bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4028: Oncogenic KRAS mutations induce PD-L1 overexpression through MAPK pathway activation in non-small cell lung cancer cells
Authors: Miura Yosuke , Sunaga Noriaki , Kaira Kyoichi , Tsukagoshi Yusuke , Osaki Takashi , Sakurai Reiko , Hisada Takeshi , Girard Luc , Minna John D. , Yamada Masanobu
Published: July 2016

The proto-oncogene KRAS is a frequently mutated oncogenic drivers in non-small cell lung cancer (NSCLC) and druggable molecular targets for KRAS-mutated NSCLC have been under investigation. Recent studies have reported promising results of nivolumab, a programmed death 1 (PD-1) immune-checkpoint-inhibitor antibody, for previously treated NSCLC patients and suggested that the programmed-death ligand 1 (PD-L1) is a candidate surrogate marker for the anti-PD-1 therapy. By microarray analysis, we found that short hairpin RNA (shRNA)-mediated stable knockdown of mutant KRAS downregulated the expression of the CD274 gene (encoding PD-L1) in the NSCLC cell lines, H358 (-2.68 fold-change) and H441 (-1.98 fold-change). Additionally, we confirmed that small interfering RNAs (siRNAs) targeting mutant KRAS, but not wild-type KRAS, reduced CD274 mRNA expression in these cell lines. As expected, the CD274 expression levels were higher in these cell lines when examining the expression in a panel of NSCLC cell lines by quantitative RT-PCR analysis. Furthermore, CD274 mRNA expression was downregulated by MEK and ERK inhibitors (U0126, FR180204, selumetinib) in H358 and H441 cells. On the other hand, the expression analysis also revealed that CD274 mRNA levels vary among KRAS-mutated cell lines. These results suggest that oncogenic KRAS mutations induce PD-L1 overexpression through activation of the MAPK pathway in NSCLC cells, whereas other unknown mechanisms that modulate PD-L1 expression underlie in different KRAS-mutated NSCLCs.

Citation Format: Yosuke Miura, Noriaki Sunaga, Kyoichi Kaira, Yusuke Tsukagoshi, Takashi Osaki, Reiko Sakurai, Takeshi Hisada, Luc Girard, John D. Minna, Masanobu Yamada. Oncogenic KRAS mutations induce PD-L1 overexpression through MAPK pathway activation in non-small cell lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4028.
Abstract 4053: A unique nutrient mixture suppresses ovarian cancer growth of A-2780 by inhibiting invasion and MMP-9 secretion
Authors: Roomi M. Waheed , Rath Matthias , Niedzwiecki Aleksandra
Published: July 2016

Ovarian cancer is the deadliest gynecological malignancy in women, and fifth leading cause of death. The American Cancer Society estimated that it would claim 14,250 lives in 2013. Despite the advances made in chemotherapy and surgery, the average time of clinical remission is approximately 2 years and the 5-year survival rate is 45%. Thus, there is an urgent need for the development of a novel therapeutic approach to ovarian cancer treatment. We investigated the effect of a unique nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract on human ovarian cancer cell A-2780 in vivo and in vitro. Athymic female nude mice (n = 12) were inoculated by I.P. with 2×106 cells in 0.1ml PBS and randomly divided into two groups. Group A (n = 6) was fed a regular diet and group B (n = 6) a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and tumors that developed in the ovary were excised, weighed and processed for histology. In vitro, A-2780 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and antibiotics. At near confluence, cells were treated with NM in triplicate at concentrations between 0-1000 μg/ml. Cell proliferation was measured by MTT assay, MMP-9 secretion by gelatinase zymography, invasion through Matrigel and morphology by H&E staining. All control mice (Group A) developed large ovarian tumors, whereas 5 out of 6 mice in group B developed no tumors, and one, a small tumor. Zymography demonstrated only MMP-9 expression, which NM inhibited in a dose dependent fashion, with virtual total block at 250 μg/ml concentration. NM significantly inhibited invasion through Matrigel with total block at 250 μg/ml concentration. MTT showed dose dependent inhibition of cell proliferation with NM and H&E staining showed no morphological changes below 500 μg/ml NM. These results suggest that NM has therapeutic potential in treatment of ovarian cancer by significantly suppressing tumor growth and by inhibiting MMP-9 secretion and invasion of A-2780 ovarian cancer cells.

Citation Format: M. Waheed Roomi, Matthias Rath, Aleksandra Niedzwiecki. A unique nutrient mixture suppresses ovarian cancer growth of A-2780 by inhibiting invasion and MMP-9 secretion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4053.
Abstract 4062: Human mammary tumor virus (HMTV) is a breast cancer pathogen
Authors: Holland James F. , Melana Stella , Nartey Teiko , Tripodi Joseph , Jaffer Shabnam , Nayak Anupna , Pogo Beatriz GT
Published: July 2016

Human mammary tumor virus (HMTV) a betaretrovirus 90-98% homologous to Mouse Mammary Tumor Virus (MMTV), the causative agent for breast cancer in mice, has been detected in approximately 40% of American women's breast cancers. Mouse DNA contamination has been definitively excluded by contamination-free guarantee techniques. In Western European countries and their former colonies, age standardized rates (ASR) of breast cancer incidence (47 to 92 per 100,000 per year) are higher than Asian incidence (29 to 43 ASR/y). This difference could be explained by excess HMTV-related breast cancer incidence. In 7 West European, American and Oceania countries, 30 to 60% contain HMTV, while in 5 Asian nations HMTV associated breast cancers range from 0 to 22%. Different indigenous murine species with disparate MMTV burdens parallel these findings: mus domesticus in the West with much MMTV in its genome, and mus castaneus or mus musculus in the East with less. HMTV is found in cells in milk of 8% of unselected American mothers and in milks of 21% of women previously biopsied for unconfirmed cancer suspicion.

