bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2993: Dichloroacetate inhibits cell proliferation and induces apoptosis in human endometrial cancer cell lines
Authors: Clark Leslie H. , Zhou Chunxiao , Bae-Jump Victoria
Published: July 2016

Objective: Dichloroacetate (DCA) is a well-tolerated, oral drug used in the treatment of hereditary lactic acidosis. DCA has been found to have anti-tumorigenic activity in various non-gynecologic cancers through its inhibition of pyruvate dehydrogenase, leading to profound mitochondrial defects in cancer cells. Thus, we sought to assess the effects of DCA on human endometrial cancer cell lines.

Methods: Three endometrial cancer cell lines (KLE, Ishikawa, and ECC-1) were treated with varying concentrations of DCA from 0.1uM to 100 uM. Cell proliferation was measured using the MTT assay. Cell cycle progression was assessed by Cellometer. Apoptosis was evaluated by the Annexin V-FITC assay using Cellometer. Western immunoblotting was performed to assess effects of DCA on cell cycle and cellular stress proteins. In addition, cellular stress was measured using a reactive oxygen species assay. All experiments were performed in triplicate.

Results: DCA inhibited cell proliferation in a dose-dependent manner in all three endometrial cancer cell lines following 72 hours of exposure. The IC50 was 30 uM for KLE, 78 uM for Ishikawa, and 72 uM for ECC-1. Treatment with DCA resulted in G1 phase cell cycle arrest in all three cell lines. DCA induced apoptosis in all three cell lines. Western blotting analysis demonstrated that DCA increased cyclin D1 expression, decreased CDK4 expression, and increased PERK expression after 24 hours of DCA exposure. A dose dependent increase in reactive oxygen species was seen in all three cell lines. At a maximum dose of 100 uM DCA, ROS production compared to controls for KLE increased 3.2-fold (p = 0.002), Ishikawa increased 1.8-fold (p = 0.003), and ECC-1 increased 2.1-fold (p = 0.002).

Conclusions: DCA effectively inhibited cell proliferation via G1 cell cycle arrest and induced apoptosis in human endometrial cancer cell lines. DCA induced significant cellular stress as evidenced by a dose dependent increase in reactive oxygen species. Further evaluation of DCA as a targeted metabolic therapy in endometrial cancer is warranted.

Citation Format: Leslie H. Clark, Chunxiao Zhou, Victoria Bae-Jump. Dichloroacetate inhibits cell proliferation and induces apoptosis in human endometrial cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2993.
Abstract 3027: Dual inhibition of PKC and p53-MDM2 or PKC and mTORC1 are novel efficient therapeutic approaches for uveal melanoma
Authors: Carita Guillaume , Frisch Dit Leitz Estelle , Dahmani Ahmed , Raymondie Chloe , Cassoux Nathalie , Piperno-Neumann Sophie , Némati Fariba , Halilovic Ensar , Jeay Sebastien , Wylie Andrew , Emery Caroline , Roman-Roman Sergio , Schoumacher Marie , Decaudin Didier
Published: July 2016

Uveal melanoma (UM), although rare in incidence, is the most common cancer of the eye in adults. Many UM patients develop metastases for which no curative treatment has been identified and therefore novel therapeutic approaches are urgently needed. UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway. This has led to the use of PKC inhibitors as a rational targeting strategy to treat UM tumors. Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors. However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents. Employing a large panel of UM patient-derived xenograft models, several PKC inhibitor-based combination studies were performed using the PKC inhibitor AEB071 (Sotrastaurin). When combined with AEB071, the targeted agents CGM097 (p53-MDM2 inhibitor), RAD001 (Everolimus, mTORC1 inhibitor) and MEK162 (Binimetinib, a MEK inhibitor) demonstrated greater activity in the UM patient-derived xenograft models than their activity as single agents. Importantly, tumor regressions were observed in several UM models with AEB071 + RAD001 and AEB071 + CGM097 co-treatments. Follow-up in vitro studies in UM cell lines using AEB071 combined with either CGM097 or RAD001 provided a more detailed mechanistic understanding of their combination activity and confirmed their ability to induce cell death. Together, these preclinical studies reveal that combining PKC and p53-MDM2 inhibitors or PKC and mTORC1 inhibitors may provide significant clinical benefit for patients with UM.

Citation Format: Guillaume Carita, Estelle Frisch Dit Leitz, Ahmed Dahmani, Chloe Raymondie, Nathalie Cassoux, Sophie Piperno-Neumann, Fariba Némati, Ensar Halilovic, Sebastien Jeay, Andrew Wylie, Caroline Emery, Sergio Roman-Roman, Marie Schoumacher, Didier Decaudin. Dual inhibition of PKC and p53-MDM2 or PKC and mTORC1 are novel efficient therapeutic approaches for uveal melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3027.
Abstract 3034: Overcoming gefitinib resistance in non-small-cell lung cancer by inducing epidermal growth factor receptor (EGFR) degradation via methionine 790 oxidation
Authors: Leung Elaine Lai-Han , Fan Xing-Xing , Wong Maria Pik , Jiang Zhi-Hong , Liu Zhong-Qiu , Yao Xiao-Jun , Lu Lin-Lin , Zhou Yan-Ling , Yau Li-Fong , Tin Vicky Pui-Chi , Liu Liang
Published: July 2016

