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Clinical Cancer Research Vol. 12, 4590-4597, August 1, 2006
© 2006 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Early Detection of Chemoradioresponse in Esophageal Carcinoma by 3'-Deoxy-3'-3H-Fluorothymidine Using Preclinical Tumor Models

Smith Apisarnthanarax1, Mian M. Alauddin2, Firas Mourtada3, Hisanori Ariga1, Uma Raju1, Osama Mawlawi4, Dongmei Han2, William G. Bornmann2, Jaffer A. Ajani5, Luka Milas1, Juri G. Gelovani2 and K.S. Clifford Chao1

Authors' Affiliations: Departments of 1 Radiation Oncology, 2 Experimental Diagnostic Imaging, 3 Radiation Physics, 4 Imaging Physics, and 5 GI Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: K.S. Clifford Chao, Department of Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-563-2300; Fax: 713-563-2368; E-mail: cchao{at}mdanderson.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Early identification of esophageal cancer patients who are responding or resistant to combined chemoradiotherapy may lead to individualized therapeutic approaches and improved clinical outcomes. We assessed the ability of 3'-deoxy-3'-18F-fluorothymidine positron emission tomography (FLT-PET) to detect early changes in tumor proliferation after chemoradiotherapy in experimental models of esophageal carcinoma.

Experimental Design: The in vitro and ex vivo tumor uptake of [3H]FLT in SEG-1 human esophageal adenocarcinoma cells were studied at various early time points after docetaxel plus irradiation and validated with conventional assessments of cellular proliferation [thymidine (Thd) and Ki-67] and [18F]FLT micro-PET imaging. Imaging-histologic correlation was determined by comparing spatial Ki-67 and [18F]FLT distribution in autoradiographs. Comparison with fluorodeoxyglucose (FDG) was done in all experiments.

Results: In vitro [3H]FLT and [3H]Thd uptake rapidly decreased in SEG-1 cells 24 hours after docetaxel with a maximal reduction of over 5-fold (P = 0.005). The [3H]FLT tumor-to-muscle uptake ratio in xenografts declined by 75% compared with baseline (P < 0.005) by 2 days after chemoradiotherapy, despite the lack of change in tumor size. In contrast, the decline of [3H]FDG uptake was gradual and less pronounced. Tumor uptake of [3H]FLT was more closely correlated with Ki-67 expression (r = 0.89, P < 0.001) than was [3H]FDG (r = 0.39, P = 0.08). Micro-PET images depicted similar trends in reduction of [18F]FLT and [18F]FDG tumor uptake. Autoradiographs displayed spatial correlations between [18F]FLT uptake and histologic Ki-67 distribution in preliminary studies.

Conclusions: FLT-PET is suitable and more specific than FDG-PET for depicting early reductions in tumor proliferation that precede tumor size changes after chemoradiotherapy.

Esophageal cancer is one of the most lethal human malignancies. Achievement of pathologic complete response after neoadjuvant chemoradiotherapy in esophageal cancer patients has been shown to be associated with improved clinical outcomes (1–4). Methods to reliably evaluate and predict the proportion of residual cancer in the esophagus after chemoradiotherapy, however, are currently lacking. The ability to predict which patients will respond to chemoradiotherapy or develop resistance to it before surgery would be invaluable for individualizing therapeutic approaches and avoiding overtreatment and undertreatment. Therefore, the need for a noninvasive tool that accurately predicts response early during the course of preoperative therapy is widely acknowledged.

Conventional anatomic imaging modalities, such as computed tomography and endoscopic ultrasonography, depict cancer response as changes in tumor size and composition, which may occur only after weeks or months following therapy, and are not ideally suitable for early prediction of response in esophageal cancer. Computed tomography is inaccurate and endoscopic ultrasonography is not always feasible in this setting (5). Positron emission tomography (PET), with the glucose analogue [18F]fluorodeoxyglucose (FDG), addresses these inherent limitations in anatomic imaging, but may be limited in distinguishing proliferating tumor cells from inflammatory tissue (6–8). Other radiolabeled molecular markers may improve the specificity and accuracy of early assessment of treatment response.

