Chemoradionuclide Therapy with ¹⁸⁶Re-labeled Liposomal Doxorubicin in Combination with Radiofrequency Ablation for Effective Treatment of Head and Neck Cancer in a Nude Rat Tumor Xenograft Model

Concordant benefits for solid tumor therapy could be obtained by combining radiofrequency ablation with PEGylated liposomes encapsulating both doxorubicin and rhenium 186 for combination chemoradionuclide therapy.

Abstract

Purpose:

To determine the therapeutic efficacy of rhenium 186 (¹⁸⁶Re)–labeled PEGylated liposomal doxorubicin (¹⁸⁶Re–liposomal doxorubicin) in combination with radiofrequency (RF) ablation of human head and neck squamous cell carcinoma (HNSCC) xenograft in nude rats.

Materials and Methods:

This investigation was approved by the animal care committee. Sixty nude rats with subcutaneously implanted HNSCC xenografts (six per group) were treated with (a) RF ablation (70°C for 5 minutes), (b) PEGylated liposomes, (c) liposomal doxorubicin, (d) ¹⁸⁶Re–PEGylated liposomes (1295 MBq/kg), (e) ¹⁸⁶Re–liposomal doxorubicin (555 MBq/kg), (f) PEGylated liposomes plus RF ablation, (g) liposomal doxorubicin plus RF ablation, (h) ¹⁸⁶Re–PEGylated liposomes plus RF ablation, or (i) ¹⁸⁶Re–liposomal doxorubicin plus RF ablation. Six rats did not receive any treatment (control group). Tumor uptake in ¹⁸⁶Re therapy groups was monitored with small-animal single photon emission computed tomography for 5 days. Therapeutic efficacy was monitored for 6 weeks with measurement of tumor volume, calculation of the percentage injected dose of fluorine 18 fluorodeoxyglucose (FDG) in tumor from small-animal positron emission tomography (PET) images, and determination of viable tumor volume at histopathologic examination. Significant differences between groups were determined with analysis of variance.

Results:

The average tumor volume (±standard deviation) on the day of therapy was 1.32 cm³ ± 0.17. At 6 weeks after therapy, control of tumor growth was better with ¹⁸⁶Re–liposomal doxorubicin than with liposomal doxorubicin alone (tumor volume, 2.26 cm³ ± 0.89 vs 5.43 cm³ ± 0.93, respectively; P < .01). The use of RF ablation with liposomal doxorubicin and ¹⁸⁶Re–liposomal doxorubicin further improved tumor control (tumor volume, 2.05 cm³ ± 1.36 and 1.49 cm³ ± 1.47, respectively). The tumor growth trend correlated with change in percentage of injected dose of FDG in tumor for all groups (R² = 0.85, P < .001). Viable tumor volume was significantly decreased in the group treated with ¹⁸⁶Re–liposomal doxorubicin plus RF ablation (0.54 cm³ ± 0.38; P < .001 vs all groups except ¹⁸⁶Re–liposomal doxorubicin alone).

Conclusion:

Triple and dual therapies had an observable trend (¹⁸⁶Re–liposomal doxorubicin plus RF ablation > ¹⁸⁶Re–liposomal doxorubicin > liposomal doxorubicin plus RF ablation > liposomal doxorubicin) of improved tumor growth control and decreased viable tumor compared with other therapies. FDG PET could be used as a noninvasive surrogate marker for tumor growth and viability in this tumor model.

© RSNA, 2011

Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.11110361/-/DC1

Introduction

Clinical management of advanced head and neck squamous cell carcinoma (HNSCC) primarily includes surgical resection, chemotherapy, and radiation therapy (1,2). Long-term survival rates for these patients are generally low (∼40%) owing to local-regional recurrence and distant metastases (1,2). Chemotherapy is limited by the inhomogeneous drug distribution in solid tumors (3). Combination therapies such as chemo- and radiation therapy appear to have a substantial effect on local-regional tumor control and overall survival rate (4–6). However, the persistently high mortality rate leaves considerable room for better treatment alternatives.

The management of advanced disease may be improved by using liposomes to deliver a high concentration of a drug to the tumor to prevent local recurrence. PEGylated liposome drug carriers have prolonged circulation time, an improved pharmacokinetic profile compared with that of free drug, and a higher tumor accumulation owing to the enhanced permeability and retention effect (7,8). Liposomes can also carry therapeutic radionuclides such as rhenium 186 (¹⁸⁶Re) for tumor therapy (9–11). PEGylated liposomal doxorubicin (Doxil; Johnson & Johnson, New Brunswick, NJ) has been approved for the treatment of various cancers (12,13). Preclinical and clinical HNSCC studies with liposomal doxorubicin have shown improved therapeutic efficacy (14,15) and enhancement of the effects of radiation therapy (16).

