bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4444: Deep learning of the immune synapse
Authors: Sidhom John-William , Pardoll Drew M. , Baras Alexander S.
Published: August 2019

Artificial intelligence is poised to revolutionize every aspect of human life, finding applications in everything from self-driving cars to diagnosing cancer. In fact, almost any task that involves pattern recognition can be formulated in a way that modern AI algorithms can be used to achieve super-human performance. The immune synapse is a highly complex interaction between several proteins and peptides that allows for a constant surveillance of foreign invaders. However, modeling these interactions is extremely difficult as the combinations of interactions is simply intractable. In immuno-oncology, the study of this interaction is crucial as anti-tumor responses rely on sensitive and specific recognition of tumor-specific antigens. Implications of accurately predicting and modeling these interactions in immune-oncology range from improved and potent vaccine design to biomarkers for predicting response to immunotherapy. Our group has developed a variety of deep learning models to model the signal transmission within the immune synapse. At the core of all architectures designed, convolutional layers, similar to ones used to learn features in images, are used to learn motifs within the sequencing data for a predictive or descriptive purpose.We first present AI-MHC, an applied deep convolutional neural network for class-specific MHC binding algorithm that achieves state-of-the-art performance in both Class and Class II predictions. By incorporating ‘meaning’ of the allele within the network, we are able to model the interaction of allele and peptide within the context of a neural network. We take these concepts further in the development of DeepMANA, a deep learning framework which combines sequence-specific information about an allele/peptide pairing to not only predict binding affinity for any allele with a known protein sequence but also provide an antigen ‘quality’ score, based on the “non-self/foreign-ness” of a neoantigen. We observe in three previously published immunotherapy clinical trials, these quality neoantigens are enriched in long-term survivors or responders. Finally, we present DeepTCR, a collection of unsupervised and supervised deep learning algorithms capable of revealing structure in T-cell receptor repertoire. We demonstrate that DeepTCR achieves state-of-the-art performance in clustering antigen-specific TCR’s and is capable of learning a predictive signature in TIL repertoire of mice treated with various immunotherapies.These types of AI technologies could yield an entire new area of biomarker discovery as well as improve our understanding of the complex interaction occurring at the immune synapse that is ultimately required for a successful anti-tumor response.

Citation Format: John-William Sidhom, Drew M. Pardoll, Alexander S. Baras. Deep learning of the immune synapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4444.
Abstract 446: Plasma genome sequencing identifies prostate cancer patients that are sensitive to platinum-based therapy
Authors: Ramesh Naveen , Sei Emi , Tsai Pei Ching , Logothetis Christopher , Corn Paul , Aparicio Ana , Zurita Amado J. , Navin Nicholas E.
Published: August 2019

Castration-resistant prostate cancer (CRPC) is a lethal disease. A subset of these patients present with atypical clinical characteristics and aggressive disease behavior. These patients, classified as Aggressive Variant Prostate Cancer (AVPC), may derive benefit from platinum-based chemotherapy. However, the identification of AVPC patients is often challenging due to heterogeneity in clinical presentations and potentially in biological drivers, which may not be fully reflected in a biopsy obtained from a single tumor site. To address these challenges, we developed an unbiased genome sequencing method called PEGASUS (Plasma Exome and Genome Analysis by Size-Selection and Unbiased Sequencing) that enables the simultaneous detection of copy number aberrations and exome mutations from circulating-tumor DNA (ctDNA). We applied this method to plasma specimens obtained from 160 CRPC patients with and without AVPC participating in a randomized trial of cabazitaxel alone or in combination with carboplatin. We were able to successfully isolate ctDNA (>2 ng) for genomic profiling using PEGASUS in 91/160 (57%) CRPC patients. Among these 91 CRPC patients, we detected common prostate cancer mutations as well as genomic copy number changes in genes such as RB1, PTEN and TP53 and several other genes associated with DNA repair. One of the most salient features that distinguished the patients’ outcomes was whether they had diploid or aneuploid genomes detected by whole genome profiling of the ctDNA. Patients with aneuploid ctDNA had worse progression-free and overall survival. Overall, our data shows the feasibility of performing whole-genome and exome profiling of ctDNA in CRPC patients to characterize the molecular features associated with distinct clinical presentations. Candidate markers associated to benefit from platinum-based chemotherapy are being evaluated. These results pave the way for future clinical applications in biomarker discovery to assist treatment decisions in prostate cancer patients.