HMTV isolated from primary cultures of metastatic breast cells is able to infect In vitro, human mammary epithelial cells, B and T lymphocytes and dendritic cells. MCF10A breast cell line infected with HMTV shows molecular changes associated with epithelial-mesenchymal transition, such as up-regulation of vimentin, and down-regulation of E-cadherin.

HMTV sequences were also shown to be more prevalent in inflammatory, gestational and familial breast cancers than in unselected sporadic samples, consistent with association with more aggressive breast cancer phenotypes. To establish a direct connection between the presence of HMTV sequences and metastatic breast cancer, we have found HMTV sequences in 94% of primary cultures of metastatic breast cells obtained directly from effusion fluids. HMTV present in primary breast tumors has been demonstrated in their metastatic axillary nodes.

Taken together the results suggest an association between the presence of HMTV sequences and breast cancer aggressiveness. HMTV poses a new and challenging dimension in breast cancer research involving causation, diagnosis, molecular mechanisms, epidemiology, therapy and prevention.

Citation Format: James F. Holland, Stella Melana, Teiko Nartey, Joseph Tripodi, Shabnam Jaffer, Anupna Nayak, Beatriz GT Pogo. Human mammary tumor virus (HMTV) is a breast cancer pathogen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4062.
Abstract 423: Toxicity and efficacy probability intervals design for phase I dose-finding in oncology trials
Authors: Li Daniel H. , Whitmore Jim , Ji Yuan
Published: July 2016

We propose a toxicity and efficacy probability interval (TEPI) design for dose-finding in phase I oncology studies. This approach incorporates efficacy outcomes to inform dosing decisions to optimize both efficacy and safety. Decision rules can be prespecified, allowing for transparency to clinicians and non-statisticians. The TEPI design is an extension over the modified toxicity probability interval (mTPI) design, which uses only dose-limiting toxicity (DLT) data for dose escalation. The TEPI method is motivated by recent promising data from cancer immunotherapies, for which response or a biomarker for response can be observed in the same interval as a DLT.

The TEPI method is based on a two-way decision table constructed by combining the target probability intervals of DLT and efficacy. It uses a beta/binomial hierarchical model to compute the posterior probabilities of intervals for toxicity and efficacy. The dosing decision depends on the joint unit probability mass of toxicity and efficacy data, which follows the Bayes rule under independent beta prior distributions. TEPI can pre-calculate all dosing actions for any possible combination of toxicity and efficacy outcome at a certain time point during a trial. For example, dose escalation or de-escalation decisions at time of 6, 9, and 12 treated patients are provided in Table 1.

In the current era of potent novel agents in oncology, phase I trials that simultaneously optimize antitumor activity and safety may accelerate development of these therapies. The proposed TEPI method recognize this needs, and is appealing to clinicians because all dose escalation or de-escalation can be prespecified. Simulation studies have been performed to evaluate the performance of TEPI, and compared to other designs. No. of RespondersNo. of Treated Patients at Current Dose LevelNo. of DLTs01-45-660-1EUES2-3SSS4-6DUDUDU01-67-990-2EUES3-4SSS5-9DUDUDU0-12-78-12120-3EUES4-6SSS7-12DUDUDU

Table 1. The letters in cells are computed based on the decision rules under the TEPI method and represent different dose-finding actions. EU: Unacceptable efficacy; DU: Unacceptable safety. Target toxicity probability = 0.3 and target response rate probability = 0.5.

Citation Format: Daniel H. Li, Jim Whitmore, Yuan Ji. Toxicity and efficacy probability intervals design for phase I dose-finding in oncology trials. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 423.
Abstract 4350: Human oral microbiome and prospective risk for pancreatic cancer: a population based, nested case control study
Authors: Fan Xiaozhou , Alekseyenko Alexander V. , Wu Jing , Jacobs Eric J. , Gapstur Susan M. , Purdue Mark P. , Abnet Christian C. , Stolzenberg-Solomon Rachael , Miller George , Ravel Jacque , Hayes Richard B. , Ahn Jiyoung
Published: July 2016

Background: A history of periodontal disease and the presence of circulating antibodies to selected oral pathogens have been associated with increased risk of pancreatic cancer in prospective studies; however, relationships of oral microbes with pancreatic cancer have not been directly evaluated. We conducted a prospective study to examine the relationship of oral microbiota with subsequent risk of pancreatic cancer.

Methods: We selected 361 incident pancreatic cancer cases and 371 matched controls from two prospective cohort studies, the ACS Cancer Prevention Study (CPS) II and the NCI Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). From pre-diagnostic oral wash samples, we characterized the composition of the oral microbiota using bacterial 16S rRNA gene amplification and sequencing. Logistic regression analysis was used to examine the association between oral microbiota and risk of pancreatic cancer, controlling for the random effect of cohorts and other covariates, age, race, sex, smoking status, alcohol consumption, body mass index, and history of diabetes.

