bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4315: Enrichment of xenotransplantable clonal cells in CD38high/CD138+ cells of multiple myeloma patients
Authors: Kim Dongkyoon , Weissman Irving L.
Published: February 2011

Multiple myeloma is a plasma cell neoplasm characterized by frequent relapse following initial therapy, implying the presence of a therapy-resistant and tumor-initiating cell population. While tumor-initiating cells have been reported for human multiple myeloma, the data provide conflicting information regarding the surface immunophenotype of these cells. Here, we analyzed bone marrow myeloma and B-lineage cells of patients for fractionation, examined engraftment of myeloma cells in various mouse models, and finally investigated myeloma-initiating population by examining the repopulation of human myeloma cells in recipient mice. The clonality of repopulating cells was also determined by RT-PCR of immunoglobulin gene variable regions. First, the examination of immunophenotype of patients’ bone marrow cells allowed us to fractionate myeloma/plasma cell-enriching CD138+/CD38high plasmacytic cells and CD19+CD38low/− B lymphocytic cells in 82% (31/38) of human samples. Then, we transplanted patient bone marrow cells into RAG2−/−γc−/−, NOD/scid or NOD/scid/γc−/− mice with/without human fetal bone pieces and examined the engraftment of myeloma cells. While human bone-bearing mice engrafted myeloma/plasma cells of 10 out of 19 samples, none of 18 samples gave rise to the repopulation of clonal myeloma/plasma/B cells in human bone-free immunodeficient mice. Further analysis of fractionated cells demonstrated that CD38high/CD138+ cells led to production of clonal human antibodies (2/10 samples) and a repopulation of clonally-related CD19+CD38low or CD138+CD38+ B lineage cells (5/10 samples) in grafted human bone of recipient mice. However, CD19+CD38low/−CD138− cells did not engraft clonally-related cells in human bone-bearing mice (0/9 samples). The analysis of CD38high/CD138+ myeloma cells demonstrated that more than 70% of patients still have heterogeneous plasmacytic cells in the expressions of CD45, CD19 or CD27. Our results suggest that human bone marrow microenvironment may be required for the survival and the engraftment of primary myeloma cells into recipient mice and that CD38high/CD138+/− plasmacytic cells but not CD19+CD38low/− B cells enrich clonal myeloma cells engrafting into recipient mice which may be responsible for tumor-initiation of multiple myeloma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4315.
Abstract 4394: Development of parallel conjugation and assay methodologies to screen for antibodies with optimal properties for use as antibody-drug conjugates
Authors: Lyon Robert P. , Ryan Maureen C. , Sutherland May , Meyer Brad , Yu Changpu , Kostner Heather , Miyamoto Jamie , Benjamin Dennis R.
Published: February 2011

The activity of antibody-drug conjugates (ADCs) on cancer cells can be affected by a multitude of factors, such as binding affinity, rate of internalization, subcellular trafficking, and efficient drug release within the target cell population. Consequently, the properties of an ideal antibody for drug delivery are not necessarily the same as those for a therapeutic naked antibody. Furthermore, the use of indirect assays involving the use of secondary antibodies to screen for optimal ADCs can be misleading, since crosslinking on the cell surface can lead to altered downstream events, and the affinity of the secondary antibody constrains the dynamic range of the assay. When seeking candidate antibodies directed against a novel antigen for ADC therapy, it is therefore most desirable to screen a large panel in the form of ADCs and evaluate their cytotoxic activities, since these results provide a direct measurement of parameters that can affect cytotoxic activity. However, when dealing with microgram quantities of a large number of antibodies as is typical of an antibody discovery campaign, the yields from conventional conjugation methodologies are limiting. We developed a novel approach that addresses this issue and have successfully applied it to the discovery of optimal ADCs out of a large panel of candidate antibodies. The technology is currently being applied to the discovery of ADCs against antigens of interest for targeted drug delivery.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4394.
Abstract 4412: In vitro activity of the novel tubulin active agent BAL27862 in MDR1(+) and MDR1(−) human breast and ovarian cancer variants selected for resistance to taxanes
Authors: Duran George E. , Lane Heidi , Bachmann Felix , Sikic Branimir I.
Published: February 2011

