Citations

Abstract Bisphosphonates reduce the incidence of skeletal-related events (fractures, pain, hypercalcemia) in patients (pts) with bone metastases from breast cancer. By inhibiting osteoclast function and subsequent bone turnover they may inhibit the growth of bone (and other) metastases. Their role in preventing or delaying the development of bone (or other) metastases in pts with early breast cancer is uncertain. Three previous trials of oral clodronate have given mixed results. The largest trial (placebo-controlled) and a smaller open-label trial suggested that oral clodronate benefitted pts with improved bone metastases-free survival (BMFS) and overall survival (OS), but a third open-label study showed no benefit with an apparent detrimental effect on survival. Studies of IV zoledronate in open-label trials in early breast cancer have also shown mixed results: the AZURE trial in pts with node positive breast cancer showed no benefit in disease-free survival (DFS) or OS with a possible effect in pts aged over 60. The ABCSG-12 trial in GnRH (plus tamoxifen or anastrozole) treated women with early breast cancer showed a small DFS benefit for those receiving IV zoledronate but no BMFS or OS benefit. Methods: B-34 is a prospective, randomized, double-blind, phase III clinical trial in pts with stage 1, 2 or 3 breast cancer assessing oral clodronate 1600mg daily for 3 years compared to placebo given alone or in addition to adjuvant chemo- or hormone therapy. Stratification is by age (<50, +50), number of positive nodes (0, 1–3, 4+) and ER/PR status. The primary end-point is DFS. Secondary endpoints are the incidence of skeletal metastases, OS, relapse-free survival, incidence of non-skeletal metastases, and incidence of skeletal morbid events. Results: 3323 patients were accrued — 1662 in Group A and 1661 in Group B. As of March 31, 2011, 54 pts were declared ineligible: 23 in Group A and 31 in Group B. The average time on study is 101.3 months. Patient characteristics were evenly distributed throughout both groups. Approximately 75% of pts in each group had negative axillary lymph nodes and some 78% in each group were ER and/or PR positive. About 64% of pts were age 50 or over. Oral clodronate was generally tolerable, toxicities observed being mainly due to the concomitant systemic chemotherapy. One pt in Group A had a 1 mm area of osteonecrosis on the palatal taurus. Compliance, as expected with trials of oral medications, has been a problem. 1910 pts completed 3 years of therapy. Event rates were slower than anticipated. This final analysis assesses a total of 598 events in the two arms. Conclusions: This trial is the largest placebo-controlled study of an oral bisphosphonate in patients with early breast cancer and will provide further information on the role of bisphosphonates in breast cancer management. Supported for NSABP by NCI U10CA12027, −37377, 69651, 69974; and Bayer Schering Pharma Oy; for ECOG by U10CA021115; for NCCTG by U10CA25224; for SWOG by U10CA38926. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S2-3.

