bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2713: Small-molecule FASN inhibitors promote growth inhibition growth and apoptosis of breast cancer
Authors: Lupu Ruth , Guerrico Anatilde Gonzalez , Khurana Ashwani , Heuer Timothy , Kemble George , KVP Chandra Mohan
Published: May 2015

Increased de novo lipogenesis due to enhanced expression of several lipogenic enzymes has been found in many types of human cancers. Fatty acid synthase (FASN), the key enzyme in the de novo lipogenesis pathway, is one of the most commonly over-expressed lipogenic enzymes. Studies in the past decade have recognized FASN overexpression as a molecular marker for poor prognosis and malignant phenotype of many cancers. Tumor-associated FASN, by conferring growth and survival advantages rather than functioning as an anabolic energy-storage pathway, has also been suggested to play an active part in the development, maintenance, and metastatic progression of human cancers. More recently, FASN has been implicated in contributing to cellular resistance to several anticancer treatments. A variety of agents have been developed to target lipogenic enzymes and the key regulators involved in lipid metabolism in cancer cell for therapeutic purposes. The development of several FASN inhibitors has been reported from both academic labs and industries. When used in in vitro, xenograft and genetically induced mouse model studies, these inhibitors have supported FASN as an excellent target. A new generation of FASN inhibitors was developed at 3-V Biosciences that are highly specific, reversible inhibitors of FASN. One compound in this series, TVB-2640, is orally bioavailable and has progressed into Phase 1 clinical studies. The effect of two anti-FASN drugs in this series, TVB-3150 and TVB-3199 were tested for their effect on breast cancer cells. We demonstrated that the EC50 is between 9-50 nM, depending on the drug and the tested cell line. We then demonstrated that the drugs inhibited the anchorage-dependent and -independent growth of FASN expressing cells in a dose dependent manner. The apparent mechanism by which the FASN inhibitors promote inhibition of growth is by inducing apoptotic cell death in a time- and dose-dependent manner that involves regulation of apoptotic and pro-apoptotic proteins. Furthermore, the combination of the anti-FASN drugs with small molecule inhibitors of the antiapoptotic proteins Bcl-2 and Bcl-xL significantly increased the apoptotic effect in a synergistic manner compared to either of the synthetic inhibitors alone. In vivo studies demonstrate tumor growth inhibition without toxicity. Clinical development of a first-in-class, orally active, potent, and reversible FASN inhibitor as a novel cancer therapeutic is ongoing.

Citation Format: Ruth Lupu, Anatilde Gonzalez Guerrico, Ashwani Khurana, Timothy Heuer, George Kemble, Chandra Mohan KVP. Small-molecule FASN inhibitors promote growth inhibition growth and apoptosis of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2713. doi:10.1158/1538-7445.AM2014-2713
Abstract 2719: Use of abiraterone against a luminal androgen receptor breast cancer cell line
Authors: Rao Krishna A. , Modur Vishnu , Ghosh Sumana
Published: May 2015

Background: Data from this pre-clinical model suggests that the LAR TNBC subtype is unique and may respond to novel hormonal agents such as bicalutamide or abiraterone. The LAR subtype represents 11 % of all TNBC (which represent 20% of all breast cancers). Thus, positive studies of abiraterone could potentially extend the application of abiraterone to an additional 2% of all breast cancer patients, who could be treated in the metastatic, neoadjuvant, and adjuvant setting (for five years) with hormonal maintenance therapy. We, therefore, conducted studies of abiraterone on LAR TNBC cell line, MDA-MB-453. .

Methods: i). Cell proliferation- Cell proliferation will be done by plating 500,000 cells in triplicate Petri dishes and measuring cell number over a six day period between parent cells and cellstreated with the following varying concentrations of abiraterone: 100 ng/ml, 250 ng/ml, and 400 ng/ml. ii).Cell cycle effects- Cells were incubated for 24 hours with abiraterone and then lifted from the plate with trypsin/EDTA (0.5%/0.1%) and fixed overnight in 80% ethanol. This was followed by staining in PBS with 10 ug/ml of propidium iodide for twenty minutes at room temperature. Flow cytometry was performed in our facility, and data was obtained on the percentage of the cell population in various points of the cell cycle. iii). Apoptosis and necrosis assay (Annexin-V-PI assay)- Cells were harvested at 72 hours post-seeding and incubation with abiraterone and washed with ice-cold PBS, followed by staining with 1ug/ml of PI and 5ul of Alexa Fluor® 488 annexin V in 100ul of annexin binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). After the incubation at room temperature for 15 minutes, 400ul of ice-cold annexin binding buffer was added and stained cells were analyzed by Becton-Dickinson FACSCalibur Flow Cytometer.

Results: Abiraterone caused a significant impact on cell proliferation and promoted apoptosis in the MDA-MB-453 population. Cell cycle effects were also noted with abiraterone treatment.

Conclusion: Abiraterone has activity against MDA-MB-453, a breast cancer cell line known to express luminal androgen receptor. The results suggest that further studies may be warranted with this agent against this unique subtype of breast cancer.