Findings: Carriage of oral pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were associated with higher risk of pancreatic cancer (adjusted Odds Ratio [OR]p.g = 1·59 and 95% confidence interval [CI] = 1·15-2·20; ORa.a = 2·19 and 95%CI = 1·15-4·15, respectively). Greater relative abundance of phylum Fusobacteria was associated with decreased pancreatic cancer risk (OR per percent increase of abundance = 0·92, 95%CI = 0·87-0·98). Risks related to these phylotypes remained after exclusion of cases that developed within two years of sample collection, reducing the likelihood of reverse causation in this prospective study.

Interpretation: The study provides evidence that the oral mcirobiota plays a role in the etiology of pancreatic cancer and may point to accessible means for prevention of this disease.

Citation Format: Xiaozhou Fan, Alexander V. Alekseyenko, Jing Wu, Eric J. Jacobs, Susan M. Gapstur, Mark P. Purdue, Christian C. Abnet, Rachael Stolzenberg-Solomon, George Miller, Jacque Ravel, Richard B. Hayes, Jiyoung Ahn. Human oral microbiome and prospective risk for pancreatic cancer: a population based, nested case control study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4350.
Abstract 436: Glycodelin expression in endometrial carcinoma
Authors: Koistinen Hannu , Pasanen Annukka , Hautala Laura
Published: July 2016

Glycodelin is a lipocalin protein mainly expressed in well-differentiated epithelial cells in reproductive tissues, including endometrium and seminal vesicles. Previously, glycodelin has been shown to induce differentiation of endometrial carcinoma cells, which was associated with reduced xenograft tumor growth. Glycodelin is highly expressed in secretory endometrium, but not in proliferative or postmenopausal endometrium. While glycodelin is weakly, if at all, expressed in cancer tissues and, when expressed, associated with better prognosis, results regarding the expression of glycodelin in endometrial carcinoma are discordant. Therefore, we studied the expression of glycodelin in endometrial carcinoma and hyperplasia by immunohistochemical staining using highly validated antibody developed in house. Furthermore, the levels of mRNA were addressed utilizing in silico transcriptomics databases.

Glycodelin transcripts were more abundant in normal uterus than in uterine carcinomas. Glycodelin staining in endometrial carcinoma sections was also much lower than that in secretory phase endometrium. Interestingly, in some endometrial hyperplasia samples glycodelin levels were much lower in hyperplastic regions of the sample as compared to those having normal histology.

In conclusion, our data show that glycodelin is not highly expressed, if at all, in endometrial carcinoma tissues. This is in keeping with the reduced expression of glycodelin in postmenopausal endometrium and association of glycodelin in epithelial differentiation.

Citation Format: Hannu Koistinen, Annukka Pasanen, Laura Hautala. Glycodelin expression in endometrial carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 436.
Abstract 4569: Use of ACPD and ICA-1 as inhibitors of atypical proteinkinase C-zeta (ζ) and iota (ι) in metastasized melanoma cells
Authors: Ratnayake Wishrawana S. , Acevedo-Duncan Mildred
Published: July 2016

The number of melanoma cases report is increased every year. Malignant melanoma is very common among all Caucasian populations worldwide. New cases in these populations are expected to be doubled every 10-20 years. According to the NIH Surveillance, Epidemiology and End Results (SEER) program, 73,870 new cases and 9,940 deaths were reported in 2015 in USA [http://seer.cancer.gov/statfacts/html/melan.html (11/10/2015)]. It is not totally understood which intracellular chemicals are involved in certain signaling pathways and which governs the metastasis of melanoma cancer cells. Even though PKC- iota (ι) was not reported in normal melanocytes, it was detected in high amounts in both transformed melanocytes and melanoma metastases. PKC- zeta (ζ) was also reported in both normal melanocytes and melanoma metastases [Melanoma Res. 12:201-209 (2002)]. We believe these atypical PKCs play an important role in cell motility of melanoma therefore we tested two inhibitors for them. The objective of this study was to test the inhibition effectiveness of ACPD [Diabetes. 63:2690-2701 (2014)] on both PKC-ι and PKC-ζ and ICA-1 [Int. J. Biochem. Cell Biol.43:784-794(2011)] as an inhibitor of PKC-ι. SK-MEL-2 metastasis melanoma cells and PCS-200-013 normal melanocyte cells were cultivated and treated with ACPD and ICA-1 in separate sets of flasks for three consecutive days while taking the cell count for every 24 hrs. Preliminary results of this experiment shows statistically significant decrease in cell number in SK-MEL-2 cells while no change in PCS-200-013. Future investigations will involve examining the effects of ACPD and ICA-1 on cell motility. Our preliminary results confirms the cell population of melanoma cells have an inversely proportional relationship with the drug concentrations. In conclusion, ACPD and ICA-1 are capable of decreasing the cell proliferation of melanoma cancer cells.

