bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4045: c-Met inhibition synergistically enhances death receptor-induced apoptosis via upregulation of DR5 in papillary thyroid cancer
Authors: Bavi Prashant , Bu Rong , Hussain Azhar R. , Ahmed Maqbool , Thangavel Saravanan , Al-Sobhi Saif , Al-Nuaim Abdulrahman , Amin Tarek , Ahmed Mohammed , Uddin Shahab , Al-Kuraya Khawla S.
Published: August 2014

The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are overexpressed and/or activated in variety of human malignancies. Previously we have shown that c-Met was overexpressed in Middle Eastern Papillary Thyroid Cancer (PTC) and significantly associated with poor prognoses. In this study, we investigated the functional link between c-Met/AKT signaling pathway and Dearth Receptor 5 (DR5) in a large cohort of PTC patient samples, a panel of PTC cell lines, and xenografts in a NUDE mouse model by using tissue microarray, MTT, flow cytometry and DNA fragmentation assays and western blotting. Immunohistochemistry staining showed a significant association of p-Met with DR5 in PTC patient samples. The PHA665752, an inhibitor of c-met tyrosine kinase, inhibited cell proliferation and induced apoptosis in a dose dependent manner in all cell lines studied. PHA665752 treatment resulted in dephosphorylation of c-Met, AKT and its downstream effector molecules FOXO1, GSK-3 and pBad. Additionally, treatment of PTC cell lines with PHA665752 resulted in activation of caspases-9 and 3, cleavage PARP and apoptosis. Moreover, HGF, a ligand of c-Met, stimulated the growth of all PTC cell lines via activation of c-MET and AKT. In addition, pre-treatment of PTC cell line with PHA665752 abrogated the HGF stimulated growth and activation of c-met and AKT further suggesting the critical role of c-met and AKT pathway in PTC pathogenesis. Furthermore, c-Met inhibitor PHA665752 up-regulated DR5 expression in PTC cell line cells, and synergistically potentiated death receptor-induced apoptosis with TRAIL. Finally, co-treatment with PHA665752 and TRAIL causes more pronounced effect on PTC xenograft tumor growth in NUDE mice. Our data suggest that c-Met/HGF pathway may be a potential target for therapeutic intervention for treatment of PTC with c-Met inhibitor alone or in combination of conventionally therapeutic agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4045. doi:10.1158/1538-7445.AM2011-4045
Abstract 4047: TRIM32 facilitates cell growth, migration and anti-apoptosis
Authors: Kano Satoshi , Hatakeyama Shigetsugu , Fukuda Satoshi , Hatakeyama Hiromitsu
Published: August 2014

Introduction: Tripartite motif (TRIM) proteins are characterized by the presence of a RING finger, one or two zinc-binding motifs named B-boxes and an associated coiled-coil region. Most TRIM proteins have been reported to have a role in the ubiquitination process. Furthermore, several TRIM family members are involved in various cellular processes such as transcriptional regulation, cell growth, apoptosis, development and oncogenesis. TRIM32 also has a RING-finger domain and functions as an E3-ubiquitin ligase. In the previous study, it has been reported that TRIM32 protein expression is elevated in a mouse skin carcinogenesis model and in human skin squamous cell carcinoma and TRIM32 mRNA expression is elevated in human head and neck squamous cell carcinoma. However, the involvement of TRIM32 in carcinogenesis has not been fully elucidated. In this study, we studied the function of TRIM32 in the progression of carcinogenesis.

Methods: We established the cell lines overexpressed TRIM32 wild type and the deletion mutant of the RING-finger domain by the infection of retrovirus vectors. Using these cell lines, we analyzed the effect of TRIM32 on cell growth, transforming activity, cell motility and apoptosis induced by cisplatin. Additionally, we investigated whether siRNA for TRIM32 effects on these functions. Furthermore, we examined the expression level of TRIM32 protein in oral carcinoma samples using immunohistochemical method and analyzed the relationship between TRIM32 and clinical presentation.

Results: Overexpression of TRIM32 promoted cell growth, transforming activity and cell motility, whereas a dominant negative mutant of TRIM32 lacking the RING-finger domain inhibited these effects. In addition, we found that TRIM32 suppressed apoptosis induced by cisplatin in HEp2 cell lines, originating from human laryngeal squamous cell carcinoma. In contrast, siRNA for TRIM32 suppressed cell proliferation, cell motility and anti-apoptosis. In clinical research, a high-expression group of TRIM32 presented more advanced stage (clinical stage, T stage and neck lymph node metastasis) than a low-expression group.

