WEDNESDAY, MAY 19, 2010

Speaker abstracts are listed in order of presentation. A gap in the sequence of abstracts, indicates that an abstract was not received at the time of this posting.

Keynote Address

The Cancer Genome and Therapeutics

J. Michael Bishop, MD, University of California, San Francisco

Cancer arises from the malfunction of genes. The malfunction has many origins, but takes only two forms: gain and loss of function. This unifying view of cancer has launched efforts to create an inventory of all the genetic and epigenetic lesions associated with each form of human cancer. Although the inventory is far from complete, a few tentative principles have emerged. First, each type of cancer may have a distinctive, but not entirely unique genetic fingerprint. Second, individual cancers may contain dozens of malfunctioning genes, only a few of which may be shared among tumors of the same type. And third, commonalities among tumors of the same type become more apparent when considered in terms of the signaling pathways affected by malfunctioning genes. These principles have spawned the pursuit of therapeutic "magic bullets" that might attack the outlaw cancer cell without harming innocent bystanders.

The nature of magic bullets for cancer will be dictated by the forms of genetic malfunction under attack. Gain of function is most readily attacked with inhibitors, and several examples are already in clinical use. Loss of function could be remedied by restitution or bypass, neither of which presently seems praticable. A third approach can address either form of malfunction: the identification of therapeutics that create a synthetic-lethal interaction solely with the genetic malfunction, examples of which are emerging. Despite anticipated advantages, magic bullets may still have to be used in combination. Ways in which this might be achieved include targeting two renegade pathways in concert, targeting two nodes in the same pathway, or bimodal therapy aimed at the same molecular target. These are early days in the search for magic bullets for cancer. But we have reason to imagine a day when genetic fingerprints of cancer could be used to guide the treatment of individual patients. In order to realize this dream of "personalized cancer medicine," the pharmaceutical industry may have to cope with fundamental changes in the market place, and regulatory agencies may have to change their ways. The principal impediment may prove to be cost.

Session I: Biology (Processes Leading to Cancer)

Lgr5 Stem Cells in Self-Renewal and Cancer

Hans Clevers, MD, PhD, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences; University Medical Centre Utrecht, The Netherlands

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. Current models state that 4-6 crypt stem cells reside at the +4 position immediately above the Paneth cells in the small intestine; colon stem cells remain undefined. Lgr5/Gpr49 was selected from a panel of intestinal Wnt target genes for its restricted crypt expression. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. In addition, Lgr5 was expressed in rare cells in several other tissues including the hair follicel and stomach. Using an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Lgr5+ve crypt base columnar cell (CBC) generated all epithelial lineages over a 14 month period, implying that it represents the stem cell of the small intestine and colon. Similar obserations were made in hair follicles and stomach epithelium. The expression pattern of Lgr5 suggests that it marks stem cells in multiple adult tissues and cancers.We have now established long-term culture conditions under which single crypts undergo multiple crypt fission events, whilst simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ve stem cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the Lgr5+ve stem cell hierarchy is maintained in organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche. The same technology has now been developed for the Lgr5+ve stomach stem cells.

Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in in Lgr5+ve stem cells leads to their transformation within days. Transformed stem cells remain located at crypt bottoms, while fueling a growing microadenoma in stomach, small intestine and colon. These microadenomas display unimpeded growth and develop into macroscopic adenomas within 4-6 weeks. When APC is deleted in short-lived Transit Amplifying (TA) cells using a different Cre mouse, the growth of the induced microadenomas rapidly stalls. Even after 30 weeks, large adenomas are very rare in these mice. We conclude that stem cell-specific loss of APC results in progressively growing neoplasia. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the “cancer stem cell”-concept.

New Cancer Targets Emerging from Studies of the von Hippel-Lindau Tumor Suppressor Protein

William G. Kaelin, Jr, MD, Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Boston, MA and Howard Hughes Medical Institute, Chevy Chase, MD

Inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL) is linked to the development of various tumors including clear cell renal carcinoma, which is the most common form of kidney cancer. pVHL is the substrate recognition component of an E3 ubiquitin ligase that targets the alpha subunit of the heterodimeric transcription factor HIF for degradation. In the presence of oxygen HIFα is hydroxylated on one (or both) of two prolyl residues by the 2-oxoglutarate-dependent dioxygenase EglN1 (also called PHD2). This modification creates a binding site for pVHL, which then targets HIFα for proteasomal degradation. When oxygen levels are low, or pVHL is crippled, HIFα accumulates, binds to HIFα, and transcriptionally activates genes that promote survival in a low oxygen environment, including VEGF. VEGF inhibitors have now demonstrated activity against kidney cancer. There are 3 HIFα paralogs. Our recent data suggest that HIF2α is a kidney cancer oncoprotein whereas HIF1α, surprisingly, is a tumor suppressor and is lost in a significant fraction of these tumors.

