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Experimental Studies |
1 From the Minimally Invasive Tumor Therapy Laboratory (G.D., M.A., G.D.G., K.E.S., J.B.K., S.N.G.), Department of Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Boston, MA 02215; and DATA Group, Department of Radiology, Massachussets General Hospital, Harvard Medical School, Boston, Mass (E.F.H.). Supported by grants from the National Cancer Institute, National Institutes of Health, Bethesda, Md (RO1-CA87992-01A1) and Bracco, Milan, Italy. Received March 27, 2002; revision requested June 4; final revision received September 24; accepted October 14. Address correspondence to S.N.G. (e-mail: sgoldber@caregroup.harvard.edu).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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MATERIALS AND METHODS: Subcutaneous R3230 mammary adenocarcinoma (1.1–1.4 cm) was implanted in female Fischer rats. Initially, 35 tumors were randomized into four experimental groups: (a) conventional monopolar RF (70°C for 5 minutes) alone, (b) liposomal doxorubicin (1 mg) alone, (c) RF ablation followed by liposomal doxorubicin, and (d) no treatment. Ten additional tumors were randomized into two groups that received a 90°C RF dose either with or without liposomal doxorubicin. Tumor growth rates and the defined survival endpoint, the time at which the tumor reached 3.0 cm in diameter, were recorded. The effect of treatments on endpoint survival and tumor doubling time were analyzed by means of the Kaplan-Meier method and analysis of variance statistics.
RESULTS: Differences in endpoint survival and tumor doubling time in the six groups were highly significant (P < .001). Endpoint survivals were 9.1 days ± 2.5 for the control group, 16 days ± 3.7 for tumors treated with 70°C RF alone, 16.5 days ± 3.2 for tumors treated with liposomal doxorubicin alone, and 26.6 ± 5.3 days with combined treatment. For 90°C RF ablation, endpoint survivals were 16.6 days ± 1.2 and 31.5 days ± 3.0 without and with liposomal doxorubicin (P < .01). Mean endpoint survival and tumor doubling times for the three RF levels (0, 70°C, and 90°C) were all significantly different (P = .01). Additionally, animals that received combined liposomal doxorubicin and 90°C RF ablation survived longer than did animals that received combined liposomal doxorubicin and 70°C RF ablation (P < .01).
CONCLUSION: Combined RF ablation and liposomal doxorubicin retards tumor growth and may increase animal survival compared with that with either therapy alone or no therapy.
© RSNA, 2003
Index terms: Adenocarcinoma, 00.32 • Animals • Breast neoplasms, experimental studies, 00.1269 • Chemotherapy • Experimental study, 00.1269 • Radiofrequency (RF) ablation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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Recently, there has been considerable interest in the use of RF ablation in conjunction with chemotherapy for the treatment of malignant tumors (22–25). Previously, local hyperthermia was used to increase the effectiveness of chemotherapy by increasing blood flow, membrane permeability, local drug uptake, and metabolism in solid tumors (26,27). In our prior work, we demonstrated that combined RF ablation with commercially available long-circulating polyethylene glycol–coated liposomal doxorubicin preparation (Doxil, Alza Pharmaceuticals, Mountain View, Calif) increases the extent of local tumor destruction when compared with either RF ablation or intravenous doxorubicin alone in an animal tumor model (24), as well in human liver tumors (28). To our knowledge, however, the potential effect of systemic chemotherapy combined with RF ablation on tumor growth and survival is unknown. The purpose of this study was to determine whether RF ablation in combination with intravenous liposomal encapsulated doxorubicin can decrease tumor growth rates and thereby potentially increase endpoint survival over RF ablation or doxorubicin alone in an animal tumor model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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Experiments were performed with a well-characterized (22,24,29) established R3230 mammary adenocarcinoma cell line (Center for Molecular Imaging Research, Massachusetts General Hospital, Boston). Fresh tumor was initially harvested from a live carrier. Within 30 minutes of this tumor explantation, the tumor was homogenized with a tissue grinder (model 23; Kontes Glass, Vineland, NJ) with aseptic technique and suspended in 7 mL of Roswell Park Memorial Institution (RPMI) 1640 medium (Biomedicals, Aurora, Ill). One tumor was implanted into each animal with direct visualization by using 0.2–0.3 mL of tumor suspension injected slowly via an 18-gauge needle into the mammary fat pad of 49 female Fischer 344 rats (mean weight, 150 g ± 20 [SD]; age range, 13–14 weeks) (Taconic Farms, Germantown, NY), the strain of animals from which this tumor was derived initially. Animals were maintained on an ad lib diet (standard rodent chow 8664, Harlan-Teklab, Baltic, Conn) and were monitored every 2–3 days to measure tumor growth. Tumor size was determined with consensus (G.D., M.A.) by measuring longitudinal and transverse diameter with mechanical calipers. Only solid nonnecrotic tumors (as determined at ultrasonography at the time of treatment) that were 1.1–1.4 cm in the largest diameter (n = 35) were used for this study. Tumors were grown for 10–14 days until the desired size was achieved, at which point they were randomized to one of the treatment arms.
