bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3023: Rapid methylation profiling of multiple genes at the same time using the methylation ligation-dependent macroarray (MLM)
Authors: Benhattar Jean , Guilleret Isabelle
Published: June 2012

Background: Aberrant methylation of multiple promoter CpG islands is a common event in human cancers. Methylation profile of cancer-related genes has extraordinary potential in basic research as well as translational medicine. The aim of this study was to replace the tedious and inefficient bisulfite-based methods with a simple technology which could permit a rapid and robust analysis of large numbers of genes in large numbers of tumor samples simultaneously.

Materials and methods: Genomic DNA was first sonicated, then couples of primers for each specific gene promoter were annealed to this DNA. In a one-step reaction, the CfoI methylation-sensitive enzyme digested unmethylated DNA, whereas the Taq ligase joined primers annealed to the methylated sequence. Tagged ligation products were amplified by PCR and finally hybridized to a nylon membrane macroarray. Methylation Ligation-dependent Macroarray (MLM) allows the establishment of DNA methylation profiles on many samples (up to 21) and on many gene promoters (up to 45) in a simple reaction in 2-3 days. To validate this novel method, we used it to detect aberrant methylation in 14 cell lines and in DNA samples of 40 patients with a colorectal cancer.

Results: In tumor cell lines, among the 42 genes investigated 23.7 (11-32) were found methylated whereas only 9 have been observed methylated in a normal fibroblast cell line (BJ). In normal colorectal tissues, about 12/40 gene promoters were found methylated; in colorectal cancers 16 to 36 genes were methylated. Methylated gene promoters identified in colorectal cancers were ADAMTS18 (78%), APC (28%), CDKN2A (20%), DKK2 (50%), GPx3 (48%), Mal (72%), MLH1 (30%), NELL1 (72%), SFRP4 (35%), SFRP5 (52%) and WRN (20%):

Conclusion: The MLM is a fast, non expensive and reliable technology that profiles the DNA methylation status of >40 promoter CpG islands in various human samples. This technology can be used in research for rapid methylation profiling of numerous samples and for identification of new epigenetic biomarkers, but also in clinic for rapid cancer diagnosis, for predictive response to cancer therapy and for distinction between progressive and non-progressive diseases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2011-3023
Abstract 3041: Focal amplification of chromosome 9p13 is associated with progression risk and activation of multiple oncogenes in oral precancers
Authors: Towle Rebecca M. , Tsui Ivy , Garnis Cathie
Published: June 2012

Oral squamous cell carcinoma, the most common form of head and neck cancer, has a dismal five year survival rate of ∼50%. This is largely due to the late stage of diagnosis and high rates of recurrence. A better understanding of the mechanisms driving early oral tumorigenesis is an essential first step towards increasing survival rates, which have not improved in decades. Specifically, by characterizing genetic alterations in early oral premalignant lesions (OPLs), we will better understand mechanisms of disease progression and discover targets for timely therapeutic intervention that could ultimately improve oral cancer outcomes.

Methods: Whole genome DNA copy number profiling was undertaken on a rare panel of OPLs with extensive longitudinal data (including known progression status for invasive disease). Recurring alterations were characterized and gene lists were refined using independent tumor-associated gene expression datasets. The functional significance of remaining gene candidates was then assessed using lentiviral shRNA and expression vectors in cell model systems.

Results: Analysis of genomic data from OPLs identified four focal recurrent alterations. Three of these alterations were associated with previously identified oncogenes: cMYC, EGFR, and KRAS. The fourth was a novel 2.04 Mbp amplification at chromosome 9p13. Based on our results and publicly available gene expression data, four candidate genes were found in this region: VCP, DCTN3, STOML2, and TLN1. Independent manipulation of each of these genes in cell models revealed changes in oncogenicity (based on changes in rate of proliferation and the degree of anchorage independence), suggesting a critical role in malignant processes associated with cancer progression.

Conclusion: We have shown that a recurring, focal 9p13 amplicon in OPLs is associated with progression to invasive oral cancer. Further, our data indicate that this region spans multiple genes with oncogenic potential. Taken together, our results suggest that a single amplification event may dysregulate multiple genes that drive progression to invasive oral cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3041. doi:10.1158/1538-7445.AM2011-3041
Abstract 3068: Detection of K-RAS mutations in Korean patients using PNAClampTM KRAS
Authors: Cho Minhye , Choi Jae-Jin , Oh Miae , Park Heekyung
Published: June 2012

Mutation of the K-RAS gene located in codons 12 and 13 may play an important role in prognosis of related diseases and responses to certain treatments targeting the pathway. Recent studies suggest that in colon cancer patients, K-RAS mutation status is a strong predictor of success in therapeutics of several tyrosine kinase inhibitors. Therefore, it is important to detect K-RAS mutations fast and accurately.

