3'-Deoxy-3'-18F-fluorothymidine PET predicts response to (V600E)BRAF-targeted therapy in preclinical models of colorectal cancer.
Journal: 2013/April - Journal of nuclear medicine : official publication, Society of Nuclear Medicine
ISSN: 1535-5667
Abstract:
Selective inhibition of oncogenic targets and associated signaling pathways forms the basis of personalized cancer medicine. The clinical success of (V600E)BRAF inhibition in melanoma, coupled with the emergence of acquired resistance, underscores the importance of rigorously validating quantitative biomarkers of treatment response in this and similar settings. Because constitutive activation of BRAF leads to proliferation in tumors, we explored 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET to noninvasively quantify changes in tumor proliferation that are associated with pharmacologic inhibition of (V600E)BRAF downstream effectors and that precede changes in tumor volume.
METHODS
Human colorectal cancer (CRC) cell lines expressing (V600E)BRAF were used to explore relationships between upregulation of p27 and phosphorylation of BRAF downstream effectors on small-molecule (V600E)BRAF inhibitor exposure. Athymic nude mice bearing (V600E)BRAF-expressing human CRC cell line xenografts were treated with a small-molecule (V600E)BRAF inhibitor (or vehicle) daily for 10 d. Predictive (18)F-FLT PET was conducted before changes in tumor volume occurred. Correlations were evaluated among PET, inhibition of phosphorylated MEK (p-MEK) and phosphorylated-ERK (p-ERK) by Western blot, tumor proliferation by histology, and small-molecule exposure by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS).
RESULTS
Treatment of CRC cell lines with PLX4720 reduced proliferation associated with target inhibition and upregulation of p27. In vivo, PLX4720 treatment reduced (18)F-FLT uptake, but not (18)F-FDG uptake, in Lim2405 xenografts before quantifiable differences in xenograft volume. Reduced (18)F-FLT PET reflected a modest, yet significant, reduction of Ki67 immunoreactivity, inhibition of p-MEK and p-ERK, and elevated tumor cell p27 protein levels. Both (18)F-FLT PET and (18)F-FDG PET accurately reflected a lack of response in HT-29 xenografts, which MALDI imaging mass spectrometry suggested may have stemmed from limited PLX4720 exposure.
CONCLUSIONS
We used preclinical models of CRC to demonstrate (18)F-FLT PET as a sensitive predictor of response to (V600E)BRAF inhibitors. Because (18)F-FLT PET predicted reduced proliferation associated with attenuation of BRAF downstream effectors, yet (18)F-FDG PET did not, these data suggest that (18)F-FLT PET may represent an alternative to (18)F-FDG PET for quantifying clinical responses to BRAF inhibitors.
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J Nucl Med 54(3): 424-430

<sup>18</sup>F-FLT PET predicts response to <sup>V600E</sup>BRAF-targeted therapy in preclinical models of colorectal cancer

Methods

Human colorectal cancer (CRC) cell lines expressing BRAF were used to explore relationships between up-regulation of p27 and phosphorylation of BRAF downstream effectors upon small molecule BRAF inhibitor exposure. Athymic nude mice bearing BRAF-expressing human CRC cell line xenografts were treated with a small molecule BRAF inhibitor (or vehicle) daily for ten days. Predictive F-FLT PET was conducted prior to changes in tumor volume. Correlations were evaluated among PET imaging, inhibition of p-MEK and p-ERK by western blot, tumor proliferation by histology, and small molecule exposure by MALDI imaging mass spectrometry (IMS).

Results

Treatment of CRC cell lines with PLX4720 reduced proliferation associated with target inhibition and up regulation of p27. In vivo, PLX4720 treatment reduced F-FLT uptake, but not F-FDG uptake, in Lim2405 xenografts prior to quantifiable differences in xenograft volume. Reduced 18F-FLT PET reflected a modest, yet significant, reduction of Ki67 immunoreactivity, inhibition of p-MEK and p-ERK, and elevated tumor cell p27 protein levels. Both F-FLT PET and F-FDG PET accurately reflected a lack response in HT-29 xenografts, which MALDI IMS suggested may have stemmed from limited PLX4720 exposure.

Conclusions

We utilized preclinical models of CRC to demonstrate F-FLT PET as a sensitive predictor of response to BRAF inhibitors. Since F-FLT PET predicted reduced proliferation associated with attenuation of BRAF downstream effectors, yet F-FDG PET did not, these data suggest that F-FLT PET may represent an alternative to F-FDG PET for quantifying clinical responses to BRAF inhibitors.

INTRODUCTION

BRAF is the most frequently mutated protein kinase in human cancer (1). A variety of activating mutations in BRAF exist, though the V600E mutation, which substitutes glutamic acid for valine, is the most commonly observed (2). This mutation results in constitutive activation of BRAF and associated downstream effectors within the mitogen-activated protein kinase (MAPK) pathway (3). Tumor expression of BRAF correlates with increased proliferation, aggressiveness, and poor prognosis (4, 5). Furthermore, growth and proliferation of tumors that express BRAF tend to depend on MAPK pathway activity, illustrating the attractiveness of pharmacological inhibition of BRAF in these tumors (6). A majority of melanomas (7) and thyroid cancers (8) express BRAF. Expression of mutant BRAF has been observed in other solid tumors, such as colon cancer (~15%) (1, 9). Recent studies have shown that inhibition of mutant BRAF with therapeutic small molecules (e.g., PLX4032) leads to reduced proliferation and tumor regression in melanoma (10, 11). In this disease, reduced proliferation following PLX4032 stems from inhibition of BRAF effectors (e.g., p-MEK, p-ERK) and up-regulation of cell cycle inhibitors (e.g., p21, p27) (12, 13).

The relationship between BRAF inhibition, reduced proliferation, and clinical response in BRAF melanoma suggests that non-invasive imaging metrics of proliferation may represent promising biomarkers of efficacy in this setting. Analogously, recent studies have associated proliferation with acquired resistance to BRAF inhibitors in melanoma (14). Additionally, clinical results evaluating BRAF inhibition in other solid tumors, such as colon cancer (15), have been less promising for reasons that may include resistance-mediated proliferation (16). The widely used PET tracer 2-deoxy-2-(F)fluoro-D-glucose (F-FDG) has been utilized to document clinical response to BRAF inhibition in melanoma (10, 17), though tissue uptake of this tracer reflects a host of metabolic processes only tangentially related to proliferation. In contrast, PET imaging with 3′-deoxy-3′-F-fluorothymidine (F-FLT) measures proliferation more directly by targeting thymidine salvage, which is related to DNA synthesis.

