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Experimental Studies |
and
Peter J. Tarcha, PhD
1 From the Department of Radiology, University Hospitals of Cleveland and Case Western Reserve University, 11100 Euclid Ave, Cleveland, OH 44106-5056 (J.R.H., A.A.E., N.T.S.); Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Mass (Y.W.); and Department of Advanced Drug Delivery, Abbott Laboratories, North Chicago, Ill (P.J.T.). Received November 18, 2004; revision requested January 6, 2005; revision received February 11; accepted March 15. Address correspondence to A.A.E. (e-mail: Exner{at}uhrad.com).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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MATERIALS AND METHODS: All animal studies were performed in compliance with the Case Western Reserve University Institutional Animal Care and Use Committee guidelines. Three studies were performed to investigate (a) in vitro dissolution of 5-FU–laden polymer implants in saline and bovine serum, (b) tissue distribution of 5-FU and its metabolite, 5-fluorouridine (5-FUrd), in the ablated liver tissue of rats (n = 4), and (c) efficacy of combined approach (n = 4) compared with that of ablation alone (n = 6) for VX2 liver tumor model in rabbits. Characterization of 5-FU release in vitro and distribution of 5-FU in rat liver tissue were analyzed by using high performance liquid chromatography; in vivo efficacy was assessed by using computed tomography and pathologic examination.
RESULTS: Results of the in vitro dissolution study showed that a 75% release of 5-FU occurred in 2 days when exposed to bovine serum and in 9 days when exposed to phosphate-buffered saline. In the ablated rat liver, the 5-FU level was higher at the center and lower at the periphery of the tissue both at 24 hours (41.0 mg per kilogram tissue vs 15.0 mg per kilogram tissue, respectively) and at 48 hours (8.0 mg per kilogram tissue vs 2.0 mg per kilogram tissue, respectively). The 5-FUrd concentration was twofold higher peripherally than centrally and was higher at 48 hours than at 24 hours. In rabbits, local delivery of 5-FU immediately after RF ablation provided a significant (P < .05) reduction in tumor size compared with ablation alone (1.80 cm3 ± 0.28 [standard error] vs 3.53 cm3 ± 0.52, respectively; P = .034) and a more than 20-fold reduction in tumor size compared with the control (1.80 cm3 ± 0.28 vs 41.95 cm3 ± 11.58, respectively; P = .018).
CONCLUSION: Combined treatment by using 5-FU polymer implants and RF ablation shows uniform sustained release of 5-FU for 48 hours at least 8 mm from the edge of the ablation zone and appears to be successful at controlling the growth of an experimental tumor in rabbits appreciably better than does ablation alone.
© RSNA, 2005
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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With the goal of reducing recurrences, we have developed a treatment therapy that combines RF ablation with local release of 5-fluorouracil (5-FU) from interstitially implanted biodegradable polymer rods. Thus, the purpose of our study was to evaluate the use of 5-FU–laden polymer implants as an adjunct to RF ablation for tumor treatment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Chemicals and Polymers
The chemical agents 5-FU and 5-fluorouridine (5-FUrd), both of which were purchased from a commercial manufacturer (Aldrich Chemical, Milwaukee, Wis), and perchloric acid (Mallinckrodt, Hazelwood, Mo) were obtained. Water was purified with a 0.2-µm filter (Milli-Q; Millipore, Billerica, Mass) before use. The biodegradable polymer that was used for the formulation and extrusion of the 5-FU–laden polymer implants was a 1:5 equivalent ratio copolymer of 1,3 bis(p-carboxyphenoxy propane) with sebacic acid. This copolymer had an apparent weight average and number average molecular weight of 108000 and 24000, respectively, as determined by comparing gel permeation chromatography results with polystyrene standards. This polymer was prepared by the Specialty Products Division of Abbott Laboratories and was stored at –80°C.
Equipment
Drug formulation and extrusion work were performed in an isolator chamber designed to provide minimal human exposure to oncolytic drugs. Drug blending into the polymer melt and extrusion of the formulated controlled release material was performed by using a bench scale (5 g mixing chamber) twin-screw compounder (Micro-Compounder; DACA Instruments, Goleta, Calif). The compounder was fitted with a cylindric exit orifice and was coupled to a variable speed conveyor belt.