Experimental and Control Groups
Forty-nine animals were used in this study. Initially, 35 rats were randomly divided into four groups. To avoid bias due to an overestimated or underestimated difference between treatments, we used simple random allocation in a controlled trial, as described previously (30). The four groups included (a) tumors treated with RF ablation alone at 70°C tip temperature (n = 7), (b) tumors treated with a 1-mg intravenous injection of liposomal encapsulated doxorubicin alone (n = 9), (c) tumors treated with RF ablation followed by intravenous injection of liposomal encapsulated doxorubicin (n = 10), and (d) no treatment (n = 9). These parameters were selected to permit direct comparison with those in prior studies to assess the acute effects of liposomal doxorubicin on RF ablation (24).
As a next step, four additional animals received treatments identical to those of the four groups outlined in the initial experiment. These four animals were sacrificed at 10 days after treatment to document differences in gross and histopathologic findings among the groups at a defined time point. Tumor growth measurements from these four animals were not included in statistical analysis of the groups, as they were not followed up to the defined survival endpoint. Subsequently, 10 additional tumors were divided randomly into two groups that received a higher RF heating dose (tip temperature 90°C) with (n = 5) and without (n = 5) adjuvant liposomal doxorubicin intravenous injection. This last experiment was performed to determine whether the RF thermal dose influenced tumor growth or endpoint survival.
RF Application
Conventional monopolar RF was applied by using a 500-kHz RF generator (model 3E; Radionics, Burlington, Mass) (Fig 1). 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 by liberally applying electrolytic contact gel. Initially, the 1-cm tip of a 21-gauge electrically insulated electrode (SMK electrode; Radionics) was placed at the center of the tumor. RF ablation was applied for 5 minutes with the generator output titrated to maintain either a tip temperature of 70°C ± 2 (mean, 90.4 mA ± 25.8; range, 48–160 mA) or 90°C ± 2 (mean, 142.6 mA ± 34.5; range, 110–170 mA). This standardized method of RF application has been demonstrated previously to provide reproducible coagulation volumes with use of this conventional RF system (22,24). Thus, RF output was varied from animal to animal to maintain a constant uniform thermal dose of RF energy. A thermocouple at the tip of the RF electrode constantly measured the local ablation temperature, thereby enabling proper generator manipulation. Parameters of the RF ablation procedure, including tip temperature, tissue impedance, and applied current, were recorded at baseline and thereafter at 60-second intervals for the duration of RF application.
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Endpoint Survival, Tumor Analysis, and Pathologic Studies
Tumors were measured in a longitudinal and transverse diameter with a mechanical caliper (G.D., M.A.) every 2–3 days until they reached 3.0 cm in largest diameter, at which point the animals were sacrificed. This surrogate for endpoint survival was selected because of requirements of the institutional animal committee, which mandated sacrifice at this point on the basis of animal size and tumor burden as dictated by the U.S. Department of Agriculture Animal Welfare Act (31–32). Any animal with tumors weighing more than 10% of its body weight, corresponding to a tumor diameter of 3 cm in this model, was considered moribund and underwent mandatory sacrifice. For all measurements, anesthesia was induced, as described previously, to permit accurate measurements. The largest diameter measured at every time point was recorded and plotted. Animals were sacrificed with intraperitoneal injection of pentobarbital (Nembutal; Abbot Laboratories, North Chicago, Ill) overdose (0.2 mL/kg). Tumors were then excised and sectioned, and the extent of visible coagulation at gross pathologic examination was measured with calipers (G.D. and M.A. concurred), as were the longitudinal and transverse diameters of the tumor itself. Histopathologic studies included staining for mitochondrial enzyme activity by incubating thin representative tissue sections for 30 minutes in 2% 2,3,5–triphenyl tetrazolium chloride, or TTC, at room temperature. This latter test is used to identify irreversible cellular injury during the early stages of RF-induced tissue necrosis (33).
