bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4162: The importance of age-related cytokines in chronic lymphocytic leukemia
Authors: Yoon Ju-Yoon , LaFarge Sandrine , Dawe David , Lakhi Sunjay , Kumar Rajat , Marshall Aaron , Gibson Spencer , Johnston James B.
Published: August 2014

Chronic lymphocytic leukemia (CLL) is a disease of the elderly with the median age at diagnosis being 72 years. Prognosis is highly variable with the relative survival progressively worsening after age 65 years. In this study we have evaluated whether the age-related cytokines, IL-6, IL-8 and TNF-alpha, play a role in the poor survival of elderly patients with CLL. The plasma levels and biological effects of the age-related cytokines, IL-6, IL-8 and TNF-alpha, were evaluated in 193 CLL patients of varying ages. Patients were chosen randomly to reflect the age spectrum and sex distribution of the Manitoban CLL population. As predicted from population studies, the survival of patients progressively worsened with age, with the primary causes of death being CLL or one of its complications (infection or second tumors). Cytokine levels were increased in CLL compared to 37 age- and sex-matched controls. A third of patients aged </=65 years had increased IL-6, compared to 10% in patients < 65. A high correlation was found between the plasma levels of these three cytokines with age and Beta2-microglobulin (a measure of tumor burden). The plasma levels of the cytokines also correlated with each other, suggesting they increased in parallel. In contrast, the standard CLL prognostic markers IgVH mutational status, ZAP-70, CD38 and Rai stage did not correlate with cytokine levels or age. Patients with increased IL-6 or IL-8 had a poorer survival than those with normal levels. For IL-6, this difference was more marked for those </=65 years. Furthermore, focusing on older patients, multivariate analysis with backward selection of variables showed IL-6 and Beta2-microlgobulin to be significant predictors of survival, while the mutational status and Rai stage were not significant. The cause of death was due to CLL-related causes and not to frailty or cardiovascular disease. However, increased cytokine levels correlated with a higher lifetime incidence of cardiovascular disease. Thus, one source of cytokines may be cardiovascular disease, although CLL cells also secreted low amounts of IL-6 and IL-8. The two cytokines, in turn, significantly increased the adhesion of leukemic cells to stromal cells, suggesting that these cytokines may influence the microenvironment, which would contribute to poor survival.

In summary, IL-6 and IL-8 are predictors of survival in CLL, where IL-6 is a stronger predictor of survival in older patients than mutational status or Rai stage. Importance of IL-6 and IL-8 may be associated with their role in facilitating the leukemic-stromal cell interaction.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4162. doi:10.1158/1538-7445.AM2011-4162
Abstract 4169: Loss of GPR30 expression identifies estrogen receptor positive endometrial carcinoma patients with poor outcome
Authors: Krakstad Camilla , Trovik Jone , Wik Elisabeth , Engelsen Ingeborg B. , Øyan Anne M. , Stefansson Ingunn M. , Kalland Karl-Henning , Akslen Lars A. , Salvesen Helga B.
Published: August 2014

In industrialized countries, endometrial cancer is the most common pelvic gynecologic malignancy, with a 2% to 3% lifetime risk. Expression of estrogen receptor (ER) and progesterone receptor (PR) is known to be associated with good prognosis and response to anti-hormonal therapy. Still, up to 20% of receptor positive cancers recur. The G-protein coupled receptor GPR30 has been suggested as an alternative estrogen receptor. We have studied a prospective series of 472 endometrial cancer patients by immunohistochemistry and 76 for mRNA expression levels by oligonucleotide array. We identify that loss of GPR30 is significantly correlated with non-endometrioid histology and high histologic grade both at the mRNA and protein levels (all p-values≤0.006). In ER positive patients (n=363), loss of GPR30 significantly correlates with high FIGO stage, non-endometrioid histology and high histologic grade (all p-values≤0.005). Loss of GPR30 reduces the 5-year survival of ER positive patients from 93% to 76% (p=0.003) and identifies a subgroup of patients with poor prognosis that until now has been evaluated as favorable survivors. Examination of the gene expression profile for ER positive tumors with and without loss of GPR30, indicates mechanisms involved related to GPR30 expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4169. doi:10.1158/1538-7445.AM2011-4169
Abstract 4171: The role of ATM and 53BP1 as predictive markers and therapeutic targets in advanced stage cervical cancer
Authors: Roossink Frank , Wieringa Hylke W. , Noordhuis Maartje G. , Kok Mirjam , ten Hoor Klaske A. , de Bock Geertruida H. , de Jong Steven , van der Zee Ate G. J. , Schuuring Ed M.D. , Wisman G. Bea A. , van Vugt Marcel A.T.M.
Published: August 2014

Chemoradiation is the standard of care for advanced stage cervical cancer patients and induces cytotoxicity by inducing high levels of DNA breaks in the tumor. The ability of tumor cells to repair therapy-induced DNA damage may counteract the effects of chemoradiation. The pathway responsible for DNA double strand break repair is called the ‘DNA damage response’.

