bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A1: Clinical and biological significance of GSK-3β inactivation in breast cancer: An immunohistochemical study
Authors: Armanious Hanan , Deschenes Jean , Gelebart Pascal , Ghosh Sunita , Mackey John , Lai Raymond
Published: June 2012

Glycogen synthase kinase 3β (GSK-3β), recently found to be functionally abnormal in various types of human disease, is negatively regulated by the PI3K/Akt signaling pathway. Since Akt is constitutively activated in a subset of breast cancer, we hypothesized that GSK-3β is inappropriately inactivated in these cases. In this study, we aimed to assess (1) the overall frequency of GSK-3β inactivation in breast cancer; (2) if there is an association between Akt activation and GSK-3β inactivation; and (3) whether there is correlation between GSK-3β inactivation and various pathologic and clinical parameters. The expression of the phosphorylated form of GSK-3β (pGSK-3β) and Akt (pAkt) were used as surrogate markers of GSK-3β inactivation and Akt activation, respectively. Immunohistochemistry applied to paraffin-embedded tissues was used to assess 72 consecutive invasive mammary carcinomas, of which 50 were estrogen receptor (ER)-positive. Overall, pGSK-3β and pAkt were positive in 34 (47.2%) and 35 (48.6%) cases, respectively. These two markers were significantly correlated with each other in the overall group and in the ER-positive subgroup (p=0.01 and 0.003, Spearman, respectively). Importantly, pGSK-3β, but not pAkt, significantly correlated a worse clinical outcome in this cohort (p=0.004, Log rank). In summary, evidence of GSK-3β inactivation was found in approximately half of the invasive mammary carcinomas. Our data suggest that this abnormality is likely attributed to Akt activation and that GSK-3β inactivation confers a worse clinical outcome.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A1.
Abstract A10: Off-target toxicity in white blood cells by AG-012986, a pan-CDK inhibitor, is mediated through inhibition of p38 MAPK signaling pathway
Authors: Lee Dong U. , Jessen Bart
Published: June 2012

Cyclin-dependent kinases (CDKs) have been an attractive target for cancer research because these kinases are predominantly active during cell division. AG-012986 is a small molecule pan-CDK inhibitor that has in vitro and in vivo antitumor properties. This compound was stopped during development due in part to rapid bone-marrow independent white blood cell toxicity in non-human primate and rodent species, and the potential for acute and delayed immunosuppression in humans. Because peripheral leukocytes are largely nonproliferating cells, it was hypothesized that the effects of AG-012986 was due to an off-target mechanism and not driven by the intended pharmacology. Using primary human peripheral blood mononuclear cells (PBMCs) and T-cells, we show that AG-012986 has a toxicity profile with greater specificity and more rapid cell death compared to the prototypical ATP-competitive kinase inhibitor, staurosporine. The cell toxicity mechanism was caspase-dependent and contained the hallmarks of apoptosis as indicated by cells staining double positive for propidium iodide and annexin-V. PBMCs or T-cells treated with AG-012986 and acutely stimulated either pharmacologically or through the T-cell receptor (TCR), exhibited decreased cellular toxicity compared to non-stimulated control cells. Interestingly under activating conditions with the pan-CDK compound, T-cells did not undergo significant cell division, as measured by BrdU incorporation, and were also rescued from apoptosis. This indicates that the pharmacology of AG-012986 was functioning as expected but that the toxicity due to its off-target effect had now been decoupled through cellular activation. Investigating the downstream signaling network showed that phosphorylation of p38 MAP kinase was impaired in the presence of AG-012986. Thus we hypothesize that AG-012986 may target the upstream kinase MKK3 and/or MKK6 for inhibition under basal conditions, leading to cell death in T cells and perhaps other white blood cells.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A10.
Abstract A11: Regulation of CD133 by the tubulin deacetylase HDAC6 can alter cancer cell state
Authors: Mak Anthony B. , Kittanakom Saranya , Chen Ginny I. , Mazitschek Ralph , Gingras Anne-Claude , Stagljar Igor , Moffat Jason
Published: June 2012

The CD133 antigen AC133/1 is a putative marker for enriching certain stem cells and cancer progenitor cells. Despite its wide application, the function and regulation of its expression remains unclear. Given the presence of its transcript in more differentiated cells, epigenetic regulation of CD133 transcripts alone cannot vouch for its utility as a marker. Therefore, understanding the regulation of CD133 protein expression is important for further defining the cellular state of tumor-initiating cells.

We used the MAPLE system (Mak et al, Mol Cell Proteomics, 2010) to identify bona fide protein interaction partners of CD133. We present the microtubule-associated histone deacetylase 6 (HDAC6) as a physical interaction partner of CD133. This interaction was validated by co-immunoprecipitation/Western Blot analyses and by a membrane yeast two-hybrid (MYTH) assay. In order to understand the functional consequence of the HDAC6 and CD133 interaction, we used lentiviral short-hairpin RNAs to knockdown HDAC6 in multiple cell types, including the human epithelial colorectal adenocarcinoma lines Caco- 2 and HT-29 and in the retinoblastoma cell line Weri-Rb-1. Interestingly, we observed a significant reduction of CD133 protein expression. This reduction was shown to be dependent on HDAC6 deacetylase activity using a deacetlyase deficient HDAC6 mutant as well as when using the small molecule inhibitor of HDAC6, tubacin.

