bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 270: Modulation of EGFR localization and transcriptional activity
Authors: Bitler Benjamin G. , Goverdhan Aarthi , Schroeder Joyce
Published: February 2011

The triple negative (ER-, PR-, Her2-) or basal-like breast cancers are the most difficult forms of breast cancer to treat, because the cancer is often chemoresistant and highly metastatic. As these cancers progress from early to late stage the 5-year survival rate drops dramatically. Many proteins are responsible for this progression, including MUC1 and the Epidermal Growth Factor Receptor (EGFR). MUC1 is a heavily O-glycosylated transmembrane protein that is over expressed in 90% of all breast and ovarian cancers. In normal cells, MUC1 is only found on the apical surface of epithelium, but in cancer, MUC1 is found to be mislocalized. As a result, MUC1 plays a role in promoting tumorigenesis and metastases, by interacting with other oncogenes such as EGFR. EGFR is overexpressed in about 60% of the most aggressive form of breast cancer (triple negative). In an EGFR-dependent mouse model of breast cancer, the loss of Muc1 directly correlates to the loss of cyclin D1. Additionally, blocking MUC1 and EGFR interactions significantly inhibited tumor progression in a xenograft and s spontaneous mouse model of breast cancer. Recently, a novel trafficking pathway of EGFR has been discovered, which was described as EGFR trafficking to the nuclear compartment. Following entry into the nucleus EGFR can act in an oncogenic manner by aberrantly activating transcription of genes such as, cyclin D1 and v-myb. In this study we discovered a novel regulator function of MUC1 in EGFR trafficking and transcriptional activity. We found that MUC1 expression in basal-like breast cancer promotes the accumulation of nuclear EGFR and mediates EGFR's interaction with chromatin. Additionally, we determined that EGFR's interaction with the cyclin D1 promoter and upregulation of the cyclin D1 protein was MUC1-dependent. The main focus of anti-EGFR therapeutics have been its tyrosine kinase activity, however this study reveals that EGFR's subcellular localization should also be considered in the development of future cancer therapeutics.

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 270.
Abstract 2720: Glycosylation of Mucins Expressed in Pancreatic Adenocarcinoma
Authors: Remmers Neeley , Rothermund-Franklin Christy , Caffrey Thomas , Hollingsworth Michael A.
Published: February 2011

Glycosylation of mucins expressed by pancreatic adenocarcinoma

There were an estimated 42,470 new cases and 35,240 deaths due to pancreatic adenocarcinoma (PA) for 2009, ranking PA as the third leading cause of cancer related death. Virtually all long term survivors are diagnosed early but only 7% are detected in the early stages of disease thus highlighting the need for an early diagnostic test. CA19-9, the only current clinical serum-based assay used to monitor PA, detects an oligosaccharide blood group antigen Sialyl Lewisa (sLeA) with the structure (Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAc), which can be present on glycolipids or as an O-linked oligosaccharide on glycoproteins, and is expressed at relatively low levels on the surfaces of normal epithelial cells. The full spectrum of proteins on which the CA19-9 epitope is expressed has never been defined. CA19-9 is differentially expressed on mucins, which are extensively glycosylated. The CA19-9 test lacks high specificity for PA, as CA19-9 is commonly elevated in patients with benign chronic pancreatitis and other malignant diseases. Different epithelial tissues and cell types produce distinct combinations of mucin-type core proteins, and the expression patterns of these are generally altered during different disease processes including malignant transformation. Mucin expression is deregulated in cancer, which results in aberrant expression and glycosylation of mucin core proteins. We employed immunohistochemistry and immunofluorescence to show the unique mucin and glycosylation patterns seen in tissue specimens from patients diagnosed with PA. Deglycosylation of samples immunoprecipitated from cancer cell lines and pancreatic cancer patient tissue specimens was used to enable detection of mucin core proteins. Immunoaffinity chromatography was used to isolate CA19-9 from patient ascites and serum samples and mass spectrometry was used to analyze the core proteins. Results show that pancreatic cancers expressing the mucin core proteins MUC1, MUC4 and MUC5AC carry the CA19-9 epitope. Preliminary results suggest that a strategy to detect the CA19-9 tumor antigen and its cognate mucin core protein(s) will improve the performance characteristics of this assay.

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 2720.
Abstract 2721: Real-time and label-free aptasensor of VEGF for cancer diagnostics
Authors: Cho Hansang , Sinha Raghu , Lee Luke P.
Published: February 2011

Early detection of cancer markers is a critical demand for clinical research, particularly in preventing cancer cells from establishing themselves in a metastatic stage. We have developed a stable, sensitive, and selective detection method utilizing a nanoplasmonic ‘aptasensor’ (aptamer-based sensor) of vascular endothelial growth factor (VEGF) for breast cancer diagnostics. Nanoplasmonic aptasensors are prepared by fixing gold nanoplasmonic particles (GNPs) on an amino-silane coated glass slide and coating poly-L-lysine (PLL) on top of GNPs, which provides the GNP with a strong positive-charged surface and a spacing layer avoiding quenching. To accomplish high selectivity, VEGF-specific aptamers are immobilized on the PLL-GNP surface by an electrostatic force. To monitor the event of target binding, the aptamer is conjugated with a fluorescent molecule, Cy-3, which has an absorption peak wavelength (λ = 552 nm) that matches with a localized surface plasmon resonance wavelength of the 80 nm in diameter GNP (λ ≈ 550 nm). In the absence of target VEGF molecules, Cy-3 is spaced in a relevant coupling distance from the GNP surface for the surface plasmon resonance to amplify an incident light and enhance the fluorescent signal of Cy-3. However, once VEGF molecules bind to the aptamer-Cy-3, the conformational change of aptamer occurs, leading to its subsequent detachment from the GNP surface, which results in no signal enhancement and, thus, a loss in the fluorescence intensity. Therefore, the concentration of VEGF molecules can be estimated by measuring the amount of signal decrease in the fluorescence intensity.