Tumor radiofrequency (RF) ablation is achieved with coagulation necrosis due to direct application of temperatures of more than 50°C via needle electrodes. This minimally invasive procedure has been used to treat various tumors (17). A major drawback of RF ablation is the inability to completely destroy the tumor (18). To overcome this problem, RF ablation has been used in combination with liposomal doxorubicin (19–21) and other therapies (22–25), resulting in larger volumes of tumor destruction.

RF ablation with liposomal doxorubicin not only increases the RF ablation–induced coagulation necrosis and intratumoral accumulation of liposomal doxorubicin but also accelerates apoptosis (19,26,27). We hypothesized that concordant benefits for solid tumor therapy would be obtained by combining RF ablation with liposomes encapsulating both doxorubicin and ¹⁸⁶Re. RF ablation would not only destroy the necrotic center of the tumor but also increase the tumor accumulation of ¹⁸⁶Re-labeled liposomal doxorubicin (¹⁸⁶Re–liposomal doxorubicin), thus increasing the targeting efficiency and potentially improving the distribution of intravenously injected liposomes in the tumor. Beta particles from the ¹⁸⁶Re radionuclide with a maximum energy of 1.07 MeV and tissue penetration depth of 2–4 mm (28) would improve the local therapeutic efficacy by killing the cancer cells in the perimeter of the RF ablation field and the remaining solid tumor, reducing the chances of local-regional recurrence. The purpose of this study was to determine the therapeutic efficacy of ¹⁸⁶Re–liposomal doxorubicin in combination with RF ablation in human HNSCC xenograft in nude rats.

Materials and Methods

Animal and Tumor Model

Animal experiments were approved by the animal care committee. During all procedures, animals were anesthetized with inhalation of 1%–3% isoflurane (Vedco, St Joseph, Mo) in 100% oxygen. Male rnu/rnu athymic nude rats (age: 4–5 weeks, weight: 75–100 g; Harlan, Indianapolis, Ind) were inoculated subcutaneously with 5 × 10⁶ SCC-4 tumor cells (ATCC, Manassas, Va) in 0.20 mL of saline at the base of the neck (29). The length (l), width (w), and depth (d) of each tumor was measured with digital calipers and tumor volume calculated as follows: (π/6)lwd (30).

Liposome Preparation

Control PEGylated liposome containing ammonium (pH) gradient and with a similar lipid composition and diameter as liposomal doxorubicin was prepared. Liposomes containing 1,2-distearoyl-sn-glycero-phosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) 2000] (Avanti Polar Lipids), and cholesterol (Calbiochem, Gibbstown, NJ) (weight ratio, 3:1:1) were manufactured by means of extrusion (9,31,32). Procedures are detailed in Appendix E1 (online).

Radiolabeling Procedure

Commercially available liposomal doxorubicin (Doxil) and PEGylated liposome were labeled with ¹⁸⁶Re perrhenate (3.7–5.18 GBq [100–140 mCi]; University of Missouri Research Reactor, Columbia, Mo) by using N,N-bis(2-mercaptoethyl)-N’,N’-diethylethylenediamine (ABX, Radeburg, Germany) (10,32). Procedures are detailed in Appendix E2 (online).

Experiment Design

Sixty nude rats with tumor volumes of approximately 1.3 cm³ were randomly separated into 10 therapy groups, with six rats per group. The control group did not receive therapy. Four groups received monotherapy with liposome (PEGylated liposome, liposomal doxorubicin, or ¹⁸⁶Re–PEGylated liposome) or RF ablation. Four groups received dual therapy of PEGylated liposome plus RF ablation, liposomal doxorubicin plus RF ablation, ¹⁸⁶Re–PEGylated liposome plus RF ablation, or ¹⁸⁶Re–liposomal doxorubicin. One group received triple therapy with ¹⁸⁶Re–liposomal doxorubicin plus RF ablation. For combination therapies, liposomes were administered immediately after RF ablation. All liposome formulations were administered via the tail vein. The lipid dose for PEGylated liposome and liposomal doxorubicin was 52 mg/kg. The doxorubicin dose was 6.5 mg/kg. The intravenous therapeutic doses for ¹⁸⁶Re–liposomal doxorubicin and ¹⁸⁶Re–PEGylated liposome were previously determined as 555 MBq/kg and 1295 MBq/kg, respectively.

Tumor volumes were measured with calipers on alternate days for 6 weeks after therapy. Four ¹⁸⁶Re therapy groups underwent imaging with use of small-animal (“micro”) single photon emission computed tomography (SPECT) for 5 days after therapy to determine tumor uptake. Therapeutic efficacy was determined from tumor volume measurements (group averages and intergroup growth trend) and fluorine 18 fluorodeoxyglucose (FDG) small-animal positron emission tomography (PET) before and 3 days, 2 weeks, 4 weeks, and 6 weeks after therapy. From image analysis using a volume of interest, the percentage injected dose in the tumor was determined by A.S., a medical physicist with 4 years of experience in image analysis. The volume of interest was drawn to cover the entire tumor. The percentage injected dose of FDG in the tumor was correlated with tumor volume measured with calipers. At 6 weeks or if tumor volume reached 9 cm³, all animals were sacrificed and excised tumors were stained with hematoxylin-eosin and MIB1 (for Ki67 expression) to determine viable and proliferating tumor volume. Response of each individual tumor to treatment was classified as complete response (>20% decrease in tumor volume compared with that at baseline), partial response (±20% decrease in tumor volume compared with that at baseline), or progressive disease (>20% increase in tumor volume compared with that at baseline) to determine therapeutic efficacy of each treatment group.