Citation Format: Naveen Ramesh, Emi Sei, Pei Ching Tsai, Christopher Logothetis, Paul Corn, Ana Aparicio, Amado J. Zurita, Nicholas E. Navin. Plasma genome sequencing identifies prostate cancer patients that are sensitive to platinum-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 446.
Abstract 4462: Sensitivity and reproducibility of onco-panel sequencing across multiple laboratories and technologies
Authors: Xu Joshua , Gong Binsheng , Jones Wendell , Johann Don ,   Onco-panel Working Group of the Sequencing QualityControl Phase 2 (SEQC2) Consortium
Published: August 2019

Onco-panel sequencing targets sequencing reads to few small regions of the genome and thus is better enabled to detect of rare but clinically relevant sub-clonal mutations. Accurate diagnosis and subsequent tailoring of therapy depends on thorough characterization of tumor mutational spectra. The lack of tumor reference samples, comprehensive approaches to assessing reproducibility and detection sensitivity, and standard practice guidelines is hindering the development of onco-panel sequencing and the realization of its benefits in cancer diagnosis and treatment. As a component of the FDA-led SEQC project phase 2 (SEQC2), the Onco-panel Working Group has designed and characterized a set of reference materials. These materials were created using cell line gDNA pooling and dilution to enable such comprehensive assessments of targeted mutation detection approaches. To this end, a cross-lab evaluation of eight Pan-Cancer panels and five ctDNA liquid biopsy assays is currently underway. Each panel was tested in three independent laboratories. To further evaluate methods to boost reproducibility, synthetic spike-in controls and synthetic plasma were also incorporated into the testing samples. Contrived samples for liquid biopsy testing were prepared through enzymatic fragmentation and size selection to mimic cell-free DNA. The onco-panels tested represent two target enrichment approaches (amplicon based and capture based) and sequencing technologies (multiple Illumina platforms and Ion Torrent). Of note, the liquid biopsy assays employ various molecular barcoding techniques to improve their detection sensitivity of rare mutations. Extensive lab QC data has been collected along with sequencing data. Performance measures have been devised to evaluate reproducibility and sensitivity of the variant/mutation calling results that are produced by the panel providers. Meta-analysis of performance measures will then be conducted for cross panel comparison. This comprehensive study will yield insights into factors underpinning sensitivity and reproducibility of onco-panel sequencing. Quantitative performance metrics and actionable data analysis recommendations will be presented to the targeted sequencing panel community.

Citation Format: Joshua Xu, Binsheng Gong, Wendell Jones, Don Johann, Onco-panel Working Group of the Sequencing QualityControl Phase 2 (SEQC2) Consortium. Sensitivity and reproducibility of onco-panel sequencing across multiple laboratories and technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4462.
Abstract 4465: The effect of insurance status on childhood and adolescent cancer survival using data from the National Cancer Database
Authors: Wang Xiaoyan , Ojha Rohit P. , Johnson Kimberly J.
Published: August 2019

Background

Differences in access, delivery or utilization of health care may impact childhood and adolescent cancer survival. In this study, we evaluated the impact of insurance coverage on survival among children and adolescents diagnosed with cancer, and explored potential mechanisms.

Methods

We obtained data from 58,421 individuals diagnosed with any cancer type at ≤ 19 years old from 2004-2010 from the National Cancer Database. We examined associations between insurance status at diagnosis or initial treatment and stage at diagnosis, treatment received (any vs. none), and all-cause mortality using logistic regression, Cox proportional hazards regression, restricted mean survival time and mediation analyses for all cancers combined and by cancer type. We evaluated the moderation effect of age on cancer death in association with insurance status using the likelihood-ratio test.

Results

Children and adolescents with Medicaid, unknown insurance, and those who were uninsured were more likely to be non-White, from lower income counties, be diagnosed at an advanced stage, and less likely to receive any treatment than those with private insurance. Those with Medicaid, unknown insurance, and who were uninsured had a 27% (95%CI, 1.22 to 1.33), 39% (95%CI, 1.26 to 1.53), and 32% (95%CI, 1.20 to 1.46) higher hazard of death during 5 years of follow-up versus those with private insurance. Survival at 60 months was modestly lower for these groups than those who were privately insured at 1.73 months (95%CI, -2.07 to -1.38), 2.48 months (95%CI, -3.29 to -1.68), and 2.13 months (95%CI, -2.91 to -1.34). The higher mortality risk and lower survival months in non-privately insured cases were observed for most cancer types. In mediation analyses, for Medicaid and uninsured patients, earlier diagnosis for staged cancers was estimated to reduce the survival deficit by approximately one fourth and one half. Treatment did not account for the insurance associated survival difference.

Conclusion

Enhancing existing insurance coverage and ensuring equal and early diagnosis may help to improve survival overall for children and adolescents diagnosed with cancer. Future qualitative studies are needed to understand institutional and family level barriers to diagnosis and care of children with cancer and to further explain the underlying mechanisms for differences in childhood survival by insurance status.

Citation Format: Xiaoyan Wang, Rohit P. Ojha, Kimberly J. Johnson. The effect of insurance status on childhood and adolescent cancer survival using data from the National Cancer Database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4465.
Abstract 4469: Clinical outcomes among patients treated with abiraterone acetate for advanced prostate cancer with pre-existing cardiovascular conditions
Authors: Lu-Yao Grace , Nikita Nikita , Keith Scott , Gandhi Krupa , Rebbeck Timothy R. , Cullen Jennifer , Nightingale Ginah , Mann Mark , Chapman Andrew
Published: August 2019

Background: Advanced prostate cancer patients with pre-existing cardiovascular diseases (CVD) are often excluded from clinical trials of abiraterone acetate (AA). Therefore, the clinical outcomes of these patients are uncertain. This study was undertaken to examine their outcomes - changes in the rate of hospitalization and short-term mortality after AA initiation by pre-existing CVD conditions.

