bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4574: Combining multimodal biomarkers as an immunogram to guide immunotherapy use: A proof of concept
Authors: Sbarrato Thomas , Sudre Lucie , Vanhille Laurent , Outters Pernelle , Angelova Mihaela , Mlecnik Bernhard , Vasaturo Angela , Bindea Gabriela , Fredriksen Tessa , Lafontaine Lucie , Bruni Daniela , Galon Jérôme , Fieschi Jacques
Published: August 2019

Our recent understanding of the immune contribution to fight cancer has deeply modified the standard of care of cancer patients. As an example, immunotherapies by immune checkpoint inhibitors (ICI) anti-PD1/PDL1 are now approved in several cancer indications, such as Non-Small Cell Lung Cancers or melanoma. However, ICI are less effective for other high incidence indications like colorectal cancer (CRC). Here, the heterogeneity as well as the relatively low degree of patient response to those immunotherapies have highlighted that factors, present in the tumor microenvironment (TME), may limit or boost the efficacy of treatment. In this context, the comprehensive assessment of these factors could be key to stratify patients and allow the selection of the optimal treatment.

In order to help clinical researchers and biopharmaceutical companies to measure the immune contribution to drug efficacy, HalioDx has developed the Cancer Immunogram, a solution based on Blank CU et al. Science (2016). Our multi-parameter approach encompassing a unique range of immune scoring assays is based on the analysis and the understanding of the immune contexture of tumors and offers a personalized and dynamic “fingerprint” of tumor-immune system interaction.

Here, we provide a Proof of Concept for the Cancer Immunogram in the context of CRC by combining the following technologies and biomarkers: Tumor Mutational Burden (Tumour foreignness), DNA mismatch-repair deficiency (MSI), TCR Sequencing (T Cell Clonality), Immunoscore® Colon (Immune Cell Infiltration), Immunosign® (Tumor Sensitivity to Immune Effectors), Halioseek® PDL1/CD8, Brightplex T-Cell Exhaustion Panel (Immune Checkpoints) and Brightplex MDSC Panel and Treg detection (Immune Suppression). We show that the Cancer Immunogram allows to identify patient specific patterns which might improve the prediction of the response to therapy. We believe that the Cancer Immunogram will help researchers and clinicians to personalise treatments in order to improve patients’ outcomes and response to cancer treatment.

Citation Format: Thomas Sbarrato, Lucie Sudre, Laurent Vanhille, Pernelle Outters, Mihaela Angelova, Bernhard Mlecnik, Angela Vasaturo, Gabriela Bindea, Tessa Fredriksen, Lucie Lafontaine, Daniela Bruni, Jérôme Galon, Jacques Fieschi. Combining multimodal biomarkers as an immunogram to guide immunotherapy use: A proof of concept [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 4574.
Abstract 4578: Macrophages are important source of PD-L1 and PD-L1 expressing on central M2 macrophages leads to the poor prognosis of NSCLC patients: Via macrophage landscape analysis for NSCLC patients with tumor PD-L1 negative
Authors: Cao Lili , Che Xiaofang , Qu Xiujuan , Qiu Xueshan , Li Zhi , Yang Bowen , Wang Shuo , Liu Yunpeng
Published: August 2019

PD-1 antibody has achieved significant clinical efficacy for advanced non-small cell lung cancer (NSCLC), but the ORR is not optimistic for the non-selected patients. PD-L1,as the therapeutic target, is still controversial to be a predictor of efficacy.It was noticed that some patients with tumor cell PD-L1 negative could still benefit from anti-PD-1/PDL1 antibody therapy.Besides the tumor cells, PD-L1 was also known to express in immune cells in TME. Therefore, in this study, we aimed to clarify the factors that affect the PD-L1 expression in TME, and their functions on the NSCLC prognosis and the effect on anti-PD-1/PD-L1 therapy.With the multiple quantitative immunofluorescence staining on 94 tissue specimens of NSCLC patients with PD-L1 expressing negative on tumor cells, we surprisingly found that macrophages,especially M2 macrophages,were the main source of PD-L1 for patients with tumor cells PD-L1 negative.Futhermore, the expression of PD-L1 in central M2 macrophages(M2 macrophages that infiltrate into tumor islets),was important for the poor prognosis of NSCLC patients,suggesting that the expression of PD-L1 in macrophages may also be associated with the benefit of anti-PD-1 therapy, which is an important factor to be considered in evaluating the efficacy of immunotherapy in the future.

Note: This abstract was not presented at the meeting.