Abstract Purpose/Background: The first-line chemotherapeutic temozolomide (TMZ) does not offer any therapeutic benefit in some cases of glioblastoma (GBM) and emergence of resistance leads to tumor progression in almost all patients. While the expression of O-6-methylguanine-DNA-methyltransferase (MGMT) correlates well with TMZ resistance, its expression varies among GBM tumors. The goal of this study is to identify additional genes with potential roles in TMZ resistance. Materials and Methods: Five primary GBM xenografts were generated from patient samples, and subsequent secondary models for resistance were developed by subjecting tumor-bearing mice to escalating doses of TMZ. Triplicate samples from parental and TMZ-resistant xenografts were analyzed for gene expression using the Illumina BeadChip microarray platform. The top 100 differentially expressed genes for each line were considered candidates. Genes were selected for qRT-PCR validation after ontological and pathway analysis. A lentivirus-mediated shRNA knockdown was performed to validate the role of several candidate genes in TMZ resistance in U87 and primary GBM12 cell lines. Knockdown was confirmed with qRT-PCR and western blots. TMZ responsiveness was assessed using CyQUANT and Neurosphere assays. Results: Upregulated MGMT expression was found in 3 of the 5 xenografts (GBM12TMZ, GBM14TMZ and GBM28TMZ) and was mechanistically linked with TMZ resistance in these lines using O6-benzylguanine to reverse the resistance phenotype. Differential expression of other DNA damage response genes included CCND2, CDKN1A, GADD45B, UBB as well as apoptosis regulators NFKB2 and BIRC3. In lines with unchanged MGMT expression (GBM22TMZ and GBM39TMZ), changes in anti-apoptosis genes NFKB2 and MAP3K1, transcription factor SRF and signal transduction gene PRKCDBP were validated by qRT-PCR. In GBM12TMZ, in addition to MGMT upregulation, expression of Ubiquitin B (UBB) was found to be the most significantly down-regulated gene both on microarray (Log2FC = −4.55, Fold change = 0.043) and qRT-PCR (Log2RQ =-4.04, RQ = 0.061). To test a possible role of UBB in TMZ resistance, knockdown was achieved in U87 (knockdown efficiency=91.2%) and in primary xenograft line GBM12 (knockdown efficiency=84.4%). The CyQUANT assay for U87UBB-KD and U87NT treated with escalating doses of TMZ revealed no significant difference in cell survival (10 M TMZ relative survival= 89.7% for U87UBB-KD and 79.2% for U87NT; p-value=0.53). Similarly, a neurosphere assay for GBM12UBB-KD and GBM12NT also yielded no significant difference in cell survival (10 M TMZ relative survival= 25.13% for GBM12UBB-KD and 23.35% for GBM12NT; p-value=0.83). Conclusions: We have used a TMZ resistance xenograft model to identify candidate TMZ resistance genes. The current data provides evidence on the role of MGMT in the development of acquired TMZ resistance while ruling out UBB as potential TMZ resistance gene in GBM. Additional studies are underway to assess the functional significance of other candidate genes, particularly those with differential expression in the setting of constant MGMT expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B68.

Abstract Heat shock protein (HSP) 90 is a molecular chaperone that modulates stability of several client proteins, many of which are key regulators of signaling pathways in glioblastoma multiforme (GBM). The potential for targeting multiple effectors makes HSP90 an appealing therapeutic target for GBM. The biological activity of HSP990, a novel HSP90 inhibitor (Novartis Pharmaceuticals) as single agent was evaluated in a panel of short-term explant cultures derived from primary GBM xenografts (n=10). HSP990 treatment resulted in a dose-dependent growth inhibition in the all xenograft lines, with an IC50 ranging from 5 to 30 nM. A 72 h exposure to HSP990 (10–30 nM) resulted in a dose-dependent decrease in the HSP90 client proteins Akt, Cdc2 and Cdc25C, a decrease in associated post-translational modifications p-Akt (S473), p-CDC2 (Y15), p-CDC25 (S216), and increased expression of HSP70. Additionally, a dose-dependent increase in HSP990-induced apoptosis (Annexin-V positivity), was observed within 24–72 hours of drug treatment. GBM tumors harbor a subpopulation of glioma stem cells that grow as neurospheres in neurobasal media and are considered important for tumor maintenance and progression. The effects of HSP990 were tested in neurospheres derived from 4 of the xenografts tested above (GBM12, GBM39, GB59, GBM12TMZ). These cultures demonstrated potential for tri-lineage differentiation upon serum challenge and had robust secondary neurosphere formation, demonstrating two hallmark features of cancer stem cells. In a bulk-culture neurosphere formation assay, co-incubation with 5 to 10 nM HSP990 was highly effective at suppressing neurosphere formation in all 4 xenograft lines. Notably, in GBM12 neurosphere cultures maintained in either serum-free media or challenged with serum, 30 nM HSP990 was equally effective at promoting up-regulation of HSP70, but exhibited less effect on Akt or Cdc2 in serum-free cultures as compared to cultures supplemented with serum. Incubation of serum-free neurosphere cultures with 5 nM HSP990 (5 days) resulted in increased glioneuronal differentiation exhibiting elevated GFAP, Oct-4 and beta-tubulin expression compared to untreated controls. To test ‘self renewal’ GBM12 neurospheres were treated with HSP990 (10, 30 nM) for 7 days followed by dissociation and plating equal numbers of viable cells at low density in fresh drug-free neurobasal media. Interestingly, HSP990 treatment did not affect the number of secondary neurospheres (surviving fraction: control = 1.00, 10 nM = 1.30, 30 nM = 1.05). Treatment of GBM12TMZ flank tumor xenografts with weekly HSP990 (13 mg/kg P.O.) resulted in a modest 9 day delay in tumor re-growth (40.3 and 49.3 respectively) compared to placebo treatment. In conclusion, HSP990 (a) had anti-proliferative, pro-apoptotic effect on differentiated serum-supplemented xenograft cultures (b) a pro-differentiation effect on serum-free GBM stem cell cultures (c) markedly suppressed neurosphere formation with prolonged drug exposure but not with more limited drug exposure (d) inhibited GBM growth in vivo. Thus, HSP990 may inhibit GBM growth both through direct growth inhibition and through pro-differentiation effects on GBM stem-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B100.