Citation Format: Wishrawana S. Ratnayake, Mildred Acevedo-Duncan. Use of ACPD and ICA-1 as inhibitors of atypical proteinkinase C-zeta (ζ) and iota (ι) in metastasized melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4569.
Abstract 4586: FGFR2 amplification in serous ovarian cancer
Authors: Tyulyandina Alexandra , Tsimafeyeu Ilya , Demidova Irina , Gikalo Marina , Tjulandin Sergei
Published: July 2016

Fibroblast growth factor receptor (FGFR) signaling pathway plays an important role in biology of cancer. Approximately 7% of ovarian cancer (OC) exhibits FGFR1 amplification. Recently the overexpression of FGFR2 was found in 95% of clear cell OC. Up to date there is no data about FGFR2 status in OC. 54 paraffin-embedded blocks from 18 patients with serous OC were analyzed by FISH to identify FGFR2 amplification. Scoring for amplification and polysomy level was adopted from previous studies for gastric cancer (Su X, et al. BJC 2014). Material from each patient had 3 samples: from primary tumor, from primary metastatic lesions, and from relapse. Intratumoral FGFR2 amplification heterogeneity was assessed in sections from all cases with FGFR2 amplification, and was defined as the presence of areas with different FISH scores within the same tumor in full sections.

Amplification was observed in 3 patients (15.7%). Interestingly, in one case amplification was observed in primary ovarian tumor but not in the metastatic nor in relapse samples. In two other cases the FGFR2 amplification was detected in relapse samples and in primary metastatic samples but not in ovary. High-level polysomy (HLP) was observed in 10 patients (55.5%). In all of those patients HLP was revealed in the samples of relapse. In 3 patients HLP was found in all 3 samples, in 3 cases HLP was observed only after relapse, in 4 cases HLP in metastasis sample was the same as in relapse sample, but it wasn't observed in ovary samples. Eight of 13 FGFR2 amplified ovarian cancers or tumors with HLP (61.5%) displayed intratumoral heterogeneity within full sections. In 5 (27.8%) cases neither amplification nor high level polysomy was observed. In conclusion, this is the first study of FGFR2 FISH in serous ovarian adenocarcinoma, demonstrating a FGFR2 amplification and HLP in primary tumor, primary metastases and relapse. Furthermore, we found evidence for intratumoral heterogeneity of FGFR2 amplification and HLP in about 61.5% of ovarian cancers. This study was supported by grant from the “Oncoprogress Foundation”. FGFR2 amplification and high-level polysomy in patients with serous cancer (13 patients)N (%), patientsPrimary tumor (N samples)Primary metastasis (N samples)Relapse (N samples)Amplification3 (15.7%)122High level polysomy10 (55.5%)3410

Citation Format: Alexandra Tyulyandina, Ilya Tsimafeyeu, Irina Demidova, Marina Gikalo, Sergei Tjulandin. FGFR2 amplification in serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4586.
Abstract 4615: eIF4B is a key effector of oncogenic Pim and PI3K/Akt/mTOR signaling pathways during Abl-mediated cellular transformation
Authors: Chen Ke , Li Jianning , Chen Jilong
Published: July 2016

Abl oncoproteins, including v-Abl, Bcr-Abl and Tel-Abl, are activated non-receptor tyrosine kinases that are associated with various malignant transformations of hematopoietic cells. Expression of Abl kinase constitutively activates multiple signaling pathways that are critically involved in the regulation of cell proliferation and survival. Despite extensive studies, the precise mechanism by which these Abl oncoproteins induce hematopoietic malignances is not fully elucidated. eIF4B plays a critical role in Abl-mediated cellular transformation, but the precise mechanisms are largely unknown. Here we show that eIF4B is a convergent substrate of JAK/STAT/Pim and PI3K/Akt/mTOR pathways in Abl transformants. Both pathways regulated the phosphorylation of eIF4B in Abl-transformed cells, and such redundant regulation was responsible for the limited effect of single inhibitor on Abl oncogenicity. Surprisingly, persistent inhibition of one signaling pathway induced the activation of the other pathway and thereby restored the phosphorylation levels of eIF4B. Simultaneous inhibition of the two pathways impaired eIF4B phosphorylation more effectively, and synergistically induced apoptosis in Abl transformed cells and inhibited the growth of engrafted tumors in nude mice. Similarly, the survival of Abl transformants exhibited a higher sensitivity to the pharmacological inhibition, when combined with the shRNA-based silence of the other pathway. Interestingly, such synergy was dependent on the phosphorylation status of eIF4B on Ser422, as overexpression of eIF4B phosphomimetic mutant S422E in the transformants greatly attenuated the synergistic effects of these inhibitors on Abl oncogenicity. In contrast, eIF4B knockdown sensitized Abl transformants to undergo apoptosis induced by the combined blockage. Collectively, the results indicate that eIF4B integrates the signals from Pim and PI3K/Akt/mTOR pathways in Abl-expressing leukemic cells, and is a promising therapeutic target for such cancers.

Citation Format: Ke Chen, Jianning Li, Jilong Chen. eIF4B is a key effector of oncogenic Pim and PI3K/Akt/mTOR signaling pathways during Abl-mediated cellular transformation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4615.
Abstract 4652: Effects of anti-DLL4 treatment on non-small cell lung cancer (NSCLC) human xenograft tumors
Authors: Brunner Alayne , Cattaruzza Fiore , Yen Wan-Ching , Yeung Pete , Fischer Marcus , Cancilla Belinda , O’Young Gilbert , Tam Raymond , Liu Yu-Wang , Gurney Austin , Lewicki John , Hoey Tim , Wang Min , Kapoun Ann M.
Published: July 2016

Background: Non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancers, the leading cause of cancer-related deaths. Notch signaling has been shown to play an important role in lung cancer initiation and progression. Delta-like ligand 4 (DLL4) activates the Notch pathway and is important for cancer stem cell (CSC) survival. Demcizumab (OMP-21M18) is a humanized IgG2 anti-DLL4 antibody currently being tested in a Phase 2 trial in combination with pemetrexed and carboplatin for first-line treatment of patients with NSCLC. Previously, OMP-21M18 in combination with its mouse anti-DLL4 surrogate has been shown to inhibit tumor growth, decrease cancer stem cell frequency, and cause dysfunctional sprouting of new vessels resulting in an anti-angiogenic effect in patient-derived tumor xenograft (PDX) models in breast, colon, ovarian, and pancreatic cancers. Here we show results from NSCLC PDX models.

