Final Report Summary - ANGIOTOX (Histopathologic and Mechanistic Assessment of Angiogenesis Inhibitor Related Toxicities: A Cross-Sectoral, Multi-disciplinary Approach.)
www.angiotox.com
Coordinator contact details : annettebyrne@rcsi.ie
Project Logo: ( See AngioTox Periodic Report 2 Attachment)
Inhibition of angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a well-established therapeutic strategy against cancer. Inhibitors of angiogenesis have been developed to block tumour growth and metastasis, and a number of these inhibitors are now clinically approved. However, contrary to initial expectations, angiogenesis inhibitors can cause a range of toxicities in patients. AngioTox is a cross-sectoral collaboration, responding to a pressing need to understand the mechanisms of toxicity associated with angiogenesis inhibitor treatment. The goal of the consortium was to facilitate comprehensive histopathologic and mechanistic assessment of angiogenesis inhibitor related toxicities following treatment with bevacizumab, sunitinib and sorafenib.
We undertook histopathologic, imaging and state-of-the-art ‘omic and biomarker profiling analyses in pre-clinical and clinical samples to gain insight into toxicity pathways and interrogate novel early safety biomarkers.
In order to comprehensively assess toxicologic response patterns associated with angiogenesis inhibitor treatment in animal models rodents were treated at clinically relevant doses. Tissue was collected at time of sacrifice according to GLP grade necropsy protocols and delivered to partners at ONCO and PATHEX for comprehensive histopathologic assessment (WP2), and to partners at VUMC, ROCHE & BTS for mechanistic and safety biomarker ‘omic studies . More than 500 pre-clinical samples from animal studies were processed for histopathology studies. A digital slide library was also prepared for all of the 500 slides and tissue microarrays further created. A clinically relevant and novel multi-modality molecular imaging approach was also developed to mechanistically interrogate off-target cardiovascular effects.
The Angiotox team was able to successfully recapitulate most of the common clinically observed toxicity patterns in pre-clinical models; Evidence of hypertension and compromised cardiovascular function were observed; A decrease in Left Ventricular Ejection Fraction was measured in both rats and mice over the course of the study. Alterations in cardiac metabolism were indicated by i) increased 18F-Fluoro-dexoyglucose (FDG) uptake and metabolism and ii) increase in fatty acid uptake and storage (FTHA- Positron Emission Tomography (PET) signal increase and higher numbers of lipid vesicles in the cardiomyocytes). Plasticity in cardiac energy substrate usage has previously been observed during hypertension with increased reliance on glucose as a metabolic substrate. This may explain the increase in FDG uptake following treatment. An imbalance in cellular metabolism as a result of off-target kinase inhibition by sunitinib may also lead to increased uptake and storage of free fatty acid by the myocardium. Additionally, a decrease in left ventricular perfusion may be the result of ‘on-target/ off-target’ sunitinib/sorafenib effects on cardiac vessels. Findings from proteomic studies [Liquid Chromatography-Mass Spectrometry (LC-MS), Reverse Phase Protein Array (RPA), ELISA and Western Blotting] further provided evidence for major metabolic aberrations (mitochondrial dysfunction, increase in glycolytic enzymes, increase in lipid storage) following treatment with anti-angiogenic TKIs. A major finding emerging from the work of the AngioTox consortium indicates that metabolic PET may represent an early cardiotoxicity biomarker strategy for patients treated with anti-angiogenic TKIs. Plans to validate this finding in patients are now in preparation. Sub-clinical thyroid effects, skin toxicity (at high doses), mild proteinuria were all further observed in pre-clinical models following comprehensive histopathologic assessment. Data also confirmed that platelet function is compromised by antiangiogenic treatment, since both in vitro and ex vivo platelet aggregation were significantly impaired by both sunitinib and sorafenib.This effect was unrelated to treatment induced thrombocytopenia. However, no clear relation between the percentage of decreased aggregation and recorded bleeding events in patients was observed. Bevacizumab also significantly interfered with platelet aggregation in vitro. However in patients, no decreased aggregations were found after a single administration of bevacizumab. In addition, no interference in platelet adherence to endothelial cells was observed following the application of angiogenesis inhibitors.
