ß2-Adrenergic Signalling Promotes Cell Migration by Upregulating Expression of the Metastasis-Associated Molecule LYPD3.

Metastasis is associated with poor prognosis in breast cancer. Although some studies suggest beta-blockers increase survival by delaying metastasis, others have been discordant. This study provides both insights into the anomalous findings and identifies potential biomarkers that may be treatment targets. Cell line models of basal-type and oestrogen receptor-positive breast cancer were profiled for basal levels of adrenoceptor gene/protein expression, and ß2-adrenoceptor mediated cell behaviour including migration, invasion, adhesion, and survival in response to adrenoceptor agonist/antagonist treatment. Protein profiling and histology identified biomarkers and drug targets. Baseline levels of adrenoceptor gene expression are higher in basal-type rather than oestrogen receptor-positive cancer cells. Norepinephrine (NE) treatment increased invasive capacity in all cell lines but did not increase proliferation/survival. Protein profiling revealed the upregulation of the pro-metastatic gene Ly6/PLAUR Domain-Containing Protein 3 (LYPD3) in norepinephrine-treated MDA-MB-468 cells. Histology confirmed selective LYPD3 expression in primary and metastatic breast tumour samples. These findings demonstrate that basal-type cancer cells show a more aggressive adrenoceptor-ß2-activated phenotype in the resting and stimulated state, which is attenuated by adrenoceptor-ß2 inhibition. This study also highlights the first association between ADRß2 signalling and LYPD3; its knockdown significantly reduced the basal and norepinephrine-induced activity of MCF-7 cells in vitro. The regulation of ADRß2 signalling by LYPD3 and its metastasis promoting activities, reveal LYPD3 as a promising therapeutic target in the treatment of breast and other cancers.

A novel spontaneous model of epithelial-mesenchymal transition (EMT) using a primary prostate cancer derived cell line demonstrating distinct stem-like characteristics.

Cells acquire the invasive and migratory properties necessary for the invasion-metastasis cascade and the establishment of aggressive, metastatic disease by reactivating a latent embryonic programme: epithelial-to-mesenchymal transition (EMT). Herein, we report the development of a new, spontaneous model of EMT which involves four phenotypically distinct clones derived from a primary tumour-derived human prostate cancer cell line (OPCT-1), and its use to explore relationships between EMT and the generation of cancer stem cells (CSCs) in prostate cancer. Expression of epithelial (E-cadherin) and mesenchymal markers (vimentin, fibronectin) revealed that two of the four clones were incapable of spontaneously activating EMT, whereas the others contained large populations of EMT-derived, vimentin-positive cells having spindle-like morphology. One of the two EMT-positive clones exhibited aggressive and stem cell-like characteristics, whereas the other was non-aggressive and showed no stem cell phenotype. One of the two EMT-negative clones exhibited aggressive stem cell-like properties, whereas the other was the least aggressive of all clones. These findings demonstrate the existence of distinct, aggressive CSC-like populations in prostate cancer, but, importantly, that not all cells having a potential for EMT exhibit stem cell-like properties. This unique model can be used to further interrogate the biology of EMT in prostate cancer.

The localization of pre mRNA splicing factor PRPF38B is a novel prognostic biomarker that may predict survival benefit of trastuzumab in patients with breast cancer overexpressing HER2.

Cancer biomarkers that can define disease status and provide a prognostic insight are essential for the effective management of patients with breast cancer (BC). The prevalence, clinicopathological and prognostic significance of PRPF38B expression in a consecutive series of 1650 patients with primary invasive breast carcinoma were examined using immunohistochemistry. Furthermore, the relationship(s) between clinical outcome and PRPF38B expression was explored in 627 patients with ER-negative (oestrogen receptor) disease, and 322 patients with HER2-overexpressing disease. Membranous expression of PRPF38B was observed in 148/1388 (10.7%) cases and was significantly associated with aggressive clinicopathological features, including high grade, high mitotic index, pleomorphism, invasive ductal carcinoma of no specific type (IDC-NST), ER-negative, HER2-overexpression and p53 mutational status (all p < 0.01). In patients with ER-negative disease receiving chemotherapy, nuclear expression of PRPF38B was significantly associated with a reduced risk of relapse (p = 0.0004), whereas membranous PRPF38B expression was significantly associated with increased risk of relapse (p = 0.004; respectively) at a 5 year follow-up. When patients were stratified according to ER-negative/HER2-positive status, membranous PRPF38B expression was associated with a higher risk of relapse in those patients that did not receive trastuzumab therapy (p = 0.02), whereas in those patients with ER-negative/HER2-positive disease that received trastuzumab adjuvant therapy, membranous PRPF38B expression associated with a lower risk of relapse (p = 0.00018). Nuclear expression of PRPF38B is a good prognostic indicator in both ER-negative patients and ER-negative/HER2-positive BC (breast cancer) patients, whereas membranous localisation of PRPF38B is a poor prognostic biomarker that predicts survival benefit from trastuzumab therapy in patients with ER-negative/HER2-overexpressing BC.

