bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4593: Glycoengineered antibodies for click chemistry applications
Authors: Sehgal Drishti , Kalscheuer Stephen , Sadhukha Tanmoy , Panyam Jayanth
Published: July 2017

Antibody-drug conjugates (ADC) hold considerable promise as anticancer agents. A critical determinant of the effectiveness of ADCs is the chemistry that is used to conjugate the payload. Currently used approaches include primarily conjugation to either side-chain amine or carboxylic acid groups or conjugation to thiols. Because these reactions are not site specific and not easily controlled, these chemistries can result in reduced affinity for the target antigen. Further, these conjugation reactions lack selectivity and can result in heterogeneous mixtures of products that differ in the sites and stoichiometry of modification. We investigated a glycoengineering strategy that enables the introduction of artificial azide groups in the antibody without affecting their antigen affinity. This is based on the observation that glycosyltransferases can incorporate non-natural sugars (e.g., azido mannose) at different sites on an IgG molecule. The azide groups inthese artificial sugars are then available to react with alkynes through copper-catalyzed ‘click’ chemistry or with strained alkynes such as dibenzyl cyclooctyne (DBCO) allowing for biorthogonal, copper-free ‘click’ chemistry. Because the sugars are added reproducibly and at asite that does not affect antigen binding, the glycoengineering technology would overcome problems associated with traditional conjugation strategies. Using this approach, azide groups were introduced in anti-CD133 and anti-perlecan antibodies. Further, the azide groups were available to react with various DBCO conjugates including fluorophores, drug molecules and nanoparticles. Importantly, the addition of artificial sugar and subsequent azide-alkyne reactiondid not affect the affinity of the antibody for the target antigen. Conjugation of nanoparticles to antibodies using this approach resulted in enhanced cellular uptake of the nanoparticles. Similarly, conjugation of a cytotoxin to the antibody resulted in enhanced cell kill in vitro. Weexpect that this glycoengineering strategy will prove to be a unique platform technology that will have a significant impact on antibody-based therapeutics.

Citation Format: Drishti Sehgal, Stephen Kalscheuer, Tanmoy Sadhukha, Jayanth Panyam. Glycoengineered antibodies for click chemistry applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4593. doi:10.1158/1538-7445.AM2017-4593
Abstract 4628: Selection of Smad2 for TGF-β to suppress dendritic cells by STAT3/c-Ski-induced repression of Smad3
Authors: Yoon Jeong-Hwan , Bae Eunjin , Sudo Katsuko , Takanashi Masakatsu , Han Jin Soo , Park Seok Hee , Weinstein Michael , Nakae Susumu , Yamashita Tadashi , Lee In-Kyu , Ju Ji Hyeon , Sumida Takayuki , Kuroda Masahiko , Miyazawa Keiji , Kato Mitsuyasu , Mamura Mizuko
Published: July 2017

The mechanisms how Smad-mediated transforming growth factor-β (TGF-β) signalling regulates dendritic cells (DCs) remain largely unknown, despite its crucial regulatory roles in the differentiation and activation of DCs. Here, we show that STAT3/c-Ski-induced downregulation of Smad3 during differentiation selects Smad2 as the specific TGF-β receptor-regulated Smad (R-Smad) to suppress the immunogenicity of DCs. Smad3 transcribed by unphosphorylated Smad2/3 prevented the progenitor cells from DC differentiation with repression of Flt3, IRF4 and STAT3. Repression of Smad3 by synergy of phosphorylated STAT3 and c-Ski was essential for DC differentiation. TGF-β suppressed DC activation via Smad2, hence DC-specific Smad2 deletion enhanced anti-lymphoma effector functions of cytotoxic T lymphocytes (CTLs) and type 1 helper T (Th1) cells in a mouse EL4 lymphoma model. Our findings reveal stepwise regulatory roles of R-Smads in DC regulation: Smad3 inhibits differentiation and Smad2 suppresses immunogenicity.

Citation Format: Jeong-Hwan Yoon, Eunjin Bae, Katsuko Sudo, Masakatsu Takanashi, Jin Soo Han, Seok Hee Park, Michael Weinstein, Susumu Nakae, Tadashi Yamashita, In-Kyu Lee, Ji Hyeon Ju, Takayuki Sumida, Masahiko Kuroda, Keiji Miyazawa, Mitsuyasu Kato, Mizuko Mamura. Selection of Smad2 for TGF-β to suppress dendritic cells by STAT3/c-Ski-induced repression of Smad3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4628. doi:10.1158/1538-7445.AM2017-4628
Abstract 4633: Crosstalk between MDSCs and Th17
Authors: Singh Brijendra
Published: July 2017

