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Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573–86. ©2016 AACR.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by poor clinical outcomes and limited treatment options. Transcriptional analysis has divided TNBC into different molecular subtypes (BL1, BL2, M and LAR) that were characterized with different signaling pathways and differed in response to similar neoadjuvant chemotherapies [1]. In a sporadic TNBC phase II study, prexasertib monotherapy showed modest single agent activity, which suggested that combination therapy is needed to maximize the benefit from prexasertib (LY2606368), a selective CHK1 inhibitor [2]. Here we leveraged a large cohort of TNBC patient-derived xenograft (PDX) models developed with nude mice to find biomarker/pathways/combination strategies related with prexasertib. Parametric Gene Set Enrichment Analysis (PGSEA) has been shown as a powerful sample-level analysis to generate enrichment scores for pathways [3]. First we confirmed that our collection of PDX models captured the heterogeneities of TNBC disease, with similar compositions of molecular subtypes as the TCGA dataset. The different molecular subtypes of PDX models also showed similar pathway changes as TNBC disease, such as increase of cell-cycle pathway in BL1-subtype, Glycolysis in BL2-subtype and ECM pathway in M-subtype. Molecular subtype analysis of a single-mouse (n=1) PDX trial evaluating antitumor activity of prexasertib showed that majority of M-subtype showed resistance to prexasertib and the other subtypes showed differential sensitivity. Within the BL1-subtype, the resistant models also showed high expression of stem-cell like features. Further PGSEA analysis based on KEGG signaling pathways revealed that the PI3K/mTOR pathway was transcriptionally elevated in resistant models, which was consistent with the additive effect of the combination therapy of prexasertib and PI3K/mTOR inhibitor (LY3023414) in this PDX trial. These data provide rationale for the development of combination therapy of prexasertib and PI3K/mTOR inhibitor for TNBC. 1. Lehmann, B.D., et al., Refinement of Triple-Negative Breast Cancer Molecular Subtypes: Implications for Neoadjuvant Chemotherapy Selection. PLoS One, 2016. 11(6): p. e0157368. 2. Karzai, F., et al., A phase II study of the cell cycle checkpoint kinases 1 and 2 (CHK1/2) inhibitor (LY2606368; prexasertib) in sporadic triple negative breast cancer (TNBC). Annals of Oncology, 2016. 27. 3. Kim, S.Y. and D.J. Volsky, PAGE: parametric analysis of gene set enrichment. BMC Bioinformatics, 2005. 6: p. 144. Citation Format: Yan Ding, Jiangang Liu, John N. Calley, Hui-Rong Qian, Philip W. Iversen, Philip J. Ebert, Richard P. Beckmann, Gregory P. Donoho, Ricardo Martinez, Wenjuan Wu, Aimee Bence Lin, Emma Bowden, Amit Aggarwal. PI3K/AKT signaling pathway is transcriptionally elevated in prexasertib-resistant TNBC PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2586.

Anti-EGFR antibodies, such as cetuximab, are effective therapies for many late-stage colorectal cancer (CRC) patients; unfortunately, many tumors are initially unresponsive while others show initial efficacy but eventually develop acquired resistance. Genomic studies of patient tumors, cell lines, and xenograft models have identified putative anti-EGFR resistance markers, including mutations in KRAS, NRAS, BRAF, PIK3CA, and the EGFR extracellular domain, as well as amplifications in ERBB2 and MET. In order to further confirm and identify new resistance mechanisms to anti-EGFR treatment in CRC, we performed retrospective genomic profiling of 25 CRC patients treated at Samsung Medical Center from 2006-2015. Patients received cetuximab containing chemo regimens with varying duration of responses, including acquired resistance cases. Our analysis identifies mutations in receptor tyrosine kinases, such as EGFR, NTRK1, and PDGFRA, as well as RAS/MAPK pathway genes that affect cetuximab response. We also uncover genomic alterations in ERBB2 and c-KIT as potential novel mechanisms regulating sensitivity to anti-EGFR antibodies. Additional genomic analyses of acquired resistance tumors and in vitro studies of a patient-derived cell line provide added insights into clonal selection and signaling pathways that bypass the EGFR blockade. Overall, our study elucidates important new facets in the landscape of anti-EGFR resistance mechanisms. Note: This abstract was not presented at the meeting. Citation Format: Steven M. Bray, Jeeyun Lee, Seung Tae Kim, Philip J. Ebert, John N. Calley, Isabella H. Wulur, Thejaswini Gopalappa, Swee Seong Wong, Hui-Rong Qian, Jason C. Ting, Jiangang Liu, Melinda D. Willard, Amit Aggarwal, Ruslan D. Novosiadly, Hee-Cheol Kim, Christoph Reinhard. Intrinsic and acquired resistance to cetuximab in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4104. doi:10.1158/1538-7445.AM2017-4104

