Project Details

Patients with macroscopic Stage III malignant melanoma are a heterogeneous group of patients with an average overall survival of less than 30%. Previously it has not been possible to predict which patients will achieve longer term survival.We recently completed a molecular profiling study which has identified a gene signature capable of predicting those patients whose survival will be good and those whose survival will be poor.

The purpose of this project is to gain a greater understanding of the biological programs that underpin these genetic signatures and to develop the gene signature further as a diagnostic tool for routine clinical use. The project will use a combination of molecular genetic and immunohistochemical techniques to adapt the current gene profile for routine use, to validate itsa pplication in independent patient cohorts and to assess itsfeasibility in routine clinical care. The expected outcome will be that this project will generate a tool which will be clinically useful for clinicians treating this fatal form of skin cancer.

References

John T, Black MA, Toro TT, Leader D, Gedye CA, Davis ID, Guilford P J, CEBON JS. Predicting Clinical Outcome through Molecular Profiling in Stage III Melanoma. Clin Cancer Res 2008;14: 5173-80

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Project Details

We are currently undertaking an analysis of the transcriptome and genome of melanoma stem cells. These studies are identifying molecules that are differentially expressed on putative stem cells but not their progeny. The transcriptome analysis is enabling a rational identification of pathways which are differentially activated in stem-like cells. Critical molecules within these would appear to be prime targets for anti-cancer therapy, so long as they are not also critical for the self-renewal of somatic stem or germ cells. It should be possible to determine whether currently available anti-cancer drugs such as receptor tyrosine kinase inhibitors act on these cells. Such drugs have established safety records and can be tested in the cancer colony forming assays. This assay is potentially suitable for adaptation to a high-throughput screening assay and the feasibility of this will be explored during the course of this project. Promising pharmacological agents will subsequently be assessed in a murine model.

References

Gedye, C., Quirk, J., Browning, J., Svobodova, S., John, T., Sluka, P., Dunbar, P.R., Corbeil, D., Cebon, J., and Davis, I.D., Cancer/testis antigens can be immunological targets in clonogenic CD133(+) melanoma cells. Cancer Immunol Immunother, 2009.

Cebon J, Geyde C, John T, and Davis ID. Immunotherapy of advanced or metastatic melanoma. Clinical Advances in Hematology and Oncology, 5:994-1006,2007

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Project Details

Cancer cells recapitulate many behaviors of pluripotent embryonic cells such as unlimited proliferation, the capacity to self renew and to migrate. Previously, a search for new embryonic genes also expressed in cancer identified Embryo-Cancer Sequence A (ECSA), also named Developmental Pluripotency Associated-2 (DPPA2). ECSA/DPPA2 has been shown to be expressed in human pre-implantation embryos and primordial germ cells (PGCs) but is rapidly down regulated upon differentiation. ECSA/DPPA2 transcripts in normal tissues are limited to testis, placenta, bone marrow and thymus but expressed in a variety of tumors including colorectal cancers and melanoma but most notably in Non-Small Cell Lung Cancers (NSCLC). Transcripts for a panel of Cancer Testis antigens (CTAs) investigated in the same NSCLC tissues, demonstrated enrichment of CTAs in ECSA/DPPA2 positive tumors. IHC demonstrated nuclear localization for ECSA/DPPA2 in subpopulations of basally located cells consistent with cells occupying a 'stem-cell niche'. Antibodies to ECSA/DPPA2 protein were detected in the sera of patients with NSCLC, but not in healthy controls. The restricted expression in normal tissues, expression in tumors with co-expression of CTAs and spontaneous immunogenicity, indicate that ECSA/DPPA2 is a promising target for antigen specific immunotherapy in NSCLC. This project will define ECS-A/DPPA2 further in human lung cancer. In particular, it will focus on defining lung cancer stem cell surface markers to enable sorting and purification of these cells.