Prediction of Pathological Stage in Patients with Prostate Cancer: A Neuro-Fuzzy Model.

The prediction of cancer staging in prostate cancer is a process for estimating the likelihood that the cancer has spread before treatment is given to the patient. Although important for determining the most suitable treatment and optimal management strategy for patients, staging continues to present significant challenges to clinicians. Clinical test results such as the pre-treatment Prostate-Specific Antigen (PSA) level, the biopsy most common tumor pattern (Primary Gleason pattern) and the second most common tumor pattern (Secondary Gleason pattern) in tissue biopsies, and the clinical T stage can be used by clinicians to predict the pathological stage of cancer. However, not every patient will return abnormal results in all tests. This significantly influences the capacity to effectively predict the stage of prostate cancer. Herein we have developed a neuro-fuzzy computational intelligence model for classifying and predicting the likelihood of a patient having Organ-Confined Disease (OCD) or Extra-Prostatic Disease (ED) using a prostate cancer patient dataset obtained from The Cancer Genome Atlas (TCGA) Research Network. The system input consisted of the following variables: Primary and Secondary Gleason biopsy patterns, PSA levels, age at diagnosis, and clinical T stage. The performance of the neuro-fuzzy system was compared to other computational intelligence based approaches, namely the Artificial Neural Network, Fuzzy C-Means, Support Vector Machine, the Naive Bayes classifiers, and also the AJCC pTNM Staging Nomogram which is commonly used by clinicians. A comparison of the optimal Receiver Operating Characteristic (ROC) points that were identified using these approaches, revealed that the neuro-fuzzy system, at its optimal point, returns the largest Area Under the ROC Curve (AUC), with a low number of false positives (FPR = 0.274, TPR = 0.789, AUC = 0.812). The proposed approach is also an improvement over the AJCC pTNM Staging Nomogram (FPR = 0.032, TPR = 0.197, AUC = 0.582).

SPAG5 as a prognostic biomarker and chemotherapy sensitivity predictor in breast cancer: a retrospective, integrated genomic, transcriptomic, and protein analysis.