Citation Format: Krishna A. Rao, Vishnu Modur, Sumana Ghosh. Use of abiraterone against a luminal androgen receptor breast cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2719. doi:10.1158/1538-7445.AM2014-2719
Abstract 2731: PELP1 promotes DNA double-strand break repair via alternative-NHEJ: Implications in therapy resistance
Authors: Krishnan Samaya R. , Nair Binoj C. , Sareddy Gangadhara R. , Mann Monica , Vadlamudi Ratna K.
Published: May 2015

Estrogen Receptor positive breast cancer is commonly treated with hormonal therapy; however, many patients acquire therapy resistance, a major clinical problem. Recent evidence suggests that the accumulation of chromosomal abnormalities via increased error-prone Alternative-Non-Homologous End Joining (A-NHEJ) DNA repair pathway could contribute to breast cancer therapy resistance. Proline, Glutamic acid, Leucine rich Protein-1 (PELP1), an oncogenic co-regulator for transcription factors, is commonly overexpressed in breast cancer, and its deregulation has been implicated in therapy resistance. Our recent studies identified PELP1 as a novel substrate of the DNA damage response (DDR) kinases. In this study, we discovered that PELP1 regulates the A-NHEJ pathway, and we developed a novel peptide drug that targets PELP1-A-NHEJ axis for the treatment of therapy resistance. Our results suggest that PELP1 is phosphorylated at Ser1033 by DDR kinases and phosphorylated PELP1 co-localizes with the gamma-H2AX foci. Using Homologous Recombination (HR) and NHEJ pathway specific reporter assays, we found that PELP1 knockdown decreases NHEJ, with no apparent effect on the HR pathway. Further, using A-NHEJ specific reporter cell line, we found that PELP1 knockdown decreased the frequency of A-NHEJ, while PELP1 overexpression increased the repair frequency. PELP1's ability to regulate A-NHEJ pathway was further confirmed by sequencing repaired plasmids and by quantitating the degree of end resection. More importantly, metaphase chromosome spreads revealed gross chromosomal abnormalities in PELP1 deregulated breast cancer cells. Mechanistic studies revealed that PELP1 interacts with Mre11, and modulates the degree of end resection at the DNA double strand breaks. Mapping studies identified the C-terminus of PELP1 as the binding site for Mre11. Using yeast based genetic screen composed of 10 million peptides, we identified a unique peptide, PIP3 that binds the C-terminus of PELP1. We developed a stapled peptide of PIP3 (sPIP3), which functioned as a potent cytotoxic agent for breast cancer cells with little activity on normal cells. Further, sPIP3 efficiently inhibited the A-NHEJ pathway and significantly, reduced survival and promoted apoptosis of therapy resistant model cells. Collectively, these findings suggest that PELP1 plays a critical role in the A-NHEJ pathway and sPIP3 represents a novel drug for the treatment of therapy resistant breast cancer.

Citation Format: Samaya R. Krishnan, Binoj C. Nair, Gangadhara R. Sareddy, Monica Mann, Ratna K. Vadlamudi. PELP1 promotes DNA double-strand break repair via alternative-NHEJ: Implications in therapy resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2731. doi:10.1158/1538-7445.AM2014-2731
Abstract 275: Clinical and demographic comparison between bladder cancer and upper urinary tract urothelial carcinoma in Taiwan
Authors: Tung Min-Che , Chen Hsin-An , Su Chih-Ming , Huang Ming-Te , Chiou Hung-Yi , Wang Yuan-Hung
Published: May 2015

Objective Urothelial carcinoma (UC) which is derived from the urothelium of the urinary tract includes cancers of bladder, renal pelvis and ureter. The aim of this study was to compare clinical and demographic features between bladder and upper urinary tract urothelial carcinoma in Taiwan.

Material and methods The present study recruited a total of 736 histopathological confirmed UC cases, which consisted of 470 bladder cancer and 266 upper urinary tract UC (UTUC) cases. Clinical and demographic characteristics were collected by structured question interview and medical chart review. This study was approved by the institutional review boards of the collaborated hospitals. The effects of risk factors on UC risk were estimated by odds ratios (ORs) and its 95% confidence intervals (CIs) using unconditional logistic regression. All statistical analyses were performed using Statistical Analysis Software for Windows, version 9.1 (SAS Institute, Cary, NC).

Results The frequencies of cigarette smoking and alcohol consumption in bladder cancer cases were significantly higher than that in UTUC cases. Compared with bladder cancer cases, UTUC cases also had a significantly higher frequencies of advanced tumor stage (T2-4) and poorly differentiation (G3). In a multivariate- adjusted logistic regression, we found the advanced stage (T2-4) was a significant prognostic factor of overall survival for bladder cancer and UTUC cases, especially the risk of the T4 stage in UTUC cases.

Conclusion Our results showed that some clinical and demographic features between bladder and UTUC cases were different in Taiwan.

Keywords: bladder cancer; ueothelial carcinoma; upper urinary tract

Citation Format: Min-Che Tung, Hsin-An Chen, Chih-Ming Su, Ming-Te Huang, Hung-Yi Chiou, Yuan-Hung Wang. Clinical and demographic comparison between bladder cancer and upper urinary tract urothelial carcinoma in Taiwan. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 275. doi:10.1158/1538-7445.AM2014-275
Abstract 2754: Induction of Sp1 factor by CYP1B1 inhibits TRAIL-mediated apoptotic pathway
Authors: Kwon Yeo-Jung , Park Nahee , Shin Sangyun , Ye Dong-Jin , Hong Mihye , Chun Young-Jin
Published: May 2015