Conclusions: In this study, our experimental results in vitro were supported by clinical research. These findings suggest that TRIM32 is a novel oncogene that promotes tumor growth, metastasis and resistance to anti-cancer drugs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4047. doi:10.1158/1538-7445.AM2011-4047
Abstract 4048: Histological image profiling identifies aggressive glioblastoma phenotype with poorer survival
Authors: Liu Yuexin , Shmulevich Ilya , Fuller Gregory , Zhang Wei
Published: August 2014

The stratification of glioblastoma (GBM) patients into clinically relevant subtypes is an essential step in the direction of personalized genomic medicine. In contrast to currently employed time- and cost-intensive genomic methodologies, we describe here a novel cost-effective approach capable of detecting predictors of invasive disease in GBM based on profiling of histological images. Unsupervised statistical analysis of imaging feature data first distinguishes two major clusters with strikingly different phenotypes. The subsequent Kaplan-Meier survival analysis of these two clusters shows that patients who had tumors in cluster 2 (which we refer to as aggressive subtype for the purpose of clarity) had poorer overall survival. Among the differentially varied features, the aspect ratio parameter, which is a strong indicator of cell movement, exhibits larger values in the aggressive subtype, suggesting greater invasiveness of this subset. Based on the initial dataset, a predictive model comprised of informative features was constructed using a weighted voting algorithm. Cross-validation of this sample set revealed a predictive accuracy of over 90% in correctly classifying these two groups, with specificity of ∼94% and sensitivity of ∼90% as determined by the receiver operating characteristic curves method. In a manner similar to gene biomarkers, some of imaging features can be used as diagnostic and prognostic factors in GBM. To identify the molecular events underlying the identified phenotypes, we performed genome-wide expression data analysis of the same sample set. Among the marker genes identified as overexpressed in the aggressive subtype were CHI3L1, CD44 and TNFRSF1A, which have been previously associated with poorer survival in GBM. In addition, pathway analysis of differentially-expressed genes indicates significant activation of the glioma invasiveness signaling and NF-κB signaling pathways; most of the altered genes in both pathways are more highly expressed in the aggressive GBM subtype and are thus potentially linked to the poorer survival of this group. Our novel approach has the potential to unify the imaging and genomic dimensions of GBM for clinical stratification.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4048. doi:10.1158/1538-7445.AM2011-4048
Abstract 4049: AP2A1 is required for UV-induced EGFR nuclear translocation
Authors: Hsu Sheng-Chieh
Published: August 2014

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is involved in cell proliferation, differentiation and survival. Aberrant expression and activation of EGFR have shown to cause various types of human and frequently associated with poor prognosis, metastasis and chemoresistance. Interestingly, this cell membrane receptor, which has been extensively studied, not only transduces signals from the cell membrane, but can also translocate into the nucleus and play a role in gene regulation, DNA repair and chemoresistance. Nuclear translocation of EGFR could be induced by ligand-dependent and ligand-independent manners including ionizing radiation (IR), cisplatin and UV treatment. However, the molecular mechanisms of EGFR nuclear translocation induced by DNA damage agents remain unclear. Here, we identify EGFR interacts with AP2A1 upon UV treatment using proteomic approach. The AP2A1 is the alpha 1 adaptin subunit of the adaptor protein 2 (AP2) complex found in clathrin coated vesicles. Knock-down AP2A1 expression inhibits the UV-induced EGFR nuclear translocation. Thus, we demonstrate here AP2A1 is required for UV-induced EGFR nuclear translocation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4049. doi:10.1158/1538-7445.AM2011-4049
Abstract 4054: Cell viability, apoptosis and ploidy analyses on a multi-laser, versatile flow cytometry analyzer – the EclipseTM
Authors: Song Yong
Published: August 2014

Cell viability, apoptosis and ploidy analyses are essential tools in cancer research involving in vitro and in vivo studies. Assays for cell count and viability, apoptosis and cell cycle analysis are described in this report. Jurkat or Chinese hamster ovary cells were treated with various reagents such as anti-CD95, taxol, or brefaldin A to induce apoptosis. Apoptotic cells were labeled with Avenxin-V conjugated with Pacific Blue or FITC to determine externalization of phosphatidylserine to the cell surface. In combination with cell impermeant dyes, such as 7-aminoactinomycin D (7-AAD) or propidium iodide (PI), live, early apoptotic and late apoptotic/dead cells were labeled and analyzed on an Eclipse equipped with the 405 and 488 lasers. The iCyt® Eclipse TM is a compact bench top flow cytometry analyzer with an autoloader that supports a wide variety of multi-well plate and test tube formats. The system can be equipped with up to 4 lasers (405, 488, 561 and 642nm) and provides simultaneous detection of 5 colors, forward scatter, side scatter, electronic volume (EV) and cell concentration. The EV parameter enables accurate cell volume (size) measurement and allows normalization of fluorescent intensity on a per volume or surface area basis. Apoptotic cells were also detected with a phycoerythrin-labeled anti-active caspase 3 antibody after fixation and permeabilization. Cell cycle analysis with G0/G1, S and G2/M phase statistics plus cell size was achieved by labeling cells with 4′,6-diamidino-2-phenylindole (DAPI) in a cell permeabilization solution and analyzing on the Eclipse. Together with its intuitive software, the Eclipse flow cytometry analyzer provides a rapid, accurate and cost effective tool for most flow cytometry applications such as cell viability, apoptosis and cell cycle analysis.