To detect a minority of mutant K-RAS among abundant wild-type alleles, we have developed a highly sensitive and simple method for the detection of K-RAS mutations using PNA-mediated real-time PCR clamping, PNAClampTM. PNAClampTM relies on the following two unique properties of PNA probes: 1) PNA-DNA duplexes generally have greater thermal stability than the corresponding DNA-DNA duplex; 2) PNA oligomers are not recognized by DNA polymerases and consequently can serve as a sequence-selective clamp during PCR amplification. This method is fast and accurate with a detection limit of approximately 0.1% mutant alleles using 10 ng to 25 ng of normal DNA as a template. The turnaround time for performance of this assay is only 2 hrs. The PNAClampTM method facilitates the detection of mutations in more than 1000-fold excess of wild-type DNA samples.

K-RAS mutations were detected in 28.3% (36/127) of patients. In detail, 29 cases had mutations in codon 12 and 7 cases had mutations in codon 13. To confirm those results, the same clinical samples were analyzed with a direct sequencing assay. Concordant results were obtained from 124 (97.6%) of the 127 clinical samples in the two assays. The discrepancy was observed in three cases. For comparison, the three clinical samples were also analyzed using DxS K-RAS Mutation Kit (DxS Ltd., UK) to give the same outcome. In this study, we will discuss development of PNAClampTM KRAS and its optimization for higher sensitivity. The PNAClampTM KRAS may serve a rapid and reliable alternative for K-RAS mutation detection in the clinical field.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3068. doi:10.1158/1538-7445.AM2011-3068
Abstract 3081: Tristetraprolin Controls let-7 regulator-Lin28 mRNA stability
Authors: Kim Chae Won , Vo Mai-Tram , Kim Hong Kyeung , Kim Hyo Jeong , Lee Hyun Hee , Yun Nal Ae , Lee Sun Young , Min Young Joo , Park Jeong Woo , Cho Wha Ja
Published: June 2012

The RNA binding protein tristetaprolin (TTP) regulates expression of many cancer-associated genes through binding to AU-rich elements (AREs) in the 3’ untranslated region (3’UTR) and facilitating rapid mRNA decay. In this study, we investigated the role of TTP in the control of micro RNA let-7 expression through regulation of Lin28 expression. TTP overexpression significantly suppressed the growth of PA1 human ovarian cancer cell line through down-regulation of CDC34 expression. However, CDC34 does not have the ARE motif within its 3’UTR, suggesting that TTP can not directly regulate CDC34 expression. Instead, CDC34 expression was regulated by let-7. Interestingly, we found that TTP regulates the expression of Lin28 which has been reported to be involved in maturation of let-7. Lin28 mRNA contains two AREs (ARE1 and ARE2) within the 3’UTR, and TTP destabilized a luciferase mRNA containing Lin28 ARE1 and directly bound to the ARE1 of Lin28 mRNA. These studies established Lin28 mRNA as a physiological target of TTP and demonstrate that TTP can control the expression levels of microRNA let-7 through regulation of Lin28 mRNA stability.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3081. doi:10.1158/1538-7445.AM2011-3081
Abstract 3085: The IFN inducible p53 target gene TRIM22 binds to the translation initation factor eIF4E and represses translation through disturbing the assembly of the eIF4F complex
Authors: Petersson Jessica , Ageberg Malin , Olofsson Tor , Gullberg Urban , Drott Kristina
Published: June 2012

TRIM22 (Staf50) is an interferon (IFN)-inducible protein as well as a p53 target gene. It belongs to the TRIM (TRIpartite Motif) family proteins, characterized of a conserved tripartite motif consisting of a RING finger, B-box and a coiled-coil domain. The TRIM family includes more than 70 proteins, involved in various biological processes; e.g. apoptosis and cell proliferation. Consistently, several TRIM family members are involved in cancer. TRIM22 has been suggested to be an antiviral protein, capable of restricting HIV-, hepatitis B- and encephalomyocarditis replication. The molecular mechanisms for TRIM22 are unclear, but the antiviral activity may be dependent on the ubiquitylation capacity of TRIM22. We have previously shown it to suppress the clonogenic growth of leukemic U-937 cells, suggesting anti-proliferative or cell death-inducing effects. Now, we demonstrate that TRIM22 binds to the translation initiation factor eIF4E, and inhibits the binding of eIF4E to eIF4G, thus disturbing the assembly of the eIF4F complex, vital for cap dependent translation of mRNA. As a consequence, TRIM22 represses translation, as shown by decreased reporter protein levels, as well as global protein synthesis. Pulse-chase and cycloheximide experiments reveal that the repressive effects on the reporter protein is not due to increased protein degradation. Taken together, our data show TRIM22 to repress the binding of eIF4E to eIF4G, suggesting a novel translation regulating mechanism, possibly mediated in response to p53 and/or IFN signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3085. doi:10.1158/1538-7445.AM2011-3085
Abstract 3090: Inhibition of p53 DNA binding function by the MDM2 acidic domain
Authors: Cross Brittany L. , Cheng Qain , Li Baozong , Yuan Zhi-Min , Chen Jiandong
Published: June 2012