Because deregulated proliferation is one of the hallmarks of cancer and a high tumor proliferative rate is associated with aggressive biological behavior and poor response to therapy (9, 10), imaging the proliferative state of cancer cells is of great interest. Recently, 3'-deoxy-3'-18F-fluorothymidine ([18F]FLT), a pyrimidine nucleoside analogue, has been validated in several in vitro and in vivo studies as a promising PET radiopharmaceutical for imaging proliferation and monitoring tumor response (11–19). The basis of FLT-PET as a surrogate measure of cancer cell proliferation is dependent on the monophosphorylation of FLT by the thymidine kinase 1 (TK1) enzyme, which results in intracellular trapping of FLT (20). TK1 is virtually absent in quiescent cells but differentially expressed in the late G1 and S phases of the cell cycle (21, 22).

Although FLT-PET has been used to assess proliferation and response to therapy (11, 12, 15–19), no study to date has examined FLT-PET for monitoring response to chemotherapy in combination with radiation. We hypothesize that FLT-PET depicts early changes in the proliferative state of esophageal cancer after chemoradiotherapy. To test this hypothesis, we developed experimental in vitro and murine models of esophageal cancer and used a treatment regimen consisting of docetaxel, a semisynthetic taxane (23), and irradiation. Docetaxel in combination with irradiation has been investigated in preclinical studies (23–28) and used in the clinic to produce an ~25% pathologic complete response rate in esophageal cancer (29–31). The main objective of this study is to determine whether FLT-PET is suitable and more specific than FDG-PET as a surrogate marker of chemoradioresponse for depicting early reductions in tumor proliferation that precede tumor size changes.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Radiopharmaceuticals. Methyl-3H-thymidine ([3H]Thd; specific activity, 65 Ci/mmol), methyl-3H(N)-3'-fluoro-3'-deoxythymidine ([3H]FLT; specific activity, 3.4 Ci/mmol), and 5,6-3H-2-fluoro-2-DDG ([3H]FDG; specific activity, 60 Ci/mmol) were purchased from Sigma-Aldrich (Milwaukee, WI), Moravek Biochemicals (Brea, CA), and American Radiolabeled Chemicals, Inc. (St. Louis, MO), respectively. [18F]FDG was purchased from PET NET-CTI (Knoxville, TN); specific activity was estimated to be >2 Ci/µmol. [18F]FLT was synthesized by radiofluorination of the 3-N-Boc-1[5-O-(44'-dimethoxytrityl)-3-O-nosyl-2-deoxy-ß-D-lyxofuranosyl]-thymine precursor as previously described (32). Analytic HPLC showed the product to be >99% radiochemically pure. Radiochemical yield was 7%, and specific activity was estimated to be >2 Ci/µmol.

Cells and cell culture. SEG-1 cells, derived from a human Barrett's-associated adenocarcinoma of the distal esophagus (33), were maintained and grown in DMEM/F12 culture medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine. Cultures were kept in a humidified 5% CO2 incubator at 37°C. For cell cycle analysis and radiotracer uptake experiments, 1 x 106 cells were seeded onto 60-mm culture dishes, and 5 x 105 cells were seeded onto six-well plates, respectively.

In vitro treatment with chemoradiation. A single dose of 10 nmol/L docetaxel (Fluka, Sigma Chemical Co., St. Louis, MO) was added to the culture medium. Twenty-four hours after docetaxel treatment, the cells were irradiated with a single dose of 8 Gy using a 137Cs source (3.7 Gy/min). All control wells were treated with PBS. The concentration of docetaxel and dose of radiation were chosen based on previously published work by Dunne et al. (24) and preliminary data demonstrating cytostatic doses of docetaxel and radiation (data not shown).

In vitro uptake of radiotracers. Cellular uptake of radiotracers was determined according to methods previously described (34) with some modifications. At 0, 4, 12, and 24 hours after docetaxel treatment and 4, 12, 24, and 48 hours after irradiation, 0.2 µCi [3H]Thd, [3H]FLT, or [3H]FDG was added to the medium of each well and incubated for 60 minutes. Untreated wells served as controls, which were incubated with the radiotracers as described for the treated cells. After incubation, the medium was rapidly removed and rinsed with cold PBS. The cells were harvested by gentle scraping, centrifuged, and weighed and assayed for radioactivity after removal of excess supernatant. The cell pellets and the media samples were solubilized with Soluene 350 and mixed with a liquid scintillation cocktail (Ultima Gold, Packard, Meridian, CT). Radioactivity was measured using a liquid scintillation counter (Tri-Carb 2100TR, Packard) and expressed as dpm. Radiotracer uptake was expressed as cell-to-media ratio: (dpm/g cell) / (dpm/mL medium). Experiments were done and repeated in triplicate.

