bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5702: A targeted re-sequencing assay for molecular profiling of somatic mutations from plasma cell-free dna (cfdna) for bladder cancers
Authors: Dong Liqin , Khetrapal Pramit , Rodney Simon , Tan Wei Shen , Rezaee Sheida , Winter Patricia de , Kelly John , Feber Andrew
Published: July 2017

Bladder cancer (BC) is the 5th most common cancer in Western societies with a rising global incidence. The major challenge to improving patient outcomes is to better identify patients at risk for recurrence, progression, and metastasis, and to monitor treatment response. A non-invasive monitoring assay is needed in order to interrogate the molecular features and clinical tumor heterogeneity of bladder cancers, which will potentially lead to better stratified patient management.

We developed a targeted amplicon-based re-sequencing workflow for detecting and tracking genomic mutations in cfDNA using dual molecular indexed primers for specific amplification of a panel of 20 of known (TERT, FGFR3, PIK3CA, HRAS) and novel BC mutations. Only 3hr time is needed for the total workflow for the library generation and followed by next generation sequencing (NGS) performed on Illumina MiSeq sequencing platform. Data analysis and variants calling was performed.

After a thorough test, optimization, and validation of the targeted panel and the workflow on selected samples of matched bladder tumor-blood pairs and controls, 45 samples were included for testing the workflow, which includes samples of 16 matched bladder tumor-blood pairs, technical duplicates of cfDNA DNA Reference Standards (Horizon Discovery) with mutations at known allelic frequencies. Technical duplicates of cfDNA extracted from blood from patients with metastatic BC (pre and post chemotherapy) were also included in this study. Somatic variants from matched tumor-blood pairs were detected. We showed that detection sensitivity at 1.0% allelic frequency was achieved. Our preliminary results demonstrated that we were able to detect mutation burden from cfDNA samples and track mutation burden change in response to chemotherapy for patients with metastatic BC.

Here we developed a sensitive, non-invasive targeted NGS workflow to detect somatic mutations for bladder cancers from both tumor DNA and cfDNA samples. Our early results demonstrated that we can detect and track mutational changes from patients with metastatic BC.

Citation Format: Liqin Dong, Pramit Khetrapal, Simon Rodney, Wei Shen Tan, Sheida Rezaee, Patricia de Winter, John Kelly, Andrew Feber. A targeted re-sequencing assay for molecular profiling of somatic mutations from plasma cell-free dna (cfdna) for bladder cancers [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 5702. doi:10.1158/1538-7445.AM2017-5702
Abstract 5703: An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of ESR1 mutations in metastatic breast cancer
Authors: Liu Jiasu , Wang Yaohuai , Sun Shengrong , Yu Dehua Derek
Published: July 2017

Activating mutations of estrogen receptor α (ESR1) are attributed to acquired resistance to estrogen-deprivation therapy in patients with metastatic breast cancer (MBC). Thus, detection of these mutations is clinically critical for monitoring early progression and effective therapeutic options in treatment of BC patients under hormonal therapy. However, current investigations for detection ESR1 mutations mainly focus on digital PCR technology due to low abundance of the cfDNA in plasma samples, which is not a currently feasible method for clinical diagnosis.

Herein, we developed a multiplex allele-specific real-time (ARMS) PCR assay for detection of ESR1 four mutations (Y537S, Y537C, Y537N, and D538G) in a single tube reaction. In this real-time PCR system, the wild-type ESR1 DNA is completely blocked by a modified probe with a higher annealing temperature, and the mutant gene is selectively amplified by the modified ARMS primers. The assay was found to be highly specific and sensitive. With this assay, as low as 0.1% mutant DNA template in the background of a total of 10ng wild-type genomic DNA could be detected.

By using this assay, we analyzed the ESR1 mutations in 50 Chinese MBC FFPE and plasma samples. The ESR1 mutations were detected in 3 FFPE and 6 plasma samples, which were confirmed by pyrophosphate sequencing.

The multiplex allele-specific real-time PCR assay provides a rapid and reliable diagnostic tool for accurate detection of ESR1 mutations with potential for clinical application.

Citation Format: Jiasu Liu, Yaohuai Wang, Shengrong Sun, Dehua Derek Yu. An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of ESR1 mutations in metastatic 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 5703. doi:10.1158/1538-7445.AM2017-5703
Abstract 5704: Ultrasensitive mutational analysis in cell-freeDNA by targeted next-generation sequencing with molecular tags
Authors: Tam Nga Wan Rachel , Diala Irmina , Chen Xiaoji , Darbonne Walter
Published: July 2017

Circulating cell-free DNA (cfDNA) in plasma offers a non-invasive approach to monitor tumor molecular profiling in real-time at multiple time-points, detection of emerging genomic alterations associated with drug resistance and clarifying cancer prognosis and diagnosis of cancer recurrence or progression. Therefore, it is crucial to improve our current NGS technology to detect low frequency alleles in cfDNA.

There are many technical challenges in NGS technology, including base errors generated from library preparation and sequencing, making it hard to distinguish real mutations from false positives produced from amplification sequencing and base-calling processes.

We have adopted a more sensitive approach to overcome this challenge. We used short, random DNA sequences called unique molecular tags (UMTs) during library preparation to label input DNA. This technique can remove background errors, false positives during data analysis, providing high confident mutation calls with better sensitivity down to 0.1% (depends on DNA input amount).