EglN1 is a 2-oxoglutarate-dependent dioxygenase, which is a recently recognized superfamily of enzymes that can be inhibited with drug-like small molecules. EglN1 inhibitors are currently being tested for the treatment of anemia, while drugs that activate EglN1 (thereby lowering HIF levels) are being explored as anticancer agents. We recently discovered that EglN2 (PHD1), an EglN1 paralog, indirectly regulates Cyclin D1 in a HIF-independent manner. Inhibition of EglN2 leads to loss of Cyclin D1, decreased proliferation, and impaired tumorigenesis in vivo. Regulation of Cyclin D1 by EglN2 requires EglN2 catalytic activity and impaired proliferation in cells lacking EglN2 can be rescued by exogenous Cyclin D1 or by loss of pRB. Therefore Cyclin D1 causes, and does not merely correlate with, the proliferation defect in cells lacking EglN2. RBP2 (JARID1A) is a pRB-binding protein linked to differentiation control by pRB. A number of JmjC-containing proteins, including RBP2, are 2-oxoglutarate-dependent dioxygenases that serve as histone demethylases. Our preliminary data suggest that inhibition of RBP2 leads to impaired proliferation, promotion of differentiation, and loss of tumorigenesis.

Deconstructing Metastasis

Joan Massagué, PhD, Memorial Sloan-Kettering Cancer Center, New York, New York

Metastasis is a multi-stage process that selects for circulating cancer cells that can infiltrate, survive in, and colonize distant organs. Cancer cell dissemination may be followed by a protracted period of latency before relapse in one or more organs, as in breast cancer, or by a swift colonization of multiple organs, as in lung adenocarcinoma. We have sought to incorporate this varied biology into experimental models and clinical gene expression data sets to deconstruct metastasis. Searching for mechanisms that prime breast cancer cells for infiltration of different organs, we found few requirements for cancer cell extravasation through fenestrated bone marrow capillaries, several key requirements (EGFR ligands, COX2, TGFb-induced angiopoietin-like 4) for infiltration through the non-fenestrated walls of lung capillaries, and several additional mediators (sialyl transferase ST6GalNac5, and others) for infiltration though the blood-brain barrier for cerebral metastasis. Searching for mechanisms that allow infiltrated breast cancer cells to survive as latent disease, we found that a hyperactive SRC pathway supports the responsiveness of breast cancer cells to SDF1/CXCL12 as a survival factor in the marrow microenvironment. Searching for mechanisms that underlie the rapid metastasis of lung adenocarcinomas to brain and bone, we uncovered an involvement of the WNT/TCF pathway and of its target genes HOXH9 and LEF1. We also find that circulating tumor cells can avidly re-infiltrate their tumors of origin, in a process of “tumor self-seeding”. This process selects for, and expands highly aggressive cancer cell subpopulations. The striking disparities in the natural progression of different cancers bring into focus important questions about the evolution of metastatic traits, the molecular mediators involved, and treatment opportunities for the prevention of metastasis in a personalized manner.

Loss of the Tumor Suppressor SNF5 Leads to Aberrant Activation of the Hedgehog-GLI Pathway

Zainab Jagani, PhD1, Lorena Mora-Blanco3, Courtney G. Sansam3, Boris Wilson3, Phuong T.L. Nguyen3, Justin Klekota1 , Pablo Tamayo4, Yoon-Jae Cho2,4, Scott L. Pomeroy2, Jill P. Mesirov4, Heinz Ruffner5, Tewis Bouwmeester5, Sarah Luchansky1, Joseph Kelleher1, Markus Warmuth1, William R. Sellers1, Charles W. M. Roberts3, and Marion Dorsch1, 1Novartis Institutes for BioMedical Research, Cambridge Massachussetts, 2Department of Neurology, Children’s Hospital Boston, 3Department of Pediatric Oncology, Dana-Farber Cancer Institute, Division of Hematology/Oncology, Children’s Hospital Boston, Harvard Medical School, Boston MA, 4Broad Institute of Harvard and M.I.T, Cambridge MA, 5Novartis Institutes for BioMedical Research, Basel, Switzerland

The Hedgehog (Hh) pathway plays an important role during development, and its aberrant activation can drive tumorigenesis. To investigate the mechanism by which GLI1, a critical effector of Hh signaling, regulates Hh pathway activation, we searched for proteins that interact with GLI1. We report that the chromatin remodeling protein and tumor suppressor Snf5 (Smarcb1/Ini1), which is inactivated in the majority of malignant rhabdoid tumors (MRT) interacts with GLI1. We show that Snf5 localizes to Gli1 regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway both in vitro and in vivo. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli activated gene expression profile in primary MRTs, and using shRNA mediated depletion of GLI1, we demonstrate that GLI1 drives the growth of SNF5-deficient MRT cells. Therefore, our studies reveal that Snf5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a novel targetable mechanism contributing to the growth of MRT cells.

