Preclinical study of treatment response in HCT-116 cells and xenografts with (1) H-decoupled (31) P MRS.

The topoisomerase I inhibitor, irinotecan, and its active metabolite SN-38 have been shown to induce G(2) /M cell cycle arrest without significant cell death in human colon carcinoma cells (HCT-116). Subsequent treatment of these G(2) /M-arrested cells with the cyclin-dependent kinase inhibitor, flavopiridol, induced these cells to undergo apoptosis. The goal of this study was to develop a noninvasive metabolic biomarker for early tumor response and target inhibition of irinotecan followed by flavopiridol treatment in a longitudinal study. A total of eleven mice bearing HCT-116 xenografts were separated into two cohorts where one cohort was administered saline and the other treated with a sequential course of irinotecan followed by flavopiridol. Each mouse xenograft was longitudinally monitored with proton ((1) H)-decoupled phosphorus ((31) P) magnetic resonance spectroscopy (MRS) before and after treatment. A statistically significant decrease in phosphocholine (p = 0.0004) and inorganic phosphate (p = 0.0103) levels were observed in HCT-116 xenografts following treatment, which were evidenced within twenty-four hours of treatment completion. Also, a significant growth delay was found in treated xenografts. To discern the underlying mechanism for the treatment response of the xenografts, in vitro HCT-116 cell cultures were investigated with enzymatic assays, cell cycle analysis, and apoptotic assays. Flavopiridol had a direct effect on choline kinase as measured by a 67% reduction in the phosphorylation of choline to phosphocholine. Cells treated with SN-38 alone underwent 83 ± 5% G(2) /M cell cycle arrest compared to untreated cells. In cells, flavopiridol alone induced 5 ± 1% apoptosis while the sequential treatment (SN-38 then flavopiridol) resulted in 39 ± 10% apoptosis. In vivo (1) H-decoupled (31) P MRS indirectly measures choline kinase activity. The decrease in phosphocholine may be a potential indicator of early tumor response to the sequential treatment of irinotecan followed by flavopiridol in noninvasive and/or longitudinal studies.

The use of gadolinium for arterial interventions.

BACKGROUND: Gadolinium (Gd) has been traditionally used as a non-nephrotoxic alternative to iodinated contrast for digital subtraction angiography (DSA) in patients with chronic renal insufficiency. However, its use has been questioned on the basis of reports of nephrotoxicity and its recent association with nephrogenic systemic fibrosis (NSF), a potentially lethal complication. Recently available data are conflicting with respect to the true safety profile of intra-arterial Gd. The purpose of this study was to examine the risk of contrast nephropathy and NSF after Gd exposure in a large population of azotemic patients undergoing DSA. METHODS: A comprehensive database encompassing data on all patients who underwent DSA between June 2003 and December 2007 at the New York Presbyterian Hospital was retrospectively reviewed. Patients receiving Gd either alone or in combination with iodinated contrast during DSA were identified and further analyzed. Acute renal failure (ARF) was defined as an elevation in serum creatinine (Cr) by >0.5 mg/dL within 48 hours of exposure. Clinical follow-up was conducted through chart reviewing as well as telephonic interviews with patients and their primary care physicians. RESULTS: A total of 153 patients underwent 179 exposures to Gd either alone (33%) or in combination (67%) with iodinated contrast. Mean follow-up duration was 27.1 months. The mean Cr level was 1.94 ± 0.78 mg/dL and 1.96 ± 1.1 mg/dL before and after DSA, respectively. There were 20 (11.2%) instances of ARF. The mean Cr level before DSA was higher in patients who developed ARF versus those in the non-ARF group (2.7 ± 1.1 mg/dL vs. 1.9 ± 0.7 mg/dL, p = 0.004). In the ARF group, 12 patients had a return to baseline renal function, four experienced irreversible renal deterioration, and four needed dialysis (4.5% incidence of irreversible renal failure). There were 19 deaths at the time of this study (12.4%). The highest risk for the development of ARF after Gd exposure occurred in patients with Cr levels of >3.0 mg/dL before DSA and in those receiving >0.4 mmol/kg of Gd. For patients who received iodinated contrast in combination with Gd, there was a trend toward a higher risk for developing ARF as compared with those receiving only Gd. Finally, there were no instances of NSF identified in any of the patients who received intra-arterial Gd. CONCLUSIONS: Although Gd has the potential to cause kidney injury similar to iodinated contrast, the risk of irreversible renal failure and the requirement for dialysis is low. Life- or limb-threatening interventions should not be avoided in this patient cohort because of preexisting elevations in Cr. These data should help guide the use of Gd in patients with chronic renal insufficiency.