We will present data showing validation of this NGS platform using commercially available DNA control, archival FFPE tissue and cfDNA matched samples and demonstrate robust sensitivity and specificity by using the off-the-shelf Archer Reveal ctDNA panel for Illumina covers 28 oncogenes. This molecular-tagged NGS panel can detect mutations down to 0.1% allelic frequency. This robust NGS platform is being used in our lab to analyze cfDNA samples, enabling the development of a non-invasive method to overcome existing challenges to provide molecular understanding of patient’s tumor evolution in real time, and aid in the development of personalized therapies for cancer patients.

Citation Format: Nga Wan Rachel Tam, Irmina Diala, Xiaoji Chen, Walter Darbonne. Ultrasensitive mutational analysis in cell-freeDNA by targeted next-generation sequencing with molecular tags [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 5704. doi:10.1158/1538-7445.AM2017-5704
Abstract 5707: STAT3 and STAT5a are potential therapeutic targets in castration-resistant prostate cancer
Authors: Mohanty Sambit K. , Yagiz Kader , Luthringer Luthringer , Amin Mahul B. , Alkan Serhan , Cinar Bekir
Published: July 2017

Background: Currently, there is no effective pharmacotherapy for metastatic castration-resistant prostate cancer (mCRPC). The mechanisms underlying mCRPC are not clearly understood, thus hindering rational-based drug design. Evidence suggests that signal transducer and activator of transcription 3 (STAT3) and 5a/b (STAT5a/b), key components of the JAK/STAT pathway, play a significant role in aggressive PC. However, expression of STAT3/5 in prostate tumor samples have not been studied. Here, we evaluated the possible role of STAT3/5a in aggressive PC.

Materials and Methods: Expression of STAT3 and STAT5a in high grade PC (HGPC, n=15) and in benign prostatic hyperplasia (BPH, n=15) tissue sections were evaluated by immunohistochemistry (IHC) according to the protocol approved by Institutional Review Board. Age ranged from 59 to 95 years (median = 81) in the former and 57 years to 86 years (median = 68 years) in the latter category. In addition, the effects of STAT inhibitor, Pimozide, on androgen-sensitive LNCaP and its castration-resistant subline C4-2 PC cell growth were assessed in vitro.

Results: A strong nuclear immunoreactivity for STAT3 and STAT5a in 93% (n=14) and 80% (n=12) of HGPC cases, respectively, were observed. Only one BPH case showed strong STAT3 expression. Focal and weak positivity for STAT3 and STAT5a were noted in 47% (n=7) and 67% (n=10) of BPH cases. None of the BPH cases showed strong STAT5a expression. HGPC cases showed significantly higher STAT3 positivity compared with BPH (p=0.01). No significant differences were observed for STAT5a expression between HGPC and BPH group (p=0.4). However, multifocal or diffuse and strong STAT3 (p<0.0001) and STAT5a (p<0.0001) expression was significantly higher in HGRPC than BPH. Pimozide exposure significantly inhibited LNCaP cell proliferation at 10 μM and C4-2 cell proliferation at 20 μM concentration.

Conclusion: Our results demonstrate that increased expression of STAT3 and STAT5a may serve as a predictive biomarker for efficacy of the JAK/STAT targeted therapy. Since JAK/STAT and androgen receptor signaling are functionally synergistic in contributing PC progression, the inhibition of JAK/STAT-alone or in combination with AR may lead to a novel treatment modality for mCRPC.

Citation Format: Sambit K. Mohanty, Kader Yagiz, Luthringer Luthringer, Mahul B. Amin, Serhan Alkan, Bekir Cinar. STAT3 and STAT5a are potential therapeutic targets in castration-resistant prostate 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 5707. doi:10.1158/1538-7445.AM2017-5707
Abstract 5714: AXL and GAS6 Co-expression in lung adenocarcinoma as a prognostic classifier
Authors: Seike Masahiro , Noro Ritaro , Kim Cheol-Hong , Zou Fenfei , Kubota Kaoru , Gemma Akihiko
Published: July 2017

AXL, a receptor tyrosine kinase implicated in cell survival, proliferation, and migration, is also associated with acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor therapy. However, its prognostic significance in lung adenocarcinoma (AD) remains unclear. We therefore evaluated the prognostic significance of the expression of AXL and/or its ligand, growth arrest-specific 6 (GAS6), in completely resected lung AD. We evaluated the relationship between AXL, GAS6, and vimentin expression, as determined by immunohistochemistry (IHC) analysis, with overall survival and disease-free survival in 113 patients with stages I-III lung AD. Protein expression was also assayed using western blot analysis in 10 lung AD cell lines. AXL-positive (AXL+), GAS6-positive (GAS6+), or AXL+/GAS6+ staining was significantly associated with vimentin-positive (vimentin+) expression. AXL+/GAS6+ and vimentin+ showed a negative tendency toward an association with EGFR mutation. AXL+, GAS6+, or AXL+/GAS6+ statuses significantly correlated with poor overall survival. In stage I cases, an AXL+/GAS6+ status significantly correlated with poor overall survival and disease-free survival, especially in cases with wild-type EGFR. In multivariate analysis, AXL/GAS6 classifications in stage I as well as in stages I-III lung AD were found to be independent factors for poor patient outcomes. Unlike lung AD cell lines with mutant EGFR, those with wild-type EGFR closely correlated with AXL and vimentin expression by western blotting. AXL+ and GAS6+ expression is relevant to a poor prognosis in resected lung AD patients at stage I. AXL/GAS6 might serve as crucial predictive and prognostic biomarkers and targets to identify individuals at high risk of post-operative death.

