bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3477: Metabolic pathway analyses identify proline biosynthesis as a promoter of liver carcinogenesis
Authors: Ding Zhaobing , Ericksen Russell E. , Steckel Michael , Haegebarth Andrea , Gruenewald Sylvia , Han Weiping
Published: August 2018

Cancer cells undergo dramatic metabolic reprogramming, largely to meet the requirements of sustained proliferation. These metabolic pathways are tightly regulated by various oncogenes, such as Myc and Ras. However, it has been difficult to directly and effectively inhibit these oncogenic signaling cascades with pharmacological compounds. Therefore, focusing on the downstream metabolic pathways that enable indefinite growth may provide a therapeutic opportunity. To identify metabolic enzymes required for hepatocellular carcinoma (HCC) tumorigenesis, we compared gene expression profiles of normal liver tissue to the Morris Hepatoma and DEN (Diethylnitrosamine)-induced HCC models as well as a liver regeneration model. PYCR1 (Pyrroline-5-Carboxylate Reductase 1), an enzyme in the proline biosynthesis pathway, was identified as one of the top up-regulated genes in the HCC models. An increase in PYCR1 protein levels in tumor samples versus normal liver tissue was confirmed by Western blot. shRNA-mediated knockdown of PYCR1 in HCC cell lines (Huh-7, Hep3B, HepG2 SNU-398, and MH3924a) potently reduced cell proliferation in vitro and tumor growth in vivo. Conversely, overexpression of PYCR1 enhanced the proliferation of Hep3B and AML12 cells. Importantly, PYCR1 expression was not elevated in regenerating tissues, and knockdown in a non-tumorigenic cell line did not influence proliferation. Additional enzymes in the proline biosynthesis pathway were also altered in tumors. For instance, ALDH18A1 was up-regulated and knockdown by shRNA decreased proliferation of human HCC cell lines. Metabolomic analyses suggested that the metabolism of glucose was disrupted in PYCR1 knockdown cells, with reduced fluxes through glycolytic and pentose phosphate pathways. Clinical data demonstrated that PYCR1 expression was increased in HCC, and expression levels correlated with increasing tumor grades and were independent predictors of clinical outcome. Overall, our data suggest that enhanced expression of proline biosynthetic enzymes promotes cell proliferation, and that inhibiting PYCR1 or ALDH18A1 may be a novel therapeutic strategy to target HCC.

Citation Format: Zhaobing Ding, Russell E. Ericksen, Michael Steckel, Andrea Haegebarth, Sylvia Gruenewald, Weiping Han. Metabolic pathway analyses identify proline biosynthesis as a promoter of liver carcinogenesis [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 3477.
Abstract 3481: Elucidating the metabolic crosstalk between lymphatic endothelial cells and breast cancer using 1H NMR metabolomics
Authors: Acevedo-Acevedo Suehelay , Millar Douglas C. , Palecek Sean P.
Published: August 2018

Breast cancer is the second most commonly diagnosed cancer among women worldwide. Patient death is typically caused by metastasis development rather than the primary tumor. Metastasis in breast cancer has been shown to occur via blood and lymphatic vessels. Research shows that breast cancer cells ‘educate' lymphatic and blood endothelial cells to support tumor growth by stimulating growth factor secretion and directing tumor dissemination. In addition, cell metabolism is altered during malignant transformation. Cancer cells have increased energy and macromolecule biosynthesis requirements to sustain rapid proliferation. However, it remains unclear how tumor endothelial cell metabolism is altered and how metabolism contributes to tumor metastasis. Therefore, this study aims to use 1H NMR metabolomics to identify breast cancer-endothelial cell metabolic interactions in a high-throughput manner. Firstly, we studied if in vitro co-culture with breast cancer altered endothelial cell metabolism. We identified distinct metabolic profiles for lymphatic endothelial cells (LECs) grown in monoculture or co-culture with three different breast cancer cell lines. Principal component analysis revealed LECs co-cultured with breast cancer cells clustered separately from control LECs. Interestingly, LECs co-cultured with triple negative breast cancer cells clustered together with LECs co-cultured with HER2+ breast cancer cell lines indicating that the metabolic changes occurring in these LECs were similar. One-way ANOVA with Tukey's HSD post-hoc analysis revealed 17 significantly different metabolites between all five conditions analyzed including: lactate, glucose, phosphocholine, aspartate, acetate, glycerophosphocholine, lysine and methionine. Quantitative metabolite set enrichment analysis revealed 13 metabolic pathways that were significantly enriched between control LECs and LECs co-cultured with breast cancer cell lines. Enriched pathways included: glycolysis, gluconeogenesis, pyruvate metabolism and protein biosynthesis. Together, these results indicate that culturing LECs with certain breast cancer lines causes significant changes in endothelial metabolism. Alterations in endothelial cell metabolism in response to breast cancer co-culture can further our understanding of tumor-vascular interactions and may lead to identification of metabolic biomarkers and a better understanding of breast cancer metastasis.