Personalized therapy is becoming a dominant cancer therapeutic strategy, Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed to treat non-small-cell lung cancer (NSCLC) patients with EGFR mutation but they only inhibit EGFR phosphorylation, acquired-resistance still remains unavoidable without EGFRT790M removal. Here, we report a new treatment strategy to overcome such drug resistance by selectively inducing EGFRT790M degradation via specific stimulation of methionine 790 (M790) oxidation. The basal reactive oxygen species (ROS) levels in EGFRT790M containing gefitinib-resistant NSCLC cell lines were substantially high and 63 clinical lung tumors showed higher NADPH oxidase 2 (NOX2) expression than 61 normal lung tissues which may contribute to high basal ROS in cancer. Interestingly, only NOX3 was upregulated by sanguinarine, a pharmacological agent to elevate ROS, resulted in EGFR over-oxidation and degradation as well as apoptosis. By contrast, such responses were lacking in EGFRWild-type (WT) cells, suggesting a new promising drug development strategy with wide therapeutic window between EGFRT790M and EGFRWT. Selective EGFRT790M degradation was manipulated by REDOX imbalance between NOX3 and methionine reductase (MsrA). When NOX3 was activated, NADPH, the key substrate of MsrA, was depleted to generate excessive ROS, resulting in inhibition of MsrA physiological reduction function on oxidized-methionine, thus localized methionine 790 oxidation

of EGFRT790M occurred. Furthermore, the in vivo tumor suppression effect of sanguinarine, NOX3 upregulation and EGFR degradation were confirmed. These findings indicate that specific inducing EGFRT790M degradation via manipulating REDOX imbalance between NOX3 and MsrA would be promising strategy for treating gefitinib-resistant NSCLC.

Citation Format: Elaine Lai-Han Leung, Xing-Xing Fan, Maria Pik Wong, Zhi-Hong Jiang, Zhong-Qiu Liu, Xiao-Jun Yao, Lin-Lin Lu, Yan-Ling Zhou, Li-Fong Yau, Vicky Pui-Chi Tin, Liang Liu. Overcoming gefitinib resistance in non-small-cell lung cancer by inducing epidermal growth factor receptor (EGFR) degradation via methionine 790 oxidation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3034.
Abstract 3040: A novel methoxyflavanone from a Chinese medicinal herb (Perilla frutescens) induces G2/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma cells
Authors: Mohamed Amer Ali Abd El Hafeez , Fujimura Takashi , Kamei Rikiya , Hirakawa Noriko , Baba Kenji , Nakano Miyako , Ono Kazuhisa , Kawamoto Seiji
Published: July 2016

Background: Perilla frutescens is a common traditional Chinese medicinal herb used for a variety of diseases, e.g. allergic asthma, cough and intestinal disorders. Flavonoids isolated from P. frutescens such as rosmarinic acid, caffeic acid and apigenin have been reported to inhibit proliferation of a variety of cancer cell lines. We recently identified a novel methoxyflavanone named PDMF (Perilla-derived methoxyflavanone) from P. frutescens as a candidate for anti-allergic drug. The objective of the study is to investigate antitumor potency of PDMF on A549 human lung adenocarcinoma and to elucidate its carcinostatic mechanisms.

Methods: Effect of PDMF on cell proliferation and viability of A549 cells were assessed by BrdU incorporation assay and trypan blue dye exclusion test. Cell cycle and apoptosis analyses were carried out by flow cytometry. To elucidate antitumor mechanisms of PDMF, expression and activation status of cell cycle/apoptosis regulators (p53/p21/BAX/caspases) were thoroughly analyzed by immunoblotting.

Results: Stimulation with PDMF not only triggered G2/M cell cycle arrest but also induced apoptosis of A549 cells in a dose-dependent manner. Mechanistically, PDMF stimulation significantly induced protein levels of p53 and its phosphorylated active forms (pSer15) accompanied by induction of p21 and BAX. We also found that proapoptotic caspase molecules (caspase 3, 8, and 9) were all activated upon stimulation with PDMF.

Conclusion: These results suggest that this Perilla-derived methoxyflavanone represents a useful cancer-preventive phytochemical that triggers G2/M arrest, apoptosis, and activation of p53 pathway.

Citation Format: Amer Ali Abd El Hafeez Mohamed, Takashi Fujimura, Rikiya Kamei, Noriko Hirakawa, Kenji Baba, Miyako Nakano, Kazuhisa Ono, Seiji Kawamoto. A novel methoxyflavanone from a Chinese medicinal herb (Perilla frutescens) induces G2/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3040.
Abstract 3048: Drug repurposing: Validation of sulfasalazine as a radiosensitizer in melanoma by blocking system Xc−
Authors: Førde Hilde Elise , Sleire Linda , Espedal Heidi , Heggdal Jan Ingemann , Selheim Frode , Pedersen Paal-Henning , Enger Per Øyvind
Published: July 2016

Melanoma is a cancer that has become increasingly frequent over the last decades in the western world. Initial treatment for primary and locoregional melanoma is surgery. In metastatic disease, systematic treatment and recently immunotherapy has been the mainstay. At this stage however, the prognostic outlook is still bleak. Thus, new treatments are urgently needed.