Citation Format: Masahiro Seike, Ritaro Noro, Cheol-Hong Kim, Fenfei Zou, Kaoru Kubota, Akihiko Gemma. AXL and GAS6 Co-expression in lung adenocarcinoma as a prognostic classifier [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 5714. doi:10.1158/1538-7445.AM2017-5714
Abstract 5719: Long intergenic non-coding RNAs: a new independent risk predictors in multiple myeloma
Authors: Samur Mehmet K. , Gulla Annamaria , Fulciniti Mariateresa , Samur Anil Aktas , Szalat Raphael , Shamas Masood , Magrangeas Florence , Minvielle Stephane , Anderson Kenneth , Parmigiani Giovanni , Avet-Loiseau Hervé , Munshi Nikhil
Published: July 2017

RNA has diverse sets of regulatory functions and a recent analysis of RNA repertoire has identified a large numbers of non-coding transcripts. One of which, long intergenic non-coding RNA (lincRNA) with transcripts longer than 200 nucleotides, are located between the protein coding genes and do not overlap exons of either protein-coding or other non-lincRNA genes. lincRNAs have been considered to provide regulatory functions, however, their precise role in cellular biology remains unclear. Here, we have studied lincRNAs using uniformly treated patients to show their impact on survival outcome in MM. We performed RNA-seq on CD138+ MM cells from 360 newly-diagnosed patients and 18 normal plasma cells (NPM) and analyzed for lincRNA and protein coding genes. MM patient data for clinical characteristics, cytogenetic and FISH as well as clinical survival outcomes were also analyzed and correlated with lincRNA data. Our data showed expression of 951 lincRNAs (median TPM > 1) with 351 lincRNAs differentially expressed between MM and normal plasma cells. Using only the expressed lincRNAs, we applied log rank tests for quartile 1 (Q1) versus Q2 through Q4 and Q4 versus Q1 through Q3 in order to identify under- and overexpressed prognostic genes, respectively. Four under and seven overexpressed genes were selected for final model. We used Más-o-menos for final predictive model, which simply calculates the risk score, by using expression values. The Kaplan-Meier estimates of EFS at 4 years were 53.3% (95% CI, 45.1% to 63.1%) and 32.6% (95% CI, 25.1% to 42.2%), and OS at 4 years were 93.2% (95% CI, 88.9% to 97.6%) and 71.1% (95% CI, 62.9% to 80.3%) in our patients having a low or high risk score. When applied to patient cohort separated by other risk categorization including minimal residual disease status (MRD), cytogenetic risk status (del17p, t(4;14) and t(14;16)) and International Staging System (ISS), lincRNA signature was able to further identify patients with significant differential survival outcomes. In summary, we report that lincRNAs have an independent effect on survival outcome in MM and provides rational for its use in risk stratification as well as to understand biological impact.

Citation Format: Mehmet K. Samur, Annamaria Gulla, Mariateresa Fulciniti, Anil Aktas Samur, Raphael Szalat, Masood Shamas, Florence Magrangeas, Stephane Minvielle, Kenneth Anderson, Giovanni Parmigiani, Hervé Avet-Loiseau, Nikhil Munshi. Long intergenic non-coding RNAs: a new independent risk predictors in multiple myeloma [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 5719. doi:10.1158/1538-7445.AM2017-5719
Abstract 5724: Circulatory microRNA expression profiling between HCV-infected African & Caucasian Americans: implications for racial health disparities
Authors: Devhare Pradip , Steele Robert , Bisceglie Adrian M. Di , Kaplan David E. , Ray Ratna B.
Published: July 2017

The purpose of the study: Hepatocellular carcinoma (HCC) is global problem and incidence of HCC is increasing in last several years. Hepatitis C virus (HCV) infection is one of the major cause towards development of HCC. HCV infection is highly prevalent in African American population compared to other ethnic groups and these people are less likely to naturally clear HCV. Higher incidence of HCC and mortality was noted in HCV infected African Americans as compared to Caucasians. However, the explanation for this disparity and the molecular mechanisms behind this are currently unknown. Circulating microRNAs in the blood are emerging as biomarkers for pathological conditions. Differential expression of miRNAs among ethnic groups would be important for optimizing personalized treatment strategies.

Experimental procedures: In this study, we assessed the differential expression of circulatory miRNAs from HCV infected African Americans and Caucasians by cancer specific miRNA array profiling. Expression of significantly altered miRNAs was validated by qRT-PCR.

Results: We identified increased expression of miR-146a, miR-150 and miR-155 in HCV infected African Americans patient sera as compared to sera of Caucasians. Further analysis demonstrated that these miRNAs were significantly elevated in African Americans diagnosed with HCV-mediated HCC. Higher expression of miR-150 was also noted in cirrhosis and HCC in African Americans, which may serve as predictor of liver disease progression in this population.

