bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-023: Describing prostate cancer heterogeneity through single cell analysis with mass cytometry
Authors: Roditi Laura De Vargas , Jacobs Andrea , Fankhauser Christian , Poyet Cedric , Wild Peter , Bodenmiller Bernd
Published: August 2018

Background

Single-cell analysis has emerged as a crucial approach to detect variations in genetic and proteomic expression. Prostate cancer is characterized by multiple genomic alterations leading to the complex nature of the disease. The heterogeneity at the proteomic level is not yet well characterized, yet intra-tumor heterogeneity can impair correct diagnosis and treatment, by driving therapy resistance. Meanwhile inter-tumor heterogeneity can strongly impact personalization of treatments highlighting the importance of characterizing both intra- and inter-tumor heterogeneity. Using single-cell mass cytometry data, we identify the proteomic profiles of the different cellular subpopulations found in prostate cancer that are common across a cohort of 50 patients while also describing the variations across patients, thus thoroughly characterizing prostate tumor heterogeneity.

Methods & Results

Mass cytometry experiments allow quantification of dozens of proteins simultaneously at the single-cell level, for thousands of cells. The high dimensionality of this data represents a challenge for data visualization and clustering, which require computational approaches for interpretation of the results. We developed a new computational method to address this task and analyzed tissue sections from surgically removed prostates from 50 patients using mass cytometry with a comprehensive set of 40 antibodies to jointly identify surface markers, enzymes, transcription factors and functional readouts.

Conclusions

Here, we present for the first time an analysis of prostate tumor cell heterogeneity by mass cytometry coupled to a newly developed computational method that improves visualization and clustering of such high-dimensional data. This study was conducted by analyzing prostate cancer samples from fifty patients at the single-cell level using mass cytometry to estimate the abundance of forty different proteins simultaneously for each cell. Our results show that immune, stromal and epithelial cells can be comprehensively distinguished across all patients. A closer look at the epithelial compartment reveals multiple subpopulations which we describe in terms of their protein expression patterns to characterize the intra- and inter-tumor heterogeneity in prostate cancer. This work demonstrates how single cell phenotypic profiles of prostate tumor cells, taken from surgically removed prostates, can be used to quantify the varying heterogeneity during tumor progression.

Citation Format: Laura De Vargas Roditi, Andrea Jacobs, Christian Fankhauser, Cedric Poyet, Peter Wild, Bernd Bodenmiller. Describing prostate cancer heterogeneity through single cell analysis with mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-023.
Abstract LB-040: New conditional Kras-alleles generated by CRISPR-based genome editing to model tumor initiation
Authors: Zafra Maria Paz , Schatoff Emma M. , Han Teng , Dow Lukas E.
Published: August 2018

KRAS is the most frequently mutated oncogene in human cancers, and specific cancer types show a clear bias in the types and frequency of Kras alterations. However, we do not have a clear understanding of how distinct Kras mutations dictate tumor biology and response to therapy. The generation of conditional animal models, such as Lox-Stop-Lox (LSL) KrasG12D and LSL-KrasG12Vgeo mice, have provided important tools to dissect the impact of Kras mutation in tumor development, but these models alone do not recapitulate the spectrum of Kras alterations in human cancer. To better reflect the Kras mutational landscape in human cancers, we have developed a series of critical LSL-Kras mouse models that recapitulate common Kras alterations observed in colorectal (G13D), pancreatic (G12R), and lung cancer (G12C), allowing the direct interrogation of the effects of these Kras mutations on disease phenotype and treatment response. To do this, we targeted embryonic stem cells (ESCs) carrying the LSL-KrasG12D allele, by co-transfection of a modified high-fidelity Cas9 (Cas9-HFz), a LSL-Kras-targeted sgRNA, and an HDR template carrying the new mutation. While Cas9-HFz displayed a slight decrease in homology-directed repair (HDR) efficiency in comparison with wildtype Cas9, it enabled high specificity editing of only the LSL allele. Targeted deep sequencing revealed a significant reduction in ‘off-target' events in wildtype Kras locus in Cas9-HFz transfected samples compared to unmodified Cas9. We next generated transgenic mice by blastocyst injection and produced mice carrying each LSL-Kras variant, a pancreas specific Cre (p48-Cre) and a far-red fluorescent Cre-reporter (mKate2). We are now analyzing the impact of specific Kras alterations in tumor initiation in distinct tissues such as the pancreas, colon and lung. Together with available LSL-KrasG12D and LSL-KrasG12V already available, our new alleles will serve as critical tools for better dissect the genetic factors that dictate disease initiation, progression, and response to therapy.

