The Development of an Angiogenic Protein "Signature" in Ovarian Cancer Ascites as a Tool for Biologic and Prognostic Profiling.

Advanced ovarian cancer (AOC) is one of the leading lethal gynecological cancers in developed countries. Based on the important role of angiogenesis in ovarian cancer oncogenesis and expansion, we hypothesized that the development of an "angiogenic signature" might be helpful in prediction of prognosis and efficacy of anti-angiogenic therapies in this disease. Sixty-nine samples of ascitic fluid- 35 from platinum sensitive and 34 from platinum resistant patients managed with cytoreductive surgery and 1st-line carboplatin-based chemotherapy- were analyzed using the Proteome ProfilerTM Human Angiogenesis Array Kit, screening for the presence of 55 soluble angiogenesis-related factors. A protein profile based on the expression of a subset of 25 factors could accurately separate resistant from sensitive patients with a success rate of approximately 90%. The protein profile corresponding to the "sensitive" subset was associated with significantly longer PFS (8 [95% Confidence Interval {CI}: 8-9] vs. 20 months [95% CI: 15-28]; Hazard ratio {HR}: 8.3, p<0.001) and OS (20.5 months [95% CI: 13.5-30] vs. 74 months [95% CI: 36-not reached]; HR: 5.6 [95% CI: 2.8-11.2]; p<0.001). This prognostic performance was superior to that of stage, histology and residual disease after cytoreductive surgery and the levels of vascular endothelial growth factor (VEGF) in ascites. In conclusion, we developed an "angiogenic signature" for patients with AOC, which can be used, after appropriate validation, as a prognostic marker and a tool for selection for anti-angiogenic therapies.

Angiogenesis-related pathways in the pathogenesis of ovarian cancer.

Ovarian Cancer represents the most fatal type of gynecological malignancies. A number of processes are involved in the pathogenesis of ovarian cancer, especially within the tumor microenvironment. Angiogenesis represents a hallmark phenomenon in cancer, and it is responsible for tumor spread and metastasis in ovarian cancer, among other tumor types, as it leads to new blood vessel formation. In recent years angiogenesis has been given considerable attention in order to identify targets for developing effective anti-tumor therapies. Growth factors have been identified to play key roles in driving angiogenesis and, thus, the formation of new blood vessels that assist in "feeding" cancer. Such molecules include the vascular endothelial growth factor (VEGF), the platelet derived growth factor (PDGF), the fibroblast growth factor (FGF), and the angiopoietin/Tie2 receptor complex. These proteins are key players in complex molecular pathways within the tumor cell and they have been in the spotlight of the development of anti-angiogenic molecules that may act as stand-alone therapeutics, or in concert with standard treatment regimes such as chemotherapy. The pathways involved in angiogenesis and molecules that have been developed in order to combat angiogenesis are described in this paper.

VEGF directly suppresses activation of T cells from ovarian cancer patients and healthy individuals via VEGF receptor Type 2.

The role of vascular endothelial growth factor (VEGF) in tumor angiogenesis is well characterized; nevertheless, it is also a key element in promoting tumor evasion of the immune system by downregulating dendritic cell maturation and thus T cell activation. We sought to investigate the possible direct effect of VEGF on T cell activation and through which type of VEGF receptor (VEGFR) it exerts this effect. Circulating T cells from healthy donors and ovarian cancer patients were expanded in cultures with anti-CD3 and IL-2 with or without VEGF for 14 days, and the number of T cells was assessed. Cultured T cells were also tested for their cytotoxic activity in a standard 4-hr (51) Cr-release assay, and the expression of VEGFRs 1, 2 and 3 was assayed by flow cytometry, immunocytochemistry and Western blotting. To assess the ability of activated T cells to secrete VEGF, levels in culture supernatants were measured by enzyme linked immunosorbent assay. The addition of VEGF in cultures significantly reduced T cell proliferation in a dose-dependent manner. Protein expression studies demonstrated that CD3(+) T cells express VEGFR-2 on their surface upon activation. Experiments with anti-VEGFR-2 antibodies showed that the direct suppressive effect of VEGF on T cell proliferation is mediated by VEGFR-2. We also showed that VEGF significantly reduced the cytotoxic activity of T cells and that activated T cells secrete VEGF in the culture environment. Overall, our study shows that T cells secret VEGF and expresses VEGFR-2 upon activation. VEGF directly suppresses T cell activation via VEGF receptor type 2.