Melanoma is considered to be a radioresistant cancer. The mechanisms underlying radioresistance are multiple and incompletely characterized. In some tumors, radioresistance is mediated by increased synthesis of anti-oxidants that scavenge reactive oxygen species (ROS), induced by radiotherapy. Glutathione (GSH) is an anti-oxidant synthesized from cystine and constitutes a major defense system against oxidative stress in mammalian cells. Cystine is taken up through system Xc− (SXC), an anti-port transmembrane protein with catalytic subunit xCT. Sulfasalazine, a drug approved in the 1950s for treatment of rheumatoid arthritis and inflammatory bowel disease, has been shown to block SXC. Based on previous reports by others, and our own recent findings, we hypothesize that: I) xCT represents a mechanism for radioresistance and is expressed in radioresistant melanoma cancer and II) xCT inhibitors can act as radiosensitizers to potentiate the efficacy of radiotherapy. Expression of the catalytic subunit of SXC, xCT, was found in tissue micro array of primary and secondary melanoma biopsies. In addition, SAS treatment dramatically reduced cystine-uptake and GSH levels in melanoma cells in vitro, and markedly increased the levels of reactive oxygen species (ROS). Furthermore, SAS and radiation synergistically increased DNA double-strand breaks and increased glioma cell death, whereas adding the anti-oxidant N-acetyl-L-cysteine (NAC) reversed cell death. Moreover, SAS and irradiation synergistically reduced subcutaneous melanoma tumor growth in vivo, compared to controls or either treatment alone. Thus, future experimental studies are warranted to validate SAS as a radiosensitizer in the treatment of metastatic melanoma. Future studies will also be aimed at assessing the effect on pulmonary melanoma metastasis.

Citation Format: Hilde Elise Førde, Linda Sleire, Heidi Espedal, Jan Ingemann Heggdal, Frode Selheim, Paal-Henning Pedersen, Per Øyvind Enger. Drug repurposing: Validation of sulfasalazine as a radiosensitizer in melanoma by blocking system Xc−. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3048.
Abstract 3086: Cyclopamine tartrate, an anti-cancer agent targeting Hedgehog signaling, strongly interferes with mitochondrial function and suppresses aerobic respiration
Authors: Alam Md Maksudul , Sohoni Sagar , Kalainayakan Sarada Preeta , Garrossian Massoud , Zhang Li
Published: July 2016

The Hedgehog (Hh) pathway is a major regulator of many fundamental processes in mammalian embryonic development. These processes include stem cell maintenance, determination of cell fate, tissue polarity, cell survival, cell differentiation and proliferation. Although this pathway is usually silenced in adult tissues, constitutive activation of the Hh pathway can lead to tumorigenesis. This aberrant characteristic can be observed in different types of human cancer including basal cell carcinomas, medulloblastoma, lung cancer, ovarian cancer, gastrointestinal cancer, breast and prostate cancer. This pathway has been shown to regulate proliferation of cancer stem cells and increase tumor invasiveness. Targeted inhibition of Hh signaling pathway is shown to be effective in cancer treatment and prevention. Therefore, the discovery of Hh pathway inhibitors can pave the way of finding effective cancer treatments. The steroidal alkaloid cyclopamine was the first compound found to inhibit Hh signaling and has been invaluable for understanding the function of Hh signaling in development and cancer. Cyclopamine tartrate (CycT) is an improved analogue of cyclopamine with increased water solubility and bioavailabilty. We performed numerous experiments to examine the effect of CycT in inhibiting Hh signaling on an array of non-small-cell lung cancer (NSCLC) lines. We discovered that CycT has a novel activity in disrupting mitochondrial function and aerobic respiration. Our data show that CycT causes a substantial decrease in aerobic respiration of NSCLC cells, similar to the effect of glutamine deprivation. Mitotracker staining showed that CycT causes mitochondrial fragmentation by increasing mitochondrial fission, which is indicated by the localization of DRP1 at fission sites. Further experiments showed that CycT increases ROS generation, which is presumably a consequence of mitochondrial hyperpolarization. Concomitantly, CycT causes apoptosis and suppress proliferation of NSCLC cells.

Citation Format: Md Maksudul Alam, Sagar Sohoni, Sarada Preeta Kalainayakan, Massoud Garrossian, Li Zhang. Cyclopamine tartrate, an anti-cancer agent targeting Hedgehog signaling, strongly interferes with mitochondrial function and suppresses aerobic respiration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3086.
Abstract 3090: Molecular structure of the major G-quadruplex formed in the PDGFR-β promoter nuclease hypersensitivity element and its binding with small molecules
Authors: Lin Clement , Agrawal Prashansa , Chen Yuwei , Kalarn Salil , Deng Nanjie , Hurley Laurence , Yang Danzhou
Published: July 2016

Overexpression of platelet-derived growth factor receptor β (PDGFR-β) is associated with multiple cancers, making PDGFR-β an attractive target for anticancer drugs. Few strategies other than molecular targeting of the PDGFR-β protein or its cognate ligand have been reported for developing inhibitors for the PDGFR-β signaling pathway. DNA G-quadruplexes (G4s) formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity, and stabilization of these G4s by small-molecule compounds could serve as a mechanism for cancer therapeutics. We have shown that the major G4 formed in the PDGFR-β promoter is from the central four G-runs, adopting an intramolecular parallel-stranded structure with a broken G-strand and contains three 1-nucleotide (nt) chain-reversal loops and one lateral loop. The novel folding of the PDGFR-β G4 highlights the inherent stability of the 1-nt loops in parallel-stranded G4 structures. Elucidating the structure of the major PDGFR-β promoter G4 is important for designing small-molecule drugs to specifically target this structure to inhibit gene transcription. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the potassium solution structure of this major G4 formed in the PDGFR-β promoter. Our structure showed a unique 3’ capping structure involving three guanine nucleotides in the lateral loop. This novel capping structure is determined by the specific loop sequence as well as the broken-stranded PDGFR-β G4 folding pattern. The unique 3’ capping structure could be specifically recognized by proteins and small molecule ligands. Unrestrained molecular dynamic calculations showed that this major PDGFR-β G4 and its 3’ capping structure are stable in aqueous environment on the ns time-scale. We also investigated the binding of small-molecules to the PDGFR-β G4 using a combination of NMR, circular dichroism (CD), and fluorescence based methods to identify compounds that can selectively bind the PDGFR-β G4 over other classic parallel-stranded G-quadruplexes, such as the c-MYC promoter G4. Our study demonstrates the structural diversity in promoter G4s which may enable specific recognition and the modulation of gene expression by small-molecule drugs.

