bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1679: Genome-wide methylation analysis in Barrett's esophagus and esophageal adenocarcinoma
Authors: xu Enping , Gu Jian , Hawk Ernest T. , Huang Maosheng , Wang Kenneth K. , Ajani Jaffer A. , Wu Xifeng
Published: August 2012

Esophageal carcinogenesis (EAC) is a multiple-step process involving the accumulation of multiple genetic and epigenetic changes. Barrett's esophagus (BE) is a precursor of EAC. In this study, we performed genome-wide methylation profiling of BE and EAC tissues to identify novel tumor suppressors involved in esophageal carcinogenesis and potential biomarkers for the malignant progression of EAC. Using Illumina's HumanMethylation27 BeadChip microarray, we examined the methylation status of 27,578 CpG sites in 94 normal tissues, 77 BE, and 117 EAC tissues. Hierarchical clustering analysis showed an excellent separation of normal tissues from BE and EAC tissues; however, the clustering of BE and EAC tissues was less clear, suggesting the methylation pattern of BE and EAC was similar and methylation occurs early during the progression of EAC. At the individual gene level, we recapitulated the major tumor suppressors that previously reported to be hypermethylated in BE and EAC, including SFRP1, APC, WIF1, etc. We also identified a large number of novel hypermethylated genes in BE and EAC tissues, in particular, genes of several family proteins, including ADAM peptidase proteins, cadherin and procadherin, and potassium voltage-gated channels. We used pyrosequencing to validate selected genes in these families. For example, in normal, BE and EAC tissue, the methylation frequency of PCDH9 was 0.20%, 24.6% and 29.5% respectively, and the methylation frequency of ADAMTS2 was 4.69%, 37.3% and 32% respectively. In general, there were significant correlations between array and pyrosequencing results. The identified differentially methylated genes may become potential biomarkers for the prediction and early detection EAC among BE patients and molecular targets for prevention and therapy of EAC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1679. doi:1538-7445.AM2012-1679
Abstract 1691: A primer dose followed by low-bolus dose schedule of tumor-targeting of S. typhimurium A1-R is effective for tumor-targeted therapy
Authors: Tome Yasunori , Zhang Yong , Momiyama Masashi , Maehara Hiroki , Kanaya Fuminori , Tsuchiya Hiroyuki , Tomita Katsuro , Bouvet Michael , Hoffman Robert M. , Zhao Ming
Published: August 2012

The S. typhimurium A1-R mutant, which is auxotrophic for leu-arg, and selected for high antitumor virulence, has been shown to be effective as monotherapy against human prostate, breast, pancreatic, and fibrosarcoma tumors. In the present study, we developed a strategy to maximize efficacy and minimize toxicity for bacterial tumor treatment in immunocompetent mice with subcutaneous Lewis lung carcinoma expressing red fluorescent protein (RFP). A small dose of S. typhimurium A1-R bacteria was first administered to tumor-bearing animals (the primer dose, 1×106 cfu i.v.) as pre-treatment followed by a high dose (1×107 cfu i.v.) 4 hours later. Compared with non-primer-dose bacteria treatment, the primer-dose strategy resulted in smaller tumors and no observable side effects. The serum lever of TNF-α was elevated in the primer-dose arms compared to non-primer dose arms. Vessel destruction by S. typhimurium A1-R was observed in immunocompetent mice expressing nestin-driven green fluorescent protein (ND-GFP), which is selectively expressed in nascent blood vessels. Vessel destruction was enhanced in the primer-dose arms compared to non-primer-dose arms. The primer dose may activate the immune system and induce cytokines in the mouse necessary for invasion of the tumor by the bacteria allowing a relatively small subsequent dose to be effective and safe.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1691. doi:1538-7445.AM2012-1691
Abstract 1698: ERG protein expression and genomic rearrangement status in primary and metastatic prostate cancer - A comparative study of two monoclonal antibodies
Authors: Tan Shyh-Han , Braun Martin , Goltz Diane , Shaikibrahim Zaki , Vogel Wenzel , Boehm Diana , Scheble Veit , Dobi Albert , Fend Falko , Wernert Nicolas , Kristiansen Glen , Perner Sven
Published: August 2012