HDAC6 promotes de-acetylation of alpha-tubulin to influence re-cycling of cell surface proteins. Furthermore, when we performed a CD133 MYTH screen against a cDNA library isolated from an adult human brain, we identified the late endosomal- and lysosomal-associated marker CD63 as an interaction partner of CD133. In fact, treatment of Caco-2 cells with tubacin resulted in trafficking of CD133 into endosomes for lysosomal degradation as determined by increased co-localization with CD63.

In an attempt to study the affect of HDAC6 knockdown on cell viability, we noticed that CD133 knockdown significantly impacted cell proliferation and clonogenicity of Caco-2 cells. We determined that the cause of this is likely due to increased differentiation as determined by the level of alkaline phosphatase activity and by quantitative realtime PCR for markers of differentiated colon cells. To assess the tumorigenic potential of Caco-2 cells with reduced CD133 expression in vitro we performed soft agar colony-forming assay, which resulted in the loss of anchorage independent growth, a well-known hallmark of cancer. Together, these results suggest that CD133 can exercise a function in establishing and maintaining a primitive cancer cell state and loss or reduction of its expression marks more differentiated cancer cells that may be nontumorigenic.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A11.
Abstract A12: Cardiac hormone receptor A, NPRA, as a prognostic marker and target for prostate cancer therapy
Authors: Wang Xiaoqin , Patel Ronil , Hellerman Gary , Coppola Domenico , Mohapatra Shyam S. , Mohapatra Subhra
Published: June 2012

Management of prostate cancer (PCa), the third leading cause of death among American men, is limited by lack of prognostic markers and therapies for targeting androgen-independent PCa. Herein we report that NPRA, the receptor for the cardiac hormone atrial natriuretic peptide, is a potential prognostic marker and therapeutic target for PCa. NPRA is abundantly expressed in the androgen-independent PCa cell lines, PC3 and DU145, but not in normal PrEC cells and only weakly in RWPE and benign prostate hyperplasia (BPH) cells. NPRA is more highly expressed by the tumorigenic TRAMP-C1 and -C2 and PTEN-CaP2 PCa cell lines than the nontumorigenic TRAMP-C3 and PTEN-P2 PCa cell lines. NPRA showed positive correlation with clinical staging in a human PCa tissue microarray (TMA). Prostate tumor cells implanted in mice deficient in atrial natriuretic peptide receptor A (NPRA-KO) failed to grow, and down-regulation of NPRA by siNPRA or NPRA inhibitor, iNPRA, induced apoptosis in PCa cells and reduced tumor burden in mice. The mechanism of iNPRA-induced anti-PCa effects was linked to NPRA-induced expression of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine overexpressed in prostate cancer and associated with tumor survival. SiNPRA significantly reduced MIF expression. Using the TRAMP spontaneous PCa model, we found that NPRA expression correlated with MIF expression during PCa progression and treatment of TRAMP-C1 xenografts with iNPRA reduced MIF expression. MIF was also seen in the human PCa TMA. This suggests that NPRA promotes PCa development by regulating MIF and may represent a prognostic marker and target for PCa therapy.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A12.
Abstract A13: Discovery of selective inhibitors of the kinase and metastatic activities of the activated Cdc42-associated kinase (ACK1)
Authors: Olabisi Oyenike , Kholodovych Vladyslav , Kamal Alam Nur-E- , Stewart-Singleton Safirah , Andayah Christina , Wood Richard , Welsh William
Published: June 2012

Activated Cdc42-associated kinase (ACK1) is a non-receptor tyrosine kinase and an effector protein of Cdc42. Previous reports have demonstrated that ACK1 is a critical factor in the cellular transformation of a number of cancer cell lines. ACK1 also contributes to cellular motility, and overexpression in certain cell lines results in enhanced motility. The significance of tyrosine kinase proteins such as ACK1 in cancer-related signaling pathways, and the implications of metastasis in the prognosis of cancer patients, point to the importance of the discovery of a potent yet selective inhibitor for this protein. As a result, we are engaged in a study focused on applying computational approaches to identify novel inhibitor molecules of ACK that will also hamper its ability to promote motility of cancer cells.

We employed customized in-house computational tools to retrieve approximately 50 small-molecule hits from our chemical databases that may bind with high affinity to the ACK1 ATP binding pocket. These hits were further screened for drug-like properties (Lipinski's Rule of 5) and rescored using GOLD docking to the ACK1 crystal structure (PDB3EQR). After rescoring, the top 25 hits were then tested for in vitro cytotoxicity in a variety of cancer cell lines. Compounds were specifically examined for their impact on the kinase activity of purified ACK1 and for their ability to modulate its metastatic properties in wound healing and transwell migration assays.

Our screening has revealed that several compounds have the ability to inhibit the in vitro kinase activity of ACK1 and also inhibit the in vitro growth of cancer cells. Ongoing immunofluorescence, cellular transformation, and cellular motility studies suggest that the selected compounds may modulate the effect of ACK1 on the motility of cancer cells. We are actively proceeding with specific structural modifications of our initial inhibitor compounds in order to find molecules which possess increased cytotoxicity to cancer cells and improved specificity for the inhibition of ACK.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A13.
Abstract A14: EGFR mutation status and the efficacy of gefitinib in combination with paclitaxel and carboplatin as neoadjuvant treatment in locally advanced cancer of the cervix: A study from eastern India
Authors: Pandey Ritwik , Mukhopadhyay Ashis
Published: June 2012

Purpose: To study the incidence of EGFR mutations (Exon 19-21) in locally advanced cervical cancer and to assess wether gefitinib, in combination with paclitaxel and carboplatin leads to better outcomes in patients who harbored EGFR mutations. Also, to study the response rates of paclitaxel and carboplatin as neoadjuvant treatment in locally advanced cervical cancer.

