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Chemotherapy combined with the angiogenesis inhibitor bevacizumab (BVZ) is approved as a first-line treatment in metastatic colorectal cancer (mCRC). Limited clinical benefit underpins the need for improved understanding of resistance mechanisms and the elucidation of novel predictive biomarkers. We assessed germline single-nucleotide polymorphisms (SNPs) in 180 mCRC patients (Angiopredict [APD] cohort) treated with combined BVZ + chemotherapy and investigated previously reported predictive SNPs. We further employed a machine learning approach to identify novel associations. In the APD cohort IL8 rs4073 any A carriers, compared to TT carriers, were associated with worse progression-free survival (PFS) (HR = 1.51, 95% CI:1.03–2.22, p-value = 0.037) and TBK1 rs7486100 TT carriers, compared to any A carriers, were associated with worse PFS in KRAS wild-type (wt) patients (HR = 1.94, 95% CI:1.04–3.61, p-value = 0.037), replicating previous findings. Machine learning identified novel associations in genes encoding the inflammasome protein NLRP1 and the ER protein Sarcalumenin (SRL). A negative association between PFS and carriers of any A at NLRP1 rs12150220 and AA for SRL rs13334970 in APD KRAS wild-type patients (HR = 4.44, 95% CI:1.23–16.13, p-value = 0.005), which validated in two independent clinical cohorts involving BVZ, MAVERICC and TRIBE. Our findings highlight a key role for inflammation and ER signalling underpinning BVZ + chemotherapy responsiveness.

Patient-derived tumor xenograft (PDX) mouse models have emerged as an important oncology research platform to study tumor evolution, mechanisms of drug response and resistance, and tailoring chemotherapeutic approaches for individual patients. The lack of robust standards for reporting on PDX models has hampered the ability of researchers to find relevant PDX models and associated data. Here we present the PDX models minimal information standard (PDX-MI) for reporting on the generation, quality assurance, and use of PDX models. PDX-MI defines the minimal information for describing the clinical attributes of a patient's tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of PDX models in cancer research. Adherence to PDX-MI standards will facilitate accurate search results for oncology models and their associated data across distributed repository databases and promote reproducibility in research studies using these models. Cancer Res; 77(21); e62–66. ©2017 AACR.

// Bruce Moran 1, 10, * , Sudipto Das 1, 10, * , Dominiek Smeets 2, 3 , Gillian Peutman 2, 3 , Rut Klinger 1, 10 , Bozena Fender 4 , Kate Connor 1, 5, 10 , Matthias Ebert 6 , Timo Gaiser 6 , Jochen HM Prehn 5 , Orna Bacon 5, 7 , Elaine Kay 7 , Bryan Hennessy 7 , Verena Murphy 8 , Bauke Ylstra 9 , Diether Lambrechts 2, 3 , Annette T. Byrne 5 , William M. Gallagher 4, 10, ** and Darran P. O’Connor 1, 10, ** 1 Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland 2 Department of Oncology, Laboratory of Translational Genetics, VIB Center for Cancer Biology, Leuven, Belgium 3 Department of Oncology, Laboratory of Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium 4 OncoMark Ltd., NovaUCD, Belfield Innovation Park, Dublin, Ireland 5 Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland 6 Department of Internal Medicine, University of Heidelberg, Mannheim, Germany 7 Department of Pathology, Beaumont Hospital, Dublin, Ireland 8 Cancer Trials Ireland, Dublin, Ireland 9 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 10 Cancer Biology and Therapeutics Laboratory, UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College, Dublin, Ireland * These authors have contributed equally to this work ** Senior co-authors Correspondence to: Darran P. O’Connor, email: darranoconnor@rcsi.ie Keywords: targeted bisulfite sequencing, methylation, epigenetics, FFPE, tumor preservation Received: July 20, 2016 Accepted: April 03, 2017 Published: May 30, 2017 ABSTRACT DNA methylation is altered in many types of disease, including metastatic colorectal cancer. However, the methylome has not yet been fully described in archival formalin-fixed paraffin embedded (FFPE) samples in the context of matched fresh-frozen (FF) tumor material at base-pair resolution using a targeted approach. Using next-generation sequencing, we investigated three pairs of matched FFPE and FF samples to determine the extent of their similarity. We identified a ‘bowing’ pattern specific to FFPE samples categorized by a lower CG proportion at the start of sequence reads. We have found no evidence that this affected methylation calling, nor concordance of results. We also found no significant increase in deamination, measured by C>T transitions, previously considered a result of crosslinking DNA by formalin fixation and a barrier to the use of FFPE in methylation studies. The methods used in this study have shown sensitivity of between 60-70% based on positions also methylated in colorectal cancer cell lines. We demonstrate that FFPE material is a useful source of tumor material for methylation studies using targeted sequencing.

Background: Combining bevacizumab and chemotherapy produced superior response rates compared with chemotherapy alone in metastatic breast cancer. As bevacizumab may cause hypertension (HTN) and increase the risk of cardiac failure, we performed a pilot study to evaluate the feasibility and toxicity of a non-anthracycline-containing combination of docetaxel with cyclophosphamide and bevacizumab in early stage breast cancer patients. Methods: Treatment consisted of four 3-weekly cycles of docetaxel and cyclophosphamide (75/600 mg/m2). Bevacizumab was administered 15 mg/kg intravenously on day 1, and then every 3 weeks to a total of 18 cycles of treatment. Serum biomarker concentrations of vascular endothelial growth factor (VEGF), cardiac troponin-I (cTnI), myeloperoxidase (MPO), and placental growth factor (PlGF) were quantified using enzyme-linked immunosorbent assay (ELISA) in 62 patients at baseline and whilst on treatment to determine their utility as biomarkers of cardiotoxicity, indicated by left ventricular ejection fraction (LVEF). Results: A total of 106 patients were accrued in nine sites. Median follow up was 65 months (1–72 months). Seventeen protocol-defined relapse events were observed, accounting for an overall disease-free survival (DFS) rate of 84%. The DFS rates for hormone receptor positive (HR+) and triple-negative (TN) patients were 95% versus 43%, respectively. The median time to relapse was 25 (12–54) months in TN patients versus 38 (22–71) months in HR+ patients. There have been 13 deaths related to breast cancer . The overall survival (OS) rate was 88%. The 5-year OS rate in HR+ versus TN was 95% versus 57%. None of the measured biomarkers predicted the development of cardiotoxicity. Conclusions: We observed a low relapse rate in node-positive, HR+ patients; however, results in TN breast cancer were less encouraging. Given the negative results of three large phase III trials, it is unlikely that this approach will be investigated further. Trial Registration ClinicalTrials.gov Identifier: NCT00911716.

