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DOI: 10.1148/radiol.2352031856
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(Radiology 2005;235:469-477.)
© RSNA, 2005

Experimental Studies

Combination Radiofrequency Ablation with Intratumoral Liposomal Doxorubicin: Effect on Drug Accumulation and Coagulation in Multiple Tissues and Tumor Types in Animals1

Muneeb Ahmed, MD, Zhengjun Liu, MD, Anatoly N. Lukyanov, PhD, Sabina Signoretti, MD, Clare Horkan, MBBCh, Wayne L. Monsky, MD, PhD, Vladimir P. Torchilin, PhD and S. Nahum Goldberg, MD

1 From the Department of Radiology, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA 02215 (M.A., Z.L., C.H., W.L.M., S.N.G.); Department of Medical Oncology, Dana Farber Cancer Institute–Harvard Medical School, Boston, Mass (A.N.L., V.P.T.); and Department of Pharmaceutical Sciences, Bouve College of Health Sciences, Northeastern University, Boston, Mass (S.S.). From the 2003 RSNA Annual Meeting. Received November 18, 2003; revision requested January 21, 2004; final revision received August 2; accepted August 17. Supported by a grant from the National Cancer Institute, National Institutes of Health, Bethesda, Md (RO1-CA87992-01A1), and Bracco Research, Milan, Italy. Address correspondence to S.N.G. (e-mail: sgoldber@caregroup.harvard.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To determine whether use of radiofrequency (RF) ablation combined with intravenously (IV) administered liposomal doxorubicin, as compared with use of RF ablation or doxorubicin alone, facilitates increased tissue coagulation and interstitial drug accumulation in animal models.

MATERIALS AND METHODS: The institutional animal care and use committee approved this study. In experiment 1, multiple canine sarcomas were implanted in seven mildly immunosuppressed dogs and grown to a mean diameter of 4.8 cm. Tumors were assigned to three treatment groups: internally cooled RF ablation (12 minutes, 2000-mA pulsed technique) followed by IV liposomal doxorubicin (10 mg per animal) (n = 6), RF ablation alone (n = 6), and liposomal doxorubicin alone (n = 4). In experiment 2, the livers and kidneys of 10 rabbits and the thigh muscles of 10 rats were randomly assigned to one of two treatment groups: conventional RF ablation (90°C ± 2, 5 minutes) followed by IV liposomal doxorubicin (5 mg per rabbit, 1 mg per rat) or RF ablation alone (n = 5, each). Coagulation diameter and interstitial doxorubicin concentration (tissues were homogenized in acid alcohol, with doxorubicin extracted for 24 hours at 5°C and quantified with fluorimetry) were measured 48 hours after treatment and compared. Multivariate analysis of variance and subsequent pairwise t tests ({alpha} = .05, two-tailed test) were performed.

RESULTS: Data are means ± standard errors of the mean. A larger diameter of tumor destruction was observed in canine sarcomas treated with RF ablation–liposomal doxorubicin (3.7 cm ± 0.6) compared with that in tumors treated with RF ablation (2.3 cm ± 0.1) or liposomal doxorubicin (0.0 cm ± 0.0) alone (P < .01). A new finding was a completely necrotic red zone (1.6 cm ± 0.7) surrounding the central RF ablation–induced white coagulation zone. Greater but nonuniform drug uptake was observed particularly in this red zone (77.0 ng/g ± 18.2) compared with uptake in the central zone (15.1 ng/g ± 3.2), peripheral area of untreated tumor (38.9 ng/g ± 8.0), and tumors treated with liposomal doxorubicin alone (43.9 ng/g ± 6.7 for all regions) (P < .01 for all individual comparisons). In experiment 2, use of combined therapy led to increased coagulation in all tissues (liver: 17.6 mm ± 3.1, P = .03; kidney: 11.0 mm ± 3.1, P = .03; muscle: 13.1 mm ± 1.3, P < .01) compared with use of RF ablation alone (liver, 13.4 mm ± 1.5; kidney, 7.9 mm ± 0.7; muscle, 8.6 mm ± 0.5). Combined therapy, as compared with liposomal doxorubicin therapy alone, was also associated with increased doxorubicin accumulation in liver, kidney, and muscle (1.56 µg/g ± 0.34, 4.36 µg/g ± 1.78, and 3.63 µg/g ± 1.43, respectively, vs 1.00 µg/g ± 0.18, 1.23 µg/g ± 0.32, and 0.87 µg/g ± 0.53, respectively) (P ≤ .01 for all individual comparisons).

CONCLUSION: Use of RF ablation combined with liposomal doxorubicin facilitates increased tissue coagulation and interstitial doxorubicin accumulation in multiple tissues and tumor types and may be useful for treatment of large tumors and achieving an ablative margin within the untreated tissue surrounding RF ablation–treated tumors.

