bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1511: Identification of metastatic subsets of pancreatic cancer stem cells possessing metabolic features of pluripotent stem cells
Authors: Hsieh Chi-Che , Liao Wen-Ying , Chen Chih-Lung , Lin Pei-Yu , Shen Chia-Ning
Published: August 2015

The current cancer chemotherapy can mainly cause pancreatic cancer remission but often fails to cure cancer due to the existence of cancer stem cells in pancreatic ductal adenocarcinomas (PDACs). Therefore, development of methods targeting pancreatic cancer stem cells could lead to improvement in PDAC therapeutic. However, the key characteristics of pancreatic cancer stem cells remain to be determined. Accumulating evidences revealed the similar features shared by pluripotent stem cells and cancer stem cells, we therefore hypothesize acquisition of pluripotent characteristics during the reprogramming process is superficially reminiscent of the dysplastic transformation proposed in cancers. In the current work, comparative genome-wide profiling analysis was conducted on embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and different subpopulation of pancreatic cancer cells. We initially revealed expression of pluripotent transcriptional factors and surface markers such as Oct4, Sox2, Lin28, CD133, ABCG2 and CD44 was significantly higher in ABCG2+ CD44+ pancreatic CSC subpopulation which was correlated with their drug resistance and high metastatic potentials. Metabolic pathway analysis was further performed and identified that, very similar to ESCs/iPSCs, the metastatic ABCG2+CD44+ subpopulations had evaluated levels of of GLUT5 and KHK and expressed higher levels of ST6Gal1. Utilizing tumor-engraft mice and spontaneous pancreatic cancer mice, we actually revealed fructose diet enhanced cancer drug resistance and CSC-mediated cancer metastasis possibly via up-regulating ST6Gal1 as knockdown of ST6Gal can affect self-renewal, invasion potential and ABCG2 efflux ability of ABCG2+CD44+ CSC subpopulations. In summary, the current work elucidated that pancreatic ABCG2+CD44+ CSC subpopulations possess metabolic features of pluripotent stem cells including the capability to efficiently to utilize fructose and to perform 2,6 sialylation via sialyltransferase ST6Gal1 which critically mediated cancer drug resistance and metastasis. Hopefully, the findings can lead to developing efficient treatments for patients of PDAC in the near future.

Citation Format: Chi-Che Hsieh, Wen-Ying Liao, Chih-Lung Chen, Pei-Yu Lin, Chia-Ning Shen. Identification of metastatic subsets of pancreatic cancer stem cells possessing metabolic features of pluripotent stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1511. doi:10.1158/1538-7445.AM2015-1511
Abstract 1517: G0-like cells support an oral cancer stem cell pool by transitioning to a JARID1B-high state
Authors: Facompre Nicole , Belden Zachary , Gimotty Phyllis , Rustgi Anil , Herlyn Meenhard , Nakagawa Hiroshi , Basu Devraj
Published: August 2015

Oral squamous cell carcinomas (OSCCs) likely contain a subset of tumor cells whose stem cell-like molecular and functional properties help sustain tumor growth. There is growing evidence that this cancer stem cell (CSC) pool exists in dynamic equilibrium with other subpopulations with tumorigenic potential. To characterize this plasticity in OSCC, we examined the developmental potential of tumor cells in a “G0-like” cell state previously shown to have high growth capacity and innate drug resistance in other tumor types. G0-like cells in OSCC exhibited hallmark molecular traits of low reactive oxygen species (ROS), high Hes1, and low total Akt levels. However, despite lacking high levels of CD44 and other stem cell markers, they prominently displayed stem cell-like functional properties of in vitro sphere formation and in vivo tumorigenicity. To evaluate the contribution of G0-like cells to the oral CSC pool, a GFP reporter system was used to isolate CSCs based on high expression of JARID1B, an H3K4me3 demethylase associated with multiple normal and malignant stem cell phenotypes. JARID1B-high OSCC cells were distinct from the G0-like subset, highly expressed CD44 and ALDH1, and possessed both molecular and functional CSC traits. Silencing JARID1B expanded the G0-like subset but abrogated its functional CSC properties, suggesting these cells mediate such functions by returning to a JARID1B-high CSC state. The G0-like and JARID1B-high subsets were further distinguished by widely disparate levels of PI3-kinase pathway activation, which corresponded to distinct responses to cytotoxic and targeted therapies. These findings reveal a JARID1B-mediated mechanism underlying a contribution of G0-like cells to a CSC pool. Further they may provide a novel basis for OSCC therapy by simultaneously targeting the CSCs and a subset G0-like cells that appear uniquely primed to return to a CSC state.

Citation Format: Nicole Facompre, Zachary Belden, Phyllis Gimotty, Anil Rustgi, Meenhard Herlyn, Hiroshi Nakagawa, Devraj Basu. G0-like cells support an oral cancer stem cell pool by transitioning to a JARID1B-high state. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1517. doi:10.1158/1538-7445.AM2015-1517
Abstract 1532: A Microbiomic analysis in African American with colonic lesions reveals bacterial markers with potential diagnostic value
Authors: Brim Hassan , Varma Sudhir , Laiyemo Adeinko , Sharif Zaki , Lee Edward L. , Ashktorab Hassan
Published: August 2015

Background: Increasing evidence suggests a role of the gut microbiota in colorectal carcinogenesis. However, no specific bacteria have been unequivocally linked to either initiation or progression of colorectal cancer (CRC).

