SSX Family Research Articles

Higher Expression Levels of SSX1 and SSX2 in Patients with Colon Cancer: Regulated In Vitro by the Inhibition of Methylation and Histone Deacetylation.

Background and Objectives: Colon cancer (CC) has a high mortality rate and is often diagnosed at an advanced stage in Saudi Arabia. Thus, the identification and characterization of potential new cancer-specific biomarkers are imperative for improving the diagnosis of CC by detecting it at an early stage. Cancer-testis (CT) genes have been identified as potential biomarkers for the early diagnosis of various cancers. Among the CT genes are those belonging to the SSX family. In order to assess the usefulness of SSX family genes as cancer biomarkers for the detection of early-stage CC, the goal of this research was to validate the expressions of these genes in patients with CC and in matched patients with normal colons (NCs). Materials and Methods: RT-PCR assays were used to analyze the SSX1, SSX2, and SSX3 family gene expression levels in 30 neighboring NC and CC tissue samples from male Saudi patients. Epigenetic alterations were also tested in vitro using qRT-PCR analysis to determine whether reduced DNA methyltransferase or histone deacetylation could stimulate SSX gene expression via 5-aza-2'-deoxycytidine and trichostatin treatments, respectively. Results: The RT-PCR results showed SSX1 and SSX2 gene expression in 10% and 20% of the CC tissue specimens, respectively, but not in any of the NC tissue specimens. However, no SSX3 expression was detected in any of the examined CC or NC tissue samples. In addition, the qRT-PCR results showed significantly higher SSX1 and SSX2 expression levels in the CC tissue samples than in the NC tissue samples. The 5-aza-2'-deoxycytidine and trichostatin treatments significantly induced the mRNA expression levels of the SSX1, SSX2, and SSX3 genes in the CC cells in vitro. Conclusions: These findings suggest that SSX1 and SSX2 are potentially suitable candidate biomarkers for CC. Their expressions can be regulated via hypomethylating and histone deacetylase treatments, subsequently providing a potential therapeutic target for CC.

Deficiency of primate-specific SSX1 induced asthenoteratozoospermia in infertile men and cynomolgus monkey and tree shrew models

Primate-specific genes (PSGs) tend to be expressed in the brain and testis. This phenomenon is consistent with brain evolution in primates but is seemingly contradictory to the similarity of spermatogenesis among mammals. Here, using whole-exome sequencing, we identified deleterious variants of X-linked SSX1 in six unrelated men with asthenoteratozoospermia. SSX1 is a PSG expressed predominantly in the testis, and the SSX family evolutionarily expanded independently in rodents and primates. As the mouse model could not be used for studying SSX1, we used a non-human primate model and tree shrews, which are phylogenetically similar to primates, to knock down (KD) Ssx1 expression in the testes. Consistent with the phenotype observed in humans, both Ssx1-KD models exhibited a reduced sperm motility and abnormal sperm morphology. Further, RNA sequencing indicated that Ssx1 deficiency influenced multiple biological processes during spermatogenesis. Collectively, our experimental observations in humans and cynomolgus monkey and tree shrew models highlight the crucial role of SSX1 in spermatogenesis. Notably, three of the five couples who underwent intra-cytoplasmic sperm injection treatment achieved a successful pregnancy. This study provides important guidance for genetic counseling and clinical diagnosis and, significantly, describes the approaches for elucidating the functions of testis-enriched PSGs in spermatogenesis.

SSX2 promotes the formation of a novel type of intranuclear lamin bodies.

SSX proteins are normally restricted to spermatogenic cells, but ectopic expression can be observed in many types of human cancer. We recently demonstrated that SSX family members may contribute to tumorigenesis by modifying chromatin structure and, in specific settings, compromise chromatin stability. Here, we used normal and tumorigenic breast epithelial cell line models to further study the effect of ectopic expression of SSX2 on nuclear organization. We show that SSX2 induces the formation of a novel type of nucleoplasmic lamin bodies. Ectopic expression of SSX2 in various breast epithelial cell lines led to the formation of a previously undescribed type of intranuclear bodies containing both A and B type lamins but no other components of the nuclear lamina. SSX2-expressing cells contained a highly variable number of lamin bodies distributed throughout the nuclear space. SSX2-mediated establishment of intranuclear lamin bodies could not be linked to previous molecular interactions of SSX proteins, including polycomb proteins and the Mediator complex, but was, however, dependent on S-phase progression. These results reveal a novel interaction between SSX2 and lamins in the nucleoplasmic space. They further suggest that SSX2 promotes the formation of chromatin neighborhoods supporting the organization of lamins into nuclear bodies. We speculate that this may have implications for the organization and functional regulation of chromatin in cancer cells. Our study contributes to the further understanding of the biology of SSX proteins in tumorigenesis.