BACKGROUND: Proliferation markers and profiles have been recommended for guiding the choice of systemic treatments for breast cancer. However, the best molecular marker or test to use has not yet been identified. We did this study to identify factors that drive proliferation and its associated features in breast cancer and assess their association with clinical outcomes and response to chemotherapy. METHODS: We applied an artificial neural network-based integrative data mining approach to data from three cohorts of patients with breast cancer (the Nottingham discovery cohort (n=171), Uppsala cohort (n=249), and Molecular Taxonomy of Breast Cancer International Consortium [METABRIC] cohort; n=1980). We then identified the genes with the most effect on other genes in the resulting interactome map. Sperm-associated antigen 5 (SPAG5) featured prominently in our interactome map of proliferation and we chose to take it forward in our analysis on the basis of its fundamental role in the function and dynamic regulation of mitotic spindles, mitotic progression, and chromosome segregation fidelity. We investigated the clinicopathological relevance of SPAG5 gene copy number aberrations, mRNA transcript expression, and protein expression and analysed the associations of SPAG5 copy number aberrations, transcript expression, and protein expression with breast cancer-specific survival, disease-free survival, distant relapse-free survival, pathological complete response, and residual cancer burden in the Nottingham discovery cohort, Uppsala cohort, METABRIC cohort, a pooled untreated lymph node-negative cohort (n=684), a multicentre combined cohort (n=5439), the Nottingham historical early stage breast cancer cohort (Nottingham-HES; n=1650), Nottingham early stage oestrogen receptor-negative breast cancer adjuvant chemotherapy cohort (Nottingham-oestrogen receptor-negative-ACT; n=697), the Nottingham anthracycline neoadjuvant chemotherapy cohort (Nottingham-NeoACT; n=200), the MD Anderson taxane plus anthracycline-based neoadjuvant chemotherapy cohort (MD Anderson-NeoACT; n=508), and the multicentre phase 2 neoadjuvant clinical trial cohort (phase 2 NeoACT; NCT00455533; n=253). FINDINGS: In the METABRIC cohort, we detected SPAG5 gene gain or amplification at the Ch17q11.2 locus in 206 (10%) of 1980 patients overall, 46 (19%) of 237 patients with a PAM50-HER2 phenotype, and 87 (18%) of 488 patients with PAM50-LumB phenotype. Copy number aberration leading to SPAG5 gain or amplification and high SPAG5 transcript and SPAG5 protein concentrations were associated with shorter overall breast cancer-specific survival (METABRIC cohort [copy number aberration]: hazard ratio [HR] 1·50, 95% CI 1·18-1·92, p=0·00010; METABRIC cohort [transcript]: 1·68, 1·40-2·01, p<0·0001; and Nottingham-HES-breast cancer cohort [protein]: 1·68, 1·32-2·12, p<0·0001). In multivariable analysis, high SPAG5 transcript and SPAG5 protein expression were associated with reduced breast cancer-specific survival at 10 years compared with lower concentrations (Uppsala: HR 1·62, 95% CI 1·03-2·53, p=0·036; METABRIC: 1·27, 1·02-1·58, p=0·034; untreated lymph node-negative cohort: 2·34, 1·24-4·42, p=0·0090; and Nottingham-HES: 1·73, 1·23-2·46, p=0·0020). In patients with oestrogen receptor-negative breast cancer with high SPAG5 protein expression, anthracycline-based adjuvant chemotherapy increased breast cancer-specific survival overall compared with that for patients who did not receive chemotherapy (Nottingham-oestrogen receptor-negative-ACT cohort: HR 0·37, 95% CI 0·20-0·60, p=0·0010). Multivariable analysis showed high SPAG5 transcript concentrations to be independently associated with longer distant relapse-free survival after receiving taxane plus anthracycline neoadjuvant chemotherapy (MD Anderson-NeoACT: HR 0·68, 95% CI 0·48-0·97, p=0·031). In multivariable analysis, both high SPAG5 transcript and high SPAG5 protein concentrations were independent predictors for a higher proportion of patients achieving a pathological complete response after combination cytotoxic chemotherapy (MD Anderson-NeoACT: OR 1·71, 95% CI, 1·07-2·74, p=0·024; Nottingham-ACT: 8·75, 2·42-31·62, p=0·0010). INTERPRETATION: SPAG5 is a novel amplified gene on Ch17q11.2 in breast cancer. The transcript and protein products of SPAG5 are independent prognostic and predictive biomarkers that might have clinical utility as biomarkers for combination cytotoxic chemotherapy sensitivity, especially in oestrogen receptor-negative breast cancer. FUNDING: Nottingham Hospitals Charity and the John and Lucille van Geest Foundation.

HAGE in Triple-Negative Breast Cancer Is a Novel Prognostic, Predictive, and Actionable Biomarker: A Transcriptomic and Protein Expression Analysis.