In vitro we observed robust radiation-induced G1 and G2 cell cycle arrests in cervical cancer cell lines, despite the expression of HPV oncogenes, which impact the DNA damage response through rapid p53 degradation. Moreover, the DNA damage checkpoint protein ATM and its substrate 53BP1 are required for proper induction of cell cycle checkpoint arrests. Yet, when long-term survival effects were measured, inhibition of ATM, but not 53BP1, strongly sensitized cervical cancer cells to irradiation. To validate the putative role of ATM and 53BP1 in advanced stage cervical cancer we investigated presence and activity of ATM as well as 53BP1 in relation to response-to-treatment in a large, well documented, series of advanced stage cervical cancer patients (n=375). We found that high levels of ATM were related to poor response to chemoradiation (P=0.013). Importantly, high phosho-ATM immunostaining, reflecting active ATM, predicted poor responses to chemoradiation even better (P=0.004). In addition, high phospho-ATM levels predicted shorter disease-specific survival (P=0.020).

Although presence of phosphorylated 53BP1 did relate to phospho-ATM (P=0.001), it was not significantly related to any clinicopathological features or survival.

Combined, our data suggest the prognostic value of ATM signaling for responses to chemoradiation and point at ATM inhibition as a potential therapeutic target to increase treatment efficacy in advanced stage cervical cancers.

Acknowledgements

This research is supported by the Dutch Cancer Society (RUG 2007-3719) and the Netherlands Organization for Scientific Research (NWO-VENI 916.76.062).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4171. doi:10.1158/1538-7445.AM2011-4171
Abstract 4174: Variation in UDP glucouronyltransferase (UGT) genes associated with prostate cancer mortality
Authors: Kohli Manish , Mahoney Douglas , Chai High , Hillman David , Rider David , Costello Brian , Cerhan James
Published: August 2014

Background: We investigated the association of germline genetic variation in hormone biosynthesis and metabolism genes with prostate cancer specific mortality in a cohort of advanced prostate cancer patients. Methods: We genotyped 852 single nucleotide polymorphisms (SNPs) from 97 genes in a cohort of 267 advanced prostate cancer patients at the time of progression to castration recurrence (CRPC) during on-going androgen ablation. Tagging SNPs with minor allele frequency (MAF) of >5% and r2 ≥0.8 were selected from HapMap, NIEHS and Seattle SNP databases. Medical records were queried for cause of death. The primary endpoint of time to prostate cancer specific mortality (PCSM), was pre-defined as time from development of CRPC to death from prostate cancer progression. Principle components analysis was used for gene-levels tests, and to account for multiple testing, we calculated the false discovery rate (FDR). For SNP level results, Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using cox regression. Results: The median age of the cohort was 72 years at CRPC. 43% had a Gleason score (GS)=8-10, 33% a GS=7, and 24% a GS<7. After a median follow up of 1.8 years (IQ range: 0.8-3.3 years), 139 patients died, of which 107 were due to prostate cancer progression. In unadjusted gene level analyses, UGT1A7 (p=0.0059; FDR=0.19), UGT1A10 (p=0.0017; FDR=0.17) and UGT1A3 (p=0.0037; FDR=0.18) were associated with PCSM. After adjusting for age and GS, SNPs strongly associated with PCSM are listed in the Table.

Conclusions: Variation in UGT genes involved in hormone metabolism yield prognostic information in CRPC. Further validation is needed to develop these as prognostic biomarkers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4174. doi:10.1158/1538-7445.AM2011-4174
Abstract LB-179: The effects of hypoxia and reactive oxygen species on the methylation status of the polo-like kinases
Authors: Ward Alejandra , Hudson John W.
Published: August 2014

The family of polo-like kinases (Plks) consisting of five known members are key regulators of important cell cycle processes, such as mitotic entry, centrosome duplication, spindle assembly, and cytokinesis. Plk1 is typically overexpressed in many cancers, while Plks 2–4 deregulation is often associated with tumorigenesis and malignancy. Plk2 has been shown to be downregulated via promoter DNA hypermethylation in blood neoplasms. Furthermore, in hepatocellular carcinoma (HCC), we and others have shown Plks 2–4 are downregulated through epigenetic modifications, while concurrent decreased Plk 1 promoter methylation was correlated with increased expression. As changes in epigenetic modifications have been associated with external environmental stimuli, we hypothesized that the Plks may be important targets whose disregulation could contribute to malignancy. We therefore examined the status of promoter methylation and expression levels of the Plk family members under hypoxic conditions, and upon exposure to reactive oxygen species (ROS). Interestingly both wild-type and Plk4 heterozygous murine embryonic fibroblasts (MEFs) exposed to ROS displayed significant promoter methylation post treatment. Correspondingly, Plk4 transcript levels were reduced to 90% with no detectable Plk4 protein after exposure to ROS. Since ROS and hypoxic conditions are associated with liver function and normal metabolism, we next examined the promoter methylation status for the individual Plks and other markers in HepG2 and Hep3B liver cell lines. We found increased hypermethylation of the promoter region as well as decreased transcripts levels for both Plk1 and Plk4 post ROS treatment in both cell lines. No detectable methylation changes were detected in SAOS (bone sarcoma, p53 null) cells, while U2OS (p53 wild type) displayed increased methylation and decreased transcript levels. This suggests that the response to ROS might be dependent upon p53 status and origin of cell line. Similar to the case with ROS, hypoxia treatment also induced hypermethylation of Plk4 and decreased in transcripts in both HepG2 and Hep3B cells, while Plk1 levels were only reduced in HepG2 cells. Our results suggest that changes in the cellular environment may be a key regulator of Plk methylation status that in turn leads to deregulation. This has important implications in HCC and other cancers, where these epigenetic alterations could be contributing factors in tumourigenesis and disease progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-179. doi:10.1158/1538-7445.AM2011-LB-179
Abstract LB-182: Preoperative serum level of homocysteine is associated with survival of breast cancer among Korean women
Authors: Lee Yunhee , Lee Sang-Ah , Lee Kyoung-Mu , Lee Ji-Young , Sung Hyuna , Li Lian , Park Sue K. , Yoo Keun-Young , Noh Dong-Young , Ahn Sei-Hyun , Choi Ji-Yeob , Kang Daehee
Published: August 2014

