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The aim was to investigate MAGE-A4 and MAGE-A1 protein expression in high-grade endometrial cancer and determine its correlation with histologic subtype, FIGO stage, presence of vascular invasion, disease free, and overall survival. Immunohistochemical staining was performed by using 77B (MAGE-A1) and 57B (MAGE-A4) monoclonal antibodies on paraffin-embedded sections from high-grade endometrial cancers diagnosed in University Hospital Split between 1998 and 2011 (n=77). Median follow-up time for survivors was 48 mo. MAGE-A4 was found to be expressed in 33% of endometrioid type endometrial cancers grade 3 and in 27% of serous and clear cell carcinomas. MAGE-A1 was found to be expressed in 93% endometrioid endometrial cancer grade 3 and 86% of serous and clear cell carcinomas. Univariate analysis showed that positive immunohistochemical staining for MAGE-A4 was associated with decreased disease free and overall survival in patients with high-grade endometrial cancer. Multivariate analysis showed an association between MAGE-A4 overexpression and decreased disease free but not overall survival in high-grade endometrial cancer. No correlation was found between MAGE-A1 immunohistochemical expression and patient survival. There was no significant correlation between MAGE-A4 and MAGE-A1 expression and histologic subtype, FIGO stage, lymph node metastasis, muscular infiltration, and lymphovascular invasion. MAGE-A4 immunohistochemical expression is associated with decreased disease free and overall survival in patients with high-grade endometrial cancer. Our findings suggest that MAGE-A1 may be expressed in the epithelial cells of the normal endometrium. MAGE-A1 is highly expressed in high-grade endometrial cancer, with no impact on survival.

Background: Photodynamic therapy (PDT) represents a palliative treatment resulting in induction of inflammatory reactions with importance for the development of an antitumor immunity. Cancer/testis antigens (CTAs) have been associated with poor prognosis in different types of cancer, including head and neck squamous cell carcinoma (HNSCC)., Materials and Methods: Tumor tissue samples before and after PDT were evaluated for the expression of four different CTAs by immunohistochemistry. Expression intensity and subcellular expression pattern were assessed., Results: Before PDT, expression of any CTA was detectable in 91%. Comparing the overall expression of CTAs, a decreased expression of all melanoma-associated antigens (MAGEs) post-treatment and a slightly increased expression of New York esophageal squamous cell carcinoma 1 (NY-ESO-1) was visible. The simultaneous cytoplasmic and nuclear expression of pan-MAGE or MAGE-A3/A4 correlated with reduced treatment-failure-free-survival (TFFS)., Conclusion: This study investigated the impact of PDT on CTA expression in HNSCC, detecting modified expression patterns after PDT. These changes may have been caused by immunological pressure or epigenetic regulation of CTA expression., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p < 0.0001), 46.6 versus 50.0 versus 109.0 for MAGE-A3/A4 (p = 0.0074) and 13.3 versus 50.0 versus 100.2 months for NY-ESO-1 (p = 0.0019). By multivariate analysis, these factors were confirmed as independent markers for poor survival. HNSCC patients showing protein expression of MAGE-A family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients., (© 2014 UICC.)

Bladder cancer is a common urinary malignancy and a prevalent cause of cancer-related death. Current therapies of early stage non-muscle-invasive bladder cancer (NMIBC) are frequently associated with undesirable toxicities and recurrence. Active antigen-specific immunotherapy may provide a valid therapeutic option for patients with NMIBC. Cancer-testis antigens (CTA) expressed in various tumour types and in a limited range of healthy tissues may represent potential targets for specific immunotherapy. MAGE-A10 is probably the most immunogenic antigen of the MAGE-A family. We evaluated the expression of MAGE-A10 in NMIBC. Seventy-nine patients undergoing surgical treatment for NMIBC were enrolled in the study. MAGE-A10 gene expression was assessed by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed on paraffin-embedded sections. MAGE-A10 gene was specifically expressed in one-third of NMIBC (n = 24: 32.43%). Gene expression was correlated with high tumour grade. MAGE-A10 protein was exclusively detectable in nuclei of tumour cells. More importantly, MAGE-A10 protein was also more frequently detectable in high-grade tumours (p = 0.0001) and in stage T1 tumours invading subepithelial tissue or lamina propria (p = 0.01). A strong correlation between MAGE-A10 staining score and tumour grade and stage could accordingly be observed. These data indicate that MAGE-A10 expression is a feature of aggressive NMIBC and might be used as a novel target for specific immunotherapy of these cancers., (Copyright © 2012 UICC.)

