced in higher grade and metastatic tumours. Ectopic expression of NR4A1 in MCF-10A, 226L, PMC42 and ZR-75-1 cells led to reduced ability of the cells to migrate, while no differences were observed in their proliferation and apoptotic index. NR4A1 expression altered the ability of the MCF-10A cells to adhere to the extracellular matrix and affected cell surface expression of integrins. CONCLUSIONS: NR4A1 acts as an antimigratory factor in two normal mammary epithelial and two breast cancer cell lines tested. It is therefore possible that NR4A1 acts as an antimigratory factor in breast tumours, and further studies should be conducted to understand the mechanisms involved.

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.

Expression and serum immunoreactivity of developmentally restricted differentiation antigens in epithelial ovarian cancer.

Cancer-embryo antigens or developmentally restricted differentiation antigens (DRDAGs), such as PLAC1 (CT92) and developmental pluripotency associated-2 (DPPA2/CT100), are expressed in pluripotent embryonic cells. They are also recognized as cancer-testis antigens (CT) which are proteins normally expressed only in the human germ line but that are also present in a significant subset of malignant tumors. These antigens may prove to be markers of 'repopulating' cells with stem cell-like characteristics and could be critical targets for immunotherapy in epithelial ovarian cancer (EOC). Our objective was to define the frequency of expression and immunogenicity of PLAC1 and DPPA2 in EOC and correlate expression with clinical outcome. One-step reverse transcriptase PCR was performed on 101 EOC samples and a panel of normal tissues. Expression of PLAC1 and DPPA2 in the EOC specimens was 21/101 (21%) and 31/101 (31%) respectively. In normal tissues, PLAC1 expression was restricted to the placenta while DPPA2 expression was restricted to the placenta and testis. Immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) were also performed on a subset of specimens. Humoral immunity was demonstrable in 2/12 serum samples from patients whose tumors expressed DPPA2. There was no demonstrable antibody response to PLAC1 in patients with PLAC1 positive tumors. The presence of PLAC1 and DPPA2 did not have a statistically significant effect on recurrence-free and overall survival. The tissue-restricted expression of PLAC1 and DPPA2, their expression in a significant proportion of EOC patients, and their potential to represent markers of stem cells make DRDAGs attractive targets for antigen-specific immunotherapy in EOC.

ECSA/DPPA2 is an embryo-cancer antigen that is coexpressed with cancer-testis antigens in non-small cell lung cancer.

PURPOSE: Cancer cells recapitulate many behaviors of pluripotent embryonic cells such as unlimited proliferation, and the capacity to self-renew and to migrate. Embryo-cancer sequence A (ECSA), later named developmental pluripotency associated-2 (DPPA2), is an embryonic gene initially isolated from pluripotent human preimplantation embryos. We hypothesized that ECSA/DPPA2 would be quiescent in most normal tissues but expressed in cancers and may therefore be a useful target for immunotherapy. EXPERIMENTAL DESIGN: ECSA/DPPA2 expression was examined in a panel of normal and tumor tissue by reverse transcription PCR, quantitative real-time PCR, and immunohistochemistry. A panel of 110 non-small cell lung cancers (NSCLC) were further investigated for the presence of ECSA/DPPA2 transcripts and several cancer testis antigens (CTA). Sera from 104 patients were analyzed for spontaneous ECSA/DPPA2 antibody production by ELISA and Western blot. RESULTS: ECSA/DPPA2 transcripts were limited to normal testis, placenta, bone marrow, thymus, and kidney but expressed in a variety of tumors most notably in 30% of NSCLC. Enrichment for CTAs in ECSA/DPPA2-positive NSCLC was observed. Immunohistochemistry confirmed nuclear and cytoplasmic localization in subpopulations of cells with coexpression of the CTA MAGE-A3. Antibodies to recombinant ECSA/DPPA2 protein were detected in the sera of 4 of 104 patients with NSCLC but not in healthy controls. CONCLUSIONS: The restricted expression in normal tissues, expression in tumors with coexpression of CTAs, and spontaneous immunogenicity indicate that ECSA/DPPA2 is a promising target for antigen-specific immunotherapy in NSCLC.

Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas.

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2-fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT-PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5-fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children.

Aberrant promoter methylation of multiple genes during pathogenesis of bladder cancer.

