Bacillus Calmette-Guerin (BCG) induces superior anti-tumour responses by Vδ2+ T cells compared with the aminobisphosphonate drug zoledronic acid.

Vδ2+ T cells can recognize malignantly transformed cells as well as those infected with mycobacteria. This cross-reactivity supports the idea of using mycobacteria to manipulate Vδ2+ T cells in cancer immunotherapy. To date, therapeutic interventions using Vδ2+ T cells in cancer have involved expanding these cells in or ex vivo using zoledronic acid (ZA). Here, we show that the mycobacterium Bacillus Calmette-Guérin (BCG) also causes Vδ2+ T-cell expansion in vitro and that resulting Vδ2+ cell populations are cytotoxic toward tumour cell lines. We show that both ZA and BCG-expanded Vδ2+ cells effectively killed both Daudi and THP-1 cells. THP-1 cell killing by both ZA and BCG-expanded Vδ2+ cells was enhanced by treatment of targets cells with ZA. Although no difference in cytotoxic activity between ZA- and BCG-expanded Vδ2+ cells was observed, BCG-expanded cells degranulated more and produced a more diverse range of cytokines upon tumour cell recognition compared to ZA-expanded cells. ZA-expanded Vδ2+ cells were shown to upregulate exhaustion marker CD57 to a greater extent than BCG-expanded Vδ2+ cells. Furthermore, ZA expansion was associated with upregulation of inhibitory markers PD-1 and TIM3 in a dose-dependent manner whereas PD-1 expression was not increased following expansion using BCG. Intradermal BCG vaccination of rhesus macaques caused in vivo expansion of Vδ2+ cells. In combination with the aforementioned in vitro data, this finding suggests that BCG treatment could induce expansion of Vδ2+ T cells with enhanced anti-tumour potential compared to ZA treatment and that either ZA or BCG could be used intratumourally as a means to potentiate stronger anti-tumour Vδ2+ T-cell responses.

Adjuvant bevacizumab for melanoma patients at high risk of recurrence: survival analysis of the AVAST-M trial.

Background: Bevacizumab is a recombinant humanised monoclonal antibody to vascular endothelial growth factor shown to improve survival in advanced solid cancers. We evaluated the role of adjuvant bevacizumab in melanoma patients at high risk of recurrence. Patients and methods: Patients with resected AJCC stage IIB, IIC and III cutaneous melanoma were randomised to receive either adjuvant bevacizumab (7.5 mg/kg i.v. 3 weekly for 1 year) or standard observation. The primary end point was detection of an 8% difference in 5-year overall survival (OS) rate; secondary end points included disease-free interval (DFI) and distant metastasis-free interval (DMFI). Tumour and blood were analysed for prognostic and predictive markers. Results: Patients (n=1343) recruited between 2007 and 2012 were predominantly stage III (73%), with median age 56 years (range 18-88 years). With 6.4-year median follow-up, 515 (38%) patients had died [254 (38%) bevacizumab; 261 (39%) observation]; 707 (53%) patients had disease recurrence [336 (50%) bevacizumab, 371 (55%) observation]. OS at 5 years was 64% for both groups [hazard ratio (HR) 0.98; 95% confidence interval (CI) 0.82-1.16, P = 0.78). At 5 years, 51% were disease free on bevacizumab versus 45% on observation (HR 0.85; 95% CI 0.74-0.99, P = 0.03), 58% were distant metastasis free on bevacizumab versus 54% on observation (HR 0.91; 95% CI 0.78-1.07, P = 0.25). Forty four percent of 682 melanomas assessed had a BRAFV600 mutation. In the observation arm, BRAF mutant patients had a trend towards poorer OS compared with BRAF wild-type patients (P = 0.06). BRAF mutation positivity trended towards better OS with bevacizumab (P = 0.21). Conclusions: Adjuvant bevacizumab after resection of high-risk melanoma improves DFI, but not OS. BRAF mutation status may predict for poorer OS untreated and potential benefit from bevacizumab. Clinical Trial Information: ISRCTN 81261306; EudraCT Number: 2006-005505-64.