ATR Is a Therapeutic Target in Synovial Sarcoma

Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by expression of SS18-SSX fusions, where treatment options are limited. To identify therapeutically actionable genetic dependencies in SS, we performed a series of parallel, high-throughput small interfering RNA (siRNA) screens and compared genetic dependencies in SS tumor cells with those in >130 non-SS tumor cell lines. This approach revealed a reliance of SS tumor cells upon the DNA damage response serine/threonine protein kinase ATR. Clinical ATR inhibitors (ATRi) elicited a synthetic lethal effect in SS tumor cells and impaired growth of SS patient-derived xenografts. Oncogenic SS18-SSX family fusion genes are known to alter the composition of the BAF chromatin-remodeling complex, causing ejection and degradation of wild-type SS18 and the tumor suppressor SMARCB1. Expression of oncogenic SS18-SSX fusion proteins caused profound ATRi sensitivity and a reduction in SS18 and SMARCB1 protein levels, but an SSX18-SSX1 Δ71-78 fusion containing a C-terminal deletion did not. ATRi sensitivity in SS was characterized by an increase in biomarkers of replication fork stress (increased γH2AX, decreased replication fork speed, and increased R-loops), an apoptotic response, and a dependence upon cyclin E expression. Combinations of cisplatin or PARP inhibitors enhanced the antitumor cell effect of ATRi, suggesting that either single-agent ATRi or combination therapy involving ATRi might be further assessed as candidate approaches for SS treatment. Cancer Res; 77(24); 7014-26. ©2017 AACR.

SSX2 regulates focal adhesion but does not drive the epithelial to mesenchymal transition in prostate cancer

Prostate cancer is the most commonly diagnosed malignancy for men in the United States. Metastatic prostate cancer, the lethal form of the disease, has a life expectancy of approximately five years. Identification of factors associated with this transition to metastatic disease is crucial for future therapies. One such factor is the SSX gene family, a family of cancer/testis antigens (CTA) transcription factors which have been shown to be aberrantly expressed in other cancers and associated with the epithelial to mesenchymal transition (EMT). We have previously shown that SSX expression in prostate cancers was restricted to metastatic tissue and not primary tumors. In this study, we have identified SSX2 as the predominant SSX family member expressed in prostate cancer, and found its expression in the peripheral blood of 19 of 54 (35%) prostate cancer patients, with expression restricted to circulating tumor cells, and in 7 of 15 (47%) metastatic cDNA samples. Further, we examined SSX2 function in prostate cancer through knockdown and overexpression in prostate cancer cell lines. While overexpression had little effect on morphology or gene transcript changes, knockdown of SSX2 resulted in an epithelial morphology, increased cell proliferation, increased expression of genes involved in focal adhesion, decreased anchorage independent growth, increased invasion, and increased tumorigenicity in vivo. We conclude from these findings that SSX2 expression in prostate cancer is not a driver of EMT, but is involved in processes associated with EMT including loss of focal adhesion that may be related to tumor cell dissemination.

