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Clinical and Developmental Immunology
Volume 2010 (2010), Article ID 891505, 8 pages
http://dx.doi.org/10.1155/2010/891505
Research Article

Potential Target Antigens for a Universal Vaccine in Epithelial Ovarian Cancer

1Department of Gynecological Oncology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands
2Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RBGroningen, The Netherlands
3Department of Epidemiology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RBGroningen, The Netherlands
4Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RBGroningen, The Netherlands

Received 7 June 2010; Accepted 16 July 2010

Academic Editor: Wmartin Martin Kast

Copyright © 2010 Renee Vermeij et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The prognosis of epithelial ovarian cancer (EOC), the primary cause of death from gynaecological malignancies, has only modestly improved over the last decades. Immunotherapeutic treatment using a cocktail of antigens has been proposed as a “universal” vaccine strategy. We determined the expression of tumor antigens in the context of MHC class I expression in 270 primary tumor samples using tissue microarray. Expression of tumor antigens p53, SP17, survivin, WT1, and NY-ESO-1 was observed in 120 (48.0%), 173 (68.9%), 208 (90.0%), 129 (56.3%), and 27 (11.0%) of 270 tumor specimens, respectively. In 93.2% of EOC, at least one of the investigated tumor antigens was (over)expressed. Expression of MHC class I was observed in 78.1% of EOC. In 3 out 4 primary tumors, (over)expression of a tumor antigen combined with MHC class I was observed. These results indicate that a multiepitope vaccine, comprising these antigens, could serve as a universal therapeutic vaccine for the vast majority of ovarian cancer patients.

1. Introduction

Epithelial ovarian cancer (EOC) is the most common cause of death in gynaecologic malignancies [1]. Most ovarian cancer patients are asymptomatic until disease has metastasized and therefore two-thirds of all patients are diagnosed with advanced stage disease [1, 2]. Although the majority of patients with advanced disease achieve complete clinical response rates due to the current therapy of aggressive cytoreductive surgery and platinum-taxane-based chemotherapy, more than 90% develop tumor recurrence, resulting in five-year survival rates of only 30% [3].

These records express the need for a new and improved therapy for EOC. The significance of the immune response for the clinical course of EOC has led to attempts to modulate it artificially with (antigen-specific) immunotherapeutic strategies [4]. Presentation of tumor antigens in the context of MHC molecules on tumor cells is critical for the efficacy of targeted immunotherapy [5]. Thus far, approaches at therapeutic vaccination in cancer patients including administration of peptide pulsed dendritic cells, recombinant viral vectors encoding tumor antigen, DNA-fusion vaccine and single peptide vaccine have not shown consistent, and high percentages of clinical successes [612]. Most clinical studies on immunotherapy targeted one antigen, limiting the use of such vaccines to those patients with (over)expression of that specific tumor antigen. Immunization using a cocktail of antigens has been proposed as a “universal” vaccine strategy. Whereas solid tumors often show heterogeneous protein expression, multiantigen vaccines may have greater therapeutic potential and compensate for tumor antigen-loss variants [13, 14]. The ability to target multiple antigens, may also improve the immunogenicity of therapeutic vaccines [13, 15]. Therefore, discovery of multiple tumor antigens in EOC may provide opportunities for multiantigen immunotherapeutic strategies that can induce sufficient clinical responses. Tumor antigens that are inherently immunogenic and oncogenic in ovarian cancer are p53 [1618], Sperm Protein 17 (SP17) [14, 19, 20], Wilms’ tumor gene 1 (WT1) [2123], survivin [2426], and NY-ESO-1 [12, 27, 28].

The presence of an (heavy) chain and -microglobulin is a prerequisite for the formation of a stable MHC class I complex [29]. Such stable MHC class I complexes are required for presentation of the tumor antigenic peptides [30].

No reports have been published describing tissue microarray staining of p53, SP17, survivin, WT1, and NY-ESO-1 with MHC class I expression in EOC. Further knowledge of the expression of multiple tumor antigens in the context of MHC class I expression is necessary to develop strategies to increase clinical efficacy of multiantigen immunotherapy in EOC.

The aim of the present study was to investigate the expression of SP17 and NY-ESO-1 and overexpression of p53, WT1, and survivin together with -microglobulin and the α-chains, HLA-A and HLA-B/C, in tumor samples obtained from a large well-documented cohort of primary EOC patients using tissue microarray.

