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Although natural killer (NK) cells are well known for their ability to kill tumors, few studies have addressed the interactions between resting (nonactivated) NK cells and freshly isolated human tumors. Here, we show that human leukocyte antigen class I low tumor cells isolated directly from patients with advanced ovarian carcinoma trigger degranulation by resting allogeneic NK cells. This was paralleled by induction of granzyme B and caspase-6 activities in the tumor cells and significant tumor cell lysis. Ovarian carcinoma cells displayed ubiquitous expression of the DNAX accessory molecule-1 (DNAM-1) ligand PVR and sparse/heterogeneous expression of the NKG2D ligands MICA/MICB and ULBP1, ULBP2, and ULBP3. In line with the NK receptor ligand expression profiles, antibody-mediated blockade of activating receptor pathways revealed a dominant role for DNAM-1 and a complementary contribution of NKG2D signaling in tumor cell recognition. These results show that resting NK cells are capable of directly recognizing freshly isolated human tumor cells and identify ovarian carcinoma as a potential target for adoptive NK cell-based immunotherapy. [Cancer Res 2007;67(3):1317-25]

IFN-gamma regulates the immunogenicity of target cells by increasing their expression of HLA class I molecules. This facilitates the T cell receptor-mediated recognition by CD8(+) T cells but decreases target cell sensitivity to lysis by NK cells due to engagement of inhibitory NK receptors. In this study, short-term tumor cell lines from patients with advanced ovarian carcinomas were established. We demonstrate the paradoxical finding that IFN-gamma treatment of these short-term ovarian carcinoma cell lines (OVACs) resulted in resistance of tumor cells to lysis by peptide- and allospecific CD8(+) T cells. Blocking experiments revealed that this phenomenon was dependent on enhanced inhibitory signalling via CD94/NKG2A receptors expressed on the effector cells. This was associated with increased expression of HLA-E mRNA and HLA-G at the protein level in IFN-gamma-treated OVACs. Furthermore, pulsing of untreated OVACs with the leader sequence peptide of HLA-G protected these cells from lysis by CTLs, thus mimicking the inhibitory effect of IFN-gamma. This study provides evidence that CD94/NKG2A receptors play an important role in regulating T cell activity against tumors and shows that IFN-gamma modulation of target cells may shift the balance of triggering and inhibitory signals to T cells, turning off their cytolytic activity.

Ovarian cancer is usually found at a late stage when the prognosis is often bad. Relative survival rates decrease with tumor stage or grade, and the 5-year survival rate for women with carcinoma is only 38%. Thus, there is a great need to find biomarkers that can be used to carry out routine screening, especially in high-risk patient groups. Here, we present a large-scale study of 64 tissue samples taken from patients at all stages and show that we can identify statistically valid markers using nonsupervised methods that distinguish between normal, benign, borderline, and malignant tissue. We have identified 217 of the significantly changing protein spots. We are expressing and raising antibodies to 35 of these. Currently, we have validated 5 of these antibodies for use in immunohistochemical analysis using tissue microarrays of healthy and diseased ovarian, as well as other, human tissues.

Studies of multiple markers in tumors are required for adequate biological characterization. We have characterized the expression of multiple proteins in human ovarian tumors using the technique of 2‐dimensional gel electrophoresis (2‐DE/PDQUEST). Tumor cells were prepared from the tissue of 22 ovarian tumors. Large variations were observed between tumors in the expression of various polypeptides, indicating heterogeneity in gene expression. An increase in the spot density of 2 cell‐cycle‐related proteins, PCNA and OP18/stathmin, was observed in carcinomas. Borderline tumors expressed low levels of these proteins. Significant increases in the levels of nm23, GST‐π, elongation factor 2 and triose phosphate isomerase were recorded in ovarian carcinomas. Furthermore, decreases in the levels of tropomyosin‐2 and lamin C were observed in malignant as compared with benign tumors. The pattern of expression of 9 protein markers was examined in individual tumors. All malignant tumors showed simultaneous alterations in the expression of 5 or more of these proteins, whereas no benign tumor showed alterations in the expression of more than 3 polypeptides. Borderline tumors showed alterations in 0 to 6 markers. We conclude that the simultaneous analysis of multiple polypeptides, which can be achieved by 2‐DE, is useful for characterization of gene expression and diagnostic studies in ovarian tumors. Int. J. Cancer 73:678–683, 1997© 1997 Wiley‐Liss, Inc.