Cell cycle analysis. At various time points (0, 4, and 24 hours after docetaxel treatment and 24 hours after irradiation), cells were harvested by trypsinization, resuspended in growth medium, resuspended in 2 mL PBS, and fixed by the addition of ice-cold 95% ethanol to 60% while being vortexed. Cells were stored in ethanol overnight at –20°C before staining, then washed and resuspended in PBS containing 10 µg/mL propidium iodide and 250 µg/mL RNase A. Cells were incubated at 37°C for 15 minutes before flow cytometric analysis using a Coulter EPICS Profile instrument (Beckman Coulter, Inc., Fullerton, CA). Cell cycle distributions were determined from histograms using Multicycle (Phoenix Flow Systems, San Diego, CA).

Animal tumor model. Male Swiss nu/nu mice from our own specific pathogen-free mouse colony were used at an age of 5 months. Mice were housed three to five per cage; fed sterilized, pelleted food (Agway, Inc., Syracuse, NY), and sterile water ad libitum; and exposed to a 12-hour light/dark cycle. The experimental protocol was approved by the Institutional Animal Care and Use Committee. Mice were maintained in a fully accredited animal facility (American Association for Accreditation of Laboratory Animal Care) and in accordance with present regulations and standards of the U.S. Department of Health and Human Services. Solitary SEG-1 tumor xenografts were generated in the right hind limbs of the mice by 10 µL i.m. injection of a single-cell suspension of 1 x 106 tumor cells. After tumors had grown to a mean diameter of 8 mm as measured in three orthogonal diameters with Vernier calipers, mice were randomized to treatment versus control groups. Mice were treated with either docetaxel alone, radiation alone, docetaxel followed by radiation, or PBS (n = 8).

Chemoradiation treatment of xenografts. Mice were treated with docetaxel and radiation as described previously (26). Tumors were locally irradiated 24 hours after docetaxel treatment (33 mg/kg i.v. via tail vein) with a single dose of 15 Gy using a small animal 137Cs irradiator. Irradiations were given under ambient air breathing conditions at a dose rate of 5.4 Gy/min. During irradiation, the mice were mechanically immobilized (unanesthetized) on a jig, and the tumor was centered in a 3-cm-diameter circular field. Tumor response to treatment was assessed by tumor growth delay as previously described (26).

Immunohistochemical staining for proliferation. Excised tumors were fixed in 10% neutral buffered formalin and embedded in paraffin blocks, from which 4-µm sections were cut for H&E and immunohistochemical staining. A section from each specimen was labeled using the monoclonal antibody B56 (BD PharMingen, San Diego, CA), which recognizes the nuclear proliferation associated Ki-67 antigen (35), and visualized using the Vectastain ABC kit with 3,3'-diaminobenzidine (Vector Laboratories, CA) and hematoxylin counterstain. Ki-67 expression was quantified according to previously described methods with modifications (36). The fraction of Ki-67-labeled tumor cells was assessed by microscopic examination of stained sections at x400 magnification. Five fields of nonnecrotic areas were randomly selected in each specimen, and in each field, the numbers of Ki-67-labeled cells were recorded as numbers per 200 to 300 nuclei scored and expressed as labeling index percentages (LIKi-67).

Radiotracer uptake in tumor tissue. In SEG-1-bearing mice, tumor uptake of [3H]FLT or [3H]FDG was assessed at 1, 2, and 4 days after chemoradiotherapy (n = 5). A bolus injection of [3H]FLT or [3H]FDG was administered to the mice i.v. via the lateral tail vein at a dose of 1 µCi in 200 µL. The mice were sacrificed 60 minutes after radiotracer injection by cervical dislocation. Muscle and tumor tissue were rapidly harvested, weighed, and solubilized with Solvable (Perkin-Elmer, Boston, MA). Radioactivity in the samples was measured using a Tri-Carb 2100TR liquid scintillation counter (Packard), and the results were expressed as percentage injected dose per gram of tissue (%ID/g). Uptake of [3H]FLT was compared with LIKi-67 within the same tumors.

