Robotic Stereotactic Body Radiation Therapy for the Adjuvant Treatment of Early-Stage Breast Cancer: Outcomes of a Large Single-Institution Study.

Purpose: Advancements in breast radiation therapy offer innumerable benefits to patients and the health care system. Despite promising outcomes, clinicians remain hesitant about long-term side effects and disease control with accelerated partial breast radiation therapy (APBI). Herein, we review the long-term outcomes of patients with early-stage breast cancer treated with adjuvant stereotactic partial breast irradiation (SAPBI). Methods and Materials: This retrospective study examined outcomes of patients who received diagnoses of early-stage breast cancer treated with adjuvant robotic SAPBI. All patients were eligible for standard ABPI and underwent lumpectomy, followed by fiducial placement in preparation for SAPBI. Using fiducial and respiratory tracking to maintain a precise dose distribution throughout the course of treatment, patients received 30 Gy in 5 fractions on consecutive days. Follow-up occurred at routine intervals to evaluate disease control, toxicity, and cosmesis. Toxicity and cosmesis were characterized using the Common Terminology Criteria for Adverse Events version 5.0 and Harvard Cosmesis Scale, respectively. Results: Patients (N = 50) were a median age of 68.5 years at the time of treatment. The median tumor size was 7.2 mm, 60% had an invasive cell type, and 90% were estrogen receptor positive, progesterone receptor positive, or both. Patients (n = 49) were followed for a median of 4.68 years for disease control and 1.25 years for cosmesis and toxicity. One patient experienced local recurrence, 1 patient experienced grade 3+ late toxicity, and 44 patients demonstrated excellent cosmesis. Conclusions: To our knowledge, this is the largest retrospective analysis with the longest follow-up time for disease control among patients with early breast cancer treated with robotic SAPBI. With follow-up time for cosmesis and toxicity comparable to that of previous studies, results of the present cohort advance our understanding of the excellent disease control, excellent cosmesis, and limited toxicity that can be achieved by treating select patients with early-stage breast cancer with robotic SAPBI.

Effects of mechanical stretch on collagen and cross-linking in engineered blood vessels.

It has been shown that mechanical stimulation affects the physical properties of multiple types of engineered tissues. However, the optimum regimen for applying cyclic radial stretch to engineered arteries is not well understood. To this end, the effect of mechanical stretch on the development of engineered blood vessels was analyzed in constructs grown from porcine vascular smooth muscle cells. Cyclic radial distension was applied during vessel culture at three rates: 0 beats per minute (bpm), 90 bpm, and 165 bpm. At the end of the 7-week culture period, harvested vessels were analyzed with respect to physical characteristics. Importantly, mechanical stretch at 165 bpm resulted in a significant increase in rupture strength in engineered constructs over nonstretched controls. Stress-strain data and maximal elastic moduli from vessels grown at the three stretch rates indicate enhanced physical properties with increasing pulse rate. In order to investigate the role of collagen cross-linking in the improved mechanical characteristics, collagen cross-link density was quantified by HPLC. Vessels grown with mechanical stretch had somewhat more collagen and higher burst pressures than nonpulsed control vessels. Pulsation did not increase collagen cross-link density. Thus, increased wall thickness and somewhat elevated collagen concentrations, but not collagen cross-link density, appeared to be responsible for increased burst strength.

There is no correlation between erectile dysfunction and dose to penile bulb and neurovascular bundles following real-time low-dose-rate prostate brachytherapy.