Expansion of myeloid-derived suppressor cells (MDSCs) within the tumor is a known barrier to successful cancer immunotherapy. MDSCs are heterogeneous population of immature myeloid cells with potent immunosuppressive activity. Emerging evidences in the recent years define the role of immunosuppressive activity of MDSCs on different subsets of CD4+ T cells, however the exact effect of MDSCs on inflammatory Th17 cells during tumor progression is not clear. Here we demonstrate the immunosuppressive effect of MDSCs on Th17 cells. We further show that the interaction between MDSCs and Th17 is not unilateral and reveal that Th17 cells can promote the immunosuppressive effect of MDSCs. MDSCs, either isolated from tumor bearing mice or generated in vitro from bone marrow (BM-derived MDSCs), can both suppress IL-17A expression. Our data also demonstrate that IFNg, produced by Th17 cells is essential for the immunosuppressive activity of BM-derived MDSCs on IL-17A expression and further that the IFNgR1-driven signaling is important for Nos2-mediated immunosuppressive effect of BM-derived MDSCs on IL-17A expression. Understanding the molecular mechanism of the interplay between MDSCs and Th17 cells may help to develop more specific targeted therapy for cancer immunotherapy and improve therapeutic efficacy of the existing therapies against cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Brijendra Singh. Crosstalk between MDSCs and Th17 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4633. doi:10.1158/1538-7445.AM2017-4633
Abstract 4639: HLA and NKG2D ligand abnormalities and IFN-γ unnresponsiveness in human colorectal carcinoma cell lines
Authors: Liang Yi-Hsin , Lin Yen-Cheng , Chen Kuo-Hsin , Kao Chiu-Hwa , Cheng Ann-Lii , Yeh Kun-Huei , Chang Chien-Chung
Published: July 2017

Background: Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, has shown promising efficacy on wild-type RAS metastatic colorectal cancer (CRC) patients. Cetuximab demonstrates anti-cancer effect via NK cells and subsequent immunogenic cell death. Chemotherapy or targeted therapy may change immune surface markers.

Methods: We characterized a panel of 6 CRC cell lines with different genetic features. The impacts of interferon-γ (IFN-γ) on cell surface expressions of major histocompatibility complex (MHC) class I (including subtypes), MHC class II (including subtypes), MHC class 1 related molecule A (MICA, the NK cell ligand), Intercellular Adhesion Molecule 1 (ICAM-1, the immunoglobulin superfamily), EGFR, and Chondroitin Sulfate Proteoglycan 4 (CSPG-4, pericyte attaching marker) before and after treatment with IFN-γ (100 U/ml, 48 h) were explored in selected cell lines

Results:

Among six human colon cancer cell lines analyzed, DLD-1, HCT15, and LoVo

exhibited no expression of HLA-A and HLA-B,C. Colo-320 demonstrated both low HLA-A and HLA-B,C expression and irresponsive to IFN-γ stimulation. In contrast, both HT-29 and SW-480 showed low HLA-A and HLA-B,C expression, but the expression of both HLA-A and HLA-B,C was corrected after IFN-γ stimulation. All six cell lines possessed low MHC class II expression and irresponsive to IFN-γ stimulation. All six cell lines possessed low MIC expression and irresponsive to IFN-γ stimulation. All six cell lines showed low ICAM-1 expression, but only HT-29 and SW-480 were responsive to IFN-γ stimulation. LOVO and SW-480 demonstrated high EGFR expression and irresponsive to IFN-γ stimulation. Only HT-29 possessed high CSPG-4 expression.

Conclusion:

These findings reveal that regardless of their genetic features, all six CRC cell lines possess a phenotype that may allow them to escape cytotoxic T and NK cell attack.

Table

Cell LineMSICIMPKRASBRAFPI3KCATP53DLD-1MSI+G13DWTE545KS241FHCT-15MSI+G13DWTE545KS241FLOVOMSI-G13DWTWTWTCOLO-320MSS-WTWTWTR248WHT-29MSS+WTV600EP449TR273HSW-480MSS-G12VWTWTR273H

Citation Format: Yi-Hsin Liang, Yen-Cheng Lin, Kuo-Hsin Chen, Chiu-Hwa Kao, Ann-Lii Cheng, Kun-Huei Yeh, Chien-Chung Chang. HLA and NKG2D ligand abnormalities and IFN-γ unnresponsiveness in human colorectal carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4639. doi:10.1158/1538-7445.AM2017-4639
Abstract 4643: Plasma thioredoxin reductase 1 as a diagnostic marker for non-small cell lung cancer
Authors: Kang Xiaozheng , Dong Chaoran , Shen Bocun , Yan Wanpu , Dai Liang , Yang Yongbo , Yin Hanwei , Xu Guobing , Zeng Huihui , Chen Ke-Neng
Published: July 2017

Non-invasive diagnostic markers for patients with suspicious non-small cell lung cancer (NSCLC) may provide needed guidance on invasive diagnostic and therapeutic decisions. Thioredoxin reductase 1 (TrxR1) is a pivotal intracellular redox sensor and antioxidant enzyme, and plays an important part in tumor growth, progression, metastasis, and chemotherapy resistance. The goal of this study is to test the feasibility of developing plasma TrxR1 as a novel diagnostic marker for NSCLC. The plasma TrxR1 activity was determined spectrophotometrically by monitoring the NADPH-dependent production of 2-nitro-5-thiobenzoate at 412nm and at 37°C. All detection kits and automatic analyzer possess independent intellectual property rights, and have been widely used in breast cancer screening. In this study, the plasma levels of TrxR1 were measured in 45 treatment-naïve NSCLC patients and 10 individuals with benign tumor-like pulmonary diseases. Taking plasma TrxR1 activity of 4 as reference, the overall concordance rate was 73.3%, while those of CEA, CA199, CA125, NSE, CYFRA21-1 and SCC were 19.0%, 7.1%, 16.7%, 35.7%, 24.4% and 2.5%, respectively. Furthermore, using TNM staging (AJCC 7th ed.) as classifier, the concordance rates of TrxR1 in local, local regional and advanced stage subgroups were 66.7% (20/30), 77.8% (7/9) and 100% (4/4), respectively. A multipoint linear fitting correlation model was created, and the equation was TrxR1 = 5.821 + 0.396*Stage. Besides, the concordance rates of TrxR1 in benign tumor-like pulmonary diseases was 60% (6/10). Our data suggest that plasma TrxR1 activity is a useful non-invasive diagnostic marker for NSCLC, and there may be a linear correlation between TrxR1 activity and pathological stage.