RF Ablation

RF ablation was performed with a 21-gauge noninternally cooled straight needle electrode with a 1-cm exposed tip placed in the center of the tumor under visualization and palpation (A.S. performed RF ablation under guidance of G.D.D., a radiologist with >10 years of experience in RF ablation). The electrode was powered by a 500-kHz, 200-W RF generator (ML-1; Valleylab [now Covidien], Boulder, Colo). To complete the electrical circuit, the rat was placed on a standard grounding electrode; electrolytic gel was used to ensure proper contact. RF ablation was applied for 5 minutes at a mean temperature (±standard deviation) of 70°C ± 2. This setup creates a coagulation diameter of 1 cm (26), which only partially ablates the tumor and provides for the testing of additional therapies to treat residual tumor.

Imaging Protocol and Image Analysis

Micro-SPECT.—High-resolution parallel hole and multipinhole collimators were used to acquire anteroposterior planar, lateral planar, and SPECT images by using a micro-SPECT scanner (Flex; Gamma Medica, Northridge, Calif) after injection of ¹⁸⁶Re–liposomal doxorubicin and ¹⁸⁶Re–PEGylated liposome. A standard source (∼0.26 MBq) of ¹⁸⁶Re–liposomal doxorubicin or ¹⁸⁶Re–PEGylated liposome was placed in the field of view for image quantification. Regions of interest were drawn around the tumor and standard on lateral planar images to determine the percentage of injected dose in tumor per gram of tissue. Image analysis was performed by A.S., who has 4 years of experience in SPECT and PET image quantification. The ratio of tumor to standard counts was determined at each time point for all animals that received ¹⁸⁶Re–liposomal doxorubicin with and without RF ablation.

Micro-PET.—Rats (four per group) were fasted overnight before undergoing FDG PET, and their body temperature was maintained with heating pads and thermal lamps for 1 hour before injection of FDG to reduce brown fat uptake. FDG in saline (9.25 MBq, 0.25 mL) was injected via the tail vein. Rats underwent PET 1 hour after injection (R4; Concorde Microsystems, Knoxville, Tenn), with the tumor placed in the center of the field of view in both axial and longitudinal directions for 10 minutes. Images were reconstructed (matrix size, 128 × 128 × 63; pixel size, 0.85 × 0.85; section thickness, 1.2 mm), displayed, and analyzed with software supplied with the scanner. The volume of interest was drawn around the tumor, and the percentage injected dose of FDG in the tumor was determined.

Histopathologic and Immunohistochemical Examination

Excised tumors were sliced along the longest diameter and stained with hematoxylin-eosin for histopathologic examination (performed by L.M.M., a pathologist with >25 years of experience). Tumors were also stained with MIB1 to determine Ki67 expression by using standard immunohistochemical techniques. Hematoxylin-eosin–stained slides were scanned with a laser scanner (Hewlett-Packard, Palo Alto, Calif), and images were analyzed by A.S. with use of ImageJ (33) (National Institutes of Health, Bethesda, Md) to determine viable tumor volume. With use of the threshold function in ImageJ, the region stained with hematoxylin was outlined and the area determined (http://rsbweb.nih.gov/ij/docs/examples/stained-sections/index.html). A viability factor was calculated by dividing the area stained with hematoxylin by the total area of the tumor section. Final tumor volume was multiplied by this viability factor to determine the viable tumor volume. The procedure is detailed in Appendix E3 (online).

Statistical Analysis

One-way analysis of variance (SAS software; SAS Institute, Cary, NC) was used to assess differences in tumor volume (performed with the Kruskal-Wallis test at the end of the study), percentage injected dose, and viable tumor volume between groups. Statistical analysis was performed by T.J.P., a biostatistician with 30 years of experience. Model assumptions of homogeneity of variance and bell-shaped distribution were examined to ensure valid analyses. Furthermore, the Kruskal-Wallis test showed similar results for the day 42 tumor volume data among the groups of interest. The Pearson method was used to determine the correlation between (a) tumor volume and percentage injected dose of FDG in tumor at 6 weeks or at necropsy and (b) viable tumor volume and percentage injected dose of FDG in tumor at 6 weeks or at necropsy. Correlations took into account group differences when appropriate. P < .05 was considered indicative of a statistically significant difference.