Abstract 1423: SSX-2 expression and function in metastatic prostate cancer

Abstract Background: The SSX family of proteins, whose function and aberrant expression in prostate cancer isn't fully understood, may lead to disease progression from localized to metastatic disease. Cancer-testis antigens (CTAs) are a group of proteins whose expression is restricted to germline cells among normal somatic cells. CTAs have been found to be aberrantly expressed in many different cancers. Due to their restricted expression to tumor cells among MHC class I-expressing cells, there has been interest in CTAs as tumor-specific targets for CD8+ T cell-directed therapies. Amongst the CTA genes is the Synovial Sarcoma X chromosome breakpoint (SSX) gene family. The SSX family consists of ten highly homologous members, several of which have been detected in a wide variety of cancers. We have previously shown that SSX expression is confined to metastatic prostate tissue, and is not detected in primary prostate tumors. The function of the SSX family is largely unknown, and further insights into its function may reveal avenues for future directed therapies. Methods: We determined the identity and prevalence of SSX family members in metastatic prostate cancer tissues and in peripheral blood samples of patients with prostate cancer. Specifically, cDNA from metastatic prostate tissues was examined by PCR with primers specific for each SSX family member; SSX family member expression was detected in peripheral blood samples using rt-PCR. To further understand SSX-2 function, prostate cell lines with SSX-2 expression knocked down or overexpressed were evaluated in vitro for morphological changes, tumorigenicity (soft agar colony formation), proliferation, and invasion (scratch test). Finally using qPCR, expression levels of markers of the epithelial to mesenchymal transition (EMT) were examined in the knocked down and overexpressed cell lines. Results: SSX-2 was found to be the dominant family member expressed in prostate cancer, with 47% (7/15) of metastatic cDNA samples expressing SSX-2. SSX-1 was detected in one sample (1/15), and no additional family members were detected. SSX-2 mRNA was similarly detectable in the peripheral blood of 17 of 33 (51%) patients with recurrent prostate cancer and 0% (0 of 10) control blood donors without prostate cancer. In in vitro functional studies, SSX-2 knockdown of the 22Rv1 cell line led to cell morphology changes, increased cell proliferation, and a decrease in soft agar colony formation. Knockdown was also accompanied by altered expression of genes involved in EMT (Twist-1, ZEB1/2, Slug, vimentin). Complementary changes were observed in the RWPE-1 normal prostate epithelial cell line transfected to overexpress SSX-2. Conclusions: Due to the preferential expression of SSX-2 in disseminated prostate cancer, as well as the effects of SSX knockdown and overexpression on EMT-related genes, we believe that SSX-2 is involved in the EMT process, and potentially in the progression of prostate cancer to metastatic disease. Citation Format: Jordan E. Bloom, Douglas G. McNeel. SSX-2 expression and function in metastatic prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1423. doi:10.1158/1538-7445.AM2015-1423

Cancer testicular antigens as a prognostic markers of chronic lymphocytic leukemia.

6575 Background: CLL is the most common leukemia in adults and has a variable course and prognosis. Clinical staging systems and the use of biomarkers such as cytogenetics and the IGVH mutational status remain the gold standard for stratification. Cancer testis antigens (CTAs) expression analyses have shown prognostic impact in other hematological malignancies such as acute leukemias and multiple myeloma. With the analysis of CTAs we aim to discover additional molecular markers that can help us predict the clinical course and outcome in CLL patients. Methods: We analyzed the expression by RT-PCR of 39 known CTAs including members of the MAGE, GAGE, MAD-CT and SSX CTAs families in a group of 59 CLL patients seen at our institution. The Moffitt Cancer Center Total Cancer Care (TCC) database was used to correlated these findings with relevant clinical and molecular markers such as Rai stage, demographic data, initial WBC count, IGVH mutational status, CD38 expression, cytogenetics by FISH analysis and treatment outcomes. Overall survival (OS) and progression free survival (PFS) were calculated using Kaplan Meier curves with a SPSS statistical software Results: Deletion 13q was the most common gene abnormality in 35/59 (59.3%). 29/59 (49.1%) required at least one line of treatment. MAGE family CTAs were present in 53/59 (83.9%), MAD-CT in 24/59 (40.7%), SSX in 14/59 (23.7%) and GAGE in 12/59(20.3%). 35/59 (59.3%) CLL patients expressed 2 or more MAGE family CTAs. No differences were noted in the Rai staging, initial white blood cell count, B symptoms, extranodal disease, presence of CD38, or unmutated IVGH. The OS was not significantly different among all CTAs families with a tendency towards a better OS those expressing the SSX family (p=0.63). Additionally, CLL patients with single expression of MAD-CT1 (p=0.05) and MAGE-B2 (p=0.038) antigens correlated with improved survival. Conclusions: Patients with SSX family have a trend towards improved survival. When analyzed by individual antigen, MAD-CT1 and MAGE-B2 expression was associated with improved survival. Further studies with a larger number of patients are needed to assess the real value of the expression of CTAs in CLL.