© RSNA, 2005


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Radiofrequency (RF) ablation has gained considerable attention as an image-guided, minimally invasive alternative to surgical resection for the treatment of focal malignancies of the liver, kidney, bone, and adrenal gland; reports of its utility at a wide range of tissue sites are continually increasing (1–6). However, enthusiasm for RF ablation has been somewhat tempered by reports of less than satisfactory long-term success rates in the treatment of larger (>3.5 cm) tumors. In a relatively recent study of RF ablation of hepatocellular carcinoma, local recurrence in 65% of lesions larger than 3.5 cm in diameter and in 75% of tumors larger than 5.0 cm was reported (7). For the percutaneous RF ablation of metastases originating from colorectal tumors, local recurrences of 35%–89% have been reported for lesions larger than 2.5 cm (8,9). The incidence of local residual tumor in the tissue surrounding the RF ablation zone in both large and small tumors suggests that foci of untreated tumor persist.

One potential strategy to overcome the current limitations of performing RF ablation alone is the addition of adjuvant intravenous liposomal chemotherapy (10–14). In several relatively recent studies, both increased intratumoral drug accumulation and increased local tumor coagulation were documented when liposomal doxorubicin was intravenously administered in conjunction with RF ablation (10–13). Goldberg et al (10) combined RF ablation with intravenous liposomal doxorubicin administration in a rat mammary adenocarcinoma model and observed a significant increase in coagulation compared with the degree of coagulation seen following RF ablation alone. Subsequently, D’Ippolito et al (12) observed reduced tumor growth in animals that received combined RF–liposomal doxorubicin treatment compared with those that received either RF ablation alone or liposomal doxorubicin alone. Additionally, Monsky et al (13) observed a fivefold increase in the intratumoral uptake of liposomal doxorubicin in tumors treated with RF ablation compared with those treated with the doxorubicin only. Preferential liposome accumulation occurred in a peripheral zone surrounding the ablated area, where continued residual tumor growth is the greatest for focal malignancies treated with conventional RF treatment paradigms (13).

Translation of this combined-treatment paradigm to clinical practice has also been reported: The results of one study (14) showed a 25%–30% volumetric increase in tumor coagulation and a reduction in residual patent intratumoral vessels with use of combined therapy. These preliminary results highlight the potential of combining RF ablation with adjuvant liposomal chemotherapeutic agents to achieve greater and more complete treatment.

Although these findings generate substantial interest in administering adjuvant chemotherapies in conjunction with RF ablation specifically and in other focal high-temperature hyperthermic therapies in general, much of the preliminary experimental work has been performed in single rat mammary adenocarcinoma models (10–13). Given that RF ablation is now being increasingly applied to a wider range of tumor and tissue types, further confirmation of these preliminary findings in more varied models, including different tissue sites, different tumor types, and more clinically relevant tumor sizes, is required. Therefore, the purpose of this study was to determine whether RF ablation combined with intravenous liposomal doxorubicin administration, as compared with RF ablation alone or doxorubicin therapy alone, results in increased tissue coagulation and interstitial drug accumulation in a large canine sarcoma model and in normal rabbit and rat tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Experimental Design
Approval from the institutional animal care and use committee of Beth Israel Deaconess Medical Center was obtained prior to the initiation of the experiments. A total of 42 ablations were performed in four animal models: subcutaneously implanted canine transmissible venereal sarcoma, normal rabbit liver tissue, normal rabbit kidney tissue, and normal rat thigh muscle tissue. This study was performed in two experimental phases:

Experiment 1: comparison of tumor coagulation and intratumoral doxorubicin accumulation in canine venereal sarcomas.—In experiment 1, a total of 16 treatments were performed in subcutaneous canine venereal sarcomas. The effect of combined RF ablation–liposomal doxorubicin therapy, as compared with the effect of RF ablation alone or intravenous liposomal doxorubicin therapy alone, on tumor coagulation and intratumoral doxorubicin accumulation was evaluated. RF ablation, intravenous liposomal doxorubicin administration, animal sacrifice, and gross pathologic analysis of coagulation were performed. Comparisons of interstitial doxorubicin accumulation were made in three treatment groups: six tumors treated with combined RF ablation–liposomal doxorubicin therapy, four tumors treated with intravenous liposomal doxorubicin only, and six tumors treated with RF ablation only. Four untreated tumors served as a control group. In this large animal tumor model, we performed quantification of liposomal doxorubicin in three zones of each tumor, where applicable: the central white zone of RF ablation–induced coagulation, the peripheral red zone of necrosis, and the distant area of untreated tumor.

Experiment 2: comparison of coagulative necrosis and intratumoral doxorubicin accumulation in three normal tissue models.—In experiment 2, a total of 30 RF ablations—10 in each model—were performed in normal rat thigh muscle, normal rabbit liver, and normal rabbit kidney tissues. In each model, RF ablation of the designated tissue followed by intravenous liposomal doxorubicin administration 30 minutes later was performed in five animals. In another five control animals in each model, only RF ablation was performed. RF ablation, intravenous liposomal doxorubicin administration, animal sacrifice, and gross pathologic analysis of coagulation were performed (as described in following text). Interstitial doxorubicin accumulation was compared in tissues treated with intravenous liposomal doxorubicin with and without RF ablation.