Note: For research use only. Not for use in diagnostic procedures.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4054. doi:10.1158/1538-7445.AM2011-4054
Abstract 4110: NanoString multiple target profiling identifies AEG-1 as an essential predictor of erlotinib outcome in EGFR-mutant non-small cell lung cancer (NSCLC)
Authors: Taron Miquel , Benlloch Susana , Rosell Rafael , Sanchez Jose Javier , Costa Carlota , Santarpia Mariacarmela , Viteri Santiago , Gasco Amaya , Massuti Bartomeu , Gimenez-Capitan Ana
Published: August 2014

Background: Low BRCA1 mRNA expression significantly prolongs progression-free survival (PFS) to erlotinib in EGFR-mutant NSCLC patients (p). In order to explore other genes potentially involved in erlotinib resistance, we used NanoString, an integrated digital technology, to examine expression levels of 48 genes in a single reaction. Several genes involved in homologous recombination repair were included in the 48-gene assessment, including the E3 ubiquitin ligases RNF8, RNF168 and RAP80.

Methods: CodeSets (Reporter and Capture probe sets) for 48 genes were custom-designed by NanoString Technologies. 100ng of total RNA for each sample was mixed with the reagents and hybridized at 65° overnight. Samples were processed and analyzed with the nCounter Prep Station (NanoString Technologies). Data analysis was performed adjusting the expression values according to positive control values and normalizing the data according to housekeeping genes.

Results: In 43 EGFR-mutant NSCLC p treated with erlotinib, response rate was 67.6%, PFS was 16 months (m), median survival (MS) was 29 m. Three genes were significantly associated with PFS: PIAS1, DBC1 and AEG-1. Median PFS was not reached in p with low levels of AEG-1, while it was 18 m for p with intermediate levels and 5 m for p with high levels (P=0.002). In the multivariate analysis, the hazard ratio for high levels of AEG-1 was 14 (P<0.001).

Conclusions: AEG-1 activates the NF-κB and PI3K/Akt pathways, induces the transcription factor LSF, which upregulates TS, ALDH3A1 and Met, conferring resistance to several cytotoxic drugs. AEG-1 mRNA expression predicts outcome to erlotinib-treated EGFR-mutant NSCLC p and could have important implications for the optimal management of NSCLC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4110. doi:10.1158/1538-7445.AM2011-4110
Abstract 4111: EGFR gene mutation as a predictive marker of the response to pemetrexed-based chemotherapy in non-small cell lung cancer
Authors: Nobuyuki Koyama , Sadakata Rinako , Miyazawa Hitoshi , Hagiwara Koichi , Kobayashi Kunihiko
Published: August 2014

[Background] Pemetrexed (PEM) is an antimetabolite that inhibits thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase, and one of standard anti-cancer agents against non-small cell lung cancer (NSCLC), particularly non-squamous cell carcinoma, whereas there is insufficient evidence for clinical practice. [Methods] We retrospectively studied 111 NSCLC cases treated with PEM-based chemotherapy in our institute to analyze the correlation of clinicopathological characteristics with the therapeutic effect of PEM. Furthermore, the validation of identified factors from the analysis was also conducted, using in vitro assays. [Results] The therapeutic effect of PEM plus carboplatin was comparable to that of PEM plus cisplatin from χ2 test. Kaplan-Meier survival analysis and logrank test showed that in the patients receiving PEM-based chemotherapy, epidermal growth factor receptor (EGFR) gene mutation was significantly correlated with the therapeutic effect and progression free survival but not overall survival. From IC50 of PEM, NSCLC cells with mutant EGFR were significantly sensitive to those with wild-type EGFR. [Conclusion] EGFR gene mutation can be a predictive marker of the response to PEM-based chemotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4111. doi:10.1158/1538-7445.AM2011-4111
Abstract 4112: De novo EGFR T790M mutation modifies outcome to second-line erlotinib in non-small cell lung cancer (NSCLC) according to metastatic site and upfront chemotherapy
Authors: Rosell Rafael , Molina Miguel Angel , Sanchez Jose Javier , Taron Miquel , Benlloch Susana , Moran Teresa , Carcereny Enric , Cardenal Felipe , Massuti Bartomeu , Magri Ignacio
Published: August 2014

EGFR T790M mutation is associated with shorter progression-free survival (PFS) (12 months [m] vs 18 m; P=0.02). We hypothesized that the site of metastases (mets) and/or prior chemotherapy could also influence outcome in these p.