With high affinity and specificity, the aptasensor of VEGF requires a small amount of sample (10 μL) for a single detection and incubation time of 1 hour at a room temperature. Also, the aptasensor achieves VEGF detection from 100 nM (2 μg/mL) to 100 pM (2 ng/mL) with a signal decrease up to 50%. In contrast, there is no discernible signal change in a non-specific binding protein solution (100 nM bovine serum albumin) or a strong ionic solution (1.4 M NaCl). Furthermore, the aptasensor is applicable to VEGF detection in human fluids, owing to the biological stability of a fully 2′-O-methyl nucleotide-based aptamer. We can detect VEGF molecules present in saliva and serum of breast cancer patients by observing signal decrease corresponding to the concentration of 100 nM (2 μg/mL) VEGF and a consistent decrease with concentration variation from 1 to 100 % in saline. The aptamer-based nanoplasmonic sensor may be useful for not only basic studies on angiogenesis but also clinical research in evaluating the effectiveness of drug candidates that inhibit VEGF secretion.

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 2721.
Abstract 2726: Salivary biomarkers for breast cancer detection
Authors: Zhang Lei , Xiao Hua , Karlan Scott , Zhou Hui , Feng Ziding , Elashoff David , Akin David , Yan Xinmin , Chia David , Gross Jenny , Karlan Beth , Wong David T.
Published: February 2011

Objectives:

The high burden of breast cancer in women worldwide underscores the unmet potential of biomarker for early detection. New strategies and biomarkers for early detection of breast cancer are sorely needed. In this study, we have conducted a prospective sample collection and retrospective blinded validation (PRoBE design) to evaluate the performance and translational utilities of salivary biomarkers for the non-invasive detection of breast cancer.

Methods:

The Affymetrix HG U133 Plus 2.0 Array and 2D-DIGE were used to profile transcriptomes and proteomes in saliva supernatant from patients with invasive ductal carinoma and matched controls respectively. The discovered transcriptomic and proteomic candidate biomarkers were subjected to a clinical validation using an independent sample set of 31 breast cancer (invasive ductal carcinoma, stages I and II) and 62 healthy controls. Transcriptomic biomarkers were validated by qPCR and proteomic biomarkers were validated by quantitative immuno-blot.

Results:

Significant variations of salivary transcriptomic and proteomic profiles were observed between breast cancer patients and healthy controls. Eleven mRNA biomarker candidates and two protein biomarker candidates were selected for independent validation. Eight mRNA biomarkers and one protein biomarker have been validated for breast cancer detection, yielding ROC-plot AUC values between 0.665 and 0.959. Six biomarkers have known associations with breast cancer.

Conclusion:

Salivary biomarkers possess discriminatory power for the detection of breast cancer, with high specificity and sensitivity. This report provides proof of concept of salivary transcriptomic and proteomic biomarkers for the non-invasive detection of breast cancer. The salivary biomarkers' discriminatory power paves the way for a pivotal clinical validation.

Supported by NIH grant U01DE016275, U01DE017790 and R21CA126733 (DTW).

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 2726.
Abstract 2727: Predicting lung cancer risk based on a novel proteomic signature of bronchial lesions
Authors: Rahman S.M. Jamshedur , Gonzalez Adriana L. , Seeley Erin H. , Zimmerman Lisa J. , Zhang Xueqiong J. , Li Ming , Kikuchi Takefumi , Chaurand Pierre , Manier M. Lisa , Olson Sandy , Shah Ronak , Miller Aloson , Putnam Joe B. , Miller York E. , Franklin Wilbur A. , Carbone David P. , Shyr Yu , Caprioli Richard A. , Massion Pierre P.
Published: February 2011

A biomarker signature to identify individuals at-risk for lung cancer would be of great clinical value. We hypothesized that the bronchial epithelium expresses a subset of proteins that once increased confer an elevated risk for lung cancer. We have therefore developed a proteomic approach to assess the risk for lung cancer. From our previous studies, we selected 11 matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) features to distinguish normal bronchial epithelium and low grade preinvasive lesions from high grade preinvasive lesions and invasive lung tumors. We tested this signature in an independent validation set consisting of bronchial specimens from 60 patients. The prediction accuracy was 78% with 73% sensitivity and 81% specificity. There was a 19-fold increased risk of having lung cancer for individuals with a signature intensity above the third quartile (95% CI, 5.65-64.81). We identified 10 of the 11 features corresponding to 7 proteins, thymosin ß4, ubiquitin, acyl-coA binding protein, cystatin A, S100A11, cytochrome c and macrophage migration inhibitory factor. The identity of these proteins was validated by immunohistochemistry, Western blotting and MALDI imaging mass spectrometry (IMS). MALDI IMS localized the candidate biomarker proteins specifically to high grade preinvasive lesions and invasive tumors. This novel proteomic signature may facilitate the selection and monitoring of high risk individuals for lung cancer early detection and chemoprevention strategies.