Results

Liposome Characterization

The mean diameters of liposomal doxorubicin and control PEGylated liposomes were 87.3 nm ± 8.5 and 91.3 nm ± 11.8, respectively. The total lipid concentration of PEGylated liposomes was 26.89 mg/mL. Labeling efficiencies of ¹⁸⁶Re–liposomal doxorubicin and ¹⁸⁶Re–PEGylated liposome were approximately 80%.

Tumor Growth Trend

The average tumor volume on the day of therapy was 1.32 cm³ ± 0.17 (Table). No significant differences in time to reach 7 cm³ were found between control and PEGylated liposome groups (Fig 1). Monotherapies excluding liposomal doxorubicin (RF ablation, ¹⁸⁶Re–PEGylated liposome, PEGylated liposome plus RF ablation) controlled tumor growth and extended the time required to reach 7 cm³ to 19 days. Dual therapy (¹⁸⁶Re–PEGylated liposomes plus RF ablation) initially improved tumor control (up to day 10 after therapy), but tumor growth increased rapidly after 10 days and reached 7 cm³ on day 21.

Tumor Response to Therapy

^*Data are averages ± standard deviations.

^†Tumor response was determined at necropsy. Data are numbers of rats.

Figure 1:

Graph shows effect of different treatment modalities on HNSCC tumor growth. Groups receiving liposomal doxorubicin (LD) in various combinations had significantly smaller tumor volumes compared with control group and those treated with other modalities. L = liposome, Re = ¹⁸⁶Re, RFA = RF ablation.

Groups receiving liposomal doxorubicin in various combinations had better tumor control than all treatment groups without liposomal doxorubicin; tumor growth did not reach 7 cm³ by 6 weeks. Liposomal doxorubicin alone showed good tumor control for 21 days after therapy. From day 21 to 35 after therapy, the tumor growth curve increased marginally. After day 35, tumor volume increased rapidly and reached 5.43 cm³ ± 0.93 on day 42.

Dual therapies (liposomal doxorubicin plus RF ablation and ¹⁸⁶Re–liposomal doxorubicin) had an almost horizontal tumor growth curve. The average tumor volume was 2.05 cm³ ± 1.36 for liposomal doxorubicin plus RF ablation and 2.26 cm³ ± 0.89 for ¹⁸⁶Re–liposomal doxorubicin alone. The addition of a second treatment modality such as RF ablation or ¹⁸⁶Re radionuclide therapy increased the therapeutic efficacy of liposomal doxorubicin and decreased the tumor volume by almost 50% at 6 weeks after therapy.

Triple combination therapy of ¹⁸⁶Re–liposomal doxorubicin plus RF ablation did not result in a significant increase in tumor volume at 6 weeks. Triple combination therapy provided the best tumor growth control, with an average tumor volume of 1.49 cm³ ± 1.47 at 6 weeks (P < .001 vs all groups except liposomal doxorubicin plus RF ablation and ¹⁸⁶Re–liposomal doxorubicin). Although average tumor volumes at 6 weeks after therapy were not significantly different between dual and triple combination therapies, further analysis of the tumor growth patterns for individual animals in each group revealed that a decrease in tumor volume was seen in more rats (four of six) in the ¹⁸⁶Re–liposomal doxorubicin plus RF ablation group than in the dual therapy groups (Table).

Micro-SPECT

¹⁸⁶Re–liposomal doxorubicin accumulation was increased in rats treated with RF ablation compared to those treated with ¹⁸⁶Re–liposomal doxorubicin alone (Fig 2). At baseline, the percentage injected dose in tumor per gram of tissue for ¹⁸⁶Re–liposomal doxorubicin plus RF ablation (1.55% ± 0.51) was not significantly different from that for ¹⁸⁶Re–liposomal doxorubicin alone (1.22% ± 0.2). By 2 days after therapy, the percentage injected dose in tumor per gram of tissue for ¹⁸⁶Re–liposomal doxorubicin increased to 2.61% ± 0.74 when administered with RF ablation compared with 1.78% ± 0.66 for ¹⁸⁶Re–liposomal doxorubicin alone. At 5 days, the percentage injected dose in tumor per gram of tissue decreased to 1.07% ± 0.25 with RF ablation and to 0.79% ± 0.31 without RF ablation. Tumor accumulation of ¹⁸⁶Re–liposomal doxorubicin increased 1.4 ± 0.08-fold when administered with RF ablation. The highest tumor accumulation of ¹⁸⁶Re–liposomal doxorubicin was observed 1 day after therapy, irrespective of RF ablation, and this increased accumulation is consistent with the circulation time of ¹⁸⁶Re–liposomal doxorubicin (32).

Figure 2a:

Planar images of ¹⁸⁶Re–liposomal doxorubicin accumulation in tumor with and without RF ablation (RFA). (a) Lateral planar images show accumulation of ¹⁸⁶Re–liposomal doxorubicin in tumor (T) with RF ablation (upper panel) and without RF ablation (lower panel) from 0 to 116 hours. A known amount of radioactivity from a standard source (S) was placed in field of view for image analysis. Accumulation of ¹⁸⁶Re–liposomal doxorubicin in tumor increased with RF ablation. Color scale represents image intensity (black = 0%, yellow = 100%). H = heart, K = kidney, L = liver, Sp = spleen. (b) SPECT images obtained with multipinhole collimator at 44 hours show increased tumor accumulation of ¹⁸⁶Re–liposomal doxorubicin after RF ablation.