PURPOSE: We evaluated the relationship between the onset of erectile dysfunction and dose to the penile bulb and neurovascular bundles (NVBs) after real-time ultrasound-guided prostate brachytherapy. METHODS AND MATERIALS: One hundred forty-seven patients who underwent prostate brachytherapy met the following eligibility criteria: (1) treatment with 125I brachytherapy to a prescribed dose of 160 Gy with or without hormones without supplemental external beam radiation therapy, (2) identification as potent before the time of implantation based on a score of 2 or higher on the physician-assigned Mount Sinai Erectile Function Score and a score of 16 or higher on the abbreviated International Index of Erectile Function patient assessment, and (3) minimum follow-up of 12 months. Median follow-up was 25.7 months (range, 12-47 months). RESULTS: The 3-year actuarial rate of impotence was 23% (34 of 147 patients). An additional 43% of potent patients (49 of 113 patients) were using a potency aid at last follow-up. The penile bulb volume receiving 100% of the prescription dose (V(100)) ranged from 0-0.05 cc (median, 0 cc), with a dose to the hottest 5% (D(5)) range of 12.5-97.9 Gy (median, 40.8 Gy). There was no correlation between penile bulb D(5) or V(100) and postimplantation impotency on actuarial analysis. For the combined right and left NVB structures, V(100) range was 0.3-5.1 cc (median, 1.8 cc), and V(150) range was 0-1.5 cc (median, 0.31 cc). There was no association between NVB V(100) or V(150) and postimplantation impotency on actuarial analysis. CONCLUSION: Penile bulb doses are low after real-time ultrasound-guided prostate brachytherapy. We found no correlation between dose to either the penile bulb or NVBs and the development of postimplantation impotency.

Feasibility of vitrification as a storage method for tissue-engineered blood vessels.

It is well established that, in multicellular systems, conventional cryopreservation results in damaging ice formation, both in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation, we performed a feasibility study using vitrification (ice-free cryopreservation) to cryopreserve tissue-engineered blood vessels. Fresh, frozen, and vitrified tissue-engineered blood vessels were compared using histological methods, cellular viability, and mechanical properties. Cryosubstitution methods were used to determine the location of ice in conventionally cryopreserved engineered vessels. Ice formation was negligible (0.0 +/- 0.0% of vessel area) in the vitrified specimens, and extensive (68.3 +/- 4.5% of vessel area) in the extracellular matrix of frozen specimens. The metabolic assay and TUNEL staining results indicated that vitrified tissue had similar viability to fresh controls. The contractility results for vitrified samples were >82.7% of fresh controls and, in marked contrast, the results for frozen samples were only 10.7% of fresh controls (p < 0.001). Passive mechanical testing revealed enhanced tissue strength after both freezing and vitrification. Vitrification is a feasible storage method for tissue-engineered blood vessel constructs, and their successful storage brings these constructs one step closer to clinical utility.

Age effects on vascular smooth muscle: an engineered tissue approach.

Tissue engineering of blood vessels offers a potential new therapy for patients with vascular occlusive disease. In addition, tissue engineering technologies offer the opportunity to study the biology of vascular cells in a biomimetic, three-dimensional environment. A model for vascular tissue engineering was used to study the effects of vascular cell age on extracellular matrix (ECM) deposition, cellular mitosis, and protein synthesis under controlled conditions in vitro. Blood vessels were grown using a three-dimensional polyglycolic acid (PGA) mesh that was seeded with either infant or adult porcine vascular smooth muscle cells. Mechanical forces in the form of pulsatile radial distension were applied for the duration of the 7-week growth period. Overall, infant cells exhibited higher levels of cellular proliferation, ECM deposition, and remodeling activity than cells derived from adult animals. In addition, vessels cultured from infant cells had enhanced physical properties compared to vessels cultured from adult cells. The differentiation state of the smooth muscle cells in the infant and adult constructs was unchanged from the native state. However, the levels of immature pro-collagen, although undetectable in the vessels grown from adult cells, were similar in native vessels and in vessels grown with infant cells. These studies have important implications for the study of aging and vascular disease and remodeling, as well as for the field of tissue engineering.

Blood vessels engineered from human cells.

Tissue engineering has made considerable progress in the past decade, but advances have stopped short of clinical application for most tissues. We postulated that an obstacle in engineering human tissues is the limited replicative capacity of adult somatic cells. To test this hypothesis, the effectiveness of telomerase expression to extend cellular lifespan was assessed in a model of human vascular tissue engineering. Telomerase expression in vascular cells isolated from elderly patients enabled the successful culture of engineered autologous blood vessels. Engineered vessels may one day provide a source of bypass conduit for patients with atherosclerotic disease.

Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts.

