bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2328: Loss of 14-3-3zeta impairs oncogene-induced mammary tumorigenesis and metastasis.
Authors: Joshi Sonali , Yang Jun , Wang Qingfei , Li Ping , Wang Hai , Pickering Brian , Xiong Yan , Parker-Thornburg Jan , Behringer Richard , Yu Dihua
Published: August 2014

The 14-3-3 family proteins are highly conserved in eukaryotes and can interact with signaling proteins by binding phosphorylated Ser/Thr motifs. Studies in our laboratory have demonstrated that 14-3-3zeta is overexpressed in greater than 40% of breast cancer patients and is a strong indicator of poor patient prognosis, however the role of 14-3-3zeta in an in vivo context, especially its role in cancer, remains to be examined. We generated a 14-3-3zeta knockout mouse model to characterize the role of 14-3-3zeta in breast cancer progression. Loss of 14-3-3zeta resulted in neonatal lethality in the C57Bl/6 genetic background due to respiratory failure. Outbreeding to CD-1 or backcrossing to FVB/N background rescued the neonatal lethality. Crossing 14-3-3zeta knockout mice with MMTV-PyMT and MMTV-neu transgenic mice revealed that loss of 14-3-3zeta prolonged tumor latency and reduced lung metastasis as compared to MMTV-PyMT and MMTV-neu mice. Mechanistically, loss of 14-3-3zeta suppressed tumor proliferation and angiogenesis and promoted apoptosis. Reverse phase protein array analysis suggested that loss of 14-3-3zeta suppressed the Akt and Erk pathway and upregulated the expression of the tumor suppressor p53. Our results demonstrate an important role for 14-3-3zeta in normal development and cancer. Additionally our 14-3-3zeta knockout mouse model will also enable future studies on the role of 14-3-3zeta in other physiological and pathological processes.

Citation Format: Sonali Joshi, Jun Yang, Qingfei Wang, Ping Li, Hai Wang, Brian Pickering, Yan Xiong, Jan Parker-Thornburg, Richard Behringer, Dihua Yu. Loss of 14-3-3zeta impairs oncogene-induced mammary tumorigenesis and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2328. doi:10.1158/1538-7445.AM2013-2328
Abstract 2353: Novel inhibitors of PIM-1, PIM-2, and PIM-3 protein kinases: medicinal chemistry leading to AZD1208.
Authors: Lamb Michelle L. , Dakin Les A. , Block Michael H. , Chen Huawei , Code Erin , Dowling James E. , Feng Xiaomei , Ferguson Andrew D. , Green Isabelle , Hird Alexander W. , Howard Tina , Huszar Dennis , Keeton Erika K. , Lyne Paul D. , Pollard Hannah , Rooney Michael , Read Jon , Wu Allan J. , Zhang Tao , Zheng Xiaolan
Published: August 2014

The PIM serine/threonine kinase family, composed of three highly homologous members, PIM-1, PIM-2 and PIM-3, are upregulated in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. Over-expression of PIM-1 or PIM-3 has also been observed several solid tumors, in particular prostate, pancreatic, gastric, bladder and hepatocellular cancers. PIM kinases are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways, thereby mediating cell proliferation and survival. We have identified novel, potent and highly selective inhibitors of the PIM family kinases. The synthesis, X-ray crystallographic binding mode, and SAR of this benzylidene-1,3-thiazolidine-2,4-dione series are described. Examples from this series exhibit single digit nanomolar potency against all three PIMs, and have been shown to be selective across a panel of more than 440 kinases, with inhibition found for only approximately 3% of the panel, and at least 10-fold selectivity over kinases outside the PIM family. The compounds additionally are stable in rat microsomes, have high aqueous solubilities, and are not potent against the hERG ion channel. The series has antiproliferative activity in a panel of AML cells, has excellent pre-clinical pharmacokinetic properties, and lead compounds have shown strong tumor growth inhibition in vivo in mouse AML xenograft models. From this series, AZD1208 has recently entered Phase I clinical trials for AML and solid tumors.

Citation Format: Michelle L. Lamb, Les A. Dakin, Michael H. Block, Huawei Chen, Erin Code, James E. Dowling, Xiaomei Feng, Andrew D. Ferguson, Isabelle Green, Alexander W. Hird, Tina Howard, Dennis Huszar, Erika K. Keeton, Paul D. Lyne, Hannah Pollard, Michael Rooney, Jon Read, Allan J. Wu, Tao Zhang, Xiaolan Zheng. Novel inhibitors of PIM-1, PIM-2, and PIM-3 protein kinases: medicinal chemistry leading to AZD1208. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2353. doi:10.1158/1538-7445.AM2013-2353
Abstract 2361: SNP (-617G>A) in ARE-like loci of the NRF2 gene: a new biomarker for prognosis of lung adenocarcinoma.
Authors: Okano Yasuko , Ichikawa Yasushi , Miyagi Yohei , Natsumeda Yutaka , Ishikawa Toshihisa
Published: August 2014

Purpose: Accumulating evidence suggests nuclear factor (erythroid-derived 2)-like 2 (NRF2) plays an important role as cellular defense, tumor suppression, and oncogenesis. However, little information is available as to the genetic polymorohisms of the NRF2 gene and their clinical relevance. We aimed to validate the NRF2 gene as a prognostic biomarker in lung cancer.