PURPOSE: The expression of HAGE as a novel prognostic and predictive tool was assessed in 1,079 triple-negative breast cancers (TNBC). EXPERIMENTAL DESIGN: HAGE protein expression was investigated in an early primary TNBC (EP-TNBC; n = 520) cohort who received adjuvant chemotherapy (ACT) and in a locally advanced primary TNBC cohort who received anthracycline combination Neo-ACT (n = 110; AC-Neo-ACT). HAGE-mRNA expression was evaluated in the METABRIC-TNBC cohort (n = 311) who received ACT and in a cohort of patients with TNBC who received doxorubicin/cyclophosphamide Neo-ACT, followed by 1:1 randomization to ixabepilone (n = 68) or paclitaxel (n = 64) as part of a phase II clinical trial. Furthermore, a cohort of 128 tumors with integrated HAGE gene copy number changes, mRNA, and protein levels were analyzed. RESULTS: In patients with EP-TNBC, who were chemotherapy-naïve, high HAGE protein expression (HAGE(+)) was associated with a higher risk of death [HR, 1.3; 95% confidence interval (CI), 1.2-1.5; P = 0.000005] when compared with HAGE(-) cases. Patients who received ACT and expressed mRNA-HAGE(+) were at a lower risk of death than those who were mRNA-HAGE(-) (P = 0.004). The expression of HAGE was linked to the presence of tumor-infiltrating lymphocytes (TIL), and both features were found to be independent predictors for pathologic complete response (pCR, P < 0.001) and associated with prolonged survival (P < 0.01), following AC-Neo-ACT. In patients with residual disease, HAGE(+) had a 2-fold death risk increase (P = 0.018) compared with HAGE(-). CONCLUSIONS: HAGE expression is a potential prognostic marker and a predictor of response to anthracycline treatment in TNBC. A prospective clinical trial to examine the therapeutic value of HAGE for TNBC cases is warranted.

Data Mining of Gene Arrays for Biomarkers of Survival in Ovarian Cancer.

The expected five-year survival rate from a stage III ovarian cancer diagnosis is a mere 22%; this applies to the 7000 new cases diagnosed yearly in the UK. Stratification of patients with this heterogeneous disease, based on active molecular pathways, would aid a targeted treatment improving the prognosis for many cases. While hundreds of genes have been associated with ovarian cancer, few have yet been verified by peer research for clinical significance. Here, a meta-analysis approach was applied to two carefully selected gene expression microarray datasets. Artificial neural networks, Cox univariate survival analyses and T-tests identified genes whose expression was consistently and significantly associated with patient survival. The rigor of this experimental design increases confidence in the genes found to be of interest. A list of 56 genes were distilled from a potential 37,000 to be significantly related to survival in both datasets with a FDR of 1.39859 × 10(-11), the identities of which both verify genes already implicated with this disease and provide novel genes and pathways to pursue. Further investigation and validation of these may lead to clinical insights and have potential to predict a patient's response to treatment or be used as a novel target for therapy.

Controlling the dynamics of cell transition in heterogeneous cultures using surface chemistry.

Developing materials that can preferentially select defined cancer cell populations for biological characterization will greatly enhance our understanding of cancer cell growth, differentiation, and invasion. The transitional events between epithelial and mesenchymal phenotypes are particularly crucial, as primary tumors and secondary metastasis are generally epithelial in nature, whereas circulating mesenchymal cells derived from primary epithelial cells appear to facilitate the spread of disease and its resistance to therapy. This study describes an amino-functionalized material, which promotes the enrichment of an epithelial phenotype from a single cell line containing both epithelial and mesenchymal subpopulations of cancer cells. The isolation and transitional control of such subpopulations using functional materials will advance understanding of the disease process, have a significant impact on the downstream development of new targeted cancer therapeutics, and also be applicable to tissue engineering and regenerative medicine.

Prostate cancer: Important steps and considerations in the design of therapeutic vaccines.

Vaccine-based immunotherapy can increase the overall survival of patients with advanced prostate cancer. However, the efficacy of vaccine-elicited anticancer immune responses is heavily influenced by the physical, nutritional, and psychological status of the patient. Given their importance, these parameters should be carefully considered for the design of future clinical trials testing this immunotherapeutic paradigm in prostate cancer patients.

Novel prostate acid phosphatase-based peptide vaccination strategy induces antigen-specific T-cell responses and limits tumour growth in mice.