Animal Models
Experiment 1.—The experiment 1 procedures were performed by using a well-characterized and established canine transmissible venereal sarcoma cell line that is identical to the animal model used in previous works (15–17). Seven female Labrador dogs (retired breeders, 30–40 kg; Team Associates, Canterbury, Conn) were mildly immunosuppressed—with 25 mg of cyclosporin (Neoral; Novartis Pharmaceuticals, East Hanover, NJ) per kilogram of body weight administered twice daily—from 5 days before tumor transplantation (by M.A. and Z.L.) until the experiments ended. Tumor transplantation was performed as described in earlier RF ablation studies (16,18) using this particular subcutaneous tumor model.

In an attempt to maximize the usable tumor yield, each animal received four subcutaneous tumor implants in the back, for a total of 24 attempted tumor implantations. The tumors were monitored weekly (by M.A. and Z.L.) for 12–14 weeks until the desired 4–5-cm size was achieved. Although each animal had four tumors implanted, owing to variations in tumor size growth, only two tumors per animal were used. Treatments were randomized by dog, and when tumors were used in dogs receiving doxorubicin, treatments were randomized by tumor to receive either RF ablation or no RF ablation. For the ablation experiments, the animals were intubated and anesthesia was maintained with isoflurane (Forane; Baxter Healthcare, Deerfield, Ill). Cardiac and respiratory parameters and arterial blood gas levels were monitored throughout the procedures.

Experiment 2.—The experiment 2 procedures were performed in normal left thigh muscles in 10 Fischer 344 rats (Taconic, Germantown, NY) that weighed 150 g each and in 10 New Zealand white rabbits (Milbrook Breeding Company, Amherst, Mass) that weighed 3–4 kg. For all procedures performed in rats, anesthesia was induced by intraperitoneally injecting a mixture of ketamine (Ketaject, 50 mg/kg; Phoenix Pharmaceuticals, St Joseph, Mo) and xylazine (Xyla-Ject, 5 mg/kg; Phoenix Pharmaceuticals). When necessary, booster anesthetic injections of one-tenth the initial dose were administered intraperitoneally every 30–60 minutes. For all procedures performed in rabbits, the animals were anesthetized with a combination of 35 mg/kg of ketamine, 2.5 mg/kg of xylazine, and 0.75 mg/kg of acepromazine maleate (Phoenix Pharmaceuticals) administered intramuscularly. Continued anesthesia was maintained with 2% isoflurane (Isoflo; Abbott Laboratories, North Chicago, Ill) or a one-half dose booster of ketamine, as required. Analgesia was induced with 0.03 mg/kg of buprenorphine (Buprenex Injectable; Reckitt and Colman Pharmaceuticals, Richmond, Va) administered intramuscularly.

Ten animals (five animals in each treatment group) were randomly assigned to be treated with RF ablation with or without liposomal doxorubicin. Each rabbit underwent a single RF ablation in the liver and a single RF ablation in the left kidney, and each rat underwent a single RF ablation in the left thigh muscle.

RF Ablation Procedure
For the small-animal (rats and rabbits) experiments, monopolar RF energy was applied by using a 500-kHz RF generator (model CC-1; Radionics, Burlington, Mass). To complete the RF circuit, the animal was placed on a standardized metallic grounding pad (Radionics). Contact was ensured by shaving the animal’s back and liberally using electrolytic contact gel. For each RF ablation performed in the rat and rabbit models (by M.A., Z.L., and C.H.), the 1-cm tip of a 21-gauge electrically insulated, noncooled electrode was placed at the center of the designated tissue area by using ultrasonographic (US) guidance. To permit RF energy deposition, the distal 1-cm tip of this needle was not insulated. RF energy was applied for 5 minutes, with the generator output titrated to maintain a designated tip temperature (60°C ± 2 [standard deviation] in rats, 90°C ± 2 in rabbits). These parameters were chosen because they have been previously shown to facilitate ablation of a zone of tissue measuring 6–7 mm and thereby allowed us to measure the effect of combined therapy (19,20). A thermocouple at the tip of the RF electrode constantly measured the local ablation temperature and thereby enabled proper generator adjustment. Parameters of the RF ablation procedure, including tip temperature, tissue impedance, and applied current, were recorded at baseline (by M.A. and C.H.) and thereafter at 60-second intervals for the duration of the RF energy application for all experiments performed in each animal model.