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 2727.
Abstract 273: Exosome targeted receptor activation (ExTRAcrine) signaling: A new mode of EGF receptor ligand signaling
Authors: Beckler Michelle Demory , Higginbotham James N. , Gephart Jonathan , Coffey Robert J.
Published: February 2011

Epidermal growth factor receptor (EGFR) ligands have been shown to act in an autocrine, paracrine and juxtacrine manner. We report a new mode of signaling - via exosomes. Exosomes are 30-100 nm nano-vesicles that are produced by intralumenal budding into multivesicular bodies (MVB); they are released from many cell types after fusion of the MVB with the plasma membrane. Our studies showed that human breast and colorectal cancer cell lines differ in the composition of EGFR ligands in exosomes isolated from conditioned medium by established methods. By fluorescent-activated vesicle sorting (FAVS; Mol Cell Proteomics 7: 1651-1667, 2008), we show that a single HCA-7-derived exosome contains TGF-α, amphiregulin (AR) and HB-EGF. Ligands localized in exosomes are signaling competent with the ectodomains presented on the outside and cytoplasmic tails on the inside, and exist as stable transmembrane proteins. To explore the biological function of individual ligands, we isolated exosomes from MDCK cells individually stably expressing TGF-α, AR or HB-EGF. We assessed the invasive capacity of these exosomes by incubating them with a breast cancer-derived cell line (LM2-4175) in a Boyden chamber assay and measured the number of invading cells 72 hours later. Exosomes purifed from MDCK cells singly expressing AR induced invasion of LM2-4175 recipient cells 4-fold more than TGF-α or HB-EGF exosomes and 15-30-fold more than recombinant ligands TGF-α, HB-EGF or AR). The number of invading cells exposed to AR exosomes was significantly inhibited by an AR neutralizing antibody, suggesting that the invasive capacity of recipient cells is dependent upon exosomal AR engagement with recipient cell EGFR. Fixed and live cell imaging by confocal microscopy showed that AR exosomes are internalized by recipient cells. Further analysis showed that exosomes isolated from mutant KRAS DLD-1 colon cancer cells increased invasion of LM2-4175 cells 5-fold more than exosomes isolated from cells in which mutant KRAS was eliminated by homologous recombination (DKs-8 cells) and this correlated with increased levels of exosomal AR present in DLD-1 cells compared to DKs-8 cells. We provide the first evidence that EGFR ligands are present in exosomes and demonstrate that AR exosomes confer a greater invasive effect on recipient cells than TGF-α and HB-EGF exosomes or recombinant EGFR ligands. We propose ExTRAcrine signaling as a novel mode of EGFR ligand signaling with important diagnostic and therapeutic implications.

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 273.
Abstract 2735: Diagnosis of prostate cancer without tumor cells using differentially expressed genes in the tumor microenvironment
Authors: Jia Zhenyu , Wang Yipeng , McClelland Michael , Sawyers Anne , Yao Huazhen , Rahmatpanah Farahnaz , Xia Xiao-Qin , Xu Qiang , Koziol James , Carpenter Philip , Wang-Rodriquez Jessica , Simoneau Anne , Meyskens Frank , Sutton Manuel , Lernhardt Waldemar , Beach Thomas , Mercola Dan
Published: February 2011

Over one million prostate biopsies are performed in the U.S. every year. However, pathology examination is not definitive in a significant percentage of cases due to missing the tumor or equivocal structures. Given that the tissue near tumors will experience expression changes, biopsies could reveal the presence of a nearby tumor even if the biopsy contains no tumor. In order to identify reliable examples of such genes we compared gene expression profiles (Affymetrix U133plus2) from 15 volunteer biopsy specimens to 13 specimens containing largely tumor-adjacent stroma. More than a thousand significant expression changes were found and thereafter filtered to eliminate possible aging-related genes and genes also expressed at detectable levels in tumor cells. Classifiers were constructed based on 114 unique candidate genes (131 Affymetrix probe sets) and tested on 380 independent cases, including 255 tumor-bearing cases, 125 nontumor cases (normal biopsies, normal autopsies, remote stroma as well as pure tumor adjacent stroma). The classifier predicted the tumor status of patients with an average accuracy of 97.4% (sensitivity = 98.0% and specificity = 89.7%). These results indicate that the prostate cancer microenvironment exhibits reproducible changes useful for detecting “presence of 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 2735.
Abstract 2736: Overexpression of L-type amino acid transporter-1 in breast cancer cells and tissues: potential association with growth and progression of tumors
Authors: Liang Zhongxing , Cho Heidi T. , Liang Ke , Zhu Aizhi , Huang Ke , Wu Hui , Hong Samuel , Goodman Mark M. , Shim Hyunsuk
Published: February 2011

