bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3773: Tapping CD4 T cells for cancer immunotherapy
Authors: Wälchli Sébastien , Myhre Marit R. , Mensali Nadia , Fåne Anne , Lislerud Kari , Kvalheim Gunnar , Gaudernack Gustav , Inderberg Else M.
Published: July 2017

T-cell based immunotherapy represents an attractive strategy for the treatment of cancer. Whereas cellular anti-tumor immune responses have typically been attributed to CD8 T cells, CD4 T cells play a critical role in tumor elimination and the priming and maintenance of CD8 T-cell responses. Recent findings have highlighted new opportunities for CD4 T cells in cancer immunotherapy. We have isolated CD4+ T cells reactive against tumor antigens from patients who experienced clinical benefit from treatment with cancer vaccines targeting antigens such as hTERT, survivin and frequent neoantigens such as frameshift mutated TGFβRII. Strong T-cell responses against the vaccine or unrelated cancer antigens suggesting epitope spreading correlated with enhanced survival and tumor regression in late stage cancer patients. These HLA class II restricted T-cell clones recognised target cells loaded with long peptides or protein and for some CD4+ T cell clones we could also show direct tumor recognition. TCRs were expressed in expanded third-party T cells by mRNA electroporation or retroviral transduction and tested for functionality. Both CD8+ and CD4+ T cells expressing the TCRs produced TNF-α, IFN-γ and redirected T cells had the capacity to kill following co-incubation with their targets. Selecting highly functional CD4+ T-cell clones reactive against tumor-associated or -specific antigens from patients with clinical responses after treatment with immunotherapy is a successful method for identifying highly functional HLA class II restricted TCRs for adoptive immunotherapy. These HLA class II-restricted TCRs may be of therapeutic value both in haematopoietic malignancies and in melanoma where tumor cells frequently express HLA class II. Furthermore, combining HLA class I- and class II-restricted TCRs for T-cell redirection may provide a more potent therapeutic effect in adoptive T cell therapy.

Citation Format: Sébastien Wälchli, Marit R. Myhre, Nadia Mensali, Anne Fåne, Kari Lislerud, Gunnar Kvalheim, Gustav Gaudernack, Else M. Inderberg. Tapping CD4 T cells for cancer immunotherapy [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 3773. doi:10.1158/1538-7445.AM2017-3773
Abstract 3782: Genetic analysis using a novel high-purity enrichment system for circulating tumor cells independent of epithelial cell antigen
Authors: Takagi Hidenori , Kozuka Masahiro , Ito Hiroshi , Kim Soo Hyeon , Hirai Mitsuharu , Fujii Teruo
Published: July 2017

Background

Genetic analysis of circulating tumor cells (CTCs) is useful as a liquid biopsy. However, there are 3 challenging issues in processing of CTC samples for clinical use of the analysis: [1] numerous residual blood cells in processed samples, [2] loss of CTCs that do not express epithelial markers, and [3] very laborious process. Here, we developed a novel system capable of overcoming all of these problems.

Methods

Our CTC analysis system is composed of 3 steps: [1] filtering of whole blood followed by immunostaining and magnetic labeling of cells trapped on the filter, [2] depletion of white blood cells (WBCs) in the cells recovered from the filter by magnetic separation, and [3] trapping the resultant cells at an observation chamber in a microfluidic enrichment device using dielectrophoresis, followed by recovering them as a concentrated sample after fluorescence microscope observation. SNV and CNV analysis was conducted using the collected concentrated sample, and the system performance was substantiated. Genetic mutations of the cells in the collected sample were detected by the Quenching Probe method. On the other hand for CNV analysis, cells in the collected concentrated sample were immobilized on slide glass and analysis of gene amplification conducted by FISH.

Results

With SNV analysis, our system successfully detected EGFR, KRAS or PIK3CA mutations of cancer cell lines spiked in 8 mL of whole blood (Table). The detection sensitivity of our method was 1 cell/mL, and both the cancer cells and their genetic mutations were detected within 9 hours of starting the processing of whole blood. Additionally, with CNV analysis, HER2 gene amplification was confirmed with cell lines spiked in 8 mL of blood. This detection result was obtained within 48 hours.

Conclusion

Our system would be useful for the analysis of gene mutations in a wide range of CTC types independent of certain epithelial antigens, such as CK and EpCAM, and can be used for various genetic analyses.

Table Results of spiking test for SNV analysisSpiked cell linesExpected count[cells/8ml]No. of detected spiked cells [cells]No. of detected remained WBCs [cells]Detected genotypesNCI-H197512979EGFR L858RNCI-H16508225EGFR exon19 deletionSW6208648KRAS codon12, 13SNU-1124154KRAS codon12, 13MCF-777341PIK3CA G1633A

Citation Format: Hidenori Takagi, Masahiro Kozuka, Hiroshi Ito, Soo Hyeon Kim, Mitsuharu Hirai, Teruo Fujii. Genetic analysis using a novel high-purity enrichment system for circulating tumor cells independent of epithelial cell antigen [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 3782. doi:10.1158/1538-7445.AM2017-3782
Abstract 3791: Clinical significance of EpCAM-negative and CEA-positive circulating tumor cells in gastric carcinoma
Authors: Miki Yuichiro , Yashiro Masakazu , Okuno Tomohisa , Kitayama Kishu , Tamura Tatsuro , Toyokawa Takahiro , Tanaka Hiroaki , Muguruma Kazuya , Hirakawa Kosei , Ohira Masaichi
Published: July 2017

It has been reported that the examination of circulating tumor cells (CTCs) is beneficial for predicting tumor stage or treatment response. The epitherial cell adhesion molecule (EpCAM) and cytokeratin has been used as epithelial markers for the identification of CTCs. Since the epithelial-mesenchymal transition is one of malignant phenotype of cancer cells, it is necessary to investigate other markers which might detect mesenchymal CTCs.

