bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 461: Dichotomous roles of Dicer1 in rhabdomyosarcoma and angiosarcoma
Authors: Hanna Jason A. , Garcia Matthew R. , Drummond Catherine J. , Go Jonathon C. , Finkelstein David , Rehg Jerold E. , Hatley Mark E.
Published: July 2017

Pediatric and adult angiosarcomas are rare and highly aggressive soft tissue sarcomas with an extremely poor prognosis. Due to the rarity of this disease especially in children, the molecular drivers and optimized treatment strategies for patients are lacking, highlighting the need for genetic and in vivo animal models. MicroRNAs are a class of small RNAs that regulate gene expression and are often dysregulated in cancers including sarcomas. DICER1 is required for microRNA biogenesis and germline DICER1 mutations result in a cancer predisposition syndrome associated with increased risk of benign and malignant tumors including rhabdomyosarcoma (RMS), a pediatric soft tissue sarcoma resembling developmentally arrested skeletal muscle. Here we show Dicer1 expression is required for tumorigenesis in a mouse model of RMS driven by activation of oncogenic Smoothened by Cre recombinase expressed from the adipose protein 2 (aP2) promoter (aP2-Cre). Unexpectedly, in studying the role of Dicer1 in RMS, we found that Dicer1 deletion with aP2-Cre leads to aggressive angiosarcoma. Angiosarcoma development was independent of the Smoothened oncogene and other genetically engineered oncogenes or tumor suppressor loss, providing the first in vivo mouse model of biallelic Dicer1 loss alone driving tumorigenesis. Angiosarcomas in aP2-Cre;Dicer1Flox/- mice histologically and genetically resemble human angiosarcoma and were enriched for microRNA-23 target genes including the oncogene Ccnd1 as well as Adam19, Plau, and Wsb1 that promote invasiveness and metastasis. The aP2-Cre;Dicer1Flox/- model provides a simple in vivo animal model to study angiosarcoma for novel therapeutics and the molecular mechanisms of cancer initiation, progression, and metastasis. In addition, our results demonstrate DICER1 and microRNAs play major and opposing roles in sarcomagenesis.

Citation Format: Jason A. Hanna, Matthew R. Garcia, Catherine J. Drummond, Jonathon C. Go, David Finkelstein, Jerold E. Rehg, Mark E. Hatley. Dichotomous roles of Dicer1 in rhabdomyosarcoma and angiosarcoma [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 461. doi:10.1158/1538-7445.AM2017-461
Abstract 4611: A 3D in vitro culture-based method to visualize and quantify effects of immuno-modulatory drugs
Authors: Daszkiewicz Lidia , Yan Kuan , Klop Maarten , Price Leo
Published: July 2017

BACKGROUNDUnleashing the full potential of cancer immunotherapy is hampered by a lack of effective in vitro testing methods. 2D cultured cells poorly reflect the in vivo situation and lack the tumour microenvironment whereas humanized animal models are slow, have low throughput and are expensive. 3D cultures, on the other hand, more faithfully reproduce the organization of a tissue, recapitulating the cell-cell connections and cellular heterogeneity found in the tumour and allow for a cost-efficient testing of drug candidates. To address the unmet need of immunotherapy cancer drug developers for robust assays that incorporate the human immune system, with functional read-outs in a high throughput (384well plate) format, we developed an in vitro platform based on 3D co-cultures of cancer tumoroids and immune cells. This assay is designed to test the capacity of small molecules, bi-specific antibodies, checkpoint modulators, vaccines and combinations of thereof to enhance anti-cancer immune responses. METHODSCancer cells of different sources were grown in a hydrogel to form tumoroids and then HLA-matched PBMCs pre-conditioned via different protocols to achieve activated or exhausted T cells, were added in the presence or absence of immune-modulatory drugs. T cell infiltration and subsequent tumoroid killing was quantified using 3D high-content imaging. After ‘optical sectioning’ 3D image stacks were reconstituted and morphometric analysis was performed with OMiner software. RESULTS AND CONCLUSIONS Addition of immune checkpoint inhibitors, pembrolizumab and ipilimumab, led to enhanced T cell infiltration and cytotoxicity of exhausted T cells. These results showed that automated 3D image and data analysis enables discrimination of immune-tumour cell interactions depending on activation status of T cells and permits the visualization of T cell re-gain of function in the presence of checkpoint modulators. A 3D environment allows the different cell types to engage in a more realistic setting than when cells are grow in a monolayer and enables the quantification of spatially resolved information, which is of an utmost importance when studying tumor-immune cell relationships. This new and innovative platform empowers immunotherapy drug developers with a powerful tool to evaluate drug performance. Furthermore, drug combinations can be studied to determine synergistic effects with other checkpoint inhibitors of standard of care compounds in an efficient manner.

