fast facts: johns hopkins oncology portfolio

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JOHNS HOPKINS ONCOLOGY PORTFOLIO Fast Facts JOHNS HOPKINS TECHNOLOGY TRANSFER AACR ANNUAL MEETING 2013

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Johns Hopkins available oncology technologies and inventors.

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Page 1: Fast Facts: Johns Hopkins Oncology Portfolio

Johns hopkins oncology portfolio

fast facts

Johns hopkins Technology Transfer aacr annual MeeTing 2013

Page 2: Fast Facts: Johns Hopkins Oncology Portfolio

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personalized Medicine k.o.l.Methods and Biomarkers for the Detection and Diagnosis of Pancreatic Neuroendrocrine Tumors and Pancreatic Cysts Biomarkers for the Detection and Diagnosis of Medulloblastoma and Brain Cancer

sTephen Baylin, M.d.Virginia and D.K. Ludwig Professor for Cancer ResearchProfessor of Oncology and MedicineDeputy Director, Sidney Kimmel Comprehensive Cancer Center

ivan Borrello, M.d. Associate Professor of OncologyMedical Director of the Johns Hopkins Cell Therapy Lab

luis alBerTo diaz, Jr., M.d.Associate Professor of OncologyDirector of Translational Medicine Ludwig Center for Cancer Genetics and Therapeutics

chrisTine ann iacoBuzio-donahue, M.d., ph.d. Professor of Pathology, Oncology, and SurgerySidney Kimmel Comprehensive Cancer Center

andrew feinBerg, M.d., M.p.h.King Fahd Professor of Medicine, Oncology, Molecular Biology & GeneticsChief, Division of Molecular Medicine; Director, Center for EpigeneticsProfessor of Biostatistics

isaac kindeM.D. Ph.D. Candidate The Johns Hopkins University School of Medicine

sTeven leach, M.d.Paul K. Neumann Professor in Pancreatic CancerProfessor of Surgery, Oncology, and Cell BiologyVice Chair for Academic Affairs, Department of Surgery

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Page 3: Fast Facts: Johns Hopkins Oncology Portfolio

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williaM g. nelson, M.d., ph.d.Director, Sidney Kimmel Comprehensive Cancer Center

Ben ho park, M.d. ph.d.Associate Professor of OncologySidney Kimmel Comprehensive Cancer Center

venu raMan, ph.d. Associate Professor of Radiology and Oncology The Johns Hopkins University School of Medicine

saraswaTi sukuMar, ph.d. Barbara B. Rubenstein Professorship in Oncology ; Professor of Pathology; Codirector of the Breast Cancer Program Sidney Kimmel Comprehensive Cancer Center

suzanne l. Topalian, M.d.Professor of Surgery and Oncology Director of the Melanoma Program at the Sidney Kimmel Comprehensive Cancer Center

vicTor velculescu, M.d., ph.d.Professor of OncologyDirector of Cancer GeneticsLudwig Center for Cancer Genetics and Therapeutics

elias zaMBidis, M.d., ph.d.Assistant Professor of Oncology and Pediatrics Sidney Kimmel Comprehensive Cancer CenterInstitute for Cell Engineering

Johns hopkins aacr presenTaTionsAll Johns Hopkins inventor-led AACR presentations and sessions.

presenTaTion aBsTracTsAbstracts for all Johns Hopkins inventor-led AACR sessions.

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Bert Vogelstein, M.D.Clayton Professor of Oncology and PathologyInvestigator, Howard Hughes Medical InstituteDirector, Ludwig Center for Cancer Genetics and TherapeuticsSidney Kimmel Comprehensive Cancer Center

Research InterestsDr. Vogelstein’s laboratory has identified a series of genetic alterations which, in concert, convert a normal epithelial cell to a malignant one. These genetic alterations affect a specific subset of oncogenes and tumor suppressor genes. The goals of Dr. Vogelstein’s current research include the following:

• Identification of other genes which, when mutated, contribute to human tumorigenesis.

• Delineation of the pathways through which these genes act.• Development of targeted therapies based on this knowledge.• Development of new diagnostic approaches based on the genes

responsible for neoplasia.

pErsonAliZED MEDicinE, k.o.l.

Kenneth Kinzler, Ph.D.Professor of OncologyDirector, Ludwig Center for Cancer Genetics and TherapeuticsSidney Kimmel Comprehensive Cancer Center

Research InterestsDr. Kinzler’s laboratory has focused on the genetics of human cancer. They have identified a variety of genetic mutations that underlie cancer, including mutations of the APC pathway that appear to initiate the majority of colorectal cancers and IDH1/2 mutations that underlying many gliomas. In addition, they have developed a variety of powerful tools for analysis of expression and genetic alterations in cancer. Most recently, they have pioneered integrated whole genome analyses of human cancers through expression, copy number, and mutational analyses of all the coding genes in several human cancer types including colorectal, breast, pancreatic and brain. The identification of genetic differences between normal and tumor tissues provide new therapeutic targets, new opportunities for the early diagnosis of cancer, and important insights into the neoplastic process.

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Technology PortfoliosMethods and Biomarkers for the Detection and Diagnosis of pancreatic neuroendrocrine tumors and pancreatic cysts

• C11331: Methods and Biomarkers for the Detection of Pancreatic Neuroendrocrine Tumors

• C11545: Novel Molecular Diagnostic for Pancreatic Cystic Lesions• C11616: Digital Ligation for the Detection of Rare Genetic Variants• C11769: Novel Biomarker for Diagnosis of Pancreatic Cysts

Biomarkers for the Detection and Diagnosis of Medulloblastoma and Brain cancer

• C11276: Biomarkers for the Detection and Diagnosis of Medulloblastoma

• C11618: Novel Molecular Diagnostic for Aggressive Brain Tumors• C11619: Novel Molecular Diagnostic for Brain Cancer

licEnsing AssociAtE

Keith Baker Senior Director, LicensingPhone: 410-516-4563 Email: [email protected]

AAcr prEsEntAtionssun, Apr 7, 8:30 - 9:40 AMAP01. Seventh Annual AACR Team Science Award.

Mon, Apr 8, 1:00 - 5:00 pM2225/6 - The molecular architecture of p85α as determined by SAXS and chemical cross-linking

Nickolas Papadopoulos, Ph.D.Associate Professor of Oncology Director of Translational Genetics, Ludwig Center for Cancer Genetics and Therapeutics Sidney Kimmel Comprehensive Cancer Center

Research InterestsDr. Papadopoulos is working to identify the genetic changes underlying the development of pancreatic neuroendocrine tumors. Dr. Papadopoulos’ current focus is on cancer genomics. He was part of the interdisciplinary team that was first to sequence all of the protein coding genes and determine genetic alterations and construct expression profiles in multiple tumors of four different common human cancers. Recent efforts involved the identification of genetic alterations that drive tumorigenesis using a new generation of sequencing technologies. He has developed an in-house sequencing pipeline based on massively parallel sequencing. He discovered the identification of novel, signature mutations in ovarian clear cell carcinomas and pancreatic neuroendocrine tumors. Dr. Papadopoulos’ most recent efforts focus on the translation of the information derived from genomic studies to diagnostics, especially early detection.

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Stephen Baylin, M.D.Virginia and D.K. Ludwig Professor for Cancer ResearchProfessor of Oncology and MedicineDeputy Director, Sidney Kimmel Comprehensive Cancer Center

BiographyStephen B. Baylin, M.D., is deputy director of The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins and the Virginia and D.K. Ludwig professor of oncology and medicine. He attended Duke University, where he earned his medical degree in 1968 and completed his internship and first year residency in internal medicine. He then worked for two years at the National Heart and Lung Institute of the National Institutes of Health (NIH). In 1971, Dr. Baylin joined the departments of oncology and medicine at The Johns Hopkins University School of Medicine. Dr. Baylin is a member of the Stand Up 2 Cancer Dream Team, Bringing Epigenetic Therapy to the Forefront of Cancer Management.

Research InterestsDr. Baylin’s research has contributed heavily to the concept that epigenetically mediated loss of gene function is a major player in the progression of human cancer. He is attempting to understand the abnormalities of chromatin and methylation assembly that may account for the appearance of epigenetic abnormalities during tumor development and how it mediates the transcriptional repression. In collaboration with the Vogelstein-Kinzler lab, Dr. Baylin has identified that an interaction between DNMTs is required in colon cancer cells to maintain the abnormal promoter methylation and silencing of important tumor suppressor genes. These studies are contributing to a more complete picture of the machinery that mediates aberrant promoter methylation in cancer and to the translational goal of targeting reversal of abnormal gene silencing as a cancer prevention and/or therapy strategy.

Technology Portfolio• C11952: Novel Combination Therapy for Advanced Cancer• C09971: Cell Line Overexpressing DNA Methyltransferase• C09856: A New Tumor Suppressor Gene• C04309: GATA-4/5 as Epigenetic Biomarkers for Lung and

Esophageal Cancers• C04028: Functional Identification of Methylated Cancer

Genes• C03982: Genomic Screen for Epigenetically Silenced Genes

Associated With Colorectal Cancer & Novel Biomarkers for the Detection and Diagnosis of Human Colorectal Cancer

• C03453: Methylated CpG Island Amplification (MCA)

licEnsing AssociAtEAditi MartinLicensing AssociatePhone: 410-516-4566 Email: [email protected]

AAcr prEsEntAtionsMon, Apr 8, 12:45 - 1:15 PMBringing epigenetic therapy to the forefront of cancer management

Tues, Apr 9, 3:05 - 3:20 PM4619 - Epigenetic therapy and sensitization of lung cancer to immunotherapy

Wed, Apr 10, 8:00 AM - 12:00 PM4666/9 - A phase 2 study investigating the safety, efficacy and surrogate biomarkers of response of 5-azacitidine (5-AZA) andentinostat (MS-275) in patients with triple-negative advanced breast cancer

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BiographyIvan Borrello, M.D., is an associate professor of oncology at The Johns Hopkins University School of Medicine and practices at the Sidney Kimmel Comprehensive Cancer Center. His expertise is in bone marrow transplants, hematologic malignancies, immunotherapy, and multiple myeloma. Dr. Borrello earned his medical degree at the Medical College of Virginia School of Medicine. He completed his residency at the University of Chicago and his fellowship at Johns Hopkins Hospital.

Research InterestsDr. Borrello’s clinical interests focus in multiple myeloma and his research interests consist of developing immune-based strategies for the treatment of myeloma. His research focuses on two major aspects: 1) developing novel approaches to adoptive T cell therapy and 2) overcoming mechanisms of immune suppression. The T cell approach focuses on marrow infiltrating lymphocytes (MILs). Dr. Borrello has shown these cells to possess greater anti-tumor efficacy than blood T cells as well as other traits that make them more ideal for therapeutic use. This work led to the development of a clinical infrastructure to grow the cells and the development of clinical trials including a randomized Phase 2 study for high-risk myeloma. Additional applications of MILs have been in the allogeneic transplant setting where his lab has shown that expansion of these cells from the patients (and not the donors) can impart highly specific anti-leukemia benefits with clinical studies to start in the next year. In terms of immunosuppressive approaches, his group made the observation that commonly used PDE5 inhibitors such as Viagra and Cialis can be effectively and safely used to inhibit the immunosuppression generated by myeloid derived suppressor cells (MDSCs). The clinical results from the first study confirm the preclinical observations that these drugs can modulate immune function. Additional clinical trials are currently underway to examine the anti-tumor efficacy of these drugs in multiple myeloma and head and neck cancer.