Heme sensor Nuclear Receptor Subfamily 1 Group D Member 1 (NR1D1/REVERBA) and its target gene Aryl Hydrocarbon Receptor Nuclear Translocator-Like (ARNTL/BMAL1) regulate circadian rhythm and angiogenesis. Given their involvement in the interplay between clock disruptions (e.g. by mutations or sleep deprival), cancer risk and vascular biology, we hypothesized that these transcription factors contribute to resistance against anti-angiogenic therapy with vascular endothelial growth factor (VEGFA) neutralizing antibody (bevacizumab, abbrev. Beva) in colorectal cancer (CRC). REVERBA bound to a predicted -672 bp Retinoic Acid Receptor-Related Orphan Receptor Alpha (RORA)-responsive element (RORE) adjacent to a BMAL1 DNA-binding motif (E-box) in the proximal promoter of the human VEGFA gene. REVERBA, indirectly and together with BMAL1, increased VEGFA mRNA and protein expression. Conversely, REVERBA siRNA and REVERBA-antagonist hemin decreased VEGFA synthesis. Beva treatment inhibited CRC growth in a C57BL/6J Apc min/+ genetically modified mouse model (GEMM) and BALB/c nu/nu mouse HCT116 xenografts, without attenuating BMAL1 levels or microvessel density (MVD). In CRC patient tumors (n=74), high BMAL1 protein expression was associated with clinical non-response to combination chemotherapy with Beva (*p=0.0061) and reduced progression-free survival (PFS) [*p=0.0223, Hazard Ratio (HR)=1.69]. In addition, single nucleotide polymorphisms (SNPs) in the BMAL1 (ARNTL) gene correlated with shorter overall survival (OS) [rs11022780, *p=0.014, HR=1.61] and PFS (rs7396943, rs7938307, rs2279287) in patients undergoing combination chemotherapy with Beva. Thus, BMAL1 is associated with Beva resistance in CRC. Since the REVERBA-BMAL1-VEGFA axis can be pharmacologically targeted, interference with this signaling pathway could circumvent resistance to anti-angiogenic therapy in CRC. Funding Statement: This study was supported by grants to EB from the Deutsche Krebshilfe (#108287 and #111086). FE received a fellowship from the “Studienstiftung des deutschen Volkes”. JB was supported by the Translational Physician Scientist (TraPS) Program of the Medical Faculty Mannheim of the Heidelberg University. WW was awarded a fellowship from the Chinese Scholarship Council (#201408080116). ME was supported by the State of Baden-Wurttemberg for “Center of Geriatric Biology and Oncology (ZOBEL) – Perspektivforderung” and “Biology of Frailty - Sonderlinie Medizin”. ATB and ME received funds from the European Commission Seventh Framework Programme (Contract No. 278981 [ANGIOPREDICT]) and are supported by the European Commission Horizon 2020 Programme (Contract No. 754923 [COLOSSUS]). ATB is further supported by Science Foundation Ireland under grant 13/CDA/2183 ”Coloforetell”. HJL was partly funded by the National Cancer Institute (grant number P30CA014089), the Gloria Borges WunderGlo Foundation -The Wunder Project, the Dhont Family Foundation, the San Pedro Peninsula Cancer Guild, the Daniel Butler Research Fund, and the Call to Cure Research Fund Declaration of Interests: The authors declare no conflicts of interest. The reagent mBeva was obtained on the basis of a material transfer agreement (MTA#10082012) from Roche Diagnostics GmbH (Penzberg, Germany). HJL has received clinical trial financial support from Merck Serono and Roche and honoraria for advisory board membership and lectures from Bayer, Boehringer Ingelheim, Genentech, Merck Serono and Roche Ethics Approval Statement: Xenograft studies conformed to the 2010/63/EU guidelines and were approved by the Dept. of Health and Children, Dublin, Ireland (B100/3654) and University College Dublin Animal Research Ethics Committee (P12-27). Therapy studies were conducted in accordance with ethical guidelines (Declaration of Helsinki) and approved by the local ethics committees for each participating site. All patients provided written informed consent for the analysis of molecular correlates.

Angiogenesis is a key tumor microenvironment (TME) event underpinning tumor growth and metastasis. Nevertheless, the relatively poor performance of anti-angiogenic therapies in clinical trials compared to pre-clinical studies implies that classical subcutaneous xenograft models have limited predictive potential in this setting. To address this issue, we established orthotopic surgical resection models of breast cancer, which replicate the phenotype of clinical post-resection micro-metastasis. To demonstrate the power and precision of these models, we recapitulated the BETH adjuvant trial (NCT00625898) where the addition of bevacizumab (BVZ) to chemotherapy plus trastuzumab (Trast) failed to provide additional benefit. SCID mice were orthotopically implanted with bioluminescent Her2+ MDA-MB-231 or HCC1954 cells and tumors resected c.5 weeks later. Following resection, mice were treated with 10 mg/kg Trast +5 mg/kg paclitaxel (PAC) IP once weekly for 6 cycles +/− weekly BVZ (5 mg/kg IP). Metastasis was monitored by imaging. Using these models our data confirms that the addition of the anti-angiogenic antibody BVZ to adjuvant Trast + chemotherapy provides no additional benefit compared with Trast + chemotherapy alone. Previous studies using non-resection subcutaneously engrafted xenografts failed to predict this outcome. Our results provide compelling evidence for the utility of cell line xenograft resection models to predict clinical outcome for TME targeting agents.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.

Increased copy number alterations (CNAs) indicative of chromosomal instability (CIN) have been associated with poor cancer outcome. Here, we study CNAs as potential biomarkers of bevacizumab (BVZ) response in metastatic colorectal cancer (mCRC). We cluster 409 mCRCs in three subclusters characterized by different degrees of CIN. Tumors belonging to intermediate-to-high instability clusters have improved outcome following chemotherapy plus BVZ versus chemotherapy alone. In contrast, low instability tumors, which amongst others consist of POLE-mutated and microsatellite-instable tumors, derive no further benefit from BVZ. This is confirmed in 81 mCRC tumors from the phase 2 MoMa study involving BVZ. CNA clusters overlap with CRC consensus molecular subtypes (CMS); CMS2/4 xenografts correspond to intermediate-to-high instability clusters and respond to FOLFOX chemotherapy plus mouse avastin (B20), while CMS1/3 xenografts match with low instability clusters and fail to respond. Overall, we identify copy number load as a novel potential predictive biomarker of BVZ combination therapy., Increased copy number alterations, indicative of chromosomal instability, is associated with poor cancer outcome. Here, metastatic colorectal cancer patients displaying intermediate-high CIN associate with improved outcome following chemotherapy and bevacizumab treatment, suggesting CIN as a predictive biomarker.

Purpose Patients with metastatic colorectal cancer (mCRC) have limited benefit from the addition of bevacizumab to standard chemotherapy. However, a subset probably benefits substantially, highlighting an unmet clinical need for a biomarker of response to bevacizumab. Previously, we demonstrated that losses of chromosomes 5q34, 17q12, and 18q11.2-q12.1 had a significant correlation with progression-free survival (PFS) in patients with mCRC treated with bevacizumab in the CAIRO2 clinical trial but not in patients who did not receive bevacizumab in the CAIRO trial. This study was designed to validate these findings. Materials and Methods Primary mCRC samples were analyzed from two cohorts of patients who received bevacizumab as first-line treatment; 96 samples from the European multicenter study Angiopredict (APD) and 81 samples from the Italian multicenter study, MOMA. A third cohort of 90 samples from patients with mCRC who did not receive bevacizumab was analyzed. Copy number aberrations of tumor biopsy specimens were measured by shallow whole-genome sequencing and were correlated with PFS, overall survival (OS), and response. Results Loss of chromosome 18q11.2-q12.1 was associated with prolonged PFS most significantly in both the cohorts that received bevacizumab (APD: hazard ratio, 0.54; P = .01; PFS difference, 65 days; MOMA: hazard ratio, 0.55; P = .019; PFS difference, 49 days). A similar association was found for OS and overall response rate in these two cohorts, which became significant when combined with the CAIRO2 cohort. Median PFS in the cohort of patients with mCRC who did not receive bevacizumab and in the CAIRO cohort was similar to that of the APD, MOMA, and CAIRO2 patients without an 18q11.2-q12.1 loss. Conclusion We conclude that the loss of chromosome 18q11.2-q12.1 is consistently predictive for prolonged PFS in patients receiving bevacizumab. The predictive value of this loss is substantiated by a significant gain in OS and overall response rate.