Introduction and Objective: Overexpression of the ERG protein is highly prevalent in prostate cancer (PCa) and most commonly results from gene fusions involving the ERG gene. Recently, an N-terminal epitope targeted mouse and a C-terminal epitope targeted rabbit monoclonal anti-ERG antibody have been introduced for the detection of the ERG protein. Independent studies reported that immunohistochemical (IHC) stains with both monoclonal anti-ERG antibodies (ERG-MAbs) highly correlate with the underlying ERG gene rearrangement status. However, a comparative study of both antibodies has not been provided so far. Here, we are the first to compare the mouse ERG-MAb to the rabbit ERG-MAb for their concordance on the same PCa cohort. Furthermore, we assessed if the ERG protein expression is conserved in lymph node and distant PCa metastases, of which a subset underwent decalcification. Methods: We evaluated tissue microarrays of 278 specimens containing 265 localized PCa, 29 lymph node, 30 distant metastases, and 13 normal prostatic tissues. We correlated the ERG protein expression with the ERG rearrangement status using an ERG break-apart fluorescence in-situ hybridization (FISH) assay and IHC of both ERG antibodies. Results: ERG protein expression and ERG rearrangement status were highly concordant regardless of whether the mouse or rabbit ERG-MAb was used (97.8% versus 98.6%, respectively). Of interest, both ERG antibodies reliably detected the ERG expression in lymph node and distant PCa metastases, of which a subset underwent decalcification. If an ERG protein expression was present in localized PCa, we observed the same pattern in the corresponding lymph node metastases. Conclusions: This is the first study to comprehensively compare the two available ERG-MAbs. By demonstrating a broad applicability of IHC to study ERG protein expression using either antibody, this study adds an important step towards a facilitated routine clinical application. Further, we demonstrate that the clonal nature of the ERG rearrangement is not restricted to the genomic level, but proceeds in the proteome. Together, our results simplify future efforts to further elucidate the biological role of ERG in PCa.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1698. doi:1538-7445.AM2012-1698
Abstract 1700: The prognostic marker SHP-1 promoter 2 hypermethylation can detect lymph node micrometastases in early stage NSCLC
Authors: Vinayanuwattikun Chanida , Chantranuwatana Poonchavist , Mutirangura Apiwat , Sriuranpong Virote
Published: August 2012

Introduction; Despite adequate surgical management of stage I non-small cell lung cancer, many patients still relapse and die of their disease. Micrometastases within lymph nodes might not be detected by standard H&E method. The authors conducted the study of epithelial methylation marker, SHP-1 Promoter 2 (SHP1P2) methylation as a potential molecular marker to determine its association with a high risk of disease relapse in stage I resectable non-small cell lung cancer (NSCLC). Method; Lymph nodes from resectable NSCLC stage II-IIIA were examined as a test set. All study participants had stage I NSCLC and underwent curative resection during 2001-2007 and had follow-up at The King Chulalongkorn Memorial Hospital, Bangkok, Thailand. No adjuvant treatment was given. Recurrence of disease within 40 months after resection was defined as high risk. Results: In stage II-IIIA, 75 lymph nodes were analyzed. SHP1P2 methylation was significantly higher in lymph nodes containing H&E detectable metastasis compared with histologically negative nodes (p=0.008). One hundred and ninety-eight lymph nodes from 23 patients with stage I non-small cell lung cancer were analyzed in this study. Seven patients (30%) relapsed within 40 months. Median follow-up period from surgery to relapsed of disease or censored if no relapse of disease was 56 months (range 6-109 months). All clinical/pathologic features were similar except for a larger primary tumor size in the early relapse group. Median tumor size is 5 cms (range 2-11.5) vs 2.5 cms (range 1.5-5.7) in high and low risk group respectively. Multivariate logistic-regression analyses included demographic data (sex, age, staging, tumor grading, size, type of surgery and number of resected lymph node) and SHP1P2 methylation status were done. A high absolute level of SHP1P2 methylation (>58%) in analyzed resected lymph node was associated with early relapse, sensitivity 71% and specificity 99% (HR 0.43-1.04; p=0.07 (univariate); p=.002 (multivariate) with an odds ratio of recurrent cancer of 32). Conclusion: SHP1P2 methylation of resected lymph node of stage I NSCLC treated with curative intent of surgery is associated with early relapsed of disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1700. doi:1538-7445.AM2012-1700
Abstract 1705: Diagnosis of chronic lymphoid leukemia on unstained blood smears using Raman microspectroscopy and supervised classification
Authors: Happillon Teddy
Published: August 2012

Chronic Lymphoid Leukemia (CLL) is a blood cancerous disease characterized by the proliferation of lymphocytes (lymphocytosis). This is the most common leukemia, preferentially affecting people aged over 50 years old. It is incurable and in most cases shows no clinical signs. Thus, it is often discovered by chance during a blood test. If necessary, morphological and immunological studies are led by analyzing blood smears colored with May-Grünwald Giemsa, by making a complete blood count and by computing a Matutes scores. These studies are necessary because it is impossible to distinguish a healthy cell from a cancerous one only using a conventional microscope even if the cells are stained. In order to establish the diagnosis of the disease reliably and automatically, a new approach based on the Raman microspectroscopy is introduced in this study. Based on the interaction between matter and light, this technology acquires a real molecular and biochemical signature of the analyzed sample in a label-free manner (no staining or marking of the sample). Recently, Raman microspectroscopy has shown a promising potential to highlight the pathophysiological states in both human and animal since differences between different pathophysiological states occur at the molecular level. From unstained blood smears, obtained by spreading a drop of blood of CLL patients and healthy persons on standard glass slides, Raman spectra are acquired on the nucleus of white blood cells. Using a supervised classification algorithm such as the “Support Vector Machine” (SVM), a spectral model is established to distinguish spectra obtained from healthy or pathological cells. This model is computed using a population of spectra called “training set” and consists of 513 spectra from 6 CLL patients and 347 spectra from 4 healthy persons. Then, a classification of the “validation set” composed of 2820 spectra from 33 CLL patients and 1074 spectra from13 healthy individuals was achieved. A sensitivity of 84.84% and a specificity of 84.61% were obtained on the patient. Finally, a “blind set” composed of 721 spectra from 8 cases is classified. The sensitivity and specificity are respectively equal to 80% and 100%. The results obtained on the differentiation between healthy and pathological cells show the real potential of Raman spectroscopy coupled with supervised classification for the diagnosis of CLL. Future studies will concentrate on the prognosis, and if possible the prediction of CLL.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1705. doi:1538-7445.AM2012-1705
Abstract 1710: Quantitative 3D mapping of total choline in human breast cancer using high-speed MR spectroscopic imaging at 3T
Authors: Posse Stefan , Zhang Tongsheng , Royce Melanie , Dayao Zoneddy , Lopez Susan , Sillerud Laurel , Eberhardt Steven , Lomo Lesley , Lee Sang-Joon , Rajput Ashwani , Russell John , Casey Linda , Bolan Patrick
Published: August 2012