MDM2 is an important regulator of p53, acting in part by promoting p53 ubiquitination. However, MDM2 has also been implicated in regulation of p53 through ubiquitination-independent mechanisms. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding, possibly by interacting with the p53 DNA binding domain. ARF binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild type conformation and allows p53-MDM2-SUV39H1 complex to bind DNA. These results provide further evidence for an ubiquitination-independent mechanism of p53 regulation by MDM2, and reveal how MDM2-interacting repressors gain access to p53 target promoters and repress transcription.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3090. doi:10.1158/1538-7445.AM2011-3090
Abstract 3092: FBW7 targets hypoxia inducible factor-1α (HIF-1α) for proteasomal degradation during hypoxia
Authors: Cassavaugh Jessica , Hale Sarah , Wong Cheung , Lounsbury Karen M.
Published: June 2012

Hypoxia Inducible Factor-1α (HIF-1α) mediates expression of genes associated with endothelial cell-mediated angiogenesis and is associated with poor outcomes in a variety of cancers. In normoxia, HIF-1α is ubiquitinated and degraded through interactions with the E3 ubiqutin ligase, von Hippel-Lindau (vHL); however, little is known about the negative regulation of HIF-1α in hypoxia. FBW7, an E3 ubiquitin ligase, has been shown to interact with several transcription factors including those phosphorylated by glycogen synthase kinase 3β (GSK3β). The current study tested the hypothesis that phosphorylation of HIF-1α by GSK3β increases the FBW7-mediated ubiquitination and degradation of HIF-1α, thereby resulting in suppression of the hypoxia-mediated angiogenic response in SKOV-3 ovarian cancer cells. HIF-1α protein and VEGF transcript levels in hypoxia were increased when GSK3β activity was inhibited and were reduced by expression of constitutively active GSK3β (GSK3S9A). Additionally, expression of GSK3S9A increased HIF-1α ubiquitination. Conditioned media from SKOV-3 cells expressing shRNA against GSK3β (shGSK3) had an enhanced effect on endothelial tube formation in a Matrigel matrix, suggesting that GSK3β exerts an inhibitory effect on hypoxia-mediated angiogenesis. Overexpression of the β and γ isoforms of FBW7 decreased HIF-1α stabilization, and HIF-1α interacts with FBW7 by co-immunoprecipitation. Furthermore, knockdown of FBW7 using siRNA resulted in increased HIF-1α levels in hypoxia. These data suggest a new mechanism for negative regulation of HIF-1α during hypoxia that utilizes phosphorylation by GSK3β and interaction with FBW7 leading to ubiquitination and proteasomal degradation. Results of this study better define the signaling pathways necessary for HIF-1α-mediated signaling and may identify new targets that mediate angiogenesis in disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3092. doi:10.1158/1538-7445.AM2011-3092
Abstract 3107: Semaphorin 4D – plexin B1 signaling in pancreatic ductal adenocarcinoma reflects tumor aggressiveness
Authors: Shingo Kato , Kubota Kensuke , Shimamura Takeshi , Kobayashi Noritoshi , Watanabe Seitaro , Shinohara Yoshiyasu , Ishiguro Hitoshi , Nakaigawa Noboru , Kubota Yoshinobu , Maeda Shin , Nakajima Atsushi
Published: June 2012

Purpose: Semaphorins are a large family of proteins that provide axonal guidance in neuronal tissues and regulate cell motility in many types of cells. In recent studies, Semaphorin 4D (Sema4D) has been demonstrated to enhance the motility and invasiveness of cancer cells through its receptor, plexin B1, in vitro and also to increase the metastatic potential of breast cancer in vivo. However, the expression patterns and functions of Sema4D in pancreatic cancer remain unknown.

Experimental Design: We examined the expressions of Sema4D and plexin B1 in surgically resected specimens of pancreatic ductal adenocarcinoma by immunohistochemistry, and also the effect of Sema4D stimulation on the invasiveness of pancreatic cancer cells.

Results: Immunohistochemical staining revealed that Sema4D was expressed on the T cells and B cells in the cancer stroma. Plexin B1 was expressed in the cancer cells, whereas its expression was only faintly detected in the normal pancreatic ductal cells. Tumors with higher expression levels of Sema4D showed a significantly higher tendency towards lymph node metastasis (p = 0.0068), distant metastasis (p = 0.036), extrapancreatic nerve plexus invasion (p = 0.0073), and a poor prognosis (p = 0.024). Increased expression of both Sema4D and plexin B1 was associated with an additive worsening of the prognosis, independent of the tumor-node-metastasis classification and the World Health Organization tumor grade (p = 0.045). Stimulation with Sema4D caused a significant increase in the invasiveness of pancreatic cancer cells, and the knockdown of plexin B1 by siRNA could block the enhanced invasive activity by Sema4D.