For the canine model, a 1-cm-tip, internally cooled RF electrode (Radionics) was inserted into the subcutaneous tumor by using US guidance. To complete the electric circuit, a second 1-cm-tip, internally cooled RF electrode, serving as the electric ground, was placed in a second tumor of the same size. This approach was used to minimize the risk of grounding pad burns on the animal. This model also enabled an equal amount of energy to be deposited within both tumors so that we could simultaneously treat two tumors in the same animal equally. Each tumor was treated with a single application of RF energy. RF energy was applied for 12 minutes (by M.A., Z.L., and S.N.G.) at an initial generator output of 2000 mA (200 W). If impedance increases were observed, the current output was automatically reduced according to a previously designed pulsing algorithm that optimizes energy deposition and tissue coagulation (21).

Intravenous Liposomal Doxorubicin Administration
Specified doses of polyethylene glycol–stabilized, long-circulating liposomal doxorubicin (Doxil; ALZA Pharmaceuticals, Palo Alto, Calif) were intravenously administered (by M.A. and S.N.G.) by means of injection through intravenous catheters in the canine (10 mg per animal) and rabbit (5 mg per animal) models. In the rat model, liposomal doxorubicin was administered (by M.A. and S.N.G.) by means of direct femoral injection (1 mg per animal). Prior to the injection, the right inguinal crease was incised and the femoral vein was exposed by using blunt dissection. The agent was injected with direct visualization; then pressure was applied to the vein for 1 minute to prevent excessive bleeding from the injection site. Incision margins were then sutured together by using 2–0 silk (Ethicon, Somerville, NJ). To avoid any artificial increase in observed intratumoral doxorubicin concentration as a result of the release of doxorubicin from the liposomes in the tumor during the application of focal hyperthermia, liposomal doxorubicin was injected 20–30 minutes after the RF ablation procedure (11,22,23).

Quantification of Doxorubicin in Tissue Samples
The fluorescent properties of doxorubicin were used to quantify this agent in tissue samples. Forty-eight hours after treatment, the animals were sacrificed (by M.A. and Z.L.) by means of pentobarbital (Nembutal, 0.2 mL/kg; Abbott Laboratories) overdose. The 48-hour time point was selected as a standardized end point time for all animals in this study because the results of prior studies have demonstrated that peak differences in interstitial doxorubicin accumulation can be identified 48–72 hours after administration (11). The specified tissues were harvested, weighed, and homogenized in acid alcohol (0.3N hydrochloric acid, 70% alcohol), and doxorubicin was extracted for 24 hours at 5°C (by M.A., A.N.L., and W.L.M.). The doxorubicin extracted from all tissue homogenate supernatant samples was quantified at fluorimetry by using an excitation wavelength of 470 nm and measuring the intensity of emission at 590 nm (24,25), and the concentrations were plotted on a standard curve of liposomal doxorubicin serially diluted in acid alcohol. Results of our earlier work (11) showed that the effectiveness of this technique is equivalent to that of silver nitrate extraction techniques for quantifying both bound and unbound forms of doxorubicin.

Quantification of Coagulative Tumor Necrosis and Histopathologic Examinations
Induced coagulative necrosis—that is, tumor destruction—was measured 48 hours after treatment according to the results of prior studies (10). The animals were sacrificed with pentobarbital overdose, and tumors were excised and sectioned (by M.A. and S.N.G.). The histopathologic examinations included, in addition to hematoxylin-eosin stain analyses, staining for mitochondrial enzyme activity, which was performed by incubating representative tissue sections for 30 minutes in 2% triphenyltetrazolium chloride at room temperature (S.S., 11 years experience). The latter test was used to identify irreversible nonspecific cellular injury (26) during the early stages of RF ablation–induced tissue necrosis (19,20), and its findings have previously been shown to correlate with histopathologic findings of RF ablation–induced tumor coagulation (10,19). Gross measurements of tumor destruction were performed in both triphenyltetrazolium chloride–stained tissue sections and unstained sections, and the extent of visible coagulation was measured with calipers. Coagulation diameter—that is, the longest measurement perpendicular to the inserted electrode (23)—was determined by consensus between the two observers who jointly measured and verified the findings (M.A. and S.N.G.). Previous study investigators have documented close correlation between the gross pathologic and histopathologic findings of RF ablation–induced coagulative necrosis (27).

