Citations

Abstract Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5-year survival rate of 20-30%. Mitotane is the only approved drug for the treatment of patients with ACC. It often carries significant toxicities which result in the discontinuation of treatment. There are no approved second line therapies. The increased incidence of ACC in the cancer predisposition syndrome, Li-Fraumeni, suggests the involvement of the p53 pathway in ACC pathogenesis. Our analysis of the gene expression profiles of 19 ACC samples identified dysregulation of the G2/M transition and the activity of the p53 modulator, MDM2 as important in ACC pathogenesis. Polo-like kinase-1 (PLK1) is involved in the G2/M transition and acts to promote MDM2 activity through its phosphorylation. We observed that PLK1 inhibition by siRNA results in up to a 70% reduction in viability in the ACC cell lines SW-13 and H295R. Therefore, we studied PLK1 inhibition as a potential therapeutic strategy. We used the small molecule inhibitor, BI-2536, to inhibit PLK1 function in SW-13 and H295R. Drug-dose response curves demonstrated that both cell lines are sensitive to pharmacological inhibition of PLK1 (IC50 doses of 0.0094815 μM and 0.062805 μM for SW-13 and H295R, respectively.) Murine xenograft studies demonstrated that BI-2536 resulted in a statistically significant reduction in tumor growth in SW-13 but not H295R. Examination of p53 protein levels in the presence of the drug showed a dose-dependent reduction in p53 levels in SW-13, which carries a homozygous p53 mutation. The same was not true in H295R, which is p53 wild-type. To test the hypothesis that BI-2536 was decreasing mutant p53 levels by promoting its proteasomal degradation, both cell lines were treated with previously determined inhibitory concentrations of BI-2536, either alone or in combination with the proteasome inhibitor, MG132. Western blot analysis showed recovery of p53 protein when cells were concomitantly treated with MG132, supporting a role for BI-2536 as a regulator of proteasomal degradation. Both ACC cell lines are relatively insensitive to MDM2 inhibition by nutlin-3 (NCI60 GI50 values range from ∼4 uM to ∼2 uM versus ACC cell lines @13-15uM), possibly because PLK1 stimulates MDM2 and renders nutlin-3 ineffective. We therefore assessed the ability of BI-2536 to sensitize ACC cell lines to the effects of the MDM2 inhibitor. BI-2636 did show synergy with nutlin-3, shifting the IC50 in SW-13 from 19.78 μM to 6.45 μM and from 12.75 μM to 2.84 μM in H295R. These results demonstrate that inhibition of PLK1 alone or in combination with an MDM2 inhibitor warrants further investigation as a treatment for patients with ACC. Cells with mutant p53 are more sensitive to the growth inhibitory effects of BI-2536. In the context of wild-type p53, the combined inhibition of both PLK1 and MDM2 results in loss of viability. Further clinical development of PLK1 as a target should take p53 mutation status into account. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 978. doi:1538-7445.AM2012-978

Abstract Rationale Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is becoming standard of care for the sampling of mediastinal adenopathy. The need for a safe, effective, accurate procedure makes EBUS-TBNA ideal for mastery training and testing. Objectives The Endobronchial Ultrasound Skills and Tasks Assessment Tool (EBUS-STAT) was created as an objective competency-oriented assessment tool of EBUS-TBNA skills and knowledge. This study demonstrates the reliability and validity evidence of this tool. Methods The EBUS-STAT objectively scores the EBUS-TBNA operator’s skills, including atraumatic airway introduction and navigation, ultrasound image acquisition and optimization, identification of mediastinal nodal and vascular structures, EBUS-TBNA sampling, and recognition of EBUS/computed tomography images of mediastinal structures. It can be administered at the bedside or using combination of low- and high-fidelity simulation platforms. Two independent testers administered the EBUS-STAT to 24 operators at three levels of EBUS-TBNA experience (8 beginners, 8 intermediates, and 8 experienced) at three institutions; operators were also asked to self-assess their skills. Scores were analyzed for intertester reliability, correlation with prior EBUS-TBNA experience, and association with self-assessments. Measurements and Main Results Intertester reliability between testers was very high (r = 0.9991, P < 0.00005). Mean EBUS-STAT scores for beginner, intermediate, and experienced groups, respectively, were 31.1, 74.9, and 93.6 out of 100 (F2,21 = 118.6, P < 0.0001). Groups were nonoverlapping: post hoc tests showed each group differed significantly from the others (P < 0.001). Self-assessments corresponded closely to actual EBUS-STAT scores (r2 = 0.81, P < 0.001). Conclusions The EBUS-STAT can be used to reliably and objectively score and classify EBUS-TBNA operators from novice to expert. Its use to assess and document the acquisition of knowledge and skill is a step toward the goal of mastery training in EBUS-TBNA.