Human cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and is known as a major enzyme for estradiol 4-hydroxylation. It has been reported that CYP1B1 expression is higher in the tumor tissues than the normal ones, especially in hormone-related cancers such as breast, ovarian and prostate. Previously, we identified that CYP1B1 induces cell proliferation by activation of Wnt/β-catenin signal pathway. To explore the role of CYP1B1 on cancer cell proliferation further, we investigated whether CYP1B1 blocks apoptotic pathways. Using Western blot and RT-PCR, the expression of TRAIL was examined in MCF-7 cells, and we found that CYP1B1 overexpression or treatment with a CYP1B1 inducer, 7, 12-dimethylbenz[α]anthracene (DMBA) caused suppression of TRAIL and its receptors (DR4 and DR5) expression. However, when cells were treated with CYP1B1 siRNA or a selective CYP1B1 inhibitor, tetramethoxystilbene (TMS), expression of TRAIL and its receptors were significantly increased. Sp1, a zinc finger-containing transcription factor, has been reported to suppress TRAIL gene expression by binding TRAIL promoter. Interestingly, CYP1B1 overexpression and DMBA induced Sp1 expression and TMS was able to prevent DMBA-induced Sp1 expression. Sp1 knockdown with siRNA suppressed c-FLIP, an inhibitor protein of TRAIL-mediated apoptosis while it enhanced TRAIL and DR4 expression. These results were reversed by Sp1 overexpession. Moreover, attenuation of Sp1 DNA binding activity using mithramycin A, a Sp1 binding inhibitor, resulted in recovery of TRAIL expression suppressed by DMBA. Taken together, these data suggest that CYP1B1 prevents apoptosis via TRAIL suppression and this is mediated by Sp1 induction.

Citation Format: Yeo-Jung Kwon, Nahee Park, Sangyun Shin, Dong-Jin Ye, Mihye Hong, Young-Jin Chun. Induction of Sp1 factor by CYP1B1 inhibits TRAIL-mediated apoptotic pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2754. doi:10.1158/1538-7445.AM2014-2754
Abstract 276: Appalachian mountaintop mining particulate matter induces malignant transformation and tumorigenesis of human lung epithelial cells
Authors: Luanpitpong Sudjit , Luo Juhua , Kneuckles Travis , Hendryx Michael , Rojanasakul Yon
Published: May 2015

Lung cancer is the leading cause of cancer death, and after smoking, environmental and occupational exposure is the major cause. West Virginia (WV), the biggest coal mining state in Appalachia, ranks the third highest rate of lung cancer incidence in the United States. Our epidemiological studies suggest that living near WV mountaintop coal mining (MTM) activities is a contributing factor of the high lung cancer risk. The purpose of this study was to investigate the effect of MTM particle exposure on cell transformation and tumorigenesis of human lung epithelial cells. Air particulate matter (PM) collected within one mile of active MTM site in Edwight, WV (PMMTM), consisting of silica (Si, 48%) and molybdenum (Mo, 29%) as major constituents, was used for cell exposure studies. As control, PM collected from the neighboring areas that do not have coal mining in Green Bank, WV (PMCON) was used. Chronic exposure (3 months) of non-tumorigenic human lung epithelial BEAS-2B cells to sub-cytotoxic concentration (1 μg/mL) of PMMTM, but not PMCON, was found to induce malignant transformation of the cells, as indicated by loss of contact inhibition and anchorage-independent cell growth. The PMMTM-transformed cells also induced tumor formation in vivo using a xenograft mouse model. Exposure to Mo (1 μg/mL), but not Si, similarly induced malignant transformation and tumorigenesis of the lung epithelial cells, suggesting that Mo may be a key contributing factor of PMMTM pathologic effects.

Citation Format: Sudjit Luanpitpong, Juhua Luo, Travis Kneuckles, Michael Hendryx, Yon Rojanasakul. Appalachian mountaintop mining particulate matter induces malignant transformation and tumorigenesis of human lung epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 276. doi:10.1158/1538-7445.AM2014-276
Abstract 2762: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations
Authors: Yamada Tadaaki , Carbone David Paul , Takeuchi Shinji , Fujita Naoya , Yano Seiji
Published: May 2015

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, show favorable effects in EGFR mutant lung cancer. However, responders eventually develop acquired resistance almost without exception, and secondary T790M mutations in EGFR account for approximately 50% of the acquired resistance. Recent studies indicated that EGFR mutant lung cancer cells are dependent on EGFR/Akt signaling for survival even after acquired T790M secondary mutation. Akt kinase-interacting protein1 (Aki1) is a scaffold protein that binds to wild-type EGFR after EGF ligand stimulation, maintains signaling specificity, and mediates activating signal to the PI3K/PDK1/Akt pathway. We hypothesized that Aki1 may play important roles in signal transduction in EGFR mutant lung cancer cells. Here, we showed that Aki1 constitutively associates with mutant EGFR in lung cancer cells with (H1975) or without (PC-9 and HCC827) T790M secondary mutation. Silencing of Aki1 inhibited phosphorylation of Akt and S6, and induced apoptosis of EGFR mutant lung cancer cells. Treatment with Aki1 siRNA markedly inhibited growth of H1975 cells in a xenograft model. Moreover, silencing of Aki1 inhibited H1975 cell growth in combination with new generation EGFR-TKIs in vitro. Aki1 was frequently expressed in tumor cells of EGFR mutant lung cancer patients (53/56 cases), including those with acquired resistance to EGFR-TKI treatment (7/7 cases). Our data suggest that Aki1 may be a key mediator of survival signaling from mutant EGFR, and may therefore be an ideal target for EGFR mutant lung cancer patients, especially those with acquired EGFR-TKI resistance due to EGFR T790M secondary mutation.