Citation Format: Xiaozheng Kang, Chaoran Dong, Bocun Shen, Wanpu Yan, Liang Dai, Yongbo Yang, Hanwei Yin, Guobing Xu, Huihui Zeng, Ke-Neng Chen. Plasma thioredoxin reductase 1 as a diagnostic marker for non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4643. doi:10.1158/1538-7445.AM2017-4643
Abstract 4644: A cell-line based TP53 expression pattern control panel with interobserver variability and next-generation sequencing analysis
Authors: Zhou Yaling , Raiciulescu Sorana , Syed Viqar , Malafronte Patrick , Wells Justin , Grantham Mica , Allgaeuer Michael , Gong Heping , Moncur Joel , Xing Deyin , Andersen John D.
Published: July 2017

Introduction: TP53 gene mutation status is known to be manifested through distinct p53 immunohistochemical staining patterns; diffuse, wild-type and null. This stratified staining pattern is routinely utilized in subtyping ovarian and endometrial cancer subtypes and distinguishing low- and high-grade serous carcinoma.

Experimental Procedure: Three ovarian cancer cell lines were used in the construction of an immunohistochemical control panel slide that highlights each respective TP53 expression pattern. Cell lines were expanded, harvested and resuspended in agarose gel and subsequently sectioned onto a microscope slide. Procured tissue specimens were submitted along with either standard control tissue or the TP53 cell line control panel to pathologists of varying experience. Next-generation sequencing analysis was performed on cases in which there was discordance in expression pattern interpretation.

Results: Interobserver variability was assessed on 44 samples between five raters of varying experience. The results were similar with the use of the TP53 expression panel (Fleiss kappa= 0.6 vs 0.53). Next-generation sequencing analysis demonstrated low-frequency variant mutations in cases in which there was reviewer discordance.

Conclusion: This study suggests the addition of a TP53 cell line expression pattern control panel could potentially increase diagnostic accuracy for ovarian and endometrial cancer subtypes and distinguishing ovarian low-grade and high-grade serous carcinoma. The cell line control has refined quality assurance potential for antibody optimization.This cell line control panel has the potential to be utilized in routine immunohistochemical staining with difficult to interpret p53 expression patterns directed to submission for next-generation sequencing.

Citation Format: Yaling Zhou, Sorana Raiciulescu, Viqar Syed, Patrick Malafronte, Justin Wells, Mica Grantham, Michael Allgaeuer, Heping Gong, Joel Moncur, Deyin Xing, John D. Andersen. A cell-line based TP53 expression pattern control panel with interobserver variability and next-generation sequencing analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4644. doi:10.1158/1538-7445.AM2017-4644
Abstract 4657: LOX and ZFPM2 as novel diagnostic biomarkers for malignant pleural mesothelioma
Authors: Kim Minkyu , Kim Hyun-Won , Jung Soon-Hee , Oh Sung Soo , Jeong Yangsik , Choi Jong-Whan
Published: July 2017

Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer being developed on outlayer of lung tissues and caused by mostly occupational exposure to asbestos. Poor prognosis needs to develop therapeutic drug as well as early diagnostic biomarker. Currently, mesothelin (MSLN), osteopontin (OPN), and fibulin 3 (FBLN3) have been reported as potential diagnostic biomarkers for MPM. In this study, we first performed bioinformatics analysis for public database to find diagnostic biomarkers for MPM. From the analysis using Cancer Cell Line Encyclopedia (CCLE) and Gene Expression Omnibus (GEO) databases, included were 7 genes involving LOX, LOXL1, LOXL2, ZFPM2, THBS2, SULF1, CDH11 identified as potential diagnostic biomarkers. These genes showed a similar diagnostic ability to FBLN3 or MSLN as MPM biomarker candidates. Further molecular approach using quantitative real-time polymerase chain reaction (QPCR) confirmed the higher mRNA expression of these candidates in MPM cell lines and patient samples. Moreover, two particular genes, LOX and ZFPM, showed MPM specific patterns of mRNA expression that were further confirmed in protein level by western blot assay. Together with biological approach, biostatistical analysis of receiver operating characteristic (ROC) analysis using the GEO database revealed significantly higher diagnostic potential of LOX and ZFPM2 genes compared to the FBLN3, one of the best diagnostic biomarkers currently reported. From this study, we believe these genes together with FBLN3 and MSLN would become novel potential biomarker candidates for MPM diagnosis.

