Eliot T. McKinley

Molecular Imaging of Therapeutic Response to Epidermal Growth Factor Receptor Blockade in Colorectal Cancer

Purpose: To evaluate noninvasive molecular imaging methods as correlative biomarkers of therapeutic efficacy of cetuximab in human colorectal cancer cell line xenografts grown in athymic nude mice. The correlation between molecular imaging and immunohistochemical analysis to quantify epidermal growth factor (EGF) binding, apoptosis, and proliferation was evaluated in treated and untreated tumor-bearing cohorts. Experimental Design: Optical imaging probes targeting EGF receptor (EGFR) expression (NIR800-EGF) and apoptosis (NIR700-Annexin V) were synthesized and evaluated in vitro and in vivo. Proliferation was assessed by 3′-[18F]fluoro-3′-deoxythymidine ([18F]FLT) positron emission tomography. Assessment of inhibition of EGFR signaling by cetuximab was accomplished by concomitant imaging of NIR800-EGF, NIR700-Annexin V, and [18F]FLT in cetuximab-sensitive (DiFi) and insensitive (HCT-116) human colorectal cancer cell line xenografts. Imaging results were validated by measurement of tumor size and immunohistochemical analysis of total and phosphorylated EGFR, caspase-3, and Ki-67 immediately following in vivo imaging. Results: NIR800-EGF accumulation in tumors reflected relative EGFR expression and EGFR occupancy by cetuximab. NIR700-Annexin V accumulation correlated with cetuximab-induced apoptosis as assessed by immunohistochemical staining of caspase-3. No significant difference in tumor proliferation was noted between treated and untreated animals by [18F]FLT positron emission tomography or Ki-67 immunohistochemistry. Conclusions: Molecular imaging can accurately assess EGF binding, proliferation, and apoptosis in human colorectal cancer xenografts. These imaging approaches may prove useful for serial, noninvasive monitoring of the biological effects of EGFR inhibition in preclinical studies. It is anticipated that these assays can be adapted for clinical use.

Imaging Biomarkers Predict Response to Anti-HER2 (ErbB2) Therapy in Preclinical Models of Breast Cancer

Purpose: To evaluate noninvasive imaging methods as predictive biomarkers of response to trastuzumab in mouse models of HER2-overexpressing breast cancer. The correlation between tumor regression and molecular imaging of apoptosis, glucose metabolism, and cellular proliferation was evaluated longitudinally in responding and nonresponding tumor-bearing cohorts. Experimental Design: Mammary tumors from MMTV/HER2 transgenic female mice were transplanted into syngeneic female mice. BT474 human breast carcinoma cell line xenografts were grown in athymic nude mice. Tumor cell apoptosis (NIR700-Annexin V accumulation), glucose metabolism [2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography ([18F]FDG-PET)], and proliferation [3′-[18F]fluoro-3′-deoxythymidine-PET ([18F]FLT-PET)] were evaluated throughout a biweekly trastuzumab regimen. Imaging metrics were validated by direct measurement of tumor size and immunohistochemical analysis of cleaved caspase-3, phosphorylated AKT, and Ki67. Results: NIR700-Annexin V accumulated significantly in trastuzumab-treated MMTV/HER2 and BT474 tumors that ultimately regressed but not in nonresponding or vehicle-treated tumors. Uptake of [18F]FDG was not affected by trastuzumab treatment in MMTV/HER2 or BT474 tumors. [18F]FLT-PET imaging predicted trastuzumab response in BT474 tumors but not in MMTV/HER2 tumors, which exhibited modest uptake of [18F]FLT. Close agreement was observed between imaging metrics and immunohistochemical analysis. Conclusions: Molecular imaging of apoptosis accurately predicts trastuzumab-induced regression of HER2+ tumors and may warrant clinical exploration to predict early response to neoadjuvant trastuzumab. Trastuzumab does not seem to alter glucose metabolism substantially enough to afford [18F]FDG-PET significant predictive value in this setting. Although promising in one preclinical model, further studies are required to determine the overall value of [18F]FLT-PET as a biomarker of response to trastuzumab in HER2+ breast cancer.

