Hideyuki Tominaga

Biologic Correlation of 2-[18F]-Fluoro-2-Deoxy-D-Glucose Uptake on Positron Emission Tomography in Thymic Epithelial Tumors

PURPOSE The usefulness of 2-[(18)F]-fluoro-2-deoxy-D-glucose ([(18)F]FDG) positron emission tomography (PET) can help predict the grade of malignancy and staging in thymic epithelial tumors. However, no satisfactory biologic explanation exists for this phenomenon. The aim of this study was to investigate the underlying biologic mechanisms of [(18)F]FDG uptake. PATIENTS AND METHODS Forty-nine patients with thymic epithelial tumors who underwent [(18)F]FDG PET were included in this study. Tumor sections were stained by immunohistochemistry for glucose transporter 1 (GLUT1), glucose transporter 3 (GLUT3), hypoxia-inducible factor-1 alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), microvessels (CD31 and CD34), cell cycle control marker (p53), and apoptosis marker (bcl-2). We also conducted an in vitro study of [(18)F]FDG uptake in a thymic tumor cell line. RESULTS There was a positive correlation between [(18)F]FDG uptake and GLUT1 (P < .0001), HIF-1alpha (P = .0036), VEGF (P < .0001), microvessel density (MVD; P < .0001), and p53 (P = .0002). GLUT3 and bcl-2 showed no positive correlation with [(18)F]FDG uptake. The expression of Glut1, HIF-1alpha, VEGF, CD31, CD34, and p53 was also significantly associated with the grade of malignancy and poor outcome in thymic epithelial tumors, whereas this relationship was not observed in GLUT3 and bcl-2. Our in vitro study demonstrated that upregulation of GLUT1 and HIF-1alpha was closely associated with [(18)F]FDG uptake into thymic tumor cell. CONCLUSION [(18)F]FDG uptake in thymic epithelial tumors is determined by the presence of glucose metabolism (GLUT1), hypoxia (HIF-1alpha), angiogenesis (VEGF and MVD), and cell cycle regulator (p53).

Prognostic significance of L-type amino-acid transporter 1 expression in surgically resected pancreatic cancer

Background:The expression of L-type amino-acid transporter 1 (LAT1) is tumour-specific and has been shown to have essential roles in cell growth and survival. However, little is known regarding the clinical significance of LAT1 expression in pancreatic cancer. This study was conducted to determine the prognostic significance of LAT1 expression.Methods:A total of 97 consecutive patients with surgically resected pathological stage I–IV pancreatic ductal adenocarcinoma were retrospectively reviewed. Tumour sections were stained by immunohistochemistry for LAT1, CD98, Ki-67 and vascular endothelial growth factor (VEGF), and microvessel density was determined by CD34 and p53.Results:L-type amino-acid transporter 1 and CD98 were highly expressed in 52.6% (51/97) and 56.7% (55/97) of cases, respectively (P=0.568). The expression of LAT1 within pancreatic cancer cells was significantly associated with disease stage, tumour size, Ki-67, VEGF, CD34, p53 and CD98. L-type amino-acid transporter 1 expression was confirmed to be a significant prognostic factor for predicting poor outcome by multivariate analysis.Conclusion:L-type amino-acid transporter 1 expression is a promising pathological marker for the prediction of outcome in patients with pancreatic cancer.

Transport of 3-Fluoro-l-α-Methyl-Tyrosine by Tumor-Upregulated L-Type Amino Acid Transporter 1: A Cause of the Tumor Uptake in PET

l-3-18F-α-methyl tyrosine (18F-FAMT) has been developed as a PET radiotracer for tumor imaging. Clinical studies have demonstrated the usefulness of 18F-FAMT PET for the prediction of prognosis and the differentiation of malignant tumors and benign lesions. 18F-FAMT exhibits higher cancer specificity in peripheral organs than other amino acid PET tracers and 18F-FDG. The accumulation of 18F-FAMT is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L highly upregulated in cancers. In this study, we examined the interaction of 3-fluoro-l-α-methyl-tyrosine (FAMT) with amino acid transporters to assess the mechanisms of 18F-FAMT uptake in PET. Methods: We applied in vitro assays using established mammalian cell lines stably expressing LAT1 or a non–cancer-type system L isoform LAT2. The inhibitory effect on l-14C-leucine uptake and the induction effect on efflux of preloaded l-14C-leucine were examined for FAMT and other amino acid tracers. FAMT transport was compared among cell lines with varied LAT1 expression level. Results: FAMT prominently inhibited LAT1-mediated l-14C-leucine uptake in a competitive manner but had less of an effect on LAT2. In the efflux experiments, FAMT induced the efflux of preloaded l-14C-leucine through LAT1, indicating that FAMT is transported by LAT1 and not by LAT2. Among amino acid–related compounds examined in this study, including those used for PET tracers, the compounds with an α-methyl group such as FAMT, 2-fluoro-l-α-methyl-tyrosine, 3-iodo-l-α-methyl-tyrosine, and l-α-methyl-tyrosine were well transported by LAT1 but not by LAT2. However, l-methionine, l-tyrosine, 3-fluoro-l-tyrosine, 2-fluoro-l-tyrosine, and O-(2-fluoroethyl)-l-tyrosine were transported by both LAT1 and LAT2, suggesting that the α-methyl moiety is responsible for the LAT1 selectivity of FAMT. FAMT transport rate and LAT1 protein level were well correlated, supporting the importance of LAT1 for the cellular uptake of FAMT. Conclusion: Distinct from other amino acid PET tracers, because of its α-methyl moiety, FAMT is selective to LAT1 and not transported by LAT2. This property of FAMT is proposed to contribute to highly tumor-specific accumulation of 18F-FAMT in PET.