In this study, we utilized preclinical models of CRC to demonstrate F-FLT PET as a sensitive predictor of response to BRAF inhibitors. In a BRAF-sensitive model, F-FLT PET predicted tumor growth arrest and reduced proliferation associated with attenuation of BRAF downstream effectors that was undetectable with F-FDG PET. In another model, F-FLT PET accurately reflected a lack of in vivo response that appeared to stem from limited drug exposure in tumor tissue. Our data suggests that F-FLT PET represents an alternative to F-FDG PET for quantifying clinical responses to BRAF inhibitors in BRAF-expressing solid tumors.

MATERIALS AND METHODS

In Vitro Studies

HT-29 (ATCC HTB-38) human CRC cell lines were obtained from ATCC and Lim2405 cells were provided by Dr. Robert Whitehead, Ludwig Institute for Cancer Research. Cell lines were maintained as sub-confluent monolayer cultures in 10-cm plates in a 95% humidity, 5% CO2, 37°C atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM; Mediatech). Growth medium was supplemented with 10% fetal bovine serum (Atlanta Biologicals) and 1 mg/mL gentamycin sulfate (Gibco). PLX4720 was synthesized as described (18) and was prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO) and aliquoted to achieve final drug concentrations as noted.

Lim2405 and HT-29 cells were propagated to 50% confluency in 6-cm plates. Cells were treated with PLX4720 (0, 10, 250, 1000, 5000 nM) for 24 h and prepared for flow cytometry as described (19). Propidium iodide (PI)-stained cells were analyzed by flow cytometry (FACStar PLUS, Becton-Dickinson). Data analysis was performed using CellQuest software (Becton-Dickinson) by manually gating to define and quantify sub-G0, G1, S, and G2/M populations.

In Vivo Studies

All studies involving animals were conducted in compliance with federal and institutional guidelines. Cell line xenografts were generated in 5-6 week old female athymic nude mice (Harlan Sprague-Dawley) following subcutaneous injection of 1×10 cells on the right flank. Palpable tumors were detected within 2 to 3 weeks post-implantation. Experiments commenced once tumor volumes reached 150-200 mm. For treatment, tumor-bearing mice were administered 60 mg/kg PLX4720 or saline vehicle by oral gavage (100 μL total volume) daily. PET imaging was conducted on day 3 for F-FDG, 16-20 h following the second PLX4720 dose, and day 4 for F-FLT, 16-20 h following the third PLX4720 dose, prior to changes in volume between vehicle-treated and PLX4720-treated tumors. For longitudinal volume assessment, xenograft-bearing mice received a single 60 mg/kg dose of PLX4720 for 10 consecutive days.

Radiopharmaceutical Synthesis

F-FLT was prepared from F-fluoride in a two-step, one-pot reaction as previously described (19, 20) using a GE TRACERlab FX-FN automated module. Aqueous F-fluoride was eluted with Kryptofix-222 and K2CO3 in CH3CN/H2O into the reaction vessel. Three sequences of heating (110°C) with He(g) flow resulted in dry F-fluoride/Kryptofix-222/K2CO3. The cyclic precursor 2,3′-anhydro-5′-O-benzoyl-2′-deoxythymidine (ABX Advanced Biochemical Compounds) was added in DMSO and reacted for 10 min at 160°C. The benzoyl-protecting group was removed from the labeled intermediate by basic hydrolysis (0.25 mol/L NaOH, 50°C, 10 min). The reaction mixture was purified on a semi-preparative C-18 high-performance liquid chromatography column eluting with 10% ethanol/10 mmol/L sodium phosphate buffer and sterilized by 0.2 μm membrane filtration. Radiochemical identity, purity, and specific activity were determined by analytic high-performance liquid chromatography. The F - FLT product was obtained with average radiochemical purity of 98.3% and specific activity of 345.5 TBq/mmol. F-FDG was synthesized in the Vanderbilt University Medical Center Radiopharmacy and distributed by PETNET. The average radiochemical purity of the product was 98.5% and specific activity was more than 37 TBq/mmol.

Imaging

PET imaging was performed using a dedicated small animal PET scanner (Concorde Microsystems Focus 220) as described (19). Briefly, mice were maintained under 2% isofluorane anesthesia in 100% oxygen at 2 L/min and kept warm via a circulating water heating pad during the PET scan. Mice were administered 7.4-9.3 MBq of F-FLT intravenously and allowed free access to food and water during a 40 minute uptake period followed by a 20 min PET acquisition. For F-FDG, mice were fasted for approximately 6 h prior to imaging and warmed in a heated (31° C) chamber for 1 h prior to F-FDG injection and during the uptake period to minimize brown fat uptake of F-FDG. Mice were administered 7.4-9.3 MBq of F-FDG intravenously and allowed free access to water during a 50 minute uptake period followed by a 10 min PET acquisition.

High-resolution ultrasound measurement of xenograft volumes was conducted as previously described (21). Briefly, 3-dimensional ultrasound imaging data sets were collected for each xenograft using a Vevo 770 ultrasound microimaging system (VisualSonics). Mice were anesthetized using 2% isoflurane in oxygen and remained on a heated stage during the course of imaging. Xenografts were covered in warmed (37°C) Aquasonic 100 ultrasound gel (Parker Laboratories) and centered in the imaging plane. Three-dimensional B-mode data were acquired by automated translation of the 30-MHz ultrasound transducer along the entire length of the xenograft.

For PET imaging, scans were reconstructed using OSEM3D/MAP as described (22). The resulting three-dimensional reconstructions had a voxel size of 0.474 mm in the x-y plane and 0.796 mm inter-slice. ASIPro software (Siemens) was used to manually draw three-dimensional regions of interest (ROIs) around the tumor volumes in the reconstructed images. F-FLT uptake was quantified and expressed as percent injected dose per gram of tissue (%ID/g) by dividing the ROI activity by the injected dose and multiplying by 100. For volumetric analysis of xenografts, ultrasound data was imported into Amira 5.2 (Visage Imaging). Tumors were manually segmented and volume was determined by summation of the in-plane segmented regions and multiplying by the inter-slice spacing.