INTRODUCTION We report initial experience with 3D mapping of tCho to monitor treatment response in patients undergoing neoadjuvant chemotherapy to predict outcome [1]. METHOD Eleven patients aged: 28 - 77 years with biopsy-confirmed, infiltrating ductal carcinoma (IDC) were studied before and during neoadjuvant therapy. Data were collected on a 3T MR scanner equipped with 8- and 16-channel breast array using PRESS volume prelocalized 3D high-speed Proton-Echo-Planar-Spectroscopic-Imaging (PEPSI) [2] with MEGA lipid suppression (TR/TE=2000ms/135ms, matrix size up to 32×16×8, voxel size=1cc, scan time: 10 minutes including a water reference scan). Spectral quantification was performed using LCModel-based spectral fitting in reference to tissue water [3] using a customized basis set with empirically modeled Lorentzian singlet peaks to account for broad and irregular line shapes of residual lipid signals, relaxation correction and automated spectral quality thresholds. RESULTS Strongly elevated tCho with mean concentrations up to 5.1 mmol/kg was measured in 7 patients with single and multi-centric enhancing lesions (Table 1). Decreases in tCho concentration and number of voxels with detectable tCho were measured in 4 patients who underwent neoadjuvant therapy. DISCUSSION AND CONCLUSION This study demonstrates feasibility of quantitative 3D mapping of tCho in invasive breast carcinoma and assessment of regional tCho changes during neoadjuvent therapy. The long-term goal is to utilize 3D high-speed MRSI as an early predictor of treatment failure in women undergoing neoadjuvant therapy. REFERENCES [1] Danishad, A., et al. NMR Biomed. 2010; 23: 233-241. [2] Posse, S., et al., Magn Reson Med, 1997. 37(6): p. 858-65. [3] Provencher et al MRM 1993. GRANT SUPPORT: NIH, DoD, UNM Cancer Center.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1710. doi:1538-7445.AM2012-1710
Abstract 1712: Ki67 index is an independent prognostic factor in pleural mesothelioma
Authors: Ghanim Bahil , Torok Szilvia , Hoda Mir , Filipits Martin , Ostoros Gyula , Setinek Ulrike , Fillinger Janos , Dome Balazs , Klepetko Walter , Berger Walter , Hegedus Balazs
Published: August 2012

Aims: Malignant pleural mesothelioma is a devastating cancer with increasing incidence. Despite the fact that for establishing the diagnosis a number of immunohistochemical markers are needed, there is only one pathological prognosticator accepted and utilized, namely the histological subtype. Accordingly, we have investigated the prognostic significance of the Ki76 labeling index in malignant pleural mesothelioma. Patients and Methods: Ki67 index has been determined by assessing the labeled nuclei per high power field and correlated in a retrospective manner with patients’ clinical history and outcome. The prognostic power of the routine proliferation marker Ki67 was first determined in a test cohort (TC) of paraffin-embedded tissue samples from 55 patients. The independence of prognostic power against basic clinicopathological characteristics has been determined by multivariate analysis. Next, we measured the prognostic power in an independent validation cohort (VC) of 42 patients from another center. Results: Median Ki67 index was found to be 10 for both the test and validation. Patients whose tumor sample was categorized by low Ki67 index (≥ 10) had significantly longer survival times than those with higher Ki76 indices (in the test cohort: hazard ratio [HR] 2.49, 95% confidence interval [CI] 1.30-4.79, p<0.01; in the validation cohort: HR 2.54, CI 1.26-5.13, p<0.01). In the multivariate analyses of the test cohort (n=54), we could prove the prognostic power of Ki67, independent from gender, age, treatment, stage and histology. Treatment (HR 4.02, CI 1.82-8.90, p<0.01) and Ki67 index (HR 2.19, CI 1.07-4.47, p=0.03) were the remaining independent significant prognosticators in the multivariate Cox regression model, whereas biphasic histology showed a clear tendency to worse prognosis when compared to the epitheloid subtype (HR 1.96, CI 0.94-4.07, p=0.07). Conclusion: Our study identifies Ki67 index as an independent, reproducible prognostic factor in malignant pleural mesothelioma and warrants its further investigation and clinical application in patients with this malignancy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1712. doi:1538-7445.AM2012-1712
Abstract 175: Atypical PKC knockdown alters TGFβ signaling potential
Authors: Gunaratne Adrian , Guglielmo Gianni Di
Published: August 2012