Bevacizumab (bvz) is a first choice anti-angiogenic drug in oncology and is primarily administered in combination with chemotherapy. It has been hypothesized that anti-angiogenic drugs enhance efficacy of cytotoxic drugs by “normalizing” abnormal tumor vessels and improving drug penetration. Nevertheless, the clinical relevance of this phenomenon is still unclear with several studies over recent years suggesting an opposing relationship. Herein, we sought to develop a new computational tool to interrogate anti-angiogenic drug scheduling with particular application in the setting of colorectal cancer (CRC). Specifically, we have employed a mathematical model of vascular tumour growth which interrogates the impact of anti-angiogenic treatment and chemotherapeutic treatment on tumour volume. Model predictions were validated using CRC xenografts which underwent treatment with a clinically relevant combinatorial anti-angiogenic regimen. Bayesian model selection revealed the most appropriate term for capturing the effect of treatments on the tumour size, and provided insights into a switch-like dependence of FOLFOX delivery on the tumour vasculature. Our experimental data and mathematical model suggest that delivering chemotherapy prior to bvz may be optimal in the colorectal cancer setting.

Nature Reviews Cancer 17, 254–268 (2017) In the online html version of this article, Joan Seoane's affiliations were not correct. He is also a member of the EurOPDX Consortium and is at the Vall d'Hebron Institute of Oncology, 08035 Barcelona, the Universitat Autonoma de Barcelona, 08193 Bellaterra,and the Institucio Catalana de Recerca i Estudis Avancats (ICREA), 08010 Barcelona, Spain.

Background: Glioblastoma (GBM), being a highly vascularised and locally invasive tumour, is an attractive target for anti-angiogenic and anti-invasive therapies. The GBM/endothelial cell response to gossypol/temozolomide (TMZ) treatment was investigated with a particular aim to assess treatment effects on cancer hallmarks. Methods: Cell viability, endothelial tube formation and GBM tumour cell invasion were variously assessed following combined treatment in vitro. The U87MG-luc2 subcutaneous xenograft model was used to investigate therapeutic response in vivo. Viable tumour response to treatment was interrogated using immunohistochemistry. Combined treatment protocols were also tested in primary GBM patient-derived cultures. Results: An endothelial/GBM cell viability inhibitory effect, as well as an anti-angiogenic and anti-invasive response, to combined treatment have been demonstrated in vitro. A significantly greater anti-proliferative (P=0.020, P=0.030), anti-angiogenic (P=0.040, P

28 EORTC – NCI – AACR Symposium on Molecular Targets and Cancer Therapeutics 29 November 2016 - 02 December 2016

Background: One of the key events in the progression of cancer metastasis is the migration of tumor cells through the vascular endothelium. Tumors induce abnormal endothelial permeability to promote cancer cell invasion. Recently, inhibition of tumor-induced vascular permeability has been shown to attenuate metastasis in vivo (Tichet M et al. Nat Commun. 2015;6:6993). Low molecular weight heparins (LMWHs) inhibit metastasis in vivo (Klerk et al. J Clin Oncol. 2005;23(10):2130-5) but may cause bleeding complications when used at effective doses. Consequently, LMWHs are unlikely to be used for prevention of metastatic progression unless bleeding side-effects can be diminished. To address this issue, we have recently demonstrated that a LMWH/statin combination (tinzaparin and simvastatin) attenuates abnormal vascular permeability and reduces epithelial cell transmigration at concentrations unlikely to cause bleeding side-effects in vivo. (Kevane et al. Res Pract Thromb Haemost. 2017;1(1)). Here, we aim to interrogate the anti-metastatic effect of the LMWH/statin combination (tinzaparin and simvastatin) in an orthotopic surgical resection model of triple negative breast cancer implementing a LMWH/stain dose that does not increase bleeding risk. Methods: NOD/SCID mice were surgically implanted with 5x105 MDA-MB-231-LUC2 triple negative breast cancer cells in the right inguinal mammary fat pad. Tumors were allowed to grow until they reached 250mm3. Subsequently, primary tumors were surgically resected. 5 days post-resection, mice were randomized into 4 groups (group 1: 0.5% methylcellulose (vehicle), group 2: 150IU/kg TNZ, group 3: 3mg/kg SVS and group 4: TNZ (150IU/kg) and SVS (3mg/kg)) and treated daily for 4 weeks. Post-resection metastatic progression was monitored by weekly bioluminescence imaging (BLI) [IVIS Spectrum]. Animals that displayed metastatic progression or significant regrowth of primary tumour were euthanized. At time of death, apical lymph nodes and lungs were removed and fixed in 10% formalin for quantification of metastatic burden Results: Prior to study commencement it was determined that a combination of 150IU/kg TNZ and 3mg/kg SVS did not significantly increase bleeding time in a cohort of tumour naïve animals. Following primary tumor resection, BLI assessment of metastatic progression revealed that 150IU/kg TNZ, 3mg/kg SVS and combination therapy of 150IU/kg TNZ and 3mg/kg SVS significantly (P= 0.0373, P=0.0086 and P=0.0409 respectively N=11) increased the time to metastasis compared to the vehicle treated animals. Conclusion: For the first time we demonstrate that tinzaparin and simvastatin may be combined to significantly delay time to metastasis while diminishing the associated bleeding risk of LMWHs in a clinically relevant mouse model of aggressive triple negative breast cancer. Citation Format: Ian S. Miller, Liam P. Shiels, Grace Buckley, Kate Connor, Annette T. Byrne, Fionnuala Ní Ainle. Interrogating the in vivo anti-metastatic action of tinzaparin and simvastatin in an orthotopic surgical resection mouse model of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2012.

There is strong pharmaceutical development of agents targeting the IL-17-TH17 pathway for the treatment of psoriasis (Ps) and psoriatic arthritis (PsA). Lung cancer accounts for 28% of all cancer-related deaths worldwide, and roughly 80% of patients with newly-diagnosed non-small cell lung cancer (NSCLC) present with metastatic disease, with a poor prognosis of around 12 months. Therefore, there is a high unmet medical need for the development of new and potent systemic treatments in this deadly disease. The emergence of immunotherapies such as anti-PD-1 or anti-PDL1 as candidate therapies in non-small cell lung cancer (NSCLC) indicates that targeting critical immuno-modulatory cytokines including those within the IL-17-Th1/Th17 axis may have proven benefit in the treatment of lung cancer. Areas covered: In this review we describe the current evidence for aberrant IL-17-Th1/Th17 settings in cancer, particularly with regard to targeting this axis in NSCLC. We further discuss the current agents under pharmaceutical development which could potentially target this axis, and discuss the current limitations and areas of concern regarding the use of these in lung cancer. Expert opinion: Current evidence suggests that moving forward agents targeting the IL-17-Th1/Th17 pathway may have novel new oncoimmunology indications in the treatment paradigm for NSCLC.

Background/Aims: This study is aimed at analyzing the survival rates and prognostic factors of stage IV colorectal cancer patients from 3 European cohorts undergoing combination chemotherapy with bevacizumab. Methods: Progression free-survival (PFS) and overall survival (OS) were analyzed in 172 patients using the Kaplan-Meier method and uni- and multivariable Cox proportional hazards regression models. Results: The median PFS was 9.7 and the median OS 27.4 months. Patients treated at centers in Germany (n = 97), Ireland (n = 32), and The Netherlands (n = 43) showed a median PFS of 9.9, 9.2, and 9.7 months, OS of 34.0, 20.5, and 25.1 months, respectively. Patients >65 years had a significantly shorter PFS (9.5 vs. 9.8 months) but not OS (27.4 vs. 27.5 months) than younger patients. High tumor grade (G3/4) was associated with a shorter PFS, T4 classification with both shorter PFS and OS. Fluoropyrimidine (FP) chemotherapy backbones (doublets and single) had comparable outcomes, while patients not receiving FP backbones had a shorter PFS. In multivariable analysis, age and non-FP backbone were associated with inferior PFS, T4 classification and therapy line >2nd were significantly associated with poor PFS and OS. Conclusion: The observed survival rates confirm previous studies and demonstrate reproducible benefits of combination bevacizumab regimens. Classification T4, non-FP chemotherapy backbone, and age >65 were associated with inferior outcome.