References

John T, Cabellero O, Svobodová S, Kong A, Chua R, Browning J, Fortunato S, Gedye CA, Davis ID, Altorki N, Simpson AJ, Monk M and CEBON JS. ECSA/DPPA2 is an embryo-cancer antigen that is co-expressed with Cancer-Testis antigens in Non-Small-Cell Lung Cancer. Clin Cancer Res, 14(11):3291-8, 2008

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Project Details

Although cancer is often a genetically heterogeneous disease, it has become clear that certain tumours are driven by specific pathways that render them particularly sensitive to targeted therapies. These include somatic mutations in the c-kit gene in gastrointestinal stromal tumours (GIST) as a predictor of response to imatinib. In addition, activating epidermal growth factor receptor (EGFR) mutations are a useful predictor of benefit to small molecule tyrosine kinase inhibitors of EGFR such as erlotinib in non-small cell lung cancer (NSCLC). Recently, antibodies targeting EGFR such as cetuximab or panitumumab have shown benefit in patients with advanced colorectal cancer (CRC) who have failed chemotherapy. Importantly, the benefit associated with these treatments is restricted to those patients with k-ras gene wild type tumours, indicating that the k-ras gene is an important predictive marker of benefit of this class of agents.

Although these genetic changes allow accurate prediction of patient groups who will derive greater degrees of benefit from particular therapies, inevitably treatment resistance will evolve. In some cases, the mechanism of resistance has been defined. For example in GIST secondary activating mutations in c-kit overcome the inhibition of c-kit activity induced by imatinib. Similarly, in NSCLC, the T790M mutation overcomes EGFR inhibition induced by tyrosine kinase inhibitors. In the case of cetuximab or panitumumab, mechanisms of resistance to treatment have not been defined. We propose to undertake a project aimed at identifying mechanisms of resistance to anti-EGFR antibodies in colon cancer. We propose an approach involving both in vitro and in vivo studies.

1. We will evaluate mechanisms of resistance of tumour cells to anti-EGFR antibodies in vitro Two colon cancer cell lines, LIM1215 and Difi, which are WT for K-Ras and sensitive to anti-EGFR antibodies will be continuously cultured in the presence of panitumumab for 4-6 weeks, or until a 50% reduction in cell viability is observed. Surviving cells will be passaged and cultured in a 2-fold higher concentration of the drug for a subsequent 4-6 weeks. DNA from surviving clones will be extracted and screened for mutations in the known hotspots in the K-Ras gene. The mutational status of other putative determinants of panitumumab resistance, BRAF and PIK3CA will also be determined by direct sequencing. Changes in expression of EGFR and PTEN will be determined by western blot.

2. We will then aim to validate these findings in vivo. Whilst the gold standard method to determine resistance mechanisms would involve repeat biopsies of metastatic tissue, this is rarely practical in real life. Hence we propose to isolate and study circulating tumour cells which are likely to be representative of the changes occurring in metastatic tissue. We would aim to study circulating tumour cells before therapy and after the development of resistance to anti-EGFR antibodies. The strategy we will adopt will be to use a FACS-based approach to select out tumour cells from PBMCs using the Ep-CAM cell surface marker. DNA will be extracted from circulating tumour cells and screened for mutations in the K-Ras, BRAF and PIK3CA genes.

Other biomarker studies

Several other studies related to biomarkers relevant to the use of targeted therapies could be developed, based on the existing trial portfolio. These depend on the interests of the candidate and whether they plan to embark on a PhD or DMedSc degree. These would involve evaluation of biomarkers relevant to the targeted therapy being used in the ATTAX2 study, the Vengeance study and the Radichol study.

We would be happy to discuss possible options for projects in more detail. To contact Niall Tebbutt please call 9496 5763.

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Project Details

DNA methylation (an epigenetic modification) is an important mechanism by which expression of tumor suppressor genes is inactivated in cancer. Through utilization of genome-wide DNA methylation screening assays we have identified a number of novel gene promoters that are methylated in colon cancer. The goal of this project is to exogenously express a subset of these genes in tumor cells and determine the effect on tumor cell growth, differentiation and survival. This is an open-ended / high risk project that would be suited to a more experienced candidate.

References

HDACs and HDAC inhibitors in colon cancer. JM Mariadason. Epigenetics. 2008 Jan-Feb;3(1):28-37.

HDAC4 promotes growth of colon cancer cells via repression of p21. AJ Wilson, D-S Byun, S Nasser, LB Murray, K Ayyanar , D Arango, M Figueroa, A Melnick, G Kao, LH. Augenlicht and JM. Mariadason. Mol. Biol. Cell. 2008. 19: 4062-4075.