Materials and Methods: From January 2008 to April 2010, 108 patients of inoperable locally advanced carcinoma of the cervix (Stage Ib – II only) were prospectively evaluated for EGFR mutations (exon 19-21) and were randomized to receive paclitaxel (175 mg/m2) and carboplatin (AUC 5) with gefitinib (For mutation +ve patients) or without gefitinib (for mutation -ve patients). Patients were assessed for incidence of EGFR mutation, response rates to treatment and conversion to operable disease after receiving neoadjuvant chemotherapy. The patients who were still inoperable after this treatment were offered chemoradiation.

Results: 30 of 108 patients (30.5%) were positive for EGFR mutations. 50 of 78 patients who received paclitaxel and carboplatin had a more than partial response to treatment giving a response rate of 64.1 %. 20 of 78 (25.6%) patients had a stable disease while 8 of 70 (11.4%) patients had a progressive disease. 25 of 30 patients who were EGFR mutation +ve had a response to paclitaxel + carboplatin + gefitinib giving this combination a response rate of 83.3%. This response rate was significantly higher (p=0.001) as compared to mutation negative patients. Only 1 of 30 (0.03%) patients in this subgroup had a progressive disease. 20 of 25 (80%) patients in the mutation +ve group had an R0 resection as compared to 35 of 78 (44.8%) patients in the mutation -ve group (p=0.02).

Inference: Approximately one-third of the cervical cancer patients were positive for EGFR mutations. These patients when given gefitinib in combination with chemotherapy had a significantly higher response rate and conversion to operability. Also that paclitaxel + carboplatin is an effective neoadjuvant regimen for locally advanced cancer of cervix giving a response rate of about 65% and high conversion to operability.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A14.
Abstract A16: Thrombin promotes hepatocellular carcinoma metastasis via mediating osteopontin-dependent tumor growth and adhesion activity
Authors: Xue Yu-Hua , Zhang Xiao-Fei , Dong Qiong-Zhu , Ye Qing-Hai , Qin Lun-Xiu
Published: June 2012

In our previous study, osteopontin (OPN) was identified as one of the leading genes that promote the metastasis of hepatocellular carcinoma (HCC), and a potential therapeutic target for HCC metastasis. The serine protease thrombin can interact with OPN and modify its biologic activity. To explore the role of thrombin alone or collaborating with OPN in HCC, we investigated the thrombin level and its correlation to the prognosis of 72 HCCs with different OPN backgrounds using real-time PCR and Western blot, and evaluated the effects of OPN fragments formed by thrombin cleavage on in vitro proliferation and adhesion of HCC cells. We found that the expression level of thrombin was positively related to the metastatic potentials of HCC cell lines, and was remarkably overexpressed in HCCs compared with the adjacent nontumor tissues. In addition, HCC tissues from patients with recurrent disease displayed much higher thrombin levels, particularly in those with OPN overexpressed. In the immunohistochemical study of 230 HCCs, only in those with a higher OPN level, a significant association was found between the higher thrombin level and the overall survival (OS; P < 0.01) and time to recurrence (TTR; P < 0.0001) of patients. Assays in vitro demonstrated that thrombin could promote the proliferation and adhesion of OPN+ HCC cells. And N-terminal of OPN showed better effects on the proliferation and adhesion abilities of HCC cells than the intact protein. Thrombin treatment could activate FAK pathway of OPN+ HCC cells, and this activation was blocked by the inhibition of integrin β1. Conclusions: Thrombin plays an important role in OPN-mediated aggressive phenotype and poor prognosis of HCC, which may be through the activation of the integrin β1 -FAK signaling. So, thrombin might be a potential therapeutic target to combat HCC progression in thrombin+/OPN+ patients.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A16.
Abstract A17: A role for the endocytic regulators Rab25 and Rab coupling protein (RCP) in K-Ras dependent colorectal cancer cells
Authors: Rooney Claire , Barry Simon
Published: June 2012