An approach for automatic classification of grouper vocalizations with passive acoustic monitoring.

Grouper, a family of marine fishes, produce distinct vocalizations associated with their reproductive behavior during spawning aggregation. These low frequencies sounds (50-350 Hz) consist of a series of pulses repeated at a variable rate. In this paper, an approach is presented for automatic classification of grouper vocalizations from ambient sounds recorded in situ with fixed hydrophones based on weighted features and sparse classifier. Group sounds were labeled initially by humans for training and testing various feature extraction and classification methods. In the feature extraction phase, four types of features were used to extract features of sounds produced by groupers. Once the sound features were extracted, three types of representative classifiers were applied to categorize the species that produced these sounds. Experimental results showed that the overall percentage of identification using the best combination of the selected feature extractor weighted mel frequency cepstral coefficients and sparse classifier achieved 82.7% accuracy. The proposed algorithm has been implemented in an autonomous platform (wave glider) for real-time detection and classification of group vocalizations.

Treatment patterns, outcomes, and resource utilization of patients with metastatic melanoma in the U.K.: the MELODY study.

BACKGROUND: Advanced melanoma is an aggressive disease with a poor prognosis. Approved therapy is limited in the U.K. and, until recently, no treatment had improved survival over best supportive care. A deeper understanding of current clinical practice will help new agents find a place in future treatment pathways. OBJECTIVES: To document U.K. clinical practice for the treatment of patients with unresectable stage III/IV (advanced) melanoma. METHODS: MELODY (melanoma treatment patterns and outcomes among patients with unresectable stage III/IV disease: a retrospective longitudinal survey) compiled registries of consecutive patients with malignant melanoma (any stage) between 1 July 2005 and 30 June 2006 from France, Italy and the U.K. Patients with advanced melanoma and ≥ 2 months of follow-up were eligible for analysis. RESULTS: There were 220 eligible patients identified in the U.K., of whom 117 (53.2%) received systemic therapy outside of clinical trials. Over half of these patients received dacarbazine as first- or second-line therapy. Healthcare-resource utilization was extensive and patients had short survival times: 1- and 2-year survival rates after first-line systemic treatment were 45.5% [95% confidence interval (CI) 37.1-53.6] and 24.7% (95% CI 17.7-32.3), respectively. CONCLUSIONS: Systemic and palliative treatments used to manage advanced melanoma in the U.K. are associated with considerable healthcare resource utilization and poor short-term survival.

Lenalidomide enhances the anti-prostate cancer activity of docetaxel in vitro and in vivo.

BACKGROUND: In this study, we investigated the effects of combining lenalidomide and docetaxel on in vitro and in vivo models of prostate cancer as a potential strategy for treatment of castrate resistant prostate cancer (CRPC). METHODS: The effects of combining lenalidomide and docetaxel on proliferation, apoptosis, invasive potential, anchorage independent growth, and p53 activation in the PC3 and DU145 prostate cell lines were investigated. The effects of the lenalidomide and docetaxel combination on LNCaP prostate cancer cell growth and invasiveness in vitro was also studied. The combination of these two agents was finally tested on a xenograft model of PC3 tumor growth in nude mice. RESULTS: Lenalidomide decreased the IC(50) of docetaxel by up to 50% (P < 0.05) and also decreased invasion in PC3, LNCaP, and DU145 cells and anchorage independent growth in PC3 cells (P < 0.01). Apoptosis in lenalidomide/docetaxel-treated cells was increased by 2.2-fold over single agent docetaxel and a corresponding increase in p53, p38, and BAD activation was observed in Western blots (P < 0.001). When PC3 challenged mice were treated with lenalidomide and docetaxel, median survival increased from 48 to 59 days and the rate of tumor growth was significantly reduced (P < 0.05). CONCLUSIONS: Lenalidomide may be a promising candidate for combination with docetaxel in the treatment of CRPC.

Supernatants derived from chemotherapy-treated cancer cell lines can modify angiogenesis.