Citation Format: Maria Paz Zafra, Emma M. Schatoff, Teng Han, Lukas E. Dow. New conditional Kras-alleles generated by CRISPR-based genome editing to model tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-040.
Abstract LB-043: Generation of clonal CRISPR/Cas9-edited human iPSC derived cellular models and its applications in physiologically relevant assays
Authors: Revankar Chetana M. , Willems Erik , Lacambacal Rex , gokirmak Tufan , Dizon Jordan , Webb Jacquelyn , Vega Raquel , Piper David
Published: August 2018

Induced pluripotent stem cell (iPSC)-derived cells are widely used in disease models, safety testing and phenotypic and functional assays. The CRISPR/Cas9 system has revolutionized genome editing thus enabling rapid generation of disease models. Cutting edge technologies like genome editing paired with iPSC-derived cell models have reshaped our approach to disease modeling in vitro. Although, CRISPR/Cas9 has had huge impact on generation of complex cellular models, there are challenges in replicating disease-in-a-dish. One of the challenges is the implementation of genome editing tools in iPSC, particularly issues associated with editing tools delivery, cell recovery and clonal isolation of genome-edited cells. Here, we describe a workflow that facilitates the generation and isolation of clonal CRISPR-edited iPSCs through the use of a novel toolset. We observed high genome editing efficiency using an iPSC line that stably expresses the Cas9 protein and obtained improved survival of the iPSCs after delivery of the editing tools through culture in StemFlex™ medium, a novel PSC culture system. Furthermore, we have demonstrated that single edited iPSCs can be isolated through the combination of an optimized cell sorting method and expanded as single cell clones with reagents that support single cell survival. Through this workflow we have generated several iPSC lines carrying disease relevant SNP mutations. These tools and workflows not only contribute to improved success in the derivation of homogenous genome edited human iPSC clones but also provide novel alternatives to study disease-causing mutations in vitro. Additionally, we have also tested these cell models in commonly used cell health assays. These cell models can be further used to generate organoids to mimic conditions found in tissues. iPSC- derived cell models not only permit the study of disease mechanism in the cell types specifically affected in the disease but also deepen our understanding of the molecular mechanisms and in turn help development of novel therapeutics.

Citation Format: Chetana M. Revankar, Erik Willems, Rex Lacambacal, Tufan gokirmak, Jordan Dizon, Jacquelyn Webb, Raquel Vega, David Piper. Generation of clonal CRISPR/Cas9-edited human iPSC derived cellular models and its applications in physiologically relevant assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-043.
Abstract LB-145: Enhancement of checkpoint inhibitor therapy by beta-alethine
Authors: Taub Floyd , Wright Suzin , Guth Amanda
Published: August 2018

Beta-alethine (BA) is a small molecule drug that has demonstrated impressive anti-tumor immune responses against melanoma, particularly when combine with low dose anti-PD1. Here we report on the dose of BA that optimally enhances anti-PD1, the efficacy of BA combined with various immune checkpoint inhibitors (ICIs) in a murine bladder cancer and we propose a potential mechanism of action of BA to explain its synergistic effect. We tested 3 different doses of BA that were logs apart (30 mg/kg, 30 ug/kg, and 600 ng/kg) either alone or in combination with low dose (10 ug per mouse for a total of 3 doses 3 days apart) anti-PD1 in the DBA mouse Cloudman melanoma mouse model. The lowest dose of BA (600 ng/kg) in combination with anti-PD1 resulted in cures in 33% of the mice when treatment was initiated when tumors were of substantial size (average size of 30 mm2). Previous work starting with smaller tumor sizes (average 20 mm2) resulted in an 80% cure rate when a low dose of BA was used in combination with low dose anti-PD1. Analysis of blood from treated animals demonstrated that the combination led to decreased expression of multiple checkpoint molecules on both T cells and myeloid cells and to a decrease in tumor promoting inflammatory macrophages. We further demonstrated efficacy of combining BA with other ICI therapies in a s.c. MBT2 murine bladder cancer study in C3H mice. In these studies, we saw that BA was synergistic with a-PD1 at all doses tested (30 mg/kg, 30 ug/kg and 600 ng/kg weekly s.c.). The effect was seen even when only 4 weekly doses of BA were given. Interestingly, tumor growth increased when BA was discontinued, a phenomenon observed in previous compassionate use veterinary studies. Synergy with OX40 and CTLA4, were also seen, but further studies of dose and schedule are required for optimization. Our results demonstrate that a low dose of BA is optimal to enhance the efficacy of anti-PD1 and that this observed combinatorial effect is conserved in at least two different tumor types in different mouse strains. Based on analysis of T and myeloid cells in treated mice, we hypothesize that BA works via inducing pleiotropic changes that result in downregulation of multiple immune checkpoint molecules on T cells and on myeloid cells making it less feasible for these cells to use other pathways of immune suppression as compared to single agent anti-PD1 therapy and thus increase potential for cures.

Citation Format: Floyd Taub, Suzin Wright, Amanda Guth. Enhancement of checkpoint inhibitor therapy by beta-alethine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-145.
Abstract LB-177: Quantification of cell signaling proteins by immuno-MALDI
Authors: Popp Robert , Zahedi René P. , LeBlanc André , Mohammed Yassene , Aguilar-Mahecha Adriana , Pötz Oliver , Basik Mark , Batist Gerald , Borchers Christoph H.
Published: August 2018

Introduction. To improve cancer patient stratification and overcome the drawbacks of currently used approaches to assess signaling pathway proteins, we set out to develop immuno-matrix assisted laser ionization/desorption (iMALDI) assays combined with a phosphatase-based phosphopeptide quantitation (PPQ) approach to measure PI3K/AKT/mTOR pathway activity. Specifically, we targeted the C-terminal tryptic peptides of AKT1 and AKT2 due to their involvement in full kinase activation.