Methods and Results: Anti-DLL4 treatment was tested in a series of NSCLC PDX models. Because DLL4 inhibition has been shown to have effects on the tumor as well as the vasculature, the combination of OMP-21M18 (targeting human DLL4) and 21R30 (antibody targeting mouse DLL4) treatment in the PDX models was used to model demcizumab treatment in humans. Treatment with anti-DLL4 in combination with chemotherapy inhibited tumor growth in a series of NSCLC PDX models. Additionally, a tumorigenicity assay showed a decrease in the frequency of tumor-initiating cells following treatment with anti-DLL4 and chemotherapy. Gene expression analysis of tumor samples provided insights into the mechanism of action.

Conclusions: Anti-DLL4 treatment in a panel of NSCLC PDX tumor models in vivo showed inhibition of tumor growth and a decrease in the frequency of tumor-initiating cells. Mechanism of action and gene expression analysis of these models treated with anti-DLL4 will be presented. These findings provide additional evidence supporting demcizumab as an effective treatment for NSCLC patients.

Citation Format: Alayne Brunner, Fiore Cattaruzza, Wan-Ching Yen, Pete Yeung, Marcus Fischer, Belinda Cancilla, Gilbert O’Young, Raymond Tam, Yu-Wang Liu, Austin Gurney, John Lewicki, Tim Hoey, Min Wang, Ann M. Kapoun. Effects of anti-DLL4 treatment on non-small cell lung cancer (NSCLC) human xenograft tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4652.
Abstract 4724: Novel and selective inhibitors of histone deacetylases (HDAC) 1 and 2 synergize with DNA methyltransferase inhibitor azacitidine in acute myeloid leukemia (AML)
Authors: Min Chengyin , Quayle Steven N. , Huang Pengyu , Shearstone Jeffrey R. , Jones Simon S. , Yang Min
Published: July 2016

AML is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. Outcomes for patients with AML remain generally poor, highlighting the need for novel treatment options. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and the DNA methyltransferase inhibitor VIDAZA™ (azacitidine) is approved by European Medicines Agency for the treatment of elderly patients with AML. Numerous clinical studies are ongoing to investigate the benefit of combining azacitidine with other investigational agents in AML.

HDAC inhibitors are promising agents for the treatment of AML. Combination studies with HDAC inhibitors plus DNA methyltransferase inhibitors have suggested beneficial clinical activity in AML. However, the toxicity profiles of non-selective HDAC inhibitors in the combination settings limit the clinical benefit. In this work, we describe the preclinical development of highly selective HDAC inhibitors, which are hypothesized to have improved safety profiles than non-selective HDAC inhibitors, for combination therapy in AML. We demonstrate that selective HDAC1/2 inhibitors are highly effective and efficacious both as single agents and in combination with azacitidine in preclinical models of AML, including established AML cell lines, bone marrow samples freshly derived from AML patients and in vivo xenograft models of human AML. At the molecular level, gene expression profiling, DNA methylation mapping and chromatin immunoprecipitation analysis were performed on AML cells treated with either an HDAC1/2 inhibitor, azacitidine, or the drug combination. Molecular signatures and genes involved in myeloid cell differentiation, apoptosis and cell cycle arrest were identified as potential mediators of the combinatorial effects of HDAC1/2 inhibition with azacitidine. Together, these findings support the clinical evaluation of selective HDAC1/2 inhibitors in combination with azacitidine in AML patients.

Citation Format: Chengyin Min, Steven N. Quayle, Pengyu Huang, Jeffrey R. Shearstone, Simon S. Jones, Min Yang. Novel and selective inhibitors of histone deacetylases (HDAC) 1 and 2 synergize with DNA methyltransferase inhibitor azacitidine in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4724.
Abstract 4983: Aminoflavone acts as an immunomodulator of tumor growth in a breast cancer mouse model
Authors: Callero Mariana Alejandra , Rodriguez Cristina Elisa , Sólimo Aldana , Bal de Kier Joffé Elisa , Loaiza Pérez. Andrea
Published: July 2016

