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Purpose: We recently reported that high thymidine phosphorylase (TP) expression is accompanied by low tumor thymidine concentration and high 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) uptake in four untreated lung cancer xenografts. Here, we investigated whether this relationship also holds true for a broader range of tumor models.Procedures: Lysates from n = 15 different tumor models originating from n = 6 institutions were tested for TP and thymidylate synthase (TS) expression using western blots. Results were correlated to [18F]FLT accumulation in the tumors as determined by positron emission tomography (PET) measurements in the different institutions and to previously published thymidine concentrations.Results: Expression of TP correlated positively with [18F]FLT SUVmax (ρ = 0.549, P < 0.05). Furthermore, tumors with high TP levels possessed lower levels of thymidine (ρ = - 0.939, P < 0.001).Conclusions: In a broad range of tumors, [18F]FLT uptake as measured by PET is substantially influenced by TP expression and tumor thymidine concentrations. These data strengthen the role of TP as factor confounding [18F]FLT uptake. [ABSTRACT FROM AUTHOR]

Background: Recent studies have shown that 3′-deoxy-3′-[F] fluorothymidine ([F]FLT)) uptake depends on endogenous tumour thymidine concentration. The purpose of this study was to investigate tumour thymidine concentrations and whether they correlated with [F]FLT uptake across a broad spectrum of murine cancer models. A modified liquid chromatography-mass spectrometry (LC-MS/MS) method was used to determine endogenous thymidine concentrations in plasma and tissues of tumour-bearing and non-tumour bearing mice and rats. Thymidine concentrations were determined in 22 tumour models, including xenografts, syngeneic and spontaneous tumours, from six research centres, and a subset was compared for [F]FLT uptake, described by the maximum and mean tumour-to-liver uptake ratio (TTL) and SUV. Results: The LC-MS/MS method used to measure thymidine in plasma and tissue was modified to improve sensitivity and reproducibility. Thymidine concentrations determined in the plasma of 7 murine strains and one rat strain were between 0.61 ± 0.12 μM and 2.04 ± 0.64 μM, while the concentrations in 22 tumour models ranged from 0.54 ± 0.17 μM to 20.65 ± 3.65 μM. TTL at 60 min after [F]FLT injection, determined in 14 of the 22 tumour models, ranged from 1.07 ± 0.16 to 5.22 ± 0.83 for the maximum and 0.67 ± 0.17 to 2.10 ± 0.18 for the mean uptake. TTL did not correlate with tumour thymidine concentrations. Conclusions: Endogenous tumour thymidine concentrations alone are not predictive of [F]FLT uptake in murine cancer models. [ABSTRACT FROM AUTHOR]

Purpose: The aim of the study was to investigate the potential of diffusion-weighted magnetic resonance imaging (DW-MRI) and 3'-dexoy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography (PET) as early biomarkers of treatment response of 5-fluorouracil (5-FU) in a syngeneic rat model of colorectal cancer liver metastases.Procedures: Wag/Rij rats with intrahepatic syngeneic CC531 tumors were treated with 5-FU (15, 30, or 60 mg/kg in weekly intervals). Before treatment and at days 1, 3, 7, and 14 after treatment rats underwent DW-MRI and [18F]FLT PET. Tumors were analyzed immunohistochemically for Ki67, TK1, and ENT1 expression.Results: 5-FU inhibited the growth of CC531 tumors in a dose-dependent manner. Immunohistochemical analysis did not show significant changes in Ki67, TK1, and ENT1 expression. However, [18F]FLT SUVmean and SUVmax were significantly increased at days 4 and 7 after treatment with 5-FU (60 mg/kg) and returned to baseline at day 14 (SUVmax at days -1, 4, 7, and 14 was 1.1 ± 0.1, 2.3 ± 0.5, 2.3 ± 0.6, and 1.5 ± 0.4, respectively). No changes in [18F]FLT uptake were observed in the nontreated animals. Furthermore, the apparent diffusion coefficient (ADCmean) did not change in 5-FU-treated rats compared to untreated rats.Conclusion: This study suggests that 5-FU treatment induces a flare in [18F]FLT uptake of responsive CC531 tumors in the liver, while the ADCmean did not change significantly. Future studies in larger groups are warranted to further investigate whether [18F]FLT PET can discriminate between disease progression and treatment response. [ABSTRACT FROM AUTHOR]