Antibodies and Immunoblotting

In vitro cell samples were collected from 10-cm plates following 48 h of PLX4720 exposure. For immunoblotting, media was removed and cell monolayers were washed with PBS prior to the addition of 450 μL lysis buffer (7 mL CelLytic M lysis buffer (Sigma), mini protease inhibitor cocktail (Roche), 100 μL phosphatase inhibitor cocktail 1 and 2 (Sigma). Protein concentrations were normalized using a BCA assay. Frozen tumor samples were subsequently homogenized and diluted to 1 μg/μL in lysis buffer. All samples were vortexed and centrifuged to collect the final cell lysate.

For western blotting, 20-40 μg of protein was loaded into 7.5-12% SDS PAGE gels and resolved by electrophoresis. Membranes were immunoblotted with antibodies to p-MEK1/2 (Cell Signaling #9154S), total MEK (Cell Signaling #9126), p-ERK1/2 (Cell Signaling #4370S), total ERK (Cell Signaling #4695), p27 (Cell Signaling #3686S), and β-tubulin (Novus Biologicals #NB600-936). The membranes were imaged on a Xenogen IVIS 200 using Western Lightning Plus-ECL (PerkinElmer) substrate.

Immunohistochemistry

Immediately following sacrifice, excised tumors were fixed in 10% formalin for 24 h and transferred to 70% ethanol. Samples were then blocked in paraffin and sectioned prior to immunostaining for Ki67 (Dako #M7240) and TK1 (Abcam #ab57757). Tissues were evaluated by an expert GI pathologist (MKW). Stained samples were imaged at 40x magnification and analyzed for expression of histological markers. The index for each marker was expressed as the average number of positive cells per field based on counting ≥ 3 random fields per tumor section and 3 sections per condition.

In Vitro Studies

HT-29 (ATCC HTB-38) human CRC cell lines were obtained from ATCC and Lim2405 cells were provided by Dr. Robert Whitehead, Ludwig Institute for Cancer Research. Cell lines were maintained as sub-confluent monolayer cultures in 10-cm plates in a 95% humidity, 5% CO2, 37°C atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM; Mediatech). Growth medium was supplemented with 10% fetal bovine serum (Atlanta Biologicals) and 1 mg/mL gentamycin sulfate (Gibco). PLX4720 was synthesized as described (18) and was prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO) and aliquoted to achieve final drug concentrations as noted.

Lim2405 and HT-29 cells were propagated to 50% confluency in 6-cm plates. Cells were treated with PLX4720 (0, 10, 250, 1000, 5000 nM) for 24 h and prepared for flow cytometry as described (19). Propidium iodide (PI)-stained cells were analyzed by flow cytometry (FACStar PLUS, Becton-Dickinson). Data analysis was performed using CellQuest software (Becton-Dickinson) by manually gating to define and quantify sub-G0, G1, S, and G2/M populations.

In Vivo Studies

All studies involving animals were conducted in compliance with federal and institutional guidelines. Cell line xenografts were generated in 5-6 week old female athymic nude mice (Harlan Sprague-Dawley) following subcutaneous injection of 1×10 cells on the right flank. Palpable tumors were detected within 2 to 3 weeks post-implantation. Experiments commenced once tumor volumes reached 150-200 mm. For treatment, tumor-bearing mice were administered 60 mg/kg PLX4720 or saline vehicle by oral gavage (100 μL total volume) daily. PET imaging was conducted on day 3 for F-FDG, 16-20 h following the second PLX4720 dose, and day 4 for F-FLT, 16-20 h following the third PLX4720 dose, prior to changes in volume between vehicle-treated and PLX4720-treated tumors. For longitudinal volume assessment, xenograft-bearing mice received a single 60 mg/kg dose of PLX4720 for 10 consecutive days.

Radiopharmaceutical Synthesis

F-FLT was prepared from F-fluoride in a two-step, one-pot reaction as previously described (19, 20) using a GE TRACERlab FX-FN automated module. Aqueous F-fluoride was eluted with Kryptofix-222 and K2CO3 in CH3CN/H2O into the reaction vessel. Three sequences of heating (110°C) with He(g) flow resulted in dry F-fluoride/Kryptofix-222/K2CO3. The cyclic precursor 2,3′-anhydro-5′-O-benzoyl-2′-deoxythymidine (ABX Advanced Biochemical Compounds) was added in DMSO and reacted for 10 min at 160°C. The benzoyl-protecting group was removed from the labeled intermediate by basic hydrolysis (0.25 mol/L NaOH, 50°C, 10 min). The reaction mixture was purified on a semi-preparative C-18 high-performance liquid chromatography column eluting with 10% ethanol/10 mmol/L sodium phosphate buffer and sterilized by 0.2 μm membrane filtration. Radiochemical identity, purity, and specific activity were determined by analytic high-performance liquid chromatography. The F - FLT product was obtained with average radiochemical purity of 98.3% and specific activity of 345.5 TBq/mmol. F-FDG was synthesized in the Vanderbilt University Medical Center Radiopharmacy and distributed by PETNET. The average radiochemical purity of the product was 98.5% and specific activity was more than 37 TBq/mmol.

Imaging

PET imaging was performed using a dedicated small animal PET scanner (Concorde Microsystems Focus 220) as described (19). Briefly, mice were maintained under 2% isofluorane anesthesia in 100% oxygen at 2 L/min and kept warm via a circulating water heating pad during the PET scan. Mice were administered 7.4-9.3 MBq of F-FLT intravenously and allowed free access to food and water during a 40 minute uptake period followed by a 20 min PET acquisition. For F-FDG, mice were fasted for approximately 6 h prior to imaging and warmed in a heated (31° C) chamber for 1 h prior to F-FDG injection and during the uptake period to minimize brown fat uptake of F-FDG. Mice were administered 7.4-9.3 MBq of F-FDG intravenously and allowed free access to water during a 50 minute uptake period followed by a 10 min PET acquisition.