This study aims to clarify the clinical significance of EpCAM negative and CEA positive CTCs in patients with gastric cancer.

Forty patients who underwent operation for gastric cancer were enrolled in this study. A total of 10ml peripheral blood samples were obtained preoperatively. Informed consent to participate in this study was obtained from all of the patients before their surgery. This study was approved by the Human Ethics Review Committee of our University. After collection of mononuclear cell fraction, these cells were stained by PAPI, anti-CD45, anti-EpCAM, anti-cytokeratin, and anti-CEA (CD66e) antibody. We enumerated the number of cells which showed EpCAM, cytokeratin, and CEA positive expression among CD45 negative cells. ROC analyses were performed for investigating predictive value of these cell counts for distant metastasis. We examined association between these cell counts and clinicopathological features.

ROC analyses revealed that the AUC for predicting distant metastasis using EpCAM, Cytokeratin, and CEA positive cell counts were 0.583, 0.638, and 0.769, respectively. The median number of EpCAM negative and CEA positive cells was 231 (range: 0-61897). The cell counts of EpCAM negative and CEA positive cells were significantly associated with Tumor depth (p=0.0084), lymph node metastasis (p=0.0107), and tumor size (p=0.023).

CEA might be useful marker for detecting high risk of recurrence in patients with gastric cancer.

Citation Format: Yuichiro Miki, Masakazu Yashiro, Tomohisa Okuno, Kishu Kitayama, Tatsuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. Clinical significance of EpCAM-negative and CEA-positive circulating tumor cells in gastric carcinoma [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 3791. doi:10.1158/1538-7445.AM2017-3791
Abstract 3802: A novel uracil-DNA glycosylase, UdgX, as a new biochemical tool to directly detect uracils in DNA
Authors: Stewart Jessica , Wei Shanqiao , Datta Madhurima , Varshney Umesh , Bhagwat Ashok
Published: July 2017

The enzyme activation-induced deaminase (AID) is essential to the B lymphocyte development and antibody maturation. It converts cytosines in DNA to uracil. The unregulated expression of AID in many B cell cancers is correlated with an increased load of mutations. AID also promotes DNA strand breaks and is required for chromosomal translocations such as IgH/Myc in these tumors. We have previously showed that B cell cancer cell lines and patient tumors which express AID at high levels also have high levels of uracil in their genomic DNA. We accomplished this by excising the uracils using a uracil-DNA glycosylase (Ung) and labeled the resulting in an abasic site with an aldehyde reactive chemical (ARP). ARP contains a biotin tag and it was detected using a fluorescently labeled streptavidin. The results from this assay showed B cell tumors have as much as 100-fold higher levels of genomic uracils. Further development of this assay has allowed the detection of uracils in the blood plasma and buffy coat of B cell cancer patients. This “liquid biopsy” assay technique has the potential to work as a simple surveillance tool for B cell cancer patients who are in remission. In addition, we are exploring the possibility of a direct detection of uracil in DNA, which would provide greater sensitivity to this assay and simplify it. A unique uracil-DNA glycosylase was recently isolated from M. smegmatis, UdgX, that binds covalently to uracils in DNA. We are developing multiple ways to detect uracil-DNA-UdgX complexes and amplifying the resulting signal. We expect that this will allow us to directly visualize uracils in B-NHL tumors and detect uracils in circulating B-NHL tumor DNA from a drop of patient plasma.

Citation Format: Jessica Stewart, Shanqiao Wei, Madhurima Datta, Umesh Varshney, Ashok Bhagwat. A novel uracil-DNA glycosylase, UdgX, as a new biochemical tool to directly detect uracils in DNA [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 3802. doi:10.1158/1538-7445.AM2017-3802
Abstract 3808: TM4SF1 regulates cell cycle and ROS status in bladder cancer cells by targeting FOXO3a via PPARγ-SIRT1 negative feedback loop
Authors: Cao Rui , Wang Gang , Qian Kaiyu , Chen Liang , Hu Qingxuan , Xiao Yu , Wang Xinghuan
Published: July 2017