Transforming growth factor beta (TGFβ) regulates many important cellular processes including growth control and cell migration. In epithelial tumor cells, TGFβ signaling is fundamentally altered and induces epithelial to mesenchymal transition (EMT) leading to increased migratory potential. We have recently reported that the inhibition of atypical PKC isoforms (aPKC) extends TGFβ induced Smad signaling by altering receptor trafficking. Here we report that siRNA targeting of aPKC in A549 lung adenocarcinoma cells extends temporal P-Smad2 signaling but does not alter the transcript levels of Snail and Slug, as assessed by qPCR analysis. Interestingly, TGFβ was not observed to have as robust an increase in MMP9 and PAI-1 levels in aPKC-silenced cells compared to cells transfected with control siRNA. To further analyze the functional consequences, we assessed EMT by immunofluorescence microscopy analysis as well as immunoblotting for epithelial and mesenchymal cell markers. We found that TGFβ-dependent EMT was significantly reduced in aPKC silenced cells. We also uncovered that in addition to increased P-Smad2 signaling, P38 MAPK phosphorylation was increased and extended in aPKC silenced cells. Finally, we discovered that aPKC phosphorylates Par6 on a conserved serine residue that has been shown to regulate EMT and metastasis in breast cancer. Taken together, we believe that aPKC regulates several aspects of TGFβ signaling, including P-Smad2, P-P38 and P-Par6 levels. Consequently, convergence of these signals in aPKC silenced cells leads to reduced EMT and migratory potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 175. doi:1538-7445.AM2012-175
Abstract 1751: A phase I study of lenalidomide in combination with bevacizumab, sorafenib, temsirolimus, or 5-fluorouracil, leucovorin, oxaliplatin (FOLFOX) in patients with advanced cancers
Authors: Subbiah Ishwaria M. , Kurzrock Razelle , Naing Aung , Piha-Paul Sarina , Falchook Gerald S. , Hong David S. , Wheler Jennifer J. , Fu Siqing , Laday Shell , Tsimberidou Apostolia M.
Published: August 2012

Introduction: Patients with advanced cancer have limited therapeutic options. We conducted an investigator-initiated 4-arm phase I study of lenalidomide (len) combined with bevacizumab (bev), sorafenib (soraf), temsirolimus (tem), or 5-fluorouracil, leucovorin, oxaliplatin (FOLFOX) in patients with advanced cancers. Patients and Methods: Patients were treated with escalating doses of len (5, 10, 20 mg daily) combined with bev (5, 7.5, 10 mg/m2 on days 1, 15); soraf (200, 400mg daily); tem (15, 20, 25mg on days 1, 8, 15, 22); or FOLFOX (oxaliplatin 65, 85 mg/m2 on day 1). The escalation phase (“3+3”) was followed by an expansion phase per arm. Response was assessed by RECIST v.1. Patients were to receive enoxaparin prophylaxis. Results: Overall, 101 patients were enrolled; and 59 were treated in the dose escalation phase (4 arms) and are included in this analysis (169 cycles to date). Diagnoses were colorectal cancer (n=19), sarcoma (n=5), ovarian (n=4), lung (n=3), hepatocellular carcinoma (n=3), melanoma (n=4), neuroendocrine (n=2), prostate (n=2) and other (n=17). The median number of cycles/patient was 2 (range, 1-10). Pretreatment characteristics and outcomes are shown in Table. Two DLTs were noted: Grade 3 transient ischemic attack (TIA) while on enoxaparin in the len + FOLFOX arm and Grade 3 acute renal failure (ARF) in the len + tem arm. Three pts (two on enoxaparin prophylaxis and one not) had venous thrombosis while on study. Fifteen of 59 (25%) pts had prolonged stable disease (SD) for > 4 months, including 7 (12%) for > 6 mos. The len+soraf and len+tem arms have reached the maximum dose level tested; currently, expansion phase). The lev+bev and len+FOLFOX arms are in the dose escalation phase. Conclusion: Lenalidomide combined with bev, soraf, tem or FOLFOX is well tolerated and it is associatd with antitumor activity in patients with advanced solid tumors. Anticoagulation phophylaxis is needed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1751. doi:1538-7445.AM2012-1751
Abstract 1754: A pharmacokinetics/pharmacodynamics study of sequence specificity of the PARP inhibitor, olaparib (O) with carboplatin (C) in recurrent women's cancers NCT01237067
Authors: Lee Jung-min , Hays John , Annunziata Christina M. , Minasian Lori , Zujewski JoAnne , Squires Jennifer , Nielsen Deborah , Houston Nicole , Moorshead David , Cedillo Mario , Kohn Elise C.
Published: August 2012

