bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5300: Dopamine prevents malignant tumor induced thymic involution in mice
Authors: Sarkar Chandrani , Chakroborty Debanjan , Yu Hongmei , Dasgupta Partha Sarathi , Basu Sujit
Published: February 2011

T cell functions are significantly impaired during tumorigenesis and the thymus being the major site of T cell maturation is severely affected by tumor development. Also, a high incidence of premature thymic involution is seen especially in childhood cancer, which often rebounds after curative treatment. Most importantly, this thymic involution may be one of the important causes for T cell defects in cancer. Recently, it has been reported that high concentration of vascular endothelial growth factor-A (VEGF-A) produced during cancer may induce thymic involution. We here show for the first time that dopamine treatment prevents thymic involution in mice bearing murine T cell lymphoma and dopamine in turn acts by inhibiting the actions of VEGF-A on bone marrow derived T progenitor cells. This finding is important as it suggests a new adjuvant therapy for the treatment of childhood cancer in which high incidence of thymic involution has been reported.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5300.
Abstract 5304: Serum vascular endothelial growth factor in advanced breast cancer patients might be a key role of inhibitory effect on lymphokine activated killer cells
Authors: Suzuki Eiji , Hirose Makiko , Suzuki Noriko , Muta Mariko , Sekine Susumu , Kitagawa Dai , Horiguchi Kazumi , Aruga Tomoyuki , Ymashita Toshinari , Kuroi Katsumasa
Published: February 2011

Previously, we reported that serum from stage IV breast cancer patients diminished the cytotoxic activity of lymphokine activated killer cells (LAKs) (Abstract #4639 AACR in 2008). Thus, we next tested what the critical role for the inhibitory effect on LAKs was. Based on the study of protein detection in the serum using Cytokine Antibody Array (Ray Biotech), serum from stage IV patients seemed to have higher level of vascular endothelial growth factor (VEGF) as compared to early stage patients. To confirm this by more accurate way, we analyzed the serum level of VEGF by using ELISA in 24 breast cancer patients. When the serum level of VEGF was cut off by median (420 pg/ml), 11 patients with higher level of VEGF out of 12 are included in the patients whose serum inhibited the cyototoxic activity of LAKs. Therefore, we hypothesized that VEGF might be one of the key role for inhibitory effect on LAKs and neutralization of serum VEGF from the patients whose serum inhibited the cyototoxic activity of LAKs could have no inhibitory effect on LAKs. Briefly, PBMCs from a healthy volunteer were used as an effector cell for all patients’ serum incubation. After activation of the PBMCs using IL-2, LAKs were prepared and incubated with the individual patient's serum and washed. The LAKs were incubated with human breast cancer cell line MCF-7 as a target cell and LDH assay was performed to detect cytotoxicity of the LAKs. Fourteen out of 24 patients serum (serum 1) showed inhibitory effect on LAKs and we picked up these patients to test blocking study of serum VEGF. Briefly, serum from these 14 patients was incubated with neutralization antibody of VEGF (R16Y, Santa Cruz Biotechnology) (serum 2) before mix with LAKs. After incubation of serum 1 and serum 2 with LAKs, target cell cytotoxicity of LAKs from incubation with serum 1 showed 30-50% reduced level as compared to control LAKs activity. Interestingly, target cell cytotoxicity of all LAKs from incubation with serum 2 did not show such inhibitory effect (mean cell cytotoxicity of LAKs was 33.48% and 57.27%, serum 1 and serum 2, respectively, control; 55.73%). These data suggest that high level of serum VEGF in breast cancer patients might be a key role of inhibitory effect on LAKs and neutralization of the VEGF could help adoptively transferred LAKs as cancer immunotherapy in the patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5304.
Abstract 5309: Programmed Death Ligand-1 expression on the cell surface is regulated by Costimulatory Molecule CD80 expressed by cancer vaccines
Authors: Haile Samuel T. , Bosch Jacobus , Agu Nnenna , Ostrand-Rosenberg Suzanne
Published: February 2011

With the lack of effective treatments for metastatic cancers, the employment of immunotherapies, cell-based vaccines particularly, to activate a patient's own immune system is a promising approach for the treatment of disseminated metastatic cancer. Many tumor cells constitutively express or are induced by Interferonγ (IFNγ) to express the inhibitory molecule Programmed Death Ligand 1 (PDL1). Since many cell-based vaccines are derived from tumor cells, the expression of PDL1 could reduce the therapeutic efficacy of the vaccines. PDL1 binds to its receptor Programmed Death 1 (PD1) on T cells and can signal for either apoptosis of T cells or can prevent their activation. As determined by flow cytometry using three anti-PDL1 monoclonal antibodies (mAb) that detect distinct PDL1 epitopes, and human lung, uveal melanoma, cutaneous melanoma, and breast cancer cells, the over-expression of CD80 blocks constitutive and IFNγ-induced cell surface expression of PDL1. Quantitative analysis of CD80 and PDL1 expression indicates an inverse relationship between PDL1 and CD80. CD80+ tumor cells contain intracellular PDL1, as assessed by flow cytometry, suggesting that CD80 regulates PDL1 at the protein level. Tumor cells transfected with CD80 constructs lacking a cytoplasmic domain or with an irrelevant non-signaling cytoplasmic domain, retain the ability to block PDL1 expression at the cell surface indicating that CD80 does not modulate PDL1 transcription via intracellular signaling. These results suggest that in addition to serving as a costimulatory molecule, CD80 facilitates T cell activation by blocking coinhibitory signals and preventing T cell apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5309.
Abstract 5320: Deletion of TGFß signaling in Gr-1+CD11b+ myeloid cells attenuates breast adenocarcinoma progression
Authors: Pang Yanli , Yan Hannah H. , Li Chaoyang , Achyut B. R. , Yang Li
Published: February 2011

