bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4347: γH2AX foci formation in lymphocytes in vivo is increased one hour after very low dose X-radiation in three young children
Authors: Halm Brunhild M. , Franke Adrian A. , Turner Helen C. , Lai Jennifer F. , Zohrabian Vatche , DiMauro Robert
Published: August 2012

Objective: Computer tomography (CT) involves ionizing X-irradiation which can cause DNA damage from free radicals. CT use has risen dramatically due to its high speed and diagnostic value but it leads to an estimated lifetime cancer risk of up to 4% with highest risk for children. Double strand breaks, the most serious DNA damage caused by X-rays and other types of ionizing radiation, leads to histone H2AX phosphorylation which causes recruitment of repair proteins to DNA damage sites. γH2AX foci therefore represent quantitatively double strand breaks and can be assayed by a fluorescent immunostaining procedure. This γH2AX assay has been shown to be useful as a reliable biodosimeter for X-irradiation in vitro, in animals, and in adults. Recently also children who received cardiac catheterization procedures involving fluoroscopy with intermediate X-irradiation doses showed a dose response in this assay. We tested whether the γH2AX assay can detect changes when children receive X-irradiation at very low doses (<3 mSv) from CT scans. Methods: For this pilot study, three children aged 3 to 21 months and scheduled for a medically indicated CT were recruited through the emergency department at Kapi'olani Medical Center (Honolulu, Hawaii). These children donated blood immediately before and 1 hour after their CT exam (1.57- 2.86 mSv). Blood was immediately processed after the second blood draw and lymphocytes were isolated and fixed. For a blind analysis, the samples were coded in duplicate and shipped to Columbia University, New York, where the lymphocytes were immunolabeled with an anti-human γH2AX monoclonal antibody. The average number of γH2AX foci per cell nucleus was determined for each of the duplicate samples. Results: The γH2AX assay showed that the fluorescent frequencies were dose dependently higher in all patients 1 hour after CT exposure as compared to before CT (p=0.046 by paired t-test). Conclusion: In this pilot study we observed a significant induction of γH2AX foci in lymphocytes after very low dose ionizing X-irradiation (1-3 mSv) in vivo in children one hour after CT.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4347. doi:1538-7445.AM2012-4347
Abstract 4349: Therapeutic radiation induces an epigenetic DNA methylation response
Authors: Antwih Deborah A. , Gabbara Kristina M. , Fasih Aisha , Ruden Douglas M. , Zielske Steven P.
Published: August 2012

Therapeutic radiation results in a cascade of cellular events culminating either in repair of DNA damage and cell survival, or failure to repair damage and cell death. Gene expression is partially under epigenetic control in the form of DNA methylation. We investigated whole genome DNA methylation profiles of MDA-MB-231 breast cancer cells following therapeutic radiation to determine genes and pathways that are differentially methylated. MDA-MB-231 cells were treated with a single dose of 0, 2, or 6 Gy and incubated at 1, 2 4, 8, 24, 48, and 72 hrs before obtaining DNA extracts. DNA was subjected to whole genome DNA methylation profile analysis using an Infinium BeadArray which has the ability to determine CpG methylation at 450,000 genomic sites and covering all genes. Compared to unirradiated cells, 2 and 6 Gy irradiated cells showed 419-1661 CpG sites with decreased methylation and 514-2657 CpG sites with increased methylation, depending on time and dose. The number of hypomethylated genes in 2 Gy irradiated cells increased over time while the number of hypermethylated genes decreased in the first 8 hrs and then increased to a maximum at 72 hrs. In 6 Gy irradiated cells, no pattern was detected, although there was overall more DNA methylation changes than in 2 Gy irradiated cells, indicated a dose effect. Gene ontology analysis using Genomatix Genome Analyzer showed significant changes in DNA repair, cell cycle, apoptosis, and cell survival pathway gene methylation. DNA repair genes were differentially methylated as early as 1 hr after irradiation and sustained through 72 hrs. Epigenetic changes persisted after DNA repair, as shown by gamma-H2AX immunofluorescence, had taken place. Cell cycle, cyclin-dependent kinase, and cyclin-dependent kinase inhibitor genes were differentially methylated 4-24 hrs post-irradiation, correlating with a G2 cell cycle arrest as determined by flow cytometry. These data suggest that epigenetic regulation in the form of DNA methylation is integrated with the cellular response to radiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4349. doi:1538-7445.AM2012-4349
Abstract 4366: miR-126 reduces the tumorigenicity of melanoma cells in vitro and alters the melanoma microenvironment
Authors: Gremel Gabriela , Mulrane Laoighse , Paul Toby , Williams Vivienne , Gallagher William M. , Pontén Fredrik
Published: August 2012