2. Materials and Methods

2.1. Patients

Since 1985, the Department of Gynecological Oncology of the University Medical Centre Groningen (UMCG) prospectively stores all clinicopathologic and followup data of epithelial ovarian cancer patients in a computerized database. Tumor samples from 361 patients were collected on a tissue microarray. This tissue microarray contains primary ovarian tumor tissue obtained before chemotherapy of 270 patients. Patients with borderline or nonepithelial tumors were excluded. Primary treatment for all patients consisted of surgery and adjuvant chemotherapeutic treatment consistent of platinum-based regimens and others. Since 1995, platinum-based chemotherapy was supplemented by taxanes.

In the current study, the 270 EOC patients were selected for tumor antigen analysis who underwent primary surgery between 1985 and 2006 and of whom sufficient paraffin-embedded ovarian tumor tissue and complete followup data were available. In a nonselected subgroup of 183 primary EOC patients, MHC class I expression was analyzed. These data are partly previously published by our group [29].

Patients were surgically staged according to FIGO (International Federation of Gynecology and Obstetrics) classification [31]. Optimal and suboptimal debulking was defined as the largest residual tumor lesions having a diameter of, respectively, 2 cm or 2 cm. Histology of all tumors was determined according to World Health Organization criteria [32].

All relevant data were filed in a separate anonymous database in which patients were given unique codes to protect patient identity. Database management was restricted to two people with access to the larger database containing all patients’ characteristics. Due to these procedures, no additional patient or institutional review board approval was required according to Dutch Law.

2.2. Tissue Microarrays

Tissue microarrays were constructed as described previously [17]. Four cores of 0.6 mm² were taken by biopsy and placed by a tissue microarrayer (Beecher Instruments, Silver Spring, MD, USA) on a recipient paraffin block. Using a microtome, 4  m sections were cut from each tissue microarray block and applied to aminopropyltriethoxysilane-coated slides. All arrayed samples were H&E-stained to confirm the presence of tumor tissue.

2.3. Immunohistochemical Staining of Tissue Microarrays

Tissue microarray sections were deparaffinized in xylene and rehydrated through graded concentrations of ethanol to distilled water. The sections were boiled for 15 minutes in a microwave to accomplish antigen retrieval. Endogenous peroxidase was blocked by incubation of sections for 30 minutes in 0.3% hydrogen peroxide. Primary antibodies, antigen retrieval buffers, and detection methods used are provided as supplementary data (Table 1). Sections were counterstained with hematoxylin. All control experiments gave satisfactory results.

tab1
Table 1: Antibodies used for immunohistochemical staining.
2.4. Scoring

Evaluation of immunostaining was independently performed by two observers blinded to the clinical data. Agreement between the two observers was 90%. Contradictory outcomes were reviewed by a gynecological pathologist and were reassigned by approval of all parties.

Immunostaining for p53, HLA-A, HLA-B/C, and -m was scored as described in previous studies [17, 29, 30]. The immunohistochemical reaction for SP17 [33], WT1 [34, 35], survivin [24, 36, 37], and NY-ESO-1 [38] was semiquantitatively graded into four classes based on the frequency of nuclear staining for SP17, WT1, and survivin, and cytoplasmatic staining in NY-ESO-1 in ovarian cancer cells: negative = no/very low frequency ( 5%) immunopositive cells; + = low frequency ( 5–25%); ++ = moderate frequency (25%–50%); +++ = high frequency (50%–75%); ++++ = very high frequency (75–100%). The cutoff was `a priori` chosen for scoring; cases with low frequency or higher were considered positive for tumor antigen expression.

2.5. Statistical Analysis of Data

Statistical analysis was carried out using the SPSS 16.0 software package fow Windows (SPSS Inc., Chicago, USA). All cases with 2 evaluable cores were excluded from analysis.

3. Results

3.1. Patients

Tumor samples from 270 consecutive primary ovarian cancer patients (median age 56.9 years, range 16–89) treated at the UMCG between 1985 and 2006 were available (Table 2). The majority of patients presented with serous histology, advanced stage, and/or high-grade disease. First-line chemotherapy regimens were platinum based in 90 (34.2%) patients and platinum and taxane based in 108 (41.1%) patients. Other regimens were given to 25 (9.5%) patients, while 40 (15.2%) patients did not receive chemotherapy because of early stage disease, comorbidity, or treatment refusal.

tab2
Table 2: Patient and tumor characteristics.
3.2. Tumor Antigen (Over)Expression in EOC

P53, SP17, survivin, WT1, and NY-ESO-1 (over)expression was observed in 48.0%, 68.9%, 90.0%, 56.3%, and 11.0% of tumors, respectively (Table 3). In 93.2% tumors, at least one of the investigated tumor antigens was (over)expressed (Table 4). Expression of only one tumor antigen was found in 40 (15.2%) tumors, 70 (26.6%) tumors expressed two antigens, 70 (26.6%) tumors expressed three antigens, 58 (22.1%) tumors expressed four antigens, and 7 (2.7%) tumors expressed all five investigated tumor antigens. Absence of expression of any antigen was seen in 18 (6.8%) patients. Nonevaluable primary tumors due to core loss during staining procedures or absence of tumor tissue ranged from 19 (7.4%) for SP17 staining to 41 (15.2%) for WT1 staining. Several specific combinations of tumor antigen expression cover high percentages of EOC patients, varying from 95.5% (214/224) combining two antigens to a maximum coverage of 98.2% (216/220) combining four antigens (Table 5).