The serum level of homocysteine is suggested to be related to inflammation, cardiovascular disease, and cancer. This study aimed to evaluate preoperative serum homocysteine level as biomarker for breast cancer prognosis. We measured serum homocysteine levels using the fluorescence polarization in 370 participants who were newly diagnosed and histopathologically confirmed breast cancer from hospitals located in Seoul, Korea between 2004 and 2007. Univariate and multivariate analyses were used to investigate association between serum homocysteine level and disease free survival. The median concentration of homocysteine was 10.09 ng/mL and interquartile range was 8.4–12.6. Kaplan-Meier curves showed that the elevated concentration of homocysteine was associated with poor disease-free survival (log rank p = 0.02). In multivariate analysis using the Cox's proportional hazard regression model, patients with greater than median levels of homocysteine showed increased risk of breast cancer recurrence compared to the patients with lower levels of homocysteine after adjusting for factors related to prognosis (hazard ratio = 2.03; 95% confidence interval, 1.13–3.64; p = 0.02). These findings suggest that higher level of homocysteine may be associated with the poor prognosis of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-182. doi:10.1158/1538-7445.AM2011-LB-182
Abstract LB-183: Association of aberrant promoter methylation of novel genes with clinicalpathology and prognosis of urothelial carcinoma
Authors: Li Ching Fei , Lee Te-Chang , Wang Sheng-Shin Alexander , Pu Yeong-Shiau
Published: August 2014

DNA methylation profiles are one of valuable biomarkers to molecularly define cancer subtypes that may serve as better indicators for disease progression, treatment and prognosis. In the present study, we investigated the aberrant promoter methylation of 5 tumor-related genes, CCND1, CHFR, FAM51A1, LGALS4, and RBBP7, which were selected from pooled DNA methylation profiles of urothelial carcinoma (UC) with various clinicopathological parameters. DNA was extracted from 90 patients with confirmed UC and 10 adjacent non-tumor controls. Promoter aberrant methylation of these 5 genes was determined by quantitative methylation-specific PCR (qMSP). Our results showed that methylation levels were higher in UC as compared with controls in promoter of CHFR, FAM51A1, LGALS4, and RBBP7 (6.24 vs. 4.27; 0.66 vs. 0.01; 7.47 vs. 2.14; 0.45 vs. 0.01, respectively). Among UC patients, the levels of promoter methylation of CCND1, CHFR and LGALS4 were significantly associated with tumor stages (p< 0.01), whereas that of FAM51A1 was inversely associated with tumor stages (p=0.012). Tumors with poor differentiation showed higher levels of promoter methylation on CCND1, CHFR and LGALS4 genes than tumors with moderate differentiation (p<0.05). UC patients with highly methylated CCND1, CHFR, LGALS4 or RBBP7 tended to have a poor survival than patients with low levels of methylated gene (p<0.02). This is the first report to show the association of promoter methylation of these 5 genes with UC tissues. We anticipate that the methylation status of these novel marker genes may have great clinical implications for further classifying UC patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-183. doi:10.1158/1538-7445.AM2011-LB-183
Abstract LB-184: Bmi1 regulates cell fate via tumor suppressor WWOX repression in small cell lung cancer cells
Authors: Kimura Masaki , Akita Nobuhiro , Takenobu Hisanori , Kimura Hideki , Nakagawara Akira , Kamijo Takehiko
Published: August 2014