Induction of tumor-antigen-specific T cells in active cancer immunotherapy is generally difficult due to the very low anti-tumoral precursor cytotoxic T cells. By improving tumor-antigen uptake and presentation by dendritic cells (DCs), this problem can be overcome. Focusing on MAGE-A3 protein, frequently expressed in many types of tumors, we analyzed different DC-uptake routes after additional coating the recombinant MAGE-A3 protein with either a specific monoclonal antibody or an immune complex formulation. Opsonization of the protein with antibody resulted in increased DC-uptake compared to the uncoated rhMAGE-A3 protein. This was partly due to Fcγ receptor-dependent internalization. However, unspecific antigen internalization via macropinocytosis also played a role. When analyzing DC-uptake of MAGE-A3 antigen expressed in multiple myeloma cell line U266, pretreatment with proteasome inhibitor bortezomib resulted in increased apoptosis compared to γ-irradiation. Bortezomib-mediated immunogenic apoptosis, characterized by elevated surface expression of hsp90, triggered higher phagocytosis of U266 cells by DCs involving specific DC-derived receptors. We further investigated the impact of antigen delivery on T-cell priming. Induction of CD8(+) T-cell response was favored by stimulating naïve T cells with either antibody-opsonized MAGE-A3 protein or with the bortezomib-pretreated U266 cells, indicating that receptor-mediated uptake favors cross-presentation of antigens. In contrast, CD4(+) T cells were preferentially induced after stimulation with the uncoated protein or protein in the immune complex, both antigen formulations were preferentially internalized by DCs via macropinocytosis. In summary, receptor-mediated DC-uptake mechanisms favored the induction of CD8(+) T cells, relevant for clinical anti-tumor response.

Recent studies indicate that ER/PR/HER-2-negative (triple-negative, TN) breast cancers may be "CTA-rich" tumors, suggesting the possibility of CTA-based cancer vaccines as a treatment option for patients bearing these tumors. MAGE-A10 together with NY-ESO-1 is probably the most immunogenic CTA, representing a potentially highly attractive target of active specific immunotherapies. Paraffin-embedded tumor sections were collected retrospectively from 165 breast cancer patients diagnosed between 2002 and 2003. Immunohistochemical staining for MAGE-A10 and NY-ESO-1 was performed. The expression of MAGE-A10 and NY-ESO-1 was correlated with other clinicopathological variables. MAGE-A10 expression (score ≥ 2+) was detected in 105/164 (64%), and NY-ESO-1 expression (score ≥ 2+) was observed in 14/164 (8.5%) patients. No correlation between MAGE-A10 and NY-ESO-1 expression and tumor size, tumor grade, Ki-67 and lymph nodes status was detectable. MAGE-A10 expression was significantly associated with ER-negative (P = 0.002), PR-negative (P = 0.002) and HER-2-negative (P = 0.044) tumors. We clearly showed that MAGE-A10 is frequently expressed in the group of TN patients, where the majority (85.7%) of tumors express this CTA. Because of limited therapeutic options for the triple-negative breast cancer, the frequent expression of MAGE-A10 CTA in these cancers may offer the opportunity for a much needed additional treatment for this group of patients.

Background: Tumor markers are genes or their products expressed exclusively or preferentially in tumor cells and cancer-testis antigens (CTAs) form a group of genes with a typical expression pattern expressed in a variety of malignant neoplasms. CTAs are considered potential targets for cancer vaccines. It is possible that the CTA MAGE-A4 (melanoma antigen) and MAGE-C1 are expressed in carcinoma of the oral cavity and are related with survival., Methods: This study involved immunohistochemical analysis of 23 patients with oral squamous cell carcinoma (SCC) and was carried out using antibodies for MAGE-A4 and MAGE-C1. Fisher's exact test and log-rank test were used to evaluate the results., Results: The expression of the MAGE-A4 and MAGE-C1 were 56.5% and 47.8% without statistical difference in studied variables and survival., Conclusion: The expression of at least 1 CTA was present in 78.3% of the patients, however, without correlation with clinicopathologic variables and survival., (Copyright © 2011 Wiley Periodicals, Inc.)