Methods. Following trypsin digestion of cell lysate, stable isotope-labeled standard (SIS) peptides are added. After splitting the solution, one aliquot is treated with phosphatase. Bead-coupled antibodies enrich the non-phosphorylated target peptides, which are washed and spotted onto a MALDI plate, and acidic MALDI matrix elutes the peptides. The resulting light/heavy ratios of both aliquots allow the calculation of protein expression levels and peptide phosphorylation stoichiometry.

Results. The iMALDI assays were validated for linear range and accuracy, as well as interference screening, requiring only ~50 µg of total cell or tissue lysate protein to quantify both AKT1 and AKT2 expression levels and phosphorylation stoichiometry. CVs of technical replicates were found to consistently be below 10%. We were able to quantify AKT1 and AKT2 from various cell lines and fresh frozen tumor samples, including SW480 and HCT116 colon cancer cell lines, MDA-MB-231 breast cancer cells, and colon cancer and breast cancer tumor lysates. Further, a direct comparison of matched fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues showed that the expression levels and phosphorylation stoichiometry quantified differ depending on the tissue preservation method, and that phosphorylation stoichiometries above 30% occur in only a small subset of samples. A direct comparison of four pairs of normal and adjacent tumor tissues showed elevated AKT1 phosphorylation stoichiometry of ~40% in a colorectal cancer liver metastasis, and significantly elevated AKT1 (3.6-fold) and AKT2 (2.2-fold) expression levels in a surgical breast tumor sample.

Future directions. In a next step, patient-derived mouse xenograft tissue samples, collected after specific drug treatment, will be analyzed, and the AKT results will be correlated to genomic data to answer the hypothesis whether the AKT expression levels and phosphorylation stoichiometries correlate with response to treatment.

Citation Format: Robert Popp, René P. Zahedi, André LeBlanc, Yassene Mohammed, Adriana Aguilar-Mahecha, Oliver Pötz, Mark Basik, Gerald Batist, Christoph H. Borchers. Quantification of cell signaling proteins by immuno-MALDI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-177.
Abstract LB-292: p95HER2-T cell bispecific antibody for breast cancer treatment
Authors: Vicario Rocío , Ruiz Irene Rius , Morancho Beatriz , Morales Cristina Bernadó , Herter Sylvia , Freimoser-Grundscholber Anne , Somandin Jitka , Sam Johannes , Ast Oliver , Barriocanal Águeda Martinez , Luque Antonio , Escorihuela Marta , Varela Ismael , Nuciforo Paolo , Fasani Roberta , Peg Vicente , Rubio Isabel , Cortés Javier , Serra Violeta , Escrivá Santiago , Sperinde Jeff , Chenna Ahmed , Huang Weidong , Winslow John , Scaltriti Maurizio , Baselga Josep , Tabernero Josep , Umaña Pablo , Bacac Marina , Saura Cristina , Klein Christian , Arribas Joaquín
Published: August 2018

T cell bispecific antibodies (TCBs) are engineered molecules that include, within a single entity, binding sites to the T cell receptor and to a tumor-specific or a tumor-associated antigen. The receptor tyrosine kinase HER2 is a tumor-associated antigen in ~25% of breast cancers. Clinical evidence shows that TCBs targeting HER2 may result in severe toxicities, likely due to the physiologic levels of HER2 in normal epithelia. Approximately 40% of HER2-positive tumors express detectable levels of p95HER2, a carboxy-terminal fragment of HER2. We previously developed different antibodies that specifically recognize p95HER2 but not full-length HER2 and hypothesized that a TCB antibody recognizing p95HER2 would be exquisitely specific for tumor cells. Here, we describe the development of such p95HER2-TCB and show that it has a potent anti-tumor effect on p95HER2-expressing breast cancers. Since p95HER2 is not expressed in normal tissues, in contrast with HER2-TCB, it has no effect on non-transformed cells expressing physiological levels of HER2. These data paves the way for the safe targeting of a subtype of HER2-positive tumors with a novel tumor-specific TCB.