For a tumor to constantly proliferate, malignant cells require nutrients, especially glucose and amino acids. There is abundant evidence that tumor growth depends heavily on glucose uptake. The L-type amino acid transporter 1 (LAT1) is a major nutrient transport system responsible for the transport of large neutral essential amino acids. In the present study, we analyzed expression levels of LAT1 mRNA and protein with RT-PCR, Western blot and immunohistochemical staining. The results showed that normal breast tissues or low malignant cell lines expressed low levels of LAT1 mRNA and protein, while LAT1 was overexpressed in highly malignant cancer cell lines and high grade breast cancer tissues. In addition, we found higher expression levels of LAT1 in breast cancer tissues were consistent with high stages of breast cancer. Furthermore, we demonstrated that blockade of LAT1 with its inhibitor, 2-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (BCH), or knockdown of LAT1 with transfection of specific siRNA inhibited proliferation of breast cancer cells. A variety of 11C- and 18F-labeled amino acids have been studied for potential use in positron emission tomography (PET) oncology. A non-natural, not-metabolizable leucine analog, anti-1-amino-3-[18F]fluorocyclobutane-1-carboxylic acid ([18F]FACBC), has shown superior tumor/normal brain ratio. We tested whether [18F]FACBC would also be an excellent PET tracer for malignant breast cancer imaging using an orthotopic breast cancer animal model and an experimental animal model of breast cancer metastasis. Our results support that [18F]FACBC could be a useful PET tracer for non-invasive imaging of breast cancer. These findings suggest that overexpression of LAT1 is necessary for proliferation of breast cancer cells and may contribute to progression of tumors. LAT1 may represent a potential target for diagnosis of breast cancer.

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 2736.
Abstract 274: Cited2, a novel EGFR-induced coactivator, plays a key role in lung cancer progression
Authors: Chou Yu-Ting , Hsu Chiung-Fang , Kao Yu-Rung , Wu Cheng-Wen
Published: February 2011

Aberrant expression and function of epidermal growth factor receptor (EGFR) are reported in most lung cancer cases and are correlated with poor prognosis. However, the role of many downstream effectors associated with abnormal EGFR signaling is not clear in cancer progression. Cited2, a novel transcriptional co-activator was identified to be induced by hyperactive EGFR mutant L858R in lung cancer cells. Overexpression of Cited2 in CL1-0 lung cancer cells allowed cell to survive in serum free condition. Knockdown of Cited2 attenuated cell growth, leading to G1/S cell cycle arrest. Through microarray analysis, we identified that expression of E2F3 and its downstream gene targets were blocked in Cited2-silenced cells. Overexpression of Cited2 enhanced E2F3 expression, supporting that Cited2 regulates E2F3 expression. By chromatin immunoprecipitation assay, we found Cited2 bound to E2F3 promoter. In addition to affecting cell growth, knockdown of Cited2 promoted cell senescence with flat and enlarged cell morphology, and increased senescence-associated ß-galactosidase activity. Through western blot analysis, we found Cited2-silencing induced p53 and p21 expression. Overexpression of Cited2 further enhanced tumor growth in nude mice. Clinical analysis showed that Cited2 was highly expressed in lung cancer but not in normal tissues. These data demonstrate that Cited2 plays a key role in lung cancer 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 274.
Abstract 2745: Expression of immunoglobulin gene in myeloblasts from patients with acute myelogenous leukemia and its distinct VKJK recombination pattern
Authors: Qiu Xiaoyan , Sun Xiaoping , He Zhiqiao , Lin Pei , You M. James , Chen Lei , Dai Chongwen , Yin C. Cameron
Published: February 2011

Immunoglobulin (Ig) gene expression is normally restricted to B lymphocytes and plasma cells. Immunoglobulin gene rearrangement has been used in the diagnosis and monitoring of response to therapy in B-cell lymphoma and leukemia. However, during the last decade, our group and others have found that Ig is also expressed in many non-hematopoietic cells, especially epithelial cancer cells. Moreover, we have reported that non-hematopoietic cancer-derived Ig expression is associated with poor prognosis, though the mechanism of its function in the survival of cancer cells is not yet known.

In this study, we assessed the expression of Ig heavy chain (gamma and mu) and light chain (kappa) in myeloblasts (CD33+CD19-CD138-, sorted by FACs) from 16 patients with acute myelogenous leukemia (AML) and 6 AML cell lines by RT-PCR, ELISA, and immunohistochemistry. As control groups, we also evaluated IgG, IgM and IgK transcripts in monocytes and neutrophils (sorted by FACs) from 15 patients with non-hematopoietic malignancies and 8 healthy individuals. We have found that Ig gamma chain was expressed in 6 of 14 AML patients, but in none of the 23 controls. All of the variable region sequences of AML-derived Ig gamma chain showed diversity, similar to B cells-derived Ig gamma chain. In contrast, Ig mu chain was expressed in 7 of 14 AML cases, 3 of 15 monocytes and 1 of 10 neutrophils from non-hematopoietic cancers, and 4 of 8 monocytes and 2 of 8 neutrophils from healthy controls. All of the variable region sequences of AML-derived mu chain from any individual showed monoclonal pattern, but different individuals showed different sequences. Kappa light chain was expressed in 15 of 16 AML cases, 11 of 15 monocytes and 3 of 10 neutrophils from patients with non-hematopoietic cancers, and 1 of 8 monocytes and 1 of 8 neutrophils from healthy controls. Interestingly, the AML-derived Ig kappa transcripts showed unique VKJK rearrangement pattern with several predominant VKJK usages. In particular, the VK1-5/JK1-3 recombination was observed in 93/155 of VKJK sequences from 15 AML cases.