Citation Format: Lili Cao, Xiaofang Che, Xiujuan Qu, Xueshan Qiu, Zhi Li, Bowen Yang, Shuo Wang, Yunpeng Liu. Macrophages are important source of PD-L1 and PD-L1 expressing on central M2 macrophages leads to the poor prognosis of NSCLC patients: Via macrophage landscape analysis for NSCLC patients with tumor PD-L1 negative [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 4578.
Abstract 4589: SRC drives invasion of luminal, but not basal, bladder cancer
Authors: Wehrenberg Bryan , Ochoa Andrea , Choi Woonyoung , McConkey David
Published: August 2019

Preventing progression from non-muscle invasive to muscle invasive bladder cancer (BC) is a major clinical priority. This transition is defined by tumor invasion through the bladder epithelium basement membrane into the lamina propria and muscle wall. Inhibition of SRC, a pro-invasion, non-receptor tyrosine kinase that signals as part of a complex with Focal Adhesion Kinase (FAK), may prevent BC progression. We hypothesize that SRC inhibition will disrupt migration and invasion of BC cells. Using The Cancer Genome Atlas (TCGA) mRNA expression data of human bladder tumors, we generated a classifier to subtype tumors as either “luminal” or “basal” and compared SRC expression between the groups. We then sequenced a panel of 30 bladder cancer cell lines. Each cell line was given a subtype call based on our luminal- basal classifier, and levels of SRC mRNA expression were examined. To determine the impact of SRC inhibition on luminal and basal subtypes, we exposed BC cell lines to the SRC-Family Kinase inhibitor, AZD0530. The impact of SRC inhibition on proliferation was measured by MTT assay. Transwell assays were used to examine the effect of SRC inhibition on migration, collagen invasion, and Matrigel invasion. Phosphorylation of SRC downstream targets after AZD0530 treatment was assessed by western blot. The role of SRC target and signaling partner FAK in migration was determined by siRNA knockdown and transwell assay. Luminal human bladder tumors had significantly greater SRC mRNA expression than basal tumors (p<0.001). SRC expression was also enriched in luminal BC cell lines (p<0.05). Inhibiting SRC with AZD0530 had minimal impact on proliferation with no subtype specific effects. Migration and collagen invasion of all luminal cell lines was significantly reduced by AZD0530 treatment (p<0.05). The majority of luminal models capable of invading Matrigel were also inhibited by AZD0530. In contrast, invasion and migration of the majority of basal cell lines was not significantly reduced by SRC inhibition, and was significantly increased in several cases (p<0.05). Both luminal and basal models exhibited reduced phosphorylation of SRC downstream targets when exposed with AZD0530. siRNA knockdown of FAK had no impact on migration in a basal cell line. Enrichment of SRC expression in luminal tumors and significant reductions in luminal cell line invasion after AZD0530 treatment suggest SRC promotes invasion in luminal BC. These results suggest AZD0530, and other SRC-Family Kinase inhibitors, could serve as effective anti-progression agents in luminal BC.

Citation Format: Bryan Wehrenberg, Andrea Ochoa, Woonyoung Choi, David McConkey. SRC drives invasion of luminal, but not basal, bladder 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 4589.
Abstract 4591: Overexpression of p62/IMP2 can promote cell migration in hepatocellular carcinoma (HCC) via activating Wnt/ β-catenin pathway
Authors: Xing Mengtao , Shi Jianxiang , Li Pei , Qin Jiejie , Zhang Xiaojun , Ma Yangcheng , Wang Xiao , Francia Giulio , Zhang Jianying
Published: August 2019

There are three members in the family of insulin-like growth factor 2 mRNA binding proteins (IMP): IMP1, IMP2 and IMP3. The IMPs are oncofetal proteins, expressed during embryogenesis and lost in most tissues in adults. However, IMPs were found overexpressed in various cancers. IMPs share similar protein structure in mammals and have similar functions: to bind specific mRNAs, regulate their expression, transportation, and degradation. Although p62/IMP2 was first reported as a tumor-associated antigen in HCC around 20 years ago, we still know little about the role of p62/IMP2 in HCC progression. Our previous studies found that p62/IMP2 was not only overexpressed in HCC tissues, but also overexpressed in HCC cell lines. To explore the biological roles of p62/IMP2 in HCC progression, p62/IMP2 was knockout in two p62/IMP2 positive HCC cell lines (SNU449, HepG2). Due to the low expression level of p62/IMP2 in SNU449, we performed the transfection experiment to overexpress p62/IMP2 in this cell line. The Wound healing assay and transwell migration assay indicated that overexpressed p62/IMP2 in both cell lines showed the ability that could promote the cell migration significantly (p<0.05). On the contrary, the lack of p62/IMP2 expression can reduce the cell migration ability (P<0.05). After analyzing the HCC expression data from Gene Expression Omnibus (GEO), the high and low IMP2 expression groups were set up based on the median expression of p62/IMP2 from GSE 14520. Three genes were selected in differential gene expression analysis with a cancer-metastasis related gene profile, including CTNNB1, ACTR2, VASP (LogFC > 0.3, Adj. p<0.001). Then we performed a western blotting analysis to explore the effect of overexpressed p62/IMP2 on Wnt/β-catenin pathway-related proteins. Overexpressed p62/IMP2 significantly enhance the expression of Wnt and β-catenin, whereas inhibiting the expression of Gsk3b and E-cadherin. Moreover, the migration ability of SNU449 and HepG2 cells were significantly reduced (p<0.05) after cultured with 10mM Wnt/β-catenin signaling pathway inhibitor XAV939 for 24h. In summary, our data showed that overexpressed p62/IMP2 can enhance the migration ability of HCC cells via activating Wnt/β-catenin signaling pathway.