tab3
Table 3: Expression levels of antigen and MHC class I components.
tab4
Table 4: Expression of single or multiple antigens in EOC.
tab5
Table 5: Expression of specific antigen combinations in EOC.
3.3. Immunostaining MHC Class I

Coexpression of HLA-A and -m or HLA-B/C and -m was observed in 98 (53.6%) and 136 (74.7%) of the tumors, respectively (Table 3). Positive MHC class I expression, defined as HLA-A and -m and/or HLA-B/C and -m coexpression, was observed in 143 (78.1%) tumors.

3.4. Coexpression of Tumorantigens and MHC Class I in EOC

Of all EOC positive for p53, SP17, survivin, WT1, or NY-ESO-1, 82.5%, 82.8%, 77.0%, 80.9%, and 80.0% were also positive for MHC class I, respectively (Table 6). In 78.4% of tumors (over)expressing one or more tumor antigens, also expression of MHC class I was found. Furthermore, 74.3% of all tumors coexpressed MHC class I and at least one tumor antigen.

tab6
Table 6: Coexpression of MHC class I components with tumorantigens.

4. Discussion

In a large well-documented cohort of representative EOC patients, (over)expression of at least one of the tumor antigens p53, SP17, survivin, WT1, or NY-ESO-1 was observed in over 90% of the tumors. To our knowledge, this is the first study on the expression of multiple tumor antigens in a large cohort of EOC. Only a minority (6.8%) of the tumors did not express one of the selected tumor antigens. About 75% of the EOC tumors expressed both, one of the tumor antigens and MHC class I. This observation underlines the relevance of designing a multiepitope vaccine consisting of p53, SP17, NY-ESO-1, survivin, and WT1 for the immunotherapeutic treatment of ovarian cancer.

This inventory tissue microarray study enables us to analyze the expression of five well-known tumor antigens in EOC, in correlation to MHC class I expression. Tissue microarray is a practical and powerful tool for high-throughput analysis of tumor tissue identifying targets in human cancers [39]. P53, SP17, NY-ESO-1, survivin, and WT1 are immunogenic target antigens in EOC. Rates of observed (over)expression of p53, SP17, survivin, and WT1 in 48.0%, 68.9%, 90.0%, and 56.3% of EOC patients, respectively, are in agreement with previous studies [16, 24, 40, 41]. NY-ESO-1 expression was seen in 11.0% of tumors in our cohort which is in agreement with the results of others [42, 43]. However, Odunsi et al. observed NY-ESO-1 expression in 43% of EOC patients [38, 44]. This difference in expression might be explained by considerable methodological variability among the different studies. The type of study design, antibodies and assays used to study NY-ESO-1 expression, determination of cutoff points for aberrant NY-ESO-1 expression, and the definition of study end points vary greatly among different studies. Immunohistochemical analyses of tumors have shown heterogeneous NY-ESO-1 expression [45]. Since expression of NY-ESO-1 is mostly focal and nonuniform, tissue microarrays containing large numbers of tumor tissue are essential to determine NY-ESO-1 expression in EOC. Our sample size of 270 EOC patients might be more potent to distinguish between positive and negative NY-ESO-1 expression in EOC compared to 143 EOC patients analyzed by Odunsi et al.

We previously reported on the expression of tumor antigens EGFR and Her-2 in our large well-documented cohort of representative EOC, using tissue microarray [46]. EGFR and Her-2 overexpression was observed in 7.0% and 5.2% of EOC, respectively. The expression of EGFR and Her-2 has been extensively studied in ovarian cancer [47, 48]. Aberrant activity of these antigens is important in tumor growth and development [49, 50]. Therefore, EGFR and Her-2 were considered to be attractive targets for immunotherapeutic strategies in EOC. Because of the low expression levels in EOC, therapeutic potential of vaccines targeting EGFR and Her-2 is limited. As the existing repertoire of known antigens in EOC is relatively small, we performed our innovative study on five highly expressed tumor antigens which may provide opportunities for multiepitope immunotherapeutic strategies targeting the majority of EOC patients.