The microRNA (miRNA) miR-126 is highly expressed in endothelial cells and often reported as down-regulated in cancer. The present study thoroughly examines the functional relevance of miR-126 in melanoma progression and suggests a central role of this miRNA as prognostic marker and modulator of the melanoma microenvironment. Initially, the expression of miR-126 was quantified in two independent, isogenic cell line model series of melanoma progression. To further explore its functional role, two stable over-expression cell lines were generated and subjected to an extensive series of in vitro assays. Subsequently, the prognostic relevance of miR-126 was assessed via in situ hybridization on a large melanoma tissue microarray cohort. In both melanoma cell line systems, miR-126 expression was inversely correlated with the level of tumorigenicity. Over-expression of miR-126 in vitro resulted in a significant decrease in colony formation, both in the presence and absence of anchorage, while cell viability remained unaffected. Using a three-dimensional spheroid invasion assay, over-expression of miR-126 significantly reduced cell invasiveness. In addition, a substantial reduction in tumor cell adhesion to VCAM-1 coated micro-channels was observed when the adhesive capacity of miR-126 over-expressing cells was examined under shear stress using the Cellix's Microfluidic Platform. Using in situ hybridization on tissue microarrays containing melanoma tissue, miR-126 expression was evident in the tumor vasculature whereas melanoma cells did not appear to express miR-126. Analysis of the degree of vascular staining revealed a significant association between lower vascular miR-126 expression and lower T-stage. In addition, high vascular miR-126 scores were significantly associated with prolonged overall survival, in particular in high grade tumors (T-stages 3 and 4). Overall, miR-126 over-expression was shown to trigger a less aggressive phenotype in melanoma cells in vitro. Nonetheless, its clinical relevance in melanoma may primarily be mediated by the tumor vasculature. Depletion of miR-126 has previously been linked to a number of tumor relevant phenotypes, including inflammation. Vascular up-regulation of the miR-126 target protein VCAM-1 may mediate increased tumor infiltration of lymphocytes and macrophages, which are known to play a crucial role in tumor development and prognosis. This renders miR-126 an exciting target for closer investigation with regard to its role in the melanoma microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4366. doi:1538-7445.AM2012-4366
Abstract 4424: Roles of the primary bile acid receptor FXR in liver repair and tumorigenesis
Authors: Meng Zhipeng , Wang Xiaoqiong , Huang Wendong
Published: August 2012

Bile acids promote processing of dietary fat and regulate glucose homeostasis in liver through its receptor, FXR. In our study, we demonstrate that bile acid/FXR signaling also plays a role in the tissue homeostasis and repair as well as hepatocarcinogenesis. Either decreased bile acid level or loss of FXR leads to impaired liver regeneration after carbon tetrachloride-induced liver injury or 70% partial hepatectomy, which indicates that FXR is an essential liver protector by regulating liver cell proliferation and death. Indeed, FXR-/- mice spontaneously develop liver cancer when they are aged due to their chronic liver injury and deregulated repair. In addition, FXR expression is down-regulated in human liver tumors compared with non-tumor regions, and the hepatocarcinogenesis in FXR-/- mice can recapitulate the process of human liver cancer initiation and progression. Therefore, FXR-/- mice provide a unique animal model for liver cancer study. For instance, we generated IFNα-/-FXR-/- mice to clarify the tumor suppressor role of the cytokine IFNα, the function of which was ever under debate. FXR-/- mice can be also used to identify or predict more differentially expressed genes in human liver cancer. For example, we found that one liver-rich miRNA, miR-194, which is down-regulated in the liver tumors of FXR-/- mice, is an important liver epithelial cell marker and prevents cancer metastasis, which has not been described before. In conclusion, ablation of FXR increases susceptibility to liver injury and tumorigenesis, and the FXR-/- mice provide a unique animal model for human liver cancer study.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4424. doi:1538-7445.AM2012-4424
Abstract 4429: An investigation on p21-activated protein kinase 1 inhibitor, IPA-3, in hepatocellular carcinoma
Authors: Wong Leo LY , Lam Ian , Wong Tracy Y N , Lai Benny WL , Zhou Yuan , Hung Red , Ching Wilson YP
Published: August 2012

Background: Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and is associated with poor prognosis due to metastasis and tumor recurrence. However, apart from Sorafenib, there is no proven effective systemic chemotherapy for HCC yet. Our previous study showed that overexpression of p21-activated protein kinase 1 (PAK1) is commonly found in HCC, which is coupled with a more aggressive tumor behavior. In this study, we examined the effect of an allosteric inhibitor of PAK1, IPA-3, on hepatocarcinogenesis. Methods: Human HCC line H2M was treated with various dosages of IPA-3 or vehicle control in order to measure the phospho-PAK1 level. The cells were also subjected to different functional assays, including proliferation assay, transwell assay and colony formation assay, to examine the anti-cancer effect of IPA-3. Tumor xenografts in nude mice were used to examine the in vivo activity of IPA-3. Results: Pretreatment with IPA-3 dose-dependently suppressed fetal bovine serum (FBS)-, and tumor necrosis factor (TNF)-α-induced PAK1 activity. The inhibitory effect was causally accompanied with suppression on cell migration, and enhancement of cell spreading. Furthermore, IPA-3 treatment on HCC cell line demonstrated a suppression of cell growth. The concomitant increase in apoptosis and delay in DNA synthesis had suggested a mechanism explaining the inhibition of HCC proliferation. In vivo, intraperitoneal administration of IPA-3 to subcutaneous xenograft mouse model significantly reduced the tumor growth rate. Conclusion: Taken together, these data showed that IPA-3 restrains cancer cell tumorigenicity and metastasis, thus, suggesting the potential use of IPA-3 in cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4429. doi:1538-7445.AM2012-4429
Abstract 4444: BRCA1 Circos: A visualization tool for BRCA1 missense variants
Authors: Jhuraney Ankita , Velkova Aneliya , Vallée Maxime P. , Carvalho Renato S. , Tavtigian Sean V. , Monteiro Alvaro N.
Published: August 2012