Abstract Background: In estrogen receptor positive (ER+) breast cancer human epidermal growth factor receptor 2 (HER2) expression is associated with resistance to tamoxifen (Tam). Endoxifen is the most abundant active metabolite of Tam, resulting in part from cytochrome P450 2D6 (CYP2D6) metabolism. While Tam has been extensively used to model proliferative ER/HER2 cross-talk, there are no published data regarding endoxifen's effect on ER/HER2 nongenomic nor ER genomic signaling. Methods: Using ER+ cells (MCF7) and ER+/HER2+ cells (MCF7-HER2, BT474) we compared the effects of Tam and endoxifen on proliferation using crystal violet assays, Erk1/2 phosphorylation by western blotting, and E2-stimulated ER-regulated gene expression of amphiregulin (AREG), c-Myc (MYC) and progesterone receptor (PGR) by real-time RT-PCR assays. To examine the anti-proliferative affects of endoxifen compared to Tam in vivo we performed E2-stimulated MCF7-HER2 xenograft studies in athymic nude mice. Endoxifen doses of 25 (low) and 75 mg/kg (high) were used based on our prior pharmacokinetic studies showing achievable steady state levels of 100 nM and 1 uM, respectively. Z-Endoxifen HCl was synthesized by NCI. Results: Endoxifen, unlike Tam, potently inhibits growth in both ER+ and ER+/HER2+ breast cancer cell lines at concentrations observed in human CYP2D6 intermediate and extensive metabolizers (>40 nM). Tam (10-1000 nM) stimulates phosphorylation of Erk1/2 in stark contrast to endoxifen at similar doses. Endoxifen (100-1000 nM) significantly decreases E2-stimulated transcription of ER-regulated genes in MCF7-HER2 cells; AREG 2-3 fold, MYC 1-2 fold, and PGR 2-4 fold. Conversely, Tam (10-1000 nM) maintains or stimulates transcription; AREG 2-3 fold, MYC 0.5-2.5 fold, and PGR 0-3 fold. After 7 weeks of treatment of MCF7-HER2 xenografts, both high (p=0.001) and low dose endoxifen (p=0.01) resulted in greater antitumor activity than Tam, with high dose superior to low dose endoxifen (p=0.024). MCF7-HER2 xenografts that progressed on Tam to 200% growth at 7 weeks were randomized to either continued Tam or endoxifen. Four weeks after randomization endoxifen was superior to Tam (p<0.0001) with tumor size decreased by ≥ 20%. No significant toxicity was observed.Conclusions: We have demonstrated in both ER+ and ER+/HER2+ breast cancer cell lines that in contrast to Tam, endoxifen potently inhibits growth (both in vitro and in vivo), does not activate Erk1/2 signaling, and inhibits E2-stimulated transcription of ER-regulated genes. Endoxifen exhibited potent antitumor activity in MCF7-HER2 xenografts refractory to Tam. These data support the ongoing NCI sponsored phase I studies of Z-endoxifen at Mayo Clinic and NCI. 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 2283. doi:10.1158/1538-7445.AM2011-2283