BAL27862 is a synthetic small molecule that induces apoptosis due to the inhibition of tubulin polymerization via a potentially new mechanism of action. The activity of this novel compound was evaluated in human breast and ovarian cancer cell line variants selected for resistance by continuous exposure to taxanes. BAL27862 retained activity against MDR1 (ABCB1)/P-glycoprotein-mediated resistance variants, unlike vinca alkaloids and taxanes. Activity was also observed in the taxane-resistant ovarian 1A9/TxTP50 tumor cell line, which overexpresses TUBB3 and was approximately 35x resistant to taxanes. Moreover, BAL27862 retained activity in ovarian cancer cells with acquired mutations in the class I (M40) β-tubulin isotype (1A9/PTX10, Phe270->Val; 1A9/PTX22, Ala364->Thr) which confer taxane resistance. The MCF-7/TxTP50 breast tumor variant was 9x resistant to taxanes, and 5x resistant to epothilone B, yet retained sensitivity to BAL27862, the vinca alkaloids (vinblastine and vincristine), colchicine and anthracyclines. These cells have significantly reduced levels of the taxane-binding target, the class I β-tubulin isotype, and reduced levels of total α- and β-tubulin relative to wild-type cells, alterations which may be responsible for a 34% reduction in bound BODIPY-paclitaxel as detected by FACS analysis. Evidence of resistance to BAL27862 (3x) was only detected in the ovarian OVCAR-3/TxTP5 variant, which was also resistant to taxanes (8x), the vinca alkaloids (4x) and colchicine (2x) and ixabepilone (2x). Finally, silencing TUBB3 using a specific siRNA sensitized cells to taxanes but did not affect BAL27862 activity in both wild-type MCF-7 and OVCAR-3 cells relative to appropriate controls, suggesting that TUBB3 status may not influence BAL27862 response. Thus, BAL27862 is a new agent with a distinct in vitro activity profile against a broad panel of human breast and ovarian tumor cell lines resistant to standard tubulin-interacting agents. These data strongly support further development of BAL27862 as a novel anticancer agent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4412.
Abstract 446: Cross-talking of hypoxia stress with IGF-1R signaling in cancer stem cell formation (germline lineage)
Authors: Huang Yen-Hua , Chen Yi-Chun , Chiang Shu-Liang , Ling Thai-Yen , Chang Te-Sheng
Published: February 2011

Recent advances in cancer research suggest that there exist cancer stem cells (CSCs) in tumors. The resources of CSCs are still controversial; but the CSCs were originated from its stem cells have been hypothesized (like glioma CSCs and intestine CSCs). The niche stress such as hypoxia effect may provide external signals in stem cell transformation. However, mechanism in regulation of hypoxia-induced stem cell transformation still remains unclear. Our preliminary observations in human pluripotent testicular tumors (seminomas and embryonal carcinomas) showed a high expression level of hypoxia-inducible protein HIF-1α/HIF-2α as well as IGF protein in tissues. This observation strongly highlights the cross-talking of niche hypoxic stress and IGF-1 signaling in transformation of germline stem cells into pluripotent cancers. To address this point, we have successfully established a serum-free stem-niche cell co-culture system to generate pluripotent germline stem cells from neonatal mouse testis (AP+GSCs), and uncovered the role of IGF-1/IGF-1R signaling in germ cell pluripotency. Interestingly, while comparing with the normoxia (21 % O2), the AP+GSCs cells showed in a dramatically increase of alkaline phosphatase activity (AP), cell proliferation, HIF-1α, HIF-2α, Oct-4 protein expression, and pluripotent gene expression (such as Oct-4, Sox2, Nanog, Klf-4, and c-Myc) under hypoxia condition (5 % O2). Moreover, the IGF-1 and IGF-1R expression were also significantly increased under hypoxia. Further analysis by using PPP (a specific inhibitor of IGF-1R phosphorylation) and/or LY294002 (a specific PI3K inhibitor) treatment dramatically reduced the Oct-4 expression as well as HIF-1α/HIF-2α protein of AP+GSCs. These observations suggested the regulation of Oct-4/HIF-2α expression by IGF-1/IGF-1R signaling. In summary, our results demonstrated an up-stream regulation of stemness (Oct-4, Sox2, Nanog, and Klf-4) and tumor factor (c-Myc) by hypoxia which was cross-talking with IGF-1/IGF-1R signaling in stem cell transformation under hypoxia condition.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 446.
Abstract 4473: PF-04691502, a potent and selective mTOR/PI3K dual inhibitor, demonstrates in vitro and in vivo antitumor activity in non-small cell lung carcinoma cells
Authors: Mehta Pramod P. , Walls Marlena , Baxi Sangita M. , Li Chunze , Batugo Minerva , Bagrodia Shubha , Cheng Hengmiao , Marx Mattew A. , Yin Min-Jean
Published: February 2011