Endocytic transport impacts a wide range of cellular functions, including growth factor signaling, cell polarity, and cell-cell and cellmatrix adhesion. As such, deregulated endocytosis is emerging as a key feature of tumorigenesis. Growth factors and their receptors are important regulators of normal cell survival and proliferation and aberrant growth factor signaling is one of the fundamental elements of tumor growth and progression. Upon activation at the plasma membrane, growth factor receptors are internalized and either trafficked to the lysosome for degradation, or returned to the cell surface via the process of endosomal recycling. Furthermore, growth factor receptors can signal from endocytic compartments en route to the lysosome, and throughout the recycling process. Downstream of growth factor stimulation, signaling from Ras GTPases to effectors such as the mitogen activated protein kinase (MAPK) pathway has also been shown to occur at endocytic compartments. Elucidation of the key regulators of the endocytic process therefore presents a novel opportunity to target certain growth factor signaling pathways. Two such key regulators are the small GTPase Rab25, and its interacting partner Rab coupling protein (RCP). Rab25 and RCP are amplified in certain breast and ovarian cancers, and this amplification is associated with increased aggressiveness of these cancers. Additionally, in mutant p53 cells RCP has been implicated in enhanced EGFR and integrin signaling, resulting in increased invasion. Here, we provide evidence for the importance of Rab25 and RCP in colorectal cancer (CRC) cell lines. Using siRNA to specifically knockdown RCP and Rab25 in a panel of CRC cell lines we have found that RCP and Rab25 knockdown significantly impacts on the proliferation of these lines. Within the panel of CRC cell lines, we have identified subpopulations of K-Ras dependent and K-Ras independent lines. K-Ras dependency appeared to be independent of K-Ras mutation status, and those lines which depend on K-Ras for proliferation also require RCP and Rab25. We are currently investigating the consequences of RCP knockdown on growth factor receptor signaling and subsequent Ras activation. These findings highlight the importance of the regulation of recycling endocytosis in colorectal cancer, and indicate a dependence of colorectal cancer cells on this process.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A17.
Abstract A2: Receptor status in breast cancer and reproductive history: Is there any relation?
Authors: Basu Chinmay K. , Pandey Ritwik , Mukhopadhyay Soma , Mukhopadhyay Ashis
Published: June 2012

Objective: Molecular profiling studies using immunohistochemistry have identified subtypes of breast cancer that can be approximately classified by estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu (HER-2) expression. These molecular subtypes and their combinations taken together with reproductive history of woman may help prognostication of breast cancer cases. We report a series of 102 cases of breast cancer treated in our institution over last five years (2004-08).

Methods: Paraffin-embedded primary breast cancers from 102 patients with invasive and ductal carcinoma were studied immunohistochemically. Markers along with reproductive history were analysed.

Result: 78 ductal 12 lobular cases were found. 43 cases were postmenopausal. Hormone receptor positive cases were 63 (61.7%), ER+ 34 (33.3%), and PR+ 29 (28.4%). HER-2/neu + cases were 34 (33.3%). There were 11 cases of triple negative (basal-like) breast cancer. Significant inverse correlation was found between HER-2/neu and hormone receptor. Early age at menarche was only found to be associated with risk of HER-2neu+, whereas breastfeeding had protective effect. Both late ages at menopause, hormone therapy was associated with poor prognosis. Higher age at first child birth was significantly associated with poorer outcome and no difference in risks with parity was noticed. HER-2/neu was not associated with response rate but their prognosis was worse. Triple negative had 3 survivors at three years. Significant associations were observed between HER-2/neu and increasing number of involved and metastases (p = 0.01). Analysis demonstrated that HER-2/neu overexpression (p = 0.02) and age (p = 0.01) were independent predictors for disease-free survival (DFS).

Conclusion: Hormone receptor positive cases are associated with better prognosis than HER-2/neu positive cases. For node positive patients, HER-2/neu overexpression was a significant predictor of DFS. Reproductive history had significant bearing in prognosis and receptor expressing subsets of breast cancer which may be clear by further study.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A2.
Abstract A20: FGFR1-amplified squamous cell lung cancers depend on FGFR1
Authors: Sos Martin L. , Thomas Roman K.
Published: June 2012

In lung adenocarcinomas of never-smokers EGFR mutations or EML4-ALK gene fusions are frequent genomic events that both expose prime therapeutic targets. By contrast, squamous cell lung cancer (SQLC) is almost invariably associated with smoking; in this subgroup of patients, no attractive therapeutic target has so far been identified. In order to identify such a therapeutically exploitable target in SQLC, we performed high-resolution genomic analyses of over 300 lung tumor specimens. Using 6.0 Affymetrix SNP-arrays we identified high-level focal amplifications of FGFR1 in 10% of over 150 primary SQLC specimens. Importantly, FGFR1 amplification was almost exclusively restricted to SQLC of smokers and almost never observed in adenocarcinomas. We also performed high-throughput cell line screening of 82 genomically annotated lung cancer cell lines using the FGFR inhibitor PD170374 and identified the presence of focal FGFR1 -amplifications as the strongest predictor of PD170374 activity. Biochemical kinase profiling across 108 kinases suggested that PD170374 was highly selective with only one of the kinases being inhibited to a similar extend as FGFR1 itself. Treatment with PD173074 induced apoptosis and inhibited MAPK- and, to a lesser extend, PI3K signaling in cell lines with focal amplification of FGFR1. In order to validate FGFR1 as a critical target in FGFR1 -amplified cells we employed retroviral expression of the gatekeeper mutant allele of FGFR1 (V561M) and thus the PD173074-sensitive cells were rendered resistant. Additionally, lentiviral-mediated knockdown of FGFR1 in FGFR1 -amplified cell lines confirmed FGFR1 as a critical target for FGFR1 inhibition in these cells. Finally, successful treatment of mice engrafted with FGFR1 -amplified lung cancer cell lines with PD173074 confirmed its on-target activity in vivo. Overall, our work demonstrates the functional relevance of amplification of FGFR1 in lung cancer and suggests that inhibition of FGFR with targeted therapeutics might be an effective strategy in squamous cell carcinoma patients with focal amplification of FGFR1.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A20.
Abstract A21: Expression of placental growth factor in hepatocellular carcinoma and peritumoral liver and its prognostic significance
Authors: Sun Hui-Chuan , Xu Hua-Xiang , Zhu Xiao-Dong , Tang Zhao-You
Published: June 2012