Citation Format: Mengtao Xing, Jianxiang Shi, Pei Li, Jiejie Qin, Xiaojun Zhang, Yangcheng Ma, Xiao Wang, Giulio Francia, Jianying Zhang. Overexpression of p62/IMP2 can promote cell migration in hepatocellular carcinoma (HCC) via activating Wnt/ β-catenin pathway [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 4591.
Abstract 4592: CD44v6 promotes aggressive phenotypes mediated through activating EGFR pathway via COL11A1/PTPRG axis
Authors: Chen Joanne Jeou-Yuan , Guo Jing-You
Published: August 2019

Cell surface receptor CD44, an important player in stem cells and cancer biology, is expressed in multiple isoforms via alternative splicing. CD44 isoforms containing variant exon 6 (CD44v6)-encoded sequences are frequently overexpressed in human tumors. In many cancers, CD44v6 serves as a marker of cancer stem cells and accounts for the metastatic susceptibility of the tumors. CD44v6 predicts for poor prognosis in several cancers. However, the actions and underlying mechanisms related to CD44v6-mediated malignant phenotypes remain to be established. In this study, we performed cDNA microarray and Gene Set Enrichment Analysis (GSEA) of the genome-wide expression profiles in cells ectopically expressing the standard form CD44 (CD44s) or CD44v6, and showed that pathways related to cell migration, invasion and metastasis are enriched in CD44v6-expressing cells which are in contrast to cell growth regulatory pathways identified in CD44s cells. In support, overexpression of CD44v6 promoted cell migration and invasion. In silico analyses of genes preferentially regulated in CD44v6-expressing cells against a multi-cancer metastasis-associated gene expression signature dataset and the gene set indicative of embryonic stem cell identity, Collagen XIA1 (COL11A1) was identified as the top candidate in CD44v6-upregulated genes. COL11A1, encoding the α1 chain of the minor fibrillar collagen XI, has been found to be highly expressed in a variety of cancers, and is implicated in the processes of metastasis, angiogenesis, and drug resistance. COL11A1 expression is correlated with cancer aggressiveness and progression, and lymph node metastasis. Using gain- and loss-of-function approaches, we further demonstrated that CD44v6 drives tumor cell invasion in part mediated through the induction of metastasis-related gene COL11A1. Upon examining the involvement of receptor tyrosine kinase pathways, we showed that EGFR is constitutively activated in CD44v6-expressing cells, and that COL11A1 supports CD44v6-mediated EGFR activation via suppressing the expression of PTPRG, an EGFR negative regulator. Our data thus suggest that CD44v6 promotes aggressive phenotypes mediated through activating EGFR pathway via COL11A1/PTPRG axis.

Citation Format: Joanne Jeou-Yuan Chen, Jing-You Guo. CD44v6 promotes aggressive phenotypes mediated through activating EGFR pathway via COL11A1/PTPRG axis [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 4592.
Abstract 4599: PD-1/PD-L1 axis in cancer cells contributes to cellular EMT in lung adenocarcinoma
Authors: Su Wen-Pin , Chang Li-Chan , Yan Jing-Jou , Su Wu-Chou
Published: August 2019

Background: Since intrinsic PD-1 receptor functions promote tumor growth was reported, we will investigate the role of PD-L1 in lung adenocarcinoma cells, and their impact on clinical outcome.

Materials and methods: Lung adenocarcinoma CL1-5 cells, derived from CL1-0 cells have more invasive ability. We prepared PDL1-overexpression human lung adenocarcinoma cell line, derived from CL1-0 cells (CL1-0-PD1) and from PC14PE6 cells (PC14PE6-PD-L1). We observed the cellular morphology, and invasiveness; epithelial-mesenchymal transition (EMT) markers and regulators were also evaluated. To explore interaction between PD-1 and PD-L1, we treated the CL1-0, CL1-5, CL1-0-PDL1 cells, PC14PE6 cells, and PC14PE6-PD-L1 cells with anti-PD-1 antibody, and then evaluated cellular invasion. We also suppressed PD-1 to test whether PD-1/PDL-1 interaction contributed to the EMT change. Further, we evaluated cellular proliferation and chemosensitivity by MTT assay and colony formation assay. Finally, we correlated PD-L1 expression with clinical outcome in patients’ tumor specimen.

Results: CL1-5 cells possessed higher PD-L1 expression than parental CL1-0 cells. CL1-0 cells with PD-L1 overexpression had more expression of EMT regulators and mesenchymal markers. Overexpression of PD-L1 in another lung adenocarcinoma PC14PE cells (PC14PE6-PD-L1 cells) become more aggressive. On the contrary, down-regulation of PD-L1 in CL1-5 cells and PC14-PE6-PD-L1 cells became indolent. Besides through the observation from confocal microscopy, PD-L1 expression was more co-localized with vimentin and slug, individually in CL1-5, CL1-0-PD-L1 and PC14PE6-PD-L1 cells. Therefore, PD-L1 promoted EMT in human lung adenocarcinoma cells. After adding anti-PD-1 antibody in CL1-5, CL1-0, and CL1-0-PDL1 cells, migration and invasion ability were decreased. Similar phenomenon was found when PD-1 antibody treatment in PC14PE6 cells, and PC14PE6-PD-L1 cells. Silencing PD-1 by siRNA also disrupt the cellular aggressivenss in CL1-0-PD-L1 and PC14PE6-PD-L1 cells. These results indicated PD-1/PD-L1 axis regulated cancer cells migration and invasiveness. PD-L1 expression also decreased cellular proliferation and had little influence on chemsensitivity. Finally, we found that higher PD-L1 expression was correlated with lymph node metastasis in clinical specimen.