Methods: This population-based study identified PCa patients from the linked Surveillance, Epidemiology and End Result (SEER)-Medicare files diagnosed between 1/1/1991 and 12/31/2013 and treated with AA in 2011-2014. The primary endpoints were 6-month overall mortality and changes in hospitalization rates following AA initiation. Major pre-existing conditions are listed in the Table 1. The changes in hospitalization rate were measured by the incidence rate ratios (IRR), post treatment rate divided by pre-treatment rate.

Results: Among 2,845 patients treated with AA (median age 75 years), 1,924 (67.6%) of these patients had at least one pre-existing CVD condition. Compared to pre-treatment period, the hospitalization incidence post-AA increased by 58% to 88%. The crude risk of 6-month overall mortality was between 21.4% to 25.6%.

Table 1:Risk of Hospital Visits and 6-month Overall Mortality among AA Patients by Pre-Existing CVD condition.Pre-existing CVD conditionNo. hospitalizations per 6-months prior to AAIRR for hospitalizations (95% CI)Crude 6-month post AA MortalityAcute Myocardial Infraction0.651.88 (1.48-2.40)25.6%Atrial Fibrillation0.561.58 (1.36-1.83)24.4%Congestive Heart Failure0.591.72 (1.54-1.92)23.4%Stroke0.521.67 (1.40-2.01)22.1%Ischemic Heart Disease0.471.78 (1.63-1.95)21.4%No CVD0.251.87 (1.59-2.20)15.8%

Conclusion: All patients experienced significant increase in hospitalization after initiation of AA. Our study highlights the importance of carefully monitoring patients after prescribing AA.

Citation Format: Grace Lu-Yao, Nikita Nikita, Scott Keith, Krupa Gandhi, Timothy R. Rebbeck, Jennifer Cullen, Ginah Nightingale, Mark Mann, Andrew Chapman. Clinical outcomes among patients treated with abiraterone acetate for advanced prostate cancer with pre-existing cardiovascular conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4469.
Abstract 4472: The hippo pathway effector TAZ regulates ferroptosis in renal cell carcinoma
Authors: Yang Wen-Hsuan , Chi Jen-Tsan
Published: August 2019

Ferroptosis is a newly appreciated lipid peroxidation-dependent regulated cell death with relevance for many human diseases, which may be a tumor suppression mechanism and may have antitumor potential. Elucidation of the mechanism is therefore important. Here, we reported that ferroptosis sensitivity is significantly influenced by cell density. The effectors of the Hippo pathway, YAP/TAZ, are the molecular sensors of cell density that regulate cell proliferation, survival, and organ size determination. Therefore, we determined the potential role of YAP/TAZ in regulating ferroptosis sensitivities. RCC cell lines and RCC PDX tumor cells mainly express TAZ that undergoes density-dependent nuclear translocation. Importantly, the removal of TAZ confers ferroptosis resistance, while the expression of constructively active TAZ sensitizes cells to ferroptosis. Mechanistically, we found that TAZ knockdown reduced ferroptosis through the repression of its target gene, EMP1. EMP1 affects ferroptosis through the regulation of NOX4 and lipid peroxidation. Collectively, we have shown that the ferroptosis can be regulated by non-genetic factors, such as cell density, through the Hippo pathway and suggest the therapeutic potential of ferroptosis-inducing agent, such as erastin, for TAZ-activated tumors.

Citation Format: Wen-Hsuan Yang, Jen-Tsan Chi. The hippo pathway effector TAZ regulates ferroptosis in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4472.
Abstract 4502: The role of A-to-I RNA editing in diversifying the microRNA functions in cancer
Authors: Liang Han
Published: August 2019

Both miRNAs and A-to-I RNA editing, a widespread nucleotide modification mechanism, have recently emerged as key players in cancer pathophysiology. However, the functional impact of RNA editing of miRNAs in cancer remains largely unexplored. Here, we systematically characterized the miRNA editing profiles of ~9000 samples across 20 cancer types from miRNA sequencing data of The Cancer Genome Atlas and identified adenosine-to-inosine (A-to-I) RNA editing hotspots. Focusing on the RNA editing hotspot in miR-200b, a key tumor metastasis suppressor, we found that the miR-200b editing level correlates with patient prognosis opposite to that pattern for the wild-type miR-200b expression. We further experimentally showed that in contrast to wild-type miRNA, the edited miR-200b can promote cell invasion and migration through its impaired ability to inhibit ZEB1/ZEB2 and acquired concomitant ability to repress new targets including LIFR, a well-characterized metastasis suppressor. In another example, an ADAR2-catalyzed RNA editing site within the miR-379 is under-edited in tumors relative to normal tissues, with a high editing level being correlated with better patient survival times across cancer types. We demonstrate that in contrast to wild-type miRNA, edited miR-379 inhibited cell proliferation and promoted apoptosis in diverse tumor contexts in vitro. Importantly, through nanoliposomal delivery, edited miR-379 mimics significantly inhibited tumor growth and extended survival of mice. Our study indicates a role of RNA editing in diversifying miRNA function during cancer progression and highlights the translational potential of edited miRNAs as new class of cancer biomarkers and therapeutics.