Citation Format: Tadaaki Yamada, David Paul Carbone, Shinji Takeuchi, Naoya Fujita, Seiji Yano. Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2762. doi:10.1158/1538-7445.AM2014-2762
Abstract 2782: The effect of ERRFI1 as a novel AKT regulator on cell proliferation and response to therapy is cell context dependent
Authors: Hou Junmei , Bhinge Kaustubh N. , Wang Liewei
Published: May 2015

ERRFI1, a negative regulator of the EGFR family, is known to inhibit EGFR activation along with downstream of EGFR signaling. It is recognized as a tumor suppressor. However, ERRFI1 has multiple functions, and here, we present a novel function of ERRFI1. We found that it can directly interact with AKT and positively regulate AKT activity through regulation of AKT473 site by inhibiting PHLPP, the phosphatase access to AKT. The effect of this regulation on cell proliferation and on response to chemotherapy is cell context dependent, e.g. EGFR levels. We hypothesize that in cancer cells with high EGFR expression, down regulation of ERRFI1 enhances EGFR signaling, while on the other hand, increases AKT dephosphorylation. The strength of these two opposing signals will determine the eventual cellular phenotypes. In cancer cells with low EGFR expression, the direct effect of ERRFI1 on AKT is dominant, resulting in activation of AKT. To test these hypotheses, we knockdown ERRFI1 in multiple human cancer cell lines with different EGFR levels. Knockdown of ERRFI1 in high EGFR expressing cells resulted in an increase of cell proliferation and resistance to gemcitabine treatment, and this effect was due to the dominant negative effect of ERRFI1on EGFR, which was confirmed by increased EGFR signaling. Knockdown of ERRFI1 in cell lines with moderate level of EGFR also increased cell proliferation and resistance to gemcitabine, although biochemically, down regulation of ERRFI1 reduced AKT pS473 levels, but increased ERK/MAPK signaling. The addition of EGFR inhibitor, gefitinib, overcame gemcitabine resistance due to downregualtion of ERRFI1. In contrary, in cell lines with low level of EGFR, ERRFI1 positively regulated AKT signaling via directly binding to AKT and blocking PHLPP access to AKT. Therefore, down-regulation of ERRFI1 inhibited cell proliferation and sensitized cells to gemcitabine. In summary, we identified a novel function of ERRFI1 as a positive AKT regulator and its effect on cell proliferation and response to therapy is cell context dependent, e.g. EGFR levels. These results provide us insights into the dual roles of ERRFI1 in regulation of EGFR and AKT pathway and would help us to better individualize cancer therapy.

Citation Format: Junmei Hou, Kaustubh N. Bhinge, Liewei Wang. The effect of ERRFI1 as a novel AKT regulator on cell proliferation and response to therapy is cell context dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2782. doi:10.1158/1538-7445.AM2014-2782
Abstract 2785: LYN is a new prognostic and therapeutic target in non-small cell lung cancer
Authors: Kim Yu Jin , Sung Minjung , Park Min Jeong , Shin Young Kee , Choi Yoon-La
Published: May 2015

SRC family kinases (SFKs) have a strong rationale as a clinical strategy in non-small cell lung cancer (NSCLC). Unfortunately, the reviewed study showed disappointing activity of SFK inhibitor dasatinib in molecularly unselected patient cohort. Therefore, the studies in NSCLC should be investigated to identify patients more likely to benefit from dasatinib either as monotherapy or in combination with other agents. Here, we considered that LYN may play specific roles in lung cancer and therefore analysed survival prognosis according to the expression of LYN and the effect of dasatinib on the inhibition of cell growth and LYN activity. We evaluated LYN expression by immunohistochemistry on 446 NSCLCs using paraffin-embedded tissues. Elevated LYN expression level of NSCLCs, especially in adenocarcinoma, female, and non-smoker, affected poor overall survival (OS, P=0.036). In lung adenocarcinoma cell lines, LYN expression impacted on cell proliferation, migration and invasion. Dasatinib treatment resulted in decreased cell viability in most of these cell lines. Also, we found that dasatinib suppressed active tyrosine phosphorylation of LYN. In the resistant cell lines, H23 and SK-LU-1, which showed elevated epidermal growth factor (EGFR) and MET activities, combined treatment of EGFR or MET inhibitor with dasatinib revealed the synergistic effects on decreased cell viability. Our findings highly suggest that LYN has potential as a new selected target molecule of SFKs in the treatment of NSCLCs.

Citation Format: Yu Jin Kim, Minjung Sung, Min Jeong Park, Young Kee Shin, Yoon-La Choi. LYN is a new prognostic and therapeutic target in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2785. doi:10.1158/1538-7445.AM2014-2785
Abstract 2794: CD8+ natural regulatory T cells as antitumor immune effectors in carcinomas of breast and lung
Authors: Sawant Anandi , Lee Carnella , Schafer Cara , Deshane Jessy , Ponnazhagan Selvarangan
Published: May 2015

CD8+ T cells are cytolytic effectors of adaptive immunity against invading pathogens, and under various pathological conditions including cancer. In syngeneic, immunocompetent murine models of epithelial cancers, including breast and lung cancer, we observed up-regulation in frequencies of a unique CD8+ T cell population, characterized as CD8+CD25-Foxp3+, referred here as CD8+ natural regulatory T cells. This regulatory cell population was present in very high numbers during initial stages of tumor growth. However, a drastic reduction in CD8+ natural regulatory T cells was observed as tumor burden increased, and also with the onset of metastasis to distant sites, including bone in the case of breast cancer. Upon further investigation of these cells, we observed that they are highly cytotoxic towards the cancer cells. Adoptive transfer of these CD8+ natural regulatory cells obtained from mice with minimal tumor burden resulted in significantly reduced tumor growth in the tumor-bearing recipients.