Aminoflavone (AFP464, NSC 710464), an antitumor agent which recently entered phase II clinical trials, acts against estrogen-positive breast cancer (ER+). AFP464, which has a unique mechanism of action by activating aryl hydrocarbon receptor (AhR) signaling pathway, decreased tumor growth rate in an estrogen dependent, tamoxifen-sensitive mammary cancer murine model (M05) syngeneic in BALB/c developed in our animal facility. Considering that AhR has recently emerged as a physiological regulator of the innate and adaptive immune responses, it is our aim to investigate whether AFP464 modulates the immune response in our mouse model. Cytometric analyses of splenic cells obtained from AFP464-treated and control animals, at three different times after treatment (13, 21 and 41 days), revealed that MDSCs were lower in animals treated with AFP464 than in the vehicle control group. On the contrary (On the other hand) no differences in macrophages, NK, CD8+ T, and CD4+ T cells were observed between these two groups. We also found a diminished quantity of MDSCs in the inflammatory infiltrate, isolated from tumors treated with AFP464. According to these results, we also observed a higher cytotoxic activity of splenic cells from the AFP464-treated group compared with the control one (13 days: 1,50 ± 0,12 vs 0,74 ± 0,18; 21 days: 2,4 ± 0,3 vs 1,16 ± 0,2; 41 days: 2,97 ± 0,2. vs 1,07 ± 0,2 p < 0,05). After an adoptive transfer of splenic cells to mice bearing M05 tumors, we observed that tumors were significantly smaller in mice that received splenic cells from AFP464-treated mice compared with controls (p < 0,05). Finally, we also investigated the effects of AFP464 on metalloproteinase, arginase and iNOS activities in peritoneal macrophages. We found a time-dependent modulation: at 13 day after treatment AFP464 induces an anti-tumor M1 profile, which was gradually reverted to a pro-tumoral M2 profile, at 41 days after treatment. These data suggest that AFP464 modulates the immune response in order to collaborate with its anti-tumor activity and places the immune system as a novel target for this anti-cancer agent to strengthen the rationale for its inclusion in breast cancer treatment regimens.

Citation Format: Mariana Alejandra Callero, Cristina Elisa Rodriguez, Aldana Sólimo, Elisa Bal de Kier Joffé, Andrea Loaiza Pérez. Aminoflavone acts as an immunomodulator of tumor growth in a breast cancer mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4983.
Abstract 4994: SEA-CD40: from bench to bedside
Authors: Gardai Shyra J. , Neff-LaFord Haley , Epp Angela , Yang Jing , Manley Thomas , Law Che-Leung
Published: July 2016

SEA-CD40 is a non-fucosylated, humanized IgG1 monoclonal antibody directed against human CD40, a co-stimulatory receptor of the TNF receptor superfamily. The consequence of enhanced SEA-CD40/FcγRIIIa binding is potent immune stimulatory activity. CD40 receptor ligation induces multiple pathways; to pave the road for identification of a specific activity signature in the clinical setting, in vitro preclinical assays were developed to monitor the immune modulatory activity of SEA-CD40. Human PBMCs stimulated with increasing concentrations of SEA-CD40 were assessed for immune changes including cytokine production, cellular activation, and modulation of cellular subsets. SEA-CD40 PBMC stimulation elicited a unique set of cytokines including MIP-1β, MCP-1, and IL-8. In addition to inducing cytokines, specific immune cell changes were also observed including up-regulation of stimulatory molecules on monocyte/ macrophages, activation of NK cells, and changes in cellular subsets such as deletion of B-cells. While some of these changes were common across the other CD40 therapeutic antibodies being tested in the clinic, SEA-CD40-specific changes were identified. These changes included a reduction in the immune dampening cytokine IL-10, induction of Th1 CXCR3 positive cells, and reduction of T-regulatory cells, all potentially contributing to an antitumor immune response.

The specific in vitro SEA-CD40 signature was also observed in vivo in cynomolgus monkeys. SEA-CD40 treatment induced the same signature cytokines observed in vitro and elicited the same cellular changes including depletion of B-cells, and activation of CD8+ T-cells. A CD40 receptor occupancy assay was also created to correlate receptor engagement with activity. Interestingly, while SEA-CD40 is rapidly cleared from plasma, it is detectable on the surface of antigen-presenting cells for up to 3 weeks.

The initial starting dose for SEA-CD40 clinical trials was calculated using the minimal anticipated biological effect level (MABEL). SEA-CD40 cytokine induction was the most sensitive preclinical marker of biologic activity and was, therefore, used for MABEL dose calculation. In the ongoing phase 1 First-In-Human clinical trial, this preclinical SEA-CD40 activity signature is being monitored in adult patients with advanced solid tumors (study NCT02376699). Establishing a clear immune biomarker strategy from pre-clinical research to clinical trials is vital for tracking the activity of our immuno-oncology drugs in patients and for identifying a safe and efficacious regimen.

Citation Format: Shyra J. Gardai, Haley Neff-LaFord, Angela Epp, Jing Yang, Thomas Manley, Che-Leung Law. SEA-CD40: from bench to bedside. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4994.
Abstract 5011: Strong FGFR3 staining is a marker for FGFR3 fusions and poor prognosis in diffuse gliomas
Authors: Granberg Kirsi J. , Annala Matti , Lehtinen Birgitta , Kesseli Juha , Haapasalo Joonas , Yli-Harja Olli , Visakorpi Tapio , Haapasalo Hannu , Nykter Matti , Zhang Wei
Published: July 2016