Figure 2b:

Planar images of ¹⁸⁶Re–liposomal doxorubicin accumulation in tumor with and without RF ablation (RFA). (a) Lateral planar images show accumulation of ¹⁸⁶Re–liposomal doxorubicin in tumor (T) with RF ablation (upper panel) and without RF ablation (lower panel) from 0 to 116 hours. A known amount of radioactivity from a standard source (S) was placed in field of view for image analysis. Accumulation of ¹⁸⁶Re–liposomal doxorubicin in tumor increased with RF ablation. Color scale represents image intensity (black = 0%, yellow = 100%). H = heart, K = kidney, L = liver, Sp = spleen. (b) SPECT images obtained with multipinhole collimator at 44 hours show increased tumor accumulation of ¹⁸⁶Re–liposomal doxorubicin after RF ablation.

Micro-PET

Micro-PET images from rats treated with ¹⁸⁶Re–liposomal doxorubicin alone and in combination with RF ablation are shown in Figure 3. Micro-PET images revealed increased FDG uptake in tumor from baseline to 6 weeks after therapy for the control group and all therapy groups except liposomal doxorubicin combination therapies (Fig E1, online). Conversely, micro-PET images of liposomal doxorubicin combination therapy groups revealed an almost constant uptake of FDG in tumor up to 6 weeks, suggesting that the tumor volume and, thus, the number of cancer cells did not change from baseline—an indication of tumor growth control. The percentage injected dose of FDG in tumor increased over time for groups where the therapy failed to control tumor growth (Fig 4). For groups receiving liposomal doxorubicin plus RF ablation, ¹⁸⁶Re–liposomal doxorubicin, and ¹⁸⁶Re–liposomal doxorubicin plus RF ablation, the percentage injected dose of FDG in tumor was not significantly different from that at baseline, indicating stable disease (P = .175–.501). Comparison of the tumor growth trend (Fig 4a) and percentage injected dose of FDG in tumor (Fig 4b) revealed a correlation between the two measurements (R² = 0.85, P < .001).

Figure 3:

Micro-PET images in rats treated with triple versus dual combination therapy. Images were obtained in rats treated with ¹⁸⁶Re–liposomal doxorubicin alone (lower panel) and in combination with RF ablation (RFA) (upper panel) before and after therapy. FDG uptake in tumor (arrows) was lower with combination of ¹⁸⁶Re–liposomal doxorubicin and RF ablation (complete response) than with ¹⁸⁶Re–liposomal doxorubicin alone (partial response).

Figure 4a:

Graphs show change in (a) tumor volume and (b) percentage injected dose of FDG in tumor (%ID) for 42 days after therapy. All therapy groups showed correlation between tumor volume and percentage injected dose (R² = 0.85, P < .001). L = liposome, LD = liposomal doxorubicin, Re = ¹⁸⁶Re, RFA = RF ablation.

Figure 4b:

Graphs show change in (a) tumor volume and (b) percentage injected dose of FDG in tumor (%ID) for 42 days after therapy. All therapy groups showed correlation between tumor volume and percentage injected dose (R² = 0.85, P < .001). L = liposome, LD = liposomal doxorubicin, Re = ¹⁸⁶Re, RFA = RF ablation.

Viable Tumor Volume at Histopathologic Examination

Histopathologic examination of microscopic slides of tumor specimens did not reveal any unique pattern among the different therapy groups (Fig 5a, Fig E2 [online]). Histopathologic comparison of specimens stained with hematoxylin-eosin and MIB1 antibody for Ki67 immunolocalization indicated that the nonnecrotic cells on hematoxylin-eosin slides corresponded to the presence of Ki67, an indication of growing cells (Fig 5b).

Figure 5a:

Comparison of hematoxylin-eosin staining and Ki67 immunolocalization in representative tumor specimens. (a) Paraffin sections (4 μm thick) obtained from middle of tumor along its long-axis diameter were processed with routine procedures for hematoxylin-eosin staining (top); corresponding sections were used for Ki67 immunolocalization, counterstained with MIB1 (bottom). Tumors from all therapy groups were characterized by lobules of pleomorphic cancer cells, with pleomorphic nuclei separated by thick bands of collagen. Each lobule had a central necrotic region surrounded by basophilic cancer cells. All specimens contained ample tumor cells in periphery of tumor, whereas central regions were largely necrotic. Nevertheless, focal areas of viable tumor cells were scattered throughout necrotic regions. Regions stained with hematoxylin (purple) corresponded to presence of Ki67 (dark brown). LD = liposomal doxorubicin, Re =¹⁸⁶Re, RFA = RF ablation. (b) Photomicrographs of specimens stained with hematoxylin-eosin (top) and Ki67 immunolocalized specimens (bottom) from control group show that viable tumor cells are also proliferating tumor cells. (c) Graph shows correlation between viable tumor area determined with hematoxylin-eosin–stained specimens (H&E) and corresponding Ki67 immunolocalization (n = 12, R² = 0.89, P < .01), confirming that viable tumor cells were proliferating.