The Intestinal Stem Cell Gene Expression Programme Predicts Colorectal Cancer Recurrence

Eduard Batlle, PhD, CREA & Oncology Programme. Institute for Research in Biomedicine (IRB-Barcelona) Barcelona, Spain

Mature differentiated cells of the intestinal tract are constantly renewed by the progeny of Intestinal Stem Cells (ISCs). Here, we report that the organization of most human colorectal cancers (CRCs) is reminiscent of that of the normal intestinal epithelium. CRC cells display phenotypes similar to either ISCs or intestinal differentiated cells and recreate the formation of crypt-like structures within tumors. We have developed a method to purify normal intestinal cell populations including ISCs. Comparison of the expression profiles of different crypt cell types versus CRC patient samples led us to identify a gene programme shared between normal ISCs and the most aggressive CRCs. We demonstrate that high expression levels of ISC genes in primary tumors identify CRC patients at high risk of disease relapse after therapy. Our results imply that the acquisition of an ISC gene programme is a central process in the development of metastatic and recurrent CRC.

Session II: Genetics and Epigenetics (Cancer Susceptibility)

“BioBank Japan” Project Toward the Personalized Medicine

Yusuke Nakamura, MD,PhD, Human Genome Center, Institute of Medical Science, The University of Tokyo

The “Biobank Japan” project started in June 2003 by the support of the Japanese government. The aims of this project are 1) discovery of genes susceptible to diseases, or those related to effectiveness or adverse reactions of various drugs, 2) identification of molecular targets for evidence-based development of drugs or diagnostic tools, 3) identification of the important genetic information that can be applied for establishment of “Personalized Medicine” and 4) studies on gene-environment interaction for prevention of diseases. To achieve these goals, we planned to collect of DNAs, sera and clinical information from 310,000 cases who have either of 47 common diseases. As the research resource bank, we constructed “BioBank Japan” that consisted of facilities that are able to store DNAs (the maximum capacity of 1,000,000 tubes) and sera (the maximum capacity of 3,000,000 tubes in liquid nitrogen). So far, we have obtained a written informed consent from more than 200,000 patients (a total number of disease cases is nearly 300,000) from 66 hospitals participating in this project. We have been performing the genome-wide association studies using 610,000 SNPs, which can cover most of our genome, to identify genes of medical importance, particularly those associated with severe adverse reactions caused by various drugs. In addition, we have developed a fully-automated SNP typing machine that enables us to make genotyping of multiple SNPs within a very short period and to provide a chance to have the “point-of–care” treatment to patients. Accumulative data and the new tool should provide us the basis to identify genes associated with common diseases and to establish the personalized treatment.

VEGF Pathway Genes: Novel Functional Variations

Laia Paré, MSc, Patrick Evans, Shiwei Duan, Sonal Kashyap, Wei Zhang, M. Eileen Dolan, Federico Innocenti, Department of Medicine, The University of Chicago, Chicago, IL

Vascular endothelial growth factor (VEGF), its receptors and signaling effectors play a central role in tumor-induced angiogenesis. Targeting tumor angiogenesis is an established antitumor therapy. Studies on germline variation of VEGF have proposed that heritable variation might predict efficacy of angiogenesis inhibitors as well as predisposing factors for cancer. Our objective was to identify novel genetic information in the VEGF pathway and functional single nucleotide polymorphisms (SNPs) that are associated with changes in gene expression.

Twenty-three genes were prioritized based upon their biological significance in endothelial function and VEGF signaling. These genes were resequenced, with full coverage for ATK1, FRS2, KRAS, MAPK11, MAPK3, NRAS, PGF, PIK3R5, VEGFB and standard coverage for CRK, FLT1, GRB2, ITGAV, ITGB5, MAP2K6, MAPK1, MAPK14, NRP1, PIK3C2A, PIK3C2B, PRKCA, PRKCE, RAF1. Unrelated HapMap DNA samples from the African Yoruba (YRI) people (n=24) and from European CEPH families (CEU) (n=23) were used for resequencing conducted through a grant obtained by the NHLBI Genotyping and Resequencing Service. In the same HapMap samples, mRNA expression analysis was performed using the Affymetrix GeneChip Array in lymphoblastoid cell lines (LCLs). This exploratory analysis used a linear regression in PLINK to select common SNPs (minor allele frequency > 5%) significantly associated with mRNA expression in each population.

We identified a total of 3.555 variants, of which 634 were not previously available in dbSNP build 128. Overall, 485 SNPs showed significant associations with mRNA expression in the CEU or YRI cell lines. Only PIK3C2B, PIK3R5, and PRKCE had SNPs associated with expression in both CEU and YRI.

We discovered novel functional SNPs in the VEGF-pathway genes that are associated with the variation of gene expression in HapMap LCLs. This work provides fundamental information to select SNPs for further testing in molecular and clinical genetic studies of angiogenesis inhibitors and cancer risk in patients of different ethnicities.