Vascular endothelial growth factor targeted therapy is a promising treatment for hepatocellular carcinoma (HCC), but its clinical benefit is often accompanied by acquired resistance. Antiplacental growth factor (PlGF) therapy is effective in animal study with less resistance. Role of PlGF in the progression of HCC is not clear. In this study, we investigated PlGF expression in tumor and peritumoral liver tissues from 105 HCC patients by immunohistochemistry in tissue microarrays. Intratumoral and peritumoral PlGF mRNA expression was analyzed in another cohort containing 37 patients. Peritumoral PlGF expression was significantly higher than intratumoral PlGF expression (P < 0.001). Intratumoral PlGF expression was not associated with patients’ overall survival (OS) or time to recurrence (TTR). However, peritumoral PlGF expression, which was associated with tumor size (P = 0.002), presence of intrahepatic metastasis (P = 0.005), TNM stage (P = 0.048), and BCLC stage (P = 0.001), was an independent risk factor for OS (P < 0.001) and TTR (P = 0.001). Prognostic value of peritumoral PlGF expression was validated in a validated cohort. Furthermore, we inferred that elevation of PlGF in peritumoral liver might be induced by hypoxia. And we found that peritumoral PlGF expression was associated with hypoxia-inducible factor 1α (HIF-1α) (P = 0.003) and PlGF expression was elevated in hepatic cell line L02 under hypoxia in vitro. These findings indicate that high peritumoral PlGF expression was associated with tumor recurrence and survival after resection of HCC. PlGF could be a target of adjuvant therapy and deserves further investigations.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A21.
Abstract A23: Frequent ASS1 deficiency in bladder cancer and sensitivity to pegylated arginine deiminase (ADI-PEG20): A potential novel therapeutic strategy
Authors: Allen Michael , Syed Nelofer , Crook Tim , Jones Louise , Lemoine Nicholas R. , Berney Dan , Szlosarek Peter
Published: June 2012

Tumors deficient in argininosuccinate synthetase (ASS1), a key enzyme involved in arginine synthesis, are auxtrophic for arginine and sensitive to amino acid deprivation. Based on reports of ASS1 negativity and arginine auxotrophy of prostate and renal cancers, we investigated the role of ASS1 and arginine depletion in bladder cancer, a common urological malignancy with limited treatment options for advanced disease. First, we screened 8 bladder cancer cell lines revealing very low mRNA levels and low or absent ASS1 protein in 4 of 8 cell lines. The ASS1 negative cell lines were all hypermethylated at the ASS1 promoter, compared with the ASS1 – positive cell lines, which were hypomethylated. An analysis of 48 patients with bladder cancer revealed 65% were ASS1 -negative by immunohistochemistry, with evidence for ASS1 methylation as a mechanism for ASS1 inactivation. There was no correlation between ASS1 loss and the type, grade or stage of bladder tumor. Next, we exposed two ASS1 -negative and two ASS1 -positive bladder cancer cell lines to pegylated arginine deiminase (ADI-PEG20), an arginine depleting agent that is currently undergoing evaluation in several clinical trials in patients with solid malignancies. The ASS1-negative, but not the ASS1 -positive, bladder cancer cell lines were sensitive to ADI-PEG20 treatment. Current work is focussed on determining whether there is enhanced cell killing when ADI-PEG20 is combined with cisplatin-based chemotherapy. In summary, ADI-PEG20 is a potential novel therapeutic approach in the treatment of ASS1 – negative bladder cancer and warrants further investigation in the clinic.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A23.
Abstract A25: Variations of human papillomavirus type 16 in cervical, lung, and skin cancers in Sichuan, China
Authors: Zhang Jian , Wang Tao , Han Min , Yang Zhihui , Zhang Min , Liu Lunxu , Zhou Bin , Chen Yue , Hou Qiannan , Wang Yanyun , Lai Ting , Zhang Lin , Hu Huaizhong , Xi Mingrong
Published: June 2012

Human papillomavirus (HPV) is closely related with many kinds of cancers such as cervical, lung, and skin cancers, etc. HPV 16 showed intratypic variations that were known to differ in oncogenic potential and geographic distribution. This study was designed to analyze sequence variants in oncogenes E6, E7 and L1 of HPV 16 in cervical, lung, and skin cancers in Sichuan, China. Materials from 138 non-malignant controls, 122 cervical cancer patients, 104 lung cancer patients and 104 skin cancer patients were analyzed by PCR and direct sequencing. The infection rates of HPV 16 in cervical, lung, skin cancers were respectively 68.9%, 17.3%, 5.8%. The Asian prototype was the most prevalent variant in cervical cancer in Sichuan (40%). Compared with that detected in cervical cancer, lung and skin cancers showed more monotonous variations, but several mutations on L1 genes existed only in lung or skin cancers such as G6818C, C7011A, and C7017A.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A25.
Abstract A28: Multiplexed immunofluorescence in formalin-fixed paraffin-embedded prostate cancer
Authors: Sevinsky Christopher , Dinn Sean , Pang Zhengyu , Li Qing , Sarachon Brion , Rothney Megan , Colligan Bruce , Douglass Larry , Carter Julia , Graff Jeremy , Ginty Fiona
Published: June 2012