Transmembrane 4 superfamily member 1 (TM4SF1) is a member of tetraspanin family, which locates in the cell membrane, functions as signal transducer to regulate cell development, growth and motility. Currently, TM4SF1 has been reported to be overexpressed in liver cancer, pancreatic cancer and colorectal cancer, but its role in bladder cancer (BCa) has not been illustrated. We have found that TM4SF1 was strongly upregulated in human bladder cancer tissues at both transcription and translation levels, compared with the paracancerous tissues and normal bladder tissues. This result was corroborated with our transcriptome analysis. Moreover, the tissue array data showed that TM4SF1 closely related with survival rate of BCa as the patients with high expression of TM4SF1 have a poorer prognosis. Furthermore, in vitro and in vivo studies suggested that proliferation of BCa cells was significantly suppressed by knockdown of TM4SF1. Moreover, downregulation of TM4SF1 induced G0/G1 phase cell cycle arrest and apoptosis possibly via induced reactive oxygen species (ROS) status in BCa cells. In addition, we observed that phosphorylated/total SIRT1 proteins were considerably reduced in the BCa cells lacking TM4SF1. In contrast, PPARγ as well as its target phosphorylated/total FOXO3a were strongly increased, an important downstream transcriptional factor of PPARγ/SIRT1 negative feedback loop, regulating cell cycle, apoptosis and ROS metabolism. Therefore, we treated the TM4SF1-silencing BCa cells with GW9662 (antagonist of PPARγ) and resveratrol (activator of SIRT1), showing a recovery of the G0/G1 phase cell cycle arrest and scavenge of ROS status. Interestingly, the increase of phosphorylated/total FOXO3a by knockdown of TM4SF1 could also be rescued by using GW9662 and resveratrol. In conclusion, our results suggested that TM4SF1 may regulate cell cycle and ROS status of BCa cells by targeting FOXO3a via the PPARγ-SIRT1 negative feedback loop.

Note: This abstract was not presented at the meeting.

Citation Format: Rui Cao, Gang Wang, Kaiyu Qian, Liang Chen, Qingxuan Hu, Yu Xiao, Xinghuan Wang. TM4SF1 regulates cell cycle and ROS status in bladder cancer cells by targeting FOXO3a via PPARγ-SIRT1 negative feedback loop [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 3808. doi:10.1158/1538-7445.AM2017-3808
Abstract 3818: Identification of a FGFR3-TACC3 fusion in esophageal cancer
Authors: Mizukami Takuro , Sakai Kazuko , Naruki Saeko , Taniyama Tomoko , Horie Yoshiki , Izawa Naoki , Tsuda Takashi , Fujino Takashi , Boku Narikazu , Yasuda Hiroshi , Fukunaga Tetsu , Nakajima Takako Eguchi , Nishio Kazuto
Published: July 2017

Aberrant activation of fibroblast growth factor (FGF) signaling has been found to contribute to carcinogenesis. Recently, activating FGFR fusions are detected in several malignancy. However, limited information is available regarding the existence of FGFR fusions in esophageal squamous cell cancer (ESCC) and gastric cancer (GC). In this report, we explored major FGFR1, FGFR2, and FGFR3 fusion transcripts in ESCC and GC specimens (n = 74 and 114, respectively) using a most comprehensive next-generation sequencing panel. We detected a targetable fusion between exon 18 of FGFR3 and exon 11 of TACC3 at a frequency of 1.4% (1/74) in ESCC, whereas other FGFR fusions were not detected in the ESCC or GC specimens analyzed. Furthermore, split FGFR3 signals were apparent in this specimen by fluorescence in situ hybridization (FISH) with break-apart probes. The patient, a 64-year-old man with unresectable T4bN3M1 poorly differentiated ESCC of stage IV, was treated with palliative chemoradiotherapy followed by taxane chemotherapy. The patient died of cancer progression, with his overall survival time having been 9.5 months.

Our results provide sequence information that should prove useful for development of simple and cost-effective diagnostic assays as an alternative to FISH for patients with ESCC harboring the FGFR3-TACC3 fusion.

Citation Format: Takuro Mizukami, Kazuko Sakai, Saeko Naruki, Tomoko Taniyama, Yoshiki Horie, Naoki Izawa, Takashi Tsuda, Takashi Fujino, Narikazu Boku, Hiroshi Yasuda, Tetsu Fukunaga, Takako Eguchi Nakajima, Kazuto Nishio. Identification of a FGFR3-TACC3 fusion in esophageal 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 3818. doi:10.1158/1538-7445.AM2017-3818
Abstract 3820: Hepatitis B virus gene pre-S2 mutant as a high risk serum marker for hepatoma recurrence after a hepatic resection
Authors: Huang Wen-Ya
Published: July 2017

Background: Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S2 mutant large HBV surface antigen (LHBS) is highly associated with HCC. This study aimed to evaluate the serum pre-S2 mutant LHBS as a high-risk recurrence marker in HCC patients after a hepatic resection.

Methods: The pre-S gene chip was used to analyze 175 patients with HBV-related HCC who underwent a hepatic resection between 2008 and 2012. Thirty independent pre-S clones in every patient were analyzed for a semi-quantitative detection.

Results: Multivariate regression analysis showed that the serum pre-S2 mutant level (≥ 5%) and the American Joint Committee on Cancer (AJCC) tumor stage were two independent high-risk factors for HCC recurrence. A Cox proportional hazards analysis also showed that the combined evaluations of the AJCC stage and serum pre-S2 mutant level provided a reliable predictive marker for HCC recurrence after primary surgery. We developed a Cox prediction model, which indicated the recurrence-free survival rates along with the time after surgery. This model was validated in an independent HCC cohort. Receiver operating characteristic curve analysis revealed that the model showed close sensitivities in the main and validation cohorts (AUC values: 0.741 and 0.704, respectively).

Conclusions: The relative level of pre-S2 mutant in serum is, independently of tumor stage, an important predictive high-risk marker for HCC recurrence after a primary hepatic resection. The combined evaluations of the AJCC tumor stage and serum pre-S2 mutant level potentially serve as a convenient high-risk predictive marker for HCC prognosis.