There was also an inverse relationship between CD8+ natural regulatory T cells and plasmacytoid dendritic cells (pDC) as tumor burden increased. Additionally, depletion of pDC, either using depletion antibody or by using a transgenic mouse model lacking pDC, showed increased numbers of CD8+ natural regulatory T cells that resulted in decreased tumor burden. Analysis of a possible molecular mechanism on the role of CD8+ natural regulatory T cells as anti-tumor effectors demonstrated that these cells express PD-1. Furthermore, PD-L1, a ligand for PD-1, is expressed by both pDC and cancer cells in vivo. Ongoing experiments are focused on deciphering a possible role of PD-1 and PD-L1 interaction in determining the fate of CD8+ natural regulatory T cells in the presence of pDC and cancer cells through in vitro assays and using transgenic murine models and depletion antibodies for both PD-1 and PD-L1. Collectively, these data suggest a novel anti-tumor immune population that has a potential to be used as immunotherapy for cancer in conjunction with conventional treatments.

Citation Format: Anandi Sawant, Carnella Lee, Cara Schafer, Jessy Deshane, Selvarangan Ponnazhagan. CD8+ natural regulatory T cells as antitumor immune effectors in carcinomas of breast and lung. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2794. doi:10.1158/1538-7445.AM2014-2794
Abstract 281: Work history and cancer mortality risks in 90,268 United States radiologic technologists
Authors: Liu Jason J. , Doody Michele M. , Little Mark P. , Freedman D Michal , Alexander Bruce H. , Kitahara Cari M. , Sigurdson Alice J. , Lee Terrence , Rajaraman Preetha , Miller Jeremy S. , Kampa Diane M. , Simon Steven L. , Preston Dale L. , Linet Martha S.
Published: May 2015

Objectives: There have been few studies on medical radiation work history factors and cancer mortality. In this nationwide prospective cohort study of United States radiologic technologists, we expanded our previous evaluation of work history and cancer mortality risks, by extending follow-up by 11 years and examining a broader range of cancers.

Methods: Using Cox proportional hazards models, we estimated cancer mortality risks through 2008 according to work history and other data derived from questionnaire responses provided during 1983-89 by 90,268 U.S. radiologic technologists. We controlled for potential confounding by age, birth year, pack-years of cigarettes smoked, body mass index, race, and gender.

Results: There were 9,564 deaths and 3,329 deaths from cancer. After controlling for potential confounding factors, we found that technologists who began working before 1950 had marginally increased risk of dying from all cancers (p-trend=0.08), as well as increased risk of dying from female breast cancer (p-trend=0.01) and stomach cancer (p-trend=0.01) compared to those who began working in 1970 or later. Other work history factors, including starting work at a younger age, working greater number of years, or holding patients for x-rays more often, were not consistently associated with increased cancer mortality risks.

Conclusions: We found that radiologic technologists who worked before 1950 had increased risk of dying from certain cancers, which may reflect higher occupational radiation exposures before the late 1950s.

Note: This abstract was not presented at the meeting.

Citation Format: Jason J. Liu, Michele M. Doody, Mark P. Little, D Michal Freedman, Bruce H. Alexander, Cari M. Kitahara, Alice J. Sigurdson, Terrence Lee, Preetha Rajaraman, Jeremy S. Miller, Diane M. Kampa, Steven L. Simon, Dale L. Preston, Martha S. Linet. Work history and cancer mortality risks in 90,268 United States radiologic technologists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 281. doi:10.1158/1538-7445.AM2014-281
Abstract 2828: High expression of heat shock protein 27 in metastases is correlated with response to bevacizumab monotherapy in patients with metastatic melanoma
Authors: Schuster Cornelia , Akslen Lars A. , Straume Oddbjørn
Published: May 2015

Background: Angiogenesis is a hallmark of cancer and essential for tumor growth and metastases. It is known that melanomas are often highly vascularized. The main objective of this study was to identify predictive markers of anti-angiogenic treatment in patients with metastatic melanoma who were treated with bevacizumab monotherapy in a phase II clinical trial. As previously published, this treatment is well tolerated and showed disease control (CR, PR and SD for at least 6 months) in 31% of the patients. Lactate dehydrogenase, M-stage and early hypertension are predictive markers.

Methods: 35 patients were enrolled in an open-label single arm phase II clinical trial at Haukeland University Hospital, Norway, and were treated with bevacizumab 10mg/kg q14 until disease progression or intolerable toxicity (ClinicalTrials.gov: NCT00139360). Paraffin embedded tissue of primary tumors and metastases as well as blood samples were available for investigation. Tissue sections (5µm) were stained with primary antibodies for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and heat shock protein 27 (HSP27). A staining index (SI) was used to quantify expression in primary tumors and metastases, as the product of staining intensity (0: absent, 1: weak: 2: moderate, 3: strong) and area (0: no positive tumor cells, 1: <10% positive tumor cells, 2: 10-50% positive tumor cells, 3: > 50% positive tumor cells). In addition, enzyme linked immune assay (ELISA) was performed for VEGF165 in plasma samples as well as bFGF and HSP27 in serum samples.