Citation Format: Minkyu Kim, Hyun-Won Kim, Soon-Hee Jung, Sung Soo Oh, Yangsik Jeong, Jong-Whan Choi. LOX and ZFPM2 as novel diagnostic biomarkers for malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4657. doi:10.1158/1538-7445.AM2017-4657
Abstract 4660: HPRT as a biomarker for tumor classification and its variability of expression in breast cancer
Authors: Townsend Michelle H. , Felsted Abigail M. , Clarke Jacob , Robison Richard A. , O'Neill Kim L.
Published: July 2017

The purpose of this study is to investigate the differential expression of HRPT between various

breast cancer tissues in comparison to healthy breast tissue in order to evaluate its potential use

as a diagnostic tool. Breast cancer is the most common malignancy diagnosed in women and

remains incurable in the metastatic setting requiring accurate and reliable tools for early

diagnosis. We analyzed HPRT, a purine salvage pathway enzyme responsible for synthesizing

90% of the available GMP and IMP in cellular maintenance. Due to its critical role in

maintaining necessary nucleotides for cell proliferation, we hypothesized an increase in the

expression of HPRT in cancer. Tissues from 48 patients with breast carcinoma were stained

using standard immunohistochemistry (IHC) techniques. We examined infiltrating ductal

carcinoma, margin of carcinoma, hyperplasia, atypical hyperplasia, adenosis, collagen fibers

tissues, and normal breast tissue in order to determine expression between various breast cancers.

Briefly, tissues were treated with a polyclonal anti-HPRT antibody along with a GADPH

positive control and a universal negative control. Tissues were incubated with an HRP-polymer

conjugated antibody, and DAP substrate which reacts to antibody-antigen binding sites and

results in a color change. Adenosis and hyperplasia are benign breast conditions and are early

signs of breast carcinoma development. Hyperplasia tissues, which are in a state of rapid

proliferation, showed significant upregulation of HPRT and adenosis tissues showed

approximately 42% upregulation. This HPRT upregulation in pre-stage 1 tissues indicates that it

may be used as a biomarker to detect early stages of breast cancer. Expression of HPRT was also

found to be upregulated in 65% of infiltrating duct carcinoma showing high expression in active

malignant areas. In marginal tissue located on the perimeter of primary tumors only 31% of

tissues show upregulation of the protein. Because the levels of HPRT in marginal tissue is

significantly lower than those found in primary tumors, HPRT may serve as a useful diagnostic

tool when evaluating successful tumor removal. These data suggests HPRT as a useful diagnostic

tool when identifying tissue with a high proliferation rate indicative of potential cancer

development. Additionally, HPRT may also be used as a tool for pathologists to evaluate the

success of tumor removal.

Citation Format: Michelle H. Townsend, Abigail M. Felsted, Jacob Clarke, Richard A. Robison, Kim L. O'Neill. HPRT as a biomarker for tumor classification and its variability of expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4660. doi:10.1158/1538-7445.AM2017-4660
Abstract 471: HER2 regulates PARP-1 expression by suppressing the let-7a microRNA in HER2+ breast cancer
Authors: Yang Eddy Shih-Hsin , Wielgos Monicka , Rajbhandari Rajani , Wei Shi , Nozell Susan
Published: July 2017

Background:

HER2+ breast cancers are sensitive to PARP inhibition and express elevated levels of the PARP-1 protein. However, the mode of regulation of PARP-1 expression by HER2 is not well understood. MicroRNAs are small non-coding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression. In this study, we investigate whether PARP1 expression in human breast cancer cells is regulated by HER2 regulation of microRNAs.

Methods:

Human HER2+ breast cancer cell lines BT-474 and SKBR3 were used in this study. MDA-MB-231 (non-HER2 expressing breast cancer cells) stably transfected with a HER2 wild-type plasmid (231 HER2) or the vector control (231 NEO) were also utilized. To identify candidate microRNAs regulated by HER2 overexpression, we performed the nCounter miRNA Expression Assay. MicroRNA and mRNA expression were validated via qRT-PCR analysis in breast cancer cell lines or patient primary tumors. Cells were also transfected with a HER2 siRNA, a let-7a mimic, and an inhibitor of let-7a as well as their respective controls. Western blot analysis and firefly luciferase assays were used to determine whether the 3’UTR of PARP1 was being directly targeted by the let-7a microRNA.

Results:

HER2 did not regulate PARP-1 at the mRNA level but increased PARP-1 protein in HER2+ breast cancer cells. Specifically, ectopic HER2 overexpression correlated with increased PARP-1 protein levels in the 231 HER2 cell line. Conversely, silencing HER2

reduced PARP-1 protein levels in the BT-474 and SKBR3 cell lines. NanoString nCounter analysis revealed that the HER2+ breast cancer cell lines expressed low levels of the let-7a microRNA. Further, let-7a expression was upregulated after HER2 knockdown in the two native HER2+ breast cancer cell lines. The let-7a mimic also reduced both PARP1 protein expression and luciferase activity in the 231 HER2 and BT-474 cell lines, whereas the let-7a inhibitor reversed these effects in the 231 NEO cell line. Importantly, human HER2+ breast tumors expressed higher levels of PARP-1 and lower levels of let-7a; whereas, the HER2- breast tumors expressed lower levels of PARP-1 and higher levels of let-7a. Further, overexpression of the let-7a mimic reduced cell proliferation in HER2+ breast cancer cells.

Conclusions:

These results suggest that let-7a regulates PARP-1 expression in HER2+ breast tumors. Let-7a may also be a potential therapeutic target and predictive biomarker of PARPi sensitivity in HER2+ breast cancer patients.