Conclusion: Sema4D could play a role in the interactions between the tumor cells and the tumor microenvironment in pancreatic cancer. In the context of numerous attempts currently ongoing to target the tumor stroma, our findings provide insight into new molecular targets for the development of treatments for pancreatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3107. doi:10.1158/1538-7445.AM2011-3107
Abstract 3124: Tetraspanin CD151-mediated signaling network between cell-matrix and cell-cell adhesions
Authors: Hong In-Kee , Lee Hansoo , Byun Hee-Jung , Park Iha , Kim Young-Myeong
Published: June 2012

CD151 has been suggested to promote cancer invasion and metastasis by regulating adhesion-dependent signaling events through its association with laminin-binding α3β1/α6β1 integrins. Previously, we have demonstrated that homophilic interactions of CD151 stimulate integrin-dependent signaling to c-Jun through the FAK-Src-MAPKs pathways in human melanoma cells. In this study, we found that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the membrane region, leading to formation of α3β1/α6β1 integrins-CD151-small GTPases complexes. Adhesion receptor complexes-associated small GTPases were found to be activated by β1 integrins-CD151 complexes-stimulating adhesion events such as interactions of integrins with laminin and homophilic interactions of CD151 on two contacting cells, implicating CD151-mediated signaling network between cell-matrix and cell-cell adhesions. Adhesion-dependent activation of small GTPases appeared to be mediated by the FAK-Src pathways. Meanwhile, EGF and phorbol ester treatment further enhanced CD151-β1 integrin complexes-induced cell motility and MMP-9 expression, along with activation of p38 MAPK and JNK, indicating a positive signaling cross-talk between CD151-β1 integrin adhesion receptor, EGF receptor, and PKC. Using siRNAs and dominant-inhibitory mutant expression constructs for various intracellular signaling mediators, we found that integrin-linked kinase (ILK), PAK1-interacting exchange factor beta (β-PIX), and p21-activated protein kinase 1 (PAK1) were involved in CD151-β1 integrin adhesion receptor complex signaling pathways. Also, MEK3/6 and MEK7 were found to participate in CD151-β1 integrin signaling cascades, upstream of p38 MAPK and JNK, respectively. Taken together, our data strongly suggest that β1 integrin-CD151 adhesion receptor complexes activate c-Jun AP-1 factor via the signaling cascades involving the FAK-Src, ILK-β-Pix, small GTPases-PAK1, MEK3/6-p38 MAPK, and MEK7-JNK pathways, leading to increased cell motility and MMP-9 expression in human melanoma cells.

Key words: tetraspanin, CD151, integrin, adhesion receptor, cell signaling

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3124. doi:10.1158/1538-7445.AM2011-3124
Abstract 3126: Intercellular communication in the urothelium: Evidence for a switch in gap junction regulation during differentiation
Authors: Hinley Jennifer S. , Kirkwood Lisa A. , Southgate Jennifer
Published: June 2012

Cells have a requirement for intercellular communication in order to maintain normal tissue polarity and homeostasis. Knowledge of the signaling molecules transferred through specific gap junctions is limited; however aberrant expression of connexins, the protein subunits of gap junctions, has been linked to many disorders including neoplastic transformation of brain and colorectal tissues. A number of reports have documented changes in connexin expression associated with bladder cancer.

The aims of this study were 1) to define the connexins expressed by the epithelial lining of the human urinary tract (urothelium) in situ and in vitro; 2) to determine if expressed connexins assemble into functional gap junctions and 3) the contribution of specific connexins to urothelial tissue physiology.

Established culture systems for normal human urothelial (NHU) cells were used to replicate proliferative (regenerative) or differentiated cell phenotypes. RT-PCR was used to screen normal urothelial tissue and NHU cell cultures for expression of connexin genes. Immunochemical techniques were employed to determine connexin expression and localization in situ and in vitro. Dye-transfer techniques were used on cultured cells to confirm that assembled gap junctions supported functional cell-cell communication.

Of the 20 human connexin genes, six were expressed in situ and by differentiated cultures, whilst two were expressed only in proliferative culture conditions. NHU cells were capable of dye transfer in both proliferative and differentiated cultures, indicating the presence of functional gap junctions. Immunofluorescence microscopy demonstrated that the switch in medium from a low (0.09mM) to near physiological (2mM) calcium concentration resulted in increased assembly of gap junctions containing connexin-43. Connexin-32 was only expressed by differentiated cultures, as shown by RT-PCR and immunochemistry, where it was localized to intercellular borders indicating a functional role.

This data represents the first systematic characterization of connexin family expression in the urothelium and provides evidence that urothelial cells alter their connexin expression upon differentiation to produce specific channels. Connexin-32 has been reported to be involved in terminal differentiation of human mammary gland and future work will focus on characterizing the role of connexins in defining the differentiated urothelial phenotype.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2011-3126
Abstract 3141: Analysis of the endocrine treatment predictive protein HOXB13 in premenopausal breast cancer
Authors: Jerevall Piiha-Lotta , Jansson Agneta , Källström Ann-Christine , Landberg Göran , Fernö Mårten , Stål Olle
Published: June 2012

