Reduction In Ki67 Research Articles

Exercise Neuroprotection in a Rat Model of Binge Alcohol Consumption

Excessive alcohol intake produces structural and functional deficits in corticolimbic pathways that are thought to underlie cognitive deficits in the alcohol use disorders (AUDs). Animal models of binge alcohol administration support the direct link of high levels of alcohol consumption and neurotoxicity in the hippocampus and surrounding cortex. In contrast, voluntary wheel running enhances hippocampal neurogenesis and generally promotes the health of neurons. We investigated whether voluntary exercise prior to binge alcohol exposure could protect against alcohol-induced cell loss. Female Long-Evans rats exercised voluntarily for 14 days before undergoing 4 days of binge alcohol consumption. Brains were harvested immediately after the last dose of alcohol and examined for various histological markers of neurodegeneration, including both cell death (FluoroJade B) and cell birth (Ki67) markers. Rats that exercised prior to binge exposure were significantly less behaviorally intoxicated, which was not a result of enhanced hepatic metabolism. Rats that exercised prior to binge alcohol consumption had reduced loss of dentate gyrus granule cells and fewer FluoroJade B positive cells in the dentate gyrus and associated entorhinal-perirhinal cortex compared to nonexercisers. However, exercise did not protect against cell death in the piriform cortex nor protect against alcohol-induced decreases in cell proliferation, evidenced by a similar alcohol-induced reduction in Ki67 labeled cells between exercise and sedentary rats. We conclude that exercise can reduce behavioral sensitivity to ethanol intoxication and protect vulnerable brain areas from alcohol-induced cell death. Exercise neuroprotection of alcohol-induced brain damage has important implications in understanding the neurobiology of the AUDs as well as in developing novel treatment strategies.

Increased estrogen sulfatase (STS) and 17β-hydroxysteroid dehydrogenase type 1(17β-HSD1) following neoadjuvant aromatase inhibitor therapy in breast cancer patients

Aromatase inhibitors (AIs) are considered the gold standard for endocrine therapy of estrogen receptor (ER) positive postmenopausal breast cancer patients. The therapy may enhance therapeutic response and stabilize disease but resistance and disease progression inevitably occur in the patients. These are considered at least partly due to an emergence of alternative intratumoral estrogen production pathways. Therefore, in this study we evaluated effects of exemestane (EXE) upon the enzymes involved in intratumoral estrogen production including estrogen sulfatase (STS), 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), and estrogen sulfotransferase (EST) and correlated the findings with therapeutic responses including Ki67 labeling index (Ki67). 116 postmenopausal patients with invasive ductal carcinoma, stage II/IIIa, were enrolled in JFMC34-0601 clinical trials between March, 2006 and January, 2008. EXE of 25 mg/day was administered according to the protocol. Pre- and posttreatment specimens of 49 cases were available for this study. Status of STS, EST, 17beta-HSD1, ER, progesterone receptor (PgR), human epidermal growth factor receptor type 2 (Her2), and Ki67 in pre- and post-specimens were evaluated. Specimens examined before the therapy demonstrated following features; ER+ (100%), PgR+ (85.7%), and Her2+ (77.6%). After treatment, the number of Ki67, PgR, and ER positive carcinoma cells demonstrated significant decrement in clinical response (CliR) and pathological response (PaR) groups. Significant increment of 17beta-HSD1 and STS immunoreactivity was detected in all groups examined except for STS in PaR. EST showed significant increment in nonresponsive groups. Alterations of Ki67 of carcinoma cells before and after therapy were subclassified into three groups according to its degrees. Significant alterations of intratumoral enzymes, especially 17beta-HSD1 and STS, were correlated with Ki67 reduction after neoadjuvant EXE therapy. This is the first study demonstrating significant increment of STS and 17beta-HSD1 following AI neoadjuvant therapy of postmenopausal ER positive breast carcinoma patients. This increment may represent the compensatory response of breast carcinoma tissues to estrogen depletion.

Abstract A49: Evidence for gene demethylation in human colon cancer by black raspberries

Abstract Black raspberries (BRBs) exhibit cancer preventative effects in the human colon, in part, by reducing the growth rate and increasing the apoptosis of human colon tumor cells. Recently, we have initiated investigations to determine whether BRBs also cause re-expression of genes in human colon cells that are silenced by aberrant hypermethylation. Cultured human colon cancer cell lines HCT116, Caco2 and SW480, were treated with an anthocyanin-rich (AC) fraction of BRBs every day for five days. AC treatment caused a decreased activity of DNA methyltransferases in a substrate-antibody assay (p<0.05) and a reduction of DNMT1 protein expression. Direct analysis of methylation changes in the promoter region of the suppressor gene p16 (CDKN2A) by Sequenom MassARRAY/EpiTYPER demonstrated a decrease in methylation of all eight p16 promoter CpG units, each unit containing 1–3 CpG sites. All cell lines treated with AC had a significant increase in apoptosis and a decrease in cell proliferation as compared to untreated cells (p< 0.05). These in vitro observations were confirmed by the examination of normal and tumorous tissue samples taken from 20 colon cancer patients who received daily dietary intervention of freeze-dried BRB powder (60g/day) in drinking water for an average of 3 weeks. DNA from the tissue samples was analyzed by MassARRAY which revealed an overall decrease in p16 methylation in both normal and tumor tissues (p<0.05). Immunohistochemical analysis of p16 and DNMT1 in the same tissue samples showed that BRB treatment increased p16 expression and decreased DNMT expression (p<0.05), both correlating with decreased methylation of the promoter region of the gene. In addition, the expression levels of Ki-67 and c-myc, both markers of cell proliferation, were reduced in berry treated samples, however, only the reduction in Ki-67 was significant (p<0.05). These data suggest that BRBs are capable of reversing aberrant methylation in both normal and tumorous human colon tissues at tolerable amounts in the diet. This effect appears to be caused by inhibition of DNMT family enzymes, and most likely DNMT1. Supported by NCI grant CA103180 and USDA grant 38903-03560. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A49.