Abstract Background: First in human studies of Z-endoxifen hydrochloride (E), the active metabolite of tamoxifen (T), are underway in metastatic breast cancer (BC). Previous data have demonstrated the superiority of aromatase inhibitors (AI's) over T in estrogen receptor (ER) + BC. Using an in vivo aromatase expressing model (MCF7/AC1), we compared the antitumor activity of E with T and Letrozole (L), as well as the antitumor activity and global gene expression changes of E with T in an L-resistant model. Methods: MCF7/AC1 tumors were stimulated with androstenedione. Once tumor size reached 300 mm3, mice (30/group) were randomly assigned to one of five treatment groups: control (daily, po), T (500 μg/day, sc), endoxifen 25 mg/kg/day p.o.(LDE) endoxifen 75 mg/kg/day p.o. (HDE) or letrozole, 10 μg/day s.c for 4 weeks. Tumors were harvested from control, T, and E groups while the L group continued treatment until the development of resistance defined as an increase in tumor volume of at least 300% from day 1. Mice with L-resistant tumors were randomly assigned to T (n=4) or E (n=5) for 4 weeks and then sacrificed. Gene expression in L-resistant tumors was quantified using Affymetrix U133+2 and changes in gene expression profiles [comparing T and E with L-resistant (n=3)] were analyzed. Genes identified as significantly different were confirmed by real-time RT-PCR assays. Results: At the 4 week time point, both doses of E and L resulted in greater anti-tumor activity than control (Wilcoxon rank sum test: all p < 0.0001); however, tumor burden did not differ between T and control (p=0.095). HDE resulted in significantly less tumor burden than T (p=0.002) but was similar to L. In mice that continued on L, resistance developed at 24 weeks in 9/25 mice. These mice were randomly assigned to either T (n=4) or E (n=5) for 4 weeks. Tumor volume (expressed as a% of its size prior to randomization) was significantly different comparing E (73.3%; range: 69.3 to 80.75%) versus T (148.39%; range: 114.07 to 165.99%) (Wilcoxon rank sum test p=0.016). Compared to control, microarray studies identified 1518 unique probe sets regulated by E (p<0.001) compared to 441 for T including estrogen-regulated genes such as progesterone receptor (PGR) and amphiregulin (AREG) that were significantly down-regulated in the E group [PGR (−6.2 fold, p=0.000008) and AREG (−3.2 fold, p=.0006) but unchanged or up-regulated in the T group (PGR unchanged and AREG +9.2 fold p=0.00002). These findings were confirmed by RT-PCR. Conclusions: Using the MCF7/AC1 model previously used to show the superiority of AI's over T, HDE demonstrated similar antitumor activity to L and was superior to T. In cells resistant to L, E was superior to T and gene expression changes demonstrate that E down-regulates while T activates estrogen regulated genes. These findings support the ongoing development of E for the treatment of ER+ BC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD01-06.

Background Combined severe proximal left anterior descending and proximal left circumflex coronary artery disease, or left main equivalent (LMEQ) disease, defines a prognostic high-risk angiographic subset of patients with chronic ischemic heart disease. While numerous observational and randomized clinical trials showed prolonged survival in surgically compared with medically treated patients with left main coronary artery disease, relatively few observational studies compared surgical and medical therapies in patients with LMEQ disease. The present report of 912 patients with LMEQ disease in the Coronary Artery Surgery Study (CASS) Registry extends the originally published 5-year surgical and medical group survival analysis to more than 16 years of follow-up and permits analysis of LMEQ patient subgroups. Methods and Results The CASS Registry contains 912 patients with LMEQ disease, defined as combined stenoses of ≥70% in the proximal left anterior descending coronary artery before the first septal perforator and proximal circumflex coronary artery before the first obtuse marginal branch, initially treated with either surgical or nonsurgical therapy. The 15-year cumulative survival estimates were 44% for the 630 patients in the surgical group and 31% for the 282 patients in the medical group. Median survival in the surgical group was 13.1 years (12.7 to 14.1 years, 95% confidence limits) compared with only 6.2 years (4.8 to 7.9 years) in the medical group (difference, 6.9 years; P <.0001). Median survival was also significantly longer in the surgical group stratified by age, sex, anginal class, left ventricular (LV) function, and coronary anatomy. However, coronary artery bypass graft (CABG) surgery did not significantly prolong median survival in patient subgroups with (1) normal LV systolic function, even if a significant right coronary artery stenosis (≥70%) also was present, and (2) mildly abnormal (LV score, 6 to 10) LV systolic function. The 15-year cumulative survival in patients with normal LV systolic function in the surgical and medical groups was 63% and 54%, respectively. Median survival was >15 years in both the surgical and medical groups ( P =NS). In patients with normal LV systolic function and right coronary artery stenosis ≥70%, the 15-year cumulative survival was also similar in the surgical and medical groups (63% and 53%, respectively). Median survival was >15 years in both the surgical and medical groups ( P =NS). The 15-year cumulative survival estimates in all subgroups were affected by convergence of the surgical and medical group survival curves caused by a disproportionate increase in late surgical group mortality. Overall, 26% of patients in the medical group ultimately underwent CABG surgery. If all medical group patients had survived long enough, about 65% would be estimated to have had surgery by 15 years. When the CASS Registry patients with LMEQ disease who participated in the randomized trial or who were randomizable were analyzed, CABG surgery did not prolong the 15-year cumulative survival estimates compared with nonsurgical therapy for randomized (71% versus 67%, respectively) and for randomizable patients (62% versus 92%, respectively) with an LV ejection fraction ≥50%. Conclusions This report, which extends follow-up of more than 16 years in CASS Registry patients with LMEQ disease, shows that CABG surgery prolongs life in most clinical and angiographic subgroups. However, median survival was not prolonged by CABG surgery in patients with normal LV systolic function, even if a significant right coronary artery stenosis (≥70%) also was present or in patients with an LV ejection fraction ≥50% who participated in the CASS randomized trial or who were randomizable. These results extend our understanding of the natural history of LMEQ disease and permit a more accurate estimate of long-term surgical and medical group survival. These long-term results should allow clinicians to make more informed decisions about the best choice of treatment available for patients with similar clinical and angiographic features.