MAGE-A10 is a highly immunogenic member of the MAGE-A family of cancer/testis tumor-associated antigens (C/T TAAs). Studies performed with broadly reactive antibodies have helped to initially characterize this TAA. However, no specific reagents have been developed so far, thus preventing a thorough analysis of its expression in healthy and tumoral tissues. We have produced MAGE-A10 gene product in soluble recombinant form, and we have used it to generate specific monoclonal antibodies (mAbs). One of these reagents, recognizing an epitope located at the COOH terminus of the MAGE-A10 gene product, was used to stain a multitumor tissue microarray comprising more than 2,500 paraffin-embedded specimens including healthy tissues, benign tumors and malignancies of different histological origin. MAGE-A10 protein was identified as an intranuclear protein of an apparent molecular weight of 70 kDa, expressed in normal spermatogonia and spermatocytes but in no other healthy tissue. Most importantly, this C/T TAA appears to be expressed in high (>50%) percentages of cancer cells from a number of malignancies, including lung, skin and urothelial tumors. Unexpectedly, high expression of MAGE-A10 TAA at the protein level was also detectable in gynecological malignancies and stomach and gall bladder cancers. The characterization of MAGE-A10-specific reagents might set the stage for the development of targeted active immunotherapy by clarifying potential indications and by allowing the selection of patients eligible for treatment and the monitoring of its effectiveness., (Copyright © 2010 UICC.)

Background: Despite diagnostic and therapeutic advances in head and neck cancer, the 5-year survival of patients with laryngeal cancer has not improved in the last 30 years. Several recent studies indicate that specific targets for immunotherapeutic approaches can be useful in the control of cancer. There is considerable interest in the expression of cancer testis antigens in human cancers since they may serve as the basis for an immunologic approach to therapy., Methods: We evaluated by immunohistochemical analysis the expression of cancer testis antigens MAGE-A4 (57B), MAGE-C1 (CT7-33), MAGE-A1 (MA454), MAGE-A3 (M3H67), MAGE-C2 (CT10.5), NY-ESO-1 (E978), and GAGE (GAGE) in squamous cell carcinoma (SCC) of the larynx., Results: A total of 63 cases (57 men and 6 women) of laryngeal SCC were available for this study. The findings were correlated with the clinical course and laboratory data. Expression of at least 1 cancer testis antigen was detected in 42 of 63 of the laryngeal SCCs (67%). In 34 of 42 of the positive cases (81%) there was simultaneous expression of ≥2 cancer testis antigens. There was significant correlation between antigen expression and advanced tumor stage (stage III/IV) in cases with reactivity to only 1 antibody (p = .01) as well as in the cases with reactivity to ≥2 primary antibodies (≥2 mAbs, p = .04). There was no association between survival and expression of any of the analyzed antigens., Conclusions: We find a high incidence of cancer testis antigen expression in SCCs of the larynx, which was correlated with advanced clinical stage. Our data indicate that cancer testis antigens could be valuable vaccine targets in laryngeal tumors, especially in those with a worse prognosis., (Copyright © 2010 Wiley Periodicals, Inc.)

Aim: To immunohistochemically evaluate the expression of MAGE-A1, MAGE-A, and NY-ESO-1 cancer/testis (C/T) tumor antigens in medullary breast cancer (MBC) tumor samples and to analyze it in relation to the clinicopathological features., Methods: This retrospective study included samples from 49 patients: 40 with typical MBC and 9 with atypical MBC. Tumor specimens were obtained from patients operated on in the University Hospital for Tumors and the Sisters of Mercy University Hospital, Zagreb, Croatia, from 1999 to 2005. Standard immunohistochemistry was used on archival paraffin-embedded MBC tissues., Results: MAGE-A1, MAGE-A, and NY-ESO-1 antigens were expressed in 33% (16/49), 33% (16/49), and 22% (11/49) of patients, respectively. No difference between the groups with and without C/T tumor antigen expression in age at diagnosis, tumor size, axillary lymph node metastasis, adjuvant therapy, and HER-2 expression was identified. Significantly more patients died in the MAGE-A-positive group than in the MAGE-A-negative group (P=0.010), whereas a borderline significance was found between MAGE-A1-positive and the MAGE-A1-negative group (P=0.079) and between NY-ESO-1-positive and NY-ESO-1-negative group (P=0.117). Overall survival, as evaluated by the Kaplan-Meier curves, was lower in MAGE-A1- (P=0.031), MAGE-A- (P=0.004), NY-ESO-1-positive groups (P=0.077)., Conclusion: Expression of C/T antigens may represent a marker of potential prognostic relevance in MBC.