The frequent expression of Ig in AML cells and the preferential usage of certain VKJK rearrangements by AML-derived Ig kappa light chain suggest a role of Ig in AML pathogenesis and a potential target for AML therapy.

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 2745.
Abstract 275: The shedding of epidermal growth factor receptor is mediated by MMP-2 or MMP-9 and stimulated by fibronectin in MDA-MB-468 cells
Authors: Vazquez-Quinones Luis E. , Valle Blanca L. , Cabrera-Toro Krizia L. , Torres-Rodríguez Sheila , Ozuna-Vázquez Hazel , Pérez-Torres Marianela , Cora Elsa M.
Published: February 2011

Activation of the epidermal growth factor receptor (EGFR) is essential for cell proliferation and tissue homeostasis. Alterations in the ERBB signaling pathway have been detected in several human cancers. Soluble isoforms of EGFR generated by proteolytic cleavage of the membrane bound receptor have been identified in several cell lines. The shedding of EGFR in MDA-MB-468 cells generates a fragment of approximately 110 kDa that comprises the extracellular domain plus four amino acids of the transmembrane domain of the full-length receptor. The shedding of EGFR in MDA-MB-468 is activated by two ligands of the receptor (EGF, TGF-alpha), fetal bovine serum, an inhibitor of phosphatases (pervanadate) and by an activator of metalloproteases (i.e. APMA). However, the proteolytic activity that cleaves EGFR is unknown. Studies using metalloprotease inhibitors have suggested that matrix metalloproteases (MMPs) are involved in the shedding of EGFR. Here, we show that the shedding of EGFR increased in MDA-MB-468 cells treated with activated MMP-2 and MMP-9. Similarly, we observed cleavage of the receptor when EGFR (Sigma) or membrane preparations from MDA-MB-468 cells were incubated in vitro with activated MMP-2 or MMP-9. This cleavage was blocked when antibodies against MMP-2 or MMP-9 were included in the incubation mixture. Since fetal bovine serum increases the shedding of EGFR in MDA-MB-468 cells, we evaluated whether other serum components in addition to EGF and TGF-alpha have similar effect. Our results showed that the shedding of EGFR increased when fibronectin (Sigma) was added to MDA-MB-468 cells growing in serum free culture medium. This increase in the shedding of EGFR by fibronectin was dose and time dependent and was abolished when antibodies against α1β5 integrin was added to the incubation medium. Together, these results strongly suggest that the shedding of EGFR is mediated by MMP-2 and MMP-9 and therefore, it is of physiological relevance.

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 275.
Abstract 2750: ARQ 621, a novel potent and selective inhibitor of Eg5: Preclinical data and early results from a clinical phase 1 study
Authors: Rosen Lee , Chen Lin-Chi , Iyengar Tara , Goldman Jonathan , Lahr Sandra , Chen Chang-rung , France Dennis S. , Chan Thomas C.K. , Schwartz Brian , Abbadessa Giovanni , Von Hoff Daniel D.
Published: February 2011

Background: ARQ 621 is an allosteric, potent and selective inhibitor of Eg5, a microtubule-based ATPase motor protein involved in cell division. Eg5 inhibition is recognized as a potential therapeutic strategy in cancer supported by the observation that over-expression of Eg5 causes genomic instability and tumor formation in mice. In this clinical trial we sought to evaluate the safety and pharmacokinetics of ARQ 621 in patients with solid tumors.

Methods: Patients (pts) are being enrolled into this multi-cohort phase 1 trial at the initial i.v. dose of 10 mg/m2/week. Drug is administered weekly intravenously over 1-2 hours. Cohorts of 3 or 6 patients are based on a 3+3 dose escalation schedule and dose is increased according to a modified Fibonacci scheme. Treatment continues until disease progression or unacceptable toxicity.

Results: Recently expanded preclinical data with ARQ 621 shows anti-tumor activity with potencies in the low nanomolar range across a much wider range of human cancer cell types than previously reported, including colon, NSCLC, gastric, and hematologic cancer cell lines. Furthermore, no evidence of bone marrow toxicity and DNA damage is evident with ARQ 621 as we have seen with a leading Eg5 inhibitor. As of November 19, 2009, 18 pts (60% male; median age 60 yrs, ECOG PS 0 (N=3), PS 1 (N=13), PS 2 (N=2) were enrolled. The most common tumors treated with ARQ 621 were colorectal (N=6) and breast (N=2). Treatment-emergent adverse events (TEAEs) were reported in 13 pts. The most common (> 10%) include fatigue (27%), redness at infusion site 3 (16%), and anemia 3 (16%). Three pts experienced serious AEs (1 each of anemia, sepsis, pneumonia) reported as not drug related. Three pts (cholangiosarcoma, liposarcoma and colorectal carcinoma) remained stable for > 4 months.

Conclusions: Preclinical studies continue to elucidate the potential indications for Eg5 inhibition as well as the combinability of ARQ 621 with other molecularly targeted therapeutics. To date, therapy with ARQ 621 appears well tolerated, with no dose-limiting toxicity observed at doses and frequencies much higher than those achieved with the leading Eg5 inhibitors of comparable potencies. Dose-limiting toxicity has not been reached, and recruitment continues to identify the maximum tolerated dose and a recommended Phase 2 dose. Additional safety, pharmacokinetic, and preliminary efficacy data will be available for presentation.