Statistical Analyses
For all experiments, coagulation diameter and interstitial doxorubicin accumulation were measured, and the results were compared by using statistical analysis. All data are given as means ± standard errors of the mean. In experiment 1, we performed a multivariate mixed analysis of variance (ANOVA) of all three regions of drug uptake and performed univariate analysis only if the treatment effect revealed at multivariate analysis was significant. Herein, we have included the P values calculated at multivariate analysis in our results. For experiment 2, we performed an initial repeated-measures ANOVA. A univariate paired t test was performed for each organ only if the treatment effect revealed at the repeated-measures analysis was significant. All analyses were performed by using the model assumption of normality.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Combined RF Ablation–Liposomal Doxorubicin Therapy in Canine Transmissible Venereal Sarcomas
Tumor coagulation.—Multivariate ANOVA, with controlling for multiple comparisons, revealed a P value of .025. Significant increases in gross coagulation were observed in the canine transmissible venereal tumors treated with RF ablation followed 30 minutes later by intravenous liposomal doxorubicin administration, as compared with the tumors treated with RF ablation alone (P < .05, Table 1). At gross pathologic observation, an increased rim of red tissue surrounded the central white region of coagulation. The mean size of the overall area of coagulation (ie, white central area of coagulation plus red rim area) was observed to increase from 22.7 mm ± 1.4 with RF ablation alone to 36.5 mm ± 5.9 with combined RF ablation–doxorubicin therapy (P < .05, Fig 1a). A similar increase in the overall diameter of the central white zone, representing direct RF ablation–induced coagulation, was observed in the canine tumors treated with the combined therapy (20.9 mm ± 1.7), as compared with the overall diameter of this zone in the canine tumors treated with RF ablation alone (15.3 mm ± 2.2) (P < .05, Table 1). Triphenyltetrazolium chloride staining revealed a lack of mitochondrial enzyme activity in both the white and the red zones of coagulation.


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  		TABLE 1. Coagulation in Canine Sarcoma Observed with Combination RF Ablation-Intravenous Liposomal Doxorubicin Therapy versus That Achieved with RF Ablation Alone

 


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  		Figure 1a. Pathologic specimens show gross coagulation in tumor and normal kidney tissue treated with RF ablation only and with combination RF ablation-liposomal doxorubicin (Doxil) therapy. (a) Two canine transmissible venereal sarcomas: one treated with RF ablation alone (top) and the other treated with combination RF ablation-liposomal doxorubicin therapy (bottom). The combined therapy led to significant increases in overall coagulation 48 hours after treatment. Although the central white zone of RF ablation-induced coagulation (arrows) is similar in both tumors, increased peripheral necrosis (arrowheads) is observed in a red zone of the tumor treated with the combined therapy (bottom) compared with a much smaller peripheral zone in the tumor treated with RF ablation alone (top). (b) In normal kidney tissue treated with the combination therapy, a central zone of RF-induced coagulation (white arrows) is surrounded by a wide zone of peripheral necrosis (black arrows) similar to the increased peripheral necrosis observed in a.

 


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  		Figure 1b. Pathologic specimens show gross coagulation in tumor and normal kidney tissue treated with RF ablation only and with combination RF ablation-liposomal doxorubicin (Doxil) therapy. (a) Two canine transmissible venereal sarcomas: one treated with RF ablation alone (top) and the other treated with combination RF ablation-liposomal doxorubicin therapy (bottom). The combined therapy led to significant increases in overall coagulation 48 hours after treatment. Although the central white zone of RF ablation-induced coagulation (arrows) is similar in both tumors, increased peripheral necrosis (arrowheads) is observed in a red zone of the tumor treated with the combined therapy (bottom) compared with a much smaller peripheral zone in the tumor treated with RF ablation alone (top). (b) In normal kidney tissue treated with the combination therapy, a central zone of RF-induced coagulation (white arrows) is surrounded by a wide zone of peripheral necrosis (black arrows) similar to the increased peripheral necrosis observed in a.

 
Histopathologic examination results confirmed that the white zones of both the tumors treated with combined RF ablation–liposomal doxorubicin therapy and those treated with RF alone were composed of cells that had undergone thermal coagulation—that is, these cells showed condensation of pyknotic nuclei, an increased number of cytoplasmic eosinophilia, and crenulation and streaming of the cellular morphology (27). In the tumors treated with the combined therapy, the enlarged red (rim) zone was largely composed of cells that had undergone classic coagulative necrosis, with elimination of visible nuclei. Admixed between these necrotic cells were variable patchy areas of hemorrhage that ranged from extrusion of single cells to 1–2-mm regions of confluent hemorrhage. With both treatment types (RF ablation alone and combined therapy), early granulation tissue was seen at the interface between normal and ablated tissues. However, hypervascular neovascularity was more prominent in the combined-therapy group.

Doxorubicin accumulation.—Multivariate ANOVA, with controlling for multiple comparisons, revealed an overall P value of .003. Significant increases in intratumoral liposomal doxorubicin accumulation were observed in the tumors treated with combination RF ablation–liposomal doxorubicin therapy, as compared with the accumulation observed in the tumors treated with intravenous liposomal doxorubicin alone (Table 2). In the combined-therapy group, the greatest liposomal doxorubicin accumulation occurred in the red zone surrounding the central zone of RF ablation–induced coagulation (77.0 ng/g ± 18.2), as compared with the accumulation in the central zone (15.1 ng/g ± 3.2) and in the peripheral untreated tumor area beyond the red zone (38.9 ng/g ± 8.0) (P < .001 for each comparison). This triphasic distribution pattern was not seen in the canine sarcomas treated with intravenous liposomal doxorubicin alone. A mean of 43.9 ng/g ± 6.7 of liposomal doxorubicin was extracted from all portions of the tumors (Table 2). Additionally, no significant difference in intratumoral doxorubicin accumulation in the peripheral tumor tissue beyond the treated tumor was noted between the sarcomas treated with combination RF ablation–liposomal doxorubicin therapy and those treated with intravenous liposomal doxorubicin alone (P = .584) (Table 2).