Immune response in ovarian cancer: how is the immune system involved in prognosis and therapy: potential for treatment utilization.

Ovarian cancer is one of the leading causes of cancer-related death among women. Resistance to the disease occurs in more than 70% of the cases even after treated with chemotherapy agents such as paclitaxel- and platinum-based agents. The immune system is increasingly becoming a target for intense research in order to study the host's immune response against ovarian cancer. T cell populations, including NK T cells and Tregs, and cytokines have been associated with disease outcome, indicating their increasing clinical significance, having been associated with prognosis and as markers of disease progress, respectively. Harnessing the immune system capacity in order to induce antitumor response remains a major challenge. This paper examines the recent developments in our understanding of the mechanisms of development of the immune response in ovarian cancer as well as its prognostic significance and the existing experience in clinical studies.

Mapping of human autoantibody binding sites on the calcium-sensing receptor.

Previously, we have demonstrated the presence of anti-calcium-sensing receptor (CaSR) antibodies in patients with autoimmune polyglandular syndrome type 1 (APS1), a disease that is characterized in part by hypoparathyroidism involving hypocalcemia, hyperphosphatemia, and low serum levels of parathyroid hormone. The aim of this study was to define the binding domains on the CaSR of anti-CaSR antibodies found in APS1 patients and in one patient suspected of having autoimmune hypocalciuric hypercalcemia (AHH). A phage-display library of CaSR peptides was constructed and used in biopanning experiments with patient sera. Selectively enriched IgG-binding peptides were identified by DNA sequencing, and subsequently, immunoreactivity to these peptides was confirmed in ELISA. Anti-CaSR antibody binding sites were mapped to amino acid residues 41-69, 114-126, and 171-195 at the N-terminal of the extracellular domain of the receptor. The major autoepitope was localized in the 41-69 amino acid sequence of the CaSR with antibody reactivity demonstrated in 12 of 12 (100%) APS1 patients with anti-CaSR antibodies and in 1 AHH patient with anti-CaSR antibodies. Minor epitopes were located in the 114-126 and 171-195 amino acid domains, with antibody reactivity shown in 5 of 12 (42%) and 4 of 12 (33%) APS1 patients, respectively. The results indicate that epitopes for anti-CaSR antibodies in the AHH patient and in the APS1 patients who were studied are localized in the N-terminal of the extracellular domain of the receptor. The present work has demonstrated the successful use of phage-display technology in the discovery of CaSR-specific epitopes targeted by human anti-CaSR antibodies.

Activating autoantibodies against the calcium-sensing receptor detected in two patients with autoimmune polyendocrine syndrome type 1.

CONTEXT: Autoimmune polyendocrine syndrome type 1 (APS1) is an autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE) gene. Hypoparathyroidism occurs in 80% of patients with APS1 and has been suggested to result from an autoimmune reaction against the calcium-sensing receptor (CaSR) in parathyroid cells. Anti-CaSR binding antibodies have previously been detected in patients with APS1. OBJECTIVE: The aim of this study was to determine whether anti-CaSR antibodies present in APS1 patients could modulate the response of the CaSR to stimulation by Ca(2+). RESULTS: The results indicated that two of the 14 APS1 patients included in the study had anti-CaSR antibodies that stimulated the receptor. These antibodies were detected by their ability to increase both Ca(2+)-dependent extracellular signal-regulated kinase phosphorylation and inositol phosphate accumulation in human embryonic kidney 293 cells expressing the CaSR. CONCLUSION: An important implication of the present results is that although the majority of APS1 patients do not have CaSR-stimulating antibodies, there may be a small but substantial minority of patients in whom the hypoparathyroid state is the result of functional suppression of the parathyroid glands rather than their irreversible destruction.