Citation Format: Rocío Vicario, Irene Rius Ruiz, Beatriz Morancho, Cristina Bernadó Morales, Sylvia Herter, Anne Freimoser-Grundscholber, Jitka Somandin, Johannes Sam, Oliver Ast, Águeda Martinez Barriocanal, Antonio Luque, Marta Escorihuela, Ismael Varela, Paolo Nuciforo, Roberta Fasani, Vicente Peg, Isabel Rubio, Javier Cortés, Violeta Serra, Santiago Escrivá, Jeff Sperinde, Ahmed Chenna, Weidong Huang, John Winslow, Maurizio Scaltriti, Josep Baselga, Josep Tabernero, Pablo Umaña, Marina Bacac, Cristina Saura, Christian Klein, Joaquín Arribas. p95HER2-T cell bispecific antibody for breast cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-292.
Abstract LB-314: The good drug, the bad barrier and the handy peptide: Improved treatment of experimental melanoma brain metastases using a synthetic peptide
Authors: Aasen Synnøve Nymark , Espedal Heidi , Holte Christopher Florian , Keunen Olivier , Karlsen Tine Veronica , Tenstad Olav , Baghirov Habib , Olberg Dag Erlend , Bjerkvig Rolf , Sarkar Gobinda , Thorsen Frits
Published: August 2018

Patients with brain metastases usually have a poor prognosis. Regardless of the constant progress in novel drug establishments, a crucial obstacle is the delivery of drugs across the blood brain barrier (BBB) and into the metastatic neoplasms. Different study designs to achieve temporary BBB opening have previously been studied. Here, we describe a synthetic peptide termed K16ApoE, which is able to transiently open the BBB for drug-delivery into experimental brain metastases.We conducted a systemic study of the ability of the peptide to open the BBB by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in nonobese diabetic/severe combined (nod/scid) mice. Further, we studied the mechanisms of the peptide using flow cytometry, electron microscopy and dynamic positron emission tomography (PET). The biodistribution of the peptide was studied in blood plasma and organs using I-125 labeled K16ApoE. Finally, a treatment study using the peptide in combination with the B-RAF inhibitor dabrafenib was conducted.An opening of the BBB for up to 60 mins was clearly demonstrated by DCE-MRI. Microscopy showed that the peptide disrupted brain endothelial cell monolayers by reducing the barrier properties of the cells. The treatment study demonstrated that the group of animals receiving K16ApoE followed by dabrafenib had smaller tumor volumes than the other two animal groups.In conclusion, the synthetic K16ApoE peptide in combination with dabrafenib decreased the number of experimental brain metastases. Thus, the current strategy could have the potential to improve the treatment of patients with brain metastatic disease.

Citation Format: Synnøve Nymark Aasen, Heidi Espedal, Christopher Florian Holte, Olivier Keunen, Tine Veronica Karlsen, Olav Tenstad, Habib Baghirov, Dag Erlend Olberg, Rolf Bjerkvig, Gobinda Sarkar, Frits Thorsen. The good drug, the bad barrier and the handy peptide: Improved treatment of experimental melanoma brain metastases using a synthetic peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-314.
Abstract LB-324: A whole chromosome aberration phenotype in non-WNT/non-SHH tumors predicts outcome within standard-risk medulloblastomas from the HIT-SIOP-PNET4 clinical trial
Authors: Goschzik Tobias , Schwalbe Ed C. , Hicks Debbie , Figarella-Branger Dominique , Doz Francois , Rutkowski Stefan , Gustafsson Goran , Lannering Birgitta , Pietsch Torsten , Clifford Steve C.
Published: August 2018

Introduction: Standard-risk medulloblastoma (SR-MB; 50-60% of patients) is currently defined by the absence of high-risk (e.g. metastatic disease, large-cell/anaplastic histology, MYC amplification) disease features. 75-85% survival rates are achieved, however the identification and validation of novel prognostic biomarkers will be essential to improve risk-adapted therapies, aimed at increased survival and reduced treatment-related late-effects.

Experimental procedures: We undertook comprehensive analysis of the pan-European HIT-SIOP-PNET4 prospective clinical trial (2001-2006; age 4-21 years at diagnosis), encompassing central clinical and radiological review, and annotation of molecular pathological features to the WHO (2016) classification. Methods were developed and/or adapted to assess methylation-dependent molecular subgroup (MassArray), copy number aberrations (molecular inversion probe array) and mutational status in scant archival material previously refractory to analysis (n=136). Independent prognostic markers/predictive models for non-WNT/non-SHH patients (n=91) were identified by multivariate analyses, and validated in a representative independent cohort (n=70).

Results: WNT (n=28; 21%) and Group 4 (n=76; 56%) tumors were enriched in SR-MB compared to published disease-wide estimates. Favorable-risk WNT disease was confirmed in patients <16.0 years at diagnosis. All events in SHH (4 of 17) occurred in tumors with TP53 mutation and/or chr17p loss. In non-WNT/non-SHH tumors, a novel whole chromosomal aberration (WCA) phenotype characterized by chr7 gain, chr8 loss, and/or chr11 loss, was identified in 42% of patients (38 of 91). This phenotype predicted an excellent prognosis (100% 5-year PFS) and its incorporation into novel survival models out-performed current risk-stratification schemes for SR-MB.

Conclusion: A favorable-risk WCA phenotype identifies a large proportion (42%) of non-WNT/non-SHH SR-MB patients for whom therapy de-escalation should be considered in future biomarker-driven risk-adapted clinical trials; remaining patients (58%; 68% 5-year PFS) might benefit from more intensive and/or novel therapies.