Conclusion: The differential expression of miRNAs suggests that these miRNAs and their target genes could be useful to gain further mechanistic insight of racial disparity associated with HCV pathogenesis.

Citation Format: Pradip Devhare, Robert Steele, Adrian M. Di Bisceglie, David E. Kaplan, Ratna B. Ray. Circulatory microRNA expression profiling between HCV-infected African & Caucasian Americans: implications for racial health disparities [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 5724. doi:10.1158/1538-7445.AM2017-5724
Abstract 5726: A novel tumor-promoting role for miR-4516 in glioblastoma
Authors: Cui Tiantian , Gray Ashley , Liu Ziyan , Geurts Marjolein , Robe Pierre , McElroy Joseph , Bell Erica Hlavin , Chakravarti Arnab
Published: July 2017

Background: Glioblastomas are the most aggressive high-grade brain tumors, which are often life-threatening due to their location and rapid growth. Although survival rates differ depending on a variety of genetic and environmental factors, the average survival rate for a glioblastoma (GBM) patient is less than 15 months. Although the mechanisms of tumorigenesis are still being elucidated, miRNAs are promising candidates to explore as novel and prognostic biomarkers in GBM. Here we demonstrate a novel role for miR-4516 in promoting growth and migration of GBM and establish the molecular mechanisms mediating these functions. Methods: Formalin-fixed, paraffin-embedded tissue blocks (n=268) were collected for all patients and total RNA was isolated. miRNAs were analyzed simultaneously using the nCounter human miRNA v2 assay (NanoString Technologies; Seattle,WA). Functional characterization studies were conducted in vitro and in vivo. The effect of miR-4516 on GBM cell growth and motility were evaluated by cell proliferation assay, migration and invasion assay, and Annexin-V assay. Realtime PCR, immunoblotting, and 3’ untranslated region luciferase assays were used to analyze miR-4516 targets and signaling pathways. Intracranial injection will be performed to investigate the role of miR-4516 in tumor growth in vivo. Results: Univariate analysis showed that miR-4516 expression in GBM patients was inversely correlated with overall survival (FDR=0.002, p=1.02E-05). Knockdown of miR-4516 blocked tumor growth and induced cell apoptosis. Tumor cell growth, migration and invasion were induced in both transient miR-4516 overexpressed GBM cells (LN229, LN18, and U87) and stable miR-4516 overexpressed GBM cells (U87-EGFRvIII). These miR-4516 tumor-promoting effects were mediated in part via direct targeting PTPN14 and CDKN1A. Investigation of the other miR-4516 targets and in vivo functional study are in process. Conclusion: Taken together, these results suggest that miR-4516 acts as a prognostic biomarker for GBM patients. Funding Information: 1R01CA169368 (PI: Houghton; Co-I:Chakravarti); 1R01CA11522358 Multiple-PI R01: Chakravarti (PI); Xia (PI); 1R01CA1145128 Baroukhim (PI); Chakravarti (Co-PI) 7/2015-6/2020; R01CA108633 (PI:Chakravarti); 1RC2CA148190 (Scientific PI: Chakravarti)

Citation Format: Tiantian Cui, Ashley Gray, Ziyan Liu, Marjolein Geurts, Pierre Robe, Joseph McElroy, Erica Hlavin Bell, Arnab Chakravarti. A novel tumor-promoting role for miR-4516 in glioblastoma [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 5726. doi:10.1158/1538-7445.AM2017-5726
Abstract 5733: Expression levels of UL16 binding protein 1 and natural killer group 2 member D in patients with gastric cancer
Authors: Yoshimura Kiyoshi , Inoue Moeko , Asao Tetsuhiko , Fuse Masanori , Wada Satoshi , Kuramasu Atsuo
Published: July 2017

UL16 binding protein 1 (ULBP1) expressed on the tumor cell surface binds to the natural killer group 2 member D (NKG2D) receptor presenting on natural killer (NK), CD8+ T, and gamma delta T cells. However, the roles of ULBP1 and NKG2D expression and associated immune responses in gastric cancer are unclear. Here, we investigated the relationships between ULBP1 and NKG2D expression and clinical outcomes in patients with gastric cancer. The expression levels of ULBP1 and NKG2D were examined in human gastric cancer cell lines and gastric cancer tissues from 98 patients who underwent surgery from 2004 to 2008. MKN-72 cells expressed ULBP1 with ULBP2, -5, or -6. NKG2D was expressed at a higher level after activation on T cells and NK cells. Among tissue sections positive for NKG2D expression, six cases were positive for CD8 and CD56. In all tissues, NKG2D-expressing cells were typically aCD8+ T cells. Patients with NKG2D expression in tumors had significantly longer overall survival (OS) than patients without NKG2D expression in tumors (p = 0.0217). The longest OS was observed in patients positive for both ULBP1 and NKG2D, whereas the shortest OS was observed in patients negative for both ULBP1 and NKG2D. The interaction between ULBP1 and NKG2D may improve OS in patients with gastric cancer and may have applications in immunotherapy for induction of adaptive immunity in patients with cancer. Moreover, ULBP1 and NKG2D may be useful as prognostic biomarker in gastric cancer.