Citation Format: Suehelay Acevedo-Acevedo, Douglas C. Millar, Sean P. Palecek. Elucidating the metabolic crosstalk between lymphatic endothelial cells and breast cancer using 1H NMR metabolomics [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 3481.
Abstract 3486: Metabolomics in estrogen receptor or triple-negative breast cancer
Authors: Hu Jennifer J. , Takita Cristiane , Lee Eunkyung , Wright Jean
Published: August 2018

Breast cancer is the most frequently diagnosed cancer in women. Metabolic syndrome and obesity may contribute to triple-negative breast cancer (TNBC) and there are obesity-related alterations in metabolites. Therefore, we pilot-tested metabolomics in ER-negative or TNBC using urine samples from 60 breast cancer cases. In total, 475 metabolites were quantified in different super-pathways: 147 in amino acid, 38 in carbohydrate, 27 in cofactors and vitamins, 12 in energy, 50 in lipid, 31 in nucleotide, 17 in peptide, and 153 in xenobiotics. Our data in 13 ER- and 47 ER+ showed that urinary beta-alanine is higher (3.35+3.73 vs. 1.82+2.26) in ER- breast cancer patients. Our data also show, the first time, that the urinary beta-alanine was higher in TNBC (3.60+4.33; N=8) compared to other subtypes (1.93+2.32; N=52). Beta-alanine is a nonessential amino acid that has previously been shown to be metabolized into carnosine, which functions as an intracellular buffer. In breast cancer cells, beta-alanine reduces both tumor cell migration and proliferation without acting in a cytotoxic fashion. It also significantly increased breast cancer cell sensitivity to doxorubicin. Our data on xanthine are also consistent with the previous study on breast tumor that slightly higher level was observed in ER- patient (1.41+1.35 vs. 1.16+0.88). The level of xanthine was significantly higher in TNBC compared to other subtypes (1.67+1.69 vs. 1.14+0.84). More importantly, our pilot data also showed that urinary mannose levels were significantly higher in ER- tumor (2.21+1.90 vs. 0.90+1.21 p=0.032) and TNBC (2.19+1.77 vs. 1.03+1.38, p=0.037), which is consistent with a previous study that reported that serum mannose levels can differentiate patients with metastatic and localized breast cancer. Metabolic drugs for cancer, such as glutaminase inhibitors, are currently under development and being tested in clinical trials. Therefore, if our data are validated, metabolomics of ER- or TNBC will contribute to innovative discoveries that will impact breast cancer prevention and precision medicine targeting metabolomic pathways.

Citation Format: Jennifer J. Hu, Cristiane Takita, Eunkyung Lee, Jean Wright. Metabolomics in estrogen receptor or triple-negative breast cancer [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 3486.
Abstract 3487: Bioenergetic profiling of cancer cell lines: Quantifying the impact of glycolysis on cell proliferation
Authors: Romero Natalia , Swain Pamela M. , Kam Yoonseok , Rogers George , Dranka Brian P.
Published: August 2018

Fast proliferating cells require tight regulation to achieve a balance between the use of nutrients for ATP production (through glycolysis and oxidative phosphorylation) and the use of intermediate metabolites to sustain the increased biosynthetic activity. Cancer cells, but also high proliferative non-transformed cells exhibit high glycolytic activity during rapid proliferation even in the presence of normal oxygen concentrations in culture. However, despite the high glycolytic activity, the role of glycolysis is not necessary as a major contributor of ATP but to allow nutrient assimilation into biosynthetic precursors. Using Agilent Seahorse extracellular flux analysis, we have developed a cell-based assay which allows simultaneous measurement of the two-main cellular metabolic pathways to calculate the total rate of cellular ATP production as well as the fractional contribution from each pathway. The assay allows for real time changes in total ATP production rate to be quantified, and also the relative source of that ATP after exposure to drugs or changes in extracellular fuel supply. When we applied this new assay to a panel of 20 cancer and highly proliferative cell lines, we found that even in cell lines considered highly glycolytic, ATP production from glycolysis never represents more than 65% of total energy production and between 30-50% for most of the cell lines analyzed. The correlation between glycolytic ATP contribution to total ATP production and other cell phenotypes such as proliferation rate and motility was also analyzed. The use of this assay will allow for improved characterization of the bioenergetic profile of cancer cell variants, discrimination between normal and cancer cell types, and allow researchers to better understand the role of aerobic glycolysis in cell proliferation.

Citation Format: Natalia Romero, Pamela M. Swain, Yoonseok Kam, George Rogers, Brian P. Dranka. Bioenergetic profiling of cancer cell lines: Quantifying the impact of glycolysis on cell proliferation [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 3487.
Abstract 3489: Comprehensive characterization of the SLC transporters in human cancer
Authors: Wang Ruiping , Zhang Shaojun , Futreal Andy , Wang Linghua
Published: August 2018

Metabolic reprogramming has now been well recognized as one of 10 hallmarks of cancer. The plasma membrane transporters, that enable efficient cellular metabolism and aid in nutrient sensing, are receiving increasing attention in recent years. Membrane transporters include 3 main classes: ABC transporters, p-type ATPases, and the solute carrier family (SLC). The SLC group of transporters includes nearly 400 members organized into 52 families that mediate the transport of a wide variety of substrates across biological membranes. Until recently, little was known about the role of these SLC transporters in the mechanisms of cancer development and progression. Yet, the genomic landscape and clinical relevance of SLC transporters in human cancer have not been investigated systematically. We here performed a comprehensive characterization of 396 SLC transporters by integrating multi-platform data across over 30 tumor types from The Cancer Genome Atlas. We observed frequent dysregulation of SLC transporters across cancer types and identified cancer type- and subtype-specific expression signatures. We identified significant correlations of these SLC transporters with the activity of cancer metabolic pathways and explored their complex regulatory networks. We also investigated the role of SLC transporters in tumor metastasis. We further evaluated the potential clinical relevance of these SLC transporters and identified multiple SLC transporters that were significantly associated with patients' clinical outcomes. Our study provided an in-depth understanding of the role of SLC transporters in cancer metabolic reprogramming, elucidated their mechanisms in cancer development and metastasis, and provided novel biomarkers and potential therapeutic targets that can be further developed for future clinical applications.