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  		TABLE 2. Intratumoral Liposomal Doxorubicin Accumulation in Different Zones of Canine Sarcomas Treated with Combination RF Ablation-Liposomal Doxorubicin Therapy versus Intravenous Liposomal Doxorubicin Alone

 
Experiment 2: Combined RF Ablation–Liposomal Doxorubicin Therapy in Three Normal Tissue Models
Tumor coagulation.—Multivariate ANOVA, with controlling for multiple comparisons, revealed P values of .007 for treatment effect and .02 for organ type. Significant increases in coagulation were observed in all tissues treated with RF ablation followed by intravenous liposomal doxorubicin administration compared with the coagulation observed in tissues treated with RF ablation alone (P < .05, Table 3). In all three normal tissues (normal rabbit liver, normal rabbit kidney, and normal rat thigh muscle), an expanded peripheral red rim surrounding the central region of white coagulation was identified at gross pathologic observation (Fig 1b).


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  		TABLE 3. Coagulation Induced by Combination RF Ablation-Liposomal Doxorubicin Therapy versus That Induced by RF Ablation Alone in Three Normal Tissue Types

 
Although tissue response to injury was similar among the different types of tissues, histopathologic analysis revealed a difference in the pattern of response to thermal injury between the normal tissues and the canine tumors. Specifically, in all three normal tissues, although the white zone had features of thermal coagulation, the red zone had a pronounced inflammatory reaction at its inner marginal interface with the zone of coagulation (Fig 2), and this reaction was qualitatively greater than that observed in the tumors. Furthermore, in the combined-treatment group, a distinct zone of coagulative necrosis, with occasional thrombosed vessels, was identified peripheral to the inflammatory reaction. Thus, for the normal tissues, the expanded red zone consisted of two components.



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  		Figure 2a. Histopathologic changes observed with combination RF ablation-intravenous doxorubicin therapy. (a) Hematoxylin-eosin-stained section (magnification, x20) of canine sarcoma tissue from the center of a tumor treated with combination therapy shows a central zone of RF ablation-induced coagulation. The tumor tissue surrounding the RF ablation-induced coagulative zone is characterized by cells that have undergone classic coagulative necrosis. (b) Hematoxylin-eosin-stained section (magnification, x4) of normal rabbit liver tissue treated with combined therapy shows three distinct histopathologic zones: The white zone (zone A) represents the thermal coagulation that is observed with RF ablation alone. Zone B, the tissue undergoing coagulation necrosis and surrounding the central RF zone, has a pronounced inflammatory reaction (arrows) at its inner marginal interface. Zone C is normal untreated liver tissue.

 


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  		Figure 2b. Histopathologic changes observed with combination RF ablation-intravenous doxorubicin therapy. (a) Hematoxylin-eosin-stained section (magnification, x20) of canine sarcoma tissue from the center of a tumor treated with combination therapy shows a central zone of RF ablation-induced coagulation. The tumor tissue surrounding the RF ablation-induced coagulative zone is characterized by cells that have undergone classic coagulative necrosis. (b) Hematoxylin-eosin-stained section (magnification, x4) of normal rabbit liver tissue treated with combined therapy shows three distinct histopathologic zones: The white zone (zone A) represents the thermal coagulation that is observed with RF ablation alone. Zone B, the tissue undergoing coagulation necrosis and surrounding the central RF zone, has a pronounced inflammatory reaction (arrows) at its inner marginal interface. Zone C is normal untreated liver tissue.

 
Doxorubicin accumulation.—Multivariate ANOVA, with controlling for multiple comparisons, revealed an overall P value of .01. Significant increases in intratumoral liposomal doxorubicin accumulation were observed in all tissues treated with combination RF ablation–liposomal doxorubicin therapy, as compared with the accumulation observed in tissues treated with intravenous liposomal doxorubicin alone (P < .01, Table 4). In the normal rabbit liver, rabbit kidney, and rat muscle tissues, mean ratios of intratumoral liposomal doxorubicin accumulation in tissues treated with combination RF ablation–liposomal doxorubicin therapy to accumulation in tissues treated with intravenous liposomal doxorubicin alone were 1.59 µg/g ± 0.38, 3.50 µg/g ± 0.99, and 3.47 µg/g ± 1.64, respectively (Table 4).