Citation Format: Han Liang. The role of A-to-I RNA editing in diversifying the microRNA functions in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4502.
Abstract 4506: MDM2 alternative splicing is regulated by microRNA binding to control p53 activity
Authors: Montes Matias , Goodwin Andrew , Chandler Dawn S.
Published: August 2019

Alternative splicing of the MDM2 is an important means by which p53 is upregulated to combat the deleterious effects of genotoxic stress. One splice variant, MDM2-ALT1, is activated in response to genotoxic stress and is comprised of only the two terminal coding exons 3 and 12 and therefore lacks a p53 binding domain. This variant has been identified in a number of human tumors, including invasive carcinoma of the breast and lung, as well as soft tissue sarcomas. Despite its pervasiveness in tumors and the therapeutic possibilities it presents, there is very little known about the regulators of that govern this alternative splicing event.

Our lab has identified a specific microRNA that is expressed at high levels in normal cells but decreased under conditions of genotoxic stress and in rhabdomyosarcoma cells, both of which are characterized by the expression of the MDM2 spliced isoform. Hence, expression of the microRNA is inversely correlated with the splice variant MDM2-ALT1. Though recent studies have shown that splicing factors can be regulated through miRNA-mediated gene silencing and indirectly influence splicing choices, we were unable to identify any splicing regulators of MDM2 to be targeted by the microRNA. Interestingly, the microRNA has been reported as one of the miRNAs that is reimported to the nucleus but has not been ascribed a role in the nuclear space. We therefore wanted to test the hypothesis that the microRNA can directly bind to the MDM2 pre-mRNA to drive splicing choices in a regulated fashion. To this end, we have identified predicted binding sites for the microRNA in the regulated region of MDM2, within and near exons 3 and 12. We have shown that the microRNA binds specifically to these regions in MDM2 in normal cells and has decreased binding after genotoxic stress. Furthermore, we have shown that overexpression of the pre-microRNA can alleviate the induction of the MDM2-ALT1 isoform in response to genotoxic stress. We propose that microRNA binding to the MDM2 pre-mRNA regulates its splicing and plays a role in the DNA damage response and the pathway to tumorigenesis.

Our work implicates for the first time that a miRNA would be acting as a splicing factor, a novel function for this class of non-coding RNAs and a novel method of regulation of the p53-MDM2 axis. Furthermore, our discoveries reveal miR-regulated splicing as a possible strategy to activate p53 and sensitize cancer cells to conventional cancer therapies.

Citation Format: Matias Montes, Andrew Goodwin, Dawn S. Chandler. MDM2 alternative splicing is regulated by microRNA binding to control p53 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4506.
Abstract 4515: Utilizing nucleic-acid scavengers (NASs) to inhibit proinflammatory and proinvasive signaling in triple-negative breast cancer
Authors: Eteshola Elias O. , Naqvi Ibtehaj A. , Gunaratne Ruwan , Moreno Angelo , Nair Smita K. , Sullenger Bruce A.
Published: August 2019

Breast cancers (BC) remain the most lethal malignancies amongst women worldwide and the second leading cause of cancer-related mortalities in the US. Subtype heterogeneity and aggressive invasive potential are believed to be the major contributors of these outcomes. BC lacking canonical histological receptors (i.e. ER/PR/HER2), called triple-negative (TNBC), are notoriously aggressive, difficult-to-treat, and metastatic. It has been observed that the degree of inflammation-driven tumorigenesis tends to correlate with increased levels of cell-free DNA (cfDNA) in cancer patient sera. Our lab had previously shown that nucleic-acid scavengers (NASs) could be used to block the pro-inflammatory and pro-invasive/metastatic signals (e.g. DAMPs) elicited by these cfDNA/RNA likely through innate immune sensors such as of the toll-like receptors (TLRs). Recently, we showed that treatment with the cationic polymer NAS, PAMAM-G3, elicited a drastic reduction in metastatic tumor burden to the liver in an immune-competent murine model of pancreatic cancer (I. Naqvi & R. Gunaratne et al., Molecular Therapy 26, 2018). Ongoing work has shown that both chemotherapy-derived TNBC conditioned media and a TLR9 agonist greatly increased TNBC in vitro invasion and was significantly inhibited upon treatment with PAMAM-G3. To elucidate the mechanism by which this NAS works in these tumor settings, our lab has developed several PAMAM-G3 derivatives, including biotin, IR-, and near-IR fluorophore labeled molecules. These molecules will allow us to conduct DAMP capture and characterization experiments, as well as perform in vitro and in vivo live imaging experiments to gain better insights into NAS PK/PD properties. Mechanistic insight into NAS anti-metastatic and anti-inflammatory capabilities will enhance our basic understanding of metastatic progression and its interplay with the immune system. Moreover, these principles will aid in the development of novel of anti-metastatic therapies to improve TNBC patient outcomes.

