bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 357: Phosphorylated histone H3 mediates epigenetic regulation of Brf1 and Pol III genes in alcohol-associated breast cancer
Authors: Lu Lei , Fang Zeng , Li Wen , He Zhimin , Zhong Shuping
Published: August 2018

Approximately 3.6% human cancers worldwide derive from chronic alcohol drinking, including cancers of breast and multiple other organs. Alcohol intake is consistently associated with an increased risk of BC (breast cancer). Alcohol consumption is more pronounced in ER+ BC cases than in ER- cases. However, the molecular mechanism remains to be determined. RNA polymerase (Pol) III genes, such as 5S rRNA and tRNAs, are elevated in both transformed and tumor cells. Brf1 (TFIIIB-related factor 1), a subunit of TFIIIB complex, specifically regulates Pol III gene transcription. Our studies have demonstrated that Brf1 expression and Pol III gene transcription are increased in alcohol-induced mice liver tumor. Alcohol-induced deregulation of Pol III gene transcription may be fundamental to the development of BC. We have found that alcohol-increased Pol III gene transcription in ER+ BC cells is significantly higher than in ER- normal breast cells and BC cells. The induction is ER dependent. The cellular levels of Brf1 protein and mRNA are increased in ethanol-treated MCF-7 cells. Ethanol markedly stimulates phosphorylation of MSK1 (MSK1ph) and histone H3 (H3ph). Our previous studies demonstrated that MSK1 mediates H3ph. MSK1 mediates ERα expression. Inhibition of MSK1 signal reduces alcohol-induced ERα expression to decrease cellular level of Brf1 and Pol III genes. Repression of Brf1 expression decreases alcohol-induced anchorage-independent cell growth and tumor formation. Furthermore, our analysis indicates that ethanol treatment increases occupancy of ERα to Brf1 promoter. The results by using Co-IP and ChIP assays indicate that MSK1, ERα and Brf1 form a complex to mediate Pol III gene transcription. Studies of human biopsies of BC patients indicate that Brf1 expression is increased in human cases of BC. High expression of Brf1 displays longer overall survival period after Tamoxifen treatment. Co-localization analysis indicate that MSK1ph and H3S10ph localizes in nuclear with Brf1. These results support the idea that alcohol activates MSK1 and induces epigenetic modification of H3ph to enhance ERα and Brf1 expression and Pol III gene transcription, resulting greater phenotypic changes. Together, our studies indicate that ethanol-induced epigenetic modification of H3ph may play a critical role in alcohol-induced cell transformation and alcohol-associated ER+ BC. The project is supported by NIH grants: AA017288, AA021114, AA02324 and AA024169

Citation Format: Lei Lu, Zeng Fang, Wen Li, Zhimin He, Shuping Zhong. Phosphorylated histone H3 mediates epigenetic regulation of Brf1 and Pol III genes in alcohol-associated 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 357.
Abstract 3573: Novel DNA vectors encoding a chimeric antigen receptor mediate long term expression without genomic integration
Authors: Schmidt Patrick , Bozza Matthias , Berger Aileen , Luckner-Minden Claudia , Tuch Alexandra , Zörnig Inka , Jäger Dirk , Harbottle Richard
Published: August 2018

Adoptive immunotherapy is one of the most encouraging therapeutic strategies for the treatment of a range of cancers. One particularly promising avenue of research is the functional introduction of Chimeric Antigen Receptors (CARs) into naive Human T-Cells for autologous-immunotherapy. Currently, the genetic engineering of these cells is achieved through the use of proprietary integrating vector systems such as lentiviruses or the sleeping beauty transposon system which present a risk of genotoxicity associated with their random genomic integration.

We have invented a novel DNA Vector platform for the safe and efficient generation of genetically engineered T-Cells for Human Immunotherapy. This DNA vector system contains no viral components and comprises only clinically approved sequences, it does not integrate into the target cell's genome but it can replicate autonomously and extrachromosomally in the nucleus of dividing human primary cells. These DNA Vectors offer several advantages over currently used vector systems; they are not subject to commercial licences, they are cheaper and easier to produce, and they can more quickly genetically modify human cells without the inherent risk of integrative mutagenesis.

In preclinical experiments we have successfully generated genetically engineered human T-Cells expressing the CAR receptor against several epitopes and have demonstrated their viability and capability in targeting and killing human cancer cells which express these epitopes. The long term anti-tumor activity of our DNA-CAR-T cells has been confirmed in vivo using xenotransplanted cell lines in immunodeficient mice.

We believe that this novel DNA Vector system provides a unique and innovative approach to this exciting therapeutic strategy for cancer therapy. We estimate that this novel methodology will provide a simpler method of CAR T-cell manufacturing, resulting in a 10-fold reduction in the cost of the CART-product.