Background: The prognostic value of cancer testis antigens in pharyngeal cancer is understudied., Methods: We recruited 90 patients who were treated for pharyngeal cancer. Monoclonal antibodies 57B and B9.8.1.1 were used for detection of MAGE-A and NY-ESO-1 genes., Results: MAGE-A and NY-ESO-1 gene products were detectable in 70.0% and 33.3% of pharyngeal tumors, respectively. No correlation was established between MAGE-A and NY-ESO-1 expression and TNM staging at presentation. Survival analysis showed a trend toward a shorter 5-year disease-free survival in the group of patients with MAGE-A-positive tumors (log-rank test, p = .122). In contrast, a trend toward a prolonged 5-year disease-free survival was observed in the group of patients with NY-ESO-1-positive tumors (log-rank test, p = .219)., Conclusion: In a large population of patients with pharyngeal cancer and available 5-year survival data, prognosis tended to be poorer with MAGE-A expression and better with NY-ESO-1 expression, but the correlations did not reach statistical significance.

Immunohistochemical expression of MAGE-A1, MAGE-A3/4 and NY-ESO-1/LAGE-1 cancer testis antigens (CTA) was assessed in 24 fetal testes from 15th to 36th week of gestation. Three monoclonal antibodies were used for immunohistochemical staining: 77B recognizing MAGE-A1, 57B recognizing multiple MAGE-A CTA, and D8.38 recognizing NY-ESO-1/LAGE-1. Expression of MAGE-A1 was not observed in fetal testis samples, whereas multi-MAGE-A and NY-ESO-1/LAGE-1 specific reagents stained pro-spermatogonia in all samples with different expression levels during the period of fetal development observed. Significant expression of MAGE-A3/4 and almost continuous expression of NY-ESO-1 in fetal testes after 22nd week of gestation suggested their important role in the development of sex cords and pro-spermatogonia in particular.

Besides their variable presence in fetal and adult germ cells, CT antigens have occasionally been detected in placental tissue. However, these data are scarce and solely based on mRNA analyses; nothing is known about their presence at the protein level. Here, we analyzed the expression of various CT antigens in placental tissues from gestational age week 5 to week 42 using monoclonal antibodies to various antigens of the MAGE-A and -C families, NY-ESO-1, as well as GAGE. We show that CT antigen expression in placenta varies widely for the various antigens, ranging from completely negative to abundant. Since little is known about the function and biology of CT antigens, interpretation of this highly variable expression pattern is purely speculative. However, our data indicate that the various CT antigens have different functions during placental development.

Production of indoleamine 2,3-dioxygenase (IDO) by tumor cells, leading to tryptophan depletion and production of immunosuppressive metabolites, may facilitate immune tolerance of cancer. IDO gene is also expressed in dendritic cells (DC) upon maturation induced by lipopolysaccarides or IFN. We investigated IDO gene expression in melanoma cell lines and clinical specimens as compared to mature DC (mDC). Furthermore, we explored effects of L-kynurenine (L-kyn) and 3-hydroxyanthranilic acid (3-HAA) on survival and antigen-dependent and independent proliferation of CD8(+) cells. We observed that IDO gene expression in cultured tumor cells and freshly excised samples is orders of magnitude lower than in mDC, providing highly efficient antigen presentation to CD8(+) T cells. Non toxic concentrations of L-kyn or 3-HAA did not significantly inhibit antigen-specific CTL responses. However, 3-HAA, but not L-kyn markedly inhibited antigen-independent proliferation of CD8(+) T cells induced by common receptor gamma-chain cytokines IL-2, -7 and -15. Our data suggest that CD8(+) T cell activation induced by antigenic stimulation, a function exquisitely fulfilled by mDC, is unaffected by tryptophan metabolites. Instead, in the absence of effective T cell receptor triggering, 3-HAA profoundly affects homeostatic proliferation of CD8(+) T cells.

