Rebekah R. White

Notch Promotes Radioresistance of Glioma Stem Cells

Radiotherapy represents the most effective nonsurgical treatments for gliomas. However, gliomas are highly radioresistant and recurrence is nearly universal. Results from our laboratory and other groups suggest that cancer stem cells contribute to radioresistance in gliomas and breast cancers. The Notch pathway is critically implicated in stem cell fate determination and cancer. In this study, we show that inhibition of Notch pathway with γ‐secretase inhibitors (GSIs) renders the glioma stem cells more sensitive to radiation at clinically relevant doses. GSIs enhance radiation‐induced cell death and impair clonogenic survival of glioma stem cells but not non‐stem glioma cells. Expression of the constitutively active intracellular domains of Notch1 or Notch2 protect glioma stem cells against radiation. Notch inhibition with GSIs does not alter the DNA damage response of glioma stem cells after radiation but rather reduces Akt activity and Mcl‐1 levels. Finally, knockdown of Notch1 or Notch2 sensitizes glioma stem cells to radiation and impairs xenograft tumor formation. Taken together, our results suggest a critical role of Notch signaling to regulate radioresistance of glioma stem cells. Inhibition of Notch signaling holds promise to improve the efficiency of current radiotherapy in glioma treatment. STEM CELLS 2010;28:17–28

Lower frequency of peritoneal carcinomatosis in patients with pancreatic cancer diagnosed by EUS-guided FNA vs. percutaneous FNA.

BACKGROUND Studies have suggested an increased risk of peritoneal seeding in patients with pancreatic cancer diagnosed by percutaneous FNA. EUS-FNA is an alternate method of diagnosis. The aim of this study was to compare the frequency of peritoneal carcinomatosis as a treatment failure pattern in patients with pancreatic cancer diagnosed by EUS-FNA vs. percutaneous FNA. METHODS Retrospective review of patients with non-metastatic pancreatic cancer identified 46 patients in whom the diagnosis was made by EUS-FNA and 43 with the diagnosis established by percutaneous FNA. All had neoadjuvant chemoradiation. Patients underwent restaging CT after completion of therapy, followed by attempted surgical resection if there was no evidence of disease progression. RESULTS There were no significant differences in tumor characteristics between the two study groups. In the EUS-FNA group, one patient had developed peritoneal carcinomatosis compared with 7 in the percutaneous FNA group (2.2% vs. 16.3%; p<0.025). No patient with a potentially resectable tumor in the EUS-FNA group had developed peritoneal carcinomatosis. CONCLUSIONS Peritoneal carcinomatosis may occur more frequently in patients who undergo percutaneous FNA compared with those who have EUS-FNA for the diagnosis of pancreatic cancer. A concern for peritoneal seeding of pancreatic cancer via percutaneous FNA is warranted. EUS-guided FNA is recommended as the method of choice for diagnosis in patients with potentially resectable pancreatic cancer.

Antidote-mediated control of an anticoagulant aptamer in vivo

Patient safety and treatment outcome could be improved if physicians could rapidly control the activity of therapeutic agents in their patients. Antidote control is the safest way to regulate drug activity, because unlike rapidly clearing drugs, control of the drug activity is independent of underlying patient physiology and co-morbidities. Until recently, however, there was no general method to discover antidote-controlled drugs. Here we demonstrate that the activity and side effects of a specific class of drugs, called aptamers, can be controlled by matched antidotes in vivo. The drug, an anticoagulant aptamer, systemically induces anticoagulation in pigs and inhibits thrombosis in murine models. The antidote rapidly reverses anticoagulation engendered by the drug, and prevents drug-induced bleeding in surgically challenged animals. These results demonstrate that rationally designed drug-antidote pairs can be generated to provide control over drug activities in animals.

Developing aptamers into therapeutics

The idea of using nucleic acid molecules as therapeutic agents was conceived in the 1970s with the development of antisense strategies. Antisense compounds are single-stranded nucleic acids that, in principle, disrupt the synthesis of a targeted protein by hybridizing in a sequence-dependent manner to the mRNAs that encode it. The mechanism of inhibition by nucleic acid aptamers is fundamentally different. Aptamers are single-stranded nucleic acids that directly inhibit a protein’s function by folding into a specific three-dimensional structure that dictates high-affinity binding to the targeted protein. Through iterative in vitro selection techniques, aptamers can be generated that bind essentially any protein (or small molecule) target. A high-affinity, specific inhibitor can theoretically be made to order, provided that a small quantity of pure target is available. Because they inhibit the activity of existing proteins directly, aptamers are more similar to monoclonal antibody or small molecule drugs than to antisense compounds, and this property greatly increases the number of clinical indications that are potentially treatable by nucleic acid–based compounds. Aptamers have been generated against a wide variety of targets, a complete discussion of which is beyond the scope of this article (for review, see ref. 1). As with any molecular therapeutic approach, the inhibitor is only as effective as the target is important. Through their ability to specifically inhibit a molecule of interest, aptamers have already proven useful as reagents for target validation in a variety of disease models. The next step, therapeutic utility, will depend on the efficacy of these novel compounds in humans. In this Perspective, we will focus on the development and status of aptamers as therapeutic molecules.

Neoadjuvant chemoradiation for localized adenocarcinoma of the pancreas.