Neural stem/progenitor cells derived from adult hippocampal dentate gyrus can be abundantly prepared from mice treated with trimethyltin

Glucocorticoids, hormones that are released in response to stress, induce neuronal cell damage. The hippocampus is a primary target of glucocorticoids in the brain, the effects of which include the suppression of cell proliferation and diminished neurogenesis in the dentate gyrus. Our previous study found that melatonin, synthesized primarily in the pineal, pretreatment prevented the negative effects of dexamethasone, the glucocorticoid receptor agonist, on behavior and neurogenesis in rat hippocampus. In the present study, we attempted to investigate the interrelationship between melatonin and dexamethasone on the underlying mechanism of neural stem cell proliferation. Addition of dexamethasone to hippocampal progenitor cells from eight-week old rats resulted in a decrease in the number of neurospheres; pretreatment with melatonin precluded these effects. The immunocytochemical analyses indicated a reduction of Ki67 and nestin-positive cells in the dexamethasone-treated group, which was minimized by melatonin pretreatment. A reduction of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and G1-S phase cell cycle regulators cyclin E and CDK2 in dexamethasone-treated progenitor cells were prevented by pretreatment of melatonin. Moreover, luzindole, a melatonin receptor antagonist blocked the positive effect of melatonin whereas RU48, the glucocorticoid receptor antagonist blocked the negative effect of dexamethasone on the number of neurospheres. Moreover, we also found that dexamethasone increased the glucocorticoid receptor protein but decreased the level of MT1 melatonin receptor, whereas melatonin increased the level of MT1 melatonin receptor but decreased the glucocorticoid receptor protein. These suggest the crosstalk and cross regulation between the melatonin receptor and the glucocorticoid receptor on hippocampal progenitor cell proliferation.

Randomised Placebo Controlled Trial Studying Short Term Biological Effects of the Combination of Letrozole and Zoledronic Acid on Invasive Breast Cancer.

Abstract To determine whether the addition of Zoledronic Acid to endocrine therapy increases apoptosis or decreases proliferation in early invasive breast cancer, a placebo controlled randomised trial comparing 14 days treatment with Letrozole or Letrozole and Zoledronic Acid pre-operatively was performed.PatientsIn total 109 postmenopausal women with early invasive hormone receptor positive breast cancer were randomised (1:1:1) to either placebo, Letrozole 2.5mg/day or Letrozole with Zoledronic Acid 4mg single dose intravenously 2-4 days before definitive surgical excision. Epithelial proliferation and apoptosis were measured on paired baseline and surgical biopsy specimens (after 14 days of treatment) using Ki67 and Activated Caspase 3 immunohistochemistry. Alterations in angiogenic markers (VCAM/VEGF and CD31) were also studied. The primary endpoint was fall in Ki67 between diagnosis and surgical excision.ResultsOverall 109 women were enrolled but paired biopsies were only available for 101 patients. PlaceboLetrozoleLet + Zoln323435Absolute Ki67 change (median,range)-0.8 (-12,12)8.6 (-14,37)12.9 (-12,29) Caspase 3 change (median,range)0.1 (-3.8, 9.3)0.4 (-2.7, -4.1)0.2 (-10.9, -14.4) Absolute change (Cell turnover index)-0.3 (-142, -59)18.9 (-201, 192)17.7 (-14, 379) Statistically significant reductions in Ki67 and Cell Turnover Index were seen with Letrozole and Let & Zol (p ≤ 0.001) but there was no significant different between Letrozole and Letrozole plus Zoledronic Acid groups (p = 0.26). Apoptosis did not change between the three groups.ConclusionLetrozole reduces proliferation by 70% when used for 14 days prior to surgery. Zoledronic Acid administration prior to surgery is safe but when administered as a single dose at a median of 3 days before surgery did not significantly increase apoptosis or decrease proliferation compared to Letrozole alone. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2009.