Citation Format: Lidia Daszkiewicz, Kuan Yan, Maarten Klop, Leo Price. A 3D in vitro culture-based method to visualize and quantify effects of immuno-modulatory drugs [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 4611. doi:10.1158/1538-7445.AM2017-4611
Abstract 4620: Signaling cascade of metastasis-associated macrophages determines breast cancer distal metastasis
Authors: Qian Bin-Zhi
Published: July 2017

Macrophages are abundantly found in the tumor microenvironment and enhance malignancy. At distal metastatic sites, our previous studies identified a distinct population of metastasis-associated macrophages (MAMs) that promote tumor cell extravasation, seeding and persistent growth. These macrophages were derived from inflammatory monocytes recruited by CCL2/CCR2 chemokine signaling and directly promote tumour cell extravasation through VEGF production. Our recent studies identified two subsequent signaling pathways that mediate the retention and function of MAMs after recruitment that are critical for tumour cell survival and persistent growth. Specifically, activation of CCR2 signaling turns on CCL3 expression in MAMs. Genetic deletion of CCL3 or its receptor CCR1 in macrophages reduces lung metastasis, and MAM accumulation in tumor-challenged lung. Adoptive transfer of wild type monocytes restores lung metastasis in Ccl3 deficient mice. Mechanistically, Ccr1 deficiency prevents MAM retention in the lung by reducing direct MAM-tumour cell interactions. On the other hand, MAMs turn on cell surface expression of FMS-like tyrosine kinase 1 (FLT1) upon differentiation from monocytes. Using several genetic models of Flt1 deficiency, we show that macrophage specific FLT1 signaling is critical for breast tumor distal metastatic potential. FLT1 specific inhibitory antibody significantly inhibits the metastatic seeding and persistent growth but does not affect the recruitment or retention of MAMs. Furthermore, we identified that FLT1 signaling regulates a set of downstream inflammatory response genes including Colony Stimulating Factor 1 (CSF1) a central regulator of macrophage biology. Using a genetic gain-of-function approach we show that CSF1 mediated autocrine signaling in MAMs is downstream of FLT1 and can restore the tumor-promoting activity in MAMs even when FLT1 is inhibited. Together, our data indicates CCL3/CCR1 and FLT1 signaling pathways are critical in regulating MAMs and promoting breast cancer distal metastasis, and suggests the therapeutic potential of targeting these pathways in treating metastatic disease.

Note: This abstract was not presented at the meeting.

Citation Format: Bin-Zhi Qian. Signaling cascade of metastasis-associated macrophages determines breast cancer distal 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 4620. doi:10.1158/1538-7445.AM2017-4620
Abstract 4623: Transcriptional analysis of the functional differences between M1 and M2 macrophages to identify new targets for myeloid cell modulation in pancreatic tumors
Authors: Yang Kimberline R. , Armstrong Todd D. , Jaffee Elizabeth M.
Published: July 2017

Macrophages exhibit dynamic plasticity in their function in response to the accumulating genetic alterations that occur during tumorigenesis, resulting in the establishment of an immune tolerized tumor microenvironment. However, depending on their environmental cues, they can switch between two functional phenotypes: pro-inflammatory/anti-tumorigenic (M1) or anti-inflammatory/pro-tumorigenic (M2). The importance of these two macrophage subsets is well-recognized; however, identifying and distinguishing M1 from M2 macrophages in patient tumors still remains a challenge. Prior studies have utilized transcriptome profiling of artificially skewed M1 and M2 macrophages from healthy donors to identify individual genes or gene clusters unique to each macrophage subset. However, these studies are limited to surface marker expression and gene expression profiles alone to distinguish subsets and lack functional validation of M1 and M2 macrophages. Surface marker and gene expression profiling of human M1 and M2 macrophages provide an incomplete characterization, as functionality is an important feature distinguishing these macrophage subsets. To improve the characterization of M1 and M2 macrophages, we performed a thorough three-step validation of artificially skewed M1 and M2 macrophages from healthy donor monocytes using surface marker expression, gene expression profiling, and multi-analyte ELISA analyses. Using our validation assays, we tested previously published protocols for their efficacy in generating M1 and M2 macrophages. While M1 macrophages using these protocols generated as expected M1 characteristics, M2 macrophages lacked hallmark functional features, particularly IL-10 secretion. This necessitated the generation of an optimized protocol to skew M1 and M2 macrophages that exhibited distinctive surface marker expression, gene expression profiles, and cytokine and chemokine production for their respective subset. We then performed RNA-sequencing to interrogate the transcriptional landscape of functional M1 and M2 macrophages and have found 1,472 transcripts that have at least a two-fold change in differential expression between M1 and M2 macrophages across 4 different donors. These results represent the most in-depth characterization of human M1 and M2 macrophages and provide insights into better biomarkers and targets for novel immunotherapy approaches.

Citation Format: Kimberline R. Yang, Todd D. Armstrong, Elizabeth M. Jaffee. Transcriptional analysis of the functional differences between M1 and M2 macrophages to identify new targets for myeloid cell modulation in pancreatic tumors [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 4623. doi:10.1158/1538-7445.AM2017-4623
Abstract 4626: Macrophages alter heparan sulfate-modifying enzyme levels, growth factor activity and cancer cell growth in 3D hydrogels
Authors: Costa Fabio Henrique Brasil da , Navone Nora M. , Farach-Carson Mary C. , Carson Daniel D.
Published: July 2017