Homeobox B13 (HOXB13), part of the two-gene expression index H:I, has proven its capacity as a predictive factor of tamoxifen benefit in breast cancer. We have previously shown that in addition to mRNA expression, the protein shows predictive value in postmenopausal patients. High levels were associated with decreased tamoxifen (Tam) benefit and may indicate endocrine resistance, whereas Tam-treated patients with low expression had a longer recurrence-free survival. Here, we have analyzed HOXB13 protein expression in premenopausal breast cancer patients. We quantified the levels of HOXB13 with immunohistochemistry in tumor samples from 487 patients sampled in duplicates on tissue micro arrays. Patients were diagnosed with stage II invasive breast cancer and randomized to Tam or no endocrine treatment. HOXB13 protein analysis was successful in 367 patients. Data were correlated with clinicopathological variables. Using Kaplan Meier analysis to investigate Tam benefit in patients with tumors expressing estrogen receptor (ER) α, progesterone receptor, or both, patients with high HOXB13 levels unexpectedly benefited from Tam (hazard ratio for recurrences 0.43, 95% CI 0.28-1.01, p=0.053). Corresponding numbers for a lower expression were 0.69 (95% CI 0.44-1.07, p=0.1). For further analysis, we stratified the patients based on tumor ERβ status, which seemed to be an indicator of the performance of HOXB13 as a treatment predictive factor. Interaction between HOXB13 expression and benefit from Tam in the ERβ- group was close to significant (p=0.06), and corresponding p-value for the ERβ+ patients was 0.11. In conclusion, endocrine treatment prediction for premenopausal breast cancer patients using the protein levels of HOXB13 does not seem to be as straight forward as in postmenopausal patients. Based on our results, ERβ may be an additional determinant in predicting benefit from Tam-treatment using HOXB13 protein expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3141. doi:10.1158/1538-7445.AM2011-3141
Abstract 3146: The involvement of Foxp3 or CD34 expression and tumor lymphocyte infiltration in the prognosis of breast carcinoma patients
Authors: Rico María J. , Rozados Viviana R. , Alasino Carlos M. , Roggero Eduardo , Nocito Ana L. , Scharovsky O Graciela
Published: June 2012

Breast cancer is a heterogeneous disease with different known biological subclasses some of which are associated with immune cell infiltration. Studies in mice have shown that thymus-derived CD4+CD25+ regulatory T cells (Treg; FOXP3+ lymphocytes) inhibit the antitumor immune response. The presence of inflammatory cells may represent an immune response to the tumor, but could also potentially exhibit pro-tumor effects including stimulation of tumor invasion or angiogenesis. Angiogenesis has an important role in the progression of breast cancer. Microvessel density has been found to be strongly associated with features of tumor aggression as larger size and poor differentiation. It was observed that different characteristics of tumors like immune cells infiltration, number of Tregs and blood vessels density would be useful tools to anticipate the response to the therapy. The aim of this study was to analyze different cellular characteristics of primary tumors of patients with breast cancer and their relationship to the response to therapy. Samples of breast carcinomas (Stage I and II) (n=14) were analyzed. Foxp3 and CD34 expression was determined immunohistochemically on formalin-fixed, parafin-embedded tumor sections. Also, peritumor and intratumor lymphocytes infiltration was evaluated by hematoxylin-eosin staining. For quantification in 20 high magnification fields, a 0 (nule expression) to 6 (high expression) score was utilized. Confocal microcopy for Foxp3 was also used. Lymphocytes were mainly found in the intratumor (IT) area. Twelve over 14 patients (DF) that had a disease free survival of approximately 5 years showed an IT score for lymphocyte infiltration [median (range): 4 (2-6)] higher than that of 2/14 patients that relapsed (R) [2 (2-2)], p<0.05. For Foxp3 expressing lymphocytes, the IT score was low in DF [2 (0-6)] and R [2 (2-2)] patients. Interestingly, some tumor cells were Foxp3+. Vascular density, evaluated through CD34 expression, was low [DF: 2 (1-4); R: (1-2)]. The relationship between the presence of Tregs cells, and the vascular density in the intratumor area, with prognosis is still controversial. Our present results show that Foxp3+ or CD34 IT expression could not predicts patients prognosis. Disease free patients showed higher intratumor lymphocyte infiltration than relapsed patients, a fact that could reflect the involvement of the host anti-tumor immune response.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3146. doi:10.1158/1538-7445.AM2011-3146
Abstract 3152: Distribution of hormone receptors (estrogen receptor, progesterone receptor and androgen receptor) in epithelial malignancies
Authors: Basu Gargi D. , Kemkes Ariane , Feldman Rebecca A. , Arguello David R. , Wright Alan , Loesch David , Ashfaq Raheela
Published: June 2012

The importance of steroid hormone receptors to the biology of breast cancer was recognized over 40 years ago. New insights into hormone receptor biology and the increasing array of proteins that can modify their function have already translated into better therapies for breast cancer. The responsiveness of a tumor to hormone therapy is an important parameter in cancer management. Besides breast cancer, other cancers also express steroid hormone receptors. Hence, the purpose of this study was to capture the relative distribution of hormone receptors in all types of cancer including breast cancer.

In a total cohort of 9,491 patient samples, hormone receptors ER, PR and AR were assayed by immunohistochemistry on a Ventana platform using antibodies ER (SP 1), PR (I E 2) and AR (AR 27). The slides were read manually by pathologists using the cutoff of (>=1+ and >=10% as positive) for AR, PR and ER. Based on these cutoffs, samples were deemed positive or negative.

