Quantitative and textural analysis of magnetization transfer and diffusion images in the early detection of brain metastases

Purpose: The sensitivity of the magnetization transfer ratio (MTR) and apparent diffusion coefficient (ADC) for early detection of brain metastases was investigated in mice and humans. Methods: Mice underwent MRI twice weekly for up to 31 days following intra-cardiac injection of the brain-homing breast cancer cell line MDA-MB231-BR. Patients with small cell lung cancer underwent quarterly MRI for a year. MTR and ADC were measured in regions of metastasis and matched contralateral tissue at the final time-point and in registered regions at earlier time-points. Texture analysis and linear discriminant analysis were performed to detect metastasis-containing slices. Results: Compared with contralateral tissue, mouse metastases had significantly lower MTR and higher ADC at the final time-point. Some lesions were visible at earlier time-points on the MTR and ADC maps: 24% of these were not visible on corresponding T2-weighted images. Texture analysis using the MTR maps showed 100% specificity and 98% sensitivity for metastasis at the final time-point, with 77% sensitivity 2-4 days earlier and 46% 5-8 days earlier. Only 2/16 patients developed metastases, and their penultimate scans were normal. Conclusion: Some brain metastases may be detected earlier on MTR than conventional T2; however, the small gain is unlikely to justify ‘predictive’ MRI.The authors gratefully acknowledge the Cambridge Institute Biological Resources Unit for expert animal care and technical assistance, the Histopathology Core Facility, Drs Joe Frank and Diane Palmieri for providing the cell line, the advice of Dr. Dan Tozer, and the support of Cancer Research UK [grant number C14303/A17197], the Brian Cross Memorial Trust, the Addenbrooke’s Charitable Trust, the University of Cambridge, Hutchison Whampoa Ltd, the Cambridge Experimental Cancer Medicine Centre, and the NIHR Cambridge Biomedical Research Centre.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1002/mrm.2625

Gemcitabine Mechanism of Action Confounds Early Assessment of Treatment Response by 3'-Deoxy-3'-[18^{18}F]Fluorothymidine in Preclinical Models of Lung Cancer

3'-Deoxy-3'-[$^{18}$F]fluorothymidine positron emission tomography ([$^{18}$F]FLT-PET) and diffusion-weighted MRI (DW-MRI) are promising approaches to monitor tumor therapy response. Here, we employed these two imaging modalities to evaluate the response of lung carcinoma xenografts in mice after gemcitabine therapy. Caliper measurements revealed that H1975 xenografts responded to gemcitabine treatment, whereas A549 growth was not affected. In both tumor models, uptake of [$^{18}$F]FLT was significantly reduced 6 hours after drug administration. On the basis of the gemcitabine concentration and [$^{18}$F]FLT excretion measured, this was presumably related to a direct competition of gemcitabine with the radiotracer for cellular uptake. On day 1 after therapy, [$^{18}$F]FLT uptake was increased in both models, which was correlated with thymidine kinase 1 (TK1) expression. Two and 3 days after drug administration, [$^{18}$F]FLT uptake as well as TK1 and Ki67 expression were unchanged. A reduction in [$^{18}$F]FLT in the responsive H1975 xenografts could only be noted on day 5 of therapy. Changes in ADC$_{mean}$ in A549 xenografts 1 or 2 days after gemcitabine did not seem to be of therapy-related biological relevance as they were not related to cell death (assessed by caspase-3 IHC and cellular density) or tumor therapy response. Taken together, in these models, early changes of [$^{18}$F]FLT uptake in tumors reflected mechanisms, such as competing gemcitabine uptake or gemcitabine-induced thymidylate synthase inhibition, and only reflected growth-inhibitory effects at a later time point. Hence, the time point for [$^{18}$F]FLT-PET imaging of tumor response to gemcitabine is of crucial importance.The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking (www.imi.europa.eu) under grant agreement number 115151, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution. This work was also supported by the Deutsche Forschungsgemeinschaft (DFG), Cells-in-Motion Cluster of Excellence (EXC1003 – CiM), University of Munster (Münster, Germany)

Quantitative and textural analysis of magnetization transfer and diffusion images in the early detection of brain metastases.