Positron emission tomography imaging and biodistribution of vascular endothelial growth factor with 64Cu-labeled bevacizumab in colorectal cancer xenografts.

Vascular endothelial growth factor (VEGF) is considered to be a major angiogenic factor responsible for the development of tumor vasculature. The aim of this study was to image VEGF expression with 64Cu‐labeled anti‐VEGF antibody (bevacizumab) non‐invasively, and to see whether or not the expression was correlated with tumor accumulation in colorectal cancer xenografts. Bevacizumab was conjugated with the bifunctional chelator 1, 4, 7, 10‐tetraazacyclododecane‐1, 4, 7, 10‐tetraacetic acid (DOTA) and radiolabeled with 64Cu. In vivo biodistribution studies and positron emission tomography (PET) imaging were performed on mice bearing human colorectal cancer (HT29) xenografts after injection of 64Cu‐DOTA‐bevacizumab, which showed clear accumulation of 64Cu‐DOTA‐bevacizumab in the tumor (22.7 ± 1.0 %ID/g, 24 ± 0.2 %ID/g, 19.0 ± 2.5 %ID/g at 24, 48 and 72 h, respectively). Tumor accumulation of 64Cu‐DOTA‐bevacizumab was significantly correlated with VEGF expression as measured by western blot (ρ = 0.81, P = 0.004). Vascular endothelial growth factor blocking with unlabeled bevacizumab significantly reduced tumor accumulation of 64Cu‐DOTA‐ bevacizumab (9.7 ± 1.2 %ID/g, P < 0.001) at 48 h. Interestingly, the blood concentration of VEGF in the mice treated with excess fold of bevacizumab was significantly higher than those without at 48 h (25.5 ± 4.6 %ID/g vs 6.5 ± 2.1 %ID/g, P = 0.0016). Liver uptake decreased from 24 h (17.2 ± 1.7 %ID/g) to 48 h (13.0 ± 4.2 %ID/g) and 72 h (10.6 ± 1.5 %ID/g) due to hepatic clearance of the tracer. The present study successfully showed 64Cu‐DOTA‐bevacizumab as a potential PET tracer for non‐invasive imaging of VEGF expression in colorectal cancer xenografts. (Cancer Sci 2011; 102: 117–121)

18F-FMT Uptake Seen Within Primary Cancer on PET Helps Predict Outcome of Non–Small Cell Lung Cancer

L-[3-18F]-α-methyl tyrosine (18F-FMT) is an amino-acid tracer for PET imaging. We evaluated the prognostic significance of 18F-FMT PET in patients with non–small cell lung cancer. Methods: Ninety-eight patients (80 men and 18 women; age range, 42–82 y; median age, 69 y) with stage I–IV non–small cell lung cancer were enrolled in this study. They included 57 with adenocarcinoma, 31 with squamous cell carcinoma, 5 with large cell carcinoma, and 5 with other conditions. The median follow-up duration was 17.0 mo. A pair of PET studies with 18F-FMT and 18F-FDG was performed, and tracer uptake by the primary tumor was evaluated using the maximal standardized uptake value (SUVmax). Overall survival and disease-free survival were calculated by the Kaplan–Meier method. The prognostic significance was assessed by univariate and multivariate analyses. Results: The best discriminative SUVmax cutoffs for 18F-FMT and 18F-FDG in the primary tumors were 1.6 and 11, respectively. In the univariate analysis, a high SUVmax was significant in predicting poor overall survival for 18F-FMT (P = 0.0129) and 18F-FDG PET (P = 0.0481). According to histologic types, 18F-FMT and 18F-FDG uptake were a stronger prognostic predictor in adenocarcinoma than in nonadenocarcinomatous disease. Patients with a high SUVmax for 18F-FMT showed significantly worse disease-free survival rates than those with a low SUVmax, and multivariate analysis confirmed that a high SUVmax for 18F-FMT was an independent and significant factor in predicting a poor prognosis in patients with adenocarcinoma (P = 0.0191). Conclusion: Uptake of 18F-FMT in primary tumors was an independent prognostic factor in patients with pulmonary adenocarcinoma.