Abstract C197: A phase 1 safety and pharmacokinetic (PK) study of the PI3K inhibitor XL147 (SAR245408) in combination with erlotinib in patients with advanced solid tumors

Abstract Background: The PI3K pathway is frequently dysregulated in cancer cells and has been implicated as a mediator of resistance to EGFR inhibitors. In preclinical xenograft models, the oral selective Class I PI3K inhibitor XL147 augments the anti-tumor efficacy of EGFR inhibitors, including erlotinib. Methods: Patients (pts) with advanced solid tumors are enrolled in successive cohorts of 3 to receive escalating doses of XL147 in combination with erlotinib. Erlotinib is dosed qd, and XL147 is dosed once daily on Days 1–21 of each 28-day cycle. Dose limiting toxicities (DLTs) are determined using Cycle 1 safety data. Tumor response is evaluated every 8 weeks. Results: As of 01 Aug2009, 18 pts with advanced solid tumors have been enrolled: NSCLC (7), CRC (5), HCC, HNSCC, biliary tract, carcinoid, ethmoid, and esophageal cancer (1 each). Pts have been treated at 6 dose levels up to 400 mg XL147/150 mg erlotinib. No DLTs or study treatment-related SAEs have been reported. The MTD has not yet been established. The most frequently reported related AEs were rash (40%), fatigue (30%), diarrhea, nausea, and vomiting (each 20%). Two events of Grade 3 thrombosis (both unrelated) were reported. Based on preliminary data, the PK of XL147 in combination with erlotinib is consistent with that observed for XL147 given alone (Exelixis Study XL147-001). Likewise, XL147 does not appear to alter the PK of erlotinib as the PK parameters of erlotinib are similar to published single agent values. Modulation of PI3K pathway and EGFR signaling by XL147/erlotinib has been demonstrated in PBMCs by reductions in phosphorylation of AKT, the AKT substrates PRAS40 and GSK3 , and ERK. In skin and tumor biopsies, robust post-dose reductions were evident in phosphorylation of AKT, the mTOR substrate 4EBP1, ERK, and EGFR. For example, in tumor biopsies from a subject with an ethmoid tumor (400 mg XL147/150 mg erlotinib), reductions in pAKT-T308 (43%), p4EBP1 (44%), pERK (43%), and pEGFR (40%) were evident at Day 15 post-dose, with a corresponding reduction in Ki67 (47%) and induction of apoptosis (2.7-fold). One EGFR inhibitor-naïve NSCLC pt (no EGFR mutations detected in archival tumor sample, 200 mg XL147/150 mg erlotinib) had a confirmed PR with a 59% reduction in the sum of target lesions. Nine pts continued on study ≥ 12 weeks, including 2 pts who remained on study ≥ 24 weeks. Conclusions: The combination of XL147 and erlotinib is generally well tolerated at doses up to 400 mg XL147/150 mg erlotinib, with no apparent drug-drug PK interaction or emergent toxicities, and results in robust simultaneous inhibition of PI3K and EGFR signaling. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C197.

Abstract B247: A phase 1 safety and pharmacokinetic (PK) study of the PI3K inhibitor XL147 (SAR245408) in combination with paclitaxel (P) and carboplatin (C) in patients with advanced solid tumors

Abstract Background: Upregulation of PI3K pathway signaling is associated with tumor resistance to P and C. The oral selective Class I PI3K inhibitor XL147 potentiates the antitumor efficacy of P and C without exacerbated toxicity in preclinical xenograft tumor models. Methods: Patients (pts) enrolled in successive cohorts of 3 receive intravenous P/C on Day 1 of each 21-day cycle, and oral XL147 qd. This study consists of two parts. In Part A, P and C will be dose-escalated up to 175 mg.m2 and AUC 6, respectively, in pts with solid tumors (MTD expansion in endometrial and ovarian cancer). In Part B, P and C will be dose-escalated up to 225 mg.m−2 and AUC 6, respectively, in NSCLC pts. Dose limiting toxicities (DLTs) are determined using Cycle 1 safety data. Tumor response is evaluated every 6 weeks. Results: As of 01Aug2009, 15 pts with advanced solid tumors have been enrolled: breast (5), cervical (2), vulvar, tonsillar, parotid, peritoneal, colorectal, esophageal, melanoma, and tongue (1 each). Pts have been treated at 5 dose levels up to 400mg XL147/175mg.m−2 P/AUC 6 C. The MTD has not yet been established. Four pts experienced one or more SAEs. Of these, one pt experienced Grade 4 thrombocytopenia which was assessed as related to study treatment (XL147 and P/C). The most frequently reported related AEs were neutropenia, anemia, fatigue (each 31%), and peripheral neuropathy (23%). Three pts have experienced a confirmed PR with the following maximal reductions (sum of tumor target lesions): 63% (tonsillar), 45% (esophageal), and 42% (cervical). In addition, 11 pts continued on study ≥ 12 weeks, with 3 pts remaining on study ≥ 24 weeks. All PRs occurred in pts who had been previously treated with a platinum-containing regimen. In addition, a platinum-naïve pt with triple-negative, inflammatory breast cancer (mutant p53 and LKB-1) experienced regression of cutaneous lesions after two cycles. When given in combination with P/C, XL147 PK was consistent with that of XL147 given alone (Exelixis Study XL147-001). Likewise, XL147 does not have an apparent effect on PK of P or C. Pharmacodynamic modulation of the PI3K pathway was evident in tumor biopsies at plasma exposures consistent with those associated with PI3K inhibition in tumors in an ongoing single-agent Phase 1 trial of XL147 (Exelixis Study XL147-001). For example, post-dose reductions in pAKT-T308 (76%) and p4EBP1 (69%), with associated reductions in pERK (70%) and Ki67 (33%), and induction of apoptosis (2.3-fold), were evident in paired tumor biopsies from the pt with cervical adenocarcinoma (dosed at 200mg XL147/175mg.m−2 P/AUC 6 C) who experienced a PR. Conclusions: XL147 at doses up to 400 mg in combination with paclitaxel at 175 mg.m−2 and carboplatin at AUC 6 is generally well tolerated, with no apparent drug-drug PK interaction or emergent toxicities, and results in robust pharmacodynamic activity in tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B247.