Conclusion: Lung adenocarcinoma cells with higher PD-L1 expression promote EMT via PD-1 receptor. PD-L1 expression lowers proliferation rate, but has little impact on chemosensitivity. Lung cancer patients with high PD-L1 expression in tumor have higher lymph node metastasis.

Citation Format: Wen-Pin Su, Li-Chan Chang, Jing-Jou Yan, Wu-Chou Su. PD-1/PD-L1 axis in cancer cells contributes to cellular EMT in lung adenocarcinoma [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 4599.
Abstract 4603: To analyse PIMT function in glioblastoma cell invasion
Authors: Belkourchia Fatima , Desrosiers Richard
Published: August 2019

The protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT) is an enzyme able to repair abnormal L-aspartyl residues in damaged proteins. Among examined tissues, PIMT shows the highest level in brain. U87-MG cells are a commonly used grade IV cellular model to study the most frequent brain tumor, the glioblastoma. Previously, we reported that PIMT amount increased when U87-MG cells were detached from the extracellular matrix. Recently, our laboratory also showed that PIMT possessed pro-angiogenic properties. Together, these PIMT features led us to postulate that PIMT could play a critical role in glioblastoma growth. To investigate PIMT involvement during glioblastoma growth, we analyzed its role in U87-MG cell viability, adhesion, migration, invasion, clonogenic properties and the reorganization of the actin cytoskeleton. For instance, PIMT inhibition by siRNA reduced cell migration and invasion by different assays: in wound healing assay, in Boyden chambers coated with gelatin and in Matrigel invasion assay. Conversely, in stably transfected U87-MG cells overexpressing wild-type PIMT, cell migration, invasive capacity and colony formation significantly increased. However, in stably transfected cells with the gene encoding for the inactive PIMT D83V, in spite of its overexpression, the migration and invasion remained similar to that observed in control cells transfected with the empty plasmid. In all these conditions, cell viability was unaffected. Finally, the reorganization of the actin cytoskeleton is regulated by PIMT protein level. Overall, these data enlighten the importance of PIMT level and mainly of its catalytic activity in migration and invasion of malignant glioma U87-MG cells and its possible contribution in cancer cell invasion during glioblastoma growth.

Citation Format: Fatima Belkourchia, Richard Desrosiers. To analyse PIMT function in glioblastoma cell 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 4603.
Abstract 4604: Kidney injury molecule-1 regulates metastasis in renal cell carcinoma
Authors: Lee Jasper C. , Sarabusky Marie A. , Champagne Audrey , Lucien Fabrice , Gunaratnam Lakshman
Published: August 2019

Over 30% of patients with renal cell carcinoma (RCC) present with metastases, with median survival of 2 years. Kidney injury molecule 1 (KIM-1) is a cell-surface glycoprotein expressed by >85% of RCC tumours and may serve as an early biomarker for RCC detection. Here we sought to determine if tumour KIM-1 plays a role in RCC cell extravasation and metastasis to lungs. Methods: We overexpressed KIM-1 in Renca cells (murine RCC) using stable transfection of an expression plasmid, or silenced endogenous KIM-1 in human 769-P and 786-O RCC cells using shRNA. We studied extravasation using the chorioallantoic membrane (CAM) model. We injected ~1x105 Renca or 769-P cells into blood vessels of chicken embryos (n=6), and measured percentage of cells that exited the capillary bed into surrounding spaces after 24 hours. To study lung metastasis, we injected ~5x105 Renca or 786-O cells intravenously through the tail vein of syngeneic BALB/c (n=10/group) or immune deficient Rag1-/- [BALB/c] (n=5/group) mice, and manually counted metastatic nodules in excised lungs after 17 days. Finally, we analyzed the publicly available Cancer Genome Atlas Illumina RNA-Seq dataset to determine if KIM-1 mRNA expression predicts overall survival in patients with clear cell RCC. Results: In CAM experiments, extravasation efficiency was significantly decreased in KIM-1pos 769-P cells compared to KIM-1neg 769-P cells (30.08% vs. 47.94%; p=0.0004), and in KIM-1pos Renca cells compared to KIM-1neg Renca cells (47.78% vs 60.73%; p=0.042). BALB/c mice injected with Kim-1pos Renca cells or Kim-1pos 786-O cells had significantly fewer lung metastases compared to mice injected with Kim-1neg Renca cells or Kim-1neg 786-O cells, respectively (257 vs 429, p=0.02; 131 vs 339 p=0.001). The inhibitory effect of KIM-1 expression in Renca and 786-O cells on numbers of lung metastases was similar in Rag1-/- mice, suggesting that the observed effects were not dependent on adaptive immunity. In the TCGA database, patients with the lowest vs. highest tertile of KIM-1 RCC expression has significantly higher mortality (unadjusted HR 1.82, 95% CI 1.23-2.70; p=0.0034). Conclusion: KIM-1 is a negative regulator of cell extravasation and inhibits the development of lung metastases in mice, and KIM-1 expression in human RCC may predict better survival. Deciphering the underlying mechanisms may lead to novel therapies.