High-resolution ultrasound measurement of xenograft volumes was conducted as previously described (21). Briefly, 3-dimensional ultrasound imaging data sets were collected for each xenograft using a Vevo 770 ultrasound microimaging system (VisualSonics). Mice were anesthetized using 2% isoflurane in oxygen and remained on a heated stage during the course of imaging. Xenografts were covered in warmed (37°C) Aquasonic 100 ultrasound gel (Parker Laboratories) and centered in the imaging plane. Three-dimensional B-mode data were acquired by automated translation of the 30-MHz ultrasound transducer along the entire length of the xenograft.

For PET imaging, scans were reconstructed using OSEM3D/MAP as described (22). The resulting three-dimensional reconstructions had a voxel size of 0.474 mm in the x-y plane and 0.796 mm inter-slice. ASIPro software (Siemens) was used to manually draw three-dimensional regions of interest (ROIs) around the tumor volumes in the reconstructed images. F-FLT uptake was quantified and expressed as percent injected dose per gram of tissue (%ID/g) by dividing the ROI activity by the injected dose and multiplying by 100. For volumetric analysis of xenografts, ultrasound data was imported into Amira 5.2 (Visage Imaging). Tumors were manually segmented and volume was determined by summation of the in-plane segmented regions and multiplying by the inter-slice spacing.

Antibodies and Immunoblotting

In vitro cell samples were collected from 10-cm plates following 48 h of PLX4720 exposure. For immunoblotting, media was removed and cell monolayers were washed with PBS prior to the addition of 450 μL lysis buffer (7 mL CelLytic M lysis buffer (Sigma), mini protease inhibitor cocktail (Roche), 100 μL phosphatase inhibitor cocktail 1 and 2 (Sigma). Protein concentrations were normalized using a BCA assay. Frozen tumor samples were subsequently homogenized and diluted to 1 μg/μL in lysis buffer. All samples were vortexed and centrifuged to collect the final cell lysate.

For western blotting, 20-40 μg of protein was loaded into 7.5-12% SDS PAGE gels and resolved by electrophoresis. Membranes were immunoblotted with antibodies to p-MEK1/2 (Cell Signaling #9154S), total MEK (Cell Signaling #9126), p-ERK1/2 (Cell Signaling #4370S), total ERK (Cell Signaling #4695), p27 (Cell Signaling #3686S), and β-tubulin (Novus Biologicals #NB600-936). The membranes were imaged on a Xenogen IVIS 200 using Western Lightning Plus-ECL (PerkinElmer) substrate.

Immunohistochemistry

Immediately following sacrifice, excised tumors were fixed in 10% formalin for 24 h and transferred to 70% ethanol. Samples were then blocked in paraffin and sectioned prior to immunostaining for Ki67 (Dako #M7240) and TK1 (Abcam #ab57757). Tissues were evaluated by an expert GI pathologist (MKW). Stained samples were imaged at 40x magnification and analyzed for expression of histological markers. The index for each marker was expressed as the average number of positive cells per field based on counting ≥ 3 random fields per tumor section and 3 sections per condition.

MALDI IMS

Immediately following PET imaging, xenograft-bearing mice were sacrificed, placed in 50mL conical tubes containing de-ionized water and subsequently frozen using dry ice. Transverse sections prepared for MALDI IMS as described (23, 24). Mass spectra were acquired in positive-ion mode using a MALDI LTQ XL linear ion trap mass spectrometer. An average of 15 laser shots/scan was used to produce a mass spectrum every 200 μm laterally across the tissue. Two dimensional ion density images were extracted from raw data using ImageQuest (version 1.0.1; Thermo Scientific, San Jose, CA, USA). False color ion ion intensity maps were co-registered with white light images to visualize drug concentration in the mouse tissues (23).

Statistics

Statistical significance of data were evaluated using the non-parametric Wilcoxon Rank Sum (Mann-Whitney U) tests in GraphPad Prism 4. Data were considered significant if P < 0.05.

Statistics

Statistical significance of data were evaluated using the non-parametric Wilcoxon Rank Sum (Mann-Whitney U) tests in GraphPad Prism 4. Data were considered significant if P < 0.05.

RESULTS

In vitro sensitivity of CRC cells to PLX4720

The BRAF expressing Lim2405 and HT-29 cell lines exhibited marked sensitivity to PLX4720, characterized primarily by concentration-dependent G1-arrest and decreased S-phase (Figure 1A) as measured by PI flow cytometry. Other cell cycle phases were only modestly affected, suggesting that primary effect of PLX4720 exposure in these cell lines was diminished DNA synthesis.

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In vitro PLX4720 exposure decreases proliferation and leads to elevated p27 levels in human CRC cell lines harboring BRAF

Lim2405 and HT-29 cells expressing BRAF exhibited marked sensitivity to PLX4720. (A) PI flow cytometry demonstrated concentration-dependent G1-arrest and decreased S-phase fractions. Western blot analysis of lysates collected from HT-29 (B) and Lim2405 (C) cells following 48 hr in vitro PLX4720 exposure (0, 10 nM, 100 nM, 250 nM, 1 μM, 5 μM) are shown. In both cell lines, p-MEK was inhibited at concentrations greater than 250 nM. Additionally, increased p27 protein expression was observed at PLX4720 concentrations greater than 250 nM.

Reasoning that F-FLT PET measures cellular proliferation in a S-phase dependent manner, we utilized Lim2405 and HT-29 cells to explore the relationship between inhibition of BRAF downstream effectors and cell cycle inhibitors. As expected, PLX4720 treatment resulted in reduced p-MEK by 50% or greater at concentrations of approximately 250 nM and above for both Lim2405 (Figure 1B) and HT-29 (Figure 1C). Levels of p27 were inversely related to p-MEK following PLX4720 exposure.