Aim: To analyze the taxonomic composition and functional potential of the gut microbiome in African Americans with colorectal lesions with the goal of detecting markers of diagnostic value.

Materials & Methods: DNA extracts from 10 stool samples of colon adenoma patients, 10 healthy subjects’ stool samples and from 10 CRC tumors and their matched normal tissues were analyzed for their microbiota composition and genomic content using Illumina MiSeq. Metagenomic Linkage Groups (MLGs) were established and those with high discriminative power between healthy and neoplastic specimens were analyzed for their genetic content. Metagenomic reads from stool samples were mapped against bacterial genes from tissues and vice-versa to identify common markers with discriminative power.

Results: The microbiota in both cancer vs. normal tissues and adenoma vs. normal stool samples were different at the 16S rRNA gene level. The metagenomic analysis led to 5 statistically significant MLGs in each set of samples. There was little overlap between the stool-based and tissue-based significant MLGs. Mapping stools reads to annotated tissue genes and vice-versa revealed a panel of 9 bacterial markers with statistically significant discriminative power between normal and adenoma in the stool dataset and visually striking (though non-significant) discriminative power between normal and tumor samples in the tissue dataset.

Conclusion: We defined MLGs with discriminative power among cancers vs. normal and adenomas vs. normals and identified 9 bacterial markers that can potentially be used as a panel for non-invasive colorectal cancer screening tests. These findings will need to be validated in a larger population of patients at different stages of the carcinogenic process and from different ethnic/racial backgrounds.

Citation Format: Hassan Brim, Sudhir Varma, Adeinko Laiyemo, Zaki Sharif, Edward L. Lee, Hassan Ashktorab. A Microbiomic analysis in African American with colonic lesions reveals bacterial markers with potential diagnostic value. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1532. doi:10.1158/1538-7445.AM2015-1532
Abstract 1536: Effect of ascites on tumor response in advanced stage ovarian cancer
Authors: Kim Soochi , Kim Anna , Kim Hee Seung , Gwak HyeRan , Jo UnTek , Song Yong Sang
Published: August 2015

Background: Ascites can create a progressive microenvironment for tumor cells, whereas there is few evidence on the role of ascites on tumor response in ovarian cancer. Thus, we investigated cellular response by ascites in ovarian cancer cell line, and evaluated the effect of ascites on clinical outcomes in patients with advanced-stage ovarian cancer.

Methods: Moreover, we collected clinico-pathologic database of 277 patients with stage IIIC-IV ovarian cancer, and investigated factors affecting overall response (OR) and survival. Furthermore, we collected ascites from four patients with stage IIIC-IV serous adenocarcinoma of the ovary, and cell migration, invasiveness and proliferation by adding ascites were evaluated in SKOV-3.

Results: OR rates were not different between patients with ascites and those without ascites (77.8% vs. 89.1%; P = 0.094), and no ascites was associated with better progression-free survival with marginal significance (37.4 vs. 27.1 mons; P = 0.056) in spite of no effect of ascites on overall survival. In multivariate analyses, no ascites was a favorable factor for reducing the risk of non-OR with marginal significance (adjusted OR, 0.286; 95% CI, 0.080-1.018; P = 0.053), whereas ascites was not an independent factor affecting survival. Moreover, ascites increased migration and invasiveness of SKOV-3 (Figs. 1A and 1B). Although proliferation of SKOV-3 was decreased for the first 24 h, it increased in time-dependent manner till 72h (Fig. 1C).

Conclusions: Ascites may contribute to invasion, migration and proliferation of ovarian cancer cells. Our clinical findings also support that ascites may affect overall response after primary treatment, suggesting the possibility that ascites can act as a tumor microenvironment related with tumor cell survival.

Citation Format: Soochi Kim, Anna Kim, Hee Seung Kim, HyeRan Gwak, UnTek Jo, Yong Sang Song. Effect of ascites on tumor response in advanced stage ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1536. doi:10.1158/1538-7445.AM2015-1536
Abstract 1538: Levels and enzyme activity of CD73 in primary samples from cancer patients
Authors: Huang Qihui , Durham Nicholas M. , Sult Erin , Wu Yuling , Liu Jenny , Holoweckyj Nicholas , Iciek Laurie , Hollingsworth Robert , Hall Brett , Herbst Ronald , Leow Ching Ching , Sachsenmeier Kris
Published: August 2015

CD73 is an ectonucleotidase that converts extracellular AMP to Adenosine. Within the tumor microenvironment, adenosine produced by CD73 is believed to have an immunosuppressive effect upon anti-tumor immunity. CD73 is found on lymphoid and myeloid cells, and blockade of this enzyme has been shown to alter the suppressive capacity of cells. Expression of surface CD73 as well as soluble CD73(sCD73) levels were measured in serum, peripheral blood mononuclear cells, and tumor samples from healthy and cancer patient donors. Enzymatic activity of both surface CD73 and sCD73 was determined and differences in levels across varying tumor types and disease status were quantified. In addition, CD73 levels and activity were measured after incubation or treatment with anti-CD73 antibody, MEDI9447. These studies suggest a potential role of surface and sCD73 as a pharmacodynamic and/ or predictive biomarker of anti-CD73 immunotherapy.