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  		TABLE 4. Liposomal Doxorubicin Accumulation Observed with Combination RF Ablation-Liposomal Doxorubicin Therapy versus That Observed with Liposomal Doxorubicin Therapy Alone in Normal Tissues

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
Given that thermal ablation is most often used as a stand-alone therapy, these strategies usually enable one to take advantage of only those temperatures that are sufficient by themselves to induce coagulative necrosis (>50°C) (28–30). However, this approach can be limiting because there is a rapid exponential decrease in temperature in tissues surrounding the RF electrode during ablation, and, thus, only relatively small volumes of coagulation are usually produced (28). Nevertheless, with this pattern of heat distribution, there are large zones of sublethal heating created during RF application that currently are not being used to achieve sustained treatment effect. Indeed, modeling study results demonstrate that if the threshold for cell death was decreased by as little as 5°C, the diameter of tumor coagulation could potentially be increased by more than 1 cm in diameter (31). Hence, lowering the temperature threshold at which cell death occurs by combining sublethal temperature with cell exposure to chemotherapeutic agents could potentially increase the volume of tumor destruction. Indeed, this type of synergy, which facilitates improved tumor destruction owing to the combining of heat and chemotherapy, has been well documented for longer (>45 minutes) thermal exposures at hyperthermic temperatures (43°–45°C) (32,33).

Furthermore, investigators in preliminary studies exploring the combination of adjuvant intravenous liposomal doxorubicin administration and RF ablation have documented increases in both tumor necrosis and intratumoral drug accumulation in one rat breast tumor model, with both processes apparently occurring in tissues heated to 45°–50°C (10–12). However, prior to the widespread clinical adoption of this combined technique, further characterization of this synergy in larger and more clinically relevant animal tumor models and in normal tissue surrounding tumor, where the greatest effect is required, is needed.

In this study, clinically meaningful increases in overall coagulation were observed in the larger canine sarcoma tumors and in the normal rabbit and rat tissues treated with combination RF ablation–intravenous liposomal doxorubicin therapy, as compared with the areas of coagulation observed in the tumors and normal tissue treated with either RF ablation alone or intravenous liposomal doxorubicin alone. These coagulation increases are consistent with and more dramatic than earlier published data in smaller rat adenocarcinoma models (10–12). Such gains in peripheral tumor necrosis may ultimately prove to be clinically important in that they address one of the primary limitations to the long-term success of RF ablation: continued residual tumor in tissue surrounding the RF ablation zone (7,8,27). These results also demonstrate the nonspecific nature of this synergistic effect observed with combination therapy and its potential application in a wide range of tissue types. This is particularly important because evidence suggests that the peripheral margins of tumors contain areas of normal tissue within which areas of microscopic residual tumor reside (34,35) and that the inability to treat these areas satisfactorily with conventional RF ablation therapy alone is a leading cause of post–RF ablation residual tumor growth (7,8,27). Indeed, an increase in overall tumor destruction of the magnitude that we observed (1.2 cm) could potentially facilitate a reduction in the number of RF energy applications by more than sixfold (36).

In earlier work with combination RF ablation–liposomal doxorubicin therapy, increases in both overall drug accumulation in the tumor and tumor destruction—and the fact that these increases occur peripheral to the central RF zone—have been documented (13). Monsky et al (13), using autoradiographic imaging of radiolabeled liposomes, demonstrated a fivefold increase in intratumoral uptake of liposomes in tumors treated with RF ablation, as compared with tumors treated with only the drug. However, the use of a small- animal model prevented them from isolating the actual doxorubicin concentration in specific zones of treatment (ie, central zone of RF coagulation, surrounding zone of increased tumor destruction, and unaffected tissue beyond the treatment margins) and from quantifying drug accumulation such that it could be directly compared with histopathologic findings. The results of our study accomplish this aim and demonstrate that the greatest drug concentration is found in a red peripheral zone of hemorrhage and frank coagulative necrosis.

We postulate that the observed increase in doxorubicin accumulation, an agent known to bind and be intercalated between nuclear DNA (37,38), is largely responsible for the specific changes in coagulative necrosis and the elimination of nuclei that are seen in only this zone. In addition, these findings have not been found in control tumors treated with RF ablation alone or in other tissues treated with lasers, in which the red zone represented the area of thermal coagulation and hyperemia—not the area of coagulative necrosis and hemorrhage (39).

Interestingly, small amounts of doxorubicin were observed in the central RF ablation zone, possibly owing to drug diffusion or drug delivery through residual persistent patent vasculature (40). The latter cause, which is most often attributed to a "heat-sink" phenomenon (41,42), is one of the postulated mechanisms behind the residual tumor growth that has been reported in up to 33% of RF-ablated tumors (7,8,42). Hence, the presence of doxorubicin in residual tumor foci may potentially limit the persistent growth of incompletely treated tumor cells.