Rapid Functional Dissection of Genetic Networks Via Tissue-Specific Transduction and RNAi in Mouse Embryos

Geulah Livshits, BS, Slobodan Beronja, PhD, Scott Williams, PhD, and Elaine Fuchs, Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, New York

Using ultrasound–guided in utero infections of fluorescently-traceable lentiviruses carrying RNAi or Cre-recombinase into mouse embryos, we have obtained highly efficient, non-invasive, selective transduction of surface epithelium. Progenitors stably incorporate and propagate the desired genetic alterations. Importantly, we achieve epidermal-specific infection using small generic promoters of existing lentiviral shRNA libraries, thus enabling rapid assessment of gene function and complex genetic interactions in skin morphogenesis and disease in vivo. Skin is a classic model for tumorigenesis, and we adapt this technology to devise a new quantitative method to ascertain whether a gene confers a growth advantage or disadvantage. Using α1-catenin as a paradigm, we uncover new insights into its role as a widely expressed tumor suppressor and reveal physiological interactions between Ctnna1 and Hras1-Mapk3 and Trp53 pathways in regulating skin proliferation and apoptosis. Our studies illustrate the strategy and its broad applicability for investigations of tissue morphogenesis, lineage specification and cancers.

Genetics and Epigenetics of Colon Cancer

Manuel Perucho, PhD, Sanford-Burnham Medical Research Institute, La Jolla, California and Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Barcelona

Microsatellite instability (MSI) generated by DNA mismatch repair (MMR) deficiency is a critical determinant of tumor cell fate driving colon cancer through a mutator phenotype pathway. Silencing of hMLH1 MMR gene in MSI positive tumors, illustrates the epigenetics-genetics sequence in cancer: epigenetic alterations precede and determine the occurrence of genetic alterations driving gastrointestinal tumorigenesis. However, the mutator phenotype is dominant over the gradual accumulation of DNA hypermethylation in determining the genotypic features that govern the phenotypic peculiarities of colon cancer of the mutator pathway.

Analysis of the same gastrointestinal cancers provided another example of the epigenetics-genetics sequence underlying colon cancer development: there was a gradual acumulation with aging of demethylation alterations as revealed by the increased amount of DNA hypomethylation in tumors from older gastrointestinal cancer patients. In contrast, genetic alterations appeared age-independent, but correlated with the extent of DNA demethylation. Thus, we concluded that DNA demethylation precedes diplody loss in a significant subset of gastrointestinal cancers. We propose that progressive demethylation occurs during aging by accumulation of errors of methylation replication in gastrointestinal stem cells. Once this methylation "wear and tear" affects some genomic sites, the risk of errors in chromosome segregation increases, leading to genomic damage and eventually to cancer development.

FRIDAY, MAY 21, 2010

Speaker abstracts are listed in order of presentation. A gap in the sequence of abstracts, indicates that an abstract was not received at the time of this posting.

Session V: Targeted Therapies I

Synthetic Lethal Approaches to Cancer Therapy

Alan Ashworth, PhD, The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, Fulham Road, London, UK

A critical link exists between genomic instability and cancer development. This instability can manifest as small changes at the nucleotide level or as gross chromosomal alterations. Mutations in the genes that encode DNA damage response proteins are responsible for a variety of genomic instability syndromes including Hereditary Non-Polyposis Colorectal Carcinoma, Bloom syndrome, Ataxia-telangiectasia, BRCA1 and BRCA2 mutated breast and ovarian cancers and Fanconi anaemia. Similarly epigenetic silencing of genes associated with the maintenance of genomic stability have also been implicated in the pathogenesis of cancer. Here, I discuss how different tumours may be classified not only by tumour site but also by the type of underlying genetic instability. This type of classification may assist in the optimization of treatment regimens as well as informing the development of new therapeutic approaches in particular based on “synthetic lethality”.

Identifying the Mechanism of Synergy of Drug Combinations Using A Data Driven Network Reconstruction Approach

Zeynep H. Gümüş1,2, Fernando Siso-Nada, Ada Gjyrezi3, Paul McDonagh4, Iya G. Khalil, PhD4, Paraskevi Giannakakou3 and Harel Weinstein1,2 . 1Department of Physiology and Biophysics; 2The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine; 3Division of Hematology and Medical Oncology,Weill Cornell Medical College; 4Gene Network Sciences, Inc.

We present an integrated experimental and computational approach designed to identify the key cellular components that either contribute to or drive therapeutic synergy of drug combinations with anticancer activity in order to develop better biomarkers and personalized therapeutic strategies. The approach includes (i) quantification of drug synergy in high throughput transcriptome experiments, (ii) data-driven reverse engineering and forward simulation technology to develop an in silico model predictive of drug synergy and to generate hypotheses and (iii) utilization of databases of interaction and functional information to create initial validation, followed by (iv) experimental validation. The goal is to develop an integrated framework that aids in understanding the mechanistic details of drug synergy to design better combination drugs that show efficacy in the appropriate tumor subtypes. We illustrate this approach with an application to the analysis of transcriptome changes in cells exposed to a synergistic anticancer drug combination: farnesyl transferase inhibitors (FTIs) and taxanes.