Citation Format: Clement Lin, Prashansa Agrawal, Yuwei Chen, Salil Kalarn, Nanjie Deng, Laurence Hurley, Danzhou Yang. Molecular structure of the major G-quadruplex formed in the PDGFR-β promoter nuclease hypersensitivity element and its binding with small molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3090.
Abstract 3102: Identify potential therapeutic drugs on head and neck cancer through systemic bioinformatics approach
Authors: Yin-Ju Chen , Chiou Jeng-Fong
Published: July 2016

Head and neck cancer (HNC) is one of the ten most frequent cancers worldwide. In Taiwan, with the the rising incidence in the past decade, HNC has become the fourth male malignant disease since year 2009. The standard therapy methods of HNC are surgery, radiation, chemotherapy or the combination of these modalities. Overall 5-year survival rate in patients with HNC is one of the lowest among common malignant neoplasms treatment of this disease and has not significantly improved over the past decades. To improve the identification of potential therapeutic drug process, integrating the signature of diseases, genes, and drugs was analyzed through bioinformatics method to find therapeutic candidates through comparison of gene signatures between disease and drug treatment. Here, we integrated HNC gene signatures (including 202 oral squamous carcinomas and 69 normal control species) and connectivity map drug database, and after querying, the drug shown with negative correlation may serve as therapeutic candidates to reverse oral cancer signature. Four potential small molecules with antitumor properties including natural product extracts or clinical therapeutic drugs in other diseases were identified. One of the natural product extracts was found it inhibited cell migration ability, cell invasion capacity and cancer stem cell sphere formation capability. Furthermore, the compound was demonstrated that tit regulated the epithelial-mesenchymal transition markers, suppressed the expression of stemness-related transcription factors (SOX2, Oct-4, and NANOG), and increased the expression of cell differentiation marker (CK18). The compound can also increase cell sensitivity to chemodrug- and radiation treatment then then suppress tumor growth in vitro and in vivo. Taking together, our study demonstrates a novel approach in drug development, by a bioinformatics method to effectively identify potential compounds for head and neck cancer therapy.

Citation Format: Chen Yin-Ju, Jeng-Fong Chiou. Identify potential therapeutic drugs on head and neck cancer through systemic bioinformatics approach. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3102.
Abstract 3112: HMGB1 and its isoform are sensitive and specific biomarkers to detect asbestos exposure and to identify mesothelioma patients
Authors: Napolitano Andrea , Antoine Daniel J. , Pellegrini Laura , Baumann Francine , Pagano Ian , Pastorino Sandra , Goparaju Chandra M. , Prokrym Kirill , Canino Claudia , Pass Harvey I. , Carbone Michele , Yang Haining
Published: July 2016

Millions of people have been potentially exposed to asbestos, the primary cause of malignant mesothelioma (MM). Presently, no reliable biomarkers are available to identify among potentially exposed people, those individuals who have actually been exposed and who are at high risk of MM. High Mobility Group Box Protein-1 (HMGB1) is a key mediator of asbestos-induced inflammation and MM pathogenesis. Recently, HMGB1 hyper-acetylation has been functionally associated to its active release by inflammatory cells. Here, we compared the serum levels of total and hyper-acetylated HMGB1 in individuals professionally exposed to asbestos, MM patients and healthy unexposed controls. HMGB1 serum levels reliably distinguished asbestos-exposed individuals and MM patients from unexposed controls. Moreover, the levels of total and hyper-acetylated HMGB1 were significantly higher in MM patients compared to asbestos-exposed individuals, and did not vary with tumor stage, suggesting that early lesions are also associated to increased HMGB1 levels. At a cutoff value of 2.00 ng/mL, the sensitivity and specificity of hyper-acetylated serum HMGB1 in differentiating MM patients from asbestos-exposed individuals was 100%, outperforming, in parallel experiments, other previously proposed biomarkers: osteopontin, fibulin-3, and mesothelin. When comparing MM patients to patients with other non-MM cytologically benign or malignant pleural effusion, the combination of two biomarkers, HMGB1 and fibulin-3, provided the highest sensitivity and specificity in differentiating these two groups. We propose total and hyper-acetylated HMGB1 as valuable biomarkers to differentiate MM patients from individuals professionally exposed to asbestos and from unexposed people.