Citation Format: Qihui Huang, Nicholas M. Durham, Erin Sult, Yuling Wu, Jenny Liu, Nicholas Holoweckyj, Laurie Iciek, Robert Hollingsworth, Brett Hall, Ronald Herbst, Ching Ching Leow, Kris Sachsenmeier. Levels and enzyme activity of CD73 in primary samples from cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1538. doi:10.1158/1538-7445.AM2015-1538
Abstract 154: Identification of TGFβ-regulated long noncoding RNAs in mammary epithelia: lncRNA-HIT mediated TGFβ-induced EMT and breast cancer metastasis
Authors: Richards Edward , Zhang Gu , Permuth-Wey Jennifer , Li Zhu-Peng , Challa Sridevi , Kong William , Su Dan , Coppola Domenico , Mao Wei-min , Sellers Thomas , Cheng Jin Q.
Published: August 2015

Background: Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. TGFβ signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown.

Methods: Genome-wide lncRNA profile was performed using NCode™ Mouse Noncoding Microarray chip from Life Technologies, which contains 10,802 lncRNAs and 25,178 protein-coding genes. Total RNA was isolated from mouse mammary epithelial NMuMG cells, which had been treated with TGFβ for 0, 12 and 24 hours, and was used to hybridize the microarray. The role of a top TGFβ-upregulated lncRNA, lncRNA-HIT, in EMT and metastasis was further characterized in cell culture and orthotopic breast cancer models

Results: Among 10,802 lnRNAs profiled, 680 were up-regulated and 633 were down-regulated during TGFβ-induced EMT, respectively. Further, we identify that lncRNA-HIT (HOXA antisense transcript induced by TGFβ) mediates TGFβ function, i.e., depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth and metastasis. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma.

Conclusions: These data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.

Citation Format: Edward Richards, Gu Zhang, Jennifer Permuth-Wey, Zhu-Peng Li, Sridevi Challa, William Kong, Dan Su, Domenico Coppola, Wei-min Mao, Thomas Sellers, Jin Q. Cheng. Identification of TGFβ-regulated long noncoding RNAs in mammary epithelia: lncRNA-HIT mediated TGFβ-induced EMT and breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 154. doi:10.1158/1538-7445.AM2015-154
Abstract 1542: Targeting KIF3a and primary cilia differentially affects sonic hedgehog sensitivity and the rate of glioblastoma progression
Authors: Hoang-Minh Lan , Siebzehnrubl Dorit , Deleyrolle Loic , Ugartemendia George , Futch Hunter , Griffith Benjamin , Breunig Joshua , Semple-Rowland Susan , Reynolds Brent , Sarkisian Matthew
Published: August 2015

Adults diagnosed with glioblastoma multiforme (GBM) typically have a median survival less than 15 months. GBM patient biopsies and derived cell lines contain subsets of cells extending primary cilia, microtubular antennae that mediate signaling events reported to influence cell proliferation and tumor cell growth. However, it remains unclear how cilia contribute to the phenotypic characteristics of GBM. To determine whether cilia influence GBM tumor progression, we generated several stable patient-derived cell lines devoid of cilia by expressing a dominant-negative form of Kif3a (dnKif3a), a protein normally essential for ciliogenesis. Depending on the line, mice receiving intracranial implants of dnKif3a-expressing cells survived shorter, as long as, or significantly longer than mice implanted with control cells. To determine if ciliated cells derived from different GBM tumors share similar sensitivities to sonic hedgehog (SHH), a mitogen present in adult brain that activates receptor signaling cascades within cilia, we exposed control and dnKif3a-expressing cells in vitro to vehicle or recombinant SHH. Interestingly, only the cell line that prolonged survival in vivo displayed significant increases in proliferation following exposure to SHH, an effect that was blocked by either dnKif3a or pretreatment of the cells with cyclopamine, an inhibitor of the SHH cascade. All cell lines tested appeared to respond to SHH as evidenced by activation of downstream SHH signaling components in their cilia. Collectively, our data suggest that strategies to disrupt primary cilia in GBM patients could lead to inhibition or enhancement of tumor growth potentially by altering cell sensitivity to the extracellular environment.

Citation Format: Lan Hoang-Minh, Dorit Siebzehnrubl, Loic Deleyrolle, George Ugartemendia, Hunter Futch, Benjamin Griffith, Joshua Breunig, Susan Semple-Rowland, Brent Reynolds, Matthew Sarkisian. Targeting KIF3a and primary cilia differentially affects sonic hedgehog sensitivity and the rate of glioblastoma progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1542. doi:10.1158/1538-7445.AM2015-1542
Abstract 1550: Significance of ectopic lymph node-like structures in neuroendocrine tumors of the small bowel
Authors: Cives Mauro , Strosberg Jonathan , Simons Stacey A. , Coppola Domenico
Published: August 2015