In vivo sensitivity of Lim2405 xenografts to PLX4720

Mice bearing Lim2405 xenografts were used to evaluate early, post-treatment F-FLT-PET to predict changes in xenograft volume stemming from small molecule BRAF inhibitor exposure. Mice were treated daily with PLX4720 over a 10-day treatment course. PLX4720 treatment resulted in significant growth arrest of Lim2405 xenografts that was detectable using high-resolution ultrasound imaging by day 7 (Figure 2A). F-FLT PET imaging of xenograft-bearing mice was conducted on day 4, when there was no quantifiable difference in xenograft volume between PLX4720-treated and vehicle-treated cohorts. We found F-FLT uptake to be significantly reduced in PLX4720-treated Lim2405 xenografts (9.9 ± 1.4 %ID/g, N = 5) compared to similar vehicle-treated controls (14.9 ± 1.5 %ID/g. N = 5) (Figures 2B/2C). Analogously, pre-treatment/post-treatment comparisons of individual mice showed a statistically significant decrease following PLX4720 exposure, but not following vehicle exposure group (Figure S1). Unlike F-FLT PET, F-FDG PET was not significantly different between vehicle-treated and PLX4720-treated Lim2405 xenograft tumors (Figure S2). A subset of mice were immediately sacrificed following imaging for correlative analysis that included preparation of whole-animal transverse thin sections for MALDI IMS; this technique was used to evaluate relative PLX4720 tissue concentrations and the spatial distribution in Lim2405 xenograft-bearing mice at the time of imaging. Pseudocolor maps of the MALDI ion intensity corresponding to the parent molecular mass of PLX4720 confirmed the presence of un-metabolized drug in tumors and adjacent non-tumor tissues such as the bowel. No background ion intensity was observed in vehicle-treated mice (Figure 2D).

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Reduced in vivoF-FLT PET precedes quantifiable, PLX4720-dependent differences in tumor volume and correlates with drug exposure in Lim2405 xenografts

Mice bearing Lim2405 xenografts were treated daily with PLX4720 or vehicle. (A) Over a 10 day regimen, PLX4720 treatment induced significant xenograft growth arrest detectable by treatment day 7. F-FLT PET was carried out on treatment day 4 when there was negligible difference in tumor volume between PLX4720-treated and vehicle-treated controls (blue box). (B) Representative transverse images of vehicle-treated and PLX4720-treated cohorts collected on treatment day 4 (xenograft denoted by arrowhead). (C) F-FLT PET was significantly lower in PLX4720-treated xenografts compared to vehicle-treated controls. Immediately following PET, mice were sacrificed and thin, transverse sections of vehicle-treated and PLX4720-treated mice were prepared for MALDI IMS. (D) Pseudocolor maps of ion intensity corresponding to the parent molecular mass of PLX4720 demonstrate minimal signal intensity in vehicle-treated animals. In contrast, treated animals demonstrated considerable PLX4720 ion intensity, with the drug primarily localized to tumor tissue (dotted line). Other tissues exhibiting PLX4720 ion intensity included the bowel, consistent with oral administration.

Western blot analysis of imaging-matched Lim2405 xenograft tissues harvested shortly following imaging on treatment day 4 illustrated that reduced F-FLT PET was associated with approximately 50% inhibition of p-MEK and p-ERK (Figure 3) and an approximately 2-fold up-regulation of p27 levels. Using immunohistochemistry, we observed a 25% reduction in TK1 positivity in PLX4720-treated Lim2405 xenografts compared to vehicle-treated controls (Figures 4A/4B). Similarly, Ki67 immunoreactivity was also reduced in PLX4720-treated tumors (Figures 4A/4B), although to a lesser extent than observed for TK1.

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Early F-FLT PET predicts inhibition of BRAF downstream effectors and p27 up-regulation

Western blot analysis quantifying BRAF effector molecules and p27 in F-FLT PET imaging-matched Lim2405 xenografts (seeFigure 2). Compared to vehicle controls, xenografts harvested from PLX4720-treated cohorts at treatment day 4 exhibited reduced levels of p-MEK and p-ERK and elevated levels of p27.

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Early F-FLT PET agrees with tumor cell TK1 levels and Ki67 immunohistochemistry

Immunohistochemistry analysis of F-FLT PET imaging-matched Lim2045 xenografts (seeFigure 2). (A) Representative TK1 and Ki67 immunohistochemistry images of xenografts harvested at treatment day 4 are shown for both vehicle-treated and PLX720-treated tumors. (B) PLX4720-treated xenografts exhibited approximately 25% fewer TK1-positive cells per field compared to vehicle controls (34.3% vs. 58.8%, p = 0.0041). A modest, though statistically significant (p < 0.001), reduction in the number of Ki67 positive cells per field was observed for PLX4720-treated xenografts compared to vehicle controls (77.4% vs. 89.5%).

In vivo sensitivity of HT-29 xenografts to PLX4720

Analogous to studies with Lim2405 xenografts, mice bearing HT-29 xenografts were treated daily with PLX4720 or vehicle over a 10-day treatment course. No difference in xenograft volume was observed between PLX4720-treated and vehicle-treated mice over the 10 day treatment course. (Figure 5A). Unlike Lim2405 xenografts, at day 4, F-FLT uptake was similar comparing PLX4720-treated and vehicle-treated HT-29 xenografts (vehicle: 2.21 ± 0.06 %ID/g, N = 5; PLX4720: 1.97 ± 0.14 %ID/g, N = 5) (Figure 5B), suggesting that F-FLT PET accurately predicted lack of response. Similarly, no difference in F-FDG uptake was observed between PLX-4720-treated and vehicle-treated HT-29 xenograft bearing mice (Figure S3A). PLX4720 treatment did not affect relative p-MEK or p-ERK levels in HT-29 xenografts, as determined by Western blot of tumor tissues, harvested immediately following imaging on treatment day 4 (Figure 5C). Surprisingly, unlike similarly treated Lim2405 xenografts, appreciable levels of PLX4720 were not detected in HT-29 xenografts of treated mice, although drug was detected in adjacent bowel (Figure 5D). In agreement with F-FLT PET, neither TK1 levels (Figure S3B) nor Ki67 immunoreactivity (Figure S3C) were significantly affected by PLX4720 treatment in HT-29 xenografts.

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F-FLT PET predicts lack of response to PLX4720 in HT-29 xenografts

Mice bearing HT-29 xenografts were treated daily with PLX4720 or vehicle. (A) Over a 10 day regimen, no difference in tumor volume was observed between PLX4720-treated and vehicle-treated. Tumor F-FLT PET (B) and phosphorylation of BRAF effectors (C) were similar between vehicle-treated and PLX4720-treated HT-29 cohorts. (D) Representative transverse MALDI IMS pseudocolor maps of ion intensity corresponding to the parent molecular mass of PLX4720 demonstrate minimal signal intensity in vehicle-treated animals. Treated animals demonstrated little PLX4720 ion intensity within tumor tissue (dotted line), yet PLX4720 ion intensity was observed in the bowel.