Uniquely within AngioTox we further aimed to determine if unknown downstream effects on target tumour receptor kinases or off-target receptor kinase inhibition in normal cells and tissue is related to toxicity of treatment. No clinical data are currently available describing how angiogenesis inhibitors may disturb downstream signalling in normal cells (tissues) and tumours, but it is known from in vitro assays that these agents inhibit multiple kinases. We thus implemented RPA technology to interrogate this question; Analyses of tissue (heart, kidney, liver, brain, large and small intestine and skin) from mice treated with anti-angiogenic TKIs revealed a number of important off-target signaling proteins perturbed by treatment. These proteins may represent promising markers that might ultimately be utilised for toxicity prediction. Putative markers are currently being validated (where feasible) in clinical samples.
In association with collaborators at Dublin City University (DCU) & the All Ireland Clinical Oncology Research Group (ICORG)] we sought to identify a panel of serum cardiotoxicity biomarkers and also to determine if individual or multiple biomarkers are associated with cardiotoxicity in breast cancer patients undergoing anti-angiogenic therapy. Our findings have revealed serum biomarkers which may have utility in predicting patients at risk of decreased LVEF following treatment with bevicizumab.
Finally, several high-end recruitment and secondment opportunities have been realized within the context of the AngioTox programme with an extensive and comprehensive training module provided to all Fellows. In particular, specialized secondments within AngioTox have provided several opportunities for high-end training of academic researchers in an industry setting.
Findings from the AngioTox programme may now be utilized by academic, clinical and industry-based investigators to facilitate improved screening of angiogenesis inhibitor toxicologic parameters, inform clinical drug dosing regimens and to ultimately facilitate the development of more specific and potent angiogenesis inhibitors, thus significantly improving patient care. New safety biomarkers discovered within the lifetime of this project are currently being prospectively validated in patients.
Coordinator contact details : annettebyrne@rcsi.ie
Project Logo: ( See AngioTox Periodic Report 2 Attachment)
Inhibition of angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a well-established therapeutic strategy against cancer. Inhibitors of angiogenesis have been developed to block tumour growth and metastasis, and a number of these inhibitors are now clinically approved. However, contrary to initial expectations, angiogenesis inhibitors can cause a range of toxicities in patients. AngioTox is a cross-sectoral collaboration, responding to a pressing need to understand the mechanisms of toxicity associated with angiogenesis inhibitor treatment. The goal of the consortium was to facilitate comprehensive histopathologic and mechanistic assessment of angiogenesis inhibitor related toxicities following treatment with bevacizumab, sunitinib and sorafenib.
We undertook histopathologic, imaging and state-of-the-art ‘omic and biomarker profiling analyses in pre-clinical and clinical samples to gain insight into toxicity pathways and interrogate novel early safety biomarkers.
In order to comprehensively assess toxicologic response patterns associated with angiogenesis inhibitor treatment in animal models rodents were treated at clinically relevant doses. Tissue was collected at time of sacrifice according to GLP grade necropsy protocols and delivered to partners at ONCO and PATHEX for comprehensive histopathologic assessment (WP2), and to partners at VUMC, ROCHE & BTS for mechanistic and safety biomarker ‘omic studies . More than 500 pre-clinical samples from animal studies were processed for histopathology studies. A digital slide library was also prepared for all of the 500 slides and tissue microarrays further created. A clinically relevant and novel multi-modality molecular imaging approach was also developed to mechanistically interrogate off-target cardiovascular effects.