Citation Format: Patrick Schmidt, Matthias Bozza, Aileen Berger, Claudia Luckner-Minden, Alexandra Tuch, Inka Zörnig, Dirk Jäger, Richard Harbottle. Novel DNA vectors encoding a chimeric antigen receptor mediate long term expression without genomic integration [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 3573.
Abstract 3579: RegCAR-TTM: Enabling next-generation CAR-T therapy
Authors: Zhang Xu , Tang Ke , Song Qingxuan , Chen Si , Kasbek christopher , Wang Tianjiao , Huang Jun
Published: August 2018

Chimeric Antigen Receptor (CAR) T-Cell Therapy has been emerging as a means of powerful cancer immunotherapy with its exceptional therapeutic efficacy, however, the full potential of CAR-T therapy is hampered by its severe side effects. Thus, CAR-T therapy that is controllable for optimal efficacy and safety is in need developing. To enable controllable CAR-T therapy, we have established a technology platform, RegCAR-TTM. In RegCAR-TTM, aptazymes are generated by in vitro selection/evolution of combinatory nucleic acid libraries and are used as molecular switches for controlling CAR expression. Aptazymes are RNA molecules consisting of an aptamer module and a ribozyme module, in which ribozyme self-cleavage can be allosterically regulated by the aptamer-ligand (e.g. a small molecule drug) interaction. With an aptazyme switch incorporated into the untranslated region (UTR) of a CAR gene, CAR expression can be temporally and dose-dependently controlled at mRNA level by a ligand, which may result in controllability of CAR-T efficacy and safety. Using RegCAR-TTM technology, we have generated aptazyme switches responsive to FDA-approved small molecules drugs with IC50 at micromolar level. Thus, next generation CAR-T therapies with aptazyme switches would be controllable by safe oral small molecules drugs. In conclusion, we have developed a technology platform (RegCAR-TTM) to generate aptazyme switches for enabling controllable CAR-T therapy.

Citation Format: Xu Zhang, Ke Tang, Qingxuan Song, Si Chen, christopher Kasbek, Tianjiao Wang, Jun Huang. RegCAR-TTM: Enabling next-generation CAR-T therapy [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 3579.
Abstract 358: miRNAs/Ago2/YY1/PcG protein/DNMT complex mediates epigenetic silencing of locus genes and associates with bortezomib-induced apoptosis of leukemia
Authors: Wang Ju-Ming , Chu Yu-Yi , Ko Chiung-Yuan , Wang Shao-Ming , Lin Pin-I , Wang Han-Ying , Lin Wen-Chi , Wu Dong-Yu
Published: August 2018

miRNAs are a class of non-coding RNAs, which bind to the 3'-untranslated regions of their target mRNAs to repress translation. However, the details of miRNA-mediated epigenetic regulation targeting genomic DNA remained largely unknown. Here, we demonstrated that transcription factor CCAAT/enhancer binding protein delta (CEBPD) was responsive to the anticancer drug bortezomib, is a highly selective reversible inhibitor for leukemia treatment, and resulting in caspase activation and apoptosis. Interestingly, following the identification of CEBPD-induced miRNAs, we found that miR-744, miR-3154 and miR-3162 feedback to suppress the transcription of CEBPD itself and genes adjacent to its 5'-flanking region, including protein kinase DNA-activated catalytic polypeptide (PRKDC), minichromosome maintenance-deficient 4 (MCM4) and ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), upon bortezomib treatment. Additionally, we previously demonstrated that the Yin Yang 1 (YY1)/polycomb group (PcG) protein/DNA methyltransferase (DNMT) complex is important for mediating epigenetic silencing of the CEBPD gene; we further found that Argonaute 2 (Ago2) interacts with YY1 and binds to the CEBPD promoter. Moreover, we revealed that miRNA binding plays a key role for YY1/PcG group protein/DNMT complex-mediated epigenetic gene silencing and is associated with bortezomib-induced methylation on genomic DNA. Altogether, we provided new insights for miRNA-mediated epigenetic regulation in the case of bortezomib-induced cell death in leukemia.

Citation Format: Ju-Ming Wang, Yu-Yi Chu, Chiung-Yuan Ko, Shao-Ming Wang, Pin-I Lin, Han-Ying Wang, Wen-Chi Lin, Dong-Yu Wu. miRNAs/Ago2/YY1/PcG protein/DNMT complex mediates epigenetic silencing of locus genes and associates with bortezomib-induced apoptosis of leukemia [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 358.
Abstract 3581: Convergence of the specificity and cross-reactivity of multiple T cell receptors isolated against a single cancer peptide/HLA complex
Authors: Sharma Preeti , Harris Daniel T. , Hager Marlies V. , Kranz David M.
Published: August 2018

Adoptive T cell therapy using T cell receptors (TCRs) or chimeric antigen receptors (CARs) requires careful selection of the receptor so as to target only the specific cancer antigen, without cross-reactivity toward structurally similar antigens. In this study, we used two TCRs (T1 and RD1) that bind to the HLA-A2 restricted melanoma antigen MART-1 (ELAGIGILTV). TCR-T1 was initially isolated from a melanoma-reactive T cell line specific for MART-1 whereas TCR-RD1 was derived completely by in vitro engineering, switching the specificity of the well-studied A6 TCR from its cognate viral peptide (Tax) to MART-1 by directed evolution. Despite their distinct origins, engineered high-affinity forms of both TCRs had remarkably similar fine-specificities as determined using a panel of MART-1 variant peptides. This binding signature allowed us to conduct a bioinformatic scan of the human and mouse proteomes for peptides that were homologous to MART-1. Our analysis resulted in identification of a self-peptide (TM74B: IIAGLGILTV) that also bound to both T1 and RD1 TCRs. T1-transduced T cells (CD4+) from HLA-A2/Db transgenic (AAD) mice were potently activated by MART-1, and cross-reacted (at 1000-fold higher doses) with the TM74B peptide. CD8+ T cells transduced with the T1 TCR were activated by AAD+ antigen presenting cells, indicating recognition of an endogenous peptide, possibly TM74B. To extend these studies, we have shown that T1-transduced AAD T cells (CD4+) are also stimulated by murine MART-1 (EAAGIGILIV) and that transfer of T1-transduced T cells into AAD mice did not result in overt toxicity. Thus, the AAD mouse could be used to study efficacy and toxicity of T1 TCR in syngeneic models. We are currently developing a B16/AAD tumor model to compare recognition of B16 melanoma by MART-1 directed TCRs and CARs. In summary, the convergence of specificity of T1 and RD1 suggests that TCRs against a cancer peptide antigen are focused on the same exposed features of the peptide. Furthermore, the results with RD1 suggest that it is possible to engineer TCRs completely in vitro, and that these TCRs can recapitulate the fine-specificities of conventional TCRs isolated from T cell clones.