Citation Format: Ruiping Wang, Shaojun Zhang, Andy Futreal, Linghua Wang. Comprehensive characterization of the SLC transporters in human cancer [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 3489.
Abstract 3519: Disrupting glutamine metabolism depletes glutathione and sensitizes atypical teratoid/rhabdoid tumor to carboplatin
Authors: Wang Sabrina Z. , Poore Brad , Alt Jesse , Rais Rana , Allen Sariah , Orr Brent , Slusher Barbara , Eberhart Charles , Raabe Eric , Rubens Jeffrey
Published: August 2018

Atypical teratoid rhabdoid tumors (AT/RT) are the most common malignant brain tumors of infancy. Recent high-throughput molecular analyses identified 3 distinct subgroups of AT/RT. Preliminary studies show that subgroups with elevated MYC expressions are particularly aggressive, with 18.5% 5-year survival. MYC is challenging to directly target, but MYC increases tumor cells' reliance on glutamine for their metabolic demands. We hypothesize that MYC-driven ATRT can be targeted by inhibiting glutamine metabolism. 6-Diazo-5-oxo L-norleucine (DON) is a glutamine analogue that was well tolerated in a phase I pediatric clinical trial, but DON has never been tested in MYC-driven pediatric tumors. Approximately 1/3 of human AT/RT express high MYC protein (immunohistochemistry for c-MYC expression on 22 human AT/RT, H-score > 1 standard deviation over the median for high c-MYC expression). DON slows cell growth and induces apoptosis in high MYC-expressing AT/RT while low MYC-expressing cells are resistant (MTS assay and Western blot for c-PARP, respectively). Transducing low MYC-expressing cell lines with lentivirus with MYC plasmid to force MYC expression confers sensitivity to DON therapy (decreased cell growth per MTS assay, increased apoptosis per Western for c-PARP). DON administered intraperitoneally 30mg/kg weekly nearly doubles median survival in orthotopic xenograft models of AT/RT (p<0.001 by log rank test; median survival extended from 21 to 36 days). Metabolic flux experiments tracing 15N and 13C labeled glutamine reveal that DON reduces intracellular glutathione. We combine DON with carboplatin because glutathione is critical for detoxification of carboplatin and find that the combination works synergistically to further slow cell growth and induce higher rates of apoptosis (MTS assay, MUSE Cell Viability Assay). This study takes a novel approach to targeting cancer metabolism and serves as the first targeted therapeutic strategy addressing the MYC-expressing subgroup of AT/RT. These findings support the rationale for potential future clinical trials testing glutamine analogues against the MYC subgroup of AT/RT as well as other deadly MYC-driven tumors.

Citation Format: Sabrina Z. Wang, Brad Poore, Jesse Alt, Rana Rais, Sariah Allen, Brent Orr, Barbara Slusher, Charles Eberhart, Eric Raabe, Jeffrey Rubens. Disrupting glutamine metabolism depletes glutathione and sensitizes atypical teratoid/rhabdoid tumor to carboplatin [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 3519.
Abstract 3528: Anti-CD47 immunotherapy regulates T cell metabolism and hypoxia in the tumor microenvironment
Authors: Feliz-Mosquea Yismelin R. , Stirling Elizabeth , Cook Katherine L. , Wilson Adam , Bharadwaj Manish , Molina Anthony J. , Yamaleyeva Liliya , Triozzi Pierre L. , Soto-Pantoja David R.
Published: August 2018

Dysfunction of infiltrating CD8+ effector T cells can be induced by hypoxia and aberrant tumor metabolite uptake in the microenvironment causing an “exhausted” T cell phenotype that limits anti-tumor immunosurveilance. Hence- therapeutic strategies aimed at improving T cell bioenergetics have the potential to reinvigorate T cell responses to reduce tumor burden. CD47 is a widely expressed receptor that controls phagocytic activity by engaging its counter receptor, SIRPα, in macrophages. Also autonomously or by binding to its ligand Thrombospondin-1 CD47 activation can control cell fate under stress. Our prior work shows that targeting CD47 on CD8+ T cells enhanced cytotoxicity against cancer cells. Moreover, depletion of CD8+ T cells in murine models reversed the anti-tumor effect of anti-CD47 therapy. Our new data shows that targeting CD47 reduced the growth of B16 melanoma tumors by approximately 50% (1607 ± 213.7, saline vs. 815.1 ± 67.8, CD47 (-) *p<0.02 n=5-6). Further examination of tumors by live photoacoustic imaging showed that tumors of animals treated with anti-CD47 had reduction in oxygen tension and reduced vascularity when compared to control. This suggests that the reduction in tumor hypoxia by CD47 may shift metabolic competition in the microenvironment to increase metabolite availability allowing optimal activation of cytotoxic T cells. Cell respirometry measurements using an Agilent Seahorse bioanalyzer showed increased levels of mitochondrial function and glycolytic flux in CD47 null T cells when compared to WT. Moreover, these cells showed increased mitochondrial density and increased levels of the mitochondrial biogenesis regulator, PGC1-a. Treatment with CD47 antibody enhanced Pmel-1 CD8+ T cell effector function by reducing B16 melanoma target cell viability by over 60%. Anti-CD47 treatment of these T cells also resulted in upregulation of cell bioenergetics, suggesting that targeting CD47 may impact T cell metabolism to enhance cytotoxic activity against cancer cells. Therefore, our studies show a new role of CD47 immunotherapy regulating immunometabolism of T cells to enhance effector function which may lead to improvement of clinical outcomes in melanoma patients.