Background: Recent studies have shown that 3′-deoxy-3′-[F] fluorothymidine ([F]FLT)) uptake depends on endogenous tumour thymidine concentration. The purpose of this study was to investigate tumour thymidine concentrations and whether they correlated with [F]FLT uptake across a broad spectrum of murine cancer models. A modified liquid chromatography-mass spectrometry (LC-MS/MS) method was used to determine endogenous thymidine concentrations in plasma and tissues of tumour-bearing and non-tumour bearing mice and rats. Thymidine concentrations were determined in 22 tumour models, including xenografts, syngeneic and spontaneous tumours, from six research centres, and a subset was compared for [F]FLT uptake, described by the maximum and mean tumour-to-liver uptake ratio (TTL) and SUV. Results: The LC-MS/MS method used to measure thymidine in plasma and tissue was modified to improve sensitivity and reproducibility. Thymidine concentrations determined in the plasma of 7 murine strains and one rat strain were between 0.61 ± 0.12 μM and 2.04 ± 0.64 μM, while the concentrations in 22 tumour models ranged from 0.54 ± 0.17 μM to 20.65 ± 3.65 μM. TTL at 60 min after [F]FLT injection, determined in 14 of the 22 tumour models, ranged from 1.07 ± 0.16 to 5.22 ± 0.83 for the maximum and 0.67 ± 0.17 to 2.10 ± 0.18 for the mean uptake. TTL did not correlate with tumour thymidine concentrations. Conclusions: Endogenous tumour thymidine concentrations alone are not predictive of [F]FLT uptake in murine cancer models. [ABSTRACT FROM AUTHOR]

Purpose: To develop a sensitive analytical method to quantify gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) and its metabolites 2′,2′-difluorodeoxyuridine (dFdU) and 2′,2′-difluorodeoxycytidine-5′-triphosphate (dFdCTP) simultaneously from tumour tissue. Methods: Pancreatic ductal adenocarcinoma tumour tissue from genetically engineered mouse models of pancreatic cancer ( KP C and KP C) was collected after dosing the mice with gemcitabine. F NMR spectroscopy and LC-MS/MS protocols were optimised to detect gemcitabine and its metabolites in homogenates of the tumour tissue. Results: A F NMR protocol was developed, which was capable of distinguishing the three analytes in tumour homogenates. However, it required at least 100 mg of the tissue in question and a long acquisition time per sample, making it impractical for use in large PK/PD studies or clinical trials. The LC-MS/MS protocol was developed using porous graphitic carbon to separate the analytes, enabling simultaneous detection of all three analytes from as little as 10 mg of tissue, with a sensitivity for dFdCTP of 0.2 ng/mg tissue. Multiple pieces of tissue from single tumours were analysed, showing little intra-tumour variation in the concentrations of dFdC or dFdU (both intra- and extra-cellular). Intra-tumoural variation was observed in the concentration of dFdCTP, an intra-cellular metabolite, which may reflect regions of different cellularity within a tumour. Conclusion: We have developed a sensitive LC-MS/MS method capable of quantifying gemcitabine, dFdU and dFdCTP in pancreatic tumour tissue. The requirement for only 10 mg of tissue enables this protocol to be used to analyse multiple areas from a single tumour and to spare tissue for additional pharmacodynamic assays. [ABSTRACT FROM AUTHOR]

Unfortunately, the original version of Figs. 4, 5 and 6b in the article [1] contained errors in the n numbers as indicated on the columns. Please note that column heights and error bars in the original figures and data in the ESM tables are correct and statistical tests are valid. These corrections do not affect any results or conclusions in this article. [ABSTRACT FROM AUTHOR]