Signs of anin situinflammatory reaction in scars of human American tegumentary leishmaniasis

Skin inflammation plays an important role during the healing of American tegumentary leishmaniasis (ATL), the distribution of cells in active lesions may vary according to disease outcome and parasite antigens in ATL scars have already been shown. We evaluated by immunohistochemistry, 18 patients with 1- or 3-year-old scars and the corresponding active lesions and compared them with healthy skin. Small cell clusters in scars organized as in the active lesions spreaded over the fibrotic tissue were detected, as well as close to vessels and cutaneous glands, despite a reduction in the inflammatory process. Analysis of 1-year-old scar tissue showed reduction of NOS2, E-selectin, Ki67, Bcl-2 and Fas expression. However, similar percentages of lymphocytes and macrophages were detected when compared to active lesions. Only 3-year-old scars showed reduction of CD3(+), CD4(+) and CD8(+)T cells, in addition to reduced expression of NOS2, E-selectin, Ki67 and BCl-2. These results suggest that the pattern of cellularity of the inflammatory reaction observed in active lesions changes slowly even after clinical healing. Analysis of 3-year-old scars showed reduction of the inflammatory reaction as demonstrated by decrease in inflammatory cells and in the expression of cell-activity markers, suggesting that the host-parasite balance was only established after that period.

Fluvastatin reduces proliferation and increases apoptosis in women with high grade breast cancer

The purpose of this study is to determine the biologic impact of short-term lipophilic statin exposure on in situ and invasive breast cancer through paired tissue, blood and imaging-based biomarkers. A perioperative window trial of fluvastatin was conducted in women with a diagnosis of DCIS or stage 1 breast cancer. Patients were randomized to high dose (80 mg/day) or low dose (20 mg/day) fluvastatin for 3-6 weeks before surgery. Tissue (diagnostic core biopsy/final surgical specimen), blood, and magnetic resonance images were obtained before/after treatment. The primary endpoint was Ki-67 (proliferation) reduction. Secondary endpoints were change in cleaved caspase-3 (CC3, apoptosis), MRI tumor volume, and serum C-reactive protein (CRP, inflammation). Planned subgroup analyses compared disease grade, statin dose, and estrogen receptor status. Forty of 45 patients who enrolled completed the protocol; 29 had paired Ki-67 primary endpoint data. Proliferation of high grade tumors decreased by a median of 7.2% (P = 0.008), which was statistically greater than the 0.3% decrease for low grade tumors. Paired data for CC3 showed tumor apoptosis increased in 38%, remained stable in 41%, and decreased in 21% of subjects. More high grade tumors had an increase in apoptosis (60 vs. 13%; P = 0.015). Serum CRP did not change, but cholesterol levels were significantly lower post statin exposure (P < 0.001). Fluvastatin showed measurable biologic changes by reducing tumor proliferation and increasing apoptotic activity in high-grade, stage 0/1 breast cancer. Effects were only evident in high grade tumors. These results support further evaluation of statins as chemoprevention for ER-negative high grade breast cancers.

The human placenta releases substances that drive lung cancer into apoptosis

BackgroundAs there is no optimal treatment of non small cell lung cancer due to its resistance to common chemotherapeutics, we investigated the effect of human placenta-conditioned medium on tumor tissue. The human placenta constitutes a mixture of maternal and fetal origin and displays a variety of immunomodulatory aspects.MethodsFreshly resected non small cell lung cancer tissues were incubated with placenta-conditioned medium in a short-term tissue culture model and A549 cells were challenged, respectively. Term placenta was used for producing conditioned medium and HOPE-fixed stimulated tumor tissue was analyzed for expression of caspase-3 and Ki67 via immunohistochemistry. The effects of conditioned medium on squamous cell carcinoma were further compared to physiological concentrations of Carboplat/Gemzar.ResultsConditioned medium caused in 2 of 3 cases elevated expression of caspase-3 and reduced expression of Ki67 in 3 out of 3 cases, while the chemotherapeutic agents caused no comparable expression of caspase-3 or reduction of Ki67. In cell culture up to 50% of karyopyknosis was investigated and even sterile-filtrated medium caused widespread reduction of Ki67 on protein level.ConclusionHuman placenta releases substances that mediate apoptosis and reduce proliferation in tumor tissue and cell culture. As even sterile-filtrated medium caused the mentioned effects we hypothesize one or more soluble mediators. The detailed way of promoting apoptosis and nature of these mediators need to be elucidated in further studies.

Combined Src and aromatase inhibition impairs human breast cancer growth in vivo and bypass pathways are activated in AZD0530-resistant tumors.