Endovascular aneurysm repair in nonagenarians is safe and effective.

OBJECTIVES: Advanced age is a significant risk factor that has traditionally steered patients away from open aneurysm repair and toward expectant management. Today, however, the reduced morbidity and mortality of aortic stent grafting has created a new opportunity for aneurysm repair in patients previously considered too high a risk for open surgery. Here we report our experience with endovascular aneurysm repair (EVAR) in nonagenarians. METHODS: Retrospective chart review identified all patients>90-years-old undergoing EVAR over a 9-year period at our institution. Collected data included preoperative comorbidities, perioperative complications, endoleaks, reinterventions, and long-term survival. RESULTS: 24 patients underwent EVAR. The mean age was 91.5 years (range 90-94) among 15 (63%) males and 9 (37%) females. Mean abdominal aortic aneurysm diameter was 6.3±1.1 cm. Eight patients (33%) were symptomatic (pain or tenderness). There were no ruptures. Fourteen patients (58%) had general anesthesia while 10 (42%) had local or regional anesthesia. Mean postoperative length of stay was 3.2±2.4 days (2.8±1.9 days for asymptomatic vs 4.1±3.2 days for symptomatic, P=.29). There was one perioperative mortality (4.2%). There were two local groin seromas (8.3%) and six systemic complications (25%). One patient required reintervention for endoleak (4.2%). There were no aneurysm related deaths beyond the 30-day postoperative period. Mean survival beyond 30 days was 29.7±18.0 months for patients expiring during follow-up. Cumulative estimated 12, 24, and 36-month survival rates were 83%, 64%, and 50%, respectively. Linear regression analysis demonstrated an inverse relationship between the number of preoperative comorbidities and postoperative survival in our cohort (R2=0.701), with significantly decreased survival noted for patients presenting with >5 comorbidities. Those still alive in follow-up have a mean survival of 36.1±16.0 months. CONCLUSION: This is the largest reported EVAR series in nonagenarians. Despite their advanced age, these patients benefit from EVAR with low morbidity, low mortality, and mean survival exceeding 2.4 years. Survival appears best in those patients with ≤5 comorbidities. With or without symptoms, patients over the age of 90 should be considered for EVAR.

Carotid artery stenting is safe and associated with comparable outcomes in men and women.

OBJECTIVE: Historically, large randomized controlled studies looking at carotid endarterectomy (CEA) have indicated an increased perioperative risk for women when gender subgroup analysis was performed. However, the outcomes of carotid stenting in women as compared to men have not been adequately investigated. We sought to compare the safety and efficacy of carotid angioplasty and stenting (CAS) when performed in women as compared to men. METHODS: Procedures, complications, demographics, co-morbidities, and follow-up data from carotid stenting procedures performed in a bi-campus division were entered into a prospective database and then retrospectively supplemented with stored angiographic image data and reviewed. Arterial anatomic characteristics evaluated using angiographic images were: common carotid/internal carotid lesion length ratio, common carotid/internal carotid diameter, index lesion length, common carotid/internal carotid artery tortuosity, and lesion and aortic arch calcification. Outcomes compared included groin complications, postoperative pressor requirements, length of stay, restenosis, stroke, myocardial infarction (MI), and death. RESULTS: Between 2003 and 2008, 228 patients underwent 238 procedures. Cerebral protection devices and self-expanding stents were placed in all patients. A total of 97 percutaneous interventions performed in 93 women were compared with 141 interventions in 135 men. Mean age in women was 71.8 +/- 9.2 years, in men was 72.2 +/- 9.1 years (P > .99); 44.3% of women and 34.7% of men had symptomatic disease (P = .14). Preoperative demographics and co-morbidities did not differ significantly between genders, with the exception of hypertension (83.0% of males vs 96.7% of females, P = .001), and history of coronary artery bypass grafting (31.8% of males vs 16.1% of females, P = .01). There were no significant differences seen in anatomic arterial characteristics, though there was a trend towards women having larger internal carotid to common carotid diameter ratios (0.65 vs 0.62) and more plaques isolated to the common carotid segment (9.5% vs 6.9%). There were no significant differences seen in overall 30-day peri-procedural stroke rate (2.1% in women and 4.2% in men, P = .48), death rate (0 % vs 0.7%, P > .99), or cardiac events (3.2% vs 0.7%, P = .3). The combined 30-day stroke, death, and MI rate was 5.7% for males compared to 5.4% for females (P > .99). There were no differences observed in the long-term survival, stroke-free survival, or restenosis between genders. CONCLUSION: Despite previous concerns over adverse outcomes in women undergoing carotid endarterectomy, from our data, carotid stenting appears to be a safe modality in women with equivalent outcomes when compared to men.