Background: C, O, and C+O have documented activity in BRCA1/2mut+ or BRCA-like breast and ovarian cancers (Br/OvCa). The effect of drug sequence on DNA damage and cell death is unknown. Our in vitro modeling suggests that exposure to O prior to C (O>C) reduced double stranded DNA damage and cell loss. Our trial tests the hypothesis that C>O causes more cellular injury. Methods: Eligible pts have recurrent women's cancers, good end organ function, and evaluable disease. A 3+3 dose escalation run-in defined the O tablet dose in combination with C (AUC 4). The study will now randomize pts to schedule A or B (below), allowing intra-pt and inter-cohort analysis of PK/PD endpoints. PBMCs will be collected C1/2 for measurement of platination and DNA damage/repair; DNA repair protein integrity will be assessed on archival tissues. Clinical benefit, response frequency and duration, and toxicity will be ascertained. Mutation carriers will undergo progression biopsy for analysis of BRCA1/2 re-expression mutations. Results: The phase I portion is complete. 12 women (8Ov/3Br/1Endometrial[En]) have been accrued on 3 dose levels (DLs). Median prior number of regimens was 5. No DLT have been seen in 5 evaluable pts on DL3 (O tablet 200mg q12h/C AUC4 [1 pt pending]). Gr3 AEs included neutropenia (44%), and gr2 were anemia (56%), fatigue (22 %), C hypersensitivity (22%), thrombocytopenia (11%), and nausea (11%). 1 pt (DL2; stable disease) died of unrelated bleeding during C4. Clinical benefit has been observed in 8 evaluable pts with PR in 2 BRCA1mut+ OvCa (9+, 11+ mo), stabilization > 4mo in 6 pts (4OvCa,1BrCa, 1EnCa), and 3 are TETE. Conclusions: O in tablet formulation at 200mg q12h x 7d with C AUC4 q21d is active and tolerable, with observed interactive marrow suppression. Subsequent women will be randomized to examine schedule dependence of activity and toxicity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1754. doi:1538-7445.AM2012-1754
Abstract 176: Kinase activity of tumor-derived exosomes as a potential biomarker for response to treatment
Authors: van der Mijn Johannes C. , Jimenez Connie R. , Piersma Sander R. , Dekker Henk , Labots Mariette , Schutte Lisette M. , Smit Egbert F. , Broxterman Henk J. , Tannous Bakhous , Wurdinger Tom , Verheul Henk M.W.
Published: August 2012

Introduction Exosomes are nanometer-sized vesicles secreted by tumor cells after fusion of multivesicular bodies with the plasma membrane that reflect tumor cell biology. Exosomes contain distinct classes of molecules including mRNA, miRNA and proteins. Because they can be isolated from blood they are promising candidate biomarkers. Diagnostic biomarkers for selection of patients for treatment with tyrosine kinase inhibitors are urgently warranted. We hypothesize that exosomes contain tumor derived kinases that may serve as biomarkers for treatment with kinase inhibitors. Methods The U87 glioma cell line with or without EGFRvIII (U87ΔEGFR) was used as an in vitro model in this study. Cell viability was studied with the MTT-assay. Exosomes were isolated from 32h conditioned media either serum-free or containing 10% exosome-deprived serum and from 5-9mL of fresh human serum from 3 cancer patients. The isolation procedure consisted of subsequent centrifugation at 500G, 2000G, 10.000G and 100.000G. The exosome pellet was treated with proteases for 1h at 37°C to digest soluble proteins, washed in PBS and lysed. Western blots for phospho-EGFR (y1068), EGFR, phospho-AKT, AKT, phospho-ERK, ERK, Alpha-Tubulin and exosome markers CD63, ALIX and CD81 were performed. Results The sensitivity of U87ΔEGFR to erlotinib (ERL) was significantly higher compared to parental U87 cells with IC50s of 1µM and 5.4µM (p<0.001), respectively. Treatment with 1 μM ERL resulted in reduced phosphorylation of EGFR, AKT and ERK in U87ΔEGFR, but not in U87 cells. The overexpression of constitutively active EGFRvIII mutant increased the phosphorylation of EGFR and downstream AKT and ERK in cells as well as in exosomes. Trypsin treatment only affected membrane proteins, where AKT and ERK levels were not altered. Exosomes from U87ΔEGFR cells treated with 0, 1 and 10µM ERL reflected the phosphorylation patterns of the cells with dose-dependent effects on AKT and ERK. Phosphorylated AKT and ERK could also be detected in exosomes from the cancer patients. Conclusion We here show that kinase activity and treatment response are reflected by exosomes derived from tumor cells in vitro. In addition we found that phosphorylated proteins can be detected in exosomes derived from blood of patients with cancer. Our aim is to further develop exosomal kinase activity analysis as an early biomarker for treatment response.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 176. doi:1538-7445.AM2012-176
Abstract 1760: Development of bisphosphonate prodrugs of doxorubicin for targeting bone metastases that are cleaved pH dependently or by cathepsin B
Authors: Hochdörffer Katrin , Schäfer-Obodozie Cynthia , Schächtele Christoph , Kratz Felix
Published: August 2012

