Purpose Treatment of metastatic colorectal cancer frequently includes antiangiogenic agents such as bevacizumab. Size measurements are inadequate to assess treatment response to these agents, and newer response assessment criteria are needed. We aimed to evaluate F-18-FDG PET-derived texture parameters in a preclinical colorectal cancer model as alternative metrics of response to treatment with bevacizumab. Materials and methods Fourteen CD1 athymic mice injected in the flank with 5x106 LS174T cells (human colorectal carcinoma) were either untreated controls (n=7) or bevacizumab treated (n=7). After 2 weeks, mice underwent F-18-FDG PET/CT. Calliper-measured tumor growth (Delta(vol)) and final tumor volume (Vol(cal)), F-18-FDG PET metabolically active volume (Vol(met)), mean metabolism (Met(mean)), and maximum metabolism (Met(max)) were measured. Twenty-four texture features were compared between treated and untreated mice. Immunohistochemical mean tumor vascular density was estimated by anti-CD-34 staining after tumor resection. Results Treated mice had significantly lower tumor vascular density (P=0.032), confirming the antiangiogenic therapeutic effect of bevacizumab. None of the conventional measures were different between the two groups: Delta(vol) (P=0.9), Vol(cal) (P=0.7), Vol(met) (P=0.28), Met(max) (P=0.7), or Met(mean) (P=0.32). One texture parameter, GLSZM-SZV (visually indicating that the F-18-FDG PET images of treated mice comprise uniformly sized clusters of different activity) had significantly different means between the two groups of mice (P=0.001). Conclusion F-18-FDG PET derived texture parameters, particularly GLSZM-SZV, may be valid biomarkers of tumor response to treatment with bevacizumab, before change in volume.

Weevaluatedmagnetic resonance imaging (MRI) voxel heterogeneity following trastuzumab and/or cisplatin in aHER2+ esophageal xenograft (OE19) as a potential response biomarker. OE19 xenografts treated with saline (controls), monotherapy, or combined cisplatin and trastuzumabunderwent 9.4-TMRI. TumorMRIparametricmapsof T1 relaxation time (pre/post contrast), T2 relaxation time, T2* relaxation rate (R2*), and apparent diffusion coefficient obtained before (TIME0), after 24 hours (TIME1), and after 2 weeks of treatment (TIME2) were analyzed. Voxel histogram and fractal parameters (from thewhole tumor, rim and center, and as a ratio of rim‐to‐center) were derived. Tumorswere stained for immunohistochemical markers of hypoxia (CA-IX), angiogenesis (CD34), and proliferation (Ki-67). Combination therapy reduced xenograft growth rate (relative change, +0.58 ± 0.43 versus controls, +4.1 ± 1.0;P = 0.008).More spatially homogeneous voxel distribution between the rim to center was noted after treatment for combination therapy versus controls, respectively, for contrast-enhanced T1 relaxation time (90th percentile: ratio 1.00 versus 0.88, P = 0.009), T2 relaxation time (mean: 1.00 versus 0.92, P = 0.006; median: 0.98 versus 0.91, P = 0.006; 75th percentile: 1.02 versus 0.94, P = 0.007), and R2* (10th percentile: 0.99 versus 1.26, P = 0.003). We found that combination and trastuzumab monotherapy reducedMRI spatial heterogeneity and growth rate compared to the control or cisplatin groups, the former providing adjunctive tumor response information. Translational Oncology (2017) 10, 459–467 Department of Health, and the Singapore Ministry of Health's National Medical Research Council under its NMRC Research Training Fellowship (Dr. Connie Yip). The authors have no conflict of interest to declare. Received 5 February 2017; Revised 21 March 2017; Accepted 21 March 2017 © 2017 The Authors. Published by Elsevier Inc. on behalf of Neoplasia Press, Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.tranon.2017.03.006 460 MRI of intratumoral voxel heterogeneity as a potential response biomarker Yip et al. Translational Oncology Vol. 10, No. xx, 2017 Introduction Esophageal cancer is the eighth commonest cancer worldwide [1]. Outcome remains poor with a 5-year overall survival rate of 18% in all patients [2]. Neoadjuvant chemotherapy and chemoradiation have been shown to improve survival in patients with resectable cancer [3–5]. The addition of trastuzumab, an anti‐human epidermal growth factor receptor 2 (HER2) monoclonal antibody, to standard chemotherapy improves overall survival in HER2-positive advanced gastroesophageal adenocarcinoma compared to chemotherapy alone [6]. One of the challenges in clinical practice is how best to image the spatial and temporal intratumoral changes with treatment.Qualitative (decrease in metabolic activity) and semiquantitative (standardized uptake value: uptake/[injected dose/patient weight]) metabolic assessment with [F] fluorodeoxyglucose positron emission tomography has improved on the sensitivity and specificity of computed tomography size-based