Micro-PET imaging and analysis. Untreated and chemoradiotherapy-treated mice were imaged on a micro-PET R4 scanner (Concorde Microsystems, Knoxville, TN). The animals (n = 2) were first anesthetized and injected with 7 MBq (200 µCi) of either [18F]FLT or [18F]FDG. Sixty minutes after injection, the animals were positioned in the field of view of the scanner and imaged for 15 minutes. Care was taken to reproduce the animal positioning between the [18F]FLT and [18F]FDG imaging sessions. The acquired list data was then histogrammed into a single frame using Fourier rebinning, and the images were reconstructed into a 128 x 128 x 63 (0.72 x 0.72 x 1.3 mm) matrix using ordered subset expectation maximization. All corrections for attenuation, scatter, dead time, and randoms were applied to generate quantifiable images. Regions of interest were then drawn over the tumor and muscle tissue without including their respective visible boundaries and an average value within the regions of interest was generated. A ratio of tumor-to-muscle (T/M) was calculated to eliminate the dependency on the injected activity. This ratio was evaluated before and after treatment as an indicator of treatment response.

Imaging-histologic correlation with autoradiography. Autoradiography was done only on tumors from mice injected with [18F]FLT. Upon completion of the micro-PET imaging studies, tumors were removed from mice and immediately frozen and processed. Sliced 8-µm sections were fixed in cold acetone, dried, and exposed to a multipurpose storage phosphor screen (Amersham Biosciences, Piscataway, NJ) overnight. A Typhoon 9400 storage phosphor screen imager (Amersham Biosciences) was used to generate digital autoradiographs and detect the distribution of radionuclide activity localized in each section. After complete radioactive decay of [18F]FLT, these same sections were processed for Ki-67 immunohistochemical staining as previously described (31, 32). Spatial correlations between regions of high, intermediate, and low intensity [18F]FLT uptake and Ki-67 expression were examined.

Statistical analysis. Statistical comparisons between controls and treated groups were calculated using the two-sided Student's t test for changes in tumor size and LIKi-67. Single-factor ANOVA was used to compare in vitro and in vivo radiotracer uptake before and after treatment. Correlations between histologic and radiotracer uptake findings were tested using linear regression analysis. All quantitative data were reported as mean ± SE unless otherwise specified. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Cellular uptake of radiotracers in vitro after chemoradiotherapy. Figure 1 shows the cellular uptake of [3H]Thd, [3H]FLT, and [3H]FDG in SEG-1 cells after treatment with docetaxel and radiation. The data were compared with untreated cell uptake in percentage. Although baseline cellular uptake of [3H]FLT was 5.6-fold lower than that of [3H]Thd (3.92 ± 0.18 versus 21.80 ± 0.22 cell/medium dpm, respectively), the dynamics of [3H]FLT uptake after treatment (Fig. 1B) closely resembled that of [3H]Thd uptake (Fig. 1A). Similar to [3H]Thd, docetaxel initially increased [3H]FLT uptake from a baseline of 3.92 ± 0.18 to a peak of 6.34 ± 0.08 at 12 hours (+62%, P = 0.008). The uptake subsequently declined rapidly to 2.92 ± 0.16 (–26%, P = 0.005) at 24 hours and continued to steadily decrease to 0.68 ± 0.06 at 72 hours after docetaxel treatment (24 hours after irradiation) compared with baseline uptake (–83%, P = 0.006).


Figure 1
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  		Fig. 1. Cellular uptake of [3H]Thd, [3H]FLT, and [3H]FDG in SEG-1 cells and cell cycle analysis after docetaxel and radiation in vitro. Plated SEG-1 cells were treated with 10 nmol/L docetaxel (0 hour) followed by 15 Gy irradiation (XRT; 24 hours). A, [3H]Thd cellular uptake served as the gold standard measure of proliferative activity. B, [3H]FLT and [3H]FDG cellular uptake. C, flow cytometric analysis of cell cycle distributions. Experiments were done in triplicates and repeated. Points, mean; bars, SE. P values were derived by ANOVA.