Citation Format: Wen-Ya Huang. Hepatitis B virus gene pre-S2 mutant as a high risk serum marker for hepatoma recurrence after a hepatic resection [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 3820. doi:10.1158/1538-7445.AM2017-3820
Abstract 3834: Tracking CAD-ALK gene translocation in urine and plasma of a colorectal cancer patient treated with ALK blockade
Authors: Siravegna Giulia , Sartore-Bianchi Andrea , Mussolin Benedetta , Palmieri Laura , Tosi Federica , Cassingena Andrea , Novara Luca , Buscarino Michela , Corti Giorgio , Crisafulli Giovanni , Bartolini Alice , Erlander Mark , Nicolantonio Federica Di , Siena Salvatore , Bardelli Alberto
Published: July 2017

A metastatic colorectal cancer (mCRC) patient carrying CAD-ALK translocation achieved partial response to an experimental ALK inhibitor and then progressed after 5 months. We studied whether urine cell-free, trans-renal DNA (tr-DNA) could be used to monitor tumor burden and patient’s response. A NGS panel was developed to interrogate 52 common cancer gene rearrangements and 14 frequently mutated genes in cancer patients. A TP53 p.R248W mutation and the CAD-ALK genomic breakpoint (rearrangement) were identified in the tumor tissue and matched plasma circulating tumor DNA (ctDNA) Urine samples were longitudinally obtained from the patient during ALK inhibitor treatment in parallel with blood. To detect the CAD-ALK translocation in urine tr-DNA we designed ultra-short (51 bp amplicon) primer pairs spanning the genomic breakpoint as a unique tumor marker. This approach allowed the non-invasive monitoring of the gene fusion in urine with amounts paralleling tumor burden. Of note, CAD-ALK gene fusion was apparent in urine tr-DNA before radiological confirmation of disease progression. The same strategy was applied to plasma ctDNA and the results were compared. To detect point mutations in urine tr-DNA, we exploited a peptide nucleic acids (PNA)-CLAMP PCR coupled with droplet digital PCR (ddPCR) analysis to specifically suppress amplification of wild-type DNA fragments. Custom PNA probes were designed for TP53 codon 248, and a ddPCR assay was optimized to detect the TP53 p.R248W mutation, which was then identified in all urine tr-DNA samples, with absolute copies correlating with tumor burden throughout ALK inhibitor treatment. In conclusion, we find that urine tr-DNA can be exploited to non-invasively monitor tumor burden by detecting tumor-specific gene fusions as well as point mutations.

Citation Format: Giulia Siravegna, Andrea Sartore-Bianchi, Benedetta Mussolin, Laura Palmieri, Federica Tosi, Andrea Cassingena, Luca Novara, Michela Buscarino, Giorgio Corti, Giovanni Crisafulli, Alice Bartolini, Mark Erlander, Federica Di Nicolantonio, Salvatore Siena, Alberto Bardelli. Tracking CAD-ALK gene translocation in urine and plasma of a colorectal cancer patient treated with ALK blockade [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 3834. doi:10.1158/1538-7445.AM2017-3834
Abstract 384: The implications of splicing variant of AIMP2 lacking exon 2 among various cancer types: An analysis of the ICGC/TCGA database and clinical validation
Authors: Kim Dong Chan , Kim Ryul , Kim Daeyoon , Song Hyojin , Shin Dong-Yeop , Kim Inho , Ahn Kwang-Sung , Kwon Nam Hoon , Kim Sunghoon , Yoon Sung-Soo , Koh Youngil
Published: July 2017

Aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMP) is the multiple tRNA synthetase complex protein called the multi-tRNA complex (MRC). In cancer, the splicing variant of AIMP2 derives a several signaling cascades, which are crucial for cancer proliferation. Detecting an exon-2 depleted splicing variant (AIMP2-DX2) is an issue of growing importance in cancer therapy. This study suggests the evidence for interrelation between the AIMP2-DX2 and cancer development. We analyzed AIMP2 and AIMP2-DX2 gene expression and their ratio on 7 commercial cancer cell lines and Multiple myeloma patient derived 536MM cell line by RT-PCR and targeted RNA sequencing. Extended this profile, the distribution of AIMP2-DX2/AIMP2 ratio and AIMP2-related major cancer pathways were analyzed using the samples in the ICGC/TCGA database. Over 23 cancer types, 753 samples were used in WTS analysis. In the DEG set analysis, 10 pre-defined major cancer pathways were analyzed among 16 cancer types. Some cancer types, especially acute myeloid leukemia (AML) showed most significant association with AIMP2-DX2 in terms of cancer signaling pathways. We focused on clinical implications of AIMP2-DX2/AIMP2 ratio in the ICGC/TCGA database. 19 AML samples were used, Overall survival (OS) showed that patients with AIMP2-DX2/AIMP2 ratio higher than Q1 shows poor OS and Most of the genes including MEK1/2, ERK, MNK1/2 in this pathway had positive association with AIMP2-DX2/AIMP2 ratio. In colon carcinoma and hepatocellular carcinoma, OS curves had a tendency in a similar way to AML. For the clinical validation of the prognostic value of AIMP2-DX2, 51 AML patients were included in this analysis. The correlation between AIMP2-DX2 expression and survival outcomes was investigated in clinical validation cohort of AML. The AIMP2-DX2-positive group had significantly inferior OS rate and had worse RFS compare to AIMP2-DX2-negative group. Our sequential data shows that the AIMP2-DX2/AIMP2 expression and their ratio can possibly be an indicator to measure malignancy of various cancer types.