Aim: To evaluate the possible prognostic role of the expression of MAGE-A4 and NY-ESO-1 cancer/testis antigens in women diagnosed with invasive ductal breast cancer and determine the expression of HER-2 antigen., Methods: The expression of MAGE-A4, NY-ESO-1, and HER-2 antigens was evaluated immunohistochemically on archival paraffin-embedded samples of breast cancer tissue from 81 patients. All patients had T1 to T3, N0 to N1, M0 tumors and underwent postoperative radiotherapy and, if indicated, systemic therapy (chemotherapy and hormonal therapy). The antigen expression in women who were disease-free for 5 years of follow up (n=23) was compared with that in women with either locoregional relapse (n=30) or bone metastases (n=28). Patient survival after 10 years of follow up was assessed., Results: The three groups of women were comparable in terms of age, type of operation, tumor size, tumor grade, number of metastatically involved axillary lymph nodes, Nottingham prognostic index (NPI), progesterone receptor (PR) status, and adjuvant hormonal therapy. Estrogen receptors (ER) were positive in 13 women in the 5-year relapse-free group vs 8 in locoregional relapse and 7 in bone metastases group (P=0.032). There were significantly fewer women who received adjuvant chemotherapy in the 5-year relapse-free group than in other two groups (7 vs 23 with locoregional relapse and 25 with bone metastases; P<0.001). This group also had a significantly better 10-year survival (14 women vs 1 with locoregional relapse and 1 with bone metastases; P<0.001). The three groups did not differ in the NY-ESO-1 or HER-2 expression, but the number of patients expressing MAGE-A4 antigen was significantly lower in the group with locoregional relapse (P=0.014). In all groups, MAGE-A4 antigen expression was associated with the NY-ESO-1 antigen expression (P=0.006), but not with tumor size and grade, number of metastatically involved axillary lymph nodes, or the ER and PR status. MAGE-A4-positive patients had a significantly longer survival than the MAGE-A4-negative patients (P=0.046). This was not observed with NY-ESO-1 and HER-2 antigens., Conclusion: Our results suggest that the MAGE-A4 antigen may be used as a tumor marker of potential prognostic relevance.

Objective: To investigate immunohistochemical expression of MAGE-A and NY-ESO-1/LAGE-1, cancer testis antigens in prostate tissues showing evidence of malignant transformation or benign hyperplasia., Methods: 112 prostate samples from patients undergoing surgery at the Urology Clinic at the Zagreb Clinical Hospital Center from 1995 to 2003 were investigated in this study. Of these, 92 carcinoma samples were obtained by radical prostatectomy, and 20 benign prostatic hyperplasia samples by transvesical prostatectomy. Three monoclonal antibodies were used for immunohistochemical staining: 77B for MAGE-A1, 57B for multi-MAGE-A and D8.38 for NY-ESO-1 expression., Results: Expression of MAGE-A1 was observed in 10.8% of carcinoma samples, whereas multi-MAGE-A and NY-ESO-1/LAGE-1 stained 85.9% and 84.8% of samples. Immunohistochemical staining was only detectable in the cytoplasm. A significant heterogeneity could be observed within a same tissue sample where areas with strong positivities coexisted with cancer testis antigens negative areas. Interestingly, a majority of 57B positive cases were also found to be D8.38 positive (correlation coefficient r=0.727 (P<0.01)). Cancer testis antigens expression was neither significantly correlated with PSA values nor with Gleason score. In benign prostatic hyperplasia tissues MAGE-A1 expression was detected in 5%, while 57B and D8.38 staining was observed in 15% samples, and in all cases percentages of positive cells were always <10%., Conclusion: Our data underline the peculiar relevance of cancer testis antigens expression in prostate cancers, with potential implications regarding both diagnosis and therapy., (Copyright (c) 2005 Wiley-Liss, Inc.)