Tumor progression in both primary and metastatic niches often involves the activation of associated or “reactive” stroma similar to that seen in a wound healing response. The stromal participants include fibroblasts, endothelial and immune cells, including macrophages. Cancer-associated fibroblasts (CAFs) and tumor-associated macrophages communicate with local cells including cancer cells and the surrounding stroma to induce epithelial-to-mesenchymal transition, immunosuppression and increased production of extracellular matrix (ECM) components, growth factors, and ECM-remodeling enzymes. Heparan sulfate (HS)-modifying enzymes produced by tumor-associated stroma play key roles in the delivery of heparin-binding growth factors (HBGFs) and cytokines, serving to enhance tumorigenesis, foster growth and recruit other bystander cells to sites of metastasis. Through the use of a novel, biomimetic, 3D collagen/hyaluronic acid hydrogel system developed in our labs, this study focused on interactions between primary human macrophages and cancer cells, specifically on how this dialogue impacts the production of the secreted HS-modifying enzymes, heparanase (HPSE) and endosulfatase 1 (SULF1). We studied the impact of co-cultures containing macrophages and endometrial or prostate cancer cell lines, as well as patient-derived xenografts, on the levels of HPSE and SULF1. We also assessed cancer growth characteristics in the presence of macrophages. Taken together, our observations indicate cancer cell responses to macrophages are cell type specific, and we demonstrated significant functional consequences of interactions between macrophages and HS-mediated signaling. We are currently determining if macrophage polarization to classically activated (M1) or alternatively activated (M2) phenotypes alters cancer cell responses in 3D. This system is being used to determine novel approaches to intervene in HBGF delivery to limit cancer growth in a more physiologically relevant in vitro system.

Citation Format: Fabio Henrique Brasil da Costa, Nora M. Navone, Mary C. Farach-Carson, Daniel D. Carson. Macrophages alter heparan sulfate-modifying enzyme levels, growth factor activity and cancer cell growth in 3D hydrogels [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 4626. doi:10.1158/1538-7445.AM2017-4626
Abstract 4627: Prognostic significance and population shift of peripheral monocytes in patients with oropharyngeal squamous cell carcinoma
Authors: Takahashi Hideyuki , Sakakura Koichi , Chikamatsu Kazuaki
Published: July 2017

Evidence has accumulated that chronic inflammation plays a critical role in carcinogenesis, tumor growth, and metastasis. To date, several inflammatory biomarkers are considered as potential prognostic factors in various cancers. The aim of the present study was to investigate the prognostic significance of the pretreatment levels of inflammatory biomarkers in patients with oropharyngeal squamous cell carcinoma (OPSCC). A total of 67 patients newly diagnosed as OPSCC was finally included. The neutrophil, lymphocyte, monocyte and platelet counts recorded before treatment was initiated, then lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were calculated. These inflammatory biomarkers and clinical parameters including age, differentiation, T-stage, N-stage, TNM-stage, smoking, and drinking were compared with progression-free survival (PFS) and overall survival (OS) using cox regression. Elevated monocyte count and advanced T-stage were significantly associated with poor PFS; in addition, these were independent prognostic factors for PFS. Elevated monocyte count and low LMR were significantly associated with poor OS; moreover, low LMR was an independent prognostic factor for OS. Of note, monocyte count and LMR were associated with prognosis more intensively in patients with p16-negative OPSCC. Following these results, we evaluated the peripheral blood mononuclear cells of 14 patients with OPSCC collected before treatments. CD14+ CD86+ HLR-DR+ cells were gated as monocytes, then divided into three subsets according to the expression of CD14 and CD16. The proportion of intermediate monocytes was lower and that of classical monocytes was higher in OPSCC patients than in healthy donors, especially in patients with elevated monocyte count. Furthermore, the expression level of PD-L1 was higher in patients than in healthy donors on all subsets. In conclusion, our data suggests that elevated monocyte count and low LMR seem to be useful biomarkers for predicting prognosis of OPSCC; furthermore, the population shift of monocytes may relate to poor prognosis of OPSCC.

Citation Format: Hideyuki Takahashi, Koichi Sakakura, Kazuaki Chikamatsu. Prognostic significance and population shift of peripheral monocytes in patients with oropharyngeal squamous cell carcinoma [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 4627. doi:10.1158/1538-7445.AM2017-4627
Abstract 4630: Development of selective and potent CDK8 inhibitors that increase NK cell activity, which translates in tumor surveillance
Authors: Hofmann Marco H. , Engelhardt Harald , Carotta Sebastian , Arnhof Heribert , Scharn Dirk , Kerenyi Marc , Mayer Moritz , Gmaschitz Gerhard , Egger Georg , Engelhardt Christian , Sanderson Michael , Impagnatiello Maria A. , Schnitzer Renate , Pearson Mark , McConnell Darryl , Kraut Norbert , Moll Jürgen
Published: July 2017

Background: Cyclin-dependent kinase 8 (CDK8) is part of the mediator complex that can either positively or negatively influence transcription. CDK8 is known to phosphorylate signal transducer and activator of transcription 1 (STAT1) at the position Ser727. STAT1 activity is regulated by JAK-mediated phosphorylation of tyrosine701 which leads to dimerization, nuclear translocation and IFN-γ induced phosphorylation mediated by CDK8. Introduction of an alanine mutation at the phosphorylation site STAT1-S727 results in enhanced NK cell cytotoxicity accompanied by increased levels of perforin and granzyme B (Putz et al. 2013).

Method: Here we present the discovery and development of potent and selective CDK8 inhibitors guided by crystallography. The inhibitory effect of optimized compounds BI 9811 and BI 1347 on STAT1 phosphorylation and perforin release was investigated in the human NK cell line NK-92MI. Direct effects on cancer cells were furthermore analyzed in a broad panel of cell lines. The compound BI 1347 was profiled in vivo in the orthotopic B16-F10 melanoma mouse model.