Results: 100% of the patients with high cytoplasmic HSP27 expression in metastases had either complete remission or partial response. Conversely, none of those with low HSP27 SI responded to treatment with bevacizumab monotherapy (Mann-Whitney U-test, p=0.044). All patients with high SI in primary tumors showed a non-significant trend to better treatment response. In contrast, there was no association between cytoplasmic VEGF staining index in primary tumors or metastases and response. Neither cytoplasmic nor nuclear staining for bFGF in primary tumors or metastases could predict response to treatment. Measurements of HSP27 and bFGF in serum and VEGF165 in plasma did not show any correlation to treatment response.

Conclusion: High staining index of HSP27 in metastases is significantly associated with response to treatment with bevacizumab monotherapy in patients with metastatic melanoma.

Citation Format: Cornelia Schuster, Lars A. Akslen, Oddbjørn Straume. High expression of heat shock protein 27 in metastases is correlated with response to bevacizumab monotherapy in patients with metastatic melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2828. doi:10.1158/1538-7445.AM2014-2828
Abstract 2836: Development of a companion diagnostic IHC assay for the biomarker-driven selection of C4.4a positive patients
Authors: Schneider Claudia , Couto Joseph , Zhu Yifei , Liao Zhiming , Pytela Robert , Hiscox Alton , Wittemer-Rump Sabine , Forssmann Ulf , Linden Lars , Willuda Joerg , Forler Daniel , Nelson Matthew , Finnern Ricarda , Krahn Thomas , Asadullah Khusru
Published: May 2015

C4.4a (LYPD3), a GPI-anchored cell surface protein, has been identified previously as a cancer- and metastasis-associated cell surface protein. It is expressed in a variety of cancer indications and, particularly, in the squamous cell subtype of non-small cell lung cancer (NSCLC) and head and neck cancer. Targeting C4.4a with a specific antibody-drug conjugate showed a potent and selective antitumor activity in various human xenograft models and in patient-derived NSCLC tumor models (Willuda J et al., AACR 2014). Since it has been previously shown in vivo that the expression of C4.4a is required for the response to anti-C4.4a treatment, we initiated the development of an IHC assay that might be used to detect C4.4a expression in patient tumor samples. For the development of this companion diagnostic IHC assay, a novel monoclonal rabbit anti-C4.4a antibody was generated. Since C4.4a is post-transcriptionally modified by shedding, we aimed to identify an antibody that binds to a similar epitope as the therapeutic C4.4a antibody. The immunizations of rabbits with specific peptide sequences of C4.4a and with the full length C4.4a recombinant protein produced several antibody candidates. Those antibody candidates were characterized in regard to their staining characteristics, domain binding, and specificity for C4.4a. Furthermore, the staining pattern of the antibody candidates and the therapeutic C4.4a antibody were compared side by side to ensure that stainings using the newly generated antibodies are predictive for anti-C4.4a treatment. The most promising antibodies were selected to set up and optimize an IHC assay on the Ventana staining platform. In summary, we developed a companion diagnostic IHC assay that might be used to detect C4.4a expression in patient tumor samples.

Citation Format: Claudia Schneider, Joseph Couto, Yifei Zhu, Zhiming Liao, Robert Pytela, Alton Hiscox, Sabine Wittemer-Rump, Ulf Forssmann, Lars Linden, Joerg Willuda, Daniel Forler, Matthew Nelson, Ricarda Finnern, Thomas Krahn, Khusru Asadullah. Development of a companion diagnostic IHC assay for the biomarker-driven selection of C4.4a positive patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2836. doi:10.1158/1538-7445.AM2014-2836
Abstract 2840: Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology
Authors: Bordeaux Jennifer , Chandrasekaran Krupa , Beruti Sue , Nerenberg Mike , Ramos Corinne , Rimm David , Lameh Jelveh , Dakappagari Naveen
Published: May 2015

With the advent of multiple HER-2 targeting agents, the need for a diagnostic test that accurately predicts the levels of HER2 in newly diagnosed breast cancer patients has never been greater. The goal of this study was to develop an immunofluorescence based assay to objectively and reproducibly quantify HER-2 protein level in Formalin-Fixed Paraffin-Embedded (FFPE) specimens using AQUA Technology and to provide a binary readout, thus eliminating reflex testing and enabling quick treatment decisions.

Methods: Three well known HER-2 antibody clones, A0485, CB11, and SP3 were evaluated across a three-log dilution series under four different antigen retrieval conditions on a tissue microarray (TMA) containing 80 breast cancer cases with known central HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) amplification status. The top six immunofluorescence assay combinations out of 108 conditions were quantified by AQUA scoring based on the their ability to accurately separate negative and positive cases using receiver operating characteristic (ROC) analysis. These top six assay conditions were then assessed on an independent TMA containing 94 breast cancer cases and their AQUA scores were compared to HER2 protein levels determined by reverse phase protein microarray (RPMA®), HER2 mRNA levels assessed by RNAscope® as well as to the current “gold standard” IHC and FISH amplification.

Results: Top three conditions clearly segregating HER2 positive and HER2 negative breast cancer cases were selected and analyzed on an independent TMA. Conditions associated with both SP3 and A0485 antibodies demonstrated high correlations with both HER2 protein levels determined by RPMA analysis and HER2 mRNA levels assessed by RNAscope (spearman's rho >0.85).