Technology Portfolio• C04580: PDE Inhibitors in Immunotherapy• C10428: Cellular Therapy for Hematological Malignancies• C12054: A Safer and More Effective T Cell Cancer

Immunotherapy

Ivan Borrello, M.D. Associate Professor of OncologyMedical Director of the Johns Hopkins Cell Therapy Lab

licEnsing AssociAtEPauline CallinanSenior Licensing AssociatePhone: 410-516-5496Email: [email protected]

Page 8: Fast Facts: Johns Hopkins Oncology Portfolio

Luis Alberto Diaz, Jr., M.D.Associate Professor of OncologyDirector of Translational Medicine Ludwig Center for Cancer Genetics and Therapeutics

BiographyLuis Diaz, M.D., is director of translational medicine at the Ludwig Center for Cancer Genetics and Therapeutics at The Johns Hopkins Kimmel Cancer Center. Dr. Diaz is also director of a new clinic for patients with metastatic colorectal cancer. This clinic focuses on the treatment of all aspects related to these patients, including support with pain, nutrition, psycho-social issues, and access to clinical trials and cutting-edge therapies. Dr. Diaz is also the chief medical officer and cofounder of Personal Genome Diagnostics.

Dr. Diaz received his medical degree from the University of Michigan Medical School, and completed his general medicine training at Johns Hopkins followed by a fellowship in medical oncology at The Johns Hopkins Kimmel Cancer Center.

Research InterestsDr. Diaz’s clinical interests include gastrointestinal cancers including colorectal cancer and pancreatic cancer. The focus of Dr. Diaz’s research is two-fold. The first is translating novel and often high-risk therapeutics with unique mechanisms of action from the lab to patients. One example is using live bacteria to target and destroy solid tumors. This approach, termed bacteriolytic therapy, is being testing in clinical trials at Johns Hopkins and the University of Pennsylvania in humans and canines with advanced solid tumors. His second area of research includes a novel test that measures tumor-derived DNA in the bloodstream. The blood test, based on the unique genetic fingerprint contained within the genome of every cancer, can detect the presence of tumor and track its progress. The ultimate goal of this work being early detection of cancer with a simple blood test before it becomes lethal.

Technology Portfolio• C12191: PapGene Test• C11618: Novel Molecular Diagnostic for Aggressive Brain

Tumors• C11545: Novel Molecular Diagnostic for Pancreatic Cystic

Lesions• C11331: Methods and Biomarkers for the Detection of

Pancreatic Neuroendrocrine Tumors• C11229: Enhanced Delivery of Nanoparticles to Tumors• C10690: Methyl-BEAMing: Digital Quantification of DNA

Methylation

licEnsing AssociAtEKeith Baker Senior Director, LicensingPhone: 410-516-4563 Email: [email protected]

AAcr prEsEntAtionsMon, Apr 8, 1:00 - 5:00 pM2205/14 - Integrated next generation sequencing and patient-derived xenografts to personalized cancer treatment

tues, Apr 9, 1:35 - 1:55 pMDetecting resistance to targeted therapies in circulating tumor DNA

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BiographyChristine Ann Iacobuzio-Donahue, M.D., Ph.D., earned a B.S. in biology from Adelphi University, followed by both an M.D. and Ph.D. degree from Boston University School of Medicine. While in Boston, she spent three years investigating how colorectal cancers develop the ability to invade through the colon and spread to other organs (metastasize). Following her education at Boston University, she moved to Baltimore, Maryland where she completed a residency in anatomic pathology at The Johns Hopkins Hospital. While in residency, she spent one additional year of research training with Drs. Scott Kern and Ralph Hruban studying the molecular genetics and gene expression profiles of pancreatic cancer.

Research InterestsDr. Iacobuzio-Donahue’s research focuses on developing new drugs tailored specifically to the unique features of pancreas cancers, and on understanding how pancreas cancers grow and spread to other organs.She has used a technique called “gene expression profiling” to study the genes of pancreas cancers. To date, Dr. Iacobuzio-Donahue’s work has identified more than 100 genes that are specifically “turned on” in the pancreas where a cancer is present. Using this approach, Dr. Iacobuzio-Donahue has identified a specific gene as a potential target for treating pancreatic cancer. Her research has identified that 99 percent of pancreas cancer cells turn on this particular gene. Following this promising breakthrough, Dr. Iacobuzio-Donahue and her collaborators have begun work to create a designer drug that will specifically act against pancreas cancer cells that activate the gene. Once this designer drug is synthesized, the study phase will commence to prove that the drug can kill the cancer cells. The study phase will use actual pancreas cancer cells growing in culture or as xenografted tumors (human pancreas cancers growing in laboratory mice). The preliminary data generated from these studies will form the basis for clinical trials in patients battling pancreatic cancer.

Technology Portfolio• C04482: Loss of Imprinting of IGF2 Alters Intestinal

Maturation and Tumorigenesis

Christine Ann Iacobuzio-Donahue, M.D., Ph.D. Professor of Pathology, Oncology, and SurgerySidney Kimmel Comprehensive Cancer Center

inventor fact sheets9 inventor fact sheets9

licEnsing AssociAtE

Keith Baker Senior Director, LicensingPhone: 410-516-4563 Email: [email protected]

AAcr prEsEntAtionssun, Apr 7, 7:00 - 8:00 AM Meet-the-Expert SessionME47. Considerations for Sequencing Analyses of Cancer Progression and Metastasis

tues, Apr 9, 1:00 - 5:00 pM4006/20 - Smad6 upregulation provides an alternative mechanism for BMP inactivation in SMAD4 wild type pancreatic cancers

tues, Apr 9, 1:00 - 5:00 pM3580/2 - Acetaldehyde and drug hypersensitivities of Fanconi anemia defects: Implications for cancer initiation, prevention, and therapy

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Andrew Feinberg, M.D., M.P.H.King Fahd Professor of Medicine, Oncology, Molecular Biology & GeneticsChief, Division of Molecular Medicine; Director, Center for EpigeneticsProfessor of Biostatistics

BiographyAndrew Feinberg, M.D., M.P.H., studied mathematics and humanities at Yale University in the Directed Studies honors program, and he received his B.A., M.D., and M.P.H. from Johns Hopkins. His honors include election to the American Society for Clinical Investigation, the Association of American Physicians, the Institute of Medicine of the National Academy of Sciences, and the American Academy of Arts and Sciences, as well as membership on the ISI most-cited authors list, a MERIT Award of the National Cancer Institute, a Doctor of Philosophy (Hon. Caus.) from Uppsala University (Sweden), and the President’s Diversity Recognition Award of Johns Hopkins University.

Research InterestsDr. Feinberg studies the epigenetic basis of normal development and disease. Early work involved the discovery of altered DNA methylation in cancer, as well as common epigenetic (methylation and imprinting) variants in the population that may be responsible for a significant population-attributable risk of cancer. This led to a major cancer epigenetics translational study to introduce epigenetic testing for colon cancer risk. His lab is also pioneering genome-scale technology for epigenetics research and is applying this to human disease including cancer. This work led to the discovery of CpG island “shores,” and that aberrant methylation in cancer involves roughly equal gains and losses of DNA methylation at these shores, and involves much the same sequences involved in normal differentiation of widely disparate tissues. He also discovered Large Organized Chromatin K9-modifications, or “LOCKs,” which are tissue-specific regions of lysine modification in histone H3, that may provide a mechanistic basis for epigenetic memory during cell division, as well as aberrant epigenetic programming in cancer. Dr. Feinberg’s lab is also developing new paradigms for the intersection of developmental biology, molecular biology and mathematics. A result of this work is a new model for an epigenetic role in evolution. This model has made specific predictions about the epigenetic basis of cancer that have proven correct experimentally, and may lead to radically new approaches to early diagnosis and therapy.

Technology Portfolio• C11353: Methylation Anti-Profiling a Novel Approach to

Diagnosing Cancer• C11137: Enhancing the Differentiation Potential of Induced

Pluripotent Stem Cells• C10889: Using Stem Cells to Identify Cancer-causing Genes; to

Perform Regenerative Medicine and to Identify New Anti-aging Drug Targets

• C10533: Novel Screen for Hypermethylated Genes as Biomarkers to Diagnose/Prognose Colorectal Cancer

licEnsing AssociAtE

Keith Baker Senior Director, LicensingPhone: 410-516-4563 Email: [email protected]

AAcr prEsEntAtionstues, Apr 9, 1:50 - 2:10 pMWhole genome analysis of DNA methylation in human cancer

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BiographyIsaac Kinde is a M.D. and Ph.D. candidate at The Johns Hopkins University School of Medicine. He is working under the expert guidance of Luis Diaz, M.D.; Nickolas Papadopoulos, Ph.D.; Kenneth Kinzler, Ph.D.; and Bert Vogelstein, M.D. In 2012 he was named one of Forbes’ Magazine’s 30 Under 30 Rising Stars in Healthcare. Recently his work with the Luis Diaz, M.D. on the PapGene Test (JHU Invention C12191) was referenced in the New York Times and the Baltimore Sun and he was interviewed by USA Today for a featured story. Mr. Kinde will also keynote the 2013 BIO International Convention in Chicago, IL.

Research InterestsMr. Kinde is developing techniques to improve the accuracy of DNA sequencing technology and demonstrating that it might be used to detect cancers arising from the colon, pancreas, and ovaries in a simple, noninvasive manner. Already, several patents have been applied for and he’s been published in Science Translational Medicine, Nature, and other journals.

Technology Portfolio• C12191: PapGene Test• C11953: Rapid Genome Sequencing Method for Prenatal

Diagnosis• C11475: Novel Diagnostic Tool for Rare Disorders

Isaac KindeM.D. Ph.D. Candidate The Johns Hopkins University School of Medicine

licEnsing AssociAtE

Keith Baker Senior Director, LicensingPhone: 410-516-4563 Email: [email protected]

Page 12: Fast Facts: Johns Hopkins Oncology Portfolio

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Steven D. Leach, M.D.Paul K. Neumann Professor in Pancreatic CancerProfessor of Surgery, Oncology, and Cell BiologyVice Chair for Academic Affairs, Department of Surgery

BiologySteven D. Leach, M.D., is the Paul K. Neumann Professor in Pancreatic Cancer, and professor of surgery, oncology and cell biology. He is also vice chair for academic affairs in the department of surgery, and a member of the McKusick-Nathans Institute of Genetic Medicine. Dr. Leach graduated from Princeton University in 1982 and received his M.D. degree from Emory University in 1986. He completed his residency in general surgery and a two-year research fellowship in pancreatic cell biology at Yale University. This was followed by a surgical oncology fellowship at the University of Texas M.D. Anderson Cancer Center. Dr. Leach joined the Vanderbilt University faculty as an assistant professor of surgery in 1995, and became an associate professor of surgery and cell biology in 1999. He was recruited to join the faculty at Johns Hopkins in July 2000 as the first Paul K. Neumann Professor.