Experimental Design: We prepared genomic DNA from 392 Japanese patients with primary lung cancer and detected a SNP (c.-617C>A) in the ARE-like loci of the human NRF2 gene by the rapid genetic testing method we developed in this study. We analyzed an association between the NRF2 gene and overall survival.

Results: We detected homozygous (A/A) and heterozygous (C/A) alleles with the SNP (c.-617C>A) in 24 (6.2%) and 147 (38.0%) patients, respectively. The frequency of the homozygote (A/A) was four-fold higher in females than in males (11.0% versus 2.7%). Multivariate logistic regression models including standard lung cancer factors revealed that the homozygous alleles (c.-617A/A) were significantly related to gender. Interestingly, lung cancer patients carrying the homozygous alleles (c.-617A/A) exhibited remarkable survival over 1,700 days after surgical operation (log-rank p=0.021).

Conclusion: The homozygous allele (c.-617A/A) in the NRF2 gene is considered to be a prognostic biomarker for assessing both the gender-related risk of lung adenocarcinoma in the non-smoking sub-population and their overall survival.

Citation Format: Yasuko Okano, Yasushi Ichikawa, Yohei Miyagi, Yutaka Natsumeda, Toshihisa Ishikawa. SNP (-617G>A) in ARE-like loci of the NRF2 gene: a new biomarker for prognosis of lung adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2361. doi:10.1158/1538-7445.AM2013-2361
Abstract 2375: Prognostic significance of the alternatively spliced ACTN4 variant in high-grade neuroendocrine pulmonary tumors.
Authors: Miyanaga Akihiko , Honda Kazufumi , Tsuta Koji , Masuda Mari , Miyamoto Akitomo , Shinagawa Shingo , Tsuda Hitoshi , Asamura Hisao , Gemma Akihiko , Yamada Tesshi
Published: August 2014

Background: High-grade neuroendocrine tumours (HGNTs) of the lung manifest a wide spectrum of clinical behaviour, but no method of predicting their outcome has been established.

Materials and methods: We newly established a monoclonal antibody specifically recognizing the product of the alternatively spliced ACTN4 transcript (namely, variant actinin-4), and used it to examine the expression of variant actinin-4 immunohistochemically in a total of 609 surgical specimens of various histological subtypes of lung cancer.

Results: Variant actinin-4 was expressed in 55% (96/176) of HGNTs, but in only 0.8% (3/378) of non-neuroendocrine lung cancers. Expression of variant actinin-4 was significantly associated with unfavorable overall survival in HGNT patients (P = 0.00021, log-rank test). Multivariate analysis using the Cox proportional hazards model showed that expression of variant actinin-4 was the most significant independent negative predictor of survival in HGNT patients (hazard ratio, 2.18; P = 0.000714) after the presence of lymph node metastasis (hazard ratio, 2.30; P = 0.00012).

Conclusions: Expression of variant actinin-4 is an independent prognostic factor for patients with HGNTs. This protein has high affinity for filamentous actin polymers and likely promotes aggressive behaviour of cancer cells. The present clinical findings clearly support this notion.

Citation Format: Akihiko Miyanaga, Kazufumi Honda, Koji Tsuta, Mari Masuda, Akitomo Miyamoto, Shingo Shinagawa, Hitoshi Tsuda, Hisao Asamura, Akihiko Gemma, Tesshi Yamada. Prognostic significance of the alternatively spliced ACTN4 variant in high-grade neuroendocrine pulmonary tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2375. doi:10.1158/1538-7445.AM2013-2375

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2383: Clinical significance of intratumoral HER2 heterogeneity in gastric cancer.
Authors: Lee Hye Seung Seung , Lee Hee Eun , Park Kyoung Un , Nam Soo Kyung , Park Do Joong , Kim Hyung-Ho
Published: August 2014

Aim: To evaluate the clinical significance of intratumoral HER2 heterogeneity in gastric cancer (GC).

Methods: A total of 322 GC tissues were evaluated by HER2 immunohistochemistry (IHC), of which 73 with IHC 2+ or 3+ were subjected to fluorescence in situ hybridization (FISH). Also, 3-5 distinct spots in each case showing different HER2 staining intensity were evaluated individually for comparing IHC staining intensity with gene copy number (GCN). Minimum, average, and maximum FISH scores were generated for each case.

Results: Intratumoral heterogeneity of HER2 overexpression and gene amplification were 54 and 30 of 73 cases with IHC 2+ or 3+, respectively. These cases were characterised by diffuse or mixed Lauren type, HER2 IHC 2+, and low-level amplification. Kaplan-Meier survival analysis revealed that the heterogeneous overexpression was significantly associated with longer disease-free survival times than the homogeneous, and the high average GCN was most associated with poor outcome. Also, there was a strong correlation between the IHC and FISH results for each spot. Quantitative PCR analysis of the cancer tissues and the cell-free plasma showed that HER2 gene copy by quantitative PCR on tissue correlated well with those by FISH, but plasma HER2 level was not.Conclusions: Considering the high incidence of intratumoral HER2 heterogeneity in GC, accurate HER2 assessment would require larger tissues and more detailed guidelines. The guidelines should include the recommendation that FISH-scoring areas be selected with reference to a corresponding IHC slide. Also, the definition of HER2-positive tumours should be reassessed considering the intratumoral heterogeneity.