 
The cellular uptake dynamics of [3H]FDG after treatment was characterized by a delayed and less pronounced decrease compared with [3H]Thd and [3H]FLT (Fig. 1B). A statistically significant reduction in [3H]FDG uptake was not observed until 36 hours after docetaxel (12 hours after irradiation). [3H]FDG uptake declined by 31% compared with baseline (P = 0.01) at 36 hours and continued to gradually decrease an additional 10% compared with baseline (P = 0.004) at 72 hours after docetaxel (48 hours after irradiation).

Cell cycle analysis. To examine the effects of docetaxel and irradiation on cell cycle distribution, flow cytometric analysis was done on SEG-1 cells in vitro after treatment (Fig. 1C). The percentage of cells in G2-M phase was elevated from a baseline of 20.61% to 61.88% at 48 hours after docetaxel treatment (24 hours after irradiation), whereas cells in G0-G1 phase were markedly reduced to 8.09% from a baseline of 39.43%. The effect of docetaxel and radiation on S-phase cells was similar to the alterations in [3H]Thd and [3H]FLT cellular uptake. The percentage of cells in S phase initially increased to 47.46% at 12 hours after docetaxel from a baseline of 39.96% and subsequently declined to 30.03% at 48 hours after docetaxel treatment (24 hours after irradiation).

Effect of chemoradiotherapy on xenograft tumor size and proliferation. The antitumor effect of docetaxel and radiation on SEG-1 tumor-bearing mice, assessed by tumor growth delay, is shown in Fig. 2A . Although the untreated group tumors continued to increase in size during the first few days after treatment, the tumors in the docetaxel plus irradiation group maintained stable sizes: 0 days (pretreatment), 7.98 ± 0.05 mm; 2 days, 8.12 ± 0.08 mm; and 4 days, 7.96 ± 0.12 mm. A significant reduction in tumor size was observed at 8 days with a maximal decline at 10 days in this treatment group (P = 0.03).


Figure 2
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  		Fig. 2. Effect of docetaxel and radiation on growth and tumor proliferation in SEG-1 tumor xenografts. A, tumor growth delay after vehicle (no treatment), 33 mg/kg i.v. docetaxel, 15 Gy irradiation, or combination of docetaxel plus irradiation (n = 8 mice/group). B, changes in tumor proliferation (Ki-67) up to 96 hours after docetaxel plus irradiation (n = 5 mice/time point). Columns, mean; bars, SE. C, typical Ki-67-stained histologic sections of SEG-1 tumors 12 to 96 hours after docetaxel plus irradiation treatment are shown at x40 magnification (x10 eyepiece). Differences in brown staining, differences in proliferation. Blue staining, hematoxylin counterstaining.

 
Because there was no significant change in tumor size in the first 4 days after chemoradiotherapy, we stained and quantified histologic sections of SEG-1 tumors for Ki-67 to assess temporal changes in tumor proliferation after treatment during this time period (Fig. 2B-C). The baseline LIKi-67 of pretreatment SEG-1 tumors was high at 92.10 ± 1.49%. A statistically significant decrease in tumor proliferation by 11% (P = 0.02) was observed as early as 4 hours after treatment compared with pretreatment LIKi-67 (data not shown). LIKi-67 in treated tumors continued to decline rapidly by 87% to 12.43 ± 1.05% at 96 hours compared with untreated tumors (P < 0.001).

Ex vivo uptake of [3H]FLT and [3H]FDG in tumors after chemoradiotherapy and correlation with proliferative markers. Treatment with docetaxel plus radiation resulted in decreases for both [3H]FLT and [3H]FDG tumor uptake (Fig. 3 ). Absolute tumor uptake of [3H]FLT after treatment was characterized by a statistically significant steep decline compared with pretreatment tumors at 1 day (–72%, P = 0.006), followed by a continued stable decline (2 days, –75%; 4 days, –76%; P < 0.005). The decrease in [3H]FDG tumor uptake after treatment, however, was less pronounced, reaching a statistically insignificant decrease by 15% at 4 days compared with pretreatment tumors. Figure 3A summarizes changes in [3H]FLT and [3H]FDG tumor uptake; in addition, tumor size and Ki-67 expression changes are charted for correlation of radiotracer uptake with independent measures of tumor response. The [3H]FLT T/M uptake ratio decreased by 57% at 1 day, 80% at 2 days, and 88% at 4 days in treated tumors compared with that of pretreatment tumors (ANOVA, P < 0.001; Fig. 3B). In contrast, the [3H]FDG T/M uptake ratio gradually declined and did not reach statistical significance.