BACKGROUND: There is evidence that tumours produce substances such as cytokines and microvesicular bodies bearing bioactive molecules, which support the carcinogenic process. Furthermore, chemotherapy has also been shown to modify these exudates and in doing so, neutralise their tumourigenic influence. METHODS: In the current study, we have investigated the effect of chemotherapy agents on modifying the cytokine profile and microvesicular cargo of supernatants derived from cancer cell lines. In addition, we have explored the effect of these tumour-derived supernatants on angiogenesis, and how chemotherapy can alter the supernatants rendering them less pro-angiogenic. RESULTS: Herein, we show that supernatants contain a rich cocktail of cytokines, a number of which are potent modulators of angiogenesis. They also contain microvesicular bodies containing RNA transcripts that code for proteins involved in transcription, immune modulation and angiogenesis. These supernatants altered intracellular signalling molecules in endothelial cells and significantly enhanced their tubulogenic character; however, this was severely compromised when supernatants from tumours treated with chemotherapy was used instead. CONCLUSION: This study suggests tumour exudates and bioactive material from tumours can influence cellular functions, and that treatment with some chemotherapy can serve to negate these pro-tumourigenic processes.

An intra-patient placebo-controlled phase I trial to evaluate the safety and tolerability of intradermal IMM-101 in melanoma.

BACKGROUND: IMM-101 is a heat-killed innate and adaptive immune-activating mycobacterial product; a phase I study aimed to determine its safety and tolerability in individuals with melanoma. PATIENTS AND METHODS: An intra-patient placebo-controlled study evaluated the safety and tolerability of three doses, namely, 0.1 (1 mg/ml), 0.5 (5 mg/ml) and 1.0 mg (10 mg/ml) of IMM-101 in stage III or IV melanoma. Each dose was administered in ascending order to one of the three cohorts. RESULTS: Based on observations from patients administered the 0.1-mg dose, it was considered appropriate to proceed with dosing the patients in the 0.5-mg dose cohort and then the 1.0-mg cohort (n = 6 per cohort). Treatment-emergent adverse events that would be considered typical of a post-vaccination state (including joint pains/aches, headaches and influenza-like symptoms) occurred at all dose levels, along with injection site reactions. These were mainly mild in intensity, resolved in a matter of days and responded well to supportive care. During post-study follow-up, two clinical responses (15%) were observed in patients with stage IV disease. CONCLUSION: IMM-101 is safe and well tolerated and there is a rationale for studying IMM-101 at a nominal 1.0-mg dose to complement conventional cytotoxic therapy for patients with advanced cancer.

T regulatory cells, the evolution of targeted immunotherapy.

T regulatory cells are able to suppress anti-tumour immunity in pre-clinical models and in patients. This review highlights the important discoveries in Treg immunology critical to the evolution of targeted immunotherapy. We also describe the therapeutic applications that are currently being assessed and their future potential.

Supernatants from lymphocytes stimulated with Bacillus Calmette-Guerin can modify the antigenicity of tumours and stimulate allogeneic T-cell responses.

BACKGROUND: Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis. METHODS: In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines. RESULTS: BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th(1)-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th(1)-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement. CONCLUSION: These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility.

Anti-tumour activity of afatinib, an irreversible ErbB family blocker, in human pancreatic tumour cells.

BACKGROUND: The combination of the reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib with gemcitabine obtained FDA approval for treating patients with pancreatic cancer. However, duration of response is often limited and there is currently no reliable predictive marker. METHODS: We determined the sensitivity of a panel of human pancreatic tumour cell lines to treatment with afatinib, erlotinib, monoclonal antibody (mAb) ICR62, and gemcitabine, using the Sulforhodamine B colorimetric assay. The effect of these agents on cell signalling and cell-cycle distribution was determined by western blot and flow cytometry, respectively. RESULTS: At 200 nM, ICR62 had no effect on growth of these tumour cells with the exception of BxPC-3 cells. BxPC-3 cells were also sensitive to treatment with afatinib and erlotinib with respective IC(50) values of 11 and 1200 nM. Compared with erlotinib, afatinib was also more effective in inhibiting the growth of the other human pancreatic tumour cell lines and in blocking the EGF-induced phosphorylation of tyrosine, EGFR, MAPK, and AKT. When tested in BxPC-3 xenografts, afatinib induced significant delay in tumour growth. CONCLUSION: The superiority of afatinib in this study encourages further investigation on the therapeutic potential of afatinib as a single agent or in combination with gemcitabine in pancreatic cancer.