Citation Format: Hye Seung Seung Lee, Hee Eun Lee, Kyoung Un Park, Soo Kyung Nam, Do Joong Park, Hyung-Ho Kim. Clinical significance of intratumoral HER2 heterogeneity in gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2383. doi:10.1158/1538-7445.AM2013-2383
Abstract 2384: A new, highly sensitive IHC test for the detection of HER2 protein in breast cancer (BC) tissue.
Authors: Pedersen Hans Christian , Thomsen Anni , Jensen Kristian , Jakobsen Karen , Wellings Bob , Dalgliesh Gillian , Tosh Kerrie , Harbron Chris , Walker Jill
Published: August 2014

HER2 protein over-expression occurs in 15-20% of invasive BC. Trastuzumab and other HER2-targeted agents have mainly been investigated in HER2 over-expressing BC (HercepTest™ 2+/3+). Analysis of clinical study NSABP-31 suggests patients with HER2 non-over-expressed BC (HercepTest™ 0, 1+, 2+ and/or HER2 FISH negative) may benefit from anti-HER2 therapy. Other novel agents are under investigation in HER2 non-over-expressed BC including AZD8931. No IHC assays are optimized for reliable detection of HER2 in the non-over-expressed range. We have therefore developed a highly sensitive, robust HER2 IHC test with a low limit of detection (HER2Low Test).

Methods and results:

The HER2Low Test uses a novel, high affinity anti-HER2 rabbit monoclonal antibody. Antibody specificity was demonstrated by western blotting using cell lysates expressing HER1, HER2, HER3 or HER4. Analytical sensitivity experiments on BC cell lines showed an ability to differentiate lower levels of HER2 than HercepTest™. Three HER2Low Test staining categories were defined: Low, Medium and High, reflecting differing levels of HER2 expression. A comparative study between HER2Low Test and HercepTest™ on 182 AZD8931 clinical trial BC samples with appropriate ethical approval and consent (Table) showed a redistribution of HercepTest™ scores 0, 1+ and 2+ into HER2Low Test categories Low, Medium and High, demonstrating increased analytical sensitivity of the HER2Low Test. HER2Low Test reproducibility and repeatability studies showed intra-run agreement of 97%, inter-run agreement of 96%, inter-instrument agreement of 87% and observer-to-observer agreement of 89%.

Conclusion:

Presented here is a novel, reproducible and highly sensitive HER2 IHC assay. This is the first study to demonstrate accurate differentiation across a wide range of HER2 expression levels in non-over-expressed BC. This assay may supplement existing HER2 testing methods for the accurate segmentation of HER2 non-over-expressing BC patients.

HER2Low Test score and conventional HercepTest™ (HcT) score in the tested samples from a phase II clinical trial (NCT00900627" locator-type="CLINTRIALGOV">NCT00900627) HER2Low Test Total Low Medium High HcT 0 11 34 12 57 (31.3%) 1+ 4 24 36 64 (35.2%) 2+ 0 11 50 61 (33.5%) 3+ (not included) 0 0 0 0 (0.0%) Total 15 (8.2%) 69 (37.9%) 98 (53.9%) 182

Citation Format: Hans Christian Pedersen, Anni Thomsen, Kristian Jensen, Karen Jakobsen, Bob Wellings, Gillian Dalgliesh, Kerrie Tosh, Chris Harbron, Jill Walker. A new, highly sensitive IHC test for the detection of HER2 protein in breast cancer (BC) tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2384. doi:10.1158/1538-7445.AM2013-2384
Abstract 241: Tumor initiating cells and FZD8 play a major role in drugresistance and tumor progression in triple negative breast cancer.
Authors: Reddy Kaladhar B. , Yin Shuping , Daniel R. Bonfil , Banerjee Sanjeev , Sarkar Fazlul , Sethi Seema
Published: August 2014

Triple-negative breast cancer (TNBC) is usually associated with poor outcome in part due to lack of benefit from targeted therapy. Previous studies have shown that neoadjuvant chemotherapy before surgery was effective in the minority of women with TNBC; in contrast, the majority who had a residual tumor after treatment had a relatively poor outcome. In order to identify the mechanism by which residual cancer cells survive chemotherapy, we performed gene expression profile using CRL2335 TNBC cell line before and after treatment with cisplatin plus TRAIL. We found a significant increase in the expression of FZD8, one of the Wnt receptors, and its downstream targets LEF1 and TCF in residual tumor cells after treatment with cisplatin plus TRAIL compared with untreated controls. Inhibition of FZD8 by siRNA in the presence of cisplatin plus TRAIL reduced β-catenin and survivin levels, and increased apoptosis compared to scrambled siRNA-treated cells. In vivo, data demonstrate that cisplatin plus TRAIL treatment, significantly reduces tumor volume in nod/SCID mice. However, we found that cisplatin plus TRAIL treatment predominantly eliminated non-tumor initiating cells (non-TICs), as demonstrated by whole-body fluorescent imaging of mice injected with mammosphere-forming cells stably transfected with DsRed. This led to TIC enrichment in residual tumors, as confirmed by immunostaining for TIC markers. Moreover, an increase in FZD8 expression was observed in residual tumors treated with cisplatin and TRAIL. Taken together, our findings suggest that FZD8-mediated Wnt-signaling may play a major role in mediating resistance to chemotherapy, making it a potential target to enhance chemotherapeutic efficacy in patients with TNBC.