Figure 5b:

Comparison of hematoxylin-eosin staining and Ki67 immunolocalization in representative tumor specimens. (a) Paraffin sections (4 μm thick) obtained from middle of tumor along its long-axis diameter were processed with routine procedures for hematoxylin-eosin staining (top); corresponding sections were used for Ki67 immunolocalization, counterstained with MIB1 (bottom). Tumors from all therapy groups were characterized by lobules of pleomorphic cancer cells, with pleomorphic nuclei separated by thick bands of collagen. Each lobule had a central necrotic region surrounded by basophilic cancer cells. All specimens contained ample tumor cells in periphery of tumor, whereas central regions were largely necrotic. Nevertheless, focal areas of viable tumor cells were scattered throughout necrotic regions. Regions stained with hematoxylin (purple) corresponded to presence of Ki67 (dark brown). LD = liposomal doxorubicin, Re =¹⁸⁶Re, RFA = RF ablation. (b) Photomicrographs of specimens stained with hematoxylin-eosin (top) and Ki67 immunolocalized specimens (bottom) from control group show that viable tumor cells are also proliferating tumor cells. (c) Graph shows correlation between viable tumor area determined with hematoxylin-eosin–stained specimens (H&E) and corresponding Ki67 immunolocalization (n = 12, R² = 0.89, P < .01), confirming that viable tumor cells were proliferating.

Comparison of the microscopic viability factor for hematoxylin-eosin–stained and immunohistochemical specimens revealed a positive correlation between the two values (n = 12, R² = 0.89, P < .01) (Fig 5c).

Figure 5c:

Comparison of hematoxylin-eosin staining and Ki67 immunolocalization in representative tumor specimens. (a) Paraffin sections (4 μm thick) obtained from middle of tumor along its long-axis diameter were processed with routine procedures for hematoxylin-eosin staining (top); corresponding sections were used for Ki67 immunolocalization, counterstained with MIB1 (bottom). Tumors from all therapy groups were characterized by lobules of pleomorphic cancer cells, with pleomorphic nuclei separated by thick bands of collagen. Each lobule had a central necrotic region surrounded by basophilic cancer cells. All specimens contained ample tumor cells in periphery of tumor, whereas central regions were largely necrotic. Nevertheless, focal areas of viable tumor cells were scattered throughout necrotic regions. Regions stained with hematoxylin (purple) corresponded to presence of Ki67 (dark brown). LD = liposomal doxorubicin, Re =¹⁸⁶Re, RFA = RF ablation. (b) Photomicrographs of specimens stained with hematoxylin-eosin (top) and Ki67 immunolocalized specimens (bottom) from control group show that viable tumor cells are also proliferating tumor cells. (c) Graph shows correlation between viable tumor area determined with hematoxylin-eosin–stained specimens (H&E) and corresponding Ki67 immunolocalization (n = 12, R² = 0.89, P < .01), confirming that viable tumor cells were proliferating.

Viable tumor volume (Fig 6b) calculated from the microscopic viability factor (Fig 6a) and the tumor volume on the day of necropsy revealed that 50% of the tumor volume was viable in the control and therapy groups, with tumor volumes reaching 7–9 cm³. In the liposomal doxorubicin group, 45% of the tumor volume was viable; this decreased to 41% and 30% when used in combination with RF ablation and ¹⁸⁶Re, respectively. Only 28% of the tumor volume in the triple therapy group was viable. Viable tumor volumes for liposomal doxorubicin plus RF ablation, ¹⁸⁶Re–liposomal doxorubicin, and ¹⁸⁶Re–liposomal doxorubicin plus RF ablation were significantly decreased compared with that in the control group (P < .0001), revealing the therapeutic efficacy of these combination treatments. The percentage injected dose of FDG in tumor at necropsy (Fig 6c) showed correlation with the calculated viable tumor volume (R² = 0.77, P < .01).

Figure 6b:

Analysis of hematoxylin-eosin–stained tumor sections for tumor viability. (a) Bar chart shows that 50% of tumor section area in control group was viable, represented by the viability factor. (b) Viability factor determined from image analysis was applied to tumor volume measured at necropsy to calculate viable tumor volume, which indicates treatment efficacy. Bar chart shows that rats treated with combination therapies of liposomal doxorubicin, which had a smaller viability factor, showed a significant decrease in viable tumor volume compared with control animals (P < .0001, one-way analysis of variance) and those treated with liposomal doxorubicin alone (P < .05, one-way analysis of variance). (c) Bar chart shows percentage injected dose of FDG in tumor (%ID) from micro-PET images obtained before necropsy. There is good correlation with calculated viable tumor volume (b), indicating that FDG could potentially be a useful marker of tumor viability (R² = 0.77, P < .01). L = liposome, LD = liposomal doxorubicin, Re = ¹⁸⁶Re, RFA = RF ablation.