Citation Format: Dong Chan Kim, Ryul Kim, Daeyoon Kim, Hyojin Song, Dong-Yeop Shin, Inho Kim, Kwang-Sung Ahn, Nam Hoon Kwon, Sunghoon Kim, Sung-Soo Yoon, Youngil Koh. The implications of splicing variant of AIMP2 lacking exon 2 among various cancer types: An analysis of the ICGC/TCGA database and clinical validation [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 384. doi:10.1158/1538-7445.AM2017-384
Abstract 3863: A subset of poorly prognostic pediatric posterior fossa ependymomas exhibit lowered H3K27me3 and DNA hypomethylation and show epigenetic similarities with H3K27M mutant diffuse intrinsic pontine gliomas
Authors: Venneti Sriram , Bayliss Jill , Mukherjee Piali , Lu Chao , Jain Siddhant , Chung Chan , Martinez Daniel , Sabari Benjamin , Margol Ashley , Panwalkar Pooja , Paroloia Abhijit , Pekmezci Melike , Eachin Richard Mc , Cieslik Marcin , Tamrazi Benita , Garcia Benjamin , Rocca Gaspare La , Santi Mariarita , Lewis Peter , Hawkins Cynthia , Melnick Ari , Allis C David , Thompson Craig B. , Chinnaiyan Arul , Judkins Alexander R.
Published: July 2017

Pediatric posterior fossa ependymomas are poorly understood childhood brain tumors and have no effective treatments. The biology of these tumors is obscure as recent sequencing efforts suggest that they lack recurrent genetic alterations. A subset of these tumors termed PF-A ependymomas exhibits CpG-island hypermethylation implicating epigenetic alterations in their pathogenesis. Through comprehensive analyses of histone modification, we discovered global H3K27me3 reduction in a subset of these tumors. Tumors with lowered H3K27me3 showed many clinical and biologic similarities with PFA-ependymomas. Global reduction in H3K27me3 is likewise observed in pediatric gliomas that bear histone H3K27M mutations termed diffuse intrinsic pontine gliomas (DIPG) that also arise in the posterior fossa of young children. Analyses of ependymomas with reduced H3K27me3 and H3K27M mutant DIPGs showed many similarities in DNA methylation and enrichment of H3K27me3 in many genomic loci important for neuroglial specification. Combined integrative analysis of both tumor types uncovered common epigenetic deregulation of select factors that control radial glial biology and radial glia in the developing posterior fossa showed reduced H3K27me3. Finally, PF ependymomas with lowered H3K27me3 were more invasive radiologically and exhibited poor prognosis in three independent cohorts (P<0.001 in all three cohorts, collective n>300). These data have clinical implications for biomarker development and to inform epigenetic approaches to treat PF ependymomas.

Citation Format: Sriram Venneti, Jill Bayliss, Piali Mukherjee, Chao Lu, Siddhant Jain, Chan Chung, Daniel Martinez, Benjamin Sabari, Ashley Margol, Pooja Panwalkar, Abhijit Paroloia, Melike Pekmezci, Richard Mc Eachin, Marcin Cieslik, Benita Tamrazi, Benjamin Garcia, Gaspare La Rocca, Mariarita Santi, Peter Lewis, Cynthia Hawkins, Ari Melnick, C David Allis, Craig B. Thompson, Arul Chinnaiyan, Alexander R. Judkins. A subset of poorly prognostic pediatric posterior fossa ependymomas exhibit lowered H3K27me3 and DNA hypomethylation and show epigenetic similarities with H3K27M mutant diffuse intrinsic pontine gliomas [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 3863. doi:10.1158/1538-7445.AM2017-3863
Abstract 3865: Targeting the epigenetic modifier HMGA2 in lethal adult and pediatric gliomas inhibits invasion, growth and tumorigenicity
Authors: Kaur Harpreet , Ali Sabeen Z. , Guo Huizi , Akhtarkhavari Sepehr , Rodriguez Fausto , Eberhart Charles G. , Raabe Eric H.
Published: July 2017

Glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG) are highly infiltrative, incurable adult and pediatric brain tumors characterized by the presence of tumor stem-like cells. Although GBM and DIPG have different epigenetic drivers, we found increased expression of an epigenetic modifier and stem cell factor HMGA2 in both tumors by immunohistochemistry and western blotting. HMGA2 is a DNA-binding protein that regulates transcription during normal embryogenesis and in cancer stem cells. We hypothesized that HMGA2 contributes to high grade glioma tumorigenicity through its ability to alter the transcription of many genes. Lentiviral short hairpin RNA (shRNA)-mediated reduction of HMGA2 in multiple patient-derived GBM cell lines significantly reduced invasion in transwell assay (shHMGA2 vs. shControl, P<0.01). Similarly, shRNA mediated suppression of HMGA2 in DIPG cell lines reduced proliferation (BrdU incorporation), reduced invasion (transwell assay), inhibited clonogenicity (soft agar assay) and increased apoptosis (cleaved caspase-3 immunofluorescence, shHMGA2 vs. shControl, P<0.001). Pharmacological inhibition of HMGA proteins using the DNA minor-groove binding drug Netropsin significantly inhibited invasion (transwell assay), decreased proliferation (BrdU incorporation) and increased apoptosis of DIPG neurospheres (P<0.01). Mice bearing orthotopic GBM xenografted cells transduced with HMGA2 shRNA lived significantly longer (108 days) compared to control shRNA (67.5 days, P<0.0001 by log-rank analysis). To investigate the mechanism of HMGA2-mediated malignancy, we discovered high levels of CD44 (a known mediator of invasion and stemness) that correlated with high HMGA2 expression in DIPG and GBM tumors. Suppression of HMGA2 led to decreased CD44 expression while ectopic expression of HMGA2 increased CD44 expression in GBM cell neurospheres. Knockdown of CD44 in HMGA2-high GBM neurospheres reduced invasion (P<0.01). Our studies suggest an oncogenic role of HMGA2 and its downstream effectors in gliomas and identify this important pathway as a potential therapeutic target. We conclude that minor groove binding agents similar to Netropsin may be effective agents for HMGA2-expressing pediatric and adult high grade gliomas.