Citation Format: Jasper C. Lee, Marie A. Sarabusky, Audrey Champagne, Fabrice Lucien, Lakshman Gunaratnam. Kidney injury molecule-1 regulates metastasis 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 4604.
Abstract 4613: Mathematical modeling of tumor growth in mouse models
Authors: Zhang Chao , Jiang Xiaoqian , Guo Sheng , Li Qixiang
Published: August 2019

A variety of tumor models are used in preclinical oncology drug discovery, including patient-derived xenografts (PDXs), cell line-derived xenografts (CDXs), and syngeneic homografts. Tumors in mouse models exhibit heterogeneous growth patterns with or without drug treatment, which is further complicated by measurement errors. Mathematical modeling of tumor growth helps us better understand such patterns and analyze drug efficacy in transplanted tumor models. Many mathematical models have been proposed to describe tumor growth curves, each with certain assumptions and equations, suitable for specific situations or data sets1-5. It is yet unknown which models are best in fitting the growth curves for a large collection of mouse models. In this study, we systematically evaluated a set of mathematical models including exponential models, logistic and Gompertz model, biphasic models, dynamic carrying capacity model, and power law model on more than 50,000 tumor growth datasets collected from in vivo efficacy studies in PDXs, CDXs and syngeneic models. A set of metrics were used to evaluate the goodness of fit, including Akaike Information Criterion (AIC), root mean square error (RMSE), coefficient of determination, mean absolute relative error, etc. We also proposed a new composite metric to combine many criteria for easy selection of growth models. Our results show that tumor growth patterns are less complex under vehicle treatment, and different mathematical models have relatively small difference in modeling the growth curves. Tumor growth patterns are more diverse under drug treatment. Tumors may shrink or completely remit, though some may grow in the first days after inoculation before drug effect takes place. Still some tumors stop growing or even shrink before drug resistance develops and resume fast growing. Therefore, different mathematical models are needed to describe the different patterns. Finally, we present summary statistics to give an overall picture on the applicability of the mathematical models, and suggest statistical models for subsequent analysis for drug efficacy evaluation and biomarker discovery.

Citation Format: Chao Zhang, Xiaoqian Jiang, Sheng Guo, Qixiang Li. Mathematical modeling of tumor growth in mouse models [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 4613.
Abstract 462: Results from multiple datasets including the TCGA reveal limited clinical significance of molecular subtypes in bladder cancer
Authors: Lahorewala Sarrah S. , Morera Daley S. , Wang Jiaojiao , Lokeshwar Vinata B.
Published: August 2019

INTRODUCTION AND OBJECTIVE: Molecular subtypes have been suggested to predict outcome in patients with muscle invasive bladder cancer (MIBC). The major subtypes reported have been “luminal” and “basal”; however, there is little consensus regarding subtype-signatures and the clinical parameters used to evaluate outcome. The objective of this study was to examine using multiple datasets, if molecular subtypes are distinct entities within MIBC and whether they predict clinical outcome.

METHODS: We analyzed transcriptome data from all 402 MIBC samples in The Cancer Genome Atlas (TCGA) and 151 high-grade MIBC samples from three datasets in Oncomine™. Transcript levels were also measured in 52 bladder tumor specimens with clinical follow-up (cohort-1). Samples were subtype-scored using gene panels (GP-11, GP-30, BASQ) consisting only of those genes common among published studies. Correlation of subtypes to recurrence/progression-free survival (R/PFS), metastasis, cancer-specific survival (CSS) and overall-survival (OS) was examined by logistic regression, Cox proportional Hazards model and Kaplan-Meier analyses.

RESULTS: Based on GP-11 subtype-scoring, >75% of the MIBC TCGA-dataset samples were mixed, i.e. neither pure-luminal nor pure-basal. When tumors were categorized as pure-luminal, pure-basal, or mixed, the subtypes could not predict OS or R/PFS. Consistent with published studies, when tumors were categorized as luminal or basal the subtypes predicted OS (P=0.051); sensitivity: 53.9%; specificity: 62%. Subtypes also correlated with tumor-grade (P=0.0005); most low-grade MIBC cases (16/21) in the TCGA-dataset were luminal. However, MIBC is rarely low-grade and subtypes could not predict OS (P=0.131) when only high-grade cases were included. Subtypes were not significant prognosticators in multivariate analyses. GP-30 and BASQ panels validated these results. In the Oncomine-dataset and cohort-1, subtypes could not predict metastasis, CSS, or OS.

CONCULUSIONS: Our study of multiple datasets reveals that molecular subtypes may reflect tumor-heterogeneity but are likely not distinct entities within MIBC. Furthermore, molecular subtypes have limited prognostic capability for MIBC patients. Although there is a need to individualize patient care, further examination into the molecular subtypes of MIBC is needed before their incorporation into clinical practice.

