Physiologically Based Pharmacokinetic (PBPK) Modeling to Predict PET Image Quality of Three Generations EGFR TKI in Advanced-Stage NSCLC Patients.

INTRODUCTION: Epidermal growth factor receptor (EGFR) mutated NSCLC is best treated using an EGFR tyrosine kinase inhibitor (TKI). The presence and accessibility of EGFR overexpression and mutation in NSCLC can be determined using radiolabeled EGFR TKI PET/CT. However, recent research has shown a significant difference between image qualities (i.e., tumor-to-lung contrast) in three generation EGFR TKIs: 11C-erlotinib, 18F-afatinib and 11C-osimertinib. In this research we aim to develop a physiological pharmacokinetic (PBPK)-model to predict tumor-to-lung contrast and as a secondary outcome the uptake of healthy tissue of the three tracers. METHODS: Relevant physicochemical and drug specific properties (e.g., pKa, lipophilicity, target binding) for each TKI were collected and applied in established base PBPK models. Key hallmarks of NSCLC include: immune tumor deprivation, unaltered tumor perfusion and an acidic tumor environment. Model accuracy was demonstrated by calculating the prediction error (PE) between predicted tissue-to-blood ratios (TBR) and measured PET-image-derived TBR. Sensitivity analysis was performed by excluding each key component and comparing the PE with the final mechanistical PBPK model predictions. RESULTS: The developed PBPK models were able to predict tumor-to-lung contrast for all EGFR-TKIs within threefold of observed PET image ratios (PE tumor-to-lung ratio of -90%, +44% and -6.3% for erlotinib, afatinib and osimertinib, respectively). Furthermore, the models depicted agreeable whole-body distribution, showing high tissue distribution for osimertinib and afatinib and low tissue distribution at high blood concentrations for erlotinib (mean PE, of -10.5%, range -158%-+190%, for all tissues). CONCLUSION: The developed PBPK models adequately predicted the image quality of afatinib and osimertinib and erlotinib. Some deviations in predicted whole-body TBR lead to new hypotheses, such as increased affinity for mutated EGFR and active influx transport (erlotinib into excreting tissues) or active efflux (afatinib from brain), which is currently unaccounted for. In the future, PBPK models may be used to predict the image quality of new tracers.

Pre-treatment tumor-infiltrating T cells influence response to neoadjuvant chemoradiotherapy in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) is a disease with dismal treatment outcomes. Response to neoadjuvant chemoradiation (CRT) varies greatly. Although the underlying mechanisms of CRT resistance are not identified, accumulating evidence indicates an important role for local antitumor immunity. To explore the immune microenvironment in relation to response to CRT we performed an in-depth analysis using multiplex immunohistochemistry, flow cytometry and mRNA expression analysis (NanoString) to generate a detailed map of the immunological landscape of pretreatment biopsies as well as peripheral blood mononuclear cells (PBMCs) of EAC patients. Response to CRT was assessed by Mandard's tumor regression grade (TRG), disease-free- and overall survival. Tumors with a complete pathological response (TRG 1) to neoadjuvant CRT had significantly higher tumor-infiltrating T cell levels compared to all other response groups (TRG 2-5). These T cells were also in closer proximity to tumor cells in complete responders compared to other response groups. Notably, immune profiles of near-complete responders (TRG 2) showed more resemblance to non-responders (TRG 3-5) than to complete responders. A high CD8:CD163 ratio in the tumor was associated with an improved disease-free survival. Gene expression analyses revealed that T cells in non-responders were Th2-skewed, while complete responders were enriched in cytotoxic immune cells. Finally, complete responders were enriched in circulating memory T cells. preexisting immune activation enhances the chance for a complete pathological response to neoadjuvant CRT. This information can potentially be used for future patient selection, but also fuels the development of immunomodulatory strategies to enhance CRT efficacy.

Thymidine phosphorylase in cancer cells stimulates human endothelial cell migration and invasion by the secretion of angiogenic factors.