response assessment in the neoadjuvant setting [7]. However, with the advent of hybrid positron emission tomography/magnetic resonance imaging (MRI) scanners, there has been renewed interest in the additional potential ofMRI for assessing esophageal cancer [8,9]. MRI reflects the soft tissue anatomy well [10], has no radiation burden, and offers a multiparametric capability beyond anatomical evaluation. For example, diffusion-weighted and dynamic contrast-enhanced MRI sequences reflecting intratumoral water diffusion (a surrogate for cellular volume) and vascularization (a surrogate for angiogenesis), respectively, have shown clinical potential following chemotherapy and/or chemoradiation in esophageal cancer [11–14]. We hypothesize that conventional chemotherapy (cisplatin) and targeted therapy (trastuzumab) cause distinctive phenotypic and biological changes within the tumor spatially over the course of treatment, reflecting their specific mechanisms of action and downstream effects. This treatment-related change may be captured by image heterogeneity analysis on a per-voxel basis, also known as image texture analysis. We suggest that in vivo spatial changes in the image texture may augment standard size-based response evaluation and complement histopathological evaluation in clinical practice [15]. Thus, as proof of principle, we aimed to evaluate the sequential changes in intratumoral MRI spatial heterogeneity following trastuzumab and/or cisplatin therapy in a HER2-expressing esophageal adenocarcinoma xenograft (OE19) and to compare this with histopathological changes in angiogenesis, hypoxia, and cellular proliferation. Materials and Methods Xenograft Model All experiments were approved by our institutional review board and performed in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986. TheHER2-expressingOE19 cells were cultured in Table 1. MRI Acquisition Parameters Parameters T1-Weighted T2-Weighted Pulse sequence Rapid acquisition rapid echo with variable repetition time (RARE-VTR) Multislice mu Respiratory gating No No Repetition time (ms) 193.44, 478.4, 878.44, 1555.502, 500

Weevaluatedmagnetic resonance imaging (MRI) voxel heterogeneity following trastuzumab and/or cisplatin in aHER2+ esophageal xenograft (OE19) as a potential response biomarker. OE19 xenografts treated with saline (controls), monotherapy, or combined cisplatin and trastuzumabunderwent 9.4-TMRI. TumorMRIparametricmapsof T1 relaxation time (pre/post contrast), T2 relaxation time, T2* relaxation rate (R2*), and apparent diffusion coefficient obtained before (TIME0), after 24 hours (TIME1), and after 2 weeks of treatment (TIME2) were analyzed. Voxel histogram and fractal parameters (from thewhole tumor, rim and center, and as a ratio of rim‐to‐center) were derived. Tumorswere stained for immunohistochemical markers of hypoxia (CA-IX), angiogenesis (CD34), and proliferation (Ki-67). Combination therapy reduced xenograft growth rate (relative change, +0.58 ± 0.43 versus controls, +4.1 ± 1.0;P = 0.008).More spatially homogeneous voxel distribution between the rim to center was noted after treatment for combination therapy versus controls, respectively, for contrast-enhanced T1 relaxation time (90th percentile: ratio 1.00 versus 0.88, P = 0.009), T2 relaxation time (mean: 1.00 versus 0.92, P = 0.006; median: 0.98 versus 0.91, P = 0.006; 75th percentile: 1.02 versus 0.94, P = 0.007), and R2* (10th percentile: 0.99 versus 1.26, P = 0.003). We found that combination and trastuzumab monotherapy reducedMRI spatial heterogeneity and growth rate compared to the control or cisplatin groups, the former providing adjunctive tumor response information. Translational Oncology (2017) 10, 459–467 www.transonc.com Trans la t iona l Onco logy Volume 10 Number xx Month 2017 pp. 459–467 459 Address all correspondence to: Connie Yip, Department of Radiation Oncology, National Cancer Centre, 11 Hospital Drive, Singapore 169610. E-mail: connie.yip.s.p@singhealth.com.sg Funding acknowledgements: This work was supported by financial support from the Department of Health via the National Institute of Health Research Biomedical Research Centre award to Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust, the Comprehensive Cancer Imaging Centre funded by the Cancer Research UK and Engineering and Physical Sciences Research Council in association with the Medical Research Council and Department of Health, and the Singapore Ministry of Health's National Medical Research Council under its NMRC Research Training Fellowship (Dr. Connie Yip). The authors have no conflict of interest to declare. Received 5 February 2017; Revised 21 March 2017; Accepted 21 March 2017 © 2017 The Authors. Published by Elsevier Inc. on behalf of Neoplasia Press, Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.tranon.2017.03.006 Introduction Esophageal cancer is the