Germline mutations in BRCA1 have been associated with a significantly increased risk of developing breast and ovarian cancers. A large number of variants have been identified in BRCA1 and are reported as pathogenic, non-pathogenic, or as variants of uncertain significance (VUS). Test results reporting pathogenic or non-pathogenic variants can be used by physicians and genetic counselors to help patients in making important clinical decisions. Around 10-15% of the reported BRCA1 and BRCA2 variants are clearly pathogenic and 5% of these reported variants are VUS. Results reporting a VUS can cause anxiety and make it difficult to advise the patient on further clinical intervention to reduce their risk of developing the disease. Many of these VUSs are non-synonymous single amino acid changes leading to missense changes. A number of previous studies have focused on assessing the functional impact of these changes on the BRCA1 protein. BRCA1 is a gene product with pleiotropic functions and therefore, different functional assays are used in these studies to analyze the variant's impact on function. However, information on the functional impact of VUS is currently not consolidated and collecting available information on missense variants depends on multiple database searches and literature review. Moreover, methods to incorporate functional data into multifactorial models have recently been developed highlighting the need for a common resource to facilitate analysis. Realizing the need for a resource that facilitates the global visualization and analysis of these variants we have developed a visualization tool for multidimensional data using Circos (http://circos.ca) to aggregate the data from all published studies that have assessed these missense variants. Circos plots have been used in the literature to represent genomic data and we have used the same concept to illustrate variants in BRCA1. We have included ∼700 variants for which we have 30 different functional experiments from various studies assessing over 150 missense variants. Results are color coded to uniformly depict various degrees of functional impact on the BRCA1 protein function so that information for any variant can be obtained in a snapshot without having to perform extensive literature searches. Therefore, we believe clinical geneticists and genetic counselors can use this research tool as a quick reference to analyze all the missense variants that have been identified along with associated information to characterize them when faced with the challenge of advising a patient with these types of VUSs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4444. doi:1538-7445.AM2012-4444
Abstract 4448: Fecal miRNA test using microRNA expressions of exfoliated colonocytes for colorectalcancer screening
Authors: Koga Yoshikatsu , Yasunaga Masahiro , Matsumura Yasuhiro
Published: August 2012

Background & Aims: To reduce the mortality rate of several cancers, the development of a screening test is needed. We have been developing new screening methods for colorectal cancer (CRC) by applying molecular biological tools to exfoliated colonocytes. In the last few years especially, we have reported the fecal RNA test including the CRC-related gene expression analysis. Here, we investigated the potential of a fecal miRNA test for the early detection of CRC. Methods: CRC patients (n = 299) and healthy volunteers (n = 116) with no abnormalities detected by screening colonoscopy were enrolled in this case control study. The miRNA expressions in the colonocytes of CRC patients (n = 47) and healthy volunteers (n = 35) were analyzed in the training study and the miRNA expressions in the colonocytes of CRC patients (n = 252) and healthy volunteers (n = 81) were validated in the validation study. First, CRC related oncogenic miRNAs for CRC detection were selected by TaqMan MicroRNA Array v3.0 (corresponded to miRBase release 14). Next, the expressions of the most suitable oncogenic miRNAs were analyzed by quantitative real-time PCR in the training study. Lastly, the sensitivity and specificity of fecal miRNA test was validated by quantitative real-time PCR in the validation study. Results: According to the results of miRNA array, 20 miRNAs were selected as candidate of miRNA analysis. In the training study, significant differences in the relative expression level of miR-17, -18a, -19a, -19b, -20a, -92a, -106a, -135a, -135b, and -146a were observed between the CRC patients and the healthy volunteers (P < 0.01). On the other hand, there was no significant difference in the relative expression level of miR-21, -183, -223, and -454* between the CRC patients and the healthy volunteers (P > 0.1) AUC of ROC curve using miR-17, -18a, -19a, -19b, -20a, -92a, -106a, -135b, and -146a were more than 0.7. The overall sensitivity and specificity in the training study using these miRNAs were 70.2% (33/47) and 74.3% (26/35), respectively. The overall sensitivity and specificity in the validation study were 67.5% (170/252) and 75.3% (61/81), respectively. Conclusions: We selected the suitable miRNAs for CRC detection in exfoliated colonocytes. Fecal miRNA test may be useful for CRC screening.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4448. doi:1538-7445.AM2012-4448
Abstract 4466: Local regulation of steroid hormones in the breast is related to single nucleotide polymorphism of steroid metabolism genes
Authors: Lee Oukseub , Chatterton Robert T. , Shidfar Ali , Heinz Richard , Fought Angela , Denise Scholtens , Khan Seema A.
Published: August 2012