Citation Format: Elias O. Eteshola, Ibtehaj A. Naqvi, Ruwan Gunaratne, Angelo Moreno, Smita K. Nair, Bruce A. Sullenger. Utilizing nucleic-acid scavengers (NASs) to inhibit proinflammatory and proinvasive signaling in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4515.
Abstract 4521: Mutant Kras expression triggers clonal acinar cell expansion as an early step in pancreas cancer
Authors: Artandi Steven E. , Neuhöfer Patrick , Roake Caitlin M. , Kim Stewart , Lu Ryan , Charville Greg
Published: August 2019

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States with the lowest 5-year survival rate of any major cancer, in part due to late detection of the disease. Studies indicate that pre-invasive lesions - pancreatic intraepithelial neoplasias (PanINs) - progress to PDA. The cell of origin for PDA is still under debate. While lineage-tracing experiments in mice indicate that PanINs originate from acinar cells, cell culture studies suggest that ductal cells can give rise to PanINs. In addition to these important questions regarding cancer initiation, mechanisms of cellular renewal in the exocrine pancreas remain poorly understood. The enzyme telomerase is intimately associated with cancer, and with stem cells and tissue renewal. Telomerase is required for long-term cell proliferation through its critical role in maintaining telomeres. The core enzyme consists of the RNA component TERC and the reverse transcriptase TERT. Moreover, telomerase is activated in over 90% human cancers and genome wide association studies have linked polymorphisms in the Tertgene with an increased risk of pancreas cancer. To analyze the role of TERT-expressing cells in the pancreas during homeostasis, regeneration and tumorigenesis, we generated a mouse line that expresses the regulated CreERT2 recombinase under the endogenous TERT promoter (TertCreERT2/+). Crossing these mice to a reporter strain enables the permanent labeling of TERT-expressing cells and their progeny upon tamoxifen injection. We find that rare TERT+ cells exist throughout the pancreas within the acinar and islet cell populations, but not in ductal cells. We show that rare TERT+ acinar cells clonally expand to renew the acinar compartment during homeostasis and during pancreas injury. To understand how these TERT+ acinar progenitor cells respond to mutant Kras, we crossed TertCreERT2/+mice with KrasLoxStopLox-G12D/+mice. We find that activation of KrasG12D in TERT-expressing cells in adult mice promotes accelerated acinar clone formation and these acinar lesions ultimately convert to PanIN pre-invasive lesions. Our data indicate that TERT-expressing cells represent an acinar progenitor cell population. Furthermore, these progenitor cells respond to mutant Kras first through a regenerative response yielding expanding acinar clones, followed by conversion to ductal PanIN lesions. We propose that acinar cell expansion is an early step in pancreatic tumorigenesis that precedes ADM and PanIN formation.

Citation Format: Steven E. Artandi, Patrick Neuhöfer, Caitlin M. Roake, Stewart Kim, Ryan Lu, Greg Charville. Mutant Kras expression triggers clonal acinar cell expansion as an early step in pancreas cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4521.
Abstract 4522: An in vitro 3D immune exclusion tumor model engineered in a layer-by-layer fashion
Authors: Hribar Kolin C. , Kleinheinz Tracy , Klementowicz Joanna , Kaufman Susan , Blake Robert A. , Eidenschenk Celine
Published: August 2019

Recent immunotherapy failures in clinical trials have elucidated the affects of the tumor stroma on attenuating therapeutic response. In some cancers, the stroma consists of a collagen-rich fibrotic zone on the tumor periphery that traps infiltrating CD8+ T cells, allowing the tumor to evade immune cell-mediated death. Typically, animal models are employed to study complex physiology. However, known challenges to these methods include cost, duration and throughput. Thus, we sought to develop a 3D in vitro model that recapitulates the immune exclusion phenotype in a high throughput manner. We utilized a novel 3D cell culture platform, VersaGel and Symphony, to pattern tumor-stroma-immune compartments in a layer-by-layer fashion in 96-well plates. VersaGel is a chemically modified extracellular matrix that is amenable to light-induced crosslinking using the Symphony apparatus. Moreover, Symphony allows for discrete layers to be formed with any VersaGel/cell mixture, with defined 3D gel thicknesses (eg. 100µm, 250µm, 500µm) and areas in the plate (eg. 2mm, 4mm, or 8mm diameter discs).