Targeting Epigenetic Misregulation in Synovial Sarcoma

Like many sarcomas, synovial sarcoma is driven by a characteristic oncogenic transcription factor fusion, SS18-SSX. In this issue of Cancer Cell, Su etal. elucidate the protein partners necessary for target gene misregulation and demonstrate a direct effect of histone deacetylase inhibitors on the SS18-SSX complex composition, expression misregulation, and apoptosis.

Targeting SSX cancer–testis antigens for immunotherapy

SSX family proteins are differentially expressed in prostate tumor tissues, and could provide potential targets for cancer treatment using immune therapies, according to a study published in Cancer Research.

Expression and Immunotherapeutic Targeting of the SSX Family of Cancer–Testis Antigens in Prostate Cancer

Recent U.S. Food and Drug Administration approval of the first immunotherapy for prostate cancer encourages efforts to improve immune targeting of this disease. The synovial sarcoma X chromosome breakpoint (SSX) proteins comprise a set of cancer-testis antigens that are upregulated in MHC class I-deficient germline cells and in various types of advanced cancers with a poor prognosis. Humoral and cell-mediated immune responses to the SSX family member SSX2 can arise spontaneously in prostate cancer patients. Thus, SSX2 and other proteins of the SSX family may offer useful targets for tumor immunotherapy. In this study, we evaluated the expression of SSX family members in prostate cancer cell lines and tumor biopsies to identify which members might be most appropriate for immune targeting. We found that SSX2 was expressed most frequently in prostate cell lines, but that SSX1 and SSX5 were also expressed after treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine. Immunohistochemical analysis of microarrayed tissue biopsies confirmed a differential level of SSX protein expression in human prostate cancers. Notably, SSX expression in patient tumor samples was restricted to metastatic lesions (5/22; 23%) and no expression was detected in primary prostate tumors examined (0/73; P < 0.001). We determined that cross-reactive immune responses to a dominant HLA-A2-specific SSX epitope (p103-111) could be elicited by immunization of A2/DR1 transgenic mice with SSX vaccines. Our findings suggest that multiple SSX family members are expressed in metastatic prostate cancers which are amenable to simultaneous targeting.

Abstract A66: Expression and immunotherapeutic targeting of the SSX family of cancer-testis antigens in prostate cancer

Abstract Members of the SSX family of proteins were among the first cancer-testis antigens (CTA) identified. Characterized by tissue-restricted expression to MHC class l-deficient germline cells and frequent ectopic expression in tumors of various histological origin, CTA are highly tumor-specific antigens. SSX proteins comprise a family of ten members with mRNA expression often found in advanced-stage disease and associated with worse patient prognosis. We have previously shown that humoral and cell-mediated immune responses to SSX2 can arise spontaneously in patients with prostate cancer, which combined with the expression of these proteins in MHC class l-negative tissue, makes SSX proteins potentially attractive targets for tumor immunotherapy. In the current study, we evaluated SSX family member expression in prostate cancer cell lines and tumor biopsies to identify therapeutic targets for vaccine therapy in prostate cancer. We found by RT-PCR and qRT-PCR that SSX1, SSX2, and SSX5 were frequently expressed in prostate cancer cell lines and could be induced by treatment with epigenetic modifying agents (EMA) such as 5-aza-2′-deoxycytidine. Furthermore, we observed differential SSX protein expression in paraffin-embedded prostate cancer biopsies on tissue microarray by immunohistochemical staining. Interestingly, SSX expression in patient samples was restricted to metastatic prostate cancer lesions (5/22; 23%), while expression in primary prostate tumors was not observed (0/73). Finally, we determined that it is possible to elicit cross-reactive immune responses to a dominant SSX epitope p103–111 shared by the different SSX family members by immunization of A2/DR1 transgenic mice with SSX peptides or a plasmid DNA vaccine encoding SSX2. Overall, these results suggest that multiple SSX family members are expressed in prostate cancer and can be simultaneously targeted using immunotherapeutic vaccines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A66.