Inhibitors of fibroblast growth factor receptors (FGFRs) have recently arisen as a promising treatment option for patients with FGFR alterations. Gene fusions involving FGFR3 and transforming acidic coiled coil 3 (TACC3) have been detected in diffuse gliomas and other malignancies, and fusion-positive cases have responded well to FGFR inhibitors. As high FGFR3 expression has been detected in fusion positive tumors, we sought to determine the clinical significance of FGFR3 protein expression level and its potential to indicate FGFR3 fusions. We thus performed FGFR3 IHC on tissue microarrays containing 676 grade II-IV astrocytoma and 116 grade II-III oligodendroglial tumor specimens. Selected cases were further analyzed using targeted sequencing. Moderate-to-strong FGFR3 staining was detected in all tumor grades, was more common in females, and associated with poor survival. Targeted sequencing identified FGFR3-TACC3 fusions and an FGFR3-CAMK2A fusion in 10 of 12 strongly stained cases (staining specificity 86%), whereas no fusions were found in 12 negatively-to-moderately stained cases (staining sensitivity 100%). Fusion-positive cases were predominantly female and negative for IDH, TP53, and EGFR/PDGFRA/MET alterations. Importantly, FGFR3 staining revealed intratumoral heterogeneity, with subclonal negative staining in a subpopulation of fusion-positive cases. Taken together, strong FGFR3 protein expression is indicative of FGFR3 fusions and may serve as a cost-effective predictive marker for FGFR-inhibitor-based treatment regimens.

Citation Format: Kirsi J. Granberg, Matti Annala, Birgitta Lehtinen, Juha Kesseli, Joonas Haapasalo, Olli Yli-Harja, Tapio Visakorpi, Hannu Haapasalo, Matti Nykter, Wei Zhang. Strong FGFR3 staining is a marker for FGFR3 fusions and poor prognosis in diffuse gliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5011.
Abstract 59: Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer
Authors: lee doyeon , Yu SungSook , Jung Dawoon E.Jung1 , Lee YeoSong , Choi Beom Ku , Lee Yong Chan
Published: July 2016

Helicobacter pylori (H.pylori) infection of the gastric mucosa is strongly associated with chronic gastritis, peptic ulcer disease and gastric cancer. Individuals infected with H.pylori possessing CagA protein produce more reactive oxygen species (ROS) and increase the risk of developing gastric cancer. Sirtuins are NAD+ dependent deacetylases that regulate cellular metabolism in response to stress. Recently, mitochondrial sirtuin, SIRT3, is known to be a tumor suppressor via its ability to suppress ROS and regulate hypoxia inducible factor 1α (HIF-1α). However, it has not been investigated whether increased ROS production by H.pylori is regulated by SIRT3 via HIF-1α regulation and is mediated through intracellular CagA. We aimed to investigate the correlation between SIRT3, ROS and HIF-1α in H.pylori infected gastric epithelial cells. In this study, we showed that SIRT3 deficient AGS cells exhibited augmented HIF-1α protein stabilization and transcriptional activity in hypoxic condition. We also found that H.pylori CagA downregulated SIRT3 activity and induced ROS production through HIF-1α stabilization, similar to hypoxic condition in gastric epithelial cells. Overexpression of SIRT3 inhibited the stabilization of HIF-1α protein and attenuated the increase in HIF-1α transcriptional activity in hypoxia. Moreover, H.pylori CagA attenuated HIF-1α stability and transcriptional activity in SIRT3 overexpressed gastric epithelial cells. Taken together, these findings provide a valuable insight into the potential role of SIRT3 in the development of gastric cancer by regulating the HIF-1α protein.

Citation Format: doyeon lee, SungSook Yu, Dawoon E.Jung1 Jung, YeoSong Lee, Beom Ku Choi, Yong Chan Lee. Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 59.
Abstract 689: Downregulation of TIMP2 via HIF-1á/miR-210/HIF-3á regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma
Authors: Kai Alan KL , Chan Lo Kong , Lo Regina CL , Lee Joyce , Wong Carmen CL , Wong Jack CM , Ng. Irene OL
Published: July 2016

Cancer metastasis is a multi-step process that involves a series of tumor-stromal interaction, including extracellular matrix (ECM) remodeling which requires a concerted action of multiple proteolytic enzymes and their endogenous inhibitors. This study investigated the role of tissue inhibitor of metalloproteinases 2 (TIMP2) in the context of hepatocellular carcinoma (HCC) metastasis. We found that TIMP2 was the most significantly downregulated member among the TIMP family in human HCCs. Moreover, TIMP2 underexpression was frequent (41.1%; 23/56) in human HCCs as compared to the corresponding non-tumorous livers and was significantly associated with direct liver invasion into the adjacent liver parenchyma and poorer survival outcomes of the HCC patients. Furthermore, stable silencing of TIMP2 in HCC cell lines enhanced the cell invasive ability and ECM degradation associated with formation of invadopodia-like feature, suggesting that TIMP2 is a negative regulator of HCC metastasis. Furthermore, using orthotopic tumor xenograft model, we demonstrated that ectopic expression of TIMP2 open reading frame in the highly metastatic HCC cell line MHCC-97L not only significantly reduced the incidence of tumor microsatellite formation and venous invasion in the primary hepatic tumor xenografts, but also pulmonary metastasis, suggesting that both extrahepatic and intrahepatic metastasis in HCC was suppressed by TIMP2 expression. Mechanistically, TIMP2 suppression in a hypoxic environment was induced through a regulatory feedback circuit consisting of HIF-1á, miR-210 and HIF-3á. Taken altogether, our findings established that TIMP2 was frequently downregulated in human HCCs and its downregulation was associated with aggressive behavior and poorer patients’ outcome. Its suppression was under the regulation of a novel feedback circuit consisting of HIF-1á/ miR-210/ HIF-3á. Overall, our study has provided solid in vitro and in vivo evidence that TIMP2 is an important regulator of ECM degradation and HCC metastasis. These findings demonstrated that perturbation of the dynamic HIF-1a signaling circuit by miR-210 inhibitor abolished TIMP2 downregulation and suppressed HCC cell invasion. They also support the notion that targeting against HIF-1á signaling is a promising direction to tackle the hypoxic responses in HCC elicited by transarterial chemo-embolization (TACE) treatment to HCC patients.