Citation Format: Sarrah S. Lahorewala, Daley S. Morera, Jiaojiao Wang, Vinata B. Lokeshwar. Results from multiple datasets including the TCGA reveal limited clinical significance of molecular subtypes in bladder 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 462.
Abstract 4623: Notch3 in lung adenocarcinoma pathogenesis and heterogeneity
Authors: Marini Kieren D. , Leung Stanley , Lee Alex , Sweet-Cordero Alejandro
Published: August 2019

Lung cancer is the leading cause of cancer related death worldwide. Non-small lung cancer accounts for 85% of diagnoses and the predominant subtype is lung adenocarcinoma (LUAD). LUAD has a less than 20% response rate to conventional chemotherapy and despite identification of several therapeutic targets, the majority of diagnoses continue to lead to death, highlighting the need for better treatments. Notch signaling in mammals is complex, with multiple receptors, ligands and modifiers. Notch ligands at the surface of one cell interact with receptors on an adjacent cell, triggering cleavage and nuclear translocation of the intracellular domain which interacts with Rbpj/Csl to control the transcription of target genes. In LUAD, significant evidence suggests that Notch plays an oncogenic role.

Previously, we identified a population of Tumor Propagating Cells (TPC), which had greater capacity for tumor formation and are enriched after treatment with cisplatin. The TPC population was defined by the expression of CD24, ITGB4 and was Notch high. Further functional validation showed that specifically Notch3 contributes to this propagation phenotype and is key in establishing functional intra-tumoral heterogeneity. However, the TPC population is still heterogeneous and likely only a subset of these cells are true TPCs. Furthermore, the gene expression signature of the mouse TPCs has prognostic significance in human LUAD, however the TPC population hasn’t been defined in human patients, limiting our ability to target the Notch pathway therapeutically.

To assess the heterogeneity of the mouse TPC population we performed FACS on tumors from KrasLSLG12D; Tp53flox/flox; YFPLSL twelve weeks after tumor initiation via AdCre. Tumors were sorted for bulk population (YFP+Lin-) and for TPC (YFP+Lin-CD24+ITGB4+Notchhi) population and single cell sequencing was performed using the 10X genomics platform. We hypothesize that aberrant Notch3 signaling is a critical pathway for survival in at least some LUAD and that Notch activation leads to gene expression changes that are important for driving human LUAD maintenance. Patient-derived xenografts (PDX) more closely recapitulate important aspects of human cancer, particularly with regards to the role of intratumoral heterogeneity and self-renewal pathways. To characterize Notch3 expressing cells in human LUAD, we performed FACS on our cohort of LUAD PDXs and sorted the Notch3+ population. We found expression of Notch3 and expression of Notch ligand Jagged2 in all LUAD tumors regardless of driver mutation.

Citation Format: Kieren D. Marini, Stanley Leung, Alex Lee, Alejandro Sweet-Cordero. Notch3 in lung adenocarcinoma pathogenesis and heterogeneity [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 4623.
Abstract 4626: Use of integrated genomic analyses in patient-derived tumor model to discover new clinical indications for the multikinase inhibitor drug candidate, DBPR216
Authors: Kuo Ching-Chuan , Jiaang Weir-Torn , Ou Jing-Jim , Chen Chiung-Tong , Hsu Shu-Ching , Shih Chuan , Lin Li-Mei , Sun Manwu , Wang Yi-Hsin , Huang Zih-Ting , Chang Jang-Yang , Ueng Shau-Hua
Published: August 2019

Developing realistic preclinical models using clinical samples that reflect complex tumor biology is critical to advancing cancer research. Patient-derived preclinical tumor models are the optimal tool for understanding drug action patterns and resistance mechanisms. In order to improve the capability of drug R&D in our institute (NHRI-IBPR), we have generated several patient-derived xenograft (PDX) models and characterized the genomic signature and responsiveness to standard-of-care (SOC) therapy. DBPR216, an orally bioavailable multikinase inhibitor, showed potent effect for treatment of gastrointestinal stromal tumors (GISTs) and acute myeloid leukemia (AML) through targeting of c-KIT and FLT-3, respectively. In order to further discover other indications of DBPR216 for clinical application, we investigated the anti-tumor effect of DBPR216 in several in-house PDX models. Among them, we found that DBPR216 was effectively to suppress PDX tumor growth in the immuno-deficient mice in two colorectal adenocarcinoma PDX models, C008 and C015. These PDX models showed similar genomic features with original tumor samples from patients when test using targeted sequencing of cancer related genes. To further identify if DBPR216 is superior to other kinase inhibitors and SOC therapy, we used in vitro PDX cell proliferation assay to quickly examine the anti-tumor effect of DBPR216 compared to a panel of therapeutic drugs. The result demonstrated that DBPR216 appeared to be superior in potency to kinase inhibitors (Regorafenib, Afatinib, Sunitinib, and Imatinib) and SOC therapy (Irinotecan, 5-FU, and Oxaliplatin). Combining the kinase profiling of DBPR216 and mutational analysis of C008 and C015 PDX models, we proposed that DDR2, FLT1, PDGFRα, PDGFRβ, RET, and SRC may be the potential targets of DBPR216 in these PDX models, and need further elucidation. Taken together, we found that DBPR216 exhibits potent anticancer effect against colorectal cancer and may bring the better opportunity than Regorafenib, a therapeutic agent for metastatic colorectal cancer in clinical. DBPR216 is now under preclinical development for further IND submission.