Citation Format: Tobias Goschzik, Ed C. Schwalbe, Debbie Hicks, Dominique Figarella-Branger, Francois Doz, Stefan Rutkowski, Goran Gustafsson, Birgitta Lannering, Torsten Pietsch, Steve C. Clifford. A whole chromosome aberration phenotype in non-WNT/non-SHH tumors predicts outcome within standard-risk medulloblastomas from the HIT-SIOP-PNET4 clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-324.
Abstract LB-357: Transcriptional landscape of stroma progression in the breast tumor microenvironment
Authors: Utama Raditya , Bastian Anja , Sadagopan Narayanan , Jin Ying , Antoniou Eric , Huang Yin , Lee Peter , Atwal Gurinder
Published: August 2018

The breast tumor microenvironment of primary and metastatic sites is a complex milieu of differing cell populations. However, the genomic expression landscape of the tumor stroma and its role in mediating cancer progression and informing effective therapies is not well understood. Here we obtained and cultured 52 cell-sorted stromal tissue samples across 37 patients from normal, primary tumor, and metastatic tumor sites. Deep RNA-seq was performed on poly(A)+ transcripts using a multiplexed barcoding sequencing strategy to increase yield and obviate batch effects. A conservative linear model of expression covariates revealed significant (q<0.05) differential expression of protein-coding genes and lncRNAs in the stroma (80 transcripts in normal versus primary and 108 transcripts in primary versus metastatic). The majority of these genes were found to be associated with pattern formation, embryogenesis, and the epithelial-mesenchymal transition. Among the genes that were silenced in the epithelial cells, IHC staining was initially done for C2orf88, ALOX5AP, and UNC5C which are important in PKA binding, immune response, apoptosis, and neural development. Furthermore, survival analyses of 827 luminal breast tumor samples from TCGA demonstrated the predictive power of the stromal gene expression in informing clinical outcome. Together these results highlight the evolution of stromal gene expression in breast tumors and suggest novel therapeutic strategies targeting the microenvironment.

Citation Format: Raditya Utama, Anja Bastian, Narayanan Sadagopan, Ying Jin, Eric Antoniou, Yin Huang, Peter Lee, Gurinder Atwal. Transcriptional landscape of stroma progression in the breast tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-357.
Abstract LB-386: MicroRNA-164a activates the Wnt pathway to promote breast cancer metastasis
Authors: Kantono Melvin , Sun Wei , Guo Beichu
Published: August 2018

Breast cancer is the most common type of malignant tumor among women, and is the second leading cause of cancer-related death in women. The triple negative breast cancer (TNBC) remains is one of the major contributors of breast cancer mortality. TNBCs are a type of aggressive breast cancer that exhibits a high rate of distant metastasis and poor survival compared with other breast cancer subtypes. Although immunotherapy with PD-1 and PD-L1 blockade have achieved durable responses in a number of tumors, including TNBC. However, the overall response rates were low. Thus, elucidating the mechanisms responsible for tumor metastasis and the interaction of tumor cells with their microenvironments is critical for developing effective therapeutic approaches to treat metastatic breast cancer. Our results suggest that miR-146 can induce oncogenic transformation and tumor metastasis in the context of breast cancer. We found that miR-146a is highly expressed in TNBC cells, and is critical for the expansion of tumor-initiating cells in breast cancer growth and metastasis. Our preliminary data further indicate that miR-146a promotes breast tumorigenesis by targeting negative regulators of Wnt pathway, which is frequently altered in various tumors. The goal of this study to develop novel therapeutic approaches that could ultimately lead to effective treatment for breast cancer, particularly TNBC.

Citation Format: Melvin Kantono, Wei Sun, Beichu Guo. MicroRNA-164a activates the Wnt pathway to promote breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-386.
Abstract SY32-01: Knowledge management and decision support for commonly tested susceptibility mutations
Authors: Parmigiani Giovanni , Braun Danielle , Hughes Kevin S.
Published: August 2018

Challenges: Germline testing for inherited genetic susceptibility to cancer has become cheaper and widely used. Multigene panels now routinely test 25 to 123 genes. Health care providers, including clinicians and genetic counselors, must interpret results, assess the risk of various cancers, and advise on clinical management strategies. Aside from a small number of well-known genes, clinicians lack simple and reliable tools to personalize prevention decisions for individuals who test positive. Evidence on the types of cancer associated with susceptibility genes, and on the magnitude of increased risks associated with a mutation, is emerging at a fast rate. However, a large number of combinations of cancer type and gene variant needs to be considered. No comprehensive databases exist, information is dispersed over a vast number of published studies, the quality of the studies is uneven, and the data presented are seldom directly applicable to precision prevention decisions, which require absolute risk estimates. Thus, when assessing cancer risks for mutation carriers, clinical practice can take little advantage of the impressive scientific advancements in this field.