Class I Histone deacetylases are upregulated in human colonic tumors and are linked to reduced cell differentiation in vivo and in vitro. AJ Wilson, DS Byun, HJ Smartt, K L’Italien, Y Sowa, D Arango, LH Augenlicht and JM Mariadason. J. Biol. Chem. 2006 May 12;281(19):13548-58.

PIK3CA/PTEN mutation status predicts response of colon cancer cells to cetuximab. M Jhawer, S. Goel, YH Ling, D-S Byun, A Wilson, S Nasser, J Shin, D Arango, L Augenlicht, R Perez-Soler, JM Mariadason. Cancer Res, 2008, Mar 15;68(6):1953-61

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Project Details

HDAC3 is a transcriptional co-repressor that is overexpressed in several cancers. Knockdown of HDAC3 causes growth arrest of colon cancer cells in vitro, suggesting this gene plays a role in tumor progression. We now wish to determine whether HDAC3 plays a similar role in colon cancer progression in vivo. In order to do this we have obtained a mouse that will enable us to genetically inactivate HDAC3 in the colon (HDAC3Lox/Lox). These mice will be crossed to Apc mutant mice which are genetically predisposed to develop colon cancer. This PhD project is to investigate the effect of genetic inactivation of HDAC3 on the normal physiology of the colon and on the incidence of colon cancer. These studies will provide insight into the molecular mechanisms that drive colon cancer and thereby contribute to the identification of novel proteins that can be targeted for the treatment of this disease.

References

HDACs and HDAC inhibitors in colon cancer. JM Mariadason. Epigenetics. 2008 Jan-Feb;3(1):28-37.

HDAC4 promotes growth of colon cancer cells via repression of p21. AJ Wilson, D-S Byun, S Nasser, LB Murray, K Ayyanar , D Arango, M Figueroa, A Melnick, G Kao, LH. Augenlicht and JM. Mariadason. Mol. Biol. Cell. 2008. 19: 4062-4075.

Class I Histone deacetylases are upregulated in human colonic tumors and are linked to reduced cell differentiation in vivo and in vitro. AJ Wilson, DS Byun, HJ Smartt, K L’Italien, Y Sowa, D Arango, LH Augenlicht and JM Mariadason. J. Biol. Chem. 2006 May 12;281(19):13548-58.

PIK3CA/PTEN mutation status predicts response of colon cancer cells to cetuximab. Jhawer, M., Goel, S., Wilson, A.J., Montagna, C., Ling, Y.H., Byun, D.S., Nasser, S., Arango, D., Shin, J., Klampfer, L., Augenlicht, L.H., Soler, R.P., and Mariadason, J.M. Cancer Res, 68(6): p. 1953-61 2008

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Project Details

The MAX study, was a randomised phase III multi-centre study conducted by the AGITG in Australia, New Zealand and the United Kingdom with a total of 471 patients randomised. The primary objective of the study was to compare progression-free survival (PFS) for patients receiving the capecitabine arm versus capecitabine plus bevacizumab or capecitabine, mitomycin C plus bevacizumab. The primary analysis of the study will be presented at ASCO 2009.

Over 80% of patients participating in the study gave consent for tissue collection for use in biomarker studies. No other studies involving bevacizumab have involved significant rates of tissue banking and thus the ability to address the role of tissue biomarkers with this targeted agent. Tissue samples are currently being collected at Austin Health. We propose to use the tissue samples to address relevant biologic questions relating to the use of capecitabine and bevacizumab in patients with mCRC.

The most valuable projects will involve assessment of novel biomarkers and their role as predictors of benefit with bevacizumab. A current project is examining the role of other VEGF family members (VEGF B-D) as predictors of resistance. However, possible other relevant biomarkers yet to be examined include angiopoietins, FGFs and HIF1-alpha and LDH. In addition to hypothesis driven research, an exploratory analysis using array technology with mRNA derived from tumour samples may also be possible.

Other biomarker studies:

Several other studies related to biomarkers relevant to the use of targeted therapies could be developed, based on the existing trial portfolio. These depend on the interests of the candidate and whether they plan to embark on a PhD or DMedSc degree. These would involve evaluation of biomarkers relevant to the targeted therapy being used in the ATTAX2 study, the Vengeance study and the Radichol study.

We would be happy to discuss possible options for projects in more detail. To contact Niall Tebbutt please call 9496 5763.