Ablation in rat livers was accomplished by using a 50-W RF generator (model RFG-3C; Radionics, Burlington, Mass) and a cooled 1-cm exposed tip, 19-gauge needle electrode (model 01803; Radionics). Ablation in rabbits was performed with a 200-W RF generator (model CTRF; Radionics) that was also equipped with a 1-cm exposed tip, 19-gauge needle electrode (model PED[10][2]K; Radionics).
Formulation and Extrusion of 5-FU Implants
The drug 5-FU was ground into a fine powder with a mortar and pestle. After the polymer was added to the compounder, which was set to a temperature of 80°C and a mixing speed of 140 rpm, the 5-FU powder was added to create a mixture that contained 15%–20% 5-FU by weight. After all of the drug had been added, the compounder was run for 2 minutes to ensure complete mixing. The mixture was then extruded onto a moving conveyer belt that could be set to produce strands ranging from 0.042 to 0.025 inches (0.107 to 0.064 cm) in diameter depending on the conveyer belt speed. After the strands had solidified, they were cut into 4–5-inch (10–13-cm) segments and stored at –80°C until used.
High Performance Liquid Chromatography and Preparation of Samples
A reverse-phase column (C 18; Regis Chemical, Morton Grove, Ill) was used for high performance liquid chromatographic analysis. The mobile phase consisted of deionized water that was injected at a flow rate of 0.6 mL/min, with an injection volume of 10 µL. The ultraviolet detector was set at 266 nm. For calibration standards, 13 mg of 5-FU was dissolved in deionized water in a 50-mL volumetric flask to make a 2.000-mmol/L stock solution. Sequential dilutions of the stock solution gave standard solutions of 1.000, 0.200, 0.050, 0.010, and 0.001 mmol/L. The standard solutions of 5-FUrd were similarly prepared at concentrations of 2.000, 1.000, 0.200, 0.050, 0.010, and 0.001 mmol/L.
In Vitro Drug Release
The 5-FU–laden polymer implants were weighed and placed in sterile 50-mL conical tubes (Falcon; Becton Dickinson, Franklin Lakes, NJ) that were filled with 20 mL of either 0.1 mol/L phosphate-buffered saline (pH 7.4) or fetal bovine serum. The tubes were placed on a shaker (Gyromax 929; Amerex Instruments, Lafayette, Calif) that was adjusted to 37°C and 120 rpm. Periodically, 0.1 mL of dissolution medium was transferred into a vial and diluted 20 times with deionized water. Aliquots of 0.1 mL of fresh phosphate-buffered saline or fetal bovine serum were added to the conical tubes each time to maintain a constant volume. The phosphate-buffered saline samples that had been diluted 20 times were then filtered through a 0.2-µm syringe filter and analyzed with high performance liquid chromatography.
Animals
Four male Sprague-Dawley rats (Harlan-Sprague-Dawley, Indianapolis, Ind) that weighted between 350 and 450 g and 15 male New Zealand white rabbits (Covance, Princeton, NJ) that weighted approximately 3 kg were obtained. All animals were cared for in compliance with the regulations of Case Western Reserve University and the principles of Laboratory Animal Care published by the National Institutes of Health. All animal procedures were performed by the authors and followed the approved protocol of the Institutional Animal Care and Use Committee at Case Western Reserve University.
RF Ablation and Polymer Implantation in Rats
Four rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (Henry Schein, Melville, NY) (50 mg per kilogram body weight). The abdomen was cleaned, and 0.1 mL of bupivacaine (Marcaine; Sanofi-Aventis, Bridgewater, NJ) was injected subcutaneously at the incision site. The liver was then exposed through an incision in the abdomen, and RF ablation was performed (N.T.S.) for 2 minutes at the center of a lobe of suitable size. The liver tissue was ablated with a 19-gauge needle electrode at 90°C, which resulted in an ablation diameter of 1.5 cm and an ablation volume of approximately 1.8 cm3. In two animals, 5-FU–laden polymer implants that weighed between 1.4 and 1.6 mg were implanted immediately after ablation; implantation was performed by suturing the implants into the cavities generated by the ablation probe (N.T.S.). Two additional rats received 5-FU–laden implants without ablation. In these animals, a needle track was generated in the lobe of each liver by using an 18-gauge needle (Becton Dickinson) to aid the insertion of the implant. After implantation, the abdominal cavities were closed, and the animals were allowed to recover.