TGFß is a very important mediator in tumor progression. However, the contribution of TGFß signaling in tumor-infiltrating inflammatory cells in this process is unclear. Gr-1+CD11b+ myeloid cells are overproduced in tumor host. They infiltrate into tumor tissues and contribute to immune suppression, tumor angiogenesis and metastasis. Data from our studies demonstrate that Gr-1+CD11b+ myeloid cells are recruited into the invasive front of breast adenocarcinomas. Tumor-infiltrating Gr-1+CD11b+ cells exhibit a significantly elevated TGFß signaling, including increased expression of TGFß type I and type II receptor, TGFß1 and TGFß3 ligands, as well as latent TGFß binding protein. They also produce a high level of multiple matrix metalloproteinases (MMPs) such as MMP13, MMP14, MMP2 and MMP9. Interestingly, genetic deletion of the gene encoding TGFß type II receptor (Tgfbr2) in Gr-1+CD11b+ cells significantly attenuates the production of MMP14 and MMP2. These in vitro studies were conducted by transduction of Gr-1+CD11b+ cells from Tgfbr2flox/flox mice (loxP-flanked Tgfbr2) with adenovirus expressing DNA recombinase. To further investigate if TGFß signaling plays an important role in Gr-1+CD11b+ myeloid cells mediated breast tumor progression, mice with conditional knockout of Tgfbr2 in Gr-1+CD11b+ cells are generated through cross breeding of Tgfbr2flox/flox mice with LysM-Cre mice expressing Cre in myeloid cells. These mutant mice are viable and appear to be normal. Southern blotting showed that Tgfbr2 deletion is restricted to bone marrow, spleen and lymph node but not other organs. Sorted Gr-1+CD11b+ myeloid cells but not T or B lymphoid cells exhibited over 85% of Tgfbr2 deletion. This specific TGF-ß inactivation in Gr-1+CD11b+ cells allows us to investigate its effect on tumor growth and angiogenesis, as well as metastasis. We observed a significantly attenuated 4T1 tumor growth in these knockout mice compared with the control littermates. We postulate that TGFß signaling in host derived Gr-1+CD11b+ cells is an important part of TGFß effect in tumor progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5320.
Abstract 5327: Upregulation of the expression of nonclassical MHC class I HLA-G antigen in human breast cancer cell lines and tissue
Authors: Jiang Xianpeng , Phillips Jeff , Head Jonathan F. , Barnett Brian , Elliott Robert L.
Published: February 2011

HLA-G is a nonclassical MHC class I molecule with immunotolerant properties. HLA-G is normally expressed by trophoblastic cells during pregnancy and protects the fetus from the maternal immune system. HLA-G is also overexpressed in tumors and plays a role in immune evasion of cancer. In this study, we used RT-PCR and immunohistochemistry (IHC) to examine HLA-G expression in normal mammary epithelial and breast cancer cell lines, normal breast tissue, and human breast cancer tissue. RT-PCR showed that normal epithelial MCF-12A cells had no HLA-G mRNA expression, while the breast cancer cell lines MCF-7, T47D and MDA-MB-231, and drug-resistant cell line NCI/Adr-Res had various levels of HLA-G mRNA expression. Furthermore, three mRNA isoforms were expressed in breast cancer cell lines. Twelve normal breast tissues from plastic surgery and 38 breast cancer tissues from mastectomy were examined by IHC. While 60.5% (23 of 38 samples) of breast cancers had medium to strong staining to HLA-G, only 33% (4 of 12 samples) of normal breast tissues had weak staining, the difference was significant (p<0.05). HLA-G staining was diffusely distributed in the membrane and cytoplasm of the cancer cells and mammary epithelia. Rare staining was found in fibroblasts, vascular endothelia or inflammatory cells. In conclusion, breast cancer cells over-express HLA-G mRNA and protein in cell lines and tumors. The role of HLA-G over-expression in breast cancer-related immune evasion remains to be determined.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5327.
Abstract 5330: Phosphatidylserine-targeting antibody enhances survival benefit of androgen deprivation therapy in mice bearing syngeneic prostate cancer
Authors: Yin Yi , Kavlie Anita , Thorpe Philip E.
Published: February 2011

Purpose: Androgen deprivation therapy (ADT) remains the cornerstone treatment for advanced prostate cancer despite being principally palliative in nature. New treatment strategies aimed at enhancing tumor immunogenicity and damaging tumor vasculature could potentially improve prostate cancer response to ADT. We recently showed that targeting phosphatidylserine by antibodies resulting in enhanced tumor immunogenicity and tumor vascular damage. Bavituximab, a chimeric version anti-phosphatidylserine antibody, is being tested in clinic trails for different cancers. Here we tested the hypothesis that the fully human anti-phosphatidylserine antibody 1N11 can enhance the antitumor properties of ADT in prostate cancer

Experimental Design: Efficacy of ADT and 1N11 against prostate cancer was assessed in C57BL/6 mice bearing syngeneic RM1 prostate tumor by using castration and mouse chimeric 1N11(mch1N11). Effects on mouse survival were assessed. In vivo localization of 1N11,effects on immune cell infiltration, vasculature density were also estimated by histology.