Methods: The T790M mutation was assessed in 129 advanced NSCLC p by TaqMan assay in the presence of a peptide-nucleic acid designed to inhibit the amplification of the wild-type allele.

Results: De novo T790M mutations were identified in 35% (45 of 129) of EGFR-mutant p before receiving erlotinib. The T790M mutation was detected more frequently in p with bone mets (35.6% vs 16.7%; P=0.03). PFS was 20 m for 58 p with a deletion in EGFR exon 19 (del 19) but without the T790M mutation vs 12 m for 23 p with both del 19 and the T790M mutation (P=0.03), but with no difference in MS between these two groups (31 m vs 29 m; P=0.56). PFS was 15 m for 26 p with the L858R mutation but without the T790M mutation vs 16 m for 22 p with both the L858R and T790M mutations (P=0.83), with no difference in MS between these two groups (27 m vs 21 m; P=0.81). When p with T790M were divided according to the presence of brain mets, PFS was 1 m for 4 p with brain mets vs 13 m for 41 p without brain mets (P=0.002), while MS was 6 m for p with brain mets vs 36 m for those without (P=0.009). No effect on PFS or MS was observed in p with the T790M mutation according to lung, liver, pleura or bone mets. In the multivariate analysis, the presence of the T790M mutation did not increase the risk of short MS (HR, 1.3; P=0.49), while having received prior chemotherapy was associated with longer MS (HR, 0.48; P=0.02).

Conclusions: The de novo T790M mutation is a marker for poor prognosis in p with brain mets. Upfront chemotherapy can play a role in the management of NSCLC p with the de novo T790M mutation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4112. doi:10.1158/1538-7445.AM2011-4112
Abstract 4118: Expression of CD133 as a prognostic marker of non-small cell lung cancers
Authors: Mizugaki Hidenori , Konishi Jun Sakakibara , Kikuchi Junko , Kikuchi Eiki , Moriya Jun , Kubota Kanako , Kinoshita Ichiro , Oizumi Satoshi , Akita Hirotoshi Dosaka , Matsuno Yoshihiro , Nishimura Masaharu
Published: August 2014

[Background] CD133 is a membrance glycoprotein with five transmembrance loops. Previous reports have suggested that CD133-positive subpopulation of multipotent cells with extensive proliferative and self-renewal abilities has biological features as a cancer stem cell. In addition, it has been reported that the presence of CD133-positive cells was associated with a significantly poorer prognosis compared with the CD133-negative cells in some solid tumors, including colon cancer, hepatocellular carcinoma and glioma. However, little is known about the relationship between the expression of CD133 and clinical and clinicopathological characteristics in lung cancer.

[Aim and Method] We conducted immunohistochemical assessment of 161 surgically resected non-small cell lung cancers (NSCLCs) at Hokkaido University Hospital between 1982 and 1994 to evaluate the correlation between CD133 expression and various features, including clinical and clinicopathological characteristics.

[Results] Normal alveoli were used as negative controls and Bowman's capsule basement membrane as positive controls. CD133 expression was significantly correlated with advanced p-Stage (p=0.040). The Kaplan-Meier method demonstrated that NSCLC patients with CD133 expression in tumor cells showed poor prognosis compared to those without CD133 expression in p-Stage II, III and IV (p=0.0098). CD133 expression alone was an independent factor for unfavorable prognostic in those patients (Hazard Ratio=3.157, p=0.015).

[Conclusion] CD133 expression was correlated with p-Stage and was an independent factor for unfavorable prognosis in patients with p-Stage II, III and IV NSCLCs. CD133 expression may be a novel prognostic marker for NSCLCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4118. doi:10.1158/1538-7445.AM2011-4118
Abstract 412: Role of Interferon Regulatory Factor (IRF)-3 in lung tumor microenvironment
Authors: Shirota Hidekazu , Klinman Dennis M.
Published: August 2014

Cytokines present in the tumor microenvironment influence whether a cancer progresses or is rejected. For example, IFNs promote anti-tumor immunity and thus contribute to tumor rejection whereas IL-10 and TGFb can inhibit anti-tumor immunity and thereby contribute to tumor survival. We find that IFNbeta levels are significantly up-regulated in the microenvironment surrounding lung tumors. In the B16 F10 mouse melanoma lung metastasis model, CD11c+ alveolar cells respond to tumor challenge by rapidly up-regulating their expression of IFNbeta. This induction of IFNbeta was totally absent in IRF3 KO mice but intact in MyD88 and TRIF KO mice. Challenge studies indicate that IRF3 KO mice develop more lung metastasis and have shorter survival than controls. These findings suggest that CD11c+ cells play an important role in tumor recognition, and that they mount an IRF3/IFNbeta pathway specific response that slows tumor progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 412. doi:10.1158/1538-7445.AM2011-412
Abstract 4122: Multi-center validation of a lymph node metastasis gene-expression signature for head and neck squamous cell carcinomas
Authors: Speel Ernst-Jan M. , Leusink Frank K.J. , van Hooff Sander R. , Kummer J Alain , van Diest Paul J. , Koole Ronald , Roepman Paul , van den Brekel Michiel W.M. , Merkx Thijs A. , Jansen Jeroen C. , Schuuring Ed M.D. , Brakenhoff Ruud H. , Baatenburg de Jong Robert J. , Slootweg Piet J. , Holstege Frank C.P. , Takes Robert P.
Published: August 2014