The formation of extranodal tertiary lymphoid follicles and lymphoid cell aggregates has been described in nonmalignant conditions including autoimmune diseases and chronic inflammation as well as in cancer. Tumor-induced lymph node-like structures (LLS) participate in the host's antitumor immune response by driving intratumoral immune cell priming. We evaluated the host immune response by immunohistochemistry in 60 well differentiated small bowel NETs. A miRNA microarray analysis was also performed to identify a molecular signature of LLS presence. In 13/60 samples the immune response was organized as ectopic LLS. When present, LLS were localized at the invasive edge of the tumors and contained germinal centers in different stages of maturation. CD21+ dendritic cells were present within the follicular germinal centers, while CD3+ T cells were dispersed in the parafollicular cortex zone surrounding the CD20+ B cell area of the follicles. Time to progression of metastatic tumors was compared in an exploratory cohort of 5 patients with strong evidence of LLS vs 5 patients with no evidence of LLS and was substantially longer in the former group (94 vs 28 months; p = 0.15). Updated survival and miRNA microarray analysis will be presented at the meeting.The understanding of the functional nature of LLS may provide new insights into the pathogenesis of the disease, possibly furnishing a new tool for prognostic stratification and prediction of response to immune-based treatments.

Citation Format: Mauro Cives, Jonathan Strosberg, Stacey A. Simons, Domenico Coppola. Significance of ectopic lymph node-like structures in neuroendocrine tumors of the small bowel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1550. doi:10.1158/1538-7445.AM2015-1550
Abstract 1559: Progranulin A9D mutation functionally leads to the cytoplasm retardation of angiogenin
Authors: Sheng Jinghao , Chen Yongdui , Li Siqi , Dong Haojie , Chen Guangdi , Xu Zhengping
Published: August 2015

Neurodegenerative diseases are progressive neurological disorders with selective neuron loss in particular regions of brain, including frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and many others. Gene defects have been recognized as a prominent factor in the etiology and pathogenesis of neurodegenerative diseases. To date, hundreds of genetic variants that located in dozens of genes have been associated with susceptibility to various neurodegenerative diseases. Among them, more than 60 mutations in progranulin (PGRN) have been reported in FTLD, AD, ALS and PD. One of the mutations in PGRN gene exon 1 introduces a charged amino acid in the hydrophobic core of the signal peptide at residue 9 (PGRN A9D), and results in cytoplasmic mis-sorting of this protein. However, the mechanism of actions of this mis-sorting remains elusive. To address this issue, we first examined the subcellular distribution of PGRN with A9D mutation in human neuronal-like cells (SH-SY5Y). The results have shown that PGRN A9D mutation failed to undergo maturation and accumulated in the stress granules. Interestingly, this mutation also induces redistribution of cellular angiogenin (ANG), a stress response factor and neurodegenerative diseases related protein, from nuclear to cytoplasm. PGRN was also directly interacted with ANG in stress granules. Furthermore, the cytoplasmic retardation of ANG by PGRN A9D mutation abolished ANG-enhanced rRNA transcription and cell survival. Taken together, these results indicate that PGRN with A9D mutation changed localization of ANG as well as cell growth and survival, suggesting that PGRN and ANG act in concert to regulate the progress of neurodegenerative diseases.

Citation Format: Jinghao Sheng, Yongdui Chen, Siqi Li, Haojie Dong, Guangdi Chen, Zhengping Xu. Progranulin A9D mutation functionally leads to the cytoplasm retardation of angiogenin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1559. doi:10.1158/1538-7445.AM2015-1559
Abstract 1576: Analysis of HSP10 as a putative biomarker of breast cancer
Authors: Shaheed Sadr-ul , Hadjisavvas Andreas , Sokratous Kleitos , Loadman Paul , Sutton Chris , Kyriacou Kyriacos
Published: August 2015

Introduction. Detection of the very earliest stages of breast cancer remain challenging and the differentiation of those carcinomas that develop into aggressive invasive forms from those that remain benign has not been achieved. Consequently, removal of the tumor by total mastectomy, local excision (LE) plus adjuvant radiotherapy (RT), or LE alone remain the main treatments, even if they prove unnecessary. Previously, using proteomics analysis, we identified proteins that were significantly changed in expression between matched normal and tumor tissues from Cypriot patients with different stages of the disease. Heat shock protein 10 (HSP10), which is responsible for the mitochondrial protein folding, was one of the proteins identified. A range of methods were used to verify HSP10 as a possible biomarker for breast cancer.

Materials and Methods. Protein extracts from breast cancer cell lines and breast tissue biopsies from Cypriot patients with different stages of the disease were analysed by (i) Western Blotting (WB) and (ii) multiple reaction monitoring mass spectrometry (MRM MS) using two proteotypic peptides unique to HSP10, in proteins extracted form fresh frozen as well as from FFPE breast tissues. Tissue microarray analysis (TMA) was carried out on core biopsies from the Leeds Breast Tissue Bank.

Results. WB indicated that HSP10 was ubiquitously and evenly expressed across breast cancer cell lines representative of different phenotypes. WB and MRM MS revealed that expression was significantly increased in invasive carcinoma compared to normal tissue, but was patient specific for fibroadenoma and DCIS cases. TMA of 360 samples indicated a strong correlation between staining and type of breast cancer (invasive ductal and lobular), grade and ER positive phenotype.

Discussion. Proteomics analysis using iTRAQ quantitation indicated an average of 1.6 fold increase in HSP10 in invasive carcinomas compared to matched normal tissue. By comparison, WB and MRM MS analysis indicated a significantly amplified discrimination in the expression of HSP10 as has been observed with other biomarker candidates. HSP10 protein levels were in agreement with mRNA results observed in breast cancer cell lines reported in the Cancer Cell Line Encyclopaedia. In conclusion, HSP10 represents an excellent biomarker candidate for further validation.