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway plays a critical role in the regulation of cell growth, proliferation and survival in cancer. In non-small cell lung carcinomas cells (NSCLCs), the PI3K/AKT/mTOR pathway is frequently activated through either the amplification/mutation of PIK3CA (which encodes the p110α subunit of PI3K), or the functional loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In addition, the PI3K/AKT/mTOR pathway plays a critical role downstream of epidermal growth factor receptor (EGFR) in a defined NSCLC population; in particular, those harboring the EGFR mutations. Dual inhibition of PI3K and mTOR therefore represents a high therapeutic value for NSCLC patients. PF-04691502 is a dual-specificity inhibitor of PI3K and mTOR which shows potent and selective activity in biochemical, cell, and animal studies. In this study, we show PF-04691502 inhibits phosphorylation of AKT (at both S473 and T308) and S6 ribosomal protein (S6RP) in a variety of NSCLC lines harboring a PIK3CA mutation or EGFR mutation, subsequently resulting in anti-proliferative activity in the NSCLC lines. Furthermore, we demonstrate that PF-04691502 is capable of inducing tumor growth inhibition in four NSCLC xenograft models including an Erlotinib resistant line, NCI-H1975. In these murine xenograft models, decreased phosphorylation of AKT, p70S6K, and S6RP was observed in treated tumors in a manner consistent with the drug levels achieved. Therefore, PF-04691502 exhibits potent inhibition in the PI3K/mTOR axis and leads to both in vitro and in vivo anti-tumor activity in NSCLCs. Moreover, our data suggests that PF-04691502 may have clinical benefit to NSCLC patients including those found to be resistant to Erlotinib treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4473.
Abstract 4478: BAY 80-6946, a highly potent and efficacious class I PI3K inhibitor, induces complete tumor regression or tumor stasis in rat xenograft tumor models with PIK3CA mutant or PTEN deletion
Authors: Liu Ningshu , Haegebarth Andrea , Bull Cathy , Schatz Christoph , Wiehr Stefan , Hauff Peter , Pichler Bernd J. , Mumberg Dominik , Jenkins Susan , Schwarz Thomas , Ziegelbauer Karl
Published: February 2011

We report on BAY 80-6946, a highly selective and potent pan class I PI3K inhibitor currently in Phase I clinical trials. The PI3K pathway is aberrantly activated in many tumors either by growth factor receptor tyrosine kinases, or by the genetic mutation and amplification of key pathway components. BAY 80-6946 was evaluated in a panel of human xenograft tumor models. This compound was administered every second (Q2D) day or once weekly using intravenous (i.v.) bolus dosing. BAY 80-6946 was well tolerated with a MTD of 10 mg/kg (Q2D) in athymic rats. In the rat KPL-4 breast tumor model (PIK3CAH1047R and HER2O/E (protein levels)), BAY 80-6946 showed robust and dose-dependent tumor growth inhibition with complete tumor regressions (10/10 CRs) observed at the 3.0 and 6.0 mg/kg doses. The animals remained tumor-free until termination of the study on day 65. BAY 80-6946 also demonstrated complete tumor stasis in the human H460 large cell lung carcinoma and HCT116 colorectal carcinoma models (bearing PIK3CA (E545K and H1047R respectively) along with mutations in KRAS), In U87MG human glioma tumor model (PTEN-deletion) dosed Q2D schedule partial regressions (PRs) were observed. The robust in vivo anti-tumor activities for this class I pan PI3K inhibitor correlated very well with the potent and sustained pharmacodynamic inhibition of p-Akt in tumors, which allowed several i.v. dosing schedules including a once weekly schedule. Thus, BAY 80-6946 given at 6mg/kg, BID once weekly demonstrated similar efficacy as given every other day (Q2D) at 6 mg/kg. Further investigation of pharmacodynamic biomakers revealed that i.v. bolus injection of BAY 80-6946 1 and 6 mg/kg produced a significant dose-dependent reduction in [18F]FDG uptake measured by decrease in SUV in rat H460 tumors supporting the clinical exploration of [18F]FDG imaging to monitor pharmacodynamic drug activity in patients.

In conclusion, BAY 80-6946 is a highly efficacious and well tolerated PI3K inhibitor in preclinical rat tumor models expressing HER2, PIK3CA mutant and PTEN-deletion. BAY 80-6946 is currently in phase I clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4478.
Abstract 4558: 6-Nitroquinoline as a metabolically-activated probe for direct fluorescence detection of hypoxia
Authors: Rajapakse Anuruddha , Sarkar Ujjal , Linder Collette , Daniels J Scott , Gates Kent S.
Published: February 2011

Hypoxic (oxygen poor) cells in tumors play an important role in cancer biology. For example, the hypoxic environment may select for cells that are incapable of undergoing apoptosis. In addition, there is recent speculation that cancer stem cells that may be responsible for metastases are harbored in the hypoxic niche of tumors. Indeed, tumor hypoxia correlates with poor patient prognosis. Accordingly, detection of tumor hypoxia may soon become an important part of the clinical characterization of human cancers. Fluorescent probes for the direct detection of hypoxia in biological systems could provide a useful complement to the radiochemical imaging and immunohistochemical staining methods that are commonly used in this field. Building on our longstanding interest in metabolically-activated, hypoxia-selective antitumor agents, we set out to develop a probe that would be metabolized under hypoxic conditions into a product with known utility as a fluorescent reporter. We selected the molecule 6-nitroquinoline as a forum to explore the fundament chemistry, biochemistry and biology underlying the development of hypoxia-selective, bioreductively-activated fluorophores. We expected that hypoxia-selective reduction of the nitro group in this probe would generate the fluorophore 6-aminoquinoline. First, we confirmed that 6-nitroquinoline is not fluorescent. We next examined whether enzymatic metabolism converted this probe to a fluorescent product. The xanthine/xanthine oxidase enzyme system was employed for the one-electron reductive activation of the probe. We find that incubation of the probe with xanthine oxidase in the presence of a stoichiometric excess (5 equiv) of the substrate xanthine generates a substantial (approximately 30 fold) increase in fluorescence at 530 nm, as expected for generation of the 6-aminoquinoline product. Mass spectrometric analysis confirms that 6-aminoquinoline is generated under these conditions. In contrast, only a small increase in fluorescence at 530 nm is observed under aerobic conditions. Experiments that varied the amount of xanthine substrate used to “fuel” the reduction reaction revealed the presence of intermediate fluorescent products that generate an emission maximum at about 430 nm. This is likely due to 6-hydroxylamino and dimerized azoxy metabolites. The enzyme NADPH: cytochrome P450 reductase similarly converts 6-nitroquinoline to fluorescent products in a hypoxia-selective manner. In conclusion, we have established that 6-nitroquinoline can serve as a direct-light-up fluorescent probe for the detection of hypoxia and we have characterized the mechanisms by which this agent is converted to fluorescent products.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4558.
Abstract 4599: The RING E3 ligase BCA2 is regulated by estrogen and co-expressed with estrogen receptor in breast cancer cells
Authors: Kona Fathima R. , Bacopulos Stephanie , Amemiya Yutaka , Seth Arun K. , Burger Angelika M.
Published: February 2011