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 2750.
Abstract 2753: Change in tumor size by RECIST correlates linearly with progression-free and overall survival in phase I studies
Authors: Jain Rajul K. , Lee J. Jack , Hong David , Gong Jing , Naing Aung , Wheler Jennifer , Kurzrock Razelle
Published: February 2011

INTRODUCTION: The Response Evaluation Criteria in Solid Tumors (RECIST) sets cutoff values for complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). The efficacy of novel anticancer therapies is traditionally described as the percentage of patients with responses in each of these 4 categories. However, the boundary values defining these categories are arbitrarily set. We therefore study the relationship between change in tumor size by RECIST and both progression-free and overall survival (PFS and OS), and evaluate if boundaries exist or if alternatively the relationship is a continuous parameter.

METHODS: We analyzed 468 participants on 24 phase I trials at MD Anderson from 10-1-04 to 6-30-08. Best response (best change by RECIST while on study) is correlated with PFS and OS by Somers' rank correlation. Kaplan-Meier survival curves of patients binned by response are also calculated (Table). Finally, a Cox model with no covariates is fit to the survival data, and analysis of best response versus the model residual is conducted.

RESULTS: Rank order testing demonstrated a correlation between best response and survival (Dxy = −0.56 for PFS and −0.34 for OS). Kaplan-Meier analysis also demonstrated a strong correlation (p < 1×10−6, Table). Residual analysis of the Cox model (Methods) demonstrated a near linear relationship between changes in tumor size from −100% to +25% and survival; patients with a change of +25% or greater did uniformly poorly. Landmark analyses at multiple time points demonstrated identical trends regardless of the landmark.

CONCLUSION: Given the linear relationship between best response to treatment and both PFS and OS, evaluating specific patient responses, for example through mechanisms such as waterfall plots, may be more valuable than aggregate CR, PR, SD, and PD rates. Additionally, the cutoffs for CR, PR, SD, and PD as defined by RECIST may not have special significance regarding survival in this population.

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 2753.
Abstract 2755: Effect of food on pharmacokinetics of zibotentan (ZD4054) in healthy subjects aged >65 years and ≤65 years
Authors: Tomkinson Helen , Kemp John , Wollseifen Thomas , Oliver Stuart , Morris Thomas
Published: February 2011

The pharmacokinetic (PK) profile of zibotentan (ZD4054), a small-molecule, specific endothelin A (ETA) receptor antagonist, was evaluated under fed and fasting conditions in two Phase I studies in healthy male subjects.

These two studies were open-label crossover studies in which half the subjects received a single zibotentan dose following a high fat meal and again after a 7-day washout in a fasted state. The other half received a single zibotentan dose in a fasted state initially and again 7 days later following a high fat meal. Study 1 (AstraZeneca study 4045IL/013) included nine subjects aged >65 years who received single-dose zibotentan 25 mg and study 2 (AstraZeneca study D4320C00028) included 30 male subjects aged ≤65 years who received single-dose zibotentan 10 mg. AUC and Cmax were log-transformed prior to analysis using an analysis of variance model fitting for the effects of sequence, subject, period and treatment.

In subjects aged >65 years who received zibotentan 25 mg, there was no difference in AUC values between the fed (GLS mean 15041 ng.h/ml) and fasted groups (GLS mean 14740 ng.h/ml; treatment ratio of fed/fasted [90% CI], 1.02 [0.8-1.30]). However Cmax was lower for fed (GLS mean 1005 ng/ml) than fasted subjects (GLS mean 1262 ng/ml; treatment ratio of fed/fasted [90% CI], 0.8 [0.58-1.08]). In subjects aged ≤65 years who received zibotentan 10 mg, there was a statistically significant decrease in AUC for fed subjects (GLS mean 4031 ng.h/ml) compared with fasted subjects (GLS mean 4711 ng.h/ml; treatment ratio of fed/fasted [90% CI], 0.86 [0.80-0.91]), and Cmax for fed subjects (GLS mean 357 ng/ml) compared with fasted subjects (GLS mean 470 ng/ml; treatment ratio of fed/fasted [90% CI], 0.76 [0.71-0.81]). In both studies, median tmax was 4 hours in the fed group versus 2 hours in the fasted group.

In study 2, a food effect on AUC was excluded because the corresponding 90% CIs were within the predefined limits of 0.8-1.25. However, a food effect could not be excluded for Cmax, because the lower limit of the 90% CI was below the predefined margin. The reduction in Cmax under fed conditions was consistent with a slower rate of absorption indicated by median tmax being delayed by 2 hours under fed conditions. The reduction in Cmax is not considered to be clinically relevant as efficacy is likely to be driven by AUC or time above a particular concentration, therefore future studies are being conducted without any food restrictions.

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 2755.
Abstract 2757: NT5C2 single nucleotide polymorphisms affects survival and response in de novo AML patients with normal karyotype
Authors: Fyrberg Anna , Lofti Kourosh , Paul Esbjorn , Paul Christer , Nahi Hareth , Gréen Henrik
Published: February 2011

The nucleoside analog cytosine arabinoside (Cytosar®, AraC) is one of the corner stones in the treatment of acute myeloid leukemia (AML). Activated AraC is metabolized to inactive compounds by several intracellular enzymes. The levels of some of these enzymes have been correlated to effect of AraC and clinical outcome in patients with AML.