Glioblastoma (GBM) is the most common and most malignant tumour of the central nervous system. Despite recent advances in understanding the biology of GBM, the disease still remains in desperate need of effective treatment options resulting in long-term improvements in overall patient survival. Molecularly targeted therapies, anti-angiogenic therapy and immunotherapy are promising avenues under investigation as future therapeutic options. Molecular imaging (MI) is an essential tool in the development of these targeted treatments, both preclinically and clinically. MI facilitates the preclinical study and interrogation of potential therapies. MI further supports non-invasive, longitudinal monitoring of therapy response and allows the study of emergence of treatment resistance via an imaging-guided therapeutic approach.

Photodynamic therapy (PDT) is an established treatment modality for cancer. ADPM06 is an emerging non-porphyrin PDT agent which has been specifically designed for therapeutic application. Recently, we have demonstrated that ADPM06-PDT is well tolerated in vivo and elicits impressive complete response rates in various models of cancer when a short drug-light interval is applied. Herein, the mechanism of action of ADPM06-PDT in vitro and in vivo is outlined. Using a drug and light combination that reduces the clonogenicity of MDA-MB-231 cells by90%, we detected a well-orchestrated apoptotic response accompanied by the activation of various caspases in vitro. The generation of reactive oxygen species (ROS) upon photosensitizer irradiation was found to be the key instigator in the observed apoptotic response, with the endoplasmic reticulum (ER) found to be the intracellular site of initial PDT damage, as determined by induction of a rapid ER stress response post-PDT. PDT-induced apoptosis was also found to be independent of p53 tumor suppressor status. A robust therapeutic response in vivo was demonstrated, with a substantial reduction in tumor proliferation observed, as well as a rapid induction of apoptosis and initiation of ER stress, mirroring numerous aspects of the mechanism of action of ADPM06 in vitro. Finally, using a combination of (18) F-labeled 3'-deoxy-3'-fluorothymidine ((18) F-FLT) nuclear and optical imaging, a considerable decrease in tumor proliferation over 24-hr in two models of human cancer was observed. Taken together, this data clearly establishes ADPM06 as an exciting novel PDT agent with significant potential for further translational development.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Introduction: Most patients with metastatic colorectal cancer (mCRC) have a limited benefit from the addition of bevacizumab to standard combination chemotherapy. However, a subset of patients benefits substantially, highlighting an unmet clinical need for a predictive biomarker of response to bevacizumab. Previously, we demonstrated that losses in chromosome 5q34, 17q12 and 18q12.1-21.1 have a significant correlation with progression free survival (PFS) in patients treated with, but not without bevacizumab. These data warrant extensive validation. Patients: For validation, two cohorts of patients that received bevacizumab were analysed, one of 121 mCRC patient samples of the European multicenter study Angiopredict and one of 81 mCRC patient samples from the Italian multicenter study MoMA. A third cohort was included of 90 mCRC samples from patients that did not receive bevacizumab. Genome wide copy number aberrations were correlated with PFS, Results: Loss of chromosome 18q11.2-18q21.1 most significantly associated with PFS in both cohorts that received bevacizumab (Angiopredict: HR=0.61, p=0.016; MoMa: HR=0.55, p=0.019). No significant association with PFS was observed in the cohort that did not receive bevacizumab (HR=0.85, p=0.67). Patients of all three cohorts without an 18q11.2-21.1 loss had similar PFS regardless of treatment (Angiopredict, median PFS 211 days; MoMa, median PFS 232 days; no bevacizumab, median PFS 189 days). No significant associations with PFS were found for chromosome 5q34 and 17q for any of the cohorts. Conclusion: We conclude that loss of chromosomal region 18q11.2-18q21.1 is consistently predictive for PFS in patients receiving bevacizumab. 18q11.2 loss provides an increase in median PFS of 83 days, compared to patients without this loss. No significant increase was found for patients that did not received bevacizumab. Citation Format: Erik van Dijk, Hedde Biesma, Martijn Cordes, Dominiek Smeets, Maarten Neerincx, Sudipto Das, Verena Murphy, Anna Barat, Orna Bacon, Jochen H.M. Prehn, Johannes Betge, Gaiser Timo, Bozena Fender, Gerrit A. Meijer, Deborah A. McNamara, Rut Klinger, Miriam Koopman, Matthias P.A. Ebert, Elaina W. Kay, Bryan T. Hennessey, Henk M.W. Verheul, William M. Gallagher, Darran P. O'Connor, Cornelis J.A. Punt, Fotios Loupakis, Diether Lambrechts, Annette Byrne, Nicole C.T. van Grieken, Bauke Ylstra. Loss of chromosome 18q11.2-18q12.1 is predictive for progression-free survival in metastatic colorectal cancer patients treated with bevacizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2579.

e15584Background: Chemotherapy combined with the angiogenesis inhibitor bevacizumab (bvz) is approved as a first-line treatment in the setting of metastatic Colorectal Cancer (mCRC) since 2004, but...

Overexpression of HER-2 in breast cancer is frequently associated with expression of EGFR, and EGFR expression influences response to HER-2 inhibition. The aim of this study was to examine the effects of combining dual inhibition of EGFR and HER-2, using trastuzumab, gefitinib and lapatinib, in HER-2 overexpressing breast cancer cells. Combination proliferation assays were performed in two HER-2 positive breast cancer cell lines, SKBR-3 and BT-474. Trastuzumab combined with lapatinib was also tested in BT-474 xenografts. In proliferation assays, dual targeting with trastuzumab and gefitinib or lapatinib showed synergy or additivity in both SKBR-3 and BT-474 cells. Trastuzumab (10 nM) or gefitinib (5 µM) alone did not induce significant apoptosis, whereas lapatinib (0.75 µM) induced significant apoptosis in SKBR-3 cells. Trastuzumab combined with lapatinib further enhanced apoptosis induction. Trastuzumab (10 nM) and gefitinib (5 µM) induced apoptosis comparable to lapatinib alone (0.75 µM), suggesting that inhibition of both EGFR and HER-2 may be required to induce apoptosis in these cells. Pre-treatment with trastuzumab and gefitinib or lapatinib enhanced response to chemotherapy in vitro. The combination of trastuzumab and lapatinib also effectively blocked tumour growth in vivo. Dual targeting of EGFR and HER-2, by combining trastuzumab with EGFR/HER-2 tyrosine kinase inhibitors, may improve response in HER-2 overexpressing breast cancer cells that also express EGFR.

Bioluminescent imaging (BLI) is a non-invasive imaging modality widely used in the field of pre-clinical oncology research. Imaging of small animal tumour models using BLI involves the generation of light by luciferase-expressing cells in the animal following administration of substrate. This light may be imaged using an external detector. The technique allows a variety of tumour-associated properties to be visualized dynamically in living models. The increasing use of BLI as a small-animal imaging modality has led to advances in the development of xenogeneic, orthotopic, and genetically engineered animal models expressing luciferase genes. This review aims to provide insight into the principles of BLI and its applications in cancer research. Many studies to assess tumour growth and development, as well as efficacy of candidate therapeutics, have been performed using BLI. More recently, advances have also been made using bioluminescent imaging in studies of protein-protein interactions, genetic screening, cell-cycle regulators, and spontaneous cancer development. Such novel studies highlight the versatility and potential of bioluminescent imaging in future oncological research.