PURPOSE: The sensitivity of the magnetization transfer ratio (MTR) and apparent diffusion coefficient (ADC) for early detection of brain metastases was investigated in mice and humans. METHODS: Mice underwent MRI twice weekly for up to 31 d following intracardiac injection of the brain-homing breast cancer cell line MDA-MB231-BR. Patients with small cell lung cancer underwent quarterly MRI for 1 year. MTR and ADC were measured in regions of metastasis and matched contralateral tissue at the final time point and in registered regions at earlier time points. Texture analysis and linear discriminant analysis were performed to detect metastasis-containing slices. RESULTS: Compared with contralateral tissue, mouse metastases had significantly lower MTR and higher ADC at the final time point. Some lesions were visible at earlier time points on the MTR and ADC maps: 24% of these were not visible on corresponding T2 -weighted images. Texture analysis using the MTR maps showed 100% specificity and 98% sensitivity for metastasis at the final time point, with 77% sensitivity 2-4 d earlier and 46% 5-8 d earlier. Only 2 of 16 patients developed metastases, and their penultimate scans were normal. CONCLUSIONS: Some brain metastases may be detected earlier on MTR than conventional T2 ; however, the small gain is unlikely to justify "predictive" MRI. Magn Reson Med 77:1987-1995, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.The authors gratefully acknowledge the Cambridge Institute Biological Resources Unit for expert animal care and technical assistance, the Histopathology Core Facility, Drs Joe Frank and Diane Palmieri for providing the cell line, the advice of Dr. Dan Tozer, and the support of Cancer Research UK [grant number C14303/A17197], the Brian Cross Memorial Trust, the Addenbrooke’s Charitable Trust, the University of Cambridge, Hutchison Whampoa Ltd, the Cambridge Experimental Cancer Medicine Centre, and the NIHR Cambridge Biomedical Research Centre.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1002/mrm.2625

Response Monitoring with [18F]FLT PET and Diffusion-Weighted MRI After Cytotoxic 5-FU Treatment in an Experimental Rat Model for Colorectal Liver Metastases.

PURPOSE: The aim of the study was to investigate the potential of diffusion-weighted magnetic resonance imaging (DW-MRI) and 3'-dexoy-3'-[(¹⁸)F]fluorothymidine ([(¹⁸)F]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 [(¹⁸)F]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, [(¹⁸)F]FLT SUV_mean and SUV_max were significantly increased at days 4 and 7 after treatment with 5-FU (60 mg/kg) and returned to baseline at day 14 (SUV_max 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 [(¹⁸)F]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 [(¹⁸)F]FLT uptake of responsive CC531 tumors in the liver, while the ADC_mean did not change significantly. Future studies in larger groups are warranted to further investigate whether [(¹⁸)F]FLT PET can discriminate between disease progression and treatment response.The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking (www.imi.europa.eu) under grant agreement number 115151, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution

Response Monitoring with [18F]FLT PET and Diffusion-Weighted MRI After Cytotoxic 5-FU Treatment in an Experimental Rat Model for Colorectal Liver Metastases.

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.The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking (www.imi.europa.eu) under grant agreement number 115151, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution

Thymidine Metabolism as Confounding Factor of 3'-Deoxy-3'-[18F]Fluorothymidine Uptake after Therapy in a Colorectal Cancer Model.