Citation Format: Sadr-ul Shaheed, Andreas Hadjisavvas, Kleitos Sokratous, Paul Loadman, Chris Sutton, Kyriacos Kyriacou. Analysis of HSP10 as a putative biomarker of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1576. doi:10.1158/1538-7445.AM2015-1576
Abstract 1583: Circulating tumor cells isolated from non-small cell lung cancer patients using the in vivo CellCollector Technology
Authors: Krahn Thomas , Gasiorowski Lukasz , Dyszkiewicz Wojciech , Dworacki Grzegorz , Zabel Maciej , Hoon Dave S. , Herold Stefanie , Nowack Bjoern , Tucholsky Johannes , Schumann Christian , Lücke Klaus
Published: August 2015

Access to tumor tissue is often limiting and archival samples may not be reflective of the current disease. Alternative approaches using liquid biopsies are being explored to support personalized treatment of cancer patients. Circulating tumor cells (CTCs) detached from primary tumors or metastases into the blood may reflect the current molecular tumor profile providing the opportunity to monitor molecular changes in the tumor. As CTCs are rare cells in the bloodstream and most in vitro approaches for capturing CTCs are based on small samples volumes (5 - 10 ml), accurate and reliable quantification of CTCs is difficult to achieve. We report on a comprehensive clinical study using a novel CellCollectorTM for in vivo isolation of CTCs.

Eighty four stage I - IIIB non-small cell lung cancer (NSCLC) patients and 7 healthy individuals were screened for CTCs using a CellCollectorTM (Gilupi, Germany) device. Molecular analysis of a patient with known mutation in the KRAS gene (G12D) was performed to confirm the predictive value of CTCs as “liquid biopsy”. CTCs were detected in 65 out of 84 NSCLC patients (77.4%) (median: 13 CTCs; range from 0 - 300). For comparison, using the U.S. Food and Drug Administration-cleared CellSearch® system only fifteen samples (17.8%) in this cohort were positive (median: 0 CTCs; range from 0-300). In all paired samples, the CellCollectorTM detected the same or higher numbers of CTCs. Healthy individuals were devoid of any cells classified as CTC. Investigating the KRAS status on CTCs captured by the CellCollectorTM, we were able to substantiate the G12D mutation that has earlier on been identified in the tissue of the primary lung tumor.Conclusions: The CellCollectorTM is a promising novel device for the in vivo isolation of CTCs and the device effectively isolated CTCs from NSCLC patients of all stages including the non-metastatic setting. The implementation of the CellColletorTM into clinical practice has the potential for early disease detection. Furthermore, this approach allows the molecular characterization of CTCs for possible therapeutic targets and early response to treatment which might be used in the setting of personalized cancer therapies and clinical development.

Citation Format: Thomas Krahn, Lukasz Gasiorowski, Wojciech Dyszkiewicz, Grzegorz Dworacki, Maciej Zabel, Dave S. Hoon, Stefanie Herold, Bjoern Nowack, Johannes Tucholsky, Christian Schumann, Klaus Lücke. Circulating tumor cells isolated from non-small cell lung cancer patients using the in vivo CellCollector Technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1583. doi:10.1158/1538-7445.AM2015-1583
Abstract 1592: CXCR4 pathways in CTCs: from bioinformatics to immunophenotype
Authors: Kallergi G. , Tsintari V. , Sfakianakis S. , Zervakis M. , Mavroudis D. , Georgoulias V.
Published: August 2015

Introduction: Bioinformatics’ analysis regarding gene expression in Normal tissue vs cancer tissue, Normal blood vs cancer patients’ blood and Normal blood vs cancer tissue revealed a subgroup of 24 genes which potentially could be overexpressed in CTCs. Among these genes are: CXCR4, a chemokine receptor which is involved in tumor metastasis and, JUNB a transcription factor participating in CXCR4 pathway. The objective of this study was to confirm the results of bioinformatics’ analysis in samples from breast cancer patients and therefore to evaluate the expression of CXCR4 and JUNB in CTCs.

Methods: Triple staining immunofluoresence with panytokeratin/CXCR4/JUNB antibodies were performed in SKBR3, MDA-MB231, MCF7 and Hela cell lines. The same experiments were performed in PBMCs from normal (n = 10.) subjects and in PBMCs (n = 55) from untreated metastatic breast cancer patients.

Results: Since CXCR4, as a chemokine receptor is also expressed on hematopoietic cells, its expression on CTCs was quantified using the ARIOL system. We used cell lines and both normal vs patients’ PBMCs in order to establish an expression pattern of both molecules. Quantification experiments among cell lines revealed that CXCR4 was overexpressed in SKBR3 following the subsequent hierarchy SKBR3>MCF7>MDA-MB231>Hela. Accordingly, the expression pattern of JUNB in cell lines was SKBR3>Hela>MCF7>MDA-MB231 with higher expression in SKBR3 and lower in MDA-MB 231. Statistical analysis revealed significant differences in the expression of both molecules between healthy donors’ PBMCs and patient's PBMCs.