Our preliminary observations indicate that the frequency of ER is highest in breast (40.8%), followed by ovary (25.0%), and uterus (14.9%), with marginal frequencies in lung (4.0%) and peritoneum (2.2%). The most common cancer types with negative ER expression are lung (14.7%), colon (13.3%), pancreas (10.5%), breast (9.4%) and skin (4.2%) The cancer distribution of PR mimics that of ER, albeit with a slightly lower prevalence (breast 25.5%, ovary 22.0%, uterus 13.3%, lung 5.6%, and pancreas 3.9%). The most common cancer types in which PR was negatively expressed are lung (14.0%), colon (12.6%), breast (12.2%), pancreas (9.5%) and ovary (7.6%) The frequency of AR, surprisingly, is higher in breast cancers (45.7%) as opposed to prostate cancer (5.8%). Other cancer types with overexpression of AR are ovary (18.5%), uterus (3.4%) and lung (3.0%). In contrast, low expression of AR is most often found in lung (14.3%), colon (12.0%), pancreas (9.7%), breast (9.6%), and skin (3.7%). All distributional differences by HR status were analyzed by Pearson's chi-square (χ2) test and showed statistical significance (p< 0.001).

To our knowledge, this is the first study involving a large patient pool in which hormone receptors have been investigated in a single clinical laboratory with standardized IHC. Cancer is a heterogeneous disease with different molecular drivers regulating its growth, survival and response to therapy. This study shows that hormone receptors are frequently expressed in cancer types outside of breast, which merits the inclusion of therapeutic strategies using hormone therapy in such tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3152. doi:10.1158/1538-7445.AM2011-3152
Abstract 3154: Human plasma, prostate and urine levels of α-, γ-, and Δ-tocopherols and their metabolites before and after supplementation with a γ-tocopherol rich vitamin E mixture
Authors: Goodin Susan , Lee Mao-Jung , Kim Isaak , DiPaola Robert , Yang Chung S.
Published: June 2012

In order to determine the blood/tissue levels of tocopherols and their metabolites, we analyzed samples from 5 prostate cancer patients who underwent prostatectomy by HPLC-ECD. These patients had a mean PSA of 6.37 (range 1.99 to 10.54) prior to surgery and a Gleason Score of 7. The plasma α-tocopherol (α-T) levels, 22.02 ± 3.85 μM (mean ± S.D., n = 10), were in the normal range. As expected, the levels of γ-T were lower and those of Δ-T were even lower. The prostate tocopherol levels were 6-14 times lower than plasma levels. Substantial amounts of carboxyethyl hydroxychromans (CEHC) and carboxymethylbutyl hydroxylchromans (CMBHC) were observed in the urine. These metabolites were also found at low levels in the plasma and prostate. In a pilot study, prostate cancer patients received no supplementation or 2 capsules of γ-TMT (a γ-T-rich dietary supplement, containing 128 mg α-T, 200 mg γ-T and 71 mg Δ-T per capsule) daily for 7 or 14 days before prostatectomy. Blood and urine samples were collected from each subject before γ-TMT supplementation and on the day of surgery. The plasma and prostate tocopherol/metabolite levels, especially those from γ-T and Δ-T, were increased in patients who received γ-TMT supplementation (but not in the patients who did not). The effect on the urine levels of tocopherol metabolites was even more evident; high levels of γ- and Δ- forms of CEHCs and CMBHCs were observed in patients who received γ-TMT supplementation. Tocopherols were not excreted in the urine even with supplementation. These results are consistent with our understanding that γ-T and Δ-T are more extensively metabolized than α-T. The rather high CEHC and CMBHC levels in the urine make them useful biomarkers for tocopherol intake and metabolism in future human studies (supported by NIH grants CA120915 and CA133021).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3154. doi:10.1158/1538-7445.AM2011-3154
Abstract 3162: Assessing the therapeutic and diagnostic potential of human thymidine kinase 1 in leukemia
Authors: Estevez Dagoberto , Whitehurst Robert A. , Sharp Daniel W. , Robison Richard A. , O'Neill Kim L.
Published: June 2012