Primary Trastuzumab Resistance—More than Just Proliferation?

Jason A. Wilken, PhD, Kristy T. Webster, B.S., and Nita J. Maihle, Ph.D., Yale University, New Haven, Connecticut

Trastuzumab (Herceptin) is the first FDA-approved therapeutic that targets a HER-family receptor tyrosine kinase (HER2/ErbB2/neu). Although trastuzumab has proven to be effective in the treatment of HER2-positive breast cancer, a substantial proportion of patients will not respond to trastuzumab-based regimens (primary resistance), and those who do respond will often lose clinical benefit (secondary resistance). While a number of studies have examined the mechanisms of secondary trastuzumab resistance, few have examined the phenomenon of primary trastuzumab resistance. Some potential mechanisms underpinning trastuzumab resistance are common to primary and secondary resistance, while others appear distinct to primary models of trastuzumab resistance. Our studies, along with the results of others demonstrate that trastuzumab induces subtle changes in the phenotype of neoplastic cells (i.e., breast and ovarian), including changes in HER receptor gene expression, even when these tumor cells are not growth inhibited by trastuzumab. These phenotypic changes include inhibition of metastatic dissemination, reliance on alternative growth and survival signaling pathways, and de novo sensitization of tumor cells to other therapeutic regimens. Our studies, in addition to recent reports by others, demonstrate that most previous studies on trastuzumab resistance have relied on in vitro models that are not representative of a chemonaive, HER2-positive carcinoma. Together, these observations suggest that the traditional definition of drug “resistance” in the context of biologically-targeted therapeutics such as trastuzumab needs to be reconsidered.

These studies were supported by the NIH and by Susan G. Komen for the Cure.

Technology Workshop

Technology Platforms Enabling Both Discovery and Translational Research in Cancer Biology

Brian A. Pollok, PhD, CSO, Life Technologies Corporation

Advanced research into the molecular complexities underlying the initiation, establishment, and metastasis for the many types of human cancers requires an ever-evolving suite of technologies enabling more sensitive and comprehensive analysis of tumor cells. The presentations in this workshop will describe the application of new technologies aimed probing the characteristics of human cancer at the genomic (base sequence and epigenetic marks), transcriptomic, proteomic, and cellular levels. Integration of these technologies is still challenging; a group discussion of how best to utilize technology to develop a more holistic description of human cancer will be considered.

Circulating Tumor Cells

Klaus Pantel, MD, PhD, Institute of Tumor Biology, University Medical Center Hamburg Eppendorf, Hamburg, Germany

Early spread of tumor cells is usually undetected by current imaging technologies. Thus, various methods have been developed to detect and characterize single circulating tumor cells (CTC) in the peripheral blood and disseminated tumor cells in the bone marrow of cancer patients. Interestingly, the bone marrow is a common homing organ and putative reservoir for disseminated cancer cells derived from various organ sites including breast, prostate, lung and colon. Peripheral blood analyses, however, are more convenient for patients than invasive bone marrow sampling and many research groups are currently focusing on the clinical utility of CTC for assessment of prognosis and monitoring of systemic therapy. Encouraging data support the assumption that the presence of CTC is correlated to an unfavorable clinical outcome, and monitoring CTC during and after systemic adjuvant therapy might provide unique information on the efficacy of new anti-cancer drugs. Moreover, molecular analysis of CTC provide new insights into the selection of tumor cells and resistance mechanisms in cancer patients undergoing systemic therapies with important implications for the future clinical management.

Surgery with Molecular Navigation - Development of Fluorescently Labeled Biological Probes to Detect Tumor Margins, Atherosclerosis and Peripheral Nerves

Quyen T. Nguyen, MD, PhD,1 , Mike Whitney2, Emilia S. Olson3,4, Todd A. Aguilera3,4, Tao Jiang2,3,Miriam Scadeng5, Leslie Ellies6, Roger Y. Tsien2,3 Departments of 1Otolaryngology, 3Pharmacology, 5Radiology, and 6Pathology, 2Howard Hughes Medical Institute and 4Medical Scientist Training Program,University of California at San Diego, La Jolla, CA

Two major goals of surgery are the accurate identification of the margin between diseased and normal tissue and the preservation of peripheral nerves. We describe the use of novel peptide-based approaches to assist the operating surgeon accomplish these two goals. Two common types of surgery where the margin between diseased and normal tissue is important are cancer surgery and surgery for atherosclerotic disease. We have recently developed a novel strategy to visualize diseased tissue using activatable cell penetrating peptides (ACPP). ACPPs are fluorescently labeled polycationic peptides coupled via a cleavable linker to a neutralizing polyanionic peptide. Upon exposure to proteases characteristic of diseased tissue such as gelatinases or thrombin, the linker is cleaved, dissociating the inhibitory peptide and allowing the activated polycationic peptide to bind the diseased tissue. We show increased ACPP uptake by tumor tissue compared to normal tissue, and increased ACPP uptake by atherosclerotic plaques compared to normal vessels in animal models. In animal models of cancer, quantitative PCR for human Alu sequences show reduced residual tumor cells following surgery performed with ACPP guidance. Furthermore, there is improved tumor-free survival in animals following ACPP-guided tumor excision compared to excision of tumor using standard surgical technique.