Photodynamic therapy (PDT) is now a well-recognized modality for the treatment of cancer. While PDT has developed progressively over the last century, great advances have been observed in the field in recent years. The concept of dual selectivity of PDT agents is now widely accepted due to the relative specificity and selectivity of PDT along with the absence of harmful side effects often encountered with chemotherapy or radiotherapy. Traditionally, porphyrin-based photosensitizers have dominated the PDT field but these first generation photosensitizers have several disadvantages, with poor light absorption and cutaneous photosensitivity being the predominant side effects. As a result, the requirement for new photosensitizers, including second generation porphyrins and porphyrin derivatives as well as third generation photosensitizers has arisen, with the aim of alleviating the problems encountered with first generation porphyrins and improving the efficacy of PDT. The investigation of nonporphyrin photosensitizers for the development of novel PDT agents has been considerably less extensive than porphyrin-based compounds; however, structural modification of nonporphyrin photosensitizers has allowed for manipulation of the photochemotherapeutic properties. The aim of this review is to provide an insight into PDT photosensitizers clinically approved for application in oncology, as well as those which show significant potential in ongoing preclinical studies.

Resistance to bevacizumab (BEV) in glioblastoma is believed to occur via activation of molecular networks including the mTOR/PI3K pathway. Using an MR/PET molecular imaging biomarker approach, we investigated the response to combining BEV with the mTOR/PI3K inhibitor BEZ235.Tumours were established by orthotopically implanting U87MG-luc2 cells in mice. Animals were treated with BEZ235 and/or BEV, and imaged using diffusion-weighted-MRI, T2-weighted and T2*-weighted before and after administration of superparamagnetic iron oxide contrast agent. Maps for changes in relaxation rates (ΔR2, ΔR2* and apparent diffusion coefficient) were calculated. Vessel size index and microvessel density index were derived. 3'-Deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) PET and O-(2-[(18)F]fluoroethyl)-L-tyrosine ([(18)F]FET) PET were further performed and tumour endothelium/proliferation markers assessed by immunohistochemistry.Treatment with BEV resulted in a pronounced decrease in tumour volume (T2-weighted MRI). No additive effect on tumour volume was observed with the BEV/BEZ235 combination compared with BEV monotherapy. The Ki67 proliferation index and [(18)F]FLT uptake studies were used to support the observations. Using ΔR2* and ΔR2 values, respectively, the BEV/BEZ235 combination significantly reduced tumour microvessel volume in comparison to BEV alone. Decreased microvessel density index was further observed in animals treated with the combination, supported by von Willebrand factor (vWF) immunohistochemistry. [(18)F]FET uptake was decreased following treatment with BEV alone, but was not further reduced following treatment with the combination. vWF immunohistochemistry analysis showed that the mean tumour vessel size was increased in all cohorts.Assessing MR imaging biomarker parameters together with [(18)F]FET and [(18)F]FLT PET provided information on mechanism of action of the drug combination and clues as to potential clinical responses. Following translation to clinical use, treatment with a BEV/BEZ235 combination could reduce peritumoral oedema obviating the requirement for steroids. The use of hypothesis-driven molecular imaging studies facilitates the preclinical evaluation of drug response. Studies of this kind may more accurately predict the clinical potential of the BEV/BEZ235 combination regimen as a novel therapeutic approach in oncology.

Background: The single hotspot mutation AKT1 [G49A:E17K] has been described in several cancers, with the highest incidence observed in breast cancer. However, its precise role in disease etiology remains unknown.Methods: We analyzed more than 600 breast cancer tumor samples and circulating tumor DNA for AKT1 E17K and alterations in other cancer-associated genes using Beads, Emulsions, Amplification, and Magnetics digital polymerase chain reaction technology and targeted exome sequencing.Results: Overall AKT1 E17K mutation prevalence was 6.3 % and not correlated with age or menopausal stage. AKT1 E17K mutation frequency tended to be lower in patients with grade 3 disease (1.9 %) compared with those with grade 1 (11.1 %) or grade 2 (6 %) disease. In two cohorts of patients with advanced metastatic disease, 98.0 % (n = 50) and 97.1 % (n = 35) concordance was obtained between tissue and blood samples for the AKT1 E17K mutation, and mutation capture rates of 66.7 % (2/3) and 85.7 % (6/7) in blood versus tissue samples were observed. Although AKT1-mutant tumor specimens were often found to harbor concurrent alterations in other driver genes, a subset of specimens harboring AKT1 E17K as the only known driver alteration was also identified. Initial follow-up survival data suggest that AKT1 E17K could be associated with increased mortality. These findings warrant additional long-term follow-up.Conclusions: The data suggest that AKT1 E17K is the most likely disease driver in certain breast cancer patients. Blood-based mutation detection is achievable in advanced-stage disease. These findings underpin the need for a further enhanced-precision medicine paradigm in the treatment of breast cancer.

During placental development, vessel formation occurs initially by vasculogenesis and subsequently by branching and nonbranching angiogenesis. We investigated vascular endothelial growth factor (VEGF)-A, angiopoietin (Ang)-1 and -2 transcript profiles, and the protein products that they encode in placentas from normotensive pregnancies throughout pregnancy. In addition, we compared these genes in placentas from normotensive women and those with preeclampsia during the third trimester. Quantitative real-time PCR analysis demonstrated that VEGF-A and Ang1 mRNA increased in a linear pattern by 2.5 (not significant) and 2.8%/wk (P = 0.034), respectively, whereas Ang2 decreased logarithmically by 3.5%/wk (P = 0.0003). Ang2 mRNA was 400- and 100-fold higher than Ang1 and VEGF-A, respectively, in the first trimester and declined to 20-fold and 7-fold in the third. Ang2 protein (ELISA) decreased by 4.7%/wk (P = 0.0001), whereas Ang1 and VEGF-A were undetectable. In preeclampsia compared with normotensive pregnancy, only VEGF-A mRNA increased significantly, by 3-fold (P = 0.006). This increase may be related to low oxygen tension, as VEGF-A is up-regulated by hypoxia. In situ hybridization and immunohistochemical studies revealed that VEGF-A was localized in cyto- and syncytiotrophoblast and perivascular cells, whereas Ang1 and Ang2 were only in syncytiotrophoblast and perivascular cells in the immature intermediate villi during the first and second trimesters, and mature intermediate and terminal villi during the third trimester. These data suggest that these molecules may play important roles in placental biology and chorionic villus vascular development and remodeling in an autocrine/paracrine manner. The tight correlation between Ang2 mRNA and protein indicates that regulation of placental vascular development occurs at the transcriptional, and not translational, level.