Results: Our data showed that castration of RM1 tumor bearing mice increased the percentage of tumor vessels with exposed phosphatidylserine from 60% to 93%. Castration, when combined with mouse chimeric 1N11, led to significant survival befit versus individual treatments. The mean survival time was 16 days with control antibody. The prolongation in survival was 15 days with the combination, as compared with 3 days with mch1N11, 5 days with castration alone. Toxicity to the mice was not observed. A dramatic enhancement of tumor infiltration by a variety of immune cells and disruption of vasculature implied the combination to be more immunostimulatory and antivasculitic.

Conclusion: Combined treatment with 1N11 and ADT improves survival of mice bearing prostate cancer. These results provide the foundation for using ADT plus fully human 1N11, or bavituximab, to treat prostate cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5330.
Abstract 5333: Novel Ab treatment tool for CRC
Authors: Shapira Shiran , Kraus Sarah , Kazanov Dina , Stahel Rolf , Benhar Irai , Arber Nadir
Published: February 2011

Background: We have shown that CD24, a cell surface molecule, is overexpressed early in the multistep process of colorectal neoplasia (Gastroenterology, 2006) hence, it can serve as a target for early detection and immunotherapy. The anti-CD24 murine SWA11 Mab (generous gift from Prof. Sthal, Zurich University) binds to CD24 and inhibits tumor cell growth, in vitro and in vivo, in a time- and dose-dependent manner (Cancer Research, 2008). However, murine Mabs have limitations because of their xenogeneic nature and due to the human anti-mouse antibody (HAMA) response.

Aim: To produce an improved, chimeric form of the SWA11 Mab.

Methods: Mammalian vector pMAZ-IgH for human γ1 heavy-chain and pMAZ-IgL for human κ light chain expression were designed. The variable region genes encoding the murine anti-CD24 SWA11 MAb were cloned for expression as chimeric γ1/kappa antibody into the mammalian pMAZ-IgH and pMAZ-IgL expression vectors and co-transfected into HEK293 cells. Stable clones were screened and selected for IgG production, analyzed by whole-cell and antigen-based ELISA, and Western blotting.

Results: The new chimeric IgG derivative of the anti-CD24 SWA11 Mab possesses similar affinity and specificity (ELISA and Western blot), but has improved activity in inhibiting cell growth.

The chimeric Mabs also have the ability to induce complement-dependent cytotoxicity, causing up to 60% cell lysis in the presence of complement. Conclusions: We had successfully generated a chimeric IgG derivative of the anti-CD24 SWA11 Mab that can serve as a novel therapeutic tool for cancer cells harboring CD24.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5333.
Abstract 5337: Phase 1 study of bavituximab in advanced solid tumor malignancies: Final results
Authors: Ibrahim Nuhad , Wong Lucas , Stopeck Alison , Rosen Lee S. , Gerber David E. , Shan Joseph S.
Published: February 2011

Background: Bavituximab, a novel human chimeric IgG1 monoclonal antibody which targets the membrane phospholipids, phosphatidylserine complexed with β2-glycoprotein I on tumor vasculature, produces host effector cell-mediated destruction of tumor vasculature and enhancement of anti-tumor immunity (He et. al. Clin Cancer Res 2009). We are reporting on an open label dose escalation Phase 1 trial to examine the safety, pharmacokinetics and pharmacodynamics of bavituximab in adults with refractory advanced solid tumor malignancies. Methods: Patients received bavituximab monotherapy, intravenously, up to 4 doses of either 0.1, 0.3., 1 or 3 mg/kg. 26 patients with a median age of 57.6 yrs (range of 37-80 yrs) who had metastatic breast cancer (n=13), colorectal cancer (n=4), pancreatic cancer (n=3), or other solid tumors (n=6), were treated with a total of 86 doses of bavituximab. Results: 6 serious adverse events were observed in 5 patients, with 1 drug-related SAE (pulmonary embolism) at the 3 mg/kg dose. The most common AEs seen in ≥15% of patients were: nausea, fatigue; headache, constipation, dizziness cough, dyspnea, anemia, and chest pain. Pharmacokinetic measures indicated T1/2 of bavituximab ranged from 28 to 47 hrs, and Cmax on Day 0 ranged from 2 to 56 ug/ml with no accumulation seen following multiple dose administration. Coagulation testing revealed no platelet or factor abnormalities, D-dimer levels were elevated in the majority of patients at baseline and increased with disease progression. The presence of a lupus anticoagulant-like activity (prolonged aPTT or DRVVT-an in vitro test artifact) was observed only in patients treated at the highest dose tested of 3 mg/kg. Overall, 8/26 patients showed stable disease as measured by Response Evaluation Criteria in Solid Tumors (RECIST) (4/14 subjects at 0.1 and 0.3 mg/kg and 4/12 subjects in 1 and 3 mg/kg dose groups). Conclusions: Bavituximab was safe and well tolerated when given as monotherapy. Based on pharmcokinetics, the maximum recommended dose for Phase II trials will be weekly at 3 mg/kg, intravenously.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5337.
Abstract 5346: A novel immunotoxin comprising quadruple RNase tethered to an internalizing anti-TROP-2 humanized MAb shows potent cytotoxicity against diverse solid tumors in vitro
Authors: Cardillo Thomas M. , Rossi Edmund A. , Arrojo Roberto , Chan Eric , Goldenberg David M. , Chang Chien-Hsing
Published: February 2011