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. In United States alone, it was estimated that nearly 137,000 people were diagnosed, and more than 50,000 were dead from the disease each year. CRC primary tumors often metastasize to liver which accounts for most of CRC death. The molecular mechanism of tumor metastasis is complicate and remains poorly understood. In this study, we have collected 79 human CRC samples, and 60 of them are paired primary tumor and liver metastatic surgically resected samples. Molecular characterization of these samples was conducted by exome and RNA sequencing, as well as SNP6.0 analysis. Genetic analysis revealed no significant difference in genetic alterations including common oncogenic mutations, whole genome mutations and copy number variations between primary and their matched liver metastatic tumors. To further characterize the molecular mechanism of metastatic progression, we have assembled gene correlation networks by utilizing a genome-wide interrogation of co-regulated networks based on RNA sequencing data of these CRC samples. Computational analysis of these correlation networks has identified gene signatures of immune-suppression, epithelial-mesenchymal transition (EMT) and angiogenesis as the key events and potentially synergistic drivers associated with CRC metastasis. Further independent cohort validation using published datasets verified that these specific gene networks of tumor microenvironment were progressively up-regulated throughout the carcinogenesis, and represented distinct biological processes. These gene networks were capable of discriminating the previously categorized CRC molecular subclasses. In addition, we also showed an association of type I interferon network with good clinical outcome of CRC, and gene networks of EMT, angiogenesis, immune-suppression and T cell exhaustion are closely associated with the poor patient outcome. We further demonstrated that the networks of EMT and angiogenesis were related to innate anti-PD-1 resistance, and the networks of immunosuppression and T cell exhaustion were associated with resistance to radiation and checkpoint blockade. Overall, we conclude that a genome-wide interrogation of co-regulated networks utilized in this study represents a valuable strategy to identify molecular mechanisms of cancer metastasis, and gene networks of immune-suppression, EMT and angiogenesis are among the key events associated with CRC metastasis. Citation Format: Jiangang Liu, Yong Beom Cho, Hye kyung Hong, Song Wu, Philip J. Ebert, Steven M. Bray, Swee Seong Wong, Jason C. Ting, John N. Calley, Catherine F. Whittington, Shripad Bhagwat, Emma Bowden, Amit Aggarwal, Christoph Reinhard, Robert Wild, Do-Hyun Nam, Woo Yong Lee, Sheng-Bin Peng. Molecular characterization of primary tumor & the paired liver metastatic biopsies of colorectal cancer reveals a critical role of immunosuppression, EMT & angiogenesis in cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 849. doi:10.1158/1538-7445.AM2017-849

Therapeutic targeting of the VEGF pathway has not matched the efficacy suggested by preclinical tumor models. To date, the limitations of this form of therapy stem either from inherent or acquired resistance of the tumor vasculature to the reduction of VEGF signaling. Previous studies have described morphological phenotypes of different blood vessel types in human and mouse tumors. Recapitulation of these vascular phenotypes with a surrogate Ad-VEGFA164 model has shown differential sensitivity of these vascular subclasses to inhibitors of the VEGF pathway. The goal of the current study was to identify molecular markers for the various subtypes of pathological vessels and to assess whether similar molecular signatures are found in blood vessels in mouse tumor models and human disease. Using the Ad-VEGF-A164 flank model, microarray analysis was performed to identify molecular markers at each time-point, representing distinct stages of vessel development and maturation. We identified unique gene signatures at the various time-points and confirmed differential expression of the genes by qRT-PCR and/or immunohistochemistry. We found that vascular markers such as CD31, Ang2, and Tie1 were pan-endothelial markers at all time-points. However, other markers such as CD34, MECA-32, vWF, SMA, Tie2, CD105, and AQP1 were expressed on subclasses of endothelial cells. Expression of the molecular markers in tumors models revealed different subclasses of tumor vessels in patient-derived xenografts which were sensitive or resistant to anti-VEGF-A or anti-VEGFR-2 treatment. These vascular subtypes were also found in tissues from human breast and gastric cancers. Together these results indicate that multiple molecular markers can be used to identify unique pathological subclasses of tumor vessels. Some of these vascular subtypes may be insensitive to inhibitors targeting the VEGF pathways and their signatures may be useful biomarkers to predict vascular sensitivity to anti-angiogenic therapy. Citation Format: Beverly L. Falcon, Marguerita O'Mahony, Julie Stewart, Jiangang Liu, Janice A. Nagy, Qi Xue, Yong Pan, Cynthia Jeffries, Emma Hatten, Tim R. Holzer, Harold F. Dvorak, Aejaz Nasir, Bronek Pytowski, Laura Benjamin, Mark T. Uhlik. Identification of molecular markers of pathological vascular subtypes with differential sensitivity to therapies targeting the VEGF pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4810. doi:10.1158/1538-7445.AM2014-4810