Preservation of peripheral nerves is one of the most important goals of any surgical procedure as accidental transection of peripheral nerves lead to significant morbidity for patients. Typically, peripheral nerves are identified by their relatively constant relationship to nearby structures as well as by their typical appearance of being elongated whitish, glistening structures. However, in many instances such as tumor involvement or infection, identification of peripheral nerves using these criteria can be difficult. We describe several novel peptides that have increased affinity to peripheral nerves. We show improved ease of nerve identification and dissection following systemic administration of fluorescently labeled peptides.

Next-Generation Sequencing Technologies

John D. McPherson, PhD, Ontario Institute for Cancer Research, Toronto, Ontario Canada

Nucleotide sequencing has undergone a revolution in the past five years. Capillary electrophoresis-based sequencers used for the Human Genome Project have given way to next-generation sequencing (NGS) platforms for large-scale and comprehensive genome analyses. These new platforms vary in their capacity and read length from approximately one million ~500 base reads to hundreds of millions of shorter reads (50-125 bases). Ten-fold or greater human genome coverage can now be achieved in a single sequencer run on some platforms in a week or less. Large-scale sequencing of single template molecules is now possible as well. Most, if not all, genomic analyses have been adapted to NGS platforms. Analysis of data generated is becoming a bottleneck as unprecedented amounts of data are generated in a short period of time.

The NGS arena continues to evolve with current platforms improving rapidly and new instruments debuting in 2010. The cost per base is dropping quickly making large-scale targeted gene sequencing or whole genome sequencing in clinical applications possible. Programs are emerging for frontline sequencing of important genes in cancer therapy providing the potential for individualized therapies.

An overview of current NGS technologies, applications and future directions will be presented.

Breast Cancer Genomics: Normal Tissue and Cancer Markers

Michèl Schummer, PhD1, J. David Beatty, MD1,2, Scott Karlan, MD3, Beth Karlan, MD3, Nicole Urban, ScD1. 1Fred Hutchinson Cancer Research Center, 2Swedish Cancer Institute, Seattle, Washington, USA, 3Cedars Sinai Medical Center, Los Angeles, California, USA

Breast cancer research has largely focused on cancer tissue or cell lines. Studies of cancer in the context of normal breast tissue, both adjacent to the cancer and from healthy individuals are scarce. The latter however are crucial for the discovery of biomarkers for early detection of breast cancer. We therefore collected two sets of well-characterized tissues: one from breast cancer patients with favorable vs. poor outcome (cancer and adjacent normal tissue) and the other from healthy women undergoing reduction mammaplasty. Potential target genes were selected from mining publicly available expression data (identifying 134 genes for quantitative PCR) and augmented by genes from a commercial PCR array.

Transcript expression analysis identified over 46 genes that discriminate between the cancer and healthy control tissues. Several of these distinguish between cancers with poor and good outcome. Surprisingly, the vast majority of them are expressed equally in poor-outcome cancers and healthy controls, indicating that molecular profiles might not be able to distinguish aggressive cancer from healthy breast tissue. But they might hold value for prognosis. Only few genes had elevated expression in poor-outcome cancers and these warrant further investigation as markers for early detection, particularly in blood.

A second finding was that 20% of the histologically normal breast tissue removed from a distant site in a breast with cancer displayed a cancer-like expression profile, while 80% were genetically similar to the reduction mammaplasty control group. This finding suggests that some regions of histologically normal breast tissue are predisposed to becoming malignant and that normal-appearing tissue with malignant signature might warrant treatment to prevent new primary tumors.

Science Alliance Workshop – Writing for Scientific Publication

Publishing is critical to the scientific profession yet training and guidance on the topic is very limited. Brooke Grindlinger, PhD, former Science Editor at the Journal of Clinical Investigation, will cover what makes a good paper, discuss strategies for selecting the appropriate journal, and provide an overview of the review process and how to navigate resubmissions.