Ribonucleases (RNase) are potential anti-tumor agents, and their potency can be enhanced by recombinant fusion or chemical conjugation with tumor-targeting antibodies, as have been demonstrated for ranpirnase (Rap), an amphibian RNase, conjugated to LL2, a murine anti-CD22 MAb (Newton et al., Blood 2001; 97: 528-35) or fused to a humanized anti-CD74 MAb (Chang et al., Blood 2005; 106: 4308-14). We have now applied the Dock-and-Lock (DNL) method to link four copies of Rap site-specifically to the carboxyl termini of a panel of tumor-targeting humanized antibodies, in particular, hRS7, an internalizing, humanized, anti-TROP-2 MAb. We combined Rap-DDD2, a fusion protein comprising Rap and the dimerization and docking domain (DDD) of cAMP-dependent protein kinase (PKA), with hRS7-IgG-AD2, a fusion protein comprising two AD2 peptides derived from A-kinase anchoring proteins (AKAPs) having each AD2 appended to the carboxyl terminus of the heavy chain. Since Rap-DDD2 is isolated as a dimer, the resulting DNL complex, designated E1-Rap, contains four copies of Rap tethered to a bivalent hRS7 IgG. In the present study, we compared the in vitro growth inhibitory properties of E1-Rap against a variety of carcinoma cell lines. In breast (MDA-MB-468), cervical (ME-180), and pancreatic (BxPC-3 and Capan-1) tumor lines, all of which express high levels of TROP-2, E1-Rap was very potent, showing EC50 in the subnanomolar range (5 to 890 pM), which was 1,000- to 100,00-fold higher than untargeted Rap or the combination of Rap and hRS7. In cell lines expressing moderate levels of TROP-2, such as the three prostate cancer lines (PC-3, DU 145, and LNCaP), E1-Rap was less potent, but still showed EC50 in the nanomolar range (1 to 890 nM). The cell binding data obtained for these solid cancer cell lines suggest that the sensitivity of a cell line to E1-Rap appears to correlate with its TROP-2 expression on the cell surface. No toxicity was observed for E1-Rap in the prostate cancer line, 22Rv1, which fails to bind hRS7. Based on these results, further in vivo studies are warranted to better determine the efficacy of E1-Rap for future development as a new candidate therapeutic for TROP-2-positive solid tumors, including breast, cervical, pancreatic, and prostatic carcinomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5346.
Abstract 5351: Human papillomavirus prevalence in bladder cancer: Molecular and pathological analysis
Authors: Shigehara Kazuyoshi , Kawaguchi Syohei , Mizokami Atsushi , Sasagawa Toshiyuki , Maeda Yuji , Konaka Hiroyuki , Kumaki Misako , Namiki Mikio
Published: February 2011

Objective: We preformed a molecular and pathological analysis to elucidate the prevalence of human papillomavirus (HPV) in bladder cancer.

Patients and Methods: 72 cases of bladder cancer treated at Kanazawa University Hospital were enrolled in this study. As a negative control, 10 tissue samples including pathologically normal transitional cell (TC) epithelium were used. DNA was extracted from each frozen sample, and DNA quality was confirmed by amplifying the β-globin gene using PCR. Samples were tested for HPV-DNA by modified GP5+/6+ PCR, and the genotype was determined using a Hybri-Max HPV genotyping kit. Moreover, in situ hybridization (ISH) was performed to confirm presence of HPV-DNA in tumor tissue using the paraffin-embedded tissue of each sample. We compared the categorical characters such as patient's age, gender, tumor grade and tumor stage between HPV positive group and negative group.

Results: Mean age of 72 patients (58 men and 14 women) was 65.2 years. 15 cases were grade1 (G1), 49, grade2 (G2) and 8, grade3 (G3). β-globin gene could be amplified by PCR test in all bladder cancer samples and normal TC epithelium. HPV-DNA was detected in 14 cases (19%) of bladder cancer samples, and was not detected in all normal TC epithelium. The grade of HPV-positive tumor indicated that 7 cases were G1 and 7 cases were G2. HPV genotypes indicated high-risk type in all cases, as follow; HPV16: 6 cases, HPV18: 4 cases, HPV33: 3 cases and HPV56: 1 case. On ISH analysis, high-risk HPV genome signals were observed in the nuclei of most of the tumor cells in all HPV positive cases. On the other hands, high-risk HPV signals were not observed in the HPV negative cases and normal TC epithelium. Type 6/11 genome signals were not shown in all samples.

We compared the categorical characters between HPV positive cases and HPV negative cases. HPV-DNA was detected frequently in younger age and low grade (G1) tumor (p<0.05).