Mortality from lung cancer is important worldwide. Recently, epigenetic aberration of lung cancer, not only genomic DNA methylation but also chromatin modification, has become an important target for lung cancer research, although previous research has demonstrated that lung cancer develops as a result of both environmental and genetic factors. Here, we demonstrated that an epigenetic regulator/polycomb group protein Bmi1 is more highly expressed in small cell lung cancer than in non-small-cell lung cancer by immunohistochemical analysis. In vitro experiments indicated that Bmi1 reduction by lentivirus-derived shRNA significantly suppressed proliferation, colony formation, and in vivo tumor formation. Importantly, apoptosis was induced by Bmi1 depletion in small cell lung cancer cells. Furthermore, a tumor suppressor WWOX was identified as a Bmi1 target in the cells by chromatin immunoprecipitation assay and quantitative real-time PCR assay; WWOX had a role as a tumor suppressor in SCLC cells; therefore, the Bmi1/WWOX pathway could be a new candidate for a new therapeutic approach for small cell lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-184. doi:10.1158/1538-7445.AM2011-LB-184
Abstract LB-186: Somatic Y641 mutations in EZH2 alter the balance of di- and tri-methylation of H3K27 in diffuse large B-cell lymphomas
Authors: Diaz Elsie , McCabe Michael T. , Jiang Yong , Chen Stephanie , Pappalardi Melissa , Smitheman Kimberly , Della Pietra Anthony , Graves Alan , Kruger Ryan , Brandt Martin , Sweitzer Sharon , Schwartz Benjamin , Tummino Peter J. , Creasy Caretha L.
Published: August 2014

EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) responsible for methylating histone H3 on lysine 27 (H3K27) and repressing transcription of target genes. PRC2 activity is essential for maintaining the self-renewal capacity of embryonic and adult stem cells and the dynamic regulation of this activity is critical for proper development and differentiation. Dysregulation of H3K27 methylation is implicated in tumorigenesis and occurs through multiple mechanisms. Elevated levels of EZH2 are known to correlate with poor prognosis in a number of solid tumors including prostate and breast. Inactivating mutations in UTX, an H3K27 demethylase which acts in opposition to EZH2, have been described in several tumor types including multiple myeloma, esophageal squamous cell carcinoma, and renal cell carcinoma. More recently, somatic mutations in EZH2 were identified in myelodysplastic syndrome (MDS), follicular lymphoma (FL), and GCB diffuse large B cell lymphoma (DLBCL). While the mutations in MDS are often homozygous and encompass missense, nonsense, and frame shift mutations at multiple regions of the protein, the mutations in DLBCL and FL are heterozygous and occur at a single residue (Y641) suggesting that the effect of these mutations on PRC2 activity could be quite different between MDS and lymphoma. Utilizing a biochemical approach with recombinant PRC2 containing either wild-type or mutant EZH2, we demonstrate that Y641 mutants exhibit an altered substrate preference. In contrast to wild-type EZH2 which prefers an unmodified or mono-methylated K27 residue, Y641 mutants act primarily on a di-methylated H3K27 with little activity for unmodified or mono-methylated K27. Consistent with these biochemical data, we find that when compared to wild-type lymphoma cell lines those harboring Y641 mutations have elevated levels of H3K27me3 and reduced H3K27me2. To further characterize the substrate specificity of PRC2 complexes containing either WT or mutant EZH2, we utilized an epigenetic peptide library which contained unmodified and modified peptides from histone H2A, H2B, H3 and H4. In addition, we have conducted cell-based studies to understand the effect of these mutations on H3 methylation and the regulation of EZH2 target genes. These data and the implications of these findings for the treatment of FL and DLBCL will be discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-186. doi:10.1158/1538-7445.AM2011-LB-186
Abstract LB-189: Fbxo7 functions in the proliferation and maturation of hematopoietic cells
Authors: Randle Suzanne J. , Meziane El Kahina , Lomonosov Mikhail , Laman Heike
Published: August 2014

The F-box protein Fbxo7 specifically enhances the levels of cyclin D-Cdk6 complexes, the predominant G1 kinase active in hematopoietic cells, which is a commonly activated oncogenic pathway in lymphoid neoplasms. In addition, Fbxo7 is itself abundantly expressed in high-grade B and T cell malignancies. Previously we have tested the ability of Fbxo7 to act as an oncogene in the hematopoietic compartment in adoptive transfer experiments using p53 null hematopoietic stem cells over expressing Fbxo7. Animals reconstituted with these cells develop T cell lymphoma, supporting the idea that Fbxo7 can have tumor promoting activity in vivo. However, in experiments presented here, to investigate the transforming capacity of Fbxo7 in B lymphocytes, we found that overexpression of Fbxo7 in the mouse pro-B cell line Ba/F3 did not transform cells nor enhance their proliferation. Instead, the stable reduction of Fbxo7 expression increased proliferation and decreased cell size, which were attributable to shortening of G1 phase, decreased p27 levels and increased Cdk2 activity. These results suggest that Fbxo7 normally restrains proliferation in pro-B cells. Reducing Fbxo7 expression also changed the cell surface marker phenotype to resemble a more immature state of B cell maturation. To investigate the activities of Fbxo7 in hematopoietic cells in a more physiological setting, we have generated an in vivo model of reduced Fbxo7 expression by engineering a mouse with an insertional mutation of the LacZ gene into the FBXO7 locus. Phenotypic characterization of these mice will also be presented, and these data support the idea that Fbxo7 regulates the proliferation and the maturation of different precursor cell populations, including pro-B cells and pro-erythroblasts. We propose that Fbxo7 has the capacity to function as a tumor suppressor and also as an oncogene depending on the cell type and its stage of differentiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-189. doi:10.1158/1538-7445.AM2011-LB-189
Abstract LB-193: APOBEC3 catalyzed genomic mutations in breast cancer
Authors: Burns Michael B. , Lackey Lela , Land Allison , Rathore Anurag , Refsland Eric , Harris Reuben
Published: August 2014