Background: The inability of current diagnostic tools to detect small lymph node metastases in patients with Head and Neck Squamous Cell Carcinoma (HNSCC) leads to a rate of undetected metastases that is too high to refrain from elective neck treatment, resulting in overtreatment. The aim of this study is validation of a gene expression signature for distinguishing metastasizing from non-metastasizing HNSCC on a large multi-center cohort.

Material and methods: Samples of oral cavity and oropharyngeal cancer were collected from all head and neck oncological centers in the Netherlands. Gene expression was analyzed with a DNA microarray representing 696 previously reported predictive genes. The negative predictive value (NPV) was assessed on the whole cohort (n=222), on a subset including only clinically node negative (cN0) tumors (n=143) and on the most relevant subset that included T1 and T2, cN0 oral cavity tumors (n=101). Histological examination was used as gold standard for nodal status.

Results: Overall, the NPV of the signature was 72 %. The signature performed better on the cN0 subset (NPV 85%) and very well in the clinically most relevant subset of T1 and T2, cN0, oral cavity SCC (NPV 89%).

Conclusion: Combining the results of clinical and radiological examination with the gene expression signature decreases the rate of undetected nodal metastasis to 11 % in the relevant group of early stage cancers of the oral cavity. This result should be considered sufficient to refrain from elective neck treatment in these patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4122. doi:10.1158/1538-7445.AM2011-4122
Abstract 4132: Predictive classifiers for response to c-Met targeted therapies in lung and colon cancer
Authors: Drubin David , Catlett Natalie L. , Park Jennifer , Van Hooser Aaron A. , Frushour Brian P. , Kenney Renee Deehan
Published: August 2014

A fundamental challenge in the development of cancer therapies is the co-development of companion biomarkers to select patients most likely to respond to treatment prior to the availability of clinical outcomes data. We developed a methodology that leverages large scale public ‘omics data to identify candidate predictive biomarker panels to identify patients with a greater likelihood of responding to targeted therapies. We applied this approach to generate mechanism-based predictive biomarkers using our causal Knowledgebase as a substrate, as well as standard gene expression-based biomarkers for c-Met targeted therapy use in lung and colon cancers, and validated the methodology using clinical data. Gene signatures for c-Met activation in lung and colon were applied to individual lung and colon cancer training set patients to stratify them by c-Met activation levels. Data from patients identified as having high and low levels of c-Met activation were used to develop mechanism-based and gene expression-based classifiers to predict response to c-Met targeted therapies in lung and colon cancer. Validation of these classifiers requires transcriptomic data with associated clinical outcomes from c-Met inhibitor trials. In the absence of such, we verified this general approach using data from Inflammatory Bowel Disease (IBD) patients treated with the TNF inhibitor Infliximab, for which published colon transcriptomic data and associated clinical outcomes data are available {GSE16879} We developed a gene signature to assess TNF pathway activation and stratify a training set of IBD patients, then generated gene expression and mechanism-based classifiers from patients with high and low TNF signaling. These classifiers were tested on an independent test set of IBD patients treated with Infliximab {GSE12251}; the mechanism and gene expression-based classifiers predicted responders with positive and negative predictive values of 85%/88% and 70%/100% respectively. This method can be applied to other targets, pathways and diseases and may facilitate companion diagnostic development by enabling the selection of appropriate patient populations prior to treatment and clinical trial participation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4132. doi:10.1158/1538-7445.AM2011-4132
Abstract 4133: Is mitochondrial DNA copy number associated with interval colorectal cancers
Authors: Thyagarajan Bharat , Barcelo Helene , Shaukat Aasma
Published: August 2014