Vascular Endothelial Growth Factor A (VEGF-A) is the predominant growth factor expressed by tumor cells to drive angiogenesis and solid tumor growth. Antiangiogenesis therapies have been clinically demonstrated to be effective, however, there are still not effective means to preselect those patients most likely to derive optimal benefit. To identify blood vessel markers that may differentiate patients responsive to VEGF pathway blockade, we used a well-established in vivo model to generate pathological angiogenesis following the delivery of adenovirus engineered to express VEGF-A164 (Ad-VEGF) to a localized subcutaneous site within the flank of a nude mouse. This model provides a temporally controlled induction of angiogenesis and subsequent remodeling and maturation of the vasculature. This enabled us to study discrete subclasses of vessel which have been previously shown to have differential sensitivities to anti-angiogenic therapy. Here we report gene expression profiling on a time course of the Ad-VEGF flank model which was used to characterize molecular and genetic changes associated with these distinct vessel subtypes. Using a nearest centroid-based classification algorithm we identified unique endothelial cell specific gene signatures representing ‘early’, ‘middle' and ‘late' stages of vessel development and maturation. To evaluate the utility of these gene signatures in predicting sensitivity to anti-VEGF therapy, we profiled a publically available gene array dataset generated from a mouse xenograft model following anti-VEGF treatment with our vessel subtype gene signatures. A majority of the early and middle vessel genes were significantly reduced in bevacizumab-treated animals, demonstrating they respond to VEGF pathway inhibition. To further evaluate the potential value of these gene signatures as predictive biomarkers we profiled a dataset of 248 gastric tumors using hierarchical clustering. Our analysis shows the middle vessel signature was highly expressed in a mesenchymal molecular subtype of gastric cancer. Interestingly, the majority of the mesenchymal gastric tumors were ‘diffuse' subtype in Lauren's classification. Together, our data demonstrate the potential usefulness of our vessel subtype gene signatures in identifying tumor subtypes that may benefit from this anti-angiogenic therapy. Citation Format: Jiangang Liu, Beverly L. Falcon, Janice A. Nagy, Shou-Ching S. Jaminet, Dan Li, Thompson N. Doman, Sudhakar Chintharlapalli, Bronek Pytowski, Mark T. Uhlik, Harold Dvorak, Laura Benjamin, Anthony S. Fischl. Bioinformatics analysis of an Ad-VEGF flank angiogenesis model identifies vessel subtype gene signatures: Implications for anti-VEGF therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2359. doi:10.1158/1538-7445.AM2014-2359

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole genome sequencing (WGS) study of 88 matched HCC tumour/normal pairs, 81 of which are HBV positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find β-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumour suppressor (35.2%). The Wnt/β-catenin pathway, altered in 62.5% of cases, is likely to act as the major oncogenic driver in HCC. TP53 alterations appear to cause increased levels of genomic arrangement and chromosomal instability. We identified chromothripsis in 5 HCC genomes (5.7%) recurrently affecting chromosomal arms 1q and 8q. We also identified recurrent HBV integration events at the known and putative cancer-related genes such as TERT, MLL4 and CCNE1, which showed upregulated gene expression in tumour versus normal tissue. The frequently altered genes and pathways in HCC reflect classical cancer hallmarks. This study identified several prevalent and actionable mutations that provide a path towards therapeutic intervention of the disease. Citation Format: Mao Mao, Hancheng Zheng, Zhengyan Kan, Jiangchun Xu, Xiao Liu, Shuyu Li, Thomas Barber, Zhuolin Gong, Huan Gao, Ke Hao, Melinda Willard, Robert Hauptschein, Paul Rejto, Julio Fernandez, Guan Wang, Qinghui Zhang, Bo Wang, Ronghua Chen, Jian Wang, Nikki Lee, Wei Zhou, Zhao Lin, Zhiyu Peng, Kang Yi, Shengpei Chen, Lin Li, Xiaomei Fan, Jie Yang, Rui Ye, Jia Ju, Kai Wang, Heather Estrella, Shibing Deng, Ping Wei, Ming Qiu, Isabella Wulur, Jiangang Liu, Mariam Ehsani, Chunsheng Zhang, Andrey Loboda, Wing Kin Sung, Amit Aggarwal, Ronnie Poon, Sheung Tat Fan, Jun Wang, James Hardwick, Christoph Reinhard, Hongyue Dai, Yingrui Li, John Luk. Whole genome sequencing reveals genetic landscape of hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-229. doi:10.1158/1538-7445.AM2013-LB-229