Abstract Poly (ADP-ribose) polymerase (PARP) inhibition can enhance the efficacy of temozolomide (TMZ) and has been shown to significantly prolong survival in orthotopic GBM xenografts. Here the effects of PARP inhibitor AG014699 (AG699) on the efficacy of TMZ was evaluated and corresponding pharmacokinetic and pharmacodynamic studies were performed in the MGMT methylated GBM12 primary GBM xenograft line. AG699 effectively sensitized GBM12 cells to TMZ in vitro in a neurosphere formation assay: IC50 of 1.56 M for TMZ when combined with 1 μM AG699 compared to 8.16 M TMZ when used alone). Using these same cells, orthotopic xenografts were established and mice were randomized to 3–28 day cycles of placebo or TMZ (50 mg/kg days 1–5) with or without AG699 (1 mg/kg IP days 1–5). Although this model is responsive to other TMZ/PARP inhibitor combinations, the addition of AG699 was ineffective (median survival: placebo 16 days, TMZ 68 days and TMZ/AG699 81 days, p=0.883). In contrast, AG699 significantly potentiated TMZ-induced tumor regrowth delay in a flank tumor model with GBM12 (median time for tumors to reach endpoint: placebo 32 days, TMZ 86 days and TMZ/AG699 121 days, p<0.01). To more clearly understand the discrepancy between intracranial and flank studies, drug accumulation and target inhibition were assessed. Plasma and tissue distribution studies in mice bearing GBM12 orthotopic xenografts showed that 0.5 h after AG699 treatment its level in brain and tumor xenograft was much lower compared with that in liver (see table). In an assessment of catalytic activity, PARP activity in liver was inhibited more than 95% of control at least 2 h after AG699 treatment, while it was inhibited less than 50% and 30% in orthotopic xenograft tissue and normal brain, respectively (Table). Collectively, these data support the hypothesis that the efficacy of AG699 combined with TMZ is limited in the orthotopic model due to limited drug accumulation within the brain, and the mechanisms of which are under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A77.