Background: The pattern of steroid hormone levels in nipple aspirate fluid (NAF) significantly differs from systemic levels, but may be a better predictor of breast cancer risk than the systemic environment. We hypothesize that local production and clearance of estrogen precursors or estrogen in the breast is affected by genetic variation in the enzymes involved in steroid transport, biosynthesis, and metabolism, thereby contributing to breast cancer risk. We examined the effect of functional single nucleotide polymorphism (SNP) on the concentrations of the NAF steroid hormones, as compared to serum levels. Methods: NAF was collected from both the contralateral unaffected breast of breast cancer patients and controls (blinded study of 265 women). Selection criteria for SNPs were; 1) known/potential functional significance on activity of the resulting enzyme. 2) Expression in breast tissue. 3) Caucasian population frequency of the minor alleles. gDNA was extracted from the plasma buffy coat by a Qiagen kit, followed by TaqMan genotyping assays (Applied Biosystems). The NAF and serum level of estradiol (E2), estrone (E1), progesterone (P4), testosterone (T), androstenedione (A4), and dehydroepiandrosterone (DHEA) were measured by immunoassay. Hormone levels were compared across genotype groups using the ANCOVA in SAS (age-adjusted significance p < 0.05). Results: (See the Table) Conclusions: There is no data yet to show that NAF hormone content is related to breast cancer risk. This hypothesis will be tested in our on-going study. Locally regulated biosynthesis and clearance of hormones from the breast seems likely. Our data support such a phenomenon, since NAF correlations with functional enzyme SNPs are more frequent than with serum. The significant relationships between SNPs and NAF hormones, not seen with the same serum hormone, support the notion that they are determinants of breast cancer risk.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4466. doi:1538-7445.AM2012-4466
Abstract 447: Has the expression of the Wnt-pathway in regard to cMYC and β-Catenin a predicitive influence on the kind of metastasis in colon cancer
Authors: Strohäker Jens , Falch Claudius , Hann-von-Weyhern Claus , Adam Patrick , Königsrainer Alfred , Fend Falko , Brücher Björn LDM
Published: August 2012

Background: cMYC as well as β-Catenin are downstream-targets of the Wnt-pathway and both have an important early function within the carcinogenesis and the progression of colon cancer. Additionally cMYC was found to be associated with a worse prognosis in other neoplasms. The goal of our investigations was the evaluation of the Wnt-pathway on primary non-metastatic colon cancer compared to lymphatic, hepatic and peritoneal metastasis in regard to the expression of cMYC and β-Catenin. Methods: 98 patients with adenocarcinom of the colon and a postoperative pT2/3-status, who underwent surgical resection between 2006 and 2008 at the University of Tübingen had been included within the study collective. 53 patients had no metastasis (control group), while 45 patients revealed primary metastasis: lymphatic metastasis (n=20), hepatic spread (n=20) and multiple metastasize (n=5). cMYC and β-Catenin was coloured on a Tissue-Micro-Array by immunohistochemistry with consecutive semiquantitative analysis. Results: 17 of 98 patients (17%) had been positive for cMYC. 5 out of these 17 (29%) revelaed metastasis. 16 out of the 17 cMYC-positive patients showed a nuclear accumulation of β-Catenin (94%) (p=0.027). 40 out of 81 (49%) cMYC negative patients revealed metastasis. No significant correlation between nuclear positive cMYC and the rate or kind of metastasis was found (p=0.133). In 29 out of 81 cMYC-negative patients (36%) a strong expression of β-Catenin was found. Conclusion: cMYC is well known for a worse prognosis in case of an overexpression in patients suffering from lymphoma. A correlation between the expression of cMYC and the kind of metastasis and / or survival nor prognosis in patients with colon cancer was not able to show (p=0.211). Additonally no correlation between the nuclear exprimed β-Catenin and different ways of metastasis was found (p=0.202).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 447. doi:1538-7445.AM2012-447
Abstract 4538: Semiquantitative detection of aberrant methylation as a tumor biomarker in the serum and plasma of patients with gastric cancer
Authors: Lee Jae-Hyuk , Kim Jin-Sook , Lee Kyung-Hwa , Lee Ji-Shin , Choi Chan , Juhng Sang-Woo
Published: August 2012