Treatment options for patients with advanced prostate cancer remain limited and rarely curative. Prostatic acid phosphatase (PAP) is a prostate-specific protein overexpressed in 95% of prostate tumours. An FDA-approved vaccine for the treatment of advanced prostate disease, PROVENGE® (sipuleucel-T), has been shown to prolong survival, however the precise sequence of the PAP protein responsible for the outcome is unknown. As the PAP antigen is one of the very few prostate-specific antigens for which there is a rodent equivalent with high homology, preclinical studies using PAP have the potential to be directly relevant to clinical setting. Here, we show three PAP epitopes naturally processed and presented in the context of HHDII/DR1 (114-128, 299-313, and 230-244). The PAP-114-128 epitope elicits CD4(+) and CD8(+) T-cell-specific responses in C57BL/6 mice. Furthermore, when immunised in a DNA vector format (ImmunoBody®), PAP-114-128 prevents and reduces the growth of transgenic adenocarcinoma of mouse prostate-C1 prostate cancer cell-derived tumours in both prophylactic and therapeutic settings. This anti-tumour effect is associated with infiltration of CD8(+) tumour-infiltrating lymphocytes and the generation of high avidity T cells secreting elevated levels of IFN-γ. PAP-114-128 therefore appears to be a highly relevant peptide on which to base vaccines for the treatment of prostate cancer.

Development of an hydrophobic fluoro-silica surface for studying homotypic cancer cell aggregation-disaggregation as a single dynamic process in vitro.

Under normal conditions the detachment of anchorage-dependant cells from their extracellular matrix typically induces programmed cell death which is mediated through a pathway referred to as anoikis. However, a resistance to anoikis in cancer enables the migration of cells from the primary tumour and the establishment of aggressive metastatic disease. Although cancer cell aggregation is known to be an important mechanism within anoikis resistance, research into the underlying mechanisms that govern this process remain problematic as commercially available tissue culture material can only sustain 2D monolayer or 3D aggregate/spheroid cultures. This necessitates the development of a system that can accommodate for cancer cell aggregation-disaggregation as a single dynamic process, without the disruption of passaging cells between alternate substrates. This study describes a procedure for modifying tissue culture polystyrene (TCP) to produce a fluoro-silica (FS) surface which preferentially promotes the deposition of a distinct profile of proteins/factors from serum which mediate the transient aggregation of human breast cancer cell lines. This modified surface therefore provides an experimental platform for better understanding cancer cell aggregation-disaggregation events in vitro, and their influence on the establishment of metastatic disease in patients with cancer.

Identification of novel breast cancer-associated transcripts by UniGene database mining and gene expression analysis in normal and malignant cells.

Breast cancer is a heterogeneous and complex disease. Although the use of tumor biomarkers has improved individualized breast cancer care, i.e., assessment of risk, diagnosis, prognosis, and prediction of treatment outcome, new markers are required to further improve patient clinical management. In the present study, a search for novel breast cancer-associated genes was performed by mining the UniGene database for expressed sequence tags (ESTs) originating from human normal breast, breast cancer tissue, or breast cancer cell lines. Two hundred and twenty-eight distinct breast-associated UniGene Clusters (BUC1-228) matched the search criteria. Four BUC ESTs (BUC6, BUC9, BUC10, and BUC11) were subsequently selected for extensive in silico database searches, and in vitro analyses through sequencing and RT-PCR based assays on well-characterized cell lines and tissues of normal and cancerous origin. BUC6, BUC9, BUC10, and BUC11 are clustered on 10p11.21-12.1 and showed no homology to any known RNAs. Overall, expression of the four BUC transcripts was high in normal breast and testis tissue, and in some breast cancers; in contrast, BUC was low in other normal tissues, peripheral blood mononuclear cells (PBMCs), and other cancer cell lines. Results to-date suggest that BUC11 and BUC9 translate to protein and BUC11 cytoplasmic and nuclear protein expression was detected in a large cohort of breast cancer samples using immunohistochemistry. This study demonstrates the discovery and expression analysis of a tissue-restricted novel transcript set which is strongly expressed in breast tissue and their application as clinical cancer biomarkers clearly warrants further investigation.

The helicase HAGE expressed by malignant melanoma-initiating cells is required for tumor cell proliferation in vivo.