Approaches: This presentation describes ongoing work to develop tools that could facilitate the translation into clinical practice of research results that would otherwise be difficult to access, systematize, interpret, and convert into actionable knowledge. The long-term goal should be to develop a clinical decision-support system that allows a clinician to enter important factors about a patient with a mutation and immediately receive patient-specific absolute risk estimates. The foundation for the clinical decision-support system needs to be laid by systematically collecting and curating studies on gene cancer associations and, ideally, developing a public access knowledge base for researchers and health care providers. These steps need computational tools for efficient implementations of this plan and for sustainable long-term updating of the knowledge base and decision-support system. The ask2me.org project and the data science behind it are attempting to achieve some of these steps. This project will be used as an illustration throughout.

Impact: Progress in this area will have a direct clinical impact by providing patient-specific absolute risk estimates. These risk estimates will, in turn, lead to more carefully personalized prevention and management for individuals undergoing susceptibility testing, and will help us optimize screening and behavioral interventions to reduce risk.

Citation Format: Giovanni Parmigiani, Danielle Braun, Kevin S. Hughes. Knowledge management and decision support for commonly tested susceptibility mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY32-01.
Abstract SY36-02: Heterogeneity in molecular subtype of hepatocellular carcinoma
Authors: Hoshida Yujin
Published: August 2018

Hepatocellular carcinoma (HCC) mortality keeps rising with emerging etiologies such as nonalcoholic fatty liver disease and hepatitis C after viral cure. Through the efforts of high-throughput and comprehensive molecular characterization of HCC tumors, it has been revealed that HCC tumors can be classified into several subtypes (e.g., aggressive or indolent subtype) according to their molecular characteristics and clinical phenotypes. Ongoing basic and clinical studies have suggested their potential implication in therapeutic decision making. Systematic and integrative characterization of heterogeneity in intra/intertumor molecular features as well as host genetic, environmental, and socioeconomic factors is expected to facilitate development of precision care strategies for HCC patients.

Citation Format: Yujin Hoshida. Heterogeneity in molecular subtype of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY36-02.
Abstract A18: Predictive and prognostic significance of RECQ1 expression in breast cancer
Authors: Parvathaneni Swetha , Lu Xing , Li Xiao Ling , Chaudhary Ritu , Lal Ashish , Madhusudan Srinivasan , Sharma Sudha
Published: August 2018

RECQ1, also known as RECQL or RECQL1, is critical for DNA replication and repair. Germline mutations in the RECQ1 gene predispose to hereditary breast cancers. Overexpression of RECQ1 has been reported in a variety of human cancers including breast cancers. Our recent studies have identified a novel function of RECQ1 in regulation of gene expression and suggest that RECQ1 contributes to tumor development and progression, in part through regulating the expression of key genes that promote invasion and metastasis of cancer cells. Additionally, our data suggest that RECQ1 expression may serve as a predictive marker of clinical outcome. Preclinically RECQ1 depletion in breast cancer cells resulted in doxorubicin sensitivity, DNA double-strand breaks accumulation, S-phase cell cycle arrest, and apoptosis. Utilizing METABRIC cohort to investigate clinicopathologic significance of altered RECQ1 expression in sporadic breast cancers, we found that in ER-positive tumors that received endocrine therapy, low RECQ1 was associated with poor survival; in contrast, in ER-negative tumors that received adjuvant anthracycline-based chemotherapy, high RECQ1 was associated with poor disease-free survival. Taken together, our data provide the first preliminary evidence that RECQ1 is a promising prognostic and predictive biomarker in breast cancers.

Citation Format: Swetha Parvathaneni, Xing Lu, Xiao Ling Li, Ritu Chaudhary, Ashish Lal, Srinivasan Madhusudan, Sudha Sharma. Predictive and prognostic significance of RECQ1 expression in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A18.
Abstract A54: A cellular and molecular atlas of the human breast for dissecting mechanisms of cell and tissue function
Authors: Sayaman Rosalyn W. , Carr Ambrose , Thi Kate , Wan Zhenmao , Pe’er Dana , Bissell Mina J. , Hines Curt
Published: August 2018

A fundamental question in biology, central to our understanding of cancer, is determining how cells coordinate and cooperate to form and maintain tissues. Developing systems-wide knowledge of the numerous cell interactions within a tissue requires robust identification of the cell types it comprises. It demands comprehensive information about each of these cell types, including the genes they express and proteins they make. Unfortunately, the cellular composition and arrangement of cells in many complex tissue systems has yet to be fully resolved, and this includes the breast. This has resulted in a crucial knowledge gap where we know very little about the elaborate biology of how different normal cell types interact, and the consequences these interactions have on each other and the tissue as a whole. Using immunofluorescence and advanced flow cytometry, we have recently developed methods to resolve and isolate every known cell type in the normal human breast. This includes several different luminal and epithelial fractions, myoepithelial cells, adipocytes, leukocytes, pericytes, erythrocytes, adipose-derived mesenchymal stem cells, vascular smooth muscle cells, and both lymphatic and vascular endothelial cells—12 different types in all. Successful modification of this multiparameter FACS procedure allowed purification of enough cells and RNA to perform next-generation sequencing (NGS) from even the rarest of populations at high depth. Here, we present global transcriptome analysis of these different cell types. These data have clarified cell-type differences and revealed new insights, including the identification of genes and gene families contributing to the unique phenotype of each cell type and the delineation of lineage-specific marker expression. Our analysis has also exposed how the cell types each contribute to their individual microenvironments through production of extracellular matrix proteins and other secreted factors. Moreover, identification of cognate ligand and receptor pairs expressed by these cells has unveiled an elaborate paracrine and autocrine signaling network that has implications towards interpreting the biologic interactome underlying tissue homeostasis. Future studies are aimed at establishing coculture models and determining the biochemical processes essential for cell-coordination and tissue maintenance, and developing knowledge of how these processes go awry in breast cancer.