In vitro sensitivity of CRC cells to PLX4720

The BRAF expressing Lim2405 and HT-29 cell lines exhibited marked sensitivity to PLX4720, characterized primarily by concentration-dependent G1-arrest and decreased S-phase (Figure 1A) as measured by PI flow cytometry. Other cell cycle phases were only modestly affected, suggesting that primary effect of PLX4720 exposure in these cell lines was diminished DNA synthesis.

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In vitro PLX4720 exposure decreases proliferation and leads to elevated p27 levels in human CRC cell lines harboring BRAF

Lim2405 and HT-29 cells expressing BRAF exhibited marked sensitivity to PLX4720. (A) PI flow cytometry demonstrated concentration-dependent G1-arrest and decreased S-phase fractions. Western blot analysis of lysates collected from HT-29 (B) and Lim2405 (C) cells following 48 hr in vitro PLX4720 exposure (0, 10 nM, 100 nM, 250 nM, 1 μM, 5 μM) are shown. In both cell lines, p-MEK was inhibited at concentrations greater than 250 nM. Additionally, increased p27 protein expression was observed at PLX4720 concentrations greater than 250 nM.

Reasoning that F-FLT PET measures cellular proliferation in a S-phase dependent manner, we utilized Lim2405 and HT-29 cells to explore the relationship between inhibition of BRAF downstream effectors and cell cycle inhibitors. As expected, PLX4720 treatment resulted in reduced p-MEK by 50% or greater at concentrations of approximately 250 nM and above for both Lim2405 (Figure 1B) and HT-29 (Figure 1C). Levels of p27 were inversely related to p-MEK following PLX4720 exposure.

In vivo sensitivity of Lim2405 xenografts to PLX4720

Mice bearing Lim2405 xenografts were used to evaluate early, post-treatment F-FLT-PET to predict changes in xenograft volume stemming from small molecule BRAF inhibitor exposure. Mice were treated daily with PLX4720 over a 10-day treatment course. PLX4720 treatment resulted in significant growth arrest of Lim2405 xenografts that was detectable using high-resolution ultrasound imaging by day 7 (Figure 2A). F-FLT PET imaging of xenograft-bearing mice was conducted on day 4, when there was no quantifiable difference in xenograft volume between PLX4720-treated and vehicle-treated cohorts. We found F-FLT uptake to be significantly reduced in PLX4720-treated Lim2405 xenografts (9.9 ± 1.4 %ID/g, N = 5) compared to similar vehicle-treated controls (14.9 ± 1.5 %ID/g. N = 5) (Figures 2B/2C). Analogously, pre-treatment/post-treatment comparisons of individual mice showed a statistically significant decrease following PLX4720 exposure, but not following vehicle exposure group (Figure S1). Unlike F-FLT PET, F-FDG PET was not significantly different between vehicle-treated and PLX4720-treated Lim2405 xenograft tumors (Figure S2). A subset of mice were immediately sacrificed following imaging for correlative analysis that included preparation of whole-animal transverse thin sections for MALDI IMS; this technique was used to evaluate relative PLX4720 tissue concentrations and the spatial distribution in Lim2405 xenograft-bearing mice at the time of imaging. Pseudocolor maps of the MALDI ion intensity corresponding to the parent molecular mass of PLX4720 confirmed the presence of un-metabolized drug in tumors and adjacent non-tumor tissues such as the bowel. No background ion intensity was observed in vehicle-treated mice (Figure 2D).

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Reduced in vivoF-FLT PET precedes quantifiable, PLX4720-dependent differences in tumor volume and correlates with drug exposure in Lim2405 xenografts

Mice bearing Lim2405 xenografts were treated daily with PLX4720 or vehicle. (A) Over a 10 day regimen, PLX4720 treatment induced significant xenograft growth arrest detectable by treatment day 7. F-FLT PET was carried out on treatment day 4 when there was negligible difference in tumor volume between PLX4720-treated and vehicle-treated controls (blue box). (B) Representative transverse images of vehicle-treated and PLX4720-treated cohorts collected on treatment day 4 (xenograft denoted by arrowhead). (C) F-FLT PET was significantly lower in PLX4720-treated xenografts compared to vehicle-treated controls. Immediately following PET, mice were sacrificed and thin, transverse sections of vehicle-treated and PLX4720-treated mice were prepared for MALDI IMS. (D) Pseudocolor maps of ion intensity corresponding to the parent molecular mass of PLX4720 demonstrate minimal signal intensity in vehicle-treated animals. In contrast, treated animals demonstrated considerable PLX4720 ion intensity, with the drug primarily localized to tumor tissue (dotted line). Other tissues exhibiting PLX4720 ion intensity included the bowel, consistent with oral administration.

Western blot analysis of imaging-matched Lim2405 xenograft tissues harvested shortly following imaging on treatment day 4 illustrated that reduced F-FLT PET was associated with approximately 50% inhibition of p-MEK and p-ERK (Figure 3) and an approximately 2-fold up-regulation of p27 levels. Using immunohistochemistry, we observed a 25% reduction in TK1 positivity in PLX4720-treated Lim2405 xenografts compared to vehicle-treated controls (Figures 4A/4B). Similarly, Ki67 immunoreactivity was also reduced in PLX4720-treated tumors (Figures 4A/4B), although to a lesser extent than observed for TK1.

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Early F-FLT PET predicts inhibition of BRAF downstream effectors and p27 up-regulation

Western blot analysis quantifying BRAF effector molecules and p27 in F-FLT PET imaging-matched Lim2405 xenografts (seeFigure 2). Compared to vehicle controls, xenografts harvested from PLX4720-treated cohorts at treatment day 4 exhibited reduced levels of p-MEK and p-ERK and elevated levels of p27.