Citation Format: Preeti Sharma, Daniel T. Harris, Marlies V. Hager, David M. Kranz. Convergence of the specificity and cross-reactivity of multiple T cell receptors isolated against a single cancer peptide/HLA complex [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 3581.
Abstract 359: IWS1 is essential for mouse development
Authors: Orlacchio Arturo , Foray Claudia , Sheetz Tyler , Reese Erika , Amari Foued , Stark Aaron E. , Perle Krista La , Sanidas Ioannis , Tsichlis Philip N. , Palmieri Dario , Coppola Vincenzo
Published: August 2018

Iws1 (Interacts with Spt6) was originally identified in Saccharomyces Cerevisiae as a binding partner of Spt6, a histone H3/H4 chaperone.As a transcription factor, Iws1 plays a key role in defining the composition of the RNA polymerase II (RNAPII) elongation complex and in modulating the production of mature mRNA transcripts.Iws1 is phosphorylated, following stimulation with growth factors, by Akt3 and Akt1 at Ser720/Thr721 in human cells (Ser667/Thr668 in the mouse).Preliminary evidence supports a critical protumorigenic role of Iws1 and its phosphorylation in several types of malignancies such as lung cancer (NSCLC), mammary adenocarcinomas and melanomas.For example, it has been shown that, while IWS1 expression in normal human lung is very low, dramatically increases in many NSCLCs. Interestingly the level of IWS1 phosphorylation is variable between tumors and correlates with Akt3 expression.Moreover data from the TCGA database show that Iws1 is expressed at higher levels in tumors than in normal mammary gland specimens and also that basal and HER-2-enriched tumors express the highest levels. The genetic depletion of Iws1 in human mammary adenocarcinoma cell lines inhibits anchorage-independent growth in soft agar and mammosphere formation and growth. Similar experiment revealed that human melanoma cells were extraordinarily sensitive to the knockdown of Iws1. These experiments identify mammary adenocarcinomas and melanomas as tumors potentially addicted to Iws1. However, Iws1 biological functions are poorly characterized and there is no reported in vivo investigation about this RNA-processing factor.To begin to shed some light on the function of this protein at organismal level, we performed a thorough analysis of the expression of Iws1 and generated Iws1-deficient mice.The systematic analysis of expression in mouse tissues shows that Iws1 is ubiquitously expressed. We also report that ablation of Iws1 causes lethality at very early stages of mouse development. In fact, Iws1-deficient mouse embryos die at pre-implantation stage. Overall, these results show that Iws1 is an essential gene for mouse development and suggest a critical function in mammary adenocarcinomas as well as in melanoma.

Citation Format: Arturo Orlacchio, Claudia Foray, Tyler Sheetz, Erika Reese, Foued Amari, Aaron E. Stark, Krista La Perle, Ioannis Sanidas, Philip N. Tsichlis, Dario Palmieri, Vincenzo Coppola. IWS1 is essential for mouse development [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 359.
Abstract 3594: DEK overexpression in breast epithelial tissue creates a pro-oncogenic environment
Authors: Johnstone Megan , Cheek Jon , Vinnedge Lisa M. Privette
Published: August 2018

Dense epithelial breast tissue, as detected by mammography, is correlated with a four- to six-fold increased risk for developing breast cancer (BC). It is currently not possible to distinguish dense breast tissue from that which will develop into cancer. Therefore, the need to identify novel markers will improve breast cancer screening and reduce expensive overdiagnosis and overtreatment. Previous research has failed to define reliable diagnostic biomarkers that specify dense breast tissue from that which has the possibility to transform into malignant tissue. DEK, a chromatin binding and DNA repair protein, is upregulated in the majority of solid cancers including breast cancer. Previous publications from our lab demonstrate, in vitro, that DEK overexpression promotes proliferation, increased motility, and epithelial polarization in part through canonical WNT/β-catenin signaling. In vivo experiments with a DEK knockout murine model display a mitigated tumor development response as compared to wild-type mice. Given that DEK is highly expressed in over 60% of BC, we generated a conditional mammary epithelial specific doxycycline-regulated Dek overexpressing (DEK OE) murine model. We find that DEK OE leads to dense epithelial breast tissue, largely due to lobuloalveolar hyperplasia. Longitudinal findings demonstrate that DEK OE results in transformation of mammary epithelial tissue with microtumor development. DEK OE tissue sections display increased transcription of Wnt genes, with upregulation of downstream WNT signaling targets such as AXIN and β-catenin. Conversely, longitudinal repression of DEK OE with doxycycline displays normal tissue morphology and organization. Our data suggest that DEK OE stimulates hyperplasia of mammary epithelium, due to increased proliferation, similar to dense breast tissue as detected by mammography, and may increase risk for spontaneous transformation and BC development later in life.