Conclusions: HPV-DNA was detected in 19% of bladder cancer. Especially, HPV-DNA was frequently detected in low grade bladder cancer generated in the younger patients. In these cases, we demonstrated HPV-DNA was presented in the tumor tissue by ISH. We consider HPV infection can be one of etiological factors of bladder tumor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5351.
Abstract 5368: Increase in viral load, viral integration and gain of telomerase genes during uterine cervical carcinogenesis can be simultaneously assessed by the HPV MLPA-assay
Authors: Speel Ernst-Jan M. , Theelen Wendy , Herfs Michael , Reijans Martin , Simons Guus , Meulemans Els V. , Baldewijns Marcella M. , Ramaekers Frans CS , Somja Joan , Delvenne Philippe , Hopman Anton HN
Published: February 2011

Oncogenic human papillomavirus (HPV) infection is the most important risk factor in cervical carcinogenesis with high viral load, viral integration into the host genome, and gain of the telomerase-related genes TERT and TERC being factors associated with progression to cancer. A recently developed multiparameter HPV MLPA-assay (Theelen et al. Int J Cancer 2009, Epub Aug 26) that allows the simultaneous assessment of these factors was applied to a series of normal and (pre)malignant frozen uterine cervical samples, and tested for its ability to identify high grade cervical lesions with both high sensitivity and specificity.

A total of 67 cervical samples from normal cervix, as well as different (pre)malignant stages was analysed by the MLPA-assay and validated by quantitative PCR, the PapilloCheck, and fluorescence in situ hybridization for the individual parameters.

Only 5 out of 37 (14%) normal samples or low grade cervical lesions (CIN1 and condyloma) showed either a HPV16 viral load higher than 25 copies per cell, viral integration, and/or gain of one of the telomerase related genes, whereas for the high grade cervical lesions (CIN2/3 and squamous cell carcinoma) one or more of these parameters were found in 25 out of 30 cases (83%). As a result, the HPV MLPA-assay combines a sensitivity of 83% with a specificity of 86% in frozen cervical specimens, making it a valuable screening tool for cervical (pre)neoplasia.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5368.
Abstract 5408: Exploiting the defective p53/MDM2/p14ARF pathway in neuroblastoma by targeting checkpoint kinase Chk1 with AZD7762
Authors: Xu Hong , Wei Xiao , Cheung Irene Y. , Cheung Nai-Kong V.
Published: February 2011

Introduction: A defective p53/MDM2/p14ARF pathway seems to be the principal mode of checkpoint dysfunction in metastatic neuroblastoma following DNA damage, which probably accounts for chemoresistance after initial treatment response. Checkpoint kinase inhibitor AZD7762 (AstraZeneca, currently in clinical trials) has the potential to enhance the action of DNA-damaging agents in some advanced solid malignancies. In this study, we assessed the potential synergy between AZD7762 and chemotherapeutic agents in the in vitro and in vivo treatment of neuroblastoma cells which harbor aberrations in the p53/MDM2/p14ARF pathway.

Experimental Design: AZD7762 was tested in combination with chemotherapeutic agents both in vitro and in vivo against a representative panel of neuroblastoma cell lines. Ten were p53/MDM2/p14ARF pathway-defective (four with p53 mutations, three with MDM2 amplification, and three with p14ARF deletion) and three were wild type. Using these lines, p53/p21 functional assay, drug induced cytotoxicity, S/G2 checkpoint abrogation, and therapy of xenografts were examined. Synergism with chemotherapy was quantified using combination index (CI). CI<1 indicates synergism, CI=1 additive effects, and CI>1 antagonism.

Results: Based on p53/p21 functional assays, all four p53 mutant lines failed to show endogenous p21 induction after DNA damage (defined as p53 pathway nonfunctional). Similarly, p53 was nonfunctional in two of the three MDM2 amplified and one of the three p14ARF deleted cell lines. In cytotoxicity assays, p53 nonfunctional lines were more resistant to DNA-damaging agents when compared to p53 functional lines. Synergy between AZD7762 and DNA-damaging agents (cisplatin, doxorubicin, etoposide, melphalan, SN38 and topotecan) was strong in all p53 nonfunctional lines (average CI values: 0.44 - 0.63), but not in p53 functional lines (average CI values greater than 1.0). In contrast, gemcitabine showed even stronger synergy with AZD7762 regardless of p53 functional status (CI values: 0.11 - 0.47). AZD7762 treatment resulted in the abrogation of DNA damage-induced S/G2 checkpoint arrest, and in the potentiation of antitumor activity of DNA-damaging agents in four mouse xenograft models.

Conclusions: AZD7762 potentiated both in vitro and in vivo the antitumor effects of DNA-damaging agents against neuroblastoma. These results suggest that the addition of AZD7762 may reverse or prevent drug resistance, especially if a defective p53/MDM2/p14ARF pathway is the underlying mechanism.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5408.
Abstract 5413: Maximal Power Design and Analysis of Drug Combination Studies: Applications to Vorinostat with Cytosine Arabinoside and Etoposide and Other Combination Studies
Authors: Fang Hong-Bin , Ross Douglas D. , Edelman Martin J. , Sausville Edward A. , Li Wei , Tan Ming T.
Published: February 2011

Drug combinations are the hallmark of cancer therapy. Preclinical experiments on multi-drug combinations are important steps to bring the therapy to clinic. A statistical approach for evaluating the joint effect of the combination is necessary because even in vitro experiments often demonstrate significant variation in dose-effect. Such variation needs to be accounted for in the experimental design and analysis. Our research has developed a maximal power (MP) design and interaction index surface (IIS) analysis methods for in vitro and in vivo (e.g., xenograft) combination studies so that the joint effect of a combination can be estimated efficiently and the most synergistic combination can be identified. We demonstrate that these statistical methods and software have resulted in the identification of highly synergistic dose combinations that could have been missed with classic methods.