Citation Format: Kaladhar B. Reddy, Shuping Yin, R. Bonfil Daniel, Sanjeev Banerjee, Fazlul Sarkar, Seema Sethi. Tumor initiating cells and FZD8 play a major role in drugresistance and tumor progression in triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 241. doi:10.1158/1538-7445.AM2013-241
Abstract 2429: Airway necrosis after salvage esophagectomy.
Authors: Tanaka Norimitsu , Hokamura Nobukazu , Tachimori Yuji
Published: August 2014

Background: Recently definitive chemoradiotherapy (CRT) without surgery has been accepted as an alternative treatment for esophageal cancer. However, unfortunately, the tumors of 5-33% patients were resistant to CRT and persistent disease was detected soon after treatment, and 16-54% patients initially showed a response but locoregional disease was discovered later. Salvage esophagectomy is the sole curative intent treatment for patients with persistent or recurrent locoregional disease after definitive CRT for esophageal carcinoma. However, salvage esophagectomy is a very high-risk operation, and airway necrosis is a fatal complication.

Methods: Between 1997 and 2007, 49 patients with thoracic esophageal squamous cell carcinoma underwent salvage esophagectomy after definitive CRT. All patients received more than 50 Gy of radiation plus concurrent chemotherapy for curative intent. We retrospectively compared patients with and without airway necrosis. Additionally, airway necrosis was categorized primary necrosis and secondary necrosis according to the cause of necrosis, and operative procedures related to airway necrosis were investigated.

Result: Overall 23of 49 patients had at least one complication (morbidity: 46.9%), and 5 died during their hospitalization (mortality: 10.2%). Airway necrosis occurred in five patients (10.2%), of four patients (80%) died during their hospitalization. Airway necrosis seemed to be closely related to operative procedures, such as resection of bronchial artery (BA), cervical and subcarinal lymph node dissection (LND) and reconstruction route. Primary necrosis occurred in 3 patients with resection of BA, cervical LND, or subcarinal LND. And bronchogastric fistula following necrosis of gastric conduit occured in 2 patients reconstructed through posterior mediastinal route.

Conclusions: Airway necrosis is a highly lethal complication after salvage esophagectomy. It is important in salvage esophagectomy to take airway blood supply into consideration sufficiently and to reconstruct through retrosternal route to prevent bronchogastric fistula.

Citation Format: Norimitsu Tanaka, Nobukazu Hokamura, Yuji Tachimori. Airway necrosis after salvage esophagectomy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2429. doi:10.1158/1538-7445.AM2013-2429
Abstract 2477: Design, synthesis and biological evaluation of substituted monocyclic pyrimidines as potent microtubule targeting antitumor agents that circumvent Pgp and βIII-tubulin-mediated resistance.
Authors: Gangjee Aleem , Mohan Rishabh , Hamel Ernest , Bai Ruoli
Published: August 2014

Tubulin binding agents are the most effective drugs in p53 resistant cancer cell lines. Although microtubule disrupting agents like paclitaxel are highly successful, they have major limitations against multidrug resistance (MDR) tumors. Overexpression of P-glycoprotein (Pgp) and/or βIII-tubulin can severely limit their clinical utility as cancer chemotherapeutic agents. Antitubulin agents like paclitaxel and other taxoids also have poor water solubility, and this hampers their administration to patients since these compounds must be formulated with polyethoxylated castor oil. This medium can cause hypersensitivity reactions and requires prolonged administration times. Recently, we reported analogs with the pyrrolo[3,2-d]pyrimidine scaffold that circumvent these drawbacks. To explore the structure-activity relationships of these compounds, including simplification of the pyrrolo[3,2-d]pyrimidine scaffold, we designed conformationally flexible substituted monocyclic N-methyl-4-methoxyanilino pyrimidines 1-6 from the corresponding 4-chloro analogs by treatment with N-methyl-4-methoxyaniline followed by N6-alkylation for 2-6 . Compounds 1-6 inhibited tubulin assembly (4, IC50 = 1.5 ± 0.2 μM) and binding of colchicine to tubulin (4, 75% inhibition at 1 μM, 95% at 5 μM with 5 μM [3H]colchicine). Four of these compounds inhibited the growth of human cancer cells in culture with nanomolar IC50’s from 15 to 62 nM. These compounds are also active in paclitaxel resistant tumor cells overexpressing either βIII-tubulin or Pgp, thus apparently overcoming two important mechanisms associated with clinical resistance to paclitaxel. Thus, we have identified simplified substituted monocyclic pyrimidines containing a novel structural scaffold for inhibition of tubulin assembly and colchicine binding and which have potent cytotoxic activity against human cancer cells in culture.

Citation Format: Aleem Gangjee, Rishabh Mohan, Ernest Hamel, Ruoli Bai. Design, synthesis and biological evaluation of substituted monocyclic pyrimidines as potent microtubule targeting antitumor agents that circumvent Pgp and βIII-tubulin-mediated resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2477. doi:10.1158/1538-7445.AM2013-2477
Abstract 2481: Synthesis and characterization of PEGylated chlorin e6 sensitizers for the photodynamic killing of human ovarian cancer cells.
Authors: Ghogare Ashwini , Ghosh Goutam , Kimani Stanley , Bartusik Dorota , Rudshteyn Benjamin , Hasan Tayyaba , Greer Alexander
Published: August 2014

Pegylated chlorin e6 photosensitizers have been synthesized with triethylene glycol attached at the ester bond(s) for a 1:1 conjugate, a 2:1 conjugate, and a 3:1 conjugate. These chlorin sensitizers were studied for hydrolytic stability and solubility. Our studies included an analysis of ovarian OVCAR-5 cancer cell uptake, localization, and phototoxicity. Because of our interest in fiber-based photosensitizer and singlet oxygen delivery methods, desirable aspects of the 3:1 chlorin e6 conjugate make it a potential sensitizer for incorporation into a fiber optic device, particularly in the area of ovarian cancer photodynamic therapy.