Citation Format: Ching-Chuan Kuo, Weir-Torn Jiaang, Jing-Jim Ou, Chiung-Tong Chen, Shu-Ching Hsu, Chuan Shih, Li-Mei Lin, Manwu Sun, Yi-Hsin Wang, Zih-Ting Huang, Jang-Yang Chang, Shau-Hua Ueng. Use of integrated genomic analyses in patient-derived tumor model to discover new clinical indications for the multikinase inhibitor drug candidate, DBPR216 [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 4626.
Abstract 4627: Mutant p53 regulation in a Li-Fraumeni mouse model
Authors: Terzian Tamara , Plehaty Molly , Sobhani Nema , Stevens Wanida , Marasligiller Stefan , Srinivas Tara , Mylavarapu Rohan , Clarke Yvonne , Arbab Farinaz , Podell Brendon , Bronson Roderick
Published: August 2019

The p53 tumor suppressor is maintained mainly at low physiological levels in normal cells by its two major inhibitors, Mdm2 and Mdm4. Mdm2 is a E3 ubiquitin ligase while Mdm4 binds to p53 and inhibits its transactivation activity. Deletion of either inhibitor results in p53-dependent embryonic lethality in mice. p53 is one of the most commonly mutated genes in human cancer. While we have gained considerable knowledge about wild-type p53 regulation and mode of action, our understanding of the oncogenic activity of mutant p53 lags behind. We know that mutant p53 is regulated in the same manner as wild-type p53. In a mouse model of Li-Fraumeni syndrome, Mdm2 binds and inhibits the hot spot mutant p53R172H (p53R175H in humans). Here we show that Mdm4, like Mdm2, regulates p53R172H stability in vivo, and that endogenous Mdm2 cannot compensate for this stabilization. Thus, mutant p53 mice that lack Mdm4 have stable high levels of p53R172H and display accelerated tumor development and an increased range of tumor types, compared to mutant p53 mice. Furthermore, when we compared mutant mice with the loss of two different alleles of both negative regulators, Mdm2 and Mdm4, we observed an even higher level of p53. This finding reveals that several tissues can tolerate high expression of oncogenic p53 and indicates a collaborative activity of these inhibitors in regulating mutant p53. The analysis of these mice uncovered new and unexpected functions of mutant p53 that we will discuss for the first time at the AACR 2019 meeting.

Citation Format: Tamara Terzian, Molly Plehaty, Nema Sobhani, Wanida Stevens, Stefan Marasligiller, Tara Srinivas, Rohan Mylavarapu, Yvonne Clarke, Farinaz Arbab, Brendon Podell, Roderick Bronson. Mutant p53 regulation in a Li-Fraumeni mouse model [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 4627.
Abstract 4632: A new mouse model for rapid identification of key factors driving prostate cancer progression and invasiveness
Authors: Riedel Maria , Bakiri Latifa , Berthelsen Martin F. , Borre Michael , Vendelbo Mikkel H. , Wagner Erwin F. , Thomsen Martin K.
Published: August 2019

Prostate Cancer is amongst the most frequently diagnosed malignancies and the second leading cause of cancer-related death in men worldwide accompanied by an increasing incidence. Until now its heterogeneity has been a major challenge in the establishment of good in vivo models for fast validation of potential driver genes.

Classic in vivo models are costly, time-consuming and often target the majority of the prostate epithelium. We successfully introduced a novel prostate cancer mouse model based on CRISPR/Cas9 technology, ensuring multiplexed gene editing.

In this model, specific in vivo gene editing was obtained in murine prostate epithelium cells of a transgenic mouse strain, harboring the CRISPR associated protein 9 (Cas9) endonuclease. Prostate epithelium cells were transduced by an Adeno-associated virus (AAV), carrying multiple single guide RNAs (sgRNA).

Genetically different viral constructs were designed, each expressing different sgRNA combinations against Pten, representing the main driver in prostate cancer, tumor suppressor Trp53 and either one of the AP1 transcription factor subunits Junb or Fos, or tumor suppressor Smad4.

Since viral transduction occurs in only a few cells, edited cell clones can clonally expand and undergo a natural selection process. Furthermore, the simultaneous gene knockouts reflect the human scenario of tumor heterogeneity.

Mouse prostates were isolated and analyzed three to nine months post-injection to obtain insight on whether or not a gene appears crucial for tumor development in a specific tissue and wherein different gene combinations correlate with tumor severity. Histological analysis revealed increased proliferation, increased AKT activation, as well as invasiveness in samples with multiple gene knockouts compared to controls with single Pten knockout. Furthermore, loss of SMAD4 accelerated cancer progression when compared to the loss of the AP-1 transcription factor. Surprisingly, knockout of Trp53 was rarely observed in prostate samples while it occurred frequently in other tumor types in the same model system. This indicates that knockout of Trp53 may not be required for prostate cancer initiation.

Overall, we established an in vivo model system of simultaneous, multiplexed gene editing in the prostate epithelium to address the biological cross-talk between various, altered pathways in prostate cancer initiation and progression.