Citation Format: Megan Johnstone, Jon Cheek, Lisa M. Privette Vinnedge. DEK overexpression in breast epithelial tissue creates a pro-oncogenic environment [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 3594.
Abstract 3602: Validation and implementation of next-generation sequencing in liquid biopsies by a novel NGS platform: Focus on non-small cell lung cancer
Authors: Bisgin Atil , Sonmezler Ozge , Boga Ibrahim
Published: August 2018

Introduction: Liquid biopsy as a novel and non-invasive test for the surveillance of cancer is rapidly growing, by next-generation sequencing (NGS) technologies with multi-gene panels.

Objective: The aim of this study was to describe the validation and implementation of a cell free tumor DNA (cfDNA) from the patients with non-small cell lung cancer (NSCLC), to sequence for 19 genes and copy number variation analyses for 5 genes via a novel NGS platform; GeneReader NGS System.

Materials and Methods: We performed the complete NGS workflow with modifications, and sequenced the cfDNA extracted from 100 NSCLC patients. The multi-gene panel includes 19 lung cancer related genes (AKT1, ALK, BRAF, DDR, ERBB2, ESR1, KIT, KRAS, MAP2K1, NRAS, NTRK, PDGFRA, PIK3CA, PTEN and ROS) to sequence and additional copy number variations of 5 genes (RICTOR, EGFR, MET, FGFR1 and ERBB2) that all were selected to represent the most actionable genes for the clinical oncology and molecular diagnostics. The sequencing was performed using a novel NGS platform (GeneReader NGS System, Qiagen).

Results: The validation process and the implementation of genetic testing from the cfDNA by this novel NGS system have been utilized for the clinical use. After quality assessment, the data from this NGS system was bioinformatically processed and a tertiary analysis was performed using QCI-Analyze and QCI-Interpret bioinformatics tools. The data was then reviewed on alterations and those reported in different mutation databases. The clinically relevant alterations were identified and reported due to the testing indications. Overall, actionable variants in EGFR, ALK, KRAS, PIK3CA, MET, FGFR1 and ERBB2 genes were detected and reported in 62 % of all NSCLC patients while the majority were in EGFR.

Conclusions: For each of the 100 samples tested thus far, at least one variant was detected showing the high sensitivity and specificity of both the multi-gene panel and this novel NGS platform. Our evaluation yielded good results for the actionable multi-gene panel selected and provides a comprehensive NGS analysis and workflow for this new platform.

Citation Format: Atil Bisgin, Ozge Sonmezler, Ibrahim Boga. Validation and implementation of next-generation sequencing in liquid biopsies by a novel NGS platform: Focus on non-small cell lung 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 3602.
Abstract 3623: The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation
Authors: Veitonmäki Niina , Thagesson Mia , Werchau Doreen , Hägerbrand Karin , Smedenfors Kristine , Månsson-Kvarnhammar Anne , Rosén Anna , Johansson Maria , Furebring Christina , Norlén Per , Ellmark Peter
Published: August 2018

ATOR-1015 is a CTLA-4 x OX40 bispecific immune activating antibody developed for tumor-directed immunotherapy. ATOR-1015 binds both targets simultaneously, promoting cell-cell interactions expected to enhance the immuno-stimulating effect of the compound. The mode of action of ATOR-1015 is thought to be a combination of regulatory T cell (Treg) depletion and effector T cell activation. It can be seen as a next generation CTLA-4 antibody with tumor-directed activity and augmented Treg depletion. The ability to induce ADCC of human Treg was investigated using an FcγR expressing reporter assay demonstrating superior effect of ATOR-1015 compared to the monospecific antibody counterparts. Further, ATOR-1015 has been shown to induce activation of T cells in the presence of CTLA-4 expressing cells, ability that is not observed when combining the monoclonal counterparts. Syngeneic tumor models in vivo using human transgenic mice cross-reacting with both targets demonstrate that ATOR-1015 reduces tumor growth and prolongs survival. Further, ATOR-1015 treatment demonstrates superior increase in the intratumoral CD8+ T cell/Treg ratio compared to the monospecific counterparts, without affecting systemic T cells. This tumor directed immune activation is demonstrated to be due to the tumor localization abilities of ATOR-1015. In conclusion, ATOR-1015 is a next generation CTLA-4 antibody with tumor directed activity with augmented T-reg depletion. It is currently in GLP manufacturing of clinical material and will start clinical trials in the second half of 2018.