Citation Format: Harpreet Kaur, Sabeen Z. Ali, Huizi Guo, Sepehr Akhtarkhavari, Fausto Rodriguez, Charles G. Eberhart, Eric H. Raabe. Targeting the epigenetic modifier HMGA2 in lethal adult and pediatric gliomas inhibits invasion, growth and tumorigenicity [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 3865. doi:10.1158/1538-7445.AM2017-3865
Abstract 3869: Cell surface vimentin targeted mAb 86C increases sensitivity to temozolomide mediated cell death in glioma stem cells
Authors: Noh Hyangsoon , Yan Jun , Gabrusiewicz Konrad , Li Shulin
Published: July 2017

Glioblastoma multiforme (GBM) is one of the most aggressive brain tumor in pediatric patients. The current standard of care including radiation and chemotherapy is not effective in most patients due to multiple factors such as resistance and poor blood-brain barrier (BBB) penetration. The development of strategies to reduce resistance and to increase sensitivity to chemotherapy may ameliorate the problems. Glioma stem cells (GSCs) are considered the source of relapse and chemoresistance. Sensitization of temozolomide TMZ resistance in GSC is therefore integral for therapeutic efficacy. Our lab has discovered that cell surface vimentin (CSV) is presented on patient-derived GSCs. In this study, we demonstrate that the treatment of GSCs with anti-CSV antibody (86C) sensitizes them to TMZ. We found that the combination of 86C and TMZ induced additional antitumor effects in 8 out of 12 GSCs. Mechanistic study of the four resistance GSCs revealed slow re-surface rate of CSV from CSV- GSCs and low CSV expression on GSCs as possible contributing factors. GSCs with rapid CSV resurfacing from CSV- GSCs was more sensitive to combination treatment compared to GSCs with a slow recovering from CSV- GSCs. Furthermore, the metabolism study shows these four resistance cells have high intrinsic mitochondria activity compared to sensitive cells. The combination of TMZ with 86C may represent a valuable strategy to reverse GSC chemoresistance.

Citation Format: Hyangsoon Noh, Jun Yan, Konrad Gabrusiewicz, Shulin Li. Cell surface vimentin targeted mAb 86C increases sensitivity to temozolomide mediated cell death in glioma stem cells [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 3869. doi:10.1158/1538-7445.AM2017-3869
Abstract 3871: Modeling the chromatin and transcriptional landscape of MYC and MYCN driven neuroblastoma in zebrafish
Authors: Zimmerman Mark W. , He Shuning , Zhu Shizhen , Yang Song , Zhou Yi , Zon Leonard I. , Look A Thomas
Published: July 2017

Elevated expression levels of MYC family genes are frequently observed in human cancer cells and correlate with tumor aggressiveness and poor prognosis. In neuroblastoma 40% of all cases are high-risk, of which 20% harbor amplification of the MYCN proto-oncogene. In high-risk cases lacking MYCN gene amplification, high expression levels of c-MYC (MYC) are often present and are associated with unfavorable histology and a poor survival. Unlike MYCN amplification, which is frequently observed in the presence of segmental chromosomal aberrations, MYC overexpression is not associated with genetic abnormalities or somatic mutations. In order to study this newly defined subgroup, we have created a novel transgenic zebrafish model in which overexpression of MYC alone in the peripheral sympathetic nervous system (PSNS) drives early-onset neuroblastoma in nearly every fish by seven weeks of age. The tumors resulting from MYC overexpression arise in the interrenal gland, which is the fish counterpart of the adrenal medulla, and are histologically identical to human neuroblastoma. We next performed the Assay for Transposase Accessible Chromatin (ATAC) sequencing and RNA-seq to identify open chromatin regions that correlate with activation of gene transcription. Lineage specific genes essential for neuronal precursor cell identity, such as PHOX2B, HAND2, and TFAP2A are highly expressed in both MYC-expressing and MYCN-amplified human neuroblastoma cell lines and are actively transcribed in zebrafish models of MYC and MYCN driven neuroblastoma. Furthermore, these studies reveal shared and differential regulatory of effects of MYC relative to MYCN activity in maintaining the malignant phenotype of neuroblastoma in vivo. Additional insight into the mechanisms of aberrant transcriptional regulation will inform the future design and use of therapeutic strategies targeting transcription in this high-risk malignancy of childhood.