The Breast Cancer Associated gene 2 (BCA2) protein contains a RING H2 finger and a zinc finger near the N-terminus. The presence of the RING domain allows BCA2 to function as an E3 ligase. RING finger proteins play critical roles in mediating the transfer of ubiquitin and ubiquitin-like modifiers to substrates; such modifications are pivotal in regulating protein homeostasis and basic cellular processes such as gene-transcriptional regulation. The RING domain in BCA2 is known to act as an E3 ligase in its auto-regulation, and is suspected to play a role in the modification of other proteins. In a cohort of 945 primary invasive breast cancers examined for estrogen receptor-alpha (ER) and BCA2 expression, 67% were found to be ER-positive, 74% of which also exhibited strong nuclear BCA2 protein expression (p = 0.004). In this study, we examined potential mechanism(s) underlying BCA2 and ER co-expression at transcriptional and protein levels. Estrogen-responsive and non-responsive breast cancer cell lines were used as model systems. To investigate whether BCA2 is an estrogen responsive gene, BCA2 and ER-positive T47D breast cancer cells were cultured in medium containing charcoal-stripped fetal calf serum supplemented with 10 nM and 100 nM 17β-estradiol respectively over a 72-hour time course. Northern and Western blots showed a 17β-estradiol-associated induction of BCA2 mRNA and protein within 72 hrs. Moreover, when ER is ectopically or stably expressed in ER and BCA2-negative MDA-MB-231 cells, prominent BCA2 expression is detected, suggesting that ER is responsible for induction of BCA2 transcription. To investigate a possible interaction of BCA2 and ER at a transcriptional level, bioinformatic analysis of the BCA2 promoter region revealed ER and PR binding sites as well as possible sites of general transcription factors.

Together our data indicates that BCA2 is upregulated in response to estrogen and that BCA2 expression can be rescued by the introduction of ER, suggesting that ER and BCA2 may be participating in crosstalk at the transcriptional level. Further investigations into this phenomenon are necessary to determine the possibility of the development of drugs targeting BCA2 in order to manipulate ER.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4599.
Abstract 4626: The mechanism and structure localization relationship studies of a small organic molecule as a fluorescent tumor marker
Authors: Kang Chi-Chih , Chang Ta-Chau
Published: February 2011

BMVC, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide, is a novel organic molecule with special tumor recognition characteristic. BMVC appears bright fluorescence in the nucleus of cancer cells while only weak fluorescence in the cytoplasm of normal cells. The strong fluorescence of BMVC in the nucleus is mainly due to significant enhancement of BMVC fluorescence upon binding to DNA. Therefore, these distinct properties of BMVC in cells allow us to differentiate cancer cells from normal cells. It appears that BMVC is a potential fluorescent tumor marker.

It is of interest to elucidate the reason why BMVC can act as a potential fluorescence tumor marker. Confocal microscopic images by merging with organelle trackers suggested that BMVC mainly localizes in the nucleus and mitochondria of cancer cells, but in the lysosome of normal cells. Several leading experiments suggested that the major pathway for BMVC uptake is endocytosis. In addition, the carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and microinjection experiments showed that the destination of endocytosed BMVC is trapped in the lysosome of normal cells. However, it is not clear why BMVC is not trapped in the lysosome of cancer cells. Since the lysosomal membrane permeabilization (LMP) could sensitize cancer cell to death, we performed the cathepsin immunofluorescence and vacuolization experiments to elucidate whether there is LMP difference between cancer and normal cells. Our results showed that the LMP indeed plays an important role for trapping BMVC in the lysosome of normal cells, but releasing BMVC to the nucleus and mitochondria of cancer cells.

Furthermore, the structure localization relationship (SLR) studies show that BMVC derivatives with large hydrogen bonding capacity (HBC) are less lysosomal membrane permeable in normal cells. In addition, we found the more lipophilic of BMVC derivatives the more mitochondria localization in cancer cells. It is likely that the HBC and the lipophilicity are two important characters in determining intracellular localization of BMVC derivatives in cells.