In Vivo Rat Samples
At 24 hours after implantation, two rats (ie, one that had undergone RF ablation and one that had not undergone RF ablation) were sacrificed (N.T.S.), and the livers were harvested (N.T.S.); this process was repeated at 48 hours for the remaining two rats. The harvested livers were sectioned for analysis, as demonstrated in Figure 1 (Y.W.). Individual liver samples were transferred into a 1.5-mL microtube and were homogenized with a pellet pestle tissue grinder (Kontes Glass, Vineland, NJ) for 3 minutes. Next, 1.0 mL of deionized water was added to each homogenized sample, and the tubes were shaken at 240 rpm for 12 hours. Three drops of 10% HClO4 were added to each tube, and the tubes were shaken for an additional 1 hour. The microtubes were then centrifuged, and the supernatant was filtered through a 0.2-µm syringe filter. The filtered solutions were subsequently used for high performance liquid chromatographic analysis (Y.W.) of the drug concentration, and the tissue concentrations of 5-FU and 5-FUrd were determined.
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Rabbit Liver VX2 Tumor Model
A total of 15 male New Zealand white rabbits were anesthetized by using a combination of xylazine (AnaSed; Lloyd Laboratories, Shenandoah, Iowa) (5.0 mg per kilogram body weight), ketamine (Ketaset; Fort Dodge Animal Health, Overland Park, Kan) (50.0 mg per kilogram body weight), acepromazine (Henry Schein) (2.0 mg per kilogram body weight), and atropine (Henry Schein) (0.2 mg per kilogram body weight), all of which were administered as an intramuscular injection.
Before tumor implantation, the abdomen was shaved and cleaned with povidone iodine solution (Betadine; Purdue Frederick, Norwalk, Conn), and 0.5 mL of a 10% bupivacaine solution was administered subcutaneously at the incision site. Next, a small midline incision was made, and the medial lobe of the liver was exposed and exteriorized (N.T.S., A.A.E.). The capsule of the medial lobe was punctured with a scalpel blade, and a small piece of VX2 tumor (2 mm3) was inserted into the liver parenchyma. A small piece of retroperitoneal fat was then removed and sutured on top of the implantation site. The abdominal muscles and skin were closed with an absorbable suture. During the recovery period, all animals received two intramuscular injections of buprenophrine (Buprenex; manufacturer, city, state/country) (0.5 mg per kilogram body weight)—once soon after surgery and once 24 hours later for pain management. If the animals were later perceived to be in pain, additional buprenophrine was given as needed. The rabbits also received a subcutaneous injection of 25 mL of 0.9% saline during recovery and 24 hours after surgery.
RF Ablation of VX2 Liver Tumor in Rabbits
Fourteen days after tumor implantation, the rabbits were anesthetized again according to the protocol described earlier. The medial lobe of the liver was exteriorized and examined for tumor growth (A.A.E., N.T.S.). Examined tumors measured approximately 0.5–1.0 cm in diameter and extended into the liver parenchyma. For the RF ablation procedure, a grounding pad was placed beneath the exteriorized medial lobe of the liver. The tumor was then pierced with the electrode, and the tissue was ablated at 90°C (standard deviation, 3) for 5 minutes (N.T.S.). Three experimental groups were studied. In the first group (n = 5), the tumor was not ablated at 14 days; this group served as the untreated control group. In the second group (n = 6), the tumor was ablated at 14 days, and the animals were allowed to recover as previously described. In the third group (n = 4), the tumor was ablated at 14 days, and 5-FU–laden polymer implants were inserted. For this group, three implants, each measuring approximately 7.0 mm long and 0.5 mm in diameter, were placed together into the hole created by the needle electrode. Retroperitoneal fat was then sutured on top of this area to retain the implants. In one rabbit, the polymer implant was inadvertently damaged during implantation; this rabbit was thus taken from the 5-FU group and placed into the ablation-only group, which resulted in the uneven distribution within the groups. All experiments were ended 28 days after tumor implantation.