This study was designed to evaluate if single nucleotide polymorphisms (SNPs) in genes coding for enzymes involved in the inactivation of AraC affects the therapeutic response in AML-patients treated with AraC in combination with anthracyclines. Three SNPs were investigated in 96 de novo AML patients with normal karyotype and correlated to response and overall survival. Two SNPs were located in the cytidine deaminase gene (CDA, A79C, rs2072671 and C-451T, rs532545), deaminating and inactivating AraC to its corresponding uridine derivative, AraU. The third SNP (A7G, rs10883841) was in the NT5C2 gene coding for the cytosolic 5′-nucleotidase II (cN-II), responsible for dephosphorylation and hence inactivation of AraC monophosphate. All SNPs were in Hardy-Weinberg equilibrium and the allele frequencies were 34.4, 34.4 and 18.8% for CDA A79C and C-451T, and NT5C2 A7G, respectively. Patients that were wild type (A/A) for NT5C2 A7G were found to have a borderline significant higher response rate (51 complete responders of 60 patients with A/A vs 20 complete responders of 30 with A/G or G/G, p=0.06) and longer survival (median survival 1.66 years compared to 0.87 years, log-rank p=0.08) compared to the rest of the patients. The two CDA SNPs (A79C, C-451T) were found to be in linkage disequilibrium. Although statistically not significant, patients that were found to be homozygous for the alternate genetic variant of the CDA SNP had a shorter survival compared to patients carrying the wild type or heterozygous genetic variants.

In conclusion, our findings suggest that certain NT5C2 (cN-II) genotypes might affect the survival after chemotherapy and may provide useful information for treatment strategies, classification and individualized chemotherapy, and eventually used as prognostic marker.

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 2757.
Abstract 2760: Phase I study of MK-0646, the humanized monoclonal antibody against IGF-1R in combination with gemcitabine or gemcitabine + erlotinib (E) for advanced, previously untreated pancreatic cancer
Authors: Shroff Rachna T. , Varadhachary Gauri R. , Bhosale Priya , Ukegbu Lerma , Overman Michael J. , Wolff Robert A. , Abbruzzese James L. , Javle Milind M.
Published: February 2011

BACKGROUND: MK-0646 binds to IGF-1R and blocks its interaction with the IGF-I/ II ligands, enhances gemcitabine (G)-induced apoptosis and inhibits the MEK/ Erk and the PI3-kinase/ Akt signaling pathways which are important in cellular proliferation, survival and drug resistance in pancreatic cancer. Receptor cross-talk between IGF-1R and EGFR and enhanced IGF-1R-induced activation of the PI3-kinase/ Akt pathways mediate resistance to anti-EGFR agents such as E. The combination of IGF-1R and EGFR antagonists has demonstrated synergy in preclinical pancreatic cancer models.

METHODS: Patients with stage IV, previously untreated pancreatic cancer, ECOG performance status (PS) 0-1, adequate hematologic and organ function were enrolled. MK-0646 was escalated in two dose levels using 3+3 statistical design. Uncontrolled hyperglycemia or cardio-respiratory conditions were exclusions. Blood was collected at study entry for IGF-1 and IGFBP-3 levels. Arm A: G 1000 mg/m2 over 100 min, weekly x 3, MK-0646 over 60 min: 5 or 10 mg/kg, weekly x 4; Arm B: G 1000 mg/m2 over 100 min, weekly x 3, MK-0646 over 60 min: 5 or 10 mg/kg, weekly x 4 + E 100 mg daily. Cycles were repeated q 4 weeks. Radiologic responses were measured with RECIST (study radiologist PB). Study endpoints: maximal tolerated dose, progression-free survival, response rate, toxicity and overall survival.

RESULTS: 22 pts enrolled, 15 males, 2 prior Whipple, 20 PS 1, 2 PS 0. Median of 2 cycles (range 1-8) administered as of 9/09. Hematologic toxicity was common: grade 3 or 4 neutropenia (n=9) or thrombocytopenia (n=6). Other grade 3 toxicities: hyperglycemia (n=2), fatigue (n=2), ALT (n=3). Dose limiting toxicity reached in Arm B at dose level 2 (MK-0646 at 10 mg/kg + E + G): febrile neutropenia (n=1), hepatic transaminitis (n=1). Radiologic responses: 5 PR (including 1 hepatic CR), 7 PD, 8 SD, 1 not evaluated, 1 not treated. TTP range (based on 12 pts) was 4 to >36 weeks. Ranges for: IGF-1 levels, 28-280 ng/mL; IGFBP-3 levels, 900-4000 ng/mL. The range of IGF-1 to IGFBP-3 ratios for patients with PR and SD was .027-.036 versus a range of .034-.058 for patients with PD.

CONCLUSION: MK-0646 is tolerable in a dose of 10 mg/kg with G 1000 mg/m2 and at 5 mg/kg with G 1000 mg/m2 and E 100 mg. Promising anti-tumor activity was noted. A randomized phase II study will be conducted with the control arm of G+E.