Statistical Analysis
For all experiments, tumor diameter was measured and results compared statistically. All data are provided as mean plus or minus SD. On the basis of studies that demonstrated exponential tumor growth patterns, tumor growth rates and doubling times were also calculated by means of regression analysis of an exponential model (34). The Kaplan-Meier method was used for endpoint survival analysis. Given the absence of censoring of our data, one-way analysis of variance was then performed on the survival endpoints for each animal for the comparisons reported. Pairwise t tests (
= .05; two-tailed test) based on the least square means were subsequently performed only if the overall P values were significant. This approach was justified because all but one animal reached the study endpoint, with the remaining censored animal surviving longer than 45 days, 4 SDs beyond the mean endpoint survival growth of any group. We then used two-way analysis of variance to determine the contributions of RF ablation and intravenous injection of liposomal doxorubicin to endpoint survival. One-way and two-way analyses of variance were also performed to determine the contribution of RF ablation and intravenous liposomal doxorubicin to the parameter of tumor doubling time.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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Kaplan-Meier analysis of endpoint survival among the six groups of animals showed that differences were significant (P < .001, log-rank test) (Fig 2). Both main effects (ie, the presence of RF heating or intravenous liposomal doxorubicin injection) were significant (P < .001 for each compared with no treatment). Pairwise comparisons indicated that, on average, the mean endpoint survival times with the three RF levels were all significantly different. However, their interaction was also significant (P = .01), which indicates that the magnitude of the positive effect of intravenous injection of liposomal doxorubicin depended on the amount of RF heating (70°C or 90°C). Conversely, the differences in mean endpoint survival times between the lower 70°C and the 90°C RF heating levels were only observed in the presence of doxorubicin.
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Histopathologic Examination
In the four animals sacrificed 10 days after therapy, large differences in tumor size were observed among the distinct treatment arms (Fig 3), in keeping with growth rates presented in Figure 1. The smallest tumor was noted in the group treated with combined intravenous liposomal doxorubicin and RF ablation. At histopathologic examination, viable cells were noted in all samples, a finding expected on the basis of the experimental design. Well-demarcated coagulation was observed after RF ablation, while patchy necrosis was noted in tumors treated with doxorubicin alone. In this latter group, however, a subjective increase in cellular necrosis, compared with the control tumors, was seen in these ill-defined areas. As expected, all tumors at experimental endpoint showed greater than 90% viability, with those undergoing RF ablation showing 2–5-mm central zones of necrosis. No viable tumor was identified in one animal who survived to day 60 (treated with 90°C RF ablation and doxorubicin).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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Several recent studies have demonstrated a complementary interaction when combining RF ablation and concurrent administration of the chemotherapeutic agent doxorubicin. In early studies with a R3230 rat breast adenocarcinoma model, Goldberg et al observed increases in coagulation necrosis diameter from 6.7 to 11.4 mm when free doxorubicin was directly injected intratumorally before RF administration in a rat breast adenocarcinoma model (33). When a liposomal form of doxorubicin with increased blood circulation times and greater tumoral specificity was used in conjunction with RF ablation, even greater coagulation necrosis (13.5 mm) was achieved (24). In a randomized pilot clinical study, Goldberg et al administered liposomal doxorubicin 24 hours before RF ablation in 10 patients with 18 intrahepatic lesions with varied diseases (primary hepatocellular carcinoma, colorectal metastases, neuroendocrine metastases, and breast cancer metastases) (28). With this combination of RF ablation with adjuvant liposomal doxorubicin therapy, the authors were able to achieve increases of 25%–30% in coagulation volume in all tumors that received combination therapy compared with RF ablation alone. More important, follow-up imaging studies demonstrated that this particular form of adjuvant therapy resulted in more complete tumor destruction, as coagulation progressed over time to include residual tumor foci at tumor margins and patent intratumoral blood vessels (28).