Citation Format: Kiyoshi Yoshimura, Moeko Inoue, Tetsuhiko Asao, Masanori Fuse, Satoshi Wada, Atsuo Kuramasu. Expression levels of UL16 binding protein 1 and natural killer group 2 member D in patients with gastric 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 5733. doi:10.1158/1538-7445.AM2017-5733
Abstract 5750: USP15 inhibits HPV16 E6 degradation and catalytically inactive USP15 has reduced inhibitive activity
Authors: Tokuda Aoi , Yoshimoto Masafumi , Yaginuma Yuji
Published: July 2017

HPVs can be classified as either high-risk or low-risk depending on the transforming potential of the virus. Most cervical cancers and a significant proportion of other anogenital cancers have been associated with infection by a small number of high-risk genital HPV types. Tumorigenesis induced by high-risk genital HPVs has been linked to the expression of two viral oncoproteins, E6 and E7, which cooperate in cellular immortalization and transformation processes. HPV16 E6 is an oncoprotein that causes the development of high-risk type human papilloma virus (HPV) and induces cancers, including oropharyngeal, anal, and cervical cancers. The stability of E6 is essential for its complete function as an oncoprotein. Using the yeast two-hybrid system, we identified ubiquitin-specific protease 15 (USP15) as an HPV16 E6-interacting protein. USP15 encodes a protein that consist of 952 amino acids and functions as a deubiquitinating enzyme. Deubiquitination is an essential process that releases ubiquitin chains from ubiquitin-protein conjugates. Aberrations in the ubiquitin-proteasome system have been recently connected to the pathogenesis of several human protein degradation disorders, including cancer and neurodegenerative diseases; as such, the proteasome is now considered to be important in the mechanism of carcinogenesis.

USP15 cleaves polyubiquitin chains of HPV16 E6 and/or ubiquitin precursors. Immunohistochemical analysis showed that USP15 was highly expressed in normal cervical squamous cells, and cervical cancer cells. Our results indicated that USP15 can increase the level of HPV16 E6 by inhibiting E6 degradation. USP15 dose-dependently inhibited the degradation of HPV16 E6. In contrast, catalytically inactive mutants of USP15 had a reduced inhibitory effect on E6 degradation. In particular, USP15 mutants of all three cysteine boxes and the NHL mutant of the KRF box had a drastically reduced inhibitive effect on HPV16 E6 degradation. In addition, HPV16 E6 mRNA was not induced by USP15; therefore, HPV16 E6 appears to be post-translationally regulated. These results suggest that USP15 has the ability to stabilize E6 as a deubiquitinating enzyme, and HPV16 E6 can affect biological functions as an oncoprotein in infected human cells.

Citation Format: Aoi Tokuda, Masafumi Yoshimoto, Yuji Yaginuma. USP15 inhibits HPV16 E6 degradation and catalytically inactive USP15 has reduced inhibitive activity [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 5750. doi:10.1158/1538-7445.AM2017-5750
Abstract 5751: Induction of cancer stem cell like characteristics by overexpression of CDX1
Authors: CHOI SANG IL , Hong Kyoung-Ok , Park Mi Ree , Na Yun Hee , Cho Soo-Jeong
Published: July 2017

Intestinal metaplasia (IM) has been regarded as the most significant risk factor for gastric cancer (GC). However, the molecular mechanisms of the pathogenesis of IM and the progression of GC remain unclear. The aim of this study was to evaluate the role of CDX1 in IM and GC using stable CDX1 overexpression in HFE 145 gastric epithelial cells, and its expression in patient samples. For CDX1 overexpression, we generated lentiviral CDX1 and infected is to HFE 145 cells. We found that CDX1 overexpression induced an increase of IM related markers such as villin and SI and a decrease of gastric epithelial marker such as MUC 5AC. Overexpression of CDX1 in HFE 145 cells caused an increase in cell proliferation (P<0.05) and showed 2.7-fold higher clonogenic ability than that of control cells. The expression of CDX1 induced in vitro invasiveness 1.9-fold higher compared to HFE 145 control cells with statistical significance (p=0.004), augmented the expression of MMP-9 and phosphorylation of FAK and paxillin. Overexpression of CDX1 also induced anchorage-independent colony formation and spheroid formation, and enhanced the expression of stem cell markers including SOX2, Oct4, NANOG and c-Myc in HFE 145 cells, suggesting CDX1-induced stem cell like phenotype of gastric epithelial cells. HFE 145 cells infected with CagA-positive Helicobacter pylori (G27 wild type) caused an increase in expression of CDX1 compared to HFE145 cells infected with CagA-negative H. pylori (G27 ΔCagE). Using luciferase promoter assay, we confirmed that the expression of CDX1 was regulated by the CagA secreted from H. pylori. In clinical practice, the Operative Link on Gastric Intestinal Metaplasia Assessment (OLGIM) staging system is often used to evaluate the degree of IM in biopsy samples or surgical specimen. We measured expression of CDX1, SI, and villin adjusted by glyceraldehyde 3-phosphate dehydrogenase expression using densitometry in 9 patients with various OLGIM stage (2 in stage 0, 1 in stage 1, 2 in stage 2, 1 in stage 3, 3 in stage 4). We were able to find out that the patients with higher OLGIM stage (stage 3-4) tended to have higher expression of CDX1 (3.935 vs. 0.416, p=0.160), SI (9.739 vs. 0.311, p=0.088), and villin (7.785 vs. 0.868, p=0.119) compared to patients with lower OLGIM stage (stage 0-2). Taken together, these results suggest that CDX1 overexpression could be one of the causes of GC tumorigenesis and progression. Further investigation on CDX1 molecule would encourage development of specific target for cancer treatment.