The first study is the combination of vorinostat (suberoylanilide hydroxamic acid) combined with ara-C and with etoposide in leukemia cell lines. The doses in the experiment were generated by the MP design and the data analyzed using the IIS approach so that synergistic, additive and antagonistic interaction dose regions are identified. Cytotoxic antagonism resulted when vorinostat was combined concomitantly with ara-C; however, when vorinostat was given first followed by a drug-free interval before ara-C treatment, this sequential combination was mostly synergistic. Etoposide combined with vorinostat was additive to synergistic, and the synergism became more pronounced when etoposide was given post a drug-free interval after vorinostat. These findings are used in designing the CTEP trial (NCI 6829: Phase I trial of vorinostat in combination with cytarabine and etoposide in patients with advanced acute leukemia and high-risk myelodysplastic syndromes, PI: Gojo/Ross). The interim results on toxicity and response have been consistent with the model.

Another study utilized a novel thiazolidine compound plus Sorafnib where initial experiments using classic methods failed to identify synergistic combinations. Subsequent experiments using the MP methodologies demonstrated significant synergistic drug combinations. The SynStat R program for the design and analysis of drug synergy is available at http://www.umgcc.org/research/biostat_software.htm).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5413.
Abstract 542: Inoculated carcinoma associated fibroblasts and hormone independent murine mammary tumor growth
Authors: Sahores Ana , Fabris Victoria , Vanzulli Silvia , Lanari Claudia , Lamb Caroline A.
Published: February 2011

Murine mammary carcinomas from the MPA-murine breast cancer model express estrogen and progesterone receptors and transit through different stages of hormone dependence. Hormone dependent (HD) tumors only grow in the presence of medroxyprogesterone acetate (MPA) and hormone independent (HI) variants are those that grow without hormone supply. In co-culture studies we have demonstrated that carcinoma associated fibroblasts (CAF) from HI tumors (CAF HI) can stimulate epithelial cell proliferation better than those from HD tumors (CAF HD). However, in vivo, CAF-HI inoculated together with purified epithelial cells (EPI) only increased tumor growth of EPI HI and they were not able to replace the hormone requirement of EPI HD. Thus, the aim of this study was to investigate the role of CAF HI enhancing HI tumor growth and to elucidate why CAF HI failed to stimulate HD cells in vivo. For this purpose murine C4-HI tumors were transplanted in BALB/c-wt (wt) or in BALB/c-GFP (GFP) mice. Mice were subcutaneously inoculated with EPI HI alone or co-mingled with CAF HI-wt (in GFP mice) or CAF HI-GFP (in wt mice) and the resulting tumors were excised at different time points. Fluorescence analysis of frozen sections indicated that inoculated CAF HI-GFP were detected 7 days after their inoculum in wt mice whereas they were no longer detected at later time points. To elucidate how CAF HI are able to enhance tumor growth when co-inoculated with EPI HI cells, we quantified polymorphonuclear (hematoxilin and eosin) and mast cells (toluidine blue staining) in the EPI HI + CAF HI group and in the EPI HI group and no significant differences were observed. In similar experiments when cells were inoculated intradermically to evaluate angiogenesis, and animals were sacrificed after different time points, we observed that on day 5, there was a significantly higher number of vessels in the co-inoculated group (Control 1.1±0.1; CAF HI 1.8± 0.8; EPI HI 2.0±0.7; EPI HI + CAF HI 3.3±0.5 p<0.05). In summary, we conclude that the inoculated CAF fail to induce HD growth because they do not persist in the tumor mass, although they may participate during the first stages of tumor growth favoring angiogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 542.
Abstract 5425: Characterization of synergistic antiproliferative effects of combined γ-tocotrienol and celecoxib treatment on neoplastic mouse mammary epithelial cells in vitro
Authors: Shirode Amit B. , Sylvester Paul W.
Published: February 2011