Citation Format: Eddy Shih-Hsin Yang, Monicka Wielgos, Rajani Rajbhandari, Shi Wei, Susan Nozell. HER2 regulates PARP-1 expression by suppressing the let-7a microRNA in HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 471. doi:10.1158/1538-7445.AM2017-471
Abstract 4715: Protective anti-tumor immune responses in breast cancer depend on tumor mutation rate
Authors: Miller Lance D. , Routh Eric D. , Chou Jeff W. , Thomas Alexandra
Published: July 2017

The relationship between tumor antigenicity and anti-tumor immune responses in breast cancer are poorly understood. Metrics that can accurately predict tumor immunogenic potential could guide therapeutic decisions for breast cancer patients. We examined the relationship between tumor immune subclasses and patient overall survival in the context of the rate of nonsynonymous mutations in breast cancers. These analyses were conducted using RNAseq and exome sequencing data from the Cancer Genome Atlas (TCGA) breast cancer cohort (n=1,098 patients). A previously described tumor-immune classification system, based on the relative abundance of infiltrating effector immune cells, was employed for assigning patients to favorable, weak or poor immunogenic dispositions (FID, WID or PID, respectively). In breast tumors exhibiting low mutation rates (LMR), no significant survival differences were observed among patients comprising the immune subclasses. By contrast, in the context of tumors with high mutation rates (HMR), the immune subclasses exhibited highly significant survival differences (p<0.001). Patients with FID tumors achieved 100% survival at 10 years as compared to those with PID tumors who displayed a 10-year survival rate of 44%. These survival differences were independent of molecular subtype composition, with the majority of the excellent-outcome FID subclass comprising predominantly of Basal-like, HER2-enriched and Luminal B breast tumors. In the smaller subset of HMR Luminal A breast tumors, the immune subclasses also exhibited significantly different survival outcomes (p=0.01), with patients of the poor-outcome Luminal A PID subclass experiencing a 10-year survival rate of 22%. By Cox proportional hazards regression analysis, the immune subclasses contributed significant additive prognostic information in the presence of AJCC stage (p=0.003). A mutation rate of 1.63 mutations per megabase of sequenced DNA was found to be an optimal threshold for distinguishing immune-relevant low and high mutational load. These findings fit a model where mutational burden, reflective of tumor antigenicity, is a major determinant of clinically-relevant breast tumor immunogenicity. This relationship between immunogenic tumor subclasses, overall survival and mutational burden may offer opportunities for therapeutic stratification of breast cancer patients.

Citation Format: Lance D. Miller, Eric D. Routh, Jeff W. Chou, Alexandra Thomas. Protective anti-tumor immune responses in breast cancer depend on tumor mutation rate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4715. doi:10.1158/1538-7445.AM2017-4715
Abstract 4721: Novel lincRNA SLINKY is a prognostic biomarker in kidney cancer
Authors: Gong Xue , Siprashvili Zurab , Eminaga Okyaz , Shen Zhewei , Sato Yusuke , Kume Haruki , Homma Yukio , Ogawa Seishi , Khavari Paul A. , Pollack Jonathan R. , Brooks James D.
Published: July 2017

Clear cell renal cell carcinomas (ccRCC) show a broad range of clinical behavior, and prognostic biomarkers are needed to stratify patients for appropriate management. We sought to determine whether long intergenic non-coding RNAs (lincRNAs) might predict patient survival. Candidate prognostic lincRNAs were identified by mining The Cancer Genome Atlas (TCGA) transcriptome (RNA-seq) data on 466 ccRCC cases (randomized into discovery and validation sets) annotated for ~21,000 lncRNAs. A previously uncharacterized lincRNA, SLINKY (Survival-predictive LINcRNA in KidneY cancer), was the top-ranked prognostic lincRNA, and validated in an independent University of Tokyo cohort (P=0.004). In multivariable analysis, SLINKY expression predicted overall survival independent of tumor stage and grade [TCGA HR=3.4 (CI, 2.1-5.4), P < 0.001; Tokyo HR=9.2 (CI, 2.2-43), P = 0.003], and by decision tree, ROC and decision curve analysis, added independent prognostic value. In ccRCC cell lines, SLINKY knockdown reduced cancer cell proliferation (with cell-cycle G1 arrest) and induced transcriptome changes enriched for cell proliferation and survival processes. Notably, the genes affected by SLINKY knockdown in cell lines were themselves prognostic and correlated with SLINKY expression in the ccRCC patient samples. From a screen for binding partners, we identified direct binding of SLINKY to Heterogeneous Nuclear Ribonucleoprotein K (HNRNPK), whose knockdown recapitulated SLINKY knockdown phenotypes. Thus, SLINKY is a robust prognostic biomarker in ccRCC, where it functions possibly together with HNRNPK in cancer cell proliferation.

Citation Format: Xue Gong, Zurab Siprashvili, Okyaz Eminaga, Zhewei Shen, Yusuke Sato, Haruki Kume, Yukio Homma, Seishi Ogawa, Paul A. Khavari, Jonathan R. Pollack, James D. Brooks. Novel lincRNA SLINKY is a prognostic biomarker in kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4721. doi:10.1158/1538-7445.AM2017-4721
Abstract 4729: Association of mitochondrial DNA copy number variation in peripheral blood leucocytes with risk and prognosis of gallbladder cancer
Authors: Chandra Abhijit , Kazmi Hasan R. , Satyam Leena K. , Kumar Saket
Published: July 2017

Gallbladder Cancer (GBC) is the most common malignancy of the biliary tract with very high incidence in Northern India, especially around Gangetic belt. Late diagnosis and poor prognosis are the important features of this disease. For improving treatment strategies and outcomes, novel non invasive prognostic biomarkers are needed. Variations in peripheral blood mitochondrial DNA (mtDNA) copy number in various cancers are widely studied but information in relation to gallbladder malignancy is unknown. In this pilot study, we investigated leucocytes mtDNA copy number variation in relation to risk and prognosis of GBC.