Citation Format: SANG IL CHOI, Kyoung-Ok Hong, Mi Ree Park, Yun Hee Na, Soo-Jeong Cho. Induction of cancer stem cell like characteristics by overexpression of CDX1 [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 5751. doi:10.1158/1538-7445.AM2017-5751
Abstract 5761: C-Fos drives non-tumorigenic head and neck squamous cell carcinoma cells to tumorigenic by promoting cancer stem cells like properties
Authors: Muhammad Naoshad , Bhattacharya Sourav , Steele Robert , Phillips Nancy , Ray Ratna B.
Published: July 2017

Purposes of the study: Head and neck squamous cell carcinoma (HNSCC) is the sixth most widespread cancer and leading cause of cancer related death world widely. Regardless of the advancement in treatment processes the overall survival rate of patients having this cancer has not substantially improved in the past few decades. Development of tumor and its progression exclusively resides in a small population of cancer cells, termed as cancer stem cells (CSCs). This population of cells can self-renew and re-form the ordered organization of tumors. CSCs contribute to HNSCC and may offer prominent therapeutic strategy for the management of HNSCC. We previously observed that c-Fos was highly up-regulated in the HNSCC sphere forming cells. In the present study, we explored the role of c-Fos in the tumor initiation in relation with cancer stemness and EMT.

Experimental procedures: We examined whether overexpression of c-Fos has an effect on tumorigenicity and stemness. For this, c-Fos was overexpressed in non-tumorigenic HNSCC cell lines and tumor growth was investigated. We also examined the expression of different CSC marker genes expression by Western blot analysis.

Summary: Our results demonstrated that exogenous expression of c-Fos in non-tumorigenic MDA1386Tu cells makes the cells tumorigenic in nude mice. Mechanistic investigations showed that c-Fos overexpression in MDA1386Tu cells enhanced epithelial-mesenchymal transition (EMT)/CSC properties and increased numbers of oralspheres when grown in ultra-low binding plate.

Conclusions: Collectively, our data strongly demonstrate that c-Fos plays a critical role in the modulation of EMT and cancer stem cell reprogramming in HNSCC cells and enhance tumor growth.

Note: This abstract was not presented at the meeting.

Citation Format: Naoshad Muhammad, Sourav Bhattacharya, Robert Steele, Nancy Phillips, Ratna B. Ray. C-Fos drives non-tumorigenic head and neck squamous cell carcinoma cells to tumorigenic by promoting cancer stem cells like properties [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 5761. doi:10.1158/1538-7445.AM2017-5761
Abstract 5763: Investigating the carcinogenic potential of various types of mineral fibers in the development of mesothelioma
Authors: Larson David , Tanji Mika , Napolitano Andrea , Pastorino Sandra , Carbone Michele , Yang Haining
Published: July 2017

Malignant mesothelioma is a very aggressive tumor and has been linked to occupational and environmental exposure to carcinogenic mineral fibers such as asbestos. It was discovered recently that individuals carrying germline BAP1 mutations are predisposed to mesothelioma in presence of even minimal exposure to carcinogenic mineral fibers. Mesothelioma causes about 3,200 deaths per year in the U.S. and over 40,000 deaths per year worldwide. Most malignant mesothelioma studies have focused on carcinogenesis due to occupational exposure to the six regulated industrial mineral fibers collectively called ‘asbestos’. Besides this family of six regulated asbestos minerals, there are other non-regulated fibers that possess physical and chemical structures that are similar to industrial asbestos, and may also be carcinogenic. However, many of those mineral fibers are unknown for their carcinogenesis. We conducted serious studies on this topic and established in vitro and in vivo assays to help determine the carcinogenesis of various types of mineral fibers. We found that high mobility box 1 protein (HMGB1) is a key initiator of asbestos-induced inflammation, which plays critical roles in the development and growth of mesothelioma, and that HMGB1 may be a good indicator for fiber carcinogenesis and a biomarker for fibers exposure. Moreover, we found that specific immune system changes that occur after exposure to carcinogenic fibers, which may be a useful tool to help interrogate the carcinogenic potential of mineral fibers. In particular, the ability to induce an M2 macrophage response appears related to the potent tumor-inducing capacity of carcinogenic mineral fibers.