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 2760.
Abstract 2779: Relationship of mammographic density with breast cancer subtypes
Authors: Gierach Gretchen L. , Lissowska Jolanta , Garcia-Closas Montserrat , Yang Xiaohong R. , Figueroa Jonine D. , Anzick Sarah , Wesolowska Ewa , Brinton Louise A. , Meltzer Paul S. , Boyd Norman F. , Sherman Mark E.
Published: February 2011

Background: Mammographic density (MD), a measure of the amount of radiologically dense breast tissue, is one of the strongest breast cancer risk factors. Studies have found that high MD increases risk for both estrogen receptor (ER)-negative and positive breast cancers, but data relating MD to breast cancer subtypes are limited.

Methods: We used data from the population-based Polish Breast Cancer Study to explore relationships between MD and breast cancer characteristics. This analysis includes 227 invasive breast cancers, which were available as frozen samples and used for mRNA and cytogenetic profiling, and fixed tissues, which were prepared as tissue microarrays for immunohistochemical analysis. Pre-treatment mammograms of the unaffected breast were retrieved for 184 (81%) cases ages 28-75 years. Craniocaudal views of digitized films were used to assess percent MD with Cumulus, a computer-assisted thresholding method. Analysis of variance (ANOVA) models were used to test the null hypothesis of no mean difference in MD between the breast cancer subtypes.

Results: Tumor subtypes were classified by key immunohistochemical markers as luminal A (ER+ and/or progesterone receptor (PR)+, human epidermal growth factor receptor-2 (HER2)-; n=129), luminal B (ER+ and/or PR+, HER2+; n=6), HER2-expressing (ER-, PR-, HER2+; n=13), basal-like (ER-, PR-, HER2-, cytokeratin 5+, and/or HER1+; n=23), and unclassified (negative for all five markers; n=8). Five cases did not have interpretable staining for all five markers and were excluded. MD values were approximately normally distributed. Preliminary ANOVA models revealed no differences in mean percent MD by tumor subtypes (p=0.29) as follows: luminal A, 27.5% (95% confidence interval (CI): 24.9-30.2); luminal B, 27.0% (95% CI: 14.8-39.2); HER2-expressing, 27.2% (95% CI: 18.9-35.5); basal-like, 24.6% (95% CI: 18.4-30.8); and unclassified, 15.7% (95% CI: 5.2-26.3). In analysis of covariance models adjusted for age there was marginal evidence of elevated MD with the HER2-expressing subtype (p=0.04), but we cannot exclude the possibility that this is a chance finding.

Conclusions: Our results do not provide strong support for an association between percent MD and breast cancer subtypes. To identify additional clues as to the biology underlying the MD-breast cancer relationship, ongoing analyses will relate MD to mRNA and cytogenetic profiles and data for protein expression via immunohistochemistry available for additional markers as previously reported.

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 2779.
Abstract 278: Regulation of constitutive Met phosphorylation in breast cancer by EGFR
Authors: Mueller Kelly L. , Haddad Ramsi , Yang ZengQuan , Ethier Stephen P. , Boerner Julie L.
Published: February 2011

Breast cancer remains a major health problem, despite advances in detection, diagnosis, and treatment. Four genomic subtypes of breast cancer have been identified: luminal A and B, HER2 positive, and basal type breast cancers. Three of these subtypes help to define the proper targeted therapeutic as breast cancers of luminal subtypes tend to be estrogen receptor positive and treatable with tamoxifen while breast cancers of HER2 positive subtype are treatable with trastuzumab. The fourth subtype, the basal type breast cancers, has a poor prognosis and has yet to have a molecular target identified. One molecule highly expressed in basal type breast cancers is epidermal growth factor receptor (EGFR). However, targeting EGFR kinase activity with small molecule kinase inhibitors has yet to show efficacy in the treatment of breast cancers. Recently, a panel of EGFR expressing breast cancer cell lines was characterized as sensitive or resistant to EGFR kinase inhibitors. One mechanism of resistance to EGFR kinase inhibitors was found to be the constitutive activation of Met, another molecule associated with basal type breast cancers. Here, the mechanism of constitutive Met activation in EGFR tyrosine kinase inhibitor resistant breast cancer cells is explored. Previously, constitutive Met activation was found to associate with acquired EGFR inhibitor resistance in lung cancer via genetic amplification of Met. In the EGFR TKI resistant breast cancer cells with constitutive Met phosphorylation, Met was not amplified or significantly overexpressed. In addition, constitutive Met phosphorylation was found to occur independent of ligand (hepatocyte growth factor (HGF)) stimulation, as HGF was not expressed in the breast cancer cell lines and inhibiting HGF association with Met via an inhibitory antibody failed to reduce Met phosphorylation. These data suggest a ligand-independent mechanism for Met constitutive phosphorylation. Interestingly, ligand-independent Met activation can be mediated by Met association with other membrane molecules including integrins, tetraspanins and receptor tyrosine kinases, including EGFR. Co-immunoprecipitation studies demonstrated that EGFR and Met co-associate and that this association occurs independent of EGFR or Met kinase activities. In addition, Met phosphorylation was reduced with EGFR TKIs as well as with downregulation of EGFR expression. Taken together, these data suggest that one potential mechanism of EGFR TKI resistance in breast cancer cells is the association and transphosphorylation of Met by EGFR.

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 278.