Antiestrogens are used to treat estrogen receptor (ER)-alpha-positive breast cancers and cause a p27-dependent G(1) arrest. Estrogen-bound ER recruits Src to mediate proteolysis of p27 and drive cell proliferation. Here, we tested the antitumor efficacy of combined Src and aromatase inhibition for ER-positive breast cancer. Antiproliferative effects of the aromatase inhibitor, anastrozole, and Src inhibitor, AZD0530, alone or in combination were tested in vitro and in vivo on aromatase-transfected MCF-7Arom5 xenografts. Xenografts were analyzed by immunohistochemistry and proteomic analysis to identify potential biomarkers of drug response and resistance. AZD0530 and anastrozole together increased p27 and caused greater G(1) cell cycle arrest than either drug alone. AZD0530 monotherapy initially retarded xenograft growth in vivo, but drug resistance rapidly emerged. Combined anastrozole/AZD0530 reduced drug resistance and showed greater antitumor efficacy in vivo with greater Src and epidermal growth factor receptor inhibition and a greater increase in p27 and reduction of Ki-67 than either drug alone, supporting further evaluation of these putative predictors of response to combined Src/aromatase inhibition in vivo. Anastrozole alone stimulated Src activity both in vitro and in vivo. AZD0530-resistant tumors showed activation of bypass pathways including MEK and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin, raising the possibility that MEK, mammalian target of rapamycin (mTOR), or PI3K inhibitors may augment Src inhibitor efficacy. These data support clinical investigation of anastrozole-AZD0530 therapy for postmenopausal ER-positive breast cancer. Loss of p27 and increased Ki-67 may predict response and further clinical studies should evaluate for activation of bypass pathways including MEK and PI3K pathways during Src inhibitor therapy.

MEK/ERK inhibitor U0126 affects in vitro and in vivo growth of embryonal rhabdomyosarcoma

We reported previously that the disruption of c-Myc through mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibition blocks the expression of the transformed phenotype in the embryonal rhabdomyosarcoma (ERMS) cell line (RD), thereby inducing myogenic differentiation in vitro. In this article, we investigate whether MEK/ERK inhibition, by the MEK/ERK inhibitor U0126, affects c-Myc protein level and growth of RMS tumor in an in vivo xenograft model. U0126 significantly reduced RMS tumor growth in RD cell line-xenotransplanted mice. Immunobiochemical and immunohistochemical analysis showed (a) phospho-active ERK levels were reduced by U0126 therapy and unaltered in normal tissues, (b) phospho-Myc and c-Myc was reduced commensurate with phospho-ERK inhibition, and (c) reduction in Ki-67 and endothelial (CD31) marker expression. These results indicate that MEK/ERK inhibition affects growth and angiogenic signals in tumor. The RD-M1 cultured xenograft tumor-derived cell line and the ERMS cell line TE671 responded to U0126 by arresting growth, down-regulating c-Myc, and initiating myogenesis. All these results suggest a tight correlation of MEK/ERK inhibition with c-Myc down-regulation and arrest of tumor growth. Thus, MEK inhibitors may be investigated for a signal transduction-based targeting of the c-Myc as a therapeutic strategy in ERMS.

Fluvastatin has biologic effects on stage 0 and 1 breast cancer.

Abstract Abstract #4122 Introduction: Statins are safe, reduce cardiovascular risk, and impact pathways critical to cancer progression. We and others have shown lipophilic statins cause apoptosis and growth suppression in vitro and in vivo, and though epidemiologic data are mixed, statin effect appears most evident in estrogen receptor (ER) negative or grade 3 disease. To look for a direct biologic effect of lipophilic statins, we conducted a perioperative pilot window trial in women with breast cancer (BC).&amp;#x2028; Methods: 40 subjects with stage 0,1 BC were randomized to high dose (80mg/day) or low dose (20mg/day) fluvastatin for 3-6 weeks prior to surgery. Paired tissue (core biopsy and surgical specimen), peripheral blood and MRI were obtained. Primary endpoint was Ki-67 (proliferation) change. Secondary endpoints included cleaved caspase-3 (CC3, apoptosis), longest diameter (LD) by MRI, and C-reactive protein (CRP) change. Subgroup analyses was planned by grade (3 vs. 1,2), statin dose; and ER status. Immunohistochemistry (IHC) on paraffin tissue used standard streptavidin biotin methods. A single breast pathologist reviewed all slides; a single radiologist read all MRIs, both blinded to timepoint.&amp;#x2028; Results: Median serum cholesterol decreased by 16% (-23% and -12% for high and low dose, respectively p=0.012), indicating drug effect and compliance. 29 patients had sufficient tumor for paired IHC, 14 and 15 were grade 3 and 1,2, and 10 and 19 were ER - and +, respectively. In grade 3 (73% of which were ER-) vs. 1,2 tumors, there was a significant decrease in Ki-67, -7.2% (interquartile range (IQR) -13.4%, 0% ) vs. -0.3% (IQR -3%, .8%), respectively, p=0.04. CC3 (apoptosis) increased, 60% vs. 13% for grade 3 vs. 1,2 tumors, respectively, p=0.015. ER- and ER+ cases had a similar reduction in Ki67 with a median drop of 2% (IQR -13.4%, 1%) and 1.2% (IQR -6.6%,0.8%), respectively, p=0.56. While CC3 was increased in ER- vs. + (55% vs. 29%), the difference was not statistically significant. There was no dose dependent effect on Ki-67or CC3.There was no evidence of Ki67 or CC3 change when all grades were analyzed together (median drop 1.2%) and no change in CRP. Of 14 subjects with paired MRIs, 4 grade 3 cases showed a significant decrease in LD, marked ductal dilatation and increased necrosis.with statin exposure.&amp;#x2028; Conclusions: A lipophilic statin, fluvastatin, reduced cholesterol and had measurable biologic changes (reduced proliferation, size and increased apoptosis) in stage 0,1 BC after only 3-6 weeks of exposure, specifically in the grade 3 subset. Results support the study of statins for chemoprevention for women at risk for or with stage 0 grade 3 BC, where new agents are needed. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4122.