Research InterestsDr. Leach’s lab studies epithelial progenitor cells in developing, regenerating and neoplastic pancreas, using both mouse and zebrafish model systems. By characterizing how pancreatic progenitor cells are regulated, he hopes to provide for their eventual therapeutic manipulation, both as probable “cells of origin” for pancreatic cancer, and also as a resource for cell replacement therapy in diabetes. Current studies in the lab include the following:

• Identifying and characterizing a novel progenitor population in adult mouse and zebrafish pancreas

• Utilizing Cre-mediated cassette exchange to interrogate transcription factor function in developing zebrafish pancreas

• Using single cell transcriptional profiling to reveal previously unrecognized heterogeneity among pancreatic epithelial cell types

• Identifying novel targets of the pancreas-specific Ptf1 transcriptional complex, using genome-wide screening techniques

• Developing a novel in vitro model of pancreatic intra-epithelial neoplasia (PanIN)

• Further developing zebrafish models of pancreatic cancer, and employing these models to functionally annotate the human pancreatic cancer genome

Technology Portfolio• C11508: Novel Renewable Source of Beta Cells for Diabetes

Treatment and Research

licEnsing AssociAtE

Rachel CassidyPortfolio DirectorPhone: 410-516-4562Email: [email protected]

AAcr prEsEntAtionssat, Apr 6, 1:00 - 3:00 pMED04. Change for the Worse: Premalignant Metaplasia in the Stomach, Esophagus, and Pancreas

tues, Apr 9 8:00 AM -12:00 pM2867/9 - TH17 Cells Promote Early Pancreatic Tumorigenesis

Wed, Apr 10 8:00 AM - 12:00 pM5008/6 - A Morphologically Distinct Cancer Initiating Cell in Human and Murine PanIN and Pancreatic Cancer

Page 13: Fast Facts: Johns Hopkins Oncology Portfolio

BiologyWilliam G. Nelson, M.D., Ph.D., was named the Marion I. Knott Director and professor of oncology and director of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins in December 2008. Dr. Nelson received a bachelor’s degree in chemistry from Yale University in 1980. In 1987, Dr. Nelson completed his medical degree and Ph.D. training at The Johns Hopkins University School of Medicine, and completed an internal medicine residency training and medical oncology fellowship at The Johns Hopkins Hospital. Dr. Nelson is a professor of oncology, urology, pharmacology, medicine, pathology, and radiation oncology. He specializes in the treatment and research of prostate cancer.

Research InterestsDr. Nelson and his lab discovered the most common known somatic genome alteration in human prostatic carcinoma cells. The DNA lesion, hypermethylation of deoxycytidine nucleotides in the promoter of a carcinogen-defense enzyme gene, appears to result in inactivation of the gene and a resultant increased vulnerability of prostatic cells to carcinogens. Studies in the laboratory have been directed at characterizing the genomic abnormality further, and at developing methods to restore expression of epigenetically silenced genes and/or to augment expression of other carcinogen-defense enzymes in prostate cells as prostate cancer prevention strategies. The laboratory also studies the role of chronic or recurrent inflammation as a cause of prostate cancer. Genetic studies of familial prostate cancer have identified defects in genes regulating host inflammatory responses to infections. A newly described prostate lesion, proliferative inflammatory atrophy (PIA), appears to be an early prostate cancer precursor. Current experimental approaches feature induction of chronic prostate inflammation in laboratory mice and rats, and monitoring the consequences on the development of PIA and prostate cancer.

Technology Portfolio• C11663: 5hmC: A Novel Marker for Cancer Detection• C11598: Improved Method for Identification of DNA

Methylation and Biomarkers for Prostate Cancer Detection• C04871: COMPARE-MS: A Novel Technique for Rapid,

Sensitive, And Accurate Detection of DNA Methylation• C04328: Antibody to Human DNA Methyltransferase 1• C05053: Small Molecules Epigenetic Inhibitors for Cancer

Prevention and Treatment

William G. Nelson, M.D., Ph.D.Director, Sidney Kimmel Comprehensive Cancer Center

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licEnsing AssociAtE

Aditi MartinLicensing AssociatePhone: 410-516-4566 Email: [email protected]

AAcr prEsEntAtionssun, Apr 7, 1:00 - 5:00 pM312/10 - A mouse model of chronic prostatic inflammation using a human prostate cancer-derived isolate of Propionibacterium acnes

sun, Apr 7, 1:30 - 3:00 pM SS07. SU2C Innovative Research Grants: Cutting-Edge Research With An Eye Towards Translation

Mon, Apr 8, 1:00 - 5:00 PM1773/11 - Nucleotide-resolution genomic breakpoint analysis of TMPRSS2-ERG rearrangements in prostate cancer by target-capture next-generation sequencing

Mon, Apr 8, 3:40 - 4:25 pMDiscussant: The future of preventive medicine: Racial and ethnic considerations

Tues, Apr 9 10:30 AM - 12:30 PM CCOS03. Prostate Cancer Screening: Now and in the Future

Tues, Apr 9, 1:00 - 5:00 pM3614/6 - Are circulating testosterone and PSA levels associated in a nationally representative sample of men without a diagnosis of prostate cancer?

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Ben Ho Park, M.D. Ph.D.Associate Professor of OncologySidney Kimmel Comprehensive Cancer Center

BiographyBen H. Park, M.D., Ph.D., is an associate professor of oncology with a joint appointment in the Whiting School of Engineering. He attended The University of Chicago receiving his B.A. degree in biology in 1989 and The University of Pennsylvania School of Medicine where he received his M.D. and Ph.D. degrees in 1995. Dr. Park trained in internal medicine and hematology/oncology at The Hospital of The University of Pennsylvania prior to coming to Johns Hopkins where he completed a post-doctoral fellowship in cancer genetics in the laboratory of Drs. Kenneth Kinzler and Bert Vogelstein. In 2002, Dr. Park joined the faculty at The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins in the Breast Cancer Research Program.

Research InterestsThe Park Laboratory’s goal is to discover and develop novel means for treating breast cancer. The lab is developing techniques to identify genes involved with hormonal and chemotherapeutic drug resistance, as well as analyzing the genetic effectors of growth and hormone receptor mediated breast carcinogenesis. Using powerful molecular genetic techniques, the lab is identifying genes whose biallelic inactivation leads to clinical drug resistance. The lab hypothesizes that there are genes whose inactivation in a recessive manner can lead to clinically relevant drug resistance. The lab is also trying to understand pathogenic mechanisms of growth/hormone receptor signaling. The continuous exposure of breast tissue to estrogens and other growth factors likely plays a role in the carcinogenic process that transforms a normal breast epithelial cell into a cancer. The lab is trying to elucidate the molecular mechanisms of aberrant receptor signaling that contributes to this process.

Technology Portfolio• C10037: K-ras Knock in Human Breast Non-tumorigenic

Breast Epithelial Cells• C10575: MMSET Knock Out and Knock Down Multiple

Myeloma Cells• C04945: Multiple Inversion Flip Flop (MIFF) Vectors• C04885: Novel Compounds that Target Estrogen Receptor

(ER) Positive. Tamoxifen-resistant Breast Cancers• C11859: A Method to Identify “driver” Functional Mutations

Causing Cancer• C03819: Genetic Disruption of PPAR(delta) Decreases

Tumorigenicity of Human Colon Cancer Cells

licEnsing AssociAtE

Keith Baker Senior Director, LicensingPhone: 410-516-4563 Email: [email protected]

AAcr prEsEntAtionssun, Apr 7, 1:10 - 1:30 pMSY11-01 - Plasma tumor DNA: Changing the paradigm for administering systemic therapies

Wed, Apr 10, 8:00 AM - 12:00 pM5331/14 - A novel function of p21 in inhibition of epithelial-mesenchymal transition through microRNAs

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BiographyVenu Raman, Ph.D., is associate professor of radiology and oncology. Dr. Raman has gained significant recognition in the area of developmental breast cancer biology. His work on deciphering the role of HOXA5 and Twist in breast cancer formation is widely recognized both nationally and internationally. Dr. Raman is an expert developmental and cancer molecular biologist, who has worked in the molecular imaging field for 10 years. Dr. Raman has significant experience in generating preclinical cancer xenograft models as well as developing chemotherapeutic molecules for treating cancers. He has been intimately involved with the JHU ICMIC since its inception, and has played a key role in the integration of molecular biology techniques with imaging.

Technology Portfolio• C11442: Novel and More Effective Treatment for Lung

Cancer• C10955: Molecular Markers of Breast Cancer Stem Cells• C10939: Clinically Relevant Tissue Specific Fluorescent

Metastatic Human Breast Cancer Cell Lines• C10873: Novel Small Molecule to Treat Cancer• C10075: Twist Overexpression Promotes Chromosomal

Instability in the Breast Cancer Cell Line MCF-7• C05061: Novel Breast Cancer Cells That Stably Express a

New Orange-red Fluorescent Protein Provides Facile Tracking of Metastatic Progression In Live Animal Model Systems

Venu Raman, Ph.D.Associate Professor of Radiology and Oncology The Johns Hopkins University School of Medicine

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licEnsing AssociAtE

Rachel CassidyPortfolio DirectorPhone: 410-516-4562Email: [email protected]

AAcr prEsEntAtionssun, Apr 7, 1:00 - 5:00 pM734/23 - Water diffusion decreased in low collagen containing hypoxic regions of breast cancer xenograft

Mon, Apr 8, 1:00 - 5:00 pM1493/21 - The Twist box is required for Twist1-induced prostate cancer metastasis

tues, Apr 9, 1:00 - 5:00 pM3745/1 - Validation of the co-expression of breast cancer stem cell markers with HIF-1α in tumors

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BiographySaraswati Sukumar, Ph.D., is the Barbara B. Rubenstein Professor of Oncology and Professor of Pathology, and a preceptor in the human genetics and pathobiology graduate programs. She serves as the principal investigator of the DOD Center of Excellence, NCI’s Specialized Program of Research Excellence (SPORE) and of the AVON Breast Cancer Foundation. Dr. Sukumar has worked in the breast cancer field since her postdoctoral training at National Cancer Institute. She joined her first faculty position at the Salk Institute in La Jolla, CA in 1989, and then moved to the Johns Hopkins Oncology Center in 1994 as associate professor to assume the position of director of basic research at the newly formed breast cancer research program. She was promoted to professor in 2001. Dr. Sukumar has participated and chaired many grant review committees in the NIH, DOD, and Susan G Komen Foundation (SGKF). She is currently a scientific advisory board member of the SGKF for the Cure. In 2011, she received the BioMaryland LIFE Award.

Research InterestsDr. Sukumar’s laboratory is identifying gene alterations to study the consequences of these changes to the cellular machinery and to translate this knowledge to early detection and novel therapies for breast cancer. The lab has identified a large number of candidate oncogenes and tumor suppressor genes by SAGE and microarray analysis. Among these, they are currently analyzing tight junction proteins, Claudin-3, -4 and 7 and the homeotic genes, HOXA5 and HOXB7. HOXA5 is a potent transcriptional regulator of the progesterone receptor and the p53 gene, and its expression is lost in breast cancer cells. Few HOXA5 minus mice survive; the females have defects in lactation. HOXB7, on the other hand, is over expressed in breast cancer, and induces production of growth factors in cancer cells.

Technology Portfolio• C12014: A Highly Sensitive, Blood Based Test for Methylation

Detection in Cancer• C11625: Markers to Differentiate Between ER Subtypes,

Predict Treatment Outcome and Risk of Reoccurrence in Breast Cancer

• C11436: HOXB7 a Therapeutic Target for Drug Resistant Breast Cancer

• C11063: PIK3CA Mutants Transgenic Mouse• C04936: HEYL, A Novel Oncogene• C04922: HOXB7 Inhibition to Prevent/Overcome Drug

Resistance in Breast Cancer

Saraswati Sukumar, Ph.D. Barbara B. Rubenstein Professorship in Oncology Professor of Pathology Codirector of the Breast Cancer Program at the Sidney Kimmel Comprehensive Cancer Center

licEnsing AssociAtE

Aditi MartinLicensing AssociatePhone: 410-516-4566 Email: [email protected]

AAcr prEsEntAtionstues, Apr 9 8:00 AM - 12:00 pM3123/21 - The Notch pathway inhibits TGF-β signaling in breast cancer through HEYL-mediated crosstalk

tues, Apr 9 8:00 AM - 12:00 pM3122/20 - HMGA1 reprograms breast cancer cells by inducing transcriptional networks involved in an epithelial-mesenchymal transition, stemness, and metastatic progression

tues, Apr 9, 1:00 - 5:00 pM3641/6 - Genome-wide methylation patterns suggest differences in breast cancer biology in American women of African and European ancestry

tues, Apr 9, 1:00 - 5:00 pM3726/12 - Combinations of HDAC inhibitor, chemotherapeutic agent and retinoic acid induce growth arrest, differentiation and tumor regression in preclinical models of breast cancer

Wed, Apr 10, 8:00 AM - 12:00 pM4759/4 - HOXB7 functions as a co-activator of estrogen receptor in the development of tamoxifen resistance

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Suzanne L. Topalian, M.D.Professor of Surgery and Oncology Director of the Melanoma Program at the Sidney Kimmel Comprehensive Cancer Center

inventor fact sheets17 inventor fact sheets17

BiographySuzanne Topalian, M.D., is a surgical oncologist who attended Tufts University School of Medicine and completed her surgical residency at Thomas Jefferson University Hospital in Philadelphia. She spent 21 years in the Surgery Branch of the National Cancer Institute where her work in basic cancer immunology and translational cancer immunotherapy garnered international attention. Dr. Topalian was recruited to Johns Hopkins jointly by the departments of surgery and oncology in 2006, to become the founding director of the melanoma program in the Sidney Kimmel Comprehensive Cancer Center. She seeks to develop immunotherapies for melanoma and other cancers by defining tumor-associated proteins (antigens) and discovering optimal ways to formulate them into vaccines, and by developing methods to enhance anti-tumor immunity with monoclonal antibodies targeting immune-modulating molecules displayed on T lymphocytes. She also serves as the chief science officer of the Melanoma Research Alliance.

Research InterestsDr. Topalian has led the clinical development of immunomodulatory monoclonal antibodies to treat patients with melanoma and other solid tumors. Her findings suggest that patients with treatment-refractory advanced metastatic cancers can respond to the blockade of the T cell co-receptors programmed death-1 (PD-1) and B7-H1. Her laboratory has been responsible for conducting correlative immunological studies in these patients and has characterized the pharmacodynamics of anti-PD-1 and anti-B7-H1. Since animal models show that blockade of PD-1 and B7-H1 synergizes with cancer vaccines, Dr. Topalian has continued to search for optimal tumor antigens, investigating melanoma-associated mitochondrial mutations and recently describing post-translationally modified phosphopeptides as a new cohort of tumor antigens recognized by human CD4+ T cells.

Technology Portfolio• C11952: Novel Combination Therapy for Advanced Cancer• C10691: Novel Peptides as a Melanoma Vaccine

licEnsing AssociAtEPauline CallinanSenior Licensing AssociatePhone: 410-516-5496Email: [email protected]

AAcr prEsEntAtionssat, Apr 6, 10:00 AM - 12:00 pM ED33. Cancer Immunology for the Non-Immunologist - Tutorial

sun, Apr 7, 10:15 - 10:50 AMImmune checkpoint blockade: Unleashing the immune system against cancer

sun, Apr 7, 1:00 - 5:00 pM446/1 - Differential expression of immuno-regulatory genes associated with PD-L1 display: Implications for clinical blockade of the PD-1/PD-L1 pathway in melanoma

tues, Apr 9, 3:05 - 3:20 pM4619 - Epigenetic therapy and sensitization of lung cancer to immunotherapy

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BiographyVictor Velculescu, M.D. Ph.D., is chief scientific officer and cofounder of Personal Genome Diagnostics. Dr. Velculescu is internationally known for his genomic discoveries in human cancer with more than 90 published papers in peer-reviewed journals. Dr. Velculescu and his colleagues were the first to perform genome-wide sequence analyses in human cancer. He is professor of oncology and director of cancer genetics at the Ludwig Center at Johns Hopkins University. Dr. Velculescu has a B.S. from Stanford University, and M.D., Ph.D. degrees and postdoctoral training from Johns Hopkins University.

Research InterestsDr. Velculescu’s laboratory is focused on developing and applying genome-wide approaches for identification of molecular differences between human cancer cells and normal cells. These alterations can exist at the level of the genome, involving mutations of specific genes underlying tumorigenesis, or at the level of the transcriptome, involving differential expression of genes in cancer cells. Using genomic tools Dr. Velculescu’s lab has developed over the past several years, including high throughput sequencing approaches, serial analysis of gene expression (SAGE), and a novel method called Digital Karyotyping, Dr. Velculescu has begun to elucidate the genetic and gene expression differences important in neoplasia. Future work will focus on systematically using these technologies to identify previously undiscovered oncogenes and suppressor genes involved in colorectal cancer. The identification of such genetic alterations will provide insight into the pathogenesis of cancer and open up new possibilities for diagnostic and therapeutic intervention.

Technology Portfolio• C11331: Methods and Biomarkers for the Detection of

Pancreatic Neuroendrocrine Tumors• C11276: Biomarkers for the Detection, and Diagnosis of

Medulloblastoma• C10471: Global Genetic Alterations Analysis & Novel

Diagnostic/Therapeutic Biomarkers for Breast and Colorectal Cancer

• C10199: Methods for Identifying Genetic Signatures in Tumors for Personalized Medicine

• C04724: Mutational Analysis of the Serine-threonine Kinome in Colorectal Cancers

Victor Velculescu, M.D., Ph.D.Professor of OncologyDirector of Cancer GeneticsLudwig Center for Cancer Genetics and Therapeutics

licEnsing AssociAtEKeith Baker Senior Director, LicensingPhone: 410-516-4563 Email: [email protected]

AAcr sEssionMon, Apr 8, 1:00 - 5:00 pM LB-75/8 - Blood-based molecular detection of acquired resistance to anti-EGFR therapies in colorectal cancer patients

sun, Apr 7, 1:00 - 5:00 pM798/5 - Clonal analysis and clinical translation of pancreatic adenocarcinoma genomes

sun, Apr 7, 3:15 - 5:15 pM MS.MCB10.01. Genome-Based Discovery in Human Cancer

Mon, Apr 8, 1:00 - 5:00 pM2205/14 - Integrated next generation sequencing and patient-derived xenografts to personalized cancer treatment

tues, Apr 9, 1:00 - 3:00 pM SY30. Blood-Based Molecular Analysis of Cancer

tues, Apr 9, 2:25 - 2:45 pMDetection of chromosomal alterations in cell free DNA of cancer patients

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BiographyElias Zambidis, M.D. Ph.D., is an assistant professor of oncology and pediatrics at The Johns Hopkins University School of Medicine. Dr. Zambidis earned his M.D./Ph.D. in the Medical Scientist Training Program (MSTP) at the University of Rochester. He did his pediatrics residency in the department of pediatrics at Washington University in St. Louis, Missouri, and his clinical/research fellowships in pediatric hematology/oncology at The Johns Hopkins Hospital and the National Cancer Institute.

His clinical expertise is in pediatric oncology specializing in hematologic malignancies, developmental hematopoiesis, blood and bone marrow transplantation (BMT), and stem cell biology and therapeutics.

Research InterestsThe Zambidis lab studies the formation of pluripotent stem cells and hematopoietic, endothelial and cardiac differentiation with a focus on the potential therapeutic use of pluripotent stem cell-derived cells. Projects include studying the developmental biology of hematopoiesis as well as improving the differentiation of human embryonic stem cells and human induced pluripotent stem cells to hematopoietic lineages including erythrocytes and lymphocytes; improving the differentiation of human pluripotent stem cells to endothelial cells capable of forming the vasculature system; and improving the differentiation of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) to the cardiac lineage. The research team also studies the developmental biology of gastrulation and cardiogenesis in model organisms and explores potential applications of pluripotent stem cell-derived cardiac in tissue engineering, regenerative medicine, cardiotoxicity screening and novel drug discovery.

Technology Portfolio• C11206: Clinically Safe Human Induced Pluripotent Stem

Cells from Hematopoietic Cells• C11071: High Quality Human Pluripotent Stem Cell (iPSC)

Lines• C10919: Cell Culture Media - Pluripotent Stem Cells to

Cardiac Lineages

Elias Zambidis, M.D., Ph.D.Assistant Professor of Oncology and Pediatrics Sidney Kimmel Comprehensive Cancer CenterInstitute for Cell Engineering

Jeanine Pennington Licensing AssociatePhone: 410-516-5680Email: [email protected]

licEnsing AssociAtE

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Johns hopkins AAcr prEsEntAtions

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prEsEntAtion ABstrActs312/10. A mouse model of chronicprostaticinflammationusing a human prostate cancer-derived isolate of propionibacterium acnesDebika Biswal Shinohara1, Ajay Vaghasia2, Shu-Han Yu3, William G. Nelson4, Angelo M. De Marzo5, Srinivasan Yegnasubramanian6, Karen S. Sfanos3. 1Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD; 2Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD; 3Department of Pathology, Johns Hopkins University, Baltimore, MD; 4Sidney Kimmel Comprehensive Cancer Center and Departments of Pathology and Environmental Health Sciences, Johns Hopkins University, Baltimore, MD; 5Department of Pathology and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD; 6Sidney Kimmel Comprehensive Cancer Center and Department of Environmental Health Sciences, Johns Hopkins University, Baltimore, MD

Background: A causative role for infectious agents and chronic inflammation in prostate cancer etiology has been difficult to establish in humans. To study this potential link in an in vivo system, we have developed a mouse model of long-term bacteria-induced chronic inflammation of the prostate using a prostatectomy-derived strain of Propionibacterium acnes that may be of particular relevance to prostate cancer.

Methods: Prostates of C57BL/6J mice were inoculated, via urethral catheterization, with PBS (control) or a strain of P. acnes (PA2) isolated from human prostatectomy tissues. Animals were assessed at 2 days, 1 week, 2 weeks, or 8 weeks post-inoculation via histology and immunohistochemistry (IHC).

Results: PA2 inoculation resulted in severe acute and chronic inflammation confined to the dorsal lobe of the prostate. Chronic inflammatory infiltrates persisted for at least 8 weeks post-inoculation. Inflammatory lesions were associated with an increase in the Ki-67 proliferative index, and diminished Nkx3.1 and androgen receptor (AR) production. Interestingly, the observed response required live bacteria. Additionally, both IHC and in situ hybridization assays for P. acnes indicated a potential intracellular persistence of P. acnes in prostate epithelial cells.

Conclusions: To our knowledge, this is the first mouse model of long-term prostatic inflammation induced by P. acnes, and more generally, any prostatectomy tissue-derived bacterial isolate. This model may serve as a valuable preclinical model of chronic prostatic inflammation that can be used to mechanistically study the link between inflammation and prostate cancer and other prostate disease.

446/1. Differential expression of immuno-regulatory genes associated with pD-l1 display: implications for clinical

blockade of the pD-1/pD-l1 pathway in melanomaGeoffrey D. Young, Tracee L. McMiller, Haiying Xu, Shuming Chen, Alan E. Berger, Jinshui Fan, Robert A. Anders, Christopher Cheadle, Drew M. Pardoll, Suzanne L. Topalian, Janis M. Taube. Johns Hopkins, Baltimore, MD

Background: Blockade of the immunosuppressive PD-1/PD-L1 pathway has shown promising clinical results in patients with treatment-refractory solid tumors including melanoma. PD-L1, a protein displayed by many tumors, ligates the PD-1 co-inhibitory receptor on activated T cells. We previously found that IFN-γ, a potent inducer of PD-L1, was expressed by TILs in PD-L1(+) but not PD-L1(-) melanomas, creating an immunosuppressive microenvironment by a mechanism that we term “adaptive immune resistance” (Taube et al., Science Transl Med 2012). In the current study, we assessed other factors associated with PD-L1 expression in melanoma through differential gene expression analysis, in order to better understand the biology of this pathway and identify potential targets for combination immunotherapies.

Methods: PD-L1(+) vs. (-) melanomas including immune cell infiltrates (n=11) were laser-capture microdissected (LCM) from formalin-fixed paraffin embedded (FFPE) specimens and analyzed by whole genome array analysis with cDNA-mediated Annealing, Selection, extension and Ligation (DASL), a

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novel technology that allows for gene expression analysis with partially degraded mRNAs obtained from FFPE specimens. Differentially expressed genes were submitted to the NIH functional analysis tool DAVID to search for enrichment in biologically related groups. Multiplex quantitative (q)RT-PCR was used to validate differential expression of genes of interest and to examine other candidate genes in new set of PD-L1(+) vs.(-) melanomas (n=11).

Results: DASL/DAVID analysis of PD-L1(+) vs. PD-L1(-) melanomas uncovered genes of interest in several immunologically relevant pathways, including “Defense Response” (p<1.5E-9) and “T-cell activation” (p<2.2E-5). Multiplex qRT-PCR to examine genes of interest from the DAVID analysis along with other potentially relevant candidate genes was normalized to the GUSB housekeeping gene or to CD45 (pan-leukocyte marker), revealing over-expression of genes associated with immunosuppression (PD-1, PD-L1, LAG-3, IL-10), activated CD8 T cells (CD8A, IFN-γ, lysozyme, perforin, CCL5/RANTES), and antigen presenting cells (CD163, TLR3, CXCL1) in PD-L1(+) melanomas (p<0.10).

Conclusions: These experiments identified groups of functionally related, differentially expressed immuno-regulatory genes in PD-L1(+) melanomas. These factors may coordinately create an immunosuppressive tumor microenvironment, by synergizing to enhance PD-L1 expression on tumor cells and to inhibit T cell function. Immunohistochemical and in vitro functional validation assays of candidate gene products are currently underway in order

from over 60 surgical samples from a Stand up to Cancer (SU2C) sponsored clinical trial. Sorted tumor populations from each patient have been interrogated with CGH arrays and whole exome sequencing. In addition we have interrogated the genomes of sorted PDA populations from a SU2C phase II trial of liver metastases from 35 patients with advanced previously treated disease, as well as those from a series of rapid autopsy samples. The genes targeted by the somatic aberrations in each tumor genome are overlaid onto a collection of 33 PDA specific maps, containing 763 genes of interest, developed as part of the Metaminer Oncology initiative.An example of the translational potential of these data includes the detection of a homozygous deletion of STAG2 in an aneuploid tumor population present in the primary and in each metastatic site of a rapid autopsy case. Targeted resequencing identified somatic mutations in a small number of additional sorted samples from our patient cohorts. Strikingly genetic and histopathologic analysis of tumors induced by transposon insertion in a KrasG12D genetically engineered mouse model showed that disruption of STAG2 promotes the development of PDA and its progression to metastatic disease. To assess the clinically significance of STAG2 expression in human tumors we screened a TMA containing a collection of 344 specimens obtained from resected patients. In normal tissue nearly all ductal cells stained with a high intensity. There was a statistically significant loss of STAG2 expression in the tumor tissue (Wilcoxon-Rank test). In univariate Kaplan Meier analysis nearly complete STAG2 positive staining (> 95% of nuclei positive)

to identify new ways to overcome adaptive immune resistance in synergistic treatment combinations with PD-1/PD-L1 blockade.

798/5. clonal analysis and clinical translation of pancreatic adenocarcinoma genomesMichael T. Barrett1, Elizabeth Lenkiewicz1, Evers Lisa1, Pedro Perez-Mancera2, David Tuveson2, Daniela Aust3, Christian Pilarsky4, Meraj Aziz1, Richard Posner1, Ramesh Ramanathan5, Victor Velculescu6, Amy Kramer7, Jeffrey Drebin7, Daniel D. Von Hoff5. 1TGen, Scottsdale, AZ; 2Cambridge Research Institute, Cancer Research UK, Cambridge, United Kingdom; 3Institute of Pathology, University Hospital Dresden, Dresden, Germany; 4Department of Surgery, University Hospital Dresden, Dresden, Germany; 5Virginia G. Piper Cancer Center, Scottsdale Healthcare, Scottsdale, AZ; 6Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD; 7Perelman Center for Advanced Medicine, University of Pennsylvania, Philadelphia, PA

A fundamental challenge for the study and translation of pancreatic ductal adenocarcinoma (PDA) genomes in patients in vivo is the presence of varied admixtures of reactive stroma, inflammatory cells, and necrosis within the tumor. Furthermore biopsies frequently contain multiple clonal populations of neoplastic cells that cannot be distinguished on the basis of morphology alone. To overcome the limitations of histopathology based methods we are using DNA content based flow cytometry to identify and purify distinct clonal PDA populations

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was associated with a survival benefit of median survival of 6.41 months (p = 0.031). Interestingly, the survival benefit of adjuvant chemotherapy was only identified in the group of patients with a STAG2 staining of less than 95% (median survival benefit 7.65 months; p = 0.028). Multivariate Cox Regression analysis showed that STAG2 is an independent prognostic factor for survival in PDA patients. We propose that our unbiased clonal profiling of PDA genomes provides a unique and highly efficient framework to identify clinically relevant genomic events in PDA

sy11-01. plasma tumor DnA: changing the paradigm for administering systemic therapiesJulia A. Beaver, Patricia Valda, Danijela Jelovac, Michaela J. Higgins, Ben H. Park. Johns Hopkins Univ., Baltimore, MD, Massachusetts General Hospital, Boston, MA, Johns Hopkins Univ., Baltimore, MD

The ability to accurately detect somatic cancer DNA alterations from cell free circulating plasma tumor DNA (ptDNA) using next generation genomic technologies has many potential applications for clinical oncology. The initial use of this technology has been to detect point mutations in peripheral blood that could serve as predictors of response to various targeted therapies. Thus the development of a “liquid biopsy” that could easily match the mutational status of a given cancer with a specific therapy had great therapeutic implications. For example, the ability to assess certain epidermal growth factor receptor (EGFR) mutations via blood versus an invasive lung

biopsy would allow for the rapid determination of whether a patient would be a candidate for small molecule EGFR inhibitors such as gefitinib or erlotinib. We initially sought to evaluate the feasibility of detecting PIK3CA mutations in circulating ptDNA from patients with metastatic breast cancer using a novel technique of digital polymerase chain reaction (PCR) called BEAMing (for Beads, Emulsions, Amplification, Magnetics). We demonstrated that BEAMing is both sensitive and specific for identifying PIK3CA mutations in metastatic breast cancer patients, though we also confirmed the work of other groups that PIK3CA mutation status can change with progression to metastatic disease. An overview of these data will be presented. While these studies demonstrated the feasibility of using ptDNA in metastatic patients as a liquid biopsy for detecting specific cancer mutations, we are now focusing efforts to exploit the quantitative nature of digital PCR technology. Specifically, by analyzing the amount of ptDNA before and after primary breast surgery in early stage breast cancer patients, we will identify which patients truly have residual microscopic disease post surgery and therefore would be the most appropriate candidates for additional systemic therapies. Thus, in contrast to the current paradigm of “over” treating breast cancer patients in the adjuvant setting, detection of ptDNA using next generation digital PCR technologies will enable us to make rational decisions regarding which patients truly have need for adjuvant systemic therapies. Preliminary data and ongoing studies will be discussed as well as the implementation of these technologies in future clinical trials.

1773/11. nucleotide-resolution genomic breakpoint analysis of tMprss2-Erg rearrangements in prostate cancer by target-capture next-generation sequencingChristopher Weier, Michael Haffner, Timothy Mosbruger, David Esopi, Jessica Hicks, Qizhi Zheng, William B. Isaacs, Angelo M. De Marzo, William G. Nelson, Srinivasan Yegnasubramanian. Johns Hopkins University, Baltimore, MD

TMPRSS2-ERG translocations occur in approximately 50% of prostate cancers and therefore represent one of the most frequently observed structural rearrangements in all cancers. However, little is known about the genomic architecture of such rearrangements. We therefore designed and optimized a pipeline involving target-capture of TMPRSS2 and ERG genomic sequences coupled with paired-end next generation sequencing to resolve genomic rearrangement breakpoints in TMPRSS2 and ERG at nucleotide resolution in a large series of primary prostate cancer specimens (n = 83). This strategy showed >90% sensitivity and specificity in identifying TMPRSS2-ERG rearrangements, and allowed identification of intra- and inter-chromosomal rearrangements involving TMPRSS2 and ERG with known and novel fusion partners. Our results indicate that rearrangement breakpoints show strong clustering in specific intronic regions of TMPRSS2 and ERG. The observed TMPRSS2-ERG rearrangements often exhibited complex chromosomal architecture associated with several intra- and inter-chromosomal rearrangements. Nucleotide resolution analysis of

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breakpoint junctions revealed that the majority of TMPRSS2 and ERG rearrangements (~88%) occurred at or near regions of microhomology or involved insertions of one or more base pairs. This architecture implicates nonhomologous end joining (NHEJ) and microhomology mediated end joining (MMEJ) pathways in the generation of such rearrangements. These analyses have provided important insights into the molecular mechanisms involved in generating prostate cancer-specific recurrent rearrangements.

2205/14. integrated next generation sequencing and patient-derived xenografts to personalized cancer treatmentElena Garralda1, Keren Paz2, Pedro P. López-Casas1, Siân Jones3, Amanda Katz2, Lisa M. Kann3, Fernando López-Rios4, Francesca Sarno5, Fátima Al-Shahrour1, David Vasquez2, Elizabeth Bruckheimer2, Samuel V. Angiuoli3, Luis A. Diaz6, Alfonso Valencia1, Victor E. Velculescu6, David Sidransky2, Manuel Hidalgo1. 1Spanish National Cancer Research Centre (CNIO), Madrid, Spain; 2Champions Oncology, Baltimore, MD; 3Personal Genome Diagnostics, Inc., Baltimore, MD; 4Laboratorio Dianas Terapéuticas, Hospital Universitario Madrid-Sanchinarro, Madrid, Spain; 5Centro Integral Oncológico Clara Campal, Hospital Universitario Madrid-Sanchinarro, Madrid, Spain; 6Ludwig Center for Cancer Genetics and Therapeutics, Johns Hopkins Kimmel Cancer Center, Baltimore, MD

Background: The knowledge of actionable somatic genomic alterations present in human tumors is enabling the new era of

personalized cancer treatment. The great intellectual challenge lies in linking confirmed mutations to protein function. Personalized tumor graft models (Avatars) can aid in the process of genomic analyses interpretation to ultimately move from molecular profile to medication. Methods: Using massive parallel sequencing we performed whole exome sequencing analysis of tumor and matched normal blood samples of 23 patients (pts) with advanced solid tumors (7 lung cancer, 7 pancreatic cancer, 1 neuroendocrine tumor, 2 glioblastoma, 1 uveal melanoma, 2 melanomas and 3 colon cancer) to identify putatively actionable tumor-specific genomic alterations. Avatar models generated by direct engraftment of tumor samples from the pts into immunocompromised mice were used as an in vivo platform to test proposed treatment strategies. Results: Successful exome sequencing analyses has been obtained for 21 pts (1 patient died prematurely, 1 sample was insufficient). Tumor specific mutations (Muts) and copy number variations were identified ranging from 5 to 952 and 0 to 36 respectively. All samples profiled contained clinically meaningful genomic alterations. A successful Avatar model was generated for 10 out of 17 pts. Two engraftment failures corresponded to EGFR mutant lung tumors resected while pts were receiving erlotinib, which initially grew but then regressed. Some of the most relevant drugabble alterations were: KRAS, CHEK1, FGFR2, IGF1R, MET, BRCA1, XPC, NOTCH, CREB3LB, GNA11, SMAD4 and EGFR. In occasions druggable alterations such as muts in NF1, PTPRC, PI3KA and DDR2 failed to provide any benefit when a targeted

drug was tested in the Avatar and accordingly treatment of the pts with these drugs was not effective. In one case, loss of STK11 lead to testing of mTOR and SRC inhibitors resulting in tumor regression both in the Avatar and in the clinic. At present time 10 pts have received a personalized treatment: 2 pts, as expected based on the Avatar model, did not response; 4 pts resulted in durable partial remissions and 4 pts are currently on treatment with disease stabilization. In one of the EGFR mutant lung pts the genomic analysis revealed traces of an acquired mutation and allowed decision making at an earlier time point, prior to relapse. Overall, there was a remarkable correlation between drug activity in the Avatar and clinical outcome in the pts, in terms of drug resistance and sensitivity. Conclusion: The detection of actionable tumor-specific genomic alterations in the clinical setting is feasible. However predicting treatment response to known oncogenes is complex and requires detailed information of how different genetic backgrounds function. Avatar models are a powerful investigational platform for therapeutic decision making and help to guide cancer treatment in the clinic.

2225/6 the molecular architectureofp85αasdetermined by sAXs and chemical cross-linkingEvan T. Brower1, Sandra B. Gabelli1, Qing Wang1, Raghothama Chaerkady1, Christopher E. Berndsen2, Robert N. Cole1, Jonathan M. Backer3, Mathias Schäfer4, Andrea Sinz5, Kenneth W. Kinzler1, Bert Vogelstein1, L. Mario

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Amzel1. 1Johns Hopkins University School of Medicine, Baltimore, MD; 2James Madison University, Harrisonburg, VA; 3Albert Einstein College of Medicine of Yeshiva University, New York, NY; 4Universität zu Köln, Cologne, Germany; 5Martin-Luther-Universität Halle-Wittenberg, Halle, Germany

The p85α protein, encoded by PIK3R1, is the regulatory subunit of phosphatidylinositol 3-kinase alpha (PI3Kα). The role of p85α extends to regulation of PTEN-phosphatase activity, the unfolded protein response, and additional cellular pathways. PIK3R1 was recently identified as frequently mutated in glioblastomas and endometrial cancers. Small-angle X-ray scattering (SAXS) and chemical cross-linking were used to complete the first investigation of the shape and molecular organization of full length p85α. p85α is elongated in shape and exists in a conformation conducive to p110α binding. The p85α structural data was utilized to generate the first model of the full length PI3K complex. Cancer associated PIK3R1 mutations are localized to interfaces between p85α domains, and in the context of the PI3K complex, are clustered at regions comprising the interface of the catalytic and regulatory subunits. A potential interface was discovered between the RhoGAP domain of p85α and the p110α kinase domain. GTPases may enhance PI3K activity by binding to the RhoGAP domain, thereby attenuating the interaction between the RhoGAP and the kinase domain. These data provide the structural groundwork required for a better understanding of the regulatory mechanisms mediated by p85, and may facilitate

the development of future drugs targeting the PI3K pathway.

3122/20. hMgA1 reprograms breast cancer cells by inducing transcriptional networks involved in an epithelial-mesenchymal transition, stemness, and metastatic progressionSandeep N. Shah, Leslie Cope, Amy Belton, Weijie Poh, Saraswati Sukumar, David Huso, Linda Resar. Johns Hopkins University, Baltimore, MD

Despite advances in our ability to detect and treat breast cancer, it remains a leading cause of death in women with cancer worldwide, and the incidence is rising. Approximately 15-20% of all cases are classified as triple negative breast cancer (TNBC), a subtype that is frequently associated with rapid progression and poor outcome. TNBC refers to the lack of detectable markers for the estrogen receptor (ER), progesterone receptor (PR), and Her2/neu amplification. These tumors do not respond to our most effective and least toxic therapies, including hormonal therapy (tamoxifen) or trastuzumab. We are studying molecular pathways that lead to tumor progression in TNBC and can be targeted with novel therapies. Our focus is the high mobility group A1 (HMGA1) oncogene. HMGA1 is highly expressed during embryogenesis, with low or undetectable levels in differentiated, adult tissues. HMGA1 is enriched in virtually all high-grade (poorly differentiated) cancers studied to date, including TNBCs, and high expression portends a poor prognosis in breast and other

cancers. To investigate the role of HMGA1 in tumor progression in breast cancer, we silenced HMGA1 expression in TNBC cell lines (MDA-MB-231, Hs578T) using lentiviral-mediated delivery of short hairpin RNA. Strikingly, proliferation was markedly impaired, and many cells underwent apoptotic cell death within 5 days following HMGA1 knock-down. Surprisingly, cell morphology also changed dramatically, whereby the fibroblast-like, spindle-shaped cells became cuboidal and epithelial-like, consistent with a mesenchymal-epithelial transition, or MET. E-CADHERIN mRNA was induced, while both SNAIL and VIMENTIN were repressed in the knock-down cells, also consistent with MET. In addition, silencing HMGA1 blocked migration, invasion, and the formation of tumor foci in the lungs following tail vein injection of MDA-MB-231 cells. Moreover, both primary tumorigenesis and metastatic progression to the lungs were markedly inhibited in MDA-MB-231 cells with knock-down of HMGA1 following implantation in mammary fat pads. Furthermore, silencing HMGA1 blocked primary and secondary mammosphere formation, indicating that HMGA1 is required for this stem cell property. Tumorigenesis experiments at limiting dilutions showed that silencing HMGA1 depletes the tumor initiator/cancer stem cell pool. Using global gene expression analysis, we identified an HMGA1 signature of differentially-regulated genes in the control cells compared to knock-down cells. We also found that the HMGA1 signature is highly enriched in embryonic stem cells. Together, these findings indicate that silencing HMGA1 reprograms invasive, mesenchymal TNBCs into

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non-invasive, epithelial-like cells with slower growth and an altered gene expression signature. Studies are now needed to determine how to target HMGA1 in therapy.

A3123/21. the notch pathway inhibitsTGF-βsignalinginbreast cancer through hEyl-mediated crosstalkHan Liangfeng1, Adam Diehl1, Nguyen K. Nguyen1, Preethi Korangath1, Teo Weiwen1, Zhe Zhang1, Scott Kominsky1, Pedram Argani1, Goran Landberg2, Saraswati V. Sukumar1. 1Johns Hopkins Univ. School of Medicine, Baltimore, MD; 2The University of Manchester, Manchester, United Kingdom

Acquired resistance to Transforming growth factor-β (TGF-β) is a key step in the early stages of tumorigenesis. Mutations of TGF-β signaling components, often found in other cancers, are rare in breast cancer, and little is known about the development of this resistance in breast cancer. On the other hand, activation of Notch pathway is known to play a substantial role in promoting breast cancer development. We hypothesized that crosstalk between these two pathways occurs through HEYL, a basic helix-loop-helix (bHLH) transcription factor, and a known direct target of Notch. We found that HEYL is overexpressed in 40% of breast cancers. Expression of Notch increased HEYL expression while knockdown of RBP-J, a critical mediator of Notch, reduced HEYL expression in HS578T and MDAMB-231 breast cancer cells. Taking into account the contradictory biological effects of Notch and TGF-β signaling, we sought to

examine whether HEYL inhibits the TGF-β pathway. TGF-β treatment or Smad3 overexpression significantly increased the luciferase activity of the TGF-β responsive reporter vector, p3TP-Luc, and the P15 (CDKN2B) gene promoter, but this transactivation was strongly inhibited by HEYL. Bimolecular fluorescence complementation assays confirmed the interaction between Smad 3 and HEYL; immunoprecipitation assays with deletion constructs showed that the Basic domain of HEYL interacts with the MH2 domain of Smad3, and that their interaction is necessary for HEYL to inhibit TGF-β signaling. In addition, using a tet-off inducible breast cancer cell model, HEYL was shown to transcriptionally up-regulate many genes that drive metastasis and tumor angiogenesis. Supporting this concept, HEYL/HER2 double transgenic mice showed nearly 50% increase in lung metastasis (20% vs 46%) compared to HER2/neu mice. Similarly, lung colonization occurred in 100% of mice injected with MDAMB231 while HEYL-shRNAs reduced incidence to 50%. Therefore, HEYL promotes breast cancer growth and metastasis in Smad-dependent and Smad-independent mechanisms.3580/2. Acetaldehyde and drug hypersensitivities of fanconi anemia defects: implications for cancer initiation, prevention, and therapySoma Ghosh, Surojit Sur, Sashidhar R. Yerram, Carlo Rago, Anil K. Bhunia, M. Zulfiquer Hossain, Bogdan C. Paun, Yunzhao R. Ren, Christine A. Iacobuzio-Donahue, Nilofer A. Azad, Scott E. Kern. Johns Hopkins Medical Institutions, Baltimore, MD

Studies of cells harboring Fanconi anemia (FA) pathway defects have aided clinical understanding of inherited cancer risks and therapeutic strategies. Here, we observed a novel and large (27X) hypersensitivity of BRCA2- and PALB2-null genotypes to the epidemiologically important ethanol metabolite, acetaldehyde. This prominent acetaldehyde sensitivity may hold evolutionary and clinical significance. Interrogation of a novel panel of cells engineered to be null for various FA genes also revealed two classes of chemical hypersensitivities: the shared and divergent phenotypes. Prominent chemical hypersensitivities to various interstrand crosslinking (ICL) agents in vitro (melphalan, mitomycin C, and cisplatin), to γ-radiation in vitro, and to mitomycin C in vivo were essentially similar among the tested genotypes. A large divergence of responsiveness existed, however, between the cell lines when using the PARP inhibitor KU0058948, the topoisomerase II inhibitor etoposide, and acetaldehyde. These results indicate that, toward some agents, not all FA defects are necessarily equivalent; this divergence among phenotypes may dissect functions differing among FA genes and may presage differing clinical and epidemiological implications. We additionally present the first engineered PALB2-null human cancer cells. The results suggest new applications in cancer epidemiology, prevention, and targeted therapy.

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3614/6. Are circulating testosterone and psA levels associated in a nationally representative sample of men without a diagnosis of prostate cancer?Sarah B. Peskoe1, William G. Nelson1, Corinne E. Joshu1, Sabine Rohrmann2, Katherine A. McGlynn3, Elizabeth A. Platz1. 1Johns Hopkins University, Baltimore, MD; 2University of Zurich, Zurich, Switzerland; 3National Cancer Institute, Rockville, MD

Background: PSA-based prostate cancer screening is controversial, in part, because of imperfect specificity, especially for aggressive disease. PSA production by prostate luminal epithelial cells is under androgenic regulation. However, the association between circulating androgen and PSA levels is unclear in general populations. Knowledge of this association may enhance clinical decision-making for an elevated screening serum PSA.

Methods: Included were 378 men, 40-85 years who participated in the National Health and Nutrition Examination Survey in 2001-04, who did not have a prior prostate cancer diagnosis, and who had not had a recent biopsy, exam, or infection of the prostate. Serum levels of PSA (Hybritech), testosterone, androstanediol glucuronide (AAG), estradiol, and sex hormone binding globulin (SHBG; carries testosterone and estradiol in circulation) (immunoassay) were previously measured. Free testosterone was estimated by mass action. We calculated geometric mean PSA levels and 95% confidence intervals by hormone quintiles. We applied

sampling weights, and adjusted for age and race/ethnicity, and also for BMI, waist circumference, smoking, diabetes, and mutually for the hormones. We stratified by age, race/ethnicity, and adiposity. We estimated the OR of a mildly elevated PSA (≥2.5 ng/mL) per hormone quintile by logistic regression.