The Angiotox team was able to successfully recapitulate most of the common clinically observed toxicity patterns in pre-clinical models; Evidence of hypertension and compromised cardiovascular function were observed; A decrease in Left Ventricular Ejection Fraction was measured in both rats and mice over the course of the study. Alterations in cardiac metabolism were indicated by i) increased 18F-Fluoro-dexoyglucose (FDG) uptake and metabolism and ii) increase in fatty acid uptake and storage (FTHA- Positron Emission Tomography (PET) signal increase and higher numbers of lipid vesicles in the cardiomyocytes). Plasticity in cardiac energy substrate usage has previously been observed during hypertension with increased reliance on glucose as a metabolic substrate. This may explain the increase in FDG uptake following treatment. An imbalance in cellular metabolism as a result of off-target kinase inhibition by sunitinib may also lead to increased uptake and storage of free fatty acid by the myocardium. Additionally, a decrease in left ventricular perfusion may be the result of ‘on-target/ off-target’ sunitinib/sorafenib effects on cardiac vessels. Findings from proteomic studies [Liquid Chromatography-Mass Spectrometry (LC-MS), Reverse Phase Protein Array (RPA), ELISA and Western Blotting] further provided evidence for major metabolic aberrations (mitochondrial dysfunction, increase in glycolytic enzymes, increase in lipid storage) following treatment with anti-angiogenic TKIs. A major finding emerging from the work of the AngioTox consortium indicates that metabolic PET may represent an early cardiotoxicity biomarker strategy for patients treated with anti-angiogenic TKIs. Plans to validate this finding in patients are now in preparation. Sub-clinical thyroid effects, skin toxicity (at high doses), mild proteinuria were all further observed in pre-clinical models following comprehensive histopathologic assessment. Data also confirmed that platelet function is compromised by antiangiogenic treatment, since both in vitro and ex vivo platelet aggregation were significantly impaired by both sunitinib and sorafenib.This effect was unrelated to treatment induced thrombocytopenia. However, no clear relation between the percentage of decreased aggregation and recorded bleeding events in patients was observed. Bevacizumab also significantly interfered with platelet aggregation in vitro. However in patients, no decreased aggregations were found after a single administration of bevacizumab. In addition, no interference in platelet adherence to endothelial cells was observed following the application of angiogenesis inhibitors.
Uniquely within AngioTox we further aimed to determine if unknown downstream effects on target tumour receptor kinases or off-target receptor kinase inhibition in normal cells and tissue is related to toxicity of treatment. No clinical data are currently available describing how angiogenesis inhibitors may disturb downstream signalling in normal cells (tissues) and tumours, but it is known from in vitro assays that these agents inhibit multiple kinases. We thus implemented RPA technology to interrogate this question; Analyses of tissue (heart, kidney, liver, brain, large and small intestine and skin) from mice treated with anti-angiogenic TKIs revealed a number of important off-target signaling proteins perturbed by treatment. These proteins may represent promising markers that might ultimately be utilised for toxicity prediction. Putative markers are currently being validated (where feasible) in clinical samples.
In association with collaborators at Dublin City University (DCU) & the All Ireland Clinical Oncology Research Group (ICORG)] we sought to identify a panel of serum cardiotoxicity biomarkers and also to determine if individual or multiple biomarkers are associated with cardiotoxicity in breast cancer patients undergoing anti-angiogenic therapy. Our findings have revealed serum biomarkers which may have utility in predicting patients at risk of decreased LVEF following treatment with bevicizumab.
Finally, several high-end recruitment and secondment opportunities have been realized within the context of the AngioTox programme with an extensive and comprehensive training module provided to all Fellows. In particular, specialized secondments within AngioTox have provided several opportunities for high-end training of academic researchers in an industry setting.
Findings from the AngioTox programme may now be utilized by academic, clinical and industry-based investigators to facilitate improved screening of angiogenesis inhibitor toxicologic parameters, inform clinical drug dosing regimens and to ultimately facilitate the development of more specific and potent angiogenesis inhibitors, thus significantly improving patient care. New safety biomarkers discovered within the lifetime of this project are currently being prospectively validated in patients.