Citation Format: Alan KL Kai, Lo Kong Chan, Regina CL Lo, Joyce Lee, Carmen CL Wong, Jack CM Wong, Irene OL Ng. Downregulation of TIMP2 via HIF-1á/miR-210/HIF-3á regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 689.
Abstract 727: TSLP expression and high serum TSLP level indicate a poor prognosis in gastric cancer patients
Authors: Watanabe Joji , Saito Hiroaki , Miyatani Kozo , Ikeguchi Masahide , Umekita Yoshihisa , Otani Shinji , Tsujitani Shunichi
Published: July 2016

Purpose: Thymic stromal lymphopoietin (TSLP) plays an important role in promoting tumor survival, by manipulating the immune response and angiogenesis. However, the clinical significance of TSLP in gastric cancer is unclear.

Method: Immunohistochemistry was used to investigate TSLP expression in non-cancerous gastric mucosa and gastric cancer tissue from patients with gastric cancer. Serum TSLP levels were measured using an enzyme-linked immunosorbent assay.

Results: Tumors with TSLP expression were significantly larger than those without TSLP expression. TSLP expression was observed more frequently in advanced (T2/T3/T4) than in early (T1) gastric cancer and in stage 3/4 than in stage 1/2. Lymph node metastasis, liver metastasis, positive peritoneal lavage cytology, lymphatic

invasion, and vascular invasion occurred significantly more often in TSLP-expressing than in non-expressing tumors. The prognosis of patients with TSLP-positive tumors was significantly worse than that of patients with TSLP-negative tumors. Patients with high serum TSLP concentrations also had a significantly worse prognosis than those with low concentrations. Multivariate analysis identified serum TSLP level as an independent prognostic indicator.

Conclusion: TSLP is closely related to the progression of gastric cancer and may predict

survival in these patients.

Citation Format: Joji Watanabe, Hiroaki Saito, Kozo Miyatani, Masahide Ikeguchi, Yoshihisa Umekita, Shinji Otani, Shunichi Tsujitani. TSLP expression and high serum TSLP level indicate a poor prognosis in gastric cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 727.
Abstract 768: The ATP6V1C2 (a vacuolar-ATPase gene) is a novel early prognosticator for colorectal cancer
Authors: Zumwalt Timothy J. , Shigeyasu Kunitoshi , Weng Wenhao , Okugawa Yoshinaga , Miyoshi Jinsei , Goel Ajay
Published: July 2016

Background: Vacuolar (v) -ATPases are expressed in certain tissues and cell types, and hypothesized to detoxify intracellular environments by expelling protons across plasma membranes, thereby contributing to acidified extracellular environments of solid tumors. Acidic microenvironments suppress anti-cancer cytotoxic T-cells and activate metalloproteinases, promoting tumor cell survival, motility and invasion. Scarce nutrients, hypoxia, and accelerated metabolism that favor pathways that produce excess intracellular protons by promoting glycolysis and lactic acid fermentation, lead to selection of tumor cells that adapted to and countered toxic/acidic cytoplasm. It is unclear whether colorectal cancers (CRCs) upregulate proton-expelling mechanisms, and whether associated genes are prognostic biomarkers.

Aim: We hypothesized that adaptations through upregulation of proton transporting genes is prognostic for CRC. This study aims to identify transcriptional adaptations that CRCs undergo in response to acidic microenvironments and evaluate their prognostic value in multiple cohorts of CRC patients.

Methods: Comprehensive microarray data from 222 CRCs obtained from The Cancer Genome Atlas were used to screen for genes upregulated in CRCs vs. normal mucosae, and stage III and IV vs. early staged tumors. Filtered genes involved in metabolite transport were tested for their ability to predict survival using Kaplan-Meier survival analysis with a stringent median cutoff. A second cohort was used to corroborate initial findings. A third cohort of matching polyps and adenomas was used to determine if candidate gene upregulation can be detected in pre-cancerous lesions. Functional analysis was performed using siRNA knockdown to determine if the candidate gene is critical for CRC proliferation.

Results: Progressive upregulation of ATP6V1C2 from early to late stage CRCs was detected in two separate multinational patient cohorts, where its high expression was correlated with shorter overall survival. Receiver operating characteristic (ROC) curve analysis confirmed that ATP6V1C2 expression successfully discriminated between cancerous and non-cancer tissues. Fisher's exact test confirmed that high ATP6V1C2 expression correlated with advanced depth of invasion (T stage), and distant and liver metastasis. ATP6V1C2 expression was increased in pre-cancerous polyps. SiRNA knockdown of ATP6V1C2 inhibited cell proliferation in high expressing cell lines (HCT116 and HCT15) but not in low expressing RKO and HT29.

Citation Format: Timothy J. Zumwalt, Kunitoshi Shigeyasu, Wenhao Weng, Yoshinaga Okugawa, Jinsei Miyoshi, Ajay Goel. The ATP6V1C2 (a vacuolar-ATPase gene) is a novel early prognosticator for colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 768.