Data Collection and Analysis
To determine the efficacy of therapy, we monitored the general health and weight of each rabbit throughout the study, and computed tomographic (CT) scans were obtained immediately before the endpoint (day 28) by using a CT scanner (MX-8000 IDT; Philips, Bothell, Wash) at the University Hospitals of Cleveland. Scanning parameters included helical acquisition, a 3-mm section thickness, 120 kVp, 270 mA, and a 180-mm field of view. During the first scanning procedure, no contrast agent was administered. At the endpoint, scanning was performed after a 3-mL intravenous injection of iohexol (Omnipaque; Nycomed Amersham, Princeton, NJ) was administered. Ablation zone volumes were calculated by measuring the area of the apparent hypovascularized regions on each transverse section and by integrating these measurements with regard to section thickness (A.A.E. with consultation of J.R.H., who had more than 20 years of experience in CT of the body).
To estimate the actual volume of the tumor at the endpoint, we used a digital camera (DC-5000; Kodak, Rochester, NY) to acquire images of the ablated tumors. At the experimental endpoint, the rabbits were euthanized, and their livers were resected. The resected livers were then sectioned with a scalpel through the perceived center of the ablated lesion parallel to the electrode track. Photographs were taken to display the cross section of the lesion. The average volume of the ablation zone was determined from these images by measuring the height and width of the total perceived ablation zone and by calculating the mean of three measurements for each parameter. An average cylindric volume was then calculated on the basis of these measurements, and the results were compared with CT data.
Statistical Analysis
Statistical analysis was performed with two commercially available software programs (InStat, GraphPad, San Diego, Calif, and Excel, Microsoft, Redmond, Wash). Mean tumor volumes ± standard errors were calculated for each treatment group. A two-tailed, unpaired Student t test was used to determine if significant differences (P 
.05) existed between rabbits treated with the combined treatment and those treated with ablation alone. A second, unpaired t test was conducted to determine if a significant difference existed between untreated and treated animals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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After comparing the efficacy of liquid agents with that of RF ablation, authors have reported greater effectiveness of RF ablation (14–16,17). A consortium of experienced and recognized individuals reported favorable outcomes regarding RF treatments but reemphasized the issue of local tumor recurrence (18). Tumor lesions in 35 (40%) of 88 patients demonstrated local tumor recurrence at follow-up imaging. These authors emphasized the numerous critical factors that affected recurrence, such as large lesion size, irregular lesion contour, increased vascularity, and less than optimal image visualization and guidance (14).
The relationship between lesion size and recurrence has been discussed by several authors, including Goldberg et al (12) and Solbiati et al (3). Solbiati et al (3) reported a series of 172 lesions treated with RF ablation. In their study, recurrence rates varied remarkably depending on the size of the lesion, with recurrence rates of 16% in lesions measuring less than 3 cm and 56% in lesions measuring more than 3 cm. From the radiologic and pathologic correlations that were observed in their small series of patients, Goldberg et al (12) concluded that, because of the irregular anatomic contours of large lesions, matching the probe placement within the lesions to optimize the ablation treatment is difficult. Such a mismatch commonly leaves a rim of viable tumor tissue at the periphery of the lesion site. In a study of patients who underwent preoperative RF ablation and subsequent tumor resection, Scudamore et al (19) confirmed similar findings in the periphery of large lesions.
Most authors report on the use of imaging guidance to direct probe placement for ablation treatments and to achieve tumor-free margins between the target lesion and normal liver. Despite using careful guidance control, Goldberg et al (12) noted that, in their series of 22 cases, there were six incompletely treated lesions and four instances of residual tumor in a thin peripheral rim. Lewin et al (20) advocated the use of magnetic resonance (MR) imaging, which because of its superior tissue resolution, can be used to monitor tissue changes relative to heat coagulation; recurrence rates for their study, however, have not yet been reported. Hansler et al (21) also used MR imaging guidance and reported recurrence rates with MR imaging that were similar to those reported by other authors who used different modalities despite efforts to accurately visualize tissue destruction. Although the current recommendation of virtually all authors is to include a 5–10-mm margin of treatment around the lesions, findings to date have shown similar local recurrence rates regardless of the guidance method used.