Cancer immunotherapy requires the induction of HLA class I restricted cytotoxic T lymphocytes (CTL) specific for tumor associated antigens (TAA). While a number of TAA have been identified, there is an urgent need for the development of adjuvants capable of stimulating CTL responsiveness. Previously, we reported the capacity of immunopotentiating reconstituted influenza virosomes (IRIV) to enhance CTL responses specific for synthetic peptides simultaneously added to cultures in soluble form. This effect was based on IRIV mediated activation of CD4(+) T cells. Here we investigated the "in vitro" immunogenicity of a novel virosome formulation coupling in a single reagent the adjuvant power of IRIV to the capacity of liposomes to efficiently encapsulate synthetic peptides. As a model epitope we chose L(27)Melan-A/Mart-1(26-35) HLA-A0201 restricted peptide from a melanoma-associated antigen widely used in tumor immunotherapy. The reagent thus developed induced the proliferation of CD4(+) T cells characterized by a T helper 1 cytokine profile and CXCR3 expression. Most importantly, it significantly enhanced the generation of L(27)Melan-A/Mart-1(26-35) specific CTL, as compared to soluble peptides, in particular at low nominal epitope concentrations (<1 microg/ml). These effector cells were able to efficiently kill HBL melanoma cells expressing Melan-A/MART-1 and HLA-A0201. The adjuvant effects observed were also detectable in the absence of CD4(+) T cells. Taken together our results suggest that this highly immunogenic antigenic formulation might qualify for clinical use in active, antigen-specific, melanoma immunotherapy.

Objective: To assess the effects of the culture of melanoma cells in 3-dimensional (3D) architectures on their immunorecognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens., Summary Background Data: Growth in 3D architectures has been shown to promote the resistance of cancers to treatment with drugs, cytokines, or irradiation, thereby potentially playing an important role in tumor expansion. We investigated the effects of 3D culture on the recognition of melanoma cells by antigen-specific HLA class I-restricted CTLs., Methods: Culture of HBL melanoma cells expressing Melan-A/Mart-1 tumor-associated antigen and HLA-A0201 on poly-2-hydroxyethyl methacrylate (polyHEMA)-coated plates resulted in the generation of aggregates of 400- to 500-microm diameters containing on average 30,000 cells and characterized by slower proliferation, as compared with monolayer (2-dimensional) cultures. HLA-A0201 restricted Melan-A/Mart-127-35-specific CTL clones were used to evaluate tumor cell immunorecognition measured as specific IFN-gamma production. Comparative gene and protein expression in 2D and 3D cultures was studied by real-time PCR and flow cytometry, respectively. Overall differences in gene expression profiles between 2D and 3D cultures were evaluated by high-density oligonucleotide array hybridization., Results: HLA-A0201 restricted Melan-A/Mart-127-35 specific CTL clones produced high amounts of IFN-gamma upon short-term (4-24 hours) coincubation with HBL cells cultured in 2D but not in 3D, thus suggesting altered antigen recognition. Indeed, Melan-A/Mart-1 expression, at both gene and protein levels, was significantly decreased in 3D as compared with 2D cultures. Concomitantly, a parallel decrease of HLA class I molecule expression was also observed. Differential gene profiling studies on HBL cells showed an increased expression of genes encoding molecules involved in intercellular adhesion, such as junctional adhesion molecule 2 and cadherin-like 1 (>20- and 8-fold up-regulated, respectively) in 3D as compared with 2D cultures., Conclusions: Taken together, our data suggest that mere growth of melanoma cells in 3D architectures, in the absence of immunoselective pressure, may result in defective recognition by tumor-associated antigen-specific CTL.

We have comparatively evaluated the proliferative response of CTL induced in metastatic melanoma patients upon immunization against Melan-A/MART-1(27-35) tumor associated antigen (TAA) to IL-2, IL-7 or IL-15 cytokines, sharing a receptor common gamma-chain (c gamma-c cytokines). Twenty-eight CTL clones were generated from CD8+ T cells obtained from 3 patients during the contraction phase of immune response following a successful vaccine mediated expansion of specific effectors. All clones were able to kill tumor cell lines expressing HLA-A0201 and Melan-A/MART-1, and displayed phenotypic characteristics of effector/memory (CD45RA-/CCR7-) or CD45RA+/CCR7- effector cells in intermediate to late developmental stage (CD28-/CD276+/-) CTL. Proliferative responses could be elicited or enhanced by IL-2 and IL-15, but not IL-7, in the absence or in the presence of T-cell receptor (TCR) triggering, respectively. Accordingly, only IL-2 and IL-15 were able to promote the survival of the CTL clones under investigation. While all clones expressed high amounts of receptor c gamma-c (CD132), lower, but detectable, expression of IL-7 receptor alpha chain was also observed. CD8+ cells from one of the patients treated were obtained 6 months after the last vaccine boost and were cultured in the presence of Melan-A/MART-1(27-35) and each of the 3 cytokines under investigation. Consistent with data from CTL clones, expansion of Melan-A/MART-1(27-35) tetramer positive cells was only observed in the presence of IL-2 or IL-15 but not IL-7. Instead, when CD8+ cells from the same patient were sampled shortly (14 days) after an additional vaccination only IL-2 was able to promote the expansion of Melan-A/MART-1(27-35) tetramer positive cells. Taken together these data suggest a selective responsiveness of TAA-specific CTL to different c gamma-c cytokines., (Copyright 2005 Wiley-Liss, Inc)

Degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) was investigated in the presence of cultured human fibroblasts, and acellular supernatants obtained from these cells; the possible effect of substrate degradation on in vitro immunorecognition was also addressed. In the presence of fibroblasts, gp100(280-288) was degraded to free amino acids with a half-life of less than 4 min; hydrolysis data support the hypothesis that substrate degradation was mainly caused by the activity of cell-expressed amino- and carboxypeptidases. Gp100(280-288) was also degraded in the presence of acellular supernatants: under these conditions, the hydrolysis pattern was similar to that observed in the presence of whole cells, but degradation kinetics was slower. As a result of these phenomena, immunorecognition of gp100(280-288)-specific cytotoxic T lymphocyte (CTL) clones was severely hampered, and was totally suppressed after 90 min. In conclusion, the high activity of fibroblast-expressed proteases, and the presence of wide-scope soluble enzymes, may explain, at least in part, the low activity of peptide-based antineoplastic vaccines, as well as the transient effectiveness of subcutaneously administered peptides in general.

Background/aims: Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy., Methods: In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3., Results: Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161-169 and the MAGE-3 sequence 271-279. In a patient with a particularly high frequency of MAGE-1 sequence 161-169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA-CCR7)., Conclusions: The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma.

The melanoma-associated antigen (MAGE) gene family of cancer-testis antigens is expressed in certain malignant neoplasms and the testis, but not in other healthy tissues. The aim of this study was to determine the usefulness of immunohistochemical staining with the 57B anti-MAGE-A4 mouse monoclonal antibody (MAb) in testicular biopsy specimens from patients with nonobstructive azoospermia and obstructive azoospermia (OA). Fifty-four cases of Sertoli cell only (SCO), 30 cases of spermatocytic arrest, 15 cases of hypospermatogenesis, and 10 testicular biopsy specimens with OA (normal spermatogenesis) were evaluated. Immunohistochemistry was performed using the 57B MAb, which primarily recognizes the MAGE-A4 antigen in paraffinized tissues. The cells were quantitated by a computerized image analysis system. Testicular biopsy specimens with normal spermatogenesis exhibited strong nuclear and cytoplasmic MAGE-A4 staining of spermatogonia and weak staining of spermatocytes, but not spermatids or Sertoli or Leydig cells. No staining was detected in SCO cases. In five cases of SCO with focal spermatogenesis, spermatogonial cells that were initially missed by hematoxylin and eosin staining were detected by MAGE-A4 immunohistochemistry. Immunostaining with the 57B MAb greatly enhanced identification of spermatogonia in cases of spermatocytic arrest and hypospermatogenesis. The number of MAGE-A4-positive spermatogonia was significantly decreased in hypospermatogenesis, as opposed to the OA group (12.1 +/- 4.3 and 30.3 +/- 10.0, respectively). The number of MAGE-A4-positive primary spermatocytes was significantly increased in early maturation arrest, as compared with the OA group (48.2 +/- 10.8 and 16.9 +/- 9.8, respectively). The 57B anti-MAGE-A4 MAb is a useful marker for the detection and quantitation of spermatogonial germ cells. It also facilitates automated image analysis and provides greater accuracy in the histopathologic evaluation of testicular biopsy specimens.