Figure 3
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  		Fig. 3. Tumor uptake of [3H]FLT and [3H]FDG after docetaxel and radiation. A, effect of docetaxel plus irradiation on [3H]FLT and [3H]FDG uptake in excised SEG-1 xenografts (n = 5 mice/time point per radiotracer) shown up to 4 days after treatment. Tumor size and Ki-67 expression were also shown as independent measures of tumor response for correlation. Data are expressed as percentage uptake compared with untreated control tumors. B, tumor uptake of [3H]FLT and [3H]FDG normalized to muscle uptake and expressed as T/M uptake ratio. Columns, mean; bars, SE. *, P < 0.005, using ANOVA. Correlation between mean [3H]FLT (C) and [3H]FDG (D) T/M uptake ratios and Ki-67 labeling index within individual SEG-1 tumor xenografts.

 
To further investigate the specificity of FLT and FDG uptake for changes in tumor proliferation after chemoradiotherapy, we compared the radiotracer uptake for individual tumors with the corresponding changes in LIKi-67 within the same tumors (Fig. 3C-D). A strongly positive correlation was found between [3H]FLT uptake in SEG-1 tumors and LIKi-67 (r = 0.89, P < 0.001). [3H]FDG tumor uptake, however, weakly correlated with LIKi-67 (r = 0.39, P = 0.08).

Micro-PET studies. Figure 4 shows representative coronal and transverse micro-PET images of SEG-1 tumor-bearing mice at 1 hour after injection of [18F]FLT and [18F]FDG in untreated and 2 days postchemoradiotherapy animals. The micro-PET imaging data were in agreement with the ex vivo radiotracer uptake studies. Both [18F]FLT (T/M uptake ratio 8.79) and [18F]FDG (T/M uptake ratio 5.36) clearly visualized untreated control tumors implanted into the right hind limbs of the mice. Chemoradiotherapy markedly reduced tumor uptake of [18F]FLT after 2 days, decreasing the T/M uptake ratio by –58%. The decrease in [18F]FDG T/M uptake ratio was less pronounced (–17%) compared with [18F]FLT reduction. The size of treated tumors did not significantly change in these mice, which was consistent with the tumor growth delay studies.


Figure 4
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  		Fig. 4. [18F]FLT and [18F]FDG micro-PET imaging of SEG-1 tumor-bearing mice. Coronal (top row) and axial (bottom row) micro-PET sections of mice 1 hour after injection with [18F]FLT (left columns) or [18F]FDG (right columns) before treatment and 2 days after chemoradiation (CRT; n = 2 mice/group). Implanted tumors (arrows) are located on the right (Rt) hind limbs of mice. Color scale, high (red) and low (purple) radiotracer uptake. Tumor uptake of radiotracers is normalized to muscle radiotracer uptake and expressed as T/M uptake ratio. *, bladder.

 
Imaging-histologic spatial correlation. Figure 5 shows preliminary data that the spatial distribution of [18F]FLT uptake in untreated and chemoradiotherapy treated tumors correlated well with the spatial distribution of Ki-67 expression. The overall intensity of [18F]FLT uptake in the autoradiographs of treated and 2 days postchemoradiotherapy tumors seemed consistent with the observed micro-PET images. Regions of high-intensity [18F]FLT uptake in the autoradiographs exhibited prominent staining for Ki-67. This positive spatial correlation was also observed in intermediate- and low-intensity areas of [18F]FLT uptake.