Establishment of stable cell lines with high expression of heterodimers of human 4F2hc and human amino acid transporter LAT1 or LAT2 and delineation of their differential interaction with α-alkyl moieties.

Abstract System L is a major transport system for cellular uptake of neutral amino acids. Among system L transporters, L-type amino acid transporter 1 (LAT1) is responsible for the nutrient uptake in cancer cells, whereas L-type amino acid transporter 2 (LAT2) is a transporter for non-cancer cells. In this study, we have established HEK293 cell lines stably expressing high levels of human LAT1 and LAT2 forming heterodimers with native human 4F2hc of the cells. We have found that l-[14C]alanine is an appropriate substrate to examine the function of LAT2, whereas l-[14C]leucine is used for LAT1. By using l-[14C]alanine on LAT2, we have for the first time directly evaluated the function of human LAT2 expressed in mammalian cells and obtained its reliable kinetics. Using α-alkyl amino acids including α-methyl-alanine and α-ethyl-l-alanine, we have demonstrated that α-alkyl groups interfere with the interaction with LAT2. These cell lines with higher practical advantages would be useful for screening and analyzing compounds to develop LAT1-specific drugs that can be used for cancer diagnosis and therapeutics. The strategy that we took to establish the cell lines would also be applicable to the other heterodimeric transporters with important therapeutic implications.

Clinical significance of L-type amino acid transporter 1 expression as a prognostic marker and potential of new targeting therapy in biliary tract cancer

BackgroundThe expression of L-type amino acid transporter 1 (LAT1) has been described to play essential roles in tumor cell growth and survival. However, it remains unclear about the clinicopathological significance of LAT1 expression in biliary tract cancer. This study was conducted to determine biological significance of LAT1 expression and investigate whether LAT1 could be a prognostic biomarker for biliary tract cancer.MethodsA total of 139 consecutive patients with resected pathologic stage I-IV biliary tract adenocarcinoma were retrospectively reviewed. Tumor specimens were stained by immunohistochemistry for LAT1, Ki-67, microvessel density determined by CD34, and p53; and prognosis of patients was correlated. Biological significance of LAT1 expression was investigated by in vitro and in vivo experiments with LAT inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) using cholangiocarcinoma cell line.ResultsIn total patients, high LAT1 expressions were recognized in 64.0%. The expression of LAT1 was closely correlated with lymphatic metastases, cell proliferation and angiogenesis, and was a significant indicator for predicting poor outcome after surgery. LAT1 expression was a significant independent predictor by multivariate analysis. Both in vitro and in vivo preliminary experiments indicated that BCH significantly suppressed growth of the tumor and yielded an additive therapeutic efficacy to gemcitabine and 5-FU.ConclusionsHigh expression of LAT1 is a promising pathological marker to predict the outcome in patients with biliary tract adenocarcinoma. Inhibition of LAT1 may be an effective targeted therapy for this distressing disease.

Imaging and biodistribution of Her2/neu expression in non-small cell lung cancer xenografts with 64Cu-labeled trastuzumab PET

Non‐small cell lung carcinomas (NSCLC) overexpress the Her2/neu gene in approximately 59% of cases. Trastuzumab, a humanized monoclonal antibody, interferes with Her2 signaling and is approved for the treatment of Her2/neu overexpressing breast cancer. However, its therapeutic use in Her2/neu overexpressing NSCLC remains obscure. The present study aimed to determine the role of 64Cu‐labeled trastuzumab positron emission tomography (PET) for non‐invasive imaging of Her2/neu expression in NSCLC. Trastuzumab was conjugated with the bifunctional chelator 1, 4, 7, 10‐tetraazacyclododecane‐1, 4, 7, 10‐tetraacetic acid (DOTA) and radiolabeled with 64Cu. The molecular specificity of DOTA‐trastuzumab was determined in NSCLC cell lines with Her2/neu overexpression (NCI‐H2170) and negative expression (NCI‐H520). Imaging of Her2/neu expression was performed in NCI‐H2170 tumor‐bearing mice with 64Cu‐DOTA‐trastuzumab PET and 64Cu‐DOTA‐IgG. In vitro studies revealed specific binding of DOTA‐trastuzumab in the Her2/neu positive NCI‐H2170 cells, while no binding was seen in the Her2/neu negative NCI‐H520 cell line. Biodistribution and PET studies revealed a significantly high accumulation of 64Cu‐DOTA‐trastuzumab in the Her2/neu overexpressing NCI‐H2170 tumor at 24 and 48 h post‐injection (21.4 ± 1.4% and 23.2 ± 5.1% injection dose/gram (% ID/g), respectively). PET imaging of Her2/neu negative NCI‐H520 tumors showed much less uptake of 64Cu‐DOTA‐trastuzumab (4.0% ID/g). The NCI‐H2170 tumor uptake of 64Cu‐DOTA‐trastuzumab was significantly higher than that of 64Cu‐DOTA‐IgG (P < 0.0001). 64Cu‐DOTA‐trastuzumab showed a very clear image of a Her2/neu positive tumor and appeared to be effective as a PET tracer for imaging of Her2/neu gene expression in NSCLC, suggesting its potential clinical use for identifying patients that might benefit from trastuzumab‐based therapy.