We provide first evidence that several antigen combinations can be used in a multiepitope vaccine for EOC treatment, since different antigen combinations cover high percentages of EOC patients. Vaccines comprising a mixture of, for example, p53, SP17, and survivin or combining survivin, WT1, and NY-ESO-1 cover the vast majority of EOC patients. Maximum coverage of EOC patients can be obtained by a vaccine comprising four antigens p53, SP17, survivin, and WT1.

Single antigen vaccines targeting p53 [51], SP17 [40], NY-ESO-1 [52], survivin [11], and WT1 [22] have been described to generate tumor antigen-specific cytotoxic T-cell lymphocytes (CTLs) able to lyse autologous tumor cells. One can envision that multiepitope vaccines may enhance immunogenicity, improving clinical efficacy of the immunotherapeutic vaccine.

Multiepitope vaccines should preferably contain multiple MHC class I-presented CTL epitopes derived from different target antigens together with a tumor-specific MHC class II-presented T-helper epitope. This will reduce the risk of immune-driven selection of antigen-loss variants of the tumor. Next, given the pivotal role of T-helper cells in promoting the primary and secondary CTL responses through the induction of DC maturation and the production of cytokines, the inclusion of T-helper epitopes in a multiepitope-based vaccine will have strong beneficial effects [6]. For example, p53-specific T-helper cells induced upon p53 specific immunization might fulfil this role [53].

Important advantages of well-defined multiepitope vaccines over nondefined vaccines, such as tumor lysate vaccines, are their defined nature [6, 54], lack of suppressive inducing antigens [5557], simple way of manipulation to prevent dominance of one antigen over the others [6, 58], universal applicability [6, 59], easiness to make in a standardized procedure [59, 60], possibility to combine with other strategies [59], and limited autoimmune toxicity [55, 61].

Moreover, administration of a multiepitope vaccine as a single mixture offers advantages including: (1) injection of a limited volume, (2) lower number of skin sites with local toxicity due to injection site reactions, and (3) lower chance of error and contamination with the preparation of one versus multiple epitope preparations [13, 14].

In contrast, previous studies showed that administration of multiple epitopes at one injection site could lead to a more vigorous response to just one of the involved antigens [62, 63]. We reasoned that this disadvantageous result might be due to immunodominance of one antigen over the other. Preclinical studies might be helpful in designing the optimal combination of multiantigen vaccines, trying to predict and/or prevent immunodominance. In contrast, the synergy between antigens included in a multiepitope vaccine might induce immune responses with increased potency compared with the response induced by the same epitopes individually [6]. Separate injection sites for all of the involved antigens may result in a significant increase in the magnitude of the antigen-specific T-cell response. It still holds true that several multiantigen combinations cover high percentages of tumors. The most favourable vaccine, based on (pre)clinical studies concerning immunodominance, can be used for treatment of EOC patients.

MHC class I downregulation was observed in 21.9% of tumors. Loss of MHC class I molecules on tumor cells, which may lead to immune escape, is often restricted to one or a few alleles. Targeting multiple epitopes restricted by different class I molecules of the patient will likely circumvent such an escape mechanism [6]. The tumor-associated antigens p53, NY-ESO-1, and WT1 epitopes are presented both by MHC class I and II (according to listing at http://www.cancerimmunity.org/, update September 2008). As a result, p53, WT1, and NY-ESO-1 [64] can function as both CTL and T-helper cell targets.

Considering the importance of the expression of MHC class I by tumor cells for immune recognition by T cells, several regimens could be added in the multiepitope vaccine to enhance MHC class I expression. Treatment with IFN-gamma is known to upregulate MHC class I [10, 65, 66]. Another possibility would be the addition of demethylating agents to the multiepitope vaccine, since DNA hypermethylation, common in human tumors, may result in the loss of MHC class I expression [67, 68].

The most promising finding that emerges from this study is that the vast majority of EOC patients present one or more tumor antigens. Furthermore, if tumor cells present one of our investigated tumor antigens, it is likely to express MHC class I as well. Therefore, a vaccine comprising the investigated tumor antigens is capable of targeting tumor cells of the vast majority of EOC patients. Since several combinations of tumor antigens cover the majority of EOC patients, different institutes can attribute personally preferred antigens to their multiepitope vaccine.

In summary, we are first to show that multiepitope immunotherapy combining tumor antigens p53, SP17, survivin, WT1, and/or NY-ESO-1 might be a promising new therapeutic vaccination strategy in ovarian cancer.

Conflict of Interest Statement

The authors declare that there are no conflicts of interest.