GFP-labeled MC38-ova (ovalbumin antigen) colon carcinoma cells were grown in 100µm thick 3D VersaGel with or without CFP-labeled mouse embryonic fibroblasts (MEF) layered on top for 4 days. After, OT-1-RFP CD8+ cells were added to the culture medium to assess their penetration depth and tumor cell death over 3 days. 3D cell analysis through the optically clear VersaGel was performed in situ using high content confocal microscopy, effectively quantifying RFP/CFP/GFP-labeled cells and DRAQ7-labeled dead cells using Z-stack analysis. In the control (tumor-only condition), OT-1 cells fully penetrated the tumor layer and induced cell death. In the experimental condition (with fibroblasts), it was found that the fibrotic stromal layer limited OT-1 penetration to the periphery of the 3D tumor, and, moreover, the total OT-1 cells increased relative to the control. Future analysis may include the utility of wild-type T cells and novel therapeutics targeting the stroma to activate the immune response. The results suggest a novel 3D immune exclusion model that integrates with high content confocal analysis and is amenable to immunotherapeutic screening and translational assessment.

Citation Format: Kolin C. Hribar, Tracy Kleinheinz, Joanna Klementowicz, Susan Kaufman, Robert A. Blake, Celine Eidenschenk. An in vitro 3D immune exclusion tumor model engineered in a layer-by-layer fashion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4522.
Abstract 4532: Differential protein and metabolite regulation in urothelial carcinoma highlights changes in immunity and receptor signaling pathways
Authors: Gethings Lee , King Adam , Peck Andrew J. , Plumb Robert S.
Published: August 2019

Bladder cancer is ranked as one of the tumours posing high morbidity and mortality, with a pathogenesis which is still not well understood. A combination of genetic and lifestyle factors, including smoking, are known to contribute towards increasing the probability of encountering cancer. Here, we describe a multi-omic approach to reveal molecular factors that may be involved in these biomolecular processes. Plasma samples consisting of healthy controls and those from patients diagnosed with bladder cancer were prepared for proteomic, metabolomic and lipidomic analyses. Label-free LC-MS data were acquired using a QTof platform utilizing a data independent (DIA) workflow termed SONAR. Small molecule analysis consisted of using Progenesis QI for data processing to provide normalised values prior to statistical analysis. Unsupervised MVA of the data showed clear distinction between cohorts. OPLS-DA was used to filter for features of significant correlation and covariance prior to identification using metabolite and lipid specific databases. Identifications matching the following criteria, mass accuracy <5 ppm, ANOVA p <0.05, %CV <30 and fold change >2 were considered for further interrogation. SONAR-based analysis indicates that this method of data acquisition enabled over an order of magnitude more specificity than equivalent methodologies with the same resolution. Proteomic data were processed and searched against a Uniprot Homo sapien database, containing reviewed entries and limited to 1% FDR. Additionally, we also searched the data against a spectral library and comparatively analysed the results from both workflows. A number of significant proteins with differential regulation were exhibited for a number of protein groups involved in antigen and lipid binding. Proteins occurring in a minimum of two out of three replicates and with ANOVA p <0.05 were considered. Biological significance of the results was established by merging data from all three omic experiments and performing pathway analysis. A number of significant pathways including complement activation, B cell mediated immunity and receptor signalling were identified as key pathways.

Citation Format: Lee Gethings, Adam King, Andrew J. Peck, Robert S. Plumb. Differential protein and metabolite regulation in urothelial carcinoma highlights changes in immunity and receptor signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4532.
Abstract 4535: The mTOR complex 2 promotes glioblastoma migration via the interactions with multiple actin-binding and microtubule-associated proteins
Authors: Chantaravisoot Naphat , Wongkongkathep Piriya , Pacharakullanon Narawit , Tamanoi Fuyuhiko , Loo Joseph A. , Pisitkun Trairak
Published: August 2019

The mechanistic target of rapamycin complex 2 (mTORC2) is one of the two multiprotein complexes in the mTOR signaling pathway. It regulates several cellular activities such as cell survival, lipid metabolism, DNA damage control, and cell motility. The mTORC2 has been reported to be involved in the promotion of metabolic reprogramming, cancer migration, and cancer invasiveness in glioblastoma. However, underlying mechanisms and roles of this particular complex in glioblastoma migration has not been completely elucidated. Previously, we characterized the mTORC2-associated interactome by affinity purification-mass spectrometry (AP-MS) technique, identifying new RICTOR-interacting partners. In addition, we also investigated the localization of RICTOR under different culture conditions and found that it correlates with migration ability of the cancer cells. When mTORC2 activity is high resulting in increased migration, RICTOR tends to localize around the cell membrane. In this work, we further determined crucial players that interact with mTORC2 to control actin cytoskeleton and microtubule dynamics to promote migration. The results from quantitative proteomic analysis of mTORC2-associated interactome suggested that the amount of certain actin-binding proteins and microtubule-associated proteins significantly changed depending on mTORC2 activity level including Gelsolin, Myosin-9, Plectin, and MAP1B. When mTORC2 signaling was inhibited resulting in reduced migration, these proteins were found to be less interacting with mTORC2. Moreover, the results also indicated that active mTORC2 links both actin cytoskeleton and microtubules together to promote migration in glioblastoma cells. Depletion of RICTOR and inhibition of mTORC2 disrupted the connection between microtubules and cell membrane. Taken together, this work provided a plausible mechanism underlying the regulation of enhanced migration in brain cancer cells.