eighth commonest cancer worldwide [1]. Outcome remains poor with a 5-year overall survival rate of 18% in all patients [2]. Neoadjuvant chemotherapy and chemoradiation have been shown to improve survival in patients with resectable cancer [3–5]. The addition of trastuzumab, an anti‐human epidermal growth factor receptor 2 (HER2) monoclonal antibody, to standard chemotherapy improves overall survival in HER2-positive advanced gastroesophageal adenocarcinoma compared to chemotherapy alone [6]. One of the challenges in clinical practice is how best to image the spatial and temporal intratumoral changes with treatment.Qualitative (decrease in metabolic activity) and semiquantitative (standardized uptake value: uptake/[injected dose/patient weight]) metabolic assessment with [F] fluorodeoxyglucose positron emission tomography has improved on the sensitivity and specificity of computed tomography size-based response assessment in the neoadjuvant setting [7]. However, with the advent of hybrid positron emission tomography/magnetic resonance imaging (MRI) scanners, there has been renewed interest in the additional potential ofMRI for assessing esophageal cancer [8,9]. MRI reflects the soft tissue anatomy well [10], has no radiation burden, and offers a multiparametric capability beyond anatomical evaluation. For example, diffusion-weighted and dynamic contrast-enhanced MRI sequences reflecting intratumoral water diffusion (a surrogate for cellular volume) and vascularization (a surrogate for angiogenesis), respectively, have shown clinical potential following chemotherapy and/or chemoradiation in esophageal cancer [11–14]. We hypothesize that conventional chemotherapy (cisplatin) and targeted therapy (trastuzumab) cause distinctive phenotypic and biological changes within the tumor spatially over the course of treatment, reflecting their specific mechanisms of action and downstream effects. This treatment-related change may be captured by image heterogeneity analysis on a per-voxel basis, also known as image texture analysis. We suggest that in vivo spatial changes in the image texture may augment standard size-based response evaluation and complement histopathological evaluation in clinical practice [15]. Thus, as proof of principle, we aimed to evaluate the sequential changes in intratumoral MRI spatial heterogeneity following trastuzumab and/or cisplatin therapy in a HER2-expressing esophageal adenocarcinoma xenograft (OE19) and to compare this with histopathological changes in angiogenesis, hypoxia, and cellular proliferation. Materials and Methods Xenograft Model All experiments were approved by our institutional review board and performed in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986. TheHER2-expressingOE19 cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO) supplemented with 2 mM L-glutamine and 10% fetal bovine serum. Cells were incubated at 37°C in a humidified environment with 5% carbon dioxide. Approximately 5 × 10 OE19 cells, in serum-free media mixed with Cultrex basement membrane extract (Trevigen Inc., Gaithersburg, MD) 1:1 to a final volumeof 8mg/ml,were injected subcutaneously into the right flanks of 6to 8-week-old female severe combined immunodeficient mice (Charles River Laboratories International, Inc.). Animals were monitored, and bidimensional measurements were obtained using a digital caliper. Once the tumors reached aminimumdiameter of 8mm, the animals were treated with intraperitoneal sterile saline (control group), cisplatin 4mg/kg body weight once a week (cisplatin monotherapy group), trastuzumab 20 mg/kg twice a week (trastuzumab monotherapy group), or a combination of cisplatin 4 mg/kg once a week and trastuzumab 20mg/kg twice a week (combination therapy group). Tumors were excised after 2 weeks of therapy and were fixed in 10% buffered formalin before being embedded in paraffin for immunohistochemistry. In Vivo Imaging MRI was performed with a 9.4-T MRI system (Bruker, Karlsruhe, Germany). The animals in each of the four groups (controls, cisplatin, trastuzumab, and combination) were anesthetized using inhalational isoflurane (2%-4%) and 1 l/min oxygen during MRI. A high-resolution T1 relaxation time map with and without intravenous administration of gadopentetate dimeglumine 0.1 mmol/kg (Magnevist; Bayer HealthCare Pharmaceuticals Inc., Germany), T2 relaxation time map, R2* map, and apparent diffusion coefficient (ADC) map were generated from the MRI acquisitions. Table 1 shows the MR acquisition parameters. Each animal was imaged at three time points: before treatment (TIME0), 24 hours after the first intraperitoneal therapy injection (TIME1), and after completion of 2 weeks of intraperitoneal treatment (TIME2). Image Analysis All the MR parametric maps were analyzed using in-house software implemented under the MATLAB (The MathWorks Inc., Natick, MA) platform.Whole