References

  1. A. Jemal, R. Siegel, J. Xu, and E. Ward, “Cancer statistics, 2010,” Ca: A Cancer Journal for Clinicians. In press. View at Publisher · View at Google Scholar · View at PubMed
  2. C. H. Holschneider and J. S. Berek, “Ovarian cancer: epidemiology, biology, and prognostic factors,” Seminars in Surgical Oncology, vol. 19, no. 1, pp. 3–10, 2000. View at Publisher · View at Google Scholar · View at Scopus
  3. M. A. Bookman, “Standard treatment in advanced ovarian cancer in 2005: the state of the art,” International Journal of Gynecological Cancer, vol. 15, no. 6, pp. 212–220, 2005. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  4. F. Coilinson and G. Jayson, “New therapeutic agents in ovarian cancer,” Current Opinion in Obstetrics and Gynecology, vol. 21, no. 1, pp. 44–53, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  5. H. T. Khong and N. P. Restifo, “Natural selection of tumor variants in the generation of “tumor escape” phenotypes,” Nature Immunology, vol. 3, no. 11, pp. 999–1005, 2002. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  6. J. H. Kessler and C. J. M. Melief, “Identification of T-cell epitopes for cancer immunotherapy,” Leukemia, vol. 21, no. 9, pp. 1859–1874, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  7. I. M. Svane, A. E. Pedersen, K. Nikolajsen, and M.-B. Zocca, “Alterations in p53-specific T cells and other lymphocyte subsets in breast cancer patients during vaccination with p53-peptide loaded dendritic cells and low-dose interleukin-2,” Vaccine, vol. 26, no. 36, pp. 4716–4724, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  8. J. Kuball, M. Schuler, and M. Schuler, “Generating p53-specific cytotoxic T lymphocytes by recombinant adenoviral vector-based vaccination in mice, but not man,” Gene Therapy, vol. 9, no. 13, pp. 833–843, 2002. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  9. A. G. Menon, P. J. K. Kuppen, and P. J. K. Kuppen, “Safety of intravenous administration of a canarypox virus encoding the human wild-type p53 gene in colorectal cancer patients,” Cancer Gene Therapy, vol. 10, no. 7, pp. 509–517, 2003. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  10. C. Chaise, S. L. Buchan, and S. L. Buchan, “DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance,” Blood, vol. 112, no. 7, pp. 2956–2964, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  11. T. Tsuruma, Y. Iwayama, and Y. Iwayama, “Clinical and immunological evaluation of anti-apoptosis protein, survivin-derived peptide vaccine in phase I clinical study for patients with advanced or recurrent breast cancer,” Journal of Translational Medicine, vol. 6, article 24, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  12. C. S. M. Diefenbach, S. Gnjatic, and S. Gnjatic, “Safety and immunogenicity study of NY-ESO-1b peptide and montanide ISA-51 vaccination of patients with epithelial ovarian cancer in high-risk first remission,” Clinical Cancer Research, vol. 14, no. 9, pp. 2740–2748, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  13. K.A. Chianese-Bullock, S. T. Lewis, N. E. Sherman, J. D. Shannon, and C. L. Slingluff Jr., “Multi-peptide vaccines vialed as peptide mixtures can be stable reagents for use in peptide-based immune therapies,” Vaccine, vol. 27, no. 11, pp. 1764–1770, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  14. J. M. Straughn Jr., D. R. Shaw, and D. R. Shaw, “Expression of sperm protein 17 (Sp17) in ovarian cancer,” International Journal of Cancer, vol. 108, no. 6, pp. 805–811, 2004. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  15. J. M. Kirkwood, S. Lee, and S. Lee, “Immunogenicity and antitumor effects of vaccination with peptide vaccine +/- granulocyte-monocyte colony-stimulating factor and/or IFIN-α2b in advanced metastatic melanoma: eastern cooperative oncology group phase II trial E1696,” Clinical Cancer Research, vol. 15, no. 4, pp. 1443–1451, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  16. H. W. Nijman, A. Lambeck, S. H. van der Burg, A. G. J. van der Zee, and T. Daemen, “Immunologic aspect of ovarian cancer and p53 as tumor antigen,” Journal of Translational Medicine, vol. 3, article 34, 2005. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  17. P. de Graeff, J. Hall, and J. Hall, “Factors influencing p53 expression in ovarian cancer as a biomarker of clinical outcome in multicentre studies,” British Journal of Cancer, vol. 95, no. 5, pp. 627–633, 2006. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  18. A. Lambeck, N. Leffers, and N. Leffers, “P53-specific T cell responses in patients with malignant and benign ovarian tumors: implications for p53 based immunotherapy,” International Journal of Cancer, vol. 121, no. 3, pp. 606–614, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  19. M. Chiriva-Internati, F. Grizzi, and F. Grizzi, “A NOD/SCID tumor model for human ovarian cancer that allows tracking of tumor progression through the biomarker Sp17,” Journal of Immunological Methods, vol. 321, no. 1-2, pp. 86–93, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  20. T. Nakazato, T. Kanuma, T. Tamura, L. S. Faried, H. Aoki, and T. Minegishi, “Sperm protein 17 influences the tissue-specific malignancy of clear cell adenocarcinoma in human epithelial ovarian cancer,” International Journal of Gynecological Cancer, vol. 17, no. 2, pp. 426–432, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  21. Y. Oka, A. Tsuboi, Y. Oji, I. Kawase, and H. Sugiyama, “WT1 peptide vaccine for the treatment of cancer,” Current Opinion in Immunology, vol. 20, no. 2, pp. 211–220, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  22. G. Li, Y. Zeng, and Y. Zeng, “Human ovarian tumour-derived chaperone-rich cell lysate (CRCL) elicits T cell responses in vitro,” Clinical and Experimental Immunology, vol. 148, no. 1, pp. 136–145, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  23. H. J. Stauss, S. Thomas, and S. Thomas, “WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells,” Blood Cells, Molecules, and Diseases, vol. 40, no. 1, pp. 113–116, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  24. C. Cohen, C. M. Lohmann, G. Cotsonis, D. Lawson, and R. Santoianni, “Survivin expression in ovarian carcinoma: correlation with apoptotic markers and prognosis,” Modern Pathology, vol. 16, no. 6, pp. 574–583, 2003. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  25. L. Sui, Y. Dong, M. Ohno, Y. Watanabe, K. Sugimoto, and M. Tokuda, “Survivin expression and its correlation with cell proliferation and prognosis in epithelial ovarian tumors,” International Journal of Oncology, vol. 21, no. 2, pp. 315–320, 2002. View at Scopus
  26. N. Zaffaroni, M. Pennati, and M. Pennati, “Expression of the anti-apoptotic gene survivin correlates with taxol resistance in human ovarian cancer,” Cellular and Molecular Life Sciences, vol. 59, no. 8, pp. 1406–1412, 2002. View at Publisher · View at Google Scholar · View at Scopus
  27. J. Matsuzaki, F. Qian, and F. Qian, “Recognition of naturally processed and ovarian cancer reactive CD8+ T cell epitopes within a promiscuous HLA class II T-helper region of NY-ESO-1,” Cancer Immunology, Immunotherapy, vol. 57, no. 8, pp. 1185–1195, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  28. Y.-T. Chen, M. Hsu, and M. Hsu, “Cancer/testis antigen CT45: analysis of mRNA and protein expression in human cancer,” International Journal of Cancer, vol. 124, no. 12, pp. 2893–2898, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  29. N. Leffers, M. J. M. Gooden, and M. J. M. Gooden, “Down-regulation of proteasomal subunit MB1 is an independent predictor of improved survival in ovarian cancer,” Gynecologic Oncology, vol. 113, no. 2, pp. 256–263, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  30. N. Leffers, A. J. A. Lambeck, P. de Graeff, A. Y. Bijlsma, T. Daemen, A. G. J. van der Zee, and H. W. Nijman, “Survival of ovarian cancer patients overexpressing the tumour antigen p53 is diminished in case of MHC class I down-regulation,” Gynecologic Oncology, vol. 110, no. 3, pp. 365–373, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  31. “Cancer Committee of the International Federation of Gynaecology and Obstetrics. Staging announcement: FIGO Cancer Committee,” Gynecological Oncology, vol. 25, pp. 383–385, 1986.
  32. R. E. Scully, “Histological typing of ovarian tumours,” in World Health Organisation. International Histological Classification of Tumours, pp. 11–19, Springer, Berlin, Germany, 2004.
  33. F. Grizzi, P. Gaetani, and P. Gaetani, “Sperm protein 17 is expressed in human nervous system tumours,” BMC Cancer, vol. 6, article 23, 2006. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  34. G. Acs, T. Pasha, and P. J. Zhang, “WT1 is differentially expressed in serous, endometrioid, clear cell, and mucinous carcinomas of the peritoneum, fallopian tube, ovary, and endometrium,” International Journal of Gynecological Pathology, vol. 23, no. 2, pp. 110–118, 2004. View at Publisher · View at Google Scholar · View at Scopus