Citation Format: David Larson, Mika Tanji, Andrea Napolitano, Sandra Pastorino, Michele Carbone, Haining Yang. Investigating the carcinogenic potential of various types of mineral fibers in the development of 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 5763. doi:10.1158/1538-7445.AM2017-5763
Abstract 5766: High-content microscopy-based screening of colorectal organoids
Authors: Rindtorff Niklas T. , Betge Johannes , Sauer Jan , Miersch Thilo , Zhan Tianzuo , Heigwer Florian , Scholl Claudia , Ebert Matthias , Fischer Bernd , Boutros Michael
Published: July 2017

High-content screening of cells has become a widespread approach for cellular assays due to its capacity to capture complex biological processes. However, conventional cell culture is limited with respect to cell and tissue architecture. Organoids are a unique model system for the intact and diseased intestinal epithelium. The 3D model can be used for the functional study of cancer development and, potentially, prospective therapeutic testing of drugs in patient derived tumor organoids. Here, we present a high-content microscopy based screening workflow to study organoid self-organization and growth with up to single cell resolution. After seeding of organoid fragments in a basal membrane extract, screening plates are incubated to allow for organoid formation. Subsequent treatment and incubation is followed by staining and imaging on a high-throughput microscopy platform followed by automated image analysis using open-source software. Profiling of both complete organoids and their individual architecture enables the quantitative description of population and tissue heterogeneity in the context of various perturbations. We generated four distinct colon organoid lines from mice carrying mutations of APC and KRAS in different combinations. These are profiled for differential phenotypic responses to a library of >1000 drug-like compounds. Also, this methodology is used to screen for clinically relevant differential treatment responses in patient derived tumor and normal colon organoids. Hence, based on this work we are able to analyze gene-drug interactions in early colon cancer development and drug response of patient derived colorectal cancer organoids.

Citation Format: Niklas T. Rindtorff, Johannes Betge, Jan Sauer, Thilo Miersch, Tianzuo Zhan, Florian Heigwer, Claudia Scholl, Matthias Ebert, Bernd Fischer, Michael Boutros. High-content microscopy-based screening of colorectal organoids [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 5766. doi:10.1158/1538-7445.AM2017-5766
Abstract 5773: Automation and miniaturization of 3D tumor models for compound screens
Authors: Kowalski Michael , Hill Kayla , Raksakulthai Vipat , Prasauckas Kristin , Jones-Roe Tara
Published: July 2017

Three-dimensional (3D) systems of cell culture can provide a more representative model of solid tumors and more physiologically relevant outputs from drug screens than two-dimensional cell cultures. A diverse array of 3D models have been used to investigate tumor physiology and susceptibility to chemotherapeutics but all of these models present challenges to establish and manipulate at the high throughputs required for screens. Cancer spheroids are large clusters of cells formed in suspension to replicate the gradients of gases, nutrients, and drugs seen by solid tumors. In contrast, Matrigel® or other hydrogel cultures are often used to replicate native cell morphology and polarity or to enable studies of 3D structure formation or cell migration. To overcome manual challenges and improve reliability, we automated the plating, drug treatment, and analysis of these 3D models. We were able to form consistent cancer spheroids in both hanging drop and low attachment plates and used imaging and flow cytometry to gain a more complete understanding of the cytotoxic drug response in this model. Cells were also plated on top of and embedded within Matrigel and cell growth and apoptosis induction was measured with standard and confocal imaging approaches. Both 3D models were miniaturized to 384-well format, thereby enabling the throughput required for large-scale screens to identify novel cancer treatments.

Citation Format: Michael Kowalski, Kayla Hill, Vipat Raksakulthai, Kristin Prasauckas, Tara Jones-Roe. Automation and miniaturization of 3D tumor models for compound screens [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 5773. doi:10.1158/1538-7445.AM2017-5773
Abstract 5781: Establishment of a 3D ex-vivo assay as a preclinical drug testing platform for personalized cancer therapy
Authors: Dhar Sumeer , Chiovaro Francesca , Tanos Tamara , Hejhal Tomas , Heilemariam Seife , Kelm Jens , Irmisch Anja , Levesque Mitchell
Published: July 2017

In vitro cell based drug testing tools have been widely used in drug discovery and early development to evaluate novel drug entities for further evaluation in preclinical in vivo models. However, a poor correspondence with in vivo models has compelled oncologists to pursue complementary in vitro strategies with better outcomes. Within this realm, a 3D ex vivo platform is being extensively used to evaluate the activity of cytotoxic drugs, targeted molecules, and antibodies. To increase the translational value of this model, maintenance of the tumor microenvironment (immune and other stromal cells) is highly pertinent. We have established a 3D ex vivo assay as a patient drug testing platform for personalized medicine therapies in collaboration with our clinical partners.

In this study we setup the protocols for obtaining single cell suspensions from biopsy and/or tumor resection samples from patients presenting with Urothelial cancers, Renal cell carcinoma, Pancreatic, NSCLC, Ovarian, and Melanomas to generate 3D microtumors in 96 well format cultured over a period of 7-15 days. The microtumors were monitored for growth characteristics, biomarker phenotype and drug activity profile. As expected the growth characteristics for each tumor varied, corresponding very well with clinical disease progression. Subsequently, these microtumors were assessed using a standard clinical immunohistochemistry diagnostics approach to evaluate disease specific biomarkers and were compared to the clinical diagnostics profile of the patients. The biomarker profile from 3D derived tumor samples showed concordance with the patient diagnostics profile. In the instances were clinical information and treatment regimens were available, the microtumors were tested and followed with single and pairwise drug treatments. The ex vivo 3D treatment outcome clearly reflected the clinical outcome. Currently, this platform is being used to develop 3D ex vivo immune-competent models to study immunomodulatory therapies. Based on the overall data we conclude that the 3D ex vivo assay system offers a highly pertinent platform to perform chemosensitivity testing providing predictive information on the clinical outcome, which enables oncologists to redefine individualized chemo/targeted treatments.