tumor volumes of interest (VOIs)were delineated by a single observer (C. Y.). Tumor volumes were derived from the T1 images. Three-dimensional differential analysis of the tumor rimand tumor centerwas also performed given the inhomogeneous tumormorphology.The tumor rim was defined as the outer 3.5mm(T1,T2,R2*maps) or outer 4.6mm(ADC map) of a tumor, reflecting the slightly different voxel size of the ADC map. For tumorswith volumesbelow themedian value, a 2.3-mmrimwas required to allow adequate sampling of the rim versus center on the ADC maps. First-order statistical histogram and fractal analysis of T1, T2, R2*, and ADC voxels were analyzed, and the following parameters were derived for whole tumor, tumor rim, and tumor center on each parametric map, Table 1. MRI Acquisition Parameters Parameters T1-Weighted T2-Weighted Diffusion-Weighted R2* Pulse sequence Rapid acquisition rapid echo with variable repetition time (RARE-VTR) Multislice multiecho (MSME) Echo planar Multigradient echo (MGE) Respiratory gating No No No Yes Repetition time (ms) 193.44, 478.4, 878.44, 1555.502, 500

We evaluated magnetic resonance imaging (MRI) voxel heterogeneity following trastuzumab and/or cisplatin in a HER2+ esophageal xenograft (OE19) as a potential response biomarker. OE19 xenografts treated with saline (controls), monotherapy, or combined cisplatin and trastuzumab underwent 9.4-T MRI. Tumor MRI parametric maps of T1 relaxation time (pre/post contrast), T2 relaxation time, T2* relaxation rate (R2*), and apparent diffusion coefficient obtained before (TIME0), after 24hours (TIME1), and after 2weeks of treatment (TIME2) were analyzed. Voxel histogram and fractal parameters (from the whole tumor, rim and center, and as a ratio of rim‐to‐center) were derived. Tumors were stained for immunohistochemical markers of hypoxia (CA-IX), angiogenesis (CD34), and proliferation (Ki-67). Combination therapy reduced xenograft growth rate (relative change, ∆ +0.58±0.43 versus controls, ∆ +4.1±1.0; P=0.008). More spatially homogeneous voxel distribution between the rim to center was noted after treatment for combination therapy versus controls, respectively, for contrast-enhanced T1 relaxation time (90th percentile: ratio 1.00 versus 0.88, P=0.009), T2 relaxation time (mean: 1.00 versus 0.92, P=0.006; median: 0.98 versus 0.91, P=0.006; 75th percentile: 1.02 versus 0.94, P=0.007), and R2* (10th percentile: 0.99 versus 1.26, P=0.003). We found that combination and trastuzumab monotherapy reduced MRI spatial heterogeneity and growth rate compared to the control or cisplatin groups, the former providing adjunctive tumor response information.

42 Background: Trastuzumab (T) is the standard of care in advanced HER2+ gastroesophageal (GE) adenocarcinoma with response in 47% of patients. We evaluated its pathological effects in a HER2+ OE19 xenograft model aiming to improve future treatment intensification/response assessment. Methods: SCID mice (n = 23) bearing subcutaneous OE19 tumors were treated with intraperitoneal saline (control), Cisplatin 4mg/kg once a week (C), Trastuzumab 20mg/kg twice a week (T) or Cisplatin and Trastuzumab (CT) for 2 weeks. Volumetric tumor response was measured by MRI. Excised tumors were stained for IHC markers of hypoxia (Pimonidazole), proliferation (Ki-67) and angiogenesis (CD34). Hypoxic (HF) and proliferative fractions (PF) were defined as the ratios of tumor area positively stained for Pimonidazole/Ki-67 to area of whole tumor section. Microvessel density (MVD) was defined as the average number of blood vessels/endothelial cells identified within 0.01cm2field at x100 magnification. Pathological assessment was performed for whole tumor, centre and rim. Mean±SD are presented. Kruskal-Wallis test was used to compare HF, PR, MVD and volumetric change between groups; significance at < 0.05. Results: Tumor growth was reduced in CT (+83% from baseline MRI) compared to control (+600%), C (+275%) and T (+129%) groups (p= 0.015). Monotherapy treated tumors had lower HF (C 0.08±0.03, T 0.09±0.07) compared to CT (0.16±0.06) and control (0.15±0.04) (p= 0.039). T and CT treated animals had higher MVD (T 23.5±12.7, CT 37.3±10.3) compared to control (18.1±10.4) and C (18.1±7.5) (p= 0.014). Whole tumor PF was lower in the monotherapy groups but this was not significant (p =0.123). PF and MVD were higher in tumor centre compared to rim in T and CT groups compared to control but there was no spatial difference in hypoxia distribution. Conclusions: Tumor control was improved with combination treatment but no synergistic pathological effect was seen; CT treated tumors were more hypoxic/angiogenic compared to monotherapy with greater cellular density/angiogenesis within tumor centre. This could be due to predominant early cytostatic effect and ongoing angiogenic normalisation with CT therapy at 2 weeks.