Various potential mechanisms have been postulated to explain the synergy between focal thermal therapy and liposomal doxorubicin. These include local hyperthermia–induced nonspecific vascular changes that alter vessel permeability and induce reversible vascular stasis (36,37), the independent cytotoxic effect of both the chemotherapeutic agent and its liposomal carrier, and heat-induced damage to cellular multidrug-resistant protein pumps (24,38,39). Additionally, Monsky et al (25) showed that RF ablation has the unique potential to function as a focal targeting mechanism to guide the accumulation of liposome-encapsulated chemotherapeutic agents. These investigators used autoradiography to visualize intratumoral distribution of radio-labeled liposomal doxorubicin after RF ablation and demonstrated concentration of the agent in tumor tissue immediately surrounding the central RF zone, with a fivefold increase in liposomal doxorubicin uptake in tumors treated with RF ablation compared with that in untreated tumors.
While previous studies demonstrate the potential advantage of adjuvant liposomal doxorubicin to increase the volume of tumor destruction obtained from RF ablation (24,28,33), findings in the current study clearly identify the possible gains in reduced tumor growth and increased endpoint survival that this combination treatment yields over no treatment or individual therapies alone. Results with the combined RF ablation and liposomal doxorubicin protocol used in the current study show mean endpoint animal survival of 26.6 days compared with 16.5, 16.0, and 9.1 days with liposomal doxorubicin alone, RF ablation alone, and no treatment, respectively. Similarly, tumor doubling times of 21.3, 14.6, 12.7, and 10.2 days were noted for these four respective groups. To our knowledge, ours is the first study that documents increased endpoint survival with combination liposomal doxorubicin and RF ablation. More specifically in our study, observed increases in RF ablation–induced doxorubicin uptake (25) were translated into tangible gains of reduced tumor growth and increased animal endpoint survival.
Although findings at Kaplan-Meier analysis demonstrated that differences in RF thermal dose (ie, 70°C or 90°C tip temperature) produced decreases in tumor growth when RF ablation was the sole treatment, an increase in thermal dose (ie, the higher 90°C RF) significantly improved the magnitude of endpoint survival in the presence of liposomal doxorubicin. This underscores the importance of a thermal energy dose effect on possible coagulation as a paradigm of combined RF ablation and concurrent adjuvant liposomal doxorubicin. Thus, findings in our study also support the need for ascertaining the optimal parameters for thermal delivery to achieve maximum potential coagulation. Clearly, future investigation should also focus on identification of the optimal RF energy delivery parameters that will provide the greatest efficacy for this treatment combination.
This study also represents a necessary next step in tumor ablation therapy research. Well-controlled studies with appropriately stratified homogeneous patient populations are needed to determine the optimal role of ablation for clinical oncologic management. Thermal strategies have been performed most often in heterogeneous patient and tumor populations with variable sample and tumor sizes and varying RF administration techniques. These differences result in clinical studies that are often limited in the identification of potential response differences between treatment paradigms (40). Hence, there remains a need for validation of these procedures, which for some techniques will likely be performed best in a straightforward manner with relevant animal models.
Our study design helped to further minimize a major criticism of many previous thermal ablation studies. Gross macroscopic measurement of the zone of coagulation necrosis is an adequate endpoint during the process of optimization to achieve maximum volumes of coagulation and to compare various RF devices (22,31,41), but such studies cannot by design answer the question of completeness of tumor destruction and the effect of residual untreated disease. Hence, defined optimal administration parameters for combination therapy should also be subjected to animal survival and tumor growth rate studies in an attempt to determine the potential usefulness in clinical trials. In the current study, viable populations of cells that continued to grow were identified in apparently completely ablated regions.