Citation Format: Sumeer Dhar, Francesca Chiovaro, Tamara Tanos, Tomas Hejhal, Seife Heilemariam, Jens Kelm, Anja Irmisch, Mitchell Levesque. Establishment of a 3D ex-vivo assay as a preclinical drug testing platform for personalized cancer therapy [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 5781. doi:10.1158/1538-7445.AM2017-5781
Abstract 5785: Characterization of a novel microfluidic in vitro model of the blood-tumor and blood-brain barrier
Authors: Griffith Jessica , Terrell-Hall Tori , Ammer Amanda , Lockman Paul
Published: July 2017

Background: There is a need for a cost-effective and reliable in vitro model of the blood-tumor barrier (BBB) and blood-brain barrier (BTB) for primary and metastatic lesions in brain. Current models do not accurately recapitulate physiological conditions and are unreliable with up to a 100-fold variance in predicting drug permeability into brain tumors.

Methods: Human umbilical vein endothelial cells (HUVECs) were cultured with either CTX-TRD2 rat astrocytes (BBB) or Met-1 murine breast cancer cells (BTB) in a microfluidic chip, which contained chambers separated by a porous wall. Shear stress was induced on HUVECs by perfusion of media through the apical chamber, and permeability was characterized by the diffusion of fluorescent tracers, one of which is subject to P-gp efflux. Data was compared to in-vivo work.

Results: Permeability of free Texas Red was significantly higher (p<0.05) in BTB chips (kin= 13.1 ± 1.3 X 10-3) than in the BBB chips (Kin=2.5 ± 0.3 X 10-3). There was a similar trend observed in larger markers, but these were not significantly different. The movement of Rhodamine 123 was also restricted, indicating functional P-gp efflux in vitro, similar to data from published in vivo studies. In the BBB model, diffusion of Rho123 increased 14-fold in the presence of verapamil (kin=14.7 ± 7.5 X 10-3) and eight fold with the addition of Cyclosporine A (kin=8.8 ± 1.8 X 10-3). In the BTB model, this increase was also observed, but to a lesser extent, as only 3 and 2-fold increases in permeability were observed, respectively. The magntidue of permeability changes were not significantly different from in-vivo models in both passive diffusion and active efflux experiments.

Conclusion: The novel microfluidic chip is a cost-effective in vitro model which recapitulates the BTB and BBB physiology and can predict permeability more reliably than current in-vitro models.

Citation Format: Jessica Griffith, Tori Terrell-Hall, Amanda Ammer, Paul Lockman. Characterization of a novel microfluidic in vitro model of the blood-tumor and blood-brain barrier [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 5785. doi:10.1158/1538-7445.AM2017-5785
Abstract 5787: Validation of spheroids with long-term incubation for high-throughput screening
Authors: Parry Jesse J. , Garner Steven M. , Cavedine Lee R. , Cracchiolo Alyssa M. , Epkins Brogan A. , Burkart Samantha C. , Sindelar Timothy J. , Wageman Charles R. , Warrior Usha , King Alastair J.
Published: July 2017

The OncoPanel™ service has established 100 genomically-characterized human tumor cell lines as targets for the screening of compounds in 3D spheroid models. We sought to (1) expand the cell line capability of the service and (2) validate the cell lines in a long-term (10-day) assay. For the cell line expansion, cells were plated at two densities in 384-well, spheroid-promoting plates, and were incubated for 3 days to allow formation of spheroids. The growth media were then removed and replaced with media containing the hypoxia probe, Lox-1. Twenty-four hours later, the ability to form spheroids was determined by bright-field, high-content imaging, and fluorescent detection of the Lox-1 probe in the hypoxic core of the spheroids. Cells were then seeded in assay plates at pre-determined densities, along with a time zero (T0) plate, and were incubated for 4 days to allow spheroid formation. Spheroid formation was verified by bright-field, high-content imaging, as previously mentioned. The growth media were removed and replaced with fresh media, followed by the addition of staurosporine over a range of 10 concentrations, using acoustic liquid transfer. At this time, the T0 plate was processed for subsequent doubling calculations. The assay plates were incubated for 10 days, re-imaged, and the spheroids were lysed using CellTiter-Glo® 3D viability reagent. Data were analyzed as the relative cell count, where the measured bioluminescence intensity was transformed to percent of control, as compared with a vehicle-treated control. Cellular response parameters were calculated using nonlinear regression to a sigmoidal single-site dose response model. A vehicle CV of <30% was required, as one criterion, for each cell line to pass quality control. Based on our tests of spheroid formation in the expansion cell lines, we have added 36 cell lines to the spheroid- or loose aggregate-forming cell line panel, with 20 cell lines validated in the long-term assay. Through the use of high-throughput screening and high-content imaging, we can verify and validate spheroid formation and growth in a 384-well format, with which we can test agents for drug discovery projects and provide value for potentially greater therapeutic outcomes.

Citation Format: Jesse J. Parry, Steven M. Garner, Lee R. Cavedine, Alyssa M. Cracchiolo, Brogan A. Epkins, Samantha C. Burkart, Timothy J. Sindelar, Charles R. Wageman, Usha Warrior, Alastair J. King. Validation of spheroids with long-term incubation for high-throughput screening [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 5787. doi:10.1158/1538-7445.AM2017-5787