Mapping of melanin-concentrating hormone receptor 1 B cell epitopes predicts two major binding sites for vitiligo patient autoantibodies.

The melanin-concentrating hormone receptor 1 (MCHR1) has been identified as a B cell autoantigen in vitiligo with antibodies to the receptor detectable in binding and function-blocking assays. Two epitope domains (amino acids 1-138 and 139-298) have been previously identified. In this study, we aimed to further define the epitope specificity of MCHR1 antibodies using phage-display technology and to identify the epitopes recognised by receptor antibodies detected in MCHR1 function-blocking assays. Antibody reactivity to MCHR1 peptides 51-80, 85-98, 154-158 and 254-260 was identified by phage-display and subsequently confirmed in phage ELISA in 2/12, 5/12, 3/12 and 6/12 of vitiligo patients, respectively. The results suggest that major autoantibody epitopes are localised in the 85-98 and 254-260 amino acid regions of MCHR1 with minor epitopes in amino acid sequences 51-80 and 154-158. Antibodies with MCHR1 function-blocking activity were determined to recognise epitope 254-260, this being the first epitope to be reported as a target site for antibodies that block the function of the receptor.

The calcium-sensing receptor is a target of autoantibodies in patients with autoimmune polyendocrine syndrome type 1.

CONTEXT: Autoimmune polyendocrine syndrome type 1 (APS1) is an autosomal recessive disorder caused by mutations in the autoimmune regulator gene. Hypoparathyroidism occurs in 80% of patients with APS1 and has been suggested to result from an autoimmune reaction against the calcium-sensing receptor (CaSR) on parathyroid cells. However, the detection of CaSR antibodies in APS1 remains controversial, with some studies disputing the relevance of the receptor as an autoantigen. OBJECTIVE: The aim of this study was to analyze a defined set of APS1 patient sera for the presence of CaSR antibodies using different assay systems. RESULTS: APS1 patients and individuals with other autoimmune disorders along with healthy subjects were tested for antibody binding to the CaSR. In an immunoprecipitation assay with the CaSR expressed in human embryonic kidney 293 cells, 12 of 14 (85.7%) APS1 and two of 28 (7.1%) Graves' disease patients were considered positive for CaSR antibodies. The prevalence of receptor antibodies was significantly greater than that in the cohort of healthy individuals only in the APS1 patient group (P < 0.0001). In a flow cytometry assay, seven of 14 (50.0%) APS1 patient sera showed binding to the extracellular domain of the CaSR. The prevalence of receptor antibodies in the APS1 patient group was significantly greater than that in the group of healthy controls (P = 0.023). No CaSR antibodies could be detected in any patients or controls using a radiobinding assay. CONCLUSION: The CaSR is an autoantigen in APS1, but detection of antibodies against the receptor appears to be influenced by the assay system used.

Autoantibody responses to melanocytes in the depigmenting skin disease vitiligo.

Vitiligo is an acquired hypomelanotic disorder characterised by circumscribed depigmented macules in the skin resulting from the loss of functional melanocytes. Population surveys have shown a prevalence ranging from 0.38 to 1.13%. The frequent association of vitiligo with autoimmune diseases, together with studies demonstrating that vitiligo patients can have autoantibodies and autoreactive T lymphocytes against pigment cells supports the theory that there is an autoimmune involvement in the aetiology of the disease. Although the pathogenic mechanisms of T cells have recently been well studied in vitiligo, the role of autoantibodies in the disease remains obscure. However, even if antibodies to melanocytes are not an agent of the disease, identifying their target antigens could provide for the development of diagnostic tests that are not yet available for vitiligo and could serve as markers for important T cell responses in patients with the disease.

Analysis of allelic variants in the catalase gene in patients with the skin depigmenting disorder vitiligo.