Bone metastases occur in up to 80 % of patients with advanced breast or prostate cancer and significantly affect the quality of life. Most frequently, bone metastases cause bone fractures, severe pain and hypercalcaemia. Current options for treating of bone metastases are surgery, radiotherapy, chemotherapy, hormone therapy, and the administration of bisphosphonates. These treatments are primarily palliative and can improve the quality of life in some cases, however without increasing the survival time of the patients. Bisphosphonates are presently the most active inhibitors of bone degradation, and they can reduce bone pain, bone destruction and tumor growth. Their therapeutic effects are due to the high affinity between bisphosphonates and the apatite structure of bone and bone metastases. In order to improve the outcome of treating bone metastases, new therapeutic approaches are urgently required. As bone-seeking agents, bisphosphonates show an uptake in the bone of 20-60% of the applied dose, and in general the uptake in bone metastases is 10-20-fold higher than in healthy bone tissue. In addition, bone metastases are characterized by an acidic pH and the over-expression of several proteases including cathepsins, matrix metalloproteases, plasmin and uPA. Surprisingly, no prodrug approaches have been reported to date that exploit the bone targeting effect of low-molecular weight bisphosphonates with a concomitant release of a bound drug in bone metastases. To this purpose, we developed water-soluble bisphosphonate prodrugs of doxorubicin for targeting bone metastases by conjugating a thiol-bearing aliphatic bisphosphonate with the 6-maleimidocaproyl hydrazone derivative of doxorubicin (1) that is cleaved pH dependently, or by attaching a maleimide derivative of the dipeptide Val-Ala-PABC (PABC = p-benzyloxycarbonyl) to the aliphatic bisphosphonate (2). Incubation studies with cathepsin B and 2 at 37 °C showed a cleavage of doxorubicin after two hours with a half time of ∼ 45 min and 1 released doxorubicin quantitatively at pH 5.0 after 2 h. The plasma stabilities (human and mouse) of both prodrugs were in the range of 3-6 h at 37 °C. Binding studies of 1 and 2 with a major component of bone, i.e. hydroxyapatite, demonstrated binding of 80-90 % of both prodrugs after 4 h at 37 °C. Finally, in an orientating toxicity study in nude mice the maximum tolerated dose (MTD) of 1 was three-fold higher compared to conventional doxorubicin whereas 2 showed essentially the same MTD as doxorubicin.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1760. doi:1538-7445.AM2012-1760
Abstract 1762: Highly potent anti-cancer ‘salan’ Titanium(IV) complexes: Structure - function relationship
Authors: Meker Sigalit , Braitbard Ori , Manna Cesar , Weiss Esther , Tshuva Edit , Hochman Jacob
Published: August 2012

Cisplatin is a widely used platinum-based metallo-chemotherapeutic drug that is considered an efficient treatment mainly for testicular and ovarian cancers. However, its narrow activity range and severe side effects trigger studies of other potent transition metal complexes. Two titanium(IV) anti-cancer agents that have been previously studied extensively as cisplatin alternatives are titanocene dichloride and budotitane. Despite their high activity, these complexes failed clinical trials mainly due to their poor water stability and formation of unidentified aggregates in water solutions. Our research group focuses on development of new, better-suited families of anti-tumor Ti(IV) complexes lacking Cp or diketonato ligands, and their investigation as anti-tumor agents. We recently reported the synthesis, characterization, and cytotoxicity of “salan” type Ti(IV) complexes [1]. The well defined hydrolytic behaviour, high stability, and high cytotoxicity of these compounds are strongly correlated to their particular structure. This enables fine tuning of complex properties by structural modifications. Herein we elaborate on the investigation of these salan titanium(IV) compounds. The correlation between the complex properties: structure, hydrolytic stability and cytotoxicity will be discussed, revealing insights on the biological mechanism of their cytotoxicity, suggesting an apoptotic cell death pathway.Additionally, further biological evaluation is presented, evincing that these compounds are markedly more active than cisplatin towards human and murine cancer-derived cell lines, including colon, ovarian, lung, cervix, pancreas, leukaemia, skin and breast, with activity also against cisplatin-resistant and multi-drug-resistant cells, and with minor effect on primary murine cells. In vivo experiments are now under way to determine both safety, as well as the anti-cancer activity of these complexes. [1] D. Peri, S. Meker, C. M. Manna, E. Y. Tshuva, Inorg. Chem., 2011, 50, 1030.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1762. doi:1538-7445.AM2012-1762
Abstract 177: MiR-10b promotes migration and invasion in pancreatic cancer cells
Authors: Ouyang Haoxu , Korc Murray
Published: August 2012

Pancreatic ductal carcinoma (PDAC) is the fourth leading cause of cancer-related death in Unites States. Due to its aggressive local invasion and metastatic nature, the median survival for patients with PDAC is only 6-month. There is therefore an urgent need to improve our understanding of the pathobiology of this malignancy. In this regard, microRNAs (miRs) have emerged as potentially important regulators in PDAC progression by acting on multiple signaling pathways involved in proliferation, apoptosis, invasiveness and metastasis. MiR-10b is an important RNA implicated in the facilitation of cancer cell invasion and metastasis in several cancers. We have previously shown that miR-10b is upregulated in pancreatic cancer cells, and that high levels of miR-10b are associated with decreased time to metastasis following therapy, enhanced resistance to gemcitabine, and decreased overall survival. In the present study, we have sought to characterize the mechanisms of action of miR-10b in PDAC. We found that overexpression of miR-10b promoted migration and invasion of PANC-1 cells in vitro, without enhancing their proliferation. Microarray analysis revealed that TIP30, a tumor and metastasis suppressor, was downregulated by miR-10b overexpression. Validation experiments using luciferase readout assays showed that TIP30 is a direct target of miR-10b. Taken together, these observations suggest that miR-10b plays an important role in PDAC and may be a novel therapeutic target in this deadly malignancy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 177. doi:1538-7445.AM2012-177
Abstract 1780: Synthesis and antitumor activity of proximicins A, B and C
Authors: Brucoli Federico , Natoli Antonino , Marimuthu Preethi , Schätzlein Andreas
Published: August 2012