In summary, the LMP and SLR studies of BMVC and its derivatives demonstrated a new direction for further design and synthesis of better fluorescent tumor markers and also provided a new platform for cancer selective drug through the combination of a cancer targeting molecule and anticancer drug.

Reference:

[1] C. C. Kang, C. C. Chang, T. C. Chang, L. J. Liao, P. J. Lou, W. Xie, E. S. Yeung, Analyst 2007, 132, 745.

[2] L. J. Liao, C. C. Kang, I. S. Jan, H. C. Chen, C. L. Wang, P. J. Lou, T. C. Chang, Analyst 2009, 134, 708.

[3] N. Fehrenbacher, M. Jaattela, Cancer Res 2005, 65, 2993.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4626.
Abstract 4652: Identification and validation of a novel nine-gene signature predicting clinical outcome in malignant melanoma
Authors: Brunner Georg , Heinecke Achim , Reitz Martina , Lippold Andrea , Berking Carola , Suter Ludwig , Atzpodien Jens
Published: February 2011

Incidence of malignant melanoma is rapidly increasing - with a doubling rate of 10-20 years. Conventional histopathological and clinical staging (based on Breslow tumor thickness, lymph node status, and ulceration) is largely inadequate for predicting clinical outcome of malignant melanoma. On the other hand, molecular prognostic markers are not yet available. Here, we identified a nine-gene signature which is closely associated with overall survival of melanoma patients.

To identify prognostic genes we correlated gene expression profiles of 136 primary melanomas with patient overall survival using Cox regression analysis: Initially, a comparative analysis of 20 high-risk vs. 20 low-risk primary melanomas with a clinical follow-up of more than 20 years (training cohort) was performed using whole-genome DNA microarray analysis and yielded 92 prognostically relevant candidate genes. Technical and statistical validation using TaqMan Array real-time RT-PCR and correlation of gene expression with patient overall survival reduced candidate gene number to 11. Expression of these 11 genes was further analyzed in an extended group of 91 primary melanomas (study cohort), yielding a nine-gene signature with prognostic significance. This novel prognostic melanoma gene signature was successfully validated using an independent set of 45 primary melanomas (validation cohort).

A risk score, based on the expression of the nine genes of the signature (KRT9, SPINK7/ECG2, KBTBD10, DCD, HES6, COL6A6, PIP, SCGB1D2, SCGB2A2), or any subgroup thereof, predicted patient overall survival in the study cohort (p = 0.0004, hazard ratio 3.83), independently of conventional AJCC 2002 staging. When combining gene expression score and AJCC staging, approximately two thirds (29/45, 64%) of patients with AJCC intermediate prognosis (i.e. stages IIA, IIB, and IIIA) were reclassified into good prognosis, exhibiting a long-term overall survival probability of 95%. Misclassification rate of all patients classified into good prognosis (low-risk gene score combined with AJCC stages I IIA/B, or IIIA) was very low at 4.6% and 6.25% in the training and validation cohorts, respectively.

The prognostic value of this novel signature-based risk score is its ability to identify patients at low risk, not identified by AJCC staging. This re-classification may allow these patients to stay treatment-free while experiencing excellent long-term survival. The remaining patients are risk patients and are in need of adjuvant therapies.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4652.
Abstract 4703: Lack of expression of argininosuccinate synthetase in human cancer tissue: A biomarker for sensitivity to arginine depletion with pegylated arginine deiminase
Authors: He Wei , Chen Jianhe , Zheng Yuhui , Dai Zaoming , He Yan , Prestayko Archie , Thomson James , Wu Bor-Wen , Bomalaski John
Published: February 2011

There is increasing awareness of metabolic abnormalities in cancer that can be exploited with new forms of treatment. In addition, there is a need for biomarkers that would help select such tumors for targeted therapies. One such metabolic abnormality is the requirement of arginine for growth by certain cancers. These cancers lack expression of argininosuccinate synthetase (ASS), the rate limiting step in the metabolism of citrulline to arginine in the Krebs urea cycle, and must obtain arginine from the circulation. It has previously been shown that hepatocellular carcinoma and metastatic melanoma have a high frequency of lack of expression of ASS. As a result, these cancers are susceptible to arginine deprivation with pegylated arginine deiminase (ADI-PEG 20) both in pre-clinical models and in phase 2 clinical trials. Similar lack of ASS expression in human tissue samples and sensitivity to arginine depletion with ADI-PEG 20 in pre-clinical models have been found in a high percentage of pancreatic, prostate, and platinum-resistant ovarian cancers. In order to explore potential other cancers that may lack ASS expression we have examined breast, lung and gastric cancer microarray samples for ASS expression by immunohistochemistry. Our results indicated that 46/111 (41.4%) breast carcinoma, 28/90 (31.1%) lung carcinoma and 68/121 (56.2%) gastric cancer were ASS negative. To explore this further, a panel of 40 ATCC tumor cell lines representing 12 different human cancer types were screened for ADI-PEG 20 induced growth inhibition using the MTS assay. Significant inhibition of proliferation in a dose dependent manner after 48 hours of ADI-PEG 20 treatment was found in 19 out of 40 tumor cell lines screened, including breast, lung, gastric, sarcoma, ovarian, prostate and pancreatic carcinomas and chronic myelogenous leukemia. Lack of ASS expression in those responding cell lines were confirmed by Western blot. These findings suggest that ASS may serve as a biomarker for targeted therapy with ADI-PEG 20. Since a high percentage of patients with breast, lung or gastric cancer lack expression of ASS, these tumors may be sensitive to arginine depletion therapy. Clinical trials of ADI-PEG 20 in these cancers are being planned.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4703.
Abstract 4738: Utilizing genotype imputation for the augmentation of sequence data
Authors: Fridley Brooke L. , Jenkins Greg , Deyo-Svendsen Matthew , Hebbring Scott , Freimuth Robert
Published: February 2011