Angiogenesis is essential for tissue development, wound healing and tissue perfusion, with its dysregulation linked to tumorigenesis, rheumatoid arthritis and heart disease. Here we show that pro-angiogenic stimuli couple to NADPH oxidase-dependent generation of oxidants that catalyse an activating intermolecular-disulphide between regulatory-RIα subunits of protein kinase A (PKA), which stimulates PKA-dependent ERK signalling. This is crucial to blood vessel growth as ‘redox-dead’ Cys17Ser RIα knock-in mice fully resistant to PKA disulphide-activation have deficient angiogenesis in models of hind limb ischaemia and tumour-implant growth. Disulphide-activation of PKA represents a new therapeutic target in diseases with aberrant angiogenesis.

e15108 Background: Standard CT imaging is limited for determining neoadjuvant treatment response in esophageal cancer but there is increasing interest in assessing multiparametric MRI (mpMRI) in this role.We evaluated changes in mpMRI during Trastuzumab and Cisplatin therapy in a gastroesophageal adenocarcinoma xenograft model (OE19) expressing human epidermal growth factor receptor 2 (HER2). Methods: 3 groups of female severe combined immunodeficient mice (n = 15) were injected subcutaneously with 5x106 OE19 cells. Animals were treated with intraperitoneal (IP) saline twice a week (Control), Cisplatin 4mg/kg once a week (Group C) or Trastuzumab 20mg/kg twice a week (Group T) for a total of 2 weeks. Animals were scanned on a 9.4T MR imaging system (Bruker, Germany) at 3 time points: before (t0), 24 hours after first IP treatment (t1) and on completion of treatment (t2). Contrast-enhanced T1-weighted (T1w), T2-weighted (T2w), diffusion weighted (DW) and T2*-weighted (T2*) sequences were acquired. Early (t1 - t0) and late (t2 - t0) parameter, tumour volume and image heterogeneity changes were compared between the groups using Kruskal-Wallis test. Results: A reduced growth rate was observed in Group T (Δvolume = 106%) compared to Control (680%) and Group C (305%) (p = 0.041) between t2-t0. Significant changes in DW and T2* (t2 - t0) were also observed. DW skewness increased in Group T (107%) but decreased in Group C (-28%) and Control (-89%) (p = 0.048). A greater reduction in T2* entropy (Group T -7.1% vs Group C -0.2% vs Control -2.2%, p = 0.032), T2* 50thpercentile (-18% vs 4% vs 15%, p = 0.034) and T2* 75th percentile (-13% vs 0.7% vs 21%, p = 0.024) were observed in Group T. A greater increase in T2* uniformity was found in Group T (48%) compared to Group C (3%) and Control (18%) (p = 0.032). There were no significant differences between groups at early t1-t0 assessment. Conclusions: Significant changes were found in DW and T2* parameters in animals treated with Trastuzumab, Cisplatin and Control, possibly reflecting the different drug mechanisms of action & tumour control, on completion of but not early in treatment. These findings offer potential new clinical imaging biomarkers for response assessment.

Lung cancer is the leading cause of cancer death in the United States. It is estimated that more than 228,000 new cases will be diagnosed in 2013, accounting for approximately 159,000 or 27% of all cancer deaths. Survival in these patients remains poor despite advances in surgery, definitive radiotherapy, and chemotherapy for primary and metastatic non-small cell lung cancer. Five-year relative survival rates remain at 27% for regional disease and 54% for node-negative disease. With the increasing personalization of therapy, there remains a need for better prognostic and predictive markers to direct patient management in lung cancer. Hypoxia and angiogenesis play an important role in the development and progression of lung cancer. Targeted and non-targeted imaging techniques in the preclinical and clinical setting, combined with advanced postprocessing techniques to assess tumor heterogeneity, may enable clinicians to better characterize lung tumors, and to predict and assess response to treatment. In this review, we summarize our current understanding of angiogenesis in lung cancer and discuss the available imaging techniques to assess this in the preclinical and clinical setting.