Results: Highly potent and selective CDK8 inhibitors were identified with an IC50 of below 10 nM in a biochemical kinase assay, which translated in a potent down regulation of the STAT1- Ser727 signal and in increased perforin and granzyme B secretion. BI 9811 and BI 1347 were highly selective for CDK8, as tested in a broad kinase panel and showed no cytotoxic activity on NK cells and most cancer cell lines, which distinguishes this compound class from published CDK8 inhibitors. A representative molecule out of this compound class demonstrated in vivo biomarker modulation and survival increase in the murine B16-F10 melanoma mouse model.

Conclusion: We developed potent CDK8 inhibitors that show activation of NK cells that translates into biomarker modulation (pSTAT1Ser727) and in vivo efficacy.

Citation Format: Marco H. Hofmann, Harald Engelhardt, Sebastian Carotta, Heribert Arnhof, Dirk Scharn, Marc Kerenyi, Moritz Mayer, Gerhard Gmaschitz, Georg Egger, Christian Engelhardt, Michael Sanderson, Maria A. Impagnatiello, Renate Schnitzer, Mark Pearson, Darryl McConnell, Norbert Kraut, Jürgen Moll. Development of selective and potent CDK8 inhibitors that increase NK cell activity, which translates in tumor surveillance [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 4630. doi:10.1158/1538-7445.AM2017-4630
Abstract 4632: Exosome-mediated transfer of miR-183-5p from tumor cells to macrophages contributes to regulate TAMs phenotypes and promote tumor progression and metastasis
Authors: Guo Jian , Wang Wei , Liu Yan , He Huiwen , Zhang Chen , Chen Chong , Luo Yunping
Published: July 2017

Tumor-associated macrophages (TAMs) perform supportive roles in promoting tumor progression and metastasis. In tumor microenvironment, the phenotypes of TAMs are regulated by a variety of factors, such as cytokines, ligends and products of metabolism. Recently, Exosome-mediated transfer of miRNAs is proved to be a significant way for intercellular communications. Whether exosomal miRNAs derived from tumor cells contribute to TAMs’ phenotypes remains unclear. In this study, we aim to identify the miRNAs in exosomes transferred from tumor cells to macrophages and explore how these miRNAs regulate the TAMs phenotypes. In the beginning we transferred CY3-labled miRNA NC to 4T1 mouse breast cancer cells and then co-cultured with bone marrow-derived macrophages (BMDMs). The CY3 fluorescence could be observed in BMDMs after 48 hour, suggesting that miRNAs could be transferred from tumor cells to macrophages. Next, we performed miRNAs sequencing in either BMDM or exosomes derived from 4T1 cancer cells. We analyzed and compared the miRNAseq data by bio- informatics and found that there were 30 of the most abundant miRNAs in 4T1 exosomes, and nearly all of which were rich in BMDMs. However, it was found interestingly that miR-183-5p was extremely low in BMDMs. Furthermore, we treated BMDMs with exosomes isolated form 4T1 cell conditioned medium, and then we found importantly the miR-183-5p in BMDMs was increased significantly, while cultured with exosome-depleted 4T1 conditioned medium, the expression of miR-183-5p in BMDMs was not changed. Meanwhile, we isolated TAMs from 4T1 mouse breast tumor while spleen macrophages from normal mouse as a control. The expression of miR-183-5p in TAMs was much higher than that in spleen macrophages. Moreover, it was determined that expression of pro-inflammatory cytokine TNF and IL-6 in BMDM were increased significantly in BMDMs treated with 4T1-derived exosomes. Correspondingly, transferred miR-183-5p mimic into BMDMs, the expression of TNF and IL-6 were also increased. Finally we co-cultured 4T1 cells with BMDMs transferred with miR-183-5p, and the migration and invasion of 4T1 cells were significantly enhanced. Taken together, our study demonstrate that miR-183-5p that high expression in 4T1 exosomes can transferred into macrophages to regulated their phenotypes and promote tumor progression and metastasis in breast tumor microenvironment.

Citation Format: Jian Guo, Wei Wang, Yan Liu, Huiwen He, Chen Zhang, Chong Chen, Yunping Luo. Exosome-mediated transfer of miR-183-5p from tumor cells to macrophages contributes to regulate TAMs phenotypes and promote tumor progression and 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 4632. doi:10.1158/1538-7445.AM2017-4632
Abstract 4636: p53 governs the tumoricidal activity of M1 macrophages in lung cancer
Authors: Hsiao Yi-Jing , Chen Hsuan-Yu , Su Kang-Yi , Ho Bing-Ching , Chen Jeremy J.W. , Yang Pan-Chyr , Yu Sung-Liang
Published: July 2017

Macrophages in a tumor microenvironment have been characterized as M1- and M2-polarized subtypes. M1 macrophage is associated with favorable clinical outcome of lung cancer patients, but the detail molecular mechanism is not thoroughly understood. Herein, we elucidate the anti-tumor mechanism of M1 macrophages by in vitro and in vivo assays. M1 conditioned media (CM) significantly reduced the tumor size in the xenograft mouse model via intratumoral administration. The M1-altered differential expression was profiled by microarray and the pathway analysis indicated that p53 was a critical regulator. Lung cancer cells harboring wild-type p53 (wt-p53) went apoptosis after M1 CM culture, but cells with mutant p53 did not. Additionally, M1 CM enhanced wt-p53 accumulation through reducing p53 ubiquitination and increasing protein stability. Moreover, we identified a p53 interacting protein STAT1 involved in M1-induced apoptosis and p53 accumulation. The M1 CM not only enhanced the interaction between STAT1 and p53 but also up-regulated and activated STAT1. We found that STAT1-Y701 phosphorylation is required for the p53 stability. The interaction of STAT1-Y701F and p53 was reduced compared to wild-type STAT1. To reveal the upstream signaling, we found that knockdown of IFNAR2, JAK1 and TYK2 attenuates M1-induced apoptosis. Taken together, STAT1 signaling is essential for M1 CM-induced apoptosis and p53 accumulation. Our findings imply that p53 and M1 macrophages play important roles in the immune surveillance in lung cancers.