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Early F-FLT PET agrees with tumor cell TK1 levels and Ki67 immunohistochemistry

Immunohistochemistry analysis of F-FLT PET imaging-matched Lim2045 xenografts (seeFigure 2). (A) Representative TK1 and Ki67 immunohistochemistry images of xenografts harvested at treatment day 4 are shown for both vehicle-treated and PLX720-treated tumors. (B) PLX4720-treated xenografts exhibited approximately 25% fewer TK1-positive cells per field compared to vehicle controls (34.3% vs. 58.8%, p = 0.0041). A modest, though statistically significant (p < 0.001), reduction in the number of Ki67 positive cells per field was observed for PLX4720-treated xenografts compared to vehicle controls (77.4% vs. 89.5%).

In vivo sensitivity of HT-29 xenografts to PLX4720

Analogous to studies with Lim2405 xenografts, mice bearing HT-29 xenografts were treated daily with PLX4720 or vehicle over a 10-day treatment course. No difference in xenograft volume was observed between PLX4720-treated and vehicle-treated mice over the 10 day treatment course. (Figure 5A). Unlike Lim2405 xenografts, at day 4, F-FLT uptake was similar comparing PLX4720-treated and vehicle-treated HT-29 xenografts (vehicle: 2.21 ± 0.06 %ID/g, N = 5; PLX4720: 1.97 ± 0.14 %ID/g, N = 5) (Figure 5B), suggesting that F-FLT PET accurately predicted lack of response. Similarly, no difference in F-FDG uptake was observed between PLX-4720-treated and vehicle-treated HT-29 xenograft bearing mice (Figure S3A). PLX4720 treatment did not affect relative p-MEK or p-ERK levels in HT-29 xenografts, as determined by Western blot of tumor tissues, harvested immediately following imaging on treatment day 4 (Figure 5C). Surprisingly, unlike similarly treated Lim2405 xenografts, appreciable levels of PLX4720 were not detected in HT-29 xenografts of treated mice, although drug was detected in adjacent bowel (Figure 5D). In agreement with F-FLT PET, neither TK1 levels (Figure S3B) nor Ki67 immunoreactivity (Figure S3C) were significantly affected by PLX4720 treatment in HT-29 xenografts.

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F-FLT PET predicts lack of response to PLX4720 in HT-29 xenografts

Mice bearing HT-29 xenografts were treated daily with PLX4720 or vehicle. (A) Over a 10 day regimen, no difference in tumor volume was observed between PLX4720-treated and vehicle-treated. Tumor F-FLT PET (B) and phosphorylation of BRAF effectors (C) were similar between vehicle-treated and PLX4720-treated HT-29 cohorts. (D) Representative transverse MALDI IMS pseudocolor maps of ion intensity corresponding to the parent molecular mass of PLX4720 demonstrate minimal signal intensity in vehicle-treated animals. Treated animals demonstrated little PLX4720 ion intensity within tumor tissue (dotted line), yet PLX4720 ion intensity was observed in the bowel.

DISCUSSION

Current imaging criteria for evaluating therapeutic response are primarily based upon anatomical response criteria, such as Response Evaluation Criteria in Solid Tumors (RECIST) or similar guidelines. Since relevant cellular and molecular changes may precede changes in size and occur within hours of treatment, RECIST criteria and associated conventional imaging methods may be ill-suited for assessing early responses of tumors to therapy. These limitations have led to the development of newer criteria, such as PERCIST (25) that recognize the potential utility of F-FDG PET to predict early response. For example, F-FDG PET has shown clinical utility to predict response for BRAF in melanoma (26), although that was not the case in this study which utilizes preclinical models of CRC. A drawback of F-FDG, however, is that its uptake reflects numerous metabolic processes. For this reason, more specific imaging biomarkers of proliferation may be better suited to predict treatment response in certain clinical scenarios (27).

Compared to F-FDG PET, F-FLT PET exhibits increased proliferation specificity (28, 29) and may be a more suitable option in some settings for evaluating response to targeted therapy. Uptake of F-FLT reflects proliferation by reporting the activity of thymidine salvage, a mechanism that provides dividing cells with DNA precursors from the extracellular environment. Upon internalization, F-FLT is monophosphorylated by the cytosolic enzyme TK1, resulting in intracellular trapping and accumulation (30-32). TK1 expression is a product of E2F transcription, which can be activated by MAPK signaling (33). For example, Solit and colleagues reported that F-FLT PET correlated with response to a MEK inhibitor in melanoma xenografts (34). Given this, we hypothesized that F-FLT PET would also be sensitive to BRAF inhibition in tumors whose proliferation was associated with constitutive MAPK pathway activity.

In this study, we utilized preclinical models of CRC to explore F-FLT PET as a metric to predict response to a BRAF inhibitor. In a BRAF-sensitive model, we found that F-FLT PET predicted tumor growth arrest and reduced proliferation associated with attenuation of BRAF downstream effectors. Notably, in the same model F-FDG PET failed to predict drug sensitivity. In another model that exhibited in vitro but not in vivo sensitivity, F-FLT PET accurately reflected a lack of in vivo response that correlated with limited drug exposure in tumor tissue. Through the use of MALDIIMS, we were able to document that the sensitive model, Lim2405 xenografts, exhibited significantly greater levels PLX4720 in tumor tissue at the time of F-FLT PET than the insensitive model, HT-29 xenografts. Though both models were treated and imaged similarly, we anticipate that differences in vasculature and/or stroma may have contributed to the observed differences in exposure. Regardless, F-FLT PET effectively reflected tumor proliferation in both models.

Other preclinical results have documented the sensitivity of CRC cell lines to BRAF inhibition, though clinical results evaluating this approach in patients with colon cancer have been less promising (15). Clinically, it remains unclear whether the lack of single agent efficacy in BRAF colon tumors stems from resistance, or as with our study, a lack of adequate exposure, or potentially other reasons. A recent preclinical study found that combined BRAF and EGFR inhibition results in improved efficacy in this setting (16). Importantly, since combination therapy led to reduced MAPK activity and proliferation, our results suggest that F-FLT PET represents a potentially superior, quantitative biomarker to evaluate the efficacy of analogous combination regimens in this setting.

CONCLUSION

We utilized preclinical models of CRC to demonstrate F-FLT PET as a sensitive predictor of response to a BRAF inhibitor. Since F-FLT PET predicted reduced proliferation associated with attenuation of BRAF downstream effectors, while F-FDG PET did not, we anticipate this imaging metric may represent an alternative to F-FDG PET for quantifying clinical responses to BRAF inhibitors.