IFN-γ regulates the immunogenicity of target cells by increasing their expression of HLA class I molecules. This facilitates the T cell receptor–mediated recognition by CD8+ T cells but decreases target cell sensitivity to lysis by NK cells due to engagement of inhibitory NK receptors. In this study, short-term tumor cell lines from patients with advanced ovarian carcinomas were established. We demonstrate the paradoxical finding that IFN-γ treatment of these short-term ovarian carcinoma cell lines (OVACs) resulted in resistance of tumor cells to lysis by peptide- and allospecific CD8+ T cells. Blocking experiments revealed that this phenomenon was dependent on enhanced inhibitory signalling via CD94/NKG2A receptors expressed on the effector cells. This was associated with increased expression of HLA-E mRNA and HLA-G at the protein level in IFN-γ–treated OVACs. Furthermore, pulsing of untreated OVACs with the leader sequence peptide of HLA-G protected these cells from lysis by CTLs, thus mimicking the inhibitory effect of IFN-γ. This study provides evidence that CD94/NKG2A receptors play an important role in regulating T cell activity against tumors and shows that IFN-γ modulation of target cells may shift the balance of triggering and inhibitory signals to T cells, turning off their cytolytic activity

Protein patterns in six samples from primary vaginal cancers, in five from normal vaginal tissue and in five primary cervical cancers, were analysed using two-dimensional polyacrylamide gel electrophoresis (2-DE). Protein expression profile was evaluated by computer-assisted image analysis (PDQUEST) and proteins were subsequently identified using matrix-assisted laser desorption/ ionisation mass spectrometry. The aim was to analyse the protein expression profiles using the hierarchical clustering method in vaginal carcinoma and to compare them with the protein pattern in cervical carcinoma in order to find a helpful tool for correct classification and for increased biomedical knowledge. Protein expression data of a distinct set of 33 protein spots were differentially expressed. These differences were statistically significant (Mann -Whitney signed-Ranked Test, Po0.05) between normal tissue, vaginal and cervical cancer. Furthermore, protein profiles of pairs of primary vaginal and cervical cancers were found to be very similar. Some of the protein spots that have so far been identified include Tropomyosin 1, cytokeratin 5, 15 and 17, Apolipoprotein A1, Annexin V, Glutathione-S-transferase. Others are the stress-related proteins, calreticulin, HSP 27 and HSP 70. We conclude that cluster analysis of proteomics data allows accurate discrimination between normal vaginal mucosa, primary vaginal and primary cervical cancer. However, vaginal and cervical carcinomas also appear to be relatively homogeneous in their gene expression, indicating similar carcinogenic pathways. There might, further, be a possibility to identify tumour-specific markers among the proteins that are differentially expressed. The results from this study have to be confirmed by more comprehensive studies in the future. Primary carcinoma of the vagina (PCV) is a rare disease affecting predominantly postmenopausal women (Pecorelli, 2001). Histologically, the majority of PCV consist of squamous cell carcinomas (Pecorelli, 2001). Owing to the rarity of this disease, little is known about the aetiological and prognostic factors. Like cervical carcinomas, PCV has been shown to be associated with HPV, but only in about 50% of the cases (Daling and Sherman, 1992;Hildesheim et al (1997)). The prognosis for PCV is quite poor with an overall 5-year survival rate of about 50%, which is worse than for cervical carcinoma (Pecorelli, 2001). Early detection is crucial for the prognosis.It has been suggested that vaginal and cervical carcinomas have common aetiology since vaginal tumours often occur as second primary malignancy in patients with a history of cervical dysplasia and/or neoplasia or hysterectomy due to these disorders (Choo and Anderson, 1982;Benedet et al, 1983;Brinton et al, 1990;Eddy et al, 1991;Kirkbride et al, 1995). In the clinical situation, it is sometimes difficult to discriminate between cervical and vaginal carcinomas, especially in patients with prior cervical disease. As 95% of the recurrences of cervical carcinoma occur within 5 years, ma...

Defective expression of HLA class I molecules is common in tumor cells and may allow escape from CTL-mediated immunity. We here investigate alterations in expression of HLA class I and their underlying molecular mechanisms in ovarian cancer patients. The HLA class I and HLA-A2 expression levels on noncultured tumor cells of 12 patients diagnosed with ovarian carcinoma were investigated by flow cytometry. Molecular analyses of antigen-processing machinery (APM) components were done in metastatic cancer cells, and the HLA genotype was determined in both these and the primary tumor. HER-2/neu-specific immunity was evaluated by enzyme-linked immunospot assays. The metastatic tumor cells from all patients expressed low levels of HLA class I surface antigens. In six of nine HLA-A2+ patients, HLA-A2 expression was heterogeneous with a subpopulation of tumor cells exhibiting decreased or absent HLA-A2 expression. One patient-derived tumor cell line completely lacked HLA-A2 but exhibited constitutive expression of APM components and high HLA class I expression that was further inducible by IFN-gamma treatment. Genotyping showed a haplotype loss in the metastatic tumor cells, whereas tumor tissue microdissected from the primary tumor exhibited an intact HLA gene complex. Interestingly, HLA-A2-restricted HER-2/neu-specific T-cell responses were evident among the lymphocytes of this patient. Abnormalities in HLA class I antigen expression are common features during the progression of ovarian cancer, and haplotype loss was, for the first time, described as an underlying mechanism.