In the present study, synergistic antiproliferative effects of combined treatment of γ-tocotrienol and celecoxib on +SA mouse mammary epithelial cells were evaluated and characterized for their mechanism(s) of action. Earlier studies have shown that these effects are mediated partly by inhibition of PGE2, which is a direct indicator of COX-2 activity in cells. Since 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD+-dependent oxidation of 15(S)-hydroxyl group of prostaglandins and has been considered a key enzyme in biological inactivation of prostaglandins, the effects of these drugs on intracellular levels of 15-PGDH were assessed. Consistent with earlier findings, western blot analysis showed increased levels of 15-PGDH by individual treatment of effective doses of both γ-tocotrienol (3.5 μM) and celecoxib (20 μM). Combined treatment with subeffective doses of γ-tocotrienol (0.25 μM) and celecoxib (2.5 μM) showed increased levels of 15-PGDH as compared to the same individual doses when treated alone. These studies indicate that the antiproliferative effects are mediated by inducing the expression of 15-PGDH, which leads to enhanced inactivation of PGE2. Similar to earlier studies which showed that these effects were associated with a suppression of Akt and NFκB activity, the present studies showed that treatment with 3.5 μM γ-tocotrienol alone induced a relatively large decrease in phospho-PDK-1 and phospho-Akt levels, but had no effect on PI3K p85, PTEN and phospho-PTEN levels. In contrast, treatment with 20 μM celecoxib alone was associated with a relatively large decrease in PI3K p85 levels. Combined treatment with subeffective doses of γ-tocotrienol and celecoxib caused a large decrease in PI3K, phospho-PDK-1, phospho-Akt and phospho-GSK-3β levels as compared to vehicle treated controls. In addition, a large decrease in phospho-IKK-α/β, phospho-IκB-α, and phospho-NFκB levels was observed in the combination treatment group relative to the same subeffective doses when given alone. In conclusion, these studies show that synergistic antiproliferative effects of combination treatment of γ-tocotrienol and celecoxib are associated with two different mechanisms; 1) a COX-2-dependent mechanism of induction of 15-PGDH leading to enhanced inactivation of PGE2; and 2) a COX-2-independent mechanism of attenuation of EGF-dependent PI3K/PDK-1/Akt and NFκB mitogenic signaling pathways. Further studies are required to evaluate the role of prostaglandin and EGF receptors in mediating these two independent mechanisms. This study was supported by grants from NIH (CA86833) and First Tech International Ltd.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5425.
Abstract 543: Stromal impact on tumor invasiveness and gelatinolytic activity at the invasive front
Authors: Hadler-Olsen Elin , Ravuri Chandra , Ljones Hilde , Rikardsen Oddveig , Steigen Sonja , Svineng Gunbjørg , Winberg Jan-Olof , Uhlin-Hansen Lars
Published: February 2011

The aim of this study was to explore the impact of tumor stroma on invasiveness and expression of gelatinases in xenograft carcinomas.

Xenograft tumors of various human carcinoma cells were established either in the tongue or the skin of BALB/c Nude mice. Cell lines originating from oral and skin SCCs were used, as well as a cell line foreign to both organs. Gelatinolytic activity in the tumors was investigated by a novel in situ zymography technique which enables high image resolution. In vivo and in vitro expression of various proteolytic enzymes were analyzed at transcriptional and protein level using RT-qPCR, immunohistochemistry, Western blotting and SDS-PAGE substrate zymography.

Tongue tumors showed an invasive growth pattern, whereas the skin tumors established from the same cell lines were non-invasive. Although the repertoire of proteolytic enzymes differed between the cell lines, the tongue tumors of all cell lines showed strong gelatinolytic activity, especially at the invasive front. In contrast, the skin tumors showed only weak gelatinolytic activity.

Our results suggest that invasiveness as well as activity of proteolytic enzymes in the tumors is more dependent on the tumor microenvironment than on inherent properties of the cancer cells.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 543.
Abstract 5430: Epigenetic targeting by histone deacetylase inhibitors reduces viability and induces apoptosis in human embryonal carcinoma cells
Authors: Bhinder Arvinder , Varma Vivek , Abbaoui Besma , Clinton Steven K. , Mortazavi Amir
Published: February 2011

The disruption of the epigenetic regulation, including the acetylation status of nucleosomal histones, impacts gene expression; and has been implicated in the pathogenesis of cancer. Histone deacetylase inhibitors (HDACIs) are being evaluated for anticancer activity based upon their ability to induce differentiation, inhibit cancer cell proliferation, and enhance sensitivity to apoptotic signaling. Although, germ cell tumors (GCT) have a high cure rate, effective agents with less toxicity are desired. Most critically, those with relapsed/refractory disease need new and effective second line agents. Serendipitously, in murine studies of prostate carcinogenesis with a novel HDACI developed at the Ohio State University we observed significant testicular atrophy, with essentially no histopathologic toxicity to other organs [Cancer Res 2008; 68:(10)]. This observation led us to investigate the in vitro effects of HDACIs (our novel HDACI and suberoylanilide hydroxamic acid - SAHA) in the NTERA-2, an embryonal carcinoma cell line derived from a human testicular cancer. We evaluated their dose- and time-dependent effects on cell viability (MTS assay), apoptosis (caspase-3/7 activity, and PARP cleavage); and HDAC inhibition (immunoblotting). Assessment of cell signaling pathways (EGFR, and ErbB2) and anti-apoptotic molecules (Bcl-2) were performed by immunoblotting. Treatment of NTERA-2 cells with HDACIs at 0.1 - 10 µM concentrations showed a time- and dose-dependent reduction in cell viability. The IC50, at 72 hours of treatment, was 0.2 µM for our novel HDACI compared to 1.5 µM for SAHA. Both agents significantly induced apoptosis, but SAHA was less potent. Our novel HDACI significantly down-regulated the expression of EGFR, ErbB2 and Bcl-2 along with increased acetylation of histones H-3 and H-4, in a dose-dependent manner. These effects were more potent than observed with SAHA. In conclusion, HDACIs, especially our novel HDACI, appear to be potent inhibitors of cell viability and inducers of apoptosis in NTERA-2 human embryonal carcinoma cells. Our studies suggest that chromatin hyperacetylation, resulting in a coordinated change in recruitment of epigenetic modulators and transcription factors including tumor suppressor genes may result from targeted HDACI in germ cell cancers. The further preclinical development of HDACIs for the treatment of advanced germ cell tumors is underway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5430.
Abstract 5438: A novel therapeutic combination with synergistic antitumor activity in colon cancer: The dual tyrosine kinase inhibitor lapatinib and the histone deacetylase inhibitor LBH589
Authors: LaBonte Melissa J. , Wilson Peter M. , El-Khoueiry Anthony , Lenz Heinz-Josef , Ladner Robert D.
Published: February 2011