Citation Format: Rosalyn W. Sayaman, Ambrose Carr, Kate Thi, Zhenmao Wan, Dana Pe’er, Mina J. Bissell, Curt Hines. A cellular and molecular atlas of the human breast for dissecting mechanisms of cell and tissue function [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A54.
Abstract A58: Application of evolutionary principles to control ER+ breast tumors
Authors: Garcia Libia , Gillies Robert J. , Gatenby Robert A.
Published: August 2018

Introduction: Estrogen receptor-positive (ER+) breast cancers are initially treated with estrogen deprivation, usually using estrogen receptor modulator drugs (SERM) that are administered at near maximal dose until progression. However, in many of ER+ tumors, preexisting resistant populations that do not express ER on immunohistochemistry are observed. Furthermore, a number of other resistance mechanisms to SERM drugs have been observed including overexpression of MDR1 cell-membrane systems, which consume energy to extrude the drug from the cell. While the vast majority of ER+ tumors respond to SERM drugs, resistant populations can rapidly emerge due to preexisting or acquired phenotypic strategies. Herein, we examine nonconventional treatment strategies, based on the Darwinian evolutionary dynamics that govern tumor evolution, designed to maintain tumor control while delaying evolution of resistance.

Materials and Methods: Different cohorts (20, 24, 4, and 41) of Nu/nu mice were injected in the mammary fat pad with 5*106 MCF7 cells. Prior to the cell injections, an estrogen pellet was subcutaneously implanted in mice. When tumors reached 300 mm3, mice were randomly distributed in treatment groups using different algorithm that combined tamoxifen, paclitaxel, and periods of no treatment. Tumor volumes were monitored by MRI, using T2-weighted images. At the end of the experimental time, tumors were collected and stained for IHC studies with H&E, CD31, SMA, MDR1, and ER.

Results: Treatments in which tamoxifen therapy was interrupted by none-drug periods permitted tumor control equal to that of high-dose standard treatment while maintaining a large population of ER+ cells. Algorithms in which tamoxifen and paclitaxel were combined achieved tumor control equal to standard therapy and decreased MDR1 expressions.

Conclusions: Treatment algorithms designed to exploit evolutionary principles to delay or prevent emergence of resistant populations maintained tumor control using lower cumulative drug doses than the standard-of-care therapy while maintaining populations of cells that are sensitive to the therapy.

Note: This abstract was not presented at the conference.

Citation Format: Pedro M. Enriquez-Navas, Libia Garcia, Robert J. Gillies, Robert A. Gatenby. Application of evolutionary principles to control ER+ breast tumors [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A58.
Abstract A63: Characterizing the role of ancestry-specific variants of the Duffy Antigen Receptor for Chemokines (DARC) gene in the breast tumor microenvironment
Authors: Martini Rachel N. , Jenkins Brittany D. , Davis Melissa B.
Published: August 2018

Chemokines play an important role in cancer, as they direct infiltrating immune cells and mediate angiogenesis, tumor progression, and direct metastasis to distal sites. This work focuses on an atypical chemokine receptor, named the Duffy Antigen Receptor for Chemokines (DARC). DARC is a nonsignaling receptor that can bind two structural classes of inflammatory chemokines, and mainly acts as a regulator of chemokine homeostasis by removing chemokines from circulation when expressed on red blood cells, or moving them from sites of inflammation into circulation to recruit appropriate immune cell types. The purpose of this study is to investigate how polymorphic variants of the DARC gene found across global populations influence ancestry-specific immune responses, and how these changes in the immune response influence the breast tumor microenvironment. A well-studied example of this is a variant restricted to recent decedents of sub-Saharan Africa, known as the Duffy-Null allele. This variant removes expression of DARC from red blood cells, causing those individuals carrying the Duffy-Null allele to lose the ability to sequester chemokines in circulation. In terms of immune response, Duffy-Null status is shown to be predictive of chronic low white blood cell counts observed in individuals of African descent. By using 1000 Genomes Project variant calling data, we have been able to identify additional gene variants restricted to varying global populations. Our initial study is focused on variants found in the regulatory region of the gene, as these variants will likely change how the DARC gene will be expressed. Preliminarily, using data from the Genotype-Tissue Expression project (GTEx), we have been able to show that our DARC gene variants are also tissue-specific expression quantitative trait loci, showing significant changes in tissue-specific gene expression in association with our variants of interest. By studying these variants with panels of human breast cell lines, ancestry-specific cell lines, and human tissue, we will be able to identify these ancestry-specific changes in immune response, and resulting breast tumor microenvironment.