Background: Prior data (Barron et al. Cancer Res. 2014 74:4065-77) suggests that pre-diagnostic exposure to aspirin can have significant effects on breast tumor biology and patient outcome. It has been proposed that aspirin inhibition of COX-2 may suppress lymphangiogenesis and metastasis (Karnezis et al Cancer Cell. 2014. 21:181-95). Here, we sought to recapitulate pre-diagnostic aspirin exposure in rodent models of Her2+ breast cancer and elucidate mechanisms of action. We also determined the effect of aspirin on tumor stroma, using a co-culture system of human tumor and mesenchymal stem cells (MSC). Methods: NOD/SCID mice were orthotopically implanted with Her2+ MDA-MB-231 or HCC1954 cells. 48hr later, animals began a daily low dose [30mg/kg or 120mg/kg] of aspirin, until tumors reached 250mm3. They were then resected. 3 weeks later, HCC1954 implanted animals were treated with trastuzumab (15mg/kg) and paclitaxel (5mg/kg) for 6 weeks. Primary tissues were analysed by immunohistochemistry to assess VEGF-C, -D, COX-2, LYVE1 and CD31. RNAseq was performed on tumours to identify aspirin perturbed molecular pathways. To determine the stromal response to aspirin, patient derived MSCs were cultured either alone or with HCC1954 cells and exposed to aspirin (2.5 or 7.5mM). Secreted VEGF-C was quantified. A tubule formation assay was performed to determine the impact of aspirin on angiogenesis. Pro-angiogenic protein expression was investigated using a human angiogenesis array platform. Results: A significant delay in tumor growth was observed in both tumor models following aspirin treatment (p Conclusion: We have successfully recapitulated pre-treatment aspirin response in surgical resection models of Her2+ breast cancer, with IHC analysis confirming the impact of treatment on angiogenic and lymphangiogenic factors. RNAseq analysis implicates aspirin mediated alterations in cellular metabolism. Our data further reveals increased response to aspirin in stromal cell populations. Citation Format: Ian S. Miller, Sonja Khan, Liam P. Shiels, Sudipto Das, Bruce Moran, Finbarr P. Leacy, Paul M. Loadman, Robert S. Kerbel, Darran O' Connor, Kathleen Bennett, Róisín M. Dwyer, Annette T. Byrne. Mechanistic interrogation of pre-treatment low dose aspirin effects in HER 2 positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1254. doi:10.1158/1538-7445.AM2017-1254

Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos. Untreated, MPM has a median survival time of 6 months,and most patients die within two years of diagnosis. There is an urgent need to identify new therapies for treating MPM patients.

ObjectiveTo evaluate the accuracy of the most commonly used anthropometric-based equations in the estimation of percentage body fat (%BF) in both normal-weight and overweight women using air-displacement plethysmography (ADP) as the criterion measure.DesignA comparative study in which the equations of Durnin and Womersley (1974; DW) and Jackson, Pollock and Ward (1980) at three, four and seven sites (JPW3, JPW4 and JPW7) were validated against ADP in three groups. Group 1 included all participants, group 2 included participants with a BMI2 and group 3 included participants with a BMI≥25·0 kg/m2.SettingHuman Performance Laboratory, Institute for Sport and Health, University College Dublin, Republic of Ireland.SubjectsForty-three female participants aged between 18 and 55 years.ResultsIn all three groups, the %BF values estimated from the DW equation were closer to the criterion measure (i.e. ADP) than those estimated from the other equations. Of the three JPW equations, JPW3 provided the most accurate estimation of %BF when compared with ADP in all three groups.ConclusionIn comparison to ADP, these findings suggest that the DW equation is the most accurate anthropometric method for the estimation of %BF in both normal-weight and overweight females.

Background Glioblastoma (GBM) is the most common and malignant primary brain tumour having a median survival of just 12–18 months following standard therapy protocols. Local recurrence, post-resection and adjuvant therapy occurs in most cases. New method U87MG-luc2-bearing GBM xenografts underwent 4.5 mm craniectomy and tumour resection using microsurgical techniques. The cranial defect was repaired using a novel modified cranial window technique consisting of a circular microscope coverslip held in place with glue. Results Immediate post-operative bioluminescence imaging (BLI) revealed a gross total resection rate of 75%. At censor point 4 weeks post-resection, Kaplan–Meier survival analysis revealed 100% survival in the surgical group compared to 0% in the non-surgical cohort (p = 0.01). No neurological defects or infections in the surgical group were observed. GBM recurrence was reliably imaged using facile non-invasive optical bioluminescence (BLI) imaging with recurrence observed at week 4. Comparison with existing method(s) For the first time, we have used a novel cranial defect repair method to extend and improve intracranial surgical resection methods for application in translational GBM rodent disease models. Combining BLI and the cranial window technique described herein facilitates non-invasive serial imaging follow-up. Conclusion Within the current context we have developed a robust methodology for establishing a clinically relevant imageable GBM surgical resection model that appropriately mimics GBM recurrence post resection in patients.

Current therapeutic strategies against glioblastoma (GBM) have failed to prevent disease progression and recurrence effectively. The part played by molecular imaging (MI) in the development of novel therapies has gained increasing traction in recent years. For the first time, using expertise from an integrated multidisciplinary group of authors, herein we present a comprehensive evaluation of state-of-the-art GBM imaging and explore how advances facilitate the emergence of new treatment options. We propose a novel next-generation treatment paradigm based on the targeting of multiple hallmarks of cancer evolution that will heavily rely on MI.

Background: Breast cancer is the leading cause of cancer in women, with 1.8 million patients diagnosed globally each year. In early stage disease, anthracycline regimens are commonly employed, but the risk of cardiotoxicity is high. Replacing anthracyclines with taxanes in combination with cyclophosphamide may improve response [1] and Bevacizumab (BVZ) may provide addtional benefit. However, as BVZ can cause hypertension and increased risk of cardiac failure, a rationale exists for studying biomarkers of cardiotoxicity in this setting. Methods: We assessed a panel of biomarkers in n = 57 early stage breast cancer patients whom underwent docetaxel/cyclophosphamide/BVZ therapy on the TC-Avastin Trial (NCT00911716). Serum was collected prior to treatment and at 3 months. Cardiotoxicity was defined as decline in left ventricular ejection fraction ≥ 10% to Results: Changes in serum MPO or cTnI were not related to cardiotoxicity when analysed in the larger cohort. However, a “cut-off” PlGF fold change of 0.99 could differentiate patients who developed cardiotoxicity from those that did not. 74% of cardiotoxicity patients, compared to only 35% of non-cardiotoxicity fell below the 0.99 cut-off. Conversely, 65% of non-cardiotoxicity patients, compared to 25% of cardiotoxicity patients fell above the cut-off (p = 0.0042). Conclusions: Our data suggest that decreased PlGF at 3 months may predict cardiotoxicity. These observations require further validation. This work was funded by AngioTox, an EC FP7 IAPP Marie Curie Award (Grant Agreement 251528). [1]Jones et al., J. Clin. Oncol.27, 1177-1183 (2009); [2] Putt et al., Clin. Chem 61, 1164-1172 (2015). Citation Format: David W. Murray, Alice C. O’Farrell, Giuseppe Gullo, Ashwini Maratha, Patrick Dicker, Mairin Rafferty, Verena Murphy, Bonnie Ky, William M. Gallagher, Norma O’Donovan, John Crown, Annette T. Byrne. Analysis of putative serum biomarker candidates for cardiotoxicity prediction in a cohort of early stage breast cancer patients treated with docetaxel, cyclophosphamide, and bevacizumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1828.