Citation Format: Yismelin R. Feliz-Mosquea, Elizabeth Stirling, Katherine L. Cook, Adam Wilson, Manish Bharadwaj, Anthony J. Molina, Liliya Yamaleyeva, Pierre L. Triozzi, David R. Soto-Pantoja. Anti-CD47 immunotherapy regulates T cell metabolism and hypoxia in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3528.
Abstract 354: Role of KAT5 in lung tumorigenesis
Authors: Pan Gilbert , Heo Eunyoung , Ando Mariko , Fujii Masanori , Kobayashi Ikei , Tenen Daniel G. , Watanabe Hideo , Kobayashi Susumu S.
Published: August 2018

Epigenetic modifications in oncogenesis have long been an area of interest in cancer research. Histone modifications are crucial in activities such as transcriptional activation, gene silencing, and epigenetic cellular memory. In particular, lysine acetylation via lysine (K) acetyltransferases (KATs) have been implicated in cancer development. Interestingly, KAT5, also known as Tip60, has been reported to possess both tumor promoting and tumor suppressing properties depending on the context of malignancy. Herein we report that KAT5 contributes to tumorigenesis in EGFR-mutated lung cancer, and Kat5-knockout mice models demonstrate significantly reduced lung tumor burden. To probe the aberrant modification of KAT5, we demonstrated that KAT5 binds to and is phosphorylated by oncogenic EGFR in co-immunoprecipitation experiments. Next, to investigate whether KAT5 is involved in cell proliferation and survival, H1975 cells harboring L858R-T790M double-activating mutations were transfected with doxycycline inducible shRNA targeting KAT5 (shKAT5). Following treatment, shKAT5 cells were observed to have suppressed proliferation rates. Pharmacological inhibition using TH1834, a known KAT5 inhibitor, also suppressed proliferation rates in shKAT5 cells; in contrast BEAS-2B cells, an immortalized normal bronchial cell line, surprisingly exhibited increased viability compared to the transformed H1975 cells, supporting KAT5's context-dependent role in normal and abnormal cell homeostasis. To further investigate KAT5 in lung tumorigenesis in vivo, we generated EGFR-mutant conditional Kat5 knockout mice using a tetracycline-induced Cre/loxP system. Following doxycycline treatment for 10 weeks, isolated mice lungs for EGFRTL/CCSP-rtTA/Cre/Kat5F/F possessed significantly lower tumor volume compared to EGFRTL/CCSP-rtTA/Cre/Kat5W/F and EGFRTL/CCSP-rtTA/Cre/Kat5W/W mice lungs. Hemotoxylin and eosin staining showed no evident hyperproliferation in lungs isolated from EGFRTL/CCSP-rtTA/Cre/Kat5F/F mice whereas lungs isolated from EGFRTL/CCSP-rtTA/Cre/Kat5W/W and EGFRTL/CCSP-rtTA/Cre/Kat5W/F did, signifying that KAT5 has a potential regulatory role in cellular proliferation. RNA-Seq and ChIP-Seq analysis of shKAT5 H1975 cells identified downstream targets involved in tumorigenic pathways. Further studies are in progress to validate the mRNA and protein expression levels of reported targets. Taken together, these data offer insight into a KAT5 mediated oncogenic pathway that can provide novel therapeutic approaches in treating lung cancer.

Citation Format: Gilbert Pan, Eunyoung Heo, Mariko Ando, Masanori Fujii, Ikei Kobayashi, Daniel G. Tenen, Hideo Watanabe, Susumu S. Kobayashi. Role of KAT5 in lung tumorigenesis [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 354.
Abstract 3552: Induction of store operated calcium entry (SOCE) suppresses glioblastoma growth by inhibiting the Hippo pathway transcriptional coactivators YAP/TAZ
Authors: Liu Zhijun
Published: August 2018

Glioblastomas (GBM) are the most aggressive brain cancers without effective therapeutics. The Hippo pathway transcriptional coactivators YAP/TAZ were implicated as drivers in GBM progression and could be therapeutic targets. Here, we found in an unbiased screen of 1650 compounds that amlodipine is able to inhibit survival of GBM cells by suppressing YAP/TAZ activities. Instead of its known function as an L-type calcium channel blocker, we found that amlodipine is able to activate Ca2+ entry by enhancing store-operated Ca2+ entry (SOCE). Amlodipine as well as approaches that cause store depletion and activate SOCE trigger phosphorylation and activation of Lats1/2, which in turn phosphorylate YAP/TAZ and prevent their accumulation in the cell nucleus. Furthermore, we identified that protein kinase C (PKC) beta II is a major mediator of Ca2+-induced Lats1/2 activation. Ca2+ induces accumulation of PKC beta II in an actin cytoskeletal compartment. Such translocation depends on inverted formin-2 (INF2). Depletion of INF2 disrupts both PKC beta II translocation and Lats1/2 activation. Functionally, we found that elevation of cytosolic Ca2+ or PKC beta II expression inhibits YAP/TAZ-mediated gene transcription. In vivo PKC beta II expression inhibits GBM tumor growth and prolongs mouse survival through inhibition of YAP/TAZ in an orthotopic mouse xenograft model. Our studies indicate that Ca2+ is a crucial intracellular cue that regulates the Hippo pathway, and that triggering SOCE could be a strategy to target YAP/TAZ in GBM.