Limits of [18F]-FLT PET as a Biomarker of Proliferation in Oncology

Background Non-invasive imaging biomarkers of cellular proliferation hold great promise for quantifying response to personalized medicine in oncology. An emerging approach to assess tumor proliferation utilizes the positron emission tomography (PET) tracer 3’-deoxy-3’[18F]-fluorothymidine, [18F]-FLT. Though several studies have associated serial changes in [18F]-FLT-PET with elements of therapeutic response, the degree to which [18F]-FLT-PET quantitatively reflects proliferative index has been continuously debated for more that a decade. The goal of this study was to elucidate quantitative relationships between [18F]-FLT-PET and cellular metrics of proliferation in treatment naïve human cell line xenografts commonly employed in cancer research. Methods and Findings [18F]-FLT-PET was conducted in human cancer xenograft-bearing mice. Quantitative relationships between PET, thymidine kinase 1 (TK1) protein levels and immunostaining for proliferation markers (Ki67, TK1, PCNA) were evaluated using imaging-matched tumor specimens. Overall, we determined that [18F]-FLT-PET reflects TK1 protein levels, yet the cell cycle specificity of TK1 expression and the extent to which tumors utilize thymidine salvage for DNA synthesis decouple [18F]-FLT-PET data from standard estimates of proliferative index. Conclusions Our findings illustrate that [18F]-FLT-PET reflects tumor proliferation as a function of thymidine salvage pathway utilization. Unlike more general proliferation markers, such as Ki67, [18F]-FLT PET reflects proliferative indices to variable and potentially unreliable extents. [18F]-FLT-PET cannot discriminate moderately proliferative, thymidine salvage-driven tumors from those of high proliferative index that rely primarily upon de novo thymidine synthesis. Accordingly, the magnitude of [18F]-FLT uptake should not be considered a surrogate of proliferative index. These data rationalize the diversity of [18F]-FLT-PET correlative results previously reported and suggest future best-practices when [18F]-FLT-PET is employed in oncology.

Volume of preclinical xenograft tumors is more accurately assessed by ultrasound imaging than manual caliper measurements

Objective. The volume of subcutaneous xenograft tumors is an important metric of disease progression and response to therapy in preclinical drug development. Noninvasive imaging technologies suitable for measuring xenograft volume are increasingly available, yet manual calipers, which are susceptible to inaccuracy and bias, are routinely used. The goal of this study was to quantify and compare the accuracy, precision, and inter‐rater variability of xenograft tumor volume assessment by caliper measurements and ultrasound imaging. Methods. Subcutaneous xenograft tumors derived from human colorectal cancer cell lines (DLD1 and SW620) were generated in athymic nude mice. Experienced independent reviewers segmented 3‐dimensional ultrasound data sets and collected manual caliper measurements resulting in tumor volumes. Imaging‐ and caliper‐derived volumes were compared with the tumor mass, the reference standard, determined after resection. Bias, precision, and inter‐rater differences were estimated for each mouse among reviewers. Bootstrapping was used to estimate mean and confidence intervals of variance components, intraclass correlation coefficients (ICCs), and confidence intervals for each source of variation. Results. The average deviation from the true volume and inter‐rater differences were significantly lower for ultrasound volumes compared with caliper volumes (P = .0005 and .001, respectively). Reviewer ICCs for ultrasound and caliper measurements were similarly low (1%), yet caliper volume variance was 1.3‐fold higher than for ultrasound. Conclusions. Ultrasound imaging more accurately, precisely, and reproducibly reflects xenograft tumor volume than caliper measurements. These data suggest that preclinical studies using the xenograft burden as a surrogate end point measured by ultrasound imaging require up to 30% fewer animals to reach statistical significance compared with analogous studies using caliper measurements.

Quantitative Preclinical Imaging of TSPO Expression in Glioma Using N,N-Diethyl-2-(2-(4-(2-18F-Fluoroethoxy)Phenyl)-5,7-Dimethylpyrazolo[1,5-a]Pyrimidin-3-yl)Acetamide