Background: Epigenetic silencing of tumor-related genes, due to CpG island methylation, is considered to be an important mechanism for the development of many tumors, including gastric cancinoma (GC). DNA methylation of multiple CpG sites in promoter regions of several tumor related genes, such as hMLH1, p14, COX2, and APC has been analyzed in GC so for. Circulating tumor DNA in serum/plasma has been demonstrated to reflect the biological characteristics of tumors. Recently, the presence of gene promoter methylation in serum/plasma DNA has also been demonstrated in patients with cancers of lung, head and neck, liver, and breast. So, in the present study, we attempted to determine the feasibility and the clinical correlations of detecting tumor-associated aberrant methylation in the serum/plasma of patients with gastric cancer. Materials and Methods: We examined promoter methylation of 6 genes using methylation-specific PCR (MSP) and methylation-sensitive high-resolution melting (MS-HRM) in paired serum, plasma and tumor samples of 50 gastric cancer patients. The tumor and the paired serum/plasma were investigated for aberrant methylation in BLU, DAPK1, GADD45G, MGMT, p15 and p16. The sensitivity of the HRM analysis was tested by using dilutions of fully methylated DNA into unmethylated DNA. Results: MSP was used to assess gene methylation in tumor samples. Promoter methylation in BLU, DAPK1, GADD45G, MGMT, p15, and p16 were detected in 47%, 37%, 59%, 32%, 14%, and 43% of tumor tissues by MSP. MS-HRM was used to assess gene methylation in serum/plasma samples concurrently. In the serum/plasma of gastric cancer patients, BLU, DAPK1, GADD45G, MGMT, p15, and p16 were methylated at frequencies of 26%, 21%, 23%, 11%, 9%, and 17%, respectively. Nineteen (38%) gastric tumor samples displayed concurrent methylation in three or more tumor genes. Aberrant methylation in one or more genes was found in 74% (37/50) serum samples and 70% (35/50) plasma samples. However, there was not a significant relationship of gene methylation between gastric tumor tissues and serum/plasma samples.Conclusion: These results suggest that aberrant promoter methylation in serum/plasma can be detected in a substantial proportion of gastric cancer patients. Detection of DNA methylation in serum/plasma may be a biomarker for early detection of gastric cancer. MS-HRM is powerful technique for the analysis of promoter methylation. Application of HRM analysis to large amount of clinical samples proves to be a fast and high-throughput way to investigate the epigenetic status of genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4538. doi:1538-7445.AM2012-4538
Abstract 4548: Prognostic value of occult metastases detected by cytokeratin 20 and mucin 2 mRNA quantification in sentinel lymph nodes from colon cancer patients
Authors: Nordgård Oddmund , Oltedal Satu , Aasprong Ole Gunnar , Søreide Jon Arne , Søreide Kjetil , Tjensvoll Kjersti , Gilje Bjørnar , Heikkilä Reino , Guriby Marianne , Lothe Ragnhild A. , Smaaland Rune , Kørner Hartwig
Published: August 2012

BACKGROUND: Lymph node (LN) metastasis status is among the strongest prognostic factors in colon cancer. Despite this, 20-30% of LN negative patients experience disease recurrence. These patients may have occult LN metastases that are not detected by routine microscopy. The aim of the present study was to investigate the prognostic value of occult metastases detected by cytokeratin 20 (CK20) and mucin 2 (MUC2) mRNA quantification in sentinel lymph nodes (SLNs) from patients curatively resected for colon cancer. METHODS: Resection specimens from consecutive patients undergoing surgery for localized colon cancer were subjected to ex vivo SLN mapping. The isolated SLNs were examined for the presence of metastases by routine HES staining and quantitative CK20 and MUC2 mRNA measurements. Primary tumor microsatelite instability- (MSI), KRAS and BRAF mutation status were determined for the included patients. Recurrence-free and disease-specific survivals were analyzed by Kaplan-Meier survival estimates and log rank tests. RESULTS: SLNs were identified in 206 (97%) of the 213 included patients. Routine histological analysis of SLNs and other regional lymph nodes revealed LN metastases in 63 patients (pN+), of which 42 (67%) had metastases detected in one or more SLNs (sensitivity 67%, false negative rate 33%). When analyzing the SLNs by CK20 and MUC2 mRNA quantification, occult metastases were suggested in 86 (60%) and 52 (36%) of the 143 otherwise LN negative (pN0) patients, respectively. Survival analysis with a median 3.6 year follow-up revealed a significant prognostic value of MUC2 mRNA quantification in SLNs from all the patients, but not for the pN0 patients. For CK20 mRNA levels in SLNs we observed no statistically significant survival differences. Patient stratification according to primary tumor MSI, KRAS mutation and BRAF mutation status did not reveal any significant survival differences related to the suggested occult SLN metastases, neither. CONCLUSIONS: Occult SLN metastases detected by quantitative CK20 and MUC2 mRNA measurements did not demonstrate any prognostic value in this study.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4548. doi:1538-7445.AM2012-4548
Abstract 4550: NT5E promoter methylation is a favorable breast cancer epigenetic biomarker
Authors: Nigro Cristiana Lo , Monteverde Martino , Lee Sonia , Lattanzio Laura , Syed Nelofer , Garrone Ornella , Merlano Marco , Hatzimichael Eleftheria , Briasoulis Evangelos , Thompson Alastair , Crook Tim
Published: August 2012