Citation Format: Mark W. Zimmerman, Shuning He, Shizhen Zhu, Song Yang, Yi Zhou, Leonard I. Zon, A Thomas Look. Modeling the chromatin and transcriptional landscape of MYC and MYCN driven neuroblastoma in zebrafish [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 3871. doi:10.1158/1538-7445.AM2017-3871
Abstract 3873: Novel non-genetic mechanisms drive rhabdomyosarcoma tumorigenesis
Authors: McEvoy Justina , Dyer Michael , Chen Xiang
Published: July 2017

Most pediatric solid tumors originate from perturbations during development, however the mechanisms that drive these disruptions and promote tumor progression are still poorly understood. We believe that epigenetic deregulation of cancer genes and developmental pathways is a common driver in pediatric cancers, especially those with a low mutation rate. To test this novel idea, we are focusing on rhabdomyosarcoma (RMS), a developing muscle tumor with striking genetic differences between two of the major subtypes. We hypothesize that differential epigenetic deregulation of differentiation programs and cancer genes between the major subtypes are essential for tumorigenesis. Indeed, our preliminary integrative analysis reveal distinct epigenetic landscapes between the two subtypes particularly at the loci of HOX cluster genes A-C, including an oncogenic HOXC long non-coding RNA (lncRNA) called HOTAIR. Knockdown of HOTAIR led to derepression of HOXD10, a candidate tumor suppressor, and a decrease in RMS proliferation, migration, and invasion. Thus, we believe HOX gene deregulation is required for tumorigenesis, and their implication in several adult cancers suggests this currently unappreciated mechanism is likely to be more broadly relevant. In addition, we identified a group of RMS specific novel lncRNAs, of which one demonstrates an essential role in cell survival. Our innovative approach to integrate epigenetic data sets from tumor types with varying genetic differences shed light on the mechanisms that drive tumorigenesis, which may lead to identifying previously unknown potential therapeutic targets. Since current therapeutic options have not changed over the last twenty-five years, these data will especially provide a strong translational impact for children with RMS, whose survival outcomes are less than 20% in the case of metastatic disease.

Note: This abstract was not presented at the meeting.

Citation Format: Justina McEvoy, Michael Dyer, Xiang Chen. Novel non-genetic mechanisms drive rhabdomyosarcoma tumorigenesis [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 3873. doi:10.1158/1538-7445.AM2017-3873
Abstract 3881: The expression of CD98hc might be a marker of radiation resistance in HNSCC
Authors: Kawasaki Yohei , Omori Yasufumi
Published: July 2017

Introduction: Head and Neck squamous cell carcinoma(HNSCC) represents an aggressive malignancy. Although new advanced therapeutic strategies have been applied to date, the 5-year survival rate have not been considerably improved. Generally speaking, high radio-sensitive HNSCC has good prognosis, however, there are radio-resistant HNSCC. Recently, Sanne et al, have reported CD98 is one of the marker of Cancer Stem Cell. Moreover, the over expression of CD98hc is high associated with different cancers including the colon cancer, renal cancer. CD98 is heterodimeric protein that comprises a heavy chain and a light chain. The CD98 heavy chain is type 2 linkage to one of 6 amino acid transporters. We hypothesize that irradiation may contribute to overexpression of CD98hc and expansion Cancer Stem Cell population in HNSCC.

Methods:cell culture: Ho-1-u-1(Mouth floor), Sa3(Gingiva), HSC2, HSC3,HSC4(tongue) were used for this experiment. These cell lines were exposed to 60 Gy (2Gy/day) to establish stable cell lines to radiation.Flow cytometry: For analysis of CD98-positive fraction, cell pellets were incubated with FITC-conjugated anti-human CD98hc mAb at the dilution of 1:11 at 4°C for 15min.Sphere formation assay: Cells were cultured in a serum-free medium. 1x104 cells were seeded in 80μl of serum free RPMI1640 medium. The recipe of which was mentioned above. Containing 1.0% agar on 100μl of a solidified serum free RPMI1640 was poured on the top of each well. The cells were incubated for 20 days in the same atomosphere as that for the monolayer culture.Migration Assay: Radius 24 well Cell Migration (Cell Biolabs, INC.) were purchased. The experiment was conducted according to the decided plotocol.

Result: In radiation resistant cells, spindle-shaped and large cytoplasmic cells were founded. After irradiation, the expression of CD98hc increased in all cell lines. In Ho-1-u-1, the population was divided into two distinct subpopulations showing a higher and a lower expression of CD98hc. Irradiated cancer cells exhibit an efficient ability to developed numerous large sphere. Irradiation had enhanced self-renewal of Cancer Stem Cells. Cell motility was higher in radio-resistant cells than their parental cells.

Conclusion: Radio-resistant cells overexpressed CD98hc. In other words, CD98hc may be a marker of radiation resistance. Since incomplete radiation therapy can expand Cancer Stem Cell population, so elucidation of mechanism of radiation resistance will improved radiotherapy. CD98hc might be the target for more efficient treatment.