In this work, we present evidence that an endogenous DNA mutating protein, APOBEC3, is massively overexpressed in a high proportion of breast cancers and that this overexpression may be responsible for the genetic variability seen in these cancers. Multiple driver mutations are required for cancer development and recent genome-wide approaches have revealed that many cancers, including breast, harbor staggering numbers of mutations, both point mutations as well as gross chromosomal rearrangements. The molecular origins of most of these somatic aberrations remain unclear. In this work, we test the hypothesis that APOBEC3, an endogenous DNA mutator, is a causative factor driving these mutations. One clue derives from the fact that C/G-to-T/A transition mutations, both within and outside of methyl-C-p-G dinucleotide motifs, predominate in subsets of many tumor types, including breast cancer. We demonstrate that APOBEC3, a DNA cytosine deaminase, is a likely endogenous source of mutation in breast cancer. We show, using quantitative reverse transcriptase PCR (qRT-PCR), that APOBEC3 is overexpressed in a majority of commonly available breast cancer cell lines (34/44) when compared to MCF-10A and hTERT-HMEC control lines. This overexpression phenotype persists when examining primary breast tumors (24/37) using patient-matched normal tissues as controls. APOBEC3 is predominantly localized to the nuclear compartment when overexpressed, as seen using fluorescently tagged APOBEC3. Additionally, its catalytic DNA cytosine to uracil deamination activity is dramatically elevated in this compartment in multiple breast cancer cell lines, as quantified using a FRET-based DNA oligonucleotide cleavage assay. This presentation will discuss the potential impact of this potent endogenous mutagen on the breast cancer genome and the implications for tumor evolution and therapy resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-193. doi:10.1158/1538-7445.AM2011-LB-193
Abstract LB-28: RAF inhibitors activate MAPK pathway through relief of RAF auto-inhibition
Authors: Holderfield Matthew , Merritt Hanne , Chan John , Wallroth Marco , Tandeske Laura , Hardy Steve , Hekmat-Nejad Mohammad , McCormick Frank , Nagel Tobi
Published: August 2014

Mutations in the RAS/RAF/MEK/ERK pathway are among the most common in human cancers, and RAF small molecule kinase inhibitors are now being tested for the treatment of BRAFV600E tumors, with promising initial results. However, both clinical and pre-clinical data indicate that selective RAF inhibitors have poor efficacy in wildtype BRAF tumors. Moreover, these inhibitors actually promote phosphorylation of downstream targets, MEK and ERK when wildtype RAF is activated by upstream components of the pathway. Here, we show that biochemical activation of wildtype BRAF and CRAF by selective RAF inhibitors is exquisitely sensitive to ATP concentrations in an in vitro kinase assay. While BRAFV600E kinase activity remains high at ATP concentrations in excess of 1mM, wildtype BRAF and CRAF reach maximum kinase velocity at ∼1–10 mM ATP and are inhibited by more than 80% in the presence of 1mM ATP. Selective small molecule RAF inhibitors block the ATP concentration dependent inhibition of wildtype RAF, demonstrating that inhibition requires RAF kinase activity and suggests RAF auto-phosphorylation. Furthermore, while in its auto-inhibited state, wildtype RAF kinase activity cannot be increased by RAF inhibitors, but is rather decreased in a dose dependent manner consistent with ATP competitive inhibition kinetics, demonstrating that binding compound does not reverse RAF auto-inhibition. Together, these results suggest a novel mechanism that can account for the paradoxical activation of MAPK pathway due to wildtype RAF inhibition whereby wildtype RAF exists in an auto-inhibited state at physiological conditions through auto-phosphorylation. In the presence of a RAF inhibitor wildtype RAF auto-phosphorylation and consequential auto-inhibition of RAF is therefore relieved to activate the MAPK pathway leading to enhanced proliferation. This study offers new insight into RAF biochemistry and enzyme kinetics with significant implications for drug development strategies and therapeutic use of RAF inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-28. doi:10.1158/1538-7445.AM2011-LB-28
Abstract LB-284: The novel therapeutic monoclonal antibody VGX-100 neutralises VEGF-C and inhibits tumor growth and metastasis in subcutaneous and orthotopic models of human cancer.
Authors: Baldwin Megan E. , Tester Angus M. , Phelan David , Klupacs Robert
Published: August 2014

Angiogenesis and lymphangiogenesis are important processes facilitating tumor growth and metastasis. Growth factors that stimulate blood and lymphatic proliferation within tumors are therefore potential targets for anti-cancer therapies. Proof of concept of the clinical utility of anti-angiogenic drugs was first established by the FDA/EMEA-approved drug bevacizumab (Avastin®) which blocks VEGF binding to its receptors VEGFR-1 and VEGFR-2, the latter being the key receptor signaling for angiogenesis. However, patients treated with bevacizumab may be refractory or develop resistance to bevacizumab, suggesting upregulation of alternative pro-angiogenic proteins that allow tumors to bypass the inhibition of VEGF signalling. VEGF-C is a logical candidate for inducing resistance to bevacizumab via this mechanism since it is also a ligand for the angiogenic receptor VEGFR-2 and for VEGFR-3 which is upregulated on tumor-associated vascular endothelium.