Citation Format: Zhijun Liu. Induction of store operated calcium entry (SOCE) suppresses glioblastoma growth by inhibiting the Hippo pathway transcriptional coactivators YAP/TAZ [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 3552.
Abstract 3584: Characterization of a novel class of engineered (TCR) fusion constructs (TRuCTMs) aimed to treat solid tumors
Authors: Patel Ekta , Ding Jian , Thorausch Nikolaus , Krishnamurthy Janani , Choudhary Rashmi , Weiler Solly , Le Bonnie , Tavares Patrick , Zieba Adam , Quinn Justin , Wang Yan , Schamel Wolfgang , Scarfo Irene , Maus Marcela , Baeuerle Patrick , Getts Dainel , Hofmeister Robert
Published: August 2018

T cells expressing chimeric antigen receptors (CARs) have demonstrated remarkable clinical benefit in certain hematological malignancies albeit with side effect such as cytokine release syndrome, on Target off tumor response and limited efficacy in solid tumors. Here, we present the preclinical evaluation of a novel T cell engineering platform designed to overcome potential hurdles of CARs and other T cell receptor modalities. Specifically, we have generated novel T Cell Receptor Fusion Constructs (TRuC™s) that fuse binder domains to subunits of the T cell receptor. These constructs when introduced using lentiviral technology, integrate into endogenous TCR, reprograming T cells to target tumor antigen in a non-major histocompatibility complex (MHC) restricted fashion and harnessing the full spectrum of TCR signaling. TRuC™ variants were constructed by recombinant fusion of an scFv or sdAb to various TCR subunits via a flexible linker sequence. Likewise, CD28ζ and 41BBζ CARs were generated using the same binders for side-by-side comparison. We demonstrated that TRuC™ variants can effectively reprogram T cells to recognize tumor surface antigens in a non-MHC-restricted fashion. TRuC™s are distinct from CARs in their ability to activate T cells through the entire TCR without additional costimulatory domains. In vitro, TRuC™ T cells were equally potent as CAR T cells in eliminating tumor cells. Compared to CAR-T cells, TRuC-T cells produced lower cytokine levels and proliferated less. Despite these difference, TRuC™ T cells were more efficient in clearing tumors in subcutaneous Raji and MSTO-211H mesothelioma models. Our findings support the development of TRuC-T cells for the treatment of solid tumors.

Citation Format: Ekta Patel, Jian Ding, Nikolaus Thorausch, Janani Krishnamurthy, Rashmi Choudhary, Solly Weiler, Bonnie Le, Patrick Tavares, Adam Zieba, Justin Quinn, Yan Wang, Wolfgang Schamel, Irene Scarfo, Marcela Maus, Patrick Baeuerle, Dainel Getts, Robert Hofmeister. Characterization of a novel class of engineered (TCR) fusion constructs (TRuCTMs) aimed to treat solid tumors [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 3584.
Abstract 3605: Next generation sequencing from cerebral spine fluid yields actionable targets in leptomeningeal carcinomatosis
Authors: Baumgarten Louisa von , Reischer Anna , Kumbrink Jörg , Jung Andreas , Liebmann Sibylle , Ormanns Steffen , Metzeler Klaus , Holch Julian , Heinemann Volker , Kirchner Thomas , Westphalen Benedikt
Published: August 2018

Background and Rationale: Leptomeningeal carcinomatosis is a cause of major morbidity and mortality in many solid malignancies. Therapeutic options are limited and include radiation therapy as well as intrathecal administration of cytotoxic agents. Accordingly, novel diagnostic and therapeutic modalities are needed in order to improve the prognosis of patients diagnosed with leptomeningeal carcinomatosis. Liquid biopsies are safe and offer great potential to define actionable targets in a variety of cancers. However, a role for liquid biopsies from cerebral spine fluid (CSF) has not been defined to date.

Patients and Methods: CSF samples from ten consecutive patients diagnosed with leptomeningeal carcinomatosis were collected as part of clinical routine and evaluated by next generation sequencing. All cases were discussed in the Molecular Tumorboard (MTB) as part of the “Molecular Diagnostics and Therapy” unit at the Comprehensive Cancer Center Munich.

Results: Samples from patients diagnosed with cholangiocarcinoma (1), ewing's sarcoma (1), breast (5), lung (2) and gastric cancer (1) were submitted for panel sequencing. Sequencing was technically successful in nine out of ten patients and circulating tumor DNA (ctDNA) was found in all of these nine patients. Of note, ctDNA was also detectable in cases in which microscopy did not reveal malignant cells in the CSF sample (4/10 cases). Tumorigenic alterations were found in seven out of these nine patients. Importantly, NGS of cerebral spine fluid detected previously unknown and therapeutically relevant alterations in this cohort. Case discussion in the Molecular Tumorboard led to therapeutic recommendations in three cases and targeted therapy was ultimately initiated in one case.