Citation Format: Andrea Napolitano, Daniel J. Antoine, Laura Pellegrini, Francine Baumann, Ian Pagano, Sandra Pastorino, Chandra M. Goparaju, Kirill Prokrym, Claudia Canino, Harvey I. Pass, Michele Carbone, Haining Yang. HMGB1 and its isoform are sensitive and specific biomarkers to detect asbestos exposure and to identify mesothelioma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3112.
Abstract 3128: Detection of genetic and epigenetic DNA markers in urine for the early detection of primary and recurrent HCC
Authors: Jain Surbhi , Hann Hie-Won , Chen Sitong , Lin Selena Y. , Chang Ting-Tsung , Hu Chi-Tan , Song Wei , Su Ying-Hsiu
Published: July 2016

The purpose of this study was to explore the potential of urine DNA biomarkers for the early detection of primary and recurrent hepatocellular carcinoma (HCC). HCC is an aggressive disease with a 5-year survival rate of 26% in early-stage cancers, and a mere 2% in later stages, with an approximate 50% recurrence rate in the first 2 years of treatment. The most commonly used screening biomarker for HCC is serum alpha-fetoprotein (AFP), which detects only 40-60% of cases. We have previously shown that urine contains fragmented, circulation-derived, cell-free DNA that can be used for detection of cancer-related DNA markers, if a tumor is present. In order to detect circulation-derived, cell-free DNA markers in urine, we have developed short amplicon (∼50 bp) PCR-based assays for the three most frequent hotspot mutations in TP53 (codon 249), TERT (-124, promoter), and CTNNB1 (hotspot in exon 3, codons 32-37), and for aberrant DNA methylation in GSTP1 (mGSTP1) and RASSF1A (mRASSF1A). This five marker panel has an area under the receiver operating curve of 0.94 (92.2% sensitivity at 80% specificity) in distinguishing primary HCC (n = 77) from non-HCC (n = 91) patients by a logistic regression-based combination algorithm. Furthermore, these 5 DNA markers scored 42 of the 45 (93.3%) AFP-negative (< 20 ng/mL) HCC urine samples “positive” in this study population. To validate the urine DNA test for the early detection of recurrent HCC, an on-going blinded study was conducted. Ten patients with treated HCC were monitored for recurrence of HCC with serum AFP and urine DNA markers. Urine samples were collected from these patients at visits with a hepatologist, barcoded, and tested for the five DNA markers. Of the 10 patients, 4 developed recurrence during the study. The HCC-specific urine DNA markers were detected in 3 out of these 4 patients six months prior to MRI diagnosis, and detected concurrent with MRI diagnosis in the other patient. In conclusion, HCC DNA markers can be detected in urine of patients with HCC by short-amplicon, PCR-based assays. Furthermore, we have demonstrated that the urine DNA test detected at least 40% more HCC in an open-labeled study as compared to serum AFP alone, and has the potential to become the first line of screening for HCC in high risk populations.

Citation Format: Surbhi Jain, Hie-Won Hann, Sitong Chen, Selena Y. Lin, Ting-Tsung Chang, Chi-Tan Hu, Wei Song, Ying-Hsiu Su. Detection of genetic and epigenetic DNA markers in urine for the early detection of primary and recurrent HCC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3128.
Abstract 3131: Expression of FGF19/FGFR4 related biomarkers in hepatocellular carcinoma
Authors: Lin Zhong-Zhe , Jeng Yung-Ming , Hsu Chiun , Tsai I-Lun , Chen Kuan-Yu , Hu Fu-Chang , Hsu Chih-Hung , Hsu Hey-Chi , Cheng Ann-Lii
Published: July 2016

Background:

Fibroblast growth factor 19 (FGF19) and fibroblast growth factor receptor 4 (FGFR4) signaling play critical roles in hepatocarcinogenesis. This study explored the expression and clinical significance of FGF19/FGFR4 related signaling molecules in hepatocellular carcinoma (HCC).

Method:

We examined the mRNA expression of FGF19, FGFR4, klotho-beta (KLB), cyclin D1 (CCND1), and FGF4 in 151 surgically resected, primary unifocal HCC using quantitative real-time PCR analysis. The correlation of gene amplification and mRNA overexpression for FGF19 and FGF4 was investigated using a real-time PCR based copy number assay. FGF19 amplification was verified using fluorescence in situ hybridization. Univariate and multivariate analyses were performed to evaluate the prognostic value of these biomarkers for tumor recurrence and survival of patients.

Results:

Overexpression of FGF19, FGFR4, KLB, CCND1, and FGF4 mRNA was detected in 40%, 32%, 26%, 15%, and 35% of 151 tumors, respectively. In multivariate analyses, large tumor size (> 12.7 cm) and advanced tumor stage (stage ≥ II) independently predict worse patient survival. Using generalized additive models, we found mRNA expression of FGFR4 and KLB was significantly associated large tumor size (> 12.7 cm); mRNA expression of FGF19 and KLB was significantly associated advanced tumor stage (stage ≥ II). Furthermore, gene amplification of FGF19 and FGF4 significantly correlated to their mRNA overexpression (P = 0.006 for FGF19; P = 0.039 for FGF4).

Conclusions:

Overexpression of FGF19/FGFR4 related biomarkers is frequently found in HCC. Overexpression of FGF19 and FGF4 mRNA significantly correlates to their gene amplification. Expression status of FGFR4, KLB, and FGF19 may determine patient survival through their impact on tumor size (FGFR4 and KLB) and tumor stage (FGF19 and KLB).