Effects of letrozole and anastrozole on low ER expressing invasive breast carcinomas: results from a randomised trial.

Abstract Abstract #1065 Introduction:&amp;#x2028; Changes in proliferation as measured by Ki67 occur within 14 days of starting treatment with an aromatase inhibitor and have been shown to be predictors of long term outcome. The biological effect of different aromatase inhibitors on low ER expressing cancers has not been studied. This study compared changes in proliferation in breast cancers expressing low levels of ER (Allred score 2-5) following 14 days of treatment with anastrozole or letrozole.&amp;#x2028; Materials and methods:&amp;#x2028; Thirty six postmenopausal women with invasive breast cancer, classified as estrogen receptor (ER) poor by an Allred score of 2-5 on initial core biopsy, were enrolled into a randomised pre-operative trial of 14 days treatment with either 2.5 mg of letrozole or 1 mg of anastrozole. Paired biopsy tissue was available for all patients: [15 who received anastrozole and 21 letrozole] with sufficient invasive cancer present for analysis. Assessment included ER (Allred score) and proliferation (% tumor cells Ki67 positive) by immunohistochemistry. Results are presented as means (SEM) and medians (range). Due to the non-normality of the data, pre- and post-treatment Ki67 values were transformed on the log scale. Change between pre- and post- treatment scores was calculated as the difference in the logged scores; analysis is by t-test and Fisher's Exact Test. All tests are two-sided. Results were back-transformed in order to preserve the original scale.&amp;#x2028; Results:&amp;#x2028; Proliferation measured by Ki67:&amp;#x2028; Anastrozole reduced tumour cell proliferation from baseline in 10/15 cancers from a mean of 31.0% (7.1) to 20.8% (6.1). The median and mean reductions in Ki67 were 37% (range -94.0% to 92.6%) and 38% (13.9) from baseline. This equates to an average fall of 2.6 times from baseline, p=0.006, by analysis of the transformed data.&amp;#x2028; Letrozole reduced proliferation from baseline in 17/21 cancers from a mean of 39.0% (7.3) to 25.3% (6.4). The median and mean reductions were 67% (range -115.6% to 99.6%) and 46% (10.6) from baseline. This equates to an average fall of 3.13 times from baseline, p=0.0002, by analysis of the transformed data.&amp;#x2028; There was no evidence of a significant difference between drugs in reduction of proliferation, the number of cases showing a fall in proliferation, or the absolute Ki67 index at 14 days. Only 3/15 (anastrozole) and 2/21 (letrozole) tumours had post-treatment absolute Ki67 indices of ≤1%. There were no significant differences in changes in proliferation between tumours with ER score 2-3 vs score 4-5.&amp;#x2028; Conclusions:&amp;#x2028; 1. Both anastrozole and letrozole significantly reduced proliferation in invasive cancers with low ER expression.&amp;#x2028; 2. There were no evident significant differences between drugs.&amp;#x2028; 3. These data suggest that aromatase inhibitors are of value even in low-ER expressing cancers, either on their own or perhaps as part of combined therapy.&amp;#x2028; 4. Analysis of more patients and of biological response according to molecular phenotype is underway. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1065.

The Novel Aurora Kinase Inhibitor ENMD-2076 Has Potent Single Agent Activity against Multiple Myeloma (MM) in Vitro and in Vivo, and Shows Synergistic Activity in Combination with Lenalidomide

ENMD-2076 is a novel, orally-active molecule that has been shown to have significant activity against Aurora A kinase as well as multiple receptor tyrosine kinases (RTK). We investigated the single agent activity of ENMD-2076 against MM cells in vitro and in vivo, and in combination with lenalidomide. ENMD-2076 free base showed significant cytotoxicity against MM cells with a mean LC50 of 3.84±0.86 μM at 48 hours in vitro. Cytotoxicity was associated with cleavage of caspase 3, 8, 9 and PARP, and loss of mitochondrial membrane potential as early as 6 hours. ENMD-2076 free base inhibited c-kit, FGFR-1, 3 and VEGFR1 and subsequently inhibition of downstream targets phosphorylated (p)-BAD, p-Foxo1a and p-GSK-3β was observed at 6 hours. NOD/SCID mice implanted with H929 human plasmacytoma xenografts and treated for 30 days with 50, 100, 200mg/kg/d ENMD-2076 showed a dose-dependent inhibition of tumor growth (Figure 1), with minimal toxicity as assessed by the stable weight of treated animals. Immunohistochemical staining of tumors from sacrificed animals showed significant reduction in Ki67 at all dose levels of treatment compared to control tumors. An increase in cleaved caspase-3 was observed on Western blot from the lysates of H929 tumors obtained from treated animals. ENMD-2076 free base also showed synergistic cytotoxic activity when combined with lenalidomide against H929, MM1.R and MM1.S cells as assessed by MTT assay and Annexin-V/PI staining. Using the Chou-Talalay method, the combination indices (CI) were < 1 for all three cell lines across a range of concentrations of ENMD-2076 free base (0.25–1.0 μM) plus lenalidomide (2.5–10 μM) indicating synergistic activity (CI=0.362 H929; CI=0.315 MM1.R; CI=0.415 MM1.S). Our results provide rationale for the investigation of ENMD-2076 alone and in combination with lenalidomide in patients with multiple myeloma. [Display omitted]