The advancement in genotyping technology and large SNP arrays has led to an increase in the number of genome-wide association studies (GWAS) for various complex traits. These GWAS have resulted in the implication of over 1500 SNPs for association with disease traits. However, the SNPs identified from these GWAS are not necessarily the functional variants. Therefore, the next phase in GWAS will involve the validation, augmentation and refining of these putative regions or loci.

One possible “next phase” approach would be to catalog all variants found in the study subjects within the loci of interest using sequencing technology, followed by the association analysis. However, sequencing a locus in a large number of subjects is still relatively expensive. By sequencing only a portion of the samples for the follow-up of signals from GWAS, followed by imputation in the remaining samples, one can significantly reduced the cost to localize the punitive variant involved in the etiology of complex phenotypes. A potentially attractive alternative option would be to impute based only on the 1000 Genomes Project; however, this has the drawbacks of using a reference population that does not necessarily match with disease status and LD pattern.

Therefore, we conducted a study to investigate the ability to carry-out genotype imputation using a reference panel consisting of sequencing data for a fraction of the study participants. The various approaches were implemented using data from the candidate gene sequencing study conducted at the Mayo Clinic and the 1000 Genomes Project. Our results show that imputation of untyped markers based on sequencing a portion of the study participants is a reasonable, cost-saving, approach for disease mapping and the refinement of putative regions detected with GWAS. We recommend a few guidelines in designing sequencing studies to take advantage of the recent advances in genotype imputation: (1) Select the largest and most diverse reference panel for sequencing, with respect to both haplotypes and phenotypes. One can also use the 1000 Genomes Project in addition to the sequencing of a portion of the study participants; (2) Genotype as many “anchor” markers as possible, in that, the number of markers genotyped on all subjects impacts accuracy. Therefore, additional genotyping of a few common SNP markers not already genotyped on all subjects using a cost effective platform may be needed if the GWAS SNP array does not provide adequate coverage in the locus to be sequenced.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4738.
Abstract 4749: Genetic signature of malignant glioma is associated with responsiveness to autologous tumor lysate-pulsed dendritic cell vaccination + TLR agonists
Authors: Prins Robert M. , Soto Horacio , Konkankit Vera , Yong William H. , Nelson Stanley F. , Liau Linda M.
Published: February 2011

Purpose: We previously reported that the toll-like receptor (TLR)-7 agonist, imiquimod, could enhance dendritic cell activation and migration, as well as stimulate T cell-mediated anti-tumor immune responses in a murine glioma model. To translate these findings, a Phase I clinical trial was initiated to evaluate this strategy for its feasibility, safety, and induction of immune responses in patients with malignant glioma. Herein, we report the results of this phase I, dose escalation clinical trial using autologous tumor lysate-pulsed dendritic cell (DC) vaccination, supplemented with the toll-like receptor agonists, imiquimod or poly ICLC, for patients with malignant glioma.

Patients and Methods: Twenty-two glioblastoma patients (15 newly diagnosed, 7 recurrent) were enrolled into a multi-cohort dose-escalation study and treated with 1, 5, or 10 million autologous DC pulsed with tumor lysate. Three biweekly intradermal vaccinations were initially administered. Subsequently, patients received booster vaccinations together with imiquimod or poly ICLC every three months until tumor progression or lysate was depleted. Patients were monitored for adverse events, survival, and immune responses. For patients with available pre-treatment tumor and/or PBMC, microarray-based hierarchical clustering, lymphocyte immunohistochemistry, FACS and cytokine bead arrays were performed for monitoring purposes.

Results: DC vaccinations were not associated with any evidence of dose-limiting toxicity or serious adverse effects. The median survival was 35 months for newly diagnosed patients and 11 months for recurrent patients, but still increasing as several patients are still alive at this point. Typical glioblastoma gene expression signatures, previously characterized by our group, were found. Contrary to our previous reports, however, patients with proliferative (Pro) or mesenchymal (Mes) signatures exhibited prolonged survival, while patients with pro-neural (PN) signatures did not. Pre-treatment Pro, Mes or Pro/Mes tumor sections had increased CD3 and CD8 positive cells within the tumor compared with PN tumor sections.