Malignant melanoma-initiating cells (MMIC) are a subpopulation of cells responsible for melanoma tumor growth and progression. They are defined by the expression of the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5). Here, we identified a critical role for the DEAD-box helicase antigen (HAGE) in ABCB5+ MMIC-dependent tumorigenesis and show that HAGE-specific inactivation inhibits melanoma tumor growth mediated by this tumor-initiating population. Knockdown of HAGE led to a significant decrease in RAS protein expression with a concomitant decrease in activation of the AKT and ERK signaling pathways implicated to play an important role in melanoma progression. To confirm that the reduction in NRAS (Neuroblastoma RAS) expression was dependent on the HAGE helicase activity, we showed that NRAS, effectively silenced by siRNA, could be rescued by reintroduction of HAGE in cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes NRAS unwinding in vitro. We also observed using tumor transplantation in Non-obese diabetic/severe combined immunodeficiency mice that the HAGE knockdown in a ABCB5+ melanoma cell line displayed a significant decrease in tumor growth and compared with the control. Our results suggest that the helicase HAGE is required for ABCB5+ MMIC-dependent tumor growth through promoting RAS protein expression and that cancer therapies targeting HAGE helicase may have broad applications for treating malignant melanoma and potentially other cancer types.

Expression of the tumour antigen T21 is up-regulated in prostate cancer and is associated with tumour stage.

OBJECTIVES: • To define the expression pattern of the tumour antigen T21 at the protein level in prostate tissues, prostate cell lines and a panel of normal tissues. • To correlate the expression pattern of T21 in prostate cancer with clinical parameters. PATIENTS AND METHODS: • Tissue samples were collected from 79 patients presenting at clinic with either prostate cancer (63 patients) or benign prostatic hyperplasia (BPH, 16 patients). • A tissue microarray (TMA) was constructed from 44 of the prostate cancer tissues and areas of benign disease (43 patients) from these tissues were also included on the TMA. The remaining tissues (prostate cancer 19 patients and BPH 16 patients) were mounted fresh frozen onto cork boards and sectioned. • Full ethical approval was granted for all aspects of the study and informed patient consent was taken before tissue collection. • Immunohistochemistry was used on the prostate tumour TMA, the normal tissue TMA and the fresh-frozen prostate tissues. Fluorescent microscopy and flow cytometry was performed on prostate cell lines. RESULTS: • Expression of T21 was highly restricted within normal tissues with only the stomach, ovary, breast and prostate having detectable T21 expression. • T21 was significantly over-expressed in prostate cancer glands compared with benign tissue and was present in >80% of the malignant specimens analysed. • Increased expression was positively correlated to pathological stage of prostate tumours. • Additionally, T21 was associated with Gleason grade and prostate-specific antigen recurrence, although statistical significance was not reached in this restricted cohort of patients. CONCLUSION: • Taken together these results show that T21 is a potential new biomarker for advanced disease and that elevated levels of T21 appear relevant to prostate cancer development.

Immunotherapy of prostate cancer: should we be targeting stem cells and EMT?

Cancer stem cells have been implicated in a number of solid malignancies including prostate cancer. In the case of localised prostate cancer, patients are often treated with surgery (radical prostatectomy) and/or radiotherapy. However, disease recurrence is an issue in about 30% of patients, who will then go on to receive hormone ablation therapy. Hormone ablation therapy is often palliative in a vast proportion of individuals, and for hormone-refractory patients, there are several immunotherapies targeting a number of prostate tumour antigens which are currently in development. However, clinical responses in this setting are inconsistent, and it is believed that the failure to achieve full and permanent tumour eradication is due to a small, resistant population of cells known as 'cancer stem cells' (CSCs). The stochastic and clonal evolution models are among several models used to describe cancer development. The general consensus is that cancer may arise in any cell as a result of genetic mutations in oncogenes and tumour suppressor genes, which consequently result in uncontrolled cell growth. The cancer stem cell theory, however, challenges previous opinion and proposes that like normal tissues, tumours are hierarchical and only the rare subpopulation of cells at the top of the hierarchy possess the biological properties required to initiate tumourigenesis. Furthermore, where most cancer models infer that every cell within a tumour is equally malignant, i.e. equally capable of reconstituting new tumours, the cancer stem cell theory suggests that only the rare cancer stem cell component possess tumour-initiating capabilities. Hence, according to this model, cancer stem cells are implicated in both tumour initiation and progression. In recent years, the role of epithelial--mesenchymal transition (EMT) in the advancement of prostate cancer has become apparent. Therefore, CSCs and EMT are both likely to play critical roles in prostate cancer tumourigenesis. This review summarises the current immunotherapeutic strategies targeting prostate tumour antigens taking into account the need to consider treatments that target cancer stem cells and cells involved in epithelial--mesenchymal transition.

Tumour-associated antigens: considerations for their use in tumour immunotherapy.