OBJECTIVES: Fifty percent of human aortocoronary saphenous vein grafts are occluded after 10 years. Intimal hyperplasia is an initial step in graft occlusion and consists of vascular smooth muscle cell proliferation. Phosphatidylinositol 3-kinase and its downstream regulator, the inositol 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), are important regulators of vascular smooth muscle cell proliferation, migration, and cell death. This study tests whether overexpression of PTEN in aortocoronary saphenous vein grafts can reduce intimal hyperplasia. METHODS: Adult dogs underwent aortocoronary bypass grafting to the left anterior descending artery by using the autologous saphenous vein. Saphenous vein grafts were treated with phosphate-buffered saline (n = 9), empty adenovirus (n = 8), or adenovirus encoding for PTEN (n = 8). Arteriography at 30 and 90 days assessed saphenous vein graft patency. A subset received saphenous vein grafts treated with a marker transgene (beta-galactosidase, n = 3), empty adenovirus (n = 4), or adenovirus encoding for PTEN (n = 4) and were killed on postoperative day 3 to confirm expression. Vascular smooth muscle cells were isolated from canine saphenous vein infected with adenovirus encoding for PTEN, and immunoblotting and proliferation assays were performed. RESULTS: Saphenous vein graft transgene expression was confirmed by means of immunohistochemistry, immunoblotting, and polymerase chain reaction. Arteriograms revealed all saphenous vein grafts to be patent. Saphenous vein grafts treated with adenovirus encoding for PTEN demonstrated reduced intimal area compared with those treated with empty adenovirus and phosphate-buffered saline (1.39 +/- 0.11 vs 2.35 +/- 0.3 and 2.57 +/- 0.4 mm 2 , P < .05), and the intima/media ratio was lower in saphenous vein grafts treated with adenovirus encoding for PTEN (0.50 +/- 0.05 vs 1.43 +/- 0.18 and 1.11 +/- 0.14, P < .005). PTEN overexpression in vascular smooth muscle cells inhibited platelet-derived growth factor-induced phosphorylation of Akt, a downstream effector of phosphatidylinositol 3-kinase. PTEN-treated vascular smooth muscle cells demonstrated decreased basal, platelet-derived growth factor-stimulated, and serum-stimulated proliferation. CONCLUSION: This study demonstrates that PTEN overexpression in aortocoronary saphenous vein grafts reduces intimal hyperplasia. The mechanism of this antiproliferative effect in vascular smooth muscle cells is likely due to inhibition of phosphatidylinositol 3-kinase signaling through Akt, with resultant decreases in vascular smooth muscle cell growth and survival. Therefore modulation of the phosphatidylinositol 3-kinase pathway through PTEN overexpression might represent a novel therapy to prevent saphenous vein graft intimal hyperplasia after coronary artery bypass grafting.

Treatment of patients with cardiac pacemakers and implantable cardioverter-defibrillators during radiotherapy.

PURPOSE: To define the practical clinical guidelines that can be implemented by busy radiation oncology departments to minimize the risk of harm to patients with implanted cardiac pacemaker (ICP) and implantable cardioverter-defibrillator (ICD) devices during radiotherapy. METHODS AND MATERIALS: A literature review was conducted to identify the mechanism of potential damage to ICPs and ICDs from exposure to electromagnetic interference and/or ionizing radiation and to assess the published evidence of such device malfunction or failure. Recommendations for patient management were obtained from three major manufacturers. Eighty-seven radiation oncology facilities across the United States and Canada were contacted to determine current practice patterns; 75 centers responded. RESULTS: The published documentation of potential life-threatening malfunction of ICP and ICD devices exposed to electromagnetic interference and ionizing radiation is considerable. However, major discrepancies exist among manufacturer recommendations and wide variations are present among radiation oncology facilities regarding patient management precautions. CONCLUSION: Precautions are necessary to minimize the risk to patients with ICP and ICD devices during radiotherapy. Practical management guidelines are presented that can be readily adopted by any busy clinical radiation oncology practice.

Gene therapy in tissue-engineered blood vessels.