Subsequently, CTCs were detected in 17 out of 55 screened patients (31%). Mean intensity of CTCs for CXCR4 was 34.38 and it was higher than Hela and lower than SKBR3. Patients with CXCR4-positive CTCs (with mean expression higher than 95% of normal PBMCs) were 53%. In addition 84% of the examined CTCs had expression profile higher than normal PBMCs.

JUNB expression in CTCs (above 95% of normal PBMCs) was identified in 76.92% of patients. JUNB mean expression in CTCs was 26.1 and it was lower than SKBR3 and higher than MCF7. Furthermore 64.3% of the total CTCs have expression higher than Normal PBMCs.

Conclusion: CXCR4 and JUNB are highly expressed in CTCs derived from breast cancer patients, in agreement to bioinformatics’ analysis. Quantification of immunofluoresence potentially delineates a subgroup of patients with high expression of CXCR4 and JUNB that could benefit from target therapies.

Citation Format: G. Kallergi, V. Tsintari, S. Sfakianakis, M. Zervakis, D. Mavroudis, V. Georgoulias. CXCR4 pathways in CTCs: from bioinformatics to immunophenotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1592. doi:10.1158/1538-7445.AM2015-1592
Abstract 1603: High purity isolation of circulating tumor cells for next-generation sequencing
Authors: Mohd Salleh Nur Lina , Lee Yi Fang , Tan Mei Hui , Rosario Michelle A. , Soo Ross A. , Bhagat Ali Asgar , Soong Richie Chuan Teck
Published: August 2015

Background: Being able to capture circulating tumor cells (CTCs) and characterize them at a molecular level promises novel insights into the mechanisms that drive metastasis, tumor recurrence and drug resistance. However, next generation sequencing (NGS) of CTCs has been challenged by antibody-enrichment bias, low CTC purity and nucleic acid yields commonly derived from existing technologies. Here, we describe the results of a new label-free protocol for CTC enrichment and NGS analysis.

Methods: The lung adenocarcinoma cell line, NCI-H1975, with EGFR T790M and L858R mutations, and colorectal cancer cell line HCT-116, with KRAS G13D mutation, were fluorescently labelled and spiked in concentrations of 250, 50, 10 and 0 cells per 7.5 mL healthy volunteer blood in duplicate. CTCs were enriched using a modified protocol of the label-free spiral microfluidics-based ClearCell® FX system. DNA was extracted by QIAamp Micro kit and sequenced using the Ion AmpliSeq Cancer Hotspot Panel and Ion Torrent PGM system.

Results: Imaging analysis indicated that CTCs were efficiently enriched (40-70% recovery) with extremely low leukocyte background (650-1300 cells). Despite a low DNA quantity (<5ng), an average depth of 6897 reads and 97% coverage was achieved in NGS analysis. Expected mutations were detectable in samples down to 10 spiked tumor cells (0.5% CTC purity). Analysis of CTCs from clinical samples is currently on-going.

Conclusion: The new protocol generated sufficient label-free CTC purity for NGS analysis, overcoming current limitations in the field, and facilitating discovery and clinical application.

Citation Format: Nur Lina Mohd Salleh, Yi Fang Lee, Mei Hui Tan, Michelle A. Rosario, Ross A. Soo, Ali Asgar Bhagat, Richie Chuan Teck Soong. High purity isolation of circulating tumor cells for next-generation sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1603. doi:10.1158/1538-7445.AM2015-1603
Abstract 1613: β-lapachone: a novel targeted therapy for atypical teratoid rhabdoid tumors (ATRTs)
Authors: Moore Zachary R. , Stuart Sarai , Cholka Agnieszka , Venkataraman Ashwin , Xu Jingying , Erdreich-Epstein Anat , Rakheja Dinesh , Boothman David A. , Amatruda James F. , Laetsch Theodore W.
Published: August 2015

Atypical teratoid rhabdoid tumors (ATRTs) are aggressive malignant neoplasms that occur in the CNS, most commonly in children <3 years of age. Despite intensive chemo-radiotherapy and surgery, the outcomes for patients with this disease remain dismal, with ∼75% of patients dying from their disease at a median of 10 months after diagnosis. Thus, it is critical to identify and validate novel ATRT targets for new therapies. We found that the pathognomonic genetic lesion of ATRTs, loss of the SWI/SNF chromatin remodeling complex member INI1/hSNF5/SMARCB1, leads to high expression of NAD(P)H:quinone oxidoreductase 1 (NQO1), a two-electron oxidoreductase. Thus, we hypothesized that ATRTs will be vulnerable to drugs, such as ß-lapachone (ß-lap, ARQ761 in clinical form), that selectively kill NQO1-expressing cells. A panel of ATRT cell lines exhibited overall high NQO1 expression and low micromolar sensitivity to ß-lap (IC50 1.6-4 uM), with the exception of cell lines that were homozygous for the C609T NQO1 (*2) polymorphism, which exhibited no NQO1 activity and were resistant to ß-lap treatment (IC50 >15 uM). A two-hour pulse with ß-lap caused a burst of ROS-mediated DNA damage and PARP1 hyperactivation, resulting in NAD+ and ATP depletion and caspase-independent, mu-calpain-mediated apoptosis. Co-treatment with dicoumarol, a specific inhibitor of NQO1, prevented these effects by blocking the NQO1-dependent bioactivation of ß-lap. Low expression of NQO1 in normal brain and other pediatric tissues suggests a wide therapeutic window for ß-lap. These findings encourage the continued study of ß-lap and other NQO1 bioactivated therapeutics for the treatment of this recalcitrant disease. This work was supported by NIH/NCI R01 CA102972 to DAB and a REACH award from the Alex's Lemonade Stand Foundation to JFA and TWL.