Background: The Delta-like 4 (DLL4)-mediated NOTCH signaling pathway is an attractive therapeutic target in cancer. However, chronic blockade of DLL4 signalling has been observed to result in vascular toxicities, such as hepatic sinusoidal dilatation. As the underlying pathogenesis is unclear, the current study was undertaken to interrogate gene expression changes and potential safety biomarkers associated with anti-DLL4 related hepatic sinusoidal dilatation. Methods: Formalin-fixed paraffin-embedded (FFPE) liver sections were derived from male and female cynomolgus monkeys administered FDLL8566 (recombinant humanized anti-DLL4 F(ab’)2 antibody) by intravenous injection once weekly for 8 weeks at dose levels of 0, 5, 15 and 50 mg/kg/week (n = 3/sex/group). RNA was extracted from whole liver sections and laser capture-microdissected (LCM) hepatic regions with or without sinusoidal dilatation. mRNA expression was quantified using a high-throughput RT-qPCR approach, with 96 pre-validated, species-specific TaqMan gene expression assays. A non-parametric statistical test was used to assess differential gene expression between sinusoidal dilatation-affected and non-affected liver tissues. The Benjamini-Hochberg multiple testing comparison error-based False-Discovery-Rate (FDR) method was applied to calculate the adjusted p-values for each probe set. Results: Fourteen candidate genes were differentially expressed between sinusoidal dilatation-affected and non-affected cynomolgus monkey livers with an FDR adjusted p value Conclusions: This work highlights the involvement of the NOTCH pathway in the maintenance of hepatic sinusoidal homeostasis in the nonhuman primate (NHP). As preclinical toxicities in NHPs have translated to patients for other anti-DLL4 inhibitors, the changes in candidate genes and potential mechanism of toxicity identified in this study are likely to be relevant to humans. The phenotypic characteristics of hepatic sinusoidal dilatation associated with anti-DLL4 resemble the microscopic and molecular features observed in the liver following oxaliplatin treatment of patients (including involvement of angiogenesis), suggesting that similar molecular mechanisms of hepatic toxicity may exist between anti-DLL4 and oxaliplatin. This work was supported by an EU funded Industry Academia Pathways and Partnerships Marie Curie Award (AngioTox) Grant Number 251528. Citation Format: Monika A. Jarzabek, Rupal Desai, Yuda Zhu, Christina Z. de Zafra, Joe Beyer, Gary Cain, Rajiv Raja, Annette T. Byrne, Priti Hegde, Jacqueline M. Tarrant. Mechanistic insights into the pathogenesis of anti-DLL4-related hepatic sinusoidal dilatation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2096.

Background: Sunitinib is a multi-targeted agent approved for treatment of a number of cancers. However, since approval, data has continually emerged relating to toxicity profiles including hypertension, hand-foot syndrome and fatigue. Underlying mechanisms are unresolved. It has been hypothesised that the multi-parameter toxicity profile is related to ‘on-target’ kinase inhibition in ‘off-target’ tissues. Methods: To interrogate off-target effects, the Zeptosens Reverse Phase protein Array (RPA) platform was used to assess tissues obtained from mice treated with sunitinib (40 mg/kg) for 4 weeks. Additional tissue was collected for histological analyses and pathophysiologic changes assessed. Results: RPA data analyses on all organs collected (heart, kidney, liver, brain, intestine and skin) revealed the presence of differentially expressed proteins associated with damage and/or stress. Of note, Cyclin D1, MEK and phosphorylated-p21(CIP/WAF1) expression increased by 1.8-fold (p < 0.05), 2.2-fold (p < 0.1) and 1.7-fold (p < 0.1) respectively in the kidneys of sunitinib treated mice. These proteins are associated with kidney damage after chemotherapy, and induction of genes associated with renal immune response, inflammation and apoptosis. Mild proteinuria was observed in sunitinib treated animals, however no gross change in renal glomerular ultrastructure was visible via electron microscopy. Phosphorylated-FGFR1, cleaved Caspase-7, and phosphorylated-cMYC expression decreased by 0.45-fold (p < 0.05), 0.4-fold (p < 0.1) and 0.8-fold (p < 0.1) respectively in the skin of sunitinib treated mice. Down-regulation of these proteins has been associated with a perturbation of cutaneous wound healing (p-FGFR1/cleaved Caspase-7). Mice treated with high dose sunitinib (80 mg/kg) showed overt signs of skin toxicity. Histopathologic studies of the thyroid gland revealed decreased thyroid epithelial cell height (p < 0.05). Nevertheless, serum levels of triiodothyronine and thyroxine remained unchanged. An orthogonal validation study (Western blotting) is ongoing based on gene ontology, pathway analysis and observed fold changes in critical signalling pathway proteins involved in cellular stress. Conclusions: Implementation of a combined histopathologic/ RPA approach may provide a sensitive method to mechanistically elucidate the off-target toxicity sequelae associated with TKIs approved in the oncology setting. Work was supported by an EU funded Industry Academia Pathways and Partnerships Marie Curie Award (AngioTox) Grant Number 251528. RPAs were performed at Bayer Technology Services, Leverkusen, Germany. Citation Format: Alice C. O’Farrell, Adam Lafferty, Ian Miller, Rhys Evans, Maurice Cary, David Murray, Marina Alamanou, Girish Mallya Udupi, Liam Shiels, William M. Gallagher, Annette T. Byrne. Proteomic analysis of off-target toxicities following treatment with the tyrosine kinase inhibitor sunitinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3010.

Background: An overwhelming majority of phase III clinical trials in the oncology setting fail as promising results observed in the ‘pre-clinic’ are not successfully translated to patients. As such, improved models which more accurately predict outcomes are now required. Herein, we have established orthotopic surgical resection models of Her2+ breast cancer, which replicate the phenotype of clinical post-resection metastasis [1,2]. Using these models we have recapitulated the BETH adjuvant breast cancer trial (NCT00625898) where the addition of bevacizumab (BVZ) to chemotherapy plus trastuzumab (TRAST) failed to provide additional benefit in the adjuvant setting. Methods: SCID mice were orthotopically implanted with bioluminescent Her2+ MDA-MB-231or HCC1954 cells and palpable tumors resected approximately 5 weeks later. 3 weeks after resection, mice were treated with 10 mg/kg TRAST + 5mg/kg paclitaxel IP once weekly for 6 cycles with or without weekly BVZ (5mg/kg IP). Metastasis was monitored by weekly bioluminescence imaging. Results: Tumor growth and imaging data confirmed that the addition of BVZ to adjuvant TRAST + chemotherapy provided no additional benefit compared with TRAST + chemotherapy alone. Previous pre-clinical studies using inappropriate non-resection models failed to predict this response. Furthermore, Reverse Phase Protein Array analysis of treated tumors implicated HER2, VEGF, mTOR and the intrinsic mitochondrial cell death apoptotic pathways in treatment resistance. Conclusion: In summary, our data provides evidence for pre-clinical models which better predict clinical outcome in the breast cancer adjuvant setting, and which represent an important resource for interrogating resistance pathways and identifying novel biomarker signatures. [1]. Breast. 2013 Aug;22 Suppl 2:S57-65; [2]. Cancer Res. 2013 May 1;73(9):2743-8.This work was performed under Irish HPRA Authorization AE18982 and was supported by the Clinical Cancer Research Trust. ATB receives funding from the Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT Grant (CCRC13GAL). Citation Format: Liam S. Shiels, Ian S. Miller, Clare Morgan, Mattia Cremona, Robert Kerbel, Bryan Hennessey, Norma O’Donovan, John Crown, Annette T. Byrne. Recapitulating the BETH adjuvant breast cancer trial (NCT00625898) using clinically accurate orthotopic surgical resection models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3269.

Background: Photodynamic therapy (PDT) is a treatment modality for a range of diseases including cancer. The BF2-chelated tetraaryl-azadipyrromethenes (ADPMs) are an emerging class of non-porphyrin PDT agent, which have previously shown excellent photochemical and photophysical properties for therapeutic application. Herein, in vivo efficacy and mechanism of action studies have been completed for the lead agent, ADMP06. Methods: A multi-modality imaging approach was employed to assess efficacy of treatment, as well as probe the mechanism of action of ADPM06-mediated PDT. Results: Tumour ablation in 71% of animals bearing mammary tumours was achieved after delivery of 2 mg kg−1 of ADPM06 followed immediately by light irradiation with 150 J cm−2. The inherent fluorescence of ADPM06 was utilised to monitor organ biodistribution patterns, with fluorescence reaching baseline levels in all organs within 24 h. Mechanism of action studies were carried out using dynamic positron emission tomography and magnetic resonance imaging techniques, which, when taken together, indicated a decrease in tumour vascular perfusion and concomitant reduction in tumour metabolism over time after treatment. Conclusion: The encouraging treatment responses in vivo and vascular-targeting mechanism of action continue to indicate therapeutic benefit for this new class of photosensitiser.