Traditional immunohistochemistry serves as a vital diagnostic assay as it allows for semiquantitatively probing the magnitude and spatial distribution of protein expression and posttranslational modifications. Unfortunately, an accurate portrayal of disease often requires several immunohistochemical stains, which are run on separate tissue sections. The application of transcriptomics analyses in cancer classification has led to the generation of multivariate index analyses that confer diagnostic, prognostic, and predictive signatures of disease. However this method is limited by the inability consider target expression in the context of tissue (stroma, epithelium) or cell (e.g. stem/progenitor cells) type. A unifying platform that would allow for the quantitative spatial analysis of multiplexed in situ target measurements promises to exploit the best features of both systems. To this end, we have developed a novel multiplexed fluorescence immunohistochemistry assay that is capable of quantitative analysis of >25 antigens in a single formalin-fixed paraffin-embedded tissue section, as well as image and data analysis capabilities for analyzing the multidimensional data. By inclusion of targets for cell and tissue compartments, including nuclei, cytoplasm and membrane, we have demonstrated the ability to investigate subcellular protein expression, phosphorylation and co-localization in individual cells. In the present study we have stained a cohort of 79 prostate cancer patients of varying Gleason grades for a total of 24 antigens, representing key mediators of cell signaling, tumor suppressors and oncogenes, tumor associated phenotypic targets, and structural proteins. The images are precisely registered and segmented into individual cells nuclear, cytoplasmic and membrane regions and the underlying expression of a given antigen is then quantitatively determined in each compartment by automated algorithms. With the advent of this new tool, we aim to define new prognostic molecular signatures in prostate cancer.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A28.
Abstract A29: Discovery and preclinical to clinical evaluation of biomarkers for Anti-DLL4, a monoclonal antibody targeting cancer stem cells
Authors: Kapoun Ann M. , Wang Min , O'Young Gilbert , Beviglia Lucia , Liu Yu-Wang , Yeung Pete , Fischer Marcus , Smith Dave , Lewicki John , Hoey Tim
Published: June 2012

Blockade of DLL4 signaling reduces tumor growth in preclinical human xenograft models by disrupting productive angiogenesis and reducing proliferation of tumor cells. Importantly in these models, blocking DLL4 also reduces cancer stem cell (CSC) frequency in multiple solid tumor types. Currently, there exists an important need to identify and evaluate pharmacodynamic biomarkers that indicate the biological activity of agents, such as anti-DLL4, that are in clinical studies. Towards this aim, we used numerous human xenograft models derived from primary human tumors to identify and characterize biomarkers for anti-DLL4. Here, we present data from these studies and describe anti-DLL4 biomarkers in tumors, in hair follicles, and in blood. We show that several vascular-related genes are significantly upregulated by anti-DLL4 in tumors including, Egfl7, Apln, Cldn5, and Notch4. We also demonstrate significant downregulation of CSC gene signatures and modulation of differentiation genesets. A subset of the identified markers were validated at the protein level and also tested in a human skin graft model, where the human tumor is engrafted in a human microenvironment/vasculature. Finally, a DLL4-Notch gene signature was identified that showed a significant association with breast cancer survival using publicly available cancer gene sets. In surrogate tissues, we identified anti-DLL4 biomarkers in blood and in hair follicles. These markers from surrogate tissues were translated into a phase I trial of Anti-DLL4 antibody (OMP-21M18), and an interim analysis of this data demonstrates that OMP-21M18 affects the Notch pathway in patient samples.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A29.
Abstract A3: The human epidermal growth factor receptors in gastric carcinomas: A study using fluorescence in situ hybridization and immunohistochemistry
Authors: Fukuda Emy , Soares Fernando , Fregnani Jose H. , Begnami Maria D.
Published: June 2012

The human epidermal growth factor receptors (EGFR) family consists of four members: ErbB-1 (HER1), ErbB-2 (HER2), ErbB-3 (HER3) and ErbB-4 (HER4). These receptors activate numerous downstream pathways in response of extracellular ligands, regulating diverse processes including differentiation, migration, proliferation, and survival. Alterations in EGFR family members play a role in the development and progression of many human cancers. In gastric carcinomas (GC), HER1 and HER2 overexpressions are thought to be prognostic factors and targets of novel biological agents. The effect of HER3 or HER4 expression in GC has not been sufficiently studied. HER3 expression is observed frequently in advanced GC with poor prognosis and HER4 gene expression seems to be higher in GC tissue in comparison with adjacent gastric mucosa. In this study, we explored gene and protein expression of the EGFR family in GC in order to establish new potentially prognostic factors. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were carried out in 221 GC using tissue microarray. IHC positivity for EGFR was defined by score 2+ or 3+ according with intensity and frequency of membranous staining. In addition, the intensity of the cytoplasm staining was also determined for HER3 and HER4 overexpression. HER1, HER2, HER3, and HER4 overexpression was found in 3/197 (1.5%), 24/207 (12%), 121/193 (64%) and 41/183 (22%) cases respectively. FISH assay was performed according to the protocol previous described. In each case, 100 tumor nuclei were evaluated. Cases showing a gene/CEN fluorescence ratio ≥ 2 were considered positive for gene amplification. FISH detected HER1 and HER2 amplification in 1.3% and 8% of the cases. Amplifications for HER3 and HER4 were not observed. Overall, alterations of the 4 members of EGFR were significantly associated with parameters involved with tumor progression, including the depth of tumor invasion, involved lymph nodes, and tumor stage. We herein showed a strong correlation between HER2, HER3 and HER4 overexpression and low-grade tumors, corresponding to the intestinal type GC according to Lauren's classification. In addition, HER2 amplification was significantly related with worse survival. These results reveal that members of EGFR family are activated in GC. The exact molecular mechanisms involved in the HER3 or HER4 alterations in gastric cancer, especially in the intestinal type, remain unclear since amplifications of these genes were not found. Gaining further understanding into the oncogenic mechanism of EGFR family may not only help in the development of targeted therapy in gastric patients but might accelerate the acceptance of a novel taxonomy of cancer which is based on the genomic perturbations in cancer genes and cancer gene families and their response to targeted agents.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A3.
Abstract A31: Prognostic microRNA biomarkers for recurrence of stage II colorectal cancer
Authors: Sanford Tiffany , Adai Alex T. , Doleshal Martina , Langfield Laura , Choudhury Bhavna , LeBourgeois Paul , Pappas Todd , Mambo Elizabeth
Published: June 2012