To address these shortcomings, authors have continued to develop and refine RF technique to reduce tumor recurrence. Goldberg et al (22) proposed the use of a local saline injection in combination with RF ablation to increase the effective size of the ablation treatment. Clinical experience with the saline method and MR imaging guidance in human trials was reported by Kettenbach et al (23). The use of saline and MR imaging, however, did not improve the overall treatment success and/or recurrence rates. In their data, Kettenbach et al (23) noted that tumors measuring 3 cm or less in diameter were eight times more likely to be completely ablated (P = .01). Residual tumor was observed in 14 (58%) of 24 tumors that were evaluated 6–8 months after RF ablation. In their patient series, Hansler et al (21) reported on the use of saline injection and RF ablation for hepatoma treatment and found a local recurrence rate of 25%. Even with this saline approach, adjacent blood vessels presented a problem as heat sinks. Kettenbach et al (23) and Komorizono et al (24), who used saline injection as an adjunct to RF ablation, reported local recurrences in 35% of patients.
To improve the outcome of this less invasive approach, numerous authors are devising combined strategies to increase the effectiveness of RF treatments. The most promising of these treatments involves the concurrent delivery of a chemotherapeutic or other agent either by injection into the local site or by systemic administration. Goldberg et al (25) published efforts to combine the local injection of doxorubicin liquid into tumors before and after RF coagulation. In their short-term nonsurvival animal experiment, they observed that the size of tumor necrosis was definitely enhanced by the injection of the liquid doxorubicin in combination with RF ablation, as compared with the size of tumor necrosis achieved with RF ablation alone. A maximum effect of the local chemotherapeutic agent was observed when the agent was introduced within 30 minutes before or after ablation. The definition of this time factor is consistent with our combined method in which the polymer was inserted immediately after RF ablation and within the time frame described by Goldberg et al (25). While the combined approach of Goldberg et al was similar to our technique, it differs greatly in two aspects. In their study, Goldberg et al (25) injected doxorubicin in liquid form, and the animals were sacrificed after a short time period. The experiment that we conducted, however, used solid polymers and was a survival experiment that spanned 28 days.
The difference in the delivery method can be appreciated by noting the results of other studies, which have shown that the injection of liquid chemotherapeutic agents has several shortcomings (26–30). These studies on animals and humans, which were conducted without the use of RF ablation or solid polymers, were performed to evaluate the effectiveness of injecting chemotherapeutic agents as liquids or gels directly into tumors. Results showed a notable local tumor effect, but the distribution was unpredictable and the clearance of the agents over time was more rapid for a period of hours (29–31). Findings from the current study suggest that our approach provides considerable benefit with respect to uniform distribution, high local concentration, and prolonged effects.
Our method, which combines RF ablation and the use of local chemical-laden polymer implants, has shown a number of advantages in earlier studies by our group (32,33). When RF is applied to produce local heat ablation, it destroys the majority of cells locally, with some survival of cells at the margins of the treated tumor or around vessels. The physical placement of the 5-FU–laden polymer implant into the postablation site is accurate because the site retains its structural integrity during the insertion before dissolution of the polymer and dispersion of the active agent occurs. The chemotherapeutic agent diffuses uniformly from the site of insertion throughout the ablation zone and extends to the peripheral margins while maintaining elevated homogeneous agent distribution (32–34). The rate of diffusion is dependent on the amount of free 5-FU and bound 5-FUrd molecules (34), with the 5-FU molecules diffusing passively and the 5-FUrd molecules diffusing more slowly because they are partially bound to residual DNA strands. The diffusivity of the bound and unbound molecules has been defined mathematically by Qian et al (34).
The second factor affecting diffusion and distribution is the site of metabolic conversion of 5-FU to 5-FUrd. Because viable cells must convert the inactive form of 5-FU to the active form, the highest concentration of the active form will be at the edge of the ablation zone in close proximity to viable cells. At the ablation site, which is more remote from the conversion site, the concentration of 5-FUrd will be less. Diffusion of the active form would move in all directions—that is, both toward and away from the ablation site—so that small a concentration of 5-FUrd is to be expected at the center of the ablation site.