Citation Format: Niina Veitonmäki, Mia Thagesson, Doreen Werchau, Karin Hägerbrand, Kristine Smedenfors, Anne Månsson-Kvarnhammar, Anna Rosén, Maria Johansson, Christina Furebring, Per Norlén, Peter Ellmark. The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation [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 3623.
Abstract 3634: PET/CT imaging prediction of response to checkpoint blockade in advanced non-small cell lung cancer patients
Authors: Mu Wei , Qi Jin , Lu Hong , Schabath Mathew , Balagurunathan Yoganand , Tunali Ilke , Pilon-Thomas Shari , Gillies Robert J.
Published: August 2018

Immune checkpoint blockade with monoclonal antibodies directed at the inhibitory immune receptors PD-1 and PD-L1 has emerged as a one of the most successful treatment approaches in advanced Non-small Cell Lung Cancer (NSCLC) patients. However, given its adverse effects, high cost and only approximately 20% response rate, accurate response predication is import to identify patients most likely to respond. In previous work, we and others have shown that tumor acidosis, generated from high rates of glucose metabolism, was a potent inhibitor of anti-tumor immunity and that neutralization of acid improved response to checkpoint blockade. In this study, we investigated the value of pre-therapy PET/CT images on response prediction to test the hypothesis that the most metabolically active tumors would have the poorest response to checkpoint blockade. 64 patients were retrospectively curated with pathology verified NSCLC and who did not receive intervening treatments between the PET/CT imaging and initiation of anti-PD1 therapy. Responders (long-term and ongoing responders, n=22) and non-responders (non-responders and transient responders, n=42) were classified according to RECIST criteria. These patients were randomly divided into two groups (n=32 each) for training and testing, respectively. From their images, we constructed 5 different fusion images comprised of PET and CT data. We then extracted 455 quantitative features from these fusion images, as well as the original PET/CT images. We then generated an additional 975 fusion features calculated from the above 455 features, as well as some clinical features (location, age, sex, and histologic type). From these, the most informative features were selected with Receiver Operating Characteristic Curve (ROC) analysis, and these were analyzed with a combined 2-layer cascade support vector machine (cSVM) classification model to relate the features to prediction of objective response. From this we determined that the most informative features were fusion features. With the cSVM, the training set achieved accuracy and area under the ROC curve (AUROC) for classifying response of 100% and 1.00, respectively. Using this model on the test set achieved accuracy and AUROC of 89.66% and 0.86, respectively. We conclude from that PET/CT fusion images may be useful prior to initiation of therapy to predict response to checkpoint blockade in advanced non-small cell lung cancer patients.

Citation Format: Wei Mu, Jin Qi, Hong Lu, Mathew Schabath, Yoganand Balagurunathan, Ilke Tunali, Shari Pilon-Thomas, Robert J. Gillies. PET/CT imaging prediction of response to checkpoint blockade in advanced non-small cell lung cancer patients [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 3634.
Abstract 3636: The immune checkpoint, HVEM, is associated with PDL1 expression in lung cancer cell lines
Authors: Ren Shengxiang , Amar Nadav , YU Hui , Rivard Christopher , Ellison Kim , Rozeboom Leslie , Hirsch Fred R.
Published: August 2018

Background: Herpes Virus Entry Mediator (HVEM) is an important immune checkpoint in cancer recognition. HVEM expressed on tumor cell membranes activates immune cell signaling pathways leading to either inhibition of activity (BTLA) or activation of immune activity (LIGHT). This study is to investigate the prevalence of HVEM expression and its association with PDL1 expression in cell lines and patient tumor tissue.Methods: A TMA of 53 characterized NSCLC, 48 SCLC cell lines and 35 malignant tumors were evaluated for HVEM and PD-L1 expression. The IHC assay for HVEM was Dako Link48 autostainer using a polyclonal antibody from R&D Systems(AF356). PD-L1 IHC was performed on the Dako Link48 autostainer using the PD-L1 22C3 pharmDx kit. Scoring HVEM employed the H-score system while for PD-L1 the tumor proportion score (TPS) was used.Results: IHC HVEM staining was optimized and demonstrated membranous staining with little or no cytoplasmic or nuclear staining. HVEM expression in the NSCLC and SCLC cell lines and malignant tumor TMA revealed a positive H-score more than 1 was 45.3%, 58.3% and 82.9% respectively. In comparison, PDL1 TPS expression of more than 1% was in 59.2%, 16.67% and 45.71% in NSCLC, SCLC cell lines and malignant tumor respectively. A significant correlation of HVEM with PDL1 expression was found in the NSCLC cell lines (r=0.518, p<0.001), while there was no significant association of HVEM with PDL1 expression in the small cell line cohort (r=0.059, p=0.688). Conclusion: HVEM expression was found to be higher in SCLC cell lines as compared to NSCLC cell lines and substantially greater in the general malignant tumor tissues. HVEM expression is associated with PD-L1 expression in NSCLC cell lines but not in SCLC cell lines. This data suggests a potential benefit of combined PD-L1 and HVEM blockade in patients with NSCLC to improve the efficacy of immune-therapy.