Effect of Gemcitabine based chemotherapy on the immunogenicity of pancreatic tumour cells and T-cells.

PURPOSE: Chemotherapy for advanced pancreatic cancer has limited efficacy due to the difficultly of treating established tumours and the evolution of tumour resistance. Chemotherapies for pancreatic cancer are typically studied for their cytotoxic properties rather than for their ability to increase the immunogenicity of pancreatic tumour cells. In this study Gemcitabine in combination with immune modulatory chemotherapies Oxaliplatin, zoledronic acid and pomalidomide was studied to determine how combination therapy alters the immunogenicity of pancreatic tumour cell lines and subsequent T-cell responses. METHODS: Pancreatic tumour cell lines were stimulated with the chemotherapeutic agents and markers of immune recognition were assessed. The effect of chemotherapeutic agents on DC function was measured using uptake of CFSE-stained PANC-1 cells, changes in markers of maturation and their ability to activate CD8+ T-cells. The effect of chemotherapeutic agents on T-cell priming prior to activation using anti-CD3 and anti-CD28 antibodies was determined by measuring IFN-γ expression and Annexin V staining using flow cytometry. RESULTS: These agents demonstrate both additive and inhibitory properties on a range of markers of immunogenicity. Gemcitabine was notable for its ability to induce the upregulation of human leukocyte antigen and checkpoints on pancreatic tumour cell lines whilst inhibiting T-cell activation. Pomalidomide demonstrated immune modulatory properties on dendritic cells and T-cells, even in the presence of gemcitabine. DISCUSSION: These data highlight the complex interactions of different agents in the modulation of tumour immunogenicity and immune cell activation and emphasise the complexity in rationally designing chemo immunogenic combinations for use with immunotherapy.

Pre-treatment with chemotherapy can enhance the antigenicity and immunogenicity of tumours by promoting adaptive immune responses.

BACKGROUND: Some cancer patients are immuno-compromised, and it has been long felt that immune-intervention is not compatible with standard chemotherapies. However, increasing evidence suggests that standard chemotherapy drugs may stimulate beneficial changes in both the immune system and tumour. METHODS: We have assessed the expression of human leucocyte antigen class 1 (HLA1) on tumour cells before and after chemotherapy agents (cyclophosphamide, oxaliplatin or gemcitabine). In addition, we show that chemotherapy-stressed tumour cells may release cytokines that enhance the interactions between dendritic cells (DCs) and T cells into growth media. RESULTS: Here we report that some chemotherapy agents can increase HLA1 expression in tumour cells, even when expression is low. Increases were associated with killing by cytotoxic T cells, which were negated by HLA1-blockade. Furthermore, T-cell function, as indicated by increased proliferation, was enhanced as supernatants derived from tumours treated with chemotherapy augmented DC-maturation and function. CONCLUSION: There is evidence that a facet of immune surveillance can be restored by appropriate chemotherapy agents. Also, tumours exposed to some chemotherapy may secrete cytokines that can mature DCs, which ultimately enhances T-cell responses.

Investigator-initiated trials of targeted oncology agents: why independent research is at risk?