Citation Format: Ashwini Ghogare, Goutam Ghosh, Stanley Kimani, Dorota Bartusik, Benjamin Rudshteyn, Tayyaba Hasan, Alexander Greer. Synthesis and characterization of PEGylated chlorin e6 sensitizers for the photodynamic killing of human ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2481. doi:10.1158/1538-7445.AM2013-2481
Abstract 2490: Novel application for pseudopodial proteomics using excimer laserablation and two-dimensional difference gel electrophoresis.
Authors: Mimae Takahiro , Ito Akihiko , Hagiyama Man , Kondo Tadashi , Okada Morihito
Published: August 2014

We developed a novel application to conduct pseudopodia proteomics. Pseudopodia are ventral actin-rich protrusions and play functional roles in cell migrations. Identification of pseudopodia proteins leads further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies. In our application, tumor cells were placed on a fibronectin-coated porous membrane to form pseudopodia. According to the motile potentials of the cells, the cells formed pseudopodial microprocesses in the pores. An excimer laser, which was used for ophthalmic refractive surgeries, horizontally ablated cells at the membrane surface to remove the cell body. The microscopic observations and the protein expression studies suggested that the laser treatment caused no apparent damages to pseudopodia. Proteins in whole cells and pseudopodia fractions were individually solubilized, labeled with a highly sensitive fluorescent dye, and separated using two-dimensional difference gel electrophoresis. Among 2508 protein spots observed, 211 had different intensity between whole cells and pseudopodia fractions (more than 4-fold differences and p value less than 0.05). The protein enrichment depended on the pore size. Mass spectrometric protein identification revealed 46 pseudopodia-localizing proteins. The localization of novel pseudopodia-localizing proteins such as RAB1A, HSP90B, TDRD7, and vimentin was confirmed using immunohistochemical examinations. The previous studies demonstrated that these four proteins may function in the cell migration process. In addition, RAB1A was indicated to play an important role in forming and extending pseudopodial microprocesses in human breast cancer cells by overexpression and knockdown experiments. This method will provide insights into the molecular details of pseudopodia and further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies.

Citation Format: Takahiro Mimae, Akihiko Ito, Man Hagiyama, Tadashi Kondo, Morihito Okada. Novel application for pseudopodial proteomics using excimer laserablation and two-dimensional difference gel electrophoresis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2013-2490
Abstract 2492: Targeted quantitation of candidate metabolic biomarkers for hepatocellular carcinoma.
Authors: Xiao Jun Feng , Zhao Yi , Varghese Rency , Zhou Bin , Poto Cristina Di , Zhang Lihua , Tadesse Mahlet G. , Ziada Dina Hazem , Shetty Kirti , Ressom Habtom W.
Published: August 2014

The aim of this study is to compare metabolite levels in sera of hepatocellular carcinoma (HCC) patients versus cirrhotic controls. This is accomplished through targeted quantitative analysis of candidate metabolic biomarkers of HCC by using an ultra-performance liquid chromatography coupled to an electrospray ionization-quadrupole linear ion trap mass spectrometry (UPLC-ESI-QLIT MS). The targets were selected from our previous untargeted analysis, where we observed association of HCC to dysregulated levels of bile acids, lysophosphocholines (LPCs), lysophosphoethanolamines (LPEs), sphingophoslipid, long chain carnitines and amino acids. In this study, we performed quantitative analysis of three metabolites (glycodeoxycholic acid, 3beta 6beta-dihydroxy-5beta-cholan-24-oic acid, 3 sulfo-glycodeoxycholic acid) in sera of 199 participants from two cohorts (US and Egypt). The participants consist of 110 subjects (55 HCC cases and 55 cirrhotic controls) from the US and 89 subjects (40 HCC cases and 49 cirrhotic controls) from Egypt. Through a logistic regression model, we analyzed the quantitative results obtained by the UPLC-ESI-QLIT MS to evaluate the ability of the three candidate markers as a panel in distinguishing HCC cases from patients with liver cirrhosis. Receiver operating characteristic (ROC) curve obtained through the regression model yielded an area under the curve (AUC) of 0.754 in distinguishing the HCC cases from cirrhotic controls for the US cohort and an AUC of 0.709 for the Egyptian cohort. The model resulted in an AUC of 0.717 for the combined US and Egyptian cohort. We compared the diagnostic capability of these metabolites versus α-fetoprotein (AFP) in the US cohort, for which AFP measurements were available for both the HCC cases and cirrhotic controls. While AFP alone resulted in an AUC of 0.681, the combination of AFP with the three metabolites increased the AUC to 0.806. These results indicate the potential of these candidate biomarkers in combination with AFP for more accurate diagnosis of HCC in high risk population of cirrhotic patients.