Citation Format: Yi-Jing Hsiao, Hsuan-Yu Chen, Kang-Yi Su, Bing-Ching Ho, Jeremy J.W. Chen, Pan-Chyr Yang, Sung-Liang Yu. p53 governs the tumoricidal activity of M1 macrophages in lung cancer [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 4636. doi:10.1158/1538-7445.AM2017-4636
Abstract 4650: Mutation detection by target sequence analyses using tissue-specific panels in esophageal squamous cell carcinoma
Authors: Iwaya Takeshi , Endo Fumitaka , Kume Kohei , Sasaki Yasushi , Tokino Takashi , Nishizuka Satoshi
Published: July 2017

Whole-exome sequencing (WES) studies on esophageal squamous cell carcinoma (ESCC) have revealed that only a small number of frequently mutated genes, such as TP53, NOTCH1, NRF2, and ZNF750, are present in individual tumors, whereas hundreds of others less frequently mutated genes are accumulated. The mutation detection rate by target sequence analyses is an important factor for clinical sequence in the course of ESCC treatment. This study aimed to evaluate the mutation detection rates of several target sequence analyses for ESCC. Ten pairs of surgically or endoscopically acquired tumor tissues and corresponding peripheral blood mononuclear cells were obtained from ESCC patients. We used three types of target sequence panels for mutation screening in 10 ESCC patients as follows: 1) Cancer Hotspot Panel (CHPv2), targeting mutation hotspot regions in the most common 50 human cancer genes that are frequently mutated (panel size: 22,027 bp), for five patients; 2) TP53 Panel, covering all exons of TP53 (panel size: 2,359 bp), for five patients; and 3) ESCC Panel, originally designed to target 31 frequently mutated genes in ESCC (panel size: 210,570 bp), for seven patients. Four missense and two nonsense mutations in TP53 were detected in CHPv2 and the TP53 Panel. Furthermore, one missense mutation in FBXW7 and one intronic mutation in the vicinity of exon 8 in TP53 were also observed in CHPv2 and the TP53 Panel, respectively. In the ESCC Panel, 47 mutations in 17 genes were detected in seven patients. The mutation detection rates by the TP53 Panel, CHPv2, and ESCC Panel were 593.0/Mb, 63.5/Mb, and 30.4/Mb, respectively. These mutation detection rates were more than 20-fold higher than that of WES, which has been reported to be 3.1/Mb for ESCC. These results suggest that tissue-specific cancer-related gene panels are a highly effective approach for mutation detection in ESCC.

Citation Format: Takeshi Iwaya, Fumitaka Endo, Kohei Kume, Yasushi Sasaki, Takashi Tokino, Satoshi Nishizuka. Mutation detection by target sequence analyses using tissue-specific panels in esophageal squamous cell carcinoma [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 4650. doi:10.1158/1538-7445.AM2017-4650
Abstract 4651: Multiple platform evaluation of KRAS codon 12/13 reference material
Authors: Garlick Russell , Konigshofer Yves , Tomson Farol L. , Yuzuki Dale , Anekella Bharathi
Published: July 2017

An assay’s ability to detect the presence of a KRAS driver mutation is critical when attempting to determine whether a tumor may respond to anti-EGFR therapy. Currently, there are two FDA approved companion diagnostics that can be used to assess whether there are mutations in KRAS codons 12 and 13, and both include their own internal controls. However, there is a need for external reference materials that allow laboratories to validate such assays - especially, at levels near their limits of detection (LODs). We describe the design of the Seraseq™ FFPE Tumor KRAS Reference Material. This reference material is composed of 7 different 5-micron FFPE curls that contain the 7 different KRAS mutations detected by the companion diagnostics: G12A, G12C, G12D, G12R, G12S, G12V and G13D. Each curl contains a mixture of KRAS wildtype GM24385 cells and a KRAS mutant cell line in sufficient quantity for most assays. KRAS mutant frequencies of about 5% were targeted and subsequently verified by digital PCR. A 5% mutant frequency was selected because it is near the LOD for the existing companion diagnostics, and is close to the minimum reported frequency of many next generation sequencing (NGS)-based assays that test for somatic variants. The reference material was evaluated using different methods, which include digital PCR, the Roche cobas® KRAS Mutation Test, the QIAGEN therascreen® KRAS RGQ PCR Kit and several NGS-based assays. Testing revealed general compatibility with these assays in that sufficient DNA could be extracted, and concordance of the results with the expected calls was high. For example, apparent DNA yields by Nanodrop with the cobas assay exceeded the required amount by more than 3-fold and all 7 curls led to a “mutation detected” result in duplicate testing. Interestingly, not all variants were detected with the therascreen assay, which may be due to some variants being present at slightly below the claimed LODs, however DNA yields were sufficient and the correct variants were identified. Finally, variant frequencies obtained by NGS were consistent with those observed by digital PCR. We conclude that the Seraseq FFPE Tumor KRAS Reference Material may be useful as part of KRAS codon 12/13 assay validation.