  35. M. Shimizu, T. Toki, Y. Takagi, I. Konishi, and S. Fujii, “Immunohistochemical detection of the Wilms' tumor gene (WT1) in epithelial ovarian tumors,” International Journal of Gynecological Pathology, vol. 19, no. 2, pp. 158–163, 2000. View at Scopus
  36. L. Kleinberg, V. A. Florenes, I. Silins, K. Haug, C. G. Trope, J. M. Nesland, and B. Davidson, “Nuclear expression of survivin is associated with improved survival in metastatic ovarian carcinoma,” Cancer, vol. 109, no. 2, pp. 228–238, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  37. G. Ferrandina, F. Legge, and F. Legge, “Survivin expression in ovarian cancer and its correlation with clinico-pathological, surgical and apoptosis-related parameters,” British Journal of Cancer, vol. 92, no. 2, pp. 271–277, 2005. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  38. K. Odunsi, A. A. Jungbluth, and A. A. Jungbluth, “NY-ESO-1 and LAGE-1 cancer-testis antigens are potential targets for immunotherapy in epithelial ovarian cancer,” Cancer Research, vol. 63, no. 18, pp. 6076–6083, 2003. View at Scopus
  39. N. M. T. Jawhar, “Tissue microarray: a rapidly evolving diagnostic and research tool,” Annals of Saudi Medicine, vol. 29, no. 2, pp. 123–127, 2009. View at Publisher · View at Google Scholar · View at Scopus
  40. M. Chiriva-Internati, Z. Wang, E. Salati, P. Timmins, and S. H. Lim, “Tumor vaccine for ovarian carcinoma targeting sperm protein 17,” Cancer, vol. 94, no. 9, pp. 2447–2453, 2002. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  41. M. V. Barbolina, B. P. Adley, L. D. Shea, and M. S. Stack, “Wilms tumor gene protein 1 is associated with ovarian cancer metastasis and modulates cell invasion,” Cancer, vol. 112, no. 7, pp. 1632–1641, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  42. K. Milne, R. O. Barnes, and R. O. Barnes, “Tumor-infiltrating T cells correlate with NY-ESO-1-specific autoantibodies in ovarian cancer,” PLoS ONE, vol. 3, no. 10, article e3409, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  43. E. Yakirevich, E. Sabo, O. Lavie, S. Mazareb, G. C. Spagnoli, and M. B. Resnick, “Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens in serous ovarian neoplasms,” Clinical Cancer Research, vol. 9, no. 17, pp. 6453–6460, 2003. View at Scopus
  44. A. Woloszynska-Read, P. Mhawech-Fauceglia, J. Yu, K. Odunsi, and A. R. Karpf, “Intertumor and intratumor NY-ESO-1 expression heterogeneity Is associated With promoter-specific and global DNA methylation status in ovarian cancer,” Clinical Cancer Research, vol. 14, no. 11, pp. 3283–3290, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  45. M. J. Scanlan, A. O. Gure, A. A. Jungbluth, L. J. Old, and Y.-T. Chen, “Cancer/testis antigens: an expanding family of targets for cancer immunotherapy,” Immunological Reviews, vol. 188, pp. 22–32, 2002. View at Publisher · View at Google Scholar · View at Scopus
  46. P. de Graeff, A. P. G. Crijns, and A. P. G. Crijns, “The ErbB signalling pathway: protein expression and prognostic value in epithelial ovarian cancer,” British Journal of Cancer, vol. 99, no. 2, pp. 341–349, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  47. M. V. Seiden, H. A. Burris, and H. A. Burris, “A phase II trial of EMD72000 (matuzumab), a humanized anti-EGFR monoclonal antibody, in patients with platinum-resistant ovarian and primary peritoneal malignancies,” Gynecologic Oncology, vol. 104, no. 3, pp. 727–731, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  48. C.-H. Lee, D. G. Huntsman, and D. G. Huntsman, “Assessment of Her-1, Her-2, and Her-3 expression and Her-2 amplification in advanced stage ovarian carcinoma,” International Journal of Gynecological Pathology, vol. 24, no. 2, pp. 147–152, 2005. View at Publisher · View at Google Scholar · View at Scopus
  49. D. E. Milenic, K. J. Wong, K. E. Baidoo, G. L. Ray, K. Garmestani, M. Williams, and M. W. Brechbiel, “Cetuximab: preclinical evaluation of a monoclonal antibody targeting EGFR for radioimmunodiagnostic and radioimmunotherapeutic applications,” Cancer Biotherapy and Radiopharmaceuticals, vol. 23, no. 5, pp. 619–631, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  50. E. Horak, F. Hartmann, and F. Hartmann, “Radioimmunotherapy targeting of HER2/neu oncoprotein on ovarian tumor using lead-212-DOTA-AE1,” Journal of Nuclear Medicine, vol. 38, no. 12, pp. 1944–1950, 1997. View at Scopus