Cardiovascular disease is the leading cause of morbidity and mortality in Western society. More than 1 million arterial bypass procedures are performed annually in the United States, where either autologous veins or synthetic grafts are used to replace arteries in the coronary or peripheral circulation. Tissue engineering of blood vessels from autologous cells has the potential to produce biological grafts for use in bypass surgery. Ex vivo development of vascular grafts also provides an ideal target of site-specific gene therapy to optimize the physiology of the developing conduit, and for the possible delivery of other therapeutic genes to a vascular bed of interest. In this article, we demonstrate that by using a novel retroviral gene delivery system, a target gene of interest can be specifically delivered to the endothelial cells of a developing engineered vessel. Further, we demonstrate that this technique results in stable incorporation of the delivered gene into the target endothelial cells for more than 30 days. These data demonstrate the utility of the retroviral gene delivery approach for optimizing the biologic phenotype of engineered vessels. This also provides the framework for testing an array of genes that may improve the function of engineered blood vessels after surgical implantation.

Effects of polyglycolic acid on porcine smooth muscle cell growth and differentiation.

Polyglycolic acid (PGA) is commonly used as a scaffold for tissue engineering. Recent studies utilized PGA as a scaffold for vascular tissue engineering using bovine and porcine smooth muscle cells (SMCs). In engineered vessels, the SMCs displayed high rates of mitosis and dedifferentiation in areas where PGA fragments were present. We hypothesized that PGA breakdown products, sequestered within a SMC vessel at the conclusion of culture, led to increased proliferation and dedifferentiation of vascular SMCs. To test this hypothesis, the current study assessed possible means by which PGA breakdown products could lead to changes in SMC phenotype. SMCs grown in high concentrations of PGA breakdown products showed, by Western blotting, decreased expression of calponin, a marker for SMC differentiation. The same was true for SMCs grown in glycolic acid (GA), which also showed decreased expression of proliferating cell nuclear antigen (PCNA), a marker for SMC proliferation. In contrast, cells grown in varying amounts of NaCl or HCl showed little change in differentiation. We conclude that, independent of acidity or osmolality, plausible products of PGA degradation appear to induce dedifferentiation of porcine SMCs in vitro. Because of dedifferentiation and decreased mitosis, commercially available PGA may not represent an optimal scaffold for vascular tissue engineering.

Engineering porcine arteries: effects of scaffold modification.

Techniques have been developed to culture bovine or porcine vascular cells on polyglycolic acid (PGA) scaffolds to form engineered vessels. Previously, it was shown that smooth muscle cells (SMCs) that were in close proximity to PGA remnants after 8 weeks of culture had lower expression of SMC markers of differentiation and were more mitotic compared with SMCs that were distant from polymer residuals. Modifications of PGA were explored as a means to minimize residual polymer fragments after culture. To hasten degradation, polymer was treated with heat, NaOH, or gamma-irradiation. Differential scanning calorimetry, mass and tensile strength degradation, and inherent viscosity were used to assess polymer characteristics. When polymer was maintained in aqueous conditions, tensile strength of treated PGA degraded to zero within 3 weeks for each treatment. Engineered vessel constructs cultured on NaOH and gamma-treated polymer displayed smooth muscle alpha-actin throughout the vessel wall. Scaffold treatment impacted graft morphology, cellular differentiation, and mechanical integrity.

Effect of pulse rate on collagen deposition in the tissue-engineered blood vessel.

The effect of mechanical stimulation on the development of tissue-engineered blood vessels was examined. In particular, three different rates of radial distension were chosen to produce a nonpulsed environment (0 beats per minute [bpm]), an adult heart rate (90 bpm), and a fetal heart rate (165 bpm). Engineered vessels were cultured for an average of 7 weeks. Vessel walls were then analyzed for collagen content and distribution. In addition, extracellular matrix remodeling was assessed through measurement of active matrix metalloproteinase type 1 (MMP-1) and tissue inhibitor of metalloproteinases type 1 (TIMP-1) levels. Vessels grown at a distension rate of 165 bpm had significantly higher collagen levels than those grown under static conditions. MMP-1 and TIMP-1 levels were also higher under pulsed conditions as compared with nonpulsed conditions. For the 90- and 165-bpm conditions, collagen and MMP-1 levels were not significantly different. TIMP-1 levels were significantly elevated at 165 bpm, indicating an increased cellular response to mechanical stimulation. Mechanical forces and their transduction represent a means to enhance the physical properties of artificial blood vessels, possibly by affecting the rate of extracellular matrix deposition and remodeling.