Citation Format: Russell Garlick, Yves Konigshofer, Farol L. Tomson, Dale Yuzuki, Bharathi Anekella. Multiple platform evaluation of KRAS codon 12/13 reference material [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 4651. doi:10.1158/1538-7445.AM2017-4651
Abstract 4652: Profiling of immunity biomarkers in whole blood
Authors: Chenchik Alex , Komarov Andrei , Makhanov Michael , Frangou Costa
Published: July 2017

Cancer immunotherapies are rapidly changing traditional treatment paradigms and expanding the therapeutic landscape for cancer patients. However, despite the current success of these therapies, significant number of patients do not respond to immunotherapy and responders often experience significant toxicities. Therefore, there is a growing need to identify predictive and prognostic biomarkers that enhance our understanding of dynamic immunity mechanisms involved in cancer development. The genome-wide expression profiling of whole blood is an attractive method for the discovery of biomarkers due to its non-invasiveness, possibility for retrospective simple clinical site processing, and rich biological content. To this end, we have developed GeneNet, a genome-wide targeted RNA expression assay that profiles 19,000 genes based on multiplex RT-PCR followed by NGS in whole blood samples stabilized in PAXgene. The GeneNet 19K assay provides comprehensive and unbiased profiling of immune cell activation and canonical immune pathway genes for both innate adaptive and humoral immune responses. In this study, we present the performance of the assay for immunophenotyping of immune cells in blood samples from TNBC patients and assess the immune responses to complex immunomodulatory stimuli in ex vivo model system. Preliminary studies demonstrate the assay’s unparalleled specificity and sensitivity resulting in better detection of low abundance mRNA transcripts as well as an improved cost-effectiveness for high-throughput clinical applications.

Citation Format: Alex Chenchik, Andrei Komarov, Michael Makhanov, Costa Frangou. Profiling of immunity biomarkers in whole blood [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 4652. doi:10.1158/1538-7445.AM2017-4652
Abstract 4653: Genomics of regulatory crosstalk between PPAR gamma & 14-3-3 genes in breast cancer cells
Authors: Khalil Ashraf A. , Kabapy Nihal F. , Sharabi Fouad N.
Published: July 2017

PPARγ, a ligand-stimulated transcription factor with differentiation promoting activity is overexpressed in a variety of cancers. Perturbation of PPAR-γ signaling is now believed to be a strategy for treatment of several cancers, including breast cancer. A set of genes regulated by PPAR-γ ligands is expected to mediate the antiproliferative and prodifferentiation effects in cancer cells. Because 14-3-3 family of proteins shows a debatable activity and varying expression levels in different tumors, in the studies presented here we explored the transcriptional regulatory role of Pioglitazone on the seven 14-3-3 isoforms presenting in MCF-7 breast cancer cells. This study demonstrated that the potent PPAR-γ agonist, Pioglitazone exerted a regulatory role on expression of 14-3-3 genes where it upregulated 14-3-3 gamma, epsilon, zeta and tau by 3.8, 5.2, 2.7 and 739 folds, respectively. However, it had a negative regulatory effect on 14-3-3 beta, sigma and Eta by 16.94, 4.58 and 2.12 folds, respectively compared with control cells. These results correlated with growth arrest and a great increase in BRCA1 gene expression by 1076 folds. In summary, these findings are the first time showing that PPAR-γ regulates 14-3-3 genes and raises question whether PPAR-γ ligands mediate their anticancer effects via regulation of 14-3-3 proteins.

Selected References: 1. Bridges, D. and G.B. Moorhead. 2005. 14-3-3 proteins: a number of functions for a numbered protein. Sci. STKE 2005(296):re10. 2. Chearwae, W. and J. Bright. 2008. PPARγ agonists inhibit growth and expansion of CD133+; brain tumour stem cells. Brit. J. Cancer 99(12): 2044-2053

Citation Format: Ashraf A. Khalil, Nihal F. Kabapy, Fouad N. Sharabi. Genomics of regulatory crosstalk between PPAR gamma & 14-3-3 genes in breast cancer cells [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 4653. doi:10.1158/1538-7445.AM2017-4653
Abstract 4654: The primary study of the correlation between mRNA expression and protein phosphorylation of epidermal growth factor receptor EGFR in colorectal and breast cancer cell lines
Authors: Wang Hongwei , Ma Catherine , Zhou Yuan
Published: July 2017