Citation Format: Zachary R. Moore, Sarai Stuart, Agnieszka Cholka, Ashwin Venkataraman, Jingying Xu, Anat Erdreich-Epstein, Dinesh Rakheja, David A. Boothman, James F. Amatruda, Theodore W. Laetsch. β-lapachone: a novel targeted therapy for atypical teratoid rhabdoid tumors (ATRTs). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1613. doi:10.1158/1538-7445.AM2015-1613
Abstract 162: Overexpression of long noncoding RNA FTX was associated with colorectal cancer progression
Authors: Iwaya Takeshi , Sato Kei , Kume Kohei , Nishizuka Satoshi , Wakabayashi Go , Mimori Koshi
Published: August 2015

Increasing evidence suggests Long noncoding RNAs (lncRNAs) have crucial roles in cancer biology. Using public microarray database, we evaluated the association between the expression status of lncRNAs and colorectal cancer (CRC) prognosis. Among the candidate lncRNAs showed significant higher expression in CRC patients with poor prognosis, we focused on FTX on chromosome X. We validated FTX expression status in 128 primary tumor samples and corresponding normal tissues from CRC patients through quantitative real-time PCR (qRT-PCR) analysis. Primary tumor tissues exhibited higher FTX expression compared to corresponding normal tissues (p = 0.0008). In terms of overall survival, patients in the FTX high expression group (n = 74) had a significantly poorer prognosis than those in the FTX low expression group (n = 74). Multivaliate analysis of overall survival revealed that the level of FTX expression was a prognostic predictor. FTX knockdown inhibited tumor cell proliferation in CRC cell line, HT29. Our results indicate that overexpression of FTX may affect CRC progression.

Citation Format: Takeshi Iwaya, Kei Sato, Kohei Kume, Satoshi Nishizuka, Go Wakabayashi, Koshi Mimori. Overexpression of long noncoding RNA FTX was associated with colorectal cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 162. doi:10.1158/1538-7445.AM2015-162
Abstract 1635: Mechanistic investigation of neutropenia associated with palbociclib
Authors: Hu Wenyue , Sung Tae , Jessen Bart , Sacaan Aida
Published: August 2015

Bone marrow suppression, especially neutropenia, has been identified as one of the main dose-limiting toxicities for the selective CDK4/6 inhibitor, palbociclib, in the clinic. However, compared to chemotherapy-induced myelotoxicity, palbociclib-induced neutropenia is quickly reversible, uncomplicated and is proactively managed by a short duration treatment-free period between cycles, or reactively by dose delay or reduction. To aid in understanding the mechanism by which neutropenia occurs and to further differentiate it from chemotherapy- induced myelotoxicity, an in vitro bone marrow toxicity assay was utilized to evaluate the cellular mechanism and the reversibility of bone marrow suppression induced by palbociclib as a single agent or in combination with anti-estrogens.

This investigation has demonstrated that palbociclib-induced bone marrow suppression manifested through cell cycle arrest without apoptosis; by contrast, chemotherapeutic agents, including paclitaxel, doxorubicin, and carboplatin, primarily caused apoptotic cell death in the bone marrow. Following palbociclib treatment withdrawal in vitro, bone marrow cells resumed proliferation in the presence or absence of the anti-estrogen (fulvestrant); while breast cancer cells (MCF-7) remained arrested in the presence of fulvestrant. Bone marrow cells did not resume proliferation following chemotherapy treatment withdrawal. To further investigate the mechanism of reversibility, we evaluated the involvement of cellular senescence. Bone marrow cells did not enter senescence following treatment with palbociclib, anti-estrogen, or a combination of both agents. MCF-7 cells, on the other hand, became senescent following palbociclib or anti-estrogen treatment, and this effect was larger in the presence of both agents.

In summary, the current investigative work shed light on the mechanism of palbociclib- induced bone marrow suppression and clearly differentiated it from that induced by chemotherapeutic agents. These results potentially explain the quick reversibility of palbociclib-induced myelosuppression and its uncomplicated nature when compared to traditional cytotoxic agents. Moreover, the data supports the current dosing regimen in the clinic, which provides time for bone marrow cells to recover during the 1 week treatment-free period without impacting tumor efficacy. This investigative work provides valuable information to the clinicians in managing the neutropenia risk associated with breast cancer treatment.

This research was funded by Pfizer, Inc.

Citation Format: Wenyue Hu, Tae Sung, Bart Jessen, Aida Sacaan. Mechanistic investigation of neutropenia associated with palbociclib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1635. doi:10.1158/1538-7445.AM2015-1635
Abstract 1636: A model-based approach toward clinical pipeline optimization
Authors: Burton Jackson , Antebi William , Zopf Christopher J. , Bottino Dean , Teng Shu-Wen , Nolan Ryan , Chakravarty Arijit
Published: August 2015

Introduction

What is the optimal “shape” for a drug pipeline: A flute? A megaphone? A trumpet? In other words, what set of stringencies for passage of candidates to the next stage in development maximizes the value of the pipeline as a whole, and how do assays predicting drug quality influence this optimum?