Conclusions: Pro, Mes or Pro/Mes gene expression signatures may identify patients with pre-existing immunity and who may benefit from immune-based vaccination studies. However, prospective studies will need to be performed to establish the validity of these findings.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4749.
Abstract 4769: Relevance of the TRIF pathway for in vivo gp96-mediated CD8+ T cell cross presentation
Authors: Luna Ernesto , Fukata Masayuki , Strbo Natasa , Abreu Maria T. , Podack Eckhard R.
Published: February 2011

Heat shock protein gp96 is an intracellular chaperone for proteins and peptides generated during protein synthesis and degradation. Different candidate receptors including CD91, TLR2 and TLR4 have been shown as important binders in the gp96 mediated cross-priming process. Both TLR2 and TLR4 use the common MyD88 signaling pathway suggesting it is an important mediator of gp96-mediated cross-priming. Our group has produced a secreted form of hsp-gp96, a fusion protein gp96-Ig. Ggp96-Ig-transfected tumor cells secrete gp96-Ig in vivo and cross prime CD8 CTL that are specific for genuine tumors antigens (Ag) and for the surrogate Ag OVA. In this work, we studied the role of both MyD88 and TRIF signaling pathways in gp96-mediated cross presentation. Briefly, one million of ovalbumin specific T cell receptor (TCR) transgenic CD8+ cells (OT-I)-gfp, were transferred intravenously into MyD88 deficient (MyD88−/−) mice, TRIF deficient (TRIF Lps2/Lps2) mice and C57/BL6 wild type (B6-WT) mice as control. Two days later the mice were immunized intraperitoneally with two million of 3T3-gp96-mIgG1-OVA cells or 3T3-OVA cells respectively. Five days later, we obtained peritoneal cells (PECs) and splenocytes in order to analyze OT-I-gfp expansion, IFNγ production, CD107a degranulation and cytokine (IL-6, TNFα, IL-10) production by ELISA. Gp96 induced CD8 CTL (OT-I) expansion is significantly increased in MyD88−/− mice compared to WT mice and significantly decreased in TRIF deficient mice. However, the frequency of IFNγ expression in CTL is diminished in MyD88−/− while is normal in TRIF deficient mice. IL-6 and TNFα production by antigen presenting cells isolated from the peritoneal cavity, was reduced in MyD88−/− mice compared with WT and TRIF deficient mice. The data indicate that both, MyD88 and TRIF mediated signals are required for full CD8 CTL expansion and cytotoxic differentiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4769.
Abstract 4809: Targeting integrin function in melanoma cells using vinculin activators results in enhances chemosensitivity
Authors: Nelson Elke S. , Folkmann Andrew , DeMali Kris
Published: February 2011

Current therapies aimed at manipulating cell adhesion work by modulating interactions between integrins (the major adhesion receptors on the cell surface) and components of the extracellular matrix. However, a complete gain or loss of integrin function can lead to mechanism-based toxicities. An attractive alternative approach is to manipulate integrin-associated proteins. Vinculin, an actin binding protein that is recruited to the cytoplasmic tail of integrin β1, is a good candidate protein for such an approach, as it regulates the strength and lifetime of such complexes. Here we have investigated the effect that a vinculin activating peptide (VAP) has on integrin-mediated adhesion. In HeLa cells and mouse embryonic fibroblasts, VAP increased integrin-mediated signaling, as well as adhesion to fibronectin and collagen, in an integrin- and vinculin-dependent manner. These regulatory effects of VAP were dependent on the peptide's ability to bind to an amphipathic α-helix in the vinculin head; substitution of an isoleucine for alanine at position 50 within this region of vinculin abolished VAP's ability to enhance adhesion. In melanoma cells, VAP likewise increased integrin-mediated cell adhesion, and increased the cells’ sensitivity to cancer chemotherapeutics, suggesting that it might serve as an effective therapeutic agent. Overall, our findings demonstrate that integrin function can be manipulated by targeting the regulation of proteins that associate with the cytoplasmic domains of the integrins, and suggest that small molecules capable of such regulation may be employed as effective adjuvants to chemotherapy for melanoma. These findings lay the foundation for the development of a new class of therapeutics that act by regulating integrin-associated proteins.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4809.
Abstract 4826: International Lung Cancer Consortium: Pooled analysis of previous lung diseases and lung cancer risk
Authors: Brenner Darren R. , Boffetta Paolo , Duell Eric J. , Bickeböller Heike , Rosenberger Albert , Muscat Joshua E. , Yang Ping , Wichmann Erich , Schwartz Ann G. , Tjønneland Anne , Friis Søren , LeMarchand Loic , Zhang Zuo-Feng , Lazarus Philip , Field John K. , McLaughlin John R. , Wiencke John , Neri Monica , Lan Qing , Orlow Irene , Park Bernard J. , Hung Rayjean J.
Published: February 2011