The EGFR family is a group of receptor tyrosine kinases that mediates cell proliferation, migration, and differentiation. Activation of EGFR signaling is triggered by ligand-binding induced receptor dimerization. The dimerization activates the intracellular kinase domains of the receptor, resulting in autophosphorylation of C-terminal tail and the initiation of downstream signaling. EGFR overexpression is correlated with more aggressive clinical indications in multiple types of cancer, including colon, breast, lung, and head and neck carcinoma. Clinical studies show that EGFR-directed therapeutic approaches improved better outcomes of metastatic lung, colorectal, and head and neck cancers. Predictive biomarkers are being developed to deliver personalized therapies. Therefore, it is important to determine the protein as well as molecular biomarker expression for selecting targeted therapies. Here we present a panel cancer cell lines to examine EGFR expressions at a molecular biomarker level (mRNA) as well as a protein level of phosphorylated EGFR(pS1060). Colon cancer cell lines HT29, HCT116, SW620, as well as the breast cancer cell line SKBR3, were cultured and processed onto formalin-fixed paraffin-embedded (FFPE) CellMax pellets. Expression levels of EGFR mRNA in the FFPE slides were examined with RNAScope technology to visualize single RNA molecules per cell by using probes specifically against EGFR. Expression of phosphorylated EGFR was examined by the phosphospecific EGFR (pS1070) antibodies by using immunohistochemistry (IHC). EGFR mRNA expression was detected in HT29 and HCT 116 cells showing strong punctuated signals in the cytoplasm. However, no EGFR mRNA signals were detected in SW620 cells. Breast cancer cell line SKBR3 showed abundant EGFR mRNA expression. Phosphorylated EGFR were found in the cell HT29, HCT 116 and SKBR3 cells with membrane staining pattern. No phosphorylated EGFR was observed in SW620 cells using Immunochemistry staining. Together, our results demonstrated that the EGFR mRNA expression is correlated to the phosphorylation of EGFR in these cancer cell lines. We concluded that the EGFR mRNA probes, phosphorylated EGFR antibodies, and/or the cell lines can be served as the control-references for clinical biomarker screening.

Citation Format: Hongwei Wang, Catherine Ma, Yuan Zhou. The primary study of the correlation between mRNA expression and protein phosphorylation of epidermal growth factor receptor EGFR in colorectal and breast cancer cell lines [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 4654. doi:10.1158/1538-7445.AM2017-4654
Abstract 4655: Elevated level of serum miR-99a is correlated with serous epithelial ovarian cancer and can be a potential biomarker
Authors: Yoshimura Akihiko , Sawada Kenjiro , Nakamura Koji , Kinose Yasuto , Nakatsuka Erika , Mabuchi Seiji , Kimura Tadashi
Published: July 2017

Objective: There is a critical need for improved diagnostic markers to detect ovarian high grade serous cancer (HGSC). microRNAs (miRNAs) stably exist in circulating blood, reflecting tissue or organ conditions and present in circulating microvesicles such as exosomes. We aimed to identify which miRNAs are highly produced from HGSCs and analyze whether serum miRNA can discriminate patients with HGSC from healthy controls.

Methods: Secreted exosomes from ovarian cancer cell lines were collected and exosomal miRNAs extracted. miRNA microarray was performed and several elevated miRNAs specific to ovarian cancer cells were picked up. Among these, we focused on miR-99a. Sera were collected from 15 patients with HGSC (stage III and IV) and 10 healthy controls. Expression level of miR-99a was determined by miRNA RT-qPCR.

Results: miRNA microarray revealed several miRNAs were highly expressed in exosomes in patients with HGSC. In patients, serum miR-99a levels were significantly increased (4.8 fold) compared with those of healthy controls. ROC analysis showed that at the cut-off of 1.4, the sensitivity and specificity were 73 % and 80 % respectively for detecting HGSC (AUC = 0.85).

Conclusion: Exosomal miRNAs can be detected in sera of patients with HGSC and have the potential to predict ovarian cancer.

Citation Format: Akihiko Yoshimura, Kenjiro Sawada, Koji Nakamura, Yasuto Kinose, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. Elevated level of serum miR-99a is correlated with serous epithelial ovarian cancer and can be a potential biomarker [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 4655. doi:10.1158/1538-7445.AM2017-4655
Abstract 4656: Assessment of BCL-2, BCL-xL and MCL-1 in bone marrow biopsy samples using VENTANA BenchMark XT automated slide stainer
Authors: Streator Ashley , Villanueva Eneida C. , Holmes Burton F. , Zhang Liping
Published: July 2017

Immunophenotyping is essential for diagnosis and classification of most hematopoietic malignancies, and often followed by ancillary studies to understand the predictive and prognostic value of biomarkers. Immunohistochemistry (IHC) provides in-situ coupling of antigen expression with morphology of precursor B- from precursor T-lymphoblastic leukemia/lymphoma. The number of different IHC antibodies that can be applied to bone marrow (BM) biopsies is increasing concurrently with development of flow cytometry and molecular diagnostic methods.

IHC detects scattered tumor cells in Hodgkin’s Lymphoma, Multiple Myeloma (MM) and Acute Myelogenous Leukemia (AML), where key biomarkers may not be detectable by current flow cytometry methods. The goal of this study was to detect BCL-2, Bcl-xL and MCL-1 that are able to predict pre-clinical sensitivity to the drug Venetoclax. We examined the expression of these biomarkers in MM bone samples decalcified using commercially available reagents to determine whether decalcification methods impact BCL-2 family IHC Staining.

Results: MM bone samples (n=59) decalcified in Decal STAT® for 20 min, stained for BCL-2 (124) and Bcl-xL (54H6). MM bone samples (n=7) decalcified in Decal STAT® for 60 min stained for BCL-2 and Bcl-xL. Finally, eleven MM bone samples decalcified in Richard Allan Decal Solution® stained for BCL-2 and Bcl-xL. MCL-1 stained poorly with commercially available decalcification solutions. For MCL-1 to be used, additional staining optimization is required. Conclusion: BCL-2 and BCL-xL proteins can be detected in MM bone samples decalcified with commercially available solutions. With these preliminary results, future studies will pursue the potential for sensitivity to "Venetoclax (ABT-199/GDC-0199)".