Citation Format: Naphat Chantaravisoot, Piriya Wongkongkathep, Narawit Pacharakullanon, Fuyuhiko Tamanoi, Joseph A. Loo, Trairak Pisitkun. The mTOR complex 2 promotes glioblastoma migration via the interactions with multiple actin-binding and microtubule-associated proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4535.
Abstract 4538: Global and phosphoproteomic analysis of AML cell line response to phosphatase inhibitor treatment
Authors: Piehowski Paul D. , McDermott Jason E. , Hansen Joshua R. , Savage Samantha L. , Tognon Cristina E. , Agarwal Anupriya , Tyner Jeffrey W. , Druker Brian J. , Rodland Karin D.
Published: August 2019

In the United States, approximately 21,000 people are diagnosed with AML each year and over 10,000 AML related deaths are reported. Genomic analysis has revealed at least 11 genetic classes of AML, and more than 20 subsets can be assigned when considering the differentiation states of Leukaemic blasts. This variability has generated significant interest in the development of targeted therapies for AML. However, complex mutational patterns and a lack of pharmacological agents for most mutational events have challenged the development of effective treatments. Recent results from the Beat AML clinical trial revealed that response to drugs is associated with mutational status, including instances where drug sensitivity is specific to combinatorial mutational events.

To better understand the molecular mechanisms underpinning drug response, we performed global and phosphoproteomic analyses on 4 AML cell lines that carry mutations associated with AML: K562 with a BCR-ABL fusion, MOLM14 with a FLT3 mutation, HL60 with a RAS mutation, and CMK with a JAK3 mutation. These cell lines were each treated with a different kinase inhibitor drug for which they have a known sensitivity. Cell samples were collected prior to treatment and time points of 30 min, 3h, and 16 hrs. Frozen cell pellets were prepared for analysis using the workflow for multiplexed, deep-scale, proteome and phosphoproteome analysis developed by the NCI CPTAC program.

The goal of our Proteogenomic Translational Research Center is to develop drug response signatures allowing the use of baseline proteome and phosphoproteome measurements to predict patient drug response, providing a foundation for rational selection of combination therapies. Preliminary analysis of the data show a dynamic temporal response to drug treatment in both the global and phosphoproteome, Figure 1. Current efforts are focused on combining knowledge-driven and data-driven analysis approaches to identify the pathways associated with drug response.

Citation Format: Paul D. Piehowski, Jason E. McDermott, Joshua R. Hansen, Samantha L. Savage, Cristina E. Tognon, Anupriya Agarwal, Jeffrey W. Tyner, Brian J. Druker, Karin D. Rodland. Global and phosphoproteomic analysis of AML cell line response to phosphatase inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4538.
Abstract 4542: The use of saliva proteins as biomarkers to predict the risk of lymph node metastasis in oral cancer
Authors: Rossi Tatiane De , Granato Daniela Campos , Ribeiro Ana Carolina , Rivera César , Heberle Henry , Telles Guilherme Pimentel , Carnielli Carolina Moretto , Brandão Thais Bianca , Santos-Silva Alan Roger , Lopes Márcio Ajudarte , Leme Adriana Franco Paes
Published: August 2019

Background. The most common oral cancer in the world is squamous cell carcinoma (OSCC), accounting for more than 90% of all cases of cancer in the oral cavity. Thus, the research on molecular markers associated with the development and progress of human diseases has been the subject of intense research. The findings that saliva has molecular profiles indicating systemic diseases urge the study of non-invasive diagnosis using saliva as source of potential diagnosis, prognosis and predictive based on proteomics.

Methods. 204 proteotypic peptides were selected previously based on DDA data from our own studies or retrieved from the SRMAtlas. Heavy labeled synthetic peptides were synthetized and SRM method was developed using Skyline v3.6. Saliva samples were collected from patients with lymph node metastasis (n=26) and without lymph node metastasis (n=14). Saliva proteins were digested with trypsin. Samples were randomized in R environment and analyzed in a triple-quadrupole mass spectrometer (Xevo TQ-XS, Waters). A Peptide Retention Time Calibration Mixture was spiked into the samples. Data comparison between groups was performed using Wilcoxon Mann Whitney test in R environment.

Results. In the SRM method, we monitored 68 proteins and 24 of them were found decreased in patients with lymph node metastasis in comparison with patients without lymph node metastasis. The data analysis showed that at least one protein has, by itself, a higher relative risk in the development of lymph node metastasis in patients with OSCC. Two other panel of proteins presented the same relative risk described before.

Conclusions. This study indicates a panel of potential OSCC marker proteins, which can contribute to the prognostic evaluation, the patient's risk profile and the possibility of recurrence, and guide therapeutic intervention strategies.

Note: This abstract was not presented at the meeting.