Conclusions: Next generation sequencing from cerebral spine fluid is feasible in clinical practice and yields tumorigenic alterations in a large fraction of patients. Importantly, our approach demonstrated the therapeutic relevance of this approach in patients with leptomeningeal carcinomatosis.

Citation Format: Louisa von Baumgarten, Anna Reischer, Jörg Kumbrink, Andreas Jung, Sibylle Liebmann, Steffen Ormanns, Klaus Metzeler, Julian Holch, Volker Heinemann, Thomas Kirchner, Benedikt Westphalen. Next generation sequencing from cerebral spine fluid yields actionable targets in leptomeningeal carcinomatosis [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 3605.
Abstract 3608: Molecular fingerprint sequencing for minimal residual disease detection in breast cancer
Authors: Comino-Mendez Iñaki , Cutts Ros , García-Murillas Isaac , Chopra Neha , Afentakis Maria , Evans Abigail , Wheatley Duncan , Skene Anthony , Russell Simon , Varughese Mohini , Dowsett Mitch , Smith Ian E. , Turner Nick
Published: August 2018

Introduction: The substantial majority of breast cancers present with early-stage disease, although micro-metastatic disease may be established at the time of diagnosis and may ultimately result in metastatic disease recurrence. Prior studies have shown that analysis of circulating tumour DNA (ctDNA) can detect micro-scopic minimal residual disease (MRD) serving as biomarker to anticipate future cancer recurrence, although sensitivity of current assays is limited. We present a novel molecular fingerprint ctDNA assay that exploits tumour whole genome sequencing (WGS) to develop highly personalised assays.

Methods: WGS was conducted on paired tumor and germline DNA from 11 early breast cancer patients to identify tumour specific small indels (SIs) to track in plasma as individual molecular fingerprints. Patient-specific amplicon-based panels were designed and validated to identify somatic reporters with no sequencing background. DNA was extracted from plasma samples taken at pre-surgery (baseline), post-surgery, and every 3 months until 1 year and every 6 months thereafter, and sequenced to 100,000X.

Results: Molecular fingerprint assays were demonstrated to detect less than a single cancer genome, with undetectable background in control samples. Using plasma samples from patients with early stage breast cancer, we detected ctDNA in 80% of the baseline plasma time-points with 100% specificity (95% confidence limits (CI) 98.7%-100%). ctDNA was detected in all patients prior to relapse, with an observed lead-time of 15.5 months, substantially improved compared with previous techniques, detecting residual disease that was not detectable by tracking mutations using digital PCR.

Conclusions: This proof-of-principle study demonstrates that tumor-specific molecular fingerprints combined with ultra-deep plasma DNA sequencing have the potential to transform MRD detection. Identifying those patients who have undetectable recurrence would pave the way for identifying who is cured by surgery alone and who requires further therapy.

Citation Format: Iñaki Comino-Mendez, Ros Cutts, Isaac García-Murillas, Neha Chopra, Maria Afentakis, Abigail Evans, Duncan Wheatley, Anthony Skene, Simon Russell, Mohini Varughese, Mitch Dowsett, Ian E. Smith, Nick Turner. Molecular fingerprint sequencing for minimal residual disease detection in breast cancer [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 3608.
Abstract 3612: Elevated TGFa levels in chronic mylogenous leukemia (CML) patient plasma measured by a highly sensitive single molecule array immunoassay
Authors: Chenna Ahmed , Cai Li , Petropoulos Christos , Winslow John W.
Published: August 2018

Introduction: Recent reports suggest that cytokine and growth factor profiling are prognostic in myelodisplasias and CML. TGF-α and IL-6 plasma levels selectively identified CML patients failing to achieve an early molecular response (EMR), or progressed on imatinib therapy (Nievergall et al, 2016). In patients failing to achieve EMR, the median TGFα concentrations were in the low pg/mL range while IL-6 levels were sub-pg/mL or undetectable. Thus, to accurately and reproducibly measure TGFα and IL-6 levels in blood as prognostic or predictive markers of imatinib response, there is a need for highly sensitive immunoassays. Here we demonstrate the utility of using a digital immunoassay to measure sub-pg/mL levels of TGFα and IL-6 in CML plasma samples assessed for BCR-ABL fusion transcripts.

Methods: Single molecule array immunoassays (Simoa) for TGFα and IL-6 were performed on the HD-1 platform (Quanterix), qualified based on precision, reproducibility, linearity, sensitivity according to kit specifications. CML blood samples were obtained from CMBP, LabCorp, Inc, following BCR-ABL e1b2, b2b2, and b3b2 transcript fusion testing by RT-PCR.