Vitiligo is an acquired hypomelanotic skin disorder characterised by circumscribed depigmented macules resulting from the loss of functional melanocytes from the cutaneous epidermis. Conditions that might result in epidermal oxidative stress and consequently damage to pigment cells have been reported in the skin of vitiligo patients, including low catalase activity and increases in hydrogen peroxide levels. However, the cause of the decrease in catalase activity has not been equivocally determined. Several allelic variants in the catalase gene, a number of which have deleterious effects upon the expression or function of the enzyme, have been described and the aim of the present work was to assess the relevance of catalase gene variants in patients with vitiligo. Associations between ten separate allelic variants in the catalase gene and a predisposition to vitiligo were investigated in case-control studies with 166 English patients and 169 ethnically-matched controls using DNA sequencing and restriction fragment length polymorphism-polymerase chain reaction methods. Of the ten allelic variants analysed, only a C/T single nucleotide polymorphism in exon 9 of the catalase gene was associated with vitiligo. The C/T genotype was significantly over-represented in the vitiligo patient group compared with the control cohort. Of 166 vitiligo genotypes, 66 (39.8%) had the C/T variant compared to 45/169 (26.6%) control genotypes (P = 0.030). No evidence for an association between other allelic variants in the catalase gene and vitiligo susceptibility was found. The low catalase activity in vitiligo patient epidermis is more likely to result from environmental conditions such as inhibitory levels of hydrogen peroxide rather than allelic variations in the catalase gene which affect either expression or function of the enzyme.

Function-blocking autoantibodies to the melanin-concentrating hormone receptor in vitiligo patients.

Vitiligo is a common depigmenting skin disorder resulting from the loss of melanocytes in the cutaneous epidermis. Although the cause of the disease remains obscure, autoimmune mechanisms are thought to be involved. Recently, melanin-concentrating hormone receptor (MCHR)-binding autoantibodies have been identified in vitiligo patients. In the present study, we aimed to determine if MCHR autoantibodies could also affect receptor function either by direct activation or by blocking its response to melanin-concentrating hormone. The results indicated that 10/18 (56%) vitiligo patient IgG samples inhibited the function of MCHR expressed in a Chinese hamster ovary cell line. In contrast, neither control (n=20) nor SLE patient (n=10) IgG samples blocked receptor function. Compared with healthy controls, MCHR function-blocking autoantibodies were found at a significantly increased frequency in the vitiligo patient group (P=0.0004). No MCHR-activating autoantibodies were detected in any of the vitiligo patient, SLE patient or control IgG samples that were analysed. In addition, vitiligo patient IgGs were tested for MCHR autoantibodies that could mediate antibody-dependent cell-mediated cytotoxicity via the receptor. However, this could only be demonstrated in two vitiligo patient sera. Overall, this work has provided additional evidence that MCHR is a B-cell autoantigen in vitiligo and has demonstrated the existence of MCHR function-blocking autoantibodies further to the receptor-binding autoantibodies previously reported.

An insertion/deletion polymorphism in the gene encoding angiotensin converting enzyme is not associated with generalised vitiligo in an English population.

Vitiligo is an acquired hypomelanotic skin disorder characterised by circumscribed depigmented macules resulting from the loss of functional melanocytes from the cutaneous epidermis and autoimmunity has been suggested to play a role in the pathogenesis of the disease. Recently, an insertion/deletion (I/D) polymorphism of a 287-base pair repetitive sequence in intron 16 of the angiotensin converting enzyme (ACE) gene has been associated with autoimmune disease and with the development of vitiligo. In this study, the distribution of ACE gene I/D genotypes was investigated in a population of 106 English patients with generalised (non-segmental) vitiligo and 174 ethnically matched healthy controls using a restriction fragment length polymorphism-polymerase chain reaction genotyping method. No significant difference in the frequencies of II, ID and DD genotypes was detected between vitiligo patients and control subjects (P=0.35). The same result was evident for the genotype distribution in vitiligo patients with an autoimmune disease and for those without when compared with controls (P=0.33 and P=0.53, respectively). In addition, the results indicated that the D allele was not significantly over-represented in the group of patients with vitiligo compared with controls (P=0.42) and that this was also the case for patients with and without associated autoimmunity (P=0.40 and P=0.62, respectively).