Thymidine Kinase 1 (TK1), a salvage pathway enzyme responsible for recycling thymine in cells for use in DNA replication and repair, is normally tightly regulated by the cell cycle; however research studies have shown that it is over-expressed and unregulated in cancer cells. Studies linking TK1 localization to the plasma membrane of lymphoma cells have suggested that this enzyme could also be used as a molecular target for cancer diagnosis and therapy. Previous research in our laboratory showed that TK1 located on the plasma membrane of different cancer cell lines had high enzymatic activity. This study was performed to support the eventual possibility of targeting TK1 for cancer diagnosis and immunotherapy in leukemias expressing high levels of TK1 on their plasma membrane. TK1 levels on plasma membrane were analyzed using a BD FACSCanto flow cytometry on lymphoblastoid cells derived from Burkitt's lymphoma (Raji cells), acute T cell leukemia (Jurkat cells), myeloblastic cells derived from promyelocytic leukemia (HL-60 cells), and normal lymphocytes from healthy individuals. Analysis was performed on unpermeabilized cells in exponential growth phase using both a mouse monoclonal anti-TK1 Ab and a rabbit polyclonal anti TK-1 Ab. Cells were incubated with human FcR blocking reagent to eliminate non-specific binding to Fc receptors. Permeabilized cells were excluded using propidium iodide staining. Using an IgG Isotype control we were able to select proper gating (0% binding). TK1 surface expression was compared to expression of sodium potassium ATPase, a common plasma membrane marker, and pan-leukocyte marker CD45. Results confirmed high levels of TK1 expression on cancer cell lines analyzed. Flow cytometry analysis showed that binding of the mouse monoclonal anti-TK1 Ab and rabbit polyclonal anti-TK1 Ab were 93.4% (SEM=1.05, N = 19) and 99.5 % (SEM=0.67, N=19) in Raji cells; 71.1% (SEM=3.41, N=19) and 89.4 % (SEM=2.74, N=19) in HL-60 cells; 63% (SEM=1.23, N=26) and 88.6% (SEM=1.81, N=27) in Jurkat cells. Lymphoblastoid cells showed significantly higher TK1 expression when compared to that of lymphocytes obtained from healthy individuals 9.9% (SEM=2.39, N=7) and 14.9% (SEM=2.43, N=3). When induced with pokeweed mitogen there was no increase in TK1 expression on the plasma membrane of normal dividing lymphocytes 7.9% (SEM=0.54, N=3) and 10.63% (SEM=2.89, N=3). Fluorescence microscopy observations provided more evidence of TK1 association with the plasma membrane of cancer cells. These results suggest that membrane-bound TK1 has potential for both diagnosis and immunotherapy in human leukemia.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3162. doi:10.1158/1538-7445.AM2011-3162
Abstract 3176: Preclinical validation of the sensitivity and specificity of a fluorescent nanobeacon for its application in colonoscopy-based imaging of colorectal cancer
Authors: Pham Wellington , Kumagai Hironori , Hiwatari Ken-Ichiro , Tachikawa Hiroyuki , Kimura Ryoji , Nakamura Kunio , Kataoka Makoto , Yamashita Shinji , Sakuma Shinji
Published: June 2012

Colorectal cancer is the third most commonly diagnosed type of cancer and the second leading cause of cancer-related death in the U.S. Currently, the key element in the control, prevention, and treatment of this disease is recognition of its early symptoms. In this work, we describe the development and validation of a specific fluorescent nanobeacon that targets the Thomsen-Friedenreich (Tf) antigen, potential biomarkers of this disease, via the terminal carbohydrate Gal-β(1-3)GalNAc.

The imaging agent is comprised of a submicron-sized polystyrene nanosphere encapsulated with a hydrophobic fluorescent dye. The surface of the nanosphere was modified with the peanut agglutinin (PNA) and poly(N-vinylacetamide (PNVA) moieties. The former binds to Gal-β(1-3)GalNAc with high affinity, while the latter enhances the specificity of PNA for the carbohydrates. This nanobeacon detects colorectal tumors by recognizing the tumor-specific antigen through the surface-immobilized PNA, and identification is made via the fluorescence signal obtained from the coumarin-6 encapsulated in the nanosphere's core. Biorecognition by the probe has been demonstrated in cell lines and orthotopic mouse models.

Here, we assessed the sensitivity and specificity of the probe in a pre-clinical MUC1 transgenic mouse model. Of note, the probe reported that the Tf antigen expression level is high in the colon and intestine of that model compared to wild- type mice. Data obtained by fluorescence imaging using this probe correlates well with those obtained via immunohistochemistry. Most importantly, the probe was not absorbed systemically upon topical application in the host. As a result, no registered toxicity is associated with the probe.

Using the Tf antigen imaging approach, we are evaluating the binding specificity of the nanobeacon in panel of human colorectal cancer tissues. Thus far, the study has indicated quantitatively a 5-fold enhancement of the Tf expression in colon cancer tissue versus the control tissue. In sum, these data demonstrate the potential for the use of this novel nanobeacon in imaging the Tf antigen as a biomarker for the early detection and prediction of the progression of colorectal cancer. Currently, several probe optimization processes are presently being carried out in our laboratory, including the incorporation of near-infrared fluorescent dyes to enhance the signal-to-noise ratio. Detailed results of the biometric validation of the nanobeacon will be reported in a timely manner.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3176. doi:10.1158/1538-7445.AM2011-3176
Abstract 3180: Mutation detection in circulating free DNA using mutant-enriched PCR and digital PCR
Authors: Huw Ling-Yuh , Spoerke Jill , Patel Rajesh , Liu Weiqun , Raja Rajiv , Amler Lukas , Hampton Garret , Punnoose Elizabeth , Lackner Mark
Published: June 2012