Results: TGFα and IL-6 concentrations in the plasma of 77 CML patients were determined and compared to levels in healthy controls. TGFα in CML plasma was elevated and ranged ~20-fold (6.13-118.6 pg/mL), in contrast to the low levels and narrow range in healthy controls (range = 1.11-3.17 pg/mL). Median TGFα levels were 10-fold higher in CML patient plasma (median = 18.8 pg/mL) relative to healthy controls (median = 1.8 pg/mL; p <0.0001). IL-6 levels ranged broadly in CML plasma (0.12-12.6 pg/mL) relative to healthy controls (0.3-1.04pg/mL), though median levels were not significantly different (CML = 0.61 vs 0.69, p = 0.873). TGFα and IL-6 concentrations were not significantly correlated. TGFα levels appear elevated in CML samples expressing all 3 BCR-ABL transcripts, however, a larger number of samples enriched in fusion transcripts are being assessed to confirm possible correlations.

Conclusions: We have qualified novel highly sensitive single molecule array immunoassays to measure TGFα and IL-6 in the plasma of CML patients and healthy controls, and found significantly elevated levels of TGFα in CML plasma. These results support further investigation of TGFα and IL-6, using sensitive immunoassays, as potential prognostic and predictive markers of CML disease progression and treatment outcome.

Citation Format: Ahmed Chenna, Li Cai, Christos Petropoulos, John W. Winslow. Elevated TGFa levels in chronic mylogenous leukemia (CML) patient plasma measured by a highly sensitive single molecule array immunoassay [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 3612.
Abstract 3625: MET signaling upregulates PD-L1 expression in human lung and gastric cancer
Authors: Ahn Hyunkyung , Kim Sehui , Jang Ji Young , Kwon Dohee , Koh Jaemoon , Kim Young A , Chung Doo Hyung , Jeon Yoon Kyung
Published: August 2018

Background: Tyrosine kinase receptor, MET, plays an important role for the progression of tumor through diverse mechanisms. Cancer immunotherapy targeting PD-1/PD-L1 pathway shows clinical benefit in patient with PD-L1 expressing solid tumor. In our previous study on non-small cell lung cancer (NSCLC) using immunohistochemistry, MET overexpression was significantly associated with programmed cell death-ligand 1 (PD-L1) overexpression. Thus, we investigated if MET signaling pathway is involved in PD-L1 expression in tumor cells.

Method and Result: Lung adenocarcinoma cell lines including H596 (harboring MET exon 14 skipping mutation), H1993 (harboring MET gene amplification), H23 and H522 (MET wild-type), and a gastric carcinoma cell line, Hs746T (harboring MET exon 14 skipping mutation and gene amplification) were used. Hs746T cells were treated with MET inhibitor or transfected with MET siRNA and subject to microarray analysis. MET inhibition or knockdown led to changes in expression level of variable immune-related molecules, of which PD-L1 was one of the most down-regulated molecules. Stimulation of H596, H23 and H522 cells with HGF, a MET ligand, increased PD-L1 expression at the mRNA and protein level. Treatment of H1993 and Hs746T cells with MET inhibitor or MET knockdown using siRNA resulted in down-regulation of PD-L1 at the mRNA, total protein, and surface expression levels. PI3K/Akt and MAPK/Erk signaling pathways were involved in MET-mediated PD-L1 up-regulation in H1993 and Hs746T cells. Finally, the Cancer Genome Atlas (TCGA) analysis showed that MET and PD-L1 expression are significantly positively correlates with each other in patients with lung cancer (in NSCLC, rho=0.219 [p<0.001]; in adenocarcinoma, r=0.401 [p<0.001]; in squamous cell carcinoma, r=0.144 [p=0.001]) and in patients with gastric cancer (r=0.137 [p=0.005]).

Conclusion: This study demonstrates that MET signaling increases PD-L1 transcription and protein overexpression in lung and gastric cancer cells, which might contribute to immune escape of tumor via PD-1/PD-L1 pathway. MET-targeted therapy may be considered as combination therapy with PD-1/PD-L1 blockades.

Citation Format: Hyunkyung Ahn, Sehui Kim, Ji Young Jang, Dohee Kwon, Jaemoon Koh, Young A Kim, Doo Hyung Chung, Yoon Kyung Jeon. MET signaling upregulates PD-L1 expression in human lung and gastric cancer [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 3625.
Abstract 3630: Deep and durable response with dual anti-CTLA-4 and PD-1 blockade in mismatch repair (MMR) proficient endometrial cancer
Authors: Oh Michael S. , Chae Young
Published: August 2018

Immune checkpoint inhibitors have shown promising efficacy in multiple cancer types, but have yet to be extensively validated in endometrial cancer. However, the recent FDA approval of PD-1 inhibitors for mismatch repair (MMR) deficient tumors has extended use of these treatments to all cancer types. PD-L1 positivity in tumor tissue has also been shown to predict susceptibility to immunotherapy and has been used to guide use of these treatments. Despite these advances, the response to immunotherapy in endometrial cancer remains poorly understood. Here, we describe the case of a patient with MMR proficient, PD-L1-negative stage IV endometrial cancer with positive response to combination PD-1 and CTLA-4 inhibition. The patient initially presented with ascites and elevated tumor markers, and cytology from peritoneal fluid showed endometrial adenocarcinoma with squamous and mucinous differentiation and clear cell features. She underwent surgical debulking with both neoadjuvant and adjuvant cycles of docetaxel and carboplatin, but unfortunately had progressive metastatic disease within 4 months. The patient declined further chemotherapeutic agents, and thus was instead trialed on combination nivolumab and ipilimumab despite her cancer exhibiting MMR proficiency and negative PD-L1. She demonstrated deep and durable on-going partial response to this treatment that has persisted still after 10 months. Her course has notably been complicated by hypophysitis that manifested after 5 months of treatment as worsening fatigue, but she has been able to continue with her treatment regimen. This case indicates the potential existence of an endometrial cancer subtype that is sensitive to immune checkpoint blockade, but based on mechanisms other than those driven by MMR deficiency or PD-L1 positivity. Improved understanding of immunotherapy in advanced endometrial cancer is clearly needed and offers the potential to significantly enhance patient outcomes.