Citation Format: Zhong-Zhe Lin, Yung-Ming Jeng, Chiun Hsu, I-Lun Tsai, Kuan-Yu Chen, Fu-Chang Hu, Chih-Hung Hsu, Hey-Chi Hsu, Ann-Lii Cheng. Expression of FGF19/FGFR4 related biomarkers in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3131.
Abstract 314: Cell cycle phase-specific drug resistance as an escape mechanism of melanoma cells
Authors: Beaumont Kimberley A. , Hill David S. , Daignault Sheena M. , Sharp Danae M. , Gabrielli Brian , Weninger Wolfgang , Haass Nikolas K.
Published: July 2016

The tumor microenvironment is characterized by cancer cell subpopulations with heterogeneous cell cycle profiles. For example, hypoxic areas within tumors contain clusters of cancer cells that arrest in the G1 cell cycle phase. It is conceivable that cancer cells exhibit differential drug sensitivity based on their residence in specific cell cycle phases. Here, we have used two-dimensional and organotypic melanoma culture models in combination with fluorescent cell cycle indicators to investigate the effects of G1-arrest on clinically used melanoma drugs. We demonstrate that G1-arrested melanoma cells, irrespective of the underlying cause mediating G1-arrest, are resistant to induced cell death by the proteasome inhibitor bortezomib and the alkylating agent temozolomide. In contrast, G1-arrested cells were more sensitive to MAPK pathway inhibition. Of clinical relevance, pre-treatment of melanoma cells with a MAPK pathway inhibitor resulted in resistance to temozolomide or bortezomib, while in contrast pre-treatment with temozolomide did not result in resistance to the MAPK pathway inhibitor. In summary, we have established a model to study the effects of the cell cycle on drug sensitivity. Cell cycle phase-specific drug resistance is an escape mechanism of melanoma cells that has implications on the choice and timing of drug combination therapies.

Citation Format: Kimberley A. Beaumont, David S. Hill, Sheena M. Daignault, Danae M. Sharp, Brian Gabrielli, Wolfgang Weninger, Nikolas K. Haass. Cell cycle phase-specific drug resistance as an escape mechanism of melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 314.
Abstract 3145: Clinical evaluation of streck cell-free DNA blood collection tubes for liquid profiling in oncology
Authors: Medina Diaz Inga , Holdenrieder Stefan , Nocon Annette , Kobilay Makbule , Skowasch Dirk , Held Stefanie , Weiland Johanna , Stamm Claudia , Diehl Frank , Holtrup Frank
Published: July 2016

Liquid profiling based on circulating tumor DNA (ctDNA) extracted from blood has become a powerful diagnostic approach in oncology. Making ctDNA analysis broadly available requires shipping whole blood to a clinical laboratory while ensuring cell-free DNA (cfDNA) and blood cell integrity to prevent dilution of ctDNA with cellular genomic DNA. Since standard EDTA blood collection tubes cannot retain this integrity for a prolonged time, several laboratories have proposed Streck Cell-Free DNA blood collection tubes (Streck cfDNA BCTs) as an alternative for sample collection.

Qualification data for the use of Streck cfDNA BCTs in oncology is limited and mainly based on blood collected from healthy individuals as well as data extrapolated from the prenatal field. However, the data generated from these samples may not represent the unique dynamics of clinical oncology specimens and therefore a study of true clinical oncology samples is required to support the use of Streck cfDNA BCT tubes in routine practice.

In this study, we performed a clinical evaluation of cfDNA sample integrity from matched Streck cfDNA BCTs vs standard EDTA tubes from colorectal, pancreatic and non-small cell lung cancer patients (N > 50). Blood drawn into Streck cfDNA BCTs was either processed immediately or 3 days after phlebotomy. DNA quantification was followed by BEAMing digital PCR (OncoBEAM™) on KRAS, NRAS and EGFR mutations and compared to matching specimens collected in EDTA tubes.

Our results suggest that cfDNA yield as well as the genomic DNA background is not affected by prolonged storage of clinical samples in Streck cfDNA BCTs for up to 3 days. In all sample sets containing mutated ctDNA, the detected mutational load was comparable between Streck cfDNA BCTs and EDTA tubes.

In conclusion, this study represents a comprehensive clinical evaluation of Streck cfDNA BCTs vs EDTA tubes for ctDNA profiling. In conjunction with the findings of our previously presented Streck cfDNA BCT shipping condition studies, our data supports the compatibility of clinical oncology specimens collected in Streck cfDNA BCTs with the BEAMing technology.

Citation Format: Inga Medina Diaz, Stefan Holdenrieder, Annette Nocon, Makbule Kobilay, Dirk Skowasch, Stefanie Held, Johanna Weiland, Claudia Stamm, Frank Diehl, Frank Holtrup. Clinical evaluation of streck cell-free DNA blood collection tubes for liquid profiling in oncology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3145.
Abstract 3164: Molecular determination of the clonal relationship between multiple tumors in BRCA1/2-associated cancer patients has clinical relevance
Authors: Geurts-Giele Willemina R.R. , van Verschuer Victorien M.T. , van Deurzen Carolien H.M. , van Diest Paul J. , Pedrosa Rute M. , Collee J. Margriet , Koppert Linetta B. , Seynaeve Caroline , Dinjens Winand N.M.
Published: July 2016

PURPOSE

Female BRCA1/2 mutation carriers commonly develop breast cancer and ovarian cancer. It is of utmost importance to know the clonal relationship between multiple tumor localizations, (multiple primaries or metastasized disease), since prognosis and treatment differ between these tumors and their metastases. We evaluated the value of targeted Next Generation Sequencing (NGS) in the diagnostic workup of BRCA1/2 mutation carriers with ≥2 tumor localizations and uncertain tumor origins.