Further evaluation of this technique consisted of the examination of drug release characteristics of 5-FU from the polymer both in vitro and in vivo and a comparison of the success of VX2 tumor treatment by using RF ablation alone versus RF ablation and 5-FU implantation combined. Findings from the current study show that concentrations of the active metabolite or 5-FU and 5-FUrd were high at the peripheral margin, which is typically the location of tumor recurrences that are observed in the clinical series (11,12,20). This is presumably because of the disrupted circulation system of the ablated volume combined with the disrupted enzymatic activities in the ablation site, which result from tissue heating. Such physiologic findings indicate that the combination of RF ablation and chemotherapy could yield a therapeutic regimen that is far more effective than the simple summation of the two individual therapies.
Last and most important, findings showed that the use of combined treatment in the VX2 tumor was statistically superior to standard RF treatment alone and to control conditions. An incidental observation in our study was that the animals treated with the combined approach showed improved systemic effects, with an increase in weight compared with that of the RF ablation group.
To our knowledge, this study is the first to combine the use of polymers and RF ablation in the treatment of tumors; another group, however, reported a similar approach with local irradiation. Berrada et al (35) studied the benefit of combining 5-FU–laden polymers with external and interstitial irradiation in a mouse fibrosarcoma model. They found that, although the placement of interstitial seeds or 5-FU–laden polymers was effective in delaying tumor growth, the most effective method was a local treatment that combined the placement of polymers with radioactive seeds.
Despite the interesting results that have been achieved in this study regarding the combined treatment of experimental tumors, our findings should be interpreted with caution because of several limitations of this work, all of which will be addressed in future studies. First, the transplantable VX2 tumor and the abridged RF ablation treatment may not fully represent the physiologic characteristics and treatment of a spontaneously occurring tumor and therefore should not be used as such. Second, the small sample size may not be a representative sampling of the population, but the convincing results led us to use the fewest possible animals in the study. Last, the study design, including the measurement of the ablation zone dimensions at the endpoint only, provided a limited data set that did not enable us to examine the time-dependent development process. Nonetheless, we are hopeful that these data will provide a basis for further development of promising adjuvant or combination therapies to be used with RF ablation for improved treatment intervention.
Practical application: Several features of the combined treatment show promise for future clinical application. Foremost, the dissolution and release of the active agent from the 5-FU–laden polymer implants at the ablated site is predictable and homogeneous and provides a therapeutically effective concentration within the immediate area of coagulation. Second, the coagulated area retains the active agent at higher concentrations than are retained in the noncoagulated tissue, and the active metabolite persists at a higher concentration at the interface between the coagulated tissue and the contiguous liver, which is a common site of residual tumor recurrence. Third, when compared with the control conditions and RF ablation alone, combined therapy allowed considerable suppression of tumor growth over the 14-day treatment period. These results indicate that this treatment, which combines RF ablation and local delivery of a chemotherapeutic agent, is promising. Finally, the results of this study not only confirm the benefit of using drug-laden polymer implants with a separate treatment but also offer future potential for treatments that combine three techniques. Because the effects of radiation and chemotherapy are unrelated, it should be possible to combine these techniques with local RF ablation; it is logical to assume that additional benefit will be gained without an attendant increase in complications.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: 5-FU = 5-fluorouracil • 5-FUrd = 5-fluorouridine • RF = radiofrequency
See Materials and Methods for pertinent disclosures.
Author contributions: Guarantors of integrity of entire study, J.R.H., N.T.S.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; literature research, all authors; experimental studies, all authors; statistical analysis, J.R.H., A.A.E., N.T.S.; and manuscript editing, all authors
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References |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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2-cm) breast cancer treated with US-guided radiofrequency ablation: feasibility study. Radiology 2004;231(1):215–224.This article has been cited by other articles:
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  B. D. Weinberg, T. M. Krupka, J. R. Haaga, and A. A. Exner Combination of Sensitizing Pretreatment and Radiofrequency Tumor Ablation: Evaluation in Rat Model Radiology, March 1, 2008; 246(3): 796 - 803. [Abstract] [Full Text] [PDF]
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) are shown. Dimensions are in millimeters.
Deceased. 