There is a critical need to develop and rigorously validate molecular imaging biomarkers to aid diagnosis and characterization of primary brain tumors. Elevated expression of translocator protein (TSPO) has been shown to predict disease progression and aggressive, invasive behavior in a variety of solid tumors. Thus, noninvasive molecular imaging of TSPO expression could form the basis of a novel, predictive cancer imaging biomarker. In quantitative preclinical PET studies, we evaluated a high-affinity pyrazolopyrimidinyl-based TSPO imaging ligand, N,N-diethyl-2-(2-(4-(2-18F-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (18F-DPA-714), as a translational probe for quantification of TSPO levels in glioma. Methods: Glioma-bearing rats were imaged with 18F-DPA-714 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of 18F-DPA-714 (130–200 MBq/0.2 mL). Blood was collected to derive the arterial input function (AIF), with high-performance liquid chromatography radiometabolite analysis performed on selected samples for AIF correction. Compartmental modeling was performed using the corrected AIF. Specific tumor cell binding of DPA-714 was evaluated by radioligand displacement of 3H-PK 11195 with DPA-714 in vitro and displacement of 18F-DPA-714 with an excess of DPA-714 in vivo. Immediately after imaging, tumor and healthy brain tissues were harvested for validation by Western blotting and immunohistochemistry. Results: 18F-DPA-714 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain. Infusion with DPA-714 (10 mg/kg) displaced 18F-DPA-714 binding by greater than 60% on average. Tumor uptake of 18F-DPA-714 was similar to another high-affinity TSPO imaging ligand, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline, and agreed with ex vivo assay of TSPO levels in tumor and healthy brain. Conclusion: These studies illustrate the feasibility of using 18F-DPA-714 for visualization of TSPO-expressing brain tumors. Importantly, 18F-DPA-714 appears suitable for quantitative assay of tumor TSPO levels in vivo. Given the relationship between elevated TSPO levels and poor outcome in oncology, these studies suggest the potential of 18F-DPA-714 PET to serve as a novel predictive cancer imaging modality.

Quantitative, Preclinical PET of Translocator Protein Expression in Glioma Using 18F-N-Fluoroacetyl-N-(2,5-Dimethoxybenzyl)-2-Phenoxyaniline

Translocator protein (TSPO), also referred to as peripheral benzodiazepine receptor (PBR), is a crucial 18-kDa outer mitochondrial membrane protein involved in numerous cellular functions, including the regulation of cholesterol metabolism, steroidogenesis, and apoptosis. Elevated expression of TSPO in oncology correlates with disease progression and poor survival, suggesting that molecular probes capable of assaying TSPO levels may have potential as cancer imaging biomarkers. In preclinical PET studies, we characterized a high-affinity aryloxyanilide-based TSPO imaging ligand, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (18F-PBR06), as a candidate probe for the quantitative assessment of TSPO expression in glioma. Methods: Glioma-bearing rats were imaged with 18F-PBR06 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of 18F-PBR06 (70–100 MBq/0.2 mL). Over the course of scanning, arterial blood was collected to derive the input function, with high-performance liquid chromatography radiometabolite analysis performed on selected samples for arterial input function correction. Compartmental modeling of the PET data was performed using the corrected arterial input function. Specific tumor cell binding of PBR06 was evaluated by radioligand displacement of 3H-PK 11195 with PBR06 in vitro and by displacement of 18F-PBR06 with excess PBR06 in vivo. Immediately after imaging, tumor tissue and adjacent healthy brain were harvested for assay of TSPO protein levels by Western blotting and immunohistochemistry. Results: 18F-PBR06 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain, facilitating excellent contrast between tumor and adjacent tissue. Infusion with PBR06 (10 mg/kg) displaced 18F-PBR06 binding by approximately 75%. The accumulation of 18F-PBR06 in tumor tissues and adjacent brain agreed with the ex vivo assay of TSPO protein levels by Western blotting and quantitative immunohistochemistry. Conclusion: These preclinical studies illustrate that 18F-PBR06 is a promising tracer for visualization of TSPO-expressing tumors. Importantly, the close correlation between 18F-PBR06 uptake and TSPO expression in tumors and normal tissues, coupled with the high degree of displaceable binding from both tumors and the normal brain, represents a significant improvement over other TSPO imaging ligands previously evaluated in glioma. These data suggest the potential of 18F-PBR06 to elucidate the role of TSPO in oncology, as well as its potential development as a cancer imaging biomarker.

18FDG-PET predicts pharmacodynamic response to OSI-906, a dual IGF-1R/IR inhibitor, in preclinical mouse models of lung cancer