Despite the recent introduction of advanced microarray-based gene expression profiling strategies, relapse risk assessment and individual treatment recommendations remain suboptimal for breast cancer patients. DNA methylation emerges as a novel source of epigenetic biomarkers that can help improve individualised breast cancer therapy. In the light of existing preclinical and clinical data we studied NT5E (5′-nucleotidase, ecto) expression and NT5E promoter methylation (NPM) in 14 breast cancer cell lines and 259 breast cancer clinical cases. In vitro, NT5E promoter methylation (as determined by MSP and pyrosequencing) was inversely associated with NT5E expression (as determined by qRT-PCR) and azacytidine produced an increase in NT5E mRNA levels in cell lines with methylated the NT5E promoter CpG. In clinical series, patients whose resected primary tumours were found to have NT5E promoter methylation, were less likely to develop metastasis (p=0.003, OR=0.34, 95% CI 0.17-0.69). Moreover in 3 out of 4 paired samples, NT5E promoter was found methylated in primary tumours and demethylated in CNS metastases. Regarding metastatic profile, patients that progressed to non-visceral metastases were more likely to have NT5E promoter methylated in primary tumours as compared to visceral metastatic disease (p=0.01, OR=11.8). NT5E promoter CpG island was densely methylated in 16 of 21 patients who developed non-visceral metastatic disease (76%) and in 6/28 cases (21%) with visceral metastases. In time-dependent endpoints analysis, patients with tumours lacking detectable NT5E methylation had increased risk for shorter DFS (p=0.001, HR 2.7) and OS (p=0.001, HR=3). The favourable prognostic value of NT5E methylation was confirmed in ER negative (p=0.011, HR=3.27, 95% CI 1.31-8.12) and in triple negative cases (p=0.004; HR=6.2, 95% CI 1.9-20). Moreover we observed a strikingly favourable outcome to adjuvant chemotherapy in patients with tumours profiled as NT5E promoter methylated regarding DFS (p=0.0016, HR 5.1, 95% CI 1.8-14.37) and OS (p=0.0005, HR 7.4, 95% CI 2.416 –23.08), which allows speculations that NPM may convey breast cancers more amenable to chemotherapy, possibly though pro-apoptotic mechanism. We suggest that NT5E promoter CpG island methylation may serve as a promising favourable breast cancer epigenetic biomarker. Our results warrant independent confirmation by other groups before considering its investigation as a metastasis predictor in clinical trials of adjuvant breast cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4550. doi:1538-7445.AM2012-4550
Abstract 4552: Utilization of pathway activation and somatic mutation analysis in combination in fine needle aspirates to identify candidate drugs for effective treatment of breast cancer
Authors: Lee Tani , Kim Phillip S. , Harvie Gulia , Meyer Gary , Leesman Glen , Langley Emma , Liu Limin , Hoe Hicholas , Princen Fred , Selvaraj Fabiyola , Liu Xinjun , Singh Sharat
Published: August 2012

A critical hurdle in implementing prognostic and predictive biomarkers in clinical trials is our inability to perform an efficient molecular characterization of the limited amounts of available clinical samples. Here we report a comprehensive molecular analysis of fine needle aspirate (FNA) samples collected from metastatic sites of 58 breast cancer (BCA) patients. The analysis included interrogation of key receptor tyrosine kinases and their downstream signaling molecules including HER1, HER2, p95HER2, HER3, cMET, IGF1R, PI3K, Shc, AKT and ERK as well as oncogenic somatic mutational profiling. A multiplexed immunoarray CEER™ (Collaborative Enzyme Enhanced Reactive-immunoassay) platform was utilized to determine the levels of pathway protein expression and phosphorylation. All FNA collections provided sufficient materials for the combined analysis of multiplexed pathway expression/activation and somatic mutation analysis. All the FNA from metastatic sites (mFNA) collected from HER2 positive primary tumors (determined by IHC) were also CEER-HER2 positive. Over-expression of HER2 was found in 10% of mFNAs collected from BCA patients with HER2 negative primary tumors. PIK3CA mutations were found in 20% of the samples in this cohort. Statistically significant higher levels of phosphorylated AKT, ERK (downstream effector targets) as well as HER1, HER2, HER3 and IGF1R (PI3K membrane recruiter proteins) were found in mFNAs with PIK3CA mutations. Subset of PIK3CA wildtype patients also showed robust pathway signature. Hence, evaluation of biomarkers in BCA patients should include both pathway proteins as well as mutation analysis. A comprehensive disease profiling can provide insightful information on efficacy of specific agents on its intended target protein inhibition. As CEER requires minimal amount of specimen, performance of routine pathway profiling can be achieved in clinical settings. A multiplexed pathway analysis can also provide valuable information about potential drug resistance mechanisms. Furthermore, a combined mutational and pathway activation profiling can guide therapeutic strategies in clinical setting.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4552. doi:1538-7445.AM2012-4552
Abstract 4680: DNA dependent protein kinase as a new molecular target for treatment of non-small cell lung cancer
Authors: Aragane Naoko , Sato Akemi , Iwanaga Kentaro , Kobayash Naomi , Kimura Shinya , Sueoka Eisaburo
Published: August 2012