Methods

A linear programming formulation was developed for four phases of a drug pipeline: Preclinical, Phase I, Phase II, and Phase III, with variables including: the total budget for the entire pipeline (B), and the number (Ni), cost (ci), and net present value (vi) of drugs in each phase (i). The model is subject to two constraints: (1) the pipeline must reduce in size with each subsequent phase (i.e. Npre≥ N1 ≥ N2 ≥ N3), and (2) the sum total of the cost times the number of drugs in each phase must be less than or equal to the total budget (i.e. cpreNpre + c1N1 + c2N2 + c3N3 ≤ B). The overall goal is to maximize the total value of drugs progressing through the pipeline (i.e. maximize vtot = vpreNpre + v1N1 + v2N2 + v3N3).

Additionally, pipeline evolution was modeled to evaluate the implications of a “predictive” drug quality assay. Hypothetical “drug quality scores” were drawn from normal distributions which reflect industry probabilities of success between phases. Drugs that are above a cutoff stringency advance forward, with the process repeated for all phases. The cost of each phase and value for successful drugs are recorded for each simulation. The process is repeated many times, with the expected value calculated at each iteration.

Results

Beginning with a pool of drug candidates, the optimal pipeline structure should approach equal sized pipeline widths as rapidly as possible.

Monte Carlo simulations demonstrated that increasing the stringency of a “quality assay” in Phase I results in a linear increase in expected value. Additionally, if the quality test is improved, e.g. by reducing the variability of the assay, the improvement in expected value will increase in an exponential manner.

Conclusions

The simulations predict that to maximize pipeline value, drug companies should: (a) pick the winners as early as possible (i.e. Preclinical or Phase I), (b) set stringent go/no-go criteria in Preclinical and Phase I development, and (c) perform prospective clinical trial simulations (Preclinical to Phase I, Phase I to Phase II, Phase II to Phase III) to improve the ability to predict winners.

Citation Format: Jackson Burton, William Antebi, Christopher J. Zopf, Dean Bottino, Shu-Wen Teng, Ryan Nolan, Arijit Chakravarty. A model-based approach toward clinical pipeline optimization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1636. doi:10.1158/1538-7445.AM2015-1636
Abstract 1650: CYC065, a novel CDK2/5/9 inhibitor: detailed mechanistic studies, determinants of sensitivity and synergistic combinations
Authors: Saladino Chiara , Frame Sheelagh , Davis Susan , Blake David , Zheleva Daniella
Published: August 2015

Background: CYC065 is a novel, second generation aminopurine inhibitor highly selective for CDK2, CDK5 and CDK9 over other CDKs and non-CDK enzymes. CYC065 has completed IND-directed drug development and is ready for FIH studies. The unique target profile of CYC065 can be exploited in cyclin E-overexpressing tumors, including a trastuzumab-resistant Her2+ breast cancer model (Scaltriti et al, PNAS 2011; 9:3761).

The aim of this study was to gather key dosing information for this compound in advance of FIH studies, and provide understanding of the compound mechanism of action in additional key target indications.

CDK9 is a key regulator of mixed lineage leukemia (MLL)-driven leukemogenesis and Mcl-1-dependent survival and therefore represents a potential therapeutic target for AML and MLL leukaemia.

Methods: A panel of acute leukemia cell lines with different MLL status (WT, MLL translocations and MLL PTD) was used in this study. Resazurin and flow cytometry-based annexin V assays were used to determine cell viability and apoptosis induction. PI staining was used to examine cell cycle distribution. Protein levels were quantified by Western blotting and quantitative imaging. Combinations were studied using the Chou and Talalay method. The in vivo activity of CYC065 was studied in murine xenograft models.

Results: CYC065 rapidly decreased phosphorylation of Ser2 -RNAP II and expression of proteins involved in cell cycle control, survival and leukemogenesis (such as Mcl-1, Cyclin E, PNUTS, Hoxa9 and Meis 1), leading to robust and early induction of apoptosis (within 4 h). Time- and dose-dependent studies indicated that 6 h pulse treatment at 0.5-1 μM CYC065 was sufficient to cause ≥ 90% cell death in sensitive cell lines. Cell lines with MLL rearrangements were highly sensitive to CYC065, whilst sensitivity of AML cell lines with WT MLL correlated with the level of Bcl-2 family proteins. Cell lines with reduced sensitivity to CYC065 could be effectively targeted by exposure to longer, 10 h pulse treatments with CYC065, or in combination with short pulses of Bcl-2 inhibitors. In vivo, CYC065 achieved >90% TGI at well tolerated dose levels in HL60 (WT MLL) and EOL-1 (MLL PTD) xenograft models.

Conclusion: CYC065 has therapeutic potential for AML and MLL leukemia. MLL status and the levels of Bcl-2 family proteins might be useful patient stratification markers for predicting sensitivity/resistance.

Citation Format: Chiara Saladino, Sheelagh Frame, Susan Davis, David Blake, Daniella Zheleva. CYC065, a novel CDK2/5/9 inhibitor: detailed mechanistic studies, determinants of sensitivity and synergistic combinations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1650. doi:10.1158/1538-7445.AM2015-1650