Citation Format: Maria Riedel, Latifa Bakiri, Martin F. Berthelsen, Michael Borre, Mikkel H. Vendelbo, Erwin F. Wagner, Martin K. Thomsen. A new mouse model for rapid identification of key factors driving prostate cancer progression and invasiveness [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 4632.
Abstract 4641: Metabolic reprogramming of the breast contributes to a cancer promoting milieu
Authors: Marino Natascia , German Rana , Rao Xi , Sandusky George , Jacobsen Max , Cao Sha , Storniolo Anna Maria
Published: August 2019

In addition to the accumulation of pro-oncogenic mutations in the epithelial cells, the tumorigenic process involves the dysregulation of the interactions between the epithelial cells and their microenvironment, as well as alterations within the microenvironment itself. The latter is composed of endothelial cells, immune cells, fibroblasts, unaffected epithelium, and adipocytes. Increasing evidences support that cell transformation progresses in a potentially cancer-promoting microenvironment context, also known as field cancerization model. Therefore, it is critical to investigate changes occurring in both the epithelial compartment and the surrounding microenvironment to understand how breast cancer initiates.

In our preliminary study, we analyzed the transcriptome profile of microdissected breast tissue compartments (epithelium, stroma and adipose tissue) from tissue biopsies obtained from women who donated their histologically normal tissue two-to-five years before breast cancer diagnosis (here labeled pre-cancer), and matched healthy control donors. In the pre-cancer breast epithelium we detected a significant increase in lipid metabolism-related genes including lipases (HSL, LPL) and perilipins (PLIN1, PLIN4), which mediate the release of fatty acids from triacylglycerol storage, and ELOVL5 and ELOVL7, which generate oleic acid. Moreover, Acyl-CoA Synthetase Medium Chain Family Member 1, ACSM1, involved in the activation of lipoic acid, an essential cofactor for mitochondrial metabolism, is also upregulated in pre-cancer breast tissue. Upregulation of genes involved in metabolic activation is observed also in the stroma and adipose tissue compartments. Interestingly, the transcriptome profiling of the pre-cancer breast stroma shows the downregulation of genes coding for several immune cell markers as compared with the healthy controls. Immunohistochemical staining of CD45 confirms a significant reduction in immune cells in the pre-cancer versus matched healthy control breasts. This finding suggests the prevalence of an immune-suppressive environment in the breast long before the clinical diagnosis of breast cancer.

Understanding the changes occurring in the cancerized field is critical for elucidating 1) the involvement of microenvironment in cancer initiation 2) the role of histologically normal yet genetically altered surgical margins in risk of disease recurrence upon lumpectomy for early breast cancer diagnosis, and 3) the mechanisms of field cancerization in the contralateral breast.

Citation Format: Natascia Marino, Rana German, Xi Rao, George Sandusky, Max Jacobsen, Sha Cao, Anna Maria Storniolo. Metabolic reprogramming of the breast contributes to a cancer promoting milieu [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 4641.
Abstract 4649: Duplex Sequencing detects rare subclonal variants that mark early carcinogenesis and preneoplastic clonal evolution
Authors: Valentine Charles C. , Fielden Mark , Young Robert , Higgins Jake , Williams Lindsey , Li Tan , Kulkarni Rohan , Minocherhomji Sheroy , Risques Rosana , Danaher Patrick , Salk Jesse
Published: August 2019

Cancer is a disease of somatic evolution, characterized by the natural selection of genomic mutations that facilitate enhanced cell survival and proliferation. Although many thousands of tumor genomes have now been sequenced, our ability to identify early genetic patterns of clonal selection in both humans and model organisms have been hampered by inadequately sensitive methodologies for identifying mutations during the long period between their occurrence and the final outgrowth of a clinically apparent tumor.

Sensitive molecular tests are capable of measuring minute levels of cancer cells in tissue samples, however no existing method is satisfactory at scaling to high-throughput detection at the rate of one cancer-associated variant per more than a million normal cells. NGS is used to study the genetic variation in cell populations, although the accuracy of standard NGS methods limit our ability to detect sub-clones below ~1%. Duplex Sequencing (DS) is a sensitive NGS error-correction method which increases the accuracy of base calls by more than 100,000-fold. DS enables precise reconstruction of the original double-stranded source molecule while overcoming both chemical and technical artifacts that arise during library preparation and sequencing.

Here we present the use of Duplex Sequencing, in both human and mouse tissues, for measuring sub-clones at allelic fractions less than 1×10-4 for an early glimpse into pre-neoplastic evolution. We illustrate how, merely weeks after mutagen exposure, we observe emerging clones of cells bearing cancer-driving mutations in histologically normal mouse tissue. Furthermore, we illustrate how similar patterns of clonal selection can be seen in multiple otherwise healthy tissues of humans as part of normal aging. We discuss how variations in the pattern and rate of accumulation of rare cancer-associated mutations offers a new preclinical tool for evaluating carcinogenicity of chemicals, as well as a potential clinical tool for assessing life-integrated carcinogenic processes and cancer risk in humans.

Citation Format: Charles C. Valentine, Mark Fielden, Robert Young, Jake Higgins, Lindsey Williams, Tan Li, Rohan Kulkarni, Sheroy Minocherhomji, Rosana Risques, Patrick Danaher, Jesse Salk. Duplex Sequencing detects rare subclonal variants that mark early carcinogenesis and preneoplastic clonal evolution [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 4649.