Citation Format: Yohei Kawasaki, Yasufumi Omori. The expression of CD98hc might be a marker of radiation resistance in HNSCC [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 3881. doi:10.1158/1538-7445.AM2017-3881
Abstract 3884: A tumor stem cell marker DCLK1 promotes hepatocellular carcinoma by regulating β-catenin, EMT and clonogenic properties of hepatocytes
Authors: Ali Naushad , Nguyen Charles B. , Chandrakesan Parthasarathy , May Randal , Dhanasekaran Danny , Bronze Michael S. , Houchen Courtney W.
Published: July 2017

Introduction: Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related deaths worldwide with a 5-year survival rate of <15%. Chronic viral hepatitis, NASH, and cirrhosis are major risk factors for the HCC development. Thus, HCC represents a major public health issue worldwide. Currently, sorafenib is the only FDA-approved drug used for HCC treatment. Unfortunately, it increases patient's survival by only 3 months. Tumor stem cells (TSCs) are likely responsible for resistance to chemotherapy, relapse, and metastasis. We have demonstrated that a TSC marker, doublecortin-like kinase (DCLK1), is induced during hepatic injury, cirrhosis and HCC and its overexpression in HCC patient’s liver significantly reduces survival. Therefore, understanding of the mechanism(s) hepatic TSCs regulation is important for the development of effective therapy against HCC.

Methods: Hepatoma Huh7 cells and primary human hepatocytes were infected with lentiviruses expressing recombinant RFP-DCLK1 fusion protein. The RFP-DCLK1-positive cells were isolated using FACS and subjected to immunofluorescence confocal microscopy, clonogenic assay, Western blot and xenograft in mice.

Results: DCLK1-overexpressing human hepatocytes formed spheroids but not the control cells. DCLK1 overexpression induced accumulation of active β-catenin in the perinuclear and nuclear regions in Huh7 cells with concomitant increase in c-Myc. The DCLK1-negative cells lacked active β-catenin and total β-catenin was observed only in their cell membranes. Hepatic β-catenin mRNA levels were unaffected during various stages of HCC. Analysis of HCC patients’ liver tissues (n= 369) suggested that elevated DCLK1 mRNAs expression significantly correlates with increased levels of MMP2, vimentin, Zeb2, Twist1 and VEGF-C which are involved in epithelial-to-mesenchymal transition (EMT) and tumor invasion. Huh7-derived tumors also exhibited high level expression of these factors.

Conclusions: These observations suggest that DCLK1 induces tumor/cancer stem cell-like properties and EMT in liver cells. DCLK1 appears to enhance active β-catenin levels and its cellular localization to the nucleus. Thus, the DCLK1 represents an important target for HCC therapy.

Citation Format: Naushad Ali, Charles B. Nguyen, Parthasarathy Chandrakesan, Randal May, Danny Dhanasekaran, Michael S. Bronze, Courtney W. Houchen. A tumor stem cell marker DCLK1 promotes hepatocellular carcinoma by regulating β-catenin, EMT and clonogenic properties of hepatocytes [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 3884. doi:10.1158/1538-7445.AM2017-3884
Abstract 3896: Exosomes derived from CD133+ hepatocellular carcinoma (HCC) cell promote the acquisition of stem cell-like properties of the targeted HCC cell via the Akt/GSK 3β/Snail and cyclin D1 pathways
Authors: Guo Pengyi , Yu Haitao , Xie Xiaozhai , Wang Yi , Chen Gang
Published: July 2017

Backgrounds and Aims: Liver cancer stem cells are closely related to the recurrence and metastasis of hepatocellular carcinoma (HCC). However, the mechanism underlying the reciprocity between liver cancer stem cells and HCC cells in the tumor microenvironment remains unclear. Exosomes act as natural shuttles to deliver bioactive molecules that alter the behavior of the recipient cells, offering a new means of cell-cell communication for physiological and pathological processes in the liver. This study aimed to clarify whether the exosomes derived from the liver stem cells could facilitate the acquisition of stem cell-like properties of the adjacent HCC cells, thereby promoting the malignant process of HCC.

Methods: CD133+ liver cancer stem cells were isolated from the highly invasive HCC-LM3 cells or tumor tissues of HCC patients using flow cytometry and were identified using qPCR and immunofluorescence. The exosomes from CD133+ liver cancer stem cells were identified using CD133 and other stem cell markers, and they were then used to treat HepG2 and Hep3B cells. The cell viability, invasion ability, sphere formation and tumorigenesis abilities of the treated HepG2 and Hep3B cells were analyzed to reveal the effect of CD133+ exosomes, and the underlying molecular mechanism was further explored.

Results: CD133+ liver cancer stem cells were successfully isolated from HCC-LM3 cells and HCC patients’ tumor tissues, which were able to release CD133+ exosomes. The intake of CD133+ exosomes promoted the putative stem cell phenotype of Hep3B and HepG2 cells, as evidenced by significantly enhanced cell proliferation, invasion and sphere formation abilities in vitro and tumorigenesis in vivo. In additional, we found that CD133+ exosomes activated the PI3K/Akt/GSK3β/Snail and cyclin D1 pathway, thereby reducing the expression of E-Cadherin and promoting the epithelial-mesenchymal transition (EMT) of HCC cells.

Conclusions: Our results suggest that exosomes derived from CD133+ HCC cells can change the malignant behavior and promote the acquisition of stem cell-like properties of targeted HCC cells via the Akt/GSK 3β/Snail and cyclin D1 pathways.

Note: This abstract was not presented at the meeting.

Citation Format: Pengyi Guo, Haitao Yu, Xiaozhai Xie, Yi Wang, Gang Chen. Exosomes derived from CD133+ hepatocellular carcinoma (HCC) cell promote the acquisition of stem cell-like properties of the targeted HCC cell via the Akt/GSK 3β/Snail and cyclin D1 pathways [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 3896. doi:10.1158/1538-7445.AM2017-3896