Despite incomplete treatment in virtually all of the animals, endpoint survival benefits were conferred, most notably with combined therapy. Ours is the first randomized study, to our knowledge, that documents any improved endpoint survival from thermal therapy when tumors were not completely ablated from the outset. Current dictum, based on surgery, is that all tumor must be ablated completely to achieve any benefit (ie, no benefit occurs if any residual disease persists). This has important implications with regard to RF ablation strategies. Given reduced growth rates in incompletely treated tumors, our results suggest that in some limited circumstances, palliative treatment with RF techniques may be beneficial. This is consistent with the results of Gillams and Lees, which demonstrated improved outcomes in inoperable colorectal metastases to the liver in patients treated palliatively with RF ablation (42).
Some limitations of this study include the limited generalization of results given the tumor model studied, the size of the ablated foci, the RF ablation technique, and doxorubicin doses selected. Although this tumor model was selected because it is a well-characterized vascular solid adenocarcinoma (22,29), it is possible that results will vary with other tumor types (ie, hepatocellular carcinoma) and with other orthotopic tumor sites (ie, the kidney). Additionally, it must be noted that a surrogate endpoint of a tripling in tumor growth was used in lieu of allowing the animals to die of their tumor burden. This latter approach has been deemed unethical, however, and as a result, we were required by our animal care committee to use an upper tumor diameter of 3 cm as our surrogate endpoint. Furthermore, while the amount of RF applied was optimal for this model to permit the demonstration of synergy between the two methods, alternative thermal ablation protocols would have been able to destroy the entire 1.4-cm tumor without combined therapy. Additionally, it is extremely difficult to accurately assess overall volumetric changes in tumor growth from extrapolation of a single diameter measurement. Likewise, given these concerns, extrapolation to larger, more clinically relevant tumors must be made with caution. Nevertheless, findings in our study permit relative comparisons to be made, and they strongly support the validity of the overall approach of combining RF ablation and chemotherapy.
Practical application: Combination of RF ablation and doxorubicin treatments not only increases tumor coagulation volume but also reduces overall tumor growth rate, increases tumor doubling time, and improves endpoint survival in a rat adenocarcinoma model. As such, RF ablation combined with concurrently administered adjuvant doxorubicin has great potential to increase the extent and completeness of treatment for focal malignancy in the context of either primary curative therapy or palliative care. Although further study to optimize delivery parameters is required, this treatment paradigm will likely continue to receive much attention as a minimally invasive treatment for malignancy.
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FOOTNOTES |
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Author contributions: Guarantor of integrity of entire study, S.N.G.; study concepts, S.N.G., G.D.G., K.E.S.; study design, S.N.G., G.D., G.D.G., M.A., J.B.K; literature research, S.N.G., G.D., G.D.G., J.B.K.; experimental studies, G.D., M.A.; data acquisition, S.N.G., G.D., M.A.; data analysis/interpretation, S.N.G., G.D., E.F.H.; statistical analysis, S.N.G., E.F.H.; manuscript preparation, G.D., M.A., S.N.G.; manuscript definition of intellectual content, S.N.G., G.D., J.B.K.; manuscript editing, G.D., J.B.K., S.N.G.; manuscript review and final version approval, all authors
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  M. Ahmed, Z. Liu, A. N. Lukyanov, S. Signoretti, C. Horkan, W. L. Monsky, V. P. Torchilin, and S. N. Goldberg Combination Radiofrequency Ablation with Intratumoral Liposomal Doxorubicin: Effect on Drug Accumulation and Coagulation in Multiple Tissues and Tumor Types in Animals Radiology, May 1, 2005; 235(2): 469 - 477. [Abstract] [Full Text] [PDF]
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C. Horkan, K. Dalal, J. A. Coderre, J. L. Kiger, D. E. Dupuy, S. Signoretti, E. F. Halpern, and S. N. Goldberg Reduced Tumor Growth with Combined Radiofrequency Ablation and Radiation Therapy in a Rat Breast Tumor Model Radiology, April 1, 2005; 235(1): 81 - 88. [Abstract] [Full Text] [PDF]
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 M. Ahmed, W. E. Monsky, G. Girnun, A. Lukyanov, G. D'Ippolito, J. B. Kruskal, K. E. Stuart, V. P. Torchilin, and S. N. Goldberg Radiofrequency Thermal Ablation Sharply Increases Intratumoral Liposomal Doxorubicin Accumulation and Tumor Coagulation Cancer Res., October 1, 2003; 63(19): 6327 - 6333. [Abstract] [Full Text] [PDF]
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