Session VI: Targeted Therapies II

Inhibition of the Platelet-Derived Growth Factor Receptor Alpha (PDGFRα) Signaling Pathway with the Human Monoclonal Antibody MEDI-575 in Preclinical Mouse Models of NSCLC

Raffaele Baffa, MD, Kevin Schifferli, Margarita Camara, Cui Chen, Leslie Wetzel, Meina Liang, Steve Coats, Bahija Jallal, Theresa LaVallee, Yong Chang, and Philipp Steiner, MedImmune LLC, Gaithersburg, MD and MedImmune LLC, Hayward, CA

The PDGFRα pathway plays an important role in the regulation of cellular proliferation, survival and migration. Activation of PDGFRα or its ligands is involved in the pathogenesis of human cancers which leads to stimulation of the signaling pathway in tumor cells but also acts as a mediator of stromal support for cancer cell growth. The clinical benefits of small molecule drugs which target multiple receptor tyrosine kinases have validated PDGFRα as a target for therapeutic intervention in cancer patients. MEDI-575 is a novel human IgG2 monoclonal antibody that selectively targets human PDGFRα. Treatment of tumor cells with MEDI-575 significantly inhibited PDGFRα kinase activity induced by PDGF ligands but had no measurable activity against PDGFRα. MEDI-575 is currently in a phase I clinical trial for cancer patients with solid tumors including NSCLC. While reports on the frequency of tumoral expression of PDGFRα in NSCLC are variable, activated PDGFRα is frequently detected on the surrounding tumor stroma. To test the consequences of inhibition of PDGFRα by MEDI-575 in tumor or stromal cells in vivo, two NSCLC xenograft mouse models were evaluated that depend on PDGFRα signaling: i) PDGFRα-positive H1703 cells grown in athymic nu/nu mice and ii) PDGFRα-negative Calu-6 cells grown in human PDGFRα Knock-In/Knock-Out (KIKO) SCID mice. The KIKO mouse model was required to evaluate whether inhibition of PDGFRα on the tumor stroma alone would result in antitumor activity because MEDI-575 did not cross-react with mouse PDGFRα. In the H1703 nu/nu system, MEDI-575 caused tumor stasis at 10 mg/kg and tumor regressions at 20 mg/kg when dosed twice per week which correlated with a decrease in PDGFRα kinase activity. In the Calu-6 KIKO model, 10 mg/kg and 30 mg/kg of MEDI-575 both caused ~50% tumor growth inhibition which was accompanied by a decrease in stromal phosphorylated PDGFRα. To determine the PK/PD relationship of MEDI-575, the concentration of circulating drug was determined in mouse serum. Approximately 300 ug/ml of MEDI-575 was required for inhibition of pPDGFRα resulting in tumor stasis in the H1703 model and ~200 ug/ml of MEDI-575 in the Calu-6 KIKO model. Furthermore, MEDI-575 was equally active at 20 mg/kg total dose whether dosed on a once weekly schedule or a two times per week schedule in the H1703 model. Both dosing regimens produced similar steady-state drug levels over seven days. In summary, our analysis of anti-tumor activity and PK/PD relationship of the anti-PDGFRα antibody MEDI-575 indicated that blockade of autocrine and paracrine PDGFRα signaling in two mouse models of lung cancer supports further clinical evaluation of MEDI-575 in patients with NSCLC.

A Novel Inhibitor of Fatty Acid Synthase Shows Activity Against Breast Cancer Xenografts and Overcomes Resistance to Anti-HER2 Drugs

Puig T, PhD1, Urruticoechea A2, Aguilar H2, Oliveras G1, Turrado C3, Cufí S1, Benhamu B3 Ortega-Gutierrez S3, López-Rodríguez ML3, and Colomer R4 1Institut Català d’Oncologia de Girona - Institut d’Investigació Biomèdica de Girona, Girona, Spain; 2Institut Català d’Oncologia - Institut d’Investigació Biomèdica de Bellvitge, Barcelona, Spain; 3Química Orgánica I, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain; 4Centro Oncológico MD Anderson España, Madrid, Spain

The majority of HER2 positive advanced breast cancer patients develop resistance to trastuzumab-based therapies within the first year of treatment. We have previously shown that inhibition of Fatty Acid Synthase (FASN) activity, an enzyme overexpressed and hiperactivated in breast carcinomas, leads to apoptosis in breast cancer cells and that this effect is tightly linked to HER2 signalling pathways. Here we show the anticancer effect of G28UCM, the lead-compound of the first generation of a novel family of synthetic FASN inhibitors, both in vitro and in an animal model of FASN+/HER2+ breast cancer xenografts.

We analysed the cellular and molecular effects of G28UCM either alone or in combination with anti-HER drugs (lapatinib, trastuzumab, erlotinib, and gefitinib) in a HER2 dependent breast cancer model. We also used trastuzumab- and lapatinib-resistant AU565 cells to evaluate the effect of G28UCM. We further characterized the anti-cancer activity and pharmacodynamic markers in an athymic mice model of human breast cancer xenograft.

G28UCM shows marked synergic antitumoral activity combined with anti-HER drugs lapatinib, erlotinib and gefitinib. Response to co-exposure of G28UCM plus growth-signal inhibitors was significantly correlated with induction of apoptosis [cleavage of poly(ADP-ribose) polymerase (PARP)] and with the changes in the activation of HER2, MAPK and AKT, as markers of drug activity. Importantly, resistant cells displayed increased sensitivity to G28UCM compared to FASN inhibitors EGCG and C75. In the FASN+/HER2+ positive breast cancer animal model study no weight loss, cardiac or other relevant toxicity were observed after long term exposure to G28UCM. A group of G28UCM-treated mice showed tumour regression compared to control, and importantly, those tumors that responded showed inhibition of FASN activity, cleavage of PARP and decreased pHER2, pAKT and pMAPK activity.