Table 1. HVEM Expression in the studyH score01-100101-200201-300NSCLC Cell lines(n=53)29 (54.72%)23 (43.4%)1 (1.8%)0SCLC Cell lines(n=48)20 (41.67%)26 (54.17%)2 (4.17%)0Malignant TMA n=356 (17.14%)16 (45.71%)9 (25.71%)4 (11.43%)

Citation Format: Shengxiang Ren, Nadav Amar, Hui YU, Christopher Rivard, Kim Ellison, Leslie Rozeboom, Fred R. Hirsch. The immune checkpoint, HVEM, is associated with PDL1 expression in lung cancer cell lines [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 3636.
Abstract 3644: Blood tumor mutational burden (bTMB) from circulating tumor DNA (ctDNA) as a biomarker for both mutational status and tumor burden in non-small cell lung cancer (NSCLC)
Authors: Davis Andrew A. , Galvez Carlos , Pan Alan , Mohindra Nisha , Villaflor Victoria , Chae Young Kwang
Published: August 2018

Background:

Tissue tumor mutational burden (tTMB) has emerged as a viable biomarker for response to immune checkpoint blockade (ICB) in multiple histologies including NSCLC. Blood TMB (bTMB) is a novel summative measure of ctDNA genomic alterations in the peripheral blood. Few studies have examined the clinical utility and correlation of bTMB with tumor burden and survival.

Methods:

We identified 53 patients, retrospectively, who had paired blood (Guardant Health) and tissue (Foundation Medicine) next-generation sequencing testing. bTMB was determined by taking the number of reported mutations over the sequencing length of ctDNA. bTMB was calculated for research purposes and has not been approved for clinical use. From the cohort, 36 patients had baseline CT scans with measurable scores by RECIST version 1.1. Survival analyses by quartile were performed for the subset of patients (N=18 PFS, N=19 OS) treated with ICB.

Results:

bTMB was significantly correlated with baseline RECIST tumor burden estimations (p=0.010, Pearson Correlation, r=0.42, Table 1). There was a trend toward mutant allele frequency (MAF) of the highest clone in blood correlating with RECIST score (p=0.061). No significant correlation was found when comparing blood and tissue TMB scores in paired patients (p=0.70). In the subset of patients treated with ICB, higher bTMB was significantly associated with shorter PFS (HR 1.81 [1.03-3.18], p=0.038) and OS (HR 2.23 [1.09-4.58], p=0.029).

Conclusions:

This study demonstrated the feasibility and clinical utility of bTMB. Our findings may indicate that bTMB reflects tumor shedding into the blood as a surrogate for tumor burden and heterogeneity given the correlation with RECIST scores. In survival analyses, higher bTMB predicted shorter survival, which also reflects bTMB as a biomarker for tumor burden. Prospective studies are necessary to study bTMB in larger cohorts of patients treated with ICB.

Correlation CoefficientPRECIST-Blood TMB r = 0.424P = 0.010RECIST-Tissue TMB r = 0.238P = 0.198RECIST-MAFr = 0.316P = 0.061MAF-Blood TMB r = 0.182P = 0.289MAF-Tissue TMB r = 0.336P = 0.065Tissue TMB-Blood TMB r = 0.073P = 0.695

Citation Format: Andrew A. Davis, Carlos Galvez, Alan Pan, Nisha Mohindra, Victoria Villaflor, Young Kwang Chae. Blood tumor mutational burden (bTMB) from circulating tumor DNA (ctDNA) as a biomarker for both mutational status and tumor burden in non-small cell lung cancer (NSCLC) [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 3644.
Abstract 3665: Longitudinal monitoring of prostate cancer evolution by plasma genome sequencing
Authors: Ramesh Naveen , Sei Emi , Gao Ruli , Tsai Pei Ching , Logothetis Christopher , Zurita Amado J. , Navin Nicholas E.
Published: August 2018

Castration-resistant prostate cancer (CRPC) is associated with poor survival and frequent metastasis to the bone. Disease progression and evolution of therapy resistance have been challenging to study, in part due to the difficulty in collecting invasive core biopsy samples longitudinally over time. While liquid biopsy methods hold great potential for longitudinal monitoring, they are currently limited to targeted measurements of single genes, or small cancer gene panels. To address this challenge, we developed an unbiased whole-genome sequencing method called PEGASUS (Plasma Exome and Genome Analysis by Size-Selection and Unbiased Sequencing) that enables the detection of copy number aberrations and exome mutations from circulating-tumor DNA (ctDNA). We applied PEGASUS to 15 CRPC patients, which identified common driver mutations and copy number aberrations in plasma DNA including AR, MYC, RB1 and PTEN. We compared matched ctDNA and metastatic tumor samples in 8 CRPC patients, which showed a high correlation (r=0.79) of copy number profiles and moderate concordance of exome mutations (~56%), but also revealed a number of ctDNA and metastasis-specific mutations. We further applied PEGASUS to analyze longitudinal samples from 10 prostate cancer patients that were treated with chemotherapy or androgen inhibition to delineate clonal evolution in response to therapy. Clonal subpopulations and dynamics were inferred from mutation frequencies, revealing both chemoresistant and chemosensitive clones. These data showed that while a subset of point mutations were under positive and negative selection; most copy number aberrations remained stable and did not change in response to therapy. Our data show the technical feasibility of performing whole-genome and exome profiling of plasma DNA and pave the way for future clinical applications in biomarker discovery and informing clinical treatment decisions in prostate cancer patients.