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States. Effective treatment is hindered by the high incidence of drug resistance and tumor recurrence, resulting in the need to identify and exploit novel therapeutic targets and drug combinations to improve clinical efficacy. The epidermal growth factor receptor (EGFR) has been implicated in colon cancer growth, progression and chemoresistance and inhibition of EGFR has therefore become an efficacious drug target. Lapatinib is a dual tyrosine kinase inhibitor targeting EGFR and HER-2 and suppressing oncogenic signaling through the RAS-RAF-MEK-ERK and PI3K/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents demonstrating promising activity against colon cancer cells through the hyper-acetylation of histone and non-histone proteins resulting in cell cycle arrest and apoptosis. HDACi are also reported to disrupt HSP90 function, inducing the degradation of EGFR-pathway client proteins (e.g. EGFR, HER-2, BRAF). This study sought to evaluate the therapeutic potential of lapatinib in combination with the HDACi LBH589 (Panobinostat) in a panel of eight colon cancer cell lines with varying EGFR/HER-2 expression and KRAS mutational status. EGFR and HER-2 mRNA and protein expression levels were determined by quantitative PCR and Western blot. Concentration-dependent antiproliferative effects of both lapatinib and LBH589 were observed in vitro (lapatinib range 7.6-15.8 μM; LBH589 range 7.2-30 nM) using the MTS assay. The combination of LBH589 and lapatinib interacted synergistically to inhibit both cell proliferation and colony formation in all colon cancer cell lines tested. Combination treatment resulted in decreased signaling through both the PI3K and MAPK pathways as determined by decreased levels of phospho-AKT and phospho-p44/42 MAPK. Co-treatment with lapatinib and LBH589 also resulted in the rapid induction of apoptosis as early as 18 h post-treatment as determined by flow cytometry and Western blot analysis of apoptotic factors including cleaved caspase-8 and PARP. These in vitro observations were extended to a LoVo colon cancer xenograft model where combination treatment resulted in greater antitumor activity than either lapatinib or LBH589 alone, with no apparent increase in toxicity. Together these data provide scientific rationale warranting further investigation of HDACi in combination with EGFR and HER-2-targeted therapies for the treatment of colorectal cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5438.
Abstract 5459: Antitumor activity of EP80061, a small-glyco drug in preclinical studies
Authors: Nancy-Portebois Vanessa , Cabannes Eric , Petitou Maurice , Serin Guillaume , Mirjolet Jean-François
Published: February 2011

Background:

Heparan sulphate (HS) containing proteoglycans regulate the activity of many proteins (growth factors, cytokines, adhesion molecules…) involved in pathologies like cancer, inflammation, cardiovascular, metabolic and neurodegenerative diseases, as well as in viral infection. HS mimicking oligosaccharides can interfere with protein/HS interactions and thus modulate the resulting biological effects. The optimization of fully synthetic HS mimetics exhibiting anti-tumour and anti-metastatic properties is currently in progress.

Methods:

Compound selection was performed using classical in vitro assays (binding and proliferation) and a cellular angiogenesis assay (Angiokit®).

In vivo, Swiss nude mice subcutaneously (SC) xenografted with A-673 human rhabdomyosarcoma tumor cells were used to compare compounds showing significant anti-tumour activity. Compounds were intraperitonealy (IP) injected on a daily basis. The most potent compound was then evaluated on syngeneic mouse models: C57 BL/6 mice intravenously (IV) injected with B16-F10 malignant melanoma cells and Balb/c mice bearing orthotopic (OT) Renca kidney carcinoma.

Results:

Binding assays to FGF-2, PDGF-BB, VEGF-B and SDF-1α in Biacore experiments and growth factor-dependent proliferation tests led to the identification of several inhibitors. Optimized compounds exhibiting anti-angiogenic activities were selected in Angiokit® assays. EP80061 was selected for further studies on the basis of significant activity in this assay.

In A-673 SC mouse model, EP80061 was also the most powerful compound. When IP injected at 30 mg/kg/inj. for 28 consecutive days, it induced a marked tumour growth delay. At D22, the optimal T/C value (46%) was reached with a significantly lower tumour volume (p=0.0006, 1672 ± 760 and 765 ± 402 mm3 for vehicle and EP80061 treated mice, respectively). EP80061 induced also a very significant decrease in lung metastasis development in B16-F10 IV injected mice treated at 30 mg/kg/inj. (p<0.0001, 92 ± 24 and 12 ± 8 metastases for vehicle and EP80061 treated mice, respectively). Promising results were also obtained on OT Renca bearing mice where EP80061 induced a decrease of OT tumour size.

Conclusion:

Current preclinical results support EP80061 as a lead candidate for further developments of small-glyco drugs as anticancer compounds.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5459.