Insulin and the insulin-like growth factors, IGF-I and IGF-II, have been reported to exert a mitogenic effect on the preimplantation mammalian embryo. Furthermore, it has been proposed that loss of imprinting of the insulin-like growth factor II receptor gene and the consequent over-production of IGF-II may be involved in the aetiology of the Enlarged Offspring Syndrome, which occurs as an artefact of in vitro embryo production. We have previously shown that apoptosis occurs in the preimplantation bovine embryo and is influenced by in vitro culture conditions. We have therefore sought to establish the effects of insulin, IGF-I and IGF-II on apoptosis and cell proliferation in bovine blastocysts in vitro. Zygotes, obtained by in vitro maturation and fertilization of follicular oocytes, were cultured to blastocysts, with or without exogenous growth factors. Embryos were stained with propidium iodide to label all nuclei and by TUNEL to label apoptotic nuclei and analyzed by epifluorescent and confocal microscopy. IGF-I and IGF-II, but not insulin, were found to increase the proportion of embryos which formed blastocysts. Insulin decreased the incidence of apoptosis without affecting blastocyst cell number. IGF-I acted to decrease apoptosis and increase total cell number and IGF-II increased cell number alone. These data suggest roles for insulin and the IGFs as mitogens and/or apoptotic survival factors during early bovine development. Perturbation of IGF-II regulated growth may be involved in fetal oversize.

The EurOPDX Consortium is an initiative of translational and clinical researchers from 14 cancer centers and universities across 9 European countries, with the common goal of creating a network of clinically relevant models of human cancer, and in particular patient-derived xenograft (PDX) models. PDX models, which are generated by transplantation into an immunodeficient mouse of a patient's tumor obtained fresh from surgery, are broadly implemented for all cancer pathologies by translational laboratories (Tentler et al., 2012). Grafted tumors have been shown to maintain high similarities with the original patient tumor. Hence, preclinical trials in large panels of models recapitulating the intertumor heterogeneity of human cancer have been shown to reproduce data obtained in clinical trials (Bertotti et al., 2011; Julien et al., 2012). The primary goal of the EurOPDX Consortium is to share PDX models and perform collaborative multicenter and multipathology "xeno patient" trials within molecularly defined tumor subsets, resulting in hypothesis-driven personalized medicine strategies for cancer patients. Altogether, the Consortium displays a panel of more than 1,500 PDX subcutaneous and orthotopic models in more than 30 pathologies, including for instance 640 models obtained from primary tumors or metastases of colorectal cancer, 200 models of pancreatic cancer, and over 120 models of breast cancer. The characterization of models will be harmonized and will include deep molecular profiling and pharmacogenomic data corresponding to current anticancer therapies. Through implementation of a shared database and the emergence of a common biobank of patient-derived viable cancer specimens, the Consortium will use characterization data to identify novel strategies for pinpointing new targets and overcoming drug resistance, and will validate therapeutic hits in collaborative multicentric preclinical trials. Best practices for the performance of such trials in mice will be implemented, with the goal of advancing their predictivity for success in the clinic. The Consortium will also generate new PDX models and integrate complementary models such as ex vivo 2D and 3D assays to achieve research goals. Implementing PDX models in the discovery of predictive biomarkers for targeted therapies will be a key objective. This initiative will create a pipeline that will greatly accelerate and advance the development of novel therapeutic strategies and their validation for cancer treatment, through more predictive preclinical or "co-clinical" data. Through this collaborative effort that is to our knowledge, unique in Europe, and the participation of pharmaceutical companies and SMEs involved in drug development, we aim to reduce the duplication of efforts in oncology drug development, ultimately improving the quality of life and overall survival of cancer patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A8. Citation Format: Emilie Vinolo, Elisabetta Marangoni, Violeta Serra, Frederic Amant, Andrea Bertotti, Andrew V. Biankin, Anne-Lise Børresen-Dale, Alejandra Bruna, Eva Budinská, Annette Byrne, Carlos Caldas, David Chang, Robert Clarke, Els Hermans, Manuel Hidalgo, Steven de Jong, Jos Jonkers, Pedro López, Gunhild Mari Mælandsmo, Vlad Popovici, Sergio Roman-Roman, Joan Seoane, Livio Trusolino, Alberto Villanueva, on behalf of the EurOPDX Consortium. The EurOPDX consortium: Sharing patient tumor-derived xenografts for collaborative multicentric preclinical trials. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A8.

Glioblastoma multiforme (GBM), the most aggressive brain malignancy, is characterized by extensive cellular proliferation, angiogenesis, and single-cell infiltration into the brain. We have previously shown that a xenograft model based on serial xenotransplantation of human biopsy spheroids in immunodeficient rodents maintains the genotype and phenotype of the original patient tumor. The present work further extends this model for optical assessment of tumor engraftment and growth using bioluminescence imaging (BLI). A method for successful lentiviral transduction of the firefly luciferase gene into multicellular spheroids was developed and implemented to generate optically active patient tumor cells. Luciferase-expressing spheroids were injected into the brains of immunodeficient mice. BLI photon counts and tumor volumes from magnetic resonance imaging (MRI) were correlated. Luciferase-expressing tumors recapitulated the histopathologic hallmarks of human GBMs and showed proliferation rates and microvessel density counts similar to those of wild-type xenografts. Moreover, we detected widespread invasion of luciferase-positive tumor cells in the mouse brains. Herein we describe a novel optically active model of GBM that closely mimics human pathology with respect to invasion, angiogenesis, and proliferation indices. The model may thus be routinely used for the assessment of novel anti-GBM therapeutic approaches implementing well-established and cost-effective optical imaging strategies.

Background In 2006 the Royal College of Surgeons in Ireland, (RCSI), introduced the first four year Graduate Entry Programme (GEP) in medicine in Ireland in line with national policy to broaden access to medical education. One concern considered at the time, was whether the GEP students could be trained to the same standard as their undergraduate Direct Entry Programme (DEP, five/six year duration) counterparts in the shorter time frame. Since students from both cohorts undertake the same examinations in the final two years, it is possible to directly compare GEP vs DEP outcomes. The primary aim of the current study was to analyse the comparative performance of GEP and DEP students undergoing these examinations between 2008 and 2013. Methods Scores from five assessments performed during the final two years were transformed to z scores for each student and 4 scores for the penultimate year were summed to create a unit weighted composite score. The resultant scores for each of the two years were used to assess the comparative performance of GEP vs DEP cohorts and to perform sub-cohort analyses of GEP outcomes. Results In all cohorts/years examined, evidence demonstrated significantly better assessment outcomes for the GEP group for the final two years’ examinations as compared with the DEP group. In all but one cohort examined, this advantage was retained when nationality factors were excluded. Further analyses showed no difference in outcomes between GEP students having science vs. non-science backgrounds and/or between those from EU vs non-EU backgrounds. Finally, data suggested weak correlations between total composite scores and entry scores in American (r = 0.15) and Australian (r = 0.08) medical school admissions tests. Conclusions We have shown for the first time in Ireland, that graduate-entry students perform at least as well, or even better, than a corresponding undergraduate-entry group. Moreover, having a scientific background on entry to the GEP confers no advantage in final assessments. These data provide evidence of the viability of the graduate entry route into medical education in Ireland.