Figure 5
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  		Fig. 5. Spatial correlation of [18F]FLT uptake and Ki-67 expression distributions in autoradiographs of SEG-1 tumors. Middle, autoradiographs of untreated (1) and 2 days postchemoradiotherapy (2) tumors. Expression of Ki-67 by histologic examination is shown from selected areas of high (E and F), intermediate (A to D), and low (G and H) [18F]FLT intensity uptake. Magnifications of Ki-67 staining are shown as insets in each panel. Cells stained brown, positive for Ki-67; cells stained blue, counterstained with hematoxylin. Arrows in (G) outline an island cluster of nonproliferating tumor cells. Color scale indicates high (red) and low (purple) [18F]FLT uptake.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This is the first study to provide preclinical evidence that FLT-PET accurately depicts early changes in tumor proliferation after chemoradiotherapy that precede tumor volume changes in in vitro and in vivo models of esophageal cancer. Our findings suggest that FLT-PET may be useful as a noninvasive imaging modality to assess early response to chemoradiotherapy in esophageal cancer. The ability of FLT-PET to detect response to chemoradiotherapy within days of treatment (after 48 hours) may allow physicians to overcome the inherent limitations imposed by conventional anatomic imaging and individualize therapeutic approaches according to whether tumors are chemoradioresponsive or chemoradioresistant. Specifically, the early identification of chemoradiotherapy responders and nonresponders would assist in avoiding unnecessary, highly morbid surgical procedures for responders (i.e., esophagectomy) and discontinuing ineffective neoadjuvant treatment regimens and instituting alternative therapies for nonresponders, thereby reducing delays in clinical management and potentially improving clinical outcomes in esophageal cancer.

The combination of docetaxel and radiation resulted in significant reduction of FLT uptake in tumors, which was depicted in our in vitro and ex vivo uptake and micro-PET imaging studies. When normalized to muscle tissue, FLT uptake in tumors in our ex vivo study decreased in a stepwise fashion with a sharp decline as early as 1 day after treatment, followed by a stable decline over the next 3 days. [18F]FLT micro-PET showed the feasibility of imaging these changes. Importantly, these reductions in FLT tumor uptake were not secondary to decreases in tumor volume, because early reduction in FLT uptake occurred during the first few days of cytostatic tumor growth. Similar findings were reported in other FLT-PET preclinical studies that used different anticancer therapies, including chemotherapy (15, 16, 19) and radiation (12, 17). Our data lend further support to the literature showing the ability of FLT-PET to detect therapeutic alterations in tumor biology before changes in tumor size are clinically detectable. It is not clear at this point whether FLT uptake after chemoradiotherapy differs from treatment with chemotherapy alone or radiation alone. The intent of the growth delay studies was to show the superior efficacy of combined docetaxel and radiation in our tumor model rather than to compare FLT uptake changes between the different treatment regimens. Future studies examining these differences would be interesting.

We validated the specificity of FLT-PET for depicting changes in tumor proliferation by demonstrating a significantly positive correlation between FLT tumor uptake reduction and conventional assessments of cellular proliferation. The dynamics of this reduction in vitro closely resembles the changes in [3H]Thd tumor uptake after treatment, which is consistent with other in vitro comparative uptake studies of [3H]FLT and [3H]Thd (14, 20). Our data, however, are the first to establish that this correlation with Thd remains intact after anticancer treatment in vitro. The altered patterns of cell cycle distribution of SEG-1 cells after docetaxel (i.e., G2-M cell cycle arrest) were also similar to findings in other published reports using taxanes (24, 25, 37). The reduction in G1-S phase cells after docetaxel further supports the rationale for using FLT-PET to assess proliferative response after chemoradiotherapy, because cellular FLT uptake is dependent on late G1-S phase specific expression of TK1 (21, 22).

It is interesting to note the initial increase in [3H]FLT and [3H]Thd uptake immediately after docetaxel that precedes their decline (Fig. 1). The cell cycle data also show a similar increase in S-phase cells, which suggests that this finding may be explained by an accumulation of S-phase cells as cells progress through the cell cycle beyond the G2-M into S phase. This in vitro finding is in contrast to the early decreases in [3H]FLT and Ki-67 observed in tumor xenografts after chemoradiotherapy. It must be pointed out, however, that the early decreases in proliferation in vivo are observed after the administration of both docetaxel and irradiation 24 hours later. Although we did not study the proliferative status of xenografts immediately after docetaxel alone, it is possible that a transient, initial increase in Ki-67 would also have been observed similar to in vitro studies.

A unique finding in our study was the spatial correlation between FLT uptake and Ki-67 expression distribution on autoradiography. Previous studies have shown the strong correlation between overall FLT tumor uptake and histologic assessment of cellular proliferation (Table 1 ). Although preliminary, our finding is the first evidence that this correlation also exists spatially for FLT-PET with reasonably high fidelity, adding to the robustness of FLT as an imaging surrogate of tumor proliferation. Furthermore, imaging-histologic correlation also buttresses the rationale and feasibility of delivering escalated doses of radiation to regions of high tumor proliferative activity in the clinical setting through conformal image-guided radiation therapy. A recent small pilot study that staged 10 esophageal cancer patients with the aid of [18F]FLT and [18F]FDG contradicts our results, reporting a negative correlation between [18F]FLT and Ki-67 in biopsy specimens (38). The small number of patients and potential for sampling error in biopsy specimens in that study may have contributed to the discrepancy from our findings. Only a study involving more patients and tightly controlled tumor sampling protocols could resolve this discrepancy.