Prognosis with non-small cell lung cancer (NSCLC) has improved with the recent introduction of molecular targeted therapy. However, it is limited to patients with epidermal growth factor receptor (EGFR) mutations, who benefit from EGFR tyrosine kinase inhibitors (EGFR-TKI). A new molecular target is needed with other NSCLC patients. In general, chemotherapeutic agents cause DNA double-strand breaks (DNA DSB), leading to apoptosis of cancer cells; DNA repair reverses their effect, resulting in resistance to chemotherapy. DNA DSB repair can be mediated via two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). More than 90% of DSBs in mammalian cells are repaired by NHEJ, which is accomplished by core protein components including Ku70/Ku86, DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, Artemis, and Cernunnos-XLF. We recently reported that NK314, a dual inhibitor of topoisomerase IIα (Top2β) and DNA-PK, evidences a potent anti-tumor effect in adult T-cell leukemia-lymphoma (ATL). NK314 induced DNA DSBs and inhibited DNA repair mediated through degradation of the DNA-PK catalytic subunit (DNA-PKcs). In the present study, we examined the effect of NK314 in NSCLC cell lines and assessed DNA-PK as a possible molecular target for NSCLC. Twenty separate NSCLC cell lines were used, with IC50 varying from 46-193 nM. Levels of DNA-PKcs protein varied among the cell lines, whereas those of Ku70 and Ku86 did not. The inhibitory effect of cell growth was closely related to level of DNA-PKcs protein, but not to levels of other DNA-damage-activated protein kinases such as ATM or ATR. Establishment of predictive markers concomitantly with the development of a novel molecular target is indispensable for clinical application. Because detection of DNA-PKcs in peripheral blood is difficult, we investigated an alternative marker for predicting DNA-PKcs inhibitor. We previously found that heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), an RNA binding protein, interacts with DNA-PK, and we established a system for detecting hnRNP B1 mRNA in peripheral blood. We therefore examined the suitability of hnRNP B1 as an alternative biomarker for DNA-PK inhibitors. Close correlation between levels of DNA-PKcs and hnRNP B1 proteins was established using Western blot analysis with NSCLC cell lines (p=0.995). These results suggest that DNA-PK is a novel molecular target for NSCLC, and detection of hnRNP B1 might serve as an alternative molecular marker for the amount of DNA-PK inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4680. doi:1538-7445.AM2012-4680
Abstract 469: CD151 and cell motility in prostate cancer
Authors: Detchokul Sujitra , Parker Michael W. , Williams Elizabeth D. , Herdiman Olivia M. , Davis Melissa , Kee Kelvin , Bolton Damien M. , Frauman Albert G.
Published: August 2012

Prostate cancer (PCa) is one of the leading causes of cancer death in men. CD151 is a member of the tetraspanin family and is associated with regulation of migration of normal and tumour cells via cell surface microdomain formation. Previous studies from our laboratory revealed that expression of CD151 differs across histological grades of PCa and high levels of expression are linked to shorter survival, independent of Gleason score1. In vitro motility assays of human PCa cell lines suggest CD151 is a motility promoter2. We have sought to develop a number of CD151 inhibitors and examine their ability to modulate prostate cancer motility and metastasis. The human PCa cell line, LNCaP (low endogenous level of CD151), transfected with CD151 shows increased motility and invasion compared to control LNCaP, whilst CD151 siRNA knock-down (KD) of PC-3 cells (high endogenous level of CD151), reduces motility compared to control PC-3. We have conducted in silico screening with compounds predicted to bind a model of the large extracellular domain (EC2) of CD151 and found that a number of these small molecules possess bioactivity in vitro and in vivo in inhibiting prostate cancer motility and progression. LNCaP growth and migration was confirmed by stimulating with 1 and 10 nM DHT at 24 hrs in charcoal stripped FBS. It was found that cell proliferation and motility of LNCaP cells was significantly increased after stimulation by 10 nM DHT at 24 hrs (p<0.01) and 48hrs (p<0.01), whilst DHT had minimal effect on control PC-3 and DU-145 cells. We have also analyzed a set of whole genome microarray expression data from PCa cell lines PC-3 and CD151 KD PC-3, and identified differentially expressed genes linked to CD151. Significant changes were seen in 172 genes after CD151 KD in PC-3 cells. We examined the protein interaction networks of our differentially expressed genes using Cytoscape3. Functional network analysis revealed high level of connectivity surrounding genes involved in transcriptional regulation, microtubule-based movement and protein folding and complex formation. CD151 is an important promoter of cell migration and metastatic disease in PCa and may serve as a target for a novel class of anti-metastatic therapeutic agents. A systems biology approach allows for improved mechanistic interpretation of biological data and may facilitate identification of other therapeutic targets. 1 Ang J et al. Cancer Epidemiol Biomarkers & Prevention (2004) 13: 1717-21 2 Ang J et al. Oncology Reports (2010) 24(6): 1593-1597 3 Shannon P et al. Genome research (2003) 13: 2498-2504