Carotid angioplasty and stenting, success relies on appropriate patient selection.

OBJECTIVE: Carotid angioplasty and stenting (CAS) is a percutaneous alternative to carotid endarterectomy (CEA) for treating patients with carotid artery stenosis. This study sought to evaluate whether patients at increased perioperative risk for CEA may be treated with CAS while maintaining equivalent outcomes. METHODS: This study was a nonblinded, retrospective analysis of data obtained from September 2002 to present in the CAS group and from January 1997 to present in the CEA group. Two hundred thirty-one CAS and 647 CEA procedures were performed. Patients were selected for CAS based on criteria that placed them at increased risk for standard CEA surgery. Except for percentage women treated, baseline demographics did not differ between patients treated with CAS and CEA: mean age (72.0 years [range 46-94] vs 70.5 years [range 42-92], P = NS), mean follow-up (12.8 +/- 11.8 months vs 8.7 +/- 10.0 months, P = NS) and percentage women treated (41.4% vs 32.3%, P = .03). Cerebral protection devices were used in 228/231 patients treated with CAS, and each patient underwent an NIH Stroke Scale assessment 24 hours postoperatively and at 30 days follow-up by an independent observer. RESULTS: Preoperative neurologic symptoms did not differ between patients treated with CAS and CEA: amaurosis fugax (6.06% vs 6.96%, P = NS), transient ischemic attacks (13.4% vs 13.9%, P = NS), strokes (19.9% vs 14.1%, P = NS) and total symptoms (27.7% vs 30.5%, P = NS). Due to the selection of patient groups based on predefined clinical characteristics, factors associated with an increased risk of complications from standard CEA surgery were generally more prevalent in patients treated with CAS: neck irradiation (6.06% vs 1.24%, P < .001), neck dissection for cancer therapy (7.8% vs 1.5%, P < .001), prior ipsilateral CEA (15.2% vs 3.4%, P

Sorafenib inhibits growth and mitogen-activated protein kinase signaling in malignant peripheral nerve sheath cells.

Malignant peripheral nerve sheath tumors (MPNST) are soft-tissue tumors with a very poor prognosis and largely resistant to chemotherapy. MPNSTs are characterized by activation of the Ras pathway by loss of tumor suppressor neurofibromatosis type 1. In view of this, MPNST may be susceptible to inhibition of the activated Ras/Raf/mitogen-activated protein kinase pathway by the B-Raf inhibitor sorafenib. MPNST (MPNST and ST8814) and dedifferentiated liposarcoma (LS141 and DDLS) human tumor cell lines were characterized for Ras activation and B-Raf expression. Tumor cells were treated with sorafenib and examined for growth inhibition, inhibition of phospho-MEK, phospho-ERK, cell cycle arrest, and changes in cyclin D1 and pRb expression. MPNSTs were sensitive to sorafenib at nanomolar concentrations. This appeared to be due to inhibition of phospho-MEK, phospho-ERK, suppression of cyclin D1, and hypophosphorylation of pRb at the CDK4-specific sites, resulting in a G(1) cell cycle arrest. These effects were not seen in the liposarcoma cells, which either did not express B-Raf or showed decreased Ras activation. Small interfering RNA-mediated depletion of B-Raf in MPNSTs also induced a G(1) cell cycle arrest in these cells, with a marked inhibition of cyclin D1 expression and Rb phosphorylation, whereas depletion of C-Raf did not affect either. With growth inhibition at the low nanomolar range, sorafenib, by inhibiting the mitogen-activated protein kinase pathway, may prove to be a novel therapy for patients with MPNST.