Citation Format: Jun Feng Xiao, Yi Zhao, Rency Varghese, Bin Zhou, Cristina Di Poto, Lihua Zhang, Mahlet G. Tadesse, Dina Hazem Ziada, Kirti Shetty, Habtom W. Ressom. Targeted quantitation of candidate metabolic biomarkers for hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2492. doi:10.1158/1538-7445.AM2013-2492

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2499: Diagnostic serum biomarkers for canine hemangiosarcoma: a potential model of human angiosarcoma.
Authors: Choi Eunju , Chen David , Lê Cao Kim-Anh , Bielefeldt-Ohmann Helle , O'Leary Caroline A. , Hill Michelle M.
Published: August 2014

People suffering from uncommon diseases often do not benefit from the advances in medical research due to lack of research. One such disease is angiosarcoma, a highly aggressive cancer originating from blood vessels with risk factors including toxic exposure, radiotherapy and lymphedema. Angiosarcoma in dogs (commonly called hemangiosarcoma) is a very similar disease with much higher prevalence, comprising up to 7% of all canine malignancies. For both species, an accurate diagnostic method is needed for more favorable outcome.

We developed a biomarker discovery pipeline consisting of a high-throughput biomarker discovery platform and a database for data mining and statistical analysis for candidate identification. The platform screens for differential glycosylation of serum glycoproteins as potential biomarkers utilizing 20 individual lectins followed by direct coupling to tandem mass spectrometry. The database facilitates the identification of differential glycosylation and produces a ranked list of potential biomarker candidates. Using this pipeline, sera from 10 dogs with hemangiosarcoma with 10 age and sex-matched control sera were processed and analyzed.

Candidates from the database were ranked based on its ability to classify normal and cancer. The ranked list of candidates for this screen composed of glycoproteins involved in cell migration, tumor immune response, and glycoproteins reported to be altered in human and canine angiosarcoma and other cancer types. Some examples will be shown to illustrate the change in specific glycosylation structures associated with cancer as measured by binding to specific lectin(s) while binding to other lectins are not changed. These glycosylation-specific changes are potentially more specific diagnostic biomarkers than circulating proteins alone.

We are currently validating the identified candidates and pursuing the development of clinical tests to improve diagnosis and clinical outcomes of angiosarcoma for both dogs and humans.

Citation Format: Eunju Choi, David Chen, Kim-Anh Lê Cao, Helle Bielefeldt-Ohmann, Caroline A. O'Leary, Michelle M. Hill. Diagnostic serum biomarkers for canine hemangiosarcoma: a potential model of human angiosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2499. doi:10.1158/1538-7445.AM2013-2499
Abstract 2506: Histology-directed lipid MALDI MS profiling can distinguish prostate cancer from normal epithelium.
Authors: Choi Seungho , Park Jun Won , Park Weon Seo , Kim Hark Kyun , Lee Kang Hyun
Published: August 2014

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is demonstrated to be useful for profiling of common solid tumors. In this approach, mass spectra are obtained from discrete locations on 10 μm-thick cryosection tissue, guided by H&E-stained top slide. To determine if a lipid signature obtained from tissue samples could be found to assist with diagnosis, frozen surgical samples collected from 7 prostate cancer patients and 9 adjacent normal tissue samples were analyzed using matrix (DHB/CHCA)-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). When leave-one-out cross validation was performed using support vector machine, permutation p value for the cross-validated misclassification rate was statistically significant (p=0.04), suggesting that lipid profiles are clearly different between prostate cancer and adjacent normal tissue samples. One of the most striking difference between prostate cancer and normal tissue is the phosphatidylcholine (PC) 32:1 (m/z 798.6) overexpression (p=0.007, tumor/normal=2.4). In order to identify molecular mechanisms of PC 32:1 overexpression in prostate cancers, we performed immunohistochemical analyses for metabolic enzymes responsible for phosphatidylcholine biosynthesis. Prostate cancers overexpress choline kinases and phosphate cytidylyltransferases, known oncogenes which generate phosphatidylcholines, compared with adjacent normal tissue samples. Thus, lipid MALDI MS analysis captures a global alteration in lipid profile of prostate cancer tissue, potentially distinguishing cancerous epithelium from normal epithelium. To our knowledge, this study is the first comprehensive lipid profiling study in prostate cancer. This histology-directed MALDI MS approach enables rapid and sensitive identification of discriminatory lipids in cancerous epithelium of prostate cancers, even those with infiltrative growth.

We are in the process of a large sample set (Supported by Korean Ministry of Education, Science, and Technology Grant 2012K001506).

Citation Format: Seungho Choi, Jun Won Park, Weon Seo Park, Hark Kyun Kim, Kang Hyun Lee. Histology-directed lipid MALDI MS profiling can distinguish prostate cancer from normal epithelium. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2506. doi:10.1158/1538-7445.AM2013-2506

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2542: Evidence for large somatic structural events detected as autosomal genetic mosaicism in GWAS data.
Authors: Machiela Mitchell J. , Zhou Weiyin , Yeager Meredith , Jacobs Kevin , Berndt Sonja , Lan Qing , Rothman Nathaniel , Savage Sharon , Taylor Phil R. , deVivo Immaculata ,   National Cancer Institute Detectable Clonal Mosaicism Consortium, Hoover Robert N. , Tucker Margaret A. , Chanock Stephen J.
Published: August 2014