Abstract Glioblastoma multiforme (GBM) is the most common and highly malignant primary brain tumor in adults with a median survival just over 1 year, and novel therapies are urgently needed. Deregulation of the p16 (INK4A)-CDK4/6-pRb pathway are commonly found in human GBM patients and selective inhibitors of CDK4/6 may be specifically useful to suppress proliferation associated with these alterations. PD0332991 is an orally active small molecule that specifically inhibits CDK4 and CDK6. Therefore, the efficacy of PD0332991 was evaluated in the Mayo panel of primary GBM xenograft lines using short-term explant cultures. We hypothesized that expression of p15 and/or p16, endogenous inhibitors of CDK4/6 activity, would suppress the response of GBM tumors to PD0332991 treatment. To test this hypothesis, 2 GBM xenograft cell lines with wild-type p16/p15 levels (GBM5 and 28) and 2 lines without detectable p16/p15 levels (GBM22 and 43) were selected for further analysis. Tumor cells were treated with graded concentrations (10 nanoM to1 microM) of PD0332991 for 5 days and subjected to CyQUANT cell proliferation assays. Consistent with our hypothesis, treatment with 1 microM PD0332991 effectively inhibited cell proliferation to 56.6±3.5% (P<0.001) and 44.3±3.4% (P<0.001) of control in GBM22 and GBM43 cells respectively, but did not affect cell proliferation in GBM5 and GBM28 cells (98.7±15.6% and 101.8±12.0% of control, respectively). In addition, 1 microM PD0332991 induced G1-phase cell cycle arrest in GBM22 (treated 79.7±0.8% vs. control 63.4±0.5% of cells in G1 phase, P<0.001) and GBM43 (treated 70.6±1.1% vs. control 48.9±4.6% of cells in G1 phase, P<0.001) but not in GBM5 and GBM28 (treated 52.3±2.7% vs. control 54.0±5.1%, treated 52.4±4.7% vs. control 54.6±2.6%, respectively). The status of p15 and p16 was confirmed by RT-PCR, which demonstrated readily detectable expression of p15 and p16 in GBM5 and GBM28, but not in GBM22 and GBM43 cells. Additional comparative genomic hybridization (CGH) data showed that CDKN2A and 2B (genes encode for p16 and p15 respectively) are homozygously deleted in GBM22 and 43, hemizygously deleted in GBM5 and normal in GBM28. We also examined the status of CDK4, CDK6, pRb and other molecules in the p16-CDK4/6-pRb pathway by Western, RT-PCR and CGH in these GBM lines and the data will be presented. We are now testing whether shRNA knockdown and/or re-expression of p15/p16 in the relevant cell lines alters their responsiveness to PD0332991. In conclusion, CDK4/6 inhibitor PD0332991 inhibits cell proliferation and induces G1 cell cycle arrest in p15/p16- null GBM tumors, but is less effective in p15/p16 expressing tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C191.

Background —Genetic defects are being increasingly recognized in the etiology of primary cardiomyopathy (CM). Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first step in the β-oxidation spiral of fatty acid metabolism, the crucial pathway for cardiac energy production. Methods and Results —We studied 37 patients with CM, nonketotic hypoglycemia and hepatic dysfunction, skeletal myopathy, or sudden death in infancy with hepatic steatosis, features suggestive of fatty acid oxidation disorders. Single-stranded conformational variance was used to screen genomic DNA. DNA sequencing and mutational analysis revealed 21 different mutations on the VLCAD gene in 18 patients. Of the mutations, 80% were associated with CM. Severe CM in infancy was recognized in most patients (67%) at presentation. Hepatic dysfunction was common (33%). RNA blot analysis and VLCAD enzyme assays showed a severe reduction in VLCAD mRNA in patients with frame-shift or splice-site mutations and absent or severe reduction in enzyme activity in all. Conclusions —Infantile CM is the most common clinical phenotype of VLCAD deficiency. Mutations in the human VLCAD gene are heterogeneous. Although mortality at presentation is high, both the metabolic disorder and cardiomyopathy are reversible.