Objective: To evaluate MAGE tumor-associated antigen (TAA) expression in an extensive panel of normal and neoplastic tissues., Summary Background Data: TAAs of the MAGE family represent targets of active specific immunotherapy. Limited-size studies indicate that they are expressed in normal testis and tumors of different histologies. High-throughput tissue microarray (TMA) technology and MAGE TAA-specific monoclonal antibodies now allow us to comprehensively evaluate their expression in large numbers of tissues and to address clinical correlations., Methods: A TMA containing 3,520 samples from 197 different tissues and a non-small-cell lung cancer TMA including 301 specimens were stained using the MAGE TAA-specific monoclonal antibody 57B. For patients with squamous cell carcinoma of the lung, the dichotomous result (positive vs. negative) of MAGE TAA staining was used as a predictor variable along with other covariates in proportional hazard regression analysis of tumor-specific survival., Results: MAGE TAAs are expressed with frequencies ranging between 22.7% (larynx) and 50% of cases (lung) in squamous cell carcinomas from different anatomic areas and in large cell carcinomas of the lung (37.9%). The authors provide here the first description of MAGE TAA expression in basalioma (48.1%). To investigate the clinical significance of MAGE expression in a frequently positive tumor type, a non-small-cell lung cancer, TMA was then studied. In this TMA 43.2% of tumors were 57B positive. In patients with squamous cell carcinoma, MAGE TAA positivity was significantly correlated with a shorter tumor-specific survival in the proportional hazard regression analysis model., Conclusions: These data suggest novel potential therapeutic indications in different types of cancers. In lung squamous cell carcinoma, the significant association of MAGE TAA expression with poor prognosis suggests that patients with 57B-positive tumors may benefit from early, specific immunotherapy procedures.

The cancer testis (CT) family of antigens are expressed in certain malignant neoplasms and are silent in normal adult tissues, except for the testis. Expression of 2 members of this family, MAGE-A4 and NY-ESO-1, has been described recently in germ cell tumors, malignant melanomas, certain carcinomas and sarcomas. Our study is the first to describe the expression pattern of CT antigens in uterine neoplasms. Ninety-eight cases of uterine neoplasms, including 41 endometrioid, 19 papillary serous and 7 clear cell carcinomas, 22 carcinosarcomas and 9 endometrial stromal sarcomas were studied. Immunohistochemistry was carried out with the 57B monoclonal antibody that recognizes predominantly the MAGE-A4 antigen in paraffinized tissues and the D8.38 antibody that recognizes NY-ESO-1. MAGE-A4 expression was found to be present in 12% of the endometrioid adenocarcinomas, 63% of the papillary serous carcinomas and 91% of the carcinosarcomas. Within the tumor population the extent of MAGE-A4 expression was highest in the carcinosarcomas. In 12 of 22 positively staining carcinosarcomas more than 50% of the tumor cells expressed MAGE-A4. NY-ESO-1 expression was seen in 19% of the endometrioid adenocarcinomas, 32% of the papillary serous carcinomas and in 45% of the carcinosarcomas. CT antigen immunoreactivity was observed in both the carcinomatous and sarcomatous components of the carcinosarcomas and strong correlation between MAGE-A4 and NY-ESO-1 expression was present in individual cases. In summary, strong MAGE-A4 expression and to a lesser degree NY-ESO-1 expression is characteristic of the vast majority of uterine carcinosarcomas and a major subset of papillary serous carcinomas. These results suggest that CT antigen expression by these tumors may represent a novel target for immunotherapy., (Copyright 2002 Wiley-Liss, Inc.)

TAAs of the MAGE family are mostly studied as targets of specific immune responses. Their potential relevance as tumor markers has also been underlined. We used a MAb, 57B, recognizing MAGE-A4 protein in paraffin-embedded sections, to evaluate its expression in bladder cancers by employing TMA including 2,317 samples from 1,849 patients. In 2,090/2,317 cases (90.2%), immunostaining yielded interpretable results. Since for some patients more than 1 sample was available, only interpretable first biopsies (n = 1,628) were considered. MAGE-A4 protein was expressed at significantly (p < 0.001) higher frequency in squamous (25/55, 45.5%) than in adeno (4/15, 26.7%), sarcomatoid (4/14, 28.6%), small cell (5/20, 25%) or transitional cell (281/1,522, 18.5%) carcinomas. In TCCs, overall MAGE-A4 positivity was significantly correlated with invasive phenotype (p < 0.001) and high tumor grade (p < 0.0001). Clinical data from 908 TCC patients were retrospectively evaluated, revealing that strong 57B staining was highly significantly associated with decreased tumor-specific survival (p < 0.0001). These data suggest that evaluation of MAGE-A4 protein expression is useful in the identification of groups of TCCs characterized by severe prognosis, thus possibly providing indications for early MAGE TAA-targeted immunotherapy., (Copyright 2002 Wiley-Liss, Inc.)

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8(+) T cells could be observed. Tetramer staining also revealed significant increases in specific CD8(+) T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.