Several mechanisms are postulated to underlie the synergy that is observed with combined RF ablation–intravenous liposomal doxorubicin therapy. Results of extensive work combining low-temperature hyperthermic therapy (42°–43°C for 45 minutes) with liposomal agents have demonstrated similar two- to fivefold increases in interstitial drug accumulation secondary to increased vascular permeability, which in turn was secondary to endothelial injury (43–45) and temporary increases in vascular endothelial pore size (22). Thus, noncoagulative hyperthermia observed in the peripheral tissue immediately surrounding the central RF zone may augment microvascular permeability to chemotherapeutic agents. This finding is suggested by the large amount of hemorrhage that we observed in tumors in this peripheral watershed area. Additionally, sublethal hyperthermic temperatures (42°–45°C) cause the maximum blood flow to tissue to double in a reversible manner (43,46) and may also cause increased drug deposition in the periphery of the treatment zone, which is consistent with findings in a previous work by Monsky et al (13) involving the use of radiolabeled liposomes and with our findings in the canine tumors treated in this study.

Our study results further underscore the generally nonspecific nature of these mechanisms, given the increases in both coagulation and drug accumulation observed in multiple tissue and tumor types. Nevertheless, differences in overall amounts of increased coagulation and drug uptake, such as those observed between rabbit liver and kidney tissues in this study, suggest that the increases observed with combination therapy may vary in a tissue-specific manner and possibly are based on different characteristics of each organ site. Indeed, variable responses to thermal injury were documented in our study, given the greater degree of inflammatory reaction observed in normal tissues compared with that in canine tumors (or prior data using combination therapy in rat mammary adenocarcinoma tumors [10,11]). Similar tissue-to-tissue variability in RF ablation efficacy has been reported by Ahmed et al (11). Thus, further characterization in other tissues may be necessary in the future. Such characterization has enabled the demonstration of the dependence of this RF liposomal interaction on thermal dose and the potential predictability of this process for rat mammary adenocarcinoma tumors, and it will likely be required in clinical studies if we are to optimize the potential of this method.

Although the results of this study provide additional data on combination RF ablation–liposomal doxorubicin therapy, several limitations of this study must be addressed. The increases in both tissue coagulation and interstitial drug accumulation are observed to varying degrees in all of the models examined within the framework of this study. However, given the heterogeneity of tumors—in terms of tumor size, location, and type—that are currently being treated with minimally invasive therapies, the specific success of the described combination therapy for each specific tumor situation cannot be reliably predicted. For example, the smaller amounts of doxorubicin accumulation observed in the subcutaneous canine tumors could have been a result of the lower drug dose per weight that was administered (although this "low" dose was apparently sufficient to cause a dramatic increase in coagulation) compared with that administered in the other animals models, differences in drug distribution on a species-specific basis, or tissue- and organ site–specific differences in drug accumulation. However, given both the previous preliminary success reported by Goldberg et al (14) in a pilot study of a variety of liver malignancies in 10 patients and the coagulation and drug accumulation increases observed in several different environments in the current study, combination RF ablation–liposomal doxorubicin therapy, as compared with RF ablation alone, may facilitate an increase in overall tumor coagulation for various tumor types, regardless of tissue environment–specific differences.

Regardless, further research that includes the preliminary application of this combination therapy in pilot clinical studies and in other tumor sites is still required. Indeed, although the influence of RF ablation and drug characteristics on coagulation and drug accumulation has been examined in preliminary studies, further work is required to gain a better understanding of thermal dosimetry and drug pharmacokinetics in a tissue-specific fashion. Last, our analyses were based on the assumption of normality, and, thus, the reported P values may not be exact. However, to our knowledge, this assumption has been the basis of the analyses performed in all ablation studies to date.

Practical application: Use of RF ablation followed by intravenous liposomal doxorubicin administration, as compared with use of RF ablation alone, leads to significantly increased tissue destruction in several tumor and normal tissue types. Significant increases in drug accumulation occur in tumors treated with RF ablation followed by liposomal doxorubicin administration and thus suggest that this combination therapy may be effective for increasing the extent of and achieving a more complete tumor ablation in patients. This combination therapy also may be useful for producing an ablative margin within the normal tissue surrounding RF ablation–treated tumors. Furthermore, given the evidence that this interaction is due to thermal therapy rather than to factors intrinsic to RF electromagnetic energy, it is likely that the gains observed, if substantiated with further study, can be anticipated with other methods of thermal therapy (ie, laser, microwave, and focused US) as well.


   FOOTNOTES
 
Abbreviations: ANOVA = analysis of variance, RF = radiofrequency

Author contributions: Guarantor of integrity of entire study, S.N.G.; study concepts, S.N.G., M.A., V.P.T.; study design, S.N.G., M.A., A.N.L., W.L.M.; literature research, S.N.G., M.A., W.L.M.; experimental studies, M.A., Z.L., C.H.; data acquisition, S.N.G., M.A., S.S., Z.L., A.N.L.; data analysis/interpretation, S.N.G., M.A., Z.L., C.H.; statistical analysis, S.N.G., M.A.; manuscript preparation, M.A., S.N.G., W.L.M.; manuscript definition of intellectual content and editing, S.N.G., M.A., V.P.T.; manuscript revision/review and final version approval, all authors


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 

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