To clarify the role of previous lung diseases (PLDs) (chronic bronchitis, emphysema, pneumonia, tuberculosis, asbestosis and silicosis) in the development of lung cancer we conducted a pooled analysis in the International Lung Cancer Consortium (ILCCO). Data from 16 studies (9 population based case-control, 4 hospital based case-control, 2 mixed case-control and 1 cohort - 10 from North America, 5 from Europe and 1 from Asia) were pooled, including 24044 cases and 81256 controls. Study-specific effect estimates were derived using logistic regression models for case-control studies and Cox proportional hazards models for cohort studies adjusted for age, sex, education and pack-years of smoking. Effect estimates were then pooled using random effects models where summary effects for each of the PLDs were evaluated separately. Stratified analyses were conducted based on smoking status, histology and study design or control source. Heterogeneity was evaluated using the Cochrane Q statistic and Galbraith plots, and publication bias was evaluated using funnel plots as well as the Begg test. A previous history of emphysema conferred a relative risk (RR) of 2.17, 95% confidence interval (CI): 1.67-2.82 (from 15 studies, p-het<.001). A previous history of chronic bronchitis conferred a RR of 1.49, 95% CI: 1.33-1.60 (from 12 studies, p-het.=0.434). Tuberculosis was associated with a RR of 1.54, 95% CI: 1.19-1.99 (from 15 studies, p-het.=0.001). Both asbestosis (RR 2.51, 95% CI: 1.53-4.12 (from 7 studies, p-het=0.379)) and silicosis (RR 1.70, 95% CI: 1.15-2.54, (from 3 studies, p-het. p=0.476)) were also associated with lung cancer risk. When the outlying studies were removed, effect estimates changed minimally and heterogeneity was reduced. Findings were consistent in subgroup analyses by smoking status and histology (adenocarcinoma, squamous cell carcinoma and small cell lung cancer). Effect estimates among never smokers were for emphysema a RR of 2.21, 95% CI: 0.99-4.90 (from 8 studies), for chronic bronchitis 1.12, 95% CI: 0.78-1.62 (from 9 studies), for pneumonia 1.37, 95% CI: 1.10-1.70 (from 10 studies) and for tuberculosis 1.54, 95% CI: 0.99-2.39 (from 9 studies). In this analysis of data from multiple continents, PLDs were associated with an increased risk of lung cancer. The evidence among never smokers supports a direct relationship between previous lung diseases and lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4826.
Abstract 5038: AZD8055 is an effective inhibitor of mTOR kinase signaling and breast cancer growth while relieving feedback inhibition of HER-kinase signaling
Authors: Rodrik-Outmezguine Vanessa S. , Cao Nen , Chandarlapaty Sarat , She Qing-Bai , Ye Qing , Smith Paul , Guichard Sylvie , Rosen Neal
Published: February 2011

The PI3K signaling pathway is often dysregulated in human cancer. mTOR kinase is a component of this pathway that mediates activation of cap-dependent translation, other AGC-kinases and feedback inhibition of upstream components of the pathway. The natural product rapamycin binds to and inhibits the mTOR-containing TORC1, but not the TORC2 complex. Rapamycin-like TORC1 inhibitors have modest clinical activity in renal cell and other cancers, but only marginal activity in glioblastoma, prostate and breast cancers in which PI3K/AKT signaling is often activated. The limited antitumor activity of rapamycin may be due to inefficient inhibition of TORC1, induction of TORC1-dependent feedback, or lack of inhibition of TORC2. Potent mTOR kinase inhibitors could obviate these issues by inhibiting both TORC1 and TORC2 and by preventing induction of phosphorylation of AKT at Serine 473.

We have now compared the effects of the specific mTOR kinase inhibitor AZD8055 and rapamycin in breast cancer models with amplified HER2 or mutant p110alpha PI3K. As expected, AZD8055 and rapamycin both inhibit S6K and S6 phosphorylation, but only the former inhibits AKT phosphorylation at Serine 473, which is, instead, induced by rapamycin. Moreover, AZD8055 is a more potent inhibitor of 4EBP-1 phosphorylation, CAP-dependent translation, and D-cyclin expression. Thus, the mTOR kinase inhibitor inhibits mTOR functions more effectively than rapamycin. Whereas both drugs cause tumor cells to undergo autophagy and G1 arrest, only AZD8055 induces significant apoptotic cell death.

The more effective inhibition of 4EBP1 phosphorylation, cap-dependent translation and enhancement of apoptosis compared to that caused by rapamycin may be due to more complete inhibition of TORC1 function or to abolition of feedback activation of AKT by AZD8055. As previously established with rapamycin, we now show that AZD8055 causes relief of feedback inhibition of HER kinase signaling in these tumors, resulting in marked induction of both ERK and PI3K signaling. These results suggest that mTOR kinase inhibition with drugs such as AZD8055 can more effectively inhibit TOR activity than rapamycin. They therefore have the potential for increased antitumor activity, but relief of TOR kinase-dependent negative feedback could create a rationale for combination therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5038.