Citation Format: Michael S. Oh, Young Chae. Deep and durable response with dual anti-CTLA-4 and PD-1 blockade in mismatch repair (MMR) proficient endometrial cancer [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 3630.
Abstract 3660: Can the presence of fusion genes in circulating cell-free DNA serve as potential biomarkers for Ewing's sarcoma
Authors: Iwata Shintaro , Kageyama Hajime , Yonemoto Tsukasa , Itami Makiko
Published: August 2018

Background: Circulating cell-free DNA (cfDNA) is fragmented DNA derived from tumors that circulates in the blood of patients. With liquid biopsy, cfDNA has the potential to be used to monitor tumor burden in real time. Most reports have described that tumor-specific alterations in cfDNA are mutations, although little has been reported for the detection of tumor specific gene fusions. The objective of this study was to identify whether cfDNA can be used to detect tumor specific gene fusions and whether this would reflect the tumor burden of patients with Ewing's sarcoma.

Patients and Methods: Multiplex long-range genomic PCR was performed to detect patient-specific genomic breakpoints in eight patients. Probes for digital PCR were designed based on the genomic breakpoint of each patient. cfDNA was extracted from the serum of patients, which was drawn at multiple time-points (pretreatment, completion of the treatment, and/or relapse). cfDNA quantification was performed by digital PCR (BioRad Qx-200).

Results: Gene fusions in the cfDNA were detected in six (75%) of eight patients with high specificity. The relative gene fusion copy ratio (gene fusion copy number/wild KRAS copy number) was higher in the patients with M1b (extrapulmonary metastases at diagnosis) compared to the patients with M1a (pulmonary metastases at diagnosis) or M0 (no metastasis), although no significant relationship was observed between the relative gene fusion copy ratio and chemosensitivity. In each patient, the relative gene fusion copy ratio was the highest in pretreatment samples and decreased as the treatment progressed. Patients with relapse displayed an elevated relative gene fusion copy ratio in advance of clinical manifestation or elevation of lactase dehydrogenase.

Conclusion: The amount of cfDNA in the serum strongly correlated with the treatment stage and would be useful as an early biomarker for Ewing's sarcoma patients.

Citation Format: Shintaro Iwata, Hajime Kageyama, Tsukasa Yonemoto, Makiko Itami. Can the presence of fusion genes in circulating cell-free DNA serve as potential biomarkers for Ewing's sarcoma [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 3660.
Abstract 3661: Comparison of ctDNA markers in matched urine and plasma from patients with liver cancer
Authors: Lee Tai-Jung , Jain Surbhi , Steffen Jamin D. , Zhang Adam M. , Hu Chi-Tan , Hamilton James P. , Su Ying-Hsiu
Published: August 2018

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer worldwide and a leading cause of global cancer mortality. Lack of efficient early detection and limited therapeutic options are two key contributing factors for the dismal prognosis of this disease. We have previously shown that urine contains fragmented, circulation-derived cell-free DNA that can be used for detection of circulating tumor DNA (ctDNA) if a tumor is present, suggesting urine as an alternative body fluid to plasma for cancer liquid biopsy. This study compared the detectability of five known HCC-associated DNA modifications, including genetic mutations (TERT -124 G>A, TP53 249 G>T and CTNNB1 hotspot at codon 32-45) and epigenetic methylation (mGSTP1, mRASSF1A), in matched urine and plasma from patients with liver cancer. In order to increase the sensitivity of detecting DNA modifications from the short, fragmented DNA templates, specialized short amplicon PCR assays were developed. To compare the detectability of HCC ctDNA markers in urine and plasma, 26 pairs of urine and plasma samples from HCC patients were identified and tested for five HCC ctDNA markers. In this cohort, 17 of 26 (65%) HCC samples had serum AFP above 20 ng/mL and were considered AFP-positive HCC. By using our HCC DNA biomarker panel composed of 5 assays, 22/26 (84.6%) urine and 23/26 (88.5%) plasma samples were found to have at least one DNA marker detected. In the combination of our HCC DNA biomarker panel with serum-AFP, the sensitivity of HCC detection in a liquid biopsy of urine and plasma as compared to serum-AFP alone (the current most used screening marker) increased from 65% to 92.3% for both urine plus AFP and plasma plus AFP. Further studies to include non-HCC samples (such as cirrhosis) to determine the specificity of the HCC DNA biomarker panel for liquid biopsy are in progress. In conclusion, our research provides the promising potential for HCC liquid biopsy screening in at-risk populations and precision medicine by using plasma, urine, or both as body fluids.

Citation Format: Tai-Jung Lee, Surbhi Jain, Jamin D. Steffen, Adam M. Zhang, Chi-Tan Hu, James P. Hamilton, Ying-Hsiu Su. Comparison of ctDNA markers in matched urine and plasma from patients with liver cancer [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 3661.