Supplementary Material

01

Supplementary Figure 1. Pre-treatment/post-treatment comparison of F-FLT uptake in Lim2405 xenograft tumors. Pre-treatment and post-treatment F-FLT uptake was similar in vehicle-treated Lim2405 xenografts. In PLX4720-treated xenografts, F-FLT was significantly reduced for each individual mouse imaged.

Supplementary Figure 2. F-FDG PET is similar between vehicle-treated and PLX4720-treated Lim2405 xenograft tumors. Contrary to F-FLT PET (seeFigure 2, Figure S1), no statistically significant difference in F-FDG uptake was observed between vehicle-treated and PLX4720-treated Lim2405 xenografts.

Supplementary Figure 3. F-FDG PET is similar between vehicle-treated and PLX4720-treated HT-29 xenografts and agrees with immunohistochemistry markers of proliferation. (A) Similar to F-FLT PET in HT-29 xenografts, no difference was observed between vehicle-treated and PLX4720-treated HT-29 tumor xenografts using F-FDG PET. Corroborating PET, biochemical, and MALDI results (seeFigure 5), TK1 (B) and Ki67 (C) in vehicle-treated and PLX4720-treated HT-29 xenografts exhibited similar immunoreactivity.

01

Supplementary Figure 1. Pre-treatment/post-treatment comparison of F-FLT uptake in Lim2405 xenograft tumors. Pre-treatment and post-treatment F-FLT uptake was similar in vehicle-treated Lim2405 xenografts. In PLX4720-treated xenografts, F-FLT was significantly reduced for each individual mouse imaged.

Supplementary Figure 2. F-FDG PET is similar between vehicle-treated and PLX4720-treated Lim2405 xenograft tumors. Contrary to F-FLT PET (seeFigure 2, Figure S1), no statistically significant difference in F-FDG uptake was observed between vehicle-treated and PLX4720-treated Lim2405 xenografts.

Supplementary Figure 3. F-FDG PET is similar between vehicle-treated and PLX4720-treated HT-29 xenografts and agrees with immunohistochemistry markers of proliferation. (A) Similar to F-FLT PET in HT-29 xenografts, no difference was observed between vehicle-treated and PLX4720-treated HT-29 tumor xenografts using F-FDG PET. Corroborating PET, biochemical, and MALDI results (seeFigure 5), TK1 (B) and Ki67 (C) in vehicle-treated and PLX4720-treated HT-29 xenografts exhibited similar immunoreactivity.

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Acknowledgments

The authors acknowledge funding from the National Institutes of Health (R01 CA140628, RC1 145138, K25 CA127349, P50 951903, P50 CA128323, S10 RR17858, U24 {"type":"entrez-nucleotide","attrs":{"text":"CA126588","term_id":"35005625","term_text":"CA126588"}}CA126588, R25 CA136440, R01 CA46413, and P30 DK058404) and The Kleberg Foundation,. The authors acknowledge Michelle Reyzer and Richard Caprioli for assistance with MALDI IMS.

Vanderbilt University Institute of Imaging Science, Vanderbilt University, Nashville, TN
Department of Biomedical Engineering, Vanderbilt University, Nashville, TN
Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, TN
Department of Pathology, Vanderbilt University, Nashville, TN
Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN
Cell and Developmental Biology, Vanderbilt University
Department of Medicine, Vanderbilt University Medical School
Department of Veterans Affairs Medical Center, Nashville, Tennessee
Program in Chemical and Physical Biology, Vanderbilt University School of Medicine, Nashville, TN
Department of Neurosurgery, Vanderbilt University Medical Center, Nashville, TN
Corresponding Author: H. Charles Manning, Vanderbilt University Institute of Imaging Science, 1161 21st Ave. S., AA1105 MCN, Nashville, TN 37232-2310; phone: 615-322-3793; fax: 615-322-0734; ude.tlibrednav@gninnam.c.yrneh

Abstract

Selective inhibition of oncogenic targets and associated signaling pathways forms the basis of personalized cancer medicine. The clinical success of BRAF inhibition in melanoma, coupled with the emergence of acquired resistance, underscores the importance of rigorously validating quantitative biomarkers of treatment response in this and similar settings. Since constitutive activation of BRAF leads to proliferation in tumors, we explored F-FLT PET to non-invasively quantify changes in tumor proliferation that are associated with pharmacological inhibition of BRAF downstream effectors and that precede changes in tumor volume.

Methods

Human colorectal cancer (CRC) cell lines expressing BRAF were used to explore relationships between up-regulation of p27 and phosphorylation of BRAF downstream effectors upon small molecule BRAF inhibitor exposure. Athymic nude mice bearing BRAF-expressing human CRC cell line xenografts were treated with a small molecule BRAF inhibitor (or vehicle) daily for ten days. Predictive F-FLT PET was conducted prior to changes in tumor volume. Correlations were evaluated among PET imaging, inhibition of p-MEK and p-ERK by western blot, tumor proliferation by histology, and small molecule exposure by MALDI imaging mass spectrometry (IMS).

Results

Treatment of CRC cell lines with PLX4720 reduced proliferation associated with target inhibition and up regulation of p27. In vivo, PLX4720 treatment reduced F-FLT uptake, but not F-FDG uptake, in Lim2405 xenografts prior to quantifiable differences in xenograft volume. Reduced 18F-FLT PET reflected a modest, yet significant, reduction of Ki67 immunoreactivity, inhibition of p-MEK and p-ERK, and elevated tumor cell p27 protein levels. Both F-FLT PET and F-FDG PET accurately reflected a lack response in HT-29 xenografts, which MALDI IMS suggested may have stemmed from limited PLX4720 exposure.

Conclusions

We utilized preclinical models of CRC to demonstrate F-FLT PET as a sensitive predictor of response to BRAF inhibitors. Since F-FLT PET predicted reduced proliferation associated with attenuation of BRAF downstream effectors, yet F-FDG PET did not, these data suggest that F-FLT PET may represent an alternative to F-FDG PET for quantifying clinical responses to BRAF inhibitors.

Keywords: PLX4720, FLT PET, proliferation, BRAF, colorectal cancer
Abstract

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