Citation Format: Naveen Ramesh, Emi Sei, Ruli Gao, Pei Ching Tsai, Christopher Logothetis, Amado J. Zurita, Nicholas E. Navin. Longitudinal monitoring of prostate cancer evolution by plasma genome sequencing [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 3665.
Abstract 3666: Diagnostic application of novel ACE technology: Treatment response monitoring via quantification of cell-free DNA (cfDNA) in plasma from late-stage cancer patients
Authors: Kovelman Robert , Hinestrosa Juan Pablo , Ye Delia , Desmond Patrick , Nguyen Michelle , Turner Robert , Searson David , Uswatta Suren , Madsen James , Bodkin David , Bazhenova Lyudmila , Bahador Afshin , Krishnan Rajaram
Published: August 2018

We have developed a novel microfluidic technology that makes use of Alternating Current Electrokinetics (ACE) to isolate macromolecules such as high-molecular-weight DNA and protein complexes as well as nanoparticles, including cell-derived exosomes. As one of the first applications of this novel technology, we have developed procedures and assays for the isolation and quantification of cell-free DNA (cfDNA) in plasma. We show that this methodology allows for the rapid, robust, and highly quantitative assay of cfDNA in undiluted plasma. Analytical studies indicate that these ACE assays are sensitive, have a very broad dynamic range, and allow for highly effective calibration strategies, thereby enabling comparison of results over time and between laboratories. Importantly, we show correspondence between the results of ACE assays and those that employ traditional technologies such as PCR. We have applied this technology to the quantification of cfDNA in plasma from cancer patients, with an initial focus on stage III and IV non-small cell lung cancer (NSCLC). With a goal of enabling less invasive, more frequent, and lower-cost treatment response monitoring in cancer patients, we have conducted clinical studies showing that the results generated from ACE assays provide informative and useful findings related to disease progression and therapeutic alternatives. We are conducting laboratory and clinical studies oriented toward the development of this technology for in vitro diagnostic use.

Citation Format: Robert Kovelman, Juan Pablo Hinestrosa, Delia Ye, Patrick Desmond, Michelle Nguyen, Robert Turner, David Searson, Suren Uswatta, James Madsen, David Bodkin, Lyudmila Bazhenova, Afshin Bahador, Rajaram Krishnan. Diagnostic application of novel ACE technology: Treatment response monitoring via quantification of cell-free DNA (cfDNA) in plasma from late-stage cancer patients [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 3666.
Abstract 3682: Expression levels of genes in primary melanoma associated with clinically meaningful characteristics
Authors: Berwick Marianne , Gorlov Ivan P. , Orlow Irene , Ringelberg Carol , Ernstoff Marc , Parker Joel , Gerstenblith Meg , Thompson Cheryl , Hernando Eva , Busam Klaus , Lezcano Cecilia , Corrales Sergio , Leong Siok , Thomas Nancy E. , Amos Christopher I.
Published: August 2018

Melanoma survival appears to be improving; however, between 13-68% of individuals with melanoma diagnosed with Stages II-III ultimately die from their melanoma. Factors influencing melanoma survival include sex, age, clinical stage, lymph node involvement and several primary tumor characteristics, such as Breslow thickness, presence of infiltrating lymphocytes (TILs), mitotic rate and ulceration. Identification of genes whose expression in primary tumors is associated with these key tumor/patient characteristics can shed light on molecular mechanisms of melanoma survival. Here we show results from a gene expression analysis of 32 formalin-fixed, paraffin embedded (FFPE) primary melanomas with extensive clinical annotation. We extracted RNA, and measured the expression of 760 genes selected based on published evidence of association with melanoma initiation or outcome using NanoString. The Cancer Genome Atlas (TCGA) data on primary melanomas was used for validation of nominally significant associations. We identified 5 genes that were significantly associated with the presence of TILs in the combined analysis of both datasets after adjustment for multiple testing using the Benjamini-Hochberg false discovery rate method: IL1R2, PPL, PLA2G3, RASAL1, and SGK2. We also identified two genes significantly associated with lymph node status (PIK3CG and IL2RA), and two genes significantly associated with sex (KDM5C and KDM6A). We found that LEF1 was significantly associated with Breslow thickness and CCNA2 with mitosis. These results demonstrate that genetic expression in primary tumors is associated with clinically meaningful characteristics of cutaneous melanoma.

Citation Format: Marianne Berwick, Ivan P. Gorlov, Irene Orlow, Carol Ringelberg, Marc Ernstoff, Joel Parker, Meg Gerstenblith, Cheryl Thompson, Eva Hernando, Klaus Busam, Cecilia Lezcano, Sergio Corrales, Siok Leong, Nancy E. Thomas, Christopher I. Amos. Expression levels of genes in primary melanoma associated with clinically meaningful characteristics [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 3682.
Abstract 3685: The BET inhibitor INCB057643 suppresses ALDH activity by targeting the ALDH1A1 super-enhancer in high-grade serous ovarian cancer
Authors: Karakashev Sergey
Published: August 2018

The emergence of tumor cells with certain stem-like characteristics such as high aldehyde dehydrogenase (ALDH) activity due to ALDH1A1 expression contributes to chemotherapy resistance and tumor relapse. However, clinically applicable inhibitors of ALDH activity have not been reported. There is evidence to suggest that epigenetic regulation of stem-related genes contributes to the efficacy of chemotherapy. Here we show that the bromodomain and extra-terminal (BET) inhibitor INCB57643 suppresses ALDH activity by abrogating BRD4-mediated ALDH1A1 expression through a super-enhancer element and its associated enhancer RNA. INCB57643 suppressed the outgrowth of platinum-treated ovarian cancer cells in a concentration-dependent manner. Consistently, INCB57643 synergizes with carboplatin in suppressing the growth of ovarian cancer cells with BRD4 expression, which was accompanied by induction of apoptosis. These phenotypes correlate with inhibition of ALDH1A1 expression through a super-enhancer element and additional stem-related genes, including LIF, HES1 and WNT5a in promoter regions bound by BRD4. Thus, targeting the BET protein BRD4 using small molecule BET inhibitor INCB057643 is a promising strategy for targeting ALDH activity in epithelial ovarian cancer.