Proximicins A, B and C are a family of three novel aminofuran antibiotics isolated from marine actinomycetes of the genus Verrucosispora. The distinctive features of these compounds are (a) the structural similarity to the DNA-binding agents distamycin (4) and netropsin (5), and (b) the presence in their molecular framework of an unusual α-aminoacid, 4-aminofuran-2-carboxylic acid. Preliminary in vitro biological evaluation of proximicins A - C against selected carcinoma cell lines has shown that all compounds have a significantly higher cytotoxicity than netropsin and distamycin. In addition, proximicins can activate cell-cycle regulatory proteins involved in the transition of cells from G1 to S phase, and proximicin C, in contrast to distamycin, can induce up-regulation of p53 and p21 in gastric adenocarcinoma cells. The total synthesis of the proximicins A, B and C was successfully carried out starting from methyl 4-Boc-aminofuran-2-carboxylate and sufficient quantities of each compound were prepared for extensive in vitro evaluation. The anticancer activity of proximicins was investigated using cellular methods. The compounds induced apoptotic cell death in L1236 (Hodgkin's Lymphoma) and Jurkat 16 (T-cell leukemia) cells as confirmed by flow cytometric DNA fragmentation analysis. Proximicin B exhibited a strong lethal effect in both L1236 and Jurkat 16 cell lines and apoptosis induction was shown to be dose-dependent after 48 h treatment. Proximicin C induced apoptosis only in Jurkat 16 cells, with L1236 showing higher resistance to the drug. In addition, the proximicins cytotoxicity was determined against selected cancer cell lines different from those previously reported. Proximicin C exhibited significantly higher cytotoxicity against U-87 MG (glioblastoma) and MDA-MD-231 (breast carcinoma) cells compared to proximicin B. Further biological experiments are currently underway to elucidate the mechanism of action of these furan-based oligopeptides. In conclusion, given their interesting molecular framework and their significant anticancer activity, proximicins represent an attractive synthetic target and could be potentially developed into anticancer drugs of broad utility.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1780. doi:1538-7445.AM2012-1780
Abstract 1783: Trifluorothymidine incorporation into DNA strongly enforces the potential of TAS-102 and leads to remarkably prolonged survival of cancer patients
Authors: Tanaka Nozomu , Oguchi Kei , Fujita Ryoto , Suzuki Takamasa , Yamamura Keisuke , Fujioka Akio , Okabe Hiroyuki , Noguchi Kazuharu
Published: August 2012

TAS-102 is a novel oral antitumor agent, consisting of trifluorothymidine (FTD) as the active component and thymidine phosphorylase inhibitor (TPI) which prevents degradation of FTD. In a double-blind randomized phase II study conducted in Japan, TAS-102 significantly improved overall survival of metastatic colorectal cancer patients, compared with placebo (TAS-102, n = 112; Placebo, n = 57; median OS, 9.0 vs. 6.6 months; HR, 0.56; p = 0.001). We investigated mechanisms of action of TAS-102 to understand how prognosis of cancer patients was improved. In vitro study, FTD was previously reported to have two mechanisms, FTD incorporation into DNA and thymidylate synthase (TS) inhibition. In this study, we show that FTD incorporation into DNA is the main mechanism of TAS-102 and the key to potentiate anti-tumor efficacy of FTD. We found that although TS inhibition of FTD reduced rapidly after drug removal, the cytotoxicity of FTD did not diminish. On the other hand, the cytotoxicity of FdUrd diminished after drug removal. As compared to FdUrd, FTD was incorporated into DNA to a greater extent by the time drug was removed. It is thought that the FTD retained in DNA contributes to sustained growth inhibition. Therefore, FTD incorporation into DNA is considered to be essential for enforcing the potential of FTD. Additionally, we investigated the in vivo potency of TAS-102 by using daily divided oral administration since this regimen was the most optimal to maximize FTD incorporation into DNA. Interestingly, TAS-102 showed marked tumor growth delay even after end of the administration and remarkable prolonged survival of mice implanted with KM20C derived from human colon cancer. Collectively, these data suggest that TAS-102 improved survival of cancer patients primarily due to FTD incorporation into DNA. FTD is previously reported to affect DNA structure, and we speculate that TAS-102 exerts its anti-tumor effects through interfering with DNA function once adequate amounts of FTD have been accumulated in the DNA.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1783. doi:1538-7445.AM2012-1783
Abstract 2414: The inhibitory effect of heat treatment against Epithelial-Mesenchymal Transition (EMT) in human colon adenocarcinoma cell line
Authors: Adachi Satoko , Kokura Satoshi , Ishikawa Takeshi , Sakamoto Naoyuki , Okajima Manabu , Matsuyama Tatsuzo , Matsuyama Tatsuzo , Tsuchiya Reiko , Nito Yuji , Yoshikawa Toshikazu
Published: August 2012