Conclusion: Assessment of HER2 status using AQUA Technology was confirmed using two expression platforms, including both protein (RPMA) and RNA (RNAscope) supporting the utilization of the AQUA methodology for diagnostic test development.

Citation Format: Jennifer Bordeaux, Krupa Chandrasekaran, Sue Beruti, Mike Nerenberg, Corinne Ramos, David Rimm, Jelveh Lameh, Naveen Dakappagari. Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2840. doi:10.1158/1538-7445.AM2014-2840
Abstract 2841: Characterization of ALK fusions in circulating tumor cells (CTCs) of NSCLC
Authors: Lu David , Krupa Rachel , Bales Natalee , Louw Jessica , Marrinucci Dena , Dittamore Ryan
Published: May 2015

Background:

Many NSCLC patients have insufficient tumor sample or have high co-morbidities preventing access to tissue biopsies for FISH or IHC to determine ALK fusion and subsequent eligibility to Crizotinib therapy. We developed a gene/protein assay for the ALK fusion and examined CTCs and CTC subpopulation incidence and molecular characterization in newly diagnosed NSCLC patients.

Methods:

10 samples from newly diagnosed NSCLC patients prior to therapy were recruited and blood specimens were collected and shipped to Epic Sciences. 2/10 had known ALK rearrangement through tissue analysis. All nucleated cells were plated onto glass slides and subjected to IF staining and CTC identification by fluorescent scanners and algorithmic analysis. CTCs, defined as classic (CK+ CD45- w/intact DAPI nuclei and morphologically distinct), apoptotic (CK+, CD45-, non-intact nuclei) and CK- (CK-, CD45-, intact and distinct) were identified. Samples were characterized with ALK IF to assess expression. Patients with known ALK rearrangements in tissue also had their CTCs assessed with ALK FISH.

Results:

Assays for the ALK protein and ALK gene rearrangement were developed and specificity confirmed utilizing H2228 (ALK positive) & A549 (ALK negative) cells spiked into donor blood and run through the Epic Assay. Expression of ALK fusions was visualized in CTCs of 1/2 patients with known ALK rearrangements and 0/8 patients with no ALK rearrangements. ALK fusion protein expression was seen in classic, apoptotic, small and CK- CTCs.

Conclusion:

ALK fusion protein and ALK rearrangement assessment with FISH utilizing the Epic CTC platform demonstrates sensitivity and specificity to identification of patients eligible to Crizotinib. The identification of ALK in CTC subpopulations identifies unique tumor cell morphology and suggests evidence of epithelial plasticity. Further clinical studies are ensuing to determine the sensitivity and specificity of ALK in CTCs and the relevance of CTC subpopulations related to Crizotinib therapy and NSCLC progression.

Citation Format: David Lu, Rachel Krupa, Natalee Bales, Jessica Louw, Dena Marrinucci, Ryan Dittamore. Characterization of ALK fusions in circulating tumor cells (CTCs) of NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2841. doi:10.1158/1538-7445.AM2014-2841
Abstract 2850: Biomarkers for detection of exfoliated tumor cells in the peritoneal cavity in rectal cancer
Authors: Kristensen Annette T. , Solbakken Arne , Larsen Stein G. , Giercksky Karl-Erik , Hovig Eivind , Lothe Ragnhild , Lind Guro E. , Guren Marianne G. , Flatmark Kjersti
Published: May 2015

During tumor growth, and possibly by manipulation during surgery, cells from rectal tumors may be shed into the peritoneal cavity. Such cells may contribute to development of metastatic disease, in the form of peritoneal carcinomatosis. Detection of cancer cells in the peritoneal cavity at the time of primary surgery might therefore be of value for prediction of disease recurrence with subsequent prognostic implications.

We previously investigated the incidence of exfoliated cancer cells in peritoneal lavage fluid after resection of locally advanced rectal cancer (LARC). DNA from cells obtained by peritoneal lavage was analyzed with respect to mutations in hotspots of the K-RAS gene. K-RAS mutations were present in 30% of the primary tumors, and were detected in 8% of the lavage fluids examined. The presence of cells with K-RAS mutations in lavage fluid was associated with poor survival, suggesting that a molecular approach for detection of exfoliated tumor cells may be feasible, but K-RAS, although highly specific for detection of tumor cells, is insufficient as the only biomarker. Furthermore, since lavage fluid was collected only at the end of the surgical procedure, the relevance of spontaneous versus iatrogenic tumor cell exfoliation could not be determined.

Based on these encouraging results, a prospective study was initiated, in which lavage is performed before and after surgical removal of the primary tumor to provide data regarding the cause of tumor cell exfoliation and their respective contributions to patient outcome. To increase detection sensitivity, a set of highly specific DNA methylation-based biomarkers is explored. In a pilot experiment, a panel of 12 biomarkers has been evaluated in primary tumors from 17 LARC patients using quantitative methylation specific polymerase chain reaction. Methylation of six genes, CNRIP1, MAL, SPG20, FBN1, SNCA and CDO1 was present at high frequencies in this cohort (in 55-88 % of the samples analyzed), and this biomarker panel has been selected for further comparative analysis in lavage samples. The presence of K-RAS, B-RAF and PIK3CA mutations is analyzed using denaturant capillary electrophoresis of PCR-amplified genes. Results from these genetic analyses of the primary tumors and lavage samples of 50 LARC patients will be presented.