Citation Format: Rachel N. Martini, Brittany D. Jenkins, Melissa B. Davis. Characterizing the role of ancestry-specific variants of the Duffy Antigen Receptor for Chemokines (DARC) gene in the breast tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A63.
Abstract B04: Palbociclib-based combinatorial drug screening in ER-positive cell lines identifies efficacious single agents and novel combinations with synergistic interactions
Authors: Smith Rebecca , Liby Tiera , Gray Joe , Korkola James
Published: August 2018

Estrogen receptor (ER)-positive breast cancer is associated with a good prognosis, but when ER+ breast tumors progress, treatment options are limited. The combination of the CDK inhibitor palbociclib plus the ER degrader fulvestrant was recently approved for patients who have recurrences of ER+ disease. However, durable responses are not typically seen with this combination, and patients eventually progress on therapy. We sought to test other drug combinations using palbociclib as the backbone of therapy. We tested 13 different FDA-approved drugs in combination with palbocilib in a panel of 7 ER+ breast cancer cell lines (600MPE, MDAMB134vii, MDAMB415, ZR75-1, ZR75B, and MCF7). Molecularly, all lines were characterized by amplification of the CCND1 region, high levels of expression of ER, and low RNA expression of EGFR. CAMA1 showed general resistance to the drugs used, never reaching a GR50 (growth rate inhibition of 50%) even at the highest dose tested with palbociclib. CAMA1 was also resistant to the majority of the other 13 drugs, with the exception of panobinostat, vorinostat, and everolimus. Across the entire cell line set, these three compounds were the most effective as single agents. For example, the HDAC inhibitors panobinostat and vorinostat had low nanomolar GR50 values in 7/7 and 4/7 cell lines, respectively. However, significant antagonism was observed between both HDAC inhibitors and palbociclib in MCF7, ZR75B, and ZR75-1, indicating that combining these agents may not be clinically effective. Everolimus plus palbociclib and trametinib plus palbociclib combinations were significantly synergistic in 3/7 cell lines (600MPE, ZR75B, and MCF7 in both cases). Interestingly, CAMA1 and MDAMB134 never showed any evidence of synergistic interactions with any of the combinations tested, suggesting that they may be a distinct subset of ER+ breast cancers. This study indicates that HDAC inhibitors may be effective for treating ER+ tumors that have progressed on antiestrogen therapy, and that combinations of palbociclib plus everolimus or trametinib may also have efficacy.

Citation Format: Rebecca Smith, Tiera Liby, Joe Gray, James Korkola. Palbociclib-based combinatorial drug screening in ER-positive cell lines identifies efficacious single agents and novel combinations with synergistic interactions [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B04.
Abstract B05: Pathologic complete response is an independent prognostic factor for locoregional recurrence in locally advanced breast cancer patients after neoadjuvant chemotherapy
Authors: Chou Hsu-Huan , Chen Shin-Che
Published: August 2018

Background: Neoadjuvant chemotherapy (NAC) has been the standard approach for downstaging in locally advanced breast cancer and can improve breast conservation rates. Pathologic complete response (pCR) after NAC has been reported to have favorable long-term outcome. However, there is still high locoregional recurrence (LRR) rate after NAC. The aim of this study is to analyze the relationship of pCR and LRR.

Method: A retrospective study was analyzed from 2002 to 2011, and a total of 263 invasive breast cancer patients had NAC and, following mastectomy or breast conserving surgery (BCS) at Linkou Chang Gung Memorial Hospital, were enrolled. Concurrent weekly epirubicin and docetaxel was standard NAC regimen and adjuvant therapy including hormone therapy, radiotherapy, and target therapy was started postoperatively according to the pathologic characters.

Result: The median age was 48 years old (range 18-75). The mean tumor size at diagnosis was 6.0 cm (range 2.0-22.0). The median follow-up time was 54.6 months (range 9.2-126.9). After neoadjuvant chemotherapy, 200 (76%) patients underwent mastectomy and 63 (24%) patients underwent BCS. 29 (11%) patients achieved pCR in this study. In univariate analysis, age<50, negative ER, negative PR, positive HER2, luminal B (HER2 positive) and HER2 overexpression, triple-negative subtype were factors to predict pCR. In multivariate analysis, age <50, luminal B (HER2 positive), HER2 overexpression, and triple-negative subtype were factors to predict pCR. No patients in pCR group developed LRR compared with 31 (13.2%) patients with LRR in non-pCR group. In multivariate analysis, pCR and age <50 were independent prognostic factors for LRR.

Conclusion: Our findings suggested that pCR and younger age are independent prognostic factors for LRR in breast cancer patients after NAC with concurrent eprubicin and docetaxel.

Citation Format: Hsu-Huan Chou, Shin-Che Chen. Pathologic complete response is an independent prognostic factor for locoregional recurrence in locally advanced breast cancer patients after neoadjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B05.