BACKGROUND: Drug development traditionally has relied upon the complementary contributions of clinicians and scientists at academic institutions and at pharmaceutical companies. Greater regulatory burdens, increased bureaucratic requirements, restricted reimbursement, and spiralling research and development costs are exerting pressure on the drug development pipeline. The result is a de-emphasis of exploratory research, particularly independent academic research, despite its proven value in identifying new drug targets and developing innovative cancer therapies. DESIGN: An expert panel assembled by the Biotherapy Development Association-a nonprofit international forum for academic and industry researchers, patients, and government regulatory and postregulatory agencies-examined the growing schism between academia and industry and identified several causes of declining academic research. RESULTS: The authors propose solutions to sustain investigator-initiated research and provide a new model whereby expert organisations provide a forum for academia and industry to plan studies within a regulatory framework to support licensure/authorisation and reimbursement for new molecularly targeted agents and biomarkers. CONCLUSIONS: Investigator-initiated trials have led to the discovery and development of innovative, safe, and effective cancer treatments. To ensure that such research continues, action will be required on the parts of legislative and regulatory bodies, industry, universities, patient advocacy organisations, and preclinical and clinical academic scientists.

The effect of colorectal cancer upon host peripheral immune cell function.

OBJECTIVE: Colorectal cancer is immunogenic. However, it is also associated with suppression of host immunity. Identifying the mechanisms involved in immune suppression is necessary to develop future immunotherapeutic strategies. The aim of this study was to assess immune cell function in colorectal cancer patients. METHOD: A total of 80 colorectal cancer patients (41 male) prior to treatment and 38 matched controls (21 male) were recruited. Venous blood samples were taken. White blood cell composition was determined using monoclonal antibodies. Levels of cytokines IFN-gamma, TNF-alpha, IL-2, IL-10, IL-4 and IL-6 were measured from the supernatants of activated peripheral blood mononuclear cells (PBMC) following thawing and re-suspension. Peripheral blood mononuclear proliferation was measured using 3H-Thymidine. RESULTS: Stage I-III cancer patients had elevated percentages of CD8 T cell (P = 0.004) whilst stage IV patients had low total lymphocyte percentages (P = 0.016). Monocyte and NKT cell percentage decreased with advanced tumour stages (P = 0.013 and P = 0.038). Patients had lower PBMC proliferation and production of the TH1 cytokines (IFN-gamma and TNF-alpha) (P < 0.001) than that of the controls. IL-6 and IL-4 production were not significantly different. IFN-gamma and TNF-alpha concentrations reduced with tumour vascular invasion (P = 0.011 and P = 0.019). CONCLUSION: Colorectal cancer induces an immunological response, shifting the cytokine balance. The most profound changes are seen once disease has spread systemically.

T-regulatory cell modulation: the future of cancer immunotherapy?

T-regulatory cells suppress anti-tumour immunity in cancer patients and in murine tumour models. Furthermore, their activity is likely to have an effect on the effectiveness of immunotherapeutic treatments for cancer. Here we describe the current status of developing clinical strategies for modulating Treg activity in cancer patients.

Inhibition of metastatic potential in colorectal carcinoma in vivo and in vitro using immunomodulatory drugs (IMiDs).

BACKGROUND: Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma and, along with pomalidomide, are being investigated in various other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about their primary mode of therapeutic action in patients with cancer. METHODS: As part of a continuing research effort, we have investigated the effects of these agents on the metastatic capacity of murine colorectal cancer cell lines both in vivo and in vitro. Allied to these, we have studied their effects on the molecular pathways associated with metastasis. RESULTS: Results indicate that thalidomide, lenalidomide and pomalidomide significantly inhibit the metastatic capability of colorectal carcinoma cells. Anchorage-independent growth, used as a coarse indicator of transformation, was significantly reduced, as were migratory capacity and invasive competence. In addition, an in vivo experimental metastasis model also showed that treatment with the drugs resulted in a significantly lower number of metastatic pulmonary nodules relative to control mice. Allied to these cellular and phenotypic changes were alterations in molecular markers of metastasis and in intracellular signalling competency. CONCLUSIONS: These results provide evidence that in addition to their immunomodulatory effects, thalidomide, lenalidomide and pomalidomide can impair the metastatic capacity of tumours, and that this mechanism may involve alterations to cell signalling functionality.

Epitopes of the CD4 antigen and HIV infection.