The SSX Family of Cancer‐Testis Antigens as Target Proteins for Tumor 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 evolutionary history of the SSX family of human C/T-antigens

C/T-antigens are endogenous proteins expressed in normal testis, ovary and placenta, or in a variety of tumors. Such expression pattern sets the C/T antigens as promising targets for cancer vaccines. The SSX family comprises several C/T antigens. Here we applied comparative genomics techniques to study the evolution of the SSX genes. The human genomic locus includes 11 genes localized on the X chromosome in two separate regions 4 Mb apart. The recent pseudogenization of two SSX genes was demonstrated using the available expression data. The comparative analysis of the human, chimpanzee and mouse genomic loci allowed us to describe the phylogeny of the family and to reconstruct the evolutionary history of the locus in terms of elementary events.

Restoring the Functional Immunogenicity of Chronic Lymphocytic Leukemia Using Epigenetic Modifiers.

Abstract 5116 BackgroundChronic lymphocytic leukemia (CLL) is a malignancy arising from immune cells (B-lymphocytes) endowed with intrinsic antigen-presenting capabilities. Such a function however is lost during malignant transformation and CLL cells are well known for their inability to process and present antigens to the T-cell arm of the immune system. Instead, malignant CLL cells elicit a vast array of immune regulatory mechanisms conducive to T-cell dysfunction and immunosupression. Recent evidence suggests that DNA methylation inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) can have lasting cancer-specific effects on the expression of highly immunogenic cancer-testis antigens (CTAs) as well as modulating costimulatory and major histocompatability molecule (MHC) surface expression. MethodsTo investigate the potential efficacy of a combined therapy of DNMTi (5-aza-2'-deoxycytidine) and HDACi (LAQ) in human CLL we performed a gene expression microarray profile and further confirmed the expression of CTAs. Furthermore we investigated phenotypic alterations due to changes in costimulatory molecules (CD40, 80, and 86), and MHC molecules (HLA-A, B, C, and HLA-DR) in both cell lines and primary cells from patients with CLL. To confirm that alterations in molecule surface expression correlated to more potent immune stimulation we characterized the formation of the immune-synapse between the CLL antigen presenting cell and healthy T-lymphocytes in treated and untreated samples using confocal microscopy and flow conjugation assay. To demonstrate functional significance we subjected treated and untreated CLL cell lines to mixed lymphocyte proliferation assays with CD4, CD8, and NK cell subsets. ResultsAs expected we demonstrate by gene expression profile and RT-PCR that many highly immunogenic CTAs (SSX family, MAGE family, NY-ESO-1, GAGE-2 and NXF2) are significantly upregulated after treatment with an HDACi and DNMTi in combination. Interestingly, preliminary data showed interferon gamma mRNA upregulation. Consistent with this information, our data also indicates that a more proinflammatory signaling pathway is developed between the CLL B-cell and the T-lymphocyte after combination treatment demonstrated by upregulated surface expression of CD86, 80, 40, HLA-A,B,C, and HLA-DR as well as the formation of significantly more, robust, numerous, and organized actin-mediated immune-synapses, changes which were not duplicated using either drug alone. Functionally, only the combined HDACi/DNMTi treatment of CLL cells led to increased allogenic T and NK cell proliferation and cytotoxicity. ConclusionsTaken together these data indicate that combined HDACi/DNMTi may benefit current antigen specific vaccine strategies for CLL by inducing the expression of a highly antigenic CTAs, increasing CLL costimulatory capacity, repairing immunological synapse, and functionally increasing the proinflammatory status of the CLL APC. DisclosuresPinilla:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.