Citation Format: Ashley Streator, Eneida C. Villanueva, Burton F. Holmes, Liping Zhang. Assessment of BCL-2, BCL-xL and MCL-1 in bone marrow biopsy samples using VENTANA BenchMark XT automated slide stainer [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 4656. doi:10.1158/1538-7445.AM2017-4656
Abstract 467: Analysis of human hypoxia related miRNAs in MDM2 transfected prostate cancer cells
Authors: Alobid Saad Ebrahim , Venkatesan Thiagarajan , Alaseem Ali , Alhazzani Khalid , Rathinavelu Appu
Published: July 2017

Recent studies have shown the crucial role of miRNAs in regulating cancer initiation, progression and metastatic process via influencing gene expression patterns. However, the distinct mechanism by which miRNA regulates tumorigenesis remains as an area requiring detailed research investigations. In our earlier studies, we have demonstrated a positive correlation between MDM2 expression and hypoxia inducible factor -1alpha (HIF-1α) levels in both normoxic and hypoxic conditions using prostate cancer cell lines. Subsequently, we postulated that the upregulation of HIF-1α is most likely regulated by MDM2 in cancers cells that are positive for this gene expression. Hence, to confirm our hypothesis, we utilized miScript miRNA PCR Array analysis (Qiagen, USA) and compared the gene expression profile of the hypoxia signaling pathway in LNCaP (prostate cancer cells) and LNCaP-MST

(MDM2 transfected prostate cancer cells) under normoxic conditions. This study was expected to outline how MDM2 impacts the differential expression of miRNAs and leading to enhancement of tumor angiogenesis via increase in HIF-1α or other genes of the hypoxia signaling pathway. Our preliminary data indicate that hsamiR99a5p, hsamiR125b5p, hsamiR1385p, hsamiR199b5p and hsalet7c5p are significantly upregulated in LNCaP-MST cells compared to LNCaP. In addition, some of the miRNAs such as hsalet7d5p, hsalet7e5p, hsalet7f5p, hsalet7g5p and hsalet7i5p are significantly downregulated in the same cells. Among the upregulated miRNAs, hsamiR125b5p emerged as an important gene that is known to downregulate the protein levels of hypoxia inducible factor 1alpha inhibitor (HIF1AN or FIH), which can normally inhibit the transcriptional activity of HIF-1α in both normoxic and hypoxic conditions. Therefore, overexpression of hsamiR125b5p seems to be associated with the increase in HIF-1α level. From the aforementioned findings, it is confirmed that the MDM2 overexpression is impacting miRNA levels such as hsamiR125b5p that are involved in hypoxia related signaling pathway. Thus, our results offer a convincing evidence that MDM2 can promote angiogenesis and increase metastatic potential of cancers through altering the miRNA expression levels. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged).

Citation Format: Saad Ebrahim Alobid, Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Appu Rathinavelu. Analysis of human hypoxia related miRNAs in MDM2 transfected prostate cancer cells [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 467. doi:10.1158/1538-7445.AM2017-467
Abstract 4682: Genomic profiles in gene expression and methylation help define the molecular characteristics of hairy cell leukemia subtypes
Authors: Edelman Daniel C. , Stevenson Holly S. , Wang Yonghong , Arons Evgeny , Waterfall Joshua J. , Petersen David , Zhou Hong , Meltzer Paul S. , Kreitman Robert J.
Published: July 2017

Hairy cell leukemia (HCL) until recently was characterized as a single blood and bone marrow malignancy, even though clinically at least two pathologies seemed to be at work. This study sought to confirm the clinical observations using genomic evidence. HCL is a chronic mature B-cell malignancy with distinctive immunophenotype, typically expressing CD20, CD22, CD25, CD11c, CD103, CD123, annexin A1 (ANXA1), and tartrate-resistant acid phosphatase (TRAP). Purine analog therapy is highly effective, with most patients achieving durable remissions. HCL- variant (HCLv) was first identified by Cawley et al. (1980) and recently recognized by the World Health Organization as a separate cancer. HCLv lacks CD25, ANXA1, TRAP, and BRAF V600E expression, and patients respond poorly to purine analogs. In order to find detailed biological information involving these two diseases, we studied the genome-wide gene expression and methylation profiles of 75 HCL patient samples; 67 were tested by both methods. Our study results indicate that HCL and HCLv show very distinct gene expression and methylation patterns. Many genes that are differentially expressed are involved in immunological and inflammatory response pathways. Several cancer associated genes such as ANXA1 and FLT3 are down-regulated in HCLv compared to HCL. Correlation patterns between methylation and gene expression for many genes such as ANXA1 suggest that methylation plays an import role in gene expression regulation for these malignancies. This study has begun the process of identifying better biomarkers for disease discrimination between HCL and HCLv and providing potential new targets for strongly needed therapies for HCLv.

Citation Format: Daniel C. Edelman, Holly S. Stevenson, Yonghong Wang, Evgeny Arons, Joshua J. Waterfall, David Petersen, Hong Zhou, Paul S. Meltzer, Robert J. Kreitman. Genomic profiles in gene expression and methylation help define the molecular characteristics of hairy cell leukemia subtypes [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 4682. doi:10.1158/1538-7445.AM2017-4682