VGX-100 is a highly specific, fully human monoclonal antibody for VEGF-C that blocks VEGF-C binding to both VEGFR-2 and VEGFR-3. Here we demonstrate that VGX-100 has an additive effect in combination with docetaxel and/or anti-VEGF (bevacizumab) in several tumor models, suggesting that VEGF-C may be an important mediator of the resistance to existing anti-VEGF therapies. Further, we demonstrate that in an orthotopic model of prostate cancer, that inhibition of VEGF-C alone by VGX-100 monotherapy is sufficient to inhibit tumor growth and significantly reduce the incidence of tumor metastasis to local lymph nodes. These data indicate that VGX-100 has exciting potential as a cancer therapeutic by targeting a key factor involved in angiogenesis, lymphangiogenesis and tumor metastasis and is expected to complement chemotherapy and/or other anti-angiogenic compounds in the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-284. doi:10.1158/1538-7445.AM2011-LB-284
Abstract LB-285: Blocking Dll4/Notch signaling inhibits development of chronic colitis-associated colorectal cancer in mice
Authors: Duarte Antonio , Badenes Marina , Trindade Alexandre , Liu Ren , Gill Parkash S.
Published: August 2014

Colorectal cancer (CRC) is one of the most frequent cancers worldwide, often developing as a complication of inflammatory bowel diseases. The Notch pathway plays a central role in intestinal homeostasia and its deregulation contributes to tumor development. Inhibition of Notch signaling through the Dll4 ligand shows anti-tumor activity against a variety of human and rodent tumors grafted in mice.

The aim of this work was to analyze Dll4 expression in both normal intestine and intestinal tumors, and determine the effect of Dll4 inhibition by using Dll4 heterozygous mice or administering a pharmacological inhibitor (soluble Dll4-Fc) in the mouse model of chemically induced colo-rectal cancer (CRC). To induce chronic colitis and CRC we combined the administration of azoxymethane, and repeated cycles of the proinflammatory dextran sodium sulfate in the drinking water, as previously described (Neufert et al. Nat Protoc 2: 1998) in either Dll4+/− mice or wild type mice treated with sDll4-Fc at a dose of 5mg/Kg/day, IP, 3x/wk from week 10 of AOM plus DSS treatment to week 14.

Dll4 expression was detected in the vascular endothelium as well as in both absorptive and secretive epithelium of the normal intestine and colorectal (CR) polyps. Both genetical (Dll4 allelic deletion, Dll4+/−) and pharmacological inhibition by the soluble extracellular domain of Dll4 (Dll4-Fc) led to significant reductions in the average number (12.5-fold in mutants, 3.7-fold in Dll4-Fc treated), average volume (1.6-fold in mutants, 2.3-fold in Dll4-Fc treated) and overall burden (15.2-fold in mutants, 6.3-fold in Dll4-Fc treated) of CR polyps, by inducing intratumoral apoptosis and disrupting proper tumor angiogenesis., leading to the formation of immature and non-functional blood vessels. Dll4 heterozygous mutants shown a 42.5% increase in tumor vascular density and a 13.9 % decrease in smooth muscle cell vascular recruitment, displaying increased vascular permeability and reduce vascular perfusion. Dll4-Fc treated mice displayed more proeminent vascular disruption, with a 127.5% increase in tumor vascular density and a 53.2% decrease in smooth muscle cell vascular recruitment, showing extensive vascular extravasation and poor perfusion of the tumor blood vessels.

In conclusion, Dll4Fc administration in chronic colitis-related CRC appears to constitute a promising therapeutical approach, capable of inhibiting the tumorigenic events as well as targeting the tumour vasculature and preventing tumor development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-285. doi:10.1158/1538-7445.AM2011-LB-285
Abstract LB-286: Reconstitution of the human ovarian tumor-microenvironment in CD34-transplanted NOD-Scid-IL2Rγc null (NSG) mice
Authors: Walter Marc , Mookerjee Ananda , Nunez-Cruz Selene , Fang Chongyun , Facciabene Andrea , Coukos George , Scholler Nathalie , Danet-Desnoyers Gwenn
Published: August 2014