Changes in Immunogenicity of Chronic Lymphocytic Leukemia Cells Mediated by Epigenetic Modifiers

The success of immunotherapeutic treatments for B-cell chronic lymphocytic leukemia (B-CLL) hinges upon the circumvention of tolerogenic mechanisms which inhibit the formation of a robust anti-cancer immune response. Recent evidence suggests that DNA methylation inhibitors and histone deacetylase inhibitors can have lasting cancer-specific effects on the expression of highly immunogenic cancer-testis antigens (CTAs) as well as modulating costimulatory and major histocompatability molecule (MHC) surface expression. To investigate the potential efficacy of a combined therapy of methylation (5-aza-2′ytidine) and histone deacetylase (LAQ) inhibitors in human B-CLL we evaluated the expression of CTAs, costimulatory molecules (CD40, CD80, CD86), and MHC molecules (HLA-A, B, C, and HLA-DR) in both cell lines and primary cells from patients with CLL. In addition, we investigated the overall functional immunogenicity of treated B-CLL cells using mixed leukocyte reactions. Results showed that many highly immunogenic CLL-specific CTAs (SSX family, MAGE family, NY-ESO-1, GAGE-2, and NXF2) are significantly upregulated after treatment with both inhibitors. Our data also indicates that the combined effect of both inhibitors was capable of inducing a more immunogenic phenotype (increased CD86, CD80, CD40, HLA-A,B,C, and HLA-DR) in CLL cells consistently, while either drug alone was incapable of producing this effect. In addition treatment with both inhibitors was capable of increasing IFNγ release mediated by allogenic T-cells. Taken together these data indicate that a combined therapy with histone deacetylase and methylation inhibitors, possibly in combination with antigen-specific vaccine immunotherapy, may effectively induce cancer-specific antigen expression and increase costimulatory and antigen presenting capacity of the B-CLL cell.

Identification of a common HLA-A*0201-restricted epitope among SSX family members by mimicking altered peptide ligands strategy

The synovial sarcoma X breakpoint ( SSX) gene family contains nine members. The SSX proteins are CT (cancer/testis) antigens and can be expressed in many tumor types. T cell immune response against SSX protein can be detected in tumor patients and mice expressing any SSX. Screening predominant protective epitopes might improve the low immunogenicity against these “self” CT antigens. Herein, we predicted HLA-A*0201-restricted epitopes for all nine SSX family members, followed by validation with epitope molecular modeling, peptide/HLA-A*0201 affinity, and binding stability assays. We obtained four highly homologous candidate epitopes with the high immunogenicity scores designated P1, P4, P5 and P6, from the nine SSX members. Each of the four candidates could elicit strong epitope-specific CTL immune responses, but P4 could evoke more interferon γ (IFN-γ)-producing T cells and more potent CTLs that could lyse more target cells. Importantly, almost all of the four epitopes induced CTLs could cross-lyse the mutual targets both in vitro in human PBMCs and HLA-A2.1/K b transgenic mice, but P4 showed superiority to other epitopes in term of cross-cytolysis. All of these results demonstrate that P4 can induce anti-tumor immunity in a fashion superior to other candidates, and may be the “common” CTL epitope among all SSX-expressing tumors. Due to its documented responses herein, P4 has potential application in peptide-mediated immunotherapy.

An Unusual Expression: The Tumour Antigen SSX2IP Is Preferentially Expressed on the Surface of Acute Myeloid Leukaemia Cells during Early Mitosis.