Citation Format: Tatiane De Rossi, Daniela Campos Granato, Ana Carolina Ribeiro, César Rivera, Henry Heberle, Guilherme Pimentel Telles, Carolina Moretto Carnielli, Thais Bianca Brandão, Alan Roger Santos-Silva, Márcio Ajudarte Lopes, Adriana Franco Paes Leme. The use of saliva proteins as biomarkers to predict the risk of lymph node metastasis in oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4542.
Abstract 4548: The chemokine receptor CCR1 is involved in microglia stimulated glioblastoma invasion
Authors: Coniglio Salvatore J. , Ramasundaram Poornema , Fournier Neshama , Hamilton Danielle S. , Marshall Gregory , Smith Keia , Habib Diana , Merritt James R.
Published: August 2019

Glioblastoma Multiforme (GBM) is the most aggressive form of adult brain tumor with a median survival time of twelve months. GBM is highly resistant to conventional therapy which includes surgical resection of the tumor, radiation treatment and chemotherapy. GBM cells are highly motile and invasive resulting in infiltrative tumors with poorly defined borders. GBM tumors are heavily infiltrated with microglia cells which are known to stimulate GBM cell invasion. Our laboratory has previously demonstrated that microglia strongly stimulates GBM invasion both in-vitro and in orthotopic animal models. This interaction was found to be dependent on CSF-1R which is expressed on all tumor infiltrating macrophages/microglia. Blockade of the CSF-1R using compounds such as pexidartinib (PLX3397) can inhibit microglia/macrophage-stimulated GBM invasion in-vitro and in vivo. A variety of chemokines are upregulated in the GBM tumor microenvironment and facilitate “cross-talk” between microglia and GBM cells eliciting a chemotactic response. We have demonstrated that the chemotactic ligand, CCL3, is similarly upregulated in GBM tumors. We postulated that inhibition of CCL3 associated receptors such as C-C receptor 1 (CCR1) might also inhibit GBM invasion, thus, a CCR1 antagonist could prove efficacious for blockade of microglia-induced glioblastoma invasion in vitro. Many potent CCR1 antagonists have been described in the literature. We chose four of these compounds with two distinct structural cores, all with reported IC50’s of less than 200 nM for inhibition of CCR1 binding versus CCL3. We examined the ability of these antagonists to block microglia-stimulated glioblastoma invasion using an in-vitro coculture invasion assay. Using quantitative PCR arrays, we also show that expression of chemokines and chemokine receptor genes is greatly altered in GBM conditioned media-treated microglia. Understanding the pattern of tumor-associated macrophage/microglia chemokine secretion in GBM may present additional targets for chemotherapeutic intervention and enhance immunotherapy.

Citation Format: Salvatore J. Coniglio, Poornema Ramasundaram, Neshama Fournier, Danielle S. Hamilton, Gregory Marshall, Keia Smith, Diana Habib, James R. Merritt. The chemokine receptor CCR1 is involved in microglia stimulated glioblastoma invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4548.
Abstract 455: Innovations in large scale liquid biopsy analysis and the Broad/IBM Cancer Resistance Project
Authors: Cibulskis Carrie , Blumenstiel Brendan , DeFelice Matthew , Fleharty Mark , Abreu Justin , Adalsteinsson Viktor , Parida Laxmi , Hamilton Susanna , Getz Gad , Lennon Niall
Published: August 2019

Broad Genomics offers a comprehensive liquid biopsy sequencing platform designed to provide the optimal flexibility for conducting research studies in a broad range of applications including: biomarker discovery, treatment resistance monitoring, and clinical grade ctDNA profiling. By utilizing low cost, low coverage whole genome sequencing in conjunction with dual unique molecular indexed (UMI) libraries we can offer a range of analysis that allow researchers to select the most appropriate samples for whole exome profiling or for deeper coverage, higher sensitivity targeted gene panels. To date we have generated over 3000 liquid biopsy whole genome copy number profiles and purity estimates and are supporting driver projects including the Broad/IBM Cancer Resistance Project and Count Me In. The study design for the Broad/IBM effort takes advantage of the discovery potential of tissue-based sequencing combined with serial liquid biopsy analysis to elucidate resistance events by tracking clonal and subclonal populations in patient samples over time. Sourcing samples for this and other similar efforts is a major undertaking and a combination of methods for maximally broad and deep genomic profiling are required to assay patients throughout the course of care, as tumor fraction in blood fluctuates. Responding to this need, and other applications requiring increased sensitivity we have developed a high throughput, automated workflow to efficiently assay cfDNA samples with lower tumor content. Benchmarking data using healthy donor pooled cfDNA samples indicates our assay is capable of detecting > 90% of variants present at ~1% minor allele fraction with less than 1 false positive variant called per megabase. This established laboratory and analytic process forms the basis of our 2Mb, 400 gene CLIA targeted assay currently undergoing validation. Through this suite of products we hope to enable an expansion of cfDNA sequencing efforts in support of clinical and research applications. Early results from emerging studies utilizing this platform to be presented.

Citation Format: Carrie Cibulskis, Brendan Blumenstiel, Matthew DeFelice, Mark Fleharty, Justin Abreu, Viktor Adalsteinsson, Laxmi Parida, Susanna Hamilton, Gad Getz, Niall Lennon. Innovations in large scale liquid biopsy analysis and the Broad/IBM Cancer Resistance Project [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 455.