Imaging biomarkers must demonstrate their value in monitoring treatment. Two PET tracers, the caspase-3/7-specific isatin-5-sulfonamide 18 F-ICMT-11 ( 18 F-( S )-1-((1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-5-(2(2,4-difluoro-phenoxymethyl)-pyrrolidine-1-sulfonyl)isatin) and 18 F-FLT (3'-deoxy-3'- 18 F-fluorothymidine), were used to detect early treatment-induced changes in tumor biology and determine whether any of these changes indicate a response to cetuximab, administered as monotherapy or combination therapy with gemcitabine. Methods: In mice bearing cetuximab-sensitive H1975 tumors (non-small lung cancer), the effects of single or repeated doses of the antiepidermal growth factor receptor antibody cetuximab (10 mg/kg on day 1 only or on days 1 and 2) or a single dose of gemcitabine (125 mg/kg on day 2) were investigated by 18 F-ICMT-11 or 18 F-FLT on day 3. Imaging was also performed after 2 doses of cetuximab (days 1 and 2) in mice bearing cetuximab-insensitive HCT116 tumors (colorectal cancer). For imaging-histology comparison, tumors were evaluated for proliferation (Ki-67 and thymidine kinase 1 [TK1]), cell death (cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling [TUNEL]), and target engagement (epidermal growth factor receptor expression) by immunohistochemistry, immunofluorescence, and immunoblotting, respectively. Tumor and plasma were analyzed for thymidine and gemcitabine metabolites by liquid chromatography-mass spectrometry. Results: Retention of both tracers was sensitive to cetuximab in H1975 tumors. 18 F-ICMT-11 uptake and ex vivo cleaved caspase-3 staining notably increased in tumors treated with repeated doses of cetuximab (75%) and combination treatment (46%). Although a single dose of cetuximab was insufficient to induce apoptosis, it did affect proliferation. Significant reductions in tumor 18 F-FLT uptake (44%-50%; P < 0.001) induced by cetuximab monotherapy and combination therapy were paralleled by a clear decrease in proliferation (Ki-67 decrease, 72%-95%; P < 0.0001), followed by a marked tumor growth delay. TK1 expression and tumor thymidine concentrations were profoundly reduced. Neither imaging tracer depicted the gemcitabine-induced tumor changes. However, cleaved caspase-3 and Ki-67 staining did not significantly differ after gemcitabine treatment whereas TK1 expression and thymidine concentrations increased. No cetuximab-induced modulation of the imaging tracers or other response markers was detected in the insensitive model of HCT116. Conclusion: 18 F-ICMT-11 and 18 F-FLT are valuable tools to assess cetuximab sensitivity depicting distinct and time-variant aspects of treatment response., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

Noninvasive monitoring of tumor therapy response helps in developing personalized treatment strategies. Here, we performed sequential PET and diffusion-weighted MRI to evaluate changes induced by a FOLFOX-like combination chemotherapy in colorectal cancer xenografts, to identify the cellular and molecular determinants of these imaging biomarkers. Methods: Tumor-bearing CD1 nude mice, engrafted with FOLFOX-sensitive Colo205 colorectal cancer xenografts, were treated with FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin) weekly. On days 1, 2, 6, 9, and 13 of therapy, tumors were assessed by in vivo imaging and ex vivo analyses. In addition, HCT116 xenografts, which did not respond to the FOLFOX treatment, were imaged on day 1 of therapy. Results: In Colo205 xenografts, FOLFOX induced a profound increase in uptake of the proliferation PET tracer 3'-deoxy-3'- 18 F-fluorothymidine ( 18 F-FLT) accompanied by increases in markers for proliferation (Ki-67, thymidine kinase 1) and for activated DNA damage response (γH2AX), whereas the effect on cell death was minimal. Because tracer uptake was unaltered in the HCT116 model, these changes appear to be specific for tumor response. Conclusion: We demonstrated that 18 F-FLT PET can noninvasively monitor cancer treatment-induced molecular alterations, including thymidine metabolism and DNA damage response. The cellular or imaging changes may not, however, be directly related to therapy response as assessed by volumetric measurements., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