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 469. doi:1538-7445.AM2012-469
Abstract 4699: A siRNA high-throughput screening identified Wee1 as determinant of Chk1 inhibitor sensitivity
Authors: Carrassa Laura , Chilà Rosaria , Lupi Monica , Ricci Francesca , Mazzoletti Marco , Celenza Cinzia , Broggini Massimo , Damia Giovanna
Published: August 2012

Targeting Chk1 protein kinase is a valid anti cancer strategy to enhance the cytotoxic effects of radio and chemotherapy. Recent evidence disclosed a role of Chk1 in unperturbed cell proliferation and survival, implying that Chk1 inhibitors could also be effective as single agents in tumors with a specific genetic background. The effect of Chk1 inhibition on a wide panel of cancer cell lines has been herein conducted by using a specific and selective Chk1 inhibitor PF-00477736. The results showed that 2 cancer cell lines out of 16 analyzed (the ovarian cancer cell line OVCA-432 and the breast cancer cell line SK-BR3) were very sensitive to PF-00477736 treatment. To identify genes in synthetic lethality with Chk1, which may justify the sensitivity to Chk1 inhibitor of some cancer cell lines, a high-throughput screening using a siRNA library directed against 719 human protein kinases, in combination with either scramble or Chk1 siRNA, was undertaken. The ovarian cancer cell line OVCAR-5, resistant to Chk1 inhibition, was used to perform the screening. After 2 independent screenings and the validation procedure using specific esiRNAs, and the three siRNAs of each target (used in the screening as a pool), the Wee1 tyrosine kinase was identified as the most significant gene in synthetic lethality with Chk1. Treatment with both PF-00477736 and a Wee1 inhibitor (MK-1775) confirmed the remarkable synergistic effect in various human cancer cell lines (breast, ovarian, colon, prostate). Same results have been obtained with AZD-7762 Chk1 inhibitor. Isogenic cellular systems differing only for the status of p53 (the colon carcinoma cancer cell lines HCT-116 p53 +/+ and p53 -/- and the ovarian cancer cell lines A2780 and A2780/E6) showed a comparable and strong synergistic effect of the combined treatment. Interestingly, in the normal fetal human lung cell line MRC-5 the effect of the combined treatment was additive. Time lapse experiments and an evaluation of the ability of cells to form colonies, suggested that cells treated with the two inhibitors almost completely lost the ability to proliferate and failed to perform a proper cell division. A detailed molecular analysis showed that the combined treatment causes cells to undergo premature mitosis before the end of DNA replication and with damaged DNA, leading to a massive cell death by apoptosis. Taken together these preclinical data address a new promising combined targeted anticancer strategy that deserves clinical investigation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4699. doi:1538-7445.AM2012-4699
Abstract 4711: Epigenetic modulation restores expression of functional progesterone receptor in endometrial cancer cells
Authors: Yang Shujie , Xiao Xue , Zhang Yuping , Meng Xiangbing , Leslie Kimberly K.
Published: August 2012

Endometrial cancer, the most common gynecologic malignancy, is a hormonally regulated tumor. Given progesterone is a tumor suppressor in the endometrium, response to progestin-based therapy correlates positively with progesterone receptor (PR) expression. Many endometrial tumors have lost of PR, which limits the clinical application of progestin-based therapy. PRB promoter methylation was reported in 74% of endometrial cancer patients, which correlate with loss of PR expression. These clinical data suggest that restoring PR expression by epigenetic modulation might sensitize tumors to progestin-based therapy. Therefore, we hypothesized that restoring PR expression in endometrial cancer cells will inhibit proliferation and induce cell death. Epigenetic modulators, such as DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) were evaluated as effective agents to restore PR expression in a panel of endometrial cancer cells. Treatment with the DNMTi 5-aza-2′deoxycytidine (5-aza-dC) or the HDACi LBH589 alone or in combination upregulated PGR mRNA levels. At the protein level, LBH589 treatment upregulated PR by 24 hours and sustained expression for at least 72 hours, even in the presence of progestrone. The upregulated PR expression by LBH589 treatment was dose dependent; an obvious increase in PR was detected with 10 nM LBH589 when compared with control, and the expression reached the highest level at 50 nM to 100 nM. To investigate if the restored PR is functional, we examined PR nuclear localization and expression of target genes. First, after treatment with LBH589 in the absence or presence of progesterone, PR nuclear localization was confirmed by immunostaining and nuclear fractionation followed by Western blotting. Next, a progesterone response element (PRE)-luciferase reporter assay was used to directly examine PR transcriptional activity. LBH589 treatment increased PRE-luciferase activity by 8- to 10-fold in Ishikawa and ECC1 cells. Furthermore, expression of endogeneous PR target genes containing PRE (FoxO1 and cyclin D) or Sp1 element (p21 and p27) was studied in response to LBH589 treatment. Restored PR upregulated FoxO1, p21, p27 and downregulated cyclin D1, demonstrating that PR is functional as a transcription factor. Our findings reveal that epigenetic modulators can restore endogenous PR expression in endometrial cancer cell lines. This preclinical study provides strong support that strategy to re-establish PR expression will result in resensitization of endometrial tumors to progestin therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4711. doi:1538-7445.AM2012-4711