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  		Table 1. Correlation between FLT tumor uptake and histologic proliferative markers in published literature

 
Although studies have shown that changes in [18F]FDG uptake during neoadjuvant chemoradiotherapy are correlated with patient outcomes in esophageal cancer (2, 39, 40), other studies have suggested that [18F]FDG may have difficulty in differentiating between complete responses and residual disease or posttreatment inflammation (6–8). Recent animal and preliminary clinical studies have suggested that [18F]FLT may be an even more sensitive measure than [18F]FDG for tumor response (12, 15–17, 19, 41–43). For example, Yang et al. (12) recently showed larger early decreases in [18F]FLT uptake in SCCVII tumor-bearing mice after irradiation than in [18F]FDG uptake. Leyton et al. (16) showed that [18F]FLT was superior in assessing tumor proliferation compared with [18F]FDG in RIF-1 tumor-bearing mice after cisplatin treatment (r = 0.89 versus r = 0.55, respectively). Similarly, we showed early decreases in FLT tumor uptake that were more pronounced and specific for tumor proliferation than those of FDG. Furthermore, when normalizing FLT uptake to background (muscle) uptake as was done in other studies (12, 18, 19), FLT displayed a much greater T/M signal contrast than FDG. Our data provide additional evidence for the assertion that imaging tumor proliferation with [18F]FLT is more suitable for monitoring treatment response than imaging tumor glucose metabolism.

It should be noted that in our tumor models, the baseline absolute [3H]FDG tumor uptake in our in vitro and ex vivo studies was low compared with absolute [3H]FLT tumor uptake. This is in contrast to several other studies comparing [18F]FLT and [18F]FDG tumor uptake in which the absolute [18F]FDG tumor uptake was ~1.5 to 5x greater than that of [18F]FLT (15–17, 42). It is possible that some of the 3H labeled to FDG in our experiments may have exchanged with water and resulted in decreased 3H counts within the cells or tumors at baseline. However, any potential errors arising from this phenomenon would likely have been eliminated by the use of the T/M uptake ratio, as this [3H]water exchange would also have occurred in muscle tissue and thus canceled each other. It is also possible that, similar to the low baseline [18F]FDG uptake observed in the Waldherr et al. study (19), SEG-1 tumor cells in our study had low FDG uptake as well. However, this is unlikely considering the relatively high tumor uptake of [18F]FDG in our micro-PET imaging study.

In conclusion, this present study showed that FLT-PET is suitable for depicting early reductions in tumor proliferation (48 hours after chemoradiotherapy) in preclinical models of esophageal cancer after chemoradiotherapy before changes in tumor size are evident. We conclude that FLT-PET is superior to FDG-PET as a noninvasive imaging tool to assess early tumor proliferative responses to chemoradiotherapy. Additional studies are ongoing to further determine whether FLT-PET can distinguish between varying degrees of treatment response and predict for pathologic complete response as well as detect early biological changes during the regrowth phase after ineffective therapy. Imaging-pathologic studies in patients with esophageal cancer are also under way to validate the sensitivity and specificity of FLT-PET in patients receiving chemoradiotherapy. If these endeavors are successful, they may allow physicians to tailor chemoradiotherapy regimens during the early phase of a treatment course and avoid ineffective and morbid therapies.


   Acknowledgments
 
We thank Nancy Hunter, Bobby Neal, and David Valdecanas for their invaluable assistance in the animal studies; Kuriakose Abraham for his expertise in processing histologic specimens; Dr. Nicholas Terry and Nalini Patel for their help in the immunohistochemistry and flow cytometry studies; and Walter J. Pagel for his assistance in manuscript editing.


   Footnotes
 
Grant support: NIH/National Cancer Institute grants CA89198 and P50 CA97007.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/16/05; revised 4/17/06; accepted 5/22/06.


   References
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 Abstract
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 References
 

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