Background Observational and randomized studies designed to compare surgical and medical therapies in patients with left main coronary artery disease (LMCD) have shown that coronary artery bypass graft (CABG) surgery prolongs life in most patients with LMCD. The present report of 1484 patients with LMCD in the Coronary Artery Surgery Study (CASS) Registry extends the originally published 5-year surgical and medical group survival analysis to more than 16 years of follow-up and permits analysis of LMCD patient subgroups. Methods and Results The CASS Registry contains 1484 patients with ≥50% left main coronary artery stenosis initially treated with either surgical or nonsurgical therapy. The 15-year cumulative survival estimates were 37% for the 1153 patients in the surgical group compared with 27% for the 331 patients in the medical group. Median survival in the surgical group was 13.3 years (12.8 to 13.8 years, 95% confidence limits) compared with only 6.6 years (5.4 to 7.9 years) in the medical group (difference, 6.7 years; P <.0001). Median survival was also significantly longer in the surgical group stratified by age, sex, anginal class, left ventricular (LV) function, coronary anatomy, and the extent of LMCD. However, CABG surgery did not significantly prolong median survival in patient subgroups with (1) left main coronary stenosis of 50% to 59%; (2) normal LV systolic function; (3) normal or mildly abnormal LV systolic function and a right coronary artery stenosis ≥70%; and (4) a nonstenotic (≤70%) right coronary artery. The 15-year cumulative survival for patients with normal LV systolic function in the surgical and medical groups was 42% and 51%, respectively. Median survival was 14.7 years in the surgical group and >15 years in the medical group ( P =NS). In patients with normal LV systolic function and a right coronary artery stenosis ≥70%, the 15-year cumulative survival rates were also similar in the surgical and medical groups (40% and 48%, respectively). Median survival was 14.3 years in the surgical group and 14.2 years in the medical group ( P =NS). The 15-year cumulative survival estimates for all subgroups were affected by convergence of the surgical and medical survival group curves owing to a disproportionate increase in the late surgical group mortality. Overall, 25% of patients in the medical group ultimately underwent CABG surgery. If all medical group patients had survived long enough, about 47% would be estimated to have had surgery by 15 years. Conclusions This report, which extends follow-up of more than 16 years in CASS Registry patients with LMCD, shows that CABG surgery prolongs life in most clinical and angiographic subgroups. However, median survival was not prolonged by CABG surgery in patients with normal LV systolic function, even if a significant right coronary artery stenosis (≥70%) also was present. These results extend our understanding of the natural history of LMCD and permit a more accurate estimate of long-term surgical and medical group survival.

AbstractVEGF induces vascular permeability (VP) in ischemic diseases and cancer, leading to many pathophysiological consequences. The molecular mechanisms by which VEGF acts to induce hyperpermeability are poorly understood and in vivo models that easily facilitate real-time, genetic studies of VP do not exist. In the present study, we report a heat-inducible VEGF transgenic zebrafish (Danio rerio) model through which VP can be monitored in real time. Using this approach with morpholino-mediated gene knock-down and knockout mice, we describe a novel role of phospholipase Cβ3 as a negative regulator of VEGF-mediated VP by regulating intracellular Ca2+ release. Our results suggest an important effect of PLCβ3 on VP and provide a new model with which to identify genetic regulators of VP crucial to several disease processes.