Cdk4/6 Inhibitor PD 0332991 Demonstrates Cell Cycle Inhibition Via FLT-PET Imaging and Tissue Analysis in Patients with Recurrent Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) carries an unfavorable prognosis and requires new treatment strategies. The associated t(11;14) chromosomal translocation results in enhanced cyclin D1 expression and cyclin D-dependent kinase (Cdk) activity to promote cell cycle progression. PD 0332991, a specific, potent Cdk4/6 inhibitor causes G1 arrest in MCL cell lines by blocking Cdk4/6-specific phosphorylation of Rb on serine 807–811 (pSRb). We report a translational evaluation of PD 0332991 in MCL patients. A phase Ib, multicenter trial is exploring safety and pharmacodynamic effects of PD 0332991 via 18F-labeled fluorodeoxyglucose (FDG) and fluorothymidine (FLT) positron emission tomography (PET) imaging and pSRb/cell cycle biomarker analyses pre-treatment and on day 21. The maximum standardized uptake value (SUVmax) was calculated in up to 10 lesions per patient and the summed SUVmax of all lesions calculated for each patient. Enrollment has been completed as planned with 16 patients receiving PD 0332991 125 mg orally once daily, 21 of every 28 days. Mean age was 66 years (range [r], 40–80), 81% were male, 70% had stage IV disease, with a median of 3 prior regimens (r, 1–8). Currently there are 8 patients with stable disease and ongoing treatment up to 40 weeks, while 8 patients have progressed. Correlative studies have been extensively employed to demonstrate proof of mechanism. Of 10 patients evaluable for PET analyses, mean change in SUVmax for FLT-PET and FDG-PET was −54.0% (r, −3.0 to −80.4%) and −23.9% (r, 7.6 to −73.3%), respectively.Among five pre- and post-treatment biopsy samples analyzed, marked reductions of 52–100% in the percentage of pSRb were noted in day 21 biopsies (p=0.011); in contrast total Rb was maintained or was reduced by a much lesser degree than pSRb (n=5; p=0.109). Additionally, among a total of eight paired samples, six demonstrated reductions in Ki67 staining post-treatment (r, 48–97%, including the five samples with reduced pSRb staining). Two additional paired samples did not demonstrate reduced Ki67 staining post-treatment. Nonetheless, the overall reduction in Ki67 post-treatment among the eight samples reached statistical significance (p=0.036). In 7/8 of cases, the degree of Ki67 reduction post-treatment correlated with the reduction in FLT-PET SUVmax (R2 = 0.62). Principal toxicities that have been observed thus far include grade 3/4 events of neutropenia and grade 3 thrombocytopenia. In patients with MCL, PD 0332991 is well tolerated with manageable toxicities. Collectively these data demonstrate that PD 0332991 inhibits Cdk4/6, leading to reduced cell proliferation (reflected in reductions in FLT-PET SUVmax, Ki 67 status and pSRb/cell cycle biomarker analyses). As Cdk4 and Cdk6 are direct and specific targets of PD 0332991, these data provide proof of mechanism of action of PD 0332991 in patients by using cyclin D1 dependent MCL as a disease model and inform combination treatment approaches.Research Funding: Supported in part by a grant from the Lymphoma Research Foundation.

Abstract 3744: Local Transplantation Of Blood-derived Late-outgrowth Progenitor Cells Increased Reendothelialization, Inhibited Smooth Muscle Cell Proliferation And Reduced Neointima Formation After Experimental Carotid Injury

Introduction : Recent studies demonstrate the vasculoprotective effects of cell-based therapies to inhibit limit restenosis. Delivery of autologous late outgrowth endothelial progenitor cells (eEPC) result in early reendothelialization and inhibition of neointimal hyperplasia. Additional anti-inflammatory treatment may further reduce neointima generation. Methods and Results : CD34+ cells were isolated using immunomagnetic beads and cultured in endothelial cell medium to obtain eEPC. Early passage cells were transduced ex vivo by a retroviral vector expression of IL-1ra (eEPC IL-1ra) or empty vector (eEPC pLXSN) and expanded up to 46 population doublings. Expression levels were confirmed by RT-PCR. Athymic nude rats underwent carotid balloon and wire injury immediately followed by local delivery of eEPCs, eEPC IL-1ra, eEPC pLXSN or buffer for 20 minutes (n=22). Elastica van Giesson staining revealed a decrease in the intima/media ratio from 1.53 ± 0.11 to 1.13 ± 0.09 (mean ± SEM) after transplantation of eEPC (p=0.009). However, no additional reduction in neointima generation was observed after transplantation of eEPC IL-1ra. Endothelial-cell specific staining demonstrated an enhancement of reendothelialisation by eEPCs 24 hours after vascular injury. After 2 weeks a reduction of Ki67+ proliferating cells from 37.0 ± 8.5 Ki67+ cells in the control group to 11.67 ± 2.85 Ki67+ cells after eEPC transplantation (P=0.03) was found. Yet, no additional anti-proliferative effects were observed after transplantation of eEPC IL-1ra. Thus, enhancement of reendothelialization through local delivery of eEPCs may be sufficient for the inhibition of neointima generation since additional anti-inflammatory treatment using eEPC IL-1ra had no beneficial effect. Conclusion : Local delivery of blood-derived expanded endothelial progenitor cells after vascular injury contributes to endothelial regeneration, inhibits neointimal smooth muscle cell proliferation and reduces neointima generation. Autologous cell transplantation after vascular injury may be a feasible strategy for promotion of reendotheli-alisation and improvement of vascular regeneration.