Purpose: To evaluate 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography imaging (18FDG-PET) as a predictive, noninvasive, pharmacodynamic (PD) biomarker of response following administration of a small-molecule insulin-like growth factor-1 receptor and insulin receptor (IGF-1R/IR) inhibitor, OSI-906. Experimental Design: In vitro uptake studies of 3H-2-deoxy glucose following OSI-906 exposure were conducted evaluating correlation of dose with inhibition of IGF-1R/IR as well as markers of downstream pathways and glucose metabolism. Similarly, in vivo PD effects were evaluated in human tumor cell line xenografts propagated in athymic nude mice by 18FDG-PET at 2, 4, and 24 hours following a single treatment of OSI-906 for the correlation of inhibition of receptor targets and downstream markers. Results: Uptake of 3H-2-deoxy glucose and 18FDG was significantly diminished following OSI-906 exposure in sensitive tumor cells and subcutaneous xenografts (NCI-H292) but not in an insensitive model lacking IGF-1R expression (NCI-H441). Diminished PD 18FDG-PET, collected immediately following the initial treatment agreed with inhibition of pIGF-1R/pIR, reduced PI3K (phosphoinositide 3-kinase) and MAPK (mitogen activated protein kinase) pathway activity, and predicted tumor growth arrest as measured by high-resolution ultrasound imaging. Conclusion: 18FDG-PET seems to serve as a rapid, noninvasive PD marker of IGF-1R/IR inhibition following a single dose of OSI-906 and should be explored clinically as a predictive clinical biomarker in patients undergoing IGF-1R/IR–directed cancer therapy. Clin Cancer Res; 17(10); 3332–40. ©2011 AACR.

Synthesis and Structure-Activity Relationships of 5,6,7-substituted Pyrazolopyrimidines: Discovery of a novel TSPO PET Ligand for Cancer Imaging

Focused library synthesis and structure-activity relationship development of 5,6,7-substituted pyrazolopyrimidines led to the discovery of 2-(5,7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (6b), a novel translocator protein (TSPO) ligand exhibiting a 36-fold enhancement in affinity compared to another pyrazolopyrimidine-based TSPO ligand, 6a (DPA-714). Radiolabeling with fluorine-18 ((18)F) facilitated production of 2-(5,7-diethyl-2-(4-(2-[(18)F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide ((18)F-6b) in high radiochemical yield and specific activity. In vivo studies of (18)F-6b were performed which illuminated this agent as an improved probe for molecular imaging of TSPO-expressing cancers.

Inducible loss of one Apc allele in Lrig1-expressing progenitor cells results in multiple distal colonic tumors with features of familial adenomatous polyposis

Individuals with familial adenomatous polyposis (FAP) harbor a germline mutation in adenomatous polyposis coli (APC). The major clinical manifestation is development of multiple colonic tumors at a young age due to stochastic loss of the remaining APC allele. Extracolonic features, including periampullary tumors, gastric abnormalities, and congenital hypertrophy of the retinal pigment epithelium, may occur. The objective of this study was to develop a mouse model that simulates these features of FAP. We combined our Lrig1-CreERT2/+ mice with Apcfl/+ mice, eliminated one copy of Apc in leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1)-positive (Lrig1(+)) progenitor cells with tamoxifen injection, and monitored tumor formation in the colon by colonoscopy and PET. Initial loss of one Apc allele in Lrig1(+) cells results in a predictable pattern of preneoplastic changes, culminating in multiple distal colonic tumors within 50 days of induction, as well as the extracolonic manifestations of FAP mentioned above. We show that tumor formation can be monitored by noninvasive PET imaging. This inducible stem cell-driven model recapitulates features of FAP and offers a tractable platform on which therapeutic interventions can be monitored over time by colonoscopy and noninvasive imaging.

3'-Deoxy-3'-18F-fluorothymidine PET predicts response to (V600E)BRAF-targeted therapy in preclinical models of colorectal cancer.

Selective inhibition of oncogenic targets and associated signaling pathways forms the basis of personalized cancer medicine. The clinical success of V600EBRAF 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′-18F-fluorothymidine (18F-FLT) PET to noninvasively quantify changes in tumor proliferation that are associated with pharmacologic inhibition of V600EBRAF downstream effectors and that precede changes in tumor volume. Methods: Human colorectal cancer (CRC) cell lines expressing V600EBRAF were used to explore relationships between upregulation of p27 and phosphorylation of BRAF downstream effectors on small-molecule V600EBRAF inhibitor exposure. Athymic nude mice bearing V600EBRAF-expressing human CRC cell line xenografts were treated with a small-molecule V600EBRAF inhibitor (or vehicle) daily for 10 d. Predictive 18F-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 18F-FLT uptake, but not 18F-FDG uptake, in Lim2405 xenografts before 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 18F-FLT PET and 18F-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. Conclusion: We used preclinical models of CRC to demonstrate 18F-FLT PET as a sensitive predictor of response to V600EBRAF inhibitors. Because 18F-FLT PET predicted reduced proliferation associated with attenuation of BRAF downstream effectors, yet 18F-FDG PET did not, these data suggest that 18F-FLT PET may represent an alternative to 18F-FDG PET for quantifying clinical responses to BRAF inhibitors.