The SSX family includes five functionally active and highly homologous members which are only expressed in the thyroid and testis in normal tissues. However these genes are expressed in several cancers including melanoma. Of note SSX1, SSX2, or SSX4 may be fused to the SYT gene as a result of the t(X;18) translocation in synovial sarcoma. Previous investigators used yeast two-hybrid systems to elucidate the SSX2 interacting protein (SSX2IP) through its association with SSX2. SSX2IP has been shown to be ubiquitously expressed in many normal tissues. We found SSX2IP through the immunoscreening of a testis cDNA library with pooled presentation M4 and M5 acute myeloid leukaemia (AML) sera. SSX2IP was found to be preferentially recognized by 62% of acute myeloid leukaemia (AML) sera (n=22) compared to 21% of normal donor sera (n=20). By RT-PCR SSX2IP was found to be expressed by four of 12 (30%) AML patient samples at presentation but none of eight normal donor haematopoietic samples (bone marrow and peripheral blood). By immunocytochemistry (ICC) we found SSX2IP protein expression in three of six myeloid leukaemia cell lines (K562, P39 and HL60), three of nine AML samples at presentation but none of three normal donor haematopoietic samples (peripheral blood). By confocal microscopy we noticed SSX2IP was predominantly expressed on the surface of K562 cells in early mitosis. We then synchronized K562 cells (as demonstrated by flow cytometry) using either serum starvation (0.1% fetal calf serum for 5 days) in the G0 phase or using 0.3mM hydroxyurea for 3 days at the G1/S interface of the cell cycle. On release back into the cell cycle (as shown by cell counts and flow cytometry) we observed a 3hr period, post-synchronisation, at 24–26 hours for serum starvation or 14–17 hours for hydroxyurea treatment during which time SSX2IP expression peaked, as detected by ICC and confocal microscopy. We transfected K562 cells with the cell surface expressed costimulatory molecule CD80 and demonstrated that SSX2IP was almost entirely restricted in its expression to the cell surface, mimicing the expression pattern of CD80. SSX2IP has a murine counterpart with 87% amino acid sequence similarity called ADIP. ADIP is a novel Afadin- and alpha-actinin-binding protein which localises at cell-cell adherens junctions. ADIP is at least partly involved in the physical association of nectins and cadherins and has more recently been implied to play a role in vesicle trafficking from the Golgi to the endoplasmic reticulum and through the Golgi complex. The mouse protein ADIP has been shown to interact with AF6, the human equivalent of which is involved in the human MLL-AF6 translocation in AML and acute lymphocytic leukaemia. However AF6 was not found to colocalise with SSX2IP in K562 cells as determined by confocal microscopy in our studies although both were expressed. P39 cells only expressed detectable levels of SSX2IP and not AF6. We have demonstrated a novel expression of the tumour antigen SSX2IP on the surface of AML cells in early mitosis. As such SSX2IP may provide a novel antibody target for the depletion of the AML cells from harvests prior to autologous transplant, when no suitable allogeneic donor can be found.

Cancer/Testis Antigen Profiling in Multiple Myeloma Define a Cohort of Patients with Poor Prognosis Regardless of Genetic Subtypes.

Cancer/testis antigens (CTAs), comprising the MAGE, CTAG, SSX and GAGE families, are expressed in different tumor types and restricted in normal tissues to germ cells of the testis. Many CTAs contain epitopes recognized by cytotoxic T cells and are potential targets for active immunotherapy. In MM, different CTAs have been shown to be expressed in cell lines and patient cells, and are associated with more proliferative and advance stage tumors. However, correlation with survival has never been reported. We undertook a global survey of CTA expression using gene expression profiling of CD138-enriched plasma-cell (PC) RNA in 15 normal, 101 MM, 22 MGUS and 24 SMM samples using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). 22 CTA genes were differentially expressed across the samples and unsupervised clustering using these genes clearly identifies a group of patients with high CTA expression. Most CTAs have significantly higher expression in MM as compared to SMM and especially MGUS, which has similar expression as normal PCs. As these antigens are not expressed in normal PCs, an upper limit of normal expression (ULN) was derived based on mean+3SD of expression in normal PCs for each CTA. A strict cut-off of normalized expression value greater than 2xULN was used to assign positive expression for each CTA genes. Using this method, the most commonly expressed CTAs are MAGE-A3 (73% of MM), MAGE-C1 (45%), MAGE-A6 (35%), SSX-1 (21%), GAGE-5 (18%) and NY-ESO-1 (14%). We then assigned patients with expression of more than 2 of these genes as having a CTA signature and correlated with clinical parameters. 45 patents (41 MM and 4 SMM) have the CTA signature. These patients have a significantly higher PCLI (median 0.2 v 0.7, p=0.0003). In fact, there is a significant correlation between the number of positive CTAs and PCLI (r=0.59, p<0.0001). Interestingly, there is no association between patients with a CTA signature and any of the recurrent genetic abnormalities (IgH translocations, chromosome 13 deletion and TC classes), yet MM patients with this signature have a significantly shorter survival (median 459 days vs not reached, log-rank p=0.017). A prognostic hierarchy exists amongst these CTAs. Only MAGE-A6, MAGE-A12, SSX-1 and SSX-5 expression has significant prognostic implications with the SSX family conferring the worse prognosis. Using a restricted panel of 3 CTAs (SSX-1, -5 and MAGE-A12), about 28% of MM patients expressing one or more of these genes can be identified with poor survival (median less than 1 year) that is independent of genetic subtypes. The genes differentially expressed between patients with and without the CTA signature are mainly involved in cell cycle regulation. Since the expression of this large set of CTAs appears to be under epigenetic control, we speculate that the same epigenetic processes that lead to dysregulation of the cell cycle also cause ectopic expression of CTA genes. As CTA expression is rare in MGUS, epigenetic processes may therefore also mediate transformation from MGUS to MM. These poor prognosis patients represent good candidates for novel therapies targeting the epigenetic machinery.