Background and Aim: In order to improve the prognosis of patients with advanced cancer, novel interventions for metastatic disease are required. While anti-tumor effects of hyperthermia have been well studied and assessed, its effects on cancer metastasis is unknown. We reported that the production of transforming growth factor-β (TGF-β) by cancer cell lines was reduced by the heat treatment, furthermore the production of the intratumoral TGF-β was also reduced by whole body hyperthermia in mouse subcutaneous tumor model. Recently, the importance of Epithelial-Mesenchymal Transition (EMT) in the progression of many cancers is recognized and many studies have shown that transforming growth factor-β (TGF-β) can induce EMT. Therefore, it is expected that heat treatment could inhibit TGF-β-induced EMT. In this study, we investigated the inhibitory effect of the heat treatment on TGF-β-induced EMT in the human colon adenocarcinoma cells. Methods: To study the effects of heat treatment on EMT, human colon adenocarcinoma cell line, HT29 cell, was used and treated with heat (43 degrees Celsius) for 1 h followed by the stimulation of TGF-β (10ng/ml). HT29 cells were examined for morphological changes using the inverted phases-contrast microscope, and the localization of E-cadherin as the epithelial adhesion factor on the cell was analyzed with fluorescent immunostaining using confocal microscope. The EMT-related factors such as vimentin, fibronectin, ZEB-1, Snail were analyzed by RT-PCR. Furthermore, the activation of nuclear factor kappa B (NF-κB) was analyzed in the nuclear extracts using an ELISA method. Results: After treatment with TGF-β, HT29 cells changed their morphology from a typical epithelium to mesenchymal spindle-shaped, and this morphological change was attenuated by the heat treatment. The expression of E-cadherin, which is the component of epithelial adhesion, was down-regulated by TGF-β, and this effect was attenuated when HT29 cells were treated with heat before the stimulation with TGF-β. The up-regulation of EMT-related factors such as fibronectin and Snail induced by TGF-β was decreased by the heat treatment. Furthermore, TGF-β treatment caused an activation of NF-κB, and heat treatment significantly attenuated TGF-β-indueced NF-κB activation in HT29 cells. Conclusions: Our results suggest that the heat treatment could suppress TGF-β-induced EMT via the inhibition of NF-κB, supporting its potential as a treatment for cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2414. doi:1538-7445.AM2012-2414
Abstract 2415: Phenotypic stabilization of mesenchymal-like cancer cells through mesenchymal-specific DNA hypermethylation
Authors: Kozaki Ken-ichi , Kurasawa Yasuhiro , Muramatsu Tomoki , Ono Hiroaki , Ishihara Takaya , Uzawa Narikazu , Pimkhaokham Atiphan , Imoto Issei , Amagasa Teruo , Inazawa Johji
Published: August 2012

The epithelial-mesenchymal transition (EMT) plays a crucial role in normal and disease processes including tumor progression. In this study, we first classified epithelial-like and mesenchymal-like oral squamous cell carcinoma (OSCC) cell lines based on expression profiles of typical EMT-related genes using a panel of 18 OSCC cell lines. Then, we performed methylation-based and expression-based analyses of components of the Wnt signaling pathway, and identified WNT7A and WNT10A as genes silenced by mesenchymal-specific DNA hypermethylation in OSCCs. A significant association was revealed between some clinicopathologic findings and the DNA methylation status of WNT7A (Normal vs Tumor, P = 0.007; T1-2 vs T3-4, P = 0.040; I-III vs IV, P = 0.016) and WNT10A (N0-N1 vs N2-N3, P = 0.046) in the advanced stages of OSCC. Moreover, we found that E-cadherin expression in cancer cells may be positively regulated by WNT7A, whose expression is negatively regulated by mesenchymal-specific DNA hypermethylation or ZEB1 in mesenchymal-like OSCC cells. Our findings indicate that epithelial-specific gene silencing through mesenchymal-specific DNA hypermethylation may stabilize the phenotypic plasticity of cancer cells during EMT/MET.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2415. doi:1538-7445.AM2012-2415
Abstract 242: Anti-obesity drugorlistat enhances sensitivity toTRAIL by two different pathways inhormone-refractory prostate cancer cells
Authors: Fujiwara Jun , Sowa Yoshihiro , Horinaka Mano , Koyama Makoto , Wakada Miki , Miki Tsuneharu , Sakai Toshiyuki
Published: August 2012

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as one of the expected cancer therapeutics. However, since hormone-refractory prostate cancer (HRPC) cells are sometimes resistant to TRAIL, we need to develop a novel strategy to overcome. We demonstrated that the anti-obesity drug orlistat enhances the sensitivity to TRAIL in HRPC cells. The combination of orlistat and TRAIL remarkably induced apoptosis in DU145 and PC3 cells. Orlistat induced the expression of DR5, which is one of the TRAIL receptors. The suppression of DR5 with siRNA reduced the enhanced apoptosis, suggesting that the enhanced apoptosis was at least partially due to the up-regulation of DR5. Although the up-regulation of DR5 at both mRNA and protein levels by orlistat was observed in both cell lines, the activation of the DR5 promoter in DU145 cells was CHOP-dependent, but that in PC3 cells was CHOP-independent. Moreover, orlistat induced reactive oxygen species (ROS), and a ROS scavenger diminished the sensitivity to TRAIL through the suppression of the CHOP and DR5 expressions in both cell lines. These results suggest that there are two different pathways of up-regulation of DR5 by orlistat, which are ROS-CHOP pathway and ROS-direct pathway. In conclusion, orlistat enhances the sensitivity to TRAIL in the ROS mediated pathways in prostate cancer cells. We believe that the administration of TRAIL and orlistat is considered a promising strategy for cancer therapy in HRPC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 242. doi:1538-7445.AM2012-242