Comparison of l-type amino acid transporter 1 expression and l-[3-18F]-α-methyl tyrosine uptake in outcome of non-small cell lung cancer

OBJECTIVE L-Type amino acid transporter 1 (LAT1) has associated with tumor growth and poor outcome of patients with non-small cell lung cancer (NSCLC). L-[3-(18)F]-α-methyl tyrosine ((18)F-FAMT) is an amino acid tracer for positron emission tomography (PET) imaging, and (18)F-FAMT uptake is mediated by LAT1. The purpose of this study is to compare the prognostic significance of (18)F-FAMT uptake in the primary tumors with that of LAT1 expression in patients with NSCLC. METHODS Fifty-nine patients with NSCLC were enrolled in this study. All patients underwent (18)F-FAMT PET prior to resection of the tumor, and immunohistochemical staining of the resected tumors were performed to compare the (18)F-FAMT uptake and LAT1 expression. Uptake of (18)F-FAMT was evaluated using semiquantitative standardized uptake value (SUV(max)), and the cutoff value was determined to discriminate patients with high SUV(max) from those with low SUV(max). Expression of LAT1 was evaluated by the score of staining intensity through 1 to 4. SUV(max) and LAT1 expression were compared according to the clinicopathological variables. RESULTS The best discriminative cutoff value of (18)F-FAMT SUV(max) within the primary tumors was 1.6. The high SUV(max) (>1.6) in (18)F-FAMT PET was significantly associated with male, and positive LAT1 expression was significantly associated with male and nonadenocarcinoma. In the univariate analysis, high SUV(max) (>1.6) in (18)F-FAMT PET and positive LAT1 expression were significant predictor of the poor outcome. Multivariate analysis confirmed that positive LAT1 expression was an independent and significant factor for predicting poor prognosis in NSCLC (P=.035). CONCLUSION LAT1 expression is a stronger prognostic factor than (18)F-FAMT uptake in surgically resected NSCLC.

Predicting cetuximab accumulation in KRAS wild-type and KRAS mutant colorectal cancer using 64Cu-labeled cetuximab positron emission tomography.

Overexpression of epidermal growth factor receptor (EGFR) is common in colorectal cancer. However, cetuximab as an EGFR‐targeting drug is useful only for a subset of patients and currently no single predictor other than V‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation status has been established. In the present study, we investigated cetuximab accumulation in colorectal tumors and major organs using 111In‐DOTA‐cetuximab. We also evaluated the potential of positron emission tomography (PET) imaging of 64Cu‐DOTA‐cetuximab. Colorectal tumor xenografts with a different EGFR expression level and KRAS mutation status were subjected to in vivo biodistribution study and PET imaging at 48 h post‐injection of radiolabeled cetuximab. The EGFR expression levels on colorectal tumors were determined by ex vivo immunoblotting and ELISA. We found that KRAS wild‐type tumors had significantly higher 111In‐DOTA‐cetuximab accumulation than KRAS mutant tumors (P < 0.001). Based on KRAS mutation status, a strong correlation was found between 111In‐DOTA‐cetuximab tumor uptake and EGFR expression level (KRAS wild type: r = 0.988; KRAS mutant: r = 0.829), and between 64Cu‐DOTA‐cetuximab tumor uptake with EGFR expression level (KRAS wild type: r = 0.838; KRAS mutant: r = 0.927). Significant correlation was also found between tumor uptake of 111In‐DOTA‐cetuximab and 64Cu‐DOTA‐cetuximab (r = 0.920). PET imaging with 64Cu‐DOTA‐cetuximab allowed clear visualization of tumors. Both radiolabeled cetuximab had effectively visualized cetuximab accumulation in colorectal tumors with a wide variety of EGFR expression levels and different KRAS mutation status as commonly encountered in the clinical setting. Our findings suggest that this radioimmunoimaging therefore can be clinically translated as an in vivo tool to predict cetuximab accumulation in colorectal cancer patients prior to cetuximab therapy. (Cancer Sci 2012; 103: 600–605)