Citation Format: Sergey Karakashev. The BET inhibitor INCB057643 suppresses ALDH activity by targeting the ALDH1A1 super-enhancer in high-grade serous ovarian 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 3685.
Abstract 3691: Collagen 1 fiber content may predict for recurrence in non-small cell lung cancer
Authors: Kakkad Samata , Huang Peng , Brock Malcolm , Krishnamachary Balaji , Bhujwalla Zaver M.
Published: August 2018

Non-small cell lung carcinoma (NSCLC) comprises 85% of all lung cancers. Approximately 30-50% of NSCLC recur despite curative resection. There is a major unmet need to identify NSCLC that are likely to recur so that these patients may be treated with more aggressive therapies. Collagen 1 (Col1) fibers are a major structural component of the extracellular matrix (ECM) of tumors. While increased Col1 fibers have been associated with increased metastasis in breast cancer, the role of Col1 in NSCLC and its relationship to recurrence has not been previously examined. We performed a blinded second harmonic generation (SHG) microscopy study on tumor tissue obtained from 5 patients with nonrecurrent NSCLC and 5 patients with recurrent NSCLC. Col1 fibers were detected with SHG microscopy that detects an intrinsic signal derived from the noncentrosymmetric molecular structure of Col1 fibers. SHG microscopy of tumor regions was performed on the H&E sections to quantify Col1 fiber volume and inter fiber distance in the tumors. At least 10 fields of view were analyzed from 1-2 sections obtained from each tumor. We additionally performed Haralick feature analyses to further analyze Col1 fiber patterns. Adjacent sections were immunostained for CD44 and α-SMA to identify cancer associated fibroblasts (CAFs) and understand their role in these tumors. We observed a significant increase of Col1 fiber volume and a significant decrease of inter fiber distance in recurrent NSCLC. Four Haralick features (orientation, contrast, energy and homogeneity) were also significantly higher in the recurrent tumors compared to the non recurrent tumors. We are currently analyzing the CAFs in the tumor sections to understand their role in the significant differences observed in Col1 fibers between recurrent and nonrecurrent tumors. If these preliminary data are confirmed in an expanded sample set, Col1 fiber analysis may provide a companion diagnostic test that can be performed rapidly on H&E tumor sections, using a confocal microscope, to evaluate the likelihood of tumor recurrence. These studies provide new insights into the role of the tumor microenvironment in NSCLC recurrence, and identify new targets for future therapeutic strategies. This work was supported in part by NIH R35CA209960.

Citation Format: Samata Kakkad, Peng Huang, Malcolm Brock, Balaji Krishnamachary, Zaver M. Bhujwalla. Collagen 1 fiber content may predict for recurrence in non-small cell lung 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 3691.
Abstract 3694: Measuring oncogene signaling with transferrin-based PET: From bench to bedside
Authors: Evans Michael J. , Truillet Charles , Ruggero Davide , Lewis Jason S. , Aggarwal Rahul , Behr Spencer C.
Published: August 2018

Background: Non-invasive tools that measure the activity of central oncogenes could be broadly useful for cancer detection and management. We have shown that radiolabeled transferrin molecules can be used to measure mTORC1 and MYC signaling in prostate cancer models, because the transferrin receptor is a MYC target gene. The purpose of this study was to complete the preclinical assessment of 89Zr-transferrin, as well as demonstrate that clinical disease harbors avidity for transferrin using 68Ga-citrate. Methods: Human prostate cancer cell lines have been manipulated pharmacologically and genetically to alter mTORC1 and/or MYC signaling, and radiolabeled transferrin has been used to study the impact of aberrant oncogene signaling on transferrin biology. A first in man study with 68Ga-transferrin PET was also performed at UCSF using a GE SIGNA PET/MR. A dose escalation study was performed in eight patients with castration resistant prostate cancer to identify the optimal signal to noise ratio. Results: Activation of mTORC1 or MYC resulted in higher transferrin receptor expression and higher transferrin uptake into cells, as expected. Pharmacological inhibition of either oncogene suppressed transferrin uptake in vitro and in vivo. 68Ga-transferrin PET/MR resolved ~75% of lesions that were detectable by CT or bone scan. Approximately 4 mCi and >3.5 hours post injection was required to visualize tumor lesions. Conclusions: These experiments establish that like MYC, mTOR activity can be quantified in prostate cancer models with 89Zr-transferrin. Moreover, our first in human study with 68Ga-transferrin shows that human disease is generally avid for the radiotracer, but the uptake is variable among lesions, consistent with a molecularly diverse disease.

Citation Format: Michael J. Evans, Charles Truillet, Davide Ruggero, Jason S. Lewis, Rahul Aggarwal, Spencer C. Behr. Measuring oncogene signaling with transferrin-based PET: From bench to bedside [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 3694.