Reliable prediction of recurrence in stage II colorectal cancer (CRC) patients is a major unresolved clinical need as recurrence is often the ultimate cause of death in these individuals. Several mRNA and DNA prognostic markers have been evaluated and, to date, none have been recommended by oncology groups due to poor specificity and sensitivity. MicroRNA (miRNA) have been shown to regulate fundamental cellular processes and have been implicated in several cancer types, including CRC. We used a microarray platform to examine the expression of more than 600 miRNAs in tumors from stage II patients with or without recurrence. Using an exploratory sample set of 12 tumors (6 nonrecurrent and 6 recurrent), we identified several miRNAs that were significantly deregulated in tumors from recurrent patients compared to non-recurrent patients. We used RT-qPCR to interrogate the differentially expressed miRNAs on the original 12 samples plus an additional set of 39 stage II CRC tumors that were not used in the initial miRNA discovery process. Using cross-validation, we derived a miRNA-based classifier that was strongly associated with recurrence (hazard ratio (HR), 3.83; p=0.005). We further evaluated associations of MSI status and mutations in KRAS, BRAF and PIK3CA genes with recurrence (n=39). Multivariate Cox analysis revealed that only the miRNA classifier was significantly associated with recurrence (HR 3.08; p=0.03). Interestingly, the combination of the miRNA classifier and mutant KRAS status was highly predictive of recurrence (p<0.0001). These data provide strong support for miRNAs as prognostic biomarkers for stage II CRC and warrant further validation.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A31.
Abstract A35: Immunological characterization of Imprime PGG® and an anti-EGFR antibody combination treatment for cancer
Authors: Antonysamy Mary A. , Dudney Christine , Yokoyama Yumi
Published: June 2012

More promising antitumor strategies utilize combination therapies, with each strategy targeting a separate aspect of oncogenesis. Imprime PGG® (Imprime PGG) is a soluble beta-1,3/1,6 glucose polymer being developed as an adjunct to monoclonal antibody (MAb) therapy for cancer. Imprime PGG, a pathogen- associated molecular pattern isolated from the cell wall of S. cerevisiae, has shown therapeutic antitumor and survival-enhancing effects when combined with an antitumor MAb in several in vivo syngeneic and xenogeneic tumor models. Here, we evaluated and characterized the antitumor activity and functional synergy of Imprime PGG in combination with an anti-EGFR MAb, cetuximab, in two different cancers, using an EGFR+ human pancreatic tumor line, MiaPaCa-2, and an EGFR+ human ovarian tumor line, SKOV-3. The objectives of this study were: (1) Evaluate the ability of cetuximab to activate complement as measured by iC3b opsonization on tumor cells (measured by flow cytometry using a FITC anti-human iC3b MAb); (2) Characterize tumor cells for expression of the complement regulatory proteins (CRP) CD46, CD55 and CD59 (measured by flow cytometry using fluorescently-labeled CRP-specific antibodies); (3) Evaluate tumor cytolysis following treatment with cetuximab +/- Imprime PGG in the absence and presence of human peripheral blood mononuclear cells (PBMC) (using the MTT colorimetric assay); and (4) Assess the ability of cetuximab +/- Imprime PGG to induce cytokines when tumor cells were cultured with human PBMC. Key findings include: (1) in vitro cetuximab does not induce iC3b deposition on SKOV-3 and MiaPaCa-2 tumor cells over that seen with fresh serum alone. (2) Both tumor cell lines expressed all CRPs but each to a variable extent. (3) Cetuximab did not induce CDC but did induce ADCC in the presence of PBMC, and Imprime PGG further enhanced cetuximab-induced cytolysis under these conditions. (4) The combination of cetuximab and Imprime PGG inhibited the secretion of VEGF in tumor cell/PBMC co-cultures compared to levels observed with either agent alone. These studies demonstrate antitumor synergy of Imprime PGG when combined with cetuximab that may be mediated by modulation of the tumor microenvironment. Overall, the data indicate synergy between Imprime PGG and cetuximab in vitro in inducing antitumor activity in both pancreatic and ovarian tumor cells and provide rationale for such combination strategy for therapy in cancer.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A35.
Abstract A37: Efficacy of combining a novel Aurora kinase inhibitor with radiotherapy in orthotopic liver tumor model of hepatocellular carcinoma
Authors: Cheng Jason Chia-Hsien , Lin Zhong-Zhe , Chia-Hung Chou , Cheng Ann-Lii
Published: June 2012