Non-invasive monitoring of tumor therapy response helps in developing personalized treatment strategies. Here, we performed sequential positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI) to evaluate changes induced by a FOLFOX-like combination chemotherapy in colorectal cancer (CRC) xenografts, to identify the cellular and molecular determinants of these imaging biomarkers. Methods: Tumor bearing CD1 nude mice, engrafted with FOLFOX-sensitive Colo205 CRC xenografts, were treated with FOLFOX (5 fluorouracil, leucovorin and oxaliplatin) in weekly intervals. On d1, d2, d6, d9 and d13 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 d1 of therapy. Results: In Colo205 xenografts, FOLFOX induced a profound increase in uptake of the proliferation PET tracer 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), which was accompanied by increases in markers for proliferation (Ki67, TK1) and for activated DNA damage response (DDR; γH2AX), whereas the effect on cell death was minimal. As tracer uptake was unaltered in the HCT116 model, these changes appear to be specific for tumor response. Conclusion: We demonstrate that [18F]FLT PET can non-invasively monitor molecular alterations induced by a cancer treatment, including thymidine metabolism and DDR. The cellular or imaging changes may not, however, be directly related to therapy response as assessed by volumetric measurements

Carbonic Anhydrase Activity Monitored In Vivo by Hyperpolarized 13C-Magnetic Resonance Spectroscopy Demonstrates Its Importance for pH Regulation in Tumors.

Carbonic anhydrase buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3(-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3(-), using (13)C-magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3(-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3(-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity using magnetization transfer measurements, which can be explained by the more acidic extracellular pH in these tumors and the decreased activity of the enzyme at low pH. This explanation was confirmed by administration of bicarbonate in the drinking water, which elevated tumor extracellular pH and restored enzyme activity to control levels. These results suggest that CAIX expression is increased in hypoxia to compensate for the decrease in its activity produced by a low extracellular pH and supports the hypothesis that a major function of CAIX is to lower the extracellular pH.The authors acknowledge funding support from Cancer Research UK (CRUK; C19212/A16628; C19212/A911376), the National Institute for Health Research Cambridge Biomedical Research Centre and the School of Clinical Medicine at the University of Cambridge, the CRUK and Engineering and Physical Sciences Research Council (EPSRC) Cancer Imaging Centre in Cambridge and Manchester. E.M.S. is a recipient of funding from the European Union Seventh Framework Programme (FP7/2007-2013) under the Marie Curie Initial Training Network METAFLUX and has support from the Calouste Gulbenkian Foundation, Champalimaud Foundation, Ministerio de Saude and Fundacao para a Ciencia e Tecnologia, Portugal.This is the author accepted manuscript. The final version is available from American Association for Cancer Research via http://dx.doi.org/10.1158/0008-5472.CAN-15-085

Correction to: The relationship between endogenous thymidine concentrations and [18F]FLT uptake in a range of preclinical tumour models.

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

Carbonic anhydrase IX is a pH-stat that sets an acidic tumour extracellular pH in vivo

Background Tumour Carbonic Anhydrase IX (CAIX), a hypoxia-inducible tumour-associated cell surface enzyme, is thought to acidify the tumour microenvironment by hydrating CO2 to form protons and bicarbonate, but there is no definitive evidence for this in solid tumours in vivo. Methods We used 1H magnetic resonance spectroscopic imaging (MRSI) of the extracellular pH probe imidazolyl succinic acid (ISUCA) to measure and spatially map extracellular pH in HCT116 tumours transfected to express CAIX and empty vector controls in SCID mice. We also measured intracellular pH in situ with 31P MRS and measured lactate in freeze-clamped tumours. Results CAIX expressing tumours had 0.15 pH-unit lower median extracellular pH than control tumours (pH 6.71 tumour vs pH 6.86 control, P = 0.01). Importantly, CAIX expression imposed an upper limit for tumour extracellular pH at 6.93. Despite the increased lactate concentration in CAIX-expressing tumours, 31P MRS showed no difference in intracellular pH, suggesting that CAIX acidifies only the tumour extracellular space. Conclusions CAIX acidifies the tumour microenvironment, and also provides an extracellular pH control mechanism. We propose that CAIX thus acts as an extracellular pH-stat, maintaining an acidic tumour extracellular pH that is tolerated by cancer cells and favours invasion and metastasis.We are grateful for the support of CRUK [grant number C14303/A17197], the Breast Cancer Research Foundation, the Royal Society, Worldwide Cancer Research and the European Research Council [SURVIVE: 723397]. JP-T and SC received support from the Spanish Ministry of Economy and Competitiveness SAF2014-23622