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Project Details

STAT3 is one of the best studied molecules involved in oncogenic signalling and has been shown to have an essential role in malignant progression. Several proof-of-concept studies in cell-culture and animal models have demonstrated that constitutive STAT3 signalling promotes the growth and survival of tumour cells and induces tumour angiogenesis and suppresses antitumour immune responses (1, 2). Interestingly, STAT3 was shown in recent studies to be overactivated in 56-90% of colorectal carcinoma biopsies. Accordingly, STAT3 is a promising molecular target for novel cancer therapies in colorectal cancer. Small Interfering RNAs (siRNAs) are one of the newest and most powerful tools of biomedical research. This project will explore the role of STAT3 in colorectal cancer and investigate the effect of siRNA inhibition of STAT3 on growth and tumourigenicity of colorectal cancer cells. For the therapeutic development of these siRNAs, novel conjugate siRNAs that target STAT3 to tumour via a humanised monoclonal antibody (3) will be created and evaluated through in-vitro and in in-vivo models. The validation of STAT3 knockdown will be assessed in colon tumour models. Additional genes associated with colorectal cancer will also be studied with siRNA knockdown, and through immunoconjugate delivery systems. This approach aims to develop a new class of therapeutic agents for the treatment of colorectal cancer.

References

1.Yu H et al. 2004 The STATS of cancer – new molecular targets come of age. Nature Rev Cancer 4:97-105

2.Mulkeen AL, et al. 2006. Short interfering RNA-mediated gene silencing of vascular endothelial growth factor: effects on cellular proliferation in colon cancer cells. Arch Surg. 141:367-74

3.Scott AM, et al. 2007 A Phase I biodistribution and pharmacokinetic trial of humanized monoclonal antibody hu3S193 in patients with advanced epithelial cancers which express the Lewis-y antigen. Clin Cancer Res 13:3286-92

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Project Details

Our laboratory program has developed an antibody specific to the Lewis-y (Ley) protein antigen (hu3S193) and conducted first in man clinical trials with this immunotherapeutic in cancer patients: The Ley antigen is found in 30-50% of breast cancers and over 70% of all epithelial cancers [1]. In laboratory assays, the hu3S193 antibody is able to recognize and kill cells which have the Ley antigen, a key feature of anti-cancer treatments [2, 3]. Hu3S193 has potent immune effector function, and has been demonstrated to efficiently target cancer cells in mouse models, and in human clinical trials [1, 4]. This research project aims to exploit the targeting properties of hu3S193, and identify potent regulators of immune system activation for improved tumour cell killing. Molecular biology techniques will be used to design and engineer novel humanised IgG constructs with improved complement and NK cell mediated immune effector function for tumour cell killing. Structural modelling studies involving Fc-C1q binding, and FcgRIII and FcgRIIb interactions with Fc, will be used to derive novel constructs, which will be characterised in in-vitro and in-vivo systems [5]. Further studies involving Fc-FcRn interactions and modulation of pharmacokinetics of recombinant humanised IgG will also be explored. This will allow the identification of potent modified forms of hu3S193 that can be extended into clinical trials.

References

1.Scott, A.M., et al., Construction, production, and characterization of humanized anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solid tumors. Cancer Res, 2000. 60(12): p. 3254-61.

2.Clarke, K., et al., In vivo biodistribution of a humanized anti-Lewis Y monoclonal antibody (hu3S193) in MCF-7 xenografted BALB/c nude mice. Cancer Res, 2000. 60(17): p. 4804-11.

3.Clarke, K., et al., Therapeutic efficacy of anti-Lewis(y) humanized 3S193 radioimmunotherapy in a breast cancer model: enhanced activity when combined with taxol chemotherapy. Clin Cancer Res, 2000. 6(9): p. 3621-8.

4.Scott, A.M., et al., A phase I biodistribution and pharmacokinetic trial of humanized monoclonal antibody Hu3s193 in patients with advanced epithelial cancers that express the Lewis-Y antigen. Clin Cancer Res, 2007. 13(11): p. 3286-92.

5.Ramsland, P.A., et al., Structural convergence of antibody binding of carbohydrate determinants in Lewis Y tumor antigens. J Mol Biol, 2004. 340(4): p. 809-18.

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