The use of stem cell-derived human immune system NSG mice and orthotopic tumors to recapitulate the human ovarian tumor microenvironment represents a significant improvement over existing mouse models of cancer. However, the careful phenotyping of a human ovarian tumor microenvironment in NSG mice has not been reported. We characterized the human ovarian tumor microenvironment in NSG mice reconstituted with a full human immune system using cord blood-derived CD34+ cell transplantation, and injected with human ovarian tumor cells. Our findings demonstrated human B cell and T cell infiltration in both orthotopic and subcutaneous tumors. Orthotopic tumors showed a 3 to 4-fold increase of human CD3+ cell infiltration in comparison with subcutaneous tumors or spleens from tumor bearing mice, however, the overall CD4/CD8 T cell ratio was similar in both tumor sites. Tumor-infiltrating T cells included effector memory TEM cells and Treg cells. Orthotopic tumor dissociates also contained significantly more human macrophages (TAMs) than subcutaneous tumors or spleens from tumor bearing mice. While all macrophages were found to have an M1-like phenotype, TAM subsets could be distinguished from splenic macrophages by CD40 or HLA-DR expression and TNFβ secretion. Furthermore, TAMs phenotypic differences observed in orthotopic tumors were associated with faster tumor growth kinetics and metastasis. Finally, we found that, in addition to the murine origin of the tumor stroma, a significant number of mouse neutrophils and macrophages were also present. We propose that humanized mice bearing human tumors could be a valuable tool for the preclinical evaluation of targeted therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-286. doi:10.1158/1538-7445.AM2011-LB-286
Abstract LB-289: Angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma of the minor salivary glands
Authors: Gleber-Netto Frederico O. , Florencio Taynara Nunes Guedes , Sousa Silvia Ferreira , Mendonça Elismauro Francisco , Gomes Carolina Cavalieri , Aguiar Maria Cassia Ferreira
Published: August 2014

The purpose of this study was to investigate angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma (MEC) of the minor salivary glands as potential predictors of tumor behavior. Intratumoral and peritumoral neoformed (NMVD) and lymphatic microvessel density (LMVD) were evaluated by CD105 and D2–40 immunohistochemistry, respectively. D2–40, VEGF-A, and VEGF-C immunoexpression were also investigated in tumor parenchyma. Twenty-six paraffin-embedded MEC samples were included in the study. Palate (50%) and buccal mucosa (19.2%) were the main affected sites. Sixteen (61.5%) tumors were low-grade, 6 (23.1%) intermediate-grade, and 4 (15.4%) high-grade. Twenty (77%) cases were clinically T1-T2, and 6 (23%) were T3-T4. Nodal metastasis occurred in 3 (11.5%) cases and distant metastasis in 1 (3.8%). Three (11.5%) cases recurred, and 3 (11.5%) patients died from disease. NMVD mean was 19.58±13.91 (400x field) for intratumoral evaluation and 10.90±7.79 (400x field) for peritumoral evaluation. LMVD mean was 5.66±3.43 (400x field) for peritumoral evaluation and 0.54±3.43 (400x field) for intratumoral evaluation. Just 6 (23.1%) cases reveal intratumoral lymphatic vessels. Twenty-two (84,6%) cases showed D2–40 in neoplastic cells, mainly in epidermoid type. Fifteen (57.7%) cases were positive for VEGFA, and 11 (42.3%) were positive for VEGF-C. A positive association was observed between VEGF-A positivity and intratumoral NMVD (p=0.05). Cases with nodal metastasis, altogether, were related to low intratumoral NMVD, high peritumoral LMVD, D2–40 expression and absence of VEGF-A. Distant metastasis was associated with low intratumoral NMVD, high peritumoral LMVD, D2–40 positivity and absence of VEGF-A and VEGF-C expression. Recurrent cases presented low intratumoral NMVD, and absence of VEGF-A and VEGF-C expression. Low intratumoral NMVD, and D2–40 positive cells were found in all tumors of patients that died of the disease.

In conclusion, although NMVD, LMVD, D2–40, VEGF-A, and VEGF-C could not predict the prognosis of MEC, intratumoral NMVD, peritumoral LMVD, D2–40 and VEGF-A expression seems play a role in the occurrence of a worse behavior in this disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-289. doi:10.1158/1538-7445.AM2011-LB-289
Abstract LB-292: Role of TLR engagement in TIL-B and clinical implications for ovarian cancer
Authors: Nunez-Cruz Selene G. , Guerra Matthew , Connolly Denise , Gimotty Phyllis , Scholler Nathalie
Published: August 2014