The CD4 (or T4) surface antigen of human T lymphocytes is an important part of the receptor for the human immunodeficiency virus (HIV). After binding to the receptor, the HIV may enter the T cell and induce the formation of syncytia. In an attempt to identify the receptor site more closely, monoclonal antibodies (Mab's) to CD4 were tested for their ability to block HIV infection in a syncytium formation assay, and the CD4 epitopes so identified were mapped by antibody cross-blocking. The antibodies that showed strong inhibition of HIV fell into two main families while a third group of Mab's blocked syncytia formation weakly or not at all. Several different isolates of HIV as well as the laboratory strain CBL1 grown in CEM cells were used to induce the syncytia. The data indicate that only some epitopes of CD4 are important for virus binding and imply that the virus-binding site for CD4 is conserved in different isolates of HIV with substantially divergent env gene sequences. Preliminary studies of patients suggest that polymorphism of these epitopes does not play a role in determining susceptibility to infection.

How immunotherapy can enhance the response to other modalities and improve outcome and quality of life.

Early studies suggested that the induction of an effective immune response could lead to elimination of residual tumour. Over a hundred years ago Coley invented his eponymous named "toxins" that appeared to induce a strong inflammatory response, leading to tumour reduction. Subsequent attempts to enhance the immune response have essentially been on a vaccine basis, trying to induce a specific response against the tumour. Numerous vaccine approaches have claimed to give significant clinical benefit in clinical response but very few of these have survived a randomised trial. A major reason for this is the heterogeneity of many tumours, as well as the various forms of defence against an immune response that they employ. It was thought that chemotherapy and radiotherapy were mutually exclusive for immunotherapy using the vaccine approach. More recently, however, it has become appreciated that vaccine approaches may enhance subsequent responses to radiotherapy and that certain chemotherapies actually enhance responses to vaccines. It has been suggested that one of the mechanisms of action of chemotherapy is to reduce the cells that suppress T-cells. These cells primarily defend the tumour from an immunological attack, but more recently it has been suggested that the benefit may encompass other aspects, such as enhancing antiangiogenic responses. One reason why immunostimulatory approaches may be so useful in cancer is that many cancers evolve out of a chronic inflammatory environment that actively suppresses cell mediated immune responses and enhances tumour angiogenesis. An ideal cancer drug would therefore be expected to have these properties. One such drug is lenalidomide, which features include marked immune stimulatory properties as well being able to inhibit regulatory T-cells. They have also been shown to enhance anticancer activity with vaccines in both preclinical models and more recently in clinical observations, where the responses to vaccines in patients with myeloma is much higher when they are on lenalidomide than other treatments. A number of regularly used chemotherapy regimens have marked activity in modulating the immune response. These maybe of benefit and the regimens will be reviewed, which include gemcitabine, cyclophosphamide and the IMiDs.

An evaluation of a preparation of Mycobacterium vaccae (SRL172) as an immunotherapeutic agent in renal cancer.

Two studies were carried out to evaluate heat-killed Mycobacterium vaccae SRL172 as an immunotherapeutic agent for patients with metastatic, post-nephrectomy, renal cell carcinoma. In the first study, 60 patients in France and the UK received injections of SRL172, and their survival was compared with that of historical controls who had been treated either with biological response modifiers (IL-2, IFN-alpha) or chemotherapy. In the second study, 36 patients were randomised to receive treatment with IL-2 alone or IL-2 plus SRL172. Survival and adverse events related to the treatments were assessed and compared between treatment groups. The first study showed that those treated with SRL172 alone survived equally as long as those receiving IL-2 or IFN-alpha and both treatment groups survived longer than those on chemotherapy (p<0.001), a result supported by Cox's proportional hazards regression analysis. The second study, stopped early due to drug supply issues, showed that the addition of SRL172 to IL-2 made no difference to survival compared to IL-2 alone, in the limited numbers treated. Adverse events occurring in those receiving SRL172 in the first study were mild and in the second study those receiving IL-2 alone had significantly more adverse events than those receiving SRL172 plus IL-2 (p<0.001). It is concluded that SRL172 may have activity in metastatic renal cancer and has very low toxicity, making it worthy of further study.