Flavopiridol targets c-KIT transcription and induces apoptosis in gastrointestinal stromal tumor cells.

Gastrointestinal stromal tumors (GIST) are characterized by activating mutations in the c-KIT gene which confers ligand-independent activation of the KIT receptor. Imatinib mesylate has been shown to effectively block constitutively active KIT and delay tumor growth. However, resistance to imatinib mesylate is emerging as a major clinical problem and novel therapies are needed. We report that treatment of GIST cells with the transcriptional inhibitor flavopiridol, initially down-regulates the antiapoptotic proteins bcl-2, mcl-1, and X-linked inhibitor of apoptosis protein which occurs as early as 4 hours after exposure. This is followed at 24 hours by the transcriptional suppression of KIT resulting in poly(ADP-ribose) polymerase cleavage and apoptosis. To separate the apoptotic effect of KIT suppression relative to the down-regulation of antiapoptotic proteins, we used small interfering RNA-directed knockdown of KIT. Results show that focused suppression of KIT alone is sufficient to induce apoptosis in GIST cells, but not to the same extent as flavopiridol. In contrast, imatinib mesylate, which inhibits KIT kinase activity but does not suppress total KIT expression, fails to cause apoptosis. We also show that flavopiridol suppresses KIT mRNA expression through positive transcriptional elongation factor inhibition and decreases KIT promoter activity. This causes a global decrease in the level of functionally mature KIT at the cell surface, resulting in a decrease in autophosphorylation at tyrosine residues 703 and 721, which characterizes activated KIT. Our results indicate that targeting KIT expression and these antiapoptotic proteins with flavopiridol represents a novel means to disrupt GIST cell dependence on KIT signaling and collectively renders these cells sensitive to apoptosis.

High-resolution MAS NMR spectroscopy detection of the spin magnetization exchange by cross-relaxation and chemical exchange in intact cell lines and human tissue specimens.

High-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy detects resolved signals from membrane phospholipids and proteins in intact cell and tissue samples. MAS has the additional advantage of quenching spin-diffusion through a mutual "flip-flop" of neighbor spins by time-independent dipolar coupling as long as the dipolar coupling is "inhomogeneous." Under MAS, significant magnetization transfer (MT) was observed between water and each proton site in membrane phospholipid and between water and the NMR-observable protein proton signals. The MT rates between water and membrane phospholipids are lower than those between water and protein proton signals. The interaction of water to other small molecules is selective with the observation of MT from water to creatine, lactate, taurine, and glycine, but not to triglyceride, phosphocholine, choline, or myo-inositol. HR-MAS NMR allows the detection of a complete MT network between water and each proton group of creatine. Two creatine pools (one motion-restricted and one motion-free) were identified in skeletal muscle.

Water suppression without signal loss in HR-MAS 1H NMR of cells and tissues.

In cell and tissue samples, water is normally three orders of magnitude more abundant than other metabolites. Thus, water suppression is required in the acquisition of NMR spectra to overcome the dynamic range problem and to recover metabolites that overlap with the broad baseline of the strong water resonance. However, the heterogeneous cellular environment often complicates water suppression and the strong coupling of water to membrane lipids interferes with the NMR detection of membrane associated lipid components. The widely used water suppression techniques including presaturation and double pulsed field gradient selective echo result in more than a 70% reduction in membrane associated lipid components in proton spectra of cells and tissues compared to proton spectra acquired in the absence of water suppression. A water suppression technique based on the combination of selective excitation pulses and pulsed field gradients is proposed to use in the acquisition of high resolution MAS NMR spectra of tissue specimens and cell samples. This pulse sequence methodology enables efficient water suppression for intact cells and tissue samples and eliminates signal loss from cellular metabolites.