Ovarian cancer is the eighth most common cancer in women and represents the most lethal gynecological cancer, where less than a third of the women survive longer than five years after diagnosis. The identification of novel therapeutic strategies is urgently needed to improve patient prognosis. Solid and hematologic malignant cells express a broad range of Toll-like receptors (TLR). Activation of TLR signaling through recognition of pathogen-associated molecular patterns leads to the transcriptional activation of genes encoding for pro-inflammatory cytokines, chemokines and co-stimulatory molecules. Solid tumor development has been associated with TLR engagement through both growth acceleration and immune escape, and epidemiological studies reported a link between TLR expression by ovarian tumor cells and chemoresistance. We previously observed a large proportion of B cells in the immune infiltrate of mouse ovarian tumors and in human ascites from ovarian cancer patients, but the role of TLR stimulation in ovarian tumor-infiltrating lymphocyte B (TIL-B) remains unclear. The objective of this study is to investigate the functional consequences of TLR expression and engagement on TIL-B in ovarian cancer. Our work has been carried in a transgenic mouse model (TgMISIIR-Tag) that spontaneously develops tumors through the expression of SV40 T antigen under the Mullerian Inhibitor Receptor type II (MISIIR) promoter. TgMISIIR-Tag tumors resemble human serous ovarian cancer, the most common type of ovarian cancer. Microarrays and flow cytometry analyzes were used to characterize the phenotype of TIL-B after CD19 magnetic sorting ex vivo or after in vitro culture. We first found that TIL-B significantly over-expressed transcripts encoding for inflammatory cytokines, growth factors, adhesion molecules and chemokines, compared to B cells from spleens and lymph nodes of healthy or tumor bearing mice. We also showed that B cells from TgMISIIR-TAg tumors and from ascites of ovarian cancer patients over-expressed TLR2, TLR4 and/or TLR9, to compare with mouse splenic or human peripheral B cells, respectively. In addition, five days co-culture of TIL-B with ovarian tumor cells and TLR4 and TLR 9 agonists further increased TLR4 and TLR9 expression, as well as the expression of CD5 and CD1d costimulatory molecules and IL10 production. Concomitant treatment with TLR antagonists reduced TLR expression and IL-10 production of TIL-B. In vivo studies aiming to interrogate the role of TLR engagement on TIL-B in ovarian cancer are ongoing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-292. doi:10.1158/1538-7445.AM2011-LB-292
Abstract LB-293: Perfluorocarbon labeling of human α-type-1-polarized dendritic cells and conventional dendritic cells for in vivo trafficking by fluorine MRI
Authors: Helfer Brooke M. , Balducci Anthony , Kalinski Pawel , Wesa Amy
Published: August 2014

Human dendritic cells (DC) have yet to live up to their clinical promise as an immunotherapeutic intervention against cancer, in spite of their in vitro profiles of their ability to effectively prime tumor-specific T cell responses. It is still unclear which is the best route of administration for the most efficient interaction of DC with tumor-specific T cells in patients, and whether the migratory capacity and localization of DC in patients correlates with clinical responsiveness. Here we utilize a perfluorocarbon (PFC) magnetic resonance imaging (MRI) tracer agent which may enable the assessment of the migration and localization of α-type-1-polarized dendritic cells (αDC1) in the clinic. αDC1 producing high levels of interleukin-12p70 with putative strong lymph node homing capabilities, are currently being evaluated clinically for anti-cancer activity. In vivo results demonstrate the capability of 19F MRI to detect and quantify labeled cells with moderate sensitivity, detecting fewer than 1×104 cells per voxel. The lack of endogenous fluorine in the body allows highly-specific 19F MR detection of label and when paired with proton MRI provides clear and quantitative cell localization within their anatomical context.

The inertness of this tracking agent on the functional capacity of DC was examined in a panel of in vitro studies. The use of the MRI tracer with both conventional DC and αDC1 indicated similar labeling efficiencies with no observed effect on the DC's ability to stimulate T cell expansion, produce IL-12p70, process antigen or express co-stimulatory and polarizing factors. These data support the clinical translation of this tracer agent to safely track and quantify the administration and migration of human αDC1 by non-invasive 19F MRI in the treatment of cancer patients. An imminent clinical trial of PFC-labeled αDC1 will provide cell tracking data in patients, facilitating advances in greater efficacy in the next generation of DC-based cellular vaccines.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-293. doi:10.1158/1538-7445.AM2011-LB-293
Abstract LB-299: Effect of antiangiogenic therapy on tumor growth, vasculature and kinase activity in luminal and basal-like breast cancer xenografts
Authors: Lindholm Evita , Kristian Alexandr , Nalvoga Hawa , Kruger Kristi , Mælandsmo Gunhild M. , Akslen Lars A. , Engebraaten Olav
Published: August 2014

Angiogenesis is important for the growth and dissemination of solid tumors, and VEGF-A is one of the main stimulators of this process. Bevacizumab binds VEGF and increases the progression-free, but not overall, survival in breast cancer patients treated with chemotherapy. For determining factors of importance for treatment response and resistance, two different human breast cancer xenografts (luminal -and basal-like) were used, which respond differently to antiangiogenic mono -or combination therapy. Both doxorubicin and bevacizumab inhibited tumor growth significantly in basal-like xenografts, but with a superior effect when given in combination. In contrast, luminal-like tumors demonstrated no additional effect of combination therapy.

Vascular characterization demonstrated a total inhibition of endothelial cell proliferation (pMVD) and reduction in mean vascular density (MVD) in the basal-like tumors three days after bevacizumab treatment, not seen in the luminal-like tumors. At day 10, combination treatment reduced pMVD and MVD in both tumors, however only MVD lasted to the end of the experiment and in the basal-like tumors.

Analysis of kinase activity in the basal-like tumors after monotherapy demonstrated an early (day 3) increase in the phosphorylation of several targets, followed by a reduction at day 10. Furthermore, the most effective treatment in vivo was accompanied with increased phosphorylation of growth control -and proangiogenic kinase substrates, like PLCγ1, EGFR, PDGFRβ, VEGFR2, and MAP kinases.

Early inhibition of vascular proliferation and upregulation of specific signaling pathways, as described above, may therefore indicate important markers for response. Such markers may also be of value in patient stratification, for the prediction of treatment effect.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-299. doi:10.1158/1538-7445.AM2011-LB-299