Citation Format: Annette T. Kristensen, Arne Solbakken, Stein G. Larsen, Karl-Erik Giercksky, Eivind Hovig, Ragnhild Lothe, Guro E. Lind, Marianne G. Guren, Kjersti Flatmark. Biomarkers for detection of exfoliated tumor cells in the peritoneal cavity in rectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2850. doi:10.1158/1538-7445.AM2014-2850
Abstract 2859: The genetic polymorphisms of ATG5 and COL4A3 are associated with the prognosis of patients with esophageal squamous cell carcinoma
Authors: Lee Jang-Ming , Yang Pei-Wen , Chiang Tzu-Hsuan , Huang Ya-Chuan , Hsieh Ching-Yueh
Published: May 2015

PURPOSE: To investigate the association between the prognosis of patients with esophageal squamous cell carcinoma (ESCC) and the autophagy or collagen related single nucleotide polymorphisms (SNPs).

PATIENTS AND METHODS: The study was analyzed retrospectively. A total of 570 patients diagnosed with ESCC were enrolled in the study and randomly assigned to a training set (n=188) or replication set (n=382). The genotypes of 16 candidate SNPs involved in autophagy or collagen-related functions were analyzed using iPLEX Gold technology from the genomic DNA of peripheral leukocytes. The association of the genotypes and the overall survival or progression-free survival of patients was analyzed by multivariate Cox regression analysis and Kaplan-Meier survival function.

RESULTS: Among these subjects, 239 patients were with early stage (stage IIa or earlier), and 331 patients were with advanced stages (stage IIb or advanced). In patients with early stages, the genetic variants of ATG5:rs1322178, ATG5:rs3804329 and COL4A3: rs28381984 was associated with increased risk of death in the training set. The associations were confirmed in the replication set and in the combined set (HR[95 % CI]=2.47 [1.31-4.65], P=0.005 in ATG5:rs1322178; HR[95 % CI]=2.16[1.08-4.31], P=0.029 in ATG5:rs3804329; HR[95 % CI]=1.48[1.00-2.20], P=0.052 in COL4A3: rs28381984, in combined set). In cumulative effect analysis of multiple SNPs, patients carrying the unfavorable genotypes at all these 3 genes exhibited a 3.87 fold increasd risk of death in early ESCC patients (HR[95 % CI]=3.87 [1.37-10.93], P=0.011). Finally, the Kaplan-Meier survival curves were constructed for overall survival by the number of unfavorable genotypes. The overall survival duration differed significantly between those with 0, 1, 2, and 3 cumulative unfavorable genotypes (log-rank P=0.012).

CONCLUSION: The hereditary genetic polymorphisms of ATG5 and COL4A3 genes can serve as prognostic predictors of the patients with early ESCC.

Citation Format: Jang-Ming Lee, Pei-Wen Yang, Tzu-Hsuan Chiang, Ya-Chuan Huang, Ching-Yueh Hsieh. The genetic polymorphisms of ATG5 and COL4A3 are associated with the prognosis of patients with esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2859. doi:10.1158/1538-7445.AM2014-2859
Abstract 2867: EGFR expression in T1/T2 oral tongue and floor of the mouth carcinoma
Authors: Kelner Natalie , Pinto Clovis Antonio Lopes , Kowalski Luiz Paulo , Coutinho-Camillo Claudia Malheiros
Published: May 2015

Epidermal growth factor receptor (EGFR) is a molecular maker that regulates the cell growth and plays an important role in tumor proliferation, invasion and metastasis formation. Objective: In this study we examined the association of immunohistochemical expression of EGFR with clinicopathological parameters, lymph node metastasis, regional recurrence and survival in selected cases of early squamous cell carcinoma (SCC) of the oral tongue and floor of the mouth. Methods: Patients with early stage SCC of oral tongue and floor of the mouth were treated surgically at a single institution from 1980 to 2010. Specimens from 139 patients were analyzed for EGFR expression using monoclonal antibody. Results: This study included 139 patients, 96 men (69%), with ages ranging from 21 to 85 years old (mean, 58 years). The tumor site was the tongue in 105 cases (75.5%) and the floor of the mouth in 34 (24.5%). With regard to pathological T-stage, 69 cases were pT1 (52.7%) and 62 were pT2 (47.3%). EGFR was overexpressed in 18 cases (13%) and was significantly associated with pT (p=0.017), thickness greater than 4mm (p=0.017), muscular invasion (p=0.014), desmoplastic reaction (p=0.021), inflammatory infiltrate (p=0.029), lymphatic embolization (p=0.016), mode of invasion (p=0.027) and perineural invasion (p=0.007). The overexpression of EGFR was also associated with lymph node metastasis (p=0.038) and regional recurrence (p=0.014). The cancer-specific survival in 5 years decreased from 90% in the group without EGFR expression to 75.3% in the EGFR overexpression group (p=0.019). Conclusion: The EGFR overexpression was associated with more aggressive pathological features, lymph node metastasis and regional recurrence in patients with early oral tongue and floor of the mouth squamous cell carcinoma.

Citation Format: Natalie Kelner, Clovis Antonio Lopes Pinto, Luiz Paulo Kowalski, Claudia Malheiros Coutinho-Camillo. EGFR expression in T1/T2 oral tongue and floor of the mouth carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2867. doi:10.1158/1538-7445.AM2014-2867