Since their discovery, tumour-associated antigens (TAA) have provided highly inviting targets for cancer therapy, especially immunotherapy. Evidence now points to their involvement in the malignant phenotype of transformed cells and heightens their importance for being targeted by different treatments. TAA vary in their nature and pattern of expression and this influences the way therapy is directed towards them. While large numbers of these antigens have been isolated from solid tumours, fewer are linked with haematological malignancies. Those TAA found in this latter group of cancers, referred to as leukaemia-associated antigens (LAA), also appear to have significant potential for promoting the malignant phenotype and have been described in detail in terms of expression and therapy. Interestingly, the action of some of LAA in blood cancers, which are stem cell derived, could act as model for solid tumours, which are increasingly thought to be also derived from a cancer stem cell origin. In this review, TAA and their use in immunotherapy will be discussed. The nature and expression of these antigens will be described together with the events that provide tumours, including haematological cancers, with the ability to avoid immune deletion.

HAGE, a cancer/testis antigen expressed at the protein level in a variety of cancers.

The search for novel tumour antigens that are either uniquely expressed or over-expressed in a wide variety of tumours is still ongoing. Because of their expression in a broad spectrum of cancers and limited expression in normal tissues, cancer/testis antigens are considered to be potentially reliable targets for immunotherapy of cancer in general. The helicase antigen HAGE has been identified as a cancer/testis antigen. However, little is known about its expression in normal and cancer tissues. Using a newly developed antibody against HAGE, specific staining of its expression by immunohistochemistry was validated and optimised on murine tumours transfected to express the HAGE protein. The antibody was subsequently used to determine HAGE expression in normal human and cancer tissue microarrays. HAGE protein expression was confirmed in 75% (12/16) of carcinomas as compared to normal tissues, which either did not express HAGE at all or expressed HAGE at very low levels with the exception of testis. Interestingly, discrepancies were also found between mRNA analysis by real time quantitative PCR (RT-qPCR) and protein analysis by immunohistochemistry, emphasising the need to validate the expression of cancer/testis antigens at the protein level prior to the development of new vaccine strategies. HAGE is therefore proposed to be a valid candidate for designing a broad spectrum vaccine against cancer.

Tumor immunology: new perspectives.

This second meeting, organized by the Cancer Immunology Working Group of AACR, concentrated mainly on three areas of importance; these included (a) ways to increase the success rate of vaccination by improving the peptides themselves, improving tumor recognition by using costimulatory molecules or the use of antibodies; (b) how the tumor microenvironment influences T-cell activity and how it can be manipulated to favor survival and action of activated T cells; and (c) a better understanding of myeloid-derived suppressor cells, which represent an ever-growing and diverse group of cells with activities leading to the inhibition of immune responses. It has been accepted that many of these improved therapies may only benefit relatively few patients and recent but rapidly developing research is currently focusing on methods that would allow clinicians to stratify patients receiving immunotherapy into responders and nonresponders, thereby predicting those who are likely to benefit from a specific vaccine therapy; this approach may benefit future clinical trials.

Estimation of peptide concentration by a modified bicinchoninic acid assay.

Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of interpeptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 degrees C for 5 min in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 degrees C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estimation of the peptide concentration within an unknown sample.

Artificial exosomes as tools for basic and clinical immunology.

Dendritic cell derived exosomes are able to mediate and modulate immune responses in vivo by semi-direct T cell activation. T cells can eradicate primary, metastatic, relapsed tumours and ameliorate otherwise fatal viral infections. Not surprisingly activation and expansion of T cells has become one of the main focuses for immunotherapy. Using nanotechnology, we have developed targeted and traceable in vivo artificial exosomes by coating liposomes (FDA approved) with an optimized number of MHC Class I/peptide complexes and a selected specific range of ligands for adhesion, early activation, late activation and survival T cell receptors. These targeted artificial exosomes are traceable both in vitro and in vivo via fluorescent and Magnetic Resonance Imaging and facilitate imaging of specific areas by applying localised nuclear magnetic interactions of hydrogens via super paramagnetic labels. Here we show that artificial exosomes activate and expand functional antigen specific T cells at sufficient levels. This novel system has potential basic and clinical applications in immunology where the study of membrane interactions is desired.