A total of 300 histologically confirmed or fine needle aspiration cytology proven GBC patients were recruited along with equal number of age and sex matched controls. The mtDNA copy number was measured by real-time polymerase chain reaction through absolute quantification using SYBR green chemistry. We also explore prognostic utility of mtDNA content in relation to overall survival of GBC patients. Statistical analysis was done by Student’s test and survival data was compared using Kaplan Meier method. Two tailed p value less than 0.05 was considered to be significant.

We observed high mtDNA content in cases as compared with controls (mean±standard deviation, 1.21±0.88 vs. 1.02±0.76). Higher mtDNA copy number was significantly associated with increased gallbladder cancer risk (p=0.005). In this study, we compared overall survival (OS) for gallbladder cancer patients in relation to mtDNA copy number median value (median, 1.12). Kaplan Meir survival curve predicts decrease in OS (p=0.03, Hazard ratio=2.54; 95% CI = 1.79-5.86) for GBC patients in relation to high mtDNA content (>median) as compared with low mtDNA copy number (<median).

This study supports an association between increased mtDNA copy number with susceptibility to gallbladder cancer in this endemic belt. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in relation to gallbladder cancer.

Citation Format: Abhijit Chandra, Hasan R. Kazmi, Leena K. Satyam, Saket Kumar. Association of mitochondrial DNA copy number variation in peripheral blood leucocytes with risk and prognosis of gallbladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4729. doi:10.1158/1538-7445.AM2017-4729
Abstract 4754: Fractionated whole brain radiation-induced behavioral changes in athymic nude mice is associated with sustained neuroinflammation and microglial M1-phenotype
Authors: Kanji Suman , Johnson Benjamin , Witcher Kristina , Gulati Pooja Gulati , Chen Shannon , Godbout Jonathan , Nelson Randy J. , Haque Saikh , Chakravarti Arnab
Published: July 2017

The objective of this study is to study the late-chronic effects of fractionated whole brain irradiation (fWBI) on cognitive impairment and associated cellular and molecular neuro-inflammatory mechanisms using clinically relevant and reliable mouse models. Microglia are recognized as the primary innate immune component of neuroinflammation. However, microglial contributions to radiation-induced cognitive impairment are poorly understood. Here, an athymic nude (Nu/Nu) mouse model was employed to address this issue. Mice were divided into two groups: radiation treatment (XRT) and no-treatment control (CTL). The whole brain of each XRT mouse received 30 Gy (3 Gy/fraction) of radiation over two weeks. XRT and CTL mice were assessed for cognitive and behavioral changes at 1, 4, and 6 months posttreatment using the novel object recognition test (for long-term, non-spatial memory), the free running Y-Maze (for short-term, spatial memory) and Barnes Maze (for spatial learning and memory). A significant decline in novel object recognition in the XRT group (P<0.05) was seen at the 4 month time point and continued to persist at 6 months (P<0.05). No significant changes in the Barnes maze or Y-maze were seen at these time points. Expression of neuroinflammatory mediator genes, from both whole brain and isolated microglia, were measured by RT-PCR. We found that markers of the M1-phenotype, which include TNF-α, and MHC II, were significantly upregulated (P<0.05) and markers of the general M2 Phenotype, which include IL-4ra, IL-10ra, and TGF-β, were downregulated (P<0.05) at 1 and 6 month time points. These results indicate that sustained neuroinflammation and microglial M1-polarization are associated with long-term cognitive impairment induced by fWBI. Radiotherapy is the most prevalent treatment for primary and metastatic brain tumors. Whereas previous studies that identified important mechanisms underlying XRT-induced cognitive decline had used treatment regimens that were not clinically relevant ones, we have employed a clinically relevant fractionation scheme to address the cellular and molecular deregulations in association with cognitive impairment.

Citation Format: Suman Kanji, Benjamin Johnson, Kristina Witcher, Pooja Gulati Gulati, Shannon Chen, Jonathan Godbout, Randy J. Nelson, Saikh Haque, Arnab Chakravarti. Fractionated whole brain radiation-induced behavioral changes in athymic nude mice is associated with sustained neuroinflammation and microglial M1-phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4754. doi:10.1158/1538-7445.AM2017-4754
Abstract 477: Down-regulated miR-23a contributes to invasion and metastasis of cutaneous melanoma by promoting autophagy
Authors: Guo Weinan , Wang Huina , Yang Yuqi , Guo Sen , Zhang Weigang , Zhao Tao , Liu Lin , Jian Zhe , Liu Ling , Wang Gang , Gao Tianwen , Shi Qiong , Li Chunying
Published: July 2017