Figure 6a:

Analysis of hematoxylin-eosin–stained tumor sections for tumor viability. (a) Bar chart shows that 50% of tumor section area in control group was viable, represented by the viability factor. (b) Viability factor determined from image analysis was applied to tumor volume measured at necropsy to calculate viable tumor volume, which indicates treatment efficacy. Bar chart shows that rats treated with combination therapies of liposomal doxorubicin, which had a smaller viability factor, showed a significant decrease in viable tumor volume compared with control animals (P < .0001, one-way analysis of variance) and those treated with liposomal doxorubicin alone (P < .05, one-way analysis of variance). (c) Bar chart shows percentage injected dose of FDG in tumor (%ID) from micro-PET images obtained before necropsy. There is good correlation with calculated viable tumor volume (b), indicating that FDG could potentially be a useful marker of tumor viability (R² = 0.77, P < .01). L = liposome, LD = liposomal doxorubicin, Re = ¹⁸⁶Re, RFA = RF ablation.

Figure 6c:

Analysis of hematoxylin-eosin–stained tumor sections for tumor viability. (a) Bar chart shows that 50% of tumor section area in control group was viable, represented by the viability factor. (b) Viability factor determined from image analysis was applied to tumor volume measured at necropsy to calculate viable tumor volume, which indicates treatment efficacy. Bar chart shows that rats treated with combination therapies of liposomal doxorubicin, which had a smaller viability factor, showed a significant decrease in viable tumor volume compared with control animals (P < .0001, one-way analysis of variance) and those treated with liposomal doxorubicin alone (P < .05, one-way analysis of variance). (c) Bar chart shows percentage injected dose of FDG in tumor (%ID) from micro-PET images obtained before necropsy. There is good correlation with calculated viable tumor volume (b), indicating that FDG could potentially be a useful marker of tumor viability (R² = 0.77, P < .01). L = liposome, LD = liposomal doxorubicin, Re = ¹⁸⁶Re, RFA = RF ablation.

Discussion

Image-guided thermal and chemotherapies are increasingly being used for cancer treatment (34,35). It has been difficult to achieve complete ablation of large or infiltrative tumors and those adjacent to large blood vessels (18,34). New treatment strategies (eg, combination of RF ablation with chemotherapy or antiangiogenic or radiation therapy) have been developed to overcome this limitation (19,22–24,36). RF ablation in combination with liposomal doxorubicin increased not only the ablation zone but also the drug accumulation in the tumor periphery, where recurrence occurs (19). Reduced tumor growth with RF ablation and external beam radiation therapy has also been reported (24) and attributed to the increased oxygenation in the peripheral tumor owing to hyperthermia, which sensitizes the tumor to radiation therapy. Considering the synergy achieved with RF ablation and chemotherapy or radiation therapy, we investigated the potential of RF ablation and chemoradionuclide therapy. To our knowledge, no studies have reported results with a combination of RF ablation and chemoradionuclide therapy.

Concordant benefits for solid tumor therapy could be obtained by combining RF ablation with PEGylated liposomes encapsulating both doxorubicin and ¹⁸⁶Re for combination chemoradionuclide therapy. The group that received ¹⁸⁶Re–liposomal doxorubicin plus RF ablation had a minimal (1.1-fold) increase in tumor volume at 6 weeks (P < .001 vs control group). Histopathologic analysis revealed a decrease in viable tumor volume for rats treated with ¹⁸⁶Re–liposomal doxorubicin plus RF ablation compared with the control group (0.54 cm³ ± 0.38 vs 3.54 cm³ ± 0.76, respectively; P < .001). Analysis of micro-PET images showed good correlation between tumor volume and percentage injected dose of FDG. These results demonstrate that changes in percentage injected dose of FDG could be used to measure therapeutic efficacy in addition to changes in tumor volume in a preclinical model.

There was synergy between ¹⁸⁶Re and liposomal doxorubicin for chemoradionuclide therapy because the addition of ¹⁸⁶Re to liposomal doxorubicin decreased both tumor volume (P = .003) and viable tumor volume (P = .02). Increased efficacy could be due to the deposition of high local ¹⁸⁶Re β radiation dose in addition to doxorubicin cytotoxicity. A study of RF ablation combined with 20-Gy external beam radiation therapy reported that 82% of animals had complete tumor control (24). Although the addition of RF ablation to ¹⁸⁶Re–liposomal doxorubicin did not result in a significant decrease in average tumor volume or viable tumor volume compared to that with ¹⁸⁶Re–liposomal doxorubicin alone, four of six rats showed a decreasing tumor volume at 6 weeks. Possible reasons why we did not see complete tumor control as achieved by Horkan et al (24) with a combination of radiation therapy and RF ablation are as follows: (a) The tumor diameter in our study was twice that reported by Horkan et al (2.05 cm ± 0.24 vs 1.0 cm, respectively); (b) ¹⁸⁶Re radiation dose rate is not constant owing to clearance and decay and, hence, could allow for repair and repopulation of tumor cells; (c) the width of the remaining peripheral tumor is larger than the path length of the β emission of ¹⁸⁶Re and, hence, the distant cancer cells could not be treated; and (d) tumor vasculature was heterogeneous, leading to a heterogeneous distribution of liposomes in tumor.