Expression of Cancer/Testis Antigens CT10 and SSX in Multiple Myeloma: Promising Targets for Antigen-Specific Immunotherapy.

Background: The introduction of autologous and allogeneic stem cell transplantation has resulted in improved responses to treatment in patients with multiple myeloma (MM). However, despite high remission rates, recurrences are frequent due to remaining minimal residual disease. Myeloma-specific immunotherapies would represent a potentially useful treatment option in these patients. Cancer/testis (CT) antigens are highly attractive targets for T cell-mediated immunotherapy of cancer, due to their immunogenicity and restricted tissue expression. We investigated the expression of a larger number of CT antigens, the expression of which is characteristically restricted to cancer and normal testis, in patients with MM.Methods: We analyzed the expression of 9 different CT antigens (SSX-1 to SSX-5, CT10/MAGE-E1, BAGE, MAGE-3, NY-ESO-1) in 10 myeloma cell lines, 54 bone marrow samples from patients with stage II-III myeloma (plasma cell infiltration 10–100 %), and 18 bone marrow samples from healthy donors using rtPCR.Results: Myeloma cell lines showed a remarkably high expression of the majority of CT antigens. All antigens, with the exception of SSX-3, were expressed in 80–100% of cell lines. SSX-3 was not expressed in any cell line. Analyzing bone marrow samples from MM patients, we observed that CT10 showed a very high expression level with 55% of all malignant samples expressing this antigen. The SSX gene family evidenced high expression levels with 40% (SSX-1), 17% (SSX-2), 13% (SSX-4), and 15% (SSX-5) of patient samples expressing the given SSX family member. SSX-3 was not expressed in any samples. In agreement with previous findings we observed a high expression of MAGE-3 (61%), an intermediate expression of BAGE (18%), and a comparably low expression of NY-ESO-1 (7%). With the exception of SSX-4, which was expressed in 18% of healthy bone marrow samples, none of the CT antigens were expressed in non-malignant samples.Conclusions: We show here for the first time that CT10 and members of the SSX gene family are strongly and specifically expressed in multiple myeloma. Importantly, both CT10 and SSX have been shown to elicit spontaneous immune responses in patients with solid tumors. Therefore, CT10 and SSX-1, SSX-2, and SSX-5 might represent valuable targets for antigen-specific immunotherapy of multiple myeloma.

Quantification of SSX mRNA Expression in Human Bone and Soft Tissue Tumors Using Nucleic Acid Sequence-Based Amplification

The SSX family proteins have been considered new members of the cancer/testis antigens because of the restricted expression in testis among normal tissues and the activation in a wide range of cancers. Thus, they would be potential molecular targets for immunotherapeutic strategies. We have developed a competitive nucleic acid sequence-based amplification (NASBA) assay to analyze SSX mRNA expression in 211 bone and soft tissue tumors. The copy numbers of SSX mRNA per mug of total RNA in tumor tissues were widely distributed, ranging logarithmically from 0.6 to 6.6. We found that malignant tumors showed significantly higher expression of SSX mRNA than benign tumors (P < 0.0001). Further, SSX mRNA expression in stage III tumors was significantly higher than that in stage I or II tumors (P < 0.005). This NASBA assay was also more sensitive compared to immunohistochemistry using newly affinity-purified polyclonal antibody against SSX. Collectively, these results suggest that the SSX quantitative NASBA assay could provide useful information to select eligible patients for SSX-specific cancer vaccines.