Background: The occurrence of invasion and metastasis is the major cause of mortality in melanoma. Recent studies suggest that dysregulated miRNAs play critical roles in this procedure, but the underlying mechanism remains elusive. Here, we show that down-regulated miR-23a can promote invasion-metastasis cascade through autophagy in melanoma.Methods: The role of miR-23a in prognosis was assessed in a cohort of melanoma patients (n = 192) with Kaplan-Meier analysis. The effects of miR-23a overexpression were investigated using assays of invasion, migration and in a xenograft model (n = 10 mice per group). Autophagy-related target of miR-23a was confirmed by bioinformatics analysis, luciferase assays and immunoblotting. Molecular studies were performed to determine the downstream cellular and molecular mechanisms. All statistical tests were two-sided.Results: Serum miR-23a level was significantly down-regulated in melanoma patients (P< .001) and was highly correlated with poor clinical outcomes (P = .027, log-rank test). In addition, miR-23a level was remarkably decreased in metastatic melanoma tissues and cell lines (P<.05, P<.01). Moreover, miR-23a overexpression prevented the invasion and migration in vitro and lung and liver metastasis in vivo (both P< .05), by targeting ATG12 and inhibiting autophagy. Mechanically, miR-23a-ATG12 axis attenuated invasion and migration through autophagy-mediated AMPK-RhoA pathway. Finally, the down-regulation of miR-23a in metastatic melanoma was caused by RUNX2 in a transcriptional repression-manner. Conclusion: MiR-23a can act as a crucial epigenetic repressor of melanoma invasion and metastasis in an autophagy-dependent way, which indicates that miR-23a-mediated autophagy inhibition can be exploited to restrain invasion-metastasis cascade in melanoma treatment.

Note: This abstract was not presented at the meeting.

Citation Format: Weinan Guo, Huina Wang, Yuqi Yang, Sen Guo, Weigang Zhang, Tao Zhao, Lin Liu, Zhe Jian, Ling Liu, Gang Wang, Tianwen Gao, Qiong Shi, Chunying Li. Down-regulated miR-23a contributes to invasion and metastasis of cutaneous melanoma by promoting autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 477. doi:10.1158/1538-7445.AM2017-477
Abstract 4772: Stearoyl-CoA Desaturase (SCD1) regulates liver tumor initiating cells through modulating ER stress
Authors: Ma Kin Fai , Lau Eunice Yuen Ting , Ng Irene Oi Lin , Lee Kin Wah
Published: July 2017

Sorafenib is the FDA approved drug for patients with advanced hepatocellular carcinoma (HCC), but the survival benefit is only modest, partly due to drug resistance. Increasing evidence shows that tumor-initiating cells (T-ICs) are intrinsically resistant to conventional treatments, and targeting signaling pathways in T-ICs provides potential therapeutic targets for HCC. In our established enriched T-IC population, we identified the activation of a lipogenesis pathway in which expression of stearoyl-CoA desaturase-1 (SCD1), an enzyme involved in the conversion of saturated into monounsaturated fatty acids, was most significant. SCD1 overexpression is frequently found in HCC samples and is significantly associated with poorer patient survival. Using overexpression and knockdown approaches, SCD1 was found to regulate the traits of T-ICs, including tumorigenicity, self-renewal, differentiation, drug resistance and the expression of liver T-IC markers. Interestingly, SCD1 was markedly upregulated in our established sorafenib-resistant cell lines as well as patient-derived xenografts (PDTXs), and its overexpression predicts the clinical response of HCC patients to sorafenib treatment. Consistently, pharmacological inhibition of SCD1 suppressed T-IC phenotypes and enhanced sensitivity to sorafenib treatment. Using a patient-derived xenograft model, we found that a novel SCD1 inhibitor (SSI-4) in combination with sorafenib demonstrated a maximal tumor suppressive effect. Induction of endoplasmic reticulum (ER) stress-mediated T-IC differentiation can account for the enhanced sensitivity towards sorafenib treatment upon SCD1 suppression. In conclusion, SCD1-induced ER stress may specifically increase the sensitivity of liver T-ICs to the effects of sorafenib treatment. Targeting SCD1 in combination with sorafenib therapy may be a novel therapeutic regimen against HCC.

Citation Format: Kin Fai Ma, Eunice Yuen Ting Lau, Irene Oi Lin Ng, Kin Wah Lee. Stearoyl-CoA Desaturase (SCD1) regulates liver tumor initiating cells through modulating ER stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2017-4772
Abstract 4773: Dysregulated expression of pentraxin-3 in glioblastoma (GBM) stem cells: Implications for GBM invasion and progression
Authors: Wesley Umadevi V. , Clark Paul , Kuo John S. , Dempsey Robert J.
Published: July 2017

Glioblastoma multiforme (GBM) is highly invasive and resistant to current therapies. The aggressiveness of brain tumor is attributed to GBM stem cells (GSC) that release inflammatory cytokines, and exacerbate inflammation in tumor microenvironment, critically affecting GBM survival and invasiveness. The glycoprotein pentraxin-3 (PTX3), a modulator of tumor-associated inflammation, is known to be correlated with tumor grade and severity of malignancies of some cancers. However, little is known about the role of PTX3 in GBM growth and invasion. In this study, we examined the role of PTX3 in GSCs exhibiting various degree of invasiveness. From patient brain tumors, our team has isolated the small subsets of glioblastoma stem cells (GSCs) and are validated for their self-renewal and multi-lineage potential. Our studies have shown that these GSCs efficiently form orthotopic brain tumors in immune-deficient mice. Our results show that conditioned media from GSCs promote pro-angiogenic tube formation from endothelial cells. GSCs that are grown in the absence of growth factors EGF and bFGF, formed efficient xenografts in immune-deficient mice and showed increased dispersal in the mice brain. Interestingly, polymerase chain reaction (RT-PCR) and immunofluorescence staining showed upregulated expression of PTX3 in these GSCs, and correlated with the degree of invasiveness. Differentiation of GSC into astrocytes in vitro, lead to decreased expression of PTX3. In support of this, recent histochemical study has shown that PTX3 levels correlate with GBM grade and is a candidate biomarker for cancer progression. Thus, PTX3 may represent a new target and marker of inflammation for glioma malignancy.