Colorectal cancers diagnosed in the interval after a complete colonoscopy (interval colorectal cancers) may occur due to due to rapid tumor growth. We have previously demonstrated that microsatellite instability (MSI) is associated with interval colorectal cancers. Mitochondrial DNA copy number has been shown to be inversely associated with rapid tumor growth in lung and breast cancers. However, the association of mitochondrial DNA copy number with interval colorectal cancers, MSI and survival remains unknown. The aim of our study was to study the association of mitochondrial DNA copy number with interval colorectal cancers, MSI and 5-year survival. We conducted a case-control study of 63 interval and 131 frequency matched non-interval colorectal cancers at the Veterans Administration Medical Center. Interval colorectal cancers were defined as colorectal cancers that developed within 5 years of a complete colonoscopy. Archived tumor specimens were tested for mitochondrial DNA copy number and MSI status. Relative mitochondrial DNA copy number was measured by a quantitative real-time polymerase chain reaction (PCR) assay using two pairs of primers, one specific for amplification of the mitochondria- specific gene (ND1) and another specific for amplification of a nuclear gene (CD3). Multivariable logistic regression was used to evaluate the association between mitochondrial DNA copy number, MSI and interval colorectal cancers. Cox proportional hazards regression was used to evaluate the association between mitochondrial DNA copy number and survival. Median mitochondrial copy number was 5.11 (range 1.4-193). Assay reproducibility, based on 20 samples run in duplicate, was 11%. There was no association between mitochondrial DNA copy number and interval colorectal cancers (OR 0.9; 95% CI 0.66-1.22; p 0.50), but mitochondrial DNA copy number was inversely associated with MSI status (OR 0.32; 95% CI 0.18-0.55; p <.001). There was a trend towards lower overall 5-year survival with lower copy number (HR 0.89; 95% CI 0.74-1.08; p 0.07), and a significantly lower survival with lower copy number among stage IV colorectal cancers (HR 0.53; 95% CI 0.31-0.90; p 0.02). Mitochondrial DNA copy number is inversely associated with MSI and overall survival for colorectal cancer. Low mitochondrial DNA copy number may be a marker of rapid tumor growth and poor survival in colorectal cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4133. doi:10.1158/1538-7445.AM2011-4133
Abstract 414: Does hedgehog signaling activation affect the tumor immune suppression microenvironment
Authors: Yang Ling , Gu Dongsheng , Liu Hailan , Zhang Xiaoli , Xie Jingwu
Published: August 2014

As an important pathway in embryonic development, hedgehog signaling plays a major role in stem cell maintenance, cell differentiation, tissue differentiation and tissue remodeling. Amounting evidence indicate that hedgehog signaling is frequently activated in a variety of human cancer. In consistent with the stromal effect of hedgehog signaling in development, several recent studies indicate a role of hedgehog signaling in tumor-stromal interaction. The molecular mechanisms by which hedgehog signaling regulates tumor microenvironment, however, remain largely elusive. We have generated data to support that hedgehog signaling regulates tumor immune surveillance. In a mouse model of hedgehog-mediated carcinogenesis, we found that activation of hedgehog signaling is associated with an increase in myeloid-derived suppressor cell (MDSC) population, which is decreased after hedgehog signaling inhibition. Our Based on our data, it is proposed that hedgehog signaling creates an immune suppressive environment through regulation of MDSC population, which is in concert with hedgehog signaling- mediated cell proliferation to promote carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 414. doi:10.1158/1538-7445.AM2011-414
Abstract 4144: Identification of molecules involved in PpIX accumulation in brain tumor induced by oral administration of 5-ALA
Authors: Suzuki Tomonari , Wada Satoru , Eguchi Hidetaka , Wakiya Kenji , Adachi Junichi , Mishima Kazuhiko , Nishikawa Ryo , Nishiyama Masahiko
Published: August 2014

[Background and Purpose] 5-aminolevulinic acid (5-ALA) is one of leading compounds widely used in photodynamic diagnosis and therapy of solid tumors: 5-ALA is metabolically converted to protoporphyrin IX (PpIX), a fluorescent molecule in response to excitation with a laser beam of 405 nm wavelength, that accumulates specifically in tumor lesions. This is found to be very effective for intra-operative detection of malignant brain tumors such as glioblastoma multiforme (GBM). On the other hand, we occasionally encountered cancerous lesions that are apparently malignant under a white light, while showing no fluorescence of the PpIX during cyto-reductive operation of the GBM. In order to overcome this problem, we hereby searched for factors associated with either presence or absence of the PpIX fluorescence by means of comprehensive gene expression analysis.

[Materials and Methods] Thirty-one patients of brain tumor (21 GBM, 3 pilocytic astrocytoma and 7 oligodendroglial tumors), orally-administrated with 5-ALA followed by operation under a fluorescence-guide, were enrolled in this study. Tumor specimens with or without the 5-ALA induced-fluorescence were collected from these patients and snap-frozen in liquid nitrogen. Total RNAs were extracted from the frozen tissues and were subjected to a comprehensive gene expression analysis using Whole Human Genome 4×44K Oligo Microarray (Agilent). We then compared gene expression levels between specimens with or without the fluorescence among individuals and selected candidate genes of which gene expression levels significantly differ between these. For validation, quantitative gene expression analyses were conducted for the candidates with real-time RT-PCR using gene specific-primers and probes. Knockdown experiments using specific siRNAs were also performed in cell lines derived from GBM to test their biological significance.