Background: The Notch pathway is frequently activated in cancer. Pathway inhibition by γ-secretase inhibitors has been shown to be effective in pre-clinical models of pancreatic cancer, in combination with gemcitabine., Methods: A multi-centre, non-randomised Bayesian adaptive design study of MK-0752, administered per os weekly, in combination with gemcitabine administered intravenously on days 1, 8 and 15 (28 day cycle) at 800 or 1000 mg m -2 , was performed to determine the safety of combination treatment and the recommended phase 2 dose (RP2D). Secondary and tertiary objectives included tumour response, plasma and tumour MK-0752 concentration, and inhibition of the Notch pathway in hair follicles and tumour., Results: Overall, 44 eligible patients (performance status 0 or 1 with adequate organ function) received gemcitabine and MK-0752 as first or second line treatment for pancreatic cancer. RP2Ds of MK-0752 and gemcitabine as single agents could be combined safely. The Bayesian algorithm allowed further dose escalation, but pharmacokinetic analysis showed no increase in MK-0752 AUC (area under the curve) beyond 1800 mg once weekly. Tumour response evaluation was available in 19 patients; 13 achieved stable disease and 1 patient achieved a confirmed partial response., Conclusions: Gemcitabine and a γ-secretase inhibitor (MK-0752) can be combined at their full, single-agent RP2Ds.

Correction: Unfortunately, the original version of Figs. 4, 5 and 6b in the article [1] contained errors in the n numbers as indicated on the columns. Please note that column heights and error bars in the original figures and data in the ESM tables are correct and statistical tests are valid. These corrections do not affect any results or conclusions in this article.

Unlabelled: Molecular imaging allows the noninvasive assessment of cancer progression and response to therapy. The aim of this study was to investigate molecular and cellular determinants of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET and diffusion-weighted (DW) MR imaging in lung carcinoma xenografts., Methods: Four lung cancer cell lines (A549, HTB56, EBC1, and H1975) were subcutaneously implanted in nude mice, and growth was followed by caliper measurements. Glucose uptake and tumor proliferation were determined by (18)F-FDG and (18)F-FLT PET, respectively. T2-weighted MR imaging was performed, and the apparent diffusion coefficient (ADC) was determined by DW MR imaging as an indicator of cell death. Imaging findings were correlated to histology with markers for tumor proliferation (Ki67, 5-bromo-2'-deoxyuridine [BrdU]) and cell death (caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling). The expression of human equilibrative nucleoside transporter 1 (hENT1), thymidine kinase 1 (TK1), thymidylate synthase, and thymidine phosphorylase (TP) were analyzed by Western blot and immunohistochemistry. Thymidine levels were determined by liquid chromatography-mass spectrometry., Results: Xenografts varied with respect to in vivo growth rates. MR imaging and PET revealed intratumoral heterogeneities, which were confirmed by histology. (18)F-FLT uptake differed significantly between tumor lines, with A549 and H1975 demonstrating the highest radiotracer accumulation (A549, 8.5 ± 3.2; HTB56, 4.4 ± 0.7; EBC1, 4.4 ± 1.2; and H1975, 12.1 ± 3.5 maximal percentage injected dose per milliliter). In contrast, differences in (18)F-FDG uptake were only marginal. No clear relationship between (18)F-FLT accumulation and immunohistochemical markers for tumor proliferation (Ki67, BrdU) as well as hENT1, TK1, or TS expression was detected. However, TP was highly expressed in A549 and H1975 xenografts, which was accompanied by low tumor thymidine concentrations, suggesting that tumor thymidine levels influence (18)F-FLT uptake in the tumor models investigated. MR imaging revealed higher ADC values within proliferative regions of H1975 and A549 tumors than in HTB56 and EBC1. These ADC values were negatively correlated with cell density but not directly related to cell death., Conclusion: A direct relationship of (18)F-FLT with proliferation or ADC with cell death might be complicated by the interplay of multiple processes at the cellular and physiologic levels in untreated tumors. This issue must be considered when using these imaging modalities in preclinical or clinical settings., (© 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Fragment-based lead discovery has been applied to urokinase-type plasminogen activator (uPA). The (R)-enantiomer of the orally active drug mexiletine 5 (a fragment hit from X-ray crystallographic screening) was the chemical starting point. Structure-aided design led to elaborated inhibitors that retained the key interactions of (R)-5 while gaining extra potency by simultaneously occupying neighboring regions of the active site. Subsequent optimization led to 15, a potent, selective, and orally bioavailable inhibitor of uPA.