The novel FASN inhibitor G28UCM shows antitumor activity with a favourable toxicity profile. Moreover, G28UCM antitumour activity remains unchanged in trastuzumab- and lapatinib-resistant cells. G28UCM warrants further preclinical development HER2 positive breast cancer, and in breast carcinomas that have progressed on trastuzumab and lapatinib.

The Discovery and Application of Cancer Therapeutics in the Genomic Era

William R. Sellers, MD. VP/Global Head of Oncology, Novartis Institutes For BioMedical Research

The application of genomic technologies in drug development spans the breadth of discovery activities from target validation to the identification of pharmacodynamic biomarkers to the enrichment and/or selection of patients for a specific therapeutic. In the past decade, expression profiling was the “omic” technology of interest and was widely deployed in the area of target discovery and for developing prognostic markers. In many ways expression based analyses are or were still in their infancy in particular with respect to the ability to functionally interrogate biological systems with chemical entities. In the past 2-3 years however, novel analytical methods and a more robust functional annotation of expression data have allowed expression-based discoveries to emerge. More importantly, however, the ramp-up in projects designed to systematically elucidate the molecular genetics of cancer allows us to envision a more rapid transition from empirically-derived therapeutics to those attacking the fundamental pathogenesis of cancer. Here, we remain limited by the lack of annotated, high-throughput pre-clinical models systems in which therapeutic hypotheses can be robustly validated prior to embarking on clinical development.

To begin closing this translational gap, we have embarked on a collaborative Cell Line Encyclopedia project (NIBR, GNF and The Broad Institute) that aims to comprehensively collect and molecularly annotate 1000 cancer cell lines. Such cell lines will be fully characterized for expression data, copy-number data and large-scale DNA sequencing data that will be made publicly available. In addition, robotic systems have been constructed allowing high-throughput automated compound profiling of large-scale cell line collections. A second-generation set of translational models is being generated in parallel using primary human tumor implantation into mice following similar principles. These translational systems will be discussed along with their impact on the therapeutic development of IAP and PI3K inhibitors.

Validation of MEK as a Target for the Treatment of Pancreatic Cancer

Judith Sebolt-Leopold, PhD, Paul Keller, M.S., Shyam Nyati, PhD, Terence Williams, MD, PhD, Alnawaz Rehemtulla, PhD, University of Michigan, Ann Arbor, Michigan

The RAS-MAP kinase signaling cascade is a key pathway known to be activated in the vast majority of human pancreatic cancers. Among the multiple protein players critical to its biological output, the downstream kinase MEK has emerged as an attractive candidate for pharmacologic inhibition. We hypothesize that MEK inhibitors will offer clear therapeutic benefit when integrated into currently adopted chemo- and radiotherapy regimens for the treatment of pancreatic cancer.

Preclinical studies have been carried out in pancreatic xenografts showing that MEK inhibition enhances the efficacy of gemcitabine. We have also demonstrated that MEK inhibition sensitizes pancreatic tumor cells to radiation treatment. We are now shifting out attention to the design and development of preclinical model systems that will prove more clinically relevant than traditional high passage xenografts. By integrating novel mouse models with imaging readouts of drug-target interaction, treatment regimens can be optimized to maximize the sensitivity of pancreatic tumor cells to MEK inhibition. In so doing, we hope to define a MEK inhibitor-based combination and biomarker strategy amenable to rapid clinical translation in pancreatic cancer patients.

Immune Checkpoint Blockade in Cancer Therapy

James P. Allison, PhD, Ludwig Center for Cancer Immunotherapy, Howard Hughes Institute for Cancer Immunotherapy, Memorial Sloan-Kettering Cancer Center, New York, New York

It has become apparent that the effectiveness of active immunologic strategies for cancer therapy can be limited by cell intrinsic and extrinsic checkpoints that limit immune responses in order to maximize target destruction and minimize harm to normal tissues. The prototype of cell intrinsic “checkpoints” whose blockade enhances anti-tumor responses is CTLA-4 (Cytotoxic T Lymphocyte Antigen – 4), which we have extensively studied in animal models and shown to be quite effective in achieving complete tumor eradication and long lasting tumor immunity. Over 4,000 patients have been treated with an antibody to human CTLA-4 (Ipilimumab, Medarex and Bristol-Meyers Squibb). Significant responses, including complete remissions, have been observed in about 15% of metastatic melanoma patients, with overall effects on survival in about 40%. This has led to considerable effort to identify biomarkers that would be useful in determining the impact of CTLA-4 blockade on immune responses in order to identify changes that correlate with clinical responses, as well as to inform combinatorial strategies that might enhance the effectiveness of Ipilimumab. I will discuss conducted on patients treated with Ipilimumab.