METHODS

Forty-two female BRCA1/2 mutation carriers with ≥2 tumor locations with tumor material available for pathologic revision were selected. Median age at first cancer diagnosis was 48.0 years (range 30-68). Four patients with inconclusive tumor origin after histological and immunohistochemical analyses were ‘cases’; 10 patients with certain tumor origin of ≥3 tumors served as ‘controls’. Tumors of cases and controls were analyzed by targeted NGS using a panel including CDKN2A, PTEN and TP53, hotspot mutation sites for 27 different genes and 143 single nucleotide polymorphisms for detection of loss of heterozygosity (LOH). Based on prevalence of identical or different mutations and/or LOH patterns, tumors were classified as ‘multiple primaries’ or ‘one entity’.

RESULTS

In 44 tumors, 48 mutations were found; 39 (81%) concerned TP53 mutations. In all 10 controls and all 4 cases, the intrapatient clonal relationships between the tumors could be unequivocally identified by molecular analysis. In all controls, tumor origins based on molecular outcomes matched the conventional histopathological diagnosis.

CONCLUSION

In 90% of BRCA1/2 mutation carriers with multiple tumors routine pathology work-up is sufficient to determine tumor origins and relatedness. In case of inconclusive conventional pathology results, molecular analyses using NGS can reliably determine clonal relationships between tumors, enabling optimal treatment of individual patients.

Citation Format: Willemina R.R. Geurts-Giele, Victorien M.T. van Verschuer, Carolien H.M. van Deurzen, Paul J. van Diest, Rute M. Pedrosa, J. Margriet Collee, Linetta B. Koppert, Caroline Seynaeve, Winand N.M. Dinjens. Molecular determination of the clonal relationship between multiple tumors in BRCA1/2-associated cancer patients has clinical relevance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3164.
Abstract 3180: Deletions of 18q characterizes a subset of aggressive prostate cancers with metastatic potential
Authors: Hube-Magg Claudia , Kluth Martina , Graunke Maximilian , Koop Christina , Thomas Steuber , Graefen Markus , Steurer Stefan , Schlomm Thorsten , Sauter Guido , Simon Ronald
Published: July 2016

Large chromosomal deletions and rearrangements of the TMPRSS:ERG locus are hallmarks of prostate cancer. Deletion of 18q is recurrently found in prostate cancer, but the prevalence and clinical significance of this alteration have not been studied in detail as to yet. We took advantage of our large prostate cancer prognosis tissue microarray (TMA) containing more than 12,000 prostate cancers with clinical follow-up data and performed fluorescence in situ hybridization (FISH) analysis using an 18q21 probe. A total of 6,881 prostate cancers were interpretable by FISH. Deletion of 18q was found in 7.5% of cancers, and was linked to advanced tumor stage (p<0.0001), high Gleason grade (p = 0.0013) and biochemical recurrence (BCR, p<0.0001). Comparison with data from TMPRSS:ERG fusion and ERG protein expression revealed that deletions of 18q were only marginally related to an ERG-fusion negative phenotype (p = 0.0063). Multivariate analysis showed that the prognostic value of 18q deletion was independent from the established prognostic factors in all cancers (p = 0.0004) as well as in the subsets of ERG-fusion negative (p = 0.0148) and ERG-fusion positive cancers (p = 0.0264). From the clinical data attached to our TMA, we defined additional clinical endpoints beyond BCR, which represent the four hallmarks (local, local invasive, occult systemic and metastatic) of tumor growth and dissemination in prostate cancer. Deletion of 18q was linked to unfavorable outcome also according to these alternative clinical endpoints. For example, the fraction of cancers with occult systemic or metastatic dissemination increased from 33.6% in patients whose tumors lacked 18q deletion to 41.8% in patients with tumors harboring 18q deletion (p = 0.0458). In summary, the results of our study demonstrate a pivotal role of 18q deletions for prognosis in prostate cancer independent of the established prognosticators.

Citation Format: Claudia Hube-Magg, Martina Kluth, Maximilian Graunke, Christina Koop, Steuber Thomas, Markus Graefen, Stefan Steurer, Thorsten Schlomm, Guido Sauter, Ronald Simon. Deletions of 18q characterizes a subset of aggressive prostate cancers with metastatic potential. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3180.
Abstract 3200: New epigenetic mechanism of disulfiram targeting chromatin remodeling in neuroblastoma
Authors: Jacques-Ricard Simon , Raynal Noel , Armaos Gregory , Da Costa Elodie , Beaudry Annie
Published: July 2016