BACKGROUND: Thymidine phosphorylase (TP) is often overexpressed in tumours and has a role in tumour aggressiveness and angiogenesis. Here, we determined whether TP increased tumour invasion and whether TP-expressing cancer cells stimulated angiogenesis. METHODS: Angiogenesis was studied by exposing endothelial cells (HUVECs) to conditioned medium (CM) derived from cancer cells with high (Colo320TP1=CT-CM, RT112/TP=RT-CM) and no TP expression after which migration (wound-healing-assay) and invasion (transwell-assay) were determined. The involvement of several angiogenic factors were examined by RT-PCR, ELISA and blocking antibodies. RESULTS: Tumour invasion was not dependent on intrinsic TP expression. The CT-CM and RT-CM stimulated HUVEC-migration and invasion by about 15 and 40%, respectively. Inhibition by 10 µM TPI and 100 µM L-dR, blocked migration and reduced the invasion by 50-70%. Thymidine phosphorylase activity in HUVECs was increased by CT-CM. Reverse transcription-polymerase chain reaction revealed a higher mRNA expression of bFGF (Colo320TP1), IL-8 (RT112/TP) and TNF-α, but not VEGF. Blocking antibodies targeting these factors decreased the migration and invasion that was induced by the CT-CM and RT-CM, except for IL-8 in CT-CM and bFGF in RT-CM. CONCLUSION: In our cell line panels, TP did not increase the tumour invasion, but stimulated the migration and invasion of HUVECs by two different mechanisms. Hence, TP targeting seems to provide a potential additional strategy in the field of anti-angiogenic therapy.

High-grade clear cell renal cell carcinoma has a higher angiogenic activity than low-grade renal cell carcinoma based on histomorphological quantification and qRT-PCR mRNA expression profile.

Clear cell renal cell carcinoma (CC-RCC) is a highly vascularised tumour and is therefore an attractive disease to study angiogenesis and to test novel angiogenesis inhibitors in early clinical development. Endothelial cell proliferation plays a pivotal role in the process of angiogenesis. The aim of this study was to compare angiogenesis parameters in low nuclear grade (n=87) vs high nuclear grade CC-RCC (n=63). A panel of antibodies was used for immunohistochemistry: CD34/Ki-67, carbonic anhydrase IX, hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF). Vessel density (MVD - microvessel density), endothelial cell proliferation fraction (ECP%) and tumour cell proliferation fraction (TCP%) were assessed. mRNA expression levels of angiogenesis stimulators and inhibitors were determined by quantitative RT-PCR. High-grade CC-RCC showed a higher ECP% (P=0.049), a higher TCP% (P=0.009), a higher VEGF protein expression (P<0.001), a lower MVD (P< 0.001) and a lower HIF-1alpha protein expression (P=0.002) than low-grade CC-RCC. Growth factor mRNA expression analyses revealed a higher expression of angiopoietin 2 in low-grade CC-RCC. Microvessel density and ECP% were inversely correlated (Rho=-0.26, P=0.001). Because of the imperfect association of nuclear grade and ECP% or MVD, CC-RCC was also grouped based on low/high MVD and ECP%. This analysis revealed a higher expression of vessel maturation and stabilisation factors (placental growth factor, PDGFB1, angiopoietin 1) in CC-RCC with high MVD, a group of CC-RCC highly enriched in low nuclear grade CC-RCC, with low ECP%. Our results suggest heterogeneity in angiogenic activity and vessel maturation of CC-RCC, to a large extent linked to nuclear grade, and, with probable therapeutic implications.

Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium.

Regeneration and growth of the human endometrium after shedding of the functional layer during menstruation depends on an adequate angiogenic response. We analysed the mRNA expression levels of all known vascular endothelial growth factor (VEGF) ligands and receptors in human endometrium collected in the menstrual and proliferative phases of the menstrual cycle. In addition, we evaluated the expression of VEGF-A, VEGF-R2 and NRP-1 at the protein level. Two periods of elevated mRNA expression of ligands and receptors were observed, separated by a distinct drop at cycle days (CDs) 9 and 10. Immunohistochemical staining showed that VEGF and VEGF-R2 were expressed in epithelial, stromal and endothelial cells. NRP-1 was mainly confined to stroma and blood vessels; only in late-proliferative endometrium, epithelial staining was also observed. Except for endothelial VEGF-R2 expression in CDs 6-8, there were no significant differences in the expression of VEGF, VEGF-R2 or NRP-1 in any of the cell compartments. In contrast, VEGF release by cultured human endometrium explants decreased during the proliferative phase. This output was significantly reduced in menstrual and early-proliferative endometrium by estradiol (E2) treatment. Western blot analysis indicated that part of the VEGF-A was trapped in the extracellular matrix (ECM). Changes in VEGF ligands and receptors were associated with elevated expression of the hypoxia markers HIF1alpha and CA-IX in the menstrual and early proliferative phases. HIF1alpha was also detected in late-proliferative phase endometrium. Our findings indicate that VEGF-A exerts its actions mostly during the first half of the proliferative phase. Furthermore, VEGF-A production appears to be triggered by hypoxia in the menstrual phase and subsequently suppressed by estrogen during the late proliferative phase.