  51. I. M. Svane, A. E. Pedersen, and A. E. Pedersen, “Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer: report from a phase I study,” Cancer Immunology, Immunotherapy, vol. 53, no. 7, pp. 633–641, 2004. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  52. K. Odunsi, F. Qian, and F. Qian, “Vaccination with an NY-ESO-1 peptide of HLA class I/II specificities induces integrated humoral and T cell responses in ovarian cancer,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 31, pp. 12837–12842, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  53. N. Leffers, A. J. A. Lambeck, and A. J. A. Lambeck, “Immunization with a P53 synthetic long peptide vaccine induces P53-specific immune responses in ovarian cancer patients, a phase II trial,” International Journal of Cancer, vol. 125, no. 9, pp. 2104–2113, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  54. J. Galea-Lauri, J. W. Wells, D. Darling, P. Harrison, and F. Farzaneh, “Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell-based cancer vaccination,” Cancer Immunology, Immunotherapy, vol. 53, no. 11, pp. 963–977, 2004. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  55. R. R. Caspi, “Immunotherapy of autoimmunity and cancer: the penalty for success,” Nature Reviews Immunology, vol. 8, no. 12, pp. 970–976, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  56. J. M. Kirkwood, A. A. Tarhini, M. C. Panelli, S. J. Moschos, H. M. Zarour, L. H. Butterfield, and H. J. Gogas, “Next generation of immunotherapy for melanoma,” Journal of Clinical Oncology, vol. 26, no. 20, pp. 3445–3455, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  57. P. Hwu and R. S. Freedman, “The immunotherapy of patients with ovarian cancer,” Journal of Immunotherapy, vol. 25, no. 3, pp. 189–201, 2002. View at Publisher · View at Google Scholar · View at Scopus
  58. A. L. Glazyrin, J. Kan-Mitchell, and M. S. Mitchell, “Analysis of in vitro immunization: generation of cytotoxic T-lymphocytes against allogeneic melanoma cells with tumor lysate-loaded or tumor RNA-transfected antigen-presenting cells,” Cancer Immunology, Immunotherapy, vol. 52, no. 3, pp. 171–178, 2003. View at Scopus
  59. C.-F. Hung, T. C. Wu, A. Monie, and R. Roden, “Antigen-specific immunotherapy of cervical and ovarian cancer,” Immunological Reviews, vol. 222, no. 1, pp. 43–69, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  60. C. J. Kim, S. Dessureault, D. Gabrilovich, D. S. Reintgen, and C. L. Slingluff Jr., “Immunotherapy for melanoma,” Cancer Control, vol. 9, no. 1, pp. 22–30, 2002. View at Scopus
  61. M. J. Cannon, A. D. Santin, and T. J. O'Brien, “Immunological treatment of ovarian cancer,” Current Opinion in Obstetrics and Gynecology, vol. 16, no. 1, pp. 87–92, 2004. View at Publisher · View at Google Scholar · View at Scopus
  62. G. G. Kenter, M. J. P. Welters, and M. J. P. Welters, “Phase I immunotherapeutic trial with long peptides spanning the E6 and E7 sequences of high-risk human papillomavirus 16 in end-stage cervical cancer patients shows low toxicity and robust immunogenicity,” Clinical Cancer Research, vol. 14, no. 1, pp. 169–177, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  63. M. J. Palmowski, E. M.-L. Choi, and E. M.-L. Choi, “Competition between CTL narrows the immune response induced by prime-boost vaccination protocols,” Journal of Immunology, vol. 168, no. 9, pp. 4391–4398, 2002. View at Scopus
  64. N. N. Hunder, H. Wallen, and H. Wallen, “Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1,” The New England Journal of Medicine, vol. 358, no. 25, pp. 2698–2703, 2008. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  65. C. Doehn, N. Esser, and N. Esser, “Mode-of-action, efficacy, and safety of a homologous multi-epitope vaccine in a murine model for adjuvant treatment of renal cell carcinoma,” European Urology, vol. 56, no. 1, pp. 123–133, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  66. Y. Nie, G.-Y. Yang, and G.-Y. Yang, “DNA hypermethylation is a mechanism for loss of expression of HLA class I genes in human esophageal squamous cell carcinomas,” Carcinogenesis, vol. 22, no. 10, pp. 1615–1623, 2001. View at Scopus
  67. C. Sers, R. Kuner, and R. Kuner, “Down-regulation of HLA class I and NKG2D ligands through a concerted action of MAPK and DNA methyltransferases in colorectal cancer cells,” International Journal of Cancer, vol. 125, no. 7, pp. 1626–1639, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  68. A. Serrano, S. Tanzarella, I. Lionello, R. Mendez, C. Traversari, F. Ruiz-Cabello, and F. Garrido, “Expression of HLA class I antigens and restoration of antigen-specific CTL response in melanoma cells following 5-aza-2-deoxycytidine treatment,” International Journal of Cancer, vol. 94, no. 2, pp. 243–251, 2001. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus