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The Best of the AACR Journals
The AACR is proud to present “The Best of the AACR Journals,” a collection of the most-cited articles published in 2013 across the
AACR journal portfolio. We congratulate the authors of these outstanding articles for their tireless efforts and contributions to the
cancer research field.
Each journal’s section in the collection includes the most-cited review article and the four most-cited research articles based on the
total number of citations from date of publication through January 2015.
Table of Contents
Cancer Discovery
The Basic Principles of Chimeric Antigen Receptor Design
Michel Sadelain, Renier Brentjens, and Isabelle Rivière .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .1
Activating HER2 Mutations in HER2 Gene Amplification Negative Breast Cancer
Ron Bose, Shyam M. Kavuri, Adam C. Searleman, Wei Shen, Dong Shen, Daniel C. Koboldt, John Monsey, Nicholas Goel,
Adam B. Aronson, Shunqiang Li, Cynthia X. Ma, Li Ding, Elaine R. Mardis, and Matthew J. Ellis .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .2
First-in-Humans Trial of an RNA Interference Therapeutic Targeting VEGF
and KSP in Cancer Patients with Liver Involvement
Josep Tabernero, Geoffrey I. Shapiro, Patricia M. LoRusso, Andres Cervantes, Gary K. Schwartz, Glen J. Weiss, Luis Paz-Ares,
Daniel C. Cho, Jeffrey R. Infante, Maria Alsina, Mrinal M. Gounder, Rick Falzone, Jamie Harrop, Amy C. Seila White,
Iva Toudjarska, David Bumcrot, Rachel E. Meyers, Gregory Hinkle, Nenad Svrzikapa, Renta M. Hutabarat, Valerie A. Clausen,
Jeffrey Cehelsky, Saraswathy V. Nochur, Christina Gamba-Vitalo, Akshay K. Vaishnaw, Dinah W.Y. Sah, Jared A. Gollob,
and Howard A. Burris III .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .2
Amplification of the MET Receptor Drives Resistance to Anti-EGFR Therapies in Colorectal Cancer
Alberto Bardelli, Simona Corso, Andrea Bertotti, Sebastijan Hobor, Emanuele Valtorta, Giulia Siravegna,
Andrea Sartore-Bianchi, Elisa Scala, Andrea Cassingena, Davide Zecchin, Maria Apicella, Giorgia Migliardi,
Francesco Galimi, Calogero Lauricella, Carlo Zanon, Timothy Perera, Silvio Veronese, Giorgio Corti,
Alessio Amatu, Marcello Gambacorta, Luis A. Diaz Jr, Mark Sausen, Victor E. Velculescu, Paolo Comoglio,
Livio Trusolino, Federica Di Nicolantonio, Silvia Giordano, and Salvatore Siena .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .3
Identification of Targetable FGFR Gene Fusions in Diverse Cancers
Yi-Mi Wu, Fengyun Su, Shanker Kalyana-Sundaram, Nickolay Khazanov, Bushra Ateeq, Xuhong Cao, Robert J. Lonigro, Pankaj Vats,
Rui Wang, Su-Fang Lin, Ann-Joy Cheng, Lakshmi P. Kunju, Javed Siddiqui, Scott A. Tomlins, Peter Wyngaard, Seth Sadis,
Sameek Roychowdhury, Maha H. Hussain, Felix Y. Feng, Mark M. Zalupski, Moshe Talpaz, Kenneth J. Pienta, Daniel R. Rhodes,
Dan R. Robinson, and Arul M. Chinnaiyan .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .3
Cancer Epidemiology, Biomarkers & Prevention
Oral Contraceptive Use and Risk of Breast, Cervical, Colorectal, and Endometrial Cancers: A Systematic Review
Jennifer M. Gierisch, Remy R. Coeytaux, Rachel Peragallo Urrutia, Laura J. Havrilesky, Patricia G. Moorman,
William J. Lowery, Michaela Dinan, Amanda J. McBroom, Vic Hasselblad, Gillian D. Sanders, and Evan R. Myers . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .4
Cancer Survivors in the United States: Prevalence across the Survivorship
Trajectory and Implications for Care
Janet S. de Moor, Angela B. Mariotto, Carla Parry, Catherine M. Alfano, Lynne Padgett, Erin E. Kent, Laura Forsythe,
Steve Scoppa, Mark Hachey, and Julia H. Rowland .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .5
Vegetarian Diets and the Incidence of Cancer in a Low-risk Population
Yessenia Tantamango-Bartley, Karen Jaceldo-Siegl, Jing Fan, and Gary Fraser . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .5
Genetic Susceptibility Loci for Subtypes of Breast Cancer in an African American Population
Julie R. Palmer, Edward A. Ruiz-Narvaez, Charles N. Rotimi, L. Adrienne Cupples, Yvette C. Cozier,
Lucile L. Adams-Campbell, and Lynn Rosenberg .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .6
Drinking Water Arsenic in Northern Chile: High Cancer Risks 40 Years after Exposure Cessation
Craig M. Steinmaus, Catterina Ferreccio, Johanna Acevedo Romo, Yan Yuan, Sandra Cortes, Guillermo Marshall,
Lee E. Moore, John R. Balmes, Jane Liaw, Todd Golden, and Allan H. Smith .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .6
Cancer Immunology Research
Getting Personal with Neoantigen-Based Therapeutic Cancer Vaccines
Nir Hacohen, Edward F. Fritsch, Todd A. Carter, Eric S. Lander, and Catherine J. Wu .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .7
Anti-CTLA-4 Antibodies of IgG2a Isotype Enhance Antitumor Activity through Reduction
of Intratumoral Regulatory T Cells
Mark J. Selby, John J. Engelhardt, Michael Quigley, Karla A. Henning, Timothy Chen, Mohan Srinivasan, and Alan J. Korman .. .. .. .. .. .. .. .. .. .. .8
The Best of the AACR Journals
i

Table of Contents
An Abscopal Response to Radiation and Ipilimumab in a Patient with Metastatic Non–Small Cell Lung Cancer
Encouse B. Golden, Sandra Demaria, Peter B. Schiff, Abraham Chachoua, and Silvia C. Formenti .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .8
T Cells Expressing Chimeric Antigen Receptors Can Cause Anaphylaxis in Humans
Marcela V. Maus, Andrew R. Haas, Gregory L. Beatty, Steven M. Albelda, Bruce L. Levine,
Xiaojun Liu, Yangbing Zhao, Michael Kalos, and Carl H. June . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .9
PD-L1 Expression in the Merkel Cell Carcinoma Microenvironment: Association with
Inflammation, Merkel Cell Polyomavirus, and Overall Survival
Evan J. Lipson, Jeremy G. Vincent, Myriam Loyo, Luciane T. Kagohara, Brandon S. Luber, Hao Wang, Haiying Xu,
Suresh K. Nayar, Timothy S. Wang, David Sidransky, Robert A. Anders, Suzanne L. Topalian, and Janis M. Taube .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .9
Cancer Prevention Research
New Perspectives of Curcumin in Cancer Prevention
Wungki Park, A.R.M. Ruhul Amin, Zhuo Georgia Chen, and Dong M. Shin .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..10
Durable Antibody Responses Following One Dose of the Bivalent Human Papillomavirus
L1 Virus-Like Particle Vaccine in the Costa Rica Vaccine Trial
Mahboobeh Safaeian, Carolina Porras, Yuanji Pan, Aimee Kreimer, John T. Schiller, Paula Gonzalez, Douglas R. Lowy,
Sholom Wacholder, Mark Schiffman, Ana C. Rodriguez, Rolando Herrero, Troy Kemp, Gloriana Shelton, Wim Quint,
Leen-Jan van Doorn, Allan Hildesheim, and Ligia A. Pinto, for the CVT Group .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..11
MicroRNA Expression Signatures during Malignant Progression from Barrett’s Esophagus
to Esophageal Adenocarcinoma
Xifeng Wu, Jaffer A. Ajani, Jian Gu, David W. Chang, Weiqi Tan, Michelle A.T. Hildebrandt, Maosheng Huang,
Kenneth K. Wang, and Ernest Hawk .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..11
MUC1 Vaccine for Individuals with Advanced Adenoma of the Colon: A Cancer
Immunoprevention Feasibility Study
Takashi Kimura, John R. McKolanis, Lynda A. Dzubinski, Kazi Islam, Douglas M. Potter, Andres M. Salazar,
Robert E. Schoen, and Olivera J. Finn . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..12
Increased Levels of Urinary PGE-M, a Biomarker of Inflammation, Occur in Association
with Obesity, Aging, and Lung Metastases in Patients with Breast Cancer
Patrick G. Morris, Xi Kathy Zhou, Ginger L. Milne, Daniel Goldstein, Laura C. Hawks, Chau T. Dang, Shanu Modi,
Monica N. Fornier, Clifford A. Hudis, and Andrew J. Dannenberg .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..12
Cancer Research
Genome-Wide Epigenetic Regulation of miRNAs in Cancer
Constance Baer, Rainer Claus, and Christoph Plass .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..13
The Noncoding RNA MALAT1 Is a Critical Regulator of the Metastasis Phenotype of Lung Cancer Cells
Tony Gutschner, Monika Hämmerle, Moritz Eißmann, Jeff Hsu, Youngsoo Kim, Gene Hung, Alexey Revenko,
Gayatri Arun, Marion Stentrup, Matthias Groß, Martin Zörnig, A. Robert MacLeod, David L. Spector, and Sven Diederichs .. .. .. .. .. .. .. .. .. .. ..14
Androgen Receptor Splice Variants Mediate Enzalutamide Resistance
in Castration-Resistant Prostate Cancer Cell Lines
Yingming Li, Siu Chiu Chan, Lucas J. Brand, Tae Hyun Hwang, Kevin A.T. Silverstein, and Scott M. Dehm .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..14
Complex Tumor Genomes Inferred from Single Circulating Tumor Cells by
Array-CGH and Next-Generation Sequencing
Ellen Heitzer, Martina Auer, Christin Gasch, Martin Pichler, Peter Ulz, Eva Maria Hoffmann, Sigurd Lax, Julie Waldispuehl-Geigl,
Oliver Mauermann, Carolin Lackner, Gerald Höfler, Florian Eisner, Heinz Sill, Hellmut Samonigg, Klaus Pantel, Sabine Riethdorf,
Thomas Bauernhofer, Jochen B. Geigl, and Michael R. Speicher .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..15
Targeting Tumor-Infiltrating Macrophages Decreases Tumor-Initiating Cells, Relieves
Immunosuppression, and Improves Chemotherapeutic Responses
Jonathan B. Mitchem, Donal J. Brennan, Brett L. Knolhoff, Brian A. Belt, Yu Zhu, Dominic E. Sanford, Larisa Belaygorod,
Danielle Carpenter, Lynne Collins, David Piwnica-Worms, Stephen Hewitt, Girish Mallya Udupi, William M. Gallagher,
Craig Wegner, Brian L. West, Andrea Wang-Gillam, Peter Goedegebuure, David C. Linehan, and David G. DeNardo .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..15
Clinical Cancer Research
The Definition of Primary and Secondary Glioblastoma
Hiroko Ohgaki and Paul Kleihues .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..16
BRAF Inhibition Is Associated with Enhanced Melanoma Antigen Expression and a More Favorable Tumor
Microenvironment in Patients with Metastatic Melanoma
Dennie T. Frederick, Adriano Piris, Alexandria P. Cogdill, Zachary A. Cooper, Cecilia Lezcano, Cristina R. Ferrone, Devarati Mitra,
Andrea Boni, Lindsay P. Newton, Chengwen Liu, Weiyi Peng, Ryan J. Sullivan, Donald P. Lawrence, F. Stephen Hodi, Willem W. Overwijk,
Gregory Lizée, George F. Murphy, Patrick Hwu, Keith T. Flaherty, David E. Fisher, and Jennifer A. Wargo .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..17
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Table of Contents
Analysis of Tumor Specimens at the Time of Acquired Resistance to EGFR-TKI Therapy
in 155 Patients with EGFR-Mutant Lung Cancers
Helena A. Yu, Maria E. Arcila, Natasha Rekhtman, Camelia S. Sima, Maureen F. Zakowski, William Pao,
Mark G. Kris, Vincent A. Miller, Marc Ladanyi, and Gregory J. Riely .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..17
Durable Cancer Regression Off-Treatment and Effective Reinduction Therapy with an Anti-PD-1 Antibody
Evan J. Lipson, William H. Sharfman, Charles G. Drake, Ira Wollner, Janis M. Taube, Robert A. Anders, Haiying Xu,
Sheng Yao, Alice Pons, Lieping Chen, Drew M. Pardoll, Julie R. Brahmer, and Suzanne L. Topalian .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..18
An Epithelial–Mesenchymal Transition Gene Signature Predicts Resistance to EGFR and PI3K
Inhibitors and Identifies Axl as a Therapeutic Target for Overcoming EGFR Inhibitor Resistance
Lauren Averett Byers, Lixia Diao, Jing Wang, Pierre Saintigny, Luc Girard, Michael Peyton, Li Shen, Youhong Fan, Uma Giri, Praveen
K. Tumula, Monique B. Nilsson, Jayanthi Gudikote, Hai Tran, Robert J.G. Cardnell, David J. Bearss, Steven L. Warner, Jason M. Foulks,
Steven B. Kanner, Varsha Gandhi, Nancy Krett, Steven T. Rosen, Edward S. Kim, Roy S. Herbst, George R. Blumenschein, J. Jack Lee, Scott
M. Lippman, K. Kian Ang, Gordon B. Mills, Waun K. Hong, John N. Weinstein, Ignacio I. Wistuba, Kevin R. Coombes, John D. Minna,
and John V. Heymach .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..18
Molecular Cancer Research
Radiation Survivors: Understanding and Exploiting the Phenotype following Fractionated Radiation Therapy
Adeola Y. Makinde, Molykutty John-Aryankalayil, Sanjeewani T. Palayoor, David Cerna, and C. Norman Coleman .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..19
A Modified HSP70 Inhibitor Shows Broad Activity as an Anticancer Agent
Gregor M. Balaburski, Julia I.-Ju Leu, Neil Beeharry, Seth Hayik, Mark D. Andrake, Gao Zhang, Meenhard Herlyn,
Jessie Villanueva, Roland L. Dunbrack, Tim Yen, Donna L. George, and Maureen E. Murphy .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..20
The Heterochronic microRNA let-7 Inhibits Cell Motility by Regulating the Genes in the
Actin Cytoskeleton Pathway in Breast Cancer
Xiaowen Hu, Jinyi Guo, Lan Zheng, Chunsheng Li, Tim M. Zheng, Janos L. Tanyi, Shun Liang, Chiara Benedetto,
Marco Mitidieri, Dionyssios Katsaros, Xia Zhao, Youcheng Zhang, Qihong Huang, and Lin Zhang .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..20
Procollagen Lysyl Hydroxylase 2 Is Essential for Hypoxia-Induced Breast Cancer Metastasis
Daniele M. Gilkes, Saumendra Bajpai, Carmen C. Wong, Pallavi Chaturvedi, Maimon E. Hubbi, Denis Wirtz,
and Gregg L. Semenza .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..21
Activation of the FGF2-FGFR1 Autocrine Pathway: A Novel Mechanism of Acquired Resistance to Gefitinib in NSCLC
Hideki Terai, Kenzo Soejima, Hiroyuki Yasuda, Sohei Nakayama, Junko Hamamoto, Daisuke Arai, Kota Ishioka,
Keiko Ohgino, Shinnosuke Ikemura, Takashi Sato, Satoshi Yoda, Ryosuke Satomi, Katsuhiko Naoki, and Tomoko Betsuyaku . .. .. .. .. .. .. .. .. .. ..21
Molecular Cancer Therapeutics
FOX(M1) News—It Is Cancer
Marianna Halasi and Andrei L. Gartel . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..22
Genome and Transcriptome Sequencing in Prospective Metastatic Triple-Negative
Breast Cancer Uncovers Therapeutic Vulnerabilities
David W. Craig, Joyce A. O’Shaughnessy, Jeffrey A. Kiefer, Jessica Aldrich, Shripad Sinari, Tracy M. Moses,
Shukmei Wong, Jennifer Dinh, Alexis Christoforides, Joanne L. Blum, Cristi L. Aitelli, Cynthia R. Osborne,
Tyler Izatt, Ahmet Kurdoglu, Angela Baker, Julie Koeman, Catalin Barbacioru, Onur Sakarya,
Francisco M. De La Vega, Asim Siddiqui, Linh Hoang, Paul R. Billings, Bodour Salhia, Anthony W. Tolcher,
Jeffrey M. Trent, Spyro Mousses, Daniel Von Hoff, and John D. Carpten .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..23
EGFR Exon 20 Insertion Mutations in Lung Adenocarcinomas: Prevalence,
Molecular Heterogeneity, and Clinicopathologic Characteristics
Maria E. Arcila, Khedoudja Nafa, Jamie E. Chaft, Natasha Rekhtman, Christopher Lau,
Boris A. Reva, Maureen F. Zakowski, Mark G. Kris, and Marc Ladanyi . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..23
NF-kB2/p52 Induces Resistance to Enzalutamide in Prostate Cancer:
Role of Androgen Receptor and Its Variants
Nagalakshmi Nadiminty, Ramakumar Tummala, Chengfei Liu, Joy Yang, Wei Lou,
Christopher P. Evans, and Allen C. Gao .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..24
Inhibition of Mutant GNAQ Signaling in Uveal Melanoma Induces
AMPK-Dependent Autophagic Cell Death
Grazia Ambrosini, Elgilda Musi, Alan L. Ho, Elisa de Stanchina, and Gary K. Schwartz .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..24
The Best of the AACR Journals
iii

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Cancer
Cancer Discovery Discovery
Cancer Discovery
CANCER
DISCOVERY
FEBRUARY
www.aacrjournals.org
Lewis C. Cantley, PhD
Editor-in-Chief
José Baselga, MD, PhD
Editor-in-Chief
Scope
Cancer Discovery publishes high-impact, peerreviewed
articles describing major advances in
research and clinical trials. As the premier cancer
information resource, the Journal presents
Review Articles, Perspectives and Commentaries,
News stories, and Research Watch summaries
of important journal articles to its readers to
keep them informed about the latest findings
in the field. Topics span the spectrum of cancer
research and medicine from the laboratory to the
clinic and epidemiologic studies.
Review Article
The Basic Principles of Chimeric Antigen Receptor Design
Michel Sadelain, Renier Brentjens, and Isabelle Rivière
Abstract
Chimeric antigen receptors (CAR) are recombinant receptors
that provide both antigen-binding and T-cell–activating
functions. A multitude of CARs has been reported over the past
decade, targeting an array of cell surface tumor antigens. Their
biologic functions have dramatically changed following the
introduction of tripartite receptors comprising a costimulatory
domain, termed second-generation CARs. These have recently
shown clinical benefit in patients treated with CD19-targeted
autologous T cells. CARs may be combined with costimulatory
ligands, chimeric costimulatory receptors, or cytokines to
further enhance T-cell potency, specificity, and safety. CARs
represent a new class of drugs with exciting potential for cancer
immunotherapy.
Michel Sadelain
Significance: CARs are a new class of drugs with great potential for
cancer immunotherapy. Upon their expression in T lymphocytes,
CARs direct potent, targeted immune responses that have recently
shown encouraging clinical outcomes in a subset of patients with
B-cell malignancies. This review focuses on the design of CARs,
including the requirements for optimal antigen recognition and
different modalities to provide costimulatory support to targeted T
cells, which include the use of second- and third-generation CARs,
costimulatory ligands, chimeric costimulatory receptors, and
cytokines. Cancer Discov; 3(4); 388–98. ©2013 AACR.
Cancer Discov April 2013 3:388–98; Published OnlineFirst April 2,
2013; doi:10.1158/2159-8290.CD-12-0548
The Best of the AACR Journals
1

Cancer Discovery
Research Article
Activating HER2 Mutations in HER2 Gene
Amplification Negative Breast Cancer
Ron Bose, Shyam M. Kavuri, Adam C. Searleman, Wei Shen, Dong Shen, Daniel C. Koboldt,
John Monsey, Nicholas Goel, Adam B. Aronson, Shunqiang Li, Cynthia X. Ma, Li Ding,
Elaine R. Mardis, and Matthew J. Ellis
Ron Bose
Matthew J. Ellis
Abstract
Data from 8 breast cancer genome-sequencing projects identified
25 patients with HER2 somatic mutations in cancers lacking
HER2 gene amplification. To determine the phenotype of these
mutations, we functionally characterized 13 HER2 mutations
using in vitro kinase assays, protein structure analysis, cell
culture, and xenograft experiments. Seven of these mutations
are activating mutations, including G309A, D769H, D769Y,
V777L, P780ins, V842I, and R896C. HER2 in-frame deletion
755–759, which is homologous to EGF receptor (EGFR) exon
19 in-frame deletions, had a neomorphic phenotype with
increased phosphorylation of EGFR or HER3. L755S produced
lapatinib resistance, but was not an activating mutation in our
experimental systems. All of these mutations were sensitive to
the irreversible kinase inhibitor, neratinib. These findings show
that HER2 somatic mutation is an alternative mechanism to
activate HER2 in breast cancer and they validate HER2 somatic
mutations as drug targets for breast cancer treatment.
Significance: We show that the majority of HER2 somatic
mutations in breast cancer patients are activating mutations
that likely drive tumorigenesis. Several patients had mutations
that are resistant to the reversible HER2 inhibitor lapatinib, but
are sensitive to the irreversible HER2 inhibitor, neratinib. Our
results suggest that patients with HER2 mutation–positive breast
cancers could benefit from existing HER2-targeted drugs. Cancer
Discov; 3(2); 224–37. ©2013 AACR.
Cancer Discov February 2013 3:224–37; Published OnlineFirst
December 7, 2012; doi:10.1158/2159-8290.CD-12-0349
Research Article
First-in-Humans Trial of an RNA Interference Therapeutic Targeting
VEGF and KSP in Cancer Patients with Liver Involvement
Josep Tabernero, Geoffrey I. Shapiro, Patricia M. LoRusso, Andres Cervantes, Gary K. Schwartz, Glen J. Weiss, Luis Paz-Ares,
Daniel C. Cho, Jeffrey R. Infante, Maria Alsina, Mrinal M. Gounder, Rick Falzone, Jamie Harrop, Amy C. Seila White,
Iva Toudjarska, David Bumcrot, Rachel E. Meyers, Gregory Hinkle, Nenad Svrzikapa, Renta M. Hutabarat, Valerie A. Clausen,
Jeffrey Cehelsky, Saraswathy V. Nochur, Christina Gamba-Vitalo, Akshay K. Vaishnaw, Dinah W.Y. Sah, Jared A. Gollob,
and Howard A. Burris III
Josep Tabernero
Abstract
RNA interference (RNAi) is a potent and specific mechanism for
regulating gene expression. Harnessing RNAi to silence genes
involved in disease holds promise for the development of a new
class of therapeutics. Delivery is key to realizing the potential
of RNAi, and lipid nanoparticles (LNP) have proved effective
in delivery of siRNAs to the liver and to tumors in animals. To
examine the activity and safety of LNP-formulated siRNAs in
humans, we initiated a trial of ALN-VSP, an LNP formulation
of siRNAs targeting VEGF and kinesin spindle protein (KSP),
in patients with cancer. Here, we show detection of drug in
tumor biopsies, siRNA-mediated mRNA cleavage in the liver,
pharmacodynamics suggestive of target downregulation, and
antitumor activity, including complete regression of liver
metastases in endometrial cancer. In addition, we show that
biweekly intravenous administration of ALN-VSP was safe
and well tolerated. These data provide proof-of-concept for
RNAi therapeutics in humans and form the basis for further
development in cancer.
Significance: The findings in this report show safety,
pharmacokinetics, RNAi mechanism of action, and clinical
activity with a novel first-in-class LNP-formulated RNAi
therapeutic in patients with cancer. The ability to harness RNAi to
facilitate specific multitargeting, as well as increase the number
of druggable targets, has important implications for future drug
development in oncology. Cancer Discov; 3(4); 406–17. ©2012 AACR.
Cancer Discov April 2013 3:406–17; Published OnlineFirst January
28, 2013; doi:10.1158/2159-8290.CD-12-0429
2
aacrjournals.org

Cancer Discovery
Research Article
Amplification of the MET Receptor Drives Resistance to
Anti-EGFR Therapies in Colorectal Cancer
Alberto Bardelli, Simona Corso, Andrea Bertotti, Sebastijan Hobor, Emanuele Valtorta, Giulia Siravegna,
Andrea Sartore-Bianchi, Elisa Scala, Andrea Cassingena, Davide Zecchin, Maria Apicella, Giorgia Migliardi,
Francesco Galimi, Calogero Lauricella, Carlo Zanon, Timothy Perera, Silvio Veronese, Giorgio Corti,
Alessio Amatu, Marcello Gambacorta, Luis A. Diaz Jr, Mark Sausen, Victor E. Velculescu, Paolo Comoglio,
Livio Trusolino, Federica Di Nicolantonio, Silvia Giordano, and Salvatore Siena
Alberto Bardelli
Silvia Giordano
Abstract
EGF receptor (EGFR)-targeted monoclonal antibodies are
effective in a subset of metastatic colorectal cancers. Inevitably,
all patients develop resistance, which occurs through emergence
of KRAS mutations in approximately 50% of the cases. We show
that amplification of the MET proto-oncogene is associated
with acquired resistance in tumors that do not develop KRAS
mutations during anti-EGFR therapy. Amplification of the MET
locus was present in circulating tumor DNA before relapse was
clinically evident. Functional studies show that MET activation
confers resistance to anti-EGFR therapy both in vitro and in
vivo. Notably, in patient-derived colorectal cancer xenografts,
MET amplification correlated with resistance to EGFR blockade,
which could be overcome by MET kinase inhibitors. These results
highlight the role of MET in mediating primary and secondary
resistance to anti-EGFR therapies in colorectal cancer and
encourage the use of MET inhibitors in patients displaying
resistance as a result of MET amplification.
Significance: Amplification of the MET proto-oncogene is
responsible for de novo and acquired resistance to anti-EGFR
therapy in a subset of colorectal cancers. As multiple anti-
MET therapeutic strategies are available, these findings offer
immediate novel opportunities to design clinical studies. Cancer
Discov; 3(6); 658–73. ©2013 AACR.
Cancer Discov June 2013 3:658–73; Published OnlineFirst June 6,
2013; doi:10.1158/2159-8290.CD-12-0558
Research Brief
Identification of Targetable FGFR Gene Fusions in
Diverse Cancers
Yi-Mi Wu, Fengyun Su, Shanker Kalyana-Sundaram, Nickolay Khazanov, Bushra Ateeq, Xuhong Cao,
Robert J. Lonigro, Pankaj Vats, Rui Wang, Su-Fang Lin, Ann-Joy Cheng, Lakshmi P. Kunju, Javed Siddiqui,
Scott A. Tomlins, Peter Wyngaard, Seth Sadis, Sameek Roychowdhury, Maha H. Hussain, Felix Y. Feng,
Mark M. Zalupski, Moshe Talpaz, Kenneth J. Pienta, Daniel R. Rhodes, Dan R. Robinson, and
Arul M. Chinnaiyan
Dan R. Robinson
Arul M. Chinnaiyan
Abstract
Through a prospective clinical sequencing program for
advanced cancers, four index cases were identified which
harbor gene rearrangements of FGFR2, including patients with
cholangiocarcinoma, breast cancer, and prostate cancer. After
extending our assessment of FGFR rearrangements across
multiple tumor cohorts, we identified additional FGFR fusions
with intact kinase domains in lung squamous cell cancer, bladder
cancer, thyroid cancer, oral cancer, glioblastoma, and head and
neck squamous cell cancer. All FGFR fusion partners tested
exhibit oligomerization capability, suggesting a shared mode
of kinase activation. Overexpression of FGFR fusion proteins
induced cell proliferation. Two bladder cancer cell lines that
harbor FGFR3 fusion proteins exhibited enhanced susceptibility
to pharmacologic inhibition in vitro and in vivo. Because of the
combinatorial possibilities of FGFR family fusion to a variety
of oligomerization partners, clinical sequencing efforts, which
incorporate transcriptome analysis for gene fusions, are poised
to identify rare, targetable FGFR fusions across diverse cancer
types.
Significance: High-throughput sequencing technologies facilitate
defining the mutational landscape of human cancers, which will
lead to more precise treatment of patients with cancer. Here,
through integrative sequencing efforts, we identified a variety of
FGFR gene fusions in a spectrum of human cancers. FGFR fusions
are active kinases. Cells harboring FGFR fusions showed enhanced
sensitivity to the FGFR inhibitors PD173074 and pazopanib,
suggesting that patients with cancer with FGFR fusions may
benefit from targeted FGFR kinase inhibition. Cancer Discov; 3(6);
636–47. ©2013 AACR.
Cancer Discov June 2013 3:636–47; Published OnlineFirst April 4,
2013; doi:10.1158/2159-8290.CD-13-0050
The Best of the AACR Journals 3

In collaboration with the
AACR Molecular
American Society of
Preventive Oncology
Cancer
Epidemiology,
Biomarkers &
Prevention
Cancer Epidemiology,
Biomarkers & Prevention
Cancer
Epidemiology,
Biomarkers
& Prevention
April 2013
Volume 22 • Number 4
Pages 479–744
Translating Science to Populations
www.aacrjournals.org
SPECIAL CONTENT
IN THIS ISSUE!
Epidemiology Group
and the
Timothy R. Rebbeck, PhD
Editor-in-Chief
Scope
Cancer Epidemiology, Biomarkers & Prevention publishes original peerreviewed,
population-based research on cancer etiology, prevention,
surveillance, and survivorship. The following topics are of special interest:
descriptive, analytical, and molecular epidemiology; biomarkers including
assay development, validation, and application; chemoprevention and
other types of prevention research in the context of descriptive and
observational studies; the role of behavioral factors in cancer etiology
and prevention; survivorship studies; risk factors; and the science of cancer
health disparities. Besides welcoming manuscripts that address individual
subjects in any of the relevant disciplines, CEBP editors encourage the
submission of manuscripts with a transdisciplinary approach.
Review Article
Oral Contraceptive Use and Risk of Breast, Cervical,
Colorectal, and Endometrial Cancers: A Systematic Review
Jennifer M. Gierisch, Remy R. Coeytaux, Rachel Peragallo Urrutia, Laura J. Havrilesky, Patricia G. Moorman,
William J. Lowery, Michaela Dinan, Amanda J. McBroom, Vic Hasselblad, Gillian D. Sanders, and Evan R. Myers
Jennifer M. Gierisch
Abstract
Oral contraceptives may influence the risk of certain cancers.
As part of the AHRQ Evidence Report, Oral Contraceptive Use
for the Primary Prevention of Ovarian Cancer, we conducted
a systematic review to estimate associations between oral
contraceptive use and breast, cervical, colorectal, and
endometrial cancer incidence. We searched PubMed, Embase,
and Cochrane Database of Systematic Reviews. Study inclusion
criteria were women taking oral contraceptives for contraception
or ovarian cancer prevention; includes comparison group with
no oral contraceptive use; study reports quantitative associations
between oral contraceptive exposure and relevant cancers;
controlled study or pooled patient-level meta-analyses; sample
size for nonrandomized studies ≥100; peer-reviewed, Englishlanguage;
published from January 1, 2000 forward. Randomeffects
meta-analyses were conducted by estimating pooled ORs
with 95% confidence intervals (CIs). We included 44 breast, 12
cervical, 11 colorectal, and 9 endometrial cancers studies. Breast
cancer incidence was slightly but significantly increased in users
(OR, 1.08; CI, 1.00–1.17); results show a higher risk associated
with more recent use of oral contraceptives. Risk of cervical
cancer was increased with duration of oral contraceptive use
in women with human papillomavirus infection; heterogeneity
prevented meta-analysis. Colorectal cancer (OR, 0.86; CI, 0.79–
0.95) and endometrial cancer incidences (OR, 0.57; CI, 0.43–0.77)
were significantly reduced by oral contraceptive use. Compared
with never use, ever use of oral contraceptives is significantly
associated with decreases in colorectal and endometrial cancers
and increases in breast cancers. Although elevated breast cancer
risk was small, relatively high incidence of breast cancers means
that oral contraceptives may contribute to a substantial number
of cases. Cancer Epidemiol Biomarkers Prev; 22(11); 1931–43.
©2013 AACR.
Cancer Epidemiol Biomarkers Prev November 2013 22:1931–43;
Published OnlineFirst September 6, 2013; doi:10.1158/1055-9965.
EPI-13-0298
4
aacrjournals.org

Cancer Epidemiology, Biomarkers & Prevention
Research Article
Cancer Survivors in the United States: Prevalence across the
Survivorship Trajectory and Implications for Care
Janet S. de Moor, Angela B. Mariotto, Carla Parry, Catherine M. Alfano, Lynne Padgett, Erin E. Kent, Laura Forsythe,
Steve Scoppa, Mark Hachey, and Julia H. Rowland
Janet S. de Moor
Abstract
Background: Cancer survivors represent a growing population,
heterogeneous in their need for medical care, psychosocial
support, and practical assistance. To inform survivorship
research and practice, this manuscript will describe the prevalent
population of cancer survivors in terms of overall numbers and
prevalence by cancer site and time since diagnosis.
Methods: Incidence and survival data from 1975–2007 were
obtained from the Surveillance, Epidemiology, and End Results
Program and population projections from the United States
Census Bureau. Cancer prevalence for 2012 and beyond was
estimated using the Prevalence Incidence Approach Model,
assuming constant future incidence and survival trends but
dynamic projections of the U.S. population.
Results: As of January 1, 2012, approximately 13.7 million
cancer survivors were living in the United States with prevalence
projected to approach 18 million by 2022. Sixty-four percent of
this population have survived 5 years or more; 40% have survived
10 years or more; and 15% have survived 20 years or more after
diagnosis. Over the next decade, the number of people who have
lived 5 years or more after their cancer diagnosis is projected to
increase approximately 37% to 11.9 million.
Conclusions: A coordinated agenda for research and practice
is needed to address cancer survivors’ long-term medical,
psychosocial, and practical needs across the survivorship
trajectory.
Impact: Prevalence estimates for cancer survivors across the
survivorship trajectory will inform the national research agenda
as well as future projections about the health service needs of
this population. Cancer Epidemiol Biomarkers Prev; 22(4); 561–70.
©2013 AACR.
Cancer Epidemiol Biomarkers Prev April 2013 22:561–70;
Published OnlineFirst March 27, 2013; doi:10.1158/1055-9965.
EPI-12-1356
Research Article
Vegetarian Diets and the Incidence of Cancer in a
Low-risk Population
Yessenia Tantamango-Bartley, Karen Jaceldo-Siegl, Jing Fan, and Gary Fraser
Abstract
Background: Cancer is the second leading cause of death in
the United States. Dietary factors account for at least 30% of
all cancers in Western countries. As people do not consume
individual foods but rather combinations of them, the assessment
of dietary patterns may offer valuable information when
determining associations between diet and cancer risk.
Methods: We examined the association between dietary patterns
(non-vegetarians, lacto, pesco, vegan, and semi-vegetarian) and the
overall cancer incidence among 69,120 participants of the Adventist
Health Study-2. Cancer cases were identified by matching to cancer
registries. Cox proportional hazard regression analysis was conducted
to estimate hazard ratios, with “attained age” as the time variable.
Results: A total of 2,939 incident cancer cases were identified. The
multivariate HR of overall cancer risk among vegetarians compared
with non-vegetarians was statistically significant [HR, 0.92; 95%
confidence interval (CI), 0.85–0.99] for both genders combined.
Also, a statistically significant association was found between
vegetarian diet and cancers of the gastrointestinal tract (HR, 0.76;
Yessenia Tantamango-
Bartley
95% CI, 0.63–0.90). When analyzing the association of specific
vegetarian dietary patterns, vegan diets showed statistically
significant protection for overall cancer incidence (HR, 0.84; 95%
CI, 0.72–0.99) in both genders combined and for female-specific
cancers (HR, 0.66; 95% CI, 0.47–0.92). Lacto-ovo-vegetarians
appeared to be associated with decreased risk of cancers of the
gastrointestinal system (HR, 0.75; 95% CI, 0.60–0.92).
Conclusion: Vegetarian diets seem to confer protection against
cancer.
Impact: Vegan diet seems to confer lower risk for overall and
female-specific cancer than other dietary patterns. The lactoovo-vegetarian
diets seem to confer protection from cancers
of the gastrointestinal tract. Cancer Epidemiol Biomarkers Prev;
22(2); 286–94. ©2012 AACR.
Cancer Epidemiol Biomarkers Prev February 2013 22:286–94;
Published OnlineFirst November 20, 2012; doi:10.1158/1055-9965.
EPI-12-1060
The Best of the AACR Journals 5

Cancer Epidemiology, Biomarkers & Prevention
Research Article
Genetic Susceptibility Loci for Subtypes of Breast Cancer in
an African American Population
Julie R. Palmer, Edward A. Ruiz-Narvaez, Charles N. Rotimi, L. Adrienne Cupples, Yvette C. Cozier,
Lucile L. Adams-Campbell, and Lynn Rosenberg
Julie R. Palmer
Abstract
Background: Most genome-wide association studies (GWAS)
have been carried out in European ancestry populations; no
risk variants for breast cancer have been identified solely from
African ancestry GWAS data. Few GWAS hits have replicated in
African ancestry populations.
Methods: In a nested case–control study of breast cancer in the
Black Women’s Health Study (1,199 cases/1,948 controls), we
evaluated index single-nucleotide polymorphisms (SNP) in 21 loci
from GWAS of European or Asian ancestry populations, overall,
in subtypes defined by estrogen receptor (ER) and progesterone
receptor (PR) status (ER+/PR+, n = 336; ER−/PR−, n = 229), and
in triple-negative breast cancer (TNBC, N = 81). To evaluate the
contribution of genetic factors to population differences in breast
cancer subtype, we also examined global percent African ancestry.
Results: Index SNPs in five loci were replicated, including three
associated with ER−/PR− breast cancer (TERT rs10069690 in
5p15.33, rs704010 in 10q22.3, and rs8170 in 19p13.11): per allele
ORs were 1.29 [95% confidence interval (CI) 1.04–1.59], P = 0.02,
1.52 (95% CI 1.12–2.08), P = 0.01, and 1.30 (95% CI 1.01–1.68),
P = 0.04, respectively. Stronger associations were observed for
TNBC. Furthermore, cases in the highest quintile of percent
African ancestry were three times more likely to have TNBC than
ER+/PR+ cancer.
Conclusions: These findings provide the first confirmation of
the TNBC SNP rs8170 in an African ancestry population, and
independent confirmation of the TERT ER− SNP. Furthermore,
the risk of developing ER− breast cancer, particularly TNBC,
increased with increasing proportion of global African ancestry.
Impact: The findings illustrate the importance of genetic factors
in the disproportionately high occurrence of TNBC in African
American women. Cancer Epidemiol Biomarkers Prev; 22(1); 127–
34. ©2012 AACR.
Cancer Epidemiol Biomarkers Prev January 2013 22:127–34;
Published OnlineFirst November 7, 2012; doi:10.1158/1055-9965.
EPI-12-0769
Research Article
Drinking Water Arsenic in Northern Chile: High Cancer
Risks 40 Years after Exposure Cessation
Craig M. Steinmaus, Catterina Ferreccio, Johanna Acevedo Romo, Yan Yuan, Sandra Cortes, Guillermo Marshall,
Lee E. Moore, John R. Balmes, Jane Liaw, Todd Golden, and Allan H. Smith
Craig M. Steinmaus
Abstract
Background: Millions of people worldwide are exposed to
arsenic-contaminated water. In the largest city in northern Chile
(Antofagasta), more than 250,000 people were exposed to high
arsenic drinking water concentrations from 1958 until 1970 when a
water treatment plant was installed. Because of its unique geology,
limited water sources, and good historical records, lifetime exposure
and long-term latency patterns can be assessed in this area with
better accuracy than in other arsenic-exposed areas worldwide.
Methods: We conducted a population-based case–control study
in northern Chile from October 2007 to December 2010 involving
232 bladder and 306 lung cancer cases and 640 age- and gendermatched
controls, with detailed information on past exposure
and potential confounders, including smoking and occupation.
Results: Bladder cancer ORs for quartiles of average arsenic
concentrations in water before 1971 (335 μg/L) were 1.00, 1.36 [95% confidence interval (CI),
0.78–2.37], 3.87 (2.25–6.64), and 6.50 (3.69–11.43), respectively.
Corresponding lung cancer ORs were 1.00, 1.27 (0.81–1.98), 2.00
(1.24–3.24), and 4.32 (2.60–7.17). Bladder and lung cancer ORs in
those highly exposed in Antofagasta during 1958 to 1970 but not
thereafter were 6.88 (3.84–12.32) and 4.35 (2.57–7.36), respectively.
Conclusions: The lung and bladder cancer risks that we found
up to 40 years after high exposures have ended are very high.
Impact: Our findings suggest that prevention, treatment, and
other mortality reduction efforts in arsenic-exposed countries
will be needed for decades after exposure cessation. Cancer
Epidemiol Biomarkers Prev; 22(4); 623–30. ©2013 AACR.
Cancer Epidemiol Biomarkers Prev April 2013 22:623–30; Published
OnlineFirst January 25, 2013; doi:10.1158/1055-9965.EPI-12-1190
6
aacrjournals.org

In collaboration with the
Cancer
Immunology
Research
Cancer Immunology Research
Cancer
Immunology
Research
July 2013 • Volume 1 • Number 1 • Pages 1–76
Illuminating the Interplay of
Cancer and the Immune System
www.aacrjournals.org
Cancer Research Institute
Glenn Dranoff, MD
Editor-in-Chief
Scope
Cancer Immunology Research publishes outstanding original articles
reporting major advances in cancer immunology that span the discipline
from basic investigations in host-tumor interactions to developmental
therapeutics in model systems, early translational studies in patients,
and late-stage clinical trials. The journal disseminates knowledge of
immunology to the cancer research community, catalyzing crossdisciplinary
work that yields a deeper understanding of the host-tumor
relationship, more potent cancer treatments, and improved clinical
outcomes.
Review Article
Getting Personal with Neoantigen-Based
Therapeutic Cancer Vaccines
Nir Hacohen, Edward F. Fritsch, Todd A. Carter, Eric S. Lander, and Catherine J. Wu
Abstract
Despite years of preclinical efforts and hundreds of clinical
studies, therapeutic cancer vaccines with the routine ability
to limit or eliminate tumor growth in humans have been
elusive. With advances in genome sequencing, it is now
possible to identify a new class of tumor-specific antigens
derived from mutated proteins that are present only in the
tumor. These “neoantigens” should provide highly specific
targets for antitumor immunity. Although many challenges
Catherine J. Wu
remain in producing and testing neoantigen-based vaccines
customized for each patient, a neoantigen vaccine offers a
promising new approach to induce highly focused antitumor
T cells aimed at eradicating cancer cells. Cancer Immunol Res;
1(1); 11–15. 2013 AACR.
Cancer Immunol Res July 2013; 1:11–15; Published OnlineFirst April
7, 2013; doi: 10.1158/2326-6066.CIR-13-0022
The Best of the AACR Journals 7

Cancer Immunology Research
Research Article
Anti-CTLA-4 Antibodies of IgG2a Isotype Enhance Antitumor
Activity through Reduction of Intratumoral Regulatory T Cells
Mark J. Selby, John J. Engelhardt, Michael Quigley, Karla A. Henning, Timothy Chen, Mohan Srinivasan, and
Alan J. Korman
Alan J. Korman
Abstract
Antitumor activity of CTLA-4 antibody blockade is thought
to be mediated by interfering with the negative regulation of
T-effector cell (Teff) function resulting from CTLA-4 engagement
by B7-ligands. In addition, a role for CTLA-4 on regulatory T
cells (Treg), wherein CTLA-4 loss or inhibition results in reduced
Treg function, may also contribute to antitumor responses
by anti-CTLA-4 treatment. We have examined the role of the
immunoglobulin constant region on the antitumor activity of
anti-CTLA-4 to analyze in greater detail the mechanism of action
of anti-CTLA-4 antibodies. Anti-CTLA-4 antibody containing
the murine immunoglobulin G (IgG)2a constant region exhibits
enhanced antitumor activity in subcutaneous established MC38
and CT26 colon adenocarcinoma tumor models compared
with anti-CTLA-4 containing the IgG2b constant region.
Interestingly, anti-CTLA-4 antibodies containing mouse IgG1
or a mutated mouse IgG1-D265A, which eliminates binding to
all Fcγ receptors (FcγR), do not show antitumor activity in these
models. Assessment of Teff and Treg populations at the tumor
and in the periphery showed that anti-CTLA-4-IgG2a mediated
a rapid and dramatic reduction of Tregs at the tumor site,
whereas treatment with each of the isotypes expanded Tregs in
the periphery. Expansion of CD8 + Teffs is observed with both the
IgG2a and IgG2b anti-CTLA-4 isotypes, resulting in a superior
Teff to Treg ratio for the IgG2a isotype. These data suggest that
anti-CTLA-4 promotes antitumor activity by a selective reduction
of intratumoral Tregs along with concomitant activation of Teffs.
Cancer Immunol Res; 1(1); 32–42. ©2013 AACR.
Cancer Immunol Res July 2013; 1:32–42; Published OnlineFirst April
7, 2013; doi: 10.1158/2326-6066.CIR-13-0013
Research Article
An Abscopal Response to Radiation and Ipilimumab in a
Patient with Metastatic Non–Small Cell Lung Cancer
Encouse B. Golden, Sandra Demaria, Peter B. Schiff, Abraham Chachoua, and Silvia C. Formenti
Abstract
A posteriori evidence suggests that radiotherapy to a targeted
tumor can elicit an immune-mediated abscopal (ab-scopus,
away from the target) effect in nontargeted tumors, when
combined with an anti-CTL antigen-4 (CTLA-4) monoclonal
antibody. Concurrent radiotherapy and CTLA-4 blockade
induced immune-mediated abscopal effects in poorly
immunogenic preclinical tumor models and patients with
metastatic melanoma. However, no such reports exist for
patients with metastatic lung adenocarcinoma. We report the
first abscopal response in a treatment-refractory lung cancer
Silvia C. Formenti
patient treated with radiotherapy and ipilimumab (a human
anti-CTLA-4 monoclonal antibody). A posttreatment increase in
tumor-infiltrating cytotoxic lymphocytes, tumor regression, and
normalization of tumor markers was observed. One year after
treatment with concurrent radiotherapy and ipilimumab, the
patient is without evidence of disease. Cancer Immunol Res; 1(6);
365–72. ©2013 AACR.
Cancer Immunol Res December 2013; 1:365–72; Published
OnlineFirst October 9, 2013; doi: 10.1158/2326-6066.CIR-13-0115
8
aacrjournals.org

Cancer Immunology Research
Research Article
T Cells Expressing Chimeric Antigen Receptors Can Cause
Anaphylaxis in Humans
Marcela V. Maus, Andrew R. Haas, Gregory L. Beatty, Steven M. Albelda, Bruce L. Levine, Xiaojun Liu, Yangbing Zhao,
Michael Kalos, and Carl H. June
Marcela V. Maus
Abstract
T cells can be redirected to overcome tolerance to cancer by
engineering with integrating vectors to express a chimeric antigen
receptor (CAR). In preclinical models, we have previously shown
that transfection of T cells with mRNA coding for a CAR is an
alternative strategy that has antitumor efficacy and the potential
to evaluate the on-target off-tumor toxicity of new CAR targets
safely due to transient mRNA CAR expression. Here, we report
the safety observed in four patients treated with autologous T
cells that had been electroporated with mRNA coding for a CAR
derived from a murine antibody to human mesothelin. Because
of the transient nature of CAR expression on the T cells, subjects
in the clinical study were given repeated infusions of the CAR-T
cells to assess their safety. One subject developed anaphylaxis and
cardiac arrest within minutes of completing the third infusion.
Although human anti-mouse immunoglobulin (Ig)G antibodies
have been known to develop with CAR-transduced T cells, they
have been thought to have no adverse clinical consequences. This
is the first description of clinical anaphylaxis resulting from CARmodified
T cells, most likely through IgE antibodies specific to the
CAR. These results indicate that the potential immunogenicity of
CARs derived from murine antibodies may be a safety issue for
mRNA CARs, especially when administered using an intermittent
dosing schedule. Cancer Immunol Res; 1(1); 26–31. ©2013 AACR.
Cancer Immunol Res July 2013; 1:26–31; Published OnlineFirst April
7, 2013; doi: 10.1158/2326-6066.CIR-13-0006
Research Article
PD-L1 Expression in the Merkel Cell Carcinoma
Microenvironment: Association with Inflammation,
Merkel Cell Polyomavirus, and Overall Survival
Evan J. Lipson, Jeremy G. Vincent, Myriam Loyo, Luciane T. Kagohara, Brandon S. Luber, Hao Wang,
Haiying Xu, Suresh K. Nayar, Timothy S. Wang, David Sidransky, Robert A. Anders, Suzanne L. Topalian,
and Janis M. Taube
Suzanne L. Topalian
Janis M. Taube
Abstract
Merkel cell carcinoma (MCC) is a lethal, virus-associated cancer
that lacks effective therapies for advanced disease. Agents
blocking the PD-1/PD-L1 pathway have shown objective, durable
tumor regressions in patients with advanced solid malignancies
and efficacy has been linked to PD-L1 expression in the tumor
microenvironment. To investigate whether MCC might be a
target for PD-1/PD-L1 blockade, we examined MCC PD-L1
expression, its association with tumor-infiltrating lymphocytes
(TIL), Merkel cell polyomavirus (MCPyV), and overall survival.
Sixty-seven MCC specimens from 49 patients were assessed with
immunohistochemistry for PD-L1 expression by tumor cells and
TILs, and immune infiltrates were characterized phenotypically.
Tumor cell and TIL PD-L1 expression were observed in 49%
and 55% of patients, respectively. In specimens with PD-L1(+)
tumor cells, 97% (28/29) showed a geographic association with
immune infiltrates. Among specimens with moderate-severe TIL
intensities, 100% (29/29) showed PD-L1 expression by tumor cells.
Significant associations were also observed between the presence
of MCPyV DNA, a brisk inflammatory response, and tumor cell
PD-L1 expression: MCPyV(-) tumor cells were uniformly PD-
L1(-). Taken together, these findings suggest that a local tumorspecific
and potentially MCPyV-specific immune response
drives tumor PD-L1 expression, similar to previous observations
in melanoma and head and neck squamous cell carcinomas.
In multivariate analyses, PD-L1(-) MCCs were independently
associated with worse overall survival [HR 3.12; 95% confidence
interval, 1.28–7.61; P = 0.012]. These findings suggest that an
endogenous immune response promotes PD-L1 expression in the
MCC microenvironment when MCPyV is present, and provide
a rationale for investigating therapies blocking PD-1/PD-L1 for
patients with MCC. Cancer Immunol Res; 1(1); 54–63. ©2013 AACR.
Cancer Immunol Res July 2013; 1:54–63; Published OnlineFirst May
21, 2013; doi: 10.1158/2326-6066.CIR-13-0034
The Best of the AACR Journals 9

Cancer
Prevention
Research
Cancer Prevention Research
The Forefront of Prevention Science
www.aacrjournals.org
Cancer
Prevention
Research
November 2013 • Volume 6 • Number 11 • Pages 1151–1250
Scott M. Lippman, MD
Editor-in-Chief
Scope
Cancer Prevention Research is devoted exclusively to cancer prevention. The
journal publishes important original studies, reviews, and commentaries
within the major topic areas of biology of premalignancy, risk factors
and risk assessment, early detection research, immunoprevention and
chemopreventive and other interventions, including the basic science
behind them. The journal includes preclinical, clinical and translational
research, with special attention given to molecular discoveries and
an emphasis on building a translational bridge between the basic and
clinical sciences.
Review Article
New Perspectives of Curcumin in Cancer Prevention
Wungki Park, A.R.M. Ruhul Amin, Zhuo Georgia Chen, and Dong M. Shin
Dong M. Shin
Abstract
Numerous natural compounds have been extensively investigated
for their potential for cancer prevention over the decades.
Curcumin, from Curcuma longa, is a highly promising
natural compound that can be potentially used for chemoprevention
of multiple cancers. Curcumin modulates multiple
molecular pathways involved in the lengthy carcinogenesis
process to exert its chemopreventive effects through several
mechanisms: promoting apoptosis, inhibiting survival
signals, scavenging reactive oxidative species (ROS), and reducing
the inflammatory cancer microenvironment. Curcumin
fulfills the characteristics for an ideal chemopreventive
agent with its low toxicity, affordability, and easy accessibility.
Nonetheless, the clinical application of curcumin is currently
compromised by its poor bioavailability. Here, we review the
potential of curcumin in cancer prevention, its molecular targets,
and mechanisms of action. Finally, we suggest specific
recommendations to improve its efficacy and bioavailability
for clinical applications. Cancer Prev Res; 6(5); 387–400. ©2013
AACR.
Cancer Prev Res May 2013 6:387–400; Published OnlineFirst March
6, 2013; doi:10.1158/1940-6207.CAPR-12-0410
10
aacrjournals.org

Cancer Prevention Research
Research Article
Durable Antibody Responses Following One Dose of the Bivalent
Human Papillomavirus L1 Virus-Like Particle Vaccine in the Costa
Rica Vaccine Trial
Mahboobeh Safaeian, Carolina Porras, Yuanji Pan, Aimee Kreimer, John T. Schiller, Paula Gonzalez, Douglas R. Lowy,
Sholom Wacholder, Mark Schiffman, Ana C. Rodriguez, Rolando Herrero, Troy Kemp, Gloriana Shelton, Wim Quint,
Leen-Jan van Doorn, Allan Hildesheim, and Ligia A. Pinto, for the CVT Group
Mahboobeh Safaeian
Abstract
The Costa Rica HPV16/18 Vaccine Trial (CVT) showed that fouryear
vaccine efficacy against 12-month HPV16/18 persistent
infection was similarly high among women who received one,
two, or the recommended three doses of the bivalent HPV16/18 L1
virus-like particle (VLP) vaccine. Live-attenuated viral vaccines,
but not simple-subunit vaccines, usually induce durable lifelong
antibody responses after a single dose. It is unclear whether
noninfectious VLP vaccines behave more like live-virus or simplesubunit
vaccines in this regard. To explore the likelihood that
efficacy will persist longer term, we investigated the magnitude
and durability of antibodies to this vaccine by measuring HPV16-
and HPV18-specific antibodies by VLP-ELISA using serum from
enrollment, vaccination, and annual visits through four years in
four vaccinated groups; one-dose (n = 78), two-doses separated
by one month (n = 140), two doses separated by six months
(n = 52), and three scheduled doses (n = 120, randomly selected).
We also tested enrollment sera from n = 113 HPV16- or HPV18
L1-seropositive women prevaccination, presumably from natural
infection. At four years, 100% of women in all groups remained
HPV16/18 seropositive; both HPV16/18 geometric mean titers
(GMT) among the extended two-dose group were non-inferior
to the three-dose group, and ELISA titers were highly correlated
with neutralization titers in all groups. Compared with the natural
infection group, HPV16/18 GMTs were, respectively, at least 24
and 14 times higher among the two-dose and 9 and 5 times higher
among one-dose vaccinees. Antibody levels following one-dose
remained stable from month 6 through month 48. Results raise the
possibility that even a single dose of HPV VLPs will induce longterm
protection. Cancer Prev Res; 6(11); 1242–50. ©2013 AACR.
Cancer Prev Res November 2013 6:1242–1250; doi:10.1158/1940–6207.
CAPR-13-0203
Research Article
MicroRNA Expression Signatures during Malignant Progression
from Barrett’s Esophagus to Esophageal Adenocarcinoma
Xifeng Wu, Jaffer A. Ajani, Jian Gu, David W. Chang, Weiqi Tan, Michelle A.T. Hildebrandt, Maosheng Huang,
Kenneth K. Wang, and Ernest Hawk
Xifeng Wu
Abstract
Barrett’s esophagus is the precursor lesion of esophageal
adenocarcinoma, whose progression follows sequential stages. However,
the low progression rate and the inadequacy and subjective
interpretation of histologic grading in predicting Barrett’s esophagus
progression call for more objective biomarkers that can improve risk
prediction. We conducted a genome-wide profiling of 754 human
microRNAs (miRNA) in 35 normal epithelium, 34 Barrett’s esophagus,
and 36 esophageal adenocarcinoma tissues using TaqMan real-time
PCR-based profiling. Unsupervised hierarchical clustering using 294
modestly to highly expressed miRNAs showed clear clustering of two
groups: normal epithelium versus Barrett’s esophagus/esophageal
adenocarcinoma tissues. Moreover, there was an excellent clustering
of Barrett’s metaplasia (without dysplasia) tissues from normal
epithelium tissues. However, Barrett’s esophagus tissues of different
stages and esophageal adenocarcinoma tissues were interspersed.
There were differentially expressed miRNAs at different stages. The
majority of miRNA aberrations involved upregulation of expression
in Barrett’s esophagus and esophageal adenocarcinoma tissues,
with the most dramatic alterations occurring at the Barrett’s
metaplasia stage. Known oncomiRs, such as miR-21, miR-25, and
miR-223, and tumor suppressor miRNAs, including miR-205, miR-
203, let-7c, and miR-133a, showed progressively altered expression
from Barrett’s esophagus to esophageal adenocarcinoma. We also
identified a number of novel miRNAs that showed progressively
altered expression, including miR-301b, miR-618, and miR-23b. The
significant miRNA alterations that were exclusive to esophageal
adenocarcinoma but not Barrett’s esophagus included miR-375
downregulation and upregulation of five members of the miR-
17-92 and its homologue clusters, which may become promising
biomarkers for esophageal adenocarcinoma development. Cancer
Prev Res; 6(3); 196–205. ©2013 AACR.
Cancer Prev Res March 2013 6:196–205; doi:10.1158/1940-6207.
CAPR-12-0276
The Best of the AACR Journals 11

Cancer Prevention Research
Research Article
MUC1 Vaccine for Individuals with Advanced Adenoma of
the Colon: A Cancer Immunoprevention Feasibility Study
Takashi Kimura, John R. McKolanis, Lynda A. Dzubinski, Kazi Islam, Douglas M. Potter, Andres M. Salazar,
Robert E. Schoen, and Olivera J. Finn
Robert E. Schoen
Olivera J. Finn
Abstract
Cancer vaccines based on human tumor-associated antigens
(TAA) have been tested in patients with advanced or recurrent
cancer, in combination with or following standard therapy. Their
immunogenicity and therapeutic efficacy has been difficult to
properly evaluate in that setting characterized by multiple highly
suppressive effects of the tumor and the standard therapy on the
patient’s immune system. In animal models of human cancer,
vaccines administered in the prophylactic setting are most
immunogenic and effectively prevent cancer development and
progression. We report results of a clinical study that show that
in patients without cancer but with a history of premalignant
lesions (advanced colonic adenomas, precursors to colon cancer),
a vaccine based on the TAA MUC1 was highly immunogenic in
17 of 39 (43.6%) of vaccinated individuals, eliciting high levels of
anti-MUC1 immunoglobulin G (IgG) and long-lasting immune
memory. Lack of response in 22 of 39 individuals was correlated
with high levels of circulating myeloid-derived suppressor
cells (MDSC) prevaccination. Vaccine-elicited MUC1-specific
immune response and immune memory were not associated
with significant toxicity. Our study shows that vaccines based on
human TAAs are immunogenic and safe and capable of eliciting
long-term memory that is important for cancer prevention. We
also show that in the premalignant setting, immunosuppressive
environment (e.g., high levels of MDSC) might already exist in
some individuals, suggesting an even earlier premalignant stage or
preselection of nonimmunosuppressed patients for prophylactic
vaccination. Cancer Prev Res; 6(1); 18–26. ©2012 AACR.
Cancer Prev Res January 2013 6:18–26; Published OnlineFirst
December 17, 2012; doi:10.1158/1940-6207.CAPR-12-0275
Research Article
Increased Levels of Urinary PGE-M, a Biomarker of
Inflammation, Occur in Association with Obesity, Aging,
and Lung Metastases in Patients with Breast Cancer
Patrick G. Morris, Xi Kathy Zhou, Ginger L. Milne, Daniel Goldstein, Laura C. Hawks, Chau T. Dang,
Shanu Modi, Monica N. Fornier, Clifford A. Hudis, and Andrew J. Dannenberg
Patrick G. Morris
Andrew J. Dannenberg
Abstract
Elevated levels of COX-derived prostaglandin E 2 (PGE 2 ) occur
in inflamed tissues. To evaluate the potential links between
inflammation and breast cancer, levels of urinary prostaglandin E
metabolite (PGE-M), a stable end metabolite of PGE 2 , were quantified.
We enrolled 400 patients with breast cancer: controls with early breast
cancer (n = 200), lung metastases (n = 100), and metastases to other
sites (n = 100). Patients completed a questionnaire, provided urine,
and had measurements of height and weight. Urinary PGE-M was
quantified by mass spectrometry. Ever smokers with lung metastasis
who had not been exposed to nonsteroidal anti-inflammatory drugs
(NSAIDs) had the highest PGE-M levels. PGE-M levels were increased
in association with elevated body mass index (BMI; P < 0.001), aging
(P < 0.001), pack-year smoking history (P = 0.02), lung metastases
(P = 0.02), and recent cytotoxic chemotherapy (P = 0.03). Conversely,
use of NSAIDs, prototypic inhibitors of COX activity, was associated
with reduced PGE-M levels (P < 0.001). On the basis of the current
findings, PGE-M is likely to be a useful biomarker for the selection of
high-risk subgroups to determine the use of interventions that aim to
reduce inflammation and possibly the development and progression
of breast cancer, especially in overweight and obese women. Cancer
Prev Res; 6(5); 428–36. ©2013 AACR.
Cancer Prev Res May 2013 6:428–436; Published OnlineFirst March
26, 2013; doi:10.1158/1940-6207.CAPR-12-0431
12
aacrjournals.org

Cancer
Research
Cancer Research
George C. C. Prendergast, PhD
Editor-in-Chief
Scope
Cancer Research is the most frequently cited cancer journal in the
world. The journal publishes original studies, reviews, and opinion
pieces offering significance and broad impact to a diverse audience
spanning basic, preclinical, clinical, prevention, and epidemiologic
research. Cancer Research seeks manuscripts that offer pathobiological
and translational impact to inform the personal, clinical, and societal
problems posed by cancer. The main scope of the journal is captured
in its primary subsections, which focus on molecular and cellular
pathobiology, tumor and stem cell biology, therapeutics and targets,
microenvironment and immunology, prevention and epidemiology, and
integrated systems and technology.
Review Article
Genome-Wide Epigenetic Regulation of miRNAs in Cancer
Constance Baer, Rainer Claus, and Christoph Plass
Abstract
Aberrant microRNA (miRNA) expression contributes to tumorigenesis
and cancer progression. Although the number of reported
deregulated miRNAs in various cancer types is growing fast, the
underlying mechanisms of aberrant miRNA regulation are still
poorly studied. Epigenetic alterations including aberrant DNA
methylation deregulate miRNA expression, which was first shown by
reexpression of miRNAs upon pharmacologic DNA demethylation.
However, studying the influence of DNA methylation on miRNA
transcription on a genome-wide level was hampered by poor miRNA
promoter annotation. Putative miRNA promoters were identified
on a genome-wide level by using common promoter surrogate
markers (e.g., histone modifications) and were later validated as
such in different tumor entities. Integrating promoter datasets and
Christoph Plass
global DNA methylation analysis revealed an extensive influence
of DNA hyper- as well as hypomethylation on miRNA regulation.
In this review, we summarize the current knowledge of the field
and discuss recent efforts to map miRNA promoter sequences
and to determine the contribution of epigenetic mechanisms
to the regulation of miRNA expression. We discuss examples of
tumor suppressive and oncogenic miRNAs such as the miR-34 and
miR-21 family, respectively, which highlight the complexity and
consequences of epigenetic miRNA deregulation. Cancer Res; 73(2);
473–7. ©2012 AACR.
Cancer Res January 15, 2013 73:473–7; Published OnlineFirst
January 10, 2013; doi: 10.1158/0008-5472.CAN-12-3731
The Best of the AACR Journals 13

Cancer Research
Research Article
The Noncoding RNA MALAT1 Is a Critical Regulator of the
Metastasis Phenotype of Lung Cancer Cells
Tony Gutschner, Monika Hämmerle, Moritz Eißmann, Jeff Hsu, Youngsoo Kim, Gene Hung, Alexey Revenko,
Gayatri Arun, Marion Stentrup, Matthias Groß, Martin Zörnig, A. Robert MacLeod, David L. Spector,
and Sven Diederichs
Sven Diederichs
Abstract
The long noncoding RNA MALAT1 (metastasis-associated
lung adenocarcinoma transcript 1), also known as MALAT-1
or NEAT2 (nuclear-enriched abundant transcript 2), is a highly
conserved nuclear noncoding RNA (ncRNA) and a predictive
marker for metastasis development in lung cancer. To uncover
its functional importance, we developed a MALAT1 knockout
model in human lung tumor cells by genomically integrating
RNA destabilizing elements using zinc finger nucleases. The
achieved 1,000-fold MALAT1 silencing provides a unique lossof-function
model. Proposed mechanisms of action include
regulation of splicing or gene expression. In lung cancer,
MALAT1 does not alter alternative splicing but actively regulates
gene expression including a set of metastasis-associated genes.
Consequently, MALAT1-deficient cells are impaired in migration
and form fewer tumor nodules in a mouse xenograft. Antisense
oligonucleotides (ASO) blocking MALAT1 prevent metastasis
formation after tumor implantation. Thus, targeting MALAT1
with ASOs provides a potential therapeutic approach to
prevent lung cancer metastasis with this ncRNA serving as both
predictive marker and therapeutic target. Finally, regulating
gene expression, but not alternative splicing, is the critical
function of MALAT1 in lung cancer metastasis. In summary, 10
years after the discovery of the lncRNA MALAT1 as a biomarker
for lung cancer metastasis, our loss-of-function model unravels
the active function of MALAT1 as a regulator of gene expression
governing hallmarks of lung cancer metastasis. Cancer Res;
73(3); 1180–9. ©2012 AACR.
Cancer Res February 1, 2013 73:1180–9; Published OnlineFirst
December 14, 2012; doi: 10.1158/0008-5472.CAN-12-2850
Research Article
Androgen Receptor Splice Variants Mediate Enzalutamide
Resistance in Castration-Resistant Prostate Cancer Cell Lines
Yingming Li, Siu Chiu Chan, Lucas J. Brand, Tae Hyun Hwang, Kevin A.T. Silverstein, and Scott M. Dehm
Scott M. Dehm
Abstract
Persistent androgen receptor (AR) transcriptional activity underlies
resistance to AR-targeted therapy and progression to lethal
castration-resistant prostate cancer (CRPC). Recent success in
retargeting persistent AR activity with next generation androgen/
AR axis inhibitors such as enzalutamide (MDV3100) has validated
AR as a master regulator during all stages of disease progression.
However, resistance to next generation AR inhibitors limits
therapeutic efficacy for many patients. One emerging mechanism of
CRPC progression is AR gene rearrangement, promoting synthesis
of constitutively active truncated AR splice variants (AR-V) that
lack the AR ligand-binding domain. In this study, we show that cells
with AR gene rearrangements expressing both full-length and AR-
Vs are androgen independent and enzalutamide resistant. However,
selective knock-down of AR-V expression inhibited androgenindependent
growth and restored responsiveness to androgens
and antiandrogens. In heterogeneous cell populations, AR gene
rearrangements marked individual AR-V–dependent cells that
were resistant to enzalutamide. Gene expression profiling following
knock-down of full-length AR or AR-Vs showed that AR-Vs drive
resistance to AR-targeted therapy by functioning as constitutive
and independent effectors of the androgen/AR transcriptional
program. Further, mitotic genes deemed previously to be unique
AR-V targets were found to be biphasic targets associated with
a proliferative level of signaling output from either AR-Vs or
androgen-stimulated AR. Overall, these studies highlight AR-Vs
as key mediators of persistent AR signaling and resistance to the
current arsenal of conventional and next generation AR-directed
therapies, advancing the concept of AR-Vs as therapeutic targets in
advanced disease. Cancer Res; 73(2); 483–9. ©2012 AACR.
Cancer Res January 15, 2013 73:483–9; Published OnlineFirst
November 1, 2012; doi: 10.1158/0008-5472.CAN-12-3630
14
aacrjournals.org

Cancer Research
Research Article
Complex Tumor Genomes Inferred from Single
Circulating Tumor Cells by Array-CGH and
Next-Generation Sequencing
Jochen B. Geigl
Ellen Heitzer, Martina Auer, Christin Gasch, Martin Pichler, Peter Ulz, Eva Maria Hoffmann, Sigurd Lax,
Julie Waldispuehl-Geigl, Oliver Mauermann, Carolin Lackner, Gerald Höfler, Florian Eisner, Heinz Sill,
Hellmut Samonigg, Klaus Pantel, Sabine Riethdorf, Thomas Bauernhofer, Jochen B. Geigl, and Michael R. Speicher
Michael R. Speicher
Abstract
Circulating tumor cells (CTC) released into blood from primary
cancers and metastases reflect the current status of tumor
genotypes, which are prone to changes. Here, we conducted
the first comprehensive genomic profiling of CTCs using array–
comparative genomic hybridization (CGH) and next-generation
sequencing. We used the U.S. Food and Drug Administration–
cleared CellSearch system, which detected CTCs in 21 of 37
patients (range, 1–202/7.5 mL sample) with stage IV colorectal
carcinoma. In total, we were able to isolate 37 intact CTCs from
six patients and identified in those multiple colorectal cancer–
associated copy number changes, many of which were also
present in the respective primary tumor. We then used massive
parallel sequencing of a panel of 68 colorectal cancer–associated
genes to compare the mutation spectrum in the primary tumors,
metastases, and the corresponding CTCs from two of these
patients. Mutations in known driver genes [e.g., adenomatous
polyposis coli (APC), KRAS, or PIK3CA] found in the primary
tumor and metastasis were also detected in corresponding
CTCs. However, we also observed mutations exclusively in
CTCs. To address whether these mutations were derived from
a small subclone in the primary tumor or represented new
variants of metastatic cells, we conducted additional deep
sequencing of the primary tumor and metastasis and applied
a customized statistical algorithm for analysis. We found that
most mutations initially found only in CTCs were also present
at subclonal level in the primary tumors and metastases from
the same patient. This study paves the way to use CTCs as a
liquid biopsy in patients with cancer, providing more effective
options to monitor tumor genomes that are prone to change
during progression, treatment, and relapse. Cancer Res; 73(10);
2965–75. ©2013 AACR.
Cancer Res May 15, 2013 73:2965–75; Published OnlineFirst March
7, 2013; doi: 10.1158/0008-5472.CAN-12-4140
Research Article
Targeting Tumor-Infiltrating Macrophages Decreases
Tumor-Initiating Cells, Relieves Immunosuppression,
and Improves Chemotherapeutic Responses
Jonathan B. Mitchem, Donal J. Brennan, Brett L. Knolhoff, Brian A. Belt, Yu Zhu, Dominic E. Sanford, Larisa Belaygorod,
Danielle Carpenter, Lynne Collins, David Piwnica-Worms, Stephen Hewitt, Girish Mallya Udupi, William M. Gallagher,
Craig Wegner, Brian L. West, Andrea Wang-Gillam, Peter Goedegebuure, David C. Linehan, and David G. DeNardo
David G. DeNardo
Abstract
Tumor-infiltrating immune cells can promote chemoresistance
and metastatic spread in aggressive tumors. Consequently, the
type and quality of immune responses present in the neoplastic
stroma are highly predictive of patient outcome in several cancer
types. In addition to host immune responses, intrinsic tumor cell
activities that mimic stem cell properties have been linked to
chemoresistance, metastatic dissemination, and the induction of
immune suppression. Cancer stem cells are far from a static cell
population; rather, their presence seems to be controlled by highly
dynamic processes that are dependent on cues from the tumor
stroma. However, the impact immune responses have on tumor
stem cell differentiation or expansion is not well understood. In
this study, we show that targeting tumor-infiltrating macrophages
(TAM) and inflammatory monocytes by inhibiting either the
myeloid cell receptors colony-stimulating factor-1 receptor (CSF1R)
or chemokine (C–C motif) receptor 2 (CCR2) decreases the number
of tumor-initiating cells (TIC) in pancreatic tumors. Targeting CCR2
or CSF1R improves chemotherapeutic efficacy, inhibits metastasis,
and increases antitumor T-cell responses. Tumor-educated
macrophages also directly enhanced the tumor-initiating capacity
of pancreatic tumor cells by activating the transcription factor
STAT3, thereby facilitating macrophage-mediated suppression of
CD8 + T lymphocytes. Together, our findings show how targeting
TAMs can effectively overcome therapeutic resistance mediated by
TICs. Cancer Res; 73(3); 1128–41. ©2012 AACR.
Cancer Res February 1, 2013 73:1128–41; Published OnlineFirst
December 5, 2012; doi: 10.1158/0008-5472.CAN-12-2731
The Best of the AACR Journals 15

Clinical
Cancer
Research
Clinical Cancer Research
Kenneth C. Anderson, MD
Editor-in-Chief
Scope
Clinical Cancer Research publishes innovative clinical and translational
cancer research studies that bridge the laboratory and the clinic.
The journal is especially interested in clinical trials evaluating new
treatments, accompanied by research on pharmacology, and molecular
alterations or biomarkers that predict response or resistance to
treatment. The journal also prioritizes laboratory and animal studies
of new drugs and molecule-targeted agents with the potential to lead
to clinical trials, and studies of targetable mechanisms of oncogenesis,
progression of the malignant phenotype, and metastatic disease.
Review Article
The Definition of Primary and Secondary Glioblastoma
Hiroko Ohgaki and Paul Kleihues
Abstract
Glioblastoma is the most frequent and malignant brain tumor.
The vast majority of glioblastomas (~90%) develop rapidly
de novo in elderly patients, without clinical or histologic
evidence of a less malignant precursor lesion (primary
glioblastomas). Secondary glioblastomas progress from lowgrade
diffuse astrocytoma or anaplastic astrocytoma. They
manifest in younger patients, have a lesser degree of necrosis,
are preferentially located in the frontal lobe, and carry a
significantly better prognosis. Histologically, primary and
secondary glioblastomas are largely indistinguishable, but they
differ in their genetic and epigenetic profiles. Decisive genetic
signposts of secondary glioblastoma are IDH1 mutations, which
are absent in primary glioblastomas and which are associated
with a hypermethylation phenotype. IDH1 mutations are the
earliest detectable genetic alteration in precursor low-grade
Hiroko Ohgaki
diffuse astrocytomas and in oligodendrogliomas, indicating that
these tumors are derived from neural precursor cells that differ
from those of primary glioblastomas. In this review, we summarize
epidemiologic, clinical, histopathologic, genetic, and expression
features of primary and secondary glioblastomas and the biologic
consequences of IDH1 mutations. We conclude that this genetic
alteration is a definitive diagnostic molecular marker of secondary
glioblastomas and more reliable and objective than clinical criteria.
Despite a similar histologic appearance, primary and secondary
glioblastomas are distinct tumor entities that originate from
different precursor cells and may require different therapeutic
approaches. Clin Cancer Res; 19(4); 764–72. ©2012 AACR.
Clin Cancer Res February 15, 2013 19:4 764–72; Published
OnlineFirst December 3, 2012; doi:10.1158/1078-0432.CCR-12-3002
16
aacrjournals.org

Clinical Cancer Research
Research Article
BRAF Inhibition Is Associated with Enhanced Melanoma Antigen
Expression and a More Favorable Tumor Microenvironment in
Patients with Metastatic Melanoma
Jennifer A. Wargo
Dennie T. Frederick, Adriano Piris, Alexandria P. Cogdill, Zachary A. Cooper, Cecilia Lezcano, Cristina R. Ferrone,
Devarati Mitra, Andrea Boni, Lindsay P. Newton, Chengwen Liu, Weiyi Peng, Ryan J. Sullivan, Donald P. Lawrence,
F. Stephen Hodi, Willem W. Overwijk, Gregory Lizée, George F. Murphy, Patrick Hwu, Keith T. Flaherty, David E. Fisher, and Jennifer A. Wargo
Abstract
Purpose: To evaluate the effects of BRAF inhibition on the tumor
microenvironment in patients with metastatic melanoma.
Experimental Design: Thirty-five biopsies were collected
from 16 patients with metastatic melanoma pretreatment (day
0) and at 10 to 14 days after initiation of treatment with either
BRAF inhibitor alone (vemurafenib) or BRAF + MEK inhibition
(dabrafenib + trametinib) and were also taken at time of
progression. Biopsies were analyzed for melanoma antigens,
T-cell markers, and immunomodulatory cytokines.
Results: Treatment with either BRAF inhibitor alone or BRAF + MEK
inhibitor was associated with an increased expression of melanoma
antigens and an increase in CD8+ T-cell infiltrate. This was also
associated with a decrease in immunosuppressive cytokines
[interleukin (IL)-6 and IL-8] and an increase in markers of T-cell
cytotoxicity. Interestingly, expression of exhaustion markers TIM-3
and PD1 and the immunosuppressive ligand PDL1 was increased
on treatment. A decrease in melanoma antigen expression and CD8
T-cell infiltrate was noted at time of progression on BRAF inhibitor
alone and was reversed with combined BRAF and MEK inhibition.
Conclusions: Together, these data suggest that treatment
with BRAF inhibition enhances melanoma antigen expression
and facilitates T-cell cytotoxicity and a more favorable tumor
microenvironment, providing support for potential synergy
of BRAF-targeted therapy and immunotherapy. Interestingly,
markers of T-cell exhaustion and the immunosuppressive ligand
PDL1 are also increased with BRAF inhibition, further implying
that immune checkpoint blockade may be critical in augmenting
responses to BRAF-targeted therapy in patients with melanoma.
Clin Cancer Res; 19(5); 1225–31. ©2013 AACR.
Clin Cancer Res March 1, 2013 19:5 1225–31; Published OnlineFirst
January 10, 2013; doi:10.1158/1078-0432.CCR-12-1630
Research Article
Analysis of Tumor Specimens at the Time of Acquired Resistance to
EGFR-TKI Therapy in 155 Patients with EGFR-Mutant Lung Cancers
Helena A. Yu, Maria E. Arcila, Natasha Rekhtman, Camelia S. Sima, Maureen F. Zakowski, William Pao, Mark G. Kris,
Vincent A. Miller, Marc Ladanyi, and Gregory J. Riely
Gregory J. Riely
Abstract
Purpose: All patients with EGF receptor (EGFR)–mutant lung
cancers eventually develop acquired resistance to EGFR tyrosine
kinase inhibitors (TKI). Smaller series have identified various
mechanisms of resistance, but systematic evaluation of a large
number of patients to definitively establish the frequency of
various mechanisms has not been conducted.
Experimental Design: Patients with lung adenocarcinomas
and acquired resistance to erlotinib or gefitinib enrolled onto a
prospective biopsy protocol and underwent a rebiopsy after the
development of acquired resistance. Histology was reviewed.
Samples underwent genotyping for mutations in EGFR, AKT1, BRAF,
ERBB2, KRAS, MEK1, NRAS and PIK3CA, and FISH for MET and HER2.
Results: Adequate tumor samples for molecular analysis were obtained
in 155 patients. Ninety-eight had second-site EGFR T790M mutations
[63%; 95% confidence interval (CI), 55%–70%] and four had small cell
transformation (3%, 95% CI, 0%–6%). MET amplification was seen in 4
of 75 (5%; 95% CI, 1%–13%). HER2 amplification was seen in 3 of 24 (13%;
95% CI, 3%–32%). We did not detect any acquired mutations in PIK3CA,
AKT1, BRAF, ERBB2, KRAS, MEK1, or NRAS (0 of 88, 0%; 95% CI, 0%–4%).
Overlap among mechanisms of acquired resistance was seen in 4%.
Conclusions: This is the largest series reporting mechanisms of
acquired resistance to EGFR-TKI therapy. We identified EGFR T790M
as the most common mechanism of acquired resistance, whereas
MET amplification, HER2 amplification, and small cell histologic
transformation occur less frequently. More comprehensive methods
to characterize molecular alterations in this setting are needed to
improve our understanding of acquired resistance to EGFR-TKIs.
Clin Cancer Res; 19(8); 2240–7. ©2013 AACR.
Clin Cancer Res April 15, 2013 19:8 2240–7; Published OnlineFirst
March 7, 2013; doi:10.1158/1078-0432.CCR-12-2246
The Best of the AACR Journals 17

Clinical Cancer Research
Research Article
Durable Cancer Regression Off-Treatment and Effective
Reinduction Therapy with an Anti-PD-1 Antibody
Evan J. Lipson, William H. Sharfman, Charles G. Drake, Ira Wollner, Janis M. Taube, Robert A. Anders, Haiying Xu,
Sheng Yao, Alice Pons, Lieping Chen, Drew M. Pardoll, Julie R. Brahmer, and Suzanne L. Topalian
Abstract
Purpose: Results from the first-in-human phase I trial of the
anti–programmed death-1 (PD-1) antibody BMS-936558 in
patients with treatment-refractory solid tumors, including safety,
tolerability, pharmacodynamics, and immunologic correlates,
have been previously reported. Here, we provide long-term followup
on three patients from that trial who sustained objective tumor
regressions off therapy, and test the hypothesis that reinduction
therapy for late tumor recurrence can be effective.
Experimental Design: Three patients with colorectal cancer, renal
cell cancer, and melanoma achieved objective responses on an
intermittent dosing regimen of BMS-936558. Following cessation
of therapy, patients were followed for more than 3 years. A patient
with melanoma who experienced a prolonged partial regression
followed by tumor recurrence received reinduction therapy.
Suzanne L. Topalian
renal cell cancer experienced a partial response lasting 3 years
off therapy, which converted to a complete response, which
is ongoing at 12 months. A patient with melanoma achieved
a partial response that was stable for 16 months off therapy;
recurrent disease was successfully treated with reinduction
anti-PD-1 therapy.
Conclusion: These data represent the most prolonged
observation to date of patients with solid tumors responding
to anti-PD-1 immunotherapy and the first report of successful
reinduction therapy following delayed tumor progression.
They underscore the potential for immune checkpoint
blockade with anti-PD-1 to reset the equilibrium between
tumor and the host immune system. Clin Cancer Res; 19(2);
462–8. ©2012 AACR.
Results: A patient with colorectal cancer experienced a
complete response, which is ongoing after 3 years. A patient with
Clin Cancer Res January 15, 2013 19:2 462–8; Published OnlineFirst
November 20, 2012; doi:10.1158/1078-0432.CCR-12-2625
Research Article
An Epithelial–Mesenchymal Transition Gene Signature
Predicts Resistance to EGFR and PI3K Inhibitors and
Identifies Axl as a Therapeutic Target for Overcoming
EGFR Inhibitor Resistance
Lauren Averett Byers
John V. Heymach
Lauren Averett Byers, Lixia Diao, Jing Wang, Pierre Saintigny, Luc Girard, Michael Peyton, Li Shen, Youhong Fan, Uma Giri,
Praveen K. Tumula, Monique B. Nilsson, Jayanthi Gudikote, Hai Tran, Robert J.G. Cardnell, David J. Bearss, Steven L. Warner,
Jason M. Foulks, Steven B. Kanner, Varsha Gandhi, Nancy Krett, Steven T. Rosen, Edward S. Kim, Roy S. Herbst, George R. Blumenschein,
J. Jack Lee, Scott M. Lippman, K. Kian Ang, Gordon B. Mills, Waun K. Hong, John N. Weinstein, Ignacio I. Wistuba, Kevin R. Coombes,
John D. Minna, and John V. Heymach
Abstract
Purpose: Epithelial–mesenchymal transition (EMT) has been
associated with metastatic spread and EGF receptor (EGFR)
inhibitor resistance. We developed and validated a robust 76-
gene EMT signature using gene expression profiles from four
platforms using non–small cell lung carcinoma (NSCLC) cell lines
and patients treated in the Biomarker-Integrated Approaches of
Targeted Therapy for Lung Cancer Elimination (BATTLE) study.
Experimental Design: We conducted an integrated gene expression,
proteomic, and drug response analysis using cell lines and tumors
from patients with NSCLC. A 76-gene EMT signature was developed
and validated using gene expression profiles from four microarray
platforms of NSCLC cell lines and patients treated in the BATTLE study,
and potential therapeutic targets associated with EMT were identified.
Results: Compared with epithelial cells, mesenchymal cells
showed significantly greater resistance to EGFR and PI3K/Akt
pathway inhibitors, independent of EGFR mutation status, but
more sensitivity to certain chemotherapies. Mesenchymal cells
also expressed increased levels of the receptor tyrosine kinase Axl
and showed a trend toward greater sensitivity to the Axl inhibitor
SGI-7079, whereas the combination of SGI-7079 with erlotinib
reversed erlotinib resistance in mesenchymal lines expressing
Axl and in a xenograft model of mesenchymal NSCLC. In patients
with NSCLC, the EMT signature predicted 8-week disease control
in patients receiving erlotinib but not other therapies.
Conclusion: We have developed a robust EMT signature that predicts
resistance to EGFR and PI3K/Akt inhibitors, highlights different
patterns of drug responsiveness for epithelial and mesenchymal cells,
and identifies Axl as a potential therapeutic target for overcoming
EGFR inhibitor resistance associated with the mesenchymal
phenotype. Clin Cancer Res; 19(1); 279–90. ©2012 AACR.
Clin Cancer Res January 1, 2013 19:1 279–90; Published OnlineFirst
October 22, 2012; doi:10.1158/1078-0432.CCR-12-1558
18
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Molecular
Cancer
Research
Molecular Cancer Research
Karen E. Knudsen, PhD
Editor-in-Chief
Scope
Molecular Cancer Research publishes articles describing novel basic
cancer research discoveries of broad interest to the field. Studies
must be of demonstrated significance, and the journal prioritizes
analyses performed at the molecular and cellular level that reveal novel
mechanistic insight into pathways and processes linked to cancer
risk, development, and/or progression. Areas of emphasis include all
cancer-associated pathways (including cell-cycle regulation; cell death;
chromatin regulation; DNA damage and repair; gene and RNA regulation;
genomics; oncogenes and tumor suppressors; and signal transduction),
in addition to studies describing new molecular mechanisms and
interactions that support cancer phenotypes.
Review Article
Radiation Survivors: Understanding and Exploiting the
Phenotype following Fractionated Radiation Therapy
Adeola Y. Makinde, Molykutty John-Aryankalayil, Sanjeewani T. Palayoor, David Cerna, and C. Norman Coleman
Abstract
Radiation oncology modalities such as intensity-modulated and
image-guided radiation therapy can reduce the high dose to normal
tissue and deliver a heterogeneous dose to tumors, focusing on areas
deemed at highest risk for tumor persistence. Clinical radiation
oncology produces daily doses ranging from 1 to 20 Gy, with
tissues being exposed to 30 or more daily fractions. Hypothesizing
the cells that survive fractionated radiation therapy have a
substantially different phenotype than the untreated cells, which
might be exploitable for targeting with molecular therapeutics or
immunotherapy, three prostate cancer cell lines (PC3, DU145, and
LNCaP) and normal endothelial cells were studied to understand
the biology of differential effects of multifraction (MF) radiation
of 0.5, 1, and/or 2 Gy fraction to 10 Gy total dose, and a single
dose of 5 and 10 Gy. The resulting changes in mRNA, miRNA, and
phosphoproteome were analyzed. Significant differences were
Adeola Y. Makinde
observed in the MF radiation exposures including those from the
0.5 Gy MF that produces little cell killing. As expected, p53 function
played a major role in response. Pathways modified by MF include
immune response, DNA damage, cell-cycle arrest, TGF-β, survival,
and apoptotic signal transduction. The radiation-induced stress
response will set forth a unique platform for exploiting the effects
of radiation therapy as “focused biology” for cancer treatment in
conjunction with molecular targeted or immunologically directed
therapy. Given that more normal tissue is treated, albeit to lower
doses with these newer techniques, the response of the normal
tissue may also influence long-term treatment outcome. Mol
Cancer Res; 11(1); 5–12. ©2012 AACR.
Mol Cancer Res January 2013 11:5–12; Published OnlineFirst
November 21, 2012; doi:10.1158/1541-7786.MCR-12-0492
The Best of the AACR Journals 19

Molecular Cancer Research
Research Article
A Modified HSP70 Inhibitor Shows Broad Activity
as an Anticancer Agent
Gregor M. Balaburski, Julia I.-Ju Leu, Neil Beeharry, Seth Hayik, Mark D. Andrake, Gao Zhang, Meenhard Herlyn,
Jessie Villanueva, Roland L. Dunbrack, Tim Yen, Donna L. George, and Maureen E. Murphy
Maureen E. Murphy
Abstract
The stress-induced HSP70 is an ATP-dependent molecular
chaperone that plays a key role in refolding misfolded proteins
and promoting cell survival following stress. HSP70 is marginally
expressed in nontransformed cells, but is greatly overexpressed
in tumor cells. Silencing HSP70 is uniformly cytotoxic to tumor
but not normal cells; therefore, there has been great interest in
the development of HSP70 inhibitors for cancer therapy. Here, we
report that the HSP70 inhibitor 2-phenylethynesulfonamide (PES)
binds to the substrate-binding domain of HSP70 and requires the
C-terminal helical “lid” of this protein (amino acids 573–616) to
bind. Using molecular modeling and in silico docking, we have
identified a candidate binding site for PES in this region of HSP70,
and we identify point mutants that fail to interact with PES. A
preliminary structure–activity relationship analysis has revealed
a derivative of PES, 2-(3-chlorophenyl) ethynesulfonamide (PES-
Cl), which shows increased cytotoxicity and ability to inhibit
autophagy, along with significantly improved ability to extend
the life of mice with pre-B-cell lymphoma, compared with the
parent compound (P = 0.015). Interestingly, we also show that
these HSP70 inhibitors impair the activity of the anaphase
promoting complex/cyclosome (APC/C) in cell-free extracts, and
induce G 2 –M arrest and genomic instability in cancer cells. PES-
Cl is thus a promising new anticancer compound with several
notable mechanisms of action. Mol Cancer Res; 11(3); 219–29.
©2013 AACR.
Mol Cancer Res March 2013 11:219–229; Published OnlineFirst
January 9, 2013; doi:10.1158/1541-7786.MCR-12-0547-T
Research Article
The Heterochronic microRNA let-7 Inhibits Cell Motility
by Regulating the Genes in the Actin Cytoskeleton Pathway
in Breast Cancer
Xiaowen Hu, Jinyi Guo, Lan Zheng, Chunsheng Li, Tim M. Zheng, Janos L. Tanyi, Shun Liang, Chiara Benedetto,
Marco Mitidieri, Dionyssios Katsaros, Xia Zhao, Youcheng Zhang, Qihong Huang, and Lin Zhang
Lin Zhang
Abstract
The heterochronic gene let-7 serves as a tumor suppressor
microRNA by targeting various oncogenic pathways in cancer
cells. Considerable evidence indicates that reduced expression
of let-7 might be associated with poor clinical outcome in
patients with cancer. Here, we report that the expression levels
of three let-7 family members, let-7a, let-7b, and let-7g, were
significantly decreased in the patients with breast cancer with
lymph node metastasis compared with those without lymph
node metastasis. Enforced expression of let-7b significantly
inhibits breast cancer cell motility and affects actin dynamics.
Using bioinformatic and experimental approaches, four genes in
the actin cytoskeleton pathway, including PAK1, DIAPH2, RDX,
and ITGB8, were identified as let-7 direct targets. Blocking the
expression of PAK1, DIAPH2, and RDX significantly inhibits
breast cancer cell migration induced by let-7b repression.
Our results indicate that reconstitution of let-7 expression in
tumor cells could provide a novel therapeutic strategy for the
treatment of metastatic disease. Mol Cancer Res; 11(3); 240–50.
©2013 AACR.
Mol Cancer Res March 2013 11:240–250; Published OnlineFirst
January 21, 2013; doi:10.1158/1541-7786.MCR-12-0432
20
aacrjournals.org

Molecular Cancer Research
Research Article
Procollagen Lysyl Hydroxylase 2 Is Essential for
Hypoxia-Induced Breast Cancer Metastasis
Daniele M. Gilkes, Saumendra Bajpai, Carmen C. Wong, Pallavi Chaturvedi, Maimon E. Hubbi,
Denis Wirtz, and Gregg L. Semenza
Gregg L. Semenza
Abstract
Metastasis is the leading cause of death among patients who have
breast cancer. Understanding the role of the extracellular matrix
(ECM) in the metastatic process may lead to the development of
improved therapies to treat patients with cancer. Intratumoral
hypoxia, found in the majority of breast cancers, is associated
with an increased risk of metastasis and mortality. We found
that in hypoxic breast cancer cells, hypoxia-inducible factor 1
(HIF-1) activates transcription of the PLOD1 and PLOD2 genes
encoding procollagen lysyl hydroxylases that are required for the
biogenesis of collagen, which is a major constituent of the ECM.
High PLOD2 expression in breast cancer biopsies is associated
with increased risk of mortality. We show that PLOD2 is critical
for fibrillar collagen formation by breast cancer cells, increases
tumor stiffness, and is required for metastasis to lymph nodes
and lungs. Mol Cancer Res; 11(5); 456–66. ©2013 AACR.
Mol Cancer Res May 2013 11:456–466; Published OnlineFirst
February 1, 2013; doi:10.1158/1541-7786.MCR-12-0629
Research Article
Activation of the FGF2-FGFR1 Autocrine Pathway: A Novel
Mechanism of Acquired Resistance to Gefitinib in NSCLC
Hideki Terai, Kenzo Soejima, Hiroyuki Yasuda, Sohei Nakayama, Junko Hamamoto, Daisuke Arai, Kota Ishioka,
Keiko Ohgino, Shinnosuke Ikemura, Takashi Sato, Satoshi Yoda, Ryosuke Satomi, Katsuhiko Naoki, and
Tomoko Betsuyaku
Kenzo Soejima
Abstract
Patients with non-small cell lung cancer (NSCLC) that harbors
epidermal growth factor receptor (EGFR) mutations initially
respond to EGFR-tyrosine kinase inhibitors (TKI) but eventually
experience relapse. Acquired resistance to EGFR-TKIs is
strongly associated with patient mortality. Thus, elucidation of
the mechanism of acquired resistance to EGFR-TKIs is of great
importance. In this study, gefitinib-resistant cell line models
were established by long-term exposure to gefitinib using the
gefitinib-sensitive lung cancer cell lines, PC9 and HCC827.
Expression analyses indicated that both FGFR1 and FGF2 were
increased in PC9 gefitinib-resistant (PC9 GR) cells as compared
with PC9 naïve (PC9 na) cells. Importantly, proliferation of
gefitinib-resistant cells was dependent on the FGF2-FGFR1
pathway. Mechanistically, inhibition of either FGF2 or FGFR1
by siRNA or FGFR inhibitor (PD173074) restored gefitinib
sensitivity in PC9 GR cells. These data suggest that FGF2-FGFR1
activation through an autocrine loop is a novel mechanism of
acquired resistance to EGFR-TKIs. Mol Cancer Res; 11(7); 759–67.
©2013 AACR.
Mol Cancer Res July 2013 11:759–767; Published OnlineFirst March
27, 2013; doi:10.1158/1541-7786.MCR-12-0652
The Best of the AACR Journals 21

Molecular
Cancer
Therapeutics
Molecular Cancer Therapeutics
Napoleone Ferrara, MD
Editor-in-Chief
Scope
Molecular Cancer Therapeutics strives to be the top choice for publishing
the best science in the discovery and preclinical development of
novel therapeutic agents for oncology, preclinical studies of approved
therapeutics, mechanisms of drug action, mechanism of drug resistance,
biomarkers of drug response, novel models and technologies, and
occasional drug toxicity mechanisms. While the journal’s main focus is
on small molecule and protein drugs, other molecular entities may be
considered.
Review Article
FOX(M1) News—It Is Cancer
Marianna Halasi and Andrei L. Gartel
Abstract
FOXM1 is an oncogenic transcription factor of the Forkhead
family and it has a well-defined role in cell proliferation and
cell-cycle progression. Expression of FOXM1 is excluded in
quiescent or differentiated cells, but its level is highly elevated
in proliferating and malignant cells. Overexpression of FOXM1
has been reported in more than 20 types of human cancer. In
recent years, FOXM1 has been implicated in diverse cellular
processes and also a growing body of experimental data has
underlined the relevance of FOXM1 in tumorigenesis. Although
FOXM1 is under the control of three major tumor suppressors
Andrei L. Gartel
(RB, p53, and p19 ARF ), it is still active in the majority of human
cancers. The oncogenic potential of FOXM1 is mainly based on
its ability to transcriptionally activate genes that are involved
in different facets of cancer development. In this review, the
contribution of FOXM1 to each of the hallmarks of cancer will
be summarized and discussed. Mol Cancer Ther; 12(3); 245–54.
©2012 AACR.
Mol Cancer Ther March 2013 12:245–54; Published OnlineFirst
February 26, 2013; doi:10.1158/1535-7163.MCT-12-0712
22
aacrjournals.org

Molecular Cancer Therapeutics
Research Article
Genome and Transcriptome Sequencing in Prospective Metastatic
Triple-Negative Breast Cancer Uncovers Therapeutic Vulnerabilities
David W. Craig, Joyce A. O’Shaughnessy, Jeffrey A. Kiefer, Jessica Aldrich, Shripad Sinari, Tracy M. Moses, Shukmei Wong,
Jennifer Dinh, Alexis Christoforides, Joanne L. Blum, Cristi L. Aitelli, Cynthia R. Osborne, Tyler Izatt, Ahmet Kurdoglu,
Angela Baker, Julie Koeman, Catalin Barbacioru, Onur Sakarya, Francisco M. De La Vega, Asim Siddiqui, Linh Hoang,
Paul R. Billings, Bodour Salhia, Anthony W. Tolcher, Jeffrey M. Trent, Spyro Mousses, Daniel Von Hoff, and John D. Carpten
John D. Carpten
Abstract
Triple-negative breast cancer (TNBC) is characterized by
the absence of expression of estrogen receptor, progesterone
receptor, and HER-2. Thirty percent of patients recur after
first-line treatment, and metastatic TNBC (mTNBC) has a poor
prognosis with median survival of one year. Here, we present
initial analyses of whole genome and transcriptome sequencing
data from 14 prospective mTNBC. We have cataloged the
collection of somatic genomic alterations in these advanced
tumors, particularly those that may inform targeted therapies.
Genes mutated in multiple tumors included TP53, LRP1B,
HERC1, CDH5, RB1, and NF1. Notable genes involved in focal
structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1,
FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous
deletion of CTNNA1 was detected in 2 of 6 African Americans.
RNA sequencing revealed consistent overexpression of the
FOXM1 gene when tumor gene expression was compared
with nonmalignant breast samples. Using an outlier analysis
of gene expression comparing one cancer with all the others,
we detected expression patterns unique to each patient’s
tumor. Integrative DNA/RNA analysis provided evidence for
deregulation of mutated genes, including the monoallelic
expression of TP53 mutations. Finally, molecular alterations in
several cancers supported targeted therapeutic intervention on
clinical trials with known inhibitors, particularly for alterations
in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways.
In conclusion, whole genome and transcriptome profiling of
mTNBC have provided insights into somatic events occurring
in this difficult to treat cancer. These genomic data have
guided patients to investigational treatment trials and provide
hypotheses for future trials in this irremediable cancer. Mol
Cancer Ther; 12(1); 104–16. ©2012 AACR.
Mol Cancer Ther January 2013 12:104–16; Published OnlineFirst
November 19, 2012; doi:10.1158/1535-7163.MCT-12-0781
Research Article
EGFR Exon 20 Insertion Mutations in Lung Adenocarcinomas:
Prevalence, Molecular Heterogeneity, and Clinicopathologic
Characteristics
Maria E. Arcila, Khedoudja Nafa, Jamie E. Chaft, Natasha Rekhtman, Christopher Lau, Boris A. Reva,
Maureen F. Zakowski, Mark G. Kris, and Marc Ladanyi
Maria E. Arcila
Abstract
In contrast to other primary epidermal growth factor receptor
(EGFR) mutations in lung adenocarcinomas, insertions in exon
20 of EGFR have been generally associated with resistance to
EGFR-tyrosine kinase inhibitors. Their molecular spectrum,
clinicopathologic characteristics, and prevalence are not
well established. Tumors harboring EGFR exon 20 insertions
were identified through an algorithmic screen of 1,500 lung
adenocarcinomas. Cases were first tested for common mutations
in EGFR (exons 19 and 21) and KRAS (exon 2) and, if negative,
further analyzed for EGFR exon 20 insertions. All samples
underwent extended genotyping for other driver mutations in
EGFR, KRAS, BRAF, ERBB2/HER2, NRAS, PIK3CA, MEK1, and
AKT by mass spectrometry; a subset was evaluated for ALK
rearrangements. We identified 33 EGFR exon 20 insertion cases
[2.2%, 95% confidence interval (CI), 1.6–3.1], all mutually exclusive
with mutations in the other genes tested (except PIK3CA). They
were more common among never-smokers (P < 0.0001). There
was no association with age, sex, race, or stage. Morphologically,
tumors were similar to those with common EGFR mutations but
with frequent solid histology. Insertions were highly variable
in position and size, ranging from 3 to 12 bp, resulting in 13
different insertions, which, by molecular modeling, are predicted
to have potentially different effects on erlotinib binding. EGFR
exon 20 insertion testing identifies a distinct subset of lung
adenocarcinomas, accounting for at least 9% of all EGFR-mutated
cases, representing the third most common type of EGFR mutation
after exon 19 deletions and L858R. Insertions are structurally
heterogeneous with potential implications for response to EGFR
inhibitors. Mol Cancer Ther; 12(2); 220–9. ©2012 AACR.
Mol Cancer Ther February 2013 12:220–9; Published OnlineFirst
January 31, 2013; doi:10.1158/1535-7163.MCT-12-0620
The Best of the AACR Journals 23

Molecular Cancer Therapeutics
Research Article
NF-κB2/p52 Induces Resistance to Enzalutamide in
Prostate Cancer: Role of Androgen Receptor and Its
Variants
Nagalakshmi Nadiminty, Ramakumar Tummala, Chengfei Liu, Joy Yang, Wei Lou,
Christopher P. Evans, and Allen C. Gao
Nagalakshmi Nadiminty
Allen C. Gao
Abstract
Resistance of prostate cancer cells to the next-generation
antiandrogen enzalutamide may be mediated by a multitude
of survival signaling pathways. In this study, we tested whether
increased expression of NF-κB2/p52 induces prostate cancer
cell resistance to enzalutamide and whether this response is
mediated by aberrant androgen receptor (AR) activation and
AR splice variant production. LNCaP cells stably expressing
NF-κB2/p52 exhibited higher survival rates than controls
when treated with enzalutamide. C4-2B and CWR22Rv1 cells
chronically treated with enzalutamide were found to express
higher levels of NF-κB2/p52. Downregulation of NF-κB2/p52
in CWR22Rv1 cells chronically treated with enzalutamide
rendered them more sensitive to cell growth inhibition by
enzalutamide. Analysis of the expression levels of AR splice
variants by quantitative reverse transcription PCR and Western
blotting revealed that LNCaP cells expressing p52 exhibit higher
expression of AR splice variants. Downregulation of expression
of NF-κB2/p52 in VCaP and CWR22Rv1 cells by short hairpin
RNA abolished expression of splice variants. Downregulation
of expression of either full-length AR or the splice variant AR-
V7 led to an increase in sensitivity of prostate cancer cells to
enzalutamide. These results collectively demonstrate that
resistance to enzalutamide may be mediated by NF-κB2/p52 via
activation of AR and its splice variants. Mol Cancer Ther; 12(8);
1629–37. ©2013 AACR.
Mol Cancer Ther August 2013 12:1629–37; Published OnlineFirst
May 22, 2013; doi:10.1158/1535-7163.MCT-13-0027
Research Article
Inhibition of Mutant GNAQ Signaling in Uveal Melanoma Induces
AMPK-Dependent Autophagic Cell Death
Grazia Ambrosini, Elgilda Musi, Alan L. Ho, Elisa de Stanchina, and Gary K. Schwartz
Abstract
Grazia Ambrosini
Oncogenic mutations in GNAQ and GNA11 genes are found in 80%
of uveal melanoma. These mutations result in the activation of
the RAF/MEK signaling pathway culminating in the stimulation
of ERK1/2 mitogen-activated protein kinases. In this study, using
a siRNA strategy, we show that mutant GNAQ signals to both
MEK and AKT, and that combined inhibition of these pathways
with the MEK inhibitor selumetinib (AZD6244) and the AKT
inhibitor MK2206 induced a synergistic decrease in cell viability.
This effect was genotype dependent as autophagic markers like
beclin1 and LC3 were induced in GNAQ-mutant cells, whereas
apoptosis was the mechanism of cell death of BRAF-mutant cells,
and cells without either mutation underwent cell-cycle arrest.
The inhibition of MEK/ATK pathways induced activation of AMPactivated
protein kinase (AMPK) in the GNAQ-mutant cells.
The downregulation of AMPK by siRNA or its inhibition with
compound C did not rescue the cells from autophagy, rather they
died by apoptosis, defining AMPK as a key regulator of mutant
GNAQ signaling and a switch between autophagy and apoptosis.
Furthermore, this combination treatment was effective in
inhibiting tumor growth in xenograft mouse models. These
findings suggest that inhibition of MEK and AKT may represent
a promising approach for targeted therapy of patients with uveal
melanoma. Mol Cancer Ther; 12(5); 768–76. ©2013 AACR.
Mol Cancer Ther May 2013 12:768–76; Published OnlineFirst
February 26, 2013; doi:10.1158/1535-7163.MCT-12-1020
24
aacrjournals.org

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