For tumor debulking or complete tumor destruction, ¹⁸⁶Re–liposomal doxorubicin may be injected at 4-week intervals because the animals had recovered from body weight loss, leukopenia, and thrombocytopenia by 3 weeks (37). The RF ablation setup could also be changed to achieve larger ablation zones and complete tumor control (19). As seen from our results, ¹⁸⁶Re–liposomal doxorubicin could be used to control tumor growth in patients without the use of other treatment options because it is not always feasible to use RF ablation around critical structures of the head and neck. The synergy achieved between ¹⁸⁶Re and liposomal doxorubicin could be through two possible mechanisms of action: G₂/M phase arrest and DNA double strand breaks due to DNA unwinding leading to the increased radiosensitization of the cancer cells by doxorubicin (38–43).

A limitation of this study was that histopathologic specimens were obtained only at 6 weeks for the triple combination therapy, which did not enable study of the exact mechanism of the effects or ablation zone measurement. Earlier histologic time points could provide further information. The combination of ¹⁸⁶Re–liposomal doxorubicin with RF ablation was studied in only one tumor model and with a standard RF ablation protocol and therapy administration sequence. The effects seen could hold true in other solid tumors, but further therapy studies in other tumor models are required to prove that this synergistic effect is universal. In addition, therapeutic effects with different RF ablation protocols and chemoradionuclide administration timing and sequence must be studied to achieve the optimal treatment protocol for chemoradionuclide therapy in combination with RF ablation.

Practical applications: Combination therapy with ¹⁸⁶Re–liposomal doxorubicin chemoradionuclide therapy and RF ablation has the potential for translation to treatment of patients with solid tumors. Effective treatment is possible, as demonstrated by the good tumor control achieved in a head and neck tumor model in this study. Noninvasive monitoring of the response to combination therapy with FDG PET is important to determine its efficacy in solid tumors. As shown in our study, FDG could be used as a surrogate marker of tumor growth and tumor viability in this model of head and neck cancer.

Advances in Knowledge.

• • In a head and neck model, the combination of radiofrequency (RF) ablation with rhenium 186 (¹⁸⁶Re)–labeled liposomal doxorubicin (¹⁸⁶Re–liposomal doxorubicin) resulted in good tumor growth control (average tumor volume, 1.49 cm³ ± 1.47) compared with other therapies tested (average tumor volume range, 2.05 cm³ ±1.36 to 11.11 cm³ ± 2.32).

• • Histopathologic analysis of tumors revealed that treatment with a combination of ¹⁸⁶Re–liposomal doxorubicin and RF ablation resulted in a significantly smaller viability factor (average, 0.28 ± 0.04) and viable tumor volume (average, 0.54 cm³ ± 0.38) compared with other therapies tested.

Implications for Patient Care.

• • When combined with RF ablation, ¹⁸⁶Re–liposomal doxorubicin provided better tumor control than liposomal doxorubicin through the deposition of a high local ¹⁸⁶Re radiation dose in the tumor in addition to the increase in the ablation zone and increased accumulation of liposomal doxorubicin.

• • Because the toxicity associated with ¹⁸⁶Re–liposomal doxorubicin chemoradionuclide therapy was limited and recoverable, this therapy in combination with RF ablation may potentially offer additional therapy options for patients with unresectable tumor.

Disclosures of Potential Conflicts of Interest: A.S. Financial activities related to the present article: RF equipment was provided to institution in kind from Valleylab (Covidien). Financial activities not related to the present article: none to disclose. Other relationships: none to disclose. G.D.D. Financial activities related to the present article: RF equipment was provided to institution in kind from Valleylab (Covidien). Financial activities not related to the present article: none to disclose. Other relationships: none to disclose. A.B. No potential conflicts of interest to disclose. W.T.P. No potential conflicts of interest to disclose. L.M.M. Financial activities related to the present article: none to disclose. Financial activities not related to the present article: received payment for development of educational presentations from Elsevier. Other relationships: none to disclose. T.J.P. Financial activities related to the present article: none to disclose. Financial activities not related to the present article: received money for grants or grants pending for various research projects. Other relationships: none to disclose. B.A.G. Financial activities related to the present article: RF equipment was provided to institution by Valleylab (Covidien). Financial activities not related to the present article: none to disclose. Other relationships: none to disclose.