Radiotherapy is one of the treatment options for hepatocellular carcinoma (HCC), but the unsatisfactory results are mainly due to insufficient dose to the tumor from hepatic intolerance. As compared to advanced technology of radiotherapy, the experience in the combined use of the effective molecular target and radiotherapy remains limited. This study was aimed to assess the effect of combining a novel Aurora kinase inhibitor, VE465, with radiotherapy, by testing the in vitro and in vivo models of human HCC. Human HCC cell line Huh-7, was treated with combined irradiation and the low- toxic dose of VE465. A significant effect on enhancing the radiation induced cell death was found in clonogenic assay. VE-465 induced proliferation blockade, histone H3 (Ser10) dephosphorylation, mitotic disturbance, and apoptosis in Huh-7 cells. The in vivo efficacy of combining VE465 with radiotherapy was investigated in the ectopic xenograft subcutaneous tumor and orthotopic liver tumor models of severe combined immunodeficient mice. Pretreatment with daily i.p. administration of VE465 (20 mg/kg) significantly enhanced the therapeutic effect of radiotherapy (5 Gy per day for 5 days) on Huh-7 derived ectopic xenograft tumor model by 54%. This differential tumor suppression correlated with the modulation of intratumoral biomarker stainings associated with histone 3 phosphorylation inhibition and apoptosis regulation. Furthermore, VE465 improved intrahepatic tumor control and survival of radiotherapy (4 Gy per day for 5 days) treated Huh-7 derived orthotopic liver tumor model by 17.2%, reduction in tumor size. The synergistic effect of combining VE465 with radiotherapy in HCC was associated with histone3 phosphorylation inhibition and cell cycle regulation. We conclude that VE465 is a potent inhibitor of Aurora kinase with clinical value in therapeutic strategies for radiotherapy to HCC.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A37.
Abstract A38: Therapeutic effect of epigallocatechin-3-gallate and silibinin on ATM dynamics in prostate cancer cell line LNCaP
Authors: Farooqi A. A. , Rashid Sadia , Mashadi Taskeen M. , Bhatti Shahzad , Saleem M.
Published: June 2012

Epigallocatechin-3-gallate (EGCG) is a major ingredient of green tea (GT) and silibinin, the active component of silymarin presumably hold a potential to prevent pathogenomics. Prostate cancer exacerbation is triggered by fusion transcripts formed because of genomic instability induced by juxtapositioning of two genes. An abolished ATM dynamics challenges integrity of DNA. In agreement with this assumption ATM and ATR were impaired in LNCaP cell line to confirm a tight interaction of ATM and ATR with the expression profile of TMPRSS2-ERG. Abolished ATM enhanced the expression of the fusion transcript. Similarly blunting of ATR downregulated the expression the fusion transcript giving a notion that ATR is involved in the chromosomal translocation. LNCaP cell lines were analyzed for the effect of EGCG and silibinin on the expression profile of TMPRSS2- ERG. In this particular study treatment of the LNCaP cell line with EGCG and Silibilin recapitulated ATM expression and activity and downregulated the fusion transcript expression. These results underscore the therapeutic effect of EGCG and SB in attenuating the exacerbation of the disease.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A38.
Abstract A4: Withaferin A induces apoptosis in imatinib-resistant CML cells by repressing the Akt signaling pathway
Authors: Chen Ching-Yi , Hung Wen-Chun
Published: June 2012

Although imatinib therapy of chronic myelogenous leukemia is effective, the resistance to imatinib challenges the treatment of this disease. Therefore, search of novel drugs to overcome imatinib resistance is a critical issue in clinic. Withaferin A (WA), an extract of Withania somniferia, exhibits anticancer activity on a number of solid tumors. In this study, we investigate the effect of WA on imatinib-sensitive and -resistant CML cells.

WA at low concentrations induces autophagy in imatinib- sensitive K562 cells. Co-treatment of chloroquine suppresses autophagy and switches WA-treated K562 cells to apoptosis. This data indicates that autophagy protects K562 cells from apoptosis induced by WA. However, we find that WA triggers caspase activation and apoptosis in imatinib-resistant T315I-positive cells and this effect is associated with downregulation of Akt activity. Treatment of the AKT inhibitor LY294002 also causes apoptosis in imatinib-resistant T315I- positive cells. Ectopic expression of constitutively active Akt reverses WA-induced apoptosis and caspase activation in imatinib-resistant T315I-positive cells. Molecular study demonstrates that WA represses the Akt signaling pathway by decreasing Akt expression. We find that WA causes Akt degradation through the ubiquitin- and proteasome-dependent pathway. More importantly, WA also induces AKT downregulation and apoptosis in primary CML cells.

Taken together, our results suggest that imatinib-resistant T315I-positive cells are more addicted to Akt-dependent survival pathway and are more sensitive to WA. Therefore, WA can be useful for the treatment of imatinib-resistant CML.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A4.