Possible role for vascular cell proliferation in cerebral vasospasm after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: During vasospasm after subarachnoid hemorrhage (SAH), cerebral blood vessels show structural changes consistent with the actions of vascular mitogens. We measured platelet-derived vascular growth factors (PDGFs) in the cerebrospinal fluid (CSF) of patients after SAH and tested the effect of these factors on cerebral arteries in vivo and in vitro. METHODS: CSF was sampled from 14 patients after SAH, 6 patients not suffering SAH, and 8 normal controls. ELISA was performed for PDGF-AB, transforming growth factor-beta1, and vascular endothelial growth factor. A mouse model was used to compare cerebral vascular cell proliferation and PDGF staining in SAH compared with sham-operated controls. Normal human pial arteries were incubated for 7 days in vitro, 2 groups with human blood clot and 1 with and 1 without PDGF antibodies. RESULTS: PDGF-AB concentrations in CSF from SAH patients were significantly higher than those from non-SAH patients and normal controls, both during the first week after SAH and for all time points measured. Smooth muscle and fibroblast proliferation was observed after SAH in the mouse model, and this cellular replication was observed in conjunction with PDGF protein at the sites of thrombus. In human pial arteries, localized thrombus stimulated vessel wall proliferation, and proliferation was blocked by neutralizing antibodies directed against PDGFs. CONCLUSIONS: Vascular mitogens are increased in the CSF of patients after SAH. Proliferation of cells in the vascular wall is associated with perivascular thrombus. Cellular proliferation and subsequent vessel wall thickening may contribute to the syndrome of delayed cerebral vasospasm.

Exploring the role of alanine in the structure of the Lac repressor tetramerization domain, a ferritin-like Alacoil.

We are interested in the determinants that specify the structure of antiparallel coiled coils. Antiparallel coiled coils often contain alanine as an important interfacial packing residue; structures containing alanine at certain well-defined positions in the heptad-repeating unit are referred to as Alacoils. Two types have been identified, containing alanine at either the g position of the heptad repeating unit (defined as the d position in the Richardson nomenclature), referred to as a rop-like Alacoil, or the e position (a position in the Richardson nomenclature), referred to as a ferritin-like Alacoil. The Lac repressor tetramerization domain forms an antiparallel four-chain coiled coil, which falls into the second class of Alacoils based on recent crystal structures. The role of alanine in such structures has not yet been explored experimentally. We test the importance of alanine at the e positions on the oligomeric state and stability of the isolated coiled-coil domain of Lac repressor by testing the effect of mutations at this position. We find that mutation to leucine is tolerated and its moderately stabilizing effect is most likely a consequence of plasticity of this motif. The effects on stability of the mutations to either serine or glutamine can be largely accounted for by helix propensity differences between these residues and alanine. The ability of the helices to adjust to such mutations, while maintaining the basic fold of this coiled coil, was further tested by making the same changes at the more highly exposed g position. Leucine at the g positions also causes an increase in stability, presumably by subtle rearrangement of the helices to allow partial desolvation of this side-chain.

Tissue engineering of the lymphatic system.

The field of tissue engineering has seen tremendous expansion in the last decade. In the last several years, tissue-engineering strategies to treat diseases of skin, cartilage, bone, bladder, blood vessel, tendon, and other tissues have been described. However, tissue-engineering approaches to treat diseases of the lymphatic system are currently nonexistent. We propose that acellular tissues, either native or engineered, could be exploited as a platform for the study of lymphatic biology, and for lymphatic tissue engineering. While speculative, this type of experimental model system could prove powerful for dissecting molecular and cellular events surrounding tumor invasion of lymphatics, as well as lymphangiogenesis. Scaffolds seeded with genetically engineered lymphatic cells could also be implanted to repopulate lymphatic vasculature. In the future, the lymphatic system will surely be added to the list of tissues and organs that prove amenable to tissue-engineering therapies.