Pediatric neuroblastoma is one of the most common extra cranial cancers in children. Despite an improvement in survival with the currently available therapies, neuroblastoma with an amplification of the transcription factor MYCN have a very poor prognosis. New therapeutic approaches must be developed to increase the survival of patients. One such approach is epigenetic drug therapy. Neuroblastoma, like many other pediatric cancers, contains several epigenetic alterations at the level of DNA methylation and histone modifications. In a screening of FDA approved drugs, we discovered some molecules having characteristics of epigenetic drugs that were unknown until now. Our study seeks to demonstrate the efficacy of these molecules in the treatment of neuroblastoma cell lines. Following preliminary tests, one of the molecules approved by the FDA stood out: disulfiram, a medication approved for the treatment of chronic alcoholism. We treated neuroblastoma cell lines (MYCN amplified: IMR-32, N91 and SK-N-DZ; MYCN non-amplified: SK-N-AS and SK-N-SH) for 48 hours with disulfiram at clinically relevant concentrations (from 10 nM to 50 μM). Our results demonstrate a 50% growth inhibition (IC50) of 50nM for the cell lines tested. In addition, after analysis by flow cytometry, we found a cell cycle block in G2/M. We also observed a decrease in the transcription factor MYCN and a reduction in acetylation of several histone marks by Western blots analysis. Further studies are underway to determine the mechanism of action of disulfiram and we also plan on doing RNA sequencing and ChIP sequencing. This study will evaluate the efficacy of disulfiram alone as well as in combination with other epigenetic drugs, such as DNA methyltransferase inhibitor decitabine, for the treatment of neuroblastoma.

Citation Format: Simon Jacques-Ricard, Noel Raynal, Gregory Armaos, Elodie Da Costa, Annie Beaudry. New epigenetic mechanism of disulfiram targeting chromatin remodeling in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3200.
Abstract 3218: Disruption of the CD39 immune checkpoint pathway increases the efficacy of various anticancer therapies in syngeneic mouse models
Authors: Lapierre Marion , Dejou Cécile , Paturel Carine , Michaud Henri-Alexandre , Gros Laurent , Bensussan Armand , Eliaou Jean-François , Bastid Jeremy , Bonnefoy Nathalie
Published: July 2016

The CD39-CD73-adenosine pathway is an emerging regulator of the immune antitumor response. CD39 is expressed within tumors and the tumor microenvironment by several cell population including immune and cancer cells. In tumor tissues, the pathway leads to the accumulation of immunosuppressive adenosine together with decreased levels of immunoactivating peritumoral ATP. We reported previously that CD39 blockade increased T cell and NK cell-mediated cytotoxic activity in vitro and disclose, during this meeting, the development of the first human-CD39-blocking humanized antibody (S. Augier et al., Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy). Here we demonstrated that this pathway is involved in tumor-induced resistance to various cancer therapies in syngeneic mouse melanoma, colon cancer and fibrosarcoma models. We used therapy-resistant mouse models or inefficacious treatment regimens in the CD39 knockout mice to assess the capacity of CD39 to affect the response to chemotherapies, tumor associated antigen (TAA)-targeting antibodies and immunomodulators such as anti-PD1 antibodies. We achieved increased response rates, increased response duration and some complete and long lasting tumor regressions in the CD39 deficient context. These preclinical proof-of-concept studies highlight the role of the CD39 immune checkpoint pathway in limiting the efficacy of various anticancer therapies in syngeneic mouse models and thereby support the potential clinical value of the humanized CD39-neutralizing antibody under development.

Citation Format: Marion Lapierre, Cécile Dejou, Carine Paturel, Henri-Alexandre Michaud, Laurent Gros, Armand Bensussan, Jean-François Eliaou, Jeremy Bastid, Nathalie Bonnefoy. Disruption of the CD39 immune checkpoint pathway increases the efficacy of various anticancer therapies in syngeneic mouse models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3218.
Abstract 3229: Microglia/macrophages activation status in diffuse gliomas
Authors: Galatro Thais F. , Sola Paula , Oba-Shinjo Sueli M. , Eggen Bart J. , Marie Suely K.
Published: July 2016

Diffuse gliomas are primary brain tumors characterized by infiltrative growth and high heterogeneity, which renders the disease mostly incurable. Advances in genetic analysis have characterized molecular and epigenetic alterations leading to impact on patients’ overall survival and clinical outcome, particularly in glioblastoma (GBM). However, glioma tumorigenicity is not controlled uniquely by its genetic alterations. The crosstalk between tumor cells and the surrounding microenvironment plays a crucial role in modulating glioma growth and aggressiveness. The most abundant non-neoplastic cells in this microenvironment belong to the myeloid lineage, comprising resident microglia, with central nervous system (CNS)-tailored functions, and infiltrating monocytes/macrophages from the bone marrow. Understanding the dynamics between tumor and myeloid cells and the correlation between oncogenic molecular alterations in the tumor and the inflammatory activation status of innate immunity cells would elucidate potential treatment alternatives. Our large cohort of human glioma consisted of both astrocytoma and oligodendroglioma tumors, ranging from grades II to IV. Using high-throughput DNA sequencing and immunohistochemistry, we have classified GBM samples in proneural, classical and mesenchymal. Next, we evaluated the activation status of microglia/macrophages within these samples. Despite the great heterogeneity, we observed higher levels of myeloid markers (IBA1, CD11b and CD68) in astrocytic tumors compared to oligodendrocytic ones and to non-neoplastic (NN) tissue. Anti-inflammation markers, such as CD163, are also more abundant in astrocytomas, as well as in the mesenchymal and classical GBM subtypes, while pro-inflammation markers, such as IL1-beta, show a more widespread expression throughout samples. Our preliminary data suggests an immune-suppressive and growth supportive characteristic for tumors with worse clinical outcome, linked to an activation profile of myeloid cells. Further exploring the characteristics and dynamics of innate immune cells within the microenvironment of different grades and subtypes of glioma is crucial for the better understanding of the disease progression.

Citation Format: Thais F. Galatro, Paula Sola, Sueli M. Oba-Shinjo, Bart J. Eggen, Suely K. Marie. Microglia/macrophages activation status in diffuse gliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3229.