PURPOSE: The aims of our study were to elucidate the role of methylation of a large panel of genes during multistage pathogenesis of bladder cancer and to correlate our findings with patient age and other clinicopathologic features. EXPERIMENTAL DESIGN: We studied the methylation status of 21 genes by quantitative methylation-specific PCR in an evaluation set of 25 tumor and 5 normal samples. Based on methylation frequency in tumors and normals in gene evaluation set, we selected 7 candidate genes and tested an independent set of 93 tumors and 26 normals. The presence or absence of methylation was evaluated for an association with cancer using cross-tabulations and chi(2) or Fisher's exact tests as appropriate. All statistical tests were two-sided. RESULTS: Most primary tumors (89 of 93, 96%) had methylation of one or more genes of independent set; 53 (57%) CCNA1, 29 (31%) MINT1, 36 (39%) CRBP, 53 (57%) CCND2, 66 (71%) PGP9.5, 60 (65%) CALCA, and 78 (84%) AIM1. Normal uroepithelium samples from 26 controls revealed no methylation of the CCNA1 and MINT1 genes, whereas methylation of CRBP, CCND2, PGP9.5, and CALCA was detected at low levels. All the 7 genes in independent set were tightly correlated with each other and 3 of these genes showed increased methylation frequencies in bladder cancer with increasing age. PGP9.5 and AIM1 methylation correlated with primary tumor invasion. CONCLUSION: Our results indicate that the methylation profile of novel genes in bladder cancers correlates with clinicopathologic features of poor prognosis and is an age-related phenomenon.

Cancer-testis (CT) antigen expression in medulloblastoma.

Medulloblastoma is the most common childhood malignant tumor of the central nervous system. Treatment of medulloblastoma requires harmful therapy and nevertheless carries a poor prognosis. Due to their presence in various cancers and their limited expression in normal tissues, CT antigens are ideal vaccine targets for tumor immunotherapy. CT antigens, such as MAGE and NY-ESO-1, have been employed in clinical trials in various malignancies but little is known about their presence in medulloblastoma. We analyzed 25 medulloblastomas for the expression of a panel of CT antigens by RT-PCR and immunohistochemistry. Messenger RNA expression in the samples was as follows: GAGE 64%, MAGEA3/6 56%, SYCP1 44%, SLCO6A1 32%, MAGEC1 28%, MAGEC2 28%, MAGEA4 28%, NY-ESO-1 20%, MAGEA1 16%, and TPTE 0%. All cases except one (96%) were positive for mRNA expression of at least one CT gene. However, CT antigen expression was scarce on a protein level. Immunoreaction to monoclonal antibody E978 (NY-ESO-1) was negative in all cases; MA454 (MAGEA1), 57B (MAGEA4), M3H67 (MAGEA3/6), CT10#5 (MAGEC2) and #23 (GAGE) were each positive in 1 case, while the highest incidence of positive immunostaining, albeit heterogeneous, was seen with CT7-33 (MAGEC1) in 3 out of the 25 cases. The absence of correlation between mRNA and protein expression in medulloblastoma has not been observed in other tumors and further studies addressing the biology of CT antigens are necessary to investigate the present discrepant results.

Prognostic impact of cancer/testis antigen expression in advanced stage multiple myeloma patients.

This study aims to analyze the expression of 14 cancer/testis (CT) antigens in multiple myeloma (MM) to identify possible prognostic markers and therapeutic targets. The expression of MAGEA1, MAGEA2, MAGEA3/6, MAGEA4, MAGEA10, MAGEA12, BAGE1, MAGEC1/CT7, the GAGE family, LAGE-1, PRAME, NY-ESO-1, SPA17 and SSX1 was studied by RT-PCR in 15 normal tissues, a pool of 10 normal bone marrow samples, 3 normal tonsils and bone marrow aspirates from 6 normal donors, 3 monoclonal gammopathies of undetermined significance (MGUS), 5 solitary plasmacytomas, 39 MM samples (95% advanced stage) and the MM cell line U266. MAGEC1/CT7 was expressed in bone marrow aspirates from one MGUS and one plasmacytoma. The frequencies at which CT antigen were found to be expressed in MM patients were MAGEC1/CT7 77%, LAGE-1 49%, MAGEA3/6 41%, MAGEA2 36%, GAGE family 33%, NY-ESO-1 33%, BAGE-1 28%, MAGEA1 26%, PRAME 23%, SSX-1 26%, MAGEA12 20.5%, MAGEA4 0%, and MAGEA10 0%. Cox's regression model showed that GAGE family expression and having >6 CT antigens expressed were independent prognostic factors when all patients were analyzed. However, MAGEC1/CT7 expression was the only independent prognostic factor when non-transplanted patients where analyzed. Based on our findings, MAGEC1/CT7, MAGEA3/6 and LAGE-1 are good candidates for immunotherapy, since together they cover 85% of our MM cases. Furthermore, expression of the GAGE family, >6 CT antigens and MAGEC1/CT7 seem to have impact on MM prognosis.