AbstractBackground: The use of neoadjuvant preoperative chemoradiotherapy CRT for pancreatic cancer has been advocated for its potential ability to optimize patient selection for surgical resection and to downstage locally advanced tumors. This article reports our experience with neoadjuvant CRT for localized pancreatic cancer. Methods: Since 1995, 111 patients with radiographically localized, pathologically confirmed pancreatic adenocarcinoma have received neoadjuvant external beam radiation therapy EBRT; median, 4500 cGy with 5-flourouracil–based chemotherapy. Tumors were defined as potentially resectable PR, n = 53 in the absence of arterial involvement and venous occlusion and locally advanced LA, n = 58 with arterial involvement or venous occlusion by CT. Results: Five patients 4.5% were not restaged due to death n = 3 or intolerance of therapy n = 2. Twenty-one patients 19% manifested distant metastatic disease on restaging CT. Twenty-eight patients with initially PR tumors 53% and 11 patients with initially LA tumors 19% were resected after CRT. Histologic examination revealed significant fibrosis in all resected specimens and two complete responses. Surgical margins were negative in 72%, and lymph nodes were negative in 70% of resected patients. Median survival in resected patients has not been reached at a median follow-up of 16 months. Conclusions: Neoadjuvant CRT provided an opportunity for patients with occult metastatic disease to avoid the morbidity of resection and resulted in tumor downstaging in a minority of patients with LA tumors. Survival after neoadjuvant CRT and resection appears to be at least comparable to survival after resection and adjuvant postoperative CRT.

Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2

Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factor-mediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent.

Predictors of a true complete response among disappearing liver metastases from colorectal cancer after chemotherapy

During chemotherapy, some colorectal liver metastases (LMs) disappear on serial imaging. This disappearance may represent a complete response (CR) or a reduction in the sensitivity of imaging during chemotherapy. The objective of the current study was to determine the fate of disappearing LMs (DLMs) and the factors that predict a true CR.

Randomized trial of vinorelbine compared with fluorouracil plus leucovorin in patients with stage IV non-small-cell lung cancer

PURPOSE This prospective randomized trial was performed to compare the effectiveness of intravenous vinorelbine tartrate with intravenous fluorouracil and leucovorin (5-FU/LV) on the primary end points of survival, quality of life (QOL), and relief of cancer-related symptoms in patients with advanced non-small-cell lung cancer (NSCLC). Secondary end points included tumor response rates and time to treatment failure. In addition, the safety of both treatment regimens was evaluated in this multicenter study. PATIENTS AND METHODS Two hundred sixteen patients with stage IV NSCLC were enrolled onto this study from 18 centers. Vinorelbine was administered at a dose of 30 mg/m2/wk. 5-FU/LV was administered at a dose of 425 mg/m2 and 20 mg/m2, respectively, for 5 consecutive days every 4 weeks. Patients with progressive disease or toxicity were removed from study while responding and stable patients were continued on therapy. RESULTS The median survival time of patients who received vinorelbine was 30 weeks, with 25% of patients alive at 1 year, compared with a median survival time of 22 weeks and 16% of patients alive at 1 year for those treated with 5-FU/LV (P = .03, log-rank test). This improvement in survival was associated with a higher objective response rate (12% v 3%) and time to treatment failure (10 weeks v 8 weeks) for vinorelbine versus 5-FU/LV. The dose-limiting toxicity of vinorelbine was granulocytopenia, with 54% of patients experiencing grade 3/4 granulocytopenia. Nonhematologic toxicity of vinorelbine was generally grade 1 or 2. The most common grade 3 toxicities were related to injection-site reactions. CONCLUSION This trial confirms the efficacy of vinorelbine in patients with advanced NSCLC. The clinical activity and relatively favorable toxicity profile of this agent make it a reasonable and useful treatment option in the management of patients with this disease.

Crystal structure of an RNA aptamer bound to thrombin.

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.

Long-term survival in 2,505 patients with melanoma with regional lymph node metastasis

ObjectiveTo examine the long-term outcomes of patients with melanoma metastatic to regional lymph nodes. Summary Background DataRegional lymph node metastasis is a major determinant of outcome for patients with melanoma, and the presence of regional lymph node metastasis has been commonly used as an indication for systemic, often intensive, adjuvant therapy. However, the risk of recurrence varies greatly within this heterogeneous group of patients. MethodsDatabase review identified 2,505 patients, referred to the Duke University Melanoma Clinic between 1970 and 1998, with histologic confirmation of regional lymph node metastasis before clinical evidence of distant metastasis and with documentation of full lymph node dissection. Recurrence and survival after lymph node dissection were analyzed. ResultsEstimated overall survival rates at 5, 10, 15, and 20 years were 43%, 35%, 28%, and 23%, respectively. This population included 792 actual 5-year survivors, 350 10-year survivors, and 137 15-year survivors. The number of positive lymph nodes was the most powerful predictor of both overall survival and recurrence-free survival; 5-year overall survival rates ranged from 53% for one positive node to 25% for greater than four nodes. Primary tumor ulceration and thickness were also powerful predictors of both overall and recurrence-free survival in multivariate analyses. The most common site of first recurrence after lymph node dissection was distant (44% of all patients). ConclusionsPatients with regional lymph node metastasis can enjoy significant long-term survival after lymph node dissection. Therefore, aggressive surgical therapy of regional lymph node metastases is warranted, and each individual’s risk of recurrence should be weighed against the potential risks of adjuvant therapy.