Recent evidence indicates human mosaic abnormalities may be more common than previously expected. We performed an analysis on blood and buccal DNA of 30,918 individuals from a collection of 54 cancer-related studies using a modified mosaic alteration detection algorithm on renormalized B-allele frequencies and log2 relative probe intensity ratios from commercially available Illumina SNP microarray chips. 391 autosomal abnormalities >2Mb in size were detected in 232 (0.75%) individuals. The distribution includes copy-neutral loss of heterozygosity constituting 192 (49%) events, copy gains 85 (22%) events, and copy losses 102 (26%) events. There was no difference between blood and buccal sources of DNA. An overall analysis by age confirmed our earlier finding (p=8.93e-07), which was also observed in controls (p=0.0086). Similarly, an overall analysis by sex confirmed mosaic abnormalities are more frequent in males than females (p=0.0016); and this association was also observed in a control-only analysis (p=0.0019). Two or more distinct autosomal events were observed in 43 individuals. Comparable to earlier studies, the most common events were 4q copy neutral loss of heterozygosity, 9q copy neutral loss of heterozygosity, 13q14 deletion and 20q deletion. 13q14 deletion was observed in cancer-free controls and individuals with epithelial cancers. Cancer specific analyses for bladder, endometrium, stomach, lung (in nonsmoking women from Asia), osteosarcoma, and prostate cancer revealed no significant association with risk for cancer. These results replicate the prevalence and age/sex relationships from our previous findings in an analysis of 57,853 individuals and further suggest the importance of studying mosaic abnormalities in the general human population. The findings underscore the importance of thorough characterization of germline DNA in parallel with somatic characterization. Our results provide further support for age-related genomic senescence and could represent the tip of the iceberg for smaller somatic events.

Citation Format: Mitchell J. Machiela, Weiyin Zhou, Meredith Yeager, Kevin Jacobs, Sonja Berndt, Qing Lan, Nathaniel Rothman, Sharon Savage, Phil R. Taylor, Immaculata deVivo, National Cancer Institute Detectable Clonal Mosaicism Consortium, Robert N. Hoover, Margaret A. Tucker, Stephen J. Chanock. Evidence for large somatic structural events detected as autosomal genetic mosaicism in GWAS data. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2542. doi:10.1158/1538-7445.AM2013-2542
Abstract 2546: Fine-mapping identifies multiple prostate cancer risk loci at 5p15, one of which associates with TERT expression.
Authors: Kote-Jarai Zsofia , Saunders Edward , Leongamornlert Daniel , Tymrakiewicz Malgorzata , Dadaev Tokhir , Jugurnauth Sarah , Benlloch Sara , Amin Al Olama Ali , Ross-Adams Helen , Neal David ,   The UKGPCS Collaborators and The PRACTICAL Consortium, Douglas Easton, Eeles Rosalind
Published: August 2014

We have previously reported an association between prostate cancer (PrCa) risk and rs2242652 on 5p15. rs2242652 lies in intron 4 of TERT, which encodes telomerase reverse transcriptase, the catalytic subunit of the telomerase ribonucleoprotein complex. Telomerase catalyzes the de novo addition of telomere repeat sequences on to chromosome ends and thereby counterbalances telomere-dependent replicative senescence. Associations between SNPs in the TERT region and multiple cancer types have been reported; however no correlation has been observed thus far between the cancer-associated SNPs in TERT and gene expression or telomere length. To comprehensively evaluate the association between genetic variation across this region and PrCa we performed a fine-mapping analysis by direct genotyping using either a custom Illumina iSelect array (114 SNPs on iCOGS) or Sequenom MassArray iPlex (25 SNPs) followed by imputation of 1,094 SNPs in 22,301 PrCa cases and 22,320 controls in the PRACTICAL consortium. To determine independently associated variants in this region, we performed multiple stepwise logistic regression analysis; SNPs were included in the model if they were significant at P<10−4 after adjustment for other SNPs. Regression models identified multiple independent associations, reflecting the complexity of this region. These SNPs fall into four regions consisting of clusters of highly or moderately correlated variants, and there is only weak LD between SNPs in different regions. To investigate whether SNPs in any of these regions were associated with TERT gene expression, we performed qPCR assays on RNA from benign prostate tissue samples using the Fluidigm Biomark™ HD system. We found evidence that the risk reducing alleles of several variants in region one associated with elevated TERT expression, providing a plausible mechanism for the differential effect of SNPs on PrCa risk. Deep re-sequencing of these loci may help to further refine this region and facilitate selection of prospective causal variants for functional validation studies.

Citation Format: Zsofia Kote-Jarai, Edward Saunders, Daniel Leongamornlert, Malgorzata Tymrakiewicz, Tokhir Dadaev, Sarah Jugurnauth, Sara Benlloch, Ali Amin Al Olama, Helen Ross-Adams, David Neal, The UKGPCS Collaborators and The PRACTICAL Consortium, Douglas Easton, Rosalind Eeles. Fine-mapping identifies multiple prostate cancer risk loci at 5p15, one of which associates with TERT expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2546. doi:10.1158/1538-7445.AM2013-2546
Abstract 2548: Targeted sequencing to identify rare variants in colorectal cancer.
Authors: DeRycke Melissa S. , Gunawardena Shanaka R. , Middha Sumit , McDonnell Shannon K. , Riska Shaun M. , Fogarty Zachary C. , Eckloff Bruce W. , Schaid Daniel J. , Goode Ellen L. , Thibodeau Stephen N.
Published: August 2014

Colorectal cancer (CRC) is estimated to be the third most common cancer in both men and women in the US in 2012, accounting for over 50,000 deaths. Familial CRC accounts for approximately 30% of all CRC, yet only 5% of familial CRCs can be attributed to mutations in known hereditary colon cancer (HCC) genes. Many families with a strong family history of CRC do not have mutations in known CRC genes. Finding the genes underpinning CRC in these families may improve screening for disease and allow the development of more targeted therapeutics.