Citation Format: Umadevi V. Wesley, Paul Clark, John S. Kuo, Robert J. Dempsey. Dysregulated expression of pentraxin-3 in glioblastoma (GBM) stem cells: Implications for GBM invasion and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4773. doi:10.1158/1538-7445.AM2017-4773
Abstract 4776: Circulating clusters in breast cancer express cancer stem cell phenotypes
Authors: Harouaka Ramdane , Hodges Gabrielle , David Allison , Polasani Rishika , Freeman Spencer , Sanchez Katherine , Harvey Kaitlin , Wicha Max S.
Published: July 2017

Breast cancer is a leading cause of death in women and its metastatic spread to distant organs is responsible for over 90% of these deaths. Circulating tumor cells (CTCs) implicated in the metastatic process occur when cancer cells undergo cytoskeletal and phenotypic transitions that enhance motility, then penetrate into the blood circulation. While CTC dissemination has typically been considered as the spread of single cells, recent evidence suggests that collective migration results in polyclonal circulating clusters that are the main drivers of metastasis. To study circulating clusters we have developed a dual microfiltration system employing two stages of antigen-independent mechanical cell isolation. As whole blood is passed through the system, circulating clusters are trapped by the first stage while single CTCs are retained by the second stage. Characterization experiments using cell lines demonstrated a capture efficiency of 87.1% for clusters and 85.7% for single cells on their respective microfilters. This system was used to investigate circulating cells in the metastatic MMTV-PyMT syngeneic mouse breast cancer model. Both clusters and single CTCs were detected in mice with histological evidence of lung metastases (14-16 weeks old), but not in mice that had not yet developed metastatic tumors (<12 weeks old). Immunofluorescence assays were established to determine expression of CD24, CD90, cytokeratins, CD45 and DAPI. Tumor cells were identified as nucleated cells that expressed epithelial cytokeratins, but not the leukocyte antigen CD45. While PyMT cells with a CD90+/CD24+ stem cell phenotype comprise only ~1.5% of tumors, they were observed in > 50% of circulating clusters. The presence of cancer stem cells was correlated to cluster size, with smaller clusters containing a greater percentage of CD90+/CD24+ cells. Isolated clusters will be further analyzed to quantify expression levels of cancer stem cell markers in each individual constituent cell. The analysis of circulating clusters may provide crucial insight into the mechanisms of metastatic disease and provide the means to discover novel targets for therapeutic intervention.

Citation Format: Ramdane Harouaka, Gabrielle Hodges, Allison David, Rishika Polasani, Spencer Freeman, Katherine Sanchez, Kaitlin Harvey, Max S. Wicha. Circulating clusters in breast cancer express cancer stem cell phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4776. doi:10.1158/1538-7445.AM2017-4776
Abstract 4814: Testing personalized therapies for ovarian cancer using mate pair genomic analysis and patient-derived xenografts
Authors: Leventakos Konstantinos , Harris Faye R. , Yang Lin , Hou Xiaonan , Weroha Saravut , Kovtun Irina V.
Published: July 2017

Purpose: The treatment of ovarian cancer poses a challenge since 70% of patients will relapse with incurable platinum resistant disease. Although ovarian cancers lack frequently-occurring driver mutations or amplifications, the study presented herein examines the concept of individualized therapy based on mate pair sequencing and protein expression in ovarian cancer patient-derived xenografts.

Experimental Procedure: Macrodissection of patient tumor was followed by genomic DNA isolation and next-generation sequencing using an Illumina Mate Pair protocol to identify structural genomic changes and copy number variations. Secondary analyses were carried out to select potential therapeutic targets among those. The tumors were also propagated intraperitoneally in immunocompromised mice and treated with standard chemotherapy or/and targeted therapy chosen based on genomic analyses.

Results: Mate pair analysis revealed that, in general, copy number changes (amplifications, gains and losses) were common in ovarian tumors and included genes potentially targetable. Tumors from early generation patient- derived xenografts showed a landscape of genomic aberrations identical to that of original patient tumor. One PDX model in which high expression of HER2 and copy number gains at RICTOR and AKT genetic loci were revealed, was treated with either chemotherapy and Pertuzumab/trastuzumab (HER2 inhibitors), MK-8669 (mTOR inhibitor) or MK-2206 (AKT inhibitor) or chemotherapy alone. Tumor size was measured by serial abdominal ultrasound and after 28 days, the final tumor size ratio compared to baseline was 0.23, 0.36, 0.55 and 0.56, respectively, indicating that the best response was seen in the combined chemotherapy and Pertuzumab/trastuzumab cohort and that was better than in the chemotherapy only cohort.

Conclusions: Patient-derived xenografts models in conjunction with genomic and protein analyses of patient tumors are valuable for testing of combination therapies including specific targeting drugs. This approach also provides actionable information for more tailored treatment of ovarian cancer patients.

Citation Format: Konstantinos Leventakos, Faye R. Harris, Lin Yang, Xiaonan Hou, Saravut Weroha, Irina V. Kovtun. Testing personalized therapies for ovarian cancer using mate pair genomic analysis and patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2017-4814