Mutations in oncogenes such as EGFR, KRAS and PIK3CA, have been shown to have profound effects on pathway signaling and tumor growth. These genetic aberrations are potentially valuable predictors for patient stratification and therapeutic responses to targeted therapies directed against these signaling pathways. Here we evaluate the utility of circulating tumor DNA as a “liquid biopsy” for predictive diagnostics and real-time monitoring of disease. A fundamental challenge with this approach is that, due to the low frequency of the mutant tumor DNA in circulation, a highly sensitive assay is required to detect the single base pair alterations. We have designed and investigated several assay formats, including TaqMan and scorpion-ARM assays for mutation detection using a digital PCR platform. We also employed LNA/PNA blockers and thermostable restriction digestion in PCR reaction to enrich the mutant DNA. The combination of the biased amplification and digital PCR dramatically increased mutation detection 10-100 fold. To assess clinical utility of the technology, data on PIK3CA mutation screening of matched patient tumor and plasma samples from a phase I clinical trial will also be reported.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3180. doi:10.1158/1538-7445.AM2011-3180
Abstract 3185: Use of whole slide digital imaging for determination of caPCNA and Ki-67 labeling index in normal breast reductions and DCIS: Comparison of the two biomarkers using the Aperio image-based positive pixel algorithm
Authors: Sandusky George E. , Rodgers Matt S. , John Karla , Gulley Patty , Malkas Linda , Hickey Robert
Published: June 2012

While mitotic rates as well as proliferation indices have been reported as significantly different among normal breast and different grades of breast cancer, a reproducible cut-off value has not emerged. The goal of this study was to determine whether more reproducible algorithms using whole slide digital images (WSDI) can define better cut-off values for the normal breast vs. DCIS.

Design: Whole tissue sections from 10 normal breast reductions and 10 cases of DCIS were evaluated by immunohistochemistry for caPCNA and Ki67 (Mib-1). Having identified an isoform of PCNA that was uniquely expressed by cancer cells (caPCNA) and distinct from the isoform expressed by non-malignant and normal cells (nmPCNA), antibody selectively binding to caPCNA was developed and used for these studies. Stained slides were scanned using the Aperio (Vista, California) automated whole slide scanning system (Scanscope CS) and were viewed using ImageScopeTM. Single fields of view from each WSDI measuring ∼420,000 µ2 and representing the highest density of caPCNA and Ki-67 positive nuclei were selected for analysis. Different areas were analyzed in a subset of slides to determine the variability in each case, and the total nuclear labeling index was generated using the Aperio positive pixel algorithm.

Results: We analyzed the labeling indices in 10 normal breast reductions and 10 DCIS cases. Both caPCNA and Ki-67 were nuclear specific with minimal cytoplasmic staining. The mean labeling indices for the Aperio automated positive pixel counts were caPCNA (0.15) and Ki-67 (0.12) for normal breast and caPCNA (0.52) and Ki-67 (0.29) for DCIS. The correlation coefficients between automated counts were 0.653 for normal breast and 0.616 for DCIS, with an overall correlation coefficient of 0.708 for the entire series.

Conclusions: Our analysis of both biomarkers yielded a similarly increasing labeling index for DCIS cases when compared to their expression in normal breast expression. The positive pixel algorithm method has a sampling bias due to subjective selection of the area being counted. In summary, the present results show similar findings for caPCNA and Ki-67 in normal breast reductions with no evidence of breast disease and in DCIS clinical cases. Comparing caPCNA with Ki-67, and unlike traditional PCNA, caPCNA can potentially better differentiate between normal breast and DCIS, and may be a useful biomarker for early stage cancer diagnosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3185. doi:10.1158/1538-7445.AM2011-3185
Abstract 3220: Application of GastroPlus™ modeling and simulation for predicting mouse and human pharmacokinetic profiles in oncology research and development
Authors: Hosea Natilie
Published: June 2012

Pharmacokinetic modeling plays an integral role in the drug discovery and development process. In particular, modeling and simulation has broad application to predict pharmacokinetics for human and non-clinical species, and as such drug concentration at the site of action, optimal dosing regimen with respect to safety and efficacy, food effects, inter-individual variability, drug-drug interaction potential and other scenarios, all with the aim of increasing understanding of a molecule and enabling informed decisions during the drug discovery and development process. GastroPlus™ is an “off the shelf” application that has been reported to accurately predict the absorption and pharmacokinetic profile of small drug-like molecules. Herein, three case studies are described where GastroPlus™ modeling and simulation was employed to predict the absorption and pharmacokinetic profile. In the first case study, GastroPlus™ was employed to predict the pharmacokinetic profile in mouse following oral administration to facilitate dose selection for in vivo pharmacological testing in tumor bearing mice. The second case study employed the parameter sensitivity analysis mode within GastroPlus™ to determine the critical parameters limiting oral exposure of the compound in mouse. The third case study employed GastroPlus™ to predict the implication of food on the human oral exposure of a compound in exploratory development in oncology patients. In all 3 case studies, the results obtained from the modeling and simulation provided key information that led to decisions on either dose selection, chemistry strategy to improve lead matter or clinical protocol design, thus demonstrating the value of modeling and simulation in both early discovery and exploratory development for predicting absorption and pharmacokinetic profiles in the area of oncology research and development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3220. doi:10.1158/1538-7445.AM2011-3220