We completed targeted sequencing of over 2,000 genes and miRNAs in 1,500 cases of CRC from the Colon Cancer Family Registry (CCFR) in an effort to identify rare variants associated with CRC risk and to identify mutations in known CRC genes missed by conventional sequencing. Several categories of genes were included: 18 known HCC genes; 18 candidate HCC genes; 185 genes in previously reported CRC linkage peaks; 1,423 miRNAs; and 363 genes identified through whole exome sequencing of 40 affected cases in 16 families with hereditary CRC (n=2,007 total genes sequenced). Genomic DNA samples were indexed, then libraries were made using Agilent's SureSelect Targeted Enrichment kit. Samples were pooled (96 samples/pool), then each pool of samples was run on five lanes on an Illumina HiSeq2000 (average of 19.2 samples/lane). GenomeGPS, an in-house developed workflow, was used for read alignment, variant calling, and annotation.

In early analyses of the first 161 cases, the mean number of mapped reads exceeded 20 million per sample and over 90% of the target region exceeded 40x coverage. A total of 26,484 SNPs were identified and passed quality filtering. Sequencing identified known mutations in MMR genes in positive control cases as well as mutations in HCC genes in previously untested cases. Analysis is currently on-going on the remaining samples and we will report results on the full set of 1500 cases at the meeting.

Citation Format: Melissa S. DeRycke, Shanaka R. Gunawardena, Sumit Middha, Shannon K. McDonnell, Shaun M. Riska, Zachary C. Fogarty, Bruce W. Eckloff, Daniel J. Schaid, Ellen L. Goode, Stephen N. Thibodeau. Targeted sequencing to identify rare variants in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2548. doi:10.1158/1538-7445.AM2013-2548
Abstract 2549: A genome-wide association study identifies PSCA as a susceptibility locus for gastric cancer and duodenal ulcer in the Japanese population.
Authors: Tanikawa Chizu , Matsuda Koichi , Nakamura Yusuke
Published: August 2014

Through a genome-wide association analysis using a total of 7,035 duodenal ulcer cases and 25,323 controls of Japanese populations, we identified two susceptibility loci at the Prostate stem cell antigen (PSCA) on 8q24 and at the ABO blood group (ABO) on 9q34. A C-allele of rs2294008 at PSCA increased the risk of duodenal ulcer (odds ratio (OR) of 1.84 with P value of 3.92 x 10-33) in a recessive model, while it decreased the risk of gastric cancer (OR of 0.79 with P value of 6.79 x 10-12) as reported previously. A T-allele of SNP rs2294008 created the upstream translational initiation codon and affects the protein localization from cytoplasm to cell surface. SNP rs505922 on ABO also associated with duodenal ulcer in a recessive model (OR of 1.32 with P value of 1.15 x 10-10). Our finding implies the crucial roles of genetic variations on the pathogenesis of duodenal ulcer.

Citation Format: Chizu Tanikawa, Koichi Matsuda, Yusuke Nakamura. A genome-wide association study identifies PSCA as a susceptibility locus for gastric cancer and duodenal ulcer in the Japanese population. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2549. doi:10.1158/1538-7445.AM2013-2549
Abstract 2551: A genome-wide association study of HCV induced liver cirrhosis in the Japanese population identifies novel susceptibility loci at MHC region.
Authors: Koichi Matsuda , Tanikawa Chizu , Nakamura Yusuke
Published: August 2014

To identify a prognostic factor(s) for patients with chronic hepatitis C (CHC), we conducted a genome-wide association study (GWAS) using 682 hepatitis C virus (HCV)-induced liver cirrhosis (LC) cases and 1,045 CHC patients in Japan. Eight SNPs which showed possible associations (P < 1.0 x 10-5) in the GWAS stage were further genotyped using 936 LC cases and 3,809 CHC patients. We found that two SNPs within the major histocompatibility complex (MHC) region on chromosome 6p21, rs910049 and rs3135363, were significantly associated with the progression from CHC to LC (Pcombined = 9.15 x 10-11 and 1.45 x 10-10, odds ratio (OR) = 1.46 and 1.37, respectively). We also found that HLA-DQA1*0601 and HLA-DRB1*0405 were associated with progression from CHC to LC (P = 4.53 x 10-4 and 1.54 x 10-4 with OR = 2.80 and 1.45, respectively). Multiple logistic regression analysis revealed that rs3135363, rs910049, and HLA-DQA1*0601 were independently associated with the risk of HCV-induced LC. In addition, individuals with four or more risk alleles for these three loci have a 2.83-fold higher risk for LC than those with no risk allele, indicating the cumulative effects of these variations. Our findings elucidated the crucial roles of multiple genetic variations within the MHC region as prognostic/predictive biomarkers for CHC patients.

Citation Format: Matsuda Koichi, Chizu Tanikawa, Yusuke Nakamura. A genome-wide association study of HCV induced liver cirrhosis in the Japanese population identifies novel susceptibility loci at MHC region. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2551. doi:10.1158/1538-7445.AM2013-2551