[Results and Discussion] A total of 14 genes were initially selected as candidates that were associated with PpIX accumulation in GBM in the microarray analysis. Among these, CDH13 (cadherin 13) gene was expressed significantly higher in the fluorescence-positive GBMs than in negative ones. Knockdown of CDH13 gene expression in GBM cells treated with 5-ALA, attenuated the fluorescence intensity, indicating its possible role in PpIX accumulation. Further experiments are underway to elucidate molecular mechanisms of CDH13 in 5-ALA-induced PpIX fluorescence in brain tumor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4144. doi:10.1158/1538-7445.AM2011-4144
Abstract 4151: High definition circulating tumor cells in patients with non-small cell lung cancer
Authors: Wendel Marco , Bazhenova Lyudmila , Nieva Jorge , Bethel Kelly , Marrinucci Dena , Cho Edward H. , Yoshioka Craig , Luttgen Madelyn , Uson Loressa , Metzner Thomas , Kolatkar Anand , Torrey Melissa , Kuhn Peter
Published: August 2014

Circulating tumor cell (CTC) counts are an established prognostic marker in metastatic prostate, breast, and colorectal cancer. However, there is little knowledge about CTC incidence rates and morphologic heterogeneity in non-small cell lung cancer (NSCLC) and their correlation to disease progression and usability for clinical staging. Here, we present a fluid phase biopsy approach that identifies CTCs without surface receptor-based enrichment, and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. The HD-CTC assay will be used to monitor HD-CTC counts and morphology in early stage, locally advanced, and metastatic NSCLC patient over time. 150 newly diagnosed NSCLC patients are being enrolled for this project. At the time of diagnosis 50 of the patients will have early stage cancers (stage I-II); 50 patients will have locally advanced (stage III) cancers, and 50 will be patients presenting with metastatic (stage IV) disease. Blood samples from each patient will be obtained at the time of diagnosis and 6 months later. We present here an interim data set that shows an incidence rate of 78% at baseline with a median CTC count of 19.5/ml of blood (range 0-441.6). Our preliminary data demonstrates that, despite stringent morphologic inclusion criteria for the definition of HD-CTCs, our HD-CTC assay shows high sensitivity in the detection of lung cancer CTCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2011-4151
Abstract 4154: Multi-parameter molecular characterization of circulating tumor cells (CTC): Development of a CTC-endocrine therapy index (CTC-ETI)
Authors: Paoletti Costanza , Brown Martha , Muñiz Maria , Rae James M. , Thomas Dafydd G. , Hayes Daniel F.
Published: August 2014

Introduction. CTC can be reproducibly and reliably enumerated using a commercially-available, automated immunomagnetic system (CellSearch®; Veridex LLC). High levels of CTC predict rapid progression in patients with metastatic breast cancer (MBC). Only ∼ 50% of patients with estrogen receptor (ER) positive MBC benefit from endocrine therapy (ET). Patients with endocrine-refractory MBC are better palliated with chemotherapy. We have developed a multi-parameter assay using CellSearch® that may identify patients with ER positive MBC who are unlikely to benefit from ET and may be better served with chemotherapy.

Methods. CellSearch® has four fluorescence channels. Three of these are used to distinguish CTC from WBC (DAPI, anti-cytokeratin, anti-CD45). The 4th “empty” channel was used to measure ER, Bcl-2, HER2, and Ki67 expression with antigen-specific fluorescent-labeled antibodies. These four markers were chosen because of their associations with sensitivity (ER, Bcl-2) or resistance (HER2, Ki-67) to ET. Cultured human breast cancer cells (MCF-7: ER +, BCL-2 +, HER2 -, Ki67 +; MDA-MB-231: ER -, Bcl-2 -, HER2 -, Ki67 +; BT-474: ER +, Bcl-2 +, HER2 +, Ki67 +) were spiked into 7.5 ml human whole blood from normal donors and separated and characterized using the CXC CellSearch® kit.

Results. Each cell line stained appropriately for the respective markers, with appropriate negative controls, although staining was heterogeneous, even within a single cell line. Using these preliminary data, we have developed a CTC-ETI in which scores are assigned to the individual categories consisting of cell counts coupled with the relative percent and degree of cell positivity of each marker. We predict that lower scores (low or no CTC, or CTC with high % and intensity of ER and Bcl-2; low HER2 and Ki-67) will be associated with favorable response to ET.

Conclusions. CellSearch® can be used to measure the expression of 4 clinically useful and proven breast cancer biomarkers. A clinical trial is underway at U. Michigan to permit calculation of CTC-ETI in patients with MBC, which will lead to a prospective trial to determine if high CTC-ETI predicts resistance and rapid progression on ET.

Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale ™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4154. doi:10.1158/1538-7445.AM2011-4154
Abstract 4155: Simultaneous quantitation of multiple signaling molecules in individual circulating tumor cells (CTCs) by multi-color laser scanning cytometry
Authors: Wu Weiguo , Zhang Yujian , Neil Chris , Copper Geoffrey M. , Kahn Larry , Melnikova Vladislava , Woo Jacky , Davis Darren
Published: August 2014

Introduction: Recent advances in circulating tumor cell (CTC) technology provide a new alternative approach to examine proteomic and molecular characteristic of tumor cells from cancer patients. The current trend in anti-cancer drug development focuses on new compounds that modulate multiple signaling pathways. With CTC detection used as a potential liquid biopsy, the simultaneous examination of signaling molecules for multiple pathways on the same CTC could provide insight into the drug activity in tumor cells. We used proprietary laser scanner cytometry (LSC) technology and developed a multi-color immunofluorescence (IF) procedure to detect 7 biomarkers within the same CTC. We validated this new staining technology using the (1) receptor tyrosine kinase pathways based molecules included total and phospho-IGF-1R, in combination with total and phospho-EGFR, or with total and phospho-ERK1/2, or with total and phospho-AKT (S473) in HT-29 colorectal tumor cells, and (2) same marker expression in CTCs enriched from cancer patients. Purpose: To validate the new multiple marker IF staining procedure and laser scanning cytometry (LSC) platform using ligand treated or untreated tumor cells, and in CTCs enriched from cancer patients. Method: A target biomarker staining panel was combined with standard CTC staining (DAPI, CK+ and CD45-) to create a 7-color IF staining panel to examine marker expression in tumor cell lines and in CTCs from cancer patients. Cultured tumor cells and CTCs enriched from Cellsearch Profile were cytospun onto slides, stained, scanned (by LSC) and analyzed. Results: Using a simultaneous co-staining procedure and LSC-based analysis, we demonstrated that treatment of HT29 cells with IGF1 for 15-30 minutes induced significant increases in both p-IGF1R expression and p-ERK1/2 and p-AKT expression on HT29 cells. In addition, anti-IGF1R and/or anti-EGFR, or with multi-kinase inhibitor treatment leads to differential reduction of p-ERK1/2 and p-AKT. Using CTCs enriched from cancer patient, simultaneous detection of multiple signaling molecules was also observed. Summary A multi-color, LSC-based methodology was developed to quantify expression of up to 4 biomarkers in CTCs. Application of this technology in clinical studies to examine the activation status of CTCs from patients could provide important information on pathway-specific drug activity and guide dosing selection.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4155. doi:10.1158/1538-7445.AM2011-4155
Abstract 416: Mammographic density and ECM stiffness
Authors: Acerbi Irene , Alfred Au , Lopez Jose , Hwang Shelley , Weaver Valerie
Published: August 2014

Breast tissue homoeostasis depends on the complex regulation of biochemical and biophysical factors present in the extra cellular matrix (ECM). Tumor tissue in mouse and human breast is characteristically stiffer than normal tissue, and changes to the ECM contribute to this phenotype. Breast density is associated with elevated collagen content, while increased deposition of collagen is known to augment ECM stiffness. Mammographic density imaging provides a qualitative description of breast density, and high mammographic density is associated with increased risk to malignancy. However, little is known about the correlation between ECM stiffness and the density in the breast image. Previously, we have used atomic force microscopy (AFM) to characterize the changes in breast tissue stiffness with cancer progression (Levental et al., Cell 2009). We hypothesized that mammographically dense breasts would be stiffer. To further understand the biophysical regulation of breast stiffness, we investigated whether mammographic density is a predictor of ECM stiffness. Towards this goal, we studied tissue samples from prophylactic mastectomies, from women with low versus high mammographic density. Tissue samples were obtained from the peripheral quadrants as well as from the retroareolar (nipple) regions, which are known to be of higher mammographic density. We performed AFM force mapping to characterize the ECM stiffness in the different regions. Our preliminary data indicate a heterogeneous pattern of stiffness within the breast. Moreover, we found that the peripheral terminal ductal lobuli have the most compliant ECM. By contrast, the stroma stiffness in the retroareolar region was 2-fold higher than in the peripheral quadrants. This work demonstrates the technical feasibility of measuring the mechanical properties as well as defined histological characteristics within the same patient specimen. The data suggest that ECM stiffness may be greater in breast tissue of high mammographic density. The correlation between mammographic density and ECM stiffness may reveal the role of collagen status in the risk to malignancy. (supported by W81XWH-05-1-0330 and R01 CA138818-01A1 to VMW, 1U01 ES019458-01 to VMW and ZW, and P50 CA 58207 to JG, VW, SH and LC.)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 416. doi:10.1158/1538-7445.AM2011-416