Improved clinical and cell cycle response with an mTOR inhibitor, daily oral RAD001 (everolimus) plus letrozole versus placebo plus letrozole in a randomized phase II neoadjuvant trial in ER+ breast cancer

530 Background: Activation of the mTOR pathway is a key adaptive change driving endocrine resistance. RAD001, an oral mTOR inhibitor, and letrozole synergistically inhibit proliferation in breast cancer cells. Methods: 270 postmenopausal women with > T2 ER+ tumors were randomized to receive 4 mo of letrozole 2.5mg QD + RAD001 10mg QD or letrozole 2.5mg plus placebo. The primary endpoint was overall clinical response. Mandatory needle biopsies were collected immediately prior to initial treatment, and at day 15 after treatment initiation. Samples were assessed at baseline for Her2 by FISH and mutation of exons 9 and 20 of PIK3CA and exons 5–8 of TP53, and at baseline and day 15 for Ki67, phospho-Akt S473, phospho-S6, PTEN, CyclinD1, ER, PR, p53, and total Akt and S6 by immunohistochemistry. A total of 207 patients (77%) were primary marker evaluable and clinical outcome evaluable; 186 patients had an evaluable second biopsy. Results: The clinical response rate with RAD001 + letrozole was significantly superior to letrozole alone at the preplanned Alpha of 0.1 (68 vs 59%, p=0.062). This was confirmed by ultrasound (OR 58% vs 47 %, p=0.035). Pharmacodynamic changes in each treatment arm were observed. Marked downregulation in progesterone receptor and cyclin D1 were seen in response to letrozole. Phospho- S6 levels showed dramatic down-regulation only in response to RAD001. Cell cycle response, as defined by either % reduction in Ki67 at day 15 or by proportion of patients with Ki67≤2 at day 15, was also significantly higher in the RAD001 + letrozole arm (57% vs 30% for Ki67≤2 at day 15, p<0.01). The rate of grade 3/4 adverse events (Aes) was 22.6% in the RAD001 + letrozole arm vs 3.8% in the placebo + letrozole arm. Most frequent grade 3/4 Aes with RAD001 + letrozole were hyperglycemia (n=7), stomatitis (3), interstitial lung disease/pneumonitis (3) and infections (3). Conclusions: RAD001 significantly increases the efficacy of letrozole in newly diagnosed ER+ breast cancer, with regard to both clinical and cell cycle response. The increased cell cycle response rate in the RAD001 arm was found in all subsets of tumors, including PTEN-positive, PIK3CA wild-type tumors. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Novartis Novartis Novartis Novartis

Introduction: challenging established dogma

Introduction: challenging established dogma

A phase II study of the c-Met RTK inhibitor XL880 in patients (pts) with papillary renal-cell carcinoma (PRC)

15601 Background: XL880 is a novel inhibitor of receptor tyrosine kinases targeting MET and VEGFR2/KDR. (IC 50 for MET 0.4nM and KDR 0.8 nM). PRC is a rare (10–15%) subtype of kidney cancer which is notoriously resistant to systemic treatment. The familial form of PRC is associated with germline activating mutations of MET, while amplification and overexpression of MET is seen in the sporadic form. In an ongoing phase I dose escalation study of XL880 in forty patients with advanced solid tumors, five partial responses (PRs) were noted in 3 of 4 treated patients with PRC, and in two pts with thyroid cancer. The MTD in this study was 3.6 mg/kg of XL880 administered orally daily for 5 consecutive days every 2 weeks. Methods: In this phase 2 study, XL880 is administered orally as a single agent at a dose of 240 mg on days 1–5 of each 14 day cycle. Previously untreated pts with a histologic diagnosis of metastatic PRC are eligible and are stratified based on the presence or absence of MET germline mutations. Planned enrollment is 15 evaluable pts in each stratum. Inclusion criteria also include ECOG performance status &lt; 3 and absence of brain metastases. Tumor response is assessed every 8 weeks by RECIST criteria. Archival primary tumor and lymph node metastasis were collected for pharmacodynamic assessments. Results: To date, 6 pts have been enrolled and received XL880. Five pts have tested negative for MET germline mutations; results for one pt are pending. Of 3 pts with at least one post baseline tumor assessment, 1 pt has demonstrated a PR and two pts have demonstrated stable disease &gt; 3 months, one of whom has improvement in bone scan and bone pain. Hypertension has been reported in 2 pts who responded to anti- hypertensive treatment. Two SAEs have been reported as unrelated; pulmonary embolism and intratumoral hemorrhage. MET expression was detected in untreated primary and metastatic tumor. Reduction in Ki67, and an increase in TUNEL labeling were observed in the post treatment sample. Conclusions: XL880 is the 1st orally available small molecule inhibitor of MET to enter the clinic and appears to be generally well tolerated. Anti-tumor activity has been observed and XL880 may represent an active treatment option for pts with PRC. Enrollment continues and updated results will be presented. No significant financial relationships to disclose.