Results: Geometric mean PSA level increased across testosterone quintiles after age and race/ethnicity adjustment (Q1: 0.80, Q5: 1.14 ng/mL; p-trend=0.001) and after further multivariable adjustment (Q1: 0.80, Q5: 1.15 ng/mL; p-trend=0.022). The same patterns were observed for free testosterone and AAG. SHBG was not associated with PSA after age and race/ethnicity adjustment (p-trend=0.85), but was strongly inversely associated after multivariable adjustment (Q1: 1.30, Q5: 0.81 nmol/L; p-trend=0.007). Estradiol and PSA levels were not associated. While PSA levels differed by age, by race/ethnicity, and by adiposity, the same sized increasing association for testosterone and PSA was seen within strata of age, of race/ethnicity, and of adiposity. The OR of PSA ≥2.5 ng/mL per testosterone quintile was 1.52 (95% CI 1.16-1.98) after age and race/ethnicity adjustment, and 1.74 (95% CI 1.13-2.68) after multivariable adjustment; SHBG explained the OR shift. The multivariable-adjusted OR of PSA ≥2.5 ng/mL per SHBG quintile was 0.60 (95% CI 0.37-1.00).

Conclusions: In this nationally representative sample, men with higher testosterone had higher PSA, even after taking into account other hormones and modifiable factors. Men with higher SHBG had lower PSA, but only after multivariable

adjustment. This information on testosterone and SHBG may be incorporated into algorithms for determining next steps following an elevated screening PSA.Funding: MD Cigarette Restitution Fund; NIH Intramural Research Program

3641/6. genome-wide methylation patterns suggest differences in breast cancer biology in American women of African and European ancestryAllyson C. Young1, Christine B. Ambrosone1, Lara Sucheston1, Dan Wang1, Li Yan1, Song Liu1, Li Tang1, Jo L. Freudenheim2, Peter G. Shields3, Carl D. Morrison1, Kitwa Demissie4, Saraswati Sukumar5, Michael J. Higgins1. 1Roswell Park Cancer Institute, Buffalo, NY; 2University at Buffalo, Buffalo, NY; 3Georgetown University, Washington, DC; 4University of Medicine and Dentistry of New Jersey, NJ; 5Johns Hopkins University, Baltimore, MD

European-American (EA) woman have a higher overall incidence of breast cancer than African American (AA) women, yet AA woman have poorer survival outcome, even after controlling for factors related to socioeconomic status. AA women are diagnosed at a younger age with aggressive breast tumors, more frequently ‘triple negative’ due to lack of estrogen and progesterone receptor (ER and PR) expression and negative for HER-2 amplification, as well as, high proliferative indices. These ‘triple negative’ breast cancers are most lethal since hormonal- or anti-HER2 therapy are not effective; therefore, fewer treatment options

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are available. Currently, the reason for racial disparities in breast cancer biology and early age of onset in AA women is largely unknown. Previous studies using a panel of 5 cancer-associated genes demonstrated a higher frequency of methylation in ER-negative tumors from younger AA women compared to EA women. We hypothesize that DNA methylation is one potential molecular mechanism that drive differences in breast cancer etiology between AA and EA women. Utilizing the Illumina Infinium Human Methylation 450K bead array, we surveyed DNA methylation at greater than 485,000 CpG sites across the genome at single-nucleotide resolution in breast tumor DNA from 138 (58 AA, 80EA) women and normal breast tissue DNA from 124 (22 AA, 102 EA) women undergoing reduction mammoplasty. Following filtering for ambiguously mapping probes and probes containing known SNPs, unsupervised hierarchical cluster analysis of the most varied CpG loci among tumor tissues distinguished three major clusters: Normal, ER-positive tumors and ER-negative tumors. We identified 157 CpG sites that are differentially methylated between tumors from AA and EA women (Δβ>0.17, FDR<0.05). Of these, 50 CpG were uniquely differentially methylated in ER-negative tumors, while 28 CpG sites were specific to ER-positive tumors. We are currently examining these loci in more depth to determine their biological significance and potential contribution to differences in breast cancer etiology between AA and EA women. Future analyses include screening a larger cohort of 1000 FFPE tumor DNAs to effectively compare differential methylation with age at onset, and a variety

of tumor characteristics and risk factors. Funded by 1 R01 CA133264 to CBA, KD, and MJH, and by Cancer Center Support Grant CA16056 to RPCI.

3726/12. combinations of hDAc inhibitor, chemotherapeutic agent and retinoic acid induce growth arrest, differentiation and tumor regression in preclinical models of breast cancerVanessa F. Merino, Nguyen Nguyen, Helen Sadik, Sean Cho, Xian Chong Zhou, Qian Chen, Duojia Pan, Saraswati Sukumar. Johns Hopkins Univ., Baltimore, MD

The histone deacetylase inhibitor (HDACi), entinostat, is being actively explored as a new-generation epigenetic drug which can lead to the change in the expression status of genes/pathways, but has low efficacy in cancer monotherapy. All-trans retinoic acid (ATRA) induces the differentiation of various types of stem cells. Data from cell culture and xenograft models from our lab showed that a combination of entinostat (MS-275), doxorubicin and ATRA effectively decreased tumor size in three breast cancer cell line xenograft models. Here, we sought to further investigate the mechanism of action of the triple drug combination in cancer cells; in particular, its effect on the breast cancer stem cell population. We performed a comprehensive genome wide analysis of gene expression of MDA-MB-231 breast cancer cells treated with ATRA, MS-275 and doxorubicin as monotherapies and as combination therapies. We saw that the drug-response gene profile

of ATRA is very similar to DMSO (vehicle)-treated cells. Accordingly, the addition of ATRA (A) to MS-275 (MA), Dox (AD) and MS-275/Dox (MAD) displayed minimal changes in the gene expression profile of each of the other treatments. Addition of Dox to MS-275 (MD), on the other hand, potentiated the “reprograming” effect of MS-275 and affected the expression of many antitumor genes known to be related to cell cycle and growth arrest. It also altered expression of genes involved in development and inflammation. The most differentially expressed genes, validated by qPCR, were novel genes from the cancer/testis antigens and tripartite motif (TRIM) family of proteins. Interestingly, in MDA-MB-231and SUM149 cells, even the addition of low doses of doxorubicin (12.5 nM) to MS-275 increased 2 and 2.6 fold the G2 cell cycle arrest in comparison to Dox and MS-275, respectively. Despite the gene expression pattern similarity between MS-275/Dox (MD) and MS-275/Dox/ATRA (MAD) groups, we saw that MAD was more effective in inducing cell death and apoptosis in vitro and in vivo. The epithelium specific ETS transcription factor-1 (ESE-1) was differentially regulated between MAD and MD and is, in fact, part of the MS-275/ATRA (MA) signature. Using limiting dilution transplantation assays in mammary fat pads of immunodeficient mice we observed that MAD treatment in vivo most effectively targeted breast cancer stem cells (BCSC) compared to any other combination of drugs. The cancer stem cell frequency of the cells isolated from MAD treated mice was 1 in 236,570. The second most effective treatment for BCSC was MA (1 in 150,721), followed by ATRA>

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MS-275>MD>Dox>DMSO>AD. In conclusion, the reprogramming events initiated by HDACi and retinoid sensitize the cells to low doses of doxorubicin. The combination therapy may have a significant effect in decreasing breast tumor growth and recurrence.

4006/20. smad6 upregulation provides an alternative mechanism for BMp inactivation in sMAD4 wild type pancreatic cancersJacqueline A. Brosnan, Richard Morgan, Catherine M. White, Seung-Mo Hong, Shinichi Yachida, Michael Goggins, Barish Edil, Christine A. Iacobuzio-Donahue. Johns Hopkins Medical Insts., Baltimore, MD

Introduction: Genetic inactivation of SMAD4, a central mediator of TGF-β superfamily signaling, is significantly correlated with metastatic behavior in pancreatic cancer patients. However, because some patients with pancreatic cancer have genetically intact TGF-β and BMP pathway components, we investigated the role of alternative mechanisms of inactivation in promoting pancreatic cancer metastasis.

Methods: Eighteen cell lines for which the genetic status of all members of the TGF-β pathway were known were used in this study. TGF-β signaling levels were analyzed in each cell line using a luciferase reporter system under the control of a Smad Binding Element (SBE). Immunoblotting was performed on protein lysates from pancreatic cancer cell lines of known genotype. Cell lines that express high levels of Smad6 were transiently transfected with shRNA constructs targeting

Smad6; cell lines that express low levels of Smad6 were transiently transfected with a construct to constitutively express Smad6. The effects of Smad6 modulation were assessed by proliferation assay, migration assay, and invasion assay. Immunohistochemistry was performed on primary and metastatic pancreatic cancer tissues and staining intensity correlated to clinical data.

Results: Functional TGF-β and BMP signaling were eliminated in cell lines with known SMAD4 inactivation, as well as in several cell lines in which these pathways remain intact. Immunoblotting for known TGF-β superfamily antagonists in these cell lines revealed differential expression of Smad6, an inhibitory Smad. Modulation of Smad6 levels in vitro suggests that Smad6 overexpression contributes to increased levels of proliferation, migration, and invasion in SMAD4-intact cell lines. Overexpression of Smad6 did not restore TGF-β signaling, but did increase BMP response. Immunohistochemistry for Smad6 in patient samples revealed a nuclear localization pattern, suggesting that the pro-oncogenic roles of Smad6 are mediated by its ability to act as a transcription factor. High Smad6 levels correlated with worse prognosis and metastatic behavior among SMAD4-intact pancreatic cancers.

Conclusions: Smad6, an inhibitory Smad, is differentially expressed in pancreatic cancer, both in cell lines and patient samples. High levels of Smad6 in patients at autopsy associate with widespread metastasis, irrespective of SMAD4 status. Preliminary studies in vitro

support a metastasis-promoting function of Smad6: Smad6 overexpression is associated with increased levels of proliferation, migration, and invasion in SMAD4-intact pancreatic cancer cell lines. Experiments are ongoing to determine the mechanism through which Smad6 is acting in pancreatic cancer.

4619. Epigenetic therapy and sensitization of lung cancer to immunotherapyJohn Wrangle1, Wei Wang1, Alexander Koch2, Hariharan Easwaran1, Helai Mohammad1, Princy Parsana1, Frank Vendetti1, Kristen Rodgers1, Xiaoyu Pan1, Kirsten Harbom1, Cynthia Zahnow1, Janis Taube1, Julie Brahmer1, Peter Jones3, Suzanne Topalian1, Charles Rudin1, Malcolm Brock1, Drew Pardoll1, Stephen Baylin1. 1Johns Hopkins Hospital, Baltimore, MD; 2University of Gent, Gent, Belgium; 3University of Southern California, Los Angeles, CA

Epigenetic alterations driving carcinogenesis and cancer progression can be specifically targeted by the demethylating agent azacitidine (Aza) and the histone deacetylase inhibitor entinostat. While this treatment combination has been effective in a limited number of patients (pts) with treatment-refractory non-small cell lung cancer (NSCLC), we observed clinical benefit in 5 of 5 patients who received immunotherapy with PD-1/PD-L1 pathway blockade immediately following epigenetic therapy. Three of 5 pts developed partial tumor regressions (RECIST criteria, duration 10+ to 20+ mo.) and 2 pts had stable disease ≥6 mo. This compares to the objective

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response + SD rates of NSCLC to monotherapy with anti-PD-1 (18% + 5%) or anti-PD-L1 (10% + 12%). To understand how epigenetic therapy may synergize with blockade of the immunosuppressive PD-1 pathway, we used genome wide methylation and expression profiling on 8 NSCLC cell lines treated with low dose Aza. We discovered complex immunomodulatory effects of Aza with up-regulation of diverse immune related pathways including Jun/Jnk, NFKB, viral defense, type I interferon signaling, the inflammasome, antigen processing and presentation and immune evasion including up-regulation of PD-L1 expression. Multiple cancer-testes antigens were also up-regulated, thereby conferring de novo antigenicity. Supporting the idea that Aza acts specifically through inhibition and degradation of DNA methyltransferase proteins, colon cancer cells genetically haplo-insufficient for DNMT1 and devoid of DNMT3b mirror the immunomodulatory effects of Aza. Upstream events potentially controlling these pathways were defined, and prominent among them was up-regulation of the transcription factor, interferon regulatory factor 7 (IRF7), a DNA hypermethylated gene. These data were used to query hundreds of primary NSCLC samples from the Cancer Genome Atlas project (TCGA). A low basal expression signature of interferon pathway related genes was significantly associated with low IRF7 expression and promoter methylation in squamous tumors. Another hypermethylated transcription factor, PITX1, which inhibits a subset of type I interferon signaling genes, tracked with non-squamous cancers. Together, these findings support a model

in which epigenetic modulation activates innate and adaptive immune responses within the tumor microenvironment together with induction of counter-regulatory immune checkpoint ligands which can be therapeutically blocked with antibodies. Based on these findings, a clinical trial testing the efficacy of DNMT and HDAC inhibition combined with PD-1 pathway blockade is under development. This work will form the basis for an immune-classification of NSCLC, as well as biomarker discovery for a novel therapeutic paradigm combining epigenetic and immunotherapy with potentially synergistic activity against the world’s most deadly malignancy.Supported by Stand Up to Cancer.

4666/9. A phase 2 study investigating the safety, efficacyandsurrogatebiomarkers of response of 5-azacitidine (5-AZA) andentinostat (Ms-275) in patients with triple-negative advanced breast cancerRoisin M. Connolly1, Rachel C. Jankowitz2, Cynthia A. Zahnow1, Zhe Zhang1, Michelle A. Rudek1, Stacie C. Jeter1, Shannon Slater1, Penny Powers1, Antonio C. Wolff1, John Fetting1, Adam M. Brufsky2, Richard Piekarz3, Nita Ahuja1, George Somlo4, Augustin Garcia5, Steven Baylin1, Nancy E. Davidson2, Vered Stearns1. 1Johns Hopkins Univ., Baltimore, MD; 2University of Pittsburgh Cancer Institute, Pittsburgh, PA; 3Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD; 4City of Hope, Duarte, CA; 5USC/Norris Cancer Center, Los Angeles, CA

Background:In preclinical breast cancer models, combination epigenetic therapy with a DNA methyltransferase inhibitor (DNMTI) and a histone deacetylase inhibitor (HDACI) yield superior estrogen receptor (ER) re-expression and greater restoration of tamoxifen responsiveness than either agent alone. We conducted a multicenter phase II clinical trial to evaluate the DNMTI 5-azacitidine (5-AZA) and the HDACI entinostat in women with advanced breast cancer.

Methods:Women with advanced HER2-negative, either triple-negative (TN; ER/progesterone receptor [PR]/HER2-negative) or hormone-resistant breast cancer received 5-AZA 40 mg/m2 (SQ, days 1-5, 8-10) and entinostat 7 mg (PO, days 3,10) every 28 days. Primary endpoint: objective response rate (ORR) in each group. Secondary endpoints: safety, tolerability, survival, clinical benefit rate. Exploratory endpoints: pharmacokinetics, pharmacogenetics, change in candidate gene re-expression/methylation in circulating DNA and mandatory tumor samples. Patients are offered ongoing study therapy at progression with addition of hormonal therapy (optional continuation phase). Sample size: Simon two-stage design with interim analysis after 13 patients per cohort (1st stage). If ≥1 response, accrual will continue for total of 27 per cohort (2nd stage). Null hypothesis: ORR at most 5% against alternative hypothesis that is at least 20% with type I error 4% and power 90%. Preclinical TN/ ER-positive xenograft studies assessing 5-AZA impact were also performed.

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Results:Thirteen evaluable patients were enrolled in 1st stage of TN cohort. Median age was 47 (31-67), median prior chemotherapies 3 (1-5), 77% white/33% black, 77% visceral disease. Median cycles received 2 (1-4). Therapy was well tolerated, most common grade 3/4 treatment related adverse events leucopenia and neutropenia (23% each). No responses observed following 1st stage and this cohort was closed. Median 1.5 additional cycles (optional continuation phase) received by 4 patients with no responses to date. Exposure to 5-AZA (Cmax=1134±1670ng/mL; AUCINF=939±724 ng*h/mL) was slightly higher than previous studies, entinostat (Cmin=0.78±0.65ng/mL) was similar. Hormone-resistant cohort proceeded to 2nd stage as 1 partial response observed. Final results will be reported once accrual complete. Ongoing preclinical studies suggest that ER-positive is more sensitive than TN breast cancer to 5-AZA.

Conclusion:Combination epigenetic therapy with agents, dose and schedule described was well tolerated but not associated with clinical activity in advanced TN breast cancer. Correlative analyses will be presented at meeting. Promising preclinical findings suggest epigenetic therapy may be efficacious in ER-positive breast cancer.

4759/4. hoXB7 functions as a co-activator of estrogen receptor in the development of tamoxifen resistanceKideok Jin, Wei Wen Teo, Takahiro Yoshida, Sunju Park, Saraswati Sukumar. Johns Hopkins Univ.

School of Medicine, Baltimore, MD

Background: Multiple factors including long term treatment with tamoxifen are involved in the development of selective estrogen receptor modulator (SERM)-resistance in ERα positive breast cancer. Increased expression of HER family members can also directly alter cellular responses to tamoxifen, but the mechanism underlying the increased expression of the HERs is not clear. In this report we show that HOXB7 is an upstream regulator of HER2 and ER expression.

Results: Overexpression of HOXB7 results in upregulation of HER2 and ERα target genes, while knockdown of HOXB7 with siRNA in tamoxifen-resistant cell lines causes decrease of HER2 and ERα target genes expression and loss of tamoxifen resistance. To mediate upregulation of HER2 and ER-target genes occurring in tamoxifen resistance, the HOXB7-ERα complex recruits ERα coactivators and promotes HER2- and ER-target genes transcriptional activity through direct binding to the HER2 enhancer and regulatory regions of ER target genes. However, depletion of HOXB7 negatively affects the binding affinity of its cofactors to the HER2 enhancer element and ER-binding sites. Also, it is likely that miR196a controls HOXB7 expression; reducing miR196a levels in tamoxifen-resistant cells causes an increase of HOXB7 expression. Overexpression of miR196a sensitizes MCF-7 tamoxifen resistant cells to tamoxifen through downregulation of HER2 and ER-target genes while inhibition of miR196a by anti-miR196a inhibitors increases

tamoxifen resistance through the upregulation of HER and ER target genes in MCF-7 cells. Further, miRNA 196a is regulated by c-MYC which undergoes stabilization through the EGFR-HER2 signaling pathway. Depletion of c-MYC by MYC shRNA enhances tamoxifen sensitivity by increasing the level of miR196a transcripts. Also, by c-MYC ChIP assay, we found that c-MYC inhibits miR196a expression through direct binding to its promoter region.Conclusions: Overexpression of HOXB7 is a key event in the initiation and maintenance of tamoxifen resistance. These studies suggest that HOXB7 acts as a key regulator orchestrating two major groups of target molecules, ERα and HER2, in the oncogene hierarchy. Blocking MYC expression or reexpression of miR196a in TAM-R cells may provide novel strategies for reversing SERM-resistance. Therefore, we have provided additional evidence that supports the thesis that functional antagonism of HOXB7 has the potential to circumvent tamoxifen resistance.

5008/6. A morphologically distinct cancer initiating cell in human and murine panin and pancreatic cancerJennifer M. Bailey, Janivette Alsina, Zeshaan Rasheed, Ya-Yuan Fu, Florencia McAllister, Pankaj Pasricha, William Matsui, Anirban Maitra, Steven Leach. Johns Hopkins Univ. School of Medicine, Baltimore, MD

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal with a survival rate of less than 6 months for the majority of patients. In order to better understand its development we hypothesized that

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there is cellular heterogeneity in murine and human PDAC precursor lesions (PanINs). We utilized two independent models of murine PanIN (mPanIN) formation to drive oncogenic KrasG12D expression in the murine pancreas during development (KCPdx1) or specifically in adult acinar cells (KCiMist1). The mice were crossed to the mTmG reporter mouse to map the fate of cells expressing Kras (KCiMist1G) that are marked by expression of GFP. We used transmission electron microscopy on mPanIN-containing tissue to identify a morphologically distinct rare cell type present in both models of mPanIN. These cells morphologically resemble tuft cells, which are characterized by their brush-like morphology, including apically projecting microvillae and the expression of DCLK1 and AcTub. Using Flourescence Activated Cell Sorting Techniques (FACS), we were able to isolate DCLK1HI/GFP+ murine PanIN tuft cells expressed in the KCiMist1G model. We discovered that these cells have an increased ability to form PanIN spheres in vitro relative to GFP+ cells alone, indicative of a progenitor PanIN stem cell function.

Using immunofluorescent labeling on a human PanIN (n=23) and metastatic PDAC (n=10) tissue arrays, we determined that in 15% of PanIN lesions, 13-30% of the cells have detectable levels of AcTub, with a primarily apical pattern of labeling. In contrast, over 80% of metastatic PDAC cells are AcTub+, but they no longer exhibit an apical pattern of labeling. Applying our cell surface FACS strategy to four different human pancreatic cancer cell lines and three early passage human xenografts, we determined

that 2-5% of human pancreatic cancer cells have detectable levels of cell-surface AcTub and DCLK1. We have functionally analyzed the tumor-initiating capacity of cell surface AcTubHI cells using in vitro tumor sphere experiments. In this assay, cell surface AcTubHI cells show a statistically significant 100-fold increase in tumor sphere-forming capacity relative to cell surface AcTubLOW cells. We further evaluated the functional significance of cell surface AcTub by analyzing the in vivo tumor initiating capacities of cell surface AcTubHI and AcTubLOW cells from a human PDAC xenograft. Cell surface AcTubHI cells exhibited significantly increased tumor initiating frequency relative to the AcTubLOW population (**p<0.0000539). These data provide important new information regarding cellular heterogeneity in murine PanIN and human pancreatic cancer, and suggest that novel strategies targeting DCLK1 and AcTub may have potential therapeutic utility.

5331/14. A novel function of p21 in inhibition of epithelial-mesenchymal transition through micrornAsXiao L. Li1, Toshifumi Hara1, Youngeun Choi2, Murugan Subramanian1, Princy Francis1, Sven Bilke1, Robert L. Walker1, Marbin Pineda1, Yu-an Yang1, Ji Luo1, Lalage M. Wakefield1, Ben H. Park3, Thomas Brabletz4, Dipanjan Chowdhury2, Paul S. Meltzer1, Ashish Lal1. 1NIH, Bethesda, MD; 2Harvard University, Boston, MA; 3Johns Hopkins University, Baltimore, MD; 4University of Freiburg Medical Center, Freiburg, Germany

The tumor suppressor p21 inhibits cell proliferation during the stress response. However, p21 can also directly regulate gene expression by repressing specific transcription factors. Here, we identified p21-regulated miRNAs by sequencing small RNAs from HCT116-p21+/+ and HCT116-p21-/- cells. Three abundant clusters, miR-200b-200a-429, miR-200c-141 and miR-183-96-182 were down-regulated in p21-depleted HCT116 and MCF10A cells. Loss of p21 induced epithelial-mesenchymal transition (EMT) and enhanced migration and invasion in multiple model systems. Identification of genome-wide targets of the miR-183-96-182 cluster indicated that miR-183 and miR-96 repressed common targets, including SLUG, ZEB1, ITGB1 and KLF4, to inhibit EMT, migration and invasion. In turn, elevated ZEB1 levels in HCT116-p21-/- cells directly repressed miR-183-96-182 cluster transcription, revealing a feedback loop. Re-introduction of miR-200, miR-183 or miR-96 in HCT116-p21-/- cells inhibited migration and invasion. These novel findings suggest that coordinated down-regulation of three miRNA clusters upon loss of p21 in unstressed cells promotes EMT, migration and invasion.

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