Background —The Bypass Angioplasty Revascularization Investigation (BARI) included 4039 patients with multivessel coronary artery disease; 1829 consented to randomization, and 2010 did not but were followed up in a registry. Thus, we can evaluate the outcome of physician-guided versus random assignment of percutaneous transluminal coronary angioplasty (PTCA) versus coronary artery bypass graft surgery (CABG). Methods and Results —We compared the baseline features and outcomes for PTCA and CABG in the overall registry and its predesignated subgroups. We assessed the impact of treatment by choice versus random assignment by comparing the results in the registry with those of the randomized trial. Statistical adjustments for differences in baseline characteristics were made. Within the registry, nearly twice as many patients were selected for PTCA (1189) as CABG (625); mortality at 7 years was similar for PTCA (13.9%) and CABG (14.2%) ( P =0.66) before and after adjustment for baseline differences between patients selected for PTCA versus CABG (adjusted RR, 1.02; P =0.86). In contrast to the randomized trial, the 7-year mortality rate of treated diabetics in the registry was equally high (26%) with PTCA or CABG. Seven-year mortality was higher for patients undergoing PTCA in the randomized trial than in the registry (19.1% versus 13.9%, P <0.01) but not for those undergoing CABG (15.6% versus 14.2%, P =0.57). The adjusted relative mortality risk for PTCA in the randomized versus registry population was 1.17 ( P =0.16). Conclusions —BARI physicians were able to select PTCA rather than CABG for 65% of registry patients who underwent revascularization without compromising long-term survival either in the overall population or in treated diabetics.

Background —The impact of percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass grafting (CABG) on long-term mortality rates in the presence of various demographic, clinical, and angiographic factors is uncertain in the population of patients suitable for both procedures. Methods and Results —In the Bypass Angioplasty Revascularization Investigation (BARI) randomized trial and registry, 3610 patients who were eligible to receive PTCA and CABG were revascularized between 1989 and 1992. Multivariate Cox models were used to identify factors associated with 5-year mortality and cardiac mortality, with particular attention to factors that interact with treatment. Diabetic patients receiving insulin had higher mortality and cardiac mortality rates with PTCA compared with CABG (relative risk [RR] 1.78 and 2.63, respectively, P <0.001), and patients with ST elevation had higher cardiac mortality rates with CABG than with PTCA (RR 4.08, P <0.001). Factors most strongly associated with high overall mortality rates were insulin-treated diabetes, congestive heart failure, kidney failure, and older age. Black race was also associated with higher mortality rates (RR 1.49, P =0.019). Conclusions —A set of variables was identified that could be used to help select a revascularization procedure and to evaluate risk of long-term mortality in the population of patients considering revascularization.

Background —Numerous studies have shown that women undergoing coronary revascularization procedures do so at a higher risk for an adverse outcome compared with men. However, the impact of advances in technology and improvements in techniques on in-hospital and long-term outcome after revascularization in women is unclear. Methods and Results —We evaluated 1829 patients with symptomatic multivessel coronary disease randomized to CABG or PTCA in the Bypass Angioplasty Revascularization Investigation (BARI), of whom 27% were women. As expected, women were older (64.0 versus 60.5 years), with more congestive heart failure (14% versus 7%), hypertension (68% versus 42%), treated diabetes mellitus (31% versus 15%), and unstable angina (67% versus 61%) than men but had similar preservation of left ventricular function and extent of multivessel disease. Women assigned to surgery received the same number of total grafts but fewer internal mammary artery grafts (72% versus 85%, P <0.01), and those assigned to angioplasty had more intended lesions (76% versus 71%, P <0.01) successfully dilated than men. At an average of 5.4 years’ follow-up, crude mortality rates were similar in women (12.8%) and men (12.0%). The Cox regression model adjusting for baseline differences revealed that women had a significantly lower risk of death (relative risk, 0.60; 95% CI, 0.43 to 0.84; P =0.003) but not a significantly lower risk of death plus myocardial infarction (relative risk, 0.84; 95% CI, 0.66 to 1.07; P =0.16) than men. Conclusions —Although the unadjusted mortality rate suggests that women and men undergoing CABG and PTCA have a similar 5-year mortality, women have higher risk profiles; consequently, contrary to previous reports, female sex is an independent predictor of improved 5-year survival after we control for multiple risk factors.